title: Analysis of cytochrome P450 metabolites of arachidonic acid by stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry.
abstract: Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3-104 folds in standard solution and 5-138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes, demonstrating that these eicosanoids may have important roles on the pathogenesis of type 2 diabetes mellitus and myeloid leukemia.
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China.
authors.forename: Quan-Fei
authors.initials: QF
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China.
authors.forename: Yan-Hong
authors.initials: YH
authors.lastname: Hao
authors.affiliation: Department of Pharmacology, Basic Medical School, Wuhan University, Wuhan 430071, PR China.
authors.forename: Ming-Zhou
authors.initials: MZ
authors.lastname: Liu
authors.affiliation: Department of Pharmacology, Basic Medical School, Wuhan University, Wuhan 430071, PR China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Yue
authors.affiliation: Department of Cardiology, Renmin Hospital of Wuhan University, Wuhan 430060, PR China.
authors.forename: Jian
authors.initials: J
authors.lastname: Ni
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China.
authors.forename: Bi-Feng
authors.initials: BF
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, PR China. Electronic address: yqfeng@whu.edu.cn.
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Feng
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003903:Deuterium; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism; D015777:Eicosanoids / Q000378:metabolism; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007553:Isotope Labeling; D007951:Leukemia, Myeloid / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008297:Male; D009206:Myocardium / Q000378:metabolism; D009928:Organ Specificity; D017207:Rats, Sprague-Dawley; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D015777:Eicosanoids; D006893:Hydroxyeicosatetraenoic Acids; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; C040776:5,6-epoxy-8,11,14-eicosatrienoic acid; C050715:8,9-epoxyeicosatrienoic acid; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D003903:Deuterium; D015126:8,11,14-Eicosatrienoic Acid
keywords: 2-Dimethylaminoethylamine; Arachidonic acid; Mass spectrometry analysis; Stable isotope probe