Abstract P201: Separation of Postprandial Lipoproteins: Purification of Chylomicrons Using an ApoB100 Immunoaffinity Method

Abstract

Introduction: Elevated fasting and postprandial triglyceride-rich lipoproteins (TRL) are associated with increased risk for atherosclerosis. In the postprandial state circulating lipids consist of dietary fat transported from the intestine, by chylomicrons (containing ApoB48) and fat transported from the liver, in very low-density lipoproteins (VLDL, containing ApoB100). Research into the roles of endogenous versus dietary fat has been hindered because current methods are inadequate to fully separate these particles. Specifically, chylomicron fractions have considerable contamination from VLDL. Hypothesis: We hypothesized that the generation of resin crosslinked to specific ApoB100 antibodies unique to VLDL would allow purification of chylomicron particles. Proof-of-principle experiments will determine: 1) individual contributions of newly synthesized fatty acids (de novo lipogenesis (DNL)), to triglycerides found in VLDL and chylomicrons; and 2) the proportion of labeled acetyl-CoA (precursor-pool value (PPV)) used to synthesize new palmitate. A difference in DNL and PPV for the purified VLDL and chylomicron fractions will confirm the successful separation of these particles. Methods: Polyclonal antibodies were produced against the C-terminus of the ApoB100 protein and crosslinked to resin generating ApoB100-specific affinity columns. To yield purified chylomicron and VLDL particles we used this technique in conjunction with stable-isotope tracer methodology in a human feeding study. Two healthy volunteers consumed a standardized diet accompanied by oral dosing of 1- 13 C-acetate every 30 minutes for 8 hours. Plasma collected hourly was used for the isolation of TRL, which was applied to the affinity resin and purified chylomicrons and VLDL collected. Triglycerides were isolated and derivatized. The enrichment of methyl-palmitate was measured by GC/MS and Mass Isotopomer Distribution Analysis was used to calculate DNL and PPV. Results: Western blots confirmed the separation of chylomicrons and VLDL by the described method. From the purified TRL fractions, the DNL-AUC (%DNL x 5 hours) values for subject 1 were chylomicron 4% and VLDL 34%, an 88% difference, and for subject 2 were chylomicron 8% and VLDL 83%, a 90% difference. The PPV calculated for each fraction in subject 1 were chylomicron 10% and VLDL 13% and in subject 2 were chylomicron 10%, VLDL 12%. On average, the PPV in the chylomicron fraction were 18% less than the VLDL fraction. Conclusions: In conclusion, results demonstrate that the immunoaffinity method effectively separated postprandial lipoproteins showing fractionated particles carry unique information regarding tissue of origin . Future applications include investigating the respective contribution of fat and sugar to atherosclerosis risk.