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Hydroxyeicosatetraenoic acids (HETE) are dramatically increased under brain ischemia and significantly affect post-ischemic recovery. However, the exact mechanism of HETE increase and their origin under ischemia are poorly understood. HETE might be produced de novo through lipoxygenase (LOX) -dependent synthesis with possible esterification into a lipid storage pool, or non-enzymatically through free radical oxidation of esterified arachidonic acid (20:4n6). Because HETE synthesized through LOX exhibit stereospecificity, chiral analysis allows separation of enzymatic from non-enzymatic pools. In the present study, we analyzed free HETE stereoisomers at 30 sec, 2 min, and 10 min of ischemia. Consistent with previous reports, we demonstrated a significant, gradual increase in all analyzed HETE over 10 min of brain ischemia, likely attributed to release of the esterified pool. The R/S ratio for 5-HETE, 8-HETE, and 15-HETE was not different from a racemic standard mix, indicating their non-enzymatic origin, which was in opposition to the inflamed tissue used as a positive control in our study. However, 12(S)-HETE was the predominant isoform under ischemia, indicating that ∼90 % of 12-HETE are produced enzymatically. These data demonstrate, for the first time, that 12-LOX is the major LOX isoform responsible for the enzymatic formation of the inducible HETE pool under ischemia. We also confirmed the requirement for enzyme inactivation with high-energy focused microwave irradiation (MW) for accurate HETE quantification and validated its application for chiral HETE analysis. Together, our data suggest that 12-LOX and HETE-releasing enzymes are promising targets for HETE level modulation upon brain ischemia., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cardiovascular disease is the leading cause of mortality in patients with renal failure. Red blood cells (RBCs) are potential reservoirs for epoxy fatty acids (oxylipins) that regulate cardiovascular function. Hemoglobin exhibits pseudo-lipoxygenase activity in vitro. We previously assessed the impact of single hemodialysis (HD) treatment on RBC epoxy fatty acids status in circulating arterial blood and found that eicosanoids in oxygenated RBCs could be particularly vulnerable in chronic kidney disease and hemodialysis. The purpose of the present study was to evaluate the differences of RBC epoxy fatty acids profiles in arterial and venous blood in vivo (AV differences) from patients treated by HD treatment. We collected arterial and venous blood samples in upper limbs from 12 end-stage renal disease (ESRD) patients (age 72±12 years) before and after HD treatment. We measured oxylipins derived from cytochrome P450 (CYP) monooxygenase and lipoxygenase (LOX)/CYP ω/(ω-1)-hydroxylase pathways in RBCs by LC-MS/MS tandem mass spectrometry. Our data demonstrate arteriovenous differences in LOX pathway metabolites in RBCs after dialysis, including numerous hydroxyeicosatetraenoic acids (HETEs), hydroxydocosahexaenoic acids (HDHAs) and hydroxyeicosapentaenoic acids (HEPEs). We detected more pronounced changes in free metabolites in RBCs after HD, as compared with the total RBC compartment. Hemodialysis treatment did not affect the majority of CYP and CYP ω/(ω-1)-hydroxylase products in RBCs. Our data indicate that erythro-metabolites of the LOX pathway are influenced by renal-replacement therapies, which could have deleterious effects in the circulation., (Copyright © 2022. Published by Elsevier Ltd.)

Dietary PUFA and their effects on adipose tissue have been well studied, but oxylipins, the oxygenated metabolites of PUFA, have been sparsely studied in adipose tissue. To determine the oxylipin profile and to examine their potential importance in various adipose sites, female and male rats were provided control, high linoleic acid (LA), or high LA and high α-linolenic acid (LA + ALA) diets for six weeks. Analysis of gonadal (GAT), mesenteric (MAT), perirenal (PAT), and subcutaneous adipose tissues (SAT) revealed higher numbers of oxylipins in MAT and SAT, primarily due to 20-22 carbon cytochrome P450 oxylipins, as well as metabolites of cyclooxygenase derived oxylipins. LA oxylipins made up 75-96% of the total oxylipin mass and largely determined the total relative amounts between depots (GAT > MAT > PAT > SAT). However, when the two most abundant LA oxylipins (TriHOMEs) were excluded, MAT had the highest mass of oxylipins and exhibited the most sex differences. These differences existed despite comparable PUFA composition between depots. Dietary LA increased oxylipins derived from n-6 PUFA, and the addition of ALA generally returned n-6 PUFA oxylipins to levels similar to control and elevated some n-3 oxylipins. These data on oxylipin profiles in adipose depots from different anatomical sites and the effects of diet and sex provide fundamental knowledge that will aid future studies investigating the physiological effects of adipose tissue., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Inflammation is a physiological response to exogenous and endogenous stimuli and, together with demyelination and immune system activation, is one of the key features of multiple sclerosis (MS). Arachidonic acid (AA) metabolism by cyclooxygenase (COX) and lipoxygenase (LO) enzymes leads to the production of proinflammatory eicosanoids, and stimulates cytokine production and activation of microglia and astrocytes, thereby contributing to MS pathology. Current therapies target the immune system but do not specifically target AA-related inflammatory pathway. Corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs) are frequently associated with immunomodulatory therapies to treat flu-like adverse effects. Few clinical and mounting preclinical data in MS show that AA metabolism contributes to immune system activation, demyelination and motor disabilities, and administration of NSAIDs reduces these symptoms. The beneficial effect of NSAIDs seems to be a prerogative of COX-2 selective inhibitors and suggests that NSAIDs selective for COX-2 may be more effective than mixed COX-1/2 inhibitors., (© 2013 Elsevier Ltd. All rights reserved.)

Knowing threshold changes in brain lipids and lipid enzymes during dietary n-3 polyunsaturated fatty acid deprivation may elucidate dietary regulation of brain lipid metabolism. To determine thresholds, rats were fed for 15 weeks DHA-free diets having graded reductions of α-linolenic acid (α-LNA). Compared with control diet (4.6% α-LNA), plasma DHA fell significantly at 1.7% dietary α-LNA while brain DHA remained unchanged down to 0.8% α-LNA, when plasma and brain docosapentaenoic acid (DPAn-6) were increased and DHA-selective iPLA(2) and COX-1 activities were downregulated. Brain AA was unchanged by deprivation, but AA selective-cPLA(2), sPLA(2) and COX-2 activities were increased at or below 0.8% dietary α-LNA, possibly in response to elevated brain DPAn-6. In summary, homeostatic mechanisms appear to maintain a control brain DHA concentration down to 0.8% dietary DHA despite reduced plasma DHA, when DPAn-6 replaces DHA. At extreme deprivation, decreased brain iPLA(2) and COX-1 activities may reduce brain DHA loss., (Published by Elsevier Ltd.)

Polyunsaturated fatty acids of nutritional value may affect cell functions after their release from cell lipid storage sites, especially phospholipids, and specific oxygenation by cyclooxygenases, lipoxygenases and cytochrome P(450). The end-products, namely prostanoids, leukotrienes, and mono-, di- and tri-hydroxy derivatives exhibit a variety of biological effects, especially on vascular cells, leukocytes and platelets. This paper reviews some results obtained with blood platelets as target cells, showing that various lipoxygenase end-products, mainly mono- and di-hydroxy derivatives, are inhibitors (IC(50) in microM range) of arachidonic acid-induced aggregation either at the cycloxygenase or thromboxane receptor site level., (Copyright 2010 Elsevier Ltd. All rights reserved.)

This work employed immunocytochemistry (ICC) techniques to study the effect of topically applied fish oil and ketoprofen on cyclooxygenase (COX-2) and lipoxygenase (LOX) within freshly excised porcine ear skin. Maintained in Hanks buffer immediately post excision, full thickness membranes were mounted in Franz diffusion cells and dosed with 1 ml of individual formulations containing ketoprofen, fish oil or both. At different timepoints, the diffused areas were recovered and relative activities of COX-2 and LOX determined. It was found that the fish oil formulation qualitatively inhibited the expression of both COX-2 and LOX enzymes. As expected, ketoprofen had no effect upon LOX expression but a significant decrease on COX-2 expression was observed. The formulation containing both fish oil and ketoprofen proved to be the most effective at inhibiting the expression of both COX-2 and LOX. Considered together with data from earlier papers, a mechanism of EPA permeation enhancement by ketoprofen may be elucidated and also show the ability of such a formulation to inhibit these enzymes and thus indicate the efficacy of such a formulation.

Cyclooxygenase and lipoxygenase arachidonate products, including prostaglandins (PGs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), are known to modulate inflammation within tissues and can serve as important etiologic factors in carcinogenesis. Eicosanoid content in tissues is typically determined either as a single molecular species through antibody-based assays or by high-performance liquid chromatography after addition of an exogenous substrate such as arachidonic acid. Unfortunately, the methods currently in use are either time-consuming or complicated. Here we report a method for simultaneously identifying eicosanoids appearing as endogenous bioactive lipids in in vivo settings using LC/MS/MS. The analyses indicate marked differences in endogenous eicosanoid content between malignant tissue types suggesting a need for selective therapeutic approaches. As a demonstration of the utility of the method, we present data to show that the technique can be used to distinguish eicosapentaenoic acid-derived formation of PGE(3) from PGE(2) in murine prostate tissue. The method has also been applied to an examination of endogenous eicosanoid metabolism in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral cancer in hamsters demonstrating the inflammatory nature of this type of cancer with elevated levels of both PGE(2) and LTB(4). In addition, the concentration of the eicosanoid 13-hydroxyoctadecadienoic acid was 67.6% lower in DMBA treated specimens than in control specimens. Thus, our method provides a powerful tool for measuring modulation of eicosanoid metabolites in various preclinical and clinical tissues and may be useful in studies of the endogenous changes in eicosanoid metabolism at various stages of cancer development.

Prostate cancer poses considerable threat to the aging male population as it has become a leading cause of cancer death to this group. Due to the complexity of this age-related disease, the mechanism(s) and factors resulting in prostate cancer remain unclear. Reports showing an increase risk in prostatic cancer with increasing dietary fat are contrasted by other studies suggesting the beneficial effects of certain polyunsaturated fatty acid (PUFA) in the modulation of tumor development. The n-6 PUFA, gamma-linolenic acid (GLA), has been shown to suppress tumor growth in vitro. Therefore, using the Lobund-Wistar (L-W) rat model of prostate cancer, we tested the hypothesis whether dietary supplementation of GLA could suppress tumor growth and development in vivo. Prostatic adenocarcinomas were induced in two groups of L-W rats, the experimental group (N-nitroso-N-methylurea, NMU/testosterone propionate, TP) and the GLA group (NMU/TP/GLA fed) undergoing similar treatment but fed a purified diet supplemented with GLA. Our findings revealed a decrease in prostate growth in the NMU/TP/GLA-fed group as determined by weight, tissue size, DNA content and prostate-specific antigen (tumor marker of prostate cancer). Comparison between the two groups showed a significant increase in 5S-hydroxyeicosatetraenoic acid and prostaglandin E(2) in the NMU/TP group. These increases paralleled the increased protein expression and activity of cyclooxygenase-2 as well as increased activity of 5-lipoxygenase. Taken together, the findings showed that intake of GLA-enriched diet does reduce prostatic cancer development in L-W rats and could serve as a non-toxic adjunct in management of human prostatic cancer.

The eye must contain highly evolved programs to limit inflammation and promote wound healing as an errant response can lead to blindness. However, pathways that protect the delicate visual axis and account for its atypical inflammatory responses remain to be clearly defined. Hence, research efforts have been initiated to elucidate the role of the anti-inflammatory LXA4 circuits in the eye. LXA4 is formed in healthy and injured corneas and both its receptor and 12/15-lipoxygenase are predominantly expressed in epithelial cells. An essential role for LXA4 in preserving ocular function is supported by 12/15-LOX deficient mice that exhibit a phenotype of impaired wound healing and LXA4 formation. A novel epithelial bioaction role for LXA4 has been uncovered in the cornea as topical LXA4 promotes wound healing and limits the sequelae of injury. These emerging studies indicate that the LXA4 circuit may hold a fundamental role in maintaining an ocular environment that actively restricts inflammation while promoting wound healing.

Dietary arachidonic acid (AA) and eicosanoids influence neoplastic cell (NC) growth, differentiation and apoptosis. Plasma membrane fatty acid and cyclooxygenase (COX) and lipoxygenase (LOX) products were investigated in lung alveolar carcinoma cells from mice fed on different diets. Two groups were fed on a basic diet plus 6% of: corn oil (rich in 18:2n-6; CO) and on olein oil (rich in 18:1n-9; O), respectively. Control group (C) received commercial diet. NC fatty acids were analyzed by GLC, and apoptosis by flow cytometry and microscopy. In NC from CO group AA levels and LOX metabolites were increased, whereas COX metabolites decreased. NC from CO compared to O group diet showed a higher count of apoptosis and increased LOX:COX ratio. High levels of AA and decreased COX eicosanoids has been involved in anti-tumoral mechanisms by increasing tumor cell apoptosis. Present data emphasizes the implications of the dietary fatty acids on the neoplastic process in this tumoral model.

Based on the clinical observation that humans with visceral adiposity have higher plasma aldosterone levels than controls, we postulated that endogenous fatty acids can be oxidized by the liver to form stimuli of the adrenal cortex. Although we could show that hepatocytes produced adrenal stimuli from linoleic acid in vitro, the yield was very small. To facilitate the elucidation of chemical structures, we incubated a large amount of linoleic acid with lipoxygenase, then treated the hydroperoxide with cysteine and iron. The major product of this process was 12,13-epoxy-9-keto-10-trans-octadecenoic acid. This epoxy-keto compound stimulated aldosterone production at concentrations from 0.5 to 15 microm. At higher concentrations, it was inhibitory. The epoxy-keto-octadecenoic acid exhibited the chromatographic characteristics of one product of the incubation of linoleic acid with hepatocytes. The results are consistent with the postulated conversion of linoleic acid to stimuli of aldosterone production. This may be a mechanistic link between visceral obesity and hypertension in humans., (Copyright 2002 Elsevier Science Ltd.)

There is increasing evidence that endocannabinoids play roles in a number of physiological and pathological processes ranging from the regulation of food intake to the inhibition of cancer cell proliferation. Consequently, multiple investigations into endocannabinoid metabolic disposition have been initiated. Such studies have begun to shed light on the mechanisms that regulate the endogenous cannabinoid system. In addition, they have identified a number of novel, endocannabinoid-derived lipids. In the future, these studies may form the foundation of efforts designed to subtly manipulate endocannabinoid tone in vivo to achieve therapeutic benefits without the profound side-effects observed with synthetic cannabinoid treatment. In addition to the well-studied hydrolytic mode of endocannabinoid metabolism, accumulating data suggest that these lipids are also susceptible to oxidative metabolism by a number of fatty acid oxygenases. These include the cyclooxygenases, lipoxygenases, and cytochrome P450s known to be involved in eicosanoid production from arachidonic acid. The available evidence concerning endocannabinoid oxidation is reviewed and the potential biological significance of this mode of metabolism is considered., (Copyright 2002 Published by Elsevier Science Ltd.)

In human colorectal carcinoma Caco-2 cells, sodium butyrate (NaBT) induces the expression of the reticulocyte, 15-lipoxygenase-1 (15-LO-1) and causes these cells to undergo differentiation and apoptosis. 15-LO-1 is also expressed in human colorectal epithelium with a significant higher expression observed in colorectal tumors. In this study, we have prepared stable Caco-2 cells that expressed 15-LO-1 under control of an inducible promoter. These cells provide a model system to study regulation of 15-LO-1 activity in colorectal cells without the interfering presence of NaBT and are useful to study the biological function of 15-LO-1. The expressed 15-LO-1 was highly active as measured in cell lysates, but we were unable to detect metabolism in intact cells. The addition of calcium to the media for the Caco-2 cells was required for 15-LO-1 to translocate from the cytosol to the membrane which is frequently a requirement for lipoxygenase activity. Despite the addition of calcium and translocation, little lipoxygenase activity was detected with intact cells. However, after removal of phenol red, a common constituent of cell culture media, we were able to detect 15-LO-1 activity in the transfected Caco-2 cultured cells. Thus the presence of calcium and the absence of antioxidants present in commonly used culture media are required for expressed 15-LO-1 to be catalytically active and to permit an examination of its biological effects., (Copyright 2001 Harcourt Publishers Ltd.)

Involvement of arachidonic acid cyclooxygenase (COX) and lipoxygenase (LOX) metabolites in platelet aggregation and coagulation induced by two varieties of cancer cells of murine transplantable tumors was studied. A lung alveolar carcinoma (LAC) and a fibrosarcoma (FS), induced platelet aggregation and plasma coagulation (P<0.05). Pretreatment of both tumor lines with a COX inhibitor did not block the tumor cell induced platelet aggregation (TCIPA). COX [12(S)-HTT] and LOX [12(S)-HETE], metabolites of washed platelets (WP), alone or co-incubated with LAC or FS cells, were analyzed. We observed higher 12(S)-HETE release with respect to 12(S)HHT when WP were co-incubated with LAC cells. With both neoplastic cell (NC) lines prothrombin time (PT) was shortened. Pretreatment of NC with iodoacetic acid, soybean trypsin inhibitor or Factor X-deficient plasma increased the PT. These results indicate that AA metabolites play a role on the procoagulation and platelet aggregation induced by mesenchymal and epithelial murine cancers.

A simple and efficient method is described to remove hemoglobin (Hb) from human term placental cytosol to study dioxygenase and co-oxidase activities of lipoxygenase. In the untreated samples, 70%-80% of the linoleic acid-dependent dioxygenase and co-oxidase activities were found to be associated with the pseudo-lipoxygenase activity of Hb. Zinc sulfate (0.5 mM) precipitated >97% of the Hb present in the cytosol. The dioxygenase activity of the ZnSO4 treated cytosol exhibited a Vmax value of 313 nmoles linoleic acid hydroperoxide formed/min/mg protein and a K(M) of 1.4 mM for linoleic acid. The ZnSO4 treated cytosol displayed co-oxidase activity toward benzidine, dimethoxybenzidine, guaiacol, pyrogallol, tetramethylbenzidine and tetramethyl-p-phenylenediamine. Nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, butylated hydroxyanisole, butylated hydroxytoluene and gossypol caused concentration dependent inhibition of dioxygenase and co-oxidase activities. These results suggest ZnSO4 precipitation of Hb from cytosol does not alter the functional characteristics of the human term placental lipoxygenase.

In vitro enzymatic and non-enzymatic polyunsaturated fatty acid peroxidation was significantly inhibited in a dose dependent manner by purified anthocyanin, a deep-red colour pigment from carrot cell culture. The kinetics showed that anthocyanin is a non-competitive inhibitor of lipid peroxidation. Anthocyanin has been found to be a potent antioxidant compared to classical antioxidants such as butylated hydroxy anisole (BHA), butylated hydroxy toulene (BHT) and alpha tocopherol. This natural agent, in addition to imparting colour to the food, might prevent autooxidation of lipids as well as lipid peroxidation in biological systems.

Although there have been numerous topical applications of plant extracts having flavonoids known as anti-inflammatory compounds, only a few studies were reported concerning effects of flavonoids on epidermal cyclooxygenase/lipoxygenase. In this investigation, effects of naturally occurring flavonoids on epidermal cyclooxygenase/lipoxygenase were studied using five selected derivatives: flavanone, apigenin (flavone), quercetin (flavonol), amentoflavone and ginkgetin (biflavone) because eicosanoids generated in the epidermis are believed to be involved in various biological activities of the skin. Microsomal and cytosolic fractions were obtained from guinea-pig epidermal homogenate by centrifugation and used as a source for cyclooxygenase and lipoxygenase. It was found that quercetin inhibited both cyclooxygenase and lipoxygenase, being more potent against lipoxygenase, while flavanone and apigenin did not show any inhibition. Amentoflavone, one of the biflavones tested, showed potent and selective inhibitory activity on cyclooxygenase (IC50 = 3 microM) which was comparable to indomethacin (IC50 = 1 microM). In contrast, structurally similar ginkgetin possessed weak inhibitory activity on cyclooxygenase. The in vivo effects of these flavonoids on the normal and diseased skin remain to be studied.

A role of prostaglandins (PGs) and leukotrienes (LTs) in the pathogenesis of nasal polyps has been recently suggested. Cyclooxygenase (CO) products (thromboxane B2, PGE2 and 6-keto PGF1 alpha) and lipoxygenase (LO) products (LTB4 and LTC4) were investigated by radioimmunoassay in polyps, hypertrophic turbinates and nasal mucosa from 14 patients with non-allergic (n = 6), allergic chronic rhinitis (n = 6) and aspirin-sensitive asthma (ASA) (n = 2), who underwent polypectomy. In all tissues CO metabolite levels were found higher than LO products (P < 0.01). Nasal polyps showed a significantly lower (P < 0.05) arachidonic acid (AA) metabolism in comparison to nasal mucosa. In polyps of allergic patients significantly higher LTB4 levels (P < 0.001) and a tendency to produce higher amounts of CO products in comparison to non-allergic subjects were observed, whereas in turbinates of non-allergic patients LT levels were significantly higher in comparison to those of allergic ones (P < 0.01). In ASA patients a decreased CO/LO ratio was found supporting the hypothesis of an imbalance of AA metabolism in this syndrome. These findings seem to indicate that the occurrence of nasal polyps may represent the result of different chronic inflammatory stimuli, regulated in part by AA metabolites.

Adhesion of tumor cells (TC) to endothelial cells (EC) is necessary for movement of TC out of the interstitium to form metastatic deposits. This interaction may be influenced by proadhesive molecules such as lipoxygenase products of arachidonic acid metabolism. We studied the effect of inflammatory stimuli, A23187 calcium ionophore, n-formyl-methionyl-leucine-phenylalanine (FMLP) and phorbol myristate acetate (PMA) on TC-EC interaction. Adherence of metastatic breast tumor cell line (MCF-7), choriocarcinoma cell line (JEG-3), and non metastatic pituitary cell (GH-3) were assayed as the number of radiolabeled TC attached to EC (cpm/well). TC and EC were incubated with A23187, FMLP, and PMA for varying time periods. Lipoxygenase products (LTB4, 5-HETE) were measured under basal and stimulated conditions using RP-HPLC and RIA. There were no differences in basal adherence of TC lines to EC. When EC were incubated with stimuli, there were significant increases in the numbers of MCF-7 and JEG-3 cells adherent to EC compared to GH-3. Light and phase contrast microscopy confirmed that TC were attached to EC. Upon stimulation, GH-3 preferentially produced prostaglandins (PGI1(2)) while MCF-7 and JEG-3 produced lipoxygenase products (LTB4 and 5-HETE). Pre-incubation of MCF-7 and JEG-3 with the lipoxygenase inhibitor nordihydroguiaretic acid resulted in partial inhibition of adhesion to EC. Our data strongly indicate a role for lipoxygenase products of arachidonic acid in adherence of TC to EC.

The effects of tumor necrosis factor alpha (TNF-alpha) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [3H]AA. TNF-alpha differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-alpha (10(-8)M) significantly increased the cyclooxygenase pathway, with prostaglandin E2 (PGE2) being a major product. However, at the lowest concentration (10(-10)M) of TNF-alpha, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-alpha was increased to 10(-9) M leukotriene B4 (LTB4), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10(-6) M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-alpha (10(-8)M) and A23187 (10(-6)M) caused an inhibitory effect on each agent-induced PGE2 or 15-HETE production.

To evaluate the clinical usefulness of steroids for septic lung injury, we investigated the effects of methylprednisolone (MP) on this disorder using an experimental rat model of cecal ligation and puncture (CLP). While 92% of the rats that underwent CLP (CLP rats) died within 30 h, those given high-dose MP (30 mg/kg) just after the operation (CLP + MP rats) survived for a significantly longer period (p < 0.01). Concentrations of endotoxin (ET) in arterial blood were significantly higher in the CLP + MP rats than in the CLP rats, while those in the bronchoalveolar lavage fluid (BALF) were significantly lower. Alveolar macrophages (AM) obtained from the CLP rats (CLP-AM) generated more O2-than did AM from sham-operated rats (sham-AM) following stimulation. However, the administration of MP did not reduce the upregulated generation of O2-by CLP-AM. While CLP-AM produced less leukotriene (LT)B4 than did sham-AM following stimulation with A23187, the administration of MP further reduced LTB4 production. When AM were cultured with [3H]arachidonic acid (3H-AA), the uptake of the isotope and the 3H release were significantly less in CLP-AM than in sham-AM. The administration of MP did not cause recoveries in the uptake and release of 3H-AA by CLP-AM. Although the survival time of CLP rats was significantly prolonged and the translocation of ET into BALF was reduced by steroid administration, the steroid effects were not explained by those on altered AM function. The upregulated generation of O2- and reduced LTB4 production from CLP-AM were not reversed by the treatment of this drug.

The stoichiometry of the oxygenation reaction of cis,cis-eicosa-11,14-dienoic acid catalyzed by prostaglandin endoperoxide synthase and soybean lipoxygenase has been investigated by using steady-state initial rate measurements. The rate of product formation (conjugated diene hydroperoxy and hydroxy derivatives) was followed spectrophotometrically at 235 nm, and the rate of oxygen consumption was measured polarographically. The ratio of the two rates, d[conjugated diene/-d[O2], is 2/1 for the prostaglandin endoperoxide synthase catalyzed reaction and 1/1 for the lipoxygenase reaction. The 2/1 ratio can be explained by two interrelated routes, each of which results in formation of the conjugated diene hydroxy derivative of the acid. One route, initiated by hydrogen atom abstraction from the acid by Compound I, results in formation of the conjugated diene hydroperoxy derivative. The latter is converted to the hydroxy derivative by regenerating Compound I from the native enzyme. The other route involves direct oxygen atom insertion into the acid by the tyrosyl radical form of Compound I. The decrease in absorbance at 235 nm obtained in the presteady-state phase suggests that during the initial contact of hydroperoxide and enzyme an epoxy-hydroxy fatty acid-enzyme complex may be formed.

Cauda epididymal spermatozoa were examined for their capacity to synthesize prostaglandins (PGs) and hydroxy acids and the role of nitric oxide (NO) on cyclooxygenase and lipoxygenase enzyme activity was determined. Sperm suspensions were incubated in the presence or absence of 300 microM sodium nitroprusside (NP) and 20 micrograms/ml hemoglobin (Hb) and then incubated with [14C]arachidonic acid (AA) for 60 min. The cyclooxygenase and lipoxygenase products of AA metabolism were measured for their radioactivity. It was found that mouse spermatozoa could synthesize PGE2 and 5-hydroxyeicosatetraenoic acid (5-HETE) under these experimental conditions. The synthesis of these two metabolites was 100% stimulated by NP, the releaser of NO, and the response to NP was completely prevented by Hb, a scavenger of NO. These data provide evidence that mouse spermatozoa can synthesize PGE2 and 5-HETE in the presence of AA in vitro and that NO is involved in the production of AA metabolites in the male gamete.

PUFA and their eicosanoid metabolites are potent biological modifiers. They have beneficial effects in a number of diseases, which may result in part from their direct actions on neutrophils as well as from their ability to modulate eicosanoid biosynthesis. A consideration of their interactions with other cell types, e.g. lymphocytes and macrophages, is beyond the scope of this review. Small alterations in structure can result in large changes in the neutrophil response. This will have important implications for the further development and use of fatty acids for therapeutic purposes.

Products of arachidonic acid (AA) metabolism via the lipoxygenase pathways may have key roles in the maintenance of pregnancy and the onset of labor. We have determined whether calcium ionophores can modulate the rate of biosynthesis within the uterus of five important arachidonate lipoxygenase metabolites, i.e. leukotriene B4 (LTB4), LTC4, 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE. Amnion, chorion, and decidual cells were isolated, grown to confluence and incubated with ionomycin. The production of LTB4, LTC4, 5-HETE, 12-HETE, and 15-HETE was determined using specific radioimmunoassays. Cell-specific, concentration-related stimulatory actions of ionomycin on 5-HETE, 12-HETE, 15-HETE, and LTC4 but not LTB4 production were found. A23187 had effects similar to ionomycin. Hence elevation of intracellular calcium levels can result in enhanced intrauterine production of arachidonate lipoxygenase metabolites that may affect pregnancy outcome.

ESWL is a safe and effective first-line treatment for urinary tract stone disease (UTSD) in children. The major complications arising from this procedure were upper urinary tract obstruction and ureteral colic. It was shown that prostaglandin synthetase inhibitors were effective in the treatment of urethral colic. The aim of this study was to measure urinary and plasma prostaglandin E2 (PGE2)- and leukotriene C4 (LTC4)-like activity in the patients who underwent ESWL before and after the treatment and investigate the role of cyclooxygenase (CO) and lipoxygenase (LO) products in early and late complications of ESWL. Urinary PGE2-like activity were increased 1 h after ESWL. (1.19 +/- 0.12 vs 1.59 +/- 0.15 g/ml, p < 0.02). The plasma values were decreased significantly after the treatment (16.7 +/- 1.7 vs 11.6 +/- 1.2 g/ml, p < 0.005). Urinary and plasma LTC4-like activities were found to be significantly decreased in the post-ESWL samples (0.58 +/- 0.006 vs 0.39 +/- 0.04, p < 0.002; 8.6 +/- 0.9 vs 4.2 +/- 0.6, p < 0.001, respectively). In conclusion, ESWL may stimulate the release of PG from the urinary tract resulting in increased peristaltism and the passage of stone fragments into the bladder. As this group of drugs has also nephrotoxic effects, they can be given prophylactically only to selected patients.

Malignant cells show increased urokinase (UK) activity and decreased peroxidation of essential fatty acids (EFA). In order to explore this phenomenon the effect of UK on the lipoxidase activity was spectrophotometrically investigated. Decreased lipoxidase activity was obtained with increased UK concentrations (r = -1.000, p < 0.0001). This proteolytic effect of UK on lipoxidase was eliminated with the addition of the UK inhibitor leupeptin. These results suggest that the increase in UK activity in malignant cells may decrease the lipoxidase activity and thus peroxidation of EFA. The effectiveness of a given EFA in killing cancer cells would therefore depend on the modulation of the lipoxidase activity by the UK-type plasminogen activator.

One of the mechanisms by which corticosteroids may modify acute graft vs host disease (GvHD) is via inhibition of arachidonic acid (AA) metabolism. Leukotriene B4 (LTB4) is a product of that pathway which may take part in the pathogenesis of GvHD through the stimulation of T-lymphopoiesis and T-lymphocyte activation. LTB4 is a metabolite of AA (20:4n-6). Alternate dietary sources of polyunsaturated fatty acids (PUFA), specifically eicosapenteinoic acid (20:5n-3) (EPA) and docosahexaenoic acid (22:6n-3) (DHA), shift the LTs formed with a decrease in LTB4 an increase in LTB5. LTB5 is a less potent agonist than LTB4 and this results in a theoretical decrease of LTB4 mediated events. Supplementation of in vitro bone marrow cultures with EPA or DHA had no detrimental effect on myeloid colony formation. Dietary EPA/DHA supplementation in mice with induced GvHD appeared to be safe and well tolerated. The LTB4:LTB5 ratio shifted from 7.65 +/- 1.75 in control-fed animals to 1.03 +/- 0.18. Fish-oil-supplementation did not compromise engraftment or stem cell content. Alone, this therapy was unable to modify GvHD.

The effects of interleukin-1 alpha (IL-1 alpha) on arachidonic acid (AA) metabolism were studied in the human osteosarcoma cell lines, G292 and SaOS-2. The cells were prelabeled with 3H-arachidonic acid. Radiolabeled metabolites were measured by reversed-phase high-pressure liquid chromatography with a radioactive detector. Indomethacin inhibited prostaglandin E2 (PGE2) production without affecting lipoxygenase (LO) products in G292 cells. In the G292 cells, IL-1 alpha (50 U/ml) induced a 10-fold increase in PGE2 production at all the incubation times tested, and a significant two-fold increase in 5 hydroxyeicosatetraenoic acid (HETE) formation after 48 h. These effects were not seen in SaOS-2 cells under identical conditions. These results suggest that, although some osteosarcomal cell lines may not respond directly to IL-1 with effects on AA metabolism, the mechanism of its action in others may involve modulation of both cyclooxygenase (CO) and LO pathways.

N-phenyllinoleamide (NPLA) is a useful marker for adulterated oil samples associated with cases of toxic oil syndrome (TOS). To date, NPLA has not reproduced the human poisoning episode in experimental animal models and, thus, its pathological role in the syndrome remains controversial. The present report describes the effect of NPLA on the lipoxygenase metabolism of exogenous arachidonic acid (AA) in mouse peritoneal macrophages (MPM). Results show that MPM cells exposed to 1mM NPLA for 2 h, when subsequently incubated with exogenous 3H-AA, undergo a significant increase in the biosynthesis of 3H-12-hydroxyeicosatetraenoic acid (3H-12-HETE) whereas levels of 3H-15-HETE are relatively stable. These data indicate that NPLA selectively potentiates the lipoxygenase metabolism of exogenous AA, supporting the possible implication of lipid peroxidative processes in the ethiopathology of TOS, although the relatively high NPLA concentration required 'in vitro' makes it unlikely that this xenobiotic could be directly related to human toxicity.

Fish oil was fed to rats in combination with an equal amount of olive, sunflower or linseed (flax) oil in semisynthetic diets for 3 weeks. Following stimulation of isolated neutrophils with calcium ionophore the levels of leukotrienes (LT) were determined by HPLC. Graphical presentation of the resultant data show a direct linear relationship between LTB production and substrate concentration with no preferential conversion of n-3 or n-6 substrates. In addition the results highlighted the greater conversion of eicosapentaenoic acid (EPA) and arachidonic acid (AA) to 5-hydroxy metabolites in stimulated neutrophils. There is no suggestion in our results of inhibition of any of the enzymatic conversion steps between EPA or AA and LTB production by any of the dietary fatty acids except by altering the EPA/AA ratio in neutrophil membranes.

Platelet activating factor (PAF) production and platelet-lipoxygenase activity were studied during hemodialysis (HD) with cuprophane membranes. Six patients were treated with first-use dialyzers (FU), and 6 patients with reused dialyzers (RU). In a random and double-blind design, 2 HD were performed for each patient, with or without BN 52021 pretreatment, a selective PAF antagonist. Platelet and leukocyte counts were performed before pretreatment and 30 min before HD starting (T-30), at the beginning of HD (T0) and after 15 and 30 min of HD (T15, T30). PAF production was analyzed by direct phase HPLC. To determine platelet-lipoxygenase activity, 12-HETE was detected by reverse phase high performance liquid chromatography (HPLC) after blood stimulation by the ionophore A23187. In the FU group, PAF and 12-HETE were produced during the first 30 min of HD. After BN 52021 pretreatment, PAF production was suppressed and platelet-lipoxygenase activity reduced. In the RU group, neither PAF nor 12-HETE production occurred, and BN 52021 had no effect. We conclude that PAF, which was involved in both platelet and leukocyte activation that occurred during hemodialysis, can be considered as a bio-incompatibility marker.

Leukotriene B4 (LTB4) is a potent chemotactic compound for neutrophils and is thought to be an important mediator of myocardial ischemia-reflow injury. We have measured LTB4 in rabbit cardiac tissue following ischemia-reflow using a sensitive and specific gas chromatographic-mass spectrometric (GC-MS) assay. The concentration of LTB4 in rabbit myocardium following 45 min ischemia and 3 h reflow was 48.7 +/- 12.5 pg/g, significantly higher than in non-ischemic tissue from the same animal (17.5 +/- 3.9 pg/g). These concentrations were at least an order of magnitude lower than previously reported values assessed by radioimmunoassay (RIA). Compared with the GC-MS method, RIA greatly overestimated LTB4 concentrations in cardiac tissue. The capacity of post-ischemic myocardium to produce lipoxygenase products, LTB4, 5-, 12- and 15-HETEs was also assessed following incubation of myocardium ex vivo with calcium ionophore. In all animals ischemic cardiac tissue produced greater amounts of LTB4, 5-, and 12-HETEs than non-ischemic myocardium and 12-HETE was the major product. Neutrophils that have accumulated in the injured tissue may be a major source of these products. However, in contrast to cardiac tissue, isolated rabbit neutrophils stimulated with A23187 produced 5-HETE as the major product with very little 12-HETE formed. These latter findings suggest that cells other than neutrophils may contribute to the production of lipoxygenase products during myocardial ischemia-reflow injury.

Platelet lipid composition, c arachidonic acid (AA) metabolism by platelets (stimulated with thrombin), serum thromboxane (Tx)B2 production and plasma lipid composition were investigated in 53 healthy females (18-45 years) and 65 males (19-45 years) with similar dietary habits. In males, serum TxB2 production and cholesterol platelet membrane levels were found significantly higher (p less than 0.001 and p less than 0.05) than in females. No differences were observed between the two groups in the AA conversion through cyclo-oxygenase and lipoxygenase pathways or in the platelet phospholipid fatty acid composition. These findings indicate that in males the platelet proaggregatory capacity is greater than in females and the higher platelet TxB2 production does not depend on a larger AA availability or on enzyme activation for its conversion. The increased TxB2 production may be, at least in part, induced by functional differences such as a different membrane cholesterol content inducing, in its turn, an increased microviscosity and/or higher number of platelet receptors for thrombin.

The effects of two specific 5-lipoxygenase inhibitors AA-863 and U-60,257 (piriprost) on the growth of two human glioma cell lines, U-343 MGa and U-251 MG were investigated. Both monolayer cultured cells and spheroids were studied. The results of the monolayer studies showed potent and dose dependent inhibitory effects on the proliferation of glioma cells (IC50/one week treatment/of AA-863: 9.0 microM, IC50 of U-60,257: 40.0 microM). The experiments made on the tumor spheroids suggested an inhibitory effect on proliferation and volume growth already at lower doses (AA-863: 0.4-2.0 microM, U-60,257: 1.0-5.0 microM), a dose range where effects were not found in monolayers. At higher doses (AA-863: 10.0-30.0 microM, U-60,257: 30.0-90.0 microM) the experiments with spheroids failed to demonstrate a further inhibitory effect on spheroid volume, probably attributed to phenomena such as swelling of cells, dissociation of spheroid structure and development of necrosis. The clearly dose dependent inhibitory effect on the proliferation of human glioma cells in monolayer culture and the inhibitory effects on spheroid growth with these specific inhibitors indicate a role for lipoxygenase products in the growth of gliomas.

Lipoxygenase-pathway metabolites of arachidonic acid are produced in pancreatic islets. They are are implicated in insulin release, since nonselective inhibitors of lipoxygenases inhibit glucose-induced insulin release. We studied the interplay in insulin release between glucose and selected icosanoids formed in 5-, 12- and 15-lipoxygenase pathways. Effects on immunoreactive insulin release of 10(7) to 10(6)-12-(R)-HETE, 12-(S)-HETE, hepoxilin A3, lipoxin B4, LTB4 or LTC4 were tested individually in 30-min incubations of freshly isolated young adult Wistar rat pancreatic islets, in the presence of 5.6 mM or 23 mM glucose. Basal insulin release (at 5.6 mM glucose) was stimulated by LTC4 and hepoxilin A3 (304% and 234% of controls at 5.6 mM glucose alone, respectively), inhibited by 12-(S)-HPETE (56%), and was not affected by 12-(R)-HETE, 12-(S)-HETE, lipoxin B4 or LTB4 (111%, 105%, 106% and 136%, respectively). Insulin release evoked by 23 mM glucose (190-320%) was inhibited (50-145%) by all icosanoids tested, except LTC4 (162%). We conclude that, among the lipoxygenase products tested, only leukotrienes and hepoxilin are candidates for a tonic-stimulatory influence on basal insulin release. Since glucose promotes icosanoid formation in islets, the observed inhibition of glucose-induced insulin release by lipoxygenase products suggests the existence of a negative-feedback system.

We have compared the metabolism of (3H) arachidonic acid by monolayers of human amnion, cells obtained prior to or following labor at term. Radiolabel was either added exogenously or previously incorporated into cellular phospholipid pools to compare metabolism of arachidonic acid from different substrate sources. Cells obtained both prior to and following labor synthesized metabolites co-chromatographing on HPLC with di- and mono-HETEs and also a metabolite with polarity corresponding to a epoxyeicosatrienoic acid. Both types of cells were able to synthesize PGE2 when (3H) arachidonic acid was added exogenously. However, only those cells obtained following labor synthesized PGE2 from (3H) arachidonic acid incorporated into intracellular pools. These findings suggest that the cyclooxygenase and PGE2 isomerase enzymes are present in amnion prior to delivery but that exogenous arachidonic acid would be required for PGE2 synthesis at that time as the enzymes do not appear to be linked to a source of endogenous arachidonic acid. At the time of parturition, there may be a switching on of an enzyme system to generate arachidonic acid from intracellular pools specifically for PGE2 synthesis or alternatively coupling of such a system to a cyclooxygenase-PGE2 isomerase system resulting in PGE2 synthesis. These findings raise intriguing new possibilities for the regulation of eicosanoid synthesis in amnion which may include membrane topography, substrate pool-enzyme linking and regulation of specific phospholipase enzymes.

Pulmonary hypertension and foreign body granulomas are recognized sequelae of chronic intravenous drug abuse. We have recently described the development of transient pulmonary hypertension and increased permeability pulmonary edema after the intravenous injection of crushed, suspended pentazocine tablets in both humans and dogs. To determine the role of vasoactive substances in the development of this transient pulmonary hypertension, we measured pulmonary hemodynamics and accumulation of arachidonic acid metabolites in dogs during the infusion of indomethacin, a cyclooxygenase inhibitor, diethylcarbamazine (DEC), a lipoxygenase inhibitor, and FPL 55712, a receptor antagonist for leukotriene C4/D4 (LTC4/D4). Following the intravenous administration of crushed, suspended pentazocine tablets (3-4 mg/kg of body weight), mean pulmonary artery pressure increased from 14 +/- 2 mmHg to 30 +/- 6 mmHg (p less than 0.05) at 60 secs with a concomitant increase in plasma concentrations of 6-keto-PGF1 alpha from 187 +/- 92 pg/ml to 732 +/- 104 pg/ml and thromboxane B2 from 206 +/- 83 pg/ml to 1362 +/- 117 pg/ml (both p less than 0.05). Indomethacin prevented the increase in both cyclooxygenase metabolites, but had no effect on the pulmonary hypertension. In contrast, DEC had no effect on the increase in cyclooxygenase products, but blocked the pulmonary hypertension. FPL 55712 did not effect either the increase in cyclooxygenase metabolites or the pulmonary hypertension. We conclude that the transient pulmonary hypertension, induced by the intravenous injection of crushed, suspended pentazocine tablets, is not mediated by cyclooxygenase products but may be mediated by lipoxygenase product(s) other than LTC4/D4.

We have investigated the effect of the heat shock response on the leukotriene generation, chemotaxis, and generation of oxygen radicals of human polymorphonuclear granulocytes (PMNs) by preincubating the PMNs at 42 degrees C. Subsequently, the different test systems were performed at 37 degrees C. As we confirmed by the release of lactate dehydrogenase and beta-glucuronidase the elevated temperatures did not result in cytotoxic or degranulating processes. After heat shock treatment the generation of leukotrienes induced by the Ca(++)-ionophore A23187, fMLP or opsonized zymosan was inhibited in a time and temperature dependent manner (preincubation phase) as was measured by HPLC-analysis. In contrast, the conversion of 14C-arachidonic acid revealed the generation of LTB4, 5-HPETE and 5-HETE solely as a result of the preincubation at 42 degrees C without any further stimulation. In addition, the chemiluminescence response induced by opsonized zymosan and the chemotaxis against C5a and LTB4 was clearly inhibited after heat shock treatment. With regard to enzyme activities of the heat treated PMNs the protein kinase C activities were enhanced whereas the LTD4-dipeptidase and the LTB4-omega-hydroxylase were not affected.

The major role of renal eicosanoid synthesis appears to be a protective one. In the cortex, prostaglandin synthesis minimises potential anoxic and ischaemic damage by vasodilatation. In the medulla, prostaglandin synthesis appears to stabilise the corticomedullary solute gradient and may play a role in cell volume regulation. Mono-oxygenase production at this site, by modifying blood flow and cellular active transport processes could again serve a protective function against anoxia and ischaemia. The release of erythropoietin also appears to be prostaglandin dependent. It is likely that leukotrienes released from inflammatory cells within the kidney will affect renal haemodynamics and capillary permeability as in other tissues.

The effects of arachidonic acid metabolites, analogues and cyclooxygenase/lipoxygenase inhibitors were tested on an "in vitro" bovine model of endothelial cell (EC)-polymorphonuclear leukocyte (PMN) adhesion. Arachidonic acid blocked adhesion at 10(-5)M, a dose which also induced aggregation of PMN. Lower doses did not affect either EC-PMN adhesion or PMN aggregation. Various cyclooxygenase pathway metabolites were inactive in the EC-PMN adhesion assay, with the exception of prostaglandin A2 and prostaglandin B2 which significantly suppressed adhesion at 10(-5)M. Of the synthetic analogs tested, 6 alpha-carbaprostacyclin I2, (5Z)-9 beta-ethynyl-calcium salt (U-64,567E) was inhibitory at 10(-5)M. The cyclooxygenase inhibitors acetylsalicylic acid, indomethacin and ibuprofen were inactive. Products of the lipoxygenase pathways, leukotriene B4 (LTB4), 5-hydroxyeicosatetraenoic acid (5-HETE) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) exhibited variable inhibitory activity at 10(-5)M only. Paradoxical effects were observed with the putative lipoxygenase inhibitors 4,7,10,13-eicosatetraynoic acid (4,7,10,13 ETYA), 5,8,11,14-eicosatetraynoic acid (5,8,11,14 ETYA) and nordihydroguairetic acid (NDGA), which also suppressed EC-PMN adhesion at 10(-5)M. The dual cyclooxygenase-lipoxygenase inhibitor, BW755C was inactive. Bovine PMNs did not respond chemotactically to LTB4 although they were able to synthesize the 5-lipoxygenase products LTB4 and 5-HETE.

Fatty acids are labeled with 18O in the carboxyl group during ester hydrolysis in H2(18)O. We utilized this principle to develop a novel mass spectrometric method for study of the turnover of arachidonic acid in intact cell systems. Analysis of 18O incorporations was by negative ion chemical ionization GC-MS. The use of deuterium-labeled exogenous substrates allowed the metabolic fate of exogenous and intrinsic compounds to be distinguished. Thus, it was shown that the preferred route of 5-lipoxygenase metabolism of exogenous arachidonic acid in ionophore-stimulated human neutrophils is via direct reaction with the enzyme and not via incorporation into cellular lipids. The re-uptake of 5-HETE was also studied. For investigation of intracellular reactions which involve ester hydrolysis, the 18O labeling method is unique in providing direct evidence of the intermediate metabolic pathway of endogenous and exogenous products associated with the arachidonic acid cascade.

Rat spleen lymphocytes prelabelled with [14C] arachidonate were suspended in fresh medium in the presence or absence of exogenous non-radioactive fatty acids added in ethanol. It was found that fatty acids stimulate thromboxane B2, PGE2, HHT and HETE formation. The effect is specific for unsaturated fatty acids. It occurs at 50 micron concentrations and is apparent after 20 minutes incubation. Unsaturated fatty acids may serve an important role in the regulation of prostaglandin and hydroxy fatty acid metabolism in vivo. This may indicate a mechanism for the action of fatty acids on the immune response.

The in vitro effects of aspirin and other non steroidal antiinflammatory drugs (NSAIDs) on 12-HETE formation were evaluated in platelet rich plasma (PRP) stimulated with collagen. Aspirin (1 X 10(-4)-1 X 10(-3) M) inhibited 12-HETE production in PRP. Inhibition of 12-HETE formation was detected also in PRP incubated with indomethacin and BW 755C. Sodium salicylate, instead, did not reduce 12-HETE synthesis. Aspirin (3 X 10(-3) M) did not modify 12-HETE synthesis in similarly challenged washed platelet (WP) preparations, inspite of its effect on the cyclooxygenase pathway. It may be concluded that aspirin and other NSAIDs, not only affect the cyclooxygenase pathway, but also inhibit 12-HETE synthesis. In addition it is shown that the 12-HETE synthesis is reduced by plasma and that this effect is enhanced in the presence of aspirin.