DOCTORAL DISSERTATION SERIES title m mcr »to,mi m onto* mmmM-MWM m / mm m cmrm mm &mu m •tm arm m m m rm m s m mt AUTHOR Shy-pit) CMVIj IINIWBSITV DEGREE MI0II6M Sffift CPU, DATE /fSt ILL. PUBLICATION NO. mi III Thyy UNIVERSITY UNIYtwl MICROFILMS /M A UU ADDA a ir u ia a u the effect of a g e , d i e t , and carbon t e t r a c h l o r i d e -i n d u c e d LIVER INJURY ON THE CHOLESTEROL CONTENT OF BLOOD AND CERTAIN other tissues in the albino rat By Slng-pao Chiang A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 1951 ACKNOWLEDGMENT The writer is greatly indebted to Dr. Carl A. Hoppert, Professor of Biochemistry, for suggesting the problem and for much advice and encouragement throughout the course of the experimental work and for invaluable assistance with the preparation of the manuscript. Thanks are due Mr. Leo Klever, Foreman of Caretakers, Vitamin Assay Laboratory, for indispensable helo in caring for the rats during the investigation. CONTENTS INTRODUCTION ........................................... 1 LITERATURE REVIEW.................... .................. 3 Cholesterol Levels as Influenced by Various Factors • Age and S e x ................ • 3 3 Certain Physiological Conditions. • • • • • • • • • 5 Dietary Factors • • • • • • • • • • . . • • • • • • 7 production of Arteriosclerosis in Experimental Animals • • • • • . • • • • • • • « . . . . . • « 10 Carbon Tetrachloride-induced Liver Injury • • • • • • 12 Absorption. • • • • • . • « • • . . . . • • • « . . 12 Biochemical Changes After Carbon Tetrachloride Injury. .................... ..12 Effects of Dietary Factors. • • • • . • • • • • • • 15 Protective and Therapeutic Means. « • • • • • . « . 16 Mechanism of Action by Carbon Tetrachloride • • • • 19 EXPERIMENTAL PROCEDURE ................................. 20 General • • • • • • • • • • • • . • • • • • « « . . . 20 Composition of the Diets Employed • • • • • • • • • . 2 1 Administration of Carbon Tetrachloride, • • • • • • • 2 1 Method of Obtaining Blood and Tissues • • • • • • • • 22 Analytical Methods. • • • • • • • • • • • • • • • • • 22 Water Content of the Liver. • • • • • • • • « . . ♦ 22 Nitrogen Content of the Liver • • • • • • • • • • • 2 3 Total Fat in the Liver. • • • • • • • • • . . . • . 2 3 Cholesterol in Blood and Tissues. • • • • • • • • • 2tl± Esterified Fatty Acids in Blood Plasma. • • • • • • 25 CONTENTS RESULTS .............. DISCUSSION. . . . . . . SUMMARY" .......... LITERATURE CITED. . . . APPENDIX (Detailed data) CONTD INTRODUCTION Cholesterol, one of the most Important naturallyoccurring sterols, is present In all cells of animal organ­ ism both in the free state and in the form of fatty acid esters* Its origin is endogenous as well as exogenous* It can be absorbed from dietary sources, synthesized from simple molecules such as acetate, acetaldehyde, acetone, pyruvate, isovalerate and short fatty acids as shown by Bloch, Rittenberg, Brady, and Gurin (1, 2, 3> k-)» and destroyed during metabolic processes* Under normal conditions the quantity or the ratio of the free to the combined forms present in the blood and tissues varies only within narrow limits, for absorption, synthesis, and destruction are so accurately attuned* However, this is not the case If the individual is under the disturbing influences of unbalanced diet, or impairment of any of the organs* In dietary fatty livers or when large quantities of cholesterol are y:iven to animals the sub­ stance accumulates in the liver largely as cholesterol esters* When the parenchyma of liver is extensively damaged the concentration of cholesterol esters in the blood plasma falls and renal lesions appear. In biliary obstruction both free and ester cholesterol in the blood rise, and the dis— proportion between free and ester forms Increases as the obstruction persists# In patients with diabetes mellltus and nephritis a general rise in all of the lipids of the blood occurs. Hypercholesterolemia has been held responsible for arterio­ sclerosis which often accompanies diabetes. The general opinion is that arteriosclerosis must be connected with a disturbance of cholesterol metabolism# Much work has been done in connection with this disease using the chick, rabbit, dog and sometimes the guinea pig and golden hamster as the experimental animal. The rat, although much used In other metabolic studies, has not been considered quite appropriate for this purpose. Therefore only scanty data are available from the literature# It was the object of the present investigation to study the effect of age, of diets both adequate and Inadequate in essential nutrients, and of carbon tetrachloride-induced liver damage on cholesterol levels In blood, liver, heart, and kidney of albino rats. LITERATURE REVIEW I, Cholesterol Levels as Influenced by Various Factors A* Age and Sex Early in 1928 Shope (5) reported from his observation on calves that changes in serum cholesterol and cholesterol ester content as related to age were of two types: first a marked and rather rapid increase from birth for a relatively short period and then a less marked and more gradual decline with advancing age. He noticed that changes with age were more uniform and regular in male than in female animals. Page ejt al (6 ) did not find variations of age in men from 20-90 years a determinable influence on either the amount or the composition of the plasma lipids. Turner and Steiner (7 ) reported, from weekly determinations of serum cholesterol in several patients over a period of 7 -li+ months, that the level of cholesterol is remarkably constant. In 1937 Sperry (8 ) concluded from a long-term study on 23 healthy adult persons that the variation in a given individual over considerable periods of time is much less than the variation among individuals and that in most persons in health the serum cholesterol seems to be maintained at a constitutional level which is characteristic for each individual. In 1930 he and Webb (9) made further studies on the same subjects and Ij. observed that In 8 of the H 4. men and 1 of the 6 women there was no appreciable change; in 6 men and 6 women increases of 15-30 percent were found. They concluded that serum cholesterol concentration increases with age in some persons but the increase is not an obligatory concomitant of ageing. Foldes and Murphy (10) noticed from their study of 20 young healthy adults and 2 0 old patients with no known disorder of lipid metabolism that practically no difference was found in the total cholesterol, cholesterol esters, and phospho­ lipid phosphorus of the two age groups. Gram and Leverton (11) studying the serum cholesterol in women of 5 different age groups, 18-70, found the increase of cholesterol in serum with increasing age to be significant. Keys e_t al (12) made a cross-sectional examination of 1 ,14.92 men from 1 7 -7 8 years old and 56L|. women from 17-30 years old. They found that over the age range 1 7 - 3 0 the cholesterol values from men and women were not significantly different as to averages, individual variability, and age trend. Over this range there was an average increase per year amounting to 2.2 mg. of total cholesterol per 100 ml. of serum. For the age range 1 7 - 7 8 years in men there was a pronounced curvi­ linear relation between age and serum cholesterol concen­ tration with a maximum in the sixth decade. The evidence, they believe, points clearly to rising values for serum cholesterol from youth through middle age to lower values in the oldest persons in the population samples. However, they did not postulate that the cholesterol level actually 5 declined in old age. Kountz at al (13) reported, from studying 9 1 2 patients aged l4.0 -8 £, that the females showed an average blood-cholesterol level of 2 3 7 mg. percent, the males 196 mg. percent. Atherosclerosis appeared earlier and more frequently among the men. Kornerup (II4.) observed from studies made on 8 7 men and 3 6 women from 1 9 - 9 6 years and 9 6 children from 1 - 1 6 years that sex and age differences were small and generally insignificant, but the serum free cholesterol levels of elderly men were significantly higher i than those of young men. With rats, Mounier £t al (15?) reported similar blood cholesterol levels for both young and old animals. B. Fasting. Certain Physiological Conditions Sure, Kik and Church (16) observed that in the albino rat during fasting there was a marked decrease of blood fatty acids and lecithin, but no change in the concen­ tration of blood cholesterol. Entenman e_b al (17) observed that there was no lipemia, or rise in the levels of total cholesterol, total fatty acids, and phospholipids during acute fasting or chronic undernutrition. Schettler (18) reported from his studies on mice that liver total choles­ terol rose on the second day of fasting but dropped by the sixth day. In the other tissues of the animals the choles­ terol remained unchanged. Pregnancy and menstruation. Okey and Stewart (19) noticed that blood cholesterol in women tends to be high just 6 preceding menstruation, with a fall near the time of onset and a rise afterwards. Lea (20) reported that non-pregnant female mice had considerably less blood cholesterol than males, and pregnant females had about the same as males* Hypothyroidism and hyperthyroidism. Johnson and Riegel (21) and Poldes and Murphy (22) founu that the blood cholesterol of dog and man Increased significantly in hypo­ thyroidism and fell to very low values in hyperthyroidism, but the cell lipid values were relatively constant. Fleischmann and Shumacker (23) obtained similar results at an earlier date, concluding that thyroxine having no specific effect on cholesterol metabolism influenced only a shift of cholesterol to and from the blood plasma. Horlick and Havel (2l|_) noticed that cholesterol and propylthiouracil alone or combined failed to produce atherosclerotic vas­ cular lesions in the rat, but cholesteroleraia of 2 to 3 times normal values resulted with cholesterol or propyl­ thiouracil feeding and approximately 6 times normal with combined feeding. Development of tumors* Bennett (25) reported early in 1922 that the outer, more actively growing portions of the tumor contained more cholesterol than the central portion. Jowett (26) noticed that pure malignant tissues are higher in phosphatide and cholesterol than are malig­ nant tissues admixed with normal tissues. Malignant tissues also showed a high proportion of bound cholesterol. Knudson et al (27) observed that during irradiation 7 with ultraviolet light the cholesterol content or the skin of rats was increased, practically all of the increase being in the ester form; the tumors showed a high cholesterol content, about 8 0 percent being in the free form; and the total blood cholesterol was somewhat below the normal. Khaletskaya (28) reported that in mice the blood cholesterol varied with the stage of the development of the tumor, showing a gradual increase from the beginning, reaching the highest (about 3 0 percent above the normal) at papilloma stage, and dropping to near normal, when definite cancer had developed* C. Dietary Factors General nutrition. Bloor (29) demonstrated that in dogs single overfeedings with fat or carbohydrate generally resulted in high plasma phospholipid and fat in the post absorptive state, whereas the cholesterol level was not affected. Schmidt et al (30) in a study of the blood choles­ terol levels of 1 0 - 2 0 normal persons during 1 9 ^4-2 -1914-7 * noticed a decrease in total, free, and esterified plasma cholesterol. Lowered fat metabolism due to poor nutrition was held responsible. Recently Schettler (31) reported an increase in total plasma cholesterol in 5 0 men and £ 0 women since 191+7-191+9• He believed the improved nutrition normal­ ised the previously low cholesterol values* Fat. Treadwell and Eckstein (32) observed that fat contents of diets fed to rats did not influence the blood cholesterol, and the neutral fat, phospholipid and choles- 8 terol of the liver. Similarly Alfin-Slater ejt al (33) noticed that the amount of cholesterol synthesized in the liver under the different dietary conditions was the same* However considerable differences were noted by Schettler (34) in mice fed plant oils and those fed animal fats. The former showed an increase in the total blood cholesterol, especially the esterified fraction, accompanied by a lowering in the organs; whereas in the latter there was no definite evidence of hypercholesterolemia. The same author (35) observed an increase in organ cholesterol when cholesterol, and sodium and potassium salts of organic acids were fed to white mice on a basal diet poor in protein and rich in fat. Harris <*t al (36) reported that a high fat-low carbo­ hydrate diet raised the serum cholesterol level in patients with high blood pressure, and that injection of insulin decreased the serum fat and cholesterol levels. Vitamins. There is much controversal information regarding vitamin A and cholesterol metabolism. Lasch (37) reported that vitamin A given 3 times a day to human beings, in doses of 4 0 #0 0 0 -8 0 , 0 0 0 international units caused an increase in the serum cholesterol (ester fraction) within 5-10 days. Chalier ejfc al (38) found that in guinea pigs deprived of vitamin A the blood cholesterol remained within normal, limits. Extra vitamin A increased the blood choles­ terol level but heavy doses decreased their olood cholesterol to mere traces. Usuni (3 9 ) noticed an increase in serum cholesterol in rabbits receiving a diet deficient in vita­ min A. Knapp and Blackberg (40) using rats reported that 9 deficiencies in vitamin A and in vitamin B complex and in caloric intake produced lesions in the eyes resembling senile arteriosclerosis in human subjects. Choline, methi­ onine, and meso-lnositol have been found effective in lowering blood and liver cholesterol (ip., l±2) . Forbes (t|_3) reported that administration of nicotinic acid caused an increased production of cholesterol in the fatty livers. The effects of biotin upon fat synthesis and storage of cholesterol in liver have been reported by Gavin, and McHenry (I4J4.) and Okey e_t al (i+5)* Biotln when given to rats, caused fatty livers which were characterised by a high content of cholesterol. This condition could be pre­ vented by the simultaneous feeding of egg white, lipocaic, or inositol. When rats are fed a biotin-deficient and cho­ lesterol-rich diet they failed to store excess liver cho­ lesterol ester. They believed biotin must be connected with cholesterol metabolism. Myasnikov (I4.6 ) noticed that in experimental cholesterol atherosclerosis of rabbits, vitamin C reduced blood cholesterol and inhibited the growth of fatty deposits in the aorta. Vitamin E was found by Dam (14-7) without effect on the deposition of choles­ terol in the aorta in rabbits. Max at al (I4.8 ) noticed that in general tissue and blood cholesterol levels in young rats were not altered by either low or high intakes of vitamin E. However a deficiency caused deposition of cholesterol in aorta. Favorable factors in treating high blood pressure and 10 arteriosclerosis* Roffo (49) observed a marked decrease In blood cholesterol In patients Injected with an extract from eggplant* Similar properties were found to exist in the artichoke* The author believed the action due to their content of magnesium and potassium salts* However Wilkinson et al (50) could not confirm this from their studies* Recently a rice-fruit diet (5l) was reported to have a marked effect in lowering the serum cholesterol, but the specific mechanism remains unknown* Rodbard ejfc al (52) found that restriction of dietary intake even to the point of emaciation gave no protection against atheromatosis or hypercholesterolemia in chicks on a diet supplemented with cholesterol• D* Production of Arteriosclerosis in Experimental Animals Species differences have to be considered in producing experimental arteriosclerosis in chicks, guinea pigs, rab­ bits and dogs* In chicks the condition may be produced either by cholesterol feeding or diethylstilbesterol administration* Considerably more cholesterol is deposited after cholesterol feeding than after estrogen treatment* Feeding cholesterol to guinea pigs results in anemia and an increase in the free cholesterol content of liver, spleen, heart, lungs and blood as shown by Okey (53)• A high cholesterol diet will Induce arteriosclerosis* Member e_t al (54) reported that cholesterol fed with cholic or glycocholic acid to rabbits Increased the choles­ 11 terol content of the blood and aorta more than the same amount of cholesterol fed alone* Capritti and Magnanl (55) observed that addition of pyridoxins to cholesterol further increased the free end esterified levels in rabbits and also the arteriosclerosis was induced more rapidly* The production of arteriosclerosis in dogs requires feeding of high cholesterol diet and thiouracil* It is usually necessary to keep the dog in good health by allowing plenty of exercise and maintaining a hearty appetite (56)* Cholesterol feeding to rats produced fatty liver, the cholesterol ester and total lipid concentration increasing after a few days and reaching a maximum after 250 days* No changes occur in the kidney, heart, brain and blood. Hypercholesterolemia in blood is produced only by rather drastic means and then for a short duration only, Byers and Friedman (57) noticed a marked rise in free-cholesterol in plasma after an intravenous injection of a cholesterol suspension* A gradual fall lasting over I4.8 hours followed each injection* Cholesterol esters rose moderately while the free cholesterol concentration was falling* They re­ ported later (58) that ligation of the bile duct resulted in 2 0 0 -14.00 percent increase In plasma cholesterol, and oral administration of large amounts of cholic acid daily for 3 days resulted in extensive hypercholesterolemia. Dent and Hayes (59) noticed that in animals whose adrenals were Intact, ascorbic acid caused an increase in serum choles­ terol, There are no reports in the literature about athero­ sclerosis being produced In the rat. 12 II* Carbon Tetrachloride-induced Liver Injury Carbon tetrachloride has long been known for its ability to produce fatty liver and hepatic cirrhosis# It is therefore much used to produce hepatoma in cancer studies* The damage it produces and means of protection and pre­ vention have often been investigated* A. Absorption Robbins (60), by injection CCl^ into the stomach, small Intestine, or colon of dogs, found no absorption from the stomach, some from the colon, and most from the small Intestine* He noticed also that the rate of absorption was increased by giving ethyl alcohol or fat at the s ame time* Analysis of tissues Indicated that bone marrow contained the most CCl^, with the liver content next highest* Small quantities were found In blood, brain, kidney and lungs* Practically all the CCl]^ was excreted through the lungs; none was found In urine* B* Biochemical Changes After Carbon Tetrachloride Injury Kretchmer e_t al (6 1 ), by feeding mice 0 * 1 ml* of i+O percent CC1 |^ in olive oil every I4. days, found an appreciable initial decrease in the cytoplasmic succinic oxidase activity of the liver cells* However after 200 days or at the time of tumor Induction the value was only slightly below normal* Bodansky ejs al (62) found the liver damage from CCl^ did not reduce the acetyl-choline esterase activity of the plasma or liver in the rabbit although it did in the rat* 13 They believed this species difference Is due to differences in the choline-ester-hydrolyzing-enzymes In the plasma and liver In each species* Hirvatashi (63) found the aspara­ ginase activity of the liver, lung, and kidney much depressed In phosphorus and CC1|^ poisoning* Richter (64) showed that the liver of rats given 0 * 1 ml* of CCl^ intraperitoneally 21+ hours previously had impaired ability to methylate nicotinamide and guanidoaoetic acid, to form urea from (NH^gSOj^ *n t*10 presence of ornithine, to hydroxylate phenylalanine, and to conjugate morphine* The oxygen uptake of slices, the succinoxidase, and choline oxidase activities, and the organic phosphate content were higher them normal In the livers of CCl^-treated rats* Kurihara (65) showed that the vitamin C contents of the aqueous humor, crystalline lens, liver, spleen, and suprarenal gland of rabbit were greatly reduced when the liver was damaged by CCl^ and other liver poisons. Rosin and Doljanske (6 6 ), by injecting 0*1 ml* of CCl^ per 100 g* body weight Intraperitoneally Into young rats, showed that about one hour later the pyroninophilic granules had com­ pletely disappeared from hepatic cells, Indicating disturbed protein metabolism as one of the first effects of CCl^ poisoning. Valori (67)* by giving rats and guinea pigs CCl^ in oil solution subcutaneously found very low liver glycogen values In the guinea pig, but only moderately low in the rat* The muscle, and kidney glycogen values were reduced more In the rat than In the guinea pig* The water Ik content of the liver in both species was not changed apprec­ iably* but the Increase in liver fat and decrease in liver protein were much greater in the guinea pig than in the rat* Guimaraes Villela and Mells (6 8 ) obtained a higher phosphatase content of the blood of rats poisoned with CClj^ or other toxicants. Dervillee et al (6 9 )* by injecting 0.15-0*75 ml. CCl^ per kg. into rabbits* observed a small Increase in total blood cholesterol. Chebotarev (70) found that CCl^ given to horses resulted in a decrease of the number of erythro­ cytes* sugar* cholesterol, fibrinogen, calcium, chlorides* and alkaline reserve of the blood, and an increase of bilirubin, non-protein nitrogen, and lactic acid in the blood. Liver glycogen was decreased. De Senarclens (71) gave subcutaneous injections of CCl^ to rats kept on a diet rich in cholesterol. The liver at death showed granulomatous lesions composed of vacuolated reticulum cells and containing lecithin. and blood lipids increased. Blood cholesterol Pierce and Gofman (72) investi­ gated the effect of CCl^ injections on serum cholesterol levels and lipoproteins of the S^. 3 -1 2 , 1 2 -2 0 , and 2 0 -2 lp groups in normal and cholesterol-fed rabbits. They found a marked increase in all classes of lipoproteins and in cholesterol in the non-cholesterol-fed rabbits, with a gradual decrease to control levels after cessation of CCl^ injections. No macroscopic atherosclerosis developed in this group. In the cholesterol-fed rabbits cholesterol and all classes of 15 lipoproteins increased not only during CCl^ injections but also continued to do so after the cessation of CCl^ injections* Very large quantities of serum lipoproteins and cholesterol developed by the end of 10 weeks, and at this time all animals had developed atherosclerosis. The authors suggested that the increase in the normally occurring Sf 12-20, and Sf 2 0 -214. classes of lipoproteins may occur as a result of impaired function of the degradation and synthetic system (possibly in the liver) involved in the metabolism of these molecules* C. Effects of Dietary Factors Diet exerts an enormous influence upon the extent of CCl^ injury* Barrett, Best, MacLean and Ridout (73) demonstrated that rats maintained on a diet low in lipo­ tropic factors or choline developed very fatty livers during the 20-day period after the administration of CClj^, whereas animals treated similarly but given a liberal supplement of choline had almost normal livers suggesting that choline or other lipotropic factors are essential for the removal of the excess fat caused by CClj^ poisoning. Post e_t al (7U-)» found that growth inhibition and lipodosis of the liver caused by CCl^ injections in rats was essentially the same whether the amounts of yeast fed were grossly inadequate, or adequate. However when large amounts of brewers yeast were added to the basal diet the harmful effects of CGlj^ were considerably moderated. The amount of food consumed 16 also influenced the severity of the cirrhosis. 8 -1 1 Rats fed g. of food daily had more severe liver lesions than those fed II4. g. daily. Bollman (75>* 76) studying the in­ fluence of dietary factors on the resistance of rats to CC1|^ found that a diet containing 6 percent protein, 79 percent carbohydrate, 1 3 percent fat and 2 percent salt mixture gave the best survival time. In diets of the same caloric Intake replacement of the fat by either protein or carbohydrate was found to afford protection to the liver. Drill and Loomis (77» 78) demonstrated that supplements of methionine decreased the degree of liver damage produced by hepatic toxins in protein-depleted animals, but not in animals receiving a normal amount of protein. Later to­ gether with Belford, they studied the effect of protein and carbohydrate intake on liver injury by CCl^ and by bromsulfalein-retention tests found a greater degree of hepatic dysfunction in animals fed the normal protein diet than those receiving a diet low in protein and high in carbo­ hydrate. The incidence of necrosis was also much less in the latter group. Campbell and Kosterlitz (79) also found the least damage in rats on a protein-free and carbohydratehigh diet. However, Hoffbauer (80) did not get good results from either a high protein or a high carbohydrate diet# D. Protective and Therapeutic Means Minot (81) observed that dogs fed well-balanced diets showed no outward signs of intoxication even with large doses of CCl^, but those on low-calcium diets frequently 17 died with, convulsions when given similar doses* The symptoms were relieved by repeated Intravenous injections of CaCl2 * Ravina (82) also found treatment with Ca (CaCl2) to be successful in CCl^ intoxication. Forbes, Leach and Williams (83) showed that administration of sulfanilamide to rats retarded development of liver cirrhosis from chronic CCl^ poisoning, and that para-aminobenzoic acid did not decrease the protective action of this drug in acute CCl^ poisoning# Miklos Szabo (81j.) observed that the protective effect of glucose against liver damage by CClj^ was considerably enhanced when thiamine was administered in liberal amounts* Dillard, Spence, and Forbes found (8fp) that the admin­ istration of large amounts of either glucose or sucrose prior to anesthesia with CCl]^ exerted no inhibitory effect on the development of fatty livers. However, administration in sufficient amounts after anesthesia greatly reduced or completely inhibited the fatty infiltration. The degree of liver necrosis was, on the whole, not effected by the various supplements employed. The authors suggested that carbohydrate administration through inhibition of fatty infiltration of the liver may enable that organ to regen­ erate when otherwise it would not be possible. De Carvalho Lima and Koch-Weser (86) found that complete protection was afforded by 100 g. of sodium xanthate, but the loss of body weight was accentuated. Aschkenasy and Rolland (8?) obtained no significant protective action from the admin­ istration of methionine or thiamine or both in chronic and 18 aouta poisoning of* adult rats with CCl^, although earlier workers, Beattie et (88) clained their treatment with methionine was successful. Drill and Loomis (8 9 ) also round methionine supplements not to be beneficial to dogs with liver injury produced by acute or chronic CCljj. poisoning# Leites and Yakusheva (90) tried the effect of pancreatic lipotropic factor in both guinea pigs and rats# They found no improvement in guinea pigs, but obtained a drop of 20 percent of liver lipids in rats with daily administration of 0.3-0.5 g* of a preparation from pancreatic tissue, and both cholesterol and glycogen decreased slightly. Inositol and insulin did not seem to have a similar effect# De Dominicis (91) found nicotinamide lowered the mortality rate by increasing the resistance to some phases of CCl^ liver damage in guinea pigs# Hove (92) observed that D-e(- tocopherol gave excellent protection against CCl^ toxicity in rats on a 10 percent casein-vitamin E-free diet# Nevertheless DI Bella (93) did not notice any difference In the toxic effect of CClj^ between the «<-tocopherol-Injected guinea pigs and the controls# that l5/*-g« vitamin Per Papper ejt al (94, 95) showed 6* body weight of rat given prior to administration of CCl^ ameliorated the effect of this hepatoxin# The liver weight was less, the quantity of fat deposited in the liver was decreased, and histo­ logic changes in the liver were also modified. analysis, however, did not show any difference. Chemical Also It was not effective when its administration followed that of 19 toxic compound. The authors were of the opinion that the vitamin is active through mechanisms whereby it prevents accumulation of fat in liver cells and retards the necrotic process* E* Mechanism of Action by Carbon Tetrachloride Opie (96) postulated that CClj^ acts by lowering the osmotic pressure of the cells of liver and of kidney to the level of the medium that surrounds them, but is restored when recovery from the injury occurs* Hirade and Minomiya (97) believed that the toxic action is due to its combination with sulfhydryl groups of protein molecules, especially enzyme proteins* EXPERIMENTAL PROCEDURE I . General The animals used for the study of the effects of age and tumor growth were obtained from the Rodent Laboratory, Department of Zoology, and those for the dietary factors and the CCl^-induced liver injury were from the Vitamin Assay Laboratory, Department of Chemistry, Studies of the cholesterol content of blood, liver, heart and kidney were made in aged rats (5 - 3 7 months old), and 11 tumor rats (H 4.-2 I4. months old). Similar analyses were performed in young rats fed 6 different rations: stock, a diet of good nutritive value, a diet of somewhat lower value, two choline deficient diets, and a rachitogenic diet, and in animals with CClj^-induced liver injury either by periodic exposure to CCl^ vapors or by intraperitoneal injection of a solution of the compound in mineral oil. Nitrogen, moisture and total fat in the liver and the esterified fatty acids in the plasma were also determined* 21 II. Composition of the Diets Employed Stock Diet Yellow c o m meal (ground) Ground wheat Milk powder (whole) Linseed oil meal Alfalfa Brewers yeast Iodized salt Rico Diet* 32.5 2 5 .0 22.5 10.0 6.0 3.0 1.0 Diet I Sucrose Yeast Casein Pat (lard) Milk powder Alfalfa Salt mixture Iodized salt 66*0 30.0 3.0 1.0 Diet II 59.5 6.0 10.0 5*0 i5.o 2.0 2.0 0.5 Low Choline Diet I Casein Lard Yeast Sucrose Salt mixture Codliver oil Pine rice Milk powder (whole) Alfalfa Iodized salt Sucrose Yeast Casein Fat (lard) Milk powder Alfalfa Salt mixture Iodized salt 72.5 3.0 10.0 5.0 5.0 2.0 2.0 0.5 Low Choline Diet II 12.0 10.0 6.0 66.0 1+.0 2.0 Casein Lard Yeast Sucrose Salt mixture Codliver oil 12.0 20.0 6.0 56.0 4.0 2.0 Rachitogenic Diet Cornmeal (yellow table meal) Wheat gluten Yeast Casein C aCO o NaCl 69.0 20.0 l+.O 3.0 3.0 1.0 * The old and the tumor rats -jere fed this ration. III. Administration of Carbon Tetrachloride Carbon tetrachloride was administered in two different ways. By the first method about 1 ml. CClj^ was introduced into a glass jar of Lj. liter capacity, and the rat was then 22 put Into til© jar until it showed signs of nervous strain and Intention to fight off the vapor. This usually took 2-lj. minutes, and the exposure was repeated 3 times a week uritil the experiment was terminated. By the second method different concentrations of CClj^ in mineral oil (0,033-0.132 ml. CCl^ per 100 g. body weight) were injected Intraperitone&lly twice weekly for two weeks. IV. Method of Obtaining Blood and Tissues The old and tumor rats were generally sacrificed within a day or two after they were received from the Rodent Laboratory. Definite periods of feeding had to be allowed for the study of the effect of various diets and of carbon tetrachloride injury. In order to avoid the influ­ ence of the absorption of food all the animals were fasted 1 2 - 1 6 hours before they were anesthetized with ethyl ether. Plasma was obtained by centrifuging oxalated blood drawn from heart puncture (lithium oxalate being used as the anticoagulant)• The liver, heart, and kidney were removed and weighed Immediately. They were then macerated Into uniformly fine particles In a Waring blender and made to definite volumes with distilled water. was extracted. An aliquot of each The details of extraction and analysis are given below. V. Analytical Methods A. Water Content of the Liver A portion of liver was weighed and dried at 50-5>5° C. 23 under reduced pressure until constant weight was obtained. The loss in weight was calculated as percent moisture in the liver. B. Nitrogen Content of the Liver Nitrogen was determined according to the semi-micro Kjeldahl method described by Clark (98), except that H 2 O2 and KOH were used instead of ethyl alcohol and NaOH respec­ tively. One milliliter of the blended liver suspension was digested for 3 hours with 0.75 g« of a mixture of HgO and K 2 S0^ ( 8 g. HgO and 100 g. anhydrous K^SOj^) and 1.5 ml. of concentrated H 2 S0 ^. The digest was then cooled, 2 drops of 30 percent H 2 O2 added, and the mixture again heated until it became clear and colorless. For distillation a $6 percent solution of KOH containing 5 percent Na2 S2(>3 (crystalline) was used. The condensate was collected in a flask contain­ ing 5 ml. of I4. percent boric acid. Titration was carried out with 0.02 M. HC1 using a mixture of methylene blue, 0 . 0 5 percent, and methyl red, 0 . 1 percent, as indicator. C. Total Fat in the Liver Dried liver from the moisture determination was ground and weighed in a thimble for Soxhlet extraction using an­ hydrous ethyl ether as the solvent. After 12 hours* ex­ traction the solvent was evaporated off and the residue dried at 50-55° C. under reduced pressure until constant weight was reached. The weight of the r esidue was taken as the total fat in the liver. 2b D* Cholesterol In Blood and Tissues Extraction of cholesterol from blood and tissues was accomplished by adding slowly a definite volume of plasma (usually 2 ml*) or an aliquot of the blended tissue sus­ pension (5 ml. In most cases) to approximately 30 m l • of a 1-1 mixture of ethanol and acetone while the flask was being rotated* A fine precipitate of protein resulted, and the flask containing both the precipitate and the extract was then brought to boiling on a water bath to insure complete extraction* After cooling to room temper­ ature this was made to a definite volume, usually £0 ml*, and aliquots were used for the determination of cholesterol and the esterified fatty acid* The latter was determined only for blood plasma* Cholesterol was determined according to Sperry’s revised method (99)* For the estimation of free cholesterol the alcohol-acetone extract was used directly, whereas hydrolysis with 50 percent aqueous KOH at l+.2° C* for 30 minutes followed by acidification with 10 percent acetic acid was the first step in the determination of the total cholesterol* Precipitation with digitonin was carried out in slightly acidic solution by the addition of a drop of 10 percent acetic acid and 2 m l • of a 0*5 percent digitonin solution in 50 percent alcohol* After standing 10-12 hours to assure complete precipitation the precipitate was centri­ fuged, washed once with ether-acetone mixture and twice with ether, and dried at 110-115° C* for 1 hour* The dried 25 digitonide was then dissolved In glacial acetic acid and the Lieberraann-Burchard reaction applied for the colorimetric estimation of cholesterol. A mixture of ice-cooled acetic anhydride-concentrated H 2S0^ (20-1) was added for the pro­ duction of the bluish-green color which was allowed to develop in darkness at a constant temperature of 25° C. Thirty minutes later the transmission was read in a photelometer supplied with a red filter. The color of a standard cholesterol solution was developed every time a series of determinations was made* The content of cholesterol was read from a standard curve prepared by the use of standard cholesterol solutions. E. Esterified Fatty Acids in Blood Plasma The esterified fatty acid in blood plasma was deter­ mined according to the method of Bauer and Hirsch (100), the principle of which is based upon the conversion of fatty acid esters into the corresponding hydroxamic acids by hydroxylamine hydrochloride and NaOH, and their sub­ sequent conversion into colored ferric salts. The reactions may be represented by the following equations: RCOORi + NH2 0H —■» RC0NH0H 3 RC0NH0H -f Fe( 0 1 0 ^ ) 3 ----- > 3 HCIO^ + R ^H FefRCONHO)^ An aliquot (usually 5 ml.) of the alcohol-acetone extract of the blood plasma was evaporated to dryness at 60° C.; 15 ml* freshly redistilled ethyl ether was added, followed 26 by 0*3 ml* of 2*5 percent NaOH In 95 percent alcohol and 0*3 ml* °f NHgOH^HCl (2*5 percent In 95 percent alcohol)* The contents were thoroughly mixed after the addition of each reagent* The solution was then evaporated just to dryness at 60° C* Color was developed by the addition of a solution of ferric perchlorate in 95 percent alcohol which was prepared one hour before use by a 1-20 dilution with 95 percent alcohol of a stock solution containing 2 g* ferric perchlorate, 20 ml* 71 percent perchloric acid and 10 ml* distilled water* After 20 minutes the transmission was read in a photelometer with a green filter. The amount of fatty acid esters was read from a standard curve which was prepared using tristearin as the standard* RESULTS Table 1 presents the data obtained from analyzing aged animals. The age ranged from 5 to 36.5 months and the distribution of the two sexe3 was about equal. In the liver the moisture content ranged from 6 i^ . 5 to 73*8 percent, total fat from 5 . 5 9 to 1 5 . 7 g* P«** g* nitrogen, and the total cholesterol from 2 . 3 3 to I4..6 2 mg. per g. of wet tissue, of which the cholesterol esters made up I4.O. 2 per­ cent for the male and 2 6 . 6 percent for the female as shown in the detailed data in the appendix. In the blood plasma total cholesterol varied from Ul|-.2 to 1 0 2 mg. per 1 0 0 ml. Free cholesterol averaged 25*4 percent for the male and 28.14. percent for the female. The esterified fatty acid in the blood had a wide range, 0 . 1 5 to 1 1 . 5 milliequivalents per liter. The cholesterol content of heart and kid­ neys ranged from 0 . 9 9 to i+..50 and 3 * 2 9 to 7 * 1 3 nig. per jrtm of the wet tissue respectively. There seemed to be little change due to age and sex except that the esterified cholesterol content of the liver of the female rats was lower than that of males. The results obtained from studying 11 tumor rats are assembled in Table 2 in the order of increasing size of the tumor. The liver moisture ranged from 67.2-72.0 per­ cent, total fat 5 .7 3 -1 6 . 3 g. per g. nitrogen, and cholesterol H VO O' . vn ro F ro ro CD . vn OJ OJ ro ro ro OJ ro o -0 ro . ro cn -0 o . ro O' 'O H o H ->i . CD . OJ H -0 F . to « H H vn ro O' h • O O' CD ->i ro . -J ro Oj . o ro ON O' O' nO -0 o • • -0 'jJ F v0 CD ro ro OJ o vn H H Number of animals 3 3 3 3! 3 Sex oj ro o H H vn Age in month* o Oj CD -o H H ♦ • H ro vn •.P- -•p- •ON vn . NO vn o> ro ro O) O' CD • . O • • CD -O -0 CD H H O -r f h ro -r v o • • OJ * H OJ . O' CD CD vn ->) ro -0 -0 CD o o o H n0 OJ . F -0 Oj • ro . CD o o “0 OJ F O • O' OJ v0 . -0 OJ H • o . NO CD CD -F" s0 vD • O' o oa • vn n0 vO . o • CD CD . vn • -O OJ vn vn o o O -o O' Oj • OJ oj -r F ro o F . ro s O o o o CD • O' o • CD Oj o VJ\ o OJ H ON . -.P- .O' O' h ro . O i v0 O' 'O . O' Oj H 00 . • « ro O' ro OJ 0D CD h ro vn OJ oo ro .vn F ro « o ro O OJ On ro vn ro -o VA H h ro M ro o HH oro •• ro ro ro O' -J -j F CD ■po H -0 o ro A> v0 • -0 hj o vno i -0 . m CD ro vn h H ro ro -F"OJ i vn f ro • to o . o o . o vn O' -j H OJ . vn H vn ro n0 o> . H • >o ro -P" Oo H . H Percent moisture in liver O O' F Total fat in liver g./g. nitrogen ro OJ O' H . s0 . s0 vn •P" Liver cholesterol mg. per gram O' O' o O' ro Blood cholesterol mg./lOO ml. plasma H CD O' . vn o Oj . vn ro O' O' H • H . . o o H . Oj Oj ro o . H vn • . OJ . O' ro s0 ro . H ro ON -0 F vn -j F . F H vn on F F f• • • • • CD ■F* F H -J -F" O o O' on ro o F F . • CD -0 OJ . o> n0 CD OJ o ro Body weight in grama (average) . o on cn • o o O' CD vn O' • o> H on . CD H H . ro on OJ -0 . -0 -0 • vn . F O' VJT . O' OJ v0 . ■F- vn H Blood esterified fatty acids m.eq./liter Heart cholesterol mg. per grim Kidney cholesterol mg. per grim o ^ W o t* OO OV tJ Q f-H > » 2! ►aa % CO >w 25* t) o W25 H Uo 2! W to O W H CD 25 W >w QO « o a o > 25 *-3f 3 Mg ► 3 ► 3 > t-* PJ H ro 03 H vO ro 03 -0 -f=~ ro ro ro K ro ro H ro H . • vn O . 0 H F~ . H 00 •P" -F- vn oj IO OJ ro vn ro 00 vO H vn O ro ro p- vO To F" O' vn pF* . Oj nO 0 -0 ro . vn 0 O' . O' —0 . •'J CD 0 . 0 H H H H O H O . O' . to Oj ro O' . vO vO . £r O' 00 . H -J 0 . 0 vn vn 00 . . . -j O' p- OJ VA) O' O . . 00 OJ . Oo 0 H H 0 -0 Oj O Oj . O O H H vn O V>J H CD ro . -«0 SO H . OJ H -P* . ■p. -T . vn 03 0 ro . ro ■P" CD . H O' -r . VJO 03 O' vO ro • v£> p- Oj . O -0 -0 03 O ro . H . . VjJ O vn . 0 H . Oj H . 10 . . H vn ■P ro F" vn . vn vn O' p• O' 0 . 0 . ro H 03 0 O' OJ 10 . ro . . ro . vn O' sO -J -Cr O' H 03 O' O' . percent moisture in liver .H Total fat in liver g./g. nitrogen 03 03 Liver cholesterol mg, per grain >0 O' . o Blood cholesterol mg./lOO ml. plasma IV) . •r Blood esterified fatty acids m.eq./liter . o Heart eh''sterol mg. per gravn .ro 03 vn vn Oj w ►3 ro . vO OJ aw > O . o -j I .64 H . O' . 00 vn . OJ . O . vn J=r . H . . p. vO v£> 03 • 0 O' O' H OJ O' O vn O' H O' 03 H • O' H H Tumor weight in grams ~sl . fO OJ . 3.34 H ro Body weight in grams OJ 6.13 H e H -<0 1.29 F" ■r . . O' ro •T sO 16.5 ro F" . V0 Age in months VJl 3.1+482.2 . ro vO . Animal number Sex 7.38 . ro 0 . 0 . p- . ro H vn . O Oj vO Ul 1.98 . XT O' OJ OJ fo OJ 12.0 00 -j ■^1 . H vn • ■^1 0 . ro Oj oj 3.90 78.1 OJ . OJ IO vn 0 ro 03 . vn 11.3 . 67.5 . 33.6 H vD 9.78 vn H 68.4 vn • vn 35.7 O O • O ro 284 ro Oj Oj Oj 0 287 P 17.0 ro Oj 0 264 cr O' ro P 16.5 Vo OJ ro 328 -3 ro Oj 03 OJ oj Kidney cholesterol Tumor ch^esterol mg. per grAm tr* ro ro mg.per ,gra»n ro vO 30 2 *1 0 -ij.* 3 6 mg. perc£ri?»df wet tissue with an average of 23.6 percent for the ester form. The blood cholesterol (total) ranged from 58.lj.-l67 mg. percent, of which 29.3 percent was in the free form. The esterified fatty acid also varied greatly, 2 .0 -1 8 . 3 milliequivalents per liter. The cholesterol content of heart and kidney ranged from 1 .0 5 to 2 .21+ and 14..60 to 7 * 3 8 mg. per grd*» respectively . The tumor tissue showed a cholesterol content of 0.85-3*3l+ mg. per< being higher than that of heart and lower than that of kidney tissue. It approximated that of the liver. Table 3 shows the effect of six different diets. In a comparison of the stock, experimental diets I and II there seemed to be little or no difference in the liver cholesterol, blood esterified fatty acids, and heart cholesterol. However in the animals fed the poorer diet the liver showed a slightly lower moisture content, being 6 5 . 5 percent as compared to 6 7 * 6 and 6 7 * 8 percent for the stock and experimental diet I respectively. The total fat was 21.5 g« per* g. nitrogen as compared to 1 2 .14- and 15«9 g. per g. nitrogen respectively. The blood cholesterol was 8 6 .14. mg. per 1 0 0 ml. as compared to 6 6 . 2 and 8 0 . 5 mg. Animals on the stock diet seemed to have a higher kidney cholesterol content (5 « 9 8 mg. per than the others (lj. . 0 8 and 14..614- mg. per The low choline diet II seemed to exert more influence than the low choline diet I. On a 10—day period the liver moisture content was lowered to 62.2 by diet CII whereas 31 TABLE 3 Blood cholesterol mg./lOO ml. plasma Total fat in liver g./g. nitrogen (wks. or da) Final wt. (ave) g. Initial wt. (ave) g. 11 EFFECT OF DIET ON CHOLESTEROL CONTENT OF BLOOD, HEART, AND KIDNEY IN YOUNG RATS H O U © 5 67.6 15.9 2.38 80.5 5.5 1.89 4.08 3 48 139 II 5 65.5 21.5 2.69 86. 4 5.8 2.05 4.64 esterol I r© -< u 0 00 C Ut CL © ^ t£) ■H £ Heart mg. per Blood esterified fatty acids m.eq./liter 136 Percent moisture in liver 48 Duration 4 Number or animals 4 rH O u © -«p £ p . r©H i OO O h u 0 © CL u *-p © . © > to •H • H6 Q l-J (a) Comparison of stock, experimental diets I and II 67.8 12.4 2.18 66.2 S 3.6 48 152 5 1.13 5.98 (b) Low choline effect and experimental diets I and II 4 34 45 Cl 10 d 68.6 18.4 1.72 86.2 5.62 1.42 3.56 4 35 55 CII 10 d 62.2 29.0 2.25 75.8 7.62 1.28 5.39 4 34 125 Cl I 10 d 3 w 69.0 26.0 4.08 85.0 9.75 1.90 6.44 4 34 98 Cl II 10 d 4 w 65.3 27.2 7.53 65.8 14.5 1.85 4.74 1*. 36 142 CII I 10 d 3 w 58.4 33.4 6.49 53.0 12.0 2.62 6.15 3 123 CII II 10 d 4 w 48.7 66.1 6.24 92.5 10.0 1.46 5.13 35 (c) Rachitogenic diet 4 50 119 s 3 69.5 13.0 2.74 76.0 7.20 1.84 6.29 8 48 77 R 3 70.0 13.5 2.05 79.0 6.90 1.86 5.59 «■ S - stock diet I - experimental diet I II - experimental diet H Cl - low choline diet I CII - low choline diet II R rachitogenic diet 32 little change was observed in animals fed diet Cl* The total fat content of the liver showed similar trends* values increased from I2*J*-29.0 g. per g* nitrogen being found with CII, and from -18.0 g. per g* nitrogen with Cl# The esterified cholesterol in liver was greatly increased for both groups of animals fbd diets Cl and CII, averaging 7 1 * 6 percent as shown in the detailed data in the appendix* When the animals which had been fed Cl and CII for 10 days were transferred to experimental diets I and II marked differences in moisture, total fat and total choles­ terol (mostly in the ester fraction) in the liver and ester­ ified fatty acids in the blood were observed* For animals fed CII previously the moisture content was decreased to ij.8#7 percent with diet II and to 5>8*1± percent with diet I* The fat content increased to 33*1* and 66*1 g* Der g* nitro­ gen and liver cholesterol 6 *i+9 end. 6 *2 lj. mg* per grdjn or diets I and II* The blood cholesterol was 92*5 mg* percent In animals fed diet II and 53*0 mg* percent In those fed diet I whereas the esterified fatty acid Increased to 10*0 and 12*0 milliequivalents per liter respectively* There seemed to be little difference in those animals previously fed Cl and changed to diet I and II# Only the total fat in liver Increased to 26*0 and 2 7 * 2 g* per g* nitrogen and the esterified fatty acids of the blood Increased to 9*75 and 1 4*5 milliequivalents per liter respectively. A comparison of results obtained from the stock and the rachitogenic diets showed very little difference in the liver constituents and the cholesterol levels of blood and 33 tissues analyzed* The data obtained from exposing young rats to carbon tetrachloride vapors are presented in Table l\.m Prolonged treatment of 11 weeks showed a decrease in liver moisture both with diets I and IT* Total fat was influenced more with diet II than diet I, being 28*14 and 2 I4..7 g* per g* nitrogen respectively whereas the stock diet had very little effect* Prom the detailed data shown in Table XIII in the appendix there seemed to be quite an individual difference in liver cholesterol* One animal each on diet I and II had very high cholesterol contents* 26*7 and 11.1 mg* pfr-grajnrespectively (mostly in ester form), and one animal each had only 8*6£ and 5*32 mg* per graanrespectively* The rest did not differ too much from those on the same diets without CCl^ treatment* Blood cholesterol was In­ creased more by the poorer diet than by the better one. The value for the former was 94*0 and 72*1 mg* per 100 ml* the latter, Kidney cholesterol was also higher In those fed diet II* Intraperltoneal Injection of CCl^ was employed in order to achieve more uniform dosage and the results ob­ tained are shown in Table Here again the total fat in liver increased, especially when the dosage was increased and the animals were fed the poorer diet* cholesterol showed a similar trend* The liver Increasing the dosage beyond the level 0*066 ml* per 100 g* body weight seemed to have little additional effect other than to raise the TABLE 4 EFFECT OF CCLj.-INDUCED LIVER INJURY ON CHOLESTEROL CONTENT OF BLOOD7 LIVER, HEART, AND KIDNEY OF YOUNG RATS (Administered by exposing to CCl^ vapors three times a week Tor 11 weeks) * bO — . tt *rl q . • © > © — .. • P n 0 g © X> •H P •H G * U) x ^ • • G 3 > at - • P t* rH G © > •H G l-l © bO •p « •H O e g © -p > G -H © rH O G G © *H G O •H G p -P «H at ( j Vh • r-t O G • P © © 1 — C E P G oe xi u 0 « G p * © • ► bO •H £ rH O G © -P m © e © © rH CL • O XJ O O O 1 —1 Ti \ O • O bO rH £ q b. P • Q 7 239 s 7 0 .1 13.6 3.36 61.2 14- 257 I 60*7 2^.7 10.£0 2^5 II 6 1 .6 2 8 .I4. 6.73 8 3 sc 3 n 56 at g -P • O bO E-» •O © •rl p •H H G G tJ © © • H P P O -H © © rH • \ >> • xt rH O G © 4- •H O G ® - p © JE E < 1 2. 00 m c Xi r-4 O £ O 0 e cr* p G O P © 0 © • rH 8 p . • © bO © £ OO G © p. S ® e w •H 5.91 1.27 5.22 72.1 1 2 *14.0 1*28 3.Q8 9 I4..O 8.70 1.1? 7.59 CQ CQ mortality rate which was 6 0 percent in the case of 0 * 1 3 2 ml* per 100 g* body weight* deal* Blood cholesterol varied a great No definite trend could however be observed* The kidney cholesterol Increased quite rapidly at the lowest level 0 * 0 3 3 ml* per 1 0 0 g*, then dropped to a normal value as the dosage of CCl^ was further increased* Finally at 0 * 1 3 2 ml* per 1 0 0 g* body weight it increased again* No change was observed in the heart cholesterol and in the esterified fatty acid of the blood plasma* TABLE 5 effect of OF c c l ^- i n d u c e d l i v e r injury on the blood, liver, heart, and kidney cholesterol of young content rats (Administered by intraperitoneal injection twice weekly for two weeks) •0 43 a u to n 3 -—» • © > « rl s H o • -p 5* © Q 6 3 u 60 r— '» • • > at • 43 :* u •H 43 •H *3 G id ____ ... •a •H © •rH £ l._ , A o •H -P at FH ■p CJ © o a o o U © • o x> 3 43 01 • H> O O rH \ -d h • rH O ■rH 43 © •H ,_Q o E ^ © 43 > G *H © rH o ?h g © H > ■H G rH © 60 g o •H t i 43 43 vH © td Vh • rH O Fh © — 43 ft O' »—i a o o - X~ u o © CL. FH © • 43 • > to •rl ^ O 60 __ Qt____ __fcl_____ ..-k3________ « E rH « 2 5 © a. 43 n • rH O © © •rl g •rl ft "C « 5o * ■a o O o i—i o • O 60 rH B in © «H 43 43 O *H m © rl © _ * © 43 o* O 43 © O © • rH 5-1 6 ___CQ________ « 43 E «i © u rH ©O C XX fc. o © p. 43 16 © © © B rH O U © 43 r ? r f. Jt Hi 2 oo O >4 © s ex © G • Tt 60 •H E __ Hi. 2 128 152 .033 S 66,9 1 6 .1 4.92 8 0 .4 7.6 2.23 6 .3 8 2 122 150 .033 I 68,9 13.5 3.49 1 0 4 .0 10.4 2.25 6.38 1 120 139 .033 II 64.2 28.2 6.67 101.0 7.5 2.76 6.55 2 113 128 .066 S 7 0 .0 1 6 .2 2.46 68.5 5.5 1.30 5.87 4 108 118 .066 I 65.9 18.7 3.71 7 2 .1 9.5 1.39 5.55 3 101*. 108 .066 II 60.9 30.2 4.57 85.0 3.8 1.25 4.50 1 90 117 .099 I 66.5 2 4 .8 5.oo 5 0 .0 4.0 1.66 4*62 2 95 110 .099 II 64. 6 27.3 4.97 76.5 4.6 1.49 4.71 1 121 118 .132* I 65.6 2 4 .1 6.55 69.2 2.0 1.74 8.12 1 82 *97 .132 II 69.5 22.5 6.00 164.0 7.2 1.63 6 .4 2 •K*Mortality was 6 0 percent on tills level DISCUSSION The results obtained from studying old animals demon­ strated that generally there is little or no influence of age and sex on the blood cholesterol, liver fat, and total cholesterol in the liver, heart, and kidney. However, the ester cholesterol in the liver is lower in the female than in the male, being 26.6 percent and Lt-0*2 percent respective­ ly. This agrees well with the findings of Okey e_b al (101)* The cholesterol esters of the liver in the female rat may be more mobilizable than in the male due perhaps to the synthesis of sex hormones required during each estrus cycle* Mounier e>t al (15) found similar values for blood cholesterol in both young and old rats. In the present study the variation was large, 1+lj..2-102 mg. percent, although no definite increase or decrease due to age was observed. Two female animals, 2 3 -214. months, had blood cholesterol values as high as 100 and 101+. mg. percent. Probably in the rat there is also a constitutional level characteristic for each animal as suggested by Sperry (8) for human beings. Some rats might be influenced by age much more than others, thus contributing to the wide range observed. The oldest rats of this group, 28-36.5 months of age, showed values of 57.0-90*0 mg. percent. Keys eti al (12) reported similar findings from an investigation made on human 37 subjects living in Minnesota* The results of the study of 11 rats with spontaneous mammary tumors showed that the blood cholesterol was gen­ erally high althougjh the variations were considerable. The blood cholesterol of rats No. 327 and No. 302 was 138 and 167 rag. percent respectively. They were the highest values observed in this investigation. Rats No. 2 3 8 and No. 338 had comparatively large tumors. Their blood cholesterol was 65>.0 and 5>8.i|. mg. percent respectively. ations might be offered. Two explan­ These two rats probably had not been eating well due to their poor physical conditions caused by the advanced stage of the tumor growth, or they might belong to the class that has low blood cholesterol by constitution. The esterified fatty acids in the blood generally increased with the blood cholesterol. The differences in results witn stock ration, diet I and diet II were observed only in the fat content of the liver and the blood cholesterol. The liver fat was appre­ ciably higher with the poorer diet. Prom the composition of the two experimental diets it is clear that they differ only in the amount of yeast and milk powder. Yeast com­ prised 3 percent of the former and 6 percent of the latter whereas the content of milk powder was % percent and 1$ per­ cent respectively. A diet comparatively low in vitamin B complex supplied chiefly by the yeast, and low in protein might be expected to lead to an Increase in the fat content the liver. Diet XI was chosen for study because in human dietaries deficiencies of a multiple nature rather than single deficiencies are encountered and they commonly involve the B complex vitamins and protein. Probably the combination of slight deficiency in the B complex and protein also results in a disturbance in the mechanism of maintain­ ing the usual blood cholesterol level. The influence of the low choline diets manifested itself in a higher fat content, a lower percentage of moisture and an increased ester cholesterol content of the liver. The blood cholesterol was normal, A comparison of the two choline diets showed that low choline diet II produced more severe symptoms, higher fat and ester cholesterol contents and lower percentage of moisture in the liver. The dif­ ference in the composition of the two diets was chiefly in the percentage of fat, being twice as high with the CII (20 percent) as with the Cl diet (10 percent). This con­ firms that choline deficiency is produced more easily with a high fat diet, other constituents being the same. In the study of influence of diets I and II on rats previously maintained on the choline diets several differ­ ences were noted. Only small differences were observed in effect of diets I and II on animals previously fed Cl. However with those previously fed CII the feeding of diet II resulted in a lower moisture and higher fat content in the liver and also in higher blood cholesterol values. This would indicate that diet CII had produced more severe changes . than diet Cl. It also follows that the deviation from the normal was much greater in the case of diet II than with diot I as indicated by the fact that the liver fat was approximately twice as high. The experiment with rachitic animals showed that there was no apparent effect on the cholesterol metabolism because all of the tissues studied were of essentially normal cholesterol content. It is quite likely that in view of the characteristically low consumption of food no diffi­ culties involving cholesterol and fat metabolism would be encountered. The 11-week period of exposure to CCl^ vapors of young rats resulted in a slightly less degree of fatty liver on the diet I. This is probably due to the greater intake of food in this group of animals. Those on stock diet showed even less degree of fatty liver. Post at al (7U-) found that rats fed larger amounts of food developed less severe cirrhosis by CCl^ poisoning. Probably the rats fed the stock diet and diet I ate more nutritious food and there­ fore were in better condition to resist and to recover from the exposure to CClj^* On the whole the condition of the rats was not made worse by prolonged exposure. It Is quite likely that as in the case of many other toxic substances rats might develop a capacity for making a successful adjust­ ment to the CCl^. Intraperitoneal Injections of CCl^ at different levels showed that the degree of fatty liver produced Increased as the concentration of CCl^ was raised from 0.033 to ko 0*066 ml. per 100 g* body weight* At each of these two levels the fat content of the liver of animals fed diet I was lower than those fed the poorer diet* Beyond the two levels mentioned above no further change was observed except that the blood cholesterol slightly decreased, probably due to the impairment of the appetite and consequent low food intake* With concentration as high as 0*132 ml* per 100 g* body weight, the mortality rate was very high* Tnus it indicates a concentration of 0*066 ml* per 100 g* body weight would be more suitable for the experimental study of liver damage by CCl^ in rats* Prom the results of this study it may be concluded that in the rat the cholesterol content in the blood, liver, heart, and kidneys does not appear to be much in­ fluenced by age and sex, except that the ester fraction in the liver is lower in the female than that in the male* Rats with spontaneous mammary tumors in general showed a tendency toward higher blood cholesterol values* Diet was observed to markedly affect liver fat, and liver cholesterol, and moderately affect blood cholesterol in the rat* Diet II, somewhat low In B complex and protein, markedly increased the liver fat and raised the blood cholesterol* Choline deficient diets greatly increased the fat and cholesterol content (ester fraction) of the liver. A rachitogenic diet had no influence on the distribution of cholesterol in the tissues studied. Carbon tetrachloride produced fatty liver and increased the liver cholesterol content (ester fraction)* Deviations from the normal were greater in animals fed diet II, probably due largely to the smaller Intake of less nutritious food* SUMMARY The analysis of the cholesterol content of blood, liver, heart, and kidney tissues of i+5 rats from months of age showed no appreciable difference with regard to age and sex, except that the average ester fraction of liver cholesterol in female rats was considerably lower than that in the male* Similar studies with 11 rats with spontaneous mammary tumors showed that the blood cholesterol levels were generally high although considerable variations occurred* A comparison of the effect of the stock diet, experimental diets I and II showed that diet II which contained less B complex and protein produced a moderate degree of fatty liver and slight Increase In blood cholesterol. The feeding of choline deficient diets I and II resulted in fatty livers, decrease In moisture content and in­ crease In liver cholesterol, mainly the ester fraction. No appreciable change was noted In the blood cholesterol, although a decrease has been reported in the literature. Diet II containing the larger amount of fat produced the more severe symptoms. When rats previously maintained on choline deficient diet I were fed experimental diets I and II relatively small differences were observed in the fat content of the liver* With choline deficient diet II differences occurred in which the deviation from the normal was markedly greater with the diet of lower nutritional value* Rats fed a rachitogenic diet showed a normal cholesterol distribution In the tissues studied* Liver injury induced by periodic exposure to CCl^ vapors resulted in fatty liver and an increase in liver choles­ terol (ester cholesterol mainly)* The degree of tissue change was influenced by diet being greatest in the case of diet II which was of lower nutritional value* Intraperitoneal injection of CCl^ in mineral oil pro­ duced similar results* A dosage of 0*066 ml. per 100 g* body weight was found most suitable for experimental study* LITERATURE CITED 1* Bloch, K. and D. Rittenberg. On the utilization or acetic acid for cholesterol formation. J. Biol. Chem., 1*45:625-636 (19*4-2) ♦ 2. Borek, E. sand D. Rittenberg. The metabolism of acetone by surviving rat liver. J. Biol. Chem., 179:8 *4.3 -8*45 (19*4-9) • 3. Brady R. 0. and S. Gurin. The biosynthesis of radio­ active fatty acids and cholesterol. J. Biol. Chem., 186:*4.6 1 -*4.69 (1950). 14-. Brady, R. 0. and S. Gurin. The synthesis of radioactive cholesterol and fatty acids in vitro. J. Biol. Chem., 189:371-377 (1951). 5. Shope, R. E. The effect of age on thetotal and com­ bined cholesterol of the blood serum. J. Biol. Chem., 80:1*41-1*4-9 (1928). 6 . Page, I. H., E. Kirk, W. H. Lewis, Jr., W. R. Thompson and D. Van Slyke. Plasma lipids of normal men at dif­ ferent ages. J. Biol. Chem., 111:613-639 (1935)* 7. Turner, K. B. and A. Steiner. A long-term study of the variation of serum cholesterol in man. J. Clin. Invest., 18:*4.5 -14.9 (1939). 8 . Sperry, W. M. The concentration of total cholesterol In the blood serum. J. Biol. Chem., 117:391-395 (1937). 9. Sperry, W. M. and M. Webb. The effect of increasing age on serum cholesterol concentration. J. Biol. Chem., 187:107-110 (1950). 10. Poldes, P. P. and A. J. Murphy. Distribution of cho­ lesterol esters and phospholipid phosphorus in normal blood. proc. Soc. Exp. , Biol, and Med., 62:215-218 (19*4.6) . 11. Gram, M. R. and R. M. Leverton. Interrelation of age serum cholesterol, and basal metabolism of women. Fed. Proc., 8 :38*4 (19*49). 1+5 12. Keys, A., 0* NIckelsen, E. V. 0. Miller, R. R. Hayes and R. L. Todd. The concentration of cholesterol in the blood serum of noimial man and its relation to age* J. Clin. Invest., 29:131*7-1353 (1950). 13* Kountx, W. B., A. Sonnenberg, L. Hofstatter and G. Wolff. Blood cholesterol levels in elderly patients. 1. Relation of age, sex, basal metabolic rate, cardiac decompensation, and coronary and peripheral sclerosis to blood cholesterol levels in the aged. Biol. Symposium, 11:79-86 (191+5). 11*. Kornerup, V. Concentration of cholesterol, total fat, and phospholipid In serum of normal men. Report of a study with special reference to sex, age. and con­ stitutional type. Arch. Internal .Med., 85:398-1+15 (1950). 15. Mounier, M., A. Farchadi and A. Maulbetsch. Blood cholesterol in the rat. Compt. rend. soc. phys. hist, nat. Genene, 58: 21+1+-21+8 (191+1) • 16. Sure, B., M. C. Kik and A. E. Church. The influence of fasting on the concentration of blood lipids In the albino rat. J. Biol. Chem., 103:1+17-1+23 (1933)* 17* Entenman, C., G. W. Changus, G. E. Gibbs and I. L. Chaikoff. The response of lipid metabolism to alter­ ations in nutritional state. 1. The effects of fasting and chronic undernutritlon upon the post absorptive level of the blood lipid. J. Biol. Chem., 131+:59-69 (191+0 ). 18. Schettler, G. Cholesterol and phosphatide in the starving mouse. Arch. ges. Physiol., 251:398-1+03 (191+0). 19. Okey, R. and D. Stewart. Effect of sterol content of the diet upon cyclic variations In blood cholesterol In women. J. Biol. Chem., 97:xxxix (1932). 20. Lea, A. J. A sex difference in blood cholesterol In mice. Endocrinology, 1+2:1+77-1+79 (191+8). 21. Johnson, J. and C. Riegel. The effect of experimental hyperthyroidism and hypothyroidism on the concentration of cholesterol In hepatic bile. Surgery, 5:260-266 (1939). 22. Foldes, F. F. and A. J. Murphy. Distribution of cho­ lesterol, cholesterol esters, and phospholipid In blood In thyroid disease. Proc. Soc. Exp. . Biol, and Med., 62:218-223 (191+6). A 1+6 23m Fleischmann, W. and H. B. Shumacker, Jr. Relationship between serum cholesterol and total body cholesterol in experimental hyper-hvpo-thyroldism. Bull. Johns Hopkins Hosp., 71:175-183 (191+2). 21+. Horllck, L. and L. Havel. The effect of feeding propyl­ thiouracil and cholesterol on the blood cholesterol and arterial intima in the rat. J. Lab. Clin. Med., 33: 1 0 2 9 - 1 0 3 6 (191+8). 25. Bennett, C. B. The cholesterol content of cancers in rats. J. Biol. Chem., 17:13-11+ (1922). 26. Jowett, M. Phosphatide and cholesterol.contents of normal and malignant human tissues. Biochem. J., 25: 1991-1998 (1931). 27. Knudson, A., S. Sturges and W. R. Bryan. Cholesterol content of skin, blood, and tumor tissue in rats irradiated with ultraviolet light. J. Biol. Chem., 128:721-727 (1939). 28. Khaletskaya, F. M. Blood cholesterol in experimental tumors in mice. (Arkh. patol. 191+8, No. 2, 1+0—i+3•) C. A., 1+2:71+19 (191+8). 29. Bloor, W. B. Diet and the blood lipids. II. Effect of occasional overfeeding on the post absorptive level. J. Biol. Chem., 103:699-705 (1933). 30. Schmidt-Thorne, Jr., G. Schettler and H. Goebel. parison of blood cholesterol during 191+2-191+7. 2. physiol. Chem., 283:63-68 (191+8). Com­ 31. Schettler, G. The Influence of nutrition on the cho­ lesterol content of the blood. Klin. Wochschr., 28: 565-566 (1950). 32. Treadwell, C. R. and H. C. Eckstein. Sterol metabolism In young white rats. IV. The effect of high- and lowfat diets on the cholesterol metabolism of four gen­ erations of white rats. J. Biol. Chem., 11+0:35-1+2 (191+1). 33. Alfin-Slater, R. B., M. C. Schotz, F. K. Shimoda and H. J. Deuel, Jr. Incorporation of deuterium into plasma and liver cholesterol of the rat under different dietary conditions. Fed. Proc., 10:155 (1951). 31+. Schettler, G. The cholesterol metabolism of the mouse. I. Effect of various fats and oils, without added cho­ lesterol, on the blood and organ cholesterol content. Biochem. Z., 319:31+9-358 (191+9). hi 35* Schettler, G. Electrolyte and cholesterol deposition* Z. ges. inn* Med*, 5:736-743 (1950). C. A., 45:611 (1951)* 36* Harris, I*, C. E* Vernon, N* Jacob and M. E. Harris* Total serum-fat in hypertony* Lancet, 257:283-285 (1949) • 37 • Lasch, P* lesterol* The effect of vitamin A upon serum cho­ Klin* Wochschr,, 13:1534-1536 (1934). 38* Charller, J*, M* Jenune, F* Simon and P* Alacoque* Relation between vitamin A and blood cholesterol. Compt* rend* soc. biol*, 129:606-608 (1938). 39* Usuni, K* Experimental studies on the influence of diet on the formation of biliary and renal calculi. II* Feeding a diet deficient in fat-soluble vitamins* 1* The quantitative change in K, Na, Ca, Mg, total cholesterol and total fatty acids in the blood* Japan J* Gastroenterology, 2:226-230 (1930)* C* A*, 25:1559 (1930). 140 * Knapp, A* A. and S* N. Blackberg* Ocular pathology in various types of dietary deficiencies with partic­ ular emphasis on arteriolarsclerosis* U* S* Naval Med. Bull., *41:1339-1344 (19*4-3). 41. Best, C, H. and J* H. Ridout* The effects of choles­ terol and choline on liver fat* J. Physiol., 86:343352 (1936). *4-2 * Herrmann, G. R. Some experimental studies In hyper­ cholesterolemia* Exptl. Med. and Surg*, 5 :114-9 - 1 5 9 (1947). *4.3 * Forbes, J. C* Vitamin B complex and fat metabolism* J. Nut., 2 2 :3 5 9 -3 6 *4- (19*41). *4 4 . Gavin, G. and E* VI. McHenry. The effects of biotin upon fat synthesis and metabolism* J. Biol. Chem., 1141:619-625 (1941). 45. Okey, R., R. Pencharz, S. Lepkovsky, E. R. Vernon, D. Jerome and M. Marquette. Dietary constituents which may influence the use of food cholesterol. 1. Eggs: Biotin and avidin* J. Nut., 44:83-99 (1951). 46. Myasmkov, A. L* Influence of certain vitamins on cholesterolemia and the development of experimental atherosclerosis* Klin. Med* U. S. S. R. 28, No* 2, 3-10 (1950 ). c. a*, 44:9024 (1950 ). *4 7 . Dam, H. Ineffectiveness of vitamin E in preventing cholesterol deposition in the aorta. J* Nut., 28: 289-295 (1944). 1*8 U.8 • Marx, W., L. Marx, Deuel* Effects of concentrate and of aorta of the rat* E. R. Meserve, P. Shimoda and H. J. the administration of a vitamin E cholesterol and bile salt on the Arch. Path., L|_7:IjJ+O— (191+9). 1+9. Roffo, A* H* Eggplant in decholesterolization. Bol. Inst* med* expt* estnd. cancer, 2 0 :2 1 2 -581+ (191+3 ). 50* Wilkinson, C* P., Jr*, R. S* Jackson and w. C* Vogel. Effect of feeding dried eggplant (Solanum Melongena) on plasma cholesterol. Proc. Soc. Exptl. Biol. Med., 71:626-658 (191+9)* 5l• Chapman, C. B. and T. B. Gibbons. sion. Medicine, 29:29-69 (1920). Diet and hyperten­ 22. Rodbard, S., C. Bolene and L. N. Katz. Hypercholes­ terolemia and atheromatosis in chicks on a restricted diet containing cholesterol. Circulation, l+:l+3-l+2 (1921)* 53. Okey, R. Cholesterol injury in the guinea pig. Biol. Chem., 126:179-190 (191*1+). J. 21+. Member, S., M. Burger and E. Oppenheim. Experimental arteriosclerosis. VI. Effects of various bile acids on cholesterol levels. Arch. Path., 38:210-211+ (191+1+). 22. Capretti, G. and B. Magnani. Vitamin and cholesterolinduced arteriosclerosis. Giorn. clin. med. (Pauna), 31:989-1001 (1920). C.A., 1+2:31+78 (1921). 26. Bereans, B., J. D. Davidson and L. L. Abell. The early lesions of canine arteriosclerosis. Arch. Path., 2l: 278-297 (1921). 57. Byers, S. 0. and M. Friedman. Hypercholesterolemia in the rat after intravenous administration of cho­ lesterol. J. Biol. Chem., 177:81+1-81+6 (191+9). 28. Byers, S. 0. and M. Friedman. Extreme hypercholester­ olemia following administration of cholic acid to the bile duct ligated rat. Fed. Proc., 10:22 (1921). 29. Dent, F. M. and R. L. Hayes. Hypercholesterolemia as a possible adrenal response to ascorbic acid. Fed. Proc., 10:17 (1921). 60. Robbins, B. H. The absorption, distribution, and excretion of CClj, in dogs under various conditions. J. Pharmacol., 3y :203-216 (1929). 61. Kretchmer, N., K. K. Tsuboi and C. P. Barnum. Succinoxidase studies of the liver cells of mice fed CCl^. Cancer Research, 7:71i+ (191+7). A 49 62. Ellis, S., S. Sanders and 0. Bodansky. Effect of CCli, liver damage in the rabbit and rat on acetyl choline ^ esterase activity. J. Pharmacol* Exptl. Therap., 91: 255-263 (1947). 63. Hiwataski, D*, et al. Amidase activity. I. Liver asparaginase. J. Exptl. Med., I4JL:384-397 (191+1). 61+. Richter, M. L. Effect of administration of CCl^ to rats on certain reactions of the liver in vitro. J. Pharmacol. Exptl. Therap., 102:94-97 (1951). 65* Kurihara, K., Sei-i-kai. The effect of liver poisons on the vitamin C content of rabbit organs. Med. J., 5 6 :No. 6 , 8 6 6 - 8 7 4 (1937) • C. A., 3 1 :8701 +. (1937). 6 6 . Rosin, A. and L. Doljanski. Pyroninophilic structures of liver cells in CClh poisoning. Proc. Soc. Exptl. Biol. Med., 62:62-61+ T l 9 4 6 ) • 67. Valori, P. Glycogen in the liver, muscles, and kidneys of rats and guinea pigs with fatty liver degeneration caused by CCl^,. The relation of glycogen, water, fat, and protein. Sprimentale, 99:550-583 (1948). C. A., 44:4585 (1950). 6 8 . Giumaraes Villela, G and M. S. Mello. pbbephat&se in experimental poisoning. 509-516. Changes in blood 0 Hospital 1949# 69. Derville, P., R. Castagnon and P. Choraerean-Lamotte. Changes in blood cholesterol in rabbits after Ingestion of CCljj.. Compt. rend. soc. biol., 127:61-63 (1938). 70. Chebotarev, R. S. Toxic effects of CCli, of horses. Veterinariya, 9:41 (1947). C. A., 42:980 (1948). 71. de Senarclens, P., Schweiz. Lipoid granulomatosis in liver (of rat). Path. Bakt., 5:150-177 (1942). C. A., 37:5137 (1943). 72. Pierce, P. T. and J. W. Gofman. The effect of CCIk poisoning on serum lipoproteins associated with athero­ sclerosis. Circulation, 4:29-33 (1951). 73. Barrett, H. M., C. H.. Best, L. L. MacLean and J. H. Ridout. The effect of choline on the fatty liver of CCl^ poisoning. J. Physiol., 97:1036 (1939). 74. Post, J., D. P. Earle, Jr., A. T. Patek, Jr., and J. Victor. Effects of yeast and food intake on experi­ mental CCli, cirrhosis of the liver in the rat. Am. J. Path., IB:661-672 (1 9 4 2 ). 50 75* Bollman, J# L. The Influence of dietary factor's on the resistance of* rats to CClh. Collected Papers Mayo Clinic and Foundation, 3 7 :8 1 /4 .-8 1 7 (19/4-6). 76. Bollman, Y. L. Influence of dietary factors on the resistance of rats to CC1,. • Conf. on Liver Injury Trans., 5:11-16 (19 /4.6 ). ^ 77* Drill, V. A. and T. A. Loomis. Effect of methionine supplements on hepatic injury produced by CCli,. Science, 103:199-201 (19/4-6). ^ 78. Drill, V. A., T. A. Loomis and J. Belford. Effect of protein and carbohydrate intake on liver injury by CCI k . Toxicol., 2 9 :1 8 0 -1814- (19/4-7). 79. Campbell, R. M. and H. W. Kosterlitz. The effects of short-term changes In dietary protein in the response of the liver to CCli, injury. Brit. J. Exptl. Path., 29:1/4-9-1/4-9 (19/4-8). ^ 80. Hoffbauer, F. W. Liver injury produced by CClj,. The influence of diet. Conf. on Liver Injury Trans., 5: 21-29 (19/4-6). 8 l ♦ Minot, A. S. in doga. The effect of Ca on the toxicity of CCli, Am. J. Physiol., 81:502 (1927). 82. Ravina, A. Intoxication with CCIk and its treatment with Ca. Presse med., 141:8 0 9 - 8 1 0 (1933)* C. A., 28; 2798 (193/4-) • 93. Forbes, J. C., B. E. Leach and G. Z. Williams. Pro­ tective action of sulfanilamide against liver cirrhosis from chronic poisoning with CCli • Proc. Soc. Exp. . Biol. Med., 51:14-7-/4-8 (19/4-2). ^ 8 /4.. MIklos Szabo. Intravenous administration of glucose and vitamin B-i in protection against liver damage. Z. ges. exptl. Med., 108:3 5 Z4 --3 6 2 (1940) • 85. Dillard, G. H. L., H. Y. Spence and J. C. Forbes. Effect of food on liver and fat of animals following CCl^ poisoning. Virginia Med. Monthly, 7 1 :l5 /4--l58 (1 9 /4/4-)• 86. de Carvalho Lima, L. P. and D. Koch-Weser. Experimental study of certain antitoxic substances counteracting the toxic action of CCli,. Rec. inst. Adolfo Lutz, 3:261271 (1943). C. A., 39:4156 (19/4-5). 9 7 . Aschkenary, A. and G. J. Rolland. Action of methionine and thiamine on blood and liver changes in experimental CCli, poisoning. Compt. rend. soc. biol., 1 4 1 : 1 1 6 3 - 1 1 6 5 (1947). 51 88. Beattie, J., H. P. Herbert, C. Wechtel and C. W. Steele, Studies on Hepatic function. I* CClj, poisoning treated with casein digest and methionine, Brit, Med, J,, 1:209-211 (19144). 89. Drill, V. A, and T, A, Loomis, Methionine therapy in experimental liver injury produced by CClj,. J. Phar­ macol., 90:138-114.9 (1914-7). ^ 90. Leites, S. M. and T. S, Yakucheva, The action of pan­ creatic lipotropic factor in. experimental toxic fatty infiltration of the liver, Arkh. Patol., ll:No. 4> 144-14.8 (1949). C. A., 144:1611 (1950). 91. G-iuseppe De Domonicis, Nicotinic acid and CClj^ in­ toxication. Raso,. ~\in. terap. sci, affini,, 4 8 :1 8 9 1 9 4 (1949). C. A., 45:3945 (1951). 92. Hove, E. L. Interrelation between a-tocopherol and protein metabolism. Ill, The protective effect of vit­ amin E and certain nitrogen compounds against CClj, poisoning in rats. Arch. Biochem., 17:487-474 (1948), 93. Salvatore Di Bella. protective action of a-tocopherol in CCli, poisoning. Boll. soc. ital. biol. spar., 25: 1267 (1949). C. A., 45:253 (1951). 94* Popper, H., D. Koch-Weser and P. B. Szanto. Protective effect of vitamin Bnp on hepatic injury produced by CCIk . Proc. Soc. Exp. Biol. Med., 71:888-690 (1949). 95. Koch-Weser, D., P. B. Szanto, E. Parbir and H. Popper. Further Investigation on the effect of vitamin B 1 2 concentrate upon hepatic injury produced by CClh. J. Lab. Clin. Med., 36:694“704 (1950). 96. Opie, E. L. Effect of injury by toxic agents upon osmotic pressure maintained by cells of liver and of kidney. J. Exp. Med., 91:285-294 (1950). 97. Kirade, J. and A. Ninomlya. The mechanism of the toxic action of organic halogen comoounds. J. Biochem. (Japan), 37:19-34 (1950). C. A., 44:3149 (1950). 98. Clark, P. E. Semi-micro quantitative organic analysis. Academic Press, Inc., New York, (1943). 99. Sperry, W. M. and M. Webb. A revision of the Schoenheimer-Sperry method for cholesterol determination. J. Biol. Chem., 187:97-106 (1950). 100. Bauer, F. C., Jr., and E. P. Hirsch. A new method for the colorimetric determination of the total esterified fatty acids in human sera. Arch. Biochem., 2.0 z 2l\2 - 249 (1949). 52 • Okay, R., H, L. Gillum and E, Yokela, Factors affecting cholesterol deposition in the tissues of rats. 1. Differ­ ences in the liver lipids of males and females, J, Biol, Chem,, 107:207-212 (19314-). APPENDIX EFFECT OF AGE ON LIVER CONSTITUENTS OF MALE RATS % Rat no. Age in mos. Body wt. gms. Liver wt. gms. 138 191 2# 219 5 10 12.5 13.5 383 578 330 356 14.5 19.8 8.59 9.46 3.73 3.42 2.60 2.63 218 14 14 14 14.5 1*22 345 456 10.9 10.9 10.6 8.23 15 15 15 16 432 331* 11.3 222 232 235 254 223 229 230 231 16.5 16.5 16.5 16.5 16.5 486 154 162 163 159 160 20 30 30 33 34 233 220 252 IkS liver in body wt. Percent moisture 69.1 6 6 ,7 N mg./g. wet wt. Total lipids Cholesterol % gm./gm. gra./ Total Free Ester dry wet wt. gm. mg. fa mg./£ % basis N 13.8 20.4 31.6 18.7 .214 .320 .487 .274 6.49 11.0 3.01 2.54 65.9 68,1* 33.0 29.0 32.5 31.5 1 5 .0 8 .7 0 4 .4 2 2.56 3.16 2.33 2.16 70.0 68.9 66.0 67.2 29.7 29.4 33.5 34.9 22.0 26.0 17.2 27.9 .314 .378 .261 .415 70.4 66.7 67.9 68.3 29.8 33.8 28.9 31.7 16.5 14.0 30.8 22.2 .234 1 0 .5 1 1 .7 1 2 .5 2.62 3.15 3.40 3.16 8 .91 9.76 10.4 11.2 10.6 1.84 2.87 3.11 3.15 3.22 6 7 .4 34.3 32.6 32.4 29.1 31.6 23.0 28.5 26.1 25.3 26.5 2 .1 4 68.7 31.4 29.7 28.1* 31.5 470 9 .4 2.80 2.65 3.41 2.00 7 0 .0 308 10.3 9.9 10.7 10.5 21.9 21.4 25.6 19.4 28.3 381 31*4 409 340 335 356 329 484 354 404 67.6 6 8 ,4 67.9 68.0 71.5 73.8 69.3 3 1 .0 4.56 3.10 2.49 29.8 45.4 10,6 12.8 7.80 11.9 4.43 3.27 3.94 4.30 2.52 1.90 2.44 2.73 43.2 41.9 7.85 6.05 15.7 10.2 3.54 3.18 3.89 3.59 1.81 1.96 2.13 43.2 49.6 .368 .373 .390 9.97 12.9 11.4 12.8 12.3 3.67 3.65 3.09 3.32 3.43 2.93 2.09 1.70 1.57 2.25 .318 .306 .358 .263 .403 10.1 10.3 12.6 8.35 13.2 3.55 4.10 5.14 2.50 3.65 .2 0 4 •454 .325 .342 .422 3 8 .0 36.4 4 0 .6 2 0 .2 42.7 45.0 52.7 34.4 EFFECT OF AGE ON CHOLESTEROL CONTENT OF BLOOD, HEART, Rat no# Age in mos# Body wt. gms. Blood Cholesterol Free mg,% Total Ester mg. % mg. % 17.5 10.0 62.0 60,0 65.0 50.0 15.9 10.3 15.5 17.5 70.0 51.0 70.0 69.0 96.0 62.6 44.2 62,0 Eaterified fatty acid m.e. /liter 1.49 73.1 90.0 1.33 7.25 4.00 77.3 79.8 77.8 74.7 3.25 2.00 k.25 8.25 14 14 lk i4.5 422 232 15 15 15 235 16 1)32 331) 31(4 1)09 29.7 7.5 7.7 254 228 229 230 231 16.5 16.5 16.5 16.5 16.5 1(86 340 335 356 329 26.9 11.7 13.7 13.9 15.7 154 162 163 159 160 20 30 30 33 34 481) 351) 40 k 56.0 57.0 78.0 0 .8 4 308 8 0 .0 0.52 1)70 77.0 0.51 6 1 .5 wt. gras. 1 .0 0 218 67.5 50.5 50.5 53.0 % in 0 .1 5 383 578 330' 356 145 222 . Uf i'UUJU iu\iw Kidney body wt. Total choles­■ wt. terol gras. % in body wt. Total oholes' terol "2/4 5 10 12.5 13.5 3U5 1)56 38l muhjij Heart 138 191 255 219 233 220 252 and 0.86 4.75 1.00 .261 .258 .248 3.57 2.24 1.38 0.99 .268 1 .1 8 1.22 0.97 3.39 i. M ii .37 0.95 .296 .281 .237 .249 .268 .318 .285 .281 3.65 1.04 1.56 1.3 8 1 .4 6 1 .5 0 0 .81 1 .0 8 52.7 83.1 87.6 1 .2 5 1.16 1.06 0.98 1.15 60.3 77.2 72.8 74.2 74*4 ii.5 1.50 1.75 1.75 3.25 1.12 0.96 0.91 0.93 0.96 .251 .282 .272 .275 .292 0.52 0.98 1.26 1.17 1.26 1.26 1.42 .260 .331 .312 .409 .302 1 .2 4 1 .3 8 1.19 1.22 4.17 3.37 3.23 4.17 2.98 2.20 3.48 1.78 2.05 .5 75 .603 .5 40 .576 2.22 1.99 2.33 1.86 .526 .577 .511 .488 3.29 3.97 4.02 5.55 2.22 1.93 1.95 2.70 .514 .578 .577 .661 5.60 4.45 3.72 4.07 1.95 2.00 1.76 2.20 2.04 .402 4.38 4.30 4.66 4.18 3.67 2.42 2.25 2.75 2 .4 2 2.57 .588 .526 .619 .620 .500 .635 .680 .786 .547 7.13 4.87 6.18 4.46 4 .4 0 5.04 4.56 4.40 4.72 EFFECT UE Percent moisture 3.34 3.19 3.1? 70.7 70.5 66,4 68,5 30.3 29.5 29.5 31.0 3.48 3.05 3.56 3.42 69.3 68.7 68.9 66.9 2.87 3.61 3.27 69.7 199 6 197 11 198 11 221 12.5 252 354 289 237 11.2 11.8 9.20 7.50 4 .4 1 234 22Ji 216 217 14 15 15.5 15.5 182 206 212 189 6.33 6.30 7.55 6 .5 0 236 237 225 226 227 16 16 16.5 16.5 16.5 197 201 198 108 196 5.64 7.30 6.50 7.14 7.43 144 142 196 195 153 17 267 18 18 218 253 223 176 179 161 20 20 321 318 9.58 6.67 16.7 12.98 7.08 23.7 244 211 313 342 245 7.12 5.95 10.9 13.7 10.7 4 28.5 28.5 36.5 311 wwnw N ng./g. wet wt. Liver wt. gms. Rat no* U X ¥£.n % Body wt. gms. Age in mos. ALrlS UW liver in body wt. Cholesterol Toted H o l d s % gm./gm. wet wt. go./ gm. N Total mg. £ 20.6 20.3 17.7 21.2 .292 .288 .267 .301 9.6k 9.76 9.06 9.72 3 .1 8 28.6 35.3 33.3 30.8 25.2 13.5 21.2 12.2 .362 .197 .308 .183 28.6 26.5 1 4 .6 3.79 70.0 69.2 70.0 32.4 31.7 32.7 33.1 32.3 3.73 3.06 5.37 4.C4 2.23 69.9 68.5 70.7 64.2 73.8 30.0 32.0 25.2 28.2 31.6 14.2 20.7 16.9 29.6 19.5 2.92 2.82 6.20 4.02 4.37 70.5 70.0 68.1 60.9 72.2 33.7 32.8 33.5 33.1 32.4 27.4 22,8 20.8 14.5 16.9 3 .8 0 7 0 .0 dry basis 14.4 12.5 Free mg.^ ^.ster # 3.40 4.46 2.79 2.03 27.2 12.7 5.59 9.12 5.95 3.19 3.12 2.49 2.71 2.03 2.02 1.96 2.02 26.3 25.2 21.2 24.8 .410 1 2 .7 .378 .209 .208 .179 11.9 6.39 6.29 5.54 3.16 2.73 2.89 2.68 2.33 1.89 2.54 1.94 2.00 2 .0 4 40.2 7.0 32.8 25.4 12.4 .203 .302 6.78 3.38 9.1+4 9.53 2 .81 2.64 2.23 11.4 31.8 .240 .461 .265 1 6 .3 .389 .326 .306 11.5 9.95 9.14 7.19 7.23 .238 .234 8.39 2.54 2.03 3.45 2 .98 3.27 3.48 2.22 4.11 \n Ul EFFECT OF AGE ON CHOLESTEROL CONTENT OF BLOOD, HEART, AND KIDNEY OF FEMALE RATS Rat no. Age in mos. Body wt. gras. Blood Cholesterol Free Total Ester mg. % mg. % mg. % 199 197 196 221 6 11 11 12.5 234 22k 216 217 11* 15 15.5 15.5 236 237 225 226 227 m Esterified fatty acid m.e./liter wt. gras. 0.73 0.96 1.02 0.86 % in body wt. 237 12.0 6 1 .0 80.3 0.68 0.98 0.98 3.75 182 206 212 189 21.7 17.3 15.9 22.6 64.5 61.5 46.7 60.0 66.3 71.8 66.0 62.4 2.25 5.00 2.75 1.75 0.63 0.68 0.63 0.65 16 16 16.5 16.5 16,5 197 201 198 188 196 27.7 10.0 15.8 15.3 17.3 82.0 50.5 60.0 70.0 74.0 66.2 80.2 73.7 78.1 76.6 4.25 3.25 6.25 6.25 1.75 0.67 0.63 0.64 0.83 .313 .323 .431 .423 17 267 11*2 196 195 153 18 18 218 1.25 1.10 1.25 of55 0.39 0.83 0.3 3 0.92 0.37 0.95 .311 .380 .296 .271 .267 253 223 178 179 161 23.7 6,00 5.25 0.69 0.73 0.97 1.12 1.09 .283 .346 .309 .327 •445 20 20 & k 28.5 28.5 36.5 252 351* 72.0 50.0 60.0 289 92.0 60.0 60.0 74*0 58.0 311 321 313 21*4 211 313 342 245 26.0 39.0 104. 100. 90. 55. 61 . 77.0 6 1.0 Kidney Heart 0 .5 9 0.95 1 .6 9 0.81 .290 .271 .353 *363 .3^6 .330 .297 •344 .340 % in Total wt. choles­ gras. terol body wt. Total choles terol 3.19 2.2 7 1.97 1.32 1.49 1.97 1.87 1.33 .591 .557 •6k? .561 5.69 5.22 4.56 2.11 0.99 1.12 1.20 1.10 1.27 1.13 1.45 .605 .617 .536 .718 3.93 3.78 5.07 4.60 1.50 1.62 0.99 1.65 1.41 1.15 1.21 1.15 1.39 1.52 •584 .602 .581 .740 .776 5.56 4.06 3.82 4.17 3.28 4.47 1.58 1.41 2.06 1.73 1.67 .592 .647 .670 .539 .525 1.32 1.28 .542 .655 .579 .565 4.09 3.91 6.02 .842 4.56 2.18 2.46 3.95 1 .4 0 1.83 1.36 2 .6 9 4.50 1.81 1.93 2,00 "t/S 5 .1 5 4.51 5 .3 2 8.64 4.40 5.55 4.31 6 .1 4 vn EFFECT OF TUMOR ON LIVER CONSTITUENTS OF TUMOR RATS Age Body Liver % Percent N Total lipids Rat Sex in wt. wt. liver moisture mg./g. * gm./gm. gm./ no. mos. gms. gms. in wet dry Jjet wt! gm. wt. basis N wt. Mg* per,gr*w Percent cholesterol esterified wet basis cholesterol Free Total 238 F 14.0 239 7.64 3.19 7 0 .0 29.7 11.9 .170 239 F 14*5 242 9.79 if.03 7 2 .0 28.0 22.7 .315 276 F 2i|.0 259 6.98 2.69 68.9 33.9 20.2 .293 287 F 17.0 28^ 9.J+0 3.31 67.5 31.2 23.7 .351 302 F 15.5 214-6 9.99 if.05 67.6 29.3 20.3 308 M 15.5 I4.70 9.57 2.03 67.8 33.4 327 F 1 9 .0 232 7.79 4.22 70.C »\i C D t F 16,5 26k 11.5 lf.36 334 F 18.5 279 1 1 .0 335' F 19.5 249 338 F 1 8 .5 328 1.94 2.17 1 0 .8 1.33 2.03 34.5 1.47 2.10 30.0 U.3 3.18 3.44 7.5 .301 10.3 2 .2 0 2 .7 6 20.2 25.0 .369 11.0 2 .7 8 4 .3 6 36.2 31.5 22.4 .320 10.2 3.29 4.23 22.2 68 .4 30.4 20.3 .297 2.58 3.90 33.8 3.94 67.2 25.2 27.7 .412 1 .7 2 2.11 1 8 .4 8 .9 4 3.59 67.0 34.1 18.7 .276 3.10 2.41 3.98 39.4 7 .1 6 2 .18 69.1 47.7 19.2 .278 5.83 3.08 3.32 7.2 5.73 11.3 8.64 9.78 16.3 vn ->i TABLE VI CHOLESTEROL CONTENT IN BLOOD, HEART, KIDNEY AND TUMOR OP TUMOR RATS Age Body Rat Sex mos. wt. no. gms. Heart Blood Tumor Kidney X Esterified wt. Cholei Chole- Wt. Chole- wt. fatty acid gms. body sterol gms. body sterol gms. body sterol Free Total Ester m.e./liter wt. mg. wt. mg. % wt. mg. mg* % mg. % % i Cholesterol 238 F 14. 239 20.7 65.0 69.2 4.0 .59 .25 1.69 1.27 .53 4.86 65.9 27.5 2 .2 4 239 F 14.5 242 27.0 67.5 60.0 3.5 .61 .25 1.76 1.2? .53 4.99 44.0 1 8 .2 3.07 276 F 24. 259 28.3 106. 73.3 12.8 .71 .27 1.31 1.44 .56 4.86 59.4 22.9 2.94 287 F 17. 284 19.5 76.3 16.5 .83 .29 1.29 1 .6 0 .56 6.13 33.6 11.8 3.34 302 F 15.5 246 45.5 167 . 72.8 18.3 .77 .31 1.35 1 .2 7 .52 5.50 39.5 16.1 2.56 308 M 15.5 470 23.5 76.1 11.0 1.01 .22 1.94 2 .08 •44 5.06 70.3 15.0 2.69 327 F 19. 232 34.0 13 8 . 75.4 1 0 .5 .70 .30 2.24 1.39 .60 6.2 3 33.4 14.4 1.87 328 F 16.5 264 34.0 155. 78.1 1 2 .0 .74 .28 1.98 1 .3 8 .52 7.38 35.7 13.5 1.64 334 F 18.5 279 24.4 91. 73.2 2 .0 .73 .26 1 .1 8 1.45 .52 4 .6 0 39.4 14.1 2.64 335 F 19.5 249 38.5 96.0 59.8 2 .4 .80 .32 1.05 1.38 .56 5.07 17.3 336 F 18.5 328 2 1 .0 58.4 6 4 .0 5.0 .90 .27 1.47 1.58 .48 4.75 100 . 82.2 98.3 6 .9 6 2.37 3 0 .5 .85 Vn. 00 TABLE VII COMPARISON OF EFFECTS OF THE THREE DIETS ON LIVER CONSTITUENTS OF YOUNG RATS Body Rat Sex Diet wt* no. gms. & lli8 1 A7 10/ 212 245* F I 21+6* M I M M F F I I I I 125 n5 148 155 24 7* F II 214-8 M II 320 322 M F F II II II 156 127 li+3 196 146 137 133 329 330 331 332 F F M M Stock Stock Stock Stock 137 161 11+9 159 316 317 316 319 321 Liver % wt. liver gms. wt. ^ N 4 Total lipids Mg* per percent Percent mg./g. * , _ / ~ 7 cholesterol esterified moisture wet £ g®./8». © W cholesterol basis wet wt# gm.N Free Total 12.2 3.87 68'.6 26.9 21.3 .311 11.6 2.11+ 2.6 2 18.3 19*1 1+.55 69.3 26.9 19.3 .279 1 0 .1+ 1.98 2.19 9.5 3.81+ 4.26 1+.25 4.06 68.8 66.7 66.2 26.5 32.1 28.1 30.7 26.5 37.0 30.5 33.3 .386 .538 .1+57 .503 14.5 16.7 16.3 1 6 .1+ 1.86 3.02 2.09 2.53 3.51+ 5.85 2.92 6.08 1+7.1+ 1+3.3 28.4 48.3 12.6 4.1+5 66.5 2 3 .6 27.5 •1+11+ 17.6 1.81+ 3.32 44.5 1 8 .6 5.1+9 65.1 22.3 36.4 .559 25.1 1.91 2.80 31.8 4.39 3.58 3.77 65.5 65.6 65.1+ 30.3 31.0 29.8 37.6 1+5.8 1+5.2 .571+ .697 .692 19.0 22.1+ 23.2 2.20 3.72 1+.1+6 1+.C6 1+.25 50.1 1+.68 7.30 4.53 9.1+1+ 6.33 9.32 5.86 67.2 34.5 32.6 25.6 26.7 22.3 .332 .331 .351 .1+31 9.62 10.1 13.7 16.1 208 4*81 4.89 6.29 6.29 6.1+2 4*91 5.oi 6.41 68.7 67.0 69.2 6 8 .0 * Determined on composite of two animals I - Experimental diet I II - Experimental diet II 1 8 .2 21+.3 29.3 2 .1 6 2 .1 6 2.1+0 1.92 2.25 2.88 3.06 2.52 3.00 3.3 1+9.1 25.0 H.5 23.8 24.9 COMPARISON OP THE EFFECT OF THE THREE DIETS ON BLOOD, HEART, AND • KIDNEY CHOLESTEROL OF YOUNG RATS Body Sex wt. Rat no. gms. Blood Diet Cholesterol Free Total Ester mg. % mg. % mg. % 21+5* F 21+6* M liiS 1 67( ±0 212 Kidney Heart Esterified fatty acid m.e./liter wt. gms. * body wt. Total wt. choles­ gms. terol mg* /g % body wt. Total choles­ terol mg. k I .11.5 65.9 82.5 29.0 0.96 .27 1.13 1.99 .63 5.70 I 7.5 46.7 83.9 23.0 1.38 .20 1.48 2.72 .65 5.21 0.1*1+ 0.1*0 0,1*8 0.57 .35 .35 .32 .37 2.55 1.45 1.87 1.72 0.90 0.9 2 1.06 4.45 1 .1 6 .72 .80 .72 .75 4.44 208 M M F F 125 115 11*8 155 I I I I 13.0 18.3 14.5 14.5 67.5 81.8 81.7 90.8 80.7 76.6 82.2 81+.3 6.0 2.1 5.1+ 8.5 21+7* F II 17.5 74.3 76.1+ 13.5 0.89 .31 1.57 0.88 .67 5 .00 II 11.5 72.5 81+.1 11.3 1.19 .35 1.79 2.63 .78 5.71 II II II 17.0 18.5 19.5 75.0 85.8 98.3 77.3 78.1+ 80.2 9.5 5.0 3.0 0.56 0.53 0.1+2 .38 .39 .32 1.91 1.77 2.46 1.03 1.13 1.03 .71 .83 .78 5 .0 2 5 .0 2 3.38 15.5 20.0 21.0 52.5 77.9 62.5 71.7 70.5 71+.3 66,1* 58.1 5.5 7.0 1.0 1.0 o.i+i* .32 1.55 1.55 0.99 1.01 1.16 1.35 .72 .63 .78 .85 6.1*8 5.1+5 6.1+6 5.51+ 316 317 318 319 320 321 322 M F F 156 127 11*3 196 11+6 137 133 329 330 331 332 F F M M 137 161 11+9 159 21*8* M Stock Stock Stock Stock 3 0 .0 # Determined on pooled sample of two animals I - Experimental diet I II- Experimental diet II 0.1*1* 0.5c 0.55 .27 .31+ .35 1 .2 8 1.42 3.44 4*40 TABLE IX EFFECT OF CHOLINE DEFICIENT DIET AND EXPERIMENTAL DIETS I AND II ON LIVER CONSTITUENTS OF YOUNG RATS Body Liver % Percent N Mg. per-grxm> Percent Total lipids Rat Sex Diet wt, wt, liver moisture mg./g, % dry gm./gm. gm./ cholesterol esterified no. gms, gms, wt. wet cholesterol ho040 ua ■w% t vh - erm. W*1 wet basis wt. Free Total W 269* F Cl 270*"* M Cl 271* M CII 272* F CII 288* M Cl, I Cl, I CII, I CII, I Cl, II Cl, II CII, II CII, 138 II 10^ 289* F 290* M 291* F 292“ M 293* F 291). M 295* F 51 1,0 45 41 56 49 58 57 143 132 113 113 151 155 135 128 115 79 92 107 126 V f V V f 6,05 6,44 66.9 24.6 33.6 .505 20.5 0.39 1.47 73.5 4.31 5.02 70.2 27.2 31.1 •443 16.3 0.52 1.98 73.8 2,81 2,59 64.1 21.6 43.0 .670 31.0 0,60 2.26 73.5 10,23 8,89 60.3 28.0 46.4 .754 26.9 0.83 2.24 63.0 9.66 3.53 70.3 33.8 22.3 .318 2.91 3.33 12.5 7.60 3.36 67.7 22.7 40.0 .591 26,0 3.26 4.84 32.6 15.0 4.91 59.5 26.6 53.9 .906 34.0 2.67 7.15 62.1 12.5 4*75 57.3 29.9 56.6 .987 32.9 2.69 8,01 66.4 12,6 6.50 64.2 22,2 48.8 .760 34.2 3.10 8.72 65.5 7.02 3.53 66.6 30.7 41.6 •625 20.3 1.98 4.27 43.6 6,89 2,65 52.0 21.5 68.3 1.31 61.0 1.98 6.67 70.3 45.3 24.0 80.4 1.71 71.3 2.93 5.81 49.6 13.7 5.67 9.41 * Composite of two rats Cl 269^-272* rats were put on choline deficientdiet CII for 10 days and then sacrificed I The rest # were put on choline deficient diet for II i-i to the specific ration - Choline deficient diet - Choline deficient diet - Experimental diet I - Experimental diet II I II EFFECT OF CHOLINE DEFICIENT DIET ON BLOOD, HEART, AND KIDNEY CHOLESTEROL OF YOUNG RATS Body Rat Sex wt* gms. no* Blood Diet Cholesterol Free % 269* F 51 Total Ester mg. % mg. % Kidney Heart Esterified fatty acid m.e./liter wt. gms. % body wt. Total wt. choles­ gms. terol mg. /g % body wt. Total choles­ terol ng. /g Cl 23.3 67.5 65.5 5.00 O .48 .56 1.60 1.13 1.23 3 .4 0 Cl 37.5 106.0 6 4 .6 6.25 0.34 .32 1.25 0.94 1.09 3 .7 2 CII 2 4 .2 75.8 68.0 8.75 0.50 .48 1.28 1 .0 4 1.00 5.1*5 CII 24*2 75.8 68.0 6.50 O .48 .43 1.29 0.98 0.84 5.33 Cl, I Cl, I CII, I CII, I Cl, II Cl, II CII, 18.5 85.0 78.2 1 0 .5 1.10 .40 1.67 2.10 0.76 6.1*4 12.0 85.0 85.7 9 .0 0.94 .42 1.94 1.94 0.86 6.45 17.5 59.0 70.4 1 2 ,0 1 .0 8 .35 1.81 2 .3 0 0.75 6 .0 9 13.5 1+7.0 71.2 1 2 .0 0*64 .24 3.31 1.96 0.75 6.21 1+2.5 65.8 35.4 1 7 .0 0.7 0 .36 2.03 1.76 0.91 5.02 31.5 65.3 52.0 1 2 .5 0.67 .34 1.70 1.94 0.93 4 .4 7 23.5 89.2 73.6 1 1 .0 0.45 .36 1.56 0.93 0.74 4 .3 0 31.5 95.3 67.1 9.0 0.87 .36 1.77 1.98 0.8 2 5.97 43 270" M 271* M 272* F 288* M 289* F 290* M 291* F 45 41 56 1+9 56 57 11+3 132 113 113 151 155 135 138 292* M 293''' F 115 79 92 10 7 29/; M 126 TT ±x 295* F 133 lO^ CII, II # Determined on pooled sample of two animals Cl - Choline deficient diet I CII - Choline deficient diet II I - Experimental diet I II - Experimental diet II TABLE XI EFFECT OF RACHITOGENIC DIET ON LIVER CONSTITUENTS OF YOUNG RATS Rat Sex no, Body Liver # Diet wt. wt* liver gms* gms* wt* Percent N Total lipids Mg..per gram Percent moisture mg*/g* at „ /„ a./a. cholesterol esterified wet basis wet wt, N wet basis cholesterol wt* M M T J.? S s s 128 123 110 5.98 4*84 4.36 4.67 3.93 3.96 70.5 F M s R R R 114 71 87 89 5.01 3.05 3.31 3.75 285* F R 89 323 32k 325 32 6 309 310 311 F F Free Total 6 8 ,9 7 0 .0 34.8 31.8 33.1 21.3 33.5 27.7 .302 .1*87 .396 8.70 2.1*8 15.3 2.78 12.0 2.85 3.81 3.81* 3.52 34.9 27.6 21.8 4.40 4.30 3.81 4.22 6 8.7 72,1 6 8 .3 6 9 .4 33.1* 36.2 35.5 32.9 33.4 29.2 33.2 '26*6 .526 .1*57 .1+28 .383 15.8 12.9 12.8 11.6 2.86 1.16 1.93 1.1+0 1+.07 1.59 1.93 1.77 2 9 .6 2 7 .0 0.0 20.9 6.67 3.65 7 0 .6 33.2 21*.7 .31*9 10.5 2.91* 1+.22 30.3 liji 8.54 3.29 6 9 .6 31*.0 18.6 .268 7.90 3.06 3.20 1+.4 7.05 4.27 6 9 .0 30.5 36.3 .526 17.5 1.72 2.90 1*0,6 6.7 0 4*44 7 0 .0 30.2 25.8 .369 11*.3 2.12 2 .6 5 20.0 5.76 3.92 70.1 29.2 2 7 .8 .539 1 8 .1* 1.65 2 .8 5 1+2.0 6.93 4 .1 2 78. !* 1*0.3 27.1 .31*6 8.60 3.01 3.81* 2 7 .8 Qli 286* M R 113 297 M R _ VI 298 '“ F R 299 " F R O O F R 80 85 74 77 74 73 88 62 ■fr Determined on composite of two rats S - Stock diet R - Rachitogenic diet t O' TADJj-Ej AJ.X EFFECT OF RACHITOGKNIC DIET ON BLOOD, HEART, AND KIDNEY CHOLESTEROL IN YOUNG RATS Rat no* Sex 323 32b 325 326 M 309 310 311 . 285'" Body wt. Diet gms. F F 128 123 110 11b F F M F IK •1 Blood M 297"’ M 296" F 299" F 300* F Kidney Cholesterol Wt. Total Fatty acid Wt. * i gras. ooay cnoj.es UOTJ ?X escerinea gras. body Free Total Ester ma./ liter wt. mg. wt. mg. fg mg.^ mg. % mg. £ Cholesterol s I k .5 16.0 27.0 17.5 70.8 58.3 87.5 87.5 79.5 72.5 69.2 80.0 3.0 2 .5 12.0 11.5 71 87 89 89 R R R R 18.9 28.0 21.0 25.0 75.5 98.5 77.5 7k .2 75.0 71.6 72.9 66.3 13.0 b .5 6 .0 11.5 1M> 113 80 85 lb 77 lb 13 88 62 R 32.0 85.0 62.3 10.0 R 17.5 56.7 69.2 R 22.0 77.5 .60 •£2 .1+6 . 1+8 •1+7 .42 .42 .1*2 1.73 1.5k 2.10 1.96 1.03 1.11*. 0.93 0.8 9 .81 .93 .85 .78 6.31 6.k0 6.10 6.37 . 1+0 .57 .1+0 .1*6 .1*2 4 7 1.1+6 .80 l.k 5 1.62 1.58 3.2k 0.66 0.71*. 0.79 1.48 .93 .85 .89 .81 k.55 5.18 5 .io 6 .6 3 .99 .38 2.22 2.58 1.00 5.62 6.8 .61 .37 1.85 1.42 .86 6.03 71.6 3.2 .62 •41 1.9k 1.19 .79 6.85 R 3k. 0 101.7 66.5 3.0 .57 .39 1.7k 1.38 .91*. 5.07 R 21.0 62.5 66. I*. 2.6 •6b •1+3 240 1.36 .91 5.52 S S S Ql. 286" Heart # Determined on pooled sample of two rats S - Stock diet R - Rachitogenic diet TABLE XIII EFFECT OF STOCK, EXPERIMENTAL DIETS I AND II ON LIVER CONSTITUENTS OF RATS (34 MONTHS OLD) EXPOSED TO CCL^ VAPORS 3 TIMES WEEKLY FOR II WEEKS Body Liver % Percent N^ Total lipids Rat Sex Diet wt# wt# body moisture mg./g# ..... r* " no. gms# gms# wt# wet % ^ 7 g#/g# g./g. wt# basis wet wt. N M M F s 321 25 6 253 268 F 2k 9 M 250 M 251 F s s s s 209 221 2k0 170 6.56 8.78 7.97 7.60 2 77 278 2 79 280 I I I I •212 302 281 23k 6#k2 14.8 11.5 7.33 II II II 25 9 210 2 66 265 266 267 F M M F 273 F 274 M 275 M S s M8* Percent cholesterol esterified „ m , cholesterol Fre® Total 68.8 70.6 69.7 33.5 31.6 34.5 274 26.0 18.7 .399 .372 .268 11.9 2.64 11.8 2.07 7.77 2.42 3.45 2.94 2.92 23.5 29.5 17.0 3.1k 3.97 3.32 4.47 70.7 694 68.3 734 31.8 30.9 31.6 27.8 49.3 26.8 29.1 33.2 .698 .386 .k!2 453 21.9 12.5 13.0 16.3 1.40 3.14 1.69 1.96 2.51 3.67 3.20 2.84 44*2 144 47.2 30.9 3.13 4.91 4.10 3.13 564 62.0 6l .6 6 I4..O 31.6 27.5 29.6 31.3 46.2 44.7 44.0 45.0 .820 .7 22 .714 .704 26.0 26.2 24.1 22.5 1.57 3.38 4.28 8.65 4.51 26.7 2.33 3.11 53.5 50.5 83.1 25.0 13.0 5.02 6.58 3.13 9.68 3.60 58 4 63.9 62.5 28.0 30.9 31.0 51.9 53.2 51.2 .889 .834 .822 31.7 27.0 26.5 2.93 11.1 2.30 5.32 2.69 3.76 73.6 56.8 31.0 9.85 3.07 10.3 M 3 8.05 3.18 S - Stock diet I - Experimental diet I II - Experimental diet II TABLE XIV EFFECT OF CCL^-INDUCED LIVER INJURY ON BLOOD, HEART, AND KIDNEY CHOLESTEROL OF YOUNG RATS {ADMINISTERED BY EXPOSURE FOR 11 WEEKS) Body Rat Sex wt* Diet gms* no* Blood Heart Kidney t Cholester Esterified Wt. % Cholesterol Wt. totsl total gms. body fatty acid gms* body Free Total Ester m.s,/liter wt. wt. mg. /(J mg. /$ mg.# nig* % Cholesterol i 26^ 266 267 268 M M F F 321 256 253 209 249 M M F 21+0 250 251 277 278 278 280 F M M F 273 F 271+ M 275 M 221 170 212 S s s s s s s 281 231+ I I I I 259 210 266 II II II 302 20.0 31.5 29.5 26.0 7 0 .8 50.8 98.3 70.8 9.0 1+3.1+ 13.5 W .5 Xi4-.lt- 50.0 3 .0 5it.o 26.6 75.0 33.3 78.14 30.1 91.0 2 4 .2 117.0 15.8 75.8 15.8 89.2 S - Stock diet I - Experimental diet I II - Experimental diet II 7 1 .7 3 8 .0 7 0 .0 6 3 .3 1+.00 1+.0 •91+ 0-.3 3.5 .75 .70 7 9 .3 6 8 .9 7 1 .6 2.3 7.5 15.8 .65 .75 .57 •31+ 91+.1+ 61+.5 57.5 62.8 l+.o 17.5 11.5 6.5 .62 .89 .83 .69 .29 .29 .30 .30 79.3 79.1 82.3 6.5 10.8 8.8 .76 .29 .29 .32 .81 .61 .86 .29 .32 .30 .31+ .39 .31 1.1+2 1*08 1.26 i.ia 1.16 1 .2 0 1 .2 0 1.10 1 .3 9 1 . 31+ 1 .2 7 2.03 1.60 1.56 1.09 •61+ .63 .62 .52 I4.8 I4 5.10 4.89 1.58 1.56 1.13 .72 .65 .67 5.27 1.36 2.09 1.88 1.31+ .61+ .69 .67 .57 0.90 .36 .62 .69 1 .0 7 1 .2 8 1.31 1.16 1.82 4 .9 1 5.55 5.90 1+.16 3.31+ kM 3.60 11.20 6.35 5.22 TABLE XV EFFECT OF STOCK, EXPERIMENTAL DIETS I AND II ON LIVER CONSTITUENTS OF RATS INJECTIONED INTRAPERITONEALLY WITH DIFFERENT CONCENTRATIONS OF CCL^ TWICE WEEKLY FOR 2 WEEKS CCIl Body Liver % Peroent Rat Sex Diet cone, wt. wt. liver moisture no. ml,/ gms. gma. wt# 100 g. body wt. 2^0 21*1 21*2 6.21 F M F M M S S I I II 0.033 0.033 0.033 0.033 0.033 M M F F M s s I I I 0 .066 0.066 0.066 0,066 0.066 263 M 261* M I II II II 0.066 0,066 0.066 0.066 113 99 303 F 301* F 305 M 306 F 307 M I II II I II 0.099 0.099 0.099 117 115 105 0.132 0.132 118 6.68 97 6.48 243 2kk 2 ^6 257 258 259 260 261 M 262 F 123 182 8.73 146 7 .8 6 166 7.34 ii*4 8.11 126 5 .10 130 136 123 5.15 4.55 4.74 102 4 .61 111 112 5.0 6 t&'/s* wet wt* .378 1 1 .8 3.50 38.9 29.1 20.3 14.6 12.4 1 .8 2 2 6 .0 2 5 .2 ,601 .382 .362 2.05 2.74 64*2 2 7 .0 49.5 .761 2 8 .2 2 .6 2 7 0.0 7 0 .0 30.3 31.6 32.7 3M .491 .512 2.27 2 .1 6 2 .1 6 69.3 64,6 31.9 29.1 68.1 6 9 .6 2 6 .2 4.04 3.96 3.85 4.52 Total lipids M g . p 9 r .$r&, Percent % dry g./g. g./g. cholesterol esterified basis wet wet N T 7 cholesterol Dasxs n ™------« pree ^otal wt. 2 6 .2 5.05 4.79 5.38 4.42 5.63 65.1 67.3 65.7 5.87 7.18 4.1*4 4.56 5.23 6.35 4.48 65.4 55.4 5.15 4.40 8 .0 8 6 .0 2 66.5 62.3 5.46 5.19 5.67 6.69 S - Stock diet I - Experimental diet I II - Experimental diet II K 6 0 .0 6 7.2 66 .8 65 .6 69.5 36 .8 4 6 .0 .708 1 6 .2 1 6 .2 2 1 .6 36.7 33.8 .546 .515 17.3 14.5 2.95 2.19 21.5 2 .44 31.7 .658 43 .0 63.2 1.14 5 2 .6 .879 45.3 .674 2 9 .2 4 8 .2 25.4 28.5 28.7 26.5 49.4 44.4 46.1 41.4 3 1 .6 35.5 3 0 .6 27.9 30.6 .725 .794 .665 .693 .596 4 0 .8 28.7 2.13 2.04 2 1 .2 2.6 0 24 .8 31.2 2 .1*2 2 .0 2 23.3 2.71 2.54 2.74 24.1 22.5 14.0 68.4 37.3 4*11 5.75 3.27 3.71 6.67 40.7 2.49 2 .1*2 4.05 3.41 8 .9 0 10.1 4 6 .6 1 4 .0 2 .9 8 26.5 4.42 5.32 4.94 3.46 4 4 .8 4 0 .0 5 .oo 4.73 5.21 6.55 6.0 0 2 6 .0 58.7 2 4 .8 51.6 52.3 47.9 61 .2 54.3 TABLE XVI EFFECT CF CCL^-INDUCED LIVER INJURY ON CHOLESTEROL IN BLOOD, HEART, KIDNEY OF RATS INJECTED INTRAPERITONEALLY WITH DIFFERENT CONCENTRATIONS OF _____________________________ CCL^ TWICE WEEKLY FOR 2 WEEKS________________________________ Body S#X no. ens. CClj, Diet °1 _______Blood___________ ______Heart__________ /* Cholesterol ml./ — oody mg. f mg. 2kl F M 123 182 F 146 166 M 2^3 s s ^ wt. gM# % % Mt* 2i|0 Ester- $ Wt‘ Cholesterol Wt. i Cholesterol ^ns. body tot-1 mg< 2 3.0 87.5 73.4 73.6 .033 .033 .033 .033 .033 12.5 25.5 111.0 37.5 98*5 29.0 100.7 77.0 61.9 70,1 82.8 10.3 5.0 13.3 7.5 7.5 .54 .63 .59 .65 .54 5.0 .42 6 .0 7 .0 7 .0 .438 .346 6.4 0 2 .0 5 2.7 6 1.31 .790 1.07 .743 6.36 6.55 .333 .331 .338 .285 .304 1.43 1.17 1.00 .795 .98 .754 5.83 5.95 1.8 9 .691 6.92 1.48 .97 •98 .797 .87 .853 5.96 5.28 .87 •88 .97 .86 4 .0 2 M M F F M 126 130 136 123 102 s s I I I *066 •066 •066 •066 •066 43.0 62.7 15.8 74.2 43.3 74.2 24.1 95.8 17.5 43.3 31.4 78 .3 41.7 74.7 59.5 15.5 .43 .46 .35 .31 261 262 263 264 M F 111 112 113 99 I 19.1 20.0 21,6 29.1 75.0 II II II •066 •066 •066 •066 95.8 95.0 74.2 68*8 77.4 69.3 7.3 5.8 3.0 2.5 .47 .33 .41 .31 .423 .295 .363 .313 1.21 1.51 1.13 1.10 117 115 105 I .099 17.2 50.0 II .099 22.5 104.0 II .099 14.0 4 9 .0 4.0 2*0 118 65.6 78.4 61.4 I 9.5 69.2 86.3 II .132 43.0:164.0 .359 .391 .371 .393 .392 1.66 1.70 1.28 1.74 73.8 2.0 7.2 .42 .45 .39 •46 .38 305 306 307 97 •132 6.80 1 .1 2 .768 256 257 258 259 260 30k .97 .789 1.14 .627 k .404 144 F F M F F mg- .391 .375 M 303 wt* 2.34 2.13 2.54 244 M M k m.eq./ liter I I II 242 Kidney S - Stock diet I - Experimental diet I 64.1 7.2 1 .63 .784 .785 .858 .870 1.01 •864 .96 .835 .95 .904 ..76 •644 .83 .856 5.97 3.60 4.47 5.4-3 4.62 4.52 4.90 8.12 6.42