12-hydroxy-5-8-10-14-eicosatetraenoic-acid and 20-hydroxy-5-8-11-14-eicosatetraenoic-acid

Arachidonic acid-derived lipid mediators play crucial roles in the development and progression of cardiovascular diseases. Eicosanoid metabolites generated by lipoxygenases and cytochrome P450 enzymes produce several classes of molecules, including the epoxyeicosatrienoic acid (EET) and hydroxyeicosatetraenoic acids (HETE) family of bioactive lipids. In general, the cardioprotective effects of EETs have been documented across a number of cardiac diseases. In contrast, members of the HETE family have been shown to contribute to the pathogenesis of ischemic cardiac disease, maladaptive cardiac hypertrophy, and heart failure. The net effect of 12(S)- and 20-HETE depends upon the relative amounts generated, ratio of HETEs:EETs produced, timing of synthesis, as well as cellular and subcellular mechanisms activated by each respective metabolite. HETEs are synthesized by and affect multiple cell types within the myocardium. Moreover, cytochrome P450-derived and lipoxygenase- derived metabolites have been shown to directly influence cardiac myocyte growth and the regulation of cardiac fibroblasts. The mechanistic data uncovered thus far have employed the use of enzyme inhibitors, HETE antagonists, and the genetic manipulation of lipid-producing enzymes and their respective receptors, all of which influence a complex network of outcomes that complicate data interpretation. This review will summarize and integrate recent findings on the role of 12(S)-/20-HETE in cardiac diseases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Animals, Genetically Modified; Cardiomegaly; Cardiovascular Diseases; Cytochrome P-450 Enzyme System; Heart Failure; Humans; Hydroxyeicosatetraenoic Acids; Myocardial Ischemia; Receptors, G-Protein-Coupled

Immunotherapy of cancer is a burgeoning field of research since the realization that our immune system intrinsically has the capacity to restrict tumor occurrence and progression. Though strategies to maximize antitumor T-cell activation are well established, the efficacy of these therapies is limited by an insufficient knowledge of the intricate tumor microenvironment and its capacity to thwart antitumor immunity. Chen and colleagues now uncover a novel immunosuppressive pathway in non-small cell lung carcinoma. Overexpression of cytochrome P450F2 in cancer cells increases production of 20-hydroxyeicosatetraenoic acid, which instructs the expression of immunosuppressive molecules in cancer-associated fibroblasts by binding the GPR75 receptor and activating STAT3/c-Jun signaling. This work proposes several innovative therapeutic anchor points that may improve the efficacy of existing immunotherapies. See related article by Chen et al., p. 4016.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Cancer-Associated Fibroblasts; Carcinoma, Non-Small-Cell Lung; Catalysis; Fibroblasts; Hot Temperature; Humans; Immunosuppression Therapy; Immunosuppressive Agents; Lung Neoplasms; Phenotype; Stromal Cells; Tumor Microenvironment

Post-exercise hypotension (PEH) is the phenomenon of lowered blood pressure after a single bout of exercise. Only a fraction of people develops PEH but its occurrence correlates well with long-term effects of sports on blood pressure. Therefore, PEH has been suggested as a suitable predictor for the effectivity of exercise as therapy in hypertension. Local vascular bioactive lipids might play a potential role in this context. We performed a cross-over clinical pilot study with 18 healthy volunteers to investigate the occurrence of PEH after a single short-term endurance exercise. Furthermore, we investigated the plasma lipid profile with focus on arachidonic acid (AA)-derived metabolites as potential biomarkers of PEH. A single bout of ergometer cycling induced a significant PEH in healthy volunteers with the expected high inter-individual variability. Targeted lipid spectrum analysis revealed significant upregulation of several lipids in the direct post-exercise phase. Among these changes, only 15- hydroxyeicosatetranoic acid (HETE) correlated significantly with the extent of PEH but in an AA-independent manner, suggesting that 15-HETE might act as specific PEH-marker. Our data indicate that specific lipid modulation might facilitate the identification of patients who will benefit from exercise activity in hypertension therapy. However, larger trials including hypertonic patients are necessary to verify the clinical value of this hypothesis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonic Acid; Biological Variation, Population; Blood Pressure; Cross-Over Studies; Dinoprostone; Exercise; Female; Healthy Volunteers; Heart Rate; Humans; Hydroxyeicosatetraenoic Acids; Hypertension; Lipid Metabolism; Male; Pilot Projects; Post-Exercise Hypotension; Thromboxanes

Circulating endothelial progenitor cells (EPCs) are recruited from the blood system to sites of ischemia and endothelial damage, where they contribute to the repair and development of blood vessels. Since numerous eicosanoids including leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) have been shown to exert potent pro-inflammatory activities, we examined their levels in chronic diabetic patients with severe cardiac ischemia in conjunction with the level and function of EPCs.. Lipidomic analysis revealed a diabetes-specific increase (p<0.05) in inflammatory and angiogenic eicosanoids including the 5-lipoxygenase-derived LTB (4.11±1.17 vs. 0.96±0.27 ng/ml), the lipoxygenase/CYP-derived 12-HETE (117.08±35.05 vs. 24.34±10.03 ng/ml), 12-HETrE (17.56±4.43 vs. 4.15±2.07 ng/ml), and the CYP-derived 20-HETE (0.32±0.04 vs. 0.06±0.05 ng/ml) the level of which correlated with BMI (p=0.0027). In contrast, levels of the CYP-derived EETs were not significantly (p=0.36) different between these two groups. EPC levels and their colony-forming units were lower (p<0.05) with a reduced viability in diabetic patients compared with non-diabetics. EPC function (colony-forming units (CFUs) and MTT assay) also negatively correlated with the circulating levels of HgA1C.. This study demonstrates a close association between elevated levels of highly pro-inflammatory eicosonoids, diabetes and EPC dysfunction in patients with cardiac ischemia, indicating that chronic inflammation impact negatively on EPC function and angiogenic capacity in diabetes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; AC133 Antigen; Aged; Antigens, CD; Body Mass Index; Cell Survival; Chromatography, Liquid; Diabetes Mellitus; Eicosanoids; Endothelial Cells; Female; Flow Cytometry; Glycoproteins; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipids; Male; Middle Aged; Myocardial Ischemia; Peptides; Stem Cells; Tandem Mass Spectrometry; Vascular Endothelial Growth Factor Receptor-2

Active metabolites of arachidonic acid (AA), eicosanoids, strongly influence renal homeostasis. The aims of this study were to measure perioperative variations in lipoxygenase (LOX)-derived 5-, 12- and 15-hydroxyeicosatetraenoic (HETE) acids levels, and to examine whether (i) dynamics of these eicosanoid generation changes during the first 5 min of renal allograft reperfusion, (ii) examined HETE acids may influence perioperative 20-HETE generation, and (iii) LOX HETE may serve as perioperative markers of early post-transplant allograft function.. Sixty-nine kidney recipients were divided into early, slow and delayed graft function (EGF, SGF and DGF, respectively) groups. Blood was taken directly before, and in the consecutive minutes of graft reperfusion. HETE concentrations were measured using liquid chromatography. Creatinine levels were measured during the perioperative period, as well as during follow-up visits (first post-transplant year).. Our results demonstrated significant differences in the concentrations and dynamics of HETE changes between the examined groups. Moreover, observed changes in HETE concentrations were strongly associated with post-transplant graft function and perioperative 20-HETE synthesis. Application of cut-off limits for newly introduced markers, that is 71.72 ng/mL for 5-HETE(5), 12.3 ng/mL for 12-HETE△(5-0) and -6.1 ng/mL for 15-HETE△(5-0), resulted in 72.5-81.5% sensitivity and 50-54% specificity for SGF/DGF prediction. Moreover, mixed model analysis revealed that recipients classified according to results of 5-HETE(5) and 15-HETE△(5-0) significantly differ in 1-year post-transplant allograft function (P  =  0.03 and P  < 0.05, respectively), however, not in the frequency of acute rejections' episodes (P = 0.91 and P = 0.31, respectively).. We hereby report that human kidney transplantations are accompanied by significant changes in LOX AA metabolism, which strongly influences and predicts early (1 year) post-transplant graft function.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonic Acid; Biomarkers; Female; Graft Survival; Humans; Hydroxyeicosatetraenoic Acids; Kidney Transplantation; Lipoxygenase; Male; Middle Aged; Perioperative Period; Retrospective Studies; Time Factors; Transplantation, Homologous

Endothelins (ETs) are a family of three peptides (ET-1, ET-2, ET-3) that are implicated in the physiological control of vascular smooth muscle cell (VSMC) and myocardial contractility and growth. ET-1 is vasoactive peptide that acts via ET-A receptors coupling inducing vascular smooth muscle cell contraction. ET-1 is involved in the development and maintenance of hypertension. Aim of this study was to investigate whether ET-1 can induce vascular smooth muscle cell proliferation through arachidonic acid (AA) metabolites formed via cytochrome P¬450 (CYP-450). VSMC proliferation was measured by [3H]thymidine incorporation in cultured cells treated by ET-1 (10 to l00 nmol/L) in presence of different inhibitors of CYP-450 (17-ODYA 5 μmol/L), lipoxygenase (LO) (baicalein 20 μmol/L) and cyclooxygenase (COX) (indomethacin 5 μmol/L). ET-1 (10 to 100 nmol/L) induced VSMC proliferation and this effect was attenuated by CYP-450 inhibitor (17-ODYA) and lipoxygenase (LO) inhibitor (baicalein) but not by cyclooxygenase (COX) inhibitor (indomethacin). CYP-450 and LO metabolites of AA, 20-hydroxyeicosatetraenoic acid (HETE) and 12-HETE increased [3H]thymidine incorporation in VSMC. Inhibitors of MAP kinase (PD-98059 50 μmol/L) and cPLA2 (MAFP 50 μmol/L) attenuated ET-1 as well as 20-HETE induced VSMC proliferation. These results suggest AA metabolites via CYP-450 mediates ET-1 induce VSMC proliferation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Cell Proliferation; Cells, Cultured; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; DNA; Endothelin-1; Enzyme Inhibitors; Flavanones; Hydroxyeicosatetraenoic Acids; Indomethacin; Mitogen-Activated Protein Kinases; Myocytes, Smooth Muscle; Rats; Receptors, Phospholipase A2; Thymidine

Reactive oxygen species (ROS) contribute to various models of hypertension, including deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Recently, we have shown that ROS, generated by cytochrome P-450 1B1 (CYP1B1) from arachidonic acid, mediate vascular smooth muscle cell growth caused by angiotensin II. This study was conducted to determine the contribution of CYP1B1 to hypertension and associated pathophysiological changes produced by DOCA (30 mg/kg) given subcutaneously per week with 1% NaCl + 0.1% KCl in drinking water to uninephrectomized rats for 6 wk. DOCA-salt treatment increased systolic blood pressure (SBP). Injections of the selective inhibitor of CYP1B1, 2,3',4,5'-tetramethoxystilbene (TMS; 300 μg/kg ip every 3rd day) initiated at the 4th week of DOCA-salt treatment normalized SBP and decreased CYP1B1 activity but not its expression in the aorta, heart, and kidney. TMS also inhibited cardiovascular and kidney hypertrophy, prevented the increase in vascular reactivity and endothelial dysfunction, and minimized the increase in urinary protein and K(+) output and the decrease in urine osmolality, Na(+) output, and creatinine clearance associated with DOCA-salt treatment. These pathophysiological changes caused by DOCA-salt treatment and associated increase in vascular superoxide production, NADPH oxidase activity, and expression of NOX-1, and ERK1/2 and p38 MAPK activities in the aorta, heart, and kidney were inhibited by TMS. These data suggest that CYP1B1 contributes to DOCA-salt-induced hypertension and associated pathophysiological changes, most likely as a result of increased ROS production and ERK1/2 and p38 MAPK activity, and could serve as a novel target for the development of agents like TMS to treat hypertension.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antihypertensive Agents; Aorta; Aryl Hydrocarbon Hydroxylases; Blood Pressure; Cardiomegaly; Cytochrome P-450 CYP1B1; Desoxycorticosterone; Disease Models, Animal; Diuresis; Endothelium, Vascular; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Hypertension; Kidney; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle, Smooth, Vascular; Myocardium; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; p38 Mitogen-Activated Protein Kinases; Proteinuria; Rats; Rats, Sprague-Dawley; Sodium Chloride, Dietary; Stilbenes; Superoxides; Time Factors; Vasoconstriction; Vasodilation

The role of eicosanoids (metabolites of arachidonic acid) in prostate diseases is receiving increased attention. We investigated the relationship between the concentrations of urinary free acids of 12- and 20-hydroxyeicosatetraenoic acids (12- and 20-HETE) and the benign prostatic hypertrophy (BPH) and prostate cancer (Pca). Urinary concentrations of 12-HETE and 20-HETE of BPH and Pca patients were significantly higher than normal subjects. After removal of the prostate gland, the urinary concentrations of these eicosanoids decreased to concentrations similar to the normal subjects. These results suggest that urinary free acids of 12-HETE and 20-HETE indicate an abnormality of the prostate gland.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Aged; Chromatography, Liquid; Humans; Hydroxyeicosatetraenoic Acids; Male; Middle Aged; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Spectrometry, Mass, Electrospray Ionization

The monohydroxylated metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), is a potent vasoconstrictor of cerebral microvessels. 20-HETE formation is substantially elevated in the cerebral spinal fluid (CSF) in the rat subarachnoid hemorrhage (SAH) model. The presence of 20-HETE in human CSF has not been demonstrated. Therefore, it was the purpose of this study to determine if HETE metabolites are present in human CSF after SAH.. CSF samples were collected daily from four SAH patients over 15 days. HETE metabolites were separated by HPLC with identification by ion-trap MS/MS and quantification via single quadrupole MS operating in negative single ion monitoring mode.. Two major metabolites were identified as 12-HETE and 20-HETE. 20-HETE maximal concentrations were 2.9 and 0.7 ng/ml at approximately 70 h in the two patients with symptomatic cerebral vasospasm (SV) after SAH. Concentrations of 12-HETE in these patients peaked at 21.9 ng/ml and 2.8 ng/ml. Concentrations of 20-HETE and 12-HETE were non-detectible in the majority of the samples obtained from two matched SAH patients without SV.. This study is the first to demonstrate that 20-HETE and 12-HETE are present in the CSF of SAH patients at physiologically relevant concentrations. Based on this information future prospective studies will allow for the delineation of the role of these metabolites in the pathogenesis of SAH.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Aged; Arachidonic Acid; Brain Ischemia; Cerebral Arteries; Cerebrospinal Fluid; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Mass Spectrometry; Middle Aged; Neurochemistry; Subarachnoid Hemorrhage; Time Factors; Vasospasm, Intracranial

Wound-induced injury of 3T6 fibroblast cultures initiated a repair process stimulated by fetal calf serum (FCS) that restored the integrity of cell cultures. In these experimental conditions, FCS induced arachidonic acid (AA) release and eicosanoid production. Our results show that the inhibition of the cyclooxygenase (COX) and/or cytochrome P-450 pathways significantly decreases the wound closure, whereas that of the lipoxygenase pathway does not modify the wound repair process. Both EP(1) and EP(4) receptors of prostaglandin E(2) (PGE(2)) mediate PGE(2) stimulated 3T6 fibroblast wound closure. Our data suggest that calcium and cAMP are involved in the signaling event induced by PGE(2) during the 3T6 fibroblast wound repair process. On the other hand, we show that ketoconazole, a cytochrome P-450 inhibitor, hinders the wound closure induced by FCS in wounded 3T6 fibroblast cultures. 12 and 20 Hydroxyeicosatetraenoic acids (HETEs), which are key AA metabolites synthesized by cytochrome P-450, partially revert the effects of ketoconazole on the wound repair process. Thus, the COX and cytochrome P-450 pathways of the arachidonate cascade are involved in 3T6 fibroblast wound closure.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Blood Proteins; Cell Line; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dinoprostone; Enzyme Inhibitors; Fibroblasts; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Mice; Prostaglandin-Endoperoxide Synthases; Receptors, Prostaglandin E; Receptors, Prostaglandin E, EP1 Subtype; Receptors, Prostaglandin E, EP4 Subtype; Signal Transduction; Wound Healing

Norepinephrine (NE) stimulates phospholipase D (PLD) through a Ras/MAPK pathway in rabbit vascular smooth muscle cells (VSMC). NE also activates calcium influx and calmodulin (CaM)-dependent protein kinase II-dependent cytosolic phospholipase A(2) (cPLA(2)). Arachidonic acid (AA) released by cPLA(2)-catalyzed phospholipid hydrolysis is then metabolized into hydroxyeicosatetraenoic acids (HETEs) through lipoxygenase and cytochrome P450 4A (CYP4A) pathways. HETEs, in turn, have been shown to stimulate Ras translocation and to increase MAPK activity in VSMC. This study was conducted to determine the contribution of cPLA(2)-derived AA and its metabolites (HETEs) to the activation of PLD. NE-induced PLD activation was reduced by two structurally distinct CaM antagonists, W-7 and calmidazolium, and by CaM-dependent protein kinase II inhibition. Blockade of cPLA(2) activity or protein depletion with selective cPLA(2) antisense oligonucleotides abolished NE-induced PLD activation. The increase in PLD activity elicited by NE was also blocked by inhibitors of lipoxygenases (baicalein) and CYP4A (17-octadecynoic acid), but not of cyclooxygenase (indomethacin). AA and its metabolites (12(S)-, 15(S)-, and 20-HETEs) increased PLD activity. PLD activation by AA and HETEs was reduced by inhibitors of Ras farnesyltransferase (farnesyl protein transferase III and BMS-191563) and MEK (U0126 and PD98059). These data suggest that HETEs are the mediators of cPLA(2)-dependent PLD activation by NE in VSMC. In addition to cPLA(2), PLD was also found to contribute to AA release for prostacyclin production via the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Finally, a catalytically inactive PLD(2) (but not PLD(1)) mutant inhibited NE-induced PLD activity, and PLD(2) was tyrosine-phosphorylated in response to NE by a MAPK-dependent pathway. We conclude that NE stimulates cPLA(2)-dependent PLD(2) through lipoxygenase- and CYP4A-derived HETEs via the Ras/ERK pathway by a mechanism involving tyrosine phosphorylation of PLD(2) in rabbit VSMC.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Aorta; Benzylamines; Cells, Cultured; Cytosol; Enzyme Activation; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Imidazoles; Male; Muscle, Smooth, Vascular; Norepinephrine; Phospholipase D; Phospholipases A; Phospholipases A2; Rabbits; Recombinant Proteins; Sulfonamides; Transfection

Arachidonic acid metabolites such as prostaglandins, thromboxanes, and leukotrienes are well known modulators of intestinal vascular perfusion, motility, and electrogenic ion transport. We investigated the effect of different hydroxyeicosatetraenoic acids (HETEs) from cytochrome P450- and lipoxygenase-dependent arachidonate metabolism on electrogenic chloride secretion in rat distal colon. Using conventional Ussing techniques, basolateral 12-HETE significantly decreased basal short-circuit current (I(sc)) and inhibited furosemide-sensitive Cl(-) secretion stimulated by either dibutyryl cAMP, prostaglandin E(2), or theophylline in a concentration-dependent manner (IC(50) = 1.5 nM). These data were underlined by significant inhibition of J(net)(Cl) in unidirectional (36)Cl flux measurements. Direct regulation of the basolateral Na(+)-K(+)-2Cl(-) cotransporter or the Na-K-ATPase could be excluded because 12-HETE had no effect on furosemide-sensitive K(+) secretion induced by epinephrine, or ouabain-sensitive Na(+) reabsorption stimulated by aldosterone. Inhibitors of Ca(2+)-activated and voltage-gated K(+) channels such as apamin, charybdotoxin, and dendrotoxin did not affect secretagogue-dependent I(sc) and its regulation by 12-HETE. In contrast, glibenclamide significantly attenuated the effect of 12-HETE on secretagogue-induced I(sc), whereas chromanol 293B, an inhibitor of cAMP-dependent K(+) conductance, had an additive effect. We speculate that 12-HETE, like glibenclamide, affects intestinal Cl(-) secretion by inhibiting basolateral K(+)(ATP) channels. In contrast to these findings, neither 5-HETE nor 20-HETE had any effect on basal I(sc) or cAMP-dependent Cl(-) secretion.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Bucladesine; Chlorides; Colon; Furosemide; Hydroxyeicosatetraenoic Acids; Male; Ouabain; Potassium; Potassium Channel Blockers; Rats; Rats, Wistar; Sodium

We recently reported that an enzyme of the cytochrome P-450 4A family is expressed in the glomerulus, but there is no evidence that 20-hydroxyeicosatetraenoic acid (20-HETE) can be produced by this tissue. The purpose of present study was to determine whether glomeruli isolated from the kidney of rats can produce 20-HETE and whether the production of this metabolite is regulated by nitric oxide (NO) and dietary salt intake. Isolated glomeruli produced 20-HETE, dihydroxyeicosatrienoic acids, and 12-hydroxyeicosatetraenoic acid (4.13 +/- 0.38, 4.20 +/- 0.38, and 2. 10 +/- 0.20 pmol. min-1. mg protein-1, respectively) when incubated with arachidonic acid (10 microM). The formation of 20-HETE was dependent on the availability of NADPH and the PO2 of the incubation medium. The formation of 20-HETE was inhibited by NO donors in a concentration-dependent manner. The production of 20-HETE was greater in glomeruli isolated from the kidneys of rats fed a low-salt diet than in kidneys of rats fed a high-salt diet (5.67 +/- 0.32 vs. 2.83 +/- 0.32 pmol. min-1. mg protein-1). Immunoblot experiments indicated that the expression of P-450 4A protein in glomeruli from the kidneys of rats fed a low-salt diet was sixfold higher than in kidneys of rats fed a high-salt diet. These results indicate that arachidonic acid is primarily metabolized to 20-HETE and dihydroxyeicosatrienoic acids in glomeruli and that glomerular P-450 activity is modulated by NO and dietary salt intake.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System; Diet, Sodium-Restricted; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney Glomerulus; Male; Mixed Function Oxygenases; NADP; Nitric Oxide Donors; Rats; Rats, Inbred Lew

Renal cortical microsomes of the cynomolgus monkey, Macaca fascicularis, were incubated with [14C] arachidonic acid in the presence of NADPH. The two main metabolites were identified as 20-hydroxyeicosatetraenoic acid (20-HETE) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) by capillary GC-MS and by chiral phase HPLC. 12(S)-HETE was formed in highly variable amounts by the very same preparation of microsomes on different occasions. The microsomal biosynthesis of 12(S)-HETE was increased several-fold by addition of Ca++, NADPH (or NADH) and ATP, and the biosynthesis was reproducible in the presence of these agents. The biosynthesis was unaffected by proadifen (SKF-525A), however, and was inhibited by nordihydroguaiaretic acid. Some preparations of renal medullary microsomes and cytosol also contained 12(S)-lipoxygenase activity, which was not stimulated by Ca2+, NADPH and ATP and thus resembled the well-known platelet enzyme. Significant formation of 12(R)-HETE could not be detected. In conclusion, 20-HETE was identified as the main cytochrome P-450-derived metabolite and 12(S)-HETE as the main lipoxygenase product in microsomes of monkey renal cortex.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Kidney Medulla; Macaca fascicularis; Microsomes; NADP