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The influence of the thyroid hormone on cholesterol metabolism in rats and mice.

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The influence of the thyroid hormone on cholesterol metabolism in rats and mice.

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Content I THE INFLUENCE OF THE THYROID HORMONE ON CHOLESTEROL METABOLISM IN RATS AND MICE i A Dissertation Presented to the Faculty of the Department of Biochemistry and Nutrition i * University of Southern California In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by Samuel B. Weiss i , June 1954 UMI Number: DP21559 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI DP21559 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest* ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 Ph. 0 Bio '54 W4^f This dissertation, written by SAMUEL BERNARD WEISS under the direction ofJ^X.sGuidance Committee, and approved by all its members, has been pre sented to and accepted by the Faculty of the Graduate School, in partial fulfillment of re quirements for the degree of D O C T O R O F P H IL O S O P H Y Dean Com m ittee on Studies fat.. Chairman ACKNOWLEDGMENTS It is with sincere gratitude that I acknowledge the guidance and invaluable assistance of Dr. Walter Marx. I wish to express my gratitude to Dr. Lore Marx for her generous and valuable technical assistance in preparing and interpreting the histological preparations used in part of this work.. I wish to thank the National Institutes of Health and the Life Insurance Medical Research Fund for the award of Predoctoral Fellowships, and the Allan Hancock Founda tion, University of Southern California, and the Laboratory Division of the Los Angeles County Hospital for the facilities that were made available. I would also like to express my appreciation to the Faculty of the Department of Biochemistry and Nutrition for their many kindnesses and helpful suggestions during my graduate career* Finally, to my family, and my many close friends, I express my sincerest gratitude for their moral support which has helped me through many difficult periods during this long undertaking. TABLE OP CONTENTS CHAPTER I. INTRODUCTION .......................... II. HISTORICAL REVIEW ..................... Biosynthesis of cholesterol ......... Sites of synthesis............ . . . Cholesterol precursors ........... . Cholesterol breakdown in vivo ....... Thyroid activity and tissue cholesterol distribution ...................... Plasma........................... Carcass .......................... The influence of thyroid on sterol metabolism ................ . < Synthesis...................... Excretion and degradation ...... III. METHODS AND MATERIALS ................. Cholesterol deposition studies ....... Treatment of animals ............... Chemical analysis ................. Cholesterol balance studies ...... Balance experiment 1 ........ . Treatment of animals ............. CHAPTER ’ PAGE Chemical analysis ........... 20 Balance experiment II .................... 21 Treatment of animals ..... .......... 21 Chemical analysis ..................... 21 Experiments using radioactive cholesterol • 22: i * Preparation of C-^-cholesterol....... 22 Randomly labelled cholesterol ....... 22 Purification .............. 23 4-C^-cholesterol.................... 24 Cholesterol suspensions ............... 24 Treatment of animals ............. 25 Chemical fractionation ....... ..... 26 Uonsaponifiable matter ............... 26 Saponifiable matter ......... 27 i Determination of Cl4................ 27 Analysis of crystalline derivatives • • 27 Dry combustion to BaCG3 ••••••• 27 Preparation of BaCOg samples for counting ....................... 30 Counting and self absorption ..... 32 Analysis of amorphous material ....... 32 Direct mount technique............ . 32 V CHAPTER PAGE Conversion of counts from direct mount to the BaCOg basis...... 32 Counting procedure .......... 34 IV. RESULTS............................... 36 Cholesterol deposition studies ........... 36 Cholesterol balance experiments .......... 40 Experiment I ........................ 40 Experiment I I ..................... 42 Experiments using radioactive cholesterol . 45 V. DISCUSSION OF RESULTS................. 52 VI. SUMMARY............* .................... 61 BIBLIOGRAPHY.................................... 64 LIST OP TABLES TABLE PAGE I. Composition of Purified Basic Diet......... 16 II. Experimental Groups......... 17 III. Composition of Modified Purina Chow Diet ... 19 IV. Effects of Thyroid and of Choline Plus ! Inositol in Rats Fed a Purified Diet Containing 20 Per Cent Fat............... 37 V. Effects of Thyroid and of Choline Plus Inositol on Cholesterol Distribution and Liver Lipids in Rats Fed 2 Per Cent Cholesterol and 0.25 Per Cent Bile Salt . , 38 VI. Effect of Thyroid on the Total Cholesterol Balance of Mice Fed a Restricted Diet Containing 0.65 Per Cent Cholesterol .... 41 VII. Effect of Thyroid on the Cholesterol Content of Carcass and Excreta of Mice Fed a Restricted Diet Containing 0.65 Per Cent Cholesterol........................ 43 VIII. Effect of Thyroid on the Total Cholesterol Balance of Mice Fed Ad Libitum on a Diet Containing 0.15 Per Cent Cholesterol .... 44 TABLE IX. X. XI. XII. Effect of Thyroid and Thiouracil on the Cholesterol Content of Carcass and Excreta of Mice Fed Ad Libitum on a Diet Containing 0.15 Per Cent Cholesterol ................. The Distribution of C- * - 4 in Hypo- and Hyper thyroid Mice 48 Hours After Intravenous 14 Administration of 4-G -Cholesterol . • . . The Distribution of C14 in Hypo- and Hyper thyroid Mice 95 Hours After Intravenous Administration of 4-C^4-Cholesterol • . • • The Distribution of C^4 in Hypo- and Hyper thyroid Mice 48 and 96 Hours After Intravenous Administration of Randomly Labelled Cholesterol................ vii page: 46 48 49 50 LIST OP FIGURES FIGURE 1. Dry Combustion Apparatus............. . • 2. BaG03 Plating Apparatus. ................. 3. Self Absorption Correction Curve for BaCOg • 4. Conversion Factor Curve From Counts as Obtained by Direct Mount to Counts as Determined by the BaCOg Basis CHAPTER I INTRODUCTION - Since Anitschkow first induced experimental athero sclerosis by feeding rabbits cholesterol, (1-4), investi gators have attempted to prevent, inhibit or treat the lesions induced by cholesterol. The use of thyroid hormone and organic and inorganic iodides, have yielded good responses (5-10) and compounds of this type have been con sidered for clinical use in atherosclerosis. Experimental findings of this nature are largely responsible for the current interest in investigations involving thyroid-sterol interrelationships. In spite of the general interest in cholesterol- induced atherogenesis in the past, somewhat less attention has been directed toward the influence of the thyroid hormone on other tissues containing this sterol. Actually, the effect of the thyroid hormone on tissue cholesterol and the mechanism by which it influences sterol metabolism, as well as atherogenesis, are inadequately understood and await further clarification. It has therefore been the purpose of this investigation to examine further the relationship between the thyroid hormone and tissue cholesterol, in both rats and mice, and to understand the mechanism by which this 2 / endocrine agent exerts its action. In 1950, Marx et al. (11) observed that rats fed a diet containing 1 per cent cholesterol and 0.4 per cent thyroid, deposited a significant amount of sterol in the liver although the plasma cholesterol content was reduced practically to normal levels. The possibility was con sidered that in the hyperthyroid state the requirement for lipotropic substances is increased. In order to study this interrelationship between thyroid and lipotropic agents, the effects of choline and inositol on cholesterol and lipid distribution were investigated in normal and hyperthyroid rats fed a diet high in cholesterol and fat. It was observed that thyroid hormone was capable of mobilizing hepatic cholesterol and lipid deposited in these cholesterol-fed rats, and it became of interest to investigate the nature of this phenomenon. The subsequent experiments reported in this dissertation were undertaken in order to study the mechanism responsible for the increased rate of hepatic cholesterol removal observed in the hyperthyroid state. Mice were chosen for this study in order to facilitate the chemical analyses of whole carcass and excreta. Balance experiments were initially performed in normal and hyperthyroid animals. These animals were fed a 3 diet containing a high cholesterol content so that the nature of this sterol disposal, i.e., excretion, destruction! or changes in tissue distribution, might be ascertained. However, the action of thyroid on sterol metabolism is complex, It was necessary, to select the proper nutritional and dietary conditions in these experiments so that the metabolic state of the experimental animals would be favorable for studying the catabolic aspects of this problem. It was felt that tracer experiments would not be subject to the limitations of choosing the proper nutri tional and dietary conditions. Therefore, the last phase of this work was conducted with isotopic carbon. Radio active cholesterol was administered to thyroid and thio- uracil treated mice to determine the extent of sterol degradation and the nature of the products formed. CHAPTER II HISTORICAL REVIEW Following the discovery of cholesterol in the late 18th century, the importance of this compound as a con stituent of normal tissue was doubted by most investigators, since it had been detected only in pathological and necrotic tissue (12, 13). Some five decades later this attitude was modified, as cholesterol was shown to exist normally in brain (14), in blood (15) and other organs. However, it was not until the 20th century that the synthesis of cholesterol was demonstrated in mammalian organisms, by the use of balance experiments (16-20). Much of the interest in the biological activity of cholesterol stems from its non-specific action as an atherogenic agent. In 1833, Lobstein (21) invented the term arteriosclerosis and used it in a generic sense to include a number of different morbid processes, all of which produce lesions and thickening of the intimal wall. Marchand (22), in 1904, coined the term atherosclerosis to designate a particular type of intimal arteriosclerosis. The prefix ethers . (Greek, athere, meaning mush) was selectee to designate the amorphous lipid accumulation in the intima. 5 It is the lipid-laden intimal plaques ‘ of atherosclerosis which reduce in size the lumina. of the arteries, and which, therefore, may impair the blood'supply to heart, brain and other vital organs, which in turn may cause chronic illness and death. As early as 1847, histo-pathologic work by Vogel (23) identified cholesterol in atherosclerotic plaques, and Mettenheimer (24) noted that the lipoidal mass was doubly refractive due to the presence of cholesterol esters. Biochemical evidence was contributed by Windaus (25) early in the 20th century, who showed that the concentrations of free and esterified cholesterol were increased in athero matous aortas. In 1908, Ignatowski (26) and Saltykow (27) first successfully induced true atherosclerosis experimentally by feeding rabbits diets of meat, milk or eggs. In these experiments, it was not clear which dietary factor was responsible for the lesions. A few years later, Anitschkow (3) and Wacker and Hueck (28) induced typical lesions by giving rabbits pure cholesterol in oil, thereby proving that the sterol was in fact the atherogenic stimulus. Research endeavors which have tried to establish a specific relationship between deranged sterol metabolism and athero sclerosis date from these discoveries. 6 Within recent years, considerable information has been forwarded concerning the biosynthesis and degradation of cholesterol in mammalian tissue. Many of the cholesterol I studies in the past were stimulated' by the belief that atherosclerosis began with a deranged sterol metabolism (29, 30). Though general knowledge of steroid metabolism is .still insufficient to allow valid conclusions in support of such a tenet, information about cholesterol metabolism per se is steadily being gathered. Biosynthesis of Cholesterol Sites of synthesis. The sites of cholesterol synthesis in living matter are no less ubiquitous than the distribution of the sterol itself in the animal organism. With the possible exceptions of adipose (31) and adult brain (32) tissue, every organ or; tissue that has been examined, is able to synthesize cholesterol. Bloch, Boreck and Rittenberg (33) were the first to show cholesterol synthesis in vitro by the incubation of liver slices with heavy water, deuterio-labelled acetate and doubly-labelled acetate. Since then, other investigators have demonstrated the in vitro synthesis of this sterol from 14 C -acetate in the following tissues: the gut, gonadal 7 tissue, kidney, adult skin, baby skin, one day-old rat brain, rabbit, fetus and the arterial wall (32, 34-37). In these experiments, the biosynthesis of cholesterol was demonstrated with tissue slices only. More recently, this synthesis has been accomplished in homogenates and in water-soluble preparations from rat liver mitochondria (38-40). Cholesterol precursors. Bloch et al. (33) showed that rat liver slices were able to convert acetate to cholesterol and long chain fatty acids. Acetone (41, 42), pyruvate (43, 44), acetaldehyde, aeetoacetate (45), isovalerate (46), butyrate, hexanoate and octanoate (43) have also been identified as active pre cursors for these syntheses. Results obtained to date have shown acetate to be a carbon source for both the isooctyl side chain and the polynuclear moiety of the sterol. A number of individual carbon atoms have been traced to either carboxyl or methyl carbon atoms of acetic acid. Direct proof has been furnish ed that the entire side chain of cholesterol originates from acetic acid (47-49). Five of the side chain carbon atoms are derived from methyl groups of acetic acid and three from acetate carboxyls. The location of acetate 'methyls (Cg^, Cg2> ^24* Cgg, anc* ^27) an<^ ac©tate j carboxyls (Cgg, C23 and C25) In the isooctyl moiety has I ( ! been established (48). A similar but incomplete character-; I ! ization of the sterol nucleus has been reported by Cornforth t , and coworkers (49). ' 4 1 I j The similarities in some structural features between ’ | the steroids and various natural products composed of j ; isoprene units, raised the possibility that acetate was ' converted to cholesterol by way of five carbon units, t j which might be at least formally related to isoprene. , 1 ; ! Bonner and Arreguin (50) obtained evidence for a utiliza- i | tion of acetate in the synthesis of natural rubber by j t isolated guayule leaves. Carrying forward this singular j < idea; Langdon and Bloch (51, 52) recently showed the , conversion of radioactive squalane, biosynthetically pre- 1 pared from labelled acetate, to cholesterol in the tissues j of the mouse. The data suggest that squalene, a dihydro- j triterpene, is an immediate precursor in the pathway of ; cholesterol synthesis. The absence of label in isolated i fatty acids in these experiments, precludes a pathway of ; two-carbon intermediaries in the conversion of squalene j to cholesterol. These observations have been confirmed by j i Tomkins e_t al. (53). Animals which had previously been fed 1 natural squalene showed a marked dilution in the isotopic t 9 labelling of cholesterol as compared to animals on a squalene-free diet, when liver slices were incubated with radioactive acetate. Both carbons of acetate were utilized in squalene synthesis. Squalene is a normal constituent of rat liver, and although it occurs in small concentrations in hepatic tissue it is regenerated at a rapid rate (52). Cholesterol Breakdown In Vivo Page and Menschick (54), in 1932, Schoenheimer and Breusch (20), in 1933, and Cook (55), in 1937, showed by means of balance experiments that the animal organism can destroy or modify cholesterol under conditions of a relatively high cholesterol intake. These investigators were aware however, that the intestinal flora in mammals was capable of metabolizing sterols (56, 57). I In 1943, Bloch at al. (58), with the aid of deuteri um-labelled cholesterol, showed that one of the products of conversion was cholic acid, a finding recently confirmed by Byers and Biggs (59) and Zabin and Barker (60). Gould (61) and Chaikoff at al. (62) gave radioactive cholesterol to mice and rats, by oral and intravenous administration, and found an appreciable amount of isotope in the expired CO2 and the fecal acid fraction. Respiratory carbon dioxide was found to be contributed by the side chain, but not from 10 the ring system (63, 64). Kritchevsky, Kirk and Biggs (65) fed C14-cholesterol to rats by stomach tube and found activity in body fats and glycogen. Cholesterol has also been shown to be a precursor of progesterone (66) and adrenocortical hormones (67) and it is possible that it may furnish the source from which other steroid hormones, i.e., sex hormones, are derived. These experiments indicate that cholesterol is metabolized ini mammalian tissues and that this sterol is converted or broken down into various degra dation products. Thyroid Activity and Tissue Cholesterol Distribution Plasma. It has been demonstrated that an increase in thyroid activity is associated with a decrease in plasma cholesterol concentrations, in both man and experimental animals (9, 10, 68-70). This inverse relationship of the thyroid hormone concentration to the plasma cholesterol level and to plasma lipids in general is well established (71). The production of arterial lesions in dogs, morpho logically similar to those in human atherosclerosis, was accomplished by Steiner and Kendall (72) by the administra tion of thiouracil concurrently with cholesterol feeding. The possibility that thyroxine exerts a protective effect 11 on atherosclerosis by preventing the development of a hyper- cholesterolemic condition has been suggested by Turner (7). Page and Bernhard (73), however, found that an organic iodide protected against cholesterol-induced athero sclerosis in rabbits, although it was without depressing effect on hypercholesterolemia and hyperlipemia. Similarly, both thyroid and thyrotropic hormones lower blood cholester ol, yet the latter stimulates cholesterol deposition in the aorta under increased sterol feeding (74, 75). These find ings led some investigators to suggest that the thyroid hormone affects atherogenesis via its influence on tissue cholesterol, rather than by its effect on hyperlipemia. Carcass. There are conflicting reports in the literature con cerning the influence of thyroid hormone on tissue sterol. According to a number of investigators thyroid powder or thyroxine elicited an increase in the cholesterol content of various organs, in particular of the liver (76-78), of the skin (79), and of the whole carcass (80-82). On the other hand, in cases of an accumulation of cholesterol in tissues, particularly in the liver and aorta, produced by dietary or endocrine mechanisms, thyroid was reported to cause a removal of the deposited sterol (5, 7, 83-85). 12 The Influence of Thyroid on Sterol Metabolism Synthesis. In 1949, Karp and Stetten (86), with the use of deuterium-labelled water, demonstrated a stimulation of cholesterol synthesis, as a consequence of thyroid feeding, in mammalian tissue. More recently, these findings have been confirmed by Byers and coworkers (87) and Marx et al. (88). Furthermore, it appears that the stimulation of sterol synthesis, in the hyperthyroid state, cannot be attributed to an increase in the metabolic rate, but more likely to a specific action of thyroxine itself (88). Excretion and degradation. Bosenman, Friedman and Byers (89) have shown a direct relationship between thyroid activity and biliary cholesterol output. This group suggested that the rate of biliary sterol excretion may be employed as an indicator of hepatic cholesterol synthesis (90). Friedman et al. (91) have also observed, in rats fed a sterol-free diet and under ad libitum conditions, an increased rate of fecal cholesterol excretion in the hyperthyroid state, as com pared to both normal and hypothyroid animals. These observations appear to confirm the stimulation of tissue 13 sterol synthesis by thyroid administration and may also account ,for,' at least in part, the mobilization and removal of tissue cholesterol observed under the influence of this endocrine agent (5, 7, 81, 83-85). In 1938, Hurxthal and Perkins (92) reported that balance experiments performed with mice on a sterol-free diet indicated that the thyroid hormone stimulated cholester ol destruction. Fleischman and Wilkins (93), in 1941, found that thyroid hormone administration to human cases of cretinism and dwarfism caused a reduction in serum cholester ol without a corresponding increase in cholesterol excre tion. These investigators suggested that either destruction of the sterol or changes in its distribution, i.e., between plasma and tissue, may have taken place. The action of thyroid hormone on tissue cholesterol and sterol metabolism in general, as indicated by the above reports, is multifold and confusing. This hormone appears to stimulate not only cholesterol synthesis but, also sterol destruction and excretion. At the same time, con tradictory reports exist as to the effect of thyroid on cholesterol deposition in vivo. Therefore, the elucidation and clarification of this hormonal-metabolic relationship appeared to be warranted. The investigations undertaken in the present thesis have aimed towards this end, and it is 14 sincerely hoped that these findings have helped to further the understanding of this perplexing biochemical problem. CHAPTER III METHODS AND MATERIALS ! I ' I i j j Cholesterol Deposition Studies , i j 1 Treatment of animals. I j Female rats (University of Southern California J i | istrain), 27 to 31 days old and weighing about 60 grams, I ! . ! I were divided into seven groups. Body weights were recorded I ! ' : ;weekly throughout the experimental period. , : I All animals were fed, ad libitum, a purified basic ; ,diet containing 20 per cent fat, 18 per cent casein and 10.25 per cent bile salt. The composition of this basic ! diet is given in Table I. ' i j The experimental groups were supplemented with the 'following components, either alone or in combination: i ; (a) 2 per cent cholesterol, (b) 0.4 per cent desiccated :thyroid, and (c) choline and inositol; the latter were j i 'each fed at a level of either 0.25 per cent or 1.0 per cent , t * of the diet. The dietary supplements of the experimental < i • groups are summarized in Table II. ! t i i Animals were autopsied under nembutal anesthesia 1 ;after an experimental period of seven weeks. Blood was ! :drawn from the inferior vena cava. Liver, and in some TABLE I COMPOSITION OP PTJRIPIED BASIC DIET Dietary component Per cent Salt mix 4.50 Casein ^ 18.00 Wesson oil 20.00 Celluflour 4.00 Sucrose 28 ,26 Corn starch 25.00 Bile salt 0.25 l/ Vitamins were added to the diet in a casein mix* Vitamins per kilogram of diets thiamine, 50 mg.} riboflavin, 20 mg.} pyridoxins, 20 mg.} calcium pantothenate, 60 mg.} nicotinic acid, 60 mg.} folio aoid, 10 mg.} para-aminobenzoio aoid, 200 mg.} 2-methyl naphthoquinone, 5 mg. In addition, the following were given once weekly orally (by syringe)* 550 I.TJ. vitamin A, 55 I.TJ. vitamin D and 0.66 micrograms vitamin B^. TABLE I I EXPERIMENTAL (SOUPS Dietary Supplements Group Z f o Cholesterol 2/ G */ - Thyroid * Choline + Inositol Number •25/^ Each 1. 0^ Bach of rats I - - mm mm 7 II + • • mm- m 6 III - - mm 7 IV - m ♦ - 9 V + + - 7 Via ♦ - + - 8 VIb ♦ - - ♦ 7 Vila ♦ + + - 9 VI lb * ♦ mm + 5 j ]/ ♦ indicates: present in diet • { -indicates! absent from diet* ■ 2/ Cholesterln - Merck and Co., Rahway, N. J* ; s/ Thyroid Powder - U «S J? • - Armour and Co. , Chicago, Illinois ♦ 18 1 I instances lungs and adrenals, were excised as quickly as 1 j possible and minced with scissors. Chemical analysis. The minced tissues were extracted using a Soxhlet ' I apparatus, first with 95 per cent alcohol and then with ether. Total lipid and cholesterol contents were determined; i i the former, by drying aliquots of the Soxhlet extracts to constant weight, and the latter, according to a modified Schoenheimer-Sperry-Chaney procedure (94). The aorta was taken for histologieal examination which was made as soon as possible to avoid loss of cholesterol during storage in formalin (11). Cholesterol Balance Studies Balance experiment 1. Treatment of animals.- Female mice, Swiss Colony strain and 5 to 6 weeks of age, were used. All animals were fed Purina chow supplemented with 1.0 per cent sulfasuxidine and 0.25 per cent streptomycin for one day prior to the balance period to prevent or re duce cholesterol destruction by the intestinal flora (57). The composition of the diet used during the preliminary and experimental periods is given in Table III. TABLE I I I COMPOSITION OP MODIFIED PURINA CHOVT DIET Dietary component Per cent Purina oho* (ground) 82.00 Yeast Anheuser-Busch, "strain G" 10,00 Dextrin 2.00 Wesson oil 4.50 Desiooated ox bile (U«S«P.) 0.25 Sulfasuxidine ^ 1.00 2/ Streptomycin — 0.25 }/ Sulfasuxidime, Sharp and Dohme, Division of Merok and Company, Incorporated• 2/ Streptomycin Hydrochloride, E. R. Squibb and Sons. I The total cholesterol balance was then determined as I 1 ifollows: Mice were separated into two groups and placed j I I |individually in two liter beakers equipped with false ; i Ibottoms and screen lids which served as metabolism cages. j ; iBoth groups were fed a diet containing Purina chow, 0.65 j ( j jper cent cholesterol, the antibacterial drugs mentioned J jabove and some other supplements as indicated. In addition, ione group received 0.4 per cent thyroid powder in its feed. \ j ! Animals in this “thyroid” group were also given one initial j injection of 50 micrograms of thyroxine. All animals ! i j received the same amount of food twice daily, a total of I s < | 3 grams per day, in food cups equipped with special tops to j i prevent spilling, and all excreta were collected for the I - 'i jentire experimental period of 16 days. ' 1 1 I At the end of the balance period the animals were ; I ,autopsled as indicated above. At the end of the pre- ! treatment period, and just prior to the beginning of the i I ! balance period, additional animals from each group were > |sacrificed (“onset controls”). j i i i Chemical analysis.- The liver, the remaining carcass| i and the excreta were hydrolyzed in 30 per cent KOH (w/v) isolution in 50 per cent (v/v) ethyl alcohol, for 3, 5, and >9 hours respectively, and, then extracted with diethyl I jether. The solid residues remaining after hydrolysis of j 2 1 the feces were then re-extracted for 20 hours in the Soxhlet apparatus, first with 95 per cent ethyl alcohol (v/v) and then with ether. The total cholesterol content of the extracts was then determined as described above. Balance experiment II. Treatment of animals.- Male mice, Swiss Colony strain, and 6 to 7 weeks of age were used. Prior to the experimental period, all animals were fed Purina chow supplemented with 0.5 per cent thiouracil for 12 days and then divided into two groups. One group continued on the same thiouracil regimen while the other group was given 0.4 per cent desiccated thyroid with the thiouracil containing diet, both for a period of 3 days. The mice were then placed in the individual metabolism cages described above and the total cholesterol balance was determined for a period of 4 days. During this time, each animal was allowed to feed ad libitum, and each group continued on either the thiouracil or thyroid regimen as indicated above. Animals were sacrificed at the end and just prior to the beginning of the balance period as indicated above. Chemical analysis.- The methods employed for tissue hydrolysis, cholesterol extraction and determination were the same as those described above Experiments Using Radioactive Cholesterol Preparation of C - * - 4-cholesterol. Randomly labelled cholesterol.- Radioactive cho lesterol, randomly labelled, was prepared biosynthetically by the incubation of carboxyl-labelled acetate with rat liver slices (33). Young adult rats, weighing between 180 and 250 grams were sacrificed by a sharp blow to the head, their livers excised rapidly and placed in cold saline. Liver slices, approximately 0.5 mm. thick, were cut free hand with a razor blade and a homemade holder and placed in a tared beaker containing saline. In a typical experiment, 2.5 grams of tissue, transferred from the beaker, were suspend ed in 15 ml. of Krebs’-Ringer-Phosphate buffer at pH 7.4, in a modified 125 cc. Erlenmeyer flask, to which had been added 8.2 mg. of radioactive acetate containing 1 me. per mM. • The mixture was incubated at 38° C, in an atmosphere of 100 per cent oxygen, under constant shaking using a Warburg apparatus adapted to hold 125 cc. Erlenmeyers. Following incubation for 3 hours, the tissue was digested with 30 per cent K0H (w/v) in 50 per cent ethyl alcohol (v/v), and - cholesterol was extracted with ether and isolated as the digitonide. The digitonide was cleaved with hot pyridine (95). The cholesterol was recovered by adding ether to the pyridine solution and centrifuging the i mixture for 20 minutes. The supernatant ether was decantedj | into a separatory funnel, washed with 3 N H2SO4, with J distilled water, dried over anhydrous Na2S©4 and evaporated! 1 to dryness in vacuo. The white residue was dissolved in a I | minimum of hot methanol and allowed to crystallize slowly | at room temperature. The long white crystals obtained had a melting point of 146-147° G and looked identical with : those obtained from commercial cholesterol when crystallized J • j in a similar fashion. An overall yield of 72 mg. of sterol1 jwas isolated from 24 grams of liver tissue; the product ' i I i had an activity of 150,000 counts per minute per mg. of ! t I carbon when counted as BaCOg. J ! I j ; I Purification.- The radioactive product, as obtained ' ! 1 ; above, was diluted with unlabelled cholesterol and purified i : ! i by means of the dibromide (96). The sterol was dissolved in 1 . . . , ! hot ethanol and that solution cooled in an ice-water bath. I 1 I , A 1 N alcoholic solution of bromine was added by capillary J I ! pipette with constant stirring until a slight yellow color ; 1 1 persisted. The fine crystalline solid, which precipitated ; 1 i | from the solution, was collected in a sintered glass filter,' i ' 1 washed twice with 10 cc. portions of ice-cold ethanol, and j 24 dried in a vacuum desiccator in the dark. The dry product was transferred to a 250 cc. round bottom flask and dissolved in 60 cc. of benzene whereupon 10 cc. of a 20 per cent Nal (w/v) solution, in absolute ethanol, was added and the mixture refluxed for two hours. After heating, the, dark brown solution was washed with an aqueous solution of sodium sulfite and then with water to remove all salts. The benzene solution was dried, evaporated to dryness under vacuum, resuspended in hot methanol and allowed to. crystallize at room temperature. A melting point of 147- 148° C was registered for this compound. The final yield of purified radioactive sterol was 52 mg. with an activity of 80,000 counts per minute per mg. of carbon. 4-(A4-cholesterol.- The synthesis of this compound, and its purification, was accomplished by the reduction of the enol acetate of A 4-cholesten-3-one labelled at C^ with C14, and by chromatography using silica gel, as described by Schwenk et al. (97). Dr. I. Zabin, of the University of California, Los Angeles, California, prepared this material, and it had a specific activity of 133,000 counts per minute per mg. of carbon. Cholesterol suspensions. Suspensions of cholesterol, for intravenous 25 administration to mice, were prepared using Tween-20 (64). Radioactive sterol was weighed in a vial (l cc. capacity) and one drop of Tween was added per mg. of cholesterol used Small amounts of chloroform were also added to aid solution A finger tube (partially filled with ethanol) was adapted to hold the vial, and as the tube was heated with a micro- burner the alcohol vapors bathed the vial. At the same time, a jet of % Sas was directed at the Tween solution to help remove the volatile chloroform vapors. When all the chloroform had been removed and the solution was quite clear, the vial was placed in a hot water bath and the Tween mixture brought to proper volume with saline. The vial was corked, shaken thoroughly to insure complete mix ing and allowed to cool. Tween-sterol preparations, of this type, when freshly prepared were completely clear and stable at room temperature for a few hours. Animals were injected with this preparation as quickly as possible and fresh suspensions were made for each experiment. Treatment of animals. The mice employed in this experiment were the same as the animals used in Balance experiment II. At the end of the pretreatment period, emulsions of radioactive cholesterol, either randomly labelled or labelled in the A ring only, were injected intravenously by way of the tail j vein into both hypo- and hyperthyroid animals. These mice | were placed in the two liter beaker metabolism cages I described above, and allowed to feed ad libitum on either i I the thiouracil or thyroid containing diet. In some j 1 l i i : instances, these animals were placed in an all glass j i t j metabolism apparatus, designed and constructed for the | collection of expired carbon dioxide and excreta, to i I determine whether isotopic carbon was being expired in j this manner. At the end of 48 and 96 hours respectively, j * t ! mice from both groups were sacrificed and autopsied,' and i i the nonsaponifiable and saponifiable fractions from liver, j r I , carcass and excreta were analysed for radioactivity. j ! Chemical fractionation. ! ; i Nonsaponifiable matter.- The procedure employed , j i | for the handling and hydrolysis of tissues and excreta | » . ^ ] was the same as described for the balance studies above, ! i however, the extraction and determination of cholesterol .! was modified in the following manner. I After alkaline hydrolysis, tissues and excreta were I i extracted with ether until no further radioactivity could j ; ; * 1 ' be detected in the ether extract. The combined ether ! extracts were washed thoroughly with distilled water, the j ' washings being added to the*aqueous residue, then evaporated 27; to dryness and adjusted to volume in 1:1 alcohol-acetone. ' Digitonin, 1.0 per cent (w/v), in 80 per cent alcohol (v/v) was added to aliquots of this solution and cholesterol was determined gravimetrically as the digitonide. The isotope content of the digitonide was determined as described below. Saponifiable matter.- The alkaline aqueous phase, remaining above, was neutralized with concentrated H2SO4 in an ice bath and the pH was adjusted to 1. This solution’ i was re-extracted with ether, the combined extracts were washed with distilled water, dried over anhydrous NagSO^, and then evaporated to dryness. The residue was adjusted to volume with alcohol-acetone 1:1 and the total saponifi able matter was determined by drying aliquots of this solution to constant weight. The content of this fraction was determined as described below. De termination of C^. Analysis of crystalline derivatives. Dry combustion to BaCO?;. - Organic compounds such as sodium acetate, cholesterol and cholesterol digitonide were combusted directly to BaCOg. A modification of a procedure described by Niederl and Niederl was employed I 28 1 j for dry combustion (98). A diagram of the apparatus used « i | is given in Figure 1. 1 | I ! The combustion train consists of the oxygen tank, i { i 1 I I; the bubble counter and U-tube, II; the movable bunsen ' burner with wing top attachment, III; the stationary 1 Sargent heating unit, IV; the Vycer combustion tube with i j | side arm, V; and the CO© collecting tube, VI. j I ♦ l i Material for combustion was placed in a platinum boat (c) and moved into position in the combustion tube. : i . The tube was corked and the system was flushed with Og • for two minutes at a normal flow rate of two bubbles per i second. Traces of C02 or moisture in the pre-heating ■ system were removed by soda lime (a) and anhydrous calcium ■ 1 1 chloride (b). Heat was slowly applied to the boat with ' the bunsen burner until all the combustible material had j 1 charred and disappeared, at which time the heat was r increased to a “blasting”'temperature. The areas under the; boat, and to the right and left of the boat, were blasted I ! separately for five minute intervals to insure complete combustion. Gaseous combustion products, which pass over the silver and platinum screens (120 mesh) (d, e) maintain-j ; ed at a temperature of 850° C, were bubbled through a col- 1 . lecting tube containing a saturated solution of Ba(0H)g in 2 per cent KOH (w/v) precipitating carbon dioxide as VyV vV\ DRY COMBUSTION APPARATUS FIGURE I barium carbonate. Upon complete combustion, the precipi tate was centrifuged, washed five times with 5 cc. portions of CO2 free water and twice with methanol. Preparation of BaCQx samples for counting.- The BaCOg, as obtained above, was ready to be plated for counting. A tared planchet was attached to one end of a hollow aluminum tube, four inches in length and 1 inch in diameter. By stretching a 3 cm. band of thin surgical tubing over the outside diameters of both aluminum tubing and planchet, the latter was held firmly in place. A diagram of the apparatus is given in Figure 2. Approximately 2 cc. of methanol was added to the BaCOg and by vigorous stirring the sample was finely sus pended. This suspension was transferred into the planchet by pipette, and the entire aluminum tube with planchet attachment was centrifuged for 15 minutes. After decanting the supernatant, the planchet was removed, air dried under a 60 watt lamp, and then heated at 110° C for 20 minutes in a drying oven. BaC^Og samples, prepared by this method and stored in a desiccator did not exchange CO2 with that of the surrounding medium to any appreciable extent. A continual check for loss of radioactivity showed a constant number of counts, within the limits of error of the counting proced- u n a s s e m b l e d ALUMINUM TUBING SURGICAL TUBING PLANCHET BaC03 PLATING APPARATUS FIGURE 2 ure, for over a period of 1§ years. Counting and self absorption.- The samples prepared above had an area of 5.31 sq. cm. and were counted in a windowless Q,-gas counter tube, Model EX7, Nuclear Corporation, Chicago. Samples ranged from 25-60 mg. per planchet in thickness and were corrected for self absorp tion to 10 mg. per planchet, as indicated in Figure 3. All samples contained counts at least three times the back ground, and a minimum of 2048 counts per sample was taken. It was not necessary to correct for coincidence in these experiments. Analysis of amorphous material. Direct mount technique.- For tissue and fecal saponifiable fractions, duplicate aliquots of each fraction were dried directly on aluminum disks fitted with lens paper, over an area of approximately 4.5 sq. cm. (99). Conversion of counts from direct mount to the BaCO?; basis. - Samples of radioactive saponifiable material, ranging from 2.5-39 mg., were directly plated and their activities determined as described below. Duplicate samples were oxidized to C02, converted to BaC(>3 and the activity of the BaCOg determined. All barium BaCO, WEIGHT M G - P E R 5 -3 SQ.CM. 33 80' 40' 20 10 0 2 0*4 RATIO OBSERVED COUNTS TO ACTUAL COUNTS SELF ABSORPTION CORRECTION CURVE FOR B*COs FIGURE 3 1 carbonate counts were corrected to a standard mass of 10 mg { as described above. The values for the ratio: i i i corrected counts from barium carbonate j counts from directly mounted material i j I were determined for a number of samples. A plot of these j values as ordinates against mg. of saponifiable material I used in the direct mount as abscissa is shown in Figure 4. * ! From this empirical curve, the factor for converting counts j obtained from a direct mount to the activity that would ; have been obtained had the of the saponifiable material : been measured after conversion to BaCOs can be read. ' Counting procedure.- Saponifiable material, I mounted directly, was counted in a Q-gas chamber as des- i cribed above. Samples ranged from 8 to 20 mg. per planchet I I and the counts observed were converted to counts as BaCO, ! O : by the correction factor eurve described above. Due to the i , lower activity obtained in the saponifiable fractions, a ' minimum of 4096 counts per sample was taken to insure a counting error of less than 5 per cent. 35 0 o % CD co < o UJ c c cc o o CO h- z 3 O o 0. < CO DC O M* O Ul > CC ui <o CD o (0 30- 3 O O i" o * T f CD O h- q: o i — 0 2 1 CO a: ai > Z o o 2-5 20 0 10 20 30 40 50 SAPONIFIABLE MATTER MG-PER 4-5 SQ-CM. CONVERSION FACTOR CURVE FROM COUNTS AS OBTAINED BY DIRECT MOUNT TO COUNTS AS DETERMINED BY THE BaC0& BASIS FIGURE 4 CHAPTER IV } j f 1 j RESULTS j i I ' Cholesterol Deposition Studies | I I | Thyroid feeding produced the symptoms typical of hyperthyroidism, such as an increased nervousness, an increased respiratory rate, a shaggy coat, an increased i food and water intake and fecal excretion, but the level j of the hormone given was not high enough to be severely , toxic. Body weight gains were similar in all groups and j I I not different from those of normal rats. For the groups 1 ! ' j not supplemented with cholesterol, the results are ! j summarized in Table IV, ; i ! j The results indicate that the purified high-fat j ; diet lacked sufficient quantities of lipotropic substances,! J I in spite of a casein content of 18 per cent, to prevent a 1 significant lipid and cholesterol deposition in the liver, j { The addition of either thyroid or choline and inositol to ; 1 ! j the basic diet reduced these values towards normal levels, ! I 1 The effects of thyroid and of choline and inositol i > ■ in rats fed the high-cholesterol diet are summarized in j ! Table V. The animals receiving cholesterol and bile salt | i ■ alone showed a moderate increase in plasma cholesterol but ; TABLE 17 EFFECTS OF THTRQID AND OF CHOLINE PLUS INOSITOL IN RATS FED A PURIFIED DIET CONTAINING 20 PER CENT FAT Group Dietary supplement Plasma cholesterol mg, f a Cholesterol % Liver Total lipids % Wei^it, f a of body weight 0* None 60 i 3 •21 t .02 6.9 ± A 3 .3 ± .1 I None 60 t 4 1.12 t •12 21.5 ± 1.3 5.5 t .3 III •4 % thyroid 65 ± 7 .34 t .01 9.5 t 1.3 5 J6 t .1 IV •25 f a of each choline and Inositol 80 ± 8 .30 * *02 6.7 t J5 4.4 1 .1 * Data for a control group of 17 rata fed Purina chow (Marx et a l - 1951)* table: v EFFECTS OF THYROID AND OF CHOLINE PLUS INOSITOL ON CHOLESTEROL DISTRIBUTION AND LIVER LIPIDS IN RATS FED 2 PER CENT CHOLESTEROL AND 0.25 PER CENT BILE SALT Group* Dietary supplement Plasma cholesterol mg. % Cholesterol % Liver Total lipids % Weight, $ of body weight II None 107 i 7 10.0 t .8 37 t 2 7.6 t 2.6 V Thyroid 74 ± 8 5.4 t J5 29 t 3 9.5 ± 1.2 ^ a 0.25 f o choline + inositol 253 t 26 7.6 t .8 26 t 1 5 .9 ± .2 VIb 1*0 % oholine + inositol 171 t 27 4.3 t .7 19 i 1 5.0 ± .6 VIIa Thyroid + 0.25 % choline + inositol 165 t 24 5.9 • .6 21 i 1 7 s t a VII* Thyroid + 1.0 % oholine + inositol 151** 2.9 ± .6 14 t 1 5.9 t .1 * All groups received cholesterol plus bile salt • ** Plasma from five animals pooled. 39 ; an extreme accumulation of hepatic sterol and total lipids. The liver weight was also markedly elevated. Thyroid feeding caused a significant drop in plasma and liver cholesterol and in liver lipids. The former approached normal levels but the liver values were still markedly elevated. In spite of this reduction in hepatic lipid fractions, thyroid hormone produced a further rise I in liver weight. As a consequence of the administration of choline and inositol, the plasma cholesterol was markedly elevated; this effect was greater with the lower dose of the lipo tropic factors. In the liver, the values for the sterol and total lipid content, and for the weight of this organ were significantly reduced; the hepatic response was correlated with the dose level. The effects of a supplementation with thyroid powder together with choline and inositol were roughly additive excepting the action of thyroid on liver cholesterol which was not significantly modified by the lower dose level of lipotropic substances. It is noted that under the conditions of these experiments even a combination of the endocrine and dietary lipotropic agents was not sufficient to prevent, a significant accumulation of cholesterol and lipid in the liver. 40 In all groups, the aorta was macroscopically normal, Histological examination revealed that administration of | thyroid to rats fed cholesterol and bile salt had a J j tendency to cause a deposition of small amounts of i cholesterol crystals diffusely scattered through the i | aortic endothelium, and a swelling of the latter. Choline i and inositol, at a level of 0.25 per cent, did not signifi cantly influence this phenomenon. Cholesterol Balance Experiments I i i Experiment I. i ! The values for the overall cholesterol balance were J i , i ' calculated from the total cholesterol input (carcass at ] | onset plus food consumed) and the total cholesterol re- J i ' ; i covered (carcass at end plus excreta)’ . The results of the | i • 1 I 1 j , first balance experiment are summarized in Table VI. j In this experiment, hyperthyroid and normal animals \ > were pair-fed restricted amounts of a diet containing 0.65 | per cent cholesterol. It was observed that in the group ^ supplemented with thyroid, the cholesterol output was significantly smaller than the sterol input; furthermore, a recovery of only 80 per cent was realized in this group as compared to one of 94 per cent in the controls. When the effects of thyroid on the cholesterol TABLE V I EFFECT OF THYROID ON THE TOTAL CHOLESTEROL BALANCE OF MICE FED A RESTRICTED DIET CONTAINING 0,65 PER CENT CHOLESTEROL Total cholesterol balance Experiment Group No.“of Input Recovery P for mice (A) ( b 'J % difference mg* I Normal 6 520 502 94. 1 1 < 0*02 Thyroid 8 515 250 80. ± 5 < 42 content of the excreta are examined separately, a similar trend is observed (Table VII). Under the conditions of this experiment, the hyperthyroid group showed a decrease in the rate of cholesterol excretion as compared with normal mice. However, the hormone treatment had only a minor effect on the carcass cholesterol content and these values were not significantly modified in the two groups. Differences between the liver and plasma cholesterol levels of the two groups were not significant, apparently because the experimental period was too short to permit the effects of thyroid along these lines to become manifest. These values therefore, are not shown separately in Table VII, but are Included in the carcass cholesterol figures. Experiment II. In experiment II, the cholesterol balance of hyper thyroid mice was compared with that of thiouracil-treated animals under a different dietary regimen,i.e., when fed ad libitum a diet relatively low in cholesterol. The results of the balance are summarized in Table VIII. The data obtained indicate that the alteration of the nutritional state of the mice profoundly modified the action of the thyroid hormone on the cholesterol balance. In the hyperthyroid group, the cholesterol recovery was i TABLE V I I j EFFECT OF THYROID ON THE CHOLESTEROL CONTENT OF CARCASS AND EXCRETA OF j f MICE FED A RESTRICTED DIET CONTAINING 0.65 PER CENT CHOLESTEROL | f Total cholesterol content Experiment Group No. of of oaroass of food eaten of exoreta/24 hrs•/ mioe Onset End per 24 hrs • gm. body weight mg. mg. mg. mg. I Normal 6 35 .5 » 1.6 36.7 t 1,6 17 .8 1.44 t ,03 Thyroid 8 37.6 i 1.0 34.0 1 1.8 17.2 1.29 t .09 t / 44 TABLE VIII EFFECT OF THYROID ON THE TOTAL CHOLESTEROL BALANCE OF MICE Fp AD LIBITUM ON A DIET CONTAINING O.IS PER CENT CHOLESTEROL Experiment Group No ." of mioe Total oholesterol balance Input Recovery P for (b) Per cent difference II Thiouracil 7 83.0 85.0 103 t 5 Thyroid 8 92,0 115 125 t 4 0.001 found to be significantly larger than the sterol input, and larger than that for the hypothyroid animals. A re covery of 125 per cent was recorded for the mice on the thyroid regimen as compared to that of 103 per cent for hypothyroid animals. The results here are in contrast to the data recorded in experiment I where a reverse relation ship was observed. When the effects of thyroid on the cholesterol con tent of excreta are examined, a similar trend is observed (Table IX). It is interesting that under conditions of a low cholesterol intake, and of an ad libitum food consump tion, the hyperthyroid group showed a significant increase in sterol excretion. Any destruction or conversion that may have also occurred was apparently obscured, in contra distinction to the results observed in the first balance experiment. The feeding of hormone or the anti-thyroid supplements had only slight effects on carcass cholesterol levels. Experiments Using Radioactive Cholesterol Hypo- and hyperthyroid mice were administered intra venously with either 4-C^-4-cholesterol or randomly labelled cholesterol. The distribution of C14 in the digitonide precipitable and saponifiable fractions of carcass and j TABLE IX EFFECT OF THYROID AND THIOURACIL ON THE CHOLESTEROL CONTENT OF CARCASS AND. EXCRETA OF MICE FED AD LIBIT DM ON A DIET CONTAINING 0.15 PER CENT CHOLESTEROL Total oholesterol content Experiment Group No. of " of 'oaroa8s ' ........ .'of food eaten " ' "of exoreta/24 hrs./ mioe Onset End' per 24 hrs. gm. body weight mg. mg. mg. mg. II Thlouracil 7 66.0 1 4 59.0 t 2 4.4 .29 t ,01 Thyroid 8 62.0 - 3 64.0 t 2 7.5 .53 1 .02 47 < excreta is summarized in Tables X, XI, and XII. The results show that, after 48 or 96 hours, a ! i considerable amount of the administered isotope could not • be recovered in the digitonide precipitable fraction of either group; however, a significantly lower percentage j was recovered, in this fraction, in the thyroid group than ; in the controls. Although the saponifiable fractions of j both groups contained a considerable quantity of isotopic , carbon, the total activity of this fraction was signifi cantly greater in the hyperthyroid group; this effect was * observed more clearly after 96 hours. With either 4-cA4- cholesterol or randomly labelled cholesterol, the recovery of total counts was smaller in thyroid fed animals, suggesting that under the influence of thyroid a larger quantity of the administered compound was metabolized to J products which were neither of a saponifiable or nonsapon- ifiable nature. This incomplete isotope recovery, in both experiments, may indicate degradation of the steroid nucleus as well as of the isooctyl side chain. After 48 or 96 hours, less isotopic material remained in the carcass of thyroid fed mice; this same group showed ! a tendency for an increased excretion of radioactive material in both fractions. In a few experiments the respiratory carbon dioxide TABLES X THE DISTRIBUTION OP C14 IN HYPO- AND HYPERTHYROID MICE 48 HOTRS AFTER INTRAVENOUS ADMINISTRATION OP 4-C14-CH0LESTER0L Recovery of administered C*-4 in Recovery of administered C*4 Total Endoorlne Mouse digitonide preoipitable fraction in saponifiable fraction oounbs supplement no* Liver Carcass Excreta Total- Liver Carcass Excreta tobal reoovered % «n s £ % £ £ £ £ £ Thiouraoil 25 14 51 14 79 •64 16 3 20 99 Thiouraoil 29 15 61 14 90 .61 7 6 14 104 Thyroid 25 7 39 28 74 •98 7 11 19 93 Thyroid 30 10 51 13 74 •37 7 15 22 96 TABUS XI THE DISTRIBUTION OP C14 IN HYPO- AND HYPERTHYROID MICE 96 HOURS AFTER INTRAVENOUS ADMINISTRATION OP 4-C14-CHOLESTEROL Endocrine supplement No. of mioe Reoovery of administered C 4 in digitonide preoipitable fraction 14 Reoovery of administered C in saponifiable fraotion Total counts reoovered Carcass Excreta fatal Careass Exoreta Total Thiouraoil 4 i 61 t 3 & 26-2 i 87 i 1 i 10 t z i 6 ! 6 i . 15-2 % 103 t 1 Thyroid 5 36 £ 2 34 i 2 70 t 3 8 t 1 16 t 2- 24 t 1 94 t £ s P for 1 difference -<*001 .02 .001 .4 < .01 < .01 <.01 TABLE XIX THE DISTRIBUTION OP C14 IN HYPO- AND HYPERTHYROID MICE 48 AND 96 HOURS AFTER INTRAVENOUS ADMINISTRATION OF RANDOMLY LABELLED CHOLESTEROL Mouse Hours Endocrine Recovery of administered C^4 in Recovery of administered C^4 Total no. supplement digitonide preoipitable fraction in saponifiable fraction oounts Liver Caroass Excreta Total Liver Carcass Excreta Total recovered J i E . i r ~ E i % i MM “i r “ : 17 48 Thiouracil 14 51 9 74 0.5 11 5 17 i 9! | 19 48 it 11 48 14 73 0.1 8 3 11 84 , 16 48 Thyroid 7 45 15 67 0.4 6 5 11 78 ! 18 48 n 6 48 7 61 0.0 10 2 12 73 j 10 96 Thiouraoil 4 49 6 53 , 0.2 3 2 ' 5 58 ; 12^ 96 t! 11 54 1 2 ' : 77 0.2; 7 4 11 88 | 16 96 R 6 37 10 52 0.1 4 2 6 58 I 21 96 R 10 47 9 66 0.3 8 6 14 80 * 1 13 96 Thyroid 5 42 16 63 0.3 5 5 10 73 ; 1 14 96 R 4 29 10 43 0.3 7 5 12 55 ; 20 96 R 5 28 31 64 0.4 5 16 21 85 ! tn o was collected and analysed for radioactivity. No signifi cant amount of isotope could be detected in this fraction either with the use of randomly labelled cholesterol nor with 4-C^-cholesterol. The results obtained using randomly labelled cholesterol (Table XII) are not as clearly defined as those obtained using cholesterol labelled in the A ring only. Since the initial radioactive experiments were conducted with the former type of compound, the difficul ties encountered in working with isotopic materials and in the preparation and administration of cholesterol emulsions is believed to be the primary factor responsible for the large variations obtained in these early experi ments. CHAPTER V DISCUSSION OF RESULTS The feeding of a high-cholesterol diet produced results qualitatively similar to those observed previously by a number of investigators (100). Under the conditions of this study, the fat and cholesterol accumulation in the liver was remarkable, in particular, if the pronounced in crease in liver weight is taken into consideration. The relation of the thyroid hormone to the plasma cholesterol level, again confirmed by the present work, is too well established to warrant further discussion (71). The influence of thyroid on the sterol content of other tissues does not always follow the pattern of an inverse relationship, since a number of investigators have reported an increase in tissue cholesterol content (76-82) and others a decrease in cholesterol deposition (5, 7, 83-85), under the influence of thyroid powder or thyroxine. How ever, the results described here support the latter obser vations. In spite of a relative deficiency of dietary lipotropic substances, the thyroid hormone exhibited a significant lipotropic activity, as far as hepatic total lipids and sterol were concerned. The thyroid treatment was unable, however, to prevent a severe lipid and cholesterol accumulation in the livers of the cholesterol- fed animals. This is in line with earlier observations of Marx et al. (11) and with results obtained by Forbes (101) who found that thyroxine did not prevent a deposition of cholesterol in the liver unless the choline intake was adequate. On the other hand, Handler (84) found that thyroid hormone mobilised hepatic cholesterol even in choline-deficient rats. In rats fed a low-cholesterol diet, the lipotropic substances did not significantly alter the plasma cholesterol level, although a pronounced effect on liver sterol and total lipid concentrations were observed, con firming previous observations of Handler (84) and other workers (102-104). In the group fed the high-cholesterol diet, however, choline and inositol caused a remarkable increase in plasma cholesterol, more pronounced at the lower level of administration. The reports in the literature concerning the effects of lipotropic substances on plasma sterol levels are contradictory. A number of investigators have reported a reduction of plasma cholesterol as a result of choline and/or inositol administration (105-107). Others have found that these substances, either alone or in combination did not modify plasma sterol levels in cholesterol-fed i normal animals (108, 109), in hypothyroid (110) or thyroid- eetomized-hypophysectomized dogs (111) or in partially hepatectomized rats (112). The results presented in this dissertation cannot be readily compared with these earlier findings, since the experimental conditions and the animal species used were different. However, our results are in , line with those of Stamler, Bolene @t al. (113), who demonstrated that choline and inositol significantly aggravated the hyperlipemia and hypercholesterolemia pro duced in cholesterol-fed chickens, and with the more recent observations of Ridout et al. (114). This group reported that in the absence of choline, only a moderate increase in serum cholesterol occurred in cholesterol-fed rats, but that the presence of choline caused large increas es in the serum cholesterol. The appearance of the diffuse scattering of cholesterol crystals observed in the aortic wall of mo&t of the rats fed thyroid was different from that of a foeal accumulation in atheromatosis. This phenomenon was neither correlated with the plasma cholesterol level nor modified by dietary choline and inositol. The results appear to indicate that thyroid hormone administration exhibited a depressing action on tissue sterol concentration, and that this action on hepatic total 55 lipids and cholesterol was significantly enhanced by choline and inositol. However, the question whether thyroid hormone ;is capable of mobilizing liver total lipids and cholesterol .in the absence of lipotropic substances cannot be answered unequivocally by these experiments, since the basic diet was not completely free of lipotropic factors although it was deficient in this respect. That cholesterol is metabolized and destroyed by the animal organism has been established by a number of workers (20, 54, 55, 58-65). The observations reported 14 here on the sterol balance and C -cholesterol experiments, confirm these previous findings and indicate that the mechanism responsible for the destruction or modification of this compound can be stimulated by the thyroid hormone. The data are in agreement with reports by Hurxthal and Perkins (92), who observed a stimulation of cholesterol destruction in mice by thyroid administration. Fleischman and Wilkins (93) also suggested that the thyroid hormone might enhance cholesterol destruction when they found a decrease in plasma cholesterol but no corresponding in crease in fecal sterol excretion, after treating hypo thyroid patients with thyroxine. Other investigators have shown that administration of the thyroid hormone causes a reduction of tissue cholesterol concentrations, in particular of plasma and liver, elevated as a consequence of hypothyroidism or j i cholesterol feeding, for example (5, 7, 71, 83-85). The ! above mentioned results suggest that the mechanism ; t responsible for this phenomenon includes reactions leading | to a destruction or conversion of cholesterol, in addition 1 to the processes causing a removal and excretion of the | sterol, as shown by Friedman £t al. (91), and the studies here. However, other phases of cholesterol metabolism are < also influenced by the thyroid hormone, and the above ! mentioned phenomenon may easily be obscured by other mechanisms, unless special conditions are chosen favorable for its demonstration. In the second balance experiment, for example, thyroid feeding did not stimulate sterol destruction, but, rather, sterol synthesis. Also, the i endocrine supplement significantly stimulated cholesterol excretion, in contradistinction to the results where the I total caloric intake was restricted. Friedman and co- ! I workers (91) observed a similar effect, and interpreted | the increase in sterol excretion to be a consequence of an increased rate of hepatic synthesis (86-88). It is obvious, therefore, that the thyroid hormone may influence several phases of cholesterol metabolism j simultaneously, and that the experimental conditions determine which effect predominates in a given case. Under the conditions of the first balance experiment reported above, where restricted feeding was employed, the total caloric intake was inadequate for the hyperthyroid animal; therefore, a condition of relative starvation or semi starvation existed. During starvation, the rate of hepatic sterol synthesis is markedly reduced, as shown by Tomkins and Chaikoff (115). Furthermore, the cholesterol intake was relatively high, a situation which also tends to reduce endogenous cholesterol synthesis, in line with reports by Gould and Taylor (61, 116), Tomkins et al. (117) and Frantz and coworkers (118). Thus, two factors operated which would depress mechanisms responsible for cholesterol synthesis, and any effect which thyroid hormone would otherwise have had in this direction may easily have been obscured by these circumstances. Therefore, the experi mental conditions were favorable for a demonstration of the influence of this hormone on cholesterol destruction or conversion, which apparently predominated. In the second balance experiment, on the other hand, the total caloric intake was more nearly adequate in the hyperthyroid group (ad libitum feeding), and the sterol intake was relatively low. Therefore, the major action of 5 8 i I the hormone was directed towards stimulating cholesterol | I synthesis (especially in the liver) and cholesterol ! excretion, and these effects predominated, obscuring any j influence the hormone might have had on cholesterol j destruction. It was not necessary to employ conditions of j restricted feeding and a high cholesterol intake in order | ! ' j to demonstrate the influence of thyroid on sterol conversion I : j in the tracer experiments. The results observed using i 1 ; radioactive cholesterol support the idea that the thyroid , I ' i | hormone acts as a lipotropic agent, at least in part, by i | stimulating cholesterol destruction or conversion to f l compounds of a saponifiable nature. It was concluded that i 1 | the higher isotope content of fecal acidic products, found ; i i , ; in the excreta of hyperthyroid mice, was primarily a I > I ; reflection of the stimulatory effect of this hormone on 1 ■ I tissue sterol catabolism. Bloch et al. (58) and other 1 | ' J ; investigators (59, 60) have shown the formation of bile i i ! acids from cholesterol. It is reasonable to believe that a major part of the radioactivity located In the saponi- | fiable fractions was due to the presence of labelled bile acids, derived from the administered isotopic sterol. Complete recovery of the administered isotope was i obtained for thiouracil-fed animals in the experiments ; I • conducted for 96 hours with 4-C^^-cholesterol. In these same experiments only 94 per cent of the injected counts were recovered for the hyperthyroid group. The small per centage of unrecovered counts for the thyroid-fed group cannot be considered significant since the limit of error for the counting procedure is of the same approximate magnitude. However, the data is suggestive in that it may indicate a degradation of cholesterol, under the influence of thyroid hormone, to products which are neither non-saponifiable or saponifiable in nature. Furthermore, the observations reported here demonstrate that thyroid hormone also stimulates the excretion of digitonide precipitable material, in addition to its effect on cholesterol conversion. The increased rate of excretion of radioactive non-saponifiable material by hyperthyroid mice (presumed to be primarily cholesterol) is in agreement with the data of Friedman £t al. (91), and indicates another possible mechanism for the lipotropic action of thyroid. The data obtained from the isotope experiments performed with randomly labelled sterol follow a pattern similar to the results obtained with nuclear labelled cholesterol. In neither the thiouraoil or thyroid treated mice was complete recovery of radioactivity from randomly 60 labelled cholesterol achieved. It has been shown by a number of investigators (60, 63, 64) that the isooctyl side chain, of the cholesterol molecule, is more suscept ible to biological oxidation than t^he polynuclear moiety of this compound, thus accounting for the large losses of activity when labelled cholesterol, biosynthetically pre pared from C-^-acetate, was administered to hypo- and hyperthyroid mice. Due to the initial difficulties in working with radioactive carbon the data from these early experiments, using randomly labelled cholesterol, are not reliable. CHAPTER VI ; SUMMARY ; i : | The relation of thyroid to cholesterol metabolism j ; was studied using three types of experimental approach: ; ! (l) deposition studies, (2) balance experiments, (3) ! . studies using radioactive cholesterol. ; I I (1) Plasma and liver cholesterol and liver total , ' lipids were determined in rats fed a purified diet con- ; ! taining 20 per cent fat, 18 per cent casein and 0.25 : per cent bile salt, and supplemented with the following components, either alone or in combination: (a) 2 per- , cent cholesterolj (b) 0.4 per cent thyroid U.S.P; (c) 1 choline and inositol, either 0.25 or 1.0 per cent each. It was observed that thyroid diminished significant-! I ■ ly the moderate hypercholesterolemia and the severe I ' cholesterol and lipid accumulation in the liver of ' I cholesterol-fed rats. [ Choline and inositol produced a marked hypercho- ' : lesterolemia, and, at the same time, a significant drop in hepatic sterol and lipoid values in the group fed the i high-cholesterol diet. . I When the endocrine and dietary agents were combined,! i 1 ■ their effects on plasma sterol were essentially addictive._; 62 ' They enhanced each other’s effects, as far as the mobilization of hepatic cholesterol and total lipid was concerned, except at the lower level of the lipotropic substances which did not significantly modify the action of the thyroid hormone. (2) The total cholesterol balance was determined in mice fed 0.4 per cent thyroid with the diet, in normal controls, and in mice fed 0.5 per cent thiouraoil. It was observed that when a modified Purina diet containing 0.65 per cent cholesterol was offered in restricted amounts, (paired feeding), significantly less cholesterol was recovered, than the amount “put in”, from the hyperthyroid mice as compared to the normal controls. These results were interpreted to indicate that thyroid stimulated the destruction or chemical conversion of cholesterol. Under the conditions of these experiments, administration of thyroid caused a significant reduction in cholesterol excretion. However, when a diet low in cholesterol was fed ad libitum, an entirely different metabolic pattern was observed. The total cholesterol output of the hyperthyroid mice significantly exceeded the total amount “put in”. These results were interpreted to indicate, in agreement with earlier reports that thyroid stimulates endogneous 63 cholesterol synthesis. Under these conditions, the hormone significantly enhanced cholesterol excretion. (3) Hypo- and hyperthyroid mice were given, intra venously, Tween suspensions of radioactive cholesterol. In the first experiment 4-C14-cholesterol was used and in the second experiment randomly labelled cholesterol was employed. At the end of 48 and 96 hours, animals were sacrificed and the carcass and excreta were examined for radioactivity. 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Creator Weiss, Samuel B. (author)

Core Title The influence of the thyroid hormone on cholesterol metabolism in rats and mice.

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Degree Doctor of Philosophy

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-602523

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