Analysis Of Arachidonic Acid Metabolites Research Articles

A novel, fast and sensitive supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method for analysis of arachidonic acid metabolites.

The development of a rapid, sensitive and reliable method for the quantification of bioactive arachidonic acid metabolites (AA-metabolites) in biological samples is quite challenging due to the minute concentration, short half-life and their structural complexity arising from different isomers. In this study, a simple, fast and environmentally friendly supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) method was developed and validated for simultaneous measurement of five (PGD2, PGE2, PGF2α, 6KetoPGF1α and LTB4) AA-metabolites in biological samples. These analytes were extracted by protein precipitation followed by separation and quantification. The analysis was completed within 3 minutes. The matrix matched linear calibration ranged from 0.5-100 ng mL-1 (r2 ≥ 0.995), whilst, the limit of quantification of PGD2, PGE2, PGF2α, and LTB4 was 0.5 ng mL-1 and was 2.5 ng mL-1 for 6KetoPGF1α. The interday and intraday precisions of the method were less than 15% while the accuracy of most of the analytes varied between 83 and 109%. Finally, as a proof of concept, the method was successfully applied for the determination of eicosanoids in human samples, which expands the possibility to explore physiological states, disease phenotypes, and novel biomarkers.

Relation between Ocular Comfort, Arachidonic Acid Mediators, and Histamine

ABSTRACTPurpose: Contact lenses are associated with discomfort during wear. This may be the result of stimulation of the ocular surface and production of pro-inflammatory mediators which are then released into the tears. This study examined changes in the concentration in tears of arachidonic acid metabolites (AAM) prostaglandins, cysteinyl leukotrienes, and resolvin-D1, as well as histamine in a general contact lens population in the morning and evening.Method: Tears were collected twice a day (morning and evening) for up to 10 days on two different occasions (with and without contact lens wear) from 30 experienced contact lens wearers for analysis of AAM and a separate group (N = 33) for analysis of histamine. Ocular comfort was rated subjectively on an ordinal scale at each time of tear collection. Tears were analyzed using commercial immunoassay-based kits. Statistical analysis was performed using linear mixed model test.Results: Ocular comfort decreased from morning to evening with and without contact lenses (p = 0.001), and the difference in comfort in the evening was greater with contact lens wear (80.9 ± 16.2 vs. 75.5 ± 16.8; p = 0.008). The total concentration of PGs (10.7 ± 10.8 ng/ml), cysteinyl leukotrienes (8.7 ± 0.38 ng/ml), resolvin-D1 (1.6 ± 0.5 ng/ml), or histamine 13.8 ± 10.4 ng/ml) did not change during the day or during contact lens wear (p > 0.05).Conclusions: Prostaglandins, cysteinyl leukotrienes, resolvin-D1, or histamine concentrations did not alter in relation to changes in comfort of the eye during the day or during contact lens wear. These results suggest that release of these mediators is not responsible for contact lens discomfort.

Separation of 5-Lipoxygenase Metabolites Using Cyclodextrin-Modified Microemulsion Electrokinetic Chromatography and Head Column Field-Amplified Sample Stacking

A cyclodextrin-modified microemulsion electrokinetic chromatography method employing head column field-amplified sample stacking was developed for the analysis of arachidonic acid metabolites of the lipoxygenase pathways. The influence of the concentration of boric acid, the surfactant sodium dodecyl sulfate, the co-surfactant 1-butanol and the oil phase octane as well as the pH of the background electrolyte, the separation voltage and the separation temperature was studied. The optimized microemulsion consisting of 20 mM boric acid buffer, pH 9.0, 3.0 % (m/v) sodium dodecyl sulfate, 0.5 % (v/v) octane, 5.0 % (v/v) 1-butanol and 15 mM α-cyclodextrin enabled the separation of 20-hydroxy-leukotriene B4, leukotriene B4, 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 12(S)-hydroxy-5,8,14-cis-10-trans-eicosatetraenoic acid, 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid as well as the internal standard prostaglandin B1 in <10 min employing a separation voltage of 17.5 kV at a temperature of 23 °C. A matrix peak from solid-phase extraction sample workup co-migrated with 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid affecting peak integration. The addition of 5 % (v/v) 2-propanol to the microemulsion resulted in the separation of this eicosatetraenoic acid and the matrix components at the expense of analysis time and peak resolution between the diastereomers 6-trans-leukotriene B4 and 6-trans-12-epi-leukotriene B4. In summary, the MEEKC method appeared to be especially suitable for the more polar arachidonic acid metabolites.

Sepsis-associated changes of the arachidonic acid metabolism and their diagnostic potential in septic patients*

Sepsis-associated changes of the arachidonic acid metabolism and the utility of arachidonic acid metabolites for the diagnosis of sepsis have been poorly investigated so far. Therefore, the primary objective of our study was to screen for differentially regulated arachidonic acid metabolites in septic patients using a lipopolysaccharide whole-blood model and to investigate their diagnostic potential. Prospective, observational, single-center, clinical study. Intensive care unit at University Hospital Leipzig. Thirty-five patients (first cohort 25 patients, second cohort 10 patients) meeting the criteria for severe sepsis or septic shock were enrolled. Eighteen healthy volunteers (first cohort 15 subjects, second cohort 3 subjects) were enrolled as controls. None. Arachidonic acid and its metabolites were investigated in supernatants of nonactivated (baseline) and lipopolysaccharide-activated heparinized whole blood of healthy subjects (n=15) and septic patients (n=25) by solid phase extraction and subsequent liquid chromatography-tandem mass spectrometry. Arachidonic acid, arachidonic acid analogues, and the cyclooxygenase-associated metabolites prostaglandin E2, 11-hydroxyeicosatetraenoic acid, and thromboxane B2 were identified as differentiating metabolites between septic patients and healthy subjects. Some of these compounds, including arachidonic acid, its analogues, and the cyclooxygenase metabolites prostaglandin E2 and thromboxane B2 differed at baseline. The inducibility of arachidonic acid and the cyclooxygenase metabolites 11-hydroxyeicosatetraenoic and prostaglandin E2 were reduced by 80% to 90% in septic patients. The degree of the inducibility was associated with severity of sepsis and clinical outcome. A reduced inducibility of COX-2 but preserved inducibility of mPGES-1 on gene expression level were confirmed in an independent cohort of septic patients (n=10) by quantitative reverse-transcription polymerase chain reaction compared to healthy controls (n=3). Arachidonic acid metabolism is markedly affected in patients with sepsis. Our data suggest that the analysis of arachidonic acid metabolites in an in vitro whole blood activation model may be a promising approach for risk estimation in septic patients that has to be further evaluated in subsequent large-scale clinical studies.

Chemopreventive effect of boswellic acid and curcumin on 7,12-dimethyl benzanthracene-induced hamster cheek pouch carcinogenesis

To evaluate the chemopreventive effects of boswellic acid and curcumin on 7,12-dimethyl benzanthracene(DMBA)-induced oral carcinogenesis in the hamster cheek pouch model. Male Syrian golden hamsters (6 - 8 weeks old, 80 - 130 g in weight) were randomly divided into seven groups, with group A serving as the untreated negative control. The left cheek pouch of the remaining hamsters was topically treated with 0.5% DMBA in mineral oil three times a week for 6 weeks. They were then randomized to six groups with group B serving as a positive control and receiving no further treatment. Groups C-G were treated topically with 5, 10 mg/L boswellic acid, 5, 10 µmol/L curcumin, or the combination of 5 mg/L boswellic acid and 5 µmol/L curcumin three times per week for 18 weeks. The animals were injected with bromodeoxyuridine intraperitoneally at 50 mg/kg 2 h prior to killing. At the 25 th week all the hamsters were sacrificed and cheek pouch tissue was harvested. One half of the tissue was snap frozen in liquid nitrogen for analysis of arachidonic acid metabolites, and the other half was fixed in 10% phosphate-buffered saline(PBS)-buffered formalin for histopathological examination. Six-weeks of DMBA followed by 18-weeks of topical application of boswellic acid and curcumin, both boswellic acid (5, 10 mg/L) and curcumin (5, 10 µmol/L) significantly inhibited the incidence from 93.8% to 73.9% (P > 0.05), numbers from 2.19 ± 0.98 to 1.13 ± 0.81 (P < 0.01) and size of visible tumors. Microscopically the incidence of squamous cell carcinoma and BrdU index were also significantly suppressed by boswellic acid and curcumin. Both boswellic acid and curcumin were effective in preventing oral carcinogenesis in DMBA-induced hamster cheek pouch model.

Molecular analyses of choline and ethanolamine plasmalogens in the blood plasma of human and rats

s / Chemistry and Physics of Lipids 163S (2010) S34–S43 S43 11,12and 14,15-dihydroxieicosatrienoic acids (DHETs) on cul- tured cell supernatants. Thus, we performed a simultaneous analysis of arachidonic acid metabolites from the three pathways. Deuterium labeled PGE2-d4, LTB4-d4, 5-HETE-d8, 12-HETE-d8, 15- HETE-d8, 13-HODE-d11, 5,6-EET-d11, 14,15-EET-d11were used as internal standards. Solid phase extraction was used for sample preparation. The recoveries were from 25% to 100% depending of the metabolites. The LC/MS/MS method used gradient on a C18 column, electrospray ionization in negative ion detection mode. The method was optimized for the best sensitivity and separa- tion within 20min. The linear ranges of the calibration curves were 0.1–200ng/ml for PGE2, LTB4, 5-HETE, 12-HETE, 15-HETE, 13- HODE, 11,12-EET, 11,12-DHET and 14,15-DHET and 1–200ng/ml for LTD4, 20-HETE, 5,6-EET, 8,9-EET and 14,15-EET. The advan- tages of this method include minimal sample preparation, high sensitivity and elimination of the problem associated with thermal instability for gas chromatography analysis. doi:10.1016/j.chemphyslip.2010.05.129

Simultaneous eicosanoid profiling and identification by liquid chromatography and hybrid triple quadrupole-linear ion trap mass spectrometry for metabolomic studies in human plasma / Multiparametrische Bestimmung und Identifizierung von Eikosanoiden mit Flüssigchromatographie in Kombination mit Hybrid-Quadrupol/lineare Ionenfallen Massenspektrometrie für Metabolom-Studien in Humanplasma

Abstract Eicosanoids play a key role in many physiological and pathological processes and might therefore serve as interesting diagnostic targets. Methods for the analysis of arachidonic acid metabolites in cells and body fluids require high sensitivity and specificity because of the very low concentrations, similar chemical structures and short half-lives of these metabolites. We established a mass spectrometric method for the simultaneous identification and quantification of arachidonic acid metabolites in human plasma samples using solid phase extraction followed by liquid chromatography (LC) and hybrid triple quadrupole-linear ion trap (QqLIT) mass spectrometry. Quantitative analysis was performed using the 4000 QTrap tandem mass spectrometer in multiple reaction monitoring (MRM) mode. As part of an independent data acquisition experiment MRMs were used as survey scans, which dependently triggered enhanced product ion (EPI) scans. Compound identification was carried out by library search using a library based on EPI spectra of standard components (prostaglandins, thromboxanes, leukotrienes and isoprostanes). The newly developed compound library enables the verification of known and structural elucidation of unknown eicosanoid metabolites in human plasma. In conclusion, our mass spectrometric method allows the simultaneous identification and quantification of arachidonic acid metabolites in one single LC-MS/MS run.

CPGES/p23 is required for glucocorticoid receptor function and embryonic growth but not prostaglandin E2 synthesis.

A number of studies have identified cytosolic prostaglandin E(2) synthase (cPGES)/p23 as a cytoplasmic protein capable of metabolism of prostaglandin E(2) (PGE(2)) from the cyclooxygenase metabolite prostaglandin endoperoxide (PGH(2)). However, this protein has also been implicated in a number of other pathways, including stabilization of the glucocorticoid receptor (GR) complex. To define the importance of the functions assigned to this protein, mice lacking detectible cPGES/p23 expression were generated. cPGES/p23(-/-) pups die during the perinatal period and display retarded lung development reminiscent of the phenotype of GR-deficient neonates. Furthermore, GR-sensitive gluconeogenic enzymes are not induced in the prenatal period. However, unlike GR-deficient embryos, cPGES/p23(-/-) embryos are small and a proliferation defect is observed in cPGES/p23(-/-) fibroblasts. Analysis of arachidonic acid metabolites in embryonic tissues and primary fibroblasts failed to support a function for this protein in PGE(2) biosynthesis. Thus, while the growth retardation of the cPGES/p23(-/-) pups and decreased proliferation of primary fibroblasts identify functions for this protein in addition to GR stabilization, it is unlikely that these functions include metabolism of PGH(2) to PGE(2).

15(S)-Lipoxygenase-2 Mediates Arachidonic Acid-stimulated Adhesion of Human Breast Carcinoma Cells through the Activation of TAK1, MKK6, and p38 MAPK

The dietary cis-polyunsaturated fatty acid, arachidonic acid, stimulates adhesion of metastatic human breast carcinoma cells (MDA-MB-435) to the extracellular matrix, but the molecular mechanisms by which fatty acids modify the behavior of these cells are unclear. Exposure to arachidonic acid activates multiple signaling pathways. Activation of p38 mitogen-activated protein kinase (p38 MAPK) is required for increased cell adhesion to type IV collagen, and this activation is sensitive to inhibitors of lipoxygenases, suggesting a requirement for arachidonic acid metabolism. The goals of the current study were to identify the one or more key metabolites of arachidonic acid that are responsible for activation of p38 MAPK and to elucidate the upstream kinases that lead to p38 MAPK activation. High performance liquid chromatographic analysis revealed that MDA-MB-435 cells metabolize exogenous arachidonic acid predominantly to 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE). Immunoblot analysis with antibodies specific to 15(S)-lipoxygenase-1 (LOX-1) and 15(S)-lipoxygenase-2 (LOX-2) demonstrated the expression of 15-LOX-2, but not 15-LOX-1, in these tumor cells. A LOX inhibitor, nordihydroguaiaretic acid, attenuated production of 15(S)-HETE and inhibited the phosphorylation of p38 MAPK following exposure to arachidonic acid. In contrast, overexpression of LOX-2 sensitized the cells to the addition of arachidonic acid, leading to increased activation of p38 MAPK. Addition of exogenous 15(S)-HETE to MDA-MB-435 cells stimulated cell adhesion to type IV collagen and activated the p38 MAPK pathway, including the upstream kinases transforming growth factor-beta1-activated protein kinase-1 (TAK1) and MAPK kinase 6. Transfection of these cells with a dominant negative form of TAK1 blocked arachidonic acid-stimulated p38 MAPK phosphorylation. These data demonstrate that 15(S)-LOX-2 generation of 15(S)-HETE activates specific growth factor receptor-related signaling pathways, thereby initiating signal transduction events leading to increased cell adhesion to the extracellular matrix.

Role of 11,12,15-Trihydroxyeicosatrienoic Acid as a New Endothelium-Derived Relaxing Factor

3 The endothelium regulates vascular tone through the release of relaxing factors including nitric oxide, prostacyclin, epoxyeicosatrienoic acids and others not yet identified. The purpose of this study was to characterize the 15-lipoxygenase product, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), as a new endothelial relaxing factor. In phenylephrine-constricted rabbit aorta, acetylcholine induced a dose-dependent relaxation in the presence of cyclooxygenase blockade with indomethicin (10 μM) and nitric oxide blockade with nitro-arginine (30 μM) which was eliminated by pretreatment with cinnamyl-3,4,-dihydroxy-a-cyanocinnamate (CDC)(1 μM) a specific lipoxygenase inhibitor, and miconizole (1 μM) a cytochrome P450 inhibitor. Additionally, arachidonic acid (1 nM - 10 μM) relaxed precontracted rabbit aorta, and this relaxation was maximally inhibited by 50% either by increasing the potassium (K + ) concentration from 4 mM to 20 mM or by the addition of the K + channel blockers, apamin (100 nM) plus charybdotoxin (100 nM). In patch-clamp studies of inside-out patches of rabbit aortic smooth muscle, we have identified a 20 pS K + channel, which is activated by increased calcium concentrations and blocked by apamin (1 μM). Structural analysis of arachidonic acid metabolites from rabbit aorta by gas chromatography/mass spectometry has revealed two 15-lipoxygenase product regioisomers; 11,12,15-THETA and 11,14,15-THETA. The synthesis of these metabolites was inhibited by CDC and miconizole. The addition of the 11,12,15-THETA fraction to precontracted rabbit aorta resulted in a concentration-dependent relaxation, which was blocked by elevated K + . The other regioisomer was without effect. These results suggest that 11,12,15-THETA, a 15-lipoxygenase metabolite, represents a novel endothelium-derived relaxing factor, which induces relaxation possibly through the activation of apamin-sensitive K + channels located in the vascular smooth muscle.

Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits.

We examined the role of cyclooxygenase-2 (COX-2) in the late phase of ischemic preconditioning (PC). A total of 176 conscious rabbits were used. Ischemic PC (six cycles of 4-min coronary occlusions/4-min reperfusions) resulted in a rapid increase in myocardial COX-2 mRNA levels (+231 +/- 64% at 1 h; RNase protection assay) followed 24 h later by an increase in COX-2 protein expression (+216 +/- 79%; Western blotting) and in the myocardial content of prostaglandin (PG)E(2) and 6-keto-PGF(1alpha) (+250 +/- 85% and +259 +/- 107%, respectively; enzyme immunoassay). Administration of two unrelated COX-2 selective inhibitors (NS-398 and celecoxib) 24 h after ischemic PC abolished the ischemic PC-induced increase in tissue levels of PGE(2) and 6-keto-PGF(1alpha). The same doses of NS-398 and celecoxib, given 24 h after ischemic PC, completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that COX-2 activity is necessary for this phenomenon to occur. Neither NS-398 nor celecoxib lowered PGE(2) or 6-keto-PGF(1alpha) levels in the nonischemic region of preconditioned rabbits, indicating that constitutive COX-1 activity was unaffected. Taken together, these results demonstrate that, in conscious rabbits, up-regulation of COX-2 plays an essential role in the cardioprotection afforded by the late phase of ischemic PC. Therefore, this study identifies COX-2 as a cardioprotective protein. The analysis of arachidonic acid metabolites strongly points to PGE(2) and/or PGI(2) as the likely effectors of COX-2-dependent protection. The recognition that COX-2 mediates the antistunning and antiinfarct effects of late PC impels a reassessment of current views regarding this enzyme, which is generally regarded as detrimental.

Quantitation of Arachidonic Acid Metabolites in Small Tissue Biopsies by Reversed-Phase High-Performance Liquid Chromatography

Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 ± 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0.0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.

Co-oxidation of acrylonitrile by soybean lipoxygenase and partially purified human lung lipoxygenase

1. Human lung lipoxygenase (HLLO) was partially purified by concanavalin-A (ConA) affinity chromatography that provided an easy and rapid one-step procedure for the removal (⩾ 96%) of haemoglobin from cytosol. 2. HLLO exhibited dioxygenase activity towards arachidonic acid (AA) and linoleic acid (LA). The dioxygenase activity towards LA varied ∼ 12-fold (48-591 nmol/min /mg protein) among different human lung samples examined. 3. Reverse-phase HPLC analysis of AA metabolites indicated the predominance of 15-lipoxygenase in human lung cytosol. 4. HLLO exhibited co-oxidase activity towards benzidine (BZD) and several other model compounds. The co-oxidase activity towards BZD was significantly inhibited by several lipoxygenase inhibitors. 5. HLLO and soybean lipoxygenase (SLO), used as a model enzyme, metabolized acrylonitrile (ACN) to 2-cyanoethylene oxide (CEO) and ultimately to cyanide. 6. HLLO was a ∼ 6-fold better catalyst than SLO in converting ACN to cyanide. The generation of cyanide by HLLO was dependent on the concentration of enzyme and the reaction was inhibited by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) and the anti-oxidant butylated hydroxytoluene (BHT). 7. Under optimal assay conditions, the covalent binding of HLLO-generated reactive intermediate(s) from [14C]ACN to protein and DNA (nmol equivalent bound/15/min mg/HLLO/mg bovine serum albumin or calf thymus DNA) was observed at ∼ 1.20 ± 0.13 and 2.20 ± 0.50 respectively. Both protein and DNA binding were inhibited by NDGA, butylated hydroxyanisole (BHA) and BHT.

Characterization of arachidonic-acid-metabolizing enzymes in adult Schistisoma mansoni

Schistosoma mansoni has previously been reported to synthesize a wide range of eicosanoids including prostaglandins, leukotrienes and hydroxyeicosatetraenoic acids (HETEs). Our analysis of arachidonic acid metabolites synthesized by microsomal and cytosolic extracts from adult S. mansoni using thin-layer chromatography and radioimmunoassay techniques indicate the presence of a soluble, enzymatically active lipoxygenase (Lox) and the absence of any cyclooxygenase (Cox) activity. The S. mansoni Lox activity catalyzed the formation of a 15-hydroxyeicosatetraenoic acid (15-HETE)-like species. This activity was calcium-independent and inhibitable by inhibitors of mammalian and plant Lox. The conversion of linoleic acid to a 13-hydroxyoctadecadienoic acid (13-HODE)-like product by S. mansoni extracts indicates that the parasite Lox-homologue is similar to mammalian 15-Lox. Immunoblot analysis of S. mansoni extracts using antisera to different mammalian lipoxygenases detects two immunoreactive proteins with molecular weights similar to plant and mammalian lipoxygenases. In addition, polymerase chain reaction (PCR) amplification of Lox-like sequences from S. mansoni genomic DNA using degenerate primers based on conserved plant and mammalian Lox sequences, generated two PCR products which hybridized to a human 15-Lox cDNA probe. While the role of eicosanoid production in the physiology of S. mansoni is not known, eicosanoids may be essential for normal physiological processes as is the case in other invertebrates. Interestingly, 15-HETE has previously been shown to have immunosuppressive effects in mammals, and this may be related to the ability of the parasite to overcome host immune responses.

Reverse-Phase High-Performance Liquid Chromatography Analysis of Arachidonic Acid Metabolites in Plasma after Stimulation of Whole Blood ex Vivo

Following the stimulation of heparinized blood ex vivo, aliquots of plasma were denatured with organic solvents containing the internal standards prostaglandin (PG) B2 and 19-hydroxy-PGB2. Precipitated material was removed by centrifugation and the supernatants were directly analyzed by reverse-phase HPLC with on-line extraction and uv detection. Stimulation of blood with A23187 lead to the formation of both leukocyte and platelet arachidonic acid metabolites as the 5-lipoxygenase products leukotriene (LT) B4, 5-hydroxy-eicosatetraenoic acid (5-HETE), 20-hydroxy-LTB4 and 20-carboxy-LTB4, the 12-lipoxygenase product 12-HETE, and the cyclooxygenase product 12-hydroxy-heptadecatrienoic acid (HHTrE) were detected in plasma; in some plasma samples LTC4 and/or LTE4 were also detected. Stimulation of blood with zymosan lead to the synthesis of LTB4 and 5-HETE as major products and of 12-HETE. Recoveries of 20-hydroxy-LTB4, LTB4, 5-HETE, 12-HETE, and HHTrE added to plasma were high (≥90%) while those of LTC4 and LTE4 were lower (50-70%); however, washing of the precipitated protein pellet resulted in >90% recoveries for all metabolites including the cysteinyl-leukotrienes. Levels of arachidonic acid metabolites in native plasma samples stored at −20°C were stable for at least 28 days, while significant loss of material was observed over the same period of time in denatured plasma samples. Finally, we made the critical observation that the capacity for A23187- (but not zymosan-, ionomycin-, or LPS and FMLP-) induced arachidonic acid metabolite synthesis in blood decreased by 80% within 1 h of blood collection. The profiling of 5-lipoxygenase products in synovial fluid from an arthritic patient illustrates the applicability of the method to protein-rich matrices other than plasma. An application of the method is shown through the demonstration of the inhibition of LTB4 formation ex vivo following the in vivo administration of a LT synthesis inhibitor to rabbits.

Viral Infection of Bovine Bronchial Epithelial Cells Induces Increased Neutrophil Chemotactic Activity and Neutrophil Adhesion

1. Acute bronchitis secondary to viral infection is associated with an influx of neutrophils. We hypothesized that bronchial epithelial cells are capable of releasing neutrophil chemotactic activity in response to viral infection. 2. To test this hypothesis, primary cultures of bovine bronchial epithelial cells were inoculated with a bovine respiratory pathogen, bovine herpes virus-1. 3. Supernatants collected from inoculated cells, before signs of toxicity, demonstrated significant neutrophil chemotactic activity using a blind well chamber neutrophil chemotaxis assay. Lipoxygenase inhibitors markedly reduced the amount of neutrophil chemotactic activity released after bovine herpes virus-1 inoculation. Analysis of arachidonic acid metabolites in cell supernatants by reverse-phase h.p.l.c. confirmed that leukotriene B4, a potent neutrophil chemoattractant, was released. 4. We also confirmed that adhesion of neutrophils to bovine herpes virus-1-inoculated bronchial epithelial cells was increased and mediated in part by the neutrophil integrin, LFA-1. 5. Thus, virally infected airway epithelial cells release leucocyte chemoattractants and hence adhesive interactions, functions that are likely to be important in the inflammatory acute response to viral infection.

Kinetic studies on arachidonate 5-lipoxygenase from rat basophilic leukemia cells.

Arachidonate 5-lipoxygenase of a 10,000 x g supernatant from RBL-1 cell homogenate was studied by a continuous assay measuring enzyme-catalysed oxygen consumption. Parallel HPLC and TLC analysis of arachidonic acid metabolites revealed that the oxygen consumption measured is solely due to 5-lipoxygenation of arachidonic acid. Oxygen consumption by this lipoxygenase was strictly dependent upon Ca2+, ATP and 5-HPETE. Removal of any of these three cofactors caused a complete inhibition of enzyme activity. Addition of the missing cofactor instantly restored the 5-lipoxygenase-dependent consumption of oxygen which remained linear for 10-20 s. Later on the velocity of the reaction decreased and after 2-3 min the enzyme became inactivated. Kinetic data were obtained from the initial velocity of the reaction using constant and saturating concentrations of CaCl2 and ATP. From Lineweaver-Burk plots substrate inhibition is evident for arachidonic acid concentrations greater than 45-50 microM. Km(app) for arachidonic acid is 182 +/- 16 microM (mean +/- SD, n = 5) and Vmax(app) is 425 +/- 140 nmol O2/(min x mg protein) (mean +/- SD, n = 5).

Conversion of arachidonic acid into 12-oxo derivatives in human platelets. A pathway possibly involving the heme-catalysed transformation of 12-hydroperoxy-eicosatetraenoic acid

Analysis of arachidonic acid metabolites in human platelets by reverse-phase HPLC with radioactivity and UV detection revealed, besides Thromboxane B 2 (TXB 2), 12-hydroxy-heptadecatrienoic acid (HHT) and 12-hydroxy-eicosatetraenoic acid (12-HETE) previously described, two peaks of unidentified material absorbing at 280 nm. This material was purified by straight-phase HPLC and characterized by UV spectroscopy and gas chromatography-mass spectrometry. Three carbonyl compounds were identified: 12-keto-5,8,10,14-eicosatetraenoic acid and two geometric isomers of 12-oxo-5,8,10-dodecatrienoic acid. In a 5 min incubation at 37 °C in the presence of 9 μM arachidonic acid, the yield was of 0.5 to 1% of added arachidonic acid for the ketonic compound and of 4 to 7% for the sum of the two isomeric fatty acid aldehydes in comparison to 10 to 13% and 25 to 28% for TXB 2 and 12-HETE, respectively. Because the three compounds carry a carbonyl group at position 12, their relationship with the 12-lipoxygenase pathway was investigated. It was found that the three compounds were formed when 12-hydroperoxy-eicosatetraenoic acid (12-HPETE) was incubated with intact or heat denaturated platelets or hemoproteins, strongly suggesting that these carbonyl compoundds are products of a heme-catalysed transformation of 12-HPETE.

High-performance liquid chromatographic analysis of arachidonic acid metabolites by pre-column derivatization using 9-anthryldiazomethane

Arachidonic acid (AA) metabolites produced by washed human platelets and rat macrophages were analyzed by high-performance liquid chromatography (HPLC) using a pre-column derivatization method. The reagent, 9-anthryldiazomethane, used in this study and AA metabolites derivatized by the reagent were purified by gel permeation chromatography (PG-peak C column), prior to normal-phase HPLC analysis. A sample containing eleven derivatives (12-, 15- and 5-hydroxyeicosatetetraenoic acid, 12- l-heptadecatrienoic acid, leukotriene B 4, prostaglandins B 2, D 2, E 2 and F 2α, thromboxane B 2 and 6-keto-prostaglandin F 1α) was separated on a normal-phase column (PG-pak B); the detection limit is better than 100 pg for all components.

The analysis of arachidonic acid metabolites in normal, uninvolved and lesional psoriatic skin

Collection of exudate from suction bullae is a commonly used method for sampling human skin for mediator analysis. It is satisfactory on skin of normal structure but is unreliable on lesional psoriatic skin in which there are major structural changes and excessive scaling. Collection of exudates from abraded sites was found to be a suitable alternative method for psoriatic skin. Arachidonic acid and 12-HETE, but not PGE 2, were significantly higher in exudate from abraded lesional psoriatic skin (494 ± 88, 45.9 ± 4.2 and 9.6 ± 1.8 ng/ml respectively, mean ± sem, n = 5) compared to uninvolved skin (154 + 38, 18.5 + 5.1 and 7.7 ± 1.9 ng/ml) or skin of normal volunteers (119 ± 37, 14.5 ± 6.7 and 4.5 ± 1.6 ng/ml, n = 7) which were similar. The coefficient of variation for exudate collection and mediator analysis was usually less that 55%. The analysis of lipoxygenase and cyclooxygenase products was simplified by the use of chlorobutane to exctract preferentially arachidonic acid and HETEs from neutral aqueous solutions.