University of Southern California Dissertations and Theses

Factors Affecting Cholesterol Absorption And Metabolism

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Factors Affecting Cholesterol Absorption And Metabolism

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Content FACTORS AFFECTING CHOLESTEROL ABSORPTION AND METABOLISM by Arthur F. Wells A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry and Nutrition) June 1959 UNIVERSITY O F SOUTHERN CALIFORNIA G RAD U AT E S C H O O L UNIV ERSITY PARK LOS A N G E L E S 7. C A LIFOR N IA This dissertation, written by ....... Ar^hur .F..Wells............ under the direction of h.d~3...Dissertation C o m mittee, and approved by all its members, has been presented to and accepted by the Graduate School, in partial fulfillment of requirements for the degree of D O C T O R O F P H I L O S O P H Y s ' / ) ' / j/ , / l c ’ t / Dean yj. ; - D a te............................... DISSERTATION COMMITTEE ACKNOWLEDGEMENTS The investigations reported herein were begun under the sponsorship and guidance of the late Professor H. J. Deuel, Jr. The memory of his warm personal friendship and outstanding intellectual ability will always remain an inspiration. To Professor Roslyn Alfin-51ater, whose encouragement, friendship and counsel has made possible the completion of this work, my deepest and sincerest gratitude. Thanks are also due to the members of the staff of the Harry J. Deuel, Jr. Laboratory for their assistance in many ways. I should like to extend my thanks to the other members of my guidance committee, Professors Ershoff, Bavetta, Mehl, Geiger, Saunders and Berson for their helpful and understanding assistance. Financial assistance in the form of a fellowship from the Proctor and Gamble Company, Cincinatti, Ohio, during part of this work, is also gratefully acknowledged. TABLE OP CONTENTS PAGE INTRODUCTION............................................. 1 HISTORICAL REVIEW ..... ............................ 3 Biosynthesis of Cholesterol ....... ......... 3 Absorption of Cholesterol ........... ....... 6 Effects of Plant Sterols on Cholesterol Absorption. 9 Catabolism of Cholesterol.................... 13 Homeostatic Mechanisms in Cholesterol Metabolism . 15 The Effects of Essential Patty Acid Deficiency on Cholesterol Metabolism .... 17 Statement of the Problem ........ 20 MATERIALS AND METHODS ................ 22 Animals ........... 22 Diets.... ................................. 22 Supplements............................................. 25 Extraction of Lipids.......... ....................... 2 5 From liver .. .................... 25 Prom plasma . . . . . . . . . . . . . 26 Determination of Cholesterol .... 26 Determination of Total Lipids ................ 29 Preparation of a Methyl Linoleate Concentrate . . . 29 Preparation of Sterol Esters ......... ...... 31 EXPERIMENTAL AND RESULTS............. 33 iii iv PAGE Experiment l.-The Effects of Plant Sterols on the Mobilization of an Excess of Cholesterol from the Liver................. .....................33 Experiment 2.-The Effects of Soy Sterols on the Absorption of Cholesterol Esters ............. 41 Experiment 3 .-The Effects of Esterified Plant Sterols on the Absorption of Cholesterol and Cholesteryl Linoleate ...... ............. 44 Experiment 4.-The Effect of Feeding Cholesterol and Plant Sterols on Alternate Days............. 48 Experiment 5.-The Effects of Essential Fatty Acid Deficiency on Cholesterol Absorption and Metabolism ................................ 51 Experiment 6 .-The Effects of Bile Salts and /S-sitosterol on Cholesterol Levels in Essential Fatty Acid-Deficient and Normal R a t s ...........54 Experiment 7.-The Effects of the Addition of P-sitosterol to the Diet of Rats During the period of Depletion of Essential Fatty Acids . 57 DISCUSSION............................. ............ 5 9 SUMMARY .........................................6 6 LITERATURE CITED .................................... 70 LIST OF TABLES TABLE I. Composition of Diets II. Cholesterol and Total Lipid Levels in the Livers of Rats Fed 3% Soybean Sterols With and Without Fat at Various Periods After Removal from a Diet Containing 1% Cholesterol III. Half-lives and Apparent Specific Rate Constants for the Removal of Excess Total, Esterified or Free Cholesterol from the Livers of Xorxnal Rats .......... IV. Plasma and Liver Cholesterol Levels of Rats Receiving Various Cholesterol Esters With and Without the Addition of Soy Sterols . V. Plasma and Liver Cholesterol Levels of Rats Fed Diets Containing Cholesterol or Cholesteryl Linoleate With and Without the Further Addition of Soy Sterols or Soy Steryl Palmitate . . . ....... VI. Plasma and Liver Cholesterol Levels of Rats Fed Cholesterol and Soy Sterols on Alternate D a j y s . . . . • ® ® . . . VII. Body Weight Gains and Liver and Plasma Cholesterol Levels of Essential Fatty Acid-Deficient Rats Fed Cholesterol or Methyl Linoleate ....... ......... VIII. Plasma and Liver Cholesterol Levels of Essential Fatty Acid-Deficient and Mormal Animals Fed Bile Salts and 5-sitosterol. IX. Plasma and Liver Cholesterol Levels of Rats Fed ^ 8 -sitosterol During the Period of Depletion of Essential Fatty Acids . . PAGI 2 3 35 4:0 42 45 50 52 56 58 v V.-J INTRODUCTION The study of cholesterol, its chemistry and bio chemistry, was begun more than a century and a half ago. Since that time cholesterol has probably received more attention from chemists, biochemists, physiologists and other workers in the field of medicine than any other single chemical compound. The implication of cholesterol in the genesis of atherosclerosis has added further impetus to research in the field of cholesterol absorption and metabolism. Atherosclerosis is a disease characterized by the deposition of cholesterol and other lipid in the blood vessel walls. Complete blockage of a blood vessel may have serious consequences. Elucidation of the mechanisms involved in atheroma formation is far from complete, but available information provides increasing evidence that diet may be an important factor. While many dietary components have been shown to alter cholesterol metabolism and/or distribution, the mechanisms by which these changes are effected are still to be determined. In many cases dietary changes, recommended for the control of hypercholesterolemia, involve the lipid part of the diet. These changes range from complete elimination of lipid from the diet to the substitution of vegetable oils for animal fats. The use of highly unsaturated vegetable oils, such as corn oil, in place of animal fats has received some experimental support and has been widely advocated for the control of atherosclerosis. Such a substitution results in an increased amount of essential fatty acids in the diet and, at the same time provides small amounts of plant sterols, which have been shown to interfere with choles terol absorption. The present work undertakes a more thorough study of the separate actions of both plant sterols and essential fatty acids on the absorption and metabolism of choles terol. It attempts further, to characterize more completely the aberrations in cholesterol metabolism produced by a deficiency in essential fatty acids. HISTORICAL RiiVIJEW The study of cholesterol dates back almost two centuries when Poullitier de la Salle (1) first isolated it from gallstones in about 1769. Over a century elapsed before even the empirical formula, C2 7 H4 QO, was determined (2) and it was not until 1932 that the structural formula (I) was completely elucidated (3,4). Ho I Biosynthesis of Cholesterol Until the advent of isotope tracer techniques, the study of cholesterol biosynthesis was limited to balance experiments. These early experiments (5,6) served to show that cholesterol could be synthesized in the animal body but did not provide evidence of either the site or mechanism of the synthesis. When isotopes became available, progress in this aspect of the study of cholesterol was greatly accelerated. It soon became apparent that acetate was an excellent pre- 3 cursor for cholesterol synthesis (7,8), and that both carbon atoms of acetate were utilized (9,10). During the past six or seven years, methods have been evolved for the complete degradation of cholesterol and the recovery of each carbon atom separately (11-15). Results obtained by these techniques have confirmed that all the carbon atoms of cholesterol may be provided by acetate and that their arrangement is in accord with earlier proposals (1 1 ) that a repeating five-carbon subunit, containing three methyl- and two carboxyl-carbon atoms, are united somehow to form cholesterol. The nature of such a five-carbon subunit is still unknown. However, acetoacetate appears to be on the path way to this hypothetical five-carbon compound, since it has been shown to be incorporated intact into cholesterol (16). It has been proposed that acetoacetate or its coenzyme A derivative then reacts with a molecule acetyl-coenzyme A to form a six-carbon compound which then loses a carbon-atom before condensing to form, eventually, cholesterol. The six-carbon compound, (2 -8 -dihydroxy-y# methyl- valeric acid (mevalonic acid) has been found to be a very active precursor for cholesterol synthesis (17). The mechanism of formation of mevalonic acid from acetoacetate and acetate is still obscure. It appears possible that f3 -hydroxy-^-methylglutanyl-coenzyme A may be a precursor for mevalonic acid synthesis and that the poor results obtained with free -hydroxyl - A methyl- glutarate (18) are due to the lack of an activating system. After the formation of mevalonic acid, the next few steps in the biosynthetic pathway are unknown. The next known intermediate is squalene. In 1926, Channon (19) noted that the feeding of squalene to rabbits caused an increase in liver cholesterol. In 1953 Langdon and Bloch (2 0 ,2 1 ) demonstrated that squalene was, indeed, an inter mediate in cholesterol synthesis. They isolated labeled squalene from the liver of a rat fed labeled acetate and subsequently recovered labeled cholesterol from a mouse fed this labeled squalene. Cyclization of squalene gives a 30-carbon sterol, lanosterol (22). Between lanosterol and cholesterol, three methyl groups are lost, the double bond in the side chain is saturated, and the 8-9 double bond is shifted to the 5-6 position. The three methyl groups are probably eliminated first to give zymosterol (23), followed by rearrangement of the ring unsaturation to give desmosterol (24). Finally saturation of the double bond in the side chain gives cholesterol. The synthesis of cholesterol from acetate appears to require the participation of enzyme systems of both the microsomes and the soluble supernatant fractions (25). 6 The cofactors were shown to be Mg++, DPN and ATP. Cholesterol is synthesized in many tissues but the liver appears to be one of the most important sources of endogenous cholesterol. Not only is the In vitro synthesis of cholesterol more rapid in liver than in many other tissues (26), but also, the liver is the sole source of plasma cholesterol (27,28). The importance of the liver as a site of synthesis of cholesterol is emphasized by the work of London and Greenberg (29), who found that after acetate injection, the peak occurrence of labeled cholesterol, in other tissues, was observed at a time when no labeled non-steroid precursors were present. The inference was made that the C-^ cholesterol found must have been derived from the plasma. Absorpt ion ojf cholest erol« The occurrence of cholesterol in milk and eggs as well as in all animal tissues results in its involuntary inclusion in the diet of all but the strictest vegetarians. Even in the vegetarian, excretion of cholesterol in the bile assures its presence in the intestinal contents. It was very early recognized that cholesterol is absorbed via the lymph (30), and more recent experiments using labeled cholesterol have confirmed that this is the exclusive route of absorption (31). Not all of the cholesterol present in the intestinal lumen is absorbed. The percentage of ingested cholesterol absorbed appears to depend on the amount fed. Thus Friedman, Byers and Shibata (32) found that 41% of a 50 mg dose was absorbed whereas only 34% was absorbed when the dose was increased to 100 mg. Similar results were obtained by Lin, Karvinen and Ivy (33) who found that of a single dose of cholesterol administered, a maximum of 92 mg could be absorbed by a rat, no matter how large the dose. The cholesterol of lymph is esterified to approxi mately 70%. This ratio of free to esterified cholesterol does not change regardless of whether free or esterified cholesterol is fed (30), and during the absorption of labeled cholesterol, the label is found in both the free and ester fractions in the lymph. The presence of a cholesterol esterifying system in the intestinal mucosa, suggested by the foregoing results, has been demonstrated. Swell, Treadwell and their collaborators have made extensive studies of the choles terol esterase of intestinal mucosa (34) as well as that found in pancreatic juice (35,36). An interesting aspect of their findings is that bile enhances the enzymatic activity of cholesterol esterase (37). This may account, in part at least, for the finding that bile salts are necessary for the absorption of cholesterol (38), although their well known role as a dispersing agent for lipids in the intestinal lumen may also be important. The esterification of cholesterol in the intestinal mucosa suggests that fatty acids might also be important in this connection. Dietary fat has been shown to enhance the absorption of cholesterol as measured by decreased fecal excretion of cholesterol (39) or by an increase in plasma cholesterol level (40). Unsaturated free fatty acids are more active in this respect than their respective glycerides, whereas palmitic and stearic acids increase cholesterol excretion (39). A recent communication of Swell, Treadwell and associates (41) describes the presence of a considerable pool of free cholesterol in the intestinal mucosa. Using labeled cholesterol in thoracic duct cannulated animals, they have followed the course of cholesterol absorption over a period of 48 hours. They suggest that the followin sequence of events occurs during the absorption of cholesterol^ (1 ) ingested cholesterol is dispersed in the intestinal lumen with the aid of bile salts, and any cholesterol esters are split by pancreatic cholesterol esterase, (2 ) the free cholesterol liberated is absorbed into the intestinal mucosa and rapidly mixes with the pool of endogenous cholesterol, (3) a major portion of the 9 cholesterol is esterified, (4) the esterified cholesterol plus some free cholesterol, protein, phospholipid and triglyceride are utilized to form chylomicrons of a rather definite composition which are then discharged into the lymph. A somewhat similar sequence of events has been proposed by Glover and Morton (42). These authors however, assign a much more prominent role to protein and a more minor one for cholesterol esterification. They view the transfer of cholesterol across the intestinal wall as occurring through a series of transfers of cholesterol from one liproprotein to another. In their view, esteri- fication occurs only when the transport mechanism for free cholesterol is exceeded, the esterified cholesterol forming a temporary reserve until the peak of absorption is over. The absorption of sterols displays considerable specificity; the phytostero'ls and saturated sterols are much less readily absorbed than cholesterol, although there is little difference in structure or fat solubility among the various sterols tested. Effects of Plant Sterols on Cholesterol Absorption. In view of the marked specificity of the absorption of cholesterol, it was, perhaps, inevitable that studies should be undertaken of the effects of the poorly absorbed plant sterols on the absorption of cholesterol. In 1951, 10 Peterson (43) reported that, in the chicken, the increase in plasma and liver cholesterol associated with cholesterol feeding was markedly reduced by the addition of soybean sterols when both sterols were fed at 1%. Subsequent reports have confirmed these results in the rabbit (44), while in the rat, where plasma cholesterol is little affected by cholesterol feeding, plant sterols inhibit the increase in liver cholesterol (45). In addition, results obtained by Alfin-Slater, Wells, After good, Melnick and Deuel (46) showed that the inhibition of the rise in liver cholesterol was not a result of a redistribution to other tissues of the animal. Peterson (47) reported that for best inhibition of cholesterol absorption, the optimum ratio of plant sterol to cholesterol appeared to be between 2 : 1 and 3 : 1 and that esterification of the soybean sterol with capric acid abolished its effect. This report is somewhat surprising in view of the relative lack of specificity of cholesterol esterase (48), but may merely indicate a deficiency of esterase in the chicken. The effects of plant sterols appear to be due to the inhibition of absorption of cholesterol. Recoveries of cholesterol from thoracic duct lymph was significantly depressed by the simultaneous inclusion of soy sterols or ^-sitosterol (45,49). The recovery of cholesterol in 11 liver and feces of rats prefed cholesterol or cholesterol plus soy sterols has also been studied (50). In this case a smaller amount of labeled material was recovered from the liver and a larger amount from the feces, when soy sterols were included in the pretest diet or were fed with the test dose. The mechanism by which plant sterols prevent the absorption of cholesterol is still under active study. Two theories have been advanced to account for their action. Davis (51) has suggested that an unabsorbable sitosterol-cholesterol complex is formed, while Swell e_t at ( 52, 53) proposed a competition with cholesterol for the absorptive system in the intestinal wall. A more recent report from the same group (54) strengthens the latter theory. In this study tritium labeled ^-sitosterol was fed to rats and substantial amounts of this substance were found in the intestinal wall six hours later. The absorption of plant sterols has been a matter of much concern. Apparent absorption of as much as 5 0% has been recorded. However, absorption values of this magnitude have been obtained by balance experiments and represent disappearance of sterol as measured by gravi~ metric or colorimetric methods. These experiments do not preclude the possibility that the plant sterols were de graded by bacterial action in the intestinal lumen to a 12 form not precipitable with digitonin or reactive in the colorimetric assays. The disappearance of tritium labeled ^-sitosterol has been noted (54). It could not be accounted for as sterol in intestinal contents, feces, intestinal wall or lymph. In this experiment, less than O.6/0 of a 44 mg dose of f2 -sitosterol was recovered from the lymph over a 48 hour period. The only other experiment using labeled (3 -sitosterol is that of Gould (57) who concluded that about 4% was absorbed in the rat and that the absorption of ^-sitosterol in man was aibout 1 0 %’ that of cholesterol. The efficacy of soy sterols and -sitosterol in reducing plasma cholesterol levels in hypercholesterolemic human patients has been the subject of many reports (58- 62). Generally, results have been promising, with re ductions of up to 5Q% being observed in some cases. Unfortunately, not all patients respond to this treatment. Wilkinson (62) found no reduction in plasma cholesterol levels in patients treated with £3 -sitosterol. There are probably many factors involved in the action of plant sterols including the timing of the dosage in relation to ingestion of food and also the fineness of dispersion of these sterols. The day to day and week to week variations in plasma cholesterol levels also pose a problem so that control experiments are necessary for proper evaluation. 13 It must be emphasized that a reduction of the plasma cholesterol level does not necessarily indicate a re gression in the atherosclerosis. While the addition of plant sterols to the diet of rabbits or chickens receiving 1% of cholesterol retards the appearance of atheroma, it has not yet been demonstrated that plant sterols have any effect on the regression of previously formed atheroma. Catabolism of Cholesterol. The major end product of cholesterol metabolism is bile acid. The formation of bile acid involves reduction of the length of the side chain of the cholesterol molecule by three carbon atoms, the oxidation of the terminal carbon in the remaining side chain to a carboxyl group, the saturation of the double bond, the epimerization of the 3-OH group to the alpha configuration, and the further hydoxylation of the nucleus at the 7 and/or 1 2 position. Hydroxylation at the 6 position also appears probable. The bile acids are generally excreted in the bile as either glycine or taurine conjugates. In the rat, the major bile acids are the taurine conjugates of cholic acid (3 0C 7 OL 12 -trihydroxy- cholanic acid) and chenodeoxycholic acid (3 & -, 7 CX -di- hydroxycholanic acid) (63). In man cholesterol is con verted mainly to glycocholic acid. Some taurocholic: and a small amount of glycodeoxycholic acid are also formed (64). 14 Bile acids isolated from feces are usually not conjugated and many products are found, presumably as a result of bacterial action. 1 A After the administration of cholesterol-4-C to rats, 1 A it was found that at least 90% of the C m the feces was excreted via the bile (65). The small amount of label excreted by the intestinal wall was mostly in the unsapon- ifiable fraction, while of the material excreted in the bile not more than 1 0% was in the unsaponifiable fraction. Other metabolic products of cholesterol of profound biological significance, but of relatively minor signifi cance from a quantitative point of view, are the steroid hormones. A discussion of the biological, transformations involved is beyond the scope of this review. The con version of cholesterol to 7-dehydrocholesterol and sub sequently to vitamin Do is another biologically important pathway of cholesterol metabolism but is also of minor quantitative significance. In addition to catabolic mechanisms in the animal tissues, the intestinal flora is also active in degrading r w S V cholesterol. It has been shown that ^ and /V * cholestenols as well as coprostanol (66,67) are present in the feces of man and the rat. The intestinal flora is also responsible for various alterations in the excreted bile acids. Homeostatic Mechanisms in Cholesterol Metabolism. The various aspects of cholesterol metabolism, such as absorption, synthesis, catabolism and distribution are in equilibrium in the normal animal. The equilibrium is maintained by a system of regulatory mechanisms, which tend to maintain tissue concentrations of cholesterol at a constant level in the face of changes in diet or environment. It is only when the capacity of one or more of these mechanisms are exceeded that changes in tissue cholesterol are noted. The hepatic synthesis of cholesterol is dependent on the cholesterol content of the diet. Feeding cholesterol results in a marked inhibition in hepatic cholesterol synthesis in vivo (6 8 ) and in vitro (69). The inhibition of cholesterol synthesis has been correlated with increased liver total (6 8 ) or free (70) cholesterol. The block in cholesterol synthesis appears to occur before the mevalonic acid stage. The use of mevalonic acid rather than acetate as a substrate in in vitro studies resulted in relatively much less inhibition of cholesterol synthesi following cholesterol feeding (71). In addition to its effect on hepatic synthesis of cholesterol, the level of cholesterol in the diet has a marked effect on its absorption. The proportion of cholesterol absorbed decreases as the level in the diet increases until a maximum level of absorption is attained (33). Since the conversion of cholesterol to bile acids represents the major pathway of catabolism of cholesterol, regulation of this reaction is an important factor in the maintenance of tissue cholesterol levels. The rate of this reaction appears to be controlled by the level of bile acids in the body. Bile acids are actively reabsorbed from the intes tine. If reabsorption of bile acids is prevented by cannulation of the bile duct, bile acid production is tremendously accelerated (72). It would thus appear that the destruction of bile acids in the intestinal lumen by the intestinal flora may play an important role in the regulation of tissue cholesterol levels. The regulation of the distribution of cholesterol between plasma and liver is poorly understood. In some species, such as the rat, this mechanism is extremely active and maintains plasma cholesterol at near normal levels even when liver levels are increased ten to twenty- fold. In other species, such as the chicken or rabbit, the regulation is not nearly so precise and increases in liver cholesterol are almost invariably followed by in creased plasma levels. The importance of the liver in the regulation of plasma levels is emphasized by the report of Friedman, Byers and Michaelis (28) that endogenous plasma cholesterol 17 is almost completely derived from the liver. Liver homogenates from starved rats show a drastic reduction in cholesterol synthesis (73). The reduced synthesis appears to be due to the formation of an in- hibitory substance since the addition of homogenate from a starved rat to normal liver depressed synthesis. The depression of cholesterol synthesis after inanition must be considered when testing the effects of deficiencies of essential nutrients. The Effects of Essential Fatty Acid Deficiency on Cholesterol Metabolism. The classical studies of Burr and Burr (74,75) demonstrated the need for essential fatty acids by the rat. The essential fatty acid deficiency syndrome was charac terized by skin lesions, renal lesions and a cessation of growth. It was concluded that linoleic and possible arachidonic acids were the essential fatty acids. Symptoms of essential fatty acid deficiency have been produced in other species (76,77) but the only human disorder, traceable to essential fatty acid deficiency with any degree of certainty, is infantile eczema (78). The suggestion that atherosclerosis may be related to a deficiency of essential fatty acids is based mainly on deductive reasoning. Many investigators (79-83) have reported a reduction in plasma cholesterol levels when 1 8 highly unsaturated vegetable oils were fed and an increase in those levels when the more saturated animal fats were fed. The effects of the more saturated vegetable oils, such as palm or coconut oil, and of artificially hydro genated vegetable oils were similar to those of animal fats, while unsaturated fish oils were similar in action to vegetable oils. Two important assumptions are involved in the derivation of the essential fatty acid deficiency theory of atherosclerosis from the facts presented. First, it is assumed that a reduction of the plasma cholesterol level is beneficial and an increase detrimental. Second, the assumption is made that the active factor in the unsat urated fats is the essential fatty acids rather than unsaturation per se. Arguments have been advanced against this theory. It is almost impossible to produce an essential fatty acid deficiency in the adult of any species. The plasma levels of essential fatty acids are no different in patients with atherosclerosis than in normal persons (84). Neither of these arguments eliminate the possibility of an impaired utilization of essential fatty acids, which could be benefited by increased intake simply by operation of the Law of Mass Action. Clarification of the role of essential fatty acids in cholesterol metabolism is necessary, before 19 the relative merits of the various arguments can be properly evaluated. An understanding of the role of any essential nutrient is usually best obtained by a study of changes in the metabolic processes of an animal deficient in that nutrient. This approach has been used by Alfin-Slater, Aftergood, Wells and Deuel (85,86) in their investigation of the effects of essential fatty acid deficiency on cholesterol metabolism. These investigators found that animals placed on a fat-free diet exhibited an increase in liver total cholesterol and a decrease in plasma cholesterol level. The increase in liver cholesterol was evident after only one week on the fat-free diet and was almost entirely confined to the cholesterol ester fraction. The alterations in liver cholesterol level were corrected by administration of methyl linoleate but not by methyl oleate. More recent work by Mukherjee and Alfin-Slater (87) has shown that the livers of essential fatty acid- deficient rats synthesize cholesterol in vitro at a much o w s ie a w » i» .-Aivrmir i i t w u m i slower rate than those from normal rats. It would appear, therefore, that accumulation of cholesterol in the livers of essential fatty acid-deficient rats is not a result of an increased synthesis. The decreased level of cholesterol in the plasma of 2 0 essential fatty acid deficient rats suggests an impairment of cholesterol distribution, but in view of the marked reduction in cholesterol synthesis it seems unlikely that impaired distribution alone can account for the increased liver cholesterol levels. The alternative is a decrease in the rate of excretion of cholesterol or its metabolites. Statement of the Problem. The use of unsaturated vegetable oils in place of animal fats, has been widely advocated for the prevention and control of hypercholesterolemia and atherosclerosis. Their effects on hypercholesterolemia have been variously ascribed to their content of essential fatty acids, to their content of plant sterols or to a combination of the two. The object of the present work was to study in more detail the separate actions of each of these factors, plant sterols and essential fatty acids. The feeding of plant sterols has been shown to inhibit the absorption of cholesterol, but whether this is due to the formation of an unabsorbable complex in the intestinal lumen, or to the inhibition of some enzymatic mechanism involved in cholesterol absorption, is still open to question. One phase of this work deals with this question, as well as with the effects of esterification of either cholesterol or the plant sterols on the inhibition of cholesterol absorption. 21 Another phase of the work deals with the mechanisms involved in the accumulation of cholesterol in the livers of essential fatty acid-deficient rats. The finding that in spite of this accumulation, or perhaps because of it, the rate of synthesis of cholesterol is markedly reduced, suggests that decreased catabolism or excretion of cholesterol may be responsible for the elevated liver cholesterol level. In this work a nutritional, "whole animal" type of approach has been used. The levels of plasma and liver cholesterol have been utilized as an index of cholesterol absorption, under various conditions of diet and deficiency state. A consideration of the results obtained has led to the advancement of hypotheses of the mechanism of action of both plant sterols and essential fatty acids. MATERIALS AND METHODS Animals Male albino rats of the University of Southern California strain were used throughout. Although plasma cholesterol levels in the rat show only minimal response to changes in diet, the concentration of liver cholesterol is very sensitive to dietary alterations and therefore acts as a good analytical tool. In addition, the ready availability, from our own colony, of animals of uniform genetic background and well defined dietary requirements, recommended their use. In the selection of animals for experiment, care was taken to randomize animals from the same litter among the various groups. At least six animals were used in each group. Diets A large number of diets were used throughout the course of this work, however, all diets used were modifi cations of one or the other of the two basic diets shown in Table 1. 22 23 TABLE I COMPOSITION OF DIETS Component Fat Free 15% Cottonseed Oil Caseini/ 2 0 . 0 0 24.00 Sucrose 71.52 52.52 Salt Mixture—/ 4.00 4.00 Cellulose!./ 4.00 4.00 Dry Vitamin Mix—/ 0.19 0.19 Vitamin A, D and E Mixture^!/ 0.05 0.05 Choline Chloride!,/ 0.24 0.24 Cottonseed Oil—/ mm m i* m 15.00 24 Footnotes to Table I i/V o r experiments not involving the production of an essential fatty acid deficiency, Lactic casein, Challenge Dairy Co., Los Angeles, California was used. For experiments involving the production of an essential fatty acid deficiency, Vitamin-free Test Casein, General Biochemicals Inc., Chagrin Falls, Ohio, was used. i/we sson Modification of the Osborne and Mendel Salt Mixture (Science 75, 339 (1932)); Nutritional Bio chemicals Corp., Cleveland, Ohio. ^/solka-Floc, Brown Co., San Francisco, California. £/The dry vitamin mixture consisted of 38.57% p-amino benzoic acid, 31.88% inositol, 12.75% ascorbic acid, 4.59% thiamin hydrochloride, 3.82% niacin, 3.82%, calcium pantothenate, 1.72% riboflavin, 1.72% pyr.idoxi.ne hydrochloride, 0.64% folic acid, 0.32% menadione, 0.16% biotin and 0.00004% vitamin B-j^; Merck and Co. and Nutritional Biochemicals Corp. %/nh e mixture consisted of one part Nopsol solution (containing 100,000 I.U. Vitamin A and 20,000 I.U. vitamin D2 per gram); Nopco Chemical Co., Harrison, New Jersey, and three parts Mixed Tocopherol Concentrate, 34%; Distillation Products Industries, Rochester, New York. £/wh en added to the 15% cottonseed oil diet, the mixture was first thoroughly dispersed in the oil. For addition to the fat-free diet, the mixture was first dispersed on a portion of the casein. Z/u. C •P., Merck and Co., Rahway, Mew Jersey. 8 / — Refined, winterized, cottonseed salad oil. Kindly provided by Best Foods, Inc., Bayonne, New Jersey. 25 Supplementation of these diets was carried out on the basis that fat-soluble materials were substituted for cottonseed oil when the 15% cottonseed oil diet was used, otherwise additions were made at the expense of sucrose. Supplements Cholesterol - U.S.P., Merck and Co., Rahway, Mew Jersey. Mixed Soy Sterols - Distillation Products Industries, Rochester, New York. ^3 «sitosterol - Kindly provided by Eli Lilly Co., Indianapolis, Indiana. Bile Salts - A commercial preparation of mixed bile saltss Difco Laboratories, Detroit, Michigan. Extraction of Lipids t — «m iM i'M ii.aa r. lUir.ttCM t n i A 3 ( > - H D From Liver.-The method used was essentially that of wwtfii in i " i i>i i p Thompson et al (8 8 ). The liver, together with 30 ml. of 61% ethanol, and 100 ml. of Skellysolve B (b.p. 63-69°C) was homogenized in a Waring Blendor for two 5 minute periods separated by a 5 minute interval. The homogenate was then transferred quantitatively to a separatory funnel and the aqueous-alcohol phase, containing the suspended tissue homogenate was returned to the blendor and re homogenized for 5 minutes with a second 100 ml. portion of Skellysolve B. A third extraction was carried out in 26 a similar fashion. The combined extracts were concentrated under reduced pressure, filtered into volumetric flasks and adjusted to volume (usually 250 ml.). Suitable aliquots were used for determination of cholesterol and total lipid. From Flasma.-Flood was obtained by cardiac puncture and treated with heparin to prevent clotting. FTasma was obtained by centrifugation of thp^heparinized blood. Proteins were precipitated and cholesterol extracted by the addition of ethanol-acetone (1 : 1 by volume) at a ratio of 14 volumes of the mixture per volume of plasma. The alcohol:acetone was added from a syringe with sufficient force to ensure complete mixing. After the precipitated proteins were removed by centrifugation, suitable aliquots of the supernatant extract were used for the determination of free and total cholesterol. Petermination of_ Cholesterol Free and total cholesterol were determined by the Nieft and Deuel (89) modification of the Sperry-Schoen- heimer method. An aliquot of extract estimated to contain 0.1 to 0.5 mg. of cholesterol was transferred to a 15 ml. centri fuge tube and evaporated to dryness at 60°C by means of a gentle stream of air. 27 For the determination of total cholesterol, choles terol esters were hydrolyzed by addition of 1 ml. of ethanol-acetone (1:1 by volume) and 2 drops of 33% (W/F) potassium hydroxide solution and heating in stoppered tubes at 60°C for 45 minutes with frequent agitation. Following hydrolysis the solution was neutralized to phenolphthalein by the dropwise addition of 15$ acetic acid and the volume was adjusted to 3 ml. by the addition of ethanol-acetone. For the determination of free cholesterol the hydrolysis procedure was omitted. The evaporated residue was brought up to a volume of 3 ml. with ethanol-acetone and one drop of 15$ acetic acid was added to ensure an acid reaction and prevent spontaneous hydrolysis. The ensuing steps in the determination of free and total cholesterol are the same. Cholesterol was precipitated as the digitonide by the addition of 1 ml. of digitonin solution (0.5$ in 50$ aqueous ethanol) and the mixture was allowed to stand overnight. The precipitated cholesterol digitonide was separated by centrifugation (3,000 r.p.m. for 20 minutes) and the supernatant liquid discarded. The precipitate was washed with 3 ml. of anhydrous ether by thorough mixing and centrifugation,the ether was decanted, and the washed precipitate was dried at 60°C with a gentle stream 28 of air. After the cholesterol digitonide was thoroughly dried it was split by the addition of 0.5 ml. of glacial acetic acid and by heating at 60°C in a stoppered tube until solution was complete. The solution was then allowed to cool, 3 ml. of chloroform was added, the tubes were stoppered again and the contents brought to 35°C* Color reagent was prepared by the dropwise addition of one volume of concentrated sulfuric acid to nine vol umes of acetic anhydride maintained in an ice bath. One ml. of this reagent was added to the solution and the mixture was maintained at 35°C with occasional shaking for exactly 10 minutes. At the end of that time, the tube was transferred to an ice bath to stop the reaction and cooled for at least 1 0 minutes before reading. The color intensity was determined in a IClett- Summerson colorimeter using a #62 filter (maximum trans mission at 620 mg,). The reagent blank consisted of acetic acid, chloroform and color reagent in the same proportions as was used in the determinations. Cholesterol values were obtained by comparison with a standard curve, which was determined simultaneously by similar treatment of known amounts of cholesterol ranging from 0.05 to 0.50 mg. 29 Determination of Total Lipids An aliquot of the tissue extract was evaporated almost to dryness on a hot plate, care being taken to prevent charring. Approximately 5 gm. of anhydrous sodium sulfate to remove moisture and 30 ml. of Skellysolve B were added. The solution was decanted through a Whatman #42 filter paper into a tared flask. The original flask and contents were rinsed five times with small portions of Skellysolve B, these rinsings being added to the first filtrate. The filtrate was evaporated to dryness on a hot plate, held overnight in an air oven at 8 8 °C and the lipid subseonently weighed. Preparation of a Methyl hinoleate Concentrate Since methyl linoleate was required in large amounts in this investigation, a simple method of preparation was evolved which provided methyl linoleate of reasonable purity. The starting material used was safflower oil'". Lin- oleic acid comprises approximately of the fatty acids of this oil and it contains no linolenic acid. Safflower oils which had an iodine value of less than 140 were rejected. '"We are indebted to the Pacific Vegetable Oil Co., San Francisco, California for supplying the Safflower Oil used in this preparation. The fatty acids of safflower oil were first converted to their methyl esters, This was accomplished by heating the oil to 60°C arid adding, with vigorous shaking, 25 ml. per 100 gi, of oil of a solution of sodium methoxide in methanol, prepared by dissolving 1,2 p . of metallic sodium in 100 ml. of absolute methanol. The mixture was maintained at 60°C for two hours and then allowed to cool overnight, The dark red, glycerol-rich layer which settled out was removed in a separatory funnel and the methyl ester layer was washed repeatedly with distilled water, care being taken to prevent the formation of emulsions, Hot water was used for the last few washes to ensure the complete removal of soaps. The methyl esters were then dried over anhydrous sodium sulfate and filtered, The mixed methyl esters were fractionated by pre cipitation with urea. Urea forms a crystalline complex with the straight chain esters of fatty acids. For a single chain length, the complex forms preferentially with the more saturated members of the series. Thus, by using an amount of urea less than required to precipitate all the methyl esters present, methyl stearate and oleate may be precipitated leaving the methyl linoleate in solution. To each 100 gnu of the mixed methyl esters of safflower oil was added 175 gm, of urea and 175 ml. of absolute ethanol. The mixture was held at 50°C for one hour, then allowed to cool overnight. The precipitate was then filtered off on a large Buchner funnel and washed with two or three small portions of absolute ethanol. The filtrate and washings were transferred to a large separatory funnel and sufficient distilled water was added to render the esters insoluble. The aqueous alcohol phase was drawn off and the esters were washed with distilled water and dried over anhydrous sodium sulfate, Final purification was effected by distillation under reduced pressure in a simple, all glass apparatus, The first and last portions of the distillate were discarded. The purity of the product, based upon Iodine Value determinations, was consistently found to be between 92 and 94f«, This is a conservative estimate since the cal culation was based on the assumption that the impurity consisted solely of methyl oleate, Overall yield, based on the original oil, was approximately 2 %, Preparation of Sterol Esters The fatty acid esters of cholesterol and of mixed soybean sterols were prepared for use in the feeding experiments. These esters were obtained by first pre paring the acid chloride of the fatty acid and reacting that with the sterol. 32 The acid chloride was obtained by the addition of oxalyl chloride to the fatty acid in a molar ratio of 2.5 moles oxalyl chloride to each mole of fatty acid. After the initial spontaneous reaction had subsided the mixture was heated on a water bath under reflux, for one hour. Excess thionyl chloride was removed under reduced pressure and the fatty acid chloride was purified by distillation in an all glass apparatus at a pressure of about 1 mm. of Hg. The sterol ester was obtained by the addition of 90% of the theoretical amount of sterol to the fatty acid chloride and maintaining at 80~90°C for 30 minutes. At the end of that time the mixture was diluted with water and boiled for a few minutes to hydrolyze the excess fatty acid chloride. The mixture was extracted with ether three times and the ether solution washed three times with 2% potassium hydroxide to remove free fatty acids. The solution was washed with water several times and dried over sodium sulfate. Finally the solution was treated with charcoal to remove colored impurities, and the re sulting solution evaporated to give the pure sterol ester. Yields were almost quantitative and the purity, as judged b3r total cholesterol determination and absence of material precipitating with digitonin, was excellent. EXPERIMENTAL AND RESULTS EXPERIMENT 1.-The Effect of Plant Sterols on the Hobiliza- tion of an Excess of Cholesterol from the Liver. Although it has been shown repeatedly that the feeding of plant sterols is effective in preventing the increase in liver cholesterol levels induced by cholesterol feeding, little work has been done on the effect of plant sterol administration on the removal of elevated concen trations of cholesterol already present in the liver. The demonstration of an accelerated rate of removal of excess cholesterol from the liver could result from either an hitherto unknown systemic action of plant sterols, or the trapping of excreted bile cholesterol in the intestinal lumen thereby preventing its reabsorption. An experiment was designed to test the effect of plant sterols, in the presence or absence of fat, on the removal of an excess of cholesterol from the livers of rats prefed cholesterol. Weanling rats were fed the 15% cottonseed oil diet to which was added 1% cholesterol, for a period of six weeks. At the end of that time a representative group of animals 33 34 were sacrificed and liver cholesterol and total lipid levels were determined. The remaining rats were divided into four dietary groups as follows: 1 ) 15% cottonseed oil, 2 ) 15^ cottonseed oil and 3% soy sterols. 3) fat free, 4) fat free plus 3% soy sterols. All diets were cholesterol free. Representative subgroups of each dietary group were sacrificed at the end of 5, 10, 17 and 32 days on the respective diets and liver cholesterol and total lipid levels were determined. The results shown in Table II indicate that removal of cholesterol from the diet resulted in a rapid fall in liver cholesterol levels. However, neither fat nor plant sterols altered significantly, the rate of this process . The levels of total lipid in the liver also decreased during the experimental period but no significant differ- ences between dietary groups were observed. The data obtained were subjected to further analysis in an attempt to obtain a quantitative measure of the rate of mobilization of excess cholesterol from the liver. Since, at each time interval studied, no significant differences 'were found among dietary groups, the values obtained were pooled to give a time curve for which each point, except the first, represented the average of 38 to 40 values. TABLE II CHOLESTEROL A HD TOTAL LIPID LEVELS IN' THE LIVERS OF MTS FED 3 % SOYBEAN STEROLS WITH AND WITHOUT FAT AT VARIOUS PERIODS AFTER REMOVAL FROM A DIET CONTAINING 1% CHOLESTEROL Dietary Group Days on Choi esterol Free Diet Liver 1 / F t L © Liver Total - . . - 1 / i/lpld—- Liver Cho Freei/ les t:erol Total 1 / o / ( 9 )“ gm % m mv/g m Control 0 1 0 .72 • f 0.47 7.97 + 0.44 2.78 - h 0 . 1 1 14.69 ■ f 1.74 15% Cottonseed Oil 5 (8 ) 1 0 . 1 2 0.32 6.36 + 0.22 2.58 • I — 0.19 8.06 + 1.46 15% Cottonseed Oil plus 3% Soy Sterols 5 (1 0 ) 11.05 — 0.3 9 7.42 + 0.74 2.31 — L 0.15 8.91 4 - 1.19 Fat Free 5 (1 0 ) 10.58 * 5 * 0.41 6.31 + 0.67 2.55 a . 0. 27 8.47 + 2.18 Fat Free plus 3% Soy Sterols 5 (1 0 ) 11.13 • r 0.55 5.04 0.41 2 . 2 2 4 - 0.13 7.66 4 - 1.37 15% Cottonseed Oil 1 0 (9) 10.36 i 0.40 4.23 + 0.32 2 . 08 i. 0 . 1 2 4.95 + 0.98 15% Cottonseed Oil plus 3% Soy Sterols 1 0 (1 0 ) 10.34 4 - 0.39 5.07 0.41 2.25 + 0 . 1 2 4.90 + 0.92 Fat Free 1 0 (1 0 ) 10.28 ± 0.39 4.14 4 0.43 2.03 - r 0 . 1 1 4.72 + 1.06 Fat Free plus 3% Soy Sterols 1 0 (1 0 ) 11.51 4 - 0.65 4.20 + 0.37 2 . 0 0 + 0. 04 3.44 + 0.36 co O l 32 The acid chloride was obtained by the addition of oxalyl chloride to the fatty acid in a molar ratio of 2.5 moles oxalyl chloride to each mole of fatty acid. After the initial spontaneous reaction had subsided the mixture was heated on a water bath under reflux for one hour. Excess thionyl chloride was removed under reduced pressure and the fatty acid chloride was purified by distillation in an all glass apparatus at a pressure of about 1 mm. of Hg. The sterol ester was obtained by the addition of 90$ of the theoretical amount of sterol to the fatty acid chloride and maintaining at 80-90°C for 30 minutes. At the end of that time the mixture was diluted with water and boiled for a few minutes to hydrolyze the excess fatty acid chloride. The mixture was extracted with ether three times and the ether solution washed three times with 2% potassium hydroxide to remove free fatty acids. The solution was washed with water several times and dried over sodium sulfate. Finally the solution was treated with charcoal to remove colored impurities, and the re sulting solution evaporated to give the pure sterol ester. Yields were almost quantitative and the purity, as judged by total cholesterol determination and absence of material precipitating with digitonin, was excellent. EXPERIMENTAL AND RESULTS EXPERIMENT 1.-The Effect of Plant Sterols on the Mobiliza tion of an Excess of Cholesterol from the Liver. Although it has been shown repeatedly that the feeding of plant sterols is effective in preventing the increase in liver cholesterol levels induced by cholesterol feeding, little work has been done on the effect of plant sterol administration on the removal of elevated concen trations of cholesterol already present in the liver. The demonstration of an accelerated rate of removal of excess cholesterol from the liver could result from either an hitherto unknown systemic action of plant sterols, or the trapping of excreted bile cholesterol in the intestinal lumen thereby preventing^ its reabsorption. An experiment was designed to test the effect of plant sterols, in the presence or absence of fat, on the removal of an excess of cholesterol from the livers of rats prefed cholesterol. Weanling rats were fed the 15$ cottonseed oil diet to which was added 1% cholesterol, for a period of six weeks. At the end of that time a representative group of animals 33 34 were sacrificed and liver cholesterol and total lipid levels were determined. The remaining rats were divided into four dietary groups as follows: 1 ) 15% cottonseed oil, 2) 15% cottonseed oil and 3% soy sterols, 3) fat free, 4) fat free plus 3% soy sterols. All diets were cholesterol free. Representative subgroups of each dietary group were sacrificed at the end of 5, 10, 17 and 32 days on the respective diets and liver cholesterol and total lipid levels were determined. The results shown in Table II indicate that removal of cholesterol from the diet resulted in a rapid fall in liver cholesterol levels. However, neither fat nor plant sterols altered significantly, the rate of this process. The levels of total lipid in the liver also decreased during the experimental period but no significant differ ences bet\?een dietary groups were observed. The data obtained were subjected to further analysis in an attempt to obtain a quantitative measure of the rate of mobilization of excess cholesterol from the liver. Since, at each time interval studied, no significant differences were found among dietary groups, the values obtained were pooled to give a time curve for which each point, except the first, represented the average of 38 to 40 values. TABLE II CHOLESTEROL AND TOTAL LIPID LEVELS IN THE LIVERS OF MTS FED 3% SOYBEAN STEROLS WITH AND WITHOUT FAT AT VARIOUS PERIODS AFTER REMOVAL FROM A DIET CONTAINING 1% CHOLESTEROL Dietary Group Days on Cholesterol Free Diet I T ' * ■ ' 1 *~r i ‘ — Liver T,t 1/ II L. O ~*n# Liver Total - - -A/ Uiipid—' Liver Cholesterol Free!/ 1 Totali/ Control 0 (3)2/ gm 10.72 + 0.47 7.97 + 0.44 mg 2.78 /gn • * 0 . 1 1 mg 14.69 /gm i 1,74 15% Cottonseed Oil 5 (8 ) 10.12 + 0.32 6.36 + 0 . 2 2 2.58 ~r 0.19 8.06 + 1.46 15% Cottonseed Oil plus 3% Soy Sterols 5 (10) 11.05 0.3 9 7. 4 2 + 0.74 2.31 j l 0.15 8.91 + 1.19 Fat Free 5 (10) iO.SS + 0.41 6.31 + 0.67 2.55 4 - 0.27 8.47 + 2.18 Fat Free plus 3% Soy Sterols 5 (1 0 ) 11.13 * s - 0.55 5.04 0.41 2 . 2 2 -f 0.13 7.66 _ 1.37 15% Cottonseed Oil 1 0 (9) 10.36 * 0.40 4.23 + 0.32 2.08 “j- 0 . 1 2 4 .95 + 0.98 15% Cottonseed Oil plus 3%’ Soy Sterols 1 0 (1 0 ) 10.34 + 0.39 o . 07 + 0.41 2.25 .j. 0 . 1 2 4.90 + 0.92 Fat Free 1 0 (1 0 ) 10.28 v 0.39 4.14 4 0.4 3 2.03 • ) * 0 . 1 1 4.72 + 1.06 Fat Free plus 3% Soy Sterols 1 0 (1 0 ) 11.51 -T 0.65 4.20 4 - 0.37 2 . 0 0 * 0 .04 3.44 + 0.36 CO O l TABLE I T - C o n t in u e d Dietary Group Days on Cholesterol Free Diet Liver Ft . 1/ Liver Total Lipid-/ I Liver Cho Free!./ lesterol To tali/ gm / ° mg/gm mg/gm 15% Cottonseed Oil 17 (S) 9.11 + 0.42 4.32 +0.36 1.96 + 0.05 2.77 4 0.15 15% Cottonseed Oil 17 (10) 10.20 + 0.5 0 4.21 4 0.20 1.7 3 + 0.05 2.64 + 0.19 plus 3% Soy Sterols Fat Free 17 (10) 8.71 + 0.55 4.67 4 0.26 2 . 0 2 + 0.08 3.53 4 0.35 Fat Free plus 17 (10) 8.15 + 0.63 4.05 + 0.2 3 2.15 + 0.11 2.72 4 0.29 3% Soy Sterols 15% Cottonseed Oil 32 (10) 10.13 + 0.26 4.10 4 0.2 0 1.93 4 0.04 2.59 4 0.12 15% Cottonseed Oil 32 (9) 11.36 + 0.44 3.51 4 0.11 1.75 + 0.07 2.29 + 0.07 plus 3% Soy Sterols Fat Free 32 (9) 11.52 + 0.36 3.41 + 0.09 1.70 4 0.07 2.38 4 0.15 Fat Free plus 32 (8 ) 9.45 4 0.75 4.17 4 - 0.42 1 .9 o 4 0.15 2.32 4 0.32 3% Soy Sterols ±/Including standard error of the mean. —/Number of animals per group are shown in parentheses. ^ 05 37 The assumption, that the process of mobilization of excess cholesterol from the liver should conform to an apparent first order reaction, seemed valid, and on that basis, the logarithm of the amount of excess cholesterol was plotted against time. Preparation of such a graph necessitated the determination of the normal level of cholesterol in the liver. Previous experience with normal animals indicated the normal total cholesterol level to be in the range of 2 . 1 0 to 2.40 mg. per gram of liver. A "normal” value of 2.25 mg. of cholesterol per gram of liver was used to calculate tentative excess total cholesterol values at the 0, 5, 10 and 17 day periods. These values were then plotted on a logarithmic scale, against time, and the value for the 32 day period was read off the extrapolated line. The value for the 32 day period obtained from this extrapolation, was subtracted from the actual level at that period to give an apparent "normal" level of 2.35 mg. of total cholesterol per gram of liver. This value was then used to recalculate "excess total cholesterol" at all time periods. The logarithms of the resultant values of excess total cholesterol were then plotted against time, the slope of the curve was determined graphically and the specific rate constant and half-life of the process were calculated. Similar calculations were made for excess esterified 38 and free cholesterol fractions. The apparent "normal” level of esterified cholesterol was obtained by calcula tions similar to those used in the determination of the "normal” total cholesterol level, while the "normal1 ’ level of free cholesterol was obtained by difference. Figure 1 shows the adherence of the values to straight lines in all three cases, indicating the probable validity of the initial assumption that the removal of excess cholesterol should follow apparent first order reaction kinetics. The apparent specific rate constants and half-lives for the removal of excess total, esterified or free cholesterol from the liver of normal rats, as well as the apparent normal levels of these components are shown in Table III. It must be emphasized that all groups of animals were normal and that, even where fat-free diets were fed, no deficiency of essential fatty acids was likely, since all groups were prefed for six weeks on a diet containing 15% of cottonseed oil. Under these circumstances, a deficiency of essential fatty acids would not be expected to develop in 32 days. LOG EXCESS C H O L E S T E R O L 39 -H O O E X C E SS TOTAL CHOLESTEROL □ O EXCESS E STER C H O L E S T E R O L E X C E S S FREE CHOLE ST EROL 0 32 28 16 20 8 0 4 TIME IN DAYS FTGIJKL 1 40 TABLE III HALF-LIVES AND APPARENT SPECIFIC RATE CONSTANTS FOR THE REMOVAL OF EXCESS TOTAL, ESTPURIFIED OR FREE CHOLESTEROL FROM THE LIVERS OF NORMAL RATS Choi es tero'l Fract ion Apparent ' ‘normal'’ value Specific Rate Constant Half-life mg/gm day-J- days Total cholesterol 2.35 0.18 2 4.3 Ester cholesterol 0.54 0.186 4.2 Free cholesterol 1.81 0 . 1 2 1 6.4 41 EXPERIMENT 2,- The Effects of Soy Sterols on the Absorption of Cholesterol Ester\s. The mechanisms through which cholesterol is absorbed from the intestine are still obscure. The presence of an active cholesterol esterase in rat pancreatic juice has led to the suggestion that hydrolysis of cholesterol esters, in the intestinal lumen, is a prerequisite for cholesterol absorption. If cholesterol is absorbed only in the unesterified form, the addition of plant sterols to a diet containing esterified cholesterol should prevent the increase in liver cholesterol level usually associated with the ingestion of such a diet. Weanling rats were fed the basal 15$ cottonseed oil diet to which was added cholesterol acetate, cholesteryl oleate or cholesteryl linoleate at a level to provide 1$ of cholesterol in the diet. Additional groups of animals were fed the three cholesterol ester-containing diets to which was added 3$ of soy sterols. Table IV shows the plasma and liver cholesterol levels attained after six weeks of feeding these diets. No significant changes were noted in the plasma cholesterol levels regardless of diet. However, the lower liver cholesterol and total lipid levels in the groups receiving soy sterols indicate that soy sterols were effective in inhibiting the absorption of cholesterol, fed as choles- TABLE IV PLASMA AND LIVER CHOLESTEROL LEVELS OP RATS RECEIVING VARIOUS CHOLESTEROL ESTERS WITH AND WITHOUT' THE ADDITION OF SOY STEROLS Addition to Basal 15/ Cottonseed Oil Diet Liver 1 / ) < L o • — Liver Total - f Lipidi/ Liver Q , \ Freei/ iciesterol Totali/ Plasma Freei/ Cholesterol Total—/ 2 / Acetate—' gm % mg/gm mg/gm mg% mg/ Cholesteryl 9.57 I0 * 2 7 10.03 + 0.41 3.92 +0 . ^3 20.79 +1.06 24.7 +2 . 1 83.3 + 0.7 Cholesteryl Acetate—/ 8 . 0 1 4.44 2 .42 3.82 25 .1 83.3 plus 3% Soy Sterols + 0.31 + 0.35 + 0.C9 +0.33 +1.7 + 5 .! Cholesteryl Oleate2/ 9.34 + 0.40 8.81 + 0 . 6 6 3.76 + 0.19 15 .95 +1.3.0 it; o iuol; + 1 .4 83.1 + 6 .0 Cholesteryl 0 1 eate—/ 8.58 4.56 2.24 3.00 21.3 73.6 plus 3% Soy Sterols + 0.28 +0.39 + 0.09 +0.19 + 0 .7 + 4.5 Cholesteryl, Linoleate2/ 8.32 9.74 4.09 21.32 23.2 84.3 + 0.22 + 0.44 +0.18 +1.45 +1.7 +4 . 6 Cholesteryl 7.12 5 .03 2 .36 3.52 2 1 . 2 6 8 . 1 Linoleate.2/ plus 3% Soy Sterols + 0.48 + 0.39 +0 . 1 1 +0.60 +1 . 8 + 3.4 i/lnc luding the standard error of the mean- All values are averages for ten animals per group. “/Fed at a level to provide 1% cholesterol in the diet. to 43 terol ester. The liver cholesterol levels attained in the absence of soy sterols show that cholesteryl acetate and cholesteryl linoleate were both absorbed equally well and both better than cholesterol oleate. 44 EXPERIMENT 3.-The Effect of Esterifled Plant Sterols mi the Absorption of Cholesterol and Cholesteryl Linoleate. wt\-^bv . aw* iwiwiiMa»N<w Ai ’ miL— n^»w n. i n *wi w »i'>nwn M 1 <m . .Mann I 1111 an I ■ IWIII It has been shown that, in the chicken, esterified plant sterols do not inhibit the absorption of cholesterol (47). In the same experiment, it was found that choles terol esters were poorly absorbed. Those results suggest that cholesterol is absorbed only in the unesterified form and that only unesterified plant sterols interfere with cholesterol absorption. In the rat, however, cholesterol esters are absorbed. Absorption presumably occurs after hydrolysis, in the intestinal lumen, by the action of the active cholesterol esterase of the pancreatic juice. The non-specific nature of this esterase, which also hydrolyzes esters of plant sterols, suggests that, in the rat, plant sterols should inhibit the absorption of cholesterol. Nine groups of rats approximately five weeks of age, were fed the basal 15*' cottonseed oil diet, supplemented with cholesterol, cholesteryl linoleate, soy sterols or soy steryl palraitate, singly and in combinations. After four weeks on these diets, the animals were sacrificed. Table V shows the results of liver and plasma cholesterol determinations. As was found in previous studies, no significant differences were evident in plasma cholesterol levels regardless of the diet fed. The TABLE V PLASMA AND LIVER CHOLESTEROL LEVELS OF RATS FED DIETS CONTAINING CHOLESTEROL OR CHOLESTERYL LINOLEATE WITH AND WITHOUT THE FURTHER ADDITION OF SOY STEROLS OR SOY STERYL PALMITATE Addition to Basal jbiver Liver Total Liver Cholesterol — --— —— ...—_ Plasma Cholesterol 15/1 Cottonseed Oil Diet TJ t. i./ Lipidi/ Freei./ Total £/ Fr e ei./ To tali./ 5 S 3 1 ^ i 7 r > mg/gm mg/gm mgif mg% None 11 o 33 4.58 2 . 0 1 2.30 2 1 . 1 71.2 ± ° ‘ 5 2 4 0 * C 9 40.06 40. 0o 42.5 43.7 1% Cholesterol 13.62 9.37 2.76 15 . 64 2 1 . 2 68.4 4 0 . 5 5 I0 " 8 1 40.24 42.91 4 !. 3 45. 8 5% S03' Sterols 1 2 . 1 2 4.72 1.59 1.82 zP.,7 74.1 * 3 - 0 .47 4-0 .21 4°. OS 4 0.05 • C O 46.5 Cholesteryl 1 o 0 7 i,u« ^ / 10.69 3.41 17.73 19.6 70.7 Linoleatei/ '3-0.44 -3-0.87 40.11 4l. 52 40.9 44.0 1% Cholesterol plus 11.34 4.31 1.57 1 . 8 6 20.9 78.9 c$> Soy Sterols ± ° ‘ 7 4 40. 1 7 40.05 40.05 42. 3 47.8 Cholesteryl 11. 54 CD CO 0 2.15 3.75 22.3 81.8 Linoleate£/ plus -3-0. 84 40.4 0 40.03 40 . 34 42.4 410.2 o% Soy Sterols Cn TABLE V-Continued Addition to Basal 15/ Cottonseed Oil Diet Liver Wt.i/ Liver Total T- ‘-I/ Lipid—' Liver Cl Fr e ei/ tolesterol Totali/ Plasma C Freei/ hoiest erol Totali/ gm C / mg/gni mg/gm mg/ mg% Soy Steryl Palmitate—^ 1 1 . 0 1 4 .4 9 2 . 06 2 . ‘ - " I & 20.4 83.2 + 0 . 6 5 + 0 . 23 + 0 .18 + 0 . 2 1 +1 . 6 +8.2 1% Cholesterol plus , Soy Steryl PaImitated 11.76 5 .64 o o c 4.18 19.2 72 .7 *0.63 +0.31 +0 . 1 1 +0 .48 +1 . 1 +4.8 Cholesteryl 12.87 6.60 2 .16 4.S6 23.5 83.1 9 / Linoleate^/ plus 0 / Soy Steryl Palmitate^/ + 0.92 + 0.54 + 0 .06 • O + I +2.7 +5 .4 i / Including the standard error of the mean. All values are averages for ten animals per group. —' ^Incorporated in the diet at a level sufficient to provide 1% of cholesterol. 2 /lnc orporated in the diet at a level sufficient to provide 3% of soy sterols. cn 47 addition of cholesterol or cholesteryl linoleate to the diet increased markedly and to an equal degree, the liver levels of cholesterol and total lipid, while the addition of free soy sterols, either alone or in combination with cholesterol, resulted in a small but significant decrease in the liver cholesterol level. Although the addition of soy steryl. palmitatc alone to the diet had no effect on liver cholesterol levels, soy steryl palmitate was effective in inhibiting the absorption of both free cholesterol and cholesteryl linoleate, but less so than the free soy sterol. The absorption of cholesteryl linoleate was inhibited, but not completely, by both free soy sterols and soy steryl palmitate. 48 EXPERIMENT £. -The Effect of Feeding Cholesterol and Plant Sterols on Alternate Days♦ Two theories have been evolved to account for the inhibitory action of plant sterols on cholesterol absorp tion. One theory suggests the formation of an unabsorbable complex of plant sterol and cholesterol in the intestinal lumen, while the other proposes the inhibition of some enzymatic reaction in the absorption process. If plant sterols display their inhibitory effect on cholesterol absorption in the absence of mixing of the two sterols in the intestinal lumen, the first of these theories becomes much less tenable. One group of weanling rats was fed the basal 15/£ cottonseed oil diet alternated daily with the same diet supplemented 'with 1% of cholesterol. A second group alternately received diets supplemented with 3% soy sterols and with 1% cholesterol. An eight hour fast period intervened between diet changes. The animals were allowed to eat, ad libitum, from 4:00 P.M. until 8:00 A.M. At that time the food container was removed from the cage and the animal was allowed only water until 4:00 P.M., at which time the alternate food was offered until 8:00 A.M. the next day. The animals grew normally on this schedule, which was continued for six weeks. A second replication of the experiment was performed with the levels of 49 cholesterol and soy sterols raised to 2% and 6%, respect ively. The results of both experiments are shown in table VI. In both cases the levels of liver cholesterol and total lipid were significantly less where the animal received the soy sterol dietary regimen rather than a sterol free diet. TABLE VI PLASM AND LIVER CHOLESTEROL LEVELS OF RATS FED CHOLESTEROL AND SOY STEROLS ON ALTERNATE DAYS Diets Alternated Liver Ft ^-/ h i * amj Liver Total Lipid Liver Cl Fr e et/ lolesterol Totali./ Plasma C Freei./ holesterol Totali./ g m V F m g / g m m g / g m m g / m g / l/ Cholesterol and Sterol Free 9.(9 + 0.56 6. S3 +0 .30 2.70 + 0 .10 8.38 +0.93 17.3 +1.2 72.7 + 3.8 1/ Cholesterol and 3/ Soy Sterols 10.30 + 0.49 5 .29 +0. 37 2.40 + 0.06 4 .57 i0,46 22.0 +1.3 84.1 +4.1 2% Cholesterol and Sterol Free 11.39 + 0.38 8.04 +0.45 2.90 +0 .13 13.58 +1.70 17.6 + 0.8 5 6.9 +6.8 2% Cholesterol and 6% Soy Sterols 10.87 + 0.65 5.03 +0 .23 2.26 + 0.08 3.41 +0 .3 o 21.3 +1.3 57.7 +6.7 -/Including the standard error of the mean. All values are averages for ten animals per group. cn o 51 EXPERIMENT 5.-The Effects of Essential Fatty Acid Defi ciency on Cholesterol Absorption and Metabolism. The work of Alfin-S.later et_ al, (85, 86) has shown that cholesterol metabolism is deranged in the essential fatty acid-deficient rat. The derangement is manifested by an increase in liver cholesterol levels and a decrease in plasma cholesterol levels. Studies were undertaken to explore further the effects of essential fatty acid deficiency on cholesterol absorption and metabolism. Weanling male rats wore fed a fat free diet for twenty weeks. At that time they exhibited severe skin symptoms of essential fatty acid deficiency and had not increased in weight for a four week period. They were then p l a c e d o n d i e t s s u p p l e m e n t e d with c h o l e s t e r o l , methyl linoleate or the two combined, and continued on these diets for an additional six weeks. The results of plasma and liver cholesterol deter minations shown in Table VII. The curative effect of linoleate is evidenced by the increased body weight, the decreased liver cholesterol levels and the increased plasma cholesterol levels of the animals receiving either methyl linoleate or cholesteryl linoleate. However, the weight gain was substantially less when linoleate was fed as the cholesterol ester than when it was fed as the methyl ester. The addition of cholesterol to the diet of essential TABLE YII BODY WEIGHT GAINS AM) LIVER AND PLASM CHOLESTEROL LEVELS OF ESSENTIAL FATTY ACID-DEFICIENT RATS FED CHOLESTEROL OR METHYL LINOLEATE Supplements added to basal fat-free Diet Gain in Body Wt.l/ Liver Total Lipidi/ Liver Ch Freei./ olesterol Totali./ Plasma C Freei./ 'holesterol Totali/ gm o ' / O mg/gm mg/gm mg% mg% None 0. 0 9.28 I1-14 2 . 59 + 0.07 6.08 + 0.49 8.7 I1*6 o 9.2 + 5.1 0 .76% Methyl Linoleate 65 .8 i3-1 11.49 + 1.36 o 4 o - L • * x S - + 0.05 4.50 + 0.23 2 2.4 + 0.9 82.6 + 2.3 1.68% Cholesteryl O / LinoleatqL/ 38 .8 ±4 •1 15.10 + 0. 65 3.20 + 0. 08 15 .36 + 0.95 21.3 + 0.8 87. 0 + 2.7 0.7 6% Methyl Linoleate plus 1% Cholesterol 5 5.8 + 4 . { 13. 01 +1.79 2 .4 2 + 0.10 8.21 + 0 .74 19 .8 + 0.6 79 .4 +1.7 1% Cholesterol -7.3 +3. 9 i . 1. t 0 + 1 • 21 2 .95 + 0.10 7 .88 + 0.37 10.9 + 1.3 53 .4 +4.2 1% Cholesterol plus 1% Bile Salts 7.5 + 5.2 14.12 + 1.16 2 . 6 9 + 0.16 16.54 + 2.41 21 .8 + 3.0 96.3 +9 .8 1 / —' Including the standard error of the mean. All values are averages for eight animals per group. 2/Equivalent to 1% cholesterol and 0.76% methyl linoleate. 53 fatty acid-deficient rats resulted in only a very small increase in the liver cholesterol level. The further addition of bile salts increased substantially the depo sition of cholesterol in the liver and, in addition, increased the plasma cholesterol levels» Supplementation of the diets of essential fatty acid-deficient animals with both cholesterol and methyl linoleate resulted in only a minimal increase in the liver cholesterol concentrations, whereas the liver cholesterol levels of animals fed cholesteryl linoleate was con siderably elevated. 54 EXPERIMENT 6. -The Effects of Bile Salts and y fl-sitosterol on Cholesterol Levels in Essential Fatty Acid-Defic.ient and Normal Rats. The preceding experiment has shown that, in the essential fatty acid-deficient rat, the addition of bile salts to the diet promoted the absorption of dietary cholesterol. In the absence of dietary bile salts, cholesterol was very poorly absorbed. Whereas bile salts promote cholesterol absorption, the plant sterol, ^-sito sterol, prevents the absorption of cholesterol. The use of these two substances, therefore, appeared to offer a means of studying the reabsorption of endogenous choles terol in the essential fatty acid-deficient rat. One group of weanling male rats was maintained for a twenty week pre-experimental period on the basal fat-free diet. A second group of littermate animals was fed the basal 15% cottonseed oil diet during the pre-experimental period. At the end of this time, when the animals re ceiving the fat-free diet exhibited severe symptoms of essential fatty acid deficiency, each group was subdivided into three subgroups. One subgroup was continued on the pre-experimental diet, a second subgroup received the pre- experimental diet plus 1% bile salts and the third sub group received the pre-experimental diet supplemented with l/o bile salts plus 3% -sitosterol. All groups 55 were maintained on these diets for six weeks. The results of plasma and liver cholesterol deter minations are shoxsrn in Table VIII. Although the plasma cholesterol levels of the animals receiving the fat-free diet were lower than those of the animals receiving the 15% cottonseed oil diet, supplementation of either diet with bile salts or bile salts and ^-sitosterol had no significant effect on plasma cholesterol levels. The addition of 1% bile salts to the fat-free diet caused a considerable increase in the already ele\rated liver cholesterol level. The addition of the same supplement to the 15% cottonseed oil diet resulted in a very much smaller, albeit significant, increase in the liver cholesterol level. When both 1% bile salts and 3% f3 -sitosterol were fed to the essential fatty acid-deficient animals, the in crease in the liver cholesterol level found with the diet containing 1% bile salts only, was completely prevented and, in fact, the liver cholesterol level was lower in these animals than in those receiving no supplement. The liver cholesterol levels of the animals receiving the 15% cottonseed oil plus 1% bile salt diet were not changed by the further addition of 5% -sitosterol. TABLE VIII PLASM AND LIVER CHOLESTEROL LEVELS OF ESSENTIAL FATTY AC ID-DEFICIENT AND NORMAL ANIMALS FED BILE SALTS AND {3-SITOSTEROL Dietary Group Liver Liver Total Liver Cholesterol Plasma Cholesterol Ft. V Lipidi,/ Freei./ Totali/ Freei./ Totali/ gm of /O mg/gm mg/gm mg$ mg$ Fat Free 9.04 9.28 2.59 6.08 8.7 59.2 - 5 " 0 o + 3 I1”14 +0.07 + 0 .49 +1.6 +5.1 Fat-free plus 7.46 8.01 3 .71 11.38 9.1 58.6 1$ Bile Salts + 0 • o S + 0 . 4 8 + 0 . 4 7 + 1 .98 + 1.2 +1.0 Fat-free plus 1% Bile 7.20 6.19 2 . 07 3 . SO 11.7 69 . 6 Salts plus 3% ■4-0.56 + 0 .37 + 0.10 + 0 . 3 3 + 1.9 + 5 .1 ^-sitosterol 15$ Cottonseed Oil 10.42 5.11 1.77 2 .12 22 .8 94.7 + 0 . 2 4 + 0 . 0 8 + 0 . 0 5 + 0 . 0 6 + 0.3 +3 .8 15$ Cottonseed Oil plus 8 . 77 5 . 67 2.29 2 .79 18.8 78. 6 1% Bile Salts •10. 39 + 0.34 I0'14 +0 .14 + 1 .5 + 4.3 15$ Cottonseed Oil plus 9.91 4 . 8 7 2 .4 0 2.74 24 . 0 94.3 1$ Bile Salts plus + 0.79 + 0 .18 + 0 . 0 7 + 0 . 0 6 + 2 . 0 + 6.1 3$ ^-sitosterol i / ~ Including the standard error of the mean. All values are averages for six animals per group. CD EXPERIMENT 7_. -The ETTects of the Addition of ^-sitosterol to the Diet of Rats During the Period of Depletion of Essent ial Fatty Acids - The preceding experiment has shown that the supple mentation of essential fatty acid-deficient rats with bile salts plus ^-sitosterol resulted in a marked reduction in liver cholesterol levels. This effect was probably due to the ^-sitosterol component, since bile salts alone had the opposite effect. A study of the effects of incorpor ating ^-sitosterol into the fat-free diet during the essential fatty acid depletion period was undertalcen. Two groups of rats were selected at weaning and placed on fat-free diets which were identical except that one contained 3$ ^-sitosterol. After ten weeks on these diets the animals were sacrificed and plasma and liver choles terol levels were determined. The results obtained are shown in Table IX. The addition of ^-sitosterol to the fat-free diet resulted in liver cholesterol levels which were almost normal and much below the levels of the animals receiving the unsupplemented fat-free diet. More over, the low plasma cholesterol level associated with essential fatty acid deficiency was reduced still further by the addition of ^-sitosterol to the diet. TABLE IX PLASM AND LIVER CHOLESTEROL LEVELS OF RATS FED ^-SITOSTEROL DURING THE PERIOD OF DEPLETION OF ESSENTIAL FATTY ACIDS Addition to Basal Liver Liver Total Liver Cholesterol Plasma Cholesterol Fat-Free Diet Vt .1/ Lipidi/ Freei/ Totali/ Fre ei/ Totali/ gm % mg/gm mg/gm mg% mg% None 8.30 8.66 2.15 3.97 14.7 62.4 •4-0 . 44 + 0.S2 +0.04 +0.23 + 1.2. + 3.2 CO f-sitosterol 8 .18 6.46 1.S7 2.74 7.2 49.4 + 0.24 + 0 .24 +0.0 3 +0.09 + 1.6 + 3.2 — Including the standard error of the mean. All values are averages for ten animals per group. U1 CD DISCUSSION" The results obtained in Experiment 1 indicate that soy sterols have no significant effect on the metabolism and excretion of stored cholesterol. If cholesterol is excreted unchanged, its reabsorption should be prevented by soy sterols in the intestinal lumen. The fact that soy sterols had no effect suggests either a minimal excretion of unchanged cholesterol or a compensatory increase in endogenous synthesis. The excretion of u n m e t a b o l i z e d c h o l e s t e r o l must h a v e b e e n s m a l l , in a n y event, since, in the early stages of dietary treatment when cholesterol disappearance was greatest, the capacity of the liver for cholesterol synthesis must have been small due to the inhibitory effect of the still-high level of cholesterol present. Experiment 1 has yielded valuable information about the cholesterol mobilization process itself. The removal of excess cholesterol from the liver follows apparent first order reaction kinetics. This seems to hold true whether free cholesterol, esterified cholesterol or total cholesterol is studied. Thus, the rate of excess 59 60 cholesterol mobilization appears to depend on the amount of excess present, although the available evidence does not permit judgement of whether the controlling factor is free cholesterol, ester cholesterol or total choles terol. In fact, liver cholesterol esterase may be sufficiently* active to provide a constant equilibrium between free and ester cholesterol. The somewhat longer half life for free cholesterol may indicate that the degredation of ester cholesterol involves a preliminary hydrolysis. Experiments 2 and 3 provide evidence for an active in vivo action of cholesterol esterase in the intestinal lumen. The effectiveness of both soy sterols and soy steryl palmitate in inhibiting the absorption of choles terol and cholesterol esters suggests that both choles terol esters and plant sterol esters are hydrolyzed in the intestinal lumen. However, the still slightly elevated liver' cholesterol levels following xhe feeding of cholesterol esters in combination with soy sterols, indicates that a small portion of the cholesterol ester may have been absorbed without prior hydrolysis. The failure of plant steryl palmitate to inhibit completely the absorption of cholesterol may have been due to incomplete hydrolysis in the intestinal lumen, thereby reducing the effective amount of soy sterol present. 61 Alternate feeding of cholesterol and plant sterols, separated by an eight hour fasting period, should effectively prevent the intimate mixing of the two sterols in the intestinal lumen. Even under these circumstances, however, soy sterols were effective in preventing the absorption of cholesterol. These results indicate that the inhibitory action of plant sterols does not result from the formation of an unabsorbable cholesterol-plant sterol complex in the intestinal lumen. The site of the inhibitory action of plant sterols appears to be localized in the intestinal wall. This view receives additional support from the recent finding (5-1) that an appreciable amount of labeled ^-sitosterol may be recovered from the i n t e s t i n a l w a l l s e v e r a l h o u r s a f t e r i t was fed. T h e same report indicated that only traces of the labeled ^-sito sterol appeared in t h e lymph. The experiments reported here provide no information concerning the mechanisms, within the intestinal wall, by which the absorption of cholesterol is prevented. However, the localization of the action of plant sterols within the intestinal wall and the structural similarity of the inhibitory substance to cholesterol suggest that the effect of plant sterols may be due to a competitive inhibition of some enzyme system concerned with choles terol absorption. The results reported here are not 62 incompatible with such an hypothesis. The conversion of cholesterol to bile acids appears to be markedly reduced in the essential fatty acid deficient rat. Mukherjee and Alfin-Slater (87) have shown that the rate of cholesterol biosynthesis in the livers of essential fatty acid-deficient rats is less than one-tenth that found in the livers of normal animals. The concentration of cholesterol in the livers of these animals, however, is increased rather than decreased, suggesting that the rate of metabolism or excretion is also decreased. Since the quantitatively most important products of cholesterol metabolism are the bile acids, an impairment in the conversion of cholesterol to bile acids would appear to be the most likely explanation. The results of Experiment 5 of this investigation lend further support to such a view. Bile salts are necessary for the absorption of cholesterol. In the essential fatty acid-deficient rat, dietary cholesterol was not absorbed unless bile salts were added to the diet, indicating a deficiency in the endogenous supply of bile salts. Bile salts are required for the action of cholesterol esterase. If hydrolysis of cholesterol esters is an obligatory step in their absorption, the absorption of cholesteryl linoleate by the essential fatty acid- 63 deficient animal would be expected to be extremely- limited. This however, is not the case. Cholesteryl linoleate is absorbed to a much greater extent than is free cholesterol even when the free cholesterol is fed with methyl linoleate. The possibility of absorption of cholesterol esters without prior hydrolysis has already been discussed in relation to the failure of soy sterols to inhibit completely their absorption. In the essential fatty acid-deficient rat, the absorption of cholesteryl linoleate appears to require neither the prior hydrolysis of the ester bond nor the presence of bile salts. The smaller weight gain of the rats that received cholesteryl linoleate compared to that of the animals that received e i t h e r m e t h y l l i n o l e a t e or m e t h y l linoleate plus cholesterol may have been due to either, a reduced absorption of linoleate when fed as cholesteryl linoleate or, to an impaired utilization of, or increased requirement for, linoleate. The presence of unabsorbed endogenous cholesterol in the intestinal lumen of essential fatty acid-deficient rats is demonstrated by the results obtained from Experiment 6 (Table VIII). The addition of bile salts to the fat-free diet resulted in an increase in liver cholesterol levels. That this increase was mediated by increased absorption resulting from bile salt feeding is 64 demonstrated by the fact that the further addition of ^ “Sitosterol prevented the increase. The slightly increased liver cholesterol levels resulting from bile salt feeding to normal animals ’ was probably due to some effect 011 cholesterol metabolism in the tissues rather than to an increased absorption, since the further addition of ^-sitosterol to the diet had no effect on these levels. In any event, changes in cholesterol absorption due to the feeding of either bile salts or ^-sitosterol are probably within the range that can be compensated for, in the norma1 an :mo1, by changes in the rato of synthesis. The lover level of cholesterol in the livers of animals receiving' a fat-free diet with bile salts and ^-sitosterol, as compared to the level observed in those animals receiving the fat-free diet alone, indicates that some of the endogenous cholesterol in the intestinal lumen is absorbed. This view' is strengthened by the results of Experiment 7. The addition of (3 -sitosterol to the diet of rats during the period of depletion of essential fatty acids largely prevents the accumulation of cholesterol in the livers of these animals. Alfin-Slater ct al. (85,86) have suggested an impair ment in the movement of cholesterol between liver and plasma. The present finding, that feeding ^3-sitosterol to animals being depleted of essential fatty acids results 65 in lower plasma cholesterol levels than are found in animals fed the fat-free diet, provides support for their argument. Since the principal sources of plasma choles terol are the liver cholesterol and dietary cholesterol being transported to the liver, elimination of the latter produces a fall in the overall level of plasma cholesterol, unless it can be compensated for by an increased release of cholesterol from the liver. The increase in the plasma cholesterol level found when cholesterol and bile salts were fed to essential fatty acid deficient animals (Experiment 5), suggests that movement of cholesterol from the plasma into the liver may also be impaired to some extent. The alterations in cholesterol metabolism, resulting from a deficiency of essential fatty acids appear to involve at least two factors. The transfer of cholesterol between liver and plasma is impaired and the conversion of cholesterol to bile acids and their subsequent excretion is markedly reduced. The excretion of unchanged choles terol, however, does not appear to be altered. The decreased rate of cholesterol biosynthesis, in these animals, may be a secondary effect, resulting from the inhibitory action of the elevated liver cholesterol con centration . SUMMARY Studies were undertaken to determine the effects of plant sterol feeding and of essential fatty acid deficiency on the absorption and metabolism of cholesterol in the rat. During the course of these investigations, additional information has been obtained concerning the absorption and metabolism of cholesterol in the normal, unsupplemented animal. It has been found that, in the normal rat fed cholesterol, an excess of cholesterol in the liver rapidly disappears when cholesterol feeding is discontinu.ed. The disappearance of excess cholesterol conforms to apparent first order reaction kinetics, with a half-life of four to six days. Plant sterols have no effect on the rate of this reaction. Plant sterols completely inhibit the absorption of dietary free cholesterol when the ratio of plant sterol to cholesterol is 3:1. The absorption of cholesterol esters is also effectively, and almost completely, pre vented. In addition, esterified plant sterols inhibit the absorption of both free and esterified cholesterol. 66 67 While the results of these experiments indicate that cholesterol esters are almost completely hydrolyzed in the intestinal tract of the normal rat, some evidence has been obtained which appears to indicate that small amounts of cholesterol esters may be absorbed without prior hydrolysis. In an attempt to resolve the question of whether or not plant sterols exert their effect on cholesterol meta bolism by combining with cholesterol, in the intestinal lumen, to form an unahsorbable complex, cholesterol and plant sterols were fed on alternate days. Under these conditions, cholesterol absorption was still prevented. It is suggested that the sequence of events by which plant sterols inhibit cholesterol absorption involves the absorption of the plant sterols into the intestinal wall, where they inhibit some enzymatic mechanism concerned with cholesterol absorpti.cn. The plant sterols are re tained in the intestinal wall for some time, but even tually they must be excreted into the intestinal lumen, since their transfer into the lymph is extremely limited. The absorption of cholesterol by the essential fatty acid-deficient rat has been investigated. It has been found that these animals are unable to absorb dietary free cholesterol, unless bile salts are supplied in the diet. Supplementation with bile salts is not required for the absorption of cholesteryl linoleate. These results appear 68 to indicate that conversion of cholesterol to bile acids is extremely limited in the essential fatty acid-deficient rat. 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Asset Metadata

Creator Wells, Arthur F. (author)

Core Title Factors Affecting Cholesterol Absorption And Metabolism

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Advisor Slater-Alfin, Roslyn B. (committee chair), Ershoff, Benjamin H. (committee member), Marx, Walter (committee member), Saltman, Paul (committee member), Saunders, Paul R. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-50048

Unique identifier UC11357943

Identifier 5904406.pdf (filename),usctheses-c18-50048 (legacy record id)

Legacy Identifier 5904406.pdf

Dmrecord 50048

Document Type Dissertation

Rights Wells, Arthur F.

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