Apolipoprotein C-III (apoC-III) is an important regulator of lipoprotein metabolism. Radioisotope and stable isotope kinetic studies show differing results in relation to the kinetics of apoC-III in HDL. Kinetic analysis of HDL apoC-III may be difficult because of its low concentration, as well as the presence of other apoproteins at higher concentration, in the HDL fraction. We used IntralipidT (IL), known to preferentially extract apoC proteins from plasma, as a means of extracting apoC-III from HDL before apoprotein separation by isoelectric focusing gel electrophoresis for the measurement of tracer enrichment. Protein purity was assessed by an isoleucine-to-leucine (Ile/Leu) ratio, as apoC-III contains no isoleucine. We compared apoC-III kinetics in 14 men using a bolus infusion of deuterated leucine. The Ile/ Leu ratio for IL-extracted HDL (IL-HDL) apoC-III (3.0 6 0.7%) was not different from that of VLDL apoC-III (2.6 6 0.6%) but was significantly lower than that of untreated HDL apoC-III (9.0 6 2.9%) (P , 0.001). The isotopic enrichment curves and fractional catabolic rates (FCRs) for IL-HDL apoC-III were not different from those of VLDL apoC-III. In contrast, HDL apoC-III had significantly lower isotopic enrichments and FCRs than IL-HDL apoC-III (P , 0.001).In conclusion, this simple IL method can be used to isolate apoC

Stable isotope labeled amino acids are commonly used as endogenous tracers to study the metabolism of lipoproteins. The determination of isotopic enrichment of particular amino acids in apolipoproteins is carried out by gas chromatography mass spectrometry (GC-MS). This report describes a robust and sensitive derivative for analysis of d 3leucine by GC-MS and its utility in studying the metabolism of human lipoproteins. The trifluoromethyloxazolinone (oxazolinone) derivative of leucine was formed in a rapid single step procedure using a mixture of trifluoroacetic anhydride (TFAA) and trifluoroacetic acid (TFA). Analysis of the oxazolinone by negative ion chemical ionization GC-MS gave excellent sensitivity and precision, which enabled accurate determination of low levels of isotopic enrichment from small amounts of protein. For example, enrichments between 0.05% and 100% in 100 pg leucine can be measured with a coefficient of variation of Ͻ 3%. To demonstrate the utility of this procedure, we measured d 3 -leucine enrichment in apolipoprotein B (apoB) isolated from VLDL and LDL as well as apoA-I isolated from HDL by gel electrophoresis and western blotting. The derivatization procedure gave excellent enrichment data from a single intravenous bolus dose of 5 mg/kg, from which the fractional catabolic rate and production rate of the lipoproteins were calculated. In conclusion, the oxazolinone derivative provides a robust and simple procedure for the sensitive analysis of isotopic enrichment for metabolic studies of human lipoproteins.