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Effects of sex hormones on cholesterol metabolism

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Effects of sex hormones on cholesterol metabolism

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Content EFFECTS OF SEX HORMONES ON CHOLESTEROL METABOLISM by Yu Min Chen A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry) June 1960 UNIVERSITY O F SO U TH ER N CALIFORNIA GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES 7. CALIFORNIA This dissertation, written by .................. under the direction of h.%3....Dissertation Com mittee, and approved by all its members, has been presented to and accepted by the Graduate School, in partial fulfillment of requirements for the degree of D O C T O R OF P H I L O S O P H Y Dean D ate..... June J..960. DISSERTATION COMMITTEE v t/ Chairman ACKNOWLEDGMENTS I should like to express my everlasting gratitude and appreciation to Professor Roslyn B. Alfin-Slater, under whose guidance and sponsorship the present work was done, and to the late Dean Harry J. Deuel, Jr., for his original sponsorship of this work and for his assistance in my coming to the United States. I am sincerely grateful to Professors John W. Mehl Ronald F. Brown, Walter Marx and Paul Saltman for their understanding and helpful criticism. My thanks, too, are due to Dr. Supravat Mukherjee for the studies of cholesterol biosynthesis, to Dr. Takao Ushiyama for his help in the studies of serum lipoprotein patterns, to Dr. Sol Bernick for his histological analysis to Dr. Richard D. Coleman for his help and encouragement, and to Professor Jerome Berson and the late Professor Ernest Geiger for their serving originally on my research committee. I am deeply indebted to United States Public Health Service for their financial support of this investigation. TABLE OF CONTENTS ACKNOWLEDGMENTS CHAPTER PAGE I. INTRODUCTION .......................... I Sex differences in ketosis .......... 3 Essential fatty acids: requirements and sex differences ............. 4 Sex differences in cholesterol metabolism....................... 7 The serum cholesterol level, the ratio of serum cholesterol to serum phospholipids (c/p) and the serum lipoprotein patterns as related to sex and sex hormones in experimental chickens ......... 7 The serum cholesterol level, the ratio of serum cholesterol to serum phospholipids (c/p) and the serum lipoprotein patterns, as related to sex and sex hormones in experimental rabbits and rats . 11 iv CHAPTER _ PAGE The serum cholesterol level, the ratio of serum cholesterol to serum phospholipids (c/p) and the serum lipoprotein patterns as related to sex and sex hormones in humans ........................ 15 The biosynthesis of cholesterol as influenced by sex and sex hormones 17 Statement of the problem........... 20 II. EXPERIMENTAL............................ 22 Materials and Methods ................. 22 Animals .......... 22 Diets ...................... 22 Gonadectomy Procedures ............... 23 Orchidectomy ...................... 23 Ovariectomy ........................ 23 Preparation of hormone solutions . . . 27 Estradiol benzoate and testosterone propionate........................ 27 Preparation of stock solutions . . 27 Dilution of stock solution .... 28 V CHAPTER PAGE Preparation of mixed hormone solutions....................... 28 Estrogenic-combined sex hormone solution ............. 29 Androgenic-combined sex hormone solution ............. 29 Ethinyl estradiol and methyl testosterone ..................... 30 Extraction of cholesterol and lipids . 30 Extraction of plasma cholesterol . . 30 Extraction of liver cholesterol and lipids........................ 31 Extraction of adrenal cholesterol and lipids........................ 32 Extraction of cholesterol and lipids from kidneys, testes and ovaries . 32 Determination of cholesterol ......... 33 Determination of total lipids .... 35 Determination of serum and plasma phospholipids ........................ 36 Preparation of reagents ............. 36 Standard phosphate solution . . . 36 vi CHAPTER PAGE Digesting solution ............... 37 Color reagent ................... 37 Procedure .......................... 37 Starch block electrophoresis of serutn lipoproteins ........................ 38 Preparation of the starch block . . 38 Preparation of sodium barbital buffer ........................ 38 Preparation of the starch block . . 39 Equilibration of starch block . . . 40 Electrophoresis of serum sample on starch block . ............... 40 Staining of paper for protein and lipids........................ 41 Extraction and determination of protein .......................... 42 Extraction and determination of cholesterol and phospholipids . . . 42 Column chromatography of liver lipids . 45 Determination of iodine value (Wijs) . 46 Biosynthesis of cholesterol ......... 47 Incubation ........................ 47 vii CHAPTER PAGE Extraction of cholesterol and assay of content............. 48 Plan of Experiments................... 50 Effects of gonadectomy on cholesterol metabolism.......................... 51 Effects of gonadectomy on the choles terol and total lipid concentrations of tissues of rats fed cottonseed oil d i e t .......................... 51 Effects of gonadectomy on the choles terol and total lipid concentrations of tissues of rats fed the reference cottonseed oil diet containing cholesterol........................ 52 Effects of gonadectomy on the cholesterol and total lipid concentrations of tissues of rats fed a fat-free diet . 52 Effects of gonadectomy on serum lipo protein patterns in rats fed the reference cottonseed oil diet with and without cholesterol ............. 53 viii CHAPTER PAGE Effects of gonadectomy on the per centage distribution of liver lipid fractions and the degree of their unsaturation in rats fed a reference cottonseed oil diet with and without cholesterol................. 54 Effects of gonadectomy on the biosynthesis of cholesterol . . . 54 Effects of Sex Hormone Administration on Cholesterol Metabolism ............. 55 Effects of estradiol benzoate on the cholesterol and total lipid concentra tions of tissues of male rats fed the reference cottonseed oil diet with and without cholesterol ........ 55 Effects of testosterone propionate on the cholesterol and total lipid con centrations of tissues of female rats fed the reference cottonseed oil diet with and without cholesterol .... 56 CHAPTER ix PAGE Effects of estrogenic-combined sex hormones on the cholesterol and total lipid concentrations of tissues in male rats fed a reference cottonseed oil diet with and without cholesterol................... 58 Effects of adrogenic-combined sex hormones on the cholesterol and total lipid concentrations of tissues in female rats fed a reference cottonseed oil diet with and without cholesterol....................... 59 Effects of testosterone propionate on the cholesterol and total lipid con centrations of tissues of male rats fed a reference cottonseed oil diet with and without cholesterol ........... 61 Effects of estradiol benzoate on the cho lesterol and total lipid concentrations of tissues of female rats fed a refer ence cottonseed oil diet with and without cholesterol ............. 62 X CHAPTER PAGE Effects of ethinyl estradiol on the cholesterol and total lipid concen trations of tissues in male rats fed a fat-free diet................ 63 Effects of methyl testosterone and the cholesterol and total lipid concen trations of tissues in female rats fed a fat-free diet ........... 63 Effects of sex hormone administration on the plasma phospholipid level and the plasma c/p ratio in male and female rats fed the reference cottonseed oil diet with and without cholesterol and fed the fat-free diet........ 64 Effects of sex hormone administration on the percentage distribution of liver lipid fractions and the degree of their unsaturation............... 65 Effects of sex hormone administration on the biosynthesis of cholesterol . . 65 III. RESULTS AND DISCUSSION................. 67 xi CHAPTER PAGE Effects of Gonadectomy on Cholesterol Metabolism.......................... 67 Body weight ........................ 67 Tissue cholesterol and total lipid concentrations ................... 69 Serum lipoprotein patterns ......... 81 Liver lipid patterns............... 90 Biosynthesis of Cholesterol ......... 92 Effects of Sex Hormone Administration on Cholesterol Metabolism ......... 95 Body weight ........................ 95 Plasma cholesterol level ........... 99 Liver cholesterol and total lipid levels ................... 109 Adrenal cholesterol and total lipid levels ................... 119 Kidney cholesterol and total lipid levels ................... 123 Cholesterol and total lipid levels in testes and ovaries............. 126 Plasma phospholipid level and c/p ratio ............ 130 xii CHAPTER PAGE Liver lipid patterns............... 134 Biosynthesis of cholesterol ........ 143 Histology .......................... 147 Comparison between the Effects of Gonadectomy and Sex Hormone Adminis tration on Cholesterol Metabolism . . 148 Possible Mechanisms in the Effects of Sex Hormon.es on Cholesterol Metabolism in Rats ........... 154 IV. SUMMARY AND CONCLUSIONS.................. 160 BIBLIOGRAPHY.................................... 164 LIST OF TABLES TABLE PAGE I. Composition of Diets C and CC ........ 24 II. Composition of Diet F F ................. 26 III. Effect of Gonadectomy on the Body Weight of Rats Fed Different Diets ........ 68 IV. Effect of Gonadectomy on the Plasma Cholesterol Concentrations of Male and Female Rats as Influenced by Diet . 70 V. Effect of Gonadectomy on the Concentrations of Liver Cholesterol and Total Lipids of Male and Female Rats as Influenced by Diet .............................. 71 VI. Effect of Gonadectomy on the Concentrations of Adrenal Cholesterol and Total Lipids of Male and Female Rats as Influenced by Diet .............................. 72 VII. Effect of Gonadectomy on the Ratio of Alpha-Lipoprotein Cholesterol to Beta- Lipoprotein Cholesterol in Rat Serum . 82 VIII. Effect of Gonadectomy on the Phospholipid TABLE xiv PAGE Concentration of Lipoproteins in Rat Serum.............................. 83 IX. Effect of Gonadectomy on the Ratio of Cholesterol to Phospholipids in the Lipoproteins in Rat Serum . T . . . . 84 X. Effect of Gonadectomy on the Percentage Distribution and the Degree of Unsatura tion of the Different Fractions of Liver Lipids in Rats Fed a 15 per cent Cotton seed Oil Diet With and Without 1 per cent Cholesterol for 6 W e e k s ......... 91 XI. Effect of Gonadectomy on the Biosynthesis of Cholesterol from Acetate-l-C^ in Rat Liver Slices...................... 93 XII. Effect of Sex Hormone Administration on the Body Weight of Male Rats Fed Different Diets................... 97 XIII. Effect of Sex Hormone Administration on the Body Weight of Female Rats Fed Different Diets................... 98 XIV. Effect of Sex Hormone Administration on the Plasma Cholesterol Concentration of XV TABLE PAGE Male Rats Fed Different Diets .... 100 XV. Effect of Sex Hormone Administration on the Plasma Cholesterol Concentration of Female Rats Fed Different Diets . . . 101 XVI. Effect of Sex Hormone Administration on the Cholesterol and Total Lipid Concen trations of Liver of Male Rats as Influenced by D i e t ................... 110 XVII. Effect of Sex Hormone Administration on the Cholesterol and Total Lipid Concentrations of Liver of Female Rats as Influenced by Diet .............................. Ill XVIII. Effect of Sex Hormone Administration on Adrenal Cholesterol and Total Lipid Concentrations of Male Rats as Influenced by Diet .............................. 120 XIX. Effect of Sex Hormone Administration on Adrenal Cholesterol and Total Lipid Concentrations of Female Rats as Influenced by D i e t ................... 121 xvi TABLE PAGE XX. Effect of Sex Hormone Administration on Kidney Cholesterol and Total Lipid Con centrations of Male Rats as Influenced by Diet ................................. 124 XXI. Effect of Sex Hormone Administration on Kidney Cholesterol and Total Lipid Concentrations of Female Rats as Influenced by D i e t ................... 125 XXII. Effect of Sex Hormone Administration on Testis Cholesterol and Total Lipid Contents of Male Rats as Influenced by Diet .......................... 127 XXIII. Effect of Sex Hormone Administration on Ovary Cholesterol and Total Lipid Contents of Female Rats as Influenced by D i e t ................... 128 XXIV. Effect of Sex Hormone Administration on the Plasma Phospholipid Level and Plasma C/P Ratio in Rats as Influenced by Diet....... .......................... 131 xvii TABLE PAGE XXV. Effect of Sex Hormone Administration on the Percentage Distribution and the Degree of Unsaturation of the Different Fractions of Liver Lipids in Rats Fed Different Diets.................... 135 XXVI. Effects of Sex Hormone Injection on the Biosynthesis of Cholesterol from 1-C^-Acetate in Rat Liver Slices . . 144 LIST OF FIGURES FIGURE PAGE 1. Effect of Gonadectomy on Plasma Cholesterol level (mg%) 73 2. Effect of Gonadectomy on Liver Cholesterol Level (mg per gram)................... 74 3a. Effect of Sex Hormone Administration on Plasma Cnolesterol Level (mgX) . . 102 3b. Effect of Sex Hormone Administration on Plasma Cholesterol Level (mg%) . . 103 3c. Effect of Sex Hormone Administration on Plasma Cholesterol Level (mg%) . . 104 4a. Effect of Sex Hormone Administration on Liver Cholesterol Level (mg per gram).................................. , 112 4b. Effect of Sex Hormone Administration on Liver Cholesterol Level (mg per gram) . 113 4c. Effect of Sex Hormone Administration on ' Liver Cholesterol Level (mg per gram) . 114 5. Effect of Sex Hormone Administration on Plasma Phospholipid Level (mg%) . . 132 xix FIGURE PAGE 6. Effect of Sex Hormone Administration on the Percentage Distribution of Phospholipids in Liver Lipids ......... 136 CHAPTER I INTRODUCTION It has been well known that young women during their reproductive years have a lower incidence of coronary atherosclerosis and a lower mortality resulting from that disease than young men of comparable age (1-8). This resistance to this disease in young women tends to disappear in the decades following menopause (8). Simi larly, eunuchs manifest less extensive coronary athero sclerosis at autopsy than do normal men (9,10). Healthy ovarectomized women experience coronary atherosclerosis of a severity resembling that of men (11,12). These observations point to the fact that sex hormones play an important role in atherogenesis. The chemical aspects of atherogenesis are thought to be intimately associated with lipid metabolism, especially cholesterol metabolism (13-16). Deposits of lipids are found in the arteries and aorta of patients suffering from atherosclerosis in its various manifesta tions. In these deposits, 40-65 per cent of the 1 phospholipid-free fat is cholesterol (16). Furthermore, there is a change in the blood lipid or blood cholesterol levels and blood lipid patterns in atherosclerotic patients (13-15). Therefore, a derangement in lipid or cholersterol metabolism may be concerned with athero genesis. As a result, there has been much interest in the possible correlation between lipid or cholesterol levels or lipid patterns of the blood and atherogenesis. The relationship between cholesterol metabolism and atherosclerosis has also been studied in experimental animals. Among the animal species used, the chicken is the only vertebrate other than human known to have a high incidence of spontaneous atherosclerosis, and, therefore, it is very useful in these studies (13). Atherogenesis can be induced experimentally in several different species of animals other than the chicken, such as the monkey, the dog, the rabbit, and the rat by feeding atherogenic diets under conditions where there is interference with other, probably endogenous, factors (17-24). Much valuable information has been obtained from these studies relating cholesterol metabolism to athero sclerosis in experimental animals, but many of the results are contradictory and depend on experimental conditions, species of the animal, age of the animal and sex of the animal. One of the most challenging problems in this area is concerned with the role of sex hormones. It is well known that sex differences exist in lipid or cholesterol metabolism in the human and various species of experi mental animals. Sex differences in ketosis. Deuel and Gulick reported that the human female develops a much greater ketosis during fasting than does the male (25). A similar sex difference in ketonuria has been demonstrated in the exogenous ketonuria produced when acetoacetic acid, butyric acid,or other ketogenic acids are given as their sodium salts to fasting rats and guinea pigs (26). Beach and co-workers confirmed this sex difference in ketonuria in alloxan diabetes in rats (27). A similar sex differ ence was also noted in the endogenous ketonuria occurring in the rats with fatty livers produced by fasting without the administration of supplementary ketogenic acids (28). Moreover, the high ketonuria noted in the normal female rat was reduced, following ovarectomy, to the value found in the normal male animals (29). Essential fatty acids: requirements and sex differences. In studies using rats, it has been reported by Alfin-Slater et al. that the administration of a fat- free diet to male rats caused a reduction of blood cho lesterol (30,31); but there is a concomitant increase of liver and adrenal cholesterol which can reach twice the normal level. The presence of a saturated fat in the diet yields results similar to the fat-free diet after the first week. After four weeks the amount of choles terol in the livers of these animals is considerably less than those on a fat-free diet, although it is still increased over the normal (30). The increased cholesterol concentration in the liver of rats on essential fatty acid low diets is found to be confined almost exclusively to the ester fraction (31). These authors postulated that essential fatty acids are necessary for the proper esteri- fication and transport of cholesterol from the liver (30). Mead and his co-workers have indicated that ingested polyunsaturated fatty acids appear in the blood largely as sterol esters and phospholipids, whereas oleic and stearic acid appear largely as triglycerides, and slowly appear in the phospholipids (32). Sinclair considered that human diets in most civilized countries are becoming increasingly deficient in essential polyethenoid fatty acids (arachidonic acid) due to hydrogenation and hardening of food fats and to other manufacturing processes (33). As a result, the cholesterol in the diet is esterified with abnormal or unusually saturated fatty acids. These abnormal esters are not readily disposed of, and may cause atheromata. Similarly, Sinclair believes that abnormal phospholipids containing abnormal or unusually saturated fatty acids are less readily disposed of, and are retained in the plasma. They increase the coagulability of the blood, and contribute to coronary and cerebral thrombosis.- Kritchevsky and co-workers have demonstrated that the feeding of corn oil (34), which contains a high proportion of essential fatty acids, is able to suppress the deposition of cholesterol in the aorta of rabbits to a marked extent, even when the animals are subjected to such a severe strain as is afforded by the presence of 3 per cent cholesterol in the diet. On the other hand, the shortening (a partially hydrogenated vegetable oil) 6 does not appear to alleviate the condition. The essential fatty acids are necessary for the synthesis of phospholipids and phospholipids may be important for the transportation of cholesterol due to the fact, as explained by Ahrens and Kunkel(35,36), that the hydrophilic character of phospholipids and their ability to diffuse may hold the hydrophobic cholesterol in solution. A sex difference exists in rats in the requirement for the essential fatty acids for growth. Greenberg et al. (37,38), and Deuel et al. (39) concluded that the daily requirement of linoleate for male rats probably exceeds 200 mg. Greenberg et al. (37-39) and Anisfeld et al.(40) found that the daily requirement of linoleate for the female rat does not exceed 100 mg and probably varies between 20 and 50 mg. This sex difference in rats in the requirement for the essential fatty acids can be also shown by the fact that female rats on fat-free diets develop the essential fatty acid deficiency symptoms much more slowly than do male rats (41). Cheng and her co-workers reported that although the protective effect of cottonseed oil against x-ray irradiation injury in adult rats is the same in both male and female rats, in young rats the effect is more pro nounced in male rats than in female rats (42). They also showed that the survival of the male rats on a fat- free diet is much lower than that of the female animals on the same dietary regime probably because of the higher linoleate requirement of the male animal. Sex differences in cholesterol metabolism. Sex differences exist in many phases of cholesterol metabolism in man and several different species of experimental animals. There are differences in the serum or plasma cholesterol levels, the ratio of serum cholesterol to serum phospholipids (c/p) and the serum lipoprotein pat terns in each species studied as far as the sex and sex hormones are concerned. The serum cholesterol level, the ratio of serum cholesterol to serum phospholipids (c/p) and the serum lipoprotein patterns as related to sex and sex hormones in experimental chickens. The chicken is the only verte brate other than man known to have a high incidence of spontaneous atheroscloerosis(13); investigations in chickens have yielded valuable information which might be associated to the problem of atherosclerosis as related to sex and sex hormones. Lorentz, Entenman, and Chaikoff reported that in the cockerel, mature rooster, and immature female bird before puberty, the plasma choles terol concentrations and the plasma c/p ratios are low (43). No significant differences in the level of cholesterol, phospholipids or total fatty acids of the blood of male birds examined at various age periods were demonstrated (43-45). In the female bird at puberty and in hens during the laying period, changes occur in the lipid composition of the plasma which have no counterpart in humans (43). The total lipid concentration of plasma is greatly increased chiefly attributable to the accumula tion of neutral fat, less to changes in phospholipids, and least to increase in the concentration of cholesterol (43). The plasma c/p ratio is much lower than in the male or immature female birds (43). The effects of sex hormone administration in chicks have been well established by many groups of investigators. Entenman, Lorentz, and Chaikoff reported that the administration of estrogens in both cockerels and immature female birds causes chemical changes resembling i the alterations exhibited by the females at puberty (46). The concentrations of cholesterol, phospholipids, and neutral fat are greatly increased. Chaikoff et al.also reported that the administration of estrogens in both cockerels and immature female birds causes anatomical changes resembling the alterations exhibited by the females at puberty (44). Pick et al. (47) have shown that the administration to cockerels of estrogen combined with cholesterol feeding inhibits prophylactically the development of coronary atherosclerosis, but has no perceptible effect on the formation of lesions in the aorta. A close correlation between the level of the plasma c/p ratio and the degree of coronary atherogenesis has been demonstrated. In spite of the hypercholester olemia, there is a much greater rise in phospholipids in the estrogen-treated birds than in birds fed cholesterol diet only. A much lower plasma c/p ratio is produced. Moreover, Katz et al. (14), and Pick et al. (48) showed that the administration of estrogen to cockerels causes a regression of the coronary atherosclerosis induced by the cholesterol supplemented diet. The atherogenic diet 10 has no effect in the egg-producing hen (14). However, surgically ovariectomized hens, completely lacking endoge- i nous estrogen secretion, do develop grossly elevated plasma c/p ratios and extensive coronary atherosclerotic plaques in response to cholesterol feeding (49-51). Thus, sexually mature male and female chicks exhibit, similar to human beings, a significant sex difference in their susceptibility to coronary atherosclerosis. Stamler et al. administered androgen simultaneously with estrogen to cockerels fed on cholesterol diet (52). Androgen tends to prevent the feminizing effects of the estrogen but does not interfere with the inhibitory action of estrogen on cholesterol-induced coronary athero- genesis. A more recent work reported by Katz et al. showed that the administration of testosterone inhibits hyper cholesterolemia but does not protect cholesterol-fed cockerels from aortic or coronary atherosclerosis (53). Castration of male or female chicks, with or without testosterone feeding, does not influence atherogenesis or hypercholesterolemia. The administration of testosterone in both male and female chicks also causes a lower plasma 11 c/p ratio. The serum lipoprotein patterns in chicks are not well established. However, it has been shown by Katz, Stamler, and Pick that estrogen apparently affects a sig nificant reduction of alpha-lipoprotein in cockerels, a change precisely opposite to that induced in humans (49, 50,54). Estrogen-treated cockerels fed on cholesterol and cottonseed oil diet also exhibit apparent decreases in plasma low density beta-lipoproteins of the 20 to 400 class. Somewhat different results have been reported by Hillyard, Entenman, and Chaikoff (55), indicating that both cholesterol feeding and stilbestrol administration independently induce a great increase in the total serum lipoproteins in cockerels although one fraction correspond ing to alpha^-lipoprotein is decreased in stilbestrol- treated chicks. The serum cholesterol level, the ratio of serum cholesterol to serum phospholipids (c/p) and the serum lipoprotein patterns, as related to sex and sex hormones in experimental rabbits and rats. In both rabbits (56) and rats (57,58), the male has a lower serum cholesterol level than the female. 12 Fillios and Mann showed that the male rabbit has a significantly lower average serum cholesterol level than the female, both before and after cholesterol feeding (56). The cholesterolemic response in rabbits to choles terol feeding is augmented by estradiol administration and by orchidectomy and is lowered by testosterone administra tion and by ovariectomy. Ludden et al. reported in some earlier papers that both testosterone propionate and estradiol propionate appear to inhibit hypercholesterolemia and atherosclerosis of the aorta in female rabbits but not in the male rabbits fed cholesterol (59,60). Rossi and Giaquinto observed that testosterone causes a regression of aortic atherosclerosis in the rabbit (61). That diethyl-stilbestrol reduces the plasma cholesterol (62) and that ovariectomy is followed by an elevation of the plasma cholesterol (63) in rabbits have also been reported. In rats, it has been shown by Aftergood et al. that the female has a slightly higher plasma cholesterol level but slightly lower liver cholesterol level than the 13 male (57). Fillios also observed that female rats have higher plasma cholesterol levels than males (58). Fillios studied the effects of castration and sex hormone administration on the plasma cholesterol level in rats (58). In rats fed Purina Chow, gonadectomy of both males and females causes an increase in plasma choles terol. The administration of estradiol dipropionate for 21 days to males causes an increase in plasma cholesterol, but there is no change in plasma cholesterol after giving estradiol to females and testosterone propionate to males and females. The administration of testosterone to both gonadectomized males and females decreases plasma choles terol but the administration of estradiol dipropionate to both gonadectomized males and females increases plasma cholesterol. In rats fed a 5 per cent cholesterol diet with cholate and treated with sex hormones for 28 days, the administration of estradiol dipropionate to both intact and gonadectomized males and females causes an increase in plasma cholesterol; the administration of testosterone propionate to both intact and gonadectomized females causes a decrease in plasma cholesterol. However, the administration of testosterone propionate to both 14 intact and gonadectomized males causes no change in plasma cholesterol; and gonadectomy of males causes no change but gonadectomy of females causes a decrease in plasma choles terol. 4 It has been demonstrated by Mbskowitz et al. that in rats fed 2 per cent cholesterol diet (64), the administration of estradiol benzoate causes a higher serum cholesterol and a higher serum lipid phosphorus together with a lower ratio of serum cholesterol to serum lipid phosphorus at the end of 18 weeks as compared with rats without estradiol administration. Castration causes no effect; the administration of testosterone causes a slightly lower serum cholesterol level but no change in serum lipid phosphorus, thus producing a slightly lower ratio of serum cholesterol to serum lipid phosphorus. A correlation is observed between the presence of the terminal elevation of the serum cholesterol-lipid phos phorus ratio and the incidence of coronary lesions. That the elevation of the serum cholesterol-lipid phosphorus ratio is associated with an increased incidence of coronary atherosclerosis is also observed in rabbits by Burger et al. (65) and in dogs by Davidson et al. (66). 15 Contrary to the above, Levin (67) and Pekkarlnen et al. (62) reported that diethyl-stilbestrol reduces the serum cholesterol in rats. Boyd and McGuire also showed that hexestrol (a synthetic non-steroid estrogen) decreases the serum cholesterol in rats (68). The serum cholesterol level, the ratio of serum cholesterol to serum phospholipids (c/p) and the serum lipoprotein patterns as related to sex and sex hormones in humans. Young women during reproductive years have a lower incidence of coronary atherosclerosis (1-8), a lower serum cholesterol level, and a lower serum c/p ratio than young men of comparable age (18,14,69,70). This phenome non in young women disappears as they become older. After menopause, women have similar or higher serum cholesterol levels and a higher c/p ratio than do men. Women from the age of 3 to 23 have higher serum cholesterol levels and higher serum c/p ratio than do men of the same age (69). There is an elevation of the serum cholesterol level and the serum c/p ratio during pregnancy (5). During the menstrual cycle, the serum cholesterol and serum c/p ratio are decreased when ovulation occurs but the serum choles terol reaches a maximum and serum c/p ratio is increased 16 after the menstrual period (71). It has been reported that estrogens depress the serum c/p ratio in pre- and post-menopausal women (72,73) and in patients (15,74-83) with coronary atherosclerosis by increasing serum phospholipid concentrations or by depressing serum total cholesterol levels. In comparison to estrogens, androgens have been reported to increase the serum cholesterol level and the serum c/p ratio in patients with coronary atherosclerosis (14,75). It has been shown that there is an alteration in serum lipoprotein patterns in atherogenesis (4-6,14, 75,76,78,83). Normal young women have less beta-lipo- protein, less beta-lipoprotein-bound cholesterol, and more alpha-lipoprotein and alpha-lipoprotein bound-cho lesterol than young men of comparable age and than older men and women. Patients with myocardial infarctions show an exaggeration of these differences in the serum lipo protein patterns between normal young women and young men of comparable age. Administration of estrogen to survivors of myo cardial infarctions and to normal controls not only cause 17 a decrease In serum cholesterol concentration and serum c/p ratio but also cause a decrease in beta-lipoprotein- bound cholesterol and an increase in alpha-lipoprotein- bound cholesterol (75,76,78,83). Opposite effects are produced following the administration of androgens (75, 78) . Significant differences between the serum lipo protein distributions of eunuchs and of non-castrated men have also been demonstrated (84). The biosynthesis of cholesterol as influenced by sex and sex hormones. Although the biosynthesis of cho lesterol from acetate has been well established (85), studies concerning the effects of sex and sex hormones on the biosynthesis of cholesterol are rare and incon sistent. In rats, estrogen has been reported by Rosenman et al. to depress hepatic biosynthesis of cholesterol, as evidenced by the biliary cholesterol concentration, with out affecting plasma cholesterol values (86). Boyd and McGuire showed that hexestrol depresses hepatic biosyn thesis of cholesterol in rats (68). In whole animal synthesis experiments with castrated rats, Landon and Greenberg have reported a decrease in turn-over of cho- 18 lesterol (87), whereas Dugal and Saucier observed an increased incorporation of labeled acetate into liver cholesterol (88). Fillios reported recently that the administration of estradiol dipropionate to castrated male and female rats increases (89), and the administration of testo sterone propionate decreases the biosynthesis of choles terol from acetate-l-C^ 24 hours after the injection of 14 acetate-l-C into the blood. Female animals have higher rates of biosynthesis of cholesterol than do males (the rate of the biosynthesis of cholesterol was measured by counting the activity of the cholesterol isolated from the plasma). Another recent report by Rubin and White shows that rat liver slices from normal female rats incorporate approximately twice as much total radioactivity from acetate-l-C^ into the digitonin-precipitable sterols as do those from normal male rats (90). Liver slices obtained from female rats 4 weeks after castration show a significant reduction in their extent of incorporation. Castration of the male rat, or treatment of the castrated animal with testosterone propionate, does not alter the capacity of liver slices to incorporate radioactivity of 19 14 acetate-l-C into the digitonin-precipitable sterol fraction. In later experiments, however, they showed that castration of the female, or treatment of the latter with estradiol does not alter significantly the extent of incorporation from that seen with liver slices from normal female rats. By using liver slices, Aftergood has demonstrated that in rats on a 15 per cent cottonseed oil diet, male rats have higher rate of biosynthesis of cholesterol than female (91). Mukherjee and Alfin-Slater observed that the biosynthesis of cholesterol by liver slices is markedly reduced in rats receiving a fat-free diet. Later on, Coleman, Chen and Alfin-Slater (93) corroborated the work of Aftergood. In addition, they found that gonadec tomy of both male and female rats decreases the rate of biosynthesis of cholesterol. In rats on a fat-free diet, the intact female has a slightly higher rate of bio synthesis of cholesterol than the intact male; although gonadectomy of males has no effect on cholesterol bio synthesis, gonadectomy of females results in a decreased biosynthesis. 20 Statement of the problem. Sex differences in cholesterol metabolism exist in man and in other animal species. Sex hormones exert their effects on cholesterol metabolism to a greater extent in man, chick, and other species fed an atherogenic diet. However, the role of sex hormones in cholesterol metabolism is still uncertain. In many cases the results obtained from animal experi mentation are contradictory. Moreover, most of the work done in this field has been centered on blood lipid chemistry. It is impossible to interpret results solely from the response of blood lipids, neglecting the response of other tissue lipids, especially liver lipids. Using the albino rat of the U.S.C. strain as the experimental animal, an investigation has been undertaken to study the effects of sex hormones on cholesterol metabolism from the following standpoints: 1. The effects of sex hormones on the levels of cholesterol in plasma, liver and other tissues; 2. The relationship between plasma cholesterol and liver cholesterol as affected by sex hormones; 3. The relationship between the plasma phospholipids and liver phospholipids as affected by sex hormones; 21 The effects of sex hormones on the serum lipo protein patterns of rats and comparison with results reported from human experiments; The composition of liver lipids (cholesterol esters, triglycerides and phospholipids) as affected by sex hormones; The effects of sex hormones on the biosynthesis of cholesterol; The relationship between the diet and the effects of sex hormones on cholesterol metabolism; The similarities and differences between the effects of gonadectomy and those of sex hormone administration on cholesterol metabolism. CHAPTER II EXPERIMENTAL Materials and Methods I. Animals. Male and female albino rats of the University of Southern California strain were used in all experiments. II. Diets. Stock diets were either Purina Laboratory Chow, Rockland commercial pellet, or a synthetic diet containing 15 per cent cottonseed oil. The experimental diets were: A. 15 per cent cottonseed oil (CSO) diet. This was also used as a control diet. It is designated as C. B. 15 per cent cottonseed oil diet supplemented with 1 per cent cholesterol. This diet was essentially the same as the 15 per cent cotton seed oil diet except that 1 per cent of cho lesterol replaced 1 per cent of sucrose. It is designated as CC. 23 The composition of diets C and CC is given in Table 1. C. Fat-free diet. The composition of the fat- free diet is given in Table 2. It is desig nated as FF. III. Gonadectomy procedures. A. Orchidectomy. The animal was anaesthetized under ether. An incision large enough to permit the extrusion of the testicle was made in the tip of the scrotum. A single silk ligature was placed around the spermatic cords, and the testes, together with the epididymis, were excised. The wounds were closed; silk sutures were used for the muscle layers and wound clips for the skin. The clips were removed after a week. B. Ovariectomy. The animal was anaesthetized under ether. A single longitudinal skin incision was made in the dorsal midline at the level of the lower poles of the kidneys. The skin was then retracted and the ovaries were exposed on both sides through the muscle wall 24 TABLE 1 COMPOSITION OF DIETS C AND CC Component C CC - ______________________(per cent)________(per cent) Sucrose 52.30 51.30 Casein, commerciala 24.00 24.00 Cottonseed Oil 15.00 15.00 Celluflourb 4.00 4.00 Salt Mixture0 4.00 4.00 Cholesterol^ ------ 1.00 Bile Saltse 0.25 0.25 Cholinea 0.24 0.24 a. Lactic casein, Challenge Dairy Co.* Los Angeles, Cal. b. Solka-Floc, Brown Co., San Francisco, Cal. c. Osborne and Mendel (Science 75., 339 (1932)), Wesson modification; Nutritional Biochemical Corp., Cleveland, Ohio. d. U.S.P.; Merck Co. e. Difco Laboratories, Inc., Detroit, Mich. 25 TABLE 1 (continued) Component C CC (per cent) (per cent) Vitamin Mixture^ 0.19 0.19 NopsolS 0.012 0.012 Alpha-Tocopherol Acetate*1 0.012 0.012 f. The vitamin mixture consisted of 38.57 per cent p- amino-benzoic acid, 31.88 per cent inositol, 12.75 per cent ascorbic acid, 4.59 per cent thiamine hydrochloride, 3.82 per cent niacin, 3.82 per cent Ca-pantothenate, 1.72 per cent riboflavin, 1.72 per cent pyridoxine, 0.64 per cent folic acid, 0.32 per cent menadione, 0.16 per cent biotin, and 0.0004 per cent vitamin B^2» Merck Co. and Nutritional Bio chemicals Corp. g. The Nopsol solution contains 100,000 I.U. of vitamin A per gm and 20,000 I.U. of vitamin D per gm, Nopco Chemical Co., Harrison, N. J. h. Nutritional Biochemical Corp., Cleveland, Ohio. 26 TABLE II COMPOSITION OF DIET FF Component Per cent Sucrose 71.86 Casein, Vitamin-testa 19.98 Celluflourb 4.00 - Salt Mixture0 4.00 Vitamin B mixture** 0.16 Nopsole 0.012 Alpha-Tocopherol Acetate^ 0.012 a. General Biochetnicals, Inc. b. Solka-Floc, Brown Co., San Francisco, Cal. c. Osborne and Mendel (Science 7J), 339 (1932)), Wesson modification; Nutritional Biochemical Corp., Cleveland, Ohio. d. The water-soluble vitamin mixture had the following composition: choline chloride, 52.94 per cent; p- aminobenzoic acid, 26.47 per cent; inositol, 13.24 per cent; riboflavin, 1.90 per cent; calcium pantothenate, 1.77 per cent; nicotinic acid, 1.60 per cent; thiamine hydrochloride, 0.95 per cent; pyridoxine hydrochloride, 0.71 per cent; folic acid, 0.26 per cent; and biotin, 0.03 per cent. Vitamin B^2 (dissolved in ethyl alcohol) was added to the extent of 0.0003 per cent. Menadione was added to the vitamin B mixture to the extent of 0.13 per cent. e. The Nopsol solution contains 100,000 I.U. of vitamin A per gm and 20,000 I.U. of vitamin D per gm. Nopco Chemical Co., Harrison, N. J. f. Nutritional Biochemicals Corp., Cleveland, Ohio. 27 just below the dorsal muscle mass. Another two incisions were made through the muscle wall; each was of the minimum length which con veniently allowed the extrusion of the ovary. After ligation of the upper horn of the uterus with silk, the ovary, together with its sur rounding fat, the oviduct, and a small portion of the uterus were excised. The wounds were closed in the same way as that in orchidectomy. IV. Preparation of hormone solutions A. Estradiol benzoate and testosterone propionate. 1. Preparation of stock solutions. A known amount of hormone was dissolved in a minimum amount of alcohol-ether (3:2) (v,v) in a tared flask. Sesame oil was added. The solution was mixed well and warmed in a water bath and the alcohol- ether was removed under vacuum. The resulting solution was weighed, together with the flask, and the hormone concentra tion of the sesame oil solution was calcu lated and expressed as mg per gm of 28 solution. 2. Dilution of stock solution. Solutions con taining three different levels of each of these hormones were prepared by diluting an aliquot of the stock solution to a definite volume with sesame oil. The con centrations of these solutions were as follows: Hormone Concentration (mg/ml) Low level Medium level High level Estradiol benzoate 0.80 1.60 3.20 (E.B.) Testosterone pro- 8.33 16.67 33.3 pionate (T.P.) In the studies of cholesterol biosynthesis in liver slices, these hormones were dis solved in ethylene glycol instead of in sesame oil for rats fed on a fat-free diet. The low level of each hormone was used. 3. Preparation of mixed hormone solutions. In addition to the hormone solutions mentioned above, two mixed sex hormone 29 solutions in different proportions were prepared as follows: (E.B. 10) Estrogenic -combined sex hormone solution -------- a------------------— ------- (T p x > This solution was prepared in sesame oil solution so that the concentration of estradiol benzoate in the mixed solution was the same as that of the low level of the estradiol benzoate solution (0.80 mg/ml) and the concentration of testo sterone propionate in the mixed solution was 1/10 of that of the low level of the testosterone propionate solution (0.833 mg/ml). (T.P. 10) Androgenic-combined sex hormone solution ------ — (E.B. 1 ) This solution was prepared in sesame oil solution so that the concentration of testosterone propionate in the mixed solu tion was the same as that of the low level of the testosterone propionate solution (8.33 mg/ml) and the concentration of 30 estradiol benzoate in the mixed solution was 1/10 of that of the low level of the estradiol benzoate solution (0.08 iqg/ml). All hormone solutions were kept under refrigeration when not in use. B. Ethinyl estradiol and methyl testosterone. For rats on a fat-free diet, two different male and female hormones were administered by mixing the hormone with the diet. Ethinyl estradiol (E.E.) was used for male rats and methyl testosterone (M.T.) was used for female rats. 24 mg of ethinyl estradiol were mixed with 6.72 kg fat-free diet, and 800 mg methyl testosterone were mixed with 10.08 kg fat- free diet. The hormone was dissolved in ethyl alcohol and then mixed with diet. V. Extraction of cholesterol and lipids. A. Extraction of plasma cholesterol. At the end of the experimental period, the rats were sacrificed under nembutal anaesthesia, and the blood was withdrawn from the heart or the bifurcation of the aorta with a heparinized 31 syringe. Plasma was obtained by centrifugation of whole blood at 3000 rpm for 20 minutes. Fourteen volumes of ethyl alcohol-acetone (1:1) (v,v) were added to each volume of plasma by means of a syringe under sufficient pressure to facilitate mixing; this precipi tated the plasma proteins, and extracted the cholesterol. The resulting suspension was centrifuged for 10 minutes at 3000 rpm and the supernatant was decanted and stored in a stoppered bottle for subsequent analysis. B. l Extraction of liver cholesterol and lipids. The liver was quickly extirpated, trimmed, and blotted. The following procedure based on a method of Thompson, et al. (94) was then employed. The tissue together with 30 ml of ethanol-water mixture (2:1) (v,v) was placed in a Waring Blendor; then 100 ml of Skelly- solve B (b.p. 63.3-69.3°C) were added and the mixture was homogenized for 5 minutes. Follow ing a 5-minute stationary period, the mixture was rehomogenized for 5 minutes. Then the contents were transferred quantitatively to a separatory funnel, the layers separated, and the homogenate layer was re-extracted twice more with 100 ml of Skellysolve B. The extracts were pooled, concentrated by evapora tion on a water bath, filtered into a volu metric flask, and adjusted to a definite volume. Aliquots were taken for lipid and cholesterol determinations. Extraction of adrenal cholesterol and lipids. A Soxhlet apparatus was used for the extrac tion of adrenal cholesterol and lipids. The pooled adrenals from each group were placed in a Soxhlet thimble and crushed carefully with a metal spatula and then refluxed for 8 hours with alcohol-ether mixture (3:2) (v,v). The extract was then transferred quantitatively into a volumetric flask and made up to volume. Extraction of cholesterol and lipids from kidneys, testes and ovaries. The extraction of cholesterol and lipids from kidneys, and testes was the same as that for the liver. In cases where the testes were very small due to 33 sex hormone administration, a micro-Waring Blendor was used, and the tissue was frozen with dry ice and then homogenized with Skellysolve B added with dry ice. The extraction of cholesterol and lipids from ovaries was the same as that for adrenals. VI. Determination of Cholesterol. The determination of cholesterol was performed by a modified Sperry-Schoenheimer method as reported by Nieft and Deuel (95). An aliquot of the tissue extract containing approximately 0.1 to 0.5 mg of cholesterol was evaporated to dryness in a 60° C constant tempera ture aluminum block by means of a stream of air. One ml of ethyl alcohol-acetone (1:1) (v,v) and 2 drops of 33 per cent potassium hydroxide were added; the tubes were corked and the samples were incubated at 60° C for 45 minutes with fre quent shaking. The solution was then neutralized to a phenolphthalein end point with 15 per cent acetic acid. Then 2 ml of ethyl alcohol-acetone 34 mixture were added. The cholesterol digitonide was precipitated by the addition of one ml of 0.5 per cent (w/v) digitonin solution (in 50 per cent (v/v) ethanol), and allowed to stand at room temperature overnight. The precipitate was then centrifuged at a speed of 3000 rpm for 20 minutes. The supernatant liquid was carefully decanted and the precipitate washed with 3 ml of anhydrous ether, added forcefully from a syringe to facilitate mixing. The tubes were then centrifuged at 3000 rpm for 10 minutes and the supernatant ether was discarded. The precipitate was dried with air at 60° C, 0.5 ml of glacial acetic acid was added, and the mixture was heated at 60° C until the precipitate was com pletely dissolved. Three ml of chloroform were added, and the tubes were brought to a temperature of 35° C. The color reagent, previously prepared by adding one ml of concentrated sulfuric acid to each 9 ml of chilled acetic anhydride, was then added. The tubes were incubated with occasional shaking for 10 minutes at 35° C at which time the 35 tubes were placed In an ice bath for at least 10 minutes prior to reading. The color intensity was then read in a Klett-Summerson photoelectric colorimeter at 620 mp. A reagent blank consisting of acetic acid, chloroform, and color reagent was used to set the zero point on the colorimeter. Values were read from a standard curve determined previously. Standards were always run with each series of samples. For the determination of free cholesterol, the same procedure used for total cholesterol was followed, with the exception that the hydrolysis with potassium hydroxide was omitted. VII. Determination of total lipids An aliquot of the petroleum ether extract of the tissue was evaporated to dryness in an Erlenmeyer flask on a water bath under a stream of air. The 30 ml of Skellysolve B and approxi mately 5 gm of anhydrous sodium sulfate were added and the contents well shaken. The solution was filtered through Whatman #42 filter paper into a tared Erlenmeyer flask and rinsed with five 36 successive portions of Skellysolve B, passing the rinsings through the filter paper and com bining the filtrates. The filtrate was evaporated to dryness on a water bath under a stream of air, dried overnight in an air oven at 88° C, and the lipids were weighed. VIII. Determination of serum and plasma phospholipids Unheparinized blood was centrifuged at 3000 rpm for 20 minutes to obtain serum. The serum or plasma was then extracted for lipids with ethyl alcohol-acetone (1:1) (v,v) according to the method mentioned before for the extraction of plasma cholesterol. The serum or plasma phospho lipids were determined by the method of Lowry et al.(96) as modified by Mehl et al. (97). The details of this method are as follows: A. Preparation of reagents. Standard phosphate solution. 100 mg of dried KH2PO4 was dissolved in distilled water and made up to 100 ml. For the working standards, 1 ml of this stock solution was diluted to 100 ml. The concentration of this solution 37 was 10 y per ml. Digesting solution. A small flask was half filled with distilled water. To this was slowly added 32.5 ml of 70 per cent per chloric acid, and 139 ml of concentrated sulfuric acid. The solution was diluted with distilled water to 1000 ml. Color reagent. 13.60 gm of Na acetate-3 t^Q and 2.5 gm of ammonium molybdate were dis solved in 1 liter of distilled water. 1 ml of 10% ascorbic acid was added to 10 ml of this mixture just before use. B. Procedure. 1 ml of the alcohol-acetone extract of serum or plasma was pipetted into a clean calibrated centrifuge tube and was evaporated to dryness in a water bath under a stream of nitrogen. 2 ml of digesting solution were added to each tube, and the tubes were placed in a metal rack and heated in the oven at 165° C for 36 hours. The tubes were then cooled and 10 ml of color reagent were added to each tube and mixed immediately. The samples were 38 then placed in a 38° C water bath for 2 hours. The optical density was read at 820 mp on the Beckman model B spectrophotometer. A standard curve was made by using the standard solution of phosphate in amounts ranging from 0.1 ml to 2 ml which is equiva lent to 1 Y to 20 T of phosphate. A reagent blank consisting of 2 ml of digesting solution and 10 ml of color reagent was used to adjust the zero point on the spectrophotometer. Standards were always run together with samples. Phospholipids were calculated by multi plying the phosphate by 25. IX. Starch block electrophoresis of serum lipoproteins The method was the one developed by Mehl elt al. (98). The procedures were as follows: A. Preparation of the starch block 1. Preparation of sodium barbital Buffer. 206.18 gm of sodium barbital were dis solved in 9800 ml of distilled water, and 100 ml of 1 N HCl solution were added. The solution was adjusted to pH 8.6 by the adding of 1 N HC1. This solution had an ionic strength of 1. Fi. reparation of the starch block. The apparatus for making the starch block con sisted of 5 plastic plates, which when held together by rubber bands, made an open box of 38.3 cm long, 3.2 cm wide and 2.3 cm high (inside dimensions). The serum sample was applied 14 cm from the end which should connect the cathode when current was passed. An excess of Baker's purified starch was weighed, mixed well with barbital buffer, and kept in a cold room at 0° C for 6 hours. The supernatant buffer was decanted and more buffer was added. The slurry was mixed well and kept in a cool room at 0° C for another 6 hours. After this procedure was repeated once again, the starch was ready for use. The starch which had been saturated and equilibrated with buffer was packed 40 in the plastic box so that there was no - bubbling and it was solid enough to retain its shape. It was trimmed to a smooth, flat surface on the top. The whole block except for the two ends was wrapped in parafilm and sealed with Scotch cellophane tape immediately. Separate parafilm should be used for wrapping the section to which the sample will be added. B. Equilibration of starch block. The starch block was equilibrated by passing a current through it for approximately 17 hours. The voltage regulator was set at 270 v producing a voltage across the block of about 145 v. The voltage and current were recorded at the beginning and end of the equilibration period. C. Electrophoresis of serum sample on starch block. After the equilibration of the starch block, 4 ml serum were mixed with Baker's purified starch in such proportions that the degree of moistening was approximately the same as that of the starch block. The wrapping paper in the sample space was opened and the sample was packed in well. A period of approximately 24 hours was needed for the electrophoresis of the sample. The voltages were adjusted in the same way as that of the equilibration of the starch block. The voltage and current were recorded at the be ginning and end of the run. Staining of paper for protein and lipids. At the end of the electrophoresis, paper staining for protein and lipids was done. The paper strips used were filter paper strips, part no. 300-028, obtained from Beckman Instru ments, Inc., Spinco Division, Belmont, Cali fornia. The wrapping paper was removed and a wetting pattern of the separated serum sample was obtained by holding the end of the strip on one side and touching the other side of the strip to the surface of the starch block. Two strips were made for protein and two more for lipids. The strips were dried 42 in the oven at 105° C for 15 minutes, and were stained with brom phenol blue for protein and oil red 0 for lipids. From the protein stain, the approximate location of albumin and alpha-, beta- and gamma-globulius were estimated. E. Extraction and determination of protein. The starch block was then cut up into 1 cm seg ments . Protein was eluted from each segment with 3 ml of 0.85 per cent NaCl solution. 0.1 ml of the supernatant solution from each segment was assayed for protein according to Folin-Ciocalteau method (99). F. Extraction and determination of cholesterol and phospholipids. After an aliquot of the NaCl eluting solution was taken for protein assay, the different segments were dried at room temperature, and extracted with ethyl alcohol-acetone (1:1) (v,v) at least 5 times. The mixture was warmed with constant stirring during extraction and filtered. The extracts were mixed well and the volume was made up to 50 ml. 43 Because of the minute amount of cholesterol present in each segment, cholesterol was deter mined by applying Zak's method (100) modified in the following way (101): 3-10 ml of the ethyl alcohol-acetone extract were pipetted into a clean calibrated tube, and the solution was evaporated to dry ness under a stream of nitrogen on a water bath. The tubes were then cooled and to each were added 3.6 ml of glacial acetic acid. They were sealed with parafilm and heated in a water bath to dissolve the precipitate. A color reagent was made beforehand as following: 1 ml of ferric chloride solution, prepared by dissolving 1 gm ferric chloride . 6 H2O in 10 ml of glacial acetic acid, was pipetted into a 100 ml graduated cylinder and concentrated sulfuric acid was added to the 15 ml level. The solution was mixed. If no precipitate appeared, additional concen trated sulfuric acid was added up to the 100 ml level. After the dried sample was dissolved com pletely in 3.6 ml of glacial acetic acid, 2.4 ml of color reagent were added. The mixture was shaken vigorously and cooled to room temperature in 20 minutes. The tubes were shaken and after 15 minutes the optical density was read at 560 mp on the Beckman model B spectrophotometer. Two blanks were made by mixing 3.6 ml of glacial acetic acid and 2.4 ml of color reagent. Standards were mixed simultaneously. A standard curve was made by using ali quots of standard cholesterol solution ranging from a volume containing 10 X up to a volume containing 180 y . From the reading and the standard curve, the cholesterol content in the aliquot of the sample was found. The phospholipid determination was the same as the one used for serum and plasma except that 5 ml aliquot of the ethyl alcohol-acetone extract was used. 45 X. Column chromatography of liver lipids. Rat liver lipids were separated into cholesterol esters, triglycerides and phospholipids by the method of Fillerup and Mead (102) as modified by Mukherjee et al. (103). A silicic acid column was prepared and washed with 3 column volumes each of methanol, acetone, ether, and pentane, respectively. Liver lipid extracts were pooled by group in such a way that the mixture contained equal weights of liver in each sample. The pooled mixture was then evapo rated in vacuo, dried with anhydrous sodium sul fate, and redissolved in a minimum amount of pentane. An aliquot was placed in the pre washed column and eluted with different solvent systems. The first fraction, cholesterol esters, was eluted by passing through the column 10 volumes of one per cent ether in pentane. The second fraction, triglycerides, and free cholesterol, was collected by washing the column with 10 volumes of 25 per cent ether in pentane. The 46 third fraction, phospholipids, was obtained by eluting with 8 volumes of 1:1 (v,v) methanol-ether mixture and 2 volumes of absolute methanol. All the three fractions were dried with anhydrous sodium sulfate, evaporated, and weighed. XI. Determination of iodine value (Wijs). The unsaturation of each fraction was determined by its iodine value in the following way: The dried and weighed fraction was dissolved in chloroform. To an aliquot of this chloroform solution, a sufficient amount of 0.1N iodine monochloride in glacial acetic acid (Wijs reagent) was added. Blanks were run simultaneously. The glass-stoppered flasks were kept in the dark for one hour. The reaction was stoppedby the addi tion of 15 ml of 10 per cent KI solution. The mixture was diluted with distilled water and titrated with 0.1 N sodium thiosulfate. The iodine value was expressed as the number of grams of iodine absorbed by 100 grams of the individual fraction separated from the column. 47 XII. Biosynthesis of cholestrol. The method of Hotta et al. (104) was adapted in the following way: A. Incubation. The rats were sacrificed by cervical fracture. Their livers were quickly excised, trimmed, blotted, and weighed. Dupli cate portions of liver were placed in a Krebs-Ringer phosphate buffer (105) in a beaker in an ice bath. Slices were prepared with a Stadie-Riggs microtome. The slices were placed in beakers tared with a small amount of ice-cold Krebs-Ringer phosphate buffer. The beakers were reweighed with the slices and then the slices, usually weighing between 300 and 600 mg, were transferred into Erlenmeyer flasks containing 5 ml of buffer and one ml of sodium acetate-l-C^ which had been equilibrating in a water bath at 37.5° C. The flasks containing the liver slices were flushed with 95 per cent 02“5 per cent CO2 mixture, stoppered, and placed back in the water bath where they were shaken for 3 hours. 48 14 B. Extraction of cholesterol and assay of C content. The medium was decanted and the liver slices were rinsed with distilled water and hydrolyzed with 2 ml of a freshly prepared solution of sodium ethylate (0.25 gm of Na dissolved in 10 ml of 95 per cent alcohol) for 30 minutes on a steam bath. After the addition of a small quantity of water, the flask was placed on a steam bath to evaporate the alcohol. The contents were then acidified to the brom cresol green end point with concentrated HC1. Exactly 10 ml of chloroform were added, and the contents were vigorously shaken and transferred to a centrifuge tube. The chloroform phase was clarified by centrifugation. An aliquot of this chloroform solution was evaporated to dryness at 60° C in a stream of air, and the residue redissolved in 5 ml of ethyl alcohol- acetone mixture (1:1 (v,v). About 5 ml of an alcohol-acetone solution of unlabeled cholesterol (10 mg per ml) were added and 49 | ! total cholesterol was precipitated as the dlgltonide with a one per cent digltonin I solution in 50 per cent ethanol. The mixture j i was allowed to stand overnight. The following' I ! day the precipitate was centrifuged at 3000 rpm for 30 minutes and washed three times with anhydrous ether. The washings were repeatedly checked for radioactivity and only negligible counts were detected. Finally, the ethanol suspension of the digitonide was diluted to a definite volume and aliquots were plated in duplicate. The planchets were dried overnight in a desic cator and the radioactivity was measured using a gas flow counter and a Nuclear Auto scaler. All counts were corrected for background and self-absorption and the results were expressed as counts per minute per gm of liver. 50 Plan of Experiments As a result of studies in our laboratory concern ing the relationship between cholesterol metabolism and essential fatty acids (30,31,57) it was shown that the accumulation of cholesterol in the liver of rats on either essential fatty acid-deficient, cholesterol-free diets or high fat, high cholesterol diets was much greater in male animals than in female animals. It therefore seemed important to understand thoroughly the role of sex hormones in this and other phases of cholesterol metabo lism. Four methods of approach were planned: (1) gona- dectomy which results in an absence of sex hormones; (2) the administration of female hormone to the intact male animal and male hormone to the intact female animal to neutralize any effects which may be evident; (3) the administration of female hormone to the intact female ani mal and male hormone to the intact male animal to enhance any effect which may be evident; and (4) the administration of the mixtures of both male and female hormones in differ ent proportions to both male and female animals. Three experimental diets were used: a 15 per cent cottonseed oil diet which was the reference diet (cholesterol-free 51 diet), a 15 per cent cottonseed oil diet containing 1 per cent cholesterol and a fat-free diet. The Indices of cholesterol metabolism which were studied included cho lesterol determinations in plasma, liver, adrenals, kidneys, testes and ovaries, total lipid determinations in all tissues mentioned except plasma, phospholipid deter minations in plasma, hepatic cholesterol biosyntheses, separation of liver lipid fractions; separation of serum alpha- and beta-lipoprotein-bound cholesterol and phospho lipids and their determinations thereof. Effects of gonadectomv on cholesterol metabolism I. Effects of gonadectomv on the cholesterol and total lipid concentrations of tissues of rats fed cottonseed oil diet. 20 male and 20 female rats, of which half of each sex were gonadectomized at weaning, were fed a Purina Chow diet for 10 weeks post-weaning and, then, a cottonseed oil diet for 6 additional weeks. II. Effects of gonadectomy on the cholesterol and total lipid concentrations of tissues of rats fed the reference cottonseed oil diet containing choles- 52 terol. 20 male and 20 female rats, of which half of each sex were gonadectomized at weaning, were fed Purina Chow diet for 10 weeks post-weaning and then a cottonseed oil diet containing cholesterol for 6 additional weeks. III. Effects of gonadectomv on the cholesterol and total lipid concentrations of tissues of rats fed a fat-free diet. 20 male and 20 female rats, of which half of each sex were gonadectomized at weaning, were fed a fat-free diet for 20 weeks post-weaning. All animals in experiments I, II and III were arranged in such a way that each group in the same sex had a very similar average weight at weaning and litter mates were distributed in different groups as much as possible. They were weighed once a week. At the end of the experimental period, the animals were sacrificed and cholesterol in plasma, liver and adrenals and total lipids in liver and adrenals were determined. 53 IV. Effects of gonadectomv on serum lipoprotein pat terns in rats fed the reference cottonseed oil diet with and without cholesterol. 24 male and 24 female rats, of which half of each sex were gonadectomized 6 weeks post-weaning, were fed Rockland rat diet for 8 weeks post- weaning, then a cottonseed oil diet for 10 days and then the cottonseed oil diet with and without cholesterol for 6 additional weeks. They were regrouped before they were put on experimental diets for 6 weeks in such a way that each group in the same sex had a close average weight and litter mates were distributed in different groups as much as possible. All animals were weighed weekly. They were divided into 8 groups as follows: Sex Gonadectomy Experimental diet Male - C Male + C Male - CC Male + CC Female - C Female + C Female - CC Female + CC 54 All animals were sacrificed at the end of the experimental period and the lipoprotein pattern was determined in the sera of all groups. V. Effects of gonadectomv on the percentage distribu tion of liver lipid fractions and the degree of their unsaturation in rats fed a reference cotton seed oil diet with and without cholesterol. The total lipids were extracted from the livers of those rats the sera of which were analyzed for their lipoprotein patterns. The total liver lipids were pooled by group and separated into cholesterol esters, triglycerides and phospho lipids by silicic acid column chromatography. The degree of the unsaturation of each fraction was determined by measuring the iodine value. The percentage distribution of each fraction was cal culated by the assumption that the sum of the isolated fractions was 100 per cent. VI. Effects of gonadectomv on the biosynthesis of cholesterol. Intact and gonadectomized male and female rats corresponding to the experimental groups in 55 I, II and III were used for these studies. The liver slices were incubated with acetate-l-C^ 14 and the incorporation of C into cholesterol was measured according to the method of Hotta et al. (104). Effects of Sex Hormone Administration on Cholesterol Metabolism VII. Effects of estradiol benzoate on the cholesterol and total lipid concentrations of tissues of male rats fed the reference cottonseed oil diet with and without cholesterol. 80 male rats were fed a 15 per cent cottonseed oil diet until they were four months old and then put on experimental diets for 6 weeks. Different levels of estradiol benzoate in sesame oil were given twice a week by subcutaneous injection on the back. Sesame oil alone was given to control animals. Injections were initiated 10 days prior to the time the animals were fed the experimental diets. Animals were weighed once a week and the amount of hormone or sesame oil used was adjusted 56 to the body weight of each week. The groups were as follows: * VIII. Experimental Level of Estradid Benzoate diet (mg/0.1 ml/100 gm body weight) c none c 0.08 c 0.16 c 0.32 cc none cc 0.08 cc 0.16 cc 0.32 At the end of the experimental period animals were sacrificed. Cholesterol in plasma, liver, adrenals, testes and kidneys and total lipids in liver, adrenals, testes and kidneys were measured. Effects of testosterone propionate on the choles terol and total lipid concentrations of tissues of female rats fed the reference cottonseed oil diet with and without cholesterol. 80 female rats were fed a 15 per cent cotton seed oil diet until they were four months old and then given the experimental diets for 6 weeks. Different levels of testosterone propionate in sesame oil were given twice a week by subcutaneous injection on the back. Sesame oil alone was given to the control animals. Injections were started 10 days before the animals were fed the experi mental diets. Animals vrere weighed once a week and the amount of hormone or sesame oil used was adjusted to the body weight each week. The groups were as follows: Experimental Level of Testosterone diet Propionate (mg/0.1 ml/100 gm body weight) c none c 0.83 c 1.67 c 3.33 cc none cc 0.83 cc 1.67 cc 3.33 At the end of the experimental period, animals were sacrificed. Cholesterol in plasma, liver, adrenals, ovaries and kidneys and total lipids in liver, adrenals, ovaries and kidneys were measured. 58 IX. Effects of estrogenic-combined sex hormones on the cholesterol and total lipid coneetitrations of tissues in male rats fed a reference cottonseed oil diet with and without cholesterol. To avoid the feminizing effect of estrogen but to retain its influences on lipid metabolism, the estrogenic-combined sex hormones were given to the male rat. Male rats were fed Rockland rat diet for 6 weeks post-weaning and then the cottonseed oil diet until they were three months old. They were then fed the experimental diets for 6 further weeks. Hormone or sesame oil injections were given twice a week and they were started 10 days before rats were fed the experimental diets. Animals were weighed once a week and the amount of hormone used was adjusted to the body weight each week. Food consumption was also measured in certain periods. Each group contained more than 10 rats. The groups were as follows: Experimental diet Sex Hormone (amount/.1 ml/100 gm body weight) C C CC CC CC 0.08 mg E.B. + 0.083 mg T.P. 0.08 mg E.B. 0.08 mg E.B. + 0.083 mg T.P. none none Analyses of cholesterol and total lipids in tissues were done the same as in experiment series VII at the end of the experimental period. Plasma phospholipids were also determined in these groups. Effects of adrogenic-combined sex hormones on the cholesterol and total lipid concentrations of tissues in female rats fed a reference cottonseed oil diet with and without cholesterol. To avoid the masculinizing effect of androgen but to retain its influences on lipid metabolism, the androgenic-combined sex hormones were given to the female rat. Female rats were fed a Rockland rat diet for 6 weeks post-weaning and then a cottonseed oil diet until they were three months old. They were given experimental diets for 6 weeks after that time. Hormone or sesame oil injections were given twice a week and were started 10 days before rats were given the experimental diets. The animals were weighed once a week and the amount of hormone used was adjusted to the body weight each week. Food consumption was measured in certain periods. Each group contained more than 10 rats. The groups were as follows: Experimental Sex Hormone diet (amount/0.1 ml/100 gm body weight) C none C 0.83 mg T.P. + 0.008 mg E.B. CC none CC 0.83 rag T.P. + 0.008 mg E.B. CC 0.83 mg T.P. Cholesterol and total lipid levels of tissues were determined the same way as in experiment series VIII. The plasma phospholipids were also determined in these groups. 61 XI. Effects of testosterone propionate on the choles terol and total lipid concentrations of tissues of male rats fed a reference cottonseed oil diet with and without cholesterol. 40 male rats were fed a Rockland rat diet post weaning until they were two and a half months old. They they were fed a cottonseed oil diet for 10 days. Hormone or sesame oil injections were started at the beginning of these 10 days. They were given experimental diets for 6 weeks after that time. The low level of testosterone pro pionate solution was used. Rats were injected twice a week and were weighed weekly. The groups were as follows: Experimental diet Testosterone Propionate (mg/0.1 ml/100 gm body weight) C C CC CC none none 0.83 0.83 Analyses of tisues were performed as in experiment series VII. 62 XII. Effects of estradiol benzoate on the cholesterol and total lipid concentrations of tissues of female rats fed a reference cottonseed oil diet with and without cholesterol. 40 female rats were fed a Rockland rat diet post-weaning until they were two and a half months old. Then they were fed a cottonseed oil diet for 10 days. Hormone or sesame oil injec tions were started at the beginning of these 10 days. They were given experimental diets for 6 weeks after that time. The low level of estradiol benzoate solution was used. Rats were injected twice a week and were weighed weekly. The groups were as follows: Experimental diet Estradiol Benzoate (mg/0.1 ml/100 gm body weight) C C CC CC none none 0.08 0.08 Analyses of tissues were performed as in experiment series VIII. 63 XIII. Effects of ethinyl estradiol on the cholesterol and total lipid concentrations of tissues in male rats fed a fat-free diet. Since ethinyl estradiol is more effective than estradiol benzoate in oral feeding, therefore, it was used here instead of estradiol benzoate. 30 male rats were fed a fat-free diet for 10 weeks post-weaning. For 8 additional weeks, 20 of them were given a fat-free diet mixed with ethinyl estradiol and the rest were continued on the fat- free diet as controls. Some of the animals given ethinyl estradiol were sacrificed at varying intervals during the period of the last 8 weeks to observe the possible alleviation of the fatty liver. They were weighed once a week. The food consumption was measured in certain periods. Analyses of tissues were performed as in experiment series IX. XIV. Effects of methyl testosterone and the cholesterol and total lipid concentrations of tissues in female rats fed a fat-free diet. Since methyl testosterone is more effective 64 than testosterone propionate in oral feeding, therefore, it was used here instead of testo sterone propionate. 30 female rats were fed a fat-free diet for 10 weeks after weaning. The 20 of them were given a fat-free diet mixed with methyl testo sterone and the rest were continued on the fat- free diet for 8 weeks. Some of the animals given methyl testosterone were sacrificed at varying intervals during the period of the last 8 weeks to observe the degree of the fatty infiltration of the liver. The food consumption was measured in certain periods. XV. Effects of sex hormone administration on the plasma phospholipid level and the plasma — ratio in male and female rats fed the reference cotton seed oil diet with and without cholesterol and fed the fat-free diet. To determine whether there is any correlation between the plasma phospholipid level or the plasma' ratio and sex hormones, an analysis of plasma phospholipids and a calculation of 65 Q plasma — ratio were done on all groups in experiment series IX, X, XIII, and XIV, and the results were compared. Analyses of tissues were performed as in experiment series X. XVI. Effects of sex hormone administration on the per centage distribution of liver lipid fractions and the degree of their unsaturation. The pooled samples of liver extracts in experiments IX, X, XIII and XIV were subjected to silicic acid column chromatography for separation of cholesterol esters, triglycerides and phospho lipids , and the iodine values of these fractions were determined. XVII. Effects of sex hormone administration on the bio synthesis of cholesterol. The effects of sex hormone administration on the biosynthesis of cholesterol were studied in male and female rats fed on different diets. In rats fed with a cottonseed oil diet with and with out cholesterol, representative male and female rats with and without hormone treatment corre sponding to the groups in experiment series VII (low level of estradiol benzoate to males), VIII (low level of testosterone propionate to females), IX (estrogenic-combined sex hormones to males), and X (androgenic-combined sex hormones to females) were used. In rats fed a fat-free diet, the male and female used were the same age as the animals in experiment series XIII and XIV but the low levels of estradiolbenzoate and testosterone propionate in ethylene glycol were given to the hormone treated animals instead of feeding ethinyl estradiol and methyl testosterone. CHAPTER III RESULTS AND DISCUSSION Effects of Gonadectomy on Cholesterol Metabolism Body weight. The effects of gonadectomy on the body weight of male and female rats fed three different diets are shown in Table III. In all cases, gonadectomy of male rats causes a decrease and gonadectomy of female rats causes an increase in the body weight as compared with the intact male and female animals. The decrease in the body weight is more marked in the gonadectomized male fed the cottonseed oil diet containing cholesterol and the increase in the body weight is more marked in the gonadectomized female fed either the reference cottonseed oil diet or this reference diet containing cholesterol. It seems that the sex difference in body weight dis appears in the gonadectomized male and female rats so that both male and female rats have a close body weight after gonadectomy. If sex hormones are the only factor to 67 68 TABLE III EFFECT OF GONADECTOMY ON THE BODY WEIGHT OF R4 Diet Sex Number of rats Gonadectomy (at weaning) Age at sacrifice (months) C1 M 10 4.5 10 + 4.5 F 10 - 4.5 9 + 4.5 CC2 M 10 - 4.5 9 + 4.5 F 9 - 4.5 11 + 4.5 FF3 M 9 - 5.5 9 + 5.5 F 9 - 5.5 8 + 5.5 ^On7Purina Chow for 10 weeks post-weaning and then diet for 6 weeks. 9 On Purina Chow for 10 weeks post-weaning and then diet containing 1 per cent cholesterol for 6 weeks 3 On fat-free diet for 20 weeks post-weaning. TABLE III ON THE BODY WEIGHT OF RATS FED DIFFERENT DIETS Body weight Gonadectomy Age at Mean weight Per cent (at weaning) sacrifice (gm) change (months) from con trol 4.5 336 mm + 4.5 315 -6 - 4.5 230 - + 4.5 305 +33 - 4.5 400 - + 4.5 320 -20 - 4.5 252 - + 4.5 311 +23 — 5.5 251 - + 5.5 230 -8 - 5.5 190 - + 5.5 218 +5 post-weaning and then on 15 per cent cottonseed oil i post-weaning and then on 15 per cent cottonseed oil cholesterol for 6 weeks. sks pos t-wean ing. 69 influence the body weight being heavier in the male and lighter in the female, this result will be reasonably expected. Tissue cholesterol and total lipid concentrations. The effects of gonadectomy on the cholesterol concentra- tion in the plasma and on the cholesterol and total lipid concentrations in the liver and adrenals are presented in Tables IV to VI. The total cholesterol concentrations of plasma and liver are also shown graphically in Figures 1 and 2. In rats fed the reference cottonseed oil diet the intact female has the same plasma cholesterol concentra tion and a lower liver cholesterol concentration as com pared with the intact male. This latter observation agrees with that reported by Aftergood et al. (57). Gonadectomy produces little change in these values, though the total plasma cholesterol may be slightly elevated in the gona dectomized female. Fillios reported an elevation in plasma cholesterol in both gonadectomized male and female rats fed a laboratory Chow.(58). This difference in result may be due to the difference in time of gonadectomy (performed on weaning rats in this investigation and on adult rats 70 TABLE IV EFFECT OF GONADECTOMY ON THE PLASMA < OF MALE AND FEMALE RATS AS II Category*- Diet2 Sex Number of rats Intact C M 10 ! Gonadectomized C M 10 1 Intact C F 10 ! Gonadectomized C F 9 i: Intact CC M 10 Gonadectomized CC M 9 J Intact CC F 9 i: Gonadectomized CC F li i: Intact FF M 9 Gonadectomized FF M 9 Intact FF F 9 Gonadectomized FF F 8 ^Gonadectomized at weaning. ^Duration on diet C - 6 weeks. Duration on die FF - 20 weeks post-weaning. 3 Includes standard error of mean. 70 TABLE IV ADECTOMY ON THE PLASMA CHOLESTEROL CONCENTRATIONS LE AND FEMALE RATS AS INFLUENCED BY DIET Cholesterol_________ _______________ Sex Number Total3 Free2 Per cent of rats (mg per cent) (mg per free cent) M 10 90.0 ± 3.7 15.7 + 0.7 17.6 M 10 94.7 + 4.0 14.3 + 0.7 15.1 F 10 96.7 + 4.1 17.4 + 1.2 18.7 F 9 112 + 5.0 16.8 ± 1.5 14.8 M 10 76.0 + 3.6 16.0 + 1.0 21.1 M 9 89.2 + 4.2 20.4 + 1.0 22.6 F 9 125 + 3.4 25.7 + 3.0 21.0 F 11 124 + 7.4 35.2 + 2.2 31.6 M 9 44.7 3.0 5.7 ± 0.7 12.8 M 9 55.3 + 3.6 8.5 + 0.7 15.6 F 9 51.8 ± 3.1 9.5 + 1.1 18.4 F 8 52.8 + 3.0 14.2 + 1.1 26.9 ng. weeks. Duration on diet CC - 6 weeks. Duration on diet ling. ■ of mean. 71 TABLE V EFFECT OF GONADECTOMY ON THE CONCENTRATIO TOTAL LIPIDS OF MALE AND FEMALE RATS Category*- Diet2 Sex Number of rats - Intact C M 10 2.62 ± 0. Gonadectomized C M 10 2.54 + mm 0. : Intact C F 10 2.17 + 0. Gonadectomized C F 9 2.13 + 0. Intact CC M 10 28.5 + 2. Gonadectomized CC M 9 20.5 + 3. Intact CC F 9 20.2 + 3. Gonadectomized CC F 11 25.5 + 1. Intact FF M 9 5.73 + 0. Gonadectomized FF M 9 3.35 0. Intact FF F 9 2.29 + 0. Gonadectomized FF F 8 2.95 ± 0. ^-Gonadectomized at weaning. ^Duration on diet C - 6 weeks. Duration on d diet FF - 20 weeks post-weaning. 3 Includes standard error of mean. TABLE V OMY ON THE CONCENTRATIONS OF LIVER CHOLESTEROL AND OF MALE AND FEMALE RATS AS INFLUENCED BY DIET _________________ Cholesterol__________________ Total Number ,To<ialv3 , Free o cent ^ip:idSv of rats (mg/gm)J (mg/gm) free (mg/gm) 10 2.62 ± 0.09 2.13 + 0.05 81.2 54.5 + 2.6 10 2.54 + mm 0.07 2.07 ± 0.06 81.8 49.5 + 2.0 10 2.17 + 0.07 1.83 + 0.06 84.7 51.4 + 2.5 9 2.13 ± 0.09 1.84 + 0.08 86.4 44.7 + 4.2 10 28.5 + 2.5 4.05 + 0.34 14.2 132 ± 6.2 9 20.5 + 3.0 3.04 + mm 0.15 13.2 119 + 12.7 9 20.2 + 3.4 2.85 + 0.19 14.1 97.3 + 6.4 11 25.5 + 1.5 3.96 ± 0.31 15.5 138 + 5.1 9 5.73 + 0.71 1.89 ± 0.08 35.0 85.3 + 6.9 9 3.35 ± 0.23 1.93 ± 0.06 57.6 52.0 + 2.5 9 2.29 + 0.10 1.70 + 0.04 74.2 51.5 + 2.1 8 2.95 ± 0.13 1.75 ± 0.07 59.3 57.6 4.7 ning. 6 weeks. Duration on diet CC - 6 weeks. Duration on st-weaning. or of mean. TABLE VI EFFECT OF GONADECTOMY ON THE CONCENTRAT TOTAL LIPIDS OF MALE AND FEMALE RA' Category'*' Diet^ Sex Number Total of rats (mg/gm) Intact C M 10 36.0 Gonadectomized C M 10 39.0 Intact C F 10 46.0 Gonadectomized C F 9 40.3 Intact CC M 10 40.5 Gonadectomized CC M 9 49.2 Intact CC F 9 56.0 Gonadectomized CC F 11 43.6 Intact FF M 9 57.5 Gonadec t omized FF M 9 56.1 Intact FF F 9 50.1 Gonadectomized FF F 8 59.3 ^-Gonadectomized at weaning. 2 Duration on diet C - 6 weeks. Duration on di€ diet FF - 20 weeks post-weaning. ^All values were determined from pooled sample 72 TABLE VI :t om y on the concentrations of a d r e n a l cholesterol and 5 OF MALE AND FEMALE RATS AS INFLUENCED BY DIET 3 ex Number of rats Cholesterol3 Total3 lipids (mg/gm) Total (mg/gm) Free (mg/gm) Per cent free M 10 36.0 7.2 20.0 285 M 10 39.0 7.0 18.0 263 F 10 46.0 8.1 17.6 242 F 9 40.3 6.7 16.6 238 M 10 40.5 6.3 15.6 291 M 9 49.2 6.5 13.4 259 F 9 56.0 10.0 17.9 317 F 11 43.6 8.3 19.0 256 M 9 57 .5 3.5 6.1 256 M 9 56.1 4.4 7.8 308 F 9 50.1 6.1 12.1 256 F 8 59.3 3.8 6.5 291 .ng. weeks. Duration on diet CC - 6 weeks. Duration on -weaning. ned from pooled sample per group. 73 Ml Intact male M2 Gonadectomized male FI Intact female F2 Gonadectomized female CSO On 15% cottonseed oil diet for 6 weeks CSOC On 15% cottonseed oil diet containing 1% cholestrol oao ^or ^ weeks "l FF On fat-free diet for 20 weeks 020] post-weaning 200. 180- 160 _ 140. 120 A 100 80, 60- 40 20 V l l 5*1F2 M3 M2 FI F2 CSO CSOC Ml M2 FI F2 FF Fig. 1 TDffect of gonadectomy on plasma cholesterol level (mgF) Ml Intact male M2 Gonadectomized male PI Intact female P2 Gonadectomized female GSO On 15% cottonseed oil diet for 6 weeks GSOO On 15% cottonseed oil diet containing 1% cholestrol for 6 weeks PP On fat-free diet for 20 weeks post-weaning 30H 28- 26- 24- 22. 20. 18- 16- 14-. 12- 10- 8- 6* 4- 2- M1 ' ' , 1 2 FI P2 Ml M2 PI F2 Ml M2 mmmm f:F2 CSO C80C PP Pig. 2 Effect of gonadectomy on liver cholesterol level(mg per gram) 75 in the report by Fillios). Changes, due to gonadectomy are marked when rats are fed the cottonseed oil diet supplemented with choles terol. As was previously-reported by Aftergood et al. (57), the intact female has a significantly higher plasma total cholesterol concentration and a lower liver cholesterol concentration than does the intact male. The plasma free cholesterol is increased in both gonadectomized males and females as compared with the intact animals. Gonadectomy causes an increased plasma total cholesterol level in the male, but causes no change in the female. This agrees in part with the results obtained by Moskowitz et al. who reported that gonadectomy of both male and female rats fed 2 per cent cholesterol increases serum cholesterol (64). However, it is in disagreement with Fillios who reported that in rats fed a 5 per cent cholesterol diet there is no change in the plasma cholesterol in the gonadectomized male but a decrease in the gonadectomized female (58). These discrepancies may be partly explained by: (1) dif ference in time of gonadectomy; (2) difference in strain; (3) difference in age of animals at the time of starting experiments; (4) difference in the duration of experimental period. 76 Both the total and free cholesterol concentrations In liver are decreased In the gonadectomized male and Increased In the gonadectomized female. Conversely, gona- dectomy of the male causes an Increase and gonadectomy of the female causes a decrease in adrenal cholesterol content * Gonadectomy also results In a change In the total lipid concentrations in liver and adrenals. The total lipids.in the liver are decreased slightly in the gonadectomized male but increased significantly in the gonadectomized female. The total lipids in the adrenal are decreased in both gonadectomized males and females. From a comparison between rats fed the cottonseed oil diet with and without cholesterol, it is obvious that marked differences resulting from gonadectomy when an atherogenic (cholesterol-containing) diet is fed to both male and female animals. A difference in liver cholesterol concentration exists between intact male and female rats on a fat-free diet; the liver cholesterol concentration of the male is approximately twice that of the female. In the intact male fed a fat-free diet, plasma cholesterol concentrations are decreased whereas both liver and adrenal cholesterol 77 levels are elevated as compared with values observed In the Intact male fed a diet containing essential fatty acids. This confirms the work reported by Alfin-Slater et al. (30,31). Although the Intact female also has a reduced plasma cholesterol level resulting from the fat- free diet, the liver and adrenal cholesterol concentra tions are not affected. Whereas gonadectomy causes little change in the cholesterol content of the plasma of rats fed the fat-free diet, differences in cholesterol concentrations in the liver are observable; the liver cholesterol concentration of the orchidectomized male is decreased, and that of the ovariectomized female is increased as compared with unoperated controls. The adrenal cholesterol concentra tion of rats fed a fat-free diet is unchanged in the gonadectomized male and increased in the gonadectomized female. These results of liver and adrenal cholesterol con centrations indicate an increased requirement for essential fatty acids in the intact male and gonadectomized female fed the fat-free diet. Based on the assumption postulated by Alfin-Slater et al. that essential fatty acids are 78 required for the proper esterificatlon of cholesterol and its subsequent transport (30), it is possible that estrogens may spare the essential fatty acids stored in the body or may possibly potentiate the transport system in which cholesterol esterified with the essential fatty acids, is involved. In rats fed the fat-free diet, the liver lipids reflect the liver cholesterol picture very closely. In the adrenal, however, in both male and female animals, gonadectomy results in an increase in total lipids. From the foregoing results it is clear that the sex difference in the tissue cholesterol and total lipid concentrations which occur in rats is emphasized when rats are subjected to atherogenic diets; e.g., the diet con taining cholesterol or the diet containing no essential fatty acids. A comparison between the effects of gonadectomy on the cholesterol and total lipid levels of tissues in rats fed a cholesterol diet and those in rats fed a fat- free diet reveals the following: (1) In rats on both atherogenic diets, the plasma total cholesterol is slightly increased in the gonadectomized male as compared with the intact male fed the- same diet; but there is no change in the gonadectomized female as compared with the intact female fed the same diet. The plasma free choles terol is increased in the gonadectomized male and female on both atherogenic diets as compared with the intact animals on the same diet. (2) The liver total cholesterol concentration is decreased in the gonadectomized male and increased in the gonadectomized female in rats on both diets as compared with the intact animals on the same diet. The liver free cholesterol reflects the same changes as does the liver total cholesterol in the gonadectomized male and female fed cholesterol, whereas it does not change in the gonadectomized male and female fed the fat-free diet. (3) The liver total lipids show no change in the gonadectomized male but are increased in the gonadectomized female in rats fed cholesterol; they are decreased in the gonadectomized male but are unchanged in the gonadectomized female fed the fat-free diet. (4) The response of adrenal cholesterol and adrenal total lipids to gonadectomy is quite different between rats fed choles terol and those fed the fat-free diet. In rats fed cholesterol, the adrenal cholesterol is increased in the gonadectomized male but decreased in the gonadectomized 80 female. There Is no change In the gonadectomized male fed the fat-free diet but there is an Increase in the gonadectomized female. The adrenal free cholesterol shows little change due to gonadectomy in rats fed cholesterol but is increased in the gonadectomized male and decreased ' in the gonadectomized female in rats fed the fat-free diet. The adrenal total lipids are decreased in both gonadectomized male and female rats fed cholesterol, whereas they are increased in both gonadectomized male and female rats fed the fat-free diet. The process of gonadectomy involves the removal of the sex organs, which results in the absence of sex hormones, either formed by or stored in these gonadal organs. If these hormones control cholesterol metabolism, it would seem that in their absence, cholesterol values in organs where a sex difference was observed in the intact animals, would no longer be different in the gonadectomized animals. This theory is only partially substantiated in the results observed (Figures 1 and 2). In the liver cholesterol of gonadectomized male and female rats fed a fat-free diet, the values obtained are quite similar. Why other values in gonadectomized animals still 81 retain differences confirms the fact that there are many factors besides sex hormones, which influence cholesterol metabolism. Also, the adrenal glands may produce a small amount cf sex hormones which may be active to a limited extent in influencing cholesterol metabolism. The adrenal cholesterol level is a reflection of stress conditions, thus, the values obtained are not a valid reflection of dietary or hormonal influences on cholesterol levels in this organ. It is, of course, assumed that since all animals were treated comparably the influences of stress will be constant. / Serum lipoprotein patterns. The effects of gonadectomy on the serum lipoprotein patterns in rats fed the cottonseed oil diet with and without cholesterol are shown in Table VII to IX. In Table VII, in rats fed a 15 per cent cottonseed oil diet, the change in the cholesterol concentration in the beta-lipoprotein fraction due to gonadectomy is sig nificant, although gonadectomy causes little change in the cholesterol concentration in the alpha-lipoprotein fraction. The beta-lipoprotein"bourid cholesterol is decreased in gonadectomized males and increased in TABLE VII EFFECT OF GONADECTOMY ON THE RATIO OF ALPHA "LIPOPROTEIN CHOLESTEROL TO BETA-LIPOPROTEIN CHOLESTEROL IN RAT SERUM Category1 Sex Diet^ o Lipoprotein Cholesterol^ (mg in 100 ml serum) C Alpha^ C Beta Number of rats Alpha Beta Intact M C 5 46.7 5.00 9.35 Gonadectomized M C 6 48.3 2.75 17.39 Intact F C 6 43.3 4.25 9.95 Gonadec t omiz ed F C 6 50.0 6.00 8.34 Intact M CC 6 65.0 5.25 11.76 Gonadectomized M CC i 5 88.7 4.50 19.72 Intact F CC 6 122 4.75 25.70 Gonadectomized F CC 6 95.8 14.2 6.71 -^Gonadectomized at adult (6 weeks post-weaning). 2 Duration on diet - 6 weeks. 3 All values were determined from pooled sample per group. oo TABLE VIII EFFECT OF GONADECTOMY ON THE PHOSPHOLIPID CONCENTRATION OF LIPOPROTEINS IN RAT SERUM Category^- Sex Diet2 Number * 3 Lipoprotein phospholipids'' (ms in 100 ml serum) of rats Alpha Beta Intact M C 5 60.0 20.2 Gonadectomized M C 6 59.8 20.2 Intact F C 6 53.5 21.2 Gonadectomized F C 6 63.3 17.5 Intact M CC 6 61.5 14.7 Gonadectomized M CC 5 76.7 18.0 Intact F CC 6 79.0 28.8 Gonadectomized F CC 6 66.8 21.5 Gonadectomized at adult (6 weeks post-weaning). 2 Duration on diet - 6 weeks. 3 All values were determined from pooled sample per group. 00 u> TABLE IX EFFECT OF GONADECTOMY ON THE RATIO OF CHOLESTEROL TO PHOSPHOLIPIDS IN THE LIPOPROTEINS IN RAT SERUM Category^ Sex Diet2 Number of rats Alpha C/P Ratio3 Beta Intact M C 5 0.78 0.24 Gonadectomized M C 6 0.81 0.14 Intact F C 6 0.83 0.22 Gonadectomized F C 6 0.79 0.35 Intact M CC 6 1.05 0.36 Gonadectomized M CC 5 1.16 0.25 Intact F CC 6 1.55 0.17 Gonadectomized F CC 6 1.43 0.66 -^Gonadectomized at adult (6 weeks post-weaning). 2 oo Duration on diet - 6 weeks. 3A11 values were calculated from those obtained from the pooled sample per group. gonadectomized females. In rats fed the cottonseed oil diet supplemented with cholesterol, gonadectomy results in changes in'the cholesterol concentration in the beta- lipoprotein fraction in the same direction as in those animals not receiving cholesterol; whereas, the choles** terol concentration in the alpha-lipoprotein fraction is changed in an opposite direction. The alpha-lipoprotein bound cholesterol is increased in gonadectomized males but decreased in gonadectomized females. If sex hormones controlled the cholesterol content of the alpha- and beta-lipoproteins, it would be expected that the values in the gonadectomized animals of both sexes would be the same. This premise does obtain in the alpha- lipoprotein fraction of the rats fed both diets where the values after gonadectomy are similar. However, the cho lesterol content of the beta-lipoprotein fraction does not behave in this way. In gonadectomized males on both diets, there is a decrease in beta-lipoprotein cholesterol to a value less than or similar to that existing in the intact female; whereas in the gonadectomized females on both diets, there is an increase in beta-lipoprotein cholesterol to a value greater than that existing in the intact male. Therefor, it must be concluded that factors 86 other than hormonal affect the beta-lipoprotein composi tion. When the ratio of alpha-lipoprotein-bound choles terol to beta-lipoprotein-bound cholesterol ^alpha Cbeta (Table VII), is considered, in all cases, females have a higher ratio of ^alpha than do males. This ratio ^beta is increased after gonadectomy in the male and decreased after gonadectomy in the female. These values indicate that there is relatively more alpha-lipoprotein bound- cholesterol and relatively less beta-lipoprotein bound- cholesterol in the intact female than in the intact male. Gonadectomy of the male causes a change in the ratio of ^alpha approaching that seen in the female; gonadectomy Cbeta p of the female produces a ratio of alpha similar to what Cbeta is obtained in the intact male irrespective of the absolute values of the cholesterol concentration in alpha- or beta- lipoprotein fraction. The per cent of total cholesterol distributed in alpha- and beta-lipoproteins of serum in the human, dog, and rabbit were reported by Barr (15) as follows: 87 Man Dog Rabbit Per cent total cholesterol In alpha-1 ipoprotein 30 83 53 Per cent total cholesterol in beta-lipoprotein 70 17 47 Assuming that the sum of the cholesterol in alpha- and beta-lipoproteins represents the total serum choles terol, then from Table V1X, the per cent of the total cholesterol in alpha- and beta-lipoproteins in rat serum can be calculated, and they are 90 and 10 in the intact male and 91 and 9 in the intact female, respectively, in rats fed the reference cottonseed oil diet. This picture is similar to that in the dog but quite different from that in the human. This difference from humans may be one of the reasons that rats are more resistant to athero sclerosis than humans. It has been reported that young women have more alpha-lipoprotein bound cholesterol and less beta- lipoprotein bound cholesterol than do young men of com parable age (13,15,75-78, 83). Similarly, female rats fed the cottonseed oil diet have less beta-lipoprotein bound cholesterol than do male rats fed the same diet; however, there is no difference between male and female rats in alpha-lipoprotein-bound cholesterol. Rats fed the cottonseed oil diet with cholesterol reflect a more close similarity to humans in this respect: the female has more alpha-lipoprotein-bound cholesterol and less beta-lipoprotein-bound cholesterol than does the male. In humans, the administration of estrogen to the sur vivors of myocardial infarction causes an increase in alpha-lipoprotein-bound cholesterol and a decrease in beta-lipoprotein-bound cholesterol. The administration of androgen causes the opposite effect (13,15,75-78,83). In rats fed a cholestrol diet, the alpha-lipoprotein bound cholesterol is increased in the gonadectomized male and decreased in the gonadectomized female; the beta- lipoprotein -bound cholesterol is decreased in the gona dectomized male and increased in the gonadectomized female. In this respect, the alpha- and beta-lipoprotein fractions are comparable in both species. In Table VIII, it can be seen that gonadectomy influences the phospholipid concentration of lipoproteins. in rats fed the cottonseed oil diet containing cholesterol. The phospholipids of both alpha- and beta-lipoproteins are 89 Increased in gonadectomized males but decreased in gona dectomized females. In rats fed the cottonseed oil diet without cholesterol, there is no change in either alpha- and beta-lipoprotein-bound phospholipids in gonadectomized males, but the alpha-lipoprotein-bound phospholipids are decreased in gonadectomized females. As listed in Table IX, the ratio of cholesterol to phospholipids (~) in beta-lipoprotein is lower in the female than in the male and is markedly changed in rats fed both diets as a result of gonadectomy. The £ ratio of beta-lipoprotein is decreased in gonadectomized males but increased in gonadectomized females. There is no change in the £ ratio of alpha-lipoprotein due to gonadectomy. These results seem to agree with those reported by Barr who found that estrogen administration to sur vivors of myocardial infarction causes no change in the £ ratio in the alpha-lipoprotein fraction, whereas there is a drop in the £ ratio in the beta-lipoprotein fraction. The £ ratios in the alpha- and beta-lipoproteins of nor? P mal human plasma is 0.50 and 1.25 respectively (average of men and women) (15); whereas the £ ratios in the P 90 alpha- and beta-lipoproteins of the serum in rats fed the cottonseed oil diet are 0.78 and 0.24 for males and 0.83 and 0.22 for females respectively. This indi cates striking differences between the two species in this respect. Liver lipid patterns. The effects of gonadectomy on the percentage distribution and the degree of unsatura tion of the different fractions of liver lipids in rats fed the cottonseed oil diet with and without cholesterol are presented in Table X. The unsaturation is increased in the cholesterol esters and phospholipids of the liver lipids in both gonadectomized males and females on both diets as compared with the intact male and female. It had been previously reported by Coleman, Chen and Alfin-Slater that the unsaturation in the fatty acids isolated from the choles terol esters of the rat liver lipids is decreased due to gonadectomy in male and female rats fed on the cottonseed oil diet and gonadectomized at weaning (93). The animals used here were gonadectomized at adults which may account for this difference. 91 TABLE X EFFECT OF GONADECTOMY ON THE PERCENTAGE DIS UNSATURATION OF THE DIFFERENT FRACTIONS C A 15 PER CENT COTTONSEED OIL DIET WITH CHOLESTEROL FOR 6 WEI Category! Sex Diet Cholesterol Per cent esters^ I.V.3 Intact (5) M C 24.3 37.7 . Gonadectomized (6) M C 34.5 52.2 Intact (6) Gonadectomized (6) F C 25.9 20.8 F C 24.6 55.8 Intact (6) M CC 40.4 38.4 Gonadectomized (5) M CC 21.5 54.7 Intact (6) F CC 46.0 59.0 Gonadectomized (6) F CC 20.6 64.7 ^Gonadectomized at adult (6 weeks post-weaning); numb animals in the group. ^From pooled sample per group. Iodine value. \ 91 TABLE X MY ON THE PERCENTAGE DISTRIBUTION AND THE DEGREE OF HE DIFFERENT FRACTIONS OF LIVER LIPIDS IN RATS FED OTTONSEED OIL DIET WITH AND WITHOUT 1 PER CENT CHOLESTEROL FOR 6 WEEKS Cholesterol esters^ Triglycerides^ Phospholipids2 Per cent I.V.3 Per cent I.V.3 Per cent I.V.3 24.3 37.7 v 68.6 112 7.2 51.5 34.5 52.2 55.9 94.4 9.5 90.7 25.9 20.8 60.4 36.7 13.7 77.8 24.6 55.8 66.8 91.5 8.6 87.4 40.4 38.4 45.1 48.3 14.5 34.5 21.5 54.7 51.3 48.7 27.2 58.8 46.0 59.0 41.4 55.1 12.6 51.8 20.6 64.7 49.5 45.5 29.9 54.5 seks post-weaning); numbers in parentheses are numbers of Sex differences exist in the percentage distribu tion of phospholipids in the total liver lipids. In rats fed the cottonseed oil diet, the female has a higher percentage of phospholipids in the total liver lipids than does the male; this percentage is increased in the gonadectomized male but decreased in the gonadectomized female in such a fashion that both new values approach to a figure midway between the normal values of males and females. In rats fed a cottonseed oil diet with cholesterol, the male seems to have a slightly higher percentage of phospholipids than does the female, and gonadectomy of both males and females causes a great increase in this percentage. Biosynthesis of Cholesterol The effect of gonadectomy on the biosynthesis of cholesterol in liver slices of rats fed different diets is presented in Table XI. Male rats fed the cottonseed oil diet have a much higher rate of biosynthesis of cholesterol than do females. The biosynthesis of cholesterol is greatly decreased in the gonadectomized male but less markedly decreased in the gonadectomized female. In rats fed the 93 TABLE XI Category EFFECT OF GONADECTOMY ON THE BIOSYNTHESIS ACETATE-1-C14 IN RAT LIVER SI Diet C' Diet CC“ Choles terol^ (c .p.m. per gm of liver) Per cent Choles- of change terol^ from gon- (c.p.m. adectomy per gm of liver) Per cent of change from gon- adectomy Per of < froi tro Male Intact 4400 ( 12) Gonadectomized 1045 (5) Female Intact 1721 (4) Gonadectomized 1468 (4) -76 375 ( 6) 366 ( 6) 0 -15 667 ( 6) 420 (4) -37 ^Gonadectomized at weaning. ^Duration on diet - 6 weeks. 3 Duration on diet - 20 weeks post-weaning. ^All values represent mean of the group; numbers in par animals in the group. TABLE XI COMY ON THE BIOSYNTHESIS OF CHOLESTEROL FROM ^TE-1-C14 IN RAT LIVER SLICES Diet CC2 Diet FF3 Choles terol4 (c.p.m. per gm of liver) Per cent of change from gon adectomy Per cent of change from con trol diet Choles terol4 (c .p.m. per gm of liver) Per cent of change from gon adectomy Per cent of change from con trol diet 375 — -92 522 i i i 00 00 (6) (8) 366 0 -65 525 0 -50 (6) (6) 667 M •» -61 685 -60 (6) (6) 420 -37 -71 470 -31 -68 (4) (4) >st-weaning. :he group; numbers in parenthesis are numbers of 94 cottonseed oil diet supplemented with cholesterol, the biosynthesis of cholesterol is greatly decreased in both the intact and gonadectomized males and females as com pared with the corresponding animals fed on cottonseed oil diet without cholesterol. With little difference between gonadectomized and intact males, however, the biosynthesis of cholesterol is more markedly decreased in the gonadectomized female fed cholesterol as compared with the intact female fed the same diet. In rats fed ,the fat-free diet, there is also a great decrease in the biosynthesis of cholesterol as com pared with animals fed the cottonseed oil diet. Similar to the rats fed cholesterol, there is no change in the biosynthesis of cholesterol after gonadectomy in the male; but the cholesterol biosynthesis is decreased in the gonadectomized female fed the fat-free diet as compared with the intact female on the same diet. The higher rate of hepatic cholesterol biosynthesis in the male than in the female agrees with data reported by Aftergood (91) but disagrees with the results obtained by Rubin and White (90) using liver slice techniques similar to the method used in this investigation and by 95 Fillios (89) using in vivo studies in plasma, who reported that female rats have a higher rate of bio synthesis of cholesterol than do male rats. The decrease in cholesterol biosynthesis which occurs after gonadectomy of both male and female rats on the cottonseed oil diet disagrees with observations reported by Dugal and Saucier who showed an increased incorporation of labeled acetate into liver cholesterol in whole animal experiments with castrated rats (88). This is not too surprising in view of the completely different experimental conditions used. Effects of Sex Hormone Administration on Cholesterol Metabolism Body weight. The effect of sex hormone administra tion on the body weight of male and female rats fed differ ent diets is presented in Tables Xll and XIII. Since the body weight is decreased in the gona dectomized male rats and increased in the gonadectomized female rats, it would seem that androgens potentiate an increase and estrogens a decrease in body weight. As is expected, there is a significant decrease in the body weight of the male rats treated with estradiol benzoate, 98 NOTES FOR TABLES XII TO XXVI E.B. - Estradiol benzoate T.P. - Testosterone proprionate E.E. - Ethinyl estradiol M.T. - Methyl testosterone E.B. (10). Estrogenic-combined sex hormones T.P 1 (potency of ratio: E.B. * 10) T.P. 1 T.P. r!0\- Androgenic-combined sex hormones E.B. 1 (potency of ratio: T.P. 0 10\ E.B. 1 C - 15 per cent cottonseed oil diet CC - 15 per cent cottonseed oil diet plus 1 per cent cholesterol FF - Fat-free diet Experimental - Four series of experiments were included for each sex fed on different diets, and control groups were set up in each series of experi ments . 1. Different levels of E.B. to male rats fed diets C and CC. 2. Low level of T.P. to male rats fed diets C and CC. 3. Estrogenic-combined sex hormones to male rats fed on diets C and CC. 4. Oral feeding of E.E. to male rats fed a fat-free diet. 5. Different levels of T.P. to female rats fed diets C and CC. 6. Low level of E.B. to female rats fed diets C and CC. 7. Androgenic-combined sex hormones to female rats fed diest C and CC. 8. Oral feeding of M.T. to female rats fed a fat- free diet. 97 TABLE XII EFFECT OF SEX HORMONE ADMINISTRATION ON MALE RATS FED DIFFERENT Treatment Number of rats Diet3 Age at sacri fice (months) Body weight Mean Per cent weight change (gm) from control Numt of I 10 C 5.5 395 -- 9 E.B. (low level) 10 E.B. (medium C 5.5 304 -23 10 level) 10 C 5.5 299 -24 10 E.B. (high level) 10 C 5.5 299 -24 10 — 10 C 4.5 359 — 10 T.P. (low level 10 C 4.5 321 -11 10 8 C 4.5 338 — 8 "hs.B. (low level) 8 E.B. T.P. (Y-) 9 C 4.5 273 -19 10 — 8 2e.e. (oral) 10 ■^Hormone injections twice a week for 52 days. ^Oral feeding for 8 weeks. 3On diets C and CC for 6 weeks; on diet FF for 18 weeki 97 TABLE XII iORMONE ADMINISTRATION ON THE BODY WEIGHT OF MALE RATS FED DIFFERENT DIETS Body weight Body w6£&ht t s) Mean weight (gm) Per cent change from control Number of rats Diet Age at sacri fice (months) Mean weight (gm) Per cent change from control 395 — 9 CC 5.5 372 — 304 -23 10 CC 5.5 309 -17 299 -24 10 CC 5.5 305 -18 299 -24 10 CC 5.5 301 -19 359 — 10 CC 4.5 347 — 321 -11 10 CC 4.5 330 -5 338 — — 8 CC 4.5 352 — 8 CC 4.5 249 -29 273 -19 10 CC 4.5 272 -23 8 FF 5.0 270 - - 10 FF 5.0 176 -20 sek for 52 days. s; on diet FF for 18 weeks post -weaning. 98 TABLE XIII EFFECT OF SEX HORMONE ADMINISTRATION ON FEMALE RATS FED DIFFEREN Body weight Treatment Number of rats Diet3 Age at sacri fice (months) Mean Per cent weight change (gm) from control Num of — 10 C 5.5 253 10 ^.P. (low level) 9 C 5.5 268 -6 10 T.P. (medium level) 10 C 5.5 257 +2 10 T.P. (high level) 10 C 5.5 246 -3 10 -- 9 C 4.5 235 10 XE.B. (low level) 10 C 4.5 212 -10 10 9 C 4.5 241 10 XT.P. (low level) T.P. ,10x E.B. V1 * 14 C 4.5 236 -2 7 13 2M.T. (oral) 8 12 ^Hormone injections twice a week for 52 days. 9 Oral feeding for 8 weeks. 3 On diets C and CC for 6 weeks; on diet FF for 18 week 98 TABLE XIII HORMONE ADMINISTRATION ON THE BODY WEIGHT OF FEMALE RATS FED DIFFERENT DIETS Body weight Body weight t s) Mean weight (gtn) Per cent change from control Number of rats Diet3 Age at sacri fice (months) Mean weight (gm) Per cent change from control 253 wm 10 CC 5.5 260 -- 268 -6 10 CC 5.5 282 +8 257 +2 10 CC 5.5 264 +2 246 -3 10 CC 5.5 251 -3 235 — 10 CC 4.5 230 — 212 -10 10 CC 4.5 215 -7 241 — 10 CC 4.5 248 -- 7 CC 4.5 241 -3 236 -2 13 CC 4.5 271 +9 8 FF 5.0 200 — 12 FF 5.0 202 +1 tek for 52 days. s; on diet FF for 18 weeks post-weaning. 99 estrogenic-combined sex hormones or ethinyl estradiol and fed on three different diets. All levels of estradiol benzoate studied decreased the body weight of the male animals to the same degree. When androgens are administered to female rats, however, the results are inconsistent and unexpected. Very little change, if any, results from hormone treatment to female rats on all the experimental diets. Plasma cholesterol level. The effect of sex hormone administration on the plasma cholesterol level is presented in Table XIV and XV, and the plasma total cholesterol levels are also shown graphically in Figures 3a, 3b and 3c. The plasma cholesterol is significantly increased in male rats fed with and without cholesterol and injected with estradiol benzoate at different levels as compared with the uninjected controls. These data agree with those reported by Fillios (58) that there is an elevation in plasma cholesterol in estradiol dipropionate-treated male rats fed either a Purina Chow or a 5 per cent choles terol diet and also with Moskowitz et al. (64) who reported an elevation in plasma cholesterol in estradiol 100 TABLE XIV EFFECT OF SEX HORMONE ADMINISTRATION ON CONCENTRATION OF MALE RATS FED I Cholesterol^- Treatment Diet Total (mg 7.) Free (mg%) % Free Die — C 50.2+3.7 12.2+1.4 24.0 C< E.B. (low level) C 69.6+4.5 26.531.3 38.6 C< E.B. (medium level) C 78.1+6.8 22.8+1.4 29.5 C( E.B. (high level) C 70.0+2.6 17.1±4.2 24.3 CC C 102 +7 22.9+1.9 22.6 CC T.P. (low level) C 87.3+5.0 19.931.4 23.0 CC — C 69.4+3.2 21.9+1.3 31.9 CC 1E.B. (lbw level) E.B. ,10. T.P. '1 ' C 81.3+2.5 26.2+0.2 32.1 CC CC ^E.E. (oral) FI FI ^Hormone injections twice a week for 52 days. ^Oral feeding for 8 weeks. ^On diets C and CC for 6 weeks; on diet FF for ^Includes standard error of the mean. TABLE XIV ORMONE ADMINISTRATION ON THE PLASMA CHOLESTEROL RATION OF MALE RATS FED DIFFERENT DIETS Cholesterol^- Diet' Cholesterol^- Free (mg%) % Free Total (mg Free (mg %) % Free .7 12 .2+1.4 24.0 CC 96. 8+8.6 16.1+8.4 16.5 .5 26 .5+1.3 38.6 CC 166 +15 49.2+11.7 29.5 .8 22 .8+1.4 29.5 CC 170 ±13 49.6+2.7 29.4 .6 17. 1±4. 2 24.3 CC 151 +10 37.7+2.4 25.2 22 .9+1.9 22.6 CC 118 +6 28.6±2.0 24.6 .0 19 .9+1.4 23.0 CC 117 ±11 24.9±2.3 21.4 .2 21.9*1.3 31.9 CC 78. 3+3.6 18.5+1.2 24.4 CC 152 ±10 35.2+2.2 23.0 .5 26 .2+0.2 32.1 CC 148 ±17 31.7+1.6 21.6 FF FF 41. 46. 4+2.6 3+9.0 5. 0±0. 5 7.7+0.5 12.2 17.4 ice a week for 52 days. eks. 6 weeks; on diet FF for 18 weeks post-weaning, or of the mean. 101 1 TABLE XV EFFECT OF SEX HORMONE ADMINISTRATION ON CONCENTRATION OF FEMALE RATS FED Cholesterol^ Treatment Diet3 Total (mg 7.) Free (mg 7o) % Free Dii -- C 84.8+2.4 29.3+1.4 34.1 C ^.P. (low level) T.P. (medium level) T.P. (high level) C C C 68.7+3.5 61.7+2.1 72.2+1.1 15.2+2.4 12.9+1.2 22.7+0.9 21.7 21.0 32.0 C C C - - C 80.2+3.4 20.2+4.1 25.0 C XE.B. (low level) C 110 *2 30.5+1.8 28.2 C C 73.5+2.9 22.5+1.0 C 1T.P. (low level) C T.P. ,10s E.B 11 ’ C 75.3+1.0 21.4+1.9 C -- F 2M.T. (oral) F ^Hormone injections twice a week for 52 days ^Oral feeding for 8 weeks. ■^On diets C and CC for 6 weeks; on diet FF for ^Includes standard error of the mean. 101 TABLE XV ORMONE ADMINISTRATION ON THE PLASMA CHOLESTEROL ATION OF FEMALE RATS FED DIFFERENT DIETS Cholesterol^ Cholesterol^ Free (mg 7o) % Free Diet^ Total (mg %) Free (mg %) % Free 4 29.3+1.4 34.1 CC 218 +16 60.8+19.3 28.0 5 15.2+2.4 21.7 CC 125 ±13 36.7±4.2 29.6 1 12.9+1.2 21.0 CC 92.1±5.7 24.5±2.3 27.2 1 22.7+0.9 32.0 CC 94.7+18.1 30.8±4.3 32.6 ,4 20.2+4.1 25.0 CC 153 +13 31.2+3.1 20.3 30.5+1.8 28.2 CC 246 ±12 59.6 ±3.5 24.4 .9 22.5+1.0 CC 117 ±15 25.0±2.0 21.4 CC 50.1+5.2 7.5±0.6 16.0 .0 21.4+1.9 CC 98.4+1.9 26.6±1.2 27.7 FF 51.9+1.7 9.6+0.8 19.2 FF 43.0+2.1 8.0±0.4 18.6 Lee a week for 52 days eks. 6 weeks; on diet FF for 18 weeks post-weaning. Dr of the mean. Ml Untreated male M2 Male + B.B.(low level) M3 Male + "E.B.(medium level) M4 Male + E.B, (high level) El Untreated feihale E2 Female + T.P.(low level) F3 Female + T.P.(medium level) F4 Female + T.P. (high level) CSO On 15% cottonseed oil diet for 6 weeks CSOC On 15% cottonseed oil diet containing 1% cholestrol for 6 weeks 240-. 220- 200. 180. 160- 140. 120. 100- 80- 60- 40. 20- a M2 M3 M4 CSO F4 CSO Ml M2 M3 m OSOC FI F2 F4 OSOC Fig. 3a Effect of sex hormone administration on plasma cholesterol level(mg%) 103 Ml Untreated male M2 Male + T.P.(low level) Male + S.E.(oral) PI Untreated female P2 Pemale + E.B.(low level) F3 Female + M.T.(oral) CSO On 15/5 cottonseed oil diet for 6 weeks CSOC On 15% cottonseed oil diet containing 1% cholesterol for 6 weeks PF On fat-free diet for 18 weeks post-weaning 240 220- 200- 180- 160- 140 _ 120. 100- 80. 60. 40- 20- PI lF2 M2 PI Ml M3 FI CSO CSOC pp Pig. 3b Effect of sex hormone administration on plasma cholesterol level(mg%) 104 240- 220. 200- 180- 160. 140. 120- 100- sa Untreated male Male + E.B.(low level) Male + E.B. /■•lO^ Untreated female Female + T.P.(low level) Female + T.P./IOn On 15% cottonseed oil diet for 6 weeks CSOC On 15/5 cottonseed oil diet containing 1/5 cholesterol for 6 weeks Ml M2 M3 El F2 F3 CSO 6a 4a 20 p i fell |E 2l CSO CSOC Fig. 3c Effect of sex hormone administra tion on plasma cholesterol level 105 benzoate-treated male rats fed a 2 per cent cholesterol diet. The Increase In plasma cholesterol levels Is from 40 td 50 per cent In rats fed the cottonseed oil diet without cholesterol and over 50 per cent In rats fed the cottonseed oil diet containing cholesterol; this increase is not proportional to the amount of hormone administered. In general, the free cholesterol in the plasma is also increased due to the injection of estradiol benzoate. The plasma cholesterol response of female rats to testosterone propionate at different levels is just opposite to that of male rats given estradiol benzoate at different levels. Both the total and free plasma cholesterol levels are decreased in female animals injected with testosterone propionate and fed with and without cho lesterol as compared with female animals given the same diets but not receiving this hormone. Although the decrease in total plasma cholesterol is not marked in rats fed no cholesterol, it is very pronounced in rats fed the cholesterol diet; a decrease of more than 50 per cent of the control value is observed. Fillios also reported a decrease in plasma choles terol in testosterone propionate-treated female rats fed 106 either a Purina Chow or a 5 per cent cholesterol diet (58). On the other hand, Moskowitz et al. reported an increase in plasma cholesterol in testosterone propionate-treated female rats fed a 2 per cent cholesterol diet (64). The sex difference in the plasma cholesterol con centration is marked. Female rats have much higher plasma cholesterol levels than do male rats on both cholesterol- free and cholesterol-containing diets which agrees with Aftergood et al. (57) and Fillios (58). The response of plasma cholesterol levels of the female rats after testo sterone propionate treatment is as expected when the response of the male rats to estradiol benzoate is con sidered. The administration of estradiol benzoate to male rats causes an increase in plasma cholesterol approaching the value seen in the female; the administration of testo sterone propionate to female rats causes a decrease in plasma cholesterol approaching the value observed for the male. The effect of the estrogenic-combined sex hormones is similar to that of estradiol benzoate in increasing the plasma cholesterol of male rats fed diets both with and without cholesterol. In this series of experiments, the plasma cholesterol concentration of cholesterol-fed 107 male rats treated with estradiol benzoate or estrogenic- combined sex hormones is increased almost 90 per cent over that of uninjected controls. The free plasma cholesterol is increased in hormone-treated male rats on both diets; this increase is especially marked in male rats fed cholesterol. The administration of the androgenic-combined sex hormones to female rats fed with and without cholesterol does not change the plasma cholesterol level, although testosterone propionate administered alone does cause a decrease. In this series of experiments, testosterone propionate reduces plasma total cholesterol more than 50 per cent and plasma free cholesterol approximately 70 per cent. The presence of a small amount of estradiol benzoate in the androgenic-combined sex hormone prepara tion might be sufficient to antagonize the hypocholester- olemic effect of testosterone propionate. The administration of testosterone propionate to male rats fed both with and without cholesterol causes no change in the plasma cholesterol level. However, the administration of estradiol benzoate to female rats fed both with and without cholesterol increases the plasma 108 cholesterol level; It Is especially significant in animals fed cholesterol. These results agree well with the report of Fillios (58) that in rats fed either a Purina Chow or a 5 per cent cholesterol diet, the plasma cholesterol is elevated in estradiol dipropionate-treated females but remains unchanged in testosterone propionate-treated males. They also agree in part with Mbskowitz et al.(64) who observed an elevation in plasma cholesterol in testo sterone propionate-treated male rats and estradiol benzoate-treated female rats fed a 2 per cent cholesterol diet. When rats are fed a fat-free diet, the plasma cholesterol tends to decrease in the methyl testosterone- treated females but remains unchanged in the ethinyl estradiol-treated males. In humans estrogens cause a decrease and androgens an increase in plasma cholesterol (13-15,75). This is contrary to what occurs in rabbits (56), chickens (13,14, 46,47) and rats (58,64). Further differences and some similarities are found when the chicken and the rat are compared. Estrogens cause an increase in plasma choles terol in both male and female chickens and rats, whereas 109 testosterone propionate causes a decrease in plasma cho lesterol in the cockerels (53) but not in male rats here. Also, the administration of the estrogenic-combined sex hormones causes a similar effect on the plasma cholesterol level to that of estrogens alone both in male rats and cockerels (52). Liver cholesterol and total lipid levels. The effect of sex hormone administration on the cholesterol and total lipid concentrations of liver of male and female rats fed different diets is presented in Tables XVI and XVII, and the liver total cholesterol levels are also shown graphically in Figures 4a, 4b and 4c. In rats fed the reference cottonseed oil diet, there is no difference in the liver cholesterol level between the estradiol benzoate-treated male and the untreated male, or between the testosterone propionate- treated female and the untreated female. This is similar to the results obtained in the gonadectomized male and female fed the same reference diet. In rats fed the cholesterol-containing diet, how ever, the administration of both estradiol benzoate to males and testosterone propionate to females causes a decrease 110 TABLE XV EFFECT OF SEX HORMONE ADMINISTRATION ON ' CONCENTRATIONS OF LIVER OF MALE RA‘ Cholesterol4 Total4 Dief3 Treatment Total (rog/gm) Free % (mg/gm) Free lipids 1 (mg/gm) C 2.79+0.10 2.23+0.03 80.0 55.8+6.0 1E.B. (low level) E.B. (medium level) E.B. (high level) C C C 2.44+0.08 3.10+0.61 2.71+0.04 2.06+0.07 2.28+0.08 2.40+0.04 84.4 73.6 88.9 47.6+1.3 39.2+5.3 37.6+1.7 --- C 2.39+0.08 2.1610.08 90.4 47.51 .9 ^.P. (low level) C 2.55+0.07 1.9710.03 77.2 49.3+2.2! --- C 2.41+0.05 2.1410.03 88.5 37.411.5 1E.B. (low level) E.B. /10\ T.P. V1 } C 2.25+0.04 1.9010.09 84.5 33.710.8 2E.E. (oral) ^Hormone Injections twice a week for 52 days. ^Oral feeding for 8 weeks. 3 On diets C and CC for 6 weeks; on diet FF for 18 w« ^Includes standard error of the mean. 110 TABLE XVI DRMONE ADMINISTRATION ON THE CHOLESTEROL AND TOTAL LIPID riONS OF LIVER OF MALE RATS AS INFLUENCED BY DIET alesterol Total4 Cholesterol4 Total4 Free % (mg/gm) Free lipids Diet (mg/gm) Total (mg/gm) Free (mg/gm) % Free lipids (mg/gm) 2.23+0.03 80.0 55.8+6.0 CC 25.3+1.0 3.63*0.14 14.3 117+7 2.06-0.07 84.4 47.6+1.3 CC 11.8*1.2 3.57*0.08 30.2 65.1*4.0 2.28*0.08 73.6 39.2+5.3 CC 20.8*2.6 4.11+0.26 19.7 89.5*6.2 1.40*0.04 88.9 37.6+1.7 CC 18.3*1.7 4.50*0.35 24.6 81.7*5.0 2.16±0.08 90.4 47.5* .9 CC 33.4*3.0 3.71+0.15 11.1 128*8*: L. 97*0.03 77.2 49.3*2.21 CC 33.7*1.8 4.38*0.24 13.0 126*8 2.14*0.03 88.5 37.4*1.5 CC 30.4+1.8 3.89*0.84 12.8 119*9 CC 6.89*2.28 2.6930.15 39.1 51.1*4.3 ..90*0.09 84.5 33.7*0.8 CC 19.8*1.9 3.59*0.16 18.1 71.4*4.0 FF 3.99*0.33 2.11*0.07 52.9 55.4*3.0 FF 3L 45*027 2.12*0.11 61.1 33.5*1.4 i week for 52 days. seks; on diet FF for 18 weeks post-weaning. : the mean. Ill % TABLE XVII EFFECT OF SEX HORMONE ADMINISTRATION ON TH CONCENTRATIONS OF LIVER OF FEMALE RATS Treatment Diet^ Total _______ (mg/gm) Cholesterol^ Free (mg/gm) ______ Total** 7 o Free lipids Di (mg/gm) T.P. T.P. T.P. C 2.22+0.10 2.10+0.11 94.6 37.7+4.2 (low level) C 2.33+0.10 1.81+0.04 77.6 32.5+8.0 (medium level) C 2.66+0.13 1.97+0.05 74.0 50.5+4.1 (high level) C 2.62+0.07 2.23+0.05 85.1 47.7+0.9 ’ E.B. (low level) C 2.30+0.07 1.84+0.06 80.0 41.7+2.6 ( 2.54±0.07 2.07+0.05 81.5 40.1+1.1 ( T.P. T.P E.B. (low level) 2.32+0.05 2.14+0.06 92.2 34.9+1.5 < ( 2.28+0.05 2.20+0.05 96.5 38.9+3.6 ( "M.T. (oral) ^-Hormone injections twice a week for 52 days. ^Oral feeding for 8 weeks. ^On diets C and CC for 6 weeks; on diet FF for 18 w< ^Includes standard error of the mean. Ill TABLE XVII MONE ADMINISTRATION ON THE CHOLESTEROL AND TOTAL LIPID S OF LIVER OF FEMALE RATS AS INFLUENCED BY DIET esterol4 Total4 Cholesterol4 Total4 Free % g/gm) Free lipids Diet (mg/gm) Total (mg/gm) Free (mg/gm) % Free lipids (mg/gm) 10+0.11 94.6 37.7+4.2 CC 24.7 12.2 3.6810.93 14.9 103 19 8110.04 77.6 32.5+8.0 CC 19.5 12.3 2.8410.17 14.5 123 18 97+0.05 74.0 50.514.1 CC 17.4 12.0 3.2110.98 18.4 107 18 23+0.05 85.1 47.710.9 CC 14.8 +1.8 2.74+0.56 18.4 96.716.7 84+0.06 80.0 41.7+2.6 CC 25.9 12.0 3.2410.11 12.5 129 17 0710.05 81.5 40.Ill.1 CC 25.8 12.0 3.6410.46 14.1 123 +6 14+0.06 92.2 34.9H.5 CC 28.6 11.1 3.7110.16 13.0 122 17 CC 13.8 +3.07 3.3910.29 24.6 90.3-10. ( 20+0.05 96.5 38.913.6 CC 27.8 11.5 3.6510.13 13.1 115 114 FF 3.2810.24 2.0410.07 62.2 45.712.1 FF 3.6710.22 2.0810.63 ■ ■ 1, 1* .BBS* 56.6 53.710.3 l week for 52 days. ieks; on diet FF for 18 weeks post-weaning. : the mean. Ml Untreated male M2 Male - f - E.B.(low level) MJ Male + E.B,(medium level) ? , 1 4 Male + E.B, (high level) El Untreated female E2 Female - t - T.P.(low level) E3 Female - t - T.P.(medium level) F4 Female T.P. (high level) CSO On 13% cottonseed oil diet for S weeks 30.1 CSOC On 13% cottonseed oil diet containing 1% cholesterol for 6 weeks do 26. 112 24. 22- 20- 18- 16- 14- 12- 10. 8 - 6 - 4- 2 - m M2 M3 FI F2 F4 Ml M2 M3 L14 FI F3 F4 CSO CSO CSOC CSOC Fig. 4a Effect of sex hormone administration on liver cholesterol level(mg per gram) 34 - 32- 30. CSO On 15% cottonseed oil diet for 6 weeks OSOC On 15% cottonseed oil diet containing 1% cholesterol for 6 weeks FF1 On fat-free diet for IS weeks post- weaning Ml Untreated male M2 Male + T.P.(low level) M3 Male E.E.(oral) FI Untreated female F2 Female - i - E.B. (low level) F3 Female - f - M.T.(oral) 113 28, 26 - 24- 22. 20- 18- 16- 14. 12- 10- 8. 6- 4- 2- t — Ml M2 m M2 FI F2 a M' 1?3 CSO CSOC FF Fig. 4b Effect of sex hormone administration on liver cholesterol level(mg per gram) OSO On 15% cottonseed oil diet for 6 weeks CSOC On 15% cottonseed oil diet containing 1% cholesterol for 6 weeks Ml Untreated male M2 M3 Maie + E•B•(1ow level) Male ~ i - E.B*/10 N 1 I C \ J K \ K' n 71 THg F3 30- 28. 26- 24- 22. 20. 1 CO ■ —1 16 - • 3- H 12- 1 0 rH 8 . 6 _ 4- 0 - Untreated female female + T.P. Female + T.P./10>1 I • -O# - 1 - E, M] M3 F1F3 Ml M2 M3 FI TB r .y c S3 CSO CSOC Fig. 4c Effect of sex hormone administra tion on liver cholesterol level (mg per gram) 114 115 in liver cholesterol levels as compared with the un treated control animals. The effect of estrogen in decreasing the liver cholesterol level of male rats is expected as the liver cholesterol level is also decreased in the gonadectomized male. The effect of androgen in decreasing the liver cholesterol level of female rats is unexpected since the liver cholesterol level is increased in the gonadectomized female. As indicated previously, there seems to be an increased requirement for essential fatty acids in the intact male and gonadectomized female judging from the results in rats fed a fat-free diet. It is postulated, therefore, that estrogens may either spare the essential fatty acids stored in the body or possibly potentiate the transport system in which cholesterol is esterified properly with the essential fatty acids. If this is the case, in male rats fed cholesterol, where there is an accumulation of cholesterol esters in the liver, the essential fatty acids either from body stores or from the food may not be sufficient enough for the proper esterifi- cation of this excess cholesterol and thus its transport is retarded. The administration of estrogen to these male rats fed cholesterol, therefore, will result in a more 116 efficient system of cholesterol transport and In turn the liver cholesterol will be decreased but the plasma choles- terol increased as compared with the untreated males fed cholesterol. This may also be one of the reasons female rats have a higher plasma cholesterol level but a lower liver cholesterol level than do male rats. However, the action of androgen to decrease the liver cholesterol concentration in female rats fed cho lesterol must be explained in some other way since the plasma cholesterol level is also decreased by the administration of androgen. Aftergood reported that in rats fed a 15 per cent cottonseed oil diet or a 15 per cent lard diet containing 1 per cent cholesterol for two weeks, males excreted a higher percentage of cholesterol ingested from the diets in feces than did females (91). This indicates that androgens may affect an increased excretion of feces cholesterol which can result in a decrease in both the plasma and liver cholesterol levels. The different re sults in the liver cholesterol levels between the androgen-treated female and gonadectomized females fed cholesterol may be due to the fact that androgens and 117 estrogens are absent In the gonadectomized female, but there is an excess supply of androgen In the androgen** treated female to counteract the estrogen naturally secreted in the body. In rats fed a fat-free diet, the oral feeding of ethinyl estradiol to males and methyl testosterone to females causes little change in the liver cholesterol levels (and also plasma cholesterol levels). The inef fectiveness of the oral feeding of sex hormones on the cholesterol level here may be due to the relative impotency of these hormones. Liver cholesterol levels are unchanged after the administration of testosterone propionate to male rats and estradiol benzoate to female rats fed both with and without cholesterol. This indicates a fact that the effectiveness of estrogen in influencing the tissue cho lesterol level in the male and the effectiveness of androgen in influencing the tissue cholesterol level in the female are probably a result of nullifying or balancing out the hormones already present in the animal. In rats fed the reference cottonseed oil diet, the administration of the estrogenic-combined sex 118 hormones to males and the androgenic-combined sex hormones to females causes no change In the liver cholesterol levels. The administration of estrogenic-combined sex hormones causes an effect similar to, but less marked than, that of estradiol benzoate In decreasing the liver cholesterol In male rats fed cholesterol. The administra tion of androgenic-combined sex hormones does not cause a reduction in the liver cholesterol in female rats fed cholesterol similar to that obtained with testosterone propionate alone. Here, the proportion of male and female hormones seems to be an important factor in decreasing liver cholesterol levels. In general, the administration of estradiol benzoate or estrogenic-combined sex hormones to male rats fed the reference cottonseed oil diet with and without cholesterol causes a decrease in the liver total lipids; the reduction is more marked when rats are fed the cholesterol diet and given estradiol benzoate rather than the estrogenic-combined sex hormones (Table XVI). On the other hand, the administration of testosterone propionate or androgenic-combined sex hormones to female rats fed the same diets causes little change in the 119 liver total lipids. There is no change in the liver total lipids after giving testosterone propionate to males and estradiol benzoate to females in rats fed with and without cholesterol as compared with the untreated control males and females on the same diet. In rats fed a fat-free diet, the oral feeding of ethinyl estradiol to males causes a decrease and the oral feeding of methyl testosterone to females causes a slight increase in the liver total lipids. These results are similar to those obtained in the gonadectomized males and females fed the fat-free diet. Adrenal cholesterol and total lipid levels. The effect of sex hormone administration on adrenal choles terol and total lipid levels of male and female rats fed different diets is presented in Tables XVIII and XIX. The administration of testosterone propionate to both males and females and androgenic-combined sex hormones to females in rats fed the cottonseed oil diet with and without cholesterol and the oral feeding of methyl testosterone to female rats fed the fat-free diet cause a consistent and usually significant decrease in both 120 TABLE XVIII EFFECT OF SEX HORMONE ADMINISTRATION ON ADRE1 LIPID CONCENTRATIONS OF MALE RATS AS Cholesterol^*- Diet3 Treatment Total (mg/gm) Free (mg/ gm) Per cent free lipids (mg/gm) Diel -- C 37.8 4.24 11.2 280 CC ^-E.B. (low level) C 49.6 6.51 13.1 237 CC E.B. (medium level) C — — — CC E.B. (high level) C — — — — — — CC — C 53.2 8.34 15.6 131 CC ^.P. (low level) C 22.8 4.50 19.7 82.1 CC — C 39.9 5.22 13.1 186 CC E.B. (low level) E.B. AO. T.P. C 1' C 39.9 6.00 15.0 204 CC CC 2E.E. (oral) FF FF ^•Hormone injections twice a week for 52 days. 20ral feeding for 8 weeks. o On diets C and CC for 6 weeks; on diet FF for 18 weeki ^Pooled sample per group. TABLE XVIII )RMONE ADMINISTRATION ON ADRENAL CHOLESTEROL AND TOTAL )NCENTRATIONS OF MALE RATS AS INFLUENCED BY DIET Lesterol4 Total4 Cholesterol4 Total4 :ee ng/ pn) Per cent free lipids (mg/gm) Diet? Total (mg/gm) Free (mg/gtn) Per cent free lipids (mg/gm) .24 11.2 280 CC 47.5 5.87 12.4 326 .51 13.1 237 CC 62.8 8.77 14.0 356 ■ — CC 64.2 8.42 13.1 320 • CC 66.7 9.11 13.7 310 .34 15.6 131 CC 52.3 6.20 11.9 99.0 .50 19.7 82.1 CC 32.1 5.12 15.9 81.9 .22 13.1 186 CC 46.2 5.70 12.3 266 CC 58.4 6.31 10.8 226 .00 15.0 204 CC 55.5 7.82 14.1 189 FF 42.8 6.67 15.6 210 FF 40.6 6.51 16.0 219 a week for 52 days. teeks; on diet FF for 18 weeks post-weaning. * 121 TABLE XIX EFFECT OF SEX HORMONE ADMINISTRATION ON ADR LIPID CONCENTRATIONS OF FEMALE RATS Treatment Diet^ Total (mg/gm) Cholesterol4 Free Per cent (mg/gm) free Total4 lipids (mg/gm) Die C 47.5 6.43 13.5 326 CC T.P. (low level) C 45.3 5.76 12.7 296 CC T.P. (medium level) C 39.7 6.45 16.2 263 CC T.P. (high level) C 28.9 5.80 20.0 229 CC -- C 46.5 5.03 10.8 118 CC 1E.B. (low level) C 38.0 4.73 12.4 88.6 CC -- C 54.6 5.23 9.6 228 CC ■^r.p. (low level) T.P. ( 10* E.B. ^ 1 ; C 37.6 5.14 13.7 176 CC CC 2M.T. (oral) FI FI ^•Hormone injections twice a week for 52 days. ^Oral feeding for 8 weeks. q One diets C and CC for 6 weeks; on diet FF for 18 we 4Pooled sample per group. TABLE XIX ORMONE ADMINISTRATION ON ADRENAL CHOLESTEROL AND TOTAL NCENTRATIONS OF FEMALE RATS AS INFLUENCED BY DIET lesterol4 Total4 lipids (mg/gm) Diet^ a Cholesterol Total4 lipids (mg/gm) ee /gm) Per cent free Total (mg/gin) Free (mg/gm) Per cent free .43 13.5 326 CC 60.5 7.32 12.1 367 .76 12.7 296 CC 38.3 3.74 9.8 315 .45 16.2 263 CC 47.4 5.73 12.1 335 .80 20.0 229 CC 31.0 3.66 11.8 230 i.03 10.8 118 CC 64.7 6.31 9.8 120 .73 12.4 88.6 CC 66.0 6.43 9.7 118 .23 9.6 228 CC 59.6 5.43 9.1 264 CC 34.1 4.20 12.3 189 i.14 13.7 176 CC 38.9 5.60 14.4 203 FF FF 52.5 28.6 6.29 4.21 12.0 14.7 219 147 * a week for 52 days. > weeks; on diet FF for 18 weeks post-weaning. 122 adrenal cholesterol and total lipid levels. The effect of estrogen on the adrenal cholesterol and total lipid levels is not clear cut, although it can be seen that in most cases where changes occur, estrogen causes an increase in the adrenal cholesterol level and little change in the total adrenal lipid level. The administration of estradiol benzoate to male animals fed with and without cholesterol causes an increase in adrenal cholesterol. A decrease in adrenal total lipids is observed in estradiol benzoate-treated male rats fed the cholesterol-free diet. The administra tion of estrogenic-combined sex hormones produces no change in both adrenal cholesterol and total lipids of male rats fed without cholesterol, but causes an increase in adrenal cholesterol and a decrease in adrenal total lipids of male rats fed with cholesterol. The administration of estradiol benzoate to females causes a decrease in both adrenal cholesterol and total lipids in rats fed the cholesterol-free diet. This is completely unexpected and it may be due to the variable cholesterol level in the adrenal caused by stress. In rats fed a fat-free diet, the oral feeding of ethinyl estradiol to males causes no change in both 123 adrenal cholesterol and total lipids; but the oral feeding of methyl testosterone to females does cause a decrease in these values. Comparing with the results in gonadectomized experiments, it agrees well that in rats fed cholesterol, the adrenal cholesterol is increased in the gonadectomized male and the estrogen-treated male, but decreased in the gonadectomized female and the androgen-treated female. The effect of estrogen to increase the adrenal cholesterol in the male animal and the effect of androgen to decrease the adrenal cholesterol in the female animal may be a response of adrenal cholesterol to the stress of sex hormone administration to transform more or less cho lesterol into adrenal cortical hormones or sex hormones. Kidney cholesterol and total lipid levels. In all cases, there is relatively no change in the cholesterol and total lipid concentrations in kidneys due to either - dietary difference or sex hormone treatment. The results are shown in Tables XX and XXI. It appears, however, that sex difference exists in kidney cholesterol levels in rats fed the reference cottonseed oil diet with and with out cholesterol. The male rat has higher kidney 124 TABLE XX EFFECT OF SEX HORMONE ADMINISTRATION ON KIDN LIPID CONCENTRATIONS OF MALE RATS AS I Cholesterol^ Total^- Treatment Diet Total (mg/gm) Free (mg/gm) Per cent free lipids (mg/gm) Die C 4.04 3.11 77.0 27.9 CC E.B. (low level) C 4.27 3.88 90.9 33.6 CC E.B. (medium level) C 3.74 3.28 87.7 30.0 CC E.B. (high level) C 4.53 4.20 92.6 24.5 CC — C 4.56 3.75 82.2 29.0 C( XT.P. (low level) C 3.90 3.66 93.9 28.3 C< -- C 4.33 3.99 92.2 28.0 C< ^•E.B. (low level) Cl E.B ,10. T.P. V 1 * C 4.70 4.20 89.4 37.2 C 2E.E. (oral) F F Hormone injections twice a week for 52 days. 2 Oral feeding for 8 weeks. ^On diets C and CC for 6 weeks; on diet FF for 18 wee ^Pooled sample per group. 124 TABLE XX )RMONE ADMINISTRATION ON KIDNEY CHOLESTEROL AND TOTAL :entrations of male rats as influenced by diet olesterol4 Total4 Cholesterol4 Total ree g/gm) Per cent free lipids (mg/gm) Diet Total (mg/gm) Free (mg/gm) Per cent free lipids (mg/gm) .11 77.0 27.9 CC 4.57 3.51 76.8 29.7 00 00 • 90.9 33.6 CC 4.93 4.42 89.7 31.6 .28 87.7 30.0 CC 4.70 4.26 90.6 29.1 .20 92.6 24.5 CC 4.08 4.04 98.0 29.0 .75 82.2 29.0 CC 4.04 3.65 90.5 28.6 .66 93.9 28.3 CC 4.11 3.68 89.5 29.8 .99 92.2 28.0 CC 4.49 4.30 95.7 41.3 CC 5.00 4.66 93.2 30.5 o CM • 89.4 37.2 CC 4.93 4.48 90.8 32.7 FF FF 4.52 4.87 4.43 4.79 98.0 98.4 37.0 35.8 » a week for 52 days. i. weeks; on diet FF for 18 weeks post-weaning. 125 TABLE XXI EFFECT OF SEX HORMONE ADMINISTRATION ON KIDNE LIPID CONCENTRATIONS OF FEMALE RATS AS 1 Cholesterol^ Total^ Treatment Diet3 Total (mg/gm) Free (mg/gm) Per cent free lipids (mg/gm) Diet -- C 3.02 2.65 87.7 21.9 CC J-T.P. (low level) T.P. (medium level) T.P. (high level) C C C 2.91 3.01 2.71 2.76 2.79 2.46 94.9 92.7 90.9 18.4 18.6 17.9 CC CC CC - - C 4.54 3.99 87.9 33.5 CC ^E.B. (low level) C 4.23 3.87 91.5 32.7 CC — C 4.52 3.85 85.2 34.1 CC 1T.P. (low level) CC T.P. r10x E.B. V 1 J C 4.65 4.26 91.6 39.6 CC -- FF 2M.T. (oral) FF •^Hormone injections twice a week for 52 days, o Oral feeding for 8 weeks. 3 On diets C and CC for 6 weeks. ^Pooled sample per group. 125 \ TABLE XXI RMONE ADMINISTRATION ON KIDNEY CHOLESTEROL AND TOTAL NTRATIONS OF FEMALE RATS AS INFLUENCED BY DIET lesterol4 Total4 lipids (mg/gm) Diet^ Cholesterol4 Total4 lipids (mg/gm) ee / gm) Per cent free Total (mg/gm) Free (mg/gm) Per cent free 65 87.7 21.9 CC 3.44 3.14 90.3 27.8 76 94.9 18.4 CC 4.19 3.89 92.8 32.7 79 92.7 18.6 CC 4.20 4.00 95.2 25.5 46 90.9 17.9 CC 3.46 3.23 93.4 27.3 99 87.9 33.5 CC 4.41 3.96 89.8 26.8 87 91.5 32.7 CC 4.55 4.13 90.8 25.4 85 85.2 34.1 CC 4.57 4.23 92.6 43.1 CC - - — — — 26 91.6 39.6 CC 4.87 4.44 91.2 34.7 FF FF 4.69 4.35 4.36 4.07 93.0 93.5 36.2 35.7 a week for 52 days. eeks. cholesterol level than does the female. Cholesterol and total lipid levels in testes and ovaries. The effect of sex hormone administration on the cholesterol and total lipid levels in the testis of male rats and in the ovary of female rats as influ enced by the diet is presented in Tables XXII and XXIII. There is a great difference in the per cent of the free cholesterol between the testis and the ovary. The per cent of the free cholesterol is only about 20- 30 per cent in the ovary but it reaches 75-95 per cent in the testis. When represented as mg per rat, the cholesterol and total lipid concentrations in testes and ovaries are decreased in all cases due to sex hormone treatment. The administration of estradiol benzoate and estrogenic- combined sex hormones to male rats fed the cottonseed oil diet with and without cholesterol and the administra tion of ethinyl estradiol to male rats fed a fat-free diet cause a very significant decrease in the cholesterol and total lipid concentrations in testes whereas the administration of testosterone propionate to male rats fed the cottonseed oil diet with and without cholesterol 127 TABLE X> EFFECT OF SEX HORMONE ADMINISTRATION ON Ti CONTENTS OF MALE RATS AS INFI Cholesterol^ Total^ Treatment Diet3 Total Free (mg/rat)(mg/rat) Per cent free lipids (mg/rat) C 3.65 3.50 95.9 44.4 *E.B. (low level) E.B. (medium level) E.B. (high level) C C C 2.25 2.31 1.93 1.00 1.15 1.03 44.4 49.8 53.4 12.3 11.8 16.3 -- C 4.24 3.64 85.9 47.7 ^T.P. (low level) C 3.57 3.16 88.6 36.9 --- C 3.75 3.28 87.5 39.9 ^E.B. (low level) E.P. /10\ T.P. '1 J C 1.08 0.61 56.5 14.6 ^E.E. (oral) ^"Hormone injections twice a week for 52 days: ^Oral feeding for 8 weeks. 30n diets C and CC for 6 weeks; on diet FF for 18 ^Pooled sample per group. 127 TABLE XXII LMONE ADMINISTRATION ON TESTIS CHOLESTEROL AND TOTAL LIPID ’ ENTS OF MALE RATS AS INFLUENCED BY DIET olesterol4 Total4 lipids Diet (mg/rat) Cholesterol4 Total4 lipids (mg/rat) ree g/rat) Per cent free Total Free (mg/rat)(mg/rat) Per cent free 3.50 95.9 44.4 CC 4.57 4.15 90.7 30.4 1.00 44.4 12.3 CC 1.81 0.81 44.7 13.8 1.15 49.8 11.8 CC 2.00 0.88 44.0 12.4 1.03 53.4 16.3 CC 2.37 1.16 49.0 13.3 3.64 85.9 47.7 CC 4.31 3.79 88.0 45.3 3.16 88.6 36.9 CC 3.28 2.86 87.1 33.5 3.28 87.5 39.9 CC 4.80 3.56 74.1 42.1 CC 0.98 0.43 43.9 8.0 ) .61 56.5 14.6 CC 1.48 0.75 50.6 14.1 FF FF 3.28 1.04 3.00 0.75 91.5 72.0 34.8 10.6 s a week for 52 days. :. weeks; or> diet FF for 18 weeks post-weaning. 128 TABLE XX] EFFECT OF SEX HORMONE ADMINISTRATION ON OV/ CONTENTS OF FEMALE RATS AS If Cholesterol4 Total4 Treatment Diet3 Total (mg /rat) Free (mg/tat) Per cent free lipids (mg/tat) — C 1.04 0.27 26.0 12.9 XT.P. (low level) T.P. (medium level) T.P. (high level C C C 0.45 0.35 0.24 0.18 0.15 0.15 40.0 42.9 62.5 8.9 8.3 5.4 — C 0.50 0.15 30.0 8.69 1E.B. (low level) C 0.30 0.10 33.3 4.27 -- C 1.01 0.27 26.7 6.29 XT.P. (low level) T.P xlOv E.B. V1 ' C 0.42 0.15 35.7 4.00 2M.T. (oral) ^•Hormone injections twice a week for 52 days. ^Oral feeding for 8 weeks. 3On diets C and CC for 6 weeks; on diet FF for 18 v 4Pooled sample per group. 128 TABLE XXIII ONE ADMINISTRATION ON OVARY CHOLESTEROL AND TOTAL LIPID ENTS OF FEMALE RATS AS INFLUENCED BY DIET lesterol4 Total4 lipids (mg/rai) Diet3 Cholesterol4 Total4 lipids (mg/fat) ee /rat) Per cent free Total (mg/tat) Free (mg/rat) Per cent free 27 26.0 12.9 CC 1.13 0.27 23.9 14.9 18 40.0 8.9 CC 0.55 0.23 41.8 9.1 15 42.9 8.3 CC 0.18 0.11 61.1 5.5 15 62.5 5.4 CC 0.22 0.12 55.5 ‘ 7.9 15 30.0 8.69 CC 0.62 0.18 29.0 7.34 10 33.3 4.27 CC 0.32 0.09 28.1 3.00 27 26.7 6.29 CC 1.06 0.24 22.6 6.72 CC 0.33 0.11 33.3 5.35 15 35.7 4.00 CC 0.48 0.14 29.2 4.92 FF FF 1.40 0.73 0.28 0.14 20.0 19.2 7.35 5.68 a week for 52 days. eeks; on diet FF for 18 weeks post-weaning. 129 causes less change In these values. The administration of testosterone propionate and androgenic-combined sex hormones to female rats fed the cottonseed oil diet with and without cholesterol and the administration of methyl testosterone propionate to female rats fed the fat-free diet also cause a very significant decrease In the cho lesterol and total lipid concentrations in ovaries whereas the administration of estradiol benzoate to female rats fed the cottonseed oil diet with and without cholesterol causes less change in these values. The per cent of the free cholesterol over the total cholesterol in testes is decreased significantly in the estradiol benzoate- or estrogenic-combined sex hormone-treated males fed with and without cholesterol and in the ethinyl estradiol-treated males fed the fat- free diet; but there is no change in the testosterone propionate-treated males with and without cholesterol. On the other hand, the per cent of the free cholesterol over the total cholesterol in the ovary is increased in the testosterone propionate-treated g c androgenic-combined sex hormone-treated females fed with and without choles terol; but there is no change in the estradiol benzoate- 130 treated females fed with and without cholesterol and in the methyl testosterone propionate-treated females on the fat-free diet. The decrease in the per cent of the free cholesterol over the total cholesterol in the testis due to hormone treatment is a result of the greater decrease in free cholesterol, whereas the increase in the per cent of the free cholesterol over the total choles terol in ovary due to hormone treatment is a result of the greater decrease in esterified cholesterol. It is possible that the decrease in the cholesterol and total lipid contents of the testis and the ovary in all sex hormone-treated rats fed the various diets may be due to the fact that the administration of sex hormones suppresses the secretion of naturally occurred sex hor mones in the sex organs and consequently an atrophy of the sex organ occurs which in turn results in a decreased ability of the organ to synthesize cholesterol. Plasma phospholipid level and £ ratio. The P effects of sex hormone administration on the plasma phos pholipid level and the plasma ratio in male and female rats fed different diets are presented in Table XXIV; the plasma phospholipid levels are also summarized in Figure 5. 131 TABLE XXIV EFFECT OF SEX HORMONE ADMINISTRATION ON THE PLASMA PHOSPHOLIPID LEVEL AND PLASMA C/P RATIO IN RATS AS INFLUENCED BY DIET Diet Sex Hormone^ treatment Plasma^ phospholipids (mg %) Plasma^ c/p c1 M _ — 142 0.49 H - (“ ) 178 0.46 T.P. 1 F -- 146 0.50 H - (t2) 140 0.54 E.B. 1 CC1 M 112 0.67 E.B.(low level)262 0.59 E.B. -.10. T.P. ^1 ' 192 0.77 F mm m» 115 1.02 T.P. (low level) 122 0.41 (x i0) 139 0.71 E.B. 1 pp2 M - - 99 0.42 E.E. (oral) 122 0.38 F 119 0.44 M.T. (oral) 109 0.39 ^•On diet 6 weeks. 2On diet 18 weeks post-weaning. ^Hormone injections twice a week for 52 days for rats on diets C and CC; oral feeding of hormone for 8 weeks for rats on diet FF. ^Pooled i sample per group. 132 300. 280- 260- 24a 220- 200- 180- 160. 14a 12a 100. sa 60- 40- 2a HI M' Ml Untreated male M2 Male - 1 - E.B, (low level) M3 Male + E.B.,10. T.P. ^ ' M4 Male + E.E.(oral) FI Untreated female F2 Female + T.P.(low level) F3 Female + T.P.,10n F4 Female - > • M.T. (oral) CSO On cottonseed oil diet for 6 weeks OSOG On 13% cottonseed oil diet containing 1% cholesterol for 6 weeks FF On fat-free diet for 18 weeks post- weaning FI MlM2 M3 FI F2 '£L M4 FI F4 CSO C30C FF Fig. 5 Effect of sex hormone administration on plasma phospholipid level (mg??) 133 The administration of estradiol benzoate or estro~ genic-combined sex hormones to male rats on diets both with and without cholesterol increases plasma phospho lipids; this increase is more pronounced in male rats fed the cholesterol diet. The estradiol benzoate-treated male animals show a greater increase in plasma phospho lipids than do the estrogenic-combined sex hormone-treated male animals. In the male animals, the plasma £. ratio is unchanged in the cholesterol-free group; and although an increase is observed as a result of diet; the effect of the hormone injection is inconsistent in this case. There is no change in the plasma phospholipid concentration due to testosterone propionate and androgenic- combined sex hormone administration to female rats on diets with and without cholesterol. However, a significant decrease is observed in the plasma ratio in female rats F fed cholesterol and treated with testosterone propionate or the androgenic-combined sex hormones. In rats fed a fat-free diet, ethinyl estradiol causes a slight increase in the plasma phospholipids in the male rats and methyl testosterone causes a small change in the plasma phospholipids in the female rats. 134. The plasma £. ratio Is slightly decreased In both ethinyl estradiol-treated males and methyl testosterone-treated females as compared with the control animals. Although no conclusions can be drawn from the plasma £. ratio here, the effect of estrogens In Increasing the plasma phospholipids may aid In the transport of the hydrophobic cholesterol due to the hydrophilic character of the phospholipid fraction. Liver lipid patterns. The effect of sex hormone administration on the percentage distribution and the degree of unsaturation of the different fractions of liver lipids is presented in Table XXV. The percentage distribu tion of phospholipids in the liver lipids in rats fed with and without cholesterol is also shown in Figure 6. In rats fed the reference cottonseed oil diet, a sex difference is apparent as far as the percentage dis tribution of the three major lipid fractions is concerned. The female has a higher percentage of phospholipids and cholesterol esters but a lower percentage of triglycerides in the liver lipids. In the estrogenic-combined sex hormone-treated males, the percentage of phospholipids is increased to a value approaching that of the female. In TABLE XXV EFFECT OF SEX HORMONE ADMINISTRATION ON THE PERCENTAGE DISTRIBUTION AND THE DEGREE OF UNSATURATION OF THE DIFFERENT FRACTIONS OF LIVER LIPIDS IN RATS FED DIFFERENT DIETS Cholesterol^ Triglycerides^ Phospholipids^ Hormone^ esters___________________ _______ Diet Sex treatment % I.V. % I.V. 7. I.V. C1 M m* mm 14 76 71 66 16 66 E.B. /10\ T.P. 1 6 89 61 64 33 81 F — 19 62 49 59 32 58 T.P. /10x E.B. Vl' 16 42 70 39 13 48 CC1 M — 40 38 45 48 15 35 E.B. (low level) 40 41 41 54 20 58 E.B. /10v T.P. V 1 ' 40 62 40 51 19 94 F — 46 59 41 55 13 60 T.P. (low level) 28 73 59 50 13 55 T.P. /10x ft* ' 1 ' 43 48 44 46 14 52 i. r. j. F — (f) 19 62 49 59 32 58 T.P. E.B. 16 42 70 39 13 48 M — 40 38 45 48 15 35 E.B. (low level) 40 41 41 54 20 58 E.B. T.P. (f) 40 62 40 51 19 94 F T.P. 46 59 41 55 13 60 (low level) 28 73 59 50 13 55 T.P. 17 (?) 43 48 44 46 14 52 FF2 M 30 68 60 68 10 63 E.E. (oral) 11 70 40 74 49 50 F — 8 84 39 80 53 75 M.T. (oral) 8 61 50 72 42 67 *0n diet 6 weeks. 2 On diet 18 weeks post-weaning. Hormone injections twice a week for 52 days for rats on diets C and CC; oral feeding of hormone for 8 weeks for rats on diet FF. ^Pooled sample per group. Ml Untreated male M2 Male - i - E.B. (low level) I . T 5 Male + 3,B.M0s T.I-5 * T ' FI Untreated female F2 Female - i - T«P,(low level) F3 Female - i - F.P./1CK E713Y " T " CkSO On 15/3 cottonseed oil diet for 6 weeks CSOG On 15/5 cottonseed oil diet containing 1% cholesterol for 6 weeks ■T7 FI cso Offeet of M3M2 IP csocn sex hormone adminir tration on the percentage distribution of -phosnholioids in liver linids 137 the androgenic-combined sex hormone-treated females, this percentage is decreased to a value approaching that of the male. The higher percentage of phospholipids of the liver lipids in the intact females and estrogen-treated males indicates that estrogens stimulate and androgens inhibit the formation of phospholipids in the liver. The relative percentage distribution of the dif ferent lipid fractions of the liver lipids can be calcu lated as the ratio of the percentage distribution of cholesterol esters to tryglycerides to phospholipids. In the normal male rat fed a cholesterol-free diet, this ratio is approximately 1:5:1; in the normal female, this ratio is approximately 1:3:2. Assuming that the phospholipids are responsible for transport of cholesterol and triglycerides, the ratio of phospholipids to cholesterol esters (2:1) and the ratio of phospholipids to triglycerides (2:3) in the female are much more satisfactory than those ratios existing in the male, phospholipids to cholesterol esters (1:1) and phos pholipids to triglycerides (1:5). Treatment of the male animal with the estrogenic- 138 combined sex hormone preparation changes the relative proportion of lipid fractions (cholesterol esters, tri glycerides, phospholipids) from 1:5:1 to 1:11:6 which yields a ratio of phospholipids to cholesterol esters of 6:1 and a ratio of phospholipids to triglycerides of 1:2, a situation which seems to be a marked improvement over the condition existing in the untreated male. On the other hand, the ratio of cholesterol esters, triglycerides and phospholipids is changed in the female animals receiving the androgenic-combined sex hormone preparation from 1:3:2 to 1:5:1, the ratio which exists in the untreated male. In general, the female animal fed the reference cottonseed oil diet has more saturated fatty acids in combination in the three lipid fractions than does the male rat (Table XXV). This may be due to the fact that the ordinary requirement of the female for essential fatty acids is lower than that of the male. A dominantly estrogenic-mixed hormone preparation given to the male rats increases, rather than decreases, the unsaturation of the fatty acids; in the same way, a dominantly androgen- mixed hormone preparation given to the female animals 139 decreases, rather than increases, the unsaturation of the fatty acids. The reasons for this apparent anomaly are not apparent as a result of this investigation. In the cholesterol-fed animals, as is to be expected, both male and female rats have a significant increase in the percentage of cholesterol esters at the expense of more triglycerides in the male and more phos pholipids in the female. In the cholesterol-fed animals, the ratio of the percentage distribution of cholesterol esters to tri glycerides to phospholipids is approximately 3:3:1 in untreated males and approximately 4:3:1 in untreated females. It seems that cholesterol feeding masks the differences between these two ratios. Treatment of the male with estradiol benzoate and estrogenic-combined sex hormones changes this ratio from 3:3:1 to 2:2:1 in both cases, which seems to be an improvement. The presence of the small amount of testosterone propionate in the combined hormone preparation does not seem to affect the action of estradiol benzoate here. The administration of testosterone propionate and androgenic-combined sex hormones to the female 140 changes the ratio from 4:3:1 to 2:5:1 and 3:3:1 respec tively. If just the ratio of phospholipids to cholesterol esters is considered, the administration of testosterone propionate alone to the females causes a change in a favorable direction, from 1:4 to 1:2. This improvement is unexpected. The ratio of phospholipids to triglycer ides, however, is decreased from 1:3 in the untreated animal to 1:5 in the testosterone propionate-treated animal. The addition of the small amount of estrogen to the androgenic-mixed hormone preparation seems to nullify its activity in this direction. The administration of a cholesterol diet to the male causes a decrease in the unsaturation of the fatty acids in all lipid fractions as compared with the male rat fed the reference cottonseed oil diet. In the female rats fed cholesterol however, the iodine values are relatively unchanged from those obtained in the animals fed the cholesterol-free diet. In general, treatment of the cholesterol-fed male animals with estrogenic-mixed hormones increases the unsaturation and treatment of the female rats with androgenic-mixed hormones causes a slight decrease in unsaturation of the fatty acids, as 141 is shown similarly in rats fed the cholesterol-free diet. In the animals fed the fat-free diet, the ratio of the percentage distribution of cholesterol esters to triglycerides to phospholipids is approximately 3:6:1 in the untreated male and approximately 1:5:6 in the un treated female. Obviously the more beneficial ratio exists in the female. The oral feeding of ethinyl estradiol to the male changes this ratio from 3:6:1 to 1:4:5, a ratio which approaches that found in the female animal. In this changed ratio, the relative percentage of phospholipids is greatly increased and the percentages of cholesterol esters and triglycerides greatly decreased. The oral feeding of methyl testosterone to the female changes the ratio from 1:5:6 to 1:6:5. The percentage of phospholipids is relatively decreased as compared to the other two fractions although this ratio is still much better than that which exists in the untreated male. The unsaturation of fatty acids in the three lipid fractions are higher in the female than in the male. In general, the administration of estrogen to the male increases slightly the unsaturation and of androgen to the female decreases the unsaturation of the fatty acids 142 in the lipid fractions examined in a similar manner revealed in rats fed cholesterol-free diet. The exception in this group is the iodine value of the phospholipid fraction in the estrogen-treated male which is decreased rather than increased; although the percentage of the phospholipid fraction is markedly increased (from 10 to 49 per cent). In the studies of the effect of gonadectomy on the liver lipid patterns, the ratio of the three liver lipid fractions of rats fed with and without cholesterol is changed to a more favorable one in the gonadectomized male and a less favorable one in the gonadectomized female as compared to the intact male and female. From the foregoing in the studies of liver lipid patterns, it may be concluded that estrogens enhance phos pholipid production and cause a relative decrease in cholesterol esters and triglycerides in the liver lipids. In general, estrogens also increase the unsaturation of the fatty acids in cholesterol esters and phospholipids. It may indicate two essential steps in the cholesterol transport system: firstly, the proper esterification of cholesterol with unsaturated (probably essential) 143 fatty acids, and secondly, the formation of the hydrophilic phospholipids to mobilize the hydrophobic cholesterol or triglycerides. Androgens may interfere with either or both steps in this proposed transport mechanism. Biosynthesis of cholesterol. The results of the biosynthesis of cholesterol from acetate-l-C^ in rat liver slices as influenced by sex hormone treatment and different dietary conditions are presented in Table XXVI. It is obvious that, the biosynthesis of choles terol is greatly decreased due to both the cholesterol and fat-free diets in both male and female rats. This has been confirmed by other investigations (91-93,106-111). It can be observed also that the hepatic cholesterol bio synthesis in the control female is markedly lower than that which occurs in the control male on the reference cottonseed oil diet. This agrees well with the results of Aftergood (91) and Coleman et al.(93) but does not confirm the results of Fillios (89) and Rubin and White (85). Hormone administration to both male and female animals on all diets causes marked increases in choles terol biosynthesis; this enhancement of synthesis is more 144 TABLE XXVI EFFECTS OF SEX HORMONE INJECTION ON THE B] FROM 1-C14-ACETATE IN RAT LIVER 15% CSO diet (6 weeks) 15% CSO diet + 1% ct (6 weeks) Category Choles- % of change terol from sex (c.p.m. hormone per gm injection of liver) Choles terol (c.p.m. per gm of liver) % of change ° t from sex i hormone t injection Male 4500 (6) mm 662 (4) - Male + E.B. 6000 (4) +33 2750 (6) +315 Male + E.B. ( T.P. v 3700 (6) +459 Female 1721 (6) - 650 (3) - - Female + T.P. 3481 (5) +102 2362 (6) +264 Female . T.P. E.B. <^) 1318 ..<6) . +103 NOTE: 1. The % of change in the biosynthesis of choleste with the corresponding animal fed the 15% cottc 2 Numbers in parentheses are numbers of animals e 144 TABLE XXVI tE INJECTION ON THE BIOSYNTHESIS OF CHOLESTEROL ACETATE IN RAT LIVER SLICES 15% CSO diet + 1% cholesterol (6 weeks) Fat-free diet (18 weeks after weaning) Choles terol (c.p.m. per gm f liver) % of change from sex hormone inj ection % <£ change from con trol diet Choles terol (c.p.m. per gm of liver) % of change from sex hormone injection %ce£ change from con trol diet 662 (4) - -85 600 (6) - -87 2750 (6) +315 -54 3500 (6) +483 -42 3700 (6) +459 650 (3) - -62 612 (6) - -65 2362 (6) +264 -32 4000 (6) +553 +15 1318 (6) +103 synthesis of cholesterol from control diet was compared mal fed the 15% cottonseed oil diet. numbers of animals analyzed in the group. 145 marked where cholesterol biosynthesis has been depressed due to dietary conditions. As the higher tissue cholesterol level resulting from either exogenous feeding or endogenous accumulation depresses the biosynthesis of cholesterol in the tissue (92,106-111), the possibility exists that both estrogens and androgens increase the hepatic cholesterol biosynthesis as a result of their effect in increasing liver cholesterol levels. In rats fed the cottonseed oil diet, there was an increased incorporation of acetate-l-C*4 into hepatic cholesterol with an unchanged total liver cholesterol level in both estrogen-treated males and androgen-treated females as compared with the untreated control animals. Therefore, the unlabeled cholesterol level will be obviously lower in either estrogen-treated males or androgen-treated females as compared with the untreated males and females. This lowered unlabeled liver choles terol level influenced by sex hormones will in turn result in an increased incorporation of acetate-l-C^4 into hepatic cholesterol. In rats fed cholesterol or fat-free diets, the hepatic cholesterol biosynthesis is greatly depressed due to the accumulation of the 146 exogenous or endogenous cholesterol in the liver, but the effect of sex hormone in decreasing the liver cholesterol is more marked as evidenced previously in cholesterol-fed animals, therefore, the effect of sex hormones to increase the hepatic cholesterol biosynthesis will be also more marked. Fillios (89) observed in the in vivo studies in plasma an increased biosynthesis of cholesterol in the castrated male and female rats after estradiol dipropionate administration and a decreased biosynthesis of cholesterol after testosterone propionate administration. This agrees in part with the results reported in this investigation. However, since the experimental conditions used by Fillios were quite different from those used here, it is not sur prising that the results are not in complete agreement. In addition, if there is a cholesterol transport system activated by estrogens but inhibited by androgens as indi cated previously, and the cholesterol biosynthesis is located mainly in the liver, the labeled cholesterol synthesized in the liver will be transported to the plasma to a greater extent in the estrogen-treated castrated animals and to a lesser degree in the androgen-treated 147 castrated animals as compared to the untreated castrated animals. Therefore, since Fillios based his conclusions by measuring only the labeled cholesterol in the plasma, this explains also his observation that the female has a higher cholesterol biosynthesis than does the male. Rubin and White (90), using rat liver slices, showed that treatment of the castrated female rats with estradiol benzoate and treatment of the castrated male rats with testosterone propionate cause little change in the biosynthesis of cholesterol from acetate-l-C^. In this case, the dissimilarity in experimental conditions may account for the differences in results. Boyd and McQuire reported a depressed hepatic cho lesterol biosynthesis in rats after the treatment with hexestrol. The activity of this synthetic estrogen with the naturally secreted hormone was not compared.(68). Rosenman et al. (86), measuring biliary cholesterol concentration, showed a decreased cholesterol biosynthesis in rats after estrogen treatment. However, total choles terol measurements rather than isotope-labeling techniques were used, and therefore the results are subject to error. Histology. From histological examination of 148 tissues (i.e., liver, adrenals, kidneys, thyroid, pitui tary, ovaries and testes), Bemick found little difference between the hormone-treated and untreated animals (112). Comparison between the Effects of Gonadectomy and Sex Hormone Administration on Cholesterol Metabolism From the foregoing experiments one can conclude that both gonadectomy and sex hormone administration influence several aspects of cholesterol and lipid metabo lism in rats. However, there are similarities and differ ences between the effects of gonadectomy and sex hormone administration; the one is a condition where there is an absence of sex hormones and the other is one where there is an additional supply of sex hormones to balance the normal secretion of naturally occurring sex hormones. In rats fed the reference cottonseed oil diet, gonadectomy has little influence on the cholesterol levels of plasma, liver and adrenals. However, sex hormone administration causes some changes in some of these values. The plasma and adrenal cholesterol levels in the estrogen-treated male are increased to values similar to those occurring in the female. The plasma and adrenal cholesterol levels In the androgen-treated female are decreased to those values observed in the male. The administration of estrogenic-mixed hormones to males also causes an increase in plasma cholesterol, indicating that the presence of small amounts of androgen does not interfere with the activity of estrogen in increasing the plasma cholesterol level. However, the administration of the androgenic-combined hormones does not reduce the plasma cholesterol level as does the androgen given alone, indicating that the presence of small amounts of estrogen does antagonize completely the action of androgen in reducing the plasma cholesterol level. Also, the administration of estrogen to female rats increases the plasma cholesterol level, but the administration of androgen to male rats does not affect the plasma cholesterol level. In all cases, sex hormone administration causes no change in liver cholesterol levels. In rats fed the cholesterol-containing diet, both gonadectomy and sex hormone administration exert a marked effect on the cholesterol levels of plasma, liver and adrenals, although gonadectomy still has little influence on the plasma cholesterol level. The response of plasma 150 cholesterol level to the sex hormone administration in cholesterol fed rats is similar to, but more marked than, that in rats fed the cholesterol-free diet. The liver cholesterol level is decreased in both the estrogen-treated and the gonadectomized males. It is also decreased in the androgen-treated females, but increased in the gonadectomized female. In the absence of sex hormones, both the male and the female should have similar cholesterol values; and, as expected, the liver cholesterol level is decreased in the gonadectomized male and increased in the gonadectomized female to similar values. However, in the estrogen-treated male, where there is excess estrogen, if estrogen does influence the cholesterol transport system as is indicated in this investigation, the liver cholesterol level will be decreased as a result of this transport system. In the androgen-treated female, if the presence of excess androgen activates the excretion of cholesterol as indi cated from the results of Aftergood (91), therefore, a decrease in liver cholesterol is possible due to androgen treatment. Treatment of the male with the estrogenic-combined sex hormones also results in a decrease in the liver 151 cholesterol level, but it is less marked than in the male treated with estrogen alone, indicating that small amounts of androgen do partially interfere with the activity of estrogen. Treatment of the female with the androgenic-combined sex hormones causes no change in the liver cholesterol level. Here, again, the presence of small amounts of estrogen completely antagonize the activity of androgen. There is no change in the liver cholesterol level when androgen is given to male rats or estrogen is given to female rats. The adrenal cholesterol is increased in the estrogen-treated and gonadectomized males but decreased in the androgen-treated and gonadectomized females. However, the effect of sex hormone administration is more pronounced than that of gonadectomy. In rats fed a fat-free diet, there is little change in the plasma cholesterol level after either gona dectomy or oral feeding of sex hormones. However, whereas the liver cholesterol level is unaffected by the oral feeding of sex hormones, it is decreased in the gonadecto mized male and increased in the gonadectomized female to 152 values which are quite similar. The ineffectiveness of the oral feeding of sex hormones on the liver cholesterol level here may be due to the relative impotency of these hormones. The adrenal cholesterol is unchanged in both gonadectomized and estrogen-treated males. It is increased in the gonadectomized female, but is decreased in the androgen-treated female. In the studies of liver lipid patterns, in rats fed the control ration containing cottonseed oil, the female has a higher percentage of phospholipids and cholesterol esters and a lower percentage of triglycerides in the liver lipids than does the male. The percentage of phospholipids in the liver lipids is increased in the gonadectomized male and decreased in the gonadectomized female so that both values approach a similar figure midway between the normal values of the male and the female. This percentage of phospholipids is increased in the estrogenic-combined sex hormone-treated male yielding values similar to that of the control female, and decreased in the androgenic-combined sex hormone- treated female to approach the normal value found in the male. 153 In the studies of liver lipid patterns, the ratio of the percentage of phospholipids to cholesterol esters or triglycerides in the liver lipids seems important. Phospholipids may be responsible for the transport of cholesterol esters or triglycerides. If this is so, the female rat has the more favorable ratio than does the male. The ratio is improved in the estrogen-treated and gonadectomized male, but becomes less favorable in the androgen-treated or gonadectomized female. In in vitro studies using liver slices incubated with acetate-l-C^, gonadectomy of both males and females causes a significant decrease, and sex hormone administra tion to both males and females causes a significant increase in hepatic cholesterol biosynthesis in rats fed the cottonseed oil diet. As indicated previously, estrogens may enhance cholesterol transport and androgens may enhance cholesterol excretion in feces, therefore, both male and female hormones may reduce liver cholesterol levels as a result of these two different systems. In either case, the decrease in liver cholesterol level will in turn increase the biosynthesis of cholesterol, i.e., 154 the Increase in the incorporation of acetate-l-C^ into cholesterol. If the sex hormones increase the biosynthesis of cholesterol, then gonadectomy of both males and females, resulting in the absence of sex hormones, should in turn decrease the biosynthesis of cholesterol as has been shown. In rats fed the cholesterol containing diet or the fat-free diet, the effect of sex hormone administra tion on increasing the biosynthesis of cholesterol is still marked, although gonadectomy causes little change under these conditions. Possible Mechanisms in the Effects of Sex Hormones on Cholesterol Metabolism In Rats From all the experimental results obtained in studies of the effects of sex hormones on cholesterol metabolism in rats, several mechanisms may be postulated. These mechanisms are aimed to explain the over-all activities of estrogen and androgens on cholesterol or lipid metabolism. Estrogens seem to be involved in the transport system in which the proper esterification of cholesterol 155 and the mobilization of the hydrophobic cholesterol by the hydrophilic action of phospholipids is concerned. As essential fatty acids are not only required for the proper esterification of cholesterol but also for the formation of phospholipids, the actions of estrogens are probably intimately related with the essential fatty acid requirement and utilization. In the experiments in rats fed the fat-free diet, an increased requirement for essential fatty acids in the intact male and gonadectomized female is indicated. It is possible, therefore, that estrogens may either spare the essential fatty acids stored in the body or possibly potentiate the transport system in which choles terol is esterified with the essential fatty acids. Evidence accumulated in this investigation indi cates a correlation between estrogens and phospholipid metabolism. It has been shown in this investigation that estrogen increases the plasma phospholipids, results in a higher percentage distribution of phospholipids and a greater ratio of phospholipids to cholesterol esters or triglycerides in the liver lipids. Estrogens also produce a more favorable — ratio in the beta-lipoprotein fraction 156 and Increases the phospholipids in both alpha- and beta- lipoprotein fractions in the serum. It is possible, therefore, that estrogen influences the synthesis of phospholipids from essential fatty acids in the liver and the subsequent formation of the transport system involving phospholipids. The estrogen-directed transport of cholesterol would result in an increased plasma cholesterol level and a decreased liver cholesterol level--a situation which occurs in the female rat as compared with the male rat. Further, in rats fed the cottonseed oil diet containing cholesterol or the fat-free diet, the liver cholesterol is significantly reduced in the gonadectomized male and significantly elevated in the gonadectomized female as compared with the intact male and female on the same diet. Also, in rats fed the cottonseed oil diet with cholesterol, the plasma cholesterol is increased and the liver cholesterol is decreased in the estrogen-treated males as compared with control animals. In male rats fed the normal cottonseed oil diet and treated with estrogen the plasma cholesterol is increased and the unlabeled liver cholesterol is also decreased as is evidenced by the results of the hepatic cholesterol biosynthesis 157 indicated previously. All these facts can result from an estrogen influenced transport system. The possible action of androgen in antagonizing the estrogen-directed cholesterol transport system has been indicated in the studies of the percentage distribu- tion of phospholipids and the ratio of phospholipids to cholesterol esters or triglycerides and the serum lipo~ protein studies. However, the inhibition of cholesterol transport will lead not only to the decrease in plasma cholesterol but also the accumulation of cholesterol in the liver. The results in this investigation showed a decrease in both plasma and liver cholesterol levels in female rats fed cholesterol and treated with androgens. In female rats fed the reference cottonseed oil diet and treated with androgen, the plasma cholesterol level is decreased as expected but the unlabeled liver choles- terol is also decreased as evidenced by the results of the hepatic cholesterol biosynthesis indicated previously. Therefore, from all these facts, some other mechanism must be considered. The effect of androgen in increasing the excretion of cholesterol in feces has been evidenced indirectly by Aftergood (91). Therefore, both plasma 158 and liver cholesterol levels will be decreased at least partly as a result of the androgen"activated excretion of cholesterol in feces. The proportions of sex hormones within the body probably influence the cholesterol level in plasma and liver since male and female animals have both sex hormones in different proportions. When the administration of estrogen to male rats causes a significant increase in plasma cholesterol level and a significant decrease in liver cholesterol level, the administration of the estrogenic-combined sex hormones to male rats also causes similar and significant effects on these values. However, when the administration of androgen to female rats causes a significant decrease in both plasma and liver choles terol levels, the injection of the androgenic-combined sex hormones to female rats causes no change in these values. Since adrenal glands also manufacture some sex hor mones, the condition of gonadectomy cannot be considered as a complete absence of sex hormones. This may account for the fact that in several cases the cholesterol values are not similar in the gonadectomized male and female. Moreover, since adrenal glands are very sensitive to stress, the adrenal cholesterol level may be variable, and also the amount of sex hormones produced from choles terol in the adrenal glands may be variable. However, the influence of sex hormones in the regulation of some phases of cholesterol metabolism has been definitely shown in this investigation. CHAPTER IV SUMMARY AND CONCLUSIONS In view of the fact that sex differences in cho lesterol metabolism have been found to occur in some species of animals, this investigation was undertaken to study more completely the role of sex hormones in influ encing various aspects of cholesterol and lipid metabolism in rats as influenced by diet. In studies in which both male and female rats fed a reference cottonseed oil diet with and without choles terol were gonadectomized, to create an absence of sex hormones, there was little change in the plasma choles terol level. Both estrogen-treated intact male and female animals had increased plasma cholesterol levels; androgen- treated intact females had a decreased plasma cholesterol level; whereas the plasma cholesterol level of androgen- treated male rats was not different from the control value. In animals fed a cholesterol-containing diet which resulted in the accumulation of cholesterol in the 160 161 liver, decreases in hepatic cholesterol levels were observed in the gonadectomized male, in the estrogen- treated male and in the androgen-treated female. An increased liver cholesterol level occurred in the gonadec tomized female. An increased requirement for essential fatty acids in the intact male and gonadectomized female was indicated from the results in rats fed a fat-free diet. The choles terol concentrations in the liver and adrenals were greatly increased in the intact male but unchanged in the intact female; in the orchidectomized male the liver cholesterol concentration was decreased to a value approaching that obtained in the intact female and in the ovariectomized female was increased to a value approaching that obtained in the intact male. In the studies of the serum lipoprotein patterns of rats fed the cottonseed oil diet with and without cho lesterol, a hormonal control on the concentration of the components of the serum lipoproteins was revealed. There was a higher ratio of alpha-lipoprotein-bound cholesterol to beta-lipoprotein-bound cholesterol in the serum of the female than of the male; this ratio was increased in the 162 gonadectomized male and decreased In the gonadectomized female as compared with the Intact male and female. There was also a lower ratio of total cholesterol to total phospholipids in the beta-lipoprotein of the serum in the female than in the male; this ratio was decreased in the gonadectomized male and increased in the gonadectomized female as compared with the intact male and female. Under all dietary conditions tested, the plasma phospholipids were increased in the estrogen-treated male but remained unchanged in the androgen-treated female. In rats fed the control ration containing cotton seed oil, the female had a higher percentage of phospho lipids and cholesterol esters and a lower percentage of tri glycerides in the liver lipids than did the male. Gona- dectomy and the administration of estrogen to males and androgen to females reversed the sex difference in the percentage distribution of phospholipids and triglycerides in the liver lipids. The male rat fed the control ration containing cottonseed oil had a much higher rate of hepatic choles terol biosynthesis from acetate than did the female rat. Whereas gonadectomy caused a significant decrease in the 163 cholesterol biosynthesis in rats on the control diet only, the administration of sex hormones caused a sig nificant increase in the biosynthesis under all dietary conditions studied. Several theories to explain these observations are advanced. It is postulated that estrogens either spare the essential fatty acids stored in the body or possibly potentiate the transport system in which choles terol is esterified with the essential fatty acids. It is also possible that estrogens may stimulate the forma tion of the hydrophilic phospholipids which are also important for the transport of the hydrophobic choles terol. 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Asset Metadata

Creator Chen, Yu Min (author)

Core Title Effects of sex hormones on cholesterol metabolism

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1960-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Alfin-Slater, Roslyn B. (committee chair), Brown, Ronald F. (committee member), Marx, Walter (committee member), Mehl, John W. (committee member), Saltman, Paul (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-76071

Unique identifier UC11357288

Identifier 6004477.pdf (filename),usctheses-c18-76071 (legacy record id)

Legacy Identifier 6004477

Dmrecord 76071

Document Type Dissertation

Rights Chen, Yu Min

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA