Analysis of cytochrome P450 metabolites of arachidonic acid by stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry.

摘要: Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators inflammation. It is well-known that play an important role in numerous pathophysiological processes. Therefore, quantitative analysis cytochrome AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic (EETs), dihydroxyeicosatrienoic (DHETs) can provide crucial information uncover underlying mechanisms AA related diseases. Herein, we developed a highly sensitive method identify quantify HETEs, EETs, DHETs extracts biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, pair probes, 2-dimethylaminoethylamine (DMED) d4-2-dimethylaminoethylamine (d4-DMED), were utilized facilely label eicosanoids. The heavy labeled eicosanoid standards prepared used as internal for quantification minimize the matrix ion suppression effects mass spectrometry analysis. In addition, detection sensitivities DMED improved by 3-104 folds standard solution 5-138 serum compared unlabeled analytes. Moreover, good separation isomers was achieved upon labeling. established provided substantial sensitivity (limit at sub-picogram), high specificity, broad linear dynamics range (3 orders magnitude). We further quantified rat liver, heart, brain tissues human using method. results showed 19 could be distinctly detected contents 11-, 15-, 16-, 20-HETE, 5,6-EET, 14,15-EET type 2 diabetes mellitus patients 5-, 12-, 8,9-EET, 5,6-DHET myeloid leukemia had significant changes, demonstrating these may have roles pathogenesis leukemia.