8-9-epoxyeicosatrienoic-acid and 20-hydroxy-5-8-11-14-eicosatetraenoic-acid

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.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Brain; Case-Control Studies; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Deuterium; Diabetes Mellitus, Type 2; Eicosanoids; Humans; Hydroxyeicosatetraenoic Acids; Isotope Labeling; Leukemia, Myeloid; Liver; Male; Myocardium; Organ Specificity; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

It has been previously shown that the cytochrome P450 (P450) modulator, fenofibrate, protects against cardiovascular diseases. P450 and their metabolites, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) were found to play an important role in cardiovascular diseases. Therefore, it is important to examine whether fenofibrate would modulate the cardiac P450 and its associated arachidonic acid metabolites and whether this modulation protects against isoproterenol-induced cardiac hypertrophy. For this purpose, male Sprague-Dawley rats were treated with fenofibrate (30 mg·kg·d), isoproterenol (4.2 mg·kg·d), or the combination of both. The expression of hypertrophic markers and different P450s along with their metabolites was determined. Our results showed that fenofibrate significantly induced the cardiac P450 epoxygenases, such as CYP2B1, CYP2B2, CYP2C11, and CYP2C23, whereas it decreased the cardiac ω-hydroxylase, CYP4A3. Moreover, fenofibrate significantly increased the formation of 14,15-EET, 11,12-EET, and 8,9-EET, whereas it decreased the formation of 20-HETE in the heart. Furthermore, fenofibrate significantly decreased the hypertrophic markers and the increase in heart-to-body weight ratio induced by isoproterenol. This study demonstrates that fenofibrate alters the expression of cardiac P450s and their metabolites and partially protects against isoproterenol-induced cardiac hypertrophy, which further confirms the role of P450s, EETs, and 20-HETE in the development of cardiac hypertrophy.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cardiomegaly; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Fenofibrate; Gene Expression Regulation, Enzymologic; Hydroxyeicosatetraenoic Acids; Hypolipidemic Agents; Isoproterenol; Male; Rats; Rats, Sprague-Dawley

Currently, there are few biomarkers to predict the risk of symptomatic cerebral vasospasm (SV) in subarachnoid hemorrhage (SAH) patients. Mono and dioxygenated arachidonic acid metabolites, involved in the pathogenesis of ischemic injury, may serve as indicators of SV. This study developed a quantitative UPLC-MS/MS method to simultaneously measure hydroxyeicosatetraenoic acid (HETE), dihydroxyeicosatrienoic acid (DiHETrE), and epoxyeicosatrienoic acid (EET) metabolites of arachidonic acid in cerebrospinal fluid (CSF) samples of SAH patients. Additionally, we determined the recovery of these metabolites from polyvinylchloride (PVC) bags used for CSF collection. Linear calibration curves ranging from 0.208 to 33.3 ng/ml were validated. The inter-day and intra-day variance was less than 15% at most concentrations with extraction efficiency greater than 73%. The matrix did not affect the reproducibility and reliability of the assay. In CSF samples, peak concentrations of 8,9-DiHETrE, 20-HETE, 15-HETE, and 12-HETE ranged from 0.293 to 24.9 ng/ml. In rat brain cortical tissue samples, concentrations of 20-, 15-, 12-HETE, 8,9-EET, and 14,15-, 11,12-DiHETrE ranged from 0.57 to 23.99 pmol/g wet tissue. In rat cortical microsomal incubates, all 10 metabolites were measured with formation rates ranging from 0.03 to 7.77 pmol/mg/min. Furthermore, 12-HETE and EET metabolites were significantly altered by contact with PVC bags at all time points evaluated. These data demonstrate that the simultaneous measurement of these compounds in human CSF and rat brain can be achieved with a UPLC-MS/MS system and that this method is necessary for evaluation of these metabolites as potential quantitative biomarkers in future clinical trials.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Brain; Chromatography, Liquid; Humans; Hydroxyeicosatetraenoic Acids; Male; Rats; Rats, Sprague-Dawley; Reference Standards; Subarachnoid Hemorrhage; Tandem Mass Spectrometry; Vasospasm, Intracranial

The renovascular effects of cytochrome P450-dependent arachidonic acid (P450-AA) metabolites synthesized by rat and rabbit kidneys were studied in the rabbit isolated kidney under conditions of constant flow and examined for their dependency on cyclooxygenase relative to their expression of vasoactivity. Kidneys were perfused with Krebs-Henseleit solution, and perfusion pressure was raised to levels of 90 to 110 mm Hg with the addition of 2 to 3 microM phenylephrine to the perfusate. Close arterial injection of 1 to 20 micrograms of 5,6-, 8,9- and 11,12-epoxyeicosatrienoic acid (EET) dose-dependently decreased perfusion pressure. The 5,6-EET was the most potent and the only epoxide dependent on cyclooxygenase for expression of vasoactivity, being inhibited by indomethacin (2.8 microM). In contrast, 14,15-EET resulted in dose-dependent increases in perfusion pressure. The vasodilator effects of the omega- and omega-1 oxidation products, 20-hydroxyeicosatetraenoic acid (HETE) and the stereoisomers of 19-HETE, were also inhibited by indomethacin. Furthermore, the renal vasodilator responses to 5,6-EET were not inhibited by either superoxide dismutase (10 U) or catalase (40 U) and, therefore, were unrelated to the formation of oxygen radicals generated during transformation of the epoxide by cyclooxygenase. As 5,6-EET and 19- and 20-HETE are synthesized by the renal tubules and can affect movement of salt and water, expression of vasoactivity by P450-dependent arachidonic acid metabolites, and after release from a nephron segment, may represent a mechanism that couples altered renal tubular function to appropriate changes in local blood flow.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Pressure; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme System; Eicosanoids; Free Radicals; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney; Male; Prostaglandin-Endoperoxide Synthases; Rabbits; Renal Circulation; Vascular Resistance