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RADIOIMMUNOASSAY‘ FOR RAT GROWTH HORMONE; ’     T I , 7? ' ' ’ M
FURTHER STUDIES ON THEOONTR'OL OEOROWTH
HORMONE SECRETION IN THE RAT

Thesis for the Degree of Ph. D.
MTCHIGAN STATE UNIVERSITY
ELIAS DICKERMAN

1971 '

   
  

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LIBRARY
Michigan Saw

1 University, ‘ E”

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This is to certify that the

thesis entitled

RADIOIMMUNOAS SAY FOR RAT GROWTH HORMONE;
FURTHER STUDIES ON THE CONTROL OF GROWTH
HORMONE SECRETION IN THE RAT

presented by

Elias Dickerman

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Physiology

 

M r professor

Datemrl 244 1971

0-7639

 

 

 

 

     
   
   
 

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ABSTRACT

RADIOIMMUNOASSAY FOR RAT GROWTH HORMONE;
FURTHER STUDIES ON THE CONTROL OF GROWTH
HORMONE SECRETION IN THE RAT
By

Elias Dickerman

   
  
  
   
   
   
  
   
   
  
 
 

1. A double antibody radioimmunoassay was developed
for rat growth hormone (RGH) in which monkey anti-RGH
serum and goat anti-monkey gamma globulin are used. RGH
120 ug) was labeled with 1125 (1 me) under the influence
of Chloramine-T (87.5 ug). This resulted in a preparation
of non-1125 of 36.5 no I125/ug RGH; 30.2% of this prepar—
ation is recovered. Separation of RGH-I125 from free I125
and repurification of RGH-I125 was done in columns of Sepha—
dex 6-50 and G-lOO respectively, at pH 8.6; the assay of
OH was carried out at pH 7.2. Three fractions were re-
wcovered upon repurification: an aggregated fraction, an

undamaged fraction and a degraded fraction. The amount of

_.a
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A- . aggregated. and degraded RGH-I125 increased with time or
:ieiter rapid freezing. The undamaged fraction was the most
1m:

' isplace. Parallel inhibition curves were demonstrated

.én,different pituitary homogenates or plasma from
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"intact rats and a purified preparation of RGH. No inhi-
‘bition was observed when using serum from hypophysectomized
rats or purified preparations of other rat anterior pitui-
tary hormones, indicating that the anti-RGH serum used was
specific, in the rat, for GH. RGH levels of several pitui~
tary preparations were lower when determined by radioimmuno—
assay than by bioassay; the mean radioimmunoassay levels
were, however, within the 95% confidence limits of the bio-
assay results. The assay reported here has a sensitivity of
0.25 mug of RGH.

2. The anti-rat growth hormone serum was tested for
cross-reactivity with purified preparations of GH, pituitary
homogenates and plasma of other animal species. Cross-
reactions were observed with pituitary homogenates and plasma
of hamster, guinea pig, gerbil and mouse, as well as with
cat and dog plasma. Partial cross-reation occurred with puri-
fied preparations of ovine and bovine growth hormone, while
no cross-reaction was observed with purified human growth
hormone, monkey pituitary homogenate and rabbit plasma.

These results reinforce the concept of a common antigenic
structure in the growth hormone of several mammalian species,
as well as the difference in antigenicity of primate growth
hormones. Furthermore, they suggest that the radioimmuno-
assay may be employed to measure GH in the hamster, guinea
pig, gerbil, mouse, cat and dog, and perhaps other species
as well.

3. Growth hormone levels were measured by radioimmuno-

aésay in plasma and serum samples of the same origin varying

1.
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Elias Dickerman

the length of incubation time for the second antibody. In
all cases plasma GH concentrations were significantly high—
er than serum GH concentrations. The differences observed
were not the result of differential influences on the ra;
dioimmunoassay. Recovery of exogenous RGH in plasma or
serum of hypOphysectomized rats was about 100%. Comparison
of intact rat plasma and serum centrifuged at the time of
collection and incubated for O, 12, 24 or 48 hours at 4°C,
or incubated for the indicated time and then centrifuged,
showed that the former resulted in almost no disappearance
of GH, while significant decreases in GH concentration were
observed with time in the latter group; the longer the in-
terval the greater the decrease. For any given time period
less GH was recovered in the serum than in the plasma group;
serum GB at 0 hour centrifugation time was 74-81% of plasma
GH. These data suggest that immunological inactivation of
RGH takes place during, and perhaps as a result of, the pro-
cess of coagulation.

h. Plasma and pituitary GH concentrations were determined
in male and female rats from age 21 days until maturity or
old age. Pituitary and plasma concentration increased steadily
during the first 8-12 weeks of age in male and female rats,
with the sharpest increase taking place after vaginal opening
in females. These hormone levels remained elevated to 120
days with decreases in plasma observed at 180 and 240 days in
females and males respectively. In female rats of two differ—

ent ages with normal estrous cycles, mean plasma GH concentration

 

 

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in estrus was significantly higher than found in proestrus,
metestrus or diestrus, when no differences were observed.

No changes in pituitary GH occurred during the cycle. Bi-
lateral ovariectomy significantly decreased plasma GH while
elevating pituitary GH levels; daily injections of estra-
diol benzoate, on the other hand, significantly increased
plasma GH with a concomitant decrease in pituitary GH. Since
the most rapid rate of body growth takes place when pitui-
tary and plasma GH are lowest, these results suggest: a)
that GH may not play as important a role in the life of the
young rat as it does in the mature animal, or b) that the
increased utilization of GH combined with a low rate of syn-
thesis prevents the building up of pituitary and plasma
levels of GH. It would appear that the elevation in plasma
GH during estrus is due to estrogen secretion..

5. The effects of castration, thyroidectomy, testoste—
rone prOpionate ( TP )and Na-thyroxine ( T4 ) on pituitary
and plasma GH levels, metabolic clearance rate (MCR) and se-
cretion rate (SR) of GH were determined in male rats. Cas-
tration and thyroidectomy significantly decreased pituitary
and plasma GH levels. Testosterone propionate and Na-thyro-
xine, on the other hand, significantly elevated pituitary and
plasma GH 10 days after treatment. Metabolic clearance rate
was not altered by castration, but it was decreased by thyroi-
dectomy and increased by TP and T4. All of the treatments
produced significant changes in SR. Castration and thyroi-

dectomy reduced SR of GH to one half and one third respectively.

 

 

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51y, TP and T4 treatment increased SR to 1.8 and
W-s higher than that observed for controls. These

*éynthssis and release of GH from the pituitary while
2” fend T4 increase the rate at which GB is synthesized by
~;.:A .releaSed from the pituitary. These results also

.;;§ gifiggsst that proper distinction be made between MCR, which
are only to the volume of plasma cleared of hormone,

""°x§fid SR, which reflects the amount of hormone used per unit

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RADIOIMMUNOASSAY FOR RAT GROWTH HORMONE;
FURTHER STUDIES ON THE CONTROL OF GROWTH
HORMONE SECRETION IN THE RAT

BY

Elias Dickerman

A THESIS

Submitted to
. Michigan State University .
. in partial fulfillment of the requirements ' .
for the degree of . . i

  

DOCTOR or PHILOSOPHY ."Va;=.,}
Department of Physiology

- 555' _ 1971

 

     

Dedicated to my parents,
David and Sivia Dickerman,
to my wife Katherine, and , v a.

to my daughter, Beyle. 1-. .v

 

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ACKNOWLEDGMENTS

During my tenure as a graduate student I have received
assistance from a number of persons. Among those who have
been particularly helpful, Dr. A. Gjerding Olsen, Assistant
Professor of Biology at Brandeis University, Waltham, Massa-
chusetts, was one of the first. I am most grateful to him
for giving me the first opportunity to participate in research,
for his guidance throughout my first research project, for
his encouragement to pursue graduate study and for his conti-
nued interest in me and my career. At Michigan State Univer-
sity, Dr. Joseph Meites, Professor of Physiology and my advi-
sor during my study for the M.S. and Ph.D. degrees, provided
sound advice and financial assistance in conjuction with Michi-
gan State University. Equally important, he allowed me to con-
duct research to the best of my abilities and provided the oppor-
tunity to meet leading scientists in the field. My sincere
appreciation to Dr. Meites cannot be described accurately in
words.

The members of my Ph.D. committee gave much time to the
reading and correcting of the thesis manuscript. In addition,
their thought -provoking questions on examinations provided
new learning situations for me. Including Dr. Meites, these
committee members are: Dr. w. Doyne Collings, Dr. Harold D.

Hafs, Dr. H. Allen Tucker, Dr. Thomas w. Jenkins, Dr. Allan

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J. Morris and Dr. Edward M. Convay. Their help is gratefully
acknowledged.

Finally, I would like to mention the help of a co—wor-
ker, Dr. Ytschak Koch. Working with him in the laboratory
and discussing my research with him, I was stimulated to many
new insights and ideas which helped greatly in the formulation
of my thesis. His incisive contributions represent an inte-
gral part of my work. Along with Dr. Koch, my brother Dr.
Samuel Dickerman, assisted me in many hours of laboratory
experimentation and consulted with me at length throughout

the course of many conversations.

 

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TABLE OF CONTENTS

LIST OF TABLES .

LIST OF FIGURES. . . . . . . . . . . . .

INTRODUCTION . . . . . . . . . . . . . .

REVIEW OF LITERATURE . . . . . . . . . .

I.
II.

III.

Assay of Growth Hormone. . . . .

Hypothalamic Control of Anterior
(AP) Secretion of Growth Hormone

Pituitary
(GR). 0 e

Secretion of Growth Hormone During

Different Physiological States .
A. Age and GH . . .

B. Hormonal Influence on GR Secretion . .

C. Nutrition and Stress . . . .

EXPERIMENTAL METHODS AND MATERIALS . . .

II
II.

III.

IV.
V.

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I.

 

Arlimals. . e o e a e I o o e e 0

Preparation of Pituitaries, Plasma

serum. 0 I I O O O I 0 D O O l I
Assay of Growth Hormone. . . . .

A. Bioassay . . . . . . . . . .
B. Radioimmunoassay . . . . . .

Metabolic Clearance Rate (MCR) .
Methods of Statistical Analysis.

EXPERImN TS O C O C I O I O C I I O U I C

Radioimmunoassay for Rat
A. Objectives . .
. Procedures . .
. Results. . . .

. Discussion .

COED

O O O O 0

Growth Hormone. .

Page

vi

21
22
29
31
31

31
32

32
33

41
42
43
43
43

64

II.

III.

IV.

GENERAL
REFERENC
APPENDIX

vi

Study on the Cross-Reactivity of Monkey
Anti-Rat Growth Hormone Serum. . . . . . .

A. Objectives .
B. Procedures .
C. Results. . .
D. Discussion .

Differences Between Plasma and Serum

RGH Levels . . . . .

A. Objectives
B. Procedures
C. Results. .
D. Discussion

GH as a Function of Age in Male and Female
Rats: the Estrous Cycle . . . . . . . . .

A. Objectives .
B. Procedures .
C. Results. . .
D. Discussion .

I I
I I
I I
I 0

Plasma and Pituitary

I I I I I I I I I I I
I I I I I I O O I I I
I I I I I I O I I I I
I I I I I I I C I I I

Concentration,

Metabolic Clearance Rate (MGR) and
Secretion Rate (SR) of GH in the Male Rat
as Influenced by Castration, Testosterone
Propionate (TP), Thyroidectomy and Na-

thyroxine (T4) . . .

A. Objectives .
B. Procedures .
C. Results. . .
D. Discussion .

DISCUSSION . . . . .
ES 0 I I I I I I O I

I I I I I I I I I I

I I I I I I I I I I I

I
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C O I I

I I I
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105
106
109
117
123
126

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LIST OF TABLES

Table Page

1. Determination of Specific Radioactivity of
RGH-I125 I 0 O I O Q 0 I O O O O I I O I I O I 62

2. Biological and Immunological Estimates of
Potency for Male Rat Pituitary Homogenates . . 65

3. Difference Between Serum and Plasma GH Levels. . 79

u. Recovery Rates of RGH from Hypophysectomized
Plasma or Serum. . . . . . . . . . . . . . . . 84

5. Pituitary GH as a Function of Age in the
Male Rat O I I I I O C I I O I O l I I I O I O 92

6. Pituitary GH as a Function of Age in the
Female Rat: The Estrous Cycle . . . . . . . . 95

7. Plasma GH Levels After Castration,
Testosterone Propionate, Thyroidectomy
and Na-therXi-ne e o e e e e e o I e n o e e o 112

8. Metabolic Clearance Rate (MCR) and Secretion
Rate (SR) of GH in Rats After Castration,
Testosterone Propionate, Thyroidectomy and
Na-thyroxine . . . . . . . . . . . . . . . . . 115

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LIST OF FIGURES

Precipitation of RGH—Antibody Complex With
Varying Dilutions of Anti-Monkey Gamma
GlObulin Serml I I I I I I I I I I I I I I

Elution Pattern of RGH—I125 Through a 0.9
x 20 cm Column of Sephadex G-50 in 0.01M
Barbital Buffer at pH 8.6 . . . . . . . . .

Elution Pattern of RGH—1125 Upon
Repurification Through a 0.9 x 50 cm
Column Of Sephadex G-100 in 0.05M Barbital
Buffer at pH 8.6. . . . . . . . . . . . . .

Effect of Time on the Elution Pattern of
RGH—I125 Repurification Through Sephadex
G-100 I I I I I I I I I I I I I I I I I I I

Effect of Rapid Freezing (~——-——) on the
Elution Pattern of RGH—1125 Repurification
Through Sephadex G-100. . . . . . . . . . .

Left Side: Relative Radioimmunoreactivity
of the G-lOO Fractions. Right Side:
Comparison Between G-50 and G-lOO Peak II
Immunoreactivity. . . . . . . . . . . . . .

Standard Curves Obtained With G-lOO Peak I
and G-lOO Peak II RGH—1125. . . . . . . . .

Rat Growth Hormone Standards. . . . . . . . .

Inhibition Curves With Rat Pituitary
Homogenate or Plasma from Intact Rats . . .

Cross-Reaction Between Monkey Anti-Rat
Growth Hormone Serum and Other Rat Anterior
Pituitary Hormones. . . . . . . . . . . . .

Effect of Hypophysectomized Male Rat Serum
On Percent Binding. . . . . . . . . . . . .

viii

Page

36

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47

49

50

52

53
56

57

59

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Figure Page
12. Immunological Relatedness of Growth Hormones. . 72

13. Dilution-Response Curves for Pituitary
Extracts and Plasma of Guinea Pig and
Hamster I I I I I I I I I I I I I I I I I I I 73

14. Dilution—Response Curves for Gerbil, Mouse,
Cat, Dog and Rabbit Preparations. . . . . . . 74

15. Effect of Hypophysectomized Male Rat
Serum or Plasma on Percent Binding. . . . . . 80

‘ 16. Effect of Na-heparin on Percent Binding . . . . 82

17. Effect of Time on Plasma and Serum RGH
Levels I I I I I I I I I I I I I I I I I I I I 86

18. Plasma GH as a Function of Age in the Male
Rat I I I I I I I I I I I I I I I I I I I I I 93

19. Plasma GH as a Function of Age in the
Female Rat: The Estrous Cycle. . . . . . . . 96

20. Pituitary and Plasma RGH After Ovariectomy
and Estradiol Benzoate Treatment. . . . . . . 99

21. Effects of Different Treatments on Anterior
Pituitary GH Concentration in the Male Rat. . 107

. 22. Disappearance of Total Radioactivity and
ImmunoErecioitable Radioactivity
(RGH-I 25) From Plasma Following a
Single iv Injection in:
A) Thyroidectomized, B) Na-thyroxine,
‘ C) Castrated, D) Testosterone PrOpionate,
\ E) Intact Rats. . . . . . . . . . . . . . . . 110
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INTRODUCTION

It is now generally accepted that the hypothalamus
exerts a controlling influence on anterior pituitary func-
tion. Green and Harris (1949) had suggested that humoral
agents secreted from the hypothalamus into the hypophyseal
portal veins traveled to the anterior pituitary to exert
a regulatory influence on the secretion of anterior pitui-
tary hormones. Some of these agents, now known as hypo-
thalamic releasing and inhibiting factors or hormones, have
recently been detected in hypophyseal portal blood plasma,
re-inforcing the suggestion of Green and Harris (1949).

Perhaps the most important contribution in neuro-
endocrinology has been the recent reports on the elucida-
tion of the structure and synthesis of thyrotropin re—
leasing hormone by the laboratories of Guillemin and
4 Schally working independently. Apparently, Schally's
. laboratory has also determined the structure and synthe-

sized growth hormone releasing hormone. Needless to say,

y‘_-

the availability of synthetic hypothalamic releasing hor-
mOnes should permit studies heretofore not possible.

The physiological role of GH-RF has been studied by

wot-uh...

measuring hypothalamic content of GH-RF by in vivo deple-
tion of pituitary GH or in vitro release of GH from

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incubated pituitaries. A few reports on plasma or serum

GH in the rat have been published. In general, however,
.the methods available for the assay of GH have not been
sensitive enough to permit the measurement of GH in reason-
able volumes of blood plasma or serum of normal animals or
have been poorly validated and characterized. Obviously,
conclusions regarding synthesis and release of GH based

on pituitary levels of GH are subject to errors of inter-
pretation.

This thesis was concerned with the development of a
sensitive radioimmunoassay for rat GH. Using this assay
method it has been possible to determine some of the nature
a1 changes in CH which take place with age, sex, estrous cycles
and as a result of other hormonal influences. Perhaps
more importantly, it was possible to study the metabolic
clearance and secretion rates of GH in animals under dif-
ferent physiological conditions. These observations are

also reported in this thesis.

 

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REVIEW OF LITERATURE

I. Assay of Growth Hormone

The bioassay of a hormone generally reflects a
quantitative definition of its biological activity. This
definition becomes more complex for a hormone which does
not have a specific target organ, as is the case for
growth hormone. The numerous biological and metabolic
effects of growth hormone have given rise to several
assays to measure its activity. They include assays
based on a) increase in body size, b) increase in width
of the epiphyseal cartilage plate of the tibia, c) changes
in organ weights, d) changes in metabolism of nitrogen,
phosphorous, sulfur, carbohydrates and others. Only
those that have been used with regularity to measure
growth hormone in the rat will be discussed here.

The acceleration of body growth observed as a
result of injecting an animal with growth hormone has
been accepted as a suitable assay procedure for pituitary
growth hormone. Several types of animals have been used in
this assay: a) "plateaued" mature female rats, b) immature
hypophysectomized rats, c) genetically dwarf mice and
d) hypophysectomized mice.

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The use of*plateaued"female rats was first suggested
by Evans and Simpson (1931). The assay involves the use
of 5-6 month old female rats which fail to gain more than
10 grams in a 20 day period. An injection period of 15-
20 days is required for the assay. Marx gt gt. (1942)
found that a straight line relationship existed between
the logarithm of the daily dose and the response as
measured in grams of body weight increase. The assay is
not a very sensitive one as it requires a minimum of 50 ug
of bovine growth hormone per rat per day in order to
elicit an average weight increase of about 8 grams in 15
days. A daily dose of 1 mg causes an average body weight
gain of 42 grams.

The observations by Smith (1926, 192?, l930),that
hypophysectomy in the rat resulted in cessation of body
growth and that growth resumed following implantation of
whole pituitary glands, were first used by Van Dyke and
Wallen-Lawrence (1930) for the assay of growth promoting
extracts of the anterior hypophysis. Later, Walker gt gt.
(1950) observed that when young rats are hypophysectomized,
growth does not cease immediately but continues at a
slower rate until the rats reach about 30 days of age. The
assay, as it was used later, is essentially that described
by Marx gt gt. (1942) and Li gt gt. (1945). It consists
of immature female rats hypophysectomized at 26-28 days

of age. The animals are used 12-14 days after surgery and

 

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injected daily intraperitoneally for 10-14 days thereafter.
The increase in weight is recorded at the end of the injec-
tion period. The assay requires 5 times less hormone

than the "plateaued" rat bioassay to elicit a response.
However, Russell (1955) showed that when the dose is
correlated with the body weight of the animals, there is
little difference in sensitivity between the two assays.

Hypophysectomized mice were first used to assay
growth hormone by Lostroh and Li (1958). The animals
were hypophysectomized at 35 days of age and used 12-14
days later. The injection period lasted 14 days. An
initial dose of 5 ug of bovine growth hormone daily pro—
duced a body weight gain of 33% in 2 weeks. This method,
as well as that of Finss-Bech (1947) using genetic dwarf
mice, was never widely used because of the difficulty of
the operation and the lack of availability of the
animals respectively.

The most widely used assay for pituitary growth
hormone is the tibia test. It is based on the observation
of Dott and Fraser (1923) that cessation of growth of the
epiphysis follows hypophysectomy in dogs and cats.

Later, Kibrick gt gt. (1941) showed that the epiphyseal
cartilage response in young hypophysectomized rats to
injections of increasing amounts of growth hormone over

a 4-day period showed a straight line dose-response curve
when plotted on a semi-logarithmic scale. On the basis of
these studies Evans gt gt. (1943) proposed the tibia test

 

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for the bioassay of growth hormone. The test was later
re-standardized and firmly established as a method for the
estimation of growth-promoting activity by Greenspan gt gt.
(1949). The assay makes use of immature female rats
hypophysectomized at 26-28 days of age. Twelve to fourteen
days later the animals are injected intraperitoneally once
daily for 4 days. Twenty-four hours after the last
injection the animals are killed and their tibias removed,
cut and stained for histological examination. An average
of 8-10 readings of the cartilage width is taken; 4-6
animals are used per dose, with two or three dose levels
used per standard or unknown. This assay is 10-20 times
more sensitive than those described above, with a total
dose of 5 ug/rat enough to elicit a significant response.
Although the tibia test represents a large improve-
ment in sensitivity it is not sensitive enough to detect
growth hormone in reasonable quantities of the body fluids.
Contopoulos and Simpson (1957b, 1959), Srebnik gt gt.
(1959), and Dickerman gt gt. (1969a) have used the tibia
test to measure the levels of growth hormone in the plasma
of rats. Large doses of plasma were used, 8—32 ml/assay
rat/4 days and problems were observed when using the larger
doses (Srebnik gt gt., 1959). Furthermore, the use of the
tibia test in measuring growth hormone in such large
quantities of plasma is complicated by the fact that the
assay is influenced by hormones other than growth hormone.

Thus, corticotrophin and hydrocortisone inhibit cartilage

 

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7

growth (Geschwind and Li, 1955; Cargill Thompson and
Crean, 1963), and estradiol partially inhibits the effect
of growth hormone on body weight gain and on cartilage
growth (Zondek, 1937; Geschwind and Li, 1955). Thyroxine,
prolactin, testosterone and insulin stimulate cartilage
growth in hypophysectomized animals (Salter and Best,
1953; Geschwind and Li, 1955; Cargill Thompson and Crean,
1963). Diet (Simpson gt gt., 1950) and antibiotics
(Geschwind and Li, 1955) also influence the tibia test.

Other methods proposed have included the decrease
in blood urea (Russell and Cappiello, 1949; Russell, 1951,
1955), the level of non-esterified fatty acids in the
plasma (Raben, 1959), and the uptake of radioactive
sulfate by the tibial epiphysis (Murphy gt gt., 1956)
or by the cosral cartilage of hypophysectomized rats
(Collins and Baker, 1960). These methods have not gained
much acceptance to date perhaps because information con-
cerning their specificity is lacking and because of the
difficulty in applying certain biochemical procedures to
routine laboratory use.

The separation of anterior pituitary hormones by
gel electrophoresis (Lewis and Clark, 1963; Kragt and
Meites, 1966) led to the observation that the width and
depth of the growth hormone band of rats and mice was
related to their biological activity (Jones gt gt., 1965;
Lewis gt a1 , 1965a; Lewis gt gt., 1965b). More recently,

the amount of growth hormone in the stained bands has been

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measured using a densitometric technique (Yanai gt a1 , 1968;

MacLeod and Abad, 1968; Nicoll gt gt., 1969; Birge gt a1

__-,
1970, or by using a colorimetric method (Lewis gt gt.,
1969). The latter method differs from those above in

that the stained band is dissolved and the optical density
of the solution is then measured. In these studies it was
shown that the optical density of the stained band, or of
the solution of the stained band, is proportional to the
amount of protein employed for electrophoresis. This
method offers the advantages of being able to detect very
small quantities of hormone and also permits the assay of
individual pituitaries. Although the sensitivity of this
assay approaches that required to assay plasma or serum
growth hormone, quantitation may be affected by masking

of the growth hormone band by serum proteins (Orstein,
1964) which have a velocity of migration similar to that
of growth hormone (K.H. Kortright, personal communication,
1970).

The development of radioimmunological assays for
protein hormones has opened up the possibility of measuring
growth hormone levels in the blood. Although several
radioimmunoassays for rat growth hormone have been reported
(Parker gt gt., 1965; Schalch and Reichlin, 1966; Frohman
and Bernardis, 1968; Birge gt gt., 1967a; Garcia and
Geschwind, 1968; and Trenkle, 1970), few of these have
been sensitive enough to detect the levels of growth hormone

in the blood of normal rats. In general, the assays have

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differed mainly in the tracer used for iodination, the
species of growth hormone used for labeling, and the
animals used for the generation of antiserum.

II. Hypothalamic Control of Anterior Pituitary (AP)

Secretion of Growth HormgtggIGfi)

The release of growth hormone from the anterior
pituitary is now generally accepted to be under hypothalamic
control. For many years, however, the relationship and
mechanism of interaction between the central nervous
system (CNS) and the anterior pituitary remained virtually
unsolved. Several reasons accounted for this, not the
least of which were the lack of a sensitive assay for CH
as well as the related problem of the lack of a specific
target organ.

Perhaps the first observations which linked the
CNS with the control of GH release from the pituitary
were the clinical observations of Armstrong and Durh
(1922) and Frazier (1936). They observed that tumors
of the infundibulum and pituitary stalk resulted in slow
growth and retarded skeletal age in patients. In 1938,
Cahane and Cahane injured the hypothalamic area in rats
and observed a reduction of body growth. These authors
suggested a possible role for the nervous system in
controlling GH secretion.

The separation of the pituitary from any nervous

connection either by transection of the pituitary stalk

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10

or by its removal and retransplantation away from its
original location was an obvious approach to study the
relationship between the CNS and AP. In this light,
Uotila (1939) observed a temporary growth delay in rats
following stalk section. Westman and Jacobsohn (1940)
transected the pituitary stalk in rabbits but failed

to inhibit growth. These authors, however, reported
marked gonadal atrophy. Greep (1936) observed some
maintenance of growth in hypophysectomized rats which
had received pituitary transplants.

The above observations and the description by Popa
and Fielding (1933) of the hypophyseal portal system,
whose flow was later correctly described to be from the
hypothalamus to the pituitary by Houssay gt gt. (1935),
Wislocki and co—workers (1936, 1937, 1938) and by Green
and Harris (1949), led Green and Harris (1949) to suggest
that humoral agents are secreted into these hypophyseal
portal veins and travel to the anterior pituitary and
there exert a regulatory influence on the secretion of
anterior pituitary hormones. These humoral agents were
later to be known as hypothalamic "releasing factors"
(Saffran gt gt., 1955).

In reviewing the post-Green and Harris (1949)
literature concerning the existence of a growth hormone
releasing factor (CH-RF or GRF), it is of interest to
note the criteria established for the definition of a
hYDOphyseal releasing factor and try to relate it with

I.~.v*

11

the work done on GH-RF. These criteria have been adapted
from the classical criteria for defining hormones (McCann
and Dhariwal, 1966), and include the following:
(a) The existence of a hypothalamic site or sites
responsible for the control of anterior pituitary
growth hormone. This site would presumably be
concerned with the production of GH-RF and its
damage or stimulation should result in detectable
alterations in the secretion of the hormone from
the anterior pituitary.
(b) The GH-RF should be extractable from the area
or areas mentioned above.
(c) The GH-RF must be able to alter the secretion
of Oh when given to an animal whose responses to
non-specific stimuli have been blocked. This
stimulation must be dose related.
(d) The factor should be effective when applied
directly to the pituitary gland t2 2129-
(e) The factor in question should be detectable
in hypophyseal portal vessel blood.

The dependence of the anterior pituitary on the CNS
for the control of growth hormone release and the
"chemotransmitter hypothesis" have been substantiated
using a number of techniques, among them , pituitary
Stalk section, pituitary transplantation, ta ZlEEQ

explantation of pituitary, brain lesions, brain stimulation

 

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12

and brain content of GH—RF.

The stalk section studies mentioned previously,
as well as those of Greep and Barrnett (1951) and Daniel
and Prichard (1964) yielded inconclusive evidence.

This could be explained by the observation of Harris (1950)
concerning the regeneration of the portal vessels and
the possibility of incomplete sectioning of the stalk.

Pituitary transplants to hypophysectomized rats have
been able to maintain some degree of growth (Greep, 1936;
Martini and de P011, 1956; Goldberg and Knobil, 1957).
The transplants have been made in different locations
with lesser or greater success. Hertz (1959) reported
body growth in young hypophysectomized rats implanted
with 4 pituitaries underneath the kidney capsule to be
2/3 of that of intact controls. Swelheim and.Wolthuis
(1962) also observed significant growth in young hypophy-
sectomized rats given a single implant underneath the
kidney capsule. Meites and Kragt (1964) reported growth
averaging 46.6% of that of intact control in young
hypophysectomized rats bearing a single subcutaneous
implant.

The differences in degree of success in part may be
related to the site of transplantation. Halasz gt gt.
(1962, 1963) observed no significant body weight gains
in hypophysectomized rats bearing implants under the
kidney capsule or the temporal lobe of the brain. Signifi-

cant weight increases were observed when the implant was

 

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13

located in the hypophysiotrOphic area of the brain.
Nikitovitch-Winer and Everett (1958) observed that
functional restitution and revascularization of the
pituitary took place after retransplantation of the gland
from the kidney capsule to the median eminence, after a
prolonged sojourn away from the sella turcica. Other
factors which account for these differences in success may
be related to the age of the pituitary donor (Meites gt al.,
1962), the delay in transplantation after hypophysectomy
(Smith, 1961), the amount of viable anterior pituitary
tissue present in the grafts, and to post-graft immunological
factors.

Studies involving brain lesions or stimulations and
their effect on growth are perhaps better substantiated.
The growth disturbances observed by Armstrong and Durh
(1922), Frazier (1936), and Cahane and Cahane (1938)
are difficult to interpret because of the extent of the
lesions. Bogdanove and Lipner (1952), Hinton and
Stevenson (1962) and Bernardis gt a1. (1963) also
reported reduced body growth in rats bearing hypothalamic
lesions. It should be stressed, however, that extensive
cerebral lesions may also interfere with food intake and
temperature regulation (Bogdanove and Lipner, 1952;
Bernardis gt al., 1963) and thereby impair growth.
Extensive lesions could also involve areas controlling the

secretdon of other AP hormones which may affect growth.

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14

Reichlin (1960a) showed that lesions of the median
eminence and primary portal plexus of the stalk signifi-
cantly reduced the growth rate of rats. Taking into
account the possibility of altered secretions of other
hormones, Reichlin (1960b) in a subsequent study injected
vasopressin, testosterone and thyroxine into the injured
animals. Pair-fed controls were used to exclude the
effects of differences in food intake. Body growth was
not restored to normal following this treatment. In
1961, Reichlin studied the effects of massive ventral
lesions of the hypothalamus in rats on body growth and
pituitary growth hormone content as measured by bioassay.
Together with the decrease in body growth he showed that
GH content in injured animals was reduced to 15% of that
found in non-injured animals. Bach at al. (196u) found
that bilateral lesions of the paraventricular nuclei in
weanling kittens caused a reduction in growth rate and
pituitary acidophilic degranulation. On the other hand,
electrical stimulation of the paraventricular nuclei of
weanling kittens by O'Brien gt g1. (196“) caused accelera-
tion of growth as measured by body weight and tibial
length. Median eminence or pituitary stalk lesions in
monkeys have been reported to block the release of GH
which follows insulin-induced hypoglycemia (Abrams gt a;.,
1966).

The development of radioimmunological methods to

assay rat GH has allowed the measurement of GH in the

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plasma of rats after stimulation or destruction of

certain hypothalamic areas. Frohman and Bernardis (1968)
showed that rats with growth retardation subsequent to
ventromedial hypothalamic nucleus destruction have
decreased levels of growth hormone in the pituitary as well
as in the plasma. In the same year, Frohman 2; a1. (1968)
showed that electrical stimulation of the above mentioned
area resulted in significant increases in plasma GH

levels. Stimulation of the cerebral cortex had no effect
on plasma GH.

In a more elegant study, Frohman and Bernardis (1970)
studied the secretion rates of GH in rats subjected to
ventromedial hypothalamic nucleus stimulation. These
authors measured the plasma GH levels and metabolic
clearance rate (MCR) of GH to arrive at the secretory
rates. The rats subjected to hypothalamic stimulation
showed a 5-fold increase in plasma GH within 10 minutes.
No change was observed in rats subjected to cerebral
cortex stimulation. A significant increase in secretion
rate was observed in the hypothalamic stimulated group.
Bernardis and Frohman (1970) studied the effects of
electrical stimulation of several areas of the hypothalamus
in rats on plasma growth hormone concentration. They
found that only stimulation of areas within and at the
border zones of the ventromedial hypothalamic nuclei
resulted in significant increases in plasma growth

hormone. Inconsistent changes were found when anterior

r————__

16

or posterior hypothalamic areas were stimulated. The
authors attributed the latter observations to be the result
of the stimulating current spreading via intrahypothalamic
fiber systems to reach the ventromedial nucleus. The
work of these authors (Frohman and Bernardis, 1968, 1970;
Frohman gt al., 1968; Bernardis and Frohman, 1970) is
the most convincing on the existence of a site concerned
with the production of GH-RF. It suggests that the
ventromedial hypothalamic nucleus is responsible for the
control of anterior pituitary GH synthesis and release
and is therefore a strong candidate for the site of GH-RF
production. Their evidence, however, does not rule out
the possibility that the site may be concerned with the
production of other releasing factors as well.

The measurement of the GH-RF in the hypothalamus
has been carried out in three ways: a) direct measurement
of pituitary GH content in animals injected with median
eminence extracts b) measurement of the pituitary
content and release of GH into medium after incubation
with median eminence extracts, and more recently c) deter-
mination of plasma GH levels following the administration
of median eminence extracts or purified preparations of
GH—RF into recipient animals.

The first claim of detection of a factor in neural
tissue responsible for controlling the release of anterior
pituitary GH was that of Franz 22 a1. (1962) in the hypo-

thalamus of swine. Their work and conclusions, however,

J.

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17

have been criticized since standard assay procedures were
not followed and large variations in response were
obtained. Deuben and Meites (1964, 1965) were the first
to present conclusive evidence for GH-RF. They reported
that neutralized acid extracts of rat hypothalamus produced
a 4 to 6—fold increase in GH release by rat anterior
pituitary after a 6 day culture. Cerebral extract failed
to increase GH release upon culture. Deuben and Meites
(1965) alSo reported reinitiation of pituitary GH release
12 ZlEEQ by a neutralized acid extract of rat hypothalamus
after release had ceased.

The first tg vivo demonstration of GH-RF activity

 

was reported by Pecile gt gt. (1965). These authors
reported that hypothalamic extracts were able to deplete
the anterior pituitary of recipient rats of bioassayable

GH. Both the lg vitro and lg vivo observations have been

 

confirmed by several investigators using bioassay
procedures either with crude hypothalamic extracts or
purified preparations of GH-RF (Schally gt g;., 1965;
Muller gt gl., 1965; Muller and Pecile, 1965; Schally
gt gt” 1966; Dhariwal gt gt, 1966; Krulich gt gt”
1965; Machlin gt gl., 1967; Schally gt g;., 1968a).
An Lg vitro method for the quantitation of GH-RF was

 

reported by Dickerman gt gt. (1969b). These authors were
the first to show a dose-response between hypothalamic
tissue and the amount of GH released into the medium.

The GH-RF activity has been reported to be present in

_.—_

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18

several species of mammals as well as in some birds and
amphibians (see Schally gt g;., 1968b).

The large depletion of bioassayable pituitary GH
observed following administration of hypothalamic extracts
was interpreted to be the result of release of large
quantities of GH into the circulation. Sawano gt g1.
(1968) administered a highly purified preparation of pig
GH-RF into rats and measured, by bioassay, the pituitary
content and plasma concentration of "growth hormone-
like activity". They reported extremely high levels of
growth hormone in the peripheral blood. These levels were
30-50 times as high as the highest levels observed using
radioimmunological assay techniques.

More important, however, has been the failure to
confirm the observations obtained by bioassay when using
radioimmunological procedures. Neither the decrease in
rat pituitary GH nor the increase of GH in blood have
been established by radioimmunoassay (Daughaday gt g;.,
1968; Garcia and Geschwind, 1968; Schalch and Reichlin,
1968). It should be stressed, however, that plasma GH
measurements by radioimmunoassay in the sheep and rhesus
monkey (Machlin gt gl., 1967; Garcia and Geschwind, 1966;
Meyer and Knobil, 1968; Garcia and Geschwind, 1968) have
shown a significant increase subsequent to the administra-
tion of crude preparations of GH-RF. Highly purified
preparations have yielded inconclusive evidence. Garcia and

Geschwind (1966, 1968) further showed that the increase

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19

in the plasma GH of rhesus monkeys was not due to hypo-
glycemia since a glucose infusion which elevated the
plasma glucose concentration to 300 mg 5 was unable to
block the response to the sheep hypothalamic extracts
injected. These authors also reported the inability to
elicit an increase in plasma growth hormone in rabbits
injected with sheep hypothalamic extract.

To our knowledge, confirmation of the action of
hypothalamic extracts on rat anterior pituitary GH
release by radioimmunoassay has come only from the work
of Daughaday gt gt. (1970) and Wilber and Porter (1970).
The former authors were able to show a two-fold increase
in the amount of GH released into the medium upon the
addition of 1.25 crude hypothalamic equivalents per
incubated pituitary. The amount of GH released was not
impressive when compared to that evaluated by bioassay
methods (Dickerman gt g;., 1969U, but this difference
may be accounted for by the different standards used.
Specificity tests, however, indicated that lysine
vasopressin at a concentration of 1.5 U/ml was capable
of eliciting a response of similar magnitude to that
obtained with the hypothalamic extract. Wilber and
Porter (1970) collected hypophyseal portal blood and
tested the plasma for GH-RF activity tg gttgg. These
authors found that addition of hypophyseal portal plasma
to the incubated pituitaries resulted in an increased

release of GH into the medium when compared to the release

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20

observed in incubations with peripheral plasma (149%

vs 100% for controls). This constitutes the first evidence
for the existence of the GH-RF in the hypophyseal portal
circulation. However, neither Daughaday gt gl. (1970)

nor Wilber and Porter (1970) tried to establish a dose-
response relationship between the amount of hypothalamic
extract and/or hypophyseal portal plasma and the amount

of GH released into the medium. Furthermore, it is difficult
to assess the work of Wilber and Porter since they give

no indication either of the actual amounts of GH secreted
or of the validity of the radioimmunoassay in their

hands. In contrast to their work, Buse gt g1. (1970)
reported a failure to observe a decrease in pituitary

GH, as measured by radioimmunoassay, in rats following
administration of hypophyseal portal plasma.

It appears, therefore, that substantial differences
exist between biological and immunological data concerning
the existence of a GH-RF in the rat. One cannot rule
out the existence of a GH-RF in view of the strong
biological evidence. On the other hand, it would be
premature to assume that the radioimmunological assays
available for rat GH are not measuring GH. There is a
definite need to establish the relationship between the
immunological and biological activities of the rat GH
molecule, as this would enable us to better understand

these seemingly disparate findings.

 

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It seems appropriate to close this section with a
word on the chemistry of the GH-RF. The factor has been
purified from ovine (Krulich gt gt., 1965; Dhariwal gt gt.,
1966) and porcine or bovine hypothalami (Schally gt gt.,
1965; Schally gt gt., 1966; Schally gt gt., 1967;

Schally gt gt., 1968b; Schally gt gt., 1969; and Ishida

gt gt., 1965). A growth hormone inhibiting factor (GIF)
has been reported by Krulich gt gt. (1968), and by Dhariwal
gt gt. (1969) in purified fractions of sheep and rat
hypothalami. Schally gt gt. (1969), however, using a
method of assay similar to that of Krulich gt gt. (1968)
have so far found no evidence for GIF activity in extracts
of porcine hypothalamus. Most recent information

(A. Schally, personal communication, 1970) indicates that
pig GH-RF is a small polypeptide consisting of 10 amino
acids. Apparently elucidation of the structure and synthesis
of this polypeptide have been accomplished by Schally's
laboratory. At present our laboratory is in the process

of evaluating the effect of natural and synthetic GH-RF

from Dr. A. Schally on the release of GH lg vitro as

 

measured by biological and radioimmunological techniques.

Physiological States

III. Secretion of Grggth Hormone During Different

The advent of radioimmunological techniques for
assaying human growth hormone (HGH) has contributed
greatly to the understanding of the factors involved in

controlling, modifying or affecting its secretion in humans.

‘1I'IF""""""""""""""""""""""'""""""""""""
22

It is not the purpose of this review, however, to emphasize
the human condition, as this will be mentioned only where
it may help to understand what takes place in the rat and
other mammals. The reader is referred to the excellent
reviews by Glick gt gt. (1965), Glick and Goldsmith (1968)
and Glick (1968, 1969), in which the regulation of GH in
humans is treated in depth. '
A. gge and GH
Growth hormone is first detectable in the fetal
rat pituitary at day 19 of gestation (Contopoulos and
Simpson, 1957a). Baker gt gt. (1956) also reported the
ability to detect growth hormone in the pituitaries of
fetal pigs. It was later shown that the total amount of_
GH present in the rat pituitary increased with age, but
that the concentration of the hormone per mg of tissue
remained constant in rats between 10 and 630 days of
age (Solomon and Greep, 1958; Bowman, 1961). Pecile gt
gt. (1965) reported that the hypothalamic content of GH-
RF was greater in 30 day old rats than in 2 year old rats.
The above results, all of which were obtained using
biological assay methods, do not agree with those reported
by Birge gt gt. (1967a) using a radioimmunoassay for rat
GH. These authors reported increases both in content and
concentration of OH with age, with male rats having higher
concentrations than females after puberty. In males
this increase continued into old age, whereas in female

rats it plateaued at maturity. It is of interest to note,

SES

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23

however, that the authors found little or no increase in
the weight of the pituitary gland in male rats after 49
days of age, and reported an average weight of 6.70 mg
per pituitary for male Sprague-Dawley rats 147 days old
and weighing 312 grams. Garcia and Geschwind (1968),
also using radioimmunological procedures, reported
increases in concentration of pituitary OH with age but
no significant difference in concentration between male
and female rats.

Burek and Frohman (1970) have recently reported that
pituitaries from adult male rats (165 days old) were
able to synthesize more GH than pituitaries from young
male adult rats (70 days old), and the latter synthesized
more GH than pituitaries from weanling male rats (23
days old) tg gttgg. Comparison of growth hormone
synthesis between adult male and female pituitaries of
the same body weight showed that the incorporation of
3H-leucine into GH was 2- to 3-fold greater in the male
pituitaries whether expressed as dpm/mg pituitary or GH
specific activity (dpm/ug growth hormone). Pituitary
concentration of GH was similar for both sexes, and no
significant difference was found in the ability of male
and female weanling pituitaries to synthesize GH. As yet,
r10 information is available on the levels of plasma GH
with respect to age in the rat.

The levels of growth hormone in the human pituitary

have been reported by Gershberg (1957). He found no

 

 

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24

difference in concentration among fetal, adolescent and
mature male pituitaries. An increase in total content was
explained as a result of the increase in size of the
pituitary. Plasma HGH is very high in the fetus and at
parturition (Greenwood gt gt., 1964a), subsequently
declining but remaining higher in children than in adults
(Greenwood gt gt., 1964b).
B. Hormonal Influence on GH Secretion

The effect on insulin induced hypoglycemia in humans
was first reported by Roth gt gt. (1963a). They showed
that the administration of insulin in doses large enough
to lower blood glucose resulted in increased HGH in
the plasma. Later Roth gt gt. (1963b) reported that falling
blood glucose levels, as well as interference with the
metabolism of glucose by administration of 2 deoxy-D-glucose,
results in increased plasma HGH. These results were
later confirmed by Hunter and Greenwood (1964), Frantz
and Rabkin (1964), and others. Luft gt gt. (1966),
however, reported that small insulin doses which did
not cause symptomatic levels of hypoglycemia, elicited a
release of GH into the circulation. These latter results
were subsequently confirmed by Greenwood gt gt. (1966)
using graded doses of insulin.

Katz gt gt. (1967) studied the effects of insulin
induced hypoglycemia in the rat. They reported a significant
«depletion of pituitary GH and of hypothalamic GH-RF. It

had been previously shown by Krulich and McCann (1966a)

.l Card"

 

 

 

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25

that insulin induced hypoglycemia in hypophysectomized
rats resulted in increased GH-RF activity in the peripheral
blood as well as in a decreased content of GH in intact
rats (Krulich and McCann, 1966b). Katz gt gt. (1967)
concluded that hypoglycemia stimulated the release of GH-RF
from the hypothalamus which in turn stimulated the release
of GH from the pituitary. These results were confirmed by
Muller gt gt. (1967a). The above results in the rat have
not been confirmed using radioimmunoassay techniques.
Daughaday gt gt. (1968), Garcia and Geschwind (1968),
and Schalch and Reichlin (1968) all failed to observe
pituitary GH depletion or rise in plasma GH following
insulin administration to rats.

Thyroidectomy in the rat results in a degranulation of
pituitary acidophiles (Purves and Griesbach, 1946;
Schooley et al., 1966), a decrease in growth rate (Koneff
gt gt., 1949; Schooley gt gt., 1966) and a decrease in
pituitary GH content (ContOpoulos gt gt., 1958; Knigge,
1958; Solomon and Greep, 1959; Schooley gt gt., 1966;
Meites and Fiel, 1967). Meites and Fiel (1967) showed
that thyroidectomy resulted in decreased content of
hypothalamic GH-RF. They also showed that thyroxine
therapy resulted in increased GH-RF and pituitary GH.
These results have been confirmed by Daughaday gt gt.
(1968) using radioimmunological assays. Hypothyroidism
as a result of propylthiouracil or radioiodine ablation

was reported to decrease pituitary GH content.

26

The interference with body growth by estrogens has
been suspected for some time (Gaarenstrom and Levie, 1939;
Reece and Leonard, 1939), although its mechanism of action
remains uncertain. Meites (1949) showed that growth of
stilbesterol-treated rats was the same as that of pair-fed
controls, attributing the decreased growth in large part
to the decrease in food intake elicited by stilbesterol
administration. Sullivan and Smith (1957) confirmed those
findings using estradiol. Josimovich gt gt. (1967)
and Roth gt gt. (1968) reported evidence suggesting the
existence of peripheral antagonism between estrogen and GH
in the rat and humans respectively. Birge gt gt. (1967b)
reported that pituitaries incubated with diethylbesterol
caused suppression of GH release but had no effect on the
amount stored in the pituitary. Birge gt gt. (1967a)
further showed that males treated with diethylbesterol or
estradiol benzoate had a decreased pituitary concentration
of GH, while gonadectomy of female rats resulted in a
slight, but not significant, increase in GH concentration.
Plasma GH levels have been reported to be higher in female
than in male rats (Schalch and Reichlin, 1966).

The levels of GH in plasma and their relation to
estrogen are better illustrated in the human. No difference
in plasma HGH was found between males and females in the
basal state, although in the ambulatory state the females
had a higher plasma concentration (Frantz and Rabkin, 1965;

Garcia gt gt., 1967). Increase in HGH levels have been

27

observed following ovulation and during the pre-menstrual
phase. Lower levels have been found in post-menopausal
women (Frantz and Rabkin, 1965). The ingestion of oral
contraceptives is associated with elevation of plasma

HGH levels (Garcia gt gt., 1967). These elevated levels
in the female are perhaps related to the need of the body
to compensate for the peripheral antagonism of estrogen
which may reduce the ability of the body to use GH.

Androgens stimulate growth when given in small doses
(Rubinstein and Solomon, 1941). Injections of testosterone
propionate to adult female rats for 14 days resultedin
increased concentration of pituitary GH when compared to
sesame oil injected controls. Castrated male rats
treated with testosterone propionate also showed increased
concentrations when compared to castrated male rats given
estradiol benzoate or sesame oil injections (Birge gt gt.,
1967a; Daughaday gt gt., 1968).

Adrenal corticoids may have an effect similar to that
observed with high doses of androgens (Evans gt gt., 1943;
Marx gt gt., 1943; and Geschwind and Li, 1955), in which
growth of long bones ceases by the closing of the epiphysis.
The effect of corticosteroids on pituitary and plasma OH
is less substantiated. Reichlin and Brown (1960) studied
the effects of adrenalectomy on growth and pituitary GH
in the rat. Although growth is impaired they found this
to be more closely related to a reduced food intake. No

change in pituitary GH concentration was observed following

28

adrenalectomy. The administration of cortisol to
thyroidectomized rats has been claimed to produce
acidophilic regranulation and a reaccumulation of GH in

the pituitary gland (Meyer and Evans, 1964), as measured

by the tibia test bioassay. Lewis gt gt. (1965b),

however, failed to show by acrylamide gel electrophoresis,
a reaccumulation of GH following the administration of
cortisol to propylthiouracil treated rats. The findings of
Lewis gt gt. (1965b) were later confirmed by Daughaday

gt gt. (1968) using a radioimmunoassay for rat GH. These
authors also failed to observe a reaccumulation of GH after
cortisol treatment to prOpylthiouracil treated rats.

The decreased pituitary GH in rats as a result of
insulin induced hypoglycemia (Krulich and McCann, 1966b;
Katz gt gt., 1967; Muller gt gt., 1967a) has been blocked
by the administration of cortisol in large doses (Muller
gt gt., 1967b). A decrease in hypothalamic GH-RF was
observed, and it was interpreted as indicating a hypothalamic
action for cortisol. The yg gttgg work of Birge gt gt.
(1967a), however, has demonstrated a direct effect of
cortisol on hemipituitaries in tissue culture. No data is
yet available on the plasma levels of GH after cortisol
treatment in rats.

The levels of pituitary and plasma growth hormone
activity during pregnancy have been measured in the rat.
Contopoulos and Simpson (1956) found no change in pituitary
GH concentration. Later, Contopoulos and Simpson (1957b)

 

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29

showed a 2- to 3-fold increase in growth hormone activity
in the plasma of pregnant rats. This activity did not
disappear after hypophysectomy on day 12 of pregnancy
(Contopoulos and Simpson, 1959) indicating an extra-
pituitary source. These results were not confirmed by
Schalch and Reichlin (1966) using a radioimmunoassay
technique. They reported no change in plasma OH with
pregnancy. These data need to be confirmed by others.

C. Nutrition and Stress

A reduction in pituitary and plasma GH activity was
found by Srebnik gt gt. (1959) in rats fed a protein
free diet for prolonged periods of time. Starvation in
the rat has been reported to decrease hypothalamic
content of GH-RF (Meites and Fiel, 1965; Dickerman gt gt.,
l969a),and pituitary concentration of GH (Meites and Fiel,
1965; Friedman and Reichlin, 1965; Dickerman gt gt.,
19698). Dickerman gt gt.(1969a) further showed that starva-
tion resulted in decreased plasma growth hormone activity.
These results have been recently confirmed by Trenkle
(1970) using a radioimmunoassay technique.

It is of interest to note, however, that other related
species do not respond in the same manner to starvation.
Thus, Garcia and Geschwind (1968) reported elevations of
plasma growth hormone in mice and rabbits after starvation.
Machlin gt gt. (1968a) reported that plasma GH levels in pigs
increased during the first 48 hours of starvation and

subsequently fell to lower levels. No significant changes

30

were observed in the plasma GH levels of young sheep
fasted for 7 days (Machlin gt gt., 1968b). This may
indicate increased GH secretion during fasting may not take
place in all species.

The effects of a variety of stresses on rat pituitary
GH have been reported. Muller gt gt. (1967a) noted that
cold, high doses of vasopressin, epinephrine and urecholine
depleted pituitary GH.‘ Muller gt gt. (1967b) later reported
cold depletion of hypothalamic GH-RF and increase in
plasma GH—RF. It was previously indicated by Krulich and
McCann (1966c) that stresses could alter the content of
pituitary GH in the rat. Recently, Parkhie and Johnson
(1969) found that heat stress would also deplete pituitary
GH but elevate the hypothalamic stores of GH-RF. As was
the case before, radioimmunological data failed to confirm
the above reports. Schalch and Reichlin (1967, 1968) found
no increase in plasma GH in rats subsequent to exercise,
moderate or severe hypoglycemia, and cold. Daughaday
gt gt. (1968) and Garcia and Geschwind (1968) also failed
to observe the effect of cold or hypoglycemia on the content

of pituitary growth hormone.

EXPERIMENTAL METHODS AND MATERIALS

I. Animals

Experimental animals were Sprague-Dawley rats obtained
from Spartan Research Animals (Haslett, Michigan). Animals
for GH bioassays were immature female rats (Charles River
Breeding Laboratories, Wilmington, Massachusetts)
hypophysectomized at 26 days of age. They were used for
GB bioassay 14-16 days after Operation.

Experimental animals were maintained on a diet of
Wayne Lab Blox pellets (Allied Mills, Chicago, Ill.) and
fed g9 libitum. The diet of the bioassay animals was
supplemented every other day with orange slices, carrots
and sugar cubes. All bioassay and experimental animals
were housed in a temperature controlled room (75: 1° F)
with automatically controlled lighting (14 hours light
daily).

II. Preparation of Pituitaries, Plasma or Serum

The rats were killed by guillotine, and the pitui-
taries were removed, weighed individually, and homogenized
in 0.01M phosphate buffer in 0.1uM NaCl, (phospho-saline
buffer, PBS), pH 7.2, with a Sonifier cell disruptor. The

31

 

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32

individual homogenates were stored at -20°C until the day
before assay. On the day before the assay, the homogenates
were thawed and diluted to the working concentration (see
specific experiments).

Plasma was collected from blood drawn by cardiac
puncture in heparinized syringes and immediately centri-
fuged for 20 minutes at 2200 rpm in an International
Centrifuge. The plasma was pipetted off and the individual
samples were immediately frozen and kept at -20°C until
assayed, unless otherwise indicated in the specific
experiments. Serum was obtained in the same manner except
that non-heparinized syringes were used. The blood was
allowed to remain 2“ hours at #00 to allow the clot to
form and the serum to separate. The blood was then centri-
fuged as described above and the serum frozen at -20°C

until assayed.

III. Assay of Growth Hormone

A. Bioassay

Growth hormone activity was measured by the standard
tibia test of Greenspan gt gt. (1949). Aqueous solutions
of pituitary homogenates were injected intraperitoneally
once a day for 4 days. Each assay included two doses of
the control and experimental solutions, as well as two
doses of NIH-GH-SB and NIAMD-RGH-RP-l or two doses of a
purified rat GH preparation supplied by Dr. Stanley Ellis
(NASA Research Center, Ames, California) designated as

 

 

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33

HVII-38-C. NIH-GH-SB was kindly supplied by the Endocrin-
ology Study Section, NIH. All NIAMD preparations were
supplied by Dr. Albert Parlow (Department of Obstetrics
and Gynecology, School of Medicine, Harbor General Hos-
pital, 1000 West Carson Street, Torrance, California).
Four assay animals were used per dose of rat pituitary or
standard preparation.
B. gggtoimmunoassay

Assay of rat serum or plasma and pituitary growth
hormone levels was performed by radioimmunoassay. The
radioimmunoassay utilizes a double antibody method develop-
ed in this laboratory in collaboration with Drs. Ytschak
Koch and Samuel Dickerman.
Monkgyggtiserum to rat GH. Two preparations of monkey
anti-rat GH serum have been used throughout this study, a
NIAMD—A-Rat GHS-l from Dr. Parlow, and a monkey anti-rat
GH from Dr. W.H. Daughaday (Washington University School
of Medicine, St. Louis, Missouri) henceforth called DMD-1.
The anti-serums were diluted to 1:500, 1:2500, 1:5000,
1:10.000, l:20,000, l:30,000, lzu0,000, 1:50,000 and
l:60,000 in 1:600 normal monkey serum (NMS) previously
diluted in 0.05M EDTA-PBS (etheylenedinitrilo tetraacetic
acid, disodium salt) pH 7.2. The dilutions were tested for
antibody titer to determine a working concentration, 200 ul
of which would bind 50% of the radioiodinated growth hormone
added to the incubation tubes. It was found that a 1:50,000
dilution of the NIAMD-A-Rat GHS-l bound 45-58% of the

34

radioiodinated GH, whereas a l:l6,000 dilution of the DMD-
l was required to bind as much.

Production of goat antiserum against monkey gamma globulin.
(Anti-MGG). A mature female goat was obtained from the
Endocrine Research Unit through the courtesy of Dr. Nellor
and Dr. Riegle. The goat was immunized by weekly sub-
cutaneous injections of monkey gamma globulin (Immunology
Inc., Glen Ellyn, Illinois, U.S.A.) emulsified in complete
Freund's adjuvant. Four immunizations, once weekly, were
given in the following amounts: 30, 35, 40,and 80 mg of
monkey gamma globulin. Two weeks later a second 80 mg
immunization was given and the goat was bled through the
jugular vein. A volume of 500-650 ml of blood was collected.
The goat was injected with booster injections of 80 mg
every four months. Periodic bleedings at 3-h month inter-
vals were made. The goat antiserum against monkey gamma
globulin was diluted with PBS to 1:2, 1:3, 1:4, 1:5, 1:6,
1:7, 1:8, 1:10 and 1:15 and titrated for the Optimal dilu-
tion to precipitate the monkey gamma globulin. Originally
the combining dilutions of 200 ul of anti-monkey gamma
globulin and NMS (200 ul of 1:600 NHS in EDTA-PBS) were
determined empirically by observing the dilution of anti-
monkey gamma globulin which gave the greatest amount of
precipitate. The proper dilution was later verified in an
experiment in which a series of tubes at 2 different radio-
iodinated growth hormone concentrations were set up as for

an assay, the amount of NMS remaining constant while dilutions

 

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33113130111
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35

of anti-MGG were varied as per above description. It can be
seen in Figure 1 that a peak in precipitation occurred when
using a 1:7 dilution of anti-MGG. This procedure for deter-
mining the best dilution has been followed for each subse-
quent bleeding of the goat.

Radioiodtnation of ratggrowth hormone. Two highly purified
rat GH preparations were used for iodination: Dr. Parlow's
NIAMD-RGH-I-l and Dr. Ellis' HVII-38-C. The following proce-
dures apply to both, although the results presented later

in the text have been obtained using the HVII-38-C preparation.
Subsequently, the term iodination quality is used to refer

to highly purified hormone preparations.

Purified rat growth hormone was first dissolved in
double distilled water (pH adjusted to 7.5) to a concen-
tration of 1 ug/ul. Twenty ul (20 ug) of rat GH were pipet-
ted into 1 m1 serum bottles. These bottles were kept at
-20°C until iodinated. Prior to iodination they were thawed
and 100 ul of 0.5M phosphate buffer at pH 7.5 added. The
solution was mixed well and 1 millicurrie (mo) of I125 with
high specific activity was added and mixed (1125 with high
specific activity and carrier free was obtained from Cam-
bridge Nuclear, Cambridge, Massachusetts). Twenty-five u1
(87.5 ug) of Chloramine-T (35 mg/10 ml) were added to the
serum bottle, the bottle sealed with parafilm and mixed, and
the reaction allowed to proceed for #5 seconds. At the end

of the reaction time 125 ug of Sodium Metabisulfate (N825205)

(50 ul of 25 mg/lo ml solution) were added and mixed thoroughly.

36

 

 

 

 

 

 

50f

1: t_,———""‘~._l

C!

3 40.” ~
8 low RGH-1'25
I)
O!
7.... 30.

:1:

o

25 2C)“ al.v”“~——

§ high Row-1'25
g .0.

1:2 54 he Its |:I0 I52 (44
ZOOpI AMGG

Figure 1. Precipitation of RGH-antibody complex with varying
dilution of anti-monkey gamma globulin serum.
RGH-112 bound is expressed in terms of total radio-
activity.

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37

One hundred ul of transfer solution containing 100 mg KI/lO ml
in 16% sucrose was added, the solution mixed thoroughly by
withdrawing and expelling it from a plastic syringe, and trans-
ferred to a 0.9 x 20 cm column of Sephadex G-50 expanded in
0.01M barbital buffer. The serum vial was rinsed with the
same syringe used in the previous step using 100 ul of a solu-
tion containing 100 mg KI/lO m1 and 1 mg bromphenol blue in 8%
sucrose. The bromphenol solution was placed in the column to
mark the layer of material transferred in the previous step.
The outlet of the column was opened and 1 ml aliquots were
collected in tubes containing 1-2 drops of 30% bovine serum
albumin, or the equivalent, in 0.05M barbital buffer pH 8.6.
The tubes were counted individually in a manual counter using
a splash shield to prevent contamination. The peak tubes were
selected and aliquots of 0.25-0.40 ml (25-35 x 106 cpm) were
transferred, with 0.15 ml of transfer solution, to a 0.9 x 50
cm column of Sephadex G-100 in barbital buffer pH 8.6 for re-
purification. Two m1 aliquots were collected in 1% BSA-PBS
previously coated tubes. The peaks were again selected and 1%
BSA-PBS at pH 7.2 added until 100 ul gave l9-21,000 cpm (on
the day of iodination). The final dilutions were counted in-
side a Nuclear Chicago gamma counter under optimal counting
conditions. The concentrated tubes were kept frozen at ~20°C
and aliquots repurified as needed. The diluted solutions were
kept at 4°C. We have found that the diluted solution may be
kept for as long as two months at 4°C in 1% BSA-PBS without

significant alterations in the immunological activity of the

38

molecule. No attempt has been made to keep the solutions for a
longer length of time.

Procedures for radioimmunoassay. The assay was carried out in
disposable culture tubes, 12 x 75 mm (Kimble Owens, Toledo,
Ohio). The tubes were labeled in the following manner:

1. Tubes A1-5: received 500 ul of 1% BSA-PBS, 200 ul of
1:600 normal monkey serum (NMS) and 100 ul of labeled rat growth
hormone. These tubes measured the amount of non-specific bind-
ing and were used as background while counting the assay tubes.

2. Tubes A6-10: received 100 ul of labeled hormone
(total count tubes).

3. Tubes 81-5, 01-5: received 500 ul of 1% BSA-PBS,

200 ul of NIAMD-A-RatGHS-l or DMD-1 at the appropriate dilu-
tion to bind approximately 50% of the total radioactivity
added, and 100 ul of labeled hormone. The radioactivities of
these tubes was considered as 100% cpm.

4. Tubes 86-17, C6-17: received the standard solutions
of NIAMD-RGH-RP-l diluted in 1% BSA-PBS to a concentration
of 1 mug/ul. In all cases, the standards or unknowns + "x"
volume of 1% BSA-PBS was such that it equaled 500 ul. The
following volumes (ul) of the standard solution were placed
in the tubes which already contained the complementary vol-
ume of 1% BSA-PBS: 250, 125, 62.5, 31, 16, 8, 4. A second
solution diluted 10 times was used to pipette the smaller
amounts: 20, 10, 5, 2.5, 1 ul which corresponded to 2, 1,

0.5, 0.25, and 0.1 mug respectively.

The sample tubes were labeled with consecutive numbers

39

from #1 through the number that was planned for the assay.
Three or more dilutions of unknown serum, plasma or pitui-
tary homogenate were used per sample. These will be indi-
cated in the specific experiments.

The order in which the above reagents were placed in
the tubes follows:

1. A11 tubes, except A6-10, received the 1% BSA-PBS.

2. The standards and unknowns, 100 ul of labeled hor-
mone and 200 ul of anti-rat GH followed. All tubes were
mixed with a Vortex mixer following the completion of each
rack. All tubes were stored at 4°C for 72 hours.

3. At the end of 72 hours, 200 ul of the anti-MGG
solution was added to each tube. The tubes were mixed and
stored at 4°C for an additional 24 hours.

4. Upon completion of the 24 hours, the tubes were
centrifuged in an International Centrifuge PR-2 at 2,200
rpm for 20 minutes. At the end of 20 minutes each tube re-
ceived 3 m1 of PBS and was centrifuged again for an addi-
tional 20 minutes as described above.

5. The supernatant of all tubes was decanted and the
precipitate was placed in a disposable plastic jacket and
counted in a Nuclear Chicago gamma counter with an automatic
changer.

Tubes Al-lO were counted for 60 seconds each. Tubes
Bl-5, 01-5 were also counted for 60 seconds and then re-
counted to record the time it took to reach 10,000 counts.

(The non-specific binding was subtracted autbmatically)..

40

The machine was then set up to count with respect to the
time recorded above and in this way gave a direct percen-
tage reading. The standard curve was extrapolated on semi-
1ogarithmic paper and the unknowns were read out and ex-
pressed as mug of GR per tube. Multiplication by the ap-
propriate factor gave the concentration of GH in mug/ml

of serum or plasma or per mg of anterior pituitary.

To minimize variations between assays several proce-
dures were ad0pted. .

l. A large volume of NIAMD-RGH-RP-l was prepared and
diluted to a l mug/ul concentration. Aliquots of 1.2 1.4
ml were then placed in disposable culture tubes and kept at
-20° C. One tube was used/assay.

2. A stock solution of 1:500 anti-rat OR was prepared.
Large amounts of the working concentration (1:50,000 for
the NIAMD-A-Rat GHS-l) were prepared and aliquoted into
glass bottles. Each bottle contained enough for 300 tubes.

3. Aliquots of the 1:600 NMS to dilute #2 above were
stored at 4°C to be used for the non-specific binding deter-
minations.

4. The anti-MGG was diluted to its working concentra-
tion and divided so as to have enough for 300 tubes/bottle.
Following the above precedures we have been able to maintain
our level of non-specific binding constant throughout the
course of this study. All racks were kept in an ice bath

while pipetting the solutions.

 

 

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41

IV. Metabolic Clearance Rate (MGR)

The metabolic clearance rate of growth hormone under
several physiological states was studied using a single
injection of radioactive hormone. The animals used for
this study were anesthetized with Na-pentobarbital (30
mg/kg) and injected through the tail vein with 1 m1 of
PBS solution containing approximately 2.4 x 106 cpm of rat
GH-I125. Blood samples of 750 ul were obtained at 1, 5,
10, 15, 20, 30, 45, 60 and 90 minutes following the in-
jection and placed in tubes containing heparin solution.
All tubes were kept in an ice bath during the collection
and centrifuged immediately afterward in an International
Centrifuge PR-2 at 2200 rpm for 20 minutes. The plasma was
frozen and kept at -20°C until assayed 5 days later.

Two doses of plasma (100 and 50 ul) were used to
react with the anti-rat GH (DMD-l) and two doses (100 and
50 ul) to count total radioactivity per sample per time. A
high concentration of DMD-l (1:1000) was used to insure
the precipitation of most of the antibody precipitable radio-
activity in each sample.

The metabolic clearance rate of GH was determined
using the following formula, described by Tait (1963) and
Kohler gt gt. (1968a): ‘

total immuno reci itable I125 RGH injected
MCR = Z'x' - dt

in which x' represents the plasma concentration of immuno-

 

precipitable 1125 - RGH. A disappearance curve of

 

 

-1
‘d .
’ .

:rdinates

231?] seat

 

 

42

immunoprecipitable radioactivity/ml and total radioactivity/
m1 of plasma was plotted for each animal using linear co-
ordinates. The integralJ/flx'odt was determined by numeri-
cally measuring the area under the disappearance curve for
the immunoreactive RGH-1125 for each animal. The value ob-
tained was divided into the total immunoprecipitable RGH-
1125 injected to obtain MCR. The individual metabolic clear-
ance rates thus obtained were then pooled per group and an-
alyzed statistically. The secretion rate of growth hormone
was arrived at by multiplying the individual MCR times the

plasma growth hormone concentration of each animal.
V. Methods of Statistical Analysis

Serum,p1asma or pituitary samples were assayed at 2
or more different dilutions to insure accurate measurement
of growth hormone levels. The average of these dilutions
was used as the growth hormone value of that sample. Mean
and standard error of the mean were calculated using the
average values of all samples in a group. Significance of
differences between groups was determined by Student's t
test or analysis of variance, followed by Duncan's new mul-

tiple range test (Bliss, 1967).

EXPERIMENTS

I. Radioimmunoassay for Rat Growth Hormone

A. Objectives

The purpose of these experiments was to develop a
radioimmunological assay for rat growth hormone capable
of measuring GH in the body fluids. In developing the
assay, several steps have been carried out to test its
validity and specificity. These have not been grouped in
one specific section; instead, they have been placed in
those sections of this thesis which the author feels are
most closely related to the topics in question. The steps
followed in establishing the assay included: a) iodina-
tion of RGH of high specific activity, b) evaluation of
the damage caused by the iodination, 0) steps to minimize
damage after iodination, d) selection of the more immuno-
reactive portion of the labeled hormone, e) specific
activity determination, f) competition curves with rat GH
reference preparations, plasma and pituitary homogenates,
g) competition curves with serum from hypOphysectomized
animals, h) cross-reactivity of the anti-rat GH serum with
other pure rat hormones, 1) measurement of pituitary GH
in different physiological conditions by bioassay and

radioimmunoassay, and j) cross-reactivity of the anti-rat

43

44

OH with preparations from other species. Steps a-i are
included in this section.

B. Procedures

The iodination of RGH (NIAMD-RGH-I-l or HVII-38-C)
was accomplished using essentially the method of Green-
wood gt gt. (1963). Initially 2.5 ug of RGH was labeled
with 1 mo of 1131, but it was later changed to 1125 because
of its longer half-life. Since the number of atoms in a
mo of 1125 is seven times greater than in a mo of 1131, the
amount of RGH was increased 7 to 8 times greater than the
original amount used. As indicated in "Materials and
Methods", 20 ug of RGH are now labeled with 1 mo of 1125.
The reader is referred to "Materials and Methods” for de-
tails of this reaction.

C. Results

1. Iodination of RGH. Sepgration fggg the free 1125.
Separation of the RGH-1125 from the free I125 was

accomplished by placing the reaction mixture in a 0.9 x
20 cm column of Sephadex G-50 expanded in 0.01M barbital
buffer at pH 8.6. The column was previously coated with a
5% solution of BSA-barbital. Figure 2 shows a typical elu-
tion pattern obtained when 1 m1 aliquots are collected in
tubes containing 1-2 drape of 30% BSA-barbital or their
equivalent. The small volume of BSA added to the tubes is
intended to coat the glassware without producing dilution of
the collected material. In this way large amounts of hor-

mone can be repurified by transferring small volumes of the

45

material to a second column, facilitating the formation of
a small packed layer. As can be seen in Figure 2, about
50% of the recovered radioactivity is present in the hor-
mone peak, while the remainder is in the form of free 1125.
2. Iodination damggg.

a. Repurificgtion of RGH-1125 in Sgptgdex G-100.

To establish whether or not damage had taken
place during iodination, an aliquot of the RGH-1125 peak
of the G-50 elution was repurified in a 0.9 x 50 cm column
of Sephadex G-100 expanded in 0.05M barbital buffer at pH
8.6. A volume of 0.25-0.40 ml was transferred with 0.15 ml
of transfer solution to aid in the packing of the layer.
Two ml aliquots were then collected in 1% BSA-PBS coated
tubes. Figure 3 shows the elution of the RGH-1125 aliquot
through the Sephadex G-100 column. As can be seen, three
peaks are discernible. The first peak is an aggregated
form of RGH-1125; the second peak corresponds to the mole-
cular weight of growth hormone; while the third peak repre-
sents the degraded fraction and iodine. It was later learned
that the relative proportion of these peaks varied with the
source of the aliquot for repurification. Aliquots from
tubes of the ascending limb of the G-50 column contained
proportionately greater amounts of the aggregated; form of
RGH-1125 (as much as 50-60% of the total radioactivity re-
covered) than aliquots of the descending limb (generally
between 5-10% of the total radioactivity). Furthermore, it

was later observed that the interval between iodination

46

 

 

 

 

 

 

 

8 -
RGH'I'za IIZB
6 u-
o )-
2 .
)< 4m-
5
(L. L
U
2 e
o‘ 4 s :2 I6 20 24

Figure 2. Elution pattern of RGH-1125 through a 0.9 x 20 cm
columnaog Sephadex G-50 in 0.01M barbital buffer
at pH . .

47

CPM x no6
t

(3
I

 

 

 

.----==J/r\\-’J K\~"}!E-F""

v

C) E! l6i 124» 1321 4ND

Figure 3. Elution pattern of RGH-1125 upon repurification
through a 0.9 x 50 cm column of Sephadex G-100
in 0.05M barbital buffer at pH 8.6.
Peak I represents aggregated hormone, peak 11
undamaged hormone and peak III degraded hormone.

a 0'3“".
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48

and repurification, as well as the method of freezing the
labeled hormone, affected the proportion of the peaks.

b. Effect of time on repurification.

Figure 4 shows the repurification of aliquots
from the same tube of the descending limb of the G-50
separation at different times after iodination. It can be
seen that the longer the interval between iodination and
repurification the greater the proportions of polymerized
and degraded RGH-1125, with a concomitant decrease in the
amount of undamaged RGH-1125 recovered. The increase in
aggregated and degraded RGH-1125 with time has been ob-
served with all aliquots repurified regardless of their
point of origin.

c. Effect of rapid freezing on repurification.

The effect of rapid freezing versus slow
freezing on the amount of damaged hormone can be seen in
Figure 5. Aliquots from the same tube of the G-50 col-
lection were subjected to an alcohol-dry ice bath fast
freezing or were placed in the freezer (-20°C) to slow
freeze them. The samples were defrosted within a week and
repurified in Sephadex G-100. It can be seen that fast
freezing (solid line) results in an increase in the amount
of aggregated RGH-1125 recovered. No change was observed
in the quantity of degraded RGH-1125.

3. Relative immunoreactivitz of the different
RGH-1125 fractions.

a. Percentgge bindigg.

 

 

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49

 

 

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_1125

Effect of time on the elution pattern of RGH

Figure 4.
repurification through Sephadex G-100.
Note the progressive increases in aggregated and

degraded labeled hormone with increasing intervals
between time of iodination and repurification of

the labeled rat growth hormone.

50

 

 

 

 

 

 

Figure 5. Effect of rapid {ggezing( :% on the elution
pattern of RGH- I repurification through
Sephadex G-100.
Note the increase in aggregated labeled hormone
with rapid freezing.

51

The immunoreactivity of the three fractions
from the G-lOO column was tested by diluting the tubes of
each fraction in 1% BSA-PBS pH 7.2 such that 100 ul of solu-
tion contained approximately 20,000 cpm. Tubes were then
set up for each fraction to measure: a) non-specific bind-
ing, b) total counts, and c) the percentage of immunore-
activity bound by 200 ul of a 1:50,000 dilution of anti-
rat GH serum. The details in setting up these tubes are
given in "Materials and Methods; Procedure for radioimmuno-
assay". As can be seen in the left hand portion of Figure
6, the anti-rat GH serum bound 38% of the aggregated RGH-
1125, 51.5% of the undamaged RGH—1125, and only 6.5% of
the degraded RGH-1125. It is of interest to note that the
non-specific binding of the aggregated RGH-I125 was 3-4
times higher than that of the undamaged protein. The
right hand portion of Figure 6 shows a comparison of immuno-
reactivity between a G-50 first peak aliquot and a G-100
second peak aliquot. For any given anti-rat GH serum dilu-
tion less of the G-50 first peak hormone was bound. This
difference is accounted for by the contamination with aggre-
gated and degraded RGH-1125.

b. Competition curves,

Figure 7 shows the competition between G-100
first peak or G-100 second peak RGH-I125 and different
quantities of a rat GH reference standard (NIAMD-RGH-RP-l)
for the anti-rat GH serum. The set up of this test followed
that described in ”Materials and Methods" regarding standards

 

100

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1:51:10 1:20 F30 {=40 F50
G-IOO Peaks Antibody Dilution x I03

Figure 6. Left side: relative radioimmunoreactivity of the

G-100 fractions. The most immunoreactive fraction
corresponded to the undamaged labeled hormone (peakII).
Right side: comparison between G-50 and G-100 peak II
immunoreactivity. For any given antibody dilution

less of the singly purified labeled hormone ( G-50
peak I ) was bound than of the doubly purified

labeled hormone correspoding to the undamaged frac-
tion ( G-100 peak II )

 

Ucaom ON.HI Icm «CTCLCQ

 

 

53

 

ICKDF
'3 so .
8
m I-
I)
9'. 60t
'7
I - Peak!
0
I! 40 t
E
2: .
h. Pbdkll
0 r-
o- 20
F
.l I I0 I00 IOOO
mpg RGH/tube
Figure 7. Standard curves obtainegzgith G-iOO peak I

and G-lOO peak II RGH-I

One hundred percent binding in abscissa re-
presents binding obtained in the absence of
cold hormone. Ordinate shows the amount of
cold hormone added per tube. It can be seen
that when aggrgggted hormone was used as tra-
cer less RGH-I was displaced from the anti-
body-antigen complex by the addition of cold
hormone.

54

in a radioimmunoassay for each of the RGH-1125 G-100 peaks.
The competition curves are expressed with respect to their
individual 100% tubes (50% binding). As can be seen in
Figure 7, any given amount of NIAMD-RGH-RP-l displaced less
of the aggregated RGH-1125 from the antibody-antigen com-
plex. It was concluded, therefore, that the second G-100
peak was the most immunoreactive, and that the aggregated
labeled hormone is not only more difficult to bind, but
once bound, is also harder to displace.

In view of the above observations the following steps
have been adopted in the assay: a) slow freezing of the
tubes collected from the G-50 column, b) repurification of
the c-50 RGH-1125 in G-lOO columns prior to use and, c)
selection of the second fraction of the G-lOO repurifica-
tion, pooling of the tubes and dilution with 1% BSA-PBS
pH 7.2 to the desired number of cpm. The diluted solution
is kept at 4°C, at which temperature the diluted hormone
remains stable for periods of at least 2 months. If more
hormone is needed, an aliquot of the frozen 0-50 RGH-I125
is thawed, repurified, and the second fraction diluted.
Henceforth all results have been obtained using labeled
hormone from the second fraction of the G-100 repurifica-
tion step.

4. Competition curves with the 0-100 RGH-1125
gecond frggttgg.
a. Against NIAMD-RGH-RP-l or NIAMD-RGH-I-l.
Once the selection of the G-100 fraction was

 

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55

accomplished it was necessary to establish inhibition
(competition) curves with rat GH preparations to ascertain
the sensitivity and reproducibility of the assay. For
this purpose two preparations of rat GH were used: a)
NIAMD-RGH-RP-l which is a reference preparation, and b)
NIAMD-RGH-I-l which is an iodination quality material.
This was done also to have a measure of the equivalency
between these two preparations since our results would
be based on the reference preparation and not on the pure
RGH material. Both were prepared as described for standards
in "Materials and Methods". Figure 8 shows the results.
The lowest limit of detection of the assay was 0.25 mug of
the NIAMD-RGH-I-l material. In repeated assays of the
same amounts of hormone reproducibility was found to be
within 5.5%. In addition we have found that the NIAMD-
RGH-I-l is, on the average, 3.2 times more potent than the
NIAMD-RGH-RP-l.
b. Agginst rat plggma or pituitgtyhomogengtg.
Figure 9 shows the inhibition curves obtained
when using different amounts of intact rat plasma or dif-
ferent volumes of a 0.1 mg/ml solution of rat anterior
pituitary tissue. The slope of the two curves was the same

as that of the NIAMD-RGH-RP-l standard.

 

c. Against other pure rat hormones,
The specificity of the anti-rat growth hor-

mone acre for rat GH was tested using purified prepara-

tions of other rat hormones available to us, namely

56

00 ES
. (3 c)
I 1

a)
C?
I

    

- NIAMD-RGH-RP-I

J)
C)

Percent RGH-1'25 Bound
N
C)

NIAMD-RGH-I-I

l

.l i .6 I00 Iooo
mpg RGH/tube

 

Figure 8. Rat growth hormone standards.
One hundred percent binding in abscissa represents
binding of RGH-112 in the absence of cold hormone.
Ordinate shows the amount of cold hormone of iodi-
nation purity (NIAMD-RGH-I-i) or standard reference
preparation (NIAMD-RGH-RP-i) added per tube.

57

IOOP

(D
C)
I

O,
C)
I

A
C)
I

 

-— NlAMD-RGH-RP-l \

Percent RGH-1'2!5 Bound

 

 

20+- -- Raf AP. Homogenole \\
Rat Plasma \
.l I IC) ICXD lCXDCl
mpg RGH or

pl A.F? HOMOGENATE or PLASMA/tub.

Figure 9. Inhibition curves with rat pituitary homogenate
or plasma from intact rats.
One hundred percent binding in abscissa represents
binding of RGH-112 in the absence of cold hormone.
Note parallel inhibition curves obtained with the
addition of plasma and pituitary homogenatq.and a
standard rat GH reference preparation.

58

prolactin, follicle stimulating hormone (FSH) and lu-
teinizing hormone (LH), all of iodination quality. These
hormones were prepared in the same manner as the rat
growth hormone standards. The results in Figure 10 show
that none of these hormones is able to significantly inhi-
bit the reaction between the RGH-I125 molecule and its
anti-serum throughout the range of concentrations used.
The very slight inhibition observed with the larger doses
of these hormones may indicate a small degree of contami-
nation of these preparations with rat GH.

d. Against hxpophysectomized rgt serum.

To ascertain that the assay was indeed react-
ing with pituitary GH in the plasma of rats and not with a
non-specific plasma protein, competition was set up against
different amounts of hypophysectomized rat serum. Since no
inhibition took place the results have been plotted in
Figure 11 as the percentage binding of the first antibody.
The solid bar on the right side represents the mean per-
centage binding of the several dilutions of hypophysec-
tomized serum used. There was no significant difference
between the mean or any individual percentage binding and
that obtained when no hypophysectomized serum was used.

5. RGH-1125 spgcific radioactivity.
In order to determine the specific radioactivity

of the labeled hormone, the recovery of free I125 was
first determined. This was accomplished by carrying out

a sham iodination in which all of the reagents were placed

b a: (p 5
o o o o
I T I 1

Percent RGH-1'25 Bound
00
C)

 

— NIAMD-RGH-RP-I
r- - - Rat Prolactin
Raf FSH

59

  

 

F --- Rat LH
.l T to I00 looo
mpg HORMONE/tube

Figure 10.

Cross-reaction between monkey anti-rat growth
hormone serum and other rat anterior pituitary
hormones.

One hundred percent binding in abscissa represents
binding of RGH-112 in the absence of cold hormone.
No inhibition was obtained upon the addition of
pure preparations of rat prolactin, LH and FSH.

use 1‘

. "U
‘0‘

1‘uc‘a DN—h. «at: 4 lllll I...

60

  
 
   
   
       

    
   
   
   

  
  
   

   
  

  
    
  

  
  
  

 

   
 
 

 

E40: 7 77 71/
MW
§b o 3/20l525 5

pl of serum/tube

Figure 11. Effect of hypophysectomized male rat serum on

percent bind ng.

The RGH-I12 bound is expressed in terms of per-
cent of total radioactivity. Solid bar at left
shows binding obtained in the absence of serum;
solid bar at right shows the average binding ob-
tained upon the addition of different volumes or
hypOphysectomized rat serum/ tube. Note that
hyponhysectomized rat serum did not alter percent
binding.

61

in an iodination vial in their regular sequence, except

that buffer alone was used with no hormone. Table 1 shows
the counting schedule of a typical preparation. It can be
seen that 77.8% of the total iodide is recovered in the

salt fraction (10 aliquots of 1 ml each), while 14.7% is
lost through the column. The actual recovery of iodide
through the column was 84%. The calculation of the yield
and specific radioactivity of RGH-1125 is based on the 77.8%
recovery of the total iodide.

In the RGH-1125 preparation shown in the lower half of
'Table l the total radioactivity of the salt peak represents
77.8% of the unchanged 1125, since 77.8% of the total I125
is recovered in the absence of protein. By simple calcu-
lation it is seen that 100% of the unchanged iodide is rep-
resented by 3470 cpm. The remaining radioactivity (12860-
3470=9390 cpm) represents 1125 transferred to 20 ug of rat
growth hormone. The counts associated with protein repre-
sent 73% of the total iodide, or 730 uc. The specific
radioactivity is thus 730 uc 1125/20 ug RGH or 36.5 no
11125/ug RGH, a percentage transfer of 73% of the original
1125 iodide. Only 30.7% of the total protein is recovered
‘through the G-50 column and this represents 6.04 ug of pro-
tein. The 1125 labeled growth hormone, of specific radio-
aCtivity 36.5 uc/ug, contains 0.75 atom of I125/molecule
0? growth hormone (molecular weight 45,000; Ellis gt gt.,
19683),

62

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ab~.o oasm eoaooa AmNHHv someones: assess
aom.o ooam ooooeoooe coaooa AmNHHV someones:
o.H comma Heae an ace Ha beacon AmNHHV season
mmmmm mNHHos mmm
aoapmpnaopa mNHHumom
m.MH F.3H mdNH A.OHMOV QESHOO SH umOH TGHUOH AmNHHV Edadom
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63

The above calculations assumed that the specific
radioactivities of the RGH-1125 adsorbed to the reaction
vial, syringe and to the Sephadex G-50 are the same as
that eluted from the column. Furthermore, it assumed that
the specific radioactivities of the different RGH-1125
fractions obtained from the Sephadex G-100 repurification
are the same as that eluted from the first column. These
assumptions have not been verified experimentally.

6. Potency estimates by RIA or bioassgy.

In order to determine the correlation between
radioimmunological and.biological activity three pituitary
preparations were measured using both methods. A group of
male rats was gonadectomized, a second group was surgi-
cally thyroidectomized while a third group served as con-
trols. Two weeks after surgery the animals were decapitated
and their pituitaries removed, weighed, pooled and homo-
genized in phosphosaline buffer. Pituitary homogenates of
control and experimental animals were bioassayed at two
dose levels (1 and 4 mg/rat/4 days or 2 and 8 mg/rat/4
days) by the tibia test of Greenspan gt gt. (1949). Two
doses of NIAMD-RGH-RP-l (10 and 40 ug/rat/4 days) and 2
doses of NIH-GH-SB (25 and 100 ug/rat/4 days) were also
used as reference standards. Four rats were injected
per dose. Radioimmunoassays of the pituitary homogenates
were done at 4 dilutions (20, 40, 80 and 160 ul) of a
solution containing 0.0125 mg/ml (control and gonadec-

tomized) or 0.25 mg/ml (thyroidectomized). Five replications

64

of the four dilutions were done for each homogenate. The
statistical methods of Bliss (1952) were used to compute
the bioassay potencies of control and experimental groups,
95% confidence limits and any departures from parallelism.

The results are shown in Table 2. In all cases the
radioimmunoassay values were below those obtained by bio-
assay, with indices of discrimination ranging from 0.63 to
0.74. Over the range of concentrations tested the potency
estimates by bioassay and radioimmunoassay were not signi-
ficantly different since the radioimmunoassay values were
within the 95% confidence limits of the bioassay deter-
minations. It is of interest to note that gonadectomy
does not result in a significant decrease in pituitary GH
concentration when measured by bioassay. This decrease
is significant by radioimmunoassay. The decrease in pitui-
tary GH concentration due to thyroidectomy is significant
by both assays. The curves obtained with the preparations
were indistinguishable from the NIAMD-RGH-RP-l standards
in the bioassay and radioimmunoassay in shape and slape.

D. Discussion

A radioimmunological assay for rat growth hormone is
reported in which 20 ug of a purified preparation is iodin-
ated with 1 mo of iodide 1125. Under the conditions de-
scribed there is a net transfer of 73% of the original 1125
iodide to the protein, 30.2% of which is recovered through
the Sephadex G-50 column for a total yield of 6.04 ug of
RGH-1125. On the assumption that the transfer of iodide

65

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.mpdada cascadmnoo Rmo cud :wozm
.aoaao damdamumH:Mozm

.Himmumcmloz<Hz mo mpsoan>dzao ms ms commoname

 

 

ac.o Amc.siao.avmm.m m.oHa.a Acme oosasopooeaonasa
oma.o as.o Amm.moumm.mmvom.am H.mus.mm Aomv noonsH HH
ea.o Ass.amuea.eavmm.mm m.HHs.aH Acme oosaeopooeesoc
mma.o no.0 AaH.oauNe.mmvbm.Ne m.HHm.mm Aomv poopsH H
m‘ molmanae mE\mz . wE\ws mum.“ mo we. «a
smou<Hm aemmmoap N.H<Hm so mo one possesses .oxm

m.HMHQHp hp mm

I’ll. 011’?!

 

mopmaowoaos
hanpaSpHa pep cane pom monopoa do nonmedpmo Hmoamoaogseaa can Hoofimoaofimnn.m mqm<e

66

I125 is equally distributed among the 20 ug of hormone a
specific radioactivity of 36.5 no Ilzs/ug of rat hormone
can be calculated. Although we have been able to obtain
higher specific radioactivities by decreasing the amount
of hormone in the reaction, this has resulted in lower
yields as well as in greater amounts of damaged fractions.
The specific radioactivity of the RGH-1125 may be shown to
consist of 0.75 atom of Ilzs/molecule of rat growth hor-
mone. In our hands this is adequate to measure pituitary
and plasma rat growth hormone.

The behavior of the iodinated rat growth hormone
molecule is similar to that reported for human growth
hormone. Our observation that subsequent to iodination it
is possible to separate the iodinated hormone into three
fractions, which correspond to an aggregated component,
an undamaged portion and a degraded component, has been
reported for human growth hormone by Berson and Yalow (1968)
using paper chromatoelectrophoresis and by Giustina gt
gt. (1968 a,b) using gel filtration on Sephadex G-100 or
G-150. Giustina gt gt. (1968 a,b) also reported an in-
crease in the polymerized form of HGH-I125 following rapid
freezing and subsequent thawing prior to repurification.
These authors tested the radioimmunoreactivity of the three
fractions and found that the peak corresponding to the
molecular weight of the hormone (second peak) was practi-
cally all immunoreactive. The aggregated peak was about 20%

immunoreactive while no immunoreactivity was found in the

67

degraded fraction. Our results are in basic agreement with
those reported above.

We have further found that the proportion of poly-
merized and degraded RGH-1125 increases with time. This
may be due in part to the formation of "free radicals" in
the solution as a result of the low energy radiation of the
1125. To decrease this damage we have stored the diluted
hormone at 4°C in 1% BSA-PBS so that the excess protein in
the system serves as a buffer. Thus, we have been able to
utilize the diluted labeled hormone for up to 2 months
without noticing any change in the radioimmunological re-
activity of the molecule, which is interpreted as a lack
of damage. Though it is certain that growth hormone pre-
parations undergo deamidation in solution, it has been found
that the immunoreactivity is not affected by this change
(Berson and Yalow, 1968). Enzymatic degradation may also
play a part in the formation of the degraded form.

In testing the radioimmunoreactivity of the several
fractions it was found that the polymerized form was not
only less immunoreactive, but once bound more difficult to
_ displace from the antibody-RGH-1125 complex. It can be
envisioned that the aggregation of molecules can hinder
immunologically active sites from competing for the anti-
body sites; once bound, however, the same aggregation of
molecules may hinder a competing molecule from displacing
it from the antibody site. If one assumes that the specific

radioactivity of the polymerized fraction is the same as that

68

of the undamaged protein, then it must be concluded that
the actual immunoreactivity of the aggregated form is less
than that recorded experimentally since the ratio of cpm/
actual bound molecule would be greater for the aggregated
molecule. Although this has not been verified experimen-
tally, it is suggested by the observation that the non-
specific binding of the RGH-1125 polymer was 3-4 times
greater than that observed for the undamaged RGH-I125.

It can be calculated that the addition of l9-21,000
cpm of freshly labeled hormone per radioimmunoassay tube
represents approximately 0.25-0.35 mug of hormone when the
specific radioactivity is 36.5 uc/ug of growth hormone.
This amount of tracer permits a sensitivity of 0.25 mug
of pure cold hormone in an assay where 50% of the labeled
hormone is bound by a l:50,000 dilution of anti-rat growth
hormone serum.

The radioimmunoassay described above is specific for
OH in the rat since it is not affected by other rat hor-
mones or by non-specific plasma proteins. Furthermore,
parallel inhibition curves were obtained between different
rat pituitary homogenates or plasma and a purified prepara-
tion of rat growth hormone. Although there is no evidence
to show that the immunological and biologically active,
sites are the same in the GH molecule, no significant dif-
ference was found in the estimated potencies of pituitary
Preparations by radioimmunoassay or bioassay. This sug-

gests that throughout a range of GH concentrations brought

69

about by physiological manipulations the relationship be-
tween these two active sites may be of little consequence

in establishing an accurate estimate of potency.

70

II. Study on the Cross-Reactivity of Monkgy Anti-Rat

Growth Hormone Serum

A. Objectives

This study was undertaken to determine the immuno-
logical cross-reactivity between the NIAMD-A-RatGHS-l and
growth hormones, pituitary homogenates or plasma from
other animal species, in an attempt to assess the feasi-
bility of using the previously described radioimmunoassay
in measuring the growth hormone of different species.

B. Procedures

Purified preparations of ovine (NIH-GH-S8), bovine
(NIH-GH-Blz) and human (NIH-GH-HS 1216C) growth hormone;
pituitary homogenates of mouse, gerbil, guinea pig, ham-
ster and monkey; and plasma of mouse, gerbil, guinea pig,
hamster, rabbit, cat and dog were tested for their ability
to compete with RGH-I125 for the immunologically active
sites of the monkey anti-rat growth hormone antibody.

Pituitary tissue from the mouse, gerbil, guinea pig,
hamster and monkey was obtained by decapitation. The
pituitaries were weighed and homogenized separately with
a Sonifier cell disruptor in phosphosaline buffer. The
pituitary homogenates were diluted to a concentration of
0.1 mg per ml. Blood was collected by cardiac or veni-
puncture in heparinized syringes, centrifuged immediately
and the plasma separated and frozen at -20°C until assayed.
All preparations were assayed for growth hormone in dupli-

cate volumes of 0.5, 1, 2, 4, 8, 16, 31, 62.5, 125, and 250

71

or 300 ul per radioimmunoassay tube. The volume in each
tube was complemented with 1% BSA-PBS to a total volume of
500 ul. The assay was carried out as described in the
preceding section.

C. Results

The results of this experiment can be seen in Figures
12, 13 and 14. A typical standard curve for rat growth 3?
hormone is shown in each figure as the basis for comparison.

Figure 12 shows the immunological relatedness between

 

ovine, bovine and human growth hormone, as well as monkey

pituitary homogenate, with rat growth hormone. No cross- k
reactivity was observed with either the purified human
growth hormone or the monkey pituitary homogenate within
the range assayed. Partial cross-reactivity was seen with
the ovine and bovine growth hormone preparations. Neither,
however, showed a parallel relationship with rat GH, and
very flat slopes were obtained.

Figures 13 and 14 show the dilution response curves
for pituitary extracts and/or plasma of gerbil, mouse,
rabbit, guinea pig, hamster, cat and dog. All of these
preparations, with the exception of rabbit plasma, cross-
reacted significantly with the antibody to rat GH.

D. Discussion

The immunological relatedness between growth hormones,
pituitary homogenates or plasma from several animal species
and rat growth hormone was studied using a double antibody

radioimmunoassay for rat growth hormone. The monkey

72

3

a)
C)
T
’.

3

  
  

b
C)
r

_. — NIAMD—RGH-RP-l
------ NIH-GH-BIZ
-- NlH-GH-SB
-°- NIH-GH-HS I2l6C
.. -°- Monkey A.P. Homogenote

Percent RGH-1'25 Bound

N
C)
I

 

L l

.l I no :00 IOOO
mug HORMONE or
pl A.P. HOMOGENATE/tube

Figure 12. Immunological relatedness of growth hormones.

One hundred percegg binding in abscissa represents

binding of RGH-I1 in the absence of cold hormone.
No cross-reaction was obtained upon adding human GH
(NIH-GH-HS 12160) or monkey A.P. homogenate to the

tubes. Partial cross-reaction was observed with

purified ovine (NIRqGH-S8) and bovine (NIH-GH-BiZ)
GH's.

 

73

 

'00 I. ;- - - - ~
t§°‘~\ ‘ ‘ x
90 “9- \
‘~\
BC) .\ E:
\. 5
7o. ‘ 5.
0‘ .6
6£) >§0\~
, '\‘
“wag i

     

.b
C)

— NIAMD-RGH-RP-i
-'-Guinea Pig Pituitary
-I- Guinea Pig Plasma
-- Hamster Plasma

° ---- Hamster Pituitary

Percent RGH-1'25 Bound
0H (n
C) C)

to
C)
I

6
I

 

 

i .6 I60 250
mpg HORMONE or pl AP HOMOGENATE
or PLASMA/tube

Figure 13. Dilution-response curves for pituitary extracts
and plasma of guinea pig and hamster.
One hundred percggg binding in abscissa represents
binding of RGH-I in the absence of cold hormone.
Note parallel cross-reactions obtained upon the
addition of plasma or A.P. homogenates.

loo

8]
C)

O)
C)

Percent RGH-1'2:5 Bound
“3 (fl is (n
C) C) C)

6

Figure

74

  
    

— NiAMD-RGH-RP-I
’ ---Gerbil Plasma
-A- Mouse Plasma

 

 

r- -o-Cat Plasma
------ Dog Plasma

_ -x- Gerbil Pituitary “kg-T"
o o 0 Mouse Pituitary ”
-0- Rabbit Plasma

I '0 loo 300

mpg HORMONE or pl A.P. HOMOGENATE
or PLASMA/tube

l4. Dilution-response curves for gerbil, mouse,
cat, dog and rabbit preparations.

One hundred percggg binding in abscissa represents
in the absence of cold hormone.

Note parallel cross-reactions with gerbil, mouse,

cat and dog plasma or A.P. preparations. No cross-

binding of RGH-I

reaction was observed with rabbit plasma.

 

75

anti-rat growth hormone utilized was shown to cross-react
with pituitary homogenates and plasma of hamster, guinea pig,
gerbil and mouse. Furthermore, it cross-reacted with cat
and dog plasma. Partial cross-reaction occurred with puri-
fied preparations of ovine and bovine growth hormone, but

no cross-reaction took place when a purified human growth
hormone or a monkey pituitary homogenate were used. In this
study, rabbit plasma also failed to inhibit the reaction be-
tween RGH-1125 and its antibody.

The data presented here reinforce the concept of a
common antigenic structure in pituitary and plasma growth
hormone of several mammalian species, as well as the dif-
ference in antigenicity of primate growth hormones. These
data agree with those reported by Hayashida and Contopoulos
(1967) using double diffusion or immunoelectrophoresis,
Ellis gt gt. (1968a) using micro-complement fixation or
immunoelectrophoresis, Tashjian gt gt. (1968) by complement
fixation, and Garcia and Geschwind (1968) by radioimmuno-
assay. A discordant note is found in that we were unable
to show cross-reactivity with rabbit plasma. This could be
the result of handling and age of the sample assayed as
well as a characteristic of the antiserum used since it has
been shown that complete or partial cross-reaction are func-
tions of the antiserum and method employed (Hayashida and
ContOpoulos, 1967). To our knowledge, this is the first
report of cross-reactivity between hamster or gerbil growth

hormone and rat pituitary GH using a double antibody

 

76

radioimmunoassay.

These cross-reactions should make it possible to
measure GH in guinea pigs, hamsters, mice, gerbils, dogs
and cats utilizing the monkey antibody to rat growth hor-
mone. However, since neither purified GH preparations of
these species nor pure preparations of other hormones were
available to test specificity, biological correspondence
should be assessed before using the assay. The latter was

not within the scope of this study.

77

III. Qtfference Between Plasma and Serum RGH Levels

A. Objectives

Preliminary results obtained while establishing

dose-response curves for plasma suggested possible dif-
ferences in the levels of GH between plasma and serum
samples. It was the purpose of this experiment to veri-
fy the preliminary observations and ascertain the degree
of this difference in serum and plasma samples of the
same origin.

B. Procedures

Male Sprague-Dawley rats were anesthetized with ether
and bled from the abdominal aorta. The blood collected
from each animal was divided equally into two large centri-
fuge tubes one of which contained 0.1 m1 of a 10mg% solu-
tion of Na-heparin per ml of blood, while the other contained
0.1 m1 of physiological saline per ml of blood. The blood
from several animals was pooled. The heparinized tubes
were immediately centrifuged and the plasma separated from
the red blood cells by pipette. Serum was obtained by
allowing the clot to form and retract for 24 hours at 4°C
at the end of which the tubes were also centrifuged and
the serum decanted. The level of OR was determined in
triplicate volumes of 12, 25, 50, 100, 150, 200, 300, 400
and 500 ul of serum or plasma.

C. Results

Since the possibility existed that the second anti-

body did not react at the same rate in plasma and serum

78

the second antibody was incubated for 1, 2, 3 or 5 days.
The results are shown in Table 3. For any given length of
second antibody incubation the levels of GH measured in
plasma were significantly higher than those measured in
serum. No difference was found within the plasma samples
or within the serum samples with the various lengths of in-
cubation. a
From the above results it was concluded that the rate
of reaction of the second antibody was not affected by time Q

in serum or plasma. In attempting to explain the large

 

differences observed several questions were posed: a) is B
the percent binding affected in different ways by serum

and plasma, b) does Na-heparin cross-react with the anti-

rat GH serum, c) does Na-heparin synergize the levels of

GH in plasma, d) when does the loss of activity take place,

e) is this loss due to aggregation or degradation of the

GH molecule, and f) what causes it.

To answer some of these questions several tests were
done. First, the effect of serum or plasma on the percent
binding was determined by placing 100 ul of serum or
plasma of hypophysectomized male rats into RIA tubes.

Both the non-specific binding and the percent binding were
determined at l, 2, 3 or 5 days of second antibody incuba-
tion. Control tubes had no plasma or serum. The results
can be seen in Figure 15. Neither serum nor plasma altered
the percent binding of the first antibody at any time of

incubation. The controls are represented by the solid

a“?

 

 

79

TABLE 3.--Difference between serum and plasma GH levels

 

 

 

 

Male Serum Male Plasma
Days of mug GH/ml mug GH/ml
Incubationl (Mean:S.E.) (MeaniS.E.)
1 35.5:1.4 111.516.4r
2 36.1:2.3 111.317.9*
3 36.5:l.2 114.1:8.9*
5 36.9:0.8 112.518.8*
*p( 0.01

1Length of 2nd antibody in days

 

80

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

13

C:

E); F W F) 7 Fl j T 1

g a g a 3 g 3 g: 3

g 8 9 8 9 9. 9 9 9

° ° 2 ° E E

g g 5 5 5 E 5 g J:
5 O o _ O — -—

+- ° " a) a, a. a) a.

g ‘3 x x F): r: x X X

0 ° ° 8 ° 8 8 ° 3
a. a. a. a h

h- >~ >. >w x >u >

O I I I I I I

a- I 2 3 5
Length of 2nd Antibody Incubation

(Days)

Figure 15. Effect of hypophysectomized male rat serum or
plasma a? percent binding.
RGH-112 bound is expressed in terms of percent of
total radioactivity. Binding obtained in the abs-
cence of hypophysectomized plasma or serum is shown
by the solid bars. The addition of hypophysectomi-
zed plasma or serum did not alter percent binding.

 

81

bars. In all cases the non-specific binding was the same.

Next, the possible cross-reaction between Na-heparin
and the anti-rat GH serum, as well as its effect on the
antibody and the labeled hormone was tested by placing
different volumes (0.1, 0.25, 0.5, l, 2, 4, 8, 16, 31,
62.5, 125, 200 and 250 ul) of a 10 mg% Na-heparin solu-
tion into RIA tubes. Triplicates of each volume were used.
The results, shown in Figure 16, have been expressed in
terms of the percent binding. No change in binding was
observed with any amount of Na-heparln used. The average
percent binding is represented by the solid bar on the
right; the solid bar on the left represents the percent
binding of the control. This suggests that Na-heparin
neither cross-reacts with the anti-rat GH serum nor affects
the labeled hormone and antibody. In a separate experi-
ment where 100 ul of the Na-heparin solution were placed in
tubes containing the RGH standards, no difference was
found between the control and heparinized standards.

To determine whether Na-heparin synergized the levels
of GH in plasma and whether a decrease in GR activity
occurred with time, a rate of recovery test was done in
hypophysectomized plasma and serum to which known amounts
of RGH had been added. The radioimmunoassay tubes were
set up as described for standards except that 100 ul of
either serum or plasma from hypophysectomized male rats
were added per tube. Four, 8, 16, 31, 62.5 mug of RGH

were added per tube. Each amount was determined in

M

x

§§$§§$§S§§§§_d
§§S§$§S§$§§§§m~0
S§§$§§§§§§§§md
S§$§S§§S§§S§S_
§§§§$§§§$§§SN
§S§$§§§§$§§§¢
S§$§§§§§S§SS®
S§$§§§§§§$§S®_
S§§S§§§§§§§§.M
§§S§§§S§S§§§DN®
§§§§§§§§§§§§mg
S§§§§§§§§§§§§OON
§§§§§§§§§§§§§OQN
O

— u p p p b

0 AV 0
6 4. 2

258 3.3.9. tooled

| -
P

Iqu °/e Na hepari

.,g
f mmfd
. anon
ttpis
nfeben
wnmemn
uemwum
ph PVB
.ftwmtm
W on“ anna
i sowemh
Mmrnhe
i numtm .
beemmmm
Mni o t
c ohsom
rdSt
ee tnia
Psenhotg
shigiun
naeennnn
op e Men
nXone 1
ieYihanb
r tat 1
asit ert
inbnhan
e iootPe
hdt ec
.mcgrnhr
a anaO-m.
Nooibpa
bid UN
f5wMMd M
erbleflt
t1 0nm1
cIlssi a
e.aw a0
thoetlt
fGohhb o
ERtstoan

Figure 16.

83

duplicate. The second antibody was incubated for l, 3
and 5 days. It can be seen in Table 4 that eSSentially
all of the added RGH is recovered. Furthermore, Na-
heparin does not synergize the level of GH in the plasma.
No decrease in GR activity was observed with increasing
length of time in the incubation of the second antibody.
This suggested, therefore, that the disappearance of GH
took place primarily before the separation of the plasma
and serum from the red blood cells.

In order to test the above assumption an experiment
was devised in which a comparison could be made between
plasma and serum left for different times before centri-
fugation and separation from the red blood cells. Blood
was collected from intact male rats from the abdominal
aorta into two syringes: one containing 0.6 ml of a 10
mg% Na-heparin solution, the other containing physiological
saline in the same volume. A volume of 5.4 ml of blood
was withdrawn into each syringe to make a total volume of
6 ml. The 6 m1 of heparinized or non-heparinized blood
were divided into 4 groups of 3 tubes each (0.5 ml/tube).
The 4 groups corresponded to 4 different times (0, 12,

24 and 48 hours). In each group one tube was for plasma
or serum, a second tube for blood to which 20 mug of RGH
had been added, and a third tube for plasma or serum to
which 80 mug of RGH per ml were added after centrifuga-
tion. Blood was collected from 20 animals and pooled for

each tube within each time group. All tubes were kept in

 

84

msoapmGHEpopou N no omaao><m

 

 

©.HOH m.ooa m.moa m.ooa m.Noa N.HOH caho>000h & saw:
m.m H.s a.m m.m m.m H.e 0.:
m.m m.m m.m a.a m.a m.s o.m
0.5H m.mH 0.0H m.ma 0.5H o.mH 0.0H
o.mm e.am o.mm m.mm o.mm o.mn e.am
m.me m.mo m.am m.em e.aw e.aw m.~m
o.o~H o.mma m.mNH o.mmH o.mma o.omH o.mNH
scammam seapom enamnam sesaom smawnam ransom
cofipn9302H and m cofipmnsomfl and m Cofipmn:ocfl and H pood< mom was

 

doao>ooom mom was

 

 

Baton no mammaa couHEOpoommsaoazs Eopu mum mo

nouns mao>ooomrr.a mam¢9

85

an ice bath during collection. The level of GH in the
plasma or serum was determined in triplicates of three
dilutions of 200, 100 and 50 ul per RIA tube. The results
have been grouped as follows: IA: plasma or serum cen-
trifuged at 0 time and incubated for 0, 12, 24 or 48 hours
at 4°C; 18: plasma or serum centrifuged at the end of

O, 12, 24 or 48 hours of incubation; 11: plasma or

serum centrifuged at 0 time, 80 mug RGH per ml added, in-
cubated for 0, 12, 24 or 48 hours; and III: heparinized
or non-heparinized blood to which 40 mug RGH per ml were
added, centrifuged at the end of 0, 12, 24 or 48 hours of
incubation. For each group of serum or plasma the level
at 0 time was regarded as 100%. The results are shown in
Figure 17.

It can be seen that neither plasma nor serum centri-
fhuged at 0 time (groups IA and 11) produces significant de-
Crweases in GR after 12, 24 or 48 hours of incubation. On
thee other hand, centrifugation of heparinized and non-
heIDarinized blood after 12, 24 or 48 hours of incubation at
4°C} results in significant decreases in GR (groups IB and
IIJZ) which increase with time. For any given time period
leans OH is recovered from the serum groups. No definite re-
lationship could be established between the initial level
0f CEHZand the degree of inactivation. The actual level of
33 ion the serum centrifuged at 0 time was 74-81% of that
fOU-nd in the plasma centrifuged at 0 time. Fibrin formation

W88 (atmerved in the plasma samples centrifuged at 24 and 48

hours .

86

6C)"

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DD 4>

C) C)

I I I f

l —

 

Percent RGH CONCENTRATION/ml Plasma

Time “it: ass: was we: “2:: °-:.: W25: we:
Group IA IB 1: 11: IA IB 1:: m
Plasma Serum

Figure 17. Effect of time on plasma and serum RGH levels,
RGH concentration/ml plasma is expressed as per-
cent of control, where control concentration is
regarded as 100%. Groups IA= plasma or serum
centrifuged at time 0 and incubated for 0,12,24
or 48 hours. Groups IB= plasma or serum centri-
fuged at 0,12,24 or 48 hours. Groups II and III
received same treatment as IA and 13 respectively,
except that exogenous RGH was added.

87

D. Discussion

The levels of GH in serum and plasma samples of the
same origin were measured by radioimmunoassay, varying
the length of incubation time of the second antibody. In
all cases plasma GH concentrations were significantly
higher than serum GH concentrations. No change was ob-
served in plasma or serum GH levels with respect to length
of second antibody incubation, suggesting that the reac-
tion reached equilibrium within 24 hours. The differences
in OH were not the result of differential influences on
the BIA itself by plasma or serum, i.e. hypophysectomized
male rat serum or plasma did not affect the percent bind-
ing of the first antibody; the second antibody reached
equilibrium within the same time span in plasma and serum;
Na-heparin did not cross-react with the anti-rat GH serum,
affect the labeled hormone or antibody, or synergize the
levels of GH in plasma.

The recovery of exogenous RGH in plasma or serum from
hypophysectomized male rats was about 100%, suggesting
that the disappearance of immunologically active GH took
place before the separation of the plasma or serum from
the red blood cells and other blood components. A direct
comparison of intact rat plasma and serum, with and without
the addition of exogenous GH, centrifuged at the time of
collection and incubated for 0, 12, 24 or 48 hours at 4°C,
or incubated for the indicated times and then centrifuged,

showed that the former resulted in almost no disappearance

88

of GH, while the latter resulted in significant decreases
in CH concentration with time. The longer the interval,
the greater the decrease. It was also observed that for
any given time period less GH was recovered in the serum
than in the plasma group; at 0 hour centrifugation time
serum GH was 74-81% of plasma OH.

This experiment indicates a significant loss of immu- F
nologically active GH in serum and plasma when centrifu-
gation takes place after a period of time. It does not i

indicate whether the loss in activity is due to aggrega-

 

tion or degradation of the GH molecule, nor the cause of
the loss.

Somatotropin inactivation by streptokinase activated
blood plasma or plasminogen has been reported by Mirsky
gt gt. (1959 a,b), who observed that human plasmin solubi-

lized about 30% of an 1131

labeled bovine GH. Streptoki-
nase activated human plasminogen or bovine plasmin have
also been reported to result in partial hydrolysis of bo-
vine GH (Ellis gt gt., 1968b). These authors suggested
that a tissue peptidase present in crude preparations of
pituitary somatotropin was identical with plasmin. Thus,
incubation of a crude extract with traces of urokinase
eliminated an autocatalytic lag observed in crude prepara-
tions which had been extracted for only % hour. Incubation

of bovine or rat OH with plasmin resulted in fractions of

20,000 and 18,400 m.w. respectively,j The conversion of

these hormones was shown to be pr0portional to the plasmin

concentration.

89

It has been shown that the immunological activity of
the native and degraded forms of rat GH differ greatly al-
though their biological activities remain about the same
(Ellis gt gt., 1968c). Using micro-complement fixation,
the degraded hormone fixed 15% as much complement as the
native GH when an antibody to the native GH was used. On
the other hand, the native hormone fixed less than 10% as
much as the degraded GH when an anti-serum to the degraded
moeity was used.

In view of these reports, and our observations con-
cerning the differences in the degree of inactivation be-
tween plasma and serum as well as the time lag in inacti-
vation observed in the plasma, it is concluded that immuno-
logical inactivation took place and that this inactivation
was probably due to degradation of the hormone by factors
involved in the process of coagulation. It is recommended,
therefore, that plasma instead of serum samples be used to
measure OH. The samples should be collected in an ice bath

and centrifuged without delay.

Keeping in mind that the radioimmunoassay does not dis-
tinguish between degradation or aggregation of a molecule,
it is not possible to rule out the loss of GH as a conse-
quence of aggregation. Obviously, simple adsorption of OH
to the fibrin strands will also produce similar results.
Further studies must be done to clarify the nature of the

GH loss observed.

90

IV. GH as a Function of Age in Male and Female Rats: the

Estrous Cycle

A. Objectives

The relationship between age and pituitary GH in the
rat has been studied using biological (ContOpoulos and
Simpson, 1957a; Solomon and Greep, 1958; Bowman, 1961) and
radioimmunological (Birge gt gt., 1967a; Daughaday gt gt., F:
1968; Garcia and Geschwind, 1968; Burek and Frohman, 1970)
assay methods, Yielding differing results (see Review of

Literature). In none of the above reports was the estrous

 

cycle considered when relating GH and age in female rats. b
Furthermore, no report has dealt with the levels of GH in
plasma or serum with respect to age in the rat. It was the
purpose of this experiment to measure the plasma and pitui-
tary levels of GH in male and female rats of different ages,
and in the different stages of the estrous cycle.

B. Procedures

Male and female Sprague-Dawley rats of different ages
were obtained from Spartan Research Animals (Haslett,
Michigan) and housed in a temperature controlled room (75
1 10F) with automatically controlled lighting (14 hours
light daily). A group of female rats 180 days old was sub-
jected to constant illumination (C.L.) for three weeks.

Individual blood samples were taken under ether anes-
thesia via heart puncture into syringes containing 0.1 m1
0f a 100 mg% solution of Na-heparin/ml of blood withdrawn.

The blood samples were kept in an ice bath during the

91

collection and centrifuged immediately after the collec-
tion at 2200 rpm for 20 minutes. The plasma was separated
by pipette and stored at -20°C until assayed. Within an
hour after the blood was collected they were killed by
guillotine and their pituitaries removed, weighed indivi-
dually and homogenized in PBS. The individual homogenates
were also stored at -20°C until assayed.

Blood samples from male rats were collected between
10:00 and 12:00 A.M. Vaginal smears were taken daily be-
tween 8:00 and 10:00 A.M. and the female rats bled between
12:00 and 2:00 P.M. of the same day. Plasma and pituitary
OR was assayed by radioimmunoassay (RIA) at 3 different
concentrations for each individual sample. Ten animals
were used per age group or stage of cycle within a particu-
lar age group. The results were analyzed by one way analy-
sis of variance followed by the multiple range test of
Duncan (Bliss, 1967). All results are expressed in terms
of the NIAMD-RGH-RP-l standard.

C. Results

Pituitary and plasma GH was measured in male rats of
23, 33, 43, 64, 84, 104 and 120 days of age. Plasma OR was
also measured in a group of male rats of about 240 days of
ages. Pituitary GH as a function of age in the male rat is
shommiin.Table 5; Figure 18 shows the levels of plasma GH
ixiinales of different ages.

The concentration and content of pituitary GH in the

male rat increased significantly with age to about 84 days.

92

 

.m.m + same no dommopaxo mosHa> HH<

 

 

n.oue.me e.amum.ame mm.ouma.oa e.mmu~.aom oma
a.num.eo N.ONHH.abm Hm.ouam.m o.a Hm.ems sea
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94

No further increase in concentration was observed with
advancing age, although a significant increase in content
was further observed at 120 days. Equally significant in-
creases were found in plasma GH from 23 to 64 day old rats.
No significant difference was found in plasma CH of rats
64, 84, 104 and 120 days of age. At 240 days the concen-
tration of plasma OR was significantly reduced to levels 7?
similar to those found in rats of about 33 to 43 days of age. '
Pituitary and plasma GH was also determined in female

rats ranging in age from 21 to 560 days old. The rats were

 

.xW .
131.3...

‘W'

divided into the following age groups: 21, 28, 34 in which
the vaginal canal was closed (vg. canal cld.), 36 and 43
with the vaginal canal apen. Rats 60 and 120 days old were
used in determining the levels of GH in the different stages
of the estrous cycle; a 180 day old group was subjected to
constant illumination to elicit constant estrous (c..estrous,
C.L.), while a 560 day old group was found to have estrous-
proestrus types of smears exclusively. The results of this
experiment are shown in Table 6 and Figure 19.

A significant increase in pituitary content and con-
centration of GH took place from 21 to 60 days of age. The
cxnlcentration of GH did not change significantly from 60 to
if“) days, while a significant decrease was observed in the
560 day old group. Although a small increase in concentra-
ifixnl was observed during proestrus, the levels of GH did not
diflfer'significantly from those found in estrus, metestrus,

orciiestrus. In contrast, the pituitary content of GH

95

 

 

 

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97

increased to about 180 days, with a significant decrease
occurring at 560 days of age. No change in content was
observed with the different stages of the cycle.

Plasma concentration of GH rose significantly from
21 to 60 days of age, with the sharpest increase taking
place from 34 days with a closed vaginal canal to 36 days
with the vaginal canal opened. In normally cycling female #7
rats the mean plasma GH concentration in estrus was sig-

nificantly higher than found in proestrus, metestrus or

 

diestrus, when no differences were observed. The levels :
of GH decreased significantly in constant estrous rats of bl
180 days and more so at 560 days of age.
A direct comparison of GH levels in the pituitary and
plasma between male and female rats of approximately the
same ages failed to show any significant differences, ex-
cept for the peak in plasma GH observed during estrus.
In view of the results obtained in female rats an
experiment was designed to test the possible influence of
estrogen on the increased levels of plasma GH observed after
canalization and during estrus of each cycle. Female rats
180-200g were divided into the following groups: a) intact
(Hantrol, b) unilateral ovariectomy, a) bilateral ovariec-
txamy, d) bilateral ovariectomy plus 0.2 m1 corn oil daily,
and.e) bilateral ovariectomy plus 5 ug of estradiol ben-
zoate (E.B.) in 0.2 ml corn oil daily. The animals were treat-
ml for 2 weeks, at the end of which time plasma and pituitaries

werne collected and assayed as described in "Procedures".

98

It can be seen in Figure 20 that unilateral ovariec-
tomy did not alter anterior pituitary concentration. On
the other hand, bilateral ovariectomy with or without corn
oil resulted in increased pituitary concentration as well
as in a decrease in plasma GH. Conversely, the administra-
tion of estradiol benzoate to bilateral ovariectomized rats
resulted in a decrease in pituitary OH with a concomitant F-
increase in plasma GH levels. 7]

D. Discussion i

The relationship between age and OR was studied in 3',

 

male and female rats ranging in age from 21 to 560 days k)
old. In addition, plasma and pituitary OR was measured
in the different stages of the estrous cycle in rats of
two different ages. The concentration of pituitary GH
showed progressive increases from 21 to 60 or 23 to 84 days
in female and male rats respectively. Concomitant with
this increase in concentration was an increase in content
which continued to 180 days in the female and 120 days in
the male. The increase in content, in view of the plateau
in concentration, can be attributed to the further increase
in pituitary size. A decrease in both content and concen-
tration was found in female rats 560 days old. No signi-
ficant change in pituitary content or concentration was ob-
served with the different stages of the cycle, although a
small rise in concentration was measured during proestrus.
Together with the increase in pituitary concentration

observed during the first 8-12 weeks of age, a similar

99

 

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estradiol benzoate treatment.

100

increase was observed for plasma GH. The level of GH in
plasma rose significantly in both male and females to 60-64
days of age. Plasma GH remained elevated up to 120 days, with
a decline in concentration observed at 180 days in females
and at 240 days in males. A further decline was observed at
560 days of age in female rats showing estrous-proestrus types
of vaginal cytology. In female rats with normal estrous cy-
cles plasma GH was significantly elevated in estrus. No diffe-
rence was found in proestrus, metestrus or diestrus. For
each stage of the cycle, the levels found at 60 days of age
did not differ from those found at 120 days of age.

The data presented here regarding pituitary GR is in
basic agreement with that of Garcia and Geschwind (1968),
who measured pituitary GH concentration in male and female
rats from 5 to 75 days of age, and Burek and Frohman (1970),
who used rats of different weights ranging from 61 to #85
grams. Our results seem to differ from those of Birge gt
al.(1967a) who reported that male pituitary GH concentra-
tion continues to increase to old age, whereas female pitui-
tary concentration plateaued at maturity. No difference in
content and concentration between males and females was
found up to eight weeks of age, after which pituitary con-
tent and concentration in males were significantly higher
than in females. It is of interest to note, however, that
these authors found little or no increase in the weight of
the pituitary gland in male rats after #9 days of age. Upon
substituting their reported pituitary weights with those

 

101

reported by others and our own for animals of the same age

or weight, little or no difference in concentration is found
between male and female rats. Furthermore, when the substi-

tution is carried out it appears that male pituitary concen-

tration also plateaus between 63 and 77 days of age. If
their results had been given by these criteria, we would be
in total agreement.

The reports cited above have all utilized radioimmuno-
logical assays. With biological assay methods, Solomon and
Greep (1958) and Bowman (1961) reported increases in con-
tent but not concentration of GH in the pituitary of female
and male rats 10 to 630 days old respectively. Upon obser-
vation of their data, however, it appears that increases in
concentration were observed up to about 6-9 weeks of age,
but their significance was discounted.

This is the first study dealing with plasma or serum

GH levels with respect to age in rats, and during the estrous

cycle in females. Our results show a steady increase in
plasma GH from 21 to about 64 days of age in both male and
female rats. In addition, female rats show a significant
elevation during estrus. If one starts with the premise
that GB is indeed required for growth, and since the most
rapid rate of body growth takes place during that time when
both pituitary and plasma GH are lowest, then considera-
tion must be given to the ability of young rat pituitaries
to synthesize and release GH and to the rate at which the

body utilizes it. Of course, there is also the possibility

 

 

 

102

that during the most rapid growth phase of life in rats,
other factors may be more important for body growth than
GH. It only needs to be mentioned that removal of fetal
pituitaries (Jost, 19#7) in rabbits does not reduce birth
weight of the young. Also, rats hypophysectomized early
in life continue to grow up to about 30 days of age
(Walker gt al., 1952).

Burek and Frohman (1970) have recently reported that
pituitaries from adult male rats were able to synthesize
more GH than pituitaries from young male adult rats, and
the latter synthesize more GH than pituitaries from wean-
ling rats lg ELEQQ. If one assumes that rate of body
growth can be used as an index of utilization rate, then
these results could be interpreted as reflecting a low
synthesis rate with almost all of the GH released and
utilized by the body, thus maintaining the relatively low
levels in plasma and pituitary GH observed during this
period of rapid growth. It should be pOinted out that
these results differ from those reported in the human by
Greenwood gt gl. (l9u6a,b) in whom the highest plasma
HGH levels were found in the fetus and at parturition,
subsequently declining but remaining higher in children than
in adults. In view of the report by Gershberg (1957) in
which he found no difference in pituitary HGH concentra-
tion among fetal, adolescent and mature male pituitaries,
these plasma concentrations may reflect differences be-

tween human and rat pituitaries in their ability to synthesize

 

103

n

GH at different ages.

Our observation that ovariectomy increased while estra-
diol benzoate decreased pituitary GH concentration confirms
the previous report of Jones gt gt. (1965) and Birge gt gt.
(1967a). We have extended these experiments to include the
levels of plasma GH and found that ovariectomy decreases
while estradiol benzoate administration increases the levels ‘
of plasma GM. Similar results have been reported after the LE
administration of estrogen-progestin to humans (Garcia gt

gt., 1967). Is estrogen, therefore, responsible for the ele—

 

 

vation of GH after canalization of the vaginal Opening and

Egnxar
'—W-a
O

at estrus of each cycle, or does GH simply follow the in-
creased levels of prolactin, FSH and LH observed during the
afternoon of proestrus? If estrogen is responsible, how
does it produce this elevation and what is its significance?
Birge gt gt. (1967b) reported that diethylstilbesterol
caused suppression of GH release from pituitaries lg xttgg.
It remains to be demonstrated that this is a physiological
and not a pharmacological effect. Does estradiol benzoate
also interfere with the synthesis and release of GH 23 1119?
If such were the case, and in view of the peripheral anta-
gonism between estrogen and GH (Josimovich gt gt., 1967;
Roth gt gt., 1968) it could be argued that the increase in
plasma GE is the result of peripheral inhibition by estro—
gens which reduce the amount of GH utilized by the body per
unit time. However, it is also possible that estrogen in-

creases hypothalamic GH-RF or directly stimulates pituitary

release.

104

The data presented here strongly indicate the need for
caution in the interpretation of results based on content
or concentration of hormones in plasma or pituitary tissue.
The need for such caution assumes special importance when
dealing with GH since there is no one tissue which can be
called a "target organ" for GE to provide an indirect para—
meter for utilization rates. The results suggest that there a
may be differences in the rate of utilization and secretion [
of the hormone between very young, adult and old animals.

The physiological significance of the increased plasma GH

WE;L

at estrus or after estradiol benzoate administration may

be clarified in a future study on a) the metabolic clear-
ance and secretion rates of GH in rats as influenced by
estrogen and the different stages of the estrous cycle, and
b) the effects of estrogen on hypothalamic GH-RF and direct-

ly on pituitary GH release.

105

V. Plasma and Pituitary Concentration, Metabolic Clearance
Rate (NOR) and Secretion Rate (SR) of GH in the Mgtg
Rat as Influenged by Castration, Testosterone PrOpiongtg
(TPL, Thyroidectomy and Na-thyroxine (Tu).
A. Objectives

 

It has been known for some time that hypothyroidism

in children results in a condition known as cretinism, when

 

growth is greatly impaired. In rats, thyroidectomy results
in degranulation of pituitary acidophiles, a decrease in

growth rate, and a decrease in pituitary GH and hypothalamic

 

growth hormone releasing factor (CH-RF). Thyroxine therapy, k!
on the other hand, increases pituitary GH (Purves and

Griesbach, 19h6; Koneff gt gt., l9h9; Contopoulos gt gt.,

1958; Knigge gt gt., 1958; Solomon and Greep, 1959; Schooley

gt gt., 1966; Daughaday gt gt., 1968) and hypothalamic GH-

RF (Meites and Fiel, 1967).

Androgens in small doses stimulate growth, increase
pituitary GH in intact or castrated rats and increase pitui-
tary GH in female rats. Castration, conversely, reduces the
amount of GH in the pituitary of males (Rubinstein and
Solomon, 1941; Birge gt gt., 1967a; Daughaday gt gt., 1968;
Kurcz gt gt., 1969).

In view of the marked influence of thyroxine and to a
lesser extent androgen, on growth and pituitary concentration
of GH it was of interest to study the effects of these hor-
mones on the production and metabolic clearance rates of GH

in the rat.

106

B. Procedures

Male Sprague-Dawley rats weighing 280-320 g were ob-
tained from Spartan Research Animals (Haslett, Michigan).
The animals were divided into five groups: a) surgically
thyroidectomized, b) Na-thyroxine treated, c) castrated,
d) testosterone propionate treated, e) intact controls.
All animals were fed and watered gg libitum. The drinking
water of the thyroidectomized group was supplemented with
a solution of 2% Ca-gluconate. L-Na-thyroxine (T4) and
testosterone prOpionate (TP) were obtained from Nutritional
Biochemicals Corporation, Cleveland, Ohio. Thyroxine was
dissolved in saline and injected subcutaneously at a dose
of 10 ug/lOO g body weight/day. Testosterone propionate
(TP) was dissolved in corn oil and injected subcutaneously
at a dose of 200 ug/lOO g body weight/day. Since we were
unaware at the time of the inactivation of GH in serum
(see III. Difference Between Serum and Plasma RGH Levels)
the animals were bled every other day (non-heparinized
blood), and killed at the end of ten days of treatment.
Twenty animals were used per group. GH in pituitary homo-
genates and serum was measured at # dilutions. In light
of the inactivation of GH in serum only the pituitary
values of this group have been kept and are presented in
Figure 21.

The experiment was repeated with the following changes
incorporated: a) 10 animals were used per group, b) blood

was collected at O, 5 and 10 days of treatment between 8:00

 

 

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107

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Effects of different treatments on anterior
pituitary GH concentration in the male rat.
RGH concentration/ mg A.P. is expressed as
percent of control, where control concentna-
tion is regarded as 100%.

 

108

and 10:00 A.M., c) the blood was collected in syringes
containing 0.1 ml of a 100 mg% Na-heparin solution/ml
blood collected: a total of 1.5 ml was collected each
time, d) all collections were done under light ether anes-
thesia via heart puncture and kept in an ice bath, e) one
hour after the last bleeding, the animals were anesthe-
tized with Na-pentobarbital (30 mg/kg) in preparation for (‘
the metabolic clearance rate study described below.
Metabolic clearance rate of a hormone may be studied

by either constant infusion or single injection of hormone.

 

Since it had been previously shown that the results were L
similar with either method (Tait, 1963; Kohler gt gt.,
1968a; Coble et al., 1969; Frohman and Bernardis, 1970),

and taking into consideration the number of animals used

in this experiment, the single injection method was adopted.
Following Na-pentobarbital anesthesia the animals were in-
jected through the tail vein with 2.4 x 106 cpm of RGH-

1125 in 1 ml of PBS. Seven-hundred and fifty ul volumes

of blood were collected at l, 5, 10, 15, 20, 30, #5, 60

and 90 minutes after the injection. The blood samples

were placed in heparinized tubes and centrifuged as de-
scribed previously.

For each time sample/animal two doses of plasma were
allowed to react with a 1:1000 dilution of the DMD-l
anti-rat growth hormone serum, and two doses to count
total radioactivity. The reaction was allowed to proceed

for 72 hours and then the second antibody was added as in

109

a normal radioimmunoassay. The samples were centrifuged
and counted 24 hours later. The remainder of the plasma
aliquots were frozen and assayed 5 months later for endo-
genous growth hormone. The levels found at 5 months did
not differ with respect to time sample/animal.

Since the disappearance curves for RGH-1125 appeared
multiexponential, individual MCR's were calculated using

the formula

total immunoprecipitable RGH—I125 injected
MCR = fxl , at

as described by Tait (1963) for a system of pools. The

 

metabolic clearance rates obtained were pooled/group and
plotted in semi-logarithmic paper as shown in Figure 22.
Since in the steady state the amount of GR cleared is equal
to the amount of GH secreted, secretion rate (SR) Was arrived
at by multiplying the mean group MCR times the plasma con-
centration of GH of the individual animals in each group.

The plasma GH concentration on day 10 was used in this cal-
culation.

The data were analyzed by "t" test for paired observa-
tions or analysis of variance followed by Duncan's multi-
ple range test (Bliss, 1967).

C. Results

Figure 21 shows the effect of the various treatments
on anterior pituitary concentration of GH. The results

have been expressed as percent change from the intact

 

IOO

50

E5

6
0

cpm lml plasma x IO3
(N
C)

Figure 22.

    
 

110

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no 29 so 40
minutes

- - Total Radioactivity

— Immunoprecipitable
Radioactivity

 

To £0 50 40
minutes

Disappearance of total radioactivit and immuno-
precipitable radioactivity (RGH-112%) from plasma
following a single iv injection in: A) thyroid-
ectomized, B) Na-thyroxine, C) castrated,

D) testosterone propionate, E) intact rats.

111

control group. At the end of 10 days, castration produced

a significant reduction in pituitary GH concentration, where-
as daily subcutaneous injections of TP, at a dose of 200 ug/
100 g of body weight, caused a significant increase. The
castrated group had 72.3% while the TP treated group con-
tained 172.1% of control values. The concentration of GH

was very significantly reduced in thyroidectomized animals

to only 6.4% of control levels. Daily subcutaneous injec- .Vm
tions of T4 at a dose of 10 ug/lOO g of body weight pro-

duced a significant rise in pituitary GH concentration, to

 

213.2% of control values. The magnitude of the changes pro-
duced by thyroidectomy and Ta was significantly different
from that elicited by castration and TP respectively.

Plasma GH was measured at O, 5 and 10 days after treat-
ment. The results are presented in Table 7 and expressed
in terms of the NIAMD-RGH-RP-l standard reference prepara-
tion. It can be seen that removal of 1.5 ml of blood at
O, 5 and 10 days did not affect the level of plasma GH. In
contrast, plasma GR was significantly reduced 5 days after
castration, remaining at this low level through day 10. No
alteration in plasma GH was observed on day 5 in the TP
treated group. On day 10, however, plasma OR was signifi-
cantly elevated. Thyroidectomy caused a significant decrease
in plasma GR. This decrease was already evident on day 5;
no further change had taken place by day 10. The admini-
stration of T4 produced a significant increase in plasma

GH by day 5. In addition, a small increase was further

112

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113

observed on day 10, although the level was not signifi-
cantly higher than that observed on day 5. The magnitude
of the changes observed on day 10 produced by thyroidec-
tomy and T4 was not significantly different from that
brought about by castration and TP respectively.

The metabolic clearance rate of GH was determined
using the formula described previously. The plasma con- r?
centration of immunoprecipitable and total radioactivity -
was plotted versus time in linear coordinates for each ani-

mal in a group. The integral d/‘x' . dt was determined
I

 

 

by extrapolating the curve to the visual intercept, which EJ
occurred generally between 100 to lfiO minutes after the in-

jection, and numerically measuring the area under the dis-

appearance curve for the immunoreactive RGH-1125 for each

animal. In order to do this it was assumed that there were

no components of the curve slower than those already ob-

served at 90 minutes. It may be noted that doubling the

time for the visual intercept to 200-280 minutes resulted in

a 6-8% decrease in the calculated MCR's.

The mean disappearance curves of total and immunopre-
cipitable radioactivity/group are shown on Figure 22 plot-
ted to 45 minutes on semi-logarithmic paper. The use of a
1:1000 dilution of DMD—l anti-rat growth hormone serum with
50 and 100 ul of plasma showed 80—90% of immunOprecipitable
radioactivity present at 1 minute after the injection. At
the end of 45 minutes, only 6-17% of the immunOprecipitable

radioactivity remained in the plasma. In ascending order of

114

immunoorecipitable radioactivity remaining at #5 minutes
were: Tu, TP, control, castrated and thyroidectomized.
The disappearance of RGH-I125 injected appeared nearly
linear for the first 10-15 minutes after which it assumed
the characteristics of a multiexponential curve. In all
cases the disappearance curve of total radioactivity fol-
lowed that of immunOprecipitable radioactivity for most
of the linear part, after which it deviated significantly,
suggesting the addition and recirculation of metabolic de-
gradation products of RGH-I125 in the plasma which were
not immunoreactive.

The individual MCR's obtained as described above were
pooled/group and analyzed statistically. The results are
shown in Table 8. All half-lives (t%) were calculated
from the linear portion of the immunoprecipitable disap-
pearance curve of each animal and pooled. The ti of in-
tact controls was similar to that observed in castrated,
TP treated and thyroidectomized rats ranging from 5.6 to
6.7 minutes. Na-thyroxine, however, produced a signifi-
cant decrease in the t% of GM, averaging 4.8 minutes. The
mean MCR calculated for intact control rats was 1.29 1
0.08 ml/min and was not significantly different from that
observed in castrated rats (1.19 i 0.04 ml/min). Meta-
bolic clearance rate of GH, however, was significantly
altered in animals after thyroidectomy, TP or T4. Both TP
and T4 raised MCR to 1.70 i 0.09 and 1.86 1 0.07 ml/min

respectively. These levels were not significantly different

 

115

 

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116

from each other. Thyroidectomy, on the other hand, pro-
duced a significant reduction in MCR (1.05 i 0.08 ml/min).
Calculation of the secretory rate of GR was performed
by multiplying mean group MCR x individual plasma GR con-
centration observed on day 10. The underlying assumption
for this is that a steady state condition exists, where the
amount of GR being irreversibly cleared is equal to the ‘“
amount of GR entering the circulation per unit time. To

ascertain that this condition existed during the MCR study

the endogenous plasma GR levels were measured 5 months later.

 

They were found not to differ with respect to time sample L;
in individual animals. It may be calculated that the ad-
dition of 2.u x 106 cpm of RGH-1125, of specific activity
36.5 uc/ug RGR, represents an addition of 30-42 mug of rat
growth hormone. In animals weighing an average of 300g,
and assuming that blood volume represents 8-10% of body
weight, it may be shown that such addition represents an
increase of 1-l.5 mug/m1 blood. This increase is insigni-
ficant with respect to endogenous plasma GR concentration.
Then, by applying the Fick principle, MCR (ml/min) x plasma
concentration (mug/m1) equals the amount of GR in mug
cleared per unit time. In the steady state, this is equal
to the rate at which GR enters the circulation from the
pituitary: this is referred to as secretion rate (SR).
The results may be seen on the right hand column in Table 8.
All of the treatments used produced significant changes

in SR. Castration and thyroidectomy reduced SE to

117

approximately one half and one third respectively. Con-
versely, TP or T4 treatment increased SR to 1.8 and 2.2
times higher than that observed for controls.

D. Discussion

The data presented here on the changes in pituitary
GR as a result of thyroidectomy, thyroxine, castration or
testosterone propionate corroborate previous observations ‘1
by others (see A. Objectives). We have extended these ob- i
servations to include plasma GR levels at O, 5 and 10 days

of treatment. Since these levels, however, may lead to er-

 

roneous conclusions regarding the utilization and secretion E;
of GH, a study of the metabolic clearance and secretion
rate was also included.

Two basic assumptions must be met to carry out a valid
MCR study: a) the endogenous and tracer hormone must be
cleared from the plasma in like manner, and b) all exponen-
tial components of a disappearance curve for a single in-
jection must be taken into consideration in order to arrive
at the prOper integration of the plasma concentration of
the tracer hormone with respect to time. A third assump-
tion is necessary if secretion rates are calculated: the
system must be in a steady state during the course of the
MCR determination.

The first assumption was not tested directly in this
study. Frohman and Bernardis (1970) showed, however, that
the disappearance of RGH-I131 was not significantly differ-

ent from that of rat pituitary extract following a single

118

intravenous injection. Similar observations have been re-
ported for labeled and unlabeled human LR in monkeys
(Kohler gt gt., 1968b), human LR-I131 and endogenous human
LR after hypOphysectomy in humans (Kohler gt gt., 1968a),
and endogenous and exogenous RGH in man follwoing hypOphy-
sectomy (Refetoff and Sdnksen, 1970). It would appear,
therefore, that under most conditions endogenous and exo-
genous tracer hormone are cleared in identical manner.
Since at 90 minutes after the injection of RGH-I125
only 1-7.3% of the original immunoprecipitable radioactivity
remained in the plasma, it was assumed that all components
of the exponential disappearance curve had been included.
Had this not been the case, however, it should be noted that
doubling the time for the prOposed intercept produced only
a small change (6-8%) in the calculated MCR. Evidence in
support of the third assumption was obtained from measure-
ment of the levels of endogenous GR in all time samples
after allowing 5 months (2% half-lives) of radioactive de-
cay to take place. No difference in plasma GR levels was
found with respect to time in individual samples. As men-
tioned previously, the addition of 2.4 x 106 cpm of RGR-
1125 with specific activity 36.5 uc/ug RGH did not signi-
ficantly alter the concentration of endogenous plasma CH.
The multiexponential disappearance curve for RGH-1125
reported here agrees with the previous report of Frohman
and Bernardis (1970) for RGH-1125. This curvilinear dis-

appearance has been reported for other pituitary hormones

 

 

119

(Kohler gt gt., 1968a,b; Coble gt gt., 1969; Yen gt gt.,
1970), and it is believed to represent the distribution
of hormone in more than one compartment (Kohler gt gt.,
1968a). At least two well defined components are measur-
able: a rapid component constituting an almost linear
decay and lasting about 15-20 minutes, and a slow compo-
nent which lasts to about 100 minutes. The ti for GR Fm
calculated from the rapid component agrees with that re-
ported by Schalch and Reichlin (1966) and Frohman and
Bernardis (1970). Furthermore, the MCR for the control

 

group reported in this paper agrees very closely with that ij
reported by Frohman and Bernardis (1970) using a single

intravenous injection of RGH-I131 or rat pituitary ex-

tract, or a constant infusion of RGH-1131.

If one considers that rate of synthesis equals the
amount stored plus the amount released per unit time, and
that in a steady state secretion per unit time is equal to
the amount of hormone cleared per unit time, then it fol-
lows that any simultaneous change in storage and secretion
in the same direction of change must, therefore, repre-
sent alterations in the rate at which the hormone is being
synthesized. In light of this, then, our results suggest
that the lack of thyroxine produces a dramatic decrease in
the rate at which GR is synthesized as well as released.
Thyroxine therapy,on the other hand, greatly increases the
rate at which GR is synthesized and released. It is ob-

vious, in the context of our steady state assumption, that

120

the presence or lack of thyroxin significantly modifies
the amount of GR metabolized by the body. The report of
. Meites and Fiel (1967) makes it likely that these para-
meters are being affected through changes in the synthesis
and release of GR-RF from the hypothalamus, which in turn
modifies the synthesis and release of GR from the pituitary.
These changes are then reflected, in this particular case,
in the concentrations of plasma GR observed. Castration
and testosterone prOpionate appear to produce changes sim-
ilar to those mentioned for thyroidectomy and thyroxine
therapy respectively. These changes, however, would seem
to be lesser in magnitude than those involving thyroxine.
This is the first report showing differences in the
rate of clearance of GR in different physiological condi-
tions. The authors cited previously (Kohler gt gt., 1968a,
b; Coble gt gt., 1969) did not find alterations in MCR of
RLR and RFSR in pre- and post-menopausal women. Changes
in MCR for steroids, however, have been previously report-
ed (Tait, 1963). Does this reflect the lack of a specific
target organ for GR action, or put in a different way, does
it reflect the fact that GR is utilized by a larger number
of cells than those hormones which have a specific target
organ? This is unknown. It should be stressed, however,
that clearance rate refers to the virtual volume of plasma
from which a substance is cleared per unit time. It does
not indicate the amount of that substance which is being

cleared in that time. It seems logical to assume that the

 

 

121

total amount utilized reflects actual metabolism, while the
volume from which that amount is extracted may or may not
be more indicative of changes in circulation time, perfu-
sion pressure, or other hydrostatic characteristics which
may influence the amount extracted per ml of perfusate.
Given two groups of subjects with similar MCR's but

whose plasma concentrations of a particular hormone dif- F3
fered 10-15 fold, and assuming a steady state during the
study, would it be correct to conclude that their hormone

metabolism did not differ? We think not. However, such a

 

conclution was reached by Kohler gt gt. (1968a) and Cable g9
gt gt. (1969) concerning the metabolism of LR and FSR in
pre- and post-menopausal women. Our own results suggest
that the lack of difference in MCR does not indicate a
similarity in the amount of GR metabolized. Thus, al-
though no difference in MCR's was found between control and
castrated rats, the castrated animals were utilizing ap-
proximately half as much hormone as that used by the con-
trol animals. Similar logic may be used to argue the lack
of metabolic difference in animals whose clearance differs
significantly but whose total amount of hormone used per
unit time is the same. This brings out another important
point.

There is little doubt that the lack of thyroxine has
a more profound influence on growth than the lack of testos-
terone propionate. Yet, our results indicate that an in-

significant difference exists in the amount of GR utilized

 

122

by thyroidectomized and castrated animals, or by TP and T4
treated animals. One must consider therefore, if there is
a change in the efficiency of utilization, a change in the
amount excreted by the kidney or differences in the amount
of hormone degraded in the liver between these two groups.
Finally, the secretion rates reported in this experi-
ment differ significantly from those reported by Frohman
and Bernardis (1970). Since we are in agreement concerning
MCR, it can be seen that the difference is due to the levels
of GR measured in plasma. It is not possible to compare
the absolute levels because different standard preparations
were used for which no equivalency has been reported. Our
pituitary and plasma measurements, however, are in agreement
with those reported by Grindeland gt gt. (1970) and Takahashi
and Kipnis (1970) who used the same standards as reported

in this experiment.

 

 

GENERAL DISCUSSION

This thesis describes, among other things, the devel-
Opment of a radioimmunological assay for rat GR. This
assay cannot be considered unique; others have been reported
previously. It is, however, an assay that has been care-
fully characterized. More important, perhaps, is that it
makes use of components that are now becoming widely avail-
able, and hopefully will help set a standard for the re-
sults obtained in different laboratories.

The advantages of a radioimmunoassay over previously
available bioassay methods are obvious: it increases sen-
sitivity by 2,000-4,000 times; it permits the measurement
of hormone in small volumes of blood plasma or serum; it
allows these measurements to be made in individual animals
in a repetitive manner; and it has other advantages.

This thesis reinforces the concept of a common anti-
genic structure in the GR molecule of several mammalian
species. It suggests that the radioimmunoassay for rat GR
may be utilized in measuring pituitary and plasma GR in
other animals including the guinea pig, hamster, mouse,
gerbil, dog and cat. Evidence is also presented regarding
the lability of GR and certain precautions are suggested

to minimize alteration of the antigenic properties of the

123

 

_ ‘-___.

124

molecule in plasma and serum.

Plasma and pituitary GR was measured in male and fe-
male rats of different ages, during the estrous cycle in
females, and in male rats influenced by the lack or excess
of thyroxine and testosterone prOpionate. The results in-
dicate that in most conditions plasma concentration of GR
is a good reflection of pituitary GR concentration. Thus,
it was found that during early life (up to 60 days) pro-
gressive increases occurred in both pituitary and plasma
concentration in male and female rats; castration and thy-
roidectomy decreased pituitary and plasma GR while testos-
terone prOpionate and thyroxine administration produced
Opposite results. An exception was found in that ovariec-
tomy or estradiol benzoate treatment produced changes in
pituitary and plasma GR which were opposite to each other.

As a consequence of these results several questions
remain to be answered: is GR necessary for growth in young
animals? If not, what is responsible for growth in the
developing animal? When does GR become necessary? If it
is necessary, is the rate at which young animals synthesize
and utilize GR different from that in adult animals? What
is the function of the elevated plasma GR level observed
at estrus? How is this rise brought about? Perhaps most
important, does plasma concentration of GR reflect the
actual amounts utilized by the body?

The results obtained in the metabolic clearance and

secretion rate study shed some light on the last question.

 

125

In this small sample of groups of mature animals it would
appear that plasma GR concentration is a good reflection

of the rate of GR secretion into the circulation. However,
in view of the lack of difference between the groups af-
fected by thyroxine and testosterone prOpionate respectively,
and keeping in mind that the effects of thyroxine are more
prominent on growth and general metabolism, caution should
be exercised in equating the amount of GR secreted per
minute to that actually utilized. Of course, it may be
argued that factors such as efficiency of utilization,
tissue sensitivity, and others may account for this lack
of difference.

A final question may be asked with regard to the MCR
and SR results. It has been demonstrated that high GR
levels in the blood or implants of GR in the hypothalamus
can inhibit GR secretion by the tg gttg pituitary. What
are the effects of increased utilization of GR by body
tissues on GR secretion? Is body utilization of GR a
controlling influence on the synthesis and release of GR

from the pituitary?

 

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. .........:|

 

 

 

 

 

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9 ml. -— _.A?( ._

 

‘ .c.
{fig-w \

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1W7. “A

”L..-

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APPENDIX

CURRICULUM VITAE

CURRICULUM VITAE

NAME: Elias Dickerman

DATE OF

BIRTH: July 9, 1945

PLACE OF BIRTH: Concepcion, Chile

NATIONALITY: Chilean (permanent resident of U.S.A.)

 

 

MARITAL STATUS: Married

PRESENT ADDRESS: Department of Physiology, Michigan State
University, East Lansing, Michigan 48823

EDUCATION:

Degree Year Institution Major Eteld of Study
B.A. 1966 Brandeis university Biology
M.S. 1968 Michigan State University Physiology
Ph.D. 1971 Michigan State University Physiology

HONORS, COMMITTEE AND SOCIETY MEMBERSHIPS:

(a) Wien International Scholar, Brandeis University,
1962-1966

(b) Graduated with Honors in Biology from Brandeis

(c) Ayerst-Squibb Travel Fellow of the Endocrine
Society to the Third International Congress of
Endocrinology, Mexico City, Mexico, June 1968

d Elected Associate Member of Sigma Xi, 1968

( )
(e)
(f)

(g)

(h)

Elected as Full Member of Sigma Xi, 1969
Member of the American Association of
University Professors
Chairman, Physiology Graduate Student Committee,
Michigan State University, East Lansing,
Michigan, 1968-1969
Member, Research and Curriculum Committee of
the College of Veterinary Medicine, Michigan
State University, East Lansing, Michigan, 1968-1969

144

 

145

POSITIONS HELD:

(a) Peace Corps Language Instructor (Spanish), Brandeis
University, Waltham, Massachusetts, June-Sept. 1966

(b) Adult Basic Education Teacher, Lansing, Michigan
Public School System, Sept. 1968-Nov. 1969

(0) Teaching and Research Assistant,Department of .
Physiology, Michigan State University, East Lansing,
Michigan, Sept. 1966-Dec. 1970

TALKS PRESENTED AT SCIENTIFIC MEETINGS:

 

Meeting Date Tapic F?
Third International Congress 1968 Effects of Starvation " ----
of Endocrinology, on Plasma GR in the Rat ,
Mexico City
54th Meeting of Federation 1970 Radioimmunoassay of Rat a -
of American Societies for Growth Hormone (GR) fi
Experimental Biology ,

RESEARCH PUBLICATIONS:

(a) Negro-Vilar, A., Dickerman, E., and Meites, J. Effects
of Starvation on Pituitary FSH and Hypothalamic FSH-
Releasing Factor (FSH-RF) in Male Rats. Federation
Proceedings g1: 269, 1968.

(b) Negro-Vilar, A., Dickerman, E. and Meites, J. FSH-
Releasing Factor Activity in Plasma of Rats After
Rypophysectomy and Continuous Light. Endocrinology
§§2 939—94“, 1968.

(c) Negro-Vilar, A., Dickerman, E. and Meites, J.
Effects of Continuous Light on Hypothalamic FSH-
Releasing Factor and Pituitary FSH levels in Rats.

Proc. §2§.‘§_2. Biol. Med, tgz: 751-755, 1968.

(d) Dickerman, E., Negro-Vilar, A. and Meites, J. Effects
of Starvation on Plasma GR in the Rat. Excerpta
Medica 121‘ 73, 1968.

(e) Dickerman, B., Negro-Vilar, A. and Meites, J. Plasma
FSH-RF in Hypophysectomized Rats after Treatment
with Testosterone Propionate or Reserpine. Proceedin s
Internat. union gtysiological Scienceg‘ltt: 112, 1963.

(f) Negro-Vilar, A., Dickerman, E. and Meites, J. Removal
of Plasma FSH-RF Activity in Hypophysectomized Rats ‘
by Testosterone Propionate or Reserpine. Endocrinology (
82: 1349-1352. 1968.

(g)

(h)

(i)

(j)

(k)

(1)

(m)

(n)

(0)

(p)

(q)

(r)

(s)

146

Dickerman, E., Negro-Vilar, A. and Meites, J. In
Vitro Assay for Growth Hormone Releasing Factor-TGR-RF).
Neuroendocrinology 4:75-82, 1969.

Dickerman, B., Negro-Vilar, A. and Meites, J. Effects
of Starvation on Plasma GR Activity, Pituitary GR

and GR-RF Levels in the Rat. Endocrinology 84:814-819,
1969.

Dickerman, E. and Mack, W. N. Radioimmunoassay of Rat
Growth Hormone (GR). Federation Proceedings 29:509,1970.

Negro-Vilar, A., Dickerman, E. and Meites, J. Effects 5,
of Starvation on Hypothalamic FSH-RF and Pituitary FSH
in Male Rats. Submitted for publication , Endocrinology.

Dickerman, B., Koch, Y.. Dickerman, S. and Meites, J.
Radioimmunoassay of Rat Growth Hormone; Effects of
Castration, Testosterone Propionate, Thyroidectomy and
Thyroxine on pituitary GR Concentration. In preparation. i

 

Dickerman, E., Koch, Y., Dickerman, S. and Meites, J.
Metabolic Clearance Rate of GR in the Rat: Effect of
Different Physiological States. In preparation.

Dickerman, E. and Meites, J. Cross-Reactivity of the
NIAMD—A-RatGRS-i Monkey Anti-Rat Growth Hormone Serum.
In preparation.

Dickerman, E., Koch, 2., Dickerman, S. and Meites, J.
Studies on the Inactivation of GR by Plasma or Serum.
In preparation.

Dickerman, B., Dickerman, S. and Meites, J. Plasma and
Pituitary GR in Male and Female Rats with Advancing
Age: The Estrous Cycle. In preparation.

Dickerman, E. and Meites, J. Serum and Pituitary
Prolactin in Castration, Thyroidectomy, Testosterone
Propionate and Thyroxine in the Male Rat. In preparation.

Dickerman, E. and Meites, J. Prolactin Inhibiting
Factor (PIF) Activity in Plasma of Rats After Hypophy-
sectomy and Continuous Light. In preparation.

Dickerman, B., Dickerman, S. and Meites, J. Effect of
Pituitary Transplantation and Constant Light on Sper-
matogenesis and serum LR and FSH. In preparation.

Dickerman, 5., Dickerman, E. and Meites, J. Effects
of Raloperidol on LR, FSH, Prolactin and GR Secretion in
the Rat. gt,vivo and tg vitro studies. In preparation.

   

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