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A study of the mechanism of the protective action of cholesterol in hyperthyroidism

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A study of the mechanism of the protective action of cholesterol in hyperthyroidism

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Content A STUDY ON THE MECHANISM' OF ' THE" PROTECTIVE ACTION OF CHOLESTEROL IN HYPERTHYROIDISM A Thesis Presented to the Faculty of the Department of Biochemistry University of Southern California In Partial Fulfillment of the Requirements for the Degree Master of Science by Louis E. Wihebrenner January 1949 U M I Number: EP41300 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 com plete manuscript and there are missing pages, th ese will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI EP41300 Published by ProQ uest 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 S tates Code ProQuest LLC. 789 E ast Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 010 '41 \A/71J This thesis, written by LOU I S_ _ E_. _ _ TO NEBRENNER .... under the guidance of h .X F a cu lt y Committee, and approved -by all its members, has been presented to and accepted by the Council on Graduate Study and Research in partial fulfill ment of the requirements for the degree of MASTER OP SCIENCE .............. E.i.S.t.HogarcLna............ Dean D ate J.aimar.y-.19^-9...... Faculty Com m ittee ( ^ . Q s f P c Chairman ACKNOWLEDGEMENTS To the members of the committee my sincere gratitude for their guidance and continued interest in carrying out this experimental problem* TABLE OP CONTENTS CHAPTER PAGE I. INTRODUCTION ......................... 1 II. HISTORICAL REVIEVT......................... 3 III. PROCEDURE AND METHODS..................... 9 Treatment of animals • •••••••••• 9 Food consumption • •••••••••••• II Oxygen consumption • •••••••••.. 13 Autopsy • • • •• •• ••• •• •• ••• 15 Cholesterol determinations •••••••• 16 Preparation of samples • •••••••. 16 Cholesterol determination. 17 IV. EXPERIMENTAL D A T A .................. 19 V. DISCUSSION................ 28 VI. SUMMARY.................................... 34 BIBLIOGRAPHY...................................... 35 LIST OP TABLES TABLE PAGE I* Composition of Diets s . . ••••••••• 12 II# Oxygen Consumption - Oxygen Consumption ex pressed as ml./lOO grams of body weight/ hour .............. 21 III. Oxygen Consumption - Oxygen Consumption ex pressed as ml./lOO square centimeters of body surface area/hour • • • .......... 22 IV, Body Weights - Following a Fasting Period of 12 Hours . • • • . . • • • • • .......... 23 V# Food Consumption 24 VI. Body and Organ Weights at Autopsy • • . • • 25 VII. Organ Weights at Autopsy - Expressed as grams /lQO grams of body weight •••.•••• 26 VIII. Total Cholesterol Levels in Blood and Tissues 27 CHAPTER I INTRODUCTION The regulation of metabolism, growth and other aspects of animal physiology by the thyroid gland have been extensively studied for the past fifty years. Considering the rather widespread occurrence of hyperthyroidism, re search workers have attempted throughout many years to bring to light agents which would safely and effectively depress thyroid activity. Research work which has involved the combined efforts of clinicians, physiologists, morphologists, organic and biological chemists, has developed compounds which may be effectively used as antithyroid agents. These compounds are numerous and have a varying degree of effectiveness. It was recently observed that cholesterol would pro long the survival time of the albino rat when administered toxic doses of desiccated thyroid. Although the relation ship of cholesterol as a body constituent has been studied In cases of hyperthyroidism, the use of this compound to counteract or Inhibit the action of the thyroid gland has not been explored. With studies of previous work on the mechanism of action of other antithyroid agents in mind, the present project was carried out on the study of the mechanism of 2 the protective action of cholesterol in cases of hyper* thyroidism. The main interest was to determine if choles terol might have some action toward reducing the increased basal metabolic rate of hyperthyroid animals* CHAPTER I I HISTORICAL REVIEW Hyperthyroidism and the physiological alterations which accompany the overactivity of the thyroid gland have heen studied extensively. Magnus-Levy (1895) first showed ari alteration in the oxygen consumed and the carbon dioxide expired in a patient given desiccated thyroid (Kendall, 1929) Volt (1897) early proved that the metabolic rate of a normal animal could be increased to a hyperthyroid state by the administration of a thyroid extract (Kendall, 1929). The hypermetabolism, tachycardia, vasomotor disturb ances and nervous disorders along with alterations in the body constituents which occur with an overactivity of the thyroid gland have been studied. It was discovered that many of the results of an overactivity of the thyroid gland were opposite to those observed in underactivity of the gland. Since it is desired to regulate the function of the thyroid gland to a normal level, a search for substances which would act as antithyroid agents was begun. The presence of substances in certain foods which would cause a hypertrophy of the thyroid gland and at the same time a lowering of the basal metabolic rate has been realised for several years. Cabbage leaves when fed to rabbits were found to cause hyperplasia of the gland and lowered the metabolic rate (Webster, Clawson, and Chesney, 1928). Since Hoffmann (quoted by Marine et al.# 1932). found that the Cruciferae contained nitriles, it was sug gested by Marine and coworkers (1932) that a cyanide com pound, might be the causative factor. Several nitriles were tested and found to be goitrogenic in rabbits. Rape seed was found to be goitrogenic (Kennedy and Purves, 1941) which action was attributed to a derivative of urea con tained in the seeds. Oral administration of allylurea produced hypertrophy and hyperplasia of the gland that was identical histologically to that in rats fed rape seed. The goitrogenic activity of the sulfonamides was discovered by MacKenzie, MacKenzie, and McCollum (1941) and Astwood (1943) demonstrated the antithyroid activity of thiouracil. The goitrogenic activity of thiourea has been demonstrated in most mammals. Thyroxin is capable of over coming the goitrogenic action of thiourea and aminobenzene compounds (MacKenzie and MacKenzie, 1943). In amphibians structural changes in the thyroid gland and inhibition of the metamorphosis in Rana plplens tadpoles by thiourea- has been noted (Gordon, Goldsmith, and Charipper, 1943) and the inhibition of the thyrotropic hormone action toward the stimulating of the metamorphosis in Rana claml- tans- by thiouracil (Hughes and Astwood, 1944). Reduction of the basal metabolic rate, lessened food intake and retardation of growth in young rats by sulfa- guanidine (Astwood, 1943), thiourea (Leathern, 1945) and thiouracil has been observed. The decrease in the basal metabolic rate by thiourea and methylthiourea was found to be directly proportional to the thyroid gland hyper plasia (Christensen, 1945). Animals lose the ability to retain iodine in the gland when administered most of these compounds (Astwood and Bissel, 1944), thereby failing to form the thyroid hormone as shown using radioactive iodine in thyroid tissue slice studies (Franklin and Chaikoff, 1943; Keston^ Goldsmith, Gordon, and Charipper, 1944)# Other substances have bden used to counteract effects of hyperthyroidism caused by thyroxin or desiccated thyroid. A great deal of work has been carried out by two Japanese workers with a substance known to them as ! , yakrltonn which is contained in the water-soluble fraction of liver extract. They found,that yakriton has an inhibiting effect toward the action of thyroxin in decreasing the metamorphosis time in tadpoles (Horiuti and Ohsako, 1934). The inhibition of this action of thyroxin was also observed by Saegesser (1932) by using the lipoid fraction of blood serum. The growth and gonadal development of young rats is retarded by the administration of the thyroid hormone (Drill, Overman, and Leathern, 1943) and these retardations have been counteracted by the feedings of whole dried liver powder (Ershoff, 1947). Later experiments have shown that the 6 substance in liver responsible for this counteraction or inhibition of the thyroid hormone is contained in the water- insoluble fraction of the liver extract. The exact nature of this constituent is yet unknown. The survival time of rats fed desiccated thyroid was increased by the feeding of yeast (Ershoff and Hershberg, 1945), or whole liver (Ershoff, 1947; Betheil, Wiebelhaus, and Lardy, 1947), The hormone of the adrenal cortex has also been shown to counteract some effects found in experimental hyperthyroidism. The rise in the basal metabolic rate and the hypertrophy of the adrenal cortex which are found upon administration of thyroxin is prevented by rather large doses of desoxycorticosterone to the guinea pig (Haen, Langfeld, and Oehme, 1959) , The effects in animals which are encountered in experimental hyperthyroidism.are numerous. It has been found that younger animals are more resistant to body weight changes and to a rise in the basal metabolic rate (Belasco and Murlin, 1941), The levels of cholesterol in the various glands and tissues in hyperthyroidism has also been deter mined, The blood cholesterol level has been found to be high in myxedematous patients which level could be lowered by the administration of thyroid hormone, A low level of blood cholesterol was found in hyperthyroid patients (Barbier and Pequiquot, 1938), The total body and serum 7 cholesterol was determined in thyroidectomized, normal and thyroxin treated rats and rabbits. It was found that the two do not run parallel in levels but rather shift of the cholesterol to the serum in hyperthyroidism and from the serum in hyperthyrodism (Fleischmann and Shuraacker, 1942)* A relationship between the serum cholesterol and the basal metabolic rate was observed by Epstein and Lande in 1922, Use of the cholesterol level as a diagnostic value in the determination of thyroid activity has been emphasized (Mason, Hunt, and Hurxthal, 1930; Hurxthal, 1933) which value seems to bear a reciprocal relationship to the basal metabolic rate. This has been disputed by workers who found some correlation but too much overlapping of the values to be of importance (Man, Gildea, and Peters, 1940), It has been suggested that the hypocholesterolemia sometimes found in hyperthyroidism is due to some factor other than thyroid activity (Cutting, Rytand, and Tainter, 1934; Schmidt, and Hughes, 1938). The protective action of cholesterol in thyrotoxi cosis has been studied only recently. It was found that a diet high in cholesterol significantly prolonged the sur vival time in rats which were administered toxic doses of desiccated thyroid and in some cases the weight loss was reduced (Marx, Meserve, and Deuel, 1948; Ershoff and Marx, 1948). The mechanism of action of cholesterol has not been explored.* Hoffmamand Hoffmann (1945) suggested that lyso- leeithln which is produced in excessive amounts during hyperthyroidism may be responsible for some of the toxic effects observed* Cholesterol and the steroid hormones of the adrenal glands are believed under normal conditions to neutralize the toxic effects of lysolecithln* Since during the prolonged administration of thyroid the sterol reserves of the body may become depleted and these toxic effects be come apparent, cholesterol added to the diet might counter act some of these toxic effects* In the experimental work that follows emphasis was placed upon studies concerning the basal metabolic rate of hyperthyroid rats* It was felt that if cholesterol was an antithyroid substance in some sense of neutralizing the effects of administered thyroid hormone, this effect might be demonstrated, in differences of metabolic rate between animals fed thyroid and those fed similar diets with choles terol added* CHAPTER I I I PROCEDURE AND METHODS I. TREATMENT OP ANIMALS Thirty-two male albino rats of the University of Southern California strain twenty-eight days of age were taken from litters containing four or more males. The animals were numbered in the conventional manner and placed four to a cage with litter mates being evenly distributed among the four groups to be designated as:I. basal, II* basal plus cholesterol, III* basal plus thyroid, and IV. basal plus thyroid plus cholesterol. Each experimental group contained eight animals* All animals were fed a stock diet (see Table I) during a training period for the determination of the oxygen consumption. The training period was continued for five weeks* For the first three weeks the rats were placed for a two hour period three times a week In the wire cages in the open vessels of the oxygen consumption apparatus. During the last two weeks of the training period, trial oxygen consumption data were taken until the figures ob tained were fairly constant. This training period waa necessary to accustom the animals to the apparatus so they would not become over excited when placed In the vessels 10 and the readings obtained would be as accurate as possible* When constant oxygen consumption data for all animals were obtained, the animals were placed on their respective experimental diets. All animals received food and water ad libitum. At the end of the third week of the experimental period the animals were separated to two animals per cage because it was felt that with fewer animals per cage there would be less chance for excitement and a better chance for all animals to obtain sufficient food and water. The animal weights were recorded every ten days. However, the weights recorded just prior to the oxygen consumption determinations were the weights observed after 12 hours fasting and were usually several grams lower than those obtained when the animals were allowed to eat. All animals were fed a basal diet throughout the experimental period consisting of the nutrients necessary for normal growth and development with additions for the experimental groups as follows: I. Basal diet. No change (See Table I) IIA. Basal plus cholesterol I_. The diet was the same as the basal diet except that it contained one percent cholesterol and was fed to the rats during the first three weeks of the experimental period. (See Table I) IIB. Basal plus cholesterol II. The diet was the 11 same as the basal diet except that it contained two per cent cholesterol and was fed to the rats during the last six weeks of the experimental period* (See Table I), III* Basal plus thyroid. The diet was the same as the basal diet except that~ it contained 0*15 percent desic cated thyroid and was.fed throughout the experimental period. (See Table I). IVA. Basal plus thyroid plus cholesterol I_* The diet was the same as the basal diet except that it contained 0.15 percent desiccated thyroid and one percent cholesterol* This diet was fed to the animals during the first three weeks of the experimental period. (See Table I). IVB. Basal plus thyroid plus cholesterol II. The diet was the same as the basal diet except that it contained 0.15 percent desiccated thyroid and two percent cholesterol. This diet was fed to the animals during the last six weeks of the experimental period. (See Table I). All diets contained 0.25 percent bile salts. In the case of the diets containing cholesterol it was felt this would aid in the absorption of the bile salts. II. FOOD CONSUMPTION The food consumption of the animals was determined at two day intervals by weighing the food given the animals I TABLE I COMPOSITION OP DIETS Ingredients Stock 1 II IIB nr IVA IVB percent percent percent percent percent percent percent Whole wheat, ground 34.0 32.50 32.50 32.50 32,50 32.50 32.50 Oats, ground 34.0 32.75 32.75 32.75 32.60 32.60 32.60 Skim milk, powdered 15.0 10.00 10.00 10.00 10.00 10.00 10.00 Alfalfa meal 4.0 4.00 4.00 4.00 4.00 4.00 4.00 Yeast, strain "O’ * 1 2.0 9.50 9.50 9.50 9.50 9.50 9.50 Wesson oil 8.0 8.00 7.00 6.00 8.00 7.00 6.00 Fortified oil2 2.0 2.00 2.00 2.00 2.00 2.00 2.00 Sodium chloride 0.5 0.50 0.50 0.50 0.50 0.50 0.50 Calcium carbonate Cholesterol3 Bile salts4 Thyroid3 0.5 0.50 0.50 0.50 0.50 o;50 0.50 1.00 2.00 1.00 2.00 0.25 0.25 0.25 0.25 0.15 0.25 0.15 0.25 0.15 ^ Brewer's yeast, strain MG", Anheuser-Busch Inc., St. Louis, Mo. 2 Fortified oil containedel600 I.U. of vitamin A and 160 I.U. of vitamin D per gram of oil. 3 Cholesterin C.P;, Amend Drug and Chemical Co., New York, N.Y. 4 Sodium glycocholate (pure), City Chemical Corp., New York, N.Yl 5 Desiccated thyroid powder, USP, Armour and Co., Chicago, 111. 13 and subtracting the food remaining* This was carried out for two weeks between the sixth and ninth weeks of the experimental period. Care was taken to measure all food spilled from the food cups by collecting on paper in the bottom of the cages and weighing. The food consumed was expressed as the number of grams of food eaten per 100 grams of body weight per day* III, OXYGEN CONSUMPTION The apparatus used to determine the oxygen consump tion of the rats was developed in this department (Mason and Winzler, 1947)^, Essentially It consists of individual glass chambers of approximately two liter capacity for each rat* Each chamber Is connected with a water manometer* Each animal is placed In a wire cage built not to cramp the animal but to reduce its capacity to move around to a minimum. The cage is placed on a platform in the chamber to keep the animal above the sodium hydroxide which is pipetted into the bottom of the chamber to absorb the carbon dioxide exhaled by the animals. Twenty-five ml* of 35 percent sodium hydroxide was found to be adequate for this absorption* The chambers were made air tight with stopcock grease and placed in a large tank of water during I wish to ext;end my thanks to Dr. Richard J. Winzler for making available the oxygen consumption apparatus used for the determinations in this work. 14 the oxygen consumption determination* The water was maintained at the temperature of thermal neutrality of the rat, that is, 270C. A positive pressure of oxygen was then placed in the chambers which pressure could be observed in the manometers* The oxygen consumption of the rats was measured by noting the change in pressure per unit of time and multiplying by a calibration constant* Prom 15 to 30 minutes were allowed for temperature equilibration after placing the chambers containing the rats In the water* Twelve to 16 five minute readings were taken from the manometers for the calculation of the oxygen consumption* The animals were fasted overnight before each series of determinations. The tests were made at the same time, each day to minimize the variation due to the time of day. The ml. of oxygen consumed are obtained by taking the mean of the three lowest manometer readings which are assumed to correspond more closely to the resting oxygen * consumption, and multiplying this mean value with a constant which incorporates body weight and vessel capacity (Mason and Winzler, 1947)* The results are expressed In terms of ml. of oxygen consumed per 100 grams of body weight per hour. The oxygen consumption has also been calculated as the cubic centimeters of oxygen consumed per 100 square 1 5 centimeters of surface area per hour. The surface area of the rats was calculated from the formula developed by- Lee, 1929, where: Surface Area = K x weight 0#60 and K is equal to 12.54 £ 0,05. IV. AUTOPSY For autopsy the animals were injected with 0.15 ml. of nembutal (60 mg. per ml.) per 100 grams of body weight to produce surgical anesthesia. An incision was then made through the body wall of the abdomen and the diaphragm to expose the heart. Approximately five ml. of blood were drawn by means of a cardiac puncture for the determination of the blood cholesterol level. The liver, kidneys, testes, adrenal glands, and seminal vesicles were removed and weighed. The liver and adrenal glands were immediately prepared for the determination of cholesterol.-*- ^ The help of Dr. Lore Marx for her guidance and aid in collecting the autopsy data is sincerely appreciated. V. CHOLESTEROL DETERMINATION 1 6 Preparation of samples. a. Blood. The blood which was drawn from the animals was first centrifuged at 2500 rpm for 20 minutes and then the serum was pipetted off. The cholesterol was extracted from the serum by adding 14 parts of 1:1 ethyl alcohol-acetone solution. This mixture was centrifuged to sediment the precipitated protein and the supernatant was decanted for the detezmination of cholesterol. b* Liver. A sample of approximately 1-2 grams of liver was ground with a mortar, and the brei was placed in a tared 25 ml. volumetric flask. The flask plus the liver was weighed to determine the weight of the sample. Approximately 18 ml. of 1:1 ethyl alcohol-acetone_solution was. added to the flask and the mixture refluxed for 10 minutes at 65°C. to extract the cholesterol. The flask was then cooled and the contents were made to volume. This was filtered and set aside for the determination of choles terol. c. Adrenal glands. The adrenal glands from two animals were pooled for this determination. The adrenal 4 glands were minced and placed in a tared 25 ml. volumetric flask. The flask plus the adrenals was weighed to deter mine the weight of the sample. Approximately 18 ml. of 17 1:1 ethyl alcohol-acetone was added to the flask and the mixture refluxed for 10 minutes at 65°€. to extract the cholesterol. The flask was then cooled and the contents were made to volume.' This was filtered and set aside for the determination of cholesterol. * Cholesterol detelimination. The cholesterol levels were determined by. a modified Schoenheimer - Sperry - Chaney procedure (Marx, Nieft, and Meserve, 1948)^. An aliquot of the ethyl alcohol-acetone extract of each sam ple was taken which was estimated to contain between 0.1 and 0.9 mg. of cholestero 1. The aliquots were evaporated to dryness and then the samples were saponified with 33 percent potassium hydroxide at 60°C. The samples were neutralized with 15.percent acetic acid with phenolphthalein as the indicator. The cholesterol was then precipitated with digitonin and centrifuged to sediment the precipitate the supernatant was discarded. The precipitate was dried and then dissolved in glacial acetic acid. Three ml. of chloroform were added and the color developed with a 1:9 sulfuric acid-acetic anhydride mixture. The amount of cholesterol was determined from the readings of the color density as obtained from the Klett-Summerson photoelectric ^ I wish to express my appreciation to Mr. Prank Shimoda for his aid in the determinations of cholesterol. 1 8 colorimeter using a standard calibration curve* All samples were carried through this procedure in duplicate. The standard curve was obtained by the determination of known amounts of cholesterol. The photoelectric color imeter readings for 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 0.9 mg. of cholesterol were determined and plotted against the cholesterol concentrations. CHAPTER IV EXPERIMENTAL DATA The experimental results obtained during a feeding period of nine weeks are summarized in Tables II to VIII, The oxygen consumption of these animals was deter mined at three week intervals. These data are shown In Table II, expressed as the oxygen consumption in ml, per 100 grams of body weight per hour (Mason and Winzler, 1948), and Table III, expressed as the oxygen consumption in ml. per 100 square centimeters of body surface area per hour (Lee, 1929), The weights of the animals which were obtained prior to the oxygen consumption determination and after 12 hours of fasting are given in Table IV. The food consumption data obtained during the seventh and eighth weeks of the experimental period are shown in Table V. These data are expressed as the average of the food eaten, in grams per animal per day, and as the average of the food consumed in grams per 100 grams of body weight per day. At the end of the experimental period the animals were sacrificed and the average weight of the livers, kid neys, testes, seminal vesicles and adrenal glands recorded. These results are summarized in Tables VI and VII. 20 The total cholesterol levels In the blood, liver and adrenal glands were determined and these data are shown in Table VXIT* The standard error of the mean was calculated in all tables by the formula: S „ r ^ d2 "nC^IT where Md” is the deviation from the mean, and "n" is the number of observations* TABLE II OXYGEN CONSUMPTION Oxygen consumption expressed as ral./lOQ grams of body weight/hour Diets Initial 3 weeks 6 weeks 9 weeks Q,Og Sm QOg SM qo2 SM ^Og SM I 123.0 .-4.0 106.0 t2.6 107.8 *4.1 — — IIA and IIB 121.0 *2.2 105.8 ~ 3*0 104.9 *3.1 mm mm mm mm III 121.7 ■ -2.1 166.® -4.4 161.9 *5.9 159.6 *6.0 IVA and IVB 120.7 -3.9 157.1 ±3.0 144.2 * 2.9 143.8 14.2 - Diets IIA and IVA were fed during the first three weeks of the experimental period and, diets IIB and IVB were fed during the remainder of the experimental period. TABLE III OXYGEN CONSUMPTION Oxygen consumption expressed as ml./lOO sq. cm, of surface area/hour Diets Initial 3 weeks 6 weeks 9 weeks ^°2 SM «*°2 SH ^°2 SM Q°2 Sm I 81.5 ± 3.3 73.1 t 2.7 78.5 ±2*9 «•«» ««» IIA and IIB 79.5 11.7 70.8 ±1.6 75.4 t 2.6 <mmm mm «■ » III 81*1 H.9 109.6 ±3.4 109.8 14.5 115.2 ! ■ 4.4 IVA and IVB i 78.7 ±2.7 98.6 ±2.0 98.3 ±2.9 102,8 ±3.7 Diets IIA and IVA were fed during the first three weeks of the experimental pefciod and diets IIB and IVB were fed during the remainder of the experimental period. to to TABLE IV ‘ BODY WEIGHTS Following a fasting period of 12 hours Diets Initial 9 weeks Average gain in weight during the experimental period Weight in grams SM Weight in grams SM Gain in weight in grams % I 158.1 1 10.5 279.6 113.2 121.5 1 5.1 IIA and IIB 161.8 * t 6.8 270.2 * 13.6 108.5 17.4 III 167.5 1 4.4 247.1 t 7.6 79.6 16.6 IVA and IVB 157.5 1 7.0 229.6 111.2 72.1 17.5 to 03 TABLE V POOD CONSUMPTION Diets Average grams of food consumed/ animal/day Range of grams of food consumed/ animal/day Grams of food consumed/lOO grams of body weight/day I 15.5 10.8 - 22.5 5.7 IIB 15.5 12.8 20.0 5.9 III 18.3 13.8 27.0 7.4 IVB 18.9 15.0 - 23.3 8.2 The food consumption was determined during the seventh and eighth weeks of the experimental period during which time diets IIB and IVB were fed. w » £ » TABLE VI BODY AND ORGAN WEIGHTS AT AUTOPSY Diets Average body weight Average organ weights in grams in grains liver Kidney Testes Seminal vesieles Adrenal glands I 290,1 11.99 2.44 2.80 1.28 0.0329 II 274.4 13.51 2.38 2.78 1.29 0.0327 III 250.6 12.39 3.07 2.63 1.06 0.0367 IV 235.8 12.65 3.07 2.68 0.87 ' 0.0359 10 Ol TABLE VII ORGAN WEIGHTS AT AUTOPSY Expressed as grains of organ per 100 grams of body weight Diets Liver Kidney Testis Seminal vesicles Adrenal glands I 4.14 0.84 0.97 0.44 0.0114 II 4.92 0,87 1.01 0.47 0.0119 III 4.95 1.25 1.05 0.42 0.0147 IV 5.36 1.32 1.17 0.37 0.0152 10 0> TABLE V III TOTAL CHOLESTEROL LEVELS IN BLOOD AND TISSUES Diets Blood Liver Adrenal gland mg./lOO. ml. SM Percent SM Percent SM I 61.0 ^5.0 0.205 -0.013 4.595 tO.325 II 66.8 ±4.1 0.793 ± 0.077 , 7.808 t0.386 III 53.5 -2.4 0.243 t0.146 4.283 ±0.215 IV 58.5 -3.3 0.805 t0.010 7.360 tO. 680 to -a CHAPTER V DISCUSSION The data secured from the determination of the oxygen consumption showed a significant increase over the normal in rats fed thyroid. However, the oxygen consumption of animals fed thyroid plus cholesterol was consistently less than that of animals fed thyroid alone. This decrease in oxygen consumption cannot be attributed to a reduction in the intake of food, and thus of thyroid hormone, as this was found to be practically identical in these two groups (See Table V), The difference in oxygen consumption ex pressed as ml, of oxygen consumed per 100 grams of body weight per hour, (See Table II) was statistically signifi cant when tested according to Fisher, 1938^, When comparing the data for the groups fed thyroid alone and the groups fed thyroid plus cholesterol, it was found that for the ob servation at six weeks the |> value was less than 0,02 and at nine weeks less than 0,05, According to this method of The formula for the comparison of two means for a significant difference as developed by Fisher is as follows 1 nl 4 n2 and T - . . ..XH? s E s S T T - i K < Z < d l > 2 4 s ( O b ) 8 ) N (nj * l)(ng * 1) ni ♦ ng - 2 where ”mt t equals the mean of the observations, ’ ’n-i0 and nn 2 W equals the number of observations minus one, and M ” equals the difference from the mean. For calculation of ”p” values ”n” is equal to n 2 # l , p” values are found in table using "TM and "n" for reference (see Fisher, p, 177, 1938). 29 . comparing two groups or data, the differences between these values are statistically significant* The significance of these data is further indicated by the fact that in the tests carried out on the sixth and ninth weeks, no single observation on Group IV equalled or exceeded the average of Group III during the corresponding periods* In the tests at 3 weeks, there was only a single Instance of overlapping of the results of £roup IV over the average of Group III* If however, the statistical signi ficance Is tested on the basis of the difference of the means being greater than three times the square root of the sum of the squares of the standard error of the means, then no significant difference exists between Groups III and IV. For the three, six and nine weeks observations these values are 15*9, 19*7 and 21.9 respectively, while the differences of the means for the corresponding periods is 8.9, 17.7 and 15.8, which in all cases is less than the value required to be considered significant. Recalculation of the oxygen consumption on the basis of ml. of oxygen consumed per 100 square centimeters of body surface area per hour (See Table III) give £ values which are also statistically significant according to Fisher for the observations of three and nine weeks, that Is £- values less than 0.05. The groups fed thyroid plus choles terol had a markedly higher oxygen consumption than either 30 the control group or the group fed cholesterol alone. Thus the addition of cholesterol to the thyroid died did not depress the high oxygen consumption sufficiently to approach normal levels. The results concerning the body weights (See Table IV) agree with findings of previous investigators (Cameron and Carmicheal, 1920 and 1921; Sciaky, 1938; Belasco and Murlin, 1941) in which the administration of the thyroid hormone caused a decrease in body weights. As was to be expected, the groups receiving thyroid hormone consumed markedly more food but showed significantly less gain In body weight. Addition of cholesterol to the diet apparently did not appreciably modify these results. It was observed that the body weights of the group fed cholesterol alone in the diet were slightly less than the corresponding group fed a cholesterol-free diet. According to Sperry and Stayanoff, 1935, rats fed a diet high in cholesterol tended to utilize their food less efficiently than rats fed a normal diet. This may be the reason for the reduction In the weight gain In the groups fed cholesterol as compared to their respec tive control groups. Another factor that should be considered is that the fat portion of the diet was reduced by an amount corresponding to the cholesterol added to the diet (See Table I). The observation that animals fed a diet high In 31 cholesterol grew less well (See Table IV) is in agreement with the findings of Sperry and Stayanoff, 1935. However, the results of the determination of the food consumption (See Table V) show that animals on the diets containing cholesterol consumed the same amount of food as their respective controls. This is in contradiction with the findings of the aforementioned investigators. The results on organ weights are in agreement with the findings of previous investigators (See Tables Viand VII). When based on 100 grams of body weight there appears to be a slight hypertrophy of the liver and kidney and a definite hypertrophy of the adrenal gland in the animals fed thyroid. This agrees with the findings of Cameron and Carmicheal In 1920 and 1921. The dose level of the thyroid hormone was not high enough to significantly decrease the weight of the sex organs. According to the work reported by Cohen in 1935 on male rats In hyperthyroidism there is a relative Increase in testicular and adrenal weight and a decrease in the weight of the seminal vesicles. The weights obtained In the present work are in agreement with these findings. In the rats fed cholesterol in addition to thyroid there was no reduction in the hypertrophy of these organs. On the con trary the liver weight was further increase. A similar effect of dietary cholesterol on the liver weight was noted 32 when the groups fed the thyroid-free diets were compared (Groups I and II)* This is in agreement with earlier ob servations (Anitschkow and Chalatow, 1913), Prom the observations of the weights of the organs In the control and the experimental groups no conclusions can be drawn as to the mechanism of the action of cholesterol in protection against hyperthyroidism* The observation that thyroid hormone slightly depresses the total blood cholesterol levels is in agreement with earlier work (Epstein and Lande, 1922; Mason, Hunt, and Hurxthal, 1930; Hurxthal, 1933; Man, Gildea, and Peters, 1940; Fleischmann and Shumaeker, 1942) In which a lower blood cholesterol level was found in hyperthyroidism* The addition of cholesterol to the thyroid diet tends to raise the blood cholesterol toward the normal level. Dietary cholesterol markedly Increases the cholesterol concentration of the liver and adrenal glands. This effect was not modified by the presence of thyroid hormone in the diet. Thus there was no correlation between the cholesterol concentration of blood and tissues* As a result of these observations, one can not consider that cholesterol definitely acts as an antithyroid agent* Although the B.M.R. was lowered in thyroid plus cholesterol- fed rats over that of the thyroid-fed rats, the level did not return to that of the normal controls. Also there is 33 little evidence that the weights of the liver and adrenal glands is significantly depressed when cholesterol is fed to the rats receiving desiccated thyroid. These findings indicate that the increased survival of hyperthyroid rats fed diets containing cholesterol (Marx, Meserve, and Deuel, 1948; Ershoff and Marx, 1948) was due to factors other than the neutralization or destruction of the thyroid hormone. CHAPTER VI SUMMARY _In rats maintained on a diet containing desiccated thyroid plus cholesterol a significant decrease in the oxygen consumption was observed, as compared to rats main tained on a diet containing desiccated thyroid alone# This effect of cholesterol was not sufficient, however, to reduce the oxygen consumption to normal levels# The reduced body weight gain of rats fed thyroid hormone was not appreciably modified by the addition of cholesterol to the diet# Liver weight was increased as a consequence of choles terol as well as of thyroid hormone in the diet, a combina tion of the two substances causing a further increase In liver weight# The hypertrophy of the kidney of hyperthyroid rats was not modified by cholesterol# The total cholesterol level of the blood was slightly depressed by thyroid hormone# Dietary cholesterol caused a slight increase in total blood cholesterol, and significant increases in total liver and adrenal cholesterol levels re gardless of the thyroid content of the diet# BIBLIOGRAPHY 35 Anitschkow, N. , and Chalatow, S.S., quoted and confirmed by Wacker, L., and Hueck, W., "Chemische und morpho- ligische Untersuchugen uber die Bedeutung des Choies ter ins im Organismus.” Arch, Exper. Path, Pham,. 74s 416, 1913, Astwood, E* B,, "The Chemical Nature of Compounds which Inhibit the Function of the Thyroid Gland,” J* Pharmacol♦, 78: 79, 1943, Astwood,. E, B., and Bissell, A,, "Effect of Thiouracil on Iodine Content of the Thyroid GlancL” Endocrinol., 34: 282, 1944. Barbier, P., and Pepquiquot, H., "Effect of thyroid Secretion on the Blood Levels of Cholesterol Esters (Influence de la secretion thyroidieune sur le taux dans le sang des esters du cholesterol. Compt. rend, Soc. de Mol., 127: 111, 1938. Belasco, I. J., and Murlin, J. R., "The Effect of Thyroxin on the Basal Metabolism and Thyroid Tissue Respiration of Rats at Various Ages." Endocrinol., 28: 145, 1941. Betheil, J. J., Wiebelhaus, V. D., and Lardy, H. D., "Studies of Thyroid Toxicity I. A Nutritional Factor which , Alleviates the Toxicity of Ingested Thyroid Substances." J. Nutrition, 34: 431, 1947. Cameron, A. T., and Carmicheal, J., "The Comparative Effects of Thyroid and Iodine Feeding on Growth in White Rats and in Rabbits". J. Biol. Chem.,45: 68, 1920. Cameron, A. T., and Carmicheal, J., "The Effect of Thyroxin on Growth in White Rats and in Rabbits." J. Biol. Chem., 46: 35, 1921. Cohen, R. S., "Effect of experimentally Produced Hyper thyroidism upon the Reproductive and Associated Organs of the Male Rat." Am. J. Anatomy, 56:,143, 1935. Christensen, B. G., "The Effect of Some Antithyroid Sub stances on the Metabolism of the Normal Rat." Acta Pharmocol. Toxicol., 1: 98, 1945. Cutting, W. C., Rytand, D. A., and Tainter, M. L., "Relation ship between Blood Cholesterol and Increased Metabolism from Dinitrophenol and Thyroid.” J. Clin. Invest., 13: 547, 1934. 36 Drill, V. A., Overman, R., and Leathern, J. H. , ’ ’Relation of the B Vitamins to Ovarian Function during Experi mental hyperthyroidism,” Endocrinol,, 32: 32V, 1943. Epstein, A, A,, and Lande, H., ’ ’The Relation of* Cholesterol and Protein Deficiency to Basal Metabolism.” Arch. Intern. Med.. 30: 563, 1922. Brshoff, B. H., ’ ’Effects of liver Feeding on Growth and Ovarian Development in Hyperthyroid Rat.” Proc. Soc. Exper. Biol. Med.. 64: 500, 1947. Ershoff, B. H., and Hershberg, D., ’ ’The Beneficial Effects of Yeast on the Cardiac Failure of Hyperthyroid Rats.” Am. J. Physiol., 145: 16, 1945. Ershoff, B. H., ’ ’Comparative Effects of Liver and Yeast on Growth and Length of Survival of the Immature Thyroid- fed Rat.” Arch. Biochem., 15: 365, 1947. Ershoff, B. H., and Marx, W., ’ ’Effects of Dietary Cholesterol on the Length of Survival of Hyperthyroid Rats.” Exper. Med and Surg., 6: 145, 1948. Fisher, R. A., ’ ’Statistical Methods for Research Workers.” 120, 1938. Fleischmann, W., and Shumacker, H. B., Jr., ”The Relation ship between Serum Cholesterol and Total.Body Cholesterol in Experimental hyper- and hypothyroidism.” Bull. Johns Hopkins Hosp., 71: 175, 1942. Franklin, A. L., and Charikoff, I. L., ”The Effect of Sulfo- nilomide on the Conversion of Inorganic Iodine to Thyroxine and Diiodotyrosine by Thyroid Slices.” J. Biol. Chem.. 148: 719, 1943. Franklin, A. L., andCharikoff, I. L., ’ ’The Effect of Sulfo- nomides on the Conversion in vitro of Inorganic Iodide to Thyroxine and Diiodotyrosine by Thyroid Tissue with Radioactive Iodine as Indicator.” J. Biol. Chem.. 152: 295, 1944. ~ Gordon, A. S., Goldsmith, E. D., and Charipper, H. A., "Effect of Thiourea'on the Development of the Amphibian.” Nature, 152: 504, 1943. Haen, A., Langfeld, H., and Oehme, C., ’ ’Relations of Thyroid arid Adrenals (TJber die Beziehungenzwlschen Schilddruse und Nebennieren).” Endokrin.. 21: 305, 1939. 37 Hoffmann, P., and Hoffmann, E. J., de Public, del Instltut., TJniversidad de Chile, 1943 1 quoted by Poldes, P. P., and Murphy, A. J., Proc. Soc. Exper. Biol. Med., 62s 218, 1946. Horuiti, I., and Ohsako, H., "The Effect of Yakriton to Counteract Thyroid Hormone." Tohaku J. Exper. Med., 505, 1934 (Reprint from Army Med. Library, 1948). Hughes, A. M., and Astwood, E. B., "Inhibition of Meta morphosis In Tadpoles by Thiouracil." Endocrinol., 34: 138, 1944. Hurxthal, L. M., "Blood Cholesterol in Thyroid Disease: Analysis of Findings in Toxic and Nontoxic Goiter before Treatment." Arch. Intern. Med., 51: 22, 1933. Kendall, E. C., "Thyroxine." Am. Chem. Soc. Monograph Series, 1929, The Chemical Catalog Co., Inc., New York. Kennedy, T. H., and Purves, H. D., "Studies on Experimental Goiters The Effect of Brassica Seed on Diets in Rats." Brit. J. Exper. Path., 22: 241, 1941. Keston, A. S., Goldsmith, E. D., Gordan, A. S., and Charipper, A. A., "The Effect of Thiourea upon the Metabolism of Iodine by Rat Thyroid." J. Biol. Chem., 153: 241, 1944. Leathern, J. H., "The Influence of Thiourea on Plasma Proteins and Organ Weight in Rats." Endocrinol., 36: 98, 1946. Lee, M. 0., "Determination of the Surface Area of the White Rat with its Application to the Expression of the Meta bolic Results." Am. J. Physiol., 89s 24, 1929. MacKenzie, C. G., and MacKenzie, J. B., "Effect of Sulfo namides and Thioureas on the Thyroid Gland and Basal Metabolism." Endocrinol., 32s 185, 1943. MacKenzie, J. B., MacKenzie, C. G., and McCollum, E. V., "The Effect of Sulfanllylguanidine on the Thyroid of "the Rat." Science, 94: 518, 1941. Man, E. B., Gildea, E. F., and Peters, J. P., "Serum Lipoids and Proteins In Hyperthyroidism." J. Clin. Invest., 19s 43, 1940. “ Marine, D., Baumann, E. J., Spence, A. W., and Cipra, A., "Further Studies on Etiology of Goiter with Particular Reference to the Actions of Cyanides." Proc. Soc. Exper. Biol. Med., 29: 772, 1932. 38 Marx, W., Nieft, M., and Meserve, E. R. To be published. Mason, G,, and Winzler, R, J# To be published, Marx, W., Meserve, E. R*, and Deuel, H, J,, Jr., “Protective Action of Dietary Cholesterol in Experimental Thyro toxicosis ,M Proc, Soc, Exper, Biol, Med,, 67: 385, Mason, R, L., Hunt, H, M,, and Hurxthal, L„ “Blood Choles terol Values in Hyperthyroidism and Hypothyroidism - Their Significance.” Hew England J. Med.. 203: 1273 Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemistry Interpretation”• Second Ed,, 1946, 497. New York. Saegesser, M,, “Die Schutzfunktion des Organisraus bei Thyreopathie." Klin, Wochensch., 12: 672, 1932, Schmidt, L. H., and Hughes, H. B., ”The Free and Total Cholesterol Content of Whole Blood and Plasma as Related to Experimental Variations in Thyroid Activity.” Endocrinol., 22: 474, 1938, Sciaky, I., “Experimental Hyperthyroidism in Different Animals (Hyperthyroidisms experimental chez differentes especes animales).” Am. d*anat. path., 15: 165, 1938. Sperry, W. M., and Stayanoff, V. A., "Effects of Long- continued Cholesterol Feeding in Rats.” J. Nutrition, 9: 131, 1935. ~ Webster, B., Clawson, T. A., and Chesney, A. M., "Endemic Goiter in Rabbits III. Effects of Administration of Iodine.” Bull. Johns Hopkins Hosp.,43: 291, 308, 1928. V .

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Creator Winebrenner, Louis E (author)

Core Title A study of the mechanism of the protective action of cholesterol in hyperthyroidism

Degree Master of Science

Degree Program Biochemistry

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 Marx, Walter (committee chair), [illegible] (committee member), Deuel, H.J. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-776514

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