Mass spectrometric analysis of 5-hydroxyeicosatetraenoic acid (5-HETE) in the perfusate of the isolated rat lung.

The quantitative analysis of 5-hydroxyeicosatetraenoic acid (5-HETE) is one means by which to assess the activation of the 5-lipoxygenase pathway of arachidonic acid in biologic fluids including lung perfusates. Using combined gas chromatography-mass spectrometry (GC-MS) and an oxygen-18-labeled 5-HETE internal standard, a method of analysis has been developed that involves conversion of 5-HETE into its pentafluorobenzyl ester (PFB) which is purified and separated from 12- and 15-HETE PFB esters by thin-layer chromatography. Following isolation and trimethylsilyl ether formation, analysis on a short 5-m capillary GC column circumvents problems of thermal degradation. Negative ion chemical ionization results in abundant production of carboxylate anions (RCOO-) with little subsequent fragmentation; as little as 40 pg of 5-HETE (s/n approximately 10:1) could be detected in 5 ng of 1,1-[18O]2-5-HETE. Studies of the isolated rat lung perfused with a 4% albumin buffer revealed a significant increase in 5-HETE (750 pg/ml perfusate) when injury was induced by addition of glucose oxidase to the perfusion buffer (0.6 U/ml). This stimulated production of 5-HETE could be reversed by prior perfusion of the lung with the drug piriprost, an inhibitor of the 5-lipoxygenase cascade.