Measurement of 20-hydroxyeicosatetraenoic acid in human urine by gas chromatography-mass spectrometry.

Abstract

Arachidonic acid can be metabolized by cytochrome P450 enzymes to a range of compounds that play a central role in the regulation of vascular tone, renal function, and blood pressure (1)(2). In the vasculature, smooth muscle cells produce 20-hydroxyeicosatetraenoic acid (20-HETE) as a major product of CYP450 metabolism. 20-HETE can cause vasoconstriction by inhibition of potassium channels and is thought to contribute to the vasoconstrictor action of hormones such as angiotensin II and endothelin (3)(4). Despite the physiologic importance of CYP450 metabolites of arachidonic acid, very little is known about the regulation of the concentration of 20-HETE in biological fluids or the relationship of these concentrations with physiologic state in healthy individuals. This has in part been attributable to the lack of reliable sensitive and specific assays to measure endogenous concentrations of these compounds. Gas chromatography–mass spectrometry (GCMS) has been used successfully to measure 20-HETE in biological samples. However, available methods rely on one or more thin-layer chromatography steps (5)(6), and for human urine the presence of interfering peaks can be a problem (7). An alternative procedure has recently been reported that uses a sensitive fluorescent HPLC assay (8), although this may lack the specificity of MS. We have developed a simplified and reliable method for the analysis of urinary 20-HETE and analyzed 20-HETE concentrations in 24-h urine samples from a group of 30 healthy individuals. Our method involves the use of a single solid-phase extraction cartridge containing both reversed-phase and strong anion-exchange packing followed by HPLC separation before derivatization and GCMS analysis. We have found that preparation of the tert -butyldimethylsilyl derivative (tBDMS), as originally used by Prakash et al. (6), gives …