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July 1960 Ps aN il 





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ae 


CORLL 
Proceedings 


DUKE UNIVERSITY LIBRARY 


SEP 6 1960 


DURHAM, N. C. 


SYMPOSIA & SPECIAL REPORTS 


Published quarterly by the 
FEDERATION OF AMERICAN SOCIETIES 
for EXPERIMENTAL BIOLOGY 








AN IMPORTANT STATEMENT ON 
BACTERIAL SENSITIVITY TESTING 





WITH THE NITROFURANS 


The individual 
FuROXONE, FuRACIN—are not interchangeable either in 
clinical application or in susceptibility testing. Although 
chemically related, these compounds differ to a highly 
significant degree in their range of antibacterial activity 
as well as in solubility, diffusion rate, and other physical 
characteristics, For this reason, SENsI-Discs* containing 
each of these nitrofurans are provided for appropriate 
disc plate testing. Results are valid only for the compound 


tested. Cross-interpretation will lead to erroneous con- 


clusions. 


nitrofurans — ALTAFUR, 


FURADANTIN, 











Nitrofuran 


Antibacterial Spectrum 


Clinical Application 


For Disc Plate 
Test Use 





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FuRADANTIN® 


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Furoxone® 


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Furacin®, 


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Wide. Particularly 
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Wide. Highly active 
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Wide. Especially 
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Wide. Encompasses 
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Systemic infections, 
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respiratory tract and 
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Urinary tract infections. 
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Enteric infections. 
(Minimal systemic 


absorption.) 


Used topically only. 


ALTAFUR 
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FURADANTIN 
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FUROXONE 
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FURACIN 
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*Available from the Baltimore Biological Laboratory (Division of Becton, Dickinson & Co.), Baltimore 18, Md. 


NITROFURANS—a unique class of antimicrobials 





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July 1960 





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FEDERATION PROCEEDINGS 





Federation 
TOCedines 


Published by 


FEDERATION of AMERICAN SOCIETIES 
for EXPERIMENTAL BIOLOGY 


American Physiological Society 

American Society of Biological Chemists 

American Society for Pharmacology and Experimental ‘Therapeutics 
American Society for Experimental Pathology 

American Institute of Nutrition 


American Association of Immunologists 


EDITORIAL BOARD 


Karu H Beyer, JR. 
K. M. BrInKHOUS 


a ane FEDERATION PROCEEDINGS is published quarterly. The 


CALDERON Howe March issue consists of the Abstracts (Part I) of the papers 


FRANK W. PuTNAM presented at the scientific sessions, and the Program (Part II) 


ARNOLD E. SCHAEFER of the Annual Meeting of the Federation. The July and 


Mitton O. LEE 
Managing Editor 


December issues contain symposia and other special papers pre- 
sented at the Federation meeting. The September issue contains 
Sara F. LESLIE the membership list and other matters pertinent to the Con- 
Executive Editor stituent Societies of the Federation. 


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eign) payable in advance. Single ‘ 

Issues: March, Part I (Abstracts) 
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July, $2.00; September, $5.00; De- 
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orders should be sent to: Federation 
Proceedings, 9650 Wisconsin Ave- 
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American Societies for Experimental Biology. Printed at 





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; July 1 
FEDERATION PROCEEDINGS Volume 19 ‘ 


viii 











‘olume 19 


July 1960 





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Virus Diseases: Some Aspects of Host and Tissue Specificity... .. .F. B. Bang editors: 


Infectious Diseases: Bacterial. 


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Gastrointestinal Diseases: Portal Hype rtension.............. _.C.G. Child, IT D. A. Rytand 
Gastrointestinal Diseases: Hepatic Coma... . . ET 2 ee na .. JS. Sherlock W. P. Creger 
Cardiovascular Disease: Arterial Hypertension. . _M. Sokolow and P. J. Sanazaro 

Cardiovascular Disease: Pulmonary Hypertension . Se eer A. A. Liebow editorial committee: 
Cardiovascular Disease: Perinatal Cireulation......... OE TA Nero Uh ..d. Lind i 
Diseases of the Kidney tenal Failure................ ....0. P. Merrill R. oui coke 
Surgical Diseases of the ~ sei a ae _.W.L. Valk and W. S. Witus G. J. Dammin 
Hem: tology: The Megaloblastic Anemias. . . RERUN er ates ie we L. Mollin ~ - payee 
Hematology: Control of Red Cell Production RRR eee _ J. Erslev ©. 3. ian 
Nutrition and Nutritional Diseases................. N. Jolliffe and R. a Goodhart “ae here 
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Allergy and Immunology: Delayed Hypersensitivity and Homografting 453 pages 


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Neoplastic Diseases: Hormone-producing or Hormone-dependent Tumors 


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Psychiatry: Behavioral Problems in the Adolescent : 
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Special Therapeutics: Physiology of Diureties................... .K. H. Beyer May, 1960 
Radiology: Reaction to Short-term Radiation in Man............. H. B. Gerstner 
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Pediatrics: Congenital Enzyme Defects........ J. D. Gerrard and A. M. Marko 


volume 22 


editors: 
V. E. Hall 
F. A. Fuhrman 
A. C. Giese 
editorial committee: 
J. M. Brookhart 
H. W. Davenport 
J. Field 
V. E. Hall 
H. Hoagland 
G. K. Moe 


710 pages 
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March, 1960 


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Annual Review of yeas 


Physiology and Medicine, A Transition Period........... .....K.C. Mele 
Nuclear Function and Nuclear-Cytoplasmic Interactions.....D. M. Prescott 
Physiological Aspects of Genetics................... ccc eee eens G. W. Beadle 
ee ee 2a, ss GD “vag wR aon he CDS RIMMED T. R. Forbes 
I oe he vg hob dee aaa ea SSO ka J.S. Nicholas 
Kidney, Water and E ee es ee ee eS 4. Leaf 


Comparative Physiology: Fuel of Muscle — 
. 1. Drummond and E. C. Black 


Comparative Physiology: Blood Pigments.......................... C. Manwell 
a ct RS a ak og 8 aie" de nie wan tod J. F. Perkins, Jr. 
RUE OS a ee gen Oe Ne CN ee eT A. S. Harris 
07 Se a Oe ayo ayy a J. M. Crismon 
Blood Volume Regulation........ Ek. B. Reeve, T. H. Allen and J. E. Roberts 
I I oe ig ng We am kg ewe RED C. A. M. Hogben 
Nerve Excitation and Synaptic Transmission....C. Spyropoulos and I, Tasaki 
Somatic Functions of the Nervous System....... J. P. Segundo and C. Galeano 
Visceral Functions of the Nervous System.....................0.. F. P. Brooks 
Higher Functions of the Nervous System.........W. D. Neff and J. M. Goldberg 
Sg RRR ON ee er re eee re Sear F. Crescitelli 
Adenohypophysis and Adrenal Cortex..........W. F. Ganong and P. H. Forsham 
CREE brie caer are ere re D. H. Solomon and J. T. Dowling 


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FEDERATION PROCEEDINGS Volume 19 











July; 





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‘olume 19 July 1960 





WIiIRA 








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FEDERATION PROCEEDINGS 


xi 





Dependable instrumentation is vital 


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measurement accuracy is greatly increased. 

The DS5-5 Scintillation Well Counter is illustra- 
ted here with the 132A Analyzer Computer, which 
combines a precision scaler and gamma-ray spectrom- 
eter in a single compact chassis. They form a system 
of unusual reliability and sensitivity for such clinical 
studies as blood and plasma volume measurements, 
red cell mass and survival studies, diagnosis of 
pernicious anemia, fat digestion and absorption 
studies, etc.* 

We would be pleased to discuss these instruments 


with you. 


nuclear -chicago *“Diagnostic Applications of : 


e ©ORPORATION Radioactive Isotopes” explains yrrrucarions 
335 E. HOWARD AVE., DES PLAINES, ILL. in detail the procedures for 
performing these studies as 

well as other common radio- 

isotope tests. Please ask us 

for your free copy. 





Ji 
xii FEDERATION PROCEEDINGS Volume 14 a 











COUNTER 


2 aii tn iia 


Sdlisiiicaiiaciaai 2s RN aaa 


FEDERATION PROCEEDINGS 


Volume 14 








EVERYTHING 
IMPROVED 
FOR 1960! 


THE NEW 
X-250A DENSITOMETER 
(for indicating dye injection 
curves in heart function 



































studies) 
¢ Linear = 
+ Less expensive 
+ Interchangeable filters to accommodate various dyes. , 
+ Small blood sample chamber for fast dynamic response 
- Factory trained representatives in most territories. ‘ 
€ 
t 
THE ATERS CORPORATION 
Electro-Medical Instrument Division 
P. O. Box 288, Rochester, Minn. é 
F 
f 
24 i 
Cc 
f 
sole manufacturers of the ee ROCKL Nl D”’ 
A C 
penicylinder . 
Stainless Steel Antibiotic Assay Cups A RELIABLE AND POPULAR 
C 
STANDARD” name brand. f 
Our laboratory MICE and RATS can now be f 
offered entirely free of Syphacia obvelata Y 
and Aspiculuris tetraptera (Oxyurids). 
Swiss-Webster SW 
” - 10mm | Rockland all-purpose RAP L 
sand mouse )¢.57 Black C-57BL 
© : strains \C-57 Brown C-57BR f 
oe} i . C-57 Leaden C-57L f 
e Testing the potency of antibiotics. C3H Agouti C3H ( 
e Testing the effectiveness of disinfect- 
— ene Sargent y Say oY li rat) Sherman albino [ 
~ e The AO.AC. dilution conformation strains ( Long-Evans hooded 
test: Results obtained by phenol co- C 
Oo eficient procedure. (Stuart test) rice list on request 
om Material: “ q ba, 
> Corrosion Resistant, Stainless Steel ~, r~, 
* Specials to your Specifications RK orkland Sarma 
© 1 gross to a box. 
©. Send for Price List NEW pe gy County) 
Ple 
S & L METAL PRODUCTS CORP. 
27 Lafayette St., Brooklyn, N.Y. 











xis FEDERATION PROCEEDINGS Volume 19 














ONE of these REICHERT INSTRUMENTS for 
FLUORESCENCE MICROSCOPY 
WILL APPEAL TO YOUR BUDGET 








Whether you select the inexpensive 
“Lux UV”, the convenient “FLUOREX” 
or the versatile “ZETOPAN”, you will 
enjoy the advantages of working with 
this most qualified and efficient appa- 
ratus for fluorescence microscopy, 
based on nearly a Century of Experi- 
ence by REICHERT in this field. 
Featuring the OSRAM HBO 200 Mercury 
Arc Lamp, all units are equipped with 
fingertip controlled illumination including 
centering device, quartz collector and 
field iris diaphragm. Lamp housings 
are reinforced aluminum cast and air- 
ventilated. Built-in slides or turrets 
accommodate ALL filters for general 
fluorescence microscopy and the 
Antigen-Antibody Technique. 

You can depend on our time-tested Power 
Supply for operation of the Mercury 
Lamp. We guarantee trouble-free per- 
formance and uninterrupted service 
facilities. 


Demonstrations can be arranged 
anywhere in the U.S.A. 








‘olume 19 








XUM 





“Lux UV” Illuminator 


Most economical fluorescence light source 
Can be used with almost any microscope 





“Fluorex” unit 
Most convenient for prolonged effortless 
operation. Enclosed light path. Fluorex and 
microscope form an integral unit. Can be 
used with most microscopes 





“Zetopan” 
REICHERT Research Microscope equipped 
with Double Lamp Unit for instantaneous 
transition from illumination for routine in- 
vestigation to fluorescence microscopy 


Please request our literature for complete particulars on the REICHERT apparatus for Fluorescence Microscopy. 


WILLIAM J. HACKER & CO., INC. 


P. O. Box 646 


CApital 6-8450 


West Caldwell, N.J. 








The ‘Heart’ of your Freeze-Drying System 
UNITRAP 


@ One automatically refrigerated cold trap for all freeze-drying procedures. 


the new Sedigux 






@ Freeze-dry on manifolds or electrically heated trays. 
@ Three liter effluent capacity per dehydration. 
@ Eight different vacuum drums available. 


@ Economical conversion of present equipment to automatic refrigeration. 


MANIFOLD DRYER 





The VirTis automatically refrigerated Unitrap offers a new convenience. 
To freeze-dry heat labile materials use this automatic cold trap with any required 
type of freeze-dryer and suitable vacuum pump. Sublimating water molecules are 
frozen out on cooling coils inside the stainless steel condenser . . . Coil temperature 
has a maximum low of -65° F. ... The high vacuum essential for freeze-drying 
is maintained and moisture prevented from contaminating vacuum pump oil. 


The eight different vacuum drums available for use with Unitrap represent 
three different types . . . For bulk processing only; for drying samples connected 
to a manifold; and combination bulk and manifold drying chambers. An electrically 
heated three tray rack is supplied for bulk drying procedures. These trays yield 
an even heat input to each sample, assuring uniform drying rates. 


The VirTis Unitrap provides maximum safety for high vacuum studies... 
(no glass used except in the fabrication of the vacuum gauge.) Because a high 
degree of drying efficiency is routinely obtained, the Unitrap is suitable for precise 
freeze-drying . studies to relate to production schedules, as well as for routine 
laboratory operations. 


For full information on automatic freeze-drying write — 


THE VIRTIS CO., INC. -- GARDINER, N. Y. 


July 








lence. 
juired 
Ss are 


ature 
rying 
a 


resent 
ected 
ically 

yield 


BS. 0s 

high 
recise 
putine 


July 1960 





7 
asi iad 


| 


I 








spare hours — save the day — with the 








activity 


of jliquid| samples 


up to a hundred at 
a time automatically 


with accurate count printout 


Here’s the way to save hours and days in lab- 
oratories handling large numbers of radio- 
active liquid samples (cr smaller numbers. 
maybe, of low-activity samples which count 
slowly). Answers the kind of problem that 
arises, for example, in human blood studies 
with Iron®® or in metabolic studies on test 
animals. 


Some of its unique features... 
Handles 100 15 cc samples 
Samples always accessible 


Uses virtually any well-type scintillation 
detector 


Operates with Picker Count-and-Time Printer 
or Printing ‘imer 


Constant background count because samples 
surround the detector uniformly 


Modest cost $2,000.00 


PICKER AUTOMATIC WELL COUNTER 2956 


This unique instrument is one of the compre- 
hensive Picker family of quality nuclear 
equipment. The line includes instrumentation 
for every phase of diagnostic and therapeu- 


tic use of radiosotopes. 


For particulars, call any Picker District Office 
—there’s probably one near you (see your 
local ‘phone book). Or write Picker X-Ray 
Corporation, 25 South Broadway, White 


Plains, New York. 


* 


nuclear 








FEDERATION PROCEEDINGS 


MOId 


r 
& 
— 


jonu yy 


SJFUODUINIISUI JVS 


XVii 





XVili 











New tips and tops on PYREX’ pipets 


... some of the new pipets we’ve added 
during the past year or so 


You can be pickier than ever about the pipets 
you use for biological work; we’ve added to and 
refined the entire line of PyREx pipets. 


New tips. There’s a bigger hole in the tip of No. 
7087 that lets you work faster with viscous 
liquids such as heavy creams, curds, and oils. 
Thanks to an extra-long, narrower tip on No. 
7084, you can work more easily with Warburgs, 
small flasks and tubes, and the like. Tips on 
No. 7105 pipets, designed especially for Folin 
tests of urine and blood sera, are now uniform 
in length and diameter. 

Like all PyREx tips these are stoned to a 
smooth bevel which reduces snagging and break- 
age significantly. 

New tops. No. 7086 is an AccU-RED pipet with 
a cotton plug top for safe transfer of a virus 
or pathogen; for the first time you can combine 


the strength and long-lasting marks of Accu- 
RED with cotton plugs. 

No. 7096 is a rinse-out pipet for accurate 
micro dilutions. 

All our tops are flat and smooth for precise 
finger control. They’re also fire polished for 
easy removal of lipstick and other stains. 

There are many more special pipets, and old 
standbys, listed in your PyREx labware catalog 
LG-1 and supplements . . . along with the 
world’s most complete line of lab glassware. 


CORNING GLASS WORKS 


86 Crystal Street, Corning, New York 
CORNING MEANS RESEARCH IN GLASS 





PYRE X° taboratory ware... the tested tool of modern research 


FEDERATION PROCEEDINGS 





Volume 19 




















olume 19 





July 1960 



















Model 55 FOR ELECTROCARDIOGRAPHY 
econ . New Model 100 Viso...2 recording speeds, 3 sensi- 


tivities for extreme diagnostic accuracy. 

Model 300 Visette . . . 18lb. portable ECG, “‘traveling’’ 
instrument. 

Model 55 EEG/ECG High Gain Preamplifier . . . de- 
signed for use with Sanborn ’cardiographs and multi- 
channel recorders for fetal ECG’s, EEG (monitoring), 
small muscle myograms. Fully transistorized, portable 
unit. 


Model 300 Visette 


Model 100 Viso-Cardiette 















150M 
Series 


FOR 
MULTI-CHANNEL 


Model 62 Twin Beam 


RECORDING PHOTOGRAPHIC 
DIRECT WRITING 550M Poly-Beam series . . . 6 or 8 chan- 
150M series . . . from one-channel bench- nels with interchangeable plug-in pre: 


amplifiers, single cabinet housing, optional 
lug-in monitoring unit (same as 350M). 
odel 62 Twin Beam .. . 2-channel sys- 
tem for diagnostic phonocardiograms, 
ECG’S, etc. 


PRESSURE TRANSDUCERS for 
use with these systems include types for 
differential or single-ended measurement 
of liquid or gas pressures; two sensitivities 
—1 cm/limm Hg. or 1 cm/0.1mm Hg.; 
350M Series optional adapters for use with earlier 


Sanborn systems. 
SANBORN 
diagnostic/research INSTRUMENTATION 


FOR VISUAL MONITORING 


Model 169A-OR Viso-Scope ... for ECG monitoring 
during surgery. Bright, sharp traces; UL approved for 
aoe use in presence of explosive gases. 

Viso-Scope Model 169A Viso-Scope . . . portable ’scope primarily 


for use with the model 179 Electronic Switch, Model 185 
Vector Timer. 


Model 179 Electronic Switch . . . for viewing up to four 
traces simultaneously on the Model 169A Viso-Scope. 


top portables to multi-channel systems in 
mobile cabinets; interchangeable plug-in 
preamplifiers. 


350M series . . . 6 or 8 channels, improved 
design, frequency response to 120 cps, 
single cabinet housing, interchangeable 
plug-in preamplifiers. Optional plug-in 
monitoring unit (scope, electronic “ida 
vector timer). Model 60 Twin Viso. 

2 channel mobile system. 





Model 60 
Twin Viso ” 















Model 179 
Electronic Switch 


Model 169A 
Viso-Scope 
Model 185 Vector Timer . . . for Vector loop presenta- 
tion on the 169A Viso-Scope. 

Model 569 Viso-Scope, 579 Electronic Switch and 


aor: 559 Vector Timer in a compact, oe in form for use 
Complete Visual monitoring Unit with 550M or 350M systems. 


Model 185 
Vector Timer 














FOR HEART SOUND ANALYSIS 


Model 278 High Fidelity Tape Recorder/Player... for 
accurate recording and playback of heart sounds. 


Model 256 Amplifying Stethoscope . . . high fidelity monitor- 
ing of cardiac sounds by groups of up to 13 listeners. 





Model 256 

Amplifying Stethoscope 

a and Loudspeaker 

Model 280 Rappaport-Sprague Acoustic Stethoscope .. . new, 

high efficiency stethoscope with five interchangeable chest 

pieces for various heart sounds. y 

Model 278 Tape Recorder/Player Model 280 Stethoscope 

Sanborn Branch Offices and Service Agencies 
in principal cities throughout the U. S. 


For complete information or demonstration of iS 1 s AN B ORN COMPANY 
Sanborn instruments, call your local Sanborn MEDICAL DIVISION 
‘epresentative, 175 Wyman Street Waltham 54, Mass. 


) 


FEDERATION PROCEEDINGS 






















TRANSDUCER MIXER 
A ZERO ADJ. B ZERO ADJ. 


@ L 4 


BRIDGE CAL. 








ar A ~ 
B=A 











SERIAL 


som cAauCt SPECIFY 
@ we te. 
- 4V¥ + ghee” = noc ae inden * 1 lock 
G * Oo . our GRASS 
e e acemnnesss (@) MODEL TM-1 
MODEL TM-1 ~~ « 


STRAIN GAUGE TRANSDUCER MIXER 


@ Algebraically adds the output of two gauges. 


@ Provides balance and sensitivity controls for each, and an 
equalizing control. 


© Allows two absolute pressure gauges to be used as a differ- 
ential system. 


© Batteries supply excitation voltage to both gauges. 


® Mode switch selects either gauge or their difference for 
recording. 


@ Calibrator included. 


@ Size: 5-4" x 5-%" x 3-%" Weight: 4 lbs. 











GRASS INSTRUMENT COMPANY 


101 OLD COLONY AVENUE - QUINCY, MASS. 





INSTRUMENT MAKERS TO THE MEDICAL PROFESSION SINCE 1935 


DOO! QI 
xx FEDERATION PROCEEDINGS Volume 19 



























‘olume 19 


July 1960 








XUM 





Here’s why AO Phasestar offers you more! 





wal de 


New Bertrand Lens Built-in Bertrand lens 
is now supplied as standard on all binocu- 
lar and trinocular Phasestars to provide 
mew convenience. You slide lens into 
—— in eyepiece tube and raise or 

ower to focus quickly on diffraction plate 
at back aperture-of objective. 













Interchangeable, Rotatable Bodies Your 
choice of interchangeable, rotatable, mon- 
ocular, binocular or trinocular . bodies. 
Simply loosen thumb screw to rotate (full 
360°) or interchange. Special inclined 
monocular body with vertical photo- 
graphic tube also available. 









Interchangeable Stages You loosen sin- 
gle set screw to remove and interchange 
stages... your choice of Gated or un- 
graduated mechanical or MICRO-GLIDE 
stages. 


The AO Spencer Phasestar is the most versatile phase you select the microscope and equipment you need to 

microscope ever developed. Beginning with a basic do the job. There’s no obligation, of course. 

Phasestar stand, you can select from a wide choice 

of interchangeable bodies, stages, optics and phase AO SPENCER PHASE ACCESSORIES FOR PLATELET COUNTS 

accessories to make | the specific model that meets The use of phase contrast 
i 





your exact needs...all in all, more than 50 possible for blood platelet counts Specimen Plane 
combinations or models to choose from. And whatever _ is increasing in acceptance 3mm 

the model you choose, you're assured of superior AO and application. The uy 

Spencer quality p/us matchless convenience, comfort longer working distance / 

and versatility. required with the phase 


Often particular phase applications require specific hemacytometer can be 
equipment and phase contrast objectives. Before you obtained with special AO 
select your phase microscope and accessories consult Spencer long working distance accessories that provide 
your AO Sales Representative. He's qualified to help up to 7mm working distance in air or 104mm in glass. 











Corer ew nr ee nr i na a a a > 


Dept. G202 


os e | 
A 0 I would like to review a specific phase microsco roblem. 
sy merican Optical Please have AO Sales Representative call. viciies | 


O Please send copy of Phasestar Brochure. 


Company ms - | 





Address és 
INSTRUMENT DIVISION, BUFFALO 15, NEW YORK City. Zone. a 


Me ee ee cs ce ce ee cm ce es ce ee Some came mc ame me em ees me ne anes cr cm cs ee ee cs cee ces es eee es ee ces cme eee es ee cee ee ee oe oe ed 





SPENCER 








FEDERATION PROCEEDINGS 


xxi 








NEW BECKMAN DB SPECTROPHOTOMETER 





CUTS 


The Beckman DB Spectrophotometer utilizes an 
exclusive double-beam system. This eliminates most 
adjustments required in single-beam units. Sepa- 
rate readings for reference and sample cells are 
unnecessary—you simply set the desired wave- 
length, insert the sample and reference and read the 
meter. Manual operation, wide wavelength range 
and direct readout combine with double-beam ac- 






New Push-button Recorder 





Regional Offices: Atlanta - Boston - 


tion to bring you up to 80% savings in routine 
analyses time. 


No. 68435—Beckman Model DB Spectrophotometer, for 320 to 770 
mu wavelength range, with two silica rectangular cells. 115 volts, 
50/60 cycle. . ae nee see ...... $1,700.00 
To extend range to 220 mu the following are needed: 
No. 68437A—UV Accessory Set Nani wate eins Aye 42 Mt $40.00 
No. 68437B—Hydrogen Lamp Power Supply............... $297.00 
Flame Attachment also available for use with Model DB. 


Ratio Recording Available 


Add a wavelength drive accessory and a Beckman 
Potentiometric Recorder to the Model DB and you have 
the lowest-priced true %T recording spectrophotometer 
available today! Features of the Beckman Potentio- 
metric Strip Chart Recorder make it the ideal companion 
for the Model DB. 


No. 67940—Beckman Potentiometric Recorder .....$475.00 
No. 68438A—Wavelength Drive Accessory. .............-- $75.00 


Ask your S/P Representative for complete specifications, or write... 


Scientific Products 


DIVISION OF AMERICAN HOSPITAL SUPPLY CORPORATION 
GENERAL OFFICES: 1210 LEON PLACE, EVANSTON, ILLINOIS 


Chicago - Columbus - Dallas - Kansas City 


Los Angeles - Miami - Minneapolis - New York + San Francisco * Washington 















BARBER 
COLMAN 








COLMAN 


ef 
+ 











(r —) 
Wheelco 


Instruments 
Division 


ke J 


























INDUSTRY-WIDE APPLICATIONS PROVE ACCEPTANCE 
OF BARBER-COLMAN GAS CHROMATOGRAPHS 


Play major role in research and 
development; quality control 








lop =a Oo 
Nrano, g Rs} 
C1—CS5 ALCOHOLS 
2. 
MEH, 
3 Buy, 
MED, : 4No, 
a “8 mame bot 
Tan, oy t 
° 
” 
w 
” 
= 
p=} 
& 
= 








The Chromatogram Is The Proof 
This typical chromatogram is from a 
tun of C,-C; alcohols on a Barber- 
Colman Gas Chromatograph with a 
100 foot, stainless steel, capillary col- 
umn. Column coating was Armeen 


Since their announcement a short time 
ago, Barber-Colman Gas Chromato- 
graphy systems have found their way 
into many industrial and research labo- 
ratories. Following are a few cases 
which show the magnitude of accept- 
ance accorded these chromatographs. 
New techniques and accessory items 
are constantly being developed by 
Barber-Colman ... the primary reason 
for the versatility and the fast-growing 
popularity of Barber-Colman chroma- 
tographs. 


Synthetic Coffee... 

A leading U.S. research institute has 
recently completed a project which 
may well point the way to a cup of syn- 
thetic coffee comparable in flavor and 
aroma to the real thing. Because aroma 
is responsible for from 80% to 90% of 
the flavor sensations received from 
coffee, it is necessary to separate the 
volatiles from brewed coffee in order 
to determine the components that pro- 
duce the aroma and flavor. A truly syn- 
thetic cup of coffee in the future is 
extremely likely. Barber-Colman chro- 
matographs are being used in trace 
work of this type. 


Hydrocarbons in Air... 

A major American chemical company 
has succeeded in isolating as few as 15 
parts per billion of some hydrocarbons 
in air with a chromatograph equipped 
with an argon ionization detector. The 
company’s scientists achieved with this 
sensitivity with a modified Barber- 
Colman ionization detector. The stand- 
ard 80-microcurie radium source in the 
detector was replaced with a one-curie 
tritium source. The tritium is absorbed 
in a titanium film on a stainless steel 
foil. After exciting the argon carrier 
gas used in the detector, the excited 
argon atoms impart their energy to 





molecules in the sample having ioniza- 
tion potentials of less than 11.6 e.v., 
the energy of the argon metastable 
state. Since the ionization potential of 
air exceeds 11.6 e.v., the detector is 
relatively insensitive to air. 


Perfumed Fragrances... 

One of the world’s foremost creators 
and producers of perfumed fragrances 
is using Barber-Colman Gas Chroma- 
tography for quality control to assure 
fragrance purity and uniformity. The 
extreme sensitivity of the chromato- 
graph makes possible the separation 
and graphic study of the many compo- 
nents that go to make a fragrance. 





Wheelco Model 20... 

Compact and portable for laboratory 
and industrial use. Designed to accept 
capillary or coiled packed columns. 
The extreme flexibility of Barber- 
Colman Gas Chromatographs provides 
qualitative and quantitative chemical 
analysis due to the many features and 
accessories offered . . . argon or flame 
ionization detectors, separate tempera- 
ture controls for column heater, ad- 
justable sample splitters, effluent split- 
ters, events markers and many more. 
Investigate the features of Barber-Col- 
man Gas Chromatographs that are sig- 
nificant to your requirements. Contact 
your nearest Barber-Colman sales and 
service office. 





Wheelco Instruments Division 


BARBER-COLMAN COMPANY 


Dept. G, 15127 Rock Street, Rockford, Illinois, U.S.A. 
BARBER-COLMAN of CANADA, Ltd., Dept. G, Toronto & Montreal » Export Agent: Ad. Auriema, Inc. N.Y. 


SD. Column temperature: 60°C. Cell 
temperature: 110°C. Flash heater tem- 
perature: 140°C. Pressure: 15 psi. 











XUM 








Large Equipment for 


Fluorescence Microscopy 





Made in West Germany 


Write for 


free detailed literature 


CARL 








LEIS> 





The Carl Zeiss Large Fluorescence 
Equipment fulfills to the highest de- 
gree the requirements of modern fluo- 
rescence microscopy in brightfield as 
well as darkfield. 

Exciter filter combinations make it 
possible to select the most suitable 
ultraviolet or blue-violet range of the 
spectrum emitted by a high intensity 
mercury vapor burner. In addition, a 
specially designed filter tube, with 
rotatable discs containing barrier fil- 
ters, not only protects the human eye 
but allows quick selection of filter 
combinations to obtain highest con- 
trast of the specimens under exami- 
nation. The purity of the produced 
fluorescence is not only of the opti- 
mum but of such high intensity that 
photomicrographs can be made in 
color or black-and-white. 


Another outstanding advantage is 
the fact that every existing Carl Zeiss 
objective can be employed—not only 
Achromats but also highly corrected 
objectives such as Apochromats, 
Neofluars, and Planachromats. This 
makes it possible to observe true 
colors produced by fluorescence and 
at the same time makes the purchase 
of objectives suitable for fluorescence 
unnecessary. 


GAL £ZEMS SS, NC. 





XXiVv 


FEDERATION PROCEEDINGS 








Volume 19 & July 














‘olume 19 & July 1660 





XUM 





cCARL 
Revolver Micro | ZENSs. | 


Projection Apparatus 








Made in 
West Germany 





In using microscopic slides for discussions at staff meetings or in the classroom, micro pro- 
jection apparatus fills an important need. However, micro projectors, thus far available, 
required complicated handling and were therefore unsuited for general use. 

With the new Carl Zeiss Revolver Micro Projection Apparatus, former difficulties are 
completely eliminated. Six objectives, coupled mechanically to suitable condensers, to- 
gether with special projection eyepieces, instantly project an image on the screen from low 
(macro) up to high magnifications. All objectives are parfocal and the matched condensers 
provide illumination by the Koehler Principle. Best resolution and greatest brightness of 
images are thereby assured. 

Special projection eyepieces guarantee favorable fields of view, even at long projection 
distances. A fully automatic carbon are lamp eliminates frequent manual adjustment, so 
that even untrained operators can easily handle this projection apparatus. 

The Revolver Micro Projection Apparatus can be quickly converted into a lantern slide 
projector for 2 x 2-in. slides by merely swinging a reflecting mirror, a condenser and a pro- 
jection lens into the path of the rays. 


Write for free detailed literature 






COMPLETE 
485 FIFTH AVENUE, NEW YORK 17, N. Y. SERVICE FACILITIES 


FEDERATION PROCEEDINGS 


XXV 





XXVI 


FOR THE SIMPLE 
AND SPECIFIC 


DETERMINATION OF 

















worthington 





use 
WORTHINGTON PURIFIED URICASE 
A highly purified suspension in 10% 


ammonium sulfate 
— or— 


WORTHINGTON URICASE POWDER 
A crude, soluble preparation from beef kidney 


—or— 


Best of all, for speedy routine clinical 


assay, use 


DETERMATUBE U 


A purified enzymatic assay system. Just add water, serum 


ily ia 


“vn gton 


(or diluted urine or unknown) and read at 293m 


For information write: 


FREEHOLD 


BIOCHEMICAL CORPORATION ww scesey 

















TACONIC 
FAR has 


GERMANTOWN NEW YORK 





Send for booklet 











KILL FLIES 


LIKE MAGIC! 


with PURINA 
FLY BAIT 


Available in dry, liquid, and sugar- 
base forms, Purina Fly Bait kills 
adult flies quickly and effectively. 
. . at your local 


PURINA DEALER’S 


FEDERATION PROCEEDINGS Volume 19 


























‘olume 19 July 1960 





XUM 


UE 
a PALF 





Developed and quality-tested in Purina’s own research colony, St. Louis, Missouri 


Purina 


Guinea Pig 
Chow 


e Complete ration, with stabilized 
vitamin C to eliminate need for green 
feed. Allow drinking water. 

e Checker form—it’s easy to feed and 
reduces waste. 

e Low in feeding cost—easily available 
through your Purina Dealer. 

e Good results—chart shows typical 
growth curve. Purina Laboratory aver- 
age for well-managed sows is 3.5 young 
weaned per litter. 


Call your Purina Dealer (in the Yellow 
Pages under ‘‘Feed”’) or write: 


Herb Graff 
Ralston Purina Company 
St. Louis 2, Missouri 


650 





555 


450 


GUINEA PIG 
GROWTH CURVE 


Weight in Grams 


Stort 2 3 45 6 7 8 9 10 
Weeks on Test 














FEDERATION PROCEEDINGS XXVil 








WHATMAN 








introduces three new Glass Fiber papers 








WHATMAN 
GF/A 


For normal analytical work. 


GF/B 


Similar to GF/A but twice as thick. 


GF/C 


For ultra-retention. 


Description 

GF/A—For use in a Gooch, Hirsch 
funnel for all types of precipitates that 
do not require ashing. 


GF/B—For use with those precipitates 
requiring additional retention and 
strength of filtering media. Should also 
be useful for those chromatographic 
separations requiring impregnation 
and for routine electrophoresis. 


GF/C—An ultra filter of unusually fast 
filtration characteristics. 


These papers are available through your 
regular dealer in laboratory supplies. 


Introductory sample packages contain- 
ing circles of all three grades together 
with a descriptive brochure may be 
obtained by writing directly to: 


reeve angel 


H. Reeve Angel & Co. Inc. 
9 Bridewell Place 
Clifton, New Jersey 








ast 


ur 
es. 
in- 
ner 





KUM 


TEFLON is a registered trade-mark 
of E. I. du Pont & Co., Inc. 


{ 





*Stopcocks with TEFLoN® Plugs manufactured under FiscHer & PorTER Patent No. 2,876,985 


The stopcock plugs”* are TE FLO y. Ka 


in this new line of KIMAX® Laboratory Glassware 
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THE BIOCHEMISTRY OF 
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BIOLOGICAL ORGANISATION 


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A Histochemical Contribution to the 
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Edited for the Academy of Sciences of the U.S.S.R. by 

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T. E. Paviovskaya 

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10.00 


Please write for fully descriptive leaflets 








International Scientific Publisher 
PERGAMON PRESS, Inc. 122 East 55th Street, New York 22,N.Y. 








XXX 


FEDERATION PROCEED 


Volume \i 


July 








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For complete information about the 
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Volume 19 





Jul 


















ANALYZE FOR 
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S-72A 


Volume 19 July 1960 





XUM 


RIBONUCLEASE: Chart of analysis allows precise quantitation of 
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xl viii 

















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experimental pharmacodynamics 
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Votume 16 

















17 NEW BOOKS PUBLISHED 


IN MAY AND JUNE 








Leo Alexander, Austin W. Berkeley, and Alene 
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chiatry) 


Frederic C. Bartter (With 23 Contributors)—THE 
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TAGONISTS 


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Lewis Kk. Etter—GLOSSARY OF WORDS AND 
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Kdmund C. Jaeger — THE BIOLOGIST’S 
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H. Kalmus and 8. J. Hubbard—THE CHEM- 
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H. Kk. King—THE CHEMISTRY OF LIPIDS 
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TECHNICON 


AUTOMATIC 


VARIABLE 


GRAD EN 


DEVICE 
produces any buffer requirement in pH 
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DISCRETE BUFFER CHANGEOVERS 


asanexample... 
















to pH 5.28 
to pH 4.25 
from pH 3.25 ..- usual in 


AMINO ACID CHROMATOGRAPHY 
instead the Autograd automatically sup- 
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a ¥ 
This solution ¢$ 
arrangement of pH 


values in the 
AUTOGRAD ft 
automatically 
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bel 






















The Avtograd‘ starts the column elution delivery at 
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values as graphed below. 















% OF TOTAL VOLUME, 
Oo 10 20 30 40 50 ass) eh oe 
T 1 T 













AutoGrad Buffer Gradient 
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6+ 














Discrete Buffer Changeover 
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*Reference: “Variable Gradient 
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E. A. Peterson and H. A. Sober, 
Anal. Chem., Vol. 31, No. 34,* 
May 1959. 





























Send for Brochure No. AG 

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Meetings 


The Gordon Research Conferences for 1960 will be held from June 
13 to September 2 at Colby Junior College, New London, New 
Hampshire; New Hampton School, New Hampton, New Hamp- 
shire, and Kimball Union Academy, Meriden, New Hampshire. 
The Conferences were established to stimulate research in uni- 
versities; research foundations and industrial laboratories with the 
purpose being achieved by an informal type of meeting consisting 
of scheduled lectures and discussion groups. Attendance at the 
Conferences is by application. Individuals interested in attending 
may obtain application forms and further information by writing 
to W. George Parks, Director, Colby Junior College, New London, 
New Hampshire. 


The Second Conference on the Chemical Organization of Cells: Normal 
and Abnormal will be held August 25-27, 1960 at the University of 
Wisconsin, Madison. Interested parties should write to: Dr. 
Joseph J. Lalich, Professor of Pathology, University of Wisconsin, 
426 N. Charter St., Madison 6, Wisconsin. 


First International Congress of Histochemistry and Cytochemistry will be 
held in Paris, August 28 to September 3. It is organized under the 
auspices of the ‘Société Frangaise d’Histochimie” in collaboration 
with the histochemical societies in existence all over the world, 
especially the ‘“‘American Histochemical Society”, the ‘‘Deutsche 
Arbeitsgemeinschaft fiir Histochemie”’, the ‘Société Belge d’ Histo- 
chimie”’, the Italian and Japanese histochemical societies and 
several non-autonomous sections of histochemistry. Correspondence 
should be addressed to: Dr. R. Wegmann, Institut d’ Histochimie 
Médicale, 45, rue des Saints-Péres, Paris (6), France. 


The Second International Course of Lyophilization is scheduled for 
a two-week period starting August 29 in Lyon, France. Full details 
may be obtained by writing to Dr. Louis R. Rey, Directeur des 
Cours [nternationaux de Lyophilisation, Laboratoire de Physio- 
logie, Ecole Normale Superieure, 24, rue Lhomond, Paris 5, 


France. 


The Fifth International Congress on Nutrition, organized by the U.S. 
National Committee of the International Union of Nutritional 
Sciences and the American Institute of Nutrition, will be held in 
Washington, D. C., September 1-7, 1960. Eminent nutrition scien- 
tists from all over the world will participate in the Congress, the 
purpose of which is to increase knowledge and improve standards 
of nutrition in all areas. 

The main feature of the scientific program will be an all-day 
Symposium on ‘World Food Needs and Food Resources.”’ Invited 
speakers will discuss the following topics: 

C. G. King, Chairman, morning session 
Welcome—-By Secretary of Agriculture, Ezra Taft Benson 
The World’s Increasing Population—James M. Bonner 
Problems of Foods and Nutrition with Views and Programs of 
FAO—Binay R. Sen 
Water Resources of the World—R. G. Gustavson 
Prospective World Production and Distribution of Food—B. Tt 
Shaw 
Socio-Economic Factors that Limit Needed Production and 


Consumption—George Harrar 


(Continued on page (vit) 




















m June 


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in its anatomical, physiological, embryological and clinical 
aspects. For this edition, all significant material in the literature 
up to 1959 is included. The mechanisms of hormone actions are 
fully discussed. A new chapter is added on ‘‘Miscellaneous Hor- 
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By C. DonnELL TurNER, Ph.D., Professor of Biology, Duquesne Uni- 


versity, Pittsburgh. 511 pages, 133 illustrations. 
New (3rd) Edition—Ready in August! 


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Ruch & Fulton— 
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Long-familiar to you as Fulton’s Physiology, this classic text is 
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Edited by Turopore C. Rucu, Ph.D., Professor and Executive Officer, 
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enced modern genetic concepts. Chapters cover: Genetic Control of 
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An up-to-date, dynamic approach to cytology for undergraduate 
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consideration as a text! 

By E. D. P. DERobertis, M.D., Professor and Director, Institute of Gen- 
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FUNDAMENTALS OF 
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This clearly written and beautifully illustrated text is designed for 
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such as anemia; hemostatic defects; diseases involving lymph 
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By Byrp S. Leavett, M.D., Professor of Internal Medicine; and Oscar 
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(Continued from page lvt) 
F. G. Boudreau, Chairman, afternoon session 


Greeting——By Secretary of Health, Education and Welfare, 
Arthur S. Flemming 

Major Nutrition Problems Today—H. Gounelle 

The Prospect of Meeting Protein Needs—W. H. Sebrell, je 

Food and Protection of Public Health—Abraham Horwitz 

Program of the World Health Organization and Plans for the 

Future—M. G. Candau 

The remainder of the scientific program will consist of seven 
half-day panel sessions, and special sessions of ten-minute papen 
(reporting unpublished original research) which will be presented 
concurrently with the panel discussions. 

Panel sessions and their Moderators are: 

Evaluation of Nutritional Status in Man—E. W. McHenry 

Effects of Processing and Additives on Foods—W. J. Darby 

Lipids in Health and Disease—K. Lang 

Animal Nutrition and Food Production—D. P. Cuthbertson 

Nutrition in Maternal and Intant Feeding—F. W. Clements 

Proteins and Amino Acids in Nutrition—J. F. Brock 

Three Hours Around the World—New Possibilities in Nutrition 

Research—V. Ramalingaswami 
Subjects of the special sessions of 10-minute papers are: 

Germ-Free Animals 

Lipids and Cholesterol 

Ruminant Nutrition 

Proteins and Amino Acids 

Kwashiorkor 

Vitamins 

Carbohydrates 

Mineral Nutrition 

Minor Elements 

A full program of entertainment is being arranged for all regis 
trants of the Congress, including a number of special events for the 
ladies. Social events included in the registration fee are a reception 
and buffet supper at the Shoreham Hotel, a classical concert with 
Stokowski conducting at Constitution Hall, and a folk music con 
cert of modern Americana at the Sheraton Park Hotel. 

Officers of the Congress are: 

Elmer V. McCollum—Honorary President 

C. G. King—President 

Paul Gyérgy—Chairman, Organizing Committee 

Milton O. Lee—General Secretary 
W. H. Sebrell, Jr. is in charge of the scientific program, and Floyi 
S. Daft is in charge of Congress social functions. 

Advance registration is strongly urged. For full information 
and for registration, and hotel reservation forms, write to: The 
Secretariat, Fifth International Congress on Nutrition, 9650 
Wisconsin Avenue, Washington 14, D. C. 


The National Institutes of Health, Bethesda, Maryland will be 
the scene of the roth Annual Instrument Symposium and Research Equip- 
ment Exhibit to be held October 4 through 7, 1960. 


The American Institute of Oral Biology will hold its 17th Annual 
Meeting in Palm Springs, California, October 8-12, 1960, pre 
ceding the meeting of the American Dental Association in Ls 


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Jaffe—Tumors and Tumorous Conditions of the Bones and Joints. 
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Boyd—Pathology for the Physician. By Witiiam Boyp, M.D., 
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900 pages, 7” x 10”. 489 illus. and 12 plates in color. 6th ed. $17.50 
Faust and Russell—Craig and Faust’s Clinical Parasitology. By 
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23 tables. 6th edition. $15.00 

Smith and Jones—Veterinary Pathology. By Hitron A. Situ, 
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(Continued from page lviit) 


The 67th 
Surgeons of the U. S. 
ington, D. C., October Theme will 

“The Military Role Chairman of the 
Scientific Program Committee is Captain Clifford P. Phoebus, 
MC, U.S. Navy, Astronautical Bureau of 
Medicine Navy Department, Washington 25, D. ©, 


Annual Convention of the Association of Military 
will be held at the Mayflower Hotel, Wash. 
31, 1960. 
in Medical Progress.” 


November 1 and 2, 


Director, Division, 


& Surgery, 


The celebration of the 250th anniversary of the Charité will be 
19, 1960 in connection with the 
150th anniversary of the Humboldt University. Applications for 
participation are to be directed to the Committee for the Prepara- 
tion of the 250th Anniversary of the Charité, Berlin N4, Schumann. 
Med. habil. Dagobert Miller, Secre- 


held in Berlin from November 6- 


strasse 20-21, % Dozent Dr. 
tary of the Committee. 


Awards 
Dr. Rene J. Dubos has been selected as the recipient of the $5000 
Passano Foundation Award for 1960. The presentation of the Award 


to Dr. 
bacteriology and biochemistry, notably the use of specific enzymes 


in biochemistry, the chemotherapy of tuberculosis, and _ pioneer 


Dubos is a recognition of his many and fruitful researches in | 





work on antibiotics from soil organisms. Indeed it has been said 


that Dr. Dubos’s ‘*treasoning and demonstration made penicillin 
inevitable.”” Dr. Dubos is affiliated with the Federation through 
the American Association of Immunologists. 


As a result of the joint efforts of the 
ological Chemists and the Division of Biological Chemistry of the 
American Chemical Society, it is expected that substantial funds 
will be available from various governmental sources for support 
of travel of a number of qualified biochemists to the 5th International 


American Society of Bi- 


Congress of Biochemistry to be held in Moscow, August 10-16, 1961. 


These funds will be available for use without restrictions as to age. | 


In addition, approximately $go00, raised by assessment of members | 


of the two societies, is available for support of travel of young 
American biochemists to this Congress. Application forms are 
available from the Travel Awards Committee, 5th International 
Congress of Biochemistry, 9650 Wisconsin Avenue, Washington 14, 
D. C. Completed application forms must be returned to this ad- 
dress by October 1, 1960. 

A new grant-in-aid program, through which relatively small 
research grants may be arranged quickly for competent scientists 
working in the field of alcoholism and related subjects, has been an- 
nounced by the Scientific Advisory Committee of Licensed Beverage In- 
dustries, Inc. LBI is making this program possible, in response to 
the growing need for more scientific information both as to the 
extent of alcoholism and as to its causes and treatments, through 
a grant of $500,000 over a five-year period. Since this is essentially 
a “seeding” program, it is expected that grants will range between 
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newals possibly considered. Application forms and detailed infor- 
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t., New York 17, N. Y. 





Ir. John Murlin, enthusiasm resulted in the founding 
of the American Institute of Nutrition, who was secretary of the 
founding group and the first editor of The Journal of Nutrition, 
died on March 17, 1960. 


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FEDERATION PROCEEDINGS 


Books and Journals Received 





ANATOMY OF THE LOWER CuHorpATES. James E. Crouch. Palo 
Alto, Calif.: n-p publications, 1960. Paperbound, 40 pp, 17 
drawings. $1.50 

An IntTRopucTION TO ANIMAL BroLocy. Braungart-Buddeke. St. 
Louis, Mo.: The C. V. Mosby Co., 1960. 416 pp. $6.25 

A LasporaToRY MANUAL FOR LIFE SCIENCE. Herbert Drapkin. 
Palo Alto, Calif.: n-p publications, 1960. Spiral bound, 129 pp. 
$2.40 

ANNUAL REviEW OF PuysioLoGcy. Vol. 22. Editor, Victor E. Hall. 
Palo Alto, Calif.: Annual Reviews, Inc., 1960. 725 pp. $7.00 
U.S.A. ; $7.50 elsewhere 

BIOCHEMISTRY OF HuMAN Genetics. G. E. W. Wolstenholme and 
Cecilia M. O’Connor, Editors for the Ciba Foundation. Boston: 
Little, Brown & Co., 1960. 347 pp. $9.50 

CANCER OF THE Cervix. G. E. W. Wolstenholme and Cecilia M. 
O’Connor, Editors for the Ciba Foundation. Boston: Little, 
Brown & Co., 1960. 114 pp. $2.50 

ELECTROPHORESIS IN PuysioLocy. Lena A. Lewis. Springfield, IIL: 
Charles C Thomas, 1960. 120 pp. $5.50 

ENZYMES IN HEALTH AND DisgEAsE. Edited by David M. Greenberg 
and Harold A. Harper. Springfield, Ill.: Charles C Thomas, 
1960. 459 pp. $14.50 

EVALUATION AND PRESENTATION OF SPECTRO-ANALYTICAL RESULTS, 
A. B. Calder. N. Y.: The Macmillan Co., 1960. 47 pp. $1.25 

GENERAL BioLoGy LABORATORY MANUAL. Benson Ellis. Palo Alto, 
Calif.: n-p publications, 1959. Spiral bound, 112 pp. $2.50 

GENERAL AND INVERTEBRATE ZOOLOGY: A LABORATORY MANUAL. 
Benson Ellis. Palo Alto, Calif.: n-p publications, 1959. Spiral 
bound, 250 pp. $4.00 

Lipowws AND BLoop PLATELETS. John H. Fercuson. Chapel Hill: 
The Univ. of North Carolina Press, 1960. 278 pp. $5.00 

Mera Binpinc IN Menicine. Editor, Marvin J. Seven. Phila: 
J. B. Lippincott Co., 1960. 400 pp. $13.75 

Myocarposis: PATHOGENESIS, CLINICAL AsPECTs & THERAPY WITH 
RECENT INVESTIGATIONS CONCERNING THE PRINCIPLES OF 
METABOLIC ELECTROCARDIOGRAPHY. Ferdinand Wuhrmann 
and Serge Niggli. Translated by Harvey Adelson. Springfield, 
Ill.: Charles C Thomas, 1960. 218 pp. $10.50 

PLANS AND THE STRUCTURE OF BEHAVIOR. George A. Miller, Eugene 
Galanter, Karl H. Pribram. N. Y.: Henry Holt and Co., 1960. 
226 pp. $5.00 

PROTIDES OF THE BioLoGicaAL FL urps. Edited by H. Peeters. Elsevier 
Publishing Co., 1960. Distributed in U. S. by D. Van Nostrand 
Co., Princeton, N. J. 420 pp. $15.75 

RECENT RESEARCH IN FREEZING AND Dryinc. Edited by A. S. 
Parkes and Audrey U. Smith. Springfield, Il.: Charles C 
Thomas, 1960. 320 pp. $13.75 

SIGNIFICANT TRENDS IN MEDICAL RESEARCH. G. E. W. Wolsten- 
holme, Cecilia M. O’Connor, Maeve O’Connor, Editors for 
the Ciba Foundation. Boston: Little, Brown & Co., 1960. 356 
pp. $9.50 

SOMATOSENSORY CHANGES AFTER PENETRATING BRAIN Wounns IN 
Man. Josephine Semmes, Sidney Weinstein, Lila Ghent, and 
Hans-Luka Teuber. Cambridge, Mass.: Harvard University 
Press for The Commonwealth Fund, 1960. 91 pp. $4.00 

Tue BroLocy oF Marine Anma ts. J. A. Colin Nicol. N. Y.: Inter- 
science Publishers, Inc., 1960. 707 pp. $14.00 

Tue CenTRAL Nervous SysTeM AND Benavior. Selected transla- 
tions from the Russian Medical Literature. Washington: U.S. 
Dept. of Health, Education, and Welfare, Public Health Service, 
1959. 1051 pp. 

Tue Principces OF ELECTROPHORESIS. René Audubert & Serge de 
Mende. N. Y.: The Macmillan Co., 1960. 142 pp. $7.00 

Tue Merck InpEx-SEVENTH Epition. Rahway, N. J.: Merck & 
Co., Inc., 1960. 1641 pp. $12.00 

Volume 19 

















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129 pp. 


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XUM 


hederation 


Proceedings 


Published and “Copyright 1960 by 


FEDERATION of AMERICAN SOCIETIES 
for EXPERIMENTAL BIOLOGY 


Volume 19 


July 1960 


Number 2 


CONTENTS 


Joint Session: PHARMACOLOGICAL ASPECTS OF CURRENT 
SociAL PROBLEMS 


Introductory Remarks. C. D. Leake.............. 
Control of the Environment: Atmosphere and 

Waters 2. 2, Stokennat oc. 'o: 5. So yoke tial eee 
Chemical Approaches to Radiation Protection in 

Maramals, 27) ts Path acct gehen 2 aes 
Control of Biological Fertility. N. Millman........ 
Control-of Insect Peats: . Barnett i366 os cnce soe 


SympostumM ON RADIATION PROTECTION AND RECOVERY 


Studies on Microorganisms. A. Hollaender......... 


Chemical Protection of Mammalian Tissues. J. R. 
Moaistnand DG: Tnonenty mii. 1 os eee acateiins poet 
Genetic Aspects of Implantation of Blood-Forming 
"Disstne; AI Za ROHS: SRUSSO 62 sy:2 sono ett So 5 cee § 
Ontogeny of the Blood Cells. R. F. Ruth.......... 
Advances in Radiation Immunology. T. Makinodan. 


Sympostum oN New AND NEGLECTED HORMONES 


Melatonin. A. B. Lerner and J. D. Case...........- 
Recent Advances in Parathyroid Hormone Re- 
SPRANG E20) EUNSOM as ey rcs ssa tin tet ssse bh ae 
Epiphysis Cerebri in the Control of Steroid Secre- 
to) 0 Clee 4) + (ee ea ee OR nie rarer eee Oe 


On the Hemodynamic Parameter Mediating in 
Aldosterone Secretion in the Dog. D. S. Gann, I. 
ED. Malis aR Ce ROWUIBE 6692, sate opt us gm 


Symposium ON PHysIOLOGy AND Druc ACTION 


TrtPOR CONS Fe tt eins Sab usin has Seeks 


Pharmacological Dissolution of Evoked Cortical 
Potentials. §. Goldring and J. L. O’Leary......... 


54! 
543 


549 
553 
J07 


562 
564 
S73 
579 
586 
599 
593 


601 


Physiology and Drug Action: An Electroencephalo- 
graphic Analysis. R. G. Bickford................ 


Some Actions of Anesthetics on the Nervous System. 
Dey We el 3 a 2 os a a Re oe ae 


Physiology and Drug Action: Behavioral Implica- 
UES Reg BREE hli5 oo Sj San alld wid 3 dieia pistes = 


SyMPosIUM ON INTERACTION OF MINERAL ELEMENTS IN 
NUTRITION AND METABOLISM 


Intraguctian: Ge K: Davis. <6)! iscle ice ha wee oss 


Calcium and Phosphorus Interactions in Nutrition 
and Physiology. R. H. Wasserman. ............. 


Nutritional Interactions of Zinc and Calcium. R. 


Magnesium Requirement and its Relation to 
Other Dietary Constituents. B. L. O’Dell....... 


Metabolic Relations of Manganese to Other Min- 
Chala Gr COCOMAG Sia. 865 eae aa sb ewes 


Interrelationships of Iron and Copper in the Nutri- 
tion and Metabolism of Animals. G. Matrone... . 


Interrelations of Copper, Molybdenum and Sul- 
fate Sulfur in Nutrition. R. F. Miller and R. W. 


FEDERATION BOARD PROCEEDINGS 1959-1G60.......... 
FEDERATION FINANCIAL REPORTS.................4.. 


THE 1960 FEDERATION MEETING: SCHEDULING A MEET- 
ING AND PREPARING AN INDEX BY ComPUTER. Claire K. 
Schultz and C. A. Shepherd ............... Feiss agin 


tA INN ok ivf teal be ee ees 


619 
626 


629 


635 
636 
643 
648 
655 
659 
666 
671 


678 
681 








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Ixiv FEDERATION PROCEEDINGS 


Volume 19 





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7olume 19 





Joint Session of the Federation 


PHARMACOLOGICAL ASPECTS OF CURRENT SOCIAL PROBLEMS*? 





Introductory remarks 


CHAUNCEY D. LEAKE, Chairman 
The Ohio State University, Columbus, Ohio 


- OF THE FUNCTION of our Federation is to empha- 
size unifying approaches to common biological problems. 
The fragmentation of scientific endeavor is unfortunate 
because it tends to make us think of ourselves and of 
nature about us as a lot of disjointed and totally un- 
related fragments. Actually, as we well know, all of 
nature is one amazingly and beautifully coordinated but 
complex whole. Poets have long recognized this: John 
Donne suggested that no person is an island separated 
from the rest of humanity. Similarly, none of us can 
really separate ourselves from the total environment to 
which we all belong. 

As the realization dawns upon us that our fragmenta- 
tion of science is giving way to new concepts of under- 
lying unity, we find we must view our whole scientific 
effort in a new light. The very complexity of experi- 
mental biology hastens this reappraisal. 

Scientific endeavor in a special field proceeds through 
several phases: 1) the early descriptive phase, with at- 
tempts to describe and classify natural phenomena, which 
is chiefly taxonomic; 2) the experimental phase, when 
tentative hypotheses in explanation of observed phe- 
nomena are tested and 3) the conceptual phase, when 
scientists review their basic concepts and assumptions, 
and revise them, not only in relation to the general 
accumulation of verifiable facts about ourselves and our 
environment, but also in relation to other scientific 
concepts. 

In the biological sciences, we are emerging from the 
first or fact-finding phase, and are vigorously in the 
second or experimental phase of development. It is high 
time for us to begin to try to review our concepts of 
biology as a science. When we do so, we discover that 
biology is, of course, closely related to all other aspects 
of scientific effort. 


' April 12, 1960, Chicago, III. 

* Paper by Arnold J. Lehman on ‘Control of Toxicity in Foods, 
Drugs and Cosmetics’ ; to be published in Symposium on Toxicology 
(Supplement to Fed. Proc. 19: September 1960). 


XUM 


I am often surprised how little we seem to realize the 
importance of the faith expressed in the 1847 manifesto 
of Carl Ludwig (1816-1895), Emile Du Boise-Reymond 
(1818-1896) and Hermann von Helmholtz (1821-1894) 
that all living processes are explainable in terms of 
physics and chemistry. Not that living phenomena are 
merely physics and chemistry, but rather explainable in 
the terms of these sciences. Paul Cranefield has well dis- 
cussed this matter. It was taken seriously by our Russian 
colleagues, under the influence of Ivan Sechenov (1829- 
1905), a pupil and life-long friend of Ludwig. It is not 
mere coincidence that it fitted into the implication of the 
manifesto of Marx and Engels, also issued in 1847, that 
history and economics are explainable in similar ma- 
terialistic terms. 

At that same time, Rudolf Virchow (1821-1902) 
indicated the significance of organizational levels in 
living material, when he proposed to study disease 
scientifically in its varying manifestations from cells to 
societies. In all the artificial conventionalities of our 
fragmented scientific disciplines, from astronomy to 
zoology, there is a technical unity in the order revealed 
mathematically, and there is a social unity revealed 
historically. Perhaps as we realize more fully the con- 
ceptual unity of the sciences, we may more satisfactorily 
appreciate the actual unity of nature. 

As Alan Watts puts it, “‘Nature cannot wisely be con- 
trolled in the same way in which it has been studied— 
piecemeal. Nature is through and through relational, 
and interference at one point has interminable and un- 
foreseeable effects.” The consequences of some of our 
pharmacological attempts at interfering with this balance 
in nature will be considered in this Joint Session. 

At the beginning of this year, I was appalled when I 
found that it was my responsibility to arrange a program 
for the Joint Session of the Federation. I had not realized 
that the Joint Session should reflect the science which I 
represent. The technical details of pharmacology are 
interesting to an increasing number of scientists, and 








542 FEDERATION PROCEEDINGS 


also to many practical-minded people in such fields as 
medicine, dentistry, pharmacy, nursing, veterinary 
medicine, public health, health technology and even 
psychology, law, criminology, agriculture, and sociology. 
However, this pharmacological technology is often too 
detailed and changeable to have much _ long-range 
significance. 

Rather than explore the facts involved in pharma- 
cological aspects of current social problems, I thought 
it would be more interesting to emphasize pharmacologi- 
cal factors which may operate in attempts at their con- 
trol. What can we do to make the applications of phar- 
macological knowledge more effective in regard to our 
overall social welfare? Much coordinated effort is im- 
plied. Much wisdom is required. Much information 
must be obtained. When this verifiable information be- 
comes available to the general public, it may be pos- 
sible for voluntary agreement to be reached on wise 
policies that could bring effective control of our po- 
tentially toxic and dangerous applications of pharma- 
cological science to our environment. Unfortunately, 
pharmacological activity always implies possible toxicity. 

Thinking about this, it struck me that pharmacological 
aspects of certain major social problems might make an 
interesting and, hopefully, a memorable Joint Session. 
I am grateful to my colleagues who so willingly, and 
promptly, prepared general discussions for this session. 
Further details on the toxicological aspects of these 
matters will be discussed in the special Toxicology 
Symposium. * 

We have five speakers on the Joint Session. Dr. Herbert 
E. Stockinger, of the Public Health Service, in Cincin- 
nati, will consider the pressing problem of the control 

8 April 13, 1960. Papers from this Symposium will be published 
as a Supplement to Fed. Proc. 19, September 1960. 


Volume 19 


of pollutions of airs and waters. Since this pollution is 
frequently caused by chemicals, pharmacology is in- 
volved. Harvey M. Patt of the Argonne National Labo- 
ratory, will review radiation protection, with special 
reference to pharmacological aspects of control. Nathan 
Millman of the Ortho Foundation will explore some of 
the consequences of our apparently necessary attempts 
at pharmacological control of biological fertility. Thomas 
Burnett of the Entomological Laboratories, Ontario, 
will show how many pharmacological efforts at pest con- 
trol may lead to quite unexpected and _ potentially 
disastrous effects on the delicate balance of living nature. 
Finally, Arnold J. Lehman, of the Food and Drug Ad- 
ministration, will tell us something about the current 
controversial matter of the attempt to control adequately 
the possible toxicity of foods, drugs and cosmetics, from 
the standpoint of protecting the overall optimum health 
of our people. 

Perhaps we should realize that we have a responsibility 
to control wisely the pollution of airs and waters, ration- 
ally to control radiation, sympathetically to control 
biological fertility, sensibly to control pests, and under- 
standingly to control potential toxicity in our foods, drugs 
and cosmetics. This will result in a better appreciation 
of the interrelations of our science with all the rest of 
our culture. 

In the sense that science is the part of our logics con- 
cerned with verifiable knowledge about ourselves and 
our environment, perhaps we will come again to realize 
that it is intimately related to ethics, which deals with 
our purposes, our motivations, and our relationships 
with each other, and with our environment. The success- 
ful application of our science to the achievement of our 
agreed-upon purposes depends upon effective esthetics. 
This is a matter for judgment and wisdom. 


REFERENCES 


1. CRANEFIELD, P. F. J. Hist. Med. & Allied Sc. 12: 407-423, 
1957- 

2. Donne, J. The Complete Poetry and Selected Prose, edited by C. M. 
Coffin.New York: Modern Lib., 1952. 


3. RussEL., B. The Impact of Science on Society. New York: Simon and 
Schuster, 1953. 

4. Warts, A. W. Nature, Man and Woman. New York: Mentor 
Books, 1960. 





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Control of the environment: atmosphere and water 


H. E. STOKINGER 
Public Health Service, Bureau of State Services, Cincinnati, Ohio 


a EXPLOSION and the industrial expansion 
associated with this explosion are fast resulting in pollu- 
tion of two of man’s most precious commodities, air and 
water, at a rate that is alarming and to an extent that 
is fast becoming nationwide. Those charged with the 
responsibility of protecting the nation’s health have re- 
sponded to the growing menace of pollution in two ways: 
1) the establishment of programs for collection of basic 
data on air and water quality that are to be both per- 
manent and nationwide, and 2) the minimization of ex- 
posure by regulation and control. 


CONTROL OF AIR POLLUTION 


I will try to describe the nature and extent of the pollu- 
tion problems in air and water and then the efforts that 
are being made at control. 

Sources and Types of Air Pollution. What is in the air 
over places of high population density, and what are 
the causes of concern? First and foremost—from the 
character of the air pollutants thus far identified—a 
potential hazard to health in man and animal; but this 
is not all. Eye irritation, reduced visibility and annoying, 
blanketing haze; and finally, wasteful plant and crop 
damage. 

I show in table 1 the nature of the dominant types of 
air pollutants and in the column on the right their po- 
tential effects. First, the so-called ‘oxidants’, a highly 
complex and as yet incompletely defined group, from 
simple molecules like ozone to complex peroxyaliphatic 
molecules, and organic free radicals, an increasingly 
prevalent group associated with dense vehicular traffic. 
The recipe for their formation: auto exhausts, sunlight 
and static meteorologic conditions. Potential effects on 
health are grave as suggested from experimental animal 
studies and some limited studies on man (ozone) indi- 
cated on the right. Ozone injures plants at low levels, 
very low levels indeed, a few tenths ppm. 

Hydrocarbons are also of great concern, the polycyclic 
aromatics particularly, 3,4-benzpyrene, because of their 
demonstrated carcinogenicity, the olefins because they 
represent critical smog precursors. 

The metal oxides, particularly because of their 
catalytic potentialities for smog formation, are little 
studied in this respect, by the way. They also add to re- 


XUM 


543 


duction of visibility, are potential carriers of adsorbed 
gases with enhanced physiologic effects. 

Reducing gas Vapors,—SO, and SO; are marked for 
their respiratory tract irritation and plant damage. 
Aldehydes, particularly formaldehyde and acrolein, are 
the currently implied agents responsible for eye irritation. 

Metals in a variety of forms add dangerously to body 
burden especially Pb; others—As, Cr, Ni—possibly add 
to the cancerogenic potential of smog. 

Control of Air Pollution. Briefly, ‘these are the conditions 
that prevail’—what efforts are being made to regulate 


TABLE 1. Recognized Air Pollutants and Their 
Potential Effects 


Air Pollutants Toxicologic Action 
Oxidants 


O3, NO, NO2, NOBr Respiratory tract irritants 


Organic peroxides, peroxy- Radiomimetic properties 
aldehydes (peroxy com- Immunogenic activity 
pounds) Carcinogenic activity 

Organic free radicals, air Plant damage 
ions 

Peroxynitrates Eye irritant 


Hydrocarbons 
Polycyclic hydrocarbons 
Gasolines, (olefins) 
Oil mists (naphthenes) 


Carcinogenic activity 
React with oxidants 
Counteract effect of oxidants 
Metal Oxides 
Fe20;, V205, MnOs, etc. Oxidizing catalysts produc- 
ing more potent oxidants 
Reduce visibility 


potentially injurious emissions to the air? Two broad 
attacks are being made: 7) basic data-gathering systems, 
to determine the nature and extent of the problem and 
2) engineering and legislative controls. 

Basic data-collecting is being done in both engineering 
and medical fields. In engineering, four networks of air 
sampling on a nationwide scale are now in operation 
by the Public Health Service. Enabled by Public Law 
159—Federal Air Pollution and Technical Assistance 
Act, the network became truly national, January 1957. 

Figure 1 shows the disposition of the air sampling 
stations that extend across this nation and into Puerto 
Rico, Alaska and Hawaii and now total 172. (See if 
your city is included; if not, phone, don’t write your 
Congressman.) In each state at least the largest city and 



















































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FIG. 


POLLUTION OF 


July 1960 


one nonurban area are included. At present one-half 
of each sample is extracted for organic compounds, one- 
half for sulfate, nitrate, metals, etc. Analyses are now 
being completed on a 6-year sampling for particulates; 
the results of the first 4 years of network sampling are 
summarized in ‘Air Pollution Measurements of the 
National Air-Sampling Network’, prepared by USPHS 
Taft Sanitary Engineering Center, Cincinnati, 1958. 

In addition to the national network, there is an ex- 
perimental network embracing 17 cities, for the sampling 
of gases. Data are being gathered on sulfur dioxide, 
nitrogen oxides, oxidants; to be added are hydrogen 
fluoride, aldehydes. 

There is still a third network, a precipitation network 
of 30 stations in cooperation with the Weather Bureau. 
Here data on radioactivity, chloride, sulfate, nitrate, 
calcium, sodium, etc., in rain and snowfall are gathered. 


TABLE 2. Air Pollution Standards of California for 
Community Air Quality 


AIR AND WATER 





Levels 
Air Pollutant a : ena ee en 
Adverse Serious Emergency 
Oxidants 0.15 ppm, | hr. 
O;3, NO» ‘Oxidant In- 
Hydrocar- dex’ (eye ir- 
bons ritation, 
Photochem- plant dam- 
ical Aero- | age, visibil- 
sols ity reduction) 
Carcinogens | Not applicable 
SO: Ippm,thr.or |} 5 ppm, 1 hr. | 10 ppm, 1 hr. 
0.3 ppm, 8 (broncho- (severe dis- 
hr. (plant constriction tress in 
damage) in man) man) 
SO; Mist No established | No established 
(H,:SO%*) level (1 mg/ level (5 mg/ 
m® give re- m}, few min- 
spiratory re- utes produce 
sponse in irritation) 
man) 
CO Not applicable | 30 ppm, 8 hr., | No estab- 


Particulates 


Reduction of 


visibility less 
than 3 mi. at 
70% relative 


or 120 ppm, 
1 ir. 


lished level 
(240 ppm, I 
hr. may 
embarrass 
sensitive 
persons) 


humidity 
Lead Not applicable | No level estab- | No level es- 
lished (in- tablished 
sufficient (insuffi- 
data) cient 
data) 
Ethylene No level estab- | Not applicable | Not applica- 
lished (plant ble 
and fruit 
damage at 
0.2-0.5 
ppm) 


XUM 


TABLE 3. Air Pollution Standards of California Motor 
Vehicle Exhaust Standards* 


Limits 

Hydrocarbons f 275 ppm by volume as hexane 
(0.165 mole “¢@ C atoms) 
Carbon monoxidet 1.5 “% by volume 
+ 5 ) 

* For a composite sample representative of the driving cycle. 
7 80% reduction needed. 
t 60% reduction needed. 


And yet a fourth is a baby network of 6 cities for the 
collection by high volume samplers for particulates that 
are being subjected to animal tests for carcinogenicity. 

The Air Pollution Medical Program of the Public 
Health Service is basic data-gathering also, notably in 
epidemiology—searching for associations of health effects 
on populations with air pollution. In another area of 
growing concern, body lead levels, studies are being con- 
ducted on ‘high-exposure groups’ to auto exhausts of 
leaded gasolines. The information gained here is of basic 
value in determining whether current exposures are 
adding significantly to lead body burden. 

Regulations and Controls. Now as to regulations and con- 
trols that are effective in reducing man’s exposure to 
air pollution, these are of two types—engineering and 
legislative. Improved engineering design of incinerators 
for waste disposal has aided materially in reducing 
noxious components of incinerated wastes. Engineering 
improvement of stack design and devices of stack effluent 
collection and removal have greatly minimized _ in- 
dustrial effluents. 

In automotive design, engineering has attempted re- 
duction of auto exhausts by control devices. Efforts to 
control automobile and other motor vehicle exhaust 
emissions are directed toward the reduction or elimina- 
tion of 3 exhaust constituents: 7) olefinic hydrocarbons, 
2) carbon monoxide and 3) polynuclear hydrocarbons. 
The most promising method is ‘afterburning’ these un- 
desirable combustibles in exhaust. To burn these com- 
bustibles energy must be added. Three methods of add- 
ing energy show promise: 7) use of a heat-exchanger in 
a direct-flame afterburner, 2) use of auxiliary fuel in a 
direct-flame afterburner and 3) use of an oxidation 
catalyst. The afterburners are claimed to reduce CO 
and hydrocarbons by 80-g0%, warm up instantly, but 
cost from $50-$250; deficiencies are increase of nitrogen 
oxides which catalyze formation of oxidants. Catalytic 
types are plagued by poisoning from the lead in gaso- 
line—thus short life and high replacement cost. 

Legislative Controls. The State of California which has 
the most acute smog situation in the nation—in and 
around Los Angeles and the San Francisco Bay area—I 
believe has made the most effective use of legislative 
controls and also has made the most! It now has 63 
regulations. (I will take the time to enumerate only 49!) 
The most recent, Rule No. 63, attempts to control the 
last major uncontrolled source of air pollution, auto 
exhausts, by modifying gasoline composition. Rule No. 








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63 serves notice on gasoline producers that ‘“‘no gasoline 
shall be sold after July 1, 1960, in California unless it 
has a Br number of 30 or less.” This effort at control 
stems from research done by the L. A. Air Pollution 
Control District which indicated reduction of olefin 
content of gasoline is associated with reduction of eye- 
irritating smog. 

Among the more effective regulations have been the 
outlawing of home incineration of wastes, prohibition of 
burning of public dumps and regulation of industrial 
stack effluents by trapping of effluents and limiting the 
periods of emissions. 

Air Pollution Standards. Probably the most forward step 
in air pollution control has been the development of 
community air pollution standards and standards for 
auto exhausts by the State of California. Ordered by 
Governor Brown and developed and promulgated by the 
California State Department of Health on February 1, 
1960, the standards are now awaiting enactment into 
law. 

Table 2 is an attempt to give you an idea of the air 
pollution components and their respective limits. Note 
first, the relative small number of substances for which 
standards have been set. Note next that the character of 
the limiting levels, adverse, serious and emergency are 
all ‘effect’? levels at or above which some effects may 
occur; it is not presently possible to establish a realistic 
limit for any air pollutant an absolutely ‘no effect’ level. 
Note also, that standards for many substances are not 
established on basis of health effects on man, but often 
concern eye irritation, plant damage and visibility. 

The particularly difficult question of carcinogens is 
recognized, but unsettled, because the limiting dose or 
conditions for cancerogenesis for any important carcino- 
gen is not known with sufficient certainty for either 
animal or man. 

The consideration of the relatively inert ethylene may 
surprise you; it is highly injurious to citrous plants, at 
levels often as low as 4 pphm. 

Table 3 shows the standards set to reduce hydro- 
carbons in auto exhausts by 80%, CO by 60%. 

Carbon Dioxide and Climate. Your chairman has in- 
sisted that I inject the subject of the global carbon dioxide 
blanket into this presentation because of its theoretic 
relation to world temperature control. This is a meteor- 
ologic-climatologic problem and because I am not a 
meteorologist I have had to rely on my meteorologic 
associates for guidance. 

As the theorists view the atmospheric carbon dioxide 
problem, man’s increased activities may affect his 
climate. How does this come about? Man’s increased 
burning of fossil fuels, coal and oil, adds approximately 
6 X 10° tons CO, per year to the atmosphere which 
presently contains 2.3 X 10” tons or 0.03 % of the total 
atmospheric mass. The CO, blanket regulates temper- 
ature because, although CO: molecules permit visible 
radiation to pass, they absorb the reflected infrared 
radiation to retain heat on earth’s surface and raise 


XUM 


POLLUTION OF AIR AND WATER 547 


temperature. During the past 100 years, 360 X 10° tons 
CO: or 13% more CO: has been added to the atmos- 
phere. From this increment, theory predicts 1°F rise 
in temperature. This is precisely what occurred! 

On this basis, a trillion tons of CO, added by 2000 
A.D. produces a temperature rise of 3.6°F. There is a 
complex, although sluggish, equilibrium, between vege- 
tation usage (plants grow more luxuriantly as CO; 
increases) and ocean and rock CO, exchange, that time 
does not allow considering except to say that in 1000 
years the 18-fold increase in CO, tonnage will be re- 
stored back to 10-fold greater than today; i.e. 0.3 % COz 
when the earth is calculated to be 22°F warmer, the 
oceans will increase in acidity and the ice caps melt and 
so will we. This I venture will never come to pass; other 
forms of energy, the sun, electricity, atomic energy will 
replace fossil fuels long before. (It does indicate, how- 
ever, that you should keep your eye on your Standard 
Oil New Jersey Stock.) 

SO. Global Blanket. Illustrative also of the broader 
aspects of air pollution is the current recognition of an 
SO, global blanket. Air pollution meteorologists now 
feel they have sufficient evidence from air-sampling for 
SO, and from precipitation-sampling for SO,; that SO.- 
SO;-SO, permeation of our atmosphere is global and 
is on the increase for the same reasons CO, is on the in- 
crease—burning of fossil fuels, chiefly. In the last few 
years the relative increase of SO, is greater than that 
of CO». The uniformity of the SO, blanket is far less 
than that of CO.; there are ‘hot spots’ around wood- 
pulp processing and ore-smelting plants and urban 
areas. Concentrations in the vicinity of such activities 
may reach 3—-4 ppm on the average; low spots over the 
oceans are around a few ppb. 

What the significance of the SO, blanket is for civiliza- 
tion the meteorologists don’t yet say. There are possible 
effects of serious consequence interpretable from current 
air pollution research: 7) general and widespread injury 
to certain types of foliage; 2) general obscuration of sun- 
light from SO,-SO; reaction; 3) increase in oxidant 
type air pollutants from catalytic action of SO; on auto 
exhausts. 


TABLE 4. Proposed Substances for Revised USPHS 
Drinking Water Standards 


Alkalinity 

Anionic detergents 
Benzene Sulfonates) 

Arsenic compounds 

Barium compounds 

Cadmium compounds 

Calcium (as chloride) 

Chlorinated hydrocarbon in- 
secticides 

Chromium as Cr** 

Copper compounds 

Cyanide as inorganic CN 

Fluoride (inorganic) 

Total solids 

Turbidity, color, taste, pH 


Iron compounds 

Lead compounds 

Magnesium compounds 

Manganese compounds 

Nitrate-nitrite (inorganic) 

Organic phosphate _insecti- 
cides 

Organic synthetics (C-filter, 
CHCI;-extractables) 

Phenols 

Selenium compounds 

Sulfates, inorganic 

Zine compounds 

Radioactivity 


(Alkyl 








548 FEDERATION PROCEEDINGS 


CONTROL OF WATER POLLUTION 


Data-Gathering Methods. Regrettably, because of time 
limitations, I can only present a thumbnail sketch of our 
efforts to control water quality. Like the data-gathering 
efforts regarding air pollution, the Public Health Service 
has a National Water Quality Network, the specific 
responsibility for which was established by Public Law 
660. 

Figure 2 shows the water-sampling stations spread 
across this nation. (If you had time you could count the 
50 of them.) Sampling is done on 7) the major waterways 
used for public supply, propagation of fish and wildlife, 
recreational purposes, agriculture, industry and other 
legitimate uses; 2) interstate, coastal and Great Lakes 
waters; 3) waters where government activity may in- 
fluence water quality. Samples are measured for chemical 
content, pollution of biologic origin and radioactivity. 
The U. S. Geologic Survey has been data-gathering for 
many years, chiefly on the mineral content of our waters. 
The U. S. A.E.C. performs watchdog services for radio- 
activity locally in waters receiving effluents from their 
operations. 

Drinking Water Standards. Although the methods of 
data-gathering and of control of water and air follow 
closely similar patterns, the efforts at control of water 
pollution differ in one important respect—control efforts 
have been in operation for 50 years. The first drinking 
water standards regulating the quality of water for inter- 
state carriers were developed by the Public Health 
Service in 1914, revised several times since, and now are 
undergoing thorough revision with several important 
additions as shown in table 4. More than two dozen 
chemical items are now included; only three not re- 
vised—Cr, phenol, selenium. Important additions are 
detergents, chlorinated hydrocarbons, insecticides and 
organic thiophosphate insecticides. The substances in- 


Volume 19 


cluded in the standards are those found from data- 
gathering to be the dominant pollutants either with po- 
tential effects on health or from the standpoint of 
esthetics, ie. taste, odor, color. The drinking water 
standards have proved valuable methods of control in 
limiting excesses of undesirable elements in our drinking 
water in the past, being adopted by state and local 
agencies, and should prove valuable controls in the 
future. 

The Public Health Service Committee for the Re- 
vision of Drinking Water Standards is anxiously await- 
ing the decision of the Federal Radiation Council ap- 
pointed last year by the President, which will report to 
the President, we hope shortly, on suitable levels of 
radioactivity for exposure of the population for adapta- 
tion to the drinking water standards. 

One further means of insuring safe drinking-water 
supplies is a recently constituted committee on Water- 
Coagulant Aids of the Public Health Service. Possibly 
unimpressive by title, this committee performs the im- 
portant watchdog task of seeing that coagulants used in 
treating water for drinking purposes is not harmfully 
polluted in the process. Removal of impurities in water 
is still a common means of water purification. One im- 
pressive example of how extensively water coagulant 
aids may be involved in water supplies is their recently 
suggested use to prevent silt accumulation in vast water 
impoundments on some of our waterways. Use of coagu- 
lant aids for such a purpose on such a scale requires the 
scrutiny and vigilance given to food additives. 

In conclusion I think we can safely say without fear of 
over-statement, that although our control of pollution of 
our air and water is far from complete, the problems 
have been recognized, effective measures for their resolu- 
tion have been taken on broad fronts and respectable 
accomplishments have been achieved. 





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XUM 


Chemical approaches to radiation protection 


in mammals’ 


HARVEY M. PATT 


Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 


Dinar TOXICITY, OWing to its broad political and 
social implications and viewed in perspective, adds a new 
dimension to our thinking about man and his environ- 
ment. Realistic evaluation of radiation hazards requires 
a formidable background of quantitative knowledge on 
an unprecedented interdisciplinary scale. In seeking 
such understanding, much attention has been given to 
the elementary mechanisms of radiation action, to the 
manner in which the radiant energy may be transferred 
to biological systems, and to the events which may inter- 
vene between such transfer and one or another biological 
response. Although we have come a long way in some 
respects, the basic phenomena and, in particular, the 
details of the primary physical and chemical processes, 
are only cursorily understood at best. Some years ago 
radiobiology emerged from the apparent confinement of 
direct action in the sense of classical target theory and 
embraced somewhat broader concepts which were more 
obviously concerned with chemical intermediation. This 
was, in retrospect, a sort of renaissance in which water 
was recognized as an important vehicle of energy transfer 
and, in consequence, as an important mediator in the 
development of many radiobiological effects. Although 
it now appears that the dichotemy between direct and 
indirect actions was somewhat illusory, concepts based 
on the radiation chemistry of water profoundly influ- 
enced the trend in radiobiological thought and formed 
the basis for the first favorable approaches to what may 
be called chemical protection. It is about these matters 
that I have been asked to comment with particular 
reference to mammalian systems. 

Definitions are, after all, arbitrary things, and it is 
necessary to consider what is meant by protection. The 
effects of exposure to high energy radiations are expressed 
in many ways in many systems on a virtually boundless 
time scale, from seconds to years to generations. For the 
most part, and largely because of convenience, protec- 
tion is usually evaluated in relation to acute lethality 
whether of cell or organism. As we shall see later, this 
may have little relevance to other manifestations of 
injury, for example, reproductive or hereditary anoma- 
lies. Acute lethality of mammals is itself a composite of 

‘Work performed under the auspices of the U. S. Atomic 
Energy Commission. 


549 


a number of syndromes, each with characteristic pa- 
rameters dependent on species and conditions of irradia- 
tion. Hence, modification of the hematopoietic syndrome, 
which is intimately associated with acute mortality in 
many species, does not necessarily imply protection 
against sequelae attributable to intestinal or neurological 
damage. Radiation intensity decreases essentially as 
the square of the distance from the radiation source 
and, in its most general and practical form, protection 
is a purely physical matter involving suitable shielding 
against the background of the inverse square law. A 
special instance of this, which is of considerable theoreti- 
cal as well as practical interest, is shielding of specific 
parts of an organism. Thus, the reduction of acute 
radiation lethality by physical protection of a portion 
of hematopoietic tissue has led to the successful applica- 
tion of marrow transplantation subsequent to irradia- 
tion. This might be thought of as a form of biological 
protection, although, in the strict sense, the marrow 
transplant is a restorative factor related to recovery of a 
specific physiologic system. There are other less dramatic 
examples of specific protective or restorative factors, 
e.g. foreign proteins, certain hormones and antibiotics. 
However, we shall not be concerned with this facet of 
the subject nor with the modification of radioelement 
toxicity, where attention is directed primarily to reloca- 
tion and removal of radio-elements incorporated in the 
body rather than to radiation effects as such. Our interest 
will be confined to the more or less general reduction 
in damage brought about by administration of chemical 
agents before exposure to certain ionizing radiations. 
Phenomena of this sort are usually interpreted in physico- 
chemical rather than in physiological terms. 

The subject of chemical protection has had a com- 
paratively brief but active history, the first definitive 
in vivo studies being reported by us some dozen years ago 
(26). The approach represented a logical extension of 
basic investigations by Fricke, Weiss, Dale and Barron 
among others on the radiochemical decomposition of 
water in simple chemical and biological systems (22, 27); 
if transfer of energy from water to other cellular compo- 
nents were a critical step in the initiation of radiation 
damage in vivo, it might be possible to demonstrate and 
to modify the sequence of events by appropriate chemical 








55° 


means. The main energy transfers (ionizations, excita- 
tions, free radical formation) occur undoubtedly within 
a small fraction of a second after passage of an ionizing 
particle. It was expected, therefore, that to be effective, 
agents which might alter mechanisms of energy dissipa- 
tion must be brought into play at the time of irradiation. 
In view of the key role of oxygen in enhancing many 
activated water reactions and biological radiosensitivity 
(35), attention was focused rather more on oxidants 
than on reductants. Several redox systems (dyestuffs and 
sulfhydryl compounds) were employed in an attempt to 
assess the relevance of such considerations to the in vivo 
situation. Attempts to demonstrate augmented oxida- 
tions were uniformly unsuccessful; we found, for example, 
that x-irradiation had no immediate effect on sulfhydryl 
levels in a number of tissues. Upon reflection, this was 
not unreasonable since the available sulfhydryl in tissue 
exceeds by several orders of magnitude the amount that 
could be oxidized directly by lethal doses of radiation. 
Significantly, however, it was noted that pre- but not 
postirradiation injection of large amounts of sulfhydryl 
as cysteine or glutathione greatly diminished acute radia- 
tion lethality; cystine and methionine were ineffectual. 
Although interest in the modification of radiation effects 
in complex organisms is by no means new (29), this sort 
of endeavor has become a focal point in recent years as 
increasing emphasis has been placed on the chemical 
basis of radiobiological actions. 

Certain of the facts of chemical protection have been 
reasonably well established in studies with small rodents; 
however, the mechanisms behind the facts are obscure 
or at best only circumstantial. The list of chemicals that 
manifest some protection against one or another effect 
when given prior to gamma- or x-irradiation is impres- 
sive (3, 22, 27). It includes thiols and disulfides related 
to cysteine and cysteamine ($-mercaptoethylamine), 
pharmacological agents such as cyanide, p-aminopro- 
piophenone, histamine and  5-hydroxytryptamine 
(serotonin), and various metabolites. These substances 
are not equally effective and greatest interest has 
centered on the cysteine-cysteamine group. Isothio- 
uronium derivatives which undergo rearrangement to 
mercaptoalkylguanidines, for example, S-2-aminoethyl- 
isothiouronium: Br- HBr (AET) are notable members of 
this class of protective substances (9, 32). The protection 
afforded by the cysteine-cysteamine group is a fairly 
general phenomenon which, in many of its aspects, re- 
sembles a radiation dose reduction. Protection is not 
confined to normal tissue; radio-resistance of tumors 
may also be enhanced (24). Most studies have been con- 
cerned with acute manifestations of x-radiation injury. 
There is scant quantitative information regarding the 
more chronic sequelae, although, a priori, one might 
suppose that some effectiveness would also be manifest 
here. Protection has been reported for some delayed 
effects, but the evidence is incomplete, particularly in 
regard to fertility, neoplasia, life span and certain genetic 
effects (21, 27). There is an increasing recognition of the 
fact that the initial damage in many tissues may be 


FEDERATION PROCEEDINGS 


Volume 19 


modified under appropriate conditions. This may not be 
apparent upon cursory examination and, as emphasized 
earlier (23, 25), requires careful evaluation of the dose- 
response patterns for the various biological end points. 
Generality of protection is predicated upon an effective 
distribution of the chemical within the organism and the 
singularity of the initiating mechanism. The failure to 
detect protection can be attributed in some instances to 
the selective localization of the various chemical agents 
(12, 27); however, our understanding in this area is still 
rather fragmentary. Under any circumstances, a concept 
of dose reduction does not necessarily imply protection 
against all radiation changes in so far as different pri- 
mary mechanisms may be involved in their development. 

The degree of protection with compounds such as 
cysteine and cysteamine may be fairly independent of 
x-radiation dose for a given effect. This has been shown 
for a single mode of acute lethality in mice by means of 
two closely spaced but variable exposures with cysteine 
given between (25). The dose reduction factor (1.7) is 
uniform whether cysteine is preceded or succeeded by 
100-700 r, the total radiation dose remaining the same 
in all instances. In contrast, there may be a shifting dose- 
reduction factor when the total x-radiation dose is 
varied over a sufficiently broad range to embrace dif- 
ferent acute mortality modes (7); this appears to be the 
case also with certain partial body exposures (33) and 
may be related, in part at least, to uneven distribution 
of protective compound in target tissues. Under optimal 
conditions, the dose reduction for gamma- or x-irradia- 
tion (30-day mortality, spleen atrophy, lymphopenia, 
neutropenia) may correspond to a nearly 50% decrease 
in the efficiency of the radiation. This is comparable to 
the protection afforded by simple hypoxia in the intact 
animal (10). Although only a few studies have been 
made, it is noteworthy that, as in the case of hypoxia, 
protection by some of these chemicals also appears to be 
an inverse function of linear energy transfer. Thus, 
agents such as cysteine, AET, and 5-hydroxytryptamine 
confer considerably less protection against fast neutrons 
than against gamma- and x-rays (23; personal communi- 
cation, H. H. Vogel, Jr.). In addition to type of radia- 
tion, the manner of irradiation (protraction or fractiona- 
tion of the dose) even with x-rays may be of some conse- 
quence for the degree of protection (18, 28). This is an 
important theoretical and practical point which requires 
detailed study. 

The optimal protection attainable in relation to 
toxicity of the protective agent is a crucial issue. Despite 
the claims from time to time about relative prophylactic 
indices and factors of safety, optimal protective effects 
almost invariably require dosages uncomforatably near 
the acutely lethal level regardless of compound or route 
of administration. It may be noted, for example, that 
cysteamine is more efficient than cysteine on a molar 
basis in mice, but that it is also more toxic; maximally 
tolerated doses of each give equivalent protection 
against acute radiation lethality, whereas suboptimal 
amounts seem to be completely additive. A similar situa- 





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July 1960 


tion may not apply to other protective agents, and the 
extent to which protective additivity may parallel toxic 
additivity for different combinations of chemicals re- 
quires further evaluation. Most protection studies have 
been performed in mice, mainly as a matter of con- 
venience, and extrapolation to larger species is tenuous. 
Although salutary effects with AET have been reported 
in a few monkeys (8), this has not been observed in dogs 
(5); AET toxicity appears to be severalfold greater in 
dog than in mouse, which precludes administration of 
comparable dosage for evaluation of protective efficiency. 
A rather similar situation obtains for cysteamine, which 
has profound toxicological effects in the dog (personal 
communication, D. Jacobus). Other chemical agents 
with some degree of protective activity in mice or rats 
such as the biological amines are effective only in doses 
associated with pronounced pharmacological activity 
(36); they have apparently not been studied extensively 
in other species. These remarks, in general, apply to 
single doses of the various protective compounds; re- 
peated administration, which would be necessary, for 
example, with fractionated irradiation, may pose even 
greater problems of toxicity. 

Many protection studies began in an attempt to eluci- 
date mechanisms, not of protective agents, but of radia- 
tion actions. The two are, of course, closely interwoven. 
There is the firm requirement that the protective agents 
must be given to the animal before irradiation to be 
effective; some effects of administration after irradiation 
have been reported on occasion (17, 20) but these have 
not been confirmed (31, 32). The compounds must ordi- 
narily be given by injection within minutes before the 
exposure, although there are a few exceptions (19). Some 
protection against acute lethality has been noted in mice 
after oral administration of cystamine or AET several 
hours before irradiation; however, the results are incon- 
sistent (27). The temporal requirement and the uni- 
formity of protection for a number of sequelae suggest 
that the decisive action occurs at an early stage in the 
chain of events, and there is rather general agreement 
that this is related to modes of energy transfer. Several 
radioprotection mechanisms have been proposed: a) in- 
activation of radicals derived from water, }) relative de- 
pletion of oxygen within cells, c) alteration in reactivity 
of target molecules. These possibilities need not be 
mutually exclusive and it is conceivable that other 
mechanisms, e.g. chelation, may also be operative for 
certain protective agents (16). 

The tendency to interpret protection as a simple 
competition with free radicals derived from irradiated 
water has been decreasing in recent years. Although an 
apparent association between protective efficiency and 
chemical structure in model systems and in vivo has been 
adduced as evidence in support of this sort of mechanism 
(1, 9, 30), there are cogent arguments against such a 
facile explanation for the selectivity of chemical protec- 
tors (27). It has been suggested, for example, from 
comparative studies of acute radiation lethality, that 
cysteamine derivatives may protect by reacting directly 


XUM 


RADIATION PROTECTION IN MAMMALS 551 


with radiation-induced radicals to form resonance- 
stabilized entities which react preferentially with other 
radicals (9, 30). Generalizations about radiation events 
at the molecular level from gross observations of this sort 
may, of course, be complicated by uncertainties con- 
cerning the biological fate of the various substances. 
One would like to know whether differences in efficiency 
of protection as a function of chemical structure reflect 
differences in capacity for radical capture or in distribu- 
tion and metabolism of the various agents. Moreover, 
some of these agents have been shown to protect non- 
aqueous systems (2, 6), and others have strong pharma- 
cological properties which may be relevant to the 
modifying action (36). 

There are a number of parallels between chemical 
protection and the oxygen effect (decreased radiosensi- 
tivity with irradiation under hypoxic conditions), and in 
many instances similar mec’anisms probably prevail. 
This is almost certainly the case with the methemoglobin- 
producing agent, p-aminopropiophenone. Hypoxia may, 
likewise, account for the protective effect of cyanide, 
morphine, and ethyl alcohol against acute radiation 
lethality in mice. The biological amines probably also 
owe their effectiveness to changes in tissue oxygen 
brought about by alterations in blood supply; this re- 
ceives strong support in recent pharmacological studies 
by van der Meer and van Bekkum (36). 

That some thiols, e.g. cysteine, may also exert a major 
part of their protective action zn vive by reducing cellular 
oxygen tension is suggested by several lines of evidence. 
Thus, we may note the similarity of degree of protection 
and of dependency on radiation quality for cysteine- 
cysteamine and hypoxia; the protective and toxic addi- 
tivity of cysteine and hypoxia; the potentiation of 
cysteine protection by agents producing minimal hypoxia 
and inhibition of protection by hyperoxia (23). There is 
little information of this sort about other protective 
thiols. It will be recalled, however, that the isothio- 
uronium, AET, apparently undergoes a rapid conver- 
sion to a cysteamine derivative (27); there may also be 
an in vivo requirement for cystamine action and protec- 
tion may be a result of its conversion in the body to 
cysteamine. It is of interest that rates of thiol auto-oxida- 
tion and protective activity are poorly correlated and 
that the highest rates are, in fact, shown by nonprotective 
thiols (15). This, however, may be understandable, if the 
latter are oxidized much more rapidly in transit to 
various cellular sites. Indeed, this could be a factor in 
the lower protective efficiency and toxicity of cysteine 
relative to cysteamine (32). On the other hand, it is un- 
likely that spontaneous oxidation of any of these com- 
pounds in the usual dosage could induce a severe over-all 
hypoxia in the intact animal. If hypoxia is a critical 
determinant of their action, it seems necessary to postu- 
late highly localized cellular areas of oxygen depletior 
brought about either directly or through other means. 
It must be admitted that the evidence for a hypoxic 
mechanism of cysteine and cysteamine protection is cir- 
cumstantial and that final judgment must await the 








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MOLECULAR 
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ALTERED 
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FIG. 1. Possible mechanisms of chemical intervention at various 
loci of energy transfer. A. Hypoxia. B. Mediator inactivation (e.g. 
radical or electron scavenger). C. Target alteration (e.g. mixed 
disulfide formation). 


development of satisfactory methods of measuring cellu- 
lar oxygen tensions zn vivo. 


The sensitizing action of oxygen has been thought of 


in terms of interactions between oxygen molecules and 
electrons, ionized and excited molecules, and free radi- 
cals that are formed along the tracks of ionizing particles 
(14). Interaction of oxygen with free radicals may lead 
to the formation of more toxic agents; oxygen interaction 
with ionized or excited molecules may also provoke their 
further breakdown. A corollary to the latter is the possi- 
bility that protective agents might combine with essential 
target molecules and thereby enhance their radioresist- 
ance. Detailed biochemical studies of cysteamine and 
cystamine by Eldjarn and Pihl (11, 27) have led to their 
formulation of such a concept of protection by mixed 
disulfide formation. This mechanism is predicated on 
rapid reaction of cysteine-cysteamine type compounds 
with S—-S and —-SH groups of tissue constituents. The 
formation of reversible mixed disulfides is believed to 
confer resistance against both direct and indirect actions 
of radiations, on the one hand by serving as the ultimate 
source of electrons repairing the original ionization, and 


FEDERATION PROCEEDINGS 


Volume 19 


on the other by partially shielding protein —SH groups 
from free radicals. The possibility of such protection 
against direct action is consistent with paramagnetic 
resonance studies which reveal that electrons from S—§ 
bonds can migrate to ionized sites in a peptide chain 
(13). Protection of —-SH enzymes in the dry state by 
cysteine can also be interpreted on the basis of mixed 
disulfides (6). Although protective thiols and disulfides 
can be bound to proteins in the manner described, the 
tissue and cellular distribution of such mixed disulfides 
and the postulated reaction products have not yet been 
demonstrated in the irradiated animal. Moreover, it is 
particularly noteworthy that there is no direct evidence 
that —-SH groups and S—S bonds represent the sensi- 
tive spots from the standpoint of radiotoxicity (34). 
Apropos of this, a mechanism of protection by mixed 
disulfide formation must account for the fact that avail- 
able sulfhydryl groups are associated mainly with cyto- 
plasmic constituents (4) whereas the nucleus appears to 
be considerably more radiosensitive than the cytoplasm 
(37). It is necessary also to interpret in these terms the 
dependency of protective capacity on radiation quality. 
Notwithstanding these reservations, the mixed-disulfide 
hypothesis has offered a refreshing approach to possible 
identification of target molecules and development of 
modifying agents. 

Some of the possible mechanisms of chemical interven- 
tion at various loci of energy transfer are diagrammed in 
figure 1. While it is almost axiomatic to search for an 
all-embracing mechanism, it is likely that no single 
mechanism will account for the in vivo action of all of the 
known protective chemicals. The surprising thing, how- 
ever, is that some sort of biological saturation appears to 
be a requirement for protection in all instances, and that 
the upper limit of protection seems independent of 
chemical agent and of postulated mechanism. In this 
sense, at least, we may think in terms of a common path- 
way. The possibility of a twofold enhancement of resist- 
ance to one or another radiation effect is not to be taken 
lightly even though the chemical means for achieving 
this are not practicable at present. When we reflect, 
however, that sensitivity to lethal action among biological 
objects differs by a factor of 10*-104, it is clear that we 
have a long way to go. 


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lume 19 


yrOUps 
ection 
enetic 
1 S—S 
chain 
ate by 
mixed 
ulfides 
d, the 
ulfides 
t been 
r, it is 
idence 
sensi- 
(34). 
mixed 
avail- 
1 cyto- 
2ars to 
yplasm 
ns the 
uality. 
sulfide 
ossible 
ent of 


erven- 
ned in 
for an 
single 
of the 
, how- 
ears to 
id that 
ent of 
in. this 
1 path- 
“resist- 
- taken 
lieving 
reflect, 
logical 
hat we 


tadiology 
ps-Akad. 


t. Conf. 


(Great 


ances i 
AND J. 





XUM 


July 1960 


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CONTROL OF BIOLOGICAL FERTILITY 553 


27. Pint, A. AND L. ELpyarn. Pharmacol. Rev. 10: 437, 1958. 

28. Rucu, R. anp H. Crucston. Radiation Res. 1: 437, 1954. 

29. ScHWARZ, G. Muiinchen med. Wehnschr. 56: 1217, 1909. 

30. SHapira, R., D. G. DoHERTY AND W. T. Burnett, JR. Radia- 
tion Res. 7: 22, 1957. 

31. Smiru, D. E. Radiation Res. 12: 79, 1960. 

32. STRAUBE, R. L. ann H. M. Parr. Proc. Soc. Exper. Biol. @ Med. 
84: 702, 1953. 

33. Swirt, M. N., S. T. Taxeta ano V. P. Bonn. Fed. Proc. 11: 
158, 1952. 

34. D. Te1cer. J. Histochem. 7: 267, 1959. 

35. THopay, J. M. anp J. Reap. Nature, London 160: 608, 1947. 

36. VAN DER MEER, C. anp D. W. van Bexkuo. Internat. J. Radia- 
tion Biol. 1: 6, 1959. 

37. ZIRKLE, R. E. anp W. Bioom. Science 117: 487, 1953. 


Control of biological fertility 


NATHAN MILLMAN 


Ortho Research Foundation, Raritan, New Jersey 


i CONTROL OF FERTILITY is one of nature’s busiest 
preoccupations. It is instructive for man to consider his 
own needs and efforts within the broad frame of this 
activity of the natural world about him. Malthus him- 
self, in 1799, referred to this broader view, noting that 
the control of fertility in plants and ‘irrational animals’ 
was a positive, albeit involuntary, process (16). It was 
his thesis that ‘preventive’ or ‘voluntary’ control is 
peculiar to man, and arises from that distinctive superi- 
ority in his reasoning faculties which enables him to 
calculate distant consequences of his actions. This 
optimistic evaluation of mankind will, I trust, be justified 
ultimately, and before certain drastic consequences of 
his affairs today become inevitable. 

There is value in maintaining for this discussion 
Malthus’ organization of positive-involuntary versus 
preventive-voluntary fertility control in plants, animals 
and man. On close examination, it is evident that the 
involuntary systems of fertility control extend to all 
species in nature, particularly if one includes all the 
daily phenomena of sustenance, environment, neighbors 
and so on. In a sense, the fertility control of the Four 
Horsemen extends throughout nature as an involuntary 
process—-war, famine, pestilence and death stalk the 
biological world, not man alone. The constant warfare 
of nature predates man. 

Consider, if you will for reasons of comparison, the 
army ant, and a diagram (fig. 1) of his‘ natural’ fate, as 
outlined by Schneirla (35). Note the role of natural en- 
vironment, directly related to reproduction and other 


processes. Study the lines of fertility control, to both 
augment and depress numbers in relation to the war-like 
activities of raids, emigrations, bivouacs and booty. The 
population control line at right center links cyclical 
activity, population increments, colony division, control 
of population, magnitude, behavior, etc. to all facets of 
the animal’s life. This is fertility control, positive and 
inexorable, in nature’s absolute sense, and it exists, 
perhaps more or less clearly in some cases, in all plants 
and animals—and, of course, in man. 

These ‘ positive-involuntary’ fertility checks in plants 
and animals are of such number as almost to defy com- 
pilation, and of such ingenuity that it is difficult not to 
speak of them in teleological and anthropomorphic 
terms. Such processes in the plant world and in the 
penumbrate plant-animal border area may range in 
cyclical length from days to years and even over centu- 
ries. From ‘simple’ controls governing their fertility 
(such as climatic and soil-nutritive changes, which are 
so familiar as not to need to be detailed here), they often 
range to complex inter-species controls with swifter and 
more elegant methods. The ‘strangler’ tree (Ficus sp.) of 
the Brazilian tropics seeds itself and grows as a vine over 
a host tree, strangles it, to finally stand on its own as a 
tree, and then reseeds (7). This is ecological competition 
of a sort, but also a dynamic species population control 
device. More ‘intentive’, perhaps, in limiting its own 
fertility and ultimate population to a certain number of 
trees per acre, is the activity of the guayule Parthentum 
argentatum; this plant elaborates through its roots an in- 








FEDERATION PROCEEDINGS 





Volume 19 





| REPRODUCTION | 





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FIG. 1. Fertility control patterns in the army ant. From Schneirla (35). 


hibitor (cinnamic acid), toxic to its own seedlings, but 
much less so to that of other species. James Bonner, who 
is fond of calling such substances ‘ antisocial drugs’, avers 
that plant interaction is sometimes beneficial to popula- 
tion and concludes that ‘thus chemical warfare and 
cooperation are merely one phase of the largér complex 
of interactions which determine the sociology of the plant 
community’ (1). 

Example upon example, then, we may cite interference 
with population extent by plant species upon itself, by 
species upon another. The modes may be environmental 
and adaptive as with the self-pruning willow that drops 
its self-rooting twigs in the muck of a swamp crowding 
out other species. It may be as specific as that of the 
Encelia brittlebush (2), which elaborates 3-acetyl-6- 
methoxybenzaldehyde, nontoxic to itself, sunflowers or 
barley, but severely toxic to tomatoes and other species 
which might crowd it out of its natural habitat. 

Familiar to us are similar systems in the microbiologi- 
cal world. Examples of self-limiting processes delineating 
the extent of intraspecies growth and of ‘antisocial’ 
agents operating between species and even between 


classes—i.e., antibiotics—are rife throughout nature. 


In Malthus’ ‘irrational’ animals, it is often more difhi- 
cult to distinguish between ‘ voluntary’ and ‘involuntary’ 
fertility-limiting processes. From the quietly desperate 
internecine war in cultures of bacteria, to the high or- 
ganizational processes of the army ant (35) which we 
have noted, through to the reported ability of the honey- 
bee (39) to control its own fertilization process and rate, 
the examples to be cited are myriad. The evolutionary 
lines by which they have developed have been examined 
in general and in detail (20). Examples of these develop- 
ment lines are the evolutionary reduction in reproduc- 
tive rate (42); the development of increased physical 
protection schemes to compensate for decreased egg 
number (13); and in biochemical changes affecting 
fertility regulation (36). Part of these systems are indi- 
vidual variations and devices, ranging from ‘simple’ 
protective coloration to intricate physical, physiological 
and biochemical mechanisms. In the latter case, Segal 
(36) calls for further study of such mechanisms, in order 
that man may be able to ‘invoke this direct and basic 
method so successful in nature’: The regulation of 
fertility as a survival method. 

We approach then, finally, the problem of man’s 








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direct voluntary control of his own fertility. From time 
immemorial, despite their very presence today, man has 
used desperate physiological and physical methods to 
curb his individual fertility and the population of his 
group. Some of these, such as infanticide, killing the 
aged, and abortion, still exist as tradition and supersti- 
tion among aboriginal tribes, their original reasons being 
largely lost: as an example, Devereux (6) cites the fre- 
quency of abortion in primitive societies. In some 
Polynesian centers, all pregnancies are terminated until 
the women are 34-37 years of age; in Mataco, all first 
conceptions are aborted, and so on. A partial list of the 
methods used to induce this end reads as follows: starva- 
tion, bleeding, mechanical instruments, foreign bodies, 
irritants, drugs and heat. Many of these same methods 
are used clandestinely in more civilized areas today, 
with as much danger, while legalized termination of 
pregnancy in this manner is permitted or under con- 
sideration in only a few countries (46). 

Today there are some attempts to use the knowledge 
of physiology of reproduction more humanely, as indi- 
cated by the calculation of fertile periods, or by absti- 
nence. Were these wholly satisfactory, however, there 
would be little impulse to continue research towards 
more positive methods. The pharmacological efforts to 
block sperm migration date back to early Egyptian 
dynasties as far as we know, and probably further (14). 
Present-day preparations for interfering with sperm 
transport employ agents falling within the classification 
of organic acids, organo-metallic compounds or surface 
active agents (17, 21). Actually, spermatozoa may be 
immobilized by a variety of means, including, with the 
above, anisotonic solutions, metabolic inhibitors, enzyme 
poisons, and so on. However, some of these take finite 
time to act and others are tissue irritants, and thus they 
do not find practical utilization today. Actual physical 
blockade by various occlusive devices is a large factor in 
human fertility control, as well. 

These methods are available to much of the world 
today and are in widespread use in some countries. They 
do not, however, have wide distribution in those over- 
populated countries where they are so desperately 
needed. The reasons for this situation are complex and 
involve expense, lack of education in methods, and 
government and religious attitude toward use, import 
or production (41). 

Efforts to control conception by systemic administra- 
tion of drugs have for the past 10 years received much 
publicity in both the scientific and general press. Several 
reviews delineate the approaches in this field (22, 24), 
and most classifications of such agents are based upon 
their physiological characters rather than upon chemical 
structure. The following brief discussion is a general pic- 
ture and is by no means all-inclusive. Antimetabolites 
such as the folic acid analogue aminopterin, desoxypyri- 
doxine, 6-mercaptopurine, o-diacetyl-l-serine, norleu- 
cine analogs and nitrogen mustard derivatives, have been 
examined and found wanting by safety criteria, although 
they do prevent littering in experimental animals (9, 24). 


r) 
ese tala 


CONTROL OF BIOLOGICAL FERTILITY 555 


The so-called antienzyme group includes compounds 
influencing hyaluronidase activity, thus supposedly 
interfering with fertilization (18). Several laboratories 
have found phosphorylated hesperidin to be unsuccessful 
in controlling fertility in animals (23, 44), in spite of an 
extensive clinical report on its efficacy (38). Similar 
compounds such as arbutin, esculin, rehibin and rutin 
were ineffective in animal studies (22, 27). 

Many compounds may be demonstrated to have an 
antispermatogenic effect in animals and man, notable 
among them a furane derivative (25). Drugs of this type 
are successful, but to date have required long induction 
dosage programs and have been accompanied by some 
side effects. 

Antihistaminics have been considered to influence 
implantation in animals (37), if given at the right time 
of the cycle, and might thus be capable of altering the 
course of pregnancy. In our hands, by standard methods 
of assay, oral control of fertility has not been achieved 
in rats with diphenhydramine at 15 mg/kg/day, or 
pyranisamine at 5 mg/kg/day (22). 

Various avenues for oral control of fertility are offered 
by agents altering the hormonal balance of the organism. 
Activity may be achieved through influencing spermato- 
genesis or oogenesis, by preventing ovulation or im- 
plantation, by altering the ovum during transport, by 
fairly specific interference with gonadotrophs, or by 
direct antipituitary activity. Within the realm of such 
agents are nonsteroid structures such as 2,6-dimethyl- 
hydroquinone, vaunted by some as giving positive con- 
trol in human trials in India (11, 33); it has been the 
subject of several laboratory studies in this country 
(22, 26, 32). This compound, supposedly the active 
agent of Pisum sativum, is again undergoing extensive 
clinical trials under the eye of the government of India, 
emphasizing their concern about the original studies. 

Another botanical source, but yielding positive con- 
trol of fertility in laboratory animals, is Lithospermum sp. 
(5, 31). This may inhibit the action of gonadotrophic 
hormones, but isolation and synthesis of the active factor 
has not been achieved. 

Natural steroid hormones and their‘ unnatural’ deriva- 
tives or artificial mimics successfully achieve fertility 
control in animals and man, as we have known for more 
than twenty years, even before such reports on the oral 
use of diethylstilbestrol and ethynyl-estradiol in 1938 
(28). The advent of potent progestational compounds 
capable of similar results when given orally has given 
impetus to this approach. 17a-methyl-, 17a-ethyl-and 
17a-ethynyl-1g-nortestosterones and 17a-ethynyl-5, 10- 
estraenolone have been described in laboratory studies 
(30, 34). The last compound has been reported to be 
effective in long clinical trials done outside continental 
United States (10); effectivity with proper usage cannot 
be challenged, although the degree of side reactions is 
still open to some question. Undergoing considerable 
study today is 17a-ethynyl-19-nortestosterane. The sym- 
posium on Fertility-Controlling Steroids (15), presented 
before the Federation last year, covered many aspects of 








550 


the properties and usage of these agents and further 
detail will serve little purpose here. 

What is important, however, is that the means of con- 
trolling human fertility are available today, whether by 
somewhat unsatisfactory physiological omissions, such as 
through abstinence and calculation of fertility periods; 
or by occlusive devices, or by spermicidal preparations. 
And if the oral route with synthetic steroids is not yet 
entirely ideal, it represents a beginning for systemic 
administration. 


It is important to the understanding of the scope of 


research in this field to realize that many laboratories 
are screening compounds for reproductive interference, 
and that a good portion of these achieve fertility control 


in animals. However, other physiological properties of 


these compounds may militate against their use. Only 
those few which satisfy the criteria of effectivity and 
safety (22) are considered for further work, and very few 
reach preliminary human trial. 

Whatever the methods used or finally achieved, man 
must not view in an isolated sense his striving to control 
his own fertility. Indeed, the activities of man in con- 
trolling plant and animal abundance has fundamental 
effects on his own environment and ultimately reciprocal 
complex harmonic overtones on his own fertility and 
population, as well as on theirs. 

Certain aspects of this—weed, pest and microbiologi- 
cal control—are being discussed by others in detail. By 
accident and intent, man affects the fertility of other 
species, but usually and unfortunately, he does not do 
these things with an over-all plan. It is only recently that 
we hear the phrases ‘The Thermodynamics of World 
Society’ (4) or ‘The World Organism’ (12). Whenever 
man sprays with DDT or ‘seeds’ an organism noxious to 
rabbits in Australia, or releases irradiated insects, or 
decreases the potential fertility of his soil, or (for that 
matter) alters his own fertility, the consequences are far- 
reaching. 

The fertility, and hence population, controls in nature 
of which examples have been given here, are achieved 


REFERE 


1. Bonner, J. In: Plant Life. New York: Simon and Schuster, 
1957- 
2. Bonner, J. AND R. Gray. Am. J. Botany 35: 52, 1948. 
3. Brown, H., J. Bonner ano J. Weir. The Next Hundred Years. 
New York: Viking, 1957. 
. Busu, I. Family Planning (London) 7(2): 8, 1958. 
. Cranston, E. M. J. Pharmacol. & Exper. Therap. 83: 130, 1945. 
6. Devereux, G. A Study of Abortion in Primitive Societies. New 
York: Julian Press, 1955. 
. DospzHansky, T. AND J. Murca-Pines. In: Plant Life. 
York: Simon and Schuster, 1957. 
8. Facary, R. M. The Population Explosion and Christian Re- 
sponsibility. New York: Oxford Univ. Press, 1960. 
g. Friepman, M. H. J. Am. Vet. Med. Assoc. 130: 159, 1957: 
10. Garcia, C. R., G. Pincus ano J. Rock. Am. J. Obst. & Gynec. 
75: 82, 1958. 
11. Guosu, S. AND A. Gupta. Int. med. Abstr. 16: 89, 1954. 
12. Greco, A. Population Bulletin 11: 74, 1955. 
13. Hitpemann, W. H. Am. Naturalist 93: 27, 


wo > 


New 


~I 


1959. 


FEDERATION PROCEEDINGS 


Volume 19 


by either a direct effect upon the gametes or some repro- 
ductive process, or by indirect alterations of the total 
environment, or by both. The end results may be the 
same, and the preventive processes in plants, animals 
and man have similarities which further study could 
amplify. Having learned that our activities alter nature’s 
fertility balance, and that hers can affect equally our 
own, we might agree with Toynbee that ‘ Human beings 
are no longer willing to be expendable items in nature’s 
extravagant balance sheet’ (47). 

With the ability to limit his fertility by direct pharma- 
cological methods available to him today, and the op- 
portunity to develop improved methods tomorrow, man 
finds himself in a situation nobly described by several 
authors (8, 29). His future under varying conditions of 
fertility control, food supply and other elements of his 
natural and man-made environment has been forecast 
(3, 19). Of these, increased food supply is only one ele- 
ment. The Stanford Research Institute Report to the 
Committee on Foreign Relations of the United States 
Senate (40) depicts the long-term implications of the 
dangerously increasing world population and suggests 
that a first line of attack includes improved communica- 
tions with other countries, control of human and animal 
diseases, changes in types of crops, reclamation of sewage, 
conversion of saline to fresh water, farming the ocean, 
control of plant disease and pests and attainment of new 
sources of natural and synthetic foods. 

It would be my hope that realizing, as we have seen 
here, 1) that population control is a law of nature, and, 
2) that what man does not do for himself, nature will do 
for him, we would operate within even a larger scope. 
Our efforts to increase chances of survival after birth 
and to augment generally the health and welfare of 
mankind must not diminish. But part of the overall pro- 
cedure must include ultimately a plan involving the 
knowledge of reciprocal effects of our activities and those 
going on about us in nature. Finally, then, we must use 
this analysis in achieving the optimum preservation of 
nature’s balance. 


NCES 


14. Himes, N. E. Medical History of Contraception. Baltimore: Wil 
liams & Wilkins, 1936. 

15. Hoacrianp, H. et al. Fed. Proc. 18: 1039, 1959. 

16. Martuus, T. R. An Essay on Population. London: J. M. Dent 
and Sons, 1952. 

17. Mann, T. In: Studies on Fertility, edited by R. G. Harrison. 
Oxford: Blackwell, 1958. 

18. Martin, G. J. Scvence 117: 363, 1953. 

19. Meter, R. L. Modern Science and the Human Fertility Problem. 
New York: Wiley, 1959. 

20. MEISENHEIMER, J. Geschlecht und Geshlechter im Tierreiche, vol. | 
and 2. Jena, Germany: Fischer, 1921. 

21. Mirieman, N. Ann. New York Acad. Sc. 54: 806, 1952. 

22. Mitiman, N. ann C. G. Hartman. Fertil. & Steril. 7: 110, 1956: 

23. Mi_uman, N. anv F. Rosen. Science 118: 212, 1953. 

24. Netson, W. O. Proc. Fifth Internat. Conf. on Planned Parenthood. 
London: Internat. Planned Parenthood Fed., 1955. 

25. Netson, W. O. ann E. STEINBERGER. Anat. Record 112: 367, 
426, 1952. 





aie 656 die bak 


35 


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sta 
pre 
her 
of j 
inst 
Sec 
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este 





lume 19 


repro- 
- total 
ye the 
1imals 
could 
iture’s 
ly our 
beings 
ature’s 


arma- 
ne op- 
‘, man 
everal 
ions of 
of his 
recast 
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to the 
States 
of the 
1g gests 
unica- 
animal 
ewage, 
ocean, 
of new 


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e, and, 
will do 
scope. 
- birth 
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ng the 
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ust use 
tion of 


re: Wil 


M. Dent 


ARRISON, 


Problem. 


re, vol. 1 


10, 1956: 
arenthood. 


12; 367, 





July 1960 


26. Nose, R. L. anp R. C. B. Grauam. Canad. M. A. J. 69: 576, 
1953- 
27. ParKEs, A. S. Lancet 2: 1285, 1953. 
28. Parkes, A. S., E. C. Dopps ano R. L. Nos te. Brit. M. J. 
2: 557, 1938. 
29. Pippincton, R. A. The Limits of Mankind, Bristol: Wright, 
1956. 
go. Pincus, G., M. C. Cuanc, M. X. Zarrow, E. S. E. HAFEz 
AND A. MERRILL. Endocrinology 59: 695, 1956. 
31. PLUNKETT, E. R. and R. L. Nose. Endocrinology 49: 1, 1951. 
32. Rosen, F. anp N. Mivvman. Endocrinology 57: 466, 1955. 
33. SANYAL, S. N. Int. med. Abstr. 16: 91, 1954. 
34. SAUNDERS, F. J., R. F. EpGREN anp V. A. Driv. Endocrinology 
60: 804, 1957. 
5. SCHNEIRLA, T. C. Proc. Am. Phil. Soc. 101: 
36. SEGAL, S. J. Eugen. Quart. 6: 94, 1959. 


10 ? 1957- 


CONTROL OF INSECT’ PESTS 557 


37. SHELEsNYAK, M. C. Endocrinology 54: 396, 1954. 

38. Steve, B. F. Science 116: 373, 1952. 

39. SnopcRass, R. E. In: The Hive and The Honey Bee, edited by 
R. A. Grout. Boston: Chapman and Grimes, 1947. 

40. Stanford Research Institute. Population, Food and Science. 
Population Bulletin 15: 145, 1959. 

41. Stone, A. Ann. New York Acad. Sc. 54: 769, 1952. 

42. Stuart, A. J. Overpopulation. New York: Exposition Press, 
1958. 

43. Turerscu, J. B. Am. J. Obst. & Gynec. 63: 1298, 1952. 

44. THompson, R. Q., M. SturTEvANT AND O. D. Birp. Science 
118: 657, 1953. 

45. THompson, W. S. Ann. New York Acad. Sc. 54: 733, 1952. 

46. Tietze, C. Proc. Fifth Internat. Conf. on Planned Parenthood. 
London: Internat. Planned Parenthood Fed., 1955. 

47. ToynBEE, A. Family Planning (London) 8(4): 11, 1960. 


Control of insect pests 


T. BURNETT 


Entomology Research Institute for Biological Control, Research Branch, 
Canada Department of Agriculture, Belleville, Ontario 


BY wx ONE EXAMINES THE PLANTS and animals charac- 
teristic of any large terrestrial community, one is struck 
by the great diversity of natural habitats. Numerous 
plants are distributed in a complex mosaic pattern, each 
plant supports a number of different animal species, and 
each phytophagous animal supports up to four trophic 
levels of carnivorous species. It is not surprising, there- 
fore, that Wytham Wood, less than two square miles of 
English countryside, contains at least 2,500 species of 
animals (14). As many carnivores are polyphagous 
rather than monophagous, a community is organized into 
a gigantic food-web instead of into a series of separate 
food-chains. An indication of the complexity that results 
from the diversity of habitats and myriad interrelations 
of animals is given in the representation of a fairly simple 
community by Shelford (32, diagram 6). 

The complexity characteristic of many natural com- 
munities has two important consequences. First, although 
complex food-chains and food-webs may not be the only 
mechanisms that regulate population density (14), it 
seems to be established empirically that mixed stands of 
vegetation have more stable populations than pure 
stands; perhaps interactions other than parasitism and 
predation affect population size. In the mixed hardwood- 
hemlock forest of Michigan there has been no evidence 
of insect damage for 300 years and less than 5% of the 
insect species there are conspicuous at any time (17). 
Second, complex communities tend to ward off invasion 
by foreign species. How this is accomplished has not been 
established; perhaps because all niches in a complex 


? 
avi tha 


community are filled, an invader must partially or com- 
pletely displace an indigenous species. It seems likely, 
therefore, that complexity of natural habitats contributes 
to the stability, both qualitatively and quantitatively, of 
populations. 

Ecological succession, the cccurrence of chance 
catastrophes, and the systematic activities of man, have 
so greatly modified the diversity usually associated with 
natural communities that in many areas of the earth the 
pattern of vegetation has become relatively much more 
uniform. The tendency toward monocultures results in 
simpler communities—the simplicity is only relative 
(2)—in which the number of species is considerably less, 
the lengths of the food-chains are greatly shortened (14), 
and large amounts of food are readily available for ex- 
ploitation. Furthermore, the quality of the food, in addi- 
tion to the amount, influences the reproduction and 
survival of phytophagous species (31, 33), and agricul- 
tural practices tend to provide plants suitable for insect 
development. The consequences of these changes are 
twofold. First, the simplified or secondary communities 
are rather easily invaded. For instance, Clausen (11) 
noted that most of the major insect pests of agricultural 
crops in the United States are of foreign origin. In other 
cases, as with the Colorado potato beetle, Leptinotarsa 
decemlineata (Say), or the wheat stem sawfly, Cephus 
cinctus Norton, the production of new crops over large 
areas permitted the transfer of hitherto innocuous insects 
from native plants. Second, with more uniform (i.e., 
dominant) plant cover the numbers of some phyto- 





558 


phagous species increase until, economically, the popula- 
tions reach outbreak proportions. In contrast to the 
mixed hardwood-hemlock forest of Michigan, pure 
stands of hemlock, aspen, balsam fir and pine were 
heavily attacked by individual species of forest insects 
(17). This tendency for dominant species of animals or 
plants in any locality to be reduced to a lower position 
by environmental forces has been called by Graham 
(17) the Law of Natural Compensation. Insect outbreaks 
represent one of the natural forces that tend to reduce 
aggressive tree species to a more appropriate position in 
relation to other associated species. Agricultural prac- 
tices, with their annual culture and harvest, tend to 
obscure long-term trends in the operations of these com- 
pensating forces. However, simple populations are un- 
stable for reasons other than the compensating effect of 
insect attack upon abundant plant species. The insta- 
bility of simple food-chains is shown by the fluctuations 
of the greenhouse whitefly, Tvrzaleurodes vaporariorum 
(Westw.), and its chalcid parasite Encarsia formosa 
Gahan when the propagation of host and parasite 
populations was started at different temperatures, 
densities and age-structures (7, 8). 


PESTICIDE PROBLEMS 


The number of phytophagous species in simplified 
communities is quite small. For example, about one 
hundred species of harmful arthropods occur on un- 
sprayed apple trees in England (23). The number of 
these species that are sufficiently dense to be important 
economically is only a small fraction of the total present. 
The use of modern pesticides of high degrees of lethality 
and persistence not only reduces the pest against which 
they are applied—up to 99% of the spruce budworm, 
Choristoneura fumiferana (Clemens), population is killed by 
aerial spraying with DDT (2)—but also eliminates many 
other species from the area treated. Consequently the 
community becomes even more simplified than it was 
before the application of the pesticide. This reduction in 
the previously limited complexity should lead to greater 
instability of populations of those species that are able 
to withstand or that escape the treatment. Not only is 
the number of biotic interactions that impinge on the 
phytophagous species greatly reduced, but the influence 
of other environmental factors on the remaining interac- 
tions becomes much greater—as in the example of the 
modification of host-parasite interaction. Since the intro- 
duction of chemical treatment into orchard practices in 
England the number of pest species has become far fewer 
but despite this the damage caused by the few remaining 
ones often is as serious as formerly (23). Also, the density 
of some species varies from season to season (23) and the 
importance of many species of pests varies with the vari- 
ation in the type of chemical treatment (27). 

The reasons why chemical warfare against insect 
pests has not been a complete success may be listed 
briefly as follows: 

Resistant Strains. In response to the selective pressure 


FEDERATION PROCEEDINGS 





Volume 19 


exerted by insecticides certain populations of insect pests 
have developed and others are developing resistant 
strains. The codling moth, Carpocapsa pomonella (L.), for- 
merly resistant to lead arsenate, nicotine and fluorine 
compounds, has now shown definite signs of resistance 
to DDT (g). In addition, some species of pests have re- 
cently shown resistance to organo-phosphorus pesticides 
such as parathion (9, 25). Persistence of resistance in a 
population, after exposure to the pesticide has ceased, 
apparently varies with the species (30), but the situation 
is complicated by the fact that resistance to one com- 
pound may be maintained be exposure to a second (24) 
and resistance to more than one chemical may develop 
at the same time (6). 

Natural Enemies. A pesticide may be lethal to the 
natural enemies of a pest whereas the pest itself may be 
relatively immune. Pickett et a/. (27) state that they 
believe the destruction of natural enemies of pests is of 
major importance in pest population fluctuations. Ex- 
tensive surveys of sprayed and derelict orchards in 
England showed that derelict orchards may contain up 
to one hundred species of predacious arthropods whereas 
in orchards where DDT, BHC or phosphorus insecticides 
were applied there may be a marked reduction or com- 
plete absence of beneficial insects until June, and where 
DDT is used to control the codling moth these may be 
absent throughout the summer (23). The use of DDT 
and parathion does not always lead to the complete ex- 
tinction of parasites and predators, however; parasitism 
of the oriental fruit moth, Grapholitha molesta (Busck), by 
Macrocentrus ancylivorus Rohw. remained high in the 
presence of these two insecticides (5). As with phyto- 
phagous species, the number of beneficial species has 
been reduced by the use of the newer organic chemicals 
but the numbers of some species have been unaffected 
or have increased (Chant, personal communication). 

Competition. The use of insecticides may reduce tempo- 
rarily the amount of damage caused by a pest, save the 
host plant from destruction, and thus prolong the length 
of the infestation (2). Pickett et al. (27) considered that 
the reduction of intraspecific competition, following ap- 
plication of insecticides, may allow the life cycle of a 
greater proportion of individual pests to be completed 
and this results in a prolongation of the infestation and 
more damage than would have otherwise occurred. An 
indication that insecticides may affect interspecific com- 
petition is found in the emergence records of several 
species of blackflies before and after the application of 
DDT to a stream in Algonquin Park (12). DDT was 
applied to this stream in July, 1944. After 1944 there 
was a great increase in the emergence of Szmulium vittatum 
Zetterstedt and Simulium decorum Walker and this in- 
crease may be related to the decrease in the emergence 
of Prosimulium hirtipes (Fries) and Cnephia dacotensis (Dyat 
and Shannon). 

Adjacent Communities. Use of insecticides for the control 
of forest pests sometimes results in extensive mortality 
to various forms of wildlife. For example, aerial spraying 
against the spruce budworm in New Brunswick resulted 








fre 
re 


AL 


the 
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age 














ume 19 


pests 
istant 
), for- 
orine 
tance 
ve re- 
icides 
>ina 
~ased, 
lation 
com- 
1 (24) 


velop 


o the 
ay be 
they 
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s. Ex- 
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- com- 
where 
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-micals 
ffected 
ion). 
tempo- 
ive the 
length 
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ing ap- 
le of a 
apleted 
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ic com- 
several 
ation of 
)T was 
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control 
or tality 
praying 
resulted 





July 1960 


in reduction of food supply and poisoning of fish to such 
an extent that salmon stock may be reduced by 1961 to 
25° of their pre-DDT level in some cases (19). 

Host Plant. Pesticides may directly affect the host plant 
of the pests for which the pesticide was applied. The 
effects range from visible burning or other injury to 
leaves or fruit to a change in the color or flavor of the 
products manufactured from plants. There is also the 
possibility that repeated application of insecticides may 
poison host plants by an accumulation of these materials 
in the soil (g). Fleschner (16) found that the degree of 
host resistance of citrus plants to the citrus red mite, 
Panonychus citri (McG.), ranged from almost complete 
immunity to marked susceptibility. There were indica- 
tions that deposits of field dust and of zinc-deficiency and 
DDT spray materials lowered the host-plant resistance 
to mite attack because of certain physical properties of 
these materials. There were also indications that DDT 
spray materials produced some change within the citrus 
leaves that caused a lowering of host-plant resistance 
some time after application, and that these changes were 
translocated from older to younger leaves. Hueck (18) 
found evidence that DDT directly influenced the amount 
of eggs laid by females of the fruit tree red spider, 
Panonychus ulmi (Koch). 

Human Health. There is a hazard to human health 
from the application of pesticides and from the effects of 
residues of pesticides consumed with plant produce (9). 


ALTERNATIVE METHODS OF PEST CONTROL 


In view of the expense of and the problems arising from 
the chemical control of pests it is not surprising that addi- 
tional methods were developed to supplement and mini- 
mize the use of modern pesticides. And as these problems 
deal with populations most of these methods have an 
ecological basis. 

Modified Spray Programs. The problems of plant injury 
and human health can be overcome in practice after 
initial experience with an insecticide (g). The deleterious 
results of modern pesticides may be reduced by modify- 
ing the kind of chemical and time of application so that 
biotic factors may exert their maximum effects on the 
pest population. The depression of beneficial arthropods 
following the application of sulphur may be prevented 
by using low-residue fungicides (27), and the mass 
destruction caused by DDT may be avoided by the use 
of more species-specific chemicals, such as the botanical 
insecticide Ryania for the codling moth (10). Unfortu- 
nately the results achieved by the more specific poisons 
seem to vary in some cases with geographical areas and 
these pesticides do not provide a general solution to the 
problem. Some of the general insecticides can be made 
more selective by coating each particle with a substance 
that prevents the contact action of the chemical from 
affecting beneficial insects but that dissolves when the 
particles are eaten by a phytophagous species (29). An 
txample of the integration of chemical and biological 
agents in the control of an insect pest is the use of the 


XUM 


CONTROL OF INSECT PESTS 559 


selective insecticide Systox against the spotted alfalfa 
aphid, Therioaphis maculata (Buckton) (34, 36, 37). The 
use of this systemic insecticide reduced the pest popula- 
tions but affected their natural enemies to a much lesser 
extent (adult parasites would be unaffected by the sys- 
temic poison, but many predators, such as mirids, 
anthocorids and mites, are partly phytophagous and thus 
would suffer with the pest). As the natural enemies are 
not eliminated a less thorough kill of the pest was re- 
quired to establish control of the aphid and in addition 
the entomophagous species were present if new migrants 
of the pest arrived in the area already treated. But a 
selective insecticide is not superior to a general insecti- 
cide if natural enemies are not present. 

Physical Methods. A relatively new approach to the 
control of some pests lies in the use of sterilized males 
(13, 20). In some instances very large numbers of mass- 
reared males, sterilized by radioactivity but otherwise 
normal, are released among the wild population of the 
pest species. Provided the females mate only once and 
the treated males greatly outnumber the normal males, 
the majority of the females will lay infertile eggs. If this 
procedure is followed for a number of generations the 
pest population will be greatly reduced. In the first field 
experiments of this type the screw worm, Callitroga 
hominivorax (Cqrl.), was eliminated from the island of 
Curacao in a matter of months (3) and later reduced in 
Florida (4). Another means of immobilizing adult males 
is to trap them before they have mated with females. 
In laboratory experiments, males of Aedes aegypti (L.) 
were trapped in flight on approaching the source of sine 
sounds of 300 to 800 c/s (38). 

Resistance. Because the characters of living organisms 
have a genetic basis it has been possible to crossbreed 
individuals within a species to produce a great range of 
variability. By selecting and isolating some individuals, 
varieties with characteristics desired by man can be 
formed within a species. For example, the quality of 
wheat, the yield, time of maturing and resistance to dis- 
ease, have been improved. In a similar manner, the 
resistance of plants to attack by insects can be increased, 
and may be based either on a mechanical resistance of 
plant tissue, on a nutrient condition of the plant that 
affects the rate of increase of the insect species, or on the 
presence or absence of some trace chemical or the pro- 
tective role of essential oils (22). The use of a mechanical 
basis of resistance is shown in that developed in the 
wheat varieties, Rescue and Chinook, against the wheat 
stem sawfly, Cephus cinctus. The larvae develop within 
the hollow stems of susceptible varieties but do not do 
so as successfully in the solid-stemmed resistant 
forms (15). 

Cultural Control. Closely related to the development of 
resistant plants is the role of cultural practices in the 
control of insect outbreaks because resistance often de- 
pends on the conditions under which plants are grown. 
Farstad (15) noted the increasing importance of the 
wheat stem sawfly from the first introduction of wheat 
on the Canadian prairies as cultural practices were 








modified throughout the years. The last major outbreak 
caused by below-normal rainfall and the cultural prac- 
tices of farm mechanization, strip farming, surface tillage 
with preservation of stubble, and use of combine threshers 
and of early-maturing, rust-resistant varieties that were 
seeded early—lasted from the early 1930’s until the early 
1950's. To meet this outbreak a cultural program that 
involved trap strips, shallow tillage, rotations with im- 
mune or resistant crops, and late seeding was devised. 
In some areas the infestation was reduced from 85 to 3 % 
in 1 year. Cultural practices and, with insects of medical 
importance, sanitation (21) often form the basis of any 
program designed to combat insect outbreaks. 
Biological Control. Many insect species were accidentally 


imported into this country without their complement of 


natural enemies and, once established, they often became 
more destructive than in their countries of origin. The 
object of biological control is to increase and to lengthen 
the food-chains of pests and so reduce these pests to the 
status they held in their original environments. During 
the last 60 years, about 95 species of insect parasites and 
predators were imported and became established in the 
continental United States whereas approximately 390 
species were imported and colonized in varying num- 
bers but failed to establish themselves (11). Much of the 
difficulty in the use of parasites and predators appears 
to lie in the lack of complete synchronization in the life 
cycles of host and parasite when brought together in 
their new environment. Also, many entomophagous in- 
sects, though released at the appropriate stage of the 
pest’s life cycle, appear to be unadapted to the new 
environment for reasons that are unknown. There is some 
possibility that entomophagous species are as capable as 
phytophagous species in developing strains resistant to 
modern insecticides. In laboratory experiments, a 
DDT-resistant strain of the effective oriental fruit moth 
parasite, Macrocentrus ancylivorus was developed (28); and 
the occurrence of parathion-resistant strains of phyto- 
phagous mites Panonychus ulmi (Koch) and Vasates 
schlechtendali (Nal.) and the predacious mite Typhlodromus 
occidentalis Nesbitt in the Okanagan Valley of British 
Columbia has been observed (25). The results of bio- 


logical control to date are that only a small portion of 


the most important agriculturai pests can be fully con- 
trolled by use of parasites and predators and with a 
substantial number no reduction was attained. Most 
biological control projects may be expected to yield 
partially beneficial results and if integrated with chemi- 
cal control the cost of plant protection may be reduced 
(11). Biological control through the use of parasitic and 
predacious arthropods is, with cultural practices, one 
of the two chief alternatives to chemical control. 
Microbial control of insect pests has been developed 
through the use of viruses, bacteria, fungi, protozoa and 
nematodes in place of entomophagous insects. These 


microorganisms play significant roles in the control of 


natural populations (35). Their value depends on the 
fact that they can be mass-produced in the laboratory 


560 FEDERATION PROCEEDINGS 








Volume 19 


and sprayed on field crops like an insecticide. In the use 
of some microorganisms the timing of application is very 
important. Advantages of this method are that the micro- 
organisms used are usually harmless to man and plants, 
that they are not affected by most insecticides (11) and 
that, although diseases may affect parasites and preda- 
tors directly and indirectly, they may greatly reduce 
insect numbers without seriously affecting the over-all 
effectiveness of entomophagous species (35). Microorgan- 
isms have been used successfully in a limited number of 
cases but further research will, no doubt, extend their 
utility. 

Diversity. The trend towards monocultures greatly 
simplifies community structure with the result that insect 
populations can more easily increase to outbreak propor- 
tions, and exotic species, especially with the improve- 
ment in transportation facilities, can more readily reach 
and become established in new environments. Therefore, 
what is needed to restore relative stability to popula- 
tions, both in composition and in numbers, is to recreate 
more complex communities by promoting the greatest 
possible ecological variety. The basis of ecological 
variety lies in combining natural habitats with the areas 
that produce our natural resources. It is possible, since 
many natural enemies are polyphagous, that the rich 
fauna of the more natural habitats would form reservoirs 
from which parasites and predators would attack our 
pests. Some examples of such reservoirs are hedges, 
hedgerows, railway embankments and inland water 
channels (14, 26). 


CONCLUSIONS 


The development of large areas of uniform vegetation 
has resulted in the breakdown of complex natural com- 
munities and the creation of much simpler biotic units. 
The relatively simple communities are unstable and 
usually lead to outbreaks of insect species. The use of 
mass-destructive chemicals against these pests causes the 
disappearance of many phytophagous species but the 
ones that remain often do as much damage as occurred 
before the treatment. As the relative importance of insect 
pests changes with changes in the type of chemicals used, 
it seems likely that the use of chemicals alone will not 
provide an adequate solution to the problem of pest 
control. A number of alternatives exist that supplement 
or replace the necessity of applying mass-destructive 
pesticides. But as many of these alternatives are based 
on ecological principles they imply the continued exist- 
ence of the injurious phytophagous species. The success 
achieved depends, therefore, on the density of the pest 
species that is necessary to cause economic loss. Plants 
are able to sustain some injury from insect populations 
because individual plants produce an excess of material 
that is the basic food supply of animal communities (1): 
But when insects feed on marketable produce it is neces 
sary to reduce their numbers to a minimum. This is bes! 














lume 19 


he use 
is Very 
micro- 
plants, 
1) and 
preda- 
reduce 
ver-all 
organ- 
iber of 
1 their 


sreatly 
t insect 
yropor- 
prove- 
’ reach 
-refore, 
yopula- 
ecreate 
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e areas 
>, since 
he rich 
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ick our 
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water 


retation 
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yle and 
> use of 
uses the 
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curred 
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the pest 
;. Plants 
yulations 
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ities (1) 
is neces 
is is best 





July 1960 


done by using specific chemicals that can be harmonized 
with those biotic agents that are exerting effective pres- 
sures on the pest and remaining phytophagous species. 


The immediate consequences of the use of general 


pesticides are fairly obvious but probably an even greater 
danger lies in the effect of such lethal factors on the 
nature of populations. The appearance of resistant 


eo N 


wn 


Co om 


XUM 


; Exton, C. S. 


. Farstap, C. 


REFIT 
ALLEE, W. C., A. E. Emerson, O. Park, T. ParK AND K. P. 
Scumipt. Principles of Animal Ecology. Philadelphia: Saunders, 
1949, P- 837. 


. Batcu, R. E. Ann. Rev. Entomol. 3: 449, 1958. 
. BaumuHover, A. H., A. J. Granam, B. A. Biter, D. E. Hop- 


Kins, W. D. New, F. H. DupLey anp R. C. BusHLanp. J. 
Econ. Entomol. 48: 462, 1955. 


. BAUMHOVER, A. H., C. N. Husman, C. C. SkipPpER AND W. D. 


New. J. Econ. Entomol. 52: 1202, 1959. 


. Boyce, H. R. anp G. G. Dustan. Proc. Tenth Internat. Congr. 


Entomology 4: 493, 1958. 


. Brown, A. W. A. Insecticide Resistance in Arthropods. Geneva: 


World Health Organization, 1958, p. 240. 


. Burnett, T. Ecology 30: 113, 1949. 
. Burnett, T. Can. J. Zool. 38: 57, 1960. 
. CuapMan, P. J. Proc. Tenth Internat. Congr. Entomology 3: 155 


1958. 


. CLrancy, D. W. ano H. J. McA ister. Proc. Tenth Internat. 


Congr. Entomology 3: 181, 1958. 


. Crausen, C. P. Tech. Bull. No. 1139. Washington: Dept. of 


Agriculture, 1956, p. 151. 


. Davies, D. M. Trans. Roy. Can. Inst. 59: 121, 1950. 
. Downine, R. S., J. MARSHALL, D. P. PrELou anp M. D. 


Proverss. Proc. Tenth Internat. Congr. Entomology 3: 175, 1958. 
In: Ecology of Invasions. London: Methuen, 
1958, p. 181. 

W. Entomology Newsletter, Research Branch, 
Canada Dept. of Agriculture 38: No. 1, 2 and 3, 1960. 


. FLESCHNER, C. A. J. Econ. Entomol. 45: 687, 1952. 
. GrauaM, S. A. Can. Entomologist 88: 45, 1956. 
. Hueck, H. J. 


The Population Dynamics of the Fruit Tree Red 


CONTROL OF INSECT PESTS 


561 


strains in so many species and to so many chemicals indi- 
cates the genetic adaptability of the phytophagous 
arthropods. As genetic systems govern characteristics 
other than resistance to insecticides the selective pres- 
sures inherent in the continued use of exceptionally 
lethal agents may develop populations quite unlike those 
we are now trying to control. 


SRENCES 


19. 
20. 
21. 
22. 


23. 


24. 
. Morcan, C. V. G. anp N. H. ANDERSON. Can. Entomologist 
26. 
27. 
28. 
29. 


. Rosertson, J. G. 
. Ropricuez, J. G. 
2. SHELFORD, V. E. 


Spider (Metatetranychus ulmi Koch 1836, Acari, tetranychidae) with 
Special Reference to the Influence of DDT. Leiden: N. V. Grafisch 
Bedrijf En Uitgeverigde Jong, 1953, p. 148. 

KEerSwILL, C. J. Atlantic Advocate 48: 65, 1958. 

Knipuine, E. F. J. Econ. Entomol. 48: 459, 1955- 

Lairp, M. Acta Trop. 16: 331, 1959. 

Martin, H. The Scientific Principles of Crop Protection. London: 
Arnold, 1959, p. 359- 

MassEE, A. M. Proc. Tenth Internat. Congr. 
1958. 

Metca_F, R. L. Physiol. Rev. 35: 197, 1955- 


Entomology 3: 163, 


go: 92, 1958. 

Pickett, A. D. J. Econ. Entomol. 52: 1103, 1959. 

Pickett, A. D., W. L. Putman anp E. J. LeRoux. Proc. 
Tenth Internat. Congr. Entomology 3: 169, 1958. 

PreLou, D. P. anp R. F. Gasser. Science 115: 117, 1952. 
Ripper, W. E., R. M. GREENSLADE, J. HEATH AND K. Bar- 
KER. Nature, London 161: 484, 1948. 

Can. J. Zool. 35: 629, 1957. 

Ann. Entomol. Soc. Am. 44: 511, 1951. 
Animal Communities in Temperate America. 


Chicago: Univ. Chicago Press, 1937, p. 365. 


. Situ, D. S. Can. Entomologist g2: 103, 1960. 

. Smiru, R. F. anp K. S. Hacen. Hilgardia 29: 131, 1959. 

. Stemnaus, E. A. Hilgardia 23: 197, 1954. 

9. STERN, V. M. anp R. VAN DEN Boscu. Hilgardia 29: 103, 1959. 
. SrerN, V. M., R. F. Smiru, R. VAN DEN BoscH anp K. S. 


HacEn. Hilgardia 29: 81, 1959. 


. Wisuart, G. anp D. F. Riorpan. Can. Entomologist 1: 181, 


1959. 











INTERSOCIETY SYMPOSIUM ON RADIATION PROTECTION AND RECOVERY! 





Studies on microorganisms 


ALEXANDER HOLLAENDER, Chairman 
Biology Division, Oak Ridge National Laboratory,? Oak Ridge, Tennessee 


‘he PROBLEM OF RADIATION protection and recovery 
has developed along several interesting lines during the 
last few years. Many of the aspects of protection, as 
well as recovery, have medical applications that will 
not be discussed in this symposium. We prefer to discuss 
the basic aspects of this subject and will be able to cover 
only a limited number of these. The field has broadened 
so extensively that it would be impossible to give a 
complete survey of the developments, even those in the 
last five years. 

In this and other areas of research, studies on micro- 
organisms have served as a kind of guide. It has not 
always been possible to apply the findings obtained with 
bacteria and fungi to mammals. In spite of this, informa- 
tion obtained from studies on bacteria, paramecia and 
other microorganisms has been useful in guiding the 
application of our findings to protection and recovery in 
mammals. 

I would like to discuss some of the work with micro- 
organisms that seems, to me at least, to have great 
significance in studies of the mechanism of radiation 
protection. A number of chemical compounds that 
protect against radiation damage have been listed in 
previous publications. We are now trying to improve 
our knowledge of how these compounds do their work. 

But first, I should like to talk about the effect of 
oxygen. The presence of oxygen intensifies the damage 
that ionizing radiation produces on living cells—micro- 
organisms and also higher organisms. In Escherichia coli, 
for instance, a dose-reduction factor of three can be ob- 
tained if the organism is irradiated in the absence of 
oxygen. In mammals, the dose-reduction factor can be 
as high as two if the oxygen tension is reduced to a 
minimum. The oxygen effect has been explored exten- 
sively. It is now being applied in connection with irradia- 
tion of malignant tissue, especially by a group working 
under the direction of Dr. L. H. Gray at Northwood, 
England. 

It has been found with microorganisms (6), especially 
E. coli, that the nutritional history of the organism has a 

! Chicago, Ill., April 11, 1960. 

2 Operated by Union Carbide Corporation for the U. S. Atomic 
Energy Commission. 





very definite effect on its survival. Table 1 shows the 
effect of different growth and plating media on the 
survival. Apparently, bacteria growing on incomplete 
media have available the enzymes necessary for the 
production of complicated nutrients needed for growth 
but that are not present in the medium. On the other 
hand, bacteria grown on a complete medium, irradiated, 
and then plated on a minimal medium seem to lack the 
ability to produce enzymes essential for synthesizing the 
compounds necessary for growth. This line of investiga- 
tion has been quite successful in the work with micro- 
organisms but we are not yet able to apply it to higher 
organisms. The main difficulty is that the mammal 
usually has a good reserve of nutrient material available, 
which prevents a clear-cut response. 

These rather crude studies on nutritional conditions 
have now been followed by a much more careful investi- 
gation of the nutritional conditions necessary for in- 
creasing survival. A group in our laboratory under the 
direction of Dr. George E. Stapleton (5) has tested the 
effects of a variety of nutritional conditions on the radio- 
sensitivity of FE. colt. More than eight years ago, we 
reported that £. coli grown aerobically in nutrient broth 
would give a straight-line survival curve, but grown 
anaerobically it would give a definite threshold-type 
curve (2). At that time it was thought that the response 
of E. coli under these conditions was probably caused by 
polyploid cells or a clumping of cells. These factors have 
now been eliminated in careful studies in which we have 
found that the pH of the medium in which the organism 
is growing is an important factor. For instance, EF. coli 
grown in a medium with lower pH will be much more 
resistant than the bacteria that reach a final pH of 
~ 7.5-8. There are many interesting aspects to these 
studies. They point out that the physiology of the organ- 
ism before irradiation, as well as its status after exposure, 
is important to its survival. More-detailed studies in 
this field are urgently needed for additional clues they 
could supply in regard to survival of living cells. 

We have been quite concerned about the following 
two questions regarding chemical protection in micro- 
organisms: a) If a chemical protects an organism against 
radiation killing, does it also reduce the mutation rate? 

















b) 
bu 
re 
ra 
se\ 


ge’ 
lin 
of 


X-I 


10¢ 


org 
sta 
pre 
the 
pre 
onl 
con 


by. 








the 

the 
ylete 

the 
wth 
ther 
ited, 
- the 
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rate? 


} 








July 1960 


TABLE 1. Effect of Growth and Plating Media 
on Survival in E. Coli 


Growth Irradiation Plating Survival 
medium (at ~20,000 r) medium 
Complete Yes Complete Fairly high 
Complete Yes Inorg. salts- Very low 
glucose 
Complete No Inorg. salts- Good 
glucose 
Inorg. salts- Yes Inorg. salts- Fairly high 
glucose glucose 


b) If the radiation dose is too low to produce any killing 
but high enough to produce mutation, will the chemical 
reduce the number of mutations? The basis for these 
rather different questions is found in observations by 
several investigators: that, in £. coli, the mutation rate 
is inversely proportionate to the survival ratio. In 
general, most of these studies have shown a straight- 
line relation between percentage survival and amount 
of energy absorbed. 

In our studies on the fungus, Aspergillus terreus, the 
x-ray effect assumes a definite threshold-type curve. 
The organism is usually irradiated in the range of 10,000— 
100,000 r. At less than 10,000 r no organisms are killed. 
In a study just completed, we found that, in this energy 
range, the mutation production increases in proportion 
to the amount of radiation given. Mutation production 
may also be effectively reduced by irradiating the 
organism in the presence of cysteamine. We can now 
state definitely that we can protect against mutation 
production even in the absence of any killing effect; 
the protection against mutation is not secondary to 
protection against killing (1). A chemical can protect 
only during irradiation. A quite thorough study on 
conditions controlling the response of bacteria, especially 
E. coli, to chemical protection (cysteine) has been made 
by Kohn and Gunter (4). 

It has also been shown that mutation production can 


STUDIES ON MICROORGANISMS 


563 


be reduced by treatment after exposure (3). As an 
example, I should like to cite R. F. Kimball’s work with 
paramecia in our laboratory. If irradiated Paramecium 
aurelia is kept in streptomycin under carefully con- 
trolled conditions, there is no cell division but the metab- 
olism continues. Dr. Kimball has now demonstrated 
that, if streptomycin is given after irradiation then the 
paramecia are removed from the antibiotic and allowed 
to grow, the percentage of mutation is much lower than 
if the irradiated protozoa are allowed to develop im- 
mediately after irradiation without the streptomycin. 
In other words, the postirradiation treatment has caused 
something to happen that interferes with the steps 
leading from the absorption of radiation to the final 
mutation that we observe. And so we may apparently, by 
treatment after irradiation, interfere with the process 
initiated by the radiation. There is other evidence that a 
good part of the radiation damage is not direct but 
proceeds through intermediate steps to the permanent 
genetic change (mutation) or to the death of the cell. 
There is no question that this provides an opportunity for 
interfering with the processes that lead to a permanent 
effect. This appears to be a fruitful approach to studies of 
radiation damage. There is also good evidence that some 
of the ‘permanent’ effects of certain chemicals, nitrogen 
mustards for instance, can be interfered with and 
partially prevented. 

I have given only a short survey of a few phases of the 
work in radiation protection that is going on. It should 
be emphasized that we need many more definite investi- 
gations to untangle this complicated problem of how 
radiation produces its effects and the mechanism of 
protection against it. The phenomenon is not simple 
and straightforward; it is not simply observing very 
crudely the effects. What we require is a better under- 
standing of the basic metabolism of living cells, different 
conditions that can interfere with their growth, and the 
mechanism of their repair. 


REFERENCES 


1. HOLLAENDER, A. AND A..M. McCartny. Science 130: 1420, 1959. 

2. HOLLAENDER, A., G. E. STAPLETON AND F. L. Martin. Nature, 
London 167: 103, 1951. 

3. Kimpaui, R. F., N. GarrHeR AND S. M. Witson. Genetics 42: 


661, 1957. 


XUM 


4. Konn, H. I. ann S. E. Gunter. Radiation Research 11: 732, 1959. 

5. STAPLETON, G. E. anp M. S. Encex. J. Bact. In press. 

6. Stapeton, G. E., A. J. SBARRA AND A. HOLLAENDER. J. Bact. 
79: 7, 1955: 








Chemical protection of mammalian tissues 


J. R. MAISIN' AND D. G. DOHERTY 


Armed Forces Institute of Pathology, Washington, D. C., and Biology Division, 
Oak Ridge National Laboratory,? Oak Ridge, Tennessee 


W. HAVE CHOSEN not to review exhaustively the 
voluminous literature on chemical protection but rather 
to consider the current status of the problem and discuss 
the general characteristics of radioprotective agents and 
their modes of administration and action, giving special 
emphasis to the degree of protection offered to what are 
presently considered to be the most radiosensitive tissues. 
Soon after the report by Barron et al. (13) that sulfhydry] 
enzymes, inactivated by x-irradiation in vitro, could be 
partially protected or reactivated by addition of gluta- 
thione, Patt et a/. (115) found that cysteine pretreatment 
offered protection to x-irradiated rats, and Bacq and 
Herve (g) found that cyanide pretreatment protected 
mice. These demonstrations that chemical pretreatment 
was effective in mammals spurred the search for more 
effective agents, and in the intervening years over 1000 
compounds have been tested for protective activity. 
Developrnent of the concepts in this field as well as de- 
tailed lists of references may be obtained from the many 
reviews and books that have been written on this subject 
(6, 19, 41, 44, 66, 109, 112, 113, 117). 

Three broad types of acute radiation death, each with 
its own characteristic time and syndrome, have been 
observed in whole-body-irradiated mammals (34). High, 
supralethal exposure doses of x- and gamma-radiation 
produce ‘CNS deaths’ marked by neurological syn- 
dromes and a survival time of from a few minutes to 10 
hours (23, 88). Little is known about CNS death and no 
satisfactory treatment for CNS injuries has been de- 
veloped. At lower exposure levels but above an LD199 /39 
(LDig9 in 30 days), about 1000 r in the mouse, an ‘in- 
testinal death’ occurs, most of the animals dying be- 
tween the third and the sixth day of a severe bacteremia 
resulting from major damage to the gastrointestinal 
tract. Any survivors die later from varying degrees of 
infection, hemorrhage and anemia. This later death, 
as well as that resulting from exposure to LD59—LDj9 
doses, is referred to as a‘ bone marrow or hematopoietic 
death’ and usually occurs, in mice, from the 8th to the 
16th day. The most effective protective agents that we 
In- 


! International Postdoctoral Research Fellow, National 


stitutes of Health, Bethesda, Md. 
2 Operated by Union Carbide Corporation for the U. S. 
Atomic Energy Commission. 


564 


have reduce the apparent radiation dose, as measured 
by 30-day mortality or by damage to the hematopoietic 
and gastrointestinal systems, by a factor of about two 
and result in increased survival of the animals. Fission 
neutrons are more effective in producing radiation effects 
than x- or gamma-radiation (139) especially with regard 
to intestinal damage, the relative biological effective- 
ness (N/X) for the intestine—according to Lesher and 
Vogel (89)—being greater than six. Very little work has 
been done in the chemical prevention of neutron dam- 
age; the few reports available indicate that the protec- 
tion is of the order of one third that obtained against 
x-radiation. 

The basic premise of chemical protection is that the 
presence of the chemical in the system at the time of 
irradiation in some manner reduces or prevents damage; 
but the chemical is ineffective when given after the 
damage has occurred. It may conceivably operate 
through any one or a combination of the following 
mechanisms: a) as a free radical-trapping agent to 
quench the free radicals arising from irradiation of water; 
b) by combination with sensitive biological molecules to 
inake them more resistant to radiation; c) by reducing 
oxygen tension, thus reducing the radical yield either 
directly or via a physiological response; and d) by stim- 
ulating repair processes before radiation damage, bring- 
ing about a more rapid recovery. These general ideas 
have been the basis for selection of most compounds 
tested for radiation protection. The only exception to 
the idea that the chemical must be present at the time 
of irradiation is the report by Kiinkel e¢ al. (77) that ir- 
radiated, hibernating dormice injected with cysteine 
3 weeks after irradiation and then awakened were pro- 
tected against the lethal effects. Smith’s similar work 
with the ground squirrel (128, 129), in which he used 
cysteine or 2-aminoethylisothiourea (AET) either at 3 
weeks after or within one-half hour after radiation, failed 
to protect the animals from radiation lethality. Hiberna- 
tion alone, although prolonging the survival of irradiated 
ground squirrels, had no significant effect on mor- 
tality (46). 

Since there is a large variation in short-term mortality 
of inbred mice given an LD59/3) radiation dose, it was 
suggested early that all compounds be tested at the 
MINIMUM LDjo9 39 dose (6, 20, 130). We agree with this 





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July 1960 


suggestion and believe that a compound should be con- 
sidered protective only when, in a given species, it re- 
duces the 30-day mortality at an LDj9 radiation dose by 
at least 50 % or increases the LD59 39 dose by at least 30 % 
of its value. Compounds offering less protection or those 
providing only local protection contribute to our under- 
standing of protective mechanisms but cannot properly 
be called protective. To facilitate discussion, compounds 
will be arbitrarily classified into three groups; those that 
produce anoxia, sulfhydryl and related compounds, and 
miscellaneous compounds including those acting through 
a physiological response. Only brief consideration will 
be given to the mortality results, our main purpose being 
to illustrate the relative effectiveness of the different 
classes of compounds. 

For obvious reasons most radiation work has been done 
in mice and rats, and only occasionally have the in- 
vestigations been extended to other species. Most com- 
pounds tested have been administered intraperitoneally, 
but the oral, intravenous and subcutaneous routes have 
been used occasionally, depending on the properties of 
the compound. Although a considerable amount of in- 
formation has accumulated on the short-term effects of 
radiation on chemically protected animals, much less is 
known about the effectiveness of the chemicals against 
delayed radiation damage. It seems clear, however, 
from the work of Maisin et al. (93), Hollaender et al. (67), 
and Upton et a/. (143), that chemicals are not so effective 
against chronic radiation damage as against acute effects. 


PROTECTIVE AGENTS 


Anoxia. The initial observation by Lacassagne (79) 
that anoxia reduced radiation damage to newborn mice 
was extended by Dowdy and associates (47) to rats and 
adult mice. They obtained 100% survival for rats ex- 
posed to an LDy99/39 radiation dose in an atmosphere 
of 5% oxygen and 95% nitrogen and 80% survival in 
mice. Reduction of oxygen tension by chemicals such as 
cyanide (g, 58), malononitrile (10), carbon monoxide 
(21, 76), sodium nitrite (31), hydroxylamine azide (22), 
and p-aminopropiophenone (3, 138) yields similar re- 
sults, although the cyanide findings have been disputed 
(47, 57). The physiological amines such as Pitressin, 
epinephrine (59, 137), histamine and its derivatives 
(104), and serotonin (5, 59, 84, 87)—the last being the 
most effective of the group—provide significant radi- 
ation protection. Hypothermia has also been shown to 
reduce radiation mortality in newborn mice (140), mice 
(70, 78) and rats (61). This approach alone, though in- 
formative and useful for local treatment, seems to have 
little practical value since near-toxic conditions must be 
maintained to obtain an effect. 

Sulfhydryl and Related Compounds. This class includes the 
most effective and useful compounds found so far. It 
has been exhaustively investigated ever since Patt and 
co-workers’ discovery (115) that cysteine effectively re- 
duced acute radiation lethality, raising the LD50/39 in 
ince from 765 to 1120 r at the maximum tolerated com- 


CHEMICAL PROTECTION OF MAMMALIAN TISSUE 


565 


pound dose of 1.2 gm/kg. Following this lead, Bacq (12) 
found that the closely related 2-mercaptoethylamine 
(MEA; cysteamine) was a more active protective agent, 
150 mg/kg yielding an LD509/30 in the 1200-r range. Sub- 
sequent studies established that a free amino group, as 
well as a sulfhydryl, was essential for maximum activity 
(2, 42, 43, 83) and that separation of the two groups 
could not exceed three methylene groups (43) without 
a major loss in activity. In contrast to cystine (115), the 
disulfide of MEA [bis(2-aminoethy])disulfide(cystamine) ] 
has been reported to be as active as MEA when given 
orally (7). Glutathione gives good protection at the 
LD100/30 level when given orally or subcutaneously (28, 
29), and it has been reported that the related glutamyl- 
aminoethanethiol is as effective as MEA (18). Diethyl- 
dithiocarbamate (11, 14) also gives good protection at 
the same radiation level. 

A new class of compounds, the aminoalkylisothioureas 
and their rearrangement products at neutral pH—the 
mercaptoalkylguanidines (45, 74, 75), were reported by 
Doherty and Burnett (42) and Shapira et al. (125) to 
have increased protective activity. The structural re- 
quirements of these compounds are similar to those for 
the mercaptoalkylamines (125); the conclusion was 
drawn that, in the sulfhydryl compounds, a basic group 
separated by not more than three carbon atoms was 
essential for maximum protective activity. The simplest 
members of the series, 2-aminoethylisothiourea (AET) 
and 3-aminopropylisothiourea are the most effective 
and may be given as either the isothioureas or rearranged 
with alkali to the corresponding mercaptoguanidines. 
They are about three times as effective on a molar basis 
as MEA; 1.e., in the same strain of mice, 240 mg/kg 
of MEA yields an LD50/30 of 1325 r whereas 152 mg/kg 
of 2-mercaptoethylguanidine (MEG) yields an LD50/30 
of 1425 r, the molecular weight ratio being 77 for MEA 
to 119 for MEG. AET or MEG remains fully effective 
for 1 hour after oral administration, but MEA rapidly 
loses its effectiveness when given orally. The correspond- 
ing guanidodisulfides, although effective at an LDj 90/30 
dose, do not elevate the LD50/39 to the same extent as the 
thiols regardless of the route of administration. 

A few other sulfur compounds have been found with 
protective activity lower than MEA; e.g., BAL (43) 
methyl- and ethylisothioureas (69), homocysteinethio- 
lactone (85), thioctic acid (54), and 2-aminoethylthio- 
sulfuric acid (68). Langendorff et a/. (86) reported that 
p-penicillamine and pi-homocysteine sensitized rats to 
radiation. 

Miscellaneous Compounds. Most of the substances in this 
group Offer little protection at the LD; 00/30 radiation level. 
Those producing some physiological change may be 
given a long time before irradiation. Kedrova and 
Krikhova (73) found, in agreement with previous work 
(86), that ACTH decreases the death rate. ACTH also 
may have a small effect when administered after ir- 
radiation (127). Langendorff et a/. (81) and Rugh and 
Wolff (122) showed that gonadectomy 3 months before 
irradiation has a small protective effect in mice given a 








566 


low dose of x-rays. Rugh and Clugston (121) demon- 
strated that female mice are less sensitive to small x-ray 
doses when they are in estrus than at other times. On 
the other hand, pregnancy offers no protection (136). 
Rixon et al. (119) have shown that parathyroid extract 
given 12 hours before irradiation or immediately after 
gives some protection. 

Illustrative of the wide variety of tested materials that 
have been reported to have some protective effect are 
the following: fluoroacetate (8), cobalt (111), magnesium 
(17), bacterial endotoxins (1, 134, 135, 150), homologous 
serum or plasma (141), ribonucleic acid (110), phenolic 
derivatives (80) and quinoline-1 , 4-di-N-oxide (62). 


TISSUE PROTECTION 


Recent work has been directed toward the qualitative 
and quantitative response of various mammalian systems 
in animals protected with the active compounds avail- 
able at present. By this, we hope to obtain a better under- 
standing of their modes of action. The most characteristic 
findings of these investigations in the various tissue 
systems will be reviewed. 

Hematopoietic System. Patt et al. (114) demonstrated 
that a single injection of cysteine (950 mg/kg intra- 
venously) to rats 5 minutes before 800 r of x-rays to the 
total body modified significantly the radiation-induced 
hematologic changes. The heterophils, lymphocytes and 
erythrocytes were less depressed and the recovery was 
more rapid than in the nonprotected rats. Rosenthal 
et al. (120) found that neutrophilia was higher in the 
blood of cysteine-protected rats exposed to 800 r, and 
there were more myeloid cells of all stages in the bone 
marrow. Cronkite et a/. (35) and Jennings and Tessmer 
(71) showed a striking difference in the rate of regenera- 
tion of the hematopoietic tissues in rats protected with 
glutathione. Urso et al. (144) demonstrated that, after 
450 or goo r, bone marrow cells and peripheral blood 
leukocytes were more plentiful in AET-treated mice 
than in nonprotected ones, the dose-reduction factor 
being about two. Smith (132) found that bone marrow 
cells protected with AET, irradiated in vitro, and in- 
jected into lethally irradiated mice prevented radiation 
death. Maisin and Doherty (95) found that, even after 
1500 r, some AET-treated mice showed hematopoietic 
recovery (fig. 14). No recovery was found in the MEA- 
‘reated animals (fig. 1B). Crouch and Overman (36) 
demonstrated, in Macaca mulatta pretreated with AET 
and given 650 r x-rays, a dramatic increase in the number 
of circulating reticulocytes on the 18th day. The hemato- 
crit returned toward normal by the 22nd to the 24th 
day and was complete by the 65th day. 

Lymphopoietic Tissues. This aspect of the radiation 
problem has not been extensively investigated. Hartweg 
(63) showed that cysteine pretreatment reduced the sub- 
lethal radiation effects on the blood elements and spleen 
of mice on the third day after irradiation. Hagen et al. 
(60) showed that, 72 hours after x-irradiation, the spleen 
weight of MEA-treated animals was twice that of the 


FEDERATION PROCEEDINGS 








Volume 19 





FIG. 1 A: Mouse femur protected with AET (8.8 mg i.p.) 8 days 
after exposure to 1500 r of x-rays. Note the presence of nucleated 
bone marrow cells, together with hemorrhage. X 48. B: View of 
a femur of a mouse protected with MEA, 6 days after a 1500-7 
X-ray exposure. Note the hemorrhage and the complete depletion 
of the bone marrow. X 32. 


controls. Urso et al. (144) found a similar protective effect 
on spleen weight in AET-treated mice. Van der Meer 
and van Bekkum (105) demonstrated that the spleen of 
mice was also protected by histamine and its derivatives. 
Gerbetzoff and Bacq (55) and Peterson and DuBois 
(116) showed that the thymus in MEA-treated mice was 
protected against x-radiation, and Makinodan et al. 
(100) found that the immune status of AET-treated 
animals given 950 r recovered at a rate comparable to 
untreated animals that received 475 r. 

Gastrointestinal Tract. The importance of the intestinal 
syndrome in contributing to the mortality of lethally 
irradiated animals has been long recognized and the 
effects of the sulfhydryl protective agents extensively 
examined in an attempt to explain the prompt recovery 
of protected intestinal tissue. Histological study showed 
that the mitotic activity of the small intestine of MEA- 
treated rats exposed to 700 r was higher than in the un 
treated control animals (go). After local exposure to 500 








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MEA- 
the un- 
to 500 





July 1960 


r, the MEA-protected small intestine of rats showed a 
higher mitotic activity than the unprotected intestine 
(39). Cysteine was also found to protect intestinal mitotic 
activity in sublethally irradiated rats (16). Smith and 
Tyree (131) found that cysteine pretreatment shortened 
the period of anorexia in lethally irradiated rats as well 
as prevented the deaths that occurred in untreated, 
force-fed rats. Williams and Long (149) showed that 
the mitotic activity of the intestinal mucosa was pro- 
tected 73 hours after goo r in glutathione or p-amino- 
propiophenone-treated animals. Conard (32) found that 
intestinal weight loss was less on the third day after ex- 
posure to 550-950 r when glutathione was given to mice 
before irradiation. DNA synthesis and intestinal weight 
in irradiated rats and mice was studied by Mole and 
Temple (106, 107), who found that MEA gave good 
protection after exposures to 1000 r with either end 
point. ( 

Maisin et al. (98) showed that AET has a marked pro- 
tective action on the gastrointestinal tract of mice in 
general, and particularly on the small intestine. They 
showed, by weight of organs and their contents, histology, 
mitotic count, and chemical analysis including DNA, 
RNA, and protein synthesis, that the small intestine 
was markedly protected after 1500 r. Maisin and Mout- 
schen (96) demonstrated that the number of chromosome 
anaphase fragments in the crypt cells of both AET- 
protected and ‘nonprotected mice is dose dependent and, 
in lethally x-irradiated mice given AET, the number of 
chromosome fragments was less than in nonprotected 
mice (fig. 2). The recovery of the mitotic activity in pro- 
tected mice was inversely related to the number of 
chromosome fragments. Maisin and Popp (gg) showed 
that decrease in potassium and increase in sodium of the 
small intestine (fig. 3), decrease in potassium of the 
colon wall, and increase in sodium and potassium of the 
contents of the gastrointestinal tract were completely 


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200 400 600 900 


FIG. 2. The protective effect of AET on the number of anaphase 
fragments as a function of x-ray dose in intestinal crypt cells of 
Ast mice 4 days after exposure. 


1500 


¢ 
ease tak 


CHEMICAL PROTECTION OF MAMMALIAN TISSUE 567 


or partially corrected in mice given AET before ex- 
posure to 1500 r. By electrophoresis, they also showed 
that there was a complete recovery of the esterase ac- 
tivity in mice treated with AET and given a goo-r 
x-ray exposure. This was not found in nonprotected 
mice or in mice given a bone marrow transplant. It was 
also demonstrated that AET prevents the progressive 
shortening of the small intestine that occurs in strain A-1 
mice given 1500 r (98). Maisin and Doherty (g5 and 
unpublished data) have shown by mitotic count of crypt 
cells in the small intestine of strain A-1 mice irradiated 
with goo and 1500 r and protected with AET, MEA, 
aminopropyl-N’-methylisothiourea and 2-aminobutyliso- 
thiourea, and serotonin, that AET and MEA gave the 
best protection (fig. 4). MEA was shown, by Maldague 
et al. (101), to protect the esophagus of rats exposed to 
4000 r of radiation to the chest. Similarly, Dunjic e¢ al. 
(49) found less damage to the oral pharyngeal mucosa 
in rats given MEA before 2000 r of radiation to the 
upper body than in the untreated controls. Upton et al. 
(142) found that AET completely prevented ‘oral death’ 
in mice given 1800 r to the head. 

Ovaries and Testes. At very low doses of radiation, MEA 
and cysteine protected the ovaries of the mouse to the 
extent that it delayed the onset of sterility (123). 

Contrary to the previous work of Maisin et al. (92) 
and Kaplan et al. (72), it is now well established that the 
testes are, to a certain extent, protected by MEA and 
AET. In 1959, Mandl (102) demonstrated that, in rats 
given a dose of 230 to 460 r, the reduction of the number 
of spermatogonia and prespermatocytes was partially 











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FIG. 3. Effect of AET on the concentration of sodium and 
potassium in the wall of the small intestines of mice exposed to 
1500 r (1 = one standard error). Potassium - concentration: 
@, 1500 r only; O, AET + 1500 r. Sodium concentration: 
A; 1500 r; A, AET + 1500r. 








568 FEDERATION PROCEEDINGS 


6- _.-—s 
*” AET, 16mg, ORAL 





/ 
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{ 2 3 4 5 6 7 
TIME AFTER IRRADIATION (days) 
FIG. 4. The comparative effects of AET, MEA, 2-ABT, APMT, 
and serotonin on the number of mitoses on the crypts of the small 
intestines of mice at different times after 1500 r. 


4 


inhibited with pretreatment of 30 mg of MEA. In the 
same year, Wang et al. (147) demonstrated that MEA 
was capable of preventing transient sterility in male 


mice during the 2 or 3 months after x-ray exposures of 


625 r to the whole body or 700 r to the testes alone. 
Maisin and Doherty (unpublished data) have shown that 
the transient sterility in mice protected with AET after 
total-body irradiation with goo r was reduced as com- 
pared to mice protected with bone marrow. 

Lung. Dunjic et al. (48) showed that the LD5) pulmo- 
nary syndrome, which appears after x-irradiation of the 
thorax, was increased 34.9% when MEA was given 
before irradiation. 

Liver. Fumagalli and Malaspina (53) demonstrated 
that, in rats protected with cysteamine and irradiated 
with 800 r, the glycogen content of the liver returned to 
normal after 24 hours. In the nonprotected animals res- 
toration occurred after 96 hours. Armellini ef al. (4) 
demonstrated some protective effect of the mitochondria 
under the same conditions, but there was no difference 
in the alkaline phosphatase activity as compared with the 
controls (52). 

Tumor Tissue. Chemical agents that could protect the 
mammal against the systemic effects of radiation but not 
the tumor tissue might be useful in radiotherapy. With 
regard to the sulfhydryl compounds, such action would 
depend on their being selectively distributed to the 
hematopoietic and intestinal system and not to the tumor 
tissue. A. D. Conger and Doherty (unpublished observa- 
tions) found that labeled AET given orally to mice was 
rapidly taken up by these two systems but only very 
slowly by an Ehrlich ascites tumor. Whole-body lethal 
x-irradiation caused regression of the tumor and doubled 
the survival time of the mice over that of the controls. 
O’Gara et al. (108) in a more extensive study of AET 


Volume 19 


distribution, found that the concentration of AET in 
tumor tissue varied widely depending on tumor type. 
Initial results with AET and radiotherapy in man (J. V, 
Schlosser, personal communication) indicate no decrease 
in tumor radiosensitivity. Cohen and Cohen (30) re- 
ported that MEA did not protect a transplanted mam- 
mary tumor against large doses of locally administered 
x-radiation. The similarity in marrow damage caused by 
radiation or alkylating agents prompted the use of sulfhy- 
dryl compounds as protective agents against nitrogen 
mustards. Cysteine pretreatment reduced leukopenia in 
animals given nitrogen mustard (25, 56, 148) as did 
MEA (56) and AET (118). 

Local Effects. Darcis and Gilson (37) and Darcis and 
Hotterbux (38) showed that the vagina and rectum are 
protected against local x-irradiation of the pelvis after 
application of cysteamine. Herve and Brumagne (64) 
demonstrated that cystamine administered by ionopho- 
resis prevented epilation in rats after local irradiation 
with 1000 r. Finally, Hofman (65) showed that cysteine 
injected subcutaneously or applied in ointment was 
effective against erythema of the rat tail. 

Combined Treatment. Some authors have tried combined 
treatments of various types in an attempt to increase 
the radiation tolerance. Maisin et al. (94) showed that 
liver shielding combined with MEA gave better sur- 
vival at 700 r than the chemical alone. At 850 r, a com- 
bination of shielding and MEA was better than either 
alone. In 1954 and 1955, Maisin et al. (g1) and Burnett 
and Doherty (27) demonstrated that, when MEA or 
AET was given before irradiation and isologous bone 
marrow was given afterward, the LDj99/3) Was increased 
as compared with the protection offered by either alone. 
Maisin and Doherty (95) have shown that a combination 
of a large dose of AET and a small dose of serotonin was 
highly toxic to strain A-1 mice. They also showed that a 
less toxic combination of a low dose of AET with a high 
dose of serotonin administered before 1500 r of X-rays 
gave about the same intestinal protection as a low dose 
of AET The combined treatment, however, 
brought about some spleen and hematopoietic recovery. 
The improvement of intestinal and hematopoietic re- 
covery depends on the quantity of AET present (95 and 
unpublished data, fig. 5) and may be the explanation 
for the good survival time that Wang and Kereiakes 
(146) obtained in mice irradiated with lethal and supra- 
lethal doses and treated with a combination of small 
doses of MEA, AET and serotonin. 

The protection offered by the optimal combined treat- 
ment compared with the protection obtained by the 
optimal dose of AET alone shows some improvement in 
recovery time. The combination of cysteine, MEA, and 
cystamine pretreatment with anoxia increases the 30-day 
survival over chemical treatment alone (40, 145). Other 
less successful attempts were made. Langendorff et al. 
(87) have shown that reserpine, which has a low pro- 
tective effect at the LDso/3%9 exposure level, added to 
MEA gave no added protection. Kedrova and Krikhova 


alone. 





Ju 


04: 


and 
toni 


(73 
AC 


MOD 


S 
prir 
tor 
tot 
den 
the 
peri 
tart 
prot 
etal 
on | 
rege 
does 
lishe 
Doh 
testi 
for 
mito 
dose 
dose 





lume 19 


AT in 

type. 
(]s.Ng 
crease 
0) re- 
mam- 
stered 
sed by 
ulfhy- 
rogen 
nia in 


is did 


is and 
m are 
; after 
> (64) 
opho- 
liation 
steine 
t was 


ibined 
crease 
d that 
r sur- 
| com- 
either 
urnett 
EA or 
- bone 
reased 
alone. 
nation 
in was 
that a 
a high 
X-rays 
v dose 
wever, 
covery. 
tic re- 
y5 and 
nation 
reiakes 
supra- 
small 


| treat- 
ry the 
ient in 
A, and 
30-day 
Other 
F et al. 
WwW pro- 
Jed to 
ikhova 





July 1960 





n> Oo 
45016 //” —AET, 10mg, ORAL+ 
Th \ p SEROTONIN, { ma, 
ra e [P+ {500 f 
4 a7 
Fé p 
« - 
? AET, 7mg, ORAL+ 
« SEROTONIN, 
4 “d 05 mg, IP 
4 °o 
\ He ‘ 
a o—oO 
10- 


er a 
. \ 267, {6 mg, ORAL + 
nr 1500 r 


SEROTONIN, {mg, IP + 
{500r 


TOTAL BONE MARROW COUNT x10® 


\ 
{1500 r 





01 TIME AFTER IRRADIATION (days ) 
is eee Sas ee eee ee 
FIG. 5. Recovery of bone marrow of mice irradiated with 1500 
and 450 r and treated with AET, serotonin, and AET and sero- 
tonin combined. 





(73) demonstrated in rats that a combined treatment of 
ACTH and cysteine hastened the death of the animals. 


MODES OF ACTION 


Since the chemical compounds seem to be effective 
principally in actively dividing tissues, the most sensitive 
to radiation damage, they must afford some protection 
to the nuclei. No direct evidence has been produced to 
demonstrate whether the protective effect is operative in 
the cells during the rest period, the mitotic cycle, or both 
periods. Trimethylcolchicinic acid methyl ether-p- 
tartrate, a mitotic inhibitor, is reported to be an active 
protective agent (133). Hagen et a/. (60) and Langendorff 
etal. (82) claim that MEA exerts some protective effect 
on DNA synthesis in normal liver but is ineffective in 
regenerating liver. AET at optimal doses protects but 
does not produce mitotic inhibition (Maisin, unpub- 
lished observation). Maisin et al. (97) and Maisin and 
Doherty (95), using the mitotic count in the small in- 
testine, found that there was an optimal radiation dose 
for the demonstration of maximal AET protection of 
mitotic activity and that the optimal AET-serotonin 
dose was not much more effective than the optimal AET 
dose alone. This finding suggests that, no matter what 


XUM 


CHEMICAL PROTECTION OF MAMMALIAN TISSUE 569 


the mechanism of protection, the presently available 
compounds are not able to protect actively dividing 


‘ tissue above a certain dose of radiation (2500 r in mice). 


The initial idea that radiation damage from low doses 
in mammals arose primarily from the direct interaction 
of radiation and biological material has been largely dis- 
proved by a mass of experimental evidence, and it is 
now assumed that the major damage to biological tissue 
is caused by the free radicals arising from irradiated 
water. Anoxia, by reducing the yield of free radicals 
formed from water, provides effective protection. The 
evidence favoring anoxia in the case of histamine and its 
derivatives is less direct. In 1956 van Bekkum and de 
Groot (15), and van der Meer et al. in 1958 (105), showed 
that histamine, although an excellent protective agent 
in the mouse, is not effective in a suspension of thymo- 
cytes. Their hypothesis was that it interferes with the 
blood supply to blood-forming organs, thereby lowering 
their oxygen tension; later van der Meer and van Bekkum 
(104) were able to demonstrate indirectly that anoxia was 
the zn vivo mechanism of histamine. Their evidence was 
that the time course and degree of spleen protection 
followed closely the degree of spleen anoxia, as measured 
by an implanted oxygen electrode. Gray et al. (59) had 
already suggested that anoxia explained the protective 
action of 5-hydroxytryptamine. Bacq et al. (8) proposed 
a more precise action for fluoroacetate by involving a 
reduction of the oxidative metabolism of the cells or ac- 
cumulation of citrate, which would complex with intra- 
cellular magnesium ions and affect DNase activity. 

The problem of mechanism in the case of the active 
sulfhydryl compounds is complex and has been the sub- 
ject of intensive study. Evidence has been produced in 
support of three modes of action: a) lowered oxygen ten- 
sion; b) reaction to form mixed disulfides with biological 
molecules, making them more resistant to radiation; and 
c) free radical trapping. Regardless of the mechanism, 
the most important requirement for a good protective 
compound is that it reach the radiation-sensitive sites. 
This has been established for the hematopoietic and in- 
testinal systems, by use of tracer-labeled compounds, for 
MEA by Eldjarn and Nygaard (50) and for AET by 
Bradford et al. (24) and Maisin and Doherty (95 and 
unpublished data). Selective intracellular distribution of 
certain mercaptoalkylguanidines was found, along with 
all types of binding, i.e., freely dialyzable, mixed di- 
sulfide and strongly bound by other means (28). Auto- 
radiograms with tritium-labeled AET confirm § the 
variation in tissue concentration and show clearly not 
only intercellular distribution but also penetration into 
the cytoplasm and nucleus (fig. 6) at every phase of the 
mitotic cycle. After 8 hours, most of the tritium-labeled 
compound has been eliminated, although small amounts 
could be found up to 3 days after injection (95 and un- 
published data). 

The anoxia mechanism has received little new support 
since its proposal by Mayer and Patt (103) and Salerno 
(124) in 1953, and it is probably not the most important 
mechanism. Indeed, evidence that anoxia and sulfhy- 





FiG. 6. A: Megakaryocyte of a normal mouse 30 min. after the 
injection of tritium-labeled AET. The nucleus and the cytoplasm 
are labeled. B: Section of a mouse testis 5 min. after the injection 
of labeled tritium AET. Note the labeling of the nuclei of cells 
in mitosis. 


dryl compounds are additive in their effect (40) argue 
against it. Van der Meer and van Bekkum (104) also 
indicate that MEA and the dithiocarbamates operate 
through a process other than anoxia. Eldjarn and co- 
workers (51, 126) postulate that sulfhydryl compounds, 
by forming temporary mixed disulfide bonds with the 
sensitive sulfhydryl groups of proteins, protect them from 
radiation damage. Free-radical attack on the mixed 
disulfide splits the linkage, forming a sulfonic acid and 
a sulfhydryl group, which half the time is that of the 
native protein. The mixed disulfide may also provide 
protection against the direct effect in that energy ab- 
sorbed within the protein molecule could be transmitted 
to the disulfide bond and liberated by its rupture in a 
similar manner. Unaffected molecules would be rapidly 


REFERENCES 


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430, 1959. 

2. ALEXANDER, P., Z. M. Bacg, S. F. Cousens, M. Fox, A. 

Herve anv J. Lazar. Radiation Research 2: 392, 1955. 


FEDERATION PROCEEDINGS 








Volume 19 





returned to their native state by the disulfide-reducing 
systems of the body. The experimental evidence in sup- 
port of this view is that labeled MEA is bound to proteins 
through a disulfide linkage; the active protective com- 
pounds ?n vitro rapidly form mixed disulfides with cystine, 
the less active reacting more slowly. In many instances, 
the zn vivo protection and the in vitro rate of disulfide bond 
formation may be correlated. 

The radical-trapping function of sulfhydryl com- 
pounds has been emphasized by Alexander et al. (2) 
and Doherty et al. (43), who stress the importance of 
chemical structure in producing compounds that can 
effectively compete for free radicals with biological 
molecules. Structural studies with the mercaptoalkyl- 
amines and guanidines show that the active ones differ 
from the less active in that the proximity of the basic 
group to the sulfhydryl group lowers the pK of the latter 
so that, at physiological pH, the molecules exist largely 
in the ionized form, thus making them good electron 
donors and trapping agents. The increased activity of 
the mercaptoalkylguanidines over the mercaptoalkyl- 
amines may be explained by the two additional nitrogen 
atoms, which should increase resonance stabilization 
after quenching a radiation-induced radical by electron 
donation and result in increased stability and less ag- 
gressive activity toward biological material than the 
water radicals. It is not possible at this time to resolve the 
controversy over how sulfhydryl compounds act. Indeed, 
there is no good reason why only one mechanism must be 
involved, especially in the complex mammalian system, 
to explain their action. Radical scavenging is somewhat 
more appealing because it does not limit protection to 
only those proteins with accessible sulfhydryl and di- 
sulfide groups but also could function with all other 
biological compounds and thus open the way to the 
examination of radical-scavenging agents completel: 
unrelated to sulfhydryl compounds. 


CONCLUSION 


The progress made in the last decade in chemical 
protection against radiation damage is encouraging 
Although the two best compounds, AET (33) and MEA 
(26), may be too toxic for extensive human use at hig) 
radiation dose levels (33), they should prove useful it 
furthering our understanding of the functioning of pro’ 
tective compounds in mammalian tissue. Increased effor! 
in this direction, especially with regard to the effects 0 
protective compounds in larger animals and in specifi 
systems—including tumors, should lead to the discover 
of more effective and less toxic substances. Such chem; 
icals with low toxicity and differential distribution \ 
normal and tumor tissue, could find considerable ap 
plication in radiotherapy. 


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4. ARMELLINI, C., 
1: 88, 1958. 


P. FUMAGALLI AND G. Pisanr. Radiobiol. latis 








30. 
oT, 


32. 
33: 


34 


35: 














olume 19 


ducing 
in sup- 
roteins 
> com- 
ystine, 
tances, 
e bond 


com- 
al. (2) 
ince of 
at can 
logical 
oalkyl- 
s differ 
e basic 
e latter 
largely 
lectron 
ivity of 
roalkyl- 
itrogen 
lization 
lectron 
less ag- 
an. the 
rIve the 
Indeed, 
must be 
system, 
mewhat 
ction to 
and di- 
ll other 
to the 
npletel; 


-hemical 
uraging 
id MEA 
at high 
aseful it 
r of pro: 
ed effor 
-ffects 0! 
| specifi 
liscover\ 
h chem 
ution 1) 


able ap 


. (Moseou 


abiol. lalis 





July 


~ aon 


9° 


© 


10. 
Il. 


rot 


30. 
31. 
32. 
33: 


— 
nN 


43. 


oo 


4 


45. 


uw 


YUM 


. Bacg, Z. M., G. DecHamps, P. 


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ur 
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150. 





; Rixon, BR. A. 


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Volume 19 


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Vi 


pil 








lume 19 


p. 122; 
Nature, 
<ERING. 


/, 1955. 
fed. 92: 


» 1957. 
2: 364, 


adiation 


> 1955: 
iG. In: 
onference 
Nations, 


TRAUBE, 


4: 435, 


IE. Am. 
PIE. Am. 
Radiation 
BRUuES. 
& Med. 
» W. H. 
tol. 171: 
J. Nat. 
OHERTY. 
LE. Acta 


)HAPIRA. 


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>. Exper. 
_ Am. J. 


6, 1953: 
NAGLER. 





Genetic aspects of implantation of 


blood-forming tissue’ 


ELIZABETH S. RUSSELL 
Roscoe B. Jackson Memorial Laboratory, Bar Harbor, Maine 


Por ALMOST 5 YEARS (16) it has been known that blood- 
forming tissue from one individual may, under certain 
circumstances, be implanted successfully in a different 
individual and may continue to function in the new 
host for a considerable period of time. Even earlier it 
was known, from mixed populations of erythrocytes in 
twin calves, that blood-forming tissue of two distinct 
genotypes could function permanently in a single indi- 
vidual (20). Possible implications of these findings for 
human welfare have been widely recognized, and many 
further implantation experiments have been carried out 
both in experimental mammals and in man. Aside from 
purely theoretical interest in the phenomenon and its 
complexities, experiments involving transplantation of 
blood-forming tissue tend to be directed toward solution 
of three serious biomedical problems (30): a) use of 
implants of blood-forming tissue as protection against 
lethal effects of extremely heavy doses of radiation (in- 
cluding x-irradiation administered in the hope of stop- 
ping leukemic growth); 6) use of previously injected 
homologous blood-forming cells to promote acceptance 
of other tissue (such as skin grafts or kidney transplants) 
from the same or related homologous source; or c) use 
of implanted normal blood-forming tissue in the treat- 
ment of hereditary anemia. 

In this paper discussion will center about recent de- 
velopments of two distinct types, in both of which the 
use of genetically controlled experimental animals has 
contributed greatly to understanding of potentialities for 
solution of each of these three biomedical problems. The 
first type of contribution from animals of known geno- 
type is provision of genetically-determined character- 
istics, differing between host and donor, which mav be 
ued to test implantation and persistence of injected 
tissue, and to analyze host-graft interactions. The second 
type of contribution is the demonstration that the specific 
genetic nature (both of host animal and of implanted 
hlood-forming tissue) and the genetic relation between 
donor and host, are important factors contributing to 
success of implantation and persistence of the implant. 


'This work has been supported by a contract between the 
Roscoe B. Jackson Memorial Laboratory and the U. S. Atomic 
Energy Commission and by a grant to the Roscoe B. Jackson 
Memorial Laboratory from the American Cancer Society. 


’ 
ost aad 


Since the literature in this field is very extensive, com- 
plete review here is impossible; consequently, only se- 
lected examples dealing with house mice will be pre- 
sented. Although not all of even the most significant 
recent contributions can be covered, relevant findings 
from our own research group, involving use of normal 
and hereditarily anemic mice (4, 5, 30, 33) will be 
summarized. 

The genetically-determined characteristics, allowing 
identification of implanted cells, may tag hematopoietic 
or myeloid cells in tissue, or their descendants in the 
flowing blood. Presence in bone marrow or lymphatic 
tissue of cells characteristic of the donor rather than of 
the host phenotype, indicates persistence of implanted 
cells. Homologous and heterologous donor hematopoietic 
cells in marrow and lymph nodes have been identified 
through chromosome differences in mitotic figures (12, 
13). Rat erythropoietic and myelopoietic cells and 
megakaryocytes have been recognized histochemically 
in the marrow space of previously irradiated and in- 
jected mice by a positive alkaline phosphatase reaction 
(13, 19, 49). Rat thymocytes in mouse thymus have 
been identified by agglutination reactions (13). Mouse 
and rat esterase-positive tissue proteins in homogenized 
liver, thymus, spleen, bone marrow and lymph nodes, 
and in serum, mav be distinguished bv electrophore- 
sis (26). 

Presence in the flowing blood, long after implanta- 
tion, of cells characteristic of the donor rather than host 
phenotype, indicates not only persistence of implanted 
blood-forming cells but also their autonomous differen- 
tiation and functioning in a genotypically foreign en- 
vironment. Leukocytes of rat origin in mouse blood may 
be recognized by alkaline phosphatase staining (13). 
Presence of rat thrombocytes in mouse blood has been 
demonstrated serologically by agglutination with specific 
antimouse platelet sera (36). Several different genetically 
determined features allow identification of homologous 
as well as heterologous erythroctyes. 

Heterologous Erythrocytes. Rat erythrocytes in the circula- 
tion of radiated implanted mice may be identified by 
immunological tests (18, 46), by differential mechanical 
fragility (18), and by alkali denaturation, crystallization 
or electrophoresis of hemoglobin (18). 

Homologous Serotypes. Serological tests have been used 








574 


TABLE 1. Pertinent Characteristics of W Series Anemias 

1) Difference from normal caused by substitution at a single 
genetic locus (WW? for ww) 

2) Affected animals characterized by life-long severe macro- 
cytic anemia 


Adult ww Adult WW?” 


RBC/mm# (X 10°) [1-12 6-7 
Hematocrit (%) 44-47 35740 
MCV 40-45 50-60 


3) Erythropoietic tissue maturation arrest basic to defect. 


very effectively in identifying homologous mouse eryth- 
rocytes in the circulation after irradiation and im- 
plantation (45) and in determining proportion of donor 
and recipient erythrocytes in x-irradiated mouse 
chimeras (23). 

Homologous Differences in Hemoglobins. Differences among 
mice in the electrophoretic pattern of hemoglobins have 
proved very useful in identifying homologous erythro- 
cytes in chimeras (23). Two easily distinguished types of 
hemoglobin (electrophoretically homogeneous versus 
diffuse) have been demonstrated in normal healthy 
mice, and segregation ratios presented, suggesting that 
the difference between the two hemoglobin patterns is 
controlled by a single gene substitution (14, 27). Dif- 
ferential salting out of separable hemoglobins has pro- 
vided a convenient method for detecting each hemo- 
globin in heterozygous animals and in chimeras (23). 
Linkage tests have established that the locus responsible 
for electrophoretic pattern of hemoglobin is in the first 
genetic linkage group near albinism (24, 25). Numerous 
inbred strains of mice are known to have hemoglobins 
of each of these types (25, 27, 28, 31, 48), making possible 
the identification of homologous erythrocytes in many 
different host-graft combinations. 

Other Autonomous Genetic Differences in Erythropovesis. 
Hereditary anemias are always characterized by reduced 
numbers of erythrocytes in the flowing blood, and fre- 
quently also by unusual size, shape, hemoglobin content 
or fragility of erythrocytes. These abnormalities may in- 
clude autonomous characteristics, determined by the 
intrinsic nature of the blood-forming tissue, and de- 
pendent characteristics, inflicted upon the blood cells or 
their precursors by correlative influences from other 
parts of the body. Successful transplantation of blood- 
forming tissue between normal and genetically-anemic 
individuals provides a basis for identifying autonomous 
abnormal erythrocyte characteristics. Once recognized, 
these autonomous characters become useful in dis- 
tinguishing between erythrocytes of donor (usually 
normal) and host (usually genetically anemic) origin. 
This approach has proved very successful in analysis of 
a hereditary macrocytic, normochromic anemia of 
maturation arrest produced in the mouse by action of 
deleterious genes of the W series. Features of this anemia 
critical to the present paper are given in table 1. The 
particular autonomous characteristics that have proven 
useful in identifying erythrocytes of donor origin are 
hematocrit value and mean cell volume (4, 5, 30, 33). 


FEDERATION PROCEEDINGS 





Volume 19 


From this summary it is abundantly clear that a great 
variety of genetically-determined characteristics have 
been located that make possible recognition of cells from 
both heterologous and homologous implants of blood- 
forming tissue. 

Protective effect of heterologous and homologous 
implants of blood-forming tissue after heavy total-body 
irradiation has been demonstrated repeatedly, but the 
protection afforded is seldom complete. Some, but not 
all, of the treated individuals survive the critical 30-day 
period immediately after irradiation. Others die be- 
tween 30 and 100 days after irradiation, frequently with 
a very characteristic syndrome, called ‘homologous 
disease’ (38-40), ‘secondary disease’ (3) or ‘foreign bone 
marrow reaction’ (45). It is characterized by three 
symptoms: emaciation, diarrhea and severe dermatitis 
(40). In most cases of isologous implantation, in which 
donors genetically completely compatible with the ir- 
radiated hosts are used, 100% of treated individuals 
survive, and ‘secondary disease’ is not seen (30, 33, 38- 
40, 45-47) (fig. 2, survival). 

Much evidence has accumulated indicating that death 
of host animals, after heavy irradiation and injection 
of foreign blood-forming tissue, usually results from an 
immunological reaction between host and donor. Since 
the injected tissue, especially that from adult bone mar- 
row or spleen, includes immunologically competent 
cells, it is possible to postulate both host-versus-graft 
and _ graft-versus-host reactions. Evaluation of both 
these possibilities, and understanding of the homologous 
disease syndrome, have been greatly advanced by recent 
elegant experiments (fig. 1) in which genetically con- 
trolled normal mice are used (3, 17, 21, 23, 38-41, 43, 
44, 49), including a) use of hosts and donors from inbred 
strains differing in particular named histocompatibility 
genes [alleles of the H-1, H-2, and H-3 series (21, 37)]; 
b) reciprocal transplants of F, hybrid cells into parent 
inbred-strain hosts and parent strain cells into F, hybrid 
hosts (fig. 1, 2a and 2b) and c) transplants between 
specially prepared strains in which a histocompatibility 


lost and donor from inbred 
stroins differing ot specific 
histocompotobility loci : 


@ Reciprocal tronsplonts between 
F, hybrids ond porent inbred 
stroins: 


% CC Ye 
( 
wr & C57BL/6J (CS7BL/6xF.\A))F o> comes 
¥ CD © oe n-2e H-24H-2® H-2°H-2° 


@ Special inbred strains with histo- 
compotobility gene transferred by 
repeoted bock- crossing: 


Xo >» 
C57BL/10. 02 


. —) 
iG) 
8 


C57BL/6J (C57BL/6 xA/J) F; CS7BL/IO 
H-2°H-2° H-2" H-2° H-2°H-2° H-2°H-2° 
FIG. 1. Mouse genetic combinations for study of host-graf 


interactions. 





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July 1960 


gene normally carried by one inbred strain has been 
transferred to a different inbred strain by repeated back- 
crossing. From these studies it appears that host-versus- 
graft reactions occur most often after radiation doses in 
the midlethal range, and _ graft-versus-host reactions 
after lethal radiation doses (fig. 2). This has become espe- 
cially clear in certain unilaterally incompatible implants 
between F, hybrids and parent inbred-strain animals. 
In these, the F, hybrid animal finds nothing foreign in 
tissues from its parent type, whereas parental strain 
animals react against the F; hybrid because of its foreign 
antigens. 

Trentin has introduced a generalization concerning 
mortality after transplantation that fits the majority of 
observations. According to his concept, early mortality, 
within 5-21 days after irradiation and implantation 
(fig. 2, early death) usually follows sublethal radiation 
doses, and tends to be associated with potentiality for 
host-versus-graft reaction. The host is interpreted as 
retaining sufficient immunological reactivity to reject 
the foreign blood-forming tissue, and then as dying from 
lack of functioning hematopoietic cells. Delayed mor- 
tality (21-100 days after irradiation and implantation), 
however, has been observed after heavier completely 
lethal radiation doses. He postulates that the lethal radia- 
tion dose destroys host capacity both for hematopoiesis 
and for immunological reactivity, so that there is estab- 
lishment of grafted cells, some of which differentiate 
into immunologically competent types. These cells then 
are considered responsible for a later graft-versus-host 
reaction leading to slow death through homologous 
disease. Importance of lymphoid cells in production of 
homologous disease has been demonstrated (15, 49). The 
tissue source of injected cells (bone marrow, spleen or 
fetal liver) also contributes significantly to probability 
of homologous disease (2, 9, 42, 45). Although Trentin’s 
interpretation fits many of the existing data, information 
critical for testing the mechanism suggested by his 
hypothesis is frequently lacking. Some of his assumptions 
(.e., permanent loss of host hematopoietic and immuno- 
logical capacity after lethal irradiation) need to be 
tested in many different situations, through direct deter- 
minations of persisting and functioning cell types (21- 
23, 49). 

Incidence and severity of homologous disease appears 
to vary according to the particular host-donor combina- 
tion used. In some combinations, parent-strain cells 
injected into F; hybrid hosts are accepted without com- 
plication, but in other combinations parent-strain cells 
lead to a high incidence of homologous disease. Success- 
ful implantation without homologous disease was ob- 
served with injection of homologous BALB/c blood- 
forming tissue into DBA/2 hosts subjected to irradiation 
of midlethal intensity (17). It happens that these two 
inbred strains match in their H-2 genes. This finding is 
one of many that support a second generalization con- 
cerning homologous implantation, emphasized _par- 
ticularly by Uphoff (43, 44). Action of genes of the H-2 


* 
ase tala 


IMPLANTATION OF BLOOD-FORMING TISSUE 





Injected 
Lymph Node Cells 




































8 
a O& ~ ADSL 
Bone Marrow 
EARLY DEATH @ = HOST ANTIBODY 
[A]=GrarT Sie 
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RADIATION DEATH 





Fic. 2. Differing reactions following total-body x irradiation 
and implantation of blood-forming tissue. 


series is exceedingly important in both host-versus-graft 
and graft-versus-host reactions that follow transplanta- 
tion of blood-forming tissue; some degree of mortality 
is always observed if host and donor differ in H-2 alleles, 
even if the remainder of the genotype is very similar. 
Present evidence suggests that difference between host 
and donor in genes of the H-1 and H-3 series is not a 
serious barrier to implantation of blood-forming tissue 
(44). 

Before leaving the general discussion of homologous 
disease, I would like to mention recent very valuable 
studies with parabiosis, rather than radiation and im- 
plantation of blood-forming tissue. In a study of genetic 
factors influencing success of parabiosis, Eichwald et al. 
(11) observed reactions very similar to the homologous 
disease of radiated-implanted animals and produced 
with similar genetic combinations. They found H-2 dif- 
ferences to be much more deleterious than H-1, H-3, or 
Y chromosome histocompatibility differences. The para- 
biotic intoxication syndrome appeared after mixture of 
flowing blood had ceased, and appeared to involve 
myeloid hypertrophy and almost certainly implantation 
of stem cells after exchange between parabiotic partners. 

In a few situations, mortality after heavy irradiation 
and implantation cannot be attributed to immunological 
reactions. In one exceptional experiment, in which 
normal hosts and donors were used, death was regularly 
observed after heavy irradiation even with isologous im- 
plantation, (fig. 2, radiation death). When CBA mice, 
with an LDj of 650 r, were subjected to 950 r and then 
given a standard injection of CBA bone marrow cells, 
71 % died within 100 days, without evidence of secondary 
disease (3). The high supralethal dose appears to have 








576 


resulted in radiation damage (the authors suggest this 
may involve the intestinal mucosa) that is not reparable 
by implantation of blood-forming tissue. 

Experiments in our laboratory, involving implantation 
of normal ww hematopoietic cells (usually from the fetal 
liver) into severely anemic WW* hosts have yielded in- 
formation pertinent to analysis of mortality following 
radiation and implantation. The LDsp of the anemic hosts 
in these experiments is between 200 and 300 r (33). In 
one experiment injection of completely isologous normal 
ww cells resulted in 18/18 surviving after 200 r, 2/9 after 
600 r, 1/8 after 800 r and 0/2 after 950 r (5). Whenever 
the irradiated anemic hosts survived, the injected cells 
implanted rapidly, changing the blood picture per- 
manently to that of a normal mouse. There was no evi- 
dence of homologous disease. The deaths seem to have 
been related to the extreme radiosensitivity of the WW 
anemic host, which, after these high supralethal radia- 
tion doses, cannot be counteracted even by isologous im- 
plants (fig. 2, radiation death). These last two examples 
(CBA hosts after 950 r, WWv anemic hosts) with mor- 
tality after irradiation and isologous implantation, 
demonstrate that the specific genetic nature of the host 
animal may be very important in determining its re- 
action to radiation, and this reaction, as well as the 
genetic relation between host and donor, may be critical 
for success of hematopoietic implantation. 

Irradiated WW” anemic hosts do, however, maintain 
their ability to reject homologous implants, particularly 
from inbred strains with different H-2 genes (5). Al- 
though seven of nine WW animals subjected to 200 r 
and then injected with homologous cells, survived more 
than 30 days, none developed a normal blood picture. 
With heavier radiation doses followed by homologous 
cell injections, 0/18 anemic hosts survived 500 r, 2/16 
survived 800 r and 0/12 survived 950 r. The anemics 
surviving after 800 r gave no evidence of implantation. 
The increase in mortality (2/46 surviving 600-950 r) 
over that observed with comparable radiation and 
treatment with isologous cells (3/19 surviving 600-950 r), 
and the failure of implantation in survivors, indicate re- 
tention of immunological reactivity of the host. It appears 
that different organ systems of the same individual may 
differ greatly in their relative and absolute radiosensi- 
tivity, and that these internal differences may contribute 
greatly to the complexities of radiation-implantation 
reactions. 

The second biomedical problem for which use of im- 
planted blood-forming tissue has been suggested is in the 
promotion of acceptance of transplants of other tissues, 
such as skin or kidney, from donors genetically similar to 
the hematopoietic tissue donor. All available evidence 
on induction of tolerance indicates necessity of im- 
plantation of injected hematopoietic cells (or at least of 
injected reticular, myeloid or some kind of stem cells). 
Acquired immunological tolerance was first produced 
in mice (8) by injection of cells (usually spleen) from 
adult mice of the donor genotype into fetal or newborn 


FEDERATION PROCEEDINGS 








Volume 19 


mice. As adults, these pretreated recipient mice were 
tolerant of skin transplants of donor genotype, maintain- 
ing indefinitely grafts they would otherwise have re. 
jected in g-11 days. This method was extremely suc- 
cessful in the first genetic combination tried (strain A 
into strain CBA), even though host and donor differed 
at the H-2 locus; but with other genetic combinations it 
was greatly complicated by a syndrome called ‘runt dis- 
ease’ (6, 7), which has many similarities to the homolo- 
gous disease already described. One characteristic of 
runt disease is marked lymphoid hypoplasia (7, 15). 
Current investigations of runt disease present a some- 
what confusing picture, which I will not attempt to 
elucidate completely, but the studies suggest that runt 
disease may be caused by a graft-host immunological 
reaction. Runted survivors of the disease, however, ap- 
pear to be tolerant of donor-type skin grafts (8). There 
is considerable evidence implicating injected spleen 
cells of C57BL mice as major villains in the production of 
runt disease (8, 11), indicating once more the impor- 
tance of specific genotypes (this time of the donor), in 
addition to the already recognized importance of 
genetic relation between host and donor. 

As regards treatment of adult hosts, acceptance of 
skin grafts between two inbred strains of mice has in cer- 
tain cases been promoted by impiantation of donor-type 
hematopoietic tissue into irradiated adult hosts. The 
high mortality and incidence of homologous disease ex- 
perienced with implantation of blood-forming tissue 
have tended to limit interest in this method, however, 
particularly when host and donor differ at the H-2 
locus. The method has been successful, and uncompli- 
cated by homologous disease, in transplants from BALB/c 
donors into midlethally irradiated DBA/2 hosts (300- 
600 r), but not in transplants from BALB/c donors into 
lightly irradiated (100 r, 200 r) DBA/2 hosts (17). It may 
be significant tac untreated mice of these two strains 
normally reject each other’s skin grafts relatively slowly, 
and that the two strains carry the same H-2 genes, al- 
though they have been estimated to differ at approxi- 
mately 13 other histocompatibility loci (17). Current 
experiments by Berrian (29), based on spleen and sub- 
sequent skin transplants between donors and _ adult 
hosts from certain of Snell’s isogenic resistant inbred 
lines differing only at particular histocompatibility loci, 
should be mentioned. These studies confirm the sugges 
tion that although H-2 differences are equally important 
for acceptance of all cell types tested, H-3 differences are 
important for acceptance of skin transplants, but not of 
spleen transplants. Spleen injections, from donors differ- 
ing from the nonirradiated host in H-3 genes only, appeat 
to have been accepted, since subsequent grafts of donor 
skin were permanently maintained. Thus recent studies 
suggest that something resembling tolerance may bt 
induced in adults with certain genetic combinations by 
pretreatment with donor-type spleen or other hemate 
poietic tissue. This finding increases the probability that 
preimplantation of blood-forming tissue may some daj 





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olume 19 


> were 
intain- 
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some- 
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July 1960 


become useful in man for promotion of acceptance of 
other tissues, since it removes the necessity of treating 
newborns and risking runt disease. Before this could 
happen, however, there would have to be a great in- 
crease in knowledge of the tissue compatibility genes of 
man, and their tissue specificities. Unless there is close 
relation in man between blood-group genes and tissue- 
compatibility genes, acquiring knowledge of genetic 
control of tissue compatibility in the human being may 
be very difficult. To make use of this as yet nonexistent 
knowledge for promotion of graft acceptance, we would 
have to make sure that all recipients and donors of 
blood-forming tissue, skin or kidneys were very carefully 
matched. 

There remains for discussion the question of implanta- 
tion of blood-forming tissue in the treatment of anemia. 
The possibility of this use was presented in the discussion 
of autonomous erythrocyte characteristics. Limitations 
imposed by the special radiosensitivity of WW” hosts, 
and importance of H-2 gene differences in the rejection 
of homologous normal cells by irradiated anemics have 
also been presented. Three further important contribu- 
tions have resulted from studies of normal blood-forming 
tissue implants in W series anemic hosts. In our experi- 
ments, the anemia of WW” adults can regularly be cured 
by injection of cells from the hematopoietic liver of 
normal embryos differing from the anemic adult re- 
cipient only by the W gene substitution, or from parent 
pure-strain normal embryos (4, 30, 33). In the initial 
experiments in which anemic hosts were subjected to 
200 r, implanted pure-strain cells in the F; hybrid WW? 
anemic hosts rapidly changed the blood picture to that 
of a normal mouse and maintained this picture through 
the remainder of the host lifespan, in the majority of 
cases for more than 500 days (30). Permanent implanta- 
tion without irradiation of the host was also obtained 
regularly when isologous ww normal fetal liver cells were 
injected into adult anemic WW?” hosts, into juvenile 
WW” hosts, and into lethally anemic juvenile WW hosts 
(4). The only difference from the earlier experiments 
with lightly irradiated anemic hosts was a delay in the 
establishment of a normal blood picture. The proportion 
of success with nonirradiated anemic adult hosts (20/24) 
was similar to that found after 200 r irradiation of the 
anemic hosts (16/18). The proportion of success was 
lower with juvenile WW” hosts (9/14) and still lower 
with juvenile WW hosts (4/15), probably owing in part 
to trauma of operation in these poorly viable hosts and 
in part to spontaneous death of the host (in the critical 
preweaning growth period) before implanted cells be- 
came established. 

Current experiments in our laboratory (29) demon- 
strate that, although implants from donors differing at 
the H-2 locus are regularly rejected, complete isology 
between host and donor (except for W genes) is not re- 
quired for successful implantation of blood-forming 
tissue without irradiation of the host. Two closely related 


WILMA 


IMPLANTATION OF BLOOD-FORMING TISSUE 


577 


inbred strains maintained in our laboratory, WB and 
WC, both carry H-2* but tend to reject each other’s skin 
grafts very slowly. In a very interesting experiment car- 
ried out by S. E. Bernstein, anemic WW? animals from 
(WB X C57BL)F: or (WC X C57BL)F; mice were 
used as hosts, normal ww embryos from the other hy- 
brid as donors of blood-forming tissue. In 15 of 17 cases, 
the blood picture changed permanently to that of a 
normal mouse, and in 14 of these 15 cases subsequent 
grafts of skin from the donor genotype were maintained 
permanently. Acceptance of skin grafts was also pro- 
moted with injections of blood-forming cells from one 
of the hybrid types into normal animals of the other 
hybrid type, suggesting implantation in this case where 
direct evidence could not be obtained. In a smaller ex- 
periment, ww fetal liver from the WC inbred strain has 
been injected into lethally anemic WW newborn anemic 
mice. One of seven injected WW individuals grew to 
adulthood and survived more than 6 months with a 
normal blood picture. 

All these experiments with nonirradiated anemic hosts 
fit with a hypothesis of competition between a large body 
of indigenous defective, slow-acting blood-forming tissue 
of the genetically anemic host and an initially small im- 
plant of rapidly functioning blood-forming tissue from 
the genetically normal donor (4). This phenomenon of 
increasing effect from the implant may be different 
from what would be experienced if both host and donor 
could form blood rapidly, and may be related to the 
specific nature of this anemia, known from other evi- 
dence to involve a definite maturation arrest (1, 34). 

The change in blood picture of W series anemic mice 
after implantation of normal blood-forming tissue demon- 
strates clearly that this anemia results from the genet- 
ically-controlled functioning of the blood-forming tissue 
rather than from any correlative influence from other 
parts of the body. With this knowledge at hand, it is 
now possible for us to test many other aspects of the 
physiology of these anemic animals. S. E. Bernstein (un- 
published data) found that successfully implanted ane- 
mics have a radioresistance very close to that of compara- 
ble normal mice, indicating that the extreme radio- 
sensitivity of the anemic mice is directly related to their 
defective blood formation. Previously reported experi- 
ments, done in collaboration with Keighley, Borsook 
and Lowy at the California Institute of Technology (32) 
have demonstrated that the WW" anemic mice do not 
respond to exogeneous erythropoietin but do respond 
normally to lowered oxygen. The cellular basis of this 
abnormality may be tested with implanted anemic mice. 

The implications for man of the experiments with im- 
plantation of anemic WW mice are that a) at least one 
type of hereditary anemia may be cured by implantation 
of normal blood-forming tissue, if the relation between 
host and donor is sufficiently close and of the proper 
nature; and 6) that there are genetically-controlled 
differences in the functioning of blood-forming tissue. 





1. 


4- 


. Brrurncuas, R. E., L. 


. Core, L. 
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. GLitcksoHN-WAELsCH, S., H. 


) fomey, P. . T.. 


. Makrnopan, T. 
. Nowe tt, P. C., L. J. Core, P. L. Roan ano J. G. HABER- 


London 181: 488, 1958. 


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Cancer Inst. 23: 75, 1959. 
BERNSTEIN, S. E. ano E. S. Russevy. Proc. Soc. Exper. Biol. 
& Med. 101: 769, 1959. 


. BERNSTEIN, S. E., E. S. RussEtt AnD F. A. Lawson. Transpl. 


Bull. 24: 106, 1959. 


>. BrttincHaM, R. E. anv L. Brent. Transpl. Bull. 4: 67, 1957. 
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AND L. Brent. Phil. Trans. Roy. Soc. 
London, Ser. B 242: 439, 1959. 

) BRENT AND P. B. MEDAWAR. Nature, 
London 172: 603, 1953. 


J. Am. J. Physiol. 196: 441, 1959. 


144: 198, 1956. 

LusTGRAAF AND M. STRAINER. J. 
Nat. Cancer Inst. 23: 1193, 1959. 

J. L. Hamerton, D. W. H. Barnes anv J. F. 
Loutit. Nature, London 177: 452, 1956. 

C. Conepon, A. D. CoNGER 
AND T. Makinopan. Proc. Soc. Exper. Biol. @ Med. 96: 714, 
1957- 

M. RANNEY AND B. SIsKEN. J. 
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sel 
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Th 
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acti 
test 
thy 
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lume 19 


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Ontogeny of the blood cells 


ROYAL F. RUTH 
Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 


Biseann ORGANS AND ORGAN SYSTEMS, the hemopoietic 
system provides one of the most striking and prolonged 
displays of embryonic processes. Throughout most of life 
it is characterized by extensive cellular proliferation, mi- 
gration, sequestration and differentiation. An abundance 
of morphological data and a lesser amount of isotopic 
evidence have established the quantitative importance of 
these phenomena in the adult. 

The work of Ottesen (25), Kindred (17) and Yoffey 
(30), among others, indicates that some lymphoid tis- 
sues synthesize DNA very actively, this synthesis is ac- 
companied by frequent mitotic division, and more cells 
enter the circulation than return from it. Morphological 
studies by Yoffey and isotopic studies by Fichtelius (10) 
suggest that many of the lymphocytes leave the circula- 
tion in the spleen and bone marrow. Fichtelius has pre- 
sented evidence that the site at which circulating 
lymphocytes are segregated is influenced by their place 
of origin. 

Fichtelius injected 0.75 we of P*-phosphate/gm of 
body weight into rats 24 hours before preparing a sus- 
pension of cells from the thymus or lymph nodes. Ap- 
proximately 250,000 cells in 1 ml of such suspensions 
were injected intravenously into 140-gm littermates of 
the donors. Twenty-four and 48 hours later, the thymus, 
lymph nodes and several other organs were removed 
from the recipients. The DNA of these organs was iso- 
laed and wet-ashed, and the activity was counted. 
These results were compared with the activity of the 
DNA fractions of the same organs after injection of P® 
phosphate and after injection of labeled thymus and 
lymph node cells from the rabbit. In terms of the total 
activity of the liver, mucous membrane of the small in- 
testine, bone marrow and_ spleen the homologous 
thymus cells preferentially labeled the spleen, and the 
homologous lymph node cells preferentially labeled the 
bone marrow. The differences were statistically signifi- 
cant at the 0.1 % level. The injection of P® as inorganic 
phosphate, or as labeled thymus or lymph node cells of 
the rabbit, did not produce preferential labeling of 
either spleen or bone marrow. These and corollary re- 
sults suggest that the complex exchange of lymphocytes 
between the hemopoietic tissues and the circulation 
nay be influenced by specific lymphocytic mechanisms. 

If the fate of the lymphocyte is determined by its 
place of origin, the lymphocytes of different lymphoid 
organs may be inflexibly and genetically different or 


XUM 


uw 


adaptably and functionally different. Is there any sub- 
stantial evidence that the lymphocytes are genetically 
diverse? At present we seem to have no adequate in- 
formation for the adult animal. On the other hand, there 
is evidence that the embryonic origin of the lympho- 
cytes is quite diverse and that they may even be derived 
from two different germ layers, the mesoderm and the 
endoderm. If this is so, such differently derived lympho- 
cytes may be as functionally and genetically distinct as 
any other derivatives of the mesoderm and endoderm. 

Several lymphoid organs, the thymus, some mam- 
malian tonsils, and the bursa Fabricius of birds have a 
dual origin from endodermal buds and mesenchymal 
condensations. The dual origin has been recognized for 
many years, and the possibility that the lymphocytes of 
these organs may be derived from the endoderm has 
been seriously considered. This view seems to have 
been nullified effectively by Jolly’s interpretation (16) 
that mesenchymal cells invade the endodermal epithe- 
lium and transform it into a mixed epithelium, which 
then forms lymphoid nodules. For the past 30 years, the 
lymphocytes of these nodules have usually been thought 
of as mesodermal. 

Auerbach and Ball (3) have cultured the mesodermal 
and endodermal components of the mouse thymus in the 
anterior chamber of the eye and in vitro. The endoderm 
does not produce buds in the absence of the mesoderm, 
but will do so when mesoderm is present or is added. 
More recently, Auerbach (2) found that the mesoderm 
will induce endodermal proliferation through a 20-u 
Millipore filter. This means that the first step is an in- 
ductive process conceptually comparable to the induc- 
tion of the lens, neural tube or cartilage. A convincing 
in vitro demonstration of endodermal lymphocytopoiesis 
would mark a historic advance in basic hematology. 

Pera (27) and Meyer et al. (21) have reexamined the 
earliest origins of the bursa Fabricius of the chicken. 
Their interpretations differ only slightly. Pera recog- 
nizes it on the 4th day of incubation as epithelial clumps 
present near the point at which the disintegrated caudal 
intestine connected with the cloaca. Both recognize it on 
the 5th day as a projection from the wall of the cloaca. 
Ackerman and Knouff (1) and Meyer et a/. have re- 
ported that the lymphoid nodules first appear on the 
12th day as purely endodermal proliferations. No meso- 
dermal contribution is evident. 

It is much easier to reach such a conclusion for the 





FEDERATION PROCEEDINGS 


Volume 19 











lume 19 





tt ee Wee 





July 1960 


bursa than for the thymus, because of its simpler forma- 
tion. Figures 1 and 2 show the appearance of the bursa 
and the spleen on the 12th and 13th days of incubation, 
respectively. The figures show that lymphoid follicles are 
produced by the bursal endoderm before any follicles 
appear in the spleen. In fact, comparable follicles do not 
appear in the spleen until after hatching. 

Figure 3 is a high-power view of a typical endodermal 
bud on the 12th day. Figure 4 is a comparable view of 
the spleen of the same animal on the same day. Basophilic 
blast cells can be seen in the spleen. These occur in 
groups of fewer than six cells and do not occur as folli- 
cles. In contrast, the basophilic cells of the endodermal 
buds of the bursa multiply to form definitive follicles 
by the 13th day. At this time, some basophilic cells seem 
to pass into the surrounding mesenchyme (fig. 5), as de- 
scribed by Ackerman and Knouff. 

The incidence of mitotic figures in the bursal endo- 
derm, the apparent passage of basophilic cells from the 
nodules to the mesenchyme, and the accumulation of 
chromatic debris first in the nodules (fig. 6) and then in 
the epithelium (fig. 7) signify proliferation and migra- 
tion on a large scale. We do not know where these cells 
go, what they do or how long they survive. However, 
there are good grounds for supposing that the bursal 
lymphocytes play an important role in antibody produc- 
tion. 

Bursectomy during the first 2 weeks after hatching 
sharply reduces the ability of the chickens to produce 
circulating antibodies (7, 8, 12, 13, 22). Splenectomy is 
much less effective. (Complete thymectomy is a difficult 
and traumatic operation, best suited to the dog, and 
dependent on extensive histological controls.) Since 
bursectomy does not affect body growth (B. Glick, Ph.D. 
Thesis, Ohio State University, 1955), its effects on anti- 
body production cannot be readily ascribed to general 
trauma. Whatever the mechanism, bursectomy lowers 
the responsiveness to antigens. In this context, the bur- 
sectomized animal is somewhat comparable to the new- 
born or x-irradiated animal. 

Large, basophilic cells, morphologically similar to the 
lymphoblasts of the adult animal, appear in many other 
embryonic sites before they appear in the thymus, bursa 
and tonsils. Most lymphoid organs have no known endo- 
dermal component. Their lymphocytes are presumed to 
come from cells of purely mesodermal origin. In general, 
lymphocytes are recognized by lack of good cytoplasmic 
criteria, i.e. the relative absence of crystals, granules, 
phagocytized debris and accumulations of protein. A 


Fic. 1. Bursa (B) and spleen (S) of 12-day chick embryo. Dark 
lining of lumen of bursa is endoderm. Toluidine blue, pH 4.0. 
X 30. 

Fic. 2. Bursa (B) and spleen (S) of 13-day chick embryo. Dark 
spots in the bursa are lymphoid follicles. Toluidine blue, pH 4.0. 
X 30. 

FIG. 3. Bursa of 12-day chick embryo. Formation of two primi- 
tive lymphoid follicles from endodermal epithelium. Toluidine 
blue, pH 4.0. X 850. 

FG. 4. Spleen of 12-day chick embryo. Scattered basophilic 
blast cells, but no follicles. Toluidine blue, pH 4.0. X 850. 


YUM 


ONTOGENY OF THE BLOOD CELLS 581 


morphological definition of the small, and supposedly 
mature, lymphocyte is largely a statement of what it 
lacks. The nucleus is small, dense, and has no nucleoli. 
The cytoplasm is a thin basophilic layer containing a 
few mitochondria and a few nonbasophilic areas. A large 
lvmphocyte has a large nucleus and cytoplasm, at least 
one nucleolus, and a diffuse cytoplasmic basophilia. The 
large numbers of morphologically similar lymphocytes in 
both the mesodermal and endodermal lymphoid organ 
suggest that the lack of morphological criteria may be 
poor grounds for assuming lymphocytes to be func- 
tionally equivalent or to have the same fates. On the 
contrary, there is considerable evidence that the meso- 
dermal and endodermal lymphoid organs and their 
constituent cells respond quite differently to some of the 
hormones. 

During sexual maturation the thymus becomes smaller, 
and the bursa, of all but the most primitive birds, in- 
volutes completely (5, 29). The involution of the thymus 
and the bursa can be simulated by injecting cortisone 
and some other steroids (15, 18, 28). Androgens are par- 
ticularly effective against the bursa (14). Injection of 
0.6 mg of 19-nortestosterone into the egg white on the 
5th day of incubation completely inhibits the develop- 
ment of the bursa of the chicken, according to Meyer et al. 
This inhibition is detectable within 8 hours. However, 
there are differences that seem to be fundamental be- 
tween the events of sexual maturation and the effects of 
hormonal treatments. 

The normal involution of the rat thymus is accom- 
panied by changes in the cell population of the thymus, 
lymph nodes and spleen. According to Kindred, the pro- 
portion of small thymic cells increases and the proportion 
of large lymph node and spleen cells increases. Treat- 
ment of the intact or adrenalectomized rat with cortisol 
or cortisone inhibits lymphocytopoiesis in all these 
lymphoid organs. Treatment of the intact rat with 
androgens or estrogens also inhibits lymphocytopoiesis, 
but treatment of the adrenalectomized rat is supposed to 
stimulate lymphocytopoiesis. Thus the sex hormones 
may have dual effects on the lymphoid tissues of the 
intact animal. One of these is an inhibition that is 
mediated by the adrenocortical steroids, and the other 
is a stimulation that is not (g). Continued treatment of 
the adrenalectomized rat with estrogens, however, de- 
creases the size of the thymus (6). The interpretation of 
the effects of hormone is greatly complicated by the lack 
of cytological data for the first few days after initiation 
of treatment. 


FIG. 5. Bursa of 13-day chick embryo. Basophilic blast cells 
escaping from the follicle into the mesodermal tissue. Toluidine 
blue, pH 4.0. X 850. 

FIG. 6. Bursa of 20-day chick embryo. Chromatin debris in one 
of the lymphoid follicles. Feulgen. X 850. 

FIG. 7. Bursa of 21-day chick embryo. Chromatin debris in the 
endodermal epithelium. Feulgen. X 850. 

Fic. 8. Spleen of guinea pig. F cell (F). Nucleus forms a dark 
cap on one side of large body of carbohydrate. Azure A Feulgen, 
periodic acid-Schiff’s and naphthol yellow AS. X 850, 




































































582 FEDERATION PROCEEDINGS Volume 19 
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TIME AFTER TREATMENT (days) TIME AFTER TREATMENT (days) TIME AFTER TREATMENT (days) 
FIG. 9. H and F cells of male guinea pig spleens after estradiol terol), —0.30; H (diethylstilbestrol), —0.02; F (cholesterol) 
pig sp : y 


treatment. Coefficient of regression: H (sesame oil), —4.21; H 
(sesame oil + estradiol), — 2.16; F (sesame oil), +0.31; F (sesame 
oil + estradiol), + 0.19. 

ric. 10. H and F cells of female guinea pig spleens after di- 
ethylstilbestrol treatment. Coefficient of regression: H (choles- 


In striking contrast to the thymus and the bursa, the 
lymph nodes of mammals and the lymphoid nodules of 
the spleen of mammals and birds may diminish, but 
they do not involute either before or during sexual 
maturation. This suggests that endodermal lympho- 
cytopoiesis is more susceptible to sexual maturation than 
mesodermal lymphocytopoiesis, or that the mesoendo- 
dermal organ becomes a less favorable environment for 
lymphocytopoiesis during sexual maturation. Kindred’s 
data suggest that a cellular shift from the endodermal 
to the mesodermal lymphoid organs may occur. There is 
no experimental evidence with which to decide this 
question. There is some evidence, however, that estro- 
gens not only affect the proliferation of endodermal 
lymphocytes but influence their synthetic activities as 
well. 

In 1889, Kurloff (19) reported that agranular blood 
cells of intact and splenectomized guinea pigs contain 
discrete bodies that may be as large as the cell nucleus. 
In the same year, Foa and Carbone (11) found similar 
bodies in cells of the blood and spleen and reported 
them to be especially numerous in the spleens of preg- 
nant guinea pigs. Splenectomy does not decrease the 
number of such cells in the circulation, but gonadec- 
tomy does (4). Testosterone does not increase the num- 
ber of circulating cells, but as little as g ug of estradiol 
dipropionate increases the proportion from 0.2-0.5 % 
of the total leukocytes on the 4th day after injection to 
15-26 % on the 12th day (20). In general, the increase 
becomes detectable about the 6th day. 

Figure 8 shows typical Foa-Kurloff body cells (F 
cells) in the spleen. The cells are colored with a modi- 


+2.78; F (diethylstilbestrol), + 10.03. 

Fic. 11. H and F cells of male guinea pig spleens after treat- 
ment with dinitrophenol and diethylstilbestrol. Coefficient of re- 
gression: H (dinitrophenol), —1.54; H (dinitrophenol + diethyl- 
stilbestrol), +1.52; F (dinitrophenol), +0.35; F (dinitrophenol + 
diethylstilbestrol), + 16.7. 


fied periodic acid-Schiff’s procedure for macromolecular 
carbohydrate (26). The single, large body of the mature 
F cell stains intensely; it is round or ovoid and has a 
smooth, regular surface. Sometimes the bodies contain 
an unstained area or vacuole, but otherwise they are 
homogeneous, when properly prepared. The cell nucleus 
forms a crescentic cup on one side of the body. The 
appearance of the body does not, in itself, tell us whether 
it is a product of secretion or phagocytosis. This is a 
critical point because the macrophages of the guinea 
pig, like those of other animals, frequently contain 
carbohydrate. 

The macromolecular carbohydrate of the phagocytes 
appears as heterogeneous and weakly stained masses of 
irregular shapes and diffuse boundaries. Occasional 
phagocytes contain very small granules of carbohydrate, 
which cannot be readily distinguished from the small 
bodies of immature F cells. After prolonged stimulation 
with diethylstilbestrol, some of the phagocytes contain 
bodies that appear to be phagocytized and altered F cell 
bodies. Despite these difficulties, it has been possible to 
show that the spleen cells containing heterogeneous car- 
bohydrate (H cells) and the F cells act like two separate 
cell populations. 

A comparison of 10 female and 10 male guinea pigs, 
after sham treatments (no estrogen), shows more H cells 
in the spleens of males and more F cells in the spleens of 
females. The means of H cells are 12.1 for the females 
and 39.7 for the males. The means of F cells are 32.5 
for the females and 7.7 for the males. The differences 
between sexes and the differences between éells are sig- 
nificant at the 1 % level. 





Wwe 


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July 1960 


Figure g indicates the numbers of H and F cells in the 
spleens of 13 pairs of male guinea pigs sacrificed at 1-day 
intervals 1-13 days after treatment. The control ani- 
mals received a subcutaneous injection of 0.05 ml of 
sesame oil and the test animals received a comparable 
injection containing 10 ug of estradiol/100 gm of body 
weight. The slopes of the lines indicate that there were 
decreases in the numbers of H cells in both control and 
test animals but no change in the numbers of F cells. 
The differences between cells are significant at the 1% 
level for both treatments. 

Figure 10 indicates the numbers of H and F cells in 
the spleens of 10 pairs of female guinea pigs sacrificed at 
1-day intervals 1-10 days after treatment. The control 
animals received subcutaneously a pellet of cholestercl 
and the test animals a pellet of diethylstilbestrol. The 
slopes of the lines indicate that there were no changes 
in the numbers of H cells, but the number of F cells in- 
creased greatly after treatment with diethylstilbestrol. 
The differences between cells are significant at the 1% 
level for both treatments. The differences between treat- 
ments are significant at the 1 % level for F cells but not 
for H cells. 

Figure 11 indicates the numbers of H and F cells in 
the spleens of 7 pairs of male guinea pigs sacrificed at 
2,3, 5, 7, 9, 11 and 13 days after treatment. The control 
animals received no pellet; the test animals received sub- 
cutaneously a pellet of diethylstilbestrol. Both groups 
were placed on a diet containing 0.2 % 2, 4-dinitrophenol, 
as a means of inducing a state of stress. The diet was 
begun on the day of treatment. All animals not sacrificed 
in the first few days showed a considerable loss of weight. 
The slopes of the lines indicate that there was no change 
in the number of H cells, but the number of F cells in- 
creased greatly after treatment with diethylstilbestrol. 
The differences between cells are significant at the 1 % 
level for both treatments. The differences between 
treatments are significant at the 1 % level for F cells but 
not for H cells. 

Table 1 shows the means of H and F cells in the 
spleens of 8 pairs of female guinea pigs sacrificed, at 
random intervals, 10-47 days after treatment. One 
animal of each pair received a subcutaneous pellet of 
cholesterol and an intrasplenic pellet of diethylstil- 
bestrol and the other animal received a subcutaneous 
pellet of diethylstilbestrol and an intrasplenic pellet of 
cholesterol. The correlation within pairs of animals is 
significant at the 1 % level for F cells; it is insignificant 
for H cells. The differences between treatments are in- 
significant. The correlation between cells is highly signifi- 
cant for the animals receiving a subcutaneous pellet of 
diethylstilbestrol and is insignificant for those receiving 
an intrasplenic pellet of diethylstilbestrol. The differ- 
ences between cells are significant at the 1 % level. 

Table 1 shows that several weeks’ treatment with 
diethylstilbestrol increases both H and F cells. More- 
over, these increases show a higher degree of correlation. 
The increase in H cells seems then to be a direct function 
of the increase in F cells, which precedes it. A large 


XUM 


ONTOGENY OF THE BLOOD CELLS 


583 


TABLE 1. H and F Cells of Spleens After Prolonged 
Diethylstilbestrol Treatment* 


Intrasplenic implantation 
| of diethylstilbestrol pellet 
Subcutaneous impiantation 


Subcutaneous implantation 
of diethylstilbestrol pellet 
Intrasplenic ————— 


Kind of cells of cholesterol pellet of cholesterol pellet 
Means for each group 
H 37-6 35-0 
78.6 


F 179.6 I 


* Eight pairs of female guinea pigs. 


number of these H cells appear to contain degenerating 
F bodies. This agrees with previous reports that estrogen 
stimulates the phagocytic activity of the guinea pig 
spleen, but it disagrees with a previous interpretation 
that estrogens are a specific stimulant of phagocytosis 
(23, 24). Coniinued treatment of the guinea pig with 
estrogens does stimulate phagocytosis, but part of this 
increase is secondary in time and magnitude to the in- 
crease in F cells and can hardly be considered specific. 

Figures 12-17 show the changes in the thymus 2-11 
days after subcutaneous implantation of a pellet of 
diethylstilbestrol. The cortex of the thymus is enlarged 
on the 3rd day and absent on the 5th day. In its place 
are cells with larger nuclei. Some of these cells contain 
small F bodies. On the 7th, gth and 11th days these 
bodies are progressively larger and approach the size 
found in increased numbers in the spleen from the 
7th day on. These changes occurred in animals that had 
been placed on the dinitrophenol ‘stress’ diet and had 
lost weight rapidly. None survived to the 15th day. The 
thymuses of the control animals that did not receive 
diethylstilbestrol did not show these changes. Thus the 
initial proliferation of the thymic cortex and its subse- 
quent diminution would seem to be caused by the 
estrogen. 

The initial proliferation of the thymic cortex, followed 
by the appearance of small thymic F cells, and the subse- 
quent appearance of large F cells in the spleen, suggest 
that some of the mature F cells of the spleen may originate 
in the thymus. The present data are not sufficient to es- 
tablish statistically the transfer of young F cells from the 
thymus to the spleen. They do suggest, however, that a 
decisive demonstration is feasible. The theoretical con- 
sequences make this highly desirable. The relative 
abundance of these cells in the thymus and the spleen, as 
compared to the lymph nodes and the bone marrow, sug- 
gests that the F cell may provide the first cytological 
proof of a selective transfer of thymic cells to the spleen. 

The evidence suggests that the lymphocytes arise from 
a) endoderm and }) mesoderm, and that the differences 
in origin impart differences in behavior and function. 
The application of this concept of duality to the blood 
cell population as a whole gives a novel emphasis to 
several established observations. i 

In the chicken embryo, erythrocytes and polymor- 
phonuclear granulocytes appear first in mesodermal 





16 

FIG. 12. Thymus of guinea pig 2 days after subcutaneous im- 
plantation of pellet of diethylstilbestrol Figures 12-17 stained as 
in fig. 8. XK 850. 

FIG. 13. Thymus of guinea pig 3 days after treatment. Cord- 
like arrangement of cells is characteristic of rapid proliferation in 
thymus of young guinea pig. 

Fic. 14. Thymus of guinea pig 5 days after treatment. Cords of 
cells are absent. Foa-Kurloff body cells (F) have appeared. 


areas; lymphoid follicles and eosinophilic granulocytes 
appear first in endodermal areas, such as the bursa and 
the thymus. This suggests that the mesoderm produces 
most of the erythrocytes and polymorphonuclear granu- 
locytes, and the endoderm produces most of the lympho- 
cytes and eosinophilic granulocytes. Furthermore, the 
special effectiveness of cortisone and related steroids as 


FEDERATION PROCEEDINGS 








FIG. 15. Thymus of guinea pig 7 days after treatment. Bodies 
of F cells are slightly larger. 

Fic. 16. Thymus of guinea pig 9 days after treatment. Bodies o/ 
F cells are slightly larger. 

FIG. 17. Thymus of guinea pig 11 days after treatment. Bodies 
of F cells are slightly larger, but not so large as that in fig. 8. 


ie 





inhibitors of the endodermal hemopoietic organs i 
paralleled by the ease with which these hormones simul 
taneously increase the numbers of erythrocytes and poly: 
morphonuclear granulocytes while decreasing the num 
bers of lymphocytes and eosinophilic granulocytes. We 
can conclude that the different kinds of blood cells can bt 
classified as mesodermal or endodermal, and that thos 





Volume 19 





Vo ES BS 


> 

























olume 19 


it. Bodies 
Bodies 0! 


nt. Bodies 
ig. 8. 


rgans i 
es simul 
nd poly: 
he nut 
ytes. We 
Is can be 
hat thos 


July 1960 


derived from the same germ layer will show certain func- 
tional similarities despite their morphological and chemi- 
cal differences. What implications does this conclusion 
have for the problem of radiation protection? 

The thymus, which is particularly susceptible to some 
of the steroids, can transmit the capacity to produce cir- 
culating antibody to a recipient animal but cannot pro- 
tect it from the lethal effects of preinjection irradiation. 
The bone marrow can protect the recipient animal from 
the lethal effects of preinjection irradiation. This sug- 
gests that the endodermal hemopoietic organs cannot 
produce the large population of erythrocytes, granulo- 
cytes and platelets that are required for radiation pro- 
tection but can produce antibody-synthesizing cells. 
The contrasting effects of bone marrow and spleen lead- 
ing to host-versus-graft and graft-versus-host reactions, 
respectively, suggest that the bone marrow is relatively 
rich in nonantibody-producing cells and the spleen is 
rich in both antibody-producing and nonantibody-pro- 
ducing cells. According to our hypothesis, this means 
that the protective effect is attributable to mesodermal 
cells and antibody production to endodermal cells. We 
argue that the plasma cell is probably endodermal. 

Some human beings lack y-globulins and plasma cells. 
Occasionally such patients have anomalies of the thymus, 
but there is not sufficient evidence to establish a correla- 
tion. These patients do, however, possess certain im- 
mune mechanisms. They resist viral infections, produce 
hypersensitive reactions, and destroy homologous grafts 


ONTOGENY OF THE BLOOD CELLS 585 


but cannot produce detectable circulating antibody. 
Since hypersensitive reactions can be transmitted by 
leukocytes, this suggests that the classical separation of 
immune reactions into antibody-mediated and cell- 
mediated types may reflect the endodermal origin of 
plasma cells and the mesodermal origin of the cells that 
transmit hypersensitivity. 

The evidence suggests that the blood cells arise from 
the endoderm as well as the mesoderm and that this 
difference in origin imparts differences in behavior and 
function. In addition, it implies that the endodermal 
lymphocytes may migrate to other sites before or during 
the involution of the organs in which they first appear. 
In embryological terms, the two types of lymphocytes 
have different prospective fates. This does not imply 
that they are incapable of performing the same functions, 
or of differentiating in the same directions. It only states 
that they normally react to some stimuli in different 
ways. These differences may be caused by their locations, 
inherent in their origins from different germ layers, or a 
result of location and inheritance. A proper elucidation 
of the mechanisms by which these dual populations 
arise obviously depends on studies of the early embryo. 
The possible significance of these dual populations in the 
adult may be more accessible through studies of both 
the embryonic and newborn animal. 


I express my thanks to Dr. R. Auerbach and Dr. H. R. Wolfe 
for copies of manuscripts in press and to Dr. J. D. Ebert for access 
to proof. 


REFERENCES 


1. ACKERMAN, G. A. AND R. A. Knourr. Am. J. Anat. 104: 163, 
1959. 

. AUERBACH, R. Developmental Biol. In press. 

. AUERBACH, R. ano W. D. BALL. Anat. Rec. 134: 530, 1959. 

. Basuptert, B. Rend. ist. sanita publ. 1: 1, 1938. 

. Beacu, J. R., O. W. ScHatm aAnp R. F. LuspBenusen. Poultry 
Sct. 13: 219, 1934. 

. Broun, S. E. anp B. HELLMAN. Acta anat. 20: 155, 1954- 

. Cuanc, T. S., M. S. Ruemns anp A. R. Winter. Poultry Sct. 
36: 735, 1957. 

. Cuanc, T. S., M. S. Ruermns anp A. R. Winter. Poultry Sct. 
37: 1091, 1958. 

9. Doucuerty, T. F. In: The Kinetics of Cellular Proliferation, 

edited by F. Stohlman, Jr. New York: Grune, 1959, p. 264. 

10. FicHTevius, K. E. Acta anat. 32: 114, 1958. 

i. Foa, P. U. anp T. Carsone. Beitr. path. Anat. 5: 227, 1889. 

. Guick, B. Poultry Sci. 34: 1196, 1956. 

13. Guick, B. Poultry Sci. 37: 240, 1958. 

14. Grick, B. Poultry Sct. 39: 130, 1960. 

15. HOun, E. O. Canad. J. Biochem. & Physiol. 34: 90, 1956. 

16. Jotty, J. Arch. Anat. micr. Morph. exp. 16: 363, 1915. 


2) of CO KR 


~~ 


co 


n 





wWLilhéA 


17. Kinprep, J. E. Ann. New York Acad. Sc. 59: 746, 1955. 
18. KirKPATRICK, C. M.. AND F. N. AnpreEws. Endocrinology 34: 
340, 1944. 


19. Kurtorr, M. G. Quoted by P. Ehrlich and A. Lazarus. 
In: Nothnagel’s Encyclopedia of Practical Medicine, edited by A. 
Stengel. Philadelphia: Saunders, 1905, p. 73. 

20. LepincuaM, J. C. G. J. Path. Bact. 50: 201, 1940. 

21. Meyer, R. K., M. A. Rao anp R. L. AsPInALL. Endocrinology 
64: 890, 1959. ; 

22. MUELLER, A. P., H. R. WoLFe AND R. K. Meyer. J. Immunol. 
In press. 

23. Nicot, T. anp I. D. Hetmy. Nature, London 167: 199, 1951. 

24. Nicot, T., I. D. Hetmy anp A. Apou-Zixry. Brit. J. Surg. 
40: 166, 1952. 

25. OTTESEN, J. Acta physiol. scandinav. 32: 75, 1954- 

26. Pearse, A. G. E. J. Clin. Path. 2: 81, 1949. 

27. Pera, L. Arch. ital. anat. e embriol. 63: 407, 1958. 

28. Serve, H. J. Morphol. 73: 401, 1943. 

29. Tater, A. M. Riv. biol. 18: 416, 1935. 

30. Yorrey, J. M. Ann. New York Acad. Sc. 59: 928, 1955. 








Advances in radiation immunology 


T. MAKINODAN 
Biology Division, Oak Ridge National Laboratory,' Oak Ridge, Tennessee 


By SEARCHING FOR A SUITABLE MODEL for differentiation, 
students of various disciplines have been attracted to 
antibody formation. The scope of this attraction is illus- 
trated by the variety of theories that have been formu- 
lated, which comes about through the difference in ob- 
jectives when the problem is approached by a biologist, 
a biochemist or a chemist (1, 4-7, 14, 21, 25, 40, 41, 44, 
45, 49, 52, 53, 57, 60). Although theories are numerous, 
very little factual information is available. We know 
that antigens injected parenterally into an individual 
stimulate the following sequence of events: a) the anti- 
gens are engulfed by macrophages, and 4) the antibodies 
then appear in mononuclear cells and in the circulation 
(10, 60). We do not know precisely what happens be- 
tween the two events. The use of heavily x-irradiated 
compatible animals as in vivo cultures for spleen cells 
from nonimmunized and immunized donors could enable 
us to evaluate these events quantitatively. The applica- 
bility of antibody formation as a model for differentia- 
tion therefore suggests that a brief survey of radiation 
immunology would be informative (for extensive litera- 
ture review, see references 13, 32, 55, 60). 

The three most outstanding events that stimulated in- 
terest in this field are a) the discovery, by Benjamin and 
Sluka in 1908 (3) and independently by Lawen in 1g09 
(28), that x-rays suppress primary antibody response, 
b) the atomic bomb blasts in 1945, and c) the demon- 
stration, by Jacobson and co-workers in 1949 and 1950 
(22, 23), that autologous relocation of cells from lead- 
shielded spleen of a heavily x-irradiated animal restores 
his immune machinery to almost normal capacity. The 
number of publications in the Journal of Infectious Dis- 
eases (1910-59), the Journal of Experimental Medicine 
(1g10-59) and the Journal of Immunology (1916-59) 
indicates the interest during the last 40 years. The curve 
in figure 1 shows essentially two periods of interest, the 
first in the early 1920’s and the second in the late 1940’s. 
The information accumulated during the last 10 years 
will receive main emphasis in this paper. 

TEMPORAL RELATION BETWEEN ANTIGEN 
INJECTION AND IRRADIATION 

Variations in species, type, route and concentration 

of antigen injected, time between primary and secondary 


! Operated by Union Carbide Corporation for the U. S. Atomic 
Energy Commission. 


J 


c 


86 


antigen injection, time of titration and criteria for im- 
munologic response, make it difficult to define unequiv- 
ocally the alterations induced in the antibody-produc- 
ing mechanism of animals given total-body x-irradiation. 
In general, however, the data shown in figure 2 are 
representative of those obtained recently by the different 
investigators. The patterns of curves are comparable, 
except that the secondary immune status of intact animals 
is more resistant to sublethal doses of x-rays to the total 
body than the primary immune status. 


ANTIBODY RESPONSE DURING MAXIMUM DEPRESSION 
PERIOD AFTER VARIOUS X-RAY DOSES 


Primary antibody response, regardless of differences in 
the variables mentioned, was depressed proportionately 
with increase in radiation dose. In contrast, reported 
results concerning the nature of secondary antibody 
response have differed sharply: some say that the sec- 
ondary response is equally as radiosensitive as the pri- 
mary antibody response, and others say it is more re- 
sistant than the primary (11, 19, 51, 56). This discrepancy 
encouraged us to reinvestigate this problem with the use 
of inbred animals and a wider range of x-ray dose. Our 
data, based on 70-80 mice per x-ray dose, show that, 
in the nonlethal x-ray dose range, the primary antibody 
response was noticeably depressed but the secondary 
antibody response was not (fig. 3); in the mid-lethal dose 
range, both responses were depressed. The sD5o radiation 
dose? is ~800 r for the secondary responders and ~600 
for the primary antibody responders, respectively. It 
should be emphasized here that, because studies involving 
intact animals merely reflect the result of the product of 
the number and types of cells participating in this phe- 
nomenon, extrapolation of these data to characterize the 
relative radiosensitive status of primary and secondary 
antibody-forming cells is not justified. 


CELLULAR CHANGES 


As we would expect inja field so complex, agreement 
on the sequential cellular events of antibody formation is 
not unanimous (10, 15,.16, 29, 36, 42, 48, 52, 59, 60). 
The combined observations of many investigators, how- 
ever, suggest that there are three phases of cellular 

2 Radiation dose necessary to suppress mean total antibody 
titer minus residual antibody titer by 50%. 


eve 
anc 
foll 


cap 
cap 
of 

gro 
stro 


clea 
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ype 








or im- 
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jation. 
2 are 
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e total 


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ported 
tibody 
1e sec- 
1e pri- 
ore re- 
>pancy 
the use 
e. Our 
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tibody 
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al dose 
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~600 
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ondary 


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ation is 
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s, how- 
cellular 


antibody 





July 1960 


20+ 


> 
1 


‘= NUMBER OF PUBLICATIONS 


NV 
i \aaadl\ ly m 


{90 1920 1940 {960 
Fic. 1. Relative interest in radiation ve since IgI0 

















(publications in J. Infect. Dis. 1910-1959, J. Exper. Med. 1910- 
1959, and J. Immunol. 1916-1959). 
X RAY 

lhe SE a et sin a Sea ep 
3572 ya 

- 

= 

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z 

2 

= 0 
254= a 

> N 

F = 
a 

lu 

io 
15+ \ PRIMARY 
10- 
0" © Sun 

10-5 0 5 © 15 30 40 50 


TIME OF ANTIGEN INJECTION BEFORE OR AFTER 710 r (days) 


FIG. 2. Effects of the temporal relation of 710 r and antigen 
injection on the immune status of mice (from T. Makinodan, B. 
H. Friedberg, M. G. Tolbert and N. Gengozian, J. Immunol. 83: 


184, 1959). 


events: phase I, involving eosinophils and macrophages; 
phase II, involving reticular cells and their descendants; 
and phase III, involving plasmacytes and lymphocytes. 
Associated with the suppressive effect of radiation, the 
following deleterious effects have been reported: a) the 
eosinophil response is inhibited (52); 6) the engulfing 
capacity of macrophages is not affected but the digestive 
capacity is reduced (13); c) the maturation and division 
of reticular cells to immature lymphoidal cells are 
grossly inhibited (60); and d) the lymphocytes are de- 
stroyed but the plasmacytes are relatively unaffected (9, 
6o, 61). Thus the data obtained through use of radiation 
clearly indicate that a concerted effort is now required 
toclarify the relation between phase I and phase IJ; i.e., 
whether phase I is coincidental to antibody formation or 
truly the initial phase of antibody formation. If the 
latter is true, we need to know whether specificity of 
antibody formation resides at this phase. We are also 
concerned with whether we are dealing with events in- 
volving a one-cell type a two-cell type or a multicell 
ype. 


XUM 


ADVANCES IN RADIATION IMMUNOLOGY 






















587 

« 0 
- 
S 100-0<° bam pig + 100 
z o 
< SDgq SECONDARY 
= 9 80+ ——Ab RESPONSE _} 80 
58 (0) S 
ee > 
5 c 
is - RESIDUAL PRIMARY 2] ~~ 
x < 
wi & SDgo PRIMARY ___ & 
F = 40. Ab RESPONSE (I) _ ss 
© a 
q 
w 207 + 20 
= 
a AGE CONTROL 
= fis Ne 
0 r 

0 200 400 600 800 100 


RADIATION DOSE (r) 


FIG. 3. Determination of sD; for primary and secondary anti- 
body response. 


RECOGNITION FACTOR OF ANTIBODY-FORMING MECHANISM 


The idea that the antigenic potency depends partly on 
the genetic relation between antigen and host has been 
implicitly accepted by many immunologists, but sys- 
tematic studies have been limited. Some progress has 
now been made through use of total-body x-irradiation 
(30, 31, 50). It has been found that normal adult mice 
respond to rat antigen equally as well as tosheep antigen, 
but irradiated mice respond to sheep antigen better than 
to rat antigen. Analogous results were obtained with bone 
marrow infusion studies (rat, hamster, guinea pig and 
rabbit bone marrow). Thus the more distant the genetic 
relation between host and donor, the higher the x-ray 
dose needed for persistence, temporary transplantation, 
or prolonged transplantation of the bone marrow. 
Younger nonirradiated mice responded to sheep, rabbit 
and rat antigens in decreasing order of magnitude, which 
is analogous to irradiated adult mice. These results not 
only suggest another avenue for studying the mechanism 
of antibody formation, but also offer some insight into 
problems pertaining specifically to tolerance induction 
in the newborn. 


MODIFICATION OF IMMUNE MECHANISM 
OF IRRADIATED ANIMALS 


There are three possible ways of modifying the im- 
mune status of an irradiated animal if there is no indirect 
effect of x-rays on antibody formation: a) administration 
of agents to accelerate the rate of recovery of the injured 
antibody-forming machinery, }) administration of agents 
to protect this machinery, and c) infusion of compatible 
antibody-forming cells. 

Jaroslow and Taliaferro (24) discovered that anti- 
body-forming capacity is restored in x-rayed rabbits 
when yeast and Hela extracts are injected with the 
antigen 1 day after 400 r. Subsequently, Kind and John- 
son (26) observed partial restoration of antibody-form- 
ing capacity of rabbits exposed to 400 r when bacterial 
polysaccharide endotoxin is administered no longer than 














588 FEDERATION PROCEEDINGS Volume 19 
1204 donor cells against the specific test antigen. We were 
TITER OF INTACT ANIMAL 7 therefore led to explore the use of genetically compatible, 
ae La heavily x-rayed animals as recipients of potential anti- 
— body-forming cells. Such recipients can be considered to 
be truly zn vivo tissue cultures. 
80-4 
ts IN VIVO TISSUE CULTURE 
604", The data accumulated in studies involving intact ani- 
8 mals are informative but limited. For example, it would 
404 seem almost fruitless to determine the minimum number 
of cells that participate in a given maximum antibody 
response of an intact animal. Also, it would probably be 
2.0- very difficult to demonstrate unambiguously the cellular 
relation between cause and effect in antibody formation. 





a9 038 O75 #15 30 60 120 240 
NUCLEATED SPLEEN CELLS (x10®) 


FIG. 4. Effect of x-rays on spleen cells. 


2 days after antigen injection. Since it is not yet known 
whether these compounds are effective at all radiation 
levels, how their restorative properties act cannot be 
explained. 

AET remains one of the best radiation-protective 
compounds. Our initial studies indicate that, although 
the antibody-forming machinery in 950-r mice pre- 
treated with AET is heavily damaged, the rate of re- 
covery is comparable to that of mice exposed to 475 r 
(37). These findings imply that protection of nonanti- 
body-forming cells by chemical pretreatment is impor- 
tant in hastening recovery of the immune status of heavily 
x-rayed animals. It ought to be stated here that there is 
no evidence to indicate that antibody-forming cells are 
the essential cells involved in protection against hema- 
topoietic radiation death. 

Modification of an irradiated animal’s immune mech- 
anism by an autologous relocation of spleen cells may be 
inferred from the lead-shielding studies of Jacobson and 
co-workers (22, 23). Since then, studies of cell transfer in 
irradiated animals have been limited mainly to use of 
sublethally x-irradiated, homologous recipients. The 
results demonstrated the positive role of transplanted 
splenic or lymphopoietic tissues in antibody formation 
(20, 27, 39, 47, 54). Nevertheless, the use of this model 
seems limited for quantitative studies, since the undesir- 
able foreign graft reaction occurs in sublethally irradi- 
ated recipients (2, 8, 12, 17, 18, 38, 58). The effects of 
any host-donor incompatibility, however subminimal, 
could conceivably affect the antibody formation by the 


Since we were convinced that these and other related 
problems are essential for a better understanding of the 
sequential events of antibody formation, preliminary 
studies were undertaken to establish the feasibility of the 
in vivo tissue culture method as a quantitative model 
(33-35, 46). We were able to demonstrate that a linear 
log, relation between primary or secondary antibody 
response and spleen cell number can be obtained regard- 
less of the strain of the mice and time of bleeding. The 
slopes of the curves, however, were dependent on the 
time of bleeding. Subsequently, in an attempt to sepa- 
rate the nonantibody-forming from the antibody-form- 
ing cells through a density gradient, a fourfold increase 
in activity was obtained by a fraction containing mainly 
large mononuclear cells. Work is now in progress to es- 
tablish the fate of these cells during antibody formation. 

The following information has been obtained from our 
studies now in progress. With intact mice, x-ray doses as 
high as 500 r had no depressing effect on the secondary 
antibody response, as measured by mean peak and mean 
total titers. The 6-day titer, however, was slightly de- 
creased after 500 r but not after 300 r. In contrast, our 
in vivo culture method showed that ~9g5 % of the spleen 
cell activity is destroyed after 400 r (fig. 4). Furthermore, 
12-24 X 10° spleen cells seem to be the minimum num- 
ber necessary to give a secondary antibody response com- 
parable to that of an intact mouse. If 500 r is assumed 
to be the minimum x-ray dose necessary to cause a de- 
tectable depressive effect on secondary antibody response 
in an intact mouse, these data would suggest that there 
are 1-2 X 10! spleen-like cells per gm of mouse (25 
gm). We might note in this connection that Osgood (43) 
calculated the total hemic cells in hematopoietic organs 
of a human being to be 1.1 X 107 per gm of body weight 
(70 kg). 


REFERENCES 


1. ALEXANDER, J. Protoplasma 14: 296, 1932. 

2. VAN Bexkum, D. W. anv O. Vos. J. Cell. & Comp. Physiol. 50, 
Suppl. 1: 139, 1957. 

3. Benjamin, E. ano E. 
1908. 

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oS ot os on 


33 


34 





ume 19 July 1960 
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oe 12. Drxon, F. J., W. O. WEIGLE anp M. P. DericHMILLeR. J. 
anti- Immunol. 82: 248, 1959. 
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t atk Immunol. 76: 151, 1956. 


would 18 


umber 19. 
ibody 20. 
bly be 
ular | 7" 
ation. J 49. 
elated 

of the | 23- 
inary 

of the | “* 
model 25. 
linear — 26. 


tibody f 27: 


egard- 8 
. The 
yn. the 

sepa- 


-form- | 3° : 
. Maxinopan, T. anp N. Gencozian. In: Proceedings of the 


crease 
nainly 


to es- 32. 


1ation. 
m our 
OSES as 


ondary f 34. 


| mean 
ly de- 
st, Our 
spleen 
rmore, 
) num- 
e com- 
sumed 
>a de- 
‘sponse 
t there 
ise (25 
rd (43) 
organs 
weight 


nes (and 


J. Nat. 


w IC 





XUM 


33- 


. Makinopan, T. 


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1953: 











INTERSOCIETY SYMPOSIUM ON NEW AND NEGLECTED HORMONES: 





Melatonin 


AARON B. LERNER AND JAMES D. CASE? 
Section of Dermatology, Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 


| YEARS AGO McCord and Allen (10) re- 
ported that a crude acetone extract of bovine pineal 
glands fed, or injected into, Rana pipiens tadpoles caused 
lightening of their skin. The same effect was observed 
when tadpoles were allowed to swim in water containing 
some of the acetone extract. McCord and Allen stated, 
“Thirty minutes after feeding pineal tissue, the tadpoles, 
which prior to the feeding had been uniformly dark, be- 
came so translucent that all the larger viscera were 
plainly visible through the dorsal body wall.’’ From the 
time of that report until now only a handful of papers has 
appeared on this subject. 

We became interested in the lightening effect of pineal 
extracts when we began to look for a neurogenic agent as 
the possible factor responsible for depigmentation in viti- 
ligo. Physiologic substances such as adrenaline, noradren- 
aline, acetylcholine etc. (4 13) lighten the color of frog 
melanocytes in vitro. Yet not one of them meets all the 
requirements for being the causative factor in vitiligo. 

Preliminary experiments with bovine pineal gland ex- 
tracts disclosed the presence of a substance that was ex- 
tremely potent in lightening frog skin in vitro. This sub- 
stance was named melatonin. It was possible to isolate 
this neurohormone by using first an zn vitro frog skin bio- 
assay test (9, 12) and later spectrophotofluorometric 
analysis and paper chromatography staining, with Ehr- 
lich’s reagent to locate melatonin during various frac- 
tionation procedures. The isolation steps are summarized 
as follows: 


Fresh frozen bovine pineal glands were lyophilized. 


Dried glands trimmed free of adherent tissue debris and blood 
were powdered in a blender and defatted with petroleum ether. 


Defatted pineal powder was mixed with water in a blender and 
the mixture centrifuged. 


Water layer was extracted with ethy] acetate. 


Ethyl acetate layer was evaporated to dryness. 
! Chicago, Ill., April 15, 1960. 


? Present address: Section of Dermatology, Department of 
Medicine, University of Washington School of Medicine, Seattle. 





Countercurrent distribution was done in the solvent system ethyl 
acetate: heptane: water in a 1:1:2 ratio. 


Silicic acid column chromatography of active countercurrent 
distribution fractions was carried out. 


In the extraction experiments a substance chemically 
related to melatonin but biologically inactive also was 
isolated. The inactive material was shown to be 5-meth- 
oxyindole-3-acetic acid, and melatonin was found to be 
N-acetyl-5-methoxytryptamine (5-8). From 100 gm of 
dry powder prepared from trimmed bovine pineal glands, 
1 mg of 5-methoxyindole acetic acid and 0.1 mg of mela- 
tonin were isolated. Since a single dry pineal gland 
weighs approximately 40 mg, 100 gm contains about 
2500 glands. It is evident that the amount of these two 
methoxyindoles in a single pineal gland is extremely 
small. However, the frog skin test is very sensitive, being 
able to detect about 107 gm/ml of melatonin. With 
these techniques it was possible to show that melatonin 
occurred not only in the pineal gland but also in small 
quantities in peripheral nerves of man, monkeys and 
cattle (7). It was present in various parts of the central 
nervous system of cattle and monkeys. 

From a biologic standpoint there are two unusual fea- 
tures about melatonin. It is the first example of the fact 
that N-acetylation of a physiologic amine is required for 
maximum biologic activity, and it is the first example of 
O-methylation of an hydroxyindole. These two subjects 
will be considered separately. 


N-ACETYLATION 


Generally it has been assumed that the nitrogen atom 
of physiologic amines, such as adrenaline, noradrenaline, 
serotonin and histamine as well as many antihistamines 
and tranquilizers, must have a positive charge in tissue 
fluids. That is, a primary, secondary or tertiary amine is 
needed for biologic function. Acetylation of the nitrogen 
atom results in a complete loss of the basic properties and 
the group is neutral. Hence when these compounds were 
acetylated, original biologic activity was lost; and no new 
biologic properties have been found. 

On frog skin, melatonin is a much more potent light- 
ening agent than 5-methoxytryptamine. When 0.1 mg 





of 


cr 


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July 1960 MELATONIN 591 
t) 
gS yea gh 90 soapy 
N 
i \ 
Serotonin N—Acetylserotonin 
HO; CH,COOH 2 a 2 
f | | 2 CH,O ‘egaeataiton CHO | en 
= a 7 HOS. mi a 


| 
- 
5—Hydroxyindole acetic acid 


sm 
cH,0/7 |? <—e 
: a 
Se un  , N 
H 
H 


H 
5—Methoxyindole acetic acid 5—Methoxytryptamine 


ih ) 


H 
Melatonin 6— Hydroxymelatonin 


FIG. 1. Steps in the formation and metabolism of melatonin. 


of melatonin is injected intracutaneously into man a dis- 
crete, punched-out ulcer develops at the injection site. 
Yet, 1.0 mg of 5-methoxytryptamine administered in the 
same way produces no reaction (A. B. Lerner and J. S. 
McGuire, unpublished data). 

It is also of interest that the most potent frog skin dark- 
ening substance, a-melanocyte stimulating hormone (a- 
MSH), is an N-acetyl compound. These two physio- 
logically opposite N-acetyl compounds, a-MSH and 
melatonin, are each effective in approximately the same 
concentration. 


O0-METHYLATION 


It has been assumed that O-methylation of hydroxy- 
indoles does not occur in mammalian tissue because no 
such reaction could be detected zn vitro under conditions 
that readily produced O-methylation of catechol amines 
and because no methoxyindoles were found in the urine 
after rats were given labeled serotonin (11). The finding 
of melatonin and 5-methoxyindole acetic acid in pineal 
tissue made it necessary to reopen this problem. Recently, 
Axelrod and Weissbach (1) showed that when N-acetyl- 
serotonin was incubated with bovine pineal extracts and 
j-adenosylmethionine, melatonin was formed in good 
yields. Serotonin itself was O-methylated at a much 
slower rate under the same conditions. Hence, the origi- 
nal view that O-methylation of hydroxyindoles does not 
occur was wrong because neither the right tissue nor sub- 
strate was chosen for the incubation experiments to de- 
tect an hydroxyindole O-methy! transferase. Also, the 
methoxyindoles are present in such small quantities that 
unless one knows in advance where to look for them in 
urine extracts from rats given labeled serotonin, they 
probably would not be found. 

O-methylation like N-acetylation of serotonin is re- 
quired for maximum biologic activity. N-acetylserotonin 
has much weaker lightening action than melatonin. 


YWIIM 


TABLE 1. Lightening of Frog Skin by 5-substituted Tryp- 
tamine and N-acetyltryptamine Compounds 
Numbers give the minimum effective concentration in yg 


per ml required to lighten frog skin (Rana pipiens) previously 
darkened with MSH. 


R, 
~ |]CH,;CH,NHR, 
a \N 
H 
R, Ro Min. effective 
conc. ug/ml 
H— —H 2X 107! 
H— —COCH; 5 X 10°? 
HO— —H I 
Ho— -.._—sss— COCH; 6 X 1073 
CH;0— —H 2X 1076 
CH;0— —COCH; 1 X 1077 
CH;CH,0— —H 5 X 10°? 
CH;CH20— —COCH; 1 X 1075 


TABLE 2. Lightening of Frog Skin by N-substituted 
5-methoxytryptamine Compounds 
Numbers give the minimum effective concentration In ug 


per ml required to lighten frog skin (Rana pipiens) previously 
darkened with MSH. 


CH;O 
CH.,CH,NHR 
\N 
H 
R Min. effective 
conc., ng/ml 
H— 2X 107 
CH;-— 2X10 1 
CHO— i x 107° 
CH;CO— 1-X 1077 
CH;CH:CO- rx 107! 
CH;CH.CH:CO- ie 107? 








592 FEDERATION PROCEEDINGS 


METABOLIC PATHWAYS 


Since the hydroxyindole-O-methy! transferase in pin- 
eal tissue utilizes N-acetylserotonin as a substrate much 
more effectively than serotonin and since a great propor- 
tion of injected serotonin is converted to N-acetylsero- 
tonin, it is likely that O-methylation of serotonin occurs 
after and not before N-acetylation. 5-methoxyindole 
acetic acid may be derived from melatonin via 5-meth- 
oxytryptamine because feeding this latter compound to 
rats was followed by excretion of 5-methoxyindole acetic 
acid in the urine (2). Or 5-hydroxyindole acetic acid 
may be O-methylated directly in the pineal gland to 
5-methoxyindole acetic acid. Recently Kopin et al. 
showed that intraperitoneal injection of labeled mel- 
atonin into rats resulted in urinary excretion in high 
yield of 6-hydroxymelatonin as the sulfate and glucu- 
ronide conjugates (3). Intravenous or oral administration 
of melatonin to human beings resulted in excretion of a 
compound having the same R: value on paper chroma- 
tography as Kopin’s 6-hydroxymelatonin sulfate (A. B. 
Lerner and J. S. McGuire, unpublished data). It is 
not known whether this 6-hydroxylation reaction also 
occurs in nerve tissue. A tentative scheme for the forma- 
tion and metabolism of melatonin is given in figure I. 


RELATIONSHIP OF STRUCTURE TO BIOLOGIC ACTIVITY 


It is not ordinarily a simple matter to compare the 
lightening effects of melatonin with those of other indole 
compounds because the dose-response curves for the 
various compounds may be different and because there 
are many kinds of interactions between lightening and 
darkening agents to test. However, when we determined 
only the minimum effective concentration of an indole 
compound required to produce lightening of melanocyte 
stimulating hormone (MSH)-darkened frog skin, some 
idea was obtained on the relationship of structure to bio- 
logic activity (tables 1 and 2—J. D. Case, A. B. Lerner 
and M. R. Wright, data to be published). Acetylation of 
tryptamine, serotonin, 5-methoxytryptamine and 5-eth- 


Volume 19 


oxytryptamine increased activity over the parent com- 
pounds. A methoxy group in the 5 position was better 
than a hydrogen, hydroxy or ethoxy group. 

When various groupings were put on the side chain 
nitrogen of 5-methoxytryptamine, it was found that a 
methyl or formyl group decreased activity whereas an 
acetyl, propyl or butyryl group increased activity. The 
last three compounds were all equally effective in pro- 
ducing lightening. 


EFFECT OF MELATONIN IN MAN 


Intracutaneous injection of 50 wg of melatonin into an 
adult man produced erythema that faded within an hour, 
However, when 100 wg was given, a punched-out ulcer 
appeared at the injection site 2-4 days later. No de- 
pigmentation was observed and there was no increase in 
local sweating. 

One patient, given 200 mg of melatonin intravenously, 
experienced mild sedation. Numerous metabolic tests 
done before and after the patient received 200 mg 
melatonin daily for 5 days revealed no change. 

At the present time the physiologic function of 
melatonin in man is not known. We do not know whether 
or not melatonin is the long sought for vitiligo factor. 


SUMMARY 


While pursuing investigations on the etiology of 
vitiligo, we isolated two methoxyindole compounds from 
bovine pineal glands. One of the compounds, melatonin, 
is a very potent lightening agent for frog skin. This sub- 
stance also is found in various parts of the nervous 
system of mammals including man. Melatonin probably 
is produced from serotonin by N-acetylation followed by 
O-methylation. Melatonin is converted to 6-hydroxy- 
melatonin and excreted in the urine as the sulfate and 
glucuronide conjugates. The significance of N-acetyla- 
tion and O-methylation is discussed. The role of mela- 
tonin in man is not yet known. 


REFERENCES 


1. AXELROD, J. AND H. WeissBacH. Science 131: 1312, 1960. 

2. ErspaMER, V. J. Physiol. 127: 118, 1955. 

3. Kopin, I. J., C. M. B. Pare, J. AXELROD AND H. WEIssBACH. 

Biochem. et biophys. acta. In press. 

4. Lerner, A. B. AND J. D. Case. J. Invest. Dermat. 32: 211, 1959. 

. Lerner, A. B., J. D. Case, K. Bremann, R. V. HEINzELMAN, 
J. Szmuszkovicz, W. C. AnTHONy AND A. Krivis. J. Am. 
Chem. Soc. 81: 5264, 1959. 

6. Lerner, A. B., J. D. Case anp R. V. HEINnzELMAN. J. Am. 
Chem. Soc. 81 : 6084, 1959. 

. Lerner, A. B., J. D. Cask, W. Mori ann M. R. Wricur. 
Nature, London 183: 1821, 1959. 


~I 


8. Lerner, A. B., J. D. Cask anp Y. Taxanasut. J. Biol. Chem. 
235: (June) 1960. 

g. Lerner, A. B. AnD M. R. Wricut. In: Methods of Biochemical 
Analysis, edited by D. Glick. New York: Interscience, 1960, 
P. 295. 

10. McCorp, C. P. anp F. P. ALien. J. Exp. Zool. 23: 207, 1917. 

11. McIsaac, W. M. anv I. H. Pace. J. Biol. Chem. 234: 858, 
1959- 

12. Mort, W. Anp A. B. LerNER. Endocrinology. In press. 

13. Wricut, M. R. anp A. B. Lerner. Endocrinology. 66: 599, 
1960. 





fr 


fro 
COU 


and 
U.S 


and 
Roh 





ume 19 


com- 
better 


chain 
hat a 
as an 
. The 


1 pro- 


ito an 
hour. 
ulcer 
o de- 
ase in 


ously, 
» tests 
Oo mg 


on of 
rether 
or. 


gy of 
s from 
tonin, 
is sub- 
ervous 
ybably 
ved by 
Jroxy- 
e and 
-etyla- 
mela- 


1, Chem. 


chemical 
1960, 


> 


], 1917. 
4: 858, 


66 ° 599, 





Recent advances in parathyroid hormone research’ 


PAUL L. MUNSON, Chairman 


Biological Research Laboratories, Harvard School of Dental Medicine, and Department 
of Pharmacology, Harvard Medical School, Boston, Massachusetts 


- STUDY OF THE PARATHYROID GLANDS has a distin- 
guished history and a promising future. Research in the 
field, relatively neglected for a number of years, is now 
being intensified and, indeed, it would be difficult to 
write a full review of current progress briefly. 

The purification of the hormone, 35 years after Collip’s 
original publication (10), has now progressed to the point 
that three laboratories have reported preparations 25-50 
times more potent than the best product obtained prior 
to 1957. 

Our own efforts to purify the hormone led to the 
development of a procedure involving extraction of the 
ground, frozen glands with «ilute hydrochloric acid, 
fractionation of the crude extract with ammonium sul- 
fate, and column chromatography on carboxymethyl- 
cellulose (14, 15). Figure 1 shows the results obtained 
from the first chromatogram. The middle pool of 
eluates had a specific activity of approximately 1800 
u/mg N or about 300 u per mg of peptide. Figure 2 
shows the results of rechromatography. The best fraction 
had a specific activity of 7800 u/mg N or, dividing by 
the factor 6.25, about 1250 U per mg of peptide. Although 
this product represented a substantial purification of the 
hormone, the shape of the curves both for nitrogen and 
for biological activity suggested to us that the goal of 
‘final purification’ might still be rather distant. 

The efficient procedure of Aurbach is based on extrac- 
tion with phenol (4). After fractionation with organic 
solvents, salts and trichloracetic acid, the extract was 
subjected to repeated countercurrent distribution, and a 
final product with a very high specific activity estimated 
at 2800 U per mg of peptide was obtained (6). Figure 3 
shows the result of redistribution of the active peak from 
a first countercurrent distribution. The points represent 
the values for nitrogen, the dotted line the theoretical 
distribution curve. 

Aurbach’s results were confirmed by Rasmussen and 
Craig (42), using a procedure that differed only in detail 
from that of Aurbach (6). Figure 4 shows the result of a 
countercurrent distribution. The smaller peak contained 


1 Supported in part by grants from the American Cancer Society 
and the National Institute for Arthritis and Metabolic Diseases, 
U. S. Public Health Service (A-1787). 

Frozen bovine parathyroid glands were donated by Eli Lilly 
and Co., through the courtesy of Dr. K. K. Chen and Dr. Ewald 
Rohrmann, and by Canada Packers, Ltd., through the courtesy of 
Dr. R. Witty. 


7 
ase tad 


593 


the activity. Redistribution of active material from a 
different solvent system produced the pattern shown in 
figure 5. The specific activity of the product obtained was 
reported to be 2500 to 3000 units per mg, the same as 
Aurbach’s product, and about twice that of our best 
product. In addition, it was stated that in a third counter- 
current distribution, in the ultracentrifuge, and in 
dialysis experiments, the product behaved like a single 
substance. The molecular weight was estimated to be 
7000 + 1500. Amino acid analysis yielded a minimum 
molecular weight of 8582 (41). 

The product obtained by Rasmussen and Craig from 
Aurbach’s phenol extract was named ‘Parathormone C’ 
because previously, by other methods, Rasmussen had 
isolated two other active peptide products called ‘A’ and 
‘B’ (39). The amino acid analyses, except for one 
discrepancy, were consistent with A and B being partial 
degradation products of C. The biological activity of C, 
however, was found to be higher than B, and that of B 
higher than A (41). 

It is possible that one or more of Aurbach’s or Rasmus- 
sen’s products represents the isolation of a pure polypep- 
tide hormone from the parathyroid glands, although in 
the context of previous experience with insulin, glucagon, 
ACTH and other peptide hormones, a conservative 
interpretation of the available data is probably justified. 

The biological assay method (32, 35) used for estima- 
tion of specific activities is basically the same in the three 
laboratories. Evaluation of the assay data is complicated, 
however, by the fact that the purified preparations have 
a shorter duration of action than the reference standard 
(U.S.P. parathyroid extract) and may have been reversi- 
bly inactivated during preparation (5, 34, 40, 43). At- 
tempts to compensate for these differences between un- 
knowns and standards by administration of the unknowns 
in gelatin or oil or with the addition of cysteine have not 
been uniformly satisfactory. The problems of the bio- 
logical assay have been considered in greater detail in a 
paper now in press (34). 

The ‘final purification’ of a peptide hormone is in 
itself a noteworthy achievement, if only for the ingenuity 
needed to surmount the numerous obstacles that present 
themselves along the way. The real significance of 
purification, however, is for the new pathways it opens 
for further advances in knowledge. Purification is the 
first step toward the determination of structure, the 
synthetic preparation of the hormone and ‘studies of 








594 















20) Fig. | 
NITROGEN 
ASIE 
~ 
= 
_ 
12 = < 
_40 U. STORD. ey 
10 ¢ Do 
N 
8s e 
CONTR.LEVEL 3S 
.05 wW 
4” 
jar - - ~ : . 
10 20 30 40 50 60 


TUBE NO. 


FIG. 1. Results of gradient elution chromatography on carbox- 
ymethylcellulose of an ammonium sulfate fraction (PV-100-F) 
of a cold acid extract of bovine parathyroid tissue. The dialyzed 
fraction was applied to the column in 0.01 M acetate buffer, pH 
4.68, and was eluted by gradual addition of 0.5 m NaCl in acetate 
buffer to the mixing chamber, as in procedure of Friedman and 
Munson (15). Top curve represents nitrogen concentration of the 
eluates. Open circles represent the serum calcium of assay rats 
given an aliquot of each eluate containing 25 ug N. Smooth curve 
labeled ‘ACTIVITY’ represents the estimated relative concen- 
tration of hormone in the eluates. Horizontal lines represent the 
mean values obtained in the assay for controls and for rats given 
40 u of U.S.P. extract, respectively. Numbers above the hori- 
zontal brackets represent the assayed specific activity, in U/mg N, 





= .200 
7 mm 
= e *s 
ws « C. 
. 
= 51398 « e 
S wv : 
| ee 
i) 14 26 38 56 
FIG. 3. Second countercurrent distribution of parathyroid 


hormone preparation recovered from first 100-tube distribution. 
System: n-butanol against 3% sodium chloride in, 20%, acetic 


acid. @, observed; broken line, theoretical. 


structure-activity relationships. Furthermore, it can tell 
us either that the pure hormone possesses all the biologi- 
cal activities of the crude gland extract, or that the 
extract must be searched for additional interesting active 
constituents. The method of preparation may also be a 
guide to a practical process for preparing purified hor- 
mone in good yield and ample quantity for more defini- 
tive studies of the pharmacology, mechanisms of action, 
interrelations with other hormones and possible diag- 
nostic and therapeutic uses. 

The primary site of action of parathyroid hormone was 
at first assumed to be in bone, and evidence for this view 
was provided by McLean and Bloom (30). At an early 
date, however, 1929, an alternative concept was intro- 
duced by Albright and Ellsworth (1). It was presented 
with such clarity and persuasiveness that it attracted 


FEDERATION PROCEEDINGS 








Volume 19 























“7 
UNITS/mg.N 
; rloo00 
044 + 8000 
E 
2 
r6000 
02; 4000 
7 t 2000 
(0) 
ie) r — r : 


10 20 30 40 50 60 


TUBE NO. 
of three pooled sets of eluates. (Data from Munson and Voelkel, 
reported at the annual meeting of the American Society of Bio- 
logical Chemists, Atlantic City, April 1959.) 

FIG. 2. Rechromatography of parathyroid hormone prepara- 
tion (PV-95-E) on carboxymethylcellulose by gradient elution 
(see explanation of fig. 1). The curve represents the nitrogen con- 
centration of NaCl buffer. 
Vertical bars represent the specific activity in u/mg N of blocks 
of pooled eluates. Vertical lines through the bars are the standard 
errors. First and last pools (marked O) had no detectable activity 
at the doses tested. (Data from Munson and Voelkel, reported at 
the annual meeting of the American Society of Biological Chem- 
ists, Atlantic City, April 1959.) 


successive eluates with in acetate 





0.300 


0.250; 


0.200 


0.150 


0.D.277 mu 


0.100 


0.050 








1 1 4 4 4 a | rt 1 
Tube no. 20 40 60 80 100 

FIG. 4. Countercurrent distribution of parathyroid hormone 

preparation after 100 transfers in system: 6% acetic acid—1.0% 





1.0% 
sodium chloride (6 parts) and a 1:1 mixture of n-butanol and 
n-propanol (4 parts). @—absorption at 277 mu; O—theoretical 
distribution. 


wide attention and came to dominate thinking and 
writing in the field until the early 1950's. 

The hypothesis of Albright and Ellsworth was based 
on the observation in a hypocalcemic hypoparathyroid 
patient that the serum inorganic phosphate was high, 
that a single injection of parathyroid extract resulted ina 
rapid increase in the urine phosphate, and that continued 








ocw 


you 
tom 
fron 














ume 19 


/oelkel, 
of Bio- 


repara- 
elution 
en con- 
buffer. 
f blocks 
tandard 
activity 
orted at 
Chem- 


1ormone 
i—1.0% 
nol and 
eoretical 


1g and 


; based 
thyroid 
Ss high, 
ted ina 
itinued 











July 1960 
; K=0.214 
12 re xx 
oe 
| 
0.8F 1 7 
P 
LG 
ai 
OA- 
3 
nl ! 1 1 














1 n 
80 120 160 
Tube number 
FIG. 5. Countercurrent distribution of parathyroid hormone 
preparation after 471 transfers in system composed of 12 parts 
0.1% acetic acid, 5 parts n-butanol and 3.5 parts pyridine. @— 
weight recovered; X —theoretical distribution. 


200 240 


treatment with parathyroid extract resulted in a gradual 
fall in the serum phosphate as well as a rise in serum 
calcium. It was reasoned that a primary effect on the re- 
absorption of phosphate by the renal tubule was sufficient 
to explain the rise in serum calcium. With loss of phos- 
phate into the urine, the plasma phosphate and the 
calcium times phosphate product in the plasma would 
fall, thus permitting increased resorption of bone on the 
solubility product principle, and leading to a rise in 
serum calcium as a secondary effect. 

The necessary experiments in nephrectomized animals 
to test the hypothesis, due to various experimental 
complications, did not at first provide consistent, clear- 
cut results. 

The balance of the accumulating evidence, however, 
including work carried out in the Albright laboratory 
(23), was against an obligatory role for the kidney as a 


9 - 

84 
= 
Zz 
WwW 
oO 
x 
WW 
740 
oO 
= 
Fic. 6. Serum calcium of calcium-depleted = 
young male rats following parathyroidec- oO 
tomy. Each point represents mean value 64 a 
from 6 to 38 rats. Vertical lines represent S.E. oO 
5 
WJ 
n” 

5 + 

4 





PARATHYROID HORMONE 


595 


mediator of the effect of parathyroid hormone on the 
serum calcium. A modified hypothesis was then offered 
by Albright and Reifenstein (2) which stated that the 
primary effect of parathyroid hormone was on some 
aspect of phosphate metabolism, whether renal or 
extrarenal. The effect on the serum calcium was still 
held to be secondary. 

The introduction of the technique of peritoneal lavage 
by Grollman et al. (22) made it possible to maintain the 
blood chemistry of nephrectomized animals near normal, 
and with the aid of this method, Grollman (21), in dogs, 
and Talmage et al. (45) in rats, demonstrated conclu- 
sively that parathyroid extract can increase the serum 
calcium in the absence of the kidneys. 

In the meantime, Barnicot (7), and soon after, Chang 
(g), had demonstrated, in well controlled experiments, 
that the parathyroid gland can act directly on bone. 
They found that parathyroid glands transplanted onto 
the parietal bone of mice and rats exerted a powerful 
local resorptive action. Often, most of the bone beneath 
the graft had been resorbed within 14 days. 

These two important series of experiments, on neph- 
rectomized animals and on parathyroid transplants, 
firmly established the fact that parathyroid hormone can 
act independently of the kidney and that it can act 
directly on bone. They led the way to a direct attack on 
the question of the importance of the effect of the 
parathyroid on phosphate for maintenance or elevation 
of the bone calcium. 

Our first experiments, with Dr. Oscar Iseri, attempted 
to elucidate this question by a study of the time course 
of changes in serum calcium and phosphorus immediately 








XUM 


os 


HOURS AFTER PARATHYROIDECTOMY 
T T T T T 
2 3 4 5 6 7 8 














INORGANIC 
PHOSPHORUS 






o 


@ 
SERUM, mg % 











CALCIUM 
6 4 
44 
1 HOURS AFTER PARATHYROIDECTOMY 
0 2 4 6 to lO 
(terminal) 


FIG. 7. Serum calcium and inorganic phosphorus of calcium- 
depleted young male rats following parathyroidectomy. Each 
point represents mean value for 6 rats. Vertical lines represent S.E. 





4h 





o OF 
50 
Sz 
raed BE LOW-CA DIET 
(e} 
23 CONTROL DIET 
Fo 
Sa 6+ 
| ee 
zo 
Wir 
- © 
mob 
ae 
oO 
7 ol 
= CONTROL DIET 
—_ 
S 6 
3 LOW-CA DIET 
= 
= 
c 
Ww 
w” 





PA 
L HOURS AFTER PARATHYROIDECTOMY 
. Oo 2 
ric. 8. Effect of parathyroidectomy on serum inorganic phos- 
phorus and calcium as modified by dietary calcium intake before 
the operation. Each point represents the mean value for 6 rats. 
Vertical lines represent the S.E. 


after parathyroidectomy and after injection of parathy- 
roid extract (32). 

Figure 6 shows the rapidity of the fall in serum calcium 
after parathyroidectomy of young male rats. The rats 
had been fed a calcium-free diet for 10-20 days before 
operation, and consequently the mean preoperative 
value was somewhat lower than the normal level of 
approximately 10 mg “%. A significant fall had occurred 
within one hour, and in from 2 to 4 hours the serum 
calcium had reached a level that leads to tetany and 
death in calcium-depleted rats 6-10 hours after para- 
thyroidectomy. If, as implied in Albright’s hypothesis, 
a rise in serum phosphate must precede the fall in serum 


596 FEDERATION PROCEEDINGS 


Volume 19 


calcium, the effect on serum phosphate, according to 
these data, must indeed be very rapid. 

The data in figure 7, illustrative of several similar 
experiments, show that quite the contrary result was 
observed. The serum inorganic phosphate not only 
failed to rise prior to the fall in calcium but actually fell 
slightly but significantly with the calcium during the 
early hours after parathyroidectomy. The marked rise 
in phosphate observed during tetany occurred too late 
to be a factor related to solubility products responsible 
for the fall in serum calcium. (The values for serum 
inorganic phosphate, much higher than in man, are 
normal for young rats.) 

Our experiment shown in figure 8 demonstrates that 
the same trends seen after parathyroidectomy in cal- 
cium-depleted rats were apparent in rats fed a diet 
adequate in calcium. Again no rise in serum inorganic 
phosphate preceded the fall in serum calcium. 

Similar conclusions could be drawn from experiments 
in which parathyroidectomized rats were injected with 
parathyroid extract. In the experiment on calcium- 
depleted rats shown in figure g, the extract was injected 
subcutaneously immediately after parathyroidectomy. 
The absorption of hormone in this form and injected by 
this route is delayed and does not completely prevent the 
fall in serum calcium seen in control parathyroidecto- 
mized rats 2 hours after the operation. A fall in the serum 
phosphate was also observed here at 2 hours as in the 
experiments shown previously, with no difference be- 
tween the control and injected rats. At 6 hours after the 
operation, however, both the calcium and phosphorus 
values had returned to the preoperative levels. The rise 
in serum calcium was not accompanied by a fall in 
phosphate. Rather, the effect of the extract was to 
return both the calcium and the phosphate to normal 
and, with the dose administered, to hold them there for 
at least 18 hours. 


PARATHYROID EXTRACT 








100u.,s.c. 
DEATH 
12 
UNTREATED 
SERUM r EXTRACT 
PHOSPHORUS !0 ’ 
mg. % 
8 
i 
SERUM = EXTRACT 
CALCIUM 8r 4 
mg. % - 
6F 
(S UNTREATED 
4- DEATH 
1 l l 1 l 1 








1 i 1 = 
oO 2 4 6 8 16 12° 14 (16 = 
HOURS AFTER PARATHYROIDECTOMY 

FIG. g. Serum inorganic phosphorus and calcium of calcium 

depleted young male rats following parathyroidectomy, without 

treatment (O) or with subcutaneous injection of 100 u of U.S.P 

parathyroid extract immediately after operation (@). Each point 
represents mean of 6 rats. Vertical lines represent the S.E. 


in| 


FI 
with 
setts 
rang 








lume 19 
ing to 


imilar 
lt was 
: only 
lly fell 
1g the 
-d_ rise 
0 late 
ynsible 
serum 
n, are 


>s_that 
in. cal- 
a diet 
rganic 


‘iments 
d with 
cium- 
yjected 
ctomy. 
‘ted by 
ent the 
idecto- 
- serum 
in the 
ice be- 
fter the 
sphorus 
“he rise 
fall in 
was to 
normal 
.ere for 


16 18 
sTOMY 
calcium 
, without 
of U.S.P 


ach_ point 





July 1960 


PARATHYROID HORMONE 








SERUM 
PHOSPHORUS F 
mg. % at 
L 
6r 


FIG. 10. Patient with idiopathic hypopara- r 
thyroidism. Serum inorganic phosphorus and ab 
calcium of patient of Albright and Ellsworth 
(1) during first 4 days of treatment with para- 
thyroid extract (PTE). 





597 
PTE INJECTIONS 
a — x 
’ v v 4 
T , T T i 1 T 
my PHOSPHORUS 













9 
CALCIUM - NORMAL LOWER LEVEL 
eB ee ee Te a 
CALCIUM ms 
mg. 
oe CALCIUM 
s OmOmng- - - - -- -- 
lL l i i L L 
4 5 6 7 8 9 


The question may properly be asked whether these 
responses of the rat to parathyroidectomy and to para- 
thyroid extract are peculiar to one species, and are not 
applicable to man. 

In figure 10, some of the data from the hypopara- 
thyroid patient studied by Albright and Ellsworth (1) 
have been plotted to permit study of the time course of 
the effect of parathyroid extract more readily. Before 
injection of the hormone, the patient showed not only a 





aA 




















a — 
1A 





3 4 s 6 
SERUM INORGANIC PHOSPHORUS, mg % 
FIG. 11. Serum calcium and inorganic phosphate of patients 
with hypoparathyroidism following thyroid surgery at Massachu- 


setts General Hospital, 1946-55. Shaded areas represent normal 
tanges for hospital lab. 


XUM 


DAY OF OBSERVATION 


low serum calcium but also the elevated serum inorganic 
phosphate characteristic of some hypoparathyroid pa- 
tients. Daily injections of parathyroid extract were 
begun. The serum calcium gradually rose and the serum 
phosphate fell, in harmony with the hypothesis put for- 
ward by the investigators. However, the change in serum 
phosphate did not precede the rise in serum calcium. 
Furthermore, although the serum calcium rose quite 
promptly to the normal level, the serum phosphate 
remained elevated. 

This classic picture of hypoparathyroidism, which 
includes a high serum inorganic phosphate as well as 
hypocalcemia, is far from the invariable rule, at least in 
the early stages of the disease, according to data col- 
lected by Krane (28). In figure 11 data are shown from 
patients at the Massachusetts General Hospital who 
developed hypocalcemia after thyroid surgery. Included 
were all such patients during a 10-year period from 
whom blood was collected for serum calcium and phos- 
phate determinations before institution of therapy. Only 
1 of the 10 cases showed a markedly elevated phosphate 
value. 

In figure 12, 4 cases of hyperparathyroidism presented 
by Albright et a/. are shown (3). In all cases, after re- 
moval of the adenoma or hyperplastic glands, the serum 
calcium fell. In no case was there a prior or significant 
accompanying rise in the serum phosphate. 

We have concluded from these data that in man as well 
as the rat the effect of parathyroidectomy and of para- 
thyroid hormone on the level of the serum calcium is not 
dependent on a prior change in the circulating level of 
inorganic phosphate. In seems most likely that the pri- 
mary target tissue of the parathyroid hormone for the 
regulation of the level of circulating calcium is bone 
rather than kidney and that whatever effects the hor- 






































598 FEDERATION PROCEEDINGS Volume 19 
CASE 16 PTX CASE 17 PTX 
7b } y 
i2i- 
14 CALCIUM CALCIUM 
Mg. % Mg.% + 
ee 10 
3h PHOSPHORUS 3b 
PHOSPHORUS 
2 L 2+ — 
1 | A. 1 1 | ! 1 | | | 
= Oo | 2 3 DAYS -10 Oo 1 2 3 DAYS 
FIG. 12. Serum calcium and in- 
organic phosphorus of hyper- 
PT parathyroid patients of Albright 
CASE 23 Xx CASE 25 et al. (3) before and after para- 
. y J thyroidectomy (PTX). 
13r 
14F 
Mg. % CALCIUM Mg. % 
i 9 lr CALCIUM 
10+ 
gt 
PHOSPHORUS 3/ PHOSPHORUS 
2r -) 
l ! mf 1 H | ae 8 i 1 
— 2 | 2 DAYS —- 2 8 DAYS 
mone may have on phosphate metabolism are of second- ? 
ary importance for this most crucial function of the mt 
=> 
parathyroid gland. y 
. . c 
Although this concept has not yet been incorporated 
into most textbooks it is now generally accepted by all é 
active investigators in the field. There has even been a by 
tendency in recent years to question the existence and Fs 
significance of a specific phosphaturic action of the & 
parathyroid hormone, However, there should be little z 
doubt that the effect on the urinary phosphate is real & 
and that it should be attributed to the parathyroid z | l 
hormone. The effect of extracts on phosphate excretion o L—__MINUTES | a eee 
: wrrit a4 00-200 «300 +4«400 «2500 +600 700 
is clear and reproducible in several species, even after 
FIG. 13. Time course of urinary excretion of inorganic phos- 


suitable allowance is made for the side effects on renal 
hemodynamics of contaminants of crude extracts. 
Furthermore, the original observation of Greenwald, 
made in 1911 (20), that parathyroidectomy is followed 
by a diminished output of phosphate in the urine, has 
been confirmed repeatedly. Finally, phosphaturic effects 
have been observed with highly purified preparations of 
parathyroid hormone (6, 25, 29, 41). 

Our data, with Beutner (8), in figure 13 illustrate the 
kind of effect that is observed under controlled conditions 
in rats. In this experiment, the unanesthetized rat was 
placed in the restraining cage of Kellogg et al. (24), which 
provides for collection of uncontaminated urine. A tail 
vein of the animal was cannulated and a rapid intra- 
venous infusion of physiological salt solution was con- 
tinued throughout the experiment in order to supply 
urine samples at intervals of 5-10 minutes. At the first 
arrow, the rat was removed from the cage, anesthetized 


phate by an unanesthetized rat after sham operation (1), para- 
thyroidectomy (2), control injection (3) and injection of parathy- 
roid extract (4). 


with ether, and an incision was made in the neck region. 
The rat was returned to the cage and the course of the 
phosphate excretion over the next 60 minutes described 
the artifact due to ether and trauma. After return of the 
rate of phosphate excretion to the preoperative level the 
rat was removed from the cage a second time, again 
anesthetized with ether and quickly parathyroidecto- 
mized by hot wire cautery. The artifact again appeared, 
but within 25 minutes the rate of phosphate excretion 
had fallen well below the control level. It continued to 
decline to a very low rate. At the third arrow a control 
intravenous injection of acidified physiological salt solu- 
tion was made through the cannula without any detect- 
able effect. A few minutes later a small dose of para- 





> 8 & 


lume 19 


and in- 

hyper- 
Ibright 
y para- 


 phos- 
| para- 
arathy- 


egion. 
of the 
sribed 
of the 
el the 
again 
decto- 
eared, 
retion 
ied to 
ontrol 
- solu- 
letect- 
para- 





July 1960 


thyroid extract (20 U) at a pH the same as the control 
injection was made by the same route. A significant 
increase was detected in the first urine collection 
that followed, only 8 minutes after the injection. The 
rate of phosphate excretion continued to rise sharply to a 
peak, followed by a slower return toward the base line. 
Other similar experiments on additional animals yielded 
the same pattern of results. 

Several conclusions may be drawn from the results. 
The rapid fall in urinary phosphate after parathyroidec- 
tomy, like the rapid fall in serum calcium shown earlier, 
indicates that the life of parathyroid hormone in the 
circulation is short and that parathyroid hormone must 
be secreted continuously. The rapidity of the response to 
the injection of parathyroid extract suggests rather 
strongly that the effect on the kidney must be direct, 
irrespective of whatever additional target systems may 
be involved. Although clearance techniques were not 
employed, the magnitude of the effects observed would 
seem to rule out any important change in renal hemo- 
dynamics as a dominant factor in the results. 

A similar conclusion for man was reached by Gersh- 
berg et al. (18). Glomerular filtration rate, renal plasma 
flow, and plasma and urinary phosphate were deter- 
mined simultaneously, and it was found that in all 42 
patients studied except two with hyperparathyroidism 
the percentage of filtered phosphate reabsorbed fell 
within 15 minutes after the injection. 

Recently, Lavender and coworkers (29) have demon- 
strated, by injecting small amounts of highly purified 
parathyroid hormone preparations into the renal artery 
of dogs, that the effect of the hormone is indeed a direct 
one since small doses had a phosphaturic effect only on 
the injected kidney. 

The site of action of parathyroid hormone on the renal 
tubule is also under investigation. Nicholson and Shep- 
herd (38) studied the sites of phosphate transport in the 
tubule with the aid of differential nephrotoxic agents 
and concluded that phosphate is reabsorbed in the first 
third of the proximal tubule and actively excreted by 
the distal tubule. Similar studies with parathyroid 


180-- 


160 


T 


140 


T 


FIG. 14. Serum calcium and com- 
position of milk in lactating rats, sham- 
operated, parathyroidectomized, or 
parathyroidectomized and injected with 
100 u of parathyroid extract. Clear bars 
represent mean control values drawn to 
auniform height to permit use of single 
scale. Mean value is given at top of each 
bar. Brackets represent the S.E. 


120 
100 
80 
60 


Per Cent of Controls 


40 


20 











PARATHYROID HORMONE 599 


extract (37) seemed to show that the principal mode of 
action of the hormone on the kidney was to stimulate 
active excretion of phosphate, not, as usually assumed, 
to inhibit reabsorption. This challenging conclusion will 
no doubt be subjected to critical discussion. Experiments 
using other techniques such as stop-flow analysis, now 
underway by Samiy and Hirsch in our laboratory, may 
strengthen or modify Nicholson’s conclusions. 

The possibility of a second effect of the parathyroid 
hormone on the kidney, that of stimulating the reabsorp- 
tion of calcium has been brought forward by Talmage 
(44). 

The extension by Gaillard (16) of the observations of 
Barnicot (7) and Chang (9) on the local resorptive action 
of parathyroid transplants on bone in vivo to the same 
effect on bone in tissue culture provides a promising 
avenue for research on the biochemistry of hormone 
action. In early experiments Gaillard used the rather 
crude U.S.P. extract as a source of the hormone, but 
more recently he has confirmed and elaborated on these 
observations with a highly purified hormone preparation 
supplied by Rasmussen (17). Goldhaber (19) was able 
to demonstrate essentially the same effect on bone in 
tissue culture without addition of parathyroid hormone 
when he increased the oxygen concentration in the tubes 
from that of air (used by Gaillard) to go or 100 % oxygen. 
Goldhaber currently is studying the relation of the 
parathyroid hormone effect to the effect of oxygen 
concentration, since the possibility that the effect of the 
hormone is related to the supply or utilization of oxygen 
should not be neglected. Kenny ef al. (27) showed that 
during resorption of bone in tissue culture an amount of ci- 
trate equal to 10-20 times that originally present in the tis- 
sue accumulated in the medium. The significance of citric 
and related organic acids in the process of bone resorp- 
tion and the action of parathyroid hormone has been 
and currently is under study in several laboratories. The 
status of the subject, in my opinion, still in the explora- 
tory stage, has recently been reviewed by Neuman and 
Neuman (36) and by Kenny (26). 

The available facts seem to be in accord with the con- 


C)- 10 Sham- operated E-1 Parathyroidectomized 
Control Rats Rats Gi rghayroia 


KN- 9 Porathyroidectomized Rats atract 













































XUM 





olics Mg Ca per Gm 
of Solid 


” 


Mg Cao per Gm Milk 
of Milk by 








600 


cept introduced by McLean and Urist (31) that the 
action of parathyroid hormone is on the stable fraction of 
bone mineral. The well-documented rapid equilibrium 
of calcium between blood and bone involves only a 
small labile fraction of the total bone and is inadequate to 
maintain the normal circulating level in the absence of 
the parathyroids. 

The two well-established sites of action of the para- 
thyroid hormone, bone and kidney, do not exhaust the 
list of biological systems on which the hormone may 
have a direct action. Foulks and Perry (11-13), for 
example, have presented data which they think are best 
explained by an action of parathyroid hormone on the 
distribution of phosphate between fluid compartments, a 
possibility which had been suggested by several earlier 
authors. 

An interesting effect of the parathyroids on the compo- 
sition of the milk was discovered by Toverud (46) in 
experiments on parathyroidectomized lactating rats. 
The paradoxical observation was made that as the level 
of serum calcium declined following parathyroidectomy 
the calcium concentration of the milk rose markedly. 
However, the milk appeared to be much thicker than in 
normal lactating rats, and on analysis it was found that 
the water content had decreased sufficiently to account 
for a major part although not all of the increased calcium 
concentration. 

In figure 14, one of many experiments on the effect of 
parathyroidectomy is shown. The effect of a single 
injection of parathyroid extract on the phenomenon was 
also determined. 

Thirty lactating rats were parathyroidectomized or 
sham-operated, on the 13th day of lactation. Eleven of 
the 20 parathyroidectomized rats were given subcuta- 
neous injection of 100 U of parathyroid extract immedi- 
ately after the operation. Milk samples were obtained 24 
hours later, 4-6 hours after isolation from the pups. At 
this time, the serum calcium of the parathyroidecto- 
mized mothers had fallen to the expected low level, while 
that of the injected rats had actually risen above the 
control level. The calcium concentration of the milk 
after parathyroidectomy, rose almost 50% above the 


FEDERATION PROCEEDINGS 


Volume 19 


controls while the injection of hormone prevented the 
rise. There was a 20 “% increase in the percentage of milk 
solids after parathyroidectomy which was completely 
prevented by administration of hormone. Parathyroidec- 
tomy was followed by a 20% increase in the calcium 
concentration of the milk solids. Here the effect of 
extract was equivocal. 

It has been suggested (32) that the parathyroid glands 
may secrete two hormones, one responsible for the 
effects on bone and the other for the effects on the 
kidney. The highly purified hormone preparations 
prepared in the three laboratories mentioned have now 
been tested for phosphaturic activity as well as calcium- 
mobilizing activity. All preparations contained both 
activities, and there was no evidence of even a partial 
separation, although the assays were probably not 
precise enough for a definitive test. As stated earlier, 
experience with the purification of other peptide hor- 
mones has led us to be cautious about premature con- 
clusions concerning biological purity. In any event, it 
will be of interest and importance to test purified 
preparations for their effect on the composition of milk. 


SUMMARY 


This brief review has concentrated on recent studies on 
the purification and on the sites and mode of action of 
parathyroid hormone. The relative importance of bone 
and kidney, both now shown to be primary sites of action 
of the hormone, in the regulation of the blood calcium 
has been clarified. In the last few years progress in 
purification of the hormone has been rapid. It should be 
possible in the near future to investigate its chemical 
structure and to make purified preparations available 
for further studies of mode and mechanism of action. 
In addition to the well-established effect of the hormone 
on bone and its phosphaturic action on the kidney, 
effects on urinary calcium excretion, distribution of 
phosphate between fluid compartments, and the compo- 
sition of milk have been suggested. All these effects and 
the mechanisms of action in each case will no doubt 
receive more detailed analysis in the future. 


REFERENCES 


1. ALBRIGHT, F. anp R. E_tswortu. J. Clin. Invest. 7: 183, 1929. 

2. ALBRIGHT, F. ano E. C. RetFenste1n, JR. The Parathyroid 
Glands and Metabolic Bone Disease. Baltimore: Williams and 
Wilkins, 1948. 

3. ALBRIGHT, F., H. W. SuLKowitcH AND E. BLOOMBERG. Am. J. 
M. Sc. 193: 800, 1937. 

4. Aursacn, G. D. Arch. Biochem. Biophys. 80: 467, 1959. 

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15. FriepMAN, S. AND P. L. Munson. Biochim. et biophys. acta 35: 


599, 1959- 


16. GarLtarD, P. J. Exp. Cell Res., Suppl. 3: 154, 1955. 

17. GarLtLarp, P. J. In: The Parathyroids, edited by R. O. Greep 
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23. INGALLs, T. H., G. A. DoNALDSON AND F. ALBRIGHT. J. Clin. 
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25. Kenny, A. D. Program of the 41st Meeting of the Endocrine 
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Physiol. 197: 502, 1959. 





qu 
Sul 
na 


lot 
red 
wh 


stir 





olume 19 


ed the 
of milk 
pletely 
roidec- 
alcium 
fect of 


glands 
or the 
mn the 
rations 
‘e now 
lcium- 
| both 
partial 
y not 
-arlier, 
e hor- 
e con- 
ent, it 
urified 
milk. 


dies on 
tion of 
f bone 
action 
alcium 
ress in 
uld be 
emical 
ailable 
action. 
mone 
idney, 
ion of 
-ompo- 
ts and 
doubt 


. Greep 


‘lin. En- 


A.M.A. 
J. Clin. 
ER. Am. 
1docrine 
eep and 


Am. J. 





July 1960 


28. Krane, S. M. J. Clin. Endocrinol. 17: 386, 1957. 

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and R. V. Talmage. Springfield, Ill.: Thomas, 1960. 

go. McLean, F. C. anp W. Broom. 4.M.A. Arch. Path. 32: 315, 
1941. 

31. McLean, F. C. anp M. R. Urist. Bone. Chicago: Univ. Chi- 
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32. Munson, P. L. Ann. New York Acad. Sc. 60: 776, 1955. 

33. Munson, P. L. Fed. Proc. 18: 291, 1959. 

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ALDOSTERONE SECRETION 601 


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39. Rasmussen, H. J. Biol. Chem. 229: 781, 1957. 

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1959- 

43. Rasmussen, H. ano R. G. Westav. Nature, London 178: 1173, 
1956. 

44. TALMAGE, R. V. Ann. New York Acad. Sc. 64: 326, 1956. 

45. Tavmace, R. V., J. R. Ettiotr ano A. C. ENpeErs. Endocrinol- 
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64: 336, 1956. 


Epiphysis cerebri in the control of steroid secretion’ 


GORDON FARRELL? 
Department of Physiology, Western Reserve University, Cleveland, Ohio 


a HAS NOW BEEN SOME THREE YEARS since we first 
postulated the existence of a factor specific for aldosterone 
secretion (4). This Symposium is a fine opportunity to 
review the developments in the study of this factor, and 
to attempt an evaluation of its present status. Paren- 
thetically, the name originally suggested for the factor 
‘glomerulotropin’ has met with some objection, because 
of its similarity to other terms referring to the kidney. 
Accordingly, we have suggested the term ‘adrenoglomer- 
ulotropin’. It might be convenient to retain the abbre- 
viation GTH (rather than AGTH) in order to avoid 
confusion with ACTH. 

We might consider the topic in the light of three 
questions. First, does the hormone exist? Second, as- 
suming that there is such a hormone, what is its chemical 
nature? Third, what glandular structure secretes it? 

With regard to the first question, does adrenoglomeru- 
lottropin exist, I think that we can say beyond any 
reasonable doubt that it does, or at least that something 
which can be extracted from pineal tissue does selectively 
stimulate the secretion of aldosterone. We have found 
biological activity of this type in saline, acetone and 
alcohol extracts of pineal, and we have made an effort to 
purify such preparations in the hope of being able to 
characterize the material. 

First, a word about the assay preparation. If one takes 
adog, makes a midcollicular section and removes the 


' Research supported by grants from the Cleveland Area Heart 
Society and the U. S. Public Health Service. 

* Work carried out during the tenure of an Established Investi- 
gatorship of the American Heart Society. 


+ 
avitaA 


brain substance, including the pineal and pituitary, 
cephalad to the section, and then permits the animal to 
stabilize for 3-4 hours, the secretory rates of aldosterone 
and cortisol are extremely low (1). The rate of secretion 
is determined by cannulating the lumbo-adrenal vein 
and isolating the steroids by paper chromatography. The 
secretory rate of aldosterone under these conditions is 
about one fifth that in the intact animal, and to the best 
of our knowledge is not influenced by hemorrhage or by 
the injection of a variety of drugs or extracts. Melatonin, 
MSH, epinephrine or norepinephrine, extracts of cortical 
tissue and a large number of inactive fractions obtained 
in the process of purifying GTH are without effect_on 
steroidogenesis in this preparation. However, if one 
injects carefully prepared extracts of diencephalic ‘tissue 
including the pineal, or extracts of pineal, the secretory 
rate of aldosterone returns to normal or supranormal 
levels, depending upon the quantity of material injected. 
In order to obtain a return of aldosterone secretion to 
normal one must inject extracts equivalent to about 5 
gm of pineal tissue. The secretory rate of aldosterone can 
also be increased in the assay animal by the injection of 
crude ACTH, or for that matter, of the highly purified 
6-corticotropin or of §,-corticotropin (2). The only real 
difference between the effects of injecting ACTH and of 
injecting preparations containing adrenoglomerulotropin 
is that whereas ACTH stimulates the output of both 
cortisol and aldosterone, GTH stimulates only aldos- 
terone secretion, having no effect on the secretion of 
cortisol. This characteristic is an essential ingredient in 
the definition of a hormone specific for aldosterone 








602 


secretion. Later this morning I will hazard a guess as to 
the relationship between GTH and ACTH. First, how- 
ever, I would like to report some recent efforts aimed at 
purifying the factor, using the decerebrate dog assay 
preparation to follow activity. Figure 1 is a flow sheet 
describing a procedure which results in a rather high 
degree of purification. Ethanol gives a fairly clean initial 
extract. On adjusting the alcohol concentration to 70 % 
by adding water, a highly pigmented oily precipitate 
forms. The activity remains in the supernatant, and 
can be extracted with CHCl. The residue from the 














1000 G. PINEAL TISSUE 
EXTRACT ABS. ETHANOL 
| 
1 | 
ADJUST TO RESIDUE —> 
70% — 
wa | 
SUPT. PE 
EXT. CHCls 
[— 
26 G. CHCIz RESIDUE Aq. fr.—~> 
HEXANE —WATER 
PARTITION 
| 
15.5 G. HEXANE RESIDUE Ag: tr.——— 
LARGE FLORISIL-HEXANE COLUMN 
10.5 mg. (45 X 40cm. 100cc fr.) fr. 1|O-90 


SMALL FLORISIL —- HEXANE COLUMN 
(1X 42cm. lOcc fr. ) 
FIG. 1. Flow sheet of procedure resulting in considerable purifi- 
cation of GTH. Figures at left indicate representative weights of 
various fractions obtained from 1 kg of fresh pineal tissue. 


30 


7 






25 


ALDOSTERONE, yg /100 kg body wt./hr. 


20 


a 
T 


ro) 
T 


uo 
T 








+ 





t t 


40 50 


10 20 30 


FRACTION (10cc) 

FIG. 2. Results of bio-assay of fractions obtained from a florisil 
column developed with hexane. Height of bars represents aldos- 
terone secretion rate in decerebrate dogs infused with aliquots of 
the fraction. Two or 3 dogs were used at each point. 


FEDERATION PROCEEDINGS 


Volume 19 


CHC); is partitioned between hexane and water, and the 
inert aqueous fraction is discarded. The hexane residue is 
chromatographed on a 4.5 x 40 cm florisil column, devel- 
oped with hexane. Approximately one hundred 100 ¢¢ 
fractions are collected, and tubes 10-90 are pooled. This 
large fraction contains the activity. Rechromatography 
in the same system, but using a 1 x 42 cm column (10 cc 
fractions) results in further purification. Figure 2 shows 
the results of assays of one of our more recent columns. 
Activity appears to be concentrated in the vicinity of 
tube 30. Figure 3 is from a recent column in which the 
weights of the various fractions were determined. It is 
clear that several compounds are present and incom- 
pletely separated. Assays of tubes 20-35 indicated that 
activity was present in a fraction in tubes 26-28, probably 
with some overlapping into later tubes. The fraction 
thus obtained was obviously inhomogeneous. Neverthe- 
less, it was active at a dose of 8 wg per dog, indicating a 
rather high degree of biological activity even at this 
level of purification. The potency is of the same order 
of magnitude as that of purified ACTH preparations (2). 

Further efforts at obtaining a single component have 
been hampered by the appearance of instability. Al- 
though in crude or partially purified extracts the factor 
is indefinitely stable at — 10°, especially in hexane solu- 
tion, the active fraction from the florisil column lost 
activity in one week on storage in alcohol in the re- 
frigerator. A recent effort at purification by counter- 
current distribution was a coraplete failure due to loss 
of activity. We have as yet no clues as to the nature of 
the inactivation, but since it occurred in neutral solution 
in the cold, it seems likely to involve a highly unstable 
configuration of some kind. 

The second question, what is the chemical nature of 
GTH, thus remains unanswered, except for the informa- 
tion provided by the process of purification. The material 
behaves as a lipid. It is rather weakly adsorbed by 
florisil, as compared to certain other biologically active 








soot | | [Fee F 

: 

Lg 3s 

* 400+ 5 
o 2750 

= 8 
= 300f 2425 
° L129 

o ’ 
< 200} 5 4i5 

x « 

uw Ww 
410 

100 + m4 

a 
a5 




















T T 
40 50 


o 
on 


10 20 30 
FRACTION (Occ) 
FIG. 3. Wts. of fractions obtained from final florisil column, from 
1 kg of starting material (cf. fig. 1). Assays were carried out on 
selected fractions as indicated. 





Ss ©. | =— ==) 4 AR Fe Fe 


pop oOo =O 





Tolume 19 


and the 
sidue is 
, devel. 
100 cc 
d. This 
graphy 
(10 cc 
» shows 
ylumns. 
nity of 
ich the 
d. It is 
incom- 
=d_ that 
‘obably 
raction 
verthe- 
ating a 
at this 
> order 
ons (2). 
it have 
ty. Al- 
> factor 
1e solu- 
nn lost 
the re- 
ounter- 
to loss 
ture of 
olution 
nstable 


ture of 
\forma- 
1aterial 
bed by 
7 active 


SE 
ALDOSTERONE, ng /100 kg body wt./hr. 
l 1 l 
ee a EE oe 


1 
ow 





2 


mn, from 
d out on 





July 1960 


lipids. For example, a-tocopherol comes off the 1 cm 
florisil-hexane column only after about one hundred 10 
cc fractions, whereas GTH appears at about 30 tubes. 
g-Carotene remains on the column even after 200 tubes. 
On paper chromatograms the activity is associated with 
avery nonpolar fraction, running just behind the solvent 
front, for example, in the system propylene glycol- 
hexane. A considerable number of spot tests have been 
unrewarding. No reaction is seen in the active area with 
dinitrobenzene, phenylhydrazine or Tollen’s reagent. A 
pale tan color results with Le Rosen reagent. 

I would like to digress at this point to mention a 
chance observation that may turn out to be important. 
In following the purification with bio-assay, we observed 
on several occasions that activity appeared to increase 
after chromatography. That is, the active mid-zone from 
the column appeared to be more active than the mixture 


TABLE 1. A Factor from Pineal Extracts Which 
Inhibits Steroid Secretion 





7 Aldosterone |j..... ie 
Experiment Fy Rn. nga ore, ae 
Intact animals, infused with | 8  36.043.2* | 35.9+3.3 
vehicle (saline) or extract of 
cerebral cortex 
Intact animals, infused with 5 15.841.5 | 24.642.7 
inhibitor fraction from pin- (P°<. .oor) | (P< 05) 
eal 
Decerebrate animals, infused 5 ee em = 
with vehiclet 
Decerebrate, infused with in- 8 6.80.4 
hibitor fraction from pineal (P < .oo!) 
Decerebrate, infused with 5 29.3+3.0 


adrenoglomerulotropin 
fraction from pineal 


* Mean and standard error. 

+ Animals used for assay 3 hours after decerebration. The 
aldosterone secretion rate was thus somewhat higher than in 
dogs permitted to rest for longer periods postoperatively (1). 


TABLE 2. Steroid Secretion Rates in Pinealectomized Dogs 


7 Aldosterone Raced 
Experiment | Pres 4 ae. ye mes ag 
Intact animals 9 31.2+3.7* | 32.84+3.9 
4hr. postoperative 
sham je 36.5+8.5 34.9+4.8 
pinealectomy 3 14,921.42 | 3P.2@sb2.5 
1 wk. postoperative 
sham 5 34.645.7 39.3+8.1 
pinealectomy ) 36.8+4.2 54-42%4.5 
3 wk. postoperative | 
sham me 37.9+4.6 38.9+3.9 
pinealectomy | 8 24. 12.6 39.44+3.1 
16 wk. postoperative (last | 
12 wk. on low sodium 
diet) 
sham 2 88, 105 13.3, 16 
pinealectomy i 193, 158 | 30.8, 33.4 


* Mean and standard error. 


? 
ase tana 


ALDOSTERONE SECRETION 603 


which was put onto the column. This suggested the 
presence of an inhibitor in the mixture, presumably 
separated from GTH as purification proceeded. In re- 
examining our data, it seemed that the very early 
fractions, upon assay, gave steroid secretion results 
lower than those to be expected from control assays in 
decerebrate dogs. We collected a number of such 
fractions from hexane-florisil columns and also from the 
least polar zones on paper chromatograms of partially 
purified pineal extracts, and assayed the material in 
intact dogs. Table 1 shows some of our results. There 
appears to be very little question that steroidogenesis is 
inhibited by this material. Extracts of cerebral cortex 
proved inert and served as controls. The inhibition of 
aldosterone secretion appeared to be more marked than 
that of cortisol. The implications of these observations 
remain to be explored. The presence of a factor in pineal 
extracts which inhibits steroidogenesis may help to 
explain some of the disagreement among various workers 
as to the biological effects of injecting such extracts. 
However, that the pineal actually secretes an inhibitor 
is as yet undecided. The material found on our chroma- 
tograms may be a degradation product, possibly of 
GTH itself. On the other hand, one would not be 
surprised if the pineal area actually secretes an inhibitor 
which acts on the adrenal. There is a fairly well-estab- 
lished precedent for inhibition as part of pineal 
physiology, particularly in regard to the gonads (5). Dr. 








35 35 
< 3 
30s | ALDOSTERONE ~ 430 
> : 
25+58 3425 
3 3 
20 FO 2420 
° ~ 
pe oe 
ISFQ A415 
we a 
10 +S a 410 
wi & 

- ° 
5+3 o715 

(=) 
a 
(o) fe) 











INTACT DECEREBRATE DECEREBRATE + 
CONTROLS + PERIPHERAL VEIN OF GALEN 
(3) BLOOD BLOOD 

(3) (4) 


FIG. 4. Evidence that blood obtained from vein of Galen contains 
a factor which stimulates aldosterone secretion. Activity is not pres- 
ent in peripheral blood. 


* x ——> Y —> ALDOSTERONE 
—ACTH— & 


A ==> 3B au C a> D 


E mF emp CORTISOL 


FIG. 5. An hypothesis as to the relationship between GTH 
and ACTH. GTH is postulated as stimulating aldosterone syn- 
thesis from ACTH-dependent precursors along a branch in syn- 
thetic chain. 








604 


Lerner’s elegant work on melatonin, which may be 
thought of as an inhibitor, adds weight to the concept. 
Returning to GTH, there remains the third question 
raised at the beginning: what structure secretes the 
hormone? At first glance, this would seem to be easily 
answered—GTH is found in pineal extracts, therefore 
the pineal secretes it. This may still turn out to be the 
case. However, removal of the pineal results in only a 
temporary suppression of aldosterone secretion. Table 2 
reviews some of our work on the subject. Although the 
output of the steroid drops significantly on the day of 
operation, it returns to normal levels at the end of a 
week. We examined the operative sites for evidence of 
remaining pineal tissue and were unable in most cases 
to find any. Furthermore, pinealectomized animals 
respond to sodium depletion by increasing the secretory 
rate of aldosterone to levels at least as high as those 
found in intact animals on the same regime. These 
studies seemed to indicate that the source of GTH was 
probably not the pineal gland per se, even though one 
can obtain the material from extracts of pineal. We were 
all rather confused by this observation. One of the young 
men working with me, Avery Pratt, designed an experi- 
ment which he felt would permit a decision as to whether 
GTH was actually secreted by the epiphyseal region of 
the brain. Pratt cannulated the vein of Galen in dogs, 
and collected blood from the vein for 1-2 hours. The 
vein drains the pineal as well as the anterior midbrain 
and posterior thalamus. He infused either the whole 
blood which he had collected, or its serum, into decere- 
brate assay animals at about the same rate of flow as that 
from the vein of Galen. The results are shown in figure 
4. There appears to be no question that blood from the 
vein of Galen contains a material which restores aldoster- 
one secretion in the decerebrate dog. It has no effect on 
cortisol secretion, indicating absence of ACTH. These 
findings appear to establish that some structure in the 


FEDERATION PROCEEDINGS 


Volume 19 


area drained by the vein of Galen does indeed secrete 
adrenoglomerulotropin. A reasonable possibility is that 
GTH is secreted by a structure lying ventral to the pineal 
and is present in pineal tissue either because it is stored 
there, or because some of the secretory tissue is included 
in pineal at the time of collection. 

In the few minutes remaining, I would like to intro- 
duce a theoretical concept with regard to the locus of 
action of GTH. I must emphasize that this is only a 
speculation, at best providing a framework for discussion 
(fig. 5). The ubiquitous action of ACTH on steroido- 
genesis (2) may be explained by its action early in the 
synthetic chain. GTH is postulated to act distal to a 
branching of the chain. Thus, it could stimulate aldoster- 
one synthesis but have no effect on the secretion of 
cortisol. Variations in GTH levels could induce the 
frequency observed changed in aldosterone output 
independently of that of cortisol (1). The observation 
that in man, with continued administration of ACTH, 
the initial increased aldosterone secretion returns to 
normal (3) could be due to a compensatory decrease in 
the circulating levels of GTH. Maintenance of aldos- 
terone secretion following hypophysectomy (1), in the 
presence of reduced rate of synthesis of precursors, could 
be associated with increased secretion of GTH. On the 
other hand, maintenance of very high levels of aldos- 
terone secretion may very well require the presence of 
ACTH. The action of the inhibitor previously men- 
tioned would, presumably, be prior to the point where 
the separation of pathways occurs, since it inhibits the 
secretion of both steroids. The return to normal of 
aldosterone secretion, following the initial drop after 
pinealectomy, may be due in part to removal of inhibi- 
tion or to increased ACTH secretion. For the moment, 
however, the whole concept must be considered very 
speculative, and only with time and additional work 
will the answers be known. 


REFERENCES 


1. FARRELL, G. Physiol. Rev. 38: 709, 1958. 

2. FARRELL, G. L., R. B. Fireminc, E. W. RauscHKo.s, F. M. 
Yatsu, M. McCatry anp C. H. Anperson. Endocrinology 62: 
506, 1958. 

3. Lippie, G. W., L. E. Duncan anp F. C. BartTer. Am. J. Med. 
21: 380, 1956. 


4. RauscHKos, E. W. anp G. L. FARRELL. Endocrinology 59: 526, 
1956. 

5. WurtMAN, R. J., M. D. ALtscHULE AND U. Hotmaren. Am. J. 
Physiol. 197: 108, 1959. 





sil] 
lu 


po 
diz 


ser 


195 


Eng 


’ 
ast Gack 





plume 19 


secrete 
is that 
pineal 
stored 
cluded 


| intro- 
cus of 
only a 
cussion 
eroido- 
in the 
il tord 
doster- 
ion of 
ce the 
output 
rvation 
\CTH, 
ims to 
ease in 
—aldos- 

in the 
, could 
On the 
’ aldos- 
ence of 
y men- 
_ where 
vits the 
mal of 
p after 
inhibi- 
loment, 
‘d_ very 
1 work 


59: 526, 


y. Am. J. 





XUM 


On the hemodynamic parameter mediating increase in 


aldosterone secretion in the dog’ 


DONALD S. GANN, IVOR H. MILLS? AND FREDERIC C. BARTTER 
Section on Clinical Endocrinology, National Heart Institute, Bethesda, Maryland 


-™ Loss (2, 10), supradiaphragmatic constriction 
of the inferior vena cava (16, 17) and bilateral constric- 
tion of the common carotid arteries (3) have all been 
shown to produce consistent increases in the rate of 
aldosterone secretion. The present studies were under- 
taken to see if any hemodynamic parameter might be 
common to all three of these stimuli. 


METHODS 


Acute experiments were performed on mongrel dogs 
under pentobarbital anesthesia. The lumbo-adrenal 
veins were cannulated (14) and blood samples were 
collected intermittently for determination of aldosterone 
by Mills’ modification (16) of the method of Neher and 
Wettstein (17). Blood was collected during a half-hour 
control period. The appropriate constriction was then 
applied, and a second collection was made during the 
second half hour after constriction. Mean and pulse 
pressures in brachial and lingual arteries and right 
atrium, and pressures in femoral vein and esophagus, 
were measured by means of indwelling cannulae and 
Statham pressure transducers, and recorded continu- 
ously on a Sanborn 8-channel recorder. Serial hemato- 
criti determinations were made. In all experiments 
utilizing caval or carotid constriction, blood loss was 
continuously measured and replaced by transfusion. 

Twenty-six studies were performed on normal dogs. 
Aldosterone secretion was stimulated by supradiaphrag- 
matic constriction of the inferior vena cava (16) in 10 
experiments. In 12 experiments, the stimulus was bi- 
lateral constriction of the common carotid arteries low 
in the neck, effected by means of chokers consisting of 
silk loops passed about the arteries and through double 
lumen tubes suspended above the arteries (3). The 
positions of the chokers and relevant anatomy are shown 
diagrammatically in figure 1. Four intact dogs were bled 
serially in 50 cc amounts while pressures were measured. 


RESULTS 


Aldosterone Secretion. The effects of caval and carotid 
constrictions and of blood loss on aldosterone secretion 
have been presented in detail elsewhere (3, 10, 16). 

‘Presented in part at the 41st Meeting of the Endocrine Society, 
1959. 

*Present address: St. Thomas’ Hospital, London S. E. 1, 
England. 


605 


Inferior Vena Cava Constriction. Constriction of the supra- 
diaphragmatic inferior vena cava produced diminished 
arterial mean and pulse pressures, diminished right 
atrial mean and pulse pressures, increased femoral 
venous pressure (7-15 cm H,O) and increased hemato- 
crit. Aldosterone secretion rose. All the hemodynamic 
changes may be explained as consequences of pooling of 
large quantities of blood, with transudation of fluid, 
‘below’ the constriction. An experiment representative of 
this group is shown in figure 2. Data derived from such 
tracings are shown in table 1. 





F1G. 1. Anatomy of carotid region in dog. Occ.—occipital artery. 
C.S.N.—carotid sinus nerve. I.C.—Internal carotid artery. C.S.- 
carotid sinus. E.C.A.—external carotid artery. C.C.A.—common 
carotid artery. N—nerve arising from thyro-carotid arterial junc- 
tion. T—Thyro-carotid arterial junction. 








FEDERATION PROCEEDINGS 


Volume 19 


























606 
3/3/59 CAVAL CONSTRICTION 
om HO | 
FEMORAL 40- 
VENOUS hee 
PRESSURE eon 
o- nets 
mm Hg 
BRACHIAL 240. 
: : ne : ARTERIAL 165-1 
Fic. 2. Effect of constriction of PRESSURE es es i a eee een peg Oe ae 
supradiaphragmatic inferior vena 90-4 ~—s 
cava on aldosterone secretion and 
on certain hemodynamic param- — 
eters. Paper speed is 25 mm/sec. ESOPHAGEAL 7 
at either side, 0.25 mm/sec. in PRESSURE 0-4 — aro 
center during introduction of the = 25. 
constriction. 
cm H, 0 
RIGHT ATRIAL 25 - 
PRESSURE o- perce oe UNE cgi ye ene 
NN Tamra AA 
- 3- 
ug/hr 
is - 
ALDO. 10 4 
5 4 
o wie 





TABLE 1. Effect of Constriction of the Supradiaphragmati 
Inferior Vena Cava on Arterial Pulse and Mean 
Pressures and Aldosterone Secretion 


Control Caval Constriction 


Art. pulse Art. mean Aldosterone —— — Aldosterone 

pressure pressure pe /hr. Pe enc Fee wg /hr. 
36 140 3.0 20 103 10.4 
20 131 5-3 16 go 10.3 
28 154 6.2 16 118 11.9 
40 158 5-4 32 15! 7-7 
20 99 2.9 14 7 6.0 
48 128 4-9 20 7 11.0 
28 110 i. 24 104 6.0 
36 124 5.3 32 95 20.0 
24 122 6.1 21 115 12.6 
30 141 2.9 21 135 14.7 


Common Carotid Artery Constriction. Bilateral constriction 
of the common carotid arteries ordinarily produced 
diminished intracarotid mean and pulse pressures, in- 
creased peripheral arterial mean and pulse pressures, 
and increased right atrial pulse pressure, (2-4 cm HO) 
without change in right atrial mean pressure, femoral 
venous pressure or hematocrit. Aldosterone secretion 
rose. All arterial pressure changes may be explained as 
manifestations of the Hering reflex (13), as carotid 
pressure falls. The increase in right atrial pulse pressure 
is also a part of this reflex (11). The fall in hematocrit 
which occasionally occurs probably results from transfu- 
sion with blood more dilute than that of the recipient. 
An experiment representative of this group is shown in 
figure 3. Data derived from such tracings are shown in 


table 24. All changes seen with carotid constriction are 
opposite to those seen with caval constriction, except for 
those in aldosterone and intracarotid pressure. (Pulse 
rate and respiratory rate increased transiently with 
constriction in most experiments). 

The effective pressure factor is further delineated by a 
series of experiments in which only slight constriction was 
applied to the carotid arteries. This produced diminished 
intracarotid pulse pressure while intracarotid mean 
pressure remained the same or even increased, as 
peripheral arterial pressure rose during the Hering 
reflex. An experiment representative of this group is 
shown in figure 4. Data derived from such tracings are 
shown in table 28. Thus, only diminished intracarotid 
pulse pressure is consistently associated with the increase 
in aldosterone secretion seen with both caval and carotid 
constriction. 

Blood Loss. Blood loss produced a fall in arterial pulse 
pressure before there was any significant alteration in 
mean pressure. A representative experiment is shown in 
figure 5. Atrial pulse pressure is also diminished early in 
blood loss, while mean pressure, hematocrit and venous 
pressure remain unchanged until loss is severe or pro- 
longed. These findings are in agreement with those of 
Padhi and co-workers (18). 


DISCUSSION 


Constriction of the inferior vena cava (7, 16), constric 
tion of the carotid arteries (3) and blood loss (2, 10) are 
all adequate stimuli to increases in aldosterone secretion. 
Of the variables measured, only intracarotid pulse pres 
sure is consistently altered in the same direction by each 
form of stimulus to aldosterone secretion, as show? 





dia 
the 
car 


Vat 
hot 





‘olume 19 


aA 


Ne eters 


tion are 
cept for 

(Pulse 
ly with 


red by a 
‘ion was 
ninished 
1 mean 
ised, as 

Hering 
roup 1s 
ings are 
acarotid 
increase 
| carotid 


jal pulse 
ation in 
hown in 
early in 
| venous 
or pro- 
those of 


constric- 
10) are 
cretion. 
lse pres 
by each 
; shown 





July 1960 


9/23/58 


LINGUAL 
ARTERIAL 
PRESSURE 


ESOPHAGEAL 
PRESSURE 








cmH, 0 
RIGHT ATRIAL > 











ALDOSTERONE SECRETION 


CAROTID 





607 


CONSTRICTION 


























PRESSURE 0, 
— 25 - f . 

emk,O 
FEMORAL ct 
VENOUS 20 - 
PRESSURE o- 

men Hg 
BRACHIAL 210 - 
ARTERIAL 35, 
ee ae 

wg /he. 

” 
ALDO. 304 
20-4 
104 





FIG. 3. Effect of bilateral common carotid artery constriction on aldosterone secretion and certain hemodynamic parameters. Paper 
speed is 25 mm/sec. at either side and 1 mm/sec. in the center, during application of constrictions. 


TABLE 2. Effect of Bilateral Constriction of the Common 
Carotid Arteries on Intracarotid Pulse and Mean 
Pressures and Aldosterone Secretion 


Control Carotid Constriction 
Intra- Intra- oe : Intra- 
carotid carotid Aldosterone ee =a carotid Aldoatesone 
pulse mean we/hr. Bosch mean . ; 
pressure pressure eee pressure 


A. Experiments with fall in intracarotid mean pressure 


22 130 3.9 18 114 5-9 
44 159 5:1 24 132 11.9 
32 167 5-3 26 159 10.0 
36 148 4.0 18 144 9.9 
46 145 3.7 18 142 8.6 
24 138 10.0 12 132 40.0 
33 140 20.0 30 127 27.0 


B. Experiments without fall in intracarotid mean pressure 


26 136 g.0 20 148 12.3 
35 150 13.7 18 150 55-3 
66 156 3.4 24 156 17.0 
27 165 6.0 18 168 9.0 
15 185 1%. 


5 7.5 186 21.0 


diagramatically in figure 6. It has been shown (3) that 
the increase in aldosterone secretion in response to either 
carotid or caval constriction does not occur if the thyro- 
carotid arterial junctions have previously been dener- 
vated. This has also been shown to be a buffer area in 
both dog (11) and cat (12). The frequency of impulses 


YUM 


in the nerves from buffer areas has been shown by 
Landgren (15) and Ead, Green and Neil (8) to be a 
function of the rate of change of distention of the blood 
vessel wall, essentially reflected as pulse pressure. The 
frequency is diminished at lower pulse pressures, even if 
mean pressure is maintained (8). This evidence from 
neurophysiological experiments is thus consistent with 
that presented here, and suggests a mechanism such as 
that shown diagrammatically in figure 7. It is postulated 
that nerve impulses arising at the thyrocarotid arterial 


junction in response to arterial pulsation inhibit central 


nervous release of a neurohormone thought to mediate 
increases in aldosterone secretion. With caval or carotid 
constriction or blood loss, diminished pulse pressures 
lead to reduced impulse traffic in the nerves, a release 
of inhibition and, ultimately, increase in aldosterone 
secretion. 

The inability of Davis, Kliman, Yankopoulos and 
Peterson (6) to block by dextran infusion the rise in 
secretion of aldosterone following caval constriction 
might appear to be in conflict with our conclusions. How- 
ever, similar experiments performed in this laboratory 
have yielded opposite results. In these experiments, the 
caval constriction was first shown to be effective in 
increasing the rate of aldosterone secretion. The infusion 
was then begun, and continued at a rate sufficient to 
reverse the fall in arterial pressure which ‘had followed 
caval constriction. Two such experiments are shown in 








608 


FEDERATION PROCEEDINGS 


Volume 19 


























11/25/58 CAROTID CONSTRICTION 
man Hig 
LINGUAL 240- a ' 
canoe 165-4 : SII Nesom a oy, pyar enn 
SSURE 90d ‘ 6 
em, 0 
ESOPHAGEAL = 25 + ° 
PRESSURE 0-4 yap 
“od is t Be, i 
- Ba 
om 1,0 
RIGHT ATRIAL 25- 
PRESSURE ot 
a ead ore mn et, a Seth aac edi edited adieetih ce cemadiincatiendtincati saa mm cateeatinadiietinetietind 
— 254 
cm H, 0 
FEMORAL i 
VENOUS 20 ~ 
PRESSURE J i 
mm lig 
BRACHIAL 240, ii e 
ARTERIAL 1654 == vy SSS SVAN 
PRESSURE 90. 
mg/ hr. 5 ws zis 
9- 
ALDO. 6 4 
3-4 
04 
Fic. 4. Effect of slight bilateral common carotid artery constriction on aldosterone secretion and certain hemodynamic param- 


eters. Paper speed is 25 mm/sec. at either side and 1 mm/sec. in the center, during application of the constriction. 


170. Ow fe) 
ARTERIAL —_ a 
MEAN 68 o~..., 
PRESSURE 
. 166 °o. 
mm Hg 
\ e he 
a ©@ 
anrenat| i 
PULSE ae 
PRESSURE |-41 .* 
mmig 


agen ARTERIAL PULSE 
& PRESSURE 


37 Th: 


' Gea 
| < 





___BLOOD LOSS (mi) _ 


° 50 100 150 200 250 300 350 400 


FIG. 5. Arterial pulse and mean pressures measured during pro- 
gressive blood loss. Dog, 19 kg. 








IVC CCA BLOOD 
NORMAL CONSTRICT. CONSTRICT. Loss 
ALDO 
SECRETION oe 
INTRACAROTID __AAAAA napihs AAAS 
B.P mean AMY 
PERIPHERAL ANY 
arTeriaL BP. AAA pens AAA 
RIGHT ATRIAL 
PRESSURE AANA cick ASANAAL AAAS 
FEMORAL 
V.P. 
HEMATOCRIT « T s t 


FIG. 6. Schematic representation of hemodynamic changes with 
three types of stimulus to aldosterone secretion. Only intracarotid 
pulse pressure shows the same direction of change with each stimu 
lus to increased aldosterone secretion. Reproduced by permission 
of the American Heart Association, Inc. (2a). 


figure 8. Arterial pulse pressures were not described in 
the experiments cited above (6), but transient decreases 
in mean pressure indicate that they could not have been 
held constant. 

It is interesting to consider the possible role of the 
mechanism postulated above in cardiac failure, whichis 
often associated with an increase in aldosterone secretion 


tion 
of b 
Am 


ture 
tain 
befc 
pres 
tion 
decc 
for 

ultir 








lume 19 


param- 


BLOOD 
LOSS 


i 
ages with 
racarotid 
ch stimu 
ermission 


ribed in 
ecreases 
ve been 


> of the 
whichis 
ecretion 


July 1960 





CNS Fy. Neve 


| 


ts Kw 


Arterial 
Pulse 


CNS 


| 


ALDO. ALDO. 

FIG. 7. Schematic representation of primary mechanism for in- 
crease in aldosterone secretion. On the left, the normal, unstimu- 
lated dog. The arterial pulsation gives rise to a certain level of im- 
pulse traffic in the nerves arising at the thyro-carotid arterial 
junction, thus inhibiting central nervous (CNS) release of a neu- 
rohormone which would stimulate adrenal (Adr.) secretion of 
aldosterone. On the right, diminished arterial (intracarotid) pulse 
pressure is shown giving rise to diminished impulse traffic in the 
nerves arising at the thyrocarotid junction, a release of CNS in- 
hibition, increased neurohormone secretion and_ ultimately, 
increased aldosterone secretion. 


ECAVAL CONSTRICTION 4 


Nerve 
MDUR 














20 


an 
\t 


Arterial 
Pulse 
































= 
= 16 BLOOD 
10 43 - cc/min. IN 
rt) | 1 FRONT 
Zz ' | LEG 
& 1 | 
5 tts 
be 
W 
0 a ' T T 5 | 
ie) 120 180 240 min, 
ORL ‘CONSTRIC TION? 
zB BLOOD 
; IN 
: | 28 min. 
r= ,; FRONT 
> {LEG 
uJ | 
Ss ' 
5 ia 
-_ 
n 
ro} 
re 
oo e_ 
Oo 60 240 min, 


ric. 8. Two experiments showing increase in aldosterone secre- 
tion following caval constriction and its reversal by rapid infusion 
ofblood above the constriction. Reproduced by permission of the 
American Heart Association, Inc. (2a). 


(1). Direct correlation of pulse pressure, aldosterone 
secretion and sodium excretion, in cardiac failure in 
man, have not been reported. However, a few data 
bearing on the relationship are in the published litera- 
ture. Figure 9 shows results, from Eichna ef al. (g), ob- 
tained on patients with low-output congestive failure 
before and after digitalization. In all patients, pulse 
pressure rose with compensation, as did sodium excre- 
tion. It is well known that pulse pressure is high during 
decompensation in patients with high-output failure, as, 
for example, in thiamine deficiency (20), and that it 
ultimately falls with therapy (5). These observations 





YVLIM 


ALDOSTERONE SECRETION 


609 


OUT of FAILURE 
F200 


F180 
+ 160 


IN FAILURE 


% CHANGE 
in 


PULSE PRESSURE 
+ 140 


+ 120 
- 100 














150 
as 

MEAN jo] BZ 
Na eB 
EXCRETION 2°) isp 
604 3 

uEq/min. 305 oe 
O04 Pe Ok 























FIG. g. Data from Eichna et al. (g), showing studies on patients 
in and out of congestive failure. After compensation, there was uni- 
formly increased sodium excretion and arterial pulse pressure, 
compared with values obtained during decompensation. 












































THIAMINE 
30, w¥ 
V.P. 20 - a 
aA 
cm HO 10- iii tee 
04 : 
ay (e) 1@) 
URINE er 
VOLUME 2 7 ae 
isd. 0 4 o——_O 
160- \ 
1404 \ 
BP. 1204 
100- ao 
mm Hg 
80- — 
604 
88 - 
° °. 
86 - 
aw «= 
Kg. 824 ° 
804 
78 | DAYS 
2 ae ee 


FIG. 10. Data from Blackett (4) on a patient with high-output 
cardiac failure (beriberi heart disease) treated with thiamine. A 
rise in arterial pulse pressure is among the earliest changes with 
therapy. 








610 


alone would suggest that oversecretion of aldosterone 
and sodium retention in these patients could not result 
primarily from a decrease in pulse pressure. It is possible 
that thiamine deficiency effects receptor areas directly. 
However, published data indicate quite clearly that the 
immediate response to thiamine therapy in_beri-beri 
heart disease includes a rise in pulse pressure as sodium 
diuresis occurs. This is shown in data recharted from 
Blackett (4) in figure 10. Weiss and Wilkins (20) show 
similar results in all four studies in which arterial pres- 
sures were measured during treatment. Evidence from 
patients with cardiac failure is thus not inconsistent with 
the findings in the dog. 

A ‘volume receptor’ has been postulated to be active 
in the reflex control of circulating volume (19). The 
present evidence suggests that such receptors may not 


FEDERATION PROCEEDINGS 


Volume 19 


only be multiple (16), but may be simple baroceptors, 
responding to distortion or pulse pressure changes. 


CONCLUSION 

Constriction of the inferior vena cava, constrictions 
of both common carotid arteries and blood loss are all 
adequate stimuli to increase in aldosterone secretion. In 
acute studies in dogs, only diminished intracarotid 
pulse pressure correlated with increases in aldosterone 
secretion with these stimuli. It is postulated that dimin- 
ished intracarotid pulse pressure leads to an increase in 
aldosterone secretion by reducing the impulse traffic in 
the nerve arising at the thyrocarotid arterial junction, 
thus releasing inhibition of a central center which may 
control release of a neurohormone mediating increase in 
aldosterone secretion. 


REFERENCES 


1. AXELROD, B. J., J. E. Gates, B. B. JoHNson Anp J. A. Leut- 
SCHER, JR. Brit. M. J. 1: 196, 1955. 

2. BARTTER, F. C. Scandinav. J.-Clin. & Lab. Invest. 10: 50, 1957. 

2a. BARTTER, F. C. anp D. S. Gann. Circulation 31: 1016. 1960. 

3. Barter, F. C., I. H. Mitts ano D. S. Gann. J. Clin. Invest. 
In press. 

4. BLackett, O. Australasian Ann. Med. 4: 261, 1955. 

5. Burwe.t, C. S. anp L. Dexter. Tr. A. Am. Physicians 60: 59, 
1947- 

6. Davis, J. O., B. Kuiman, N. YANKOPOULOS AND R. E. PETER- 
son. J. Clin. Invest. 37: 1783, 1958. 

7. Davis, J. O., M. M. Pecuet, W. C. Bai, Jr. anp M. J. 
Goopkinp. J. Clin. Invest. 36: 689, 1957. 

8. Eap, H. W., J. H. Green ano E. Nerv. J. Physiol. 118: 509, 
1952. 

g. Eicuna, L. W., S. J. Farsper, A. R. Bercer, D. P. EArze, 
B. Raper, E. PELLAGRiINo, R. E. ALBERT, J. D. ALEXANDER, 
H. TauBe AND S. YOUNGWIRTH. Circulation 7: 674, 1953. 


10. FARRELL, G. L., R. S. RosNAGLE AND E. W. RaAuscHKoLs 
Circulation Res. 4: 606, 1956. 

11. Gann, D. S. ano F. C. Bartrer. Am. J. Physiol. 197: 
1959- 

12. GREEN, J. H. J. Physiol. 122: 70P, 1953. 

13. Herinc, H. E. Die Karotissinus Reflexe auf Herz und Gefasse. 
Leipzig: Steinkopf, 1927. 

14. Hume, D. M. ann D. H. NeEtson. Surg. Forum; Proc. 4oth 
Congr. Amer. Coll. Surgeons, 568, 1955. 

15. LANDGREN, S. Acta physiol. scandinav. 26: 35, 1952. 

16. Mixus, I. H., A. G. T. Casper AND F. C. Barter. Science 128: 
1140, 1958. 

17. NEHER, R. and A. WerttstTEIN. J. Clin. Invest. 35: 800, 1956. 

18. Paput, R. K., E. M. Nanson ann R. B. Lynn. Ann. Surg. 148: 
827, 1958. 

19. SmitH, H. W. Am. J. Med. 23: 623, 1957. 

20. Weiss, S. AND R. W. Wikins. Ann. Int. Med. 11: 


1229, 


104, 1937. 





or, 
Ta 
an 
re] 
ass 


of 


ph 
dis 
oul 


col 
of ¢ 


dis 
dru 
int 
me 
the 
He 
dict 
of t 
It s 
ten- 


can 
Exp 





olume 19 


eptors, 


se 


‘ictions 
are all 
ion. In 
carotid 
sterone 
dimin- 
ease in 
affic in 
nction, 
h may 
ease in 


SCHKOLB 


7: 1229, 


Gefasse. 


oc. 4oth 


ence 128: 


1956. 


urg. 148: 


, 1937: 





INTERSOCIETY SYMPOSIUM ON PHYSIOLOGY AND DRUG ACTION' 





Introduction 


HALLOWELL DAVIS, Chairman 
Central Institute for the Deaf, St. Louis, Missouri 


is INTRODUCTION is written at leisure, some months 
after the actual symposium, and with all the manu- 
scripts in hand. It is a combination of what I may have 
said at the time, recalled by imperfect memory, with 
what I might have said if all of the manuscripts had been 
available before the meeting. It is thus a review or com- 
mentary rather than an introduction. 

This symposium on Neuropharmacology introduces 
the second of a series of Bi-Society sessions by this title. 
The program has been arranged by a Bi-Society com- 
mittee under the chairmanship of Dr. Harold Himwich. 
These sessions are joint sessions of the American Society 
for Pharmacology and the American Physiological So- 
ciety. The series is likely to continue for a number of 
years, with or without introductory symposium pro- 
grams such as the following. In a field developing as 
rapidly as neuropharmacology we may look forward to 
an annual or biennial symposium without becoming too 
repetitive. These symposia should provide a current 
assessment of accomplishment and of developing points 
of view. 

A year ago I pointed out that we should divide our 
general topic, on the basis of objectives, techniques, ob- 
servations and concepts, into two major divisions, Neuro- 
pharmacology proper and Psychopharmacology. The 
distinction is still valid, although, as it happens, three of 
our speakers this year will deal with neural mechanisms, 
ie. Neuropharmacology, while only Dr. Hunt will be 
concerned with the modification of the over-all behavior 
of animals, i.e. Psychopharmacology. 

Actually Dr. Hunt will give a broad and informative 
discussion of ‘problems on the behavioral assessment of 
drugs.” He will be more concerned with methods, with 
interpretation of data and with the limitations of various 
methods, than with any summary or generalization as to 
the actions of particular drugs or even classes of drugs. 
He will not present new data. I believe that, mirabile 
dictu, he will not show us a single lantern slide !.Critique 
of this sort is a very valuable type of symposium paper. 
It simply cannot be done in one or even in a series of 
ten-minute papers. 

‘Atlantic City, N. J., April 14, 1959. Organized by the Ameri- 
can Physiological Society and the Society for Pharmacology and 
Experimental Therapeutics. 


7 
avi tad 


611 


Dr. Bickford, Dr. Goldring and Dr. Brazier will all 
deal with the electrical output of the nervous system and 
its modification by drugs. They differ in the size of the 
anatomical or physiological unit with which they are 
concerned. Dr. Bickford deals with the areas and systems 
that are differentiated by electroencephalographic tech- 
niques in man and, like Dr. Hunt, will give us a critique 
of method and not present details of new observations. 
He does have some slides, some very fine ones as a matter 
of fact, which he will use to illustrate types of effect that 
can be observed, and certain broad considerations about 
the electroencephalogram in relation to the whole or- 
ganism and to the analysis of drug action. His contribu- 
tion is another excellent example of what I call the ‘tu- 
torial’ type of paper. 

Dr. Brazier’s contribution is rather more specific. She 
will show the possibilities of certain types of analysis of 
the electrical output of the nervous system—the auto- 
matic computation of average response and autocorrela- 
tion. These two methods, in spite of the elaborate in- 
strumentation which they require, should become power- 
ful additions to simple multichannel recording and to 
conventional frequency analysis. Dr. Brazier uses the 
new methods to study the action of certain anesthetics. 
Her conclusions deal with cortical responses evoked by 
way of specific and nonspecific pathways and systems. 

Even more detailed is the paper by Drs. Goldring and 
O’Leary, who are concerned with the physiological 
analysis of electrical responses of the cortex and the use 
of drugs in this analysis. The anatomical unit here is 
the neuron and its major subdivisions: the dendrite, the 
soma and the axon. Much is made also of the typical 
arrangement and orientation of the neurons of various 
classes in the cerebral cortex. The analysis is in terms of 
typical actions, particularly the action of dendrites, and 
the effects of certain drugs on their electrical responses. 
Some of the conclusions are basic to the interpretation of 
electrical records from gray matter in general and from 
the cortex in particular. The observations and the inter- 
pretations differ significantly in several respects from 
those presented last year by Dr. Grundfest. More em- 
phasis is placed on the fact that cortical neurons are im- 
bedded in and form part of the tissue of the brain and 
that drugs applied topically to the surface can syste- 








612 


matically effect one part of the dendritic system more or 
earlier than another. The interpretations are still con- 
troversial, and it is therefore appropriate that Dr. 
Goldring should present his evidence systematically and 


FEDERATION PROCEEDINGS 


Volume 19 


in some detail. The correct solution to some of the ques- 
tions that are involved is of great importance for under- 
standing both the organization of the cerebral cortex and 
the nature of the action of several classes of drug. 


Pharmacological dissolution of evoked 


cortical potentials 


SIDNEY GOLDRING AND JAMES L. O’LEARY 
Division of Neurosurgery and Beaumont-May Institute of Neurology, Washington University 
School of Medicine, St. Louis, Missouri 


TD aitaiemess: STIMULATION within the paths of each of 
several thalamic projections to the cortex elicits a cortical 
potential of characteristic form and polarity. This is il- 
lustrated most clearly by comparison of the results of 
recruiting (mid-line nuclei stimulation) and primary 
sensory (relay nuclei stimulation) responses. The pri- 
mary response of sensory projection cortex to a single 
shock is a complex diphasic (positive/ negative) potential, 
the initial positive phase of which is polyphasic, i.e. 
surmounted by a series of faster spikes. By contrast, the 
single cortical response to a stimulus applied to the mid- 
line thalamic nuclei is a predominantly monophasic 
negative potential of simpler form. Activated by serial 
stimuli delivered in slow repetition the successive tran- 
sients grow in amplitude. Experimental manipulation 
leading to analysis of these responses has related polarity 
and form of potential to the vertical distribution of the 
discharging elements within the cortex (1-3). For exam- 
ple, the initial slow positive deflection of the primary 
response signals synaptic activation (probably dendritic) 
in the deeper cortical layers; the ensuing negative one 
has been interpreted as due either to antidromic conduc- 
tion along apical dendrites or to independent synaptic 
excitation of these cell parts. An origin from the cell 
bodies of successively superficial cortical layers has been 
suggested for the brief positive spikes. In the case of the 
recruiting wave, activation of apical dendrites in the 
superficial cortical layers (ca. upper 0.5 mm) appears to 
account for the major portion of the potential. Thus 
(by inference) one system can activate both cell bodies 
and dendrites, its terminals being distributed through 
much of the cortical thickness; the other system probably 
excites mainly dendrites, its terminals being most dense 
in the superficial reaches of the cortex. 

This arrangement affords a useful point of departure 
for investigating the effects of pharmaceuticals on corti- 
cal evoked potentials, and particularly for evaluating the 
sensitivity of cortical dendrites and cell bodies to different 


drugs. Information is already available concerning the 
increased susceptibility of the negative component of 
the primary response and of the recruiting wave to anes 
thetics, convulsants and anoxia. These potentials are 
abolished before the positive wave of the primary re- 
sponse is, and in light of their inferred dendritic origin 
the observations indicate a high degree of sensitivity of 
apical dendrites to drug action and to metabolic changes. 

Hereafter we report the extension of the experimental 
approach leading to the above conclusions and compare 
the effects of drugs on dendritic (cerebral and cerebellar) 
and recruiting potentials and their accompanying slow 
negativity. The slow negativity has been studied recently 
by us (4-6) and our observations indicate the proba- 
bility that it is a separate postsynaptic process. It has an 
unusually long duration (150-300 msec) and accom- 
panies most evoked cortical potentials, having been seen 
(fig. 1) in conjunction with cerebral and cerebellar 
dendritic responses (5, 12) recruiting waves (5) and 
primary evoked potentials (5). As a background for the 
work to be described we summarize the evidence for 
considering the slow negativity a separate excitation 
process, and review the electrophysiological properties 
of that slow potential. 

Using cortical surface stimulation a threshold stimulus 
evokes the usual negative 15 msec dendritic potential. 
This is followed by a positive aftereffect which grows in 
amplitude as the size of the primary potential increases 
with increasing stimulus strength. At a voltage near 
maximum for the dendritic spike the surface positivity 
commences to be submerged by a rapidly developing 
negativity. This is the slow negative potential which we 
describe (fig. 2A, 12 v. and 15 v.). Under these exper: 
mental conditions the difference in threshold of the two 
components is probably accountable to a deeper origif 
of the slow negativity in the cortical thickness. In figurt 
2B a saline-filled pipette, having an outside diameter @ 
100 w and led from a calomel half-cell, was inserted 


7 ae — ee 


“Sn 


neg 
drit 
are 
use 
wit 
orig 
last 
the 


exc 


tive 
sub) 


the 
app 





7 
asi tad 





olume 19 


> ques- 
under- 
ex and 


ing the 
ent of 
oO anes 
als are 
ary re- 
: origin 
ivity of 
hanges. 
imental 
om pare 
*bellar) 
1g slow 
ecently 
proba- 
has an 
accom- 
en seen 
rebellar 
5) and 
for the 
nce for 
citation 
operties 


timulus 
ytential. 
TOWS in 
ncreases 
xe neat 
ositivity 
veloping 
hich we 
experi 
the two 
r origin 
n figure 
meter of 
inserted 





’ 


July 1960 


Dendritic (cerebral) 





Dendritic (cerebral) 


FIG. 1. Various evoked responses accompanied by slow nega- 
tivity. Singly evoked response in monkey. Stimulus strength was 
maximal for slow negativity. B- repetitively evoked (6/sec.) re- 
sponse in monkey at !%4 stimulus strength used in A (A and B from 


g). C: repetitively evoked (6/sec.) responses in cat (from 12). 
D: responses (6/sec.) elicited in rabbit (from 5). £. response from 
rabbit striate cortex; stimulus = light flash, 10 msec (from 10). Cal, 
in lower right-hand corner: vertical line of right angle = 500 pv; 
horizontal line = 20 msec. In this and all other figures, except 
fig. 9, straight line is baseline against which all potential changes 
are read. Excursions above the baseline are positive, below 
negative. 


through the surface cortical layer. The tip was lowered 
in 0.1 mm fractions (or less) while recording the re- 
sponses to serial stimulation at 6/sec. The polarity of the 
slow negativity was found to reverse more deeply (0.7— 
0.9 mm) than that of the preceding dendritic one (0.15- 
0.4mm) (6). Thus, on the basis of threshold differences 
(using surface stimuli) and differences in depth of origin 
in the cerebral cortex we conclude that the 15 msec 
negative potential assignable to activity in apical den- 
drites and the ensuing 150-300 msec slow negative wave 
are different neural processes. The fact that the stimulus 
used is a very local one and recording is accomplished 
within 0.5 mm of the stimulus site argues against an 
origin by fusion of many temporally dispersed short- 
lasting processes of variable latencies, and suggests that 
the slow negativity is an independent postsynaptic 
excitation of unusually long duration (6). 

The slow negativity can also be uncovered by repeti- 
tive stimulation. With successive stimuli (of a strength 
subthreshold for the appearance of this component in 
the response to a single stimulus) the slow negativity 
appears gradually, coming to replace the positive after- 


XUM 


CGRTICAL POTENTIALS 


613 










FIG. 2. A- cortical responses to stimuli of increasing strength. 
B: serially evoked responses to direct cortical stimulation (6/sec.) 
as the cortical recording electrode is lowered through the cortex. 
Note the primary potential is clearly reversed at 0.4 mm, whereas 
the slow negativity does not show complete reversal until a depth 
of 0.9 mm is reached. Straight line = baseline. Positive up. Vertical 
cal. line = 500 uv; horizontal, 30 msec. (From 6). 





Positive after-effect 
Slow negativity 





FIG. 3. Repetitively evoked dendritic potentials and accom- 
panying slow negativities. 4- 6/sec. B: 20/sec. Cal. in lower right- 
hand corner: vertical line of right angle = 500 uv; horizontal 
line = 30 msec. 


effects of the later dendritic responses (fig. 34). Upon 
increasing the stimulus frequency the slow negative 
responses to the individual stimuli summate and fuse to 
provide a smooth d.c. change which outlasts the stimulus 
period for a singularly long time (1—3 sec.) (fig. 3B). The 
graded character of this summated potential suggests 
that it has a dendritic origin. Thus far we have not ob- 
served after-positivity summation. Because of this, and 
because of the identical thresholds of after-positivity and 
preceding dendritic spike, we continue to regard the 
former as an aftereffect of the latter and not as a separate 
excitation process. 








614 


(120/80) 


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Nembutal - 69 mgm. (110/70) 





Nembutal - additional 15 mgm. (130/80) 


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FIG. 4. Effect of Nembutal on serially evoked dendritic po- 
tentials and their summed negative aftereffect. In A, 
stimulus frequencies are 5 and 20 per sec., upper and lower traces 
respectively. A: prior to Nembutal injection. B: after i.v. injection 
of 60 mg Nembutal given in two injections of 30 mg each. C: after 
an additional 15 mg Nembutal. In this figure and in fig. 6 numbers 
to right above tracing refer to blood pressure. Vertical line of cali- 
bration symbol, 500 uv; horizontal line, 20 msec. (From 4). 


Changes produced by Nembutal, procaine, metrazol 
and GAB (gamma aminobutyric acid) are described. 
GAB was applied to the cortical surface. The other 
agents were administered intravenously and controlled 
in separate experiments by anoxia (100% nitrogen in- 
halation) and hypotension (exsanguination), permitting 
evaluation of systemic factors which might contaminate 
results. It will be seen that the dendritic (cerebral and 
cerebellar) and recruiting potentials, and the slow nega- 
tivities which follow them, are affected differentially by 
the several pharmacological agents. The results obtained 
with drugs injected intravenously indicate different 
pharmacological sensitivities for transient and slow nega- 
tivity. The data obtained with GAB (applied to the 
cortical surface) suggest a different origin in the cortical 
depth for the early and late potentials. The accumulated 
results afford additional evidence that the primary po- 
tential (either dendritic or recruiting) and the following 
slow negativity are separate neural processes. 

EFFECTS OF INTRAVENOUSLY INJECTED NEMBUTAL, 
PROCAINE AND METRAZOL ON CEREBRAL DENDRITIC 
POTENTIAL AND ACCOMPANYING SLOW NEGATIVITY 


With the exception of metrazol, which was studied in 
the monkey (precentral agranular cortex), experiments 


FEDERATION PROCEEDINGS 


B and C 


Volume 1g 


were carried out upon the cat (anterior suprasylvian 
cortex). The animals were prepared under ether anes: 
thesia with procainization of the wound surfaces. Theres 
after Flaxedil (gallemine triethyliodide) and artificial 
respiration were substituted. Blood pressure and _ pulse 
were monitored continuously with a Sanborn electro- 
manometer. 

Calomel electrodes and a d.c. amplifier and oscillo- 
scope permitted undistorted recording of the slower 
potentials, and also enabled us to observe SP (steady 
potential) changes. Monopolar (critical electrode upon 
cortex and reference one upon frontal periosteum) and 
transcortical recording was employed. The stimulating 
electrode was tripolar. It was composed of three insulated 
100 uw wires arranged to form an equidistant triangle 
with tip separation of 75 u. These were led to the stimu- 
lator through a circuit (8) used to control shock artifact. 
A Grass stimulator was employed, the stimulus strength 
being set at 3-15 volts at 0.01 msec duration. 

1) Nembutal (4). The initial effect of injecting Nembutal 
was to change the SP across the cortex positively with 
respect to its state preceding the injection. With an addi- 
tional injection of Nembutal the SP continued to shift 
slowly positively. The slow potential changes resulting 
from stimulation were superimposed upon this shifting 
potential of the resting cortex. 

Nembutal was injected slowly over a I- to 3-minute 
period in equal 30 mg doses. A transient blood pressure 
drop might succeed each such injection, but after several 
minutes the pressure returned to normotensive levels. It 
was then that the records were taken. However, after 
injections amounting to 150-300 mg the pressure might 
remain low, hovering between 60-80/40-60. 

After injection of 30-60 mg the slow negativity of a 
singly evoked response was observed to replace the usual 
positive aftereffect. (Compare responses I, 2, 3 upper 
trace fig. 4A! with corresponding responses of fig. 4B.) 
The irregularity in appearance of the slow negativities 
in figure 4B is due to spontaneous accentuation of the 
ECG rhythms by Nembutal. With serial stimulation at 
20-30/sec. the successively evoked dendritic spikes pro- 
gressively decreased in amplitude, the slow potential 
change, resulting from summation of slow negativities, 
increasing significantly (cf. lower trace fig. 44 with that 
of fig. 4B). An additional 15 mg of Nembutal exagger- 
ated the change just described (fig. 4C). Indeed with 
Nembutal totaling 150-200 mg the spikes failed to be 
evoked beyond the first few of a series of stimuli, the 
response to serial stimulation thereafter consisting only 
of the initial few spikes followed by an exaggerated sum- 
mation of slow negativities lasting through the stimulus 
period and significantly into the poststimulatory trace 
(fig. 5). 

2) Procaine (4). When small amounts (15-30 mg) were 


1 In study from which figs. 4 and 6 were taken, slow negativity 
was called negative aftereffect. At that time we interpreted slow 
negativity as part of the excitation-recovery cycle of the same 
elements (apical dendrites) yielding the 15 msec negative dendritic 
potential. 








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July 1960 





FIG. 5. Effect of Nembutal (total 150 mg i.v.) on repetitively 
evoked dendritic (20/sec.) potentials and summed slow negativity. 
Cal. in lower right-hand corner: vertical line of right angle = 500 
uv; horizontal line = 20 msec. 


injected slowly, neither pulse rate nor blood pressure was 
affected. However larger amounts (60 mg) injected 
rapidly could produce cardiac arrest and death. Only 
under the latter circumstances did procaine produce a 
significant SP change. 

The effect of procaine on the stimulation record was 
the virtual opposite to that of Nembutal and this obser- 
vation is reminiscent of the known antagonism between 
the clinical effects of these drugs. At 30 mg the usual 
summation of slow negativities did not occur with 20/sec. 
serial stimulation (cf. lower trace fig. 6A! with that of 
6B and recall the exaggeration of the same summed slow 
negativities by Nembutal). This change was most marked 
immediately following an injection, partial recovery oc- 
curring later. By contrast the serially evoked dendritic 
potentials (6 and 20/sec.) were unaffected at this time 
(fig. 64 and B). However, with injection of additional 
procaine to a total of 60-100 mg the 6 and 20/sec. 
evoked potentials diminished somewhat in amplitude 
(fig. 6C). Still more procaine caused seizure activity to 
develop and reliable results could no longer be obtained. 
The blood pressure was elevated in the animal from 
which the records of figure 8 came. However, it was not 
believed that this influenced the results because corre- 
sponding findings were obtained in normotensive 
animals. 

3) Metrazol (g). One half to one minute following 
injection (50-100 mg) there was a mild increase in slow 
negativity with 6/sec. serial stimulation, virtually no 
change occurring in the dendritic potentials. With the 
onset of seizure discharge serial stimulation became 
ineffective; the dendritic potentials first diminishing in 
size and then disappearing from the tracing entirely. 
Baseline instability precluded evaluation of any changes 
in slow negativity at this time. However, in other ex- 
periments in which the paroxysmal record was char- 
acterized by alternating periods of spiking and 
quiescence, no evidence of slow negativity was observed 
with repetitive stimulation during the isoelectric part 
of the tracing. Upon cessation of paroxysmal discharge, 
the ECG was isoelectric and neither dendritic potential 
nor slow negativity could be evoked with serial stimu- 
lation. At this time the SP was usually shifted nega- 
tively 1-5 mv. With recovery of ECG and return of 
SP to the preinjection base line, serial stimulation be- 
came progressively more effective in eliciting the usual 
response. At first only a small dendritic potential followed 
each stimulus and it did not have either a positive after- 


XUM 


CORTICAL POTENTIALS 615 


Control (175/100) 


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‘ 





Procaine - 30 mgm. (160/105) 
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Procaine - total of 90 mgm. (160/105) 


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FIG. 6. Effect of procaine on serially evoked dendritic responses 
and their summed negative aftereffect. In A, B and C stimulus 
frequencies are 5 and 20 per sec. for upper and lower traces re- 
spectively. A: before the injection of procaine. B: after i.v. in- 
jection of 30 mg procaine. C- after a total of go mg. In both B and C 
procaine was given in divided doses of 15 mg each; a 5-min. interval 
separated each injection. Vertical line of calibration symbol, 500 
uv; horizontal line, 20 msec (4). 


effect or a subsequent slow negativity. As the potentials 
increased in amplitude a prominent positive aftereffect 
came to follow each, the after-positivity appearing, we 
believe, because elements yielding the slow negativity 
had not sufficiently recovered to cut into the positive 
aftereffect by algebraic summation. With further re- 
covery of SP, the dendritic potentials regained their pre- 
injection amplitude and once again were followed by 
slow negativities during serial stimulation. The failure of 
appearance of both dendritic potential and slow nega- 
tivity during paroxysm suggests active participation of 
the elements yielding these two potentials in the parox- 
ysmal discharge. 

Separate experiments were carried out to determine 
to what degree systemic factors might contribute to the 
observed potential changes. The effects of hypotension 
by exsanguination and anoxia by 100 % nitrogen inhala- 
tion were studied. Both hypotension and anoxia caused 
a reduction in amplitude of the serially evoked dendritic 
potentials before producing any change in the summed 
slow negativity. However, in neither case were changes 
observed which were comparable to the ones effected by 
agents capable of causing a transitory hypotension. In 
these instances the summed slow negativity could in- 
crease in amount while the primary potential disap- 
peared (Nembutal, fig. 5) or the summed slow negativity 








616 





Modification of the dendritic response in rabbit by 
0.1% GAB. A: series of dendritic responses evoked by repetitive 
(6/sec.) stimulation prior to GAB application. B and C are tracings 
obtained 20 secs. following application of GAB to the cortical 
surface. B: response to a single stimulus. C: series of responses to 
repetitive stimuli (6/sec.). Vertical line of right angle = 500 pv; 
horizontal line = 20 msec. (From 5). 


FIG. 7. 


could disappear while the primary potential remained 
unchanged (procaine, fig. 6). 

The series of experiments which we have just described 
indicate a different pharmacological and metabolic 
sensitivity for dendritic potential and slow negativity. 
Nembutal (representative of barbiturates) depresses the 
primary potential, but augments slow negativity (figs. 4 
and 5). Procaine, typifying local anesthetics, has little 
effect on the dendritic potential but depresses the ensuing 
slow negativity (fig. 6). The seizure discharge produced 
by metrazol also disassociates the two potentials, pri- 
mary potential recovering before slow negativity in the 
postparoxysmal period. The slow negativity is more 
resistant to anoxia and hypotension. 


EFFECTS OF SURFACE APPLICATION OF GAB ON DENDRITIC 
(CEREBRAL AND CEREBELLAR) AND RECRUITING POTEN- 
TIALS AND THEIR ACCOMPANYING SLOW NEGATIVITIES 


Following surface application of 1% GAB (rabbit 
cerebral cortex), singly- and serially-evoked negative 
dendritic spikes become reversed in sign and appear as 
positive potentials. Concurrently, the slow negativity 
shows a striking augmentation, increasing in amplitude 
four- to fivefold (fig. 7) (5). In the cerebellum (cat) GAB 
(0.25~1 %) may also reverse the directly activated spikes 
to positive polarity, but it does not produce the marked 
increase in slow negativity seen in cerebral cortex 
(fig. 8) (12). Indeed the cerebellar slow negativity may 
remain the same size or increase in amplitude only 
slightly (cf. fig. 8A with B). In addition, the effective 
concentration for reversal varies in different animals, a 
given concentration producing spike reversal in one 
animal but diminishing or abolishing the spike in 
another. 


FEDERATION PROCEEDINGS 


Before GAB 





Fic. 8. Effect of GAB on repetitively evoked (6/sec. surface 
stimulation) cerebellar dendritic potentials and accompanying 
slow negativities. B: one min. after surface application of GAB. 
C: 12 min. after cortex was irrigated. Cal. in lower right-hand 
corner: vertical line of right angle = 500 wv; horizontal line = 36 
msec (12). 


In both cerebrum and cerebellum GAB may reduce, 
reverse or abolish the dendritic spike. The critical factors 
in determining the outcome appear to be GAB concen- 
tration and stimulus strength. Note that GAB is applied 
to the cortical surface. It follows, then, that through dif- 
fusion the agent will establish a concentration gradient. 
Thus it is our opinion that a strong solution can affect 
both superficial and deep elements together, markedly 
depressing or abolishing the response, whereas a weaker 
solution, blocking activity principally in the superficial 
layers and leaving the deeper elements unaffected, will 
give reversal. Jasper, Gonzalez and Elliot (7) also con- 
clude that reversal of dendritic potential by GAB results 
from a block in the superficial cortical layers and re- 
tained activity deeper in the cortex. However, they sug- 
gest that the superficial layers actually may ke more 
sensitive than the deeper ones to GAB and imply that 
this would be the case with a uniform concentration of 
GAB throughout the entire cortical thickness. Such an 
explanation is entirely possible since other evidence indi- 
cates a high degree of susceptibility of superficial apical 
dendrites to convulsants and anesthetics. As to impor- 
tance of stimulus strength, if the stimulus is weak its effect 
will reach from the surface only into the superficial cor- 
tex, and in that circumstance the application of either a 
weak or a strong solution could depress or abolish the 
potential without a reversal of potential sign resulting. 

The relationship of potential configuration to stimulus 
strength and the dependency of spike reversal on the 
retention of deep cortical activity during simultaneous 
depression of superficial layers is illustrated in the follow- 
ing experiment (carried out with G. H. Bishop). Three 
recording electrodes were arranged by placing one on 
the surface of the cerebral cortex, one 0.2 mm subsurface, 
and the third in the immediately underlylng white 
matter (diagram, bottom of fig. g). Thus, we obtained 
records from the upper fraction (1-2), lower fraction 
(2-3), and entire cortical thickness (1-3). Stimulus 


Volume 19 





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July 1960 CORTICAI 
strengths of 5, 15 and 25 v. at 0.04 msec were employed 
to obtain responses from these three pairs of leads. 
Figure g is a comparison of the transcortical (1-3) 
record with that derived from the surface-subsurface 
(1-2) lead; figure 9B compares the transcortical trace 
with the one obtained from the subsurface to white (2-3) 
combination. Observe, that at all stimulus strengths the 
transcortical response is the sum of the potentials re- 
corded by the upper (1-2) and lower (2-3) fractions and 
that at the lowest stimulus strength (5 v.), where the 
effective stimulus is more superficial, the upper fraction 
(1-2) contributes the major portion of the transcortical 
potential (cf. fig. gA (1-2), 5 v., with fig. gB (2-3) 5 v.). 
Following surface application of 0.1 % GAB, the trans- 
cortical (1-3) potential evoked by the 5 v. stimulus is 
markedly reduced in amplitude. However, the ones 
elicited by 15 and 25 v. stimuli are reversed, the 25 v. 
response showing the largest positive potential (fig. gC). 
The amplitude reduction at 5 v. is readily explained by 
depression of activity in the upper fraction which makes 
the major contribution to the transcortical potential 
(cf. fig. gD, 5 v. with fig. gA, (1-2) 5 v.). At the same 
voltage, the deep fraction shows very little response be- 
cause the weak surface stimulus fails to activate the 
deeper cortical elements (fig. 9B (2-3), 5 v. and fig. 9£, 
5 v.). Not until the stimulus is made sufficiently strong 
to excite the deeper fraction (fig. gF, 15 v. and 25 v.) 
is reversal of the transcortical response to a positive 
potential observed (fig. gC, 15 v. and 25 v.). Of course, 
at this time the upper fraction (fig. gD, 15 v. and 25 v.) 
records a positive potential since the subsurface lead is 
nearer the major sink of current flow. 

GAB has a similar effect on recruiting responses 
studied in the rabbit, reversing the transient and aug- 
menting slow negativity (5). Stimulating electrodes were 
placed in the nucleus paracentralis and evoked serial 
(6/sec.) responses from the frontal agranular cortex. The 
recording electrodes were placed so that records from 
transcortical, upper and lower cortical fractions could be 
obtained. Direct current recording was employed. Con- 
trol experiments in which procaine, veratrine and boiling 
water were applied to the cortical surface gave results 
similar to those produced by GAB. In all instances re- 
versal of the recruiting transient to positive sign was 
associated with deep cortical negativity recorded by the 
intra-subcortical lead combinations. 

Purpura, Girado and Grundfest (g) earlier observed 
reversal of the cerebral dendritic potential with GAB. 
However, they reported failure to obtain such reversal 
of the cerebellar dendritic potential, application of GAB 
to the cerebellar cortical surface causing disappearance 
of the response. They interpreted these findings with 
respect to their concept relating polarity of evoked 
dendritic response to type of synaptic activation, specu- 
lating that the negative dendritic response indicates 
depolarization and is the sign of excitatory postsynaptic 
activation, while a potential of positive sign is due to 
hyperpolarization of the dendritic membrane and signals 
the activation of inhibitory postsynaptic elements. They 


xXUM 


. POTENTIALS 


617 


SV. .04 MSEC. 15 V. 


5 





FIG. g. Effect of GAB on cerebral dendritic potential recorded 
with fractional leads. 1, 2 and 3 are recording electrodes. S—corti- 
cal surface. W—white matter. 5 v., 15 v. and 25 v. at top of each 
vertical column indicates the stimulating voltages used to elicit the 
responses in each of those columns. The stimulus duration was 0.04 
msec. for all voltages. Vertical cal. = 500 uv; time signal = 5 msec. 
For complete details see text. 


feel that reversal of sign of the cerebral dendritic poten- 
tial to positive (GAB) results from uncovering the ac- 
tivity of inhibitory synapses by blockade of excitatory 
ones. In their view the disappearance of cerebellar re- 
sponse without reversal of sign indicates paucity of 
inhibitory postsynaptic potentials in cerebellar cortex. 
Our evidence that GAB also reverses the sign of the 
cerebellar dendritic potential (fig. 8), that surface posi- 
tivity is related to deep negativity in cerebral cortex 
(fig. 9), and that the sign of recruiting responses is also 
reversed by the application of such nonspecific depres- 
sants as boiling water, novocain or veratrine is not com- 
patible with such an interpretation. Instead, we suggest 
that the potential changes produced by GAB in both 
cerebral and cerebellar cortex find a more ready explana- 
tion in the familiar variables which influence volume con- 
ductor recording: stimulus strength, relationships of re- 
cording electrodes to sources and sinks of current flow, 
and concentration of the test agent employed. Similar 
conclusions were drawn by Jasper, Gonzalez and Elliot 
(7) from their study of GAB effect on direct and indirect 
cortical responses. 








618 


An explanation of why GAB augments slow nega- 
tivity in cerebrum (fig. 7), but leaves it relatively un- 
changed in cerebellum (fig. 8B) is found in recent studies 
of the cerebellar dendritic potential and its accompany- 
ing slow negativity (Rhoton, Goldring and O'Leary, 
unpublished data). Upon increasing stimulus strength 
from threshold to maximum, a positive aftereffect like 
that observed to follow the cerebral dendritic spike 
(fig. 24), is not recorded under the experimental condi- 
tions described in this paper. Instead, slow negativity 
appears when the stimulus strength is raised just above 
threshold for the spike, and this grows in amplitude 
along with the primary potential as stimulus strength is 
increased. Thus, in the cerebellum the slow negativity is 
never concealed by an opposing after-positivity, and for 
any given stimulus its true amplitude is evident. By con- 
trast in cerebral cortex the positive aftereffect sums 
algebraically with the slow negativity and at least par- 
tially submerges it at lower stimulus strengths. For exam- 
ple, in figure 2A a truer approximation of slow nega- 
tivity amplitude for 15 v. would be obtained by adding 
to it (arithmatically) the voltage of the after-positivity 
derived at 10 v. The GAB augmentation of slow nega- 
tivity in cerebrum, therefore, may be more apparent 
than real, for in cerebral cortex the after-positivity is 
intimately related to the dendritic potential and has the 
same superficial origin in the cortex. Therefore, following 
surface application of GAB it will be affected there to a 
far greater extent than the more deeply originating slow 
negativity; and the latter, now relatively unopposed, will 
show a significant amplitude increase. By contrast, in the 
cerebellum where a positive aftereffect does not follow 
the dendritic potential, little change in slow negativity 
is seen with surface application of GAB. 

The changes produced by GAB cannot be interpreted 


FEDERATION PROCEEDINGS 


Volume tg 


as indicating differential sensitivity of primary potential 
and slow negativity to this agent; rather it provides 
additional evidence for a different origin of these two 
potentials in the cortical depth. GAB was applied to the 
cortical surface and therefore one would expect the 
dendritic potential to show the greatest change, it having 
a more superficial origin than the slow negativity 


(fig. 2B). 


SUMMARY 


The study provides examples of how pharmacological 
agents applied to the cortical surface of cerebrum and 
cerebellum as in the case of GAB, or given intravenously 
as with Nembutal, procaine and metrazol, can be used 
to analyze the components of evoked cortical responses, 
Dendritic (cerebral and cerebellar) and_ recruiting 
(cerebral) responses and their accompanying slow nega- 
tivities were studied. The intravenously injected agents 
illustrate differences in pharmacological sensitivity of 
primary potential and slow negativity. Surface applica- 
tion of GAB, and similar application of procaine, vera- 
trine and boiling water as controls, suggest a different 
origin in the cortical thickness for the primary potential 
(dendritic or recruiting) and the slow negativity. Thus, 
although for different reasons, both sets of data give 
additional evidence for considering primary potential 
and the accompanying slow negativity as separate 
cortical processes. 

The study also emphasizes how results, which are 
derived by introducing pharmacological variables into 
electrophysiological studies, are determined by method 
of drug administration (systemic or surface cortical ap- 
plication), the concentration of the test agent, the stimu- 
lus strength and the conventions of volume conductor 
recording. 


REFERENCES 


1. Bishop, G. H. ano M. H. Crare. J. Neurophysiol. 16: 19, 1953. 

2. Bremer, F. Physiol. Rev. 38: 1006, 1958. 

3. Crare, M. H. ann G. H. Bisuop. Electroencephalog. & Clin. 
Neurophysiol. 8: 582, 1956. 

4. Gotprine, S., J. S. Metcatr, S. H. Huanc, J. Sutevps AND 
J. L. O'Leary. J. Nerv. & Ment. Dis. 128: 1, 1959. 

5. Gotprinc, S., J. L. O'Leary anv S. H. Huanc. Electroen- 
cephalog. & Clin. Neurophysiol. 10: 663, 1958. 

6. Gotprinc, S., J. L. O'Leary, D. L. Winter anp A. L. 
PEARLMAN. Proc. Soc. Exper. Biol. & Med. 100: 429, 1959. 

7. Jasper, H., S. Gonzatez anp K. A. C. Exuior. Fed. Proc. 
17: 79, 1958. 


8. Lanpau, W. 


445, 1956. 
g. O'Leary, J. 


M. Electroencephalog. & Clin. Neurophysiol. 8: 


L., S. Go_princ anp W. S. Coxe. Epilepsia In 
press. 

10. PEARLMAN, A. L., S. Go_princ AnD J. L. O'Leary. Pror. 
Soc. Exper. Biol. & Med. 103: 600, 1960. 

11. Purpura, D. P. M., M. Girrapo anv H. Grunprest. Science 
125: 1200, 1957. 

12. Ruoton, A., S. Gotprinc anv J. L. O’LEary. Am. J. Physial. 


In press. 








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RY. Prot. 
T. Science 


I. Physiol. 





Physiology and drug action: an electro- 


encephalographic analysis: 


REGINALD G. BICKFORD 


Mayo Clinic and Mayo Foundation, Rochester, Minnesota? 


Bx SHIFTING from what might be called the ‘micro’ level 
of analysis of drug-neuron interactions, presented by 
Dr. Goldring, to the more macroscopic level of analysis 
demanded by electroencephalography, it is well to admit 
from the outset that certain additional difficulties be- 
come apparent. In the first place, the subject is vast; 
almost any drug that has any demonstrable action on the 
central nervous system also has some effect on the electro- 
encephalogram, and many such actions have been in- 
vestigated. Furthermore, the literature is dispersed over 
the neurophysiologic, pharmacologic, neurologic, psy- 
chologic and psychiatric fields. Second, owing to a basic 
lack of understanding of the EEG or even the neuronal 
elements from which it arises, few general principles exist 
in the field that allow investigators to summarize con- 
veniently the vast array of data. Third, the nature of the 
EEG itself, with its sensitivity to many factors extraneous 
to the experimental situation, renders the entire field 
prone to methodologic pitfalls. Thus, it appears desirable 
to emphasize the methodologic aspects of the interac- 
tions of drugs and the EEG. 

The association of the action of drugs in the form of 
anesthetic agents with changes in electric events in the 
brain dates from the very discovery of brain waves by 
Caton, in 1875. However, in the period that followed, 
the complicating effects of anesthetic agents that were 
necessarily present in animal investigation greatly im- 
peded progress in the field. Only during the last decade 
have methods of recording been developed that allow 
one to study the action of drugs on the EEG without 
interference from these agents. 


PROCEDURAL CONSIDERATIONS 


Some of the problems that face, and often defeat, the 
naive investigator in this field are illustrated in figure 1. 
In the first place, it is a gross misapprehension to refer 
to one electroencephalographic finding associated with a 
particular pharmacologic action. The electric activity of 
the brain consists of multiple-potential fields that are 
three-dimensional and that differ considerably in ana- 
tomically separate regions and at various depths. These 
factors are summarized on the left side of figure 1, which 


! This investigation was supported in part by grants B1591 and 
Bigi7 from the U.S. Public Health Service. 

? The Mayo Foundation is a part of the Graduate School of the 
University of Minnesota. 


WIM 


619 


also draws attention to the fact that the EEG is a poten- 
tial derived from the activity of individuals of large 
neuron populations. Therefore, the form of the electric 
waves recorded depends on factors such as separation of 
electrodes and their orientation in relationship to specific 
structures. Differences also occur in spontaneous and 
evoked potentials, the latter being responses to a specified 
input to the brain. Some methods of quantitation are 
indicated on the left. 

The right side of figure 1 is concerned with problems 
related to the action of the drug itself. Some factors relate 
to the mode of application of the drug, whereas others 
concern the crossing of the blood-brain barrier and the 
possible peripheral effects of drugs in addition to those 
on the brain itself. The extremely important question of 
the dose of drugs must be considered. It is now common 
practice to use preparations that simplify the action of a 
drug by confining its effects to some relatively isolated 
part of the central nervous system. Many such studies on 
animals are misleading when they are applied to man. 
The difficulties of such inferential experiments are well 
illustrated by a large number of observations made on 
the tranquilizing agents. From a study of such observa- 
tions, it is clear that in many instances doses of drugs are 
being employed that are close to the toxic level when con- 
sidered in terms of dosage in the whole organism; there- 
fore, conclusions based on such studies may have little 
relevance to the action of tranquilizers as seen in the 
human (13). The common method of administering a 
drug is to give it according to some prepared schedule. 
However, later discussion will concern the process of 
administering a drug depending on its effect on the brain 
as measured by the EEG, this being a servosystem of 
administration. Recent interest has been shown in 
methods of self-administration of drugs, ranging from 
the operant-conditioning technics to the more familiar 
situation of drug addiction. 

Finally, the middle section of figure 1 illustrates some 
experimental or naturally occurring differences that may 
be present in the brain itself influencing the interaction 
of drugs and the EEG; such differences, in fact, often are 
specially designed to simplify the experimental pro- 
cedure. These range from the use of electric stimulation 
to activate various cortical or subcortical regions of the 
brain to studies on the isolated brain lobe (20) and the 
experimental (12) or clinical epileptic lesion. 











620 
ELECTRICAL DRUG 
ACCESS APPLICATION 
Location ~ee Indirect CNS 
t CNS 
Aggregation | peor, Direct C 
> e 
TYPE ADMINISTRATION 
Spontaneous To Schedule 
Evoked By Effect (Servo) 
Operant 
om ‘Addiction 


titot 
Quantitation DOSAGE LEVEL 


Visual 
Analyzer BRAIN Related To <— 
Time Delay STIMULATION 

ISOLATION PATHOLOGY 


FIG. 1. Procedural considerations in analysis of action of drugs 
on the EEG. 


EFFECT OF ENTIRE ORGANISM ON EEG 

Another important factor that must be kept con- 
stantly in mind in research procedures involving the 
EEG is that the latter in many ways reflects the inte- 
grated activities of the entire organism, ranging from 
chemical and physical factors to the psychologic state. 
These multiple variables are diagrammed in figure 2, 
which lists some of the more important factors that must 
be taken into account and controlled in any observations 
involving the EEG. Much of the published work con- 
cerning the EEG and the action of drugs is unreliable 
because some of these factors have been overlooked. 
They will be summarized briefly. 

The psychologic setting has a profound influence on 
the EEG; it always must be designated or an attempt 
must be made to maintain it at some required level. The 
great importance of this factor is illustrated by a recent 
personal experience. Hauser and associates (9), in our 
laboratory, noted that the alpha rhythm in 80% of 
normal persons who smoked a cigarette increased in fre- 
quency by 1-2 cycles per second (cps), as illustrated in 
figure 3, which shows an increase of 1 cps in subject A 
and 2 cps in subject B. Since this increase occurred at the 
time of cardiac acceleration, it was first attributed to 
the effect of inhaled substances, including nicotine. 
However, studies in which a glass tube containing unig- 
nited cotton was substituted for the cigarette produced 
a similar shift in alpha frequency in a number of persons, 
and our observations to the present indicate that the 
shift in alpha rhythm is related to some concomitant 
action of smoking, perhaps a shift in attention, and not 
to the effect of any inhaled agents from the cigarette 
itself. 

A serious difficulty encountered in many pharmaco- 
logical experiments is the tendency for an undisturbed 
person with his eyes closed to become drowsy, a situa- 
tion that may alter the EEG profoundly. In some recent 
observations that my associates and I made on the inha- 
lation of nitrous oxide, this factor was of such importance 
that we had to design a special performance test that was 
sufficiently difficult to keep the subject from drowsiness 
but not difficult enough to inhibit his alpha rhythm 
completely. 


FEDERATION PROCEEDINGS 


Whole 
Species 


Volume 1 9 














ANXIOUS 
AWAKE [BRAIN ENVIRONMENT | 
DROWSY , 
— CHEMICAL 
COe2 
Oz 
BRAIN INPUT Blood Sugar 
PHYSICAL 


VISUAL INPUT 
COMMUNICATION LEVEL 


Blood Pressure 


AFFERENT + 
(Stimulotion) SPECIES ALPHA Temperature 
ee PATHOLOGY 
(Deprivation) 


FIG. 2. General factors to be considered in the relationship of 
the EEG to entire organism. 


CONTROL 


Left Posterior Parietal oceipitel| 
Ae A a 














Eyes Closed 


® 2nd CIGARETTE 


Left Posterior Parietal Occipital 


Very Vv 





89 10111213 





Eyes Closed 





89 101112 {3 


CONTROL 


Left Posterior Parietai Occipital NV 


Eyes Closed 


891011213 
2nd CIGARETTE 


Left Posterior Parieta! Occipital 


als mh nfl Ant wn ae 
i peed | | WV ¥ vw WV 





Eyes Closed LSEC. | 1-20 pv. 


8 9 10111213 
FIG. 3. EEG and frequency analysis spectrum of two subjects 


(A and B) before (control) and after smoking two cigarettes. Bi- 
polar derivation with eyes closed. 


Apart from modifying the cerebral input to ensure a_ 


constant state of alertness, other changes in this parame- 
ter are of importance in the study of pharmacological 
actions. The rhythmic variation of input, known in the 
visual system as ‘photic driving,’ is a useful test that will 
be referred to later. The opposite situation, that of de- 
creased input, is probably important in the large group 


be 
ra 
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ha 
ha 
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pe 
th. 





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1 the 
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roup 





July 1960 


FIG. 4. Response to hyper- 
ventilation in a normal person, 
with changes following adminis- 
tration of glucose. Note that slow 
waves from the frontal regions 
produced by 2! min. of hyper- 
ventilation (H.V.) do not ap- 
pear with the same period of hy- 
perventilation after ingestion of 
50 mg of glucose. 





oe eer 


8-l0-—~ PALL NI NDI LPAI 


of observations on drugs that have been made with the 
subject under the influence of curarizing agents. An 
interesting example is seen in the curarized rabbit. This 
agent appears to induce a condition of abnormal sleepi- 
ness that does not develop with the same ease in the 
noncurarized, freely moving rabbit. Hence, the results 
obtained on curarized rabbits should be considered 
critically before they are assumed to represent the effects 
of drugs on a normal animal. 

A number of other factors must be carefully controlled 
during experiments dealing with the action of drugs. The 
most important of these is the level of carbon dioxide in 
the blood. The human EEG is extremely sensitive to 
hyperventilation (11); thus, a drug, by direct respiratory 
stimulation or by the development of anxiety, may pro- 
duce slow waves such as those illustrated in figure 4, 
which are more prone to occur when the amount of blood 
sugar is low. Under the heading ‘brain’ in figure 2, one 
must consider some extremely important differences 
among the EEG’s of various animals and between those 
of animals and man. Early investigators, as well as some 
more recent ones, frequently have confused the 5- to 
6-cps rhythm seen in the rabbit with the alpha rhythm 
in man; however, many of the properties of these two 
rhythms are diametrically opposed, and the former is 
now recognized as a rhythm arising mainly from rhi- 
nencephalic structures (8). The cat possesses little in the 
way of alpha rhythm comparable to the human, al- 
though the spindles appearing in records from this 
animal during sleep and barbiturate anesthesia have 
been erroneously referred to at times as alpha rhythm. 
The animals which show rhythms closely resembling 
the human alpha rhythm include the dog, a convenient 
animal, since it will allow its eyes to be held closed, and 
the monkey, which is often not so cooperative. 

Finally, the question of quantitation of the EEG should 
be considered. Methods employed for this evaluation 
range from visual judgment and classification to various 
automatic methods, the best known of which is automatic 
harmonic analysis. While the subjective reading method 
has obvious pitfalls, it is less well recognized that subtle 
errors can creep into automatic analysis. This may hap- 
pen, for instance, when rhythmic ocular movements 
that often occur in an anxious person during injection of 
a drug are fed into the analyzer and appear in the fre- 


2+ MIN. HV. 
Bohm rrr) A vAVatAta /\ Uwe 
em VY 
13 ro \ pyr 
5-70 a ee ee eee 


PHYSIOLOGY AND DRUG ACTION 621 


50 GRAMS GLUCOSE GIVEN 
24 MIN. HV. 


AAA NANA AN ray ny Ande Aa AV ere 
IVAW ne 





ONLI AYE re 


LRRD Ee yy 


SEC, 120mm 


Fre NV yy 





eee 


quency spectrum as genuine, slow-wave activity. Fre- 
quency analysis, although an extremely useful method 
for studies on drugs, may be quite misleading unless it 
is carefully monitored by visual inspection. 

A general difficulty in the interpretation of the action 
of a drug that is given systematically also should be 
noted. Since any such drug may act on peripheral organs, 
a change in the EEG related to any such administration 
must be checked carefully for its relationship to such 
indirect effects as a change in the level of consciousness 
resulting from anxiety, from the painful or unpleasant 
peripheral actions of drugs, from changes in hyperventi- 
lation resulting from anxiety, or from changes in blood 
pressure. Thus, it is hardly surprising that many would- 
be investigators have been discouraged by the difficulties 
that plague this field. Most workers have been content 
to establish a correlation between some aspect of the 
EEG and the concentration of drugs. In addition, how- 
ever, there is often an attempt to tie in psychological or 
behavioral information; the desirability of providing 
such information directly rather than by inference al- 
ready has been noted. 


EFFECTS OF DRUGS ON EEG: GENERAL SURVEY 


Rather than attempt a comprehensive coverage, I 
shall highlight some findings of interest in relationship 
to the mechanisms of drugs. A good starting point is a 
neuronal system specifically designed for the detection 
of chemical substances, namely the olfactory mechanism. 
In the human, Sem-Jacobsen and associates (19), in our 
laboratory, recorded directly from the olfactory bulb 
by means of implanted depth leads. With this system, 
signalling the receipt of olfactory information involves 
the activation of a rhythmic system of 40-50 cps (fig. 5). 
Another example of a localized response to the action 
of a drug was reported by Porter (16), who illustrated 
a focal rhythmic discharge arising in the posterior hypo- 
thalamus after administration of epinephrine. It should 
be emphasized, however, that such localized electric 
concomitants of the action of drugs appear to be quite 
rare, at least at the ‘macro’ level of recording. It is more 
usual to find the electric effects of a given drug distrib- 
uted simultaneously through various regions of the 
brain, although the pattern may vary in different parts. 

Anesthetic Agents. These agents are examples of drugs 

















622 FEDERATION PROCEEDINGS Volume 1 
9 
-3yK 
1-38 ye iia a tale ny We Yate lo gy ed 
S-Sanieypt nen petiirary ie yhed Vb Yrndtinmryneninr Fae Mtl fir Ait geile w/t 
5 Teararemeote seat ieee Ahimalieadis io bin ihteomenenmamnil 
Thay tn yy tea y hana Ma NO NEP Nee Ne 
Tae 
cue wine 
TINCT. VALERIAN SMELL 
FIG. 5. Fast rhythmic discharge occurring in human olfactory bulb with olfactory stimulation produced by sniffing tincture of 
valerian. Diagram on far left shows depth-electrode contacts 5 and 7 lying alongside olfactory bulb. From Sem-Jacobsen and associ- 
ates ( 1Q). 
it might be simply quantitated by means of an integrator 
; fe system and that this could be made to feed the anesthetic 
me “omplex p 
S 200 f 1 : : 
ape Wino agent at a rate proportional to the integrated voltage 
3 §z oo". wary, MARS output of the brain. Such a system constitutes a feed- 
SOR . ° re ° 
83s back loop capable of maintaining a relatively constant 
Sfp Burst suppression(late) depth of anesthesia (1) (fig. 
yx pa Pr ‘cee . 
Ss Te, Although good correlation has been found between 
$32 Suppression changes in the EEG and the concentration of the agent 
PSN = 
SE » in the deeper levels of anesthesia, considerable mental 
; es — = = 7 impairment can be produced by minimal concentra- 





EEG levels 





FIG. 6. Gin nce lh ii in pattern in the human EEG with 
increasing depth of anesthesia. From Martin and associates (15) 


that manifest widespread action on electric rhythms in 
the brain. While their action on the EEG varies in de- 
tail from one agent to another, those potent 
enough to cover a wide span of anesthetic depth usually 
induce changes such as those diagrammed in figure 6. 
The initial effect is often, but not always, to produce an 
increase in fast activity, such as was shown originally 
for barbiturates by Brazier and Finesinger (4). This 
phase usually is followed by a slowing in frequency, with 
a great over-all increase in amplitude. This change 
reaches a peak in light anesthesia; thereafter, a suppress- 
ing effect of the anesthetic agent is manifest in the form 
what has been called the 
A notable point of these ef- 
manifested by the 


drugs 


of an increasing degree of 
‘burst suppression’ pattern. 
fects is that the extreme variability 
normal EEG among different people 
out under the influence of anesthesia, 


becomes ironed 
and relatively 


little variability of pattern is seen in moderate and deep 
response of the 
and 


EEG to 


me that 


anesthesia. The form of the 


anesthesia suggested to my co-workers 


Anesthetic 






EEG. Brain Waves 
~~ Deep 


Moderate 


Gain Controi 
(Depth of anesthesia) 


Light 


Resting 





Tissue removal 
of anesthetic 





tions of anesthetic agents without appreciable change in 
the EEG, even though the latter is quantitated by means 
of the frequency analyzer (fig. 8). 

Arousal System. Other studies are beginning to shed 
some light on the neurophysiological basis of changes in 
consciousness produced by drugs. A great stimulus to 


this type of investigation has followed the delineation of 


the arousal system by Magoun and co-workers, and this 
influence has made itself felt in the field of pharmacology. 
French and associates (6) have shown that the multi- 
synaptic reticular system is extremely sensitive to drugs 
in contrast to the lateral sensory systems, and they re- 
gard this as an important mechanism underlying the 
behavioral manifestations of the anesthetic state. Much 
important work also has been done to measure the action 
of many types of drugs on the arousal system by Rinaldi 
and Himwich (17). instance, they showed that 
chlorpromazine has the property of blocking the arousal 
response. Elkes and co-workers (5) reported similar re- 
sults. However, the absence of hypnotic effects of this 
drug in the human and its minimal effects on the EEG 
in many patients who show excellent tranquilization 
leave considerable doubt as to whether the physiological 


For 


mechanism underlying its action has yet been dis- 
=> — “~ 
Integrator Pulses | 
—— 
oe n | FIG. 7. Diagram of system to control 
the dose of anesthetic agents in servo- 
anesthesia. Tank on left represents the 
HHH concentration of anesthetic drug in the 
brain, and alongside are diagrammatic 
bd samples of the corresponding patterns In 
the EEG. From Bickford (1). 
+ 





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July 1960 





PHYSIOLOGY AND DRUG 


ACTION 


csec., T= 50 uv. 


30% NITROUS OXIDE 














Blurred Vision, Perseveration, 


Recall Impaired 65% 

















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Fic. 8. Absence of change in EEG in patient breathing 30% nitrous oxide in whom psychological tests showed a 65°% menta 


impairment. 

laboratory by Dr. Henrie (10). 

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FIG. g. Anticonvulsant screening test in a light-sensitive epileptic patient. 


TPA "sec ree 


Photoconvulsive response to stimulation with light 


(photocell in 8th channel) is entirely suppressed after the intravenous injection of 1 gm of trimethadione (Tridione). 


covered. The importance of observing a behavioral cor- 
relate in experiments involving the activation system is 
illustrated by the work of Elkes and associates, who have 
shown that, under the influence of atropine, electro- 
graphic desynchronization changes may be blocked and 
yet the behavioral response of alerting is not affected. 
This dissociation between behavioral and electrographic 
data should be taken as a warning that an implied cor- 
relation between these two aspects of cerebral function 
cannot be taken on trust but requires experimental veri- 
fication. 

Convulsants and Anticonvulsants. Space does not permit a 
detailed review of the clinically important field of the 
actions of convulsant and anticonvulsant drugs in rela- 
tionship to the EEG. Some of the mechanisms operative 
in the latter field have been discussed by Toman and 
Taylor (21). The seizure process can be looked on 
as occupying local pathways, with the possibility of more 
generalized spread by what may be called ‘transmission’ 
pathways. Most convulsants, such as pentylenetetrazole 
(metrazol) appear to have an action on both types of 
pathway, the type that predominates being dependent 
on the mode of application of the drug to the nervous 


system. Generalization effects are well illustrated by 


Gastaut and Hunter’s (7) experiments, in which a re- 
sponse evoked by stimulation with light and at first 
confined to the visual system spreads widely under the 
influence of increasing doses of metrazol, with the even- 
tual appearance of a jerk associated with each flash. 
Anticonvulsant action also can be regarded as taking 
place at various levels in the nervous system. Thus, 
successful anticonvulsant medication may fail to change 
the localized discharging process and yet apparently 
may limit its transmission, so that clinical manifestations 
of the seizure are no longer in evidence. 

Attempts have been made in our laboratory to test 
the effectiveness of an anticonvulsant by its ability to 
suppress the photoconvulsive responses to light in light- 
sensitive epileptic patients (22) (fig. 9). By the success 
or failure of such screening tests, we have attempted to 
predict the long-term anticonvulsive properties of the 
particular drug in the patient. Such predictions are not 
always confirmed in practice, presumably because the 
factors in long-term anticonvulsive actions are somewhat 
different from those involved in immediate action of the 
drug. 

Pyridoxine Deficiency. Considerable interest has been 
evidenced recently in the study of convulsive states asso- 





FEDERATION PROCEEDINGS Volume 19 


Generalized Convulsions 2 weeks — On SMA Formula From Birth 


1-3" ee Pew) e/a nna, 
3-5 a [VY eV (Vn ly 
5-74 Maen WA ARM ae re pte ayn hes 


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2-4". Va PPADS A Wily on ren a pial red Ne fa val 
4-6 Iyer en yin Var Wn 
6-8 CAV Mile Ano necahead PAA NA WAI peared win 
8-10 + Y nya ref roy POP MIA EAT a 


I * 50 pv. 
2 weeks Later — On Evaporated Milk — No Further Convulsions 


FIG. 10. Change in EEG of a g-week- 
old baby taking a formula that was de- 
ficient in pyridoxine. Note remarkable 
normalization of the record when the 
child was given evaporated milk. Anti- 
convulsants were not given. 














MeN RNR RMR ae eee eee eee 


Control Img. Adrenochrome 

L.E -L.T. Pra nnn pref NO epee ee ots | Nv aver iV OE eee “ Nn AA me he eee 
Cat standing—eyes open- pow extended 

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SEC.) I* 100 wv. 

eee re ae eA vy WW ero \ A avaated|| A cat Aiea 
a ; a 

R.E-R.0. ~“W \M\ )\V"\eeerw 


FIG. 11. Effects of adrenochrome on the EEG of the cat. Tracing on the right was taken minutes after the intraventricular injection of 


1 mg of adrenochrome. From Schwarz and associates (48). 


DENDRITES | 


“WWM I 


Eyes Open 


NEURO — 
REGULATORY 


Rhythms 


05 sec. 





CELL BODY 


ig, 


NEURO — 
TRANSACTIONAL 


QU 





2000 py 
On Light Off 


O5 sec 
me: Ee 
Pulses 








FIG. 12. Diagram contrasting supposed functional properties in 
cell body and dendrites of the neuron. From Bickford (2). 


vsec.) r= 50 pv. 


ciated with a deficiency of pyridoxine. We have observed 
seizures resulting from use of a baby-food formula that 
was deficient in pyridoxine (fig. 10); a remarkable 
change occurred in the EEG when a nondeficient diet 
was substituted. Seizures associated with the use of 
isoniazid and the hydrazines also have been related to 
the pyridoxine mechanism; more recently, some indica- 
tions exist of a relationship with the amount of gamma 
aminobutyric acid in the nervous system (14). 

Little needs to be said about the EEG correlates of 
the ‘psychotomimetic’ drugs and their opposites, the 
tranquilizers, since the changes at relevant doses in the 
human are nearly always minimal and apparently of 
little significance in regard to specific action. By using 
special modes of administration, Schwarz and _asso- 
ciates (18) demonstrated changes such as those shown in 





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as de- 
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July 1960 


figure 11 following the intravenous injection of adreno- 
chrome in the cat. 

Role of Drugs in Study of Cerebral Function. Perhaps no 
account of the action of drugs should be closed without 
reference to some of the numerous applications of drugs 
as tools in the investigation of cerebral function. The 
complications resulting from the use of anesthetic agents 
in the early days of neurophysiology already have been 
mentioned. However, some dividends have accrued 
following the discovery that deep anesthesia produced 
by barbiturates would suppress spontaneous potentials 
and thus leave a clean field for mapping the sensory 
areas of the brain by evoked potentials carried by path- 
ways relatively unaffected by anesthesia. In the field of 
clinical electroencephalography, convulsant drugs such 
as metrazole have proved to be important agents in 
diagnosis through their effect in bringing latent spike 
foci into evidence. Likewise, study of locally applied 
convulsants such as strychnine has added greatly to the 
knowledge of epileptic lesions in addition to providing 
evidence of transmission pathways in the brain, a technic 
usually known as ‘neuronography.’ 


COMMENT AND CONCLUSIONS 


The question can be raised as to what extent the EEG 
has been successful in elucidating the neurophysiological 
or behavioral changes that accompany the action of 
drugs on the nervous system. Even when allowance is 
made for the inadequacies of access to functional regions 
of the brain, particularly in humans, the results leave 
much to be desired. Considerable success has accom- 
panied efforts to find electroencephalographic corre- 
lates of convulsant action, and much further exploration 
remains to be done, which eventually should throw 
considerable light on the neurophysiological problems 
related to epilepsy. While the grosser changes in anes- 
thesia have a reliable correlation with the EEG, a con- 
siderable degree of mental impairment above the anes- 
thetic level may be associated with little definite change 
in the EEG. However, the situation is more unsatis- 


PHYSIOLOGY AND DRUG ACTION 625 


factory when the action of psychotomimetic and tran- 
quilizing drugs is considered. In this situation, it is 
natural to consider whether a) the methods of measure- 
ment are at fault, 6) the nervous system is being asked 
the right kind of question, c) the right part of the nervous 
system is being asked the right question and d) an elec- 
tric correlation with behavior is necessarily to be ex- 
pected. Under these circumstances, it is useful to see 
whether the shortcomings of the electroencephalographic 
findings in relation to the action of drugs are paralleled 
by similar findings in other aspects of electroencephalog- 
raphy. In fact, such parallels apparently are found in 
the lack of specific changes in the EEG associated with 
such major functions of the brain as intellectual activity, 
voluntary movement and emotional change. The in- 
hibitory effects sometimes demonstrable on various 
spontaneous rhythms hardly appear to provide an ade- 
quate basis for understanding the mechanism of these 
complex mental functions. 

These thoughts suggest that the EEG perhaps is re- 
lated more to regulatory processes in the brain than to 
those processes concerned with the direct transmission 
of information. These ideas are represented in figure 12, 
which suggests that the processes dealing with transac- 
tion of information are carried out at a unit level, 
whereas neuroregulatory processes indirectly related to 
the former, and represented by the EEG, are phenomena 
located in the dendritic system of neurons. This idea is 
not altogether new, and some evidence in support of it 
can be found in the work of Bishop (3) and others. The 
time is now at hand when direct evidence obtained by 
means of a single-unit recording in the human may pro- 
vide information that can support or refute the hypothe- 
sis. 

Most of the experimental investigations of the action 
of drugs and their effect on the EEG will have to be re- 
peated at a more ‘micro’ and perhaps a more transac- 
tional level of neuronal organization before the degree 
of psychoelectric correlation that many observers believe 
exists at some level in the nervous system can be demon- 
strated objectively. 


REFERENCES 


. Bickrorp, R. G. Elec. Eng. 70: 852, 1951. 

. Bickrorp, R. G. TRE, ME-6:164, 1959. 

. Bishop, G. H. Physiol. Rev. 36: 376, 1956. 

. Brazier, M. A. B. AND J. E. FinesinGer. A.M.A. Arch. Neurol. & 
Psychiat. 53: 51, 1945. 

. Exes, J., C. ELKEs AND P. B. BRADLEY. J. Mental Sci. 100: 125, 
1954. 

6. Frencu, J. D., M. VERZEANO AND H. W. Macowun. A.M.A. 
Arch. Neurol. & Psychiat. 69: 519, 1953- 

. Gastaut, H. Ano J. Hunter. Electroencephalog. & Clin. Neuro- 
physiol. 2: 263, 1950. 

8. Green, J. D. ano A. A. Arpuini. J.] Neurophysiol. 17: 533, 
1954- 

. Hauser, H., B. E. Scowarz, G. Roto anp R. G. BickFrorpb. 
Electroencephalog. & Clin. Neurophysiol. 10: 576, 1958. 

10. Henrig, J. (Thesis). Minneapolis, Minn.: Univ. Minnesota. 

11. HEPPENSTALL, M. E. anp G. D. GreviL_e. In: Electroen- 

cephalography: A Symposium on Its Various Aspects, edited by D. 

Hill and G. Parr. London: Macdonald, 1950, pp. 127-165. 


ON = 


~ ow 


o 


12. Kerrn, H. M. anv R. G. Bickrorp. Am. J. Physiol. 179: 650, 
1954- 

13. Kitiam, K. F. In: Brain Mechanisms and Drug Action: A Sym- 
positum, edited by W. S. Fields. Springfield, Ill.: Thomas, 
1957- 

14. Kitiam, K. F. ann J. A. Bain. J. Pharmacol. & Exper. Therap. 
119: 255, 1957- 

15. Martin, J. T., A. Fautconer, Jr. AnD R. G. Bickrorp. 
Anesthesiology 20: 359, 1959. 

16. Porter, R. W. Am. J. Physiol. 169: 629, 1952. 

17. Rinatpr, F. anp H. E. Himwicu. A.M.A. Arch. Neurol. & 
Psychiat. 73: 387, 1955. 

18. ScHwarz, B. E., K. G. Waxim, R. G. Bickrorp AND F. R. 
LicHTENHELD. A.M.A. Arch. Neurol. @ Psychiat. 75: 83, 1956. 

19. SEN-JACOBSEN, C. W., R. G. Bickrorp, H. W. DopcE, JR. AND 
M. C. Petersen. Proc. Staff. Meet., Mayo Clin: 28: 166, 1953. 

20. Swank, R. L. J. Neurophysiol. 12: 161, 1949. 

21. Toman, J. E. P. ano J. D. Taytor. Epilepsia 1: 31, 1952. 

22. Wuite, P. T., R. G. Bickrorp anp A. A. Bamey. Electro- 
encephalog. & Clin. Neurophysiol. 6: 145, 1954. 








Some actions of anesthetics on the nervous system 


MARY A. B. BRAZIER 


Neurophysiological Laboratory’, 


of Electronics’, Massachusetts Institute of Technology, 


‘i FIRST POINT to which I would like to draw atten- 
tion is the effect barbiturate anesthesia has on the flexi- 
bility or pliability of the response systems of the central 
nervous system. Under this anesthetic certain pathways 
are blocked off, others are released from inhibition, re- 
sponses in the specific pathways become more rigidly 
synchronized, and the general balance of activity in the 
brain is changed. It is recognition of these effects that 
has made physiologists so wary in recent years of apply- 
ing localizing findings made under anesthesia to their 
conception of normal brain function. 

For example, the finding of French, Verzeano and 
Magoun (6) that the specific primary response to a 
sensory stimulus is followed within 8 to 10 msec by 
another response that has traveled via the reticular 
formation and nonspecific thalamic system, can only be 
adequately demonstrated in the unanesthetized animal. 
We have examined this point for responses to flash. 

Figure 1 illustrates the response to which I refer. I 
should add, perhaps, that these records are averages of 
about 150-200 responses, the average being electroni- 
cally computed by a device designed and constructed 
by my colleague, Dr. John Barlow (1). The isolated 
columns that stand at the beginning and end of each 
record are for calibration only. The flash occurs at the 
beginning of the continuous record and the lines of the 
graph paper are at intervals of 20 msec. The envelope 
gives the wave-form of the average response. 

In figure 1A the response that has traveled through 
the mid-line structures can be seen very clearly following 
the primary response in the cortex of the unanesthetized 
animal. Figure 18, recorded simultaneously, shows 
equally clearly that the response has not traveled up 
through the lateral geniculate nucleus. With a moderate 
degree of barbiturate anesthesia (13-30 mg/kg) the 
surface negative wave is not only no longer cut into by 
the positivity of the nonspecific response (fig. 1C) but it is 
also augmented. We have previously suggested (2) that this 
increase in surface negativity under light pentobarbital 
anesthesia appears to be an impairment by the drug of 

! Aided by grants from the U. S. National Institute for Neuro- 
logical Diseases and Blindness (B 369 Physiology), the U. S. Navy 
(Office of Naval Research NR 101-445) and the U. S. Air Force 
(Office of Scientific Research AF-49-(638)-98). 

? Supported in part by the U. S. Air Force (Office of Scientific 
Research, Air Research and Development Command) the U. S. 
Army (Signal Corps) and the U. S. Navy (Office of Naval Re- 


search). 


626 


Massachusetts General Hospital, and Research Laboratory 


Boston, Massachusetts 


an ascending inhibitory influence acting in the un- 
anesthetized animal through the nonspecific system. The 
sketch on the lower right superimposes the unanesthe- 
tized and anesthetized records to bring out this aug- 
mentation. 

















FIG. I. 
anesthesia. 
nucleus: no anesthesia. C- 


Averaged responses to flash in a cat with and without 
A—visual cortex: no anesthesia. B—lateral geniculate 
-visual cortex: pentobarbital anesthesia. 
D—superposition of A and C. Electrode positions verified post- 
mortem. E, F—Above records A and C are mounted 4 histograms 
showing distribution of amplitude of the responses averaged at 4 
chosen points in each. Note decrease in variability induced by 
pentobarbital (see text). From: Brazier, M. A. B. Colloquium on 
Electrophysiology of the Brain and Higher Nervous Activity. Acad. 
Sct. USSR, 1958. Electroencephalog. & Clin. Neurophysiol. 
Suppl. 13, 1960. 


Moscow, 


By use of another computer which we have been 
privileged to use in the Research Laboratory of Elec- 
tronics at M.I.T. we have been able to obtain some 
measure of the variability of the responding cortex. This 
instrument, which has been described by its designers 
elsewhere (5), not only gives the curve of the averaged 
responses, but can also display histograms showing the 
scatter of the individual responses that went to make 


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July 1960 








RESPONSE 
TO FLASH 
IN CENTER 
MEDIAN 





UNANES THETIZED 















ETHER (INCR. GAl 











CAT 405 














FIG. 2. Averaged responses recorded from implanted electrodes 
in the centre median in an unanesthetized cat, and in the same 
animal under 4 different anesthetics. With equal amplification 
there is impairment of response to single flash only with ether. 
(Electrode position confirmed post-mortem. ) 


hee | 


UNANESTHETIZED (Peak ot 55 msec) 






NEMBUTAL (Peck of 40 meec) 





CHLORALOSE (Peoks ot 29,36 & 54 msec) TRIBROME THANOL (Pecks at 37, 46 & 63 msec} 


FIG. 3. Responses to flash in the cingulate cortex in various 
anesthetics (cat). Averaged responses from the cingulate cortex of a 
cat with implanted electrodes. Pentobarbital 30 mg/kg; chloralose 
20 mg/kg; tribromethanol 50 mg/kg. Multiple peaks on wave-like 
responses suggest multiple input, and the lack of the shorter latency 
peak in the unanesthetized state suggests some release of these 
pathways by the drugs from an inhibitory restraint. 


up the average reading at each point on the curve. For 
clarity in figure 1 histograms are reproduced for only 
four of the points averaged in each curve. 

On the left (fig. 1£) in the recording from the un- 
anesthetized animal, histograms are reproduced show- 
ing the scatter of values that went to make up the aver- 
age at 15, at 25, at 35 and at 45 msec respectively after 
the flash. On the right (fig. 17), from the same cat 
under pentobarbital, histograms are shown for the 
scatter of responses at 10, 20, 30 and 40 msec. The much 
tighter distribution of values under the anesthetic can 
be very clearly seen. 


XUM 


ANESTHESIA AND THE NERVOUS SYSTEM 627 





VISUAL CORTEX (i2 msec) 





Or 


RIGHT MOTOR CORTEX (37 msec) 





LEFT MOTOR CORTEX (38 msec) 


FIG. 4. Responses to flash in reticular formation and cortex in 
light pentobarbital anesthesia (cat). Averaged responses to flash re- 
corded from 4 positions of implanted electrodes in a cat’s brain. 
The onset of the initial deflection at the visual cortex is noted. The 
onset of the nonspecific responses is less easy to define so an ap- 
proximate value for the peak of local negativity has been indicated. 




















MOTOR CORTEX 









Se AED gies a EY Se ae a. 


af 












AS OES ZR FA PS ON A 


ea Be 
Sie 




















VISUAL CORTEX RETICULAR FORMATION 














SIMULTANEOUSLY RECORDED RHYTHMS IN AN UNANESTHETIZED DROWSY CAT 














VISUAL CORTEX RETICULAR FORMATION 








FIG. 5. Autocorrelograms processed to extract from mixed 
rhythms of the cat’s electroencephalogram those components which 
hold their frequency and phase relationships for an appreciable 
time. In the awake cat the rhythms of the motor cortex are of 
different frequency and have more persistence than those of the 
other two sites depicted. The motor cortex also shows some evi- 
dence of two competing frequencies. These rhythms drop out 
when the cat falls asleep and only slow undulations, all of the same 
frequency, appear in the autocorrelogram. The curved lines of the 
graph paper are 20 msec apart and the abscissa is not time but 
length of delay at which each autocorrelation was computed. In 
these curves the value of the autocorrelation (ordinates) was com- 
puted in delay steps of 5 msec increasing from left to right. The 
autocorrelation function is, of course, highest (i.e. 1) at zero delay 
on the extreme left of the continuous curve. For further details of 
this technique see reference (4). 








628 FEDERATION PROCEEDINGS 


The question does arise however as to whether this is 
due to variation in response mechanisms only, or also 
to variation in background cortical activity into which 
the impulses enter. The latter is an important component 
in the variability seen in the unanesthetized record but 
when the histograms for all points along the curve are 
studied there is found to be a greater variability during 
the medially conducted response than during the pri- 
mary specific wave. Space limits the reproduction of all 
these histograms here but some impression of greater 
scatter can be seen in the third sample of the unanes- 
thetized record, i.e. 35 msec after the flash. 

I would not wish to suggest that the introduction of 
the anesthetic has left open only the classical specific 
route to the cortex through the geniculate, for explora- 
tion of the responsivity of mid-line thalamic nuclei has 
revealed a response to singly occurring flashes that is 
resistant to anesthetics, at least at the levels we have used. 
In fact, ether is the only agent which in our hands so 
far has depressed at all appreciably the large slow-wave 
response of the centre median to flash, though not com- 
pletely abolishing it (see fig. 2). This result is in keeping 
with the finding of Dr. Eve King (7) that ether was the 
only anesthetic of those she examined that depressed the 
recruiting response evoked by stimulation of the centre 
median. 

The wide wave-like form of this averaged response in 
all anesthetics would suggest a great scatter in time 
of the responses that went to make up the average. In 
fact, with greater resolution, i.e. by averaging more 
responses, one can show that the response in the centre 
median is bimodal as has been reported elsewhere (3). 

One is, of course, interested in whether this response 
in the centre median is passed on to the cortex, and we 
have been especially interested in cortical areas outside 
the specific receiving cortex. The averaging technique 
that we use has allowed us to see responses that we have 
been unable to detect in our cathode ray traces. Figure 
3 shows the wave-like responses to flash that we find in 
the averaged recordings made from the cingulate cortex 
of a cat unanesthetized, and in the same animal under 
each of three anesthetics. These are strikingly different 
in form from the familiar primary responses at the 
specific projection cortex. 

Taking our electrodes further down the brain stem 
we find by this averaging technique, that responses to 
single flashes are recordable also in the reticular forma- 
tion. 

Figure 4 shows responses under pentobarbital. The 
wave-like response in the reticular formation reaches a 
peak at about 20 msec after the flash. Meanwhile in the 
visual cortex impulses arriving in the specific radiations 


Volume 19 


have already caused the surface-positive deflection (be- 
ginning 12 msec after the flash). The wave-like responses 
of the nonspecific cortex reach their peaks at approxi- 
mately 37 msec. In this instance they are being recorded 
from the sensorimotor cortex. Similar responses are found 
in the reticular formation in tribromethanol anesthesia 
and in ether. 

In describing these results I should emphasize that 
this report has been restricted to single stimuli. It is 
when repetitive stimulation is used that the effect of 
anesthetic agents and the role of the reticular formation 
become most marked, for it is the recovery phase that is 
usually most severely impaired. 

In this brief note there is not time to follow this point 
further or to describe our later experiments, but I 
should perhaps emphasize that these subcortical re- 
sponses that have just been described are to be differ- 
entiated from the well-known and much later Forbes- 
Morison ‘secondary discharge’, which with varying level 
of anesthetic has its own pattern of waxing and waning, 
both cortically and subcortically. 

In closing I would like to refer to the intrinsic rhythms 
in the various regions where cells aggregate in the brain. 
When autocorrelation is used to sort out periodic com- 
ponents from the mixture of potential changes recorded 
from these sites we find different periodicities in the 
various centers in the unanesthetized cat. For the prin- 
ciples and technique of this kind of analysis, see Brazier 
and Casby (4). 

Figure 5 is an example from a cat awake but not 
alerted, showing a clear rhythm of 13 cps in its motor 
cortex. In the visual cortex such poorly defined rhythm 
as can be detected at all is 11.5 cps. A faint rhythm in 
the reticular formation has a frequency of 12 cps. When 
this unanesthetized cat becomes spontaneously drowsy 
(i.e. without any drugs), all parts of the cortex and the 
reticular formation have the same slow beat. I might 
remark in passing that we have found a similar result 
with light anesthesia, although of course at deep levels 
all rhythmic phenomena are lost. 

This result will probably not surprise electroen- 
cephalographers who are familiar with the great variety 
of patterns that the EEG may exhibit in the waking man 
and with the fact that we all become electroencephalo- 
graphically indistinguishable from each other when we 
fall asleep. The same is true of man in the anesthetized 
state, as Dr. Bickford has so clearly shown. 


The work reported here has been that of a team including Miss 
R. Carpenter, Mrs. L. King, Miss M. Magavern, Dr. J. S. Barlow, 
as well as our colleagues in the Communications Biophysics 
Laboratory at M.I.T., among whom we would especially like to 
mention Mr. Frank Nardo. 


REFERENCES 


1. Bartow, J. S. Electroencephalog. © Clin. Neurophysiol. 9: .340, 


1957: 
2. Brazier, M. A. B. Brain Mechanisms and Consciousness. Oxford: 


Blackwell, 1954, p. 163. 
3. Brazier, M. A. B. Reticular Formation of the Brain. Boston: Little, 


1958, p. 151. 


4. Brazier, M. A. B. ano J. U. Cassy. Electroencephalog. © Clin. 
Neurophysiol. 4: 201, 1952. 

5. Crark, W. Average response computer. Quart. Progr. Report, 
Mass. Inst. Technol. Research Lab. Electronics, April, 1958. 

6. Frencu, J. D., VerzEAno, M. anp Macoun, H. W. Arch. Neural. 
Psychiat. 69: 519, 1953. 

7. Kine, E. E. J. Pharmacol. G& Exper. Therap. 116: 404, 1956. 





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& Clin. 
Report, 
. Neurol. 


56. 





Physiology and drug action: behavioral implications 


HOWARD F. HUNT 
Department of Psychology, University of Chicago, Chicago, Illinois 


sbi IN TIME prevent this paper from covering 
more than a fraction of the broad range implied by the 
title: ‘Behavioral Implications’. We will concentrate on 
the use of behavior of the total organism as a tool of 
analysis—as an indicator—in the experimental study of 
drug action, so a more realistic title might be: ‘Problems 
in the Behavioral Assessment of Drugs’. 

Behavior, properly studied, can be exquisitely sensitive 
to drug and other effects, but our ability to interpret 
changes in behavior lags behind our ability to produce 
them. The lag arises partly from the weakness or in- 
completion of the theories we depend upon to transform 
events in behavior into scientific facts and partly from 
intrinsic factors that make changes in behavior, as such, 
ambiguous as to cause. The intent, however, is not to 
discern so many difficulties as to obscure the more posi- 
tive possibilities, but rather to improve perspective to 
favor the more discriminating use of sensitive behavioral 
indicators now available and to come. 

The notion that changes in the behavior of the intact 
organism can be used to explore and assess the action of 
drugs in vivo certainly is not new. Informal behavioral 
assessment must have been going on for generations; 
many of the useful drugs of antiquity as well as of the 
modern age first claimed attention by virtue of their 
gross effects on behavior. The problem however, is not 
just to find substances that influence behavior or some 
behavior that is easy to alter. Almost anything, if given 
in sufficient quantity, will produce some change in some 
sort of behavior. Rather, the challenge lies in the de- 
velopment of methods, theories and contexts of inter- 
pretation, that not only provide for precise and replicable 
measurements of changes in behavior but also that specify 
which changes are important and relevant and that 
permit close analysis of the changes, to bring out more 
clearly what they are and how they are mediated. The 
challenge is most pointed, of course, in the study of the 
‘psycho-active’ drugs aimed specifically at modification 
of pathological behavior. 

It would be nice to ke able to say that psychology had 
achieved this goal—that scientific behavioral indicators 
of unimpeachable interpretability stand ready only 
waiting to be applied. Unfortunately, this is not the case, 
though substantial beginnings have been made. Be- 
havioral technology has enjoyed a tremendous postwar 
development and almost all of the experimental methods 
for the study of behavior are candidates for service here. 


629 


XUM 


These methods have improved in variety and in quality 
and precision so that, for many infrahuman organisms 
at least, we can produce or evoke certain kinds of be- 
havior almost to order. Further, we probably are on the 
edge of a breakthrough to the imaginative application 
of these methods to human behavior. 

In the interest of simplicity, we will consider the 
problems under the following major headings: sensitivity 
and replicability of results; interpretability of results; 
problems of inter-species and inter-situation generaliza- 
tion; and the specificity of the behavioral indicators to 
particular drugs and underlying physiology. 


SENSITIVITY AND REPLICABILITY 


This is the most comforting topic to tackle first be- 
cause here we have made the greatest progress, largely 
through the development of what most of you have 
heard of as operant conditioning. Neither a new syn- 
thesis nor a new theory, it comprises a logic of analysis 
and, more obviously, a way of going about the investiga- 
tion of behavior—a productive technology combined 
with a frame of mind disinclined to speculate about the 
‘whys’ of behavior at the expense of determining the 
‘whats’. With present techniques and following a few 
straightforward rules of analysis, an investigator can 
generate great amounts of ‘hard’ data, ‘hard’ in the 
sense that the behavior changes that appear are clear 
cut and replicable. 

In traditional operant conditioning, the food- or 
water-deprived animal is placed in a chamber where a 
lever can be pressed (or a target pecked, in the case of 
a bird), on the animal’s initiative, to produce a food or 
water reward. The apparatus can be set to reward each 
depression of the lever or it can be set to yield inter- 
mittent reward in accordance with an automatically 
controlled schedule of reinforcement that makes reward 
available (if the animal makes a lever response) only 
after the lapse of a fixed or variable interval of time 
(fixed and variable interval reinforcement), only after 
the animal has made a specified number—constant or 
varied—of responses (fixed and variable ratio reinforce- 
ment), or only as the animal holds his output down to 
some predetermined low level (differential reinforce- 
ment of low rates). In this DRL schedule, only those 
responses are rewarded which follow the last preceding 
response by some predetermined minimum: interval, 








630 FEDERATION PROCEEDINGS 


say 20 seconds. As reward is continuously withheld in 
experimental extinction, the animal eventually stops 
making lever responses, of course. 

The various schedules of reinforcement produce 
characteristically different outputs of lever-pressing. 
One or several of these can be placed under stimulus- 
control by having a signal (a discriminative stimulus), 
e.g. a light, present when one schedule of reinforcement 
is in force and another signal, e.g. a sound, present when 
another schedule is in force, and so on. After the animal 
has been trained to discriminate between these condi- 
tions, both schedules with their signals may be presented 
sequentially and both thus tested during a single run 
(multiple schedules). Such a run would contain several 
different kinds of behavior, all expressed in terms of 
variations in the rate of lever-pressing, an arrangement 
of obvious utility if one is interested in bringing out 
selective effects of drugs or other experimental variables 
on different aspects or kinds of behavior. Or, animals may 
be trained on several different schedules, but without 
the exteroceptive signals, and the schedules may be put 
in force sequentially during a single testing run. In this 
mixed schedule procedure, the animal has to depend 
upon internal stimuli and stimuli produced by events 
associated with reinforcement to regulate his behavior 
(e.g. see 5). 

The utility of the multiple-schedule approach is 
nicely illustrated in an experiment by Morse and 
Herrnstein (18) which showed that sodium pentobarbital 
had a pronounced disruptive effect on behavior for a 
fixed interval reward schedule but left the behavior for 
ratio reinforcement intact. Had segments of ratio rein- 
forcement scheduling not been included during the drug 
runs to show that the subject (pigeon) was motivated 
and capable of high output, the poor performance for 
interval reinforcement might have been attributed er- 
roneously to general effects of the drug on motivation, 
activity or coordination. 

Though many, perhaps most, of the details remain to 
be worked out, this sort of use of schedules of reinforce- 


ment—to provide internal controls to bring out selective 
effects—appears very promising. In fact, Ferster and 


Skinner recently published a substantial book describing 
in detail the behavioral effects of a large variety of 
different combinations of reinforcement schedules (8). 
With these instructions, the investigator may produce 
output curves with the desired physical characteristics 
for his particular purposes, almost on a_ prescription 
basis. The major problem is to determine how these 
different outputs and changes in them should be inter- 
preted: their physical characteristics are known, but 
their psychological properties, particularly their in- 
ferential significance, are still somewhat obscure. 

Even so, behavioral indicators of this type bring out 
subtleties in drug effects on behavior hard to get at in 
any other way at the animal level. For example, am- 
phetamine though subjectively a stimulant sometimes 
increases but also sometimes decreases output for regular 
or variable interval reinforcement. Morse and Herrnstein 


Volume 19 


were able to show, however, that the drug had a pro- 
nounced excitatory effect under the special conditions 
of a ‘strained ratio’ schedule in pigeons (18). To produce 
‘ratio strain’, the number of pecking responses required 
for each reward is set so high that the bird just barely 
will work for it. Usually, each reinforcement on such a 
schedule is followed by a prolonged period of no respond- 
ing, followed by a burst of responses at a high rate 
until the reward comes, followed by another period of 
no responding, and so on. In this experiment, am- 
phetamine increased total output but did so by shorten- 
ing the periods of nonresponding. The output during 
the bursts of responding prior to each reward, though 
far short of the birds’ physiological maximum, was not 
increased and may even have been decreased. Series of 
pecks simply occurred more often, at times when the 
birds normally would not have been responding. 

Another interesting application of schedules of re- 
inforcement appears in an experiment by Sidman on 
rats comparing amphetamine and alcohol (25). He used 
a DRL schedule in which only those lever-responses 
separated from the last preceding response by at least 
21 seconds were rewarded. This schedule generates 
uniform but low rates of response and produces a char- 
acteristic distribution of inter-response times, with the 
modal interval between responses falling at around 18-21 
seconds and with a decrease in frequency above and 
below that interval. Here the rats learn to regulate their 
behavior and time their responses so that a large number 
of them are spaced at about the right interval from each 
other and are thus rewarded. Amphetamine (1.5 and 
3 mg/kg) increased overall output on this schedule by 
breaking up the timing discrimination so that the re- 
sponses came closer together, rather than by increasing 
the output of responses at or near the correct interval. 
Alcohol (1 mg/kg), on the other hand, sharply decreased 
total output but left the timing discrimination intact, 
so that the number of responses at all interresponse 
intervals was reduced fairly evently across the distribu- 
tion. 

Operant methodology has been applied profitably to 
the study of aversively controlled behavior. For example, 
in Sidman’s avoidance lever-pressing procedure the 
rat learns to press a lever to postpone a shock for some 
specified period of time, say 10 seconds. If he fails to 
press within a specified interval, again say 10 seconds, 
he gets a shock, and so on. The output of lever responses 
is a major function of the interval by which each lever 
response postpones the shock (response-shock interval) 
and the interval between shocks if no lever responses are 
made (shock-shock interval). Sidman has presented data 
indicating that amphetamine (3 mg/kg) has somewhat 
the same effect on avoidance lever-pressing as on the 
DRL lever-pressing described earlier—the timing be- 
havior tends to be disrupted and the intervals between 
responses shortened. In addition, reserpine (.1 mg/kg), 
as would be expected, sharply reduced avoidance lever 
pressing if the rat received only about 20% of the shocks 
normally due him for failing to press the lever fast 





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July 1960 


enough. This effect disappeared, however, if he was 
given all the shocks he ‘earned’, almost as if the tran- 
quilizing effect of the drug at this dose disappeared as 
the situation approached its normal stringency (23, 24). 
Sidman avoidance represents an economical and rigorous 
method for studying avoidance and other aversively con- 
trolled behavior, partly because of the precision with 
which the indicator behavior can be controlled and 
varied in sensitivity and partly because it yields such 
a large amount of avoidance data per hour of running 
time, in comparison with other methods. 

The present instrumentation with levers, targets, 
buttons and boxes was originally a matter of convenience. 
It is fast becoming a matter of tradition and custom, but 
unfortunately so because other kinds of behavior in 
varied situations are excellent for use as experimental 
indicators in species other than the rat and pigeon— 
particularly in man. In its broadest form, operant analy- 
sis has been applied fruitfully to such diverse subject 
matters as the interpretation of some aspects of our social 
order, language and speech, school learning, control 
of food and water intake, and socialization and other 
aspects of child development, to name but a few. 

Operant methodology has the greatest generality. 
It requires only a situation in which a brief, repeatable 
response can be placed under control by reward, with 
the subject left free to respond again, and where the 
dependent variable or indicator can be the rate or the 
change in rate at which the response occurs (7). The 
subject is left free to elaborate and emit his behavior in 
accordance with rewarding (or punishing) contingencies 
that are under rigorous control. Because of this, the 
strength and sensitivity of the indicator behaviors can 
be adjusted precisely to bring out clearly the main effects 
of the experimental variables, an important considera- 
tion in the study of drugs that may have subtle rather 
than gross and categorical effects. Further, the experi- 
mental animal, working out his own salvation in his own 
way, will often provide data that answer questions the 
experimenter never even thought of asking. This con- 
trasts with many other techniques in which the sensi- 
tivity of the indicator behavior is relatively fixed and in 
which the experimental arrangements and the definition 
of the response limit the information the subject can 
provide to categories anticipated by the experimenter. 

Application of operant methods has turned up a num- 
ber of ‘hard’, new findings. One example appears in the 
work of Olds and his associates who have demonstrated 
that operant lever pressing can be acquired and main- 
tained as a function of reinforcement by electrical stim- 
ulation of parts of the thalamus, hypothalamus and septal 
region through implanted electrodes, with the stimula- 
tion contingent upon depression of the lever. The re- 
sponse thus established differs in some ways from ordi- 
nary operant lever pressing, but there is little question 
but that rats will work for this electrical stimulation as 
a reward. Despite some problems in interpreting self- 
stimulation data, evidence already available indicates 
the importance of this technique for investigating not 


PHYSIOLOGY AND DRUG ACTION 631 


only behavior and central nervous system function but 
drug effects as well, through differential effects of various 
drugs on thresholds for reinforcement by self-stimulation 
in various locations in the brain (19, 20). Similarly, the 
experimental production of gastrointestinal ulcers in 
the monkey, by Brady and his associates at Walter Reed, 
is important in its own right, however difficult it may be 
to understand just how this effect comes about and just 
what its direct implications for the human ulcer patient 
may be (4). Here, one monkey of a pair restrained in 
adjoining conditioning-chairs, pressed a lever on a 
Sidman avoidance schedule to avoid electric shock by 
postponing it. The other, the control running-mate, 
had his lever disconnected so that regardless of his be- 
havior he received a shock each time the first monkey 
did. On a 6-hours-on, 6-hours-off working schedule, 
the monkey that could avert the shocks by pressing the 
lever—the ‘executive’ monkey—got the ulcer while the 
helpless control animal that received the same number 
of shocks did not. 


INTERPRETATION 


Problems of interpretation might seem to become less 
difficult as we move into the study of abnormal and emo- 
tional behavior. Many of the animal methods have an 
appealing face validity—the situational pressures and the 
reactions to them have many features analogous with 
what we see (or think we see) in human adjustment 
problems. Further, we have developed a lot of theory, 
however loose, to explain the learning of behavior pa- 
thology and emotional maladjustment. 

Much of my own work has been concerned primarily 
with behavior that might be considered as an analogue 
of or, better, as a component of animal experimental 
neurosis. Usually, experimental neuroses are produced 
by forcing animals to make discriminations beyond their 
capacities, by massive painful stimulation, by demanding 
delay and control in a complex sequential task and then 
replacing the expected reward with a traumatic stimulus, 
by restraining the animal and reducing the behavioral 
alternatives open to him in a difficult situation, or some 
combination of these. The animal that emerges is emo- 
tionally disturbed and atypical; his behavior shows all 
kinds of evidence of aversively controlled components and 
emotional conditioning (22). This behavior and _ its 
antecedents are usually so complex theoretically, how- 
ever, that even the analysis of the effects of experimental 
procedures on these states from a straight empirical 
point of view is difficult. It is hard to determine what 
aspect of the total pattern is affected by the drug, and 
how. 

Accordingly, we tried to select what appeared to be 
the simplest element in this melange—conditioned ‘fear’ 
or a conditioned emotional response—and studied it as 
it appeared in the free behavior of the unrestrained rat 
or cat. The basic methodology resembles that reported 
by Watson and Raynor almost 40 years ago in describing 
how they conditioned the child, Albert, to become emo- 








632 FEDERATION PROCEEDINGS 


tionally disturbed by a furry object presented to him 
as a conditioned stimulus in conjunction with a loud 
noise that served as the unconditioned stimulus (26). 
In the laboratory, we present an innocuous conditioned 
stimulus (a clicking noise or a light) for a period of 
several minutes, terminating it in conjunction with 
momentary but unavoidable and painful shocks to the 
feet. After a few such pairings, often after only one, the 
conditioned stimulus or signal acquires the power to 
suppress, either partially or wholly, intercurrent ongoing 
behavior by evoking what appears to be an emotional 
disturbance, as indicated by concurrent defecation. In 
the grill box (a chamber with a grill floor for administra- 
tion of shocks and a transparent wall to permit observa- 
tion), the ongoing behavior so suppressed is the explora- 
tory activity rats ordinarily show in an apparatus prior 
to conditioning. Or, adapted from a method first de- 
scribed by Estes and Skinner for demonstrating some 
quantitative properties of ‘anxiety’ (6), the ongoing 
behavior suppressed is operant lever pressing for a water 
reward in a lever apparatus. Here the conditioned fear 
is superimposed on another learned behavior that occurs 
at a regular rate which itself can be adjusted by varying 
the amount and scheduling of reward and the strength 
of the thirst drive. Thus, the symptomatic strength of the 
conditioned fear can be measured to some extent in 
terms of how severely presentation of the conditioned 
stimulus reduces the output of lever pressing. By ad- 
justing the output of lever pressing, we can calibrate the 
tests and adjust their sensitivity to reflect conditioned 
fear more precisely at various levels of strength, both 
strong and weak. In both the grill box and the lever ap- 
paratus, thoroughly conditioned rats normally show a 
tense, crouching posture—they ‘freeze’—and usually 
defecate as well upon presentation of the conditioned 
stimulus, and this pattern is easily distinguishable from 
simple inactivity, sleep, ataxia etc. We have called this 
a conditioned emotional response (CER), conditioned 
in that a neutral stimulus acquires the power to evoke it, 
emotional in that it depends upon reinforcement with 
a painful stimulus, and response in that the effects of 
the procedure appear as an alteration in behavior (13). 

Clear cut as this indicator would seem to be, inter- 
pretation of some of our research with it has proven com- 
plex, as illustrated in a segment of our research on be- 
havioral effects of electro-convulsive shock (ECS). We 
noted early that ECS had the initial effect of making the 
rats irritable and jumpy. From this, we expected that 
ECS might increase the intensity of emotionally-toned 
behavior such as the CER, just as Gellhorn found it to 
produce a recovery of an extinguished conditioned 
avoidance-response (10), on the ground that a hyper- 
irritable rat would show a more vigorous than normal 
reaction to a signal that had acquired emotional sig- 
nificance through conditioning. 

The opposite happened, of course. Rats showed a 
consistent post-ECS amnesia for a CER established in 
a grill box prior to treatment, just as extrapolation from 


Volume 19 


human data on the therapeutic use of ECS in psychiatric 
illness would suggest should happen. Experiments in 
which the CER was superimposed on a lever-pressing 
habit showed that this amnesia was selective and could 
be partial. ECS weakened or eliminated the CER, 
depending on the number and spacing of convulsions, 
but had no deleterious effect on the lever-pressing habit 
(1, 3, 13). Brady’s dissertation (2) showed that the am- 
nesia was temporary; after ECS, the CER returned, to 
appear clearly within a month after convulsions and to 
be at almost full pretreatment strength within go days, 
Thus, ECS produced a selective, partial, temporary 
amnesia for CER conditioning. 

Rather than search for additional qualifying adjec- 
tives, we investigated the nature of the postconvulsive 
changes by behavioral methods aimed at determining 
the status of the conditioning during that period when 
the treated rats did not show the CER. Here we first 
established the CER by conditioning in the grill box 
or lever apparatus, subjected all animals to ECS and 
divided the animals into two groups. The first group re- 
ceived a single CER retention test and then were put 
away to rest until retested 30 days later. Rats handled 
in this way showed the usual findings. On the first test 
after ECS, the CER was weak or absent, but it returned 
to appear at almost full pretreatment strength on the 
30-day test. The second group received an initial re- 
tention test after ECS, along with the other group, but 
subsequently received daily experimental extinction 
trials in which the signal for the CER was presented 
without shocks once daily for 13 more days. Then the 
rats rested until their second retention test 30 days after 
ECS. These animals in which the CER was subjected 
to post-ECS extinction did not recover the CER by the 
30-day test. That the extinction appeared to have pre- 
vented the recovery of the conditioning after ECS 
indicated to us that the convulsions had not produced a 
true amnesia. The CER was present and organized in the 
animal after ECS even though it might not appear in 
overt behavior; otherwise the experimental extinction 
could not have had its usual decremental effect (14). 
Some other sequel of ECS was preventing the CER from 
appearing, symptomatically, in the behavior of the 
animal. 

Subsequently, R. W. Goy’s dissertation (g) shed addi- 
tional light on this problem. In investigating the relation 
between the pretreatment strength of the CER and the 
amount of effect ECS had upon it, he took advantage of 
the fact that there are two major ways to control the 
strength of a conditioned response. One is by varying the 
number of training trials; the other is by varying the in- 
tensity of the reinforcing stimulus—in this case the shocks 
to the feet. The details of the experiment and its total 
interpretation are too complex for review here, but, in 
brief, Goy found that increasing the intensity of the 
shocks to the feet in conditioning greatly increased the 
resistance of the CER to attenuation by ECS while 
increasing the number of conditioning trials within the 
range studied had no notable effect, even when all the 





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July 1960 


CERs were brought to approximately equal symptomatic 
strength prior to ECS. 

This, together with the repeatedly verified observa- 
tion that the recovery of the CER after ECS seemed to 
parallel the disappearance of the post-ECS irritability, 
suggested that we might be dealing with a problem in 
stimulus generalization, among other things. Briefly, 
the notion was that the animals were tested for retention 
after ECS under stimulus conditions significantly altered 
from those that had obtained during training. Such shifts 
at testing, away from the training conditions, usually 
result in a weaker conditioned-response. 

More specifically, Heistad (who has done most of the 
work on this) suggested that the convulsions, following 
Gellhorn’s findings (11), produced sympathetic dom- 
inance in the autonomic system that might lead to 
changes in the subjective stimulus state of the rat at 
testing, changes particularly relevant to conditioned 
emotional disturbance. He also reasoned that chlorpro- 
mazine, thought to produce a parasympathetic dom- 
inance, might act as an antagonist to ECS. If so, chlor- 
promazine after ECS would increase the strength of a 
CER weakened by the convulsions, even though the 
normal effect of the drug on conditioned fear is the re- 
verse. And Heistad has reported findings of just this 
sort (12). The effect here is small, but the outcome was 
so unexpected within the usual view of ECS and drug 
effects that it excites interest, however tenuous the reason- 
ing leading to it. My own interest in it is heightened by 
the fact that not just any tranquilizer has this effect. 
In unpublished research, John Harvey and I have found, 
for example, that meprobamate given to rats after the 
CER has been weakened by convulsions simply weakens 
it further—the effects seem to be additive with this drug 
that has little or no effect on the autonomic nervous 
system. Thus, experimentation on postconvulsive am- 
nesia now promises to become a study of stimulus gen- 
eralization, on one hand, and a study of drug effects on 
the autonomic nervous system, on the other, if the lines 
of investigation suggested by present data, prove fruitful. 


INTER-SPECIES AND INTER-SITUATION GENERALIZATION 


Conditioned fear and experimental neuroses have 
enough elements in common with human psychiatric 
disorders to tempt us to generalize from the animal to 
the human case. These elements include traumatic 
stimulation, strong emotional disturbance, some dis- 
organization of behavior, the development of behaviors 
that attempt to minimize the amount and duration of 
painful stimulation, both conditioned and unconditioned, 
and so on. Further, convulsive treatments and some 
tranquilizing drugs appear to have beneficial effects; 
and the behaviors arise at least in part out of experience 
and are modified by it. Theoretical considerations 
counsel restraint in extrapolation from animals to 
humans, however, even though some empirical data tend 
to be encouraging. If these animal behaviors are ana- 
logues of human neurotic problems, the relation is close 


PHYSIOLOGY AND DRUG ACTION 633 


in the case of the traumatic rather than psychoneuroses. 
Though workers in this area differ in this opinion, our 
data suggest that conflict, particularly intrapsychic 
conflict, does not appear to play a critical role as such in 
the development of these states as studied so far (15, 17). 

Theoretical specifications for an animal analogue of 
psychoneurosis would seem, technically, to demand: 
a) a situation in which stimuli arising from impulses to 
action have become dangerous, through conditioning, 
so that when they occur 6) they evoke an emotional 
disturbance which interferes with the behavior that 
normally would eliminate the cause of the impulses to 
action (e.g. an animal conditioned to be so frightened 
by the sensations of thirst and the act of drinking that 
it cannot or will not drink and thus has to remain both 
thirsty and afraid). Such procedures have rarely been 
employed, but in those instances I know about the 
animals have surmounted their difficulties without de- 
veloping diffuse patterns of fixated emotional disturbance 
(21). A good animal analogue of depression is difficult 
to conceive, similarly an analogue of psychosis, even 
though it is comparatively easy to produce behavior 
that looks retarded and withdrawn by the usual trau- 
matic stimulation (17). 

In man, where we are confronted with an immensely 
complicated organization of behavior, language and 
capacities for other symbolic self-stimulation play a 
pivotal role in the regulation of behavior and in psycho- 
pathology. Though such capacity can be demonstrated 
at a rudimentary level in some animals, there is little 
evidence that they use symbolic transactions extensively 
in the regulative, self-manipulative way humans do. 
Thus, while animal data will provide many hints and 
suggest new methods and lines of inquiry into human 
behavior, it is far from clear that we can work out the 
extrapolations from the animal to the human sphere 
with the aid of more animal data and logical analysis 
alone. Human behavior, too, must be studied with pre- 
cision before we can tie the animal and human data 
together convincingly. And it is one thing to develop 
an analogy, and reason by it from species to species, but 
quite another and more difficult thing to have done the 
experimental work that demonstrates an homology. 

Interpretation of animal data and extrapolation across 
species boundaries would be considerably enhanced 
if we had a better picture of what our behavioral tests 
were testing. The situation is particularly critical with 
respect to some of the operant methods. These are ex- 
quisitely sensitive but in some cases they are so devoid 
of theoretical underpinning that extrapolation from them 
to another kind of testing situation is difficult indeed. 
More trans-situational validation—the explicit use of 
a variety of measures of the same function, with con- 
gruent results in situations where the indicator behaviors 
differ in topography—would expand the usefulness of 
some of these techniques tremendously. If all replication 
or cross-checking is restricted to minor variants of one 
methodology, the results may include substantial arti- 
facts common to all of the variants. For example, a tech- 








634 


nique depending primarily upon rate of output of a 
motor response can give very misleading results in the 
investigation of the effects of a drug that alters activity 
as a side effect, unless special procedures are introduced, 
not only to measure activity as such but also to handle 
complex interactions between activity and the indicators 
of emotion, discrimination, sensory threshold or what- 
ever it is that is the primary object of study. 


SPECIFICITY 


We would be fortunate indeed if we could always as- 
sume a sort of one to one behavior—soma parallelism, 
if we could find an indicator behavior that could be 
counted on faithfully to reflect the state of affairs in a 
particular underlying somatic system. Behavior does not 
appear to be organized in that way, however, and prob- 
ably few such specific behavioral indicators will emerge. 
In fact, a good deal of behavior appears to be organized 
around maintaining invariant relations with significant 
features of the environment in spite of fluctuations in 
underlying somatic systems. Other behavior (probably 
most of it) is a complex resultant of many interacting 
system states and can be changed by any number of 
agents. Ironically enough, one of the behaviors most 
sensitive to drug effects (operant lever pressing main- 
tained by interval reinforcement) is very undiscriminat- 
ing—almost anything will influence it. Alone, it can tell 
you that something is happening; the conjunctive use of 
other indicators with it is required to reveal what that 
something is. 


FEDERATION PROCEEDINGS 


Volume 19 


Finally, our previous experience with ECS suggests 
that the effects of drugs on many behavioral indicators 
will be relative and quantitative rather than categori- 
cal—relative not only to the dose of the drug but also, 
as is not widely recognized, relative to the psychological 
history of the indicator (16). The effect of a standard 
dose of a drug (or other experimental treatment) may 
vary considerably depending upon the values of the con- 
ditioning parameters employed in establishing and main- 
taining the behavior (intensity or magnitude of rein- 
forcement, number and distribution of training’ trials 
etc.). Sidman called this phenomenon a ‘drug-behavior 
interaction’ in discussing his finding that the effect of 
reserpine on avoidance lever-pressing tended to dis- 
appear as the rat received more shocks (24). Of course, 
‘drug-behavior interactions’ could be considered simpiy 
as embarrassments to research in this field. There is 
every reason to believe, however, that they could be 
employed systematically to analyze drug effects in a 
sensitive (and sophisticated) fashion, very much as Goy 
showed that the intensity of the pain shocks to the feet, 
rather than the number of training trials, determined the 
magnitude of the effect of ECS on conditioned fear (9). 
‘Parameter-effect’ curves could be prepared showing 
the effects of standard doses of a drug over a range of 
each of the major conditioning parameters for the in- 
dicator-behavior employed. These, if worked out sys- 
tematically, would indicate of which parameters the 
drug effect was a function and might provide valuable 
clues as to the mechanisms by which the drug effects 
were mediated. 


REFERENCES 


1. Brapy, J. V. ano H. F. Hunt. J. Comp. Physiol. Psychol. 44: 
204, 1951. 
2. Brapy, J. V. J. Comp. Physiol. Psychol. 44: 507, 1951. 
3. Brapy, J. V., H. F. Hunt anp I. Geiier. J. Comp. Physiol. 
Psychol. 47: 454, 1954- 
4. Brapy, J. V., R. W. Porter, D. G. Conrap anno J. W. 
Mason. J. exp. anal. Beh. 1: 69, 1958. 

. Dews, P. B. J. exp. anal. Beh. 1: 73, 1958. 

. Estes, W. K. anp B. F. Skinner. J. Exp. Psychol. 29: 390, 
1941. ' 

+. Ferster, C. B. Psychol. Bull. 50: 263, 1953. 

8. Ferster, C. B. ano B. F. Skinner. Schedules of Reinforcement. 
New York: Appleton, 1957. 

g. Goy, R. W. The effect of electro-convulsive shock on a conditioned 
emotional response: relation between amount of attenuation and 
strength of the conditioned emotional response. (Ph.D. dissertation). 
Univ. Chicago, 1953. 

10. GELLHORN, E., M. KessLer AND H. Minatoya. Proc. Soc. Exper. 
Biol. & Med. 50: 260, 1942. 

11. GELLHORN, E. Physiological basis of neurology and psychiatry. 
Minneapolis: Univ. Minnesota Press, 1953. 

12. Heistap, G. T. J. Comp. Physiol. Psychol. 51: 209, 1958. 

13. Hunt, H. F. anp J. V. Brapy. J. Comp. Physiol. Psychol. 44: 
88, 1951. 


Ho 


14. Hunt, H. F., P. JERNBERG AND J. V. Brapy. J. Comp. Physiol. 
Psychol. 45: 589, 1952. 

15. Hunt, H. F. ano J. V. Brapy. J. Comp. Physiol. Psychol. 48: 
395, 1955; 

16. Hunt, H. F. Ann. New York Acad. Sc. 67: 712, 1957. 

17. Hunt, H. F. Effects of drugs on emotional responses and ab- 
normal behavior in animals. In: Psychopharmacology: Problems in 
Evaluation. J. O. Cole and R. W. Gerard (editors). Washington, 
D. C.: Nat. Acad. Sci. Nat. Res. Council Publ. 583, 1959. 

18. Morse, W. H. ann R. J. HERRNSTEIN. Ann. New York Acad. S. 
65: 303, 1956. 

19. Oxps, J., K. F. Kittam ano P. Bacu-Y-Rira. Science 124: 265, 
1956. 

20. Oxps, J. Science 127: 315, 1958. 

21. Ors, L. S. Drive conditioning as a response to biogenic drive stimuli 
previously associated with painful stimulation. (Ph.D. dissertation). 
Univ. Chicago, 1956. 

22. RussELt, R. W. Brit. J. Psychol., 41:95, 1950. 

23. SipMan, M. J. Comp. Physiol. Psychol. 46: 253, 1953. 

24. Smpman, M. Ann. New York Acad. Sc. 65: 282, 1956. 

25. SipMAN, M. Science 122: 925, 1955. 

26. Watson, J. B. AnD R. Raynor. J. Exp. Psychol. 3: 1, 1920. 





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American Institute of Nutrition 


SYMPOSIUM ON INTERACTION OF MINERAL ELEMENTS IN NUTRITION 
AND METABOLISM! 





Introduction 


GEORGE K. DAVIS, Chairman 


Nutrition Laboratory, University of Florida, Gainesville, Florida 


a PRESENTING THIS SYMPOSIUM sponsored by the Coun- 
cil of the American Institute of Nutrition, we are happy 
to acknowledge the co-sponsorship of the Nutrition 
Study Section of the Division of Research Grants of the 
National Institutes of Health. A grant from the Na- 
tional Heart Institute, H-5104, has made possible the 
pre-printing of the papers and the distribution to the 
members of the American Institute of Nutrition and to 
those of you who are in attendance here today. We ap- 
preciate this interest on the part of the National Heart 
Institute and the financial help represented by the 
grant. 

In presenting this Symposium on mineral element 
interactions in nutrition, it is recognized that the need 
for these elements as a requirement for normal growth, 
development and reproduction has been recognized 
for many years. Yet the function of these individual 
elements is but imperfectly known and each year pro- 
vides exciting new information with respect to the role 
of any given element in body metabolism. 

Perhaps the most challenging frontier in mineral 
element nutrition is in the interactions which occur be- 
tween two or more of these vital nutrients. So involved 
are mineral element functions that it would presently 
seem impossible to evaluate the requirement of any 
given species for an element, such as copper, without 
considering the effects of molybdenum, sulfate, iron and 
zinc levels. As more investigations are carried out with 
the mineral elements the more certain research workers 
are that here we have additional keys that will help 
explain the intricate functioning of cellular and extra- 
cellular components of the body. 

In a single afternoon it would be ridiculous to try 


Study Section, Division of Grants, National Institutes of Health, 
and supported by Grant H-5104, National Heart Institute. 


XUM 


635 


which we class as essential in the nutrition of animals, 
including man. Consequently we have selected a few of 
these elements and asked the men you see here to pre- 
pare papers on the function and interaction of these 
elements. Those manuscripts have been printed and 
are in your hands. Many of you have had the opportunity 
to read them before coming to the Federation meetings. 
Our design is to give a maximum of opportunity for dis- 
cussion by and among the panelists. To stimulate this 
discussion we invite questions from you in the audience. 
These questions should be written and brought to us 
here at the microphones. 

This procedure is in part a trial run for the symposia 
which will be held at the Fifth International Nutrition 
Congress, September 1-7, 1960, in Washington, D. C. 
To a certain extent both you, the audience, and we, the 
panelists, are being trained in this technique designed 
to get a maximum from our time, our panelists and the 
subject under discussion. 

Each of our participants has been asked to present a 
short summary of the material he has covered in his 
paper emphasizing important aspects and areas needing 
investigation. This will be followed by discussion. The 
discussion will be summarized and the published sym- 
posium will include that summary. 

In presenting the subject of interactions of mineral 
elements in nutrition and metabolism it was natural 
that we should first consider calcium and phosphorus, 
this then led to zinc, magnesium and manganese, all of 
which have important relationships with calcium and 
phosphorus. Iron and copper interactions in hemoglobin 
formation in effect served to open the field of trace ele- 
ment interaction, and activity in the relationship of 
copper, molybdenum and sulfate sulfur is certainly 
stimulating. These mineral interrelationships are, in 
reality, just a small fraction of what is fast becoming a 
major part of nutrition. 








Calcium and phosphorus interactions 


in nutrition and physiology 


R. H. WASSERMAN 


Laboratory of Radiation Biology, Department of Physiology, 
New York State Veterinary College, Ithaca, New York 


Scene HAS SEVERAL important functions within the 
animal and to summarize, it is: ¢) an essential element in 
the conversion of prothrombin to thrombin in the blood- 
clotting mechanism, 4) a necessary ion (but replaceable 
by strontium) in promoting normal cardiac, smooth and 
skeletal muscle function, c) essential for the normal 
functioning of nervous tissue—in a low calcium environ- 
ment, nerve becomes hyperexcitable and leads to the 
formation of spontaneous impulse transmission associated 
with clinical ‘tetany’, d) implicated in the maintenance 
of normal intracellular environment and in the develop- 
ment of bio electrical potentials at the cell surface and e) 
a major constituent of the skeleton and teeth. Selecting 
the most important physiological function of calcium 
would be a meaningless contemplation since these several 
roles of calcium are essential for productive metabolism 
and for life itself; on a quantitative basis, attention is 
usually given to the hard tissues of the body because 
about 99% of the body calcium is found in bones and 
teeth. 

Phosphorus also has many-fold functions in the body 
and is: a) essential for anabolic and catabolic reactions 
in the body as exemplified by the role of phosphorus in 
high energy bond formation (e.g., adenosine triphos- 
phate); 6) a precursor for biologically significant com- 
pounds such as the phospholipids, which are important 
in the formation of cell membranes and in cellular perme- 
ability; c) a presursor in the synthesis of geneticaily 
significant substances, particularly desoxyribonucleic 
acid (DNA); d) a contributor to the buffering capacity 
of body fluids and cells and ¢) a major constituent of 
bones and teeth. Like calcium, the majority of the body 
phosphorus is located in the hard tissues; in the adult, 
about 86 % of the total body phosphorus is found in bones 
and teeth, and the balance in soft tissues. 

It appears that the primary metabolic function that is 
common to calcium and phosphorus concerns the forma- 
tion of skeletal tissue and teeth. In both intramembra- 
nous and intracartilaginous ossification, the bone. salt 
that is formed has a crystal structure and chemical com- 
position considered to be closely similar to that of 
hydroxyapatite (3Ca;(PO,)2Ca(OH)s), an extremely hard 
difficultly soluble calcium phosphate salt with a solubility 


636 


product (expressed as aca++ X anpo, = ) of about 0.49 
xX 1077 (1). The enamel and dentine of teeth also can 
attribute their hardness to the presence of insoluble 
calcium phosphate salts and these have been shown by 
x-ray diffraction to have a crystalline structure also of 
the apatite series. The formation of bones and teeth, 
therefore, is dependent upon the presence of adequate 
amounts of calcium and phosphate ions within the 
milieu of calcification, and the levels of mineral elements, 
in turn, would be related to their nutritional supply. 
The calcification process is a complex phenomenon not 
completely understood at present. Biological fluids are 
undersaturated in respect to the hydroxyapatite salt, and 
bone salt formation occurs at calcification sites due to 
the presence of specialized biochemical reactions (1, 2). 
The latest theories as to the nature of these reactions have 
been summarized by Neuman and Neuman and include 
the presence of glycolytic systems for producing a high 
local Ca X P product or crystal nucleation (epitaxy) ora 
combination of these processes. 

It would seem that an understanding of bone salt 
formation is dependent upon a knowledge of the physi- 
cal-chemical interaction of calcium and phosphate ions 
in solution and the essential conditions for precipitation 
of the insoluble salts. Also, the interrelationship of these 
ions in other biological systems would likewise be de- 
pendent upon a consideration of insoluble salt formation, 
particularly in the gastrointestinal absorption of calcium 
and phosphorus, the formation of urinary calculi, and 
kidney calcification. These concepts have been clearly 
reviewed in reference to skeletal metabolism by Neuman 
and Neuman (1) and McLean and Urist (2). Suffice it 
to re-emphasize here that calcium and phosphorus do 
form insoluble salts and that the type of salt formed would 
be related to such factors as the pH of the system, the 
presence of extraneous cations and anions, the presence 
of solubilizing organic acids (e.g., citrate) etc. It should 
be further pointed out that calcium forms relatively 
undissociated complexes with biochemically important 
phosphates, such as adenosine triphosphate, adenosine 
diphosphate and hexose phosphates (3-5); these reac- 
tions further indicate the intimate relationship of calcium 
and phosphorus in biological systems. 


WIiIRMA 





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July 1960 


It seems logical, therefore, to consider the interrelation 
of calcium and phosphorus in the nutrition and physiol- 
ogy of the animal as an entity. Rather than presenting 
an exhaustive and complete review of the subject, the 
present paper deals with the interaction of these elements 
in several physiological processes and the importance of 
these relationships in the normal and abnormal state. 
For more complete summaries of calcium and phospho- 
rus metabolism, references (1, 2, 6-12) should be 
consulted. 


GASTROINTESTINAL ABSORPTION OF CALCIUM 
AND PHOSPHORUS 


The physiological mechanism controlling the absorp- 
tion of calcium is not completely known but new insight 
into this process has been gained recently from studies 
reported by Schachter and Rosen (13). Using surviving, 
everted duodenal sacs, these investigators have shown 
that Ca*® can be transported against a concentration 
gradient from the mucosal to the serosal side of the gut 
membrane, that this process is inhibited by metabolic 
inhibitors (e.g., NaCN, NaN;, NaF, HgCh, 2, 4-dinitro- 
phenol etc.) and also inhibited by magnesium and cobalt 
ions, and that vitamin D was essential for the operation of 
the transport system. The evidence has clearly shown that 
Ca*® transport was dependent upon the integrity of the 
metabolic function of the tissue zn vitro although a specific 
active transport system for calcium could not be postu- 
lated. As pointed out by Schachter and Rosen, the 
transport of Ca*® may be an indirect result of the active 
transfer of cations in the opposite direction (serosal to 
mucosal) or the effect of a primary anion transport. A 
recent study by Rasmussen (14) has shown that the in 
vitro system was significantly influenced by the para- 
thyroid status of the experimental animal; surviving 
duodenal sacs of the normal rat were able to produce a 
greater concentration gradient than duodenal sacs taken 
from parathyroidectomized rats. Recent experimentation 
in our laboratory has shown that similar intestinal 
preparations from rats given a low calcium diet for one 
week were able to transport Ca* to a greater degree than 
rats maintained on high calcium diets; it was also 
interesting to note that the duodenal tissue was unable 
to transport Sr® against a concentration gradient. 
This and other evidence (15) suggested that the absorp- 
tive discrimination between Ca* and Sr®, has as its 
basis, the ‘active’ transport of calcium by a metabolically 
dependent system and the movement of strontium by 
physico-chemical processes (diffusion). 

Prior to the studies of Schachter and Rosen, others 
had postulated the existence of a calcium transport sys- 
tem based upon the observation that calcium absorption 
is narrowly controlled and is somewhat independent of 
dietary calcium intake. Also, it has been a frequent 
finding that animals on low calcium diets are more 
efficient absorbers of dietary calcium than animals 
raised on high calcium diets (16-18) and this adaptation 
may be mediated through the postulated ‘active’ trans- 
port system. 


XUM 


INTERACTIONS OF MINERALS 637 


The mechanism of phosphorus absorption by the 
gastrointestinal tract also is unknown but investigators 
have been hesitant to postulate a transport system. The 
phosphate ion is apparently readily absorbable and 
studies by McHardy and Parsons (19) have shown that 
increasing levels of phosphate ions in the intestinal lumen 
linearly enhanced net absorption, a response typical of a 
diffusion-like process. Earlier studies by Cohn and 
Greenberg (20) and Carlsson e¢ al (21) have shown that 
vitamin D did not enhance phosphate absorption in rats 
raised on a vitamin D-deficient, high Ca-low P diet. 
Vitamin D-deficient chicks and poults also were able to 
utilize inorganic phosphorus to the same extent as 
normal birds (22). The situation differs from phytate 
phosphorus in several species wherein this form of phos- 
phorus may not be as available as inorganic phosphate, 
and vitamin D seems to enhance the availability of 
phytate phosphorus. 


CALCIUM, PHOSPHORUS AND VITAMIN D 


The function of vitamin D in the metabolism of cal- 
cium and phosphorus has been studied extensively and 
it seems that any unqualified statement for vitamin D 
action can find proponents and opponents in the litera- 
ture and among researchers currently engaged in the 
problem. However, there is general agreement that 
primary physiological action of vitamin D is the en- 
hancement of calcium absorption by the intestinal tract; 
this was shown in balance studies performed a _half- 
century ago and has been confirmed and reconfirmed 
with the use of radiocalcium in the rachitic animal 
(23-26). The actual biochemical mechanism of vitamin 
D action is unknown but recent theory implicated 
local citrate production in the intestine as a possible 
explanation; the citrate formed within the absorbing 
cell of the mucosa could complex calcium and transfer 
the soluble, un-ionized calcium citrate parenterally 
(1). Arguments against this theory are that the vitamin 
D-dependent transport system for calcium  charac- 
terized by Schachter and Rosen was unable to trans- 
port Sr®> (15), and stable strontium was unable to in- 
hibit Ca** transport. Since the pK values for the citrate 
complexes of calcium and strontium are similar, it would 
be expected that strontium could compete with calcium 
if such a system existed. Also, it has been shown that 
calcium citrate is not absorbed by the rat to any greater 
degree than other forms of calcium (27, 28). Migicovsky 
(24) visualized a possible direct reaction of calcium with 
vitamin D or with a vitamin D derivative at the absorp- 
tion barrier but no supporting data have been forth- 
coming on this point. 

Vitamin D also apparently exerts a direct effect on the 
skeleton although this action is not properly understood. 
Carlsson (29) and Lindquist (30) demonstrated a solu- 
bilizing effect of vitamin D on bone mineral in rats on 
excessive and moderate dosages of the vitamin; others 
(31, 32) have postulated an effect—direct or indirect— 
on bone accretion. Vitamin D also has an effect on the 
urinary excretion and kidney clearance of calcium and 








638 FEDERATION PROCEEDINGS 


phosphorus as emphasized in experiments by Crawford 
et al. (33) and Litvak et al. (34), although in other situa- 
tions such effect has not been observed (35). 

The interrelationship of calcium and phosphorus in the 
diet and vitamin D action has been given much atten- 
tion In the rat, for example, a clear-cut case of rickets 
can be produced only when a high calcium-low phos- 
phorus diet or a low calcium-high phosphorus diet is 
used. When the Ca:P is about unity and the calcium and 
phosphorus content of the diet are adequate, growth and 
calcification proceed in the absence of vitamin D, al- 
though at a slower rate than in the presence of vitamin 
D (23); others have not demonstrated a vitamin D effect 
in the rat, under similar conditions, when adequate 
levels of dietary Ca and P were present (36-38). Species 
other than the rat are more dependent upon vitamin D 
for normal calcium metabolism. Gershoff et al. (39), in 
studies with the chick, investigated the influence of diets 
varying in calcium and phosphorus content and in the 
absence or presence of vitamin D on the absorption of 
Ca**. The degree of radiocalcium absorption in the 
absence of vitamin D varied with the mineral content of 
the diet; it was observed, for example, that chicks given 
a diet containing 1.14 % Ca and 1.02 % P (Ca:P ratio = 
1:1) absorbed more Ca** than birds given a diet con- 
taining 1.16% Ca and 0.27% P (Ca:P ratio = 4:1). 
In the presence of vitamin D, the absorption of Ca*® 
under both situations was the same. An earlier report 
by Migicovsky and Jamieson (24) was somewhat in 
disagreement in that the absorption of radiocalcium 
varied indirectly with the calcium content of the diet 
when vitamin D was present. In the latter studies, the 
calcium content of the diet ranged from 0.3-2.18% Ca 
whereas the phosphorus level was maintained at about 
0.95%. The different conditions could perhaps explain 
the discrepancy. Further studies by Gershoff et al. (40) 
also had shown the cat to be sensitive to vitamin D; 
severe rickets was produced in kittens without resorting 
to unusual Ca:P ratios. In fact, more severe rickets was 
observed in the feline given diets containing 1% Ca 
and 1% P (Ca:P ratio = 1) than on diets containing 
2% Ca and 0.65% P (Ca:P ratio = 3:1). Also, it was 
observed that cats that survived acute rickets during their 
growing period showed a spontaneous healing of rickets; 
this was interpreted to indicate that the vitamin D 
requirement for adult cats is low. 

The fraction of ingested calcium that is absorbed in the 
rat has been shown to vary inversely with the calcium con- 
tent of the diet using conventional balance techniques and 
also with Ca* (41). This alteration in efficiency of calcium 
absorption on different levels of dietary calcium is the 
result of physiological adaptation by a little understood 
process. Haavaldsen et al. (42) proposed that vitamin D 
is essential for the manipulation of calcium adaptation. 


CALCIUM AND PHYTATE PHOSPHORUS 


The influence of phytic acid on calcium metabolism 
has been well documented, and 


the significance of 


Volume 19 


phytate phosphorus in animal and human nutrition has 
been covered in several recent reviews (11, 12, 43). 
Phytic acid, the hexaphosphate of inositol, is present in 
a wide variety of plants and may contribute significantly 
to the total phosphorus ingested. Early studies by 
Mellanby showed that cereals that contained high levels 
of phytic acid produced a more severe rickets in dogs 
than in cereals not so rich in phytic acid, and McCance 
and Widdowson showed that the addition of sodium 
phytate to white bread caused a decreased absorption 
of calcium. More recent studies by Bronner et al. (44, 
45), using radiocalcium as a tracer, compared the 
uptake of calcium from a phytate-rich cereal (oat- 
meal) with the uptake of calcium in the presence of 
farina, a phytate-free cereal; also the effect of added 
sodium phytate to the farina cereal was studied. The 
experimental subjects were adolescent boys of inadequate 
intelligence but otherwise normal; the main source of 
calcium in the cereal diets was in the form of milk 
calcium. From observations of the Ca* levels in the 
serum of the individuals after the test meals, it was calcu- 
lated that the calcium ingested with the farina cereal 
was absorbed 1.4 times that from the oatmeal cereal 
and 2.2 times that of the farina-plus-sodium phytate 
meal. As summarized by Harris (43), important consider- 
ations in the problem are that phytate as the acid or in 
the form of a soluble salt apparently interferes with 
absorption of calcium and iron in man, that phytates 
in food substances are not as available for interference as 
free phytate salts and that the inhibitory effect of phy- 
tates can be minimized in the presence of excess calcium, 
vitamin D or a proper Ca:‘P ratio. 

It has been generally agreed that there is no significant 
problem from phytic acid in this country (11, 43) al- 
though in areas of calcium deprivation, the nutritional 
role of phytates may assume considerable importance. 
Pertinent studies have shown that vitamin D increases the 
availability of phytate phosphorus by perhaps increasing 
the level of phytase enzyme in the intestine (46). 

Sodium phytate has been extensively studied by 
Henneman and colleagues (47) as a potential therapeutic 
agent in the treatment of hypervitaminosis D, sarcoid 
with hypercalcemia, and certain renal stone syndromes. 
In a balance study with a patient with sarcoid, hyper- 
calcemia and recurrent renal stones in his single kidney, 
it was observed that the administration of sodium phy- 
tate (8.8 gm per day) decreased calcium urinary excre- 
tion and similarly increased fecal calcium excretion such 
that the calcium balance was little altered. On discon- 
tinuing phytate administration, fecal calcium decreased 
to the pretreatment level. Prolonged administration of 
sodium phytate over a 13-month period indicated that 
the suppression of excessive urinary calcium excretion 
was continuous and that, only after phytate treatment 
was stopped, did excretion values rise to their initial 
values. The general conclusion from these and other 
studies (48) was that sodium phytate may have merit in 
situations in which there is an excessive absorption of 
calcium. 


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July 1960 


CALCIUM, PHOSPHORUS AND TEETH 


The process of dentine calcification is similar to that 
for bone and is similarly influenced by various dietary 
and hormonal conditions; amelogenesis (the formation of 
enamel) is much less affected by extraneous factors (11). 

The tooth is metabolically active only during forma- 
tion but is essentially unaltered after eruption. The 
chemical composition of the tooth has been shown by 
Sobel and Hanok (49) to reflect somewhat the mineral 
levels in serum which in turn are dependent primarily 
on the nutrient composition of the diet; in order to ob- 
serve such changes due to diet, Ca:P ratios of 10.4 and 
0.123 were employed. 

In a series of studies, Haldi et al. (50) and Wynn et al. 
(51, 52) examined the effect of Ca:P ratio of the diet 
on dental caries and the chemical composition of teeth. 
In one study, the calcium content of the diet was main- 
tained constant at 0.50 and the phosphorus content was 
varied from 0.24 to 1.48%, thereby altering the Ca:P 
ratio from 2.0 to 0.33. It was observed that, as the 
phosphorus level of the diet increased with a concomitant 
reduction in the Ca:P ratio, the incidence of caries 
formation was reduced. Also, there was no observable 
alteration in the chemical composition of enamel or 
dentine of molars or incisors. The reduction of the cario- 
genicity of the diet was therefore related to either the 
increased dietary level of phosphorus or to the altered 
Ca:P ratio or both. In a more recent study, the phos- 
phorus content of the diet was maintained at 0.5%, 
whereas the calcium level was varied from 0.29 to 1.57 %; 
the Ca:P ratio of the diet therefore ranged from 0.5 to 
3.3 As the calcium content of the diet was increased, the 
number of carious lesions and the caries score were 
significantly reduced. In the analysis of the teeth, it was 
again noted that the calcium and phosphorus content of 
the diet was not reflected in the chemical composition 
of the teeth. It was concluded that the anticariogenic 
property of the diets was not related to the Ca:P ratio 
of the diet but to the absolute amounts of calcium and 
phosphorus present. Harris (36) also was of the opinion 
that the Ca:P ratio becomes important only when the 
level of available phosphorus is low; in studies with 
hamsters, on a diet adequate in phosphorus, the Ca:P 
ratio could be changed from 4.0 to 0.25 without in- 
fluencing the incidence of dental caries. 

The caries-inhibiting effect of inorganic phosphates has 
aroused interest as a possible, practical method of 
decreasing tooth decay in the human. Experiments on 
the rat and hamster had shown that added soluble 
phosphates in the diet could reduce dental caries in these 
species. Recent studies reported by McClure and Muller 
(53) and Van Reen and Glassford (54) confirmed the 
earlier studies in showing that NagHPQ, was an effective 
anticaries agent whether this salt was present in drinking 
water or in bread. Dicalcium phosphate (CaHPQO,) was 
not found to be cariostatic unless a high level of NaCl 
was present in the diet or in the salt mixture. McClure 
and Muller (loc crt) suggested that the cariostatic effect 


INTERACTIONS OF MINERALS 


639 


of these minerals was perhaps localized in the oral cavity; 
the effect may be an influence on the maturation and 
mineralization of enamel by surface contact with these 
minerals or by a buffering action. 


CALCIUM, PHOSPHORUS AND UROLITHIASIS 


The formation of urinary calculi, as well as the type 
of calculi produced, appears to be sensitive to several 
dietary factors, especially absolute levels of calcium and 
phosphorus and the Ca:P ratio. On an adequate cal- 
cium-low phosphorus diet in rats, Schneider and Steen- 
bock (55) observed that stones were readily formed and 
that they were primarily composed of calcium citrate. 

Studies by Sager and Spargo (56) on the production 
of calcium citrate urinary calculi in rats fed normal 
calcium-low phosphorus diets indicated that the primary 
causative factor was an increased absorption of calcium 
on this regime. The increased absorption of calcium was 
reflected in a hypercalcemia and an elevated urinary 
calcium level. In order to maintain ionized serum cal- 
cium within tolerable limits, the citrate concentration in 
serum was also increased. Thus, the elevated urinary 
calcium levels were paralleled by urinary citrate levels 
and the high concentrations of these urinary constituents 
resulted in the local precipitation of the calcium citrate 
stone. It was also observed by Sager and Spargo (loc cit) 
that protein depletion of the rats accelerated the forma- 
tion of the calculi. This coincides with the recent 
observation by Van Reen et al. (57) that high dietary 
levels of casein have an inhibitory effect on calcium 
citrate calculi formation in the caries-susceptible Osborn 
Mendel rat of the NMRI-D strain. In the latter studies, 
uroliths were produced on diets that contained rather 
normal calcium and phosphorus levels (e.g., Ca, 0.43 %; 
P, 0.19%; Ca:P = 2.2), and 15-20% casein; this same 
diet was ineffective in producing calculi in the Sprague- 
Dawley and Long-Evans strains. At higher levels of 
casein, as mentioned above, calculi formation was pre- 
vented; chemical analysis of urine indicated that rats fed 
diets resulting in urolithiasis excreted large amounts of 
calcium and citric acid but little phosphate and the 
urine from rats fed diets preventing calculi formation 
contained high levels of phosphate and small quantities 
of calcium and citric acid. 

Recent evidence cited by Gershoff and Faragalla (58) 
indicated that pyridoxinn deficiency in cats resulted in a 
marked increase in endogenous oxalate production that 
caused oxalate nephrocalcinosis; a similar oxaluria was 
also reported to occur in pyridoxine-deficient rats and 
the oxaluria was increased by desoxypyridoxine or 
isoniazid administration. Domanski (59) produced a high 
incidence of calcium oxalate stones in rats by adding 
ammonium oxalate and ammonium chloride to an 
otherwise normal diet; ammonium oxalate alone re- 
sulted in a relatively low incidence of calculi formation 
and ammonium chloride alone was without effect. 

The studies by Vermeulen and colleagues reported 
at the 1959 High Calcium Symposium and recently 








640 


published (60, 61) are particularly intriguing. Urinary 
stone disease was studied in the rat by inserting a small 
sterile zinc disc within the bladder; at 6 weeks after the 
disc implantation, the zinc foreign body was removed 
and weighed. Excess phosphorus as phytate, hexameta- 
phosphate and orthophosphate were added to the basal 
diet containing 1.3% Ca and 0.9% P. It was observed 
that each of the experimental regimens inhibited stone 
formation although excess phosphate may have been 
thought to increase urolith production. Also, each of the 
phosphate sources caused an elevated urinary excretion 
of phosphorus and a consequential decrease in citrate 
excretion in the urine. The mechanism of phosphorus 
action was thought of in terms of orthophosphate since 
phytate and hexametaphosphate were hydrolyzed in 
the intestine to the orthophosphate form. These investi- 
gators felt that excess dietary phosphate apparently 
interfered with calcium and magnesium absorption, thus 
inhibiting stone formation. In a corollary study, the 
basal diet was supplemented with calcium lactate and, 
as expected, the excretion of urinary calcium was ele- 
vated but stone formation was prevented. The mecha- 
nism of stone inhibition in this situation was thought to 
be the converse of the high phosphate diet; the excess 
calcium in the diet converted dietary phosphorus to the 
insoluble calcium phosphate, decreasing the absorption 
of phosphate ion and thereby reducing the calcium 
phosphate product of urine below the point of precipita- 
tion. 

It is apparent that the calcium and phosphorus ratio, 
and the absolute levels of dietary mineral, are important 
considerations in urolithiasis. However, other factors 
such as the protein level in the diet, acid-base relation- 
ships and vitamin content of the diet modify the influence 
of the mineral effects. 


CALCIUM, PHOSPHORUS AND PARTURIENT PARESIS 


Parturient paresis (milk fever) is a disease of cattle 
which occurs typically at the beginning of lactation. As 
defined by Hibbs (62), milk fever is ‘characterized by a 
sudden paralysis, gradual loss of consciousness,: and, if 
untreated, usually terminates in death.’ From a review 
of many studies, it was concluded that the hypocalcemia 
associated with this disease is the failure of the calcium 
regulatory mechanisms to mobilize mineral rapidly 
enough from the skeleton to equal the rate of secretion 
of calcium into milk. The failure to maintain serum 
calcium levels in milk fever has been generally thought 
to be due to an insufficiency of parathyroid secretion. 

The regulatory role of parathormone on calcium 
homeostasis has been reviewed recently by McClean 
(63) and involves a ‘feed-back’ mechanism. When blood 
levels of calcium fall, the parathyroids are stimulated to 
increased activity; when serum calcium is in excess, 
parathyroid activity is decreased. In the absence of the 
parathyroids, serum calcium is maintained at about 
seven mg per 100 ml by ion transfer or ion exchange 
processes on a minute-to-minute basis. The parathyroid 


FEDERATION PROCEEDINGS 





Volume 19 


glands function to maintain serum calcium levels be- 
tween the range of 7 mg % and normal (about 10 mg “%); 
the parathyroid mediated control process is slower to 
respond to alterations in serum calcium levels than the 
ion exchange process. 

Since it has been a basic assumption that parathyroid 
insufficiency is an etiological factor in milk fever, cura- 
tive regimes have been proposed that may stimulate 
parathyroid function. Using this hypothesis, Boda and 
Cole (64) fed pre-partum dairy cows that were disposed 
to milk fever on low calcium-high phosphorus diets 
(Ca:P = 1:3.3). Their results indicated that this treat- 
ment effectively prevented milk fever when fed during 
the last month of the dry period. A correlation between 
the calcium: phosphorus ratio and the incidence of milk 
fever was observed; in their experiments, there was a 
30% incidence of milk fever in the animals fed a diet 
containing a calcium-phosphorus ratio of 6:1, 15% 
incidence in cows fed a diet with Ca:P ratio of 1:1, and 
no incidence with the diet containing a Ca:P ratio of 
1:3.3. The mechanism suggested was that the low 
calcium-high phosphorus diet induced a compensatory 
hypertrophy of the parathyroid glands, and the excess 
parathormone thus released was able to prevent the 
hypocalcemia in the postparturition state. 

Luick et al. (65) attempted to delineate the physio- 
logical mechanism by which the low calcium-high 
phosphorus diets elicit their protective response in 
parturient paresis. From studies with radiocalcium the 
reservoir of mobilizable skeletal calcium was estimated 
and it was observed that the ‘mobilizable calcium pool’ 
decreased in size as the Ca:P ratio of the diet increased 
from 1:7 to 6:1, and the pool turnover rate increased as 
the ratio was similarly altered. It was further estimated 
that, in cows fed the low calcium-high phosphorus diets, 
the ‘mobilizable calcium’ was approximately 60% of 
body calcium; with the cows on the high calcium-low 
phosphorus ratio, ‘mobilizable calcium’ represented 
only 37% of body calcium. As a result of their studies, 
Luick and associates proposed that feeding low calcium- 
high phosphorus diets to pre-partum cows was more 
than a mechanism for stimulating the parathyroid glands 
to produce more hormone when serum calcium levels 
fall. It was proposed that, on the low calcium-high 
phosphorus diet, a continually greater amount of para- 
thyroid hormone was secreted during the feeding period 
and would explain the elevated serum calcium level 
that was observed on this regime. 

Another treatment proposed to reduce the incidence 
of milk fever is the injection of massive doses of vitamin 
D (62). Recent studies by Conrad et al. (66) had indi- 
cated that excessive levels of vitamin D exerted their 
protective effect mainly by increasing the rate and 
quantity of calcium absorption from the gastrointestinal 
tract. Several related effects occurred as a consequence of 
the vitamin D treatment, such as a decreased endogenous 
fecal excretion of calcium and a three-fold increase in 
net calcium retention (accretion). 

Recent attention has been given to the possible bene- 


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nous 
se in 


ene- 





July 1960 


ficial properties of aluminum hydroxide in combination 
with calcium lactate in preventing parturient paresis 
(67). Earlier Studies by Page and Page had shown that 
such an aluminum hydroxide-calcium lactate product 
increased calcium retention and decreased neuromuscu- 
lar irritability in pregnant women. When the product 
was fed to pre-partum cows for eight days prior to 
parturition at the rate of one ounce per day, a consider- 
able reduction in milk fever was observed in test herds. 
The mechanism may be due to an increased absorption 
of calcium as postulated for the pregnant women; the 
aluminum hydroxide would perhaps form an insoluble 
aluminum phosphate or adsorb other anions on the gel 
surface and thereby yield a more available calcium ion. 


CALCIUM AND CASEIN PHOSPHOPEPTIDES 


The intraintestinal digestion of protein molecules results 
in the transient formation of complex peptides; it is pos- 
sible that such peptides may be absorbed as such or may 
be further hydrolyzed to aminoacid and products. When 
casein was digested in vitro by proteolytic enzymes of the 
intestinal tract, high molecular weight peptides were 
formed that resisted further digestion (68). The resistance 
to hydrolysis was probably due to the fact that the 
peptides were phosphorylated since incubation with 
phosphatases eliminated the insensitivity of these sub- 
stances to proteolytic enzymes. It was further noted 
that these phosphopeptides behaved as effective che- 
lating agents with several metal ions. The calcium 
phosphopeptide contained 10% calcium and was highly 
soluble to the extent of about 50 gm of complex per 100 
ml solution. In animal experimentation, Mellander (69) 
observed that the calcium phosphopeptide was readily 
absorbable from the gastrointestinal tract in rats and 
chicks on a rachitogenic diet. Studies with infants had 
shown that calcium bound in this form could be absorbed 
from the digestive tract and that bone calcification oc- 
curred in rachitic children in some cases without the 
simultaneous administration of vitamin D (70). The 
high availability of calcium in milk as compared with 
inorganic calcium may be due in part to the formation 
of such a complex. 


CALCIUM, PHOSPHORUS AND RADIOSTRONTIUM RETENTION 


The interrelationship of Ca and P to radiostrontium 
metabolism bears mentioning here because of the quali- 
tative similarity of calcium and strontium and also 
because of an interesting problem that has evolved from 
the following studies. In an experiment with young and 
rapidly growing male rats, Wasserman, Comar, and 
Papadopoulou (41) had reported that increasing levels 
of calcium and phosphorus in the diet resulted in a 
proportional decrease in the retention of chronically 
ingested radiostrontium; the importance of phosphorus 
in this study was not fully recognized and therefore was 
not emphasized. Later reports by Palmer et al. (71) 
described investigations with mature rats wherein it was 
noted that an elevation of the calcium level of the diet 


INTERACTIONS OF MINERALS 641 


alone (no alteration of the phosphorus level) did not 
proportionally decrease the retention of chronically 
ingested Sr®°, The contrasting results from these two 
studies led to further experimentation, wherein it was 
verified by Wasserman and Comar (72) that, in the 
mature rat, calcium alone was unable to effect any 
appreciable reduction in radiostrontium retention, 
whereas in the young rat calcium alone was able to do 
so. It was further observed that calcium and phosphorus 
additions to the diet of the mature rat was able to 
diminish the body burden of the chronically ingested 
radionuclide. Therefore, a definite interaction was ap- 
parent between age and phosphate levels of the diet in 
determining the effectiveness of dietary calcium in 
yielding a significant effect on Sr®° metabolism. Still to 
be determined is the role of phosphorus in this situation. 
Is phosphorus acting as a precipitant (in the gastrointes- 
tinal tract) of the insoluble phosphates, is it altering 
intestinal membrane permeability, or is phosphorus 
acting at the skeletal level? 


SUMMARY AND CONCLUSIONS 


The present review has illustrated some of the inter- 
related effects of calcium and phosphorus in various 
nutritional and physiological situations. Needless to say, 
many important and significant studies went unmen- 
tioned for reasons of limitation of time and space. 
Reference should be made to the excellent material 
presented at the 1959 Symposium on High Calcium 
Intake sponsored by the American Institute of Nutri- 
tion and the National Institutes of Health for supple- 
mentary information. 

In the past, considerable emphasis was placed on the 
ratio of calcium: phosphorus in the diet as an indication 
of nutritional wholesomeness. Although the Ca:P ratio 
should be considered in assessing dietary adequacy, one 
should be cognizant of the absolute levels of these 
minerals in the diet. In fact, the ratio concept has been 
deemphasized by recent studies reported by Harris (36), 
and those by Wynn e¢ al. (51, 52) and Haldi et al. (50) 
on the cariogenicity of various diets. Yet, the ratio of 
Ca:P in the diet is still an important factor when either 
of the elements is present in deficient amounts. 

A conclusion from the studies cited here, and from 
other studies, is that there is no apparent simple, un- 
complicated relationship between the metabolism of 
calcium and phosphorus. Although the interaction of 
these elements in certain instances can be explained 
primarily on the basis of physico-chemical concepts, the 
relationship becomes exceedingly complex as biological 
factors are introduced into a given system. Obviously, 
the presence of other inorganic ions or organic entities 
would also interfere with the prediction of events from a 
knowledge of only the respective concentrations of cal- 
cium and phosphorus in the diet. In this connection, it 
became apparent that the reciprocity of effects of dietary 
calcium on phosphorus absorption and dietary phos- 








642 FEDERATION PROCEEDINGS Volume 19 


phorus on calcium absorption has not been firmly 
established. Under conditions of severe alterations of 
Ca:P ratio, there may be a depression of the availability 
of the ion present in the least amount. Under other 


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40. GersHorr, S. N., M. A. Leco, F. J. O'Connor anv D. M. 
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~I 





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1953- 

7: 203, 

D. M. 


JULOU. 


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SENDA. 


h. Buo- 


58. 
J. Nue 


J. Nu- 
J. Nu- 
58: 36, 
B: 633. 
40. 

tion 69: 


B: 526, 


CHapP- 


rol. 82: 


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Physiol. 
10: 98, 


4: 520, 
saliensts 


.NBERG. 


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Nutritional interactions of zinc and calcium 


R. M. FORBES 


Department of Animal Science, University of Illinois, Urbana, Illinois 


. THE PAST FIVE YEARS a reawakened interest in 
the role of zinc in animal nutrition has resulted in con- 
siderable clarification of the enzymic roles of this element 
(24) and of practical conditions modifying the response 
of swine and of poultry to diets of moderate zinc content. 
Two major factors which appear to affect zinc require- 
ments are the source of dietary protein and the level of 
dietary calcium. It is the purpose of this review to bring 
together the evidence concerning the calcium-zinc inter- 
relationship and to evaluate the several proposals that 
have been made to explain the mechanism of this inter- 
relationship. 


SWINE STUDIES SHOWING AN EFFECT OF CALCIUM 
ON SWINE PARAKERATOSIS 


The initial conclusive report showing a practical need 
for zinc supplements to swine rations (22), and showing 
a deleterious effect of calcium supplements which 
could be overcome by zinc, has been followed by many 
corroborative papers. Tucker and Salmon (22) found 
that hogs fed practical diets containing either hy- 
draulic or solvent process peanut meals developed 
severe dermatitis and impairment of feed intake and 
weight gain. These diets contained bonemeal and salt as 
sole mineral supplements and contained 34 to 44 ppm of 
zinc. Addition of 2% bonemeal or 1.5% CaCO; in- 
creased the symptoms, but they could be alleviated 
dramatically by a 0.02 % supplement of ZnCQs3. 

In view of the practical implications of these findings 
and the surprise that a ration containing 7-10 times the 
concentration of zinc reportedly required by the rat (8), 
it is understandable that intensive investigations in this 
area of nutrition were instituted quickly. In table 1 are 
found growth data and certain specifications of experi- 
ments reported in the literature showing that increments 
of calcium are inhibitory to the weight gain of young 
pigs and that this inhibition may be lessened by zinc 
supplementation. These experiments were not designed 
to answer questions relative to the effect of calcium on 
zinc requirement. All that may be said with certainty is 
that growth depression accompanying high calcium diets 
is decreased by zinc supplementation of diets already 
deficient in zinc. One might as logically infer that the 
unfavorable effect of excess calcium is magnified in the 
presence of a zine deficiency. 


XUM 


643 


Retarded growth is only the first of the symptoms of 
zine deficiency. Incidence of skin lesions, of the type 
termed parakeratosis, is a more specific symptom, but 
one more difficult to express quantitatively. In the report 
of Lewis et al. (10), the appearance of skin lesions closely 
paralleled the depression of weight gain. It is significant 
from the standpoint of interpreting other experiments 
that in Lewis’ work, animals receiving 28 ppm zinc and 
0.8% calcium gained well and showed a minimum of 
parakeratosis for eleven weeks, while those receiving 28 
ppm zinc and 1.2% calcium showed parakeratotic 
symptoms and depressed gain after the second week. 
During weeks 12-20 the 0.8% calcium group showed 
both symptoms of zinc deficiency, thus illustrating the 
advantage of relatively long-term experimentation. 

Luecke et al. (13) also report a close correspondence 
between parakeratosis and depressed weight gains. All 
pigs receiving 1.5% calcium and 31 ppm zine showed 
dermatosis (7 severe and 3 mild), while only 3 of 10 pigs 
receiving .98 % calcium and 29 ppm zinc showed derma- 
tosis (2 severe and 1 mild). Of the remaining 20 pigs on 
their experiment, only one showed dermatosis, and this 
was a mild case. 

An exception to the correlation between weight gain 
and dermatosis is seen in the report of Lewis e¢ al. (11) in 
which they found that addition of NaHsPQ, to provide 
0.8% P in the diet, rather than the basal level of .47 %, 
had no effect on weight gains but decreased the parakera- 
tosis to essentially the same low level as the basal + zinc 
group. It is in this paper that the suggestion is first made 
that calcium may interfere with zinc absorption. The 
data of Luecke et al. (12) also show a good correlation 
between weight gain and appearance of dermatosis, both 
being similarly affected by levels of calcium and of zinc 
in the diet. 

Among other criteria of effects of varying levels of zinc 
and calcium in the diet of growing swine are tissue levels 
of zinc, blood alkaline phosphatase levels, and endog- 
enous fecal zinc excretion. Hoekstra et al. (7) and Lewis 
et al. (10) have reported changes in tissue zinc levels as a 
result of increasing calcium content of the ration. Hoeks- 
tra et al. report that addition of bonemeal to a ration con- 
taining 80 ppm zinc lowers zinc content of liver and of 
kidney cortex, but not of spleen or pancreas. Bonemeal 
addition to a ration containing 35 ppm zinc does not 
affect the zinc content of any of these tissues. In contrast, 








644 FEDERATION PROCEEDINGS 


Lewis et al. report that additions of calcium to rations of 
35 ppm zinc content lowered the zinc concentration in 
liver, kidney cortex, hair, pancreas and bone. The 
changes observed were of the order of reductions to 65- 
69 % of values in liver, hair and bone of the ‘low-calcium’ 
animals. The physiological significance of these changes 
is obscure but may be related to differences in total feed 


TABLE 1. Summary of Growth Data Showing Stimulatory Effect 
of Zinc on High-Calcium Diets Fed to Swine 


Ca, |p, | 20. oo Remarks Ref 
Yo |” “| PPM ii /day 
Corn-soybean .98 .7 | 35 .g1 | 8-week period (13) 
meal .98) .7 | 55 |1-25 | 10 pigs per lot 
1.5) -8 | 35) -54 
1.5 | .8 | 55 |1.06 
Corn-soybean 82) .5 | 35.39 | 12-week period (11) 
meal 1.2 | .8 | 35 | .og | 7-8 pigs per lot 
1.4) .6 | 95 | .07 
1.4 | -5 | 35 | -10 
.82| .8 | 35 | .35 
82) .5 | 85 | .73 
1.40 .8 | 85 | .89 
Corn-cotton- .48 32 | .79 | 6-week period (21) 
seed meal .48 44 1.10 | 8 pigs per lot 
.67 g2 | .48 
-07 44 |1.05 
.67 80 1.19 
1.03 g2 | .12 
1.03 44 | -79 
1.03 80 1.19 
Semipurified 68 28 1.02 | g-week period (3) 
Isolated soy 1.06 28 | .83 6 pigs per lot 
Protein .68 128 1.13 
1.06 128 1.10 
Corn-soybean 5 | -5 | 28 |1.14 | 20-week period (10) 
8 .5 | 28 .8 | 6 pigs per group 
1.2| .9 | 28) .1 at 12 weeks only 
8 | .5 |128 1.20 | 1.2% Ca group 
retarded 
Corn-soybean .65) .5 | 45 |1.22 | g-week period (12) 
meal .65, .5 95 1.30 | 10 pigs per group 
1.25) -9 | 40 | -75 
.5 | -6 | 32 | .59 | 8-week period 
.5  .6 | 82 11.23 | 10 pigs per group 
1.2} .6 | 29 | .21 
1.2 | .6 | 79,|1.16 
i.9 | -6 | 31 | -17 
1.9 | .6 | 81 | .98 
Corn-soybean .64; .54) 30.17 | 6-week period (17) 
meal 1.19 .93 33. .02 | Controlled feed 
1.23 .g6) 94 | .29 intake 





(and hence zinc) intake rather than to a specific effect 
of calcium on zinc level in the tissues. The authors state, 
**Excessive calcium probably reduced the zinc content 
of these organs by hindering zinc absorption from the 
intestinal tract,” and, “‘It is likely that none of the body 
components analyzed to date represent the site at which 
zinc exerts its beneficial effect on parakeratotic swine. 
Perhaps the point of action is in the gastrointestinal tract 
rather than in the body proper.” 

Luecke et al. (12, 14) and Newland ef al. (17) have 
shown parakeratosis to be accompanied by low blood 


Volume 19 


serum alkaline phosphatase. There is a trend in their 
data for calcium to decrease blood serum alkaline phos- 
phatase, while zinc raises it markedly. 

A definite attempt to account for the observed rela- 
tionship between calcium and zinc is that of Newland 
et al. In a carefully controlled experiment involving 
individually fed weanling pigs, an attempt was made to 
measure endogenous fecal zinc excretion of pigs fed 7) 
30 ppm zinc with .64 % Ca and .54 % P, 2) 33 ppm Zn with 
1.19% Ca and .g3% P, or 3) 94 ppm Zn with 1.23% 
Ca and .g6% P. Equal feeding was an integral portion 
of the experimental design. When the pigs had been on 
these diets 8 days they were given a single injection of 
Zn®*. Collection of feces was made during days 16 
through 21, during which time the same diets were fed. 
At the 21st day the animals were slaughtered and tissues 
and excreta examined for total zinc and Zn®. Total fecal 
zinc excretion amounted to 110, 93 and 93% of the 
intake, respectively, for the three groups of pigs. Total 
zinc concentrations of blood, liver and kidney were not 
altered by the treatments, but specific activity of feces 
and of liver was higher in group 2, receiving high calcium 
and low zinc, than in the other two groups. The percent- 
age of fecal zinc of endogenous origin was calculated to 
be 6.4, 10.7 and 4.4, respectively. These figures, however, 
like those for specific activity of fecal zinc, need careful 
interpretation, since the zinc intake was 2-4 times 
greater in group 3 than in the other groups. This fact 
alone would lower the specific activity of tissue and of 
fecal zinc for group 3, aside from variation in calcium 
intake. Further, it is not clear how the facts of higher 
specific activity (and total activity) in both feces and 
tissues 13 days after a single injection led to a conclusion 
that a ‘higher rate of zinc metabolism’ exists. Actually, 
the greater specific activity of fecal zinc from the high 
calcium-low zinc fed pigs is in large part accounted for 
by the lower total zinc concentration in the feces of these 
animals, a reflection of more complete absorption of 
dietary zine or of a less complete apparent absorption of 
other dietary nutrients in the presence of high calcium. 


SWINE STUDIES NOT SHOWING RELATIONSHIP BETWEEN 
CALCIUM, ZINC AND PARAKERATOSIS 


A number of experiments with swine have not demon- 
strated a significant degree of interaction between cal- 
cium and zinc in the development of symptoms of 
parakeratosis. Results of these experiments are summa- 
rized in table 2. One of these was conducted with a 
casein-glucose semipurified basal diet containing 0.6% 
calcium and g ppm zinc (1). In this meticulous experi- 
ment the addition of CaCO; to provide a total of 1.5% 
calcium did not affect weight gain over a 6-week period. 
Pigs started to fail in feed intake during the second week; 
skin lesions appeared rapidly during the third week. On- 
set of symptoms and their eventual severity were not 
affected by calcium level. 

Bellis and Philp (2) employed a barley-fish meal- 
peanut meal ration in their experiment. Calcium fed at 


’ 
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July 1960 


levels from .75 to 1.55% failed to affect weight gains. 
In diets containing 1.55% calcium, increasing phos- 
phorus from 0.7 to 1.30% depressed weight gain and 
decreased the incidence of skin lesions. Calcium levels 
from .95 to 1.32% all were accompanied by about 
50-60 % incidence of mild parakeratosis, whereas only 
32% (13 out of 40) of the pigs fed 0.75% calcium de- 
veloped even a mild parakeratosis. Incidence of skin 
lesions in pigs fed the 1.55% calcium rations was some- 
what higher than in those fed lower levels of calcium, 


TABLE 2. Summary of Growth Data from Swine Feeding 
Experiments in which Calcium-Zinc Interrelationship 
is not Apparent 











| | 
Ration | Ca} F | au Remarks Ref. 
| % | % [ome i : 
———— re wea ae —_—— acim 
Casein-glucose | .6 | .75| 9 | .21 | 6-week period (1) 


7 pigs per group 














| .6 | .73/104 | .67 
Barley-fish meal |1.32) .7 | 20 | .80 | 5-week period (2) 
\1.15) .7 | 20| .77 | 40 pigs per group 
| <95] .7 | 20°) -87 
| .75| -7 | 20) .74 
|1.55| -7 | 20 | .77 | 4-week period 
|1.55|1.0 | 20 | .63 | 34 pigs per group 
1.55|1.3 | 20 | .63 
Corn-oats-bar- 6 | -6 | 33 | .85 | 11-week period | (5) 
ley-soybean -6 | .6 | 50 |1.17 | 10 pigs per group | 
meal-fish meal] .6 | .6 |100 |1.00 
1.0] .6 | 38 | .84 
11.0 .6 | 50 |1.03 
1.0 ‘6 |100 11.10 
1.8 | .6 | 32 | .84 
11.8 | .6 | 50 | .93 
11.8 | .6 |100 | .85 
3-0 | .6 | 33 | -53 
3-0 | .6 | 50 | .67 
13.0 | .6 |100 | .66 | 
Starch-oats- | .4| -46) 34 | -72 | 8 pigs each treat-| (25) 


| : 
soybean meal-) | ment in 4 succes-' 


fish meal | .86) .46) 34 | .72 | sive 4-week peri-| 
| .4| .46|140 | .74 ods | 
| 86) .46140 | .67 | 
Corn-soybean | ry) | | go |1.23 | 16-week period (6) 
[3 | go |1.48 10 pigs per group | 
| 5 | | 30| .89 | 
| .5 | | go |1.46 
| +7 | | 30 | -73 
“a | | 99 1-24 





and some pigs (12 of 34) developed severe lesions on the 
0.7% phosphorus diet. The authors state the opinion 
that the action of calcium is not necessarily a simple 
chemical one, that the answer may be found at the level 
of cell nutrition. 

Heigener et al. (5), in a well-planned experiment, 
found that increasing dietary calcium from 0.60 to 3.02 % 
decreased growth, and that supplements of zinc above 
the 33 ppm basal level increased growth proportionately 
at each calcium level. Their data also show that maxi- 
mum growth at any calcium level was attained with 50 
ppm zinc, and that zinc supplementation alleviated 


UM 


INTERACTIONS OF MINERALS 645 


skin lesions but that 100 ppm was no more effective in 
this respect than 50 ppm. 

The study by Hoefer e¢ a/. (6) shows that on low zinc 
rations, an increase of dietary calcium concentration 
from 0.3 to 0.7% decreases weight gain and increases 
severity of parakeratosis. Sixty ppm Zn added to these 
rations eliminated the skin syndrome and _ increased 
weight gain, but animals still gained less on the highest 
calcium level than on the two lower levels. 

Studies of zinc balance and excretion under different 
conditions of calcium nutriture in swine have been 
conducted in an attempt to reveal a relationship that 
would explain aggravation of symptoms of zinc defi- 
ciency as a result of increasing calcium intake. 

The data of Beardsley (1) show that calcium supple- 
mentation of a very low zinc diet (9 ppm) had no effect 
on zinc absorption, which averaged 68% of the intake. 
The urinary excretion of zinc was increased, however, 
by pigs fed 1.5% calcium, so that the retention of ap- 
parently absorbed zinc amounted to g1 and 76%, 
respectively, for pigs fed low- and high-calcium diets. 
Since rate of growth and tissue concentration of zinc 
were not lowered by calcium supplementation, the 
significance of the greater urinary excretion is not clear. 

The detailed balance study of Whiting and Bezeau 
(25) failed to show that calcium has an effect on absorp- 
tion of zinc or on retention of absorbed zinc. On the 
basal, 34 ppm zinc diet, 21 % of the zinc was apparently 
absorbed, irrespective of calcium level, and retention of 
absorbed zinc equalled 31 and 22% for low- and high- 
calcium diets. Thus, qualitatively the data are in good 
agreement with those of Beardsley (1), although the 
degree of zinc deficiency was much greater in the latter’s 
experiment. 

The following experiment, conducted by M. K. 
Lloyd and M. C. Bell (personal communication), 
demonstrates a beneficial effect of high calcium on 
absorption and retention of zinc from swine diets con- 
taining a moderate excess of this element. Eight York- 
shire barrows averaging 134 lb. were divided into 2 
groups of 4 pigs each and placed on rations containing 
0.5 % and 1.2% calcium. They were fed a ration of corn, 
soybean meal, meat and bonemeal, alfalfa meal and 
mineral and vitamin mix. The trace mineral mix con- 
tained an excess of zinc to supply about 140 ppm of zinc 
in each ration. An average of 29 and 59% of orally 
administered Zn® was retained on the low Ca and high 
Ca rations, respectively. Stable Zn retention on these 
rations was 31 and 45% for low Ca and high Ca, respec- 
tively. Intraperitoneal injection of Zn® gave a much 
higher retention of Zn®, with 93 and 95% retained. It 
was noted that calcium supplementation resulted in 
decreased phosphorus balance and decreased urinary 
phosphate excretion. In the work of Beardsley (1) 
calcium supplementation did not affect phosphorus 
balance, but markedly increased fecal phosphate excre- 
tion while decreasing urinary phosphate output (Forbes, 
unpublished data). 








646 
STUDIES OF CALCIUM-ZINC INTERACTION IN 
POULTRY NUTRITION 


The published experiments concerning the effect of 
calcium on zinc deficiency in poultry present a diversity 
of results, as do those for swine. The first such experi- 
ments are those of Young et al. (26) in which the effect 
on zinc requirement of increasing Ca from 1.2 to 1.8%, 
with 0.6 % phosphorus, was tested in 10-day experiments 
with chicks. The basal diet was a semipurified isolated 
soy protein-glucose diet containing 15 ppm zinc. At 
both calcium levels, 40 ppm-added zinc were required 
for optimum weight gain. Chicks on the higher calcium 
level gained as well as those on the lower, and consumed 
less feed. Results of a similar nature are presented by 
Pensack et al. (19), employing casein-glucose diets with 
6, 20 and 40 ppm zinc and 1.0 and 2.0% calcium in a 
4-week test. Twenty parts per million of zinc in diets 
containing either 1.0 or 2.0% calcium were sufficient for 
maximum weight gain. Zinc analysis of tissues from 
these chicks (Bogdenoff et a/., personal communication) 
showed that in ash of liver, bone and feathers the zinc 
content was decreased by raising dietary calcium, in- 
creased by raising dietary zinc, and increased further 
by both additions. Muscle ash zinc was decreased by 
calcium; zinc had no effect. Whole blood ash zinc was 
increased by calcium and by zinc, but effects were not 
additive. 

Investigations leading to the conclusion that zinc 
utilization by the chick is affected by dietary calcium 
include those of Morrison and Sarett (16), O’Dell e¢ al. 
(18) and Turk et al. (23). The first cited authors em- 
ployed an isolated soy protein-sucrose basal diet con- 
taining 30 ppm zinc and 1.2% calcium. Supplements 
of 0, 5 and 25 ppm zinc were made, and at each zinc 
level, 0.5 and 1.0% calcium were added. Weight gain 
after 14 days on experiment indicated that the depressing 
effect of each level of calcium supplementation was 
eliminated by the supplement of 25 ppm zinc. O’Dell et 
al., using a washed soy protein-glucose basal diet con- 
taining 1.1 % calcium, found that raising calcium to 1.6 
depressed growth and doubled incidence of hyperkera- 
tosis, and that a further increase to 2.1% calcium was 
no more detrimental. Addition of 63 ppm zinc more 
than doubled the weight gain above that obtained on the 
basal diet and eliminated incidence of hyperkeratosis. No 
effect of calcium was observed on any of the zinc- 
supplemented diets. 

Turk et al. (23) reported that chicks hatched from 
hens fed a g-ppm Zn soybean meal diet were zinc- 
deficient. The deficiency was aggravated by raising 
calcium content of the hens’ ration from 2.23 to 4.0%, 
and hatchability of eggs was decreased. Addition of 50 
ppm zinc eliminated signs of zinc deficiency. 


EFFECT OF CALCIUM ON ZINC REQUIREMENT AND 
EXCRETION OF THE RAT 


Forbes and Yohe (4), employing casein-glucose and 
isolated soy protein-glucose diets fed to weanling male 


FEDERATION PROCEEDINGS 


Volume 19 


albino rats, have investigated the effects of calcium 
supplementation on weight gain and on zinc balance. 
In their first experiment 24 rats were fed for 6 weeks 0.8 
and 1.8% calcium at zinc levels of 7, 18, 31 and 52 ppm 
in casein-glucose diets. Feed intake was equalized except 
for the animals on the basal level of zinc. Collection of 
urine and feces was made during the second and sixth 
weeks. Eighteen ppm of zinc were adequate for maxi- 
mum weight gain at both calcium levels. At each zinc 
level weight gain was less by animals receiving 1.6% 
than by those receiving .8% calcium. Although the 
difference between gains due to calcium was greatest on 
the 7 ppm zinc level, statistical analysis did not show a 
significant interaction between calcium and zinc. Bal- 
ance data showed no influence of calcium on zinc 
excretion. Of the total zinc intake, 6% appeared in 


TABLE 3. Effect of Dietary Calcium on Tissue 
Zinc Concentration 





% change in tissue zinc 





Tissue conc. accompanying References 
higher dietary calcium 

Pig kidney +11 (1) (17) 
Pig kidney cortex —12 | (7) (10) 
Pig liver —17 | or). ry) 
Pig bone —19 | (1) (10) 
Pig blood —10 | (1) (10) (17) 
Pig hair — 33 | (10) 
Rat liver oO | (4) 
Rat carcass o | (4) 
Chick liver —15*, +18f | Bogdonoff 
Chick muscle —41, —38 | (personal com- 
Chick blood +22, —10 | munication) 
Chick feathers — 36, +44 
Chick bone —25, +13 | 





* Data from animals fed 6 ppm Zn. 
+ Data from animals fed 40 ppm Zn. 


urine, and 43% appeared in feces. The total zinc reten- 
tion was, thus, 51 % as an average of all levels of zinc 
fed. In view of finding a higher zinc requirement on the 
part of rats fed a soy protein diet, and the fact that 
investigators in swine nutrition had observed a detri- 
mental effect of calcium on parakeratosis, a further 
experiment was conducted using isolated soybean pro- 
tein-glucose diets. The results of the second experiment 
were qualitatively similar to those of the first. Calcium 
depressed growth at all levels of zinc. The percentage of 
zinc consumed appearing in the feces amounted to 54%, 
there being no consistent effect of calcium on fecal zinc. 


DISCUSSION 


As one reviews the literature dealing with the effect 
of calcium on zinc utilization, several things become 
apparent. First, one must conclude in the face of the 
diverse evidence that under some, but not all, condi- 
tions there does exist a relationship between calcium 
intake and the status of zinc nutriture of swine and of 
poultry. Delineation of these conditions is requisite to an 








ume 19 


lcium 
lance. 
ks 0.8 
ppm 
xcept 
ion of 
sixth 
maxi- 
1 zinc 
1.6% 
h the 
est on 
10W a 

Bal- 

zinc 
ed in 


| com- 
on) 


reten- 
yf zinc 
on the 
t that 
detri- 
urther 
nN pro- 
riment 
alcium 
rage of 
54%, 
1 zinc. 


effect 
ecome 
of the 
condi- 
alcium 
and of 
> to an 





July 1960 


understanding of the relationship. Second, although it is 
apparent that, in the presence of suboptimum zinc 
nutriture, increasing the calcium intake may magnify 
the skin lesions and growth depression, it has not been 
demonstrated that the requirement for zinc has been 
altered. Third, of the various proposals that have been 
made in attempts to explain the apparent decrease in 
zinc utilization accompanying an increase in calcium 
intake, none of them that have been tested have pro- 
vided the answer. Fourth, although the practical solu- 
tion to the problem of parakeratosis is obvious, there 
remain intriguing problems concerned with zinc metabo- 
lism as affected by calcium/zinc ratios and by the rela- 
tionship of other dietary and environmental factors to 
the zinc supply. 

In attempting to explain the observed effect of calcium 
on zinc utilization it seems logical to consider the possible 
sites at which calcium may interfere. For example, is it 
possible that increasing calcium concentrations in the 
diet may promote a chemical reaction in the diet that 
would render the zinc less absorbable? Or, is the effect 
of calcium to produce conditions in the intestinal tract 
which are less favorable for zinc absorption? While 
neither of these hypotheses has been examined separately, 
and this would be difficult, those studies which have 
been made of zinc absorption have not revealed a 
depression in apparent or true absorption of zinc as a 
consequence of increasing the calcium level of the diet. 
Two studies (9, 20) of the effect of calcium on zinc 
solubility in aqueous solution have been conducted. 
These do show calcium to decrease zinc solubility, but 
the conditions employed have little in common with 
those present in the intestinal tract. Zinc excretion via 
the kidney has, in the two balance studies with swine, 
been shown to be increased in animals receiving high 
calcium diets. The amounts of zinc excreted in this man- 


INTERACTIONS OF MINERALS 647 


ner are small but may be sufficient to account for the 
small trend toward lower levels of zinc in tissues of 
animals fed high calcium diets, as shown in table 3. 

The results of tissue analysis for*zinc as affected by 
increased concentration of dietary calcium vary be- 
tween investigators, between species and among tissues. 
This may be accounted for either by variation between 
animals, between other dietary or environmental factors, 
or in the zinc analysis itself. A summary of available 
data on tissues showing the greatest differences is shown 
in table 3. In general, the differences observed in zinc 
content of these tissues were shown by the investigators 
cited to be statistically significant. Other tissues analyzed 
by these investigators have revealed little or no change 
in zinc concentration due to change of calcium intake. 
Recently Miller e¢ al. (15) have reported nonsignificant 
changes in zinc concentration in swine liver and kidney 
as a result of increasing dietary calcium from 0.6 to 
1.25%. It thus appears that the result of tissue analysis 
for zinc do not account for the increased severity of 
deficiency symptoms occasioned by increased dietary 
calcium concentration. 


CONCLUSIONS 


It seems most probable, on the basis of present evi- 
dence, that interference in zinc function by calcium 
occurs at the cellular level. Although a variety of 
enzymes are known to contain zinc, and other reactions 
are catalyzed by zinc, these activities do not appear to be 
depressed in tissues of zinc-deficient animals, and further- 
more, the skin lesions of zinc deficiency cannot yet be 
explained on the basis of known enzymic or catalytic 
functions of zinc. Until we can describe the biochemical 
lesion(s) of zinc deficiency we will not be able to explain 
the effect of calcium on the visible syndrome of this 
deficiency. 


REFERENCES 


1. BEARDSLEY, D. W. Ph.D. Thesis. Urbana, IIl.: University of 
Illinois, 1958. 

2. Betis, D. B. anp J. McL. Puiwp. J. Sci. Food Agr. 8: 5119, 
1957- 

3. Conrap, J. H. anp W. M. Bergson. J. Animal Sci. 16: 580, 
1957- 

. Forses, R. M. ano M. Youe. J. Nutrition 70: 53, 1960. 

5. Hercener, H., A. Micuna, H. J. Neumann, H. BEHRENS AND 
K. Hamann. Landwirtsch Forsch. 11: 185, 1958. 

6. Hoerer, J. A., R. W. Leucke, E. R. Mitrer, D. E. ULLREy, 
A. M. Pearson AND H. R. Ritcure. J. Animal Sci. 18: 1503, 
1959. 

7. Hoexstra, W. G., P. K. Lewis, Jr., P. H. Pumtuips ano R. H. 
Grummer. J. Animal Sci. 15: 752, 1956. 

8. Hove, E., C. A. EL-venyem anp E. B. Hart. Am. J. Physiol. 
119: 768, 1937. 

g- Lewis, P. K., Jr., Ph.D. Thesis. Madison, Wis.: University of 

Wisconsin, 1958. 
. Lewis, P. K., JR., W. G. Hoekstra anp R. H. Grummer. J. 
Animal Sci. 16: 578, 1957- 

11. Lewis, P. K., Jr., W. G. Hoekstra, R. H. GRUMMER AND P. 
H. Puiturps. J. Animal Sci. 15: 741, 1956. 

12. Leuckxe, R. W., J. A. Hoerer, W. S. BRAMMELL AND D. A. 
Scumipt. J. Animal Sci. 16: 3, 1957. 

13. Leucke, R. W., J. A. Hoerer, W. S. BRAMMELL AND F. 
Tuorp, Jr. J. Animal Sci. 15: 347, 1956. 


5 


XUM 


14. Leucke, R. W., D. A. Scumipt anp J. A. HoeFer. J. Animal 
Sez. 17: 1185, 1958. 

15. Miter, R. F., N. O. Price, J. H. CARTER AND C. C. Brooks. 
J. Animal Sci. 18: 1460, 1959. 

16. Morrison, A. B. ann A. P. Sarett. J. Nutrition 65: 267, 1958. 

17. NEwtanp, H. W., D. E. Utirey, J. A. HoEFER AND R. W. 
Luecke. J. Animal Sci. 17: 886, 1958. 

18. O’Dett, B. L., P. M. NEWBERNE AND J. E. Savace. J. Nutri- 
tion 65: 503, 1958. 

19. Pensack, J. M., J. M. HENson anv P. D. Bocponorr. Poultry 
Sct. 37: 1252, 1958. 

20. Roperson, R. H., P. J. ScutaBLe AND K. E. Jonnson. Poultry 
Sct. 36: 1153, 1957- 

21. STEVENSON, J. W. anv I. P. Earze. J. Animal Sci. 15: 1036, 
1956. 

22. Tucker, H. F. anp W. D. Satmon. Proc. Soc. Exper. Biol. © 
Med. 88: 613, 1955. 

23. Turk, D. E., M. L. Sunpe anp W. G. Hoekstra. Poultry Sci. 
38: 1256, 1959. 

24. VALLEE, B. L. Physiol. Rev. 39: 443, 1959. 

25. Wuitinc, F. ano L. M. Bezeau. Canadian J. Animal Sei. 38: 
109, 1958. 

26. Younc, R. J., H. M. Epwarps, JR. AnD M. B. Guus. Poultry 
Sc?. 37: 1100, 1958. 








Magnesium requirement and its relation to 


other dietary constituents 


BOYD L. O’DELL 


Department of Agricultural Chemistry, University of Missouri, Columbia, Missouri 


—_ TELEOLOGICAL CONSIDERATIONS magnesium had 
been deemed a dietary essential long before Leroy (39) 
demonstrated the fact with the mouse. Since that time a 
voluminous literature had developed on the significance 
of dietary magnesium. The scope of the present discussion 
is limited largely to the interrelationships of magnesium 
and other diet components, but an attempt has been 
made to summarize recent literature that bears on the 
quantitative requirements of various species for magne- 
sium. For purposes of orientation it seemed worthwhile 
to include a brief discussion of the gross pathology of 
magnesium deficiency. 


PATHOLOGY OF MAGNESIUM DEFICIENCY 


The classical symptoms of magnesium deficiency in 
the rat were first described by Kruse et al. (34). 
Inasmuch as the early observations on the pathology of 
magnesium deficiency, including the changes in chemical 
composition of tissues, have been adequately reviewed 
(12) they will not be repeated here. Although not all 
symptoms appear in any one species, the gross symptoms 
of magnesium deficiency include peripheral vasodila- 
tion, skin lesions, hyperirritability, muscle dysfunction, 
calcification of soft tissues, defective bones and teeth and 
retarded growth. 

The various species show many similarities in the 
nature of the pathology exhibited, but there are marked 
differences. Whereas the rat develops a marked hypere- 
mia within three to five days on a deficient diet, this 
symptom has not been observed in any other species 
studied including the rabbit (36), the pig (43), the calf 
(5) and the guinea pig (51). Moreover, skin lesions, 
which usually appear in the rat after vasodilation sub- 
sides, seldom if ever occur in other species. 

Hyperirritability is one of the most common character- 
istics of magnesium deficiency. It has been observed in 
rabbits (36), pigs (43), calves (5), chicks (1, 3) and 
ducks (68). In these species the initial phase of irritability 
is followed by convulsions and usually death. Hyperir- 
ritability is also the diagnostic symptom of the hypomag- 
nesemic disease of cattle and sheep commonly known as 
grass tetany. Although this disease is not the result of a 


1 Original work supported in part by National Science Founda- 
tion Grant No. G—3025 


648 


simple dietary deficiency of magnesium, it is mentioned 
here because the symptoms are entirely analogous to 
those described in animals severely depleted of magne- 
sium (61). In contrast to the other species the guinea pig 
is quite resistant to nervous symptoms and seldom if ever 
dies in a convulsive state. In our experience a few animals 
have been affected with tremors and convulsions but 
this has been rare indeed (51). 

While there is little evidence of gross muscle dysfunc- 
tion in the magnesium-deficient rat, other species show 
various degrees of muscle incoordination and stiffness. 
The pig develops weak pasterns, sickled hocks and a 
concave-bowed back (43). Chicks and ducklings develop 
decreased muscle tone and a tendency to squat (1, 3, 68). 
Calves fed milk diets low in magnesium become weak in 
the pasterns and manifest a peculiar gait (64). Guinea 
pigs become stiff in the hind limbs and assume an ab- 
normal stance (51). This symptom appears to be similar 
to that first described in guinea pigs fed a diet of milk 
and barley (73) but is distinct from the wrist stiffness 
syndrome which was later studied in the same laboratory 
(69). In our experience the stiffness of the hind limbs 
that occurs on a low magnesium diet is cured by feeding 
magnesium salts, but wrist stiffness is not. 

Soft tissue calcification occurs at least to some extent in 
several species but it has not been reported in the dog, 
the pig or in poultry. Although calcareous deposits in the 
kidneys of the rats are readily detected histologically, 
there are no reports of grossly visible deposits. A true 
calcinosis involving the diaphragm, endocardium and 
intima of blood vessels was observed in milk-fed calves 
(49), but this has not been confirmed in calves fed an 
artificial milk diet (5). The latter diet contained only 0.5 
mg of magnesium per 100 ml while milk contains ap- 
proximately twenty times this amount. It is now recog- 
nized that milk as the sole source of food for the calf is 
deficient in vitamins D, E and possibly A as well as iron, 
copper and manganese (64) and these deficiencies no 
doubt contributed to the lesions observed in the early 
studies. On the other hand the widely different concen- 
trations of magnesium in the two diets may have had a 
major influence on the type of pathology observed. In 
the case of severe magnesium deficiency death from 
tetany may ensue before calcinosis has an opportunity to 


XUM 








tioned 
ous to 
nagne- 
ea pig 
if ever 
nimals 
ns but 


ysfunc- 
; show 
iffness. 
and a 
evelop 
3, 68). 
reak in 
yuinea 
an ab- 
similar 
yf milk 
tiffness 
ratory 
limbs 
eeding 


tent in 
e dog, 
; in the 
tically, 
A true 
n and 
calves 
fed an 
nly 0.5 
ns ap- 
recog- 
calf is 
1S iron, 
cies no 
> early 
oncen- 
had a 
‘ed. In 
1 from 
nity to 





July 1960 


develop. The guinea pig is particularly prone to meta- 
static calcification and readily develops grossly visible 
deposits of calcium phosphate (31, 32, 42, 51). A similar 
calcinosis syndrome occurs in the cotton rat maintained 
on a low magnesium diet (10). 

Rats maintained for long periods on diets subnormal 
in magnesium develop fragile bones and defective teeth. 
Guinea pigs frequently show exostoses on the mandibles 
and the incisors become eroded, dark in color, softened 
and fragile (51). The molars are not affected in the same 
manner but commonly become overgrown and coated 
with a dark-pigmented calculus. 

The symptoms of magnesium deficiency in man are 
not known with certainty because an uncomplicated 
deficiency has not been observed. Patients frequently 
exhibit neuromuscular disorders that resemble those 
seen in deficient animals and most clinical studies have 
been undertaken because of this analogy. A clinical 
syndrome, characterized by tremors, muscle twitching, 
delirium, and occasionally by convulsions, has been 
considered to be a manifestation of magnesium de- 
ficiency (18, 19, 63). The evidence for this concept 
included low concentrations of serum magnesium, re- 
sponse to therapy with magnesium and a _ positive 
magnesium balance during treatment. Many of these 
patients were chronic alcoholics and the results may have 
been complicated by liver cirrhosis and other clinical 
factors (72). No clinical symptoms resulted when two 
normal men were maintained for 24 days on a diet that 
supplied about 13 mg. of magnesium per day (17). The 
total loss of magnesium from the body was 25.8 and 71.6 
mEq, respectively, but loss in the urine decreased sharply 
demonstrating the ability of man toconserve magnesium 
effectively. 


INTERRELATIONSHIPS OF MAGNESIUM AND OTHER 
DIETARY COMPONENTS 


Calcium. The pharmacological action of magnesium as 
a depressant of the neuromuscular system has been 
recognized for nearly a century. It is the only known 
metallic ion that will produce anesthesia (15). Moderate 
doses of magnesium salts which will cause general anes- 
thesia and loss of reflexes are counteracted immediately 
by a comparatively small quantity of calcium ion (45). 
The mechanism whereby magnesium exerts its pharma- 
cological action is not well known, but the fact that 
calcium counteracts the effect demonstrates clearly the 
physiological antagonism between the two ions. 

The idea of physiological antagonism between cal- 
cium and magnesium in the area of nutrition evolved 
from work with plants (40). This concept was given 
further support by the work of Mendel and Benedict 
(46) who showed that injection of soluble magnesium 
salts into dogs, cats and rabbits increased the concentra- 
tion of urinary calcium. Recently it has been shown 
that subcutaneous administration of magnesium sulfate 
caused a rapid increase in the rate of urinary excretion 
of radiocalcium regardless of whether the isotope was 


INTERACTIONS OF MINERALS 649 


given two or 60 days prior to treatment (8). The action of 
magnesium ion was similar to that of citrate ion and 
appeared to have a direct osteolytic effect on bone. Thus 
the accumulated evidence leaves little doubt that injec- 
tion of magnesium salts results in loss of calcium from the 
body. 

The effect of magnesium ingestion on calcium reten- 
tion is less clear. Early studies showed that doubling the 
magnesium intake of dogs increased urinary calcium 
slightly and resulted in a more negative calcium balance 
(41). When the magnesium intake of swine was doubled, 
there was also a marked increase in urinary calcium 
and a negative calcium balance resulted (25). The addi- 
tion of K,HPQO, largely counteracted the effect of 
magnesium and this was attributed to the formation of 
insoluble Mg;(PO,)2 in the intestine. In cattle main- 
tained on a_phosphorus-deficient ration the daily 
ingestion of magnesium sulfate caused a marked lowering 
of the calcium balance (53). As in the case of swine, sol- 
uble phosphate overcame the detrimental effect and 
Epsom salt fed along with Ca3(PO,)2 or bonemeal for as 
long as a year exerted no ill effect. Other investigators 
observed that the addition of 3-5 % of MgCQ; to a low- 
calcium, high-phosphorus ration produced definite im- 
provement in the condition and calcium balance of 
calves (33). In no case did the addition of magnesium 
salts have a detrimental effect on the general health or on 
calcium and phosphorus retention in cows or calves. 

High levels of dietary magnesium depressed the growth 
rate of rats fed diets low in phosphorus regardless of the 
calcium level and hindered calcium retention when both 
calcium and phosphorus were low (24). On the other 
hand there was little detrimental effect from a high mag- 
nesium intake when either the calcium or the phosphorus 
intake was high and still less when both were high. In 
contrast to these results no detrimental effect on calcium 
assimilation was observed in another laboratory when 
1~2 % of MgCOQ; was added to an exceptionally low phos- 
phorus diet regardless of the calcium content (14). Pro- 
longed administration of large doses of magnesium lac- 
tate or gluconate to dogs was not accompanied by loss of 
calcium when the phosphate intake was adequate (2). 

For the most part, studies of calcium metabolism in 
man show only slight and variable effects from ingestion 
of relatively high levels of magnesium (6, 22, 67). Tib- 
betts and Aub (65) observed that ingestion of large 
amounts of magnesium increased urinary excretion of 
calcium, but elevating the phosphorus intake counter- 
acted the effect of magnesium and brought the calcium 
into balance. In another study daily intakes of more than 
1 gm of magnesium by adults actually improved the cal- 
cium balance provided the phosphorus intake was ade- 
quate (7). 

It is evident that under some circumstances the inges- 
tion of excessive quantities of magnesium leads to loss of 
calcium from the body. However, this effect appears to 
be of little or no significance if the intakes of calctum and 
particularly of phosphorus are adequate. The effect of 
soluble phosphate in counteracting the detrimental ac- 








650 FEDERATION PROCEEDINGS 


tion of magnesium has frequently been attributed to the 
formation of insoluble magnesium phosphates, but this 
concept was questioned by Haag and Palmer (24) who 
observed that magnesium was not precipitated by phos- 
phate at pH 6.0. It is conceivable that both calcium and 
phosphate ion are metabolic antagonists of magnesium. 

The influence of calcium on magnesium metabolism 
has been studied less extensively than the converse. 
While there is no doubt that the injection of calcium salts 
gives rise to excess magnesium in the urine (47) the effect 
of calcium ingestion on magnesium retention is less clear. 
There was no effect on magnesium retention in dogs 
when calcium chloride was fed (41) but a decreased mag- 
nesium balance resulted when rats were supplied with 
water containing 1.5% of CaCl, (30). 

Tufts and Greenberg (66) were the first to report that 
a high content of calcium in the diet accentuates the se- 
verity of magnesium deficiency in the rat. Unfortunately, 
the extra calcium was supplied as the phosphate so that 
the phosphorus content of the diet was elevated along 
with the calcium and it is not clear what influence the 
increased phosphate may have had on the results. Never- 
theless, it was found that 5 mg of magnesium per 100 gm 
of the basal diet, which contained 0.40 “c of calcium and 
0.45 % of phosphorus, supported a rate growth equal to 
that of the controls. However, this level did not prevent 
vasodilation and hyperirritability. When the diet con- 
tained 1.66% of calcium and 1.00% of phosphorus, 13 
mg % of magnesium was barely able to support growth 
equivalent to that of the controls. It was concluded that 
a diet high in calcium not only increased the incidence 
and severity of convulsive attacks and hyperemia in mag- 
nesium-deficient animals but also increased the minimum 
requirement for growth. More recent work (9, 29) with 
the rat has confirmed the observation that a high level of 
dietary calcium hastens the onset of magnesium-defi- 
ciency symptoms, increases mortality and depresses the 
rate of growth when the magnesium intake is low. 

The question whether or not high calcium consump- 
tion increases the minimum requirement for growth has 
not been answered definitively. From their studies on the 
rat Hegsted et al. (29) concluded that when the magne- 
sium intake was near the minimum requirement the ef- 
fect of high calcium intake was too small to be detected. 
Recently, the effects of dietary magnesium and calcium 
levels on growth, serum cholesterol, vascular sudano- 
philia and kidney tubule calcification have been studied 
in the same laboratory (70). The basal diet contained 24 
mg % of magnesium and in some cases 1 “c of cholesterol 
and 0.3 % of cholic acid. Extra magnesium improved the 
growth rate, and in general decreased lipid deposition in 
the heart when cholesterol and cholic acid were fed. The 
antagonistic effects of calcium were less clear. The higher 
levels of dietary calcium usually depressed growth and 
intensified kidney damage slightly on the low magnesium 
diets but had no effect on lipid deposition in the vascular 
system. 

In studies on the interrelationships of calcium, phos- 
phorus and magnesium in the guinea pig, it was observed 


Volume 19 


that keeping either calcium or phosphorus relatively high 
had less effect in accentuating magnesium deficiency than 
keeping both high (42). Data collected in our laboratory 
(51) and summarized in table 1 show that high calcium 
intake has a deleterious effect in the magnesium-deficient 
guinea pig causing depressed growth and high mortality. 

Phosphorus The interrelationships of magnesium and 
phosphorus have been alluded to in the discussion of cal- 
cium and magnesium antagonism. A high intake of phos- 
phorus prevents the loss of calcium that is caused by 
excessive magnesium, but the mechanism of this effect is 
not entirely clear. 

The work of Selye and co-workers shows that excess 
phosphate will induce symptoms suggestive of magne- 
sium deficiency when animals are placed under various 


TABLE 1. Dietary Calcium and Phosphorus Levels and 
Magnesium Deficiency in Guinea Pigs* 


No Magnesium Added 0.3% Magnesium Added 





Dietary P |— ne eed eee Sees ee oe i li aco ae 
s 
” Daily Gain Mortality Daily Gain | Mortality 
gm q gm % 
— - a a | —— 
| i 
A. 0.9% calcium diets 
0.4 3.0 21 5-7 18 
0.8 0.8 50 6.0 oO 
[7 100 3.7 oO 
B. 1.7% calcium diets 
0.4 3:0 38 6.0 oO 
0.8 0.2 83 5.8 oO 
Cor | 100 4-7 o 
C. 2.5% calcium diets 
0.4 1.9 83 5. o 
0.8 100 4.8 oO 
1:7 100 5-0 oO 





* Eight-week experimental period. 


stress situations, but the results are complicated by the 
fact that potassium as well as magnesium has an amelior- 
ative effect. In one study (58) rats were fed a diet low in 
both potassium and magnesium for a period of 13 days 
and during the last 6 days they received various salts by 
stomach tube. The administration of monosodium phos- 
phate produced cardiac necrosis, nephrocalcinosis and 
muscular cramps, all of which were prevented by the 
simultaneous treatment with either MgCl. or KCl. In 
another series of experiments (56) rats maintained on a 
laboratory chow were given dihydrotachysterol and 
monosodium phosphate by stomach tube. A widespread 
calcification occurred in the heart, aorta and kidney, and 
edema and inflammation appeared around the arteries 
in the heart. The cardiac and aortic lesions were com- 
pletely prevented by administration of MgCl, and largely 
prevented by KCl. Calcification in the kidney was sig- 
nificantly inhibited by magnesium but only slightly by 
potassium. Similar results were obtained when parathy- 
roid hormone and phosphate were given (57). It is sig- 
nificant that potassium and magnesium afforded protec- 
tion when administered subcutaneously so that they 





XUM 





ume 19 


y high 
7 than 
‘atory 
Icium 
icient 
tality. 
1 and 
of cal- 
phos- 
ed by 
fect is 


EXCESS 
agne- 
arious 





by the 
nelior- 
low in 
3 days 
alts by 
_ phos- 
is and 
oy the 
Cl. In 
J ona 
1 and 
spread 
y, and 
rteries 
> com- 
largely 
as sig- 
tly by 
rathy- 
is sig- 
orotec- 
t they 





July 1960 


could not interfere with absorption. The suggestion was 
made that perhaps phosphate sensitizes and magnesium 
and potassium desensitize the body to the deleterious 
action of parathyroid hormone. 

Hogan et al. (31) observed a calcinosis syndrome in 
guinea pigs fed a purified diet that contained 0.8% of 
calcium, 0.9% of phosphorus and the quantities of the 
other minerals that were commonly supplied in purified 
diets at that time. The incidence of gross lesions and mor- 
tality was markedly decreased and growth rate increased 
when the phosphorus content of the diet was lowered to 
0.5%. Later it was shown (32) that the symptoms were 
more severe when the diet contained 1.7 % of phosphorus 
and that addition of extra potassium and magnesium 
largely alleviated the symptoms of the deficiency syn- 
drome. Magnesium appeared to be the critical nutrient 


INTERACTIONS OF MINERALS 651 


the amount of magnesium required to support the maxi- 
mum rate of gain in guinea pigs (51). This is illustrated 
by the results presented in figure 1. When the diet con- 
tained 0.4% of phosphorus, the minimum requirement 
was about 80 mg, whereas when it contained 1.7% of 
phosphorus the requirement was about 240 mg per 100 
gm of diet. 

By what mechanism does high phosphate consumption 
increase the magnesium requirement? The ingestion of 
excess phosphate by hospital patients had relatively little 
effect on magnesium absorption and no effect on mag- 
nesium balance (65). Balance studies in the rat indicated 
that the level of inorganic phosphorus did not affect mag- 
nesium absorption (44), but it should be pointed out that 
the levels of phosphorus tested were relatively low On 
the other hand balance studies in the guinea pig suggest 





T T T T T 


6.0F © - 0.4% Phosphorus 
© - 1.7% Phosphorus 
D - Deposits 





M - Mortality 





wn 
S 
— 


= 
t=) 
T 


Daily Gain, gm. (12 week period) 





FIG. 1. Effect of dietary phos- 
phorus level of magnesium re- 
quirement of guinea pigs. Twelve 
animals were started on each 
diet and the mortality (M) and 
incidence of visible deposits (D) 
at autopsy are indicated. All 
animals represented by the upper 
curve received 0.4% of phos- 
phorus and those by the lower 
curve, 1.7% of phosphorus. 








1.0F 
M - 42% 
D - 58% 
! 4 re 1 1 n ps 
5 10 20 35 60 120 150180 240 300 360 


Dietary Magnesium, mg. per 100 gm. (log scale) 


in this syndrome because soft tissue calcification occurred 
when the magnesium intake was suboptimal even though 
the potassium level was high (42). The damaging effect 
of high levels of dietary phosphorus on guinea pigs that 
consumed a diet severely deficient in magnesium is shown 
in table 1 (51). In the absence of supplementary mag- 
nesium the detrimental effect of phosphorus was in direct 
proportion to its concentration regardless of the calcium 
level. Thus, consumption of excess phosphorus as well as 
of excess calcium accentuated the symptoms of mag- 
nesium deficiency and, by most criteria, high phosphorus 
was more damaging than high calcium. Furthermore, 
their effects were additive and adjustment of the ratio 
of calcium to phosphorus was of no benefit when the 
intakes were high. Excessive dietary phosphate accen- 
tuated the symptoms of magnesium deficiency in the rat 
as well as in the guinea pig (51). 

Not only does excessive phosphate aggravate the symp- 
toms of magnesium deficiency such as soft tissue calcifi- 
cation, mortality and poor growth but it also increases 


that magnesium absorption is impaired by excessive phos- 
phate consumption (50). Table 2 shows the apparent ab- 
sorption and the retention of magnesium at two phos- 
phorus and two magnesium levels. The potassium level 
was varied along with the magnesium level, but presum- 
ably this is of no consequence for the purpose of com- 
paring the two phosphorus levels. Regardless of the mag- 
nesium intake animals that consumed the diets containing 
1.7% of phosphorus retained less magnesium than 
those that received 0.4% of phosphorus. This effect on 
retention can be explained, at least in part, by the de- 
creased magnesium absorption in animals on the high 
phosphorus diets. These observations strongly suggest 
that excess phosphate in the diet decreases absorption of 
magnesium, but the possibility should not be overlooked 
that phosphorus has a specific metabolic effect which in- 
creases the requirement for magnesium.. In this connec- 
tion it should be pointed out that many of the enzymatic 
reactions which require magnesium as a cofactor are con- 
cerned with phosphorus metabolism. 








652 FEDERATION PROCEEDINGS 


Other Components. Because of the high potassium con- 
tent of spring grass and wheat pasture on which cattle 
frequently develop hypomagnesemia and tetany, the ef- 
fect of dietary potassium level on the magnesium require- 
ment has been investigated. In one study the addition of 
5% of potassium to a purified diet that contained 0.05 % 
of magnesium decreased the rate of gain in rats, but it 
had no effect on the magnesium content of the blood. 
Furthermore, additional magnesium did not overcome 
the growth depression (54). In another investigation 
2.9% of potassium hastened the onset of magnesium- 
deficiency symptoms and depressed the growth rate of 
rats (g), but again there was no evidence that extra mag- 
nesium overcame the growth depression. Excess potas- 
sium consumption caused a slight reduction in the serum 
magnesium of sheep but the effect was not reversed by 
feeding magnesium oxide (35). Other investigators have 
observed no effect on serum magnesium and it may be 
concluded that excess potassium has no specific effect on 
the magnesium requirement (4). 


TABLE 2. Effect of Dietary Phosphorus Level on Magnesium 
Absorption and Retention in Guinea Pigs 


Dietary 0.05% Mg 0.35% Mg 
Phosphorus, % 0.4% K 1.5% K 
A. Apparent absorption, % 
0.4 31 (6)* 88 (8) 
7 —59 (6) 66 (7) 
B. Retention, mg/kg of B.W./day 
0.4 =1064 +7.0 
1.7 —34-9 a | 


* Number of animals. 


Evidence has accumulated which incriminates exces- 
sive ammonia formation in the rumen as a causative fac- 
tor in hypomagnesemia of ruminants. There is a rise in 
the ammonia content of rumen liquor when cattle are 
transferred from stall to grass feeding and the serum and 
urinary magnesium levels drop markedly. The introduc- 
tion of ammonium salts directly into the rumen reduced 
urinary magnesium and led to the hypothesis that hypo- 
magnesemia is the result of poor absorption associated 
with high ruminal ammonia (26). However, the serum 
magnesium was not changed in these animals so that 
more work is needed to establish ruminal ammonia as the 
primary causative agent. 

It has been known for many years that the source of 
carbohydrate in the diet affects the absorption of mag- 
nesium as well as of calcium. The replacement of 25% 
of starch by lactose in the diet of rats caused greater ex- 
cretion of magnesium in the urine and less in the feces 
with the net result of greater retention (52). Rats that 
ingested 60 % of galactose also excreted more magnesium 
in the urine than pair-fed animals that consumed glucose 
(27). By use of radioactive magnesium it has been shown 
that the site of action of lactose is in the intestines and not 
within the tissues (38). Although there may be no rela- 
tionship to the effect of carbohydrates, it is worthy of 
note that magnesium absorption was also enhanced by 


Volume 19 


the antibiotics, chloramphenicol and neomycin (28). In 
addition to the similarities in the absorption of calcium 
and magnesium, there are striking differences. For ex- 
ample vitamin D affects magnesium absorption only 
slightly (44). 

It is generally agreed that the magnesium requirement 
of the rat is met by about 20 mg per 100 gm of a purified 
diet. Such diets usually contain about 20% of casein and 
5% of fat. However, when rats consumed a diet that con- 
tained 10% of casein, 20% of fat, 1% of cholesterol, 
0.3% of cholic acid and 24 mg % of magnesium, they 
developed typical symptoms of magnesium deficiency, 
and the lesions were prevented by feeding four to eight 
times as much magnesium (70). In addition to the usual 
symptoms of deficiency this atherogenic diet produced 
lipid deposition in the aorta and heart valves. Signifi- 
cantly, a higher level of magnesium reduced the degree 
of lipid deposition, but had no effect on serum choleste- 
rol. Although this diet required a higher concentration of 
magnesium than normal to prevent deficiency symptoms, 
the cause cannot be attributed with certainty to choles- 
terol or any other specific dietary constituent. The nature 
of the diet, its relatively high caloric density and its low 
protein content, would tend to decrease the amount of 
food consumed and thus the daily intake of critical nu- 
trients. Nevertheless, it is significant that this combina- 
tion of nutrients increased the amount of magnesium re 
quired in the diet. 

Rats fed the atherogenic diet described above and 
given 20% alcohol as the only fluid required more mag- 
nesium in the diet than those that received water in place 
of alcohol (23). It might be said that alcohol increased 
the magnesium requirement and in one sense it did. The 
fact that alcohol supplied calories and decreased food 
consumption may well explain the need for higher levels 
of magnesium in the diet. There is also evidence to sug- 
gest a disturbance of magnesium metabolism in patients 
suffering from chronic alcoholism as indicated by an 
altered serum concentration in some patients (19). How- 
ever, Wacker and Vallee (72) reviewed the literature 
that deals with magnesium deficiency and alcoholism 
and concluded that present data do not allow a decisive 
conclusion as to any causal relationship between alco- 
holism and magnesium deficiency in man. 


QUANTITATIVE REQUIREMENTS 


The analytical methods available in the past for the 
quantitative determination of magnesium in biological 
materials have been inadequate. Consequently errors in 
estimation of the magnesium content of basal diets and 
supplements have no doubt contributed to discrepancies 
in reported requirements as have the different criteria of 
adequacy and experimental conditions employed. In any 
estimate of magnesium requirement, the concentrations 
of other dietary constituents must be considered. Particu- 
larly important in this regard are the concentrations of 
calcium and phosphorus. A summary of reported require- 
ments of various immature laboratory and domesticated 








ime 19 


). In 
cium 
Tr ex- 

only 


ment 
rified 
1 and 
t con- 
terol, 
they 
ency, 
eight 
usual 
duced 
gnifi- 
legree 
yleste- 
ion of 
toms, 
holes- 
1ature 
ts low 
unt of 
al nu- 
ybina- 
1m re 


e and 
- mag- 
| place 
reased 
1. The 
1 food 
levels 
oO sug- 
atients 
by an 
How- 
rature 
holism 
ecisive 
1 alco- 





for the 
logical 
rors in 
ts and 
pancies 
teria of 
In any 
rations 
articu- 
ions of 
equire- 
ticated 


YLIM 


July 1960 


animals is presented in table 3. When the diets contained 
the levels of calcium and phosphorus commonly used, 
0.8-1.0% and 0.5-0.7%, respectively, the approximate 
requirements, in mg per 100 gm of diet, were: the rat, 20; 
the rabbit, 35; the guinea pig, 80; the pig, 45; the calf, 
200; the chick, 35; and the duck, 50. At best these values 
are only approximations because of the paucity and vari- 
ability of the data. Considering the physiological differ- 
ences among the species represented, the requirements 
are in quite close agreement except for the calf and pos- 
sibly the guinea pig. The high requirement of the calf 
may result from poor absorption (62), but this does not 
explain the high requirement of the guinea pig because 


TABLE 3. Magnesium Requirements of Various Laboratory 














and Domesticated Animals 

! | 

Dietaryt 

| Requirement 
Species and Age Span (wk)* | in Diett Ref. 

|  mg/100 gm Ca is 

| % | % | 
Rat (4-14) | 5 O04 | O74} «66 
Rat (4-14) | 13 r7 E.0) | 66 
Rat (4-6) | 208 it | 0.7 | 37 
Rat (3-6) | 25 | 0.6-1.8 | 0.5 | 29 
Rabbit (8-18) | 30-408 1 ong 0.4 | 36 
Guinea pig (2-10) 80 0.9 | Os4e} G1 
Guinea pig (2-10) 240 Og fore | 68 
Pig (3-9) 40-50® oy ie7l@ 
Calf (0-12) | 1088 I. | 04] 5 
Calf (0-52) | 200-3008§ 1.0 | O27 64 
Chick (0-3) | 35 1 | 0.6 | I 
Chick (0-4) | 25 1.2 | 0.8 | 20 
Chick (0-2) | 40-56 a ee | 48 
Duck (0-2) | 50 L>, s@vgr .. | 086 | 68 





* Age span over which the trial was run has been estimated 
in some Cases. 

} Criterion of adequacy was growth except for those values 
marked ‘B’ in which case it was maintenance of serum mag- 
nesium level. 

t Percentages of calcium and phosphorus are approximations 
calculated from estimated composition of ingredients. 

§ Requirement based on milk-fed calves and expressed by 
the author as 1.4-2.1 gm per 100-lb body weight. Food con- 
sumption estimated (5). 


in this species about go% of the excreted magnesium 
passes by way of the kidney (50). 

No attempt will be made to review extensively the 
literature that relates to the magnesium requirement of 
adult animals. Relatively little is known about the re- 
quirements of adult monogastric animals. The literature 
on the requirements of adult cattle has been summarized 
and suggests a value for maintenance of 10 to 15 mg per 
kg of body weight (4). The average daily requirement 
for maintenance of two adult sheep fed herbage was 
about 16 mg per kg (16). 

Relatively little has been added to our knowledge of 
the human requirement since the excellent review of 
Duckworth and Warnock (13). From balance studies it 
had been concluded the diet of children of age 4-7 years 
should supply 13 mg of magnesium per kg of body weight 


INTERACTIONS OF MINERALS 


653 


(11). In infants, 2-6 months of age, positive balance was 
maintained by 10-20 mg per kg (60). Although the fig- 
ures were not to be considered minimum values, positive 
balance was maintained in adult hospital patients on 220 
mg and in active subjects on 300 mg per day (65). The 
average magnesium content of 150 American dietaries 
has been estimated at 340 mg (59) while a survey of 1000 
British families gave average values which ranged from 
160 to 380 mg, depending on economic status (13). It is 
difficult to reconcile these values with the recent results 
obtained with college women on self-selected diets (55). 
In this study the daily intake ranged from 160 to 2550 
mg and negative balances occurred in 10 of 12 subjects 
that consumed between 200 and 300 mg daily. 

In any estimation of requirement, considerations must 
be given to availability. This is particularly true when 
one deals with natural foods but there are also differences 
in availability of magnesium supplied as chemically pure 
compounds. Using maintenance of serum magnesium in 
the rat as a criterion, magnesium supplied as MgCOs, 
MgO or as it occurs in the wheat plant is only 95, 61 and 
75%, respectively, as available as when supplied as the 
sulfate (37). On the other hand balance studies in the rat 
indicated that it was equally absorbed when supplied as 
the carbonate, phosphate or phytate (44). From 65 to 
86% of the magnesium in fresh grass was retained by 
guinea pigs and from 63 to 74% by cattle (21). 


CONCLUSIONS 


The symptomatology of magnesium deficiency varies 
from species to species but the two outstanding symptoms 
are hyperirritability and metastatic calcification. Aside 
from the species differences, the prevailing symptom in 
an animal will depend to some extent on the severity of 
the deficiency and on other dietary constituents. 

Physiologically calcium and magnesium are strong 
antagonists, but from a nutritional standpoint this an- 
tagonism is less marked. When the diet is low in phos- 
phorus, excess dietary magnesium causes loss of calcium 
from the body but not when dietary phosphorus is ade- 
quate. Conversely, consumption of excess calcium accen- 
tuates the symptoms of magnesium deficiency in an ani- 
mal that consumes a deficient diet. However, there is 
only meager evidence that excess calcium increases the 
minimum amount of magnesium required to support 
growth. 

Although it may not be justifiable to refer to a calcium- 
phosphorus antagonism, phosphorus will counteract the 
deleterious effect of excess magnesium and it will accen- 
tuate the symptoms of magnesium deficiency. There is 
also evidence that excess phosphorus will increase sub- 
stantially the minimum magnesium requirement. Phos- 
phorus seems to exert its effect chiefly by decreasing ab- 
sorption of magnesium but there is limited evidence that 
it has a direct metabolic effect. 

While the source of carbohydrate in the diet exerts an 
influence on magnesium absorption, most of the other 
dietary components studied appear to have little or no 
specific effect on the magnesium requirement. 








654 


nN 


G1 & GO 


~I 


. CarRsweELt, H. E. 


. Evmsuie, W. P. 


FEDERATION PROCEEDINGS 


Volume 19 


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F., E. M. Knott ann F. W. Scuiutz. J. Nutri- 
tion 22: 53, 1941. 


. Sjottema, B. Nutrition Abst. @ Rev. 1: 621, 1932. 

. SmitH, R. H. Biochem. J. 71: 609, 1959. 

. Suter, C. ano W. O. Kuinoman. Neurology 5: 691, 1955- 
. THomas, J. 


W.. Symposium-Magnesium in Agriculture. West 
Virginia Univ., Sept. 1959. In press. 


. Trpsetts, D. M. ano J. C. Aus. J. Clin. Invest. 16: 491, 1937. 
» Torts, E. V. 


AND D. M. GREENBERG, J. Biol. Chem. 122: 715, 
1938. 


. Unveruit, F. P., J. A. Honey ano L. Bocert. J. Exper. Med. 


32: 41, 1920. 


98. VAN REEN, R. ANnp P. B. Pearson. J. Nutrition 51: 191, 1953. 
. VAN WaGTENDONK, W. J. AND R. Wutzen. In: Vitamins and 


Hormones (vol. VIII). New York: Acad. Press, 1950, p. 69. 
Virate, J. J., E. E. HELLERsTe in, D. M. HEGsteD, M. Naka- 
MURA AND A. FarBMAN. Am. J. Clin. Nutrition 7: 13, 1959. 


. Vitae, J. J., P. L. Warre, M. Nakamura, D. M. HEcstTeED, 


N. ZAMCHECK AND E. E. HELLEsTEIN. J. Exper. Med. 106: 757, 


1957- 
AND B. L. VALLEE. New England J. Med. 


259: 431 and 475, 1958. 


. Wuzen, R. anp A. M. Baurs. Physiol. Zoél. g: 508, 1936. 





EX 


by 


Ate 





me 1G 


Nutni- 


1nimal 


1955: 
8. 
25:33 
5: 23, 
sity of 
. Path. 


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. Nutri- 


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. West 


1, 1937: 
22: Jig 





ver. Med. 


» 1953- 
mins and 
». 69. 

[. NAKA- 
959: 
JEGSTED, 
106: 757; 


J. Med. 


1936. 


YIM 


Metabolic relations of manganese to other minerals' 


G. C. COTZIAS 
Medical Research Center, Brookhaven National Laboratory, Upton, Long Island, New York 


Re THEME OF THIS SYMPOSIUM is interaction and 
therefore the absence of this word from our title must be 
obvious. This omission underlines our thesis which is: 
Within the context of its biological functions, manganese 
remains unaffected by the vagaries of direct metabolic 
interaction with other metals pertinent to nutrition. 
This terse statement might become acceptable if some 
of its terms are defined. 

When we say ‘direct metabolic interaction,’ we mean 
any demonstrable displacement, replacement or modi- 
fication of action of manganese. Therefore, mere con- 
stancy of concentrations of this versus other body 
constituents does not constitute per se direct metabolic 
interaction. 

The cardinal functions of manganese occur within 
the mammalian body proper and, to be precise, intra- 
cellularly. Interactions occurring prior to the intake of 
food or even within the gastrointestinal lumen are 
therefore not germane, regardless of how intensely they 
might be reflected in the body’s metabolism. Thus 
materials which act as absorptives or cathartics, whether 
specific or nonspecific, are excluded from consideration 
here, in spite of their value in other aspects of nutrition. 

Metals pertinent to nutrition are the ones which 
occur in tissues or foods. Therefore, technetium and 
some other unusual metals are not considered, in spite 
of their being useful tools in other areas of study. 

The present effort aims to document the singular 
specificity which characterizes the pathway of manganese 
through the body. It has to be conceded, however, that 
the animal’s nutritional state with regard to manganese 
might have been a determinant in the demonstration 
of this specificity. Hence, manganese deficiency as well 
as some states of manganese excess will be touched upon 
at the outset. 

The sum total of the available evidence, as interpreted 
by ourselves at least, indicates that manganese is not a 
mere ‘trace element’ but indeed a precious metal as far 
as the economy of the body is concerned. 


EXCESSIVE MANGANESE INTAKE 


In general, high manganese intakes are well tolerated 
by animals. As was shown by Gallup, Walters and 
‘This work was performed under the auspices of the U. S. 
Atomic Energy Commission. 


65 


McOsker (1) and others (2), high manganese intakes 
might result in retention of this element in some of the 
tissues of the body, notably the liver. 

Above and beyond abnormalities in concentration of 
the element, very high manganese intakes have been 
reported to cause some other changes as well: Chornock, 
Guerrant and Dutcher (3) have reported retarded 
growth in rats the dry diet of which contained 1.73 % 
manganese. The animals in this series seemed to have 
shown negative calcium and phosphorus balance and 
some of them even developed severe rickets. Hartman, 
Matrone and Wise (4) have found evidence that massive 
manganese intakes interfere with the absorption of iron. 
Fain, Dennis and Harbough (5) suggest that low blood 
magnesium levels might be observed in cows receiving 
100 mg of manganese per kilogram diet, but, amazingly 
enough, neither lower or higher manganese intakes had 
any effect on blood magnesium. Blakemore, Nicholson 
and Stewart (6) have reported lactation tetany in cows 
feeding on high manganese pastures. These cows had 
high blood manganese levels as well. Gubler et al. (7) 
have reported that high intakes of manganese affected 
the partition pattern of copper among the various 
organs but not the total body concentration of copper in 
rats. They discuss some findings with permutations of 
diets which offered high (or normal) manganese (or 
copper) diets to rats. 

Experiences such as the ones listed above have been 
encountered at least in part by more than one group of 
investigators (2). They indicate that massive intakes of 
manganese might interfere with the absorption of some 
other minerals from the gut. As will be seen in the 
discussion of manganese deficiency, these effects are 
reciprocal. Thus it is evident that during the preparation 
of the feed or after its ingestion massive amounts of 
manganese produce changes in the state of the minerals 
in question which render them nutritionally unavailable. 

A more subtle effect of elevated manganese intakes 
was encountered by Bertinchamps and Cotzias (8; 
unpublished observations). They have discovered that 
the biliary excretion of radiomanganese is qualitatively 
affected by the amount of manganese in the diet of the 
animals. Ordinarily the radioisotope appears promptly 
in the bile of rats. Its concentration becomes maximal 
rapidly and diminishes exponentially thereafter. If the 
animals are fed diets containing 20 ppm manganese or 








656 FEDERATION PROCEEDINGS 


more, the initial series of events are qualitatively the 
same, but later an impressive second wave of radiation 
appears in the bile which reaches a maximum about go 
minutes following the injection of the isotope. This 
indicates that with low manganese intakes only one 
pathway of manganese excretion is evident, but that 
with higher intakes a second and slower route of ex- 
cretion is also utilized. It is unknown as yet whether this 
second excretory path is specific for manganese or 
whether other metals can utilize it also. 


CHRONIC MANGANESE POISONING 


This disease syndrome is mentioned here only to 
present a complete argument. It occurs primarily in 
miners who are exposed to inhalation of manganese dusts. 
It is also presumed to occur under other conditions of 
manganese loading. It is characterized by two classes 
of manifestation: a) a chronic pneumonopathy and 
6) a progressive central nervous system affliction similar 
to Parkinsonism. The pneumonopathy seems like almost 
any chronic pneumoconiosis and is due to the injurious 
effects of the manganese dust on the lung. The nervous 
affliction on the other hand is of considerable interest 
from the physiological point of view because, although 
it is indubitably a form of manganese poisoning, this 
element has been found to occur in normal concentration 
in the tissues of the victims of this disease. Writings by 
Von Oettingen (9), Rodier (10) and the author (2) 
might be consulted for details. No element other than 
manganese has been implicated as a cofactor in the 
development of this disease. 


MANGANESE DEFICIENCY 


The most reproducible manifestations of this de- 
ficiency include deceleration of the growth processes, 
induction of bone anomalies, reproductive dysfunction 
and central nervous system pathology. The mani- 
festations of the deficiency are not materially different 
in mammals as compared to birds but the latter have 
exhibited higher susceptibility. Thus they will be dis- 
cussed separately. ; 

Mammals. Following the original paper in 1931 of 
Kemmerer, Elvehjem and Hart (11), many groups of 
investigators have produced manganese deficiency or 
have observed its spontaneous appearance in a number 
of mammalian species. The detailed account of this 
large experience is not within the scope of the present 
communication but can be sought in less specialized 
treatises (2, 12). It is of interest, however, that symptoms 
attributable to manganese deficiency have tended to be 
generally reversible in adult mammals if the diet was 
supplemented with manganese salts. One exception is 
the work of Miller et al. (13) which showed that lameness 
produced in pigs feeding on deficient diets was not 
reversed by manganese supplements. As opposed to the 
reversibility of this condition in the adult mammal, 
when the deficiency is imposed early during gestation, 
irreversible manifestations are often produced, as will 
be noted below. 


Volume 19 


The precise levels at which manganese deficiency can 
be induced in mammals are rather tenuous. This is due 
to the fact that there are many independent dietary 
factors, the excess or paucity of which might affect the 
susceptibility of an animal to manganese deficiency, 
Some of these are mentioned below. 

The manganese deficient adult mammals develop 
any or all of the manifestations listed above. Smith, 
Medlicott and Ellis (14) among other investigators have 
shown that bones tend to become short, thick, light, 
fragile and deformed. The exocrine apparatus of the 
testes undergoes pronounced degenerative changes. 
Among other workers, Hill, Holtkamp, Buchanan and 
Rutledge (15) have described a pronounced ataxia in rats 
which for four generations were living on 0.03 mg per 
day per rat. These animals showed loss of equilibrium, 
backward gait and serious postural reactions to stimuli. 
Their brains, however, appeared histologically normal. 
Rudra (16) showed that manganese-deficient animals 
develop a vicarious appetite which he termed ‘manganese 
hunger’. 

Additional cardinal manifestations of manganese 
deficiency are impairment of growth and the birth of 
nonviable young in large proportions. The females tend 
to become pregnant with a frequency which is sig- 
nificantly lower than that of control animals. 

Wachtel, Elvehjem and Hart (17) have observed that 
5 ug of manganese per rat per day produced deficiency 
and that this was markedly aggravated by high calcium, 
low phosphorous diets. These diets also produced 
progressive anemia in the animals. 

Manganese deficiency of the mammalian fetus has 
been alluded to, but the impressive observations of 
Hurley, Everson and Geiger (18) merit special atten- 
tion. These investigators determined that manganese- 
deficient mothers give birth to ataxic rats if manganese 
supplements are withheld up to the 14th day of gesta- 
tion. Following the 14th day of pregnancy, luxuriant 
administration of manganese supplements failed to 
affect both the degree and the incidence of ataxia in the 
young. 

Birds. Like in the mammals, the deficiency _ has 
different implications if it be imposed on adult animals 
or prior to hatching. If the disease occurs in the adult, 
it is referred to as perosis, if it appears in the embryo, 
as chondrodystrophy. Both diseases had been observed 
prior to their becoming linked with manganese me- 
tabolism. 

Perosis was first linked to manganese deficiency by 
Wilgus, Norris and Heuser (19). While they were study- 
ing the aggravation of perosis by high calcium and high 
phosphorous diets, they encountered a salt sample 
which improved the condition. This sample contained 
manganese among other contaminants. Later work by 
these and other investigators (12, 2) substantiated the 
fact that perosis is almost synonymous to manganese 
deficiency in birds. Wilgus and Patton (20) discovered 
that very high concentrations of phosphorous and of 
calcium can produce perosis in chicks feeding on normal 


~Tszamt et 








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July 1960 


diets. They confirmed earlier reports that high con- 
centrations of these elements in perosis-producing feeds 
increase both the severity and the incidence of the 
disease. Evans, Rhian and Draper (21) have stated that 
concomitant choline deficiency markedly aggravates 
manganese deficiency. 

Chondrodystrophy occurs in chicks hatched from eggs 
laid by manganese-deficient hens. It is characterized by 
retarded growth of the embrya, edema, large abdomina 
and many stigmata due to abnormalities of bone struc- 
ture. Lyons and Insko (22) have been able to prevent 
it by either injecting 0.03 mg of manganese (as the 
sulfate) into the eggs or by feeding manganese (40 mg/ 
kg diet) to the hens. 

Gallup and Norris (23) have studied the nutritional 
availability of various forms of manganese and have 
discovered that susceptibility to perosis is to some extent 
strain-linked. Thus here, as in the case of the mammals, 
a precise statement of the deficiency levels for manganese 
is impossible. 


MANGANESE REQUIREMENTS 


Requirements of this mineral have been investigated 
with several criteria in mind. If maximum growth be an 
acceptable criterion, the following values can be con- 
sidered as valid: Smith and Ellis (24) have shown that 
approximately 300 wg per animal per day is about 
optimal for the rabbit, but this has been found to be 
suboptimal by others (12, 2). Plumlee et al. (25) con- 
sider 3.5 mg per kg diet as optimal for piglets, but 
Johnson (26) fixes this level at 0.5 mg per kg diet. 
Gallup and Norris (23) found that 0.03-0.05 mg per 
kg diet prevents perosis in chickens. 

Man’s requirements have been estimated to range 
from 3 to 9 mg per day (2) for the adult; that of children 
has been estimated as 0.2 to 0.3 mg per kilogram body 
weight per day. It is evident that here again it is not 
possible to fix accurately the levels of manganese re- 
quirement. 


MANGANESE IN RELATION TO OTHER MINERALS 


It was alluded to previously that excessive manganese 
intakes may interfere with the utilization of other 
nutrients and that excesses of some nutrients may 
interfere with the absorption of manganese. This was 
interpreted as indicating the formation of chemical 
compounds within the gastrointestinal lumen which 
render these nutrients unavailable. However, work with 
enzymes, with unicellular organisms and with organ 
slices has shown that manganese may be replaced 
by other minerals in vitro. Thus it became important 
not to rely exclusively on the above interpretation, but 
to design specific experiments to answer the questions 
of direct interaction. The necessity for such experiments 
is underlined by the observations of Thiers and Vallee 
(27). These investigators showed that when intracel- 
lular fractions from rat liver were ranked according to 


INTERACTIONS OF MINERALS 


657 


their iron content, their manganese content also became 
ranked, but in a reciprocal fashion. 

Cotzias and Greenough (28) have therefore studied 
the rate of elimination of Manganese* following ad- 
ministration of various other elements in mice. They 
injected the isotope (Mn*Cl,) in a carrier-free form 
parenterally into mice. They then determined the change 
of the concentration of the radioactivity in the bodies 
of these intact living mice by inserting them into a 
radiation sensing device. The data were plotted semilog- 
arithmically as per cents of the first count against 
elapsed time. It was found that the rate of elimination of 
radiomanganese was fairly constant from one animal 
to the next, provided that the manganese concentration 
in their diet remained constant. When the manganese 
concentration was increased the excretion rate of Mn* 
was also increased. In animals which were fed a low 
manganese diet, acceleration of the excretion rate of 
Mn* was also produced by parenteral administration of 
manganese compounds ranging in valence from Mn°® 
(= metal powder) to Mn’+ (= permanganate). 

This part of the experiments showed that one kind of 
manganese replaces another in the body very readily 
and that, therefore, a great part of the body manganese 
is readily exchangeable. This had been surmised also by 
Maynard and Cotzias (29) and Borg and Cotzias (30). 

Since manganese in the body of the mouse was 
readily available for exchange, it became opportune to 
ascertain whether it might not exchange also with 
other metals. For this study the experimental design 
was essentially identical with the one just described. 
Animals were parenterally tagged with isotope, their 
body radiation was determined, and the initial rate of 
excretion was ascertained. They were then given in- 
jections of magnesium, of members of the first transition 
group of elements, and of members of period VII. The 
amounts of metal injected were highly significant in 
relation to the estimated total mouse content for each 
metal, in several instances being multiples thereof. 
Surprisingly, only manganese affected the rate of elimina- 
tion of radiomanganese. Furthermore, the animals which 
had not responded to challenges with the nonmanganese 
metals named above did respond promptly to man- 
ganese injections when those were given later as an 
additional control. 

Since this evidence was gathered with the aid of 
intact animals the total body radiation of which was 
determined, it could not reflect the possible displacement 
of manganese from one organ to another or: from one 
intracellular organelle to another. Thus experiments 
were conducted which were identical to the ones already 
described with the exception of the end-point: the dis- 
sected organs were now tested for their content of Mn*. 
Again, only manganese displaced 
within the body. 

On the basis of these experiments, the pathway of 
manganese through the body was now considered as 
specific. A search was begun for mechanisms that might 


radiomanganese 








658 FEDERATION PROCEEDINGS 


keep manganese from meeting with other elements which 
by virtue of concentration (= magnesium) and/or 
higher binding capacity (= copper, zinc) might displace 
manganese from its sites of localization in the body. A 
protein was thus discovered by Bertinchamps and Cotzias 
(31) in human plasma, which binds manganese. More- 
over, this B, globulin is different from transferrin (the 
iron binding protein), and was therefore termed ‘trans- 
Furthermore, Borg and Cotzias (32) have 
supports 


manganin.’ 
brought forward evidence which 
the fact that mammalian and human red cells (and 
possibly other tissues) have one or more porphyrins which 


strongly 


contain manganese instead of iron. 


REFI 

1. Gattup, W. D., L. E. Watters anp D. E. McOsker. Proc. 
Oklahoma Acad. Sci. 32: 71, 1951- 

2. Corzias, G. C. Physiol. Rev. 38: 503, 1958. 

3. Cuornock, C., N. B. Guerrant anp R. A. DutcHer. J. 
Nutrition 23: 445, 1942. 

4. Hartman, R. H., G. Marrone ann G. H. Wise. J. Nutrition 
57: 429, 1955- 

5. Fain, P., J. DENNis AND F. G. HarspouGu. Am. J. Vet. Res. 13: 
348, 1952. 

6. Blakemore, F., J. 
Record 49: 415, 1937. 

7. Guser, C. J., D. S. Taytor, E. J. Ercuwarp, G. E. Cart- 
WRIGHT AND M. M. Wintrose. Proc. Soc. Exper. Biol. & Med. 
86: 223, 1954. 

8. BERTINCHAMPs, A. AND G. C. Corzias. Fed. Proc. 17: 428, 1958. 

g. Von OetTiNGEN, W. F. Phystol. Rev. 15: 175, 1942. 

10. Roper, J. Brit. J. Indust. Med. 12: 21, 1955. 

11. KeEMMERER, A. R., C. A. ELVeHyjEM AND E. B. Hart. J. Biol. 
Chem. 92: 623, 1931. 

12. UnpERWoop, E. J. Trace Elements in Human and Animal Nu- 
trition. New York: Acad. Press, 1956. 

13. Mirrer, R. C., T. B. Kerry, M. A. McCarty ann W. T. S. 
Tuorp. Proc. Soc. Exper. Biol.& Med. 45: 50, 1940. 

14. Smiru, S. E., M. Mepiicotr anp G. H. Exuis. Arch. Biochem. 
4: 281, 1944. 

15. Hitt, R. M., D. E. Ho_tkamp, A. R. BucHANAN AND E. K. 
Rut epceE. J. Nutrition 41: 359, 1950. 

16. Rupra, M. N. Nature, London 153: 11, 1941. 


A. NICHOLSON AND J. STEWART. Vet. 


Volume 19 


SUMMARY 


The sum total of the experience presented here 
indicates that there exist interactions between man- 
ganese and other important mineral nutrients but that 
these interactions probably occur outside the body 
proper. Therefore, they are not metabolic interactions, 
although they have demonstrable nutritional conse- 
quences. Within the body proper, manganese appears to 
pursue a specific path which does not normally permit 
equilibrium between it and other metals. Within the 
plasma, it is carried by a specific protein (transman- 
ganin) and other segments of its pathway through the 
body also appear to be specific for this metal. 


YRENCES 


17. WacHTEL, L. W., C. A. ELvEHjEM AND E. B. Hart. Am. J. 
Physiol. 140: 72, 1943. 

18. Hurvey, L. S., G. J. Everson anp J. F. Geicer. J. Nutrition 
60: 309, 1958. J. Nutrition 67: 445, 1959. 

19. Witcus, H. S., Jr., L. S. Norris anp G. F. Heuser. Science 
84: 252, 1936. 

20. Wiicus, H. S., JR. anp A. R. Patton. J. Nutrition 18: 35, 
1939. 

21. Evans, R. J., M. Rutan ano C. I. Draper. Poultry Sci. 22: 
188, 1943. 

22. Lyons, M. anp W. M. Insko. Kentucky Agric. Exper. Sta. Bull. 
1937, P- 37!- 

23. GaLitup, W. D. anp L. C. Norris. Poultry Sci. 18: 76, 1939. 

24. Smitu, S. E. anp G. H. Exuis. J. Nutrition 34: 33, 1947. 

25. PLumLeE, M. P., D. M. TurasHer, W. M. Bergson, F. N. 
ANDREWs AND H. E. Parker. J. Animal Sci. 15: 352, 1956. 

26. Jounson, S. R. J. Animal Sct. 2: 14, 1943. 

27. Turers, R. E. ano B. L. Vatiee. J. Biol. Chem. 226: 911, 
1957- 

28. Corzias, G. C. 
1298, 1958. 

29. Maynarp, L. S. anp G. C. Corzias. J. Biol. Chem. 214: 489, 


AND J. J. GREENOUGH. J. Clin. Invest. 37: 


1955- 

30. Bora, D. C. ann G. C. Corzias. J. Clin. Invest. 37: 1269, 1958. 

31. BertincHamps, A. J. AND G. C. Corzias. Fed. Proc. 18: 469, 
1959- 

32. Borc, D. C. ano G. C. Corzias. Nature, London 182: 1677, 
1959. 


XUM 








me 19 


here 
man- 
that 
body 
tions, 
onse- 
ars to 
ermit 
1 the 
man- 
h the 


Am. J. 
utrition 
Science 
18: 35, 
Net. 22% 
2. Bull. 


, 1939: 


4: 489, 


), 1958. 
18: 4609, 


»: 1677, 








Interrelationships of iron and copper in the 


nutrition and metabolism of animals 


GENNARD MATRONE! 
Nutrition Section of the Department of Animal Industry, North Carolina State College, Raleigh, North Carolina 


i MOST OBVIOUS area of metabolism in which iron 
and copper are known to interact is in hemopoiesis. 
On the surface it would seem reasonable to expect that 
an understanding of the basic nature of this interrelation- 
ship is at hand, since the pioneering work conducted by 
the Wisconsin workers (1) was done over thirty years 
ago. Yet despite the fact that these observations have 
been confirmed and extended by many workers (2-5) 
and that new insight has been obtained about the bio- 
synthesis of heme, the prosthetic group of hemoglobin 
(6-8), knowledge concerning the mechanism of the iron- 
copper interaction is still obscure. Thus, the reviewer has 
chosen to survey several avenues which appear to be 
promising, to the end that a reasonably definitive 
description of the problem will emerge. 

The specific areas which will be covered are as follows: 

a) Hematologic manifestation of Fe and Cu defi- 
ciency—in erythrocytes, reticulocytes and in bone mar- 
row; 6) effect of Fe and Cu on heme chromoproteins 
other than hemoglobin; c) effect of copper on iron 
absorption, iron storage and mobilization of tissue iron; 
d) iron and copper in the biosynthesis of hemoglobin 
and heme; e) effect of Fe and Cu interrelationships on 
dietary requirement of these metals. 


HEMATOLOGIC MANIFESTATION OF Fe AND Cu DEFICIENCY 


Erythrocytes. Combined iron- and copper-deficiency 
anemia has been reported to be microcytic and hypo- 
chromic in the rat (9, 10), rabbit (11), swine (12, 13) 
and the chick (Hill, personal communication). Hema- 
tologic studies designed to separate out the specific 
Fe and Cu effects are limited, and as pointed out by 
Lahey et al. (12), ‘such data as are available are difficult 
to evaluate,’ since in most instances the animals are 
made anemic by means of milk diets prior to initiation 
of copper and iron studies. Consequently, because milk 
is deficient in both iron and copper, the effects observed 
may be influenced by an initially present combined iron 
and copper anemia. Lahey ef al. reported (12) that 

? Work of reviewer and co-workers cited as ‘unpublished data’ 
supported in part by grants from Herman Frasch Foundation and 
Moorman Manufacturing Co., Quincy, Illinois. 


659 


either copper and/or iron deficiency led to a hypo- 
chromic, microcytic anemia in swine. Hill (personal 
communication) found that in chicks iron anemia and 
combined iron and copper anemia also were hypo- 
chromic and microcytic, but that copper anemia was 
normocytic and normochromic. In dogs (14, 15) iron- 
deficiency anemia is microcytic and hypochromic bnt 
copper deficiency gives rise to a normocytic and normo- 
chromic anemia. The anemia observed in ruminants 
grazing copper-deficient pastures has been reported to be 
macrocytic and hypochromic in cattle (16, 17) and 
macrocytic in sheep (18). Copper anemia in lambs, 
however, has been reported (19) to be microcytic and 
hypochromic. 

The only exception noted to the classical picture of 
iron anemia, microcytic and hypochromic erythrocytes, 
was reported by Blaxter et al. (20). They found that 
iron deficiency in calves was microcytic and normo- 
chromic. Unpublished observations made at North 
Carolina State College in connection with an iron and 
copper study with calves (21) confirm the normochromic 
character of erythrocytes in iron anemia of calves. 

The metal anemia of infants, children and adults is 
characterized as a microcytic, hypochromic type due 
to iron insufficiency (22—24). 

It is apparent from this survey of the literature that 
a composite blood picture of iron and/or copper anemias 
in animals has been described only for the pig, the dog 
and the chick. The results from two of the three species 
indicate that copper anemia, unlike iron or combined 
iron and copper anemia, is normocytic and normo- 
chromic. Despite the enormous amount of work on iron 
and copper anemias in the rat, reports of morpho- 
logical changes occurring in uncomplicated copper 
anemia were not found. It is interesting to note, how- 
ever, that Stein and Lewis (25) observed that rats with 
hypochromic, microcytic anemia resulting from a 
combined copper and iron deficiency responded to 
copper by an increase in number of red cells without 
an increase in hemoglobin concentration. Moreover, 
Smith and Medlicott (10) reported that milk anemic 
rats responded to iron by an increase in the mean 
corpuscular volume from 37 to 51 cu yu, whereas no 








660 


increase in the mean corpuscular volume was observed 
following copper administration. Furthermore, Cart- 
wright ef al. (13) reported that when iron was injected 
instead of fed orally to swine on a copper-deficient diet 
the injected iron did not prevent the development of 
anemia but the cells were normocytic and hypochromic. 
The data from the above reports suggest that a) the 
anemia of combined iron and copper deficiencies is 
dominated by the morphological characteristics of 
uncomplicated iron deficiency, and }) microcytosis may 
be a characteristic of iron deficiency (whether induced 
directly or indirectly) rather than that of uncomplicated 
copper deficiency. A decrease in erythrocyte numbers 
apparently is characteristic of iron and/or copper anemia 
(9-13, 25, 26). The one exception to this generalization 
has been reported for iron anemia in the dog where 
little change from normal was observed (14). 
Reticulocytes. The addition of copper to the diet of 
milk anemic or combined iron and copper anemic rats 
(10, 27), rabbits (11) and swine (12) elicits a marked 
increase in percentage of reticulocytes whereas iron has 
little effect. The reticulocytosis following copper therapy 
apparently is persistent whereas the _reticulocytosis 
following combined iron and copper therapy is relatively 
transitory (27). Copper anemic dogs (14) and swine (12) 
show a lower percentage of reticulocytes than those with 
iron anemia. The administration of copper to copper 
anemic dogs (28) and swine also increased the per- 
centage of reticulocytes. These results suggest that copper 
is more directly involved than iron with reticulocytosis. 
Bone marrow. From the point of view of iron-copper 
interaction the reports on rats (29, 30), swine (12, 13) 
and dogs (14, 28) are particularly informative. McCoy 
and Schultze (29) found no essential difference between 
the bone marrow of iron-deficient and copper-deficient 
rats. Their studies showed that the marrow of either 
iron or copper anemic rats was essentially hyperplastic 
having a lower lipid content and a higher content of 
nitrogen and water soluble solids than normal marrow. 
The Utah workers (12, 13) reported that either iron or 
copper deficiency brought about a normoblastic hy- 
perplasia of the bone indistinguishable from each other. 
Thus, in view of the similarity of the hematological 
changes in swine these workers suggested that a common 
factor, lack of available iron, was involved in either iron 
or copper anemia. Van Wyk et al. (14) reported that the 
morphologic alterations in the bone marrow of copper- 
deficient dogs were unlike those in the marrow of iron- 
deficient dogs. The proportion of immature forms with 
large nuclei and basophilic cytoplasm was greater in 
the marrow of the dogs deficient in copper than in 
iron-deficient dogs. In addition, these workers reported 
that the quantity of cytoplasm and the hemoglobin 
content of the late normoblasts in the bone marrow were 
greater in the copper-deficient dogs than in similar 
cells from the iron-deficient dogs. These workers con- 
cluded that ‘In the bone marrow, there was no evidence 
of a deficiency in hemoglobin content but rather evidence 


FEDERATION PROCEEDINGS 


Volume 19 


of defective development of cells of the erythrocytic 
series,’ in which copper had a role distinct from iron. 


EFFECT OF FE AND CU ON LEVELS OF HEME 
CHROMOPROTEINS OTHER THAN HEMOGLOBIN 


Cytochromes. In 1934 Cohn and Elvehjem reported 
(31) that cytochrome A was absent or markedly reduced 
in heart and liver tissues of milk anemic rats and that 
copper alone restored the intensity of the A component. 
This observation was extended by Schultze (32, 33) who 
reported that cytochrome oxidase activity of heart, 
liver and bone marrow was greatly decreased in copper- 
deficient rats and quickly regenerated with addition of 
Cu to the diet. Iron-deficiency anemia did not affect 
cytochrome oxidase activity. Since these investigations 
other workers have reported similar findings. Gubler 
et al. (34) found an 8-fold decrease in the heart tissue of 
copper-deficient swine and a 3-fold decrease in the liver. 
On the other hand, cytochrome oxidase activity of these 
tissues was not affected in iron-deficient pigs. Gallagher 
et al. (35) also reported a decrease in cytochrome oxidase 
activity in the liver, kidney and brain of copper-defi- 
cient rats. Hill (personal communication) found a de- 
crease in cytochrome oxidase activity of heart homog- 
enates from copper-deficient chicks, but iron deficiency 
effected no change in activity. 

Evidence for changes in other cytochromes is less 
definitive. Cohn and Elvehjem (31) reported a slight 
decrease in the cytochrome B and C components in the 
heart and liver tissues of doubly deficient rats, which 
was reversed only when both iron and copper were 
added to the diet. In swine (34) iron deficiency decreased 
liver and kidney cytochrome C whereas copper de- 
ficiency had little effect on kidney and a stimulating effect 
on heart cytochrome C. Beutler (36, 37) also reported 
that in iron-deficient rats a marked decrease occurred 
in both the liver and kidney cytochrome C. 

Catalase. Schultze and Kuiken (38) reported that in 
rats either copper or iron deficiency decreased catalase 
activity of liver, kidney and blood. The decreases 
observed in copper deficiency, which were more marked 
than in iron deficiency, rapidly returned to normal when 
copper was fed. Adams (39) observed a rise in liver 
catalase activity of milk anemic females and castrated 
males. Addition of iron had little effect and the effect 
of Cu was variable, but copper and manganese de- 
creased the liver catalase to normal. He suggested that 
the rise in catalase was due to a deficiency of copper 
and manganese and that these elements had a de- 
pressing effect on catalase synthesis. In two reports on 
swine (12, 34) liver catalase activity was decreased in 
iron deficiency whereas no effect of copper deficiency 
on liver catalase activity was observed in the first report 
but a decrease was noted in the second study. Neither 
iron nor copper deficiency affected catalase activity of 
the kidney and red blood cells significantly. Gallagher 
et al. (35) reported no effect of copper deficiency on 
catalase activity of rat liver. 





et a 


a5 & omel-= A 





lume 19 


ocytic 
‘on. 


orted 
duced 
1 that 
onent. 
) who 
heart, 
»pper- 
ion of 
affect 
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f these 
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is less 
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er de- 
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ported 
curred 


that in 
atalase 
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copper 
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ncy on 





XUM 


July 1960 


It is clearly apparent that a decrease in cytochrome 
oxidase activity is a basic effect of copper deficiency 
but not of iron deficiency. It is suggested, moreover, 
that the index of cytochrome oxidase activity is more 
sensitive to copper status than is hemoglobin level. It is 
interesting to note that cytochrome oxidase activity 
which is lower in zinc toxicity (excess dietary zinc 
interferes with copper metabolism) is restored by sup- 
plemental copper but not by supplemental iron (40-42). 
Recent additional evidence reported by Wainio e¢ al. 
(43) that cytochrome oxidase is a copper-enzyme which 
contains a heme prosthetic group and possibly a lipid 
fits in with the work of Gallagher et al. (44) and raises 
the possibility of a direct rationale for the effect of copper 
on cytochrome oxidase activity. The evidence presented 
suggests that cytochrome C activity is more directly 
affected by iron than by copper deficiency. The results 
on catalase activity suggest that this enzyme is affected 
by more variables than the two under consideration and 
that its usefulness in studying the mechanism of the 
iron-copper interaction may be limited. 


EFFECT OF COPPER ON IRON ABSORPTION, IRON 
STORAGE AND MOBILIZATION OF TISSUE IRON 


Despite the fact that the subject of this section has been 
under investigation since 1932 the problem has yet to 
be resolved. Elvehjem and Sherman (45) reported that 
when rats deficient in both iron and copper were fed 
iron, the iron content of liver and spleen was increased 
2 to 3-fold over that of the controls. On the other hand, 
doubly deficient rats fed iron alone at the rate of 0.5 
mg per day for 14 days and then 0.05 mg of copper alone 
for 14 days regenerated hemoglobin and simultaneously 
decreased their iron stores of liver and spleen to con- 
centrations similar to the doubly deficient controls. 
When graded levels of iron were fed to combined iron- 
and copper-deficient rats, the iron stored in the liver was 
proportional to the dose. In the presence of copper, 
hemoglobin formation was found to be proportional to 
the iron intake and the liver showed no storage until 
0.3 mg per day was fed. From these observations the 
investigators concluded that copper does not affect 
assimilation of iron but does function in the conversion 
of inorganic iron into hemoglobin. 

Cunningham (46) and Josephs (47) found no signifi- 
cant difference between the total iron content of body 
iron in rats fed a milk diet plus iron and that of rats 
fed a milk diet plus iron and copper from weaning. 
Schultze (2) in his review concluded that copper had no 
effect on iron absorption. Several years later Houk e¢ al. 
(48) reported that copper increased the absorption of 
iron, since they found that adding copper to copper- 
deficient diets increased the dietary iron utilization from 
approximately 30 to 70%, as measured by total body 
iron content. Chase et al. (49, 50) also working with rats 
reported that copper increased iron absorption based 
on Fe®® studies and total body iron content of rats. 
In rationalizing their work, which was in agreement 


INTERACTIONS OF MINERALS 


661 


with that of Houk e¢ a/. (48) but contrary to the results 
of Cunningham (46), they stated that the latter in- 
vestigator ‘fed 0.2 mg per rat per day, which is a sub- 
optimal amount.’ They (49), however, overlooked the 
fact that Houk et al. fed diets containing only 4.2 ppm 
of Fe, an amount equivalent to 0.05 mg per rat per day 
assuming an average intake of 10 gm of dry matter 
per day. 

For the rat, it appears that a direct effect of copper 
on iron absorption has not been firmly established 
because of the difficulties in sorting out direct and in- 
direct effects. In the reviewer’s opinion, the evidence 
presented suggests that an indirect effect of copper on 
iron absorption in the rat is the major factor. Body iron 
can be divided into two compartments, iron in hemo- 
globin and iron in body tissues such as the liver. When 
hemoglobin synthesis is blocked in copper deficiency, 
absorbed iron can enter only the body tissue pool, which 
in time induces the iron mucosal block, decreasing iron 
absorption. When iron can also enter the hemoglobin 
pool, such as when copper is administered, iron from the 
liver and other storage sites moves out into the hemo- 
globin pool, the iron mucosal block is lifted and more 
iron is absorbed. 

A direct effect of iron on copper absorption in the rat 
appears unlikely (46, 48-50). 

The effect of copper on iron absorption of the pig 
has been studied intensively and systematically by the 
Utah workers (12, 13, 51-56). 

On the basis of their early studies they suggested 
that ‘copper may, in some basic manner, be concerned 
wherever and whenever iron moves.’ They found marked 
morphologic and biochemical similarities between iron 
anemia and copper anemia in swine. Biochemical 
evidence for effect of copper on iron absorption and 
movement was based on the fact that low and similar 
values of serum iron were found in either iron or copper 
anemia. The per cent of uptake of radio-iron in hemo- 
globin of copper-anemic swine was only 20 to 25% of 
the dose, for iron anemic 83% and for controls 93% 
(51). Liver iron was lowest in pigs with iron anemia, 
intermediate in copper-anemic pigs and highest in the 
control livers. In a radio-iron study the livers of copper- 
deficient pigs contained a smaller percentage of the dose 
of radio-iron but in absolute amounts Fe®* absorbed 
was equal to that of the controls (51). 

In subsequent studies (55, 56), ferrokinetic data 
indicated that the transport and mobilization of tissue 
iron in copper-deficient pigs was not impaired. In fact, 
disappearance rate of injected Fe®® from _ plasma, 
plasma iron turnover rate, red cell iron turnover rate 
and red cell incorporation rate, values were all greater 
in the copper-deficient pigs than in the controls. Along 
with these findings, the investigators made the significant 
observation that the life span of the red blood cells of 
copper-deficient swine was considerably less (13 vs 63 
days) than control pigs. They (56) showed further that 
red cells of copper-deficient swine, which are lower in 
copper content, when injected into the blood of normal 








662 FEDERATION PROCEEDINGS 


swine survived almost as long as the cells of normal 
swine. These investigators concluded that copper- 
deficiency anemia in swine resulted ‘from both a short- 
ened erythrocyte survival time and limited capacity of 
the bone marrow to produce red blood cells.” They also 
suggested that copper is an essential component of 
erythrocytes. They maintained, however, that the new 
results did not contradict their previous conclusions that 
in copper-deficient animals there is an impaired ability 
to absorb iron from the gastrointestinal tract. These 
data and conclusions imply, on the surface at least, 
that the mechanism of iron transport across the mucosal 
cells of the intestine is different from the movement of 
iron across cell-wall barriers in other tissues of the body. 
It may be instructive at this point, therefore, to ra- 
tionalize, if possible, the results and conclusions of the 
work with swine in the light of present concepts of the 
mechanism of iron transfer and movement in the animal 
body. 

The familiar concept of the ferritin-mucosal block 
hypothesis of iron absorption and iron movement in the 
animal body was developed by Granick (57-59) and 
extended by Mazur and co-workers (60-63). A signifi- 





H 
S 
a 
= ees Fe’ pe 
‘S 
H 


Oxidized Ferritin 
(inactive form) 


Reduced Ferritin 
(active form) 


FIG. I 


cant feature of this concept is that there are two forms of 
ferritin, inactive ferritin in which the iron is in the 
ferric state and active ferritin which possesses some 
ferrous iron stabilized by sulfhydryl groups. A diagram- 
matic scheme illustrating these two forms as shown by 
Mazur is given in figure 1. The ferrous iron, which is 
dissociable from the biologically active form of ferritin, 
is the species of exchange in the passage of iron across 
the intestinal mucosa for iron absorption, ‘across the 
placental membrane, across the liver cell wall into the 
plasma for transport and across the membrane of cells 
storing ferritin in the bone marrow, liver and spleen. 
This hypothesis implies that essentially the same mech- 
Mazur and 


oxygen 


anism is operative wherever iron moves. 
co-workers have shown, moreover, that low 
tension favors the formation of the active form of fer- 
ritin. 

It is proposed that the results with swine are not 
necessarily at variance with the above mechanism. The 
larger turnover rate of iron in the tissues of the copper- 
deficient swine is consistent with the expectation that 
the ratio of active iron to inactive iron would be greater. 
The rationale which follows for reconciling the low 
serum iron values and lowered iron absorption in the 
copper-deficient pigs is presented purely for speculative 
considerations. In copper anemia, movement of iron into 


Volume 19 


hemoglobin is restricted because of diminished red cell 
production (56). Absorbed iron is confined largely to the 
tissue iron pool either in storage or transit. The higher 
ratio of biologically available iron brings about a 
greater exchange between iron in storage, ferritin, and 
in transit, iron bound to siderophilin. Since ferritin 
synthesis has been shown to be iron induced (58, 64-66), 
each time the iron is returned to the storage tissues more 
ferritin synthesis results. With each increase in the 
relative concentration of ferritin less net iron would be 
in transit until an equilibrium is reached, resulting in 
lower.serum iron values and in lower iron absorption 
because of the ferritin mucosal block. Offhand, it would 
seem that under copper-deficiency conditions the mu- 
cosal block might be attained more quickly with higher 
levels of dietary iron than with lower levels because 
the mucosal compartment would also be subject to the 
effects of ferrous iron from the lumen side. A schematic 
representation of these speculations is depicted as 
follows (fig. 2): this hypothesis is consistent with the 
results obtained with copper-deficient swine, e.g., lower 
plasma iron values, decreased absorption, and a slower 
rate of disappearance of radioactive iron from the liver 
and bore marrow despite a greater radio-iron turnover. 


i? — — —eFe* * = Siderophilin = Fet* —X—BHd 
i {| Iron Pool ~ 4 —" Pool 
z oe = Mucosol Storage <=Storage 
Iron Protein X Ferritin Ferritin Protein x 
Pool Pool Pool 
FIG. 2 


Unlike the pig and the rat, extensive deposits of iron 
have been reported (4, 5, 67, 68) in the liver of rumi- 
nants having copper deficiency resulting from grazing 
copper-deficient herbage. Marston reported that iron 
was deposited as hemosiderin in the tissues. This ob- 
servation is interesting in light of the report by Granick 
(59) that ‘no crystalline ferritin could be obtained from 
the liver and spleen of beef cattle, sheep and deer.’ 
Whether or not the dissimilarities between ruminants 
and swine are due to species differences is difficult to 
evaluate since it is possible that under field conditions 
other factors might markedly influence the manifestation 
of copper deficiency. In iron and copper studies (21, 69) 
with calves fed milk diets at North Carolina State 
College, level of dietary copper affected level of copper 
in serum and liver but not the level of iron in the same 
tissues, nor was an excessive level of iron found in the 
liver of any of the calves. In these studies, however, 
frank copper deficiency was not evident even though 
serum copper levels in the non-copper supplemented 
group were as low as 10-15 wg/100 ml in some cases. 
It is apparent that more information from controlled 
experiments with ruminants is necessary before de- 
finitive answers to some of the questions raised can be 
made. In any case, the evidence available indicates that 
copper does not influence iron absorption in rumi- 
nants. 


WIiIRA 








lume 19 


ed cell 
to the 
higher 
out a 
n, and 
erritin 
4-66), 
; more 
in the 
uld be 
ing in 
rption 
would 
e mu- 
higher 
ecause 
to the 
ematic 
ed as 
th the 
lower 
slower 
e liver 
‘nover. 


Hb 
ron Pool 


Storage 
Protein X 


of iron 
 rumi- 
rrazing 
it iron 
is ob- 
rranick 
d from 
deer.’ 
iinants 
cult to 
ditions 
station 
21, 69) 
State 
copper 
e same 
in the 
ywever, 
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nented 
» Cases. 
1trolled 
re de- 
can be 
tes that 
rumi- 





July 1960 


IRON AND COPPER IN THE BIOSYNTHESIS OF HEMOGLOBIN 
AND HEME 


The two main sites for hemoglobin formation are bone 
marrow and cells of the blood. The capacity of blood 
cells to synthesize hemoglobin in vitro apparently is 
greatest in reticulocytes (70-73). Rate of heme syn- 


TABLE I. Effect of Copper and Iron on Hemoglobin 
Level of Milk Anemic Rats* 


Levels of Cu 
mg/rat/day 


Fe 
mg/rat/day 0 01 025 .05 10 
gm Hb/100 ml blood 
3rd Week 
0.10 407 6.8 8.5 9.9 10.2 
0.25 Box 8.7 13.4 10.9 13.2 
0.50 8.1 [2.2 13.2 13.9 14.1 
¢th Week 
0.10 4.6 923 g.2 11.0 11.8 
0.25 5-1 10.1 14.2 1352 14.2 
0.50 g.1 13.3 13.5 14.1 14.5 





* Data from (86). 


TABLE 2. Effect of Copper and Iron on Hemoglobin Level of 
Chicks Started on Experiment at 1 Day of Age* 


Levels of Cu 
ppm dry matter of diet 


Fe 
bpm 1.80 2.20 3.20 5.20 
gm Hb/100 ml blood 
12th Day 
10 4.6 4-9 ye 7.2 
15 5-5 8.1 ve: 8.2 
20 yes 8.5 9.6 10.2 
40 9.6 10.2 11.8 10.6 
17th Day 
10 4-0 4.8 ACY 6.3 
15 6.3 5-7 7.8 11.0 
20 5.6 75 g.1 14.4 
40 8.2 8.6 10.5 14.2 
22nd Day 
10 4.1 3.2 5-2 5-5 
15 4.0 6.5 5.8 6.5 
20 5.1 5.8 6.8 10.5 
40 6.9 722 10.5 10.7 





* Unpublished data of Hill and Matrone, N. C. State College, 
Raleigh, N. C. 


thesis in vitro is highly correlated with reticulocytes or 
immature red blood cells even for avian erythrocytes 
(74, 75). Laver et al. found that hemolysates of eryth- 
rocytes of phenylhydrazine anemic chickens (many of 
which were immature cells) synthesized heme from 
glycine and succinate 8 times faster than did the hemoly- 
sates prepared from normal cells. In an_ interesting 
experiment conducted by Anderson and Tove (personal 
communications) erythrocyte hemolysates from blood 
of iron- and copper-deficient chicks that had received 


XUM. 


INTERACTIONS OF MINERALS 663 


an injection of copper 4 days prior to bleeding synthe- 
sized protoporphyrin from 6-aminolevulinic acid at a 
much greater rate than did cell hemolysates from blood 
of untreated controls, of iron injected chicks and of 
noimal controls. Addition of copper in vitro did not have 
a stimulatory effect on any of the hemolysates. An ex- 
planation of these results is that copper injected in vivo 
stimulated reticulocytosis. 

In in vitro studies with rabbit reticulocytes, it has been 
shown (73, 76, 77) that iron stimulated protein synthesis 
of hemoglobin. London et al. obtained similar results 
with rabbit reticulocytes but with duck erythrocytes 
incorporation of glycine was not stimulated although 
iron markedly stimulated incorporation of glycine into 
heme, an effect reported by others (78-80). Brown 
reported that iron was necessary for synthesis of 6- 
aminolevulinic acid in step(s) where pyridoxal phosphate 
is concerned and in steps between protoporphyrin and 
heme. Iodice et al. (81) reported that purified 6-amino- 
levulinic acid dehydrase contained 0.1% copper and 
that the anemia seen in copper deficiency might be 
related to lowering of this enzyme. Later they (82) 
reported that removal of the copper did not affect the 
activity of the enzyme. Anderson and Tove (83) re- 
ported that heme synthesis with chicken blood from 
copper-deficient chicks was stimulated by the addition 
of copper in vitro but subsequently were unable to repeat 
these results because of difficulties in producing un- 
complicated copper deficiency in chicks (Anderson and 
Tove, personal communicauon). 

That copper plays a role in red cell formation and 
cytochrome oxidase activity is well established. Whether 
these two factors are related is worthy of further in- 
vestigation. Granick (84, 85) has indicated that Oy, is 
required for coproporphyrinogen conversion to pro- 
toporphyrin and that the electron transfer system 
associated with mitochondria of red cells may be in- 
volved in the synthesis of 6-aminolevulinic acid from 
glycine and succinate. Gallagher et al. (35, 44) in a 
comprehensive investigation of the biochemistry of 
copper deficiency in the rat reported that the only 
constant and early enzymological disturbance observed 
occurred in cytochrome oxidase and_ succinoxidase 
systems. Livers of copper-deficient rats were almost 
devoid of heme a which they propose is the prosthetic 
group of cytochrome oxidase. A suppression of phos- 
pholipid synthesis was also noted in copper deficiency, 
the fault occurring in the process of condensation of 
acyl CoA and a-glycerophosphate. They suggest that 
heme a is not synthesized in copper deficiency because 
of the inability to attach the fatty acid to the pyrrole 
nucleus. Thus it is apparent that implication of an iron- 
copper interaction has not been established in in vitro 
studies. 


EFFECT OF IRON AND COPPER INTERRELATIONS ON DIETARY 
REQUIREMENTS OF THESE METALS 


A clear cut interaction of iron and copper on the 
dietary requirements of these elements has been shown 








664 FEDERATION PROCEEDINGS 


for the rat (86) and the chick (87). As shown in tables 
1 and 2, increasing levels of copper promote higher 
levels of hemoglobin with the same level of iron and 
vice versa. Mitchell and Miller concluded that .25 mg 
of iron per rat per day was sufficient providing 0.1 mg 
of copper per rat per day was fed but that 0.5 mg per 
day used by many investigators affords a margin of 
excess. Hill and Matrone tentatively estimate the mini- 
mum requirements for chicks are approximately 40 
ppm of iron and 4 ppm of copper. 

In studies (21, 69) with calves involving three levels of 
copper and three levels of iron in a factorial arrange- 
ment, copper did not have a significant effect on hemo- 
globin level even though the non-supplemented copper 
diets contained less than 0.5 ppm Cu of moisture-free 
diet. Symptoms of frank copper deficiency were not 
evident but 6 mg of copper per day was required to 
maintain normal levels of serum copper. These inves- 
tigators estimated that the minimum iron requirement 
of calves was 30 mg per day. Blaxter et a/. have reported 
that the iron requirement for calves is more than 20 mg 
and less than 100 mg per day. 

Whether or not a copper-iron interrelationship affects 
the dietary requirement of these metals for other species 
is not certain because generally either iron or copper is 
fed at a constant level. Teague and Carpenter (88), 
employing several levels of copper, present data indi- 
cating that the copper requirement of swine is between 
0.035 mg and 0.050 mg/Ib live weight. The Utah 
investigators (12, 13) in their studies with swine used 
0.5 mg copper and 30 mg iron per kg body weight per 
day. Matrone, Thomason and Bunn (unpublished data), 
using a basal milk diet containing 5 ppm Cu of moisture- 
free diet, found that the minimum iron requirement 
for pigs from 1 day of age to 6 weeks was approximately 
60 ppm. 

The effect of an iron-copper interaction on the 
requirement of these metals for man is not known 
because unequivocal copper deficiency in the adult 
human has not been reported (24), although alterations 
in the metabolism as evidenced by hypocupremia 
apparently occur in a variety of pathologic states, 
especially in infants and children in disorders in which 
metabolism of protein is disturbed (24, 89). Further 
studies are needed before the full significance and 
underlying causes of these changes will be known. 


SUMMARY 


Evidence of an iron-copper interaction is clearly 
apparent only in the finished product, hemoglobin. 
Conclusive evidence that a mutual interaction occurs 


Volume 19 


in a mechanism or biochemical-reaction sense has not 
been obtained. Iron plays a role in the synthesis of both 
heme and globin of hemoglobin. The primary role of 
copper in hemopoiesis appears to be in the formation 
and development of reticulocytes rather than in the 
synthesis of protoporphyrin and hemoglobin per se. 
Recent evidence opens up the possibility that iron and 
copper may have a mutual interrelationship in reactions 
associated with the function of cytochrome oxidase. 


AREAS FOR FUTURE INVESTIGATION 


It is apparent from this review that there are gaps in 
our knowledge of iron and copper metabolism both on 
the whole animal level and on the cellular level. Part 
of the problem is associated with difficulties in obtaining 
copper deficiency in animals, and another part may be 
due to a lack of experiments having comprehensive 
objectives in exploring Cu and Fe metabolism. Ideally, 
to understand the areas in metabolism where iron and 
copper interact it is essential that simple copper de- 
ficiency, simple iron deficiency and the double de- 
ficiency be investigated concurrently. Studies with other 
species similar to those with swine by the Utah workers 
should greatly increase our insight into the problem. 

Since copper and iron are both implicated in erythro- 
cyte formation, greater emphasis should be centered on 
bone marrow investigation both in vivo and in vitro. In 
vitro techniques utilizing erythrocytes for synthesizing 
hemoglobin applied to bloods of copper and/or iron 
deficiencies should prove illuminating. Almost all 
studies of porphyrin biosynthesis have dealt with the 
biosynthetic pathway leading to protoporphyrin 9. 
Although quantitatively this compound is the most 
important, the importance of other hemoproteins 
cannot be overlooked. This is particularly true of the 
prosthetic group of cytochrome oxidase which has been 
strongly implicated in copper deficiency. The role of 
ceruloplasmin, the copper protein of plasma, and of 
erythrocuprein (go, 91) would also appear to bear 
study. The significant finding that copper deficiency 
in swine shortens the erythrocyte life span (56) should 
stimulate investigation of this phenomenon in other 
species. 

The activity of xanthine oxidase and levels of hypo- 
xanthine in the liver and mucosa of copper- and/or 
iron-deficient animals may be worthy of investigation 
in view of the interesting work of Green and co-workers 
(62, 63). Their work indicates that rate of iron release 
from hepatic ferritin stores is limited by the level of 
hypoxanthine and xanthine in the hepatic cell. They 
postulate that the conversion of ferritin iron to the 
ferrous state is mediated by reduced xanthine oxidase. 


REFERENCES 


. Ervenyem, C. A. Physiol. Rev. 15: 471, 1935. 

. Scuuttze, M. O. Physiol. Rev. 20: 37, 1940. 

. Cartwricut, G. E. Blood 2: 111, 256, 1947. 

Marston, H. R. Physiol. Rev. 32: 66, 1952. 

. Unperwoop, E. J. Trace Elements in Human and Animal Nu- 
trition. New York: Acad. Press, 1956, p. 430. 


wtp OO NH = 


6. SHemin, D. Nat. Acad. Sci.-Nat. Research Council, Publ. 
No. 557, Washington, 1958, p. 66. 

7. Bocorap, L. Nat. Acad. Sci.-Nat. Research Council, Publ. 
No. 557, Washington, 1958, p. 74. 

8. Granick, S. Nat. Acad. Sci.-Nat. Research Council, Publ. 
No. 557, Washington, 1958, p. 84. 


WIiIRAA 








‘olume 19 


1as not 
of both 
role of 
mation 
in the 
per se, 
on and 
‘actions 
ase. 


gaps in 
oth on 
=]. Part 
taining 
may be 
hensive 
[deally, 
on and 
per de- 
dle de- 
h other 
workers 
em. 

rythro- 
ered on 
vitro. In 
resizing 
or iron 
ost all 
‘ith the 
yrin 9. 
e most 
oroteins 
of the 
as been 
role of 
and of 
o_ bear 
ficiency 
should 
1 other 


f hypo- 
and/or 
tigation 
workers 
release 
level of 
l. Theg 
to the 
oxidase. 


cil, Publ. 
cil, Publ. 


cil, Publ. 





July 1960 


23. 
24. 
25. 
26. 
27. 
28. 
2g. 
30. 
3. 
32. 
33- 
34. 


49. 
50. 


51. 


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39. Apams, D. H. Biochem. J. 54: 328, 1953. 
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INTERACTIONS OF 


52. 
53: 


54: 


55: 


56. 


57: 
58. 


59- 
60. 
61. 


62. 
63. 


64. 
65. 
66. 
67. 


68. 


69. 


70. 


72. 


73: 
74: 
75: 
76. 
77: 
78. 
79: 
80. 
81. 
82. 
83. 
84. 


85. 
86. 


87. 
88. 


MINERALS 665 
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Busu, J. A., W. N. Jensen, G. E. Cartwricut Anp M. M. 
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Cartwricut, G. E., C. J. Guster, M. E. Laney anp M. S. 
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Jensen, W. N., J. A. BusH, HELEN AsHENBRUCKER, G. E. 
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Busu, J. A., W. N. JENSEN, J. W. ATHENS, H. ASHENBRUCKER, 
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Granick, S. J. Biol. Chem. 149: 157, 1943. 

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GREEN, S. AND A. Mazur. Fed. Proc. 16: 188, 1957. 

GreEN, S., A. K. Sana, A. W. CARLETON AND A. MAzur. 
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Chem. 214: 91, 1955- 

FINEBERG, RicHARD A. AND Davip M. GREENBERG. J. Biol. 
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Marston, H. R., J. H. LEE anp I. W. McDona.p. J. Agr. 
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AND P. H. Lowy. J. Biol. Chem. 196: 669, 1952. 

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J. 70: 4, 1958. 

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Ricuert. Fed. Proc. 18: 352, 1959. 

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1958. 

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1958. 

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421, 1931. 

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Teacue, H. S. anp L. E. Carpenter. J. Nutrition 43: 389, 
1951. 


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J. Biol. Chem. 234: 40, 1959. 


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Biol. Chem. 234: 46, 1959. 








Interrelations of copper, molybdenum and 


sulfate sulfur in nutrition 


RUSSELL F. MILLER AND R. W. ENGEL 
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute, Blacksburg, Virginia 


j- ESSENTIAL ROLE of molybdenum in plant nutrition 
has been well documented. Molybdenum is known to 
function in nitrogen fixation by soil microorganisms. 
Evidence for the essentiality of molybdenum for animals 
is emerging. Molybdenum has been reported to be 
essential for growth in chicks and poults fed purified 
diets (62, 63). Whether or not a growth response to low 
levels of dietary molybdenum can be expected may 
depend upon dietary history. Thus hatchery chicks did 
not respond to the element (46) while chicks obtained 
from dams fed a diet containing reagent-grade minerals 
or the molybdenum antagonist, tungsten, responded 
moderately (46, 74). Previous dietary history may also 
account for the positive results obtained in chicks fed 
practical type diets in one laboratory (45), contrasted 
with negative results obtained by others (46, 74). 

In spite of the virtual elimination of molybdenum and 
xanthine oxidase from all tissues in the rat by feeding 
tungstate, no overt deficiency symptoms were noted 
(39). The more obvious deficiency symptoms, that can 
be demonstrated in the chick under similar dietary 
conditions, may be attributable to the greater need for 
molybdenum and xanthine dehydrogenase in the 
formation of large amounts of uric acid, the end product 
of protein metabolism in this species. 

A nutritional role of molybdenum in the growing lamb 
has been reported (28). Associated with growth stimu- 
lation was an increase in ruminal cellulose degradation, 
an implication that the molybdenum was stimulatory 
for cellulose-degrading microorganisms. Molybdenum 
is essential for the optimal activity of the enzyme, 
xanthine oxidase (17, 18, 64). 

Quite apart from the question of essentiality of 
molybdenum is the effect of high dietary levels. Molyb- 
denum toxicity and factors which influence this con- 
dition in animals will be the primary objective of this 
review. In 1938, in certain areas of England, a condition 
in cattle called teartness was described (31). It was 
established that a high intake of molybdenum was the 
cause of the disease (32, 33). The symptoms resembled 
those shown by cattle in certain copper-deficient areas 
of Holland (8). It became apparent that the forage 
contained acceptable levels of copper; however, supple- 
mental inorganic copper was found to overcome or 
prevent the symptoms of teartness (32, 33). This ob- 


666 


servation indicated that there was an interaction between 
copper and molybdenum; that is, high dietary levels 
of molybdenum produced a conditioned copper de- 
ficiency which was not overcome by the copper levels 
originally present in the feed. Conversely, in a condition 
of hypercuprosis in sheep supplemental molybdenum 
was an effective treatment (27). Molybdenum toxicity 
in ruminants has been observed in many _ locations 
(7, 11, 12, 14, 43, 48, 71, 75). Symptoms of molybde- 
nosis in cattle generally include emaciation, diarrhea, 
achromotrichia, anemia and weakness or stiffness. 
Generally, where such symptoms are observed the feed 
contains from 6 to 36 ppm of molybdenum. 

The manner in which molybdenum produces an 
apparent copper deficiency is ill defined. Using radio- 
active copper and molybdenum administered to the 
bovine and to the rat (10), an interference in bone 
metabolism due to molybdenum was observed and a 
similarity between the behavior of molybdenum and 
phosphorus was found. These workers suggest several 
possibilities to explain the toxic action of molybdenum: 
a) interference, due to a lowered liver copper, in enzyme 
systems necessary for skeletal metabolism; }) inhibition 
of these enzyme systems by molybdenum; and c) com- 
petition between phosphorus and molybdenum _ for 
deposition in the bone. 

The literature on the toxicity of molybdenum for 
experimental animals has been reviewed (29). The 
chemical nature of the molybdenum compound did not 
affect the toxicity of molybdenum to any marked extent 
as long as the molybdenum-containing compound was 
soluble. Thus, molybdenum sulfide was considerably 
less toxic than compounds in which the element oc- 
curred as the molybdate. 

In experimental studies with cattle, the feeding o 
molybdenum resulted in diarrhea, anemia, weakness 
and decolorization of the hair (7, 33). The effect with 
sheep was similar (48, 75). In addition, the wool texture 
and appearance resembled a copper deficiency (23). 
Rats did not develop anemia or diarrhea (41). Oc 
casionally, there was a bone abnormality with rat 
(10, 78). The symptoms of chronic molybdenum toxicity 
in the rabbit were similar to and difficult te distinguish 
from a copper deficiency. Diarrhea and change in the 


XUM 








between 
y levels 
per de- 
rr levels 
ondition 
bdenum 
toxicity 
ocations 
oly bde- 
iarrhea, 
stiffness. 
the feed 


uces an 
x radio- 
to the 
in bone 
1 and a 
um and 
several 
ydenum: 
enzyme 
yhibition 
c) com- 
um for 


1um for 
g). The 
did not 
d extent 
und was 
iderably 
nent oc: 


eding of 
weakness 
ect with 
| texture 
cy (23). 
41). Oc 
jith rats 
1 toxicity 
stinguish 
re in the 








July 1960 


color of the fur were usually not present, but anemia and 
alopecia developed (3). 

There is a species difference with regard to tolerance 
of molybdenum. Since copper and sulfate level in- 
fluence molybdenum toxicity, as will be pointed out 
later, it is difficult to compare results of various experi- 
ments. Of the mammals, it appears that cattle and 
sheep are the least tolerant of dietary molybdenum and 
horses and pigs most tolerant, with rats, rabbits, guinea 
pigs and poultry intermediate. Rabbits are apparently 
more tolerant of molybdenum than rats. The level of 
molybdenum required to reduce chick growth is not 
clear. Thus, it was reported (16, 61) that chicks can 
tolerate levels of dietary molybdenum as high as 2000 
ppm before growth depression resulted, and the re- 


TABLE I. Effect of Varying Sulfate Intake upon 
Blood Molybdenum Level of Sheep (25)* 


Daily SO, Intake Blood Mo 


gm pg/roo ml 
roa 114 
1.8 78 
5 oes 28 
5-7 29 


* Diet supplied 15 mg of Mo per sheep daily. 
PP 5 P k 


TABLE 2. Effect of Dietary Sulfate Level upon Rat Blood 
Molybdenum Levels (54)* 


Sulfate Added Whole Blood Mo 


ppmt pg/ml 
oO 16.6 

400 10.6 
800 9.9 
2,200 10.0 
31300 7.6 


* Diet contained 100 ppm of Mo. 
+ As equimolar mixture Na,SO, and K2SO4. 


sulting growth depression was not alleviated by the 
addition of dietary copper. In contrast, chick growth 
was inhibited by the dietary addition of as little as 200 
ppm of molybdenum (4); and turkey poult growth was 
depressed by 300 ppm of molybdenum (42). In both 
instances, supplemental copper partially alleviated the 
molybdenum-induced growth inhibition. 

The observation that whole liver counteracted the 
toxicity of molybdenum in the rat, and that this effect 
could not be accounted for on the basis of the liver 
copper content, suggested other dietary factors were 
involved in the copper-molybdenum interaction (59). 
One of the important observations pointed to the role 
of inorganic sulfate in mitigating the harmful effects of 
molybdenum (20). In earlier studies it had been es- 
tablished that a diet of lucerne hay, when compared to 
one of oaten hay, reduced the concentration of molyb- 
denum in the blood of sheep fed diets high in molyb- 
denum (19). Subsequently, it was found that the lowered 
blood molybdenum levels of sheep fed lucerne hay, 
when compared to those fed oaten hay, was due to the 


INTERACTIONS OF MINERALS 


667 


higher concentration of inorganic sulfate in the lucerne 
hay (21, 22). The molybdenum concentration of sheep 
blood reflected the molybdenum intake when inorganic 
sulfate intake was constant, but the actual blood molyb- 
denum concentration for any given molybdenum intake 
was dependent upon the amount of sulfate in the diet. 
With the molybdenum concentration of the diet con- 
stant, as sulfate concentration increased, the blood 
molybdenum concentration decreased (table 1). Similar 
results were reported with the rat (54). These results are 
summarized in table 2. 

Using sheep as the experimental animal, increased 
intakes of sulfate, at constant molybdenum intakes, 
caused an increase in blood copper concentration. 
Molybdenum and sulfate enhanced copper losses as 
measured by liver and blood concentrations of this 
element in cattle (57). The ration contained 0.03 % 
inorganic sulfate. The sulfate effect on copper loss was 
not observed, however, if the ration contained as much 
as 0.1% inorganic sulfate (15). Supplemental molyb- 
denum decreased the liver copper concentration of 
barren ewes (1). In pregnant ewes, dietary molybdenum 
increased the liver copper concentration, but this effect 
was eliminated when sulfate was fed with molybdenum. 
Sulfate additions to the molybdenum-supplemented diets 
reduced the liver copper concentration of the new-born 
lambs. A very rapid fall in liver and blood copper 
concentration was found (82) when sheep were fed a 
high-molybdenum diet (3-10 ppm) and high sulfate 
diets (0.10-0.70%). Sheep fed a basal diet containing 
5-2 ppm of copper, 0.8 ppm of molybdenum and 0.04 % 
inorganic sulfate maintained normal liver and blood 
copper concentrations (83). When fed the same diet 
supplemented to provide 5.1 ppm molybdenum and 
0.40% inorganic sulfate, there was a progressive fall in 
liver and blood copper levels, and dystrophic wool and 
hypochromotrichia were observed. With only sulfate 
added to the basal diet, liver copper levels fell but not to 
levels associated with hypocupremia. With only molyb- 
denum supplementation, the copper level was also 
depressed moderately. Enzootic hypocupremia in New 
South Wales could be explained in terms of the copper- 
sulfate-molybdenum interaction, since the levels of these 
minerals in natural herbage simulated the levels used in 
these studies. 

In the interpretation of copper retention data in 
ruminants, it is worth mentioning that internal parasites 
may have a depressing effect on copper retention as 
measured by liver and blood copper levels (5, 36, 70). 

Using rats as the experimental animal, it was reported 
that increased levels of dietary sulfate, at constant 
molybdenum intake, resulted in a decrease in blood 
copper concentration which is the reverse of that found 
in sheep. The use of a different species and of different 
experimental conditions may explain this apparent 
anomaly. 

The addition of sulfate to the diet resulted in an 
increase in the urinary molybdenum excretion, an 
increase in fecal molybdenum excretion, and molyb- 








668 


denum loss from certain tissues in molybdenum-fed 
sheep. Dietary sulfate, or methionine, increased the 
urinary excretion of molybdenum in molybdenum-fed 
sheep (67). 

The effect of sulfate upon molybdenum-fed rats has 
been reported by a number of investigators (54, 56, 78, 
79). The growth inhibition caused by 75 or 100 ppm 
of molybdenum could be mitigated by the addition of 
2,200 ppm of inorganic sulfate. The inclusion of molyb- 
denum in the diet caused an increase in the liver and 
blood levels of molybdenum and copper. This increase 
was reduced when inorganic sulfate was added to the 
diet containing molybdenum. Sulfate overcame the rat 
growth-depression caused by levels of molybdenum up 
to 1,200 ppm. It was demonstrated conclusively that the 
sulfate ion functioned specifically in mitigating the 
growth depressing effect of molybdenum in the rat and 
that the following ions were inactive: citrate, tartrate, 
acetate, bromide, chloride and nitrate. 

Inorganic sulfate reduced the molybdenum-induced 
growth depression of chicks (16, 50, 61). Supplemen- 
tation of the diet with methionine, taurine, sodium 
sulfide or sodium bisulfide was not as effective as in- 
organic sulfate in alleviating the molybdenum-induced 
growth-depression. 

Sulfate metabolism in rabbits fed diets high in molyb- 
denum has been studied. Lowered levels of S*® were found 
in the liver and bones, and the urinary excretion of S** 
was higher in rabbits fed molybdenum compared to 
those fed the control diet (30). These workers point out 
that an important factor in the beneficial effect of sulfate 
lies simply in its replacement value, since tissue retention 
of radiosulfate was depressed by dietary molybdenum. 
The higher content of total sulfur in bones in molyb- 
denum toxicity was interpreted as evidence of inter- 
ference in the ossification process and a resultant higher 
concentration of cartilage of which sulfate is an im- 
portant constituent. 

The achromotrichia reported in copper deficiency in 
the rat (40) appears to have its counterpart in chicks 
in the condition known as feather depigmentation. 
Molybdenum alone, as a copper antagonist, failed to 
induce this condition (80), but the inclusion of dietary 
sodium thiosulfate with molybdenum resulted in feather 
depigmentation which could be prevented by additional 
dietary copper (49). 

It is clear that both dietary copper and dietary sulfate 
ions function in alleviating the harmful effects of excess 
dietary molybdenum. These interesting interrelations 
have been demonstrated in a number of animal species, 
including sheep, rabbits, rats, chicks and turkeys. Sulfate 
supplementation of molybdenum-containing diets results 
in decreased molybdenum retention and in increased 
urinary excretion of molybdenum aside from the passive 
effect of increased urine volume associated with high 
sulfate intakes. The sulfate effect is not shared by other 
ions, and sulfate derived from endogenous degradation 
of sulfur amino acids is also effective in increasing urinary 
excretion of molybdenum. Thus, the effect of the sulfate 


FEDERATION PROCEEDINGS 


Volume 19 


ion is, at least in part, explained on the basis of its 
ability to reduce the tissue concentration of molyb- 
denum (76). 


EFFECT OF OTHER MINERALS ON COPPER METABOLISM 


In addition to the effect of molybdenum and sulfate, 
a number of minerals apparently can influence copper 
metabolism. High dietary levels of zinc, nickel and 
manganese limited copper storage in the liver of sheep 
(24). High manganese intake has also been noted to 
block or antagonize the limiting effect of molybdenum 
on copper retention in sheep (25), although not in cattle 
(57). Molybdenum and manganese together exhibited 
a severely limiting effect on copper retention in sheep 
when the diet was high in protein. These results suggest 
several factors, in addition to sulfate, are operative in 
influencing copper retention in this species. They are of 
further interest because the copper-molybdenum-sulfate 
imbalance in pasture herbage does not always explain 
the hypocuprosis in sheep and cattle that is seen in 
parts of England and New Zealand (2, 13). 

High levels of dietary zinc decreased rat growth and 
hemoglobin formation, liver copper concentration, and 
liver and heart cytochrome oxidase activity (47). 
Added copper partially restored the hemoglobin level 
and completely restored the cytochrome oxidase activity 
and the copper content of the liver to normal levels. 
Two groups of workers found that high levels of zinc 
produced an anemia in rats that supplemental copper 
could correct (38, 73). Molybdenum and zinc feeding 
reduced rat growth more than molybdenum feeding 
alone (38). Contrasted with the above results, which 
suggested that zinc may antagonize copper or enhance 
the antagonistic effect of molybdenum, is the report 
which suggested no interaction (44). 

It has been reported that iron sulfide limits liver 
copper storage in sheep, and it appeared that excess 
dietary calcium carbonate reduced liver copper levels 
(24). Excess dietary calcium carbonate reduced rat 
growth that could be partially overcome by supplemental 
copper (9, 51). In addition, liver copper appeared to 
be reduced by the excess calcium carbonate, an ob- 
servation which was not confirmed in the subsequent 
study. In swine, excess dietary calcium carbonate 
reduced growth, an effect that added dietary copper 
could not overcome. A significant reduction in kidney 
copper and iron concentration by feeding excess dietary 
calcium carbonate was observed (53). 


EFFECT OF PROTEIN LEVEL 
MOLYBDENUM TOXICITY 


UPON 


In addition to the mitigating effect of dietary copper 
and sulfate, dietary protein also appeared to be in- 
volved in alleviating molybdenosis. As is well established, 
low-protein intakes augment the toxicity of other 
elements, such as selenium (72) and cadmium (34). 
From a review of the literature on molybdenum toxicity 
in the rat it appears, among other variables, that the 


emeaneemeen 





XUM 





7olume 19 


s of its 
molyb- 


YLISM 


sulfate, 
copper 
cel and 
of sheep 
oted to 
odenum 
n cattle 
chibited 
n sheep 
suggest 
ative in 
y are of 
1-sulfate 
explain 
seen in 


vth and 
on, and 
y (47). 
in level 
activity 
| levels. 
of zinc 
copper 
feeding 
feeding 
_ which 
*nhance 
report 


ts liver 
t EXCess 
r levels 
ced rat 
emental 
ared to 
an ob- 
sequent 
rbonate 
copper 
kidney 
dietary 


copper 
be in- 
blished, 
f other 
n (34). 
toxicity 
hat the 





July 1960 


level of molybdenum required to cause a reduction in 
growth is greater when the dietary protein level is 
adequate or above. Generally at least 400 ppm of 
molybdenum are required to inhibit rat growth. A 
significant reduction in rat growth can be demonstrated 
with diets containing 100 ppm of molybdenum if the 
dietary protein is restricted to 10 to 12% of casein. 

The observation that methionine supplementation to 
a methionine-adequate diet alleviated a molybdenum- 
induced rat growth inhibition (37) suggested a possible 
mechanism by which extra protein may reduce molyb- 
denum toxicity. Preliminary results indicated that 
dietary protein level had a marked influence upon the 


TABLE 3. Effect of Dietary Casein Level Upon Molybdenum 
Toxicity (4 to 6 Rats/ Treatment, 6 Replicates) 


Treatment —SO:,-Mo T30#" sox, +Mot +S0:,*+Mot 
6-wk. weight gain, gm 
8% Casein 60 66 26 52 
12% Casein 109 115 74 104 
18% Casein 126 140 133 138 





* 2,000 ppm SQ, as an equimolar mixture Na2SO, and K2SO,. 
+ 100 ppm Mo as Na2MoQ,. 


TABLE 4. Effect of Dietary Casein Level upon Blood and 
Liver Cu and Mo Levels of Molybdenum-Fed Rats 
(¢ Rats/ Treatment, 2 Replicates)* 


Liver | Blood 


Treatment Cu Mo Cu Mo 


ppm dry, fat-free ppm whole blood 


8% Casein 35 29 


7 12 
12% Casein 18.5 12 3.8 6.2 
18% Casein 14 12 1.8 4.6 





* Diet was low in sulfate and contained 100 ppm of Mo. 


toxicity of molybdenum (52). Using a low-copper, low- 
sulfate basal diet, the toxicity of molybdenum, as 
measured by rat growth, increased as the casein level 
of the diet decreased (table 3). The results indicated that 
100 ppm of dietary molybdenum caused a marked 
growth depression when 8% of casein was fed. The 
growth depression could not be overcome by the ad- 
dition of 2,000 ppm of inorganic sulfate. When 12% of 
casein was fed, 100 ppm of dietary molybdenum caused 
a growth depression that could be alleviated by the 
addition of 2,000 ppm of inorganic sulfate. No growth 
depression was noted when the diet contained 18% of 
casein. The small growth stimulation, as a result of 
inorganic sulfate supplementation of the control diet, 
was not statistically significant nor was it consistent in 
all replicates (table 3). 

Methionine did not appear to be as effective in 
overcoming the growth-depressing action of molyb- 


INTERACTIONS OF MINERALS 669 


denum as was an equivalent level of sulfur as inorganic 
sulfate. The results of a limited study (table 4) indicated 
that high-protein feeding effected a reduction in the 
liver and blood molybdenum and copper levels of 
molybdenum-fed rats. Results of a carefully conducted 
balance study in humans (55), using as subjects pre- 
adolescent girls, indicated that with molybdenum intake 
constant (approximately 70 yg per subject daily), 
average molybdenum retention was increased five-fold 
when the protein intake decreased from an average of 
2.5 gm per kilogram of body weight to 0.7 gm _ per 
kilogram of body weight. The results of these balance 
studies in growing children support the view that an 
adequate dietary protein level is necessary for the 
elimination of dietary molybdenum. 


MOLYBDENUM AND COPPER AND THEIR EFFECTS UPON 
ENZYME SYSTEMS 


The activity of a number of enzyme systems---cyto- 
chrome oxidase (35, 68) succinoxidase and catalase 


TABLE 5. Effect of Dietary Molybdenum Level upon Activity 
of Rat Liver Cytochrome Oxidase and Femur Alkaline 
Phosphatase 


Treatment _ Cyt. C Oxidizedt Phenol Liberated§ 


uM /min/mg liver Ne uM/gm of bone 


Basal 0.172 26.6 


Basal + 100 ppm Mo* 0.134 26.5 
Basal + 300 ppm Mo* 0.144} 21.8 
Basal + 600 ppm Mo* 0.143 18.4 





* Molybdenum added as NazMoQ,. 

7 6 rats/treatment. 

t Significantly different from control at 1% level of prob- 
ability but not different from each other. 

§ 6 rats/treatment, 2 replicates. 


(6g)—have been shown to be reduced by a dietary copper 
deficiency in the rat. It was found (66) that the zm vitro 
addition of molybdenum reduced the aerobic oxidation 
of L-cystine by a crude enzyme preparation obtained 
from the hide of sheep. Furthermore, a crude enzyme 
preparation obtained from the hide of a black molyb- 
denum-fed sheep oxidized tyrosine and DOPA at a 
reduced rate compared to a similar enzyme preparation 
obtained from the same sheep fed a low-molybdenum 
diet. Jn vitro work indicated molybdenum inhibited the 
oxidation of p-phenylenediamine by the copper-con- 
taining protein, ceruloplasmin. 

High-level molybdenum feeding resulted in an alter- 
ation in the activity of a number of rat liver and kidney 
enzymes. Liver and blood alkaline phosphatase activity 
was increased and kidney and intestinal alkaline phos- 
phatase activity was decreased (56, 77, 79, 81). The 
addition of 100 ppm of dietary molybdenum during a 
six-month feeding trial increased liver and blood alkaline 
phosphatase activity in the rat, an effect which could 
be overcome by the oral administration of eight mg per 
day of vitamin E. Histochemical staining for alkaline 








670 FEDERATION PROCEEDINGS 


phosphatase in the liver indicated molybdenum increased 
phosphatase activity in parenchymal cells and_ that 
supplemental vitamin E produced an essentially normal 
picture (58). 
Sulfide oxidase 
molybdenum, although these workers failed to detect a 
disfunction of sulfur metabolism which could in any way 
effect of sulfate against 
It has been suggested 


activity was reduced by dietary 


account for the protective 
molybdenum toxicity in the rat. 
that the effect of sulfate may be due to the result of an 
influence of the sulfate ion on the permeability of cell 
membranes to molybdenum (26), but attempts to 
demonstrate any influence of the sulfate ion on the rate 
of diffusion of molybdenum through the red blood cell 
wall were unsuccessful (66). The hypothesis has been 
advanced that high-level molybdenum feeding produces 
a conditioned copper deficiency. A decrease in the 
activity of liver cytochrome oxidase, which has recently 
been reported (6), lends support to the above hypoth- 
esis. In these studies there was no effect of dietary 
molybdenum level above the minimum level of 100 
ppm of molybdenum (table 5). Since dietary molyb- 
denum alters the activity of alkaline phosphatase in soft 
tissues and produces bone abnormalities (3, 10, 65, 78), 
its effect on the alkaline phosphatase activity of bone 
(femur) in the rat is of interest (60). Molybdenum 
feeding was associated with a significant reduction in the 
activity of extractable bone phosphatase (table 5). 


Volume 19 


SUMMARY 


Although a conditioned copper deficiency is involved, 
the biochemical defects resulting from molybdenum- 
poisoning in animals are largely unexplained at present. 
The detoxifying action of sulfate ion is at least in part 
explained by its ability to reduce the concentration of 
molybdenum in the tissues. The protective effect of 
dietary protein is at least in part explained through the 
formation of sulfate ion during its endogenous deg- 
radation. 

Failure to explain all field conditions of hypocuprosis 
or hypercuprosis on the basis of the copper, molybdenum 
and sulfate content of the feed suggests that other 
dietary factors influence copper metabolism. There is 
some evidence that zinc, manganese and calcium (as the 
carbonate) also may be concerned with copper me- 
tabolism, and that dietary protein may interact with 
these minerals, particularly with manganese. 

Tissue cytochrome oxidase, succinoxidase and catalase 
are reduced in the tissues in simple copper deficiency; 
and preliminary results indicate that a reduction in 
cytochrome oxidase can also be demonstrated in molyb- 
denum-fed rats. 

Molybdenosis results in elevated blood and _ liver 
alkaline phosphatase and a reduction in the concen- 
tration of this enzyme in kidney, intestine, and bone. 
The many and complex interactions that are suggested 
by the studies reviewed here should provide the hasis 
for fruitful further exploration. 


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volved, 
Jenum- 
resent, 
in part 
tion of 
Tect of 
igh the 
is deg- 


uprosis 
denum 
- other 
here is 
(as the 
er me- 
>t with 


atalase 
ciency; 
tion in 
molyb- 
1 liver 
‘oncen- 
| bone. 


rgested 
e hasis 


Biopkys. 


Mc Elroy 
P- 445: 
0, 1945, 
dE. & 
p E. A. 


9, 1959. 


Nature, 
Jealott’s 
Agr. Set. 

72: 15, 
roc. Roy. 
54: 


950. 
FELD. J. 





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INTERACTIONS OF MINERALS 671 


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McCiymont. Nature, London 175: 


DISCUSSION 
Edited by GeorceE K. Davis 


Question: Will you define interaction, specifically mineral ele- 
ment interaction? 

Answer (George K. Davis): Interaction, as we must consider 
it here in this Symposium, may be interpreted to mean the dis- 
placement of one element by another in a reaction within the 
animal body. A perfect example would be the alteration of a 
biochemical reaction by the replacement in a compound of one 
element by another. Interaction must also be interpreted to in- 
clude the influence which one element or group of elements 
may have upon another element, thereby altering its metabo- 
lism either favorably or unfavorably. While we are particularly 
concerned with the reactions in the tissues of an animal, it is 
impossible to ignore changes which occur in the diet and there- 
fore the total economy of a given species. 


PHOSPHORUS AND CALCIUM 


Question: Does the mechanism of calcium transport across the 
wall of the gut account for the adjustment of animals and 
humans to very low levels of calcium in the diet? 

Answer (R. H. Wasserman): Recent work done by Dr. 
David Schachter and colleagues (Am. J. Physiol. 196: 357, 1959) 
and repeated by us at Cornell suggests a mechanism to explain 
the more effective utilization of calcium on low calcium diets. 


XUM 


An in vitro system was used in which everted gut sacs from rats 
were injected with Wilson-Wiseman solution labeled with 
Ca**®; the external incubating solution was equally labeled. 
Following the incubation of the everted sac under usual con- 
ditions, a ratio is established between the Ca*® label inside 
and outside. A ratio greater than one indicates a transference 
against a concentration gradient, and is suggestive of an 
active transport system for calcium. 

We have shown, using this technique, that the everted gut 
sacs from animals raised on a low calcium diet showed an 
inside: outside ratio of 3.2 whereas animals raised on a high 
calcium diet showed a ratio of 1.7. These results indicate a 
more efficient transport of calcium from the mucosal to the 
serosal portion of the gut by animals raised on the low calcium 
diet. 

In other experiments by Schachter and colleagues, a similar 
tendency for increased calcium transport has been shown in 
pregnant animals in contrast to nonpregnant and in young 
animals in contrast to mature animals, thus a more efficient 
transport system may represent an adaptation or increase in 
efficiency with a given dietary intake. 

It should be pointed out that the proposed ‘active’ trans- 
port system is highly specific for calcium. Magnesium will 








672 FEDERATION PROCEEDINGS 


inhibit this process and perhaps this explains some of the 
antagonism between magnesium and calcium. Barium and 
strontium are not transported by this system. 

Question: Since this technique involves a transport or move- 
ment through the gut and into a solution which represents, 
theoretically at least, the visceral cavity, to what extent can 
these results be extrapolated to the intact living animal? 

Answer (R. H. Wasserman): This of course is a question 
which may be raised of any in vitro system. However, this may 
be partially answered by seeking similarities between data 
obtained with the everted gut sac and information from the 
live animal. 

Examples of these analogies include a discrimination between 
calcium and strontium at both the duodenal level and the ileal 
level in vitro and in vivo; the preferential movement of strontium 
over calcium into the ileum under both conditions; the partial 
dependence of the movement of calcium across the intestinal 
membrane in the total animal and in the gut sac upon vitamin 
D. In the rachitic animal, the in vitro transport system activity 
is decreased and can be restored with vitamin D. Studies by 
Drs. Harrison and Harrison (Johns Hopkins) have also demon- 
strated that vitamin D is involvec in increased permeability 
of the intestinal tract to calcium. 

Question: Is it possible that the existence of active transport 
systems for trace elements would differ under conditions of 
marginal dietary deficiency as compared to systems relatively 
free of these elements? 

Answer (R. H. Wasserman): I do not believe that we can 
generalize that there is a transport system for all trace elements 
nor for all elements. A transport system for iron has recently 
been described but a transport system for other trace elements 
has not been demonstrated so far as I am aware. 

Question: What evidence do we have for a role of calcium, 
phosphorus, vitamin A and vitamin D in the formation of 
salivary calculi? 

Answer (R. H. Wasserman): I am not familiar with work 
that has been done in this area. 

Question: Would you suggest that the consumption of milk 
is the best protective mechanism against strontium-go? 

Answer (R. H. Wasserman): The best protective nutritional 
practice would be the elimination of all sources of calcium 
contaminated with radioactive strontium which would mean 
using calcium that had been buried in the earth for millions of 
years. However, in view of dietary realities and the need for 
protein and carbohydrates as well as calcium and other 
minerals, there is real merit to the concept that at the present 
time our safest food with respect to body accumulation of 
strontium-go is milk. 

Based on observations with respect to strontium and 
calcium ratios in various food products, less total strontium-go 
retention would result if all of the calcium in the diet was 
obtained from milk. 

Question: Is there any hazard in the consumption of stable 
strontium as it occurs in the diet? 

Answer (R. H. Wasserman): No. There is one report, as 
yet unconfirmed, that small amounts of strontium are necessary 
for good ossification. High strontium levels have been used 
therapeutically in osteoporosis. The action of strontium is 
unknown in this regard and there have not been consistent 





Volume 19 


increases in skeletal mineralization but the patient seems to 
feel better on the high levels of strontium. 

Question: Is phosphate more effective than calcium in elim. 
inating strontium? 

Answer (R. H. Wasserman): No. In older animals both 
calcium and phosphorus are necessary to reduce the retention 
of strontium. Elevating either calcium or phosphorus in the 
diet has little effect, but an elevation of both calcium and phos- 
phorus causes a reduction in strontium retention. In young 
animals it has been found that increased dietary levels of 
calcium decrease radiostrontium retention. 

Question: Should the recommended dietary levels for other 
minerals be conditioned on the basis of a given calcium intake? 
Would you comment on high calcium as a cause of kidney 
stones? 

Answer (R. H. Wasserman): It is very difficult to make any 
generalization here with regards to the effects of different 
calcium levels upon the status of an individual. However, the 
adaptive mechanisms such as have been described for calcium 
certainly indicate that the net retention of calcium may be 
essentially the same whether the animal is on a high or low 
dietary calcium intake. 

Answer (R. W. Engel): Dr. L. A. Maynard at one time 
brought to my attention that a ruminant on alfalfa alone with 
a rather wide ratio of calcium to phosphorus, on the order of 
5 to 1, will do quite well in terms of growth, production and 
reproduction. 

Answer (George K. Davis): Dr. C. F. Huffman of Michigan 
State has pointed out that this last statement is true only if the 
level of phosphorus and perhaps some other components in the 
alfalfa are sufficiently high. 

You will recall that at the Federation meeting a year ago a 
symposium on the effects of high calcium was held. It was 
pointed out in that symposium that the adverse effect of high 
calcium is exhibited at low levels of intake of other mineral 
elements. Increasing the intakes of such elements as iron, phos- 
phorus, iodine, manganese and zinc, above minimum levels 
will counteract the adverse affects of increased calcium in the 
diet. 

With respect to the effect of calcium on stone formation, it 
was pointed out in that symposium that the conditions where 
high calcium intake has an adverse effect associated with in- 
creased urinary calculi appear to be limited to patients, usually 
ulcer patients, who have been on a high bicarbonate intake 
which is far from a usual situation in any area let alone the 
so-called ‘stone belt’. 

Answer (R. F. Miller): In some of our swine studies in which 
the animals were on a low copper diet but with very respectable 
levels of the other trace elements, increasing the calcium re- 
sulted in poor growth and this was not corrected by increasing 
the copper level. There is of course the possibility that the diet 
was low in a mineral which was not recognized as marginal, 
but in this particular instance increasing the calcium from .65 
to 1.25% interfered with growth. 


CALCIUM 


Question: It appears that we have little information as to 
where in the body our mineral elements function. Recently 
potassium-40 has been used in estimating the total muscle mass 





WIAA 





lume 19 
ems to 
1 elim- 


s both 
ention 
in the 
1 phos- 
young 
vels of 


- other 
ntake? 
kidney 


xe any 
ferent 
*r, the 
ilcium 
ay be 
or low 


- time 
e with 
der of 
n and 


-higan 
if the 
in the 


ago a 
t was 
f high 
ineral 
phos- 
levels 
in the 


on, it 
where 
th in- 
sually 
ntake 
ie the 


which 
stable 
m re- 
‘asing 
e diet 
ginal, 
m .65 


as to 
ently 
mass 





July 1960 


of the intact animal. Is there some way in which we can measure 
the total skeletal mass in a similar fashion? Can we com- 
partmentalize any of the trace elements in a similar fashion? 

Answer (R. H. Wasserman): There is no normally occurring 
radioactive isotope of calcium. At present an attempt is being 
made to provide quantities of calcium-47, but this is the nearest 
approach to the solution of the problem which you pose. 

Answer (G. C. Cotzias): I think it is well to point out that we 
still lack basic information on how many of our nutrients func- 
tion. Until we can enlighten ourselves on the biochemical reac- 
tions within the body, we are far from understanding inter- 
actions. 

Answer (Gennard Matrone): It should be pointed out that 
when we study interactions, we must, in reality, study response 
curves rather than single points. This is especially true when 
we study more than one variable as is the case when we study 
differing ratios as well as absolute quantities of elements such 
as copper and iron. 


MOLYBDENUM 


Question: What is the relationship of dietary molybdenum to 
the optimum level of xanthine oxidase? Or in other words, is 
there a molybdenum requirement? 

Answer (R. W. Engel): While it is well demonstrated that 
molybdenum is a part of xanthine oxidase, there is real question 
as to the essentiality of xanthine oxidase in the rat. I believe 
there is more evidence for the essentiality of molybdenum in 
poultry, although even here I am uncertain as to the agree- 
ment, among investigators, on this point. It is my opinion that 
at present we do not have adequate evidence to say that there 
isa need for dietary molybdenum. 

Answer (L. C. Norris, Cornell University): While our results 
at Cornell University have not yet been published, they showed 
that when chicks were fed a purified isolated soybean protein 
diet, containing 1.9 ppm of molybdenum, supplementation 
with 0.5 ppm molybdenum increased the rate of gain an average 
of 6.4% in five comparable experiments, using duplicate lots of 
20 chicks each per treatment in each experiment. The in- 
creased gain was highly significant statistically (P_ < 0.005). 

I should point out that we have been unable to develop a 
purified casein diet with less than .24 ppm of molybdenum. 
In contrast to the results with the isolated soybean protein 
diet, the chicks fed the casein diet showed no requirement 
for additional molybdenum. I believe that Dr. W. W. Wester- 
feld (J. Nutr. 59: 539, 1956) and his group at the State Uni- 
versity of New York Medical College at Syracuse were able 
to develop a casein diet with as little as .o2 ppm of molybdenum 
and observed no requirement for additional molybdenum 
when the diet was fed to chicks. 

Answer (Allen A. Kurnick, University of Arizona): It would 
appear from our work with molybdenum and I believe also 
from the work conducted by Dr. Richert and Dr. Westerfeld 
that the first observed effect in chick tissues from birds on low 
molybdenum diets has been with xanthine dehydrogenase. 
Whenever a growth response has been observed with the 
addition of molybdenum to the diet, we have observed an 
increased activity of xanthine dehydrogenase. In 1957 we 
reported in an abstract in Poultry Science that the molybdenum 


XUM 


INTERACTION OF MINERALS: DISCUSSION 673 


in Drackett protein was less available to the chick and to the 
turkey poult but more available to the rat. 

Question: Would you describe molybdenum toxicity? 

Answer (R. W. Engel): At low levels of molybdenum intake, 
it would appear that a molybdenum toxicity is actually a copper 
deficiency. In other words, there may be adequate copper 
present and actual copper storage in an animal, but with 
molybdenum present the symptoms of a copper deficiency 
develop. 

Answer (R. F. Miller): I believe there may be a molybdenum 
toxicity induced by higher levels of molybdenum intake that is 
distinct from the copper deficiency syndrome that results from 
lower levels of molybdenum intake. Under these conditions, 
we do not see the typical copper deficiency symptoms of 
anemia, diarrhea, bone effects, hair effects (achromotrichia) 
but we do have a cessation of growth, poor condition and a re- 
duction in activity of certain enzymes (U. S. Public Health 
Bulletin 293, published in 1945, deals specifically with this 
toxicity problem). Dr. J. R. Couch’s group (Texas A. & M.) 
have reported that supplemental dietary copper did not 
alleviate a molybdenum-induced chick growth inhibition. 

Answer (Gennard Matrone): In some work with rabbits, we 
have observed that liming soybeans has improved the growth 
and development of rabbits on the soybean diet. The improved 
quality of the soybeans was associated with an increase in the 
molybdenum content, which could be obtained either by lim- 
ing or by the addition of molybdenum to the growing plants. 
Addition of molybdenum to the low molybdenum soybeans 
did not give the improved rabbit performance obtained with 
soybeans fertilized with molybdenum or with lime. On the sug- 
gestion that the improved performance was due to improved 
quality of protein, egg albumin was added to the diet but with- 
out beneficial effect. Thus we have a situation where addition 
of molybdenum improved the soybean diet when added to the 
growing soybeans, but the mechanism or the identity of the 
improvement has not been ascertained. 

Answer (R. W. Engel): I believe that evidence from our 
laboratory supports the point of view that the toxicity of 
molybdenum at least at comparatively low levels is due to an 
interference with copper utilization. Molybdenum is rapidly 
removed from the body and may entirely leave the system in a 
matter of 3 or 4 days. Molybdenum removal is not accom- 
panied by a copper loss. As soon as the moybdenum is removed 
the copper appears to function normally. 

Question: What is the relationship between molybdenum 
and bone formation? 

Answer (R. F. Miller): Our results would appear to bear out 
the hypothesis, that the reduced calcification due to excess 
dietary molybdenum may be due to an interference with the 
phosphatase enzymes and particularly bone phosphatase. One, 
of course, must make the assumption that phosphatase is neces- 
sarily a part of the bone calcification mechanism. Certainly 
the decreased calcification which accompanies molybdenum 
toxicity is a frequently observed symptom. 

Some of our results, especially with the young animal, sug- 
gest that the relationship of molybdenum to decreased bone 
formation may be through the sulfate relationship. The car- 
tilaginous matrix of bone involves the metabolism of consider- 
able sulfate (chondroitin sulfate). It is possible that molyb- 








674 FEDERATION PROCEEDINGS 


denum may adversely affect sulfate metabolism and thus pre- 
vent normal calcification. 

Answer (George K. Davis): In our own studies with molyb- 
denum toxicity, we have observed that in young animals there 
is a sharp reduction in alkaline blood phosphatase and that 
there may be an almost complete failure of bone ossification. In 
older animals, suffering from molybdenum toxicity, we have 
observed a change in bone structure which, when accom- 
panied by low copper intakes, resembles an osteoporosis in 
cattle. At the terminal stages there may be a very sharp increase 
in alkaline blood phosphatase. With copper intakes ranging 
from 10 to 50 ppm and with the development of molybdenum 
toxicity, there appears to be a change in the crystalline struc- 
ture of bone but not a decrease in the total ash content. Under 
both circumstances multiple fractures are a common result. 


ZINC 

Question: What has been the relationship between casein and 
Drackett protein with respect to zinc availability? 

Answer (R. M. Forbes): Our results indicate a lower availa- 
bility of zinc from the isolated soybean protein. In a recent 
issue of the Journal of Nutrition (J. Nutr. 70: 53, 1960) we re- 
ported that rats fed a diet of casein containing 7 ppm of zinc 
excreted 16% of the zinc in the feces. A similar diet, but con- 
taining Drackett protein showed 56% of the zinc excreted in 
the feces thus indicating a lower availability from the casein. 


MANGANESE 

Question: Divalent manganese and divalent magnesium are 
interchangeable in some in vitro enzyme systems. Is there any 
evidence that dietary manganese may eliminate a dietary mag- 
nesium deficiency? 

Answer (G. C. Cotzias): Evidence which we have at the pres- 
ent indicates that most divalent manganese in the body will 
either be rapidly changed to trivalent manganese or eliminated. 
Therefore, I do not believe that magnesium deficiency would 
be alleviated by adding excess manganese to the diet. 

Question: Manganese and magnesium have been shown to 
replace each other in many in vitro reactions; how do you inter- 
pret this in view of the statement that there are no interactions 
of manganese with other elements within the animal tissues? 

Answer (G. C. Cotzias): In the first place I disagree with the 
assumption that the zn vitro experiments are in fact a parallel to 
in vivo experiments. I believe that elements have distinct ‘per- 
sonalities’. Further, that just as you have distinct syndromes for 
toxicities caused by the different elements such as copper and 
manganese, so you should be able to show distinct deficiency 
changes. Does the body distinguish between two different ele- 
ments? In some of our investigations we endeavored to discover 
an anti-manganese. Not only could we not find an anti-man- 
ganese we discovered that we could not displace manganese 
with another element. We could only displace one kind of 
manganese, namely radiomanganese, with additional man- 
ganese. It appears to us that this specificity is due to the 
presence of a specific receptor in the plasma which we called 
transmanganin because the iron receptor was called transferrin. 

This receptor changes the divalent form to a trivalent form, 


which is stable only when it is bound through a receptor. Our 


Volume 19 


receptor is a protein but the receptor does not have to be a 
protein in all cases. 

Since the trivalent form is unstable and therefore not avail- 
able to the biochemist, he must use the abnormal form which 
is a divalent form. The divalent form of manganese may be 
confused with other divalent minerals such as copper and 
magnesium by the tissues. On the other hand the trivalent 
form found under in vivo conditions is very specific. If we were 
able to conduct our in vitro experiments with the trivalent form, 
it might be possible to reconcile many of the differences which 
at present cause confusion in our biochemistry. 

Question: It has been observed that high levels of manganese 
interfere with hemoglobin formation. Dr. Matrone’s work at 
North Carolina with baby pigs has shown that levels of 125 
ppm retarded hemoglobin formation. Somers and Shive (Plant 
Physiol. 17: 582, 1942) showed that in plants manganese could 
be antagonized by distorting the ratio with iron. Does not this 
suggest an interaction between iron and manganese in the true 
sense? 

Answer (G. C. Cotzias): I am not going to rise to the defense 
of plants, but I believe that the effect of manganese upon hemo- 
globin formation actually should come under the heading of 
toxicology. I believe that these levels of manganese actually 
create a redox system in the gut which is not present normally 
and which is responsible for the abnormalities which have 
been observed. 

Question: In a symposium conducted at these meetings, it 
has been shown that children consuming 3 and 4 milligrams of 
manganese per day showed a significant increase in the reten- 
tion of manganese when the dietetic protein was reduced to 
one fourth the previous intake. Is this not an interaction be- 
tween manganese and protein? 

Answer (G. C. Cotzias): This is quite a respectable type of 
relationship as opposed to the interaction suggested as between 
manganese and magnesium. I welcome this information and it 
is quite concordant with our own notions and results. ‘This is an 
area of most important interaction, the inorganic-organic 
type of interaction. I believe this is a very fruitful area for 
future investigation. 

Question: It appears that a protein or some other organic 
factor is involved with the absorption of iron. Is it not possible 
that this same organic factor may also hold manganese? In 
other words that one may replace the other? 

Answer (G. C. Cotzias): No, I cannot accept this because it 
goes against the quantum rules. The element which may be 
bound to a receptor site is limited by the characteristics of the 
receptor site and this is by no means a haphazard affair. It is 
quite well regulated. 

Answer (George K. Davis): I think we may resolve this situa- 
tion by pointing out that mineral elements are furnished to the 
various metabolic systems of the body through enzyme systems. 
Since enzymes are proteins, it follows then that very specific 
inorganic-organic interactions between mineral elements and 
proteins are always involved in the metabolism of the mineral 
elements 

Question: What is the relationship be.ween manganese and 
calcification as shown in perosis? 

Answer (G. C. Cotzias): I think that the role of manganese 
may be similar to that suggested for copper in that it is related 





XUM 





olume 19 


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July 1960 


to the control of an oxidation system. With a manganese de- 
ficiency there is an interference in protein synthesis, as there 
is of fat synthesis, centralized around the oxidation steps 
necessary in the synthesis of these substances. I believe that 
this is the way in which imperfect bone is formed rather than 
through a direct interaction. 


MAGNESIUM 


Question: What is the method employed to determine mag- 
nesium, especially in the presence of large amounts of phos- 
phorus? 

Answer (Boyd L. O'Dell): Dr. E. E. Pickett is conducting 
the analysis of trace minerals in connection with our work 
and he is now determining magnesium by a flame spectrophoto- 
metric method. We consider that this is a considerable improve- 
ment over the Titan yellow method which in our laboratory has 
always given higher results than those obtained by the flame 
spectrophotometric method. 

Answer (R. M. Forbes): At the University of Illinois we use 
an automatic titration method which I certainly would suggest 
if you have an automatic titrator. At present we are using a 
Sargent Malmstadt titrator which provides a convenient tech- 
nique for running magnesium and calcium at the same time. 
We remove phosphorus with zirconium nitrate. 

Question: Would you describe the mechanism of renal calculi 
development in magnesium deficiency? 

Answer (Boyd L. O’Dell): The kidney is the first organ that 
is damaged in a magnesium deficiency and this is best observed 
histologically. In the guinea pig, gross lesions occur with an en- 
larged kidney, somewhat pale in color, mottled and with a 
rough indented surface. Histologically you can see calcification 
sites in the tubules particularly in the corticomedullary area. 

Question: Would you describe magnesium anesthesia? 

Answer: (Boyd L. O’Dell): I have not observed magnesium 
anesthesia personally, but it has been described in the litera- 
ture. The magnesium salts are administered intravenously. 

Question: Are guinea pigs receiving 60 mg % of magnesium 
in the diet free of pathology? Have you fed levels of 96 to 192 
mg % magnesium in the diet and do such animals remain free 
of pathology? 

Answer (Boyd L. O’Dell) : Guinea pigs which receive go mg % 
of magnesium and 0.4% phosphorus show no gross pathology 
and grow at a maximum rate. No histopathological studies 
have been made. If the phosphorus level is 0.8 to as high as 
1.7% we do see pathology with 90 mg % of magnesium. We 
have fed much in excess of 192 mg % of magnesium to guinea 
pigs without pathology. 

Question: Has a mechanism been proposed for the action of 
divalent magnesium upon calcium and phosphate? 

Answer (Boyd L. O'Dell): I am not aware of a mechanism 
whereby magnesium might regulate the metabolism of calcium 
or phosphate. Some relationships might be suggested. Mag- 
nesium may affect the absorption and kidney excretion of 
phosphorus. There appears to be a relationship between mag- 
nesium deficiency and the development of hyperparathy- 
roidism. In magnesium-deficient animals serum phosphorus is 
elevated, possibly because of reduced excretion. There are 
some results which suggest that magnesium may act in much 
the same manner as the citrate ion. 


INTERACTION OF MINERALS: DISCUSSION 675 


Question: Would you comment on the possibility that the 
various tetanies associated with either season or pasture may 
be associated with magnesium metabolism and are these inter- 
actions? 

Answer (Boyd L. O'Dell): There certainly appear to be die- 
tary interactions in the tetanies. There are two types of tetanies 
that occur in ruminants, particularly cattle, but also in sheep. 

The so-called winter or stall tetany may be a true magnesium 
deficiency although this is far from clear. Grass tetany or the 
tetany that occurs where animals are turned from dry lot to 
lush pasture is not a true magnesium deficiency although it can 
be relieved by administration of magnesium ions and feeding 
magnesium may prevent this type of tetany. 

In the tetanies there exists a low serum magnesium level. 
There are, however, large differences between individual ani- 
mals. Older animals are more subject to these tetanies per- 
haps because the magnesium stored in the bone is less readily 
mobilized in older animals. It has been suggested that reduced 
parathyroid function may account for individual animal 
variation. 

Another suggestion relates to the high level of ammonia 
found in the rumens of animals turned from dry lot to fresh 
pasture. 

Thus the combination of lowered magnesium level in the 
spring forage, higher ammonia in the rumen, higher phosphate 
level in the forage, and perhaps other factors may result in the 
formation of a magnesium ammonium phosphate of low 
availability and therefore lead to a magnesium deficiency. 

Question: Do you know if magnesium ammonium phosphate 
has ever been found in the feces of these animals? 

Answer (Boyd O'Dell): Head and Rook (Nature 176: 262, 
1955) did some work in this area, but I do not believe they re- 
ported this phosphate as being present. 

Question: Is it possible that there may be chelating com- 
pounds in the forage for magnesium such as exist for zinc in 
some feeds? 

Answer (Boyd O’Dell): This is a definite possibility. The 
possibility of chelation is receiving considerable attention and 
although we have no direct evidence, it is a possibility that 
cannot be ruled out. 

Question: Is it possible that the magnesium chlorophyll 
combination prevents the availability of magnesium? 

Answer (Boyd O'Dell): To my knowledge this point has not 
been checked. Such a compound of course represents a chela- 
tion but the stability of the compound, so far as I know, has 
not been studied. 


COPPER AND IRON 

Question: What is the effect of adding additional sulfate to the 
diet of a ruminant animal or of a non-ruminant animal upon 
the copper and iron metabolism. 

Answer (R. W. Engel): Early Australian work demonstrated 
that in certain areas sheep fed hay prepared from oats de- 
veloped a copper toxicity. If these sheep were given alfalfa 
hay instead of oat hay but with the same intake of copper, no 
toxicity developed. The Australian workers were able to 
demonstrate that the oats were low in sulfate and alfalfa con- 
tained much larger quantities. They further demonstrated 
that feeding inorganic sulfate with the oat hay prevented the 








676 FEDERATION PROCEEDINGS 


copper toxicity. The third factor which must be remembered 
was that these diets were also low in molybdenum. 

Answer (Gennard Matrone): So far as I know there have been 
no studies to show an influence of sulfate upon iron metabolism. 

Question: Is there any justification for writing an ion as 
divalent or trivalent in representing the metabolism of iron 
when we know that neither ion could exist as such at physio- 
logical pH’s, but must be coordinated with other compounds? 

Answer (Gennard Matrone): I think such a designation is 
justified since the ionic state may vary depending upon the 
type of chelation. Iron chelated in heme has a valence of two, 
in ferritin a valence of three. Thus I think it is not only per- 
missable but necessary to state the ionic form of the element, 
although not in the sense that it is floating around as a free ion. 

Question: Injectable iron is given to baby pigs and iron nor- 
mally given to anemic infants contains copper. Would you 
comment on the advisability of adding copper to the injectable 
iron? 

Answer (Gennard Matrone): Without direct evidence, I 
could see little advantage in adding copper to such a prepara- 
tion. Copper deficiency takes much longer to develop than 
simple iron deficiency and since a baby pig has a good copper 
store at birth, a deficiency is unlikely because of the catalytic 
amounts required. 

Answer (R. F. Miller): I should like to add that most com- 
mercial iron preparations contain sufficient copper contamina- 
tion to supplement the copper intake. 

Answer (R. W. Engel): Uncomplicated copper deficiency 
is perhaps rare. The Food and Nutrition Board has not seen 
fit to establish a recommended copper allowance. In our ex- 
perience children on natural diets have an intake of approx- 
imately 1.2 mg per day. With protein at the N. R. C. recom- 
mended level, 6 of 12 children were in negative balance and 
6 in positive balance on this level of copper intake. When the 
protein level was reduced to one half of this level, all of the 
children went into negative copper balance. This lower protein 
diet averaged 1.06 mg in copper content. 

Question: It has been reported from Michigan State (J. An. 
Sct. 19: 249, 1960) that adding copper to a diet causing para- 
keratosis corrected the condition as well as zinc additions. 
Would you comment on this? 

Answer (R. M. Forbes): It is my understanding that the addi- 
tion of 125 ppm of copper to the diets which presumably were 
already adequate in copper alleviated parakeratosis and stim- 
ulated growth in spite of the fact that the diets were deficient 
in zinc. It is my understanding that there has been some 
variation in this response. 

Question: Do you know whether the mineral supplements 
used were analyzed for zinc? 

Answer (R. M. Forbes): I believe they used reagent grade 
chemicals, but it is my understanding that only the basal diets 
were analyzed for zinc. A report was not made of the zinc 
content of the diets supplemented with zinc or with copper. 

Answer (George K. Davis): I think it is only fair to point 
out that in biological experiments such variations as have been 
reported here are not uncommon. Since we must of necessity 
deal with feed ingredients produced at different times and 


perhaps even from different areas, it is always possible that an 


Volume 19 


ingredient other than that which we are studying may have a 
critical effect on the end result. 

Answer (W. G. Hoekstra, University of Wisconsin): The 
supplements as such were not analyzed for zinc, but when 
the entire rations to which the supplements had been added 
were analyzed, there was no detectable change in zinc con- 
tent. We used 150 ppm of copper as copper sulfate. I would 
like to say that we have tried using copper in a number of our 
experiments, studying its relationship to zinc and parakeratosis 
and have been unable to confirm the Michigan State work. In 
one or two instances it appeared that copper was antagonistic 
to zine, but certainly we have not been able to show the pro- 
nounced beneficial effect of copper towards parakeratosis. 

Answer (George K. Davis): In work at Florida we have seen 
results in which copper appeared to cure parakeratosis and 
other results in which it had no discernible effect. Perhaps it 
is well to call attention to the work of Dr. R. Braude and his 
group (Brit. J. Nutr. 11: 70, 1957) who reported that 250 ppm 
of copper in the diet was as effective as an antibiotic in pro- 
moting growth in young pigs. Work at other stations has been 
far from uniform in confirming these observations. 

Answer (R. F. Miller): I should like to point out that the 
protein in the diet is of exceptional importance in studying 
the effect of a mineral element. We have observed that a 
given level of molybdenum becomes less toxic as the level of 
dietary protein is increased. These results cannot be entirely 
explained by an increase in the sulfur-containing amino acids. 
Addition of extra methionine has not had the same effect as 
the addition of protein. 

More especially to the point, we have made the observation 
that the vitamin-free casein may have changed since the time 
we made our initial observation on molybdenum toxicity so 
that no longer does 100 ppm of molybdenum show growth 
inhibition when the casein level of the diet is 12%. 

Question: What is-the role of copper in the formation of 
reticulocytes? 

Answer (Gennard Matrone): I must make a conjecture in 
trying to answer this question. Recent evidence indicates that 
copper is an integral part of cytochrome oxidase. Since cyto- 
chrome oxidase functions in the terminal oxidation system 
within the metabolism of the animal, this suggests a basic role of 
copper in terms of cell formation and growth. Thus it may well 
be that copper plays a common role which is concerned not 
only with erythrocyte formation but also the formation of 


other cells. 


ZINC 


Question: Is it possible that the parakeratosis observed in 
swine and attributed to zinc deficiency is actually an allergic 
reaction? 

Answer (R. M. Forbes): I can only give an opinion. It has 
been pointed out to me that the skin lesions in parakeratosis 
and in psoriasis in humans are very similar histologically. This 
would suggest an allergenic reaction. We do not as yet have a 
biochemical lesion which can be specifically related to para- 
keratosis. 

Question: It has been stated that A.D.M. C-1 protein con- 


tains molybdenum. Is this true? 


XUM 








olume 19 
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July 1960 


Answer (Boyd L. O’Dell): We have analyzed this material 
at Missouri and it does contain molybdenum. I might also 
mention in experiments which we have conducted with chicks, 
operated on to separate urine and feces, the molybdenum in 
this protein was highly available and was excreted by the 
kidneys. 

Answer (R. F. Miller): The excretion of molybdenum in the 
urine as an indication of availability seems to be a normal 
physiological reaction with any protein source. Molybdenum is 
absorbed from the gut and is rapidly excreted through the 
kidney. 

Question: In view of the fact that toxic levels of zinc cause 
a change in phosphorus as well as calcium metabolism, do you 
think an interaction exists between zinc and phosphorus? 

Answer (R. M. Forbes): There probably is an interaction, 
but I base this on studies by Dr. Hoekstra and his group at 
Wisconsin rather than the toxicological studies mentioned. 


INTERACTION OF MINERALS: DISCUSSION 677 


Addition of sodium phosphate to diets low in zinc, but con- 
taining adequate phosphorus, so that the phosphorus level was 
doubled to a level of 0.8%, reduced the incidence of skin lesions 
in parakeratotic pigs, although growth rate was not improved. 
So far as I know this is the most clear-cut evidence of such a 
relationship. 

Question; What is the possibility of an existence of a zinc- 
vitamin D interrelationship? 

Answer (R. M. Forbes): I know of no evidence or research 
which would permit a comment on this question. 

Answer (Gennard Matrone): In experiments with rats re- 
ceiving a diet containing .75% of zinc, the animals developed 
soft rubbery bones within 4 weeks when started as young 
animals. However, our results point to an indirect effect on 
metabolism such as that referred to between copper and cell 
development rather than an effect between zinc and _ phos- 
phorus. 


CONCLUSION 


GeorcE K. Davis 


I believe that in the papers presented here and the discussion 
which you have heard, it has been well emphasized that in the 
field of mineral interaction the research is vigorous, the pos- 
sibilities are intriguing and the field is wide open. 

It certainly has been emphasized that we are just beginning 
to touch on the biochemical reactions which are responsible 
for the metabolism of the mineral elements in the body tissues. 
Elements which in the past we have ignored or assigned to a 
minor role in nutrition are now being found to be key elements 
in vital enzymatic reactions. 

As might be expected, the renewed interest in the function 
of trace elements and the interaction of trace elements in 
animals is being reflected in a renewed interest with respect 


to the action of these elements in human metabolism and there- 
fore as constituents of the human dietary. 

No longer is it possible to discuss an element in terms of 
its requirement without considering the other nutrients in the 
diet, whether these be protein, carbohydrate or other mineral 
elements. The research studies which have been reported and 
reviewed here suggest that the interactions in which these 
elements take part make up part of the complex whole that 
we call normal metabolism. 

In the over-all nutrition of human or animal, a deficiency 
or a toxic excess of any given element must be considered 
against a background of the levels of other nutrients which are 
present in the diet. 








Federation Board Proceedings 


1959-1960 


anienan COMMUNICATION IS GETTING to be a major 
problem in scientific affairs. With increasing specializa- 
tion among the various sciences, it is becoming more and 
more difficult for scientists to understand what is going 
on in fields in which they are not technically active. The 
very multiplication of terms and abbreviated alphabeti- 
cal complexes increases the difficulty of communication, 
and unless we all make some serious effort at communi- 
cating better with each other, our entire scientific effort 
may be jeopardized. It is thus appropriate that attempts 
be made to improve communication between scientists 
on general terms. This may help to focus attention on 
current significant progress in the different sciences. 

The wide extension of our scientific publication serv- 
ices, with the stereotyped character of our reports, espe- 
cially in the experimental biological sciences, is generat- 
ing serious problems. These involve not only adequate 
indexing, abstracting, and reviewing, but also the sheer 
mechanics of publication itself. We are also faced with 
an almost impossible task of preparing our libraries to 
accommodate this material. With such a flood of new 
verifiable knowledge inundating us, what kind of an ark 
of refuge can we find for ourselves? Can we weather the 
flood? 

These problems of communication are extremely prac- 
tical ones facing our Federation. With over 11,000 in 
attendance at our annual meetings, and with abstracts 
going beyond 2,500, the difficulty of programing and ar- 
ranging the meeting is ballooning to frightening propor- 
tions. 

Communication between the various Societies com- 
prising the Federation is greatly facilitated during the 
annual meeting. However, we can meet only m Atlantic 
City or in Chicago, because of the large number of our 
members and their associates who attend our annual 
meeting. This does require exceptional hotel accommo- 
dations. We are meeting for the next 3 years in Atlantic 
City. There an improved central auditorium will help 
the meetings. 

What are we going to do if our number continues to 
increase to 15,000? Perhaps we can decrease the load by 
having fall meetings for all the Societies. The fall meet- 
ings are very successful both for the physiologists and 
pharmacologists. 

The Advisory Committee has been authorized to 
study the character of future meetings and to bring 
recommendations to the Federation Board. 


NEW FEDERATION ACTIVITIES 


In connection with our annual meeting, and the prob- 
lem of adequate communication, we have inaugurated a 


~ 


/ 


new public relations program. Primarily this is designed 
to improve communication between the scientists and 
the general public. We do have a responsibility of getting 
our work across to the public, so that it may be utilized 
for public welfare. 

At the Chicago meeting we were fortunate in having 
the services of a special Public Information Committee. 
This functioned admirably under the chairmanship of 
Dr. Klaus Unna of the University of Illinois School of 
Medicine. An efficient Press Room public information 
service was developed especially for the meeting under 
the joint direction of Dr. Sidney Negus of the Medical 
College of Virginia and Mr. Ralph Rohweder of the 
National Society for Medical Research. As the members 
of the Federation become increasingly aware of our pub- 
lic information program, they may learn to use the Press 
Room facilities more satisfactorily and thus promote better 
communication with the general public. 

Scientists in general may think that they can work as 
they often formerly did, in an ivory tower. No more. 
Science is now an accepted part of our cultural pattern. 
If wise public policies are to be made on the basis of veri- 
fiable information about ourselves and our environment, 
then it is necessary that scientific information be made 
available to ordinary people in words that they can 
understand. Scientists generally will find it wise to coop- 
erate more freely with science writers so that their scien- 
tific findings can be interpreted for general public under- 
standing. 

The Federation is now actively cooperating with the 
American Medical Association on the important matter 
of promoting mutually satisfying interprofessional rela- 
tions among the health professions. This matter got off to 
a poor start, but fortunately has developed in a way that 
may bring very far-reaching and beneficial results gener- 
ally to all those who are concerned with the affairs for 
the many and growing health professions. 

While not a new matter for Federation interest, the 
problem of appropriate care for experimental animals 
remains always before us. Fortunately we are closely 
associated with the National Society for Medical Re- 
search. The activities of this organization have been well 
directed for several years by Ralph Rohweder. He is 
keenly alert to all efforts at interfering with the con- 
trolled use of experimental animals, and he watches 
carefully for the development of any fanatical opposition. 


MANPOWER SURVEY IN THE BIOLOGICAL SCIENCES 


An important new Federation activity has been the 
inauguration of a Study of manpower needs in the basic 
health sciences. This is moving rapidly and deserves dis- 


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XUM 


July 1960 


cussion in some detail. It is supported by a grant from the 
National Institutes of Health. The proposal to conduct 
an extensive manpower survey was carefully explored by 
the Advisory Committee of the Federation Board at 
meetings in Washington and also at an extended meeting 
in Laconia, New Hampshire. It has been possible to get 
it underway in a surprisingly short time. 

The survey follows some of the implications resulting 
from a preliminary study of the same sort conducted by 
the American Society for Pharmacology and Experi- 
mental Therapeutics. Here the situation is clear: there 
simply aren’t enough properly trained pharmacologists 
to undertake satisfactorily the enormous screening job 
involved in the thousands of potentially useful new chem- 
icals that are pouring upon us. The efforts of the pharma- 
cology group are thus primarily directed toward recruit- 
ment. Results have been promising. Brochures have been 
prepared for distribution to high school and college 
students, giving some indication of the career rewards in 
professional pharmacology and toxicology. Further, an 
attempt has been made to promote standards of graduate 
teaching in pharmacology in order to provide effective 
training, and grants have been obtained to aid in these 
training programs. 

These considerations led to the realization that a gen- 
eral manpower survey in the experimental biological 
sciences is clearly in order. The purposes of the survey are 
1) to estimate the needs over the next decade for experi- 
mental biologists in federal and state research and oper- 
ating agencies, in industry, in universities and colleges. 
and in private research institutions; 2) to estimate the 
training facilities necessary for meeting these needs, per- 
sonnel, equipment and space, and 3) to promote re- 
cruitment of qualified youngsters for satisfactory careers 
in experimental biological research. 

The Board appointed a well-qualified committee for 
the proposed manpower survey, under the chairmanship 
of E. B. Fred, President Emeritus of the University of 
Wisconsin. Its members include K. K. Chen of the Lilly 
Research Laboratories; Philip Handler of Duke Univer- 
sity School of Medicine; Herbert E. Longenecker, Presi- 
dent of Tulane University; John Nicholas of Yale; Wil- 
liam H. Sebrell of Columbia University School of Public 
Health, and Maurice Visscher of the University of Min- 
nesota. Ward Darley of the American Association of 
Medical Colleges, Thomas D. Kinney of Western Re- 
serve University, Howard A. Meyerhoff of the Scientific 
Manpower Commission and several other consultants 
also function with the survey committee. 

The committee has secured the services of Dr. John T. 
Cowles, who has been engaged in manpower studies for 
some time at the University of Pittsburgh, to direct the 
survey. He has all the equipment and trained personnel 
available for the kind of a survey proposed by the Federa- 
tion. Under a contract with the University of Pittsburgh, 
a broadly devised and logically organized preliminary 
program is under way. It is anticipated that the survey 
will require at least a couple of years. Its results are ex- 
pected to be far reaching. 


FEDERATION BOARD PROCEEDINGS 


679 
BIOLOGICAL HANDBOOKS 


Another new activity of the Federation deals with the 
series of Handbooks of Biological Data. These were 
started under the auspices of the National Academy of 
Sciences-National Research Council. The Federation was 
asked to administer the program and last September re- 
sponsibility for these Handbooks, henceforth to be known 
as Biological Handbooks, was transferred to us from the 
NAS-NRC. In order to guide the project, a committee 
was appointed consisting of Raymund L. Zwemer of the 
Department of State as Chairman, J. W. Heim of Wright 
Air Development Center, T. C. Byerly of the Depart- 
ment of Agriculture, George B. Brown of Sloan-Kettering 
Institute, Frank B. Rogers of the National Library of 
Medicine, J. Franklin Yeager of the National Institutes 
of Health, and Dwight E. Gray of the National Science 
Foundation. Philip L. Altman was made Director of the 
Office of Biological Handbooks. A grant from the Na- 
tional Science Foundation for support of this project 
became effective in January 1960 and further support 
has been obtained from the National Institutes of Health 
and the Department of the Air Force. 

A general biological data handbook, useful for college 
biology undergraduate and graduate students, as well as 
high school teachers, was authorized with a guiding com- 
mittee of Thurlo B. Thomas of Carleton College, C. S. 
Chadwick of George Peabody School for Teachers, and 
Tunis Baker of Hope College. A handbook on Composi- 
tion of Blood and Body Fluids is being compiled, and 
there is authorized a handbook on Growth. The com- 
mittee for the latter is composed of William C. Steere of 
the New York Botanical Garden, Nathan W. Shock of the 
Baltimore City Hospitals, and Warren Andrew of the 
University of Indiana. 

The handbook on Composition of Blood and Body 
Fluids will comprise some 116 tables making about 300 
pages, with an additional 100 pages of text. Each table 
has been reviewed by three or more different reviewers. 
A special handbook of tabular data relating to pharma- 
cology is being reviewed by pharmacologists for advice 
as to its possible usefulness. 


FUTURE ACTIVITIES OF THE FEDERATION 


At the April 10 meeting of the Federation Board in 
Chicago, there was extensive discussion of problems of 
programing for Federation meetings. It was felt that 
machine programing for meetings should be continued, 
and that extension of intersociety sessions is desirable, but 
that complete unification of the Societies’ programs 
should not be attempted. In general our Societies were 
in agreement that the machine programing experiment 
was moderately successful but not definitive. It was felt 
that it might be improved with a more satisfactory system 
of categories. The deadline for receipt of abstracts was 
changed to December 16, 1960. 

The Board received the audited financial statements 
and approved the proposed budget for 1960. We are ina 
sound financial position, thanks to excellent manage- 
ment. 








680 FEDERATION PROCEEDINGS 


The Board authorized expression of interest in the 
over-all problem of documentation, requesting the privi- 
lege of making nominations for membership in the 
United States National Committee to the International 
Federation of Documentation. 

The Advisory Committee was instructed to review the 
problem involving additional space at Beaumont House 
and to recommend action to the Board. 


The Federation has agreed to administer the details of 


awarding travel grants for the 5th International Congress 
of Biochemistry to be held in Moscow August 10-16, 
1961. The 5th International Congress on Nutrition will 
meet in Washington, D. C., September 1~—7, 1960, with 
several thousand scientists from all parts of the world. 
This congress will consider world needs and food re- 
sources, human nutritional status, food processes and 


Volume 19 


additives, lipids in health and disease, animal nutrition 
and food production, nutrition in maternal and infant 
feedings, proteins and amino acids in nutrition, and new 
possibilities in nutritional research. Our Executive Offi- 
cer, Milton O. Lee, is the General Secretary of the Con- 
gress. 

There is to be a pharmacology conference in Stock- 
holm in 1961. No details are available as yet regarding 
the International Congress of the Physiological Sciences 
to be held in Leiden in 1962. 

I sincerely appreciate the cooperation and _ willing 
spirit-which has been demonstrated by all those with 
whom I have been privileged to work, and I leave the 
Federation Board with a thank-you to you all. 


CHAUNCEY D. LEAKE 
Chairman 


LIM 








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LEAKE 
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FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY 


FINANCIAL REPORTS 


EXHIBIT A. Balance Sheet—December 31, 1959 


Assets: 
Cash in banks 
Accounts receivable 
Building and loan deposits—~—~~- - 
Inventory of books and supplies 
Prepaid expenses ~---- ——-- : 
Land, buildings and equipment (less eiiommibliaiel depre eciation of $42,762) 
Trust fund investments at cost (market value $24,736) - 


Total Assets 
Deduct—Liabilities : 
Accounts payable and accrued expenses 
Mortgage payable 
Deferred income-- 


Total Liabilities 
Net Assets, Representing Fund Capital (per Exhibit B Below) 


EXHIBIT B. Statement of Income and Expenses and Fund Capital 
for Year Ended December 31, 1959 


Income: 

FEDERATION PROCEEDINGS: 
Assessments on member societies —--~—~—~- 
Subscriptions, advertising, reprints, etc. 

Annual meeting : - 

Placement information service 

Grants and government contracts - 

Rental income 

Dividends and other income 


Total Income 


Expenses: 
Salaries, wages, Social Security, group insurance, and pension 
Printing and engraving : 
Advertising and exhibit commissions 
Projection and public address services 
Professional and other personal services 
Rentals of facilities and equipment 
Grants and fellowships 
Operation and maintenance of buildings, iliialie and equipment 
Other expenses and supplies 


Total Expenses 


Excess of Income Over Expenses 
Fund Capital: 
Balance, December 31, 1958 
Gain on sale of securities 
Balance, December 31, 1959 


681 


$152,963 
24,797 
61,999 
5,105 
14,803 
170,800 
15,568 


$446 ,035 


$ 29,461 
37,500 
227,177 


294, 138 


$151 ,897 


$ 21,784 
87,611 
149,398 
9,418 
103,79! 
7,516 
_-35329 

$382 ,847 


$167,581 
60 ,650 
28 ,998 
9,497 
13,283 
10,656 
18,800 
28 ,602 

I ,033 


$349, 100 


$ 33,747 


“I~ 
von 


$151 ie 








The 1960 Federation Meeting: scheduling a meeting 
and preparing an index by computer 


CLAIRE K. SCHULTZ AND CLAYTON A. SHEPHERD 


Applied Mathematics Department, Remington Rand Univac, Division of 
Sperry Rand Corporation, Philadelphia, Pennsylvania 


Wraes THE FEDERATION of American Societies for 
Experimental Biology first decided to experiment with 
new scheduling and indexing techniques for its 1960 
meeting, it was thought that a tool for use by the elected 
secretaries of the member societies was what was wanted. 
The tool was envisioned as being edge-notched cards 
(such as those manufactured by the Royal McBee Corp., 
Port Chester, N. Y., or the Zator Co., Cambridge, 
Mass.) or hand-sorted peek-a-boo cards (such as those 
manufactured by Jonker Business Machines, Wash- 
ington, D. C.), which could be manipulated for various 
purposes. Development of a descriptor system for use 
with these cards was considered the area for research. 
It had been decided not to prepare a subject index for 
the 1960 papers. 

To help organize and implement the proposed re- 
search project, Dr. Richard Orr! was asked to be a con- 
sultant. A proposal for a research grant was drawn up 
and subsequently approved, and a contract for the 
time and facilities of the authors was made with Rem- 
ington Rand.” It then became feasible to combine the 
research interests of the Federation with those of Rem- 
ington Rand. By computer analysis of the abstracts of 
the 1959 Federation meeting, a descriptor list for use 
with the 1960 Federation papers was compiled. Specu- 
lation about uses of a Univac I* computer for purposes 
other than compiling the descriptor list led to the con- 
cepts finally used, which are described in this paper. 
At a Federation Board meeting in June 1959, the secre- 
taries of the member societies were given a demonstra- 
tion of several hand-sorted punched-card systems and 
were introduced to the possible uses of the Univac com- 
puter. At that meeting the decision was made to forego 
experiments with punched cards for 1960 and instead 

‘Executive Director of the Institute the Advancement 
of Medical Communication, New York City. 

* In this paper Remington Rand will be used to refer to Reming- 
ton Rand Univac, Division of Sperry Rand Corp., Univac En- 
gineering Center, Philadelphia. 

* A trade-mark of Sperry Rand Corp. This note applies to all 
references to the Univac computer and Univac products discussed 
in this paper. Unityper and Synchrotape are also trade-marks of 
Sperry Rand Corp. 


or 


682 


try to develop computer techniques for handling the 
problems of the Federation. 

There was insufficient time to try more than two 
methods this year. The alternative to the computer 
method, which served as a control for part of the com- 
puter experiment, was the method for planning and 
scheduling the program used by the secretaries in the 
past. The parts of the computer experiment for which 
no alternative methods were used were those of prepar- 
ing a subject index and of arranging the abstracts into 
subject groupings for publication. 

One part of the computer experiments, scheduling 
the annual meeting, was not published or used directly 
in any way this year. Instead, it was reviewed by the 
Councils of the member societies of the Federation so 
that they could reject, modify or adopt the computer 
method. 

From the point of view of Remington Rand this proj- 
ect was undertaken as research for developing new com- 
puter techniques. From the point of view of the Federa- 
tion it was undertaken as an experiment for devising 
automatic methods of programming and for publica- 
tion activities preparatory to the annual meeting of the 
Federation. The realization of both objectives has re- 
sulted in a service from Remington Rand to the Federa- 
tion and perhaps to other organizations with similar 
problems. An evaluation of the methods described in 
this paper is being made for purposes of the Federation; 
the results will be published under the direction of Dr. 
Milton Lee, Executive Officer of the Federation. 

It is highly encouraging to the authors that 9 months 
of research have been so highly fruitful in providing an 
approach to automatically producing a ‘standard’ index 
and also in providing a technique for maximizing audi- 
ence satisfaction at a large meeting. Research along 
these lines will continue at Remington Rand. 


OBJECTIVES 

The project began officially on April 1, 1959, under a 
grant (NSF G-g246) from the National Science Founda- 
tion to the Federation. The interest of the National 
Science Foundation was to permit the Federation to 
explore techniques for programming and_ scheduling 


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July 1960 


its annual meeting other than those already in use. They 
hoped that the results of the research of the Federation 
could be applied to the problems of other scientific 
societies. 

There are a number of coordinated activities that 
precede each annual meeting of the Federation: 

1) Announcing the meeting and the rules for sub- 
mitting papers; 

2) Collecting and publishing the abstracts submitted 
or sponsored by members; 

3) Arranging the abstracts into subject groups of 
from 10 to 12 each for purposes of programming the 
annual meeting; 

4) Scheduling the sessions to avoid conflicts (when- 
ever possible) ; 

5) Publishing the program; 

6) Preparing and publishing an author index to the 
abstracts and the program; 

7) Preparing and publishing a subject index to the 
abstracts. 

Prior to and during 1960 the staff at Federation head- 
quarters performed steps 1, 2, 5, 6 and 7. The project 
was undertaken primarily to help perform steps 3 and 4, 
which previously were executed by the secretaries of 
the member societies; however, the project eventually 
included the second part of step 2, and the first part of 
step 7. For the 1960 meeting, as in the past, it was planned 
that each secretary should receive copies of the abstracts 
submitted by members of his society; the secretary 
would work directly with the abstracts to accomplish 
step 3 and, subsequently, step 4. Choosing session titles 
was to be the individual responsibility of each secretary, 
as was assigning the order of papers within the sessions. 
Also, as in the past, the printing deadline for the ab- 
stracts would follow the authors’ deadline for submitting 
abstracts by about a week, giving the secretaries little 
time for careful consideration of subject matter. Secre- 
taries of societies with a large membership would be es- 
pecially rushed. The familiar method of programming 
would be used, both as a control for the new method 
and to assure a program of no less than the usual qual- 
ity. 

The research goal was to prepare the program by 
computer in the same 1-week period that was allotted 
the secretaries and to compare the program directly 
with that produced by the secretaries. This goal proved 
to be an oversimplification. It did not take into account 
all the parameters of the problem and resulted in many 
later complications. 

One complication was the discovery that some mem- 
bers of the Federation Board preferred to continue the 
practice of scheduling separate meetings for the mem- 
ber societies during the week of the annual Federation 
meeting, while others preferred to integrate the majority 
of papers from the societies into Federation sessions. 
While a compromise between separate and intersociety 
sessions had existed for several years, the discussion 
prior to the 1960 meeting led to the request that the 


PROGRAMMING AND INDEXING BY COMPUTER 683 


computer project produce both a compromise program 
and a totally integrated program for comparison. 


PREPARING A LIST OF SUBJECT CATEGORIES 


To define the subject matter of interest to the Federa- 
tion, the abstracts of papers presented at the 1959 
Federation meeting were analyzed for their subject 
content. During the summer of 1959, all of the 2,383 
abstracts were indexed according to a dictionary de- 
veloped by the senior author and colleagues at Merck 
Sharp & Dohme Research Laboratories, West Point, 
Pennsylvania. This authority list was also familiar to 
others who assisted in the indexing. In addition, a large 
amount of indexing utilized terms used by the authors of 
the abstracts, since the dictionary was not suited exactly 
to the subject matter of the Federation, and because it 
was desirable to anticipate the cross references needed 
to translate the authors’ language to a standardized 
language. After the indexing was completed, and the 
input standardized according to the taped dictionary, 
all of the terms not found in the dictionary were evalu- 
ated. These terms were then entered in the dictionary, 
either as accepted descriptors or as cross references. 
Frequency of occurrence was given consideration dur- 
ing this process, so that for synonyms or near-synonyms, 
the ‘expression of choice,’ statistically, was made the 
accepted descriptor, and related terms became cross 
references. To analyze the product of the indexing, 
computer techniques were used which had been de- 
veloped for an earlier project (*‘A Computer Analysis of 
the Merck Sharp & Dohme Research Laboratories In- 
dexing System,” to be published in American Documen- 
tation). The goal of the computer analysis was to arrive 
at descriptors or sets of descriptors that would be broad 
enough (or specific enough) to classify the 1959 ab- 
stracts into groups of 10 to 12 each. It was assumed that 
the subject matter changed little enough from one 
meeting to the next so that the collective terms for 1959 
would be nearly adequate for the 1960 papers as well. 
Equipped with the computer-produced statistics, a 
group of scientists concerned with the project (the secre- 
taries of the six societies and Dr. Milton Lee, Dr. Ray 
Daggs, Dr. Richard Orr and Mrs. Claire Schultz) 
modified and rearranged the computer findings to 
produce a list of subject categories, later known as 
Form B (fig. 1). This was an important step. All of the 
subsequent computer experiments centered around 
Form B, and the Federation authors’ use of it. Form B 
was designed to make explicit the areas of current re- 
search in experimental biology in the fields of interest 
to the Federation. It was not intended to be a classifi- 
cation of biological knowledge; it was intended only as a 
classification of research interests. In addition, Form B 
had the important psychological function of obtaining 
the Federation authors’ participation in nearly all 
phases of the experiment; therefore, the success of the 
experiment depended basically upon the adequacy of 
the listing and its clarity to the authors. 








684 


FEDERATION PROCEEDINGS Volume ig 


FORM B. PROGRAMMING 





f (This Form must be returned with Form A to the Secretary of your Society) 


DO NOT WRITE IN THIS BOX 


| lst author ’ Bergen Sau ley . Ir 


2 
Last Name 


| Title of paper Hepalic gly cegenoly|r eheci ef vasopress I/ 
| Society to which submitted Aner: AN Pysvolegical See; ¢ hy 


Please study the subject-category list below before marking. Do not be concerned over organization of the cate- 
gories, seeming omissions, or semantics. The most specific categories are printed in italic, the most general in beld type, 
and intermediate in roman. 





Choose the two most specific categories into which your paper fits, as your first and second choices for sessions. 
If italicized categories do not fit, choose from those in roman or beld type. The computer will classify your paper to the 
degree of specificity vou indicate. Do not write in other subject categories, but use the blanks at the end of the list for 


further particularization, as explained below. 


Place the numbers 1 and 2 in boxes ({1)) at the left of your first and second choices of subject categories. 





Then, on the short lines ( ) at the right of categories, place the numbers 1, 2, 3, 4, 5 to indicate, in the order of 
your preference, the five sessions you will be most interested in attending at the meetings. These data will be used to 
avoid conflicts in sessions insofar as possible. 


Finally, in the blanks at the end of the subject-category list, please supply up to four additional descriptive terms 
(words or short phrases), which can be used, in addition to the subject categories, for further classifying and for in- 
dexing the content of your paper. The terms you supply should contain nouns used in your abstract. Generic names of 
chemical compounds and drugs should be used, rather than trade names or jargon. 


SUBJECT CATEGORIES 


Amino Acids Properdin an 


8 


C) — _) 036 C1 065 — 
[| 002 Biosynthesis _ _ C) 037 Biological Regulation ___ C) 066 Vasodilators oun 
[] 003 ~=—s- Chemistry ee C) 088 Body Temperature ___ C) 067 Cardiac Dynamics _ _ 
7] 004 Metabolism Sills () 089 ~=—s Enzymal eb C 068 Cardiac Output seliiny 
[5 005 ~=—- Nutrition news [) 040 ~~ Feed-back Cj 068 Cardiac Muscle perenne 
[) 006 Atherosclerosis _2 Mechanisms inte 0 070 Contraction cia 
[) 007 ~—s Experimental ___ [1 041 Hormonal minis 0 071 Disorders — 
— 008 ~=—s Nutrition sala [) 042 ~=Rhythms ss (0 072 Electrocardi- 

[] 009 ~=— Pathophysiology__ C 043 Bielegical Oxidation ___ ography sana 
[] 010 Behavior pail ( 044 ~—« Electron Transport —__ D 073 Excitability conan 
1 011 _— Disorders pore (1 045 ~=—s Krebs Cycle siatbel 0 074 Metabolism = 
(10012 Drug Effects _ - 046 = Oxidative 2 075 = Exper.C V Disease __ 
- 013 Excitation = ___ Phosphorylation ___ DC O76 Cells ote 
[] 014 Psychoses See [) 047 Bedy Fluids and () 077 Active Transport een 
(J 015 Inhibition ___ Sperer oa 0 078 = Cytochemistry bees 
1 016 ~— Learning salamat: 048 wy (1 079 Membranes eid 
1 017 ~—— Motivation aa 1 049 = Abnormal C080 Microsomes nape 
[) 018 Blood sia Metabolism em — 061 = Mitochondria enna 
[] 019 Bone Marrow ___ ™) 050 = Glycogen wee — 062 ~=—s Reproduction sii 
[1 020 Coagulation —__ (1) 051 Monosaccharides 0 083 — 
0 021 Disorders ___ C 052 = Oligosaccharides 9 ___ 2 084 Sulfonamides cues 
[) 022 Platelets ania C—- 053 = Cardievescular 02 085 ~—s_ Antibiotics — 
[) 023 Thrombin ___ SB yraheer, ears — 086 = Cytotoxic Drugs —— 
1 024 Erythrocytes ___ ] 054 Blood Flow sles C) 087 Connective Tieeve — 
(—D 025 Destruction ___ 0 055 Coronary sana — 088 _s Disorders ict 
CD 026 Formation ___ C) 056 Peripheral at 0 088 = Metabolism — 
[) 027 Metabolism ___ ) 057 Pulmonary ea () 090 Mucopolysac- 

C028 = Groups Seek C058 ~=s Blood Pressure 89 ____ charides ties 
— 029 ~— Leukocytes ws [) 050 H — — 081 Wound Healing — 
— 080 Leukemia «te (— 060 Shock atte C) 092 Endecrines neue 
CO 081 Proteins ee CO 061 Blood Vessels ae (1) 088 = Adrenal Cortex etal 
0 082 Albumins ___ C—- 082 ~— Blood Volume aunts C2 004 Disease States am 
CD 083 Complement ___ — 068 Cardiovascular 0D 085 Stress — 
CD 084 Globulins ___ Drugs ani 0 006 Adrenal Medulla  — 
- 085 Hemoglobin ___ — 064 Glycoeides aan (—- 067 ~—s— Anterior Pituitary - __ 


FIG. 1. Form B. 





XUM 





olume 19 


ms 


SeLESZRESSASR See Se eee oe QF 





July 1960 


— 098 
5 099 
2 100 
5 101 
Cj 102 
C 103 
0 104 
0 105 
CF 106 
6 107 
0 108 
0 109 
5 110 
0 1 
5 112 
O us 
5 114 
5 1s 
0 116 
G7 
Oo 118 
O 119 
C120 
5 121 
FD 122 
CO 123 
5 124 
125 
126 
127 
128 
129 
130 
131 
F 132 
0 133 
134 
F135 
0 136 
C137 
5 138 


OUOOOSO 


O 


0 189 


0 140 
5 141 
Oj 142 
5 143 
O14 
5 45 


2 146 
O 47 
5 148 
5 149 
5 150 
6 151 
O 152 
5 158 


ACTH 
Control 
Somatotrophin 
Glucocorticoids 
Action 
Biosynthesis 
Metabolism 
Therapy 
Insulin 
Action 


Diabetes Mellitus 


Secretion 
Mineral Corticoids 
Posterior Pituitary 
Sex Hormones 

Androgens 

Estrogens 
Thyroid Gland 


Iodine Metabolism 


Thyroxine 
Environment 
Air Pollution 
Altitude 
Hyperthermia 
Hypothermia 
Space 
Gastrointestinal Tract 
Absorption 
Liver 
Bile 
Motility 
Pancreas 
Stomach 
Secretion 
Genetics 
Metabolic Errors 
Genitourinary Tract 
Disorders 
immune Response 


Antibody Formation 


Antigen-antibody 
Reaction 

Experimental 
Disease 


Hypersensitivity 

Anaphylaxis 
Tissue Transplants 
Vaccines 


Upids 


Fats and Fatty 
Acids 
Metabolism 
Synthesis 
Transport 


es a 


PROGRAMMING AND INDEXING BY COMPUTER 


SEReeeeeen eeeeene 


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0 154 
5 155 
O 156 
0 157 
5 158 
O 159 
5 160 
5 161 


C 162 
5 163 
5 164 
6 165 
OC 166 
5 167 
5 168 
5 169 
170 
171 
172 
178 
174 
175 
6 176 
0177 


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FIG. 


Metabolism 
Synthesis 
Lymphatic System 

Microorganisms 
Endotoxins 
Luminescence 

Dhiners) Metaboliann 
Calcium, 

Phosphorus 
Magnesium 
Potassium 
Sodium 
Muscles 


Chemical Properties 
Contractile Proteins 


Contraction 
Metabolism 


Myoneural Junction 


Physical Properties 


Neuroanatomy 


Surgical Procedures 


Analgesics 
Analeptics 
Anesthetics 
Anorexic Agents 
Anticholinergic 

Agents 
Cholinesterase 

Inhibitors 
Monamine Oxidase 

Inhibitors 
Sedative Agents 

Meccan yebobogry 

Acetylcholine 
Amine Oxidases 
Autonomic N S 
Brain 


PPEE RET ETTT TEEPE nt 


1. Form B (second page). 


2 209 
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5 211 
5 212 
5 213 
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O 215 
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D 217 
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5 219 


0 220 


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os 


Isotope Piideteen 
Thermal 


Protection 
Tissue Effects 
Renal Function 
Disorders 
Diuresis 
Electrolyte 
Excretion 
Glomeruli 
Tubules 
Reproduction 
Respiratory Tract 
Respiration 
Mechanics 
O2, CO2 
Transport 
Pulmonary 
Diffusion 
Tissue 


Stress 
Tiesve Culture 
Texicity Studies 
Tumors 
Ascites 
Chemotherapy 
Growth 
Metabolism 
Radiation Effects 


Vitamin D 
Vitamin E 
Vitamin K 


685 














686 
Table 1. Number of Author Choices in Each Subject Category 
Number of Number of 
Subject Author Subject Author 
Category Choices Category Choices 
AMINO ACIDS & CARBOHYDRATES a3 
BIOSYNTHESIS 5 ABNORMAL METABOLISM 6 
CHEMISTRY 3 GLYCOGEN 2 
METABOLISM 43 MONOSACCHARIDES 10 
NUTRITION 15 OGLIOSACCHARIDES 2 
ATHEROSCLEROSIS 15 
EXPERIMENTAL 15 CARDIOVAS R_SYSTEM 19 
NUTRITION 6 BLOOD FLOW 5 
PATHOPHYSIOLOGY 4 Coronary 6 
Peripheral 23 
BEHAVIOR 3 Pulmonary 17 
DISORDERS 1 BLOOD PRESSURE 9 
DRUG EFFECTS 17 Hypertension 9 
Excitation 3 Shock 15 
Psychoses 3 BLOOD VESSELS & 
Inhibition 1 BLOOD VOLUME 6 
LEARN ING 2 CARDIOVASCULAR DRUGS 12 
MOTIVATION 1 Glycosides 6 
BLOOD 35 Vasoconstrictors 15 
BONE MARROW 27 Vasodilators 5 
COAGULAT ION 9 CARDIAC DYNAMICS 7 
Disorders 22 CARDIAC OUTPUT 10 
Platelets 13 CARDIAC MUSCLE 10 
Thrombin 11 Contraction 19 
ERYTHROCYTES 41 Disorders 1 
Destruction 6 Electrocardiography 6 
Formation 11 Excitability 4 
Metabolism 2 Metabolism 4 
GROUPS 11 EXPER. CV DISEASE 6 
LEUKOCYTES 1 
Leukemia 34 CELLS 5 
PROTEINS 7 ACTIVE TRANSPORT 26 
Albumins 7 CYT OCHEMOSTRY 20 
Complement 3 MEMBRANES 14 
Globulins 9 MICROSOMES 6 
Hemoglobin 3 MITOCHONDRIA 8 
Properdin é REPRODUCTION 3 
BIOLOGICAL REGULATION 5 CHEMOTHERAPY 8 
BODY TEMPERATURE 8 SULFONAMIDES 5 
ENZYMAL 7 ANTIBIOTICS 3 
FEED-BACK MECHANISMS 8 CYTOTOXIC DRUGS 0 
HORMONAL & 
RHYTHMS 5 iE TISSUE 14 
DISORDERS 5 
BIOLOGICAL CXIDATION 10 METABOLISM 5 
ELECTRON TRANSPORT 5 MUCOPOLYSACCHARIDES 14 
KREBS CYCLE 7 WOUND HEALING 4 
OXIDATIVE 
PHOSPHORYLAT I ON 4 
BODY FLUIDS AND SPACES 10 














From the authors’ use of Form B we note that certain 
analytical findings of the computer, ignored by the 
human beings who designed Form B, became impor- 
tant. There were two main types of human error. One 
type was ignoring certain clues from the computer 
about the relative importance of subjects. For example, 
the computer statistics made it clear that certain classes 
of enzymes were the subject of many papers, but the 
human editors rejected the class names, per se, without 
making adequate alternative provisions for papers about 
enzymes. The second main type of human error was 
dividing a particular category into all of the academically 
interesting subdivisions rather than into only those sub- 
divisions determined by the computer to be statistically 
significant. It will be shown later (table 1) that there 
were a number of subdivisions on Form B that were not 
used. In some cases the latter type of error was re- 
sponsible for this. 


METHODS AND EQUIPMENT 


A Univac I computer was used throughout the ex- 
periment. Auxiliary equipment included a Unityper 


FEDERATION PROCEEDINGS 


Volume ig 











Table 1. Number of Author Choices in Each Subject Category (cont.) 
Number of Number of 
Subject Author Subject Author 
Category Choices Category Choices 
ENDOCRINES 17 IMMUNE_RESPONSE 15 
ADRENAL CORTEX 10 ANTIBODY FORMATION 20 
Disease States re) ANT IGEN-ANT IBODY REACTION 41 
Stress 6 EXPERIMENTAL DISEASE 1é 
ADRENAL MEDULLA 3 HYPERSENSITIVITY 21 
ANTERIOR PITUITARY 5 Anaphylaxis 11 
ACTH 2 TISSUE TRANSPLANTS 18 
Control 4 VACCINES 3 
Somatotrcphin 4 
GLUCOCORTICOIDS 5 
Action 8 LIPIDS 13 
Biosynthesis 4 FATS AND FATTY ACIDS 8 
Metabolism 6 Metabolism 17 
Therapy 0 Synthesis 15 
TNSULIN 6 Transport 12 
Action 14 LIPOPROTEINS 10 
Diabetes Mellitus 5 PHOSPHOLIPIDS 20 
Secretion 3 STEROLS 2 
MINERAL CORTICOTDS 4 Blood Levels ll 
POSTERTOR PITUITARY 4 Diet 1 
SEX HORMONES § Metabolism 12 
Andorgens 8 Synthesis 7 
Estrogens 7 
THYROID GLAND & LYMPHATIC SYSTEM 3 
Iodine Metabolism & 
Thyroxine 16 
MICROORGANISMS 22 
ENVIRONMENT. 4 ENDOTOXINS 14 
AIR POLLUTION 1 LUMINESCENCE 3 
ALTITUDE 1 
HYPERTHERMIA 5 MINE: METAB OLISM 16 
HYPOTHERMIA 10 CALCIUM, PHOSPHORUS 15 
SPACE, 0 MAGNESIUM 4 
POTASSIUM 2 
SODIUM x 
GASTROINTESTINAL TRACT 12 
ABSORPT ION 15 MUSCLES 4 
LIVER 19 CHEMICAL PROPERTIES 4 
Bile 2 CONTRACTILE PROTEINS a2 
MOTILITY 7 CONTRACT TON 13 
PANCREAS 6 METABOLISM 6 
STOMACH 6 MYONEURAL JUNCTION 7 
Secretion 10 PHYSICAL PROPERTIES 1 
GENETICS 2 
METABOLIC ERRORS 8 0 
SURGICAL PROCEDURES a 
GENTTOURINARY TRACT 3 
DISORDERS 0 














typewriter for typing information onto magnetic tape 
and a Univac High-Speed Printer for printing informa- 
tion from magnetic tape. 

Form B (fig. 1) was used to gather data for each part 
of the experiment. Federation headquarters mailed it 
to each of 6,036 members for 1960. Each author was 
asked to indicate on Form B the first and second choice 
of subject categories to which his paper could be as- 
signed. This information was used in the computer pro- 
grams to determine how the 1960 papers could be classi- 
fied into groups of from 10 to 12. 

Each author was also asked to indicate on Form B, 
in the order of his preference, the five sessions he would 
be most interested in attending while at the annual 
meeting for 1960. The tabulated list of authors’ prefer 
ences was used in the computer programs to determine 
the number of authors interested in attending a par- 
ticular session, and the scheduling conflicts to be avoided. 
At the bottom of Form B, the author was asked to suggest 
up to four additional index terms for his paper. 

Form B and one copy of the author’s abstract were 
forwarded by the secretaries to Remington Rand. To 
guarantee 100% return of the B Forms, the secretaries 
filled in those few forms which were not returned by the 
authors. Form A (the copy of the author’s abstract to 


e748 2A 





f 















































lume 19 July 1960 
2) Table 1. Number of Author Choices in Each Subject Category (cont. ) 
umber of Number of Number of 
uthor Subject Author Subject Author 
1oices Category Choices Category Choices 
15 OPHARMACOL OGY 42 SYNTHESIS 23 
20 ANALGESICS 5 Amino Acid Activation & 
ret ANALEPTICS 20 Relation to DNA and RNA 9 
18 ANESTHETICS ai 
ms ANOREXIC AGENTS 0 
1 ANTICHOLINERGIC AGENTS 2 RADIATIONS 8 
ig CHOLINESTERASE GENETIC EFFECTS te) 
3 INHIBITORS 11 IONIZING 9 
MONAMINE OXIDASE ISOTOPE TECHNIQUES 4 
INHIBITORS 25 THERMAL 13 
13 SEDATIVE AGENTS 8 PROTECTION 3 
8 TISSUE EFFECTS 11 
17 . 
15 4 RENAL FUNCTION 33 
12 z DISORDERS 4 
10 13 DIURESIS 18 
20 : 7 ELECTROLYTE EXCRETION 13 
> Brain Stem 14 GLOMERULI 2 
Cerebral Cortex 17 TUBULES 19 
un Hypothalamus 10 
: Mid-brain 1 REPRODUCTION 6 
1 CATECHOL AMINES 22 
7 ELECTRICAL POTENTIAL 7 RESPIRATORY TRACT 3 
3 TPHERAL NERVES 4 RESPIRATION 18 
6 Mechanics 22 
16 02, CO2 Transport 18 
20 7 Pulmonary Diffusion 8 
“ Tissue 3 
14 6 
3 17 
16 - 
22 S 3 
15 Pe STRESS b 
4 TISSUE CULTURE 24 
2 NUTRITION 13 
1 BOHYDRATES 2 TOXICITY STUDIES 21 
ICTENCY DISEASES 6 
4 1 TUMORS 10 
4 ig ASCITES | 
ad 4 CHEMOTHERAPY 11 
lef 9 GROWTH 5 
6 TRACE ELEMENTS 3 METABOLISM 14 
Z RADIATION EFFECTS 4 
QRESITY 4 VIRUSES 35 
5 ENTEROVIRUSES 15 
: MYXOVIRUSES 45 
a PHOT OSYSTHESIS 12 PHAGE 5 
PORPHYRINS 4 VITAMINS 7 
VITAMIN A 4 
PROTEINS, POLYPEPTIDES 25 VITAMIN B 21 
: METABOLISM 8 Vitamin By2 14 
1c tape PHYSICOCHEMICAL VITAMIN C a 
PROPERITES 26 VITAMIN D 2 
iforma- STRUCTURE 24 VITAMIN E 13 
VITAMIN K 3 
ch part 
ailed it be used for photo-offset printing, fig. 2) was forwarded 
lor was f to Federation headquarters. The input stage of proces- 
| choice sing the data by the new techniques was done under a 
be as- great deal of pressure at Remington Rand because the 
ter pro- f printer’s schedule called for having the abstracts volume 
e classi- arranged, insofar as the computer was concerned, one 
day after receipt of the final mailing from the secre- 
‘orm B, taries. The computer time necessary for arranging the 
e would F abstracts volume was only 2 minutes (table 5), but 
annual Ff transcribing the data onto magnetic tape and checking 
preter’ Fit for accuracy was relatively time-consuming (25 man- 
termine | hours plus 1 hour of computer time). The newness of 
a pal- f the indexing procedure made it necessary (this first 
ivoided. J time) to do a great deal of human editing of Form B, 
y suggest F which contributed considerably to the time needed for 
bs preparing the input. The procedure for preparing the 
ict were # input was as follows: 
and. To 1) The author’s choices for programming his paper and 
cretariés § for attending the meetings were manually copied from 
d by the f Form B onto coding sheets. 
stract (0 2) Professionally trained personnel manually edited 





XUM 


PROGRAMMING AND INDEXING BY COMPUTER 


687 


the author’s indexing choices for consistency and re- 
corded the edited choices on the partially prepared 
coding sheets from step 1. (The complete magnetic- 
entry for the abstract shown in fig. 1 is illustrated as it 
appeared on coding paper in fig. 3.) 

3) The information was transferred from coding 
paper to magentic tape, using the Unityper typewriter. 
To check for transcription errors, this step was per- 
formed twice, by two different typists. 

4) The two Unityper typewriter tapes were com- 
pared, errors were corrected and all of the correct data 
were merged onto one tape. The data were arranged 
in numerical sequence, using the control number as- 
signed each abstract by the society secretary, and 
checked for completeness. 


PROGRAMMING AND OPERATION 
Format of Input Tape 


At this point we began the computer programs for 
the project, each of which is described below. Com- 
plete information about each abstract required 10 
words of computer storage space (refer to fig. 3). The 
first word is the control number assigned to the ab- 
stract by the society secretary. The first half of the next 
word contains the numbers of the two categories that the 
authors chose for programming the paper. The last 
three characters of the second word, and all of the third 
word, were used to record the author’s attendance 
choices. The remaining seven words were available for 
index entries. Index entries were used in truncated form 
if they were longer than the twelve letters available in 
the computer word. A special look-up in a tape dic- 
tionary was used to complete the entry at output time. 
Nonalphabetizing prefixes to chemical names, such as 
(g-alpha) or (1,3 bis) were transferred to the end of the 
name to facilitate sorting. If fewer than seven index 
entries were assigned to an abstract, the unused portion 
of the allotted computer space was filled with spaces. 
There were 2,526 abstracts of papers for the 1960 meet- 
ing. One block of computer tape accommodated six 
abstracts; therefore, the total amount of space required 
for the data at the time of input was 521 blocks, or ap- 
proximately one-fourth of one standard reel of mag- 
netic tape. 


The Abstracts Volume 


This part of the project consisted of arranging the 
volume of abstracts for publication. The volume was 
divided into subject groups, arranged as on Form B, 
and the author’s first subject choice was used to place 
his paper in the abstracts volume. 

The master copy of each abstract, Form A (fig. 2), 
had been filed at Federation headquarters according to 
the serial number assigned to it by the society secretary. 
The computer program needed to designate that serial 
number, the subject category to which the abstract was 
being assigned, the place it was to have within the cate- 





688 





FEDERATION PROCEEDINGS 


DEADLINE DATE: DECEMBER 31 








IMPORTANT: These in- 
structions must be followed 
COMPLETELY. Read all 
instructions before you be- 
gin typing on this special 
form. See also sample ab- 
stracts on the reverse of 
this sheet. 











1. Abstracts must be sub- 
mitted in two ways: (a) the 
final version on this special 
form for reproduction by 
photo-offset in FEDERATION 
PROCEEDINGS, and (b) 6 ver- 
batim copies (original and 5 
carbons) typed double spaced, 
on 8-1/2" x 11" paper, for 
uses of the Program Com- 
mittee. DO NOT try to make 
carbon copies when typing on 
this form. 

2. Yourentire abstract, in- 
cluding title, author(s), loca- 
tion, text and acknowledgments 
must be within the rectangle 
outlined at the right. Leave 
no top or left margin within 
the rectangle. See samples on 
reverse of this sheet. Short 
specific titles are desirable. 

Use standard abbreviations. 
Capitalize the first letter of 
trade names. 

3. Use a typewriter, preferably electric, 
with ELITE type. Clean type before using. 
Use a carbon ribbon, if possible; otherwise, a 
reasonably new, good quality, BLACK SILK 
ribbon. (A new ribbon may smudge; an old 
ribbon may be too faint.) 

4. Practice typing the abstract in a rec- 
tangle 5-1/4'"' x 4-1/4" on plain paper before 
using this special form. 

5. Single space all typing onthis form. The 
text of the abstract should be a single para- 
graph, starting with a 3-space indentation. Any 
special symbols, such as Greek letters, that 
are not on your typewriter must be drawn by 
hand in BLACK ink. Tables are permitted 
within this space. 

6. DO NOT ERASE. Remember that your 
abstract will appear in FEDERATION PRO- 
CEEDINGS exactly as you submit it; any 
erasure smudges, errors, misspellings, poor 





Abstract Reproduction Form A 


TYPE ABSTRACT HERE 4 


hyphenations and deviations from good usage 
will be glaringly apparent in the published 
abstract. 

7. Underline names and initials of authors. 
Place an asterisk (*) after the name of each 
non-member author who is co-author with a 
member. If no author is a sponsoring mem- 
ber, do NOT use an * but type (intr. by ) 
after name of last author. See samples on re- 
verse side. 

8. NOTE: Any poorly prepared abstract 
unsuitable for direct reproduction will be re- 
typed by the printer and the author will be 
charged $5. 

9. DO NOT FOLD THIS SHEET. Mail by 
first class mail, with the six copies typed on 
8-1/2" x 11" sheets to the Secretary of the 
Sponsor's Society. These must reach him NO 
LATER THAN DECEMBER 31. Use cardboard 
backing to avoid damage in the mail. 











(Sponsor's Signature) 


(Society to which abstract is submitted) 


(Sponsor must be a member of the Society to which the abstract is sent. See your Society's rules for eligibility of papers.) 


FIG. 2. Form A. 


Volume 19 








lume 19 


page 
shed 


ors. 
each 
th a 
em- 


sical 


| Feo 


ract 
: re- 
il be 


il by 
d on 
f the 
n NO 
oard 





XUM 


July 1960 


PROBLEM 
OATE 


PAGE 2% 





FIG. 3. Work sheet for forming computer words from data. 


gory, and the page on which it was to appear. A sample 
page of the computer output is shown in figure 4, as 
printed by the Univac High-Speed Printer. 

The Federation staff put the A Forms into the order 
prescribed by the computer and sent them to the prin- 
ter, who made the page layout according to the instruc- 
tions of the computer, and reproduced the abstracts 
volume by photo-offset. 

The arrangement of the 1960 abstracts volume per- 
mitted the author of a paper to choose its position in the 


CATEGORY NOe ABSTRACT NOe 


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PROGRAMMING AND INDEXING BY COMPUTER 


689 


volume. At the time of this writing, reader reaction to 
the new format is not known. The present format has 
greater utility than formerly in that it helps the reader 
to locate papers in his general area of interest. The 
feature of identifying the society to which the paper was 
submitted has been retained by placing that information 
at the head of each abstract. In addition to the subject 
index, prepared by the computer, an author index, 
prepared by Federation headquarters personnel, is 
included in the abstracts volume. 


Grouping the Papers 


The first. goal of this part of the project was to form 
groups of from 10 to 12 papers that would be related in 
subject. When the first choices of the authors were tal- 
lied, some categories, as was to be expected, contained 
more than 12 papers and others fewer than 10. The 
highest number of choices in any one category was 43, 
the lowest, zero. Complete statistics about this use of 
Form B are given in table 1. We assigned as many 
papers as possible to the author’s first-choice category, 
but some papers had to be placed in second-choice 
categories. The categories that had well over 12 papers 
required a rather exhaustive exploration of the possi- 


SFCRETARY'S NOe PAGE NO, 
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FIG. 4. Sample page of computer output for analyzing abstracts for publication. 








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Volume 19 





WIAA 


July 1960 














Table 2. Steps and Results of Programming Runs 
No. Rasis Tnput Results Time 
of of (ab- (abstracts) (minutes) 
Run |} Computing | stracts)| Assigned |Unassigned| Sort 
Author's 
1 |] first 2526 2245 281 0 5 
choice 
Author's 
2 || second 281 195 86 0 0.7 
choice* 
Replace- 
ment in 
ertinent 
Pg pants 86 43 43 2 | 0.7 
choice 
category 
Totals 2526 2483 43 2 2.9 























* Or replacement of an abstract in the first-choice 
category which could be assigned. 


bilities for second-choice assignment. The simplified 
flow chart for the computer program for this step (fig. 5) 
shows that if a first-choice category contained 20 or 
more papers, a second session was automatically created 
for that subject; this helped to reduce the number of 
second-choice placements. For the categories with 30 
or more papers, it would have been possible to create a 
third session on the subject, but for purposes of the ex- 
periment it was decided, instead, to place these papers in 
categories with fewer than 10 papers. Papers were <as- 
signed to second-choice categories by three distinct 
steps (computer runs), giving interesting differential 
statistics for each step. Table 2 shows the statistics, to- 
gether with an explanation of each step and its running 
time. 

After the computer had carried out all of the instruc- 
tions given to it for assigning and reassigning abstracts, 
there were still 43 that had not been assigned, and there 
were 89 categories with fewer than 10 papers. Human 
manipulation of these data was required to complete 
the process, but the number of categories had been 
reduced to 34% of the total number of categories. 
Next year the process can be refined so as to require even 
less human _ handling. 

To enable the secretaries and their countil members to 
compare the computer-produced sessions with the 
sessions planned by themselves, the contents of each 
session had to be made explicit as to abstracts and the 
order in which papers were scheduled. A complete 
schedule was made, separately for each of the societies, 
and jointly for the Federation, to accomplish an in- 
tegrated program. 

The idea that a computer was in competition with 
human beings needs to be avoided as carefully as pos- 
sible when comparing the computer grouping with 
that of the secretaries. One of the main purposes of this 
project was to find ways to assist the secretaries with 


PROGRAMMING AND INDEXING BY COMPUTER 


691 


their task. The purpose of the comparison, then, is to 
determine to what degree the instructions to the com- 
puter accomplished what was required. It is generally 
true that if the instructions become inadequate, they 
may be altered to obtain a more satisfactory product. 
After there has been an opportunity for the Federation 
Board to relay their feelings about this project, the level 
at which modifications may need to be made in the 
computer program will become apparent. 














Table 3. The 25 Category 
Conflicts Most Necessary 
to Avoid 

Number | Number of 
Categories of Persons 
Points | Represented 
137,138 636 92 
O44, 046 536 7h 
216,217 528 Th 
219,220 447 76 
138,140 418 64 
218,220 380 59 
199,202 370 60 
174,183 368 48 
137,140 325 53 
218,219 322 48 
242,250 322 46 
202,220 318 53 
077,079 315 50 
181,192 315 45 
199,220 307 50 
231,233 293 41 
238,239 276 39 
137,139 268 42 
230,231 258 34 
138,139 256 40 
237,238 253 35 
250,251 252 31 
043,044 249 32 
214,217 246 33 
250,252 244 33 

















692 


FEDERATION 


PROCEEDINGS Volume 1g 
































: Pair author's choices: Compute weights y \ No 
Extract author's , s ; : End of 
2 re 
Bikeuunte chbewes a bal with 2nd, lst with 3rd, for pairs of Input? 1 
2nd with 3rd, etc. choices 
Yes 
2 














Arrange list of pairs 
with their weights in 
numerical sequence 





Tabulate occurrences of 
Pe each pair, 


and add their 


+O 














weights (A) 
Arrange tabulated list Print 
of pairs in sequence ea) (B) 
according to weights (B) 




















FIG. 6. 


Scheduling the Sessions 


Objectives and problems. When all of the papers had been 
fitted into groups of between 10 and 12, the 250 sessions 
for the 1960 meeting were scheduled according to day 
(Monday through Friday) and time (morning or after- 
noon). The size and location of the room in which each 
session was to be held also was considered. The most 
important scheduling problem was to avoid conflicts 
in subject matter of sessions programmed for the same 
time. Until this year it was impossible to recognize all 
conflicts and to compare their relative importance. 
The next most important problem was the size of the 
room in which to schedule each session. Another diffi- 
cult problem was predicting attendance at individual 
sessions. 

The computer experiment was concerned with each 
of these problems but did not attempt to achieve such a 
subtle refinement as scheduling two equally popular 
sessions in adjacent rooms so as to make ‘session hop- 
ping’ convenient. 

Avoiding conflicts. Form B was used to gather the neces- 
sary information for handling these problemas by com- 
puter. As stated previously, each author was asked to 
indicate, in the order of his preference, the five sessions 
he would be most interested in attending while at the an- 
nual meeting. The computer used these data to weigh 
the importance of conflicts among these sessions. The 
author’s first choice was assigned a value of five points, 
his second, four points, and so on. In the example shown 
in figure 1 the author chose: 

1. 048 (5 points) 
2. 006 (4 points) 
106 (3 points) 
145 (2 points) 
204 (1 point) 


gh 


Therefore, a conflict between categories 048 and oc6 
was given a weight of g points. The computer was used 


to assign weights for each possible conflict for each 


Procedure for weighting categories, flow chart. 


author. The complete set of conflicts, with their weights, 


for the example above is as follows: 


048 ,006 = g points 
048,106 = 8 points 
048,145 = 7 points 
048,204 = 6 points 
006,106 = 7 points 
006,145 = 6 points 
006 ,204 = 5 points 
106,145 = 5 points 
106,204 = 4 points 


145,204 = 3 points 

The author’s five choices thus have been used to cal- 
culate the relative importance of 10 possible conflicts 
in scheduling. This was done for each of the authors 
who supplied the necessary data. The computer cal- 
culations concerning the most important conflicts this 
year are given in table 3, and a simplified flow chart of 
the three computer runs is shown in figure 6. This in- 
formation was used in scheduling the computer-produced 
sessions, which were forwarded to the secretaries for 
evaluation. The computer schedule for an integrated 
program for 1960 is given in table 4, together with the 
computer’s prediction of attendance at these meetings 
(based on the data received from authors and on the 
previous year’s registration of 10,000 persons). 

Predicting attendance. Attendance at the sessions could 
not be predicted very realistically from data about the 
interests of authors, since the number of persons attend- 
ing the meeting is four times the number of first authors 
(of those papers with more than one author), and only 
the first author checked his choice of interest. However, 
assuming authors’ interests to be a valid sample of the 
interests of the total audience, a list of the relative size 
of the audience for each of the sessions was compiled 
(table 4). This list was not used this year, but if the ac- 
tual attendance at the 1960 sessions is compared with it, 
the validity of using a similarly prepared ‘list another 
year can be estimated. 


WLM 








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July 1960 


The Index 


Objectives and problems. Neither we nor anyone else 
had ever produced a standard index by computer. The 
objectives in producing the index were 7) to utilize the 
computer, 2) to make the index more detailed and more 
carefully cross-referenced than previous Federation 
indexes, which had been poor in these respects because 
of the short period of time in which they had to be pre- 
pared. Objective 3 was not too difficult to realize, ex- 
cept that it was necessary to make the index for 1960 
in an even shorter amount of time than was available 
previously. The need to standardize authors’ terminol- 
ogy, as well as the various indexing depths at which 
they worked, added to the challenge. Early in the plan- 
ning, we thought we could use a tape dictionary to 
help standardize the authors’ input, but there was no 
way to predict exactly what terminology would be neces- 
sary in preparing the dictionary. Since during the actual 
computer run there would be no time to construct a tape 
dictionary, the B Forms were edited manually by Dr. 
Orr and Mrs. Schultz. Human editing was time-con- 
suming but gave us the advantage of familiarizing our- 
selves with the data before making final decisions. 

Procedure. The procedure we used for indexing was to 
1) read the title of the paper, 2) look at the author’s 
choice of subject categories (these were to be used as 
index entries), 3) rcad his indexing suggestions at the 
bottom of Form B, 4) read his abstract, and 5) accept, 
modify, reject or supplement his suggestions. The 
number of index entries actually used for each abstract 
varied from 2 to g, with an average of 5 to 6. The index 
for 1959 had an average of 1.5 entries per paper. 

A record of the cross references to be inserted in the 
index wascompiled on 3 by 5-inch cards as we proceeded, 
then taped for handling by the computer. The computer 
indicated whether a cross reference should read ‘See’ or 
‘See also,” depending on whether or not any abstracts 
had been indexed under that entry. For example, 
Chemotherapy—antibiotics was an entry on Form B. To 
guide readers looking for papers on antibiotics, we 
included the cross reference: 


Antibiotics, See Chemotherapy—