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Leukotriene B4 (LTB4) is a potent mediator of inflammation and plays a key function in the pathophysiology of chronic asthma. Detectable urinary levels of LTB4, arises from the activation of leukotriene pathways. In this study an ultra-fast, selective and sensitive analytical method based on semi-automatic microextraction by packed sorbents (MEPS) technique, using a new digitally controlled syringe (eVol®) combined with ultra-high pressure liquid chromatography (UHPLC), is proposed for the measurement of urinary LTB4 (U-LTB4) levels in a group of asthmatic patients (APs) and healthy controls (CTRL). Important parameters affecting MEPS performance, namely sorbent type, number of extraction cycles (extract-discard) and elution volume, were evaluated. The optimal experimental conditions among those investigated for the quantification of U-LTB4 in urine samples were as follows: porous graphitic carbon sorbent (PGC), 10 extractions cycle (10×250 μL of sample) and LTB4 elution with 100 μL of acetonitrile. The UHPLC optimum conditions resulted in a mobile phase consisting of 95% (v/v) of acid aqueous solution (v/v), and acetonitrile 5% (v/v); flow rate of 500 µL/min, and a column temperature of 37±0.1 °C. Under optimized conditions the proposed method exhibit good selectivity and sensitivity LOD (0.37 ng/mL) and LOQ (1.22 ng/mL). The recovery ranging from 86.4 to 101.1% for LTB4, with relative standard deviations (% RSD) no larger than 5%. In addition, the method also afforded good results in terms of linearity (r(2)>0.995) within the established concentration range, with a residual deviation for each calibration point below 6%, and intra- and inter-day repeatability in urine samples with RSD values lower than 4 and 5%, respectively. The application of the method to urine samples revealed a tendency towards the increased urinary LTB4 levels in APs (5.42±0.17 ng/mL) when compared to those of CTRL group (from ND to 1.9 ng/mL). Urinary measurement of LTB4 may be an interesting and non-invasive option to assess control of asthma., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Aim: To assess the pharmacokinetics, pharmacodynamics, safety and tolerability of the 5-lipoxygenase-activating protein inhibitor, GSK2190915, after oral dosing in two independent phase I studies, one in Western European and one in Japanese subjects, utilizing different formulations., Method: Western European subjects received single (50-1000 mg) or multiple (10-450 mg) oral doses of GSK2190915 or placebo in a dose-escalating manner. Japanese subjects received three of four GSK2190915 doses (10-200 mg) plus placebo once in a four period crossover design. Blood samples were collected for GSK2190915 concentrations and blood and urine were collected to measure leukotriene B₄ and leukotriene E₄, respectively, as pharmacodynamic markers of drug activity., Results: There was no clear difference in adverse events between placebo and active drug-treated subjects in either study. Maximum plasma concentrations of GSK2190915 and area under the curve increased in a dose-related manner and mean half-life values ranged from 16-34 h. Dose-dependent inhibition of blood leukotriene B₄ production was observed and near complete inhibition of urinary leukotriene E₄ excretion was shown at all doses except the lowest dose. The EC₅₀ values for inhibition of LTB₄ were 85 nM and 89 nM in the Western European and Japanese studies, respectively., Conclusion: GSK2190915 is well-tolerated with pharmacokinetics and pharmacodynamics in Western European and Japanese subjects that support once daily dosing for 24 h inhibition of leukotrienes. Doses of ≥50 mg show near complete inhibition of urinary leukotriene E₄ at 24 h post-dose, whereas doses of ≥150 mg are required for 24 h inhibition of blood LTB₄., (© 2012 GlaxoSmithKline. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.)

Acute transplant rejection is the leading cause of graft loss in the first months after kidney transplantation. Lipoxygenase products mediate pro- and anti-inflammatory actions and thus we aimed to correlate the histological reports of renal transplant biopsies with urinary lipoxygenase products concentrations to evaluate their role as a diagnostic marker. This study included a total of 34 kidney transplant recipients: 17 with an acute transplant rejection and 17 controls. LTE4, LTB4, 12-HETE and 15-HETE concentrations were measured by enzyme immunoassay. Urinary lipoxygenase product concentrations were not significantly changed during an acute allograft rejection. Nevertheless, LTB4 concentrations correlated significantly with the body temperature (P ≤ 0.05) 3 months after transplantation, and 12- and 15-HETE concentrations correlated significantly with renal function (P ≤ 0.05) 2 weeks after transplantation. In conclusion, our data show a correlation for LTB4 with the body temperature 3 months after transplantation and urinary 12- and 15-HETE concentrations correlate positively with elevated serum creatinine concentrations but do not predict acute allograft rejection.

Background: GSK2190915, a potent 5-lipoxygenase-activating protein inhibitor, prevents the synthesis of leukotrienes and 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE)., Objective: To assess the effect of GSK2190915 on the allergen-induced asthmatic responses., Methods: Nineteen eligible male subjects with mild asthma were enrolled in and completed this four-centre, double-blind, two-way crossover study (ClinicalTrials.gov NCT00748306). Subjects took GSK2190915 100 mg and placebo orally once daily for 5 days in randomized order. On Day 1 and 4 they had a methacholine challenge, on Day 3 they had an inhaled allergen challenge, and on Days 4 and 6 they had sputum induction., Results: GSK2190915 attenuated the early (0-2 h) and late (4-10 h) asthmatic responses to inhaled allergen compared with placebo. There was a statistically significant attenuation of the early asthmatic response (EAR) by GSK2190915; treatment difference of GSK2190915 vs. placebo for the minimum FEV(1) EAR was 0.408 L (0.205, 0.611). There was a statistically significant attenuation of the late asthmatic response (LAR) by GSK2190915; the treatment difference of GSK2190915 vs. placebo for the minimum FEV(1) LAR was 0.229 L (0.041, 0.417). There was a statistically significant attenuation of allergen-induced sputum eosinophil count on Day 4 following GSK2190915: mean treatment difference (95% CI) between GSK2190915 and placebo was -9.95% (-18.15%, -1.77%). Compared with placebo, GSK2190915 100 mg reduced median sputum LTB(4) by > 90% on Days 4 and 6. There was no effect on methacholine PC(20) post allergen. GSK2190915 was generally well tolerated., Conclusion and Clinical Relevance: GSK2190915 shows potential as a treatment for patients with asthma., (© 2012 Blackwell Publishing Ltd.)

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.

Clinical symptoms of patients with mastocytosis may include skin reactions, but also gastrointestinal symptoms with hyperacidity and dysmotility (e.g. ulcer, diarrhea, pain). They are mostly caused by mediators derived from activated mast cells. In order to investigate the impact of leukotrienes on the clinical symptoms excretion of leukotriene B4 (LTB4) and leukotrienes C4-D4-E4 (cysteinyl-leukotrienes) into urine was studied in 9 patients with indolent systemic mastocytosis divided into a group with high and low intensity of symptoms and in 11 healthy volunteers. Leukotriene excretion was determined by ELISA and correlated with methylhistamine excretion. Patients with systemic mastocytosis with high and low intense symptoms showed significantly higher urinary excretion of cysteinyl-leukotrienes than controls. There was a positive correlation of cysteinyl-leukotriene excretion and urinary methylhistamine excretion. LTB4 excretion was also significantly increased in patients with systemic mastocytosis compared to healthy volunteers. No correlation of urinary LTB4 excretion with urinary methylhistamine was observed. The present study demonstrates that urinary excretion of LTB4 and cysteinyl-leukotrienes LTC4-D4-E4 is clearly enhanced in indolent systemic mastocytosis Hence, determination of leukotriene excretion into urine can be used as a tool in the diagnostic and in the therapeutic monitoring of systemic mastocytosis.

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.)

Aim: Glomerular infiltration of macrophages is a characteristic alteration of renal pathology in hyperlipidaemic renal injury. Leukotriene B4 (LTB4) is a bioactive eicosanoid and macrophage and has two key enzymes for LTB4 synthesis, 5-lipoxygenase and leukotriene A4 (LTA4) hydrolase. The purpose of this study was to evaluate the role of LTB4 in accelerated hyperlipidaemic renal injury., Methods: To induce accelerated hyperlipidaemic renal injury, 8 week old male spontaneously hypercholesterolaemic (SHC) rats were fed with a high cholesterol diet for 6 weeks. LTA4 hydrolase activities in the kidney and urine LTB4 levels were examined. The effects of LTB4 antagonist (ONO-4057) were also evaluated., Results: Urinary protein and LTB4 excretion was increased by a high cholesterol diet for 6 weeks. The scores of glomerular foam cell accumulation and sclerosis, numbers of infiltrated macrophages in glomeruli and interstitial area, LTA4 hydrolase activity in renal cortex were higher in the high cholesterol diet group than the normal diet group. LTB4 antagonist treatment reduced urinary protein and LTA4 activity and attenuated renal pathological changes., Conclusion: These results suggest that LTB4 directly contributes to accelerated hyperlipidaemic renal injury and the therapeutic potential of LTB4 antagonist for renal damage induced by hyperlipidaemia., (© 2011 The Authors. Nephrology © 2011 Asian Pacific Society of Nephrology.)

The pathogenesis of Henoch-Schönlein purpura (HSP) is not clearly understood. It remains unclear how changes of lipoxin A(4) (LXA(4)) that acts as a "braking signal" in inflammatory process occur in patients with HSP. In this study, we determined the temporal changes of blood and urinary LXA(4), Leukotriene (LT)B(4) and urinary LTE(4) in 49 children with HSP. Inverse temporal changes between gradually increased blood and urinary LXA(4) and gradually decreased blood and urinary LTB(4) and urinary LTE(4) were found in patients with HSP. Furthermore, both 15-S-hydroxyeicosatetraenoic acid and LXA(4) inhibited the LTB(4)-induced chemotaxis of leukocytes and release of LTB(4) from leukocytes obtained from the patients in the active phase of HSP. In 22 children with HSP nephritis, concordant with the gradually increased severity of mesangial proliferation and proteinuria, the glomerular expressions of 15-lipoxygenase and the levels of urinary LXA(4) gradually decreased and the glomerular expressions of LTC(4) synthase and the urinary LTE(4) and LTB(4) gradually increased. The levels of blood and urinary LXA(4) in patients with HSP nephritis were lower than those in patients with purpura alone in early resolution of HSP. The levels of blood and urinary LTB(4) and urinary LTE(4) in the patients with HSP nephritis were higher than those in patients with purpura alone in early resolution of HSP. There was positive correlation between blood LTB(4) and serum C-reactive protein in 49 children with HSP. These data suggest that LTs may play a proinflammatory and profibrotic role in the pathogenesis of HSP, and insufficiency of LXA(4) may be responsible for the patients with HSP whose illness become more serious.

Cysteinyl-leukotrienes are important pro-inflammatory mediators in bronchial asthma (BA) and are derived from arachidonic acid by the action of 5-lipoxygenase. We identified a novel polymorphism, c.760 G>A (E254K), in exon 6 of the 5-lipoxygenase gene (5-LO). This substitution was detected in 11 out of 180 patients with BA, but not in any of the 150 non-allergic subjects. The frequency of c.760 G>A showed a significant difference between BA and non-allergic subjects (P=0.0007). The c.760 G>A polymorphism existed at the surface edge of the C-terminal catalytic domain, and the E-to-K substitution changed the charge of the side chain from negative to positive. Thus, our results suggest that E254K in the 5-LO might be associated with BA.

We have developed a method for measuring leukotriene B4 glucuronide, a marker of systemic leukotriene B4 biosynthesis, in human urine. This method involves the separation of two positional isomers of leukotriene B4 glucuronide by high-performance liquid chromatography, followed by hydrolysis with beta-glucuronidase and then leukotriene B4 quantification by enzyme immunoassay after purification by high-performance liquid chromatography. One of two positional isomers of leukotriene B4 glucuronide was predominantly present in urine. The concentration of the isomer increased in urine from aspirin-intolerant asthma patients after aspirin challenge. Urinary leukotriene E4 and leukotriene B4 glucuronide concentrations in 13 normal healthy adults were 94.6 pg/mg-creatinine (median) and 22.3 pg/mg-creatinine, respectively. Urinary LTE4 concentration increased during the first 3h after allergen inhalation in atopic patients. However, allergen-induced bronchoconstriction was not associated with an increased concentration of LTB4 glucuronide in urine. The method enabled us to precisely determine urinary leukotriene B4 glucuronide concentration.

Background: Acute right lower abdominal pain may present a diagnostic dilemma. Leukotrienes have been found to be elevated in familial Mediterranean fever (FMF), a disease manifesting with recurrent episodes of "acute abdomen.", Objectives: To determine whether urine leukotriene B4 (LTB4) may differentiate between an FMF attack and some other forms of acute right lower abdominal pain., Methods: The LTB4 level was determined, using a commercial enzyme-linked immunosorbent assay (ELISA), in urine samples obtained from 36 patients with acute (< 24 hours) right lower abdominal pain presenting to the emergency department, and from 18 healthy volunteers., Results: Compared with the healthy control subjects, LTB4 was significantly higher in those who had FMF (12 patients, p < 0.03). In other forms of acute right lower abdominal pain, including appendicitis (eight patients), urologic disorders (eight patients), and nonspecific abdominal pain (eight patients), intermediate levels of LTB4 were observed, not significantly different from those of either FMF patients or healthy control subjects., Conclusions: In the samples tested, urine LTB4 levels were not instrumental in differentiating FMF from other acute right lower abdominal pain.

Background: Aspirin challenge of aspirin-intolerant asthma (AIA) patients causes a significant increase in leukotriene E4 (LTE4) concentration in urine. However, knowledge on leukotriene B4 (LTB4) generation in patients with AIA is insufficient. Recent research has demonstrated that exogenously administered LTB4 is excreted as glucuronide into the urine in human healthy subjects., Objective: The purpose of this study is to estimate urinary LTB4 glucuronide (LTBG) concentration in the clinically stable condition in healthy subjects and asthmatic patients and to investigate changes in urinary LTBG concentration in patients with AIA after aspirin challenge., Methods: A provocation test was performed by intravenous aspirin challenge. After urine was hydrolysed by beta-glucuronidase, the fraction containing LTB4 was purified by high-performance liquid chromatography and LTB4 concentration was quantified by enzyme immunoassay. Urinary LTBG concentration was calculated as the difference between the concentration obtained with hydrolysis and that without hydrolysis., Results: (1) After hydrolysis, the presence of urinary LTB4 was verified by gas chromatography-mass spectrometry-selected ion monitoring. (2) The urinary LTBG concentration was significantly higher in the asthmatic patients than in the healthy subjects (median, 5.37 pg/mg creatinine [range 1.2-13] vs. 3.32 pg/mg creatinine [range, 0.14-10.5], P = 0.0159). (3) The patients with AIA (n = 7), but not those with aspirin-tolerant asthma (n = 6), showed significant increases in LTBG and LTE4 excretions after aspirin challenge. (4) When the concentrations after aspirin challenge were analysed simultaneously, a significant linear correlation was observed between urinary LTBG concentration and urinary LTE4 concentration in patients with AIA (Spearman's rank correlation test, r = 0.817, P = 0.0003)., Conclusion: LTBG is present in human urine, albeit at a concentration lower than urinary LTE4. In addition to a marked increase in cysteinyl-leukotriene production, aspirin challenge induced LTB4 production in AIA patients.

Objective: To determine urinary leukotriene B4 (LTB4) levels and their role in FMF:, Methods: Urinary LTB4 levels were studied using a commercial ELISA kit in 12 FMF patients during abdominal attacks, and 20 FMF patients during remission., Results: Urinary LTB4 levels in FMF patients during attacks were comparable to those during remission, but higher than normal levels (p = 0.03)., Conclusions: These findings suggest a persistent activation of the leukotriene pathway in FMF. Whether elevated LTB4 levels are the cause or the effect of inflammation is yet to be determined.

Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils and is thought to play a role in a variety of inflammatory responses in humans. The metabolism of LTB4 in vitro is complex with several competing pathways of biotransformation, but metabolism in vivo, especially for normal human subjects, is poorly understood. As part of a Phase I Clinical Trial of human tolerance to LTB4, four human subjects were injected with 150 nmol/kg LTB4 with one additional subject as placebo control. The urine of the subjects was collected in two separate pools (0-6 and 7-24 h), and aliquots from these urine collections were analyzed using high performance liquid chromatography, UV spectroscopy, and negative ion electrospray ionization tandem mass spectrometry for metabolites of LTB4. In the current investigation, 11 different metabolites of LTB4 were identified in the urine from those subjects injected with LTB4, and none were present in the urine from the placebo-injected subject. The unconjugated LTB4 metabolites found in urine were structurally characterized as 18-carboxy-LTB4, 10,11-dihydro-18-carboxy-LTB4, 20-carboxy-LTB4, and 10,11-dihydro-20-carboxy-LTB4. Several glucuronide-conjugated metabolites of LTB4 were characterized including 17-, 18-, 19-, and 20-hydroxy-LTB4, 10-hydroxy-4,6,12-octadecatrienoic acid, LTB4, and 10,11-dihydro-LTB4. The amount of LTB4 glucuronide (16.7-29.4 pmol/ml) and 20-carboxy-LTB4 (18.9-30.6 pmol/ml) present in the urine of subjects injected with LTB4 was determined using an isotope dilution mass spectrometric assay before and after treatment of the urine samples with beta-glucuronidase. The urinary metabolites of LTB4 identified in this investigation were excreted in low amounts, yet it is possible that one or more of these metabolites could be used to assess LTB4 biosynthesis following activation of the 5-lipoxygenase pathway in vivo.

Neutrophil activation with the release of intracellular granule contents has been observed in sickle cell disease (SCD). Because leukotriene B(4) (LTB(4)), a 5-lipoxygenase metabolite of arachidonic acid in neutrophils, is a chemoattractant and enhances neutrophil adhesion to endothelium, we assessed plasma levels of this metabolite in controls (n = 9) and individuals with SCD, SS genotype, both in basal "steady state" (n = 37) and during episodes of vaso-occlusion (n = 10) and acute chest syndrome (n = 5). Thirteen patients with SCD, SC genotype, in steady state were also studied. Although no significant differences were noted between the control (136 +/- 32 fmol/mL) and SC genotype (177 +/- 83 fmol/mL, P >.15), LTB(4) levels were markedly increased in patients with SS genotype in basal steady state (207 +/- 64 fmol/mL, P <.003) compared with those in controls. Values were further increased during vaso-occlusion (264 +/- 94 fmol/mL) and acute chest syndrome (363 +/- 124 fmol/mL). These levels were significantly different from measurements taken during steady state (P <.04 and P <.0001, respectively). No correlation was noted between LTB(4) level and total white cell or neutrophil count. Additionally, the significant correlation noted in SCD between increased levels of plasma LTB(4) and soluble L-selectin (P <.03) reflects neutrophil activation. We also observed an effect of LTB(4) on red cell-endothelial adhesion at concentrations that appear clinically relevant (1-10 pmol/mL) with concomitant up-regulation of mRNA for the endothelial vitronectin receptor. These properties of LTB(4) are relevant to disease pathophysiology, providing further evidence of the contribution of the neutrophil to the proinflammatory and proadhesive phenotype in SCD.

Sjögren-Larsson syndrome (SLS) is an autosomal recessively inherited neurocutaneous disorder caused by a deficiency of the microsomal enzyme fatty aldehyde dehydrogenase (FALDH). We report the clinical characteristics and the results of molecular studies in 19 SLS patients. Patients 1-17 show the classical triad of severe clinical abnormalities including ichthyosis, mental retardation and spasticity. Most patients were born preterm, and all patients exhibit ocular abnormalities and pruritus. Electro-encephalography shows a slow background activity, without other abnormalities. MRI of the brain shows an arrest of myelination, periventricular signal abnormalities of white matter and mild ventricular enlargement. Cerebral (1)H-MR spectroscopy reveals a characteristic, abnormal lipid peak. The degree of white matter abnormality in the MRIs and the height of the lipid peak in (1)H-MR spectra do not correlate with the severity of the neurological signs. The clinical presentation and the clinical course is strikingly similar in these patients. Patient 18 shows a mild phenotype that essentially contains the same, but less severe, clinical features. Patient 19 exhibits the typical, but very mild, dermatological and ocular abnormalities, without any clinical neurological involvement. The diagnosis of SLS was confirmed by demonstration of the enzyme defect in cultured skin fibroblasts. Furthermore, as might be predicted from the essential role of FALDH in leucotriene B(4) (LTB(4)) metabolism, elevated urinary concentrations of LTB(4) and 20-OH-LTB(4) were found in all patients studied. Molecular studies of the FALDH gene revealed eight different mutations, including three new ones: a large 26-base pair deletion (21-46del), a missense mutation (80C-->T) and an insertion mutation (487-488insA). The vast majority of SLS patients seem to be severely affected independent of their genotype.

Background & Aims: Leukotrienes are proinflammatory mediators. Ethanol inhibits the catabolism of both cysteinyl leukotrienes (leukotriene E(4) [LTE(4)] and N-acetyl-LTE(4)) and leukotriene B(4) (LTB(4)) in hepatocytes. We examined the metabolic derangement of leukotriene inactivation by ethanol in humans in vivo., Methods: LTE(4), N-acetyl-LTE(4), LTB(4), and 20-hydroxy-LTB(4) were quantified in urine samples from 16 patients with acute alcohol intoxication (mean blood ethanol, 75 mmol/L). In 9 healthy volunteers, urinary LTE(4) was determined before and after ethanol consumption (mean blood ethanol, 14 mmol/L)., Results: The excretion of LTE(4) during alcohol intoxication was 286 compared with 36 nmol/mol creatinine in healthy subjects (P < 0.01); the corresponding values for N-acetyl-LTE(4) were 101 and 11 nmol/mol creatinine, respectively (P < 0.001). This excretion of cysteinyl leukotrienes decreased when the blood ethanol concentration returned to normal. LTB(4) and 20-hydroxy-LTB(4) were detectable only in patients with excessive blood ethanol concentrations (mean, 95 mmol/L). In healthy volunteers, LTE(4) excretion increased 3-5 hours after ethanol consumption (mean peak concentration of 1.5 nmol/L compared with 0.5 nmol/L for basal values; P < 0.005)., Conclusions: Ethanol at high concentration induces increased leukotriene excretion into urine. These changes are consistent with inhibition of leukotriene catabolism and inactivation induced by ethanol, as well as with a higher leukotriene formation caused by ethanol-induced endotoxemia.

In 6 patients with Sjögren-Larsson syndrome (SLS), the urinary excretion of leukotriene B(4) (LTB(4)) and omega-hydroxy-LTB(4) was found to be highly elevated, whereas omega-carboxy-LTB(4) was absent. This abnormal pattern of urinary excretion of LTB(4) and its metabolites appears to be unique to patients with SLS and offers a new approach to the diagnosis of this disorder. Moreover, defective inactivation of LTB(4) might be of pathophysiologic significance in the disease.

The Sjögren-Larsson syndrome (SLS) is a severe neurocutaneous disorder due to fatty aldehyde dehydrogenase (FALDH) deficiency. The recent discovery of the role of FALDH in the degradation of leukotriene B4 (LTB4) opened the way to the development of a new therapeutic strategy for SLS, i.e. 5-lipoxygenase inhibition. We treated one SLS patient with zileuton during five weeks. During the treatment period we found decreased values of LTB4 and omega-OH-LTB4. The severity of the pruritus diminished, and favorable changes in the child's behavior were observed. The height of the prominent "lipid peak" of cerebral white matter (that is characteristically found on proton magnetic resonance spectroscopy in SLS patients) decreased during treatment, and increased again when treatment was stopped. In conclusion, the beneficial effects of 5-lipoxygenase inhibition in SLS are very promising and encourage further research.

Sublingual immunotherapy has been suggested for the treatment of respiratory allergies. Many controversial studies have been reported on the efficacy of sublingual immunotherapy. The aim of this prospective study was to evaluate whether sublingual immunotherapy was effective according to clinical and laboratory results in pediatric allergies. Thirty-nine allergic, grass pollen sensitive children were admitted into the study. Sublingual immunotherapy was given over a 12-month period to 21 children (mean age 10.5 +/- 3.3 years), 10 of whom had seasonal allergic rhinitis and 11 seasonal allergic asthma. During the same period, 18 children (mean age 11.1 +/- 2.5 years), 10 with seasonal allergic rhinitis and eight with seasonal allergic asthma, received placebo. Symptom scores and drug requirements were recorded and urine samples were collected to detect urinary levels of leukotrienes (Uc-LTB4 and Uc-LTE4). In patients who received sublingual immunotherapy, the symptom scores of seasonal allergic rhinitis significantly decreased, but no statistically significant changes were observed in terms of symptoms of seasonal allergic asthma. Uc-LTE4 and Uc-LTB4 levels of seasonal allergic rhinitis, with a geometric mean and 95% confidence interval (CI), were significantly decreased from 216 (103-464) and 61 (22-198) pmol/mmol creatinine to 78 (29-159) and 35 (12-118) pmol/mmol creatinine, respectively (p < 0.05 and p < 0.05). On the other hand, Uc-LTE4 and Uc-LTB4 levels for seasonal allergic asthma were 180 (92-355) and 78 (44-258) pmol/mmol creatinine and decreased to 156 (72-402) and 69 (32-254) pmol/mmol creatinine, respectively. These changes were not statistically significant (p > 0.05). According to our clinical results and urinary levels of leukotrienes, which are mediators showing the severity of allergic inflammation, it can be suggested that sublingual immunotherapy may be useful in the treatment of seasonal allergic rhinitis but not of seasonal allergic asthma.

The metabolic inactivation of leukotrienes proceeds by beta-oxidation from the omega-end. We investigated the importance of peroxisomes and mitochondria in LTB4 oxidation in vivo. LTB4 and its oxidation products were analysed after high-performance liquid chromatography separation by immunoassays and gas chromatography-mass spectrometry in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency, and healthy controls. LTB4 (median 97; range 35-238 nmol/mol creatinine) and its omega-oxidation product omega-carboxy-LTB4 (median 898; range 267-4583 nmol/mol creatinine) were present and significantly increased in the urine of all patients with Zellweger syndrome compared to the controls (P <0.01). In contrast, LTB4 and omega-carboxy-LTB4 were below the detection limit (< 5 nmol/ mol creatinine) in patients with long-chain acyl CoA dehydrogenase deficiency and healthy controls. The beta-oxidation product omega-carboxy-tetranor-LTB3 was neither detectable in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency nor in the controls (< 5 nmol/mol creatinine). Analysis of urinary leukotrienes represents an additional diagnostic tool in peroxisome deficiency disorders. Furthermore, these results clearly underline the essential role of peroxisomes in the oxidation of LTB4 in humans.

Leukotrienes have been implicated in the bronchoconstriction caused by indirect stimuli. In the present study we examined the effect of oral ABT-761, a novel 5-lipoxygenase (5-LO) inhibitor, on exercise- and adenosine (AMP)-induced bronchoconstriction in nine asthmatics. At the four 1-d, single-dose treatment periods, ABT-761 (200 mg) or placebo (P) was ingested 5 h before challenge in a double-blind, crossover fashion. At study periods 1 and 2 the subjects performed an exercise challenge and at study periods 3 and 4 an AMP challenge. Pretreatment with ABT-761 caused a significant inhibition of the maximal percentage fall of FEV1 from baseline (p = 0.037) and a reduction of the percentage fall in FEV1 (area under the curve, AUC) of 61.4 +/- 14.1% (mean +/- SEM) after exercise challenge (p = 0.021). Although pretreatment with ABT-761 did not significantly inhibit the maximal fall of FEV1 after AMP challenge (p = 0.134), the overall bronchoconstriction was significantly inhibited, the AUC being reduced by a mean (+/- SEM) of 82.7 +/- 7.2% (p = 0.012). There was no significant correlation between the protective effect against exercise and that against AMP for individual patients. The percentage change in urinary leukotriene E4 (LTE4) excretion at exercise was + 18.1 +/- 10.9% on placebo and -44.8 +/- 6.2% after ABT-761 (p = 0.017); changes at adenosine were + 38.5 +/- 27.0% on placebo and -36.7 +/- 9.8% after ABT-761 (p = 0.028). On placebo, exercise produced a marked stimulation of the ex vivo LTB4 production, whereas adenosine was associated with only a minor increase; ABT-761 caused a greater than 90% inhibition (p < 0.05 for both challenges). We conclude that ABT-761 is a potent and long-acting 5-LO inhibitor which significantly attenuates exercise- and adenosine-induced bronchoconstriction, indicating that leukotrienes are important mediators in both challenges.

Leukotrienes may mediate bronchoconstriction in asthma. Cysteinyl leukotriene production rises in vivo after allergen challenge, but few reports describe leukotriene concentrations in clinical asthma or in children. Using high performance liquid chromatography/radioimmunoassay, plasma and urinary leukotrienes in asthmatic children (aged 5-10 years) were measured during an acute exacerbation (peak expiratory flow (PEF) < 65%, n = 10) and one month later (PEF 74-169%, n = 9), and in non-atopic normal children (aged 1.3-13.2 years). In the asthmatics, geometric mean (95% confidence interval) plasma leukotriene B4 (LTB4) was 746 pg/ml (398 to 1403) acutely and 1026 pg/ml (662 to 1593) in remission, compared with 369 pg/ml (167 to 728) in the normal children (n = 14). Plasma cysteinyl leukotrienes were low or undetectable, but urinary leukotriene E4 (LTE4) was higher in the asthmatics during an acute episode (210 pmol/mmol creatinine, 101 to 454) and at follow up (179 pmol/mmol, 110 to 293), compared with the normal children (98 pmol/mmol, 81 to 118, n = 41). This persistent increase in plasma LTB4 and urinary LTE4 concentrations one month after a severe asthmatic episode suggests leukotriene production is related to chronic inflammation rather than to acute bronchoconstriction.

The pharmacodynamics, kinetics, and tolerability of a new orally active 5-lipoxygenase inhibitor were evaluated in healthy male volunteers. MK-0591, 50, 125, and 250 mg every morning and 250 mg every 12 hours, was administered for 10 days. Leukotriene B4 biosynthesis ex vivo in ionophore (A23187)-stimulated whole blood and leukotriene E4 levels in urine were determined. Leukotriene B4 production was inhibited up to 90% of baseline for 12 hours after administration at the highest dose. The degree of leukotriene B4 inhibition ex vivo in whole blood significantly correlated with plasma MK-0591 concentrations (0 to 1500 ng/ml; r = 0.73). Urinary leukotriene E4 was inhibited by > 80% at 24 hours after administration for all dose levels. Pharmacokinetics of MK-0591 were linear, with a half-life of approximately 6 hours. Very little accumulation was seen after multiple dosing. MK-0591 had no effect on testosterone levels, and good tolerability was shown at all dose levels of MK-0591 administered for up to 10 days.

Sickle cell (HbSS) disease is associated with rheological and inflammatory stresses within the microcirculation. In order to determine the role of leukotrienes in the inflammatory processes in HbSS patients, we analysed plasma and urine levels of leukotrienes (LT); LTB4, LTC4, LTD4, and LTE4 as indicators of their in vivo metabolism. Plasma and urine level samples of 15 HbSS patients in steady-state and age-matched healthy, homozygous (HbAA) controls were extracted for leukotrienes and quantitated by HPLC. Control plasma level of leukotrienes (mean +/- SEM, ng ml-1) were: LTB4, 8.95 +/- 0.26; LTC4, 7.24 +/- 0.21; LTD4, 11.42 +/- 0.40; and LTE4, 14.51 +/- 0.50. Corresponding values for HbSS patients were: LTB4, 6.15 +/- 0.42; LTC4, 13.61 +/- 1.45; LTD4, 6.44 +/- 0.51 and LTE4, 4.97 +/- 0.37. The differences were significant at P < 0.05. Urine levels (mean +/- SEM, ng mmol-1 creatinine), for controls were: LTB4, 10.60 +/- 0.35; LTC4, 360.0 +/- 9.82. Values for HbSS urine were: LTB4, 27.50 +/- 3.33; LTC4, 356.0 +/- 17.87; LTD4, 69.90 +/- 14.51. LTD4 was not detected in control urine. These results suggest that sickle cell patients may exhibit impaired ability to catabolize LTC4 in plasma during steady state conditions. This altered metabolism may contribute to the persistent stress of the microcirculation, and is probably related to the abnormal microvascular rheology of sickle blood cells.

In order to investigate the possible contribution of leukotrienes to the airway hypersensitiveness and inducibility of asthmatic spells, the probable temporal correlation of the urinary leukotriene levels (U-LTB4 and U-LTC4) were estimated along with the peripheral neutrophil counts in two groups of asthmatic children with or without an inflammatory sign of the elevated CRP. The first group consisted of 6 asthmatic children possessing inflammatory signs and symptoms of fever and peripheral leukocytosis. They all were hospitalized. The second group of 6 patients of light asthmatic attack without any signs and symptoms of acute inflammation were studied in the Outpatient Department. In the first group, U-LTB4 was as high as 258.6 +/- 88.9 ng/mmol Cr. during the attacks while U-LTB4 levels of the second group was as low as 62.2 +/- 32.20 ng/mmol Cr. The difference was statistically significant at p less than 0.01. Further mathematical analysis revealed a positive correlation of U-LTB4 to the peripheral neutrophils counts at r = 0.71. Thus, it was concluded that the elevation of U-LTB4 levels in the first group was strongly related to infections, and their asthmatic spells were thought to represent an infection-induced type. On the contrary, U-LTC4 levels in the samples during the asthmatic attacks were increased in 2 of the 6 of the first group. They both represented grave asthmatic spells. In the remainder, U-LTC4 levels did not rise enough to induce spasmodic contractions of the bronchial smooth muscles. Thus, it was also discussed that the most appropriate timing for urine collection for the study of U-LTs is some time following an asthmatic attack.

Nephrotoxic nephritis, a model system for glomerulonephritis, is characterized by glomerular inflammation, proteinuria, and a marked increase in ex vivo glomerular eicosanoid production. This study addressed whether urinary eicosanoids might serve as noninvasive markers for glomerular inflammation and damage with nephrotoxic nephritis and its accelerated variant. Accelerated nephritis, relative to simple nephritis, was characterized by more substantial glomerular inflammation, particularly that due to neutrophils. Correspondingly, accelerated nephritis was accompanied by greater proteinuria and more marked elevations in glomerular eicosanoids generated ex vivo. With respect to urinary eicosanoids, thromboxane, but not leukotriene B4, was detected in the urine of normal animals. After the induction of nephrotoxic nephritis, urinary thromboxane was moderately elevated (twofold) and urinary leukotriene B4 was variably present (three of seven animals). In accelerated nephritis, urinary thromboxane was more markedly elevated (sixfold) and leukotriene B4 was consistently present. The presence of urinary leukotriene B4 was confirmed by gas chromatography/mass spectrometry. Urinary eicosanoids together correlated with glomerular leukocyte numbers and proteinuria by linear regression. Urinary leukotriene B4 individually correlated with glomerular neutrophil numbers. Renal metabolism of leukotriene B4 to omega oxidation products by the rat kidney was not apparent. These data validate that the enhanced glomerular eicosanoid metabolism seen in nephrotoxic nephritis takes place in vivo and additionally suggest that both urinary thromboxane and leukotriene B4 may serve as noninvasive markers for glomerular inflammation and damage. In light of these and prior studies, urinary thromboxane may be a general marker of glomerular inflammation and leukotriene B4 may be a more specific index of acute inflammation.

Antipsoriatic agents have been shown to decrease skin levels of arachidonic acid and its metabolites including 12-monohydroxy-eicosatetranoic acid (12-HETE), and leukotriene B4 (LTB4). In addition, specific systemic and topical lipoxygenase inhibitors have been reported to be effective in the treatment of psoriasis. The objective of this study was to investigate the effect of a potent oral leukotriene biosynthesis inhibitor (MK886) in patients with chronic plaque psoriasis. Clinical response together with the changes of LTB4 levels in lesional skin biopsy specimens, and urinary leukotriene E4 (LTE4) excretion were evaluated. In addition, markers of inflammation, proliferation and keratinization were studied immunohistochemically. No change in clinical scores or lesional LTB4 levels were observed with a 10 1/3-day course of MK886. A statistically significant reduction in urinary LTE4 excretion was observed: mean LTE4 (ng/h) were 5.14 before treatment and 1.51 on day 11 with MK886; and 7.55 before treatment and 6.57 on day 11 with placebo treatment. Epidermal accumulation of polymorphonuclear leukocytes (PMN) tended to diminish in the MK886 treatment group. These results indicate that although a reduction (greater than 70%) in urinary LTE4 excretion was found, and a slight decrease of epidermal PMN accumulation was observed, no correlative changes in clinical scores or LTB4 levels in skin lesion were found with a short course of MK886.

Five milligrams of [5,6,8,9,11,12,14,15-3H8]-leukotriene B4 (LTB4) (1.68 Ci/mmol) were infused into a monkey over a three hour period. Twenty-five per cent of the infused 3H-activity was recovered in the urine during the twenty hours of collection. Plasma and urinary metabolite volatility studies revealed that in contrast to previously studied eicosanoids, more than 70% per cent of the infused LTB4 3H-label was converted to tritiated water. The major nonvolatile urinary metabolite of LTB4 representing 0.8% of the infused material was identified as 20-OH-LTB4. LTB4 was not excreted in the urine. Other nonvolatile metabolites of LTB4 representing less than 0.4% each of the infused material were isolated from the urine. While there was an adequate quantity of some of these metabolites for partial characterization, there was insufficient material for structural elucidation. Further studies were performed in rabbits in which either LTB4 or the structurally related compound 8,15-dihydroxyeicosatetraenoic acid (8,15-diHETE) were infused intravenously. In these rabbits the metabolism of LTB4 and 8,15-diHETE was similar to that in the monkey with greater than 80% of the infused 3H-activity converted to tritiated water. These studies suggest that leukotriene B4 and structurally related compounds undergo extensive degradation in vivo via the beta-oxidation system.

A rapid and simple method for the on-line concentration and high-performance liquid chromatographic (HPLC) purification of the leukotrienes in good yield from biological fluids is described. Readily available antisera are used in conjunction with this system to give a specific and sensitive assay for leukotrienes B4, C4 and D4 with sub-nanogram limits of detection. Tritium-labelled leukotrienes are used as internal standards, both to locate the leukotrienes post-HPLC and to accurately determine recoveries.

The intravenous administration of [3H]leukotriene C4 in the monkey Macaca fascicularis results in the biliary and urinary elimination of [3H]leukotriene D4 and [3H]leukotriene E4 in addition to more-polar metabolites. Separation of these polar metabolites and chromatographic comparison with synthetic w-oxidized leukotrienes indicated the in vivo formation of w-hydroxy-[3H]leukotriene E4 and w-carboxy-[3H]leukotriene E4. Time course studies of the [3H]leukotriene metabolite pattern in bile and urine showed that w-hydroxy-leukotriene E4 was decreasing as w-carboxy-leukotriene E4 and additional polar derivatives were increasing.

Adenosine is a renal vasoconstrictor that plays an important role in mediating renal adaptive responses to decreases in renal perfusion pressure. It is known that adenosine acts on the metabolism of arachidonic acid, but the direct repercussions of adenosine in the production of renal prostaglandins and leukotrienes have not been studied. This study was undertaken to evaluate the effect of the intrarenal infusion of adenosine upon the urinary elimination of arachidonic acid derivatives. Samples of urine were collected with lysine acetylsalicylate and determination of prostaglandins (PGs) and leukotrienes (LTs) was performed by radioimmunoassay of samples previously separated by HPLC. The infusion of adenosine decreases the urinary excretion of 6-keto-PGF1 alpha and TxB2 significantly. There was no significant change in urinary excretion of PGE2 while LTB4 and LTC4 showed a tendency to increase. These results suggest that a fall in the synthesis of PGI2 along with an increase in LTC4, which is a constrictor of mesangial cells, could be responsible for the renal vasoconstriction phase of adenosine. Therefore, it was concluded that adenosine vasoconstriction is mediated through the inhibition of the cyclo-oxygenase pathway, diminishing the synthesis of PG vasodilators.