Determination of trans-arachidonic acid isomers in human blood plasma

Endogenous trans fatty acids originate from diet, but recent studies also suggest that cis-trans isomerization of fatty acids is possible by nitrogen dioxide radical, a product of NO and nitrite oxidation. We developed a method for quantitative analysis of four trans-arachidonic acids (TAA) in human plasma using isotopic dilution gas chromatography/mass spectrometry (GC/MS) with deuterium-labeled internal standard. Esterification of the plasma fatty acid extract with pentafluorobenzyl (PFB) bromide followed by high-performance liquid chromatography purification yielded a fairly pure fraction containing TAA-PFB esters that was analyzed by GC/MS. Partial separation of the TAA isomers was obtained on various GC columns. Comparison of the retention time with the synthetic standards revealed that all four TAA isomers are present in human plasma. The mean concentration of TAA in human plasma was 20.2ng/ml. The levels of isomers were 12.48+/-1.28, 2.75+/-0.39, and 4.99+/-0.74ng/ml for 5E-AA + 11E-AA, 8E-AA, and 14E-AA, respectively. The identification of TAA in plasma suggests that isomerization of arachidonic acid occurs in vivo. Our method allows distinguishing between the dietary and the NO(2)-dependent mechanisms of trans fatty acid formation and will be useful in defining the role of TAA as an in vivo marker of nitrooxidative stress in clinical and experimental settings.