To examine the effect of diet fat type on rates of cholesterol synthesis and esterification during feeding and fasting, nine healthy male subjects were fed solid-food diets of 40% fat as predominantly either olive oil (MONO), safflower-oil margarine (POLY), or butter (SAT). At the end of each two-week diet trial, subjects were given deuterium (D) oxide orally and de novo synthesis was measured from D incorporation into cholesterol and interpreted as rates of fractional synthesis (FSR) (pools/day) into the rapidly exchangeable free cholesterol (FC) pool. Absolute synthesis rates (ASR) were calculated as the product of FSR and the FC pool. Pool size for each subject was obtained from analysis of the specific activity decay curve of an intravenous injection of 4-14C-cholesterol over nine months. Synthesis was measured over two consecutive 12-h fed periods followed by two consecutive 12-h fasted periods. Serum samples were also assayed for lathosterol concentration, an index of cholesterol synthesis. Serum cholesterol and non-HDL cholesterol concentrations were highest on the SAT diet, lowest (P<0.001) on the POLY diet and intermediate on the MONO diet, triglyceride levels were greater (P<0.03) on the SAT diet than on the POLY diet, and HDL levels were lowest (P<0.05) on the SAT diet and highest on the MONO diet. Cholesterol D enrichment and FSR during each 12-h period were greater (P<0.014) on the POLY diet than on the SAT diet; MONO enrichment and FSR were not significantly different from those on the other two diets. Similar results were obtained for rates of cholesterol esterification (P<0.001). Deuterium enrichment data suggested, and lathosterol data confirmed, that free cholesterol synthesis was greater during the fed period than during the fasted period (P<0.01); however, this could not be confirmed for rates of cholesterol esterification. Results suggest that POLY fat feeding augments de novo cholesterol synthesis without adverse effects on total serum cholesterol concentrations, and that the deleterious effects of SAT fat on serum cholesterol are not brought about by augmented de novo synthesis. Finally, the combination of deuterium incorporation and mathematical modelling produces estimates of daily cholesterol synthesis which are compatible with those invoked by more laborious techniques. / Land and Food Systems, Faculty of / Graduate

The study was performed to determine factors influencing the esteriflcation of plasma cholesterol in young and aged rats. The distribution of LCAT activity was determined following gel nitration chromatography and ultracentrifugation of whole plasma respectively. When rat plasma was fractionated on a Bio-Gel A-5 Mcolumn, LCAT activity was found to be associated with the HDL fraction. A similar result was observed upon 24 hr density gradient ultracentrifugation of the plasma. However, following prolonged 40 hr preparative ultracentrifugation, the majority of the LCAT activity was displaced into the lipoprotein-free infranatant fraction (d> 1.225 g/ml). The dissociation of LCAT from the HDL fraction occured to a smaller extent in aged rat plasma than in young rat plasma. Plasma incubation (37°C) experiments followed by the isolation of lipoproteins and the subsequent analysis of their cholesterol content revealed that in vitro net esteriflcation of free cholesterol (FC) by LCAT as well as the fractional ufilization of HDL-FC as substrate were lower in the plasma of the aged animal as compared to that of the young animal despite the fact that the total pool of FC was higher in the former. The net transfer of FC from lower density lipoproteins (d<1.07 g/ml) to HDL provided the FC (in addition to HDL-FC) for esteriflcation in the plasma of both young and aged rats, and this process was not substantially affected by aging. Substrate specificity studies indicated that HDL from young rats was a better substrate for LCAT than the HDL from aged rats. The HDL isolated from the plasma of aged rats was enriched with apo E and had a considerably higher molecular weight than the HDL from young rat plasma. The ratio of phosphatidyl choline/sphingomyelin was lower in the HDL of aged rats. These data suggest that the decreased plasma cholesterol esteriflcation in aged rats is due to changes in the composition and size of the lipoprotein substrate (HDL).

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published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The metabolic fate of cholesterol delivered to rat hepatocytes by rat plasma lipoproteins was determined. Binding and degradation of rat low and high density lipoproteins (LDL and HDL) in rat hepatocytes were studied. ¹²⁵I-labelled LDL and HDL were incubated with cells in the presence of varying concentrations of unlabelled lipoproteins for two hours at 37°C. The amount of ¹²⁵I-LDL and ¹²⁵I-HDL binding and degradation decreased by increasing concentrations of respective unlabelled lipoproteins. The presence of 50-fold excess of unlabelled LDL or HDL resulted in a reduction of ¹²⁵I-LDL and ¹²⁵I-HDL bindings by 66-82%, and degradations by 63-88%, respectively. Equilibrium dissociation constants (K [subscript d]) determined by Scatchard analysis for HDL (.15 x 10⁻⁸M) and LDL (1.04 x 10⁻⁸M) revealed that HDL have approximately 7-fold higher binding affinity for receptors on cell surface than LDL. Specific use of LDL and HDL-cholesterol for bile acid synthesis by rat hepatocytes was investigated. When LDL and HDL labelled with ³H-LDL cholesterol was transformed to bile acids mostly as lithocholic, chenodeoxy and deoxycholic acids. A technique developed for isolation of hepatocytes from rat liver was described. Once isolated by the technique most cells retained their microscopic structural integrity, and excluded trypan blue. The viability was 93%, which decreased to 86% after four hours of incubation. The presented data demonstrated that both HDL and LDL bind to specific receptors on hepatocytes and undergo proteolytic degradation in rats. The study also showed that the binding affinity of HDL to hepatic receptors was much greater than that of LDL but in total binding LDL uptake was four times greater than HDL, suggesting the presence of two specific binding sites for HDL and LDL. The first direct evidence for the preferential utilization of HDL-cholesterol for biosynthesis of bile acids in vivo is presented. This finding is compatible with the current concept of HDL as the protective lipoprotein against developing coronary heart disease. / Graduation date: 1985

The effect of increased egg consumption on plasma cholesterol levels and its distribution among lipoproteins and Apolipoprotein A-I/A-II ratios of high density lipoprotein subfractions in individuals who did and did not demonstrate response in plasma cholesterol levels was studied in 20 healthy middle-aged men (30-55 years of age) for three months. Lunch containing 3 eggs was fed all subjects during the first month classification period. Subsequently, subjects were divided into hyper and hyporesponders according to their blood cholesterol level. Thereafter, 6 eggs were fed to hypo (n=12) while hyper (n=8) continued to consume 3 eggs with lunch. The average plasma cholesterol level of the 20 subjects was increased from 185±34 to 195±34 mg/lOOml during the classification period. The mean plasma cholesterol level of the hyperresponders was significantly (p<0.025) increased from 170±42 to 199±40 mg/100ml during classification while that of the hyporesponders fell slightly. The additional 6 eggs consumed by the hyporesponders for 6 weeks did not increase the mean plasma cholesterol level. The apoprotein ratios did not change as a result of the increased cholesterol diet and were not significantly different between the two response groups. The hyper and hyporesponders differed significantly in initial plasma cholesterol parameters that diminished as the subjects were challenged with increased cholesterol consumption. These initial differences included a significantly lower low density lipoprotein cholesterol/high density lipoprotein cholesterol ratio of 2.26±0.6 for hyperresponders compared to 3.31±0.9 for hyporesponders. There were also initial differences in the percentage of whole plasma cholesterol found in the HDL fractions: hyperresponders had a greater percentage of total and free cholesterol in the HDL and HDL-2 fractions and a greater percentage of total cholesterol in the HDL-3 fraction. The present data demonstrate that increased cholesterol consumption will increase plasma cholesterol levels and that there is individual variability of plasma cholesterol response as a result of feeding cholesterol. / Graduation date: 1984

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