Sex- and enantiospecific differences in the formation rate of hydroxyeicosatetraenoic acids in rat organs.

Hydroxyeicosatetraenoic acids (HETEs) are hydroxylated arachidonic acid (AA) metabolites that are classified into midchain, subterminal, and terminal HETEs. Hydroxylation results in the formation of R and S enantiomers for each HETE, except for 20-HETE. HETEs have multiple physiological and pathological effects. Several studies have demonstrated sex-specific differences in AA metabolism in different organs. In this study, microsomes from the heart, liver, kidney, lung, intestine, and brain of adult male and female Sprague-Dawley rats were isolated and incubated with AA. Thereafter, the enantiomers of all HETEs were analyzed by liquid chromatography-tandem mass spectrometry. We found significant sex- and enantiospecific differences in the formation levels of different HETEs in all organs. The majority of HETEs, especially midchain HETEs and 20-HETE, showed significantly higher formation rates in male organs. In the liver, the R enantiomer of several HETEs showed a higher formation rate than the corresponding S enantiomer (e.g., 8-, 9-, and 16-HETE). On the other hand, the brain and small intestine demonstrated a higher abundance of the S enantiomer. 19(S)-HETE was more abundant than 19(R)-HETE in all organs except the kidney. Elucidating sex-specific differences in HETE levels provides interesting insights into their physiological and pathophysiological roles and their possible implications for different diseases.

Structural basis for altered positional specificity of 15-lipoxygenase-1 with 5S-HETE and 7S-HDHA and the implications for the biosynthesis of resolvin E4.

Human 15-lipoxygenases (LOX) are critical enzymes in the inflammatory process, producing various pro-resolution molecules, such as lipoxins and resolvins, but the exact role each of the two 15-LOXs in these biosynthetic pathways remains elusive. Previously, it was observed that h15-LOX-1 reacted with 5S-HETE in a non-canonical manner, producing primarily the 5S,12S-diHETE product. To determine the active site constraints of h15-LOX-1 in achieving this reactivity, amino acids involved in the fatty acid binding were investigated. It was observed that R402L did not have a large effect on 5S-HETE catalysis, but F414 appeared to π-π stack with 5S-HETE, as seen with AA binding, indicating an aromatic interaction between a double bond of 5S-HETE and F414. Decreasing the size of F352 and I417 shifted oxygenation of 5S-HETE to C12, while increasing the size of these residues reversed the positional specificity of 5S-HETE to C15. Mutants at these locations demonstrated a similar effect with 7S-HDHA as the substrate, indicating that the depth of the active site regulates product specificity for both substrates. Together, these data indicate that of the three regions proposed to control positional specificity, π-π stacking and active site cavity depth are the primary determinants of positional specificity with 5S-HETE and h15-LOX-1. Finally, the altered reactivity of h15-LOX-1 was also observed with 5S-HEPE, producing 5S,12S-diHEPE instead of 5S,15S-diHEPE (aka resolvin E4 (RvE4). However, h15-LOX-2 efficiently produces 5S,15S-diHEPE from 5S-HEPE. This result is important with respect to the biosynthesis of the RvE4 since it obscures which LOX isozyme is involved in its biosynthesis. Future work detailing the expression levels of the lipoxygenase isoforms in immune cells and selective inhibition during the inflammatory response will be required for a comprehensive understanding of RvE4 biosynthesis.

Novel Synthetic Analogues of 19(S/R)-Hydroxyeicosatetraenoic Acid Exhibit Noncompetitive Inhibitory Effect on the Activity of Cytochrome P450 1A1 and 1B1.

BACKGROUND AND OBJECTIVES: Cytochrome P450 (CYP) 1A1 and CYP1B1 enzymes play a significant role in the pathogenesis of cancer and cardiovascular diseases (CVD) such as cardiac hypertrophy and heart failure. Previously, we have demonstrated that R- and S-enantiomers of 19-hydroxyeicosatetraenoic acid (19-HETE), an arachidonic acid endogenous metabolite, enantioselectively inhibit CYP1B1. The current study was conducted to test the possible inhibitory effect of novel synthetic analogues of R- and S-enantiomers of 19-HETE on the activity of CYP1A1, CYP1A2, and CYP1B1. METHODS: The O-dealkylation rate of 7-ethoxyresorufin (EROD) by recombinant human CYP1A1 and CYP1B1, in addition to the O-dealkylation rate of 7-methoxyresorufin (MROD) by recombinant human CYP1A2, were measured in the absence and presence of varying concentrations (0-40 nM) of the synthetic analogues of 19(R)- and 19(S)-HETE. Also, the possible inhibitory effect of both analogues on the catalytic activity of EROD and MROD, using RL-14 cells and human liver microsomes, was assessed. RESULTS: The results showed that both synthetic analogues of 19(R)- and 19(S)-HETE exhibited direct inhibitory effects on the activity of CYP1A1 and CYP1B1, while they had no significant effect on CYP1A2 activity. Nonlinear regression analysis and comparisons showed that the mode of inhibition for both analogues is noncompetitive inhibition of CYP1A1 and CYP1B1 enzymes. Also, nonlinear regression analysis and Dixon plots showed that the R- and S-analogues have KI values of 15.7 ± 4.4 and 6.1 ± 1.5 nM for CYP1A1 and 26.1 ± 2.9 and 9.1 ± 1.8 nM for CYP1B1, respectively. Moreover, both analogues were able to inhibit EROD and MROD activities in a cell-based assay and human liver microsomes. CONCLUSIONS: Therefore, the synthetic analogues of 19-HETE could be considered as a novel therapeutic approach in the treatment of cancer and CVD.

Structural and mechanistic insights into 5-lipoxygenase inhibition by natural products.

Leukotrienes (LT) are lipid mediators of the inflammatory response that are linked to asthma and atherosclerosis. LT biosynthesis is initiated by 5-lipoxygenase (5-LOX) with the assistance of the substrate-binding 5-LOX-activating protein at the nuclear membrane. Here, we contrast the structural and functional consequences of the binding of two natural product inhibitors of 5-LOX. The redox-type inhibitor nordihydroguaiaretic acid (NDGA) is lodged in the 5-LOX active site, now fully exposed by disordering of the helix that caps it in the apo-enzyme. In contrast, the allosteric inhibitor 3-acetyl-11-keto-beta-boswellic acid (AKBA) from frankincense wedges between the membrane-binding and catalytic domains of 5-LOX, some 30 Å from the catalytic iron. While enzyme inhibition by NDGA is robust, AKBA promotes a shift in the regiospecificity, evident in human embryonic kidney 293 cells and in primary immune cells expressing 5-LOX. Our results suggest a new approach to isoform-specific 5-LOX inhibitor development through exploitation of an allosteric site in 5-LOX.

Reduced Heart Exposure of Diclofenac by Its Polymeric Micellar Formulation Normalizes CYP-Mediated Metabolism of Arachidonic Acid Imbalance in An Adjuvant Arthritis Rat Model: Implications in Reduced Cardiovascular Side Effects of Diclofenac by Nanodrug Delivery.

In this study, we tested whether the extent of drug presence in the heart contributes to the elevated cardiovascular risk of nonsteroidal anti-inflammatory drugs (NSAIDs). A fluorescently tagged nanoformulation of an NSAID with high cardiovascular (CV) risk (diclofenac) was developed as diclofenac ethyl ester (DFEE) encapsulated in traceable (cyanine-5.5-labeled) polymeric micelles (DFEE-TM) based on methoxypoly(ethylene oxide)-block-poly(ε-caprolactone)(PEO-b-PCL) (MW, 5000:3500 g/mol). Diclofenac pharmacokinetics and tissue distribution, as well as ex vivo near-infrared images of organs and whole bodies, were compared between healthy rats and rats with adjuvant arthritis (AA) following the administration of a single intravenous (iv) dose of DFEE-TM. Moreover, the biodistribution and antiarthritic activity of DFEE-TM were compared with those of free diclofenac (once-daily intraperitoneal, ip, 10 mg/kg for 7 days). The concentration ratios of cytochrome-P450-mediated cardiotoxic (20-hydroxyeicosatetraenoic acid) over cardioprotective (epoxyeicosatrienoic acids) metabolites of arachidonic acid (ArA) in the heart, kidneys, and plasma were measured as markers of cardiotoxicity. The nanocarrier was found in the joints of AA, but not in those of healthy rats. Both free diclofenac and DFEE-TM comparably controlled AA. Diclofenac delivery via PEO-b-PCL micelles reduced the accumulation of diclofenac in the heart of AA rats. Despite similar antiarthritic activity, the polymeric micellar formulation showed a reduction in the ratio of cardiotoxic-over-cardioprotective eicosanoids of ArA in the heart and plasma of AA rats. The results showed the positive effect of diclofenac prodrug nanodelivery in changing the normal biodistribution of diclofenac away from the heart, leading to lowered diclofenac-induced biomarkers of cardiotoxicity in the heart and plasma of AA rats.

Inhibition of 20-hydroxyeicosatetraenoic acid (20-HETE) glucuronidation by non-steroidal anti-inflammatory drugs in human liver microsomes and recombinant UDP-glucuronosyltransferase enzymes.

20-hydroxyeicosatetraenoic acid (20-HETE) is an arachidonic acid metabolite which is known to increase platelet aggregation and cardiovascular risk. In this study, nine non-steroidal anti-inflammatory drugs (NSAIDs) selected by chemical structures were screened to determine their effects on the glucuronidation of 20-HETE using human liver microsomes (HLMs). Then, the combined effects of the selected NSAID and genetic polymorphisms in UDP-glucuronosyltransferase (UGT) were investigated. Among the tested NSAIDs, diclofenac was the strongest inhibitor of 20-HETE glucuronidation with an IC50 value of 3.5 µM. Celecoxib, naproxen, mefenamic acid, ibuprofen, and indomethacin showed modest inhibition with IC50 values of 77, 91, 190, 208, and 220 µM, respectively, while acetylsalicylic acid, rofecoxib, and meloxicam did not inhibit 20-HETE glucuronidation. Glucuronidation of 20-HETE by UGT2B7 and UGT1A9 recombinant enzymes was significantly inhibited by indomethacin, mefanemic acid, diclofenac, ibuprofen, naproxen, and celecoxib (P < 0.001). In addition, diclofenac exhibited a competitive inhibition mechanism with the Km value of 20-HETE glucuronidation increasing from 23.5 µM to 62 µM in the presence of 3.5 µM diclofenac. Diclofenac further decreased 20-HETE glucuronidation in HLMs carrying UGT2B7*2 alleles compared with the wild-type HLMs. The results from this study would be useful in understanding the alteration of 20-HETE levels in relation to NSAID and UGT genetic polymorphisms.

Arg-513 and Leu-531 Are Key Residues Governing Time-Dependent Inhibition of Cyclooxygenase-2 by Aspirin and Celebrex.

Aspirin and Celebrex are well-known time-dependent inhibitors of the cyclooxygenases (COX). Molecular dynamics simulations suggest that Arg-513 and Leu-531 contribute to the structural mechanisms of COX inhibition. We used mutagenesis and functional analyses to characterize how substitutions at these positions influence time-dependent inhibition by aspirin and Celebrex. We show that substitutions of Leu-531 with asparagine and phenylalanine significantly attenuate time-dependent inhibition of COX-2 by these drugs. The introduction of side chain bulk, rigidity, and charge would disrupt the formation of the initial noncovalent complex, in the case of aspirin, and the "high-affinity" binding state, in the case of Celebrex. Substitution of Arg-513 with histidine (the equivalent residue in COX-1) resulted in a 2-fold potentiation of aspirin inhibition, in support of the hypothesis that the presence of histidine in COX-1 lowers the activation barrier associated with the formation of the initial noncovalent enzyme-inhibitor complex. As a corollary, we previously hypothesized that the flexibility associated with Leu-531 contributes to the binding of arachidonic acid (AA) to acetylated COX-2 to generate 15R-hydroxyeicosatetraenoic acid (15R-HETE). We determined the X-ray crystal structure of AA bound to Co3+-protoporphyrin IX-reconstituted V349I murine COX-2 (muCOX-2). V349I muCOX-2 was utilized as a surrogate to trap AA in a conformation leading to 15R-HETE. AA binds in a C-shaped pose, facilitated by the rotation of the Leu-531 side chain. Ile-349 is positioned to sterically shield antarafacial oxygen addition at carbon-15 in a manner similar to that proposed for the acetylated Ser-530 side chain.

Revising the structure of a new eicosanoid from human platelets to 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid.

Eicosanoids are critical mediators of fever, pain, and inflammation generated by immune and tissue cells. We recently described a new bioactive eicosanoid generated by cyclooxygenase-1 (COX-1) turnover during platelet activation that can stimulate human neutrophil integrin expression. On the basis of mass spectrometry (MS/MS and MS3), stable isotope labeling, and GC-MS analysis, we previously proposed a structure of 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (DXA3). Here, we achieved enzymatic synthesis and 1H NMR characterization of this compound with results in conflict with the previously proposed structural assignment. Accordingly, by using LC-MS, we screened autoxidation reactions of 11-hydroperoxy-eicosatetraenoic acid (11-HpETE) and thereby identified a candidate sharing the precise reverse-phase chromatographic and MS characteristics of the platelet product. We optimized these methods to increase yield, allowing full structural analysis by 1H NMR. The revised assignment is presented here as 8,9-11,12-diepoxy-13-hydroxyeicosadienoic acid, abbreviated to 8,9-11,12-DiEp-13-HEDE or DiEpHEDE, substituted for the previous name DXA3 We found that in platelets, the lipid likely forms via dioxolane ring opening with rearrangement to the diepoxy moieties followed by oxygen insertion at C13. We present its enzymatic biosynthetic pathway and MS/MS fragmentation pattern and, using the synthetic compound, demonstrate that it has bioactivity. For the platelet lipid, we estimate 16 isomers based on our current knowledge (and four isomers for the synthetic lipid). Determining the exact isomeric structure of the platelet lipid remains to be undertaken.

Synthesis of oxidized phospholipids by sn-1 acyltransferase using 2-15-HETE lysophospholipids.

Recently, oxidized phospholipid species have emerged as important signaling lipids in activated immune cells and platelets. The canonical pathway for the synthesis of oxidized phospholipids is through the release of arachidonic acid by cytosolic phospholipase A2α (cPLA2α) followed by its enzymatic oxidation, activation of the carboxylate anion by acyl-CoA synthetase(s), and re-esterification to the sn-2 position by sn-2 acyltransferase activity (i.e. the Lands cycle). However, recent studies have demonstrated the unanticipated significance of sn-1 hydrolysis of arachidonoyl-containing choline and ethanolamine glycerophospholipids by other phospholipases to generate the corresponding 2-arachidonoyl-lysolipids. Herein, we identified a pathway for oxidized phospholipid synthesis comprising sequential sn-1 hydrolysis by a phospholipase A1 (e.g. by patatin-like phospholipase domain-containing 8 (PNPLA8)), direct enzymatic oxidation of the resultant 2-arachidonoyl-lysophospholipids, and the esterification of oxidized 2-arachidonoyl-lysophospholipids by acyl-CoA-dependent sn-1 acyltransferase(s). To circumvent ambiguities associated with acyl migration or hydrolysis, we developed a synthesis for optically active (d- and l-enantiomers) nonhydrolyzable analogs of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC). sn-1 acyltransferase activity in murine liver microsomes stereospecifically and preferentially utilized the naturally occurring l-enantiomer of the ether analog of lysophosphatidylcholine. Next, we demonstrated the high selectivity of the sn-1 acyltransferase activity for saturated acyl-CoA species. Importantly, we established that 2-15-hydroxyeicosatetraenoic acid (HETE) ether-LPC sn-1 esterification is markedly activated by thrombin treatment of murine platelets to generate oxidized PC. Collectively, these findings demonstrate the enantiomeric specificity and saturated acyl-CoA selectivity of microsomal sn-1 acyltransferase(s) and reveal its participation in a previously uncharacterized pathway for the synthesis of oxidized phospholipids with cell-signaling properties.

Comprehensive measurements of hydroxylinoleate and hydroxyarachidonate isomers in blood samples from primary open-angle glaucoma patients and controls.

We previously reported that lower systemic antioxidant capacity is involved in primary open-angle glaucoma (POAG) and exfoliation syndrome pathogeneses as measured by ferric-reducing activity. In the present study, we measured hydroxylinoleate (HODE) and hydroxyarachidonate (HETE) isomer serum levels after sample reduction and saponification to investigate POAG pathogenesis. POAG patients (n = 198) were recruited and divided into normal- and high-tension glaucoma groups (n = 84 and 114, respectively) depending on intraocular pressure. Total HODE (/linoleic acid) and HETE (/arachidonic acid) serum levels were significantly higher in the POAG group (211.9 ± 143.0 and 181.0 ± 164.1 µmol/mol, respectively) than in controls (167.1 ± 105.2 and 132.5 ± 139.7 µmol/mol, p = 0.0025 and 0.0101, respectively). The associations between HODEs/HETEs and glaucoma were further confirmed by multivariate analyses after adjusting for differences in demographic parameters. Among the HODE isomers, the levels of 9- and 13-(Z,E)-HODEs (p = 0.0014) and singlet oxygen-specific products (i.e., 10- and 12-(Z,E)-HODEs, p = 0.0345) were higher in the POAG group than in controls, while free radical-mediated oxidation-specific products (i.e., 9- and 13-(E,E)-HODEs, p = 0.0557) demonstrated a marginal difference. Enzymatic and singlet oxygen-mediated fatty acid oxidation may be major pathways of oxidation process in glaucoma subjects.

Identification of 19-(S/R)Hydroxyeicosatetraenoic Acid as the First Endogenous Noncompetitive Inhibitor of Cytochrome P450 1B1 with Enantioselective Activity.

The overexpression of cytochrome P450 1B1 (CYP1B1) is a common characteristic of several diseases and conditions, such as inflammation, cancer, and cardiac hypertrophy. CYP1B1 is believed to contribute to pathogenesis of these diseases by mediating the formation of toxic compounds, either from exogenous or endogenous origin. We recently reported that an arachidonic acid metabolite, 19(S/R-)hydroxyeicosatetraenoic (HETE) acid, protects from cardiac hypertrophy by inhibiting the formation of toxic compounds, midchain HETEs, known to be formed by CYP1B1. This raised the question whether 19(S/R)-HETE can directly inhibit CYP1B1. In the current study, we report that 19(S/R)-HETE enantioselectively inhibits human recombinant CYP1B1 activity measured by 7-ethoxyresorufin O-deethylation assay. 19(S)-HETE is more potent than the R enantiomer (K i = 37.3 and 89.1 nM, respectively). Noncompetitive inhibition was identified as the mechanism of CYP1B1 inhibition, which underlines the potentially important physiologic role of 19(S/R)-HETE as an endogenous CYP1B1 inhibitor; to our knowledge, 19(S/R)-HETE is the first inhibitor of its kind to be reported.

Deuterated Arachidonic Acids Library for Regulation of Inflammation and Controlled Synthesis of Eicosanoids: An In Vitro Study.

The synthesis of signal lipids, including eicosanoids, is not fully understood, although it is key to the modulation of various inflammatory states. Recently, isotopologues of essential polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic positions (D-PUFAs) have been proposed as inhibitors of non-enzymatic lipid peroxidation (LPO) in various disease models. Arachidonic acid (AA, 20:4 n-6) is the main precursor to several classes of eicosanoids, which are produced by cyclooxygenases (COX) and lipoxygenases (LOX). In this study we analyzed the relative activity of human recombinant enzymes COX-2, 5-LOX, and 15-LOX-2 using a library of arachidonic acids variably deuterated at the bis-allylic (C7, C10, and C13) positions. Kinetic parameters (KM, Vmax) and isotope effects calculated from kH/kD for seven deuterated arachidonic acid derivatives were obtained. Spectroscopic methods have shown that deuteration at the 13th position dramatically affects the kinetic parameters of COX-2 and 15-LOX-2. The activity of 5-LOX was evaluated by measuring hydroxyeicosatetraenoic acids (8-HETE and 5-HETE) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Deuteration at the seventh and 10th positions affects the performance of the 5-LOX enzyme. A flowchart is proposed suggesting how to modulate the synthesis of selected eicosanoids using the library of deuterated isotopologues to potentially fine-tune various inflammation stages.

5,6-DiHETE attenuates vascular hyperpermeability by inhibiting Ca2+ elevation in endothelial cells.

Although more than 100 lipid metabolites have been identified, their bioactivities remain unknown. In a previous study, we discovered that the production of several lipid metabolites in the intestines dramatically changed in colitis. Of these metabolites, 5,6-dihydroxyeicosatetraenoic acid (DiHETE) possesses novel anti-inflammatory activity in the vasculature. In this study, we used mouse and human umbilical vein endothelial cell (HUVEC) models to elucidate the mechanisms underlying the vascular activity of lipid metabolites, particularly those related to the release of histamine, a major proinflammatory mediator that stimulates endothelial cells to produce NO, a mediator of vascular relaxation and hyperpermeability, by activating intracellular Ca2+ concentration-dependent signaling. In a mouse ear, the administration of 5,6-DiHETE did not induce inflammatory reactions, and pretreatment with 5,6-DiHETE inhibited histamine-induced inflammation, specifically vascular dilation and hyperpermeability. In an isolated mouse aorta, 5,6-DiHETE treatment did not influence vascular contraction but attenuated acetylcholine-induced vascular relaxation. In HUVECs, treatment with 5,6-DiHETE inhibited histamine-induced endothelial barrier disruption and inhibited the production of NO. Most notably, 5,6-DiHETE inhibited histamine-induced increases in intracellular Ca2+ concentrations in HUVECs. Our findings suggest that 5,6-DiHETE attenuates vascular hyperpermeability during inflammation by inhibiting endothelial Ca2+ elevation, which might lead to a novel pharmacological strategy against inflammatory diseases.

Analysis of HETEs in human whole blood by chiral UHPLC-ECAPCI/HRMS.

The biosynthesis of eicosanoids occurs enzymatically via lipoxygenases, cyclooxygenases, and cytochrome P450, or through nonenzymatic free radical reactions. The enzymatic routes are highly enantiospecific. Chiral separation and high-sensitivity detection methods are required to differentiate and quantify enantioselective HETEs in complex biological fluids. We report here a targeted chiral lipidomics analysis of human blood using ultra-HPLC-electron capture (EC) atmospheric pressure chemical ionization/high-resolution MS. Monitoring the high-resolution ions formed by the fragmentation of pentafluorobenzyl derivatives of oxidized lipids during the dissociative EC, followed by in-trap fragmentation, increased sensitivity by an order of magnitude when compared with the unit resolution MS. The 12(S)-HETE, 12(S)-hydroxy-(5Z,8E,10E)-heptadecatrienoic acid [12(S)-HHT], and 15(S)-HETE were the major hydroxylated nonesterified chiral lipids in serum. Stimulation of whole blood with zymosan and lipopolysaccharide (LPS) resulted in stimulus- and time-dependent effects. An acute exposure to zymosan induced ∼80% of the chiral plasma lipids, including 12(S)-HHT, 5(S)-HETE, 15(R)-HETE, and 15(S)-HETE, while a maximum response to LPS was achieved after a long-term stimulation. The reported method allows for a rapid quantification with high sensitivity and specificity of enantiospecific responses to in vitro stimulation or coagulation of human blood.

Isomer distribution of hydroxyoctadecadienoates (HODE) and hydroxyeicosatetraenoates (HETE) produced in the plasma oxidation mediated by peroxyl radical, peroxynitrite, hypochlorite, 15-lipoxygenase, and singlet oxygen.

Free and ester forms of unsaturated fatty acids and cholesterol are oxidized in vivo by multiple oxidants to give diverse products. Some lipid oxidation is mediated by enzymes to selectively give specific products, while others proceed randomly to produce mixtures of many kinds of regioisomers and stereoisomers. The efficacy of antioxidants against lipid oxidation depends on the nature of the oxidants and therefore the identification of oxidant is important for understanding the roles and effects of lipid oxidation and antioxidants in vivo. In the present study, the isomer distribution of hydro(pero)xyoctadecadienoates (H(p)ODEs) and hydro(pero)xyeicosatetraenoates (H(p)ETEs), the most abundant lipid oxidation products found in human plasma, produced in the oxidation of plasma by peroxyl radicals, peroxynitrite, hypochlorite, 15-lipoxygenase, and singlet oxygen were examined. It was shown that 9- and 13-(E,E)-HODEs, 13(S)-(Z,E)-HODE, and 10- and 12-(Z,E)-HODEs were specific lipid oxidation products by free radical, 15-lipoxygenase, and singlet oxygen, respectively. The isomer distribution of HODEs produced by peroxynitrite was similar to that by peroxyl radical, suggesting that the peroxynitrite mediated lipid oxidation proceeds by free radical mechanisms. The production of HODEs and HETEs by hypochlorite was very small. HODEs may be a better biomarker than HETEs since linoleates are oxidized by simpler mechanisms than arachidonates and all the HODEs isomers can be quantified more easily. These products may be used as specific biomarkers for the identification of responsible oxidants and for the assessment of oxidant-specific lipid oxidation levels and effects of antioxidants in vivo.

Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition.

Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.

Functional characterization of a common CYP4F11 genetic variant and identification of functionally defective CYP4F11 variants in erythromycin metabolism and 20-HETE synthesis.

CYP4F11, together with CYP4F2, plays an important role in the synthesis of 20-hydroxyeicosatetraenoic acid (20-HETE) from arachidonic acid. We identified 21 variants by whole exome sequencing, including 4 non-synonymous variants in Korean subjects. The proteins of the wild-type CYP4F11 and the four coding variants (C276R, D315N, D374Y, and D446N) were expressed in Escherichia coli DH5α cells and purified to give cytochrome P450-specific carbon monoxide difference spectra. Wild-type CYP4F2 was also expressed and purified to compare its activity with the CYP4F11 wild-type. Wild-type CYP4F11 exhibited the highest maximal clearance for erythromycin N-demethylase activity followed by the variants D374Y, D446N, C276R, and D315N. In particular, the CYP4F11 D315N protein showed about 50% decrease in intrinsic clearance compared to the wild type. The ability of wild-type CYP4F11 and the variants to synthesize 20-HETE from arachidonic acid was similar; the CYP4F11 D315N variant, however, showed only 68% of wild-type activity. Furthermore, the ability of CYP4F2 to synthesize 20-HETE was 1.7-fold greater than that of CYP4F11. Overall, our results suggest that the metabolism of CYP4F11 substrates may be reduced in individuals carrying the CYP4F11 D315N genetic variant and individuals carrying the common D446N CYP4F11 variant likely exhibit comparable 20-HETE synthesis as individuals expressing wild-type CYP4F11.

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl-γ-cyclodextrin-modified micellar electrokinetic chromatography.

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl-γ-cyclodextrin (HP-γ-CD)-modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate-15 mM borate buffer (pH 9.0) containing 30 mM HP-γ-CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)- and (R)-enantiomers of regioisomeric 8-, 11-, 12-, and 15-HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8- and 12-HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8-HETE was found to be a mixture of 98% (R)-enantiomer and 2% (S)-enantiomer, while the 12-HETE was a mixture of 98% (S)-enantiomer and 2% (R)-enantiomer. The present study demonstrates that the HP-γ-CD-modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.

Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential.

Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (kcat/Km) of this reaction was 31% of the kcat/Km of AA. The only major product of h15-LOX-2's reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA4, indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.

CYP Genetic Variants, CYP Metabolite Levels, and Symptomatic Carotid Stenosis in Ischemic Stroke Patients.

AIM: To evaluate the relationship between CYP genetic polymorphisms and CYP metabolite levels with carotid artery stenosis in acute ischemic stroke (IS) patients. METHODS: Eleven single nucleotide polymorphisms (SNPs) of seven CYP genes were genotyped in 136 IS patients with carotid stenosis and 158 patients without carotid stenosis. CYP plasma metabolite levels [20-hydroxyeicosatetraenoic acid (HETE), total epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DiHETEs)] were assessed in a subsample of 90 patients with carotid stenosis and 96 patients without carotid stenosis. We evaluated the relationship between assessed variants and carotid stenosis risk, variants with CYP metabolite levels, and variants in mediating the differences of CYP metabolite levels between patients with carotid stenosis and those without. Additionally, gene-gene interactions were analyzed to assess the interactive role of the assessed variants in affecting CYP metabolite levels and risk of carotid stenosis. RESULTS: The genotypes of rs17110453CC, rs751141GG, and rs9333025GG were significantly associated with carotid stenosis risk. Also these polymorphisms were associated with CYP plasma metabolite levels in patients with carotid stenosis. There was a significant gene-gene interaction between rs17110453 and rs9333025 in affecting the risk of carotid stenosis. Patients with rs17110453CC and rs9333025GG had a significantly higher risk of carotid stenosis than those with 17110453AA and rs9333025AA (OR=2.12, 95% CI: 1.13-7.26, P=0.013). CONCLUSIONS: Specific CYP450 gene SNPs and their interactions are associated with CYP450 plasma metabolite levels, which may partially explain their associations with carotid stenosis. Further studies are needed to validate our findings.