71-7428
CRIST, William Lee, 1941- SOME EFFECTS OF ENDOGENOUS AND EXOGENOUS ESTROGEN AND PROGESTERONE ON ALANINE AND ASPARTATE AMINOTRANSFERASE LEVELS IN THE PLASMA, LIVER, MUSCLE, AND UTERUS OF THE RAT.
The Ohio State University, Ph.D., 1970 Physiology
University Microfilms, A XEROXCompany, Ann Arbor, Michigan 1 SOME EFFECTS OF ENDOGENOUS AND EXOGENOUS ESTROGEN AND PROGESTERONE
ON ALANINE AND ASPARATATE AMINOTRANSFERASE LEVELS IN THE
PLASMA, LIVER, MUSCLE, AND UTERUS OF THE RAT
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
William Lee Crist, B.Sc., M,Sc.
********
The Ohio State University 1970
Approved by
Adviser Department of Dairy Science ACKNOWLEDGMENTS
I wish to especially thank my adviser. Dr. T. M. Ludwick, for his advise and guidance throughout the course of this study and for his assistance in the preparation of this manuscript.
I am grateful to the Federal Dairy Cattle Breeding Project for financial assistance in the form of a research assistantship, and to the members of the project for various types of help.
I wish to express my thanks to Dr. E. W. Brum for programming and statistical assistance.
I wish to thank Dr. W. R. Gomes for the use of the equipment in his laboratory.
Thanks to Dr. D. R. Davis for the use of his electric drill which was used in the homogenization of tissues.
I would like to express my appreciation to Mrs. Crabtree for the expedient typing of this manuscript.
To my wife and children, special thanks for the tolerance and understanding exhibited and the encouragement given during this study.
ii VITA
March 2, 1941 ...... Born - Homeworth, Ohio
1959-1964...... The Ohio State University, Columbus, Ohio
1964...... B.Sc., The Ohio State University, Columbus, Ohio
1965-1967...... Research Assistant, Department of Dairy Science, The Ohio State University, Columbus, Ohio
1967...... M.Sc., The Ohio State University, Columbus, Ohio
1967-1968...... Graduate Research Assistant, Department of Dairy Science, The Ohio State University, Columbus, Ohio
1968-1969...... Research Associate, Department of Dairy Science, The Ohio State Univer sity, Columbus, Ohio
1969-1970...... Administrative Assistant, University College, The Ohio'State University, Columbus, Ohio
FIELDS OF STUDY
Major Field: Reproductive Physiology
PUBLICATIONS
Variations in Bovine Blood Serum Transaminase Values Associated with Levels of Milk Production. W. L. Crist, D. R. Davis and T. M, Ludwick. J. Dairy Science, 49:731, 1966 (Abstract).
iii Effects of Exogenous Estrogen and Progesterone on Serum En2yme Levels
in Dairy Cows, D. R. Davis, W. L. Crist, and T. M. Ludwick, J,
Dairy Science, 49:731, 1966 (Abstract).
Effects of Season, Stage of Lactation, Stage of Gestation and Level of
Milk Production on Serum Transaminase Activity. W. L. Crist,
T. M. Ludwick, E. W. Brum, and D. R. Davis. J. Dairy Sci., 50:
998, 1967 (Abstract).
Effects of Age, Body Weight, and Stage of Gestation on Plasma Glutamic-
oxaloacetic and Glutamic-pyruvic Transaminase Activities in
Immature Holstein Cattle. L. R, Boots, W. L. Crist, D. R. Davis,
E. W. Brum, and T. M. Ludwick. J. Dairy Sci., 51:952, 1968
(Abstract).
Effects of Age, Body Weight, Stage of Gestation, and Sex on Plasma
Glutamic-oxaloacetic and Glutamic-pyruvic Transaminase Activities
in Immature Holstein Cattle. L. R. Boots, W. L. Crist, D. R.
Davis, E. W. Brum, and T. M. Ludwick. J. Dairy Sci., 52:211, 1969.
Some Relationships of Stage of Lactation and Gestation, and Level of
Milk Production to Plasma Glutamic-oxaloacetic and Glutamic-
pyruvic Transaminase Activities. L. R. Boots, T. M. Ludwick, and
W. L. Crist, J. Dairy Sci,, 52:922, 1969 (Abstract).
Effects of Ovarian Hormones on the Synthesis and Storage of Aspartate
Aminotransferase (GOT) and Alanine Aminotransferase (GPT) in the
Rat. W. L. Crist, T. M, Ludwick and D. R. Davis. J. Dairy Sci.,
53:652, 1970 (Abstract).
Iv TABLE OF CONTENTS
Page ACKNOWLEDGMENTS ...... ii
VITA...... iii
LIST OF TABLES...... vi
LIST OF FIGURES ...... x
INTRODUCTION...... 1
Chapter
I. REVIEW OF LITERATURE ...... 3
Causes of Variation in Aminotransferase Activity Stressor Factors Physiological Conditions Metabolic Effects Hormonal Influence Tissue Variations of Aminotransferase Activity Ovarian Function Hormones Produced by the Ovary Variations in the Estrogen and Progesterone Levels During the Estrous Cycle Pseudopregnancy Effects on Hormone Levels Ovariectomy Effects on Hormone Levels Estrogen and Progesterone Influence on Enzyme Levels
II. MATERIALS AND METHODS ...... 27
General Procedure Experimental
III. RESULTS AND DISCUSSION...... 33
IV. SUMMARY AND CONCLUSIONS...... 66
BIBLIOGRAPHY...... 68
APPENDIX
v LIST OF TABLES
Table Page 1. Means and Standard Errors of GOT and GPT Activities of 3 Tissues in the Rat During the 4 Stages of the Estrous Cycle and Pseudopregnancy ...... 48
2. GOT and GPT Least-Squares Means and Standard Errors for Ovariectomized and Sham-operated Rats Adjusted for Time Effects (1 to 21 Days) . . . 49
3. Least-Squares Means and Standard Errors of Tissue and Body Weight for Ovariectomized and Sham-operated Rats Adjusted for Time Effects (1 to 21 Days)...... 50
4. Subclass Means and Standard Errors for GOT and GPT Activities per mg. of Wet Weight of Uterine Tissue Determined from 1 to 21 Days Following Surgery in Ovariectomized and Sham-operated Rats...... 50
5. Subclass Means and Standard Errors for GOT and GPT Activities of Whole Uterine Tissue Determined from 1 to 21 Days Following Surgery in Ovariectomized and Sham-operated Rats...... 51
6. Subclass Means and Standard Errors for Whole Uterine Weight (mg.) for Ovariectomized and Sham-operated Rats from 1 to 21 Days Follow ing S u r g e r y ...... 52
7. GOT and GPT Least-Squares Means and Standard Errors for Hormone Treated Rats Adjusted for Time Effects (12 Hours to 5 Da y s ) ...... 55
8. Subclass Means and Standard Errors for GOT and GPT Activities per mg. Wet Weight of Liver Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats ...... 56
9. Subclass Means and Standard Errors for GOT and GPT Activities per mg. Dry Weight of Liver Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats ...... 57
vi Table Page 10. Subclass Means and Standard Errors for GOT and GPT Activities of Whole Liver Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats ...... 58
11. Subclass Means and Standard Errors for GOT and GPT Activities per mg. Wet Weight of Uterine Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats .... 59
12. Subclass Means and Standard Errors for Whole Uterine Weight (mg.) for Hormone Treated Rats from 12 Hours to 5 Days Following Treatment .... 60
13. Subclass Means and Standard Errors for GOT and GPT Activities of Whole Uterine Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in R a t s ...... 61
14. Overall Means and Standard Deviations for the Independent Variables in the Ovariectomy Study ....*..«*» .84
15. F Values for GOT and GPT Activity per ml. Plasma from Analysis of Variance on Ovari ectomized and Sham-operated Rats from 1 to 21 Days Following Surgery ...... 85
16. Subclass Means and Standard Errors for GOT and GPT Activities per ml. Plasma Determined from 2 to 21 Days Following Surgery and Adjusted for Surgical Treatment Effects in the Ovariectomy Study ...... 86
17. F Values for Whole Liver Weight and for GOT and GPT Activities in Liver Tissue from Analysis of Variance on Ovariectomized and Sham-operated Rats from 1 to 21 Days Follow ing Surgery ...... 87
18. Subclass Means and Standard Errors for Whole Liver Weight (g.) Determined from 1 to 21 Days Following Surgery in Ovariectomized and Sham-operated R a t s ...... 88
19. Subclass Means and Standard Errors for GOT and GPT Activities of Liver Tissue Determined from 1 to 21 Days Following Surgery and Adjusted for Surgical Treatment Effects in the Ovariectomy Study ...... 89
vli Table Page 20, F Values for GOT and GPT Activities in Muscle Tissue From Analysis of Variance on Ovariectomized and Sham-operated Rats from 1 to 21 Days Following Surgery ...... 90
21, Subclass Means and Standard Errors for GOT and GPT Activities of Muscle Tissue Determined from 1 to 21 Days Following Surgery in Ovariectomized and Sham- operated Rats ...... 91
22. Subclass Means and Standard Errors for GOT and GPT Activities of Muscle Tissue Determined from 1 to 21 Days Following Surgery and Adjusted for Surgical Treat ment Effects in the Ovariectomy Study ...... 92
23. F Values for Uterine Weight and GOT and GPT Activities in Uterine Tissue from Analysis of Variance on Ovariectomized and Sham-operated Rats from 1 to 21 Days Following Surgery ...... 93
24. Overall Means and Standard Deviations for the Independent Variables in the Hormone Study ...... 94
25. F Values for GOT and GPT Activities per ml. Plasma from Analysis of Variance on Hormone Treated Rats from 12 Hours to 5 Days Following Treatment ...... 95
26. F Values for GOT and GPT Activities in Liver Tissue from Analysis of Variance on Hormone Treated Rats from 12 Hours to 5 Days Following Treatment 96
27. F Values for GOT and GPT Activities in Muscle Tissue from Analysis of Variance in Hormone Treated Rats from 12 Hours to 5 -Days Following Treatment ...... 97
28. F Values for GOT and GPT Activities in Uterine Tissue from Analysis of Variance on Hormone Treated Rats from 12 Hours to 5 Days Following Treatment ...... 98
29. Subclass Means and Standard Errors for GOT and GPT Activities per ml. Plasma Determined from 12 Hours to 5 Days Follow ing Hormone Administration Adjusted for Treatment Effects ...... 99
viii Table Page 30. Least-Squares Means and Standard Errors of Tissue and Body Weight of Hormone Treated Rats Adjusted for Time Effects (12 Hours to 5 Days) ...... 100
31. Subclass Means and Standard Errors for Whole Liver Weight (g.) for Hormone Treated Rats from 12 Hours to 5 Days Following Treatment ...... 101
32. Subclass Means and Standard Errors for Whole Liver Weight and GOT and GPT Activities in Liver Tissue Determined from 12 Hours to 5 Days Following Hormone Administration Adjusted for Treatment Effects. . . . 102
33. Subclass Means and Standard Errors for GOT and GPT Activities in Muscle Tissue Determined from 12 Hours to 5 Days Follow ing Hormone Administration Adjusted for Treatment Effects...... - 103
34. Subclass Means and Standard Errors for GOT and GPT Activities of Uterine Tissue Determined from 12 Hours to 5 Days Follow ing Hormone Administration Adjusted for Treatment Effects...... 104
lx LIST OF FIGURES
Figure Page 1. Effects of Ovariectomy on Uterine Weight and GOT Activity (by mg. of Uterus and by Whole Uterus) in the R a t ...... 53
2. Effects of Ovariectomy on Uterine Weight and GPT Activity (by mg. of Uterus and Whole Uterus) in the Rat...... 54
3. Effects of 17p-Estradiol on Uterine Weight and GOT Activity (by mg. of Uterus and by Whole Uterus) in Ovariectomized R a t s ...... 62
4. Effects of 17£-Estradiol on Uterine Weight and GPT Activity (by mg. of Uterus and by Whole Uterus) in Ovariectomized R a t s ...... 63
5. Effects of Progesterone on Uterine Weight and GOT Activity (by mg. of Uterus and by Whole Uterus) in Ovariectomized R a t s ...... 64
6. Effects of Progesterone on Uterine Weight and GPT Activity (by mg. of Uterus and by Whole Uterus) in Ovariectomized Rats...... 65
x INTRODUCTION
Aminotransferase enzymes have been studied rather extensively
both in terms of their mechanisms of control and their relation to
animal function. Some relationships between aminotransferase enzymes
and stress factors, disease, environment, feeding, estrous cycle,
pregnancy, lactation, age, and other factors have been observed In
several species.
These studies have suggested several ways in which these
aminotransferases may be important indicators of metabolic or physio
logical changes or responses of animals. Aspartate aminotransferase
(GOT) and alanine aminotransferase (GPT) in the serum of animals have been used as an indirect measure of stress in several species.
Myocardial infarctions and hepatic disease In humans cause a rise in
serum GOT and serum GPT. The level of GPT in the rat liver is thought
to serve as a kind of metabolic barometer sensitive to different pressures, high enzyme activity being associated with an increased rate of gluconeogenesis and low activity with the conservation of amino acids for growth of new tissues.
Many studies have been carried out in recent years to try to explain the underlying cause of variation in enzyme levels. The effects of several hormones on aminotransferase levels have received considerable attention. Glucocorticoids cause an increase in hepatic 2
GPT in rats while estrogen and progesterone prevented this gluco
corticoid induced increase. Estrogen has been found to cause an
increase in both GOT and GPT in several tissues in the rat. The
actions of the ovarian hormones on aminotransferase activity therefore are not very clear. The objectives of this investigation were to
determine some of the relationships between the ovarian hormones and
enzyme synthesis and levels in various tissues. REVIEW OF LITERATURE
In view of the accepted relationship between certain enzymes
and hormones, it has been postulated that a definite explanation for
some of the variation in enzyme level may be a direct or indirect
result of hormone values in the blood or other tissues. Some of the
causes of variation in enzyme levels and possible relationships between
certain enzymes and hormones, particularly those produced by the ovary, will be reviewed in this section.
Causes of Variations in Aminotransferase Activities
One of the first uses of the enzymes aspartate aminotransferase
(GOT) and alanine aminotransferase (GPT) was as a diagnostic tool in human disease. LaDue, Wroblewski and Karmen (70) found that serum
GOT levels rose 2-20 fold within 24 hours after myocardial infarction.
Steinberg and Ostrow (114) reported that in over 90 percent of the cases of myocardial necrosis studied in humans, a rise in serum GOT occurred within 36 hours after infarction. These aminotransferase levels usually returned to normal within 5 days. Other workers (24) observed that experimental necrosis of lung, liver, spleen, kidney and gastrointestinal tract, as well as the heart (4, 72) was associated with increased serum GOT activity. Two conflicting reports by Pearl et al. (86, 87) indicated that myocardial damage produced in rats by 4
isoproterenol did not elevate serum GOT or GPT level, but instead the
restraint of the rats in many of these experiments caused the rise in
these enzymes.
The degree of increase in serum GPT levels in clinical patients
is used in diagnosing hepatic disorders and as an indication of the degree of severity of the disorder. Wroblewski and Ladue (126) found
that acute hepatitis causes a rise in serum GPT activity of 2 to 1000
times normal. The rise in serum GOT following the disease closely
paralleled that of serum GPT but to a lesser degree.
Other diseases have been found to increase transaminase levels.
Kuttler and Marble (69) reported that artificially induced or naturally occurring white muscle disease (muscular dystrophy) in lambs greatly increased GOT levels in the blood. Blinco and Dye (10) observed increased serum GOT in calves as well as lambs with muscular dystrophy. The serum transaminase activity appeared to be proportional to the extent of the muscle damage. It was suggested that this increased serum enzyme level may permit the objective diagnosis of white muscle disease without sacrifice of the animal. These workers
(10) also observed that serum GOT was not increased by scours and was only slightly increased by staphylococcus meningitis, toxemia and severe internal injuries.
A tying-up disease is prevalent among horses required to per form maximum physical exertion. A significant difference was found in serum GOT levels between horses that tied up and those that did not (21). It was suggested that serum GOT levels might be used as an indication of horses that might tie-up. Stressor factors, Selye (104) proposed that animals respond
in a general way to a variety of stress situations. This general adaptation syndrome involves the pituitary-adrenocortical system. It has also heen reported that certain aminotransferases increase in activity following various stress situations and this response might be a part of the general response to stress, GOT and GPT levels have been suggested to be used as an indirect measure of stress.
Many investigators (2, 21, 68, 124) employ the use of physical exercise in the study of the changes in enzyme levels due to stress.
Wegmann et al. (124) reported a significant increase in plasma GOT and GPT within 30 min. of exercise on a bicycle ergometer in healthy untrained and unadapted male students. Exposure to reduced pressure of 312 mm Hg for 30 minutes and positive acceleration of 2,5 x g on a human centrifuge for 30 minutes also significantly increased plasma
GPT activity while only lowered pressure was effective in increasing plasma GOT levels. Altland et al. (2) found that in rats walking exercise caused an increase in serum GOT and GPT levels. Altitude alone did not elevate these enzyme levels while exercise at altitude increased their activity more than exercise alone. Less change in enzyme levels occurred in trained than in untrained rats. Similar results were observed for serum GOT in horses following training and exercise (21). Swimming exercise was found by Critz and Withrow (29) to increase heart and skeletal muscle GOT of rats. No change in the
GOT levels of these tissues due to swimming was observed following adrenalectomy indicating that the adrenal corticoids were essential 6
for the exercise-induced increase in enzyme activity.
Davis (34) found that exposing rats to a temperature of 3°C for
24 or 36 hours caused an increase in GOT and GPT activity in the plasma and liver and an increase in GOT in the muscle. However these animals were taken off feed 12 hours before cold exposure. When rats were given free access to feed before and during the time of exposure a large portion of the observed change in enzyme levels xjas abolished.
Beaton (9) observed an increase in liver GPT activity of the rat following acute cold exposure for about 7 days while the blood GPT level did not change. The effects of local cold injury on GOT activity in the rabbit was studied by Penneys et al. (88). They observed a decrease in GOT activity in both the chilled and the contralateral legs and a rise in the serum levels. Ligation of the femoral artery of the chilled leg prevented the decrease in GOT activity in the unchilled leg.
Other stress factors have been found to cause a change in enzyme levels. GOT and GPT in the serum of pregnant cows increased measurably following transport by railroad (19). Stimuli such as hypoxia, hypothermia, aseptic inflammation, burn shock and septicemia were found to cause an increase in serum enzyme activity (11).
Physiological conditions. In recent years considerable interest has developed in the effects of various physiological factors on the aminotransferases, Davis and co-workers (32) found that serum GOT levels in the dairy cow tended to peak at the 15th day of the estrous cycle with a secondary peak during estrus. Roussel and Stallcup
(101) confirmed this peak at estrus. Serum GPT levels were fairly stable throughout the cycle, Davis et, al. (33) also observed that
exogenous estrogen caused an increase in serum GOT activity in the
cycling cow while exogenous progesterone had little effect. The GOT
activity in the human endometrium was found to vary during the menstrual cycle (59). The specific activity of GOT increased during
the proliferation phase of the cycle with a peak at midcycle, at about
the time of ovulation. There was a subsequent decline in specific
activity throughout the secretory phase which reached a minimum near menstruation.
Stallcup jet al_ (112) found no differences in plasma amino
transferase activities between open and pregnant cows, and likewise
Boots et: al. (17) found no significant variation due to gestation.
Other workers, however, reported that plasma aminotransferase activity
decreased with advancing stage of pregnancy in cows (28) and heifers
(16), In the rat, Harding (54) found similar effects of pregnancy on liver GPT, which declined to a level half that found in normal rats by the 21st day of pregnancy. Aminotransferase levels also have been
found to rise at parturition in dairy cows (56). Induced early
embryonic death in cattle did not change the level of serum GOT or
GPT (20).
Boots et al. (15, 17) and Stallcup et al. (112) found signifi
cant negative correlations between serum aspartate aminotransferase activity and days of lactation in the dairy cow whereas Crist et al.
(27, 28) reported a significant increase in these enzyme activities with advancing stage of lactation. Crist et al. (27, 28) also found that increased plasma aminotransferase activity was associated with 8
increased levels of milk production while reports by Boots et: al.
(15, 17) were contradictory. Rakes et al. (94) found a significant
negative correlation between milk production and a ratio of GOT to
GPT in serum. In the pseudopregnant rabbit Hafez and co-worker (52)
found that the GOT activity of the endometrium was depressed by
lactation.
Sex differences in enzyme activities have been reported by
several workers (67, 79, 91, 120). Lin al_. (71) found rat liver
tyrosine-Ql-ketoglutarate transaminase higher in males than females.
The controlling factor appeared to be testosterone. However other workers (94, 101) have reported higher serum GOT levels in cows than bulls. Boots ejt al. (16) found no significant differences between
open heifers and young bulls,
Nichol and Rosen (83) found that age of rat influenced liver
GPT levels, with the enzyme activity relatively low during the first
six weeks of life and increasing progressively until about one year of age; liver GOT activity did not show this variation. In bulls
Roussel and Stallcup (99) observed a negative correlation between age and serum GPT while Boots jst al. (16) reported significant effects of age on serum GOT and GPT activities with the enzyme levels lower
in young bulls. Other workers (53) reported no effect of age on aminotransferase levels in the ejaculate of bulls. Tyrosine amino
transferase in the liver of rats was reported by Lin and co-workers
(71) to be related to body weight while Boots at al. (16) reported no differences in plasma aminotransferase levels in bred heifers due to 9
body weight, Hankiewicz ejt al. (53) found no correlation between GOT
or GPT activity in the ejaculate of bulls due to breed, potency of
bulls, sperm survival or sperm motility. Species differences in serum
GOT and GPT have been reported (132) indicating that these enzymes are
under genetic control. Several investigators (14, 28, 99) have
observed significant seasonal variations in serum aminotransferases.
The enzyme activities are higher during the summer months than during
the winter months. Higher temperatures in the summer may be the
primary factor influencing these enzymes (14).
As indicated by the research reported here, many environmental
factors have been found to influence the aminotransferase enzymes.
However the direction and degree of variation of these enzymes due
to the various factors which were studied by different investigators
is not very consistent.
Metabolic effects. Nichol and Rosen (83) working with rats have reported on metabolic factors affecting alanine and aspartate aminotransferases. Theysuggest ,fthat the level of activity of GPT in liver can serve, in a sense, as a metabolic barometer sensitive to different pressures, high activity being associated with an increased rate of gluconeogenesis and low activity with the conserva tion of amino acids for the growth of new tissues. '1 This statement is supported by work (54, 67, 83, 98) that has indicated that growth of nev; tissues in an animal causes a depression of liver GPT but not
GOT. Tumor growth, pregnancy, partial hepatectomy and rapid growth in rats were all found to depress levels of liver GPT. When gluco- 10 corticoids, which stimulate gluconeogenesis, were administered, liver GPT activity rose (83, 97, 98). Also alloxan diabetes and starvation increased the hepatic GPT levels (67, 83, 98). Rats fed diets high in protein resulted in increased liver GPT activity compared with control animals fed a diet lacking protein (67, 83, 97,
98). This response of GPT was suggested to be due to either sub strate induction because of increased levels of amino acids or to an adrenal mediated response due to a metabolic stress associated with dietary variation. These latter observations of factors increasing liver GPT levels support the interpretation that this enzyme is rate-limiting in gluconeogenesis (98).
Diurnal variation of 3-4 fold in rat liver tyrosine amino transferase has been reported (25, 60, 127). Recently Davis (34) measured the circadian variation in GOT and GPT activity in the plasma, liver and muscle of rats. Only plasma GOT activity varied significantly.
This diurnal variation in aminotransferase level was suggested to be due to lighting regime and feeding habits of the rats.
Hormonal influence. Several hormones have been reported to influence aminotransferase levels. In the dairy cow, Davis et al. (33) found that exogenous estrogen Increased serum GOT but not GPT levels.
Progesterone produced little or no response in these enzymes. Boots
(13) found that injections of 17(3-estradiol to ovariectomized rats significantly increased GOT in the plasma, liver, uterus and muscle.
GPT activity was increased In all tissues except the uterus. Rosen et al. (97) found that thyroxine as well as 17p-estradiol caused an increase in hepatic GPT in the rat. However, the injection of 17(3- 11 estradiol or progesterone along with cortisol prevented the response of GPT that would have resulted from the administration of cortisol alone. Testosterone, insulin or growth hormone did not alter normal liver GPT levels or affect the response of this aminotransferase when injected in combination with cortisol.
Early work by Rosen and co-workers (98) indicated that rat liver
GPT activity was inducible by glucocorticoids such as hydrocortisone while GOT diawed little response. This has been substantiated by other workers (102, 103), but Sheid et al. (105) reported a 100 percent increase in GOT in liver homogenate following cortisone injections.
The increase in GPT activity following glucocorticoid administration increases substantially over the course of 48 hours and remains at a high level for several more days (83). The mechanism whereby gluco corticoids cause an increase in liver GPT activity has not been established. Work by Segal and co-workers (102), however, adds support to the hypothesis of an increased synthesis of catalytic protein.
They found that properties of the enzyme from normal and corticoid- treated animals were indistinguishable in the kinetic parameters studied and in the several steps of the purification procedure. Also a large fraction of the corticoid-induced increase in activity is blocked by the administration of ethionine.
The mechanism of aminotransferase induction by glucocorticoid hormones has been studied more extensively for aminotransferases other than GPT. One of these is tyrosine aminotransferase. The induction of this en2yme in the liver of rats,has been found to be mediated 12 entirely by adrenal hormones (64). Much of the evidence (64, 66, 89,
90, 123, 125) in this area suggests that an early action of gluco corticoids induction of tyrosine aminotransferase is an increase in
RNA synthesis. Also a synthesis of protein appears to be required for enzyme induction (30, 46, 51, 64, 65, 117, 118). The observation that actinomycin or 5-fluorouracil, if administered in large doses and long enough after enzyme induction by glucocorticoids, would cause an increase in the rate of synthesis of induced aminotransferase in the liver of rats or in hepatoma tissue culture cells, rather than the usual inhibition, led to an interesting proposal by Tomlcins at al.
(117) and Garren £t al. (46) as to the mechanism of control of enzyme synthesis. They suggested (118) that the rate of translation of the aminotransferase messenger is normally limited by a labile substance which they referred to as a ''cytoplasmic repressor'*. The mRNA for the repressor as well as the repressor itself must turn over rapidly.
The increased tyrosine aminotransferase synthesis by inhibitors of
RNA synthesis would, therefore, result from inhibition of repressor mRNA formation followed by rapid degradation of the remaining repressor messenger as well as the repressor itself. This would result in the translation of the stable aminotransferase messenger being derepressed.
Tissue variation of aminotransferase activity. Enzyme activity has been found to vary considerably among the various tissues of the body. Some species differences have also been observed in the tissue distribution of enzymes (133). One problem with trying to obtain 13
accurate relative levels of enzymes in several tissues of animals
within a species is that dietary and hormonal manipulations can lead
to changes in the concentration of enzymes (133). Thus the normal
levels of enzyme activity in various tissues which are due to genetic
control may be altered.
Aspartate and alanine aminotransferase activities were observed
in liver, kidney, heart, spleen, skeletal muscle, ventral prostate,
brain and testis of the rat in 1952 (5). The GPT activity was highest
in the liver while the GOT activity was highest in the heart, GOT
and GPT activities were found to be approximately equal in human blood
serum (62). GOT activity of whole blood hemolysates was ten times
as high as in serum. More than 90 percent of the GPT activity was
reported by Cornelius (26) to occur in the liver in all the mammals
he studied, including the cat, fox, dog, rat, bison and steer.
Zimmerman et al. (133) measured the GOT and GPT activity in liver, myocardium, kidney, skeletal muscle and brain in six species including
the guinea pig, rabbit, pigeon, chicken, rat and horse. GOT levels were found to be highest in the myocardium in all species studied
except the horse, which had the highest levels in the liver. Levels
of GOT were almost as high in liver as those in myocardium in the rabbit, guinea pig and chicken. In each species, brain GOT activity was relatively low. In the rabbit, GOT levels in the kidney and muscle were lower than in the brain while in the guinea pig and rat
this enzyme showed little difference among tissues in enzyme levels.
In the chicken and horse, levels of GOT in kidney and muscle were
somewhat higher than the GOT activity of the brain. In the pigeon, 14 all organs except the brain had high levels. GPT activity was highest in thi liver of all species studied. All other tissues in each species had relatively low enzyme levels with the exception of the kidney of the chicken and the muscle and kidney of the pigeon. These workers
(133) reported that in general, species with the highest levels of
GOT in serum had the highest values in the tissues. GPT content in the liver xras highest in species with the highest serum levels and lowest in those with the lowest serum levels.
Recently there have been several reports on the aminotransferase levels in the semen and genital tract of the male (38, 39, 100).
Alumont et_ al_. (3) found GOT activity to be about ten times greater in the epididymal fluid and seminal plasma than in the blood serum of the ram. Almost no GPT activity, however, was detected in the epididymal fluid and seminal plasma. Stallcup and co-workers (113), working with the bovine, reported the GOT activity in epididymal fluid and the seminal plasma was more than six times and three times greater, respectively, than in the blood serum. The GPT concentration in the epididymal fluid was almost twice that found in blood serum and four times that found in seminal plasma. Murdock et^ al. (80) reported the highest activity of GOT in the seminal vesicle fluid compared with epididymal and testicular fluids in the ram. Comparing GOT levels in seminal plasma and spermatozoa, they found that the activity was nearly the same for the ram while the GOT level of the spermatozoa was over twice as high as that found in the seminal plasma of the bull.
Studying the activities of GOT and GPT in the various reproductive organs of the bull, Roussel and Stallcup (100) showed that GOT activity 15
was significantly higher than GPT. Also, aspartate aminotransferase
was located primarily in the testis, ampulla, seminal vesicle,
disseminate prostate and bulbourethral gland. GPT activity was found
primarily in the testis, corpus epididymidis and disseminate prostate.
Ovarian Function
Hormones produced by the ovary. Estrogens and progestogens are
the primary hormones produced by the ovary. Androgens and relaxin
have also been found to be secreted by the ovary. Relaxin is a poly
peptide hormone reported to be secreted by the ovax*ies of several
species including rabbits, rats, mice, pigs, whales and sharks (18,
121). Zarrow and O'Connor (131) determined that it is probably
secreted by the corpus luteum. Relaxin has been called a ''hormone
of pregnancy'' (119) since it is found in increased amounts during
the late stages of pregnancy in various mammals. It has not been
found in the blood of men or nonpregnant women (119).
It has been known for some time that ovaries under abnormal
or experimental circumstances, in many mammals, produce androgens (85).
More recently there have been reports (73, 109) of low levels of
androgen production by the normal ovary. Androstenedione but not
testosterone was found in normal ovarian tissues while both of these
androgens were reported in cysts of women with various gynecological
disorders (1). There are indications that hilus cells of the ovary may be an important source of ovarian androgen in the human female
(37).
It has been known that the ovary produces estrogen and proges- 16
terone. Research concerning the rate of secretion, level in the blood
and various tissues and the source of production in the ovary of these
hormones have been hampered until recently by the lack of accurate
chemical assay techniques. Much of the evidence in these areas is
indirect.
The ovarian follicle is the primary source of estrogen produc
tion in the normal ovary. To determine the cellular site of estrogen
secretion, the histochemical demonstration of the key enzymes in the biosynthesis of the steroid hormones is often used. The theca Interna cells are thought to be responsible for the major output of estrogenic hormone by the ovary (37, 108, 122). The theca lutein cells of the corpus luteum and the interstitial cells of the ovary however, as well as the theca interna cells have been implicated as producers of estrogens (128), That the ovarian cells other than those of the
Graafian follicle can produce estrogen was demonstrated by Mandl and
Zuckerman (74, 75). After the X-irradiation of the ovaries in rats and mice sufficient to destroy all Graafian follicles, irregular cycles persisted for about six weeks, followed by continuous vaginal estrus between one and fourteen weeks and ultimately by anestrus.
The main source of progesterone secretion in mammals is normally considered to be the corpus luteum (81, 130), Erb et al. (41) recently supported this statement with work in the bovine. They measured progestins in the ovary and CL of cows following estrus and breeding, during pregnancy and post-partum. The CL contained 947, of the total progestins in 100 ovaries. There was a high correlation between the progestins in CL and total amount in the ovary. The progestins in 17 ovaries without a CL were low and often non detectable. Also in the nonpregnant cow, Gomes £it al. (47) reported a significant correlation between ovarian venous blood plasma progestins and corpora lutea concentrations of progestin.
There is adequate evidence, however, that indicates that progestins are secreted by ovarian tissue other than the CL. Researchers (106,
129) have chemically identified progesterone and its metabolites in follicular fluid. In rabbits, after coitus or LH injection, Hilliard et ol. (57, 58) observed that progestins ore secreted before ovulation occurs and that the removal of the follicles prior to gonadotrophin administration does not diminish significantly, the progestin output by the ovary. Hilliard et al. (57) proposed that, in the rabbit, at least, the interstitial cells of the ovary secreted the progestins.
That much of the progesterone secreted by the ovary during the estrous cycle of the rat comes from sources other than the CL has been strongly indicated by several researchers (44, 76, 82) who have reported that progesterone levels in the ovarian venous blood pealced during the proestrus phase of the estrous cycle. By studying the morphology of the Golgi apparatus in lutein cells, McDonald et al. (76) concluded that the luteinized granulosa cells are the source of the progesterone secreted during estrus and diestrus, but the cells that secrete progesterone during proestrus could not be identified. This suggests that the cells which produced progesterone during proestrus may have been cells other than the luteinized granulosa cells.
Variations in the estrogen and progesterone levels during the 18 estrous cycle. Until recent years, knowledge of the secretory output of the ovary during the estrous cycle has been entirely indirect and derives chiefly from (a) substitution experiments carried out in a variety of species and (b) assays of urine (43). Recently, an increasing number of reports on the level of progesterone, especially in the ovary and plasma, measured by chemical analysis have been published.
Everett (43) states that it is unwise to regard phenomena such as vaginal cornification, turgescence of vulva and sex skin, and uterine growth as direct quantitative measures of estrogen output of the ovary, since progesterone is known to modify estrogen effects and progesterone is secreted at times other than the luteal phase of the cycle, such as just prior to ovulation. Cupps ejt al. (31) report that estrogens do not appear to be stored within the body and their excretory rate is an excellent index of their secretion. Mellin ejt al.
(78) measured the total urinary estrogen excretion of a cow during the estrous cycle. They observed two peaks, the largest occurred during the last three days of the estrous cycle just prior to ovula tion. Urinary estrogen excretion dropped abruptly following ovulation.
A second peak of less magnitude was reported during days 6 through 11 of the cycle.
Few reports appear in the literature on the quantitative estimates of estrogen in the peripheral blood of mammals. In the ewe,
Norman et_ al. (84) measured estradiol-17f3, estrone and 16-ketoestradiol-
17|3 by fluorimetry during the estrous cycle. Total estrogen level 19
was found to increase from a low value of 0.14 /Lig./lOO ml plasma at
two days following ovulation to a high value of 2,53 /jg./lOO ml plasma
just prior to ovulation. Short e_t al (107) reported that estrone and
estradiol-17f3 concentrations of ovarian vein plasma during estrus were
similar to the values found during mid-cycle.
Using a vaginal tetrazolium reduction assay, Hori ej: al. (61)
recently measured the concentration of free estrogen in the ovarian venous plasma of rats. During metestrus a significant amount of
estrogen, 0.2 ng,/ml. plasma of estradiol equivalent, was measured.
The estrogen level rose sharply in the afternoon of diestrus and continued to rise to a peak before noon of proestrus of 4.5 ng./ml.
About 10 hours prior to ovulation, coinciding with LH release from the pituitary, the estrogen level declined sharply. Barnea and co-workers (8) estimated estrogen levels in the rat by following vaginal smears and uterine weight after ovariectomies performed at different stages of the estrous cycle. They reported variations in the estrogen levels during the estrous cycle similar to those observed by Hori ejt al. (61),
A relatively large number of investigations have been published in recent years on the progestin level in the blood plasma and ovaries of animals during the estrous cycle in which chemical assays have been employed. In the bovine, Gomes and Erb (49) reported that in the majority of studies in which the concentration of progesterone of the corpora lutea was measured, there was an initial rise in concentration associated with the early proliferative stages of the CL (Days 1-4 of the cycle) followed by a relatively constant level until Day 9 or 20
10. The concentration of progesterone then increased again to a peak
on days 14 and 15 followed by a decline to the next estrus. Progestin
(progesterone and 203-hydroxy-& ^-pregnene-3-one) concentration of
the bovine CL reported by Gomes et^ al. (47) demonstrated a pattern
similar to that described for only progesterone. Gomes et al. (47)
also reported a significant correlation between the concentration of
progestins in bovine CL and in ovarian vein plasma. They reported
(47) however, that progesterone levels in jugular vein plasma did not
reflect the stage of the cycle, progestin concentrations in the CL or
progestin levels in the ovarian venous plasma.
More recently, however, investigators (92, 93) have observed
changes in the level of progesterone in the peripheral blood plasma
during the bovine estrous cycle that generally parallel the reported
(47, 49) changes in progestin concentrations in the CL and the ovarian
venous plasma (35). The following levels of progesterone in nanograms
per ml in the peripheral blood of the bovine during the estrous cycle
were reported by Plotka et al. (92): estrus 10.1; 2 days 9,9; 4 days
17.7; 6 days 10.G; 8 days 17.7; 10 days 18.4; 12 days 20.4; 14 days
25.8; 5-6 days before estrus 17.1; 3-4 days before estrus 19.4; and
1-2 days before estrus 13.3.
Variations observed in the peripheral plasma progesterone levels
(111) and the ovarian venous effluent (48) of sows during the estrous
cycle were similar to those discussed for the cow. The peak in the
progesterone level was found to be on days 10 to 12 and 10 to 15, respectively, for the ovarian venous blood (48) and the peripheral
plasma (111). 21
The rat has a very short estrous cycle (four or five days) and
does not have a functional CL although low levels of progesterone have
been reported to be produced by the rat CL (23). Therefore the
progestin secretion rate during the cycle would be expected to be very
low. McDonald e£ al. (76) measured the concentration of progesterone
in the ovarian venous plasma of the rat during the estrous cycle.
From these data the secretory rate of this hormone from the ovary was
calculated. The lowest rate of secretion was during the morning of
proestrus (0,09 jig per ovary per hour) and the highest during the night
of proestrus (4.85 jig per ovary per hour). At estrus, the level had
decreased to 0.2 jig per ovary per hour. A second peak (1.64 jig per
ovary per hour) was reported in late diestrus. Similar results were obtained by Feder ejt al. (44) on the progestin levels in the arterial
plasma of rats during the estrous cycle. They reported the highest
concentration of progesterone (3.11 jig/100 ml plasma) during the pre ovulatory period. Also, they reported a small peak during diestrus.
The concentration of 20a-hydroxypregn-4-en-3-one did not vary signifi cantly during the cycle but the means ranged from 2.13 to 5.08 jig. per
100 ml. of plasma.
Pseudopregnancy effects on hormone levels. Pseudopregnancy involves the prolongation of the secretory function of the corpus luteum of ovulation and therefore a delay in the onset of the next estrus. In rats and mice, which have very short estrous cycles, the
CL becomes functional and secretes progesterone (96) causing the vagina and uteri to undergo progestational changes generally associated with progesterone action. In pseudopregnancy the endocrine balance 22
is very similar to true pregnancy (22, 119), except that there are no
developing young in the uteri. Pseudopregnancy in rats has been
reported (43, 96, 119) to last 12-14 days with the CL undergoing a
decline in function two or three days earlier (42). The termination
of pseudopregnancy is designated by the onset of the next estrus.
Pseudopregnancy may be induced in rats by stimulating the cervix of
the animal in estrus with a glass rod or other mechanical means, by
electrical stimulation, or by mating with a sterile male (119)..
Ovariectomy effects on hormone levels. Bilateral ovariectomy
removes the primary endogenous source of estrogen and progestin in most nonpregnant mammals. The genital tract atrophies and remains
infantile following ovariectomy (37). Progesterone, however, has been found in the blood plasma of ovariectomized mammals (44, 93, 95,
128) with the source of this hormone being the adrenal gland. Feder et al. (44) reported that, in the female rat following castration,
20a-hydroxypregn-4-en-3-one disappeared from the arterial plasma with
in 48 hours while progesterone was detected (0.84 jug. per 100 ml.
plasma) even 25 days after ovariectomy. Following adrenalectomy and ovariectomy no measurable progesterone was detected in the plasma.
It is not surprising that the adrenals are a source of progesterone
since the biosynthetic pathways in the production of steroid hormones in the adrenal cortex are very similar to those of the ovary. In fact, progesterone is an intermediate in the synthesis of gluco corticoids in the adrenal cortex. Stimulation of the adrenals by
ACTH treatment in ovariectomized rats was even found, by Kesko (95), to elevate the concentration of progesterone in the blood plasma. There is also evidence Chat the adrenal cortex is capable of estrogen secretion (40, 77). The adrenal production of estrogen however is thought to normally not be of any physiological significance
(128). So the possibility exists that a low level of ovarian hormone may still exist in the circulation of animals after ovariectomy. It is of interest however that the adrenal glands of rats ovariectomized after puberty decreased in weight (50, 74) rather than increasing as might be expected if the adrenal cortex increased its secretion of ovarian steroids.
Ovarian hormone influence on enzyme levels. . That the ovarian hormones estrogen and progesterone may have an influence on GOT or
GPT activity in animals was indicated by the observation that serum
GOT varied during the estrous cycle of the dairy cow (32, 101).
Pregnancy has been found to influence both of these enzymes (16, 28).
In the rat, liver GPT was reported by Harding (54) to decrease sub stantially during the latter stages of pregnancy. Both estrogen and progesterone were found to inhibit the expected rise in liver GPT activity following glucocorticoid administration. Sheid e£ al. (105) observed no effect of estradiol administration to intact rats on liver
GOT levels, but cortisone injections increased liver GOT activity about one half as much in females as males. This suggests a possible inhibition by estrogen or an enhancement of activity by androgen or a combination of both. Boots (13) reported that 17f3-estradiol injected into ovariectomized rats significantly increased GOT and GPT activity in the plasma, liver and muscle and GOT in the uterus. Awapara (6) 24
found that estrogen administered to intact male animals decreased
alanine aminotransferase and increased aspartate aminotransferase
activity in the ventral prostate. No change in either enzyme was
observed in the seminal vesicle.
Ovarian hormones have also been reported to affect the activity
of enzymes other than the aminotransferases. In a review by Knox
et al. (67) the effects of sex, castration, hormone administration to
both castrated and intact animals, pregnancy, and lactation on several
different enzymes in various tissues are presented. In eight of the
ten enzymes studied, male animals had a higher level of enzyme activity
than female animals in the respective tissues. Castration of the
male animals almost invariably decreased or did not change the level
of the enzymes reported. Such uniform results were not observed
following ovariectomy in female animals. Estrogen administration to
intact or castrate animals, likewise did not produce any consistent
change in enzyme levels. Estrogen treatment reversed the effects of
castration of females in the case of four of these enzymes, pseudo-
cholinesterase of liver and serum, p-glucuronidase of the uterus,
cysteic acid decarboxylase of liver and esterase of the uterus and
vagina. The direction of effects however were not consistent among
enzymes. Ovariectomy decreased, and estrogen increased, the activity
of the first two enzymes while these treatments had the opposite
effect on the last two enzymes. Of the eight enzymes reported, the activity of all enzymes except serum pseudo-cholinesterase increased
during pregnancy and lactation. These increases were found in
specialized tissue functioning during pregnancy and lactation and In 25 other tissues such as liver. Pregnancy and lactation may reflect the
influence of a combination of estrogen and progesterone on enzyme levels.
More recently, Singal at al. (110) studied the hormonal induc tion of phosphofructokinase in the rat uterus. Estradiol-173 induced this enzyme and while progesterone itself had no significant effect, it inhibited the observed Increase in enzyme activity in estradiol- treated rats. Eckstein and Villee (36) observed the effects of' estradiol-173 on the activities of enzymes of the Krebs cycle, enzymes of glycolysis and the dehydrogenases of the pentose cycle in the uterus of the ovariectomized rat. A high dose of estradiol decreased the activity of aconitase and of isocitric dehydrogenase while it increased the .levels of the dehydrogenases of the pentose cycle.
Ovariectomy itself did not affect the activity of any of the enzymes except to decrease glucose-6-phosphate dehydrogenase. Ovariectomy of the mouse was found by Suzuki £t al. (115) to decrease the activity of carbonic anhydrase in the uterus. Estradiol in the normal mouse increased the uterine weight and enzyme activity while progesterone produced the opposite response in both cases. Suzuki et al. (116) further reported no remarkable change in the level of this enzyme in the liver, kidney, pancreas or blood of the mouse between estrus and diestrus. In the intact mouse estradiol decreased and progesterone
Increased the carbonic anhydrase activity of the liver. At 24 days after surgery, ovariectomy had caused an increase in liver enzyme levels. Estradiol, given to the ovariectomized mouse, decreased the enzyme activity in the liver and increased it in the blood. Barker 26 and Ludwick (7) reported that ovariectomy reduced the activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and NADP+-malic dehydrogenase in the rat mammary gland during lacta tion. Estradiol increased the total activity of all three enzymes in the mammary glands of ovariectomized rats in their tenth day of lactation. Progesterone also increased the activity of all three enzymes but generally not to the degree that was observed for estradiol.
It is somewhat evident from the materials reviewed, that signifi cant relationships exist between hormones and enzymes. These relation ships are currently understood only to a small degree for certain tissues of certain species. MATERIALS AND METHODS
A total of 164 female rats of the Wistar strain were used to provide 656 tissues on which 1312 enzyme determinations were made.
Both aspartate and alanine aminotransferase activities were determined on the plasma, liver, gastrocnemius muscle and uterus of each rat.
All rats were housed in hanging wire cages in a small animal room maintained at 26 _+ 2°C. They had free access to tap water and purina Lab Chow at all times. The lighting system was set to main tain a daily schedule of 12 hours of light and 12 hours of dark.
Tissue Collection and Processing
Blood samples were collected from ether anesthetized rats by decapitation into a 50 ml beaker containing 1,0 ml of 5% sodium citrate in physiological saline. Samples were centrifuged immediately at 5,900 x g for 45 minutes in a Servall table-top centrifuge main tained at 3°C. The plasma was decanted, stored at 3°C, and assayed within 24 hours.
Liver, gastrocnemius muscles and uterus were excised immediately following decapitation and placed in a 0,1 M potassium phosphate buffer (pH 7.4) in an ice bath. Whole liver and uterus were blotted with tissue and weighed. The large posterior lobe of the liver was excised, wrapped in aluminum foil and frozen at -25°C until enzyme analysis could be determined. Both gastrocnemius muscles and whole uteri were stored in the same way.
27 28
Before enzyme analyses were determined, the liver and gastro
cnemius muscles were allowed to thaw at 3°C. A center cross section
from the lobe of the liver was removed, blotted with tissue and weighed to the nearest mg. This tissue, averaging approximately
700 mg., was used in the enzyme determinations. An adjacent cross
section of nearly equal size was removed and used to determine the
percent dry matter of the liver.
The right gastrocnemius muscle was blotted with tissue and weighed to the nearest mg. before use in the enzyme determinations.
The left gastrocnemius muscle was used to determine the percent dry matter of the muscle.
The uterus was taken from the -25°C cold room and placed in the top of a MVE Model A-8000 liquid nitrogen semen storage tank for 15 minutes. It was then removed and immediately crushed with a hammer
to facilitate the homogenization.
Liver and gastrocnemius muscle samples were homogenized in 5 volumes and uterus samples in 10 volumes of 0.1 M potassium phosphate buffer (pH 7.4) with a glass tube and a motor-driven teflon pestle.
The crude homogenate was centrifuged in a Servall Superspeed RC 2-B refrigerated centrifuge at 30,000 x g for 30 minutes. The supernatant was decanted and subsequent dilutions were made for enzyme analysis.
All enzyme analyses were made within 24 hours after the samples were thawed.
Enzyme Determination
The GOT and GPT determinations were made by the method described 29
by Karmen ( 63 ) in 1954 using NADH. The reagents used were obtained
from university laboratory stores or Calbiochem of Los Angeles,
California. These reagents were used to make up the Boehringer kits
for GOT and GPT determinations that are distributed by Calbiochem (12).
The GOT determination is a coupled reaction based on the oxida
tion of NADH as follows: GOT aspartic + a-Ketoglutaric Oxaloacetic + Glutamic acid acid ' acid acid
Oxaloacetate + NADH + H+ Malate + NAD
The procedure used in determining GOT activity is presented below:
1. A blank containing 2.5 ml of 0.042 M aspartate in 0.1 M
potassium phosphate buffer plus 0.5 ml of plasma or tissue
homogonate was used with each sample assayed.
2. The following reagents were successively pipetted into
the 4 ml sample cell:
a. 2.3 ml aspartic/phosphate buffer
b. 0.05 ml malic dehydrogenase (MDH)
(388 IU per ml of 2.8 M ammonium sulfate)
c. 0.5 ml plasma or tissue homogenate
3. The solutions in the cell were mixed with a plastic spatula.
The sample cell was placed in the cell holder of the 'Spectro-
photometer and zeroed against the blank.
4. A volume of 0.05 ml of NADH (0.012 M in 1% sodium bicarbonate)
was added to the solutions in the cell.
5. The reaction was started by adding 0.1 ml of Qf-Ketoglutarate 30
(0.16 M)
6, The reaction was allowed to proceed for at least 3 minutes.
The GPT determination is very similar to the one for GOT,
utilizing a coupled reaction based on the oxidation of NADH as presented
below: GPT alanine + Oi-Ketoglutaric Pyruvic + Glutamic acid acid acid LDH Pyruvate + NADH + H+ Lactate + NAD*
The procedure used in determining GPT is very similar to the
preceding one for GOT. The only differences are as follows:
1. A blank containing 2,0 ml of 0.11 M alanine in the
phosphate buffer with 1.0 ml plasma or tissue homogenate
was used.
2. A volume of 1.8 ml of alanine/phosphate buffer is used
instead of the 2,3 ml of aspartic/phosphate buffer.
3. Lactic dehydrogenase (302 IU per ml of 2.2 M ammonium
sulfate) replaces the malic dehydrogenase.
4. A 1.0 ml volume of plasma or tissue homogenate is used
rather than 0.5 ml.
A Beckman D-B spectrophotometer with a Seargent recorder attached was used in all determinations of the above reactions. The samples were maintained at a constant temperature of 25°C. in the cell holder
throughout the assay. The decrease in absorbance at 340 MU per unit
time was plotted as a linear regression by the recorder. From this,
the change in absorbance per minute was calculated and multiplied by
either 2000 or 1000 to obtain the units of GOT and GPT activity, respectively. This figure represents the unins of enzyme activity per 31
ml. plasma or tissue homogenate. This unit of enzyme activity is
commonly referred to as the Karmen unit (70) and was originally
defined as that amount of enzyme which at 340 Mji will cause a change
in absorbance of 0.001 per ml. serum per minute at 25°C in a 3 ml.
assay volume.
Experiments
Experiment 1. Forty rats averaging 217 grams were used to
establish the enzyme level in the plasma and 3 tissues of the normal
cycling and the pseudopregnant rat. At least 6 rats were sacrificed
in each of the 4 stages of the estrous cycle and pseudopregnancy.
The one group was made pseudopregnant by stimulation of the vagina with a glass rod. They were pseudopregnant between 9 and 15 days when sacrificed. Stage of the cycle and pseudopregnancy were determined by taking daily vaginal smears and microscopically determining the cell
types (130). An attempt was made to sacrifice an equal number of animals from each group on a given day. All rats were sacrificed in
the early afternoon.
Experiment 2. Sixty-four rats averaging 240 grams were either ovariectomized or sham-operated to determine the effects of removing the primary endogenous source of estrogen and progesterone on the pla sma and tissue aminotransferase levels. Ovariectomy was performed by the dorsal approach (130) with ether anesthesia. Four each of the ovariectomized and sham-operated rats were sacrificed on 1, 2, 3, 5,
8, 12, 16 and 21 days following surgery. Data from one ovariectomized rat sacrificed on day 16 and one sacrificed on day 21 were not used 32
because of evidence of ovarian tissue remaining. All rats in this
experiment were sacrificed in the early afternoon.
Experiment 3. Sixty rats averaging 276 grams were used to.study
the effects of replacement hormone therapy on the enzyme activities of
the ovariectomized rat. Ovariectomy was by the dorsal approach at * least 19 days before use. The experimental rats were given one of the
following injections subcutaneously on a per 100 gram of body weight
basis; 5.0 fjg, 17 g-estradiol in 0.1 ml. of corn oil, 1.0 mg. proges
terone in 0.1 ml. corn oil or 0.1 ml. corn oil. Four rats from each
treatment group were killed 0.5, 1, 2, 3, and 5 days after injection.
All animals were killed at 4 P.M. The 17 (3-estradiol and the proges
terone were obtained from the Sigma Chemical Company.
Statistical Analysis
The first experiment was analysed utilizing the least-squares analysis of variance method (45). The least-squares computer program described by Harvey (55) was used in the analysis of experiments 2 and 3. RESULTS AND DISCUSSION
EXPERIMENT 1
In Experiment 1, the levels of GOT and GPT in the liver,
gastrocnemius muscle, uterus and plasma of the rat in the four stages
of the estrous cycle and pseudopregnancy were measured. The means
and standard errors of these enzymes for the liver, muscle and uterus
are presented in Table 1. Each mean represents nine samples except
for metestrus and pseudopregnancy means which represent 7 and 6
samples, respectively. Plasma enzyme levels are not included in this
table because lysis of blood was a problem with some samples at the
time of collection.
GOT activity was highest in liver tissue and slightly lower in muscle, GPT level was also highest in the liver with muscle enzyme
activity being only about 1/4 the level found in the liver. Zimmerman
et al. (133) also reported higher aminotransferase activities in liver
than muscle with muscle GPT levels being relatively low. GOT levels were higher than GPT in all three tissues studied.
Generally there was little variation in the enzyme levels in
the tissues during the various stages of the estrous cycle and pseudo pregnancy. Least-squares analysis of variance indicated that the only values which varied significantly were GOT activity per mg. of dry weight of liver (P<0.01), GOT activity per mg. of wet weight of uterus
33 34
(P<0.01) and GPT activity per whole uterus (P<0.05). When only the
four stages of the estrous cycle were considered, GPT activity per uterus varied significantly at the 1% level.
In the liver, only GOT activity showed significant variation in the reproductive stages studied, but both GOT and GPT activities were at their lowest level during estrus. There is indirect evidence (97,
105) to indicate that estrogen and progesterone may inhibit amino transferase activity in the liver of rats. Recent reports (8, 61) indicate that estrogen is secreted from the ovary in increasing amounts starting at diestrus and peaking at proestrus. The highest peak of progesterone release has also been found to be during proestrus (44).
These hormones may be having an influence on the GOT and GPT activities of the liver the day following their peak level in the blood. This does not explain, however, the relatively high values observed for these enzymes during pseudopregnancy, when progesterone levels in the blood would be much higher than at any time during the estrous cycle,
GOT activity per mg. wet weight of uterus varied during the estrous cycle and pseudopregnancy while GPT levels were relatively constant. It is of interest that the enzyme activities for whole uterine tissue varied significantly for GPT but not GOT. The uterus apparently increased in weight enough at the time of estrus to cancel the significant GOT per mg. wet tissue variation observed, which was highest during pseudopregnancy and diestrus. Also, this increased uterine weight around estrus apparently caused the GPT activity, which was relatively stable on a per mg. tissue basis, to vary significantly throughout the cycle on a whole tissue basis. These data do not 35
support the findings of Hochman £t al, (59) who found in women, an
increase in the specific activity of the uterine endometrium during
the proliferative phase of the menstrual cycle,
EXPERIMENT 2
The overall means and standard deviations for the variables
studied are presented in Table 14. Each mean represents 62 values
except for the plasma means which contain 51 samples. Samples within
each mean, except the plasma means, included four samples from ovari ectomized and four from sham-operated rats on Days 1, 2, 3, 5, 8, 12,
16, and 21 after surgery. On Days 16 and 21 the ovariectomized groups contained three samples instead of four. The only differences in the distribution of plasma samples were that on Day 1 no samples are represented and on Day 3 the ovariectomized and sham-operated groups contain two and three samples, respectively.
The results of this experiment were analyzed by the least-squares analysis of variance method to determine the effects of ovariectomy on the enzyme activities in the various tissues. The overall effects of ovariectomy adjusted for time effects as well as the effects of ovariectomy within each time period on enzyme levels were computed.
The effects of time following surgery were also considered. The overall effects of ovariectomy, adjusted for time, on the GOT and GPT activity in the plasma and tissues studied are presented in Table 2.
The only significant variation in enzyme levels between sham-operated
(control) and ovariectomized rats was in the GOT and GPT activities 36
per whole uterus. The uterine enzyme values were only about one half
as high in ovariectomized compared to sham-operated rats. Ovariectomy
did not affect liver weight significantly but it did decrease uterine
weight (Table 3). Body weight of ovariectomized rats was significantly
greater (P<0,05) than the controls (Table 3).
Plasma enzyme levels were not significantly influenced by ovari
ectomy as previously mentioned nor was there any significant inter-
t action between the effects of time following surgery with ovariectomy
(Table 15). Wien the effects of ovariectomy within each day of
sacrifice were considered, no significant variations were observed.
The effect of days following surgery adjusted for surgical treatment
effects was significant for plasma GOT (P<0.01) and GPT (P<0.05)
activity. Significant linear and quadratic relationships for both
enzymes, and cubic relationships for GOT, were observed over time.
The least squares-means for these enzymes were generally higher for
Days 2 and 3 following surgery than for later days (Table 16).
No significant enzyme variations were noted in the liver due to ovariectomy or due to an interaction of ovariectomy and time following
surgery (Table 17). Liver weight showed a significant (P<0.05) time and surgical treatment interaction and an effect of ovariectomy within
Day 5, 16 and 21 (P<0,05). No consistent pattern of change in liver weight was observed however (Table 18). Days following surgery significantly influenced the GOT and GPT activity per mg. dry weight of liver as indicated by the F values in Table 17. Significant linear and quadratic time effects were noted. The liver aminotransferase activities declined in the later days following surgery (Table 19). 37
Similar results were found for both enzymes per mg. wet weight. The
pattern of change in enzyme activity for the whole liver was similar
to that activity expressed on a mg. basis but only the effect of time
on GOT activity was significant.
Muscle GOT activity per mg. wet weight of tissue varied signifi
cantly (P<0.05) due to the interaction between time following surgery
and the type of surgery (Table 20). On the basis of ovariectomy effects within time, GOT per mg. wet weight and per mg. dry weight of muscle
varied at the 5% level of significance within Days 8 and 12 following
surgery. The direction of variation was not consistent. Ovariectomy
increased the GOT level on Day 8 and decreased it on Day 12 (Table 21).
The overall effect of time adjusted for surgical treatment effects was highly significant for GOT and GPT activity on a per mg. wet and dry
tissue basis (Table 20). GOT levels varied in a linear fashion (P<0.05).
The highest level of enzyme activity in the muscle was on Day 2 follow ing surgery as indicated by the least-squares means in Table 22. The significant variation in plasma, liver and muscle enzyme levels due to time following surgery adjusted for surgical treatment effects may be associated with surgical stress. Large daily variation may also have contributed to these changes. Both GOT and GPT activity in plasma, liver and muscle peaked on the second day following surgery. Several investigators (98, 102, 103) have found that glucocorticoids increase liver GPT levels while there is conflicting reports (98, 105) on whether liver GOT activity is enhanced or not by glucocorticoid action.
Increased glucocorticoid levels in the blood following surgery may be 38
responsible for the observed rise in liver enzyme levels. This may
have also been the cause of the increase in muscle aminotransferase
at Day 2. The higher concentration of enzymes in the tissues would
then result in increased release of enzymes into the plasma.
The changes in enzyme activity per mg. of wet uterine tissue
for ovariectomized and sham-operated rats in the sampling days are presented in Table 4. The only significant variation observed was within Day 1 for GOT (P<0.01) and GPT (P<0.05) activity (Table 23).
The reason ovariectomy would significantly decrease enzyme activity one day following ovariectomy and not in later days is unexplained.
Dramatic changes occurred in the aminotransferase activity per whole uterus. The least-squares means in Table 2 indicate that the average effect of ovariectomy from one to 21 days reduced both amino
transferase activities of uterine tissue by about one half. There was a very highly significant (P<0.01) time and surgical treatment interaction effect on both uterine enzyme levels. Ovariectomy affected the GOT and GPT activity per uterus at the 5% level within
Day 5, and GPT within Day 3. Ovariectomy had a very highly signifi cant effect (P<0.01) within all other days (Table 23), The changes in GOT and GPT activity per uterus due to ovariectomy over a 21 day time period are indicated by the subclass means presented in Table 5.
Values for ovariectomized animals are less than those for the sham- operated rats on any given day. After Day 2, the enzyme activity for ovariectomized animals decreases with increasing number of days after surgery, thereby increasing the difference between the surgical treat- 39
ment groups in the later days.
The fact that ovariectomy had very little effect on amino
transferase activity per mg, of uterus and a large effect in terms of
enzyme activity per whole uterus would indicate that tissue weight is
changing significantly due to ovariectomy. Tissue weight was found
to decrease following ovariectomy (Table 6). Uterine weight in
castrated rats was lower (P< 0.01) than controls within each day
studied following surgery except for Day 1. This decrease in uterine weight was expected since ovariectomy is known to result in a drastic
decline in the weight of this tissue (128).
The effects of ovariectomy on uterine weight and GOT activity
over the 21 day period is illustrated in Figure 1, This graph is in
terms of percent of control, the 100% line representing the values of the sham-operated rats. These percentages were calculated using
the least-squares means of the respective subclasses. As this graph indicates, GOT activity per mg. of uterus does not vary greatly from the control line. The aminotransferase activity per whole uterus follows very closely the changes in the uterine weight. Both per centages drop rather dramatically from Day 5 to Day 8 following ovari ectomy and then continue to decline slowly through Day 21. Both uterine weight and GOT per uterus are about 40% of the level of the sham-operated rats at Day 21. The enzyme activity for the whole uterus is dependent upon the activity per mg. of tissue and the uterine weight. This graph shows very vividly that uterine weight changes following ovariectomy had most of the influence on the GOT activity of the uterus. 40
The changes in GPT activity of uterine tissue and uterine weight changes following ovariectomy compared with changes in sham-
operated animals are illustrated in Figure 2. This graph is in terms of percent of control (sham-operated) values as explained for Figure 1.
The changes observed are very similar to the changes in GOT activity
(Figure 1) indicating that the primary effect on GPT activity per uterus following ovariectomy was the change in uterine weight.
EXPERIMENT 3
The means and standard deviations of the variables studied in the hormone treatment experiment are presented in Table 24. Enzyme determinations were made on all tissue and plasma samples from 60 rats except for one muscle sample and one plasma sample that were not analyzed due to technical difficulty in the laboratory. The distri bution of experimental animals included four each of estrogen, proges terone and corn oil treated rats sacrificed 12 hours, 1, 2, 3, and 5 days following treatment. The exceptions are that one plasma sample and one muscle sample are missing from Day 2 estrogen and corn oil treated groups, respectively.
To determine if estrogen or progesterone treatment influenced the level of GOT and GPT in the three tissues and plasma of the ovari- ectomized rat, the results of this study were analyzed by the least- squares analysis of variance method. Least-squares means were computed for all of the subclasses. The overall effects of hormone treatment adjusted for time after hormone treatment were computed and 41
the interaction of hormone treatment with time after treatment effects
were considered. The effects of estrogen or progesterone within each
time period were determined as well as the effects of time adjusted
for hormone treatment effects.
The overall effects of hormone treatment adjusted for time
after treatment for the independent variables in this study are
indicated by the least-squares means in Table 7. Plasma enzyme levels
were not significantly influenced by hormone treatment (Table 25).
Liver GOT per mg. wet weight (P<0.01) and per mg. dry weight (P<0.05)
were decreased by 17p-estradiol and progesterone (Table 26). Hormone
treatment significantly influenced muscle GPT per mg. wet weight
(Table 27), with estrogen treated animals higher and progesterone
treated animals lower than controls (corn-oil treated). GOT and GPT
in the uterus per mg. wet tissue and per whole tissue were influenced
significantly by hormone treatment (Table 28). The least-squares
means in Table 7 indicate that both estrogen and progesterone increased
the enzyme activity per mg. of uterus slightly, estrogen causing the
greatest increase in activity. Progesterone increased the amino
transferase levels per uterus only slightly while estrogen increased both enzyme activities of this tissue dramatically.
Plasma GOT and GPT levels show a highly significant (P<0,01)
time effect when adjusted for hormone treatment influences (Table 25).
The least-squares means for plasma GOT and GPT activities (Table 29) are fairly constant over the five time periods except for high values of GOT on Day 1 and GPT on Day 0.5. No significant overall hormone treatment effect on plasma enzymes was observed. Estrogen or proges- 42
terone treatment within sample days also was not significant (Table 25).
The overall effects of hormone treatment adjusted for time effects on liver GOT and GPT levels are indicated by the least-squares means in Table 7. The pattern of response of both enzymes was similar although only GOT per mg. of wet (P<0.01) and dry (P<0.05) tissue were affected significantly (Table 26). Progesterone decreased the enzyme activities per mg. liver while estrogen caused an even greater decrease. The effect of estrogen and progesterone on liver weight, although not significant, was opposite to their effects on enzyme activity (Table 30). Therefore, the decrease in aminotransferase activity per liver due to estrogen and progesterone was not as great as that reported on a per mg. tissue basis.
A significant interaction of hormone treatment and time was observed for GOT per mg. liver (P^ 0.05) and GPT whole liver (P<0.05).
On a within day basis, GOT and GPT activities per mg. wet (Table 8) and dry (Table 9) tissue were decreased significantly (P<0.01) on
Day 2 due to estrogen treatment. Also on Day 2 following hormone treatment, progesterone significantly decreased GOT (P<0.01) and
GPT (P<0.05) activity but not to the degree that estrogen did
(Tables 8, 9). Comparing the enzyme levels on the various days following hormone administration within treatment group, GOT and GPT levels were low on Day 2 following estrogen treatment (Tables 8, 9).
No definite pattern of change in enzyme activity is noted following progesterone treatment. It is interesting that within the control group (corn oil treated) GOT and GPT activity per mg. liver peaked on
Day 2 (Tables 8, 9) which increased the differences in enzyme 43
activities between control and hormone treated animals. Whether the
corn oil itself caused this increase in liver enzyme activity is not
known. The decrease in aminotransferase activity due to estrogen is
not supported by work of Boots (13). He observed that estrogen caused
an increase in liver GOT and GPT activities as well as an increase
in the aminotransferase activities in plasma and muscle. The different
experimental designs of the two studies may have contributed to the
differences in the results. Boots (13) did not have control animals at each time period following treatment. His control animals were
sacrificed at the time of treatment. Some of the variation he observed may have been daily fluctuation due to factors other than hormone treatment. Rosen et al. (97) however, also reported a rise in liver
GPT activity following estrogen administration. But they found that estrogen or progesterone would inhibit the increase in liver GPT which normally occurs from cortisol administration. The results from
Experiment 1, of this study, indicate that estrogen and progesterone may cause a decrease in liver GOT activity since a low level was found at estrus during the reproductive cycle of the rat. In Experiment 2 however, no consistent change in GOT or GPT activity of the liver was observed following ovariectomy. Sheid ^ al. (105) observed that estrogen had no effect on liver GOT but cortisol administered to female rats increased liver GOT activity only one half as much as it did in male rats. The role of hormones in the control of tissue amino transferase levels appears to be far from being resolved.
The aminotransferase activities per whole liver were not signifi cantly influenced by estrogen or progesterone on Day 2 following 44 hormone treatment (Table 10), because of the significant (P<0.01) increase in liver weight on this day due to estrogen and progesterone treatment (Table 31). Liver weight was significantly lower (P<0.05), due to estrogen treatment, on Day 5 (Table 31) resulting in GOT
(P<0.01) and GPT (P< 0.05) activities per whole liver being lower than controls. GPT activity per liver tissue was increased (P< 0.05) over controls on Day 0.5 (Table 10). This was due to an Increase which was not significant, in both liver weight (Table 30) and enzyme activity per mg. tissue (Table 8) on Day 0.5.
The effects of time following hormone treatment adjusted for treatment effects were significant for GOT activity per mg, dry weight of liver (P<0.05), tissue weight (P<0.01), and GOT and GPT activities per whole liver (P^O.Ol), No pattern of change in the least-squares means of any of these variables over time following treatment was evident (Table 32).
GPT on a per mg, of wet tissue basis was the only muscle enzyme significantly (P<"0,05) influenced by hormone treatment (Table 27).
The general effect of hormone treatment on aminotransferase activities of the muscle was for progesterone to decrease and estrogen to slightly increase the levels (Table 7). No within day affects of estrogen or progesterone were observed. Both GOT and GPT in terms of per mg, wet weight and per mg. dry weight varied significantly over the days following hormone treatment (Table 27). The least-squares means
(Table 33) indicate that muscle GPT levels rose slowly to a peak at
Day 3 while GOT started relatively high on Day 0.5 and then declined before reaching a peak on Day 3. 45
Hormone treatment effects adjusted for time effects signifi
cantly influenced GOT and GPT activity per mg. uterus and per whole uterus (Tables 7, 28). The overall interaction of hormone treatment and time was significant (P<0.01) for all of the uterine enzyme variables (Table 28). The least-squares means in Table 11 indicate that estrogen significantly increased GOT activity per mg. uterus within Day 1 (P<0.05), Day 2 (P< 0.01) and Day 3 (P< 0.01) and GPT activity within Days 2 and 3 (P<0.01). Both aminotransferase levels reached a peak on Day 3 following estrogen treatment. These results agree only partially with those of Boots (13), He observed an Increase in GOT activity but not GPT following estrogen administration. Proges terone caused a slight, but significant (P< 0.05) increase in GOT activity per mg. uterus on Days 2 and 3 following hormone treatment
(Table 11), with the level of activity being the same on both days.
Estrogen dramatically increased uterine weight over controls on each of the 5 sampling days (P<0.01) while progesterone had no significant influence on uterine weight (Table 12). This increase in uterine weight due to estrogen resulted in a great increase in GOT and GPT activities per uterus (Table 13). Both enzymes activities were increased significantly (P<0.01) on Days 1, 2, and 3 and GOT activity was also increased on Day 5.
The Figure 3 illustrates the relative changes in uterine weight and GOT activity following estrogen treatment. This graph is in terms of percent of control as previously discussed for Figure 1 in Experi ment 2. The 100% line on this graph represents the values for the corn-oil treated animals. GOT activity per mg. of uterus peaks at Day 3 following estrogen treatment at 144% of control values and then
declines down to almost control levels on the fifth day. Uterine weight increases rather markedly up to Day 3 (195% of control) and
then declines rather slowly to Day 5 (147% of control). The changes
in GOT per uterus are a function of both enzyme activity (per unit
tissue weight) and tissue weight. Therefore uterine GOT activity
rises sharply to 263% of control values on Day 2, and then declines
rapidly after Day 3 to 147% of controls at Day 5. The major cause of
the increase in GOT per uterus is the change in uterine weight. This
increase in uterine weight of ovariectomized rats following 17p-
estradiol administration is well documented. Many researchers are
using uteri in ovariectomized rats to study the mechanism of action
of estrogen because of the dynamic changes that occur in this tissue
following estrogen administration.
The relative changes in GPT activities and uterine weight due
to estrogen treatment are illustrated in Figure 4. This graph is
like the previous one, and is in terms of percent of control values
(corn-oil treated animals). The changes illustrated in this graph
for GPT activity are very similar to those observed for GOT levels
in Figure 3. In fact, the GPT activity per mg. of uterus peaks at
263% of controls on Day 2, exactly the same as GOT activity.
The effects of progesterone on uterine weight and GOT and GPT
levels in the uterus are illustrated in Figures 5 and 6. These graphs are also in terms of percent of control values. Uterine weight
changes only slightly over the five day period, increasing slightly but not significantly by Day 5. Both aminotransferase activities per whole uterus followed the changes in their respective enzyme levels per mg. tissue. GOT activity as a percent of control activity was high'on Days 2 and 3 following progesterone treatment, (Figure 5),
GOT per mg. tissue being significantly higher than controls on these days. GPT activity of uterine tissue showed only small variations following progesterone treatment but peaked on Day 3 and declined to below control values on Day 5 (Figure 6).
Highly significant effects of time adjusted for treatment effects were observed on uterine aminotransferase activities (Table 28). GOT and GPT activities per mg. of wet uterine tissue increased to a peak at Day 3 as shown by the least-squares means in Table 34. The amino transferase levels for the whole uterus increased until Day 2 follow ing treatment, and then declined on Days 3 and 5 (Table 34). The results of this study do not offer any possible explanation for this time affect. Daily variations in enzyme activity and the possible stimulation by the hormone carrier (corn oil) may have contributed to these enzyme changes. 48
Table 1. Means and Standard Errors of GOT and GPT Activities of 3 Tissues in the Rat During the 4 Stages of the Estrous Cycle and Pseudopregnancy.
Karmen Units Proestrus Estrus Metestrus Diestrus Pseudo pregnant
GOT/mg. Dry Wt, Liver** 320.2 279.0 326.7 298.4 321.7 S.E. 8.6 5.7 12.4 9.6 12.0
GPT/mg. Dry Wt, Liver 156.3 139.2 171.8 148.3 177.2 S.E. 7.5 4.6 10.9 10.6 17.0
GOT/mg. Dry Wt. Muscle 245.8 240.9 271.3 244.2 203.0 S.E. 16.0 11.0 19.6 22.5 7.3
GPT/mg. Dry Wt. Muscle 37.6 31.5 36.0 32.4 28.8 S.E. 4.5 1.2 3.5 3.0 1.7
GOT/mg. Wet Wt. Uterus** 3.4 3.8 3.6 4.0 4.2 S.E. 0.3 0.3 0.4 0.3 0.2
GPT/mg. Wet Wt. Uterus 1.1 1.2 1.1 1.2 1.2 S.E. 0.1 0.1 0.1 0.1 0.1
GOT/Uterus 2852.0 3090.0 2417.0 2605.0 2622.0 S.E. 219.0 163.0 276.0 181.6 234.0
GPT/Uterus* 880.0 1021.0 732.0 749.0 779.0 S.E. 56.0 51.0 61.9 60.3 109.0
^Significant differences among the 5 means at the 5% level.
**Significant differences among the 5 means at the 1% level. Table 2. GOT and GPT Least-Squares Means and Standard Errors for Ovariectomized and Sham-operated Rats Adjusted for Time Effects (1 to 21 Days).
GOTGPT Independent Variable Ovar-X S.E. Sham-op. S.E. Ovar-X. S.E. Sham-op. S.E.
Plasma E. Units/ml. 85.1 4.4 82.9 4.1 22.7 1.1 23.1 1.0
Liver Units/mg. Wet Wt. 39.4 1.0 39.5 1.0 22.1 0.7 22.8 0.7 Units/mg. Dry Wt. 139.7 3.8 137.1 3.7 78.2 2.5 78.9 2.4 Units/Whole Tissue 383.9 11.1 375.2 10.6 215.4 7.2 216.6 7.0 (10-3)
Muscle Units/mg. Wet Wt. 46.7 0.9 46.8 0.9 5.2 0.1 5.3 0.1 Units/mg. Dry Wt. 189.9 3.6 190.2 3.4 21.0 0.5 21.5 0.4
Uterus Units/mg, Wet Wt. 7.3 0.1 7.7 0.1 1.6 0.1 1.7 0.1 Units/Whole Tissue 1427.6 63.5 2745.6 61.0 313.8 17.0 619.0 16.2 50
Table 3. Least-Squares Means and Standard Errors of Tissue and Body Weight for Ovariectomized and Sham-operated Rats Adjusted for Time Effects (1 to 21 Days).
Ovar-X. S.E. Sham-op. S.E.
Liver Wt. (mg.) « 9853 194 9606 187
Uterine Wt. (mg.)** 194 8 356 8
Body Wt. (g)* 246 4 235 4
^Significant at the 5% level. **Significant at the 1% level.
Table 4. Subclass Means and Standard Errors for GOT and GPT Activities per mg. of Wet Weight of Uterine Tissue Determined from 1 to 21 Days Following Surgery in Ovariectomized and Sham-operated Rats.
GOT3 GPTb Days Following ------Surgery Ovar-X. Sham-op. Ovar-X. Sham-op.
1 6.4 7.9 1.5 2.0 2 8.5 7.8 1.8 1.6 3 7.6 7.0 1.7 1.6 5 7.5 6.9 1.4 1.4 8 7.8 8.1 1.7 1.8 12 7.0 8.1 1.6 1.8 16 6.8 7.9 1.7 2.0 21 7.1 7.9 1.8 1.7
aStandard error of all means was 0.4,
^Standard error of means of ovariectomized animals on Days 16 and 21 was 0.2; all others were 0.1. 51
Table 5. Subclass Means and Standard Errors for GOT and GPT Activities of Whole Uterine Tissue Determined from 1 to 21 Days Following Surgery in Ovariectomized and Sham-operated Rats.
GOT3 GPTb Days Following Surgery Ovar-X. Sham-op. Ovar-X, Sham-op.
1 1698 2422 393 603 2 2008 3025 424 601 3 1658 2345 360 532 5 1680 2278 317 464 8 1413 3105 311 693 12 1093 3030 244 682 16 943 2840 235 723 21 930 2920 227 652
aStandard error of means of ovariectomized animals on Days 16 and 21 was 199; all others were 173.
^Standard error of means of ovariectomized animals on Days 16 and 21 was 53; all others were 46. 52
Table 6, Subclass Means and Standard Errors for Whole Uterine Weight (mg.) for Ovariectomized and Sham-operated Rats from 1 to 21 Days Following Surgery.
Uterine Weight (mg.)a
Surgery Ovar-X. Sham-op.
1 273 306
2** 236 386
3** 218 333
5** 223 335
8** 180 384
12** 155 378
16** 139 358
21** 130 373
Uterine weight means were significantly different at the 1% level.
aStandard error of means of ovariectomized animals on Days 16 and 21 was 26; all other means were 23. % of Control O U 100 70 80 SO 30 Figure 1. Effects of Ovariectomy on Uterine Weight and GOT and Weight Uterine on Ovariectomy of Effects 1. Figure ciiy b g o trsadb hl Uterus) Whole by and Uterus of (by mg. Activity in the Rat. the in Dy fe Surgery) (DaysAfter 10 12 418 14 20
n. nt/g Uterus Units/mg. Enz. n. Units/Uterus Enz. Wt. Uterine 22 Enz. Units/rag. Uterus
ui.cix(ic iyc* Enz. Units/Uterus
2 4 6 8 10 12 14 16 18 20 22
(Days After Surgery)
Figure 2. Effects of Ovariectomy on Uterine Weight and GPT Activity (by mg, of Uterus and by Whole Uterus) in the Rat. Table 7. GOT and GPT Least-Squares Means and Standard Errors for Hormone Treated Rats Adjusted for Time Effects (12 Hours to 5 Days).
______GOT______.______GPT______
Independent Estrogen Progesterone Corn Oil Estrogen Progesterone Corn Oil Variable Treated S.E. Treated S.E. (Control) S.E. Treated S.E. Treated S.E. (Control) S.E. Plasma Units/ml. 94.7 4.0 93.6 3.8 94.7 3.8 22.4 0.7 22.5 0.7 23.7 0.7
Liver Units/mg. Wet Wt. 57.1 1.8 59.3 1.8 65.5 1.8 22.8 1.2 24.2 1.2 26.6 1.2
Units/mg. Dry Wt. 198.3 5.7 206.2 5.7 221.2 5.7 79.2 3.7 83.9 3.7 89.7 3.7
Units/Whole Tissue(10-3) 613.5 1 6.7 611.7 16.7 633.2 16.7 246.0 8.4 248.7 8.4 254.9 8.4
Muscle Units/mg. Wet Wt. 56.8 1.2 53.3 1.2 56.5 1.2 5.2 0.1 4.8 0.1 5.0 0.1
Units/mg. Dry Wt. 238.8 5.1 225.3 5.1 238.0 5.3 21.8 0.5 20.2 0.5 21.2 0.5
Uterus Units/mg. Wet Wt. 8.8 0.2 8.2 0.2 7.4 0.2 1.8 0.1 1.7 0.1 1.6 0.1
Units/Whole Tissue 1654.5 40.6 982.0 40.6 885.5 40.6 342.6 9.5 203.7 9.5 192.2 9.5 Table 8. Subclass Means and Standard Errors for GOT and GPT Activities per mg. Wet Weight of Liver Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats.
GOT3 GPTb Days Following Estrogen Progesterone Corn Oil Estrogen Progesterone Corn Oil Treatment Treated Treated (Control) Treated Treated (Control)
0.5 60.8 62.7 68.6 27.0 25.4 25.9
1 56.4 54.6 60.4 19.8 19.0 20.9
2 48.3** 57.1** 79.5 19.9** 25.2* 34.5
3 58.0 60.7 57.7 24.7 27.1 25.8
5 61.6 61.4 61.3 22.7 24.3 26.0
Significantly different from corn oil treatment (control) at the 5% level.
Significantly different from corn oil treatment (control) at the 1% level.
aStandard error for all GOT means was 4.0.
^Standard error for all GPT means was 2.7.
Xj\ O' Table 9. Subclass Means and Standard Errors for GOT and GPT Activities per mg. Dry Weight of Liver Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats.
GOT3 GPTb
Day Following Estrogen Progesterone Corn Oil Estrogen Progesterone Corn Oil Treatment Treated Treated (Control) Treated Treated (Control)
0.5 211.0 218.4 238.7 93.3 87.6 89.8
1 191.6 185.8 201.7 67.3 64.4 69.5
2 163.2** 187.8 249.2 67.1** 82.9* 107.9
3 204.7 214.4 198.3 87.2 95.7 88.7
5 220.9 224.7 218.2 81.3 88.9 92.6
Significantly different from corn oil treatment (control) at the 5% level.
Significantly different from corn oil treatment (control) at the 1% level.
aStandard error for all GOT means was 12.8.
^Standard error for all GPT means was 8.4. Table 10. Subclass Means and Standard Errors for GOT and GPT Activities of Whole Liver Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats.
GOT (10_3)a GPT (10"3)b
Days Following Estrogen Progesterone Corn Oil Estrogen Progesterone Corn Oil Treatment Treated Treated (Control) Treated Treated (Control)
0.5 732.7 601.5* 721.1 322.7* 238.1 268.3
1 526.7 490.7 532.4 184.3 170.0 183.2
2 616.1 645.4 600.2 253.0 281.6 258.9
3 558.0 602.9 558.2 236.6 269.6 245.5
5 634.3* 717.9 754.0 233.2** 284.2 318.5
Significantly different from corn oil treatment (control) at the 5% level.
Significantly different from corn oil treatment (control) at the 17. level.
g Standard error of all GOT means was 37.2.
“standard error of all GPT means was 18.9.
Xj i CO Table 11. Subclass Means and Standard Errors for GOT and GPT Activities per mg. Wet Weight of Uterine Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats.
GOT3 GPTb
Days Following Estrogen Progesterone Corn Oil Estrogen Progesterone Corn Oil Treatment Treated Treated (Control) Treated Treated (Control)
0.5 6.6 7.5 7.3 1.4 1.7 1.6
1 9.3* 8.2 7.9 1.8 1.7 1.7
2 9.9** 8.7* 7.3 2;1** 1.7 1.5
3 10.3** 8.7* 7.1 2.2** 1.8 1.5
5 8.0 7.9 7.5 1.6 1.6 1.8
Significantly different from corn oil treatment (control) at the 57. level.
Significantly different from corn oil treatment (control) at the 1% level.
aStandard error of all GOT means was 0.4.
^Standard error of all GPT means was 0.1.
Ln \0 60
Table 12. Subclass Means and Standard Errors for Whole Uterine Weight (mg.) for Hormone Treated Rats from 12 Hours to 5 Days Following Treatment.
Uterine Weight (mg. )a
Days Following Estrogen Progesterone Corn Oil Treatment Treated Treated (Control)
0.5 174** 125 128
1 189** 121 129
2 223** 115 114
3 188** 117 111
5 157** 124 114
Significantly different from corn oil treatment (control) at the 1% level.
Standard error of all means was 11. Table 13. Subclass Means and Standard Errors for GOT and GPT Activities cf Whole Uterine Tissue Determined from 12 Hours to 5 Days Following Hormone Treatment in Rats,
GOT3 GPTb
Days Following Estrogen Progesterone Corn Oil Estrogen Progesterone Corn Oil Treatment Treated Treated (Control) Treated Treated (Control)
0.5 1155 940 943 242 211 200
1 1745** 993 1010 342** 207 212
2 2190** 1003 833 466** 194 177
3 1923** 995 788 407** 210 170
5 1260** 980 855 257 196 201
Significantly different from corn oil treatment (control) at the 1% level.
aStandard error of all GOT means was 91.
^Standard error of all GPT means was 21. % % of Control 260 • 260 240 160 180 200 220 100 120 140 Figure 3. Effects of 17 p-Estradiol on Uterine Weight and GOT Activity GOT and Weight Uterine on p-Estradiol 17 of Effects 3. Figure 5 1 (by mg. of Uterus and by Whole Uterus) in Ovariectomized Rats. Ovariectomized in Uterus) Whole by and Uterus of (by mg. 2 (Days After Hormone Treatment) Hormone After (Days 5 3 4
n. nt/g Uterus Units/mg. Enz. n. Units/Uterus Enz. Uterine Wt. Uterine IsJ C\
7, of Control 260 220 240 180 200 160 140 100 120 80 iue4 Efcso 7 -srdo nUeieWih n P ciiy w Activity GPT and Weight Uterine on 0-Estradiol 17 of Effects 4. Figure 51 .5 (by mg. of Uterus and by Whole Uterus) in Ovariectomized Rats. Ovariectomized in Uterus) Whole by and Uterus of (by mg. (Days After Hormone Treatment) Hormone After (Days 2 3 5 4 n. Units/Uterus Enz. n. nt/g Uterus Units/mg, Enz. Wt. Uterine % % of Control 120 110 100 Figure 5. Effects of Progesterone on Uterine Weight and GOT Activity GOT and Weight Uterine on Progesterone of Effects 5. Figure * 5 1 (by mg. of Uterus and by Whole Uterus) in Ovariectomized Rats. Ovariectomized in Uterus) Whole by and Uterus of (by mg. Dy fe omn Treatment) Hormone (DaysAfter 2 3 5 4 n. ht/g Uterus Uhits/mg. Enz. Tissue Wt. Tissue Units/Uterus Enz, -F> CTi
% % of Control 110 120 100 80 Figure 6. Effects of Progesterone on Uterine Weight and GPT Activity GPT and Weight Uterine on Progesterone of Effects 6. Figure Rats. (by mg. of Uterus and by Whole Uterus) in Ovariectomized in Uterus) Whole by and Uterus of (by mg. (Days after Hormone Treatment) Hormone (Daysafter Units/Uterus , z n S M ^ >n. nt/g Uterus Units/mg. ^>Enz.
Uterine Wt Uterine
SUMMARY AND CONCLUSIONS
One hundred sixty-four female albino rats were used to study
the influence of estrogen and progesterone on GOT and GPT levels in
the plasma, liver, muscle and uterine tissues. Tissue enzyme levels were determined throughout the estrous cycle, for periods after ovariectomy, and after hormone replacement therapy.
The following conclusions were made from the evaluation of
data which resulted from 1312 tissue enzyme determinations:
1. GOT levels were higher than GPT levels in all tissues.
2. Both enzymes were higher in the liver than in the muscle,
with very low activity in the uterine tissue.
3. In general, the variations in GOT and GPT activity during
the estrous cycle were small.
4. Surgical stress appeared to cause an increase in enzyme
activities in the plasma, liver and muscle. This may
have been a result of glucocorticoid release from the
adrenal gland.
5. The primary effect of ovariectomy was to decrease uterine
weight which resulted in decreased GOT and GPT levels per
whole uterus.
6. Both estrogen and progesterone decreased the enzyme
activity of the liver in the ovariectomized rat with
66 67
the greatest effect on the second day following hormone
administration.
7. Estrogen dramatically increased the total uterine GOT
and GPT activity. This was primarily a result of
increased uterine weight rather than an increase in
enzyme activity per mg. tissue,
8. The primary mode of action of estrogen on uterine GOT
and GPT levels appears to be through cellular growth •
and proliferation rather than a selective increase in
enzyme synthesis.
v BIBLIOGRAPHY
1. Aakvaag, A. and P. Fylling. Progesterone and androgens in cyst
fluid and vein blood in the human ovary. Acta. Obst. Gnec.
Scand. Suppl., 45:158, 1966.
2. Altland, P. D., B, Highman, and B. D. Nelson, Serum Enzyme and
tissue changes in rats exercised repeatedly at altitude: Effects
of training. Amer. J. Physiol., 214:28, 1968.
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1968. APPENDIX 84
Table 14. Overall Means and Standard Deviations for the Independent Variables in the Ovariectomy Study.
Independent Variable Mean Standard Deviation
Body Wt. (g) 240.1 29.1
Plasma GOT/ml. 83.2 31.3 GPT/ml. 23.1 6.4
Liver GOT/mg. Wet Wt. 39.6 7.9 GPT/mg. Wet Wt. 22.5 4.3
GOT/mg. Dry Wt. 139.2 31.8 GPT/mg. Dry Wt. 78.7 16.3
Whole Tissue Wt. (mg.) 9714.5 1253.3
GOT/Tissue (10“3) 380.8 67.0 GPT/Tissue (10”3> 216.0 37.5
Muscle GOT/mg. Wet Wt. 46.7 6.4 GPT/mg. Wet Wt. 5.2 0.7
GOT/mg. Dry Wt. 189.7 24.1 GPT/mg. Dry Wt. 21.2 2.9
Uterus GOT/mg, Wet Wt. 7.6 0.9 GPT/mg. Wet Wt. 1.7 0.3
Whole Tissue Wt. (mg.) 279.8 97.0
GOT/Tissue 2123.7 792.1 GPT/Tissue 474.0 187.2 85
Table 15. F Values for GOT and GPT Activities per ml. Plasma from Analysis of Variance on Ovariectomized and Sham-operated Rats from 1 to 21 Days Following Surgery.
Independent Variable GOT GPT
Days (D) 11.60** 5.74* Linear 21.34** 3.45* Quadratic 28.55** 19.03** Cubic 4.73* 3.60
Treatment (T) 0.14 0.06 (Ovar-X vs. Sham.) DT Interaction 0.50 1.62 T Within D 2 1.03 2.79 T- Within D 3 0.12 2.50 T Within D 5 0.02 0.03 T Within D 8 0.33 2.60 T Within D 12 1.04 0.29 T Within D 16 0.63 1.52 T Within D 21 0.03 0.01
‘^Significant at the 5% level.
^Significant at the 1% level. 86
Table 16. Subclass Means and Standard Errors for GOT and GPT Activities per ml. Plasma Determined from 2 to 21 Days Following Surgery and Adjusted for Surgical Treatment Effects.in the Ovariectomy Study.
Days Following Surgery GOT S. E, GPT S.E.
2 133.3 7.4 31.9 1.8
3 102.8 9.6 22.4 2.3
5 64.0 7.4 20.1 1.8
8 80.8 7.4 20.6 ' 1.8
12 64,3 7.4 20.4 1.8 « 16 62.4 8.0 20.1 1.9
21 80.3 8.0 24.7 1.9 87
Table 17. F Values for Whole Liver Weight and for GOT and GPT Activities in Liver Tissue from Analysis of Variance on 0variectomi2ed and Sham-operated Rats from 1 to 21 Days Following Surgery.
Units/mg. 1 Dry Tissue Independent Variable GOT GPT Whole Tissue Wt.
Days (D) 13.12** 4.98* 2.78 Linear 40.13** 14.15** 6.54* Quadratic 4.83* 5.42* 1.03 Cubic 0.65 1.69 0.02
Treatment (T) 0.24 0.04 0.84 (Ovar-X vs. Sham) DT Interaction 0.53 0.07 2.80* T Within D 1 0.16 0.00 0.25 T Within D 2 2.66 0.10 0.02 T Within D 3 0.26 0.66 0.16 T Within D 5 0.02 2.24 5.32* T Within D 8 0.17 0.02 1.29 T Within D 12 0.16 0.77 3.38 T Within D 16 0.26 1.27 4.18* T Within D 21 0.25 0.11 5.83*
^Significant at the 5% level.
**Significant at the 1% level. 88
Table 18. Subclass Means and Standard Errors® for Whole Liver Weight (g.) Determined from 1 to 21 Days Following Surgery in Ovariectomized and Sham-operated Rats,
Days Following Surgery Ovar-X. Sham-op.
1 9.7 10.1
2 8.4 8.3
3 9.3 9.6
5 10.6 8.9
8 9.8 10.6
12 10.7 9.3
16 9.2 10.9
21 11.2 9.3
g Standard error of means of ovariectomized animals on Days 16 and 21 was 0.6 ; all others were 0.5. 89
Table 19. Subclass Means and Standard Errors for GOT and GPT Activities of Liver Tissue Determined from 1 to 21 Days Following Surgery and Adjusted for Surgical Treatment Effects in the Ovariectomy Study.
Days Following Units/mg. Dry Tissue Units/Whole Tissue Surgery GOT3 GPTb GOT(10_3)c GPT(10-3)d
1 154.8 87.2 421.4 238.8
2 195.6 101.2 452.1 233.8
3 123.7 74.7 340.6 205.0
5 .. 143.5 80.5 375.1 209.4
8 128.3 68.5 379.1 203.3
12 134.1 73.5 385.0 210.4
16 110.7 72.3 333.1 216.4
2* 116.5 70.7 349.8 211.4
aStandard error of means on Days 16 and 21 was 7.9; all others were 7.4.
^Standard error of means on Days 16 and 21 was 5.2; all others were 4.8,
cStandard error of means on Days 16 and 21 was 22900; all others were 21200.
^Standard error of means on Days 16 and 21 was 15100; all others were 14000. 90
Table 20. F Values for GOT and GPT Activities in Muscle Tissue From Analysis of Variance on Ovariectomized and Sham- operated Rats From 1 to 21 Days Following Surgery.
E. Units/mg. Wet Tissue E, Units/mg. Dry Tissue Independent Variable GOT GPT GOT GPT
Days (D) 6.19** 4.36** 4.77** 4.10** Linear 4.82* 3.12 7.00* 1.96 Quadratic 2,52 3.62 1.22 5.31 Cubic 0.15 1.95 1.59 0.68
Treatment (T) 0.00 0.66 0.00 0.67 (Ovar-X vs, Sham) DT Interaction 2.46* 0.96 2.21 0.91 T Within D 1 0.03 0.07 0.02 0.00 T Within D 2 0.80 0.62 0.76 0.59 T Within D 3 3.33 2.41 3.88 2.91 T Within D 5 1.02 0.75 1.00 0.83 T Within D 8 5.12* 0.01 4.25* 0.01 T Within D 12 4.79* 1.30 4.08* 0.93 T Within D 16 2.11 1.60 1.42 1.14 T Within D 21 0.01 0.73 0.04 0.68
^Significant at the 5% level.
^Significant at the 17. level. Table 21. Subclass Means and Standard Errors for GOT and GPT Activities of Muscle Tissue Determined From 1 to 21 Days Following Surgery in Ovariectomized and Sham-operated Rats.
Units/mg. Wet Tissue Units/mg. Dry Tissue Days Following GOT3 GPTb G0Tc GPTd Surgery Ovar-X. Sham-op. Ovar-X. Sham-op. Ovar- X. Sham-op. Ovar-X. Sham-op.
1 40.0 40.5 5.4 5.5 165.8 163.9 22.4 22.3 2 51.3 54.4 6.1 6.4 202.5 214.4 23.9 25.3 3 37.9 44.1 4.5 5.2 157.7 184.7 18.8 21.8 5 48.4 45.0 4.9 4.5 194.4 180.7 19.9 18.3 8 53.0 45.3 5.2 5.1 213.7 185.5 20.8 21.0 12 43.2 50.7 5.0 5.5 181.0 208.6 21.0 22.7 16 52.4 47.1 5.1 4.6 202.0 184.3 20.0 18.0 21 47.7 47.4 5.0 5.4 205 .6 200.0 21.0 22.6
aStandard error of means of ovariectomized animals on Ihys 16 and 21 was 2.8; all others were 2.4.
^Standard error of means of ovariectomized animals on Days 16 and 21 was 0.4; all others were 0.3.
cStandard error of ovariectomized animals on Days 16 and 21 was 11.2; all others were 10.0.
dStandard error of ovariectomized animals on Qiys 16 and 21 was 1,5; all others were 1.3. 92
Table 22. Subclass Means and Standard Errors for COT and GPT Activities of Muscle Tissue Determined from 1 to 21 Days Following Surgery and Adjusted for Surgical Treatment Effects in the Ovariectomy Study.
Days Following Units/mg. Wet Tissue Units/mg. Dry Tissue Surgery GOT3 GPTb G0TC GPTd
1 AO. 3 5.5 164.8 22.3
2 52.8 6.2 208.5 24.6
3 41.0 4.9 171.2 20.3
5 46.6 4.7 187.6 19.1
8 49.2 5.1 199.6 20.9
12 46.9 5.3 194.8 21.8
16 . 49.8 4.9 193.1 19.0
21 47.6 5.2 201.1 21.8
a Standard error of means on Day 16 and 21 was 1.8; all others were 1,7 m
^Standard error of all means was 0.2.
cStandard error of means on Day 16 and 21 was 7.4; all others were 6.8 •
^Standard error of means on Day 16 and 21 was 1.0; all
others were 0.9 • Table 23. F Values for Uterine Weight and GOT and GPT Activities in Uterine Tissue From Analysis of Variance on Ovariectomized and Sham-operated Rats From 1 to 21 Days Following Surgery.
Independent Units/mg. Wet Tissue Units/Whole Tissue Variable GOT GPT GOT GPT Tissue Wt.
Days (D) 1.76 1.81 2.77* 1.53 1.48 Linear 0.04 2.30 4.79 0.43 7.53** Quadratic 0.25 0.09 0.02 0.04 0.11 Cubic 0.31 4.06 0.07 2.98 0.06
Treatment (T) 3.22 2.08 223.79** 168.46** 192.98** (Ovar-X vs. Sham) DT Interaction 2.68* 1.37 6.12** 4.46** 4.79 T Within D 1 7.65** 6.30* 3.82** 10.42** 1.06 T Within D 2 1.48 1.12 17.37** 7.43** 21.61** T Within D 3 1.42 0.17 7.93** 7.01* 12.73** T Within D 5 1.41 0.02 5.99* 5.09* 11.86** T Within D 8 0.28 0.22 48.05** 34.34** 39.71** T Within D 12 3.82 1.97 62.97** 45.25** 47.24** T Within D 16 3.91 1.81 51.72** 48.13** 38.93** T Wittiin D 21 1.66 0.00 56.94** 36.56** 48.23**
*Signi£icant at the 5% level.
**Significant at the 17. level. VO u> 94
Table 24. Overall Means and Standard Deviations for the Independent Variables in the Hormone Study,
Independent Variable Mean Standard Deviation
Body Wt. (g) 276.7 21.8
Plasma GOT/ml. 94.6 21.5 GPT/ml. 22.8 4.0
Liver GOT/mg. Wet Wt. 60.6 9.7 GPT/mg. Wet Wt, 24.5 6.0
GOT/mg. Dry Wt. 208.6 30.9 GPT/mg. Dry Wt. 84.3 18.9
Whole Tissue Wt. (mg.) 10360.0 1805.5
GOT/Tissue (10-3) 619,5 102.7 GPT/Tissue (10“3) 249.8 55.1
Muscle GOT/mg. Wet Wt. 55.6 5.5 GPT/mg. Wet Wt. 5.0 0.6
GOT/mg. Dry Wt. 234.2 25.0 GPT/mg. Dry Wt. 21.1 2.7
Uterus GOT/mg. Wet Wt. 8.2 1.2 GPT/mg. Wet Wt. 1.7 0.3
Whole Tissue Wt, (mg.) 141.8 39.4
GOT/Tissue 1174.0 445.9 GPT/Tissue 246.1 93.5 95
Table 25. F Values for GOT and GPT Activities per ml. Plasma from Analysis of Variance on Hormone Treated Rats from 12 Hours to 5 Days Following Treatment.
Independent Variable GOT GPT
Days (D) 9.16** 9.10** Linear 9.33** 17.19** Quadratic 0.12 12.64** Cubic 3.31 3.61
Hormone Treatment (T) 0.03 1.00 DT Interaction 1.34 1.77 T Within D 0.5 2.31 2.49 T Within D 1 0.31 1.87 T Within D 2 0.84 1.49 T Within D 3 0.28 0.74 T Within D 5 1.66 1.59
^Significant at the 1% level. 96
Table 26. F Values for GOT and GPT Activities in Liver Tissue From Analysis of Variance on Hormone Treated Rats From 12 Hours to 5 Days Following Treatment.
Independent Units/mg. Wet Tissue Units/mg. Dry Tissue Variable GOTGPT GOTGPT
Days (D) 1.35 3.12 3.10* 4.11** Linear 0.02 0.31 1.26 2.00 Quadratic 1.07 0.79 5.38* 0.05 Cubic 0.40 3.04 3.55 7.17*
Hormone Treatment(T) 5.99** 2.60 4.12* 1.97 DT Interaction 2.97* 1.47 2.47* 3.92 T Within D 0.5 1.03 0.09 1.25 0.12 T Within D 1 0.54 0.13 0.39 0.09 T Within D 2 16.12** 7.66** 11.90** 6.05** T Within D 3 0.17 0.21 0.40 0.29 T Within D 5 0.00 0.39 0.06 0.48
•^Significant at the 5% level.
**Significant at the 1% level. 97
Table 27. F Values for GOT and GPT Activities in Muscle Tissue From Analysis of Variance in Hormone Treated Rats From 12 Hours to 5 Days Following Treatment.
Units/mg. Wet Tissue Units/mg. Dry Tissue Independent Variable GOT GPT GOT GPT
Days (D) 2.70* 6.50** 4.08** 7.90** Linear 4.47* 8.81** 4.47* 8.31** Quadratic 2.08 15.69** 4.61* 19.96** Cubic 4.00 1.35 7.22* 3.27
Hormone Treatment(T) 2.67 3.21* 2.18 2.55 DT Interaction 0.63 0.23 0.56 0.15 T Within D 0.5 2.11 1.25 1.43 0.80 T Within D 1 0.35 1.06 0.31 0.89 T Within D 2 0.40 0.32 0.48 0.24 T Within D 3 0.48 0.49 0.66 0.41 T Within D 5 1.97 0.99 1.65 0.78
^Significant at the 57. level.
**Signifleant at the 17. level. 98
Table 28. F Values for GOT and GPT Activities in Uterine Tissue From Analysis of Variance on Hormone Treated Rats From 12 Hours to 5 Days Following Treatment.
Independent Units/mg. Wet Tissue Units/Whole Tissue Variable GOT GPT GOT GPT
Days (D) 8.59** 3.87** 7.62*-* 5.03** Linear 1.05 0.69 1.12 0.59 Quadratic 26.35** 13.25** 23.08** 17.38** Cubic 3.73 0.27 6.09* 2.15
Hormone Treatment(T) 16.10** 4.98* 106.31** 77.89** DT Interaction 4.31** 3.85** 8.41** 8.18** T Within D 0.5 1.49 2.12 1.85 1.04 T Within D 1 3.69* 0.46 22.35** 12.87** T Within D 2 11.43** 7.89** 66.27** 58.45** T Within D 3 16.27** 9.03** 44.25** 35.72** T Within D 5 0.48 0.88 5.21** 2.52
^Significant at the 5% level.
**Significant at the 1% level. 99
Table 29. Subclass Means and Standard Errors for GOT and GPT Activities per ml. Plasma Determined from 12 Hours to 5 Days Following Hormone Administration Adjusted for Treatment Effects.
Days Following Treatment GOT S.E. GPT S.E.
0.5 89.7 5.0 27.6 0.9
1 120.2 5.0 22.6 0.9
2 85.2 5.2 21.8 1.0
3 93.8 5.0 20.8 0.9
5 82.9 5.0 21.3 0.9 Table 30. Least-Squares Means and Standard Errors of Tissue and Body Weight of Hormone Treated Rats Adjusted for Time Effects (12 Hours to 5 Days).
Estrogen S.E. Progesterone S.E. Corn Oil S.E. Treated Treated (Control)
Liver Wt. (mg.) 10860 288 10360 288 9860 288
Uterine Wt. (mg.)** 186 5 120 5 119 5
Body Wt. (g.) 280 5 277 5 272 5
**Hormone treatment effects significant at the 1% level. 001 101
Table 31. Subclass Means and Standard Errors9 for Whole Liver Weight (g.) for Hormone Treated Rats from 12 Hours to 5 Days Following Treatment.
Days Following Estrogen Progesterone Corn Oil Treatment Treated Treated (Control)
0.5 12.1 9.8 10.8
1 9.4 9.0 8.9
2 12.8** 11.3** 7.7
3 9.7 10.0 9.7
5 10.3* 11.7 12.4
mff * ■ Significantly different from corn oil treatment (control) at the 5% level.
Significantly different from corn oil treatment (control) at the 1% level.
aStandard error of all means was 0.6. Table 32. Subclass Means and Standard Errors for Whole Liver Weight and GOT and GPT Activities in Liver Tissue Determined from 12 Hours to 5 Days Following Hormone Administration Adjusted for Treatment Effects.
Days Following Units/mg,, Dry Tissue Units/Whole Tissue Treatment GOT3 GPTb g o t (i o -3)c GPT(10-3)d Whole Tissue Wt.(g.)e
0.5 222.7 90.2 685.1 276.3 10.9
1 193.0 67.8 516.6 179.2 9.1
2 200.1 86.0 620.6 264.5 10.6
3 205.8 90.5 573.0 250.6 9.8
5 221.3 87.6 702.0 278.7 11.5
Standard error of all means was 7.4.
^Standard error of all means was 4.8. Q Standard error of all means was 21499.
^Standard error of all means was 10884. £ Standard error of all means was 0.4. 102 103
Table 33. Subclass Means and Standard Errors for GOT and GPT Activities in Muscle Tissue Determined from 12 Hours to 5 Days Following Hormone Administration Adjusted for Treatment Effects.
Days Following Units/mg. Wet Tissue Units/mg. Dry Tissue Treatment GOT3 GPT*3 GOT*3 GPT**
0.5 57.2 5.0 242.5 21.2
1 55.7 5.1 229.2 20.9
2 54.9 5.2 234.0 22.1
3 58.2 5.4 249.7 23.0
5 51.8 4.4 214.8 18.2
aStandard error of mean on Day 2 was 1.6; all others were 1.5.
^Standard error of all means was 0.1.
cStandard error of mean on Day 2 was 7.0; all others were 6.6.
^Standard error of mean on Day 2 was 0,7; all others were 0.6. 104
Table 34. Subclass Means and Standai'd Erx'ors for GOT and GPT Activities of Uterine Tissue Determined from 12 Hours to 5 Days Following Hormone Administration Adjusted for Treatment Effects.
Days Following Units/mg. Ret Tissue Units/Whol e Tissue Treatment GOT3 GPTb G0Tc GPTd
0.5 7.15 1.54 1012.50 217.71
1 8.45 1.74 1249.17 253.73
2 8.62 1.78 1341.67 278.99
3 8.70 1.85 1235.00 262.53
5 7.S4 1.65 1031.67 217.77
aStandard error of all means was 0.22.
^Standard error of all means was 0.06.
cStandard error of all means was 52.45
^Standard error of all means was 12.25