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This study aimed to examine the leukotriene metabolism during COVID-19. In total, 180 participants were included in this study, of which 60 were healthy controls, 60 required intensive care units (ICU), and 60 did not require intensive care (non-ICU). The serum levels of 5-lipoxygenase (5-LO), 5-LO activating protein (ALOX5AP), and cysteinyl leukotriene (CYSLT) were measured, and the mRNA expression levels of 5-LO, ALOX5AP, and cysteinyl leukotriene receptor 1 (CYSLTR1) were investigated. Compared with the control group, both the non-ICU and ICU groups had lower levels of 5-LO and mRNA expression. ICU patients had lower levels of 5-LO and mRNA expression than non-ICU patients. CYSLTR1 mRNA expression was highest in the ICU group, followed by the non-ICU group, and healthy controls had the lowest mRNA expression levels. CYSLT levels were higher in the control group than in the non-ICU and ICU groups. CYSLTR1 expression was higher in patients than in controls; therefore, selective leukotriene receptor blockers can be used as treatment options. CYSLTR1 expression was higher in the ICU group than in the non-ICU group. Furthermore, CYSLTR1 mRNA expression may be a promising biomarker of COVID-19 severity.

Background: There is need to understand biological markers and mechanisms in Gulf War illness (GWI). Goal: To examine whether and how eicosanoids - prostaglandins and leukotrienes - are altered in veterans with GWI. Methods: Seventy participants including 37 GWI and 33 healthy controls, shared exposure information, and had plasma eicosanoids assessed - prostaglandin F2 alpha ( pgf2α ), prostaglandin D2 ( pgd2 ), leukotriene B4 ( lb4 ) among others. Values were compared for GWI versus controls. Eicosanoid intercorrelations were compared in cases vs. controls. For the most significantly altered eicosanoid in GWI, exposure and symptom relations were assessed. Results: Prostaglandins and leukotrienes were depressed in GWI, strongest for pgf2α, then lb4. Eicosanoid intercorrelations differed in GWI vs. controls. Fuel-solvent, pesticide, radioactive chemicals and metal exposures related negatively to pgf2α; as, in GWI, did chemical attack and vaccines. Multivariate predictors included fuels-solvents and radioactive chemicals (negative); tetanus vaccine and herbicides (positive). Fuels-solvents and radioactive chemicals predicted lower pgf2α in cases, controls, and all participants controlled for case status. Lower pgf2α related to GWI "Kansas criteria" domains of pain, respiratory, and (borderline significantly) skin symptoms. Conclusion: Multiple eicosanoids are depressed in GWI, particularly pgf2α and lb4. Prior fuel-solvent exposures, radioactive chemicals, and (in GWI cases) vaccines were linked to lower pgf2α.

Background: Immediate hypersensitivity reactions occurring during anesthesia are classified as allergic when skin tests and mast cell tryptase are positive and as nonallergic when negative results are obtained. Cysteinyl leukotrienes (cysLTs) are potent mediators synthesized by mast cell and eosinophil that induce bronchial constriction. They could play a role in hypersensitivity reactions., Methods: cysLT C4, D4, and E4 concentrations were measured by a competition immunoassay in serial plasma samples obtained prospectively from 21 anesthetized controls and retrospectively from 34 patients who reacted at induction of anesthesia (24 with allergic and 10 with nonallergic reactions)., Results: In controls, the median (interquartile range) cysLT concentration was 0.83 (0.69 to 1.02) μg/l before anesthesia and was unchanged 30 min, 6 h, and 24 h afterward. In the patients with allergic reactions, the values were highly increased 30 to 60 min after the reaction (17.9 [7.8 to 36.0] μg/l), while the patients with nonallergic reactions had less increased values (7.3 [3.0 to 11.5] μg/l). The difference between the three groups was significant (P < 0.0001). Increased values persisted during the 24 h of observation. Concentrations were significantly higher in patients with bronchospasm (P = 0.016)., Conclusions: cysLTs appear to be an important mediator of allergic and nonallergic immediate hypersensitivity reactions. These findings might open a new field for management of patients with hypersensitivity reactions, especially nonallergic ones.

Context: The role of neuroinflammation in methanol-induced toxic brain damage has not been studied., Objective: We studied acute concentrations and the dynamics of leukotrienes (LT) in serum in hospitalized patients with acute methanol poisoning and in survivors., Methods: Series of acute cysteinyl-LT and LTB4 concentration measurements were performed in 28/101 hospitalized patients (mean observation time: 88 ± 20 h). In 36 survivors, control LT measurements were performed 2 years after discharge., Results: The acute maximum (C max ) LT concentrations were higher than concentrations in survivors: C max for LTC4 was 80.7 ± 5.6 versus 47.9 ± 4.5 pg/mL; for LTD4, 51.0 ± 6.6 versus 23.1 ± 2.1 pg/mL; for LTE4, 64.2 ± 6.0 versus 26.2 ± 3.9 pg/mL; for LTB4, 59.8 ± 6.2 versus 27.2 ± 1.4 pg/mL (all p < 0.001). The patients who survived had higher LT concentrations than those who died (all p < 0.01). Among survivors, patients with CNS sequelae had lower LTE4 and LTB4 than did those without sequelae (both p < 0.05). The LT concentrations increased at a rate of 0.4-0.5 pg/mL/h and peaked 4-5 days after admission. The patients with better outcomes had higher cys-LTs (all p < 0.01) and LTB4 (p < 0.05). More severely poisoned patients had lower acute LT concentrations than those with minor acidemia. The follow-up LT concentrations in survivors with and without CNS sequelae did not differ (all p > 0.05). The mean decrease in LT concentration was 30.9 ± 9.0 pg/mL for LTC4, 26.3 ± 8.6 pg/mL for LTD4, 37.3 ± 6.4 pg/mL for LTE4, and 32.0 ± 8.8 pg/mL for LTB4., Conclusions: Our findings suggest that leukotriene-mediated neuroinflammation may play an important role in the mechanisms of toxic brain damage in acute methanol poisoning in humans. Acute elevation of LT concentrations was moderate, transitory, and was not followed by chronic neuroinflammation in survivors.

The role of inflammation and oxidative stress is critical during onset of metabolic disorders and this has been sufficiently established in literature. In the present study, we evaluated the effects of sesamol and sesamin, two important bioactive molecules present in sesame oil, on the generation of inflammatory and oxidative stress factors in LPS injected rats. Sesamol and sesamin lowered LPS induced expression of cPLA 2 (61 and 56%), 5-LOX (44 and 51%), BLT-1(32 and 35%) and LTC 4 synthase (49 and 50%), respectively, in liver homogenate. The diminished serum LTB 4 (53 and 64%) and LTC 4 (67 and 44%) levels in sesamol and sesamin administered groups, respectively, were found to be concurrent with the observed decrease in the expression of cPLA 2 and 5-LOX. The serum levels of TNF-α (29 and 19%), MCP-1 (44 and 57%) and IL-1β (43 and 42%) were found to be reduced in sesamol and sesamin group, respectively, as given in parentheses, compared to LPS group. Sesamol and sesamin offered protection against LPS induced lipid peroxidation in both serum and liver. Sesamol, but not sesamin, significantly restored the loss of catalase and glutathione reductase activity due to LPS (P<.05). However, both sesamol and sesamin reverted SOD activities by 92 and 98%, respectively. Thus, oral supplementation of sesamol and sesamin beneficially modulated the inflammatory and oxidative stress markers, as observed in the present study, in LPS injected rats. Our report further advocates the potential use of sesamol and sesamin as an adjunct therapy wherein, inflammatory and oxidative stress is of major concern., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Autism is a neurodevelopmental disorder that clinically presented as cognitive deficits, social impairments and sensory dysfunction. An increasing body of evidence has shown that oxidative stress and inflammation are involved in the pathophysiology of autism. Recording biomarkers as measure of the severity of autistic features might help in understanding the pathophysiology of autism., Methods: This study investigates the plasma levels of 8-isoprostane and Cysteinyl leukotrienes (CysLTs) in 44 autistic children and 40 healthy controls. The recruited autistic patients were assessed for behavior, cognitive and sensory deficits by using different autism severity rating scales, including the Childhood Autism Rating Scales (CARS), Social responsiveness scale (SRS) and Short Sensory Profile (SSP). Receiver Operating Characteristics analysis (ROC) of the obtained data was performed to measure the predictive value of 8-isoprostane and Cysteinyl leukotrienes (CysLTs) as oxidative stress- related parameters. Pearson's correlations between the measured parameters was also performed., Results: The concentrations of 8-isoprostane and CysLTs in autistic patients were significantly higher than those in controls. While cognitive and social impairments did not show any significant differences, the SSP results were strongly correlated with the levels of both of the biomarkers assessed. However, autistic children showed improvements in oxidative stress status (as determined by 8-isoprostane levels) at increasing ages., Conclusion: This study indicates that 8-isoprostane and CysLTs can be used as markers for the early recognition of autistic patients through sensory deficits phenotypes which might help early intervention.

The aim of the study was to determine the effects of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF), interleukin-1-alpha (IL-1α), and nitric oxide donor (NONOate) on both in vivo and in vitro secretion of prostaglandin (PG)E2, PGF2α, leukotriene (LT)B4, and LTC4 by the bovine mammary gland. In the first experiment, tissues isolated from the teat cavity and lactiferous sinus were treated in vitro with LPS (10 ng/mL), TNF (10 ng/mL), IL-1α (10 ng/mL), NONOate (10(-4) M), and the combination of TNF + IL-1α + NONOate for 4 or 8 h. PGE2 or PGF2α secretion was stimulated by all treatments (P < 0.05) excepting NONOate alone, which did not stimulate PGF2α secretion. Moreover, all factors increased LTB4 and LTC4 secretion (P < 0.05). In the second experiment, mastitis was experimentally mimicked in vivo by repeated (12 h apart) intramammary infusions (5 mL) of (1) sterile saline; (2) 250-μg LPS; (3) 1-μg/mL TNF; (4) 1-μg/mL IL-1α; (5) 12.8-μg/mL NONOate; and (6) TNF + IL-1α + NONOate into 2 udder quarters. All infused factors changed PGE2, 13,14-dihydro,15-keto-PGF2α, and LT concentrations in blood plasma collected from the caudal vena cava, the caudal superficial epigastric (milk) vein, the jugular vein, and the abdominal aorta (P < 0.05). In summary, LPS and other inflammatory mastitis mediators modulate PG and LT secretion by bovine mammary gland in both in vivo and in vitro studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Leukotrienes (LTs), including cysteinyl-LTs (LTC4, LTD4 and LTE4) and LTB4, are potent inflammatory lipid mediators which have been involved in the pathophysiology of respiratory diseases. LC-MS/MS techniques for measuring LT concentrations in sputum supernatants, serum, urine and exhaled breath condensate (EBC) have been developed. In asthmatic adults, reported LTB4 and LTE4 concentrations in sputum range from 79 to 7,220 pg/ml and from 11.9 to 891 pg/ml, respectively. Data on sputum LT concentrations in healthy subjects are not available. In EBC, reported LTE4 concentrations range from 38 to 126 pg/ml (95% CI) in adult asthma patients and from 34 to 48 pg/ml in healthy subjects. LTB4 concentrations in EBC range from 175 to 315 pg/ml (interquartile range) in asthmatic children, and from 25 to 245 pg/ml in healthy children. Enabling an accurate quantitative assessment of LTs in biological fluids, LC-MS/MS techniques provide a valuable tool for exploring the pathophysiological role of LTs in respiratory disease and might be useful for assessing the effects of therapeutic intervention. This review presents the analytical aspects of the LC-MS/MS techniques for measuring LT concentrations in biological fluids and discusses their potential utility for the assessment of airway inflammation and monitoring of pharmacological treatment in patients with asthma phenotypes and other respiratory diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Inflammation is increasingly recognized as being of both physiological and pathological importance in the immature brain. Cerebellar pathology occurs in autism, as a neurodevelopmental disorder with genetic and environmental origins. The genesis of this disorder is still not understood but inflammation in utero or early in childhood is an environmental risk factor., Methods: Prostaglandin E2 (PGE2), cysteinyl leukotriene as two important lipid mediators together with 8 isoprostane as marker of oxidative stress were measured using ELISA in plasma of 20 male autistic patients compared to 19 age and gender matching control participants., Results: PGE2, leukotrienes and isoprostanes recorded significantly elevated levels in autistics compared to controls. Role of these measured parameters in inflammation and autoimmunity as two etiological factors in autism were discussed in details., Conclusion: Receiver Operating Characteristic (ROC) curve analysis shows satisfactory values of area under the curve (AUC) which could reflect the high degree of specificity and sensitivity of the altered PGE2, leukotrienes and isoprostanes as predictive biomarkers in autistic patients from Saudi Arabia.

Leukotrienes (LTs) are involved in the pathogenesis of lung fibrosis and were increased in exhaled breath condensate (EBC) of the patients with pneumoconiosis. However the possible influence of extra-pulmonary disorders on the EBC markers is not known. Therefore in parallel with EBC, LTs' levels in the plasma and urine were measured in patients with pneumoconiosis (45 × asbestos exposure, 37 × silica exposure) and in 27 controls. Individual LTs B4, C4, D4 and E4 were measured by liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS). In EBC, LT D4 and LT E4 were increased in both groups of patients (p<0.001 and p<0.05), comparing with the controls. Both LT B4 and cysteinyl LTs were elevated in asbestos-exposed subjects (p<0.05). Asbestosis with more severe radiological signs (s1/s2-t3/u2) and lung functions impairment has shown higher cysteinyl LTs and LT C4 in the EBC (p<0.05) than mild asbestosis (s1/s0-s1/s1). In addition, in the subjects with asbestosis, cysteinyl LTs in EBC correlated with TLC (-0.313, p<0.05) and TLCO/Hb (-0.307, p<0.05), and LT C4 with TLC (-0.358, p<0.05). In pneumoconioses, EBC appears the most useful from the 3 fluids studied.

Objective: Pulmonary function frequently deteriorates after cardiopulmonary bypass (CPB). Chronic obstructive pulmonary disease (COPD) increases risk of respiratory complications after CPB. Cysteinyl leukotrienes are important mediators of respiratory dysfunction. Their role during cardiac surgery and its lung complications is incompletely understood. We studied whether production of cysteinyl leukotrienes changes during and after cardiac surgery with CPB and differs between patients with and without COPD., Methods: Patients with (n = 9) and without (n = 10) moderate-to-severe COPD undergoing cardiac surgery with CPB were prospectively included. Plasma and urinary cysteinyl leukotriene and leukotriene B(4) concentrations were measured by enzyme-linked immunosorbent assay after anesthesia induction, at end of CPB, after CPB, and 2 hours after intensive care unit admission. Gas exchange and respiratory mechanics were also assessed., Results: Patients with COPD had larger airway resistances after CPB and chest closure (P < .001), lower ratio of arterial Po(2) to inspired oxygen fraction at intensive care unit admission (215 ± 37 vs 328 ± 30 mm Hg, P < .05), and longer postoperative mechanical ventilation (13.7 ± 5.8 vs 6.8 ± 3.4 hours, P < .01). Urinary cysteinyl leukotriene concentrations increased with time in both groups (P < .01), but more in patients with than without COPD (P < .05). Plasma cysteinyl leukotriene concentrations increased significantly between baseline and intensive care unit admission in patients with but not without COPD (P < .01). Concentrations of leukotriene B(4) in plasma and urine did not increase significantly with time and were not different between groups., Conclusions: Release of cysteinyl leukotrienes increases during cardiac surgery with CPB and is larger in patients with than without COPD. This may be related to higher lung and airway production of cysteinyl leukotrienes and neutrophil activation in patients with COPD., (Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

n-3 PUFA supplementation helps in the prevention or treatment of inflammatory diseases and CVD. However, many supplementations reported sofar are either a combination of n-3 PUFA or used large daily amounts of n-3 PUFA dosages. The present study investigated the influence of increasing dose intake of DHA on the fatty acid composition of phospholipids in neutrophils and on their capability to produce leukotrienes(LT) B4 and B5 in vitro. Twelve healthy volunteers were supplemented with increasing daily doses of DHA (200, 400, 800 and 1600 mg, each dose in TAG containing DHA as the only PUFA and for a 2-week period). At the end of each supplementation period, neutrophil fatty acid composition,and LTB4 and LTB5 production were determined by GC and liquid chromatography-tandem MS, respectively. The DHA/arachidonic acid ratio increased in a dose-dependent manner with respect to the increasing doses of DHA supplementation and was significantly different from baseline after supplementation with either 400, 800 or 1600 mg DHA. The LTB5/LTB4 ratio was significantly increased compared to baseline after supplementation with 800 and 1600 mg DHA. LTB5/LTB4 and DHA/arachidonic acid ratios were correlated (r 0.531, P<0.0001). The present data suggest that both changes in neutrophil lipid composition and LT production occurred with daily supplementation with 800 and 1600 mg DHA. The clinical benefits associated with these doses of DHA in inflammatory diseases remain to be investigated.

Objective: To evaluate the in vivo effects of firocoxib, meloxicam, and tepoxalin on prostaglandin (PG) and leukotriene production in duodenal mucosa and other target tissues in dogs with chronic osteoarthritis (OA)., Animals: 8 dogs with chronic, unilateral OA of the stifle joint., Procedures: In a crossover design, each dog received placebo (no treatment), firocoxib, meloxicam, or tepoxalin for 7 days, followed by a 21-day washout period. On the first day of treatment (day 0; baseline) and days 2, 4, and 7, samples of whole blood, synovial fluid, and gastric and duodenal mucosae were collected. Prostaglandin E2 concentrations were measured in synovial fluid of the stifle joint and after ex vivo stimulation of whole blood samples. Synthesis of PGE1 and PGE2 was measured in samples of gastric and duodenal mucosae. Concentrations of thromboxane B2 (TxB2) were measured in whole blood samples. Leukotriene B4 (LTB4) concentrations were measured in samples of whole blood (ex vivo stimulation) and gastric and duodenal mucosae., Results: Firocoxib, meloxicam, and tepoxalin significantly suppressed whole blood concentrations of PGE2, compared with baseline and placebo concentrations, at days 2, 4, and 7. Tepoxalin significantly suppressed serum TxB2 concentrations, compared with baseline, firocoxib, meloxicam, and placebo, at all 3 time points. Production of PGE1 and PGE2 was significantly lower in duodenal versus gastric mucosa. Tepoxalin significantly decreased rates of PGE1 and PGE2 in duodenal and gastric mucosae, compared with baseline rates., Conclusions and Clinical Relevance: PG production was lower in the duodenum than in the stomach. Firocoxib had a COX-1-sparing effect in vivo.

The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors. Inflammation was produced by adding carrageenan into a newly formed air pouch and prostaglandins produced. While macrophages and neutrophils are present in the inflamed pouch, little 5-LOX products are formed. Cellular 5-LOX activation was obtained by adding calcium ionophore (A23187) into the pouch thus providing a novel model to evaluate the efficacy and selectivity of 5-LOX inhibitors. Also, we described modifications to the in vitro 5-LOX enzyme and cell assays. These assays included a newly developed fluorescence-based enzyme assay, a 5-LOX redox assay, an ex vivo human whole blood assay and an IgE-stimulated rat mast cell assay, all designed for maximal production of leukotrienes. Zileuton and CJ-13,610, a competitive, non-redox inhibitor of 5-LOX, were evaluated for their pharmacological properties using these assays. Although both compounds achieved dose-dependent inhibition of 5-LOX enzyme activity, CJ-13,610 was 3-4 fold more potent than zileuton in all-assays. Evaluation of 5-LOX metabolites-by LC/MS/MS and ELISA confirmed that both compounds selectively inhibited all products downstream of 5-hydroperoxy eicosatetraenoic acid (5-HPETE), including 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE), without inhibition of 12-lipoxygenase (12-LOX), 15-lipoxygenase (15-LOX), or cyclooxygenase (COX) products. In the rat air pouch model, oral dosing of CJ-13,610 and zileuton resulted in selective inhibition 5-LOX activity from pouch exudate and ex vivo rat whole blood with similar potency to in vitro assay. These data show that the rat air pouch model is a reliable and useful tool for evaluating in vivo efficacy of 5-LOX inhibitors and may aid in the development of the next generation of 5-LOX inhibitors, such as the non-redox inhibitors similar to CJ-13,610.

Some changes take place in the spectrum of fatty acid, cholesterol and individual phospholipids' composition in the rat liver, under E-hypovitaminosis, that can play a considerable role in the cell damage. The level of cysteinyl leucotriene decreases in the blood and liver under E-hypovitaminosis and it rises to the control level under vitamin E correction. This demonstrates the influence of tocopherol on 5-LO pathway arachidonic acid.

Leukotrienes are potent inflammatory mediators synthesized locally within the cardiovascular system through the 5-lipoxygenase pathway of arachidonic acid metabolism. The leukotrienes, consisting of dihydroxy leukotriene LTB4 and the cysteinyl leukotrienes LTC4, LTD4 and LTE4, act by targeting cell surface receptors expressed on inflammatory cells and on structural cells of vessel walls. LTB, induces leukocyte activation and chemotaxis via high- and low-affinity receptor subtypes (BLT1 and BLT2), respectively. Recently, BLT, receptors were found on human vascular smooth muscle cells, inducing their migration and proliferation. Cysteinyl leukotrienes are vasoconstrictors and induce endothelium-dependent vascular responses through the CysLT, and CysLT2 receptor subtypes. There is also pharmacological evidence for the existence of further CysLT receptor subtypes. Taken together, experimental and genetic studies suggest a major role of leukotrienes in atherosclerosis and in its ischemic complications such as acute coronary syndromes and stroke. Furthermore, the effects on vascular smooth muscle cells suggest a role in the vascular remodeling observed after coronary angioplasty, as well as in aortic aneurysm. Further experimental and clinical studies are needed to determine the potential of therapeutic strategies targeting the leukotriene pathway in cardiovascular disease.

To evaluate the levels of leukotrienes (LTs), interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-gamma (IFN-gamma) in patients with eczema and observe the effects inversed by mizolastine. Serum LTB4, LTC4, IL-2, IL-4, IFN-gamma and urinary LTE4 levels were detected by enzyme-linked immunosorbent assay (ELISA) and LTB4, LTC4, LTE4 concentrations of cutis tissue were measured by reverse-phase high-pressure liquid chromatography (RP-HPLC) in 10 eczema patients and 10 healthy volunteers. Eczema patients received mizolastine 10 mg once a day for 5 days, respectively, for comparison between before and after treatment. The above markers were assayed again after treatment. Serum LTB4, LTC4, IL-2, IFN-gamma and urinary LTE4 and skin tissue LTB4, LTC4, LTE4 levels in patients are higher than those in healthy volunteers significantly (P < 0.05). But serum IL-4 level did not show significant difference between patients and normal controls (P > 0.05). Mizolastine significantly reduced serum LTB4 and IFN-gamma levels as well as skin lesion LTB4, LTC4, LTE4 concentrations. LTs are involved in the pathogenesis of eczema. Mizolastine clearly reduces LTs levels in skin lesion.

COX-inhibitors promote nasal polyps or bronchial asthma in individuals susceptible to an alteration of the pattern of the eicosanoids, especially leukotrienes and prostaglandins. This is associated with an abnormal release of eicosanoids from white blood cells. Since COX-inhibitors also protect from colorectal cancer an analogous association may be suggested. The study was performed to detect abnormal patterns of eicosanoids in white blood cells of patients with intestinal cancer compared to healthy controls. Seventy patients with intestinal cancer (stomach = 5; colon = 25; sigma = 18; rectum = 22) were compared to 62 healthy controls. Blood leukocytes from patients in complete long-lasting remission were incubated with diluent, arachidonic acid or acetylsalicylic acid. The synthesis of prostaglandin E(2) and peptido-leukotrienes was quantified using competitive enzyme-immuno-assays and calculated for individual eicosanoid patterns. The mean basal and arachidonic- or acetylsalicylic acid-modulated PGE(2) synthesis in patients was significantly higher than in controls (4.8-fold, 9.4-fold, 3.7-fold, respectively) whereas pLT was generally less elevated. We conclude that the eicosanoid-pattern of white-blood-cells from patients with intestinal cancer differs significantly from that in healthy individuals. This abnormal cellular metabolism may contribute to the manifestation of cancer and help to detect individuals at risk.

Monitoring of eicosanoid synthesis in peripheral blood cells has significant potential for improving the diagnosis and therapy of many human diseases. The quantitative relation between concentrations of prostaglandins and leukotrienes is central to the physiologic function of the eicosanoid network. Here we show that this regulation, which we call the functional eicosanoid typing (FET), fluctuates dynamically in individual living blood cells from patients, thereby limiting the accuracy with which concentration circuits of eicosanoids can transfer metabolic information. Using living cells in functional cell testing, we characterised the eicosanoid pattern score (EPS). A novel technique based on binomial errors on lipid mediator partitioning enabled calibration of in vivo biochemical parameters in molecular units. We found that eicosanoid production rates fluctuate over a time scale of about twenty minutes, while intrinsic noise decays rapidly. Thus, biochemical eicosanoid parameters, noise, and slowly varying cellular states together determine the effective FET. These results can form a basis for quantitative modelling of natural eicosanoid circuits in diagnosis of eicosanoid related diseases and design of synthetic ones for the prediction other diseases.

Purpose: To test whether mono- or dimeric X-ray contrast media (CM) may induce the de novo production of cysteinyl-leukotriens (cys-LT), that could contribute to allergic/allergy-like side effects., Materials and Methods: Leukocytes from 39 patients receiving iopromide or iotrolan for routine CT-examination were analyzed for the production of cys-LT. Histamine levels were analyzed in plasma specimens. One patient with a positive history of a previous CM-reaction did not receive CM-injection., Results: Three patients of the iopromide and five of the iotrolan group showed adverse reactions. Reactors had increased cys-LT values in samples obtained before CM-injection induced by the positive control (anti-FcepsilonRI antibodies) (6763.7 pg/ml+/-1367.3 versus 2299.8 pg/ml+/-399.2; p<0.007). Patients with versus without CM-reaction did not differ significantly with respect to their histamine values before CM-administration. In vitro iopromide (p<0.0002) and iotrolan (p<0.0008) induced significant cys-LT production as compared to IL-3 stimulation. In vivo both CM induced a significant increase 6h after CM administration (p<0.05)., Conclusion: Our findings suggest that both CM seem to induce cys-LT production. As to whether the observed increased values in pre-dose samples of patients with as compared to those without reactions could contribute to identify high risk patients should be investigated in larger patient groups in future.

Objective: To evaluate in vivo effects of tepoxalin, an inhibitor of cyclooxygenase (COX) and lipoxygenase (LOX), on prostaglandin (PG) and leukotriene production in osteoarthritic dogs., Animals: 7 mixed-breed adult dogs with chronic unilateral arthritis of a stifle joint., Procedure: Dogs were treated in accordance with a randomized 3-way crossover design. Each dog received an inert substance, meloxicam, or tepoxalin for 10 days. On day 0 (baseline), 3, and 10, dogs were anesthetized and samples of blood, stifle joint synovial fluid, and gastric mucosa were collected. Concentrations of PGE2 were measured in synovial fluid and after lipopolysaccharide stimulation of whole blood; PGE1 and PGE2 synthesis was measured in gastric mucosa. Thromboxane B2 (TxB2) concentration was measured in whole blood. Leukotriene B4 (LTB4) concentration was determined in gastric mucosa and in whole blood after ex vivo stimulation with a calcium ionophore., Results: Tepoxalin significantly decreased LTB4 concentrations in the blood and gastric mucosa at day 10 and TxB2 concentrations in the blood and PGE2 in the gastric mucosa and synovial fluid at days 3 and 10, compared with baseline values. Meloxicam significantly decreased PGE2 concentrations in the blood at days 3 and 10 and synovial fluid at day 3. Meloxicam also decreased PGE1 and PGE2 synthesis in the gastric mucosa at day 3. Meloxicam did not affect LTB4 synthesis in the blood or LTB4 concentrations in the gastric mucosa., Conclusions and Clinical Relevance: Tepoxalin has in vivo inhibitory activity against COX-1, COX-2, and 5-LOX in dogs at the current approved recommended dosage.

Background: We assessed whether nonsteroidal anti-inflammatory drugs (NSAIDs) may provoke blood basophil activation in vitro in aspirin- and NSAID-hypersensitive patients, as detected by a flowcytometric technique using the CD63 marker--flowcytometric basophil activation test (FAST) assay--in addition to the sulfidoleukotriene (sLT) release--the cellular allergen stimulation test (CAST)., Methods: Sixty aspirin- and/or NSAID-hypersensitive patients were studied. Thirty control patients without history and negative provocation challenge were also included. The percentage of activated basophils after in vitro stimulation with NSAIDs at 3 different concentrations was evaluated by an anti-CD63 phycoerythrin conjugate (FAST assay) and the amount of sLTs released in the cell supernatant by ELISA (CAST assay)., Results: For aspirin, the FAST indicated a sensitivity of 41.7%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 99.4%; for paracetamol 11.7 and 100%, for metamizol 15 and 100%, for diclofenac 43.3 and 93.3%, and for naproxen 54.8 and 74.1%. Many patients showed positive tests to more than 1 NSAID. When considering the first 4 NSAIDs, the global sensitivity increased to 66.7%, while the specificity remained at 93.3%. The addition of the CAST results still increased the sensitivity up to 73.3%, but with a decrease of the specificity to 71.4%., Conclusions: The FAST shows a high percentage of positive reactions, which may reach 60-70% when 4 NSAIDs are tested and even 88% when the test is performed within 1 month of the last clinical drug exposure and reaction. The test has a high specificity above 90%. The addition of sLT determinations yields additional information in a few isolated cases. It is suggested that this test, when properly used, may help avoid some cumbersome and dangerous provocation challenges.

Background: The role of leukotrienes (LTs) in the pathophysiology of isocyanate-induced asthma is not well known., Objective: We sought to characterize the type of airway inflammation induced by exposure to isocyanates and to investigate whether exposure to isocyanates induced an increase in LT receptor cysteinyl leukotriene ((CysLT)(1), CysLT(2) and leukotriene B(4) receptor (BLT(1))) expression, as well as a release of LT (LTC(4) and leukotriene B(4) (LTB(4))) and IL-8 in both asthmatics with isocyanate-induced asthma and healthy subjects., Methods: We investigated eight subjects with isocyanate-induced asthma and eight healthy subjects. Both groups underwent specific inhalation challenges to isocyanates in the laboratory. Induced sputum was collected before and after exposure to isocyanates. CysLT(1), CysLT(2) and BLT(1) expression was assessed by flow cytometry, whereas LTC(4), LTB(4) and IL-8 were measured in the sputum supernatants by enzyme immunoassay., Results: Exposure to isocyanates induced an increase in sputum neutrophils only in subjects with occupational asthma. There was a significant increase in CysLT(1) and BLT(1) receptor expression, as well as a release of LTB(4) and IL-8 after exposure to isocyanates compared with the baseline, only in subjects with isocyanate-induced asthma, whereas there was no increase in LTC(4). Exposure to isocyanates did not induce any change in LT receptor expression nor in the levels of LTC(4), LTB(4) and IL-8, in healthy subjects., Conclusion: The neutrophilia observed after exposure to isocyanates is likely to be related to the release of LTB(4), probably enhanced by the increased expression of BLT(1) on neutrophils as well as by the release of IL-8. The significance of the increase of CysLT1 receptor expression on neutrophils is unknown and needs further investigation.

Leukotrienes (LTs) are active lipid mediators derived in the 5-lipoxygenase pathway. LTC(4), the primary cysteinyl LT, is cleaved by gamma-glutamyl transpeptidase (GGT), resulting in LTD(4). We studied the synthesis and metabolism of LTs in three patients with GGT deficiency. LTs were analyzed in urine, plasma, and monocytes after HPLC separation by enzyme immunoassays, radioactivity detection, and electrospray tandem mass spectrometry. Analysis of LTs in urine revealed increased concentrations of LTC(4) (12.8-17.9 nmol/mol creatinine; controls, <0.005 nmol/mol creatinine), whereas LTE(4) was below the detection limit (<0.005 nmol/mol creatinine; controls, 32.2 +/- 8.6 nmol/mol creatinine). In plasma of one patient, LTC(4) was found to be increased (17.3 ng/ml; controls, 9.6 +/- 0.4 ng/ml), whereas LTD(4) and LTE(4) were below the detection limit (<0.005 ng/ml). LTB(4) was found within normal ranges. In contrast to controls, the synthesis of LTD(4) and LTE(4) in stimulated monocytes was below the detection limit (<0.1 ng/10(6) cells; controls, 37.1 +/- 4.8 cells and 39.4 +/- 5.6 ng/10(6) cells, respectively). The formation of [(3)H]LTD(4) from [(3)H]LTC(4) in monocytes was completely deficient (<0.1%; controls, 85 +/- 7%). Our data demonstrate a complete deficiency of LTD(4) biosynthesis in patients with a genetic deficiency of GGT. GGT deficiency represents a new inborn error of cysteinyl LT synthesis and provides a unique model in which to study the pathobiological coherence of LT and glutathione metabolism.

Background: We assessed the reliability of basophil activation test (FAST) and sulphidoleukotriene production (CAST) in the in vitro diagnosis of allergy to metamizol, evaluating its sensitivity and specificity., Methods: Twenty-six patients allergic to metamizol and 30 control individuals were studied. Skin tests with metamizol, FAST, and CAST were performed., Results: FAST sensitivity was 42.3% and specificity 100%. The PPV of FAST is 100% and the NPV 99.4%. The likelihood ratio for a positive value cannot be calculated because the specificity is 100% and the likelihood ratio for a negative value is 0.58. CAST sensitivity was 52%, and specificity 90%. The PPV of the test is 5% and the NPV 99.5%. The likelihood ratio for a positive result was 5.2 and that for a negative result 0.53. FAST detects a larger number of cases when patients are studied within the first 6 months after the clinical reaction (chi = 4.2, P = 0.04) than later. Together with skin tests, FAST allowed detection of 69.2% patients allergic to metamizol, the same as CAST 76%. The joint use of the three techniques allowed identification of 76.9% of cases., Conclusions: FAST and CAST are useful for the diagnosis of allergy to pyrazolones. Its usefulness clearly increases when recent reactions are studied.

Experiments on rabbits with epinephrine-induced damage to the heart showed that progression of necrotic degenerative processes in the myocardium is augmented by increased content of prostaglandin F(2alpha)and predominance of constrictive over vasodilatory effects in the E and F(2alpha)prostaglandin system. Twenty-four hours after injection of epinephrine we observed an increase in blood concentrations of myofibrillar fraction of creatine phosphokinase, serotonin and histamine, and the F(2alpha)/E prostaglandin ratio. The concentration of leukotriene B(4)increased during the urgent adaptation period, which correlated with a decrease in elastic properties of erythrocytes. In vitro passage of erythrocytes through a 2.5-micro capillary sieve sharply decreased, which played an important role in the progression of myocardial and cerebral hypoxia and ischemia. Myocardial necrosis and endothelial dysfunction developed 24 h later aggravate these pathological shifts.

Background: Polyphenolic phytochemicals inhibit vascular and inflammatory processes that contribute to disease. These effects are hypothesized to result from polyphenol-mediated alterations in cellular eicosanoid synthesis., Objective: The objective was to determine and compare the ability of cocoa procyanidins to alter eicosanoid synthesis in human subjects and cultured human aortic endothelial cells., Design: After an overnight fast, 10 healthy subjects (4 men and 6 women) consumed 37 g low-procyanidin (0.09 mg/g) and high-procyanidin (4.0 mg/g) chocolate; the treatments were separated by 1 wk. The investigation had a randomized, blinded, crossover design. Plasma samples were collected before treatment and 2 and 6 h after treatment. Eicosanoids were quantitated by enzyme immunoassay. Endothelial cells were treated in vitro with procyanidins to determine whether the effects of procyanidin in vivo were associated with procyanidin-induced alterations in endothelial cell eicosanoid synthesis., Results: Relative to the effects of the low-procyanidin chocolate, high-procyanidin chocolate induced increases in plasma prostacyclin (32%; P<0.05) and decreases in plasma leukotrienes (29%; P<0.04). After the in vitro procyanidin treatments, aortic endothelial cells synthesized twice as much 6-keto-prostaglandin F(1alpha) (P<0.01) and 16% less leukotriene (P<0.05) as did control cells. The in vitro and in vivo effects of procyanidins on plasma leukotriene-prostacyclin ratios in culture medium were also comparable: decreases of 58% and 52%, respectively., Conclusion: Data from this short-term investigation support the concept that certain food-derived flavonoids can favorably alter eicosanoid synthesis in humans, providing a plausible hypothesis for a mechanism by which they can decrease platelet activation in humans.

Objective: To compare the evolution of plasma concentrations of leukotriene (LT) B4, LTC4, LTD4, and interleukin (IL)-8 in patients with acute respiratory distress syndrome (ARDS) and in patients at risk of ARDS and to assess the value of these mediators in predicting mortality rate from ARDS., Design: A case-control study comparing ARDS patients and patients at risk of ARDS as well as survivors and nonsurvivors with ARDS., Setting: Hospital intensive care unit, laboratory, and department of hematology., Patients: Twenty-one patients with ARDS and 14 patients at risk of ARDS., Intervention: Arterial blood samples were collected on days 0, 1, and 5 after admission to the intensive care unit., Measurements and Main Results: LTs were extracted, separated by high-pressure liquid chromatography and quantified by enzyme immunoassay. IL-8 was analyzed by ELISA. Plasma concentrations of LTB4 and LTC4 plus LTD4 were significantly higher in ARDS patients than in patients at risk of ARDS during the first 24 hrs. Concentrations of IL-8 were also higher in ARDS patients than in patients at risk throughout the study, although the differences between the two groups were only significant on day 5. Only the plasma concentration of LTB4 on day 1 was a marker of ARDS (72.2% sensitivity, 84.6% specificity). A logistic regression analysis showed that LTB4 and IL-8, on day 1, were markers of mortality rate in patients with ARDS (70.0% sensitivity, 87.5% specificity)., Conclusions: LTs are elevated during the early phases of ARDS, whereas IL-8 increases throughout the study. The evaluation of LTB4 and IL-8 may be useful prognostic indices in patients with early phase ARDS after admission to the intensive care unit.

Background: The cysteinyl-leukotrienes (LTC(4), LTD(4), LTE(4)) are critical bronchoconstrictor and eosinophilotactic mediators in asthma while LTB(4) is a potent neutrophil chemoattractant. Glucocorticosteroids are front line anti-inflammatory treatment for asthma but the evidence that they reduce leukotriene (LT) synthesis in vivo is poor., Methods: In a randomised, double blind, placebo controlled, crossover trial immunoassays were used to measure ex vivo synthesis of LTC(4) and LTB(4) by calcium ionophore stimulated blood leucocytes and bronchoalveolar lavage (BAL) cells of eight normal subjects and eight patients with mild allergic asthma 4-6 hours after intravenous administration of a single 100 mg dose of methylprednisolone., Results: Ionophore stimulated synthesis of LTC(4) (but not LTB(4)) in blood granulocytes tended to be higher in asthmatic subjects (mean 9.7 ng/10(6) cells) than in normal subjects (4.2 ng/10(6) cells; p = 0.08) and intravenous methylprednisolone reduced synthesis of LTC(4) (but not LTB(4)) to normal levels (2.9 ng/10(6) cells; 95% CI for the reduction 1.0 to 12.5 ng/10(6) cells; p = 0.03). In blood mononuclear cells methylprednisolone reduced LTC(4) synthesis in asthmatic subjects from 1.26 to 0.79 ng/10(6) cells (95% CI for the reduction 0.26 to 0.79, p = 0.014) and tended to reduce LTC(4) synthesis in normal subjects from 1.51 to 0.86 ng/10(6) cells (p = 0.08). Methylprednisolone also significantly reduced synthesis of LTB(4) in mononuclear cells from both subject groups (p = 0.014). It had no effect on LT synthesis in BAL cells from either group nor on LT levels in BAL fluid., Conclusions: Intravenous methylprednisolone can reduce synthesis of leukotrienes in blood granulocytes and mononuclear cells within six hours of a single intravenous dose.

Both tachykinins and leukotrienes (LTs) have been demonstrated to be the mediators for hyperpnea-induced bronchoconstriction (HIB) of guinea pigs. We tested the hypothesis that leukotrienes modulate HIB indirectly by triggering tachykinin release. Ninety nine young guinea pigs were divided into four groups: control; LTC4; FPL 55712 (a LT receptor antagonist); and MK-886 (an inhibitor of LT synthesis). Each animal was anesthetized, cannulated, paralyzed, and artificially ventilated. The protocol included the baseline, hyperpnea, and recovery periods. Thus, animals in each group were further divided into three subgroups: baseline; recovery-3 min; and recovery-8 min. We measured dynamic respiratory compliance (Crs), forced expiratory volume in 0.1 s (FEV0.1) and maximal expiratory flow at 30% total lung capacity (Vmax30), as well as determined substance P (SP) and LT levels in plasma and bronchoalveolar lavage (BAL) during either the baseline or the recovery (3 or 8 min) period. Hyperpnea caused decreases in Crs, FEV0.1 and Vmax30, indicating HIB, in the control group at 3 min and 8 min of the recovery period. Both FPL 55712 and MK-886 significantly attenuated HIB. In the control group, hyperpnea caused significant increases in SP and LT levels in both plasma and BAL. These increases in SP levels were significantly suppressed, however, by FPL 55712 and MK-886. Compared to the control group, infusion of LTC4 did not significantly alter either HIB, SP or LT levels in most cases. An additional group of 24 animals treated with neurokinin-2 receptor antagonist, SR 48968, demonstrated that SR 48968 significantly suppressed hyperpnea-induced increases in plasma, but not in BAL, LT levels. Since FPL 55712 and MK-886 first suppress LT activities, these results suggest that suppressed LT activities attenuate HIB indirectly via reducing tachykinin release.

Leukotrienes (LTs) are cell-membrane derived lipid inflammatory mediators, synthesized and eliminated by the liver. LTs have effects on liver cells in some pathological conditions. In this study, we measured plasma endogenous and liberated leukotriene (LT) concentration in peripheral blood leukocytes stimulated in vitro by the calcium ionophore (CaA23187) and platelet-activating factor (PAF). Production of LTs was measured in type A (n=37) and type B (n=10) acute hepatitis patients and control subjects (n=10). LTs levels were measured by high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The concentration of LTB4 measured in plasma and stimulated peripheral blood leukocyte supernatants of children with hepatitis A infection was found to be statistically elevated and in positive correlation with serum alanine aminotransferase (ALT) levels. In plasma samples of hepatitis B patients, LTC4 and LTE4 were measured in significantly elevated concentrations. These results suggest that LTB4 may be a critical mediator of hepatitis A virus-induced hepatocellular injury.

A reversed-phase high-performance liquid chromatographic method with gradient elution for the separation of the mediator substances histamine and the leukotrienes C4 (LTC4), D4 (LTD4), and E4 (LTE4) is described. The detection occurs fluorimetrically after automated precolumn derivatization with o-phthaldialdehyde. All components are chromatographically separable. Because of the different excitation and emission wavelengths, only the most important biological active mediators histamine and LTC4 are determinable in one parallel chromatographic run. The method is examined by linearity and precision tests and is applicable to biological sample matrices like cell supernatants of human basophils enriched by Percoll-density gradient centrifugation and stimulated for mediator release by anti-IgE. The established method is suitable to separate the mediators from other matrix components. The determination limit for histamine is 55.0 micrograms/L and that for LTC4 16.0 micrograms/L, referring to the reference solutions. Therefore, a fast, economical method for the common determination of the most important mediators histamine and LTC4 is established. This method is also suitable for high sample amounts in routine medical analysis.

Background: The stimulation of blood basophils to release mediators in vitro is widely used for diagnosis of allergic diseases. Tryptase release and sulphidopeptideleukotriene production are both triggered by cross-bridging of adjacent IgE molecules on the surface of IgE-bearing basophils., Objective: We have compared the sensitivity of tryptase release test (TRT) and cellular allergen stimulation test (CAST) which, respectively, measure tryptase and sulphidopeptideleukotrienes that are produced upon cell stimulation by mite extracts., Methods: Blood was taken from 247 patients with allergy to mites and 137 non-allergic control subjects. We measured tryptase release from basophils after allergen challenge in vitro by sandwich radioimmunoassay. The sulphidopeptideleukotrienes production was quantified by an ELISA test based on a monoclonal antibody which recognized leukotriene T4 (LTC4) and its metabolites LTD4 and LTE4., Results: Our data show that both methods are equally effective to distinguish allergic patients from normal controls (P > 0.0001). There was a significant correlation between mite-specific serum IgE and CAST results (r = 0.69 for Dermatophagoides pteronyssinus; r = 0.73 for Dermatophagoides farinae). Correlations between IgE against mites and tryptase values appeared rather poor (r = 0.47 for Dermatophagoides pteronyssinus; 0.49 for Dermatophagoides farinae). Moreover, the data were used for the calculation of sensitivity, specificity, prevalence, and overall efficiency (Roc/Galen & Gambino analysis). The results were as follows: 71%, 87%, 64%, 76% (CAST results for Dermatophagoides farinae); 64%, 78%, 53%, 70% (TRT results for Dermatophagoides farinae)., Conclusion: The partial discrepancies observed could be interpreted as a consequence of conditions that were technically not optimal. False-positive results may be due to the action of some non-specific cytotoxic agent, false-negative results may be due to hyporesponsive basophils or the low number of cells participating in the reaction and finally, in the case of TRT, to G4 monoclonal antibody to tryptase employed.

Recent evidence suggests that generation of hydroxyl radicals in the presence of lipid membranes can lead to oxidation of arachidonic acid esterified to glycerophospholipids and the production of compounds isomeric to prostaglandins, thromboxanes, and leukotrienes. Liquid chromatography tandem mass spectrometry and multiple reaction monitoring were employed to quantitate the production of 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE), and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) in red blood cells ghosts treated with t-butylhydroperoxide (tBuOOH). Untreated red blood cell ghosts were found to contain low, but measurable quantities of these three 5-oxygenated eicosanoids as phospholipid esters. Following treatment, there was approximately a 53- and 22.5-fold increase in 5-HETE and 5-HPETE, respectively, and an 8.5-fold increase in 5-oxo-ETE. The formation of these compounds was inhibited nearly 90% by the antioxidants butylated hydroxytoluene, ascorbic acid, and resveratrol providing further evidence for free radical mediated oxidation of arachidonic acid. This analytical protocol provided sufficient sensitivity for detection of these compounds in studies in which previous analysis by high-pressure liquid chromatography with UV detection failed to detect their presence. These results reveal that the biologically active eicosanoids 5-HPETE, 5-HPETE, and 5-oxo-ETE are formed esterified to phospholipids following exposure of cellular membranes to reactive oxygen species and free radicals in a model system where intracellular antioxidant mechanisms were depleted.

Specific immunotherapy has been successfully used in the treatment of allergic diseases for years. In this study, we examined the clinical efficacy of specific immunotherapy and its effect on cysteinyl leukotriene releasability by blood leukocytes in house dust mite allergic subjects with asthma and allergic rhinitis. In an open, parallel study, 13 subjects were treated with specific immunotherapy for 12 months and five patients served as the control group. Before specific immunotherapy treatment, and 1 and 12 months after, patients were evaluated with respect to total immunoglobulin (Ig) E levels, Dermatophagoides pteronyssinus specific IgE levels, symptom scores, usage of rescue medicines, lung function tests, nasal challenge scores, skin reactivity to D. pteronyssinus and cysteinyl leukotriene releasability by blood leukocytes. At the 12th month of specific immunotherapy, total asthma symptom scores, bronchodilator requirement, number of sneezes following nasal challenge and immediate skin reactivity to D. pteronyssinus was decreased, while FEV1 increased compared to the pretreatment values (p < 0.05 for each). These parameters remained unchanged in the control group. In vitro cysteinyl leukotriene releasability by blood leukocytes in response to D. pteronyssinus antigen and anti-IgE antibody remained unchanged both in the specific immunotherapy group and the control group. However, in the treated group, there were six patients who showed at least a 50% reduction in their symptoms after specific immunotherapy. In five of these, cysteinyl leukotriene release induced by both HACM buffer and D. pteronyssinus were decreased by more than 50% of the baseline values. The other patient demonstrated only a marked decrease (more than 50%) in background. Our results suggest that specific immunotherapy is effective in the treatment of allergic rhinitis and bronchial asthma due to house dust mite allergy and that the clinical response to specific immunotherapy may be associated with decreased cysteinyl leukotriene releasibility by blood leukocytes in some patients.

Bacterial endotoxins are seen to possess strong proinflammatory activities. These substances may intensify inflammation in the airways of patients with chronic obstructive pulmonary disease (COPD) and asthma by facilitating release of various mediators from different types of cells. Sulfidoleukotrienes (sLT) cause bronchoconstriction, increase vascular permeability and stimulate mucous secretion. The aim of our study was to evaluate sLT release from peripheral blood leukocytes stimulated by Klebsiella pneumoniae lipopolysaccharide (LPS) and obtained from COPD and asthma patients. Nineteen subjects with mild or moderate stable bronchial asthma, nine patients with COPD and 10 healthy controls entered the study. Cellular allergen stimulation test (CAST)-ELISA test was performed using Bühlmann Laboratories AG kits to determine sLT production. The differences between atopic (462.57 SD = 215.89 pg/ml) and nonatopic (474.25 SD = 158.02 pg/ml) asthmatics in comparison to healthy controls (191.55 SD = 53.2 pg/ml) were statistically significant (p < 0.005) upon LPS stimulation at the concentration of 10 micrograms/ml. At lower LPS concentration (1 microgram/ml) the difference was statistically significant only between nonatopic asthmatics and healthy subjects (p < 0.02). In the COPD group the sLT production in either LPS concentration was higher than in the controls but the difference was not significant. We suppose that leukocytes obtained from asthmatics and COPD patients are more susceptible to LPS than these cells from healthy individuals. An increased sLT production upon LPS stimulation during respiratory bacterial infection may intensify inflammation, bronchoconstriction and increase nonspecific bronchial hyperreactivity.

Pentobarbital-anesthetized dogs were instrumented for hemodynamic measurements and cooled by heat exchange tubes. Through a catheter in the coronary sinus, blood samples were obtained and plasma leukotrienes measured using a high-pressure liquid chromatography technique. Hemodynamic function was significantly reduced during cooling, and during subsequent rewarming hemodynamic function was only partly restored. Leukotriene C4 and B4 were significantly increased at a body core temperature of 31 degrees C and 25 degrees C during cooling but not during rewarming (28 degrees C) and after rewarming (37 degrees C). This indicates that during decreasing body temperature elevated leukotriene levels may increase vascular permeability, inflammation, and vasoactivity and counteract temperature-dependent decrease in these functions. Disturbed microcirculation may thus diminish cardiac recovery during rewarming.

Estrogens have a beneficial effect on atherosclerosis and osteoporosis after menopause, but their exact mechanism of action is still unknown. The aim of the present study was to investigate the effects of estradiol and its metabolites catechol estrogens on arachidonic acid metabolism in vitro. Estradiol had no effect on arachidonic acid metabolism up to 33 microM in A23187-stimulated human whole blood. All catechol estrogens (2-hydroxyestradiol, 2-hydroxyestrone, 4-hydroxyestradiol and 4-hydroxyestrone) had similar kinds of actions on arachidonic acid metabolism, being over ten times more potent inhibitors of leukotriene synthesis (IC50 values 0.044-0.16 microM) than thromboxane (IC50 values 0.99-2.1 microM) and prostaglandin E2 synthesis (IC50 values 0.84-5.5 microM). It is suggested that some of the protective actions of estrogens--e.g., on atherosclerosis and osteoporosis--may be related to the inhibition of leukotriene synthesis by catechol estrogens.

1 Leukotrienes constitute a class of potent bioactive mediators known to play a pivotal role in inflammation. Since their biosynthesis has been shown to be enhanced by platelet-neutrophil interactions, leukotrienes may be involved in these interactions and the arterial response to injury. Therefore, we investigated the effects of the selective leukotriene biosynthesis inhibitor 3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2- dimethylpropanoic acid (MK-886) on the acute thrombotic and vasomotor responses after arterial injury by angioplasty. 2 Carotid arterial injury was produced by balloon dilatation in control (molecusol vehicle; n=10) and treated (MK-886, 10 mg kg(-1), i.v.; n=9) pigs. The acute thrombotic reaction following deep arterial wall injury was quantified with 51Cr labelled platelets and 111In labelled neutrophils, and the vasomotor response was determined angiographically. 3 Platelet deposition at the site of deep arterial wall injury averaged 56.4+/-11.0x10(6) platelets cm(-2) in the control group, and was significantly reduced to 18.2+/-3.8x10(6) platelets cm(-2) (P<0.005) by treatment with MK-886. Neutrophil deposition was also decreased by MK-886, from 242.8+/-36.8 to 120.9+/-20.3x10(3) neutrophils cm(-2) (P<0.01). MK-886-treated animals had a significant decrease in the postangioplasty vasoconstrictive response at the site of endothelial injury distally, from 37.5+/-3.1% in the control group to 13.5+/-2.5% (P<0.001). 4 The effects of MK-886 were associated with a profound inhibition of ex vivo leukotriene B4 (LTB4) synthesis in blood stimulated by the calcium ionophore A23187 and a significant reduction of neutrophil aggregation in whole blood (P<0.01), whereas neutrophil superoxide anion production, serum thromboxane B2 and platelet aggregation in whole blood were not influenced. 5 The relevant effects of MK-886 are primarily related to inhibition of neutrophil function and suggest an important modulatory role for leukotrienes in the pathophysiological response associated with platelet and neutrophil interactions following arterial injury in vivo.

Cross-linking IgE on basophils is known to cause both sulfidoleukotriene (sLT) generation and histamine release. We recently developed an ELISA to determine sulfidoleukotriene generation by blood mononuclear cells which employs pretreatment with IL-3 to enhance leukotriene generation (cellular antigen stimulation test, CAST). Here, we compared the CAST and whole blood histamine release in response to honey bee/yellow jacket venom (BV/YJV) in 23 patients clinically suspected of type-I allergy to these venoms. Of these, 17 were diagnosed as "definitive venom allergics," defined by a positive skin test at 100 ng/ml of venom or less. The six in whom such skin reactivity was absent were labelled "suspected venom allergics." Both venoms stimulated sulfidoleukotriene generation and histamine release also from control individuals (n = 10). In patients, insect venoms generally stimulated histamine release and sulfidoleukotriene generation in excess of the mean + 3 SD of values obtained with control individuals. However, about half of the patients reacted predominantly with either histamine release or sulfidoleukotriene generation. No overall correlation was found between threshold doses necessary to stimulate sulfidoleukotriene generation (ThsLT) and histamine release (ThHist). (Linear correlation coefficients between ThsLT and ThHist were -0.02 for honey bee venom and 0.13 for yellow jacket, n = 23). Both findings are in contrast to the concept that these responses occur in parallel. From results with "definitive venom allergics," CAST sensitivity was calculated as 100% for honey bee venom and 83% for yellow jacket, and that of the histamine release assay as 62.5% for honey bee venom and 50% for yellow jacket. Specificity of the CAST was calculated as 77% for honey bee venom and 100% for yellow jacket, and that of the histamine release assay as 44% for honey bee venom and 60% for yellow jacket. Thus, CAST results are closer to skin test results than to those of the whole blood histamine release assay.

In vitro cysteinyl leukotriene (cLT) release from blood leukocytes was measured in eight normal individuals (NI), nine patients with nasal polyps (NP) without aspirin intolerance, and eight patients with NP, asthma, and aspirin intolerance (AI). Blood leukocytes were prestimulated with interleukin-3 (IL-3) and incubated with acetylsalicylic acid (ASA) (10 and 100 micrograms/ml) together with C5a (10(-8) mol/l) for 18 h. cLT release (LTC4, LTD4 and LTE4) from blood leukocytes was measured with a competitive enzyme-linked immunoassay. Background cLT release was 259 +/- 66 pg/ml (mean +/- SEM) in the NI group, 185 +/- 33 pg/ml in the NP group, and 578 +/- 136 pg/ml in the AI group (P = 0.1). After incubation with 10 micrograms/ml ASA, cLT concentration was lower in normal subjects (346 +/- 72 pg/ml) and in patients with NP (209 +/- 53) than in patients with AI (686 +/- 75 pg/ml, P = 0.002). After incubation with 100 micrograms/ml ASA, cLT concentrations were 285 +/- 72 pg/ml in the NI group, 313 +/- 77 pg/ml in the NP group, and 654 +/- 121 pg/ml in the AI group (P = 0.04), respectively. Simultaneous incubation with ASA 10 micrograms/ml and C5a (10(-8)mol/l) resulted in a cLT concentration of 751 +/- 171 pg/ml in the NI group, 343 +/- 102 pg/ml in the NP group, and 2196 +/- 480 pg/ml in patients with AI (P = 0.0006), whereas simultaneous incubation with ASA 100 g/ml and C5a (10(-8)mol/l) resulted in 268 +/- 51 pg/ml in the NI group, 412 +/- 97 pg/ml in the NP group, and 1701 +/- 368 pg/ml in the AI group (P = 0.005). In patients with AI, cLT release from blood leukocytes is altered when compared with normals and patients with NP. The presented cLT-release assay could be of potential use in the in vitro diagnosis of AI.

This paper summarizes methodological aspects of measurements of eicosanoids in biological samples and describes some applications of such methods in studies on leukotriene formation in the human airways and the effects of drugs interfering with these compounds in asthmatic subjects. For estimations of in vivo production of eicosanoids, major, stable metabolites were selected for analysis in biological fluids. An enzyme immunoassay for LTE4 was validated for use in unextracted urine samples. To monitor thromboxane production, a radioimmunoassay for 11-dehydro-TXB2 was developed and used for measurements in samples of human plasma and urine. In vitro production of leukotrienes in chopped human lung was measured by UV-spectroscopy after extraction and separation on RP-HPLC. Corrections for losses during purification were performed with individually selected internal standards.

The influence of parenteral L-alanyl-L-glutamine dipeptide on the cysteinyl-leukotriene (cys-LT) synthesizing capacity from neutrophils was studied in patients undergoing colonic surgery. The decrease in cys-LT, observed postoperatively, could be normalized with parenteral glutamine, while the cys-LT decrease persisted in controls. We conclude that the provision of glutamine in the postoperative state improves normalization of neutrophil functions (e.g., generation of cys-LT), which is an essential prerequisite for host defences.

The present study was carried out for the purpose of measuring platelet activating factor (PAF) and leukotrienes (LTs) in middle ear fluid (MEF) or in otorrhea of children with acute otitis media (AOM), with secretory otitis media (SOM), and with chronic otitis media (COM) on the acute exacerbation. PAF, LTC4, and LTD4+LTE4 concentrations were measured by radioimmunoassay in purulent MEFs obtained from 15 ears of 15 children with AOM, in mucoid or gluey MEFs from 16 ears of 15 children with SOM, and in purulent otorrhea from 9 ears of 9 children with COM on the acute exacerbation. PAF concentrations were 25.4 +/- 9.0 ng/mg total phospholipid (TPL) in AOM, 4.9 +/- 1.9 ng/mg TPL in SOM, 20.7 +/- 15.7 ng/mg TPL in COM, demonstrating significant differences between AOM and SOM (p < 0.05) and between COM and SOM (p < 0.01). LTC4 concentrations were 92.9 +/- 73.8 pg/mg total protein (TP) in AOM, 52.0 +/- 42.5 pg/mg TP in SOM, and 28.5 +/- 11.2 pg/mg TP in COM. LTD4+LTE4 concentrations were 326.5 +/- 177.0 pg/mg TP in AOM, 288.2 +/- 144.6 pg/mg TP in SOM, and 94.0 +/- 58.1 pg/mg TP in COM, demonstrating significant differences between AOM and COM and between SOM and COM (p < 0.01). The results obtained indicated that PAF was involved in the onset of AOM and COM on the acute exacerbation, and that LTs played an important role in SOM.

Collision-induced dissociation (CID) mass spectra were recorded on underivatized, hydroxylated lipoxygenase products, including S(S)-, 12(S)- and 15(S)-hydroxyeicosatetraenoic adds (HETE), leukotriene (LT) B4, LTB5, the LTB4 metabolites 20-OH-LTB4, 20-COOH-LTB4 and 10,11-dihydro-LTB4, the LTB4 isomers 6-trans-LTB4, 6-trans,12-epi-LTB4 and S(S),12(S)-dihydroxyeicosatetraenoic acid (diHETE) as well as S(S),6(R)-diHETE, 14(S), 15(R)-diHETE and lipoxin (LX) A4 and B4. Pseudomolecular [M-H]- parent ions were generated by electrospray ionization. The ions were accelerated to 4 keV, selected by the double focusing sectors Of 2 hybrid tandem mass spectrometer, decelerated to 400 eV and focused into a collision cell containing xenon. The resulting fragment ions were mass analysed by an orthogonal time-of-flight analyser. The CID spectra obtained were structurally informative allowing isomer differentiation.

Nucleated bullfrog erythrocytes have 5-lipoxygenase (LO) and are the first non-mammalian cell to exhibit endogenous sulfidopeptide leukotriene (LT) synthesis. Non-nucleated mammalian platelets lack 5-LO, but contribute significantly to LTC4 production by transcellular synthesis. However, nucleated bullfrog thrombocytes have not been examined for 5-LO activity. Endogenous leukotriene synthesis by bullfrog thrombocytes and mixed leukocytes was analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC). Calcium ionophore activated (A23187) leukocytes demonstrated 5-LO, 12-LO, and 15-LO activity. Spectral analysis demonstrated synthesis of LTB4, LTB4 isomers, 15(S)-monohydroxyicosatetraenoic acid (HETE), 5(S),12(S)-diHETE, 5(S),15(S)-di-HETE, lipoxin A4 (LXA4) and LXB4. Thrombocytes synthesized large quantities of sulfidopeptide leukotrienes but no lipoxins. Sulfidopeptide leukotriene and LTB4 radioimmunoassay analysis and the radiological RP-HPLC profile of [3H]AA metabolism further confirmed synthesis. Incubations with [3H]LTC4 demonstrated slow and incomplete conversion to [3H]LTD4. Thrombocyte leukotriene profile changed over time revealing a significant shift from the LTC4 synthase to LTA4 hydrolase pathway, corresponding with release of large amounts of LTA4. Thrombocytes potentially play a pivotal role in inflammatory and cardiovascular responses. 5-LO activity in amphibian homologs to mammalian platelets and erythrocytes compared with the lack of activity in the mammalian counterparts may correspond to the loss of the nucleus in the evolution of these cells.