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Abstract
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 d3-leucine 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 <3%. To demonstrate the utility of this procedure, we measured d3-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.—Dwyer, K. P., P. H. R. Barrett, D. Chan, J. I. Foo, G. F. Watts, and K. D. Croft. Oxazolinone derivative of leucine for GC-MS: a sensitive and robust method for stable isotope kinetic studies of lipoproteins.
Highlights
Stable isotope labeled amino acids are commonly used as endogenous tracers to study the metabolism of lipoproteins
In this report we show that the oxazolinone derivative of leucine allows excellent precision and limits of detection for isotopic enrichment in apolipoprotein B (apoB) and apoA-I
Levels of enrichment of 0.01% can be obtained with a coefficient of variation (CV) of Ͻ6%, whereas enrichment levels above 0.1% can be determined with a CV of Ͻ2% (Fig. 4)
Summary
Stable isotope labeled amino acids are commonly used as endogenous tracers to study the metabolism of lipoproteins. In metabolic studies using stable isotope tracers, gas chromatography mass spectrometry (GC-MS) is used to measure isotopic enrichment of amino acids in apolipoproteins.
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