Abstract
Emerging evidence has shown that an abnormal postprandial accumulation of dietary fat is atherogenic. The aim of this study is to measure triacylglycerol (TAG) kinetics in endogenous and exogenous lipoproteins in both fed and fasted states using stable isotopes. Healthy volunteers were injected a bolus of 2H5-glycerol to label TAG and underwent a fasted and fed study in random order. A continuous feeding protocol was developed for the fed study. Lipoproteins were isolated by ultracentrifugation. Endogenous and exogenous particles were purified into apoB100 and apoB48 containing lipoproteins using protein G sepharose coupled to three monoclonal antibodies specific for apoB 100 (4G3, 5E11 and Bsoll 6). The kinetics of the TAG was determined by hydrolysing separated TAG to release glycerol and measuring its enrichment by gas chromatography mass spectrometry. A mathematical model was developed to calculate the fractional catabolic rate (FCR). Six volunteers (2M, 4F) were recruited, age 36.8±3.9yr, BMI 24.4±1.1kg/m2. The separation was complete for both apoB100 and apoB48 containing lipoproteins. Glycerol enrichment of TAG was measured in Sf>60, Sf 20-60 and Sf 12-20 lipoprotein fraction in both fed and fasted states. There was no difference in the FCRs in each density lipoprotein fractions of endogenous lipoproteins between the fasted and fed study. The TAG FCR for chylomicrons (Sf>60) was similar to the FCR for VLDL1 in the fed state and was significantly higher (p=0.01) than the FCR for chylomicron remnants (Sf 20-60). In conclusion, a novel immunoaffinity chromatography technique which achieves complete separation of endogenous and exogenous lipoproteins has been developed and validated. This methodology has the potential to be used to accurately quantify the kinetics of lipoprotein metabolism and could be used to provide an insight into the mechanisms which lead to abnormal postprandial lipoprotein metabolism in metabolic syndrome and type 2 diabetes.