LYMPH LIPID CLASS DISTRIBUTION OF RATS FED A THREONINE IMBALANCED DIET CONTAINING 30% CORN OIL OR 30% COCOA BUTTER By V‘ Constance M: Parks A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition 1970 ACKNOWLEDGMENTS I wish to thank the following people who have helped me in my graduate studies and in the completion of this thesis: Dr. Dr. Dr. Dr. Dr. Mrs. Mrs. Modesto G. Yang, my major professor Dorothy A. Arata Werner G. Bergen Loran L. Bieber Dena C. Cederquist Frances Murray Gizis Jenny Taylor Johnson LITERATURE REVIEW. . . . . . TABLE OF CONTENTS PAGE DEVELOPMENT OF THE CONCEPT OF AMINO ACID IMBALANCE. REGULATION OF FAT METABOLISM BY THREONINE. . . . INFLUENCE OF DIETARY FAT ON FATTY LIVERS. . . . LIPID ABSORPTION AND CHYLOMICRON FORMATION. INTRODUCTION. . . . . . PART I. LIVER FAT ACCUMULATION IN ADULT RATS FED 5% CASEIN, THREONINE IMBALANCED OR THREONINE SUPPLEMENTED DIETS WITH 30% CORN OIL OR COCOA BUTTER AS THE FAT SOURCE. . . . . . . . SECTION A. Determination of the time at which fat accumulation would be at a maximum in the livers of adult rats fed a threonine im— balanced diet containing 5% casein and 30% corn oil. . . . . . . . . . . . . . . . . Experimental procedure. . . . . . . . . . . . . . Results and discussion. . . . . . . . . . . . . . SECTION B. Fat accumulation in the livers of adult rats fed threonine imbalanced or threo— nine supplemented diets containing 30% corn oil or cocoa butter for 2 weeks. . . Experimental procedure. . . . . . . . . . . . . . Results and discussion. . . . . . . . . . . . . . SECTION C. Fat accumulation in the livers of adult rats fed threonine imbalanced or threo- nine supplemented diets containing 30% corn oil or cocoa butter for 4 weeks. . . Experimental procedure. . . . . . . . . . . . . . Results and discussion. . . . . . . . . . . . . . 2 9 13 20 30 32 33 33 33 34 .34 34 35 .35 .35 TABLE OF CONTENTS (CONT'D.) PAGE PART 2- ENDOGENOUS LIPID OF LYMPH FROM FASTED RATS PREVIOUSLY FED THREONINE IMBALANCED OR THREONINE SUPPLEMENTED DIETS WITH 30% CORN OIL OR COCOA BUTTER AS THE FAT SOURCE. . . . . .42 Experimental procedure. . . . . . . . . . . . . . . 43 ReSUlts. C C C O O O O O C O O O O O O O C O O O C O 45 Discussion. 0 O O O O O O O O O O O O 3. O O O O O C .52 PART 3. EFFECT OF FORCED FEEDING CORN OIL OR COCOA BUTTER ON THE LYMPH LIPID OF FASTED RATS PREVIOUSLY FED THREONINE IMBALANCED OR THREONINE SUPPLEMENTED DIETS WITH 30% CORN OIL OR COCOA BUTTER AS THE FAT SOURCE. . . . . .57 Experimental procedure. . . . . . . . . . . . . . . .58 ReSU-lts. O O O O O O O O O O O O O O O O O O O O O O 58 Discussion. . . . . . . . . . . . . . . . . . . . . 74 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . . 133 ii LIST OF TABLES TABLE PAGE PART 1 1. Fatty acid composition of corn oil and cocoa butter. 38 2 0 Diet compOSi-tion. O O O O O O O O O O O O O O I O O 39 3. Percent fat in the livers of adult rats fed a threo— nine imbalanced diet containing 5% casein and 30% corn oil for 2, 3 and 4 weeks as compared to normal liver fat in adult rats fed a standard laboratory chow diet. . . . . . . . . . . . . . . . . . . . . . 4O 4. Percent fat in the livers of adult rats fed threo- nine imbalanced or threonine supplemented diets containing 30% corn oil or cocoa butter for 2 weeks. 40 5. Percent fat in the livers of adult rats fed threo— nine imbalanced or threonine supplemented diets‘ containing 30% corn oil or cocoa butter for 4 weeks. 41 PART 2 6. Endogenous lymph lipid recovery. . . . . . . . . . . 54 7. Percent class distribution of endogenous lymph lipid 54 8. Percent fatty acid composition of endoqenous lymph lipid O O O O O O O O O O O O O O O O I O O O O O O O 55 9. Weight of endogenous fatty acids. . . . . . . . . . 56 PART 3 10. Lymph lipid recovered after forced feeding corn oil. 80 ll. Lymph lipid recovered after forced feeding cocoa bUtter. O O O O C O O O O O O O O O I O O O O O O O 81 12. Percent class distribution of lymph lipid recovered after forced feeding corn oil. . . . . . . . . . . . 82 13. Percent class distribution of lymph lipid recovened after forced feeding cocoa butter. . . . . . . . . . 83 14. Percent saturated fatty acids recovered from lymph lipid following corn oil forced feeding. . . . . . . 84 15. Percent saturated fatty acids recovered from lymph lipid following cocoa butter forced feeding. . . . . 85 iii TABLE l6. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. LIST OF TABLES (CONT'D) PAGE PART 3 (CONT'D) Percent fatty acids in the triglyceride fraction of lymph lipid after forced feeding corn oil. . . . . . 86 Percent fatty acids in the triglyceride fraction of lymph lipid after forced feeding cocoa butter. . . . 87 Percent fatty acids in the cholesterol ester frac- tion of lymph lipid after forced feeding corn oil. . 88 Percent fatty acids in the cholesterol ester frac— tion of lymph lipid after forced feeding cocoa butter90 Percent fatty acids in the phospholipid fraction of lymph lipid after forced feeding corn oil. . . . . . 91 Percent fatty acids in the phospholipid fraction of lymph lipid after forced feeding cocoa butter. . . . 92 TGFA after forced feeding corn oil. . . . . . . . . 94 TGFA after forced feeding cocoa butter. . . . . . . 95 CEFA after forced feeding corn oil. . . . . . . . . 96 CEFA after forced feeding cocoa butter. . . . . . . 98 PLFA after forced feeding corn oil. . . . . . . . . 99 PLFA after forced feeding cocoa butter. . . . . . . 100 iv FIGURE PART 3 1. Total lymph lipid after forced feeding corn oil. 2. Triglyceride fraction of lymph lipid after forced feeding corn Oil 0 O O O O O O O O O O I O O O O O 3. Cholesterol ester fraction of lymph lipid after forced feeding corn Oil. . . . . . . . . . . . . 4. Free cholesterol fraction of lymph lipid after forced feeding corn oil. . . . . . . . . . . . . 5. Phospholipid fraction of lymph lipid after forced feeding corn oil . . . . . . . . . . . . . . . . 6. Total lymph lipid after forced feeding cocoa butter 7. Triglyceride fraction of lymph lipid after forced feeding cocoa butter. . . . . . . . . . . . . . . 8. Cholesterol ester fraction of lymph lipid after forced feeding cocoa butter. . . . . . . . . . . 9. Free cholesterol fraction of lymph lipid after forced feeding cocoa butter. . . . . . . . . . . 10. PhOSpholipid fraction of lymph lipid after forced feeding cocoa butter. . . . . . . . . . . . . . . 11. Percent saturated fatty acids of the triglyceride fraction of lymph lipid after forced feeding corn 011 O O O O O I O O O O O O O O O O O O O O O O O 12. Percent saturated fatty acids of the cholesterol ester fraction of lymph lipid after forced feeding corn Oil 0 O O C O O O O O O O O O O O O O O O O O 13. Percent saturated fatty acids of the phOSpholipid fraction of lymph lipid after forced feeding corn 010 O O O O O C O O O O O O O O O O O O O O O O O 14. Percent saturated fatty acids of the triglyceride fraction of lymph lipid after forced feeding cocoa bUtter. C O O O O O O O O O O O O O O I O O O O O 15. Percent saturated fatty acids of the cholesterol LIST OF FIGURES ester fraction of lymph lipid after forced feeding cocoa butter 0 O O O O O C O O O O O O O O O O O 0 PAGE 102 103 104 104 105 106 107 108 108 109 110 111 112 113 114 FIGURE 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. LIST OF FIGURES (CONT'D) PART 3 (CONT'D) Percent saturated fatty acids of the phospholipid fraction of lymph lipid after forced feeding cocoa bUtter. O O C O O C C O C C O O O O O O O O O O C Group Group Group Group Group Group Group Group Group Group Group Group Group Group Group GrOUp Group Group GrOUp Group Group GrOUp Group Group I, TGFA after forced feeding corn oil. . . . II, TGFA after forced feeding corn oil. . . III, TGFA after forced feeding corn oil. . . IV, TGFA after forced feeding corn oil. . . I, TGFA after forced feeding cocoa butter. . II, TGFA after forced feeding cocoa butter. III, TGFA after forced feeding cocoa butter. IV, TGFA after forced feeding cocoa butter. I, CEFA after forced feeding corn oil. . . . II, CEFA after forced feeding corn oil. . . III, CEFA after forced feeding corn oil. . . IV, CEFA after forced feeding corn oil. . . I, CEFA after forced feeding cocoa butter. . II, CEFA after forced feeding cocoa butter. III, CEFA after forced feeding cocoa butter. IV, CEFA after forced feeding cocoa butter. I, PLFA after forced feeding corn oil. . . . II, PLFA after forced feeding corn oil. . . III, PLFA after forced feeding corn oil . . IV, PLFA after forced feeding corn oil. . . I, PLFA after forced feeding cocoa butter. . II, PLFA after forced feeding cocoa butter. III, PLFA after forced feeding cocoa butter. IV, PLFA after forced feeding cocoa butter. vi PAGE 115 116 116 117 118 119 119 120 120 121 121 .122 123 124 124 125 125 126 127 128 129 130 131 131 132 LITE RATURE REVIEW DEVELOPMENT OF THE CONCEPT OF AMINO ACID IMBALANCE Fatty livers were produced experimentally by Best and Huntsman (1932) in rats fed diets high in saturated fats. The accumulation of fat in the livers was prevented by the addition of choline to these high fat diets, thus demonstra— ting the lipotropic activity of choline. In later studies, Best and Huntsman (1935) found that liver fat accumulation was not regulated entirely by choline since when casein was added to diets low in choline, there was a "choline—like action”. The authors thus suggested that some of the amino acids of casein may be choline precursors. In other studies, Tucker and Eckstein (1937) investigated the possibility that compounds which act as precursors to choline i vivo may prevent liver fat accumulation due to choline deficiency. When .5% methionine was added to a 5% casein. 40% fat diet, liver fat levels were reduced to 11% as compared to unsupplemented control values of 20%. By supplementing low choline diets with deuterium-labeled methio— nine, du Vigneaud gt a1. (1941) showed that the methyl groups of methionine are used for synthesis of choline by the rat. Later the methionine—Sparing action of choline was prOposed by Engel (1948) and Alexander and Engel (1952). The data of Best and Huntsman (1935) suggested a lipo— trOpic activity of dietary protein. In their work, rats were prefed a hypolipotrOpic diet of mixed grains and 40% fat for three weeks. At the end of the feeding period the diet was replaced by sucrose and fed for thirteen days. The 3 replacement of the diet with sucrose resulted in a marked elevation of liver fat levels (14 vs. 23%, respectively). Adding choline (75 mg/day) to the sucrose caused the reduc— tion of liver fat to 5%. Supplementing the sucrose with ca— sein (20% of the diet) reduced liver fat levels but to a less— er degree than when the sucrose was supplemented with choline. Although the data suggested a lipotropic effect of protein, the authors postulated that proteins per fig were not lipotro— pic and the effect of casein in reducing liver fat levels in their study was due to impurities such as betaines in the protein. Channon and Wilkinson (1935) showed that proteins actually did have lipotropic activity since rats fed a 5% casein diet developed fatty livers (13% of wet weight of liver) but when the diet contained 20% casein the liver fat level was reduced to 7%. When casein was increased to 50% of the diet, the liver fat level was further reduced to 6%. The authors postulated that proteins had a lipotrOpic effect and that this effect was in proportion to the level of protein in the diet. More recent experiments have confirmed the lipotropic action of increasing levels of protein (Harper 3; 31., 1953 a, b). Beeston §E_al. (1936) showed that this lipotropic activity of protein could not be due to precursors for cho— line synthesis in the protein molecule because rats fed a 5% casein diet supplemented with choline had twice as much liver fat as rats fed a 30% casein diet. These results with protein led to studies of the lipotropic action of various amino acids. Beeston and Channon (1936) found that rats 4 fed diets containing 5% casein and 40% fat develOped fatty livers; the addition of cystine, glutamic acid, aspartic acid, serine, glycine and phenylalanine to the diet was not effec- tive in preventing liver fat accumulation. Furthermore, cystine exerted an "anti—lipotropic” effect and promoted the accumulation of fat in the liver. This increase in liver fat could be reversed by feeding additional casein. These findings were confirmed by Tucker and Eckstein (1937) using similar dietary conditions. Much work has been reported to show the influence of the amino acid threonine on liver fat accumulation and growth in rats. In initial studies by Singal gt gt. (1948), threonine was found to be limiting in casein diets for growth. Growth of rats was not normal when a 9% casein diet supplemented with histidine, valine, threonine and lysine or nicotinic acid alone was fed. Further experiments by these workers showed that of the four amino acids studied, only threonine produced normal growth when added to the 9% casein diet supplemented with tryptOphan or nicotinic acid. The authors noted that the livers from the threonine deficient rats appeared to be fatty but fat analysis was not done. In a later study, Singal gt gt. (1949) found that the addition of threonine to a 9% casein diet supplemented with cystine, choline and tryptOphan reduced the liver fat level in rats from 16.0 to 6.6%. The lipotropic activity of threo— nine was not due to an increased food consumption since there was 5.9 and 14.4% liver fat respectively without threonine supplements when the animals were pair fed. Further studies 5 with 9% casein diets demonstrated that only the L isomer of threonine prevented fatty livers and stimulated growth of rats on a deficient diet (Singal gt gt., 1953 b). The concept that one amino acid may be limiting for growth and another for liver fat deposition was established by Litwack, Hankes and Elvehjem (1952) when rats were fed a 9% casein diet supplemented with either tryptOphan or threo— nine. TryptOphan was found to be most limiting for growth and threonine the most limiting for liver fat accumulation. Singal gt gt. (1953 a) studied the effect of a threonine deficiency on liver fat accumulation. They found that a threonine devoid diet did not cause fatty livers and that maximum liver fat accumulation did not occur until the diet contained .7% DL—threonine; when threonine was increased to 1.1%, the liver fat level was normal. Large doses of choline reduced liver fat levels in these rats, but not as effective— ly as threonine. The authors concluded that there were two separate yet related phenomenon in liver fat production: (1) a primary methyl group or choline deficiency in which threonine was without effect and (2) a primary threonine deficiency in which choline was effective only when supple- mented at very high levels. The histological studies of Shils and Stewart (1954) support Singal's theory on the two types of fatty livers: rats were fed various amounts of corn and casein with and without additional choline. The imbalanced corn protein caused a periportal distribution of liver fat in the lobules while the choline deficiency resulted in the centrolobular distribution of liver fat. The relative effectiveness of methionine, choline, pro— tein, and threonine in preventing liver fat accumulation was investigated by Harper gt gt. (1954 a, b) and Lucas and Rid— out (1955). They demonstrated that the lipotropic action of methionine was mediated through the synthesis of choline. However, in a low protein diet, threonine exerted an addition— al effect, indicating its action was distinct from that of choline. The concept of amino acid imbalance was introduced by Harper gt gt. (1954 c): factors other than the absolute amount of amino acids in a protein can affect the lipotrOpic activity. When rats were fed a 9% casein diet supplemented with choline and tryptOphan, liver fat levels were normal but addition of .l% methionine caused liver fat to accumulate. The liver fat level could be reduced by supplementing threo— nine to the diet, or by pair-feeding the methionine—supple— mented rats with a control group. The addition of methio— nine caused increased protein synthesis, thus precipitating the threonine deficiency. The authors concluded that the proportion as well as the absolute amounts of amino acids in a protein affects liver fat deposition in a low protein diet. Fatty livers were thus produced by supplementing a low pro— tein diet with the most limiting amino acid or acids thus causing a deficiency of the second most limiting amino acid, usually threonine (Harper gt gt., 1954c and Winje gt gt., 1954). Deshpande, Harper and Elvehjem (1958) created a second 7 type of imbalance by supplementing the diet with the second most limiting amino acid or acids in the diet. They fed rats 6% fibrin diets in an attempt to determine the fate of the amino acids not used for protein synthesis. They found that four amino acids, leucine, isoleucine, valine and histidine, were all about equal and first limiting for growth. Addition of .4% methionine and .6% phenylalanine, the second most limiting amino acids, to the 6% fibrin diets caused a growth retardation that was reversed with the addition of all four of the most limiting amino acids. Morrison and Harper (1960) confirmed these results using 8% casein diets supplemented with L-cystine or DL-methionine. Adding .36% DL—threonine to the diet caused a depression in the growth rate which could be corrected by addition of either niacin or tryptOphan. These experiments confirmed the earlier concept demonstrated by Litwack, Hankes and Elvehjem (1952), that one amino acid might be limiting for growth and another for fat deposition. On the other hand, Harper (1959) found that under certain dietary conditions, threonine can be limiting for both growth and liver fat production. By using a 6% casein diet supple— mented with 4% of an amino acid mixture which contained tryptophan but no threonine, he showed that between .025 and .050% of L-threonine was required to overcome the growth de— pression and the fatty livers caused by the deficient diet. He concluded that the threonine originally present in the diet was made unavailable because of the imbalance of amino acids. Harper gt gt. (1954d) showed that the deposition of fat 8 in the livers of threonine imbalanced rats appeared to be a function of the age of the animal since the amount of fat in livers of weanling rats fed a 9% casein diet containing methionine and choline decreased gradually as the animals matured and the protein requirement decreased. Liver fat reached a peak of 30—40% after two weeks on the diet and then tapered off until after ten weeks the liver fat level was 15%. Harper gt gt. (1954 d) found the greatest accumula- tion of fat in the livers of adult rats after two weeks when the threonine imbalanced diet was reduced from 9 to 5% casein. The addition of either threonine or glycine to the 5% casein diet reduced the level of liver fat. Thus Harper gt gt. (1954 a) postulated that three groups of dietary factors must be considered in evaluating the lipotropic activity of proteins: (1) choline which is involved in phOSpholipid metabolism and can be partially replaced by methionine; (2) threonine and/or other essential amino acids which are re— quired for normal lipid metabolism; and (3) glycine, serine, and betaine which act in a more non—specific way by sparing essential compounds. REGULATION OF FAT METABOLISM BY THREONINE Rats fed threonine imbalanced diets develOp fatty livers but little is known about this type of fat infiltration: it may be that inadequate protein synthesis in the deficiency state may cause damage to the enzyme systems related to lipid metabolism and simultaneously lead to an increased content of fat in liver cells. In rats fed a 9% casein diet supple— mented with choline, tryptophan and methionine, that is, a threonine imbalanced diet, the activities of the mitochon— drial enzymes, succinic oxidase and choline oxidase, were higher in liver homogenates from threonine imbalanced as com— pared with control animals, while endogenous respiration and the activities of the cytOplasmic enzymes xanthine oxidase and tyrosine oxidase, were lower. The differences in endogen- ous respiration between the basal and threonine imbalanced groups were presumed to reflect a metabolic difference in oxidative pathways in the two groups (Harper gt gt., 1953 c). When a double amino acid imbalance was produced in rats by restricting tryptOphan as well as threonine in a 9% casein diet, the enzyme changes observed on the threonine imbalanced diet did not occur, but fat accumulated in the livers to the same extent as with the single deficiency (Arata gt gt., 1954). The marked reduction in endogenous oxidation seen in threonine imbalanced fatty livers, and the known involvement of the pyridine nucleotide co—enzymes in oxidation-reduction reactions (White, Handler & Smith, 1964) promoted investiga— tions to determine the activity of these compounds in the 9 10 livers of rats fed threonine imbalanced diets. Arata gt gt. (1956) reported a decrease in the concentration of pyridine nucleotides in livers from threonine imbalanced rats. Since the pyridine nucleotides are essential co-factors in fat oxidation, a deficiency in these co—enzymes could be a major factor in liver fat accumulation resulting from feeding a threonine imbalanced diet. Carroll gt gt. (1960) confirmed the fact that the effect of threonine imbalance on fatty livers varies with the age of the animal (Harper gt gt., 1954 d) and attempted to corre— late changes in certain enzyme systems and the accumulation of liver fat with time. Weanling rats developed the maxi— mum amount of liver fat after twenty—four days on the threo— nine imbalanced diet; after six weeks, liver fat levels had fallen to approximately half of the maximum. Furthermore, the activities of xanthine oxidase and malic dehydrogenase, varied with time. The activity of these enzymes decreased in the imbalanced animals for nineteen days after which the activity increased. It appeared that fat could not be mobil— ized out of the liver until after recovery of the enzyme systems. Arata, Carroll and Cederquist (1964) found changes in other enzyme systems as well. Nine percent casein threo— nine imbalanced diets were fed to weanling rats for two, four and six weeks and compared to controls supplemented with threonine. In the imbalanced rats, liver fat was highest after two weeks. The activity of the fatty acid oxidase system was depressed during this period in the imbalanced animals. The enzyme activity increased to normal as the 11 liver fat decreased to near normal in the following weeks. The levels of labile phOSphorus from adenosine diphosphate and adenosine triphosphate were lower than control values. The activity of the DPN—cytochrome c reductase system, an enzyme of the electron transport chain, was depressed in imbalanced animals after two weeks. Methfessel gt gt. (1964) found no change in the activity of malic dehydrogenase, but the activity of the enzyme system which catalyzes the DPN— mediated oxidations of TPNH by cytochrome c was lower in rats with fatty liver due to feeding choline— and threonine— deficient diets. Thus both Arata and Methfessell and co— workers presented data suggesting that some phase of the electron tranSport system may be altered by a threonine im— balance. Yoshida and Harper (1960) fed rats the same diet as Arata gt gt. and at the time of maximum liver fat accumula— tion, injected acetate-L—C14 and palmitate-l—C14 intraperi— toneally. The incorporation of G14 into body fat and neutral fat and phospholipid of liver were significantly greater in rats fed the threonine imbalanced than the threonine supple- mented diet, suggesting that the liver fat accumulated as a result of increased synthesis of fat. Viviani gt gt. (1966) studied lipid metabolism in fatty livers of rats fed a low protein rice diet deficient in and supplemented with lysine and threonine. In agreement with Yoshida and Harper, the excess in the fatty livers consisted primarily of neutral lipid, there being an increase in both glycerides and to a smaller extent cholesterol ester. The 12 amount of total phOSpholipid, proteolipid protein and plas- malogens was not changed. The effect of lysine and Lhreoninv deficiency on incorporation of acetate—Cl4 into liver neutral fatty acids was unchanged in the livers of deficient animals however, there was a pronounced decrease in incorporation of Cl4 into phospholipid fatty acids in the deficient rats. This finding, and the fact that the plasma of deficient rats had more free fatty acids and less esterified fatty acids and phospholipid in the plasma would suggest a decrease in trans— port of lipid out of the liver. This would contradict the previously mentioned theory of decreased oxidation of liver fat with a threonine imbalance. Thus, metabolic defects that singly or in combination might account for the pathogenesis of fatty liver are (Viviani gt gt., 1966) (1) increased triglyceride synthesis in the liver; (2) decreased fatty acid oxidation in the liver; (3) increased uptake by the liver of arterial blood lipids (chylomicrons and free fatty acids); and (4) decreased secretion of lipoproteins from the liver into the bloodstream. INFLUENCE OF DIETARY FAT ON FATTY LIVERS Channon and Wilkinson (1936) correlated the chemical composition and physical prOperties of a dietary fat with liver fat accumulation during choline deficiency when they fed weanling rats a 5% casein, 40% fat diet in which butter fat, beef fat, palm oil, coconut oil, olive oil, and cod liver oil were used as fat sources. The highest liver fat levels were produced by rats fed butter fat while those fed cod liver oil had the lowest levels (30.7 vs. 7.2% of wet weight of liver). The authors concluded that liver fat accumulation depended directly on the intake of C14 to C18 saturated fatty acids. Addition of choline prevented the fat accumulation; thus it was assumed that choline partici— pated in a process of desaturation. Channon gt gt. (1942) confirmed the assumption that with a choline deficient diet the extent of fat accumulation is related to the proportion of saturated fatty acids from carbon fourteen to carbon eighteen. Later, studies by Benton gt gt. (1956) confirmed the findings of Channon gt gt. that liver fat accumulation was influenced by the amount of saturated fat in a low protein diet deficient in choline. They found that when rats were fed 9% casein, 20% fat diets containing different diet fats, the liver fat levels were high when butter fat or lard was fed while the liver fat levels were low after corn oil or margarine was fed. The effects of the fats on liver fat levels were increased when the level of protein in the diet 13 14 was decreased and when choline was omitted from the diet. When the fatty acids of butter were isolated and fed as gly— cerides, liver fat levels were similar to those found after feeding butter fat. However, the solid fatty acid fraction of butter caused a much greater accumulation of liver fat than did the liquid fatty acid fraction. A higher level of choline was required to lower liver fat levels in rats fed 30% butter fat rather than 30% corn oil (Benton gt gt., 1957). Rats fed butter fat required .15% choline to reduce liver fat levels to 16.6% while those fed corn oil required .12% choline to reduce liver fat levels to 16.3%. Supplements of cystine, methionine, tryptophan or all three amino acids did not affect these values. The authors concluded that the requirement of choline varies with the type of dietary fat fed. Harper gt gt. (1953 a, 1954a) found more fat in the liver when butter fat rather than corn oil was fed to rats on a 10% casein diet, deficient in choline and threonine. The result was the same whether the level of fat in the diet was 5% or 20%. When the diet was supplemented with threonine, the liver fat was reduced to a greater degree in the rats fed corn oil than in those fed butter fat. Thus the nature of the fat in the diet appeared to influence the effect of a threonine deficiency on fat metabolism. In addition to the degree of saturation and chain length of a fatty acid, Morris gt gt. (1965) showed that the degree of hydroqenation of a dietary fat influenced the amount of fat that accumulated in livers of rats fed threonine 15 imbalanced diets. They fed weanling rats 9% casein, 30% fat diets deficient in threonine and choline. When corn oil was the fat source in the diet, liver fat levels were high but when corn oil was replaced with hydrogenated vegetable oil, liver fat levels were reduced; when corn oil was hydrogenat— ed, liver fat levels were also reduced. Since hydrogenated corn oil and un—hydrogenated corn oil have similar carbon chain lengths and hydrogenated corn oil contains 45% trans acids, the authors attributed the lipotropic action of hydro— genated corn oil to the presence of the trans fatty acids formed during the hydrogenation process and then postulated that these unnatural trans isomers may mediate a metabolic path different from that taken by natural fats and oils. The evidence presented was inconclusive however, since in addition to changes in the geometry of the double bonds to form the trans acids, there was also an increase in the amount of saturated fatty acids. To determine whether the ability of the hydrogenated fat to lower liver fat was the result of chemical treatment, Woolcock (1967) used the same 9% casein, threonine imbalanced diet as used by Morris. The fat sources (30% of the diet) were untreated natural lard, commercially treated lard in which the fat had been molecularly rearranged without changing the fatty acid composition, hydrogenated corn oil and corn oil. The commercially treated lard had no effect on liver fats as compared to either corn oil or natural lard controls. Hydrogenated corn oil again reduced liver fats. Since chemi— cal treatment of lard had no effect on liver fat levels, and 16 since the major differences between the fatty acid composi— tion of chemically treated lard and hydrogenated corn oil was the geometrical configuration of the double bond, the author concluded that the effects of hydrogenated corn oil was not due to chemical treatment, but rather to the presence of the trans isomers introduced during the hydrogenation process. To discriminate between the increase in saturation or the increase in trans isomers as the variables in the hydro— genated corn oil responsible for the reduction of liver fat, Taylor (1970) designed the following experiment in which saturation of the fat was eliminated as a variable by a com— parison of the elaidinized olive oil and natural olive oil. Olive oil after elaidinization had essentially the same fatty acid composition as the starting material except that approx- imately 90 to 95% of the oleic acid had been converted to elaidic acid. To make this compound more comparable to those fed by Morris and Woolcock, the olive oil and elaidinized olive oil were diluted 50% with corn oil. The rats fed the elaidinized olive oil, threonine imbalanced diets showed a reduction in liver fat from the olive oil fed controls, this reduction was attributable only to the presence of the elaidic acid. Such a reduction supported Morris's original hypothesis that trans isomers prevented liver fat accumulation in threonine imbalanced rats. To elucidate the means by which the trans isomer could limit liver fat accumulation in the threonine imbalanced rats, Taylor (1970) traced its metabolic pathways with radioisotOpes 17 and compared the results with that of the cis isomer. For this purpose, weanling rats were fed a 9% casein, 30% fat, .5% choline, threonine imbalanced diet. The diet fat was either a 1:1 mixture of corn oil and olive oil or a 1:1 mix— ture of corn oil and olive oil that had been elaidinized. At the end of four weeks the rats were forced fed approximate— ly 1 ml of corn oil containing 20‘pCi per 165 grams of body weight of either l-Cl4—oleic acid or l-Cl4—elaidic acid and sacrificed at four and eight hours thereafter. The tissues and serum were analyzed for radioactivity. A limited accumu— lation of liver lipid was observed in threonine imbalanced rats fed elaidinized olive oil as a result of an initial preferential uptake of the trans isomer in adipose tissue, an increase in liver lipoprotein synthesis and/or release, and a faster rate of hydrolysis of circulating lipoprotein lipid. The enhancement of lipOprotein synthesis appeared to be mediated by a preferential incorporation of the trans isomer into the liver phospholipids. The author postulated that the mode of enhancement was via (1) the physical charac- teristics of the liver lipid micelles containing the trans isomer, (2) an acceleration of triglyceride micellerization or (3) a direct influence on lipoprotein synthesis. To test the effect of the unnatural isomers present in chemically hydrogenated fats, as well as the degree of satu— ration and chain length on liver fat deposition, different dietary fats with varied fatty acid compositions were select— ed and incorporated into 9% casein, threonine imbalanced diets (Woolcock, 1967). The dietary fats selected 18 included a natural hydrogenated fat (lard), a chemically hydrogenated fat (hydrogenated corn oil), cocoa butter, plus a variety of oils, such as corn, olive, safflower and coco— nut. These fats were added to the diet at a level of 30%. The level of choline in the basal diet (.15%) proved to be inadequate for this quantity of fat in the diet and conse— quently a choline deficiency was induced. In an effort to determine the mechanism of the lipotropic action of choline, choline levels were increased to 1.0% of the diet. Threonine imbalanced rats fed hydroqenated corn oil (which contained 45% trans isomers) or cocoa butter (which contained 34% stearic acid) did not develop fatty livers. Rats fed natural corn oil, natural lard, olive oil, safflower oil, and commer- cial lard had comparable liver fat levels. Of all the di— etary fats studied, coconut oil induced the highest liver fat concentration. Choline supplementation significantly lower— ed liver fat levels for rats fed all dietary fats, but a more pronounced effect was observed in rats fed coconut oil. Analysis of fatty acid composition in livers of choline deficient rats showed an increase in linoleic and oleic acid levels. This fatty acid pattern was consistent irrespective of the dietary fat fed. A common metabolic pathway was suggested for stearic acid and the trans isomers. Since these acids have been found to OCCUpy theCX —position of the phospholipid molecule, and these same acids have been found to be preferentially incorporated into the phOSpholipid molecule in the intestinal mucosa rather than the triglyceride molecule, the author suggested that these acids do not travel 19 via the liver but are tranSported directly to other tissues, where they may have a specific function. LIPID ABSORPTION AND CHYLOMICRON FORMATION The major products of triglyceride hydrolysis by pan- creatic lipase and conjugated bile salts in the intestinal lumen are free fatty acids and monoglycerides. These prod— ucts combine with bile salts to form mixed micelles. The fatty acids and monoglycerides are then absorbed across the intestinal mucosa while the conjugated bile salts reenter the lumen and eventually are absorbed in the distal small intestine, then resecreted via liver and bile (Senior, 1964). After entry into the cell both fatty acids and monoglycerides are esterified to triglyceride. The two major metabolic routes are: (l) the acylation of fatty acids to Acyl-CoA thioesters which then react with L—0(-glycerolphOSphate to yield phosphatidic acid derivatives and (2) the direct inter- action of monoglycerides with fatty Acyl-CoA molecules to yield diglycerides and triglycerides. This latter pathway does not involve the formation of phospholipids (Isselbacher, 1965). Triglyceride, synthesized by the intestinal mucosa are then incorporated into chylomicrons by the intestinal cell. The chylomicron consists of a core of triglyceride which closely resembles the dietary fat in fatty acid composition. All of the cholesterol ester and part of the free cholesterol of the particle are also in the core. The core is envelOped in an outer layer which consists largely of phOSpholipid and with a small amount of protein and free cholesterol. The polar lipid or 1ip0protein envelope of lymph chylomicrons is 20 21 formed intracellularly but its composition changes as the result of exchanges with lipids and proteins of the extra— cellular fluid compartments (Zilversmit, 1967). After feeding corn oil, there is an increase in the percentage of protein, cholesterol, both free and esterified, and phOSpholipid as the particle size diminishes. For parti— cles with a diameter of greater than 200 mp there was 96% triglyceride, 2.8% phospholipid, .41% free cholesterol, .l4% cholesterol ester and .52% protein; particles of less than 140 mp had 88% triglyceride, 9.8% phospholipid, .98% free cholesterol, .46% cholesterol ester and 2.02% protein (Yokoyama and Zilversmit, 1965). The chylomicrons are released from the intestinal cells into lacteals. From these lacteals they are collected into lymphatic channels which empty into the cisterna chyli. This organ, situated behind the aorta, overlapping it later— ally at the level of the origin of the superior mesenteric artery in front of the vertebral column, is drained by the thoracic duct. The duct runs through the abdomen and thorax and Opens into the junction of the left internal jugular vein and the subclavian vein at the point where they drain into the left superior vena cava (Lambert, 1965). The majority of absorbed fatty acids containing ten or more carbon atoms in length are found in the lymph of the thoracic duct as previously described; fatty acids with carbon chains shorter than ten to twelve carbons are trans— ported in the portal venous blood as free fatty acids (Harper, 1967). 22 There are several mechanisms for the removal of chylo— micron fatty acids from the circulation by the liver. Direct removal mechanisms include uptake by liver cells of intact chylomicrons or chylomicron triglyceride molecules, and up— take of free fatty acids after lipolysis of chylomicron fatty acid esters in extracellular compartments of the liver. Chylomicron fatty acids might also reach the liver by an indirect mechanism, namely extrahepatic lipolysis and subse— quent tranSport of free fatty acids to the liver (Ontko and Zilversmit, 1967). In livers removed from intact rats given labeled chylomicrons, the triglyceride—Cl4 to phospholipid—Cl4 ratio was high, a finding unexpected if the liver had acquired this C14 by removal of circulating fatty acids formed by extrahepatic lipolysis. These results demonstrated the abil— ity of the liver to remove and utilize chylomicrons directly and suggest that direct removal accounts for a significant portion of the chylomicron fatty acids utilized by the liver of the intact rats (Ontko and Zilversmit, 1967). Studies in which a single labeled fatty acid was fed and the lipid class distribution of the chylomicron measured showed that the common, long chain fatty acids, with the ex— ception of stearic acid, were similarly absorbed from the intestine. In rats (Blomstrand, 1954 and Bergstrom gt gt., 1954) and in humans (Blomstrand and Ahrens, 1958 and Blom— strand gt gt., 1959) labeled palmitic, oleic and linoleic acids given singly were absorbed similarly, 90% or more of the labeled fatty acid recovered in lymph was present as tri— glyceride, and 2-6% as phOSpholipid. Stearic acid was 23 incorporated less into triglyceride and more into phOSpho— lipid in rats and humans (Blomstrand gt gt., 1959: Borgstrom, 1952 a, b). Cholesterol esters contained about 1—2% of the labeled fatty acids after feeding stearic acid to rats (Borgstrom, 1952 a), or stearic and linoleic acids to humans (Blomstrand gtht., 1959); on the other hand, the possibility of intes- tinal specificity towards particular fatty acids for choles— terol esterification has been suggested by tg gtt£g_studies. Homogenates of rat intestine incorporated polyunsaturated fatty acids into cholesterol ester much more rapidly than saturated fatty acids, whereas pancreatic homogenates utilized oleic acid more rapidly than the other fatty acids (Murthy, 1961). Others have considered the intestinal esterifying activity to be derived from the pancreas (Swell gt gt., 1950 and Hernandez gt gt., 1955) and have demonstrated a relative specificity for unsaturated fatty acids with pancreatic cholesterol esterase (Swell gt gt., 1955 and Hernandez, 1957). Studies of the fatty acid composition of the lymph chylomicrons after the ingestion of fat have suggested that the constituent fatty acids are not only derived from the recently fed fat but are derived from other sources within the animal as well (Blomstrand, 1958; Bragdon, 1960; Kayden, 19601 and Borgstrom, 1952 c). During the period following a meal of corn oil, the fatty acid composition of lymph chylo— microns rapidly changed toward the composition of the corn oil and eventually became almost identical with it (Bragdon and Karmen, 1960 and Kayden gt gt., 1960). However, in the 24 early stages of this period, after the fat content of the chyle had already increased significantly, the fatty acid composition of the chylomicrons did not closely resemble that of the fed fat and neither did that of the lymph samples collected in the later stages when the fat content of the lymph was still high. Both of these samples contained large amounts of fatty acids not present in the diet. To distinguish the effects of the relative specificities of the esterification mechanisms from the effects of varying contributions from recently fed fat and from fatty acid pools in the animal, the following study was designed (Karmen, Whyte and Goodman, 1963 a, b). Mixtures containing similar l4-labe1ed fatty acids (palmit- amounts of two, three or four C ic, stearic, oleic and linoleic acids), but with varying ratios of unlabeled fatty acids, were given by gastric intu— bation to rats with cannulated thoracic ducts and the lymph collected for 24 hours. With the exception of a slight discrimination against stearic acid, the processes of fatty acid absorption and chylomicron triglyceride formation dis- played no specificity for one fatty acid relative to another. In contrast, chylomicron cholesterol ester formation showed marked specificity for oleic acid, relative to the other three fatty acids. This specificity was not significantly altered by varying the composition of the test meal, by in- cluding cholesterol in the test meal, or by feeding the ani— mal a high-cholesterol diet for several weeks preceding the study. Considerable dilution of the dietary fatty acids with endogenous fatty acids was Observed. In one experiment, 25 43% of the chylomicron triglyceride fatty acids were of endogenous origin. Relatively more, 54%, of the cholesterol ester fatty acids were of endogenous origin. Different fatty acids fed to the rat were not incorporated to equal extents into chylomicron lecithin. There was a marked specificity for stearic acid and a lesser specificity for linOleic acid. Oleic acid was incorporated least of all into lecithin. The incorporation of different dietary fatty acids varied with the nature of the diet, but the addition of fatty acids from endogenous sources was of such magnitude as to make this change less evident, so that the over-all fatty acid pattern of lecithin was relatively constant and independent of the composition of the diet. The endogenous contribution to lecithin fatty acids, 65%, was greater than the endogenous contribution to the sterol ester or triglycer- ide fatty acids of the same chylomicron sample. This endog— enous contribution to lecithin also varied from fatty acid to fatty acid, being greatest with palmitic acid and least with oleic acid. Lecithin carried a relatively large share of stearic acid, and relatively very little oleic acid with— in the chylomicron. Many publications (Boucrot & Clement, 1965; Clement gt gt., 1965; and Verdino gt gt., 1965) suggest that during the post—prandial period triglyceride contains a high prOportion of endogenous chains, in agreement with Karmen gt gt.(l963 a, b) while others think that triglycerides contain a major— ity of chains coming from the diet (Borgstrom, 1952; Bragdon and Karmen, 1960; Mattson and Volpenhein, 1962, 1964; and 26 Savary and Constantin, 1966). Savary and Constantine (1967) suggested that if the lymph in Karmen's study (1963 a, b) was fractionated with respect to time, the endogenous con— tamination would not have been so great: as an example, a rat forced fed 200 mg oleic acid had 72, 64 or 48% dietary chains in lymphatic triglycerides, depending on whether the analysis had been performed on 0—5, 0—9 or 0—30 hour lymph samples. The intestinal lymph of rats which had been raised from weanling to four months of age on a fat—free diet or on diets containing lard or corn oil was studied (Verdino gt gt., 1965). After fasting for twenty-four hours, a lymph collection was made; the fatty acid composition represented that of the endoqenous pool of fatty acids. The fatty acid composition of the diet influenced the lipid class composition of the lymph; the endogenous lymph lipid of the corn oil group con— tained a higher percentage of phospholipid than did the groups on the lard and fat—free diets (35 vs. 23 & 24%). This was apparently at the expense of triglyceride (53 vs. 65 & 62%), indicating a greater synthesis of phospholipid when the diet was high in polyunsaturated fat. Experiments were also re— ported on the incorporation of dietary palmitic-l—C14, oleic~ l—C14 and linolenic acids and tripentadecanoin into the tri— glycerides of the intestinal lymph of animals in the above groups. The composition of endogenous lipid was found to influence both the relative extent of incorporation of fatty acids into triglycerides or phOSpholipids, and also the re— synthesis of different fatty acids into triglycerides. 27 These observations were in accord with the findings of previous studies (Karmen gt gt., 1963 a, b, c). There have been few publications concerning the source of the endogenous lipid in the lymph. Baxter (1966) found that in the thoracic duct lymph of rats eating a fat—free diet there was 6.5 mg of lipid recovered per hour. The lipid, 70% triglyceride, 24% phOSpholipid and 6% total cholesterol, reached the thoracic duct via lymphatics from the intestine, little entered from the liver. The fatty acids were derived in part from bile lipid———possibly 50%, and in part from other intraluminal materials. Studies with labeled palmitic acid indicated that little circulating free fatty acid was taken up by the mucosa and incorporated into lymph lipid. The output of esterified fatty acids in the thoracic duct lymph of fasting rats decreased by 80% when bile was removed by means of a fistula (Shrivastava gt gt., 1967). When bile salts were reinfused, there was no reversal, indi— cating that it was a loss of bile lipid and not bile salt which was responsible for the decreased lymph lipid. Before biliary drainage they resembled plasma lipids therefore, suggesting that fatty acids liberated from phoSpholipid of bile in the lumen contribute the major portion of esters of fasting lymph and lipoprotein passing from plasma into lymph provides much of the remainder. Stein and Stein (1966) suggested three sources for endogenous phOSphOllpld7 (1) serum lysolecithin, which is taken up by the mucosal cell; (2) lecithin from the bile and (3) lecithin synthesized gg_novo in the intestinal mucosal cell. 28 Lindsey and Wilson (1965) designed studies to determine whether cholesterol synthesized in the intestinal wall con— tributes to the circulating cholesterol of rats. They took advantage of previous studies, namely that feeding of cho— lesterol to the rat suppresses the synthesis of cholesterol by the liver but not by the intestine (Siperstein and Guest, 1960 and Gould gt gt., 1953) and that the intestinal lymph represents the sole means by which the intestine contributes cholesterol into the circulation (Chaikoff, 1952). Therefore, the cholesterol—fed, lymph cannulated rat was utilized for a study of the conversion of acetate-Cl4 to circulating cho— lesterol—C14. The results demonstrated that cholesterol—Cl4 appeared in the intestinal lymph of cholesterol~fed rats 14 injected with acetate—Cl4 Furthermore, this cholesterol—C did not reach the lymph via either blood or bile and must therefore have arisen in the gut wall itself. The authors concluded that cholesterol synthesized in the intestinal wall contributed to the circulating cholesterol pool. Sylven and Borgstrom (1968) measured cholesterol in the thoracic duct lymph of rats that had been fasted twenty-four hours. They found that the lymph lipid recovered during the fasting period yielded .87 pmoles/hour of cholesterol; the feeding of 800‘pmoles triolein yielded 1.65‘pmoles/hour; the feeding of 50,pmoles cholesterol and 800‘pmoles triolein yielded 5.00‘pmoles/hour. They concluded that the amount of endogenous cholesterol was dependent on the dietary fat fed. There is evidence that protein formation in the intestine is necessary for transport of long chain fatty acids from the 29 intestinal lumen. Using protein synthesis inhibitors, Issel— bacher (1965) showed the inability of the mucosal cell to trans— port reesterified lipid out of the cell into the lymphatic system. In a study by Jacobs and Largis (1969a), the mesen— teric lymph duct was cannulated and the rat fed radioactive amino acids by stomach tube. The radioactivity was then follow- ed into this lymph both as free amino acids and protein frac— tions. The dietary amino acids appeared in the lymph and were transported from the lumen as free amino acids and newly syn— thesized protein by the lymphatic system. There Was a depres— sion of lymph amino acid levels during the early phase of intestinal absorption which returned to normal in the later phase. The depression occurred at the same time the newly synthesized protein appeared in the lymph. These same work— ers (Jacobs and Largis, 1969 b) then showed the effects of the protein synthesis antagonists cycloheximide and puromycin up— on the transport of amino acids and newly synthesized protein into the lymph. After feeding a fatty meal (bovine cream) fortified with leucine-Cl4, these antagonists depressed the formation of lymph protein with a concurrent increase of amino acids. The puromycin may act at the sRNA—AA stage and cycloheximide at the peptide stage in the synthesis of protein required for lipid tranSport as related to amino acids in the cellular pool and released into lymph. INTRODUCTION Weanling rats fed a threonine imbalanced diet contain— ing 30% fat and adequate choline developed fatty livers; the accumulation of fat was greater when corn oil rather than cocoa butter was the source of fat in the diet (Woolcock, 1967). The main difference in these fats was the amount of saturated fatty acids, 63% with cocoa butter and 15% with corn oil (Table 1). To determine how these fats with different fatty acid compositions could influence liver fat levels, the lymph lipid was studied because the tranSport of lipid from the intestine may be influenced by the amino acids and fatty acids available for chylomicron formation. For these reasons, rats were fed threonine imbalanced or threonine supplemented diets with 30% corn oil or 30% cocoa butter as the fat sources. After fasting to remove residual dietary fat, the endogenous lipid of the lymph was collected and analyzed. It has been reported that endogenous lipid influences the absorption of dietary lipid and changes the class distribution of the lymph lipids. Therefore, the rats were forced fed either corn oil or cocoa butter to see how the endogenous lipid and/or the fatty acid composition of the previously fed fat would affect the absorption and incor— poration of these fats into the various fractions of the 30 31 lymph lipid. To collect the lymph, the diets had to adapted for adult rats since it was not feasible to cannulate the lymphatics of a weanling rat. Harper (1954 d) observed the greatest amount of fat in the livers of adult rats at two weeks when the amino acid imbalanced diet containing 5% fat and .15% choline was reduced from 9% to 5% casein. The pres— ent experimental diets contained 5% casein but the amount of fat was increased to 30% with a corresponding increase in cho- line to .5%. The effects of feeding these 5% casein, threonine imbal— anced or threonine supplemented diets with 30% corn oil or cocoa butter to adult rats have been described in the follow- ing three parts: part 1, the effects on liver fat accumula- tion; part 2, the effects on endogenous lymph lipid class distribution and fatty acid composition and part 3, the effects on the incorporation of corn oil or cocoa butter into the lymph lipid. PART 1 LIVER FAT ACCUMULATION IN ADULT RATS FED 5% CASEIN, THREONINE IMBALANCED OR THREONINE SUPPLEMENTED DIETS WITH 30% CORN OIL OR COCOA BUTTER AS THE FAT SOURCE 32 33 Section é. Determination of the time at which fat accumula— tion would be at a maximum in the livers of adult rats fed a threonine imbalanced diet containing 5% casein and 30% corn oil. Experimental procedure Male rats of the Sprague—Dawley strain weighing 240—260 grams were housed in individual wire bottom cages in a temperature and light regulated room. Groups of five rats were fed Diet I, a 30% corn oil, threo— nine imbalanced diet (Table 2) gg_11bitum for a period of 2, 3, and 4 weeks. Rats of the same weight were fed standard laboratory chow to serve as controls for normal liver fat accumulation. Weight gain and food consumption were record— ed. At the end of each period, the rats were lightly anesthe— tized and killed by bleeding from the abdominal aorta. Livers were dissected out, weighed and frozen until analysis. Liver moisture was determined gravimetrically and the fat extracted with diethyl ether on a Goldfisch Apparatus for three hours. Standard errors of the mean were calculated for all data. Significant differences were calculated by students' "t" test. Results and discussion There was more fat in the livers of rats fed the threonine imbalanced diet for 2, 3, or 4 weeks than in the livers of rats fed the laboratory chow but no significant differences among the 2, 3, and 4 week values (Table 3). The percent liver fat appeared to be greater at 2 weeks, and probably would have been significantly greater if there would have been more rats used in each group. There— fore, 2 weeks was the length of feeding time for Section B. 34 Section E. Fat accumulation in the livers of adult rats fed threonine imbalanced or threonine supplemented diets contain— ing 30% corn oil or cocoa butter for 2 weeks. Experimental procedure Rats weighing 240—260 grams were divided into 4 groups of 10 and fed the following diets (Table 2) for 2 weeks: I 30% corn oil, threonine imbalanced II 30% corn oil, threonine supplemented III 30% cocoa butter, threonine imbalanced IV 30% cocoa butter, threonine supplemented At the end of the feeding period the rats were killed and the livers treated as described in Section A. Results and discussion There was significantly more liver fat with group I than with group II and with group III than with group IV (Table 4); threonine was effective in lowering liver fat levels with both fat sources. However, there was no further reduction in liver fat when cocoa butter rather than corn oil was the source of fat in the diet as seen with weanling rats (Woolcock, 1967). Therefore these same diets were fed for 4 weeks (Section C) to see if more significant changes in the amount of liver fat with the different fats could be attained. 35 Section 2. Fat accumulation in the livers of adult rats fed threonine imbalanced or threonine supplemented diets contain— ing 30% corn oil or cocoa butter for 4 weeks. Experimental pgocedure The procedure of Section B was repeat— ed except that the diets were fed for 4 weeks instead of 2 weeks. Furthermore, feces were collected for 3 days, dried to constant weight and the fat extracted on the Goldfisch Apparatus with chloroform—methanol 2:1 for 6 hours; digesti- bility coefficients for the fats were then calculated. Allowances for endogenous fecal lipid were not made. Results and discussion The effects of feeding diets I, II, III and IV to adult rats for 4 weeks are shown in Table 5. Groups II and IV gained more weight and consumed more food than groups I and III reSpectively. The decreased weight gain with rats fed the imbalanced diets may be due to de— creased food consumption. This was suggested by Harper (1964) who theorized that the amino acid imbalance depresses the plasma concentration of the limiting amino acid, threonine, causing the blood amino acid pattern to resemble that of an animal fed a much more deficient diet; this may then trigger an appetite—depressing mechanism and cause food intake to fall. Both groups III and IV consumed more food than groups I and II respectively but, the weight gain did not follow this pattern. Group III gained more weight than group I while group IV gained less weight than group II. This difference in growth cannot be explained on the basis of food consumption data. 36 Corn oil had a higher digestibility coefficient than cocoa butter (88.4 i 1.1 vs. 83.0 i 1.4) which was signifi— cant at the 1% level. The high percentage of stearic acid in the cocoa butter should not have affected the digestibility since there was also a high percentage of oleic acid present; this would have formed disaturated monounsaturated triglycer— ides which are well absorbed. Korn (1961) determined the positions of the fatty acids in cocoa butter triglycerides and in chylomicrons of rats fed cocoa butter; they were both of nonrandom structure; cflrposition of cocoa butter trigly— ceride was palmitic, 39%; stearic, 47%; oleic, 13%; linoleic, 2% and [g-position was 3%, 5%, 76%, and 11% respectively; €4-position of chylomicron fat was palmitic, 36%; stearic, 35%,- oleic, 27%; linoleic, 2% and fl.position was 18%, 8%, 60%, and 17% respectively. There must be some other reason for the increased food consumption with cocoa butter fed rats (Groups III and IV). These differences were not seen with weanling rats (Woolcock, 1967). The percent liver moisture was higher with groups III and IV than with groups I and II. There was no inverse relationship between liver moisture and liver fat. More liver fat accumulated with group I than with group II and, with group III than with group IV; threonine was effective in reducing the amount of liver fat. There was more liver fat with group I than with group III, thus showing the lipo- tropic effect of cocoa butter. The effect was not additive since when both threonine and cocoa butter were present, the level of liver fat was no different that when threonine and 37 corn oil were both in the diet. Woolcock (1967) suggested that the high percentage of stearic acid in cocoa butter may have a sparing effect on the lipotropic action of threo- nine by being preferentially incorporated into the phOSpho— lipid molecule in the intestinal mucosa. Her study did not test this hypothesis therefore, the following studies were designed to observe the lymph lipid of animals which had been fed a threonine imbalanced diet with either corn oil or cocoa butter as the fat source. Animals fed a threonine supplemented diet with either corn oil or cocoa butter as the fat source served as controls. 38 Table 1. Fatty acid composition of corn oil and cocoa butter. Fatty acid (%) Corn Qtt Cocoa Butter C16 (palmitic) 13 27 018 (stearic) 2 36 Cl8:l (oleic) 28 34 Cl8:2 (linoleic) 57 3 % saturated (C16 + C18) 15 63 39 Table 2. Diet Composition Components QLOEB I Elf—OER .13. Group III 9.119.122 I! sucrose 28.8 28.8 28.8 28.8 casein 5.0 5.0 5.0 5.0 fat1 30.0 30.0 30.0 30.0 (corn (corn (cocoa (cocoa oil) oil) butter) butter) salts2 4.0 4.0 4.0 4.0 vitamin mix3 .25 .25 .25 .25 DL methionine .3 .3 .3 .3 DL tryptOphan .l .l .1 .1 choline4 .5 .5 .5 .5 alphacel 31.05 30.69 31.05 30.69 DL threonine — .36 — .36 lContaining 10 ml corn oil mix/50 g corn oil; corn oil mix; ‘X-toc0pherol acetate 75 mg/corn oil 10 ml = 75 mg d—toc0ph— erol/kilo diet. 2 Salts Wesson, L. G. "A modification of the Osborne—Mendel Salt Mixture containing only inorganic constituents” Sci. 1;, 339, 1932. 3 Vitamins were included in sucrose to provide in mg per 100 grams diet: thiamine hydrochloride riboflavin niacin pyridoxine Ca pantothenate inositol folic acid vitamin B 12 ( 1% trituration) biotin vitamin A conc. 500,000 IU/g calciferol 40,000,000 IU/g .0383 p-amino benzoic acid menadione 10 ml 50% choline chloride solution/kg [.4 bOOOOOEOU'IUI 3.; N ON I I O O O O N H 4 40 Table 3. Percent fat in the livers of adult rats fed a threonine imbalanced diet containing 5% casein and 30% corn oil for 2, 3, and 4 weeks as compared to normal liver fat in adult rats fed a standard laboratory chow diet. tht Liver fat (% dry weight) Std. lab chow diet 10.0 t .61 Threonine imbalanced diet 21.2 i 1.82 (2 weeks) 19.7 i 2.22 (3 weeks) 2 15.8 + 1.6 (4 weeks) 1Standard error of the mean. 2Significantly different from std. lab chow diet (1% level). Table 4. Percent fat in the livers of adult rats fed threo- nine imbalanced or threonine supplemented diets containing 30% corn oil or cocoa butter for 2 weeks. 95222 Diet I Corn oil, threonine imbalanced 26.8 i 1.71 II Corn oil, threonine supplemented 19.5 i 1.42 III Cocoa butter, threonine imbalanced 22.3 i 2.0 IV Cocoa butter, threonine supplemented 17.3 i 1.33 1Standard error of the mean. 2Significantly different from group I at the 1% level. 3Significantly different from group III at the 5% level. Table 5. Percent fat in the nine imbalanced or threonine 30% corn oil or cocoa butter I food consumption 405.1l (g/rat/4 wks.) : 7.4 weight gain 7.4 (g/rat/4 wks.) :2.1 liver moisture 70.7 (%) i .3 liver fat 21.4 (% dry wt.) : .8 41 livers of adult rats fed threo- supplemented diets containing for 4 weeks. Group ;3_ III ty 484.12’4 477.52'4 539.1 : 7.7 ‘:15.4 :15.6 55.12'5 16.83’4 2.2 12.9 ‘13.0 :3.8 70.55 72.63 72.0 i 2 i .8 i .7 18.32 19.63’4 16.8 + .6 + .3 + .9 p1 Standard error of the mean. Significantly different from Significantly different from Significantly different from Significantly different from (11wa group I at the 1% level. group I at the 5% level. group IV at the 1% level. grOUp IV at the 5% level. PART 2 ENDOGENOUS LIPID OF LYMPH FROM FASTED RATS PREVIOUSLY FED THREONINE IMBALANCED OR THREONINE SUPPLEMENTED DIETS WITH 30% CORN OIL OR COCOA BUTTER AS THE FAT SOURCE 42 43 Experimental procedure Diet Four groups of rats were fed the experimental diets (I, II, III & IV; Table 2) gg libitum for 4 weeks. Lymph collection To prepare a rat for surgical Operation, the food cup was removed from the cage on the last night of the feeding period. After 12 hours of fasting, the rat was anesthetized with MetOphane (brand of methyoxyfluorane from Pitman-Moore, Dow Chemical Co. Box 1656 Indianapolis, Ind.) and the thoracic lymph duct cannulated above the cisterna chyli similar to the method of Bollman, Cain and Grindlay (1948). Heating polyethylene tubing (Clay Adams N. Y., PE 60) about 8 inch from the end formed a J—shaped cannula which was then siliconized with Siliclad (Clay Adams N. Y.). After the cannula was tied into the duct, it was anchored to back muscle and skin. The abdominal muscles were sutured and the skin clipped together. The rat was then placed in a plexi- glas restraining cage and allowed twelve hours to recover with access to .9% NaCl solution and no food. If there was a steady lymph flow after the recovery period, the lymph was collected for twelve hours into a test tube set in a cold water bath. This was called the 0 hour or fasting period collection and was frozen until analysis. Lipid extraction The lipid from the whole lymph sample was extracted with 300 m1 chloroform-methanol 2:1 (Karmen gt gt., 1963 a). After addition of 50 ml .04 N H2804 to Split the extract into two phases, the lower phase was collected, dried over anhydrous sodium sulfate, evaporated to dryness on a rotary evaporator and redissolved in hexane. 44 Column chromatography Five 50 ml burettes with glass wool layered in the bottom were used for columns. A slurry of 25 ml 100—200 mesh silicic acid (Bio Sil A, Calbiochem L.A., Calif.) and hexane were poured into the column. Hexane was forced through the column with nitrogen gas to remove air bubbles and to pack the silicic acid. To condition the col— umn, it was washed with 10 ml ether, a 15:15 m1 mixture of ether and acetone and 20 m1 ether (Hirsch & Ahrens, 1958). The column was then rewashed thoroughly with hexane. The total lymph lipid sample was applied to the top of the col— umn in a small amount of hexane. The cholesterol ester fraction (CE) was eluted with 150 m1 1% ether in hexane; the triglyceride (TG) and free cholesterol (FC) fraction with 150 ml chloroform and 200 ml acetone; the phospholipid frac- tion (PL) with 150 ml methanol. All solvents were redistill— ed before use. The fractions were collected in large flasks and evaporated to 50 ml. Thin layer chromatoggaphy Completeness of the column separ— ation was checked by thin layer chromatography in hexane— diethyl ether—glacial acetic acid 90:10:1. Free fatty acids, diglycerides and monoglycerides which were eluted with the TG—FC fraction were present in trace amounts only. §pectroPhotometric determinations Determinations of FC and CE were made by the method of Zak (1954) using pure choles- terol and cholesterol oleate as standards, TG by the method of Snyder and Stephens (1959) using olive oil triglycerides as a standard, and PL by the method of Bartlette (1959) using KH2P04 as the standard. 45 Gas liquid chromatography Methyl esters Of fatty acids were prepared according to Karmen gt gt. (1963) and extracted with twice redistilled hexane. The composition of the methyl esters of fatty acids was determined by a F & M gas liquid chromatograph with flame ionization detector (F & M Scientific Corp. Avondale, Pa.). The six foot column contained Diato— port S (80—100 mesh) coated with 5% DEGS (F & M Scientific Corp. Avondale, Pa.). The Operating temperature was pro— grammed from 155°C to 210°C at 50 per minute. Peak areas were measured by triangulation and expressed as percent area. Results 1. Endogenous lymph lipid recovery Total lipid Groups I and II had 68 mg lipid; groups III and IV had 98 and 104 mg respectively (Table 6). There was a 30—36 mg increase in total lymph lipid when cocoa butter rather than corn oil was the fat source in the previously fed diet. Triglyceride (TE) The increase in total lipid was due main— ly to an increase in TG; group III and IV each had 82 mg while groups I and II had 50 and 54 mg respectively (Table 6). Cocoa butter increased the TG in groups III and IV when com- pared with groups I and II; the addition of threonine to the diets had no effect on the amount of endogenous TG in the lymph lipid. Cholesterol ester (SE) and free cholesterol (gt) CE and FC remained 4-5 mg and 1-2 mg respectively for all groups; neither the previous diet fat nor the addition of threonine 46 to the diet affected the endogenous CE and PC of the lymph lipid. Phospholipid (3t) Group II had 5 mg less PL than group I (8 vs. 13); group III had 4 mg less PL than group IV (11 vs. 15); group I had only 2 mg more PL than group III (13 vs. 11) while group IV had 7 mg more PL than group II (15 vs. 8) (Table 6). The addition of threonine to the corn oil diet caused a decrease in endogenous PL but caused an increase in endogenous PL when added to the cocoa butter diet. There was little difference between the two groups fed the threo— nine imbalanced diet but with the groups fed the threonine supplemented diet, there was more endogenous PL when cocoa butter rather than corn oil was the fat source. 2. Percent class distribution gt lymph lipid When the lipid classes were expressed as a weight percent of the total lymph lipid during the 12 hour fasting period (Table 7), group I had 7% less TG, the same % CE, 1% more FC and 6% more PL than group II. Group III had 5% more TG, 1% less CE, the same % FC, and 3% less PL than group IV- Group I had 11% less TG, 2% more CE, 1% more FC and 7% more PL than group III. Group II had 1% more TG, 1% more CE, the same % FC and 2% less PL than group IV. Considering differences of more than 5% as significant, the only differences would be less TG and more PL with group I than with groups II or III. 3. Ratio gt_phospholipid tg triglyceride Since the main differences among the groups were the percentages of TG and PL, the ratio Of mg PL/ mg TG was calculated. The ratio values for groups I, II, III and IV were respectively: 47 .26, .15, .13 and .18. Group I had much more PL in relation to the amount of TG than did group II or III. The differences between grOUps II and IV and between groups III and IV were very small. These values showed that cocoa butter as well as threonine was effective in lowering the ratio of PL/TG but the effect was not additive, when both cocoa butter and thre— onine were present, the ratio was not lower than when either cocoa butter or threonine was present. 4. Percent saturated fatty acids Fatty acids, determined by gas liquid chromatography, are listed in Table 8 and the diet fat composition is listed in Table 1. The value for percent saturated fatty acids is obtained by adding together the percent C16 and C18. Both corn Oil fed groups had a higher percentage of saturated fatty acids in the lymph lipid than was in corn Oil; the increased percentages in the lipid classes for groups I and II respectively were as follows: TG, 23 & 32%; CE, 21 & 17%; PL, 35 & 40%. The PL were more saturated than the TG or CE. Both cocoa butter fed groups had a lower percentage of saturated fatty acids in the lymph than was in the cocoa butter; the decreased percentages in the lipid classes for groups III and IV respectively were as follows: TG, 21 & 19%; CE, 24 & 25%; PL, 10 & 13%. The PL were more saturated than the TG or CE. Group I had 9% less saturated fatty acids in the TG, 5% less in PL and 4% more in the CE than group II. Group III had 2% less saturated fatty acids in the TG, 1% more in PL, and 3% less in the CE than group IV. GrOUp I had 4% less 48 saturated fatty acids in the TG, 3% less in the CE and 3% less in the PL than grOUp III. Group II had 3% more saturat- ed fatty acids in the TG, 6% less in the CE, and 5% more in the PL than group IV. Considering the great differences in the percentage of saturated fatty acids in the diet fats, there seems to be very little difference in saturated fatty acids of endogenous lymph lipid among the groups. About the only effect threonine supplementation had on saturated fatty acid incorporation into the lymph was an increase in the amount of saturated fatty acid in the TG of the corn Oil fed groups, but this increase probably would not be significant. 5. Percent fatty acid composition The percent fatty acid composition of all lipid fractions are presented in Table 8. IE C16 was incorporated into TG in preference to C18 (26—32 vs. 12-15%). There was 6% more C16 (32 vs. 26%) and 6% less C18:2 (27 vs. 33%) with group II than with group I. There was little difference between group III and group IV. Group I had 13% less C18:l (13 vs. 26%) and 16% more C18:2 (33 vs. 17%) than group III. Group II had 11% less C18:1 (13 vs. 24%) and 9% more C18:2 (27 vs. 18%) than group IV. Both corn oil groups, I and II, had less C18:l and more C18:2 than cocoa butter groups III and IV; this was the influence of the previously fed diet fat. gt C16 was incorporated into CE in preference to C18 (22—26 vs. 10~14%). There were little differences between groups I and II and between groups III and IV. Group I had 4% less C18 (10 vs. 14%), 15% less Cl8:1 (15 vs. 30%), 12% more C18:2 (22 vs. 10%) and 5% more C20:4 (22 vs. 17%) than group III, 49 group II had 4% less C18 (10 vs. 14%), 15% less C18:1 (15 vs. 30%), 15% more C18:2 (24 vs. 9%) and 4% more C20:4 (24 vs. 20%) than group IV. Both corn oil groups, I and II, had less C18, less C18:1, more C18:2 and more C20:4 than cocoa butter groups, III and IV; these changes were due to the differences in the fatty acid composition of the diet fats. No changes in the percent fatty acid composition of the CE were due to the addition of threonine to the diets. Pt. C16 and 018 were equally incorporated into PL of all groups (24-30 vs. 22—27%). Group I had 5% more C18:2 (21 vs. 16%) and 5% less C16 and C18 (C16, 28 vs. 30%; C18, 22 vs. 25%) than group II. There were no differences between groups III and IV. Group I had 5% less C18 (22 vs. 27%), 6% less C18:1 (6 vs. 12%) and 8% more C18:2 (21 vs. 13%) than group III. Group II had 6% less C18:1 (6 vs. 12%) and 6% more C16 (30 vs. 24%) than group IV. Threonine affected no change when added to the cocoa butter diet but with the corn oil diet, caused a decrease in the percent of 018:2 and an increase in the percent Of C16 and 018. In the PL, both corn oil groups had less C18:1 than cocoa butter groups but that was the only similarity; the corn Oil imbalanced group had less C18:1, less C18 and more 018:2 than the cocoa butter imbalanced group while the corn oil supplemented group had less C18:1 and more C16 than the cocoa butter supplemented group. The addition of threonine to the diet had no effect on the percent fatty acid composition of the TG, CE or PL when cocoa butter was the fat source. When corn oil was the fat source, there was an increase in the percent C16 and a 50 decrease in the percent 018:2 in the TG, There was no change in the CE, but an increase in the percent 018:2 and decrease in the percent 016 and 018 in the PL. However, these changes were 6% or less, therefore quite insignificant. The substitution of cocoa butter for corn Oil caused similar changes in the percent fatty acid composition of the TG and CE of both threonine imbalanced and supplemented diets; there was more 018:1 and less 018:2 with cocoa butter than with corn oil groups. In the PL this was not the case, the changes between the imbalanced groups and between the supple— mented groups were dissimilar. Again, the difference was 8% or less, and with only 4 rats per group, these changes can be considered insignificant. 6. Weights gt fatty acids The mg of fatty acids in each of the lipid classes, listed in Table 9, were determined by the following calculation: Mg TG x 95.6%1x % fatty acid / hours mg TGFA/ hr. Mg CE x 42.9%;x % fatty acid / hours mg CEFA/ hr. Mg PL x 74.5%1x % fatty acid / hours = mg PLFA/ hr. 2935 There were no differences between groups I and II or between groups III and IV. Group III had 8 mg more 016, 6 mg more 018, 14 mg more 018:1 and 4 mg more 020:4 per hour than group I. Group IV had 5 mg more 016, 4 mg more 018, 12 mg more 018:1 and 4 mg more 020:4 per hour than group II. There was more 016, 018, 020:4 and much more 018:1 with cocoa lCalculated by using these estimated values as the % fatty acids of the lipid structure and based on the molecular weight of the fatty acids present. 51 butter groups than with corn Oil groups. There was no change in amount of TGFA due to addition of threonine to the diets. g§§§_ There were no differences between groups I and II or between groups III and IV. Group I had .2 mg less 018:1 and .3 mg more 018:2 per hour than group III. Group II had .4 mg less 018:1 and .2 mg more 018:2 per hour than group IV. There was less 018:1 and more 018:2 with corn oil groups than with cocoa butter groups. There was no change in the amount of CEFA due to addition of threonine to the diets. 2225, Group I had .7 mg more 016, .5 mg more 018, 1.0 mg more 018:1 and .6 mg more 020:4 per hour than group II. Group IV had .6 mg more 016, .7 mg more 018, .5 mg more 018:2 and .9 mg more 020:4 per hour than group III. Group I had .5 mg more 016, .9 mg more 018:2, .4 mg more 020:4 and .4 mg less 018:1 per hour than group III. Group IV had .8 mg more 016, 1.2 mg more 018, .6 mg more 018:2 and 1.3 mg more 020:4 than group II. The addition of threonine to the corn oil diet caused a decrease in the amount of all PLFA except 018:2. The addition of threonine to the cocoa butter diet caused an increase in all PLFA except 018:1. With the change in diet fat, threonine had a reverse effect. With the corn oil imbalanced group there was more 016, 018:2, 020:4 and less 018:1 than with the cocoa butter im— balanced group while the cocoa butter supplemented group had more 016, 018, 018:2 and 020:4 than the corn Oil supplemented group. 52 Discussion A threonine imbalance affects the amount and composition of endogenous lymph lipid. Rats previously fed a corn oil threonine imbalanced diet had similar amounts of endogenous TG, 0E and F0 but more PL in the lymph; a higher ratio of PL to TG; a similar percent saturated fatty acids; a similar percent fatty acid composition; similar amounts of TGFA and CEFA but more 016, 018, 018:1 and 020:4 PLFA in the lymph than rats previously fed a corn oil threonine supplemented diet. Rats previously fed a cocoa butter threonine imbalanced diet had similar amounts of endogenous TG, CE and F0 but less PL in the lymph; a similar ratio of PL to TG; a similar per- cent saturated fatty acids; a similar fatty acid composition; similar amounts of TGFA and CEFA but less 016, 018, 018:2 and 020:4 PLFA in the lymph than rats previously fed a cocoa butter threonine supplemented diet. The imbalance affects mainly the PL of the lymph lipid but had just the opposite effect when corn Oil rather than cocoa butter was the source of fat in the previous diet. The endogenous lymph lipid is affected by the fatty acid composition of the fat in the previous diet. Rats previously fed a corn oil threonine imbalanced diet had less TG, 0E and F0; a higher ratio of PL to TG; a lower percent 018:1 and higher percent 018:2 in TG; a lower percent 018:1 and higher percent 018:2 in CB; less 016, 018, 018:1 and 020:4 TGFA, more 018:2 and less 018:1 CEFA and more 016, 018:2, 020:4 and less 018:1 PLFA in the lymph than rats previously fed a cocoa butter threonine imbalanced diet. Rats previously fed a 53 corn oil threonine supplemented diet had less TG and PL; a lower percent 018:1 and higher percent 018:2 in TG, a lower percent 018:1 and higher percent 018:2 in CE; less 016, 018, 018:1 and 020:4 TGFA, more 018:2 and less 018:1 CEFA and less 016, 018, 018:2 and 020:4 PLFA in the lymph than rats pre- viously fed a cocoa butter threonine supplemented diet. With the exception of PL, the differences between the imbalanced groups and between the supplemented groups were similar ——— due to the fatty acid composition of the diet fat. This study has shown that the amount and composition of endogenous lymph lipid was affected by a threonine imbalance but to a greater degree by the fatty acid composition of the fat in the previously fed diet. 54 Table 6. Endogenous lymph lipid recovery. £1219 (mg) Pitt 81:29.2 1‘9. _E. $221222; 00, threo. imbal. I 50 4 2 13 68 CO, threo. Suppl. II 54 4 l 8 68 CB, threo. imbal III 82 4 2 11 98 CB, threo. suppl. IV 82 5 2 15 104 Table 7. Percent class distribution of endogenous lymph lipid. Lipid (weight %) 1.3122 m It 99 £92.13 00, threo. imbal. I 73 6 3 18 CO, threo. suppl. II 80 6 2 12 0B, threo. imbal. III 84 4 2 11 CB, threo. suppl. IV 79 5 2 l4 55 Table 8. Percent fatty acid composition of endogenous lymph lipid. Fatty acid (%) gtggp (Triglyceride) t tt ttt_ t! C16 26 32 27 29 C18 12 15 15 15 C18:1 ‘ l3 13 26 24 C18:2 33 27 17 18 C18:3 or C20:l 3 l 2 l C20:4 13 13 13 14 (% saturated) (38) (47) (42) (44) (Cholesterol ester) C16 26 22 25 24 C1631 5 5 4 3 C18 10 10 14 14 C18:1 15 15 30 30 C18:2 22 24 10 9 C20:4 22 24 17 20 (% saturated) (36) (32) (39) (38) (PhOSpholipid) C16 28 30 26 24 C18 22 25 27 26 C18:1 6 6 12 12 C18:2 21 l6 13 14 C20:4 23 23 23 24 (% saturated) (50) (55) (53) (50) 56 Table 9. Weight of endogenous fatty acids. I§§§’(mg/hr) 016 018 018:1 018:2 018:3 or 020:1 020:4 C_EF_A_ (mg/hr) 016 016:1 018 C18:1 018:2 020:4 w (mg/hr) 016 018 018:1 018:2 020:4 F‘lH Group III 21 12 20 14 1 10 o s |—' 43 s 1'" U" N IV 22 12 19 14 ll PART 3 EFFECT OF FORCED FEEDING CORN OIL OR COCOA BUTTER ON THE LYMPH LIPID OF FASTED RATS PREVIOUSLY FED THREONINE IMBAL— ANCED OR THREONINE SUPPLEMENTED DIETS WITH 30% CORN OIL OR COCOA BUTTER AS THE FAT SOURCE 57 58 Experimental procedure Forced feeding After the 0 hour collection of lymph was completed (Part 2), the rat was removed from the restraining cage and lightly anesthetized. A stomach intubation of one ml of fat was given. 0f the rats fed the four diets (see Part 2), two from each group were forced fed corn oil and two were forced fed cocoa butter as shown below: forced feeding group t diet 99' pg I corn Oil, threonine imbalanced 2 rats 2 rats II corn Oil, threonine supplemented 2 rats 2 rats III cocoa butter, threonine imbalanced 2 rats 2 rats IV cocoa butter, threonine supplemented 2 rats 2 rats Lymph collection After forced feeding the rat was returned to the restraining cage and the lymph collected in a test tube set in a circulating cold water bath, then frozen until analysis. The rat was killed after the final collection. The lymph lipid was analyzed as described in Part 2. Results 1. Lipid recovety following corn oil forced feeding Total lipid The total lymph lipid recovered over the 30 hour period after forced feeding corn oil was 704.7, 610.9, 1030.6, and 839.0 mg respectively for groups I, II, III and IV (Table 10). Group I had 93.8 mg more lipid than group II and group III had 191.6 mg more lipid than group IV; both imbalanced groups had more lipid than their respective supplemented groups, but to a greater degree with the cocoa butter group. 59 GrOUp III had 325.9 mg more lipid than group I and group IV had 228.1 mg more lipid than group II, thus both cocoa butter groups had more lipid than both corn oil groups. Figure 1 shows the pattern of recovery of total lipid (mg/hr). At 4 and 8 hours more lipid was recovered with groups III and IV than I and II; at 8 hours group IV had more lipid than group III and at 12 hours groups I, II and IV all had less lipid than group III; at 30 hours the values were near fasting levels for all groups. Triglyceride (TG) The 30 hour recovery of triglyceride after forced feeding corn oil was 629.5, 557.3, 935.9 and 748.9 mg respectively for groups I, II, III and IV (Table 10). Since triglyceride made up the largest percentage of the total lipid, the recovery pattern of triglyceride (Figure 2) close- ly resembled the recovery pattern of total lipid (Figure 1). Group I had 72.2 mg more TG than group II and grOUp III had 187.0 mg more TG than group IV; there was more T0 with the imbalanced than with the supplemented diets. Group III had 306.4 mg more TG than group I and Group IV had 191.6 mg more TG than group II; both cocoa butter groups had more TG than both corn Oil groups. Cholesterol ester (0E) Over the 30 hour period after corn oil forced feeding there was 8.8, 7.9, 10.4 and 12.3 mg CE for groups I, II, III and IV respectively. There was .9 mg more CE with group I than with group II and 1.9 mg more 0E with group III than with group IV; both imbalanced groups had more 0E than both supplemented groups. Group III had 1.6 mg more 0E than group I and group IV had 4.4 mg more 0E 60 than group II; both cocoa butter groups had more CE than both corn oil groups. Figure 3 shows the greatest differences in CE recovery at 4 hours; there was much more CE with group IV than with grOUp III or group II. Free cholesterol (FC) Over the 30 hours after corn oil forced feeding, there was 5.8, 4.5, 9.5 and 6.6 mg PC for groups I, II, III and IV respectively (Table 10). There was 1.3 mg more PC with grOUp I than with group II and 3.1 mg more FC with group III than with group IV; both imbalanced groups had more FC than both supplemented groups. Group III had 3.7 mg more FC than group I and group IV had 2.1 mg more FC than group II; both cocoa butter groups had more FC than both corn oil groups. Figure 4 shows the recovery of FC, The greatest differ— ence appears to be the larger amount of PC with group III than with group I mainly at 4 and 8 hours and with group III than with group IV at 8 and 12 hours. Phospholipid (PL) The phospholipid recovered for grOUps I, II, III and IV respectively was 60.6, 41.2, 74.8 and 71.2 mg for the 30 hour period (Table 10). Group I had 19.4 mg more PL than grOUp II and group III had 3.6 mg more PL than group IV; both imbalanced groups had more PL than both supplemented groups however, probably the difference between the cocoa butter groups would not be significant. Group III had 24.2 mg more PL than group I and group IV had 30.0 mg more PL than group II; both cocoa butter grOUps had more PL than both corn oil groups. 61 Figure 5 shows the increase in PL with forced feeding and the gradual decline to near fasting levels at 30 hours for all groups except group III. At 12 hours, the PL of group III increased before declining to near fasting levels at 30 hours. 2. Lipid recovery following cocoa butter forced feeding Total lipid The total lymph lipid recovered over the 30 hour period after forced feeding cocoa butter was 545.1, 565.6, 394.5 and 455.3 mg respectively for groups I, II, III and IV (Table 11). Group II had 20.5 mg more lipid than group I and grOUp IV had 60.8 mg more lipid than group III; both SUpplemented groups had more lipid than both imbalanced groups, but to a greater degree with the cocoa butter diet. Group I had 150.6 mg more lipid than group III and group II had 110.3 mg more lipid than group IV; both corn oil groups had more lipid than both cocoa butter groups. Figure 6 shows the total lipid recovery. At 4 hours about the same amount of lipid had been recovered from all grOUps; at 8 and 12 hours group II had slightly more lipid than grOUp IV and group I had much more lipid than group III; at 30 hours all groups had near fasting level amounts of lipid. Triglyceride The 30 hour recovery of TG after forced feeding cocoa butter was 468.5, 495.0, 340.9 and 392.8 mg reSpective— 1y for groups I, II, III and IV (Table 11). Group II had 26.5 mg more TG than group I and group IV had 51.9 mg more TG than grOUp III; both supplemented groups had more TG than both imbalanced grOUps. Group I had 127.6 mg more TG than grOUp III and group II had 102.2 mg more TG than group 62 IV; both corn oil groups had more TG than both cocoa butter groups. Since TG made up the largest percentage of the total lipid, the recovery pattern of TG (Figure 7) closely re— sembled the recovery pattern of total lipid (Figure 6). Cholesterol ester The 30 hour recovery of CE after forced feeding cocoa butter was 10.2, 12.6, 9.9 and 10.2 mg respec— tively for groups I, II, III and IV (Table 11). Group II had 2.4 mg more CE than grOUp I and group IV had .3 mg more CE than group III; the supplemented group had more CE than the imbalanced group when corn oil was the fat source in the diet but there was little change when cocoa butter was the source of fat in the diet. Group I had .3 mg more CE than group III and group II had 2.4 mg more CE than group IV; there was more CE with the supplemented group when corn oil rather than cocoa butter was the diet fat but the difference between the imbalanced groups was not significant. There was little change in the recovery pattern of CE; it remained near or below fasting CE levels (Figure 7). Free cholesterol The 30 hour recovery of FC after cocoa butter forced feeding was 4.9, 4.7, 4.2 and 5.0 mg respective— 1y for groups I, II, III and IV (Table 11). The differences among the grOUpS were small. The recovery pattern of EC is shown in Figure 8. Phospholipid Over the 30 hour period, 61.5, 53.3, 39.5 and 47.3 mg PL were recovered for groups I, II, III and IV re— spectively (Table 11). Group I had 8.2 mg more PL than group II while grOUp IV had 7.8 mg more PL than group III; 63 adding threonine to the corn oil diet caused a slight de- crease in PL but adding threonine to the cocoa butter diet caused a slight increase in PL. GrOUp I had 22.0 mg more PL than group III and group II had 6.0 mg more PL than group IV, both corn oil groups had more PL than both cocoa butter groups but, to a greater degree with the imbalanced diet. The recovery pattern of PL is shown in Figure 9. There was much less PL with grOUp III at 8 hours and much more PL with group I at 12 hours than with the other groups. 3. Comparison of the lipid recovery following corn oil and cocoa butter forced feeding, Total lipid A comparison of the lymph lipid of groups pre— viously fed the same diets but forced fed corn oil (Table 10) or cocoa butter (Table 11) shows that there was 159.6 mg more lipid with group ICO than with group ICB (704.7 vs. 545.1 mg): 45.3 mg more lipid with group IICO than with group IICB (610.9 vs. 565.6 mg); 636.1 mg more lipid with group IIICO than with group IIICB (1030.6 vs. 394.5 mg); 383.7 mg more lipid with group IVCO than with group IVCB ( 839.0 vs. 455.3 mg). All groups forced fed corn oil rather than cocoa butter had more lipid and to a greater degree when cocoa butter rather than corn oil was the source of fat in the previous diet. It was interesting to note that when corn oil rather than cocoa butter was forced fed, the effect of the previous— ly fed diet on total lipid recovery was just reversed; groups ICO and IICO had less lipid than grOUpS IIICO and IVCO re— spectively while groups ICE and IICB had more lipid than 64 groups IIICB and IVCB respectively. Also, groups IICO and IVCO had less lipid than groups ICC and IIICO respectively while grOUps IICB and IVCB had more lipid than groups ICE and IIICB respectively. Triglyceride There was 161.0 mg more TG with group ICO than with group ICB (629.5 vs. 468.5 mg), 62.3 mg more TG with grOUp IICO than with group IICB (557.3 vs. 495.0 mg), 595.0 mg more TG with group IIICO than with group IIICB (935.9 vs. 340.9 mg), and 356.1 mg more TG with group IVCO than with group IVCB (748.9 vs. 392.8 mg); all groups forced fed corn oil rather than cocoa butter had more TG, and to a greater degree when cocoa butter rather than corn oil was the source of fat in the previous diet. As with the total lipid, when corn oil rather than cocoa butter was forced fed, the effect of the previously fed diet on TG recovery was just reversed; group ICO and IICO had less TG than groups IIICO and IVCO reSpectively while groups ICE and IICB had more TG than IIICB and IVCB respec— tively. Also, groups IICO and IVCO had less TG than groups ICC and IIICO respectively while groups IICB and IVCB had more TG than groups ICE and IIICB respectively. Cholesterol ester There was about the same amount of CE with group ICO as with group ICB (8.8 vs. 10.2 mg), 4.7 mg less CE with group IICO than with grOUp IICB (7.9 vs. 12.6 mg), about the same amount of CE with groups IIICO and IIICB (10.4 vs. 9.9 mg) and about the same amount of CE with groups IVCO and IVCB (12.3 vs. 10.2 mg). Groups ICO and IIICO had more CE than groups IICO and 65 IVCO respectively while group ICB had less CE than group IICB and groups IIICB and IVCB had about equal amount of CE. Both grOUps IIICO and IVCO had more CE than groups ICO and IICO respectively while group IICB had more CE than group IVCB and, groups ICB and IIICB had similar amounts of CE. The changes in CB among the groups forced fed corn oil and among the groups forced fed cocoa butter were not similar. Free cholesterol There was about the same amount of free cholesterol with groups ICO and ICB (5.8 vs. 4.9 mg), IICO and IICB (4.5 vs. 4.7 mg) and IVCO and IVCB (6.6 vs. 5.0 mg); group IIICO had 5.3 mg more FC than group IIICB (9.5 vs. 4.2 mg). Groups ICO and IIICO had more FC than groups IICO and IVCO; groups IIICO and IVCO had more FC than groups ICO and IICO respectively. There was little difference among the groups forced fed cocoa butter. Therefore, the changes in FC among the groups forced fed corn oil and among the grOUps forced fed cocoa butter were not similar. PhOSpholipid Group ICC and ICB had about the same amounts of PL (60.6 vs. 61.5 mg), grOUp IICO had 12.1 mg less PL than group IICB (41.2 vs. 53.3 mg), group IIICO had 35.3 mg more PL than group IIICB (74.8 vs. 39.5 mg), and group IVCO had 23.9 mg more PL than gnoup IVCB ( 71.2 vs. 47.3 mg). Group ICO had more PL than group IICO and groups IIICO and IVCO had about equal amounts of PL; group ICB had more PL than group IICB but group IIICB had less PL than IVCB. Groups IIICO and IVCO had more PL than groups ICO and IICO respectively while groups ICB and IICB had more PL than 66 groups IIICB and IVCB respectively. Again, most of the differences in PL among the groups forced fed corn oil are dissimilar from the differences among the groups forced fed cocoa butter. 4. Percent lipid class distribution after corn oil forced feeding In Table 12, the lipid classes are expressed as a weight percent of total lipid recovered after corn oil forced feeding. Within groups, the percent class distribution changed only slightly from 4 to 8 to 12 hours after forced feeding; among grOUps, the percent class distribution at 4, 8 and 12 hours was very similar. There was 88.4—94.7% TG, .4-l.2% CE, .4—.8% FC and 4.4—9.7% PL at 4, 8 and 12 hours. At 30 hours the TG decreased to 76.9—84.8%, the CE increased to 2.6—5.0%, the FC increased to 1.3-2.6%, and the PL in— creased to 10.7-16.1%. There was more TG and less PL with groups I and II than with groups III and IV respectively at 30 hours. The percent class distribution during the fasting period (Table 7) was very similar to that at 30 hours for IICO, IIICO and IVCO; at 30 hours, ICO had more TG and less CE and PL than during the fasting period. 5. Percent lipid class distribution after cocoa butter forced feeding In Table 13, the lipid classes are expressed as a weight percent of total lipid recovered after cocoa butter forced feeding. Within groups, the percent class distribution changed only slightly from 4 to 8 to 12 hours after forced feeding; among groups, the percent class distri— bution at 4, 8 and 12 hours was very similar. There was 83.7—91.9% TG, .7—2.8% CE, .5-1.3% FC and 6.6—12.2% PL. At 67 30 hours, the TG decreased to 75.3-79.2%, the CE increased to 5.4-6.0%, the FC increased to 1.5—1.9% and the PL increas— ed to 13.6-17.4%. The percent class distribution during the fasting period (Table 7) was very similar to that at 30 hours for groups ICB, IICB and IVCB; IIICB had less TG and more CE and PL than during the fasting period. 6. Comparison of the percent lipid class distribution after corn oil and cocoa butter forced feeding The percent class distribution for groups forced fed corn oil and cocoa butter was similar at 4, 8 and 12 hours; at 30 hours, ICC and IICO 'had a higher percent TG and a lower percent CE and PL than ICB and IICB respectively. The addition of threonine to the diet had little effect on the percent class distribution of the lymph lipid. 7. Percent saturated fatty acids in the lipid classes after corn oil forced feeding A comparison of the saturated fatty acids (C16 + C18) in the TG, CE and PL fractions after corn oil forced feeding can be made from Figures 11, 12 and 13. TG contained the lowest percent saturated fatty acids; at 4, 8 and 12 hours they contained only 13-19% and increased to 34% at 30 hours (Table 14). CE contained more saturated fatty acids than TG but less than PL; at 4, 8 and 12 hours, groups I, III and IV had 21.4—28.3% saturated fatty acids and group II had 29.7—33.6% saturated fatty acids (Table 14). At 30 hours groups II and IV had more saturated fatty acids than groups I and III respectively. The PL contained more saturated fatty acids than the other lipid classes, 42-52% in all grOUps (Table 14); there were more saturated fatty 68 acids in group IV than in grOUp III at 4, 8 and 30 hours and more in group II than in group I mainly at 12 hours. About the only effect caused by the addition of threo— nine to the diet was an increased percent of saturated fatty acids in the CE fraction when corn oil was the source of fat in the previously fed diet. 8. Percent saturated fatty acids in the lipid classes after cocoa butter forced feeding A comparison of the percent saturated fatty acids recovered from TG, CE and PL fractions after cocoa butter forced feeding can be made from Figures 14, 15 and 16. The TG contained as much as 57~62% saturated fatty acids at 4 hours; the values for all groups decreased to near or below fasting levels by 30 hours (Table 15). The CE contained 30—41% saturated fatty acids at 4, 8 and 12 hours and decreased at 30 hours (Table 15). The percent saturated fatty acids of the PL ranged from 45—65% (Table 15). There were more saturated fatty acids with group II than with group I at 4 and 8 hours, and more with group IV than with group III at 4, 8 and 30 hours. At 8 hours, the values for grOUps I and II decreased while the values for groups III and IV increased. 9. Comparison 9: the percent saturated fatty acids in the lipid classes after corn oil and cocoa butter forced feeding TG, CE and PL contained more saturated fatty acids when cocoa butter (Figures 14, 15 and 16) rather than corn oil (Figures 11, 12 and 13) was forced fed; the difference how— ever was greatest in the TG fraction. 69 10. Percent fatty acid compgsition g£_T§ after corn oil forced feeding The fatty acid composition of the TG, CE and PL were expressed as the percent of the total fatty acids present; any change less than 10% was not considered signifi— cant. The percent fatty acids in the TG fraction of the lymph lipid after corn oil forced feeding are shown in Table 16. At 4, 8 and 12 hours the individual fatty acids varied by less than 10% among comparable groups. There was 46-58% C18:2, 21—37% C18:1, 10-l7% C16 and 2—4% C18 in all groups. At 30 hours, the percent fatty acids in groups I and II were similar to those found at 4, 8 and 12 hours, but in group III and IV there was less C18:2 than with group I and II. The percents of C18:3 or C20:1 (3-5%) and C20:4 (4—6%) found only at 30 hours, did not change among the groups. 11. Percent fatty acid composition g§_T§_after cocoa butter forced feeding, The percent fatty acid composition of the TG after cocoa butter forced feeding is shown in Table 17. At 4, 8 and 12 hours the values among the groups were similar : 35—46% C18:1, 24—32% C16, 20—30% C18, and 3—11% C18:2. At 30 hours, the percent C16 remained the same as at 4, 8 and 12 hours but in all groups there was a decrease in C18 and C18:1 and an increase in C18:2. In all groups at 30 hours there was 5% C18:3 or C20:1 and 5—9% C20:4. 12. Comparison 9: the percent fatty acid composition of TC after corn oil and cocoa butter forced feeding With cocoa butter forced feeding (Table 17) there was an average of 17-21% more C18, 10—14% more C16, 7—10% C18:1 and 38—44% 70 less C18:2 than with corn oil forced feeding (Table 16) for all groups. 13. Percent fatty acid composition 9: CE after corn oil forced feeding The percent fatty acids in the CE fraction after corn oil forced feeding are shown in Table 18. The average values for all groups were 17—23% C16, l7—30% C18:2, l7-22% C20:4, 5—9% C18 and 4-7% C16:l. There was more C18:1 with group III than with group I (31 vs. 19%) and with group IV than with group II (33 vs. 20%); both cocoa butter groups had more C18:1 incorporated into CE than both corn oil groups. There were no differences due to addition of threonine to the diet. 14. Percent fatty acid composition 9: CE after cocoa butter forced feedipg The percent fatty acids in the CE after cocoa butter forced feeding are shown in Table 19. The average values for all groups were 18—21% C16, 17—26% C20:4, 14-15% C18, 9—17% C18:2 and 5-8% Cl6:l. There was more C18:1 with group III than with group I (33 vs. 24%) and with group IV than with group II (32 vs. 21%); both cocoa butter groups had more C18:1 incorporated into CE than both corn oil groups. There were no differences due to addition of threonine to the diet. 15. Comparison pf the percent fatty acid comppsition pf CE fatty acids after corn oil and cocoa butter forced feeding A comparison of the average percent CE fatty acids after corn oil and cocoa butter forced feeding show the only changes of 10% or more to be 16% more C18:2 with group ICO than with group ICB and 14% more C18:2 with group IIICO than with group 71 IIICB. When threonine was added to the diets, corn oil and cocoa butter were equally incorporated into the CE but with the imbalanced diet, more C18:2 was incorporated into the CE after corn oil rather than cocoa butter forced feeding. 16. Percent fatty acid composition 9: PL after corn oil forced feeding The percent fatty acids in the PL after corn oil forced feeding are shown in Table 20. Over the 30 hour period there was an average of 27—36% C18:2, 22-27% C16, l6—26% C18, 10—14% C18:1 and 8-13% C20:4 for all groups. The only time when there was a 10% or greater change among comparable groups was at 8 hours, there was more C18:2 with group III than with group IV (40 vs. 30%). The previously fed diet had little effect on the prOportions of fatty acids incorporated into PL. 17. Percent fatty acid composition 9: EL after cocoa butter forced feeding The percent fatty acids in the PL after cocoa butter forced feeding are shown in Table 21. Over the 30 hour period there was an average of 26—31% C18, 24—27% C16, l4—23% C18:2, 15—18% C18:1 and 10—14% C20:4. The only time when there was a 10% or greater change among comparable groups was at 8 hours, there was more C16 with grOUp III than with group I (32 vs. 19%). The previously fed diet had little effect on the prOportion of fatty acids incorporated into PL. 18. Comparison 9f the percent fatty acid composition 9: EL after corn oil and cocoa butter forced feeding A comparison of the percent fatty acid composition of PL after corn oil (Table 20) and cocoa butter (Table 21) forced feeding shows that in group ICO there was 13% more C18:2 and 10% less C18 72 than in group ICB; group IICO had only 8% more C18:2 and only 7% less C18 than group IICB; group IIICO had 17% more C18:2 than group IIICB and grOUp IVCO had 13% more C18:2 than group IVCB. The differences seen were due to the differences in the composition of the previously fed diet fat,and not the addition of threonine to the diet. 1 l9. TGFA after corn oil forced feeding The amounts of TGFA, CEFA and PLFA were calculated as described in Part 2. Of the TGFA after corn oil forced feeding (Table 22, figures 17— 20) there was more C18:1 (1517 vs. 8.1 mg/hr) and more C18:2 (26.7 vs. 16.6 mg/hr) with grOUp III than with group I. There was more C18:1 (12.5 vs. 7.5 mg/hr) and more C18:2 (21.5 vs. 14.6 mg/hr) with group IV than with group II. There was more C18:1 and C18:2 with both cocoa butter groups than with both corn oil grOUps. 20. TGFA after cocoa butter forced feeding There were no significant differences in the amounts of TGFA among the groups (Table 23, Figures 21-24). 21. TGFA, comparison pf corn oil and cocoa butter forced feeding Of the TGFA, there was more C18 (6.3 vs. .7 mg/hr) with ICB than with ICO and with IICB than with IICO (6.4 vs. .7 mg/hr). There was more C18:1 with IIICO than with IIICB (15.7 vs. 6.6 mg/hr) and with IVCO than with IVCB (12.5 vs. 7.4 mg/hr). There was more C18:2 with all groups forced fed corn oil rather than cocoa butter (I, 16.6 vs. 1.6; II, 14.6 vs. 1.8; III, 26.7 vs. 1.0; IV, 21.5 vs. .9 mg/hr). 22. CEFA after corn oil forced feeding Of the amounts of CEFA after corn oil forced feeding (Table 24, Figures 25—28) 73 there was much more C18:1 with grOUp III than with grOUp I (51 vs. 25,pg/hr) and with grOUp IV than with group II (59 vs. 23.ng/hr). There was more C18:2 with grOUp I than with group II (39 vs. 26 pg/hr) and more C20:4 with grOUp IV than with group II (37 vs. 23 pg/hr). The addition of threonine to the diet decreased the amount of C18:2 in the CE. 23. CEFA after cocoa butter forced feeding Of the amounts of CEFA after cocoa butter forced feeding (Table 25, Figures 29—32), there was more C18:1 with grOUp III than with group I (46 vs. 29,pg/hr) and with group IV than with group II (47 vs. 34 pg/hr). There was more C18:2 with group II than with group IV (28 vs. 12 pg/hr). There was more C20:4 with grOUp II than with group I (42 vs. 26 pg/hr) and with grOUp II than with group IV (42 vs. 28,pg/hr). The addition of threonine to the diet increased the amount of C20:4 in the CE after corn oil forced feeding. 24. CEFA, comparison pf corn oil and cocoa butter forced feeding Of the CEFA there was more C18 with group IICB than with IICO (23 vs. 9,pg/hr); there was more C18:1 with grOUp IICB than with grOUp IICO (34 vs. 23,pg/hr) and with group IVCO than with IVCB (59 vs. 47,pg/hr); there was more C18:1 with grOUp ICO than with ICB (39 vs. 19,pg/hr), with group IIICO than with group IIICB (39 vs. 12,ug/hr) and with group IVCO than with group IVCB (31 vs. 12 pg/hr). There was more C20:4 with grOUp IICB than with group IICO (42 vs. 23,pg/hr). 25. PLFA after corn oil forced feeding Of the PLFA after corn oil forced feeding (Table 26, Figures 33-36) there was more C18 (633 vs. 260,pg/hr), more C18:1 (350 vs. l40,ug/hr) 74 and more C18:2 (698 vs. 378 pg/hr) with group IV than with grOUp II. There was more C18:2 with group I than with group II (700 vs. 378,pg/hr). 26. PLFA after cocoa butter forced feeding Of the PLFA after cocoa butter forced feeding (Table 27, Figures 37—40) there were no significant differences among the groups. 27. PLFA, comparison 9: corn oil and cocoa butter forced feeding Of the PLFA, there was more C18:2 with group IIICO than with group IIICB (900 vs. l90,pg/hr) and with group IVCO than with group IVCB (698 vs. 235dpg/hr). Discussion A threonine imbalanced diet as compared to a threonine supplemented diet previously fed to rats affects the incor— poration of corn oil into the lymph. When forced fed corn oil, the corn oil threonine imbalanced group had more lipid due to an increase in the amount of TG, CE, FC and PL; a similar percent class distribution; a similar percent fatty acid composition; a similar amount of TGFA but more C18:2 CEFA and PLFA than the corn oil threonine supplemented group. When forced fed corn oil, the cocoa butter threonine imbal— anced group had more lipid due to an increase in TG, CE, FC but not PL; a similar percent class distribution; a similar percent fatty acid composition; a similar amount of TGFA, CEFA and PLFA than the cocoa butter threonine supplemented group. Both imbalanced groups had more lipid than both supplemented groups but when cocoa butter was the source of fat in the diet, there was no difference in the amount of 75 PL, CEFA and PLFA. When corn oil rather than cocoa butter was the source of fat in the previously fed diet there was a difference in the incorporation of corn oil into the lymph. The corn oil threonine imbalanced group had less lipid due to a decrease in TG, CE, FC and PL; a similar percent lipid class distribu— tion; a similar percent fatty acid composition except for a decreased percent C18:1 in CE; less C18:1 and C18:2 TGFA, less C18:1 CEFA and the same amount of PLFA as the cocoa butter threonine imbalanced group. The corn oil threonine SUpplemented group had less lipid due to a decrease in TG, CE, FC and PL; a similar percent class distribution; a sim— ilar percent fatty acid composition except less C18:1 in CB; less C18:1 and C18:2 TGFA, less C20:4 CEFA and the same amount of PLFA as the cocoa butter supplemented group. Both cocoa butter grOUps had more lipid, as increased percent C18:1 in CE, more TGFA (C18:1 and C18:2) and the same amount of PLFA as the corn oil groups; the only effect that was dissimilar was more C18:1 CEFA with the cocoa butter imbal— anced group and more C20:4 CEFA with the cocoa butter supple— mented group. A threonine imbalanced diet as compared to a threonine supplemented diet previously fed to rats affects the incor— poration of cocoa butter into the lymph. When forced fed cocoa butter the corn oil imbalanced group had less total lipid due to a decrease in TG and CE but also there was an increase in PL; a similar percent class distribution and percent fatty acid composition; similar amounts of TGFA and 76 PLFA but less C20:4 CEFA than the corn oil supplemented group. When cocoa butter was forced fed to the cocoa butter threonine imbalanced group there was less lipid due to a decreased amount of TG and PL; similar percent class distribution, fatty acid composition, amounts of TGFA, CEFA and PLFA than the cocoa butter threonine supplemented group. Both imbalanced groups had less lipid, mainly less TG; similar percent class distributions, fatty acid compositions and amounts of TGFA, CEFA and PLFA than both supplemented groups. The only dif— ferences were more PL and less C20:4 CEFA with the corn oil imbalanced than with the corn oil supplemented group. When corn oil rather than cocoa butter was the source of fat in the previously fed diet there was a difference in the incorporation of cocoa butter into the lymph. The corn oil imbalanced group had more lipid due to an increase in TG and PL; a similar percent class distribution; a similar fatty acid composition except for more C18:1 in CE; similar amounts of TGFA and PLFA but less C18:1 CEFA than the cocoa butter imbalanced group. The corn oil supplemented group had more lipid due to an increase in TG, CE and PL; a similar percent class distribution; a similar percent fatty acid composition except less C18:1 in CE; similar amounts of TGFA and PLFA but more C18:2, C20:4 and less C18:1 CEFA than the cocoa butter SUpplemented group. Both corn oil groups had more lipid, TG and PL; a similar class distribution; similar percent fatty acid composition except less C18:1 in CE; similar amounts of TGFA, PLFA and less C18:1 CEFA, but the corn oil supplemented group also had more C18:2 and C20:4 CEFA than 77 the cocoa butter supplemented group. Rats fed the same diet but forced fed different fats had different fatty acid compositions of lymph lipid as would be expected however, they also had different amounts of fat incorporated into the lymph. l. Rats, previously fed a corn oil threonine imbalanced diet, forced fed corn oil had more lipid, an increase in TG; a similar percent class distribution; less saturated fatty acids in all lipid classes; more C18:2 and less C16, C18 and C18:1 in TG, more C18:2 in CE and more C18:2 and less C18 in PL; more C18:2 and less C18 TGFA, more C18:1 CEFA and similar amounts of PLFA as rats forced fed cocoa butter. II. Rats, previously fed a corn oil threonine supplemented diet, forced fed corn oil had more lipid, an increase in TG, CE and PL; a similar percent class distribution; less satu- rated fatty acids in all lipid classes; more C18:2 and less C16, C18 and C18:1 in TG, similar percent CE fatty acids, more C18:2 and.1ess C18 in PL; less C18 TGFA; less C18, C18:1 and C20:4 CEFA and similar amounts of PLFA as rats forced fed cocoa butter. III. Rats, previously fed a cocoa butter threonine imbalanced diet, forced fed corn oil had more lipid, an increase in TG, FC and PL; a similar percent class distribution; less satu— rated fatty acids in all lipid dlasses; less C16, C18 and C18:1 in TG, more C18:2 in CE, and more C18:2 in PL; more C18:1 and C18:2 TGFA, more C18:1 CEFA and more C18:2 PLFA than rats forced fed cocoa butter. IV. Rats, previously fed a cocoa butter supplemented diet, 78 forced fed corn oil had more lipid, an increase in TG and PL; 'a similar percent class distribution; less saturated fatty acids in all lipid classes; less C16, C18 and C18:1 in TG, similar percent fatty acids in CE and more C18:2 in PL; more C18:1 and C18:2 TGFA, more C18:1 CEFA and more C18:2 PLFA than rats forced fed cocoa butter. The differences between the rats fed corn oil imbalanced diets and forced fed corn oil and cocoa butter were similar to the differences between the rats fed corn oil supplemented diets and forced fed corn oil and cocoa butter. The differences between the rats fed cocoa butter imbalanced diets and forced fed corn oil and cocoa butter were similar to the differences between the rats fed cocoa butter supplemented diets and forced fed corn oil and cocoa butter. From this study, it has been shown that the incorporation of fat into the lymph is influenced by (l) the balance of amino acids in the diet, (2) the fatty acid composition of the previously fed diet fat, (3) the fatty acid composition of the fat forced fed and (4) a combination of these three variables. Since fat accumulated in the livers of rats fed threonine imbalanced diets, it was thought that by analyzing the lymph lipid, it might have been possible to see a correlation be— tween liver lipid and incorporation of fat into the lymph but this was not possible. When corn oil was forced fed, threonine influenced the lymph lipid differently than when cocoa butter was forced fed. However, it was evident that a threonine imbalance did affect the incorporation of fat 79 into the lymph. Perhaps feeding the diets to rats with lymph duct cannulas and analyzing the lipid recovered would show the effect of threonine more clearly than forced feed— ing of fat. 80 Table 10. Lymph lipid recovered after forced feeding corn oil. GrOUp ICO Lipig (mg) In: (hr) :12 9:2 1:9 as 29.22;. 4 179.7 1.2 1.0 12.6 194.5 8 158.5 1.3 1.2 11.8 172.8 12 126.6 1.2 1.0 10.4 139.2 30 164.7 5.1 2.6 25.8 198.2 Total 629.5 8.8 5.8 60.6 704.7 Group IICO 4 216.9 1.4 .9 10.1 229.3 8 132.5 .9 .7 8.6 142.7 12 76.1 1.0 .6 5.8 83.5 30 131.8 4.6 2.3 16.7 155.4 Total 557.3 777.9 4.5 41.2 610.9 Group IIICO 4 293.2 1.8 2.0 18.3 315.3 8 328.1 1.3 1.9 15.1 346.4 12 191.6 1.6 1.5 17.7 212.4 30 123.0 5.7 4.1 23.7 156.5 Total 935.9 10.4 9.5 74.8 1030.6 Group IVCO 4 318.2 2.6 1.7 22.3 344.8 8 230.0 1.5 1.2 16.2 248.9 12 93.8 1.2 .9 10.3 106.2 30 106.9 7.0 2.8 22.4 139.1 Total 748.9 12.3 6.6 71.2 839.0 Table 11. Lymph lipid recovered after forced feeding cocoa butter. Group ICB £3919 (mg) 111“: (hr) 219 SF; ES .111; 2955.1 4 137.5 1.2 1.0 14.7 154.4 8 139.7 1.0 .8 11.9 153.4 12 96.3 1.0 .8 12.9 111.0 30 95.0 7.0 2.3 22.0 126.3 Total 468.5 10.2 4.9 61.5 545.1 Gropp IICB 4 130.0 1.6 .8 12.1 144.5 8 156.3 1.2 .8 13.0 171.3 12 84.3 1.3 .7 6.3 92.6 30 124.4 8.5 2.4 21.9 157.2 Total 495.0 12.6 4.7 53.3 565.6 Group,IIICB 4 151.0 1.5 1.0 10.8 164.3 8 78.8 1.1 .7 6.6 87.2 12 32.9 1.1 .5 4.8 39.3 30 78.2 6.2 2.0 17.3 103.7 Total 340.9 9.9 4.2 39.5 394.5 GroppVIVCB 4 133.5 1.2 1.1 12.8 148.6 8 116.6 1.3 1.1 12.3 131.3 12 55.2 1.4 .7 7.1 64.4 30 87.5 6.3 2.1 15.1 111.0 Total 392.8 10.2 5.0 47.3 455.3 82 Table 12. Percent class distribution of lymph lipid recover— ed after forced feeding corn oil. Group ICO Lipig (weight %) 1111.32 (hr) :2 92 52 as 4 92.4 .6 .5 6.5 8 91.7 .8 .7 6.8 12 90.9 .9 .7 7.5 30 83.1 2.6 1.3 13.0 Group IICO 4 94.6 .6 .4 4.4 8 92.9 .6 .5 6.0 12 91.2 1.2 .7 6.9 30 84.8 3.0 1.5 10.7 GrOUp IIICO 4 93.0 .6 .6 5.8 8 94.7 .4 .5 4.4 12 90.2 .8 .7 8.3 30 78.7 3.6 2.6 15.1 Group IVCO 4 92.2 .8 .5 6.5 8 92.4 .6 .5 6.5 12 88.4 1.1 .8 9.7 30 76.9 5.0 2.0 16.1 Table 13. 83 ed after forced feeding cocoa butter. Group ICB 21E§.(hr) 4 8 12 30 Grogp IICB 4 8 12 30 Group IIICB 4 8 12 30 Gropp IVCB 12 30 89.1 91.0 86.8 75.3 89.9 91.2 91.0 79.2 91.9 90.3 83.7 75.4 89.8 88.8 85.7 78.8 Lipid (weight %) F2 .6 Percent class distribution of lymph lipid recover— 84 Table 14. Percent saturated fatty acids recovered from lymph lipid following corn oil forced feeding. Group ICO Lipig (%) 1‘3": (hr) "LG. C_E & 4 18.6 22.4 41.6 8 15.0 25.2 40.6 12 16.8 26.5 38.6 30 21.8 21.4 46.3 Group,IICO 4 17.8 32.2 44.6 8 16.2 33.6 43.9 12 19.3 29.7 52.2 30 29.3 28.9 49.9 Group IIICO 4 14.6 24.1 43.6 8 12.5 28.3 40.0 12 17.9 21.5 43.7 30 33.6 25.1 47.6 Group IVCO 4 13.2 20.6 52.4 8 13.3 26.8 49.1 12 15.4 25.5 42.7 30 31.2 33.5 57.4 85 Table 15. Percent saturated fatty acids recovered from lymph lipid following cocoa butter forced feeding. Group ICB Lipig (%) "Lime (hr) T._<.3_ C_E El: 4 59.8 33.3 50.2 8 58.0 40.3 45.9 12 46.0 35.3 49.1 30 40.4 32.2 51.4 GrOUp IICB 4 58.6 33.7 58.1 8 55.4 36.0 50.7 12 50.1 32.2 49.5 30 41.6 26.5 56.4 Group7IIICB 4 57.3 40.8 45.8 8 54.5 29.6 60.2 12 46.3 37.2 57.1 30 37.8 25.7 47.0 Group IVCB 4 61.6 34.2 49.0 8 56.9 36.6 64.2 12 49.8 39.3 57.2 30 40.7 26.0 59.3 86 Table 16. Percent fatty acids in the triglyceride fraction of lymph lipid after forced feeding corn oil. Group ICO Tim: (hr) Fatty acid (%) 4 _8_ lg §_O_ Ayg. C16 17 13 15 17 15 C18 2 2 2 5 3 C18:1 27 27 29 23 26 C18:2 55 58 54 49 54 C18:3 or C20:1 3 C20:4 4 Group IICO C16 16 14 16 23 17 C18 2 2 4 7 4 C18:1 27 29 27 21 26 C18:2 55 55 53 40 51 C18:3 or C20:1 4 C20:4 6 Group IIICO C16 12 10 14 23 15 C18 2 2 4 1.1. 5 C18:1 30 30 37 29 31 C18:2 55 58 46 28 47 C18:3 or C20:1 3 C20:4 6 87 Table 16 (cont'd.) Time (hr) Gross IVCO i E 12 2.9 AYE- C16 11 11 12 21 14 C18 2 2 4 10 5 C18:1 31 32 30 29 31 C18:2 55 55 55 28 48 C18:3 or C20:1 5 C20:4 6 Table 17. Percent fatty acids in the triglyceride fraction of lymph lipid after forced feeding cocoa butter. Group ICB $122 (hr) Fatty acid (%) g 8 1g 39 Ayg, C16 31 29 24 25 27 C18 29 29 22 16 24 C18:1 35 38 46 25 36 C18:2 6 4 8 22 10 C18:3 or C20:1 5 C20:4 7 Gropp IICB C16 29 26 27 25 27 C18 30 29 23 17 24 C18:1 36 39 41 28 36 C18:2 6 5 9 21 10 C18:3 or C20:1 5 C20:4 5 Table 17 (cont'd.) GropprlIICB C16 C18 C18:1 C18:2 C18:3 or C20:1 C20:4 GrOUp IVCB C16 C18 C18:1 C18:2 C18:3 or C20:1 C20:4 [©- 30 27 38 32 30 35 88 [CD 27 27 41 28 29 4O Iimg,(hr) _1_2 26 20 43 ll 26 24 43 ll 33 16 26 15 35 14 28 24 38 Table 18. Percent fatty acids in the cholesterol ester fraction of lymph lipid after forced feeding corn oil. GrOUp ICO T133 (hr) Fatty acid (%) 4 8 1g 39 C16 18 20 20 17 C16:1 5 7 6 4 C18 5 5 7 5 C18:1 18 20 22 18 C18:2 31 33 27 28 C20:4 25 14 18 29 19 30 22 89 Table 18 (cont'd.) fling (hr) Group IICO 4 8 T2 30 éyg. C16 24 23 23 23 23 C16:1 7 7 8 5 7 C18 8 ll 7 6 8 C18:1 18 22 21 19 20 C18:2 23 24 20 24 23 C20:4 21 14 21 24 20 GropprIIICO C16 17 20 16 18 18 C16:1 5 6 5 4 5 C18 7 8 6 8 7 C18:1 30 33 31 30 31 C18:2 25 21 29 17 23 C20:4 l6 l3 13 24 17 Group IVCO C16 14 18 16 22 17 C16:1 3 5 4 5 4 C18 7 9 10 11 9 C18:1 33 33 33 32 33 C18:2 18 21 20 10 17 C20:4 26 15 18 l9 l9 90 Table 19. Percent fatty acid in the cholesterol ester fraction of lymph lipid after forced feeding cocoa butter. Group ICB Tng (hr) Fatty acid (%) fl 8 £3 32 éyg. C16 21 23 20 20 21 C16:1 6 4 6 5 5 C18 12 17 16 12 14 C18:1 27 23 26 2O 24 C18:2 13 11 14 20 14 C20:4 21 22 19 22 21 Gropp IICB C16 18 19 18 16 18 C16:1 5 5 6 4 5 C18 15 17 14 10 14 C18:1 22 20 23 19 21 C18:2 17 12 15 22 17 C20:4 23 27 24 29 26 Group IIICB C16 21 14 23 17 19 C16:1 10 8 7 5 8 C18 20 16 15 9 15 C18:1 31 33 38 31 33 C18:2 5 9 6 l4 9 C20:4 13 20 12 25 17 Table 19 (cont'd.) Group IVCB C16 C16:1 C18 C18:1 C18:2 C20:4 lb 20 14 27 22 91 [CD 19 18 32 19 Time (hr) Tg 21 7 18 34 5 14 35 13 23 14 32 19 Table 20. Percent fatty acids in the phospholipid fraction of lymph lipid after forced feeding corn oil. Group ICO Fatty acid (%) C16 018 C18:1 C18:2 C20:4 Group IICO C16 C18 C18:1 C18:2 C20:4 25 17 36 14 26 19 30 17 I0) 25 16 12 41 26 18 13 34 10 Time (hr) .12 24 15 14 35 30 22 11 27 10 30 14 27 23 25 16 36 ll 27 20 10 29 13 92 Table 20 (cont'd) Time (hr) Group IIICO 4 8 T3 3g 31g. C16 23 20 22 23 22 C18 20 20 21 25 22 C18:1 15 15 14 ll 14 C18:2 32 40 35 25 33 C20:4 10 6 7 16 10 Group IVCO C16 24 24 21 29 24 C18 28 26 22 28 26 C18:1 13 15 15 14 14 C18:2 25 30 34 21 27 C20:4 10 6 9 8 8 Table 21. Percent fatty acids in the phospholipid fraction of lymph lipid after forced feeding cocoa butter. GropprCB Tng (hr) Fatpy acid (%) fl 3 33 3g 33g. C16 24 19 25 26 24 C18 26 27 24 26 26 C18:1 12 19 20 8 15 C18:2 22 27 23 21 23 C20:4 16 9 9 20 13 Table 21 (cont'd.) Group IICB C16 C18 C18:1 C18:2 C20:4 GrOUp IIICB C16 C18 C18:1 C18:2 C20:4 Group IVCB C16 C18 C18:1 C18:2 C20:4 [A 28 30 15 18 22 24 19 18 17 24 25 19 17 15 93 [CD 23 28 18 22 10 32 29 22 11 28 36 19 11 2133 (hr) 12 24 26 16 24 11 29 28 18 13 12 25 33 20 14 32 25 11 21 12 25 22 13 20 21 27 32 12 14 14 15 21 10 27 26 18 16 14 26 31 18 14 11 94 Table 22. TGFA after forced feeding corn oil. Grouj ICO 1133 (br) Fatty acid (mg/hr) 4 8 12 C16 7.2 5.0 4.4 C18 .8 .7 .7 C18:1 11.4 10.1 8.8 C18:2 23.6 22.1 16.4 C18:3 or C20:1 C20:4 Group IICO C16 8.2 4.4 2.9 C18 1.0 .7 .7 C18:1 14.2 9.2 5.0 C18:2 28.5 17.3 9.7 C18:3 or C20:1 C20:4 Group IIICO C16 8.6 8.2 6.6 C18 1.6 1.7 1.7 C18:1 21.2 23.1 16.7 C18:2 38.6 45.5 20.9 C18:3 or C20:1 C20:4 Table 22 (cont'd.) Group IVCO C16 C18 C18:1 C18:2 C18:3 or C20:1 C20:4 Table Gropp 23. TGFA after ICB Fatty acid (mg/hr) 4 C16 C18 C18:1 C18:2 C18:3 C20:4 Grogp or C20:1 IICB C16 C18 C18:1 C18:2 C18:3 C20:4 or C20:1 10.0 9.6 11.4 1.9 95 ICD 6.1 17.6 30.1 [CD 10.0 9.6 12.8 10.8 14.7 2.0 Time (hr) Time (hr) forced feeding cocoa butter. Table 23 (cont'd.) GrOUp IIICB 4 C16 10.9 C18 9.8 C18:1 13.8 C18:2 1.6 C18:3 or C20:1 C20:4 Gr0pp IVCB C16 10.1 C18 9.6 018:1 11.3 C18:2 1.0 C18:3 or C20:1 C20:4 Table 24. CEFA after corn Gropp ICO Fatty acid Lug/hr) 4 C16 C16:1 C18 C18:1 C18:2 C20:4 23 6 6 22 38 31 96 8.0 11.0 oil forced feeding. [CD 30 11 30 50 21 Time Time (hr) 27 34 23 21 34 36 25 39 28 Table 24 (cont'd.) GrouprICO C16 C16:1 C18 C18:1 C18:2 C20:4 Group IIICO C16 C16:1 C18 C18:1 C18:2 C20:4 Group IVCO C16 C16:1 C18 C18:1 C18:2 C20:4 lb» 36 10 12 26 34 32 34 11 15 61 49 32 39 18 92 49 71 ICD 23 11 22 24 14 30 13 49 31 20 27 14 49 31 22 97 Time (hr) I—-' M 21 21 27 11 55 51 24 2O 12 41 25 22 21 26 26 23 10 4O 23 31 37 19 53 17 32 23 26 23 29 12 51 39 27 31 16 59 31 37 Table 25. Group ICB Fatty acid (pg/hr) 4 C16 C16:1 C18 C18:1 C18:2 C20:4 Group IICB C16 C16:1 C18 C18:1 C18:2 C20:4 Group IIICB 98 CEFA after forced feeding cocoa butter. 27 7 15 34 16 27 32 27 38 30 41 C16 C16:1 C18 C18:1 C18:2 C20:4 31 15 3O 47 20 I U) 23 17 23 11 22 24 21 25 15 34 18 11 2O 41 12 25 Time (br) 16 26 14 19 27 22 35 23 36 28 18 48 15 2O 34 34 37 33 2O 37 44 58 25 14 46 21 37 17 29 19 26 29 23 34 28 42 26 11 21 46 12 24 99 Table 25 (cont'd.) 2132 (br) Group IVCB 4 8 19 99 899. C16 26 29 32 26 28 C16:1 10 9 ll 5 9 018 17 26 27 13 21 C18:1 34 48 52 53 47 018:2 11 ll 8 l9 l2 C20:4 28 28 21 34 28 Table 26. PLFA after forced feeding corn oil. Group ICO Time (hr) Fatty acid (pg/hr) 4 8 18 99 919. C16 580 550 460 300 473 C18 400 340 290 200 308 018:1 210 260 270 100 210 C18:2 840 890 770 300 700 C20:4 330 150 140 150 193 Group IICO 016 480 420 330 190 355 C18 360 280 240 160 260 C18:1 170 210 120 60 140 C18:2 560 540 290 170 378 C20:4 310 150 110 110 170 100 Table 26 (cont'd.) 11mg (hr) Group IIICO 4 8 18 89 4yg. 016 790 570 740 230 583 C18 690 550 710 240 545 C18:1 520 410 470 110 378 C18:2 1070 1110 1170 250 900 C20:4 330 170 220 160 220 Group IVCO C16 1000 710 400 270 595 C18 1180 770 420 260 633 C18:1 530 450 290 130 350 C18:2 1030 910 650 200 698 C20:4 420 180 170 70 210 Table 27. PLFA after forced feeding cocoa butter. Group ICB 11mg (hr) Fatty acid (pg/hr) 4 8 18 89 49g. C16 660 420 610 240 485 C18 720 600 570 230 530 C18:1 340 410 480 70 325 C18:2 590 590 540 190 478 C20:4 440 200 210 180 258 Table 27 (cont'd.) Group IICB C16 C18 C18:1 C18:2 C20:4 Group IIICB C16 C18 C18:1 C18:2 C20:4 GrouP IVCB C16 C18 C18:1 C18:2 C20:4 H}- 630 680 340 410 190 450 470 370 360 350 570 600 460 400 350 101 ICD 560 670 430 540 230 390 350 270 140 90 650 830 440 260 130 2133 (br) 12_ 280 300 190 280 130 260 260 160 120 100 330 430 260 190 110 220 100 190 .110 180 160 90 140 150 170 200 80 9O 90 265 355 165 320 310 223 190 173 430 515 310 235 170 102 (MG/HR) TOTAL LYMPH LIPID r, 0 4 8 12 30 TIME (HRS) Figure 1. Total lymph lipid after forced feeding corn oil (*grOUp I, OgrOUp II, Dgroup III,‘group IV.) 103 NIL 50!. 405 T0 mg/hr HOURS Figure 2. Triglyceride fraction of lymph lipid after forced feeding corn oil (*grOUp I, Ogroup II, Dgroup III, ‘ grOUp IV). 104 CE mg/hr mt II I r T H 4 8 12 30 HOURS Figure 3. Cholesterol ester fraction of lymph lipid after forced feeding corn oil ( ‘ group IV). group I, O grOUp II, B group III, FC mg/hr N I n V I I TIT—1" 4 8 12 30 HOURS Figure 4. Free cholesterol fraction of lymph lipid after forced feeding corn oil (* -“grOUp IV). group I, O grOUp II, UgrOUp III, 105 SI. 4.. E g 2.. .1 Q I" l l I I 1: I 0 4 8 12 30 HOURS Figure 5. PhOSpholipid fraction of lymph lipid after forced feeding corn oil (*group I, 0 group II,’ C] group III, ‘ group IV). 106 30+- 20,. III TOTAL LYMPH LIPID (MG/HR) *1 I I77 H'T—' 4 3 12 30 TIME (HRS) Figure 6. Total lymph lipid after forced feeding cocoa butter (*group I, 0 group II, DgrOUp III, ‘ group IV), 107 E a? E (D l l l I I I II I 0 4 8 12 30 HOURS Figure 7. Triglyceride fraction of lymph lipid after forced feeding cocoa butter (*group I, 0 group II, Dgroup III, ‘ group IV). 108 1- .4» E Fa .— E 3 .2 1 l I l l l T ' I II I 8 I2 30 HOURS Figure 8. Cholesterol ester fraction of lymph lipid after forced feeding cocoa butter (*group I, 0 group II, Dgroup III, ‘group IV). Jr E a .2- E E .L. HOURS Figure 9. Free cholesterol fraction of lymph lipid after forced feeding cocoa butter (*group 1,0 group II, Dgroup III, AgrOUp IV). 109 3. g» 2.. E I- a . r . I: r II 4 8 12 30 HOURS Figure 10. Phospholipid fraction of lymph lipid after forced feeding cocoa butter (*group I, 0 group II, Dgroup III, ‘ group IV). 110 ‘I" (D 9 O < 404-— )- ’— F— < LI. 0 I— UJ ’— ‘< a: Z) ’2 20 (I) % 3 II" I I I I I: T II 4 8 I2 30 TIME (HRS) Figure 11. Percent saturated fatty acids of the triglyceride fraction of lymph lipid after forced feeding corn oil (*group I, 0 group II, DgrOUp III, ‘group IV). 111 I 40p (1) 9 O .< >_ I- p... I'- < u. 8 30- .— E 0 ID *— 3 p. I 20- 44 I T 1" r I I I II 4 - 8 I2 30 TIME (HRS) Figure 12. Percent saturated fatty acids of the cholesterol ester fraction of lymph lipid after forced feeding corn oil (*group 1: Ogroup II, B group III, A grOUp IV). 112 b- (0 9 L) .< ,_ so; I- I- 4: U. C) “J Ib- P v .< (I I_J I) 2 .0 «Ir 39 ._ [L I I If I 1 I 1* 0 4 8 I2 30 TIME (HRS) Figure 13. Percent saturated fatty acids of the phospholipid fraction of lymph lipid after forced feeding corn oil (* group I, 0 group II, Dgroup III, A group IV). 113 U) 9 r 2 55.... >- I— p. 4: U. C3 DJ E ‘5 ' C I— “II '( (D °\° I I... I I I H r 0 4 8 12 30 TIME (HRS) Figure 14. Percent saturated fatty acids of the triglyceride fraction of lymph lipid after forced feeding cocoa butter (*group I, Ogroup II, D grOUp III, ‘ group IV). 114 45.. ‘8 6 ‘< , __ I E D (II I: 35- ”'.‘\ < ,9«/ '53: ‘ o 3‘, \o P I O 25- - T I I r ”I n 4 I 12 3 TIME (HRS) Figure 15. Percent saturated fatty acids of the cholesterol ester fraction of lymph lipid after forced feeding cocoa butter (*group I, 0 group II, 0 group III, ‘ group IV). 115 65- (D E 0 < E ‘ ' < u. C n o _ g 55.. O . < C! 3 I '< w e o ’ 32 77"IIIVJ !! ' I 45. I If I I I II I II 4 8 I2 30 TIME (HRS) Figure 16. Percent saturated fatty acids of the phOSpholipid fraction of lymph lipid after forced feeding cocoa butter (*group I, Ogroup II, [jgroup III, A grOUp IV). 116 20.. E” III. E f3 I . no; - ., 8 12 '30 HOURS TGFA after forced feeding corn oil GroupI Figure 17.. (Dc16, .Cl6: 1, OClB, .C18. 1, ‘018. 2, Aczo. 4). an. 20. E” III- < I E m n \ T T 12 "30 HOURS Figure 18. GrGUp II, TGFA after forced feeding corn oil (DC16,.C16.1,0C18, .C18.1,AC18:2, Ac20.4). 117 40.. an- 20- g» III- E (D " #0 6L 0— I I I o 4 8 12 30 HOURS Figure 19. Group III, TGFA after forced feeding corn oil (D C16, I C16:1, OC18, . C18:1,‘ C18:2, A C20:4). 118 40 .. 30 .. 201. E 10L <1 I; I m _ 1F It ‘ II 4 8 12 30 HOURS Figure 20. GrOUp IV, TGFA after forced feeding corn oil (D C16, I C16:1, OC18, .C18:1, ‘C18:2, A C20:4). 119 10(- a! I TG FA mg/hr L I o 4 a 12' 730' HOURS Figure 21. Group I, TGFA after forced feeding cocoa butter (CI C16,. C16:1, Oc18, .c18:1, A C18:2, A C20:4). 15.. 10.. In I TG FA mg/hr HOURS Figure 22. Group II, TGFA after forced feeding cocoa butter (DC16, I C16:1, Oc18, .C18zl, Ac18:2, AC20:4) 120 Z?- T '5 T a! TGFA mg/hr I I v II 4 3 I2 30 HOURS Figure 23. Group III, TGFA after forced feeding cocoa butter ([3 C16,. C16:1, OC.18,.C18:1, AC18:2, A 020:4). 5:. on E 5.. '1 u. (D I— HOURS Figure 24. Group IV, TGFA after forced feeding cocoa butter (DC16, .C16z1, OC18,.C18:1, ‘C18z2, AC20:4). 121 u e T CEFA pg/hr I a...) «b... ”—1 d N w :3 HOURS Figure 25. Group I, CEFA after forced feeding corn oil (DC16, I Cl6:l, O C18,.C18:1,A C18:2, A C20:4). e 301 < " l‘g‘ . fl: . A (J 10- (. Illlllllfie'.-_8_ - __ 0'7 I HOURS @— d N a»: C: Figure 26. Group II, CEFA after forced feeding corn oil ([3 C16,.C16:l,OC18,.C18;1, Ac18:2, A C20:4). 122 CEFA pg/hr HOURS Figure 27. Group III, CEFA after forced feeding corn oil (D 016, IC.16:.1, 0018,. C18:1, A C18:2, A C20:4). 123 90. III- A 50. f; 301 E ° IL I I I I 712 I o 4 8 12 30 HOURS Figure 28. Group IV, CEFA after forced feeding corn oil ([3 016,. C16:1, 0 C18, .c18:1, A Cl8:2, A C20:4). 124 u e I C E FA ug/hr "I Figure 29. Group I, CEFA after fo ced feeding cocoa butter (CI C16,. 016:1, 0C18,. C18:1, C18:2,AC20:4). HOURS Il\\ ‘ 'é 30.. \NAI I 23 I.~ !,r E _ . \.‘" . g m M (I 4' II 12' "36 HOURS Figure 30. Group II, CEFA after forced feeding cocoa butter (CI C16, .Cl6:1,0C18,.C18:l,AC18z2, Ac20:4). 125 E 30 \ no a. .< b 0 III HOURS Figure 31. Group III, CEFA after forced feeding cocoa butter (Dom,Ic16:1.0c18,.c18:1,Ac18:2,Ac20:4). ‘ A VA. S 7— r- E 30I— N I E- . L) 10 <-ll.r 1 l d I I i '1 I 4 II I 30 HOURS Figure 32. Group IV, CEFA after forced feeding cocoa butter (0916, I C16:1, 0C18,. C18:1, A C18:2, Ac20:4). 126 900 - IIIII I 500 b g 300... ‘ DD 3. E A. -4 “1::* °' 100 \g [I I I I I7 I r71 I) 4 8 I2 30 HOURS Figure 33. Group I, PLFA after forced feeding corn oil (U C16, I C16:1, Oc18, I C18:1, Ac.18:2, Ac20:4). 127 PLFA ugfln I HOURS Figure 34. Group II, PLFA after forced feeding corn oil (CI C16, I616:1,Oc18,.c18:1,Ac18:2,A C20:4). 128 10001. 500 PLFA pg/hr 100- T 4 HOURS 6- a— d N- a a Figure 35. Group III, PLFA after forced feeding corn 011 ([3016, I C16:1,0C18,.C18:1, Ac18:2, A C20:4). 129 10110 .. 5110 _ PLFA pg/hr ‘ e e I I I H I II 4 12 30 HOURS cad Figure 36. Group IV, PLFA after forced feeding corn oil (1:1 C16, I C16:1, OC18,.C18:1, A018:2, A C20:4). 130 PLFA pg/hr 4' s 12 30 HOURS =3d Figure 37. Group I, PLFA after forced feeding cocoa butter (DC16, .Cl6:l, OC18, .c18:1, A C18:2, Ac20:4). 131 PLFA pg/hr a... 9.... ‘ N w e I 4 HOURS Figure 38. Group II, PLFA after forced feeding cocoa butter (DC16,.C16:1,20018,.C18:1,‘ C18:2, Ac20:4). PLFA pg/hr I 4 HOURS 6-1 ”-4 d N a a Figure 39. Group III, PLFA after forced feeding cocoa butter (CI C16, I C16:1,0 C18,. c18:1, A018:2, Aczo:4). 132 100 ,. PLFA 118/hr I 4 HOURS - qr- «Iqb- .- Figure 40. GrOUp IV, PLFA after forced feeding cocoa butter (C1 C16, Ic16:1. 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