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

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

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Content SOME STUDIES ON THE EFFECTS OF DIETARY CHOLINE ON THE PLASMA CHOLESTEROL CONCENTRATION IN THE RAT by Elmer H. Rice 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) June 1958 UMI Number: DP21585 All rights reserved INFORMATION TO ALL USERS th e 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 DP21585 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 48106-1346 ............ELMERH.JRICE............. under the direction o f hie G uidance C om m ittee and app ro ve d by a ll its members, has been p re sented to and accepted by the F a c u lty of the G raduate S cho ol, in p a rtia l fu lfillm e n t of re quirements f o r the degree of D O C T O R O F P H I L O S O P H Y D a te JUNE 1958 . d b;» l si wf, i This dissertation, w ritte n by \ G uidance C om m ittee k v ' r ? c . C hairm an ACKNOWLEDGEMENTS Professor Walter Marx provided me with the "facts of life" in scientific research by securing the necessary support for these studies. For this I am grateful, but I particularly wish to extend my sincere appreciation for his kindness, patience, guidance, and encouragement throughout this work. I wish to thank Dr. Henry Weimer for the con fidence he has placed in me. I wish to thank Dr. G. F. Hungerford for his kind assistance in the preparation of rats with lymph duct fistulas. To Dr. George Kunitake and to Mr. Morris Gutenstein I extend thanks for the pleasant hours of association in the laboratory and for the occasional loan of a helping hand. A special offer of thanks goes to my wife, Irene, for her help throughout my years of graduate study. I TABLE OF CONTENTS i ! * i ! PAGE ! i iINTRODUCTION ..................................... 1 1 i REVIEW OF THE LITERATURE 2 j Choline and Lipotropism 2 j Discovery and development .............. 2 Characteristics of choline deficiency fatty livers 8 j Choline and plasma cholesterol 9 ; The Relationship of Choline to Factors 1/tfhich t May Influence the Plasma Cholesterol 1 1 Concentration • •••••.. ........... 12' i Cholesterol absorption 12 ! Cholesterol synthesis • . • • 13 i Cholesterol conversion and excretion • . 13 i I Cholesterol transfer between the plasma : and tissues 15 Cholesterol carrying capacity of plasma . 16 i Cholesterol-phospholipid interrelation ships . . . . . . ............... 18 ■ STATEMENT OF THE PROBLEM AND PLAN OF ATTACK .... 20 ! MATERIALS AND METHODS............................ 23 Materials.................................. 23 V PAGE Animals.......................... 23 Diets............................ 23 Detergents...................... 23 Tween-2 0 .................... 23 Triton MR-1339 .................... 23 Radioactive cholesterol ••••••••. 2? Methods............................ 25 Surgical techniques .................... 25 Lymph duct cannulation...... 25 Bile duct ligation.......... 26 Bile duct cannulation...... 26 Intravenous injections...... 27 Drawing of blood............ 27 Chemical methods •••••••••••• 28 . 1 Ll Preparation of cholesterol-H—C- 1 * solution for injection.. 28 Extraction of blood plasma ........ 28 Extraction of other tissues and gastrointestinal tract washings. 29 Extraction of bile.......... 29 Determination of cholesterol .... 30 Determination of phospholipid phosphorus............ 30 Determination of total lipid ........ Radioactivity techniques ................ Determination of purity of radio active cholesterol •»••••• Assay of radioactivity . . ........ Plan of the Experiments ...................... The effect of choline on cholesterol absorp tion as measured by the method of lymph duct cannulation .............. The effect of choline on cholesterol absorp tion as measured by analysis of the gastrointestinal contents .......... The effect of choline on the distribution of radioactivity in the bile after the intravenous administration of solubilized cholesterol-^f-C^ • • • • The effect of choline on the hypercho lesterolemia resulting from ligation of the bile duct .................... The effect of choline on the hyperlipemia and hypercholesterolemia resulting from the intravenous administration of a detergent (Triton MR-1339) ........ vii PAGE The effect of choline on the composition of liver lipids in the rat........... *+0 RESULTS............................................. **2 The Effect of Choline on Cholesterol Absorption as Measured by the Method of Lymph Duct Cannulation.............................. *f2 The Effect of Choline on Cholesterol Absorption as Measured by Analysis of the Gastro intestinal Contents................ *+7 The Effect of Choline on the Distribution of Radioactivity in the Bile after the Intra venous Administration of Solubilized Chole s terol-^-C11* ' 53 The Effect of Choline on the Hypercholesterolemia Resulting from Ligation of the Bile Duct. . $6 The Effect of Choline on the Hyperlipemia i and Hypercholesterolemia Produced by the 1 i Intravenous Injection of a Detergent (Triton WR-1339) 59 ; The Effect of Choline on the Composition of j ► Liver Lipids in the Rat.................. 66 I DISCUSSION......................................... 69 j SUMMARY............................................. 8*f | i BIBLIOGRAPHY.................... . ____89J LIST OF TABLES TABLE PAGE I. Composition of Diets.............. ...... 2b II. The Effect of Dietary Choline on Choles terol Absorption as Determined Using Bats with Lymph Fistulas ....... *+6 III. Growth and Food Consumption of Rats Fed Diets C and D ...................... bQ IV. Body Weights and Liver Weights of Rats at the End of the Second Absorption Experiment ........................ *+9 V. Effect of Dietary Choline on Cholesterol Absorption as Determined from the Radioactivity of Gastrointestinal Con tents of Rats Fed Cholesterol-1 *-^1 * • 52 VI. Effect of Choline on the Radioactivity Appearing in Whole Bile and in the Saponifiable Fraction of Bile Following the Intravenous Injection of Choles terol-1 *-^1 * 55 VII. Effect of Choline on the Hyperlipemia Produced by Ligation of the Bile Duct................................ 57 ix PAGE VIII. Effect of Dietary Choline on the Plasma Cholesterol Concentration Before Triton Injection .................. 60 IX. Effect of Choline on the Hyperlipemia Produced by the Injection of Triton . 61 X. Effect of Choline on the Interrelation ships of Plasma Lipids in Triton- Induced Hyperlipemia ' .............. 6H- XI. Effect of Choline on the Composition of Liver Lipids . ^ .................. 67 LIST OF FIGURES FIGURE 1* Self Absorption Curve for Cholesterol-C^ Dissolved in Lymph or Cottonseed Oil • . 2. Accumulation of Radioactivity in Lymph After Feeding Cholesterol-1 *-^** to Choline- Supplemented Rats With Lymph Duct Cannulas .............................. 3. Accumulation of Radioactivity in Lymph After Feeding Cholesterol-1 *-^1 * to Choline- Deficient Rats With Lymph Duct Cannulas .............. INTRODUCTION One of the characteristic biochemical lesions of choline deficiency is the accumulation of excess lipid in the liver. A rise in free and esterified cholesterol, both of which are found in normal livers, accounts for a portion of the total increase in lipid. As a result of recent observations made in this laboratory, choline is now known to influence the concentration of cholesterol in the blood plasma. In the work to be presented here, the effect of choline on blood cholesterol has been emphasized, and the experiments to be described were designed to shed some light on possible mechanisms whereby choline exerts its effects. REVIEW OF THE LITERATURE Studies on choline range through the fields of chemistry, biochemistry, pharmacology, physiology, and microbiology. The many aspects of investigations on this compound and on compounds related to it have been reviewed by Best and Ridout (1), Griffith (2, 3> *0 9 Frame (5)9 Best and Lucas (6), McHenry and Patterson (7)» and Jukes (8). The most recent comprehensive review appeared in 195^ under the authorship of several of the most prominent workers in the field (9) • In the first part of this survey of the literature, those studies on choline have been selected which in some way are concerned with the interrelationship between choline and cholesterol. This section has been entitled * fCholine and Lipotropism.*1 * A later portion of this intro duction will deal with factors which influence the concen tration of cholesterol in the plasma, with emphasis again, on information related to choline. Choline and Lipotropism Discovery and development When certain species of animals are fed diets adequate in proteins, fats, carbohydrates, minerals, and all 3 : the known vitamins, with the exception of choline, changes result in the liver and kidneys as well as in other organs : of the body. The requirement for dietary choline has been | fully verified, and the prevention of the changes in the ^ first two organs mentioned is now generally referred to as • t the lipotropic effect of choline (10). Wilgram, Hartroft, [ and Best (11) recently suggested that the term "lipotropic1 *j be expanded to include the prevention of the accumulation j of fat not only in the liver and kidneys but also in the , i heart and blood vessels of animals that exhibit these I i lesions when choline and its precursors are excluded from : i the diet. Mann has criticized this extension of the term ! (12). It may be remarked that the term had already been i extended from its original usage which concerned only the j effects of choline on the content of liver fat (7} 13). j i The original demonstration of the effect of choline! on liver lipids was made on depancreatized dogs (I1 *). j Previous experiments had shown that such animals developed | I fatty livers (116) which could be prevented by the j i ingestion of either raw pancreas (16), or lecithin (17). I Best and his co-workers subsequently discovered that ; i pancreatectomy was not necessary in order to demonstrate ; l the production of fatty livers, but that certain dietary j i regimens gave rise to a similar condition in the rat (18). b i The diets were high in saturated fats and contained wheat, . oats, corn meal, beef fat, and bone meal. j As work progressed in the study of fatty livers [ i resulting from insufficient choline, several other factors were found to influence the production of choline defi- i \ ciency. Best and Huntsman (19) showed that dietary casein j < acted lipotropically. Channon and Wilkinson (20) confirmedj i this observation and introduced the low casein (5 per cent); i high fat (*K) per cent) diet which was widely used there- J i after in the study of choline. The fact that the low j I casein diet is inadequate in both quantity and quality of ’ protein was later noted by Channon els al. (21). Tucker and! Eckstein (22) established that methionine has a very ! i definite lipotropic action. ' Griffith and Wade (23, 2b) found that another i i condition which influenced the formation of fatty livers ! t was the age of the rat. Using weanling rats on diets which' i appeared adequate in protein content (15 to 25 per cent casein) and other dietary essentials except choline, they ' t were able to produce, within ten days, a pathological state| i characterized by severe hemorrhagic renal degeneration, | ocular hemorrhage, and regression of the thymus. At the I i same time, fatty livers developed. The symptoms were more I pronounced in males than in females, but were less severe i jin both sexes if the animals were over thirty days of age ; ■when placed on the diet. The condition was prevented by .choline and aggravated by cystine. The antilipotropic | ! action of cystine had been observed earlier by other i ! i ■workers (25)* J i i ! A very satisfactory explanation of the failure to j J I ■find a lipotropic action of casein in weanling rats was jprovided by Griffith (26) who also offered an explanation ! ' jfor the previously mentioned cystine effect. He considered : i ! 1 that the young weanling rat was in a condition of high ! 1 i * ;metabolism and therefore required a greater amount of | 1 l choline than would an older animal. By improving the ! !protein nutrition of the animal, higher levels of casein | * I ;served to stimulate metabolism and growth and thus pre- ! jcipitated a condition of such severe choline deficiency thatj not only did fatty livers appear but, in addition, the other! < ' i jsymptoms mentioned. The extra methionine supplied in the | i imoderate casein diet either was not sufficient, or could notj i Jbe converted to choline at a rate rapid enough, to overcome 1 the choline deficiency. Cystine also improved the growth * ♦ I jand metabolism of the weanling rats, probably because this j I iamino acid was present in minimal amounts in the moderate | i ■ i casein diets and was actually the limiting factor influenc- I ;ing growth. By adding supplements of cystine it was no j jlonger limiting, and the deficiency of choline became more evident. ! The problem still remaining is how to provide ] . adequate sources of protein while limiting the methionine j to levels which are not lipotropic. Hartroft (27) has ‘ reviewed this subject and has presented the diets used by ■ the Toronto group in present day studies. Fischer and ■ Garrity (28) have published the composition of a diet containing 18 per cent casein which gives reproducible ; choline deficiency symptoms. It was mentioned earlier that methionine has a lipotropic action; furthermore, this amino acid supports , growth in the absence of dietary choline. The suggestion was made by DuVigneaud and associates (29) that the lipo tropic activity of methionine may arise indirectly, by supplying methyl groups for the synthesis of choline. | Transfer of methyl groups from methionine to choline | precursors was later demonstrated with the aid of isotopic ‘ labeling (30). 1 Using the two lines of investigation described, ! prevention of fatty livers and studies on methyl transfer ■ (transmethylation), some of the findings on lipotropic effects have been explained in terms of transmethylation. It has become apparent, however, that not all lipotropic J t substances can act as methyl donors, nor can all the methyl- I donors replace choline in every instance where it is \ i ! i I required, A variety of lipotropic substances has been ! ! . i 1 i , discovered, although the number of known methyl donors is i ! 1 , small. In planning diets deficient in lipotropic agents, j , it is usually necessary to be concerned only with the j i ! choline and methionine content, for other lipotropic sub- j ' stances such as phosphoryl choline, dimethylthetin, - j j propiothetin, or betaine have not been reported to be ; ' present in the usual dietary ingredients in concentration ! ; high enough to be of significance. The experiments reviewed thus far have pointed to | \ the importance of a dietary source of labile methyl groups i i , | for the prevention of fatty livers. Such a dietary require-- j ment strongly suggests that the animals are unable to ; j • 1 ( synthesize labile methyl groups from other dietary ingre- ! I dients. In recent years, however, evidence has been ! i ; obtained which indicates that such synthesis can occur. , The exact nature of the one-carbon precursor of the methyl i I group is not known; but by the use of isotopic tracers, 1 ' carbon from the following sources has been shown to appear | ! i i in the methyl groups of choline and methionine isolated ,from rat tissues: methanol (31), formaldehyde (31, 32), | formic acid (31» 33) 5 acetone (31 *) j and- serine (35> 36). j 8: In growth studies, homocystine has been found to be able ; to replace methionine as an essential amino acid provided j choline is also present in the diet (37). Diets containing! homocystine but devoid of methionine, choline, or other ’ i known sources of labile methyl have been observed to support limited growth, however (38); such a finding is interpreted as additional evidence that synthesis of labile methyl groups does occur in the animal body. In no instance has optimum growth been demonstrated in the i absence of dietary sources of labile methyl, and it j t appears that under the best of circumstances the rate may ! be limited. i 1 Characteristics of choline deficiency fatty livers j MacLean and Best (39) and Hartroft (U-0) have t * described the changes which occur in the histological structure of rat liver as related to the length of time on j I 1 choline-deficient diets. One outstanding feature is the j formation of large fatty masses which are called lipo- ] diastemata. These appear after a period of from one to two] 1 months of choline deficiency and are formed by the fusion \ of several fat-filled cells which have ruptured. The j condition of fat accumulation in the liver can be reversed,! [ up to a certain stage, by addition of choline to the diet; ; 1 however, if the choline-deficient diet is continued for J 9 several months, the liver cells atrophy, the excess fat disappears, and growth of fibrous tissue ensues. Chemical studies of the composition of fatty livers j i produced in rats by choline deficiency have revealed that | the lipid which accumulates consists mostly of triglycer- i ides, but that the level of cholesterol is also greater than normal (*+1). This is found to be true even when no j cholesterol is included in the diet. It has also been ! shown that if cholesterol is fed, a fatty liver develops 1 i which responds to the administration of choline (*f2). ^ Larger doses of choline are required to reduce the accumula-j tion of fat in this type of fatty liver than in the former, and in either case, the effect of choline on hepatic cholesterol does not appear until more pronounced decreases in fat have occurred (^3)* Choline and plasma cholesterol Weiss, Marx, and Marx (M+-) have recently observed i that dietary choline and inositol influence the level of j cholesterol not only in the liver but also in the plasma of j the rat. The circulating cholesterol levels were higher j i when choline and inositol were included in the diet than i when these substances were omitted. These workers con- 1 sidered that the differences which they noted were ! significant only in the cases in which cholesterol was also i being fed. Their results parallel the observations made by! ' Stamler ejt al. 0+5) who demonstrated that choline and i ; inositol significantly aggravated the hyperlipemia and | ' hypercholesterolemia produced in chickens by a high cho- i : i ; lesterol intake. Weiss ej; al. also noted, however, that | I i reports in the literature are contradictory with respect to j ! the action of lipotropic agents on plasma sterol levels in conditions accompanied by hypercholesterolemia. They cited : i , references indicating that choline and/or inositol admin- j ! istration results in a reduction of plasma cholesterol ' : (MS, *+7} ^-8). Himsworth (^9) and Firstbrook (50) observed 1 i ' no effect of choline on plasma cholesterol. Lipotropic factors were also ineffective in altering the hypercho lesterolemia caused by the following: hypothyroidism (51)>' thyroidectomy-hypophysectomy in dogs (52), and partial i hepatectomy in rats (53)• McKibbon ej; al. (5*0 developed a diet which ! I - 1 i produced a fatal choline deficiency in puppies in three I , weeks or less. They observed that such animals had decreased blood levels of both total and esterified cho- i i 1 lesterol. ; i J In a paper published after the work to be reported j I here was begun, Ridout e£ al. (55) confirmed the work of j ■ Weiss ej; al. (MO. In rats fed purified diets for three 11 i i weeks, differences in plasma cholesterol levels were \ J ! > present which depended only on the presence or absence of ■ ! ' ! choline in the diet/ On a choline-free, cholesterol-free j i ration, the values for esterified plasma cholesterol | amounted to 32±9 mg./lOO ml.5 on the same diet containing j ; 1 i 0.08 per cent choline the esterified cholesterol level was 1 j 6l±6 mg./lOO ml. Doubling the amount of choline caused no j further rise, and the authors refer to the effect noted as I | | evidence that choline is required for the maintenance of I the plasma cholesterol concentration within the normal 1 1 : 1 , range. > ; ] I In the same paper, these authors have also shown ' that the addition of cholesterol to either the choline- : deficient or choline-supplemented diet influenced the post- ! ' , | prandial esterified cholesterol level in the plasma. The j 1 , peak level occurred between two and four hours after ! • i ' ! : removal of the food from the animal cages, but exceeded the normal range for the rats used only when more than O.b per .cent cholesterol was fed. Cholesterol which had accumulated' ! i Min the liver was eliminated by these authors as a source of! 1 ] • the postprandial increases in the plasma levels. While j ' pre-feeding rats a diet containing cholesterol but free of j : choline for a period of three weeks led to deposits of cholesterol in the liver, these rats when subsequently 12 given a diet free of cholesterol but containing choline showed no postprandial rise in the plasma cholesterol. The authors suggest that the postprandial rise previously noted may have been due to some influence of choline on the absorption or utilization of cholesterol. The Relationship of Choline to Factors Which May Influence the Plasma Cholesterol Concentration Byers, Friedman, and Rosenman (56) have reviewed the literature pertaining to the regulation of the plasma cholesterol concentration. They consider that the content of cholesterol in the plasma at any instant is the net result of the processes of absorption, synthesis, excretion, and destruction or conversion of this sterol in the animal body. Two other possibilities can be added which con ceivably could influence the plasma cholesterol, namely, the interchange of cholesterol between the tissues and the plasma, and the carrying capacity of the plasma itself. In their review, Byers et. al. make no specific mention of experiments concerned with the effect of lipotropic factors' on any of these processes. Cholesterol absorption Three studies on cholesterol absorption and the effects of simultaneous feeding of lecithin or choline and . 13 inositol have been found in reviewing the literature. Two of the reports (*+5> 575 are, in a sense, contradicted by the third (58). Since the conclusions concerning the effects on cholesterol absorption are all based upon the effects of these substances on the plasma cholesterol levels, they only beg the question. Cholesterol is a fat-soluble substance. It is evidently not absorbed simply by being carried in with fat, however, for ^-cholestanol and sitosterol are also fat soluble, and yet these sterols, in contrast to cholesterol, are only slightly absorbed, if at all (59? 60). Cholesterol synthesis The effect of choline on cholesterol synthesis has been studied by Guggenheim and Olson (61). Acetate labeled with carbon-l^ was fed to rats on choiine-deficient and supplemented diets. Analysis of the liver cholesterol revealed that choline did not influence the rate of in corporation of acetate into the cholesterol molecule. Cholesterol conversion and excretion Cholesterol which is present in the lower intestinal tract arises from three possible sources: diet, biliary secretions, and direct excretion through the wall of the intestine. From isotope studies using cholesterol-^-Cl^, Siperstein and Chaikoff (62) have concluded that not more than 10 per cent of the administered radioactivity which is found in the feces are derived from excretion through the intestinal wall. The majority of the excreted carbon-l^ arrives in the feces by first being secreted in the bile. That portion of the cholesterol carbon which is present in the nonsaponifiable fraction of the fecal fat is derived about equally from the bile and from direct excretion. Friedman, Byers, and Gunning found that more than half the fecal nonsaponifiable matter came by way of intestinal excretion; the rest came from bile (63). Bloch et al. have shown that cholesterol can be converted to cholic acid in the dog (6^). Using cho lesterol labeled with carbon-l^, Bergstrom found that the main isotope-containing compound in the bile of rats is cholic acid (65). He concludes that bile acids seem to be quantitatively the most important conversion and excretion products of cholesterol, metabolism. Nothing has been reported concerning the effect of choline on such conver sion or excretion. Colwell measured the volume of bile and its content of cholesterol in choline-deficient and control rats (66). He found that the deficient rats secreted somewhat less bile than the animals receiving choline. The concentration 15 of cholesterol in the bile was reported to be the same in the two groups. The interpretation of these findings in terms of the effect of choline on blood cholesterol is difficult, especially since nothing has been reported con cerning the effect of choline on the absorption or destruction of cholesterol. One might wish to postulate that reduced bile secretion would lead to lowered plasma ; cholesterol levels by virtue of the smaller amount of bile cholesterol available for reabsorption in the intestine. On the other hand, less bile secretion could be pictured as a compensatory mechanism preventing the further reduction of the blood levels and thus be an effect rather than a cause. Cholesterol transfer between the plasma and tissues Studies concerned with the influence of choline or other lipotropic agents on the interchange of cholesterol between the tissue cells and blood plasma are lacking. That transfer of the sterol molecule takes place in both directions is evidenced by numerous studies, such as the appearance of labeled cholesterol in tissues after its intravenous injection (67), or the restoration of the plasma cholesterol levels after plasmapheresis (68). The classical measurements of Schoenheimer and Rittenberg have demon strated that cholesterol, like other body constituents, , is in a dynamic state of removal and replacement in both the cells and plasma (69). The hypercholesterolemic state can be produced in normal animals without the use of dietary cholesterol, and it is evident that very large amounts of cholesterol can be transferred from the tissues and retained in the plasma ;j under certain conditions. Two examples of such conditions 1 may be cited here: ligation of the bile duct (70) and the : intravenous administration of certain detergents such as Triton WR-1339 (71). The exact mechanism or mechanisms by 1 which such high blood cholesterol levels are achieved is | not known, but the available evidence points toward increased rates of synthesis (72) and alterations in the sterol carrying capacity of the blood (735 7*0 as likely explanations in both instances. Cholesterol carrying capacity of plasma Ninety per cent or more of the cholesterol of the i plasma is carried in a combined form as part of the lipo protein molecules (75)• The presence of protein closely j associated with lipid has been demonstrated by means of the; ultracentrifuge (76), electrophoresis (77)> and solubility studies (78). Fischer and Garrity studied the effect of | choline deficiency on the electrophoretic pattern of the serum proteins (28). The albumin concentration in the sera' 17 of choline-deficient rats did not differ significantly from* that in choline-fed controls until kidney lesions appeared.. At that time the albumin concentration was lower in the deficient animals than it was in the controls. Increases in QC- and /^-globulins were observed in the sera of deficient animals which did not seem to be related to degeneration of kidney tissue. #-Globulin was not affected by the choline content of the diet. The majority of the lipid would be expected to be associated with the -globulins with some also present in the OC-globulin fractions (79). Wilgram, Lewis, and Blumenstein (80), using a i technique of ultracentrifugal analysis, have reported that the low density and AT-lipoproteins are decreased in male rats fed a high-fat, choline-deficient diet. Paper electro phoresis did not reveal any marked differences in the serum proteins of the experimental group as compared with the choline-fed control group. The observed lowering of the concentration of a lipoprotein fraction of the plasma carries with it the intriguing possibility that some molecule which normally acts as a carrier of cholesterol in the plasma may be lacking or at least in poor supply in the; choline-deficient animal. Thus, there may be no defect in : cholesterol metabolism except as it results from 18 alterations in the availability of some other components of lipoprotein molecules. These components might be a protein or a phospholipid or even a triglyceride, or combinations of these. This concept has similarly been advanced by Byers, Friedman, and Rosenman (56) with respect to the alterations in the plasma cholesterol concentration in various diseases They have summarized their review by stating, • • • changes in this liquid tissue itself y rather than in the fixed tissues of the body, determine the rate at which cholesterol leaves or accumulates in it. . . . The role of the liver is an essential but indirect one, in that either it furnishes the substance(s) changing the absorptive capacity of the plasma proteins or it produces a greater quantity of those proteins able in themselves to bind and retain greater quantities of cholesterol. Cholesterol-phospholipid interrelationships Phospholipids, particularly those containing choline, would seem to be one class of compounds which might be in short supply in the plasma of choline-deficient rats. McKibbon and Taylor (81) performed an extensive analysis of the tissues of normal and choline-deficient puppies. They found a marked decrease in the total plasma phospholipids in the deficient group, although the per centage of the phospholipids which contained choline remained unchanged. The data further suggested that sphingomyelins were replacing lecithins. 19 Although numerous studies have been made on the effect of dietary choline on the phospholipid content of such tissues as liver, intestine, and kidney in the rat (9) no reports have been found which are concerned with the effect of choline deficiency on the level of these important lipids in the blood of this species. Reports in the literature would seem to indicate that the concentrations of cholesterol and phospholipids in the plasma are in some way directly related (82, 83). It may be possible that these two substances are combined in some fixed ratio in lipoprotein molecules (S1 *-). Gould (85) speaks of one of the functions of phospholipid in this way: Plasma phospholipids do not transport fatty acids from one tissue to another as previously thought. One of their functions in plasma seems to be concerned in some way, not well understood, with the maintenance of all the plasma lipids in a state of optical clarity. STATEMENT OF THE PROBLEM AND PLAN OF ATTACK i Rats fed choline-deficient diets have lower plasma cholesterol levels than rats fed diets supplemented with i choline. This dissertation represents an attempt to • ! elucidate the mechanism or mechanisms responsible for this 1 effect of dietary choline. I I The following three possibilities were taken into consideration in planning the study: (a). Choline may be required for the^proper absorption j of cholesterol from the intestine; in the absence of choline less cholesterol is absorbed thus leading to lowered plasma cholesterol levels. The first experimental approach was a study of the absorption of carbon-l^-labeled cholesterol administered by I stomach tube to choline-deficient and choline-supplemented rats. ~ The results of such an experiment were expected to reveal not only the effect of choline on the absorption of ; exogenous cholesterol but also the effect of the lipotropic : agent on the reabsorption of endogenous cholesterol which ■ had been secreted into the intestines by way of the bile. ; (b). Choline may in some manner influence destruction ! i or conversion of cholesterol. ; The experimental investigation of this possibility consisted in the determination of the content of radio activity in the saponifiable and nonsaponifiable fractions of bile collected from choline-deficient and normal rats after the intravenous administration of cholesterol-1 +-C^1+. Any significant difference in the amount of radioactivity appearing in the saponifiable fraction of the bile from the rats fed the two diets could be taken as an indication that choline influences the metabolic conversion or destruction of cholesterol. (c). Choline may be required for the transfer of cholesterol from the liver to the plasma, and, in par ticular, it may be required for the formation of compounds necessary for carrying cholesterol in the plasma (lipo proteins) . As a means of evaluating these last hypotheses, choline-deficient and choline-supplemented rats were subjected to two procedures which are known to produce hypercholesterolemia in rats fed normal diets, i.e., ligation of the bile duct and the intravenous injection of detergents. The levels of plasma cholesterol and plasma phospholipid were determined. It was anticipated that the results of such an experiment would establish whether or not hypercholesterolemia could be achieved in the 22 choline-deficient animal; while the comparative degree of hypercholesterolemia would possibly lend support to the hypothesis that dietary choline is required for the forma tion of cholesterol-containing lipoproteins of the plasma. MATERIALS AND METHODS Materials Animals In the study of cholesterol absorption by the technique of lymph duct cannulation, the Long-Evans strain of rats was used. Rats of this strain were obtained from ,the Pacific Animal Farms, Hawthorne, California. Rats of the University of Southern California strain were used in all other experiments, including the second study of cholesterol absorption. Diets Four diets were used. These consisted of two dif ferent basal, choline-free diets (Diets A and C) and the same two diets to which choline was added (Diets B and D). ; The compositions of the four diets are shown in Table I. Detergents Tween-20. Tween-20 is a polyoxyethylene sorbitan monolaurate detergent manufactured by the Atlas Powder Company, Wilmington, Delaware. The sample used here was kindly donated by Dr. Norman Krinsky. Triton WR-1339. Triton WR-1339 is a polymeric p-isooctyl polyoxyethylene phenol. It was first f .......... TABLE I ' COMPOSITION OF DIETS Ingredients First absorption experiment only All other experiments I Choline- deficient (Basal) gm./lOO gm. diet B Choline- supplemented gm./lOO gm. diet C Choline- deficient (Basal) gm./lOO gm. diet D Choline- supplemented gm./lOO gm. diet Casein (vitamin-free) 10 10 15 15 Sucrose 72 71 56 55 Cottonseed oil 9.5 9.5 20 20 Celluflour 2.0 2.0 ■*f.O k . o Salt mixturei/ 5.0 5.0 ^.5 ^.5 Cystine 0.5 0.5 0.5 0.5 Inositol 1.0 1.0 0.1 0.1 Choline . . . . 1.0 . . . . 1.0 Vitamins 2/ 2/ 2/ 2/ f I 1/ Wesson,s modification of Osborne and Mendelfs original salt mixture (88). i ■ | 2/ Vitamin contents of the four diets— per kilogram of diet ! " " " i B]_ 10 mg. Menadione 5.0 mg. Ascorbic acid 100 mg. Calcium B2 10 mg. Biotin 0.10 mg. Niacin 60 mg. pantothenate i B$ 10 mg. PABA *+00 mg. Folic acid 10 mg. 60 mg. ; Fat soluble vitamins: A - *K)00 USP units; D - 800 USP units; E - 0.50 gm. mixed tocopherols [ £ ' 25 manufactured and supplied by the Rohm and Haas Company, Philadelphia, Pennsylvania. It is now distributed by Winthrop Laboratories, New York, New York. The sample used here was donated by Dr. Michael Schotz, Cleveland Clinic, Cleveland, Ohio. Radioactive cholesterol The radioactive cholesterol used in these studies j 1 was labeled in carbon number four of the steroid nucleus. . 11» It was prepared by Dr. Irving Zabin from H--C*1 - -cholestene- 3-one by reduction with sodium borohydride. The product obtained was purified as described by Schwenck et al. (86). The cholesterol had a specific activity of 1.3 x 10^ counts : 'per minute per milligram of carbon as measured in a gas flow counter (87). Methods Surgical techniques Lymph duct cannulation. This operation was per formed by Dr. G. F. Hungerford on rats anesthetized with nembutal. The technique used was a modification of that of * Bollman et al* (89). After surgery the animals were kept in restraining cages patterned after the design of Bollman (90). 26 The cannulated rats were given saline to drink to help promote lymph flow and reduce the clotting of lymph in the cannula. Even so, it was usually necessary to insert fine copper wire into the cannulae to remove clots which tended to form after the operation. Once the lym- \ phagogic effect of the saline had begun, clots in the lumen of the cannulae were not troublesome, provided that the | tubing had a sufficiently large inside diameter. Completely successful cannulation was obtained by using the polyethylene tubing distributed by the Clay-Adams Co. The size number was PE-50; the inside diameter was 0.58 mm., outside diameter 0.965 mm. Bile duct ligation. The abdomen of a rat anesthe tized with ether was opened by a midline incision. The bile duct was located and ligated with a cotton thread at a point in the first third of its length, in order to avoid impeding the flow of the pancreatic secretions. The incision was closed with either cotton thread or wound clips. Bile duct cannulation. The bile duct was first 1 ligated as described above. Wads of cotton moistened with ’ physiological saline solution were used to hold the lobes of the. liver away from the field of operation. After j making a short, longitudinal slit in the swollen portion of the duct proximal to the liver, the beveled end of a polyethylene cannula was easily inserted into the duct pointed toward the liver. The cannula was tied in place by passing a thread around the duct just above the point of insertion. The loose ends of the ligature were also used to hold the cannula firmly in place. The cannula was secured to the skin of the rat at the site of its exit from the body to remove strains on the above described ligature. The incision was closed with either cotton thread or wound clips, and the animal was placed in a restraining cage. Intravenous injections. Intravenous injections were made using a #26 hypodermic needle into either a tail vein or a superficial vein located just under the skin on the inside of the thigh. It was necessary to cut the skin to find this latter vessel. Drawing of blood. In the experiments in which serial blood samples were required, the following technique was employed. The tail of the animal was first cleansed with soapy water. It was then dried and given a thin coat of silicone grease. Under light ether anesthesia, a deep slit was made across a tail vein with a sharp razor blade. The oozing blood was collected in a heparinized syringe bearing a blunted #18 or #20 hypodermic needle. The silicone grease, by preventing the blood from wetting the tail, aided in the formation of drops which could easily be aspirated. By starting incisions near the tip of the tail ; and working toward the base, serial samples could be obtained. Chemical methods Preparation of cholesterol-^-C^* solution for in.iection. The method reported by Meier e j f c al. (91) was ! employed for the preparation of an aqueous solution of cholesterol. Tween 20 was first added to the dry j cholesterol-1 *-^**, one drop of Tween per 2 mg. of choles- ! terol. Sufficient acetone was added to bring the choles- i \ i terol into solution. The acetone was then removed with the; aid of a stream of nitrogen while heating the solution in a! bath of boiling alcohol. Saline was added to make a final i concentration of cholesterol of about 2 mg./ml. | Extraction of blood plasma. The method of Sperry | was used, employing a mixture of chloroform and methanol j I (92). The extract was not purified beyond filtration. j Aliquots were taken for cholesterol, phospholipid phos- ! phorus, and total lipid determination. j In instances in which cholesterol alone was determined, extraction was done by adding 1 ml. of plasma J i to l1 * ml. of a mixture of ethanol and acetone (1:1 by ; volume). I 29 Extraction of other tissues and gastro-intestinal tract washings. When it was desired to obtain total cho lesterol separate from saponifiable matter, the tissues or washings were first hydrolyzed in alcoholic potassium hydroxide. The final concentration of ethanol was about 50 per cent (v/v), and the alkali was 30 per cent (w/v). The samples were refluxed for three hours to complete hydrolysis and then extracted with four portions of petroleum ether (Skelly B, b.p. 63.3 - 69.3° 0.) in a separatory funnel. When free and esterified cholesterol values were ; desired, extraction was carried out in a Soxhlet extractor, using as solvent a mixture of ethanol and ether (3:1 by volume). The total time of extraction was eight hours. Extraction of bile. Aliquots of bile were first hydrolyzed by refluxing with potassium hydroxide at a final concentration of 30 per cent (w/v). The alkaline solution was extracted four times with ether in a separatory funnel. The aqueous residue was acidified. It was found that complete extraction of the saponifiable material from this acidic solution could not be accomplished simply by shaking with ether but that preliminary drying of the acidified residue was necessary. 30. Completeness of extraction was ascertained by comparing the sum of the radioactivities of the two frac tions with the radioactivity of a similar aliquot of bile before hydrolysis and extraction. Determination of cholesterol. The measurement of the cholesterol content of the extracts was carried out , using a modification of the Schoenheimer-Sperry method 1 published by Nieft and Deuel (93). Determination of phospholipid phosphorus. The method used for phosphorus determination was that developed by Werkheiser (9*0. Aliquots of lipid extracts were digested with sulfuric acid using perchloric acid as an aid to clear the digests. Determination of total lipid. A micro method developed by Bragdon (95) was used for the determination of the total lipid content of extracts from blood plasma. A macro, gravimetric procedure was used to deter mine the total lipid content of liver tissue. The alcohol-ether extract was made up to volume, and an aliquot was evaporated to dryness. The dried residue was dissolved in petroleum ether and filtered into a tared flask. The solvent was evaporated and the residue dried \ to constant weight in a vacuum oven. Radioactivity techniques Determination of purity of radioactive cholesterol. Because of the report that cholesterol labeled with radio active carbon can decompose on standing (96) and because the material used in these experiments had been prepared earlier, it seemed advisable to determine if the cholesterol used here showed evidence of decomposition. The material was subjected to chromatography on paper using two dif ferent solvent systems, and in both cases the results indicated a homogenous product. One migration was carried out on ^Quilon'^-treated filter paper according to the method of Kritchevsky and Kirk (97). Development of the chromatogram with either silicotungstic acid or antimony pentachloride (98) revealed a single spot which corresponded to the distance moved by a sample of pure non-radioactive cholesterol. The other migration was carried out using a mixture of 95 per cent butanol and 5 per cent pyridine as the moving phase. Again a single spot was observed. The chromatograms were cut into sections 5 mm. wide, and each section was counted. The region of radioactivity corresponded with the spot on the paper. Assay of radioactivity. Direct counting techniques were used in this work, that is, the materials assayed for 32 radioactivity were not combusted before counting. The radioactivity in lymph or bile was determined by placing volume aliquots of lymph or bile directly on tared ; aluminum counting dishes and drying the samples under a (heat lamp. The bile samples spontaneously spread evenly j 1 over the surface of the dish. To aid the spreading of the ; i * lymph, two or three drops of ammonium hydroxide were added. I Lipid extracts were counted in the following manner. The extracts were first made to volume with , petroleum ether (Skelly B) in volumetric flasks. Aliquots i were placed in test tubes, and evaporated to dryness at 60° C. To the dried residues were added known volumes of chloroform containing cottonseed oil of such a concentration that 0.2 ml. of the solution contained approximately 15 mg. of oil. After thoroughly mixing with the dried residue, duplicate aliquots were placed on tared counting dishes which had been provided with a piece of lens tissue to prevent creeping-* of the lipid up the sides of the dish : while the samples were drying. This technique was I t applicable to both the saponifiable and nonsaponifiable fractions. The purpose of adding the cottonseed oil was to f add weight to any sample which might have a negligible ; amount of solid; material. By this means, counting of any samples at or near infinite thinness was avoided. The 33 samples were initially dried under a heat lamp and then placed in a desiccator over sulfuric acid. A self absorption curve was prepared by mixing equal aliquots of a solution of cholesterol-^-C-^ in acetone with increasing amounts of non-radioactive lymph. Each of these solutions was then made to the same volume with saline and thoroughly mixed. Equal aliquots were removed and placed on counting dishes, dried, weighed, and counted as described above. The counts per minute obtained for each sample, corrected for background, were plotted against the logarithm of the weight of the sample. The best straight line was drawn through the points of this graph, and from this line the ratios were computed which are plotted as abscissas in Figure 1. Employing cottonseed oil, in place of lymph, as the non-radioactive diluent, a graph was obtained which was identical to that presented in Figure 1 for lymph. The curve of Figure 1 was used in the following way. The counts per minute obtained for an experimental sample, corrected for background, were divided by the ratio (obtained from the graph) for that weight of sample. The quotient was the counts per minute which would have been obtained if the sample had weighed 15 mg. - Weight o f cample - W } ■ 3^ 20 10 0.4 1.2 0.8 Countfl per minute at weight W Counts per minute at lf> mg. Figure 1. Self-Absorption Curve for Cholesterol- C Dissolved in Lymph or Cottonseed Oil. 35 All counting was done in a gas flow counter, and samples were counted for a minimum of 2560 counts. The standard deviation of the counting error was thus ±2 per cent. Flan of the Experiments The effect of choline on cholesterol absorption as j 1 — - ] measured by the method of lvmph duct cannulation Male, Long-Evans rats weighing between 90 and 120 gm. were divided into two groups. One of these groups was offered a synthetic choline-deficient diet (Diet A, Table I); the other group was offered the same diet to which choline had been added at a level of 1 per cent of the diet (Diet B, Table I). After a minimum period of three weeks on the diets, the animals were prepared for absorption studies by insert-; ing a polyethylene cannula into the cisterna chyli of the thoracic lymph duct. The rats were then placed in restraining cages. A twenty-four hour period was allowed for recovery of the animals from the effects of the operation and to be sure the lymph flow was continuous. At the end of this . i L . time, a dose of cholesterol-4~C^ dissolved in cottonseed oil (Wesson oil) was administered by stomach tube. 36 The quantity of oil given was between 0.5 and 0.7 ml., and the amount of cholesterol was from 3 to 5 mg. Lymph was collected in graduated centrifuge tubes over one hour or two hour intervals during a total period of twelve hours. Aliquots of the lymph samples were placed on count ing dishes and dried under a heat lamp. Counting was done as previously described. The effect of choline on cholesterol absorption as measured bv analysis of the gastrointestinal contents The results obtained from the previous experiment suggested that certain changes in procedure were advisable in order to produce a more severe choline deficiency and thus accentuate the possible effects of choline on cho lesterol absorption. The principal changes made were the following: (1). The content of protein in the diets was increased to improve the growth of the animals. (2). The quantity of choline added to the choline- supplemented diet was reduced to 0.5 per cent, since it was observed in the previous experiment that supplementation with 1 per cent choline caused some anorexia. (3)* Weanling rats were used. It has been shown that the very young rat has a greater requirement for dietary choline than older rats and that it evidences choline a?"* deficiency symptoms not present in older rats given the same diet. (k-). The technique used to measure cholesterol absorp- ; tion was different. It was felt that the variation in absorption rates observed among the rats of the first experiment was in part a consequence of the use of surgery and restraining cages, both of which might affect absorp- i tion by way of their influence on the nervous and circula tory systems (99). No surgery was performed in the second ; experiment, and except for the slight trauma of force feeding the radioactive cholesterol, the rats were not disturbed during the absorption period. Two groups of male weanling rats of the University of Southern California strain were offered, respectively, choline-deficient diet C (Table I) and choline-supplemented ! diet D (Table I), for a period of six weeks. During the first three weeks on the diets, the animals were weighed periodically, and a record was kept of their food consump- | tion. For measurement of cholesterol absorption, the following procedure was employed. The animals were starved for twenty-four hours. At the end of this period 'they were lightly anesthetized with ether, and 1.6 mg. of cholesterol- 1+— dissolved in 1.0 ml. of cottonseed oil (Wesson oil) 38 was administered by stomach tube. In addition, the animals which were on the choline-supplemented diet received, by stomach tube, 25 mg. of choline chloride dissolved in 0.25 ml. of water. The animals were returned to their cages for a period of six hours. At the end of the six hour absorp- tion period the rats were sacrificed with an overdose of ether and -the livers were removed and weighed. The gastro-j intestinal tract was then removed and the contents flushed out with hot saline. To facilitate the washing process, the intestines of each animal were washed in two sections. The first section included the small intestine and the cecum; the second section consisted of the large intestine and the rectum. The contents of the stomach were obtained as a separate sample in order to find out if the stomach could be considered to have emptied completely during the absorption period. The washed walls of the intestine and the washed stomach were combined as a single sample to be hydrolyzed and extracted. The liver constituted another sample, as did the carcass. By analyzing all these samples from each - animal it was possible to obtain an evaluation of the completeness of recovery of the administered radioactivity. 39 The effect of choline on the distribution of radioactivity ' in the bile after the intravenous administration of \ 11 + solubilized cholesterol-H— C-1 " . ♦ Male weanling rats were fed the choline-deficient I and choline-supplemented diets as in the second experiment i I (Diets C and D, Table I), After a minimum period of six : \ weeks, the rats were subjected to the bile duct cannulationj i procedure previously described. i A period of twenty-four hours was allowed for ! recovery from the operation, after which time a sample of ! I cholesterol-^-Cl^ solubilized with the aid of Tween 20 was j ! injected intravenously. Bile was then collected for \ periods of from twenty-four to forty-eight hours. Aliquots of the bile were hydrolyzed and extracted as described, and the content of radioactivity was determined in the i i i saponifiable and nonsaponifiable fractions as well as in ; samples of the untreated whole bile. j i The effect of choline on the hypercholesterolemia resulting1 from ligation of the bile duct Weanling rats were placed on choline-deficient and j ■ i supplemented diets (Diets C and D, Table I) for a period of! I six weeks. Blood samples were withdrawn at the end of the 1 feeding period, and the bile ducts of the animals were | ligated immediately afterward. A second blood sample was [ ko taken from each animal at the end of the third day after bile duct ligation. The samples were analyzed for cho lesterol and phospholipids. A third sample of blood was taken ten days after ligation from which only cholesterol could be analyzed. In order to obtain a sample large enough for analysis of cholesterol and phospholipid, equal volumes of plasma from two animals were pooled for extraction. The effect of choline on the hyperlipemia and hypercho lesterolemia resulting from the intravenous administration of a detergent (Triton WR-1^9) Weanling rats were fed either the choline-deficient or choline-supplemented diets (Diets C and D, Table I) for six weeks. At the end of the feeding period, blood samples were withdrawn as previously described and the plasma extracted by the Sperry method. The animals were then given an injection of 1 ml. of a solution of Triton WR-1339 'in water. The concentration of the Triton was 100 mg. per ml. Seventy-two hours after the injection, blood samples were taken again, the plasma was extracted, and the extracts analyzed for phospholipid, total lipid, and cholesterol. The effect of choline on the composition of liver lipids in the rat Two groups of rats received from weaning the hi • choline-deficient and supplemented diets (Diets C and D, Table I). At the end of a six week feeding period the rats were sacrificed and the livers removed quickly and weighed. Weighed aliquots of the ground livers were placed in Soxhlet thimbles and extracted with alcohol- ether mixture as described. The extracts were analyzed for; phospholipid phosphorus, total lipid, and cholesterol. j RESULTS The Effect of Choline on Cholesterol Absorption as Measured by the Method of Lvmph Duct Carmulation The lymph samples which were counted were volume aliquots of the hourly or two hourly collections. The average counts per minute obtained from duplicate samples, corrected for self-absorption, were multiplied by the appropriate factors to obtain the total radioactivity of the lymph collected during a given time period. Graphical representations of the accumulation of radioactivity in lymph are shown in Figures 2 and 3 for the twelve animals used in this experiment. A measurable amount of radioactivity appeared in the lymph of all animals by the end of two hours 5 after an initial lag period, the absorption rate increased. The rate, represented by the slopes of the lines, appears to have reached a fairly constant value by the end of four or five hours after the administration of cholesterol and to be continuing undiminished at the end of twelve hours. This observation also applies to animals number 16 and number 22, although with the scale used, the twelve hour values could not be plotted conveniently. *+3 O - ANIMAL NUMBER 4 6 a 10 HOURS A FTER CHOLESTEROL FEED IN G Figure 2. Accumulation, of Radioactivity in Lymph After Feeding Cholesterol-^f-C14* to Choline-Supplemented Rats With Lymph Duct Cannulas. Mf 40 O - ANIMAL NUMBER 5- 2 6 8 10 / 2 HOURS A F T E R CHO LESTERO L F E E D IN G Figure 3® Accumulation of Radioactivity in Lymph After Feeding ChoXesterol-^-C^ to Choline-Deficient Rats With Lymph Duct Cannulas® The total counts recovered at the end of twelve ; hours for both groups are given in Table II. Since the j i animals did not all receive the same amount of radioactive j cholesterol, the amount of radioactivity in the lymph at i the end of twelve hours is expressed in the last column as • a percentage of the administered dose. These latter ! j figures probably represent the best method of comparing the j rates of absorption of cholesterol, for much of the varia- . i • 1 bility observed in the total radioactivity was eliminated i by taking into account the size of the dose. Even so, the : 9 \ variation among the animals of both groups was very high, j and although the mean values of the per cent of administered i counts which appeared in the lymph at the end of twelve hours were different for the two groups of rats, statistical i analysis revealed a relatively high probability that this f difference was due to chance. Also shown in Table II are the body weights and liver weights of the rats. The liver weight is considered | to be a function of the body weight in the normal animal; i however, in animals which have a severe choline deficiency ; the accumulation of lipid in the liver, in part, gives rise ( to liver weights which are significantly higher than normal j i when expressed as per cent of the body weight. As shown in j ;Table II, the rats fed the deficient diet did not show this i TABLE II THE EFFECT OF DIETARY CHOLINE ON CHOLESTEROL ABSORPTION AS DETERMINED USING RATS WITH LYMPH FISTULAS Animal number Body Weight Gm. Liver weight Radioactivity Gm. % body weight Administered Recovered in lymph after 12 hours Cts. per min. Cts. per min. % activity admin. , A. Choline-supplemented group 1 262 8.77 3.35 307,000 77,300 25.2 3 280 9.2>+ 3.30 2>+>+,000 *+3,800 17.9 8 279 9.17 3.29 151,000 20, *+00 13.5 81 279 8.87 3.17 152,000 18,000 11.8 l*f 262 8.08 3.08 151,000 26,300 17 .*+ 19 28? ZiS6 2.63 .111/ 192,000 >+6,900 2>+.>+ Average 2 75 8.62 3-13±0 l8.>++2.50i/ B. Choline-deficient group 13 288 11.7 >+.06 2>+>+,000 >+9,000 20.1 16 278 8.9>+ 3.22 378,000 132,000 3^.9 21 310 9-70 3.13 2Mf ,000 >+5,600 18.7 22 28> + 11.2 3.9*+ 192,000 81,000 ^3.3 23 352 11.8 3.36 151,000 3^,300 22.7 30 338 12.5 3.68 152,000 31,>+00 20.7 Average 308 11.0 3.56+0• I6i/ 26.7+>+.09i/ 10$<p<20$ 10$cp<20$ -T 1L .With, standard, deviation of—the.mean.- *+7 effect to a pronounced degree and can be considered to be suffering from only a mild choline deficiency. The Effect of Choline on Cholesterol Absorption as Measured bv Analysis of the Gastrointestinal Contents In the next experiment to be reported, changes were made in the experimental procedure which were intended to j produce a more severe choline deficiency in one group while improving the food consumption and quality of the i diet of both groups. The changes made have been described j in the plan of this experiment. The results shown in Tables III and IV demonstrate that the choline-deficient diet used was indeed producing symptoms of choline deficiency 5 this is shown by the slower rate of growth and the heavier livers in the deficient animals. The food consumption and body weight changes of rats maintained on the two higher-protein diets (Diets C and D, Table I) for a period of three weeks after weaning j are presented in Table III. The animals represented in this table are the same animals for which data are given in Tables TV and V. Food consumption measured during the first three weeks on the diet shows that the anorexia t observed previously in the animals fed the choline- ; ! supplemented diet was not present with this diet. TABLE III GROWTH AND FOOD CONSUMPTION OF RATS FED DIETS C AND D Food con Gain in Animal sumed per Hoay weign'cs weight per number day At After Gain 100 gm. food start 28 days per day consumed A. Choline-supplemented group (Diet D) gm. 1 10.9 *+5 l8*f 5.0 b $ .b 2 10.9 * * ■ 9 17b b .5 1+0.9 3 10.0 36 158 1+3.6 b 11.5 192 5.1 M+.7 5 11.5 b2 182 5.0 1+3-1+ 6 1 0 .k- 59 185 J+-5 ^3-3 7 11.3 5*f 192 “ +•9 1 +3.8 8 11.0 51 192 5.0 1+5.9 9 11.3' ^3 190 P '3 1+6.6 10 11. i f *+9 187 b . 9 1+3.2 Averagel/ 11.0+0.2 bQ±2 18^+3 b .9 ± 0 .1 1+1+.1+0.5 B. Choline-deficient group (Diet C) gm. 11 9.8 50 l»+6 3 A 35.2 12 10.6 bo 160 *+•3 1+0.5 13 Died on 8th day b3 — — --------- l b 10.7 56 167 £.0 37.0 15 10.0 Mf 167 1+3.8 16 10.5 pb 175 *+.3 i+l.O 17 9.7 b-9 152 3.7 37.9 18 lO .b b6 173 *+.5 1+3.8 19 9.2 b9 Ib b 3> 37.1 20 8.3 b6 122 2.7 32.8 Averagei/ 9.9+0.3 b8±2 156+6 3.9to.2 38.8+1.3 30<p<M)$ p=100% p<lg P<1* 1/ | With standard deviation of the mean. TABLE IV BODY WEIGHTS AND LIVER WEIGHTS OF RATS AT THE END OF THE SECOND ABSORPTION EXPERIMENT Animal Body Liver Liver weight number weight weight as per cent of gm. gm. body weight A. Choline-supplemented group 1 246 8.07 3.28 2 215 7.53 3.50 3 162 5.65 3-56 1 + 246 8.35 3.39 5 247 7.60 3.06 6 228 7.40 3.25 7 262 8.14 3.10 8 258 8.28 3-21 9 262 8.33 3.18 Average!/ 237+11 7.7010.28 3.27+0.18 B. Choline deficient group 11 219 il .5 5.71 12 202 13.4 6.63 lb 202 14.0 6.96 15 213 13.9 6.52 16 222 14.9 7.15 17 185 12.0 6.51 18 248 17.9 7.22 19 207 11.4 5.49 20 184 14.6 7.95 Averagei / 209±7 13.7+0.7 6.68+0.25 2%<p<5% p<l# p<l% 1/ With standard i deviation of the mean. The choline-deficient animals ate somewhat less food, although the average indicates that this amounted to only about one-tenth less per day than the supplemented rats. It can be seen from the last column that not only did the choline-deficient rats eat less food, but that they also utilized the food less efficiently for purposes of weight gain. The gain in grams of body weight per 100 gm. of food eaten was 38.8 gm. for the deficient rats and M+.l gm. for those receiving the choline supplement. At the end of the six week feeding period prior to absorption studies, the rats were weighed again, and at the end of the absorption experiment the liver weights were also determined. The results of these two measurements (Table IV) again confirmed that symptoms typical of choline deficiency had been produced. The mean liver weights expressed as per cent of the body weight were more than twice as great in the choline-deficient rats as in the supplemented rats. Furthermore, the livers of the two groups were grossly different in appearance. The rats fed the deficient diet had enlarged, pale livers with blunt, rounded edges; the control group exhibited normal livers with a healthy red color and sharp edges. Chemical analysis of livers of rats fed these two diets also demonstrated the effect of choline deficiency. 51 These values will be presented later. One additional observation was made which further substantiates the conclusion that Diet C was producing severe choline deficiency. Several of the rats fed the deficient diet were noticeably sick after one week on the diet, and one animal in this group died on the eighth day. Examination revealed grossly hemorrhagic kidneys in this animal. None of the rats fed the supplemented diet exhibited any such symptoms. These findings are in keeping with the picture of choline deficiency presented by Griffith and Wade (23, 24-). The results of the second absorption experiment are presented in Table V. The radioactivity in the non- saponifiable matter extracted from the several fractions of each animal is presented as a percentage of the administered radioactivity. Only a small portion of the radioactivity remained in the stomach at the end of six hours. This was found to be true for both groups of rats and indicates that choline has no influence on the stomach emptying time great enough to affect this function for six hours. The major portion of the radioactivity appeared in the contents of the intestinal tract and in the washed walls of the intestines. The mean values for the absorp tion of cholesterol can be calculated by subtracting the ! TABLE V i I j I EFFECT OF DIETARY CHOLINE ON CHOLESTEROL ABSORPTION AS ; DETERMINED FROM THE RADIOACTIVITY OF GASTROINTESTINAL ! CONTENTS OF RATS FED CHOLESTEROL-^-C1^ Animal number Radioactivity of the nonsaponifiable fraction re covered at the end of six hours Per cent of radioactivity administered Intestinal Washed in contents testinal wall Stomach contents Liver A. Choline-supplemented group 1 59.8 39.0 2.6 6.0 2 *+0.0 >+5.7 1.6 9.2 3 31.*4 2.9 7.5 b *49.1 36.1 3.1 6.0 5 *44.1 *+6.*+ 3.3 10.>4 6 ■+5.*+ *46.1 3A 1.7 7 *+9.8 >+8.8 0.0 0.2 8 *+0.*+ *+9.*+ 2 .b *4.5 ; 9 27.0 53.“ + 2.2 • its® Averagei/ *42.913.3 *45.911.8 2 .b ± 0 .b 5.6+1.1 | B. Choline deficient group 1 i 11 27.0 57.*4 5.2 9.1 i 12 66.5 21.*+ 3.3 3.2 ! l b >49.8 3*4.0 2.8 6.0 : 15 67.7 23.2 b .6 6.6 I 16 39.1 3*4.>4 1.7 9.5 1 17 *4*+.3 >+9.6 3.1 3.8 ] 18 3*4.8 >+8.5 2.8 2.7 1 19 79.6 l*+.5 0.2 2.2 20 35.3 >+7.6 3.9 — Average!/ *49-316.0 36.715-0 3.1+0.5 >4.911.0 | 1 30$cp*.*+0# 50%<pc60% - I / j With standard deviation of the mean. j 53 percentage of radioactivity in the contents of the intestinal tract from 100. The results are 57.1 per cent for the control rats and 50.7 per cent for the choline- deficient animals. Statistical analysis of these figures indicates that they are not significantly different* A wide range of variability, as observed in the previous experi ment, is also shown by these rats. | A similarly wide range is also shown by the quantities of radioactivity appearing in the livers of both groups of rats. Statistical analysis again indicates that the means of these values are not significantly influenced by the presence or absence of choline in the diet. The Effect of Choiine on the Distribution of Radioactivity in the Bile after the Intravenous Administration of Solubilized Cholesterol-^-- Five rats were successfully treated in this experi ment, although an attempt was made to carry out .the pro cedure on three additional animals. These three animals survived the operation, but ultimately died at the time of injection of the Tween-stabilized solution of cholesterol. i The cause of death was not determined. Two choline- deficient and three choline-supplemented animals survived through twenty-four hours of bile collection. 5*+ Included in Table VI are the volumes of bile secreted, the concentration of the total solids, and the total radioactivity of the bile together with the radio activity of the saponifiable fraction. Both the latter measurements are expressed as fractions of the administered radioactivity. The volume of bile collected during the first twenty-four hour period bore no relation to the diets of the rats. Considering the variability, no conclusions can be drawn from this small sample with respect to the effect of choline on bile flow. Although the percentage of the administered radio activity which appeared in the whole bile differed slightly in the two groups of animals, no pronounced effect of choline was observed. The mean value for the two choline- deficient rats during the twenty-four hours was 20.0 per cent and the mean for the supplemented rats amounted to 19.0 per cent. That portion of the radioactivity which appeared in the saponifiable fraction consistently accounted for a very large fraction of the radioactivity of the bile. The slight difference in distribution of radioactivity in the bile fractions of the two groups of rats is not significant. These results suggest that cholesterol derived from the TABLE VI EFFECT OF CHOLINE ON THE RADIOACTIVITY APPEARING IN WHOLE BILE AND IN THE SAPONIFIABLE FRACTION OF BILE FOLLOWING THE INTRAVENOUS INJECTION OF CHOLESTEROL-^-C1^ Animal number Dietary group Volume of bile Ml. Total solids Mg./ml % of adminis tered radio activity in bile after 2b hours Whole Sapon- bile ifiable fraction 1 Choline- 13.6 31.1 2 b .0 22.9 2 deficient 8.3 26.3 16.0 15.3 Average 10.9 2 8.7 20.0 19.1 3 Choline- 13.5 27.3 18.7 17.5 b supplemented 6.8 27.8 15.2 1*+. 2 5 1^.8 28.7 22.8 21.5 Average 11.7 27.9 19.0 17.7 plasma is degraded equally well by choline-deficient and supplemented rats, as far as can be estimated by the con ditions used in this experiment. The Effect of Choline on the Hypercholesterolemia Resulting from Ligation of the Bile Duct The rats used in this experiment were placed on the experimental and control diets at weaning. Evidence of sickness appeared among the rats on the deficient diet at the end of the first week. None of the rats died, however, and none of the control rats showed evidence of discomfort. At the time of the ligation operation the livers were examined, and those of the deficient rats were found to be larger and more pale, with blunt edges. The supplemented animals had smaller, richly red livers with sharp thin edges. Included in Table VII are the concentrations of total plasma cholesterol and of phospholipids of the two groups of rats before and after ligation of the bile ducts. Each value represents the results of analysis of extracts from the pooled plasma samples of two rats. The phospho lipid concentrations were obtained by multiplying the phospholipid phosphorus concentration by twenty-five. 57 TABLE V II EFFECT OF CHOLINE ON THE HYPERLIPEMIA PRODUCED BY LIGATION OF THE BILE DUCT Concentration of plasma lipids Animal numbers Total cholesterol Before After ligation ligation 3rd. day 10th. day Phospholipids Before After liga- ligation tion 3rd. dayi Before Ratio Cholesterol: Pho s pho 1ip id After mg./lOO ml. mg./100 ml mg./100 ml. mg./lOO ml. mg./1Q0 ml. ligation ligation A. . Choline-supplemented group 1,2 75.6 158 1^2 195 375 0.39 0.^2 3,N- 5^.6 196 93.8 115 1250 |0A7 0.16 5,6 60.5 195 167 155 575 0.39 0.3^ 7,9 . 67.3 156 108 173 375 0.39 0.*+2 Average^ 6*+. 31*+. 6 176±11.1 128+16.5 159±ll +.3 6¥f+20.7 0Al±0.02 0.3310.06 B. Choline-deficient group 21,23 35.5 103 75.0 68 2^-5 jO.52 0.^2 22,2*+ ^-3.2 101 86.7 85 2^-8 0.51 OAl 25,26 51.3 95.5 67.0 165 275 ,0.31 0.35 00 C\l * \ IN C M 32.5 l*+0 90.0 118 255 0.28 0.55 Average^/ *f0.6±*+. 2 110+10.2 79.5+5-3 109±21.3 256+6.7 0.*+0+0.06 . 0.^3±0.0^-3 P<1^ 2%<p<5% 5%<v<10fo 20$<p<30$ p?90^ 20^<p<30^ With standard deviation of the mean. 58 Considering, first, the values obtained before ligation, it can be seen that the total cholesterol con centration was significantly lower in the plasma of the choline-deficient animals than in the control rats fed the supplemented diet. The phospholipid levels were also lower in the deficient group. Ligation of the bile duct resulted in an increase in the plasma cholesterol of both groups of rats although the levels attained were greater in the case of the sup plemented rats than in the deficient group. Increases also occurred in the phospholipid concen tration as the result of bile duct ligation, and again the highest levels were found among the animals receiving the choline supplement. Also shown in Table VII are the ratios of cho lesterol to phospholipid for each of the rats before and after ligation;of the bile duct. Before ligation this ratio was found to be very nearly the same for the two groups of rats. Statistical analysis indicates that the ratios were not significantly altered by ligation in either group. In summary, the results of this experiment show that the plasma cholesterol and phospholipid concentrations in rats fed diets supplemented with choline are higher than corresponding levels in choline deficient animals. 59 In addition, these differences are increased after ligation of the bile duct. The evidence indicates that the plasma cholesterol and phospholipid concentrations change in a parallel manner after ligation and this relationship holds even in the absence of dietary choline. The Effect of Choline on the Hyperlipemia and Hypercholesterolemia Produced bv the Intravenous Injection of a, Detergent (Triton WR-1^9) The injection of the detergent used in this experi ment, Triton WR-1339> was unaccompanied by any evidence of distress on the part of the rats injected. This is in contrast to the previously mentioned experience with the injection of a solution of Tween 20 in which case death of three animals occurred shortly after the injection. The values of plasma total cholesterol before Triton injection were significantly lower in the choline- deficient rats than in the choline-supplemented animals (Table VIII). This observation is in agreement with the results presented in the previous experiment. The concentrations of plasma total and free cho lesterol, phospholipids, and total lipids after Triton injection are given in Table IX. The total cholesterol concentrations were lower in the choline-deficient rats TABLE VIII EFFECT OF DIETARY CHOLINE ON THE PLASMA CHOLESTEROL CONCENTRATION BEFORE TRITON INJECTION Animal number Total cholesterol mg./lOO ml* A. Choline-supplemented group 85.0 b-2 70.1 76.3 *+5 50.^ *f6 62.5 51 71.0 Average^/ 69.1+1* .8 B. Choline-deficient group 0 63.0 ‘ *7.3 bo 27.5 b-7 29.3 57 not determined Averagei/ ^1.7+8.^- With standard deviation of the mean TABLE IX EFFECT OF CHOLINE ON THE HYPERLIPEMIA PRODUCED BY THE INJECTION OF TRITON Concentration of plasma lipids ' 1 1 Mg./lOO ml. plasma 1 Animal 1 number Cholesterol ! Total cho1 - Free cho Phospholipid Total lesterol lesterol lipid | A. Choline-supplemented group 1 1 j 25 **73 363 875 9090 1 + 2 ¥)i* 313 950 8760 1 1 + 1 + 535 *t03 820 8590 M-5 371 * 292 775 5170 ; 1 + 6 ¥L2 317 880 7220 51 369 278 750 6290 Average!^ ‘*28+26 328+19 8m±30 75101639 B. Choline-deficient group 1 I 0 263 229 550 **160 300 229 **50 ¥*80 | bo 203 1^3 375 2300 ; b7 283 250 525 **830 ! 57 250 233* V25 3530 Averagel/ 259117 213+18 l *6$+3l * 3860+36O p a # p a # p a # P<1# 1 With standard deviation of the mean. i l 62 than in the supplemented animals; however, significant increases occurred in both groups as a result of Triton injection. Free cholesterol levels showed a similar response in both groups, and the lower values again were found in the deficient rats. When the plasma phospholipid levels were compared in the two groups after Triton injection, the lower levels appeared in the deficient rats. In order to establish whether increases occurred in the phospholipids of either group, an estimate of the levels before treatment was required. The technical difficulties of drawing blood, together with the desire to avoid fatal injury to the rats, limited the amount of plasma which was available for i analysis before the injection of Triton; as a consequence, values could not be obtained for both cholesterol and phos pholipids before treatment. The mean levels of phospholip ids obtained before bile duct ligation may be used for comparison with the values after Triton in the respective groups of this experiment. The close agreement of the total cholesterol concentration before treatment in these two experiments lends support to this use of the phospho lipid values, and the comparison indicates that significant , increases in phospholipid levels occurred in both groups of ; rats as a result of Triton injection. 63 Blood drawn from the rats seventy-two hours after Triton administration showed an extremely creamy appearance indicative of an intense lipemia. It can be seen in Table IX that the plasma total lipid levels were indeed very high in both groups of rats. However, the levels reached in the choline-supplemented rats were nearly twice as great as those in the deficient animals. Further relationships between the plasma lipid constituents for the two groups after Triton injection are presented in Table X. Statistical analyses indicate that there were no significant differences between choline- deficient and supplemented rats with respect to the follow ing: the total cholesterol and phospholipid expressed as per cent of the total lipid, the ratios of free to total cholesterol, and the ratios of either free or total cho lesterol to the phospholipid concentration. A significant difference was observed, however, in the concentration of free cholesterol expressed as per cent of total lipids# In this instance, the data in the first column of Table X show that a higher percentage of free cholesterol was found in the lipids of the deficient animals than in the supplemented rats. TABLE X EFFECT OF CHOLINE ON THE INTERRELATIONSHIPS OF PLASMA LIPIDS IN TRITON-INDUCED HYPERLIPEMIA Animal Cholesterol Phospholipid number as per cent of as per cent of total lipid total lipid Free Total Ratios Free cholesterol Total cholesterol Total cholesterol Phospholipid Free cholesterol Phospholipid A. Choline-supplemented group 25 1+.0 5.2 9.6 0.77 0.5!+ 0.4-1 1+2 3.6 *+.6 10.8 0.77 0.1+3 0.33 M+ “ +.7 6.2 9.5 0.75 0.65 0.1+9 **5 5.5 7.2 15.0 0.78 0.1+8 0.38 **6 b .k 5.7 12.2 0.77 0.1+7 0.36 51 1+.1+ 5.9 11.9 0.75 0.1+9 0.37 Average!' '+.‘ +±0.3 5.8*0.'+ 11.5*0.8 0.77*0.01 0.51*0.03 0.39*0.02 B. Choline-deficient group 0 5.5 6.3 13.2 0.87 0.1+8 0.1+2 32 5.1 6.7 10.0 0.76 0.67 0.51 1+0 6.2 8.8 16.3 0.70 0.51+ 0.38 “ +7 5.2 5.9 10.9 0.88 0.51+ 0.1+8 57 6.1 7.1 12.0 0.86 0.59 0.50 Average!' 5.6±0.2 6.9±0.5 12.5*1.1 0.81*0.01+ 0.56*0.03 0.1+6*0.03 P<1 % 5*<p*iojf ko%<p^5o% 20#<.pO0# 10&.p<L20$ 1/ With standard deviation of the mean. 65 In summarizing the results obtained in this experi ment the following may be noted; (1). A lowering of the plasma cholesterol concentra- , tion solely as the result of choline deficiency was | observed, in keeping with the results of the previous ; experiment. i (2). Hats receiving the choline supplement had a more i : severe lipemia after Triton injection than did the rats of i I the choline-deficient group. i (3). A higher degree of hypercholesterolemia was reached in the choline-supplemented than in the deficient group after Triton administration. (h). The level of phospholipids was higher in the plasma of the choline-supplemented rats than in the , deficient group after Triton administration. ■ (5)* The concentration of free cholesterol was found i to have reached a value higher than the generally accepted normal limit in both groups after Triton administration. Significant differences between the two groups of rats were t ' observed, with the higher concentration in the choline- i supplemented animals. However, this lipid constituted a ’ higher percentage of the total plasma lipid in the choline- ! deficient rats. 6 6 The Effect of Choline on the Composition of Liver Lipids in the Rat The results shown in Table XI confirm the observa tions previously made by gross examination of the livers of the rats fed the diets C and D. The mean percentage of total lipids in the choline deficient liver is slightly more than four times greater than in the rats fed the supplemented diet. It is interesting to note that the liver cholesterol level bears an inverse relationship to the plasma concentration. The liver of the choline- deficient rat has a higher concentration of total cho lesterol than that of the supplemented rat, while the blood levels of deficient rats are lower than those of supple mented rats (Tables VII and VIII). The phospholipid does not show this inverse relationship. In Table VII it is shown that the mean plasma phospholipid level is less in the deficient group than in the control animals, whereas the percentage of phospholipid in the liver is unaffected by the presence or absence of choline in the diet (Table XI). All the lipids for which analyses were made showed increases as a result of choline deficiency; this is true for the free cholesterol and phospholipids since the weights of the livers were much greater in the deficient 67 TABLE XI EFFECT OF CHOLINE ON THE COMPOSITION OF LIVER LIPIDS Animal number Body weight gm. Liver weight gm. Liver % "body weight Total lipid % liver weight Phospholipid % liver weight % total lipid Total cholesterol Free cholesterol % liver weight I total lipid I liver weight % total lipid Choline-supplemented group 1 222 6.20 2.79 5.50 2.0 3? 2 200 7.85 3.93 if. 30 1.0 2*f 3 251 10.07 * 4 -.01 5.52 -- -- * f 236 9.63 * f .07 5.21 1.5 29 5 226 9.89 if.37 5.26 1.2 22 6 193 7.6* 4 - 3-96 5.09 1.0 20 7 22 7 7.60 3.35 5.50 1.8 33 8 228 9.12 if.00 7.00 1.9 26 Average 9 10 11 12 S Average 223 8.50 3.82 198 12.02 6.06 181 11.30 6.25 209 12.80 6.12 235 17.07 7.26 150 11. i f 5 7.62 136 8.66 6.51 185 12.21 6.63 B. 0.2* 4 - 2 0.2lif 0.236 0.206 0.23* 4 - 0.2*f*f 0.275 0.229 5.ifl 1.5 27 0.235 Choline--deficient group 26.2 1.6 6.1 0.580 19.3 1.6 8.5 0.570 25.i f 1.6 6.3 0.662 22.0 l.if 6.2 0.515 23.2 l.if 5.8 0.if93 18.5 1.0 5.3 0.839 22.i f l.if 6.i f 0.610 if.*f3 0.211 3-83 if. 96 0.180 if.16 if.28 0.152 2.76 3.97 0.151 2.92 *+,*f 6 0.163 3.11 if.8i 0.200 3.92 if.98 0.216 3.92 3.27 0.185 2.65 i f .*f0 0.182 3-ifl 2.21 2.99 2.61 2.33 2.12 52 0.211 0.232 0.2if9 0.237 0.200 0.3Qif 2.79 0.238 0.8Qif 1.22 0.979 1.08 0.858 1.65 1.10 rats. The greatest increases occurred in the glyceride fraction as evidenced by the fact that the phospholipids total and free cholesterol values were lower in the deficient group when expressed as per cent of the total lipid. DISCUSSION Evidence has been presented here which confirms the observations of Weiss, Marx, and Marx and of Rid out et, al. (55) that plasma cholesterol concentrations are lower in choline-deficient rats than in rats receiving diets supplemented with choline. The object of the j i studies presented in this dissertation was to explore several possible mechanisms which may be responsible for this effect of dietary choline. The level of cholesterol in the blood plasma may 1 be considered to be the net result of several metabolic processes including the synthesis, destruction, absorp tion, and excretion of cholesterol, as well as the transfer of cholesterol between the plasma and the fixed tissues. The plasma cholesterol concentration also depends upon the capacity of the plasma, itself, to hold cholesterol molecules in the circulation. The experiments' i reported here were concerned with three of these processes, i i.e., absorption, destruction, and transfer from the 1 liver to the plasma, and with the carrying capacity of the: plasma, itself. ; The first working hypothesis considered that j choline may be required for the proper absorption of 70 cholesterol from the intestine. This hypothesis was sub jected to two experimental studies. These experiments differed in two principal respects: in the method used for measuring absorption and in the severity of the deficiency in the animals receiving the low-choline diet. In the first absorption study, cholesterol-l f-C^I f was administered by stomach tube to rats bearing lymph-duct cannulas. Radioactivity which appeared in the lymph was assumed to be indicative of all the cholesterol being absorbed from the intestine. The reports of Biggs, Fried man, and Byers (100) and of Chaikoff and his co-workers (19) substantiate this assumption, since both groups report that essentially all the cholesterol which enters the animal from the intestine comes in by way of the lymphatic system rather than by way of the portal vein. The collection of lymph as separate hourly or two- hourly samples after the administration of the radioactive . cholesterol permitted an examination of the time course of cholesterol absorption. The graphs representing cho lesterol absorbed versus time obtained for the individual rats were similar in the two groups in the following respects. The rate of absorption, as indicated by the slope of the lines, began slowly and increased rapidly during the period of between two and four hours after 71 administration of the dose. After reaching a maximum value, the rate appeared to continue undiminished for the remainder of the twelve hours of measurement. No essential differences were observed between the curves obtained for the choline-deficient and the supple mented rats. A wide range of variability in the absorption rate was observed in both groups of animals.. This is evident not only from the slopes of the lines in these curves but also from the values obtained for the radio activity recovered in the lymph, expressed as per cent of the radioactivity administered, over the collection period. Statistical analysis of the latter values indicated that the mean rate of absorption in the choline-deficient rats was not significantly different from that in the supple mented animals. It is possible that the failure to demonstrate an effect of dietary choline deficiency on cholesterol absorp tion in this experiment was due to the production of only a moderate degree of choline deficiency in the rats receiving the low-choline diet. On gross inspection, the livers of these animals appeared larger and more pale than did the livers of the rats receiving the choline supplement. How ever, comparison of the liver weights, expressed as per cent of the body weight, showed that the livers of the deficient group were only slightly heavier than those of the supplemented rats. Since it is known that severe choline deficiency in the rat is characterized by pale, markedly enlarged livers due to the infiltration of lipid, it is evident that the choline-deficient diet used in this experiment produced only a moderate choline deficiency. It is concluded that, under the conditions of this experiment, moderate choline deficiency did not influence the rate of absorption of cholesterol from the intestine. The second absorption experiment was performed using rats with severe choline deficiency and comparing the absorption of cholesterol in these animals and in rats receiving the same diet supplemented with choline. In order to achieve severe choline deficiency, two principal changes were made in the experimental procedure. The protein content of the diet was increased from the level of 10 per cent of the diet to 15 per cent. This was done to improve the growth of the animals and thus create a greater demand for labile methyl groups. The second change in volved placing the rats on the diets at weaning. Griffith and Wade (23, 2*+) have shown that vigorously growing young rats have a greater requirement for choline than do older animals. Evidence has been presented that these modifica tions of the previous procedures resulted in severe choline 73 deficiency, for when compared with the rats receiving the supplemented diets, the animals of the deficient group showed poorer growth and gross evidence of enlarged, pale, ; fatty livers. In addition, analysis of the fat content of the livers confirmed the evidence obtained from gross examination. Hemorrhagic kidney lesions appeared in this I group with no evidence of kidney damage among the supple- j mented rats. It seemed possible that the method used for measur-. ing absorption in the previous experiment may have been, at least in part, responsible for the variability in absorp tion observed among the animals. Verzar and McDougall (99) report that disturbances of the nervous and circulatory systems affect the absorption rate. Since both of these systems were very likely affected by the surgical treatment involved in cannulation of the lymph duct, the use of the lymph cannula was abandoned. In its place, the method of difference was employed. In this method a known amount of ! radioactive cholesterol was fed by stomach tube, and after a six hour absorption period, the animals were sacrificed and the content of radioactivity in the gastrointestinal tract recovered and measured. The difference between the i amount of radioactivity fed and the amount recovered in the' i gastrointestinal tract was designated the amount absorbed. ! 7*+ The results again revealed a high degree of variability in the absorption of cholesterol among the animals of both groups. It is possible that nervous in fluences played a part in this experiment also. A training period in which the animals are handled often and subjected to practice feedings might reduce the variability. Statistical analysis of the results indicated that,. t under the conditions of this experiment, severe choline deficiency did not affect the rate of absorption of cho lesterol from the intestine. It is concluded that failure to absorb cholesterol at a normal rate is not responsible for the observed lowering of the plasma cholesterol levels in choline deficiency. The second working hypothesis stated that choline may, in some manner, influence the destruction or conversion of cholesterol. The experimental investigation of this : supposition was based upon the report of Siperstein and Chaikoff (62) that the major route for the removal of conversion products of intravenously administered cho lesterol is by way of the saponifiable fraction of the bile. It was anticipated that any pronounced alteration in the rate of conversion of cholesterol as a result of choline deficiency might be evidenced by a difference in the ratio of saponifiable to nonsaponifiable derivatives in the bile 75 of this group as compared with choline-supplemented animals. Choline-deficient and supplemented rats were sub- , Tk jected to bile duct cannulation, and cholesterol-4—C was injected intravenously as an aqueous solution dissolved with the aid of Tween 20. Bile was collected from these animals, and measurements were made of the carbon-1^ present in the whole bile and in the saponifiable and nonsaponi- fiable fractions of the bile. The values obtained were essentially the same for the two groups of animals. Approximately 20 per cent of the administered carbon-l^f appeared in the bile in twenty-four hours, and over 90 per cent of this radioactivity was contained in the saponifiable fraction. These findings are in agreement with those reported by Siperstein and Chaikoff (62) for rats maintained on stock diets. The observation that neither the total radio activity in the bile nor the distribution of this radio activity between the saponifiable and nonsaponifiable fractions of the bile wasc influenced by dietary choline suggests that the lipotropic agent does not alter the metabolic conversion of cholesterol. These data, then, fail to support the hypothesis that an increase in the rate of conversion or destruction of cholesterol is responsible for the lowered plasma cholesterol levels in the 76 choline-deficient rat. Further interesting speculations can arise when the data just discussed are considered in the light of the fact that the liver cholesterol content is substantially elevated in choline deficiency. Using the values presented, in Table XI for animals raised on the same diets used in this experiment (Diets C and D), it can be estimated that : i the livers of the choline-supplemented rats contained approximately 20 mg. of cholesterol. The livers of the deficient rats contained 3.7 times this amount, or 7*+ mg. Now, if it were assumed that this liver cholesterol con stitutes a metabolic pool with which the injected cholesterol-^-Cl1 * is rapidly mixed and diluted before being metabolized, then the observation that the total f radioactivity in the bile was unaffected by the absence of i dietary choline would suggest that the deficient rats secreted more material in both fractions of the bile than ! did the supplemented rats. However, since Colwell (66) has shown that the con centration of cholesterol in the bile is not influenced by : choline deficiency, and since neither the volume of bile secreted nor the total solid content nor ;the relative amount of nonsaponifiable radioactivity was found to be 1 markedly different in the two groups of rats studied here, 77 it would seem likely that the liver cholesterol did not effect an appreciably greater dilution of the cholesterol- . 1 L 4— in the choline-deficient rats. This would suggest that not all the cholesterol of the liver in the choline- deficient rat is part of a common metabolic pool. Two reports in the literature support this sup position. The first is the report by Hartroft (4-0) who has shown by histological methods that as lipid accumulates in the liver cell in choline deficiency, it does so to the point that the cell can no longer hold it, and the cell ruptures. When this occurs, a portion of the liver lipid, including the cholesterol, becomes encysted outside the cell. This finding is consistent with the contention that not all the cholesterol in the liver of the choline- deficient rat is available for immediate utilization or mobilization. The second report concerns another unique aspect of the accumulated liver cholesterol in choline deficiency; this excess cholesterol evidently does not depress the incorporation of acetate into the cholesterol molecule (61). On the other hand, Frantz and co-workers have shown that in rats which have varying levels of liver cholesterol as a result of feeding this sterol together with stock diet for varying lengths of time, the extent of incorporation of acetate into cholesterol is inversely 78 related to the cholesterol content of the liver (102), Such a difference in the effect of the accumulated hepatic cholesterol again may be interpreted as an indication that only a portion of the cholesterol of the liver is meta- bolically active. Turning next to the third working hypothesis, it was postulated that choline is required for the proper transfer of cholesterol from the liver to the plasma. Since it is known that either ligation of the bile duct or injection of Triton in rats fed normal diets promotes a rise in plasma cholesterol as the result of transfer of cholesterol from the liver to the plasma (719 85)* an examination was made of the effect of these two treatments on plasma lipids in choline-deficient rats. The levels of cholesterol and phospholipids in the plasma of deficient rats before and after bile duct ligation and Triton administration were compared with the corresponding levels in choline-supplemented rats. Choline was found to have a pronounced effect on the hypercholesterolemia which resulted from both bile duct ligation and Triton administration. Although increases occurred in the plasma cholesterol concentration of both choline-deficient and supplemented rats, the levels remained lower in the deficient animals* The effects of these two treatments on choline-deficient rats have not been reported in the literature, but the plasma cholesterol concentrations of the choline-supplemented animals are comparable to those reported for rats fed stock diets (72, 73, 7*f, 103). It is particularly interesting to note that these experiments demonstrated that it is possible to produce hypercholesterolemia in choline-deficient rats. The extent of the rise in plasma cholesterol appears to be limited by the availability of choline. One might expect that the higher content of cholesterol in the livers of the deficient rats would lead to higher levels of cholesterol in the plasma of these animals than in those receiving the choline supplement. This did not occur, however, Such a finding again points to the previously mentioned possi bility that not all the cholesterol in the liver of the choline-deficient rat is available for immediate mobiliza tion or metabolism. The effect of choline on the levels of phospho lipids in the plasma was not as distinct as for cholesterol Increases in phospholipids occurred in both groups after bile duct ligation and Triton administration. The dif ference between the plasma levels in the deficient and supplemented rats was found by statistical analysis to be highly significant only after Triton injection. As in the case of cholesterol, the higher levels of phospholipids were found in the animals receiving dietary choline. Again, since the effects of neither bile duct ligation nor Triton injection on the blood lipids of choline-deficient rats have been reported in the literature, only those phospholipid values obtained for the supplemented rats are subject to comparison with the results of other workers. The concentrations of phospholipids in this group fall within the ranges reported by Friedman and Byers for Long- Evans rats both before and after either bile duct ligation or Triton administration (73, 103)* Their animals were fed stock rations. After Triton treatment the levels of total lipids were found to be higher in both groups of rats than the generally accepted values for normal rats. Significantly lower levels were found in the deficient rats when compared with the supplemented group. Friedman and Byers have reported values for total lipids as high as 2790 mg. per 100 ml. within twenty-four hours after Triton injec tion, and these workers also report that the plasma tri glycerides continue to rise for as long as thirty-six hours after the injection of the detergent (10*+). The total lipid levels reported here for the choline-supplemented rats are nearly three times the values reported by Friedman 81 and Byers• Such excessively high values may be related either to the fact that these samples were obtained at seventy-two hours after Triton administration or that a different strain of rat was used. It is apparent from the hypercholesterolemia which resulted after bile duct ligation and after Triton injec tion that a transfer of cholesterol from the liver to the plasma had occurred in both groups of rats, The observa tion of lower plasma cholesterol levels in the deficient rats under these conditions supports the hypothesis that choline is required for this transfer. The findings suggest that the lower cholesterol levels which occurred in the plasma of the deficient rats before either of these treatments may also have been due to impaired transfer of cholesterol from the liver to the plasma. The possibility cannot be excluded that the dif ferences observed in plasma cholesterol after both treat ments may, at least in part, be the result of an unequal rate of cholesterol synthesis in the two groups of rats. Increases in the incorporation of isotopic acetate into cholesterol are known to occur following either bile duct ligation or Triton injection in stock-fed rats (72, 7*+). The extent of such an increase in synthesis may be limited in the choline-deficient rat. It is possible, of course, 82 i that alterations in both synthesis and transfer are respon- i sible for the observed effects after the two treatments. t i * With respect to the difference in plasma cho- j lesterol before either bile duct ligation or Triton t injection, it is known that the choline-deficient rat is able to incorporate acetate into cholesterol at the same j ; rate as the supplemented rat (61). Thus, synthesis is | eliminated as a factor responsible for the lowered plasma i ! levels in this instance. Impaired transfer of cholesterol r I from the liver to the plasma remains as a distinctly ! | possible explanation for the effect of choline on the ! plasma cholesterol concentration. i 1 Embodied in the third working hypothesis was the t ! proposition that choline may be required for the formation i j of compounds necessary for carrying cholesterol in the j plasma. Such compounds actually constitute a vehicle for I the transfer of cholesterol from the liver to the plasma. ! The data obtained from the experiments with bile duct ligation and Triton injection support this postulate. The j finding that choline deficiency influenced the concentra- , tion not only of cholesterol but of phospholipids and total I lipids as. well, suggests that this lipotropic agent is 1 | implicated in the formation of lipoprotein complexes. The i ! ways in which choline is involved in the synthesis of 83 lipoproteins may be several and complex, bat the simplest consideration is that it may be required for the formation of the lecithin portion of the complete lipoprotein complex. In choline deficiency the supply of choline for incorporation into lecithins is probably limited, and without this necessary portion of the lipoprotein the whole complex cannot be formed. Thus without the formation of the complex, cholesterol cannot enter the plasma, but instead remains in the liver. The data obtained here do not provide information to evaluate this consideration, and it is offered as an additional hypothesis. Viewed as a unit, the results obtained from the experiments presented here indicate the following: decreased absorption or increased destruction of cho lesterol can probably be eliminated as factors responsible for the lower plasma cholesterol concentration in the choline-deficient rat; the data support the hypothesis that choline deficiency impairs the transfer of cholesterol from the liver to the plasma. | SUMMARY | 1 I I An investigation has been made of some possible j • i ( 1 mechanisms through which dietary choline may influence I the concentration of plasma cholesterol in the rat. ! * i I The possibility that the lowered plasma choles- ; i * terol levels observed in choline deficiency may be due to j ! impaired absorption of cholesterol from the intestine has 1 i been examined by two methods. In the first experiment, ! I cholesterol-1 *--^1 * was administered by stomach tube to j ! i \ choline-deficient and choline-supplemented rats with 5 r ! thoracic lymph duct fistulas. The radioactivity contained i j in the lymph collected for a total period of twelve hours i was compared for the two groups of animals. Although j individual values varied widely in both groups, dietary choline was found to have no significant effect on the rate of cholesterol absorption as measured by this method.| In the second absorption experiment, cholesterol- \ 1 4 - 4—C was administered by stomach tube to choline- j deficient and supplemented rats, and after a six-hour ; I absorption period the quantity of radioactive nonsapon- | ; ifiable material still present in the gastrointestinal , i ' tract of each animal was determined. The amount of i I cholesterol absorbed, as calculated from the difference 8? between the radioactivity fed and that recovered in the intestinal contents, was found not to be significantly different in the two groups of rats. The results of these two experiments failed to uphold the hypothesis that the lower plasma cholesterol levels found in choline-deficient rats are the result, of decreased absorption of cholesterol from the intestine. The possibility that dietary choline may influence the plasma cholesterol content of the rat by altering the rate of conversion of cholesterol to biliary excretion products was next investigated in the following manner. Choline-deficient and supplemented rats were provided with bile duct cannulas and injected intra venously with cholesterol-^-C^. Bile collected from the two kinds of rats was found to be substantially the same in the following respects: (1) the volume of bile secreted, (2) the concentration of biliary solids, (3) the fraction of administered C*^ appearing in the whole bile, and (*+) the fraction of administered present in the^saponifiable portion of the bile. These results do not support the hypothesis under consideration, namely, that the lower plasma cholesterol levels in choline deficiency were caused by an increased rate of conversion or destruction of cholesterol. 86 On the basis of the data just described, it is suggested that only a portion of the cholesterol present in the liver of the choline-deficient rat takes part in* the metabolic pool of cholesterol. The transfer of cholesterol from the liver to the plasma constituted the third mechanism investigated. Since either bile duct ligation or the injection of Triton WR-1339 is known to be followed by pronounced increases in the rate of this transfer, groups of choline-deficient and control rats were subjected to these two treatments. Measurements were made of plasma cholesterol and phospho lipids in the two groups of rats before and after the treatments. Increased transfer of cholesterol into the plasma was evidenced by hypercholesterolemia in both choline- deficient and choline-supplemented rats as a result of either bile duct ligation or Triton injection. However, the plasma cholesterol concentration remained signifi cantly lower in the choline-deficient animals than in the control group. Increases were observed in plasma phospholipid levels in both choline-deficient and choline-supplemented rats after either treatment. Significant differences were observed between the two groups of rats only after Triton 87 : administration. | i After Triton injection, plasma free cholesterol and; i total lipid concentrations were found to be higher in both | groups of rats than the generally accepted values for j normal animals of this species. j An interpretation of these results in terms of the j possible effects of choline on the formation of lipo- j protein complexes is presented. j i The work presented in this dissertation adds to thej knowledge of the relative importance of three processes in ! i the metabolism of cholesterol which conceivably could be ' influenced by dietary choline and which, in turn, could bring about changes in the level of cholesterol in the , plasma. The data indicate that neither the absorption of i cholesterol from the intestine nor the conversion of cho- j lesterol to biliary excretion products appears to be ; affected by dietary choline deficiency. In view of these ! i i findings and the observed effect of choline on the transfer) i i of cholesterol from the liver to the plasma, it appears ' probable that an impairment of this transfer may be responsible for the lower plasma cholesterol concentrations j i observed in choline-deficient rats. BIBLIOGRAPHY BIBLIOGRAPHY 1. 2. 3. if. 5. 6. 7. 8. 9. 10. 11* 12. 13. l*f. 15. 16. Best. C. H., and Ridout, J. H., Ann. Rev. Blochem.« 8, 3^9 (1939). Griffith, W. H., J. Nutrition. 22, 239 (19^1). Griffith, W. H., Biol. Symposia. 193 (19*+1). Griffith, W. H., in Evans, E. A., The Biological Action of the Vitamins. Univ. of Chicago Press, Chicago Frame, E. G., Yale. J. Biol. Med.. 1M-. 229 (19^2). Best. C. H.. and Lucas. C. C.. Vitamins and Hormones. 1, 1 (19^3). McHenry, E. ¥., and Patterson, J. M., Physiol. Rey., 2jt, 128 (19Mf). Jukes, T. H., Ann. Rev. Biochem.. 16. 193 (19*f7). I Harris, R. S., Griffith, W. H., Myc, J. F., | Hartroft, W. S., Lucas, C. C., and Best, C. H., ■ Vitamins (N. 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Creator Rice, Elmer H (author)

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

Degree Doctor of Philosophy

Degree Program Biochemistry

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-2077

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