ANNUAL REPORT
OF
PROGRAM ACTIVITIES
U : NATIONAL HEART AND LUNG INSTITUTE
Fiscal Year 1973
Part II
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
Public Health Service National Institutes of Health
INTRAMURAL RESEARCH
NATIONAL HEART AND LUNG INSTITUTE
July 1, 1972 - June 30, 1973
INTRAMURAL RESEARCH
Project Reports
Cardiology Branch
Summary 1
1. Phosphorylation of human platelet myosin and contractile
proteins from other sources 11
2(c) Characterization of cardiac contractile proteins from
patients with idiopathic hypertrophic subaortic stenosis 12
3(c) Echocardiographic findings in patients with idiopathic
hypereosinophilic syndromes 13
4. Effect of lidocaine and of elevating arterial pressure on
the incidence of spontaneous ventricular fibrillation in
dogs during coronary occlusion 14
5. Effect of nitroglycerin on the incidence of spontaneous
ventricular fibrillation during coronary occlusion in dogs- 16
6(c) Echocardiographic assessment of secondary cardiomyopathies- 17
7(c) ASH masquerading as coronary artery disease and idiopathic
myocardial hypertrophy 18
8(c) Electrocardiographic findings in 100 patients with
asjonmetric septal hypertrophy 19
9(c) Familial incidence and genetic transmission of IHSS 20
10(c) Coronary collateral function in patients without coronary
artery disease 21
11. Visualization of acute myocardial infarction by the
Radionuclide Gallium-67 22
12. Lack of deleterious effects of vagally mediated brady-
cardia during acute myocardial ischemia in the dog 23
13(c) Effects of nitroglycerin on coronary collateral function
in patients with coronary occlusive disease 24
14. Failure of nitroglycerin to alleviate acute myocardial
ischemia or improve collateral flow when M^02 is held con-
stant in the heart-lung preparation 26
15(c) Quantification of ventricular regularization in atrial
f ibrillation — — 27
16(c) Quantitative assessment of left ventricular function in
man utilizing roentgen videodensitometry 29
17. Adrenergic influences on ventricular fibrillation threshold
and post-occlusion arrhythmias 30
18(c) Factors affecting the operative mortality in aortic
valATular disease 3 2
19(c) Mitral valve position in patients with asymmetric septal
hypertrophy 33
20. A video scanner-analog computer system for the analysis of
routine echocardiograms 35
21(c) Long-term effects of operation on obstruction and LV
hypertrophy in IHSS 36
22(c) Distribution of the cardiomyopathy in IHSS 37
23(c) Non- invasive determination of the pressure gradient in
patients with idiopathic hypertrophic subaortic stenosis 39
24(c) Echocardiographic diagnosis of IHSS by detection of
asymmetric septal hypertrophy 41
25(c) The clinical characteristics of obstructive and non-
obstructive asymmetric septal hypertrophy 42
26(c) A real time system for two-dimensional echocardiography 43
27(c) Effects of operation on the cardiac response to exercise
in patients with IHSS 45
28. Reduction in extent of myocardial infarction when nitro-
glycerin and methoxamine are administered during coronary
occlusion 46
29(c) Preoperative predictors of the long-term results of wave
replacement in patients with aortic regurgitation 47
30(c) Characterization of the cardiac response to exercise and
effects of propranolol in patients with asymmetric septal
hy pe r t r ophy 4 9
31. Cholinergic innervation of the cardiac conduction system:
autonomic and functional correlations 51
32. Electrical stability of acutely ischemic myocardium: Inde-
pendent influences of heart rate and vagal tone 53
33. The electrical stability of acutely ischemic myocardial
effects of nitroglycerin 55
34. The role of prostaglandins in the control of coronary
vascular tone 56
35. Physiological and biochemical characteristics of a new
group of inotropic agents: Analogues of angiotensin II 58
36(c) Differences in distribution of morphologic abnormalities
in patients with obstructive and non-obstructive asymmetric
septal hypertrophy (ASH) : Light and electron microscopic
ob s e rva t io ns 5 9
37(c) Significance of multiple intercalated discs in hyper-
trophied human myocardium 61
38(c) Postoperative response to intense upright exercise in
patients with ventricular septal defect and pulmonary
hypertension: relation of impairment to age at operation 62
39(c) The occurrence of a-glycogen in human cardiac muscle cells- 63
40(c) Unusual evolution of acquired infundibular pulmonary
stenosis in patients with ventricular sepal defect:
Clinical and morphologic observations 64
41(c) Ultrastructural features of myocardial cell degeneration in
patients with cardiac hypertrophy 65
42. Effects of vagal stimulation on survival during experi-
mental acute myocardial infarction 66
43(c) Evaluation of the ability of echocardiography to measure
alterations in left ventricular volume 67
44(c) Use of biventricular cineangiography in the evaluation of
patients with asymmetric septal hypertrophy 68
45(c) Echocardiography during upright exercise 69
46(c) Sustained effects of nitroglycerin ointment in patients
with angina pectoris 70
47(c) Deterioration of myocardial function following aorto-
coronary bypass operation 71
48. Increased myocardial ischemia caused by reflexly induced
hypotension during coronary occlusion in the conscious dog- 72
ii
Clinic of Surgery
Summary 73
49(c) NU-5 atomic powered pacemaker — experimental and clinical
evaluation 79
50(c) Long-term follow-up of patients with congenital aortic
stenosis treated with aortic commissurotomy 80
51. The pathophysiology of intimal hyperlasia of the venous
graft in the arterial system 81
52(c) Silastic ball variance detection 82
53(c) Light and electron microscopic evaluation of hypertrophied
right ventricular outflow tract muscle from patients with
; I ■ congenital heart disease 83
'"-']'.', 54(c) Assessment of aortic regurgitation in patients with the
use of the Doppler Bidirectional Velocitometer 84
55, Experimental production of progressive mitral stenosis 85
, . , 56. The effects of naloxone hydrochloride on ventricular
irritability ■ 86
57(c) Clinical effects of abrupt decrease in left atrial pressure
on renal sodium excretory patterns 87
58. Systemic and renal responses to a graded reduction in
cardiac output — ' 88
59. Blood volume expansion without altering hematocrit. Renal
effects in a cross-transfusion model 89
60. Systemic and renal effects of methyl prednisolone following
a graded reduction in cardiac output 90
61. Apical-aortic valvular anastomosis for diffuse left
ventricular outflow tract obstruction 91
62. Experimental evaluation of the membrane oxygenator 92
63. Transcutaneous measurement of aortic regurgitation with
a Doppler Velocitometer 93
64. A new canine model for echocardiographic evaluation of
myocardial function 94
65. A comparison of left ventricular pressure, aortic flow and
echocardiography in determining the response of left
ventricular function or propranolol 95
66. Experimental creation of infundibular pulmonic stenosis
ventricular septal defect and right ventricular hyper-
tension in puppies 96
" 67(c) A review of eighteen years experience with total correction
of tetralogy of Fallot at the NIH 97
68. The experimental creation of infundibular pulmonic stenosis
and demonstration of its hemodynamic relationships 99
69. Hemodynamic and anatomic evaluation of experimental
valvular aortic stenosis 101
70. Effects of aortic stenosis on (A) coronary blood flow and
(B) regional distribution of blood flow 102
71. Hypothermic asanguineous circulatory arrest (HACA) 103
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Summa ry — -^ — ■ 105
72. A new radiochemical esterolytic assay for human urinary
urokinase 117
73. Clinical biochemistry of the kallikrein-kinin system 119
74. Peptide biochemistry 122
75. Studies on the enzymes involved in the activation of human
plasma kallikrein: PF/dil and Hageman Factor 124
76(c) Antigen-antibody-induced release of mediators from
passively sensitized human lung: Studies on histamine and
arginine esterases 128
77. Biochemistry of the kallikrein-kininogen-kinin system 130
78. The role of prostaglandins in the vascular system 133
79. Studies on the isolation and characterization of
clostridial electron transfer proteins and other iron-
sulfur proteins 135
80(c) Effect of renal infarction on aromatic amino acid decar-
boxylase levels in serum and kidney tissue 138
81. Characterization of bovine adrenal dopamine 3-hydroxylase- 140
82. The regulation of the hydroxyindole pathway of tryptophan
me tabolism 142
83. In vivo protein synthesis in heart, aorta, and mesenteric
artery of normotensive and spontaneously hypertensive rats
84. Studies on 5,6- and 5, 7-dihydroxytryptamine — Agents that
cause degeneration of serotonergic neurons 147
85. Studies on serotonin N-acetyl-transf erase in mammalian
pineal glands 149
86. Studies on tryptophan hydroxylase — 153
87. Monoamine metabolism and blood pressure of spontaneously
hypertensive rats 155
88. Strain differences of catecholamine synthesizing enzyme
activity in the rat 158
89. cAMP dependent protein kinase in vascular smooth muscle
and its relationship to calcium uptake 160
90(c) The effects of varying salt intake and of acute saline
infusion on norepinephrine and dopamine excretion in man — 162
91(c) Clinical investigation of cardiovascular drugs 164
92. The role of endogenous prostaglandin synthesis in the
control of the coronary circulation 167
93(c) The role of renal prostaglandins in sodium homeostasis
and blood pressure regulation in man 170
94(c) Studies on the biological role of histamine: Alteration of
histamine metabolism in man and animals by salicylates and
other inhibitors 172
95(c) The biological role of histamine and monoamines: Studies
of histaminase and other enzyme activities in medullary
thyroid carcinoma and hyperlipoproteinemia 175
96. The biological role of histamine and other amines:
Participation of histamine and serotonin in inflammation — 179
97. Studies on the biological role of histamine and poly-
amines: Ornithine decarboxylase and histaminase activities
in rat thymus and other organs 182
98(c) Metabolism of hydroxyproline and collagen 185
99(c) Studies on the interrelationships between the renin-
angiotensin system, urinary and plasma kallikrein and
prostaglandins in normal volunteers and hypertensive
patients 188
100. Effects of tolbutamide on plasma renin activity (P.R.A.) — 190
101(c) Urinary and plasma kallikrein 192
102. Studies on renal catecholamine synthesis 195
103. Acute effects of alphamethyldopa on plasma renin activity- 197
104(c) Effects of steroid, thyroid and protein hormones, electro-
lytes and psychodelic agents on neural and sensory
f unc tion 199
105(c) Trace metal metabolism 206
106(c) Taste and olfaction 217
Molecular Hematology Branch
Summary 23 7
107. Metabolism of globin mRNA transcription in bone marrow
cells 239
108. The mechanism of hemoglobin biosynthesis in rabbit
reticulocyte cell-free systems 240
109(c) Regulation of hemoglobin chain synthesis in beta-
thalassemia 242
110. Evolutionary homology of components of the protein
synthesizing machinery 244
111. The mechanism of hemoglobin switching in sheep and goats — 246
112. Mechanism of action of the enzjnne RNA-directed DNA
p o lyme r as e 248
Laboratory of Technical Development
Summary 251
113. Discrete cell temperature measurement study 259
114. Development of stopped-flow micro-calorimetry for the
study of biochemical and cellular reactions; applications
to clinical biochemistry 262
115. The development of methods for investigating the mechanism
of biochemical reactions important in cardiology, pul-
monary and respiratory function, and in the circulation 265
116. An automated method for rapid bacterial and mammalian cell
growth and assay 269
117. Fluorescent complexes of proteins 273
118. Methodology in fluorescence measurements 276
119. Applications of fluorescence in biochemistry 279
120. Countercurrent chromatography: Liquid-liquid partition
chromatography without solid support (Part II) 282
121. Countercurrent chromatography: Liquid-liquid partition
chromatography without solid support (Part I) 286
122(c) Development of the spiral coil membrane blood oxygenator
and systems for long-term temporary support of pulmonary
and cardiovascular systems 290
123. Blood flow measurement using nuclear magnetic resonance
te chniques 295
124. Peltier-seebeck equilibrator 298
125. Pseudo differentiator 300
126. Measurement of picomole amounts of carbon dioxide 301
127. Calorimetric measurement of carbon dioxide 303
Laboratory of Kidney and Electrolyte Metabolism
Summary 307
128. The effect of chlorpropamide on the water permeability
response of the toad bladder to vasopressin 313
129. A study of the leakage of cyclic AMP from the toad
urinary bladder 315
130. A study of the concentration of cyclic AMP in the urinary
bladder of the toad 317
131. Function of the early distal tubule of frog 320
132. Function of the thick ascending limb of Henle's loop 322
133. The study of ion transport in renal cortical collecting
tubules 3 25
134. Mechanism of salt and water transport by proximal renal
t ubu 1 e s 328
135. Volume regulation in duck erythrocytes. Hormonal control
of cation transport in duck erythrocytes 330
136. Effect of cardiac glycosides on the excitation-contraction
system in mammalian skeletal muscle 334
Pulmonary Branch
Summary - Section on Pulmonary Biochemistry 339
137(c) Sites of action and physiologic importance to some
mediators of the type I allergic reaction in man 341
138. Models of lung growth 343
139. Mechanism of collagen and non-collagen protein synthesis
in rabbit lung cell-free systems 345
140(c) The composition and synthesis of collagen in human lung 347
Laboratory of Chemistry
Summary 3 49
141. A study of organic and bio-organic systems by magnetic
resonance 355
142. Mass spectrometry and structure of natural products 358
143. Structural characterization of natural products 361
144. X-ray crystallographic studies of molecules of interest
for theoretical reasons and for understanding of modes
of physiological actions 364
145. Theoretical and computational investigations of the
limitations of small-angle X-ray scattering 367
146. The characterization of natural materials 368
Laboratory of Biochemistiry
Summary 371
147. Studies on the anaerobic metabolism of the branched-
148. Biochemical genetics of NH^-assimilatory enzymes in
149. Kinetics and mechanisms of biochemical reactions 392
150. 1. Anaerobic metabolism of certain amino acids and other
nitrogen compounds with especial reference to the role
of B]^2 coenzyme and to electron transfer and phosphory-
lation reactions involved.
2. Methane biosynthesis and one-carbon compound
metabolism 398
vi
151. 1. Studies on pyridoxal-P and B-.^ coenz3rrae-dependent
amino mutases.
2. Studies on 3,5-diaralnohexanoate dehydrogenase 402
152. Menadlone-dependent p-nltrophenylphosphatase of
Clostridium stlcklandll 405
153. Ciliary structure and function.
1. Motility: Energy transducing proteins.
2. Mlcrotuble structure: a. Mutants resistant to drugs
which Inhibit normal microtubule assembly, b.
Tubulin affinity labeling with podophyllotoxln
derivatives 407
154. Enzyme structure and mechanisms of action and control 411
155. Regulatory proteins of glutamlne synthetase from E^. coll
separation, purification, and regulatory mechanisms 418
156. 1. Components Involved In the adenylylation and
deadenylylatlon of glutamlne synthetase from
Pseudomonas putlda
2. Preparation and characterization of E-adenylylated
glutamlne synthetase 470
157. 1. Kinetic reaction mechanism of the y-glutamyl trans-
ferase activity of E^. coll glutamlne synthetase.
2. Purification and properties of the Pjj regulatory
protein associated with the E^. coll ATP: Glutamlne
synthetase adenylyl transferase 422
158. Regulation of nitrogen metabolism In E^ coll W. The
application of continuous culture techniques 425
159. 1. Stereochemical studies of enzymatic reactions
2. Synthetic studies of organic compounds of biological
Interest 429
160. DNA synthesis In E^ coll 433
f'161. Studies on the binding site of mouse myeloma protein,
specific for the 2,4-dlnltrophenyl (DNP) group 436
162. Adenosine triphosphate - actln Interactions. Application
of a contact-labeling technique to elucidation of the
protein binding site 438
163. Studies on the self-radlolysls of chymotrypsln, labeled
with a trltlated dllsopropylphosphoryl (DIP) group. Re-
lationship of free-radical distribution to binding site
composition 440
164. Proteolytic fragmentation of fibrinogen 442
165. Structure of enzymatic fragments of fibrinogen 444
166. The Interaction of actln and myosin 446
167. Control of the Interaction of actln and myosin by native
tropomyosin ' 449
168. Resolution and characterization of the electron transport
chain of E. coll 451
169. The enzymatic propertels of SH]^ blocked myosin and its
interaction with actln and ATP 454
170. Membrane biochemistry: Chemical composition and molecular
organization of the amoeba plasma membrane 457
vii
171. Membrane biochemistry: Mechanism of membrane fusion in
amoeba in relation to endocytosis 460
172. Cytology of Acanthamoeba 463
Laboratory of Chemical Pharmacology
Summary 465
173. Acetaminophen-induced liver necrosis. I. Relationship
between hepatic necrosis and the covalent binding of an
acetaminophen metabolite to liver macromolecules 473
174. Acetaminophen-induced liver necrosis. II. Protective role
of glutathione 474
175. Acetaminophen-induced liver necrosis. III. Relationship
between severity of liver necrosis and urinary metabolites
of acetaminophen 479
176(c) Acetaicinophen-induced liver necrosis. IV. Clinical
177. Acetaminophen-induced necrosis. V. N-oxidation of
acetanilide analogues 485
178. The role of cytochrone P-450 in N-hydroxylation of N-
acetylarylamines. Studies with 2-acetylaminofluorene
179. Role of drug-metabolism in the hemolysis caused by aniline
in rats 489
180. Role of the methemoglobinemia in hemolysis caused by
aniline and acetanilide in rats 493
181. Furosemide-induced hepatic and renal tubular necrosis.
I. Occurrence of tissue lesions and modulation of the
toxicity by treatments which alter drug-metabolizing
act ivi ty 496
182. Furosemide-induced hepatic necrosis. II, Comparison of
necrosis with covalent binding of furosemide 498
183. Drug-induced necrosis in the kidney: Comparison of
mercuric chloride, p-aminophenol and furosemide 500
184. Possible role of cytochrome c^ reductase and cytochrome
P-450 in the covalent binding of halome thanes to micro-
somal protein - studies using specific antibody against
cytochrome c^ reductase and by specific destruction of
cytochrome P-450 502
185. Mechanism of renal toxicity-induced by chloroform 507
186. Relationship between covalent binding of aromatic hydro-
carbons to lungs and production of bronchial necrosis 510
187. Biochemical changes after acetaminophen and furosemide-
induced liver injury 516
188(c) Isoniazid-related hepatitis in man: Significance of ab-
normal liver function tests during prophylactic therapy
and correlation of susceptibility with acetylator pheno-
ty pe 5 18
189. Isoniazid-induced hepatic damage: Investigations of the
possible formation of an alkylating metabolite 521
190. The influence of pretreatment of rats on isoniazid
metabolism 524
191. Spironolactone and testicular cytochrome P-450: Decrease
of cytochrome P-450 and progesterone 17a-hydroxylase after
spironolactone administration 527
viii
192. Toxic effects of l,l'-dlinethyl-4,4'-dipyrldilium di-
chloride (paraquat) in the rat and rabbit 530
193. Mechanism of dimethylnitrosamine-lnduced liver toxicity 533
194. Covalent binding as a basis of toxicity of chloramphenicol:
Possible modification of structure to diminish toxicity of
chloramphenicol without loss of antibacterial activity 535
195. Drug metabolism in fetal tissue 537
196. Interaction between nitrofurazone and various tissues
197. Effect of chronic treatment with phenobarbital or 3-
methylcholanthrene on cytochrome P-450 and metabolic
activity in testes, adrenals, lungs and liver 541
198. Studies on the role of NADH in the metabolism of drugs
by cytochrome P-450 enzymes in liver microsomes 547
199. Development of a histochemical method for the assay of
glutathione in tissues 550
200. The role of electrolytes in binding, mobilization and re-
lease of norepinephrine in adrenergic neurons 552
201. Does sodium inhibit calcium-dependent mobilization of
neurotransmitter at an intracellular or extracellular
site of action 555
202. The effects of exogenous metabolites of glycolysis on the
retention of norepinephrine by rat heart slices 557
203. Studies on the potentiation of CCl/-induced hepatotoxicity
in rats by pretreatment with ethyl alcohol, isopropanol,
or acetone 559
204. Mechanisms involved in thyprotective action of Dibenamine
pretreatment against the hepatotoxicity of CCl, 562
205. Diene conjugation and lipid peroxidation as a mechanism
of hepatotoxicity 564
206. Temperature-dependent toxicity of adrenergic agonists in
mice 566
207. Studies on the in vivo covalent binding of CCl, and CCl^Br- 568
208. Studies on covalent binding as related to hepatotoxicity:
comparison of CCL, and CBrCln 574
209. Covalent binding as a biochemical mechanism for toxicities:
Covalent binding of stllbene to liver microsomes 579
210. The mechanisms of hydrazine toxicity 581
211. The role and mechanism of action of hormones in the control
of cellular processes: I. The study of the effects of
carbamylcholine on cyclic 3' ,5'-guanosine monophosphate
levels in the lung 584
212. The role and mechanism of action of hormones in the control
of cellular processes. II. The role of GTP in the
prostaglandin E-, activiation of the human blood platelet
adenyl cyclase 587
213. Purification and characterization of the enzymatic ion
transport system 589
214. Comparative pharmacology of receptor systems in smooth
ix
215. Studies on the chemical nature of the pharmacological
receptor systems in vascular smooth muscle. I. Labeling
studies of proteins associated with the a-adrenergic
receptor in rabbit aorta 595
216. Biochemical studies of the interaction of oxidant
pollutants with the lung. I. The effect of N0„ on phospho-
lipid bilayers 597
217. Physicochemical studies of lung surfactant lipoprotein 599
218. Physicochemical studies of complexes between drugs and
biomolecules. XI. Fluorescence polarization as a tech-
nique for measuring drug binding to plasma albumin 602
219. Physicochemical studies of complexes between drugs and
biomolecules. XHI. Spin label studies of horse erythro-
cyte carbonic anhydrases C and D 604
220. Physicochemical studies of complexes between drugs and
biomolecules. XIV. A spin label study of avidin 606
221. Physicochemical studies of complexes between drugs and
biomolecules. XV. A spin label study of azoester
(methyl phenyldiazene carboxylate) induced damage to
human erythrocyte ghost membranes 608
Office of the Director, Intramural Research
222. Mathematical theory of renal function 611
223. Computer simulation of renal function 614
ODIR - Section of Pathology
Summary 61 7
224(c) Alcoholism — an important but unemphasized factor predis-
posing to infective endocarditis with reemphasis on the
syndrome of pneumococcal endocarditis, meningitis and
pneumonitis in alcoholics 625
225(c) The quadricuspid semilunar valve 626
226(c) A new cause of a diastolic murmur after replacement of
the aortic valve with a caged ball prosthesis 627
227(c) Calcification of prosthetic valve anuli: A late compli-
cation of cardiac valve replacement 628
228(c) Mitral valvular disease. A clinicopathologic survey
of the conditions causing the mitral valve to function
ab no rma 1 ly 629
229(c) Pathologic anatony of the cardiomyopathies (idiopathic
dilated and hypertrophic types, infiltrative types and
endomyocardial disease with and without eosinophilia) 630
230 Lesions observed in arterial autogenous vein grafts
light and electron microscopic evaluation 631
231(c) Cardiovascular pathology in hyperlipoproteinemia
anatomic observations in 41 necropsy patients with
normal or abnormal serum lipoprotein patterns 632
232(c) Structural features of cardiac myxomas 633
233(c) Operative treatment of hypertrophic obstructive cardio-
myopathy. The case against mitral valve replacement 634
234(c) The pathologic anatomy of cardiac valve replacement 635
235(c) Structural alterations in tissue cardiac valves
implanted in hiamans and calves 636
236(c) Non-rheumatic valvular cardiac disease. A clinico-
pathologic survey of 27 different conditions causing
valvular dysfunction 637
237(c) Ankylosing spondylitis and aortic regurgitation
description of the characteristic cardiovascular lesion
from study of 8 necropsy patients 638
238(c) Cardiac ultrastructural changes induced by daunorubicin
therapy 639
239(c) Early human arteriosclerosis 641
240(c) The coronary arteries and myocardium in acute myocardial
infarction and shock 643
241(c) Pathology of Sipple's syndrome 645
242(c) Chylomicrons and the formation of foam cells in type I
hyperlipoproteinemia 647
243(c) Ultrastructural studies of Tangier disease 648
244(c) Ultrastructure and cytochemistry of glycogen in cardiac
d is eas es 649
245(c) Intermyo fibrillar and nuclear-myo fibrillar connections
in human and canine myocardium. An ultrastructural
study 650
246(c) Plasma membrane extensions in intercalated discs of human
myocardium and their relationship to partial dissociation
of the discs 651
247(c) Fatal tachyarrhythmias associated with myofibrillar loss,
mitochondrial hyperplasia and fatty change in myocardium.
A new clinico-pathologic syndrome in children 652
ODIR - Section on Experimental Atherosclerosis
Summary 653
248. Blood velocity profiles and hemodynamic stresses in the
aorta and its major branches, including the large coronary
arteries 657
249. Vascular mechanics: arterial wall properties 658
250. The topography of experimental atherosclerotic lesions
in the dog and pig 661
251. Development of canine and miniature swine experimental
atherosclerotic animal colonies 663
252. Endothelial nuclear orientation and morphology and its
relation to hemodynamic factors- 665
253. An arterial life support system for study of plasma
protein transvascular transport mechanics 667
254. Re-endothelialization of eroded vascular interface 669
255. Growth and metabolism of the aortic smooth muscle cell
and its response to various factors implicated in the
pathogenesis of atherosclerosis 671
256. The rate and mechanisms of regression of various
crystalline lipids placed in the arterial wall 672
257. Quantitation of 4 separate isotopes following oxygen
combustion of plasma lipoproteins or aorta 674
258. Aortic metabolism of plasma lipoproteins 675
xl
259 Characterization of canine plasma lipoproteins in the
normal and hyperlipemic animal 677
260 Triglyceride lipase associated with the aortic endo-
thel ium 679
Molecular Disease Branch
S ummary 681
261(c) NHLI Type II coronary intervention study 693
262(c) The study of the lipid storage disease metachromatic
leulcodys trophy 696
263(c) Enzymatic studies in tissue lipid storage diseases 700
26A(c) Glycolipids and other lipid constituents of normal
human liver 705
265(c) Tissue lipidoses: Abnormal biochemistry in tissue lipid
storage diseases 708
266(c) The biochemistry and metabolism of plasma lipoproteins:
The metabolism of plasma lipoproteins in the rat 711
267 The biochemistry of plasma lipoproteins: The structure
of plasma lipoproteins in the rat 713
268(c) The biochemistry of plasma lipoproteins: The structure
of very low density lipoproteins 715
269(c) The biochemistry and metabolism of plasma lipoproteins:
Post-heparin lipolytic enzymes and their role in normal
and abnormal lipid transport and clearance 718
270 Structure-function relationships of the plasma apolipo-
pr o t eins 722
271 1. Chemistry and physical properties of human parathyroid
hormone (HPTH) .
2. Mass spectroscopic identification of the methyl and
phenyl thiazolinone and thiohydantoin amino acids ob-
tained from the automated edman degradation of poly-
peptides and proteins.
3. Chemistry of the high density lipoprotein, apo-Lp-G-I
(A-I ) 7 24
272(c) The biochemistry and metabolism of plasma lipoproteins:
The turnover and function of very low density, low
density and high density lipoproteins 731
273(c) Tissue lipidoses: Microscopic studies in tissue lipid
storage diseases 735
274(c) The biochemistry and metabolism of plasma lipoproteins:
Studies of familial hyperlipoproteinemia 738
275 The biochemistry and metabolism of plasma lipoproteins: A
study of a differentiated mouse liver cell line 747
276 The effect of insulin and dexamethasone on the metabolism
of mammalian cells in culture 751
277 Release of histamine from mast cells 754
278 Adipose tissue metabolism: Lipoprotein lipase 756
279 Role of cyclic AMP and cyclic CMP in modulation of growth,
differentiation and aging of cells in culture 758
280 Effects of hormones on metabolism of adipose tissue
studied in vitro 760
xll
281 Investigation of the control of cyclic A^G' phospho-
diesterase (PDL) activity in fat cells 762
282 Metabolism of Guanosine 3 *, 5 '-monophosphate in lung 76A
283 Adipose tissue hormone-sensitive lipase: Mechanisms for
regulation of activity 767
Laboratory of Biochemical Genetics
284 Gene expression in neuroblastoma x glioma hybrid cells 777
285 Biochemical assays for action potential or receptor
ionophores 778
286 Selection for cells of neuronal origin synthesizing
specific transmitters 779
287 Development of cell lines with neuronal phenotypes 780
288 Study of acetylcholine receptors on muscle cells grown
in vitro 781
289 Probing neuronal acetylcholine receptors with a—
bungaro toxin 782
290 The acetylcholine receptors of the nervous system in
Aplysia 783
291 Development of cell lines with neuronal properties 785
292 Development of cell lines expressing neuronal phenotypes
by treating embryonic neuronal tissue with ethylnitro-
sourea 786
293 The storage and release of molecules required for synaptic
communication 787
294 Ultrastructural investigation of the neuronal properties
of somatic cell hybrids between neuroblastoma and other
cell types 788
295 Cell recognition and adhesion as early events in synapse
formation 790
296 Glycolipids of neuroblastoma and neuroblastoma x glioma
hybrid cells 791
297 Dissociated cell culture assay of nerve growth factor 792
298 Neural models for a two dimensional nerve net 793
299 Chemistry and function of microtubules 795
300 A general method for mapping mammalian genes using somatic
cell hybrids 797
301 Development of an in vitro model of the Lesch-Nyhan
Syndrome 798
302 Genetic analysis of A particles using somatic cell hybrids- 799
303 Genetic analysis of adenosine 3',5'-cyclic monophosphate
(cAMP) in mammalian cells 800
304 Disseminated intravascular coagulation 801
305 Genetic analysis of differentiated functions using somatic
cell hybrids 803
306 The biology of cyclic AMP in E. coli 804
307 Mechanisms in protein synthesis 806
308 Cultured cell systems for neurobiology 808
309 A histof luorescence study of cultured chromaffin cells 810
ANNUAL REPORT OF THE
CARDIOLOGY BRANCH
NATIONAL HEART AND LUNG INSTITUTE
July 1, 1972 through June 30, 1973
The experimental interests of the Cardiology Branch developed over the past
few years have continued. These relate to the pathogenesis, pathophysiology,
and treatment of coronary artery disease; the ultras true tural and molecular
mechanisms responsible for impaired contractile function of the heart; the de-
velopment of the potential capabilities of echocardiography; and the applica-
tion of echocardiographic and electron microscopic techniques to define the
basic developmental pathophysiology, genetics, and epidemiology of asymmetric
septal hypertrophy, or ASH (idiopathic hypertrophic subaortic stenosis).
CORONARY ARTERY DISEASE
Mechanisms of Sudden Death and Pharmacologic Treatment of Acute Myocardial
Infarction
I) Atropine in Acute Myocardial Infarction. Approximately 60% of deaths
caused by acute myocardial infarction (AMI) occur within the first two hours
of onset of symptoms and before medical aid arrives. Since bradycardia 1) oc-
curs in a high percentage of patients seen soon after the onset of AMI, and 2)
is believed to predispose to the development of ventricular ectopic rhythms
and ventricular fibrillation (VF) , it has been suggested that a substantial
decrease in the number of prehospital arrhythmic deaths would result if atro-
pine were administered shortly after onset of ischemic pain in patients with
bradycardia. We have performed several studies to test this hypothesis.
1) When AMI is produced in conscious closed-chest dogs by inflation of a
balloon cuff previously implanted around the left anterior coronary artery
(LAD), arrhythmias, including VF, tend to develop more frequently in dogs treat-
ed with atropine than in randomly selected control dogs. We also found that
the type of ventricular ectopic beats occurring during the early phase of AMI
in dogs can be characterized as malignant arrhythmias (those that when present
often lead to VF and death) and benign arrhythmias (those that never are asso-
ciated with the eventual development of VF) . Using this information, we demon-
strated that while benign arrhythmias are successfully suppressed by increasing
heart rate with either atropine or atrial pacing, these interventions rarely
abolish malignant arrhythmias. We next determined the influence of pronounced
and persistent bradycardia, produced by vagal stimulation, on the incidence of
benign and malignant arrhythmias during AMI. In the same closed-chest dog prep-
aration heart rate was held between 80 and 100 beats /min in the control group
and between 40 and 60 in the bradycardia group. Our findings demonstrated that
the overall incidence of arrhythmias as well as the incidence of malignant ar-
rhythmias was not increased by bradycardia during AMI. Moreover, when arrhyth-
mias occurred in the bradycardia group, they were more short-lived, with sig-
nificantly fewer arrhythmias present 45 minutes after AMI in the bradycardia
as compared to the normal heart rate dogs. Thus a) bradycardia appears to ex-
ert a favorable influence on the incidence of ventricular arrhythmias during
experimental AMI, b) increasing heart rate with atropine tends to have a dele-
terious effect on arrhythmias, c) not all ventricular arrhythmias are potenti-
ally lethal, and d) interventions may have very different effects on benign
and malignant arrhythmias, a finding indicating that the clinical efficacy of
antiarrhythmic drugs cannot be based on the assumption that a decrease in over-
all arrhythmia incidence is equivalent to a reduction in mortality.
2) Additional studies were designed to determine the mechanisms responsible
for the apparent protective effects of bradycardia and deleterious effects of
increasing heart rate on serious ventricular arrhythmias during AMI.
a) First, we found a highly significant direct correlation between per
cent increase in heart rate produced by atropine and per cent increase in is-
chemic injury (as determined by ST segment elevation recorded from intramyo-
cardial electrodes) over a heart ratf range of 30 to 180 beats/min. This find-
ing indicates that the increase in MVO2 produced by a faster rate is sufficient
to unfavorably alter the critical balance between myocardial oxygen demand and
supply, thereby producing increased ischemia.
b) Previous studies from other laboratories showed that bradycardia and
that acute myocardial ischemia lower VF threshold and increase disparity of
myocardial refractory periods, findings indicating diminished electrial stabil-
ity and leading to the now commonly accepted concept that increasing heart rate
during AMI protects against serious ventricular arrhythmias. However, the elec-
trophysiologic effects of a faster heart rate during AMI were not explored, an
important omission since elevating heart rate during AMI increases the degree
of ischemic injury. We thus measured VF threshold and disparity of refractory
periods during AMI in open-chest dogs. As we suspected, fibrillation threshold
was lower at faster heart rates and disparity of refractory periods was great-
er. Thus, lower heart rates during AMI lead to electrophysiologic changes that
protect against the development of serious ventricular arrhythmias. In addi-
tion, we found that vagal stimulation per se, independent of changes in heart
rate, enhanced electrical stability of the ventricle. This was an unexpected
result, since it commonly is believed that vagal innervation of the ventricles
is not physiologically important. To localize the cholinergic pathways respon-
sible for this stabilizing electrophysiologic effect, vinblastine, a neurotoxic
agent, was injected into the para-aortic area adjacent to the AV node. This
area contains cholinergic ganglia that supply postganglionic fibers to the ven-
tricle. Five to 7 days later, vagal stimulation caused atrial slowing but did
not change VF threshold, signifying that vagal efferents to the atria were in-
tact but those to the ventricle (responsible for increasing electrical stabil-
ity) were functionally inoperative. The anatomic correlate of this electro-
physiologic finding was obtained by studying the morphologic relationship of
the ventricular conducting system and cholinergic nerves within the ventricu-
lar septum. In control dogs Purkinje fibers were encapsulated by numerous
cholinergic nerves. However, few cholinergic fibers were found in vinblastine
treated dogs. We conclude that 1) stimulation of the vagus nerve decreases
vulnerability of the ventricle to fibrillation, and 2) this action is mediated
by a rich network of cholinergic nerves intimately related to Purkinje fibers
in the ventricular septum.
3) To determine whether the electrophysiologic effects of vagal stimulation
are of functional significance, the incidence of VF is being assessed in dogs
with and without vagal stimulation during 30 min of LAD occlusion. In one
series of experiments heart rate is allowed to slow in the vagally stimulated
dogs. While 87% of the control dogs have fibrillated after 30 minutes of AMI,
only 407o of the vagally treated dogs have. Moreover, when heart rate is held
constant (to assess the effects of vagal stimulation per se ) , 88% of control
dogs have developed VF during coronary occlusion, vihile only 45% of the vagally
stimulated dogs have.
II, Nitroglycerin and Acute Myocardial Infarction. Nitroglycerin (TNG) gen-
erally is believed contraindicated in the treatment of AMI because of the po-
tential deleterious effects of a decrease in blood pressure (and thereby coro-
nary perfusion pressure) and reflex increase in heart rate. If TNG dilates
coronary collateral vessels, however, its net effect may be beneficial. We
have now completed several studies, the results of which suggest that TNG may
favorably alter the course of AMI.
1) We first demonstrated that hypotension, either reflexly induced (by stim-
ulating the carotid baroreceptors) or induced by hemorrhage, increases the de-
gree of ischemic injury occurring during AMI. In contrast, when identical lev-
els of hypotension are produced by TNG, the degree of ST elevation (and presum-
ably, the degree of ischemia) is reduced during 15 minutes of coronary occlu-
sion. Moreover, v^en arterial pressure is held constant by simultaneous infu-
sion of the alpha receptor agonists methoxamine or phenylephrine, the current
of injury is reduced further. To confirm that the beneficial action on ST seg-
ments reflects a decrease in myocardial ischemia, another series of randomly
treated closed-chest dogs was studied. In these animals the effects of TNG
and methoxamine on development of myocardial infarction was examined after 5
hrs of coronary occlusion. Ten minutes after onset of occlusion, ST elevation
was measured in each of several previously implanted intramyocardial electrodes,
and dogs were randomized into control and treated groups. Treated dogs received
iv TNG during the remainder of the 5 hr occlusion, and arterial pressure was
maintained at preinfusion levels with iv methoxamine. Six of 13 control dogs
died; 2 of 14 treated dogs died. After 24 hours severity of infarct was as-
sessed in survivors by measurement of creatine phosphokinase (CPK) content of
myocardium subjacent to each electrode. In controls, a significant reduction
in CPK occurred in 48 of 57 electrode sites where ST elevation was present af-
ter 10 minutes of occlusion. Only 19 of 69 such sites showed significant CPK
reduction in treated dogs (p < .001). We conclude that in experimental coro-
nary occlusion TNG reduces myocardial ischemia, an effect greatly potentiated
by preventing the decrease in coronary perfusion pressure and reflex tachy-
cardia with methoxamine.
2) It currently is believed that TNG does not significantly increase myocar-
dial blood flow in patients with diseased coronary arteries. Thus, relief of
angina pectoris by TNG is ascribed to a lowering of myocardial oxygen demands.
The studies just presented, however, demonstrated that even when 2 of the more
important determinants of myocardial oxygen consumption were held constant
(arterial pressure and heart rate), TNG still reduced ischemic injury. There-
fore, to determine \Aiether TNG can increase blood flow to ischemic regions, the
capacity of coronary collaterals to supply blood to areas of potential ischemia
was assessed before and after TNG in dogs in which MVO2 was held constant, and
in patients undergoing saphenous vein bypass surgery. In the animal experiments.
we found that whenever TNG reduced the current of injury recorded by epicardial
electrodes after LAD occlusion, the reduction in ischemic injury always corre-
lated with an increase in retrograde coronary flow, a measure of the capacity
of the collaterals to supply blood to ischemic myocardium. In patients under-
going coronary artery bypass operation, flow and pressure measurements were
made from the distally (but not proximally) attached vein graft while the fi-
brillating heart was sustained by cardiopulmonary bypass. Following TNG coro-
nary collateral resistance fell. Thus, TNG can reduce ischemic injury during
experimental AMI as a result of increased collateral flow. Moreover, TNG also
is capable of diminishing resistance to collateral flow in man despite severe
multivessel disease.
3) We next studied the electrophysiologic effects of TNG during AMI produced
in the open-chest dog. VF threshold was measured during ischemia in the pres-
ence and absence of TNG infusion, and during ischemia when phenylephrine was
administered with TNG to maintain arterial pressure at control levels. We
found that TNG enhanced electrical stability of the heart during AMI; moreover
when arterial pressure was restored by simultaneous infusion of phenylephrine,
VF threshold during AMI returned to control values.
4) Finally, a highly malignant model of AMI in dogs was developed to deter-
mine whether the beneficial effects of TNG on ischemic injury, collateral re-
sistance, and electrical stability could alter the incidence of VF and death
during AMI. Twelve of 13 control dogs died from VF during 30 minutes of AMI;
in contrast, only 7 of 14 dogs treated with TNG and methoxamine died (p < .05).
As a result of these studies, we conclude that the long-standing clinical ca-
veat not to administer TNG to patients with AMI may be in error; indeed, TNG
may be uniquely valuable in the treatment of AMI, having the potential to di-
minish ischemic injury and reduce the incidence of fatal arrhythmias.
Surgical Therapy of Coronary Artery Disease
Despite the enormous number of aorto-coronary bypass grafts being performed
worldwide, little is known of the effects of operation on overall and regional
myocardial function. We have studied 22 consecutive patients who underwent
cardiac catheterization before and one week to 9 months after aorto-coronary
bypass. We found that all patients, regardless of whether grafts were patent
or occluded, improved clinically. In many angina was relieved totally. How-
ever, overall LV performance deteriorated in 10 of these patients and improved
in only one. In patients whose grafts were occluded, ejection fraction de-
creased an average of 187o (p "^ .05). Twenty-eight LV wall segments were sup-
plied by patent grafts; function improved in 6, was unchanged in 13, and dete-
rioBted in 9. Twenty-two segments were supplied by occluded grafts; none im-
proved, 14 were unchanged, and 8 deteriorated. We conclude that regardless of
graft patency, most patients improve clinically. Despite this, deterioration
of myocardial function is not uncommon, even when grafts are patent. It there-
fore is apparent that aorto-coronary bypass operations may precipitate myocar-
dial damage. Thus, the commonly employed argument that operation should be
performed in the asymptomatic or minimally symptomatic patient to save viable
muscle may not be valid.
Diet-Drug Intervention Study
One of the most important aspects of the coronary artery disease problem is
the development of a potent preventive approach. Evaluation of one such ap-
proach has been initiated in collaboration with the Lipid Metabolism Branch
and is knovn as the NHLI Type II Coronary Intervention Study. The primary
question we hope to answer is whether lowering blood cholesterol with diet and
cholestyramine in patients with Type II hyperlipoproteinemia beneficially ef-
fects the atherosclerotic process. The major criterion we will employ to an-
swer this question is whether there is regression of anatomic disease or evi-
dence for slower progression, conclusions that will be based on coronary arte-
riograms. The program is now well under way and some of the information that
has emerged to date is detailed in another section of the Annual Report.
ASYMMETRIC SEPTAL HYPERTROPHY, OR ASH (IHSS)
Over the past dozen years our understanding of the disease spectrum embrac-
ing IHSS has advanced considerably. Originally, it was considered that all pa-
tients with IHSS had a dynamic form of subaortic stenosis. Subsequently, it
was recognized that LV outflow obstruction was only one manifestation, and an
inconstant one, of a disease that is basically a cardiomyopathy. Further ad-
vances in our understanding of the genetic, epidemiologic, and pathophysiologic
aspects of this disease were frustrated by lack of a sensitive noninvasive
method for disease detection. We have found, however, that echocardiography
is an extraordinarily powerful technique for diagnosing IHSS. Employing both
echocardiography and ultrastructural analysis of myocardium obtained from pa-
tients undergoing cardiac surgery, we have determined the cause of the disease
and defined its pathophysiologic spectrimi.
1) Asymmetric septal hypertrophy (ASH) , characterized by a ventricular sep-
tum of at least 1.3 times the thickness of the posterior-basal LV free wall,
was found by echocardiographic examination in all patients with typical ob-
structive IHSS. Moreover; a) when family members who clinically were believed
to have a non-obstructive type of cardiomyopathy were studied, ASH was detected
in all, b) ASH was detected in other patients diagnosed previously as having
idiopathic non-obstructive LVH, and c) ASH was not present in patients with any
other form of heart disease, with the exception of an occasional patient with
chronic pulmonary or right ventricular hypertension (primary pulmonary hyper-
tension or pulmonary stenosis), entities which can be easily distinguished on
clinical grounds from patients with ASH. These findings confirm the hypothesis
that IHSS is only one manifestation of a disease spectrum in which obstruction
may or may not occur. They also demonstrate that ASH is the characteristic
anatomic marker of this disease.
2) IHSS is thought to occur as a familial abnormality in only 25-357o of pa-
tients. Reliability of this estimate is uncertain, however, because of the
previous lack of a sensitive and specific marker of the disease. We therefore
studied the pattern of inheritance of IHSS in 30 families, using ASH (detected
echocardiographically) as the disease marker. Of the 30 families, at least one
affected relative was found in 28. In the remaining two families we studied
only one and two members respectively. Forty-six percent of 105 first degree
relatives had ASH; an equal percentage of males and females were affected.
Penetrance was complete in nine of 10 parental pairs available for analysis.
These findings are compatible with the concept that ASH is always familial and
is transmitted as an autosomal dominant trait with a high degree of penetrance.
3) With the capacity to establish the diagnosis of ASH noninvasively, we
defined the clinical spectrum of this disease. We found that less than 10% of
patients with ASH have the obstructive form (i.e., classic IHSS). Approximate-
ly 207o of all patients with ASH would have been considered entirely normal.
An additional 607o of patients with ASH had some minor abnormalities (moderate
fatigue or dyspnea on exertion, or a nondescript soft systolic murmur, or non-
specific ST and T wave abnormalities on EKG) ; specific diagnosis would not have
been possible, however, without echocardiographic demonstration of ASH. More-
over, analysis of multiple clinical parameters failed to distinguish symptomat-
ic nonobstructed ASH patients from the obstructed group with two exceptions:
in nonobstructed ASH the murmur was softer with little variation following pro-
vocative maneuvers, and a bisferious carotid pulse was absent. Angina and syn-
cope, symptoms usually considered characteristic of obstruction, also were
common in patients without obstruction. We conclude that a) the classic ob-
structive form of ASH (IHSS) constitutes a relatively small portion of the en-
tire disease spectrum; b) most patients with ASH are asymptomatic or have only
mild symptoms; c) when symptoms are present, they cannot be used to distinguish
between patients with and without obstruction; d) the absence of typical phys-
ical findings makes clinical diagnosis of nonobstructive ASH difficult; and e)
echocardiography is the simplest and most reliable means of establishing the
diagnosis.
4) Some clinicians consider IHSS to be a diffuse cardiomyopathy and suggest
that operation to eliminate obstruction is ill-advised. However, our echocar-
diographic studies demonstrated that ventricular hypertrophy in IHSS is typi-
cally nonuniform in distribution with greatest hypertrophy invariably involv-
ing the ventricular septum. To determine whether pathologic changes are lim-
ited to the ventricular septum or are more diffusely distributed, light and
electron microscopic studies were performed on myocardium obtained at opera-
tion from 8 patients with obstructive ASH and 4 patients with nonobstructive
ASH who were either severely limited or had died of their disease. Morphologic
abnormalities were grouped into 2 categories: 1) intracellular abnormalities
(disarray of myofibrils and myofilaments), 2) cell-to-cell abnormalities (dis-
organization of muscle cells, abnormal cell contacts). The septum of all pa-
tients contained many muscle cells that displayed extensive intracellular and
cell-to-cell abnormalities. In the nonobstructed patients, cell-to-cell ab-
normalities also were present in the free LV wall. In contrast, cell-to-cell
abnormalities were not seen in the free LV wall of any of the obstructed pa-
tients. Thus, a wide distribution of the cardiomyopathic process seems to be
characteristic of severely symptomatic patients who do not have obstruction;
symptoms in these patients can be attributed to decreased contractility and
decreased diastolic compliance. In severely symptomatic patients who have LV
outflow obstruction, however, the more bizarre cellular manifestations of the
cardiomyopathic process appear confined to the septum; we believe symptoms in
these patients are due mainly to outflow obstruction and the resuling increase
in ventricular pressure work.
This belief is reinforced by results of our operative experience obtained
in collaboration with the Surgical Branch. This experience now includes 68
patients operated on for IHSS. Preoperatively, all patients had large intra-
ventricular pressure gradients at rest or with provocation and were in func-
tional classes III and IV. After septal myotomy and myectomy no resting gra-
dient was present in 49 of 53 patients studied; the remaining 4 had minimal
obstruction. LVEDP fell in most patients in whom it was elevated preoperative-
ly, and symptomatic improvement occurred in all. Of significance, all patients
but one have maintained their increased functional capacity during followup
periods ranging from one to 13 years (mean 4.5 years). In addition, 16 patients
have been studied two to 11 years after operation (mean 6.5 years) by echocar-
diography or catheterization. No patient has evidenced recurrence of LV out-
flow obstruction. It therefore is apparent that LV outflow obstruction is a
major cause of symptoms in such patients. Moreover, our results suggest that
the cardiomyopathic process in patients with the obstructive form of the dis-
ease is either not progressive, or that the rate of progression is very slow.
5) In an attempt to further extend the capabilities of echocardiography in
characterizing the patient with ASH, we studied the echocardiograms of patients
with obstructive ASH to determine whether the distance between the hypertrophied
ventricular septum and the abnormally positioned anterior leaflet of the mitral
valve present during systole (a characteristic feature of IHSS) can be used to
quantitatively predict pressure gradient. A computed "obstruction index" (in-
corporating the degree and duration of narrowing) was found to correlate excel-
lently with simultaneously determined LV outflow pressure gradients. By em-
ploying the obstructive index we can predict pressure gradient from echocardio-
graphic data alone to within 107o of the value simultaneously determined at cath-
eterization.
6) Obstruction in IHSS is produced by an abnormal forward movement of the
anterior mitral leaflet during systole. The primary cause of the abnormal leaf-
let motion, and thereby the obstruction, would appear to be the massively hyper-
trophied septum. Thus, pathologic studies have shown that the septum can cause
displacement of the papillary muscles, which would result in traction on the
anterior leaflet and lead to its abnormal anterior position during systolic
ejection. In addition, we have performed echocardiographic studies which have
demonstrated that, compared to normal, the mitral valve in patients with IHSS
lies more anteriorly even at the onset of systole (i.e., prior to ejection), a
finding also compatible with a tethering effect. The resulting narrowed out-
flow tract at onset of ejection may predispose to the production of a high ve-
locity jet between the septum and anterior mitral leaflet, resulting in a
Venturi effect and thereby contributing to the leaflet's systolic anterior move-
ment. If such a mechanism is operative, it may explain why patients with ob-
struction do not have extensive involvement of the LV by the myopathic process,
vrtiile patients without obstruction do. Thus, if two preconditions are necessary
for obstmction, i.e., a narrow outflow tract at onset of systole and a high
velocity jet during ejection, it is likely that the velocity of fiber shorten-
ing in patients with diffuse LV disease will not be sufficient to cause the
anterior mitral leaflet to move anteriorly and abut against the septum. Addi-
tional echocardiographic studies performed in the operating room demonstrated
that immediately after left ventriculomyectomy 1) the mitral valve moves into
a more normal posterior position, 2) the abnormal systolic movement of the mi-
tral valve is abolished, and 3) the outflow gradient disappears.
VALVULAR HEART DISEASE
Elucidation of the Determinants of Irreversible Myocardial Failure
1) Although most patients with rheumatic valvular disease improve following
operative intervention, there are patients in whom a dilated heart does not
regress in size and in whom any improvement in symptoms is minimal. We there-
fore initiated a study to define prospectively 1) whether a particular group-
ing of preoperative functional derangements leads to prohibitive operative
risks, and 2) what types of derangements can be reversed or improved by opera-
tive abolition of the mechanical defect. Evaluation of myocardial function
includes exercise testing and calculation of ventricular volumes, ejection frac-
tion, mean VCF, etc. (as determined by cardiac catheterization and echocardio-
graphy). In addition, biopsies are being obtained for electron microscopic
analysis as well as biochemical assessment of the contractile proteins. Our
preliminary data indicate that significant improvement in exercise capacity can
occur in patients with extremely large preoperative ventricular volumes and
very low ejection fractions; these same patients, in the absence of such a
prospective protocol, might have been denied operation in the past because of
their poor preoperative hemodynamic status. Ultrastructural analysis has iden-
tified some patients with severe degrees of myocardial cell atrophy associated
with extensive deposition of collagen. Further analysis and experimental stud-
ies are being performed to determine whether such ultrastructural changes are
good markers of irreversible myocardial failure, and whether excessive deposi-
tion of fibrous tissue represents a cause or an effect of irreversible cardiac
decompensation.
2) In addition to the above prospective study, a retrospective analysis of
89 patients with pure aortic regurgitation operated upon between 1962 and 1971
was carried out. Our goal was to determine whether any preoperative risk fac-
tors could be correlated with operative mortality and subsequent survival. Al-
though analysis is still preliminary, several factors have emerged as powerful
predictors of survival. Thus, when one simply analyzes the preoperative ECG
and employs Estes point score for assessment of LVH, 567= of patients who had
point scores > 6 were dead by 36 months; in contrast, only 357o of patients were
dead who had point scores of less than 6. Similar correlations were obtained
with LV end-diastolic, left atrial, and pulmonary arterial pressures. These
data may well form the basis of future decisions regarding the optimal time for
operative intervention in patients with aortic regurgitation.
MOLECULAR MECHANISMS RESPONSIBLE FOR CARDIAC CONTRACTION
AND FOR ALTERATIONS IN CONTRACTILITY PRODUCED BY DISEASE
The new Section on Molecular Cardiology has initiated research in two major
areas. The first concerns regulation of contractile proteins, particularly
those found in human blood platelets and fibroblasts. A membrane free prepara-
tion from human blood platelets which incorporates -^^p from i( labeled ATP has
been purified. The -^^p is uniquely incorporated into a specific light chain
on the myosin molecule and its incorporation is markedly influenced by the pres-
ence of cyclic AKP. Present studies concern the control of this phosphoryla-
tion and its significance.
8
The second major project centers on defining the molecular defect in the
cardiac contractile proteins of patients suffering from asymmetric septal hy-
pertrophy (IHSS), a genetic disease characterized by bizarre, malaligned myo-
cardial cells. The contractile proteins from operative specimens of several
patients have been purified. Particular attention is being paid to the myosin
molecule: a) Are the light chains from this myosin those of fetal or adult
muscle? b) Is the "C-protein", which is thought to influence alignment of myo-
sin molecules, present and functioning? In addition to characterizing the
structure and enzymatic activity of myosin, actin and tropomyosin and their
interrelationships also aie being studied.
Serial No. NHLI-1
1. Cardiology
2. Molecular Cardiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Phosphorylation of Human Platelet Myosin and Contractile Pro-
teins from Other Sources
Previous Serial Number: NHLI-18
Principal Investigator: Robert S. Adelstein, M,D.
Other Investigators: Mary Anne Conti, B.A.
William Anderson, B.A.
Cooperating Units: None
Project Description: Human platelet myosin, which is markedly similar to mus-
cle myosin in structure and function, contains at least two light chains (mol.
wts. 18,000 and 14,000) as well as a heavy chain (mol. wt. 200,000). Purified
extracts from washed platelets incorporate -^^P from ^-labeled ATP into the
18,000 molecular weight light chain. No incorporation is found into the myo-
sin heavy chain, platelet actin or tropomyosin. If inorganic -^^P is substi-
tuted for AT P, insignificant incorporation occurs into the light chain. The
incorporated ^^p appears to be covalently bound through an ester linkage to
serine or threonine since incubation at pH 11.8 released all the counts, where-
as incubation at pH 1.0 did not.
The phosphorylated light chain has been purified using gel filtration and
its amino acid composition has been determined.
Proposed Course of Project: The dependence of this phosphorylating system on
Cyclic-AMP is being studied. The entire system (i.e., protein kinase and phos-
phatase) all of which appears to be present in the platelet, is being purified.
Primary studies on the phosphorylated light chain, in order to determine the
exact location of the phosphorylated residue have been initiated. Finally,
the role phosphorylation plays in controlling muscle contraction in platelets
as well as in other cells, e.g. fibroblasts, neuroblastoma cells as well as
cultured cardiac cells, will be explored.
Honors and Awards: None
Publications: None
//
Serial No, NHLI-2(c)
1. Cardiology
2. Molecular cardiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Characterization of Cardiac Contractile Proteins from
Patients With Idiopathic Hypertrophic Subaortic Stenosis
Previous Serial Number: None
Principal Investigator: Robert S. Adelstein, M.D„
Other Investigators: Chester E. Clark, M.D.
Barry J, Maron, M.D.
Stephen E, Epstein, M.D,
Mary Anne Conti, B.S.
William Anderson, Jr., B.A.
Cooperating Units: None
Project Description: Studies in this laboratory have been initiated to
characterize the contractile proteins from hearts of patients suffering from
IHSS. Using small amounts of tissue removed during operation, actin, myosin
and tropomyosin have been extracted and partially purified. The myosin was
purified using gell filtration and a single peak containing all the ATPase
activity was faind.
Initial studies on the myosin molecule have focused on the number and molecular
weight of the heavy and light chains as well as its ATPase activity.
Efforts at present are being made to grow these cells in vitro and to compare
their properties to other cardiac cells grown in vitro and removed furing
operative procedures.
Proposed Course of Project: The contractile proteins of hearts from patients
suffering from IHSS will be characterized both as to structure and function.
Myosin will be studied to see whether its light cha ins are similar to adult or
embryonic cardiac myosin. The interaction of myosin and actin as well as the
ability of myosin to form aggregates will also be studied.
This latter property (ability of myosin molecules to aggregate) will be studied
by using uranylacetate staining with the aid of the electron microscope. These
findings will be correlated with biochemical studies on the content of "C"
protein, a newly found muscle protein thought to influence the aggregation of
myosin.
Honors and Awards: None
Publica t ions : None
/*»-
Serial No, NHLI-3(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Echocardiographic Findings in Patients With Idiopathic
Hypereosinophilic Syndromes
Previous Serial No,: None
Principal Investigator: Jeffrey S„ Borer, M,D,
Other Investigators: Walter L. Henry, M„Do
David C. Dale, MoD,
Cooperating Units: National Institute of Allergies and Infectious Diseases
Project Description: Echocardiography (ECHO) is an increasingly important
tool in the diagnosis and classification of primary and secondary cardiomyo-
pathies. This report deals with echocardiographic evaluation of 8 men, aged
7 to 67, with chronic idiopathic hypereosinophilic syndromes (IHS) , IHS had
been present from 5 to 140 months. No patient was referred originally because
of cardiac disease. Only one had clinical evidence of cardiac dysfunction.
Six of the 8 patients, with eosinophil counts ranging from 6900 to 94,000, had
definite ECHO abnormalities. Most prominent was significant S3mimetrical
thickening of the septum and left ventricular free wall, mean thickness being
14.3 mm+1,2 (SEM) (normal 9,4 mm+,2, p ,01), The other 2 patients, with eo-
sinophil counts of 3900 and 9500, had no abnormality but their septal and
free wall thicknesses were at the upper limit of normal. Instantaneous left
ventricular transverse dimension and velocity of circumferential fiber
shortening were measured in every patient. No uniform abnormality in maximum
velocity of circumferential fiber shortening was found. However, in 2 patients,
1 symptomatic, abnormalities in diastolic relaxation consistent with a restric-
tive defect were seen. The symptomatic patient also had transverse dimension
slightly below the lower limit of normal. In about 1/3 of fatal idiopathic
hypereosinophilic syndrome cases pathologic studies reveal endo- and myocardial
fibrosis, mural thrombi and ventricular hypertrophy, with either constricted or
dilated left ventricular cavities. Heretofore, it was believed that cardiac
involvement in idiopathic hypereosinophilic sjmdrome leads rapidly to death.
The present study suggests that ECHO may be of value in reassessing the
prevalence and natural history of cardiac involvement in idiopathic hypereo-
sinophilic syndromes. Moreover, ECHO may provide an objective parameter for
evaluation of therapy in idiopathic hypereosinophilic sjmdromes.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
1 /3
Serial No. NHT.T-4
1. Cardiology
2* Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effect of Lidocaine and of Elevating Arterial Pressure on the
Incidence of Spontaneous Ventricular Fibrillation in Dogs
During Coronary Occlusion
Principal Investigator: Jeffrey S. Borer, M.D,
Other Investigators: Lura Harrison, Ph.D.
Richard Levy, B.A.
Kenneth M. Kent, M.D., Ph.D.
Robert E. Goldstein, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: Two of the major goals in the therapy of acute myocardial
infarction (AMI) are reducing the high mortality occurring from ventricular
fibrillation (VF) during the early phase of AMI, and reducing the extent of
ischemic injury. Although lidocaine is commonly used clinically to prevent
VF after AMI, its relative efficacy has been questioned, particularly early
during AMI when mechanisms causing arrhythmias are believed to be different
from those present several hrs later (i.e. vdien patients are usually admitted
to hospital). In addition, we previously showed phenylephrine- induced
elevation of mean arterial pressure (AP) diminishes ischemic injury during
AMI in dogs. Whether such an effect reduces the incidence of arrhythmias,
however, is unknown. The present study was designed to determine the effects
of lidocaine and of elevating AP on the incidence of VF during 30 min of
coronary occlusion in open-chest dogs. AMI was produced by ligating the LAD
and first septal perforating coronary arteries. Dogs were randomly assigned
to one of 3 groups: a) control, b) lidocaine (2 mg/kg bolus, followed by 70 or
100 ug/kg/min infusion), or c) elevated AP (phenylephrine infused to maintain
AP>25 and<50 mm Hg above pretreatment levels). After 10 min of pretreatment
according to the assigned regimen, the coronary arteries were occluded.
In contrast to pre\ious studies, our preliminary data suggest that lidocaine
reduces the incidence of VF during the early phase of AMI; on the other hand,
elevating AP appears to have no protective effect. Whether deleterious effects
on pump function produced by increasing AP counterbalanced potential protective
effects in this model (in which massive anterior wall infarction was produced),
and v^ether a protective effect can be demonstrated in the presence of less
extensive infarction are unknown.
l¥
Serial No. NHLI-4
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
/5-
Serial No. NHLI-5
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effect of Nitroglycerin on the Incidence of Spontaneous
Ventricular Fibrillation During Coronary Occlusion in Dogs
Previous Serial Number: None
Principal Investigator: Jeffrey S. Borer, M.D.
Other Investigators: Robert E. Goldstein, M.D.
Kenneth M. Kent, M.D., Ph.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: Two of the major challenges in the treatment of patients
with acute myocardial infarction (AMI) are to reduce the incidence of prehos-
pital deaths and to diminish the extent of ischemic damage. We previously
showed that nitroglycerin (TNG) reduces ischemic injury and increases ventri-
cular fibrillation threshold during experimental AMI. To determine the
functional significance of these beneficial myocardial and electrophysiologic
effects, high occlusions of the LAD and septal perforating coronary arteries
were produced in open-chest dogs for 30 min. Prior to occlusion, dogs were
randomized into 2 groups: 1) control, and 2) TNG treated (0,45 meg as a
bolus followed by 0,3 mcg/min). Mean arterial pressure in the TNG group was
maintained at pretreatment levels with iv methoxamine. After 10 min. of
pretreatment according to the assigned regimen, the coronary arteries were
occluded and treatment was continued. Twelve of 13 control dogs died from
ventricular fibrillation; in contrast, only 7 of 14 dogs treated with TNG and
methoxamine died (p<0.05). We conclude that TNG may be uniquely valuable in
the treatment of the prehospital and hospital phases of AMI, having the
potential to diminish ischemic injury and reduce the incidence of fatal
arrhythmias.
Proposed Course of Project: Completed
Honors and Awards: None
Publication: None
/(>
Serial No. NHLI-6(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md,
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Echocardiographic Assessment of Secondary Cardiomyopathies
Previous Serial Number; None
Principal Investigator: Jeffrey S. Borer, M.D,
Other Investigators: Walter L, Henry, M.D.
Stephen £• Epstein, M.D.
Cooperating Units: None
Project Description: Cardiomyopathic changes frequently occur as concomitants
of several systemic diseases. Detection by conventional clinical techniques
is relatively insensitive, however, and diagnosis generally is made late in
the course of the disease. In the present study, echocardiography is being
utilized a) to define abnormalities of cardiac anatomy and function in patients
with several diseases frequently associated with cardiomyopathies, b) to
determine the evolution of these abnormalities with time, and c) to assess
the relative sensitivity of echocardiographic and clinical techniques in
detecting cardiac disease. Patients with the following diseases are being
studied: amyloidosis, chronic idiopathic hypereosinophilia, chronic alco-
holism, systemic lupus erythematosus, hemochromatosis, progressive systemic
sclerosis, carcinoid syndrome, rheumatoid arthritis, malignant neoplasia and
several neuromyopathies. Thus, far, initial evaluation has been performed in
40 patients. From our preliminary results it is clear that certain cardio-
myopathic changes are apparent from the echocardiogram before, and in some
cases long before, they are apparent by clinical assessment.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications; None
17
Serial No. NHLI-7(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: ASH Masquerading as Coronary Artery Disease and Idiopathic
Myocardial Hypertrophy
Previous Serial Number: None
Principal Investigator: Chester E. Clark, M.D.
Other Investigators: Walter L. Henry, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: Echocardiography is the most specific and sensitive
means of detecting asymmetric septal hypertrophy (ASH) , the characteristic
anatomic abnormality in IHSS. With this technique we have found that left
ventricular outflow obstruction (classic IHSS) occurs in less than 20% of
patients falling into this disease spectrum. Thus, clinical diagnosis in the
majority of patients, who have the nonobstructive form of the disease, is
difficult, since the typical physical findings present in patients with
obstructive ASH are absent. Indeed, we have found 13 patients with echocardi-
ographically detected ASH who have been carried in our clinic with the diagnoses
of coronary artery disease or idiopathic myocardial hypertrophy. Five patients
had exertional chest pain and abnormal electrocardiograms, often with
"diagnostic" Q waves compatible with old transmural infarction; these patients
were diagnosed as having coronary artery disease. Eight patients presented
with LVH detected on either the electrocardiogram, physical examination, or
both. IHSS was not suspected, and catheterization, performed in several
patients, was nondiagnostic; these patients were diagnosed as having idiopathic
myocardial hypertrophy. Correct diagnosis in all of these patients could only
be made by echocardiographic studies. We conclude that ASH is a more common
disease than previously suspected and that it can masquerade in several
different clinical costumes.
Proposed Course of Project: Continuing
Honors and Awards : None
Publications: None
/^
Serial No. NHLI-8(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Electrocardiographic Findings in 100 Patients with Asymmetric
Septal Hypertrophy
Previous Serial Number: None
Principal Investigator: Chester E. Clark, M.D.
Other Investigators: Walter L. Henry, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: The typical physical findings present in patients with
classic obstructive IHSS makes clinical diagnosis relatively easy. However,
less than 207o of patients falling into the IHSS disease spectrum have left
ventricular outflow obstruction. Thus, considerable difficulty exists in es-
tablishing the correct diagnosis in the majority of patients with this disease.
The problem is compounded by the fact that ECG findings in patients with IHSS
often are "characteristic" of coronary artery and other cardiac diseases. This
study was undertaken to define the spectrum of ECG abnormalities present in
patients with nonobstructive and obstructive IHSS. Obstruction was considered
present vflien the LV outflow gradient exceeded 30 mm Hg at rest or with provo-
cation.
The following ECG abnormalities were found: atrial fibrillation (5%), right
atrial enlargement (67o) , left atrial enlargement (287o) , WPW syndrome (67o) , first
degree A-V block (77o) , right bundle branch block (17o), left bundle branch block
(57=), left atrial hypertrophy (227o) , abnormal axis [ 0 to -90° (277o), -90° to
-180° (17o), +90° to +180° (27c)], abnormal Q waves (247o), abnormal T waves (547.)
and left ventricular hypertrophy by Estes criteria (407o) . Abnormalities were
present in 957. of patients with obstruction but in only 547o without obstruction.
Abnormalities that were more common in the obstructive than nonobstructive group
were: atrial fibrillation, first degree A-V block, left atrial hypertrophy,
left atrial enlargement, abnormal Q waves, abnormal T waves and left ventricu-
lar hypertrophy. We conclude that 1) patients with obstruction are more likely
to have abnormal ECG's than patients without obstruction, 2) there is no ECG
feature that is either diagnostic of ASH, that eliminates it from consideration
or distinguishes obstructive from nonobstructive patients, and 3) any patient
with an unusual ECG of unknown cause should be suspected of having ASH.
Proposed Course of Project: Continuing.
Honors and Awards: None
Publications: None
1 l<P
Serial No. NHTJ-9(c>
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Familial Incidence and Genetic Transmission of IHSS
Principal Investigator: Chester E. Clark, M,D.
Other Investigators: Walter L. Henry, M„Do
Stephen E. Epstein, M.D,
Cooperating Units: None
Project Description: The pattern of inheritance of asymmetric septal hj^per-
trophy (ASH), the characteristic anatomic abnormality in IHSS, was studied by
echocardiography in thirty families. The probands were four outpatients and
twenty-six patients admitted serially for diagnostic evaluation. Twenty-
eight of thirty families contained at least one member, in addition to the
proband, with ASH„ Forty-six percent of 105 first degree relatives studied
had ASH; an equal percentage of males and females were affected. Penetrance
was complete in nine of the ten parental pairs available for analysis.
Clinical parameters were unreliable for detecting ASH since only 167c. had
classic IHSS. The history was negative in 607., and the EKG was normal in
50% of those with ASH on echocardiogram. On the basis of this study we con-
cluded that ASH is always familial and is transmitted as an autosomal
dominant trait with a high degree of penetrance.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: None
A)
Serial No. NHLI-lO(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Coronary Collateral Function in Patients Without Coronary
Artery Disease
Previous Serial Number: None
Principal Investigator: Robert E. Goldstein, M,D.
Other Investigators: Lawrence Michael is, M.D.
Andrew G. Morrow, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: Clinic of Surgery, NHLI
Project Description: Intercoronary anastomoses frequently play a vital role
in sustaining coronary blood flow to regions of myocardium whose source of
blood supply has been compromised by coronary occlusive disease. Uncertainty
has remained, however, whether anastomoses must develop de novo following an
ischemic insult or whether they are capable of sustaining some degree of col-
lateral flow even in the absence of localized myocardial ischemia. Studies in
various animal species have shown considerable variability in the extent of
normal intercoronary anastomoses, depending upon the particular species studied.
We therefore have investigated the function of intercoronary collaterals in hu-
mans by measuring back pressure and back flow in occluded coronary arteries at
the time of aortic valve replacement. Only those individuals are studied who
have angiographically documented normal coronary arteries. Following aortotomy
all patients routinely have perfusion cannulae placed in the right and left
coronary ostia. The experimental measurements are made via a t-connection in
these coronary perfusion lines during brief proximal occlusion of each of the
perfusion lines prior to and following intravenous nitroglycerin. Preliminary
data indicate that back pressure and back flow is usually quite small in these
individuals, suggesting that such patients have relatively poor coronary col-
lateral function. A practical corollary of this preliminary observation is
that perfusion of the entire myocardium in most non-ischemic individuals under-
going aortic valve surgery most likely cannot be achieved by perfusing just one
coronary artery.
Proposed Course of Project: Project continuing.
Honors and Awards: None
Publications: None
I?/
Serial No. NHLI-11
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Visualization of Acute Myocardial Infarction by the Radionuclide
Gallium-67
Previous Serial Number: None
Principal Investigator: Robert E. Goldstein, M.D.
Robert J. Kramer, M.D.
Other Investigators: John W. Hirshfeld, M.D.
Gerald S. Johnson, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: Department of Nuclear Medicine
Clinical Center, NIH
Project Description: ^^Ga has been shown to accumulate in a variety of inflam-
matory and neoplastic lesions, thereby permitting identification of these ab-
normalities by radioisotope imaging. We have found that °'Ga also accumulates
selectively in recently infarcted myocardium. AMI was induced in dogs by liga-
tion of the left anterior descending coronary artery. "'Ga (3 mCi) was injec-
ted iv. Hearts were removed 24 hours later, and isotope localization was eval-
uated by an Anger camera and by autoradiography. In 5 of 7 dogs, Ga was found
to accumulate preferentially in regions of visible infarction. Tissue creatine
phosphokinase (CPK) , determined in 4 of these dogs, was markedly reduced in the
infarcted region. The infarcts of 2 dogs without preferential "'Ga uptake show-
ed lesser degrees of CPK reduction. Ga localization in regions of AMI was
also demonstrable by scanning 3 intact, closed-chest dogs. Hearts of 2 animals
with transient (20 min.) ischemia showed neither ^'Ga accumulation nor CPK de-
pression. Thus, °'Ga localization discriminates infarcted from normal or only
transiently ischemic myocardium. Unlike potassium and its analogs, "'Ga scan-
ning does not depend on resolution of "cold" areas nor would it introduce am-
biguities due to flow limitation of isotope delivery. For these reasons "'Ga
may prove particular useful in the imaging of AMI in man.
Proposed Course of Project: Project completed
Honors and Awards: None
Publications: Manuscript in prepastion.
SiSi.
Serial No. NHLI-12
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Lack of Deleterious Effects of Vagally Mediated Bradycardia
During Acute Myocardial Ischemia in the Dog
Previous Serial Number: None
Principal Investigator: Robert E. Goldstein, M.D.
Other Investigators: Eldon R. Smith, M.D.
Gary Famham, M.D.
Richard B. Karsh, M.D.
David R. Redwood, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: Recent electrophysiologic data have brought into question
the concept that in acute myocardial ischemia (AMI) vagally mediated bradycardia
leads to serious ventricular arrhythmias. To evaluate this concept more criti-
cally, AMI was produced for one hour in 48 sedated, closed-chest dogs by infla-
tion of a balloon cuff previously implanted around the LAD coronary artery.
Heart rate (HR) was controlled by electrical stimulation of the cervical vagi.
In 20 bradycardia dogs HR was maintained between 40 and 60 bpm; in 27 control
dogs, HR was held between 80 and 95. Ventricular arrhythmias were classified
as benign (R-Rpvc interval > 0.43 sec) or malignant (R-Rpvc S 0-^3 sec). Es-
cape idioventricular rhythms frequently observed in bradycardia dogs were not
counted unless R-Rpvc was < 1.0. Except after ventricular fibrillation (VF)
sustained hypotension was not observed in any animal. The number of dogs with
arrhythmias of each type is listed below. The number of arrhythmias occurring
within 10 min of release of occlusion is in parentheses.
None Benign Malignant VF
BD (total 21) 11(6) 5(4) 5(10) 0(1)
CD (total 27) 11(8) 8(5) 8(13) 0(1)
We conclude that normotensive animals do not experience an increased incidence
of ventricular arrhythmias when myocardial ischemia is accompanied by vagally
mediated bradycardia.
Proposed Course of Project: Project completed.
Honors and Awards: None
Publications: ARTICLE PUBLISHED IN A PERIODICAL:
Goldstein, R.E., Karsh, R.B., Smith, E.R., Orlando, M.
Norman, D., Farnham, G.S., Redwood, D.R. and Epstein, S.E.:
Circulation In press.
P3
Serial No. NHLI-13(c)
1. Cardiology
2. Cardiovascular Diagnosis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effects of Nitroglycerin on Coronary Collateral Function in
Patients with Coronary Occlusive Disease
Previous Serial Number: None
Principal Investigator: Robert E. Goldstein, M.D.
Other Investigators: Edward B. Stinson, M.D.
James L. Scherer, M.D.
Todd Grehl, M.D.
Ronald Senegan, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: Department of Cardiovascular Surgery
Stanford University School of Medicine
Department of Radiology
Clinical Center, NIH
Project Description: Beneficial effects of nitroglycerin during myocardial
ischemia are well documented. Nevertheless, little is known concerning the
direct influence of nitroglycerin on coronary collateral function in humans
with diseased coronary arteries. We therefore measured the ability of col-
laterals to sustain retrograde flow and back-pressure (peripheral coronary
pressure, or PCP) before and after nitroglycerin in patients undergoing sa-
phenous vein bypass. Measurements were made via the dis tally (but not proxi-
mally) attached vein graft while the fibrillating heart was sustained by car-
diopulmonary bypass and while the coronary artery into which the vein graft
opened was totally occluded proximal to the site of graft attachment. Nitro-
glycerin (100 (ig bolus and 100 to 150 (ig/min) was infused into the ascending
aorta. Baseline measurements in 27 patients revealed: aortic pressure mean
80+4 (S.E.) mm Hg, PCP 30 + 3 mm Hg, retrograde flow 6.9 + 1.9 ml/min, and
calculated collateral resistance of 63 + 18 mm Hg/ml/min. Following nitrogly-
cerin, aortic pressure decreased 19 + 27o but retrograde flow was not consis-
tently changed; thus calculated collateral resistance fell significantly by
an average of 267o (P < .05). Similarly, a fall in calculated collateral re-
sistance (mean 487o) was noted in 6 individuals in whom aortic pressure changes
were attenuated by altering systemic flow. Peripheral coronary pressure de-
creased consistently after nitroglycerin, but less than that expected from the
fall in aortic pressure. When expressed as a fraction of aortic pressure,
peripheral coronary pressure actually increased 9.5 + 4.07o (P < .02), a find-
ing consistent with enhanced flow through collateral channels. In conclusion,
although their clinical significance is uncertain, our data indicate that
1 -2^
Serial No. NHLI-13(c)
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
nitroglycerin is capable of diminishing resistance to collateral flow despite
severe multivessel involvement. Comparison of these physiologic data with pre-
operative angiographic results revealed a close correlation between the size
and extent of coronary collaterals, as assessed by a newly devised angiographic
grading system, and physiologic evidenc of collateral function. In particular,
the size and extent of collaterals showed a clear-cut inverse correlation with
calculated coronary vascular resistance. Thus the radiographic appearance of
coronary collaterals appears to be an accurate predictor of coronary collateral
function during coronary bypass surgery.
Proposed Course of Project: Project completed.
Honors and Awards: None
Publications. Manuscript in preparation.
5ir
Serial No. NHLI-14
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Failure of Nitroglycerin to Alleviate Acute Myocardial Ischemia
or Improve Collateral Flow When MVO2 is Held Constant in the
Heart-Lung Preparation.
Previous Serial Number: None
Principal Investigator: Robert E. Goldstein, M,D.
Other Investigators: B. Gregory Brown, M.D., Ph.D.
Stephen E. Epstein, M,D.
Cooperating Units: Section on Experimental Atherosclerosis, NHLI
Project Description: Nitroglycerin reduces S-T segment elevation after acute
coronary occlusion in the intact dog, an effect not mediated by changes in ar-
terial pressure or heart rate. To determine whether increased coronary collat-
eral flow contributed to this effect, canine heart-lung preparations were stud-
ied at 5, 10, and 15 minutes following acute LAD occlusion. Total coronary flow,
coronary A-V O2 differen9e, and MVO2 were measured continuously. Heart rate,
arterial pressure, and MVO2 were held constant, thereby excluding peripherally
mediated nitroglycerin action. Retrograde flow was collected from the distal
segment of the ligated LAD. Epicardial S-T segment elevation was measured with
an 8-lead electrode positioned on the ischemic zone. Seven dogs served as con-
trols and 4 dogs received nitroglycerin at 11 minutes (100 \ig bolus, then 75
|i,g/min) . We found that 1) the magnitude of retrograde flow was a strong deter-
minant of S-T segment change, suggesting that coronary collaterals protect
against ischemic injury and 2) although nitroglycerin increased total coronary
flow from 105 to 143 ml/min (P < .02), it had no significant effect on retro-
grade flow or S-T segment elevation. Since nitroglycerin does not alter col-
lateral function or ischemic ECG changes when MV02is constant, our data suggest
that nitroglycerin probably alleviates acute ischemj.a in the intact dog by al-
tering the peripheral hemodynamic determinants of MVO2 or retrograde flow.
Proposed Course of Project: Project continuing.
Honors and Awards: None
Publications: None
^
Serial No. NHLI-15(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS -NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Quantification of Ventricular Regularization in Atrial Fibril-
lation
Previous Serial Number: NHLI-124
Principal Investigator: Robert E. Goldstein, M.D.
Other Investigators: Leonard Grauer, M.D.
Paul Tecklenberg, M.D.
James Ware, Ph.D.
Martin Miller, B.A.
Stephen E. Epstein, M.D.
Cooperating Units: Biometrics Branch, NHLI
Laboratory of Applied Studies, DCRT
Clinic of Surgery, NHLI
Project Description: The ventricular response in patients with atrial fibril-
lation is characteristically irregular. Detailed scrutiny of R-R interval
lengths however, has shown a tendency in certain individuals for the length of
adjacent R-R intervals to be equal. Detection of this subtle evidence of non-
random ventricular response may be particularly useful in evaluating alterations
in the cardiac conduction system after digitalis or other pharmacologic agents
and possibly in predicting the development of digitalis excess. For this rea-
son we have developed a statistical method to quantify the extent to \>^ich equal
adjacent R-R interval lengths exceed random expectation. This method will then
be applied to electrocardiograph records (2000 consecutive R-R intervals) ob-
tained from patients in stable atrial fibrillation when digoxin therapy is with-
held and after digoxin is reinstituted. Results of the R-R intervals length
analysis will be compared with simultaneously determined serum digoxin levels.
Similar studies of the degree of regularization of the ventricular response
will also be conducted after atropine and during exercise. The purpose of our
study is 1) to observe the frequency and extent of ventricular regularization
during atrial fibrillation in the absence of digitalis toxicity, 2) to observe
whether ventricular regularization might provide an index of digitalis activity
(in addition to and possibly independent of the influence of digitalis on mean
ventricular rate), and 3) to evaluate the ability of influences other than dig-
italis to modify ventricular regularization. Such information may be helpful
in analyzing the degree of ventricular regularization in individuals suspected
of manifesting digitalis toxicity and in guiding the course of digitalis therapy
in individuals where response of the mean ventricular rate is not wholly ade-
quate as a measure of digitalis action.
Ay
Serial No. NHLI- 15(c)
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Recently, mathematical techniques and computer programs have been refined.
Use of simulated data has confirmed the sensitivity and specificity of our
index of regularization. When actual patient data are analyzed by these meth-
ods, our techniques; suggest that certain patients display a consistent tendency
toward regularization in the absence of any clinical overt evidence of digitalis
toxicity. At present this observation is being explored in greater depth.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
3ii
Serial No. NH LI- 16(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Quantitative Assessment of Left Ventricular Function in Man
Utilizing Roentgen Videodensitometry
Previous Serial Number: None
Principal Investigator: Leonard E. Grauer, M,D.
Other Investigators: William H. Schuette, B.E.E.
Willard C. Whitehouse, B.S.
Samuel B. Itscoitz, M.D.
David R. Redwood, M.D.
Cooperating Units: Biomedical Engineering and Instrumentation Branch
Division of Research Services, Television Engineering
Section, Clinical Center
Project Description; Left ventricular ejection fraction, the ratio between
stroke volume and end-diastolic volume, is widely used as an index of ventri-
cular function in patients with myocardial and valvular heart disease. How-
ever, as conventionally determined, the calculation of end-diastolic and end-
systolic ventricular volumes involves planimetry of appropriate frames of the
left ventricular cineangiogram, which is a time consuming and cumbersome under-
taking. Because of the difficulties of this method, an automated technique has
been developed that allows for the direct measurement of ejection fraction.
This technique determines the rate of wash-out of roentgen dense contrast
material from the left ventricle during cineangiography. A densitomer placed
over the image of the left ventricle continuously measures the changes in con-
trast density during the cine run. The degree to which the contrast is cleared
from the left ventricle with each systole is a function of the ejection fraction
and can be determined by finding the number of cardiac cycles (N) necessary to
wash out 507o of the contrast. The ejection fraction is then equal to i-e"^'"'-'''^
(the equation for an exponential curve). Preliminary results with this method
show an excellent correlation with data obtained by planimetry techniques.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
Af
Serial No. NHLI-17
1, Cardiology
2, Clinical Physiology
3, Bethesda, Md,
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Adrenergic Influences on Ventricular Fibrillation Threshold
and Post-occlusion Arrhythmias
Previous Serial Number; None
Principal Investigator: Lura Harrison, Ph. D.
Other Investigators: Kenneth M. Kent, M.D.
Cooperating Units: None
Project Description: The adrenergic neurohormones have been implicated in
disturbances of cardiac rhythm. Several reports have documented the
effectiveness of beta blocking drugs against certain experimental and clinical
arrhythmias. This study was undertaken to evaluate the role of a beta
blocking drug, propranolol, in controlling ventricular fibrillation during
normal conditions and following coronary artery occlusion. Thoracotomies
were performed on anesthetized dogs. The heart was exposed and the left
anterior descending coronary artery was carefully dissected. Stimulating
electrodes were placed on the left ventricle. Ventricular fibrillation
threshold (VFT) was determined by introducing a train of premature stimuli
into the left ventricle. The train of stimuli was introduced after the QRS
complex and extended throughout the vulnerable period. VFT was measured
during control conditions and following a 5 minute occlusion of the anterior
descending coronary artery. D,L-propranolol (0.05 mg/Kg) was administered
and control and post-occlusion VFT were measured again. A second series of
animals were administered D-propranolol (0.05 mg/Kg). Mean VFT during control
conditions was 55 ma (8 dogs). After administration of D,L-propranolol,
mean VFT was 125 ma. Mean post-occlusion VFT was 25 ma prior to
administration of D,L-propranolol and 25 ma after D,L-propranolol. Adminis-
tration of D-propranolol raised control VFT from a mean value of 41 ma to
45 ma. Thus, D,L-propranolol produced marked elevations in VFT during control
conditions but did not alter mean VFT during cardiac ischemia. D-propranoIol,
which has very little beta blocking activity, produced insignificant changes
in VFT.
Seven dogs were administered 6-hydroxydopamine. 6-OHDA depletes myocardial
norepinephrine stores. VFT determinations were carried out 72 hours following
6-OHDA administration. Mean VFT was 72 ma during control and 61 ma after
D,L-propranolol (0.1 mg/Kg). Following occlusion of the left anterior
descending coronary artery, mean VFT was 30 ma before D,L-propranolol and 26 ma.
after propranolol. It appears that the beta blocking properties of D,L-
propranolol are important in the elevation of VFT during control conditions.
1 30
Serial No. NHLI-17
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1972
Eighteen animals were subjected to occlusion of the left anterior descending
coronary artery at its origin. The first septal artery was also occluded.
Ten of these animals served as controls while the other 8 were treated with
6-OHDA 72 hours prior to study. Each animal was observed for a period of 30
minutes or until ventricular fibrillation occurred. Two of the 8 6-OHDA-
treated dogs fibrillated during the 30 minute study period. Eight of 10
control dogs fibrillated. It appears that the adrenergic neurohormones play
a role in development of spontaneous post-occlusion ventricular fibrillation.
Proposed Course of Project: Completed
Honors and Awards : None
Publications : None
5/
Serial No. NHLI-l8(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Factors Affecting the Operative Mortality in Aortic Valvular
Disease
Previous Serial Number: NHLI-131(c)
Principal Investigator: Walter L. Henry, M.D.
Other Investigators: Chester E, Clark, M.D.
Robert E. Goldstein, M.D.
Samuel B. Itscoitz, M.D.
Leonard E. Grauer, M.D.
David R. Redwood, M.D.
D. Luke Glancy, M.D.
Stephen E. Epstein, M.D.
Andrew G. Morrow, M.D.
Edward B. Stinson, M.D.
Charles L. Mcintosh, M.D.
Cooperating Units: Clinic of Surgery
Project Description: The purpose of this study is to define prospectively
those preoperative factors that indicate an increased operative risk or that
irreversible myocardial dysfunction has occurred. All patients 18 years old
or over admitted to the Cardiology Branch for aortic valve replacement will
be evaluated. Patients with significant involvement of other valves will not
be considered. Preoperative and 6-month postoperative assessment will be
based mainly on data obtained by cardiac catheterization (including coronary
arteriography) and echocardiography. Evaluation of myocardial function will
include ventricular volumes, LV mass, mean dv/dt, mean VCF, Vj^^jj and maxyog.
These data, as well as operative risk, will also be correlated with EKG,
x-ray, phonocardiogram, and exercise testing.
Forty-six patients have been assessed preoperatively . Twenty-five of
the forty-six have been studied also at the 6-month postoperative assessment
point. We expected a sample size of 50-100 patients. Preliminary results
suggest that many patients who in the past would not have been offered surgery
because of a suspected high risk, in fact do well following operation.
Proposed Course of Project: Continuing
Honors and Awards : None
Publications: None
3S-
Serial No. NHLI-19(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Mitral Valve Position in Patients with Asymmetric Septal
Hypertrophy
Previous Serial Number: None
Principal Investigator: Walter L. Henry, M.D.
Other Investigators: Chester E. Clark, M.D.
Stephen E. Epstein, M.D.
Cooperating Units : None
Project Description: For over a decade, it has been recognized that an unusual
form of obstruction to left ventricular outflow occurs in idiopathic hyper-
trophic subaortic stenosis (IHSS) . Pathological and echocardiographic reports
indicated that the basic abnormality in this disease was a cardiomyopathy
characterized by marked asymmetric septal hypertrophy (ASH) . Initially most
investigators considered the obstruction to be caused by obliteration of the
left ventricular outflow tract by vigorously contracting and hypertrophied
muscle. Recently, however, it has become clear that the obstruction is
caused by the anterior leaflet of the mitral valve which, during systole,
moves forward into the outflow tract and against the interventricular septum.
It also has been observed that many individuals with IHSS have the cardiomyo-
pathy but do not develop outflow obstruction. To determine why some patients
with asymmetric septal hypertrophy do and others do not have obstruction, we
measured the position of the mitral valve at the onset of systole (i.e.,
immediately before the abnormal forward motion) . Mitral valve position was
defined as a ratio of the posterior wall-mitral valve distance divided by the
septal-mitral valve distance. In normals, the mitral valve is positioned near
the posterior wall (ratio 0.28 + 0.01). In patients with ASH, the valve is
positioned much closer to the septum (ratio 0.85 + 0.08). When these patients
were subdivided according to the degree of left ventricular outflow obstruction,
we found that the proximity of the mitral valve to the ventricular septum just
prior to ejection was directly related to the magnitude of the gradient that
occurred during systolic ejection. We conclude that mitral valve position at
the onset of systole is a major factor determining the presence and severity
of left ventricular outflow obstruction in patients with asj^mmetric septal
hypertrophy. This finding is compatible with the concept that the hypertrophied
septum results in forward tethering of the anterior mitral leaflet during
isometric systole; the narrowed outflow tract results in a high velocity jet
during early systolic ejection which, via a Venturi effect, causes the mitral
leaflet to move even further forward resulting in LV outflow obstruction.
1 55
Serial No. NHLI-19(c)
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Proposed Course of Project: Continuing
Honors and Awards : None
Publications: None
3/
Serial No„ NHLI-20
1, Cardiology
2„ Clinical Physiology
3. Bethesda, Md .
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: A Video Scanner -Ana log Computer System for the Analy-sis of
Routine Echocardiograms
Principal Investigator: Walter L, Henry, M.D,
Other Investigators: James Griffith, M,S.E»E,
Cooperating Units: Electrical and Electronic Engineering Section
Biomedical Engineering and Instrumentation Branch
Division of Research Service
Project Description: A major reason for the expanding use of echocardiography
(ECHO) in cardiac diagnosis is that rapidly moving cardiac structures can be
visualized with ease and safety. Typically, ECHO studies have been limited to
qualitative observations because detailed quantitative measurements of cardiac
structure motion have been possible only through laborious and time-consuming
manual information processing. Therefore, we developed a video scanner-analog
computer system that greatly simplifies ECHO analysis. Up to 8 non-overlapping
cardiac structures first are traced manually from routine ECHO's onto trans-
parent paper. This traced record then is converted by a television (TV)
camera into a TV picture consisting of 524 video lines. These video lines
are processed sequentially by 8 individual signal detectors, each generating
a digital value proportional to the distance of each traced signal from the
beginning to the video line. The digital data is converted to analog form,
processed using analog computer techniques, and recorded on a strip chart.
Using this system, we analyzed 20 routine ECHO's and compared the results with
manual analysis. Mean velocity of circumferential fiber shortening (VCF) ,
maximum VCF, instantaneous transverse LV dimension (TLVD) , instantaneous
estimated LV volume ([TLVlfl ) and the derivative of mitral valve motion were
all accurately obtained (i>0.95). In addition, other physiological data recor-
ded on the ECHO record, such as EKG and pressures, may also be analyzed in
conjunction with cardiac structure motion (allowing, for instance, the
construction of force -velocity curves). The use of this system to analyze in
detail the motion of a variety of individual and paired cardiac structures
allows important cardiac diagnostic information to be obtained easily from
routine echocardiograms.
Proposed Course of Project: The developmental work is completed. The system now
is being used routinely.
Honors and Awards: None
Publications: None
5r
Serial No, NHLI-21(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Long-Terra Effects of Operation on Obstruction and LV
Hypertrophy in IHSS
Principal Investigator: Walter L. Henry, M.D,
Other Investigators: Stephen E. Epstein, MoD,
Chester E. Clark, M„D.
Edward B. Stinson, M.Do
Andrew G„ Morrow, MoD„
Cooperating Units: Clinic of Surgery, NHLI
Project Description: Surgical myectomy for IHSS results in symptomatic
improvement and loss of LV outflow obstruction. To determine the mechanism
by which the obstruction disappears, and the long-term effects of operation,
we used echocardiography to measure mitral valve postion, mitral valve
systolic motion, and left ventricular free wall thickness in 2 groups of
patients with IHSS: 13 patients who had myectomy performed 2 to 11 years
(mean 6.5 years) previously, and 27 nonoperated patients. Preoperative
hemodynamic data were comparable in both groups. The prominent forward
mitral valve motion in raidsystole, indicative of obstruction, was present in
each nonoperated and absent in each operated patient. Mitral valve position
at onset of systole was determined by calculating the ratio of mitral valve-
posterior left ventricular wall distance to septal-mitral valve distance. In
normals, the mitral valve is positioned near the posterior left ventricular
wall (ratio 0.28+.01), While the mitral valve is anteriorly positioned in
both IHSS groups it is more anterior in nonoperated patients (ratio 1.04 +
106) than in operated patients (ratio .66+,04, p .01). Intraoperative studies
in 6 patients revealed the mitral valve to assume a more posterior position
immediately after myectomy; this coincided with disappearance of the midsys-
tolic forward mitral valve movement. Left ventricular free wall thickness
was 13.2+.05 mm in the nonoperated patients (normal 9.4+. 02), and 11, 5+. 04 mm
in the operated (p .05). We conclude the mitral valve is tethered forward in
IHSS. Septal myectomy relieves this tethering and thereby abolishes the mid-
systolic forward mitral valve motion and hence left ventricular outflow ob-
struction. Abnormal mitral valve position and motion did not recur postop-
eratively during long-term followup. Finally, left ventricular free wall
thickening appears to regress postoperatively.
26
Serial No. NHTJ-22(c>
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Distribution of the Cardiomyopathy in IHSS
Previous Serial Number: NHLI-130(c)
Principal Investigator: Walter L. Henry, M.D,
Other Investigators: Chester E. Clark, MoD.
Stephen E, Epstein, M.D.
Cooperating Units: None
Project Description: Although symptoms in patients with IHSS are usually re-
lated to left ventricular (LV) outflow obstruction caused by a disproportion-
ately hypertrophied ventricular septum, IHSS is believed to be a diffuse car-
diomyopathy involving the entire LV. The fact that several patients in whom
LV outflow obstruction spontaneously disappeared have died as a result of
severe diffuse congestive heart failure supports such a view. Based on the
concept that IHSS is a diffuse cardiomyopathy it has been suggested that sur-
gical intervention is not justified since the diffuse myocardial deterioration
will presumably continue and eventually result in congestive heart failure
despite the surgically-induced abolition of the LV outflow gradient. This
view of operative therapy is reasonable if the hypothesis regarding the
diffuse nature of IHSS is correct. However, long-term postoperative follow-up
of over 65 patients at the NIH suggests that the marked symptomatic improvement
occurring shortly after surgery in almost all patients has been maintained for
many years.
To determine whether IHSS is a diffuse cardiomyopathy or is localized to the
interventricular septum, we are measuring LV posterior wall thickness in
sjmiptomatic IHSS patients, asj^mptomatic family members and long-term post-
operative IHSS patients. This study has become feasible since the technique
of echocardiography enables septal and LV posterior wall thickness to be
measured non-invasively as often as is necessary. Our working hypothesis is
that if the disease is not a diffuse and progressive cardiomyopathy, the mean
LV posterior wall thickness would be normal both early in the course of the
disease before LV outflow obstruction develops (for example in patients who have
echo evidence of septal hypertrophy but no other manifestations of IHSS) and in
IHSS patients who have had relief of the obstruction by surgery. While our
data are still preliminary, several trends are emerging. Thus far, mean
posterior LV wall thickness in sjraiptomatic patients with IHSS is greater than
normals and essentially equal to mean posterior LV wall thickness in patients
with other forms of LV outflow obstruction. In contrast, LV posterior wall
thickness in 16 long-term postoperative IHSS patients are all within the range
of normal. In addition, we have identified several asymptomatic family members
of IHSS patients in whom the interventricular septum is hypertrophied; unlike
1 37
NHLI-22(c)
the symptomatic propositi, however, the LV posterior wall is normal. Moreover,
mitral valve echocardiograms are normal in these subjects, suggesting that LV
outflow obstruction is not present.
These preliminary observations are consistent with the hypothesis that the
basic abnormality in IHSS is not a diffuse and progressive cardiomyopathy.
Rather, it would appear that the specific pathologic process of IHSS may be
limited to the septum and that non-specific hypertrophy of the remainder of
the LV occurs only after LV outflow obstruction has developed. If this
hypothesis is correct, it would have important implications regarding both the
basic pathophysiology of IHSS and its surgical therapy.
Proposed Course of Project: Several complete families of patients with IHSS
will be studied as well as those postoperative IHSS patients who have survived
5 years or longer. In addition, arrangements have been made to correlate pre-
operative and intraoperative echocardiographic studies with electron-microscopic
studies performed on septal and free wall biopsies.
Honors and Awards: None
Publications: None
38
Serial No. NHLI-23(c)
1. Cardiology
2. Cardiovascular Diagnosis
'3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Non-Invasive Determination of the Pressure Gradient in
Patients with Idiopathic Hypertrophic Subaortic Stenosis
Previous Serial Number: NHLI-149(c)
Principal Investigator: Walter L. Henry, M.D,
Other Investigators: Chester Clark, M.D.
D, Luke Clancy, M,D,
Stephen E. Epstein, M,D,
Cooperating Units: None
Project Description: The variable obstruction to left ventricular outflow in
idiopathic hypertrophic subaortic stenosis is thought to be caused by an
abnormal forward movement of the anterior mitral valve leaflet during ventricu-
lar systole resulting in narrowing of the aortic outflow tract between the
anterior mitral valve leaflet and the asymmetrically hypertrophied interven-
tricular septum. This abnormal leaflet motion has been demonstrated angio-
graphically and subsequently confirmed by echocardiography. The latter
technique is particularly useful since it allows a beat-by-beat recording of
both the anterior mitral leaflet motion and the mitral valve -septal distance.
The abnormal systolic mitral valve motion has been described previously as
only being seen when an LV outflow gradient is present and disappearing when
the gradient is absent.
These echo observations were made on the day prior to cardiac catheterization
and are difficult to interpret in view of both the highly variable nature of
the LV outflow obstruction and some of our own angiograms that show the leaflet
abnormality in patients without a resting LV outflow pressure gradient. More
importantly, no studies have been published correlating mitral valve-septal
distance with the degree of LV outflow obstruction in quantitative terms.
In order to determine whether the mitral leaflet motion or the mitral valve-
septal distance can be used as a non -invasive assessment of the degree of LV
outflow obstruction, echocardiograms were obtained in patients with idiopathic
hypertrophic subaortic stenosis during diagnostic cardiac catheterization^
Anterior mitral leaflet motion and the mitral valve-septal distances were
measured by echocardiography and compared with simultaneously recorded LV
outflow pressure gradients under a variety of conditions. Using data obtained
from 56 individual cardiac cycles in ten patients with variable pressure
gradients, it has been possible to compute an obstruction index which incor-
porates the degree of narrowing of the septal -mitral valve distance as well
as the duration that this narrowing occurs. This index was plotted against the
39
NHLI-23(c)
simultaneously determined LV outflow pressure gradients over a range of
pressure gradients between 0 and 160; the oDrrelation coefficient was 0.97
(P .001). This non-invasive technique provides us with a powerful tool to
determine the natural history of idiopathic hypertrophic subaortic stenosis
as well as the acute and long-term effects of pharmacologic and surgical
interventions.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: Article to be published in a periodical: NEW ENGLAND JOURNAL
OF MEDICINE.
Vfe»
Serial No. NHLI-24(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Echocardiographic Diagnosis of IHSS by Detection of
Asynnnetric Septal Hypertrophy
Previous Serial Number: NHLI-132(c)
Principal Investigator: Walter L. Henry, M.D.
Other Investigators: Chester E. Clark, M.D.
Joyce McKay, B.S.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: IHSS is a disease characterized by variable subaortic
obstruction to left ventricular outflow that in most patients can be diagnosed
at the bedside by characteristic physical findings. In other patients, par-
ticularly those without resting pressure gradients, the physical, electrocar-
diographic, and roentgenographic findings may be unimpressive and the diagnosis
difficult unless cardiac catheterization is performed. Recently we have found
that echocardiography, a noninvasive technique, can be used to measure septal
and LV posterior free-wall thickness. The present study was undertaken to
determine if echocardiography could be used to detect asj^mmetric septal hyper-
trophy, a finding that at postmortem examination has been shown to be a sensi-
tive and specific abnormality in patients with IHSS. Up to the present time,
12 normals, 14 patients with LV outflow obstruction (exclusive of patients
with IHSS) , and 62 patients with a wide variety of cardiac abnormalities have
been studied. In these patients the mean septal-posterior free wall ratio was
1.06 with only one of 88 patients having a ratio greater than 1.3. In contrast,
11 patients with catheterization-documented IHSS had a mean septal-posterior
free wall ratio of 1.6; every patient had a value exceeding 1.3. In some
patients, the septal-free wall ratio was the only evidence of IHSS short of
catheterization. The ratio has been found to be abnormal even in patients who
at catheterization have no resting gradient. Thus, this ratio is a sensitive
and specific method for noninvasively diagnosing IHSS and is well suited for
both family screening studies and followup studies of affected individuals.
Proposed Course of Project: Completed
Honors and Awards : None
Publications: ARTICLE PUBLISHED IN A PERIODICAL
Henry, W.L., Clark, C.E. and Epstein, S.E.: Asymmetric septal hypertrophy.
Echocardiographic identification of the pathognomonic anatomic abnormality
of IHSS. Circulation 47: 225-233, 1973.
1 ^/
Serial No. NHLI-25(c)
1, Cardiology
2. Clinical Physiology
3o Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Clinical Characteristics of Obstructive and Nonobstructive
Asyranietric Septal Hypertrophy
Principal Investigator: Walter L. Henry, M.D,
Other Investigators: Chester E. Clark, M„D,
Stephen E. Epstein, MoD.
Cooperating Units: None
Project Description: Since the early descriptions of IHSS, patients have been
described with features suggestive of the disease but in whom no resting or
provocable left ventricular outflow obstruction could be demonstrated. These
findings have been interpreted as indicating that IHSS is only one manifesta-
tion of a disease spectrum, i.e., hypertrophic cardiomyopathy in which
obstruction may or may not occur. Recently we have confirmed this hypothesis
by using echocardiography to identify a specific anatomic abnormality, the
presence of which is independent of outflow obstruction. Asymmetric septal
hypertrophy (ASH), characterized by a ventricular septum at least 1,3 times as
thick as the posterior-basal left ventricular free wall, was found in all
patients vh ose disorder falls within the IHSS disease spectrum. One hundred
patients with ASH were examined. Analysis of multiple clinical parameters
failed to distinguish the nonobstructive ASH patients from the obstructive
group with two exceptions: in nonobstructive ASH the murmur was softer with
little variation following provocative maneuvers and a bisferious carotid
pulse was absent. Angina and syncope, symptoms usually considered character-
istic of obstruction, were also common in patients without obstruction. We
conclude 1) obstructive and nonobstructive ASH patients cannot be distinguished
symptoraatically, 2) the absence of typical physical findings makes the clinical
diagnosis of nonobstructive ASH difficult, and 3) echocardiography is the
simplest and most reliable means of establishing the diagnosis in patients with
ASH.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
^^
Serial No. NHLI-26(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: A Real Time System for Two-Dimensional Echocardiography
Previous Serial Number: None
Principal Investigator: Walter L. Henry, M.D.
Other Investigators: James Griffith, M.S.E.E.
Cooperating Units: Electrical and Electronic Engineering Section
Biomedical Engineering and Instrumentation Branch, DRS
Project Description: During the past several years one-dimensional echo-
cardiography has proven to be a useful tool in cardiac diagnosis. Two-
dimensional echocardiography promises to be even more useful, particularly in
patients with congenital heart disease.
A system has been developed for obtaining real time, two-dimensional echo-
grams using a hand held instrument that rapidly scans a thirty degree sector
from a fixed spot on the patient's chest. This design was used because in
many individuals, satisfactory ultrasound signals only can be obtained from a
limited window located between ribs, lateral to the sternum and medial to the
lungs. In addition the system is rather small and thus an operator easily can
alter its orientation until the desired cardiac structures are seen on the
display.
The scanning device uses the same pulsed-echo transducer and signal proces-
sing techniques used for one-dimensional systems. A sector is scanned by
tilting the transducer back and forth with a crank and lever system powered by
a small DC motor. Tachometer feedback controls motor speed so that frame rate
does not change appreciably with variation in pressure between the transducer
and patient. To monitor transducer angle, a rotary variable differential
transformer (RVDT) is also attached to the motor through a level system iden-
tical to the one driving the transducer. Because of the scanner's design, the
transducer can be pressed directly against the patient's chest, using a layer
of acoustic coupling gel to ensure good ultrasonic coupling.
The echos received from the scanned sector are displayed on a CRT screen.
This display is produced by electronically synchronizing the transmitted pulses
and the transducer's angular position. This synchronization allows the elec-
tron beam in the display system to scan the CRT in such a way that its position
is always proportional to the position from which the echos are being received.
Thus, the scan is displayed on the CRT screen in real time. A television camera
^s
Serial No. NHTJ-26fc)
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
(focused on the display CRT) and a video tape are used to record the data.
This recording system allows visualization of individual cardiac structures,
including fast moving structures like the mitral valve. Another important
strength of the video system is that data are available for instant replay and
analysis.
Proposed Course of Project: Continued design improvement may require 6 months
to one year. During this time, experience in diagnosing a variety of cardiac
diseases will be gained. In addition, other two-dimensional systems will be
evaluated.
Honors and Awards: None
Publications: None
^^
Serial No. NHLI-27(c)
1. Cardiology
2. Cardiovascular Diagnosis
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effects of Operation on the Cardiac Response to Exercise in
Patients with IHSS
Previous Serial Number: None
Principal Investigator: John W. Hirshfeld, Jr. , M.D,
Other Investigators: Robert E. Goldstein, M.D.
David R, Redwood, M.D,
Andrew G. Morrow, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: Clinic of Surgery
National Heart and Lung Institute
Project Description: Operative resection of a portion of the abnormally and
disproportionally hypertrophied ventricular septum in patients with IHSS abol-
ishes left ventricular outflow obstruction, reduces left ventricular end-dia-
stolic pressure, and uniformly results in marked symptomatic improvement.
However, improved hemodynamics have been demonstrated only at rest in the
catheterization laboratory, and the conclusions that operation improves symp-
toms are based on historical information, a notoriously unreliable means of
assessing the results of cardiac surgeiy , We therefore have initiated a study
to characterize the circulatory response to mild and intense levels of exer-
cise before and after operation as well as to determine the effects of opera-
tion on objectively determined exercise capacity.
Patients with catheterization documented IHSS, who because of severe symp-
toms are referred for operation, are being studied preoperatively in the exer-
cise laboratory. Cardiac output, oxygen consumption, and pulmonary and system-
ic arterial pressures are determined at rest, at a low level of exercise, and
during an exhausting level of treadmill exercise. At another time the patient's
maximal exercise capacity (measured in terms of total body oxygen consumption)
is determined. These studies will be repeated six months after operation. At
the present time 9 patients have been studied before operation and are await-
ing postoperative assessment.
Course of Project: Continuing
Honors and Awards: None
Publications: None
Vi"
Serial No. NHLI-28
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md .
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Reduction in Extent of Myocardial Infarction When Nitro-
glycerin and Methoxamine are Administered During Coronary
Occlusion
Principal Investigator: John H. Hirshfeld, Jr., M.D.
Other Investigators: Jeffrey S. Borer, M.D,
Michael Barrett, M.D,
Robert E. Goldstein, M.D,
Stephen E. Epstein, M.D.
Project Description: Nitroglycerin (TNG) reduces ST segment elevation in
dogs during 15 minutes of coronary occlusion. Moreover, when the TNG-induced
fall in blood pressure (BP) is abolished by simultaneous administration of
methoxamine, a further decrease in ST elevation ensues. The present study
examined the effects of TNG and methoxamine on the development of myocardial
infarction after 5 hours of coronary occlusion in 30 closed-chest sedated
dogs. Acute coronary occlusion was produced by inflating a cuff previously
implanted around the LAD coronary artery. After 10 minutes of ischemia (the
intensity of which was estimated by measuring ST segment elevation recorded
by intramyocardial electrodes), dogs were randomized into control and treated
groups. Treated dogs received iv TNG during the remainder of the 5 hour
occlusion. BP was maintained at preinfusion levels with iv methoxamine. Six
control dogs died and each of the 10 survivors developed grossly visible
extensive transmural infarction. In contrast, only 3 treated dogs died and
10 of the 12 survivors had minimal or no visual evidence of infarction. This
marked reduction in the severity of the ischemic insult was more quantitative-
ly documented in survivors 24 hrs after infarction by measurement of creatine
phosphokinase (CPK) content of myocardium subjacent to each electrode. In
controls, a significant reduction (p^,05) in CPK occurred at 48 of 57 elec-
trode sites where ST elevation was observed after 10 minutes of occlusion.
Only 19 of 69 such sites showed significant CPK reduction in the treated dogs
(p^.OOl) . Each control dog had multiple sites where CPK was ^407, of CPK in
nonischemic areas, but only 2 treated dogs displayed such CPK loss. Three
treated dogs manifested no CPK reduction. Thus, administration of TNG, with
support of BP by methoxamine, markedly reduces the extent and severity of in-
farction occurring after 5 hours of coronary occlusion.
Proposed Course of Project: Project completed
Honors and Awards: None
Publications: None
^
Serial No. NHLI-29(c)
1. Cardiology
2. Cardiovascular Diagnosis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Preoperative Predictors of the Long-Term Results of Valve Re-
placement in Patients with Aortic Regurgitation.
Previous Serial Number: None
Principal Investigator: John Hirshfeld, Jr., M,D.
Other Investigators: Stephen E. Epstein, M.D.
D. Luke Clancy, M.D.
Andrew G. Morrow, M.D.
Cooperating Units: Clinic of Surgery
National Heart and Lung Institute
Project Description: The long term results of aortic valve replacement in all
patients operated upon at the NIH for pure aortic regurgitation have been re-
viewed. Of the 89 patients in the series, 50% died within four years of oper-
ation: there were 14 operative and 31 late deaths. These results are disap-
pointing and suggest that earlier operation might be advisable in some patients.
We therefore extended our studies to determine whether preoperative prognostic
indices could be developed to identify those patients who were entering into
a subgroup that would be at particular high risk following operation.
Survival curves for patients with different preoperative clinical and hemo-
dynamic parameters were constructed and compared. Symptoms, functional class,
heart size, or cardiac output did not correlate with long term survival. How-
ever, several powerful prognostic indices were identified. 1) The severity of
left ventricular (LV) hypertrophy was graded from the ECG by the Estes method.
Sixty-five percent of patients with a point score of 6 or less were living at
3 years; this contrasted with only 447o of patients living with point scores
greater than 6 (507o were dead within 14 months) . 2) Of patients whose LV end-
diastolic pressure was normal, 74% were alive at 3 years; however, only 40% of
patients whose LVEDP was greater than 20 mm Hg were alive at 3 years (50% were
dead within 20 months). 3) Of patients with a normal pulmonary arterial sys-
tolic pressure (PAP), 79% were alive at 3 years; in contrast, of patients with
PAP greater than 40 mm Hg, only 39% suirvived 3 years (50% were dead within the
first 6 months).
These data indicate that subgroups of patients with aortic regurgitation can
be identified preoperatively who will have markedly different prognoses follow-
ing aortic valve replacement. If similar information can be generated to de-
fine survival of unoperated patients, it might be possible to develop more sat-
isfactory criteria regarding optimal timing of operation relative to the course
of the patient's illness.
1 V7
Serial No. NHLI-29(c)
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
Vg
Serial No. NHLI-30(c)
1. Cardiology
2. Cardiovascular Diagnosis
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Characterization of the Cardiac Response to Exercise and
Effects of Propranolol in Patients with Asymmetric Septal
Hypertrophy
Previous Serial Number: None
Principal Investigator: John W. Hirshfeld, Jr., M.D.
Other Investigators: Robert E. Goldstein, M.D,
David R. Redwood, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: The cardiac response to upright exercise in patients with
asymmetric septal hypertrophy (ASH) has not been characterized. In addition,
although propranolol is used for the symptomatic treatment of many patients
with this disease, it recently has become apparent that patients present with
a wide hemodynamic and synq)tomatic spectrum. Thus, left ventricular outflow
obstruction may be present under resting conditions (classic IHSS), it may ap-
pear only upon provocation, or it may be entirely absent. Moreover, some pa-
tients have angina pectoris as a limiting symptom, while others report fatigue,
shortness of breath, or presyncope. The present study was undertaken to study
the circulatory response to exercise of these various subgroups and to deter-
mine if different patterns of response to propranolol exist.
Cardiac output, oxygen consumption, and pulmonary arterial pressures are
determined at rest, at a low level, and at an exhausting level of treadmill
exercise before and after propranolol. In a separate series of chronic
studies, maximal exercise capacity (measured as maximal total body oxygen
consumption) is determined and the effects of oral propranolol determined.
Fourteeen patients have been studied to date; 9 had LV outflow obstruction,
5 did not. Propranolol consistently reduced cardiac output during exercise.
The drug also reduced or had no effect on maximal exercise capacity of all pa-
tients except one. This patient was limited by severe angina pectoris and im-
proved following the drug. These preliminary data indicate that relief of LV
outflow obstruction produced by propranolol does not result in an improved
cardiac output response to exercise. Moreover, they suggest that propranolol
may improve maximal exercise capacity only in those patients who have severe
angina pectoris.
Proposed Course of Project: Continuing.
1 ^
Serial No. NHT ,7-30^0')
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Honors and Awards: None
Publications: None
SO
Serial No. NHLI-31
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Cholinergic Innervation of the Cardiac Conduction System:
Autonomic and Functional Correlations
Previous Serial Number: None
Principal Investigator: Kenneth M. Kent, M.D., Ph.D.
Other Investigators: David Jacobowitz, Ph.D.
Stephen E. Epstein, M.D.
Theodore Cooper, M.D., Ph.D.
Cooperating Units: Laboratory of Clinical Science
National Institute of Mental Health
Project Description: Vagal stimulation increases ventricular fibrillation
threshold (VFT) under ischemic and nonischemic conditions. The purpose of the
present study was to localize the cholinergic pathways responsible for this
important stabilizing effect. VFT was defined as the current required to pro-
duce VF by a 200 msec pulse delivered to the ventricle during the vulnerable
period. In 6 control dogs, vagal stimulation significantly raised VFT from
24 + 3 ma to 88 + 8 (p < .03). In another 8 dogs, vinblastine, a neurotoxic
agent, was injected into the para-aortic area adjacent to the AV node. This
area contains cholinergic ganglia that supply postganglionic fibers to the
ventricle. Five to 7 days later, vagal stimulation caused atrial slowing but
did not change VFT. Thus, vagal afferents to the atrium were intact, but
those to the ventricle, responsible for increasing electrical stability, were
functionally inoperative. The anatomic correlate of this electrophysiologic
finding was obtained by sectioning the ventricular septum of control and vin-
blastine injected dogs and staining adjacent sections with 1) pseudocholin-
esterase, to localize the ventricular conducting system and 2) acetylcholin-
esterase, to identify cholinergic nerves. In control dogs many Purkinje fibers
were surrounded by cholinergic nerves. However, few of these cholinergic
fibers were found in vinblastine treated dogs.
Since the para-aortic dissection in these animals also caused catechola-
mine depletion by interrupting ventricular sympathetic nerves, another group
of animals was studied. Total cardiac sjrmpathectomy was performed by treat-
ing the animals with 6-OH-dopamine (20 mg/kg) 5 days before the study. Cate-
cholamine depletion raised VFT significantly to 73 + 14 (p < .001). However,
vagal stimulation further enhanced electrical stability in these animals by
raising VFT to 108 + 21 ma. In a single animal in which vinblastine was
injected into the para-aortic region with subsequent cholinergic denervation.
r/
Serial No. NHLI-31
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
vagal stimulation actually lowered the VFT from 40 ma to 24 ma. Analysis of
norepinephrine content in the ventricle of this animal revealed that normal
catecholamine stores were present as opposed to the remainder of the operated
group. The lowering of VFT by vagal stimulation was therefore interpreted as
decreased electrical stability of the heart mediated by intact sympathetic
nerves and absent cholinergic fibers.
We conclude that 1) stimulation of the vagus nerve decreases vulnerability
of the ventricle to fibrillation, 2) this action is mediated by a rich net-
-work of cholinergic nerves intimately related to Purkinje fibers in the ven-
tricular septum, and 3) this effect is independent of intact catecholamine
stores which in fact appear to have opposite effects.
Proposed Course of Project: Completed
Honors and Awards : None
Publications: None
.52-
Serial No. NHLI-32
1. Cardiology
2. Clinical Physiology
3# Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Electrical Stability of Acutely Ischemic Myocardium;
Independent Influences of Heart Rate and Vagal Tone,
Previous Serial Number: NHLI-138
Principal Investigator: Kenneth M. Kent, M.D., Ph.D.
Other Investigators: Eldon R. Smith, M.D.
David R. Redwood, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: Previous investigations have shown that a slower heart
rate (HR) and myocardial ischemia independently diminish the electrical
stability of the heart. It therefore was suggested that increasing heart rate
during myocardial infarction might diminish the incidence of serious ventricular
arrhythmias. However, since increased HR during experimental acute myocardial
ischemia augments the degree of ischemia, an evaluation of the presumed
"protective" effects of increased HR on the electrical stability of acutely
ischemic myocardium was undertaken. The differences in refractory periods (RP)
of eight contiguous areas of the left ventricle were determined as a function
of HR, In non- ischemic myocardium, the disparity of RP was less at a HR of
180 than 60, However, in ischemic myocardium the disparity increased in three
of six animals as the HR was increased from 60 to 90, in seven of ten animals
as HR was increased from 60 to 120, and in all animals when the HR was increased
from 60 to 180, The increased disparity of RP is believed to favor development
of re-entrant arrhythmia. The vulnerability of the heart to develop ventricular
fibrillation was assessed by determining ventricular fibrillation threshold
(VFT). During ischemia, VFT was not only an inverse function of HR but also
was found to be independently influenced by electrical stimulation of the
cervical vagus nerves. In the absence of vagal stimulation VFT was lowered
in only one of four dogs as HR was increased from 50 to 90, but decreased
30% (P<0.01) as HR reached 120 and 74% at 180 beats/min. When vagal
stimulation was used to control HR VFT was lowered 37% as HR was increased
from 50-60 to 90 (P<0.05). We conclude that increasing HR within a
physiologic range by diminishing vagal tone during myocardial ischemia decreases
electrical stability of the ventricle by 1) increasing ischemia consequent to
the rate induced increase in myocardial oxygen requirements, and 2) a direct
electrophysiologic action of the vagus on the ventricular myocardium.
S3
Serial No. NHLI-32
!• Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Proposed Course of Project: Completed
Honors and Awards: None
Publications: ARTICLE PUBLISHED IN A PERIODICAL
Kent, Kenneth M., Smith, Eldon R., Redwood, David R., and Epstein,
Stephen E. : Electrical stability of acutely ischemic myocardium:
Independent influences of heart rate and vagal tone. Circulation 47;
291-298, 1973.
SV
Serial No. NHLI-33
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md,
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Electrical Stability of Acutely Ischemic Myocardial
Effects of Nitroglycerin
Previous Serial Number: None
Principal Investigator: Kenneth M. Kent, M.D.
Other Investigators: Eldon R. Smith, M.D,
David R. Redwood, M.D.
Stephen E. Epstein, M.D,
Cooperating Units: None
Project Description: Nitroglycerin (TNG) is effective in reducing the size of
ischemic injury following experimental coronary occlusion. In this study the
effects of TNG on the electrical stability of acutely ischemic myocardium was
assessed. Ventricular fibrillation (VF) was produced in 5 open-chest dogs by
delivering a 200 msec train of pulses to the left ventricle during the vulner-
able period. Voltage was increased until VF occurred, VF threshold (VFT) was
defined as the current in milliamps (ma) required to produce VF. Acute myo-
cardial ischemia (AMI) was produced by occlusion of the LAD coronary artery.
Heart rate was kept constant at 120/min by destruction of the A-V node and
ventricular pacing. VFT was measured during 1) nonischemia, 2) ischemia, 3)
ischemia plus nitroglycerin, and 4) ischemia plus nitroglycerin and simultane-
ous infusion of phenylephrine to restore mean systemic arterial pressure (SAP)
to control levels. Interventions were randomized. In the absence of ischemia
VFT was 77+5 ma, a value unaffected by either TNG or phenylephrine. Follow-
ing 6 min of AMI, VFT was reduced to 30 + 6 ma (p < .01). When TNG was infused
at 200 |j,g/min (causing a mean decrease in SAP of 26%), VFT after 6 min of AMI
increased from 30 + 6 to 55 + 3 ma (p < .005), Restoration of SAP during TNG
infusion by phenylephrine raised VFT further to 75 + 6 ma, a value identical
to that present in the absence of ischemia. We conclude that TNG enhances
electrical stability of the heart during experimental AMI, an effect probably
related to reduction of ischemic injury. Since VFT increases even further after
SAP is restored to control levels, it is clear that this beneficial electrophys-
iologic effect is caused by some action of TNG other than reduction in myocar-
dial O2 consumption mediated by decreased afterload.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: Manuscript in preparation.
1 ST'
Serial No. NHLI-34
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Role of Prostaglandins in the Control of Coronary Vascular
Tone
Previous Serial Number: None
Principal Investigator: Kenneth M. Kent, Ph.D. , M.D.
Other Investigators: R. Wayne Alexander, M.D. , Ph.D.
Cooperating Units: Experimental Therapeutics Branch
National Heart and Lung Institute
Project Description: The prostaglandins are a ubiquitous group of vasoactive
agents that have been shown to be physiologic regulators of blood flow in the
kidney and adipose tissue. The purpose of the present study was to assess the
role of the prostaglandins in the regulation of coronary blood flow.
Coronary blood flow was measured in anesthetized open-chest intact dogs and
isolated canine heart lung preparations by direct cannulation of the left main
coronary artery (Gregg cannula). The coronary cannula was perfused with ar-
terial blood and flow was measured with an electromagnetic flow probe in the
extracorporeal circuit. Prostaglandin activity was estimated in coronary sinus
blood by ethyl acetate extraction, separation of the prostaglandins on a silic-
ic acid column and conversion of prostaglandin E2 to B.
Coronary blood flow regulation was assessed by the magnitude of the hyper-
emic response to ischemia and hypoxia. Excess coronary flow following ische-
mia (reactive hyperemia) was computed by electronic integration of the area
under the coronary flow curve after occlusions of the left main coronary artery
which lasted 10, 15 and 20 seconds. The integral of the flow curve, above base-
line flow, following occlusion was proportioned to the length of the occlusion:
10 sec occlusion = 27 + 8 ml ; 15 sec = 49 + 14 ml ; 20 sec =62+18 ml. Pros-
taglandin synthesis was then blocked by intracoronary indomethacin, 10 ^xg/ml
(concentration sufficient for 907., inhibition of prostaglandin synthetase).
After indomethacin the reactive hyperemia was significantly decreased (10 sec
occlusion =13+7 ml, 15 sec =31+7 ml, 20 sec = 31 + ml; p < 0.025 for all
three groups). Likewise the coronary vasodilator response to hypoxia was sig-
nificantly impaired. Preliminary studies with meclofenamate 10 |i,g/ml, another
prostaglandin synthetase blocking agent, have demonstrated similar effects on
coronary vasodilation induced by ischemia and hypoxia.
To rule out the possibility that the decrease in hyperemia responses was
due to nonspecific actions of the drugs, the coronary vascular reactivity to
1 Si
Serial No. NHLI-34
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
two vasodilator agents was determined before and after indomethacin and meclo-
fenamate. The dose response curves to adenosine and exogenous prostaglandin
E2 were unchanged after indomethacin and meclofenamate.
To determine whether the decrease in myocardial flow in response to ische-
mia or hypoxia was due to^a drug induced decrease in metabolic rate and myo-
cardial O2 consumption (MVO2) ,MV02 was measured after intracoronary adminis-
tration of indomethacin and meclofenamate. Neither drug changed MVO2 i^i two
experiments in intact dogs.
In preliminary experiments ischemia and hypoxia both led to a release of
a prostaglandin -like substance (a conversion product of PGE2) from the heart.
This response was absent after infusion of indomethacin or meclofenamate.
These results suggest that the coronary vasodilation response to ischemia and
hypoxemia is dependent on intact prostaglandin synthesis.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
S7
Serial No. NHLI-35
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Physiological and Biochemical Characteristics of a New Group
of Inotropic Agents: Analogues of Angiotensin II.
Previous Serial Number: NHLI-137
Principal Investigator: Kenneth M. Kent, M.D. , Ph.D.
Other Investigators: Theodore Goodfriend, M.D.
Theodore Cooper, M.D. , Ph.D.
Cooperating Units: Department of Pharmacology
University of Wisconsin, Madison, Wisconsin
Project Description: Angiotensin II and analogues of this compound are potent
inotropic agents which have the unique property of increasing contractility of
hypoxic myocardium without subsequent deterioration of muscle function as found
when either norepinephrine or ouabain are administered under hypoxic condi-
tions. The lack of contractile deterioration may be due to the mitochondrial
effects of the angiotensins, which include increasing the rate of phosphoryla-
tion, increasing respiratory control, and increasing resistance to "aging."
The above studies were performed in isolated myocardium perfused with a
modified Krebs solution. To test the effectiveness of these compounds in in-
tact blood perfused hearts, a blood perfused canine heart-lung preparation was
developed. Unfortunately, in this preparation, the cardioactive analogues of
angiotensin II are virtually inactive. The inef fectivenss is probably due to
either the large amount of circulating renin in this preparation with the re-
sultant tachyphylaxis of angiotensin receptors, or to the cleavage of these
peptides by the many peptidases present in whole blood. To test the latter
hypothesis, the contractile effects of N-methyl and N-poly serine analogues
were studied since peptidase cleavage should be delayed. However, these com-
pounds also were ineffective in intact hearts. Although these compounds rep-
resent a unique group of inotropic agents, the potential beneficial effects in
intact hearts appears doubtful.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: ARTICLE PUBLISHED IN A PERIODICAL:
Kent, Kenneth M. , Epstein, Stephen E., Cooper, Theodore: Potentiation
of the contractile effects of norepinephrine in hypoxia. J. Clin. Invest.
51: 2459-2464, 1972
se
Serial No. NHLI-36(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Differences in distribution of morphologic abnormalities in
patients with obstructive and non-obstructive asymmetric
septal hypertrophy (ASH): Light and electron microscopic
observations.
Previous Serial Ntimber: None
Principal Investigator: Barry J. Maron, M.D.
Other Investigators: Victor J» Ferrans, M.D,, Ph.D.
Andrew G. Morrow, M.D.
William C. Roberts, M.D.
Walter L. Henry, M.D.
Chester E. Clark, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: Section on Pathology, NHLI
Clinic of Surgery, NHLI
Project Description: To determine whether pathologic changes in the heart of
patients with asymmetric septal hypertrophy (ASH) are limited to the
ventricular septum (VS) or are more diffusely distributed, light and electron
microscopic studies were made of myocardium obtained at operation from
8 patients with obstructive ASH (typical IHSS) and 4 patients with non-
obstructive ASH who were either severely limited or had died of their
disease. Morphologic abnormalities were grouped into two categories:
1) intracellular abnormalities (disarray of myofibriles and myofilaments),
2) cell-to-cell abnormalities (disorganization of muscle cells, abnormal cell
contracts). Septal biopsies of all patients contained many muscle cells that
displayed extensive intracellular and cell-to-cell abnormalities. In the
nonobstructed patients, intracellular and cell-to-cell abnormalities also were
present in biopsies of the left ventricular (LV) free wall. In contrast,
cell-to-cell abnormalities were not seen in the free LV wall of any of the
obstructed patients. These findings demonstrate that extensive morphologic
abnormalities in both obstructive and nonobstructive ASH are invariably
present in the ventricular septum. Extensive involvement of the free wall,
however, appears to be limited to the nonobstructive patients. These findings
suggest that while cardiac function is impaired by a severe diffuse cardio-
myopathy in the nonobstructive patients, functional limitation of the
obstructive patients is due largely to LV outflow obstruction.
Sf
Serial No. NHLI-36(c)
1* Cardiology
2» Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
6o
Serial No. NHTJ-RT^r^
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Significance of Multiple Intercalated Discs in Hypertrophied
Human Myocardium
Previous Serial Number: None
Principal Investigator: Barry J. Maron, M.D.
Other Investigators: Victor J. Ferrans, M.D., Ph.D.
Cooperating Units: Section on Pathology
National Heart and Lung Institute
Project Description: Multiple intercalated discs were frequently observed in
muscle cells from patients with cardiac hypertrophy of various causes. Evi-
dence is presented that these structures are the intercellular junctions of
lateral processes of muscle cells. The sizes and shapes of these processes
showed marked variations, depending in part on the plane of sectioning.
It is postulated that lateral processes develop in certain cells which have
side-to-side intercellular junctions with adjacent cells, and that localized
mechanical tensions induce their growth. These tensions may be due to shear-
ing forces exerted at side- to-side junctions when contraction occurs at differ-
ent rates or magnitudes in adjacent cells. Such forces in time could lead to
asymmetric and complementary growth of sarcomeres along the two sides of the
lateral junctions, to reorientation of these junctions, and to the eventual
formation of lateral processes.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: None
6f
Serial No. NHLI-38(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Postoperative response to intense upright exercise in patients
with ventricular septal defect and pulmonary hypertension:
relation of impairment to age at operation.
Previous Serial Number: None
Principal Investigator: Barry J. Maron
Other Investigators: David R. Redwood, M.D., John W. Hirshfeld, Jr., M.D.,
Robert E. Goldstein, M.D., Andrew G. Morrow, M.D.,
Stephen E. Epstein, M.D.
Cooperating Units: Clinic of Surgery
Project Description: The cardiac response to intense upright exercise after
closure of a ventricular septal defect (VSD) associated with pulmonary arterial
(PA) hypertension is unknown. To evaluate such pts, 11 asymptomatic subjects
were studied at rest and during intense treadmill exercise 3 to 15 yrs (mean
9.5) after operation. Four pts had operations between 5 and 10 yrs of age;
7 pts between 12 and 28 yrs. Pre-operative mean PA pressure (PAP) ranged from
24 to 77 mm Hg (avg=53 ram Hg), pulmonary to systemic flow ratio from 1.6 to 4.5,
and pulmonary to systemic resistance ratio from .17 to .46. Postoperative
mean PAP was normal at rest in 10 pts and elevated to 40 ram Hg in 1. With
intense exercise (sufficient to lower PA02 saturation to 30%) cardiac output
(GO) in 5 pts was below the normal range (i.e. <6.9 L/min/M^), The defect in
each of these had been corrected after 10 yrs of age. Moreover, the magnitude
of the abnormality in the CO response to exercise was directly related to age
at correction (CO=8.7-.13x, where x==age at operation; p<.01). Four pts
manifested an abnormal increase in mean PAP during exercise (range 49-70 mm Hg;
normal ^35 ram Hg). This abnormality was also related to age at operation
(PAP=23.3+l.lx; p<.05). These results indicate that in pts with VSD and
preoperative PA hypertension 1) late postoperative exercise hemodynamics may
be abnormal and 2) these abnormalities are related to age at operation. Since
all pts were asymptomatic, the clinical significance of these abnormalities
remains to be determined.
Proposed Course of Project: Comjieted,
Honors and Awards: None
Publications: None
^a
Serial No. NHLI-39(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Occurrence of a-Glycogen in Human Cardiac Muscle Cells
Previous Serial Number: None
Principal Investigator: Barry J. Maron, M.D.
Other Investigators: Victor J. Ferrans, M.D.
Cooperating Units: Section on Pathology
National Heart and Lung Institute
Project Description: Rosettes of glycogen were found in cardiac muscle cells
in 5 of 18 patients with aortic valvular disease and in 3 of 23 patients with
idiopathic hypertrophic subaortic stenosis. Although most of the glycogen in
each heart was of the monoparticulate type, focal areas containing glycogen
rosettes, 1700 to 2300 A in diameter, were observed in cells that were hyper-
trophied but that otherwise had normal ultrastructure, and in cells showing
myofibrillar degeneration. Identification of these rosettes as glycogen was
confirmed by staining with the periodic acid-thiosemicarbazide-silver protein-
ate method and by digestion with amylase. It is suggested that these rosettes
represent a pathologic alteration of glycogen metabolism in hypertrophied and
degenerating cardiac muscle cells.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: None
63
Project Title:
Serial No. NHLI-40(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Unusual Evolution of Acquired Infundibular Pulmonary Stenosis
in Patients with Ventricular Septal Defect: Clinical and
Morphologic Observations
Previous Serial Number: None
Principal Investigator: Barry J. Maron, M.D.
Other Investigators: Victor J. Ferrans, M.D., Ph.D.
Robert White, M.D.
Cooperating Units: Section on Pathology
National Heart and Lung Institute
Department of Radiology
Johns Hopkins Hospital, Baltimore, Md.
Project Description: Hemodynamic and cardiac morphologic studies were carried
out in patients with ventricular septal defect who showed unusual evolution of
obstruction to right ventricular outflow. Both patients developed progressive
infundibular pulmonary stenosis, one in the presence of a spontaneously closing
ventricular septal defect and the other after operative closure of the defect.
Light and electron microscopic examination of resected infundibular muscle re-
vealed hypertrophy and abnormal shapes and arrangements of cardiac muscle cells.
These abnormalities resembled those found in left ventricular outflow tract
muscle of patients with idiopathic hypertrophic subaortic stenosis. It is sug-
gested that abnormal muscle cells may be located in the infundibular area of
some patients with ventricular septal defects; growth of these abnormal cells
in the right ventricular outflow tract may then lead to localized hypertrophy
and to development of progressive infundibular obstruction.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: None
6^
Serial No. NHLI-41(c)
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Ultrastructural Features of Myocardial Cell Degeneration in
Patients with Cardiac Hypertrophy
Previous Serial Number: None
Principal Investigator: Barry J. Maron, M.D.
Other Investigators: Victor J. Ferrans, M.D., Ph.D.
Stephen E. Epstein, M.D.
Cooperating Units: Section on Pathology
National Heart and Lung Institute
Project Description: Numerous attempts to demonstrate a biochemical defect
responsible for irreversible myocardial failure have been unsuccessful. To
determine whether the primary defect under such circumstances is physical
rather than biochemical, we are studying the ultrastructural features of myo-
cardial cell degeneration in patients undergoing aortic valve replacement and
correlating these findings with postoperative hemodynamic and functional re-
sults. Myofibrillar degeneration was found in muscle cells obtained from the
left ventricle in 4 of 14 patients. Earliest evidence of cellular degenera-
tion appears to be selective loss of myosin filaments and proliferation and
spreading of Z bands. Later morphologic changes include proliferation of sar-
coplasmic reticulum and Z band-like material along the cell membrane, increased
numbers of mitochondria and glycogen granules, appearance of a-glycogen, loss
of thin (actin) filaments and thickening of the basement membrane. Degenerat-
ing cardiac muscle cells are usually surrounded by increased number of collagen
fibrils. Analysis is continuing to determine whether collagen deposition is a
secondary phenomena (serving to replace degenerating muscle cells), or repre-
sents a primary change which, perhaps by interfering with oxygen delivery to
the hypertrophied myocardium, leads to muscle cell degeneration.
As patients return for their six months postoperative evaluation, these ul-
trastructural alterations will be correlated with vrtiether or not improvement
in myocardial function occurred following valve replacement.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
^S"
Serial No. NHLI-42
1. Cardiology
2. Clinical Physiology
3. Bethesda, Md .
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effects of Vagal Stimulation on Survival During Experimental
Acute Myocardial Infarction
Principal Investigators: Richard W. Myers, M.D,
Alan S. Pearlraan, M,D,
Other Investigators: Richard Hyman, B.S.
Kenneth M. Kent, M.D, , Ph.D.
Robert E. Goldstein, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: It is commonly believed that vagally mediated brady-
cardia predisposes to ventricular fibrillation (VF) during acute myocardial
infarction (AMI). However, both bradycardia and, independently, vagal
stimulation enhance electrical stability of the myocardium during experimen-
tal AMI. To determine the functional significance of these electrophysiolo-
gic effects, we studied the influence of vagal stimulation on spontaneous
development of VF during AMI in open -chest dogs. AMI was produced by
ligating the LAD and first septal perforating coronary arteries for 30 min.
In one series of experiments, dogs were divided into 3 subgroups: control
dogs without vagal stimulation (heart rates 150-180), dogs with the vagus
stimulated to reduce heart rate to 90-100, and dogs with vagus stimulated to
reduce heart rate to 60-70. VF occurred significantly later after coronary
occlusion in both groups of vagally stimulated dogs when compared to control
animals (p<.05): mean survival time was 29 minutes in dogs with HR 60-70, 23
minutes in dogs with HR 90-100, and 15 minutes in control dogs. Survival
also was enhanced in vagally stimulated dogs, averaging 70%, 407., and 107. in
the three groups respectively. In the second series of experiments, we
determined the effect of vagal stimulation on the incidence of VF during AMI
with heart rate held constant by RV pacing. Survival times were 22 minutes
in the vagally stimulated dogs and 11 minutes in the control group (p^o05) .
Similarly, while 557. of the vagally stimulated dogs survived, only 117. of the
control animals survived (p<.05) . We conclude that in experimental AMI vagal
stimulation, with or without accompanying sinus bradycardia, protects against
development of VF.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
Serial No. NHLI-43(c)
1. Cardiology
2. Cardiovascular Diagnosis
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Evaluation of the Ability of Echocardiography to Measure Al-
terations in Left Ventricular Volume
Previous Serial Number: None
Principal Investigator: David R. Redwood, M,D.
Other Investigators: Walter L. Henry, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: Recent studies to determine whether echocardiography can
be utilized to measure end-systolic and end-diastolic left ventricular (LV)
volumes have shown a good correlation between volumes estimated by echo (gen-
erally based on the cube of the distance between the interventricular septum
and the posterior LV wall) and volumes estimated by LV angiography. However,
the sensitivity of the echo technique in detecting alterations in LV volumes
has not been studied, bearing in mind that large errors nay occur with result-
ing decreased sensitivity since volume measured by echo varies as the cube of
the transverse dimension. Accordingly, we are studying in normal volunteers
the effect on this dimension of maneuvers and pharmacologic agents known to
alter LV volume, namely tilting, atropine, nitroglycerin, phenylephrine and
methoxamine.
Thus far, seven subjects have been studied and the results indicate that the
anticipated changes in LV volume are readily detectable by means of a measure-
ment of the transverse LV dimension by echocardiography. If these preliminary
results are confirmed, the method may be applied to similar determinations of
volume changes induced by various interventions in patients with heart disease.
Proposed Course of Project: Continuing.
Honors and Awards: None
Publications: None
67
Serial No. NHLI-44(c)
1. Cardiology
2. Cardiovascular Diagnosis
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Use of Biventricular Cineangiography in the Evaluation of
Patients With Asymmetric Septal Hypertrophy
Principal Investigator: David L. Redwood, MoD,
Other Investigators: James L. Scherer, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: Diagnostic Radiology Department
Clinical Center
Project Description: Recent echocardiographic studies have demonstrated that
disproportionate hypertrophy of the interventricular septum is the hallmark of
a disease spectrum recently termed asymmetric septal hypertrophy (ASH). For
technical reasons, echoes can be obtained only from the upper part of the
septum and from the basal portion of the posterior left ventricular (LV) free
wall. Pathologic studies have shown, however, that the myopathic process in
patients with ASH is distributed nonuniformly ; in particular, the posterior
basal free wall of patients with nonobstructive ASH usually is normal, but
other portions of the LV free wall, which cannot be detected by echo, may be
grossly hypertrophied. The present study was therefore designed to delineate
the extent and degree to which the hypertrophic process involves the septum
and LV free wall in patients with ASH.
During diagnostic cardiac catheterization studies, contrast material is
injected simultaneously into both the right and left ventricles with the
patient positioned in the steep left anterior oblique (LAO) position. Since
the right and left borders of the septum are visualized simultaneously, septal
thickness can be measured as can thickness of a segment of the LV free wall.
The LV cine is repeated with the patient in the lateral position, and in the
shallow LAO position to explore LV free wall thickness in further detail.
Preliminary results in 7 patients with ASH with or without obstruction
have confirmed the finding that the septum is hypertrophied to a greater ex-
tent than the posterior LV free wall. We plan to use these techniques to
determine whether the extent of the LV involvement by the cardiomyopathic
process can be assessed.
Proposed Course of Project: Continuing
Honors and Awards: None
Publications: None
1 4i
Serial No. NHLI-45(c)
1. Cardiology
2. Cardiovascular Diagnosis
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Echocardiography During Upright Exercise
Previous Serial Number: None
Principal Investigator: David R. Redwood, M,D.
Other Investigators: Eldon R. Smith, M,D.
Seth R. Goldstein, ScD
Walter L, Henry, M.D.
Cooperating Units: Biomedical Engineering & Instrumentation Branch
Division of Research Services
Project Description: Echocardiography has been shown to be a valuable, non-
invasive technique in the diagnosis and evaluation of myocardial, valvular
and congenital heart disease. Thus far, however, studies have been limited to
patients at rest in the supine position. Studies in the upright position at
rest and during bicycle or treadmill exercise have been hampered by the inabil-
ity to 1) manually hold the transducer stationary, particularly during patent
activity, and 2) predictably and rapidly alter transducer position. An appara-
tus has been designed T«Aiich successfully overcomes these problems. The echo
transducer is mounted in a hemispherical bearing on a light weight aluminum
platform held to the anterior chest wall by elastic straps. Two hydraulic
pistons mounted on the platform at right angles to one another are attached
to this bearing and as a result move the transducer. These pistons form part
of an electrohydraulic feedback control system so designed that the piston po-
sitions are accurately controlled by a two-axis joystick potentiometer which
is remote from the patient. Thus, movement of the joystick results in & cor-
responding movement of the transducer. With this apparatus, the transducer not
only can be readily adjusted but also can be held stationary despite movement
of the patient during tilting and exercise. In addition, the voltages genera-
ted by the joystick pan be recorded on the echo tracing and utilized to provide
a permanent record of actual transducer position. With this technique altera-
tions in left ventricular volume as judged by measurement of the transverse
left ventricular dimension have been successfully determined during tilting,
drug interventions, and during bicycle and treadmill exercise. This method
thus considerably broadens the application of echocardiography in the study of
heart disease.
Proposed Course of Project: Completed
Honors and Awards: None
Publications : None
^9
Serial No. NHLI-46(c)
1. Cardiology
2. Clinical Physiology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Sustained Effects of Nitroglycerin Ointment in Patients With
Angina Pectoris
Previous Serial Number: NHLI-147(c)
Principal Investigator: Nathanial Reichek, M.D.
Other Investigators: Robert E. Goldstein, M.D.
Michael Nagel, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: We have examined the ability of TL nitroglycerin ointment
to alter time to onset of ischemic pain (exercise duration) when 12 patients
with angina exercised repeatedly on an upright bicycle ergometer. Dose of ni-
troglycerin was selected to produce a 10 ram Hg fall in resing systolic blood
pressure and/or a 10 beat per minute rise in resting heart rate. Nitroglycerin
and placebo were administered on successive days and the sequence of administra-
tion reversed in alternate patients. Nitroglycerin produced a significant in-
crease in resting heart rate (+14 bpnn and 1 hour, +17 bpm at 3 hour) and fall
in resting systolic blood pressure (-14 mm Hg at 1 hour, -16 mm Hg at 3 hour),
while placebo did not. In every patient exercise duration was greater with
nitroglycerin than with placebo at 1 and 3 hours. The differences (+2.9 min.
at 1 hour, +2.5 min. at 3 hour) were highly significant (P < .001). Nine of
12 patients were able to exercise at higher work loads after nitroglycerin.
In six of seven patients ST segment depression at equal exercise was reduced
following nitroglycerin (-0.8 mm at 1 hour, -0.9 mm at 3 hour, P < .05). Thus
nitroglycerin produced persistent changes in resting heart rate and systolic
blood pressure, associated with a sustained increase in exercise duration and
attenuation of ischemic EKG changes. In contrast to oral and sublingual ni-
trates, nitrogen-related enhancement of exercise performance was consistent and
unequivocal three hours after administration.
Proposed Course of Project: Completed.
Honors and Awards: None
Publications: None
rc
Serial No. NHLI-47(c)
1. Cardiology
2. Cardiovascular Diagnosis
3. Bethesda, Mairyland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Deterioration of Myocardial Function Following Aorto-Coronary
Bypass Operation
Previous Serial Number: NHLI-148(c)
Principal Investigator: Richard L. Shepherd, M.D.
Other Investigators: Samuel B. Itscoitz, M.D.
D. Luke Clancy, M.D.
Edward B. Stinson, M.D.
Richard L. Reis, M.D.
Gordon N. Olinger, M.D.
Chester E. Clark, M.D.
Stephen E. Epstein, M.D.
Cooperating Units: Clinic of Surgery
National Heart and Lung Institute
Project Description: Twenty-two patients underwent cardiac catheterization
before and 1 week to 9 months (avg=5 mos.) after aorto-coronary bypass. Left
ventricular (LV) end-diastolic and end-systolic volumes were measured from
single-plane (RAO) LV cineangiograms. Two groups were examined: those with
all grafts patent (10 patients, 15 grafts) and those with one or more grafts
occluded (12 patients, 22 grafts, 15 occluded). In the patent group, all 10
were clinically improved; 5 still had angina. In the occluded group, all 12
were improved; 8 still had angina. Cardiac index was unchanged in both groups.
LVEDP increased significantly (+4.4 + 2.2 mm Hg, p < .05) in the occluded
group. The relation of LV end-diastolic pressure to LV stroke work indicated
that in the patent group LV performance improved in 1, deteriorated in 3, and
was unchanged in 6 patients; in the occluded group none improved, 7 were worse,
and 4 were unchanged. Ejection fraction decreased an average of 10% in the
occluded group (p < .05); no patient improved and 5 deteriorated. In the pa-
tent group, only 2/10 patients showed improvement while 2 showed deterioration.
Twenty-eight LV wall segments were supplied by patent grafts; 6 improved, 13
were unchanged, and 9 deteriorated. Twenty-two segments were supplied by oc-
cluded grafts; none improved, 14 were unchanged, and 8 deteriorated. We con-
clude that regardless of graft patency, most patients improve clinically. De-
spite this, deterioration of myocardial function is not uncommon, even when
grafts are patent.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: None
7/
Serial No. NHLI-48
1, Cardiology
2, Clinical Physiology
3, Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Increased Myocardial Ischemia Caused by Reflexly Induced Hypo-
tension During Coronary Occlusion in the Conscious Dog
Previous Serial Number: None
Principal Investigator: George E. Thibault, M.D.
Other Investigators: Gary S. Famham, M.D. Robert E. Goldstein, M.D,
Richard W. Myers, M.D. Stephen E. Epstein, M.D.
Cooperating Units: None
Project Description: Recent evidence indicates that hemorrhagic hypotension
increases acute myocardial ischemia (AMI) during coronary occlusion, whereas
nitroglycerin induced hypotension has no such deleterious effects. Hypotension
that occurs during AMI in man, particularly when associated with bradycardia,
appears due to reflex changes causing vasodilatation, which contrasts with the
vasoconstriction accompanying hemorrhagic hypotension. This study examined
the effects of hypotension induced by reflexly decreasing vascular resistance
during AMI. AMI was produced in conscious closed-chest dogs by inflation of a
balloon cuff previously implanted around the LAD coronary artery; controlled
levels of hypotension were achieved by stimulation of the carotid baroreceptors
using externally applied negative pressure. Ischemic injury was estimated by
summating S-T elevation ( ST) from 12 previously implanted intramyocardial
electrodes. Thirteen paired hypotensive and normotensive occlusions were per-
formed in 6 dogs. Baseline mean BP was 105 (range 90-122) and HR 76 (range
50-120), with no significant difference in HR between normotensive and hypo-
tensive occlusions. In all pairs ST at 10 min was greater during the hypo-
tensive occlusions (p < .001), and there was a highly significant correlation
between the % fall in BP (9 to 487„) and the degree of ischemic injury ( ST =
.59 x 7o fall BP + 5.7; r = .75). We conclude that reflexly induced hypotension
increases ischemic injury despite the potentially beneficial effects of de-
creased vascular resistance and decreased myocardial work.
Proposed Course of Project: Completed
Honors and Awards: None
Publications: ARTICLE PUBLISHED IN A PERIODICAL. Thibault, George E. ,
Famham, Gary S., Myers, Richard W. , Goldstein, Robert E. and Epstein, Stephen
E. Increased myocardial ischemia caused by reflexly induced hypotension during
coronary occlusion in the conscious dog. Clinical Research. In press.
r5L
ANNUAL REPORT OF THE
CLINIC OF SURGERY
NATIONAL HEART AND LUNG INSTITUTE
July 1, 1972 through June 30, 1973
The clinical and laboratory programs of the Surgery Branch have, as
in past years, largely centered upon the study of operative methods for
the correction of congenital and acquired heart and lung diseases, assess-
ment of the results of such operations, and laboratory studies related to
cardiovascular physiology and pharmacology.
Is There a Natruretic Hormone? Expansion of the intravascular volume
in a normovolemic subject results in an increase in the renal excretion of
salt which is not dependent on ADH, withdrawal of aldosterone, renal inner-
vation, or an increase in filtered load. This volume-expansion natruresis,
which is secondary to decreased tubular sodium reabsorption, has been
attributed to a humoral agent. The humoral nature of this mechanism has
been suggested by cross-transfusion studies in which blood from a saline-
loaded animal was exchanged with a second animal whose blood volume was
kept unchanged. Many elaborate subsequent studies have been done which
have both refuted and corroborated the existence of such a "natruretic
hormone." Because many of these studies were done with saline and plasma-
like substances, which caused a decrease in the hematocrit of both dogs,
it has not been proved that pure intravascular expansion is the stimulus
for this salt losing phenomenon (a decrease in hematocrit is known to de-
crease sodium reabsorption). An experimental cross-transfusion model was,
therefore, developed which obviates any decrease in hematocrit and main-
tains normal plasma oncotic pressures.
Ten such sets of cross-transfusion experiments were performed. In
all studies, two dogs were connected - femoral artery to femoral vein -
with a high flow pump that transferred exactly equal volumes of blood from
one dog to the other, A one liter reservoir was connected to the system
and a mixture of blood and dextran allowed to equilibrate with both dogs'
circulating blood volume. After a prolonged period of equilibration, a
liter of blood which was a composite of the blood from both dogs plus the
mixture of blood and dextran, was removed from the reservoir and infused
into dog #1 while continuous cross -trans fusion was maintained. Plasma and
urine were collected at regular intervals from both dogs, and urine volume,
sodium excretion, creatinine clearance, osmolar and free water clearance,
and fractional sodium excretion were determined. In all cases, dog #1
(volume expanded) demonstrated a significant diuresis and natruresis.
Dog #2 (normovolemic) did not demonstrate any consistent increase in the
excretion of salt. Thus, the studies do not demonstrate a substance in
the blood of the volume expanded animal which effects natruresis in the
recipient animal .
73
Clinical Studies of the Relations of Left Atrial Pressure to the
Patterns of Renal Sodium Excretion. Shortly after the development of
operations for the relief of severe mitral stenosis it was recognized that
immediately after operation patients with this valvular malformation have
high levels of antidiuretic hormone (ADH) and a resulting increase in free
water reabsorption in the postoperative period. This has been attributed
to pressure receptors in the left atrium which Interpret the fall in left
atrial pressures to Indicate hypovolemia, and which then trigger a release
of ADH. Since left atrial receptors also can Influence an increase in
sodium excretion with elevation in atrial pressure, it was of Interest to
determine whether the reciprocal situation, retention of sodium with a de-
crease in pressure, exists.
Six patients undergoing closed mitral commissurotomy and four
patients undergoing exploratory thoracotomy through a similar incision
(control series) were studied. All patients had a complete renal profile
preoperatively, and urine and plasma were collected and analyzed for 5-7
days postoperatively. Although plasma aldosterone levels were much higher
in the commissurotomy patients preoperatively, they had the same relative
percentage rise in aldosterone levels in the postoperative period as did
the control patients. Although G.F.R. (as measured by endogenous creatinine
clearance) increased from preoperative values in the commissurotomy patients,
their total sodium excretion fell to extremely low levels (mean 3 mEq./day)
in the first three postoperative days. These data suggest that left atrial
pressure receptors do operate to effect sodium retention, and additional
patients in both groups are being subjected to continuing study.
In order to evaluate the patterns of sodium excretion associated
with changes in left atrial pressure experimentally, efforts have been made
to develop a method for the gradual production of mitral stenosis in the
dog. Initially, rings made of Amerold, a plastic which absorbs tissue
water and subsequently swells, were sutured in the left atrium immediately
above the mitral annulus. It was found that this device was unsatisfactory
because it promoted thrombus within the atrium, and the orifice of the ring
narrowed too rapidly to permit survival. Subsequently, a doughnut-shaped
flexible ring covered with segmented polyurethane has been utilized. This
is sutured into the supravalvular position and can be gradually Inflated
by an external connection with a resulting gradual decrease in "mitral
valve" area. Initial experience with this technique indicates that it is
satisfactory, and the model will be used to evaluate the effects of gradually
increasing left atrial pressure on salt and water metabolism.
Experimental Quantification of Aortic Regurgitant Flow with the
Doppler Velocitometer. The only means with which the severity of aortic
regurgitation can be quantified preoperatively in man is by means of cine-
angiographic measurements of left ventricular volume. The technique is
laborious and time consuming, and measurements are often impossible if
irregularities of cardiac rhythm occur during the study. Therefore, a
noninvasive method for estimating regurgitant flow would be a valuable
clinical tool.
74
Acute aortic regurgitation was produced in 10 dogs by punching one
or more holes in the valve leaflets under normothermic inflow occlusion.
The absolute percentage of regurgitant flow was then determined with an
electromagnetic flow probe placed around the ascending aorta and/or the com-
mon brachiocephalic trunk at its origin. To ascertain zero flow the artery
was occluded distal to the probe. Simultaneously, the regurgitant flow in
the brachiocephalic trunk was measured transcutaneously using a Doppler
bidirectional velocitometer with a fixed internal zero, applied at the
sternal notch. Regurgitant flows ranging from 10-757o of forward flow were
created. In 9 of the 10 animals, the regurgitant flow measured transcu-
taneously was within 11% (± 6%) of that measured directly with the electro-
magnetic flow probe. The difference between the two measurements was not
significant. In one dog the difference between the two measurements was
23%.
These data indicate that transcutaneous measurement of regurgitant
flow in the large arteries of the thoracic inlet, using a Doppler bi-
directional velocitometer, may be an accurate method of assessing absolute
percentage of aortic regurgitation. The study has been extended to patients
with rheumatic valvular disease undergoing operation. Aortic regurgitant
flow is assessed by the Doppler technique preoperatively, after the induc-
tion of anesthesia, and by direct application of the transducer to the
ascending aorta after it has been exposed. The measurements obtained by
this method are then correlated with measurements of forward and regurgitant
flow obtained from an electromagnetic flowmeter around the ascending aorta.
The Long-Term Results of Aortic Valvulotomy Carried Out in Young
Patients with Congenital Valvular Aortic Stenosis. Little information is
available concerning the role of aortic valvulotomy in altering the course
of a patient with congenital aortic stenosis. Many persons consider that
the operation provides only a short period of palliation and that no opera-
tion is indicated until prosthetic replacement of the valve becomes neces-
sary. The experience at this Institute has been to the contrary, and a
follow-up study of young patients with congenital aortic stenosis was made.
Thirty-eight patients (30 boys, 8 girls) with congenital valvular
aortic stenosis, ages 1-21 years, underwent aortic valvulotomy between 1957
and 1967. Each operation was performed with cardiopulmonary bypass. There
were no operative deaths. Preoperatively 30 patients had symptoms of left
ventricular dysfunction. Each patient had a parasternal thrill associated
with the aortic stenosis murmur; six patients had murmurs of aortic insuf-
ficiency. The preoperative aortic valve gradients averaged 91 mm. Hg. The
mean preoperative aortic valve area index was 0.54 cm. /M.^.
Thirty-six of the 38 patients have been evaluated postoperatively;
the mean duration of follow-up is 10 years. Thirty-two patients are asymp-
tomatic. All patients continue to have a murmur of aortic stenosis; twenty
patients also have a murmur of aortic insufficiency. Three patients have
required aortic valve replacement 9, 11, and 15 years after valvulotomy.
Thirty-three patients have had postoperative catheterizations: the average
aortic valve gradient was 27 mm, Hg, the mean left ventricular end-diastolic
7r
pressure was 13 nnti. Hg, and the mean aortic valve index was 0.76 cm.2/M. .
One cardiac death occurred five years postoperatively secondary to aortic
regurgitation, congestive failure, and myocardial infarction.
Thus, long-term relief of congenital valvular aortic stenosis may
be effected by valvulotomy with minimal operative and postoperative risk,
and continued application of the operation in young patients is indicated.
Experimental and Clinical Studies of Homologous Veins in the Arterial
System. Homologous veins are frequently utilized to replace segments of
diseased arteries, both in the leg and in the coronary arterial bed. In
both sites progressive obliteration of the lumen of the vein graft is some-
times observed. Segments of homologous vein were utilized to replace
segments of excised femoral arteries or as grafts between the aorta and
a coronary artery in dogs. The grafts were studied two days to one year
after insertion. In the femoral position, the veins showed disruption of
the endothelium, mural fibrin deposits, and edema and inflammation of the
media after only one week. By two weeks, the smooth muscle cells of the
media had disappeared and focal proliferative lesions were seen beneath an
intact endothelium; the endothelial lesions were diffuse by 12 weeks. In
the aortocoronary position veins studied at 6, 9, and 12 months revealed
medial fibrosis and extensive intimal proliferation which often cause luminal
narrowing of severe degree. Extension of the intimal proliferation from the
graft into the distal coronary artery was also observed. Aortocoronary vein
grafts obtained from patients 17 to 57 days after insertion revealed a sim-
ilar pattern of subendothelial proliferation. By electron microscopy the
lesions were found to be composed of mature smooth muscle cells and col-
lagen, primarily oriented parallel to the axis of blood flow. Damage to
the endothelial cells, followed by the deposition of fibrin and its sub-
sequent organization appear to be the cause of the proliferative lesions.
The studies are being extended to determine whether the incidence and
severity of the lesions can be influenced by the means by which the graft
is excised and preserved before blood flow through it is restored.
Systemic and Renal Responses to Graded Reductions in Cardiac Output.
Seventeen dogs undergoing a moderate saline diuresis were studied as cardiac
output was gradually decreased by microsphere embolization of the coronary
arteries. Multiple, small injections, at 20-30 minute intervals, effected
a graded and sustained reduction in cardiac output with accompanying eleva-
tion of left atrial pressure and ECG changes typical of ischemia. Small
additional reductions in cardiac output after an initial decrease to 50-60%
of control invariably resulted in sudden death or frank, irreversible cardio-
genic shock. Initial small reductions in cardiac output, which occurred
prior to an elevation in left atrial pressure, were associated with a marked
decrease in peripheral flow (measured with both an electromagnetic probe
and a Doppler bidirectional velocitometer) with sparing of the renal blood
flow and mean arterial pressure. Progressive reduction in cardiac output
lowered the renal blood flow and arterial pressure, but to a lesser extent
than peripheral flow--a situation unlike hemorrhagic shock. The diuresis
and natruresis accompanying saline infusion gradually diminished as cardiac
output fell, but oliguria was seen only after cardiac output had decreased
to 507, of control. A modest fall in glomerular filtration rate was seen at
4 7C
70% reduction in cardiac output and tubular sodium reabsorption rose
throughout the experiments .
These studies suggest that diminished peripheral flow is an earlier
and more reliable index of a progressive deterioration in cardiac output
than is arterial pressure, renal blood flow, or left atrial pressure, and
that oliguria is a manifestation of a terminal reduction in cardiac output.
Radioisotope Powered Pacemaker. For the past seven years a joint
development program with the AEC has been directed to the development of a
cardiac pacemaker utilizing a nuclear energy source. Since May 1969, 65
atomic powered units have been implanted in dogs, the last 30 of the NU-5
configuration. One unit of this series has evidenced intermittent opera-
tion, but all others have paced the experimental animals consistently and
uninterruptedly. Finally, with virtually no fanfare and scarcely any
publicity at all, the first unit was implanted into a human subject on
April 9, 1973. Four additional units will be available for human use during
this spring and summer, but their application will be reserved for young
patients with long life expectancies. The patients will be followed by a
telephone monitoring system which permits precise measurement of the stimulus
rate and the recording of a lead I ECG. The AEC has spent $4.5 million on
this project, but our contribution in blood, sweat, toil, and tears has been
even greater.
77
Serial No. NHLI-49(c)
1, Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: NU-5 atomic powered pacemaker - experimental and clinical
evaluation
Previous Serial No: None
Principal Investigator: Andrew G. Morrow, M. D.
Other Investigators: Charles L. Mcintosh, M.D.
Peter Frommer, M.D.
Joseph E. Pierce, D.V.M.
Cooperating Units: Office of the Director and Section on Lab Animal
Medicine & Surgery, NHLI
Project Description: Efforts began six years ago to develop a cardiac
pacemaker energy source (Plutonium ^^°) suitable for human implantation.
Since May 1969 sixty- five atomic powered pacemakers have been implanted in
dogs for in vivo testing, of these 30 are of the NU-5 series. The first
human implant was done April 9, 1973 in a patient with complete heart block.
Current plans include four additional human implants by the summer of 1973.
Results: One pacemaker in the 30 animals implanted with the NU-5 series
pacemakers has intermittently failed, the remainder of the pacemakers have
functioned well. Wound breakdowns have necessitated the reimplantation of
several pacemakers. The unit implanted in our patient is functioning well
at this time.
Proposed Course: Animal evaluation will continue for an indefinite time
period with those now implanted. Human implants will be evaluated over
the next decade. Publications will be submitted concerning the animal
work at NIH and the human evaluation in conjunction with Beth Israel Hospital,
Newark, New Jersey.
7f
Serial No. NHTJ-SO^c^
1. Clinic of Surgery
3. Bathe sda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Long-term follow-up of patients with congenital aortic
stenosis treated with aortic commissurotomy
Previous Serial No.: NHLI-172(c)
Principal Investigators: David M. Conkle, M.D.
Andrew G. Morrow, M.D.
Other Investigator: Michael Jones, M.D.
Project Description: Thirty-eight patients (30 boys, 8 girls) with
congenital valvular aortic stenosis, ages 1-21 years, underwent aortic
valvulotomy between 1957 and 1967. Each operation was performed with cardio-
pulmonary bypass. There were no operative deaths.
Preoperatively 30 patients had symptoms of left ventricular dysfunction. Each
patient had a parasternal thrill associated with the aortic stenosis murmur;
six patients had murmurs of aortic insufficiency. The mean preoperative
aortic valve gradient was 91 mm. Hg. The mean preoperative aortic valve
area index was 0.54 cm2/M2.
Thirty-six patients have been evaluated postoperatively; the mean duration
of follow-up is 10 years. Thirty-two patients are asymptomatic. All
patients continue to have a murmur of aortic stenosis; twenty patients have
a murmur of aortic insufficiency. Three patients have required aortic valve
replacement 9, 11, and 15 years after valvulotomy. Thirty-three patients
have had postoperative catheterizations: the mean aortic valve gradient
was 27 mm. Hg, the mean left ventricular end-diastolic pressure was 13 mm.
Hg and the mean aortic valve index was 0.76 cm2/M2. One cardiac death
occurred 5 years postoperatively secondary to aortic regurgitation, congestive
failure, and myocardial infarction.
Thus, long-term relief of congenital valvular aortic stenosis may be effected
by valvulotomy with minimal operative and postoperative risk, and continued
application of the operation in young patients is indicated. To be presented
at the annual meeting of the Society for Vascular Surgery, Toronto, Canada,
June 22 and 23, 1973.
io
Serial No. NHLI-51
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 197 2 through June 30, 1972
Project Title: The pathophysiology of intimal hyperplasia of the venous
graft in the arterial system
Previous Serial No: NHLI-170
Principal Investigators: David M. Conkle, M. D.
Michael Jones, M. D.
Other Investigators: William C. Roberts, M. D.
Victor Ferrans, M. D.
Edward B. Stinson, M. D.
Frederick H. Levine, M. D.
David B. Melvin, M. D-
Cooperating Units: Section of Pathology, NHLI and Cardiology Branch, NHLI
Project Description: Canine femoral and aortocoronary vein grafts were
evaluated from 2 to 365 days postoperatively. Femoral vein grafts showed
focal endothelial disruption, mural fibrin deposition, and medial edema
with inflammatory infiltrates during the first week; loss of medial smooth
muscle cells and focal subendothelial lesions with intact endothelium by 2
weeks, and diffuse subendothelial lesions by 12 weeks. Coronary vein grafts
studied at 6, 9 and 12 months had medial fibrosis and extensive intimal
proliferation causing up to 907o luminal narrowing. Extension of the intimal
process into the coronary artery distal to the bypass graft also compromised
the arterial lumen. Coronary vein grafts obtained from humans dying 17 and
57 days after graft insertion revealed a similar subendothelial prolifera-
tion. Electron microscopy showed that the subendothelial lesions were
composed of mature smooth muscle cells and collagen, primarily oriented
parallel to the axis of blood flow. Recurrent enodthelial cell damage,
followed by mural fibrin deposition and organization, appears to be the
cause of the subendothelial proliferative lesions.
Publications: Jones, M, Conkle, DM, Ferrans, VJ, Roberts, WC, Levine, FH,
Melvin, DB, Stinson, EB: Lesions observed in arterial autogenous vein
grafts: Light and electron microscopic observations. Circulation: In
press. (Presented at 45th Scientific Sessions of the American Heart
Association October 1, 1972).
SI
Serial No. NHLI-52(c)
L. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Silastic ball variance detection
Previous Serial Number: None
Principal Investigator: Charles L. Mcintosh, M.D.
Other Investigators: William Schuette, B.S.
Andrew G. Morrow, M.D.
Cooperating Unit: Biomedical Engineering &. Instrumentation Branch
Project Description: The technique for determining percent stroke
length of the poppet of a prosthetic valve (Starr-Edwards) was developed
in 1970 at the NIH. Other techniques for diagnosing ball variance have
a low yield (-^2570) for detecting this possible fatal complication. The
limiting factor in utilizing our technique is the ability to visualize
the barium impregnated ball on cine.
Results: Eleven patients have been studied and operated upon at the
NIH for aortic ball variance since 1970. Nine of these patients had
other criteria of ball variance, but the last two patients were thought
"normal" by existing criteria. The first patient required operation for
a diseased mitral valve, and inspection revealed obvious ball variance
of the aortic ball. The second patient was operated on for aortic ball
variance determined by this technique and the poppet was found to be
variant at the time of operation.
Proposed Course: Patients with a 1000 and 1200 series Starr-Edwards
prosthesis will be screened routinely in our clinic. This technique
may be applied to any make prosthesis if the poppet can be visualized
on cine. The description of the technique is being submitted to the
American Heart meetings and the clinical results will be submitted to
the American College of Cardiology.
62-
Serial No. NHLI-53(c)
1. Clinic of Surgery
3. Bethesda, Md,
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Light and electron microscopic evaluation of hypertrophied
right ventricular outflow tract muscle from patients
with congenital heart disease.
Previous Serial Number: NHLI-160 (c)
Principal Investigators: Michael Jones, M.D.
Victor J. Ferrans, M.D.
Cooperating Unit: Section of Pathology, NHLI
Project Description: Samples of operatively resected myocardium from
sixty patients (ages 3 to 53 years) with congenital heart disease were
submitted for light and electron microscopic evaluation. Patient
diagnoses included tetralogy of Fallot, infundibular and valvular pul-
monic stenosis with an intact ventricular septum, isolated atrial septal
defect, discrete subpulmonic stenosis with a ventricular septal defect,
double outlet right ventricle both with and without infundibular obstruc-
tion, and ventricular septal defect with aortic regurgitation.
Characteristics of hypertrophy included a two to three-fold increase in
cell size, multinucleate cells, multiple intercalated discs, dilated
transverse tubules, and structural evidence of protein synthesis. Degen-
erative changes present were institial fibrosis, cellular and myofibrillar
disorganization, Z band widening, myofibrillar degeneration, myelin figure
degeneration, and smooth endoplasmic reticulum proliferation. In addi-
tion, ultrastructural and histochemical evidence was present for abnormal
morphology of and intramitochondrial localization of glycogen. An ultra-
structural evaluation was also made of normal and thickened endocardium
present in these patients. Attempts are being made to correlate these
findings with various clinical and hemodynamic parameters.
A comprehensive discussion of the morphologic manifestations of hypertrophy,
chronic pressure and volume overload, and hypoxia is in preparation.
Publications: Jones, M., and Ferrans, V. J.: Intramitochondrial glycogen
in hypertrophied infundibular muscle of patients with congenital heart
disease. Am. J. Path. 70: 69-84, 1973.
Ferrans, V. J,, Buja, L. M., Jones, M. : Ultras true ture and cyto-
chemistry of glycogen in cardiac diseases. In Recent Advances in Studies
on Cardiac Structure and Metabolism, Vol. Ill, University Park Press,
Baltimore. In press.
S3
Serial No. NHLI-54(c)
1. Clinic of Surgery
3. Bethesda, Md,
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Assessment of aortic regurgitation in patients with
the use of the Doppler Bidirectional Velocitoraeter
Previous Serial Number: None
Principal Investigators: Lawrence L. Michaelis, M.D.
Paul R. Hickey, M.D.
Andrew G. Morrow, M.D.
Project Description: We have demonstrated in the animal research
laboratory that the Doppler Bidirectional Velocitoraeter affords an
excellent non-invasive means of assessing experimentally produced
aortic regurgitation.
We are now beginning a clinical evaluation of this diagnostic technique.
Patients with aortic regurgitation are studied with the Doppler tech-
nique preoperatively. Recordings over the sternal notch, at the point
of greatest velocity in the great arteries exiting from the thoracic
outlet, are made prior to the day of surgery on the ambulatory patient
and in the anesthesia induction room under general anesthesia. The
amount of regurgitant flow (as a percentage) is then calculated and
compared with regurgitant flow determined with an electromagnetic flow
probe about the ascending aorta prior to the onset of cardiopulmonary
bypass and valve replacement.
Project will be continued with comparative measurements of regurgita-
tion by the Doppler instrument and with measurements made with an
electromagnetic flowmeter.
0f^
Serial No. NHTJ-fiii
1 . Clinic of Surgery
3. Bethesda, Md. 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Experimental production of progressive mitral stenosis
Previous Serial Number: None
Principal Investigators: Lawrence L, Michaelis, M.D.
Kent W. Jones, M.D.
Other Investigators: Michael Jones, M.D.
Norman L. Luka, M.D.
Project Description: An effort has been made to create an animal model
of progressive mitral stenosis. Initial attention was directed towards
an Ameroid ring (a plastic which absorbs tissue water with subsequent
swelling) which was placed above the mitral annulus . This device was
found to be excessively thrombogenic and swelling was too rapid. A sub-
sequent model has been designed and implanted which looks promising.
A segmented poljmrethane covered ring is sewn into the supra-mitral area
and a circumferential internal balloon gradually inflated via an external
connection with a subsequent decreasing mitral valve area.
If this model proves successful in effecting a progressive mitral
stenosis we plan to study many aspects of the disease, i.e., effects of
gradually increasing left atrial pressure on salt and water homeostasis,
exercise tolerance and the critical level of valve area, mechanisms of
pulmonary edema, etc.
er
Serial No. NHLI-56
1. Clinic of Surgery
3. Bethesda, Md .
Project Title;
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
The effects of naloxone hydrochloride on ventricular
irritability
Previous Serial Number: None
Principal Investigators: Lawrence L. Michaelis, M.D.
Paul R. Hickey, M.D.
Other Investigators;
Cooperating Units;
William Dixon, M.D.
Thomas Clark, M.D.
Andrew G. Morrow, M.D.
Department of Anesthesia, Clinical Center
Division of Thoracic & Cardiovascular Surgery,
Bethesda Naval Hospital
Project Description: Naloxone hydrochloride (Narcan) is a narcotic
antagonist which is coming into common clinical use. Recently, in the
same 24 hour period, two episodes of ventricular fibrillation have
immediately followed the intravenous administration of small amounts of
this drug. The two cases - a patient 24 hours post-triple valve replace-
ment at the " [LI, and a patient at the Bethesda Naval Hospital who was
18 hours ' -repair of a dissecting aortic hematoma, are presented in
the papi
The patients received naloxone from different drug lots, nevertheless
remaining drug ampules from each source were obtained and tested in the
animal laboratory. Naloxone HCl from these two sources was injected into
five dogs and the vulnerability of the dog's heart to ventricular fibril-
lation was assessed using the ventricular excitability curve as an index
of vulnerability. Excitability curves were determined on a control
basis, after morphine sulphate and following naloxone administration.
Rapid intravenous injection of the drug caused a brief tachycardia and
transient rise in arterial pressure, with a run of premature ventricular
contractions observed in two of the five animals. There was no demon-
strable increase in ventricular excitability over control values.
The two detailed case presentations and the results of the laboratory
studies are presently being prepared for publication along with a
warning as to this complication and our recommendations for safeguards
in administration of the drug.
e^
Serial No. NHLI-57(c)
1. Clinic of Surgery
3. Bethesda, Md. 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Clinical effects of abrupt decrease in left atrial
pressure on renal sodium excretory patterns
Previous Serial Number: None
Principal Investigators: Lawrence L. Michaelis, M.D.
Jack G. Copeland, M.D.
Project Description: It has been appreciated for some time that
patients undergoing closed mitral commissurotomy have high levels of
ADH with resultant increase in free water reabsorption in the post-
operative period. This has been attributed to left atrial stretch
receptors which "sense" this fall in pressure as relative hypovolemia
and signal a release of ADH. Since left atrial receptors also can
influence an increase in sodium excretion with elevation in pressure,
we have been curious as to whether the reciprocal situation, retention
of sodium with a decrease in pressure, exists.
Six patients undergoing closed mitral commissurotomy and four patients
undergoing an exploratory thoracotomy through a similar incision
(control series) were studied. All patients had a complete renal
profile preoperatively and urine and plasma were collected and analyzed
for 5-7 days postoperatively. Although plasma aldosterone levels were
much higher in the commissurotomy patients preoperatively, they had
the same relative percent rise in aldosterone levels in the postoperative
period as did the control patients. Although G.F.R. (as measured by
endogenous creatinine clearance) increased from preoperative values in
the commissurotomy patients, their total sodium excretion fell to
extremely low levels (mean 3 mEq/day) in the first three postoperative
days. We feel that these data are suggestive that left atrial pressure
receptors may be operative in effecting this sodium retention. We are
awaiting further patients to finalize the study.
er
Serial No. NHLI-58
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Systemic and renal responses to a graded reduction in
cardiac output
Previous Serial No. :
Principal Investigators: Lawrence L. Michaelis, M. D.
Paul R. Hickey, M. D.
Norman L. Luka, M. D.
Other Investigators: Scott I. Allen, M. D.
Cooperating Unit: Division of Computer Research and Technology
Project Description: Seventeen dogs undergoing a moderate saline diuresis
were studied as cardiac output was gradually decreased by microsphere
embolization to the coronary arteries. Multiple, small injections, at 20-
30 minute intervals, effected a graded and sustained reduction in cardiac
output with accompanying elevation of left atrial pressure and ECG changes
of ischemia. Small additional reductions in cardiac output after a decrease
to 50-607„ of control invariably resulted in sudden death or frank, irrevers-
ible cardiogenic shock. Initial small reductions in cardiac output, which
occurred prior to an elevation in left atrial pressure, were associated with
a marked decrease in peripheral flow (measured with both an electromagnetic
probe and a Doppler bi-directional velocitometer) with sparing of the renal
blood flow and mean arterial pressure. Progressive reduction in cardiac
output lowered the renal blood flow and arterial pressure but to a lesser
extent than peripheral flow--a situation unlike hemorrhagic shock. The
diuresis and natruresis accompanying saline infusion were gradually dimin-
ished as cardiac output fell, but oliguria was seen only after cardiac out-
put had decreased to 507,, of control. A modest fall in glomerular filtration
rate was seen at 707o reduction in cardiac output and tubular sodium re-
absorption rose throughout the experiments. These studies suggest that
diminished peripheral flow is an earlier and more reliable index of a
progressive deterioration in cardiac output than is arterial pressure, renal
blood flow, or left atrial pressure, and that oliguria is a manifestation of
an extensive reduction in cardiac output.
This project will be presented at the Society for Vascular Surgery in
June, 1973 and the manuscript will be published in Surgery late in 1973.
98
Serial No. NHTJ-59
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Blood volume expansion without altering hematocrit.
Renal effects in a cross -trans fusion model
Previous Serial Number: None
Principal Investigators: Lawrence L. Michaelis, M.D.
Norman L, Luka, M.D.
Bruce A. Reitz, M.D.
Project Description: Expansion of the intravascular volume in a normo-
volemic subject results in an increase in the renal excretion of salt
which is not dependent on ADH, withdrawal of aldosterone, renal innova-
tion, or an increase in filtered load. This volume-expansion natruresis,
which is secondary to decreased tubular sodium reabsorption, has been
attributed to a humoral agent. The humoral nature of this mechanism has
been suggested by cross-transfusion studies in which blood from a saline-
loaded animal was exchanged with a second animal whose blood volume was
kept unchanged. Many elaborate subsequent studies have been done which
have both refuted and corroborated the existence of such a "natruretic
hormone." Because many of these studies were done with saline and plasma-
like substances, which caused a decrease in the hematocrit of both
dogs, it has not been proved that pure intravascular expansion is the
stimulus for this salt losing phenomenon (a decrease in hematocrit is
known to decrease sodium reabsorption). An experimental cross-transfusion
model was therefore developed which obviates any decrease in hematocrit
and maintains normal plasma oncotic pressures.
Ten such sets of cross -trans fusion experiments were performed. In all
studies, two dogs were connected - femoral artery to femoral vein - with
a high flow pump that transferred exactly equal volumes of blood from
one dog to the other. A one liter reservoir was connected to the
system and a mixture of blood and dextran allowed to equilibrate with
both dogs' circulating blood volume. After a prolonged period of
equilibration, a liter of blood which was a composite of the blood
from both dogs plus the mixture of blood and dextran, was removed from
the reservoir and infused into dog #1 while continuous cross -trans fusion
was maintained. Plasma and urine were collected at regular intervals
from both dogs, and urine volume, sodium excretion, creatinine clearance,
osmolar and free water clearance, and fractional sodium excretion were
determined. In all cases, dog #1 (volume expanded) demonstrated a sig-
nificant diuresis and natruresis. Dog #2 (normovolemic) did not demon-
strate any consistent increase in the excretion of salt. The studies thus
do not demonstrate a substance in the blood of the volume expanded animal
which effects a natruresis in the recipient animal. These data are
presently being prepared to be submitted for publication.
Serial No. NHLI-60
1, Clinic of Surgery
3. Bethesda, Md,
PHS-NIH
Individual Project Reports
July 1, 1972 through June 30, 1973
Project Title: Systemic and renal effects of Methyl Prednisolone
following a graded reduction in cardiac output
Previous Serial Number: None
Principal Investigators: Lawrence L. Michaelis, M.D.
Paul R. Hickey, M.D,
Norman L. Luka, M.D.
Other Investigators: Scott I. Allen, M.D,
Cooperating Units: Division of Computer Research and Technology
Project Description: Large doses of methyl prednisolone have been
recommended in the treatment of various forms of shock. Effects of
this drug in cardiogenic shock are variable and difficult to evaluate
in the clinical situation when multiple other drugs are used concurrently.
Urine output is often transiently improved following administration of
the drug and some investigators have attributed this to a direct renal
action of methyl prednisolone.
Eight dogs which had been subjected to a graded reduction in cardiac
output by multiple small embolizations of microspheres to the coronary
arteries were treated with large doses of methyl prednisolone (30 mg/kg)
after their cardiac output had fallen and stabilized at 50-60% of control
values. The effect of the drug on cardiac output, peripheral blood flow,
arterial pressure, and renal blood flow were determined.
Urine and plasma were collected at frequent intervals and the following
information obtained: Urine volume, sodium excretion, creatinine clear-
ance (exogenous), osmolar clearance, free water clearance, and fractional
sodium reabsorption. Preliminary analysis of the data suggests that
methyl prednisolone does not improve the depressed myocardium but may have
a direct renal effect with transient diuretic and natruretic capabilities.
<^o
Serial No, NHLI-61
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Apical-aortic valvular anastomosis for diffuse left
ventricular outflow tract obstruction
Previous Serial Number: None
Principal Investigators: John W. Brown, M.D.
Arthur J. Roberts, M.D.
Charles L. Mcintosh, M.D.
Project Description: There is no satisfactory corrective operation for a
form of left ventricular outflow tract obstruction termed "tunnel aortic
stenosis. " A prosthesis was constructed to bypass this obstruction using
a Starr-Edwards aortic ball valve (size 7A) mounted into a 20 mm. Dacron
woven graft. Apical-aortic anastomosis with occlusion of the ascending
aorta was carried out in 20 dogs (average weight of 25 Kg.) using cardio-
pulmonary bypass. Anastomosis to the apex of the left ventricle was
accomplished with a flexible cloth-covered polyure thane stent which pro-
jected 5 mm. into the cavity of the left ventricle. The end-to-side aortic
anastomosis was located at the level of the 7th and 8th intercostal arteries.
Aortic occlusion was accomplished using a soft jaw clamp placed approxi-
mately 2 cm. distal to the coronary ostia. Average cardiopulmonary bypass
time was 25 min.
Results: Nineteen of 20 dogs survived the operation, and two are
alive and well six to seven months postoperatively. The 17 postoperative
deaths were divided into eight early (3-12 hr.) deaths secondary to ventricular
arrhythmias, bleeding, and technical errors. The nine late (3-21 days)
deaths were attributed to congestive heart failure, infection, and one of
an iantrogenic hemothorax.
Proposed Course: Both surviving dogs will undergo cardiac catheteriza-
tion. An additional series of dogs using modifications of the above pros-
thesis and more detailed acute and chronic postoperative evaluation will be
studied.
^/
Serial No. NHLI-62
1. Clinic of Surgery
3. Bethesda, Md .
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Experimental evaluation of the membrane oxygenator
Previous Serial Number: None
Principal Investigators: Arthur J. Roberts, M,D.
Charles L. Mcintosh, M.D.
Other Investigators: John W. Brown, M.D.
Lynn H. Harrison, Jr., M.D.
P. David Myerowitz, M.D.
Edward Stool, M.D.
Theodore Kolobow, M.D.
Cooperating Unit: Laboratory of Technical Development, NHLI
Project Description: The Kolobow membrane oxygenator has been used for
cardiopulmonary bypass in over 60 dogs. Design and fabrication problems
of incorporating the Kolobow membrane into a prototype heart-lung machine
have been largely resolved. Cardiopulmonary bypass using partial whole
blood prime, normothermia, 1007o oxygen (6-8 1/min. flow) was employed at
flow rates of 60-80 cc . Kg. Approximately 50 animals were pumped for
three hours or less with a short-term survival (24 hours) of 707o. Long-
term survival (> 1 week) for this group was 507o. Animals pumped more
than three hours had s short-term survival of 307o, and no long-term sur-
vivors have been achieved to date. Arrhythmias, congestive failure or
pulmonary insufficiency have been responsible for the deaths in the
greater than three hour perfusion group.
The arterial p02 has ranged between 100 and 300 mm. Hg and
venous p02 30 to 45 with the present membrane. These values tend to be
well maintained for a given animal subjected to longer perfusions (>3 hours),
CO2 elimination was initially a problem in our system, but an improved
vacuum system and better quality control of membrane production now provides
arterial p02 of 33-42 and venous pC02 of 44-50 to be achieved.
Hemolysis was minimal during and immediately after bypass.
Postbypass levels of SCOT 80-120, LDH 100-150, and serum hemoglobin
20-55 mgm7o have been recorded. Hematocrit fell 87o and platelets decreased
107o during CPB. Lactic acid was 40-60 mgmT, and total protein decreased
30% with bypass.
Proposed Course: Current plans are to continue animal
studies directed toward employing this membrane clinically for both cardio-
pulmonary bypass and prolonged pulmonary assist.
f5
Serial No. NHLI-63
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Transcutaneous measurement of aortic regurgitation with
a Doppler Velocitometer
Previous Serial Number: None
Principal Investigator: Paul R. Hickey, M.D.
Lawrence L. Michaelis, M.D.
Other Investigator: Norman L. Luka, M.D.
Project Description: Acute aortic regurgitation was produced in 10 dogs
by punching one or more holes in the valve leaflets under normothermic
inflow occlusion. The absolute percentage of regurgitant flow was then
determined with an electromagnetic flow probe placed around the ascending
aorta and/or the common brachiocephalic trunk at its origin. Occlusion
distal to the probe was performed to ascertain zero flow. Simultaneously,
the regurgitant flow in the brachiocephalic trunk was measured trans-
cutaneously using a Doppler bidirectional velocitometer with a fixed,
internal zero, applied at the sternal notch. Regurgitant flows were
created ranging from 10-75% of forward flow. In 9 of the 10 animals,
the regurgitant flow measured transcutaneously was within 11% (± 6%)
of that measured directly with the electromagnetic flow probe. The
difference between the two measurements was not statistically significant.
In one dog the difference between the two measurements was 23%.
These data indicate that transcutaneous measurement of regurgitant flow
in the large arteries of the thoracic inlet, using a Doppler bi-directional
velocitometer, is an accurate method of assessing absolute percentages of
aortic regurgitation. The simplicity of this noninvasive technique makes
it promising for clinical use and studies are underway to evaluate its
accuracy in man.
Proposed Course: This data has been submitted to the Forum of the
American College of Surgeons and is presently under consideration for
presentation.
<fS
Serial No. NHTJ-64
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Reports
July 1, 1972 through June 30, 1973
Project Title: A new canine model for echocardiographic evaluation
of myocardial function
Previous Serial Number: None
Principal Investigators: P. David Myerowitz, M.D.
James Griffith, B.S.
Arthur J. Roberts, M.D.
Lynn H. Harrison, Jr., M.D.
Walter Henry, M.D.
Charles L. Mcintosh, M.D.
Cooperating Units: Biomedical Engineering & Instrumentation Branch
Cardiology Branch, NHLI
Project Description: A reliable canine model for echocardiographic
evaluation of myocardial function has not been previously reported.
Because of the configuration of the dog's chest wall, surface echo-
cardiography is difficult to obtain. In six dogs a 1.7 x 0.5 cm.,
2.25 M Hz barium titanate echo transducer was sutured to the anterior
epicardial surface of the right ventricle. This was positioned to
measure septal thickness, left ventricular posterior wall thickness
and left ventricular transverse diameter in a manner similar to that
used in clinical echocardiography. The dynamic relationships of
these structures were analyzed with a video scanner-analog computer
system. The animals were studied two to five weeks postoperatively in
the closed chest, unanesthetized state, at which time the cardiac
dimensions listed above were measured. Volume changes were estimated
from the ultrasonically determined left ventricular diameter and
compared with volume changes simultaneously measured with an electro-
magnetic flow probe placed around the ascending aorta.
Results: This technique has proved to be a dependable means of following
left ventricular dimensions in dogs echocardiographically up to three
months after insertion. In addition, the correlation between volume
changes estimated by flow probe versus echocardiography was > 0.9.
Proposed Course: The project has been completed and an abstract submitted
for the next meeting of the American College of Surgeons.
9^^
Serial No. NHLI-65
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: A comparison of left ventricular pressure, aortic flow
and echocardiography in determining the response of
left ventricular function or propranolol
Previous Serial Number: None
Principal Investigators: P. David Myerowitz, M.D.
James Griffith, B.S.
Walter Henry, M.D.
Charles L, Mcintosh, M.D.
Cooperating Units: Biomedical Engineering & Instrumentation Branch
Cardiology Branch, NHLI
Project Description: Presently, administration of propranolol, a
negative inotropic agent used in arrhythmias, is monitored by arterial
pressure. The purpose of this study was to see if echocardiographic
changes could be found which precede the hypotensive effect of the drug
and could be used in monitoring its administration. Five dogs were
instrumented with aortic flow probes, left ventricular pressure trans-
ducers and echo transducers. They were then given 15 mg. of propranolol
intravenously (10 times the dose recommended in adults) in 1 mg. incre-
ments. Left ventricular pressure, aortic flow and left ventricular
transverse dimensions were recorded.
Proposed Course: Data is presently being collected and analyzed.
9S'
Serial No. NHTJ-66
1. Clinic of Surgery
3. Bethesda, Md,
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Experimental creation of infundibular pulmonic stenosis,
ventricular septal defect and right ventricular
hypertension in puppies.
Previous Serial No.: None
Principal Investigators: Kent W. Jones, M.D.
Michael Jones, M.D.
Stephen B. Colvin, M.D.
Other Investigators: Lawrence L. Michaelis, M.D.
Andrew G. Morrow, M. D.
Project Description: The combination of infundibular pulmonic stenosis
with an intracardiac shunt and associated right ventricular hypertension
is a commonly encountered congenital cardiac malformation requiring surgical
palliation and/or correction. The purpose of this study was to surgically
create an experimental model simulating the above disorder, in order to study
and more fully understand the anatomic, pathologic and hemodynamic changes
which accompany such defects.
Foxhound puppies, weighing 6.0-8,0 Kg., were used for this model. Infundi-
bular pulmonic stenosis was achieved by placing a No . 1 silk suture through
the septal band of the crista supraventricularis and tying it anteriorly,
tight enough to elevate RV pressure to approximately one-half of systemic
level. Utilizing inflow and outflow occlusion, a ventricular septal defect
was created by removing a 5 mm. diameter core of muscular septum with an
instrument similar to a sharpened cork borer attached to suction. A Kel-F
plastic tube with flanged edges was then placed through the created defect
to maintain patency.
Results: Thirty- three (33) puppies underwent operation and 25 survived, for
an operative survival of 767o. All survivors had an easily palpable thrill
over the anterior surface of the RV from the newly created VSD. Eleven
animals have undergone left heart catheterization 4-6 weeks postoperatively
and seven have demonstrated a large left-to-right shunt by dye curve.
Proposed Course: We intend to study these dogs at 4-6 months of age using
both right and left heart catheterization to determine intracardiac pressures
and the size of the interventricular shunt. Right ventricular angiography
is planned to demonstrate the size of the infundibular outflow tract. A
systemic- pulmonary anastomosis will then be constructed and at a later date
a repeat RV angiogram will be performed to determine if the size of the out-
flow tract has changed or become atretic. The hearts will ultimately be
studied grossly and microscopically at necropsy.
Serial No. NHLI-67(c)
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: A review of eighteen years experience with total correction
of tetralogy of Fallot at the NIH
Previous Serial No.: None
Principal Investigators: Kent W.~ Jones, M.D.
Michael Jones, M.D.
Other Investigators: Andrew G. Morrow, M. D.
Project Description: Between September 1955 and April 1973, 164 patients
have undergone total operative correction of tetralogy of Fallot. Their ages
at the time of operation ranged from 2 to 41 years with a mean age of 16 years.
Five (3%) patients had isolated valvular pulmonic stenosis, 65 (407o) had only
infundibular pulmonic stenosis, and 94 (57%) had a combination of the two
lesions. Forty-five (277o) of the patients also had an atrial septal defect,
and 53 (327=) had undergone a previous palliative procedure. Thirty-four
patients died in the postoperative period for a total operative mortality of
217o; however, there were only 9 deaths following the last 100 operations, and
there have been no deaths following the last 33 procedures. Five additional
deaths occurred 6 months to 3 years postoperatively, for a late mortality of
47o.
Ninety-four (727o) of the 130 operative survivors have been followed at the
NIH. By New York Heart Association Classification, 85 (917o) are functional
Class I, 6 (67o) Class II, and 3 (37.) Class III, Ninety-three (727.) patients
have undergone postoperative catheterization. Thirty-nine (427.) have an
RVEDP>6 mm, Hg. Ten (117o) have a gradient across the RV outflow tract of
>30 mm. Hg. Fifteen (167.) have a residual interventricular shunt; 13 of these
occurred in patients operated upon prior to 1966, and in the last 87 cases,
only 2 (27.) patients have had residual shunts. Of the 53 patients who had
previous palliative operative procedures, 9 (177o) died at the time of total
correction. There was a 67. operative mortality in patients with a preoperative
QP/QS >1. 0/1.0, compared with a 237 mortality in those with a QP/QS<1 .0/1 .0 .
Ninety- five patients underwent outflow tract reconstruction with a prosthetic
gussett, and 18 (197.) died in the early postoperative period. Of the 45
patients with an atrial septal defect, 21 (477.) had the defect closed at the
time of total correction„ Three (147.) of the patients died, compared to 7 (297.)
deaths which occurred in the 24 patients with atrial septal defects which
were not closed.
Seven (47o) of the patients undergoing operation had permanent complete heart
block postoperatively requiring a pacemaker. Three (437) of these patients
died.
1 97
Serial No. NHLI-67(c)
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Five (47o) of the operative survivors have died 6 months to 3 years post-
operatively. Two had residual interventricular shunts, moderate gradients
across the RV outflow tract and elevated RVEDP. One had complete heart
block and a small residual shunt. The fourth had severe tricuspid and
pulmonic regurgitation with an RA mean of 18 and an RVEDP of 10 mm. Hg. The
fifth patient was asymptomatic and died suddenly of unknown causes.
Proposed Course: Continued review is planned.
f^
Serial No. NHLI-68
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The experimental creation of infundibular pulmonic stenosis
and demonstration of its hemodynamic relationships
Previous Serial Number: None
Principal Investigators: Kent W. Jones, M.D.
Michael Jones, M.D.
Stephen Colvin, M.D.
Other Investigators: Lawrence L. Michaelis, M.D.
Andrew G. Morrow, M.D.
Project Description: Infundibular pulmonic stenosis is a commonly
encountered congenital cardiac malformation occurring singularly, in
conjunction with valvular pulmonic stenosis, and in association with
other congenital intracardiac defects. In order to study the hemodynamic
relationships of this obstructive lesion, a surgically created model of
this disorder was utilized.
Foxhound puppies, weighing between 4.2-6.5 Kg., were subjected to a
right thoracotomy. A suture of #1 silk was placed through the septal
band of the crista supraventricularis and tied anteriorly, tight enough
to elevate the RV pressure to approximately one-half of systemic level.
Sixteen (16) of twenty-six (26) animals survived the operative procedure
for an operative survival of 62%.
The survivors were then allowed to grow over a 5-6 month period, at
which time they were subjected to a right thoracotomy for insertion of an
indwelling catheter in the pulmonary artery distal to the pulmonic valve.
Approximately one week thereafter, they underwent right and left heart
catheterization to assess the dynamics of the obstruction. Baseline LV,
RV and PA pressures were recorded simultaneously, and a baseline orifice
area was calculated by the method of Gorlin and Gorlin, using the RV-PA
pressure gradient and LV dye curve cardiac output to calculate infundibular
blood flow. After stable baseline values were obtained, the animals were
subjected to constant drip infusions of isoproterenol and calcium chloride,
and increments of intravenous acetyl strophanthadin. Cardiac output,
infundibular blood flow and orifice area were determined with each pro-
vocative measure and new baselines were obtained following each intervention.
Results: At present, ten dogs weighing 14.7 - 17.8 Kg. have undergone
catheterization studies with data being obtained from eight. The average
peak systolic gradient across the infundibular obstruction was 56 mm. Hg,
with a mean gradient of 39 mm. Hg and an orifice area of 0.33 cm . With
1 99
Serial No, NHTJ-68
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
isoproterenol infusion the area of the stenotic orifice decreased in seven
of eight dogs (117o - 61?o from baseline). All five dogs receiving calcium
chloride demonstrated a decrease in the orifice size (12-247o), as did all
eight dogs receiving acetyl strophanthadin (6-30%). Increasing doses of
acetyl strophanthadin demonstrated a progressive decrease in the stenotic
orifice size. Necropsy observations on the hearts of three dogs revealed
right ventricular hypertrophy, as evidenced by a right ventricular free
wall thickness approaching the thickness of the left ventricular free
wall. A dilated chamber was present distal to the infundibular obstruction
and two of the three dogs had pulmonary valve leaflets which were thickened
and deformed, presumably from the proximal jet of blood striking them.
In addition, the measured stenotic infundibular orifices were similar to
the calculated resting orifice area.
Conclusion: The above findings demonstrate the dynamic nature of the
obstruction in infundibular pulmonic stenosis and point out the hazards
which may be encountered in children with this disorder if given an ino-
tropic agent.
Serial No. NHLI-69
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Reports
July 1, 1972 through June 30, 1973
•Project Title: Hemodynamic and anatomic evaluation of experimental
valvular aortic stenosis
Previous Serial Number: None
Principal Investigator: Jack G. Copeland, M.D.
Other Investigators: Barry Maron, M.D.
Lawrence L, Michaelis, M.D.
Cooperating Unit: Cardiology Branch, NHLI
Project Description: In 20 dogs valvular aortic stenosis has been
created by placing spring steel clips on the aortic valve commissures.
Sequential postoperative cardiac catheterizations have confirmed that
stenosis is produced resulting in an average peak systolic gradient of
30 mm. Hg and an average valve area of .75 cm.^ (approximate 25% of
normal). Seventeen dogs were catheterized twice. Approximately one-
half of these showed valve areas which decreased between the second and
fourth week postoperatively. Pathologic examination of the valve at
one to six months postoperatively disclosed fibrotic stiff leaflets
in all hearts with small orifices (< 1 cm. 2) in those dogs which retained
their stenosis, and tears in the leaflets or slippage of the clips in
those which lost their stenosis. All hearts were hypertrophic when com-
pared in weight (Gms.)/ body weight (Kg.) to a normal series of dogs
matched for sex and breed at p ^ .001. Ultras true tural evaluation of
this tissue is presently underway. When this is completed, the study
will be terminated.
Proposed Course: Acute experiments will be done using this model to
examine the physiology of valvular aortic stenosis.
/«/
Serial No. NHLI-70
1. Clinic of Surgery
3. Bethesda, Md .
PHS-NIH
Individual Project Reports
July 1, 1972 through June 30, 1973
Project Title: Effects of aortic stenosis on (A) Coronary blood flow
and (B) Regional distribution of blood flow
Previous Serial Number: None
Principal Investigators: Jack G. Copeland, M.D.
Norman Luka, M.D.
Other Investigators: Lawrence L. Michaelis, M.D.
Project Description: Using a technique for creating valvular aortic
stenosis in dogs which has recently been improved and tested in this
laboratory, two studies of the physiology of valvular aortic stenosis
are being undertaken. Study A is involved with measurement of myo-
cardial oxygen consumption (MVO2) in acutely produced valvular aortic
stenosis. Cardiac output is varied by pacing the heart from the right
atrium and manipulating bilateral femoral A-V shunts. Coronary artery
blood flow is measured by timed collections from the coronary sinus.
Arterial and coronary sinus blood O2 content is measured as the cardiac
output is raised. An electromagnetic flowmeter is placed on the aortic
root and pressures in the LV and ascending aorta are monitored.
Proposed Course for A: At present the study is in a developmental
stage. Eventually, we plan to assess the relations of stroke work,
dp/dt, and time tension index to MVO2 on dogs with aortic stenosis
compared to those without aortic stenosis.
Proposed Course for B: Study B is in the planning stage and has not
been attempted. Its aim will be to examine dogs with aortic stenosis
during strenuous exercise. It is hoped that syncope will be produced
and that its mechanism will be apparent from monitoring cardiac output
(aortic root EMF), LV and aortic pressures, brachiocephalic blood flow
(brachiocephalic EMF) and electrocardiogram.
/o3l
Serial No. NHTJ-71
1. Clinic of Surgery
3. Bethesda, Md.
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Hypothermic asanguineous circulatory arrest (HACA)
Previous Serial Number: None
Principal Investigators: Jack G. Copeland, M. D.
Bruce A. Reitz, M. D.
Other Investigators: Arthur J. Roberts, M. D.
Lawrence L. Michaelis, M. D.
Project Description: Hypothermic asanguineous circulatory arrest (HACA)
is being evaluated. The technique could be used in infant open heart
surgery, in massive trauma requiring heroic surgery, in some standard
operations which would be technically simpler (partial hepatectomy,
replacement of the aortic arch, etc.), and in donor preservation for
cardiac transplantation. The technique consists of rapid total body
washout with Krebs solution with dextran at 0-3°C until the temperature
(middle ear and nasopharynx) falls to 10°C or less (usually < 5 min. in
20 Kg. dog), at which time circulatory arrest is instituted. The
animals' blood is collected and reinfused during rewarming. The period
of HACA has been varied from 10 min. to 3 hours. Serial monitoring
of blood gases and pH, Hct., and electrolytes has been done. Rewarming
is started with an exchange transfusion of warm blood from the animal
(diluted approx. 1:4) and is continued to a temperature of 32 C when
cardiopulmonary bypass is discontinued. Fourteen experiments have been
done. Survival is consistent after periods of up to 3 hours HACA;
survival without apparent neurologic damage occurs with up to 2 hours
HACA. Assessment of LV function and contractility in the immediate
and late postoperative period and serial evaluation of organ function
(liver, kidney, pancreas, bone marrow) with appropriate blood tests are
being done. Survivors are later sacrificed and subjected to a complete
autopsy.
Proposed Course: Study cardiac function in 5-10 more experiments.
Examine CNS, pH, p02, P'C02, during HACA. Examine arteriolar flow of retina
during cooling and warming. Perform partial hepatectomy and/or replace-
ment of aortic arch during HACA. Evaluate blood clotting before and
after HACA. Use the same technique with puppies rather than adult dogs.
/es
ANNUAL REPORT OF THE
EXPERIMENTAL THERAPEUTICS BRANCH
NATIONAL HEART AND LUNG INSTITUTE
July 1, 1972 through June 30, 1973
The goal of the Experimental Therapeutics Branch is the application of
basic biochemistry and pharmacology to understanding mechanisms of disease and
to develop new therapies for human disease. The Branch is composed of four
sections.
SECTION ON EXPERIMENTAL MEDICINE
The goal of this section is to close the gap between the laboratory and
the patient by conducting preclinical and clinical research in certain areas
of human disease, especially hypertension and the role of vasoactive sub-
stances. Our activities this year can be summarized under the following three
headings .
I. Hypertension
It is apparent that the regulation of normal blood pressure is dependent
upon striking a fine balance between a number of vasodilator and vasoconstric-
tor systems. Therefore, we have undertaken studies in each of these systems
to identify their roles in human hypertensive disease and also to define the
Interrelationships that are operative between them.
Kallikreins are enzymes which control the production of kinin peptides,
potent endogenous vasodilators. Kallikrein in urine is apparently produced in
the kidney. We have found that normal volunteers excrete on the average twice
as much kallikrein in the urine as patients with essential hypertension. The
level of urinary kallikrein varies with sodium intake. Thus, normal subjects
excrete 11.6 + 1.0 EU/day on an ad lib Na+ diet, but they excrete 33.0 + 2.5
EU/day on a 9 meq Na+ diet (p < 0.001). Patients with essential hypertension
not only excrete significantly less kallikrein (5.0 + 0.8 EU/day on ad lib
Na+) than the normals but also have a smaller increase in excretion on 9 meq
Na+ diet (9.4 + 1.8 EU/day) . Urinary kallikrein excretion is increased an
average of 7-fold in patients with primary aldosteronism and is unresponsive
to changes in dietary sodium. Kallikrein excretion is increased 2.5 times in
normals by fludrocortisone and spironolactone decreases the increased urinary
kallikrein excretion. of patients with primary aldosteronism or of normals on
a 9 meq Na+ diet. Thus, the kallikrein-kinin system is directly responsive to
levels of mineralocorticoids and measurement of urinary kallikrein may be an
accurate and economic new screening test for patients with primary aldosteronism.
The kallikrein-kinin system has potent vasodilator properties. Patients with
essential hypertension appear to have reduced basal activity of this system
and also reduced responsiveness to physiologic stimuli.
The renin-angiotensin-aldosterone system is vasoconstrictor. We have
modified established radio-immunoassay procedures to yield an assay which is
easier, and quicker to perform and which is highly specific, sensitive and
reproducible. We have established normal levels of plasma renin activity (PRA)
1 /oC
for our laboratory and have shown that urinary and plasma kallikrein levels
can be dissociated from PRA. We have also demonstrated that alphamethyldopa,
a centrally acting antihypertensive drug, lowers PRA.
The prostaglandins (PG) are endogenous unsaturated fatty acids with potent
effects on blood vessels. We have developed a radio- immunoassay capable of
measuring PC's of the A, E and F series from plasma or urine in subnanogram
amounts. We find that normal levels in man are about 250 pg/ml of plasma for
both PGA and PGE. PC's appear to mediate the hyperemic response of the coro-
nary circulation to hypoxia and/or occlusion.
Catecholamines and the adrenergic nervous system have profound effects
on blood pressure and renal function. We have found that acute infusions or
chronic administration of salt to normal humans cause an increase in urinary
dopamine, a decrease in urinary norepinephrine and a decrease in serum dopamine
6-hydroxylase (DBH) activity. The latter is the enzyme which controls the
conversion of dopamine to norepinephrine. Studies with radioactive precursors
in rats have also indicated that DBH in kidney plays a key role in salt and
water homeostasis. We have shown previously that plasma DBH levels in man do
not correlate with age, blood pressure or sympathetic nerve activity.
II. Histamine, Other Amines and Related Enzymes
We have shown previously that the enzyme histaminase is increased in the
blood of some patients with metastatic medullary carcinoma of the thyroid
(MCT) and that it is a good biochemical marker for the tumor in tissues. We
now find high levels of DOPA decarboxylase activity in MCT, normal levels of
tyrosine and tryptophan hydroxylase and no detectable ornithine and histidine
decarboxylase. The DOPA decarboxylase activity in MCT resembled that in
pheochromocytoma in its response to various inhibitors. This provides further
evidence that the diverse manifestations of Sipple's Syndrome (I.e., MCT,
pheochromocytoma and parathyroid hyperplasia) arise from single genetic dysplasia
of embryonic neural crest cells.
Thymic lymphocytes are rich in ornithine decarboxylase. This enzyme con-
verts L-ornithine to putrescine. This polyamine has been shown to be involved
in rapid cell growth and organ regeneration. We find that the ornithine de-
carboxylase activity is dependent on intact lymphocytes, falls markedly with
steroid involution and can serve as a useful biochemical marker for thymic
lymphocytes.
III. Collagen
We have developed methods for measuring the rates of collagen synthesis
and of collagen degradation in human skin. In both keloids and hypertrophic '
scars, two examples of over-abundant wound healing, the rates of both synthesis
and degradation are increased. Obviously synthesis is increased more than
degradation since the lesion increases in size. When corticosteroids are in-
jected into the lesions they become noticeably smaller and softer. Studies
performed at that time show that the rate of collagen synthesis is still as
rapid as before. This implies that the steroids must have activated collagenase.
/O^
We have shown previously that the rate of collagen synthesis is increased
in the skin of patients with scleroderma. Now we find that the rate of collagen
degradation is decreased in this disease. This provides a better explanation
for the rapid accumulation of collagen seen in the early stages of this disease.
SECTION ON BIOCHEMICAL PHARMACOLOGY
The objective of this section is to define, at molecular level, physio-
logic processes that relate to human disease and particularly to hypertension.
A wide variety of approaches to this goal have been used and are summarized
in the following four headings.
I. Regulation of Mammalian Enzyme Levels
Melatonin synthesis in the pineal gland is under neuronal control and its
regulation via induction of serotonin N-acetyltransferase is one of the most
dramatic examples of enzyme induction known in mammalian tissues. Appropriate
neuronal stimulation of the gland results in a 50 to 100-fold increase in
enzyme level. It was previously established that neuronal stimulation was via
B adrenergic receptors that in turn activated adenyl cyclase. Since many of
the effects of cAMP are mediated by protein kinases, it was of great interest
to find that pineal tissue contain relatively high amounts of protein kinase.
The kinase from bovine pineal gland was isolated. It is a typical kinase
consisting of a catalytic and a regulatory subunit. If the neuronal effect on
enzyme levels is mediated by protein phosphorylation then one or more of three
events must occur: gene activation via chromatin phosphorylation, stimulation
of ribosomal translation, or direct activation of pre-existing apoenzyme. We
found that chromatin is a good substrate for pineal protein kinase and that
its template capacity for messenger RNA synthesis is enhanced after phosphory-
lation. This was shown by a 20% increase in actinomycin D binding and a direct
20% stimulation of RNA synthesis using E.coli polymerase. The role of the
kinase in translation was more difficult to establish since we discovered that
ribosomes have a protein kinase bound to them. This kinase was removed by a
0.5 M KCl wash, isolated as a nearly pure protein and shown to be a typical
c-AMP-dependent kinase. This kinase was not one of the initiation factors
which are removed from ribosomes by the same washing procedures. The presence
of large amounts of kinase precluded measuring an effect of kinase on transla-
tion. Finally, attempts to activate serotonin N-acetyltransferase activity by
direct phosphoration of pineal homogenates (kinase + c-AMP + ATP) did not re-
sult in any increase in activity. Our tentative conclusion is that the c-AMP-
dependent protein kinase may exert its regulatory effects at both transcriptional
and translational levels.
A preliminary characterization of serotonin N acetyltransferase indicates
that it is an enzyme (mw 40 - 80,000) that requires SH group for its activity,
but does not appear to form an acetyl-enzjme intermediate.
II. Biochemistry of Neurohtunoral Amines
Increasing attention is being devoted to the terminal enzyme in norepine-
phrine biosynthesis. Dopamine-6-hydroxylase (DgH) is found in neurotransmitter
granules and is released together with the entire contents of the granule upon
sympathetic neuronal transmission. Other workers have established that the ^^f
3
enzyme occurs in a form which has molecular weight of 300,000 and contains
significant amounts of copper. During the past year we have isolated D6H from
bovine adrenal glands. Following polyacrylamide gel electrophoresis a single
band was observed with either protein specific or carbohydrate specific stain.
Electrophoresis in sodium dodecylsulfate and a reducing agent established that
the monomer molecular weight is about 75,000. Quantitative carbohydrate analy-
sis indicates that the protein is a typical glycoprotein containing 3% of its
weight as sugar residues. Pure proteins with high catalytic activity contain
about 4 atoms of copper per tetramer. Thus, DBH appears to be a tetrameric
glycoprotein containing 4 atoms of copper.
Tryptophan hydroxylase is the rate-limiting enzyme in serotonin synthesis
and is a marker for serotonergic neurons in the central nervous system. Using
5,6 and 5,7 dihydroxytryptamine (DHT) which are cytotoxic and specifically
taken up into the serotonergic neuron, we have measured regional depletion of
the enzyme tryptophan hydroxylase in rats. 5,6-DHT causes partial depletion
of tryptophan hydroxylase in all brain parts, but particularly severe (75 to
90%) depletion in mesencephalic tectum and spinal cord. In all regions the
serotonergic destruction appears to be permanent with no recovery of activity
within 60 days. 5,7-DHT appears to be even more effective causing 50 to 100%
lasting depletion of tryptophan hydroxylase in striatum, septum, pons-medulla,
hypothalamus, mesencephalon, forebrain and spinal cord.
The modification of the tryptophan hydroxylase assay to utilize 6-methyl-
tetrahydropterin and saturating substrate levels has greatly aided our study
of this enzyme. We also found that complex mixtures of cations added to buffer
stabilize the enzyme during purification and therefore significant progress is
now being made on the isolation of this enzyme from the mesencephalic tegmentum.
III. Molecular Changes Associated with Hypertension
While little evidence is available for a direct pathogenic role for cate-
cholamines in essential hypertension, it is now clear that noradrenergic nerves
(central and peripheral) are involved in blood pressure regulation. Using the
genetic spontaneous hypertensive rat (SHR) and eight control species, we have
established that rat species can have widely varying levels of each of the
catecholamine biosynthetic enzymes. No direct correlation with mean systolic
blood pressures is apparent. In vivo catecholamine synthesis studies provide
supportive evidence for central depressor systems that are mediated by nora-
drenergic fibers. Having access to 9 strains of animal showing substantial
variations in the catecholamine biosynthetic enzymes it was possible to reassess
the role of the individual enzymes by measuring in vivo catecholamine biosyn-
thetic rates. This study of norepinephrine synthesis confirms the rate-limiting
role of tyrosine hydroxylase, indicates that DBH level may have some affect,
and suggests that some factor other than enzyme level (possibly the availability
of tetrahydropterins) exerts partial control of synthesis rate.
In an unrelated study we have examined in vivo vascular protein synthesis
in SHR and the parent normotensive strain, the Kyoto-Wistar . Adult hypertensive
rats had an increased rate of actomyosin synthesis in the heart. Vascular non-
collagen protein synthesis was increased in the SHR by 13 weeks of age. Ex-
fo%
(
periments are now being extended to prehypertensive SHR in an effort to
determine whether increased protein synthesis precedes or follows the develop-
ment of hypertension.
IV. Iron-Sulfur Proteins
Iron-sulfur proteins catalyze numerous redox reactions in all forms of
life. Previous experimental work from this laboratory has focused on proper-
ties and structural details of the bacterial electron transfer proteins
ferredoxin and rubredoxin as models for the more complex enzymes. We have now
prepared an antibody to Clostridium pasteurianum rubredoxin and examined cross
reactivity with other bacterial rubredoxins. All rubredoxins bind to the
antibody but only that from £. pasteurianum forms a precipitating complex.
Further work using nuclear magnetic resonance has resolved the S protons of
cysteinyl residues of clostridial ferredoxin and provided detailed information
on the role of the cysteinyl groups in forming the iron-sulfur active center.
SECTION ON PHYSIOLOGICAL CHEMISTRY
I. Biochemistry of the Kallikrein-Kinin System
An inactive form of kallikrein has been discovered in human urine, which
is activated by high concentrations of trypsin. Following activation, the
molecular weight drops from 50,000 to 40,000 which is the molecular weight of
native, active hxnnan urinary kallikrein (HUK) . Several lines of evidence
indicate that inactive kallikrein is a kallikrein- inhibitor complex. Thus, low
concentrations of trypsin are strongly inhibited by the inactive kallikrein
and activation can occur spontaneously by prolonged dialysis or by freezing and
thawing .
A new assay of HUK has been developed which measures both the free and the
inhibitor-complexed forms of kallikrein. It involves a trypsin step to liberate
kallikrein bound to inhibitor, a heating step to destroy urokinase (which could
contribute as much as 10% to the esterase measured in the old method) , and the
replacement of tosyl-arginine methyl ester with a better substrate, carboben-
zyloxy-arginine methyl ester.
The previously reported new radiochemical esterase assay for human plasma
prekallikrein has been refined and its specificity established. In a study of
15 patients who underwent surgery, a 15-50% fall in prekallikrein was observed.
The lowest levels were reached at different times ranging from 1-4 days, and
in all patients activity began to return to normal by the 15th day.
No alteration in prekallikrein was found in 20 patients with cystic
fibrosis. This contradicts the report of another laboratory that there is
significantly less enzyme in about 50% of their patients' plasma.
The esterase assay appears to be an ideal screening test for Hageman and
Fletcher factor deficiencies and could have wide application in hematological
laboratories. With minor modification it also has been used to detect defi-
ciencies of the C'l esterase inhibitor in patients with hereditary angioedema.
5 /o9
Studies concerned with the mechanism of activation of prekallikrein in
human blood have shown a requirement for Hageman factor. The latter is commonly
activated by exposure of plasma to a negatively charged surface. Evidence has
been obtained which indicates that this activation of Hageman factor requires
both a hitherto unrecognized factor and prekallikrein and is not merely the
consequence of a non-enzymatic structural change of the molecule bound to the
surface.
Quantitative data have been obtained which show that permeability factor
elaborated when plasma is diluted 1/100 is activated Hageman factor and/or low
molecular weight fragments derived from active Hageman factor.
New procedures for the isolation of human kallikrein and prekallikrein
have been developed which permit the facile isolation of these proteins in high
yield. The procedure involves the use of affinity columns of I^-arginine-Ci2~
agarose and sheep anti-human IgG-agarose. The latter is used "to remove IgG
which has been extremely difficult to separate from plasma kallikrein.
Earlier evidence (obtained by "harsh" chemical treatment of outdated
plasma) for the occurrence of 2 forms of low molecular weight (LMW) kininogen
in human plasma has been greatly strengthened by finding the 2 LMW forms in
fresh plasma treated under "mild" conditions.
Kallikrein has been partially purified from human sweat and saliva. These
enzymes are similar (or identical?) to urinary kallikrein in as much as they
are inhibited by antibody to urinary kallikrein. In addition, all 3 enzymes
show the same spectrum of inhibition with several competitive and non-competitive
inhibitors.
Our efforts to understand the role of the kallikrein-kinin system in
kidney have led us to the finding of kininogen in blood-free rat kidney.
Hence, kidney contains its own supply of kininogen as well as kallikrein. In
preliminary experiments in rats depleted of kininogen by treatment with cellu-
lose sulfate, kininogen levels in renal vein were 150% higher than renal
artery. Plasma kininogen may, at least in part, arise from kidney.
Kinins in urine are probably produced in kidney. A column procedure has
been developed for the isolation of kinins from urine in preparation for their
assay. Kinins in human blood are rapidly inactivated in lung. In preliminary
experiments over 80% of radioactive bradykinin was destroyed in one pass through
lung. Over 50% of this destruction was due to the action of kininase II, a
peptidyl peptidase which may be identical to the angiotensin converting enzyme.
An assay for kininases in human blood has been developed. It involves the
use of radioactive bradykinin and a CM-Sephadex column which conveniently
separates the radioactive split products from unhydrolyzed kinin.
A radiochemical esterase assay for urokinase in human urine has been
developed. It is similar in principle to the kallikrein assay, but employs
acetylglycyllysine methyl ester which is a specific substrate. This assay will
open up studies on the physiological role of urokinase in kidney.
//<0
The vasoactive glycopeptide in yellow jacket venom reported last year has
been further characterized. The structure is:
sugar 1 sugar 2
1 I
Thr-Ala-Thr-Thr-Arg-Arg-Arg-Gly-Bradykinin
Sugar 1: N«Ac Galactosamine 1-2 moles. Galactose, 1 mole
Sugar 2: N'Ac Galactosamine 2-3 moles, Galactose, 2 moles
II, Prostaglandins
Chemical studies relating to the possible significance of the prostaglan-
dins of the A series have revealed that:
1) Human blood cells rapidly convert PGA to a more polar metabolite.
This finding could have a profound effect on interpretinjg the PGA values for
human plasma reported by others.
2) The absence of a PGA isomerase in human plasma has been confirmed.
However, plasma contains a non-dialyzable heat-labile factor which greatly
stimulates the isomerase activity of rabbit plasma.
3) Enzymatic dehydration of PGEj^ to PGA-, could not be demonstrated in
homogenates of various tissues. However, homogenates of rat kidney convert
PGE]^ to a less polar metabolite which cochromatographs with PGA.
High speed liquid chromatographic systems have been developed for the
separation of PGA from PGB and PGA^ from PGA2.
High speed liquid chromatographic systems have also been developed for
the rapid identification of the PTH amj.no acids. This technique complements
the previously developed gas chromatographic and mass spectrophotometic methods.
SECTION ON NEUROENDOCRINOLOGY
I. Influence of Hormones and Hallucinogens on Neural Function
We have shown that LSD-25 and marihuana do not affect the manner by which
the neuraxon is depolarized. These data, indirectly, support the concept that
these drugs carry out their effects by primarily interfering with synaptic
transmission.
Adrenocorticosteroid hormones affect sensory function for all sensory
stimuli, i.e., vision, audition, taste, smell, hearing and touch. The studies
which we carried out in the past year demonstrated the role these hormones
played in touch sensitivity. These effects appear to be mediated through the
direct role of the adrenocorticosteroid on the receptor, the neuraxon, the
brain or some combination of these three systems, not through ACTH effects.
f((
1
These steroid hormones serve to inhibit the inflow of sensory signals from the
outside world so that maximum integration of those sensory signals which are
received can take place. Removal of these hormones results in the failure of
normal integrative phenomenon and information loss. The role of ACTH in this
system is unclear.
Gonadal hormones and pituitary gonadotropins also influence neural function
as measured by visual or auditory evoked responses. Estrogen increases the
amplitude of the electrical activity of the brain. However, these neural phe-
nomena are also influenced by age and genetic factors which are independent
of gonadal hormone function. It appears that absence of the Y chromosome also
contributes to the large amplitude electrical activity of the brain observed
In phenotypic females.
Thyroid hormones also appear to alter receptor, neuraxon and brain func-
tion as indicated by effects on taste, smell and hearing. The manner and
extent of these effects are not clearly defined.
II. Metals
We have established that copper-ceruloplasmin and zinc a2 macroglobulin
complexes are metalloproteins which act as metals and that copper-albumin and
zinc-albumin complexes are the physiologically active metalloproteins in blood.
These latter macromolecular ligands are in equilibrium with micromolecular
ligands in serum, mainly the amino acids, histidine and cysteine. Metals are
conserved and protected against renal loss by their complex formation with
large molecular weight proteins. Excessively large concentrations of histidine
or cysteine in blood (in hepatitis, cancer or after histidine administration)
shift the equilibrium of macromolecular bound metal to micromolecular bound
metal and these metals are lost from the body and metal depletion occurs. This
has served as a model for Wenecke's encephalopathy. This concept can be uti-
lized for effective treatment of metal poisoning, including lead, cadmium or
mercury. It has been shown to be effective in vitro in removing cadmium from
its albumin binding site.
The immediate role of zinc in growth is through its control of food intake.
If this metal is removed from the diet man or animals will stop eating. Our
studies suggest that the endocrine and growth effects of this metal are due to
its effects on food intake rather than to any direct effect on hormone metabolism.
We have published the first studies concerning copper and zinc levels in
man between birth and 2 years of age. We have also Indicated that age-dependent
changes in total serum copper and zinc levels are due largely to increases in
serum levels of the largest macromolecular ligands of copper and zinc, cerulo-
plasmin and «£ inacroglobulin, respectively.
Zinc loss produces severe pathological changes in several organ systems.
Therefore, it is important to identify drugs or other agents which alter zinc
metabolism to specify their mode of action. 6-Azauridine and histidine produce
zinc depletion by altering the manner by which zinc is complexes with albumin.
Similar changes have been proposed for the action of growth hormone. Adrenal
cortical steroids may act via a direct role on the kidney since they do not
8 //2-
appear to alter directly the formation of the zinc-albumin complex in blood.
We have also shovm for the first time that progesterone directly affects blood
copper levels probably through induction of ceruloplasmin. Thus, both estrogen
and progesterone induce this metalloprotein.
Data in four subjects with hepatic porphyria indicate that histidine
administration either significantly inhibits the production of urinary uro-
porphyrins and coproporphyrins or inhibits the production of porphyrin pre-
cursors. Our initial data suggest that this inhibition occurs through histi-
dine inhibition of 6 amino levulinic acid and suggests that histidine plays a
role in heme synthesis.
Successful treatment of patients with idiopathic hjrpoguesia with zinc ion
is uniformly associated with the production of a zinc containing protein in
parotid saliva. These data suggest that zinc acts to induce the production
of this protein in the same manner that copper induced ceruloplasmin in the
blood of patients with Menkes Steely Hair Sjmdrome. The induction of this pro-
tein is dose dependent upon the amount of zinc ion administered. Removal of
zinc ion from these patients is associated with the loss of this protein from
saliva. This protein appears in normal parotid saliva. Since taste buds do
not contain blood vessels or lymphatics the presence of this zinc containing
parotid salivary protein probably supports taste bud function. These studies
are the first in which a specific role for saliva in taste has been observed
and they relate the role that zinc plays both in saliva and in taste.
In patients with idiopathic hypogeusia, a significant number improve with
placebo therapy. In those patients who do not respond and in whom the zinc
salivary protein is induced by zinc ion, taste acuity returns to normal fol-
lowing zinc administration. Continued administration of zinc ion is necessary
for continued production of this protein. In those patients in whom zinc ion
does not induce this protein taste function does not return to normal. These
results suggest two possible modes of therapy: (1) administration of larger
amounts of zinc ion may be effective in inducing this protein, or (2) topical
application of this protein in the oral cavity.
III. Taste and Olfaction
Difference in function of each of three major types of cells in the taste
bud has been described and related to anatomical form. In man we have related
form and function of taste buds from different papillae such that an accurate
"taste map" can now be drawn for the first time; each taste bud subserves all
taste qualities, but buds in fungiform papillae are most sensitive to salt
and sweet, buds in circumvallate papillae are most sensitive to sweet alone.
Thus, the tongue is equisensitive to sweet, the anterior portion most sensitive
to salt. Buds in palatal papillae are most sensitive to sour and bitter.
The neurochemical basis for the initial, neural events in taste, has
been described. This has been demonstrated by locating acetylcholinesterase
in the pore region of the bud and by producing hj^ogeusia and ageusia in man
following the oral introduction of anti-cholinergic agents without the pro-
duction of anesthesia. Blockade of electrical activity measured at the chorda
tympanl in rat has also been carried out by placing anti-cholinergic agents
9 ftZ
on the tongue. This is the first demonstration of the role of a neurotrans- ,
mitter at the pore of the taste bud.
Taste bud membranes have been isolated and their chemical properties
described. Specific binding of several sugars have been shown to take place at
these membranes whereas control tissue does not possess any of these properties.
Binding constants have been calculated and they agree closely with reported
data.
Miracle fruit glycoprotein (MFGP) has been isolated and purified. MFGP I
has two effects in man when placed into the oral cavity: (1) altering the
taste of all sour tastants to sweet, and (2) blocking sweetness and bitterness
normally produced by sweet and bitter tastants. The chemical properties of
MFGP have been described including its amino acid and sugar composition.
Various chemical reactions have produced changes in activity. Removal of the
terminal sugar completely inhibits the action of MFGP in altering sour tastants
to sweet whereas the blocking ability remains intact. Removal of several of
the glycosidase linkages enhances the activity of MFGP with respect to change
of sour to sweet taste by factor of 2 to 3. Through this mechanism we have
also radioactively labeled MFGP. The data suggest that MFGP is a plant lectin
and appears to be the first plant lectin isolated which has physiological
activity. This finding has served as the basis for a biochemical rather than
a bioassay for this material. Since MFGP can alter the taste of all sour tas-
tants to sweet it has ideal properties as a sugar substitute and may be im-
portant in the treatment of obesity.
Various substances when added to food or drink have been shown to alter '
intake behavior. Addition of monosodium glutamate (MSG) or NaCl decreases
sensitivity to bitter tastants. These agents reduce the bitterness of foods
and fluids. Addition of glucose increases salt intake. Since sugar is being
added in larger amounts to more foods and drinks its association with the pro-
duction of increased NaCl intake may represent an important, heretofore un-
recognized etiological cause of hypertension. Zinc depletion from food produces
anorexia and hypogeusia, and eventually marked slowing of growth and sexual
development. The problem is a much more common one than previously recognized.
This association has been found in middle-class school children in Denver,
Colorado, as well as in patients in Egypt and Iran. Correction of this abnor-
mality by supplying enough zinc in the diet is uniformly associated with the
return to normal of each abnormality.
Salt intake and hypertension have been related in man and animals for
many years. We have demonstrated that patients with hypertension prefer saltier
solutions than do normal subjects and that effective treatment of the hyper-
tension returns this abnormal propensity to normal. This suggests that some I
factor (s) which influence blood pressure also influence salt preference and
salt intake in man.
A new genetic taste marker, the fruit of the plant Antidesma bunius, has
been discovered which differentiates among people who are PTC taste blind.
Approximately 1/2 of the PTC taste blind people taste antidesma as sweet or J
sour whereas the other 1/2 taste it as intensely bitter. This trait has been I
linked in one group of patients to an XgA marker on red blood cells and in A
another group to hypertrophic subaortic stenosis. V
10 ,,^
Zinc loss by any means is associated with anorexia and hypogeusia. We
have shown this interrelationship in patients following thermal bum due to
loss of zinc rich fluids, following X-irradiation, following head trauma, after
hypothyroidism and in acute and chronic liver disease due to wastage of zinc
in the urine. The diagnosis of idiopathic hypogeusia involves several para-
meters which we have developed over the past year. These parameters include
the presence of Rudolph's sign, (localization of Technitium 99m over the area
of the nose during a routine brain scan) the infiltration of chronic inflam-
matory cells into the lamina propria of the nasal mucous membrane and the
decrease in production of a parotid salivary zinc containing protein. The
mechanism of taste loss appears to parallel and to be secondary to the loss of
the parotid salivary zinc containing protein whose normal function is to supply
nutrients to the taste bud (since it contains no blood vessels or lymphatics)
and appears to get its major nutrition from parotid saliva. In this sense we
have proposed, for the first time, a specific mechanism whereby saliva supports
taste. In patients with this disease treated with zinc ion, those who recover
taste acuity do so through induction of this protein either directly or in-
directly with zinc. Those patients who do not recover taste acuity exhibit no
change in the low concentration of this protein which they pathologically ex-
hibit. This induction is dose dependent upon the amount of zinc ion adminis-
tered.
The prediction of ultimate fertility in hypogonadal men may be related
to the type of olfactory defects present. Men with Type II hyposmia usually
become fertile with appropriate therapy whereas men with Type I hyposmia have
not become fertile in spite of any therapy. This observation clearly relates
olfactory acuity and sexual function in men.
A new approach to the diagnosis of several disease states involving
olfactory defects has been made through the development of the technique of
biopsy of the nasal mucous membrane and through the localization of Technitium
99m in the region of the nose (Rudolph's sign). These techniques have allowed
us to identify the characteristic pathology of patients with Sjogren's syndrome
and those with h5T)osmia of several different etiologies.
11 tlC
Serial No. NHTJ-72
1. Experimental Therapeutics Branch
2. Physiological Chemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: A New Radiochemical Esterolytic Assay for Human Urinary
Urokinase
Previous Serial Number: None
Principal Investigator: Toshio Imanari, Ph.D.
Other Investigators: John J. Pisano, Ph.D.
Cooperating Units: None
Project Description:
Objectives; Urokinase is an enzyme produced in kidney which has recently
assumed prominence because of its effectiveness in the treatment of thrombolytic
crises. Its use in patients with pulmonary emboli has been life-saving. Uro-
kinase is in great demand but short supply. A sensitive simple assay could
facilitate its isolation from urine. The role of urokinase in kidney is
unknown and a simple assay could stimulate study of this important enzyme.
Major Findings: A new radiochemical esterolytic assay for human urinary uro-
kinase has been developed. It is based on the previously reported principle
outlined in report NHLI-209 (1972).
The assays are performed with 10 yl of urine and %-glycyllysine methyl
ester as substrate. Specificity of the assay is indicated by the facts that
the esterase measured in urine is indistinguishable from standard urokinase in
its substrate specificity and inhibition by polypeptide protease inhibitors.
Urokinase is unstable in urine. In some specimens as much as 75% of the activity
was lost after 3 days storage at 4". Even at -15° as much as 50% of the activity
was lost.
Significance to Biomedical Research and Institute Program: This assay for
urokinase appears to be the simplest and most sensitive method yet developed.
It should be well-suited for research on the role of urokinase in the kidney
and in clinical studies. Urokinase is reported to be elevated in renal in-
farction and after hypoxic tubular damage and decreased in renal insufficiency
and in carcinomatosis.
Proposed Course of Project: The assay will be applied to the study of renal
disease, hypertension and various clinical states where the excretion might be
altered.
IIT
Serial No. NHLI-72
Honors and Awards: None
Publications: None
/i8
Serial No. imttt T-T.*^
1. Experimental Therapeutics Branch
2. Physiological Chemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Clinical Biochemistry of the Kallikrein-Kinin System
Previous Serial Number: NHLI-209 and NHLI-215
Principal Investigator: John J. Pisano, Ph.D.
Other Investigators: Toshio Imanari, Ph.D.
Tokio Kaizu, Ph.D.
Jack Pierce, Ph.D.
Jorge Guimaraes, Ph.D.
Ron Thompson, Ph.D.
Harry Keiser, M.D.
Roy Aaron, M.D.
Robert E. Wood, M.D.
Harold Newball, M.D.
Cooperating Units: Metabolic Diseases Branch, NIAMDD
Surgery Branch, NCI
Pediatric Metabolism Branch, NIAMDD
Pulmonary Branch, NHLI
Project Description:
Objectives: To characterize and develop procedures for the measurement of
components of the kallikrein-kinin system. To establish the role of the kalli-
krein-kinin system in health and disease.
Methods and Major Findings: 1) Human Urinary Kallikrein (HUK) . An inactive
form of kallikrein has been discovered in human urine which is activated by
high concentrations (1 yg/90 yl) of trypsin. Low (1 ng/90 yl) concentrations
of trypsin are inhibited. This kallikrein is also activated by prolonged
dialysis or by freezing and thawing. These data indicate that the inactive
form of kallikrein is a kallikrein- inhibitor complex. The inhibitor has a high
affinity for trypsin which is able to displace it from kallikrein. The inhibitor
may be the trypsin inhibitor previously reported in human urine. Its molecular
weight is estimated to be near 10,000 in as much as the kallikrein- inhibitor
complex has a molecular weight of 50,000 and the kallikrein contributes 40,000.
The kallikrein separated from inhibitor is indistinguishable from free kalli-
krein in (1) its chromatographic properties, (2) its spectrum of reactivity with
competitive and non-competitive inhibitors and (3) its inhibition by antibody
to free kallikrein.
ftf
Serial No. NHLI-73
A new assay for HUK has been developed which measures both free and inhibi-
tor-complexed kallikrein. The essential steps include; (1) pre- incubation with
trypsin to liberate complexed kallikrein, (2) addition of lima bean trypsin
inhibitor to inhibit residual trypsin, (3) a heating step to inactivate urokinase,
(4) the use of ^H-carbobenzyloxy-arginine methyl ester as substrate. Liberated
^H-methanol is determined by the methods described in the report NHLI-209 (1972) .
In 20 urine specimens examined, a mean of 30% (range: 0-f^5% of the kalli-
krein was excreted complexed to inhibitor.
2) Kinins in Urine. Recovery of kinins from urine is conveniently deter-
mined with the aid of 3h or l^C-bradykinin. Either an IRC-50 or CM-Sephadex
column eluted with 9 N acetic acid and 0.5 M ammonium acetate respectively may
be used for quantitative recovery of kinins from urine. Crucial steps in the
overall procedure include: (1) the collection of urine in acid which destroys
kininases in urine and prevents bacterial growth, (2) the use of plastic equip-
ment (e.g., columns, pipettes, counting vials); this gives consistently high
recoveries which were not achieved when glass equipment was employed. Using
this isolation procedure and the rat uterus bioassay from 2-79 yg kinin was
found in 24 hr collection from 7 subjects.
3) Human Salivary and Sweat Kallikrein. Human sweat kallikrein has been
partially purified by DEAE cellulose chromatography. Human salivary kallikrein
has been partially purified by acetone precipitation followed by DEAE-cellulose
chromatography. Both enzymes closely resemble human urinary kallikrein in their
spectrum of activity with various ester substrates, their inhibition by a number
of non-competitive inhibitors and in their inhibition by antibody to human
urinary kallikrein. An assay for sweat kallikrein has been developed which
employs a buffer containing gelatine and 0.15 M NaCl to stabilize the enzyme
which is present in extremely low concentrations. The principle of the radio-
chemical esterolytic assay is given in report NHLI-209 (1972) .
4) Plasma Prekallikrein. A new radiochemical esterolytic assay for human
plasma prekallikrein has been developed which is based on the principles out-
lined in Report NHLI-209 (1972) . The assay involves (1) a pretreatment of plasma
with acetone to destroy kallikrein inhibitors, (2) activation of prekallikrein
with kaolin, (3) inhibition of interfering esterases with lima bean trypsin
inhibitor, (4) the use of % tosyl-arginine methyl ester as substrate. The
esterase activity of plasma measured by this procedure is indistinguishable from
the activity of purified human plasma kallikrein in its inhibition by competative
and non-competative inhibitors, and temperature and pH optima.
Normal individuals contain 0.8 - 1.6 (mean 1.4) units of prekallikrein per
ml plasma. Plasmas from 20 patients with cystic fibrosis contain normal levels
of prekallikrein. These data contradict a report from another laboratory which
claims a significant reduction in about 50% of their patients.
Patients (15) undergoing surgery all showed a fall in plasma prekallikrein
which reached its lowest level in 1-4 days (15-75% fall, mean 50%) post surgery.
By the 5th day the levels were rising in all subjects.
lA)
Serial No. NHLI-73
The plasma of a patient with Fletcher-factor deficiency was found to
contain no prekallikrein in our assay. This confirms an earlier report that
Fletcher-factor is kallikrein.
3
5) Catabolism of Bradykinin by Human Lung and Plasma. H-phe-8-Bradykinin
administered to 2 subjects via a catheter into the pulmonary artery was over 80%
destroyed in one pass through the lung. At least 50% of the destruction was
due to kininase II which may be identical to the angiotensin converting enzymes.
Klninase II cleaves ^H-phe-arg from bradykinin.
An assay for kininases in plasma has been developed which takes advantage
of the facile separation of split products from intact kinin on a CM-Sephadex
column. Kinins are determined by incubating radioactive kinin with 30 yl plasma
for 15 min at 37", pH 7.4. The reaction is stopped with l,10,o-phenanthroline
and acetic acid.
6) Kininogen. Kininogen has been found in rat kidney perfused with saline
until free of blood. The amount found per kidney is equivalent to 1 yg brady-
kinin which is the amount of kininogen found in 0.25 ml blood.
The kidney may be a significant source of plasma kininogen as indicated by
the following experiment. The renal artery of rats depleted of kininogen by
administration of cellulose sulfate contained, 7 hours after treatment, 60% and
the renal vein 86% of the pretreatment levels. The hepatic values were artery
60% and vein 74%. Thus both tissues appear to be sources of plasma kininogen
but the kidney appears to be a richer source. The kinetics of release indicate
synthesis of kininogen in these tissues and not merely release of stores.
Significance to Biomedical Research and Institute Program; Kinins are the most
potent vasodilator substances known. They are generated in plasma and exocrine
glands by the action of kallikrein on an a-globulin, kininogen. Characterization
and quantitative measurement of these components of the kinin system is crucial
to an understanding of the role of this enzyme in health and disease.
Significant alterations in urinary kallikrein excretion have been noted in
patients with hypertension and in normal individuals on various levels of dietary
salt or mineralocorticoids. It would appear that the kallikrein-kinin system
plays an important role in the regulation of blood pressure and in renal function.
Proposed Course of Project: To continue the basic studies on the characterization
and quantitative assay of components of the kallikrein-kinin system. To establish
the role of the kallikrein-kinin in hypertension and in exocrine glands,
especially kidney and lung. To identify the coupling of kallikrein-kinin system
to other vasoactive systems including the renin-angiotensin system, mineralo-
corticoids and prostaglandins.
Honors and Awards: None
Publications: None
/d-l
Serial No. NHLI-74
1. Experimental Therapeutics Branch
2. Physiological Chemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Peptide Biochemistry
Previous Serial Number: NHLI-208, 210, 212, 216, 217
Principal Investigator: John J. Pisano, Ph.D.
Other Investigators: Hisanobu Yoshida, Ph.D.
Cooperating Units: None
Project Description:
Objectives: To develop appropriate methodology for the characterization and
assay of peptides. To discover new biologically active peptides.
Methods: The following techniques are employed: gas chromatography, high
pressure liquid chromatography, ion-exchange chromatography, droplet coimter-
current chromatography.
Major Findings: High Pressure Liquid Chromatography. Several column packings
have been evaluated for the separation of PTH-amino acids. Of the two which
seem most promising Corasil-II and Corasil-C18, the latter has given the better
results. Thus it has been possible, for the first time, to elute all the PTH
derivatives using a single column. Resolution of some of the derivatives is
not entirely satisfactory. However, even at its present state of development
the method nicely complements the gas chromatographic procedures previously
developed in this laboratory.
Droplet Countercurrent Chromatography. In a continuation of the study of
factors which affect the efficiency of this separation technique it has been
found that the angle of the columns is important for two reasons: the angle
influences droplet size and rate of passage of the droplet along the length of
the column. It appears that high molecular weight substances (e.g., insulin
M.W. 6000) are particularly sensitive to the rate of droplet movement. Thus
when the column was placed at 30° relative to the bench top, the efficiency was
almost doubled that achieved at 60° . A new droplet instrument has been con-
structed which allows the choice of an infinite number of column angles. This
will allow control of droplet size and rate of movement and promises to provide
more versatility and efficiency.
Yellow Jacket Kinin. The new vasoactive peptide isolated from yellow jacket
venom and reported last year (NHLI-216) has been further characterized and
found to contain carbohydrate. The structure of this peptide is:
^^
Serial No. NHLI-74
sugar ^ sugar (2)
Thr-Ala-Thr-Thr-Arg-Arg-Arg-Gly-Bradykinln
Sugar 1: N-AcGalactosamine 1~2 Galactose 1
Sugar 2: N-Ac«Galactosamine 2^3 Galactose 2
It may be the first vasoactive glycopeptide discovered in nature.
Significance to Biomedical Research and Institute Program: Better methods for
the characterization and measurement of vasoactive peptides are needed in order
to gain a clearer understanding of their role in the regulation of blood pressure.
Countless naturally occurring biologically active peptides are awaiting discovery.
With new and improved analytical techniques the measurement of known peptides
and identification of new vasoactive peptides will be much easier.
Proposed Course of Project: To develop, perfect and apply new methods of analy-
sis to the assay of known peptides and the discovery of new vasoactive peptides.
Honors and Awards: None
Publications:
Tamura, Z., Nakajima, T., Nakayama, T., et al: Identification of Peptides
with l-Dimethylaminonaphthalene-5-Sulfonyl Chloride. Anal. Biochem. ,
in press.
Pisano, J.J., Bronzert, T.J., Peyton, M.P., et al: e-(Y-Glutamyl) Lysine
Crosslinks: Determination in Fibrin from Normal and Factor Xlll-def icient
Individuals. Ann. N.Y. Acad. Sci., 202:98-113 (1972).
Finlayson, J.S., Mosesson, M.W. , Bronzert, T.J., et al: Human Fibrinogen
Heterogeneities. II. Crosslinking Capacity of High Solubility Catabolic
Intermediates. J. Biol. Chem. , 247:5220-5222 (1972).
/^i
Serial No. NHLI-75
1. Experimental Therapeutics Branch
2. Physiological Chemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on the Enzymes Involved in the Activation of Human
Plasma Kallikrein: PF/dil and Hageman Factor
Previous Serial Number: NHLI-213
Principal Investigator: Marion E. Webster, Ph.D.
Other Investigators: Sachiko Oh-ishi, Ph.D.
Cooperating Units: None
Project Description:
Objectives: The kallikrein in human plasma is normally found as an inactive
precursor called prekallikrein. Studies of the mechanism by which this enzyme
is activated in human blood has clearly shown that Hageman factor (HF) is re-
quired for its activation. As active HF (Factor XII) initiates the intrinsic
clotting pathway, activates a plasminogen activator and promotes platelet aggre-
gation and aggregate stability, activation of this enzyme results in both the
formation and dissociation of clots as well as the generation of kinins. Further
studies were undertaken to better understand the role of this enzyme system in
a number of pathological conditions — to clarify the role played by factors
described in the early literature, to investigate the mechanism of action of
a number of inhibitors and to investigate the mechanism by which inactive
Hageman factor is converted to active Hageman factor by contact with negatively
charged particles.
Methods: Measurement of the components of this enzyme system by bioassay or by
a radiochemical method employing (■^H)-TAMe have been previously described
(NHLI-213).
Major Findings: Miles, Wllhelm and coworkers, a decade ago, described two
permeability factors, PF/Nat (permeability factor/native) and PF/dil (permea-
bility factor/dilute) in a number of mammalian plasma. PF/Nat was found in
lower dilutions of plasma whereas PF/dil was found in dilutions of plasma 1:100
or greater. PF/Nat was distinguished from PF/dil in that it was not inhibited
by soybean trypsin inhibitor and that the increased capillary permeability
induced by it in guinea pigs lasted for 1 hour as compared to 15-20 minutes for
PF/dil. PF/dil had many properties similar to plasma kallikrein. However, in
1962 Mason and Miles demonstrated that PF/dil formed kinins from native plasma,
but not from plasma heated to 60° suggesting that PF/dil formed kinins by acti-
vation of endogenous plasma kallikrein. With the demonstration that active
Hageman factor (HF) , or fragments derived therefrom, were capable of directly
activating prekallikrein, it appeared likely that PF/dil and active HF were
1 fS.^
Serial No. NHLI-75
identical substances. However, direct experimental proof was lacking, due in
large part to the relative insensitivity of the clotting and biological kinin-
generating assays. With the development of the radiochemical method for the
determination of active HF by measuring the amount of kallikreln formed from
purified prekallikrein, the activity of these dilutions of plasma could readily
be determined. When dilutions of plasma were stored in glass tubes, two types
of esterases were generated. The direct esterase activity of the dilutions, in
the absence of prekallikrein gave a pattern of activity similar to that for
PF/Nat and like PF/Nat this esterolytic activity was not inhibited by soybean
trypsin inhibitor. On the other hand, plasma at greater dilutions was capable
of activating prekallikrein and the activity of these various dilutions of
plasma gave a pattern of activity similar to that found for PF/dil.
Further evidence to support the hypothesis that active HF or its fragments
were present in these dilutions was obtained by electrophoretic analyses on
polyacrylamide gel. In general, three main peaks of activity could be recovered
from these gels showing a pattern similar to those previously found for prepara-
tions of active HF and its fragments. An initial peak of activity, which was
near or at the origin of the gel and which might represent activation of residual
inactive HF on the gel surface; a second peak of activity which was recovered
in the globulin region and a third peak of activity in the albumin region. When
the amount of activity recovered in the globulin region was compared to that
recovered in the albumin region the percent recovered varied with the dilution
of plasma, the greater the dilution of plasma the greater was the formation of
the 30,000 molecular weight fragment (third peak).
Inhibitors in plasma also influence the generation of active HF by dilution,
particularly the C'l esterase inhibitor. This technique, therefore, not only
provides a relatively simple method for the diagnosis of hereditary angioneurotic
edema (NHLI-213) , but also provides a method for extending our knowledge of
Inhibitors for these enzyme reactions. Spermine and cytochrome C are specific
inhibitors of the clotting activity of HF in that they do not affect other
components of the clotting sequence. Both of these inhibitors prevented the
generation of active HF without affecting the activity of HF once it had been
activated or of plasma kallikrein. Similarly hexadimethrine bromide affected
only the activation step. Two other inhibitors of HF, polylysine and methylene
blue, inhibited the active enzyme. The hexyl ester of trans-AMCHA and epsilon-
aminocaproic acid (EAC) , inhibitors of plasminogen activation or of plasmin, in
some normal plasma caused inhibition of the activation phase. EAC-hexyl ester
and trans-AMCHA were without effect. Lima bean trypsin inhibitor only weakly
inhibited active HF, while two other trypsin inhibitors from soybean and from
pancreas were such potent inhibitors of plasma kallikrein that their effect on
the activation step or active HF could not be determined.
As methods for the preparation of active HF from acetone activated plasma
by adsorption and elution from supercel (NHLI-213) had resulted in multiple
forms of the active enzyme, attempts were made to prepare inactive HF. Initially
a method was devised for the determination of HF by adsorption and activation
of a negatively charged surface. In this technique human plasma (5 yl) was
/a^
Serial No. NHLI-75
added to supercel; the supercel was washed free of proteins and added to a
source of prekallikrein; the kallikrein so generated was determined by the
radiochemical method described above. Zero order kinetics were not obtained
with this procedure, but it was sufficiencly quantitative so that it could be
employed as a method for detecting inactive HF during its purification. However,
little if any activity could be recovered from plasma fractionated by a number
of procedures. In addition, an apparent complete loss of HF occurred under
experimental conditions which should not have altered its content. For example, .
adsorption of plasma with aluminum hydroxide and then with a small amount of \
celite, a procedure developed by Ratnoff and Davie as their Initial step in the
purification of HF, appeared to be completely devoid of activity. It seemed
possible, therefore, that this plasma did not lack HF, but substance(s) required
for the activation of HF on a negatively charged surface.
Further investigation of this reaction showed that when HF-deficient plasma
was mixed with the adsorbed plasma prior to its addition to the supercel, acti-
vation of HF occurred, clearly demonstrating that HF-deficient plasma contained
a substance or substances necessary either for the adsorption or activation of
HF on supercel. Additional studies showed that both inactive HF and the
substance (s) required for its activation were firmly adsorbed to the supercel
and that these latter factor(s) did not alter the adsorption of HF to supercel
but were rather required for its activation.
Attempts have been made to purify these activator (s) of HF from normal
human plasma by adsorption through a number of affinity columns. Filtration i
through a lysine-sepharose column similar to that used by Deutsch and Mertz '
for the removal of plasminogen from plasma did not adsorb the activity, suggesting
that plasmin was not an activator of HF. However, when lysine was linked to
sepharose through a 12-carbon aliphatic chain, complete removal of activity
occurred. Similarly a spermine-sepharose column was effective in adsorbing the
activity, and additional studies showed that these latter two columns had ad-
sorbed both inactive HF and its activator (s) . The activity could be eluted
from these columns with 5 M guanidine. However, elution with a guanidine gradient
caused a partial loss of activator activity and it was noted that the remaining
activator activity eluted in the same fractions as the kallikrein activity and
that prekallikrein had been converted to kallikrein during the purification
procedure.
A direct test of the possibility that plasmin, plasminogen, kallikrein
or prekallikrein were one or more of the activators of HF found in normal plasma
showed that neither plasmin nor plasminogen could activage HF in aluminum hy-
droxide adsorbed plasma. Kallikrein, however, did generate activity when added
at twice the concentration found in normal plasma. Prekallikrein, on the other f
hand, failed to generate activity, although this same concentration of prekalli-
krein could readily correct the defect found in Fletcher (prekallikrein) deficient
plasma.
Activation of HF by a negatively charged surface has previously been
thought to be by adsorption of the protein molecule in such a manner that a
rearrangement occurs which results in the formation or unmasking of an active
catalytic site. The present data, however, would indicate that HF, itself, is |
not activated by a negatively charged surface such as supercel. Also, prekallikrein,
itself, is not capable of activating inactive HF although the active enzyme .^
3
Serial No. NHLI-75
kallikrein can readily do so. Active HF can activate prekallikrein and kalli-
krein can activate inactive HF, but how do these molecules become activated on
a negatively charged surface? It would appear likely that still another as yet
unknown activator is required. Which molecule is activated first? It is
possible that further studies on this mechanism might help explain the anomoly
that HF-deficient and Fletcher deficient plasma have prolonged clotting times
in vitro but these patients have no abnormal hemorrhagic symptoms. It appears
possible that this unknown activator may activate both molecules and that
activation of either molecule may be sufficient to control the bleeding tenden-
cies. This will be particularly likely, if kallikrein, like active HF, can
convert inactive PTA (Factor XI) to active PTA.
Significance to Biomedical Research and Institute Program; The generation of
kinins by plasma kallikrein has been implicated in a number of pathological
conditions such as hereditary angioneurotic edema, gouty and rheumatic arthritis,
asthma, pulmonary edema, reactions to blood transfusions, infection by patho-
genic organisms, pancreatitis, etc. A complete understanding of this system
which is involved in both the formation and dissolution of clots as well as in
the generation of kinins, could lead to the development of specific inhibitors
which might be therapeutically useful.
Proposed Course of Project; Continued investigation of the factors necessary
for the activation of Hageman factor.
Honors and Awards; None
Publications :
Zweig, M.H., Maling, H.M. and Webster, M.E.; Inhibition of sodium urate
induced rat hindpaw edema by colchicine derivatives: correlation with
antimitotic activity. J_. Pharmacol. Exp. Therap . , 182:344-350, 1972.
Webster, M.E., Maling, H.M., Zweig, M.H. , Williams, M.A. and Anderson, W. ,
Jr.: Urate crystal induced inflammation in the rat; evidence for the
combined actions of kinins, histamine and components of complement.
Immunol. Comm., 1:185-198, 1972.
Webster, M.E., Maling, H.M., and Zweig, M.H. : Evidence for kinins, his-
tamine and complement as mediators of the inflammatory response. Protides
of the Biological Fluids. 20 (in press) .
Webster, M.E.; Section 3.1, Bioassay; Chapter 21, Kinins. In Berson,
S.A. and Yalow, R.A. (Eds.) Methods in Investigative and Diagnostic
Endocrinology , (Ln press ) .
/A7
Serial No. NHLI-76(c)
1. Experimental Therapeutics Branch
2. Physiological Chemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Antigen-Antibody- Induced Release of Mediators from Passively
Sensitized Human Lung: Studies on Histamine and Arginine
Esterases
Previous Serial Number: NHLI-187(c)
Principal Investigator: Marion E. Webster, Ph.D.
Other Investigators: Harold H. Newball, M.D.
Sachiko Oh-ishi, Ph.D.
Floyd L. Atkins, M.D.
Michael A. Beaven, Ph.D.
Cooperating Units: Pulmonary Branch
National Heart and Lung Institute
Project Description:
Objectives: Previous studies (NHLI-187(c) , 1971-1972) on the release of his-
tamine from leukocytes of normal and allergic individuals had shown that hista-
mine was released only from the leukocytes of sensitized individuals. However,
as a model of the pathophysiology of hay-fever and asthma, the passive sensi-
tization of human lung fragments as first described by Parish (Nature 215:738,
1967) appeared to provide a more direct approach to the investigation of the
role of mediators in this immediate-type allergy. The purpose of this study
is to identify mediators released from human lung during sensitization; to
determine the mechanism of their release; to identify inhibitors of their re-
lease and to assess the role of pharmacological compounds used in the treatment
of allergy.
Methods: Human lung obtained during surgery is cut into fragments (100-200 mg
wet weight), sensitized with sera obtained from individuals allergic to ragweed,
washed free of serum proteins and incubated with a purified preparation of antigen
E (0.6 ug) . Release of histamine from the sensitized lung was measured by a
sensitive enzymatic assay developed in this laboratory (Beaven et^ al. , Clin.
Chim. Acta 37:91, 1972). Enzyme (s) which hydrolyze N-substituted arginine esters
were determined by a modification of the radiochemical technique developed for
the determination of human urinary kallikrein using (3H)-TAMe as substrate
(Beaven et al. , Clin. Chim. Acta 32:67, 1971).
Major Findings: Initially, the conditions required for optimal release of his-
tamine were reinvestigated. In agreement with earlier investigators, greater
release of histamine occurred from lungs sensitized with diluted rather than
undiluted sera suggesting that undiluted sera contains proteins capable of
Serial No. NHLI-76(c)
competing with reagenic antibody for tissue binding sites. However, in our
hands, sensitization for 20 hours at room temperature resulted in greater re-
lease of histamine than incubation for 4 hours at 37". These studies have
confirmed the ant igen-ant ibody release of histamine from sensitized human lung
in vitro. In addition we have found that enzyme (s) which hydrolyze N-substi-
tuted arginine esters are also released. These arginine esterase (s) were
unstable in the diluting medium but could be stabilized by addition of albumin
(300 yg/ml) . Maximum histamine release occurs in 3-5 min and remains constant
for up to 60 min following addition of antigen. However, release of arginine
esterase activity preceded that of histamine with a peak activity at 1-5 min
followed by a second peak at 15 min. This lack of similarity in the time course
of release may indicate that the activities originate from different sites within
the tissue. In one experiment, disodium cromoglycate (100 yg/ml) inhibited the
release of arginine esterase but did not affect histamine release.
In addition the action of some mediators on isolated human bronchi has
been examined. Histamine (30-800 ng/ml) and acetylcholine (0.1-8 yg/ml) con-
tract human bronchi as does prostaglandin F2c( (5 yg/ml). However, bradykinin
in two out of three bronchi failed to contract the muscle even in doses up to
100 yg/ml. In one tissue, contractions were obtained initially with bradykinin
at 2-3 yg/ml. At a later period of time, this same tissue failed to respond
to bradykinin even when the concentration was raised to 100 yg/ml. On the
other hand, in all tissues bradykinin has suppressed the response of histamine
and acetylcholine. Quantitation of this effect in one tissue showed that brady-
kinin (10-100 ng/ml) could partially or completely suppress the contraction
given by 800 ng histamine or 8 yg acetylcholine. The antihistaminic, tripro-
lidine, blocked the response to histamine but did not prevent contraction by
acetylcholine.
Significance to Biomedical Research and Institute Program: The identification
of mediators released by human lung passively sensitized with reagenic antibody
should aid us in our knowledge of human allergies. A better understanding of
the mechanism of their release and methods for their blockade could lead to
more rational therapy.
Proposed Course of Project; Continued investigation of the mediators released
by human lung passively sensitized with reagenic antibody.
Honors and Awards : None
Publications: None
/Af
Serial No. NHLI-77
1. Experimental Therapeutics Branch
2. Physiological Chemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Biochemistry of the Kallikrein-Kininogen-Kinin System
Previous Serial Number: NHLI-214
Principal Investigator: Jack V. Pierce, Ph.D.
Other Investigators: Jorge A. Guimaraes, Ph.D.
Cooperating Units: None
Project Description:
Objectives: Purification of glandular kallikreins and components of the plasma
kinin system for characterization purposes and for production of specific anti-
serums. Preparation of purified specific antibodies for biochemical and chemi-
cal studies. Preparation of affinity adsorbents from purified antibodies and
antigens for purification and other purposes, such as devising specific bio-
chemical and radioimmunochemical assays. Applications of these purified proteins,
affinity adsorbents, and assay methods to studies of human disease states, such
as hypertension.
Major Findings: 1) Human Plasma Kallikrein and Prekallikrein. Steps (a)
through (c) of the prekallikrein purification scheme described in the previous
report (Serial No. NHLI-214) are the same. The next step (d) , affinity chroma-
tography on L-arginine-Ci2-agarose columns, has been refined. First, an optimum
flow rate duTing the adsorption step (at room temperature), was found to be
about 80 ml per cm2 cross-sectional area of the column per hour. Second, one
ml bed volume of affinity adsorbent was found capable of retaining all of the
prekallikrein present in about 60 ml of the DEAE-cellulose filtrate (1.0 ml =
0.5 ml of original plasma). However, best results were obtained when no more
than about 40 ml of filtrate per ml of bed volume was used. Third, better
recoveries were realized by using 0.01 M Tris-HCl-O. 01% Polybrene, pH 7.0, in-
stead of 0.15 M NaCl-0.01 M Tris-HCl (or sodium phosphate)-0.01% Polybrene,
pH 8.0-8.2, as previously used. Fourth, elution of the column is now performed
by the following sequence of eluents at room temperature: (i) 0.01 M Tris'HCl-
0.01% Polybrene, pH 7.0 (10 vol); (ii) 0.01 M sodium acetate, pH 5.5 (6 vol);
(iii) 0.01 M sodium phosphate, pH 7.5 (0.5 vol); (iv) distilled water (0.5 vol);
(v) 0.01 M acetic acid (0.5 vol); and (vi) 0.01 M sodium phosphate, pH 7.5.
Eluent (ii) removes impurities, eluent (iii) is used to bring the column to
no
Serial No. NHT,T-77
pH 7.5; eluents (iv) and (v) remove the prekallikrein activity; and eluent (vi)
is used to prevent a too low pH in the fraction containing the eluted activity.
A purification of 25 to 30-fold in 70 to 80% yield has been realized by this
elution sequence on 0.54 x 7.0-cm trial columns.
Sheep antiserum to the IgG fraction from the affinity chromatogram of the
DEAE-cellulose filtrate (as described in the previous report. Serial No. NHLI-
214), contained mainly antibody to IgG, but also a low titer of precipitating
antibody to human plasma kallikrein — as shown by Ouchterlony immunodiffusion
tests with either inactive kallikrein or active kallikrein which had been
highly purified by affinity chromatography on a soybean trypsin inhibitor-
agarose column. Both active and inactive forms of plasma kallikrein had been
freed of IgG by means of anti-human IgG-agarose affinity chromatography. There-
fore, the sheep was boosted with the inactive human plasma kallikrein prepara-
tion just mentioned. The antiserum taken 11 days later now had a very low
anti-IgG titer and a fairly high titer against both the immunogen and the highly
purified human plasma kallikrein preparation. The trace amounts of anti-IgG
could be readily adsorbed out, leaving only the antibody to human plasma kalli-
krein in the filtrate, by passing the antiserum over a column of human IgG-
agarose. Immunization of a second sheep against the highly purified active
human plasma kallikrein has also given a good anti-plasma kallikrein titer.
The isolation of specific antibody to human plasma kallikrein for affinity
chromatography and other uses presents a problem, since only small amounts of
highly purified human plasma kallikrein or prekallikrein are available for making
affinity adsorbents. The strategy of preparing pure antibody from an immune
precipitate formed by mixing whole antiserum with crude antigen (as has been
done successfully in the case of antibodies to human plasma kininogen and rat
urinary kallikrein), cannot be used here because the gel filtration elution
volumes of IgG and human plasma kallikrein are nearly identical and because this
kallikrein, unlike urinary kallikrein and plasma kininogens, will not adsorb to
hydroxyapatite from an 8 M urea solution of the immune precipitate, since plasma
kallikrein is highly basic. Therefore, goat anti-sheep immunoglobulin, using
purified sheep anti-human IgG as the immunogen, has been prepared.
2) Himian Plasma Kininogens. Hydroxyapatite chromatography (linear phosphate
gradient in the presence of 1 M NaCl) of the LMW kininogens from the gel fil-
tration experiment described in the previous annual report (Serial No. NHLI-214)
gave two peaks of kininogen activity in about the same ratio and at the same
phosphate eluent concentrations as previously observed for LMW kininogens iso-
lated by an entirely different scheme and from outdated, instead of fresh plasma.
This provides further evidence that LMW kininogens I and II are present as such
in fresh human blood — i.e., that LMW kininogen I is not an artifact, as several
workers have suggested. The same hydroxyapatite chromatographic method applied
to HMW kininogens (from the gel filtration experiment) gave only a small peak
followed by a long tail of activity.
Significance to Biomedical Research and Institute Program: The purification of
the components of the plasma kallikrein system is crucial to investigations of
its physiological fundtion(s). This system is activated simultaneously with
the blood coagulation and lysis system: Factor XII (Hageman Factor) of the
latter appears to be an integral part of the former. There are a number of
Serial No. NHLI-77
observations implicating the kallikrein system in inflammation, pain, immune
reactions, the carcinoid syndrome, arthritides of various etiologies, and
hereditary angioedema.
Proposed Course of Project: Human Plasma Kallikrein. We plan to isolate goat
antibody to sheep immunoglobulin on columns of sheep anti-human serum albumin
and insolubilize it on agarose. This adsorbent will then be used to adsorb
sheep immunoglobulins from the anti-human plasma kallikrein sheep serums. These
purified immunoglobulins (containing anti-human plasma kallikrein), after co-
valent attachment to agarose, can be used to remove human plasma kallikrein and
prekallikrein from crude mixtures, such as plasma. We are presently trying to
obtain highly purified human plasma prekallikrein from 3 liters of human plasma.
Among other uses, the prekallikrein will be employed to prepare sheep antiserum,
since such an antiserum may contain a class of antibodies which will bind only
to prekallikrein.
Human Plasma Kininogens. More of the purified LMW and HMW kininogens will
be prepared by the scheme described earlier (previous annual report Serial No.
NHLI-214) . Hydroxyapatite chromatography of the HMW kininogens in the presence
of 3 M NaCl will be tried, as well as electrofocusing of both LMW and HMW forms.
Human Urinary Kallikrein. Preparation of purified antibody and antigen
from an immune precipitate (formed by mixing at equivalence antiserum to human
urinary kallikrein B5 and 300 liters of ultraf iltered human urine)will be per-
formed, probably by gel filtration in 6 M guanidine hydrochloride.
Honors and Awards : None
Publications:
Loeffler, L.J. , and Pierce, J.V.: Acyl azide derivatives in affinity
chromatography. Immobilization of enzymatically active trypsin on beaded
agarose and porous glass. Biochim. Biophys. Acta, in press.
/3i
Serial No. NHT,T-7«
1. Experimental Therapeutics Branch
2. Physiological Chemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Role of Prostaglandins in the Vascular System
Previous Serial Number: None
Principal Investigator: Lauren M. Cagen, Ph.D.
Other Investigators: John J. Pisano, Ph.D.
R. Wayne Alexander, M.D., Ph.D.
Cooperating Units: None
Project Description:
Objectives; 1) To develop sensitive assays for prostaglandins (PC's) and their
metabolites in plasma and urine and to measure the levels of these compounds
in normal and pathological states.
2) To study individual enzymes of PC metabolism with respect to their
modulation by physiological and pharmacological effectors.
Major Findings: Considerable controversy now centers on whether prostaglandins
of the A series are of natural origin and on the possible role of these com-
pounds as circulating hypotensive agents. Evidence against such a role for the
PGA's is the extremely low level of PGA's found in plasma by Dr. Alexander of
this laboratory.
1) Enzjnnic dehydration of PGEj^ to PGA-j^ could not be determined in tissue
homogenates from a variety of sources. However, homogenates of rat kidney were
found to convert small amounts of PGE^ to a less polar metabolite which chromato-
graphs with PGA^.
2) The existence of a specific PGA isomerase has been held as evidence for
the occurrence of these compounds in vivo. The absence of this activity in
human plasma was confirmed. However, human plasma was found to contain a heat-
labile factor which greatly stimulates the isomerase activity of rabbit plasma.
The nature of this factor and the mechanism of this stimulation are being
explored.
3) A report that PGA is converted to more polar metabolites by human blood
cells has been confirmed. The effect of this conversion as measurement of
apparent plasma concentration of PGA, and the products of the conversion are
being studied.
4) HPLC has been applied to the separation of PGA from PGB and of PGAj^ to
PGA2 to permit their independent measurement by radioimmunoassay. PGBj^ has been
only partially separated from PGB2. /33
Serial No. NHLI-78
Significance to Biomedical Research and Institute Program: Since prostaglandins
of the A and E series are among the most potent of vasoactive substances, it Is
of great Importance that the circulating levels as well as the origin and fate
of these substances be determined.
Proposed Course of Project: 1) In addition to continuing the studies mentioned
above, the applicability of existing assays for prostaglandin synthetase to
vascular smooth muscle is being explored. In collaboration with Dr. R. Wayne
Alexander of this laboratory, the existence of this enzyme system in vascular
smooth muscle and its regulation by antl-lnf lamatory drugs will be investigated.
2) Work is now in progress to purify and identify the polar metabolites
of PGA, produced by human erythrocytes. These compounds will be tested for
pharmacological activity and methods for their assay in plasma and urine will
be developed In order to assess the possible Importance of PGA derivatives as
circulatory hormones. Concurrently, whole blood and urine will be assayed
for the metabolites. Their presence in these fluids could have a profound
effect on our understanding of the physiological role of PGA.
3) The factor or factors in human plasma responsible for the stimulation
of rabbit plasma Isomerase will be characterized. It is of particular Interest
whether such factors are components of a human prostaglandin A Isomerase.
Honors and Awards: None
Publications: None
f3'/
Serial No. NHLI-79
1. Experimental Therapeutics Branch
2. Section on Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on the Isolation and Characterization of
Clostridial Electron Transfer Proteins and Other
Iron-sulfur Proteins
Previous Serial Number: NHLI-205
Principal Investigator: Walter Lovenberg, Ph.D.
Other Investigators: William D. Phillips, Ph.D.
Kerry T. Yasunobu, Ph.D.
Cooperating Units: Central Research Department, E. I. Dupont de Nemours
and Co., Wilmington, Delaware; Department of Biochemistry
and Biophysics, University of Hawaii, Honolulu
Project Description:
Objectives: It is becoming increasingly apparent that proteins containing
iron-sulfur complexes are ubiquitous and perform a wide range of biological
functions. These complexes are now known to rival heme groups as agents for
electron transfer. Previous work on this project has characterized the
active center of the small bacterial ferredoxin, and led to the discovery and
characterization of rubredoxin from Clostridium pasteurianum. This protein
has been particularly ammenable to structured analysis. The following
structural parameters have been identified in previous reports: the complete
amino acid sequence, the 3-dimensional structure to 2.0 A by x-ray diffraction,
the nature and bond angles of iron ligands, and the electronic properties and
configuration of the iron atom.
The objectives of the work during the past year have been to: 1) obtain
antibodies to C^ pasteurianum rubredoxin and establish the immunological
properties and cross reactivity of rubredoxin from various organisms,
2) further explore the chelate structure of clostridial ferredoxin using
high resolution nuclear magnetic resonance,
3) obtain information on the role of the polypeptide in modification
of the redox properties of the iron-sulfur complex.
Methods: Previously reported methods were used for the isolation of ferredoxin
and rubredoxin from C_^ pasteurianum. Antibodies to C. pasteurianum rubredoxin
were prepared by repeated intraimascular injection of rubredoxin together with
1 /3f
Serial No. NHLI-79
Freund's adjuvant into goats. A high titer of antirubredoxin (5 mg/ml) was
obtained and was isolated from servim by specific adsorption to rubredoxin-
Sepharose. Subsequently, antirubredoxin-Sepharose columns were prepared in
order to test the affinities of various bacterial rubredoxins.
NMR studies on Cj_ pasteurianum f erredoxin were done in DoO using a
high resolution instrument and preparations of extremely pure f erredoxin.
Major Findings: Antibodies to Cj_ pasteurianum rubredoxin were obtained.
These antibodies formed single precipitin lines on ouchterlony immunodiffusion
plates only with rubredoxin and aporubredoxin from C^ pasteurianum. Rubredoxin
from other bacterial species did not form precipitates with antirubredoxin,
but all were adsorbed on antirubredoxin-Sepharose columns. Relative affinity
of the antibody for the various rubredoxins was determined by its ability
to inhibit the biological activity of the NADP-cytochrome C reductase system.
In studies on C^ pasteurianum f erredoxin using the NMR technique pre-
viously reported, we have been able to study the denaturation-renaturation
process by focusing on the 3 protons of the cysteinyl residue. These are
displaced substantially downfield by contact shifts due to their proximity to
the antiferromagnetically spin exchange coupled iron atoms. Upon addition of
dg DMSOa these contact shift proton resonances begin to broaden and decrease
in intensity. Readdition of D2O at least partially restores these resonances.
This provides an excellent example of how NMR can be used to examine preliminary
changes in protein conformation before permanent denaturation takes place.
Significance: Iron-sulfur proteins constitute perhaps the most important
group of redox enzymes and electron carriers known in biological systems.
The current work has continued to advance our knowledge of the structure and
biological role of these proteins. Based on the current work, as well as that
in previous annual reports and the current literature, we can now make some
important conclusions about the active sites of iron-sulfur proteins. It
appears that there may be only 3 basic types of active sites and that these
can occur one or more times within the total structure of the proteins. Each
active site can accept or donate only a single electron. The proposed
structures are as follows:
1) Rubredoxin - A single high spin iron atom tetrahedrally
coordinated by four cysteinyl sulfhydryl groups.
2) Plant type ferredoxin - Two high spin iron atoms that
are antiferromagnetically coupled, each being surrounded
tetrahedrally by four sulfur atoms. Two inorganic sulfides
provide bridge atoms and 4 cysteinyl sulfurs provide the
other ligands.
3) Clostrial type ferredoxin - Four high spin iron atoms are
arranged at opposing corners of a cube with the remaining
comers occupied by inorganic sulfides. Each iron atom
is also bonded to a cysteinyl sulfur atom. These 4 iron
2 /3^
Serial No. NHLI-79
atoms therefore are each also in an approximate tetrahedral
ligand field of four sulfur atoms.
If this somewhat simplified concept of iron-sulfur centers withstands the test
of time and further experimentation, then our understanding of the more com-
plex iron-sulfur proteins should be greatly enhanced.
Proposed Course of Project: Continuation of the current line of experimenta-
tion is planned with emphasis both on structural details and the redox
properties of the iron-sulfur centers.
Honors and Awards: None
Publications :
1. Eaton, W. A. and Lovenberg, W. : In Lovenberg, W. (ed.):
Iron-Sulfur Proteins. New York, Academic Press, 1973.
2. Lovenberg, W. : In Neilands, J.B. (ed.): Microbial Iron
Metabolism. New York, Academic Press, 1973.
3. Yasunobu, K. T. and Lovenberg, W. : Immunological properties
of the Clostridium pasteurianum rubredoxin. Arch. Biochem.
Biophys . , in press.
4. McDonald, C. C, Phillips, W. D. , Lovenberg, W. , and Holm,
R. H. : PMR studies on Clostridium pasteurianum ferredoxin:
Origins of contact-shifted resonances and denaturation by
dimethylsulf oxide . Proc. N.Y. Acad. Sci. , in press.
/S7
Serial No. NHTJ-80(c)
1. Experimental Therapeutics Branch
2. Section on Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effect of Renal Infarction on Aromatic Amino Acid Decarboxyl-
ase Levels in Serum and Kidney Tissue
Previous Serial Number: None
Principal Investigator: James S. Goodwin, M.D.
Other Investigators: Perry V. Halushka, M.D. , Ph.D.
Hirohiko Yamabe, M.D.
Walter Lovenberg, Ph.D.
Cooperating Units: None
Project Description:
Objectives: Aromatic L-amino acid decarboxylase catalyzes the decarboxyla-
tion of a variety of aromatic amino acids. The most significant reactions
catalyzed by this enzyme are the synthesis of dopamine and serotonin from the
corresponding amino acids in neuronal tissue.
Very high concentrations of this enzyme are also found in mammalian kidney,
though its exact location and function there are not known. There is no
enzyme activity in normal rat or human serum. The objectives of the present
investigation are:
1) To see if aromatic L-amino acid decarboxylase "leaks" into the serum
and is measurable during various types of physical insult to the kidney.
2) To localize tissue distribution of aromatic L-amino acid decarboxylase
within the kidney by causing selective destruction of tissue.
Methods: Serum was obtained from a patient undergoing acute rejection of a
transplanted kidney, and also from a patient with methacillin related inter-
stitial nephritis.
For the animal studies Sprague-Dawley rats had one of the following
surgical procedures:
1) sham operation
2) denervation of left kidney
3) ligation of left ureter
^) ligation of left upper pole artery
1
/-fS
Serial No. NHLI-80(c)
Rats were sacrificed three to five days later and aromatic L-amino acid
decarboxylase levels of sermn and both kidneys were measured.
Major Findings; 1) The first two patients described above like all normal
individuals had no measurable aromatic L-amino acid decarboxylase activity in
their sera.
2) Denervation of a rat kidney resulted in 20% decrease in enzyme activity.
Ligation of the ureter resulted in a 50 to 75% decrease in tissue activity 5
days after ligation with the appearance of small but measurable amounts of
enzyme activity in the rat serum.
Ligation of a branch of the renal artery did not result in the appearance
of enzjnne activity in the serum, although the total renal enzyme was decreased
by about 30% in the infarcted kidney. This value appeared to be related to
the degree of infarction.
Significance; On the basis of measurements in two patients, it does not appear
that kidney rejection or nephritis cause the appearance of aromatic L-amino
acid decarboxylase activity in human servim.
Since denervation gives only a 20% reduction in enzyme activity, it is
apparent that most of the dopa decarboxylase in the kidney is not associated
with nerve endings. Ligation of the ureters, a procedure that leads to the
preferential destruction of tubules before parenchymal tissue is greatly
affected, resvilted in marked decrease in activity. Also, the appearance in
rat serum of the enzyme with ureteral ligation leaves open the possibility of
a parallel finding in a human disease for which this enzyme could be a marker.
Proposed Course of Project; More samples of serum from patients with various
renal diseases are being collected. Animal experimentation using toxins that
are specific tubular poisons, such as cadmium, is planned. Also, various
techniques for isolating "tubular fractions" from kidney homogenates are being
studied.
Honors and Awards; None
Publications:
1. Yamabe, H. and Lovenberg, W. ; Decarboxylation of 3,4-dihydroxy-
phenylalanine by oxyhemoglobin. Biochem. Biophys. Res. Commun.
47: 733-739, 1972.
fli
Serial No. NHLI-81
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Characterization of Bovine Adrenal Dopamine 3-Hydroxylase
Principal Investigator: Ellen F. Wallace, Ph.D.
Other Investigators: Walter Lovenberg, Ph.D.
Mark Kranz
Cooperating Units: Department of Biology
Johns Hopkins University
Project Description:
Objectives: The objective of this project was to study the chemical and
physical properties of a pure preparation of bovine adrenal dopamine B
hydroxylase (DBH) .
Methods: DBH was isolated by a previously published procedure and was assayed
using tyramine as substrate. Following octopamine formation from tyramin,
p-hydroxybenzaldehyde was liberated by periodate and measured spectrophoto-
metrically.
Major Findings: DBH has been shown to be a very large (290,000 MW) protein
that is partly released from chromaffin granules along with catecholamines.
The remainder of the DBH is membrane-bound. It was therefore of interest to
investigate whether the enzyme is composed of smaller subunits that might
include bound carbohydrate.
An electrophoretically homogenous preparation of DBH was subjected to
disc gel electrophoresis in the presence of sodium dodecyl sulfate and mer-
captoethanol. One band that migrated with a mobility corresponding to a
molecular weight of approximately 75,000 was observed. The enzyme apparently
is composed of four identical or closely related subunits.
A positive stain for carbohydrate was obtained for both the tetramer and
the monomer bands on the acrylamide gels. A preliminary carbohydrate analysis
showed the DBH has a carbohydrate content of about 3%. The principal sugar
residues are mannose , galactose, fucose, hexose amines, and sialic acid.
The biquinoline assay for copper was performed on the purified enzyme
preparation. Copper was present in the ratio of four moles of copper per
mole of tetrame.
/4o
Serial No. NHLI-81
Significance to Biomedical Research and Institute Program; The finding that
DBH is a tetramerie glycoprotein allows for a further investigation into the
nature of binding of DBH to membrane and control of enzyme activity. It is
possible that the carbohydrate moiety is the site of DBH binding to the mem-
brane and that reversible subunit association might be involved in regulation
of activity or release from granules.
Proposed Course of Pro.iect; Experiments are planned to characterize the sub-
units of DBH. It will be of interest to determine whether the monomers are
identical and if they are enzymically active. The role of the sugar moieties
in enzyme activity will also be studied. Attempts will be made to obtain
information about the site of binding for copper.
Honors and Awards: None
Publications : None
/f^/
Serial No. NHLI-82
1. Experimental Therapeutics Branch ^
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973 J
Project Title: The Regulation of the Hydroxyindole Pathway of Tryptophan j
Metabolism |
Previous Serial Number: None
Principal Investigator: Walter M. Lovenberg, Ph.D.
Other Investigators: Joseph A. Fontana, Ph.D.
Dante Picciano, Ph.D.
James S. Goodwin, M.D.
Cooperating Units: Molecular Disease Branch, NHLI
Project Description:
Objectives: The hydroxyindole pathway of the pineal gland offers a unique
opportunity to examine mammalian enzyme regulation. The synthesis of melatonin ,
is controlled by the levels of serotonin N-acetyltransferase (NAT) which in
turn is under neuronal control. Sympathetic stimulation via the superior
cervical ganglia or the administration of a 6 receptor agonist can cause a
50 to 100-fold increase in NAT activity. Work in other laboratories has
identified cyclic AMP as the second messenger in this system. The objective
of this project was to define at a molecular level the mechanism by which cAMP
is eliciting the large increase in NAT activity.
Methods: Protein kinases were isolated by ammonium sulfate fractionation, gel
filtration and DEAE cellulose chromatography. Calf thymus chromatin was iso-
lated by standard techniques. Protein kinase activity was determined by the
incorporation of 32p into protein using y-^^T-kTV and millipore filtration.
Binding of cAMP, and actinomycin D were measured by similar techniques.
Template capacity was measured using E.coli polymerase and ^h-UTP. Serotonin
N-acetyltransferase was measured by radio-techniques described in an accompany-
ing report.
Major Findings: Both rat and bovine pineal glands have substantial amounts of I
a cAMP-dependent protein kinase. This cAMP- dependent protein kinase was
purified to homogeneity and was found to consist of a catalytic and regulatory
(cAMP binding) subunit. Chromatography on G-200 in the presence of cAMP
permitted isolation of each subunit. The catalytic subunit (cAMP independent
protein kinase) could be converted to the cAMP dependent form by addition of
the regulatory subunit. It was then reasoned that if protein phosphorylation
was mediating the changes in NAT then at least one of three processes must
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Serial No. NHLI-82
occur: 1) stimulation of gene transcription, 2) increase in rate of trans-
lation of -RNA by ribosomal protein synthesis, or 3) a direct activation of
the apoenzjnne by phosphorylation. The first possibility was examined by
isolating calf thymus chromatin and testing it as a substrate for the protein
kinase. Chromatin was a good substrate and its phosphorylation was cAMP
dependent. The effect of phosphorylation on histone binding to chromatin was
evaluated by measuring the actinomycin D binding capacity and the template
capacity. Both actinomycin D binding and RNA synthesis rate were increased
about 20% following phosphorylation. The F^ and F3 histones were the major
classes phosphorylated. The effect of kinase and ATP was not cAMP dependent.
However, it is clear that phosphorylation can change the transcribing properties
of the chromatin.
The effect of kinase on ribosomal protein synthesis was next evaluated.
It was soon apparent that rabbit reticulocyte ribosomes contained a large
amount of endogenous protein kinase. This kinase could be removed by washing
the ribosomes with 0.5 M KCl. The kinase was purified from the wash fraction
and found to be similar to other histone kinases. Gradient elution of this
enzyme from DEAE cellulose columns indicated it was distinct from all of the
protein synthesis initiation factors that are removed by a 0.5 M KCl wash. The
role of kinase in ribosomal function was difficult to evaluate because of its
endogenous nature and the fact that high energy phosphate compounds are used
in protein synthesis experiments. Preliminary studies indicate that cAMP
stimulates globin synthesis (20 to 30%) using a crude rabbit reticulocyte
system.
Phosphorylation of crude pineal extracts consistently resulted in a
slight depression of NAT activity and in no case a stimulation.
SignificanE to Biomedical Research and Institute Program; It can be concluded
from these studies that cAMP may exert an effect on NAT by stimulating protein
kinase which in turn activates both transcription and translation. Activation
of a precursor protein by phosphorylation does not occur. Therefore, the
sequence of events by which neuronal impulses can be translated into changes
in enzyme can be hypothesized as follows:
Sympathetic Nerve -*■ Norepinephrine -> Pineal ->■ Adenyl
3 Receptor Cyclase
cAMP ^ Protein ^ Increased ^ Elevated
Kinase Transcription NAT
+
Translation
Proposed Course of Project; The following experimental approaches will be
undertaken:
1) Further examination of the products of chromatin phosphorylation,
particularly the acidic proteins.
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Serial No. NHLI-82
2) Definition of the role of the protein kinase in initiation and
polypeptide synthesis.
3) Direct measurement of incorporation of amino acid into NAT and in vivo
experiments with protein synthesis inhibitors.
Honors and Awards: None
Publications:
Fontana, J. A., Picciano, D., and Lovenberg, W. : The identification and
characterization of a cyclic AMP dependent protein kinase on rabbit
reticulocyte ribosomes. Biochem. Biophys. Res. Comm. 49:1225-1232, 1972.
Fontana, J. A. and Lovenberg, W.: Pineal protein kinase: effect of
enzymic phosphorylation on actinomycin D binding by, and template
activity of, chromatin. Proc. Nat. Acad. Sci., USA 70:755-758, 1973.
/«/f
Serial No. NHLI-83
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: In Vivo Protein Synthesis in Heart, Aorta, and Mesenteric
Artery of Normotenslve and Spontaneously Hypertensive Rats
(SHR)
Previous Serial Number: None
Principal Investigator: Hirohiko Yamabe, M.D.
Other Investigators: Walter M. Lovenberg, Ph.D.
Cooperating Units: None
Project Description:
Objectives: Previous studies reported that rats with spontaneous hypertension
had increased peripheral vascular resistance and by indirect measurements had
increased wall to lumen ratios in their blood vessels. The objective of this
study was to measure the rate of in vivo incorporation of ■'-^C lysine into
smooth muscle and connective tissue proteins in SHR and control rats at various
ages. Furthermore if changes occurred to determine whether these changes
preceded or followed the development of hypertension.
Methods: The incorporation of l^C lysine into proteins of the heart, thoracic
aorta, and the mesenteric artery was measured 2 hours following I.V. injection
of 10 ycuries of ^^C lysine. In heart, the actomyosin was specifically isolated
by standard techniques. In the vessels the non-collagen proteins were isolated
following extraction with hot 5% trichloroacetic acid. The rate of incorpora-
tion was estimated by determining the specific activity of the protein (dpm/mg)
and comparing it with the measured specific activity of the serum lysine.
Wistar-Kyoto rats (the parentstrain of SHR) were used as control animals.
Major Findings: The methodology described above provided easily measured
incorporation rates. At the age of 15 months the rate of incorporation of
lysine into cardiac actomyosin and vascular non-collagenous proteins was signi-
ficantly higher in the SHR as compared to Wistar-Kyoto rats. At 13 weeks of
age the hypertensive rats also exhibited higher incorporation rates in mesen-
teric artery. Incorporation studies are now under way using younger animals.
Significance to Biomedical Research and Institute Program: It is well-known
that significant cardiac hypertrophy occurs in older SHR. It is therefore not
surprising that we observed increased cardiac protein sjmthesis in 1-year old
SHR. The real significance of these somewhat preliminary results is that
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Serial No. NHLI-83
changes appear to occur in vascular protein synthesis relatively shortly after
the onset of increased blood pressure. Since these animals show elevations
in blood pressure by 6 weeks of age it will be important to determine incor-
poration rates in animals of this age or younger. It is hoped that these
studies will shed light on the often asked question of whether changes in
peripheral resistance precede or are the result of hypertension.
Proposed Course of Project: Studies are now in progress to determine the
lysine incorporation in cardiac and vascular proteins in a wider age range of
rats. It is planned that in some of these studies amino acid incorporation
into collagen and elastin will be examined specifically.
Honors and Awards: None
Publications: None
/^
Serial No. NHLI-84
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on 5,6- and 5,7-Dihydroxytryptamine — Agents That
Cause Degeneration of Serotonergic Neurons
Previous Serial Ntunber: None
Principal Investigator: Stephen J. Victor, M.D.
Other Investigators: Walter M. Lovenberg, Ph.D.
Hans G. Baumgarten, M.D.
Cooperating Units: None
Project Description:
Objectives; 5,6-Dihydroxytr3^tamine, a hydroxylated analogue of serotonin
(a putative CNS neurotransmitter) , has recently been shown to cause degeneration
of serotonergic neurons in the brain and spinal cord of rats . These morphologic
changes are accompanied by loss of weight, loss of temperature control, hyper-
activity, hyperresponsiveness to sensory stimulation, and bizarre social be-
havior with increased aggressiveness. Similarly, loss of serotonin in various
regions of rat brain as well as spinal cord has recently been demonstrated. The
objective of this project is further clarification of the action of hydroxylated
serotonin derivatives, as well as their specificity, metabolism and mechanism
of action. Accordingly, questions examined during the past year include: 1)
the effect of 5,6-dihydroxytryptamine on regional tryptophan hydroxylase (an
enzymatic marker for serotonergic neurons) in rat brain and spinal cord; 2) the
effect of 5, 7-dihydroxytryptamine, a new serotonin analogue, on tryptophan
hydroxylase; 3) the effect of 5,6- and 5, 7-hydroxytryptamine on blood pressure
of the normotensive rat.
Methods; Tryptophan hydroxylase activity was measured by a technique described
in an accompanying report. Wistar rats were treated by standard intraventricu-
lar injection of serotonin analogues dissolved in 20 yl of physiologic saline
containing 1-5 mg ascorbic acid.
Major Findings: 1) A severe and long-lasting depletion of tryptophan hydroxylase
occurs in mesencephalic tectum as well as spinal cord 9-60 days following intra-
ventricular injection of 75 micrograms of 5,6-dihydroxytryptamine. Significant
loss of tryptophan hydroxylase also takes place in mesencephalic tegmentum,
cortex + thalamus, pons-medulla and hypothalamus. These changes are accompanied
by weight loss as well as the above behavioral changes.
/f7
Serial No. NHLI-84
2) We have observed that when 5,7-dihydroxytryptamine is injected intra-
ventricular ly, the above behavioral changes take place, but without the
accompanying weight loss. The animals were shown to tolerate a much higher
dose of 5,7-dihydroxytryptamine than 5,6-dihydroxytryptamine, suggesting that
the former drug is associated with far less toxicity, thus allowing the dose
to be doubled. A highly pronounced and long-lasting depletion of tryptophan
hydroxylase takes place in all brain regions as well as spinal cord following
intraventricular injection of 150 micrograms of 5,7-dihydroxytryptamine.
Furthermore, when 75 micrograms of 5,7-DHT was injected (the same dose as that
given for 5,6-DHT), significant loss of tryptophan hydroxylase was seen in all
brain regions and spinal cord after 12 days. This shows that these two isomers
have different modes of action on the central nervous system, and suggests
that an almost total destruction of the central nervous system serotonin tracts
can be accomplished by chemical means.
3) No significant change in blood pressure was observed following intra-
ventricular injections of 75 micrograms of 5,6-dihydroxytryptamine or 150
micrograms of 5,7-dihydroxytryptamine in the normotensive rat, despite the
fact that elevated blood pressure have previously been observed following
treatment with parachlorphenylalanine, an agent known to inhibit tryptophan
hydroxylase.
Significance to Biomedical Research and Institute Program: 5 , 6- and 5 , 7-Dihy-
droxytrypt amine are important tools in the study of the central nervous system
from a blochemdcal, morphological and behavioral standpoint. Serotonin neurons
are believed to play a role in behavior and sleep, as well as blood pressure
and temperature regulation. This system has been implicated in certain patho-
logic states, such as schizophrenia, in the mechanism of action of certain
hallucinogenic drugs, and in morphine addiction. The manipulation of sero-
tonergic neurons by chemical means can thus contribute significantly to the
elucidation of the above problems.
Proposed Course of Project; Studies on the specificity of 5,6- and 5,7-DHT
are currently in progress utilizing changes in regional tyrosine hydroxylase
as an index of effect on catecholamine neurons. Studies of the metabolism
of these agents, using l^C-labelled 5,6-DHT and 5,7-DHT are planned. Also,
studies of the effect of 5,6-DHT and 5,7-DHT on the development of experi-
mental hypertension are planned, as well as studies on the mechanism of action
of these agents.
Honors and Awards: None
Publications:
Baumgarten, H.G., Victor, S.J., and Lovenberg, W.: Effect of intra-
ventricular injection of 5,7-dihydroxytryptamine on regional tryptophan
hydroxylase of rat brain. Journal of Neurochemistry, in press, 1973.
/¥i
Serial No. NHLI-85
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on Serotonin N-Acetyl-Transf erase in Mammalian
Pineal Glands
Previous Serial Number: None
Principal Investigator: James S. Goodwin, M.D.
Other Investigators: Walter M. Lovenberg, Ph.D.
Cooperating Units: None
Project Report:
Objectives; Serotonin N-acetyl transferase is the rate-limiting enzjone in the
conversion of serotonin to melatonin. At night there is a four to six-fold
increase in melatonin production in the rat pineal, and a 20 to 40 fold
Increase in N-acetyl transferase activity. This increase is mediated through
new protein synthesis. There is a very rapid fall in N.A.T. activity in vivo
when dark-adapted rats are exposed to light. This is parallelled in vitro
by the rapid loss in N.A.T. activity when crude pineal homogenate is incubated
at 27".
The objectives of this present investigation are to determine the molecular
basis for this rapid in vivo and in vitro deactivation of N.A.T. and also to
determine whether reactivation of the N.A.T. is possible and whether such a
reactivation plays a role in the in vivo regulation of melatonin synthesis.
Specifically, our goals are:
1) purification of N.A.T.
2) test the hypothesis that an acetyl-enzyme is the active form of N.A.T.
3) test the hypothesis that a sulf-hydryl group is involved in the active
site of N.A.T.
4) test hypothesis that N.A.T. is activated via phosphorylation by cAMP
dependent protein kinase.
Methods: 1) Purification. Beef or rat pineal homogenates were put through
standard purification steps, including ammonium sulfate fractionation, Sephadex
fractionation, and affinity binding to sepharose column which had tryptamine
(a substrate for N.A.T.) covalently bound.
1 /Vf
Serial No. NHLI-85
2) Because it was necessary to measure low amounts of enzyme rapidly, a
new method (based on previous concepts) was devised for the assay of N.A.T.
In this assay the enzyme was incubated at 37° in the presence of 10"3 M
pargyline, lO'S M acetyl CoA and 2 x 10"^ M, l^C serotonin (2.0 x 10^ dpm/ymole) .
Reactions were done with a total volume of 50 yl and stopped by the addition
of 0.5 ml water. The mixture was then passed through a 0.3 x 3 cm column of
permutit which was then washed with 1.0 ml H2O. The acetylated tryptamine
appeared in the affluent which was collected while the substrate was completely
adsorbed to the column. This assay showed the appropriate responses to the
amount of tissue and time of incubation.
3) The enzyme N.A.T. is relatively labile. Another method had to be
developed to quickly separate reagents used to treat the N.A.T. preparations
before the enzyme activity was assayed. Consequently, we used the technique
of centrifuging the enzyme prep through a bed of G-25 Sephadex. All small
molecular weight compounds were retained in the Sephadex bed while most proteins
including greater than 90% of N.A.T. activity, passed through the Sephadex
bed. This method was also used in the purification steps, with increasing
pore size of Sephadex being utilized in a step-wise fashion, enabling us to
isolate the N.A.T. activity from all proteins that either were retarded in
G-50 Sephadex or were not retarded by G-lOO Sephadex. This enabled us to
approximate the size of the N.A.T. molecule. The centrifugation step is fast
(15 min) and does not result in dilution of the N.A.T., as does regular column
fractionation.
A) Other workers have found that large doses of isoproteinol to rats will
mimick the nightly 40-100 fold increase in N.A.T. activity in the pineal.
Consequently we routinely injected rats with isoproterenol (5 mg/kg) S.Q. three
hours before sacrificing, enabling them to achieve greater enzyme activity.
Major Findings: As reported by others, the level of N.A.T. in the pineal gland
of beef or rat during the day is very low (15 pmole/mg/30 min) . In the rat the
level can be increased by several hours of darkness or 2 hour prior treatment
with isoproteranol (5 mg/kg) to levels as high as 1000 pmole/mg/30 min). Since
rat pineal provide limited amounts of tissue (1 mg/rat) we attempted to purify
and concentrate the low activity from beef pineals. About 90% of the activity
in a 30,000 xg supernatant fraction of beef pineal gland homogenates precipitated
between 45 and 60% saturation with ammonium sulfate. Further purification was
obtained by differential centrifugation using Sephadex G-50 and G-150. While
the specific activity of this preparation was considerably lower than that of
the activated rat pineal gland, this did provide a large quantity of relatively
active enzyme for studies on the properties and factors related to its stability.
This enzyme preparation had a half life of about 15 minutes when incubated
at 37°, but was stable for up to 2 weeks when stored in liquid nitrogen.
The potential role of sulf-hydryl groups of the enzyme was investigated
by incubation in the presence of thiols, and by the use of reagents that react
with free SH groups. Dithio-2-nitrobenzoic acid (Elman's reagent) was a good
/SO
Serial No. NHLI-85
inhibitor of N.A.T. and the inactivated enzyme could be reactivated by the
addition of dithiothreitol. The latter compound itself was stimulatory (20%)
to the enzyme preparation, thus it appears that free sulf-hydryl groups parti-
cipate in the catalytic site.
Other acetyl transferase systems (citrate lyase) have been shown to be
active only when the enzyme is acetylated, and a sulf-hydryl group has been
identified as the acetyl acceptor. In an attempt to demonstrate a similar
phenomenon with N.A.T. several experimental approaches have been used:
1) treatment of the enzyme with acetic anhydride (0.1 M) in the presence
or absence of a reducing agent resulted in no apparent activation or inhibiton
of enzyme activity.
2) using tritHted acetyl CoA to directly demonstrate an acetylated enzjme
resulted in no specific incorporation of the label into the protein fraction.
3) hydroxylamine which would react with -S-C-CH3 groups on the enzyme is
not inhibitory when it is removed from the enzyme solution prior to assay.
It can be tentatively concluded from these studies that an acetylated
enzyme is not formed during the transfer of the acetyl group from coenzyme A
to the indole ethylamine. Further studies will be required to confirm this
conclusion.
The question of whether the dramatic changes in N.A.T. activity in the
pineal gland are totally dependent on protein sjmthesis and destruction was
examined. Since this increase in N.A.T. activity of the gland is mediated by
cAMP and the gland contains high levels of a cAMP dependent protein kinase, we
examined the possibility that phosphorylation of the enzyme extracts might
result in activation or deactivation. Using glands taken from rats 30 mln
following the onset of lighting, the homogenates were preincubated with cAMP,
ATP and protein kinase. The resulting enzymes consistently showed about a 20%
decrease in activity over the control (cAMP) and probably not for deactivation.
Another approach to this problem was to measure the in vivo Incorporation
of l^C-leucine into proteins of the pineal gland. Under basal and stimulated
(isoproterenol 5 mg/kg) conditions. No gross changes in protein synthesis
were observed, although Incorporation was easily measured.
Significance to Biomedical Research and Institute Program: The regulation of
N.A.T. activity in the pineal is an exceptional model for mammalian enzyme
regulation. This activity can be rapidly induced some 50 fold over its basal
level. The rapid turnover of the enzyme and consequently its great lability
in vitro however, have made it extremely difficult to adequately characterize
this protein. The results of the study to date indicate that this is a sulf-
hydryl enzjnne with a molecular weight in the range of 40,000 to 60,000. No
conclusive evidence could be obtained for an acetyl-enzyme intermediate although
this is still a distinct possibility. It is clear however that if this protein
could be characterized and the mechanism by which it is activated determined,
o tS-/
Serial No. NHLI-85
then the whole sequence of events by which neuronal impulses modulate enzyme
activities would be understood.
Proposed Course of Project: Further work is being directed at purifying N.A.T.,
and working with more purified preparations on the role acetyl CoA and diothio-
threitol play in stabilizing N.A.T.
Honors and Awards: None
Publications: None
/^
Serial No. NHLI-86
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on Tr3T)tophan Hydroxylase
Previous Serial Number: None
Principal Investigator: Stephen J. Victor, M.D.
Other Investigators: Walter M. Lovenberg, Ph.D.
Cooperating Units: None
Project Description:
Objectives: Tryptophan hydroxylase is the rate-limiting enzyme in serotonin
synthesis. Study of this enz3mie is thus necessary for the understanding of
physiologic control of serotonin synthesis, as well as its possible alteration
by drugs and in disease states. The ultimate objective of this project is
the isolation and characterization of this important enzyme, which has been
difficult to study in the past because of its instability, and is difficult
to assay.
Methods: The present assay for tryptophan hydroxylase is a modification of one
described recently in the literature. Tissue is homogenized in 0.05 M Tris«HCl
and 0.002 M dithiothreitol and centrifuged at 40,000 xg for 20 minutes. A
portion of the supernatant fraction containing 1.5 to 2.0 mg protein is premar-
bated with 14 ymoles of Tris«HCl (pH 7.4), 0.14 umole ot-methyltetrahydropterin
(cof actor) and 0.5 ymole dithiothreitol for 10 min at 37°. Tryptophan is added
to a final concentration of 1 wM. and the mixture is incubated for 30 min. The
reaction is terminated by the addition of perchloric acid and the mixture centri-
fuged. Two hundred yl of the supernatant fraction is added to 1.1 ml of 3 N HCl
and the native hydroxyindole fluorescence is measured.
Major Findings: Significant activity of tryptophan hydroxylase had been found
in beef pineal and rat brainstem in the past, and partial purification of these
enzymes was accomplished. Because of the availability of this new, sensitive
fluorometric assay and new understanding of the optimal type and amount of
cofactor, much larger amounts of activity have been shown in rat brain. We
have reinvestigated the level and regional distribution of tryptophan hydroxy-
lase in the central nervous system of the rat.
/sy
Serial No. NHLI-86
Activity Activity
Brain Region 5-HTP Brain Region 5-HTP
nmole/nig/30 min nmole/mg/30 rain
Septum 0.73 Remaining 0.42
forebrain
Striatum 0.21 Mesencephalic 1.22
tectum
Pons-medulla 1.62 Mesencephalic 7.04
tegmentum
Hypothalamus 1.25 Spinal cord 0.79
Most of the activity in rat brain is found in mesencephalic tegmentum, the
location of the raphe nuclei which contain the cell bodies of most of the
serotonergic neurons in the CNS. Enzyme obtained from this region can be
partially purified, and is stable to freezing, thus enabling further detailed
studies. Porcine brain tryptophan hydroxylase was about 50% that of the rat,
but it had very similar regional distribution. Significant activity has also
been found in human carcinoid tumor. Enzyme obtained from this source has
also been partially purified, and is also stable to freezing.
Significance: Serotonin is a ubiquitous biogenic amine, having a variety of
physiologic and pharmacologic effects, such as vasodilatation. It is also
believed to be an important neurotransmitter in the central nervous system,
and its neurons may have a role in sleep, in aggressive behavior and in
temperature and blood pressure regulation, as well as in hallucinogenic drug
action, morphine addiction and certain disease states. Studies of the rate-
limiting enzyme in its synthesis is vital to the understanding of its
physiologic and pharmacologic action.
Proposed Course of Project: Experiments on the isolation of this enzyme are
continuing. Kinetic studies are planned. The ultimate goal of this project
is determination of the structure of this enzyme through the use of physico-
chemical methods.
Honors and Awards: None
Publications:
Lovenberg, W. : Serotonin synthesis in brain, in "Proceedings of the Inter-
national Congress of Pharmacology", S. Karger, Basel, Switzerland, in
press.
/^
Serial No. NHT,T-87
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Monoamine Metabolism and Blood Pressure of Spontaneously
Hypertensive Rats
Previous Serial Number: None
Principal Investigator: Hirohiko Yamabe, M.D.
Other Investigators: Walter M. Lovenberg, Ph.D.
Cooperating Units: None
Project Report:
Objectives: Rats with genetic hypertension provide a useful model for the
study of this disease. The objective of this project is to clarify the role
of catecholamines in the development and maintenance of the spontaneous hyper-
tension. Catecholamine metabolism in the spontaneously hjrpertensive rat (SHR)
has been studied in the brainstem, heart, spleen, kidney and adrenal glands.
Brainstem serotonin levels and tryptophan hydroxylase activities have also been
measured since there is evidence suggesting a relationship between central
serotonergic system and the blood pressure regulation.
Methods: Catecholamine biosynthesis has been investigated by measuring 1) cate-
cholamine content, by standard methods, 2) catecholamine synthesis rate by the
a-methyl tyrosine method, 3) rate of incorporation of radioactive precursors
into catecholamines and 4) activities of enzymes related to catecholamines
(tyrosine hydroxylase, aromatic L amino acid decarboxylase and dopamine 6-
hydroxylase (DBH) . Serotonin content and tryptophan hydroxylase activity have
also been measured in the brainstem. In some experiments animals of different
ages were used, i.e., 4-5 week-old, 9-13 week-old (early hypertensive stage)
and 9 month-old (advanced stage) . Age-matched normotensive Wistar/Kyoto rats
known as the best control rats for SHR, and in some experiments Wistar/NIH rats
were used as controls.
Major Findings: Catecholamine content, synthesis rate, incorporation rate of
labelled precursors and enzyme activities of tyrosine hydroxylase and aromatic
L-amino acid decarboxylase in 9-12 week old rats were reported the preceding
year.
Additional findings in the current year are as follows:
1) Brainstem: the NE content and sjmthesis rate in SHR of 4.5 weeks of
age and 9 months of age were not lower than in Wistar-Kyoto. DBH activity in
1 /5y
Serial No. NHLI-87
SHR of 10-13 weeks of age was similar to that in Wistar/Kyoto, but lower than
in W/NIH. Serotonin content (ng/g) was 20% higher in SHR of 12 weeks of age
than in Wistar/Kyoto but tryptophan hydroxylase activity was nearly identical.
2) Heart: the NE content and synthesis rate in SHR of 4.5 weeks of age
were higher than in Wistar/Kyoto, but those in SHR of 10-12 weeks of age and
9 months of age were lower than in Wistar/Kyoto.
3) Spleen: the NE content in SHR was always lower than in Wistar/Kyoto.
Although the NE synthesis rate in SHR was slightly higher at 4.5 weeks of age,
it was substantially reduced at 10-12 weeks and 9 months of age than in
Wistar/Kyoto.
4) Kidney: the incorporation of C-tyrosine (repeated i.p. injection,
50 yCi) into norepinephrine in adult W/NIH was substantially higher than either
Wistar/Kyoto or SHR.
5) Adrenal glands: DBH activity in SHR of 11-13 weeks of age were similar
to that in Wistar/Kyoto but far lower than in Wistar/NIH.
6) Serum: serum DBH activity in Wistar/NIH was highest among these three
strains but very low and below the sensitivity of our assay method in Wistar/
Kyoto and in SHR.
In general, catecholamine synthesis in peripheral organs (heart and spleen)
of 4.5 week-old SHR seems to be higher than in Wistar/Kyoto, however this
pattern was reversed in 9-13 weeks-old and 9 month-old SHR. In no case was
catecholamine metabolism in SHR markedly different than that in W/Kyoto. DBH
activities in brainstem, adrenal glands and serum of 9 to 13 week-old SHR were
lower than in Wistar/NIH but very similar to those in Wistar/Kyoto.
Significance to Biomedical Research and Institute Program; Using the best control
rats (Wistar/Kyoto), catecholamine metabolism in SHR of various ages was
examined. In the peripheral tissues such as the heart and the spleen, cate-
cholamine metabolism was lower in adult and older SHR, which may be a change
secondary to sustained hypertension, but it was higher in young (4.5 week-old)
SHR, which might be related to the development of hypertension. It should be
noted that at as early as 6 weeks of age, there is already a significant dif-
ference of blood pressure between SHR and Wistar/Kyoto. Catecholamine metabo-
lism in the brainstem of SHR was not lower than that of Wistar/Kyoto in 4.5
weeks of age, and no evidence of the relationship between reduced catecholamine
metabolism and the development of hypertension was found.
Proposed Course of Project: Further studies on catecholamine metabolism in
blood vessels will be investigated using animals of different ages.
Honors and Awards: None
/Si
Serial No. NHLI-87
Publications:
Yamabe, H., de Jong, W. , and Lovenberg, W.M. : Further studies on
catecholamine synthesis in the SHR: catecholamine synthesis in the
central nervous system. Eur. J. Pharm., 22:91-98, 1973.
^^7
Serial No. NHLI-88
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Strain Differences of Catecholamine Synthesizing Enzyme
Activity in the Rat
Previous Serial Number: None
Principal Investigator: Hirohiko Yamabe, M.D.
Other Investigators: Walter M. Lovenberg, Ph.D.
Carl T. Hanson, Ph.D.
Cooperating Units: Laboratory Aids Branch
Division of Research Services, NIH
Project Report:
Objectives: While we were investigating the relationship between catechola-
mine metabolism and the development of the high blood pressure in spontane-
ously hypertensive rats (SHR) , it was noticed that two control normotensive
Wistar strains, i.e., Wistar/NIH (W/N) and Wistar/Kyoto (W/K) showed some
difference in catecholamine metabolism of various organs. The purpose of this
work is to determine if there are strain differences in catecholamine metabo-
lism and to establish whether there is any relationship between blood pressure
and catecholamine metabolism.
Methods: Tyrosine hydroxylase, aromatic L-amino acid decarboxylase and dopamine
6 hydroxylase activity was measured by standard technique in the brainstem and
the adrenal gland of various rat strains. Rat strains used were ACI/N, ALB/N,
M520/N, M/N, PETH/N, RHA/N, SD/N, SHR/N, WKY/N (Wistar-Kyoto) and W/N. 5 to 8
week old male animals were used. All enzyme activities are given in mymole
product formed/mg soluble protein/hour.
Major Findings:
1) Brainstem
a) Tyrosine Hydroxylase - tyrosine hydroxylase activity in the brain
stem of these strains were not very different and showed 0.47 to 0.58 mymoles
Dopa formed/hr/mg water soluble protein.
b) Aromatic L-amino acid decarboxylase - these rat strains may be
divided into two groups according to their decarboxylase activities.
/S3
Serial No. NHLI-
(1) RHA/N, SHR/N and WKY/N - activities ranged from 38 to 45
mymoles dopa decarboxylated/hr/mg water soluble protein.
(2) ACl/N, AlB/N, M520/N, OM-N, SD/N and W/N - activities ranged
from 65 to 83, which were approximately twice as much as the activity of the
first group.
c) Dopamine B-hydroxylase - the activities ranged from 4.5 (WKY/N)
mymoles octopamine formed/hr/mg 0.1% triton X soluble protein to 7.0 (RHA/N).
The activities were not very different from each other, although the range
was a little wider than seen in tyrosine hydroxylase activity. While these
strains exhibited a wide spectrum of normal blood pressure there is no rela-
tionship either between blood pressure and any of these enzyme activities.
2) Adrenal glands
a) Tyrosine hydroxylase activity ranged from 3.8 to 9.0 in the
following increasing order: RHA/N, DM/N, M520/N, SHR/N, ACl/N, AL3/N, WKY/N
and ODS/N.
b) Aromatic L-amino acid decarboxylase activity ranged from 121 to
183, in the following increasing order: OM/N, SHR/N, RHA/N, W/N, PETH/N,
M520/N, NCl/N, WKY/N.
c) DBH activity ranged very widely (50 to 193) SHR/N, WKY/N, RHA/N,
PETH/N, OM/N, W/N, ACl/N, ALB/N, M520/N. M520/N showed far higher value than
other strains.
There appears to be positive relationship between tyrosine hydroxylase activity
and aromatic L-amino acid decarboxylase activity and an inverse relationship
between either blood pressure and aromatic L-amino acid decarboxylase activity
or blood pressure and DBH activity, although these relationships are of
questionable statistical significance.
Significance to Biomedical Research and Institute Program: Comparing the
maximum velocity of these enzjnnes, tyrosine hydroxylase is clearly the rate-
limiting enzyme of norepinephrine biosynthesis in either the brainstem or
adrenal glands. There is no suggested co-relationship between any two of
these three enzyme activities except tyrosine hydroxylase and aromatic L-amino
acid decarboxylase. The inverse correlationship between brainstem enzymes and
blood pressure as suggested earlier was not seen in these different rat strains.
Proposed Course of Project: Catecholamine content of various organs in these
strains will be measured, and some enzyme activities in other organs than
brainstem and adrenal gland, especially cardiovascular systems, will be measured
to see if there is a relationship between blood pressure and these activities.
Honors and Awards: None
Publications: None
2 ^^
Serial No. NHLI-89
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: cAMP Dependent Protein Kinase in Vascular Smooth Muscle and
its Relationship to Calcium Uptake
Previous Serial Number: None
Principal Investigator: R. Wayne Alexander, M.D., Ph.D.
Other Investigators: Joseph A. Fontana, Ph.D.
Walter M. Lovenberg, Ph.D.
Cooperating Units: None
Project Description:
Objectives: The purpose of this study is to characterize the microsomal
Ca-H- uptake system in rabbit aorta in regard to the effects of cAMP and to
demonstrate a cAMP dependent protein kinase in the microsomal fraction.
Methods: Microsomes were prepared by differential ultracentrifugation from
the intimal medial layer of rabbit aortas. Calcium uptake in the presence of
oxalate was determined by ^5ca incorporation. Protein kinase activity was
determined by measuring 32p incorporation into microsomal protein or into
exogenous histone from y-^^P ATP. ^E cAMP binding to microsomal protein was
determined. Phosphorylated protein, protein bound 3h cAMP or ^5ca-H- containing
microsomes were trapped on Millipore filters and counted. Ca-Mg ATPase was
determined by measuring Pi released from ATP.
Major Findings: There is specific binding of ^h cAMP to rabbit aortic micro-
somes and there is stimulation of Ca++ uptake by cAMP in a range of 2 x 10~5
to 5 X 10~^ M. The stimulation averages 10% (p < .05) increase from a basal
value of 36+8 mu moles Ca++ uptake/mg protein/15 minutes at 2A° . A cAMP
dependent protein kinase was present in the microsomes which phosphorylated
both endogenous receptors (2.8 pmoles/mg protein/lO min) and exogenous histone
(lA pmoles/mg protein/10 min). The addition of cAMP results in no further
stimulation of the Ca-Mg ATPase.
Significance to Biomedical Research and Institute Program: This is the first
description of a stimulation by cAMP of vascular smooth muscle microsomal Ca++
uptake. The presence of a cAMP dependent protein kinase which phosphorylates
microsomal protein and the lack of effect of cAMP on the Ca-Mg++ ATPase system
suggests that the protein kinase may play a role in modulating intracellular
calcium stores and thereby affecting excitation-contraction coupling in vas-
cular smooth muscle.
1 /6o
Serial No. NHLI-89
Proposed Course of Project; Terminated
Honors and Awards: None
Publications : None
/^/
Serial No. NHLI-90(c)
1. Experimental Therapeutics Branch
2. Biochemical Pharmacology
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Effects of Varying Salt Intake and of Acute Saline
Infusion on Norepinephrine and Dopamine Excretion in Man
Previous Serial Number: NHLI-197(c)
Principal Investigator: R. Wayne Alexander, M.D., Ph.D.
Other Investigators: John R. Gill, Jr., M.D.
Hirohiko Yamabe, M.D.
Walter M. Lovenberg, Ph.D.
Harry R. Keiser, M.D.
Cooperating Units: Clinical Endocrinology Branch, NHLI
Project Description:
Objectives: The objectives are to ascertain whether dietary manipulation of
NaCl intake or acute saline infusion affects the urinary excretion of dopamine
and norepinephrine in man.
Methods: Twenty-four hour urinary dopamine and norepinephrine excretions were
determined in normal and hypertensive subjects under the following conditions:
a) ad lib salt intake, b) 9 meq Na+ diet, and c) 259 meq Na+ diet. Addition-
ally, in 7 normal subjects urinary dopamine and norepinephrine and plasma
dopamine B-hydroxylase activity were measured before and during acute I.V.
saline infusion.
Major Findings: During the salt depletion phase of the study a decrease in
urinary dopamine of up to 50% was noted. During the same phase of the study
an increase of up to 75% in urinary norepinephrine in comparison with baseline
values was observed. Conversely salt repletion resulted in a dopamine excre-
tion which was increased relative to the salt depletion state in all cases and
relative to control values in several cases. Concomitant with the increased
dopamine excretion during salt repletion there was a significant decrease in
norepinephrine excretion. In some cases this decrease was to below baseline
values. There was no significant difference between the normotensive and
hypertensive patient groups. During saline infusion an increase in urinary
dopamine excretion relative to control periods was noted. The increase was
from 2.2 to 2.8 Mg per 20 min (p < 0.05). Plasma dopamine 6-hydroxylase
decreased from 210 units/ml to 172 units/ml (p < 0.05). Changes in urinary
norepinephrine were not significant.
/6d
Serial No. NHLI-90(c)
Significance to Biomedical Research and Institute Program: The inverse re-
lationship between dopamine and norepinephrine excretion when sympathetic
activity was manipulated by varying the salt balance was unexpected. Other
Investigators have previously demonstrated relatively large amounts of dopamine
and its primary metabolite homovanllllc acid in the urine. The physiologic
role and, in fact, the source of urinary dopamine are unknown. Several
investigators have been unable to detect dopamine in the plasma of man. Our
data suggest that urinary dopamine is related, at least in part, to the sym-
pathetic nervous system and possibly is derived from renal sympathetic nerves.
Still other investigators have demonstrated that dopamine has pharma-
cologic effects in the kidney which are unique relative to other vascular beds.
Thus, dopamine dilates the renal vasculature and consequently is strongly
naturetlc. The present study suggests a possible role for dopamine In normal
sodlxun homeostasis.
Proposed Course of Project: Terminated.
Honors and Awards: None
Publications: None
/i>3
Serial No. NHLI-91(c)
1. Experimental Therapeutics Branch
2. Experimental Medicine
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Clinical Investigation of Cardiovascular Drugs
Previous Serial Number: NHLI-192(c)
Principal Investigator: David Horwitz, M.D.
Other Investigators: Harry R. Keiser, M.D.
B. Van Clineschmidt, Ph.D.
Richard Wyatt, M.D.
Christian Gillin, M.D.
Cooperating Units: Clinical Psychopharmacology Branch, NIMH
Project Description:
A. Reactivity of Human Temporal Arteries
Objectives : Increased sensitivity of arteries to the sympathetic neuromediator
norepinephrine has been widely reported in patients with essential hypertension.
Previous in vivo techniques have not been able to establish whether this resulted
from enhanced responsiveness of the receptor-smooth muscle unit or from a dif-
ferent initial state of muscle tone and a different wall-liunen ratio. In an
attempt to resolve this we have performed in vitro studies in which responses
of helical strips of biopsied human temporal arteries to graded doses of nore-
pinephrine and phenylephrine were measured. Through the use of the competitive
antagonist, phentolamine, the dissociation characteristics of alpha receptors
were also compared in hypertensive and normotensive subjects.
Major Findings: Dose-response curves were obtained from vessel strips from
eight subjects with essential hypertension and eight normotensive subjects.
They revealed no evidence of increased reactivity to norepinephrine and phenyl-
ephrine in the hypertensive subjects. Average concentrations in ng/ml of nore-
pinephrine and of phenylephrine yielding quarter-, half-, and three-quarter-
maximum responses were as follows:
NOREPINEPHRINE PHENYLEPHRINE
ED25
ED50
ED75
ED25
ED50
ED75
Hypertensive (n=8)
13
41
153
399
1170
3873
Normotensive (n=8)
10
41
163
286
1070
4452
The negative log of the molar concentration of phentolamine which reduced
the effect of a double dose of phenylephrine to that of a single dose (pA2) was
1 /^^
;d25
ED50
ED 7
15
49
159
4
14
62
Serial No. NHLI-91(c)
8.0 for the hjrpertensives and 7.9 for the normotensives; this was not statisti-
cally significant.
The effects of pretreatment with steroids were studied in six additional
normotensive subjects; four had received dexamethasone and two had received
prednisone. The results suggested increased reactivity of the vessels from the
subjects treated with prednisone whereas vessels of subjects treated with dexa-
methasone were not significantly different from those from untreated subjects.
NOREPINEPHRINE
Normotensive - Dexamethasone (n=4)
Normotensive - Prednisone (n=2)
Significance to Biomedical Research and Institute Program; This is the first
direct study of the characteristics of receptors and smooth muscle in human
essential hypertension. It demonstrated that the receptor-smooth muscle unit
of hypertensives was not hyper-reactive to sympathetic agonists. Moreover,
dissociation characteristics of alpha receptors were similar in hj^iertensive
and normotensive subjects.
Initial observations suggested that reactivity of human muscular arteries
to norepinephrine could be increased by pretreatment with prednisone.
Proposed Course of Project: Studies of vessel characteristics in Raynaud's
disease and further studies of the influence of steroids on vessel reactivity
are planned.
B, Serotonergic Mechanisms in Narcolepsy
Objectives; The etiology of narcolepsy is unknown. Normal sleep consists of
two alternating phases which have been designated as rapid eye movement (REM)
and non-rapid eye movement (NREM) sleep. Current evidence suggests that the
attacks of sleepiness and related symptoms of narcolepsy are associated with
episodes of REM sleep. In previous studies in this Branch it was shown that
parachlorophenylalanine (PCPA) , an inhibitor of serotonin synthesis, reduced
REM phase sleep by 20 to 70% in patients with migraine and the carcinoid syn-
drome; administration of 5-hydro3y tryptophan (5-HTP), the precursor of sero-
tonin, caused varying degrees of return of REM sleep in subjects receiving
PCPA. Our studies were concerned with the effects of drug-induced alterations
in serotonin levels on narcolepsy. Parachlorophenylalanine was used to reduce
brain serotonin levels. L-5-HTP was used to increase brain serotonin levels
and the peripheral decarboxylase inhibitor MK 486 was given concurrently to
enhance penetration into the brain. Administration of the monoamine oxidase
inhibitor pargyline is believed to elevate brain levels of catecholamines and
of serotonin.
f^r
Serial No. NHLI-91(c)
Major Findings: a) PCPA. Three patients with typical REM-onset narcolepsy
received parachlorophenylalanine in progressive doses to a maximum of 2 gm/day
for 25 to 35 days. Effects were monitored with standard rating forms and
frequent recordings of EEGs and eye movements. Each subject showed an increase
in REM sleep (7,10,55%); clinical effects were modest and variable. The effects
on REM sleep contrasted with those obtained in past studies in 18 patients
without sleep disorders; all of the latter showed decreases in REM sleep which
ranged up to 70%.
b) L-5-HTP and MK 486. Two of the subjects underwent courses of MK 486,
150 mg/day, both alone and together with small doses of L-5-HTP. Combined
therapy utilizing up to 200 mg/day of 5-IITP produced multiple side-effects and
mild enhancement of sedation. In previous studies hypertensive subjects tolerated
several-fold larger doses of both drugs with minimal side-effects.
c) Pargyline. The three narcoleptic subjects received pargyline in daily
doses of up to 50 mg/day. Each showed virtually complete suppression of REM
sleep and varying degrees of symptomatic improvement.
Significance to Biomedical Research and Institute Program: The results suggest
that narcoleptics respond abnormally to changes in brain levels of serotonin
but do not clearly implicate abnormalities of serotonergic pathways as the
cause of narcolepsy. The results suggest potential usefulness of pargyline for
the treatment of narcolepsy.
Proposed Course of Project: The study will be pursued in additional subjects.
Effects of cholinergic compounds will be studied.
Honors and Awards: None
Publications:
Horwitz, D., Alexander, R.W. , Lovenberg, W. , and Keiser, H.R. : Human serum
dopamine-B-hydroxylase. Relationship to hypertension and sympathetic
activity. Circulation Res. 32: 594-600, 1973.
I^i
Serial No. NHLI-92
1. Experimental Therapeutics Branch
2. Experimental Medicine
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Role of Endogenous Prostaglandin Synthesis in the Control
of the Coronary Circulation
Previous Serial Number: None
Principal Investigator: R. Wayne Alexander, M.D., Ph.D.
Other Investigators: Kenneth Kent, M.D., Ph.D.
John J. Pisano, Ph.D.
Harry R. Keiser, M.D.
Cooperating Units: Cardiology Branch, NHLI
Project Description:
Objectives: To ascertain whether prostaglandin synthesis in the dog heart
plays a role in the control of coronary circulation.
Methods ; The left main coronary artery was cannulated and perfused in two
different preparations. In the first preparation the left main coronary artery
was perfused via an extracorporeal circuit from the right femoral artery in an
otherwise intact, open chest dog. In the second preparation, a heart lung
preparation with a constant after load of 100 mm Hg, the left main coronary
artery was perfused via an extra-corporeal circuit from the main extra corporeal
aortic blood flow. The coronary sinus was cannulated in each preparation for
blood sampling. The following physiologic measurements were made:
1) coronary blood flow by means of a flow meter interposed in the extra-
corporeal coronary perfusion circuit
2) systemic arterial pressure
3) left atrial pressure
4) arterial pH, PaC02, hemoglobin saturation and coronary sinus levels of
lactate and prostaglandins (PG)
5) cardiac Mygo
6) cardiac tissue PO2 by means of an epicardial PO2 electrode
Prostaglandins were measured by means of a radioimmunoassay after organic ex-
traction, silicic acid chromatography and conversion of the PGE to PGB with
1 i('7
Serial No. NHLI-92
alkali treatment. Two maneuvers were utilized to induce changes in coronary
flow. First reactive hyperemia was induced by occluding the coronary perfusion
circuit for variable periods up to 20 seconds. Second, hypoxia was induced by
ventilating with 5% O2, 5% CO2 and 90% N2. Prostaglandin and lactate measure-
ments were made before and during the increased coronary flow resulting from
these interventions. The influence of synthetase inhibition on the increased
coronary blood flow resulting from hypoxia and from temporary coronary occlusion
was studied by giving two biochemically different PG synthetase inhibitors, \
indomethacin and meclofenamate. In the intact animal 50 mg of either drug were
infused into the coronary perfusion circuit over 15 minutes. In the heart lung
preparation 15 mg of either drug were injected into the extracorporeal circuit.
Major Findings: Both indomethacin and meclofenamate markedly attenuate reactive
hyperemia in the intact dog and in the heart lung preparation. For six animals
in which the data has been tabulated, at 10, 15, and 20 seconds of coronary
occlusion reactive hyperemia was decreased 50-55% (p < 0.025 for each interval)
after indomethacin.
In the heart lung preparation during hypoxia both the absolute increase
in blood flow and the percentage increase relative to the pre and post-hypoxla
baselines were decreased after PG synthetase inhibition.
Of particular interest was the observation that in the heart lung prepara-
tion but not in the intact animal, a progressive increase in coronary blood
flow was seen during the period of time when reactive hyperemia and hypoxia |
were induced. After each intervention the basal flow was higher than before
eventually increasing up to two fold. Over a period of 30 minutes after giving
PG synthetase inhibitors, the basal blood flow gradually decreased to or slightly
below the initial baseline flow.
Preliminary observations suggest that during reactive hyperemia a prosta-
glandin-like material is released from the heart in an amount approximately
two-fold that of baseline levels. After PG synthetase inhibition no release of
prostaglandin is seen and the basal release from the heart is decreased. No
changes in O2 consumption were seen after indomethacin.
Significance to Biomedical Research and Institute Program: This is the first
demonstration that endogenous prostaglandin synthesis may have a physiologic
role in the regulation of the coronary circulation. We believe that the pro-
gressive increase in coronary blood flow seen in the heart lung preparation is
due to a decrease in sympathetic tone plus the effects of unopposed endogenous
PG synthesis with resultant dilatation. With inhibition of PG synthesis the i
blood flow returns to levels determined solely by the metabolic needs of the
heart. This work has important implications in angina pectoris and myocardial
infarction.
Proposed Course of Project: Other prostaglandin synthetase inhibitors will be
utilized to assure that the observed effects are not non-specific. The prosta-
glandin-like materials released will be characterized and identified. The
effects of prostaglandin inhibition on the regional, i.e., endocardial-epicardial
blood flow in the heart will be studied. (
2 /£S
Serial No. NHTJ-92
Honors and Awards: None
Publications: None
I6<f
Serial No. NHLI-93(c)
1. Experimental Therapeutics Branch
2. Experimental Medicine
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Role of Renal Prostaglandins in Sodium Homeostasis and
Blood Pressure Regulation in Man
Previous Serial Number: None
Principal Investigator: R. Wayne Alexander, M.D,, Ph.D.
Other Investigators: John J. Pisano, Ph.D.
Harry R. Keiser, M.D.
Cooperating Units: None
Project Report:
Objectives: To develop methodology for measuring prostaglandins in sub-nanogram
amounts and to apply these methods to the study of the role of prostaglandins
in renal physiology and pathology in man.
Methods: A radioimmunoassay for prostaglandins has been developed. Existing
methods have been modified to permit the quantitative extraction and fraction-
ation of prostaglandins of the A, E and F series from plasma or urine. After
acidification and organic extraction the prostaglandins are chromatographed on
silicic acid columns. The PGA and PGB, PGE, and PGF fractions are eluted. An
antibody to PGA has been developed in the sheep which provides a sensitivity
in our radioimmunoassay for either PGA or PGB of 10 picograms. The primary
renal prostaglandin, PGE2, is measured as PGB2 after the alkali conversion of
PGE2 to PGB2. A radioimmunoassay for PGF2 ^ is being developed.
Major Findings: Normal values for plasma PGA in man have been found to be 200-
300 pg/ml. Normal values for plasma PGE are approximately 300 pg/ml. Normal
values of PGE in urine are being determined.
Significance to Biomedical Research and Institute Program: The development of
methods for the measurement of prostaglandins in urine and plasma of man is
essential for the elucidation of the role of these potent compounds in sodium
and water homeostasis, vasoregulatory functions and in such pathologic states
as essential and renovascular hypertension and primary aldosteronism.
Proposed Course of Project: As a necessary complementary and confirmatory
prostaglandin assay, the parallel superfused bioassay of Vane will be set up in
our laboratory in May, 1973 with the assistance of Dr. Sergio Ferreira from
Dr. Vane's laboratory. Plasma and urine levels of prostaglandins will be
measured in normals and hypertensive patients during control and experimental
conditions.
1 /70
Serial No. NHLI-93(c)
Honors and Awards: None
Publications: None
/7l
Serial No. NHLI-94(c)
1. Experimental Therapeutics Branch
2. Section on Experimental Medicine
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on the biological role of histamine: Alteration
of histamine metabolism in man and animals by salicylates
and other inhibitors
Previous Serial Number: NHLI-186(c)
Principal Investigator: Michael A. Heaven, Ph.D.
Other Investigators: Zdenka Horakova, Ph.D., Harry R. Keiser, M.D.
and Kenneth L. Kirk, Ph.D.
Cooperating Units: Laboratory of Chemistry, NIAMDD
Project Description:
Objectives: The synthesis and metabolism of histamine in man has been in-
vestigated with the objective of finding ways to increase histamine levels
in tissues in the treatment of vascular disorders. One approach was the
administration of large doses of L-histidine, the precursor of histamine, and
the other was the administration of various inhibitors of histamine metabolizing
enzymes. As described in last year's report the effectiveness of these treat-
ments was assessed by measurement of urinary histamine excretion and by study-
ing the metabolism of injected 6- H-histamine in vivo. In the course of this
year's studies one patient had a clearly abnormal pattern of labeled histamine
metabolites in urine. This finding prompted us to inquire about the patient's
medications and it was discovered that this patient ingested aspirin daily.
Further studies in patients and rats have confirmed our original observation
with aspirin and these studies are described in this report. In addition, a
series of f luorohistamine and histidine compounds produced by Dr. K. L. Kirk
have been tested for their inhibitory activity on the various enzymes related
to histamine metabolism.
Methods : Drugs were administered orally three times daily for 6 days in human
subjects or four times over a 24 hour period in rats. Single doses of drugs
were also given to rats. Labeled histamine was injected I.V., 2 hour after
the last dose of drug. 6--^H-Histamine was given to human subjects (5 yCi/kg)
and rats (50 uCi/kg) . In some studies 2 (ring carbon) -I'^C-histamine (25
uCi/kg) and H- imidazole acetic acid (50 yCi/kg) were injected into rats.
Urine was collected every 6 hours, and aliquots frozen. The labeled compounds
were assayed by 1) isotope dilution, derivatization and crystallization using
the procedures described in previous reports and publications of this
laboratory, and 2) chromatographic separation (10 pi urine) on thin-layer
cellulose plates using a butanol, ammonia, system and measurement of radio-
1 /7A
Serial No. NHLI-94(c)
activity on 2 nm segments. With 10 pi urine, the labeled metabolites separate
into sharp bands 2 to 6 mm wide and can be assayed separately on the one
chromatogram.
3
The H-imidazole acetic acid was prepared from the unlabeled material by
catalytic exchange and was purified by thin layer chromatography using the
system described above.
The various fluorohistamines and histidine derivatives were added in
concentrations of 10"^, 10"^ and 10~6 m to incubations of the following;
1) histaminase, partially purified from rat intestine or plasma from pregnant
women plus 3-%-histamine, 10"^ M; 2) partially purified preparation of guinea
pig brain histamine methyl transferase, S-adenosyl methionine--'-^C (methyl) and
histamine, 10~7 m, and 3) histidine decarboxylase partially purified from rat
fetal liver and from germ-free rat stomachs, L-histidine 2.5 x 10~5 M and L-
hlstidine-carboxyl-l^C or L-histidine-2 (ring label) -l^C, 100 nCi. Pyridoxal
phosphate, 10"^ M, was included in 1) and 3). Enzyme activity was determined
by release of %20 ^^ 1) ^^^ ■'■^002 in 3) and by formation of ■^^C-methylhistamine
in 2) and ^^C-histamine in 3) using the procedures that have been developed
in this laboratory and described in various annual reports over the past 4
years. Incubations were run with and without the fluorocompounds to assess
inhibition of enzyme activity by the compovind and without substrate to deter-
mine if the fluorocompound is a substrate for the enzyme.
Major Findings: Studies with fluorohistamine and histidine derivatives and
enzymes related to histamine. The 2, and 4, fluorohistidine and the fluoro-
histamine analogs did not inhibit histidine decarboxylase, histamine-N-methyl
transferase or histaminase activity in concentrations up to 10"^ M. Small
(< 1%) but measurable amounts of a l^C-methylated derivative was formed when
4 fluorohistamine was incubated with histamine-N-methyl transferase and S-
adenosyl methionine--'-^C. The methylated compound was not identified further
and it is not known if the labeled compound was derived from fluorohistamine or
from histamine present as a contaminant.
Inhibition of histamine metabolism by various inhibitors in man. Studies
have now been completed in 28 patients with the following results. In 8»
patients who received no drug, g--^H-histamine was recovered in urine as H-
methylhistamine (11 to 20%) , %-methylimidazole acetic acid (57 to 72%) , the
riboside of %-imidazo.le acetic acid (16 to 24%) and 1 to 4% as unchanged %-
histamine. Administration of amino guanidine (30 mg daily), a specific inhibitor
of histaminase, to 5 patients abolished the formation of imidazole acetic acid
and increased the excretion of tritiated methylimidazole acetic acid from 14
to 29%. In 3 patients the administration of chloroquin (250 mg daily) produced
drug levels that were sufficient to inhibit histamine-N-methyl transferase by
more than 90% in vitro but produced no significant reduction of methylation in
vivo. Administration of the monoamine oxidase inhibitor, pargyline (1 mg/kg,
7 days) in two patients resulted in the excretion of additional -'H-methyl-
histamine (7 and 14% before pargyline and 22 and 32% after pargyline) and a
corresponding decrease in the deaminated product -^H-methylimidazole acetic acid.
i73
Serial No. NHLI-94(c)
From these studies it appears that 1) both the two pathways of histamine
metabolism, deamination by diamine oxidase and methylation by histamine-N-
methyl transferase complte for histamine metabolism, 2) when diamine oxidase
is inhibited, histamine is metabolized by the alternative pathway; i.e.,
methylation, and 3) inhibition of methylation in vivo is not possible with
currently known inhibitors of this enzyme.
The effect of salicylates on the metabolism of 3--'H-histamine in man and
rats. Chronic administration of aspirin (3.6 g daily) in 5 subjects largely
abolished the excretion of the riboside conjugate of imidazole acetic acid
(13 to 24% to less than 4%). Acetaminophen, 3.6 g daily, had no apparent effect
on metabolism of g-^H-histamine in one subject.
In rat, aspirin and sodium salicylate, in doses of 300 mg/kg, blocked
completely riboside conjugation (n=5) . Other anti-inflammatory agents which
included phenylbutazone, acetaminophen, salicylamide, indomethacin and
phenacetin in high doses did not alter the metabolism and excretion of labeled
histamine. The inhibition of riboside conjugation by aspirin was related to
dose. As little as 10 mg/kg aspirin partially inhibited riboside conjugation
(by 20%). After 25 mg/kg aspirin, conjugation was inhibited 50%, after 50
mg/kg aspirin, 76%, and almost complete inhibition was achieved with higher
doses.
3
Injected H-imidazole acetic acid was converted (96%, n=3) to imidazole
acetic acid riboside in rat. This reaction was inhibited by sodium salicylate
or aspirin to the same degree as that observed in studies with labeled histamine.
When -^H-imidazole acetic acid and l^C-histamine was injected simultaneously,
the ratio of % to I'^C for all metabolites remained constant after all doses
of aspirin, even though riboside conjugation was suppressed by variable amounts.
The ^H- imidazole acetic acid appeared to be uniformly mixed with the endogenous
pool of imidazole acetic acid in the body.
The above studies show that salicylates specifically inhibit the conjuga-
tion of imidazole acetic acid to riboside in^ vivo, and that inhibition is
almost complete with therapeutic doses of aspirin in man.
Significance; The studies indicate that normal therapeutic doses of salicylates
inhibit riboside conjugation of histamine metabolites, although further studies
are required to determine if conjugation reactions with other sugars are blocked
by salicylates.
Proposed Course of Project; In view of the well-established findings that
aspirin markedly alters sugar metabolism, the effect of aspirin on the conjuga-
tion of some endogenous substances as well as foreign compounds is now being
studied.
Honors and Awards; None
Publications; None
/7/
Serial No. NHLI-95(c)
1. Experimental Therapeutics Branch
2. Section on Experimental Medicine
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The biological role of histamine and monoamines: Studies
of histaminase and other enzyme activities in medullary
thyroid carcinoma and hyperlipoproteinemia
Previous Serial Number: NHLI-185(c)
Principal Investigator: Michael A. Beaven, Ph.D.
Other Investigators: Floyd L. Atkins, M.D.
Harry R. Keiser, M.D.
Ronald M. Krauss, M.D.
Cooperating Units: Surgery Branch, NCI; Molecular Diseases Branch, NHLI
Project Description:
Objectives; This project continues all clinical studies of various enz3nnes,
including histaminase, in normal and disease states. These studies explore
the possible usefulness of sensitive isotopic assays in the diagnosis and
characterization of disease. In this report we describe our studies of two
disorders; medullary thyroid carcinoma and familial Type I hyperlipoproteinemia,
a disorder associated with low plasma lipolytic activity after heparin
administration.
Medullary carcinoma of the thyroid is a calcitonin producing tumor of
thyroid C-cells and frequently occurs in association with pheochromocytoma
as an inherited trait (Sipple's syndrome). The work described last year showed
that histaminase activity is a specific marker for this tumor and measurement
of this enzyme has been widely used in studies of a large family (Wampler
family) with this disease. Recently, workers in Sweden reported that mammalian
thyroid C-cells contain the aromatic amino acid decarboxylase, DOPA decarboxylase.
This report prompted us to look for DOPA decarboxylase and related enzymes in
the human tumor.
Methods : Control subjects were healthy laboratory personnel or volunteers
hospitalized at the NIH. The patients with medullary carcinoma of the thyroid
were members of a family with the inherited disease as well as patients whose
families had no history of the disease. The diagnosis of medullary thyroid
carcinoma was confirmed by histological examination of surgical and biopsy
specimens and by high calcitonin levels in plasma. Tissues for chemical
analyses were obtained at surgery.
Patients with hypertriglyceridemia were diagnosed as Type I, III, IV, and
1 /7i^
Serial No. NHLI-95(c)
V according to the classification of Fredrickson, Levy and Lees, by measure-
ment of plasma triglyceride and cholesterol concentrations and the
characterization of plasma lipoproteins by paper electrophoresis. Assays were
performed on homogenates of frozen or freshly excised tissues. Histaminase
activity was assayed by the tritium release assay described in earlier reports.
The various amino acid decarboxylases were assayed by measurement of the re-
lease of 1^C02 from L-DOPA-1-l'^C (DOPA decarboxylase) ; D-L-ornithine-1-l^C
(ornithine decarboxylase) ; L-histidine-l--'-'^C (histidine decarboxylase) .
Histidine decarboxylase was also assayed by an isotope dilution technique.
Tyrosine and tryptophan hydroxylase were determined using 3, 5--^H- tyrosine ( H
release) and L-tryptophan (serotonin formation) as substrates. Extraction and
fluorimetric procedures were used for the assay of serotonin and the catechol-
amines. The enzymatic procedure of Beaven et al. (Clin. Chim. Acta 1972) was
used for the assay of histamine. Calcitonin levels were assayed by radio-
immunoassay (Tashjian et al. , New Eng. J. Med. 283: 890, 1970) in the labora-
tory of Dr. A. H. Tashjian, Jr.
Major Findings: Enzyme activities in tumor from patients with medullary thyroid
carcinoma. The thyroid tumor had high DOPA decarboxylase activity compared to
that in adjacent thyroid or thyroid obtained from patients without the disease.
The mean (+ SEM) activity in tumor was 1306 + 247 nmoles/g/hr compared to 31 +
10 nmoles/g/hr in adjacent thyroid and 115 nmoles/g/hr in normal thyroid from
patients without the tumor. These values paralleled those of histaminase
activity which we have shown previously and is a marker for medullary thyroid
carcinoma. Ornithine decarboxylase and histidine decarboxylase activities
were not detected in tumor. Tyrosine and tryptophan hydroxylase were present
but not significantly different in tumor and normal thyroid.
The DOPA decarboxylase activity in the tumor resembled that in pheo-
chromocytoma in its behavior to various decarboxylase inhibitors. The enzyme
in both tumors decarboxylated L-DOPA, L-tryptophan and L-tyrosine to the same
degree and are inhibited by specific inhibitors of DOPA decarboxylase. The
levels of serotonin, catecholamine, histamine, in medullary thyroid carcinoma
were similar to those in adjacent thyroid.
Plasma histaminase activity as a marker for medullary thyroid carcinoma.
Serum histaminase activity has now been measured in over 300 normal subjects
and patients. In 80 normal subjects the enzyme levels have ranged from 0.3 to
6.3 units/ml (mean 1.7 + 0.8 S.D.). In 52 patients with medullary thyroid
carcinoma activity has ranged from 0.8 to 150 units/ml and in 29 of these
patients the enzyme activity was above normal (> 3.6 units/ml). In 70
relatives of these patients and 56 patients with a variety of other tumors, the
activity in all cases was in the range of normal. These data indicate that
histaminase activity is a specific marker for this tumor but may not be elevated
in all patients with the disease. Our experience with surgical treatment is
now extensive and the results indicate that measurement of this enzyme is use-
ful in detecting metastatic tumor. In 16 patients who had localized disease,
plasma enzyme activity fell to normal levels but in 9 patients activity did not
decline and in some patients increased after surgery. Metastatic tumor was
subsequently detected in all 9 patients. Calcitonin levels had returned to
9 /7<r
Serial No. NHLI-9 5(c)
normal in all but one of these patients and for this reason metastatic tumor
was not at first suspected.
Studies in patients with hyperlipoproteinemia: Heparin administration
and plasma histaminase activity. In normal subjects (n=37) a rise in plasma
histaminase activity occurred after the administration of heparin in doses as
low as 10 units/kg. The extent of this rise varied widely but in a particular
individual the response was constant and dose dependent. The studies described
in last years report showed that in 13 patients with Type I hyperlipoproteinemia
the increase in plasma histaminase after 10 units heparin/kg was significantly
less than that in normals or in patients (n=27) with other types of hyperlipo-
proteinemia. This low response was more apparent after 75 units/kg of heparin.
However, the deficiency (or defect) was not absolute. There was a small
response in half of the patients with the Type I disorder and in one patient
the response was high. The high response may indicate a genetic or biochemical
heterogeneity in this disorder, although this patient was otherwise similar
to other Type I patients.
Careful examination of the data from all patients and normal subjects
(n=65) has shown a highly significant correlation (p < 0.001, r = 0.84)
between the individual's basal plasma histaminase activity (i.e., before
heparin) and the increase in activity after heparin. A possible explanation
for this relationship is that the basal level and the rise in plasma enzyme
activity are determined by the level of histaminase activity in tissues. Thus,
high amounts of histaminase in tissues would lead to high basal enzyme activity
in plasma and a large response to heparin.
Significance; The studies in medullary thyroid carcinoma are the first demon-
stration of DOPA decarboxylase in thyroid C-cells in humans and they indicate
that monoamines may have a role in calcitonin secretion as suggested by other
workers for the C-cells in thyroid of other species. The finding of the
enzyme in both medullary thyroid carcinoma and pheochromocytoma provides
evidence that the syndrome of medullary carcinoma and pheochromocytoma result
from a heritable defect in a single cell system possibly the neuro-ectodermal
cell system. The studies also indicate that histaminase activity has a useful
role to play in the detection of metastatic medullary carcinoma.
Proposed Course of Project; The effect of alterations of monoamine metabolism
on calcitonin secretion in patients with medullary thyroid carcinoma will be
studied to determine if monoamines have an essential role in the regulation of
calcitonin secretion by the C-cells.
Honors and Awards: None
Publications:
1. Baylin, S. B. , Beaven, M. A., Keiser, H. R. , Tashjian, A. H. , Jr.,
and Melvin, K. E. W. ; Serimi histaminase and calcitonin levels in
medullary carcinoma of the thyroid. Lancet I: 455-458, 1972.
/77
Serial No, NHLI-95(c)
2. Baylin, S. B. , Beaven, M. A., Buja, L. M. , and Keiser, H. R. :
Histaminase activity: A biochemical marker for medullary
carcinoma of the thyroid. Am. J. Med. 53: 723-733, 1972.
3. Keiser, H. R. , Beaven, M. A., Doppman, J., Wells, S. , Jr., and
Buja, L. M. : Sipple's syndrome: Medullary thyroid carcinoma,
pheochromocytoma, and parathyroid disease. Combined Clinical
Staff Conference at the National Institutes of Health. Ann. Int.
Med. 78: 561-579, 1973.
4. Baylin, S. B. , Beaven, M. A., Krauss, R. M. , and Keiser, H. R. :
Response of plasma histaminase activity to small doses of heparin
in normal subjects and patients with hyperlipoproteinemia.
J. Clin. Invest., in press (August).
11%
Serial No. NHTJ-96
1. Experimental Therapeutics Branch
2. Experimental Medicine
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Biological Role of Histamine and Other Amines: Partici-
pation of Histamine and Serotonin in Inflammation
Previous Serial Number: NHLI-188
Principal Investigator: Zdenka Hor4kov4, Ph.D.
Other Investigators: Michael A. Beaven, Ph.D.
Marion E. Webster, Ph.D.
Cooperating Units: None
Project Description:
Objectives: Histamine, serotonin, the kinins and prostaglandins are all
thought to mediate the inflammatory response after tissue injury. One hypo-
thesis that ties these separate mediators together is that the release of hista-
mine and in some species, serotonin, precedes that of kinins and prostaglandins.
It has been suggested from studies with inhibitors of histamine, kinin and
prostaglandin synthesis, that histamine release mediates the initial response
and other mediators participate in the later stages of inflammation. Direct
measurements of the time course of release of all of these mediators have not
been made, and in this study the kinetics of release of the first of these
mediators, histamine, has been investigated. The studies are designed to answer
two questions: 1) if there is a correlation between the development of edema
and release of the amines after mild, moderate or severe injury and 2) if the
release of histamine is essential for the later stages of inflammation,
possibly, in the induction of kinin and prostaglandin synthesis. Accordingly,
we have studied the inflammatory response after depletion of mast-cell amines
by treatment with compound 48/80.
Methods: Mild, moderate and severe inflammation was induced by 1) immersion of
the rat paw in hot water at 53°, or SS^C for 30 sec; 2) injection of 0.1 ml 10%
sodium urate suspension, or 3) carrageenin solution. Samples of skin and sub-
cutaneous tissue exudate, and blood from aorta and popliteal vein were collected.
Edema was determined by measurement of paw volume. Histamine was assayed by
the enzymatic procedure of Beaven et al (Clin. Chim. Acta, 1972). A similar
assay is being developed for serotonin.
Major Findings: Time-Course of Inflammation and Histamine Release. Our previous
studies showed that edema developed after heating at 53° and 56°C and was accom-
panied by release of tissue histamine into tissue fluid and blood. More detailed
investigation of the kinetics of this release has now shown that the release
is closely correlated with the development of edema. After 53°C, there was a
1 (^9
Serial No. NHLI-96
delay of 30 min and then edema developed rapidly. Histamine release showed a
similar delay and rapid release. After 56°C, edema and histamine release was
immediate and rapid. Once edema was fully developed the release of amino into
the tissue fluid and plasma diminished.
The edema slowly subsided after 53°C but not after 56°C and was not asso-
ciated with further release of histamine. Injury at the higher temperature
resulted in extensive tissue necrosis and in loss of 40% of the paw. Tissue
damage was minimal after SS'C.
Carrageenin injection resulted in a rapidly developing severe inflammation.
Edema was maximum by 1 hr and then slowly subsided over the course of 24 hrs.
Histamine measurements have not been completed at this time. Uric acid in
contrast, produced a slowly developing edema (50% increase in paw volume).
There was no substantial decrease in histamine levels in the paw tissue although
the levels decreased in the wound fluid during the development of edema. No
increase in histamine levels could be detected in the blood.
Measurement of Serotonin. Because rat mast cells contain serotonin in
addition to histamine, an enzymatic isotope procedure is being developed for
the assay of serotonin. The assay is based upon the conversion of serotonin
to N-acetyl serotonin by N-acetyl transferase from rat liver and acetyl CoA
and then to melatonin by pineal methyl transferase enzyme and S-adenosylmethionine
(l^C-methyl) . The l^C-melatonin thus formed, is extracted into chloroform
which is assayed for •'•^C. Plasma proteins have been found to inhibit the
acetylation of serotonin and our current work is concerned with the development
of a simple procedure, such as micro ultra-dialysis, to prepare protein free
samples for assay.
Effect of Compound 48/80 on Inflammation and Histamine Release . Depletion
of tissue histamine by administration of the histamine liberator, compound 48/80
(1 mg/kg, i.p., 4 times over 48 hr), resulted in a greatly reduced inflammation
and tissue damage after heating at 53° or 56°C. Edema was reduced (60%) as
was the release of histamine from tissue (85%) and, as described in last year's
report, tissue damage was reduced. These rats have now been observed over the
course of 6 months and permanent damage was confined to the loss of toes which
reduced paw length to 25% (range 24 to 30%) compared to 40% loss (range 35 to
46%) in rats that did not receive compound 48/80.
Significance to Biomedical Research and Institute Program: The studies show
that histamine is released directly into tissue fluid and the circulation and
that this release is intimately associated with the development of edema. The
studies with compound 48/80 further indicate that the release of histamine has
an important influence on the later stages of inflammation, and suppression of
histamine release may be a useful approach in the treatment of inflammation.
Proposed Course of Project: Further studies of heat and carrageenin induced
inflammation will include measurements of prostaglandin synthesis. The possible
influence of histamine on prostaglandin synthesis will be examined by pretreat-
ment of animals with compound 48/80 or with combinations of antihistamine and
2 /iO
Serial No. NHLI-96
antiserotonin agents. The effect of the non-steroid anti- inflammatory agents,
aspirin, phenylbutazone and Indomethacin on the release and synthesis of the
various mediators will be investigated.
Honors and Awards: None
Publications:
Cohen, I.K., Beaven, M.A., Horakova, Z., and Keiser, H.R. : Histamine and
collagen synthesis in keloid and hypertrophic scar. Surgical Forum,
23:509 (1972).
/^/
Serial No. NHLI-97
1. Experimental Therapeutics Branch
2. Section on Experimental Medicine
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on the biological role of histamine and polyamines:
Ornithine decarboxylase and histaminase activities in rat
thymus and other organs
Previous Serial Number: NHLI-189
Principal Investigator: Floyd L. Atkins, M.D.
Other Investigators: Michael A. Beaven, Ph.D.
Cooperating Units: None
Project Description:
Objectives: The polyamines, putrescine, spermidine and spermine, are thought
to play a role in rapid tissue growth. In several systems increased polyamine
synthesis and RNA polymerase activity are associated with rapid growth. These
systems include the regenerating rat liver after partial hepatectomy and the
cardiac hypertrophy induced by stress. Studies in this laboratory have shown
that polyamines may also have an important role in the thymus possibly in
relation to the proliferation of Ijnnphocytes in this organ. The thymus has
high levels of polyamines, a rapid cell turnover, and high levels of ornithine
decarboxylase and histaminase (diamine oxidase) , two important enzymes in the
biosynthesis and metabolism of the polyamines.
In this report we describe studies which show that the ornithine
decarboxylase is located inside and histaminase outside the lymphocyte and
that in the intact lymphocyte, L-omithine is normally taken-up and converted
to putrescine. In addition, the level of ornithine decarboxylase activity in
the soluble and insoluble fractions of various tissues has been studied since
preliminary studies had indicated that a major part of the ornithine decarboxyl-
ase in tissue homogenates resides in the insoluble (nuclear) fraction whereas
in prostrate, a principle source of the enzyme, the enzyme is soluble.
Methods; Ornithine decarboxylase activity was assayed in two ways: by
measurement of the release of ^^C02 from D,L-omithine-carboxyl-l^C and by
measurement of the formation of ^^C-putrescine (by an isotope dilution
derivative procedure) from D,L-ornithine-2-l^C. Histaminase activity was
determined by measurement of tritiated water formed upon deamination of 3-%-
histamine and diamine oxidase by measurement of the deamination of I'^C-putrescine
as described by Beaven and Jacobsen (J. Pharmacol. Exptl. Therap. 176: 52,
1971).
/?<?-
Serial No. NHLI-97
Dexamethasone phosphate (500 yg/kg) was administered s.c. to deplete
thymus lymphocytes. The animals were sacrificed 24 hours after administration
of dexamethasone. Isolated lymphocytes were prepared by teasing the cells
out from the thymus and washing them in buffer.
Major Findings; 1) Studies of ornithine decarboxylase and histaminase in
rat thymus . High levels of both enzyme activities were found in thymus of
a variety of rat strains and germ-free rats. The ornithine decarboxylase
activity, and to a lesser extent the histaminase activity, declined after the
involution of thymus in older rats. After depletion of the thymus lymphocytes
by dexamethasone treatment, ornithine decarboxylase activity decreased by 60%
after one dose of dexamethasone and by more than 90% after two doses, while
histaminase activity was largely unchanged. The assay of various thymus
fractions indicated that at least 90% of the ornithine decarboxylase in thymus
was present in the lymphocyte and that 99% of the histaminase activity resided
outside these cells in a soluble form.
The activity of ornithine decarboxylase was dependent on the presence of
intact lymphocytes. This enzyme activity was largely destroyed by sonication
of the lymphocytes or extensive homogenization of the thymus. Enzyme activity
was also destroyed by freezing and thawing. When the lymphocytes were incubated
with DL-[2--'-^C] -ornithine and the labeled products separated by paper electro-
phoresis, two peaks of radioactivity, which corresponded to ornithine and
putrescine, were obtained. -^^C-labeled putrescine was found in the lymphocytes
but not in the incubation medium. When [ 2, 3-^H] -putrescine was added to the
incubation, the tritium labeled amine remained outside the lymphocytes and
was not metabolized. These results indicated that putrescine was formed with-
in the lymphocyte and that it did not readily diffuse across the cell wall.
Further characterization of the ornithine decarboxylase activity in
lymphocytes showed that the enzyme was not inhibited by 10~-3m a-methyldopa,
an inhibitor of the aromatic ly-ami no acid decarboxylase, but was inhibited,
98% and 78% respectively, by 10-3m NSD 1055 and 10"% a-hydrazinohistidine
both of which inhibit pyridoxal dependent decarboxylases such as histidine
decarboxylase and the aromatic Lr-amino acid decarboxylase.
2) Studies of ornithine decarboxylase in other tissues. In kidney, brain
and heart homogenates, a major part (> 90%) of the ornithine decarboxylase
activity, as measured by CO2 release, was associated with the particulate
fraction which was separated by low speed centrifugation. In the prostate,
however, 60 to 70% of the enzyme activity was in the soluble fraction.
In regenerating rat liver, ornithine decarboxylase activity increased 7 to
13 fold in both the soluble and particulate fraction. When enzyme activity
was at its highest level (8 hours after partial hepatectomy) only 10% of the
total activity was in the soluble fraction. The nature of the soluble and
insoluble enzyme requires further study.
3) The identity of histaminase with diaiaine oxidase in rat thymus and
other organs. Diamine oxidase has been isolated and purified from two sources,
2 /i3
Serial No. NHLI-97
pig kidney and human placenta, and has been shown to catalyze the deamination
of putrescine as well as histaminase. However, other soluble amine oxidases,
for example, soluble plasma amine oxidase, possess histaminase activity and
the identity of the histaminase activity in rat and human tissues, as
measured by the tritium release assay, was established by the following
findings. All tissues with high histaminase activity, including rat intestine,
thymus, adrenals, plasma after heparin injection and human plasma during preg-
nancy, medullary thyroid carcinoma, possess high diamine oxidase activity. The
correlation between the histaminase and the diamine oxidase activities were
highly significant. In each tissue, putrescine, a substrate of diamine oxidase,
competitively inhibited histamine deamination, and histamine inhibited
putrescine deamination. Hence, the two activities appear to be due to the
same enzyme.
Significance: Investigations in the last 10 years have shown that the thymus
is necessary for survival and development of the immune system. The role of
thymus is not completely understood. A number of studies indicate that the
thymus lymphocytes perform essential tasks in the other lymphoid organs of the
body. Measurement of ornithine decarboxylase activity or the labelling of
the polyamine pool in thymus Ijonphocytes may provide one approach in the study
of thymus derived lymphocytes in the body. The polyamines are thought to be
important in the regulation of RNA synthesis and the finding of high levels of
intracellular ornithine decarboxylase in thymus lymphocytes is compatible with
this role.
Proposed Course of Project: The role of polyamines in the living cell will be
further studied using the thymus lymphocyte as a model. Ornithine decarboxyl-
ase inhibitors will be studied in respect to their effect on RNA synthesis and
cell growth.
Honors and Awards: None
Publications:
1. Beaven, M. A. and de Jong, W. : Presence of histaminase and
ornithine decarboxylase activities in rat thjnnus. Biochem.
Pharmacol. 22: 257-265, 1973.
/S/
Serial No. NHLI-98(c)
1. Experimental Therapeutics Branch
2. Section on Experimental Medicine
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Metabolism of Hydroxyproline and Collagen
Previous Serial Number: NHLI-191(c)
Principal Investigator: Harry R. Keiser,M.D.
Other Investigators: I. Kelman Cohen, M.D.
Cooperating Units: Charles Vogel, M.D. , Solid Ttomor Centre, Uganda Cancer
Institute, Kampala, Uganda; NCI
William B. Looney, M.D., Ph.D., Division of Radiobiology
and Biophysics, University of Virginia, Charlottesville,
Va.
Project Description:
Objectives: 1. Study of collagen metabolism of skin in normal man, healing
wounds and certain disease states. 2. Study of the effects of various agents
on wound healing. 3. Further studies of protocollagen proline hydroxylase
(PPH) activity as a diagnostic test for and an index of therapeutic effective-
ness in patients and animals with hepatocellular carcinoma.
Methods : PPH activity in tissues is a good index of the rate of collagen
synthesis. PPH activity is measured in minced tissue by determining the
tritium released during hydroxylation from a biologically prepared ^H-proline
labelled substrate. With only minor modification we have utilized the same
method to measure PPH activity in blood, Collagenase activity in media
around cultured explants of tissue is a good index of collagen degradation.
This activity is quantitated by measuring the ^H-labelled fragments released
by collagenolytic splitting off of small fragments from a reconstituted gel
of biologically prepared radioactively labelled collagen fibrils. Previous
reports detail the establishment, testing and validation of these methods in
our laboratory.
Major Findings: We have shown previously that collagen synthesis as measured
by PPH activity and collagen degradation as measured by collagenolytic activity
are markedly increased in both keloid and hypertrophic scars. Since both
lesions are characterized by excessive scar formation it is apparent that the
increase in collagen synthesis is greater than the increase in degradation.
One form of therapy has been injection of a corticosteroid directly into the
lesion. We chose 5 patients with bilateral earlobe keloids and 3 patients
with large hypertrophic scars. One earlobe keloid or one end of the
1 isr
Serial No. NHLI-98(c)
hypertrophic scar in each patient was injected with 1 to 2 ml of triamcinolone
(40 mg/ml) every A to 6 weeks. The opposite earlobe keloid or the other end
of hypertrophic scar was injected with an equal amount of vehicle. When the
lesions became smaller or softer they were excised or biopsied. The PPH
activity in 5 untreated keloids, 3685 + 642 dpm/mg dry wt/hr (mean + SEM) , and
3 hypertrophic scars, 2373 + 983, was significantly (p < 0.01) higher than
that of 6 normal scars, 659 + 233, and 7 biopsies of normal skin, 450 + 119.
The PPH activity in treated keloids, 4589 + 2281, appeared higher than that
in untreated keloids but not significantly so. The PPH activity in treated
hypertrophic scars, 1477 + 935, appeared lower than that in untreated hyper-
trophic scars, but not significantly so. Thus, while corticosteroids produced
a reduction in the size of the lesions there was no significant decrease in
the rate of collagen synthesis.
Scleroderma is a disease which, during its early phases, is characterized
by increased collagen deposition in the skin. We have shown previously that
the rate of collagen synthesis in this disease is increased. In this study
we measured both PPH activity and collagenase activity in skin biopsies from
6 patients with scleroderma and 6 patients of the same ages with other diseases
but no apparent skin abnormalities. The PPH activity in the skin of the
patients with scleroderma, 410 + 18 dpm/mg/hr (mean + SEM) was significantly
(p < 0.001) higher than that of controls, 233 + 25. The collagenase activity
in the skin of patients with scleroderma, 215 + 39 cpm/10 mg dry wt tissue/
4 hr incubation (mean + SEM), was significantly (p < 0.01) lower than that of
controls, 392 + 32. Thus in scleroderma the rate of collagen synthesis is
markedly increased at the same time the rate of collagen degradation is
significantly decreased.
We have shown previously that the liver is the major source of PPH activity
in serum and that a high level of serum PPH activity in man is a good diagnostic
test for hepatocellular carcinoma. However, we could only theorize that the
high levels of serum PPH activity in patients with hepatocellular carcinoma
were due to a very rapid turnover of cells in this rapidly fatal tumor. There-
fore, we measured PPH activity in tumors from 6 groups of rats, 9 animals per
group, each group with a different strain of hepatocellular carcinoma. The
results were as follows:
Tumor
Tumor Strain Doubling Time (days)* Tumor PPH (dpm/mg/hr)
3924A 4.35 2251 + 160
5123TC 5.03 1092 + 156
7800 6.07 1088 + 197
9121 7.96 1288 + 99
9633 17.46 469 + 52
16 24.46 758 + 124
*Doubling time determined by measurement of thymidine turnover in vivo.
It is apparent that there is an inverse correlation between doubling time and
tumor PPH activity.
2 /U
Serial No. NHLI-98(c)
Significance; In keloids and hypertrophic scars the rates of collagen syn-
thesis and degradation are both increased. Since both lesions are
characterized by excessive collagen the S3mthetic process overbalances degrada-
tion. Systemic corticosteroids have been shown to decrease collagen synthesis
in normal wounds. When corticosteroids are injected into keloids and hyper-
trophic scars both lesions get smaller and softer. Our data would indicate
that the rate of collagen synthesis in these lesions remains high despite the
corticosteroids. This suggests that the steroids are producing their
therapeutic effect by stimulation of collagen degradation. The activation of
collagenase by steroids has been suggested previously by J. Houck. The data
also suggest that collagen synthesis in these lesions is not as sensitive to
control as it is in a normal wound.
We have shown previously that the rate of collagen synthesis in the skin
of patients with scleroderma is increased over that of controls. Now we show
that the collagenase activity in this skin is significantly reduced from that
of controls. Thus, collagen synthesis is increased while degradation is de-
creased. This combination of events explains more readily the rapid accumula-
tion of collagen noted in this disease and provides 2 possible therapeutic
points of attack.
The finding that the PPH activity in various strains of hepatocellular
carcinomas in rats varies inversely with the doubling time or rate of growth
of the tumor supports our hypothesis that the high levels of PPH activity in
the serum of patients with hepatocellular carcinoma are due to the rapid turn-
over of liver cells in this tumor. This suggests that following serum levels
of PPH activity in patients with this tumor may be a useful index of chemo-
therapeutic effectiveness.
Proposed Course of Project; 1. Studies of keloids and hypertrophic scars have
ended since Dr. Cohen, our plastic surgeon, has left. 2. Further information
on levels of PPH activity in patients with hepatocellular carcinoma receiving
chemotherapy have stopped since research activities and our source of specimens
were stopped by political unrest in Uganda. 3. We plan to study the effects
of certain anti-cancer drugs on the levels of PPH in rat hepatocellular carci-
nomas as a further means of evaluating the effectiveness of these agents .
4. Studies of collagen metabolism in blood vessels of normal and hypertensive
man and animals.
Honors and Awards; None
Publications;
Reiser, H.R. , Vogel, C.L. , and Sadikali, F. ; Protocollagen
proline hydroxylase in sera of Ugandans with hepatocellular
carcinoma. J. Nat. Cancer Inst. 49; 1251-1255, 1972.
/?7
Serial No. NHLI-99(c)
1. Experimental Therapeutics Branch
2. Experimental Medicine
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on the Interrelationships Between the Renin-Angiotensin
System, Urinary and Plasma Kallikrein and Prostaglandins in
Normal Volunteers and Hypertensive Patients
Previous Serial Number: None
Principal Investigator: Perry V. Halushka, Ph.D., M.D.
Other Investigators: Harry R. Keiser, M.D.
R. Wayne Alexander, Ph.D., M.D.
Harry S. Margolius, Ph.D., M.D.
Project Description:
Objectives: The role of the renin-angiotensin in soditrai homeostasis has been
extensively studied in normal volunteers and patients with various forms of
hypertension. The purpose of this study was to investigate the interrelation-
ships between the renin-angiotensin system, plasma prostaglandins and urinary
and plasma kallikrein in normal volunteers and hypertensive patients during
changes in sodium intake.
Until recently the only methods available to measure the activity of the
renin-angiotensin system were bioassay procedures which were unreliable and
tedious. In 1969 (J. Clin. Endocr. 29, 1349, 1969) a radioimmunoassay procedure
was developed to measure the activity of the renin-angiotensin system and this
new procedure proved to be more reliable and expeditious than bioassay pro-
cedures.
Methods: The previous radioimmunoassay procedures were modified to produce a
decrease in the variability and also to increase the number of unknown samples
which could be processed at one time.
The pH of plasma samples was adjusted to 6 just prior to a standard incu-
bation at 37°. By incubating the samples at the pH optimum of plasma renin the
amount of angiotensin I generated was increased per unit time and the incubation
time could be reduced to one hour. This helped to insure linear generation of
angiotensin I during the incubation without the addition of excess renin sub-
strate. The addition of this step also markedly decreased the interassay
variability.
Another important modification was the addition of deangiotensinized plasma
to the blank and standard tubes. This decreased the blank readings and also
made the standard tubes more representative of the unknown tubes. The potency
estimates for the unknown samples were obtained using a computer program
1 /92
Serial No. NHLI-99(c)
("Competitive Protein Binding Assays", O'Dell and Daughaday, Chapter 8).
The quality control sample had a mean value + S.E.M. of 2.49 + 0.13 ng/ml/hr
after 29 determinations.
Patients and normal volunteers were placed on a series of diets contain-
ing 109 meq Na for 1 week, then 9 meq Na'^ for 1 week, and finally 259 meq
Na'^ for 1 week. Each diet contained 100 meq id". At the end of each dietary
period a supine and standing (4 hr) blood sample was taken for PRA, plasma
kallikrein and plasma prostaglandins.
Major Findings; Plasma renin activity in normal volunteers and patients with
essential hypertension.
109 meq Na"*" 9 meg Na"^ 259 meq Na
Normal Volunteers Supine 1.39 (0.1-5.8)* 4.6 (1.5-8.3) 0.71 (0-2.5)
(n=8) Standing 4.7 (2.1-10.9) 16.2 (11.1-23) 3.0 (0.2-8.4)
Essential Supine 3.2 (0.43-6.3) 7.2 (0.48-19.8) 0.74 (0-2.5)
Hypertension Standing 7.3 (1.6-19.5) 15.1 (0.97-30) 2.4 (0.43-5.0)
(n=8)
*mean (range) in ng/ml/hr
Our values of PRA for the normal volunteers and patients with essential
hypertension on 9 meq and 109 meq Na diets are similar to what has been re-
ported by another group (J. Lab. Clin. Med 77^: 1025, 1971) using a similar
assay procedure. PRA values for subjects on a 259 meq Na"'"/100 meq KT*" are not
available from the literature. Measurements of plasma and urine kallikrein
have been completed in most of these normals and patients and are reported
in the annual report by Margolius et al. Plasma prostaglandin levels are
being measured as indicated in an annual report by Alexander et al. Correla-
tions and interrelationships between these three systems will be made as soon
as all individual values are determined.
Significance; It is apparent that the regulation of blood pressure is due to
a fine balance between the renin-angiotensin-aldosterone, kallikrein-kinin ,
and prostaglandin systems. All previous studies have been directed at only
one aspect of this problem. We can now study the role and interrelationships
of these systems in normals and hypertensive subjects and understand both
normal physiologic and pathologic mechanisms. This should lead to more
effective therapy for hypertension.
Proposed Course of Project; Further studies are planned using antihyperten-
sive agents, drugs which block the synthesis or action of prostaglandins and
drugs which block the actions of angiotensin II. The present study is also
being continued to identify and characterize important subgroups of hyper-
tensive patients such as those with low plasma renin activity.
Honors and Awards: None
Publications: None
2 /Sf
Serial No. NHLI-100
1. Experimental Therapeutics Branch
2. Experimental Medicine
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effects of Tolbutamide on Plasma Renin Activity (P.R.A.)
Previous Serial Number: None
Principal Investigator: Perry V. Halushka, Ph.D., M.D.
Other Investigators: Harry R. Keiser, M.D.
Cooperating Units: None
Project Description:
Objectives: Recent studies (Diabetes 19:(suppl 2) 747, 1970) have provided
evidence that the use of tolbutamide or phenformin for the treatment of adult-
onset diabetes mellitus is associated with an increased cardiovascular morbidity
and mortality when compared to the placebo or insulin treated groups.
Other studies (N.E.J.M. 286, 44, 1972) have purported to show an increased
cardiovascular morbidity and mortality in patients with essential hypertension
and an increased PRA.
This project was initiated to ascertain whether or not tolbutamide could
increase PRA.
Methods: Plasma renin activity (P.R.A.) was measured using a radioimmunoassay
as described in another annual report.
Dogs were anesthetized with pentobarbital, 30 mg/kg I.V., and a catheter
was placed in the femoral artery for monitoring systemic arterial pressure.
The left renal artery and vein were cannulated for infusing tolbutamide and
sampling renal venous blood respectively. Tolbutamide was infused in doses
of 2 mg/kg, 4 mg/kg and 8 mg/kg into the renal artery, each over a 30 minute
period and at the end of each period a renal venous sample was taken for deter-
mination of PRA.
Major Findings: Plasma renin activity was elevated above control levels in 3
of 5 dogs at the 2 mg/kgm and 8 mg/kgm doses and in 4 of 5 dogs at the 4 mg/kgm
dose. However, the mean PRA for each dose was not statistically different from
that of the control period.
The effects of tolbutamide may have been partially obscured by the fact
that the PRA had already been markedly stimulated by pentobarbital anesthesia.
l9o
Serial No. NHLI-100
Significance to Biomedical Research and Institute Program: The question about
the effect of oral hypoglycemic agents on PRA remains unanswered. There appears
to be an effect but It Is not a very large effect. Further studies along this
line would require large numbers of animals and are not warranted.
Proposed Course of Project: A protocol has been approved to study the effects
on PRA of intravenous Infusions of tolbutamide (1 gm) in diabetic patients and
normal volunteers. Another protocol has been approved for the study of the
effect of chronic oral administration of tolbutamide on PRA in diabetic patients.
Honors and Awards: None
Publications: None
f9/
Serial No. NHLI-lOl(c)
1. Experimental Therapeutics Branch
2. Section on Experimental Medicine
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Urinary and Plasma Kallikrein
Previous Serial Numbers: NHLI-79(c), 194(c), 207
Principal Investigator: Harry S. Margolius, M.D., Ph.D.
Other Investigators: David Horwitz, M.D., Ronald G. Geller, Ph.D.,
R. Wayne Alexander, M.D. .Ph.D., John R. Gill, M.D. ,
John J. Pisano, Ph.D., Jack V. Pierce, Ph.D.,
Harry R. Keiser, M.D.
Cooperating Units: 1- Ronald Brown, M.D.
2. Stephen Zinner, M.D.
Edward Kass, M.D., Ph.D.
3. Hans Brunner, M.D.
John Laragh, M.D.
4. Drori Ben-Ishay, M.D.
Department of Medicine
Vanderbilt University
Department of Medicine
Harvard University
Department of Medicine
Columbia University
Department of Medicine
Hadassah Medical School
Jerusalem
Project Description:
Objectives: To study the role of the kallikrein-kinin system in normal and
hypertensive humans and animals.
Methods: Humans. Urinary kallikrein excretion was measured with a previously
described radiochemical assay (NHLI 79c) . Plasma kallikrein activity was
measured with a modification of that procedure developed by Pisano et al.
Urinary and plasma kallikrein were measured in normal subjects, patients
with essential hypertension or primary aldosteronism during different salt
intakes. The effects of drugs and acute saline or acute water loading on
kallikrein excretion were determined.
Urinary kallikrein excretion from each kidney was measured in patients
with renovascular hypertension. Urinary levels were determined in children
from families with and without hypertensive histories and in patients with
essential hypertension categorized according to plasma renin levels.
Rats. The effects of altered sodium intake, desoxycorticosterone or
adrenalectomy on urinary kallikrein excretion were measured in normal, spon-
taneously hypertensive, and the "salt-sensitive" versus "salt-resistant"
199-
Serial No. NHLI-lOl(c)
strains of rats developed by Dahl.
Major Findings; Humans. 1) Urinary kallikrein excretion is significantly
increased (p < 0.001) during 9 meq Na intake and falls to control levels
during 259 meq Na intake in normal subjects (ad lib Na = 11.6 + 1.0 EU/24 hr,
9 meq Na = 33.0 + 2.5 EU/24 hr, 259 meq Na = 11.2 + 0.8 EU/24 hr) . Kallikrein
excretion in essential hypertensives is significantly lower than in normals
and increases less during 9 meq Na intake (ad lib Na - 5.0 + 0.8 EU/24 hr,
9 meq Na = 9.4 +1.8 EU/24 hr, 259 meq Na = 5.1 + 0.9 EU/24 hr) . Kallikrein
excretion in 9 patients with primary aldosteronism is elevated and unrespon-
sive to changes in Na intake (9 meq Na = 22.2 + 3.3 EU/24 hr, 259 meq Na =
26.4 +3.6 EU/24 hr) . Fludrocortisone increased kallikrein excretion in each
of 6 subjects from 9.6 + 2.8 EU/24 hr to 21.4 + 4.6 EU/24 hr (p < 0.025).
Spironolactone decreased kallikrein excretion in primary aldosteronism and
in normals on 9 meq Na intake. The data show that urinary kallikrein
excretion is a new diagnostic tool for detecting hyperaldosteronism in patients
with hypertension.
2) Contrary to previous reports, neither acute water nor saline loading
altered urinary kallikrein excretion in any of 11 normal volunteers.
3) Although all data is not yet evaluated, there appears to be a difference
in the amount of kallikrein excreted by the compromised vs^ the normal kidney
in 25 patients with unilateral renovascular disease, with the contralateral
normal kidney excreting very little, if any, kallikrein.
4) Studies in 396 children from 75 families have so far shown a highly
significant familial aggregation of kallikrein excretion (p < 0.001). The
data are being evaluated to determine if urinary kallikrein excretion in
children is correlated in any way with their blood pressure (Zinner et^ al. ,
New Eng. J. Med. 284: 401, 1971).
5) Kallikrein excretion was measured in the coded urines of 60 patients
with essential hypertension and with low, normal or high plasma renin activity.
The data has not been decoded at this time.
6) Plasma kallikrein levels for 19 normals averaged 2678 + 144 (Mean + SEM)
dpm/30 min incubation (range 1539 to 3588) . This was significantly lower
(p < 0.001) than that for 34 hypertensives where levels were 3356 + 122
(range 1349 to 4999) . Kallikrein levels were constant in the same individual
over several weeks. No significant difference in plasma kallikrein was noted
between supine and standing values or with alterations in dietary sodium.
Rats. In normal rats, low sodixim intake or desoxycorticosterone administra-
tion increased kallikrein excretion 2 to 3-fold after 10 days to 2 weeks thus
confirming the findings in normal human subjects. Bilateral adrenalectomy
significantly decreased kallikrein excretion (p < 0.01). In contrast,
spontaneously hypertensive rats show a decrease in kallikrein excretion with
low Na intake and an increase with high Na intake.
/93
Serial No. NHLI-lOl(c)
Although not yet completed, there is a significant difference in urinary
kallikrein excretion in the "salt-sensitive" compared with the "salt-
resistant" rats.
Significance: The above data, taken collectively, provide further strong
support for the hypothesis that the kallikrein-kinin system is actively
involved in hypertensive disease, mineralocorticoid effects and sodium
homeostasis. Our studies show that urinary kallikrein excretion is controlled
by endogenous aldosterone levels in rats and humans. Measurement of urinary
kallikrein excretion is an important new tool in screening for hyperaldo-
steronism in hypertensive subjects.
Our data indicate that the kallikrein-kinin and renin-angiotensin systems
are interdigitated. Kallikrein and renin are both present in the kidney.
Angiotensin is vasoconstrictor and anti-natriuretic. The enzyme in lung which
produces angiotensin II ("converting enzyme") is now known to be identical to
the enzyme which destroys kinins in lung (bradykininase II). Therefore, it
is possible that understanding the relationships between the kallikrein-kinin
system and renin-angiotensin-aldosterone-axis will be of fundamental impor-
tance in the pathophysiology and perhaps, etiology, of different hypertensive
disease states.
Proposed Course of Project: 1. Study the effects of drugs especially anti-
hypertensive therapy on the kallikrein-kinin system in normal and hypertensive
man. 2. Localize kallikrein within the kidney. 3. Determine more precisely
the relationships between renin-angiotensin levels and kallikrein-kinin levels.
4. Determine if other factors (renal vascular resistance, intravascular
volume, prostaglandins, etc.) can be related to kallikrein-kinin system
activity in urine, renal tissue, and blood.
Honors and Awards : None
Publications:
1. Margolius, H.S., Geller, R.G., de Jong, W. , Pisano, J.J., and
Sjoerdsma, A.: Urinary kallikrein excretion in hypertension.
Circulation Res. Suppl. II 31: 125-131, 1972.
2. Geller, R.G., Margolius, H.S., Pisano, J.J., and Keiser, H.R. :
Effects of mineralocorticoids, altered sodium intake, and
adrenalectomy on urinary kallikrein in rats. Circulation
Res. 31: 857-861, 1972.
(H
Serial No. NHLI-102
1. Experimental Therapeutics Branch
2. Experimental Medicine
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Studies on Renal Catecholamine Synthesis
Previous Serial Number: None
Principal Investigator: Osamu limura, M.D.
Other Investigators: Harry R. Reiser, M.D.
Hirohiko Yamabe, M.D.
Walter M. Lovenberg, Ph.D.
Cooperating Units: None
Project Description:
Objectives: A role for dopamine (DA) in sodium homeostasis of the kidney has
recently been suggested by several investigators. DA and norepinephrine (NE)
synthesis from l^C-tyrosine has been studied in the kidney of rats under the
following experimental conditions: denervation of the kidney, salt loading,
salt restriction, stressful stimuli and experimental high blood pressure.
Methods: l^C-tyrosine, 50 yCi in total, was injected i.p. at two hour intervals
and rats were sacrificed 2 hours after the last injection. Radioactivity in
tyrosine, dopa, DA and NE was measured following separation on columns of Dowex
50 and alumina.
Major Findings: Under the experimental conditions described above about 400
dpm/gm kidney were found in the DA fraction and about 200 dpm/gm in the NE frac-
tion. The NE synthesis rate is consistent with data obtained in other tissues
and experiments. The incorporation into DA however was much larger than antici-
pated based on known total DA content and specific activity of the tyrosine in
servim. While it is possible that the incorporation may be in a small rapidly
turning over pool, it is possible that the DA fraction is contaminated with
another metabolite of tyrosine and therefore DA incorporation rates must be
considered tentative.
The effects of the various physiologic maneuvers on catecholamine synthesis
are as follows:
1) Denervation of the kidney: In each animal, the left kidney was denervated
and the right kidney was left intact. ^^C-NE was lower in the denervated kidney
but similar incorporation of ^^C-tyrosine into l^C-DA was noted in both kidneys.
2) Salt loading and restriction (15 days) : Salt loaded rats showed lower
Serial No. NHLI-102
C-DA and ^^C-NE in the kidney compared with salt restricted rats or control
rats under normal diet.
3) Cold and immobilization stress: Immobilization stress increased ■'^^C-NE
synthesis and cold stress increased apparent ■'-^C-DA synthesis.
4) Spontaneous hypertension: Adult spontaneously hypertensive rats (SHR)
showed significantly lower l^C-NE than that of Wistar/NIH (W/N) but similar to
Wistar/Kyoto (W/K) . l^C-DA in SHR and W/N were higher than that in W/K.
Significance to Biomedical Research and Institute Program; Since catecholamines
have been postulated to play a role in Na+ homeostasis it is of significance
that salt loading changes DA and NE synthesis in the kidney. Denervation of
the kidney as expected reduces NE synthesis, but does not seem to affect DA
synthesis, suggesting a possible extraneuronal source of dopamine. It should
be noted that aromatic _L amino acid decarboxylase, the enzyme that catalyzes
the synthesis of DA is largely an extraneuronal enzyme in the kidney.
Proposed Course of Project: When all of the data has been analyzed the project
will be terminated.
Honors and Awards : None
Publications: None
m
Serial No. NHLI-103
1. Experimental Therapeutics Branch
2. Experimental Medicine
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Acute Effects of Alphamethyldopa on Plasma Renin Activity
Principal Investigator: Perry V. Halushka, Ph.D. , M.D.
Other Investigators: Harry R. Keiser, M.D.
Cooperating Units: None
Project Description:
Objectives: Alphamethyldopa (Aldometv-/) has been shown to exert its anti-
hypertensive effect primarily through the central nervous system (J. Pharm.
Pharmac. 20:409, 1968). Chronic oral treatment with alphamethyldopa (200 mg/
kg/day) in dogs has reportedly produced a significant lowering of plasma renin
activity (PRA) . The purpose of this project was to determine if the lowering
of plasma renin activity was mediated through the central nervous system.
Methods; PRA was measured using a radioimmunoassay as described in another
annual report.
Dogs were anesthetized with pentobarbital (30 mg/kg) Intravenously and
then given a continuous maintenance dose of 6 mg/kg/hr intravenously. Systemic
arterial blood pressure was measured continuously from the femoral artery and
blood samples for PRA were taken from the external jugular vein. The left
vertebral artery was exposed at the angle of the neck and clavicle and cannu-
lated. Normal saline was infused into it at a rate of 0.5 ml/min for 1 hour
during which control blood samples were taken for plasma renin activity.
Alphamethyldopa (20 mg/kg) dissolved in saline was Infused into the vertebral
artery of 4 dogs over a period of approximately one hour. Another group of
four dogs was prepared in a similar fashion but instead of alphamethyldopa
received the vehicle, (normal saline adjusted to pH 4.5). A third group (4
dogs) received alphamethyldopa (20 mg/kg) intravenously.
Major Findings; Preliminary studies using the soluble ethylester of alphamethyl-
dopa produced inconsistent results.
Within 60 minutes after the start of the infusion of alphamethyldopa into
the vertebral artery mean PRA decreased from a control value of 32.5+4.0 ng/ml/hr
(mean+S.E.M. ) to 12.2+4.4 (p<0.01). PRA remained significantly depressed for
3 hrs before returning to control levels by the fourth or fifth hour. The mean
arterial blood pressure was significantly lowered from control levels of 123.5+
0.9 mm Hg (mean+S.E.M.) to 107.5+4.4 within 1-1/2 hours after the start of the
alphamethyldopa and remained significantly lowered until the experiment was
terminated 5 hours later.
1 197
Serial No. NHLI-103
Alphamethyldopa administered intravenously significantly lowered PRA within
30 minutes from a control level of 23.4+3.4 ng/ml/hr (mean+S. E.M, ) and reached
the lowest level of activity in 1-1/2 hours (7.3+2.6) (p<.01) and remained
suppressed, returning to control levels at 3 hours. The mean arterial blood
pressure fell within 30 min from a control level of 141.9+3.1 mm Hg and remained
significantly decreased for 1-1/2 hours (121.3+6.0 mm Hg) (p<.05). Thereafter,
although the blood pressure never returned to the control values it was not
statistically significantly different from the control values.
In the dogs that received only saline the PRA (control 36.5+10.8 mg/ml/hr)
and the mean arterial blood pressure (control 151.9+3.1 mm Hg) did not change
significantly throughout the study.
Significance to Biomedical Research and Institute Program: Unfortunately, the
results of these studies do not help to localize the site of action of alpha-
methyldopa' s effect on PRA.
Thus, the important question still remains as to where centrally acting
anti-hypertensive drugs produce their PRA lowering effect, centrally or peri-
pherally.
Proposed Course of Project: Further studies are going to be carried out in an-
imals to elucidate the mechanism of the suppression of PRA by alphamethyldopa.
Experiments are just beginning in dogs which have had acute unilateral nephrec-
tomy and denervation of the contra-lateral kidney to determine if the suppressive
effect of alphamethyldopa on PRA is mediated through the central nervous system.
Additional studies are planned to study the mechanism of the lowering of
PRA by other centrally active antihypertensive agents.
Honors and Awards: None
Publications: None
/f«
Serial No. NHLI-104(c)
1. Experimental Therapeutics Branch
2. Section on Neuroendocrinology
3. Bethesda, Maryland
PHS-NIII
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effects of Steroid, Thyroid and Protein Hormones, Electrolytes
and Psychodelic Agents on Neural and Sensory Function.
Previous Serial Number: NHLI 218 (c)
Principal Investigator: Robert I. Henkin, M.D., Ph.D.
Other Investigators: N. Prakash, Ph.D.
J. Fontana, Ph.D.
M. Buchsbaum, M.D.
D. Gilbert, Ph.D.
J. Gillin, M.D.
F. Catalanotto, D.D.S.
Cooperating Units: National Institute of Mental Health, National Institute of
Neurological Diseases and Stroke, Bethesda, Maryland;
Harvard School of Dental Medicine, Boston, Massachusetts.
Project Description:
Objectives : To investigate systematically the interrelationships between
steroid hormones, protein hormones, electrolytes and psychodelic agents on
neural and sensory function with respect to the manner by which sensory signals
are detected and integrated by the sensory receptor, neuraxon and brain.
Q
Major Findings: 1. Effects of lysergic acid diethylamide (LSD-25) and A
tetrahydrocannibol (marihuana) on neural conduction in squid giant axon.
In an effort to evaluate the action of the hallucinogens LSD-25 and marihuana
on neural conduction the effects of these drugs on the basic ionic events which
give rise to the neuraxon potential in squid giant axon were investigated. Ad-
ministration of either of these hallucinogens in high concentrations did not
alter the basic ionic events which give rise to the action potential. However,
administration of amounts of LSD-25 smaller than those which were ineffective
in altering Na* or K* currents to the intact squid produced inhibition in several
behavorial parameters including touch and spontaneous swimming. These results
suggest that LSD-25 is effective in altering some aspects of neural activity
in the squid but it is ineffective in altering the Na* or K* currents of the
action potential. The apparent locus for the major effects of these drugs
appears to be at the synapse.
2. Neural correlates of age, sex and gonadal hormones. It has been
previously observed that the amplitude of the electroencephalogram (EEC) of
women is much larger than that of men. This suggested to us that there could
1 /ff
Serial No. NHLI-104(c)
be hormonal influences upon the electrical activity of the brain. In an effort
to evaluate the affects of sex steroids on neural function visual and auditory
average evoked responses (AER) were studied in 166 normal males and females
from age 6 to 60 and in 10 patients with chromatin negative gonadal dysgenesis
(45 XO) . Females of all ages and patients with 45 XO without treatment with
gonadal steroids exhibited larger amplitude AER than men. No correlation be-
tween cephalometric roentgenographs (i.e., measurement of thickness of the
skull bone or scalp skin thickness) and AER amplitude was found to explain
these differences. Younger subjects exhibited larger AER amplitude and greater
increases in amplitude with increasing stimulus intensity; latencies were shorter
and increased with increasing intensity rather than decreasing as in older
subjects. These findings suggest that levels of FSH, LH or gonadal steroids
cannot be the only determinants of the sex differences observed in the electrical
activity of the brain. From the data on patients with 45 XO, it may be hy-
pothesized that a marker to which genetic information determining female
characteristics may be related, may be associated with the female AER pattern.
3. Manual and oral sensation in patients with taste and smell loss of
several etiologies. Patients with hypogeusia and hyposmia of several etiologies
may exhibit other perceptual abnormalities in the oral cavity. To study these
possible changes, we measured manual and oral stereognosis in patients with
hypogeusia and hyposmia which occurred following influenza (n=53) , x-irradiation
to the head and neck (n=4) , trauma to the head (n=5] , and subsequent to surgi-
cal procedures not involving the head and neck (n=3) . The control group con-
sisted of 50 adult normal volunteers. Mean scores and times of the patients
groups for the 20 NIH forms used were compared to the control means.
Patients who developed taste and smell abnormalities following influenza
or x-irradiation recognized the same number of manual forms as did the controls,
but took significantly longer (p < .01) to do so. These same patients recognized
significantly fewer oral forms that did the controls (p < .01) while patients
who developed hypogeusia and hyposmia following trauma or after surgery recog-
nized approximately the same number of forms as controls. Patients with taste
and smell abnormalities of any etiology took about twice as long as controls
to recognize the oral forms but the differences were significant (p < .01)
only for those developing symptoms after influenza or x-irradiation. These re-
sults suggest that some patients with taste and smell disorders also exhibit
abnormalities in the oral perception of form whereas others do not. The
mechanisms by which these differences occur may be important to our understanding
of the sensory modalities of taste and smell.
4 . The role of adrenal corticosteroids in the control of manual and oral
sensation. Patients with decreased taste and smell acuity (hypogeusia and
hyposmia, respectively) , not uncommonly exhibit other abnormalities of oral
perception. Some complain of glossopyrosis (burning tongue) without overt
glossitis, changes in salivary consistency without measurable decreases in
salivary flow and abnormal speech patterns caused by what patients relate as
unreliable oral placement of the tongue during conversation. Others, soon
after the onset of hypogeusia and hyposmia, exhibited changes in auditory
2 ^ac
Serial No. nHLI- 104(c)
perception including decreased auditory acuity, tinnitus and vertigo. In an
effort to learn more about changes in oral perception in these patients we
studied manual and oral stereognosis in patients with hypogeusia and hyposmia
of several etiologies. In these studies, manual and oral stereognosis was
measured in 7 patients with Gushing 's syndrome (CS) and 50 normal volunteers.
Since patients with Gushing' s syndrome without treatment also exhibit hypogeusia
and hyposmia as well as other impairments of sensory detection and recognition
we also studied light touch and two point discrimination. Thresholds for light
touch on the hand and in the mouth were consistently higher in the patients with
CS than in the normal subjects. Similarly the patients with GS made significantly
more errors than did the normal volunteers and required significantly longer to
do so. Treatment of the patients with antiadrenal drugs or with surgery which
significantly lowered endogenous secretion of Cortisol resulted in a return of
light touch and manual and oral from recognition to or toward normal. These
results indicate that touch and form recognition, as taste, smell and hearing,
are regulated by carbohydrate-active steroids with respect to detection and
recognition of sensory signals.
5. The role of adrenal corticosteroids in the control of circadian
variation of taste and olfaction in normal man and in patients with adrenal
cortical insufficiency. Although circadian changes in the endogenous secre-
tion of adrenal corticosteroids and ACTH have been well known for some time as
have the relationships between concentration of adrenal corticosteroids and
several sensory functions the role which adrenocorticosteroids and AGTH play
in the regulation of circadian changes in taste and olfaction had not been
previously evaluated. In these studies taste and smell thresholds were
measured in 17 normal volunteers (9 men, 8 women) and in 5 patients with adrenal
cortical insufficiency (2 with panhypopituitarism, 3 with Addison's disease)
on adequate hormonal replacement therapy and after all therapy was withdrawn
for 4 or more days. Results indicated that there was a specific circadian
pattern of variation in both taste and smell detection in normal men and women
which closely followed in time the circadian pattern of secretion of endogenous
Cortisol. The circadian pattern of variation for women extended over a wider
range than for men although the pattern for the two sexes was similar. Patients
with adrenal cortical insufficiency on normal replacement therapy exhibited
circadian patterns of variation which could not be distinguished from the normal
volunteers. However, removal of hormonal replacement therapy resulted not only
in a significant increase in detection acuity for both taste and smell but also
the elimination of the circadian pattern of variation. This occurred both in
patients with panhypopituitarism as well as with Addison's disease which suggests
that Cortisol rather than ACTH is the major determinant of this change. Read-
ministration of hormonal replacement therapy resulted both in a decrease in
detection acuity and in the return of a circadian pattern of variation in these
patients within 24-48 hours; this pattern could not be distinguished from that
of normal subjects.
6. Effects of adrenocorticosteroids and of ACTH on sleep. To determine
the effects of adrenal corticosteroids and adrenocorticotropin (ACTH) on human
sleep 8 hour intravenous infusions of ACTH 40 U were administered to 9 healthy
volunteers (beginning at either 8 a.m., 3 p.m., or 11:30 p.m.) and to 3 patients
3 ^/
Serial No. NHLI- 104(c)
with Addison's disease beginning at 8 a.m. ACTH produced a significantly
greater reduction in REM sleep in the normal volunteers than in the patients
with Addison's disease. During the overnight infusion, approximately 4 hours
of continuous infusion of ACTH was required before REM sleep was reduced. The
REM suppressive effect of ACTH appeared to attenuate about 12 hours following
the termination of the ACTH infusion. In addition, ACTH significantly reduced
total sleep time in the volunteers following infusions beginning at 8 a.m. and
3 p.m. but not when ACTH was infused during sleep. Delta sleep was also re-
duced following the 8 a.m. infusion in the volunteers.
These results suggest that ACTH affected sleep through its effect on
adrenal corticosteroid secretion. Time appears to be an important aspect of
the effect of ACTH and adrenocorticosteroids upon the central nervous system.
(Table 1)
7. Sensory changes during the menstrual cycle. It has been commonly ob-
served that women experience changes in perception during the menstrual cycle.
However, attempts of several investigators to measure the magnitude or the
timing of these changes within the cycle have not been consistently successful.
Beiguelman studied taste sensitivity to the bitter substance phenylthiourea
(PTC) during the menstrual cycle and was unable to demonstrate any change
whereas Glanville and Kaplan reported that changes in the taste of PTC did
occur during menses. Olfactory sensitivity to the smell of the musk-like
compound exaltalide was reported to be most acute in the mid-cycle period by
Le Magnen who considered the increased acuity specific for this substance, re-
lated to its supposed sexual proclivity. Koster reported that changes in ol-
factory acuity occurred at various times during the menstrual cycle related
primarily to the length of the cycle itself. These changes were demonstrable
using m-xylol as the test stimulus leading him to suggest that these changes
were based on differences in olfactory acuity rather than to any sexual con-
natation of the stimulus. Changes in auditory acuity during the menstrual
cycle were reported usually during the luteal phase of the cycle. Other in-
vestigators reported that pathological changes in auditory acuity i.e., deafness,
occurred during the menstrual cycle in some patients again usually during the
luteal phase. This phenomenon was called pre-menstrual deafness and usually
remitted in intensity after the onset of menses.
These previous studies suggested that changes in sensory acuity for several
modalities occurred during menses but the nature of these changes and their re-
lationship to the hormonal changes which occurred during the menstrual cycle
were not clearly identified. The purpose of our present studies was to define
the sensory changes which occurred during the menstrual cycle and to attempt
to relate these changes to the hormonal changes that occur during this period.
The present study was carried out in 5 normal female subjects aged 19-23.
Body weight, basal body temperature, detection and recognition thresholds for
the taste of NaCl and for the smell of pyridine in water, detection of sinusoidal
auditory signals, tactile perception on the adductor surface of the palm of the
left hand, two point discrimination and plasma LH, by radioimmunoassay, were
measured either daily or at intervals of 2-3 days in each subject. Studies
demonstrated that sensory detection for all sensory modalities including taste.
,035.
Serial No. NHLI-104(c)
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A}3
Serial No. NHLI-104(c)
smell, hearing, touch and two-point discrimination was more acute during the
follicular phase of the menstrual cycle than during the luteal phase. These
changes were observed whether the menstrual cycle was "short", i.e., less than
28 days or "long", i.e., greater than 28 days. These changes occurred for all
sensory modalities but were more apparent for olfactory acuity than for other
sensory modalities. These effects may be related not only to the effect of
estrogen on increasing sensory acuity during the follicular phase of the cycle
but also to the effects of progesterone on decreasing sensory acuity during
the luteal phase of the cycle.
8. The role of thyroid hormone in sensory function. Taste and smell
thresholds were determined in 18 unselected primary hypothyroid patients,
each diagnosis gased upon unequivocal clinical and laboratory criteria, in-
cluding elevated levels of serum TSH. Median detection thresholds (MDT) and
median recognition thresholds (MRT) for 4 taste stimuli, NaCl, sucrose, HCl
and urea, and 2 smell stimuli, pyridine and nitrobenzene, were determined using
a forced choice - 3 stimulus drop technique for taste and sniff technique for
smell. MDT/MRT in the entire hypothyroid group for taste stimuli were: NaCl
75/90 (normal: 12/30 mM/L); sucrose 30/30 (normal: 12/30); HCl 15/22.5 (normal:
3/6); and urea 500/500 (normal: 120/150), and for smell stimuli were: pyridine
10-3/10-2 (normal: lO'^/lO'^ M/L) and nitrobenzene lO'^/lO'^ (normal: 10"^/
10"^) . Decreased detection of at least one taste stimulus occurred in 14/18
patients and one smell stimulus in 17/18. Subjective responses of hypothyroid
patients to taste and smell stimuli generally indicated markedly diminished
appreciation of the intensity of the stimuli. Symptoms of dysgeusia, including
metallic taste in the mouth, and disagreeable taste of certain foods, were
elicited in 10/18 patients and symptoms of dysosmia in 8/18. Following treat-
ment with triiodothyronine or thyroxine, both subjective and objective improve-
ment in taste and smell were marked, and occurred as early as 3 weeks after on-
set of therapy. These results show that taste and smell functions are frequently
and significantly impaired in hypothyroid patients and that these defects are
reversible with treatment.
Significance: 1 . Role of hallucinogens in neural conduction. We have shown
that LSD- 25 and marihuana do not affect the manner by which the neuraxon is
depolarized. These data, indirectly, support the concept that these drugs carry
out their effects by primarily interfering with synaptic transmission.
2. Role of steroid and thyroid hormones in neural function. A. Adreno-
corticosteroid hormones affect sensory function for all sensory stimuli, i.e.,
vision, audition, taste, smell, hearing and touch. The studies which we carried
out in the past year demonstrated the role these hormones played in touch sen-
sitivity. These effects appear to be mediated through the direct role of the
adrenocorticosteroid on the receptor, the neuraxon, the brain or some combination
of these three systems not through ACTH effects. These steroid hormones serve
to control sensory inflow inhibiting the inflow of signals from the outside
world so that maximum integration of those sensory signals which do get received
can take place. Removal of these hormones results in the failure of normal
integrative phenomenon and information loss. The role of ACTH in this system
is unclear.
6 3^o<f
I
Serial No. NHLI-104(c)
3. Gonadal hormones and pituitary gonadotropins also influence neural
function as measured by visual or auditory evoked responses. Estrogen in-
creases the amplitude of the electrical activity of the brain. However, these
neural phenomena are also influenced by age and genetic factors which are in-
dependent of gonadal hormone function. It appears that absence of the Y chrom-
osome also contributes to the large amplitude electrical activity of the brain
observed in phenotypic females.
4. Thyroid hormones and neural function. In a manner similar to the
ubiquitous effects of adrenocorticosteroids on neural function thyroid hormones
appear to effect receptor, neuraxon and brain function. They also effect sen-
sory acuity for taste, smell and hearing. The manner and extent of these effects
are not clearly defined.
Proposed Course of Project: 1. To define the role of ACTH in
sensory and neural function to evaluate if there is any role of this protein
hormone on neural function independent of its effects on stimulation of adreno-
corticosteroid secretion.
2. To define the role of thyroid function in taste, smell and hearing in
man and animals and to define the role which thyroid hormones play in receptor
function. Similarly, the role which TRH (thyroid releasing hormone) plays in
neural function in relationship to its effects on thyroid hormone secretion
will be evaluated.
3. Sensory studies in patients with various metabolic and nutritional
disorders will continue to evaluate the roles these abnormalities play in the
obtaining of various types of sensory information.
Honors and Awards : None
Publications: Henkin, R.I., Stillman, I.S., Gilbert, D.L. and Lipicky, R.J. :
Ineffectiveness of hallucinogens on altering Na-K currents in
squid giant axon. Psychopharmacologia (In press)
Henkin, R.I., Gilbert, D.L., Stillman, I.S. and DiPolo, R.:
Ineffectiveness of adrenocorticosteroids and adrenocorticotropin
in altering Na-K currents in squid giant axon. Experientia
1973 (In press) .
Catalanotto, F. and Henkin, R.I.: Manual and oral stereognosis
in patients with hypogeusia and hyposmia. Archives of Oral
Biology, 1973 (In press).
Henkin, R.I.: The role of adrenal corticosteroids in sensory
processes. In Sayers, G. (Ed.): Handbook of Physiology, 1973
(In press) .
^or
Serial No. NHLI-105(c)
1. Experimental Therapeutics Branch
2. Section on Neuroendocrinology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Trace Metal Metabolism
Previous Serial Number: NHLI 219 (c)
Principal Investigator: Robert I. Henkin, M.D., Ph.D.
Other Investigators: Eugene Giroux, Ph.D.
N. Prakash, Ph.D.
Joseph Fontana, Ph.D.
J. Levine, M.D.
R, Aamodt, Ph.D.
A.E. Jones, M.D.
G. Johnston, M.D.
C.F.T. Mattern, M.D.
D. Tschudy, M.D.
J.C. Gillin, M.D.
S. McConnell, Ms.
M. Swenberg, Ph.D.
Cooperating Units: Department of Medicine, Presbyterian Hospital, N.Y.,N.Y.,
Department of Pediatrics, Denver, Colorado; Department of
Pediatrics § Obstetrics, N.Y. Hospital, N.Y., N.Y.; De-
partment of Physiology, University of Minnesota, School of
Medicine, Duluth, Minnesota; National Cancer Institute,
Clinical Investigation Branch, National Cancer Institute,
Nuclear Medicine Branch, NIAID, Virology Branch, Bethesda,
Maryland
Objectives : To study the physiology, metabolism, biochemistry and pathology
of copper, zinc and other trace metals in physiological fluids and tissues of
normal subjects, in patients with various diseases and in animals. These stucfi.es
include the interaction between metals and their binding proteins.
Major Findings:
Biochemistry: 1 . Effect of transitional metal ions on (Na + K )ATPase activ-
ity and the uptake of norepinephrine and choline by brain synaptosomes . The
inhibition of (Na* + K*)ATPase activity of rat brain synaptosomes by transition
metal ions takes three forms: (a) Cu** and Hg completely inhibit activity at
a concentration exceeding 10 pM, (b) Zn"*"*" inhibition is intermediate with comr
plete inhibition occurring at concentrations exceeding 100 yM and (c) Co**,
Ni and Mn** inhibition never exceeds 20% even at concentrations exceeding
200 pM.
Ao6
Serial No. NHLI-105(c)
Inhibition of (Na + K )ATPase activity of rat brain synaptosomes by Cu
is rapid, irreversible and unaffected by subsequent addition of EDTA. Prein-
cubation of synaptosomes with Cu'*"'' for as short as 10 min produced complete in-
hibition of (Na"*" + K''')ATPase activity. Preincubation with Zn"*"^ for over 20 min
was required to produce complete inhibition while little or no inhibition
occurred after preincubation with Mn ■•■■*■ or Co*"^ for as long as 30 min.
Except for Mn , uptake of H norepinephrine (NE) and H choline (CH) by
rat brain synaptosomes was inhibited by those transitional metal ions which in-
hibited synaptosomal ATPase activity. For Mn"*"*" up to 300 yM had no effect on
CH uptake whereas NE uptake was depressed by as much as 54% in the presence of
10 yM of Mn"*"*. At this latter concentration (Na"^ + K''')ATPase activity was un-
affected.
2. Competition for zinc among serum albumin and amino acids. A stability
constant at pH 7.4 for the 1:1 zinc-human serum albumin complex was determined.
The effectiveness with which amino acids compete with human serum albumin
for zinc was shown to depend upon the relevant metal complex stability constants.
Studies by gel chromatography of model systems and computations involving sta-
bility constants indicate that cysteine and histidine are the only important
amino acid ligands of zinc in plasma. The amount of amino acid-complexed zinc
in normal human serum is calculated to be 1 yg zinc per 100 ml. The small
ligand-complexed fraction in plasma may be of importance in zinc transport and
probably accounts for most urinary zinc excreted in normal and pathological
conditions.
3. Macromolecular ligands of exchangeable copper, zinc and cadmium in
human serum. Dissociation of metal -protein complexes of copper, zinc and
cadmium in human seriim by histidine and glycine was studied. Distribution of
metal between protein-complexed and amino acid-complexed species as a function
of amino acid concentration is similar for solutions of human serum albumin and
native human serum, indicating that human serum albumin and the macromolecular
ligands which complex loosely bound serum copper, zinc, and cadmium have equal
avidity for these metal ions. Copper-albumin complex is the least stable,
cadmium- albumin complex the most stable and zinc-albumin intermediate with re-
spect to dissociation by histidine.
Physiology
1. Changes in total, non-diffusible and diffusible plasma zinc and copper
during infancy. Concentrations of total, non-diffusible and diffusible zinc and
copper in plasma were studied in 130 normal infants. Total plasma zinc concen-
tration in the newborn was at adult levels, fell to values just below adult levels
within the first week of life, fell further to values significantly below adult
levels at two and three months of age, returned toward adult values at four
months of age and, except for a fall to levels significantly below adult levels
about one year of age, remained at adult levels throughout the remainder of in-
fancy. On the other hand, total plasma copper concentration in the newborn was
at levels significantly below adult levels, gradually rose during the first week
of life, fell to levels significantly below adult levels at two months of age,
rose to levels within the adult range at three months of age, and rose still
2 c^iOZ
Serial No. NHLI-105(c)
higher to levels above the adult range at eight months of age, at which level
values persisted throughout the remainder of infancy. Changes in total plasma
zinc and copper during this period of time were related mainly to changes in
non-diffusible or macromolecular liganded zinc and copper,
2. Inhibition of urinary porphyrins and porphyrin precursors and excretion
of total body zinc in two types of hepatic porphyrin by histidine. Zinc and
porphyrin metabolism were studied in three patients with hepatic porphyria
[one with cutanea tarda (PCT) and two with acute intermittent porphyria (AIP)]
on a metabolic ward at NIH while ingesting a constant diet. Measurements of
urinary uroporphyrin and coproporphyrin in PCT, serum and urinary porphobilinogen
(PBG) and 6-aminolevulinic acid (6-ALA) in AIP, serum and urinary zinc and
copper and total body ^S^n were made during the following 12-16 day sequential
periods: initial control, oral administration of ^^Zn, ZnSO^ (300-400 mg/day) ,
and L-histidine (8-32 g/day) , and final control. ZnS04 significantly increased
serum and urinary zinc, but had no consistent effect on copper, porphyrins, or
porphyrin precursors. L-histidine (24-32 g/day) significantly lowered urinary
uroporphyrin and coproporphyrin in PCT and significantly lowered serum and
urinary PBG and 6-ALA in AIP; concurrently, significant decreases in biological
th of °^Zn, increases in urinary zinc excretion, decreases in serum zinc with-
out significant change in fecal ^^Zn, occurred in all patients compared to the
ZnSO^ period or to the previous control period. Withdrawal of L-histidine re-
sulted in the rapid onset of significant increases in urine and serum 6-ALA
and PBG in AIP, In PCT, urinary uroporphyrin and coproporphyrin remained sig-
nificantly decreased after L-histidine withdrawal for the remainder of the study.
Simultaneous decreases in serum and urinary porphyrin precursors in AIP suggest
that L-histidine is not acting via renal mechanisms. Thus, the observed de-
creases in porphyrins and porphyrin precursors may be related to some direct or
indirect effect of L-histidine on porphyrin metabolism or to an L-histidine-
mediated mobilization and excretion of a tightly bound tissue zinc pool.
3. Control of copper and zinc metabolism by estrogen and progesterone.
The relationships between plasma and tissue concentrations of copper and zinc
and that of several hormones, including adrenal cortical steroids, thyroid hor-
mone, growth hormone, and estrogen have been studied in man and other animals.
Changes in serum concentration of copper and zinc have been reported to occur
during the menstrual cycle and during pregnancy. However, the details of these
physiological and biochemical interrelationships have not been well characterized
and therefore the pituitary-gonadal regulation of copper and zinc metabolism
has not been clearly established.
In order to define these interrelationships, changes in the concentrations
of plasma copper and zinc were studied in intact female rats throughout the
estrous cycle, during pregnancy and pseudopregnancy, in ovariectomized female
rats before and after administration of estrogen and/or progesterone and in
ovariectomized, hypophysectomized rats before and after administration of human
chorionic gonadotropin and pregnant mares' serum gonadotropin. Changes in plasma
copper followed changes in plasma estrogen during the estrous cycle, following
ovariectomy and following administration of exogenous estrogen. Changes in
plasma copper followed changes in plasma progesterone during the estrous cycle,
following ovariectomy and following administration of exogenous progesterone.
3 A>S
Serial No. NHLI-105(c)
Changes in plasma zinc could not be consistently related to changes in either
plasma progesterone or estrogen.
4 . Growth hormone dependent changes in zinc metabolism in man. Serum
zinc and plasma growth hormone concentrations and urinary zinc excretion were
measured in 18 patients with untreated acromegaly before and after treatment
with X- irradiation. These same parameters were also measured in 4 patients with
isolated growth hormone deficiency before and after treatment with human growth
hormone. For the patients with untreated acromegaly serum zinc concentration
was significantly (p < 0.001) below normal (patients, 62 ± 3 yg/100 ml, M ± 1
SEM; controls, 92 * 2 ug/100 ml) while urinary zinc excretion was significantly
(p < 0.001) greater than normal (patients, 1318 ± 154 yg/24 hours, controls,
456 ± 23 )ig/24 hours). Plasma growth hormone levels ranged from 5 to 160
myg/ml. Following treatment serum zinc concentrations in patients with acro-
megaly increased significantly (p < 0.01) to 82 ± 5 ug/100 ml, urinary zinc
excretion decreased significantly (p < 0.01) to 580 ± 223 yg/24 hours and
human growth hormone levels fell. In patients with untreated isolated growth
hormone deficiency serum zinc concentration was elevated above normal and
urinary zinc excretion was below normal levels. Serum zinc concentration de-
creased following treatment with human growth hormone whereas urinary zinc ex-
cretion increased.
These data demonstrate that growth hormone levels in serum are inversely
related to levels of zinc in serum and directly related to urinary excretion
of zinc. These changes may be related to the manner by which human growth
hormone either directly or indirectly affects the binding of zinc to macromo-
lecular ligands in blood and thereby the urinary excretion of zinc bound to
micromolecular ligands.
5. The role of adrenal corticosteroids in the control of zinc and copper
metabolism. Serum zinc and copper concentration and urinary zinc and copper
excretion were measured in 8 patients with adrenal cortical insufficiency or
panhypopituitarism on and off hormonal replacement therapy and in 8 patients
with Gushing' s syndrome without treatment and after treatment with drugs which
suppressed endogenous secretion of adrenalcorticosteroids or after surgical
adrenalectomy. These same parameters were also measured in 10 normal volunteers
before and after administration of adrenocorticotropin (AGTH) , 40 units given
intravenously over 8 hours each day for 1-4 days or after administration of the
carbohydrate-active steroid prednisolone, 50 mg orally for 5 days. Serum zinc
and copper concentrations were also measured in 20 cats prior to and after sur-
gical adrenalectomy and after replacement with either carbohydrate-active
steroids, Na-K active steroids or both hormones. Diffusible and non-diffusible
zinc and copper were also measured in each patient group by ultrafiltration
through a membrane which allowed the passage of proteins or molecular weight
greater than 20,000 but retarded proteins of heavier molecular weight.
Patients with adrenal cortical insufficiency on adequate hormonal replace-
ment therapy exhibited sertim concentrations of zinc and copper and urinary ex-
cretion of zinc and copper which were not significantly different from that of
controls. After withdrawal of hormonal replacement therapy both serum zinc
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Serial No. NHLI-105(c)
and copper concentration increased significantly while urinary zinc and copper
excretion decreased. Significant increases in serum zinc and copper also
occurred after adrenalectomy in cats. After replacement therapy with carbohy-
drate-active steroids alone or with added Na-K active steroids semm zinc and
copper concentrations decreased to normal levels and urinary zinc and copper
excretion increased. In normal volunteers ACTH administration decreased serum
zinc and copper concentrations during the first day of administration and in-
creased urinary zinc and copper excretion as did oral administration of pred-
nisolone. Patients with Gushing 's syndrome exhibited serum zinc and copper con-
centrations which were significantly lower than normal and urinary zinc and
copper excretion which was significantly greater than normal. Treatment with
adrenal cortical suppressive agents or with surgical adrenalectomy resulted in
a return of serum zinc and copper concentrations to normal and a lowering of
urinary zinc and copper excretion.
These studies indicate that an inverse relationship exists between levels
of plasma Cortisol and serum zinc and copper concentration whereas a direct
relationship exists between plasma Cortisol and urinary excretion of zinc and
copper. These changes can be related to a direct effect of Cortisol on the
production of increases in diffusible serum zinc and copper, subsequent decreases
in both serum zinc and copper concentrations and an increase in urinary zinc
and copper excretion.
6. The role of zinc in growth and development. Zinc has been considered
to be essential for normal growth and development. However, zinc deficiency,
per se, has always been associated with anorexia which obviously limits growth
and development. In an effort to separate zinc deficiency from anorexia 4
groups of rats were studied. One group received zinc deficient diet, ad lib,
while a second was pair-fed with zinc supplemented diet to this group. A third
group received zinc supplemented diet ad lib while a fourth group was forced
fed a zinc deficient diet in an amount~as near as possible to that of the ad lib
rats fed zinc supplemented diet. Growth and organ weight of groups 1 and 2~and
of groups 3 and 4 were comparable except for the decreased size of testes in
group 1. However, serum zinc concentration and urinary zinc excretion of the
rats fed zinc deficient diet, groups 1 and 4, were similar and were low whereas
that of groups 2 and 3 were similar and were high and significantly different
from normal. These data indicate that anorexia rather than zinc deficiency per
se is the relevant factor in the production of growth retardation in zinc de-
ficiency in rats.
7. The role of zinc in the morphology of the testes and in the secretion
of testosterone. To investigate whether or not zinc plays a specific role in
the function of the gonad per se we compared the in vitro formation of testos-
terone (T) and dehydroepiandrosterone (DHEA) from 1,2 H3 cholesterol in testi-
cular tissue of zinc deficient and pair-fed rats. Morphologic evaluation of
testes from both groups of rats was also performed.
Results showed that body weight of the pair-fed and zinc deficient rats
were the same but that plasma zinc concentration and testes weight of the zinc
deficient rats were significantly lower tliat their pair-fed controls (p < 0.005,
t test). Results also showed that testicular tissue from zinc deficient and
pair-fed control rats produced the same amount of T and DHEA (Table 1).
Serial No. NHLI-105(c)
3 3
TABLE 2 Synthesis of H testosterone (T) and H dehydroepiandrosterone
(DHEA) from 1,2 H^ cholesterol by testes from zinc deficient
and pair-fed control rats
Zinc Deficient
Pair-Fed Control
T
7.2 ± 0.8""
8.83 + 2.9
DHEA
2.5 ± 0.4*
2.8 ± 0.6
*(Mean dpm/gm tissue + 1 SEM) x 10 ; * (Mean dpm/gm tissue ± 1 SEM) x 10'
Morphological examination of testicular tissue from zinc deficient rats re-
vealed a spermatogenic arrest. These studies indicate that zinc deficiency,
per se, does not directly decrease gonadal hormone secretion and suggest that
the major effect of zinc deficiency on gonadal function is through some alter-
ation of pituitary gonadotropins.
8. Role of zinc ion in LH secretion. Since testosterone secretion was
similar from the testes of zinc deficient and pair-fed rats the defect in zinc
deficiency was related to release of testosterone. This placed the defect in
the hypothalamus, the pituitary or in some combination of these two tissues.
In order to evaluate this possibility four groups of rats fed the following
diets were studied: (1) zinc deficient diet, ad lib, (2) pair-fed with (1)
with zinc supplemented diet, (3) zinc supplemented diet, ad lib, (4) force fed
zinc deficient diet in an amount similar to that eaten by group (3) . Serum
zinc concentration and urinary zinc excretion revealed groups 1 and 4 to be
zinc deficient whereas groups 2 and 3 were zinc replete. Measurement of pitui-
tary LH concentrations were similar in groups 1 and 2 but were significantly
lower in groups 3 and 4 (Table 2). These data indicate that anorexia rather
than zinc deficiency per s£ results in a significant decrease in pituitary LH
content. The pituitary LH deficit in zinc deficient rats may be altered if
caloric intake can be increased even though total body zinc is apparently
decreased.
TABLE 2 CONDITION LH (yg,mg pituitary)
Group I Zn Deficient Diet, ad lib 1.06
Group II Pair-Fed with I Zn supplemented Diet 0.82
Group III Zn Supplemented Diet, ad lib 1.40
Group IV Force Fed Zn deficient Diet 1.94
6 ^//
Serial No. NHLI-105(c)
9. Effects of L-histidine and zinc on serum and urinary zinc and copper
in rat. In an effort to obtain a satisfactory model system in which the effects
of L-histidine could be studied over a long period of time 21 male Sprague
Dawley rats were housed individually in metabolic cages and divided into 3
groups of 7 each. Group I received zinc supplemented diet alone, Group II
received zinc supplemented diet with histidine added at 3 concentrations,
1 g/kg, 5 g/kg and 7.5 g/kg. Group III received the same diet as Group II
only with additional zinc, 8 g/kg. Growth rate of the three groups of rats
were similar. However, urinary zinc excretion was significantly greater in
the histidine and histidine + zinc fed rats than in the rats fed zinc supple-
mented diet alone. These data indicate that zinc and copper excretion can be
significantly increased by feeding L-histidine in rat.
10. Measurements of salivary zinc and copper in normal man and in
patients with various disease states including disorders of taste and smell.
Because of the low concentration of zinc and copper in saliva it has been im-
practical to measure these metals in this important fluid. However, with the
use of the atomic absorption flameless sampler in which the entire sample is
combusted under control conditions it is possible to measure zinc and copper
in the range of 1 ppb instead of the 0.5 ppm which was the lower limit of
accuracy with the flame. With this technical advance it was ascertained that
parotid saliva, collected via a Lashley cup method, in normal man was fairly
uniform, independent of rafe of flow and was between 30 to 100 ppb for Zn
and 15 to 40 ppb for Cu.
In general, patients with hypogeusia which occurred following an upper
respiratory infection, following surgery or head injury exhibit significantly
lower than normal parotid salivary concentrations of zinc, between 0 to 20 ppb
whereas concentrations of copper, Na and K are within normal limits. Treatment
of patients with zinc ion produces two types of changes. One group of patients
treated with zinc ion exhibit significant increases in parotid salivary zinc
concentration to levels as high as 2 times the upper limit of norm.al whereas
the other group shows no change over those levels measured prior to treatment.
However, serum zinc levels and urinary zinc excretion increases in both groups
of patients. It is of great interest that only those patients who exhibit a
significant increase in parotid salivary zinc concentration exhibit a concomi-
tant subjective and objective improvement in taste acuity whereas those patients
in whom little or no change in salivary zinc concentration occurs exhibited little
or no subjective or objective change in taste acuity after treatment. Increases
in salivary zinc are always accompanied by a concomitant increase in protein.
These data suggest that in those patients in whom zinc ion administration pro-
duces an increase in both zinc and salivary parotid protein the zinc ion has
induced a zinc containing salivary protein. This protein has been found in nor-
mal subjects with normal taste acuity and where levels are low it is always
associated with hypogeusia, even in apparently "normal subjects" who do not com-
plain overtly of hypogeusia. This parotid salivary protein may act as a taste
supporting protein.
^a
Serial No. NHLI-105fc>
11. Copper and zinc metabolism in Schizophrenia. Zinc and copper concen-
trations were studied in blood, urine, CSF, gastric fluid, and hair of 20 patients
with unmedicated acute and chronic schizophrenia. Ceruloplasmin was measured
in blood in each patient. Mean values for the patients for each tissue studied
were as follows: serum, Zn 92 ± 3, yg/100 ml (mean ± 1 SEM) , Cu 93 ± 5, yg/100
ml; urine, Zn 286 ± 35 yg/24 hr, Cu 30 ± 3 yg/24 hr; CSF, Zn 2 ± 1 yg/100 ml ,
Cu 3 ± 1 yg/100 ml; gastric fluid, Zn 42 ± 4 yg/100 ml, Cu 7 ± 2 yg/100 ml; hair,
Zn 136 ± 6 yg/100 ml. These values are all within normal limits and suggest that
patients severely ill with acute and chronic schizophrenia do not differ from
normal in any parameter of zinc or copper concentration in any tissue studied.
Although treatment with zinc or copper chelating agents have been used with some
frequency in patients with schizophrenia this therapy cannot be based on abnor-
malities of zinc or copper levels in several tissues of these patients.
12. Alterations in copper and zinc metabolism following administration of
6-azauridine-triacetate. Various drugs alter copper and zinc metabolism in man.
6-azauridine triacetate (6-Az) is an antimetabolite which inhibits de_ novo
pyrimidine synthesis and has been used in man for treatment of a variety of
diseases. It also alters amino acid metabolism producing increases in serum
of thiol containing amino acids and of histidine. Since the relationship be-
tween these amino acids and the metabolism of copper and zinc has been under
intensive study in our laboratory over the past few years we studied 7 women
with progressive systemic sclerosis given 6-Az in order to evaluate the changes
in blood and urine copper and zinc which occurred following its administration.
The drug was given orally for periods of 7 days beginning with 3 g daily and
increasing by 3 g daily each 7 day period of a total of 4 periods and a maxi-
mum dose of 12 g daily. Significant decreases in total serum zinc and copper
were found in each patient (p < 0.05) whereas significantly increased urinary
zinc excretion occurred (p < 0.01). These changes can be directly related to
the hyper-aminoacidemia and hyper-aminoaciduria (particularly histidine and
thiol containing amino acids) which occurred following administration of this
drug. The decreased serum zinc concentration and increased urinary zinc ex-
cretion was dose related to the amount of drug administered.
13. Zn M and Zn metabolism in patients with various abnormalities of
zinc metabolism. Because of the need to study the absorption, distribution
and excretion of zinc in man Zn°^ and Zn^% have been administered intravenously
and orally to 23 patients with several abnormalities of taste and smell, with
Addison's disease, with hepatic porphyria and to those with cystinuria taking
D-penicillamine. Results are still being processed for most of these studies.
However, preliminary data suggest that in man zinc is absorbed to a large part
in the stomach and not in the duodenum as in rat. Other preliminary data in-
dicate that D-penicillamine increases the normal rate of zinc absorption from
about 30% of the injected dose to over 95% of the injected dose and is also
active in enhancing its time of excretion.
^/3
Serial No. NHLI-105(c)
14. The Environmental Protection Agency (EPA) Panel on Zinc. The EPA
has established several panels to ascertain the present state of knowledge about
various metals. These include chromium, lead, magnesium, copper, and zinc.
These panels have been set up under the auspices of the National Academy of
Science. I have been designated Chairman of the Panel on Zinc for the EPA. The
task of the panel is to publish a position paper on the one hand with respect
to possible toxic levels of this element in the environment or in other sources
such as food or water products and on the other with respect to inadequacy of
zinc content in the environment or in sources of this element reaching man.
Significance:
1. Metal-protein interactions. We have established that copper-cerulo-
plasmin and zincQi2 macroglobulin complexes are metalloproteins which act as
blood storage forms of metals and that copper- albumin and zinc-albumin complexes
are the physiologically active metalloproteins in blood. These latter macro-
molecular ligands are in equilibrium with micromolecular ligands in serum
mainly the amino acids, histidine and cysteine. The metals are conserved and
protected against renal loss by their complex formation with large molecular
weight proteins. If excessively large concentrations of histidine or cysteine
appear in blood as occurs in hepatitis or cancer or after histidine administration
then these metals are lost from the body and metal depletion occurs. This sys-
tem has allowed for a useful model of the production of Wenecke's encephalopathy
via zinc loss with histidine administration. This system can also be utilized
for the treatment of metal poisoning and may be one of the most effective forms
of treatment of metal toxicity, including lead or mercury. It has been shown
to be effective in vitro in removing cadmium from its albumin binding site.
2. Role of zinc in growth and development. The immediate major role of
zinc in growth appears to be through its control of food intake for if this
metal is removed from the diet man or animals will stop eating. Our studies
suggest that the endocrine and growth effects of this metal are due to its
effects on food intake rather than to any direct effect on hormone metabolism.
3. Age-related changes in copper and zinc. No previous data about copper
and zinc levels in man between birth and 2 years of age have been published.
We have filled in this gap. We have also suggested that age-dependent changes
in total serum copper and zinc levels are due largely to increases in serum
levels of the major transport form of copper, ceruloplasmin and to the macro-
molecular ligands which bind zinc.
4. Drug and hormonal effects on zinc metabolism. Because zinc is a criti-
cally important trace element its loss produces severe pathological changes in
several organ systems. Therefore, it is important to identify drugs or other
agents which alter zinc metabolism and it is important to specify their mode of
action, 6-Azauridine and histidine produce zinc depletion by altering the
manner by which zinc is complexed with alblumin. Similar changes have been pro-
posed for the action of growth hormone. Adrenal cortical steroids may act via
a direct role on the kidney since they do not appear to alter directly the for-
mation of the zinc-albumin complex in blood. We have also shown for the first
time that progesterone directly affects blood copper levels probably through
induction of ceruloplasmin. This means that both estrogen and progesterone
induce this metal loprotein.
9 Sl</
Serial No. NHLI-105(c)
5. Treatment of the hepatic porphyria with histidine. Data in four sub-
jects with hepatic porphyria indicate that histidine administration either sig-
nificantly inhibits the production of urinary uroporphyrin and coproporphyrin
or inhibits the production of porphyrin precursors. Our initial data suggest
that this inhibition occurs through a histidine inhibition of 6 amino acid levu-
linic acid and suggests that histidine may play some role in heme synthesis.
6. Treatment of patients with hypogeusia with zinc ion. Successful
treatment of patients with idiopathic hypogeusia with zinc ion is uniformly
associated with the production of a zinc containing protein in parotid saliva.
These data suggest that zinc acts to induce the production of this protein in
the same manner that copper induces ceruloplasmin in the blood of patients
with Menkes Steely Hair Syndrome. Previously only hormones and similar agents
have been associated with the induction of metal carrying proteins but the
present data suggest that a metal itself may be involved as an inducer of its
own carrying protein under specific conditions. The induction of this protein
is dose dependent upon the amount of administered zinc ion. In some patients
25 mg of zinc ion is too little whereas 50 mg of zinc ion is adequate to induce
this protein. Removal of zinc ion from these patients is associated with the
loss of this protein from saliva. This protein appears in normal parotid saliva.
Since taste buds do not contain blood vessels or lymphatics the presence of
this zinc containing parotid salivary protein serves to support taste bud
function. These studies are the first in which a specific role for saliva in
taste has been observed and they firmly relate the role that zinc plays both
in saliva and in taste.
In patients with idiopathic hypogeusia in those in whom this protein is in-
duced by zinc ion taste acuity returns to normal following zinc administration.
Continued administration of zinc ion is necessary for continued function and
continued production of the protein. In those patients in whom zinc ion dies
not induce this protein taste function does not return to normal. These results
suggest two possible modes of therapy: (1) since the induction of this protein
is dose-dependent with respect to zinc, and since zinc toxicity occurs at
levels 20 times the largest dose given in any of our studies, administration
of larger amounts of zinc ion may be effective in inducing this protein, or
(2) in those patients in whom this protein cannot be practically induced it
should be possible to supply this protein topically and return acuity to normal
in this manner.
Proposed Course of Project:
1. The several mechanisms by which hormones and drugs affect zinc metabolism
will be investigated.
2. The role of various diuretic agents and their cysteine and/or histidine
complexes will be studied to investigate the direct role these agents play at
the ascending loop of Henle and in the proximal tubule.
10 ^/r
Serial No. NHLI-105(c)
3. Kinetics of Zn and Zn M will be studied in man to learn more about
the metabolism of this metal.
4. The zinc containing protein in parotid saliva will be isolated and its
characteristics evaluated. The relationship between this metalloprotein and
the normal and abnormal metabolism of zinc in normal subjects and in patients
with various diseases will be investigated. Isolation of this protein should
offer a useful tool by which patients with various taste disorders may be
treated.
Honors and Awards : None
Publications :
1. Giroux, E.L. and Henkin, R.I.: Macromolecular ligands of exchangeable
copper, zinc and cadmitim in human seriam. Bioinorganic Chem. 2: 125-
133, 1972.
2. Prakash, N., Fontana, J. and Henkin, R.I.: Effect of transitional
+ +
metal ions on (Na ♦ K )ATPase activity and the uptake of norepinephrine
and choline by brain synaptosomes . Life Sci. 12: 249-259, 1973.
3. Sato, N. and Henkin, R.I.: Pituitary-gonadal regulation of copper and
zinc metabolism in the female rat. Amer. J_. Physiol . (In press).
4. Henkin, R.I,, Schulman, Joseph D., Schulman, Carol B., Bronzert, Diane:
Changes in total, non-diffusible and diffusible plasma zinc and copper
during infancy. J_. of Pediatrics 82: 831-837, 1973.
5. Slavik, M., Danilson, D., Keiser, H. and Henkin, R.I.: Alterations in
metabolism of copper and zinc following administration of 6-azauridine
triacetate. Biochemical Pharm. (In press).
11 <3/6
Serial No. NHLI-106(c)
1 . Experimental Therapeutics Branch
2. Section on Neuroendocrinology
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Taste and Olfaction
Previous Serial No.: NHLI-220 (c)
Principal Investigator: Robert 1. Henkin, M.D., Ph.D.
Other Investigators: Clark Lum, Ph.D.
Paul J. Schechter, M.D., Ph.D.
Eugene L. Giroux, Ph.D.
A. L. Larson, M.D.
Frank Catalanotto, D.D.S.
C.F. T. Mattern, M.D.
Navah Paran, Msc
Cooperating Units: Department of Psychology, Duke University, Durham, N.C.;
Presbyterian Hospital, N.Y.,N.Y.; Cambell Institute for
Food Research, Camden, N.J.; NIAID:LCI, NEI, NCI: Surg.,
NIDR:OMS, NIAMD: A 5 R, NCI: Pathological Anatomy Branch.
Project Description:
Objectives: To investigate in a systematic manner the anatomical, physiologi-
cal, pharmacological, pathological and chemical correlates of taste and ol-
faction.
Major Findings: Taste
Anatomy. 1. Architecture and function of taste buds. A. Three major cell
types comprise the 40-60 cells which make up the taste bud of man and other
animals. Type I cells comprise approximately 80% of the taste cells. Type II
cells, approximately 15% of the taste cells and Type III cells, 5% of the
cells. Although the function of these cell types are not completely understood
synapses are structured such that brain — > taste bud connections are present
in Type I and II cells whereas taste bud >■ brain connections are present
only in Type III cells. Helical bundles of tonafilaments have been observed
in Type II cells and we have suggested that their function lay in contraction
and relaxation such that the structures of the pore might be protected from
noxious or toxic substances which enter the oral cavity and which might injure
the taste bud. Using relatively primitive techniques with light microscopy two
years ago we observed what appeared to be opening and closing of fungiform
papillae in mouse tongue following the application of noxious substances such
as high concentrations of HCl. Recently using an episcope, TV camera and a
video tape recorder Mattern has demonstrated an outpouring of cell fluid from
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Serial No. NHLI-106(c)
the pore of the fungiform papilla of a mouse tongue in response to the appli-
cation of the vapor of high concentrations of HCl. This is compatible with
the proposed function of the Type II cell. Preliminary studies with reserpine
and similar agents suggest that this mechanism is controlled by adrenergic
agents since reserpine injected into the mouse blocks this action.
B. Using available clinical and physiological information we hypothesized
that the major neurochemical transmitter system in the taste bud must be cho-
linergic in nature. In an effort to evaluate this hypothesis we predicted that
acetylcholine and acetylcholinesterase had to be present at the pore of the
taste bud. Studies to demonstrate the validity of this hypothesis, using histo-
chemical electron microscopic techniques, have indeed showed the presence of
acetylcholinesterase distributed along the finger-like projections of the taste
bud cells within the taste pore. Appropriate controls, including eserin and BW
completely inhibited the acetylcholinesterase reaction. However, to date,
acetylcholine has not yet been demonstrated by autoradiography using radio-
active precursor material. In another series of experiments various ganglionic
blocking agents have been presented orally to man and taste thresholds for four
taste qualities and forced scaling values have been obtained. In these studies
the depolarizing post -ganglionic blocking agent succinyl choline was effective
in producing hypogeusia for all taste qualities. The non-depolarizing pre-
ganglionic blocking agent curare produced ageusia in man. The non-depolarizing
post ganglionic blocking agent panchoronium also produced hypogeusia as did the
drug methacholine which acts like succinyl choline in competing for acetylcho-
line sites at the post ganglionic membrane. These studies strongly suggest
that neurochemical events involving cholinergic transmission, probably involving
acetylcholine probably control the generator potential of the taste bud. Our
anatomical evidence of synapse direction suggests that this event takes place
at the Type III cell; therefore this cell is probably the receptor cell of the
taste bud. Thus the tastant-receptor complex probably forms at the plasma mem-
brane of the Type III cell. It may be hypothesized that the function of the
Type I cell, the only taste cell in which neurosecretory granules are found, is
to supply acetylcholine for normal taste bud function. The anatomical obser-
vations of synaptic direction, in part, supports this hypothesis.
C. Effects of curare on response to NaCl and HCl in rat. Female Sprague-
Dawley rats were anesthetized, their chorda tympani nerves exposed and the neural
output monitored by a recorder and counted by an EPUT meter. By this method
electrical discharges from the nerve were summed for periods of 1 sec. NaCl
and HCl of various concentrations were placed on the rat tongue and the responses
recorded before and after placement of curare, 3 mg/ml on the tongue. Prior
to placement the usual increasing frequency dose-response relationship was
observed for increasing concentrations of NaCl (30-1000 mM) and HCl (15-150 mM) .
Following placement of curare response to NaCl occurred only following placement
of a 1000 mM solution and that response was 1/3 the untreated value; response
to HCl first occurred at a 60 mM concentration and the responses were also 1/2
normal. These results in rat verify the psychophysical responses obtained in
man and indicate that taste responses in rat may be greatly inhibited by non-
depolarizing preganglionic blockage.
<?/«
Serial No. NHLI-106(c)
Chemistry
1. Purification and some properties of tlFGF, a taste-modlfylng glycoprotein
from miracle fruit. MFGP (miracle fruit glycoprotein) Is the name given a taste
modifying glycoprotein purified from "Miracle Fruit", Synsepalum dulclflcum. We
have derived a new purification scheme for MFGP which involves adsorption of the
miracle fruit polyphenols by insoluble polyvinylpyrrolidone followed by three
ion-exchange column chromatography steps. Some of the chemical and physical
properties of MFGP were determined. Our results extend those of others although
results on the carbohydrate constituents are contradictory. MFGP contains 6.3%
carbohydrate, 14.4% nitrogen and has a molecular weight of approximately 45,000.
When denatured and reduced it is cleaved into two fragments, of approximately
28,000 and 17,000 mol. wt. The amino acid and carbohydrate composition of MFGP
have been determined. Fucose xyloee, mannose and galactose have been confirmed
in acid hydrolyzates of the glycoprotein. Twenty micrograms of MFGP produces a
marked increase in sweetness of lemon and concomitantly a marked diminution of
sourness. This yield and activity is more than three times that reported by any
other group.
2. Chemistry of miracle fruit glycoprotein (MFGP) . Further Isolation and
purification of MFGP. A modification of the initial procedure used to isolate
and purify MFGP has been made to reduce the time and number of chromatography
steps previously used. These modifications consist of: (1) reducing the volume
of 0.1 M carbonate buffer used in extraction to a volume of 3 times the net
weight of the berry pulp; (2) reducing the time for extraction to 30 minutes for
stirring with PVP; (3) adjusting the pH of the extract to 6.0 prior to passing
it through the first Bio Gel MC-30 column and (4) washing the column with pH 6.0
buffer and then pH 6.5 phosphate 0.1 M buffer. The first crude carbonate extract
showed physiological activity 5 times stronger than the second extract when the
pulp was repeatedly extracted in the same manner. All the other steps and amount
of reagent used were the same as used previously.
The active fractions eluted from the Bio Gel MC-30 column were colorless
and exhibited one single band with either Commassie Brilliant blue (for protein)
or with Schiff 's base (for glycoprotein) in Disc electrophoresis. These fractions
have remained stable at 4°C for 4 months.
Further purification with chromatography on Sephadex-DEAE with NaCl linear
concentration gradient elutlon (0.05 and 0.65 M in 0.1 M carbonate buffer, pH
10.5) was carried out. The active fraction so obtained was dialyzed against
water and lyophylized. The dried protein was examined for biological activity.
A linear relationship was obtained at 4' to 100 yg between the logarithm of
amount of protein obtained (yg) v.s. relative sweetness of citric acid (C.02 M) .
3 . The effect of Smith oxidation and borohydride reduction on MFGP. The
pH of MFGP was adjusted to 4.5 with 1.0 N acetic acid. As a control 10% dis-
tilled water was added to an aliquot of this solution. Into the MFGP solutions
at pH 4.5 NalO^ was added to make solutions ranging in concentration from 10~7
to 10~3 M. The mixtures were kept at 4°C, in darkness, for 5, 15, 30, 60, 75,
120 minutes. At the end of reaction 50 pi of sample was quickly drawn and tested
by bioassay. The reaction was terminated by adding one or two drops of ethylene
glycol. The activity of all reaction mixtures were reduced from 40% to 100% in
5 to 60 minutes at various NalO^ concentrations; at 3 x 10"^ M 40% redaction
3 ^(9
Serial No. NHLI-106(c)
occurred In 5 minutes, 56% in 15 minutes, 60% in 30 to 120 min.; at 5 x 10~5 M,
60% reduction in 15 min., 75% in 45 and 75 min.; at 10"^ M 68% in 30 min. and
100% reduction in 60 min. At 3 x 10"^ M NaCl solution after 40 hours the MFGP
lost all its sugar bioactivity and the solution of 0.02 M citric acid was taste-
less (no sourness was detected). This might have occurred earlier than 40 hours.
The reducing sugar was decreased as the sugar bioactivity decreased but the pro-
tein bands were unchanged; the disc electrophoreses showed a single band iden-
tical to the control. This indicates the polysaccharide moiety plays an impor-
tant role in the taste modifying effect of MFGP. These results also suggest that
the binding effect of protein which apparently has a blocking effect was not
effected by NalO^ at the concentrations tested.
Reduction with sodium borohydride: Dialized MFGP was dissolved in 0.1 M phos-
phate buffer pH 8.0 or the pH of MFGP solution was adjusted to 8.0 with Na2HP04
solution (0.1 M). 10% of 10"^ M NaBH4 was added to this solution and kept at 4°C
for 30 min. to 27 hours. At the end of the reaction samples were drawn and
tested quantitatively for activity. The reaction was terminated by adjusting pH
of reaction mixture to 3.5. At pH 7.6, 8.0 and 8.4, all solutions increased
their activity with different optimum reaction time. At pH 8.0 the activity
reached maximum at 2.5 hours but at pH 7.6 and 8.4 more than 20 hours was required.
At pH 8.0, NaBH^ 10"^ M the maximum activity increased to about 300% over control
in 2.5 hours and remained for at least 3 hours. The activity decreased after 10
hours and returned to the level of initial activity after 27 hours. At the maxi-
mal increase in activity, the protein moiety of MFGP remained unchanged as shown
by disc electrophoresis and by Dawely assay; however, the amount of reducing
sugar was reduced 10%. As the activity decreased the content of the reducing
sugar was also continuously decreased. This suggests the possibility that one
sugar was reduced first and the change in this sugar is responsible for the in-
crease in activity. The further reduction of the other sugars, on the other
hand, may be responsible for decreasing activity.
4. Hydrolysis of MFGP. All the neutral sugars was completely liberated
in 20-40 minutes in 1.0 N trif lugloacetic acid in an evacuated sealed tube
containing N2 at 105°C. Under the same condition in 2.0 N HCl only xylose and
mannose were liberated after 1-4 hours of hydrolysis. Further studies were
undertaken to determine the sequence of polysaccharides by the alternating actions
of Smith oxidation and borohydride reduction on the various sugars. Tritiated
MFGP derivatives have been prepared for the studies in structure-activity inter-
relationships, the binding specificity of MFGP and quantitative assay by bio-
chemical means rather than by bioassay.
5. Preparation of taste bud membranes. The isolation procedure of taste
bud membranes from bovine circumvallate papillae was performed by initial treat-
ment of excised bovine circumvallate papillae with freezing, hypotonic swelling,
nitrogen pressurization, selective homogenization and filtration. The filtrate
was then treated to isolate purified taste bud membranes by differential and su-
crose gradient centrifugation from the nuclear and microsomal pellets. The iso-
lated taste bud membranes were washed three times in a Kreb-bicarbonate buffer,
pH 7.4 and dialyzed overnight against the same buffer solution in order to obtain
a sucrose free taste bud membrane suspension. The exicsed epithelial tissues
were treated in the same manner described previously in the taste bud membrane
isolation.
A 3t^
Serial No. NHLI- 106(c)
6. Measurements of specific binding of sugars to taste bud membranes.
Incubations were carried out at 30 C in plastic micro test tubes (Capacity
0.4 ml). The incubation medium contained 10 mM Kreb-bicarbonate buffer, pH
7.4 and labeled and unlabeled sugars at various concentrations. Sugars used
in the study were labeled and unlabeled sucrose, fructose, glucose, cyclamate,
and saccharine. Taste bud and epithelial membrane suspensions (7-15 pg protein)
were suspended in the incubation medium to complete a reaction volume of 100 yl.
After incubation, with shaking for 30 min. at SO'C, 50 yl of 1% polyethylene
glycol, m.w. 6000 was layered on top of the incubation mixture. The tubes
were centrifuged immediately in a Beckman microcentrifuge for 3 min. The
supernatant fluid was aspirated with a very fine tip Pasteur Pipette attached
to a vacuum line. The pellet was washed once by adding 50 yl buffer and then
the fluid was aspirated as described above. The tip of the plastic centrifuge tube
was cut off with a razor blade at a point just above the pellet and was trans-
ferred to a 10 ml liquid scintillation fluid for radioactivity counting.
Biological specificity of sugar binding on the taste bud membranes hae been
demonstrated for all sugars studied as indicated by the decrease of relative
labeled sugar binding on the taste bud membranes when concentrations of un-
labeled sugar are increased. However, there was no displacement of labeled
sugar on the epithelial tissue fraction by the addition of unlabeled sugar
indicating the non-specificity of binding to other than taste bud membranes.
The specific binding activities of sucrose, fructose, glucose, cyclamate
and saccharine on the taste bud membranes and on corresponding epithelial
tissue were placed on Scatchard plots. The resulting curves showed good
linearity for sucrose, fructose and glucose, indicating that only a primary
group of binding sites was present on the taste bud membranes. The resulting
points showed non-linearity for cyclamate and saccharine, indicating there was
a primary group of binding sites present on the initial linear portion of the
curves, and a secondary group of binding sites present on the lower non- linear
portion of the curves .
The specific binding properties of sucrose, fructose, glucose, cyclamate
and saccharine are shown in Table 1. The order of binding affinity was
sucrose > fructose > glucose > cyclamate > saccharine. The highest sugar concen-
tration bound per mg membrane protein was fructose, the lowest concentration
of sugar bound was sucrose.
Taste bud membranes isolated from taste buds of bovine circumvallate
papillae have shown specific biological activities on sucrose, fructose, glucose,
cyclamate and saccharine. The specific biological activities for sucrose,
|fructose and glucose are in the same order of magnitude as behavorial responses
'in cow, and the concentrations of specific binding affinities for sucrose,
fructose, glucose, cyclamate and saccharine are similar to the reported recog-
nition thresholds for these sugars in man.
Sid- 1
I
Serial No. NHLI- 106(c)
TABLE 1 Binding Sugars to Taste Bud Membranes
Sugar Kd\ (f
(x 10"^ M) (x 10"2 M/mg protein)
0.5
10.5
2.5
4.0
6.6
2.7
6.4
Dissociation constant
2
Sugar concentration bound per mg taste bud membrane protein
Sucrose
1.06
Fructose
1.76
Glucose
3.36
Cyclamate
1"
11.67
1"
32.57
Saccharine
1'
18.28
1"
66.23
Physiology
1 . Influence of intake of various substances on the intake of tastants
and other substances. A. The intake of dextrose influences the intake of salt.
Adult Sprague-Dawley male rats were given lab chow to which 0, 10%, 20%, 30%,
40%, 50%, and 60% dextrose was added for one or two weeks. During the intake
of this diet rats were offered a choice between 0.30 M NaCl and water. Normally
rats reject 0.30 M NaCl and prefer water. With 0 or 10% added dextrose rats
preferred water to NaCl. However, with concentrations of 20% dextrose or
greater rats took in significantly greater quantities of NaCl than under control
conditions without dextrose or with 10% added dextrose.
^S^
Serial No. NHLI-106(c)
Condition
Control - no added dextrose
10% - added dextrose
20% - added dextrose
30% - added dextrose
50% - added dextrose
60% - added dextrose
Preference for 0.30 M NaCl
1st Week 2nd Week
20 ± 5% (Mean ± 1 SEM) 27 ± 4%
25 ±4% 27 ± 5%
44 ±7% 44 ± 8%
51 ± 5%
48 ± 5%
46 ± 7%
Upon dlscontiniiatlon of added dietary dextrose in diet (60% added) more
than 3 weeks was required before a significant decrease in preference for
0.30 M NaCl occurred.
B. The intake of monosodium glutumate (MSG) and NaCl influences the intake
of bitter substances. Studies carried out in man suggested that MSG decreased
taste thresholds for bitter substances. In order to study this phenomenon
systematically male Sprague Dawley rats, 120 * 10 g (Mean ± 1 SEM) were housed
individually and divided arbitrarily into groups of 3. Each rat was fed daily
with 10 g lab chow containing 10% glucose. Their drinking behavior was observed
when offered a choice between water or quinine sulfate (1.1 x 10~^M) , water or
MSG (10~^ and lO'^M) , water or quinine sulfate plus MSG (10"^, 5 x 10"^, 10-3,
10-2m) and water or quinine sulfate plus NaCl (0.015 M) .
The results indicated that rats preferred dilute MSG (10"%), with a pre-
ference of 77.6 ± 5.5% and rejected higher concentrations of MSG (10"^M) 11.29
± 5.1%. MSG (IQ-'^M) had no effect on the bitter taste of quinine (1.1 x 10"^)
when both were added together (11.0 ± 2.7%) compared to that of the control,
water v.s. quinine (1.1 x lO'^M) alone (11.3 ± 2.7%). As the concentration of
added MSG increased the preference of quinine solution increased, 16.6 * 9.6%
for 5 X 10"^ M MSG and 43.5 ± 1.7% for 10-3 M MSG but decreased as the concen-
tration of MSG increased to 10-2 M, 21.5 ± 6.1%. With 0.015 M NaCl added to
quinine the taste preference of rat toward quinine also increased to 27.5 ± 4.0%.
^SL3
Serial No. NHLI-106(c)
2. Altered preference for sodium chloride, anorexia and changes in plasma
and urinary zinc in rats fed a zinc deficient diet. Changes in NaCl preference
and in plasma and urinary zinc were studied in weanling rats fed, ad_ libitum,
zinc deficient or zinc supplemented diets and in rats pair fed an amount of
food equal to that eaten by rats fed a zinc deficient diet. NaCl preference in
rats fed the zinc deficient diet was significantly greater than in pair fed
(i.e., food deprived) controls or in rats fed a zinc supplemented diet, ad
libitum. This alteration in preference occurred within 3 days of initiation
of the zinc deficient diet although anorexia was observed in these rats with-
in the first two days.
Concentrations of plasma zinc and 24 hour urinary zinc excretion were sig-
nificantly lower in rats fed the zinc deficient diet than in pair fed rats or
in rats fed the zinc supplemented diet ad_ libitiom. These data suggest an inter-
relationship between an increased preference for NaCl and anorexia mediated
through zinc deficiency.
3. Effect of zinc on taste preference of quinine sulfate and HCl. In order
to extend our knowledge of the manner by which zinc alters taste acuity weanling
male Sprague Dawley rats, 50 * 5 g, were arbitrarily divided into two groups.
One group of rats received zinc deficient diet (.5ppm Zn"*"*") , while a second
group, the control group, was pair-fed with the same diet supplemented with
zinc (SOppm Zn*"'') . Growth and food intake were measured for each individual
rat and means for each group were calculated. The preference behavior of the
two groups of rats were examined by the standard methods used in our laboratory
to determine preference for the noxious substances HCl and quinine sulfate.
Total fluid intake was also measured. Urinary excretion of zinc and copper was
determined throughout the course of studies by atomic absorption spectropho-
tometry and plasma zinc levels were measured at the end of the study.
Urinary zinc excretion of the pair fed rats was significantly higher than
that of the zinc deficient rats and their plasma zinc fevels were also signifi-
cantly higher. Urinary zinc excretion was significantly lower in the rats fed
zinc deficient diet compared to the pair fed rats. These differences were first
observed after the first week of experiment . At the end of 5 weeks
the plasma zinc levels of control rats was significantly higher (218.4 ± 8.5
Mg/ml) than that of zinc deficient rats (150.0 ± 15.6 P < 0.005). No signifi-
cant difference between the two groups was observed in plasma copper. The
growth of the two groups of animals were identical throughout the study.
The zinc deficient rats exhibited higher taste preferences toward quinine
sulfate (1.1 x 10"%), 39.8 * 4.9% and 0.002 M HCl, 42.4 ± 6.5% than those of
the controls, 16.6 ± 2.6% (P < 0.005) and 20.48 ± 6.6% (P < 0.05), respectively.
These studies demonstrate that zinc deficient rats develop hypogeusia;
i.e., they will accept solutions normally rejected by control, zinc replete
rats.
J^V
Serial No. NHTJ-lQ6(c)
4. The effects of histidlne monohydrochlorlde In rats. In order to develop
a model system to evaluate effects of zinc depletion 21 male Sprague Dawley rats,
270 ± 20 g, were arbitrarily divided into 3 groups. One group of rats, the
control rats (C) received zinc supplemented diet ad lib. A second group of 7
rats received the zinc supplemented diet plus histidine monohydrochlorlde at
dosage of 1.5 and 7.5 g/kg for 2, 8 and 3 weeks respectively (H) ; a third
group of 7 rats received the same diet as the H group plus zinc 8 mg/kg (as
zinc carbonate) (HZ). All animals were pair fed their respective diets with the
H group. The effects of histidine monohydrochlorlde on the rats were observed
with respect to their taste preference toward NaCl (0.3 M) , body weight, organ
weight and urinary and plasma zinc and copper content.
Total fluid intake and NaCl preference were higher in the H group than in C.
The additional zinc in the HZ group appeared to decrease the appetite of this
group resulting in less food intake and smaller body weights. Histidine at high
dosages, 5 and 7.5 g/kg significantly increased the total body weight, and the
weights of liver and kidney in H rats. No significant differences were observed
in urinary zinc excretion of rats given 1 g/kg histidine monohydrochlorlde.
However at higher doses of histidine, 5 or 7.5 g/kg, urinary zinc excretion
was significantly higher (27.19 ± 2.9 ug/24 hr and 34.88 ± 3.35 yg/24 hr for
H group, respectively, and 26.18 ± 3.5 yg/24 hr and 41.0 ± 3.9 yg/24 hr for
HZ group, respectively) than those of control rats (4.79 ± 0.49 ug/24 hr and
2.82 ± 0.28 yg/24 hr, respectively). At higher histidine doses the urinary
zinc was significantly higher in both H and HZ groups.
5. Salt taste, salt preference and hypertension in man and animals.
A. NaCl preference in spontaneously hypertensive rats; age and blood pressure
effects. Preference for 0.30 M NaCl was determined in spontaneously hyperten-
sive (SH) male rats and in normotensive Wistar/Kyoto (WI/KY) controls of the
same strain with a two bottle preference test for 16 weeks post-weaning. Pre-
ference for the salt solution in the SH rats increased as blood pressure in-
creased to the h3rpertensive range as the animals increased in age. Increased
preference and hypertension reached maximal levels at 14 weeks of age and re-
mained at these levels thereafter. No significant difference in preference or
blood pressure was observed in the normotensive controls during the same period.
SH rats in which preference for 0.30 M NaCl was measured for the first time at
14 weeks of age had hjrpertension similar to that of the SH rats exposed to the
salt solution from weaning; elevated preference for 0.30 M NaCl in these two
groups of SH rats was also similar. WI/KY normotensive controls first given
NaCl at 14 weeks of age had low levels of preference similar to that observed
in control rats given NaCl from weaning.
B. Salt preference in patients with untreated and treated essential
hypertension. NaCl preference was studied in 16 patients with essential hy-
pertension and 26 normotensive volunteers over a 2 day test period. Prior to
placement in the test group each was shown to exhibit normal detection and
recognition thresholds for the taste of NaCl. Each was placed on a constant
dry diet containing 9 mEq Na and, as the only source of fluid, given a choice
of drinking either distilled E2O, 0.15 M NaCl or some combination of the two
fluids. Patients with essential hypertension drank a significantly greater
proportion of their total fluid as saline (day 1: 35% vs 13%; day 2: 34 vs 14%)
9 <5j<r'
Serial No. NHLI-106(c)
and drank a greater total volume of fluid (day 1: 1332 vs 669 cc ; day 2: 1419
vs 824 cc) than did the normotensive volunteers. The total amound of Na"*" con-
sumed by the patients was 4.8 to 7.3 times greater than that of the normotensive
volunteers. Effective treatment of hypertension lowered mean NaCl preference,
Na"*" intake and total fluid intake in the four subjects studied under these condi-
tions. These latter findings suggest that effective treatment with antihyper-
tensive agents may play some role in altering salt appetite.
6. Taste localization in fungiform, circumvallate and palatal papillae in
man. Using a forced choice-three stimulus drop technique in which micropipettes
rather than standard sized pipettes were used as droppers it was possible to
determine detection and recognition thresholds in taste buds in single fungi-
form, circumvallate and palatal papillae in man. Results indicated that taste
buds in fungiform papillae were equisensitive to salt and sweet solutions and
less sensitive to sour and bitter solutions. Taste buds in circumvallate pa-
pillae were most sensitive to sweet solutions and less sensitive to salt, sour
and bitter solutions. Taste buds in palatal papillae, as previously observed,
were most sensitive to sour and bitter solutions and less sensitive to salt and
sweet solutions. These results indicate that taste sensitivity to sweet is
equisensitive over the entire tongue but most sensitive to salt over the anterior
2/3, the location of fungiform papillae. Sensitivity to sour and bitter is
most acute over the palate. Studies of forced scaling indicate that judgments
of taste intensity are related primarily to the number of functioning taste
buds whereas thresholds are related to the presence of any normally functioning
bud within any group of buds.
7. Antidesma taste responsiveness. Antidesma bunius is a tree which pro-
duces a purple-black fruit which when ripe is about the size of a grape. Most
people who taste this fruit say that it tastes sweet. However, a few people
who taste this fruit say that it tastes bitter. Studies were carried out to
identify the nature of these differences and to attempt to relate these differ-
ences to definable genetic characteristics. In a sample of 200 people, approx-
imately 160 stated that this berry was either sweet or sour whereas the other
40 noted that it was either tasteless or bitter. These 40 were also character-
ized by absence of response to the bitter taste of phenylthiocarbamide (PTC)
whereas the other 160 all tasted PTC as bitter. Of the 40 PTC non-taster-anti-
desma "tasters", approximately one-half found antidesma tasteless, the other
half found it bitter. Of these latter patients several family groups were
studied. These studies suggested that the bitter taste of antidesma was in-
herited as an autosomal dominant trait and that it was associated with the
XgA marker on red blood cells in one family and with hypertrophic subaortic
stenosis in another.
8. Effects of pronase on taste responsiveness in rat. In order to evaluate
the effects of alterations in the protein structure of the taste bud membrane
on the neural responses in rat the chorda tympani of rats was exposed, an
electrode slipped around the nerve and the output monitored and recorded on
an EPUT meter before and after placement of pronase, 1 mg/ml, for 5 minutes
onto the surface of the rat tongue. Neural responses were recorded, before and
after pronase, following the -placement of varying concentrations of NaCl (30-
1000 mM), sucrose (30-1000 mM) , HCl (15-150 mM) and urea (500-5000 mM) on the
10
3^i,
Serial No. NHLI-1 06(c)
surface of the tongue. Prior to placement of pronase or after the placement
of the buffer in which the pronase was kept responses to each tastant was sim-
ilar to that reported by several investigators for normal rats. After placement
of pronase responses to all tastants were attenuated and higher concentrations
of tastant were necessary to elicit threshold responses. However, once thres-
hold was reached responses were similar to those recorded prior to pronase
placement. Electron microscopic studies of taste buds after oral placement of
pronase for 5 minutes showed no anatomical changes in the region of the pore.
These studies indicate that the neural events of taste may be influenced by
modification of the manner by which tastant-receptor interaction occur as re-
lated to alteration of the protein structure of the receptor.
Pathology
Pathophysiology of taste and olfaction.
1. Taste loss and anorexia followed thermal burn. Anorexia and subsequent
decreased caloric intake commonly follow thermal injury.. In addition, there is
an accompanying loss of large amounts of fluid and electrolytes, including trace
metals. Previous work in this laboratory has demonstrated a relationship be-
tween loss of trace metals, including zinc, and anorexia and hypogeusia in man.
Other investigators have noted severe anorexia in zinc-depleted animals. Be-
cause severe decreases in tissue zinc have followed thermal injury, we wondered
if anorexia and hypogeusia were related to zinc loss in this condition. In an
effort to study these relationships, changes in zinc and copper metabolism and
taste acuity were measured concomitantly in 14 patients following thermal injury.
Eighty-four percent exhibited hypogeusia (decreased taste acuity) . Mean serum
total zinc concentration was decreased and mean urinary zinc excretion was in-
creased in the patients with hypogeusia compared to controls. An association
among hypogeusia anorexia, and altered zinc metabolism after thermal injury is
strongly suggested.
2. Taste abnormalities after X- irradiation. We have studied several
patients who developed hypogeusia following radiation therapy of different in-
tensities to several organ systems. In general, treatment of these patients
with zinc ion, 25 mg of elemental zinc given orally as zinc sulfate four times
a day, has been efficacious in returning their taste acuity toward or to nor-
mal (109.95 mg of zinc sulfate contains 25 mg of zinc ion). Since administra-
tion of zinc ion may itself produce some gastrointestinal toxicity, therapy
is always given in the middle or toward the end of each meal and with a snack
at night. In addition, we have treated some patients prophylactically with
zinc ion prior to the start of their radiation therapy. These latter patients
have generally experienced fewer subjective complaints referable to the gusta-
tory system, and none has developed as severfe hypogeusia as observed in any
of the nontreated patients. Due to possible gastrointestinal symptoms which
can occur with oral zinc therapy, the amount of prophylactically administered
zinc ion has varied considerably. The particular dosage is dependent upon
several factors of the radiation, including intensity and duration of the
therapy, site of treatment, and the development of subsequent nausea. In gen-
eral, prophylactic administration of zinc ion was particularly useful in allaying
the anorexia and dysgeusia observed in some patients following irradiation.
11 ^^7
Serial No. NHTJ-106(c)
3. Taste abnormalities In anorexia nervosa. Patients with anorexia ner-
vosa exhibit anorexia, amenorrhea, lower than normal levels of serum zinc, lower
than normal levels of urinary zinc excretion and hypogeusia and hyposmia.
Some patients also exhibit dysgeusia and dysosmia which occur after the onset
of their anorexia. Hypogeusia was measured in each patient with anorexia ner-
vosa in whom amenorrhea and lowered urinary excretion of zinc was observed.
Whether or not these changes follow the onset of the anorexia and are secondary
or whether the zinc abnormalities are the cause of the anorexia is not knovm.
However, treatment with placebo does not alter either the abnormalities of zinc
metabolism the amenorrhea, the anorexia, hypogeusia or hyposmia.
4. Relationship between disordered gustatory function, liver function and
zinc metabolism in patients with acute and chronic liver disease. Anorexia is
a prominent symptom in patients with liver disease. Since appetite, food
preference and taste acuity are manifestations of gustation a systematic eval-
uation of gustatory function was undertaken in 22 patients with acute viral
hepatitis and in 16 patients with chronic liver disease (9 alcoholic cirrhosis,
5 post-necrotic cirrhosis, 2 chronic active hepatitis). Sixteen of the patients
with hepatitis were studied throughout the course of their disease. Subjective
information about gustation and objective measurements of taste acuity (detection
and recognition thresholds and scaling judgments for NaCl, sucrose, HCl and urea)
were obtained in each patient. Anorexia, dysgeusia and increases in detection
thresholds, recognition thresholds and scaling judgments (decreased acuity) were
found in patients with acute viral hepatitis and with chronic liver disease when
compared with the responses of 47 normal volunteers. In the 16 patients with
hepatitis studied serially gustatory function (subjective and objective measure-
ments) improved significantly as their disease waned. In patients with acute
viral hepatitis and in patients with chronic liver disease, measurements of
taste acuity were correlated significantly with concentrations of serum bilirubin,
alkaline phosphatase and zinc. These parallel changes suggest that gustatory
acuity quantatively reflects some aspects of hepatic function.
5. Idiopathic hypogeusia and hyposmia. We have recently described the
syndrome of idiopathic hypogeusia with dysgeusia, hyposmia and dysosmia. This
relatively common heretofore unrecognized syndrome occurred following an influ-
enza-like illness in 87 of the first 143 patients (61%) studied at the NIH. The
evaluation of patients with this syndrome called PIHH has led us to recognize
that a specific set of diagnostic factors must be evaluated in order to make this
diagnosis. Tests used to evaluate this disease are as follows: (A) History. Per-
sistent loss of taste and smell following an upper respiratory infection.
(E) Examination of nasal airway. The tenacious, clear, usually white, mucous
blanket normally present is missing. The nasal airway is markedly patent such
that the deeper structures of the nasal cavity may be easily observed on anterior
rhinoscopy. (C) Localization of ^^TcO/, in the region of the nose following in-
travenous injection. From 30-50% of patients with PIHH exhibit accumulation of
this radionuclide in the area of the nose whereas less than 5% of patients with-
out this disease do so. (D) Abnormal nasal mucous membrane. Each patient with
PIHH exhibits a characteristic change in the nasal mucous membrane as observed
by biopsy. These changes include squamous metaplasia of the mucosal epithelium
and marked infiltration of lamina propria with chronic inflammatory cells. Ade-
quate treatment of these patients results in a return toward normal of these
^2 ^S
Serial No. NHLI- 106(c)
pathological changes. (E) Abnormal levels of parotid salivary zinc containing
protein. In each patient with this disease parotid salivary zinc is lower than
in subjects with normal taste acuity (0-20 ppb Zn , patients; 30-100 ppb Zn ,
controls) . Abnormal levels persist in those patients treated with zinc ion who
do not respond to therapy whereas in those patients who respond to therapy with
zinc there is an increase in this salivary zinc either to normal levels or to
levels as great as 2-3 times normal.
6. Abnormalities of taste and smell following head trauma. Loss of ol-
factory acuity after trauma to the head is a well recognized phenomenon and
has been estimated to occur in 1.3% to 65% of all head injuries. Blows to the
occiput have been reported more likely to produce smell loss, prestimably by a
contre-coup effect, than blows to the forehead or other sites on the head
and even trivial injuries have been shown to result in olfactory sensory deficits.
In addition, distortion of odors has been described after head trauma. Spon-
taneous recovery of olfactory acuity has been claimed in 8 to 39% of patients
suffering from post-traumatic smell loss.
We have studied patients who experienced abnormalities of taste and smell
following head trauma. For the first time quantitative estimates of the taste
and smell losses were made in these patients. Abnormalities of taste and smell
were found in each of the 29 patients who were studied. These abnormalities
Included hypogeusia, dysgeusia, hyposmia and dysosmia. This sjmdrome can occur
even after minimal head trauma and can begin months after the moment of injury.
The patients exhibited a significant decrease in total serum zinc concentration
(patients, 77 ± 3 yg/100 cc, mean ± 1 SEM, vs controls, 99 ± 2, p 0.001) and
a significant increase in total serum copper concentrations (113 - 4 yg/100 cc
vs 100 ± 2, p < 0.001) compared to control subjects. Symptoms of hypogeusia,
dysgeusia and dysosmia are frequent sequellae of head injury and are important
to the patients and to their care following trauma.
Olfaction
1. Olfactory status and response to clomiphene in male gonadotropin de-
ficiency. Administration of clomiphene citrate, 200 mg daily for 7 to 14 days
and 50 to 100 mg daily for 4 additional weeks, produced significant increases
in plasma levels of gonadotrophins and testosterone in noinnal males. Patients
with multiple anterior pituitary hormone deficiencies or with isolated gonado-
trophin deficiency and type I hyposmia (absent response to vapors at the pri-
mary olfactory area) were unresponsive to clomiphene. With clomiphene treat-
ment two of three patients with type II hyposmia (subnormal olfactory responsive-
ness) and one of two patients with normal olfaction and hypogonado trophic hypo-
gonadism had increases of levels of plasma testosterone and plasma or urinary
gonadotrophins to the normal male range. Normal spermatogenesis was demonstrated
in two cases and fertility in one. Clomiphene is effective treatment for certain
males with gonadotrophin deficiency.
2. Nasal mucous membrane biopsy in Sjogren's syndrome; A new technique
for evaluation of the pathology of the syndrome. We have recently emphasized
the frequent occurrence of xerorhinia in patients with Sjogren's sjmdrome, asso-
13 Sd^f
Serial No. NHTJ-IOfi^c^
elated with nasal crusting and hyposmia resulting from decreased nasal mucous
secretion. We hypothesized that the xerorhinia occurred via the same mechanism
as did the xerostomia; i.e., infiltration of mucous and serous glands of the
nasal mucous membrane with chronic inflammatory cells. We thus wondered whether
biopsy of the nose might not prove to be easier and associated with less morbidity
than biopsy of the lip yet provide similar information. In addition, the easy
availability of the tissue would make repeat studies practical. We have studied
15 patients with well documented SjHgren's syndrome. Biopsies of the nasal mu-
cous membrane were performed in each patient and the results compared with simi-
lar biopsies taken from the minor salivary glands of the lip. Results indicate
that pathological changes found in both tissues were similar and that morbidity
of the former procedure was minimal.
3. The localization of TCO4 in the region of the nose in SjHgren's syn-
drome. Rudolph's Sign: Xerostomia and xerorhinia have resulted in decreased
taste and smell acuity, respectively, in patients with Sjtigren's syndrome. These
patients also complain of local nasal symptoms such as dry nose, nasal crusting,
and loss of smell acuity. These changes prompted us to examine the nasal mucous
membrane following surgical biopsy and specific pathophysiologic changes were ob-
served. Rgyiew of the salivary scintigrams of these patients revealed an accumu-
lation of ^''TcO^ in the region of the nose and we vondered whether these phen-
omena might be related. The present study was undertaken to examine systemati-
cally the uptake of "^^TcO^ in the region of the nose of 14 patients with Sjo-
gren's syndrome, to demonstrate the extent and intensity of accumulation of the
nuclide in the nasal region and to attempt to explain the pathophysiology of this
event.
Eleven of 14 patients (79%) exhibited significant uptake of ^'cO^ in the
region of the nose (Rudolph's sign); 8 of these patients had a grade of 3"^ or
greater (scale 0 to 4''"). In each of the 11 patients with Rudolph's sign there
was significant infiltration of chronic inflammatory cells around the glands of
the nasal mucous membrane as seen by nasal biopsy and evaluated independently
from evaluation of the ^^"'Tc04 accumulation. This chronic inflammatory reaction
in the nasal mucous membrane appears to be the basis for the accumulation of
'^^TcO^ in the nasal region of the face for any area of acute or chronic inflam-
mation appears to take up ^'^''"■TcOi^. In a concomitant study of 40 other consecu-
tive patients in whom brain scan was carried only 2 (5%) exhibited any uptake of
99mxc04 in the region of the nose.
4. A study of the potential applications of olfactory research in man.
This study provides a comprehensive review of the feasibility of developing and
utilizing the chemical senses of the soldier. While bio-sensors and odor-sensing
devices can supplement, they cannot replace the potential performance of the nor-
mal human olfactory system. There is no current program in the Army to train sol-
diers to use their chemical senses. Experience in other countries suggests that a
soldier trained to recognize olfactory cues is more effective in the field, more
responsive to hazards, and less likely to give olfactory information to the enemy
than one whose chemical senses are underutilized. Practical testing and training
methods suited to the Army's needs have been lacking in the past, and basic know-
ledge of the mechanisms of olfaction and taste is incomplete. However, elements
of a suitable technology have been identified in agencies such as the National
Bureau of Standards and in certain industries. This technology appears suitable
for further development within the Army research and development community. This
14 ^30 ■
Serial No. NHLI-106(c)
report concludes that it is feasible for the Army to use these methods in deter-
mining classifications of personnel that require normal acuity of the chemical
senses and to institute training programs.
In addition, regular testing for normal acuity of the chemical senses may
uncover early cases of some illnesses, indicate extensive exposures to ionizing
radiation, and categorize personnel as either suitable or unfit for certain Army
duties. If administered at recruitment, these tests can be used later to docu-
ment disability claims for service-related losses of olfaction and taste.
Significance; Taste
1. Anatomy. Difference in function of each of three major types of cells
in the taste bud has been described and related to anatomy. Taste studies in man
have related form and function of taste buds in various types of papillae such
that an accurate "taste map" can now be drawn for the first time. Although all
taste buds subserve all taste qualities, buds in fungiform papillae are most sen-
sitive to sweet alone. This makes the tongue equisansitive to sweet with the an-
terior portion most sensitive to salt. The buds in the palatal papillae are most
sensitive to sour and bitter.
The neurochemical basis for the initial ^preneural events in taste, i.e., the
formation of the generator potential, has been described. This has been demon-
strated by locating acetylcholinesterase in the pore region of the bud around the
finger-like projections. This also has been demonstrated by producing hypogeusia
and ageusia in man following the oral introduction of anticholinergic agents
without the production of anesthesia. Blockade of electrical activity measured
at the chorda tympani in rat has also been carried out by placing anticholinergic
agnets on the tongue of rat. This is the first demonstration of the role of a
neurotransmitter at the pore of the taste bud.
2. Chemistry. Taste bud membranes have been isolated and their chemical
properties described. Specific binding of several sugars has been shown to take
place at the membranes whereas control tissue does not possess any of these pro-
perties. Binding constants have been calculated and they agree closely with
psychophysical data.
Miracle fruit glycoprotein (MFGP) has been isolated and purified. MFGP has
two effects in man when placed into the oral cavity: (1) altering the taste of
all sour tastants to sweet, and (2) blocking sweetness and bitterness normally
produced by sweet and bitter tastants. The chemical properties of MFGP have been
described including its amino acid and sugar composition. Various chemical reac-
tions have produced changes in activity. Removal of the terminal sugar complete-
ly inhibits the action of MFGP in altering sour tastants to sweet whereas the
blocking ability remains intact. Removal of several of the glycosidese linkages
in a manner not yet clearly defined enhances the activity of MFGP with respect to
change of sour to sweet taste by a factor of 2-3. Through this mechanism we have
also labeled MFGP radioactively. The data thus far obtained strongly suggests
that MFGP is a plant lectin and appears to be the first plant lectin isolated
which has any physiological activity. This finding has also been instrumental in
allowing us to obtain a biochemical rather than a bioassay for this material.
Since MFGP can alter the taste of all sour tastants to sweet it has ideal pro-
perties as a sugar sugstitute and is important in the treatment of obesity.
15 ^3/
Serial No. NHLI-106(c)
3. Physiology. Various substances when added to food or drink have been
shown to alter intake behavior. Addition of MSG or NaCl decreases sensitivity
to bitter tastants . It is useful to add these agents to reduce the bitterness
of certain foods and fluids. Addition of glucose increases salt intake. Since
sugar is being added in larger amounts to more and more foods and drinks its
association with the production of increased NaCl intake represents an important,
heretofore unrecognized etiological source of hypertension. Zinc depletion
from food produces anorexia and hypogeusia. Decreased zinc intake produces
decreased food intake, hypogeusia and eventually marked slowing of growth and
sexual development. This problem is a much more common one than previously
recognized. This association has been found in middle-class school children
in Denver, Colorado, as well as patients in Egypt and Iran. Correction of this
abnormality by supplying enough zinc in the diet is uniformly associated with
the return to normal of each abnormal symptom. This syndrome occurs in adults
as well as in children and deserves close attention. Salt intake and hypertens ion
have been related in man and animals for many years. We have demonstrated that
patients with hv'pertension prefer saltier solutions than do normal subjects
and that effective treatment of the hypertension returns this abnormal propensity
to normal. This suggests that some factor(s) which influence blood pressure
also influence salt preference and salt intake in man.
A new genetic taste marker has been discovered which differentiates among
those people who are PTC taste blind. This marker is the fruit of the plant
Antidesma bunius. Approximately 1/2 of the PTC taste blind people taste
antidesma as sweet or sour whereas the other 1/2 taste it as intensely bitter.
This trait has been linked in one group of patients to an XgA marker on red
blood cells and in another group to hypertrophic subaortic stenosis.
4. Pathology. Zinc loss by any means is associated with anorexia and
hypogeusia. We have shown this interrelationship in patients following ther-
mal burn due to loss of zinc rich fluids, following X-irradiation, following
head trauma, after hypothyroidism and in acute and chronic liver disease due
to wastage of zinc in the urine.
Idiopathic hypogeusia is a disease in which taste acuity is lost. The
diagnosis of this disease involves several parameters which we have developed
over the past year. These parameters include the presence of Rudolph's sign,
the infiltration of chronic inflammatory cells into the lamina propria of
the nasal mucous membrane and the decrease in production of a parotid salivary
zinc containing protein. The mechanism of taste loss appears to parallel and
to be secondary to the loss of the parotid salivary zinc containing protein
whose normal function is to supply nutrients to the taste bud (since it con-
tains no blood vessels or lymphatics) and appears to get its major nutrition
from parotid saliva. In this sense we have proposed, for the first time, a
specific mechanism whereby saliva supports taste. In patients with this
disease treated with zinc ion, those who recover taste acuity do so through
induction of this protein either directly or indirectly with zinc. Those
patients who do not recover taste acuity exhibit no change in the low concen-
tration of this protein which they pathologically exhibit. This induction is
dose dependent upon the amount of zinc ion administered.
16 S53^.
Serial No. NHLI-106(c)
Olfaction
The prediction of ultimate fertility in hypogonadal men may be related
to the type of olfactory defects present. Men with Type II hyposmia usually
become fertile with appropriate therapy whereas men with Type I hyposmia have
not reached fertility in spite of the therapy applied. This observation
clearly relates olfactory acuity and sexual function in men.
A new approach to the diagnosis of several disease states involving
olfactory defects has been made through the development of the technique
of biopsy of the nasal mucous membrane and through the localization of
Technitium ^^'" in the region of the nose (Rudolph's sign). These techniques
have allowed us to identify the characteristic pathology of patients with
Sjogren's syndrome and those with hyposmia of several different etiologies.
Proposed Course of Project:
1. The major neurochemical transmitters in the taste bud will be identi-
fied.
2. Form and function differences among taste buds in the various types
of papillae in man will be identified and characterized.
3. The taste bud receptor protein will be studied to understand the
mechanism(s) by which tastant-receptor binding takes place.
4. MFGP will be studied with respect to its mechanism and to how its
binding to the taste receptor membrane influences the initial, preneural
events of taste. The use of MFGP in low calorie foods as a treatment for
obesity will be investigated.
5. Studies of the mechanism by which various substances influence taste
and food intake will be studied. These substances include MSG, carbohydrates
of various types and NaCl .
6. The relationship between salt intake and hypertension will be studied
with respect to the manner by which intake is controlled and how it influences
blood pressure.
7. The role of zinc in taste at the membrane-receptor, in receptor enzymes
such as alkaline phosphatase and in the parotid zinc containing protein will be
studied.
8. The relationship between olfaction and sexual function will be studied.
Honors and Awards: Maryland State Lecturer in Otorhinolaryngology, 1973.
Maryland State ENT Otorhinolaryngology Society
Certificate of Merit, New York Institute of Food
Technologists, 1973
17 ^^^
Serial No. NHLI-106(c)
Publications:
McConnell, S.D. and Henkin, R.I.: Increased preference for Na* and K*
salts in spontaneously hypertensive (Sfl) rats. Proc. Soc. Exp. Biol. Med.
143: 185-188, 1973.
Cohen, K., Schechter, P.J. and Henkin, R.I.: Hypogeusia, anorexia and
altered zinc metabolism following thermal burn. JAMA 223: 914-916, 1973.
Hamilton, C.R.,Jr., Henkin, R. I., Keir, G. and Kliman, B. : Response to
clomiphene and olfactory status in male gonadotropin deficiency. Effects
of prolonged clomiphene treatment of male hypogonadotropinism. Annals
of Int. Med. 78: 47-55, 1973.
Henkin, R.I.: Handbook of Sensory Physiology, Vol. IV., Chemical Senses 1,
Olfaction (Ed. L.M. Beidler) New England J. Med. 287: 997, 1972 (Book Review)
Henkin, R.I.: Handbook of Sensory Physiology, Vol. IV., Chemical Senses 1,
Olfaction (Ed. L.M. Beidler) JAMA 222: 1314, 1972. (Book Review)
Henkin, R.I.: Handbook of Sensory Physiology, Vol. IV., Chemical Senses 1,
Olfaction (Ed. L.M. Beidler) Annals of Int. Med. 78:468,1973 (Book Review)
Catalanotto, F., Slavik, M. , Danilson, D., Keiser, H. , and Henkin, R. I.:
Changes in preference for NaCl following administration of 6-azauridine
and 6-a2auridine triacetate. Pharmacology and Therapeutics in Dentistry
(In press) 1973.
Sato, N., Haller, E.W., Powell, R.D. and Henkin, R.I.: Sexual maturation
in bulbectomized female rats. J. of Reproduction and Fertility (England)
(In press) 1973.
Henkin, R.I.: A study of circadian variation in taste and smell in normal
man and in patients with adrenal cortical insufficiency: the role of adrenal
cortical steroids. Circadian Conference, Columbia University. In Bio-
rhythms and Human Reproduction, John Wiley S Sons, N.Y. (In press) 1973.
Henkin, R.I.: Sensory changes during the menstrual cycle. Circadian
conference, Columbia University, In^ Biorhythms and Human Reproduction,
John Wiley 5 Sons, N.Y. (In press) 1973.
Schechter, P.J., Horwitz , D. and Henkin, R.I.: Sodium chloride preference
in essential hypertension. JAMA (In press) 1973.
McConnell, S.D. and Henkin, R.I.: NaCl preference in spontaneously hy-
pertensive rats: age and blood pressure effects. Amer. J. Physiol.
(In press) 1973.
18
^5/
Serial No. NHLI-106(c)
McConnell, S.D. and Henkin, R.I.: Altered preference for sodium chloride,
anorexia and changes in plasma and urinary metabolism in rats fed a zinc
deficient diet. J. of Nutrition (In press) 1973.
Henkin, R.I.: Anosmia and ageusia due to nonallergic rhinitis. JAMA
(In press) 1973 Questions 6 Answers
19 ^3^
Annual Report of the
Molecular Hematology Branch
National Heart and Lung Institute
July 1, 1972 through June 30, 1973
The mechanism of hemoglobin biosynthesis and the regulatory controls
involved in hemoglobin gene expression are being studied in human disease
states as well as in animal model systems o The objective is to understand
the molecular basis of hereditary anemias (specifically the thalassemias and
hemoglobinopathies) in order to be able to devise effective methods for
treating these diseases « The general approach has been to fractionate normal
and diseased red blood cells into the various components required for hemo-
globin synthesis and then to reconstitute the cellular components in such a
way as to reproduce the activity of che original intact cello The function of
each cellular component from the diseased cell can then be compared with its
counterpart from a normal cell. The first phase of this program has been
successfully completed, namely, the fractionation of the cytoplasm of normal
and diseased reticulocytes and the reconstitution of a cell-free system
capable of duplicating the normal and disturbed functions of reticulocyte
cytoplasm. The laboratory is now engaged in a similar attack on the nucleus
of erythrocyte precursors. In addition, two related projects have received
strong emphasis: first, a program has been established which is aimed at the
cell-free synthesis of the DNA gene for a globin chain; second, the recent
ability to induce A -♦ C globin gene -switching in tissue culture encourages
attempts to learn the mechanism of this switch in order to try to obtain
adult to fetal gene switching in humans »
A summary of our results obtained over the past twelve months for
projects continued from past years follows:
1) Initiation factors: An intensive effort has been made to purify
the four known initiation factors, IF-Ml, IF-M2A, IF-MZB, IF-M3 and the two
known elongation factors, EF-1 and EF-2, to homogeneityo IF-Ml and EF-2 are
now pure and IF-M2B and EF-1 are within 1-2 steps of purity. IF-M2A and
IF-M3 are now under attack. During these studies another factor, ribosome
dissociation factor, has been discovered.
2) Mechanism of hemoglobin initiation: Because of its unique
structure and in vivo role, the mechanism of initiation of the sheep p
chain has been investigated in detail. The p chain is 5 amino acids short
on its N-terminal end: the N-terminus is Pro. In all other globin chains
studied (human a, ^, p^, y; rabbit a and p, sheep a, p^, p^) , the chain is
initiated with a Met donated from the initiator tRNA, Met-tRNAp. This Met is
then cleaved or not depending on the globin chain. Once cleaved the next
amino acid serves as the N-terminal amino acid of the final globin chain.
However, in the case of the p chain, more than Met is cleaved: the N-terminal
dipeptide of the nascent chain is Met-Asn not Met-Pro.
3) Molecular basis of thalassemia: Active mRNA has been insolated
from the bone marrow cells of patients with thalassemia and various other
A37
hemolytic anemias. Jusc as with previous studies using reticulocyte mRNA,
the bone marrow mRNA faithfully reproduces the globm synthesizing ability
of the intact cell. Studies on the mechanism of initiation of the thalas-
semic (3 globin chain indicates that initiation appears to be normal. Thus
the molecular defect in p-thalassemia is probably in the amount, rather than
in the translation, of the P globin mRNAo
4) Hemoglobin A - C switch in sheep and goats; The A - C globin
gene switch in goats has been reproduced in tissue culture cells. Cultured
Type A bone marrow cells, in the presence of erythropoietin, partially switch
off Hb A {.0-2^2 ^ synthesis and switch on the synthesis of Hb C {0.2^2'] » This
switch has been confirmed by product identification of the globin chains by
CMC chromatography o
5) Tissue culture of bone marrow cells; Culture conditions have
been found for maintaining bone marrow cells in an active metabolic state
for several days. Erythroid precursors are maintained in number and function.
These conditions were utilized in obtaining the A — C switch described in
(4) above.
A report on the new programs established during the past twelve months
follows :
1) Synthesis of a globin DNA gene; Utilizing the enzyme RNA-
directed DNA polymerase from avian myeloblastosis virus, a DNA copy directed
from globin mRNA has been obtained. Unfortunately, this copy is not a com-
plete one. The mechanism of activity of the enzyme is now being examined in
detail in hopes of developing conditions for synthesizing a complete (and
active) DNA gene,
2) Cell-free synthesis of globin mRNA: The enzyme DNA -dependent
RNA polymerase is being purified from bone marrow and liver cells. This
enzyme is being used in an attempt to synthesize globin mRNA from bone
marrow chromatin (or DNA) in a cell-free system.
ats
Serial No. NHLI-107
1. Molecular Hematology Branch
2. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Mechanism of Globin mRNA Transcription in Bone Marrow Cells
Previous Serial Number: None
Principal Investigators: Alan W. Steggles, Ph.D.
W. French Anderson, M.D.
Other Investigators: Dante J. Picciano, Ph.D.
Cooperating Units: Dr. Joseph E. Pierce, Section on Lab Animal
Medicine and Surgery, NIH
Mr. Leonard Stuart, Ungulate Unit, NIH Animal
Center
Project Description:
Objectives: Since the molecular defect in beta-thalassemia appears to be
in the decreased amount of beta globin mRNA, a defect in the regulation of
beta-globin mRNA synthesis is suggested. The objective of this project is
to determine the normal mechanism of regulation of globin mRNA synthesis in
bone marrow cells.
Methods : Young sheep are the source of bone marrow and other tissue.
Chromatin and chromatin fractions (histones, acidic proteins, DNA) are ob-
tained by standard techniques. DNA-dependent RNA polymerase is purified by
standard enzyme purification techniques. RNA product analysis is by sucrose
gradient centrlfugation and polyacrylamide gel electrophoresis.
Major Findings: 1) The crude RNA polymerase B fraction from several tissue
sources appears to be similar. The mammalian RNA polymerase, however, is
distinctly different from E. coli RNA polymerase.
2) A "stimulatory factor" has been partially purified which increases
the activity and (?) specificity of the mammalian RNA polymerase.
Significance to Biomedical Research and Institute Research: Understanding
the mechanism of mRNA transcription in eucaryotic cells is essential to
understanding gene action.
Proposed Course of Project: Purification of the RNA polymerase and the
"stimulatory factor" followed by studies on the detailed mechanism of action
of the enzyme and the factor.
Honors and Awards: None
Publications: None
1 A39
Serial No. NHLI-108
1. Molecular Hematology Branch
2. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Mechanism of Hemoglobin Biosynthesis in Rabbit Reticulo-
cyte Cell-free Systems
Previous Serial Number: NHLI-295: NHLI-286
Principal Investigators:
William C. Merrick, Ph.D.
Norton Elson, M.D.
Hermann Graf, Ph.D.
Dante J. Picciano, Ph.D.
Ronald G. Crystal, M.D.
W. French Anderson, M.D.
Other Investigators:
Philip Prichard, Ph.D.
Jerold A. Last, Ph.D.
Cooperating Units:
Project Description:
None
Objectives : The mechanism and regulation of mammalian protein synthesis is
being investigated by utilizing hemoglobin biosynthesis as a model system.
The rabbit reticulocyte has been fractionated into many of its various com-
ponents. The separate steps required in the protein synthesizing process
can be separately studied by recombining just those components required for
each individual step.
Methods : Standard techniques of enzyme purification and of protein and
nucleic acid fractionation have been employed. Enzymatic assays have been
developed or modified from those described in the literature to examine the
individual steps in the processes of initiation and elongation of protein
synthesis. Chromatographic and electrophoretic techniques have been devel-
oped for product analysis of partial globin chains.
Major Findings: 1) Continuing efforts to purify each initiation and elonga-
tion factor to homogeneity has resulted in pure IF-Ml and EF-2, and nearly
purified IF-M2B and EF-1. IF-M2A and IF-M3 are now being emphasized.
2) A new factor, ribosome dissociation factor, has been discovered.
This factor is distinct from the known initiation and elongation factors. Its
function is related to the dissociation of the 80 S ribosome into the 60 S
and 40 S subunits.
3) Because of its unique structure and i^ vivo role, the mechanism of
initiation of the sheep Q^ chain has been investigated in detail. The 6^
1 ^<A>
I
Serial No. NHTJ-10R
chain is 5 amino acids short on its N-terminal end: the N-terminus is Pro.
In all other glob in chains studied (human a, B , B^, Y; rabbit a and B,
sheep a, B^, B^) « the chain is initiated with a Met donated from the initiator
tRNA, Met-tBtNAp. This Met is then cleaved or not depending on the globin
chain. Once cleaved the next amino acid serves as the N-terminal amino acid
of the final globin chain. However, in the case of the B^ chain, more than
Met is cleaved: the N-terminal dipeptide of the nascent chain is Met-Asn not
Met-Pro .
Significance to Biomedical Research and Institute Program: An understanding
of the mechanism and regulation of mammalian protein synthesis is important
to the understanding of overall normal cell function.
Proposed Course of Project; Purification of each initiation and elongation
factor to homogeneity. Determination of the precise role of each factor in
the translation process.
Honors and Awards: None
Publications;
1. Crystal, R.G., Nienhuis, A.W., Elson, N.A. , and Anderson, W.F.;
Initiation of globin synthesis. Preparation and use of reticulo-
cyte ribosomes retaining initiation region messenger ribonucleic
acid fragments. J. Biol. Chem., 247, 5357-5368, 1972.
2. Crystal, E.G., Nienhuis, A.W., Prichard, P.M., Picciano, D., Elson,
N.A., Merrick, W.C, Graf, H., Shafritz, D.A., Laycock, D.G.,
Last, J.A. , and Anderson, W.F.; Initiation of globin synthesis.
FEES Letters, 24, 310-314, 1972.
3. Elson, N.A. , Crystal, R.G., and Anderson, W.F.: Paper electro-
phoresis and chromatography of oligopeptides with N-terminal
methionine. Analyt. Biochem. (in press) .
4. Crystal, R.G., Elson, N.A. , and Anderson, W.F.; Initiation of
globin synthesis; Assays. K. Moldave and L. Grossman (eds) ;
Methods in Enzymology, (in press) .
5. Merrick, W.C, Graf, H., and Anderson, W.F.; Preparation of protein
synthesis initiation factors IF-Ml, IF-M2A, and IF-M2B from rabbit
reticulocytes. K. Moldave and L. Grossman (eds): Methods in
Enzymology, (in press) .
6. Prichard, P.M. and Anderson, W.F.: Preparation of rabbit reticulo-
cyte initiation factor IF-M3. K. Moldave and L. Grossman (eds);
Methods in Enzymology, (in press) .
7. Merrick, W.C, Lubsen, N.H., and Anderson, W.F.: A ribosomal
dissociation factor from rabbit reticulocytes distinct from
known initiation factors. Proc. Nat. Acad. Sci. USA (in press).
8. Anderson, W.F.; Purification and characterization of reticulocyte
initiation factors. Proc. of Internat. Colloquium on Normal
and Abnormal Protein Synthesis in Higher Animals, Paris, 1973
(in press) .
2 •^'i^/
Serial No. NHLI-109(c)
1. Molecular Hematology Branch
2. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Regulation of Hemoglobin Chain Synthesis in Beta-Thalassemia
Previous Serial Number: NHLI-317(c): NHLI-285(c)
Principal Investigators: Arthur W. Nienhuis, M.D.
Norton A. Elson, M.D.
Ronald G. Crystal, M.D.
W. French Anderson, M.D.
Other Investigators: Ramon Velez, M.D.
Patricia Canfield
Cooperating Units: None
Objectives: Beta thalassemia, also known as Cooley's Anemia, is an hereditary
disease characterized by severe anemia. The anemia is a consequence of a
low level of beta chains of hemoglobin being produced by the patient's red
blood cells which results in an excess of alpha globin chains. These excess
alpha chains precipitate in the cell and cause the cell to be lysed. No
amino acid change has been found in the hemoglobin beta-chains of thalassemic
patients. It is generally assumed, therefore, that the molecular abnormality
resides in the regulation of beta-chain synthesis. The normal mechanisms for
regulation of hemoglobin chain synthesis and the nature of the defect in
beta-thalassemic cells which allows normal alpha-chain, but limited or absent
beta-chain, production are being investigated.
Methods: Blood and/or bone marrow is obtained both from patients with beta-
thalassemia and from patients displaying a high reticulocyte count secondary
to another cause (for example, autoimmune hemolytic anemia, sickle cell anemia,
etc.). Cellular components involved in hemoglobin synthesis are isolated
and utilized in a cell-free hemoglobin synthesizing system. By substituting
each component in turn from the thalassemic cells into the non- thalassemic
cell-free system, it has been possible to test each component of the thalas-
semic system to determine which is normal and which is abnormal.
Major Findings: 1) Active mRNA has been isolated from the bone marrow cells
of patients with thalassemia and various other hemolytic anemias. Just as
with previous studies using reticulocyte mRNA, the bone marrow mRNA faith-
fully reproduces the globin synthesizing ability of the intact cell.
2) Studies on the mechanism of initiation of the thalassemia beta
globin chain indicates that initiation appears to be normal. The thalassemic
beta globin chain is initiated by Met-tRNA- and requires the normal initia-
tion factors. Elongation and termination are already known to be normal.
1 <:5ya
Serial No. NHLI-109(c)
Thus the molecular defect in beta-thalassemia is probably in the amount,
rather than in the translation, of the beta globin mRNA.
Significance to Biomedical Research and Institute Program: Beta-thalassemia
is a severe hereditary disease which affects a sizeable number of individuals
in the Mediterranean and Asiatic countries. A method for treating this
disease, which will reduce or eliminate the frequent blood transfusions
required, is needed. In addition, the techniques utilized to learn how the
rate of hemoglobin synthesis is regulated can then be applied to studying
other diseases where the defect is also in faulty regulation in the synthesis
of a gene product.
Proposed Course of Project; Attempts will be made to correct the alpha/beta
globin chain imbalance by influencing the synthesis of alpha and/or beta
chains ±n vitro in thalassemic bone marrow and reticulocytes.
Honors and Awards; None
Publications :
1. Nienhuis, A.W. , Canfield, P.H., and Anderson, W.F.; Human bone
marrow hemoglobin messenger RNA; Isolation and translation in
homozygous and heterozygous beta-thalassemia. J. Clin. Invest,
(in press) .
2. Crystal, R.G., Elson, N.A., Nienhuis, A.W. , Thornton, A.C., and
Anderson, W.F.; Initiation of globin synthesis in beta-thalassemia.
New Eng. J. Med. 288, 1091-1096, 1973.
3. Nienhuis, A.W. , Falvey, A.K., and Anderson. W.F.; Preparation of
globin messenger RNA. K. Moldave and L. Grossman (eds) ; Methods
in Enzymology (in press) .
4. Anderson, W.F.: Isolation and translation of messenger RNA from
beta-thalassemia reticulocytes. Proc. Third Conf. on Cooley's
Anemia, New York Academy of Sciences, New York, 1973 (in press).
5. Anderson, W.F.; Isolation and translation of thalassemia mRNA.
Proc. Internat. Colloquium on Normal and Abnormal Protein Synthesis
in Higher Animals, Paris, 1973 (in press).
c^'/S
Serial No. NHLI-110
1 . Molecular Hematology Branch
2. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Evolutionary Homology of Components of the Protein
Synthesizing Machinery
Previous Serial Number: NHLI-316: NHLI-288
Principal Investigators: Dante Picciano, Ph.D.
Norton Elson, M.D.
W. French Anderson, M.D.
Other Investigators: William C. Merrick, Ph.D.
Cooperating Units: None
Project Description:
Objectives: The extent of evolutionary homology of the components of the
cell's protein synthesizing machinery in organisms thoughout the phylogenetic
tree is being examined.
Methods : Cell components are isolated from organisms ranging from E.coli
to human. Interchangeability of components between species is analyzed in
cell-free assays.
Major Findings: 1) The initiation factors from rabbit liver appear to be
identical, or at least very similar, to the corresponding factors from
rabbit reticulocytes.
2) The initiator tRNA from E.coli (Met-tRNAp )will not replace the
mammalian initiator tRNA(Met-tRNA^^^^^^) in initial dipeptide synthesis
(Met -Val) directed by globin mRNA.
r
3) Globin mRNA from various sources (human, rabbit, sheep, goat, human)
can be effectively translated in a rabbit cell-free system.
Significance to Biomedical Research and Institute Program: The fact that at
least parts of the cell's protein synthesis apparatus appear to have remained
essentially unchanged during evolutionary development implies that many con-
clusions concerning the mechanism of cell function determined in lower or-
ganisms might be applicable to human tissues. By demonstrating the inter-
changeability of a component, it is possible that genetic apparatus from
lower organisms might eventually be used therapeutically in man.
Proposed Course of Project: The species and tissue specificity of the cell
components involved in protein synthesis will continue to be investigated.
1 «2/(^
Serial No. NHLI-llQ
Honors and Awards: None
Publications:
1. Picciano, D.J. , Prichard, P.M., Merrick, W.C., Shafritz, D.A. ,
Graf, H., Crystal, R.G., and Anderson, W.F.: Isolation of protein
synthesis initiation factors from rabbit liver. J. Biol. Chem.
248. 204-214, 1973.
2. Picciano, D.J., and Anderson, W.F.: Preparation of protein synthesis
initiation factors from rabbit liver. K. Moldave and L. Grossman
(eds) : Methods in Enzymology (in press) .
jx/r'
Serial No. NHLI-111
1. Molecular Hematology Branch
2. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Mechanism of Hemoglobin Switching in Sheep and Goats
Previous Serial Number: NHLI-287
Principal Investigators: Jane Barker, Ph.D.
Jerold Last, Ph.D.
Sherrill Adams, M.A.
W. French Anderson, M.D.
Other Investigators: Arthur Nienhuis, M.D.
Cooperating Units: Dr. Joseph E. Pierce, Section on Lab Animal
Medicine and Surgery
Mr. Leonard Stuart, Ungulate Unit, NIH Animal
Center
Project Description:
Objectives: An animal model is sought to study the mechanisms involved in
the regulation of gene expression for globin synthesis including the mechanism
for globin chain switching (which occurs in many higher organisms including
humans) . The best model system appears to be the switch from hemoglobin A
to hemoglobin C which occurs in goats and certain sheep when they are made
anemic. This switch appears to be induced by the hormone erythropoietin.
We hope to reproduce this globin chain gene switch in an in vitro system
and to investigate the mechanism.
Methods: Sheep and goats are made anemic by bleeding or by phenylhydrazine
injection. Samples of bone marrow cells are obtained and are grown in tissue
culture. Alterations in cellular activity are studied as a function of
erythropoietin administration and of experimental conditions. Methods are
being developed for isolating active messenger RNA from these cultured bone
marrow cells.
Major Findings: 1) The hemoglobin A to hemoglobin C switch in goats has been
reproduced in tissue culture cells. Cultured Type A bone marrow cells, in
the presence of erythropoietin, partially switch off Hb A (aaBa^) synthesis
and switch on the synthesis of Hb C (aaBa^) • This switch has been confirmed
by product identification of the globin chains.
2) Culture conditions have been found for maintaining bone marrow cells
in an active metabolic state for several days. Erythroid precursors are
maintained both in number and function. These conditions were utilized in
obtaining the A to C switch described above.
1 SLf^
Serial No. NHLI-111
Significance to Biomedical Research and Institute Research; It has been
widely suggested that a potential treatment for thalassemia as well as for
sickle cell anemia would be to induce the patient's bone marrow cells to
switch from the synthesis of hemoglobin A to the synthesis of hemoglobin F.
Such a switch should eliminate production of the abnormal hemoglobin. In
order to understand how the normal switch from hemoglobin F to hemoglobin A
occurs, as well as to understand how it might be possible to make this
switch reversible, the mechanism by which a reversible switch in an animal
model system occurs is being studied. Sheep and goats provide such a model
system for analysis. By learning the mechanism of switching in sheep and
goats, it should be possible to apply this knowledge to human patients.
Proposed Course of Project; Studies are in progress to determine this
mechanism of the A to C switch in tissue culture.
Honors and Awards; None
Publications:
1. Nienhuis, A.W. and Anderson, W.F.: Hemoglobin switching in sheep
and goats: Change in functional globin messener RNA in reticulo-
cytes and bone marrow cells. Proc. Nat. Acad. Sci., USA, 69,
2184-2188, 1972.
2. Barker, J.E., Last, J. A., Adams, S.L., Nienhuis, A.W., and
Anderson, W.F.: Hemoglobin switching in sheep and goats:
Erythropoietin-dependent synthesis of hemoglobin C in goat bone-
marrow cultures*. Proc. Nat. Acad. Sci., USA (in press).
At^Z
Serial No. NHLI-112
1. Molecular Hematology Branch
2. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Mechanism of Action of the Enzyme RNA-Directed DNA
Polymerase
Previous Serial Number: None
Principal Investigators: Amy Falvey, Ph.D.
Joel Vavich, M.D.
Judith Kantor, M.Sc.&Hyg.
W. French Anderson, M.D.
Other Investigators: Jerold A. Last, Ph.D.
Dante J. Picciano, Ph.D.
Cooperating Units: Robert Gallo, M.D. of the National Cancer
Institute
Special Virus Cancer Program, National Cancer
Institute
Project Description:
Objectives: The enzyme RNA-directed DNA polymerase, also known as Reverse
Transcriptase, from avian myeloblastosis virus (and other sources) provides
a possible means for synthesizing a DNA gene directly from isolated messen-
ger RNA. Unfortunately, the DNA product so far obtained is only a partial
copy of the mRNA template. It is desired, therefore, to learn the mechanism
of action of the enzyme in order to establish conditions whereby a complete
(and active) DNA gene can be transcribed from a globin mRNA.
Methods : 1) The enzyme is purified by standard procedures from avian
myeloblastosis virus obtained from Dr. Beard thru the National Cancer
Institute's Special Virus Cancer Program.
2) Globin mRNA is isolated from rabbit reticulocytes; DNA product
analysis is by CeSO^ density gradient centrifugation.
3) Artificial block polynucleotide templates are being synthesized by
means of various enzymatic and chemical techniques including use of the
enzyme polynucleotide phosphorylase.
4) Primers and single base oligonucleotides are prepared by enzymatic
and/or chemical means followed by purification by standard nucleic acid
fractionation techniques.
P<^
Serial No. NHLI-112
Major Findings; 1) The DNA gene product obtained from globln mRNA directed
Reverse Transcriptase action is only a partial product.
2) Mechanism studies using artifically synthesized block polynucleotides
demonstrated that the activity of the enzyme Reverse Transcriptase is greatly
affected by the composition and size of the template and primer.
Significance to Biomedical Research and Institute Program; The ability to
synthesize a DNA gene Iji vitro would be a major step towards the goal of
successful therapy of human genetic diseases.
Proposed Course of Project: Detailed studies on the mechanism of action of
the enzjnne RNA-directed DNA polymerase are being carried out.
Honors and Awards ; None
Publications; None
SiH
ANNUAL REPORT OF THE
LABORATORY OF TECHNICAL DEVELOPMENT
NATIONAL HEART AND LUNG INSTITUTE
July 1, 1972 through June 30, 1973
The Laboratory of Technical Development develops new
instruments, devices and methods that can contribute to the
solution of clinical and basic medical science problems.
Specific technological systems and devices as well as
advances in engineering and basic physical science are exploited
for the needs of research and clinical medicine. Systems are
developed and evaluated in close association with other NIH
research programs.
LIQUID LIQUID CHROMATOGRAPHY
Development of countercurrent chromatography has been
continued on two types of instruments, the flow-through coil
planet centrifuge and the elution centrifuge, previously
developed here. Versatility and advantages over the convention-
al chromatographic system have been demonstrated by applying a
variety of two-phase systems and test samples.
With the flow- through coil planet centrifuge, both simple
and gradient elution of dipeptide samples are carried out at
high efficiencies ranging from 3000 to 1300 theoretical plates.
The gradient elution technique demonstrates a solute concentrat-
ing power as high as 10 times that in the original sample solution,
which can be applied to extract a minute amount of biological
principle from a large quantity of a crude mixture. The
system also enables the prediction of the retention volume of
the solute from the partition coefficient determined by
separatory funnel technique. For separation of macromolecules ,
the optimum column configuration of a coiled helix (60°) is
chosen to stabilize low interfacial tension phase systems
without losing the column efficiency. With a sec. Butanol 1%
Dichloroacetic acid (1:1) phase system, bovine insulin is
eluted out in a few hours to demonstrate a co-existing
deaminated form which was previously reported by Craig et. al.,
with countercurrent distribution method requiring over 1000
transfers.
The elution centrifuge technique is found to be applicable
to polymer phase systems used for partition of macromolecules
and particulates. Among various column configurations examined,
the coiled helix col\amn satisfies the fundamental requirements,
that is, stability of the retained stationary phase and
efficiency of partitioning. Capability of the technique is
.057
demonstrated on partition of polynucleotides (poly U, poly C,
poly A, and poly I) , DNA and RNA by stepwise and gradient
elution. Since the conventional liquid chromatography can
hardly utilize this aqueous/aqueous phase system and the
partition entirely relies upon the time consuming counter-
current distribution method, the present method should be useful
to extend the potential of the polymer phase systems.
FLUORESCENCE METHODS
Proteins labeled with dyes have a visible fluorescence
which is partly polarized. The degree of polarization can
be altered by shortening the lifetime with a quencher.
Correlation of the lifetime, the polarization, and the rate
of change of polarization with change of lifetime allows
determination of the rotational relaxation time of the protein.
Bilirubin complexes with proteins are of practical and
theoretical interest. The fluorescence circular dichroism
and absorption spectra of bilirubin complexes with albumins
of different species and apo-myoglobins of horse and sperm
whale have been investigated showing conformational features
of both protein and ligand.
A series of solutions of specific fluorescent substances
has been developed which have known lifetimes of from 0.20
to 115 nsec. + 3%. These solutions can be used to standardize
lifetime apparatuses as well as to aid in the computation of
lifetimes .
Fluorescent impurities have been found in water held in
contact with polyethylene. The spectra and characteristics
of various types of water used in analytical procedures have
been investigated.
Stopped-flow fluorescence has been applied to the rate
of folding and unfolding of different proteins, using the
changes in quantum yield of the tryptophan emission. A study
was performed which showed that the fluorescence of low density
lipoprotein was changed in various ways by partial or total
delipidation. Silver ion fluorescence quenching of protein
fluorescence was also studied. The mechanisms involved were
found to be energy transfer and collisional quenching.
CYTOLOGICAL METHODS
Rapid assay of cell response to mitogens, lectins, and
immunoactive reagents is being explored along several lines
in collaboration with other laboratories at NIH. The capillary
cXTA
I
tube scanning equipment developed for automation of micro-
biological assays of similar agents and antibiotic testing
is now being evaluated for cell assays.
Efforts of the past year have been directed at developing
methods for growing cells within a capillary tube so that the
monitoring and assaying of their growth can be accomplished
using scattered light. This has included developing methods
for creating the proper environment for the cells, developing
techniques for handling and washing the cells within the
capillary tube and using pulse height analysis and integration
to assay their growth.
Mouse marrow granulocyte growth has been monitored and
assayed using these techniques and this technique will be
extended to study human stem cells.
A method for culturing mycoplasma has shown promise. This
is a method whereby the cells are grown in thin lines on an
agar plate.
Studies of cell adhesion utilizing mouse hepatoma cells
have also shown promise. Two methods are being looked at,
resistance to fluid drag and centrifugatal forces applied to
the cells while in the capillary environment. Both of these
methods have shown promise and it seems that cell synchroniza-
tion and separation within the capillary can alse be performed
using these methods.
It would appear that a method of observing discrete cell
responses could be observed by a method that would be responsive
to individual cell temperatures reflecting their state of
activity.
The capillary tube method is responsive to growth and
requires sequential observation to determine their response
and the method is not particularly suited to recovery of
specific cells. The proposal to explore the utility of a
method responsive to individual cell temperature to reflect
the state of metabolic or physical activity has shown some
promise. Previous efforts to demonstrate individual cell
temperatures were based on the use of a newly developed solid
state material, Barium Strontium Niobate ; it was abondoned due
to defects in the crystals available and observation that the
curie temperature was higher (45°C) than represented (35°C) .
Liquid crystal free and microencapsulated were tested but
appeared less promising in terms of sensitivity and homogeneity.
Current efforts have demonstrated apparent success by
use of the interferometric observation of the thickness of a
<PS'3
deposit of volitile silicone oil on the obverse of a thin
membrane bearing the cells and observed with a microscope.
Dynamic evaporation and recondensing the oil maintains a
dynamic state of constantly reforming oil films the thickness
of which is responsive to the temperature of the membrane.
An imaging system with a niobium foil held at its
superconducting transition temperature in a liquid helium
cryostat to measure infrared radiation from individual cells
is being constructed for comparison. The niobium transition
bolometer is a novel approach suggested by our liason physicist
on temporary duty here from the Naval Weapons Center at China
Lake California.
Improvements in materials and signal processing equipment
form the basis of a new concept of microcalorimetry applicable
to biological measurements where long equilibration times
and slow integrations are incompatible with the instability of
such systems. A scheme for isothermal calorimetry has been
analyzed and is in process of being evaluated which is based
on the idea that a pair of high efficiency thermoelectric
heat-pumping wafers can be placed face to face to enclose a
thin microcavity for the sample with the other faces in contact
with a heat sink that is only required to absorb the evolved
heat emitted by a micro sample without significant temperature
change. The unusual characteristics of the system are obtained
by supplying current to the heat pump to maintain a near zero
thermal gradient across each element by alternatly sensing and
pumping heat. An isothermal system is maintained by this means;
evolved heat is simply a function of pumping current. The
system greatly increases the speed of response and negates
thermal capacity by pumping heat rather than permitting it to
escape by conduction or warming the sensor. Thus, equilibration
after adding a sample is very rapid as the system is forced to
equilibrium by the pump current. The period can be disregarded
while the slower evolution of heat can be measured during growth
or reaction by integration of the pump current.
The device has been constructed, its electrical behavior
analyzed and is ready for application to model biological
binding, immunochemical or micrometabolic studies to evaluate
what appears to be a new concept in calorimetry by eliminating
the need for large heat sinks and thick insulation.
PHYSIOLOGICAL MEASUREMENTS
A calorimetric instrument that monitors pC02 in the
extracorporeal blood circuit during the use of a membrane
oxygenator for acute respiratory failure exploits the fact
that the reaction of 1 nl/sec of CO with LiOH releases 4 ywatts
of heat. We have built a microcalorimeter with sensitivity and
<av
stability adequate to measure 10 nl/sec. of CO2 with a 40:1
signal-to-noise ratio. Blood passes through a probe in the
extracorporeal circuit; a short length of silicone rubber
tube allows CO2 to pass from the blood, in proportion to the
blood PCO2 , through the wall of the silicone tube with a stream
of nitrogen. The nitrogen carries the CO2 through a desiccant
to a container of LiOH in the calorimeter where the heat
released by the reaction is measured. We have shown that the
system can operate for a least 7 days with baseline stability
of + 3T (equivalent) and sensitivity stability of + 2T (equiva-
lent) . The overall instrument, less carrier gas supply, is
housed in a 13 X 23 X 32 cm box; the sensor probe is at the end
of a 1 meter dual lumen tube.
The principle was further developed to measure picomoles
of acid-releasable COo in nanoliter amounts of fluid in the
study of kidney acid-base control mechanisms. We have exploited
the fact that 4 yjoule is released when 10"^ 1 of CO2 reacts
with LiOH to build a calorimetric micro CO2 analyzer. We put a
grain of LiOH on one of a pair of thermistors forming a
resistance measuring half-bridge. Nanoliter samples are injected
through a mercury drop seal into concentrated phosphoric acid
and the released CO2 is transported to reaction chamber by a
stream of Freon-12. The system noise is low enough to yield
an ultimate sensitivity of 4xl0~12 moles of CO2 .
Nuclear magnetic resonance flow measuring and tracing
instrumentation has been constructed to operate at low magnetic
fields to reduce the contribution of the protons in the tissue
overlying the flowing blood on the basis that only premagnetized
protons make a major contribution to the observed signal and
low frequencies used in low fields penetrate with less loss
than high fields. The instrumentation has not lived up to
expectations and is still being improved. While the instrumenta-
tion is being further developed in this laboratory the contract
laboratory at the Medical College of Wisconsin has confirmed
that using tap water with a 2.1 second time constant that good
signals could be obtained seven time constants later without
signal enhancement methods.
These findings suggest that NMR tag detect systems are
useful over large distances involving many time constants.
Further studies with T]^ indicated that relaxation time was
dependent upon the magnetic field and hence the frequency of
detection. With h\aman blood, T-, of 0.25 seconds was measured
with a dc magnetic field of 23.4 gauss corresponding to a
Larmor frequency of 100 kHz. Similar studies in a magnetic
field of 760 gauss with a detector operating at 3.2 megaHz gave
a T-, of approximately 0.7 seconds for fully oxygenated human
blood. A T-, of 0.4 seconds was measured in partially oxygenated
human blood. These tests and theoretical investigation suggests
that T-i is a function of frequency and percent of oxygenated
5 A^^
hemoglobin. Future studies will be designed to investigate
structures capable of providing larger magnetic fields to
study T-j^ as a function of frequency, oxygenation, so that a
practical system can be designed for use in the measurement
of human cerebral blood flow. At present the experimental and
theoretical studies suggest that it should be possible to
measure arterial and venous cerebral blood flow in the human
with NMR systems. Theoretical studies have confirmed our
experimental studies in tapered and bifurcated tubes. During
this phase of the project, flow studies were made in the
isolated perfused kidney of the dog. Continuous flow and
regional flow measurements were possible in this preparation.
Pulsatile flow was measured in the radial artery at the wrist
of an adult human volunteer. Furthermore, preliminary NMR studies
were conducted in stumptail macaque monkeys indicating that NMR
signal output levels changed in a coil located over the occipital
protuberance with deprivation of oxygen secondary to clamping
off an endotracheal airway or following hyperventilation. The
studies to date have been encouraging and have provided new
information on the effects of T-^ vs. frequency and oxygenation.
INSTRUMENTATION FOR KINETIC AND EQUILIBRIUM MEASUREMENTS
The high-speed stopped flow apparatus developed in this
laboratory has been undergoing a number of refinements to
improve its reliability, resistance to corrosion, and biological
compatibility. Three experimental instruments made to our
specifications have been built and are undergoing testing. One
of the instruments has made it possible to work out the primary
steps in the lactate dehydrogenase, its isoenzymes, creatine
phosphokinase , and transaminase reactions.
The high time resolution of this instrument has permitted
direct stopped-flow flash photolysis studies on red cells.
Thus, in Hb S the sickling process can be studied .
A further extension of the development of instruments to
study sickle cell anemia is being continued at the University
of Milan. Several instruments are being built to assist in
the assessment of hemoglobin reactions using the special cyanate
intermediates made there.
In collaboration with the Thermochemistry Section, NBS ,
three batch calorimeters have been constructed at NIH and are
undergoing testing in various laboratories to ascertain their
suitability for use in biochemical and clinical chemistry using
only 150 microliters of each reagent. The measurement of amino
acids, using decarboxylases has achieved a sensitivity of 0.2
nanomoles. Another instrument is being used to study antigen-
antibody reactions and the deoxygenation of sickle cells.
A$5
I
In order to carry out replicate experiments rapidly on
highly labile biological materials, a stopped-flow micro-
calorimeter has been constructed and is operating with a cycle
time of 5 minutes. The same sensitivity as the batch machine
is available but with the ability to handle 100 to 500 micro-
liter samples of each reagent.
SECTION ON PULMONARY AND CARDIAC ASSIST DEVICES
This section has established the value of the spiral coil
membrane lung for extended respiratory assist in pulmonary
failure in the laboratory in sheep, and clinically in man.
Recently, a patient with acute respiratory failure from
Pneumocystis carinii pneumonia was supported continuously for
9 1/2 days with the membrane lung with subsequent recovery.
He is now well and alive. This perfusion is the longest on
record and attests to the benign nature of this treatment mode,
and the superiority of the membrane lung over presently
used blood oxygenators.
We have developed a technique for producing pinhole free
silicone rubber membranes reliably, and in quantity. Membranes
of pure dimethylsiloxane gum have also been cast without
pinholes with equal reliability. These pure silicone gum
membranes have been shown to be superior in antithrombogenicity
to all pure polymers studied so far. These and other new
membranes are continuously evaluated in chronic animal studies
in sheep. This project is carried out in conjunction with an
on campus scientist from the Armed Forces Radiological Research
Institute and a guest worker from the Asahi Chemical Co., in
Japan.
Work is in progress to permit long term preservation of
an excised heart or another internal organ. Sheep hearts
have been perfused ex vivo for 3 days at 10 °C with normal
function on rewarming. Hearts have also been maintained with
a synthetic perfusate dialyzed against animal blood across
cellophane and thin silicone rubber membranes. All these
hearts exhibit a continuous electrical and mechanical activity
down to 10°C and. lower.
a^r
Serial No. NHLI-113
1. Laboratory of Technical Development
3. Bethesda, Maryland
PHS - NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Discrete Cell Temperature Measurement Study
Previous Serial No.: NHLI-105
Principal Investigator: E. Ronald Atkinson
Other Investigator: Robert L. Bovman
Cooperating Units: None
Project Description:
Objectives: Cellular metabolic processes produce heat. The heat produced
results in a temperature gradient about living cells. The objectives of this
project are (a) to determine a method of measuring this temperature gradient
about discrete cells in vitro, (b) to provide a rapid method of measurement,
and (c) to provide a non-destructive method of measurement.
Methods Employed:
(a) Analysis: The analytical portion of the project consists of simple
modeling of the thermal cell-ambient situation. Adjustable parameters are
experimentally evaluated. On the basis of the model, the instrumental
requirements for thermal and spatial resolution are established. Alternative
physical effects are considered for exploitation and a preliminary selection
made.
(b) Materials Selection: Materials having the requisite properties are
configured and tested in a manner similar to their actual use in cell
temperature determination. Measurements of living cells are made.
(c) Instrumental Design: The most promising approach is subjected to
design optimization. A prototype instrument is fabricated and evaluated.
Performance limits and problem areas are defined for this class of instrument.
Major Findings:
(1) Updated MEDLARS and Smithsonian SIE literature searches indicated that
no discrete cell temperature measurements have yet been made. A number of
microcalorimetric studies of large aggregates of cells were found. These
studies indicate thermal outputs on the order of 10"' to 10"° watts/cell
in actively metabolizing mammalian and bacterial cells of 10 to 100 micro-
meter diameter.
^sy
Serial No. NHLI- 113
(2) Several laboratories of the U. S. Atomic Energy Commission have been
canvassed for possible radioactive isotope materials to be used in
calibrating cell temperature instruments. It appears that plutonium 239
oxide microspheres are appropriate for the job. Acquisition and handling
of these microspheres is under investigation.
(3) A super-conductive bolometer cryostat has been designed and is in the
final stages of fabrication and asserably. Twenty-five micron thick niobium
foil has been acquired for the construction of the super-conductive bolometer
element .
(4) Some of the better known cholesteric liquid crystal temperature
indicators were configured for discrete cell temperature measurement. The
materials investigated thus far fell far short of the required sensitivity.
Other liquid crystals will be examined as they become available.
(5) Barlum-strontium-niobate was ground and polished to thin film
dimensions and configured on a thermal stage to test the ferroelectric
curie point thermometer concept. The strontium rich barium-strontium-
niobate tested fell far short of the required sensitivity. The material
was found to have a very gradual ferroelectric-paraelectric transition.
This gradual transition seems to be characteristic of the few non-stoichio-
metric ferroelectrics reported in the literature. A single composition
material, triglycene selenate has a curie point at the right temperature
and will be tested for adequate sensitivity for discrete cell temperature
measurement.
(6) A unique instrument, the "Teensy-Temp', has been designed, fabricated,
and is under intensive investigation for discrete cell temperature measure-
ment. The device utilizes the differential condensation rate of hexamethyl
siloxane on a cellulose nitrate membrane to measure heat output of objects
resting on the other side of the membrane. Read-out of siloxane film
thickness is by inter ferometric imaging. The "Teensy-Temp" appears to have
adequate sensitivity and may lend itself well to certain types of discrete
cell temperature measurement. Preliminary experiments with protozoa and
mouse hepatoma cells have been encouraging. A computer simulation of"reen8y-
Teap" operation has been programmed and system parameters are being optimized.
Significance to Biomedical Research and the Program of the Institute:
Cell temperature measurement is fundamental to metabolic, ontologlc , and
pathologic cytological investigations.
Proposed Course: It is proposed to continue the material investigation
to establish performance bounds and develop configurations suitable for cell
temperature measurement. An instrument incorporating one or more selected
materials will be designed, fabricated, and evaluated with a variety of
cell cultures.
^C
Serial No. NHLI- 113
Honors and Awards: None
Publications: "Microscopic Temperature Determination with Ferroelectric
Thin Film Optics" by R. L. Bowman and B. R. Atkinson. Proceedings of
the 16th Annual Technical Meeting of the Society of Photo-Optical Instru-
mentation Engineers, 17 October 1972, San Francisco, California.
3i6l
Serial No. NHLI- 114
1. Laboratory of Technical Development
3. Bethesda, Maryland
PHS - NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title:
Previous Serial No.
Development of Stopped-Flow Micro-Calorimetry for
the Study of Biochemical and Cellular Reactions;
Applications to Clinical Biochemistry
NHLI-104
Principal Investigator: Robert L. Berger
Other Investigators:
Cooperating Units:
Nadja Rehak, Frank Noble, Technical Development
Donald Young, Chief, Clinical Chemistry, NIH
G. A. Armstrong, Chief, Thermochem. Sect., NBS
Edward Prosan, Physical Chemistry Div., NBS
N. 0. Kaplan, Prof., School of Med, Univ. Calif. S.D.
Hans Krebs , Prof. Biochem., Oxford Univ.
Luigi Rossi-Bemardi , Prof. Enzymology, Univ. Milan
Mario Marini, Assoc. Prof .Biochem. , Northwestern Univ.
Norman Davids, Prof.,Eng. Mech., Penn State Univ.
Fabrication-BEMI; Research Instr. Facility, School
of Med., UCSD;, American Instr. Co.; Thermonetics ,
Inc.; Thermometries, Inc.
Project Description:
Objectives: Virtually all chemical reactions produce heat and calorimetry
has long been used to investigate them. For biological use, however, high
sensitivity, small volumes of reactants , and short equilibration times are
needed. It is the objective of this project to develop such an instrument
for use in the time range of a few seconds to 1 or 2 hours.
Methods Employed: Initial designs are constructed in this laboratory with
special assistance from commercial firms in the construction of sensors;
contracts are let, where warranted, for the development of a completed
instrument with refinements that would tax our own facilities. The instru-
ment is then tested in conjunction with other interested biochemical calori-
metrists utilizing appropriate enzymatic and cellular reactions.
Major Findings: Three of the batch type small calorimeters have been built
and two placed in different laboratories for testing. This instrument,
developed in collaboration with NBS, is identical to the new flow calorimeter
except that a small two-compartment cell is used and mixing is achieved by
rotation of the inner block. 150 ^1 of each reagent can be used. Equilibrium
Sii,X
Serial No. NHLI-114
Is achieved in 15 min. One of these units has been placed with Prof. Hans
Krebs, Oxford Infirmary, for exploratory studies of its use for decarboxyla-
tion reactions in amino acid analysis and metabolic control reactions. The
second unit has been placed with Prof. Luigi Rossi-Bernardl, University of
Milan; his report follows:
The NBS-NIH mlcrocalorlmeter has been extensively tested In our laboratory
by measuring changes resulting from (a) a standard neutralization reaction
and (b) reactions of biochemical and clinical interest. The reaction was thus
initiated by mixing 100 to 150 microliters of Reagent 1, which was contained
in a Kel-F cuvette contained in the calorimeter with 5 to 30 microliters of
the second reagent contained in a micro syringe. Satisfactory calibration
curves were obtained by this method by titrating standard base and acid.
The heat changes measured were between 5 and 100 mllllcalorles. The first
reaction of biochemical Interest studied was a thermal titration of human
oxy and deoxyhemoglobin, obtaining results in agreement with those obtained
by standard macro calorimetrlc techniques. Several reactions of clinical
significance are under study in our laboratory at the present tlmej such as
(a) agglutination reactions like those Involved in the determination of blood
groups and for the diagnosis of pregnancy and (b) the deoxygenation reaction
of normal human oxyhemoglobin and of hemoglobin from sickle cell anemic
patients.
Stop- flow mlcrocalorlmeters developed in this laboratory and a rapid batch
mlcrocalorlmeter designed by E. Prosen (NBS) have been calibrated using
HCl/NaOH (C02-fre«) reaction. The calibration heat was in the range of
0.5 - 150 meal, and the minimum volume of each reactant was 29 )il (max.
175 jil). The heat output obtained was linear (+ 2-U%) and the stability
over a period of 7 months satisfactory (K = 16.67 + 0.17 Joules/volt sec).
In order to evaluate the use of microcalorlmetry in clinical chemistry,
the following enzyme , catalyzed reactions have been investigated and their
heats of reaction as measured are listed:
Urate/urlcase 4.26 kcal/mole
Urate/uricase catalase 28.26 kcal/mole
L-glutamlc acid 3.88 kcal/mole
L-lyslne 3.47 kcal/mole
The heat corresponding to reactions catalyzed by catalase obtained on the
basis of the urate reaction is 24.0 kcal/mole at 30° C as compared to the
reported value of 24,0 + 0.3 kcal/mole.
In order to evaluate the use of microcalorlmetry for the kinetic study of
enzjrme catalyzed reactions, the urate-uricase reaction was chosen for
Investigation. The stolchlometry of this reaction Is quite well established
and the kinetic studies have been carried out by spectrophotometric methods.
Thus the results obtained using microcalorlmetry can be compared with those
already existing.
2 <9^3
Serial No. NHLI-114
Significance to Biomedical Research and the Program of the Institute: The use
of these methods as an analytical tool for clinical chemistry shows consider-
able promise as a means of improving the accuracy, precision, and thruput of
clinical tests. In addition, it makes possible the use of many new tests
for enzyme or substrate tests, antigen-antibody reactions, coagulation tests,
etc., which are not now able to be done due either to the lack of a suitable
detection method or to the fact that the present tests are long, have high
variability, and are therefore not used.
Perhaps of more importance from the long-range significance of this project
is the possibilities that the calorimeter offers for the study of many
biochemical reactions which cannot now be investigated due to a lack of a
suitable detector of the reaction. An example of current interest is the
many steps preceeding final coagulation that occurs in the forming of a
thrombus.
Proposed Course: The direct application of these methods is projected by
a variety of individuals requesting grant support. Two projects, one using
our calorimeter (NBS) and one employing our thermal titration apparatus
(Marini) are pending funding by the Automated Clinical Chemistry Section,
NIGMS. Our own interests will be directed toward instrument development as
outlined under the project on the development of methods for investigating
the mechanism of hemoglobin reactions. In addition, we will serve as
consultants to both the NBS personnel and the Northwestern group (Marini).
This will involve assisting in biochemical systems analysis and instrumental
problems. The problems of references, serum samples, etc., will be handled
by Young, NIH Clinical Center, Clinical Chemistry Section. Cooperation with
several industrial firms desiring to become active in the area will also be
provided (American Instrument Company, Division of Travenol Laboratories,
Thermonetics , Inc., and Thermometries, Inc.
Honors and Awards: Invited speaker, Symp. on Thermochemistry: Clinical
Applications of Microcalorimetry , NIOIS. Invited speaker. Academic Clinical
Laboratory Physicians and Scientists Annual Meeting.
Publications: None
»^
Serial No. NHLI-115
PHS - NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Development of Methods for Investigating the
Mechanism of Biochemical Reactions Important in
Cardiology, Pulmonary and Respiratory Function,
and in the Circulation.
Previous Serial Ho.: NHLI-103
Principal Investigator: Robert L. Berger
Other Investigators: R. Wayne Albers, NIND&S, Lab. Neural. Chem.
M. Marini, Dept. Biochem, Northwestern Univ. Med. Sch.
N. 0. Kaplan & J. Everse, U of C San Diego Med. Sch.
L. Rossi-Bernardi, Univ. of Milan
T. Asakura, J.A.McCray, & P. Smith, Johnson Founda-
tion, Univ. of Penna.
W. F. Friauf, BEIB
R. Shrager, DCRT, Lab. Physical Sciences
M. Sapoff, Thermometries, Inc.
Project Description:
Objectives: The objectives of this project are to develop new instrumentation
methods, data handling techniques and theoretical treatments for the physio-
cheaical study of the thermodynamics, kinetics and thus the mechanisms of
enzyme action in solutions and in the intact cell or cell membrane. In
particular, to study, in collaboration with other laboratories, the reactions
of hemoglobin with the respiratory gases both in the normal state and as
modified by the changes of physical factors, small molecules, various
metabolites, and genetically, such as in sickle cell anemia. The reactions
of various cellular enzymes, particularly ATPase and lactate dehydrogenase,
and their interactions, and control, in the cell are studied as they relate
to the hemoglobin reactions in cardiology, pulmonary and respiratory function,
and circulation. Where appropriate application is indicated, clinical
analytical methods are developed using these techniques.
Methods Employed: the methods used in the Investigation of the mechanisms
of enzyme action are those of pre-steady state chemical kinetics and thermo-
dynamics. Measurements of the appropriate parameters are made by building the
necessary equipment to mix solutions rapidly and follow the course of the
resulting chemical reactions by optical, thermal, glass electrode, etc.,
detectors. In general, equipment is not available, either in the literature
or commercially, for investigations in this area. Such appratus is conceived
and designed in this laboratory, together with consultants, construction being
carried out wherever most appropriate; i.e., in our shops or by commercial
forms, special university facilities, or at the several special research
laboratories such as the Jet Propulsion Laboratory. A special effort is then
1 s^r
Serial No. NHLI- 115
made to produce commercial versions of the equipment available to the public.
In pursuing these investigations, a wide variety of physical parameters must
be studied, which leads to the need for an understanding of the underlying
physical theory governing the reactions. Expert consultants and collaborators
are brought in to assist in the design, analysis, and evaluation of the
equipment, particularly as it applied to certain specific enzyme systems
under investigation.
Major Findings: The high-speed flow apparatus has been undergoing improvements
in materials to render it less prone to corrosion, vibration, and stop-valve
breakdown. Three instruments are in the field, one at the University of
California at San Diego where Kaplan and Everse are studying the initial rates
of reaction of the dehydrogenases with their coenzyme and substrates; another
at the Johnson Foundation for Medical Physics, University of Pennsylvania,
where McCray and Smith are adapting the unit to a high-energy liquid dye
laser to do stopped-flow laser flash photolysis of oxyhemoglobin with
particular emphasis on HbS. In addition, preliminary experiments indicate work
can be done directly on the red cell which would help considerably in under-
standing the sickling process. The third apparatus is in this laboratory
where continued work is being done on hemoglobin and the Ca + EGTA reaction.
It is hoped that materials and instrument electronic problems will be finished
in the near future and extensive testing can begin.
Work on the stopped-flow rapid reaction thermal and pH system has proceeded
slowly, due to the lack of personnel, but several important advances are to be
noted. A fast thermistor probe has been developed and tested. Its 1/e res-
ponse is 12 milliseconds with a glass-coated bead. An A-C Bridge has been
developed by BEMB which has a sensitivity of about 50 microdegrees using
this probe.
Some advance has been made on the problem of coating glass electrodes to
protect them from the response slowing caused by exposure to protein. A
commercial coating, a silicone, has been found to help considerably. Uncoated
response (10-907.) was 2 sec for the radiometer type G222 C in phosphate buffer
with stirring (likely time limit is the stirring). In 10 mM hemoglobin the
response was down to 10 sec or slower. After coating, it was back to 2 sec.
Automation of the pH titration system cannot proceed until the electrode
response has been considerably improved.
The new three-syringe variable ratio stopped-flow system has been completed.
A small double mixer attached to a supersil Quartz observation cell has been
built to go with it. The dimensions are such that it will fit any 1 cm
square cuvette holder. The unit has been installed in a standard Guilford
Spectrophotometer with a modified photometer head. Mixing tests and kinetic
experiments on solubilized ATPase are being conducted by Albers.
3iU
Serial No. NHLI- 115
A thermal titration system developed in this laboratory has been undergoing
intensive testing on hemoglobin, cytochrome C, and other molecules at the
Dept. of Biochemistry, Northwestern Univ. School of Medicine. Data analysis
is being carried out on the DCRT PDP-10 computer using the Shrager-Knott
MLab system. The first full analysis of any isolonic protein titrated
potentiometrically and thermally has been carried out on isoionic hemoglobin
and the results are presently being analyzed. At present the major instru-
ment problem is the development of a fast pH electrode unaffected by protein.
The thermal titration takes only 90 sec whereas the pH titration takes over
an hour. It is hoped that a combined pH-heat system can be built in the near
future .
A new stopped-flow microcalorimeter has been constructed using Peltier sensors.
A pre-incubator has been installed and a new PVC cell constructed. Testing
indicates an instrument constant of 13.4 joules/volt-sec. The lower this
number, the more sensitive the instrument is. This instrument can handle
100 to 500 microliters of each reagent. The instrument has a sensitivity
of .1 microwatt. In terms of a chemical reaction this represents 0.2 nanomoles,
A repeat can be done every five minutes. IS min are required presently between
different samples. It is now clear that the sensitivity can be increased
another order of magnitude and the time for equilibration reduced two orders
of magnitude.
Significance to Biomedical Research and the Program of the Institute:
An understanding of the basic mechanism of disease is a prerequisite to
prevention or cure. The investigation of the reaction of the respiratory
gases with hemoglobin, the red cell, and cytochrome oxidase in heart muscle
cells is fundamental to an understanding of normal cell respiration and
particularly to what has gone wrong as in the case of sickle cell anemia,
mycardial infarction, etc. It is hoped that this research will result in
instrumentation to permit the medical scientist to perform research leading
to clarification of the ways in which, for example, sickle cell anemia can be
managed by, say, the use of cynate, which we know binds to the charged
terminal ex; -amino groups of the hemoglobin molecule. The extension of such
investigations to other disease systems and possible results seem abundantly
clear in terms of preventive medicine and improvement in health care.
Proposed Course: The high-speed flow system will continually be upgraded
and extensively tested both as a stopped-flow unit and combined with the
liquid dye laser. The hemoglobin experiments in cooperation with others
will be expanded to a more comprehensive pH and temperature range as well as
to mutant hemoglobins such as HbS, Seattle and Bethesda. All of these show
widespread and physiologically important variations in their dissociation
curves, which almost assuredly can be elucidated by an understanding of the
"on" and "off" reactions coupled with the Bohr effect.
5i67
Serial No. NHLI- 115
Extensive tests are also planned for the new thetmistor stopped-flow apparatus
together with a new variable ratio stopped-flow system that will permit an
enzyme concentration to be held constant while substrate is varied in order
that Vjpgjj and Michaelis constants can be very rapidly determined.
A differential thermal titration cell will be constructed for the purpose
of eventually including the automated pH titration system also under
development.
The new driven Peltier calorimeter will be developed and tested for use in
clinical biochemistry reactions. The computer simulation and data-handling
problems will be extended to improve the effectiveness and simplicity of
the instrument. In particular, the interface between the instrument and
the PDP-10 Computer Modeling Laboratory will be expanded to achieve reasonably
fast turnaround time.
Honors and Awards:
1. Member, Advisory Board "Kinetic Center", Johnson Foundation for Medical
Physics, Dept. of Biophysics and Biophysical Chemistry, School of Medicine,
University of Pennsylvania.
2. Consultant, Biotechnology Branch, Division of Research Resources.
3. Guest Worker and Consultant to Thermochemistry Section, Physical Chemistry
Division, Inst, of Material Sciences, National Bureau of Standards.
Publications:
1. J. Everse, R. L. Berger and N. 0. Kaplan, Complexes of Pyridine Nucleotides
and their Function. In Structure & Function of Oxidation Reduction
Enzymes. Edited by A. Skeson & A. Ehrenberg, Pergamon Press, Oxford
and New York, 1972.
2. R. L, Berger, L. Carpenter, J. Everse and J. L. Kaplan, Human Isoionic
Hemoglobin: Preparation and Kinetic Properties. Anal. Ltr 6:125, 1973.
3. R. L. Berger and B. Balko, Thermal Sensor Coatings Suitable for Rapid
Response Biomedical Applications. In Temperature, Its Measurement and
Control in Science and Industry, Vol. 5, Part 3, 1973.
3i4,g
Serial No. NHLI- 116
1. Laboratory of Technical Development
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: An Automated Method for Rapid Bacterial and
Mammalian Cell Growth and Assay
Previous Serial No.: NHLI-102
Principal Investigator: Peter Carmeci
Other Investigators: None
Cooperating Units: Clinical Chemistry Laboratory
Department of Laboratory Medicine
University of Minnesota Hospital
Dr. Philip Blume
Laboratory of Infectious Diseases, NIAID
Dr. Helmut Brunner
Medicine Branch, NCI
Dr. Joan Bull, Dr. Larry Abrams
Molecular Diseases Branch, NHLI
Dr. V. Mangenello
American Instrument Company, providing
Test prototype for manufacturing.
Project Description:
Objectives: The objectives of this project are to adapt this
instrument to various requirements of biomedical research by
cooperating with potential users of the method to develop the
techniques and methods necessary to facilitate the utilization
of the capillary tube scanner for clinical and research
application. At the present time efforts have been expended in
the following five areas of endeavor.
A. Developing methods and techniques for automating antibiotic
sensitivity tests.
B. Developing methods of pulse height discrimination for
(1) segregating types of bacterial colonies during incubation,
and (2) studying the effects of growth factors and environment
on mammalian cells.
a^f
Serial No. NHLI- 116
C. Developing methods for early detection of mycoplasma
(pneumonia) .
D. Developing methods for detecting the adhesive qualities of
of hepatoma and other cells.
E. Developing methods for assaying bone marrow stem cells.
A. The efforts to date have been oriented towards developing
an inexpensive and readily automated method for performing
antibiotic sensitivity tests using the Colony Counter System
developed here. The approach has been to replace the capillary
tubes with a single plastic plate containing 14 channels that
will accept a mixture of agar and organisms. Each channel of
the plate contains a strip of antibiotic impregnated filter
paper. This paper is bonded to the side of the channel so that
the light path is not obstructed. The entire plate is covered
with an adhesive backed mylar film. The entire plate can
readily be prepared and stored prior to injection of an agar-
bacterial solution.
To date a final mold has been fabricated and 2000 plates have
been made. Dr. Blume at the Universitv of Minnesota Hospital
will use these for clinical tests. Subsequent work will be
aimed at overcoming the technical and economic difficulties of
performing these clinical tests.
B. A bread-board model pulse height discriminator has been
built and the growth of stem, myeloma, hepatoma cells has been
demonstrated. In addition, it has been found that a measurement
of total growth of all viable colonies in a capillary, i.e.,
the integration of light scattered pulses, provides another
distinctive and sensitive parameter for growth analysis. Both
of these parameters, total growth and pulse height discrimina-
tion, have been designed and fabricated in a new unit that is
presently undergoing test. Subsequent work will be to
characterize the growth of myeloma, hepatoma, and stem cells
under specific environmental conditions.
C. Previous indications have shown that myoplasma can be
detected by scattered light within 48 hours (compared to 10 day
incubation period normally required) . Growth in agar has not
been conducive to growth. Growth in broth and in thin films
of agar has been successful but not optimal. A new technique,
based on the previous experience of growing, mycoplasma on thin
films of agar, has shown more optimal growth. This consists of
introducing the mycoplasma in solution to the surface of a
thick (2 mm) surface of agar in a number of thin lines that are
optically aligned to the capillary scanner. Further testing of
$^70
Serial No. NHLI-116
this technique is under way.
D. The adhesiveness of cells to glass is related to the
characteristics of the cell surface or membrane. This
adhesiveness has been used as a measure for alterations of this
surface by gluco-cortecoids and other substances. The capillary
tube and the detection of scattered light provides a relatively
easy means for assaying the property of cell adhesiveness.
Preliminary work has shown that hepatoma cells grown in media
within a capillary tube adhere to the glass and can readily be
counted. The capillary also provides a means for washing off
the media and non -adhered cells and monitoring the growth of
adhered cells.
Two methods of assaying adhesiveness have been investigated.
The first is to wash the cells in the capillary using known
shearing forces i.e.; wash the cells using constant pressure.
The other has been to centrifuge the cells in the capillary
removing all but the well adhered cells. Both of these methods
have shown promise for adhesive assay along with the possibility
of using these methods to synchronize cell growth and for cell
separation. Studies so far have indicated that the small
immature cells have much greater adhesive qualities than larger
groups. Subsequent work will be aimed at determining which of
the techniques provides more optimal results for cell adhesion
and to investigate the use of these methods for cell separation
and synchronization.
E. The assaying of viable bone marrow stem cells normally
requires 10 days. It has been demonstrated that stem cell
growth can be detected and assayed within 3 days using capillary
tubes and the scanner. Present efforts have been towards
correlating data from the scanner with present methods of assay,
developing filling and culturing techniques, and determining
the effects of pH, type of media, temperature and oxygen upon
the growth of the cells in a capillary environment. Pulse
height analysis should give further insight into the growth
characteristics of bone marrow stem cells. These studies have
been performed in mouse marrow granulocyte growth, subsequent
work will be aimed at normal human stem cells.
Significance to Biomedical Research and the Program of the
Institute: The utilization of capillary tube scanning techniques
in bacteriology and mammalian cell cultures provides (a) means
of detection very early in the growth of colonies, and (b)
methods that are rapidly adaptable to automation and require
extremely small samples. The application to the study of cell
metabolism, metabolic defects, oncology, and clinical cell
sample assay is anticipated.
57/
Serial No. NHLI-116
Proposed Course:
A. Continued application to fully automate the antibiotic
sensitivity testing procedures by cooperating with Dr. Blume.
B. Develop hardware for pulse height analysis and investigate
its utilization for segregation of two or more organisms by
their growth rates, and characterize cell growth under varying
conditions .
C. Develop techniques to apply capillary scanning for detecting
and measuring mycoplasma (pneumonia) .
D. Investigate the potentials of the instrument for determining
cell adhesiveness, cell synchronization and cell separation.
E. Optimize this technique for bone marrow stem cell assay.
Honors and Awards: None
Publications :
Abrams , L. , Carmeci, P., Bull, J. M. , and Carbone , P. P.:
Capillary Tube Scanning Applied to In Vitro Mouse Marrow
Granulocyte Growth. J. Nat. Cancer Inst. 50: 267-270, 1973.
A7A
Serial No. NHLI-117
1. Laboratory of Technical Development
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Fluorescent Complexes of Proteins
Previous Serial Number: NHLI-263
Principal Investigator: Raymond F. Chen
Other Investigators: None
Project Description:
Objectives: Proteins have biological functions which are very
often complexes where the ligand emits light, we can study the
nature of the binding and further our understanding of protein
function. Also, proteins covalently labeled with fluorochrome
signal their physical characteristics through the properties of
their extrinsic fluorescence.
Methods Employed: Fluorescence measurements of spectra, quantum
yield, lifetime, and polarization were made according to methods
previously developed and described using Aminco-Bowman spectro-
fluorometer and TRW lifetime apparatus.
Major Findings: Work was continued on the bilirubin-albumin
system. Previously we had found that bilirubin in complex
with the albumins of several species including human, bovine,
rabbit, pig, horse, and sheep was fluorescent and developed
intense CD bands. Also we had found that fluorescence quenching
of the albumin fluorescence by the bilirubin was a convenient'
way to determine the dissociation constants. Stopped-flow
fluorescence measurements had shown that the binding was fast,
but the development of bilirubin fluorescence was measurable,
in the range of 15-100 msec. We have now continued the stopped-
flow measurements to study the binding kinetics with the differ-
ent albumins and have shown marked species difference in rates
of bilirubin fluorescence development. A major finding was
that although the binding is very rapid, there is not only a
slower development of bilirubin fluorescence but also a slower
additional quenching of protein fluorescence. In other words,
after bilirubin binds to albumins, bilirubin structure changes
as does protein conformation.
Bilirubin has also been bound to apo-myoglobins. The myoglobins
are proteins containing heme. Apo-myoglobins are the proteins
with the heme removed. It was reasoned that since bilirubin is
a tetrapyrrole breakdown product of heme, the bilirubin might
be able to curl up and bind in the heme crevice of the apo-
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Serial No. NHLI- 117
myoglobins. We have investigated this possibility with horse
and sperm whale myoglobins and find, indeed, that the binding
the bilirubin causes a strong extrinsic circular dichroism band
(CD) consistent with asymnetric binding to a specific site on
the proteins. The fluorescence of the bilirubin does not seem
to be enhanced on binding. However, the protein fluorescence
is quenched and it is suggestive of 1:1 binding. This is
supported by apparent displacement of the bilirubin by hemin,
and displacement of ANS (a dye which binds to the heme crevice)
by bilirubin.
Depolarization of fluorescence measurements have been used for
about a dozen years for following changes in shape of proteins.
Usually the method is to measure the polarization of fluorescence
as a function of temperature of viscosity and calculate the
relaxation time from the data. An alternative method has been
applied by others to depolarization studies utilizing tryptophan
fluorescence of proteins; namely, to follow the polarization
changes upon altering the lifetime of the fluorescence with
quenchers. This approach has been investigated with dye-protein
conjugates, and various quenchers. We have prepared protein
complexes containing DNS, fluorescein, anthracene, and NBD
(an oxadiazole dye) and tested the efficacy of various quenchers
including Cu"*"*", iodide, and various other anions. The advantages
and limitations of this method of utilizing fluorescence
polarization have been appreciated. On the one hand, one can
get the relaxation times at 1 temperature without changing the
viscosity. On the other hand, the precision of the measurements
depends on the precision of the lifetime measurements, and there
is a question as to whether the quencher interacts with the
protein complex to alter the very characteristics one seeks to
measure. We find generally that the method is useful where
the dyes are attached to exposed sites.
Significance to Biomedical Research and the Program of the
Institute: Bilirubin-albumin complexes occur physiologically
and ,are important in heme metabolism and certain pathological
states such as kernicterus. Furthermore, bilirubin competes
with fatty acids for binding sites on albumin, so the study of
bilirubin-albumin complexes is relevant to fatty acid transport
and metablolism. The investigation into the use of quenching
to obtain fluorescence depolarization-relaxation time data
helps to advance a biophysical method for determining protein
structure.
Proposed Course: Although part of the bilirubin work has been
written up in a symposium proceedings volume, we wish to do a
more complete write-up and publication. We also wish to do
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Serial No. NHLI-117
further experimental work on the bilirubin-myoglobin systems.
The plarization of fluorescence measurements will be studied
further using the newly arrived ORTEC single photon spectrometer,
using nanosecond polarization methods. Also, we hope to set
the instrument up to do time-resolved spectra.
Honors and Awards : None
Publications:
Chen, R. F: Bilirubin Fluorescence and Fluorescence Quenching
in Complexes with Serum Albumins of Different Species.
Abstract EVIb5/4 , Fourth International Congress of Biophysics,
Moscow, Aug. 7-14, 1972.
SilS-
Serial No. NHLl-lli
1. Laboratory of Technical Development
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Methodology in Fluorescence Measurements
Previous Serial No. : NHLI-95
Principal Investigator: Raymond F. Chen
Other Investigators: None
Cooperating Units: None
Project Description:
Objectives: Instrumentation and methods in fluorescence
spectroscopy are increasing as the number of people using these
tools increases. There is room for improvement and extension
of techniques, and we wish to make a continuing effort in this
direction.
Methods Employed: As in the past, we have taken commercially
produced instrumentation and asked ourselves how to improve or
modify the apparatus to extend its usefulness. Also we try to
point out in the literature ways to use various apparatus that
are not initially obvious.
Major Findings: 1) With the new ORTEC model 9200 single photon
spectrometer, we investigated the possibility of determining
lifetimes without need for a digital computer. The instrument
determines the shape of the lamp pulse and then determines the
convolution of the lamp pulse with the fluorescence decay of a
sample. The desired decay curve is one which would result from
a true delta function light pulse. As this is not directly
obtainable, most workers resort to a digital computer to de-
convolute the observed fluorescence curve. Our approach is to
make up a series of fluorescent solutions of known lifetime.
One then compares the curve for the unkown with the curves of
the standards until one finds the standard curve which matches.
In order to have standard solutions of known lifetime, we have
found that quinine in sulfuric acid has a lifetime which is
generally agreed to be 18.9 nsec. in 0.1 N H2SO4 . We then
discovered that Cl~ quenches the fluorescence with very strict
adherence to collisional kinetics (Stern-Volmer law) even where
the quenching is over 99% complete. Thus , addition of chloride
ion produces parallel quenching and shortening of lifetime.
^7^
Serial No. NHLI-118
Any desired lifetime from 0.20 to 18.9 nsec. can be produced
with an estimated accuracy of 3%. Also, pyrenebutyric acid in
water was found to have a lifetime of 115 nsec. It can be
quenched by KI so that its lifetime can be made to vary from 18
to 115 nsec. Thus a whole series of solutions covering the
range from 0.20 to 115 nsec. has been developed and should be
of general usefulness in photochemistry.
2) We have made some preliminary measurements with the ORTEC
instrument with mixtures of substances both of which fluoresce
at about the same wavelength. To resolve the fluorescences,
we can make our measurements at different times after the
excitation if the lifetimes of the substances differ. With such
pulsed-source fluorescence measurements, we have essentially
the same advantage which phosphorimetry has long enjoyed; namely,
elimination of the scatter, and time resolved analysis.
3) Impurities in water often are the limiting factor in the
sensitivity of fluorometric procedures. We have long known
that various plastics release such impurities into water, and
have documented this in polyethylene squeeze bottles. We
measured the spectrum of the impurity emission as a function of
duration of contact with water. Deionized and charcoal-treated
water is free of such contamination. Tap distilled water is
also pure enough to use for fluorescence procedures.
Significance to Biomedical Research and the Program of the
Institute: Our continuing interest in fluorescence methodology
is consistent with the past work of this institute where the
spectrof luorometer was first developed almost 20 years ago.
Fluorescence spectroscopy has become a widely used tool in
biomedical research.
Proposed Course: We plan to do much work with the ORTEC instru-
ment. There are some problems where it would be advantageous
to interface the instrument with a computer; how this can be
done is being pursued with Dr. R. L. Berger. We also want to
obtain an X-Y plotter for the multi-channel analyzer component
of the ORTEC and use that in conjunction with our lifetime
standards method. The question of resolving multiple components
by time-resolved fluorometric analysis will receive attention.
Honors and Awards : None
Publications:
1. Chen, R. F.: A source of fluorescent impurities: Polyethylene
containers. Anal. Lett. 5: 664, 1972.
577
Serial No. NHLI- 111
Chen, R. F.: Fluorescence Lifetime Measurements in
Biological Fields, for translation and publication in
Z Tamura et. al., editors. Fluorescence Spectrophotometry
(Keiki-bunseki) , in Japanese, Kodansha, Ltd., publishers,
Tokyo .
Si78
Serial No. NHLI- 119
1 . Laboratory of Technical Development
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Applications of Fluorescence in Biochemistry
Previous Serial Number: NHLI-96
Principal Investigator: Raymond F. Chen
Other Investigators: Dr. H. Pollard, Dr. A. Light
Cooperating Units: Reproduction Research Branch, NICHD
Laboratory of Chemical Biology, NIAMD
Project Description:
Objectives: We wish to prove our newly developed methods and
techniques by applying them to specific problems of great
biochemical interest. By so doing, we try also to solve those
problems, which are of importance in themselves.
Methods Employed: We have used methods we have previously
reported on for measuring fluorescence and fluorescence
polarization. Measurements on proteins which cause much light
scatter were done with horizontally polarized light, a method
we innovated before. Stopped-flow fluorescence was performed
with a unit interfaced with the Aminco-Bowman fluorometer.
Major Findings: 1. Stopped-flow fluorescence has been used to
investigate the re-folding kinetics of bovine and other types
of serum albumin, which are unfolded at acid pH and refold upon
jumping the pH back to neutrality. We find distinct steps which
are inconsistent with the two-state (or all-or-nothing) theory
of native vs. denatured proteins. A manuscript is under prepara-
tion describing this work.
2 . Work has continued to extend our previous observations on
the quenching of protein fluorescence by Ag"*". Briefly, there
are two main mechanisms of this quenching. One depends on
collisional quenching of silver ion with tryptophanyl groups.
The second mechanisms appears to be eragy transfer, since the
combination of Ag"*" with -SH groups results in an intense ultra-
violet absorption band which can cause quenching. We have used
quenching by Ag+ to do polarization of fluorescence measurements
on a series of protein solutions, which are partly quenched.
3.79
Serial No. NHLI- 119
In this way the dependence of polarization on lifetime is
determined; and, in conjunction with lifetime measurements, can
permit the determination of rotational relaxation times. A
manuscript has been prepared.
3. The use of intrinsic fluorescence for depolarization studies
has been investigated. When G. Weber first applied the
depolarization method to proteins, he used the fluorescence of
dyes attached to the protein. The intrinsic fluorescence of
proteins had not yet been discovered. Later, it was thought that
the intrinsic fluorescence had too short a lifetime to be used
in this method. In fact, we find, with proteins of low MW such
as RNase, pepsin, ovalbumin, beta lactoglobulin, trypsin, and
chymotrypsin, that usable Perrin plots can be obtained by
following the polarization of intrinsic fluorescence as a
function of temperature or viscosity. Altering the viscosity
at a constant temperature was found to be the best method.
4. In collaboration with Dr. H. Pollard, NICHD we have looked
at the spectrum of fluorescence of human serum low density
lipoprotein particles and lipid-depleted derivatives. The
fluorescence changes markedly upon removal of cholesterol and
phospholipids.
5. In collaboration with Dr. A. Light, NIAMD, we studied the
rate of combination of fragments of staphylococcal nuclease.
Fragments of this protein can be produced by tryptic digestion
or by synthesis. These fragments have no structure, but when
combined, they bind by non-covalent forces and assume the con-
formation and activity of the native enzyme. One fragment has
the single tryptophan of the protein. Upon recombination of
fragments, the tryptophan fluorescence increases some three-
fold. We found that the fluorescence increase as a first-order
process, even though it was obviously a second-order reaction of
two fragments. The finding was consonant with the fact that
the tryptophan was in only one of the two fragments. A manuscript
has been prepared.
Significance to biomedical research and the Program of the
Institute: These problems are of biochemical interest, and the
demonstrated usefulness of our methods will be of importance to
other workers in these general areas.
Proposed course: We wish to apply the silver ion quenching
studies to tyrosyl fluorescence and proteins containing only
tyrosine. Preliminary studies show that silver quenches
tyrosyl as well as tryptophanyl fluorescence. We plan to collate
and report the data on polarization using intrinsic protein
fluoresence. More stopped-flow experiments are planned on the
SLSO
Serial No. NHLI- 119
folding and unfolding kinetics of various proteins. Additional
improvements in the system are contemplated for temperature
variation and computerizing the output. Much time is wasted in
data reduction in kinetic studies. Investigation of DNA
complexes of fluorescent dyes such as acridines and quinine have
been performed by others, but we wish to study such systems with
the ORTEC system, and possibly by flow-oriented polarization
methods.
Honors and Awards: Invited lecturer. Symposium on Relaxation
Methods in Molecular Biology, Copenhagen, August 15-17, 1972.
Publications:
1. H. B. Pollard and R. F. Chen: Fluorescence and Circular
Dichroism Studies on Human Serum Low Density Lipoprotein
Particles and Lipid-depleted Derivatives. J. Supramolecular
Struct., In Press.
S>^1
Serial No. NHLI- 120
1. Laboratory of Technical Development
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Countercurrent Chromatography: Liquid-Liquid
Partition Chromatography without Solid Support
(Part II)
Previous Serial Number: NHLI-101
Principal Investigator: Yoichiro Ito
Other Investigators: Robert L. Bowman
Cooperating Units: None
Project Description:
Objectives: Development of the countercurrent chromatographic
system using the flow- through coil planet centrifuge:
1. Analysis of the centrifugal force field.
2. Prediction of the retention volume of the solute peak
from the partition coefficient.
3. Separation of dipeptides by a simple elution.
4. Separation of dipeptides by a gradient elution.
5. New Helical column configuration for separation of
macromolecules .
Methods Employed and Major Findings:
1. The centrifugal acceleration field produced by the flow -
through coil planet centrifuge is analyzed mathematically. The
results clearly show that the system gives a homogeneous
acceleration field at all locations on the coil holder with the
direction parallel to the line drawn through the center of the
revolution and the center of the rotation. Since the centri-
fugal field experienced by all parts of the helical column as
it rotates and revolves is the same, the helical column need
not be located coaxially with the center of revolution.
2. One of the advantages of the present technique over the
conventional liquid chromatography is that one can predict
3g<?.
Serial No. NHLI-120
the location of the solute peak from the partition coefficient
which is determined with a separatory funnel. We have obtained
a formula
V = CK / (1+K)k
R'
where Vp' denote the apparent retention voliame; C, the column
capacity; K, volume ratio of the stationary to the mobile in
the equilibrated column; and k, the partition coefficient of
solute.
The applicability of the above formula is examined on the
elution of 14 peptide samples with a n-BuOH/l%CHCl2COOH (1:1)
by using a 0.55 mm bore column of 70 m length spun at 600 rpm
with the 30.7 cm revolutional radius and by pumping the aqueous
phase at a rate of 24 ml/hr . The results show the discrepancies
between the measured and the calculated figures being less than
10% and the method should be useful for practical purposes.
3. In order to demonstrate the capability of the method, seven
dipeptide mixtures are separated with n-BuOH/1% CHCI2COOH (1:1)
phase system. A column of 140 m long, 0.55 mm i.d. tubing is
used and a flow rate of 12 ml/hr is applied at 720 rpm with
20.2 cm revolutional radius. All components are well separated
and eluted within 20 hours at efficiencies ranging from 300 0
(first peak) to 1300 (final peak) theoretical plates.
4 . Gradient elution is applied to separate the above peptide
mixture. An exponential gradient between the upper phases of
n-BuOH/CHCl2COOH/ 0 . IM ammonium formate (1:0.01:1) and of
n-BuOH/O.lM ammonium formate (1:1) under a similar running
condition by using a 70 m column at a reduced flow of 6 ml/hr.
Despite the shorter column used, all components are well
separated and eluted out within 8 hours.
The gradient elution technique is also capable of concentrating
a minute amount of solute from a large quantity of a crude
mixture. When the sample solution is diluted with the mobile
phase, 10 ml sample size (originally 0.3 ml) still yields sharp
elution peaks for the later peaks having partition coefficients
less than 0.3, where the solute concentration at peak maximum
is estimated as high as 10 times that in the applied sample
solution. The solute concentrating power becomes less efficient,
if the sample is dissolved in the stationary phase. Similar
experiments done by diluting the sample with the stationary
phase show that all peaks are evenly affected losing the
resolution at the sample size of 4 ml.
5. It has been found that low interfacial tension phase systems
used for separation of macromolecules tend to produce emulsifi-
cation in the vertical helical column. We challenge this
2 ^W
Serial No. NHLI- 120
problem by modifying column configurations as follows:
a. Vertical helix column for control.
b. Coiled helix column: The tubing is wound onto a flexible
round core which in turn is coiled around the column holder at
various angles.
c. Flat coiled helix column: The tubing is wound onto a flat
flexible strip which is coiled onto the folder at various
angles.
Studies on phase distribution diagrams of sec. -BUOH/H2O and
n-BuOH/CH3COOH/H20 (4:1:5) obtained from these three types of
columns give clear-cut results that both coiled helix columns
mounted at 60° show an excellent flat equilibrium region with
over 4 0% retention of the stationary phase, while the conven-
tional vertical helix column show emulsif ication and loss of
the stationary phase. Careful studies to compare stability of
the retained stationary phase show that the flat coiled helix
column is superior to the coiled helix column. Since the
efficiencies yielded by three types of the column are very
similar, the flat coiled helix configuration is considered to
be most suitable for low interfacial tension phase systems.
In order to demonstrate the capability of the method, bovine
insulin is partitioned with sec.-BuOH/l% CHCI2COOH (1:1) on a
60° flat coiled helix column of 0.55 mm i.d. and 70 m length,
at 720 rpm with 30.7 cm revolutional radius, the sample is
eluted out within 4 hours at a flow rate of 6.0 ml/hr. The
result confirms the previous finding of countercurrent distri-
bution method by Craig, et. al. that the sample contains a
significant amount of deaminated form, which is not detected
by either ultracentifugation or electrophoresis.
Significance to Biomedical Research and the Program of the
Institute: The results of the experiments demonstrate that
the support-free liquid-liquid partition technique is applicable
not only to low molecular weight substances but also to
macromolecules of biomedical interest. The technique also
yields high efficiency in both simple and gradient elution while
the retention volume of the solute is easily predicted before-
hand by measuring the partition coefficient. The method has
a capability of concentrating a minute amount of principle
from the crude mixture to a degree as high as 10 times, which
will provide an ease in detection and further use of the
principle for various investigations.
Proposed Course: The future plan may be:
1. Further improvement of the apparatus.
3 ^^y
Serial No. NHLI- 120
2. Separation of cells and other biological particulates.
3. Other applications of biomedical interest.
Honors and Awards: None
Publications :
1. I to, I, and Bovmian, R. L. : Countercurrent Chromatography
with the Flow- through Coil Planet Centrifuge. J. Chroma-
tography Science, in press.
^gS"
Serial No. NHLI- 121
1. Laboratory of Technical Development
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Countercurrent Chromatography: Liquid-Liquid
Partition Chromatography without Solid Support
(Part I)
Previous Serial Number: NHLI-99
Principal Investigator: Yoichiro Ito
Other Investigators: Robert L. Bowman
Cooperating Units: None
Project Description:
Objectives: Application of polymer phase systems for separation
of macromolecules .
Method Employed: The elution centrifuge has been employed in
this study. Principle and design of the apparatus were
previously described.
In order to investigate the behavior of the polymer phase
systems three types of separation columns are made, each from a
5 m long, 0.55 mm I.D. Teflon tubing as follows:
1. Straight helical column: The tubing is wound onto rigid
rod of 2 mm diameter (4 units connected in series) and mounted
onto the holder in such a way that the column axis is nearly
parallel to the axis of the holder.
2. Twisted column: The tubing is folded in two and twisted
along its length to make a rope-like structure in which the
individual strands have the appearance of a stretched helix of
small diameter. The twisted tube is then wound around the
holder as tightly as possible.
3. Coiled helix column: The tubing is wound around a flexible
core (2 mm in diameter) which is in turn tightly coiled onto
the holder.
Phase distribution diagram, which indicates the percentage of
the retained stationary phase volume within the equilibrated
<5^
Serial No. NHLI- 121
column at a given flow rate, is drawn for each type of the
column. Partition efficiency of these columns is compared
under the optimum revolutional speed given from the phase
distribution diagrams.
Polymer phase systems used in this study are composed of 5%
(w/w) dextran 500 (Pharmacia) , 4% (w/w) polyethylene glycol
6000 (Union Carbide) , and lOmM sodium phosphate buffer at various
pH to adjust the partition coefficinets of samples such as
poly U, poly C, poly A, poly I, etc. The partition coefficients
of these samples on the above phase systems have been well
studied by Per Oke Albertsson to give convenient references to
the present study.
The following general procedures are applied for partition
studies. The column is filled with the lower phase (10 mM
NaH2P04 phase system) and a sample solution (0.1 g% x 0.15 ml
dissolved in the same phase solvent) is introduced at a rate of
2.7 ml/hr while the column is spun at 1500 rpm at 18 °C. Then
the column is eluted with the upper phase of the same solvent
system under the same condition for about one hour. After
impurities such as small molecular weight substances and
particulates are eluted out, stepwise or gradient elution is
applied with the upper phase of the desired phase composition.
The effluent is continuously monitored with an LKB Uvicord II
at 260 nm.
Major Findings:
1. The fluctuating centrifugal acceleration field in the elution
centrifuge is reanalyzed on a coordinate system fixed on the
column holder. The results show that the vector rotates in the
shape of figure eight with 6 greater than 0.25 (B=r2/ri, where
rx indicates the minimum distance between the arbitrary point
and revolutional axis, and r2 , the distance between the point
and the rotational axis) , whereas the vector rotates in one
direction twice in one revolution with 3 less than 0.25. The
results suggest that with small B values (less than 0.25) the
centifugal force might spiral down the particulates or droplets
along the coiled tube in the straight helical column, if both
handedness are matched together.
2. The above possibility is tested on the two phase polymer
phase system by using right and left handed straight coiled
columns. The results show clearly that retention of the
stationary phase, either upper or lower phase, is exactly the
same in both cases, indicating no significance of such rotating
field on retention of stationary phase under applied condition.
diST
Serial No. NHLI- 121
3. Studies on phase distribution diagrams gave the following
findings.
a. Retention volume of the stationary lower phase is always
greater than that of the stationary upper phase in all types
of the column. This may be due to the affinity of upper phase
to wall surface.
b. Straight coiled column shows the greatest retention volume
of 70% for stationary lower phase and 57% for stationary upper
phase at 1700 rpm. On the other hand, both twisted and coiled
helix columns show similar retention volume for either upper
or lower phase stationary, the figure being approximately 40%
for lower phase and 3 5% for upper phase.
c. All three types of columns show flat portion of the curve
in the wide range of rpm (1000 to 1700 rpm) for stationary
lower phase, indicating optimum condition for solute partition-
ing .
4. Partition efficiency of these columns are examined by
locally introducing a sample (poly U, 0.15 mg) . A flow rate
of 2.7 ml/hr is applied under 1500 rpm. When the upper phase
is mobile, the straight coiled column gives a broad peak which
is substantially improved in the twisted column, whereas the
coiled helix column yields the sharpest peak, indicating the
highest efficiency among all. When the lower phase is mobile,
the peak becomes similar in all three types of the column with
a moderate sharpness. Since the lower phase is very viscous
and difficult to handle, further experiments are carried out
with the coiled helix column by pumping the upper phase.
5. Partition of polynucleotides with simple elution: Using
the coiled helix column, each polynucleotide sample is parti-
tioned on phase systems of various sodium phosphate composition
( 10 mM) . Poly U is eluted with NaH2P04 : Na2HP04 = 1:1, poly C
and A with Na2P04 :Na3P04 = 7.5:2.5, and poly I with Na2HP04:
Na3P04 = 1:1 volume ratio, the results being consistent with
the previous partition data by Albertsson. The results also
show the evidence that the sample occasionally contains a
significant amount of impurities, some of which are undialysable.
6. Partition of polynucleotides with gradient elution
(exponential) . The gradient elution is carried out between the
starting medium (NaH2P04) and the ending medium (Na3P04) with
a mixing chamber of 2 ml capacity. The results show the expected
elution pattern of each sample with the peak location correspond-
ing to its partition coefficient. When a mixture of two samples
are partitioned, the resulted peak pattern clearly demonstrates
^SS
Serial No. NHLI- 121
the interaction of the polynucleotides to form double or triple
strands.
Significance to Biomedical Research and the Program of the
Institute: The results of the experiments indicate that the
polymer phase systems are also applicable to countercurrent
chromatography by the elution centrifuge. While the polymer
phase systems have a great potential to separate macromolecules
and particulates under the mildest condition, their high
viscosity and extremely low interfacial tension (aqueous/
aqueous) makes it difficult to adopt the conventional liquid
chromatography, hence, the partition so far entirely relies
upon the time-consuming, low efficiency extraction systems.
The present system yields the efficiency of less than 3 0 seconds
per transfer compared with several minutes per transfer in the
Albertsson's thin layer countercurrent distribution apparatus.
Furthermore, the present technique enables gradient elution
and continuous monitoring and fractionation with the conventional
elution systems used for liquid chromatography.
Proposed Course:
1. Refinement of the apparatus.
2. Separation of cells and other biological particulates on
the polymer phase systems.
3. Other interesting application with the elution centrifuge.
Honors and Awards: Invited to present a paper at the Ninth
International Congress of Biochemistry in Stockholm, Sweden,
July, 1973.
Publications: None
^ff
Serial No. NHLI- 122 (c)
PHS - NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title:
Development of the Spiral Coil Menbrane Blood
Oxygenator and Systems for Long-Term Temporary
Support of Pulmonary and Cardiovascular Systems
Previous Serial No.: NHLI-109(c)
Principal Investigators: Theodor Kolobow
Other Investigators:
Cooperating Units:
Edward Stool, Gerald Vurek, Joseph Pierce,
Robert L. Bovnnan.
Laboratory of Kidney and Electrolyte
Metabolism
Project Description:
Objectives:
1. To optimize the design of the membrane lung perfusion system for long-
term use in the laboratory and in man. The superiority of the membrane lung
system will be documented by long-term perfusions.
2. Pinhole formation in membranes has been the single most important cause
of membrane lung failure. No commercial manufacturer has been able to
guarantee absence of pinholes in membranes they supply. A program was
instituted to devise methods to fabricate "zero defect" silicone rubber
membranes, and to procure membranes of various thicknesses, and surface
characteristics.
3. Polymers of high gas permeabilities are commercially compounded using
various additives and fillers. Tissue and blood compatibility may be signi-
ficantly improved if these additives can be reduced or eliminated. Membranes
using only pure silicone gum at the blood membrane interface will be tested
in both an in vitro and in vivo system.
4. A laboratory study is under way to establish the relative importance of
temperature and perfusate for the successful long-term hypothermic preserva-
tion of the ex vivo sheep heart. This study arises from the observation that
there are definite limitations in the present day state of the art to
preserving organs beyond 24 hr without functional impairment on reimplantation.
P.^0
Serial No, NHLI- 122(c)
Methods Employed and Major Findings:
1. The membrane lung.
The spiral membrane lung as now used by various research groups has been
independently evaluated under contract at the Utah Biomedical Test facility
and at Brown University. The standard 2-1/2 m^ membrane lung has a rated
blood flow of about 3-1/2 1/min (O2 transfer 60 cc/m^/min, and a CO2 transfer
of 50 cc/mVmin). The performance of this design appears to be well above
the performance of other stationary membrane lung systems now available, or
in the process of being developed.
A. Clinical applications.
This laboratory has been consulted on occasions about patients in acute
respiratory failure both at the Clinical Center and within the local medical
community. Of particular interest is the case of an 11-year old boy with
ALL and acute respiratory failure (presumptively diagnosed as pneumocystic
carinii pneumonia) at the Clinical Center, whose condition, despite best
medical and surgical respiratory care, became hopeless. It was then decided
to connect him to an extracorporeal membrane lung perfusion system. For the
greater part of the bypass, the major portion of blood respiratory gas
exchange was performed by the artificial lung. His pulmonary function grad-
ually improved and it was possible to wean him off the artificial lung after
9-1/2 days of continuous perfusion. Pulmonary function studies and lung
fields after bypass showed continued improvement and he was discharged two
months after termination of bypass. He is now attending school full time.
This patient is the seventh survivor on record among more than 50
bypasses for pulmonary assist with membrane lung blood oxygenation. This
bypass is almost twice the longest perfusion, with survival, reported to
date and reflects the great future potential of this treatment modality.
B . Animal research .
(1) Silicone membranes from our membrane casting facility have been
incorporated into finished membrane lungs and tested in long-term perfusions
in the sheep animal model. These membranes appear to be superior to commer-
cially available membranes in terms of absence of pinholes, excellent gas
transfer, and little or no change in blood perfusion pressure.
(2) Further evidence has been accumulated to show that extreme
respiratory alkalosis (pH 7.9 or higher) is a major factor In lung tissue
injury (pulmonary infarction) under conditions where pulmonary blood flow
has been severely impaired (such as in pulmonary embolism, shock, etc.).
Such pulmonary lesions are avoided when COo enriched gases are inhaled.
2. Membrane production.
The laboratory facility to continuously cast thin- film membranes has been
completed and performs to our highest expectations.
dff/
Serial No. NHLI- 122(c)
Silicone membranes as thin as 10 microns have been successfully cast
without pinholes. It has also been possible to co-cast up to three separate
layers consecutively, and to reinforce the third layer with fabric. More
recently, we have shown that pure silicone gum without fillers can be co-cast
with a layer of reinforced gum. Thus, blood will contact pure gum, rather
than gum with filler (see below under hypothrombogenic membranes).
It was shown earlier that silicone polymer crosslinking can be effectively
performed with organic peroxides or with high-energy radiation, both under
nitrogen. Equally effective polymer crosslinking occurs also when air
cured with organic peroxides, provided it is followed by ultraviolet post-
irradiation under nitrogen (this latter treatment is effective for thin films
only, as U. V. radiation is effectively absorbed by thick films).
Electron spin resonance spectra were studied after membrane crosslinking by
irradiation in nitrogen and in air. It was shown that methyl and methylene
radicals reacted rapidly with oxygen to form peroxide radicals. Thus,
during silicone crosslinking in air, some radicals were consumed by the
reaction with oxygen and were not used in the crosslinking reaction.
3. Hypothrombogenic surfaces.
Investigations concerned the thrombogenicity of pure polydimethylsiloxane
without the additives found in medical grade silicone rubber. This polymer,
%rtien crossl inked by ionizing radiation in an inert atmosphere, shows a
Lee White whole blood clotting time of over 45 min and a clotting time of about
90 min when air-blood interfaces are avoided.
Polymer deposition from solution appears to be a feasible method for applying
this surface to assembled extracorporeal devices, such as oxygenators.
Animal testing using "minilung" membrane artificial lung indicates clear
superiority of this coated membrane over standard commercial membranes in
regard to thromboresistance.
4. "Ex vivo" organ preservation by hopothermic perfusion.
Isolated hypothermic organ perfusion with plasma or a synthetic medium
is associated with rise in perfusion pressure and development in interstitial
edema. This study was undertaken to determine whether the untoward changes
were due to hypothermia, or the result of improper perfusion media. Donor
hearts were perfused with fresh blood (5-10° C) continuously obtained from
a donor animal, and upon perfusion, reinfused into the donor animal. At
termination of preservation, hearts were rewarmed and cardiac function
determined in a conventional manner.
(1) Preservation at 5° C was uniformly unsuccessful in 6 consecutive hearts.
(2) Preservation at 10° C for 24 hr resulted in 4 successful preservations;
two experiments failed because of technical difficulties.
3 Si9A
Serial No. NHLI- 122(c)
(3) Preservation at 10° C for 48 hr resulted in two out of two successful
preservations o
(4) Two preservations at 10° C for 72 and 96 hr were associated with a
sudden injury of the preserved heart appearing at about the 70th hour of
preservation. This finding appears to be based on imnunological rejection.
In all perfusions at 10° C we have been impressed by a lack of rise in
vascular resistance during the preservation time (except when carried out
past 70 hr) , by the limited or absent interstitial edema, and by the
surprising finding of electrical and mechanical activity of the preserved
heart at 10° C.
Significance to Biomedical Research and the Program of the Institute:
We have established in a single clinical perfusion that acute terminal
pulmonary failure can be tided over with the use of extracorporeal membrane
lung blood gas exchange. The 9-1/2 days duration of bypass suggests that
the artificial lung (membrane) can be safely employed for long-term use for
respiratory assist.
By inference, the membrane lung should be considered safe and preferable
for short-term use as well, such as in open heart surgery. As a corollary,
it also follows that use of the membrane lungs for long-term cardiopulmonary
support is feasible and awaits application.
Studies on improved membranes and surfaces are designed to make long-term
use of these devices much safer, and more efficient.
Our work in organ preservation is designed to establish parameters for
long-term in vitro organ preservation. At the same time, results are
directly applicable to organ culture and growth, tumor culture and growth,
immunochemistry, effect of cytotoxic agents on organ growth, the artificial
placenta, and others.
Proposed Course:
1. Clinical studies in long-term respiratory assistance with an extra-
corporeal membrane lung will be continued. This method is also directly
applicable to cardiac assistance in patients in need of temporary circula-
tory support and these studies will also be pursued.
2. Various silicone and silicone gum surfaces will be Incorporated in
membrane lungs and evaluated in the laboratory, and clinically if warranted.
3. We will continue our extended term extracorporeal respiratory blood gas
exchange in animals. Our goal will be to safely, and routinely, bypass
animals while on the membrane lung for periods up to one month. It is
assumed that most clinical disease entities that are to be reversible will
recover substantially within this time period to allow safe discontinuance
of extracorporeal assist.
4 3ffZ
Serial No. NHLI- 122(c)
4. We will continue to study mechanism of polydimethylsiloxane cross-
linking by electron spin resonance spectral analyses, and apply this
knowledge to preparation of thin and strong membranes.
Publications:
1. Kolobow, T. , and Spragg, R. : Dispersion Casting of Zero
Defect Silicone Rubber Membrane for Use in the Membrane
Lung . J. Association for the Advancement of Medical
Instrumentation , 1973. In press.
2. Zapol, W. , and Kolobow, T. : Isolated Extracorporeal
Placentation of the Fetal Lamb: Techniques and Observations,
Mammalian Fetus In Vitro. In press.
9.94
Serial No. NHLI- 123
1. Laboratory of Technical Development
2. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Blood Flow Measurement Using Nuclear Magnetic
Resonance Techniques
Previous Serial Number: NHLI-108
Principal Investigators: Vsevold Kudravcev, Robert L. Bowman
Other Investigators: Anthony Sances, Jr.
Joseph H. Battocletti
Cooperating Units: Medical College of Wisconsin
Milwaukee, Wisconsin, on contract to LTD
Project Description:
Objectives: In addition to last year's objective of developing
a nuclear magnetic method of studying cerebral circulation
non-invasively , this year's project includes the consideration
of capillary and shunt flow measurement in the human finger for
diagnostic purposes (Reynaud's).
Progress: The work is divided between this laboratory and a
group, under contract to this laboratory, at the Medical College
of Wisconsin directed by A. Sances. This group conducts the
biological observations using our magnetic labelling techniques.
In this laboratory, the work consisted of the following:
(1) Instrumentation development, (2) low magnetic field source
development, and (3) development of special NMR sensor probes
designed to perform in the biological environment.
Because of very weak nuclear induction, it is necessary to have
very sensitive low noise electronic devices to obtain useful
NMR response from flowing blood through skin and tissues. In
some cases, precessing proton induction may have a value of only
.25 microvolts. To receive this weak signal, we find it
necessary to use new, improved signal exciters.
During the last year, we completed the experimental development
of the above mentioned devices, and are currently constructing
and evaluating their performance.
rfs'
Serial No. NHLI- 123
Our previous NMR instrumentation was designed to use magnetic
field sources over 300 gauss in strength. To minimize
interference from proton spins distributed in the skin and
tissues, and to obtain a clear response from flowing protons only,
it was determined that the NMR detector field should be within a
range of 20-80 gauss utilizing practical moderate polarizing field
values (100 -500 gauss) . This weak homogeneous detector field
of 20-80 gauss is obtained in the simplest way by using the
following electromagnetic devices: solenoids, helmholtz coils
and the square field coil. After constructing and testing each
of these field devices, we found the square coil to be most
suitable because it allowed us to obtain the greatest volume of
homogeneous field suitable for biological application.
Several new experimental sensor probes suitable for finger blood
flow measurement were constructed and tested using our old NMR
instrumentation .
Because of the limited densitivity, of this instrumentation and
use of a relatively strong detector field, very weak responses
of poor resolution and signal to noise ratio were obtained.
These probes will be evaluated at a later date after construction
of new low field instrumentation is completed.
Major Findings: A new type of sensor proble consisting of two
induction coils placed "in line" was developed especially for
use with the time sharing NMR detection method. One of the
coils in this probe excites NMR response in flowing liquid; the
second coil placed downstream detects NMR free precession.
Critical decoupling, present in conventional cross coil NMR
probes, does not exist in this "in line" arrangement. Probes
of this arrangement are easily placed for cerebral blood flow
measurement, where cross coil probe placement is difficult.
The cooperating laboratory in Wisconsin, supported by contract
by our Institute, constructed a pulse-superregenerative NMR
detector - exciter using the circuit developed in our laboratory.
This detector was applied for cerebral blood flow measurement
in the macaque monkey and other blood flow experiments. The
results were reported at the Cerebral Blood Flow Meeting held
on November 16, 1972 in Washington, D. C. The laboratory also
successfully repeated our experiment demonstrating the possi-
bility of NMR magnetic label detection after several time
constants.
Significance to Biomedical Research and the Program of the
Institute: As described in the previous report, these non-
invasive techniques will provide practical application in
vascular disease diagnosis and therapy evaluation.
Af^
Serial No. NHLI- 123
Proposed Course: Evaluation of new instriimentation performance;
cooperation with the Medical College of Wisconsin for biomedical
application.
Honors and Awards: None
Publications :
1. Kudravcev, V., and Bowman, R. L. : Practical Aspects of
Nuclear Magnetic Resonance Applied to Blood Flow Measure-
ment. 25th ACEMB, Bal Harbour, Florida, October, 1972.
2. Battocletti, J. H. , Linehan, J. H. , Hosek, R, S., Sances , A.,
Halbach, R. E., Larson, H. D. , Itskovitz, S. M. , Evans,
R. L. , Bowman, R. L. , and Kudravcev, V. : NMR Blood Flow
Measurement in an Isolated Perfused Kidney. Proc. 25th
ACEMB, October 1972, Bal Harbour, Florida.
3. Ackmann, J. J., Larson, S. J., Sances, A., Reigel, D. H. ,
Battocletti, J. H. , Dallmann, R. L. , Bowman, R. L. , and
Kudravcev, V.: Computerized Non-Invasive Monitoring of
the Trauma Patient. 25th ACEMB, Bal Harbour, Florida,
October 1972.
4. Battocletti, J. H., Linehan, J. H. , Larson, S., Sances, A,
Bowman, R. L. , Kudravcev, V., Genthe , W.K. , Halbach, R. E.,
and Evans, S. M. : Analysis of a NMR Blood Flowmeter for
Pulsatile Flow. IEEE Trans, on Biomed. Eng., BME-19 :
403-7, November 1972.
5. Battocletti, J. H. , Sances, A., Larson, S. J., Evans, S. M, ,
Bowman, R. L. , Kudravcev, V., and Halbach, R. E. : A Review
of NMR Techniques Applied to Biological Systems. Symposium
and Workshop on the Effects of Low- frequency Magnetic and
Electric Fields on Biological Communication Processes and
6th Annual Meeting of the Neuroelectric Society. In press.
6. Battocletti, J. H., Sances, A., Larson, S. J., Halbach, R.E.,
Bowman, R. L. , Kudravcev, V., and Evans, S. M. : NMR
Detection of Low Magnetization Levels in Flowing Fluids.
IEEE Trans-Proc Magnetics Society, In Press.
Si97
Serial No. NHLI-124
1. Laboratory of Technical Development
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Peltier-Seebeck Equilibrator
Previous Serial Number: NHLI-97
Principal Investigator: Frank W. Noble
Other Investigators: Dr. Robert L. Bowman, Dr. Robert L. Berger
Dr. Nadja Rehak
Cooperating Units: None
Project Description:
Objectives: The Peltier-Seebeck Equilibrator is a new thermal
element equivalent to an electrically adjustable thermal
resistor with indication of heat flow through the resistor.
Methods Employed: A switching system operated at 60 Ha samples
and stores the Seebeck voltage on the sensor during part of
the cycle, then feeds current proportional to the stored Seebeck
voltage back into the sensor during the remainder of the cycle,
causing the sensor to heat or cool the sample by means of the
Peltier effect so as to reduce the temperature difference
between the faces of the sensor. The Peltier current, propor-
tional to the heat flow rate through the sensor, is indicated
on a meter and can be recorded on a strip chart.
Major Findings: The PSE will make it possible to measure heat
rate evolved from a sample without allowing the temperature to
rise appreciably.
Significance to Biomedical Research and the Program of the
Institute: This property could be useful, for instance, in
studying activity of bacteria where the rates are thought to be
critically related to temperature.
A second and experimentally demonstrated property of the PSE is
in speeding the thermal response of calorimeters. If heat is
introduced by means of light absorption at the sensor surface,
it is possible to increase the sensor speed by factors exceeding
two hundred. The practical utilization of this property depends
upon the calorimeter design, since the sensor can pump only heat
it can sense.
Sl^S
Serial No. NHLI-124
Proposed Course: A theoretical analysis of the PSE has been
produced in draft form preliminary to publication.
Honors and Awards: None
Publications: None
S^??
Serial No. NHLI-125
1. Laboratory of Technical Development
3. Bethesda, Maryland 20013
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Pseudo Differentiator
Previous Serial Number: None
Principal Investigator: Frank W. Noble
Other Investigators: None
Cooperating Units: None
Project Description:
Objectives: Electronic differentiation with respect to time of
blood pressure and flow signals has been used to study the
performance of the heart. The usual differentiator tends to
increase the noise content of the input signal, especially
frequency components which are harmonics of the 60 Ha power line,
Methods Employed: The Pseudo Differentiator samples the input
signal at a 3 0 Ha rate, phase locked to the power line,
subtracting the previous sample from the present sample on a
continuing basis. Since the sample time separation is constant,
the sample difference is proportional to the average derivative
throughout the sample interval. Suitable filtering tends to
reduce the high frequency noise in general. In particular,
since the sampling is synchronous with the power line at half
its frequency, the system does not respond at all to 60 Ha
or any of its harmonics.
Major Findings: A rough model operating, for convenience, at
60 He has been built and tested for immunity to a hypothetical
120 He power line frequency. The nulls at harmonics of the
line frequency are complete only at certain phase angles,
indicating that the switching time for the mechanical choppers
used is excessive.
Proposed Course: The device will be rebuilt using fast solid-
state switching to eliminate the switching-time artifact
mentioned above.
Honors and Awards: None
Publications: None
3,oO
Serial No. NHLT- 126
1. Laboratory of Technical Development
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Measurement of Picomole Amounts of Carbon
Dioxide
Previous Serial Number: None
Principal Investigator: Gerald G. Vurek
Other Investigators: L. C. Stoner
Cooperating Units: Laboratory of Kidney and Electrolyte
Metabolism - NHLI
Objectives: This project is concerned with the development of
a technique suitable to measure the total acid-releasable
carbon dioxide in nanoliter amounts of kidney tubule fluid.
The technique should have a sensitivity of 4 picomoles or less
and a precision of 5% or better.
Major Findings: Other work has established that calorimetry of
the heat released when CO2 reacts with LiOH provides a sensitive
and quantitative measure of the amount of CO2 involved.
Approximately 0.1 y joule is released per picomole. After
reviewing the various alternatives , we decided that the best
way to make the measurement was to allow the reaction to occur
in a well insulated, low thermal-mass temperature measuring
system. Thermistor bridges can be used to measure temperature
changes on the order of 2x10"^ C; if the reaction takes place
over a ten second period, the thermal capacitance must be less
than 2xl0~2 joule/°C and the thermal resistance must be at least
3xl02''c/watt. If the thermal mass can be reduced, the system
time constant can be reduced, thus decreasing the analysis time.
The work to date has been concerned with making a low thermal
mass system.
We use thermistors in the form of flakes 50/ym thick by 1 mm
square. These have been supported by their 25 ym lead wires
and on various substrates including cork, glass, and 25 ym silver
foil. The first was the most sensitive but it was too fragile
and broke when a flake of LiOH was placed on it. The last
shows promise as it offers adequate stiffness and low thermal
mass. The thermal resistance of the system depends principally
tol
Serial No. NHLI- 126
on conductive paths. These can be minimized by careful design
and by using a low thermal conductivity carrier gas. Xenon
is the best for this purpose, as it has 1/6 the conductivity
of air, but Freom 12 (CCI2F2) is a reasonable alternative
having 1/3 the conductivity of air and 1/500 the cost of Xe .
We obtained a 300 uvolt signal using Freom-12 as a carrier to
introduce 7 nl/sec . of CO2 into the reaction chamber, and using
a PAR model 121 "lock-in" A.C. resistance bridge amplifier to
extract the unbalance signal from a 50 kilo-ohm thermistor
half-bridge. The short-term noise (0.2 Ha and above) was less
than 0.2 uVP-P and the drift was less than 0.01 yV/min.
We designed the CO2 releasing chamber to be as compact as
possible in order to keep the sample-to-carrier ratio as long
as possible. Carrier gas passes over a lyl container of
concentrated phosphoric acid, through a Mg(Cl04)2 desiccant, to
the reaction chamber. The sample is injected into the acid
with a pipet inserted through a mercury drop seal . The mercury
drop prevents gas from escaping from the chamber while allowing
easy access to the acid. The total volume of the gas compart-
ment is estimated to be less than 50 yl. We use a low-power
binocular microscope to perform the sample handling manipulations
Initial tests have indicated the ultimate sensitivity to be
about 4 picomoles; one test of four successive pipettings of
8 nl aliquots of 10 mM NaHCOs gave readings with a relative
error of 3 percent.
Significance to Biomedical Research and the Program of the
Institute: This instrument will allow renal physiologists to
obtain a direct measure of the amount of carbon dioxide
transported into the lumen of various parts of kidney tubules.
That measurement is important in the program of elucidating
the role of the kidney in acid-base control. Our particular
approach, calorimetry, offers sensitivity, specificity, and
economy .
Honors and Awards: None
Publications: None
30^
Serial No. •NHLI-127
1. Laboratory of Technical Development
2. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 197 2 through June 30, 1973
Project Title: Calorimetric Measurement of Carbon Dioxide
Previous Serial Number: NHLI-107
Principal Investigator: Gerald G. Vurek
Other Investigators: Theodor Kolobow, Edward Stool
Cooperating Units : None
Project Description:
Objectives: The objective of this project is to develop an
apparatus suitable for measuring the partial pressure of carbon
dioxide in blood. The use of membrane oxygenators for temporary
pulmonary support. The instrument is to provide continuous
indication of blood pC02 without withdrawal of samples and with
good baseline and sensitivity stability.
Major Findings: The advantages of calorimetry over earlier and
more commonly used approaches have been confirmed. Calorimetric
measurement of CO2 offers reasonable specificity, sensitivity
superior to infrared methods and comparable with mass spectro-
meters, and freedom from shifts in the electrochemical potential
drifts common to the Severinghaus electrode. We had previously
shown that the measurement of the heat released by the reaction
of CO2 with LiOH was a feasible approach to the problem. The
overall chemical reaction exploited is:
CO2 +LiOH -> Li2C03 + H2O + 8.9 x 10^ joules/mole. Our
efforts of the last year have been directed toward making a
calorimeter with appropriate sensitivity and stability and
toward establishing the condition of the LiOH reactant so that
it can be used.
Calorimeter design was chosen to be as simple and compact as
possible to be compatible with the stability specification. At
a nominal CO2 flux of 10 nl/sec, the power to be measured is
40 y watts; this suggested that the system should show a
stability of one y watt or better. A set of thee concentric
aluminum cylinders forms the basic thermostat configuration. At
the center of the innermost cylinder is an aluminum disc with a
pair of 16-junction bismuth-telluride thermopiles connected in
opposition. The LiOH is contained in a cup placed against the
50 3
NHLI-127
face of one thermopile. We wound a heater element and a
resistance thennometer element around the outermost cylinder to
provide a constant temperature for the calorimeter. The set of
cylinders is surrounded by polystyrene foam and a phenolic
plastic case. Both the thermopile preamplifier and the heater -
thermostat amplifier are mounted in the case to minimize error
signals from changes in ambient temperature. The overall
dimensions of the case are 28 cm long by 8.8 cm. dia. Less than
1/2 watt is required to hold the interior at 37° when the
ambient is 20°; we have encountered some trouble when the ambient
has risen to 29° because the thermostat amplifier and thermopile
amplifier contribute enough power to cause the former to be
unneeded at such high ambients. It requires about 15 minutes
for the calorimeter to recover from the thermal transient
induced by insertion of an absorber container. The calorimeter
performs satisfactorily for the purpose of CO2 measurement.
Lithium hydroxide was chosen because it is available in highly
porous form suitable for gas absorption. It exists in either
the hydrated or dehydrated form, depending on the temperature
and pH20. The literature indicates that the material which
reacts with CO2 is LiOH.H20; we have confirmed that if the
material is dried vigorously at 120°C over Mg (C104)2/ it does
not react appreciably with CO2 . On the other hand, if enough
water vapor is present to convert all of the LiOH to the
monohydrate, the extra water vapor released by the reaction with
CO2 causes the crystaline hydrate to go into solution. Since
the silicone membrane we plan to use to separate the blood phase
from the carrier gas is more permeable to water vapor than CO2 ,
some effort has been expended on establishing the proper
humidity of the gas before it encounters the absorber. With
improper humidif ication , the calorimeter displays "overshot".
Step changes in pC02 are indicated initially as a large power
change followed by a slow fall-off; the time constant is 20
minutes or so with the steady state value 10-20 percent less
than the initial peak. This "overshot "was attributed to the
shift in equilibrium in the rate of formation of the hydrate,
which is exothermic, or if there is net loss of water, endo-
thermic. The literature indicates that if the PH2O is below
the LiOH/LiOH.H20 equilibrium value, the material will all go
to the dehydrated non-reactive form. At 37° this is llT or
about 23% RH. If some CO2 is present, it will react with any
LiOH.H20 releasing water locally and using the effective
humidity. After considerable experimentation, the best solution
seemed to be to pass the gas over Mg(CL04)2; s'^^" though this
reduces the water vapor to a very low level, there is enough
hydrate present to initiate the reaction of CO2 and the release
water is then adequate to sustain it. Storage of the LiOH
container over silica gel is not sufficient to prevent the
reaction from proceeding. There remains problems of satisfac-
tory storage of the LiOH containers; as the LiOH comes from
its stock bottle, it is about 3% LiOH.H20. When this material
2 3o</'
NHLI-127
is placed in the calorimeter, it takes about a day to achieve
a stable hydrate fraction, which is reflected in a shifting
baseline and falling sensitivity over that equilibration
period. When the starting material has been stored over silica
gel for several days , it shows a much more rapid approach to
equilibrium. Not enough data have been collected to establish
that this is the best approach. On the basis of the work we
have done, LiOH seems appropriate for the calorimetric approach.
We have designed a flow-through probe to sample the CO2 in an
extracorporeal blood flow system. This probe consists of a
modified commercial tubing connector made of polycarbonate
plastic. A one cm length of 0.06 cm OD x 0.03 cm ID silicone
rubber tube projects into the blood flow path; CO2 from the
blood passes through the wall of the silicone rubber into a
stream of nitrogen which carries it to the calorimeter. The
carrier gas passes down a pair of 1 meter long stainless steel
tubes protected with a Teflon sheath. In the unlikely event
the silicone rubber should break, carrier gas could enter the
circulation; this potential hazard is small since the flow rate
is less than 10 yl/sec. and since the event would be indicated
by a loss of signal at the calorimeter. A valve and water trap
are included in the instrument to give a visual check of carrier
flow. By selecting the appropriate valve position, the carrier
can be diverted from the calorimeter to the water trap and the
appearance of bubbles indicates carrier flow. A more fail-safe
arrangement could be made using a vacuum system. The probe
has some velocity sensitivity but the sensitivity stays
constant for flows over 250 ml/min. through a 1 cm dia. tube.
It is possible to install a thermistor on the probe to measure
sample temperature and thus correct the indicated pC02 to the
patient's temperature.
Significance to Biomedical Research and the Program of the
Institute: Long-term on-line monitoring of blood PCO2 is
becoming more useful, particularly when membrane oxygenators
are being used for treatment of acute respiratory failure. The
calorimetric approach offers the needed sensitivity and
stability together with compactness and economy.
Honors and Awards: None
Publications: None
3br
ANNUAL REPORT OF THE
LABORATORY OF KIDNEY AND ELECTROLYTE METABOLISM
NATIONAL HEART AND LUNG INSTITUTE
July 1, 1972 through June 30, 1973
The Laboratory of Kidney and Electrolyte Metabolism has
made a number of significant advances in the elucidation of
the mechanism of electrolyte transport in kidney, toad bladder,
avian erythrocyte, and heart muscle. Only the first three will
be summarized in the annual report. That concerning excitation-
contraction coupling in heart muscle is discussed in detail
in an appended individual summary.
Isolated segments of the mammalian nephron;
Studies of isolated segments of the renal tubule by a micro-
perfusion technique developed in this laboratory have been ex-
tended in the past year. The method has been adapted to permit
analysis of the mechanism of water and electrolyte transport and
of the action of diuretic agents and hormones, not only in the
proximal tubule and the cortical collecting tubule of the rabbit
as previously reported, but also in the distal nephron of the
amphibian and the thick ascending limb of the rabbit.
The proximal tubule in the mammal reabsorbs 50-70% of the
glomerular ultrafiltrate isosmotically . The mechanism by which
this occurs has not been completely characterized. There is
considerable evidence from micropuncture and microperfusion
studies that has been interpreted as indicating that the driving
force that creates the osmotic gradient for water flow is active
sodium transport with coupled passive flow of chloride along
the resultant electrical gradient. Ouabain, an inhibitor of
active sodium transport, interferes with net absorption of
fluid and lowers the transtubular potential difference from
-4 mv, (lumen negative) to 0. Since the Cl~ concentration in
the tubule lumen increases above that of bathing plasma during
the reabsorptive process in vitro, it has been suggested that
diffusive flux of Cl~, rather than active Na+ transport, may
be the driving force for fluid absorption under these circum-
stances. We have examined this thesis by studying the effects
of changes in the composition of the perfusion solution on net
fluid absorption and the transtubular P.D. We have demonstrated
that removal of HCO3 from both lumen ultrafiltrate and bathing
serum is without effect on the reabsorptive rate and P.D.
In contrast, an increase in the Cl~ concentration in the lumen
above that of plasma, effected by removal of HCO3, amino acids
and glucose from the perfusion solution alone, results in a
reversal of the P.D. (+2 mV, lumen +) . Isosmotic reabsorption
persists, albeit at a reduced rate. Similar results by others
have been interpreted as supporting the view that fluid re-
absorption is now a consequence of diffusion of Cl~ along its
3o7
concentration gradient with associated passive flux of Na down
the newly created electrical gradient. Our results are not
consistent with this interpretation since reabsorption is
inhibited by ouabain despite persistence of a Cl~ diffusion
potential. Furthermore, elimination of the Cl~ concentration
gradient and P.D. by removal of HCO3 from the bathing serum
as well, does not reduce reabsorption or affect the ouabain
sensitivity of the process. Studies are now in progress to
examine the role of other solutes on the characteristics of
reabsorption and the relationship between Na"*" transport and the
transport of amino acids and glucose.
The major regulatory function of the kidney is the
maintenance of the volume and composition of the body fluids.
The thick ascending limb of the distal nephron serves an
important role in this regard. It creates the electrolyte
gradients within the kidney necessary for both the conservation
and elimination of water. It has been thought that dilution
in this segment, a consequence of the transfer of sodium
chloride without water from lumen into blood, was effected by
an active sodium transport pump analogous to that in other
portions of the kidney and in most living cells. Since most
diuretics were thought to act by inhibiting sodium transport
in this segment, thereby accounting for the increased urinary
elimination of salt, it was difficult to understand why these
drugs did not exert extrarenal toxic effects by inhibiting
sodium transport in other cells in the body. It is now apparent
that chloride, not sodium, is actively transported in the thick
ascending limb and that it is this chloride transport mechanism
that is specifically inhibited by a number of potent diuretics.
As pointed out in a previous report, sodium chloride
without water is absorbed in the thick ascending limb by a
process involving active chloride transport. This creates an
electrical gradient, positive within the lumen, of sufficient
magnitude to account for coupled passive flow of sodium.
Ouabain, and three clinically effective diuretics; mersalyl,
ethacrynic acid and furosemide inhibit Cl~ transport and reduce
the trans tubular P.D. in the thick ascending limb. All three
diuretics are effective only when applied to the lumen surface
of the nephron. Mersalyl, which binds to protein in plasma,
has little or no effect when placed in the serum bathing the
tubule but when added to the perfusion solution eliminates
sodium chloride reabsorption and reduces the potential differ-
ence. These effects are blocked by prior or simultaneous
administration of p-chloromercuribenzoate , a sulfhydryl
reactive agent. The effect of ethacrynic acid, a drug presumed
in the past to act by inhibiting sulfhydryl catalyzed reactions
in the tubule cell, is unaltered by p-chloromercuribenzoate.
On the other hand the cysteine complex of ethacrynic acid, a
3oS
major urinary excretory product, is a considerably more potent
inhibitor of chloride transport than is ethacrynic acid alone.
The specificity of action of furosemide on active Cl~ transport
was supported by the observation that the drug is ineffective
in the cortical collecting tubule, a segment in which Na"*" but not
Cl~ is actively transported. Amiloride, on the other hand, an
inhibitor of sodium transport in other epithelial cells,
produced a rapid decrease in the transport of sodixom and
potassium in the cortical collecting tubule with a concomitant
reversal of the polarity of the P.D. The drug was ineffective
in the thick ascending limb however, lending credence to the
view that the capacity to inhibit chloride transport is a
necessary property of a diuretic that acts in this segment of
the nephron .
Toad Bladder;
This section of the laboratory continues to employ the
urinary bladder of the toad Bufo Marinus, as a model for
examining the effects of hormones and other agents on the
transport of water and electrolytes across epithelial sheets.
The bladder has been shown to respond to a variety of agents
including vasopressin and aldosterone with alterations in
transport analogous to those induced by the same agents in
the mammalian kidney. The biochemical events that accompany
changes in transport have been the subject of considerable
study. As first demonstrated in this laboratory vasopressin
increases sodium transport and osmotic water flow in the
toad bladder via the intermediacy of cyclic AMP. Similar
changes in water permeability are also induced in the
cortical collecting tubule of the rabbit by vasopressin and
cyclic AMP. In the past year studies have been initiated
to investigate the relationship between the extent of the
accumulation of cyclic AMP in isolated epithelial cells of
the toad bladder and the alterations in the physiological
response to antidiuretic hormone induced by several naturally
occurring agents; namely, PGE^, norepinephrine, and calcium.
We have previously reported that prostaglandin in low con-
centrations interferes with the hydroosmotic effect of
vasopressin but not with that of cyclic AMP. Similar effects
were observed when norepinephrine was added to the bathing
medium and when the concentration of calcium in the medium
was elevated from 2.5 mM to 10. We had initially interpreted
these results as indicative of interference with the capacity
of the enzyme adenylate cyclase to generate cyclic AMP.
Further evidence was sought in support of this view in the
present studies. Portions of intact bladders were incubated
for various time periods with and without appropriate test
agents. At the end of this period the epithelial cells were
rapidly separated from the underlying stroma, frozen in liquid
nitrogen, and their cyclic content measured by a protein
3o1
binding assay developed in the Laboratory of Biochemical Genetics.
It was noted that vasopressin increased the cyclic content of
the cells in a linear fashion for 15 minutes after which the
content remained stable for approximately one hour. This is
of interest since the increase in sodium transport induced by
the hormone peaks at an earlier time and is not maintained at
a maximal rate for the full hour. The maximal hydroosmotic
response also occurs within less than 15 minutes but is main-
tained for a longer period than is sodium transport. The
addition of norepinephrine to the bathing medium markedly
decreased the vasopressin induced accumulation of cyclic AMP
as did low concentrations (lO'^M) of PGE^^, and 10 mM calcium.
This was to be expected were the associated inhibition of
physiological response to ADH a consequence of a decrease
in the generation and accumulation of cyclic AMP as originally
proposed. A higher concentration of PGE (10~^M) however
augmented the extent of accumulation of cyclic AMP of itself
and may reflect stimulation of another cyclic nucleotide
pool within the tissue. Two drugs of current interest were
also studied, chlorpropamide and amiloride. The first, a
sulfonyl urea derivative, effective in decreasing the polyuria
of pituitary diabetes insipidus; the second, a diuretic that
increases sodium excretion by blocking reabsorption of
sodium across the luminal barrier of the distal nephron rather
than by inhibiting active sodium transport across the basal
surface of the cell. Chlorpropamide had been shown in our
laboratory and elsewhere to potentiate the hydroosmotic effect
of vasopressin but not that of cyclic AMP. It was without
effect on the extent of accumulation of cyclic AMP in toad
bladder epithelial cells exposed to vasopressin and theophylline
(an inhibitor of the enzyme that degrades cyclic AMP to an
inactive form in vivo). Amiloride, on the other hand,
decreased the acciomulation of cyclic AMP induced by vasopressin
and theophylline.
Endogenous cyclic AMP is degraded within the cell by a
cyclic nucleotide phosphodiesterase to inactive 5~AMP. The
diesterase is of importance in regulating the extent of
accumulation of the nucleotide induced by stimulation of
adenylate cyclase, the enzyme that converts ATP to cyclic
AMP. We had previously shown that diesterase activity is
altered by aldosterone, and that this may account for the
permissive effect of the steroid on the physiologic response
of the toad bladder to vasopressin (see earlier report) . It
is also probable that cyclic AMP diffuses out of cells in
vitro and that this process may also serve to regulate the
action of cyclic AMP in vivo. As a preliminary approach to
an examination of this hypothesis, the extent of leakage of
endogenous cyclic AMP from intact toad bladders into the
surrounding bathing media was examined. It was demonstrated
that in the absence of vasopressin trace amounts of cyclic
3/0
AMP appear in the bathing medium within a 60 minute period.
The addition of vasopressin is associated with a significant
increase in the rate of leakage of cyclic AMP, although the
concentration in the medium never exceeds that within the cell,
evidence that the extrusion process is passive. By devising
a technique that permitted collection of samples of medium
from either surface of the toad bladder it was further es-
tablished that the nucleotide preferentially diffused out
across the blood surface of the tissue rather than across
the urinary border. It was also noted that the rate of
accumulation of cyclic AMP in the bathing mediiom from bladders
incubated with vasopressin alone was greater or the same as
that from bladders incubated with vasopressin plus theophylline,
despite a considerably higher intercellular content of
nucleotide in the latter. It appears likely that theophylline
potentiates the effect of vasopressin on both the permeability
responses and the accumulation of cyclic-AMP not only by
limiting the breakdown of cyclic AMP but also by decreasing
the permeability of the cell membrane to the derivative.
Avian Erythrocytes ;
A number of cells have the capacity to regulate their
volume in anisotonic media. Duck erythrocytes, as first
demonstrated in this laboratory, are capable of reverting
to their original isotonic volume in either isotonic or
anisotonic media following experimentally induced perturbations
in size. Regulation is achieved by appropriate changes in
cation content and associated isosmotic shifts in water. As
noted in an earlier report, the effector for this process,
that is "the volxome controlling mechanism" is resident in
the cell membrane and is responsible for specific alterations
in membrane permeability that lead to losses or gains of
monovalent cations. In a previous report epinephrine induced,
cyclic AMP dependent, restitution of volume from an initially
shrunken state in isotonic media was described. The present
studies were directed at examining the characteristics of
the mechanism of shrinkage of swollen cells back to their
original volume. Cells were prepared by a method involving
the use of a sulfhydryl reactive agent that so alters the
permeability of the membrane as to permit rigid control of
the internal cation composition of the erythrocyte. It was
observed that swollen cells prepared by this method return
to their original volume in isotonic media only if the
potassium content exceeds that of the surrounding bath.
This is accomplished by an accelerated diffusive loss of
potassium with isosmotic water shifts. The volume regulatory
mechanism (as reflected by the enhanced permeability to
potassium) is not inhibited by ouabain though the glycoside
blocks the putative active sodium extrusion pump. The
3//'
latter had heretofore been considered the primary determinant
of both the cation content and volume of erythrocytes. Swollen
cells prepared with a high internal sodium on the other hand,
shrink in isotonic media below their original volume, and do
so by a mechanism involving only the active sodium pump. This
is blocked either by ouabain or removal of potassium from the
bathing medium which also interrupts sodium extrusion. The
results indicate that the volume controlling mechanism and
the sodium pump are independent effectors and are spatially
separate. This conclusion was supported by the results of
studies of shrinkage of swollen cells containing an excess
of both potassium and sodium. In these, sodium loss via the
pump was uninfluenced by the presence or absence of net
potassium loss through the volume regulatory system and vice
versa.
3 /A
Serial No. NHLI-128
1. Kidney & Electrolyte Metabolism
2. Electrolyte Transport
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July I, 1972 through June 30, 1973
Project Title: The effect of chlorpropamide on the water permeability
response of the toad bladder to vasopressin.
Previous Serial Number: NHLI-115
Principal Investigators: Stefan Zgliczynski, M.D.
Joseph Handler, M.D.
Jack Orloff, M.D.
Other Investigators: None
Cooperating Units: None
Project Description:
Objectives: Chlorpropamide, a sulfonyl urea derivative used for the
management of diabetes mellitus, has been shown to be efficacious in the
management of pituitary diabetes insipidus. The drug reduces urine output
in approximately two-thirds of patients with this defect in vasopressin
secretion (S. Zgliczynski, Polish Arch. Med. Wewn. 46:549, 1971). On the
basis of earlier studies including those reported from this laboratory, it
is likely, but not established, that chlorpropamide acts by stimulating
adenylate cyclase activity, thus increasing the intracellular concentration
of cyclic AMP. A recent report states that although, as demonstrated in
this laboratory, chlorpropamide alone enhances the water permeability
response of the toad bladder to vasopressin, it potentiates the inhibition
of this response by prostaglandin E^ (PGEj^) . It is the purpose of this
study to examine the effect of chlorpropamide on the effectiveness of other
inhibitors of the response to vasopressin and to examine the effect of the
same combination of agents on the response to theophylline, xdiich mimics
vasopressin by inhibiting the destruction of cyclic AMP catalyzed by the
enzyme cyclic nucleotide phosphodiesterase. Finally, the effect of
chlorpropamide will be examined on the content of cyclic AMP in the
epithelial cells of the bladder and on adenylate cyclase and cyclic
nucleotide phosphodiesterase activity of broken cell preparations. It is
anticipated that the results of this study will yield further insight into
the mechanism of action of this drug in vasopressin responsive tissues.
Ifethods: The water permeability response to vasopressin, to cyclic
AMP, and to theophylline is measured in the presence or absence of chlor-
propamide and one of the inhibitors, prostaglandin E-, (PGE-|^) , epinephrine,
or cysteine, using the sac technique of Bentley. The cyclic AMP content
of tichloroacetic acid extracts of epithelial cells scraped from toad
1 3/3
Serial No. NHLI-128
bladders incubated with some of the foregoing combination of vasopressin,
theophylline, and chlorpropamide is measured using the protein kinase
binding assay of Oilman. Adenylate cyclase activity of the 700 Xg pellet
fraction of homogenates of the epithelial cells is assayed using
5* -adenylylimido-diphophate (AMP-PNP) as substrate and alumina to separate
the cyclic AMP formed in the assay from the AMP-PNP. Cyclic nucleotide
phosphodiesterase activity is assayed by incubating homogenates of
epithelial cells with various concentrations of 3H-cyclic AMP. The
5 '-AMP formed is converted to adenosine by the addition of 5 ' -nucleotidase .
The adenosine is then separated from cyclic AMP and 5 ' -AMP by column
chromatography using DEAE sephadex.
Major Findings: We have confirmed the earlier observation that
chlorpropamide increases the water permeability response of the bladder
to vasopressin and to theophylline, but not that to cyclic AMP. Unexpected
results were obtained when similar experiments were performed in the
presence of agents (PGEj^, epinephrine, cysteine) thought to inhibit
adenylate cyclase activity in situ . In the presence of 10"'M PGE] , or
10"'M epinephrine, chlorpropamide inhibited the water permeability response
to vasopressin. Chlorpropamide does stimulate the response to vasopressin
in the presence of cysteine and stimulates the response to theophylline
in the presence of each of the three inhibitors. These results are not
readily interpretable in terms of simple effects of each agent on one
site in the adenylate cyclase mediated response to vasopressin. Direct
examination of the effect of chlorpropamide on adenylate cyclase activity
revealed no effect on the activity of the enzyme in the absence or presence
of vasopressin regardless of whether chlorpropamide was added iri vitro
or the intact tissue had been incubated with the drug before cell dis-
ruption for the assay. 10"-% chlorpropamide added in vivo or vitro
had no significant effect on cyclic nucleotide phosphodiesterase activity
of epithelial cell homogenates. Finally, in preliminary experiments,
chlorpropamide had no significant effect on the cyclic AMP content of
epithelial cells of bladders incubated with or without vasopressin. Taken
at face value, the results indicate that the effect of chlorpropamide
cannot be explained simply on the basis of interaction with adenylate
cyclase. If further studies support the initial impression that the drug
does not alter cyclic AMP levels in the cells, an alternative hypothesis
involving an effect at another step in the response to vasopressin will
be required.
Proposed Course of Project: The effect of chlorpropamide in the
cyclic AMP content of bladder epithelial cells will be examined further.
Honors and Awards: None
Publications: None
3/V
Serial No. NHLI-129
1. Kidney & Electrolyte Metabolism
2. Electrolyte Transport
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: A study of the leakage of cyclic AMP from the toad
urinary bladder.
Previous Serial Number: None
Principal Investigators: Shigeharu Urakabe, M.D.
Joseph S. Handler, M.D.
Jack Orloff, M.D.
Other Investigators: None
Cooperating Units: None
Project Description:
Objectives: The permeability and transport response of the toad
urinary bladder is mediated by cyclic-AMP generated and accumulated within
the epithelial cells of the bladder. In this in vitro study, the loss of
cyclic AMP from bladder cells into the bathing medium is examined. The
information will be of value in understanding the metabolism of cyclic AMP
in this tissue and as a guide to certain in vivo situations where cyclic
AMP levels in plasma and urine are determined to evaluate normal and
abnormal endocrine function in mammals.
Methods: In most experiments intact bladders are incubated as sheets
of tissue in Ringer's solution in erlenmeyer flasks. In some experiments
each lobe of the bilobed bladder is incubated as a sac so that the solutions
bathing the mucosal and the serosal surfaces can be collected separately.
The cyclic AMP released into the medium is determined after proteins in the
medium are removed by precipitation with trichloroacetic acid and the cyclic
AMP separated by column chromotography . Tracer 3H-cyclic AMP is used to
estimate recovery of cyclic AMP after collection from the erlenmeyer flask.
In all the experiments, paired tissue from the same animal is used for
control and experimental preparations.
Major Findings: Under control conditions (normal Ringer solution,
no added agent), approximately 0.20 pmoles of cyclic AMP per mg dry wt of
bladder leaked out of intact bladders in a 60 min period. Five to ten
times as much cyclic AMP was found in the solution bathing bladders exposed
to 75 mU/ml of vasopressin, virtually all of the cyclic AMP appearing in
the solution bathing the serosal surface of the bladder. The concentration
of cyclic AMP in the medium was always at least one order lower than that
in the epithelial cells.
1 3/^"
Serial No. NHLI-129
The possibility that the cyclic AMP appearing in the serosal solution
originated in the smooth muscle of the bladder was evaluated in two sets
of experiments using the serosal supporting stroma after removal of
epithelial cells. In the first, the stroma was prepared by scraping the
epithelial cells off the stroma with a glass slide. In the second, the
stroma was prepared by incubating the intact bladder in a solution free of
calcium and magnesium and containing 2 mM EDTA and then gently massaging
the bladder to dislodge the epithelial cells. Both cells and stroma were
then incubated further in regular Ringer solution. Vasopressin had no
effect on the release of cyclic AMP from either preparation of stroma.
When cells prepared by the second method were incubated with vasopressin
their cyclic AMP content rose as did that of their bathing medium.
10 mM theophylline alone has little effect on the cell content of cyclic
AMP and did not alter the accumulation of cyclic AMP in the bathing
medium. Although the combination of 10 mM theophylline plus 75 mU/ml
of vasopressin results in cell cyclic AMP levels about three times greater
than 75 mU/ml of vasopressin, cyclic AMP levels in the medium bathing
cells exposed to the combination were the same or lower than in the medium
bathing cells exposed to vasopressin alone. In preliminary experiments
under these same conditions in \Aich tracer cyclic AMP was added to the
bathing medium from the beginning of the incubation, over 80% of the
tracer was recovered as cyclic AMP, indicating that the failure to
accumulate cyclic AMP in the medium proportional to the accumulation in
the cells exposed to vasopressin plus theophylline may be the result of
reduced leakage of cyclic AMP out of theophylline treated cells. If this
interpretation is correct, impairment of cell membrane permeability to
cyclic AMP would be a second mechanism by v^ich theophylline elevates
intracellular cyclic AMP levels, and indicates that theophylline or
its analogs may alter the relationship between the concentration of cyclic
AMP in cells and in urine and blood.
Proposed Course of Project: Project is completed.
Honors and Awards: None
Publications: None
31^
Serial No. NHLI-130
1. Kidney & Electrolyte Metabolism
2. Electrolyte Transport
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: A study of the concentration of cyclic AMP in the urinary
bladder of the toad.
Previous Serial Number: None
Principal Investigators: Rodney Omachi, M.D.
Dianne Robbie, Ph.D.
Joseph S. Handler, M.D.
Jack Orloff, M.D.
Other Investigators: None
Cooperating Units: None
Project Description:
Objectives: Previous studies in this laboratory have established the
thesis that cyclic AMP is the intracellular mediator of the action of
vasopressin on the toad urinary bladder and other tissues that respond to
the hormone with an increase in permeability and/or an increase in the rate
of active sodium transport. In this study, extremely sensitive and reliable
techniques for measuring small quantities of cyclic AMP will be utilized to
study further the changes in the concentration of cyclic AMP in toad blad-
ders stimulated by vasopressin in the presence of other agents known to
alter the permeability response to the hormone. It is anticipated that the
information will afford a more comprehensive picture of the control of the
permeability response to vasopressin.
Methods: Urinary bladders of the toad Bufo marinus are dissected out,
cut into pieces, incubated in amphibian Ringer's solution, and then in-
cubated for periods of 3 min to 60 min in Ringer's solution containing
added agents. Epithelial cells rapidly scraped from the mucosal surface
of bladders are frozen in liquid nitrogen, thawed in cold 8% trichloro-
acetic acid containing tracer 3H-cyclic AMP for recovery, and their cyclic
AMP extracted at U°C. The protein is separated by centrifugation and
measured by the method of Lowry, et al. After separation by column
chromatography on Dowex AG 50-X8, cyclic AMP is measured using the protein
binding assay of Oilman.
Major Findings: In response to 15 mU/ml of vasopressin, a concentration
that elicits a maximal sodium transport and water permeability response,
1 3/7
Serial No. NHLI-130
cyclic AMP levels in whole bladders and in epithelial cells rise pro-
gressively for 15 min to mean values approximately double those of control
tissue. 10 mM theophylline, an inhibitor of the enzyme that catalyzes the
destruction of cyclic AMP, causes a slight rise in cyclic AMP levels in
epithelial cells from control tissue. Theophylline augments greatly the
increase in cyclic AMP levels elicited by vasopressin and of more importance,
reduces the scatter in the results. Theophylline was therefore incorporated
into subsequent protocols. Cyclic AMP levels are 41, and 166 pmoles per
mg cell protein in bladders incubated with 7.5 and 75 mU/ml of vasopressin
and are 11 pmoles per mg protein in control tissue.
Three agents known to inhibit the water permeability response to
vasopressin were studied. Norepinephrine, irfiich inhibits the increase in
water permeability produced by vasopressin inhibits the cyclic AMP response
by 637o. Prostaglandin Ej^ also inhibits the water permeability response to
vasopressin in toad bladder, but by itself increases sodium transport, as
does vasopressin. At the low concentration of 2.5 x 10"°M, PGE-i inhibits
the cyclic AMP response to vasopressin by 75% without any effect by itself
on cyclic AMP levels. At a higher concentration of 2.5 x 10"^M, however,
PGEn causes a five- fold increase in cyclic AMP levels. This is as great an
increase as that produced by vasopressin. Vasopressin plus PGE-i at the
high dose produces the same increase in cyclic AMP content as vasopressin
or PGE]^ alone. An increase in the concentration of calcium in the Ringer's
solution from 2.7 mM to 10 mM, which inhibits the water permeability
response to vasopressin, inhibits the cyclic AMP response to vasopressin
by 507o. The foregoing data are consistent with the conclusion that
norepinephrine, PGE]^, and 10 mM calcium inhibit the physiologic effects of
vasopressin by inhibiting the accumulation of cyclic AMP, in agreement with
previous findings that these agents do not inhibit the water permeability
response to exogenous cyclic AMP.
The drugs chlorpropamide and amiloride were also investigated.
Chlorpropamide, 2 mM, which enhances the water permeability response to
vasopressin, does not increase the cyclic AMP response to 10 mU/ml of
vasopressin. Similarly, in the presence of 10 mM theophylline, 1 mM,
chlorpropamide does not enhance the cyclic AMP response to 2.5 mU/ml of
vasopressin. These results do not support the hypothesis that chlor-
propamide affects adenylate cyclase or phosphodiesterase, thereby
potentiating the effects of vasopressin. Amiloride, lO'^M, which inhibits
sodium transport in the toad bladder, probably by decreasing the entry
of sodium across the apical plasma membrane, inhibits the cyclic AMP re-
sponse to vasopressin plus theophylline by 15% and 20% at 15 minutes and
30 minutes respectively (p < 0.05 and < 0.01). The physiologic signifi-
cance of the effect of amiloride on cyclic AMP metabolism remains to be
determined.
3fg
Serial No. NHLI-130
Proposed Course of Project: The effect of other agents such as
lithium and indomethacin which are reputed to modify the response of
the toad bladder to vasopressin will be examined and their effects on
cyclic AMP levels elicited by vasopressin correlated with their effects
on the permeability response to vasopressin.
Honors and Awards: None
Publications: None
^tf
Serial No. NHLI-13i
1. Kidney & Electrolyte Metabolism
2. Renal Mechanisms
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Function of the early distal tubule of frog
Previous Serial Number: None
Principal Investigators: Larry C. Stoner, Ph.D.
Maurice B. Burg, M.D.
Jack Orloff, M.D.
Other Investigators: None
Cooperating Units: None
Project Description:
The amphibian distal nephron is known to dilute the urine
and is responsible for as much as 60% of the total amphibian
nephron NaCl reabsorption. Several investigators have reported
a lumen negative P.D. for the amphibian distal nephron. Pre-
liminary results of this study show that the early distal
tubule of frog, a segment not previously studied because of its
inaccessibility to puncture exhibits a lumen positive P.D.
Objectives: The objectives of this study are to isolate
and characterize both the early and late distal nephron segments
of frog. Further, we intend to explore the possible similar-
ities between the early distal segment and the mammalian thick
ascending limb, another nephron segment characterized by lumen
positive P.D.
Methods: Previously described for use in mammalian tubule
segments. For this project a method for dissecting segments of
early distal nephrons from frog was developed.
Major findings: 1. The most surprising observation is
that the observed transepithelial potential of this nephron
segment averages 11 mv, lumen positive, a finding not previously
reported for any nephron segment except the mammalian thick
ascending limb, where the positive P.D. is taken as evidence
for the active reabsorption of Cl~.
2. Other observations which suggest that this amphibian
early distal tubule may be homologous to the mammalian thick
IM)
Serial No. NHLI-131
ascending limb are :
a. P.D. reversibly inhibited by ouabain.
b. P.D. reversibly inhibited by furoseitiide (at low con-
centrations this drug appears to be very specific for the thick
ascending limb) .
c. Perfusion with solutions made hypotonic by the omission
of part of the NaCl normally present results in increased lumen
positive P.D., suggesting that like the thick ascending limb
this segment is more permeable to Na than CI.
d. Insensitive to low concentrations of amiloride a potent
inhibitor of active sodium transport in mammalian cortical
collecting tubule and other transporting epithelia.
Proposed course of project: 1) To further explore the
similarities of this early segment of frog distal nephron to
mammalian thick ascending limb. 2) To confirm results of other
inveistigators by isolating and characterizing the amphibian
late distal nephron.
Honors and Awards : None
Publications : None
3d-l
Serial No. NHLI-132
1. Kidney S. Electrolyte Metabolism
2. Renal Mechanisms
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Function of the thick ascending limb of Henle ' s
loop
Previous Serial Number: NHLI-112
Principal Investigators: Maurice B. Burg, M.D.
Nordica Green
Jack Orloff, M.D.
Other Investigators: None
Cooperating Units: None
Project Description:
Objectives: The thick ascending limb of Henle ' s loop
is a renal tubule segment of special importance since it is
1) one of the major sites of NaCl transport which is responsible
for urinary concentration and dilution, 2) it is thought to be
a major site of action of diuretic drugs. These conclusions
had been based on indirect evidence since the segment is buried
within the kidney, inaccessible to micropuncture , and had not
previously been studied directly. The purpose of the present
investigation was to study isolated portions of this segment
directly in vitro in order to confirm the above conclusions
and to elucidate the transport mechanisms involved.
Methods: Thick ascending limbs of Henle ' s loop were
dissected from rabbit kidneys and perfused in vitro. Electrolyte
transport and the associated electrical phenomena were measured
as previously described.
Major findings: We previously found that net NaCl reab-
sorption in this nephron segment is a consequence of active CI
transport which causes the electrical P.D. to be positive in
the lumen and that Na transport is largely or entirely passive.
The most effective known diuretic drugs are furosemide,
ethacrynic acid, and the organic mercurials (such as mersalyl) .
On the basis of previous clearance and micropuncture studies
they were believed to act in the ascending Henle ' s loop. In
the present studies furosemide (IO'^m) , mersalyl (3xlO"^M) and
ethacrynic acid (lO'^M) in the tubule lumen each caused a large
1 3ii
Serial No. NHLI-132
decrease (^50%) in the electrical P.D. and net Cl flux, indi-
cating inhibition of active Cl transport.
The fall in P.D. induced by furosemide in the lumen was
rapid (<5 seconds) and iiiunediately reversible, whereas furosemide
(10~^M) in the bath had essentially no effect, Furosemide (and
the other drugs also) caused a small decrease in Na and Cl
permeability and in the electrical conductance, but these effects
are considered to be secondary to its most important effect
which is inhibition of active Cl transport.
A longer time (1 to 10 minutes) was required for ethacrynic
acid and mersalyl to lower the P.D., and the reversal was also
slower. The effect of mersalyl, however, was immediately
reversed by p-chloromercuribenzoate in the lumen (lO'^M) just
as the diuresis caused by mersalyl is reversed by p-
chloromercuribenzoate in vivo. In other biological systems
p-chloromercuribenzoate and mersalyl generally have similar and
additive inhibitory actions. Uniquely, in this tubule segment
they are antagonistic. Therefore, the effect of mersalyl on
the isolated tubule in vitro can be identified with its
diuretic effect in vivo. Mersalyl in the bath did not affect
the P.D. except at a high concentration (10~'*M) and the effect
was not reversible. Similarly, HgCl2f and high concentrations
of p-chloromercuribenzoate in the bath, also caused irreversible
inhibition of the P.D. We conclude that the mercurials have two
distinct effects: 1) the diuretic effect which is caused by
specific organic mercurials (e.g. mersalyl) at the tubule lumen
surface, and 2) a non-specific toxic effect caused by any of the
mercurials when they gain access to the cell interior.
The concentration of ethacrynic acid required to inhibit
the P.D. was high (>10~ M) . Ethacrynic-cysteine complex (a
urinary excretion product) inhibited active chloride trans-
port at a lower concentration (3xlO~^M) in the lumen than did
ethacrynic acid alone, and probably is the major active form
of the drug. Ethacrynic cysteine in the bath (10~^M) had no
effect. High concentrations of ethacrynic acid (10~^M) in the
bath caused irreversible inhibition of the P.D., presumably a
toxic effect. Serum in the bath protected against this action
of ethacrynic acid. We conclude that 1) ethacrynic acid is
converted into the ethacrynic-cysteine complex in vivo. 2) The
complex, which is less toxic than ethacrynic acid and a more
potent diuretic is secreted into tubule fluid by proximal
txibule cells. (Furosemide and mersalyl probably are similarly
secreted by proximal tubules.) 3) The ethacrynic-cysteine
complex acts in the lumen of the thick ascending limb of Henle ' s
loop to inhibit active Cl transport and to thereby diminish
NaCl reabsorption. 4) The action of the ethacrynic-cysteine
3^?
Serial No. NHLI-132
complex on the thick ascending limb is probably different from
the previously described in vitro actions of ethacrynic acid in
that it does not involve inhibition of sulfhydryl catalyzed
reactions .
-3
High concentrations of hydrochlorothizide (10 M) also
caused a small ('\-20%) inhibition of active chloride transport
in the isolated ascending Henle ' s loop. The drug caused the
P.D. to decrease whether present in the lumen or bath, but
was more rapidly effective in the former.
-4 -3
Amiloride (10 M) and acetazolamide (10 M) in the lumen
and bath had no effect on the isolated ascending Henle 's loop,
although even lower concentrations inhibit in the other nephron
segments .
Proposed course of the project: Completed.
Honors and Awards : None
Publications: Burg, M. B. and Green, N. : Function of the thick
ascending limb of Henle ' s loop. Am. J. Physiol.
224: 659-668, 1973.
32*/
Serial No. NHLI-133
1. Kidney & Electrolyte Metabolism
2. Renal Mechanisms
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The study of ion transport in renal cortical
collecting tubules.
Previous Serial Number: NHLI-113
Principal Investigators: Larry C. Stoner, Ph.D.
Maurice B. Burg, M.D.
Jack Orloff, M.D.
Other Investigators: Clifford Patlak, M.D.
Cooperating Units: Dr. Patlak is a member of the National
Institute of Mental Health
Project Description:
Objectives: The cortical collecting tubule in addition
to regulating water reabsorption by increasing its water per-
meability in response to vasopressin also actively reabsorbs Na
from and secretes K into its lumen. The present studies are
intended to expand our knowledge of transport properties of
these and other ions in cortical collecting tubule.
Methods: are the same as those described previously
except that in this study cortical collecting tubules were
maintained at 37''C instead of 23''C used in earlier studies.
Major Findings: 1) Transport of Na, K , and Cl : The
calculation of the net fluxes of Na, K and Cl from unidirection-
al fluxes, measured radioisotopically, show that 8 pM cm~-^sec~^
of sodiiom is reabsorbed by cortical collecting tubules at
37°C, a value about 1/4 that of proximal tubules of rabbit.
Roughly three-quarters of the net Na reabsorption is
accompanied by a net reabsorption of Cl~, the remainder is
balanced by the secretion of K"*".
The conclusion of earlier studies that Na and K were
actively transported has been confirmed in this study by com-
parisons of the flux ratios and the observed transepithelial
P.D. The analysis also suggests that net Cl~ transport is
not passive but also involves exchange diffusion. The per-
meability coefficients and partial ionic conductances of Na,
Sets'
Serial No. NHLI-133
K and CI have been calculated from the unidirectional flux data.
The relative permeabilities in descending order are
^Cl^^K^^^Na* ^ comparison of partial ionic conductances with
the total tissue conductance (measured electrically) revealed
that the partial ionic conductance to CI was much larger than
the total electrical conductance. It was concluded that 80%
of the unidirectional backflux of CI is not electrically active
and is best explained by the presence of CI exchange diffusion.
2) Effects of diuretics:
a) Furosemide is a potent diuretic which inhibits the
reabsorption of Na and CI in thick ascending limb of Henle ' s
loop. Concentrations of the drug 10 times that which produced
a maximal response in thick ascending limb were placed both in
the bath and lumen of collecting tubules. No effect was
observed either on the P.D. or on the ability of tubules to
transport Na and K.
b) The potassium sparing diuretic amiloride caused a
rapid reversal of polarity of the lumen negative P.D. from
-35 mv to +10 mv. Concomitantly the active transport of both
Na and K (measured with radioisotopes) was inhibited. The drug
was found to be effective only from the lumen surface of col-
lecting tubules.
3) Urinary acidification by cortical collecting tubules:
The positive P.D. which existed after the inhibition of Na and
K transport by amiloride was of interest since it was possible
that it indicated the active transport of yet another ion.
Since reversal of the polarity of the P.D. was also observed
when the presence of ouabain or replacement of sodium by choline
was used to inhibit sodium transport, the positive P.D. was not
the result of amiloride itself. The possibility that the
positive P.D. was the result of Cl transport was eliminated by
demonstrating that amiloride produced a positive P.D. when all
of the Cl was replaced by sulfate. The elimination of the
positive P.D. by acetazolamide , a carbonic anhydrase inhibitor
and sensitivity of the P.D. to the CO2 tension of the bathing
medium suggest that the secretion of hydrogen ion is responsible
for the positive P.D.
Attempts to directly demonstrate the existence of and to
further elucidate the mechanisms of this urinary acidification
process are planned.
4) When the perfusion solution (pH=7.4) of a collecting
tubule is exchanged for an acidic solution (pH<6.5) an increase
in the lumen negative P.D. from -38 to -62 is observed. From
3ii
Serial No, NHLI-133
preliminary results of the measurements of net fluxes of Na and
K, it appears that the change in P.D. is associated with a
change in the rate of K secretion and that sodium reabsorption
remains constant. This is the only experimental condition
found to date which may effect K secretion without changing
Na reabsorption.
Proposed course of project: Further experimentation may
permit us to define more precisely the relationship between the
transepithelial P.D. and the active transport of Na"*", H"*", and K
in collecting tubules and the effects of other drugs and
hormones on these.
Honors and Awards : None
Publications : None
30.7
Serial No. NHLI-134
1. Kidney & Electrolyte Metabolism
2. Renal Mechanisms
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Mechanism of salt and water transport by
proximal renal tubules.
Previous Serial Number: NHLI-111
Principal Investigators: Maurice B. Burg, M.D.
Michael D. Lutz, M.D.
Jean Cardinal, M.D.
Jack Orloff, M.D.
Other Investigators : None
Cooperating Units: None
Project Description:
Objectives: The proximal nephron isosmotically reabsorbs
about 70% of the glomerular filtrate. The mechanisms by which
this occurs have been only partially characterized. There is
a considerable body of evidence from micropuncture and micro-
perfusion studies suggesting that Na"*" is actively reabsorbed
against an electrical gradient. The transtubular potential
difference (P.D.), and the associated fluid reabsorption is in-
hibited by ouabain suggesting an active transport process.
Recently, evidence has been presented indicating that the
electrical driving force and perhaps the mechanism of fluid
transport is dependent upon the composition of the luminal
fluid. The present investigations are intended to examine
the effect of luminal fluid composition on the transtubular
P.D. and rate of fluid reabsorption, Jv
Methods: previously described.
Major findings: 1) The role of bicarbonate in fluid trans-
port has been studied using HC03-free ultraf iltrate of serum in
the lumen and HC03-free serum in the bath. The P.D. and Jy,
under these conditions was compared to those during perfusion
with otherwise identical solutions containing 25 mM of bicar-
bonate (replacing CI) . The P.D. and J^ were identical in the
two conditions, indicating that HCO3 is not essential for fluid
absorption or generation of the electrical P.D.
32g
Serial No. NHLI-134
2) Since the composition of the luminal fluid is changed
considerably by the transport processes as it transits through
the proximal tubule, we studied the characteristics of fluid
transport during luminal perfusion with a solution similar to
the fluid found by micropuncture at the end of the proximal
tubule. The perfusate was an artificial solution containing
no glucose, amino acids, or bicarbonate (chloride concentration
was higher than the bath of rabbit serum) pH was 6.7. The
P.D. was +2 mv, lumen positive with the artificial solution in
the lumen compared to -4 mv, lumen negative, with ultrafiltrate
of serum in the lumen. J^ was 40% less with the artificial
solution. Ouabain, 10~^M, added to the bath during perfusion
with the artificial solution resulted in a marked fall in Jv
with little change in the positive intraluminal P.D. Other
tubules were perfused with a similar lumen solution but with
HC03-free serum in the bath in order to eliminate the chloride
concentration gradient across the tubule wall. The P.D. was
zero, but J^ was the same as with HCO3 in the bath. Thus the
positive intraluminal P.D. is due to the chloride concentration
difference, but evidently neither the P.D. nor CI gradient acts
as a significant driving force for net Na and fluid reabsorp-
tion.
During perfusion in the absence of a chloride concentration
gradient, there was fluid reabsorption without any P.D. Ouabain
inhibited J^ under these conditions suggesting that active
electrolyte transport is involved. The identity of the activity
transported solute or solutes under these conditions remains to
be elucidated.
Proposed course of study: The role of various other solutes
in the luminal fluid in determining the characteristics of fluid
transport will be further examined, specifically, the relation-
ship between Na transport and the transport of glucose and
amino acids .
Honors and Awards : None
Publications: Lutz, M.D., Cardinal, J., and Burg, M.B.:
Electrical resistance of the renal proximal
tubule perfused in vitro. Am. J. Physiol,
in press.
3JLf
Serial No. NHLI-135
1. Kidney & Electrolyte Metabolism
2. Electrolyte Metabolism
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Volume regulation in duck erythrocytes
Hormonal control of cation transport in duck
erythrocytes
Previous Serial Number: NHLI-110
Principal Investigator: Floyd M. Kregenow, M.D.
Other Investigators: None
Cooperating Units: None
Project Description:
Previous reports (1970-71) demonstrated that duck erythro-
cytes contain a "volume controlling mechanism" which regulates
cell size in either isotonic or anisotonic media. These cells
return to their original isotonic volume in either hyper- hypo-
or isotonic medium from a previously enlarged or shrunken con-
dition. The changes in cell size involve alterations in cation
content and isomotic shifts in cell water. Conceptually the
mechanism contains a receptor, transmitter, and effector. The
latter, located in the membrane is responsible for alterations
in membrane permeability which lead to the loss or gain of
monovalent cations. Previous evidence (1969-70) (1970-71)
(1971-72) indicated that there are two effectors. One operates
when cells need to lose cations (enlarged cells) ; while the
other functions when cells must gain cations (shrunken cells) .
Objectives: To further characterize the "volume control-
ling mechanism" by:
1) determining the specificity of the "volume controlling
mechanism" and associated effector for K when this mechanism
functions in enlarged cells.
2) characterizing the other effector system responsible
for the cation movement and accumulation seen in shrunken
cells. This system is hormonally controlled in isotonic media
(norepinephrine) by a process which is mediated by CAMP,
(1969-70) (1970-71) . Characterizing this system may therefore
aid in understanding other hormonally controlled transport
33o
Serial No. NHLI-135
systems which utilize CAMP as an intracellular mediator.
Major Findings: I. To study the specificity of the volume
controlling mechanism for K when it operates in enlarged cells,
we prepared enlarged cells with various Na and K contents. The
procedure used to obtain these cells has been described in the
previous annual report; it is a modification of a method first
described by Garahen and Rega and has been used previously in
non-nucleated erythrocytes.
Only enlarged cells containing a high K content return to
their original size in the standard isotonic medium. The process
of regulation resembles that described previously. For the re-
gulatory process is not affected by the cardiac glycoside,
ouabain. K is lost from the cell only if the electrochemical
gradient is favorable. And tracer measurements indicate that K
leaves the cell because of a transient increase in an apparent
diffusional pathway. Finally, both the increase in K efflux
and the net K loss cease once the cells reach their original
isotonic voliime, despite the continued presence of a steep
gradient for K loss across the membrane.
In contrast, cells containing a high Na content fail to re-
establish their original voliome, but shrink instead until an
apparent minimal volume is reached (4/5 of normal) . Not only
is the process of cell shrinkage different but so is the process
of cation removal. The cation pump, responding to an increase
in the internal Na concentration, removes the Na rather than the
volume controlling mechanism. For both cell shrinkage and Na
loss are blocked by the cardiac glycoside ouabain, or removing
K from the extracellular medium, procedures known to inhibit the
cation pump. And Na leaves the cells now against an electro-
chemical gradient rather than with the gradient.
The fact that cells reach a minimal volume and shrink no
further is in itself interesting. It indicates that yet another
undescribed mechanism is present in these cells which limits
the reduction in cell size. These cells have an active cation
pump and an increased electrical potential (as indicated by
the chloride ratio) . Most interesting though, is the presence
in these cells of an enomous rapid bidirectional increase
in Na and K movements which are not unlike those associated
with the other effector portion of the "volume controlling
mechanism" .
Enlarged cells containing both Na and K utilize both
mechanisms, (the cation piomp and volume controlling mechanism)
to reduce their cation content. Only if Na is prevented from
leaving and there is sufficient K present will these cells re-
33/
Serial No. Iv;piLI-135
establish their original size. Otherwise, intermediate cells
act as High Na cells, provided the [Na]^, is larger than normal,
shrinking below the original isotonic volume until they reach
the minimal volume.
Thus, these studies demonstrate that the cation pump and
one of the effector portions of the "volume controlling mechan-
ism" function independently and are spatially separate within the
membrane. It is still possible, however, for two different
mechanisms to use the same membrane pathway. Accordingly, ex-
periments were designed using enlarged cells with an inter-
mediate Na and K content to eliminate this possibility. In these
cells, the rate of loss of K through the volume controlling
mechanism was not influenced by the presence or absence of Na
loss through the cation pump pathway. And visa versa, Na loss
through the cation pump pathway was not influenced by the
presence or absence of net K loss through the volume controlling
mechanism. These results would not be expected if Na and K
were competing for the same membrane pathway and they indicate
that the pathways for the two mechanisms are separate.
Finally, experiments were performed using high Na cells,
^^Na and a low Na bathing medium which allows one to say that the
pathway used by the "volume controlling mechanism" can not use Na.
That is to say, there is a specific relationship between the K
ion and this pathway which does not exist for the Na ion.
Major Findings: II. It has been suggested previously
(Kregenow, J. Gen. Physiol., Vol. 58:396, 1971 and J. Gen.
Physiol. Vol. 61, April 1973) that the other effector asso-
ciated with the volume controlling mechanism represents a
Na & K facilitated diffusion system. An alternate proposal,
(Kregenow, J. Gen. Physiol. Vol. 61, April 1973) involves an
increase in the electrical potential secondary to an increase
in K permeability (P^) relative to Pel- The K which accumulates
through this effector would do so then in response to the en-
hanced electrical gradient.
Accordingly, experiments were designed to assess which of
these proposals were correct, if either. These experiments
utilized the fact that both the ouabain insensitive net K accumu-
lation and the associated rapid bidirectional Na and K tracer
movements^ produced by this effector, depend on the extracellular
[Na] and [K] (Riddick, Kregenow, and Orloff, J. Gen. Physiol.
57:752, 1971; J. Gen. Physiol. 58:396, 1971). We also used
various pharmacological agents, the hormone norepinephrine, and
cells in which the SO4 ion has completely replaced the CI ion.
Studies to date confirm that this effector is functionally
333-
Serial No. NHLI-135
different. They also indicate, tentatively, that the first
proposal is correct.
Proposed course of project: I. Manuscript submitted,
project completed. II. To continue to pursue stated objectives,
Honors and Awards : None
Publications: None
335
Serial No. NHLI-136
1. Kidney & Electrolyte Metabolism
2. Exp. Cardiovascular Diseases
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Effect of cardiac glycosides on the excitation-
contraction system in mammalian skeletal muscle
Previous Serial Number: NHLI-119
Principal Investigators: Stephen Hajdu, M.D.
Other Investigators: None
Cooperating Units: None
Project Description:
Objectives: In last year's report (NHLI-119) a hypothesis
was advanced for the explanation of the excitation contraction
coupling (E-C) , a process which transforms the action potential
into contraction in skeletal and heart muscle. According to
this theory the calcium, the actual propagator of this process,
originates from a rather thick sheath surrounding the muscle
fiber immediately outside the electric membrane. The existence
of this layer around the muscle fiber containing the cardio-
globulin system has been histologically demonstrated by immune
fluorescence. This theory has it that the cardioglobulins keep
up an environment around the individual muscle fibers which con-
tains calcium in considerably higher concentration than in the
space between adjacent fibers. The concentration of calcium in
this pool around the fibers is accurately set and is independent
from the plasma calcium. During depolarization of the electric
membrane, calcium from this pool freely enters the fiber and
triggers the actomyosin into contraction. Since the cardio-
globulin-containing membrane accumulates calcium around the
muscle fiber in concentrations higher than in the interf ibrillar
space, its function must be that of an active ion transport sys-
tem. Due to its exposed location as an extracellular structure,
and due to its properties of taking up calcium from the extra-
cellular space and releasing calcium into the pool surrounding
the fibers in two separate steps, this system is ideally suited
for the study of the basic properties of an ion transport sys-
tem.
To furnish evidence that the cardioglobulin membrane
functions as an active calcium transport system and to under-
3V
Serial No. NHLI-136
stand its function in the larger frame of E-C coupling, the
following aspects of the system were under study:
A. Three components of the transport system were isolated
from human blood plasma (cardioglobin-A, -B, and -C) . A fourth
component, which is the product of the muscle cell itself,
complements the other three into a functional unit. Extraction
and purification of this fourth component has been delayed by
lack of time and manpower.
B. The energy for the function of this transport system is
provided from the high-energy phosphate bond contained by cardio-
globulin A. In the absence of cardioglobulin A the system does
not function. No other high-energy phosphate bond was found
that would have replaced cardioglobulin A as a source of energy.
If cardioglobulin A loses its high-energy phosphate, it can be
recharged under controlled conditions by creatine phosphate.
C. The transport system specifically selects calcium among
the many ions of the plasma. The selectivity is based on the
nuclear charge of the ions (2+ only) , and on the size of the ions,
which should be very clos§ to a relative size corresponding to
an atomic radius of 0.99 A as measured in crystalline structures.
No other ion in the blood plasma except calcium fulfills this
requirement. Among ions not present in the plasma, cadmivim is
the only one that meets this requirement.
D. If the bond between calcium and the carrier protein
were governed by the law of mass action, as is the case between
calcium and seriim proteins, the transport system would not be
able to transport against a chemical gradient. It was found,
however, that the bond in the cardioglobulin C - calcium complex,
by adsorbing 8.2 Kcal, becomes independent of mass action, i.e.
the calcium is taken up from any concentration and released
against any gradient. Since on the one hand ions in hydrated
form are generally not proportional to the size measured in
dehydrated form, while on the other hand it was found that one
of the qualities of the ion by which it is selected by the bind-
ing site is a size proportional to the dehydrated size, at least
some of the water of hydration (probably the outercoordination
shell) should be split off before the ion can be bound. Split-
ting off the loosely bound water of calcium, therefore, is the
means by which the 8.2 Kcal can be put into the bond.
E. The final requirement for an active transport system is
that it should be inhibited by any of the biologically active
cardiac glycosides. This and the mode of action of cardiac
glycosides on this transport system are the subject of the
present report.
33r
Serial No. NHLI-136
Methods employed: Since the behavior of the cardioglobulin
membrane is only ideal in skeletal muscle (impermeable to
calcium by diffusion, but efficiently transferred across the
membrane unidirectionally by an active process) , the experiments
were made on excised guinea pig diaphragms. The handling of
isolated tissues and experiments with Ca were carried out
under the same conditions as those described in our earlier
report (NHLI-119) .
Major findings: Preliminary experiments have shown that the
cardiac glycosides inhibit the E-C coupling of skeletal muscle,
thus rendering the muscle inexcitable in a period of time which
depends on the dose of the glycoside and the concentration of
the calcium of the bathing medium. A 100-fold increase of gly-
coside (1 pg-lOO ug/ml strophantidine) decreases the time re-
quired for the development of a complete block from 115 to 15
min in a calcium-free medium. At 2.5 mMol calcium the same
decrease is 420 to 180 min. If the same glycoside concentration
is used (100 ug/ml) the time required for the development of the
block decreases exponentially as the calcium of the bathing
medium decreases (within the range of 0-5 mMol calcium) . These
findings are in good agreement with the effect of glycosides
found on other transport system (K"*") .
In order to get some insight into the effect of glycosides
on the calcium transport system on the molecular level, the
effect of the drug was studied on 3 different states of the
transport system.
45
A. Effect of the drug on the uptake of Ca
Guinea pig diaphragms deprived of calcium needed for E-C
coupling were exposed to a glycoside for 10 minutes just prior
to the uptake of '^^Ca. The drug significantly reduced the
capacity of the sites to bind ^^Ca (479 - 14 cpm/mg compared to
the control, which is 775 i 19).
45
B. Effect of cardiac glycosides on Ca held by the
E-C coupling system
Diaphragms in which the calcium used for E-C coupling was
tagged with ^^^^ i^ a preliminary step showed that at the time
of the addition of the drug there is a release of a small but
significant amount of calcium, with a corresponding increase of
contractile activity. This effect of the glycoside, however,
is temporary (5-15 min) and independent from the dose of the
glycoside, since the same amount of ^^Ca was released by 4 yg/ml
ouabain as by 1000 pg/ml.
45
C. The effect of cardiac glycosides on the release of Ca
3 35^
Serial No. NHLI-136
45,
Ca-tagged diaphragms were prepared in advance and the
release of calcium was achieved either by electric stimulation,
or by use of 5 mg/ml caffein. ^S^a release was compared in the
presence or absence of 4 yg/ml ouabain. Contractile activity
and ^^ca release were both greatly diminished (caffein) or
completely abolished (electric stimulation) .
The effect of glycosides on this transport system allows
some conclusions to be drawn as to their action on transport
systems in general. The antagonistic effect of the transported
ions (calcium in this case, potassivim in the case of potassium
transport) on glycoside action can be explained as follows : The
transport system exists in two different states, one in which
the transported ion is attached to the system, and the other in
which the ion is released and the system returns empty towards
the low-ionic side of the membrane. If a glycoside can attach
itself to the transport system only in the latter case, the
higher the concentration of the transported ion, the smaller the
chance for a glycoside to find an empty transport site . Thus
there is direct competition between a glycoside and the trans-
ported ion for the same transport unit.
The inhibiting effect of the cardiac glycosides on the
calcium transport system of skeletal muscle has thus been es-
tablished. We obtained the final requirement, namely that the
cardioglobulins embedded in a membrane around the muscle fiber
are parts of an active transport system. The general depressing
effect of the glycosides on every process studied on this system
(uptake, holding the release of calcium) , however, suggests
that while there is no doubt that the drug affects some vital
point of the transport mechanism, it is probably not a direct
effect on those functions which we were able to study.
Proposed course of project: Since the molecular site of
action of cardiac glycosides has not been identified in the
above studies , experiments using the individual components of
the cardioglobulins will be designed for possible elucidation
of this long-standing question. Experimentation is already in
progress to extend the effect of cardiac glycosides demonstrated
on skeletal muscle to similar functions of the mammalian heart.
The observation of the paradox that the most positive inotropic
drugs of the heart, the cardiac glycosides, elicit the most
negative inotropic effect in skeletal muscle, reveals basic
differences between the two kinds of muscle. If these differ-
ences can be elucidated on the basis of this new model of E-C
coupling, this will reinforce our hypothesis.
Honors and Awards: None
337
Serial No. NHLI-136
Publications: Leonard, E. J., Coe , J. E., and Hajdu, S.:
Product of anti-cardioglobulin-B antibody in
the bullfrog. Circ. Res, in press.
356"
Annual Report of the
Section on Pulmonary Biochemistry
Pulmonary Branch
National Heart and Lung Institute
July 1, 1972 through June 30, 1973
The connective tissue of the lung is fundamental for lung structure and
mechanical properties. In himian pulmonary disease there is a broad spectrum
of connective tissue involvement from an excess of collagen in the fibrotic
interstitial disorders to an apparent loss or destruction of connective
tissue in the emphysematous disorders. The overall objective of this work is
to understand the control of connective tissue synthesis in the lung so as to,
ultimately, learn to selectively inhibit connective tissue synthesis in the
fibrotic disorders and to promote lung growth in the emphysematous disorders.
The primary goals for the first year were to understand the composition of
collagen in animal and human lungs; to develop in vitro systems to study the
synthesis of collagen in the lung and to develop animal models of lung growth.
All three objectives have been achieved; a summary of the ongoing projects
begun in July 1972 are as follows :
I. Composition of Lung Collagen
Both rabbit and human fetal lung collagen are composed of two primary
structures, a-j^ and a.2 chains. These have been extensively characterized by
chromatographic and electrophoretic techniques and their amino acid composition
has been determined. The ratio of a,/a2 in the hximan fetus, however, is
usually (approximately 3.5) suggesting lung collagen is a mixture of types
of collagen or that there are (similar to the maturation of globins) primitive
and adult forms of lung collagen.
II. Synthesis of Lung Collagen In Vitro
Conditions have been developed to study the in vitro synthesis of
collagen in hviman and rabbit lung tissue slices. The collagen product has
been identified by carboxjnnethycellulose chromatography, SDS-acrylamide gels,
acid pH gels, hydroxyproline content and sensitivity to collagenase. This
system has been used to examine the types of collagens synthesized in small
quantities of human lung. In addition, a cell- free system has been developed
from rabbit lung. A portion of the sjmthesized product has been identified
as collagen.
III. Animal Models of Lung Growth
A. Protein Synthesis During Development. The in vitro tissue-slice
system has been used to examine collagen and non-collagen protein synthesis
during rabbit lung development. From fetal to adult life there is a 6-fold
increase in lung collagen content (yg/mg dry wt) , yet at least a 10-fold
decrease in the rate of collagen synthesis. Non-collagen protein synthesis
decreases only 31-fold, suggesting that a greater percentage of the protein
synthesizing activity of the lung is applied toward collagen synthesis early in
35f
life compared to later in development.
B. Lung Growth Following Pneumonectomy. The same system has been used
to examine protein synthesis in rabbit lung following pneumonectomy. At 2
months, paired litter mates undergo either pneumonectomy or sham thoracotomy.
Four weeks later, the remaining lung in the pneumonectomized group is two to
four times the control in terms of dry weight, DNA content and protein content.
Preliminary evidence suggests increases in rates of collagen synthesis as
well. This model will be utilized to study lung growth on several levels.
3v^
Serial No. NHLI-137(c)
1. Pulmonary Branch
2.
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Sites of Action and Physiologic Importance of Some Mediators
of the Type I Allergic Reaction in Man
Previous Serial Number: None
Principle Investigator: Harold H. Newball, M.D.
Other Investigators: Clifford A. Hall, M.D.
Harry R. Keiser, M.D.
Marion E. Webster, Ph.D.
John J. Pisano, Ph.D.
Claude J. Lenfant, M.D.
Cooperating Units: Experimental Therapeutics Branch, NHLI
Project Description:
Ob j ectives : The Type I allergic reaction in man as exemplified by the acute
asthmatic attack is thought to be mediated by several agents including:
histamine, bradykinin, and prostaglandin Fort' ^^ sites of action of these
agents and their relative physiologic importance will influence the type and
mode of therapy of the asthmatic patient. We are presently studying the
relative effects and sites of action of bradykinin and histamine on the
respiratory system of normal subjects and asthmatics. We are, in addition,
investigating the effects of these agents on the in vitro human bronchial
smooth muscle, and the antigen-antibody induced release of mediators from the
passively sensitized lung.
Methods : Normal and asthmatic subjects have been given intravenous injections
of histamine and bradykinin. The effects of these agents on the various sites
of the respiratory triee were evaluated with conventional respiratory physiologic
techniques. The antigen-antibody induced release of mediators from passively
sensitized lung has been described by Dr. Marion E. Webster (Principle Investi-
gator) NHLI-187(C).
Major Findings : Bradykinin in vivo (1 yg/Kg) and in vitro has no physiolog-
ically significant effect on normal human bronchi. The asthmatic bronchi
in vivo responds to intravenous bradykinin by dilation. This probably
represents secondary bronchodilation from bradykinin induced adrenaline release
by the adrenals. Both normal and asthmatic subjects show evidence of brady-
kinin induced alveolar duct constriction.
1 3V/
Serial No. NHLI-137(c)
Significance to Biomedical Research and Institute Program: Since bradykinin
appears to have important physiologic effects only on the alveolar ducts,
therapeutic modalities will be directed at this site of the respiratory tree.
If bradykinin releases adrenaline in asthmatics as suggested by our data, the
reported elevated blood kinin in asthmatics might cause a continuous release
of adrenaline which may contribute to the "epinephrine fast" condition seen
during the severe asthmatic attack. All of the commonly used bronchodilators
act through the 6-2 receptor, which is unresponsive in the "epinephrine fast"
condition. This suggests the need for developing bronchodilators that act by
mechanisms other than the stimulation of the 6-2 receptor.
Proposed Course to Project: Alveolar duct constriction was previously con-
sidered unimportant as a cause of the symptom complex observed during the
acute asthmatic attack. We will attempt to confirm the physiologic implication
of alveolar duct constriction by rapid freezing techniques. We will also
investigate the relative importance of the various mediators in the asthmatic
patient. The studies of antigen-antibody induced release of mediators from
passively sensitized human lung, will also be continued.
Honors and Awards : None
Publications: Newball, H. Arterial Blood Samples Should be Stored in Ice
for Gas Analysis. JAMA, Feb. 5, 1973, 223: 696.
3/3>
Serial No. NHLI-138
1. Pulmonary Branch
2. Section on Pulmonary Biochemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Models of Lung Growth
Previous Serial Nvunber: None
Principle Investigators: Ronald G. Crystal, M.D.
Kathryn H. Bradley, M.S.
Sally D. McConnell, M.S.
Morton Cowan, M.D.
Other Investigators: None
Cooperating Units : None
Project Description:
Objectives: In order to approach the problem of control of structural protein
synthesis in the lung it is necessary to have methods to "turn on or off" the
synthesis of these proteins. Two approaches are being used: (1) studying
normal development of structural protein synthesis in the developing lung and
(2) examination of "compensatory" lung growth following unilateral pneumonectomy.
In the first, the synthesis of collagen decreases as aging progresses, in the
second, the total synthetic activity of the lung is markedly stimulated.
Methods : (A) Protein synthesis during lung development. Conditions have been
developed so that in vitro collagen and non-collagen protein synthesis in
lung slices is linear for more than 5 hours. Rabbit lungs obtained from fetal
through adult ages are used under these conditions. Following incubation^DNA,
total protein, dry weight, l^C-hydroxyproline , ^^c-hydroxyproline, l^C-proline
non-collagen protein, soluble -'-^C-proline and soluble ^^c-proline are measured
in the tissue slice. Rates of synthesis of collagen and non-collagen proteins
are calculated and compared on the basis of DNA and specific activity of
isotope in the tissue.
(B) Lung growth following pneumonectomy. Two-month-old rabbits undergo
unilateral pneumonectomy while litter mate controls undergo sham thoracotomy.
Four weeks later, the animals are sacrificed and examined as explained above.
Major Findings (A)Protein synthesis during lung development. The amount of
collagen per unit weight increases 6-fold from fetal to adult life. Yet the
rate of collagen S3mthesis per cell markedly decreases (more than 10-fold)
while the rate of non-collagen protein synthesis decreased at a slower rate
1 3^2
Serial No. NHLI-138
(less than 3-fold). The relative proportion of protein synthesis directed
toward collagen synthesis in the newborn is 5 times that of the adult,
suggesting that during periods of rapid lung growth, an increased proportion
of the protein synthesizing machinery in the lung is directed toward collagen
synthesis.
(B) Lung growth following pneumonectomy. Four weeks following pneumon-
ectomy, the remaining lung increases 2-4 times in dry weight, protein, collagen &
total DNA. The rate of protein (collagen and non-collagen) synthesis is
variably increased, but when considered on the basis of the total lung, the
amount of protein being synthesized is 2 to 4 times the control.
Significance to Biomedical Research and Institute Program; These two models
provide a means to study the lung during periods of "turning off" or "turning
on" of protein synthesis. Through a comprehensive approach at several bio-
chemical levels it should be possible to develop an understanding of the
control of structural and nonstructural protein synthesis in the lung. This
has obvious applications to the control of fibrosis in the interstitial
disorders. An understanding of lung growth will hopefully provide mechanisms
to approach the regeneration of physiological functional alveolar units in the
emphysematous disorders.
Proposed Course to Project: (A) The study of protein synthesis during lung
development is almost complete. The next steps include:
(1) Isolation and separation of individual lung cells into homogenous
populations to facilitate identification of those cells responsible for
synthesis of specific proteins.
(2) Extension of these findings to the cell-free protein synthesizing
system to study the control of synthesis of specific proteins at the trans-
lational, and eventually the transcriptional level.
(B) The study of lung growth following pneumonectomy has just begun.
Studies are in progress to more closely define the pattern of lung growth
following pneumonectomy including structural and nonstructural protein
synthesis, DNA synthesis, tRNA, ribosomal RNA and specific mRNA synthesis.
It may be possible to identify "lung growth factors" in the serum of post-
pneumonectomy animals, analogous to similar findings following hepatectomy.
Honors and Awards: None
Publications: Crystal, R. G. , Bradley, K. H. , and McConnell, S. D. Changes
in Lung Collagen Synthesis with Age. Am. Rev. Resp. Disease.
(In Press) .
3<^</^
Serial No. NHLI-139
1. Pulmonary Branch
2. Section on Pulmonary Biochemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Mechanism of Collagen and Non-Collagen Protein Synthesis in
Rabbit Lung Cell-Free Systems
Previous Serial Number; None
Principle Investigators: Ronald G. Crystal, M.D.
James F. Collins, Ph.D.
Kathryn H. Bradley, M.S.
Other Investigators : None
Cooperating Units: None
Project Description:
Objectives: While tissue-slice systems are adequate for preliminary identi-
fication of protein synthesis products, it does not have the potential to
understand the molecular basis of biochemical events. As a primary goal of
this laboratory is to understand and control the mechanisms of lung growth, it
is necessary to develop systems in which specific identification of control
mechanisms can be identified. For this reason, a cell-free protein synthesizing
system is desirable and affords an opportunity to understand the synthesis of
the lung structural proteins at several levels.
Methods : Techniques have been developed to isolate the protein synthesizing
machinery from the cells of fetal, young and adult rabbits. Using conventional
techniques we have isolated poljrribosomes , elongation factors, synthetases and
post translational enzjmes (i.e., proline hydroxylase, necessary to hydroxy late
proline incorporated into collagen chains during synthesis) . These have been
used with initiation factors and tRNA isolated from rabbit reticulocytes to
complete the cell-free system. Identification of collagen is complex in a
cell-free system, but a rapid assay using purified bacterial collagenase has
been used with success .
Major Findings: The cell-free system derived from the lung is very active in
synthesizing protein. While only 1-2% of the entire protein synthesizing
machinery in the lung is directed toward collagen synthesis, this can be
enriched to 15-20% by isolating polyribosomes from the large pieces of endo-
plasmatic reticulum recovered following homogenization of the lung. The
"initiation" of new protein synthesis on these pol3rribosomes can be accomplished
with "initiation" factors from the rabbit reticulocyte, further suggesting
that the translational mechanisms of protein synthesis in varied mammalian
1 3^C
Serial No. NHLI-139
cells are probably universal. Preliminary studies have yielded intact
ribosomal RMA from the lung with early evidence as to the isolation of lung
collagen mRMA.
Significance to Biomedical Research and Institute Program: At each stage of
development of the other projects in this laboratory, the cell-free system can
be applied to understand the control of synthesis of structural and nonstructural
proteins in the lung. Once the normal mechanisms are identified, comparison
with human pathology will be made. For example, identification of a specific
collagen mRNA in a certain fibrotic disease of the lung may help identify the
primary pathology. In addition, the cell-free system is readily adaptable to
studying control mechanisms, such as the possible inhibition of collagen
synthesis in the fibrotic disorders while non-collagen synthesis is not
disturbed.
Proposed Course to Project: A homogenous cell-free system from the lung
synthesizing collagen will be established. As we develop techniques to identify
other proteins synthesizing in the lung, this will be applied to the cell-free
system. Techniques will be developed to establish cell-free system from lung
cells cultured from human biopsy material. Investigations will concentrate on
identifying the molecular mechanisms controlling lung growth in health and
disease.
Honors and Awards : None
Publications: None
3<^
Serial No. NHLI-140(c)
1. Pulmonary Branch
2. Section on Pulmonary Biochemistry
3. Bethesda, Maryland
PHS-NHLI
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: The Composition and Synthesis of Collagen in Human Lung
Previous Serial Nximber: None
Principle Investigators: Ronald G. Crystal, M.D.
Kathryn H. Bradley, M.S.
Other Investigators: None
Cooperating Units : None
Project Description:
Objectives: The connective tissue of the lung is fundamental for lung
structure and mechanical properties. In addition, the interstitial lung
diseases, many of which result in pulmonary fibrosis, represent approximately
20% of lung diseases. Almost nothing is known about the composition of lung
collagen nor its synthesis and regulation. With the ultimate aim being an
ability to control pulmonary fibrosis through molecular mechanisms, our
laboratory is developing a baseline of information regarding collagen compo-
sition and synthesis in the human lung.
Methods : Lung tissue is obtained from fetuses following therapeutic abortion
or from patients undergoing thoracotomy. Methods have been developed to
extract intact collagen chains from human lung sequentially with 1 M NaCl, 0.5
acetic acid or 5 M guanidine. Identification of collagen is done by carbo-
cjnnethylcellulose chromatography, SDS-acrylamide gels, acid pH gels, sensi-
tivity to collagenase and hydroxyproline content. The chromatography and gel
techniques allow determination of the relative amounts of ai and a2 chains.
Conditions have been developed to use small amounts (125 mg wet weight) of
human lung to synthesize collagen in vitro.
Maj or Findings : Both a^ and a2 chains have been isolated and purified from
human fetal lung; increasing amounts are extracted with the sequential
extraction. The two chains have certain differences in specific amino acids
yet are remarkably similar in amino acid to newborn human skin. Since all
mammalian tropocollagens (the basic unit of collagen macro-structure)
molecules known are always in the form ia.j)20i.2 or (aj)^, it is expected that
there will be either Zaj^ chains for every a2 chains or all a^ chains . The
extracted collagens have an (X-^/a.2 ratio of approximately 3.5. This suggests
a heterogenous mixture of (at least two) types of collagen of the forms
(a2^)20t2+ (012^)3. Human fetal lung slices synthesize a-, and a2 chains in vitro
in the same ratio. This suggests the collagen in human fetal lung represents
j/r
Serial No. NHLI-140(c)
a mixture of primitive fetal with adult collagen or a mixture of different
forms of collagen in different structures of the lung.
Significance to Biomedical Research and Institute Program: Pulmonary fibrosis
is a common and serious form of lung disease. Through the techniques developed
in the past year, we should be able to define the composition of collagen in
normal human lung during development. With this baseline, it should be
possible to categorize the fibrotic lung disorders on the basis of the kinds
of collagens that are synthesized, in the same manner that the hemoglobin,
immunoglobulin and lipoprotein disorders have been characterized.
Proposed Course of Project: The heterogenic ity of collagen in human lung will
be defined. Attempts will be made to identify the cell types synthesizing
different collagens. Once the normal patterns of collagen synthesis are
defined, the fibrotic disorders will be examined. We are establishing a
tissue culture laboratory so that human lung fibroblasts may be cultured from
biopsy material and studied in regards to collagens being synthesized.
Honors and Awards: None
Publications: Crystal, R. G. Collagen Synthesis in the Mammalian Lung.
J. Clin. Invest., June 1973.
Bradley, K. H. and Crystal, R. G. Collagen Composition and
Synthesis in the Human Fetal Lung. J. Clin. Invest. (Submitted)
5^^
ANNUAL REPORT OF THE
LABORATORY OF CHEMISTRY
NATIONAL HEART AND LUNG INSTITUTE
July 1, 1972 through June 30, 1973
During the past year the Laboratory has engaged in a wide variety of
analyses of structures of natural products brought to us by investigators
both within and outside of NIH. Mass spectrometry and nmr remain the most
heavily used techniques and in contrast to previous years, nmr is employed
at an earlier stage of isolation. In the past, mass spectrometry was our
work horse technique in such investigations because so little sample was
required; nmr was necessarily reserved for later stages when milligrams had
been acquired by tedious purification procedures. Unfortunately, by this time
the structure was often known from other data. Nmr spectrometry was therefore
mainly of use in elucidating the structures of synthetic products which were
obtainable in larger quantities.
Our Digilab Fourier transform apparatus has changed all this. Thus,
we have been able to obtain satisfactory spectra on quantities as little as
50 pg collected from gas chromatographs. Avoiding contamination of the sample
and solvents used in workup is the major problem remaining and further work
will be done to improve such procedures. Several examples are listed below,
wherein this approach has proved very successful (phosphonoglycons,
7-oxycholesterol derivatives in disease states, etc.).
A further advantage of the Fourier transform system is that we can
efficiently study the C nmr spectra of compounds at the 10 mg level.
Considerable effort has been spent this year on acquiring experience in this
area since it holds great promise for straightf«prward structure analyses as
well as disclosing subtle interactions between C-labelled substrates (e.g.
sphingomyelin) and proteins. Phosphorus nmr is another area of investigation
improved by Fourier methods, and we have been able to investigate phosphorus
compounds in straight serum as well as elucidating the structures of several
new phosphorus analogues of amino acids.
Our involvement with the Division for Computer Research continues to be
strong since it is our conviction that the complexity of most modern
instrumentation requires that the instrument be both run and analyzed largely
by computer methods; To this end, we have acquired a GT-40 interactive
ocilloscope system which will enable us to transfer data directly to and from
the PDP-10 from our remote location. Most of the members of the Laboratory
by now have become familiar with the PDP-10 time-share concept. In this
connection the NHLI-DCRT mass spectral search routine has been enthusiasti-
cally accepted by many biochemists at NIH, in the USA ('^20 users/day), and
soon it will be transferred to the International General Electric Timeshare
network under the auspices of the Aldermaston (England) Mass Spectrometry
Data Center.
3^f
We have further committed ourselves to the task of computer searching of
data files and literature in general through a computerized microfiche system.
At present this applies only to mass spectra and x-ray crystallography. In
the latter case, all of the existing X-ray literature can be rapidly
searched by input of structure to obtain references to that particular
compound. We hope to extend these methods first to enable search by
structure or activity of Chemical Abstracts for Compounds of Biological
Activity (CBAC File) and later to as much chemical information as possible.
Further activities in collaboration with DCRT include the use of mass
spectral and other types of data to classify drugs according to their
physiological activities by the use of artificial intelligence methods. In
this way we have found that sedatives and tranquilizers separate into two
domains using this approach, and it will shortly be applied to the extensive
list of compounds which require testing for anti -cancer activity.
In this connection, our Laboratory has spent a great deal of effort in
bringing the newly acquired Finnigan GC-MS into useful operation. Problems
remain, but the end is in sight.
Some time has been spent on becoming acquainted with electrochemical
analytical methods since they seem to have such promise in the biological
area and an apparatus is being built specifically for the analysis of
dopamine and norepineephrine in urine.
Our X-ray crystal lographic facility under Dr. J. V. Silverton is in
full operation at the present and a wide variety of problems have been solved,
ranging from theoretical studies on small strained ring compounds and the
conformation of the drug viminol to studies on the validity of bi layer models
of LDH obtained from small -angle scattering.
In the area of natural products, work continues on insect trail and
alarm substances because of their basic biochemical interest and unusual
structures. An attempt has been made to isolate an active plant principle
responsible for its bitter taste to PTC-negative individuals because of its
possible importance in genetic studies.
We have completed construction of the field desorption mass spectrometer
source mentioned last year, and it will be installed shortly. Hopefully this
will extend our ability to obtain mass spectra on nonvolatile substances of
biological origin.
In the past year, either alone or in collaboration with others, members
of our Laboratory have:
1. Applied chemical ionization mass spectrometry to a wide variety of
compounds in projects throughout NIH. For the most part, this has been done
as a service to the groups involved and proof that our efforts developing
chemical ionization mass spectrometry were not misspent is evidenced by the
NIAMDD's acquisition of their own chemical ionization mass spectrometer.
3SV
2. Made extensive use of, and modified, the DCRT-NHLI mass spectral
search routines now used by several hundred investigators throughout the
USA and even in foreign countries (S. Heller, DCRT).
3. Used the above mass spectral search routine to develop new correla-
tions in mass spectrometry of general application. Using the file in
combination with Euclidean distance artificial intelligence routines, we have
grouped the mass spectral file into certain classes of chemical structure.
4. Identified phenol and benzaldehyde as unusual components of the
defensive secretion of a millipede. Identified the mold metabolite mellein
and methyl anthranilate as caste-specific compounds in male carpenter ants
(M. Blum, Univ. of Georgia).
5. Helped to elucidate the structure of the reaction product of N-acetyl-
1-lysine with pyridoxal methyl chloride, a fluorescent probe used for locating
and characterizing functional groups in lysine containing proteins (E. Miles,
NIAMDD).
6. Proved, in contrast to an earlier report, that plasticizers do not
occur at appreciable levels in human blood under ordinary circumstances.
7. Identified skatole and tridecene as a defensive substance in a
chrysopid secretion (M. Blum).
8. Identified the hormone sensitive lipase inactivator as ascorbic
acid (S. Tsai, NHLI).
9. Elucidated the structures of 2-aminoethylphosphonic acid and 1-
hydroxy-2-aminoethylphonosphonic acid as the major polysaccharide constituents
of amoeba plasma membrane (E. Korn, NHLI).
10. Studied the methane chemical ionization mass spectrometry of a
series of flavanoids (D. Kingston, Univ. of Virginia).
11. Completed an nmr study of the reaction of formaldehyde with the
imidazole side chain of histidine as a model for the inactivation of imidazole
containing proteins by formaldehyde (M. A. Marino, Univ. of Chicago).
12. Designed and constructed, with the help of our shop a field
desorption source for the MS-9 mass spectrometer.
13. Elucidated the structure of a series of cholesterol stearate-oleates
oxygenated in the 7-position. These substances were unusual lipids forired
in a diseased state of several individuals (G. Assmann, NHLI).
14. Continued work on the elucidation of the cord factor (BCG) and a
related acyl sugar sulfate (M. Goren, National Jewish Hospital, Colo, and
E. Ribi, Rocky Mountain Laboratories, Colo.).
3^1
15. Initiated work on the so-called cross-legs factor, found to be a
glyceryl galactosyl phosphoryl ethanolemine responsible for the inhibition
of mating in drosophilla (E. Levenbrook, NIAMDD).
16. Elucidated the structure of a new amino acid (a-amino 6-hydroxy
hexanoic acid) found in Crotolaria spp (E. Pant, Univ. of Allahabad, India).
17. Worked on the structure of an unusual new alkaloid found in a
species of melochia tomentosa. The structure appears to be a highly
aromatic polynuclear ring compound (G. Kapadia, Howard Univ.).
18. Developed and utilized a program for searching a microfiche file
of mass spectra directly by computer (S. Heller, DCRT).
19. Elucidated the structures of several metabolites of the plasticizer
dioctyl phthalate, using GC-MS (E. Rubin, Johns Hopkins).
20. Prepared a tabulation of the Merck Index by molecular weight for
use in mass spectrometry.
21. Designed and constructed a device for preparing small quantities
of distilled diazamethane without resorting to distillation.
22. Conducted an X-ray crystal lographic analysis of the analgesic
viminol to determine the details of its steric configuration in connection
with theories on drug activity.
23. Using high pressure liquid chromatography, studied the constituents
of acacia villosa krameria ixina. These plants contain tannens which are
strongly carcinogenic (G. Kapadia).
24. Studied the constituents of the fruits of anti-desmabunius in an
attempt to isolate the substance responsible for the bitter taste in PTC-
negative individuals (R. Henkin, NHLI).
25. Studied the constituents of bencasia bisbida, isolating tetrahydro-
amentoflavone, a hitherto unknown biflavonyl.
13
26. Studied the C nmr of the 4 diastereoisomers of 3-methylcyclo-
hexanediols dnd-jtheir benzoates in an effort to correlate their stereo-
chemistry with •^C chemical shift (H. Ziffer, NIAMDD).
1 3
27. Studied the C spectra of steroids related to five vinyl norcho-
lestane-3-one to assess interactions in certain steroids (H. Ziffer, J. Seeman,
NIAMDD).
28. Studied aspects of the biosynthesis of phenalenone pigments of
licantus through proton decoupling and relaxation studies (U. Weiss, NIAMDD;
J. M. Edwards, Univ. of Conn.).
5<ri
29. Analyzed the complex proton spectrum of phenol and its anion in
order to study changes in chemical shifts of the aromatic protons on conversion
to the anionic form.
30. Determined that the triterpenoid products of callotropsis are a
and B amyrin and 3-epimoritinol (R. Pant, Univ. of Allahabad).
13
31. Studied C enriched peptides to determine changes in conformation
with pH. In certain cases separate resonances are observed for complexed and
uncomplexed species.
32. Studied the phosphorus nmr of samples of HDL and LDL and observed
quantitative differences in the types of phospholipid present in the two
materials(G. Assmann, NHLI).
33. Studied the phosphorus nmr of a patient with periodic high phosphorus
levels using phosphorus P nmr. Definite differences were observed in
comparison with normal individuals (W. Miller, NIAMDD).
34. Determined the structures of a series of phenol and p-anisyl
borneols using C nmr.
35. Determined by X-ray crystallography the structure of [1,4,2,2]
propel lane and [3,2,2]propellane, disproving Wybergs contention that the
bonds should be in one hemisphere. Further X-ray studies in this area are
being initiated.
36. Developed and utilized cathode-ray oscilloscope techniques to
display directly X-ray crystal lographic data on the adage computer.
37. p ysicig ^^ray crystallography, determined the structure of tetracyclo
[5.5.1 .O'.O'^'] tridecane-4,8,12-trione to a high degree of accuracy.
38. Worked on the structure of [3.3.3]propellane dione. This work
is still in progress; non-standard techniques must be used (U. Weiss, NIAMDD).
39. Developed methods to evaluate by Fourier transformation methods,
models of LDH bilayers proposed by Luzzote, et al . , from small angle
scattering methods.
3^3
Serial No. NHLI-141
1. Laboratory of Chemistry
2.
3. Bethesda, Maryland
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: A Study of Organic and Bio-organic Systems by
Magnetic Resonance.
Previous Serial Number: NHLI 30
Principal Investigator: E. A. Sokoloski, B. S.
Other Investigators: None
Cooperating Units: None
Project Description:
Nuclear Magnetic Resonance Spectroscopy offers a technique to study
biochemical and organic material in solution. The technique has been
applied during the past year to a wide variety of such materials.
BIOCHEMICAL APPLICATIONS
Carbon-13 spectroscopy allows observation of the backbone of biochemical
systems and is well suited to the study of protein complexes. The lack of
sensitivity can be overcome by enrichment of selective sites.
Dr. I. Chaiken (NIAMDD) , by solid phase synthesis techniques, has accomplished
enrichment in two biochemical systems. PH versus Carbon-13„shif t data has
been obtained on (His -C . .9) » syn(l-15)peptide, (His -C . _„.
SRNase-S' -complex. Material approximately .QOIM, 90% enriched were run in
2 ml HO — D„0+5% Pdioxane in an 8mm/12mm coaxial cell system, each titration
point needing approximately 16 hours scan time. The peptide gave a smooth
titration curve with PKA = 6.0. The complex showed considerable scattering
of data points and no conclusions are yet possible for the inconsistencies.
Further^ experiments are planned. The peptide-protein complex pair
(gly -C ^ ) syn(l-15) peptide and (gly -C „ ) SRNase-S' complex have
also been finder study. Although lack of material has limited the amount of
data obtained, separate resonances for complexed and uncomplexed species
have been observed at one of the enriched sites. The hint of considerable
information from this system is seen for the near future.
3Cs:
Serial No. NHLI-141
STRUCTURAL DETERMINATION BY CARBON-13 SPECTROSCOPY
In collaboration with Dr. G. Lyle of the University of New Hampshire
a C study at natural abundance of a series of phenyl and P-anisyl
borneols has been undertaken in order to determine the site of attachment
of the aromatic nuclei on the borneol backbone. Other data suggest either
carbon 4 or 6 as the most likely site.
The CMR spectra in each case showed 3 quaternary carbons dictating
substitution at the C-4 position. The previously unpublished chemical
shift data should add to the limited amount of such data currently
available.
Carbon-13 shift data was also obtained on a series of cyclohexanediols
synthesized by Dr. H. Ziffer. In collaboration with Dr. R. J. Highet of
this Laboratory, this work has provided reference data which should prove
interesting to the general scientific community.
BIOCHEMICAL AND CLINICAL APPLICATION OF PHOSPHORUS NUCLEAR MAGNETIC RESONANCE
Phosphorus-31 another nucleus directly observable by nmr, has been
studied extensively during the past year. Material obtained from amoeba
plasma membrane has been shown to contain 2 aminoethylphosphonic acid and
l-hydroxy-2-aminoethylphosphonic acid. The downfield position of the
phosphorus resonances relative to phosphate confirmed that they contained
carbon-phosphorous bonds. Mass spectral data obtained by Dr. H. M. Pales
and proton nmr together with chemical evidence enabled the final structural
determination to be made.
31
The binding of the dinucleotide UPCA to RNase was carried out using P
nmr. Titration of the dinucleotide with increasing amounts of RNase gave an
extrapolated shift of 2 ppm. for completely bound dinucleotide. PH
titration data on the dinucleotide alone and in the presence of protein
showed a pH dependent effect at PK = 5.1 demonstrating that hydrogen ion
migration from active histidine residues 12 and 119 (PKA's 6.5 and 6.0)
to the phosphonate of the dinucleotide is not observed.
3£Z,
Serial No. NHLI-141
Samples of HDL and LDL obtained by DR. G. Assmann are also the subject
of phosphorus nmr study. Spectra obtained showed quantitative differences
in the types of phospholipid present in the two materials. Recombination
experiments may shed further light on the lipid-protein interaction.
Samples obtained by Dr. W. Miller from a patient who periodically has
exhibited elevated phosphorus levels have been examined by P nmr.
Resonances were obtained for the PO, anion and phospholipids at physiological
concnetration by scanning for periods of 16 hours. The concentration
difference between normal and elevated was observable by this technique,
suggesting that P nmr may have application for selective clinical problems.
MICRO STRUCTURAL DETERMINATION
Micro Proton Magnetic Resonance was used to characterize material
isolated from the Carpenter Ant (Camponotus Herculeanus) by workers at
the University of Georgia. Two hundred microgram quantities were examined
in the Fourier transform mode giving spectra sufficiently resolved in less
than 8 hours of scan time. Mass spectral data in combination with pmr
enabled the structure to be determined as mellein.
Honors and Awards : None
Publications :
Korn, E. D., Dearborn, D. G., Fales, H. M. , and Sokoloski, E. A.
Phosphoglycan — A Major Polysaccharide constituent of the Amoeba
Plasma Membrane. J. Biol. Chem. 248, 2257, 1973.
srr
Serial No. NHLI-142
1. Laboratory of Chemistry
2.
3. Bethesda, Maryland 20014
PHS-NIH
Individual Project Report
July 1, 1972 through June 30, 1973
Project Title: Mass Spectrometry and Structure of Natural
Products
Previous Serial Number: NHLI-35
Principal Investigator: H. M. Fales, Ph.D.
Other Investigators: G. W. A. Milne, Ph.D.
Cooperating Units: None
Project Description:
The compound isolated from urine of cancer victims (C. Levy, NCI,
Baltimore) was found to consist mostly of citric acid. The important amine
component is present in much smaller amounts than previously thought.
The chemical ionization quadrupole system has presented certain
difficulties because of source design and it cannot be yet considered fully
operational. Modifications in the computer software recently received should
help in achieving routine operation.
The commercial pyrolysis system for use with the LKB GC-MS has not
yet proved successful and is still being modified to achieve the necessary
degree of temperature control.
Interactions with the Suburban Hospital emergency toxicology center,
set up by NIMH after an earlier study in NHLI , continue, and the feasability
of operation with a quadrupole using hydrog 
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