EFFECTS OF THE STRUCTURAL ARRANGEMENT OF DIETARY TRIGLYCERIEDES ON THE LIPID COMPOSITION OF RAT TISSUES Thesis far the Deg!” o§ Ph. D‘ MICHIGAN STATE UNIVERSITY Charles Eéwarcf Eéson i964 IHESIS This is to certify that the thesis entitled EFFECTS OF THE STRUCTURAL ARRANGEMENT OF DIETARY TRIGLYCERIDES ON THE LIPID COMPOSITION OF RAT TISSUES presented by Charles Edward Elson has been accepted towards fulfillment of the requirements for Ph.D. Food Science degree in firm/96w Major profésdr Date September 8, 1964 0-169 LIBRARY Michigan State University ABSTRACT EFFECTS OF THE STRUCTURAL ARRANGEMENT OF DIETARY TRIGLYCERIDES ON THE LIPID COMPOSITION OF RAT TISSUES by Charles Edward Elson In order to determine the effects of dietary lipid structure on lipid metabolism in the rat, a lipid fraction containing 33 percent polyunsatur- ated fatty acids at thedif-ester position was isolated from lard (diet B). A second lipid containing an equal amount of polyunsaturated fatty acids esterified at theygf-position was isolated from soybean oil (diet C). The effects of these diets on rat feces, subcutaneous, perirenal, blood serum, liver and aorta lipids were compared to those of an iso-caloric, fat-free diet (diet A). Three groups of four mature males were maintained on these diets for a two week period. Liver and blood serun free and total cholesterol were determined colorimetrically. Gravimetric procedures were used to determine total lipid, neutral lipid and phospholipid content of these tissues. The gas chromatograph was used to identify the methyl esters of fatty acids from phospholipids and each neutral lipid fraction which had been separated by thin-layer chromatographic procedures. When dietary lipids supplied 40 percent of the calories, feces and blood serum lipid levels were elevated. The polyunsaturated fatty acid (PUFA) content of feces produced by diet C was slightly elevated. All diets produced highly unsaturated body fats. The liver and blood choles- terol levels were lowered by feeding diet C, this decrease occurring in the free cholesterol fraction. Liver phosPholipid, and triglyceride PUFA patterns were quite similar in the absence of dietary effects. In Charles Edward Elson blood serum, the phospholipids differed significantly in their composi- tion as compared to the neutral lipids. It was generally found that the PUFA content of the liver and aorta lipids was quite similar while the blood lipids contained significantly lower amounts of PUFA. A possible mechanism for the blood serum cholesterol lowering effects of vegetable oils, based on these experimental observations and conclusions of other investigators is suggested. EFFECTS OF THE STRUCTURAL ARRANCEMENT OF DIETARY TRICLYCERIDES. ON THE LIPID COMPOSITION OF RAT TISSUES By Charles Edward Elson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR.OF PHILOSOPHY Department of Food Science 1964 L? I \, ‘.\"\0 u'.) 1 ACKNOWLEDGMENTS The author wishes to express his deepest appreciation for the guid- ance, support and encouragement of Professor L. J. Bratzler throughout this study and for his critical review of this manuscript. Sincere appreciation and thanks are expressed to Dr. L. R. Dugan for his many suggestions, assistance and willing counsel. The author is further in- debted to Dr. A. M. Pearson for his aid and instruction in the operation of laboratory equipment. Appreciation is expressed to Dr. B. S. Schwei- gert for his advice in planning this study and his readiness to counsel the author when problems arose during his graduate study. The author is also indebted to the other members of his guidance committee, Dr. R. J. Evans and Dr. E. P. Reineke for their time and effort in his behalf. The financial aid provided by a Public Health Service Traineeship is esPecially appreciated by the author and his family. ii TABLE OF CONTENTS Page MRDD IJCT ION O O O O O O O O I O O O O O O I O O O O O O O C O O 1 REVIEW OF LITERATImE C O O O O O O O O O O O O O O O O O O O O O 3 EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . . . . . . . . . 21 Source of animals . . . . . . . . . . . . . . . . . . . . . 21 Extraction of lipids . . . . . . . . . . . . . . . . . . . 23 Thin-layer chromatography . . . . . . . . . . . . . . . . . 25 Preparation of methyl esters . . . . . . . . . . . . . . . 26 Gas chromatography . . . . . . . . . . . . . . . . . . . . 28 Cholesterol determinations . . . . . . . . . . . . . . . . 29 Statistical analysis . . . . . . . . . . . . . . . . . . . 31 RESMTS m DlscmSION . . O O O O O I O O C O O O O O O O O I O 32 SW MD CONCLIJSIONS . O O O O O O O O O O O O O O O O O O O 65 BIBLIOGRAHIY O O O O O O O O O O O O O O O I 0 APPENDIX C O O O O I O O O O O O O O O O O 0 iii Table 10 11 12 13 14 15 16 17 LIST OF TABLES Fatty acid composition of fractions crystallized from ace tone . . O C C . O O O O O O C O I O C C O O O O C O . Dietary record . . . . . . . . . . . . . . . . . . . . . Analysis of variance of lipid content of rat feces . . . ‘Multiple range test of rat feces lipid content . . . . . Fatty acid composition of rat feces neutral lipids . . . Analysis of variance of the neutral lipid content of feces 11p ids O O O 0 O O O O O O O O O C O O O I O O O O O O 0 Analysis of variance of polyunsaturated fatty acid content of subcutaneous and perirenal fats . . . . . . . . . . . Fatty acid content of subcutaneous and perirenal fat . . Analysis of variance of liver weight (g liver/100 g body Weight) C O C O O C O C I . O O C C O O O O I O O O O O 0 Analysis of variance of lipid content of liver tissue (g/100 g wet weight) . . . . . . . . . . . . . . . . . . Analysis of variance of neutral lipid content of rat liver lipids O O O O O O I I O O O O O O I O O I I O O O O O 0 Cholesterol content of rat liver . . . . . . . . . . . . Analysis of variance of rat liver cholesterol measurements 'Multiple range analysis of dietary effect on liver cholesterol levels . . . . . . . . . . . . . . . . . . . Fatty acid composition of lipid fractions isolated from livers of rats fed diet A. . . . . . . . . . . . . . . . Fatty acid composition of lipid fractions isolated from livers of rats fed diet B . . . . . . . . . . . . . . . . Fatty acid composition of lipid fractions isolated from livers of rats fed diet C . . . . . . . . . . . . . . . . iv Page 22 32 33 33 35 36 37 38 39 39 4O 40 41 41 43 44 45 Table 18 19 20 21 22 23 24 25 26 27 28 29 3O 31 32 Analysis of variance of the polyunsaturated fatty acid com- position of lipid fractions isolated from livers of rats fed diets A, B and C O C O O I O O O C C O O C C I O O O IMultiple range analysis of the polyunsaturated fatty acid content of fractions isolated from rat liver . . . . . . Analysis of variance of blood serum lipid content (mg/ml) Analysis of variance of neutral lipid content of rat blood serm lipids I O O O O O O O O O I O O O O O O O O O O O iMultiple range analysis of dietary effects on proportion of neutral lipids in rat blood serum lipids . . . . . . . . Cholesterol content of rat blood serum . . . . . . . . . Analysis of variance of rat blood serun cholesterol levels Analysis of variance of rat blood serum free cholesterol levels I O O O I O O O I O O O O O O O O O O O O O O O 0 Analysis of variance of the phosPholipid content of rat blood germ O O O O O O I O O O O I O O C O O O O O O O 0 Fatty acid composition of blood serum lipid fractions from rats fad diet A O O O O O O O O O I O O O O O O O C C O 0 Fatty acid composition of blood serum lipid fractions from rats fed diet B O O O O O O O O O O O O O O O O O O O O 0 Fatty acid composition of blood serum lipid fractions from rats fed diet C . . . . . . . . . . . . . . . . . . . . . Analysis of variance of the polyunsaturated fatty acid composition of lipid fractions isolated from rat serum of rats fed diets A, B and c I O O O O O O O I O O O O O I 0 ‘Multiple range analysis of the polyunsaturated fatty acid content of rat serum lipid fractions . . . . . . . . . . Fatty acid composition of triglycerides, cholesterol esters, monoglyceride-free fatty acids and phosPholipids isolated from pooled aorta samples . . . . . . . . . . . . . . . . Page 46 46 47 48 48 49 49 50 51 52 53 54 51 55 56 Table 33 34 35 36 37 38 39 4O 41 42 Page Analysis of variance of the polyunsaturated fatty acid content of lipid fractions isolated from aortas of rats fed diets A, B and c C C O O O C O O C O O O O O O O O O O 56 Analysis of variance of the polyunsaturated fatty acid content of triglycerides from rat blood, liver and aorta . 57 iMultiple range test of polyunsaturated fatty acid content of triglycerides from rat blood, liver and aorta . . . . . 57 Analysis of variance of the polyunsaturated fatty acid content of rat liver, blood and aorta cholesterol esters . 58 iMultiple range test for polyunsaturated fatty acid content of the cholesterol esters of rat blood, liver and aorta . . 58 Analysis of variance of the polyunsaturated fatty acid content of rat liver and blood diglycerides . . . . . . . . 59 Analysis of variance of the polyunsaturated fatty acid content of monoglycerides-free fatty acid fraction of rat liver, b100d and aorta O O O O O O O O O O O O O O O C C 0 60 ‘Multiple range test for polyunsaturated fatty acid content of the monoglyceride-free fatty acid fraction of rat blood, liver and aorta O O O O O O O O O O O O O C O I O O O O O O 60 Analysis of variance of the polyunsaturated fatty acid content of phosPholipids isolated from the serun, livers and aortas of rats fed diets A, B and C . . . . . . . . . . 61 'Multiple range test for polyunsaturated fatty acid content of the phospholipid fractions isolated from rat serum, liver and aorta I O O O O O O O C O O O O O C O O O O O O C O O O 61 vi LIST OF APPENDIX TABLES Appendix A Lipid content of rat feces . . . . . . . . . . . . . . . . B Fatty acid composition of triglycerides isolated from rat feces O O O O I O O O O O I O O O O O O O O O O O O O O O O C Fatty acid composition of diglycerides isolated from rat feces O I O O O O O O O O O O O C O O O O O O O O O O O O O D Fatty acid composition of monoglyceride-free fatty acids isolated from rat feces . . . . . . . . . . . . . . . . . . E Fatty acid composition of rat subcutaneous fat . . . . . . F Fatty acid composition of rat perirenal fat . . . . . . . . GLiveranalyses..............oo..oo.. H Fatty acid composition of triglycerides isolated from rat liver 0 O O O C O O O O I O O O I O O I O O O O O O O O O O I Fatty acid composition of cholesterol esters isolated from rat liver 0 O O O O O O O I O O O O O O O C O O O O O O O O J Fatty acid composition of diglycerides isolated from rat liver 0 C C O I C C O O O C O O O O O O O I O I O O C O O 0 K Fatty acid composition of monoglyceride-free fatty acid fraction isolated from rat liver . . . . . . . . . . . . . L Fatty acid composition of phOSpholipids isolated from rat liver 0 I O I O O O O O O O O O O I O O O O I O O O O O O O M BIOOd ser‘m lipids O O I O I O O O O O O O O O O O O O O O N Fatty acid composition of triglycerides isolated from rat b100d germ O I I O O O O I O I O O C O O O O O O O O O O O 0 Fatty acid composition of cholesterol esters isolated from rat b100d Serlm O I O O O O O I I O O O O O O O O O O O O O P Fatty acid composition of diglycerides isolated from rat bIOOd serlm O O O O O C O C O I O O O I O O O O O O O I O 0 vii Page 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 Appendix Page Q Fatty acid composition of monoglyceride-free fatty acids isolated from rat blood serum . . . . . . . . . . . . . . . 92 Fatty acid composition of phospholipids isolated from rat bIOOd serlm O O O O O O O O O O O I O O O I O O I O O O O O 93 Composition of basal and pretest diets . . . . . . . . . . 94 viii INTRODUCTION Research in the general area of public health over the past 15 years has greatly influenced today's diet, especially the lipid components of the diet. This research has generally indicated that the substitution of vegetable oils for animal fats, the addition of linoleic acid to the diet, or the reduction of fat intake would lower serum cholesterol levels. High serum cholesterol levels have, on the basis of extensive papulation stu- dies, been associated with coronary thrombosis susceptibility. Naturally, this work has had a vast influence on present dietary patterns. The food industry has seen a steady decrease in the consumption of animal fats and an increase in the consumption of fats from vegetable sources. New pro? ducts have been developed and widely advertised on the basis of their polyunsaturated fatty acid content. New markets have been created for agricultural products such as safflower seed while other markets are being drastically reduced. In view of the economic effects and perhaps, healthful effects, of this research, the present study was initiated to determine if the poly- unsaturated fatty acid content of vegetable oils is the sole factor re- sponsible for the cholesterol lowering effects. Two fats were selected, each containing 33 percent polyunsaturated fatty acids. One of the fats was from a vegetable source and contained the unsaturated fatty acids primarily at the/5’-position of the trigly- ceride molecule. The other was from an animal source and its unsaturated fatty acids were primarily esterified at thea1_-position. These fats were -1- -¢—. —.v~ introduced into rat diets in equal amounts. The effects of these diets were compared with those of an isocaloric, fat free diet in order to study their effects on blood serum cholesterol levels, liver cholesterol levels, and the fatty acid composition of lipid fractions isolated from blood, liver, aorta, lipid pools and feces. REVIEW OF LITERATURE Weinhouse and Hirsch (1940) described atherosclerosis or atheroma as essentially a focal and predominantly an intimal change and should not be confused with generalized arteriosclerosis or other circulatory disorders. Since the essential components of the human atherematous plaque appear to be intimal fibrous and lipid infiltration, it is there- fore not unnatural that most theories on its pathogenesis center around either fat, fibrin or both, but considerable controversy exists as to which component is primary and which is secondary. The filtration theory, as described by Page (1954) implies that lipid infiltration of the intima is the primary process and that the fibrous tissue present indicates the reaction to the presence of fat. This theory postulates that the centrifugal force of the circulation drives fat into contact with arterial intima. Over the years, lipid de- posits accumulate, but this is accelerated or intensified in the presence of blood lipids in excessive amounts or abnormal forms, if the intralum- inar or lateral filtration pressure is high or if the endothelium of the vessel walls is abnormally permeable. According to weinhouse and Hirsch (1940), and Rabinowitz (1960), a satisfactory explanation is at hand for the particular localization of the plaques at sites of pressure or velo- city change within the circulatory system, and for the increased severity of atherosclerosis in the presence of hypertension and such disorders of lipid metabolism as diabetes mellitus and essential xanthomatosis. The steady encroachment of the plaque on the lumen of the vessel favors -3- super-added thrombosis from circulatory stasis. They stated that choles- terol is present in high concentrations in the lipids of the atheromatous plaques, the chemical nature of which resembles that of blood plasma. Swell 25.91, (1960b) reported that in humans with coronary artery disease, the media and serum had approximately the same cholesterol es- terified fatty acid composition. Linoleic acid accounts for 40 percent of the total fatty acids. The thickened intima and the plaques had nearly the same cholesterol esterified fatty acid composition and contained sig- nificantly less linoleic and arachidonic acids and more oleic acid and saturated fatty acids. Palmitic and oleic acids comprised from 72 to 75 percent of the total fatty acids in the triglyceride fraction. The per- centages of palmitic and oleic acids were strikingly constant and were within the ranges of 29 to 35 percent and 39 to 46 percent, respectively. A relatively high level of stearic acid, 11.1 percent, was found in the media triglyceride fraction. A much lower level of polyunsaturated fatty acids was found in the media triglyceride fraction than in the esterified cholesterol fraction. Swell 35.31, (1960b) also reported that the phOSpholipid fraction was characterized by a high level of saturated fatty acids and significantly more arachidonic acid. Palmitic, stearic, and oleic acids accounted for 50 to 59 percent of the total fatty acids. . In another report, Swell gt_§1, (1960a) found that tLe composition of cholesterol esterified fatty acids of aortic media was very similar to that of serum in elderly humans with atherosclerosis. The plaque material contained a greater prOportion of saturated and monoenoic acids than did I// the serum or the media. In the media and serum, the major cholesterol ester contained linoleic acid while in the plaques and liver, cholesterol was primarily esterified with oleic acid. Evrard et al. (1962) found dissimilarities between the cholesterol esterified fatty acid patterns of plasma and atheroma. hey concluded that thisexeludes the likelihood that the cholesterol esters accumulate in the aortic intima by a simple random deposition of the plasma choles- terol esters. Swell g£_§g, (1962) stated that the aortic cholesterol esters were not of constant composition but were dependent upon the dietary fat. They suggested that derangements of cholesterol oleate metabolism may be important factors in the deposition of cholesterol esters in the aortas and liver. Mukherjee §£_al, (1958) stated that the effects of feeding different dietary fats on rat liver cholesterol levels were apparent within one weeks time. Swell g£_gl, (1960a) indicated that the serum.and liver of normal rats were distinctly different in their cholesterol esterified fatty acid composition. Swell g£_§l, (1960c) found that up to 70 percent of the total cholesterol esterified fatty acids in the serum were unsaturated, the main serum cholesterol esterified fatty said being arachidonic acid. The major fatty acids of the liver cholesterol esters were of the satur- ated and monoenoic type with palmitic acid predominating (32.5 percent). They concluded that liver and lymph cholesterol esters play an important regulatory role in blood cholesterol level and composition. Klein (1958) and Avigan and Steinberg (1958) had previously reported that rat plasma and liver cholesterol esters differ markedly in fatty acid composition. The polyunsaturated fatty acid composition of the plasma cholesterol esters was much higher than in the liver esters. Also, the cholesterol levels did not follow the same relationship to dietary fats. The liver cholesterol concentration was found to be higher when diets contained a higher prOportion of unsaturated fatty acids while the inverse was true of the blood plasma. The work of Russell g£_§1, (1962) with corn oil and lard substantiated these results. On diets with 10 percent corn oil or lard, rat livers contained 3.33 and 2.65 mg choles- terol per gram, respectively, while the cholesterol levels in the blood serum.were 71 and 110 mg percent. When the dietary fat was increased to 30 percent, the corn oil diet produced 4.37 mg/g and 67 mg percent while the lard diet produced 3.23 mg/g and 100 mg percent in the liver and blood serum. Reiser gt a}, (1963) also were in agreement that diets high in unsaturated fatty acids produced higher cholesterol levels in rat livers. On a low fat diet, the liver cholesterol level was only 2.3 mg/g, satur- ated triglycerides produced 4 and 5 mg/g and trilinolein and safflower oil diets, 7.3 and 7.8 mg/g, respectively. They stated that these re- sponses are compatible with the observed increases in fecal steroids which accompany decreases in serum cholesterol caused by unsaturated dietary fat, since the major route of cholesterol from serum to intestine, as cholesterol and bile acids, passes through the liver. They believed an increase in liver cholesterol levels on unsaturated fat diets supports the hypothesis that the mechanics by which unsaturated acids maintain lower serum cholesterol is by their influence on transport, either by forming labile esters, or by forming unsaturated phOSphatides which may aid in the transport of cholesterol esters across cell membranes. Gerson g£_gl, (1961) were in complete agreement. Okey ggpgl, (1957) and Linazasoro g£_§1, (1958) were unable to find differences between diets on liver cholesterol levels. Aftergood gt_§1, (1956) reported that rats fed 15 percent lard diets had higher liver cho- “‘ lesterol levels than those fed 15 percent cottonseed oil diets. Their w" reasoning was that possibly the difference in action of the two fats was due to the greater quantity of essential fatty acids found in cottonseed oil over that which occurs in lard. These essential fatty acids may be required for normal cholesterol transport and metabolism; an inadequate supply will tend to cause an accumulation of cholesterol in the liver. Alfin-Slater g£.§1, (1954) stated that the cholesterol concentration in the liver of rats on low essential fatty acid diets were increased. This increase over the liver cholesterol levels in rats fed 12.5 percent corn oil occurred almost exclusively in the ester fraction. The rats had free cholesterol levels of 1.75 and 1.85 mg/g and cholesterol ester levels of 0.31 mg/g and 1.30 mg/g on 12.5 percent corn oil and fat free diets, re- spectively. They concluded that cholesterol esters with saturated fatty acids may be unavailable for proper metabolism and transport. Mnkherjee and Alfin-Slater (1958) also reported that diets deficient in essential fatty acids resulted in an increased cholesterol level in rat livers. This accumulation of liver cholesterol induced a decrease in cholesterol synthesis. When essential fatty acids were added to the diet, liver cholesterol levels returned to normal and cholesterol synther sis increased. Reiser gt_gl, (1960) reported that miniature pig plasma and liver cholesterol esters were comparable as to fatty acid composition. Karmen g£_gl, (1963) may be in agreement since they reported that in humans the cholesterol esterified fatty acids were primarily of endogenous origin and that the esters showed a marked specificity for oleic acid. 0n the other hand, Swell gt_§1, (1964) reported that the cholesterol esters synthesized in rat liver microsomes bear no relationship to the cholesterol esters present in rat serum. They believed that it is possi- ble that only certain esters are released, to an appreciable extent, into the blood. In fasted rats, Swell g£_gl, (1960c) reported that cholesterol esters from serum and liver had 12.4 and 32.5 percent palmitic acid, 2.6 and 21.9 percent stearic acid, 9.8 and 20.1 percent oleic acid, 19.5 and 12.2 percent linoleic acid and 50.0 and 8.8 percent arachidonic acid. This was in agreement with Klein and Martin (1959) who also reported differ- ences in liver and serum cholesterol esterified fatty acids. These authors speculated that the differences might be due to varying turnover rates for the different esters. Mukherjee g£_§l, (1957) reported that oleic acid was absent from serum cholesterol esters while Klein and Janssen (1959) published that serum cholesterol esters contained 59 percent oleic acid. These differ- ences might have been due to analytical technique, or, more likely to the state of the rat when samples were taken (Swell_gt_§l., l960c).Trans- esterification reactions between phospholipids and cholesterol esters may occur (Glomset 3531., 1962). Brot 95g}, (1964) and Aftergood g; §_1_. / (1956) are in agreement that approximately 75 percent of the liver cho- lesterol exists in the free form. Reiser gt_§1, (1960) reported that triglycerides from the plasma, liver and depot fat of the miniature pig were not related as measured by their fatty acid composition. Furthermore, only depot fat triglycerides responded to differences in the lipid composition of the diet. The tri- glyceride composition of polyunsaturated fatty acids in perirenal fat, plasma and liver on low fat, 20 percent myristyl oleyl linoleate and 20 percent cottonseed oil were as follows: Perirenal Plasma Liver Dienoic Trienoic Dienoic Dienoic Trienoic Low fat 4.0 0.7 7.8 3.2 0.6 ZOZ‘M-OéL 3.8 0.9 8.2 2.3 0.0 20% 030 33.5 1.1 9.7 1.6 0.0 Since the level of dienoic fatty acid was twice as high in blood plasma as in other tissues, Reiser gt 31, (1960) concluded that the plasma tri- glycerides probably did not originate in the liver. In the presence of readily available carbohydrates, saturated fatty acids were utilized for energy to a greater degree than unsaturated fatty acids and therefore, were stored in depot fats to a lesser degree. Okey 35 21. (1962) were in agreement that plasma and liver lipids were less subject to changes than the adipose tissue when various diets were fed. They also reported that lauric and myristic acid tended to accumulate preferentially in the adipose tissue. -10- Conversely, Swell g£_§1, (1962) reported that, in rabbits, the nature of the dietary fat incorporated into a high cholesterol diet in- fluenced the serum and tissue cholesterol ester and triglyceride fatty acid composition. In an analysis of the phospholipid fraction from rat liver, Getz g£_§;, (1961) found 62 percent linoleic acid, 15.6 percent oleic acid and 8.9 percent palmitic acid. This work was in agreement with Dittmer and Hanahan (1959) and Macfarlane g£_al, (1960). 'When Patil and Magar (1960) fed vegetable fats to rats, the liver levelsof dienoic acid in the cholesterol ester and phospholipid fractions ‘wereelevated. The phospholipid fraction was found to have a higher con- tent of tetraenoic, pentaenoic and hexaenoic acids than the cholesterol ester and triglyceride fractions. Nestel and Steinberg (1963) reported rat liver glyceride and phospho- lipids to have the following composition; myristic acid, 1 percent each, palmitic acid 23 and 14 percent, palmitoleic acid, 3 and 7 percent, stearic acid 4 and 15 percent, oleic acid 36 and 27 percent, linoleic acid 32 percent each and arachidonic acid 1 and 5 percent, respectively. Except for differences in the stearic acid levels, these figures are in agreement with Getz g£_§l, (1961). In the analysis of liver free fatty acid and diglyceride fractions, Getz g£_§l, (1961) found high levels of palmitic acid030.6 and 23.9 per- cent, stearic acid,10.5 and 6.6 percent, oleic acid, 25.0 and 28.8 percent and linoleic acid, 19.5 and 34.1 percent, respectively. These levels for free fatty acids are in agreement, generally, with Nestel and Steinberg (1963) and Rose 95 31, (1964). -11- Nichaman §£_§l, (1963) reported that the cholesterol esters of human blood serum contained 15.5 percent palmitic acid, 19.6 percent stearic acid, 52.7 percent linoleic acid and 7 percent arachidonic acid. Mukherjee g£_§1, (1957) examined the influence of fat free rations and stock rations on the triglyceride, cholesterol ester and phospholipid fractions of rat serum. They reported that of these fractions, 49 percent was in the form of phospholipids, 33 percent as cholesterol esters and 18 percent as triglycerides. The fatty acid composition was as follows: Fatty acid Saturated 18:1 18:2 18:3 20:4* Stock ration Triglyceride 76.5 0.0 18.0 1.0 1.0 Cholesterol ester 55.8 0.0 25.0 1.5 9.8 Phospholipid 71.7 0.0 10.4 0.6 13.2 Fat free ration Triglyceride 21.0 72.0 1.6 4.3 1.1 Cholesterol ester 30.1 53.0 9.0 1.3 3.6 Phospholipid 47.3 41.2 3.5 6.7 1.3 *Fatty acid abbreviation system suggested by Dole g£_§1, (1959) Mead (1957) suggested that the synthesis of the monoenoic fatty acid might be accelerated in cases of fat deficiency to maintain a certain de- gree of total unsaturation and physical properties of the tissue lipids which under normal conditions were provided by dietary essential fatty acids and their metabolites. Mohrhauerv and Holman (1963) were in agree- ment, finding that the oleic content of the total liver lipids was increased to 40 percent when a fat free diet was fed.' -12- Okey and Lyman (1957) found that serum phospholipids showed some tendency to vary in the same direction as serum cholesterol. They sug- gested that this might be taken to indicate a connection between the level of circulating cholesterol and that of phospholipids and would be in line with the evidence that a large part of the circulating cholesterol is in combination in giant molecules containing protein, phospholipids and free fatty acids. The influences of the dietary lipid composition on serum.cholesterol levels have been intensely investigated. Kinsell g£_gl, (1952) reported the first findings that vegetable fats tended to lower serum cholesterol levels in man. Since that early "letter to the editor", the hypothesis that vegetable fats do lower serum.cholesterol levels has been supported by Ahrens g£_§l, (1954), Hardinge and Stare (1954), Bronte-Stewart g£_§l, (1956), Ahrens g£_§l, (1957a), Ahrens g£_§l, (1957b), Brozek gt_§1 (1957), Horlick and Craig (1957), Brown and Page (1958), Jolliffe 25’s}, (1959). Keys g£_§1, (1956) disagreed to some extent, explaining that the level of fat in the diet had greater effect on lowering serum cholesterol levels than did the degree of unsaturation. Pollack (1959) reported that highly unsaturated, low fat diets resulted in a higher incidence of car- diovascular disease in Japanese when compared to a highly saturated, high fat diet in Thailand. James and Lovelock (1958) reported that the assumed relationship be- tween low essential fatty acid intake and coronary artery disease resm; upon two proved claims; firstly, an inverse relationship between linoleic acid intake and blood cholesterol levels in man, and secondly, an asso- -13- ciation between low essential fatty acid intake and atheroma in animals. However, they concluded that at this stage of deve10pment in the study of coronary artery disease, it can be stated unequivocally that there is no adequate evidence to show that a deficiency of essential fatty acids is a factor in its causation. Mohrhauer and Holman (1963b) reported that on fat free diets, the subcutaneous fats contained 29.3 percent palmitic acid, 18.5 percent palmitoleic acid and 47.7 percent oleic acid. These results are in agree- ment with the findings of Mead (1957) and Reiser g£_§1, (1960). Webb g£_gl, (1961) and James g£_§1, (1961) reported that the fecal fats of humans did not closely resemble the dietary fats. Odd isomers of oleic acid and 10 hydroxy-stearic acid were present in the feces. Tests proved that stearic acid is the precursor of both Icids. The major fatty acids of the feces in free and bound forms were myristic acid, 8.9 and 4.4, palmitic acid, 55.2 and 35.3, stearic acid, 12.9 and 31.8, oleic acid (including isomer) 10.3 and 17.2, and linoleic acid, 1.3 and 2.8 percent, respectively. Hoet g£_gl, (1963) reported that the odd isomers of various fatty acids in the feces of rats are products of the intestinal bacteria. On a fat free diet, rat feces contained 27.4 mg fatty acids per gram of dry feces. The percent fatty acid composition was: lauric, 1.0; myristic, 3.4; palmitic, 38.9; palmitoleic, 2.6; stearic, 24.9; oleic, 17.9; and linoleic acid, 11.2. The fat in this case is derived from endogenous sources such as bile secretinns, desquamated mucosa cells, mucosal secre- tions and from bacteria. ‘When 21 percent corn oil was added to the diet, -14- the feces contained 44.3 percent palmitic, 23.8 percent stearic, 25 per- cent oleic and 8.5 percent linoleic acid. The fat excretion increased only slightly with increasing fat ingestion. Spritz and Ahrens (1963) reported that the neutral sterols and bile acids of feces represent more than 99 percent of the excretion products of cholesterol under normal conditions. The identification of the sterols has been hindered by multiple variations in chemical structure produced by intestinal bacteria and the complexity of substances with which sterol extracts are contaminated. The sterols were primarily cholesterol and coprostanol which accounts for 90 percent of the sterols and bile acid V/ in feces. Schoenheimer and Sperry (1934) found that cholesterol was excreted as coprosterol and dihydrocholesterol. Since only 11 percent of the total sterols was unsaturated, the cholesterol content of feces was very low. Siperstein and Chaikoff (1952) agreed that only a very small fraction of body cholesterol is excreted as non-saponifiable compounds, i.e. choles- terol, c0prosterol or dihydrocholesterol. The majority of C]-4 from a dietary cholesterol source was found as saponifiable compounds, i.e. bile acids, in feces. In studies on the activity of different segments of the rat intestine during absorption of iodine-labeled olive oil, Benson gt_gl, (1956) found maximum activity in the distal jejunum. Based on the assumption that the segment with the highest activity represents the site of maximal absorp- tion, they concluded that the distal jejunum was the primary site of fat absorption. -15- Frazer (1962) stated that the chain length of a fatty acid may affect its digestibility. Long-chain fatty acids such as palmitic and stearic acid, are poorly absorbed as compared with shorter-chain compounds. The addition of a liquid oil vehicle to the long-chain saturated fatty acids causes a marked improvement in absorption. Deuel (1955) reported the relationship between melting point and digestibility as follows: Melting point Digestibility 9C coefficient in rat Myristic acid 53 82 Palmitic acid 63 36 Stearic acid 69 16 Oleic acid 14 95 Elaidic acid 51 96 Shoreland g£_§1, (1957) reported that ruminants had no special diffi- culty in the absorption of trans acids. 0n the other hand Deuel (1955) indicated that the higher melting trans acids behaved more similarly to the more suturated fatty acids in guinea pigs. According to Frazer (1962) there is probably selective absorption or more specifically, selective rejection of certain fatty acids even though they might have low melting points. When erucic acid (melting point 33.5°C) was fed, the proportion of erucic acid in the feces fat was significantly higher than the prOportion in the fat fed. Also, as shown by James 33.51. (1961) hydroxy-fatty acids are not absorbed to any degree. Frazer and Sammons (1945), Desnuelle g£_gl. (1948), Mattson g£_§1, (1952) and Mattson and Beck (1956) have shown that pancreatic lipase did -16- not rapidly and completely hydrolyze dietary glyceride to fatty acids and glycerol. The end products were di- and mono-glycerides, fatty acids and relatively little free glycerol. Desnuelle g£_§1, (1948) and Mattson and Beck (1952) have shown that pancreatic lipase has a selective action on fatty acids in a triglyceride, the fatty acids in the one (ad ) and three (0(l) positions being preferentially liberated. Frazer and Sammons (1945) listed four sources of lipase: gastric, pancreatic and intestinal secretion and lipoclastic bacteria. Pancreatic lipase was the most active. Frazer (1960) described the differences between fat emulsion in the intestinal lumen and fat particles is the chyle: Intestinal lumen Chyle Composition Triglycerides Triglycerides Diglycerides containing long- Monoglycerides chain fatty acids Short-chain fatty acids Phosphatidyl choline Long-chain fatty acids Cholesterol esters Surface film Lower glyceride-fatty LipOprotein acid-bile salt complex He concluded that triglycerides were reformed and that long-chain fatty acids, with more than 10 carbons in the chain, are selected for this esterification. The shorter-chain fatty acids did not re-enter glycerides; nor was liberated glycerol re-utilized in the intestinal cell for glyceride synthesis. Fatty acids were also taken up into phosphatides, especially lecithin, and into cholesterol esters. For the latter, there was a pre- ferential selection of more unsaturated fatty acids. Two pathways for glyceride syntheses have been demonstrated in the intestinal cell. The -17- pathway described by Kennedy (1957) involves phosphatidic acid as an in- termediate. According to Clark and Habscher (1960), this pathway operates in the small intestine as follows: CH2 OCOR 2 R-C 0 S COA I CH2 OH I CH OCOR CHOH \ ( Q " CHZ-O-P-oH CHZ-C-P-OH l l RCOSCoA 0H 0H 2 RCOOH CHZOCOR A glycerol phosphate phosphatidic acid CH OCOR ki_, H3 P04 H3P04 1 CHZOCOR CHZOH CH2 OCOR e———' | Triglyceride CHOH ¢=¥—— CH OCOR CHZOH CHZOH glycerol 0‘ , g diglyceride Buell and Reiser (1959) proved that the free glycerol liberated from lipase hydrolysis of the di-or mono-glycerides could not be re-utilized for triglyceride or glycerol phOSphate synthesis in the intestinal mucosa. They stated, however, that there was a possibility that free glycerol can be used in the liver for O‘glycerol phosphate synthesis. A pathway described by Clark and Hubsher (1961) allowed direct incor- poration of fatty acids into lower glycerides. Therefore, according to Frazer (1962), the composition of the dietary glyceride will affect the glycerides resynthesized in the intestinal mucosa. -18- Kennedy and Weiss (1955), (1956) described a pathway allowing for the incorporation of D-d , ’diglycerides into the phospholipid, leci- thin, as outlined below: Choline CHZO-C-OR ATP RIC o o c H ADP CHZOH phosphorylcholine CTP D- d , ( diglyceride .PPi CMP-PC Cytidine diphosphate choline CNP L-Gl - lecithin According to Kennedy and Weiss (1956) a similar pathway involving the active intermediate cytidine diphosPhate ethanolaminee is reaponsible for the formation of phosphatidyl ethanolamine. In studies on the fatty acid composition of lecithin from various sources, Tattrie (1959), Hanahan g£_§1, (1960) and Marinetti g£.§1, (1960) found that there was a specific location of the saturated and unsaturated fatty acids. The saturated fatty acids were located at the“ -ester position and unsaturated fatty acids were found preferentially located at the fif-ester position of egg lecithin, bovine plasma lecithin, and rat liver lecithin. -19- Hilditch and Stainsby (1935) stated that lard triglycerides con- tained the saturated fatty acid, palmitic acid, almost exclusively at the {-position. Young (1961) reported that lard contains unsaturated fatty acids in thei-position in only 28 percent of the total trigly- cerides. Mattson §£_§1, (1964) indicated that palmitic acid is found predominantly at the fly-ester position in the triglycerides of the domes- tic pig. Myristic acid also tended to concentrate at the F-position. The anatomical position from which adipose tissue was taken did not in- fluence the distribution of fatty acids on the triglyceride molecule. Mattson §£_§1, (1964) also reported that the composition of the dietary fat had no effect on the adipose triglycerides of pigs as shown below: Fatty acid 14:0 16:0 16:1 18:0 18:1 18:2 18:3 Total F.A. Mole 7.x 1 28 3 15 3 42 2 I -ester fatty acid 5 72 4 4 12 3 o 2. F.A. in flposition 167 86 44 9 1o 11 o The fatty acid composition of triglycerides from pigs on a safflower oil diet was as follows: Fatty acid 14:0 16:0 16:1 18:0 18:1 18:2 18:3 Total F.A. mole 2" 1 3 1 8 20 54 3 ,6 -ester F.A. 2 3o 2 3 12 42 2 7. F.A. in (3 ~position 67 92 67 12 20 26 22 x The analytical error was seven percent. -20- An analysis of soybean oil by Mattson and Volpenheim (1963) revealed that the/67-ester of triglycerides was primarily occupied by unsaturated fatty acids. The composition was given as follows: Fatty acid 16:0 18:0 18:1 18:2 18:3 Total F.A. mole Z 12 4 25 51 8 fl -ester F.A. 1 o 22 69 8 7. F.A. on/ ester 3 o 29 45 33 EXPERIMENTAL PROCEDURE Source of Animals The Carworth strain of albino rats used in this study were obtained from the Michigan State University Animal Husbandry Department rat colony. Males weighing 500 g t 150 g at 18 to 24 months of age were selected since this physiological age approximates a hunan'smiddle age. (Kleiber, 1961). The rats were divided into three groups of four animals on the basis that the weights be distributed through all groups in a manner as uniform.as possible. Three rations were formulated to supply 76 Calories (wagner, 1962) daily to each rat. The basic diet consisted of low fat ingredients as suggested by Wookarand Sebrell (1945). The basic ration was found to provide 4.1 C/g by a Parr Oxygen Bomb Calorimeter gross energy determin- ation (Appendix S). The lipid portions of the rations were isolated from soybean oil and lard. In order to obtain a vegetable lipid containing 33 percent polyunsaturated fatty acids, refined soybean oil was fractionally crys- tallized from acetone at -ll°C. As shown in Table 1, the crystallizgtion involving 10 percent soybean oil in acetone (W/W) provided the required lipid. A like procedure was attempted with Open kettle rendered lard ob- tained from the Michigan State University Meat Laboratory. A fraction containing 24 percent polyunsaturated fatty acids was isolated from -21- -22- Table 1. Fatty Acid Composition of Fractions Crystallized fromAcetonex Percent soybean oil in acetone W/W 10 15 20 25 Myristic acid 1.1xx 3.2 1.7 0.3 Palmitic acid 21.0 26.5 24.1 21.0 Stearic acid \ 15.1 9.8 11.6 11.2 Oleic acid 27.3 19.3 19.3 22.6 Linoleic acid 29.5 38.6 37.1 37.3 Linolenic acid 5.9 2.5 6.2 7.5 Percent polyunsaturated F.A. 35.4 41.1 43.3 44.8 x Uncorrected area percent by gas chromatography. xxAverage of three determinations of methyl esters prepared from the fraction. acetone at -26°C. Since this lipid fraction did not meet the specifica- tions previously stated, a barrow was fed a diet containing 20 percent corn oil for 17 days. The panniculus adiposus was finely chopped and rendered under nitrogen gas in an autoclave at 121°C for two hours. The lipid layer was removed and centrifuged at 2100 rpm for 15 minutes. The lard obtained by this procedure contained 34 percent polyunsaturated fatty acids. The fatty acid composition.was shown to be myristic, 0.7; palmitic, 18.2; palmitoleic, 9.7; stearic, 7.2; oleic, 30.1; linoleic, 26.4; and linolenic acid, 7.8 percent. Differential cooling curves as described by Jacobson 25.31, (1961) were used to prove the resultant triglycerides were not composed entirely of saturated or unsaturated fatty acids. The pre- sence of extremely small amounts of diglycerides in both lipid samples was revealed by thin-layer chromatography (Mangold, 1961). -23- The first group of rats was maintained on the basic fat free ration (diet A) for a period of two weeks. The second and third groups were maintained at an iso-caloric level with the first group. Forty percent of the calories in each case was supplied by either the lard lipid frac- tion (diet B) or the soybean oil lipid fraction (diet C). The rats were caged separately, placed randomly on the racks and fed daily for a two week period. Feces samples were collected by placing a paper towel under each cage for a 24 hour period. The samples were weighed and stored under nitrogen gas at -30°C until the analyses were performed. At the end of the two week dietary regimes, the rats were decapitated 24 hours after feeding and held over a funnel in order to collect blood samples. Massaging increased the amount of blood collected from each rat. The samples were centrifuged at 2800 rpm and 5°C for 15 minutes. The blood serum was decanted and stored under nitrogen gas at -30°C. The ab- dominal cavities were Opened, and liver, aorta, and perirenal fat samples were taken. The livers were blotted on paper towels and weighed. The extraneous fatty material was removed from the aortas. Subcutaneous fat samples were taken from.the area of the last lumbar vertebra. These sank ples were stored under nitrogen at -30°C. Extraction of Lipids The lipids were extracted from the liver, blood, feces and pooled aorta samples by a modification of the method suggested and recommended by Ostrander and Dugan (1962). Each sample was placed in a VerTis "45" / -24- flask with 130 m1 absolute methanol and macerated with a VerTis "45" homogenizer for five minutes at medium speed. The sample was transferred to a Waring Blender jar with 65 ml chloroform and blended for five min- utes. The VerTis "45" flask was rinsed with 65 md chloroform.which was then added to the Haring Blender jar and blended for 30 seconds. In order to precipitate the protein in the sample,65 ml distilled water con- taining one to one and a half 3 zinc acetate was added and blended for 10 seconds. The sample was filtered by suction on a Buchner funnel using Whatman No. 1 filter paper. Nitrogen gas was directed over the sample during this process. The residue and filter paper were returned to the blender jar along with one half of a filter paper used to wipe the funnel. To this residue 100 m1 chloroform was added and the sample was blended two and a half minutes and then filtered as before. The blender jar and residue were rinsed with 50 ml chloroform. The total filtrate was trans- -ferred to a separatory funnel and the heavy chloroform layer was removed for lipid analysis. Remaining lipids in the aqueous layer were extracted with 100 ml chloroform. The solvent was removed from the chloroform layer under reduced pressure by means of a rotating flash evaporator. The lipid concentrations of the liver, feces and blood samples were calculated on a gram or ml basis. The dry samples were stored under nitrogen at -30°C. The solvent system for the blood serum and pooled aorta samples was re- duced by one-half. The phospholipids were separated from the neutral lipids by a modi- fication of a method reported by Bates (1958), Reiser $5.31, (1960) and Choudhury and Arnold (1960). Each lipid sample was dissolved in 50 m1 -25- chloroform and mixed with 10 g silicic acid and stirred with a magnetic stirrer for 10 minutes. This Operation was performed under a direct stream.of nitrogen gas. The chloroform.was filtered off by suction through a sintered glass funnel and the silicic acid was washed with four 50 ml portions of chloroform. The solvent was removed by reduced pressure and the weight of the neutral lipids was obtained. The silicic acid was then washed with four 50 m1 portions of meth- anol to remove the phospholipids. The weights of the phospholipid fraction were recorded following the removal of the methanol under re- duced pressure at 70°C. Thin-Layer Chromatography Following unsuccessful attempts to achieve the complete separation of neutral lipid classes on Florosil columns (Carroll, 1961) thin-layer chromatography was employed. Camag equipment consisting of 20 by 20 cm glass plates, spotting template, applicator and chromatographic develop- ing tank was used. Anasil B, a thin-layer chromatographic silica gel adsorbent containing a calcium sulfate binder, was slurried with distilled water in a ratio of 1:2 (HIV) and applied to the glass plates in an uni- form layer about 250 microns in thickness. The plates were allowed to dry at room temperature for 15 minutes and then were activated by heating in an oven at 105°C for one hour. The activated plates were stored in a desiccator until they were required for analytical procedures. Capill- ary tubes were used to apply the samples to the chromatOplates. The plates were developed in a equilibrated chromatographic tank utilizing .o y ‘0‘... -25- 100 ml solvent consisting of 80 m1 hexane, 20 m1 diethyl ether and one m1 concentrated acetic acid which was added to prevent excessive tailing. After the solvent traveled 16 cm up the plate, the chromatoplates were allowed to dry at room temperature. Visualization of the developed plates was accomp1ished by Spraying with five percent phosphomolybdic acid in ethanol or 50 percent sulfuric acid in 50 percent ethanol to which two percent ferric chloride was added. The plates were then charred in an oven at 105°C and the Spots were traced on acetate paper. Cholesterol, cholesterol acetate, oleic acid and triolein were used as standards for cholesterol, cholesterol esters, free fatty acids and triglyceride classes. Preparation of Methyl Esters Methyl esters of the fatty acids were made utilizing a method deve1- 0ped by MeGinnis and Dugan (1964) and Dugan and McGinnis (1964). One g fat samples were suSpended in 20 m1 diethyl ether and homogenized with :3 VerTis "45" homogenizer. The homogenate was transferred to a 125 m1 Erlenmeyer flask mounted in an ice bath on a magnetic stirrer. Under a constant stream of nitrogen gas, two and one half ml concentrated sulfuric acid were added drOpwise to the flask. The flask was stappered and stirred for 10 minutes reaction time, after which, a drop of alcoholic phenolphthalein was added and the mixture was titrated with 3.6 N methan- olic potassium hydroxide. The neutral mixture was transferred to a separatory funnel, the reaction flask rinsed with diethyl ether, and the solvent was washed with cold, distilled water. The aqueous layer was -27- discarded and the diethyl ether layer was dried over anhydrous sodium sulfate. The sample was filtered through'Whatman No. 1 filter paper and the solvent was removed under a stream of nitrogen gas. The result- ing methyl esters of fatty acids were injected into the gas chromatograph for qualitative and quantitative analysis. The neutral lipids from the liver, feces, blood serum and pooled aorta samples were separated into classes on thin-layer chromatoplates. Approximately seven mg samples were spotted across the plate and a referb ence mixture was spotted at the boundary. Following the development of the plate, a cover glass plate was taped over the absorbant surface ex- cluding the area occupied by the reference material and one neutral lipid unknown. This area was sprayed with 50 percent sulfuric acid in 50 percent ethanol plus two percent ferric chloride. The plates were charred and the various lipid classes were identified by relative Rf values and the red color developed by spots containing cholesterol. The lipid classes were scraped into 125 mu Erlenmeyer flasks and suspended in 20 mm diethyl ether. The esterification procedure was followed as in the fat analysis. The phospholipid fractions were dissolved in 20 m1 diethyl ether and esterified in the same manner. In order to evaluate the accuracy of the procedure, 4 mg of palmityl oleyl stearin, obtained through the courtesy of R. J. Vander wal, Armour and Company Research Laboratories, was analyzed by this procedure. This specific triglyceride yielded 38 percent palmitic acid, 30 percent each of oleic and stearic acids and two percent myristic acid by gas chromatographic analysis. -23- Gas Chromatography A Barber-Colman Model 20 gas chromatograph equipped with a radium ionization detector and a Barber-Colman recorder was used. For most of the analyses, the following adjustments were maintained: argon gas press- ure, 30 1b.; argon gas flow rate, 151 ml per minute; injector port and detector temperatures, 240°C; column temperature, 170°C; cell voltage, 1250v; sensitivity, 1 x 10'7 amps full scale; Split flow, 200 ml per minute; and column, 6 ft by 1/4 in. copper tubing with 12 percent ethylene glycol succinate on 60/70 mesh Anakron A or 20 percent diethylene glycol succinate with two percent phOSphoric acid on 60/80 mesh Chromasorb W. The columns were packed using an electric vibrator and then coiled to a diameter of five inches. The columns were preconditioned at 200°C with an argon flow rate of 150 ml per minute for at least 24 hours. The detection system was checked for quantitative accuracy by inject- ing aliquots of an equal mixture of the methyl esters of stearic acid, oleic acid, linoleic acid and linolenic acid, and demonstrating that under the conditions used, the peak area for each ester averaged 25 percent of the total. The results of quantitative analysis of fatty acid composition reported were taken directly from areas under the curve without corrections for possible variations in the response of the detector to different mole- cular Species. The analysis also did not include long chain methyl esters present with retention times greater than methyl arachidonate. The reten- tion times of various methyl esters (99+ percent pure) were compared to the retention times of the unknown methyl esters for qualitative analyses. -29- When standards were not available, peaks were tentatively identified by semilogarithmic plots of retention volumes against carbon number. Cholesterol Determinations The total and free cholesterol content of blood serum.were measured by the method published by Ferro and Ham.(l960). A modification of this method was used for liver cholesterol measurements. The total liver lipid extract was dissolved in 100 ml diethyl ether and duplicate 10 m1 samples were taken. The solvent was then removed from each sample under reduced pressure. The residue was dissolved in 10 m1 isopropyl alcohol and one half ml was taken for analysis. Four and one half m1 is0pr0py1 alcohol were added to the sample, which was then shaken vigorously and permitted to stand for 10 minutes. The sample was then centrifuged for 10 minutes at 3000 rpm at 5°C. For total cholesterol measurements, one m1 of the supernatant was placed in a 16 x 100 mm test tube. One half ml alcoholic potassium hydrox- ide (5 percent KOH in 95 percent ethanol) was mixed with the supernatant sample which was-then placed in a water bath at 37°C for 30 minutes. The tubes were removed from the water bath and one drop of phenolphthalein (one percent in ethanol) was added. The mixture was titrated with 10 percent acetic acid until the color disappeared, and then one additional drop was added. Free cholesterol measurements were made by placing two m1 of the iso- propyl alcohol supernatant in 16 x 100 mm test tubes. To both the neu- tralized total and free cholesterol samples, one m1 digitonin solution -30- (one g digitonin in 50 ml 95 percent ethanol and diluted to 100 ml with distilled water) and one drOp of 30 percent aluminum chloride were added. The aluminum chloride acted as a gathering agent for the digotonide pre- cipitate. The samples were mixed by swirling and allowed to stand for 30 minutes. The tubes were centrifuged at 3000 rpm for 10 minutes and the supernatant was removed by suction. The packed precipitate was washed thoroughly with 3 ml acetone, centrifuged, and the supernatant was removed by suction. The tubes were inverted and drained over a paper towel to insure that no precipitate was lost. The precipitate was broken by adding 0.2 ml distilled water and shaking. A colordevelopment mixture was prepared by mixing three volumes reagent grade acetic anhydride with two volumes reagent grade acetic acid. To 10 parts acid-anhydride stock solution, 1 part reagent grade concentrated sulfuric acid was added and the mixture was cooled to room temperature. Six ml of the color development mixture were added to the digitonide precipitate. The solution was trans- ferred to a cuvette and the peak absorption at 640 Magainst a water blank was observed within 90'? 30 seconds. Matched cuvettes were used in a Baush and Lomb Spectronic 20 calorimeter. Liver cholesterol stan- dards were prepared by adding 100 mg free cholesterol to 100 m1 isoprOpyl alcohol. Standards were then taken with 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 mg cholesterol. The standards were prepared for absorption readings by the procedure described for free cholesterol. The optical densities of the unknowns were read against a standard curve. The values obtained for free cholesterol were divided by two. All readings were then based on one percent of the total lipid extract, -31- and this factor was used in estimating the total and free cholesterol content of the samples. The values were then converted to a mg cholesterol per g wet tissue basis. Statistical Analysis The statistical analyses were based on the methods of Kempthorne (1952), Duncan (1955) and Snedecor (1956). Analysis of variance and multiple range tests were employed. RESULTS AND DISCUSSION 0n the fat free (diet A), lard (diet B) and soybean oil (diet C) diets, one rat on each died of undetermined causes before the test period was completed. The deceased rats lost little weight and showed no exter- nal signs of sickness. The caloric consumption per rat is given in Table 2. Table 2. Dietary Record Days Total Calories Final Weight Diet Rat No. on test Calories /day weight (g) change (g:) A 1 14 1064 76 499 ~51 A 2 14 1064 76 501 ~60 A 3 14 1064 76 535 ~77 A 41 12 891 74.3 457 -45 B 1 14 1064 76 515 ~41 B 2 14 1064 76 565 ~44 B 32 4 278 69.5 650 -7 B 4 14 1064 76 479 ~28 c 13 12 760 63.3 375 +11 C 2 14 1064 76 515 ~34 C 3 14 1064 76 516 ~46 C 4 14 1064 76 628 ~48 ICn the three days prior to death, caloric consumption was 57.5, 49.3 and 24.6 C/day. 20a the day prior to death, caloric consumption was 51.5 Calories. he rat stopped eating 2 days prior to death. Due to the deaths, statistical significance at the P = .05 level was not observed in many expected cases since the "F" ratio was necessarily extremely high (6.94). For that reason, data approaching significance and data indicating possible trends are reported herein. Furthermore, since the investigation was primarily designed to study the effects of -32- -33- diet on the di-, tri-, and tetraenoic (polyunsaturated) fatty acid composi- tion of the tissues, the analyses generally considered only the percentage of polyunsaturated fatty acids in each lipid component. Contrary to the report of Hoet g£.§l, (1963) increases in the fat content of the diet significantly increased the level of feces fat. How- ever, Hoet §£.§l, (1963) compared rats on fat free rations with rats on 21 percent corn oil diets. In the present study, the rations containing lipids furnishing 40 percent of the total calories produced significantly higher feces lipid levels as shown in Tables 3 and 4. Table 3. Analysis of variance of lipid content1 of rat feces. Source df M. S. F Replicates 2 10.05 2.49 Diets 2 86.69 21.46** Replicates x diets 4 4.04 **P4( .01 1Appendix A. Table 4. Multiple range test of rat feces lipid content. Number of pairs (2) (3) Shortest significant range 5.58 5.69 Diet A B C Percent lipid 3.12 1': 0.66 12.00 “t 3.28 12.80 t 2.63 Note: Any two means not underscored by the same line are significantly different. -34- Perhaps the physiological capability of the lipid absorption mechan- ism has been exceeded at this high level of dietary fat. Furthermore, age effects may have some influence on lipid absorption. Table 5 presents the fatty acid composition of the various neutral lipid fractions isolated from the feces. Cholesterol esters were not detected in the neutral lipids. Statistical examination of the effects of diet and fraction on the fatty acid composition revealed no signifi- cant differences. There appear to be major differences in the rate at which various fatty acids were absorbed. Diets containing 33 percent 'polyunsaturated fatty acids produced feces containing five percent polyunsaturates. One percent myristic acid in the diet yielded 45 percent myristic acid in the feces. This is, perhaps, explained by Hoet g£_§1, (1963) who reported that the greatest percentage of feces lipids were of endogenous origin. Frazer (1962) offers a further clue in that some fatty acids are select~ ively rejected or absorbed in the digestive process. In the present case, the author would postulate that the polyunsaturated fatty acids were pre- ferentially absorbed at the expense of palmitic and stearic acids. There was apparently a trend towards greater absorption of polyunsaturated fatty acids from diet B. The diets, A, B and C, produced feces triglycerides yielding 5.8, 3.9, and 6.8 percent polyunsaturated fatty acids, respect- ively. In the diglyceride fraction, the results were 6.4, 5.2 and 10.2 percent, respectively, and in the monoglyceride-free fatty acid fraction the percentages were 2.1, 1.7 and 33.3, respectively. The latter figure produced by diet C was postulated to be due to the structure of the dietary Fatty acid compositionlof rat feces neutral lipids Table 5. Diet C B Diet Diet A Monoglyceride & free fatty acid Diet C B Diglyceride Diet Diet A Diet B Diet C Triglyceride Diet A Fatty acid 0.6t1.0 0.4f .7 12:0 -35- NQ'QOOHN MNF: NF: $«hhwweuooeo (“LORI-400‘ 22.9+25.9 so 0000 NONHd'v-I m cu<3c>uwcn<3 49+¢+I+I+I+h+lc>c>c>c>c> «noxuww U3C>cw<3 camxc: F~F4 OOd‘IO O'\wa Eoum poumaomw moowuomum mwmwa mo mowuwmomEOo vwom auumm .mH manna n.0wm.ma o o o o anew H H.0Mm.o ¢.Dwm.o H.oww.o ~.owm.o mama ¢.aHH.HH N.§MM.mH H.wwm.ma m.§wm.oa H.~Mh.mu N-wH w.~«m.aa ~.Nwo.oa c.mwo.ha o.hwm.ma o.~wm.mm Huma ¢.NHm.~m m.awm.m n.HMH.m m.nhw.¢ H.owa.m ouwa o H.Han~ w.ow§.u h.HHh.N ~.owm.m Hume a.a«m.¢~ m.§Hm.m~ o.mflm.- n.0«H.oN n.H«N.o~ o-oH H.ofln.c H.Mwm.mm m.wwm.m¢ o.aawm.wm m.mwh.m ones 8 a H N.0Hh.0 a o-NH mmwmqaosomosm mpaom Noumm mpauoomdwwn museum owauoomwwaua vwom moum Honoumoaoso huumm okuoomawocoz m poem pom come no muo>wa Boom woumaomw msowuomum mange mo cofiuflmomaoo vqom muumm .oH edema flow-4.2 o o o a a8 a m.owu.o m.HHw.o H.owa.o m.owm.o mama fiawmfi saws;- oawflm- flee-1: Sour-won «a: o.owb.ua h.HHm.~H m.mwm.aa m.~«m.¢a n.mwm.w~ H-wH - ”m m mwfl an m mawn 5H a awe m m Ham m m Hum e o.mH o manna can-"o; «.5:- HAflsa :3 m.mwm.mu m.owm.w~ ¢.¢fiwa.o~ o.nflwm.m~ m.mwa.n~ cued n.awo.a o.mwn.ma n.0HHH.m¢ n.muws.mm «.mwn.n cued o m.mhh.~ o o.awm.o o ouma mpamwaosomosm mpaom muumm mpwuooMHwan muoumm omwuoohflwaua mwom worm Honouaoaono huumm omwuoomawoooz 0 user com mumu mo wuo>fia scum poumaonw meoauomum pwdwa mo soauwmoQEOo whom huumm .na manna -45- The statistical analysis of the polyunsaturated fatty acid composi- tion of the various rat liver fractionsis given in Table 18. Table 18. Analysis of variance of the polyunsaturated fatty acid composi- tion of lipid fractions isolated from livers of rats fed diets A, B and C. Source df MS F Replicates 3 39.35 0.47 Diets 2 31.38 0.38 Replicates x diets 6 83.56 Fractions 4 1044.15 53.06** Diets x fractions 8 11.07 0.50 Replicate x diet x fraction 36 19.68 {*P«<'.01 Appendices H, I, J, K and L. The differences found among the fractionswere analyzed as shown in Table 19. Table 19. Multiple range analysis of the polyunsaturated fatty acid content of fractions isolated from rat liver Standard error of fraction mean i 2.22 (N2 - 36) Number of pairs: (2) (3) (4) (5) Shortest significant range: 8.52 8.88 9.15 9.28 Fraction: Cholesterol Di- Free fatty acid Phospho~ Tri- ester glyceride monoglyceride lipid glyceride Means: 10.13"-*4.36 13.72'1’6.40 15. 60455.01 29.311‘4. 12 302735.57 Note: any two means not underscored by the same line are significantly different. -47- In view of the relatively small standard deviations presented for the triglyceride and phospholipid fatty acids from rat livers, the author would postulate that specific enzymatic processes apparently are respon- sible for the formation of these fractions. The hypothesis is strengthened by the lack of dietary effects on these fractions. The similarities between the liver triglyceride and phospholipid polyunsaturated fatty acid content may support the triglyceride synthesis scheme proposed by Kennedy (1957). Geyer g£_§l, (1960) reported that the fatty acid distribution on triglyceride molecules was similar to that of phospholipids in mouse fibroblasts. The dietary effects on the blood serum lipid levels were non-signifi- cant in the present study (Table 20). Table 20. Analysis of variance of blood serum lipid content1 (mg/m1) Source df MS F Replicates 2 69.88 0.60 Diets 2 335.58 2.86 Replicate x diet 4 117.21 1Appendix M. The serum lipid levels, 9.07‘k0.82, 29.12-k14.56 and 24.94t9.55 mg per ml, of rats on diets A, B and C, reapectively, do indicate that high diet- ary lipid levels tend to increase serum lipids. Slaughter immediately after feeding may have produced significant effects in this case. The variance in the case of diets B and C may have been due to the differences -43- in elapsed time following the consumption of the ration by each rat be- fore sacrifice. The neutral lipids accounted for 55.6lt5.87 percent, 10.40t6.12 per- cent and 7.97*4.78 percent of the total serum lipids of rats on diets A, B and C, respectively. These differences were highly significant as shown in Tables 21 and 22. Table 21. Analysis of variance of neutral lipid content of rat blood serum lipids1 Source df MS F Replicates 2 45.94 2.18 Diets 2 2160.02 1020.37** Replicate x diet 4 21.10 HP (7.01 1Appendix M. Table 22. Multiple range analysis of dietary effects on prOportion of neutral lipids in rat blood serum lipids. Standard error of mean t 6.50 Number of pairs (2) (3) Shortest significant range 25.55 26.07 Diet C B A Percent neutral lipid 7.97'1'4.78 lO.40'-'-'6.12 55.611187 Note: any two means not underscored by the same line are significantly different. The blood serum cholesterol levels in mg/100 ml are presented in Table 23. -49- Table 23. Cholesterol content of rat blood serum Total Esterified cholesterol Free cholesterol cholesterol Rat mg_ mg[100 ml serum mgllOO m1 serum A 1 105.4 30.4 25.0 2 140.6 58.9 81.7 3 131.3 51.4 79.9 B 1 140.6 50.0 90.6 2 131.3 45.9 85.4 4 124.3 36.5 87.8 C 2 122.6 22.5 99.1 3 94.6 17.5 77.1 4 94.9 19.5 75.4 The analysis of variance of total blood serum cholesterol levels failed to show significant dietary effects (Table 24). Table 24. Analysis of variance of rat blood serum cholesterol levels Source df MS F Replicates 2 15.83 Diets 2 335.35 1.86 'Replicate x diet 4 180.35 Form 1 9786.00 327.40** Diet x form 2 397.05 l3.28** Replicate x diet x form 6 29.89 -50- Even in the presence of the diet-form interaction, the extremely high "F" test for form (free and esterified) indicates that a statistically significant difference exists between the free and esterified cholesterol levés. The diet-form interaction was investigated by analyzing the diet- ary effects on free and esterified cholesterol (Table 25). Table 25. Analysis of variance of rat blood serum free cholestzrol levels Source df MS F Replicate 2 35.04 0.29 Diet 2 670.52 5.64 Replicate x diet 4 118.79 Although there was not a statistical difference, it was apparent that the differences in the blood serum cholesterol level tended to occur in the free cholesterol fraction. The mean total cholesterol contents were 125.77tl8.26, 132.07t8.17 and 103.60115.59 mg per 100 ml and esterified cholesterol contents were 78.87'173.47, 87.93‘1‘2.60 and 83.87‘1’8.65 mg per 100 ml in serum from rats on diets A, B and C, respectively. These results disagree with those of A1fin~Slater g£_§1, (1954) who reported that the dietary effects occurred in the esterified cholesterol fraction. It was further noted that there appears to be an inverse rela- tionship between the phospholipid content of the blood serum lipids and cholesterol content. Okey and Lyman (1957) have reported a direct rela- tionship. The inverse relationship was studied further by analyzing the variance of the phospholipid content of the rat blood serum (Table 26). -51- Table 26. Analysis of variance of the phospholipid content1 of rat blood serum. Source df MS F Replicate 2 46.66 0.42 Diet 2 439.18 3.95 Replicate x diet 4 111.13 1Appendix M. The diet had no significant effect on the phOSpholipid content on blood serum. However, examination of the dietary means reveals a trend in that the values were 3.9910.20, 26.6lt14.14 and 22.76t8.31 mg per ml for diets A, B and C, respectively. These results also do not fit the hypothesis that there is a direct relationship between blood serum choles~ terol and phOSpholipid levels (Okey and Lyman, 1957). The fatty acid composition of the lipid fractions isolated from the blood serum of rats fed diets A, B and C is given in Tables 27, 28 and 29. The statistical analysis of the polyunsaturated fatty acid composi- tions of these various fractions is given in table 30. Table 30. Analysis of variance of the polyunsaturated fatty acid composi- tion of lipid fractions1 isolated from rat blood serum of rats fed diets A, B and C. Source df MS F Replicates 2 1.58 0.04 Diet 2 59.89 1.57 Replicate x diet 4 38.04 Fraction 4 196.10 13.64** Diet x fraction 8 26.63 1.85 Replicate x diet x fraction 24 14.38 **P C .01 lAppendices N. 0. P. 0 and R. .cowuomum maeaaosomono onu ca meow oHo~Ouwaamo ucoouom H.~N pm: H4 umm N.N.H ¢.~ o o o o «now H o o o ¢.o_w 0.0 m-wa n.m H ¢.o w.~.w m.~ ~.H.w m.m ~.o.w w.n m.N.w 0.0 N-wa o.m H m.mH n.N_H w.oH m.n.w o.mH o.m.fl m.n m.h.w n.am HHwH was so u. ass as ..-.. as fie i... we to .._.. o.~ 3.. n. as 92 - «m.~H H ¢.m a.H_H w.¢ m.o.w o.~ H.N.w ¢.m m.o.w ~.m Huoa N.M.H m.mm m.c.w H.m~ o.HH.H H.o~ ¢.N H N.Ha w.¢_w m.wa o-oH m.m H o.m m.oH_w H.¢m m.m~.fl N.nm n.0H.H ¢.oo w.ma.w 0.0a ouqa o.o w 0.0 H.o.w m.o ¢.o.w 0.0 m.o w o.H ~.o_H m.o onma pwmwaosmmonm pwom muDMH omwuoomawwn noumo owguouwamgua paom ooum dououmoaoso huumm opawoomamosoz .4 pogo so mums aouw moowuompm pHoHH Eamon mooHn mo cowuwmooeoo meow huumm .NN mamas -53- H.m_fl a.m o o o o ¢00N H N o M.¢ o w N_w H N m o.“ N o o N.“ a H m.mH m H.“ N «H m N.“ m m m H.“ N m N o_w a N m o.% m o N.mH m o_H H mH m m H m «H w N.w 0 HH N o.“ N N m oH.w 0 NH H.mH H N M a on o o.“ N N N N.w m a m o.“ m N H m.“ o m o.mH o o o.“ N m o H.“ m N a H.“ N H o H M.H q H.0H o N w a mN m NH.“ m mN m H_M o «H m.o_H o o 0 HH H m mH o-oH o n H m N o 0N M.m No H NNAH 0 me e N.“ o mN o Nm.w 0 mm o-dH m o H.o.o N.o H o o m.o H m.o m.H_H o.N 0.0 H N.o onNH pHoHHosmmonm wHom huumm omHuoohHmwa nouns opwuoomeHuH vHom ooum HonoumoHono Nuumm omHuoomeocoz .m uon pom mumu Scum chHuomum pHdHH Enema mooHn mo aoHuHmomEoo pHom Nuumm .wN oHan -54- so ta oao-ofia-oe emai- 33.. Ho 5 38w dawn-so Nada s. w.m H N.N o o o o ¢-ON H o.o.H m.o o o m.o H_m.o m-mH N.N H.N.qH H.¢.H m.m H.N H N.N o.w H o.m N.N.H o.w N-wH N.H H m.HH N.w H N.N m.m H m.o H.o_H N.N N.N H c.HH H-wH N.o H ¢.NN N.N H m.m H.¢.H N.m m.H H N.N H.H H w.m o-mH m.o 14km.o 0.0 H m.H N.H H H.N N.o H m.H o.o H ¢.N H-oH m.H H m.HN m.m H m.mH ¢.N H o.m m.o H N.NH m.H H m.HH o-oH H.o_H m.oH m.mN_H H.oo N.0N H N.oo w.NH_H H.¢o o.NH H N.om o-qH N.H H N.H «.0 H N.o n.0H H m.o w.o H N.H H.N H o.N o-NH o o 0.0H HaH.¢ o H.m H m.m ouoH pHmHHotem0£m wHomINuumm omHuoohdwwn nouns mpwuouwfiwwue mwom mouw HonoumoHono Noumm owwuoomeoaoz .o uoHp pom memo Scum mcoHuomuw mHmHH Enema pooHn mo :oHuHmooaoo pHom Noumm .mN oHnt -55- The multiple range analysis of the polyunsaturated fatty acid con~ tent of the fractions is given in Table 31. Table 31. Multiple range analysis of the polyunsaturated fatty acid con~ tent of rat serum lipid fractions. Standard error of fraction mean t’1.89 (N2 = 24) Pairs (2) (3) (4) (5) Shortest significant range 7.48 7.82 8.01 8.18 Fraction: Monoglyceride di- Cholesterol Tri- PhOSpho- free glyceride ester glyceride lipid fatty acid Percent polyunsaturated fatty acid 4.66‘f2.95 5.59‘f2.80 5.98-14.47 7.691375 16.12f5.08 V Note: any two means not underscored by the same line are significantly different. These results indicate that the phospholipid fraction of rat blood ser- um contains significantly more polyunsaturated fatty acids. Geyer gghgl. (1960) have previously reported that the fatty acid distribution patterns of triglycerides and phospholipids in human blood serum were dissimiliar. The fatty acid composition of the pooled aorta samples from each dietary group is presented in Table 32. ~56- Table 32. Fatty acid composition of triglycerides, cholesterol esters, monoglyceride-free fatty acids and phospholipids isolated from pooled aorta samples. Fatty acid composition Fraction Diet 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:4 Triglyceride A 0.1 3.1 24.9 5.7 5.1 33.9 27.1 0.2 0 B 0.1 2.4 20.3 4.0 8.4 36.7 26.7 1.4 0 C 0.1 6.6 18.8 3.8 8.3 27.9 32.7 1.9 0 Cholesterol A 0.6 23.8 20.5 3.4 5.8 25.9 18.7 1.0 0 esters B 0.3 12.1 18.7 1.9 10.3 34.6 22.0 0.5 0 C 1.2 20.9 20.5 0 12.5 22.3 20.9 1.7 0 Monoglyceride A 0.1 6.7 20.3 2.7 20.7 22.9 26.6 0 0 ~free fatty B 0.2 11.4 16.4 0 21.4 23.2 26.4 T 0 acids cl 0.3 7.3 18.4 0 19.4 23.1 27.6 0.3 0 PhOSpholipids A 0.1 2.9 20.9 3.3 9.8 28.1 32.8 1.4 0.7 B T 6.1 18.3 5.2 12.1 32.7 24.9 1.1 0.5 C 0.1 4.3 17.2 2.6 10.2 28.2 36.0 0.9 0.1 13.6 percent 20:3 was detected. The analysis of variance indicated no significant effects due to diet or fraction as shown in Table 33. Table 33. Analysis of variance of the polyunsaturated fatty acid content1 of lipid fractions isolated.frommaortas of rats fed diets A, B and C. Source df MS Diet 2 8.76 0.62 Fraction 3 44.13 3.10 Diet x fraction 6 14.23 -57- The fractions tended to have different contents of polyunsaturated . + + + + fatty acrds. The mean values were 30.0-4.0, 29.1-5.0, 28.3-2.8 and 21.6- 1.6 for the triglyceride, phospholipid, and monoglyceride-free fatty acid and cholesterol ester fractions, respectively. The polyunsaturated fatty acid content of triglyceride fractions varied from tissue to tissue as shown in Tables 34 and 35. Table 34. Analysis of variance of the polyunsaturated fatty acid content of triglyceride from rat blood, liver and aorta. Source df MS F Replicates 2 16.09 1.09 Diet 2 23.87 1.62 Replicate x diet 4 14.70 Tissue 2 1454.80 64.37** Diet x tissue 4 16.89 0.75 Replicate x diet x tissue 12 22.60 *FP.( .01 Table 35. Multiple range test of polyunsaturated fatty acid content of triglyceride from rat blood, liver and aorta. Standard error of location i 3.40 (N2 = 12) Number of pairs (2) (3) Shortest significant range 10.47 10.98 Tissue: Blood Liver Aorta Percent polyunsatured fatty acid 7.69‘t3. 75 29.41t5.77 30.00’1'3.46 Note: any two means not underscored by the same line are significantly different. -53- Reiser g£_§l, (1960) reported that plasma triglycerides did not ori- ginate in the livers of miniature pigs. This conclusion was supported by the present study involving rats. Apparently, triglycerides with higher levels of polyunsaturated fatty acids were either selectively re- tained by the liver or deposited in the aorta wall. The differences in the . unsaturated fatty acid content of choles~ terol esters from various tissues are shown in Tables 36 and 37. Table 36. Analysis of variance of the unsaturated fatty acid content of rat liver, blood and aorta cholesterol esters. Source df MS F Replicates 2 18.31 0.19 Diet 2 12.48 0.13 Replicate x diet 4 97.86 Tissue 2 3138.93 50.73** Diet x tissue 4 139.69 2.26 Replicate x diet x tissue 12 61.68 **P ¢f.01 Table 37. Multiple range test for ’unsaturated fatty acid content of the cholesterol esters of rat blood, liver and aorta. Standard error of tissue mean “I 5.56 (112 --- 12) Number of pairs (2) (3) Shortest significant range 19.20 19.86 Tissue Blood Liver Aorta Percent unsaturated fatty acid 14.81’1'9.42 24.64‘r9.67 50.93‘1'6.06 Note: any two means not underscored by the same line are significantly different. -59- These results are in contrast to Swell 2;.gl, (1960b), who reported similarities between the fatty acid composition of cholesterol esters in human serum and aortas. Swell g£.§l. (1960a) reported distinctive differ- ences between rat serum and liver cholesterol esterified fatty acids. The present study indicated non-significant differences between these tissues. The present work supports the conclusion of Evrard g£_§1, (1962) that the cholesterol esterified fatty acid pattern of the serum differs from that of the aorta. The present study also supports the report of Reiser g£_§1, (1960) in that significant differences were not present between the cholesterol esterified fatty acid patterns of blood serum and liver. Swell gg_§1, (1964) reported that liver cholesterol esters bear no rela- tionship to blood serum cholesterol esters. Table 38 indicates that significant differences exist in the polyun- saturated fatty acid content of rat blood serum and liver diglycerides. The blood serum contained 5.59+3.22 percent and liver, 15.28t5.68 percent polyunsaturated fatty acids. Table 38. Analysis of variance of the polyunsaturated fatty acid content of rat liver and blood diglycerides. Source df MS F Replicates 2 5.92 0.35 Diet 2 28.08 1.67 Replicate x diet 4 16.82 Tissue 1 422.44 l4.30** Diet x tissue 2 14.16 0.48 Replicate x diet x tissue 6 29.55 **P 4 .01 -60- Significant differences were found between the polyunsaturated fatty acid content of the monoglyceride«fr:e fatty acid fraction isolated from various rat tissues (Tables 39 and 40). Table 39. Analysis of variance of the polyunsaturated fatty acid content of monoglyceride-free fatty acid fraction of rat liver, blood and aorta. Source df MS F Replicates 2 13.35 0.29 Diet 2 41.29 .91 Replicate x diet 4 45.49 Tissue 2 1258.47 1006.67** Diet x tissue 4 1.85 1.48 Replicate x diet x tissue 12 1.25 **P{('.Ol Table 40. Multiple range test for polyunsaturated fatty acid content of the monoglyceride-free fatty acid fraction of rat blood, liver and aorta. Standard error of tissue mean 1'0.79 (N2 = 12) Number of pairs (2) (3) Shortest significant range 2.43 2.55 Tissue Blood Liver Aorta Percent polyunsaturated fatty acids 4.66t2.95 16.04t4.96 23.3oiz.4o Note: any two means not underscored by the same line are significantly different. -61.. Significant differences were found in the polyunsaturated fatty acid content of the phospholipids isolated from rat serum, liver and aorta (Tables 41 and 42). Table 41. Analysis of variance of the polyunsaturated fatty acid content of phospholipids isolated from the serum, livers and aortas of rats fed diets A, B and C. Source df MS F Replicates 2 2.58 0.34 Diets 2 37.87 5.04 Replicate x diet 4 7.51 Tissue 2 681.73 33.30** Diet x tissue 4 99.42 4.86* Replicate x diet x tissue 12 20.47 *P(.05, **P< .01 Table 42. Multiple range test for polyunsaturated fatty acid content of the phospholipid fractions isolated from rat serum, liver and aortas. Standard error of mean t 3.20 (N2 = 12) Number of pairs (2) (3) Shortest significant range 9.86 10.18 Tissue Blood serum Liver Aorta Percent polyunsaturated fatty acids 16. 12’57. 14 28.78‘374.50 32.801'4.81 Note: any two means not underscored by the same line are significantly different. -62- Dietary lipids evidently had a limited effect on the polyunsaturated fatty acid composition of the phospholipid fraction. Means for the effects of diets A, B and C were 24.6, 24.8 amd 28.3 percent polyunsaturated fatty acids. Based on the scheme of phospholipid synthesis from/ , fldiglycer— ides proposed by Kennedy and weiss (1955), (1956), it is possible to explain the presence of a greater perzentage of polyunsaturates in the phospholipids isolated from rats on diet C. This eXplanation has also been suggested by Lands and Hart (1964). Brockerhoff e£_§l, (1964) fed glyceryl 1, 3 dioleate, 2 palmitate -l-C14 to rats and found no relationship between this structure and that of palmitate -l-C14 labeled triglycerides and lecithin. The author would suggest that in the presence of specific enzymes for ph05pholipid synthe- sis, the497-uncaturated triglyceride supplied by diet C would be more read- ily acceptable as a precursor for phospholipid synthesis. It has been shown in the present study that the blood serum lipids contained a higher percentage of saturated fatty acids than did the aorta or liver lipids. Fisher and Gurin (1964) reported that high density ( 71.063 and < 1.21) plasma lipoprotein isolated from rat serum contained small quantities of figmly bound long-chain saturated fatty acids. These ‘/’ authors reported that these fatty acids are in all probability, covalently bonded to the protein. In a general review of protein lipid interactions and their relation to the physical-chemical stability of concentrated mdlk, Brunner (1962) was careful not to draw the apparent conclusion that the bound lipid mole- cule associates itself only with receptive sites in the protein. If this were the case, the existence of low-density lip0proteins, such as the -63- [EV-lipoprotein of blood plasma, in which the lipid moiety constitutes more than 70 percent of the complex could not be explained. Brunner (1962) explained that in this case, most of the lipid molecules find themselves in association with other lipid molecules. The hydrocarbon moieties of , these lipids interact strongly through nonpolar bonding, i.e. van der // Waals' forces. Long chain saturated fatty acids, such as stearic, pack rather closely, whereas the distortion around the double bonds of unsatur- ated fatty acids precludes close packing. In the presence of saturated fatty acids, a more cohesive complex could then be formed. WOlf and Dugan (1964) reported that the milk fat-globule membrane contained 89.3 percent saturated fatty acids in the triglyceride fraction, 91.1 percent in the diglyceride fraction and 84.5 percent in the free fatty acid fraction. Calculations revealed that 71.2 percent of the triglycerides were tri- saturated. Mnno- and di-unsaturated glycerides contained saturates primar- ily at theldrlester. The author would then assume that the trisaturated or’dr-saturated glycerides are required to form stable complexes. Gurd (1960) reported that lipid components are exchanged or trans- ferred from the lipoprotein complexes by actual collision between lip0pro- tein complexes themselves. The exchange or transfer occurs in the result- ing collision complex which then decomposes once more into individual lipo- protein complexes. Downie g£_gl, (1963) have shown that lipid deposits occur at the bi- furcation of arteries. At these points there would be a greater physical stress on the lipoprotein complexes as described by Gurd (1960). The pre- sent study indicates that rat serum contains significantly more saturated -64- fatty acids in all of the lipid fractions. This leads the author to the conclusion that lipid fractions containing higher contents of polyunsatur- ated fatty acids form less stable lipoprotein complexes in the serum. These lipid moieties are deposited through physical processes in the more stationary tissues. The accumulation of polyunsaturated fatty acids in various tissues is then predicted to occur. Furthermore, an explanation is possible for the higher cholesterol values observed in blood serum of subjects fed animal fats. More stable lipoprotein complexes would be V/ formed due to the higher saturated fatty acid content and to theyfgLsaturb ated glycerides found in these fats. The stable lipoprotein complexes would be less likely to release cholesterol and cholesterol esters in the liver where they could be converted to and excreted as bile acids. SUMMARY AND CONCLUSIONS A study was made to determine the effects of dietary triglyceride structure on various lipid components of rat tissues and feces. A lipid containing 33 percent polyunsaturated fatty acids predominantly at the d or A tester linkage was isolated from lard and was incorporated into diet B. Another lipid fraction, containing 33 percent polyunsaturated fatty acids predominantly at the/ester linkage, was fractionally crys- tallized from soybean oil. This lipid was introduced into diet C. These u~ effects were compared with those of a fat free diet (diet A). Inclusion of lipids at the level of 40 percent of the calories sig- nificantly increased the feces lipid levels. The diets had no signifi- cant effect on the polyunsaturated fatty acid content of the feces neutral lipid classes. However, the polyunsaturated fatty acid content of feces from rats fed diet 0 tended to be higher. Apparently, there was selective absorption of polyunsaturated fatty acids at the expense of palmitic and stearic acids. There were indications that the phOSpholipid content of the feces from rats fed diet B was somewhat elevated. The diets produced highly unsaturated body fats. It was postulated that a deficient supply of saturated fatty acids in the diet forced the rats to utilize saturated fatty acids previously deposited in the body fat deposits. Diet C produced significantly lower liver cholesterol values. This decrease occurred in the free cholesterol fraction. The polyunsaturated fatty acid content of liver triglycerides and phOSpholipids was significantly higher than in the other fractions. -65- -66- Indirect evidence led to the hypothesis that there were specific enzymes involved in the synthesis of these fractions. The levels of dietary lipids tended to have a direct relationship to the level of blood lipids. The phospholipid content of the serum lipids from rats fed diets B and C was significantly higher. This trend was also indicated by the apparent increase in the phOSpholipid content of blood serum from rats fed diets B and C. The phospholipids contained a significantly higher prOportion of polyunsaturated fatty acids than did the other serum lipid fractions. Blood serum cholesterol levels were decreased by feeding diet C. This decrease occurred in the free cholesterol fraction. The lipid frac- tions of the aorta samples tended to be similar to those of the liver. In view of these results, it was postulated that fractions contain- ing the more unsaturated fatty acids form less stable lipoprotein complexes in the blood serum. These complexes would be more likely to decompose under physical stress allowing for the transfer of lipid fractions to the more stationary tissues, i.e. liver and aorta. It was further postulated that the lipid structure containing the unsaturates primarily at the /57Lester would be less likely to serve as a precursor for the trisaturates and the, -saturated mono- and di-saturated glycerides that tend to stabi- lize lip0protein complexes. This hypothesis would explain the different effects of diets B and C on blood cholesterol even though they contained equal amounts of polyunsaturated fatty acids. In the case of diet B, more stable lip0protein complexes would be formed and the level of circulating cholesterol would be elevated as compared to diet C. This may offer a clue to the cholesterol-lowering effects of vegetable oils. BIBLIOGRAPHY Aftergood, L., H. J. Deuel, Jr., and R. B. Alfin-Slater. 1956. he comparative effects of cottonseed oil and lard on cholesterol levels in the tissues of rats. J. Nutr. 62:129. Ahrens, E. H. Jr., R. Blomstrand, J. Hirsch, W. Insull, Jr., M. L. Peter- son and T. T. Tsaltas. 1957. The influence of dietary fats on serum lipide level in man. Lancet 272:943. hrens, E. H. Jr., J. Hirsch, W. Insull, Jr., R. Blomstrand, T. T. Tsaltas and M. L. Peterson. 1957. The influence of dietary fats on serum lipide levels in man. Jour. Amer. Oil Chemists Soc. 34:563. Ahrens, E. H. Jr., C. H. Blankenhorn and T. T. Tsaltas. 1954. Effect on human serum lipids of substituting plant for animal fat in the diet. Proc. Soc. Exp. Biol. Med. 86:872. Alfin-Slater, R. B., L. Aftergood, A. F. Wells and H. J. Deuel, Jr. 1954. The effect of essential fatty acid deficiency on the distribution of endogenous cholesterol in the plasma and liver of the rat. Arch. Biochem. Biophys. 52:180. Avigan, J. and D. Steinberg. 1958. 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Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:4 A 1 1.3 61.3 13.9 4.7 1.0 14.1 3.5 T T A 21 T 52.2 13.3 5.6 T 17.3 6.2 T o A 32 o 79.3 9.8 6.7 1.3 o 2.0 o o B 13 o 43.9 18.4 5.9 15.9 14.6 0.4 T o B 2 0 41.9 15.3 4.5 0.8 26.6 8.9 T 0 B 44 0.1 53.1 13.3 6.9 1.0 14.8 8.2 T 2.4 C 2 0 52.7 19.7 4.9 T 22.7 T T 0 c 35 o 53.4 14.8 2.2 2.0 17.5 9.0 0.9 o C 4 T 42.7 19.3 7.5 1.9 18.4 4.2 0.2 0 $81.7 percent 20:2 and 3.2 percent 20:3 were detected. 0.7 percent 20:3 was detected. 3 Trace 20:3 was detected. 4 0.1 percent 10:1 was detected. 5 0.1 percent 20:3 was detected. * Tentative identification. -78- Appendix C. Fatty acid composition of diglycerides isolated from rat feces. Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:4 A 1 T 71.4 10.2 5.4 1.5 7.3 4.3 T T A 21 T 73.5 4.8 3.3 T 1.4 2.3 0 4.9 A 3 0 81.1 9.8 2.9 0.5 3.6 1.3 0.4 0.2 B 1 0 64.9 14.9 6.4 3.5 9.2 1.0 T 0.2 B 22 o 66.0 11.1 4.6 1.1 10.4 4.3 0.8 0 B 4 1.7 64.3 11.4 5.1 T 6.4 2.6 T 4.7 c 23 0 78.3 12.0 2.0 1.3 5.5 0.9 T 0 c 34 0 56.4 12.4 2.0 0.8 9.7 5.9 T 0 c 4 0 62.8 14.8 3.9 1.3 6.0 1.8 T 9.6 1 * 5.6 percent 20:2 was detected. 1.6 percent 20:2. Trace of 22:1 was detected. 5.6 percent 22:1 was detected. Tentative identification. #war-a Appendix D. Fatty acid :omposition of monoglycerides-free fatty acids isolated from the feces of rats. Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:4 A 1 T 77.2 7.4 4.8 0.4 6.8 3.1 T T A 21 0 85.8 4.9 3.8 T 1.1 1.1 T 1.9 A 3 0 58.7 24.1 5.0 T 8.7 0.4 0 0 B 1 0 90.2 7.6 1.7 T T T T 0 B 2 0 72.8 11.4 2.7 1.4 7.6 3.8 T 0 B 4 0 64.3 13.6 3.2 1.1 10.3 6.2 1.3 0 c 2 0 76.9 9.1 4.4 1.9 5.8 1.8 T 0 c 32 0 65.6 5.8 8.5 0.9 4.5 3.1 0 17.7 c 43 0.4 14.5 4.5 1.3 0.4 1.7 0.7 T 51.1 5* 1.3 percent and 1.7 percent 17:0 and 20:2 were detected. 3 Trace 22:1 was detected. 21.1 percent, 4.0 percent and 0.3 percent 22:1, 22:2 and 22:3 were detected. * Tentative identification. -80- Appendix E. Fatty acid composition of rat subcutaneous fat. Fatty acid composition Rat No. 12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 A l 0 0.9 0.2 15.9 6.9 2.4 35.0 33.7 5.1 A 2 T 1.0 0.2 16.6 4.4 2.5 36.3 36.5 2.4 A 3 T 1.7 0.8 14.1 6.4 3.6 33.1 33.2 7.2 A 4* 0 1.1 0.4 14.2 6.7 3.3 35.4 33.6 5.3 B l T 1.1 0.4 16.2 6.2 4.4 32.6 33.2 5.9 B 2 T 0.8 0.3 14.8 8.5 2.2 37.2 32.9 3.1 B 3 O 1.1 0 16.5 11.4 1.2 32.8 34.2 2.3 B 4 T 1.2 0.2 17.0 5.8 4.0 35.3 32.3 3.2 C 1 T 3.7 0.5 23.3 5.9 19.1 31.1 13.7 2.1 C 2 0 1.0 0.3 14.4 7.2 4.1 35.4 33.4 4.0 C 3 T 0.8 0.2 16.3 5.4 2.8 35.0 36.5 3.1 C 4 T 0.9 0.2 18.4 5.4 3.9 34.3 33.7 3.2 *Tentatively identified. Appendix F. Fatty acid composition of rat perirenal fat. -31- Fatty acid composition Rat No. 12:0 14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3 20:4 A l 0 0.8 0.2 16.5 3.4 2.9 36.5 37.2 2.5 0 A 2 T 0.9 0.2 15.8 3.8 3.1 36.3 37.0 2.9 0 A 3 T 1.2 0.5 13.4 7.1 4.4 32.1 31.6 9.5 0 A 4 0 0.7 0.2 13.4 6.3 3.2 33.4 32.9 4.7 0 B 1 T 0.9 0.2 16.7 4.7 3.9 33.5 35.2 4.7 0 B 2 T 0.7 0.1 17.6 4.4 2.8 37.9 34.3 2.1 O B 3 0 0.8 0.3 17.4 5.6 2.9 34.3 35.2 3.5 0 B 4 T 1.0 0.2 16.1 6.6 5.4 30.7 32.5 7.7 0 C l 0.1 1.4 0.4 15.7 8.4 7.8 31.3 29.3 5.6 0 C 2 0 0.9 0.2 13.9 2.9 3.5 36.2 39.1 3.3 0 C3 0 0.7 0.2 16.0 2.9 3.7 34.2 37.2 3.5 0 C 4 T 0.8 0.3 18.1 4.7 3.7 33.8 35.0 3.6 0 *Tentative identification. -32- Appendix G. Liver analyses Liver Liver Liver weight percent percent Percent g body total neutral Rat No. weight lipids lipids A 1 12.619 2.53 5.67 23.91 A 2 17.905 3.57 3.67 17.96 A 3 17.751 3.32 3.46 15.81 A 4 9.896 2.17 4.64 19.87 B 1 11.816 2.29 5.92 23.57 B 2 16.301 2.89 6.22 28.95 B 3 28.380 4.37 3.32 42.24 B 4 13.741 2.87 7.35 14.27 C 1 20.796 6.49 3.39 25.36 C 2 13.722 2.66 6.94 52.00 C 3 12.958 2.51 5.22 40.23 C 4 20.381 3.25 5.66 20.85 -33- Appendix H. Fatty acid composition of triglycerides isolated from rat 1 iver o Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 A 1 T 3.4 28.6 2.8 2.5 32.3 29.0 0.1 A 2 T 10.8 30.5 2.9 1.9 28.1 25.4 0.5 A 31 0.1 9.8 26.5 5.3 4.1 25.3 28.0 1.0 A 4 0.1 3.7 22.1 4.5 2.5 34.6 30.7 1.3 B 1 T 3.0 25.1 3.6 4.0 30.9 32.4 0.9 B 2 T 2.0 26.5 3.4 2.3 33.4 31.0 1.1 B 3 T 2.7 28.2 3.2 3.1 35.7 27.7 0.5 B 4 T 9.5 24.9 3.1 3.1 34.1 27.6 0.8 0 1 0 2.3 32.9 4.3 4.2 25.2 31.0 0.1 c 2 0 13.2 24.1 2.4 6.5 36.4 17.4 T c 3 0 3.4 25.4 4.4 4.6 23.7 37.7 0.9 c 4 0 1.8 25.9 3.6 3.0 27.7 37.1 1.0 1* Traces of 8:0 and 10:0 were detected. * Tentative identification. -84- Appendix I. Fatty acid composition of cholesterol esters isolated from rat liver. Fattygacid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 A 1 0.2 36.8 28.2 2.3 4.9 15.8 11.9 T A 21 1.0 54.5 21.4 2.5 1.5 7.8 5.4 0 A 32 2.3 41.7 16.5 1.6 4.0 6.8 7.1 0 A 43 0.8 37.1 18.5 17.3 3.2 11.0 9.0 0.2 B 1 0 13.9 33.3 2.5 9.6 24.3 16.1 T 8 2 0.4 50.7 26.4 4.6 3.2 8.7 6.1 0 B 3 0.3 33.7 26.2 3.1 2.1 20.4 13.2 0.9 B 4 0.5 55.9 18.1 0.5 4.5 13.9 6.7 0 c 1 3.3 6.8 53.3 0.2 7.7 12.9 14.7 0.5 c 2 0 63.7 15.2 0.2 4.7 12.1 4.0 0 c 3 0 45.6 24.6 0 4.3 14.7 10.1 0 c 4 0 38.5 20.7 2.5 5.5 17.3 15.7 0 1* 0.6, 2.2 and 2.9 percent 8:0, 10:0, and 14:1 were detected. 18.5 percent 8:0 and 10:0 and 1.5 percent 14:1 were detected. Trace, 1.7 and 0.9 percent 10:0, 14:1, 15:0 were detected. * Tentative identification. Appendix J. -85- Fatty acid composition of diglycerides isolated from rat liver. Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 A 11 0.5 27.4 32.0 4.0 3.5 14.9 15.1 0.7 A 22 0.6 43.1 27.0 2.3 1.2 11.1 12.7 1.3 A 33 0.4 41.8 25.2 2.7 2.7 9.2 8.5 0 A 4 0.5 48.1 19.3 7.8 2.2 11.5 10.0 0.7 B l 0 40.9 23.3 2.3 3.3 16.0 13.2 T B 2 T 44.8 17.2 1.3 2.3 19.6 14.4 0.4 B 3 T 16.9 27.4 3.2 1.3 23.2 27.2 0.8 B 44 0.3 54.9 17.2 2.7 5.3 11.6 8.3 T C 1 0 33.2 47.1 0.1 4.8 4.2 7.7 3.0 C 2 0 68.0 14.5 0.5 3.9 9.9 3.0 0 C 3 0 34.8 21.3 1.0 6.6 16.8 19.5 T C 4 0 36.2 21.4 2.5 6.9 14.2 18.4 0.6 1* 1.8 percent 14:1 was detected. 2 Trace of 10:0 was detected. 2 9.6 percent 17:0 was detected. Trace of 10:0 was detected. * Tentative identification. Appendix K. -86- fraction isolated from rat liver. Fatty acid composition of monoglyceride-free fatty acid Fattypacid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 A 1 0 33.6 33.3 4.1 4.9 10.4 13.2 0 A 2 0.1 12.0 37.0 3.6 4.3 21.7 20.4 0.9 A 3 0.6 40.6 30.7 4.4 7.0 8.5 8.2 0 A 4 0.3 59.5 17.6 6.7 2.1 6.9 7.0 0 B 11 0 39.4 28.4 0.9 6.8 13.6 9.5 0 B 2 0.2 35.3 23.5 2.7 5.4 16.8 15.8 0.4 B 3 T "33.8 20.3 3.6 3.8 18.6 19.8 0.1 B 4 0.1 27.2 31.3 2.7 7.0 15.0 16.0 0.8 c 1 10.6 20.5 32.4 0 1.3 18.6 16.5 0 c 2 0 7.1 33.6 0 23.0 19.3 17.1 T c 3 0 4.5 18.4 0 35.9 18.3 22.5 T c 4 o 22.0 29.7 3.0 11.0 15.5 18.4 0.6 1* 2.1 percent 17:0 was detected. * tentative identification. Appendix L. ~87- Fatty acid composition of phospholipids isolated from rat liver. Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:4 A 11 0.2 0.6 30.4 0 29.2 12.9 13.4 T 13.3 A 22 T 1.5 23.6 0 26.9 12.9 16.7 T 18.2 A 33 T 1.2 26.4 0 28.1 15.3 13.6 T 15.4 A 44 T 0.8 30.0 0 26.3 14.7 14.3 T 14.0 B 15 T 1.2 23.8 0 36.0 11.3 9.0 T 19.0 B 26 T 0.5 27.7 0 31.0 10.0 11.5 T 18.9 B 37 T 0.5 23.7 0 30.7 14.2 12.3 T 18.0 B 48 T 0.7 24.5 0 32.6 10.7 11.6 T 19.6 c 19 T 1.4 26.0 0 27.2 11.3 18.8 T 15.3 c 210 T 4.0 30.4 0 32.8 11.8 13.1 T 6.6 c 311 T 0.3 24.7 0 36.6 12.3 14.2 T 11.7 c 412 T 0.5 21.8 0 31.8 12.6 17.4 T 15.8 %*Trace of 14:1 was detected. Trace of 14:1 was detected. 3 Trace of 14:1 was detected. 4 Trace of 14:1 was detected. 5 Trace of 14:1 was detected. 6 Trace of 14:1 was detected. 7 Trace of 14:1 was detected. 8 Trace of 14:1 was detected. igrace of 10:0 was detected. 12 * Tentative identification. 1.1 percent 10:0 was detected. 11Trace of 10:0 was detected. Trace of 10:0 was detected. -88- Appendix M. Blood serum lipids Total Percent Percent mg total neutral neutral phospho- Rat No. m1. serum lipids mg/ml lipids lipids lipids 4.0 39.7 9.93 24.7 62.22 37.78 3.5 31.5 9.00 16.9 53.65 46.35 3.1 25.7 8.29 13.1 50.97 49.03 4.1 170.8 41.66 14.0 8.20 91.80 4.7 61.8 13.15 10.7 17.31 82.69 4.1 133.8 32.56 7.6 5.69 94.31 4.2 118.7 28.26 14.2 11.96 88.04 4.3 139.3 32.40 11.2 8.04 91.96 1.3 18.4 14.15 7.2 3.91 96.09 -89- Appendix N. Fatty acid composition of triglycerides isolated from rat blood serum. Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 A 11 0.2 7.8 29.6 6.2 12.5 39.3 3.7 T A 22 0.5 20.8 24.5 4.5 8.8 30.6 8.4 0.5 A 33 0.7 35.5 19.9 4.9 5.5 25.1 7.7 0.7 B 14 0.5 19.0 28.4 5.1 8.1 24.5 13.4 0.5 B 2 0.6 81.7 6.9 4.1 1.9 3.2 1.6 0 B 4 0.9 61.2 11.3 3.0 4.9 11.2 3.8 3.6 c 25 4.4 56.6 9.9 1.9 2.6 7.9 5.7 0 c 36 0.3 35.7 12.8 3.0 4.2 14.2 11.6 0 c 47 1.3 58.3 11.9 2.4 4.6 12.7 6.6 1.4 * 0.7 percent 14:1 was detected. 0.8 and 0.6 percent 10:0 and 14:1 were detected. Trace of 10:0 was detected. Trace of 10:0 was detected. 10.4 percent 10:0 and 0.5 percent 11:0 were detected. 18.2 percent 10:0 was detected. Trace 10:0 and 0.8 percent 14:2 were detected. *Tentative identification. NH noun-kw -90- Appendix 0. Fatty acid composition of cholesterol esters isolated from rat blood serum. Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 A 11 1.5 71.5 10.1 5.8 1.3 6.4 1.5 0 A 22 0.5 50.9 14.0 2.5 2.7 11.5 14.0 0 A 3 1.0 73.2 9.6 1.9 1.9 4.5 8.0 0 B 13 1.3 77.8 6.1 3.3 3.0 2.4 2.8 3.2 B 2 1.3 81.7 7.2 1.0 2.5 3.5 2.8 0 B 44 3.5 77.4 6.5 0.8 1.3 2.2 ' 1.6 0 c 25 1.1 79.5 6.2 1.7 1.4 2.6 3.3 T c 3 0.5 44.5 18.8 2.0 3.5 14.4 11.8 0 c 46 2.1 68.3 11.9 1.7 5.0 6.1 4.8 0 1* 1.9 percent 14:1 was detected. 3.9 percent 14:1 was detected. Trace of 10:0 was detected. 2.6 percent 10:0 and 3.5 percent 14:1 were detected. 4.2 percent 14:1 was detected. Traces of 8:0 and 10:0 were detected. * Tentative identification. mun-Pun -91- Appendix P. Fatty acid composition of diglycerides isolated from rat blood serum. Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 A 11 0.4 58.6 16.6 2.4 5.1 13.5 2.3 0 A 22 0.3 25.0 32.5 3.6 10.9 20.0 4.7 0 A 3 1.0 76.0 11.3 1.9 1.5 5.4 8.0 0 B 13 1.4 62.3 11.9 2.1 7.1 9.6 4.1 0.9 B 2 0.5 53.4 15.4 2.6 3.2 14.8 4.8 5.3 B 44 0.8 64.4 14.8 4.1 3.3 10.4 2.3 0 c 25 18.4 38.6 11.8 1.3 0.9 6.0 10.6 0 c 3 1.1 79.8 7.7 1.5 1.9 3.2 4.4 0 c 4 0 62.3 7.6 3.4 8.4 10.2 8.0 0 * 1.0 percent 14:1 was detected. Trace of 10:0 was detected. 2.8 percent 17:0 was detected. Trace of 10:0 was detected. 12.3 percent 10:0 vas detected. * Tentative identification. UI-PWNH -92- Fatty acid composition of monoglycerides-free fatty acids isolated from rat blood serum. Appendix Q. Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 A 11 0.5 22.4 36.3 5.9 12.5 19.8 1.5 0 A 22 0.7 37.9 27.6 3.8 10.3 14.6 3.2 0.3 3 1.2 42.0 23.5 4.6 6.9 16.1 5.6 0 B 1 0.3 13.0 40.1 4.4 15.3 20.7 6.5 0 B 2 0.6 60.1 17.9 3.2 3.4 11.6 3.2 0 B 43 0.8 55.6 19.6 3.5 4.3 11.3 2.0 1.2 c 24 0.3 86.0 7.3 1.1 0.7 2.0 2.6 0 c 3 0.7 59.9 21.5 2.2 3.7 7.5 4.4 0 c 4 1.0 34.3 26.1 2.0 7.2 18.1 10.4 1.0 1* 0.8 percent 16:2 was detected. 2 1.6 percent 16:2 was detected. 2 unidentified 1.5 percent between 14:0 and 16:0. Trace of 10:0 was detected. * Tentative identification. Appendix R. blood serum. -93- Fatty acid composition of phospholipids isolated from rat Fatty acid composition Rat No. 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:4 A 11 0.8 9.8 31.6 22.1 18.5 11.1 0.1 0 4.4 A 22 1.1 10.7 37.2 0 30.1 12.6 8.3 0 0 A 33 T 3.6 37.0 0 29.0 16.8 10.9 T 2.7 B 14 0.3 4.0 30.4 0 28.7 14.1 15.3 T 7.3 B 25 1.1 11.2 26.3 0 29.8 15.6 12.5 T 2.9 B 46 0.3 6.6 28.4 0 32.8 15.7 14.7 T 1.4 c 2 0.3 9.3 23.5 0 27.5 11.1 16.7 T 11.2 c 37 0.7 20.6 21.1 0 26.7 9.8 14.1 T 6.9 c 48 2.6 18.9 20.0 1.5 27.9 13.5 12.1 T 3.6 1*Trace of 10:0 and 1.6 percent 17:0 were detected. Trace of 10:0 and 17:0 were detected. 3 Trace of 10:0 was detected. 4 Trace of 20:3 was detected. 5 Traces of 8:0, 10:0, 20:3, and 1.3 percent 17:0 were detected. 6 Trace of 20:3 was detected. 7 Trace of 10:0 was detected. 8 Trace of 10:0 was detected. *Tentative identification. -94- Appendix 8. Composition of basal and pretest diets. Ingredient Vitamin free casein Alphacel cellulose Sucrose Salt mixture Vitamin mixture Ground shelled corn Sucrose Soybean oil meal Fishmeal Alfalfa meal Dried Skimmilk Corn oil Super trace mineral salt B-vitamin supplement Vitamin A and D concentrate Basal diet Pretest diet Percent 21.10 16.45 58.45 4.00 336.275 g/lOO lb 46.100 5.000 20.000 10.000 5.000 10.000 3.000 0.500 0.125 .050 :53 (.2: $.23 ‘11 . “ w 31mg 5- ”4"?- Zé'nifi Q9.- wit—Vt- f‘" '1'" "’ I U 9?... ii