University of Southern California Dissertations and Theses

An investigation of the distribution and mechanisms of deposition of exogenous cholesterol in the rat

(USC Thesis Other)

An investigation of the distribution and mechanisms of deposition of exogenous cholesterol in the rat

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content AH INVESTIGATION OP THE DISTRIBUTION AND MECHANISMS OP DEPOSITION OP EXOGENOUS CHOLESTEROL IN THE RAT A Dissertation Presented to the Faculty of the Graduate School University of Southern California In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by Leslie Irene Rice February 1954 UMI Number: DP21555 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI DP21555 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 -1 3 4 6 “ Ph. D This dissertation, written by .............LESLIE..]S.iM3 I0E ............. . under the direction Faculty Committee, fy-I) f y -1 ^ cm Studies, and approved by all its members, has j [) been presented to and accepted by the Council ' ^ on Graduate Study and Research, in partial fu l fillm ent of requirements fo r the degree of D O C T O R O F P H I L O S O P H Y Dean D ate.. Committee on Studies / J Is* / Chairm an 'CObJjL TABLE OP CONTENTS PAGE HISTORICAL INTRODUCTION . . . . . . . . ........... 1 Introduction............ 1 Deposition of Cholesterol in Animal Tissues . . . 2 The rat . • ..................................... 3 Other species................................... 4 Experimental cholesterol atherosclerosis . . . 6 Isotopic studies . . . . . . . . .............. 9 The Cholesterol Content of Ultracentrifugally Prepared Liver Cell Fractions .. . . . . . . 10 The Role of the Reticulo-Endothelial System in Cholesterol Deposition ......................... 12 STATEMENT OP THE PROBLEM . . . . . . . . . . . . . 21 METHODS AND MATERIALS . . . . . . . . . . .......... 23 Met hods.................. 23 Lipid extraction ......................... 23 Soxhlet method............................... 23 Alkaline hydrolysis method . . . . . . . . . 24 Waring blendor method .................... 26 Extraction of plasma........................ 30 Extraction of u r i n e ........................ 30 iii PAGE Cholesterol analysis ........... ...... 30 Total lipid analysis ..... .............. 31 Deuterium analysis ......................... 32 Preparation of the cholesterol digitonide 32 Deuterium measurement.................... 33 Materials ............................. 33 A nimals...................................... 33 Diets 34 EXPERIMENTAL AND RESULTS ......................... 37 Preparation of Deuterium-Labeled Cholesterol . 37 The exchange method of Bloch and Rittenberg 37 Biological preparation from the hen’s egg • 38 Recovery of cholesterol from cholesterol digitonide ........................ 40 Deposition of Deuterium-Labeled Cholesterol in the Tissues of the Rat 43 Experiment I. Determination of the distribu tion of exogenous cholesterol in the tissues of the rat 43 Experimental procedure 43 Results . • ...................... 46 Experiment II. Comparison of the distribu- tion of deuterio-cholesterol in the tissues of rats previously fed normal and high-cholesterol diets .................. Experimental procedure Results............. • • • • .............. The Effect of Cholesterol Feeding on the Quan titative Distribution of Free and Esterified Cholesterol in Ultracentrifugal Fractions of Rat Liver Homogenates ......................... . Experiment III. Effect of duration of cho lesterol ingestion on the distribution of cholesterol in three cell fractions .... Experimental procedure... .............. . Results .................... .. Experiment IV. The distribution of cholester ol in five liver cell fractions from rats fed cholesterol for seven days .............. Experimental procedure • ............. • Results................................... Experiment V. The effect of the administra tion of reticulo-endothelial blocking agents upon the deposition of cholesterol and total lipids in the livers of cholesterol V PAGE fed rat s............. 80 Experimental procedure . . . . . . . . 80 Results........... ................. .. . 83 DISCUSSION .................................. 89 SUMMARY........................................ 107 BIBLIOGRAPHY .............................. Ill LIST OP TABLES TABLE PAGE I. The Comparison of Different Methods of Extracting Cholesterol Prom Peces . . . 25 II. The Comparison of the Waring Blendor and Alkaline Hydrolysis Techniques for the Extraction of Cholesterol Prom fiat Liver Homogenates ....... 28 III. The Efficiency of the Waring Blendor Technique on Successive Extractions of Cholesterol Prom a Rat Liver Homogenate ............. 29 IV. Composition of Diets . . . . . . . . . . . 35 V. The Effect of Cholesterol Ingestion on the Cholesterol Content of Rat Tissues . . . 47 VI. The Deposition of Deuterium-Labeled Cho lesterol in the Tissues and Peces of the fiat ................ 49 VII. The Effect of Cholesterol Feeding for Various Time Periods on the Total Cholesterol Content in Tissues and Peces of the Rat ........... 56 vii TABLE PAGE VIII* Ratio of Free to Total Cholesterol in the Liver and Plasma of Rats Fed a 1. Per cent Cholesterol D iet.................... 57 IX. Cholesterol Concentration and Deuterium Con tent in the Feces of Rats Fed Deuterio- Chole sterol................................ 59 X. Distribution of Exogenous Cholesterol in Tissues and Excreta of Rats Previously Fed a Low-Cholesterol or a High-Cho- lesterol Diet for 30 Days . . . . . . . . 61 XI. Per Cent of Ingested Deuterio-Cholesterol Incorporated into the Tissues and Feces of Rats Prefed Normal and High-Cho- lesterol D i e t s ............................ 63 XII. Distribution of Total and Free Cholesterol in Three Ultracentrifugal Fractions of Liver Homogeantes of Rats Fed 1 Per Cent Cholesterol Diet at Three Time Intervals During Experimental Period 69 XIII. Comparison of the Concentration of Free and Esterified Cholesterol in the Liver Frac tions From Normal and Cholesterol-Fed Rats 77 TABLE XIV. XV. XVI. XVII. XVIII. viii PAGE The Per Cent of Free Cholesterol in the Total Cholesterol of Liver Fractions From Normal and Cholesterol-Fed Rats . . . 79 The Effect of Intravenous Injection of Thorotrast and Trypan Red on the Liver Cholesterol in Rats Fed Cholesterol .... 85 The Effect of Intravenous Injection of Thorotrast and Trypan Red on the Plasma Cholesterol of Rats Fed Cholesterol ..... ....................... 86 The Effect of Intravenous Injection of Thorotrast and Trypan Red on the Total Liver Lipids of Rats Fed Cholesterol ...................... 88 The Deposition of Free and Esterified Deuterio-Cholesterol in the Livers of Rats Prefed Normal and High- Cholesterol Diets • 97 LIST OF FIGURES FIGURE PAGE 1. Incorporation of Deuterium into the Cho lesterol of the Xolk of the Hen's Egg . • 41 2. The Effect of Cholesterol Ingestion on the Content of Esterified and Free Cholesterol in Three Ultracentri fugal Fractions of Rat Liver Homo- genates.................. ................... 70 3. The Effect of Cholesterol Ingestion on the Percentage Distribution of Total Cholesterol in Three Ultracentrifugal Fractions of Rat Liver Homogenates . • • • 73 4. Fractionation Scheme for Rat Liver Homo- genate Prepared in 0.25 M Sucrose Solution............................... 76 HISTORICAL INTRODUCTION Introduction The cholesterol found in animal cells has been shown to be derived from two sources': synthesis within the organism itself, and deposition from animal lipids ingested in the diet. Early balance experiments showed that "animals were capable of synthesizing cholesterol when they were fed diets lacking in this substance, and it was later demonstrated that many isolated tissues could in corporate isotopically labeled precursors into the cholesterol molecule. On the other hand, it was known early in the twentieth century that dietary cholesterol was absorbed from the intestine and deposited in the blood and other tissues. The relative importance of these two sources of cellular cholesterol has long been a topic of investigation, and the recent emphasis'on the probable role of cholesterol in the process of atherogenesis has led to renewed interest in this problem. In the work to be reported here, some of the mechanisms involved in the deposition of exogenous cholesterol in the rat have been studied. 2 Deposition of Cholesterol in Animal Tissues The development of interest in the fate of cho lesterol ingested in the diet can be traced back to the beginning of the 20th century. That this dietary cho lesterol was absorbed from the gastrointestinal tract was evidenced by an increase in cholesterol content of the plasma and by the failure to recover all of‘ the ingested cholesterol in the feces. In 1915, Mueller (1) reviewed these early findings and reported that in the dog, cho lesterol was absorbed via the intestinal lymphatic system, and that prior to, or during absorption, .the dietary cho lesterol was either hydrolyzed or esterified so that a constant proportion of free to esterified cholesterol was always found in the lymph. Mueller's observations have been confirmed in recent years with the aid of isotopically labeled cholesterol (2, 3). It was found that the feeding of cholesterol re sulted in its deposition in other tissues, in addition to the blood. In 1913, Weltmann §.nd Biach (4) reported that herbivores fed cholesterol exhibited a storage of double- refracting cholesterol esters in ovaries, adrenals, kidneys and liver. In the-same year, Anitschkow and Chalatow (5) and Wacker and Hueck (6) reported independently that the 5 feeding of pure cholesterol to rabbits resulted in arterial lesions consisting of fatty plaques on the intimal surface, which were similar to the early lesions found in human » atherosclerosis. Subsequently, the effects of cholesterol feeding were studied in many species, and the responses were found to range from widespread deposition in many organs, in the case of the rabbit, to localized deposition in the liver of the rat. The rat. The first investigations of the effects of cho lesterol feeding in omnivores were made in 1929 by Schoenheimer and Yuasa (7) who found that prolonged cho lesterol ingestion caused marked increases in the liver cholesterol content of rats, cats, and mice. In 1933, several groups of investigators reported almost simul taneously that cholesterol ingestion by rats rapidly re sulted in fatty livers containing greatly elevated con centrations of cholesterol esters (8, 9, 10, 11). Increases in free (unesterified) cholesterol in this organ were small or undetectable (8, 10, 12, 13). Sperry and Stoyanoff (12) found that prolonged cholesterol feeding to the rat pro duced small increases in the free and total cholesterol of other tissues including the lungs, kidneys, adrenals, and c arc ass. However, Chanutin and Ludewig (10) and Cook and Mc'Cullagh (13) were unable to detect changes in the cho~ leaterol concentration of any organs besides the liver, even after long periods of cholesterol feeding. Signifi* cant increases in plasma cholesterol concentration oc curred in the rat when cholesterol was fed (13,14,15), although these changes were much less pronounced than those occurring in some other species such as the rabbit (13). In general, the rat has been found to respond to a high cholesterol diet by a rapid storage of cholesterol in the liver, a slower and less pronounced increaaesin plasma cholesterol content, and little or no deposition of cholesterol in the other tissues. Other species. The observation of the presence of fatty livers containing large quantities of cholesterol esters in rats fed cholesterol was extended to many other species, in* eluding the rabbit (13,16,17) guinea pig (18,19), parrot (13), chicken (20), hamster (21, 22 ), catt(7, 23),24), and mouse (23, 25, 26). Elevation of the blood cho- lesterol levels also occurred In all of these species as well as in the dog (27). In the examination of other organs, the most widespread deposition of dietary cho lesterol was reported in the rabbit (13, 16, 17, 28), where Increases in cholesterol content were observed, 5 by chemical or histological methods, in liver, blood, kidneys, spleen, lungs, heart, adrenals, bone marrow, gonads, subcutaneous tissue, blood plasma, and arteries. In other species, not including the rat, some deposition of cholesterol and lipids was observed throughout the body but to a much lesser extent than in the rabbit. This was demonstrated by Cook and MeCullagh (13) in the guinea pig. In comparing this species with the rat and the rabbit, these investigators concluded that mechanisms for handling excess cholesterol were less efficient than in the rat, but more effective .than in the rabbit in which cho lesterol accumulation in the tissues resembled a passive process. Other effects of cholesterol feeding in the guinea pig included retardation of growth and anemia accompanied by splenic enlargement (18). The influence of cholesterol-rich diets on the mouse, hamster, and cat is not so clearly defined. Some accumulation of cholesterol esters was noted by Schettler (25) in the spleen and kidneys of mice fed cholesterol. AltschuL (29) reported lipoidosis (foam cell accumulation) in various organs of hamsters fed egg yolk powder. The cat exhibited considerable cholesterol storage in the liver and little in the rest of the tissues (24). The effects of cholesterol feeding in the chicken 6 have been rather thoroughly explored by Stamler and Katz and their associates (20) who found general organ lipoid osis characterized by deposition of cholesterol esters in the heart, kidneys, intestine, carcass, liver, and aorta, with the most marked deposition in the liver and aorta. Experimental cholesterol atherosclerosis. For many years after the first discovery of cho lesterol-induced atherosclerosis in rabbits (5, 6), attempts to produce arterial lesions of comparable severity in other species by feeding cholesterol met with limited success. Bailey (30) reported the production of experimental cholesterol atherosclerosis in guinea pigs, and these observations were later both confirmed (31, 28) and denied (13); this animal has never been widely used imstudying the disease. Cook and MeCullagh (13) were unsuccessful in inducing arterial lesions in cats or parrots by feeding cholesterol. Mice also showed little or no response to cholesterol feeding in this respect (31). The hamster evidenced some fatty infiltration (22) and foam cell accumulation, and occasional calcification and necrosis (29) in the aorta, but owing to the lack of severity of the lesions, it was felt (21, 22) to be un 7 suitable for the study of experimental atherosclerosis. More recently, Steiner and Kendall (32) succeeded in demonstrating atherosclerosis in dogs when thiouracil was fed along with the cholesterol diet. The chicken is the only species besides the rabbit which has been used extensively in investigations of experimental cholesterol atherosclerosis (33, 34, 35). It is interesting to point out that none of the procedures used to produce experimental atherosclerosis in other species have been particularly effective in the rat. Rosenkrantz and Bruger 0-4) noted the absence of arterial lesions in rats whose blood cholesterol levels had been elevated by feeding egg yolk, and other investi gators (13, 28, 36) failed to detect any deleterious effects of cholesterol ingestion on the vascular system. Page and Brown (37) recently showed that even the administration of thiouracil and cholic acid (two agents which induce pronounced hypercholesterolemia) in conjunc tion with cholesterol feeding failed to result in atherosclerosis, although some lipid infiltration of the blood vessels occurred, along with considerable infiltra tion of the kidneys, liver, and heart. However, a procedure devised by Bragdon and Boyle (38), the intra venous injection of low density lipoproteins obtained from 8 the serum of cholesterol-fed rabbits by ultracentrifuga tion, caused some atheromatous-like lesions in rats. Some lipid infiltration was also produced in the arteries of rats under the following conditions: low-choline diets (39), sodium chloride-induced hypertension and cholesterol feeding (40), high doses of vitamin E (41), and treatment with thiouracil or with thyroid and cholesterol (42). However, in none of these cases were significant lesions observed. Thus, the rat seems to be the laboratory animal most resistant to experimental cholesterol atherosclerosis. Many investigators are of the opinion that this resistance is due to the possession by the rat of mechanisms for the disposal of excess cholesterol which are superior to those found in most other species. Another explanation for the failure of the rat to develop atherosclerosis in spite of occasional instances of lipid infiltration in the arteries, offered by Wilens and Sproul (43) and by Page and Brown (37), is that although cholesterol may be mobilized and deposited, the arterial tissue fails to respond to it. This hypothesis is contradicted, however, by the observa tions of Bragdon and Boyle (38) of atheromatous lesions in rats injected with lipoproteins obtained from the plasma of hypercholesterolemic rabbits; and it is evident 9 that much remains to be discovered concerning the mechanisms for handling cholesterol in the rat as compared to species susceptible to experimental atherosclerosis. Isotopic studies. It has been pointed out that no substantial in creases occur In the cholesterol concentration of tissues other than the liver and plasma in rats fed cholesterol. However, the question of whether or not exogenous cho lesterol is incorporated at all into the tissue cholesterol could not be answered until recently when isotopically- labeled cholesterol became available in sufficient quantities to be employed in feeding experiments. Van Bruggen, Hutchens, and West (44) fed C'1 ' 4- eholesterol in oil to two young male rats, and after 48 hours found radioactivity in the cholesterol of the gut, carcass, liver, skin, and brain. Gould reported (45) that upon feeding of C14-cholesterol to rats and mice, 15 per cent of the labeled cholesterol present in the body was localized in the liver, a small amount was in the blood, and the rest was distributed rather generally in the tissues. Similar investigations were carried out in rabbits by Biggs and Kritchevsky (46) and in dogs by Pavarger and 10 Metzger (47). In both species, labeled cholesterol was found in all the organs studied, with the exception of the brain. In the dog, the liver and spleen retained an important part of the labeled cholesterol (47). In the work to be reported here, a more detailed investigation has been made of the distribution of exo genous labeled cholesterol in the tissues of the rat and of the effect of previous cholesterol-feeding upon this deposition. The results of these experiments reconfirmed the existing evidence for the importance of the liver in the storage of cholesterol. The Cholesterol Content of Ultracentrifugally- With attention focused on the liver as the major site of cholesterol storage, it then became of interest to localize further the site of deposition within the liver cell. Some information concerning the lipid com position of liver cell particulates had been obtained by the application of the differential centrifugation technique of Bensley and Hoerr (48) and Claude (49), modified and standardized for rat liver homogenates by Schneider and Hogeboom (50). Cholesterol was identified in mitochondria (51, 52, 53, 54) and microsomes (53, 55), Prepared Liver Cell Fractions and very small quantities were also found in nuclei (56, 57) isolated from rat liver homogenates. The quantitative dis tribution of free and esterified cholesterol within the ultracentrifugal fractions of rat liver homogenates was studied by Chauveau _et al (56) and by Schotz et al (57); both groups of investigators employed a preliminary high speed centrifugation to separate a lipid-containing frac tion called the "free fatM by Chauveau et al (56) and the floating or "Fn layer by Schotz et al (57); this fraction was found to contain a high percentage of the small quan tity of esterified cholesterol present in the normal rat liver. Schotz and co-workers reported (57.) that 60 per cent of the unesterified cholesterol was localized in the submicroscopic particulate, or microsomal fraction, while the remainder was distributed among the mitochondrial, nuclear, and supernatant fractions. Chauveau ejt al (56) also found only small percentages of the free cholesterol in the nuclear and mitochondrial fractions, but reported that the remainder, or largest part, was almost evenly divided between the microsomal and supernatant fractions. However, this difference between the results of the two investigations can be reconciled by consideration of the fact that Chauveau at al used a centrifuge which provided a maximum centrifugal force of only 50,000 x g, at which 12 speed, they pointed out, the subraicroscppic particles were not completely sedimented. In light of this information concerning the distri bution of cholesterol within the normal rat liver cell, it became of interest to determine into which of the ultra centrifugal fractions of rat liver exogenous cholesterol was incorporated; therefore, the effect of a high-cholest- erol diet, fed for varying periods, on the distribution of free and esterified cholesterol within the rat liver cell has been determined. i The Role of the Reticulo-Endothelial j System in Cholesterol Deposition An event which has been shown to occur in the development of experimental atherosclerosis is the accumu lation of lipid-filled cells in the linings of the arte ries. Some pathologists believe that these cells may be derived from the reticulo-endothelial system which is com- | posed of (a) tissue histiocytes, (b) reticular cells of the spleen, bone marrow, and lymph glands, (c) endothelial cells lining the sinuses of the liver, spleen, adrenal and other organs, and (d) polymorphonuclear leucocytes and monocytes of the blood. The function of this system in cholesterol metabolism has been investigated, and some 13 evidence exists that it may play a specific role in the uptake of cholesterol from the blood not only by the blood vessels but by other tissues as well. In the period from 1909 to 1913, Aschoff and his students (among them Anitschkow) developed a theory that there is a system of cells, the reticulo-endothelial system, which takes up and retains fat and substances of high molecular weight from the blood stream. Anitschkow was the first to recognize that xanthoma cells or ’ ’foam" cells (to which classification belong the lipid-filled "cells found in atheroma) originate from cells of the reticulo-endothelialssystem. The importance of the foam cell in the development of atherosclerosis, both in humans and in cholesterol-fed animals, is well recognized, but pathologists have varying opinions concerning the mode of formation of these cells. (a) Duff (31) believes that foam cells are all macrophages which have engulfed lipid material deposited in the intima as a result of local injury; (b) Leary (17) and Dauber and Katz (58) are of the opinion that foam cells are lipid-filled cells which have become detached from other parts of the reticulo-endothel ial system (specifically Kupffer cells from the liver sinusoids) and which enter the blood stream and actively penetrate the endothelium of the vessels; (c) Wacker and 14 Hueck (6), Heuper (59), and Altschul (28) believe that endothelial cells, and some other cell types as well, can degenerate into foam cells. These theories, as well as others, have been reviewed, and analyzed by Duff (31), Leary (17), Altschul (28), Thannhauser (60), and Gubner and Ungerleider (61). The majority opinion seems to be that lipids are accumulated in the arterial wall for reasons not as yet defined (possibly interference with lymphatic and vasa vasorum drainage of the intercellular fluid (61), disturbance of the stable emulsion due to alteration of the normal proportion of different lipid constituents of the blood and consequent precipitation of lipids in the intercellular spaces (62, 63), or filtration of lipids from the plasma by the arterial wall (64)) prior to phagocytosis by macrophages or other elements of the reticulo-endothelial system. Cholesterol ingestion results in a reticulo endothelial response in other organs of the body in addi tion to the blood vessels. In cholesterol-fed rabbits, Payne and Duff (65) observed foam cell production in the spleen, kidneys, liver, and other parts of the reticulo endothelial system even earlier than in the aorta. Altschul (28), Duff (31), and Weinhouse and Hirsch (66) also observed generalized foam cell accumulation through- 15 ; i ! I out the organs in this species as a result of cholesterol j j , i feeding. In a detailed histological study of cholesterol- ! I fed rabbits, Leary (17) found no evidence of any activity j j f i I on the part of Kupffer cells until considerable amounts of 1 cholesterol esters had accumulated in the parenchymal l ; liver cells. This overloading was followed by phago- i l i cytosis of ester cholesterol by the Kupffer cells. In i ! guinea pigs fed cholesterol, Woerner (67) reported the j : ! ; accumulation of stainable fat in the Kupffer cells but f j very little in the parenchymal cells of the liver, while, i i , in contrast, Altschul (28) observed simple fatty degenera- t . tion of the liver cells with no foam cell formation. i j . The effects of injection of colloidal suspensions i ! of cholesterol on the reticulo-endothelial system are even j i I | more pronounced. Bevans (68) injected rabbits intra- | I • ■ | venously with colloidal cholesterol, and within 3 hours | ; i [ found small amounts of cholesterol in the Kupffer cells | ! of the liver but very little in the parenchymal cells. { j She also observed cholesterol deposition in endothelial | I ! cells in kidneys, lungs, spleen, and blood vessels and • concluded that intravenously injected cholesterol was ' I ; taken up through the body of the rabbit by the reticulo- ! endothelial system, from which it was gradually cleared j ; after injections were discontinued. Similar observations j 16 were made by Tomkins (69) in mice given subcutaneous in jections of cholesterol suspensions. She observed, histologically, that when the injected cholesterol came into contact with wandering macrophages it was converted to cholesterol esters at the cell surfaces and then taken into the cells. Bloch, Berg, and Rittenberg (70) administered suspensions of deuterium-labeled cholesterol to dogs by the intravenous route.and determined the dis tribution of the administered cholesterol in the tissues. The highest concentration of labeled cholesterol was in the lung, and the next highest was in the liver. Gashin and Moravek (71) observed a similar order of cholesterol deposition in cats injected with colloidal cholesterol. Osborn jet al (72) observed statistically significant increases in cholesterol concentration in the lungs but not in the liver or other organs of dogs injected intra venously with colloidal cholesterol. It Is interesting to compare these latter three experiments with the work of Gardner and Cummings (73) who injected crystalline silica, in suspension, intra venously into rabbits. These investigators reported that crystals of the order of 12 microns were taken up from the circulation by the lung, those of the order of 6 microns were sifted out in the spleen, while those of the order of 17 1-3 microns were removed from the circulation by the Kupffer cells in the liver. Furthermore, Pollack (74) showed that the degree of atherosclerosis induced by injection of cho lesterol suspensions became more severe as the size of the cholesterol particles in suspension was increased. These facts indicate„ that cholesterol introduced directly into the blood stream in an artificially stabilized suspension is taken up by phagoeytic cells in a non-specific manner; the relatively great phagocytic activity of the lungs explains the high concentrations of injected cholesterol deposited in this organ. However, the significance of the results observed with artificial cholesterol suspensions is rendered questionable by the more recent findings of Friedman, Byers, and Gunning (75) who injected physio logically emulsified cholesterol (hypercholesterolemic rat serum obtained by injecting rats with ligated bile ducts with sodium cholate) into rats intravenously. In this case, the excess cholesterol in the blood was removed almost entirely by the liver, while only a small amount was deposited in the rest of the organs of the abdominal and thoracic cavities. Although possible variations in the response of different species to injected cholesterol complicate the comparison, it might be concluded that the cholesterol of hypercholesterolemic serum (and, presumably, 18 of normal serum) is dispersed in such a manner as to be handled entirely differently from the artificial cho lesterol suspensions previously studied, and the role, if any, of the reticulo-endothelial system in handling physiologically emulsified cholesterol becomes even more mysterious• Other experiments designed to determine the function of the reticulo-endothelial system in cholesterol metabolism utilized the techniques of splenectomy and blockage of the system through the injection of certain dyes. A variety of results were obtained. Diminished blood cholesterol levels were observed by. Goebel and Gnoins. ski (76) and by Leites (77) in splenectomized dogs and in dogs with blocked reticulo-endothelial systems. Leites (77) further reported that oral administration of olive oil and cholesterol caused less lipemia after a preliminary blocking than before blocking. He postulated that partial blockage of the reticulo-endothelial system increased its ability to take up lipids from the blood. Gol'ber (78) found that removal of the spleen and the liver resulted in a lowering of the blood cholesterol. This effect was attributed to a diminished cholesterol synthesis due to lack of a humoral factor generated by the spleen* Contradictory findings were reported by other 19 workers. After splenectomy, increased lipid levels were observed by Leites (79) in dogs and by Takagi (80) in rabbits. The retention of cholesterol in the blood after its oral or parenteral administration was also observed in both species (79, 80). Both Takagi and Leites postulated that the increase in blood cholesterol was due to decreased destruction of cholesterol by the non-functioning reticulo endothelial system. Randles and Kundsen (81) reported that splenectomy had no effect on the blood cholesterol of rats fed either normal or high-cholesterol diets, Recently, Brown et al (82) observed an elevation of blood cholesterol in guinea pigs injected with thorotrast (colloidal thorium dioxide) and trypan red; they reported that the ratio of free to esterified cholesterol was not affected by this treatment. It can be concluded, therefore, that the role of the spleen and reticulo-endothelial system in cholesterol metabolism is probably an involvement in the exchange of cholesterol and lipids between the blood and the tissues or in the storage of cholesterol in the tissues. Considerable evidence has been produced that another lipid- soluble substance, vitamin A, is stored in the Kupffer cells of the liver (88, 84) and cannot be taken up by the liver or removed from the blood under conditions of 20 reticulo-endothelial system blockage (82, 85, 86). In studying the mechanisms by which the liver of the rat handles exogenous cholesterol, it was felt that further investigations concerning the function of the reticulo- j endothelial system should be undertaken. STATEMENT OF THE PROBLEM The purpose of the work to be reported here was to investigate the sites of deposition of dietary cholesterol in the rat and to study some of the factors involved in this deposition. Deuterium-labeled cholesterol was employ ed to determine whether and to what extent exogenous cho lesterol is incorporated into the tissue cholesterol of organs other than the liver and plasma. The possibility that overloading the animal with cholesterol might influence, qualitatively or quantitatively, the site and extent of cholesterol deposition has been investigated by comparing the accumulation of deuterio-cholesterol in the tissues of rats previously fed normal and high-cholesterol diets for 30 days. The remarkable capacity of the liver to store exogenous cholesterol evidenced in these experi ments led to an investigation of the distribution of exogenous cholesterol among the constituents of liver cells separated by differential centrifugation. Evidence in the literature suggests that the reticulo-endothelial system may be involved in the uptake of lipid material from the blood and its deposition in the tissues. This possibility has been investigated by com paring the amount of deposition of dietary cholesterol in 22 the livers of rats Injected with saline or with reticulo endothelial blocking agents. v METHODS AUD MATERIALS Methods \ Lipid extraction* Soxhlet method. Most of the animal tissues studied t in these experiments were extracted by this procedure. The organ was trimmed to remove fat, mesentery, and parts of blood vessels, blotted to remove excess moisture, ground in a mortar or minced finely with scissors, and transferred to a tared extraction thimble which was then reweighed to give the weight of the tissue. A mixture of 95 per cent ethanol and ethyl ether (3:2 by volume) was added, and extraction was carried out for 8 hours. Lipid values were expressed on a wet weight basis. The efficiency of this procedure for tissue extraction has been demonstrated in this labora tory ^ m To test for completeness of extraction of fecal cholesterol, the following experiments were performed. Feces obtained from animals which had subsisted for several months on a high-cholesterol diet were dried for 48 hours at 80° C, then powdered and mixed thoroughly in a mortar. Six weighed samples were extracted by the follow ing procedures: Samples 1, 2, and 3 were subjected to 1/ Alfin-Slater, R. B., Unpublished data, 1949 24 Soxhlet extraction with ethanol-ether mixture (3:2) for 8, 16, and 24 hours, respectively; Sample 4 was extracted in the Soxhlet apparatus for 8 hours with 95 per cent ethanol, followed by extraction for an additional 8 hour period with ethyl ether; and Samples 5 and 6 were extracted by the alkaline hydrolysis method described below, and the re sidues remaining after digestion were collected on filters and extracted in the Soxhlet apparatus with ethanol-ether (3:2) for 16 hours. Cholesterol analyses were performed on each of the extracts obtained from Samples 5 and 6 and on those obtained from Samples 1 through 4. The results (Table I) demonstrate that extraction by the Soxhlet method with a 3:2 mixture of ethanol-ether for 8 hours effectively removes the cholesterol from samples of dried fecal material. Therefore, this method was used in the experiments to be described, with the exception that a 16 hour period of extraction was used as an extra pre caution. Alkaline hydrolysis method. The material to be extracted was digested for three hours in a covered con tainer with 20 per cent (w/v) sodium or potassium hydroxide in 67 per cent (v/v) ethanol. The hydrolysate was neutralized and acidified with 12 N hydrochloric acid, cooled, and extracted five times with ethyl ether, or until 25 TABLE I THE COMPARISON OF DIFFERENT METHODS OF EXTRACTING CHOLESTEROL FROM FECES Sample number — Extraction method Cholesterol mg./gm. 1 Soxhletj ethanol-ether (3:2); 8 hrs• 40.6 2 Soxhletj ethanol-ether (3:2);16hrs. 40 .7 3 Soxhlet; ethanol-ether (3:2);24hrs. 42 .2 4 Soxhlet; ethanol 8 hrs., then ether 8 hrs • 38.4 5 Alkaline hydrolysis followed by ether extraction in separatory funnel 36.2 5R Soxhlet; ethanol-ether (3*2); 16 hrs. 3.3 39.5 6 2/ Alkaline hydrolysis followed by ether extraction in separatory funnel 39.5 6R Soxhlet; ethanol-ether (3:2); 16 hrs. 0.5 40 .0 a/ Feces from rats fed a diet including 1% cholesterol, 0 bile salt, and ZOfo fat were pooled, dried, powdered, and mixed thoroughly. Samples weighing 2 gm. were used. b/ Residue remaining after alkaline hydrolysis and extraction of Sample 5 • o/ Residue remaining after alkaline hydrolysis and extraction of Sample 6. 26 the extract was colorless. If a residue remained after hydrolysis, it was filtered off and extracted in the Soxhlet apparatus. This method was particularly effective for handling carcasses, because the alkaline digestion dissolved fur* and skin, while the solution of most of the bones was completed by acidification of the hydrolysate. Waring blendor method. This procedure, an adapta tion of the technique of Thompson e_t al (87), is useful for the extraction of lipids from liquid or semi-liquid material which cannot be conveniently handled by the Soxhlet technique nor, in cases in which.hydrolysis of ester linkages is undesirable, by the alkaline hydrolysis method. Therefore, it was used for the extraction of small amounts of liver homogenates and of ultracentrifugally prepared cell fractions. The material, to be extracted was transferred quantitatively to a Waring blendor and homogen ized for 10 minutes with a mixture of water, .ethanol, and petroleum ether (b.p. 63.3 - 69.3° C) in a ratio of 1:2:10 by volume. (The volumes used were usually 10 ml. of water, 20 ml. of ethanol, and 100 ml', of petroleum ether.) The * blendor contents were then poured into a separatory funnel, and the ethanol-water layer was separated from the ether layer, transferred back into the blendor cup, and re extracted twice for 5 minute periods with 100 ml. portions 27 of petroleum ether. The extracts were pooled, concentrated by evaporation, and filtered into volumetric flasks for % cholesterol analysis. The effectiveness of this method for extracting cholesterol from liver homogenates was tested in three experiments. In the first experiment, 4 ml. aliquots of a normal rat liver homogenate were extracted as described. The alcohol-water residues were then digested with alkali and extracted with ethyl ether. The ether extracts of the residues were found to contain only negligible amounts of cholesterol. This finding was confirmed in a second similar experiment. Furthermore, the amount of cholesterol extracted from samples of a liver homogenate by the Waring blendor method was the same, within the range of experi mental error, as the amount extracted by the alkaline hydrolysis method (Table II). In order to establish the optimum period of extraction, the cholesterol extracted , from two identical samples of liver homogenate in each of four successive extraction periods was determined. The first extraction period was 10 minutes in duration, while the rest were 5 minutes. In both of the samples the major part of the cholesterol was removed in the first 10 minute period (Table III). A small but significant quantity was removed in the second period, and only negligible amounts 28 TABLE II THE COMPARISON OF THE WARING BLENDOR AND ALKALINE HYDROLYSIS TECHNIQUES FOR THE- EXTRACTION OF CHOLESTEROL FROM RAT LITER HOMOGENATES Sample number Extraction method Cholesterol mg ./4 ml. homogenate 1 Waring blendor 1.98 1R £/ Alkaline hydrolysis 0.00 2 Waring blendor 2.09 2R b/ Alkaline hydrolysis 0.00 3 Alkaline hydrolysis 2 .18 4 Alkaline hydrolysis 2.18 w Each sample consisted of 4.0 ml. of a homogenate prepared from 3.0 ml. of 0.88 M sucrose per gm. of normal rat liver. b/ The ethanol-water residue remaining after Waring blendor treatment of the original sample • 29 TABLE III . THE EFFICIENCY OF THE WARING BLENDOR TECHNIQUE ON SUCCESSIVE EXTRACTIONS OF CHOLESTEROL FROM A RAT LIVER HOMOGENATE Extraction time Cholesterol = 5 ---- - . . SamPl9 1 . SemPle 2 mg./4ml. homogenate mg./4 ml. homogenate 10 2.29 2.46 5 0.18 0.11 5 0.04 0.00 5 0.00 0.00 Total 2.51 2.5? 30 were found In the third extract* Extraction of plasma* Aliquots of plasma were placed in 50 ml. centrifuge tubes. The plasma proteins were precipitated by the addition of 14 ml. of ethanol- acetone (1:1) for each 1 ml, of plasma. Thorough mixing of the ethanol-acetone solution with the plasma was effected by adding the solvent rapidly and forcefully from a hypodermic syringe. The tubes were then centrifuged for 5 minutes at 2500 R.P.M., and the extracts were decanted Into storage bottles for later analysis. Extraction of urine. The method to be described was adapted from one which was used by Callow et al (88) for the extraction of urinary 17-ketosteroids. Fifteen ml. of 12 N hydrochloric acid were added for each 100 ml. of urin^ and the mixture was refluxed for 15 minutes in order to hydrolyze cholesterol esters. The solution was immediately cooled in an ice bath and extracted five times with ethyl ether. The combined extracts were washed four times with 2 N sodium hydroxide solution and finally twice with water. Cholesterol analysis. A modified Schoenheimer-Sperry technique as des cribed by Nieft and Deuel (89) was used for the determina- 31 tion of total cholesterol. This method involved the hydrolysis of cholesterol esters with alkali, precipitation 2/ of the cholesterol with digitonin , splitting of the digitonide with glacial acetic acid, and addition of the Liebermann-Burchard reagent which reacted with the liberated cholesterol to produce a green color which was then read in a Klett-Summerson photoelectric colorimeter. When free cholesterol values were required, the alkaline hydrolysis step was omitted, and the unesterified cho lesterol precipitated directly with digitonin. Esterified cholesterol values were calculated from the difference between total and free cholesterol. Total lipid analysis. Total lipids were measured in the liver extracts by the following technique. An aliquot of the ethanol-ether extract was evaporated almost to dryness, taken up in petroleum ether (b.p. 63.3 - 69.3° C), and dried over anhydrous sodium sulfate. The petroleum ether extract was then decanted through Vifhatman #42 filter paper into a weighed flask. The sodium sulfate was washed five times -with 10 ml. portions of the solvent, and the filter paper was similarly treated. The extract in the weighed flask 2/ Digitonin, L. Light and Co. Ltd., Colnbrock, 32 was then evaporated to dryness, the residue dried over night at 80° C, and the flask reweighed. The difference between the weights of the flask before and after addition of the lipid residue was taken as the weight of total lipids in the aliquot used. Results were expressed as mg. total lipid per gm. of wet liver. Deuterium analysis. Preparation of the cholesterol digitonide. The tissue extract containing the deuterio-cholesterol to be analyzed was treated with potassium hydroxide at 60° C for one hour to hydrolyze the esters. The mixture was then neutralized and acidified slightly with 15 per cent acetic acid, and for each milligram of cholesterol present, one milliliter of a solution of 0.5 per cent digitonin in 50' per cent ethanol (a calculated excess) was added. The mixture was allowed to stand at room temperature over night to permit complete precipitation of the cholesterol digitonide. The precipitate was separated by centrifuga tion and washed three times with hot distilled water (or until all color was removed), three times with ethanol- acetone (1:1), and finally three times with anhydrous ethyl ether. If deuterium was to be estimated in both the free 33 and esterified cholesterol of a given extract, the solution was first acidified slightly (without preliminary alkaline hydrolysis), and digitonin .was added to precipitate the free cholesterol. After removal of the precipitate by centrifugation, alkali was added to the supernatant solu tion, and hydrolysis of the esters was carried out as described above. Deuterium measurement. The pure white cholesterol digitonide was dried in a vacuum desiccator, and duplicate portions weighing from 6 to 20 mg. were combusted over copper oxide. The resulting deuterium-enriched water samples were collected and reduced over zinc to mixtures of hydrogen and deuterium gas, and the ratios of deuterium to hydrogen were determined in a Consolidated Nier Isotope Ratio Mass Spectrometer (90). The ratios obtained for the digitonides were converted to atom per cent deuterium by referring to a standard curve and recalculated for cho lesterol by multiplying by a factor of three. Materials Animals. Female albino rats from the University of Southern California colony were used in these experiments. 34 Diets. Diet.A. Purina laboratory chow, Ralston Purina Co., St. Louis, Mo. Diet B. The basal synthetic diet is described in- Table IV. Diet C. .The basal synthetic diet (Diet B) supple- 3/ mented with 0.5 per cent ox bile • Diet D. The basal synthetic diet (Diet B) supple mented with 1 per cent cholesterol ^ in replacement for 1 per cent of the lipid. Diet E. This diet was identical to Diet D, except for the further addition of 0.5 per cent ox bile. Extract of ox bile powder, U.S.P., Armour and Co., Chicago, 111. 4/ Cholesterin, Merck and Co., Rahway, New Jersey. 35 TABLE IV COMPOSITION OF DIETS Weight of component/100 gm. diet Diet B Diet C Diet D Diet E gm. gm. gm. gm. Commercial casein 27.0 27.0 27.0 27 .0 Sucrose 29.5 29.0 29.5 29.0 Corn starch 30.5 30 .5 30 .5 30.5 Celluflorn* 3.0 3.0 3.0 3.0 Salt mix — 4.0 4.0 4.0 4.0 Fat soluble vitamin mix —^ 1.0 1.0 1.0 1.0 c/ Cottonseed oil — 5.0 5.0 4.0 4.0 Cholesterol —^ 0.0 0.0 1.0 1.0 Bile extract — 0.0 0.5 0.0 0.5 / Water soluble vitamins — ___ ___ 36 FOOTNOTES. :T0 TABLE IT a/ Wesson modification of the Osborne-Mendel salt mixture • b/ 1.0 gjn. mixed tocopherols (Mixed Tocopherols, Type 4-34, Distillation Products Industries, Rochester, N. Y.,)and 40 mg. Nopsol (a stable, -water dispersible vitamin A and D mixture with 50,000 I. IJ. of vitamin A and 10,000 I. U. of vitamin I>2 per g?n.) mixed well with 10 gm, cottonseed oil • c/ Wesson oil. d/ Cholesterin, Merck and Co., Rahway, New Jersey. e/ Extract of ox bile powder, U.S.P., Arm our and Co., Chicago, 111. i/ 250 T thiamin hydrochloride, 500 Y riboflavin, 200 Y pyridoxine hydrochloride, 2 mg. calcium pantothenate, 100 mg. inositol, 100 mg. choline chloride, 250 Y niacin, 50 Y menadione, 100 Y folic acid, 10 Y biotin, and 50 mg. paraaminobenzoic acid per 100 gm.of diet. The vitamins were mixed thoroughly with casein before adding to the other ingredients• 10 ml. of a solution of vitamin B-^ in 95 Per cent ethanol, 0.1 Y/ml., were added to the dry ingredients and mixed well before adding the lipid ingredients EXPERIMENTAL AND RESULTS Preparation of Deuterium-Labeled Cholesterol Deuterium-labeled cholesterol was prepared by two methods: (a) a catalyzed exchange reaction according to the method of Bloch and Rittenberg (91), and (b) isolation from the eggs of hens fed deuterio-acetate and deuterium oxide (92). The exchange method of Bloch and Rittenberg (91). Cholesterol was heated for 72 hours under pressure with deuterium oxide and deuterio-acetic acid (CD3COOD) in the presence of active platinum as a catalyst. Under these conditions, some of the hydrogens atoms of the cho lesterol exchanged with the deuterium of the medium, and the resulting cholesterol contained 2.6 atoms per cent deuterium. It has been demonstrated by Fukushima and Gallagher (93) that in labeled cholesterol prepared in this manner, the deuterium is distributed equally between the ring and the side chain, with 40 per cent of the isotope at C-6, 3 per cent at C-3, and 50 per cent at or among C-24, C-25, C-26, and C-27. The isotope introduced into the steroid molecule in this manner is stably bound in the sense that exchange of deuterium atoms for hydrogen atoms does not occur in an aqueous solution (91, 94)._____________ 38 The deuterio-acetic acid used in this reaction was prepared as follows: deuterated malonie acid, formed by direct reaction of malonie acid with deuterium oxide for five hours under reflux, was decomposed by heating at 165° G as described by Halford and Anderson (95). The result ing deuterio-acetic acid contained 60 atoms per cent deuterium. • The procedure used in this investigation for the preparation of deuterio-cholesterol differed from that of Bloch and Rittenberg in that methanol was found to be a more satisfactory solvent than was acetone for recrystalli zation of the cholesterol. Biological preparation from the hen’s egg. It has been shown that when deuterium oxide or deuterium-labeled acetate are administered to. rats, the isotope is incorporated into the cholesterol of the tissues (96, 97). The isotope is stably bound to the carbons of the cholesterol molecule and is located in both the nucleus and side chain (97). The hen’s egg is a tissue which is known to have a relatively high concen- , , tration of cholesterol; moreover, the egg can be obtained without sacrificing the animal. Assuming that the pre cursors involved in cholesterol synthesis are the same in 39 the chicken as in the rat, an attempt was made to prepare deuterium-labeled cholesterol by feeding deuterio-acetate and deuterium oxide to laying hens and isolating the cho lesterol from the egg yolk* Concurrently, Kritchevsky and Kirk (98)" reported that when sodium acetate-G"^^ was fed to laying hens, there was a rapid incorporation of the radio active carbon into the cholesterol of the eggs. An isotonic solution of sodium acetate, labeled in * the methyl group with 9.0 atoms per cent deuterium, dissolved in 2.5 per cent deuterium oxide was administered in the drinking water to two laying hens for 7 days. Sub sequently, only 2.5 per cent deuterium oxide was given for 19' days. Eggs were collected as available starting from the first day for a period of 44 days. . To separate the yolks from the whites, the eggs were immersed in boiling water for 10 minutes .after which the coagulated white portions were discarded. Cholesterol was extracted from the mashed yolks by Soxhlet extraction with ethanol-ether (3:2) for 8 hours. On analysis of 24 eggs, the individual yolks were found to contain an average of 16 mg. of cho lesterol per gm. of coagulated' yolk and a total of 200 to 250 mg. of cholesterol per yolk. Cholesterol was pre cipitated and isolated as the digitonide, and the deuterium concentration was determined as previously described. 40 The incorporation of deuterium into the egg yolk cholesterol of hen number 1 over the experimental period is shown in Figure 1. The isotope appeared in the cho lesterol as early as the second day of the experiment. The maximum deuterium concentration (0.6 atoms per cent) was reached on the 12th day, 5 days after withdrawal of the deuterio-acetate from the drinking water. This con centration was maintained through the 32nd day, after which there was a rapid decrease in the amount of label. Cholesterol digitonide precipitates of low (0.1 to 0.3 atom per cent), medium (0.4 to 0.6 atom per cent), and high (over 0.6 atom per cent) deuterium enrichment obtained from these eggs and from the tissues of animals used in cholesterol turnover studies in this laboratory were pooled, and the deuterio-cholesterol was recovered by splitting the digitonide with pyridine as follows. Recovery of cholesterol from cholesterol digitonide. Cholesterol digitonide, thoroughly washed and dried, was dissolved in pyridine (approximately 1 ml. of pyridine per 20 mg. of digitonide) and heated in a boiling water bath for one hour with occasional stirring. Five volumes of ethyl ether were added to the cooled solution while stirring. Cholesterol remained in solution; the digitonin A T O M S X DEUTERIUM I N CHOLESTEROL 41 071 0 - 6 0 4 - 0 3 * 0 2* 01 TIME IN DAYS A* D-ACETATE (9 0 ATOMS % EXCESS) IN 2-5% 0,0 B - 2 5% 0 ,0 INCORPORATION OF DEUTERIUM INTO CHOLESTEROL OF YOLK OF HEN’S £ 6 6 FIGURE 1 42 precipitated and was removed by centrifugation. The pre cipitate was treated again with pyridine to split any re maining cholesterol digitonide. The ether supernatant containing the cholesterol > was transferred to a separatory funnel and washed repeatedly with 3 N sulfuric acid to remove pyridine and finally with distilled water until neu* tral to litmus. The ether solution was then dried over anhydrous sodium sulfate and evaporated to dryness. The residue was dissolved in methanol and the cholesterol re crystallized from this.solvent three times. The yield from this procedure amounted to approximately 50 per cent of the theoretical. The resulting product had a melting point of 145-147° C (theoretical m.p. 148.5° C) and when tested with the Liebermann-Burchard reagent gave color intensities identical with those obtained with the ehd- 5/ lesterol used as a standard in this laboratory. h/ Cholesterin, Merck and Co., Rahway, New Jersey. 43 Deposition, of Deuterium-Labeled Cholesterol in the Tissues of the Rat Experiment I: Determination of the distribution of exo genous cholesterol in the tissues of the rat. Experimental procedure. Pour female rats weighing 180 gm. and reared from weaning on Purina chow (Diet A) were fed, for 8 days, Diet D (Table IY) in which the cho lesterol was labeled with 0.9 atom per cent deuterium. During this feeding period, the animals were kept in in dividual metabolism cages which fitted over large glass funnels. Urine was collected in a tube just beneath the stem of each funnel. Peces were deflected into a separate container by a pear-shaped glass bulb supported on glass hooks in the opening of the urine collection tube. Urines of the four animals were pooled over the 8 day period; feces were treated in the same way. The excreta were stored under ethanol-ether (3:2) in the refrigerator until analyzed. Each rat was given 10 gm. of food daily (except for the 8th day, when each received 6 gm.) in a cup which was fastened inside a larger receptacle to minimize spilling. The animals ate all of the food. The amount of labeled cholesterol ingested by each rat during the experimental 44 period was 760 mg. Pour female rats of similar age and weight which had been fed the Purina chow were sacrificed to obtain normal organ cholesterol values. At the end of the experimental period, the rats were sacrificed by administration of an anesthetic dose of Nembutal (0.15 ml. of a solution containing 60 mg. per ml./lOO gm. body weight) and withdrawal of the blood by heart puncture. The organs to be examined were quickly removed and trimmed. Liver, kidneys, lung, spleen, and brain were mashed with a mortar and pestle, while the ovaries, heart, aortas, and small intestine (after thorough washing) were minced finely with scissors. These ground tissues were then placed in individual weighed Soxhlet thimbles and extracted with ethanol-ether (3:2) for 8 hours. The adrenals were removed from their capsules before being mashed against the inside of an extraction thimble.- Livers were extracted individually, brains, kidneys, and intestines in pairs, and the other organs were pooled for the four animals. The blood was treated with heparin to prevent clotting and centrifuged to separate plasma from cells. The plasma was extracted with ethanol-acetone (1:1) as previously described. The volume of blood cells was 45 measured, and the cells of the four rats were pooled and transferred with ethanol-ether to an extraction thimble and extracted as described above. Body water was obtained from the pooled ground carcasses by lyophilization. The dried carcasses were then hydrolyzed by refluxing with ethanolic sodium hydroxide and extracting with ether as described previous ly- The combined feces were dried, powdered, and weighed. The dry material was divided into four portions and extracted for 16 hours with ethanol-ether (3:2) in the Soxhlet apparatus. . The extracts of tissues and feces were adjusted to a convenient known volume, and total cho lesterol analyses were performed on suitable aliquots. Total cholesterol was precipitated as the digitonide from the organ and feces extracts and analyzed for deu terium content. The quantities of cholesterol in the ovaries> aortas, and urine were so limited that insuffic ient cholesterol digitonide.was obtained for. a deuterium measurement. The body water was distilled over alkaline potassium permanganate to remove organic material, reduced over zinc, and the ratio of deuterium to hydrogen was determined in the resulting gas. 46 Results. The organ cholesterol values of the animals fed the diet containing 1 per cent deuterio- cholesterol are compared in Table V with those of the Purina-fed animals. Significant and variable amounts of cholesterol were deposited in the liver and plasma of the rats fed cholesterol. Less striking increases in the cho lesterol concentration, expressed as mg./gm. of tissues, occurred in the adrenal glands, lungs, and intestines. Although the cholesterol concentration in the spleen was only slightly elevated after cholesterol feeding, the total rag. of cholesterol in this organ was significantly greater in the experimental group than in the controls, owing to a slight splenomegaly in the former, an effect of cholesterol feeding which has frequently been observed in this laboratory. The lower total cholesterol value in the intestine of the group fed deuterio-cholesterol was due to more vigorous scraping of the mucosa in the process of washing and to consequent loss of tissue. Deuterium-labeled cholesterol was found in all the tissues examined, although only a negligible amount was deposited in the brain'(Table VI). The deuterium con centration was highest and almost identical in the liver, spleen, lung, adrenal, and heart and decreased respectively TABLE V THE EFFECT OF CHOLESTEROL INGESTION s/ ON THE CHOLESTEROL CONTENT OF RAT’ TISSUES Experiment I V Cholesterol Mg./gm. M'g. total 0 days £/ 8 days £/ 0 days 2/ 8 days 2/ Liver £./ 1.56 5.08 12.0 36.6 1.80 5.90 13.7 33.3 1.52 . 5.61 12 .1 37 .2 1.78 15.4 13.9 85 .9 Kidneys 4.35 4.42 5.31 5.94 Spleen 3.22 3.84 1.66 2.94 Lungs 4.98 5.92 3.90 4.65 Adrenals 45 .2 58.8 1.12 2.19 Brain 14.5 15 .9 17 .1 18.8 Small intestine . 1.90 3.02 3.65 1.85 Red blood cells $/ 1.19 1.13 7.74 6.90 Plasma <V 34.9 2/ 51.1 2.27 3.12 (27.0 - 39.0) (38.3 - 66.0) (1.75 - 2.54) (2.34 - 4.02) Ovaries 8.26 7.27 0.41 0.46 Heart 1.38" 1.55 0.70 0.88 Aorta 3.55 2.89 0.04 0.06 Carcass « ■ ■ ■ (290) y Feces 25.1 159 FOOT NOTES TO TABLE V Each of the cholesterol-fed rats received 760 mg. of cholesterol labeled with 0.9 atom per cent deuterium in a period of 8 days. Values are averages for 4 rats in each group, except in the case of the livers where individual values are given. Number of days on cholesterol diet • Values were calculated by assuming a blood volume of 6*7 ml*/ 100 gn. body weight and a hematocrit of 50 (99)* These values are expressed as mg. per cent. The values in parentheses are ranges for the 4 animals. This is a calculated value. A carcass cholesterol concentration of 0.2 per cent was assumed (100)* TABLE VI THE DEPOSITION OF DEUTERIUM-LABELED CHOLESTEROL s/ m THE TISSUES AND FECES OF THE RAT Experiment I Cholesterol i/ Tissue Mg. total Deuterium, content 2i Exogenous deposition mg. atom % mg./gm. mg. total % of ingested h/ Liver Si 36.6 0,44 2,48 17.9 2.36 33.3 0.43 2.86 . 16.1 / (18.3)— 2.12 37 .2 (2 .76)5/ (2 .41)5/ 85 .9 0.44 7.53 42.0 5.52 Average 23.6 Average 3,10 Kidneys 5.94 0.18 0,89 1.19 0.16 Spleen 2.94 0.45 1.92 1.47 0.19 Lungs 4.65 0.45 2.96 2.33 0.31 Adrenals 2.19 0.47 30.6 1.14 0.15 Brain 18.8 0.01 0.18 0.21 0.03 Intestine Red blood . cells Si Plasma */ 1.85 0.30 1.02 0.62 0.07 6.90 0.38 0.48 2.91 0.38 3.12 0.32 0.18 1.11 0.15 Heart 0.88 0.46 0.80 0.45 0.06 Carcass £/ (290) 0.25 (0.56) (80.0) (10.5) Feces 159 0.87 6.10 , 154 20.3 Total - 269 35.4 t o 50 FOOTNOTES TO TABLE VI Each of the cholesterol-fed rats received 760 mg. of cholesterol ( labeled with 0.9 atom per cent deuterium in a period of 8 days. Values are averages for 4 rats in each group, except in the case of the livers where individual values are given. Calculated by isotope dilution. This sample was lost before a deuterium measurement had been made. Therefore, the deuterium concentration of the liver cholesterol of the other animals was used in calculations for this rat • Values were calculated by assuming a blood volume of 6.7 ml./ 100 gm. body weight and a hematocrit of 50 (99) • The values in parentheses were calculated. A carcass cholesterol concentration of 0,2 per cent was assumed (100) . 0 51 in blood cells, plasma, small intestine, carcass, kidney, and brain. It is interesting that in spite of the greater quantity of cholesterol deposited in the liver of the fourth rat, as compared to the other cholesterol-fed rats, the deuterium concentration of the liver cholesterol in this rat was identical to that in the others. The deu terium concentration of the fecal cholesterol was equal, within the range of experimental error, to that of the cholesterol ingested. Cholesterol deposition was estimated by calculating the dilution of deuterium-labeled cholesterol by the cho lesterol already in the organ; the results are shown in Table VI as total mg., mg./gm. tissue, and as per cent of deuterio-cholesterol ingested. In Order to make these calculations, it was assumed that prior to absorption, the labeled cholesterol of the diet was not diluted with any unlabeled cholesterol which might have been present in the intestine and that, therefore, the absorbed cholesterol had a deuterium concentration equal to that of the cho lesterol in the diet, or 0.9 atom per cent. Of the organs studied, the liver was quantitatively the most important site of cholesterol deposition, the amount of cholesterol deposited there being almost 10 times that found in any of the other organs. ; 52 It was possible to obtain an estimate of the per centage of ingested cholesterol recovered from the tissues and feces by making the following assumptions: (a) In order to calculate the total cholesterol content of the plasma and blood cells, a blood volume of 6.7 ml./lOO gm. body weight was assumed (99). '(b) Although total carcass weights and cholesterol content were not measured, it was shown in Experiment II that the carcasses weighed approxi mately 80 per cent of the total body weight and that the cholesterol concentration was approximately 0.2 per cent, in agreement with the value reported by Lange (100). Calculating on this basis, the total carcass cholesterol was estimated. (c) Since no appreciable amounts of food were spilled or remained uneaten, the total amount of cholesterol ingested was taken to be equal to that given to the animals, or 760 mg. The quantity of labeled cho lesterol recovered from the organs, carcasses, and feces amounted to only 35 per cent of that fed (Table VI). This low recovery will be discussed later. The deuterium concentration in the body fluids was found to be. negligible. This may indicate that the amount of cholesterol which was broken down by the tissues into water was so low that dilution with the body fluids present made detection impossible by the analytical methods used. 53 Experiment II: Comparison of the distribution of deuterio- cholesterol in the tissues of rats previously fed normal and high-cholesterol diets. Experimental procedure. Two groups of 8 female / rats were used. Starting at weaning, Group I was fed Diet C (Table IV), and Group II was fed Diet E, which was identical to Diet C except that 1 per cent of the fat was replaced by cholesterol. After a 30 day period, which will be referred to as the pre-feeding period, 4 rats of each group were sacrificed, and the cholesterol content of the organs was determined. These animals were designated as Groups la and. Ila. The remaining animals (Groups lb and lib) were placed for 7 days on Diet E, the cholesterol of which was labeled with 0.85 atom per cent deuterium* During this period, the animals were kept in metabolism cages, and urine and feces were collected and pooled each day for the two groups. Pood consumption was measured daily. The cholesterol ingested by each animal during the experimental period amounted to 740 mg. for Group lb and 690 mg. for Group lib. On the fifth day, one of the rats of Group lib escaped from its cage for several hours and was dropped from the experiment. The animals were sacrificed; the cholesterol content of the organs was 54 measured; and the deuterium concentration of the cho lesterol was determined as in the preceding experiment, with the following modifications. Separate analyses of free and total cholesterol was performed on the liver and plasma of each rat in the four groups. The deuterium concentration was also deter mined for free and esterified cholesterol in the livers of the animals fed deuterio-cholesterol. Due to the small amount of cholesterol present, it was not possible to carry out a similar procedure with plasma; therefore, the extracts were pooled for each group and the total plasma cholesterol precipitated as the digitonide for deuterium analysis. The carcasses of the animals fed deuterio- cholesterol (Groups lb and lib) were pooled for each group; the body water was separated; and cholesterol was extracted as in Experiment I. During the lipid extraction process, the extracts for the two groups were accidentally combined so that it was not possible to determine the concentration and deuterium content of the cholesterol for each group. The carcasses of the rats in Groups la and Ila were treated individually by the alkaline hydrolysis method, and the cholesterol content of the extracts was determined. 55 Each day’s feces output was extracted separately for each group, while the urine was pooled over the 7 day period before extraction. The total content of cholesterol was not measured in the urine but was precipitated as the digitonide for deuterium analysis. Results. The effect of 0, 7, 28, and 55 days of cholesterol feeding upon the organ total cholesterol values is shown in Table VII. No increase in cholesterol concentration (mg./gm. tissue) occurred in lung, kidney, spleen, intestine, brain, adrenal gland, blood cells, or heart. The liver and plasma showed definite and large increases, the liver as early as 7 days and the plasma after 28 days. A significant increase in cholesterol concentration was also apparent in the carcass. A gradual enlargement of the spleen during the 35 day period was observed, although the cholesterol concentration per gram of spleen did not change. The rise in liver cholesterol could be largely accounted for by esterified cholesterol, as shown by the marked decrease in the ratio of free:total cholesterol after only 7 days of cholesterol feeding (Table VIIlO;. Parallel changes occurred in the plasma to a lesser degree. In general, it can be stated that in this experiment, whenever a marked rise occurred in the cholesterol TABLE VII THE EFFECT OF CHOLESTEROL FEEDING FOR VARIOUS TIME PERIODS ON THE TOTAL CHOLESTEROL CONTENT IN TISSUES AND FECES OF THE RAT Experiment II Tissue oholesterol values I !g. eholesterol/gn. tissue Total mg. cholesterol Tissue (Diet C) prefed 30 days (Diet E) prefed 30 days (Diet C) prefed 30 days (Diet E) prefed 30 days (Group la) (Group lb) (Group Ha) (Group lib) (Group la) (Group lb) (Group Ha) (Group lib) 1 o 7 days 0 days 7 days 0 days 7 days 0 days 7 days Liver 2.19 / 17.2 19.2 29.3 15 tS 93.0 140 190 (1.93-2.36)2/ (12.1-24.4) (17.6-20.2) (22.4-36.2) (10.8-18.8) (67.3-131.0) (109-170) (133-265) Fidneys 4.74 4.70 4.64 4.70 6.04 6.34 6.00 5.92 Spleen 3.57 3.44 3.60 3.62 1.51 1.81 2.19 2 .55 Lungs 6.64 6.68 5.76 5.49 3.48 4.29 3.85 3.75 Adrenals 46.2 43.5 48.9 32.1 0.96 1.02 1.06 0,76 Br ain- 14.2 14.6 14.6 14.8 14.8 16.0 15.7 15.8 Intestine 1.80 1.58 1.93 1.95 2.35 1.83 2.36 2.17 Blood cells V 1.31 1.29 1.41 1.26 6.44 6.42 7.00 6.13 Plasma & / 79.6 72.7 154,4 116.5 3.92 3.64 7.58 5.67 (62.3-106 .0} (50 .0-110.0) (138 .0-186 .0) (96 .8-150.8) (2.92-5,68) (2.52-5 .61) (6.47 - 8.28) (4.63-7 .33) Peart 1*48 1.83 1.56 1.56 0.70 <0.99 0.77 0.30 Vercass 1.54 (2.00)2/ 2.38 (2 .SO)®/ 179 (248)®/ (274)2./ (298)2./ Feces mmmm • • 1 1 1 9 a 1 364 347 a/ Values ere averages for 4 rats in each group, exoept Group lib which was composed of 3 rats* b/ Number of days on deuterio-cholesterol diet, c/ Values in oerentheses are ranges for each group. df Values were calculated by assuming a blood volume of 6.7 ml./lOO gn. body wei^vt and a hematocrit of 50 (99). e/ These values were arrived at by extrapolation, in order to provide a basis for further calculations. vn O ' 57 TABLE VIII RATIO OF FREE TO TCTAL CHOLESTEROL IN THE LIVER AND PLASMA OF RATS FED A 1 PER CENT CHOLESTEROL DIET Experiment II Mg. free cholesterol/mg. total cholesterol Tissue Normal diet (Diet C) prefed 30 days Cholesterol prefed diet (Diet E) 30 days Group la Group Tb 0 days 7 days Group Ila 0 days Group lib 7 days Liver 0.93 , 0.22 . . . (0.87-1.00) ~ (0.18-0.26) 0.17 0.13 (0.16-0 .19) (0.11-0 .14) Plasma 0.26 0.18 0.16 0.17 (0.24-0.28) (0.14-0.21) (0.13-0.18) (0.15-0.22) a/ Number of days on the deuterio-cholesterol diet • b/ Values given are averages and ranges for 4 rats in each group exeept Group lib which consisted of 3 rats • 58 concentration of either liver or plasma, the increase was predominantly in the esterified fraction, while the free cholesterol levels were only slightly elevated over the normal values. In contrast, the ratio of free to total cholesterol of the blood cells was not influenced by the cholesterol-rich diet but remained constant at 0.95. The fecal cholesterol of both the rats prefed Diet C, the control diet (Group lb), and those prefed Diet E, the high-cholesterol diet (Group lib), was fairly constant, ranging from 65-75 mg./gm. of dried feces after the first day on the deuterio-cholesterol diet (Table IX). This value is markedly higher than the 25.1 mg./gm. excreted by the rats fed deuterio-cholesterol in Experi ment I • Labeled cholesterol was found in all the tissues examined in Groups lb and lib (Table X). Insufficient cholesterol digitonide was obtained from the plasma of Group lb for a deuterium analysis. In the animals prefed Diet C (Group lb) the deuterium concentration of the cho lesterol was highest and almost equal in the liver (both free and esterified), adrenals, and blood cells and de creased, respectively, in the lung, spleen, intestine, carcass, kidney, and brain. These findings are essen tially in agreement with those of Experiment I. In TABLE U CHOLESTEROL CONCENTRATION AND DEUTERIUM CONTENT IN THE FECES OF RAIS FED DEUTERIO-CHOLESTEROL s/ Number of days on Cholesterol deuterio-cholesterol Mg./ga. feces Total mg. Atom % deuterium Mg. deuterio-cholesterol Group lb 1 15.7 14.9 . 0.56 9.8 2 70 .9. 56.7 0,71 47 .3 3 68.4 56.0 0.81 53.3 4 68.0 57 .1 0.81 54.4 5 73.3 65 .5 0.81 62,4 6 77 .2 64.9 0.81 61.8 7 76.0 58.5 ; -0 .81 55.7 Total 344.7 Group lib ' 1 95.4 76.3 0.11 9.9 2 60.4 34.4 - 0.15 6.1 3 77.2 ‘ 43.2 0.76 38.6 4 82 .3 59.2 - 0.75 ’ 52 .3 5 70.8 59.5, 0.85 59.5 6 65 .9 46.7 0.85 46.7 7 69.0 47.6 0.80 44 .9 Total , 258*0 W to FOOTNOTES TO TABLE IX The deuterio-cholesterol contained 0.85 &tom % deuterium. Bach rat of Group lb ingested 740 mg. of deuterio-cholesterol. Each rat of Group lib ingested 690 mg. of deuterio-cholesterol. Values were obtained on pooled samples from 4 rats . Values were obtained on pooled samples from 4 rats, from the first to the third day and from 3 rats thereafter • TABLE X DISTRIBUTION OP EXOGENOUS CHOLESTEROL ^ IN TISSUES AND EXCRETA OF RATS PREVIOUSLY FED A LOK-CHOLESTEROL OR A HIGH-CHOLESTEROL DIET FOR 30 DAYS Experiment II Total oholesterol Deuterium oontent in oholesterol Deuterio-oholesterol deposited Qroup lb y Qroup lib y Qroup lb Qroup lib Group lb Group lib Group lb Group lib m£. s&* atom ^ atom % mg./gn. ■svV* mg. mg. Liver Free 19.7 23.4 0.61 0.39 2.18 1.68 11.8 10.7 Esterified 73.0 166 0,51 0.35 8.10 10.7 43.8 68.4 Kidneys 6.54 6.92 0.16 0.14 0.89 0.77 1.19 0.98 Spleen 1.81 2.65 0.43 0.39 1.74 1.61 0.92 1.17 Lungs 4.29 3.75 0.44 0.33 2.94 2.13 2.22 M 6 Adrenals 1;02 0.76 0,50 --- 25.6 0.60 m m m m Brain 16.0 16.8 0.11 — - 0.19 --- 0.21 m m m m ' Intestine 1.83 2.17 0.32 0.32 0.59 0.73 0.69, 0.82 Red blood oells St 6.42 6.13 0.49 0.38 0.74 0.56 3.70 2.74 Plasma s/ 3.64 5.67 aeamaeae 0.46 ■ M sw a a 63.0 m m m m 3.07 Caroass (248)d/ (298)4/ (0.27)2/ (0.27 )S/ (0.63)4/ (0.79)4( (78.6)4/ (94.5)4/ Urine m m m m 0.51 0.44 aseeswaa — — m m m m Feces 364 347 0.8l£/ 0 .8 S & / m m m m Total - 345 489 258 441 */ The deuterio-oholesterol oontained 0.85 atom % deuterium. Eaoh rat of Qroup lb ingested 740 mg. of deuterio-oholesterol. Eaoh rat of Qroup lib ingested 690 mg. of deuterio-oholesterol. b/ The values given are averages for 4 rats in Qroup lb and 3 rats in Qroup lib. o/ Values were oaloulated by assuming a blood volume of 6.7 ml./lOO gn* body weight *“8 a hematoorit of 50 (99). d/ These values were estimated from those obtained for Groups la and Ha (Table VII). e/ This value was obtained from the oomblned oaroass oholesterol of Groups lb and lib. f/ Average of individual values from the third to the seventh day of deuterio-oholesterol administration p 62 Group lib, prefed the 1 per cent cholesterol diet (Diet E), a similar order of deuterium concentration in the organ cholesterol was observed, except that the highest concen tration was in the plasma cholesterol. However, in this group, the deuterium concentration of both the free and esterified liver cholesterol and of the cholesterol of the lung and blood cells was significantly lower than in these tissues of Group lb. The percentages of ingested deuterio-cholesterol incorporated into the tissues of the animals of both experiments are shown in Table XI. A comparison of the deposition in the rats of Experiment I, which weres fed Diet D containing no bile salt, and Group lb of Experiment II, fed Diet E containing 0.5 per cent ox bile, shows that the percentage of dietary cholesterol deposited in all the organs except the liver was quite similar in the two experiments. The amount deposited in the liver, however, was considerably greater in the rats fed ox bile along with the labeled cholesterol (Group lb). It is interest ing that deposition in the carcass was not augmented by the inclusion of bile in the diet. The percentage of labeled cholesterol recovered in the feces was over twice as great in the animals of Group lb, which were prefed Diet C, as in those prefed Purina (Experiment I). This TABLE XI PER CENT OF INGESTED DEUTERIO-CHOLESTEROL INCORPORATED INTO THE TISSUES AND FECES OF RATS PREFED NORMAL AND HIGH-CHOLESTEROL DIETS D-cholesterol deposition b/ Experiment II Organ Experiment I - -7---- (prefed purina) Group lb _/ Group lib 2/ (prefed normal diet) (prefed oholesterol) Diet C Diet E t i t Liver Free 1,6 1.6 Esterified 5.9 9.9 Total 3.1 7^5 llT5 Kidney 0.16 0.16 0.14 Spleen 0.19 0.12 0.17 Lung 0.31 0.30 0.21 Adrenal 0.15 0.08 Brain 0.03 0.03 --- Intestine 0,07 0.0-9 0.12 Blood cells 0.38 0,50 0.40 Plasma 0.15 — — 0.45 Heart / 0.06 / — / — — / Carcass 21 (10.5) 21 (10,6) 21 (13.7) 21 Focos 20 #3 46*6 37 *4 Total 35.4 66.0 64.1 64 FOOTNOTES TO TABLE XI a/ The values given are averages for 4 rats in Experiment I and Group lb of Experiment II and for 3 rats in Group lib, Experi ment II • l/ Each of the rats of Experiment I ingested 760 mg. of deuterio- cholesterol, while those of Groups lb and lib, Experiment II, ingested 740 mg. and 690 mg. of deuterio-cholesterol, respectively* s/ These values were estimatedj see footnote f/^, Table V and footnote e/, Table VII. 65 f difference was largely responsible for the marked difference in total cholesterol recovered in the two groups (35 per cent for Experiment I as compared to 66 per cent for Group lb of Experiment II). In Experiment II, the percentage of ingested cho lesterol deposited in the organs was almost identical in Group lb (prefed Diet C) and Group lib (prefed Diet E), with two exceptions. The amount of deuterio-cholesterol incorporated into the esterified cholesterol of the livers was significantly greater in Group lib than in Group Ibj however, the percentage deposited in the free cholesterol fraction, was identical for the two groups. The difference apparent in the incorporation of labeled cholesterol into the carcasses of the . two groups of Experiment II is-.of no significance, since these values are only approximations, as explained previously. The Effect of Cholesterol Feeding on the Quanti tative Distribution of Free and Esterified Cho lesterol in Ultracentrifugal Fractions of Rat * Liver Homogenates Experiment III: Effect of duration of cholesterol inges tion on the distribution of cholesterol in three cell fractions. 66 Experimental procedure. Twenty-four female rats, approximately 60 days old and 150 gm. in weight, which had been reared since weaning on Purina chow were used. These animals were placed for 5 days on a diet identical 6/ to Diet C except that sodium glycocholate was used instead of ox bile extract and then given an identical diet with 1 per cent of the fat replaced by cholesterol (Diet E). Groups of four animals were sacrificed after 0, 1, 2, 4, 7, and 14 days on the cholesterol diet by administration of an anesthetic dose of Nembutal and withdrawal of the blood by heart puncture. The animals were deprived of food 1 hour before sacrifice. The liver of each rat was quickly extirpated, trimmed, blotted, placed in a beaker immersed in cracked ice, and minced with scissors. Portions of the minced liver weighing 2.5 gm. were homogenized with 7.5 ml. portions of ice-cold 0.88 M sucrose solution in a modified Potter-Elvehjem homogenizer immersed in an ice bath. Four ml. of a 0.44 M sucrose solution were placed in a 13 ml. Lusteroid centrifuge tube. Eight ml. of the homogenate were taken into a 10 ml. hypodermic syringe to §/ Sodium glycocholate, Mallinckrodt Chemical Works, New York, N. Y. 67 which was attached a blunt, 4 inch long, 18 guage needle. The needle was carefully inserted to the bottom of the centrifuge tube, and the homogenate was slowly released from the syringe, forming a well-defined layer beneath the less dense 0,44 M sucrose. The procedure was repeated for each of the four livers in a group. The centrifuge tubes were centrifuged in a pre-cooled (4° C) #40 rotor of a Spinco ultracentrifuge, model L, for 2 hours at an 1/ average force of 105,000 x £ During centrifugation, a creamy material migrated to the tops of the tubes. This material was designated the floating layer, or "F" layer. This fraction and a portion of the 0.44 M sucrose solution which separated it from the other fractions were removed with a syringe and needle. Any creamy material adhering to the wall of the tube was wiped off with a cotton swab which was then combined with the , T Ff l layer for future extraction and analysis. The reddish, translucent supernatant fluid was next removed along with the remaining 0.44 M sucrose, leaving the residue which consisted of unbroken cells, cell debris, nuclei, mitochondria, and microsomes. 2/ The g values were oalculated for the centers of the tubes. 68 Each fraction, as well as a 1,0 ml, aliquot of the original homogenate, was extracted by the Waring blendor method; the extracts were adjusted to 50 ml.; and duplicate determinations of free and total cholesterol were made on each extract. The esterified cholesterol was calculated from the difference in total and free cholesterol. All values were referred to 4.0 ml. of homogenate. Results. In the livers of the normal animals sacrificed at the start of the experiment, 86 per cent of the total cholesterol was unesterified (Table XII). Ninety per cent of this free cholesterol was -contained in the residue fraction, and the remainder was evenly dis tributed between the "P" layer and supernatant fraction. The distribution of esterified cholesterol was difficult to determine, since the differences between free and total cholesterol in the different fractions were so small that the calculated values for esterified cholesterol were not significant. The sum of the total cholesterol recovered from the three fractions amounted to 85 to 100 per cent of that in the unfractionated homogenate throughout the experiment• Upon ingestion of the experimental diet, there was a rapid deposition of cholesterol in the unfractionated homogenate (Figure 2 and Table XII). The effect was________ 1 TABLE) XII DISTRIBUTION OF TOTAL AND FREE CHOLESTEROL IN THREE ULTRACENTRIFUGAL FRACTIONS OF HOIOGENATES OF RATS FED 1 PER CENT CHOLESTEROL DIET AT THREE TIME INTERVALS DURING EXPERIMENTAL PERIOD LIVER Fraction Cholesterol per 4 ml. homogenate 0 day 2 days 14 days Total Free ■Total Free Total Free mg • mg. mg. mg. mg. Original homogenate 2.40 2.06 4.08 2.27 13.40 3.44 (2.20-2 .69) (2.02-2 .13) (3.58-4.70) (2.16-2 .33) (9,60-17 ,05) (3.18-3.72) Floating layer 0.50 0.10 1.11 0.06 6.15 0.69 (0.18-0.45) (0.04-0 .18) (0.81-1.53) (0.02-0.12) (4.25-8.95) (0.48-1.13) Supernatant 0.30 0.09 0.28 0.16 0.62 0.31 (0.24-0.59) (0.07-0.13) (0.26-0.31) (0.13-0 .19) (0.53-0.19) (0.28-0,33) Residue 1.61 1.52 2.59 1.87 5.01 2.15 (1.55-1.63) (1.43-1.64) (2.43-2.82 (1.76-1.96) (4.60-6.03) (2 .05-2 .30 0> < £ > MG. C H O LESTER O L/4ML. HOM OGENATE ESTERIFIED CHOLESTEROL FREE CHOLESTEROL - 0 - ORIGINAL HOMOGENATE -•-R E S ID U E -x -" F " LAYER SUPERNATANT 4- 0 8 1 2 4 0 4 8 1 2 DAYS DAYS FIGURE 2* The Effect of Cholesterol Ingestion on the Content of Esterified and Free Cholesterol in Three Ultracentrifugal Fractions of Rat Liver Homogen- ates • 71 pronounced even after only one day of cholesterol feeding, and the cholesterol concentration increased linearly with time until the 14th day, the final day of the experiment. Most of this cholesterol was deposited in the esterified form, although a gradual rise in free cholesterol also occurred. The deposition was localized in the "P" layer and residue; the amount of cholesterol deposited in the "F" layer was almost twice as great as that in the residue (Table XII). Furthermore, the total cholesterol of the ”Fn layer underwent a 20-fold increase during the 14 day period, while only a 3-fold rise occurred, in the residue. In both of these fractions, esterified cholesterol accounted for most (over 80 per cent) of the increase in total cholesterol. Free cholesterol increased slightly in the residue and "F" layer during cholesterol feeding. Only a slight change was observed in the amount of free and esterified cholesterol in the supernatant fraction. The percentage of free cholesterol in the total cholesterol decreased with time in the whole homogenate, further emphasizing the deposition of esterified cholester ol. This change was evident in both the residue and "F" layer. 72 During the period of cholesterol ingestion, marked changes occurred in the percentage distribution of cho lesterol in the liver cell fractions studied (Figure 3). The percentage of total liver cholesterol decreased from 73 to 42 per cent in the residue, increased from 13 to 50 per cent in the nFM layer, and decreased from 13 to 5 per cent in the supernatant fraction. These changes could be attributed primarily to the large increase in esterified cholesterol in the "F* 1 layer. Experiment IV. The distribution of cholesterol in five liver cell fractions from rats fed cholesterol for seven days. In order to define more precisely the location of cholesterol in the residue fraction, a more detailed frac tionation of the livers of rats fed cholesterol for seven days was carried out. Experimental procedure. Four female rats of the same age, weight, and dietary history as those used in the preceding experiment were fed Diet C for 5 days, followed by the 1 per cent cholesterol diet (Diet E) for 7 das^s. At the end of this period the animals were weighed and sacrificed as previously described. The livers were per- &/ fused in the following manner • Sixty ml. of ice-cold 8/ The author is indebted to Dr. John Ganguly for this perfusion method. The procedure was also used by Schotz et al (57) in obtaining data on normal rats. P E R C E N T O F T O T A L CHOLESTEROL I N EAC H FRACTION 80- 70* 6 0 - F" LAYER 50- RESIDUE 4 0 - 30- 20- SUPERNATANT DAYS FIGURE 3. The Effect of Cholesterol Ingestion on the Percentage Dis tribution of Total Cholesterol in Three Ultracentrifugal Fraotions of Rat Liver Homogenates • 74 0.25 M sucrose solution were forced through the aorta with a syringe attached to a blunt 18 guage hypodermic needle which was inserted through an incision in the left ventricle. The hepatic vein was severed to provide an outlet for the blood and perfusion fluid. This procedure, which could be completed in approximately two minutes, removed most of the blood from the liver as shown by the scarcity of red cells at the bottom of the tube after centrifugation. The livers were then removed and homogenized exactly as described for the preceding experi ment, except that 0.25 M sucrose was used as the homogeni zation fluid. This was done for two reasons: (a) to decrease the time required for the fractionation procedure, and (b) to provide results which could readily be compared to those obtained for normal rats by Schotz jet al (57). All further steps of the procedure were carried out in a cold room at 3-4° C. The homogenate was filtered through single napped flannelette to remove whole cells (101). Duplicate samples of 8.0 ml. of the homogenate were then carefully layered under 4.0 ml. of 0,125 M sucrose solu tion in a 13 ml. Lusteroid tube and fractionated in the ultracentrifuge. The layered homogenate was centrifuged for one hour at 105,000 x g at 3-4° C. The residue, ”Fn layer, and 75 Supernatant-1 were separated as described for the preceding experiment. The latter two fractions were extracted immediately by the Waring blendor method. The residue was rehomogenized with 4 ml. of 0*25 M sucrose, for 5 strokes of the pestle, using a Lucite pestle made to fit the centrifuge tube. The resulting homogenate was fraction ated by a modification of the Schneider-Hogeboom method (50) as described by Schotz jet al (57). The procedure is shown diagrammatically in Figure 4. At the end of the final one hour spin at 105,000 x g, additional floating material could be seen at the top of the tubes. This material was separated and labeled MFn layer-2. Each ultracentrifugal fraction, as well as an aliquot of the original homogenate, was extracted by the Waring blendor method, and the extracts were analyzed for free and total cholesterol. The cholesterol values were expressed as mg. of cholesterol in 4.0 ml. of homogenate. Results. In Table XIII the cholesterol values for the liver fractions of the cholesterol fed rats are compared with values reported for normal rat livers by Schotz et al (57). As reported for the preceding experi ment (Table XII), cholesterol ingestion resulted in a pronounced increase in esterified cholesterol and a HOMOGENATE I I HOUR 1105,000 XG FLOATING LAYER RESIDUE SUPERNATANT-1 REHOMOGENIZE 10 MIN. 6 0 0 XG NUCLEAR RESIDUE 110 MIN. 600XG N. RESIDUE 110 MIN 600XG SUPER. SUPER. 10 MIN 5.000X G NUCLEI SUPER. MITO. RESIDUE 1 10 MIN 5 ,0 0 0 XG SUPER MITO. RESIDUE I 10 MIN 2A.000XG SUPER. I HOUR 105,000 X G SUPER. MITOCHONDRIA MICROSOMES SUPERNATANT-2 FIGURE 4* Fractionation Scheme for Rat Liver Homogenate Prepared in 0.25 M Sucrose Solution. TABLE XIII COMPARISON OF THE CONCENTRATION OF FREE AND ESTERIFIED CHOLESTEROL IN THE LITER FRACTIONS FROM NORMAL AND CHOLESTEROL-FED RATS Descript ion Mg * cholesterol/4 .0 ml. homogenate of Normal , Cholesterol-fed rats fraction rats —' Rat 1 Rat 2 Rat 3 Rat 4 Whole homogenate Free cholesterol 1.552 2.260 2 .380 Lost 2 .500 Floating layer-^l (1.067-1.836) 0.053 0.189 0.164 1.720 0.518 Floating layer-2 (0.039-0.068) 0.049 0.043 0.050 0.063 Nuclei 0.104 0.198 0.183 0.127 0.081 Mitochondria (0.061-0 .203) 0.207 0.338 0.230 0.252. 0.269 Microsomes (0.078-0.269) 0.971 1.086 1.430 1.080 0.829 S upernat ant-1 (0.782-1.125) 0.068 0.068 0.046 0.085 0.096 S upernat ant-2 (0.019-0.096) 0.108 0.041 0.040 0.052 0.049 (0.033-0.150) Esterified cholesterol Whole homogenate 0.365 (0.302-0.497) 1.140 1.860 Lost 6.780 Floating layer-1 0 .187 (0.125-0 .277) 1.730 1.736 8.500 6 .570 Floating layer-2 - - - - - 0.082 0.046 0.126 0.142 Nuclei 0.004 (0.00-0.013) 0.078 0.107 0.098 0.106 Mitochondria 0.006 (0.00-0 .032) 0.048 0.080 0.059 0.064 Microsomes 0.058 (0.026-0.074) 0.272 0.250 0.283 0.260 Supernatant-1 0.020 (0.015-0.041) 0.071 0.034 0.105 0.128 Supernatant-2 0.027 (0.012-0.040) 0.044 0.034 0.065 0.034 a/ These data are from the work of Schotz et al (57) 78 smaller rise in free cholesterol in the "F" layer. The previously observed increase of cholesterol in the "residue” fraction was shown, on further fractionation, to be localized primarily in the microsomal fraction, with only slight changes in the nuclear and mitochondrial frac tions. This increase was largely accounted for by esteri fied cholesterol. Only minor changes, of doubtful signifi cance, occurred in the supernatant fractions. Small addi tional quantities of free and esterified cholesterol were found in "F" layer-2 in the cholesterol-fed rats; this fraction was present only in minute quantities in the normal animals ^ » The ratios of free to total cholesterol, expressed as per cent free cholesterol, in each of the ultracentri fugal fractions of the cholesterol1 -fed rats are compared in Table XIV with the values for normal rats reported by Schotz et al (57). Cholesterol ingestion for 7 days pro duced a decrease in the proportion of free cholesterol in all of the cellular components. The recovery of cholesterol in the various frac tions amounted to from 87 to 99 per cent of the free and J)/ Schotz, M. C., Personal communication. TABLE XIV THE PEE CENT OF .FREE CHOLESTEROL IN THE TOTAL CHOLESTEROL OF LIVER FRACTIONS FROM NORMAL AMD CHOLESTEROL-FED RATS Fraction Per cent of total fraction cholesterol in eaoh in free form Normal diet is Cholesterol diet Homogenate 81.9 40 .3 (78.8 - 85.5) (26.7 - 56.1) F layer - 1 22 .8 10.6 (17.7 - 26.0) ( 7.3 - 16.8) F layer - 2 36.1 (28.2 - 48.6) Nuclear fraction 95 .7 58*7 (85.3 - 100) (43.3 - 71.8) Mitochondrial fraction 96 .6 80.8 Submicroscopic (85.5 - 100) (74.2 - 87.6) particulate fraction 94.2 80 .9 (91.9 - 97.6) (76.0 - 85.1) Supernatant - 1 75.2 48.6 a (45.1 - 96.9) (42.9 - 57.5) Supernatant - 2 76.2 51.4 (55.6 - 89.9 (44.4 - 59.0) 2/ These data ace from the work of Schotz et_ al (57) Values are averages of analyses on livers of 6 rats • Figures in parentheses represent range of values • 8© total cholesterol of the unfractionated homogenates. The recovery of esterified cholesterol, which was calculated from the difference between total and free cholesterol, varied considerably, owing to slight inequalities in the recovery of free and total cholesterol. Experiment V. The effect of the administration of reticulo endothelial blocking agents upon the deposition of cholestep ol and total lipids in the livers of cholesterol fed rats. Experimental procedure. Female rats weighing 160-190 gm. and reared from weaning of Purina chow diet were used. Although Brown jet al (82) successfully used the intraperitoneal route for administering admixture of trypan red and thorotrast to guinea pigs, it was found that edema resulted when this method was attempted in rats. Furthermore, according to Baillif (102), who made a de tailed investigation of thorium dioxide effects in the rat, intravenously injected thorium dioxide was collected primarily in the macrophages of the liver and spleen, whereas subcutaneously or intraperitoneally administered thorium dioxide was distributed through regional lymph nodes before entering the liver or spleen. Therefore, the intravenous route was chosen, and a technique for repeated 81 injection was devised. The tail vein was chosen as the site of injection, and the following technique was' found to be satisfactory. A horizontal nick was made with a scalpel through one of the veins of the tail of a rat under light ether anesthesia, at a distance of approximately two inches from the animal’s body, and a small wedge-shaped section of the skin was removed. A polyethylene tube , 4 inches long, was inserted into the severed vein to a distance of 1% to 2 inches. The wound, which usually bled only moderately, was disinfected with1 Tincture of Metaphen and covered with a small "Bandaid". The success of the operation was tested by injecting physiological saline into the catheter " with a syringe and 22 guage hbedle.. When the tubing was correctly, placed, no resistance to the entrance of the fluid was felt. The tail was then fastened securely with adhesive tape to a narrow wooden splint, leaving the wound and the end of the catheter untaped. A metal shield made from copper sheeting was wrapped around the wound and pro truding catheter to prevent accidental removal of the tub ing. Subsequent injections were made under light ether ^anesthesia to prevent removal of the catheter due to move-' 10/ Clay-Adams Company, Inc., polyethylene tubing, Cat. no. PE50, 0.58 mm. inside diameter and 0.965 mm. outside diameter. i 82 ; ments of the animal during injection. Coagulation of blood ' in the tube could usually be prevented by injecting a small I | quantity of heparin following administration of the test : substance. Occasional clots did form, however, and when- i : ever these could not be dislodged with a wire, it was ] ! necessary to insert a fresh catheter into the vein. i Eight rats (Group I) whose tails veins had been t j catheterized as described above were injected intravenously; 1 i twice daily with 0.5 ml. per 100 gm. body weight of a ; ^ ll/ ! : mixture of one part of 4 per cent trypan red in physio- 1 12/ 1 logical saline and 2 parts of thorotrast . A second I I j group of eight catheterized animals (Group II) was injected! ; with the same volume of 0.85 per cent sodium chloride t i I | solution and served as the control group. All animals i i were weighed daily. During the first two days of injec- i i 1 tions, the rats were maintained on the stock diet (Purina • chow) in order to establish blockage of the reticulo- i | endothelial system. Subsequently, both groups were fed a i i ! diet containing 1 per cent cholesterol and 0.5 per cent j ll/ National Aniline Division, Allied Chemical and j Dye Corporation, New York, N. Y. 12/ A stabilized colloidal suspension of thorium 1 dioxide in physiological solution. Heyden Chemical Corp., New York, N. Y. I 1 ; _________ 83 sodium glycocholate (Diet E) for 5 days, during which time the injections were continued twice daily. After 5 days of cholesterol feeding, the animals were starved for 24 hours and then sacrificed by injection- of Nembutal and removal of the blood by heart puncture. The livers were removed and weighed, and samples of each were preserved in formalin for histological examination. The remainder of each liver was extracted with ethanol- ebher (3:2 by volume) in the Soxhlet apparatus as pre viously described. Cholesterol was determined in these extracts and also in ethanol-acetone (1:1 by volume) extracts of the blood plasma. Total lipids were also measured in the liver extracts as described earlier. Sections of the formalin-preserved liver tissue were stained with hematoxylin-eosin and examined for evidence of reticulo-endothelial blockage and for lipid infiltra- tion ^ . Results. Deposition of cholesterol in the livers was completely prevented in the animals injected with thorotrast and trypan red, while the customary rise in 15/ The author is indebted to Professor Hugh Edmondson for preparation and examination of the histo logical sections. cholesterol concentration was observed in livers of the saline-injected control group (Table XV). In agreement with the preceding experiment, the increase in liver cholesterol; in the control group was primarily due to esterified cho lesterol, with a small increase in free cholesterol also occurring. The percentage of free cholesterol in total cholesterol after 5 days of cholesterol feeding was in fairly good agreement with that observed in Experiment III (51 per cent as compared to 43 per cent). The liver cholesterol values of the thorotrast- treated, cholesterol-fed animals were within the range of values observed in normal rats in this laboratory over a period of several years. The plasma cholesterol concentrations for the two groups are shown in Table XVI. Both free and esterified cholesterol were significantly higher in the saline- treated controls than in the experimental group. Comparing these values with those in Experiment I, the animals of which were the most comparable to these in dietary history, it can be seen that the average plasma cholesterol in the thorotrast-treated group was very similar to that in the Purina-fed animals, while the plasma cholesterol values of the saline-injected animals were comparable to those in the rats fed cholesterol for 8 days (Table V). It is TABLE XV THE EFFECT OF INTRAVENOUS INJECTION OF THOROTRAST AND TRYPAN RED " a/ ON THE LIVER CHOLESTEROL IN RATS FED CHOLESTEROL ~ Material injected Animal number Liver cholesterol Free Esterified Total % Free mg;/gm. mg./gm. mg./gm. % 1 2.44 2.28 4.72 51.7- 2 2.64 3.80 6.44 41.0 3 3.42 1.72 5.14 66.5 4 3.98 1.7-9 5.7-7 ' 69.0 Saline 6 3.51 4.50 8.01 43 .8 7 2.86 3.62 6.48 44.2 8 2.71 2.97 5.68 47.7 9 2.78 3.45 6.23 44.6 Average 3 .04±0 .17^/ 2.89±0 .30 6 .06±0 .24 51 ,1±S .3 11 2.36 0.16 2.52 93.6 Thoro- 12 2.38 0.16 2.54 93.6 trast 13 2.67 0.38 3.05 87 .5 +’ 14 2.39 0.21 2.60 92.0 Trypan 15 2.45 0.19 2.64 92 .8 red 18 1.91 0.14 2.05 93 .1 20 2.44 0.04 2.48 98.4 Average 2 .37±0 .09 0 ,18±0 .04 2 .55-0 .11 93 .Oil .2 a/ The animals were fed cholesterol (Diet E) during the experi mental period* b/ Standard error of the mean* TABLE XVI THE EFFECT OF INTRAVENOUS INJECTION OF THOROTRAST AND TRYPAN RED ON THE PLASMA CHOLESTEROL OF RATS FED CHOLESTEROL - Material injected Animal number Plasma cholesterol Free Esterified Total % Free mg*/l00 ml • mg./lOO ml. mg./lOO ml. 1 18.0 43 .0 61.0 29.5 2 12 .9 34.6 47 .5 27 .2 Saline 3 12 .0 39.0 41 .0 29.3 4 23.4 52 .6 76.0 30 .8 6 10.8 32 .7 43 .5 24.9 7 14.7 37 .3 52.0 28.3 8 19.2 53.8 73.0 26.3 9 14.7 43.3 58.0 25.3 Average - 15.7*1 .5 & 40.8*2 .8 56.5*4.6 27 .7*0 .8 Thoro- 11 9.3 15 .7 25.0 37.2 trast 12 5.7 26.3 32 .0 17 .8 13 10.2 33 .3 43 .5 23.4 Trypan 14 10.2 32.3 42.5 24.0 red 15 11.1 25 .9 37.0 30.0 20 13.2 17 .8 41.0 42 .6 Average - 10.oil.0 25.2i3.0 35 .2i3 .0 29.2tl.6 a/ The animals were fed cholesterol (Diet E) during the experimental j period* b/ Standard error of the mean. 87 of particular interest that the ratio of free to total cholesterol was similar and within the normal range for both groups, although individual variations were greater in.the animals injected with thorotrast and trypan red. Blockage of the retieulo-endothelial system prevent ed fatty infiltration of the liver to a large degree as shown by total lipid analysis (Table XVII). Evaluation of the degree of vacuolation shown in histological sections of the livers confirmed this finding. TABLE XVII THE EFFECT OF INTRAVENOUS INJECTION OF THOROTRAST AND TRYPAN RED ON a/ THE TOTAL LIVER LIPIDS OF RATS FED CHOLESTEROL “ Material injected Animal number Total liver lipid 1 mg./gm. 47 .5 2 67 .5 3 49.3 Saline 4 46.3 6 69.3 7 70.5 8 46.6 9 56.6 Average - 56.7 ± 3.8 11 46.0 Thorotrast 12 30.8 and 13 53.2 14 38 .8 Trypan red 15 27 .6 18 18.6 20 49 .7 Average 37.8 1 4.8 a/ The animals were fed cholesterol (Diet E) during the experimental period* Standard error of the mean* DISCUSSION Analysis of the tissues of rats fed a diet supple mented with deuterium-labeled cholesterol for 8 days showed that although the cholesterol concentration was increased significantly in the liver and the blood plasma only, exogenous cholesterol was present also in the kidneys, spleen, lungs, adrenals, intestine, red blood cells, heart, and carcass, evidently in replacement for some of the endogenous cholesterol originally present in these organs. The concentration of deuterium-labeled cholesterol was found to be highest in the liver and adrenals, and slightly lower in the spleen, lungs, plasma, blood cells, and urine; the isotope content of the cholesterol of the intestine and carcass was consistently less than in the above organs, while with the exception of the brain in which only negligible incorporation of exogenous cholester ol occurred, that of the kidneys was the lowest of all the tissues examined. These data agree in part with those of Van Bruggen, Hutchens, and West (44) who fed C^-cholesterol to rats; of the five tissues studied (liver, carcass, skin, gut, and brain), they found the highest specific activity in the \ liver cholesterol. However, these investigators reported 90 that a significant amount of labeled cholesterol was pre- 14/ sent in the brain — * while, as mentioned above, only a negligible amount of incorporation into this organ was found in the present study, which is in accord with pre vious reports of a very low rate of turnover of cholesterol in the adult rat brain (103-105). The order of decreasing concentration of labeled cholesterol found in various organs in the rat was similar to that found by Biggs and Kritchevsky (46) in rabbits fed tritium-labeled cholesterol, except for the values obtained for the kidneys. The cholesterol of the rabbit kidney had a specific activity of the same order of magnitude as the liver, adrenal, lung, and other organs, while in the rat, the activity of the kidney cholesterol was markedly and consistently lower than in the other organs. This difference between the two species is difficult to inter pret at this time, but it may be an indication that the rat kidney turns over cholesterol at a slower rate, as compared to the other organs, than does the kidney of the rabbit. The amount of labeled cholesterol deposited in the livers of rats fed cholesterol without added bile extract 1^/ A private communication from one of the authors indicates that this result may have been due to contamina tion. 9^ (Experiment I), expressed as percentage of the total quan tity of labeled cholesterol present in the body, amounted to 18 per cent which compared favorably with Gould’s ■j A figure of 15 per cent (45) in rats fed C -cholesterol. The addition of bile extract to the diet (Experiment II) caused a marked increase in deuterio-cholesterol deposition in the liver; this observation provides further evidence- for the Importance of bile in the absorption of cholesterol which has been demonstrated by other investigators (106- 108). However, the deposition of deuterio-cholesterol in other tissues was not augmented as a result of this in creased absorption, further demonstrating the remarkable capacity of the liver for the storage of excess cholesterol entering the body. The varied individual response to cholesterol inges tion, evidenced by the different quantities of labeled cholesterol present in the separate livers (Experiment I), was also observed by Kritchevsky, Kirk, and Biggs (109) in rats fed G14-cholesterol. An interesting observation was the large discrepancy between the average plasma cholesterol values in the rats prefed the synthetic control diet containing 0.5 per cent bile extract (Group la, Experiment II) and those prefed Purina diet (Experiment I) (79.5 mg. per cent contrasted to 92 34.9 mg. per cent), while only a small difference was ob served in the liver cholesterol values (2.2 mg./gm. as compared to 1.7 mg./gm.). Analysis of the bile prepara tion employed showed it to contain a small amount of cholesterol (approximately 10 mg./gm.), but it is hardly possible that the ingestion of this quantity of cholesterol could lead to such a marked increase in plasma cholesterol content, particularly without causing a concurrent increase in liver cholesterol levels. It is more likely that the bile extract induced an elevation in plasma cholesterol through the action of cholic acid, which, as Byers and Friedman (110) have shown, produces this effect when ad ministered to rats. There was also a marked difference in the total amount of ingested cholesterol recovered from the carcasses and excreta of the rats prefed Purina diet from those prefed the synthetic control diet containing bile extract. This difference was due principally to a lower cholesterol content in the feces from the rats reared on Purina diet, although the total weight of fecal material collected over the period of feeding of labeled cholesterol was almost the same in the two groups. In view of the evidence for greater cholesterol absorption in the rats prefed the synthetic diet with bile extract, it is improbable that 92 less cholesterol destruction could have occurred in the tissues in this group, thus accounting for greater recovery of the ingested cholesterol. It seems more likely that differences in the intestinal flora brought about by the two diets, may have led to more bacterial destruction (26, 111, 112) of cholesterol in one case than in the other. Another possible explanation of this difference is based upon the fact that bile acids are known to be a metabolic sand product of cholesterol (70, 113). If an equilibrium between bile acids and cholesterol can be postulated, the presence of bile acids in the diet should decrease the amount of cholesterol which is converted, thereby increas ing the cholesterol content in the feces. The presence of considerable quantities of labeled cholesterol in tissues in which no significant increase in cholesterol concentration occurred (red blood cells, spleen, lungs, etc.) and the fact, that the concentration of labeled cholesterol was approximately equal in the plasma and in these tissues may be indications that exogenous cholesterol mixes with the cholesterol already present in the plasma and becomes indistinguishable from endogenous cholesterol in exchanging with the cholesterol already present in the tissues. Gould (114) has shown in dogs and Biggs et al, (115) in man, that free 94 cholesterol of the red blood cells, and it might be assumed that a similar interchange occurs with the other organs, particularly in view of the fact that the cholesterol in most of the tissues is in the unesterified state (116)* Gould (114) has concluded, on the basis of unpublish ed data, that the movement of cholesterol from the plasma to the tissues is an irreversible process. If thisvere the case, one would predict that the addition of exogenous cholesterol to the plasma would lead to increased cho lesterol content in the tissues owing to increased deposi tion, unless this deposition were counteracted by (a) stimulation of cholesterol destruction in these tissues or by (b) depression of cholesterol synthesis. The evidence for increased destruction of cholesterol under conditions of cholesterol feeding or excess plasma cholesterol is indirect in nature. Cook (117) found that when the absorp tion of dietary cholesterol was facilitated by the addition of fat to the diet, a greater amount of cholesterol destruc tion occurred, as shown by an increase in the fecal excre tion of an acid which Cook believes to be a metabolic pro duct of cholesterol. Friedman, Byers, and Gunning (75) reported that the content of biliary cholic acid, which has been shown to be an important metabolic product of cholesterol (70, 113), vras elevated in rats after intra 95 venous injection of hypereholesterolemic serum. However, in consideration of the substantial evidence for a rapid uptake of excess plasma cholesterol by the liver (114), and the evidence in favor of the ability of the liver to destroy cholesterol (118), it can be assumed that a large part of the excess cholesterol was broken down in the liver, itself, and no conclusions about the effect of cho lesterol feeding upon the rate of destruction, if any, in the other tissues can be drawn at this time. G-ould (114) and Tompkins et al (119) have shown that the ingestion of excess cholesterol results in inhibition of hepatic cholesterogenesis as measured by incorporation of isotopic ace.tate into the organ cholester ol. However, a similar influence of dietary cholesterol upon the synthesis of cholesterol in other organs has not been demonstrated. It is felt, therefore, that the most likely explanation for the presence of labeled cholesterol in the tissues in which a concurrent elevation in cho lesterol does not occur is that the exogenous cholesterol becomes mixed with and indistinguishable from the endogen ously formed cholesterol in the plasma, and that the circulating plasma cholesterol is in equilibrium with the tissue cholesterol. 96 Evidence that a similar exchange of free cholesterol also occurs between the plasma and liver can be found in the observation that after 7 days of deuterio-cholesterol ingestion, a large proportion of the free cholesterol originally present in the liver had been replaced by labeled free cholesterol (Table XVIII), This condition obtained in rats prefed either normal or high-cholesterol diets• Although some increase in the free cholesterol content of the liver was noted during the first week of cholesterol ingestion, after this period, the free cho lesterol concentration leveled off, while deposition of cholesterol esters continued. It may be postulated that a rapid adjustment was made by the liver to the increased influx of cholesterol from the plasma, resulting in a speeding up of the esterification process and a shift in equilibrium toward the esterified condition. It is also possible that the lack of a proportionate increase in free cholesterol to total cholesterol content may have been a consequence of depression of cholesterol synthesis in the liver, caused by ingestion of the high-cholesterol diet. The observation that after 7 days of deuterio- cholesterol administration the deuterium concentration of the free cholesterol was equal, within the limits of TABLE XVIII THE DEPOSITION OF FREE AND ESTERIFIED DEUTERIO-CHOLESTEROL IN THE TISSUES OF RAIS PREFED NORMAL AND HIGH-CHOLESTEROL DIETS s/ Previous diet Organ Cholesi 0 days —' (1) ;erol cot 7 days^/ (2) >tent of organ Net increase (2-1) Deuterium content of cholesterol Deuterio- cholesterol deposited (3) mg. 26* atom $ mg. Low cholesterol Liver (Diet C) Free 14.3 26.0 + 5.7 0.51 12 .0 Esterified 1.25 73.0 +71.7 0.51 43 .8 Plasma 3.92 3.64 - 0.32 --- High cholesterol Liver (Diet E) Free 24.1 23.2 - 0.9 0.39 10.6 Esterified 115 .0 164.0 +49.0 0.35 67.5 Plasma 7.58 5.67 - 1.91 0.46 3.07 a/ The deuterio-cholesterol contained 0.85 atom per cent deuterium. Each rat prefed Diet C ingested 740 mg. of deuterio-cholesterol. Each rat prefed Diet E ingested 690 mg. of deuterio-cholesterol • b/ This diet was fed for 30 days prior to administration of labeled cholesterol, o/ Number of days on deuterio-cholesterol diet. 98 experimental error, to that in the esterified cholesterol of the liver (Table XVIII) might be explained by assuming that (a) excess labeled cholesterol enters the liver from the plasma in the free state and is then rapidly esterified or that (b) there is a constant dynamic equilibrium between the free and esterified cholesterol of the liver. A consideration of the figures for esterified cho lesterol in Table XVIII leads to the observation that in the animals prefed the normal diet, the quantity of labeled esterified cholesterol present in the liver account ed for only slightly more than half of the total increase in esterified cholesterol content as compared to the control group. However, in the rats prefed the high-cholesterol diet, all of the esterified cholesterol deposited over the 7 day experimental feeding could be accounted for by deuterio-cholesterol. Interpretation is difficult, for whereas in the case of the cholesterol-prefed group, one might conclude that exogenous cholesterol was selectively filtered from the blood and stored in the liver, it is apparent that in the rats prefed the control diet, a large quantity of unlabeled cholesterol was also stored. Owing to the increase in individual variations in liver cho lesterol content after longer periods of cholesterol in- j gestion observed in this and in other experiments in this 99 laboratory, it is felt that the observations on the rats prefed the normal diet are of greater significance and should be considered more seriously in formulating inter pretations than the data obtained on the rats prefed cho lesterol. Therefore, the more acceptable hypothesis is that a mixture of cholesterol of endogenous and exogenous origin is removed from the plasma and stored in the liver, and that the liver does not distinguish between these two sources of cholesterol. Since the elevation in plasma cho lesterol concentration caused by cholesterol feeding in the rat is much smaller than the increase in liver cholesterol content, it is necessary to assume that only small increases in plasma cholesterol concentration are required to initiate cholesterol deposition in the liver. The previous feeding of a 1 per cent cholesterol diet for 30 days had little effect upon the deposition of deuterio-cholesterol in the kidney, spleen, intestine, or feces. However, there was an appreciable decrease in the amount of labeled cholesterol found in the lungs and in the red blood cells of the cholesterol prefed animals, which may indicate that cholesterol ingestion caused a slight depression of turnover rate in these organs. The deuterium content of the liver cholesterol, both free and esterified, was lower in the cholesterol prefed group, owing to dilu 100 tion of the exogenous cholesterol by cholesterol already present in the plasma and liver. In contrast to these findings in the rat, Biggs and Kritchevsky (94) reported that in rabbits the deposition of exogenous cholesterol was significantly increased in all tissues examined, after feeding of high-cholesterol diets. The question of whether cholesterol is removed from the plasma and stored in the liver without change in the protein binding or in the relationship of free to esteri fied cholesterol can be considered on the basis of informa tion obtained in the liver fractionation experiment. Analysis of cell fractions obtained by differential centri fugation of liver homogenates showed that the excess cho lesterol esters found in the liver of cholesterol-fed rats were associated with a fraction (the T , Fn layer) which migrated centripetally in a medium of 0.125 M sucrose. This floating material can be observed at the top of the centrifuge tube after high-speed centrifugation of a liver homogenate. Simple observation shows that only small amounts of the "F" layer can be separated from homogenates of normal rat livers, while the fatty infiltration result ing from cholesterol ingestion leads to large increases in the amount of this material. Chauveau e_t al (56), who examined a similar fraction obtained from normal rat livers, r i also found that it contained a high proportion of the cho- J i : i lesterol esters. , 1 i The floating material has been shown to contain also! ! phospholipids and neutral fats (56). Krinsky (120) recent-* j ly reported that a large portion of the esterified vitamin J 1 A of rat liver is associated with the "P" layer. Chauveau j and co-workers (56) were unable to detect any nitrogen in ! this fraction and, consequently, referred to it as "free fat". Krinsky found very small amounts of nitrogen present' ! which, however, might have arisen from contamination from | other cell fractions. I The relationship of the centripetally migrating I 1 I ; material to the structure of the unbroken cell is extremely! hypothetical at this time. In an earlier investigation, ! I : Fallade and Claude (121) postulated that a centripetally I migrating material obtained by centrifugation of liver | homogenates was derived from the structure in the intact ! cell known as the G-olgi apparatus. The possibility has also been suggested (122) that the floating material con- ! sists of the fat droplets which can be observed micro- | scopically in the cytoplasm and which are known to increase, » j ; in size and number in fatty livers. Neither of these : hypotheses has been further substantiated. i The lack of significant amounts of protein in the I I !_ . . __________ University ot Southern Cadifosui 102 floating material with, which cholesterol esters are assoc iated leads to several speculations. (a) Cholesterol esters may become dissociated from the proteins with which they have been shown to be associated in the blood and are stored in the liver dissolved in other lipids. In view of the large increase in fat droplets which can be observed histologically in fatty livers due to cholesterol feeding, it is felt that these are probably the site of cholesterol storage and the source of the material which migrates centripetally in the centrifuge. (b) Cholesterol absorbed from the intestine may enter the blood in the form of chylomicrons (minute lipid droplets unassociated with protein) which are removed by the liver and stored, (c) Finally, it cannot be overlooked that loosely assoc iated complexes of lipids and proteins might be split by the mechanical processes of homogenization or ultracentri fugation. When the complete differential centrifugation procedure was carried out on the livers of cholesterol-fed rats, somewhat more cholesterol was found in the "P” layer and less in the combined particulates than when only the simple separation into "residue11, supernatant, and "F" fractions was accomplished in one high-speed centrifugation. Although this demonstrates that some lipid was separated from the particulates by repeated washing and centrifuga 103 tion, the marked difference in the ratio of free to ester cholesterol in the "F" layer and in the particulates of the livers of both normal and cholesterol-fed rats shows that the lipid complexes in these fractions are significantly different. In contrast to the apparently protein-free condition of the cholesterol esters (if it can be assumed that this is not due to mechanical separation of the lipid-protein complexes), the free cholesterol is found associated primarily with the submicroscopic fraction, and owing to the proportionately higher protein content of this fraction, might be assumed to be bound to protein. The small increases in esterified cholesterol con tent in the nuclear, mitochondrial, and microsomal frac tions over the period of cholesterol feeding might at first glance be attributed to an adsorption phenomenon. However, an examination of the data reveals that although deposition of cholesterol was markedly higher in the unfractionated livers of rats 3 and 4 than in rats 1 and 2, this difference was not manifest by a corresponding increase in cholesterol concentration in the particulate fractions of the livers of rats 3 and 4, which might be expected if adsorption (or, in the case of the nuclei, contamination with unbroken cells) were the explanation. 104 Since the difference in cholesterol deposition of rats 3 and 4 as compared to rats 1 and 2 was reflected only in the floating layer, the importance of this fraction as the major site of cholesterol deposition is further emphasized. The difference in response to a high-cholesterol diet of rats injected with saline or with a mixture of thorotrast and trypan red is assumed to he caused by blockage of the reticulo-endothelial system in animals treated with the latter mixture. These substances are known to be stored.by the reticulo-endothelial system, and, when injected intravenously, have been shown to be selectively segregated by the macrophages.of the liver and spleen (102). Effects on other metabolic systems have not been reported. The free and esterified cholesterol values in the plasma and liver, as well as the total lipid content of the liver, were lower in the rats with blocked reticulo-endo thelial systems than in the saline-injected controls. These differences were very significant statistically, when analyzed by the Irt, f test (123). However, the ratios of free to total cholesterol in the plasma were within the normal range and almost identical in the two groups. In the absence of data on rats treated similarly, but fed a low-cholesterol diet, it is not possible to state whether, 105 in this experiment, blockage of the reticulo-endothelial system caused a lowering of blood cholesterol values below normal or prevented the increase which usually occurs upon ingestion of cholesterol. It is evident that treatment with thorotrast and trypan red effectively prevented the deposition of free and esterified cholesterol and total lipid in the liver* Some of the possible mechanisms through which this phenome non might have been effected are the following: (a) Block age of the reticulo-endothelial system may inhibit a process of active phagocytosis necessary for the removal of cho lesterol from the blood by the Kupffer cells lining the sinusoids of the liver. It is conceivable that lipo proteins may be taken up from the blood by the liver in such a manner, prior to separation of the lipid and protein molecules and their further metabolism. (b) Cholesterol may be stored in the Kupffer cells, themselves, as has been postulated for vitamin A (83, 84), in which case overloading these cells with colloidal material would reduce the amount of storage. However, in view of the observation that cho lesterol ingestion by the rat results in a large increase in the amount of fat droplets visible microscopically in the parenchymal cells of the liver and that this increase is partly prevented by simultaneous administration of 106 reticulo-endothelial blocking agents, and since it is felt that the fat droplets are probably the source of the ’ ’ F" layer, in which a large portion of the excess cholesterol in the liver is accumulated during cholesterol ingestion, it seems unlikely that the cholesterol is stored preferen tially in the Kupffer cells. (c) Blockage of the reticulo endothelial cells may prevent some hitherto undefined re action necessary for the storage of cholesterol. (d) In terference with the reticulo-endothelial system may cause alterations in the physical state of the plasma cholesterol, ^.g., the size of the particles with which it is associated, so that it is taken up by organs other than the liver. The failure of the blood cholesterol to rise in the cholesterol-fed rats treated with thorotrast is in agree- * ment with Leites1 observations (77) on dogs treated simi larly. Three possible explanations for this finding, are the following: (a) excess cholesterol entering the blood from the intestine may be deposited in tissues other, than the blood and liver, possibly the fat depots; (b) destruc tion of cholesterol may be stimulated or synthesis may be retarded; (c) absorption from the intestine may be retarded by reticulo-endothelial blockage; however, there is at pre sent no evidence that this system Is involved in the absorp tion process. SUMMARY An investigation has been made of the distribution of exogenous cholesterol in the tissues of the rat. Special emphasis has been placed on the quantitative dis tribution of free and esterified cholesterol in ultra- centrifugally separated fractions of the livers of rats fed cholesterol. A method has been described for the biological preparation of deuterium-labeled cholesterol from the egg yolks of hens fed deuterium oxide and deuterio-acetate. Deuterio-cholesterol has been prepared in quantity using this method and also the platinum-catalyzed exchange re action according to Bloch and Rittenberg. The ingestion of a synthetic diet supplemented with 1 per cent of deuterium-labeled cholesterol for 7 days led to a significant increase in cholesterol concentration in the liver and blood plasma only. However, exogenous cho lesterol was present also in all of the other organs examined, with the exception of the brain in which only a negligible amount was detected. The concentration of deuterio-cholesterol was highest in the liver and adrenals, slightly lower in the spleen, lungs, plasma, blood cells, and urine, and 108 decreased successively in the small intestine, carcass, kidney, and brain. The amount of exogenous cholesterol deposited in the liver amounted to 18 per cent of the total quantity of labeled cholesterol found in the body. The addition of bile extract to the diet caused a marked increase in this percentage but did not influence the deposition of cholesterol in the other tissues. The previous feeding of a diet containing 1 per cent cholesterol for 30 days had little effect on the■ deposition of deuterio-cholesterol in the kidney, spleen, or intestine. However, there was a decrease in the amount of exogenous cholesterol in the lungs and red blood cells, which may indicate that cholesterol feeding results in a depressed turnover of cholesterol in these tissues. It has been postulated that exogenous cholesterol, upon entering the plasma, becomes mixed with and indis tinguishable from the endogenous cholesterol already pre sent, and that the cholesterol of the plasma is in equil ibrium with the tissue cholesterol. The remarkable capacity of the liver of the rat to store exogenous cholesterol led to an investigation of the distribution of cholesterol in the livers of rats fed a 1 per cent cholesterol diet for 0 to 14 days. Three 109 constituents of the liver homogenates separated by- differential centrifugation were studied in this respect, A rapid and almost linear increase in esterified cholesterol concentration was observed in the centripetally migrating, or floating, fraction. A less pronounced de position of esters occurred in the residue fraction, which was composed of.nuclei, mitochondria, and microsomes. A more gradual and less significant increase in the content of free cholesterol was observed in the floating layer and residue. No significant changes in cholesterol con centration were found in the supernatant fraction. During the period of cholesterol ingestion, the percentage of total liver cholesterol decreased from 73 to 42 per cent in the residue, increased from 13 to 50 per cent in the floating layer, and decreased from 13 to 5 per cent in the supernatant fraction. These changes could be attributed primarily to the large increase in esterified cholesterol content of the floating layer. A more detailed fractionation of the homogenates of the livers of rats fed the high-cholesterol diet for 7 days showed that the increase in esterified cholesterol concentration previously observed in the combined parti culates, or residue fraction, was localized primarily in the microsomal, or submicroscopic particulate, fraction. 110 Individual differences in the quantities of cho lesterol deposited in the livers of the rats studied were reflected in the floating layer only, further emphasizing the importance of this fraction as the major site of cho lesterol deposition. The possible relationship of the floating material to the structure of the intact liver cell and the signifi cance of the association of exogenous cholesterol with this fraction have been discussed. The role of the reticulo-endothelial s;ystem of the rat in the storage of dietary cholesterol was investigated by comparing the cholesterol concentration in the livers of rats injected with saline or with a mixture of thoro- trast and trypan red, reticulo-endothelial blocking agents. The free and esterified cholesterol values in the plasma and liver, as well as the total lipid content of the liver, were significantly lower in the rats treated with thoro- trast and trypan red than in the saline-injected controls. Several possible explanations for these observations have been suggested. BIBLIOGRAPHY 1. Mueller, J. H., J. Biol. Chem., 22, 1 (1915). 2. Biggs, M. W., Friedman, M., and Byers, S. 0., Proc. Soc. Exptl. Biol. Med., 78, 641 (1951). 3. Chaikoff, I. L., Bloom, B., Siperstein, M. D., Kiyasu, J. Y., Reinhardt, W. 0., Dauben, W. G., and Eastham, J. F,, J. Biol. Chem., 194, 407 (1952). 4. Weltmann, 0., and Biach, P., Z. exptl. Pathol. Therap., 14, 367 (1913). 5. Anitschkow, N., and Chalatow, S., Centr. allgem. Pathol, u. pathoi. Anat., 24, 1 (1913). 6. Wacker, L., and Hueck, W., Munch, rated. Wochschr., 60, 2097 (1913). 7. Schoenheimer, R., and Yuasa, D., Z. physiol. Chem., 180, 5 (1929). 8. Okey, R., Proc, Soc. Exptl. Biol. Med., 50, 1003 (1933). 9. Blatherwick, N. R., Medlar, E. M., Bradshaw, P. J., Post, A. L., and Sawyer, S. D., J. Biol. Chem., 105, 93 (1933). 10. Chanutin, A., and Ludewig, S., jJ. Biol. Chem., 102, 57 (1933). 11. Best, C. H., and Ridout, J. H., J. Physiol., 78, 415 (1933). 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.r 24. 25. 112 Sperry, W. M., and Stoyanoff, V. A., jT. Nutrition, £, 131 (1935). Cook, R. P., and McCullagh, G. P., Quart. J. Exptl. Physiol., 29, 283 (1939). Rosenkrantz, J. A., and Bruger, M., Arch. Pathol., 42, 81 (1946). Weiss, S. B., Marx, L., and Marx, W., Endocrinology, 50, 192 (1952). Aylward, P. X., and Stott, W., Biochem. _J., 31, 2055 (1937). Leary, T., Arch. Pathol., 32, 507 (1941). Okey, R., and Greaves, V. D., J. Biol. Chem., 129, 111 (1939). Cook, R. P., Biochem. J., 30, 1630 (1936). Stamler, J., Bolene, C., Levinson, E., Dudley, M., and Katz, L. N., Am. J. Physiol., 155, 470 (1948). Goldman, J., Arch. Pathol., 49, 169 (1950). Marx, W., Marx, L., and Deuel, H. J., Jr., Am. Heart J., 42, 124 (1951). Schoenheimer, R., Z. physiol. Chem., 180, 1 (1929). Menschick, W., and Page, I. H., Z. physiol. Chem., 218, 95 (1933). Schettler, G., Biochem. Z ., 319, 349, 444 (1949). 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 113 Wainfan, E., Henkin, G., Rice, L. I., and Marx, W., Arch.. Biochem. and Biophys., 58, 187 (1952). Steiner, A. and Domanski, B., Am. J. Med. Sci., 201, 820 (1941). Altschul, R., Selected Studies on Arteriosclerosis, Charles C. Thomas, Springfield, 1950. Altschul, R., Am. Heart J., 40, 401 (1950). Bailey, C. H., Proc. Soc. Exptl. Biol. Med., ,13, 60 (1915). Duff, G. L., Arch. Pathol., 20, 8l (1935). Steiner, A., and Kendall, P. E., Arch. Pathol., 42, 433 (1946). Lindsay, S., and Chaikoff, I. L., Arch. Pathol.., 49, I 434 (1950). Dauber., D. V., and Katz, L. N., Arch. Pathol., 54, 937 (1942). Katz, L. N., Circulation, 5, 101 (1952). Chalatow, S. S., Centr. allgem. Pathol. u. pathol. Anat., 25, 197 (1914). Page, I. H., and Brown, H. B., Circulation, 6_, 681 (1952). Bragdon, J. H., and Boyle, E., Am. J. Pathol., 28, 527 (1952). 114 39. Hartroft, W. S., Ridou't, J. H., Sellers, E. A., and Best, C. H., Proc. Soc. Exptl. Biol. Med., 81, 384 (1952). 40. Wissler, R. W., Proc. Inst. Med., Chicago, 19, 79 (1952). 41. Marx, W., Marx, L., Meserve,-E. R., Shimoda, F., and Deuel, H. J., Jr., Arch. Pathol., 47, 440 (1949). 42. Marx, W., Marx, L., and Shimoda, P., Proc. Soc. Exptl. Biol. Med., 73, 599 (1950). 43. Wilens, S. L., and Sproul, E. E., Am. J. Pathol., 14, 177 (1938). 44. Van Bruggen, J. T., Hutchens, T. T.,. and,,West, E. S., Federation Proc., 10, 263 (1951). 45. Gould, R. G.,,Circulation, 2, 467 (1951). 46. Biggs, M. W., and Kritchevsky, D., Circulation, 4, 34 (1951). 47. Favarger, P., and Metzger, E. F., Helv. Chim. Acta., 35, 1811 (1952). 48. Bensley, R. R., and Hoerr, N. L., Anat. Record, 60, 449 (1934). 49. Claude, A., J. Exptl. Med., 84, 51 (1946). 50. Schneider, W. C., and Hogeboom, G. H., J. Biol. Chem., 185, 123 (1950). 51. Bensley, R. R., Anat. Record, 69, 341 (1937). 115 52. Chargaff, E., J. Biol. Chem., 142, 491 (1942). 53. LazaroY/, A., Biol. Symposia, 10, 9 (1943). 54. Swanson, M. A., and Artom, C., J. Biol. Chem., 187, 281 (1950). 55. Kretchmer, N., and Barnum, C. P., Arch. Biochem. and Biophys., 31, 141 (1951). 56. Chauveau, J., Clement, G., Clement-Champougny, J., and LeBreton, E., Arch, sci. physiol., _5, 305 (1951). 57. Schotz, M. C., Rice, L. I., and Alfin-Slater, R. B., J. Biol. Chem., 204, 19 (1953). 58. Dauber, D. V., and Katz, L. N., Arch. Pathol., 56, 473 (1943). 59. Hueper, W. C., Medicine, 20, 397 (1941). 60. Thannhauser, S. J., Lipoidoses, Diseases of the Cellular Lipid Metabolism, Oxford University, Press, New York (1950). « 61. Gubner, R., and Ungerleider, H. E. , Am. J. Med., €>, 60 (1949). 62. Bloch, B., Brit. J. Dermatol., 43, 61 (1931). 63. Schaaf, P., Zentr. f. Haut. u. Geschlechtskr., 35, 193 (1930). 64. Wilens, S. L., Science, 114, 389 (1951). 65. Payne, T. P. B., and Duff, G. L., Arch. Pathol., 51, 116 379 (1951). 66. Weinhouse, S., and Hirsch, E. F., Arch. Pathol., 50, 856 (1940). 67. Woerner, A., Proc. Ain. Diabetes Assoc., 10, 180 (1950). 68. Combined Staff Clinics, Am. J. Med., j5, 103 (1949). 69. Tomkins, E. H., Arch. Pathol., 42, 299 (1946). 70. Bloch, K., Berg, B., and Rittenberg, D., J. Biol. Chem., 149, 511 (1943). 71. Cashin, M. F., and Mor&vek, B.., Am. J. Physiol., 82, 294 (1927). 72. Osborn, M., Womack, N., Daum, K., and Kridelbaugh, W., Arch. Pathol., 52, 546 (1951). 73. Gardner, L. U., and Cummings, D. E., Am. J. Pathol., 9, 751 (1933). 74. Pollack, O. J., Am. Heart J., 38, 459 (1949). 75. Friedmen, M., Byers, S. 0., and Gunning, B., Am. J. Physiol., 172, 309 (1953). 76. Goebel, F., and Gnoinski, H., Compt. rend, soc. biol., 96, 1483 (1927). 77. Leites, S., Bjochem. Z., 186, 391 (1927). 78. Gol'ber, L. M., Fiziol. Zhur. S.S.S.R., 36, 600 (1950). 79. Leites, S., Biochem. Z., 186, 436 (1927). 80. Takagi, K., Hagoya Igakkai Zasshi, 43, 575 (1936). 81. Randles, F. S., and Knudson, A., J. Biol. Chem., 76, V 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 117 89 (1928). Brown, H. Z., Philips, I., and Kagan, B. M., Metabolism, Clin, and Exptl., ±t 349 (1952) . Uotila, U., and Simola, P. E., Arch, path. Anat. (Virchowjjs), 301, 523 (1938). Hirt, A., and Wimmer, K.,. Klin. Wochschr., 19, 123 (1940). Lasch, P., and Roller, D., Klin. Wochschr., 15, 1636 (1936). Wendt, H., and Konig, D., Klin. Wochschr.,. 16, 1255 (1937). Thompson, S. Y., Ganguly, J., and Kon, S. K., Brit. J. Nutrition, J3, 50 (1949). Callow, N. H., Callow, R. K., and Emmens, C. W., Biochem. J., 32, 1312 (1938). Nieft, M. L., and Deuel, H. J., Jr., J«. Biol. Chem., 177, 143 (1949). Alfin-Slater, R. B., Rock, E. M., and Swislocki, M., Anal. Chem., 22, 421 (1950). Bloch, K., and Rittenberg, D., J. Biol. Chem., 149, 505 (1943). Rice, L. I., Alfin-Slater, R. B., and Deuel, H . J., Jr., Proc. Soc, . Exptl. Biol. Med., 80, 562 (1952). 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 118 Fukushima, D. K., and Gallagher, T. F., J. Biol. Chem., 198, 861 (1952). Biggs, M. W., and Kritchevsky, D., Circulation, 4, 34 (1951). Halford, J. 0.,. and Anderson, L. C., J. Am. Chem. Soc., 58, 736 (1936). , Rittenberg, D., and Sehoenheimer, R., _J. Biol. Chem., 121, 235 (1937). Bloch, K., and Rittenberg, D., J. Biol. Chem., 145, 625 (1947). Kritchevsky, D., and Kirk, M., Proc. Soc. Exptl. Biol. Med., 78, 200 (1951). Farris, E. J., and Griffith, J., Q., The Rat in Laboratory Investigation, J. B. Lippincott, Philadelphia, 1949, pp. 410, 413. Lange, W., J. Am. Oil ChemistsT Soc., 27, 414 (1950). Hogeboom, G. H., Schneider, W. C., and Striebach, M., £• Biol. Chem., 196, 111 (1952). Baillif, R. N., Am. J. Anat., 92, 55 (1953). Waelsch, H., Sperry, W. M., and Stoyanoff, V. A., SL* Biol. Chem., 155, 291 (1940). Srere, P. A., Chaikoff, I. L., Treitman, S. S., and Burstein, L. S., J. Biol. Chem., 182, 629 (1950). |105. i jl06. '107. 108. ;io9. i i ! ;no. i , 111. i 112. t Ins. i i ! ,114. '115. i I |ll6. ! I 1117. 119 Alfin-Slater, R. B., Deuel, H. J., Jr., Schotz, M. C«,; and Shimoda, P., Federation Proc., j?, 144 (1950). | I Sehoenheimer, R., Biochem. Z., 147, 258 (1924). j i Loeffler, K., Z. physiol. Chem., 178, 196 (1928). ! Si’ perstein, M. D., Chaikoff, I. L., and Reinhardt, j w * 1* Chem., 198, 111 (1952). j Kritchevsky, D., Kirk, M. R., and Biggs, M. W., Metabolism, Clin, and Exptl., 1, 254 (1952). Byers, S. 0., and Friedman, M., Am. _J. Physiol., 168, I I 138 (1952). ! I Dam, H., Biochem. J., 28, 820 (1934). j Baumgartel, T., Klin. Wochschr., 22, 297 (1943). I Bergstrom, S., Kgl. Fysiograf. Sallskap. Lund, . Forh.-, 22, 1 (1953). | Gould, R. G., Am. J. Med., 11, 209 (1951). j i Biggs, M. W., Kritchevsky, D., Colman, D., Gofman, j J. W., Jones, H. B., Lindgren, P. T., Hyde, G., 1 and Lyon, T. P., Circulation, 6, 359 (1952). i ! Peters, J. P., and Van Slyke, D. D., Quantitative { I Clinical Chemistry, Vol. I, Williams and Wilkins, . I Baltimore, 1946, p. 422. ‘ Cook, R. P., Polgar, N., and Thompson, R. 0., , Biochem. J., 43, ix (1948). j 118. 119. 120. 121. 122. 123. Meier, J. R., Siperstein, M. D., and Chaikoff, I £• Blol> Chem., 198, 105 (1952). Tomkins, G. M., Sheppard, H., and Chaikoff, I. L J. Biol. Chem., 201, 137 (1953). Krinsky, N. I., and Ganguly, J., _J. Biol. Chem., 227 (1953). Pallade, G. E., and Claude, A., J. Morphol., 85, 71 (1949). Schneider, W. C., and Hogeboom, G. H., Cancer Research, 11, 1 (1951). Fisher, R, A., Statistical Methods for Research Workers, Oliver and Boyd, London, 1936. 120 . L. , 202, 35, University of Southern'CalSfeEmi?

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

An investigation of carotene metabolism in the alloxan treated rat

PDF

Chemical, physicochemical, and biological studies on the mucoproteins of plasma and serum

PDF

Competitive antagonists of thyroxine and structurally related compounds

PDF

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

PDF

A comparative study of the utilization of jojoba and cottonseed oil in the rat

PDF

A comparison of the turnover of cholesterol in rabbits on a normal and high cholesterol diet

PDF

Carbohydrate metabolism in thiamine deficiency.

PDF

A nutritional evaluation of the essential fatty acids in the rat

PDF

Adrenalectomy and fat absorption

PDF

A study of the effect of glycogen on the oxidation of butyrate by rat liver slices

PDF

A comparison of glucose tolerance in normal rats and those with fatty livers

PDF

A study of the ketone body metabolism of excised rat tissues with respect to labile phosphate content

PDF

Cholinesterase levels in the sera of rabbits with livers experimentally poisoned by carbon tetrachloride

PDF

A study on the comparison of the cholesterol content of rat liver homogenates before and after incubation

PDF

A method for the analysis of deuterium-labeled tissue cholesterol: Attempts to study the relation of the thyroid to the turnover of cholesterol in rats

C<super>14</super> stearic acid metabolism during fat absorption in the rat.

PDF

C14 stearic acid metabolism during fat absorption in the rat.

PDF

Comparison of results of phasic and chromatographic methods for carotene analysis.

PDF

Some studies on the effects of dietary choline on the plasma cholesterol concentration in the rat

PDF

A study of methods of cholesterol analysis

PDF

An investigation of factors involved in the inception of blood coagulation.

Asset Metadata

Creator Rice, Leslie Irene (author)

Core Title An investigation of the distribution and mechanisms of deposition of exogenous cholesterol in the rat

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

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

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Deuel, H.J. (committee chair), [illegible] (committee member), Marx, Walter (committee member), Wehl, John W. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-602082

Unique identifier UC11354010

Identifier DP21555.pdf (filename),usctheses-c17-602082 (legacy record id)

Legacy Identifier DP21555.pdf

Dmrecord 602082

Document Type Dissertation

Rights Rice, Leslie Irene

Type texts

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

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

Repository Name University of Southern California Digital Library

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