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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.)

Snoring is the most characteristic symptom of obstructive sleep-disordered breathing (SDB) and recurrent wheezing is the most common clinical manifestation of asthma. The purpose of the present review is to outline the impact of SDB and recurrent wheezing/asthma on sleep quality and to summarize the epidemiologic and pathophysiologic evidence supporting an association between the two disorders. Enlarged tonsils and adenoid or obesity predispose to obstructive sleep apneas and hypopneas which are accompanied by arousals, restless sleep, and frequently daytime sleepiness, inattention, hyperactivity, and academic difficulties. Subjects with history of wheezing are also at risk for sleep disturbance and daytime cognitive dysfunction. Asthmatic children have more frequent snoring, apneas, and hypopneas during sleep than non-asthmatic subjects and tonsillar hypertrophy mediates at least in part this epidemiologic association. In addition, preliminary evidence indicates that treatment of sleep apnea with adenotonsillectomy results in improved control of coexisting asthma. Elevated concentrations of leukotrienes and oxidative stress markers have been detected in the exhaled breath condensate of children with asthma and probably contribute to bronchoconstriction. Moreover, sleep apneic children have increased expression of leukotrienes and leukotriene receptors in adenotonsillar tissue. Viral respiratory infections may induce inflammation and oxidative stress in the asthmatic airway enhancing not only bronchospasm, but also biosynthesis of leukotrienes within pharyngeal lymphoid tissues, which promote adenotonsillar enlargement and sleep apnea. In conclusion, taking under consideration the epidemiologic association between obstructive SDB and asthma, when one of the two disorders is diagnosed, the possibility of the other disease being present should be entertained. Pediatr. Pulmonol. 2011; 46:1047-1054. © 2011 Wiley Periodicals, Inc., (Copyright © 2011 Wiley Periodicals, Inc.)

Background: Infiltration of inflammatory cells in bronchial mucosa and glandular hypersecretion are hallmarks of asthma. It has been postulated that exhaled breath condensate (EBC) mirrors events in epithelial lining fluid of airways, such as presence of local inflammation as well as glandular hypersecretion. It is also well known that eosinophil cationic protein (ECP) and cysteinyl-leukotrienes (cys-LT) are released by circulating inflammatory cells when triggered by antigen stimulation in asthma patients., Objectives: The aim of this study was to evaluate whether chlorine and/or cys-LT in EBC would reflect changes of exposure of airborne pollen in patients with asthma., Methods: EBC and serum were collected from 23 patients with allergic asthma during a pollen season and repeated 5 months later during a period with no aeroallergens. Chlorine was measured by means of a sensitive coulometric technique and cys-LT by an EIA technique. Serum ECP was measured and lung function tests were performed and symptoms noted during both occasions., Results: Significantly higher concentrations of chlorine in EBC (p = 0.007) and ECP in serum (p = 0.003) were found during the pollen season compared to post-season. Chlorine levels tended to be higher in patients who reported of chest symptoms compared to those who denied symptoms during the pollen season (p = 0.06). Areas under the receiver-operated characteristic curves (AUC(ROC)) were compared and similar discriminative power to identify exacerbations of asthma was recorded by chlorine in EBC (range 0.67-0.78) and ECP in serum (range 0.64-0.78)., Conclusion: It is concluded that chlorine in EBC and ECP in serum decreased significantly post-season, and this is suggested to mirror the decrement in airborne antigen. It is furthermore proposed that chlorine in EBC and ECP in serum tend to have a similar capacity to identify seasonal variations in airborne pollen in patients with asthma.

Background: Inhaled endotoxin is known to induce airway inflammation, causing bronchial hyperreactivity., Objective: We characterized the response to lipopolysaccharide-inhalation by measuring exhaled nitric oxide (eNO) and inflammatory mediators., Patients and Methods: A total of 43 adult volunteers (13 asthmatics, 30 healthy controls) inhaled stepwise LPS every 30 min up to a cumulative dose of 100 microg (2.5, 10.5, 42, 45 microg). After each provocation and up to 24 h later, FEV(1) was determined; the procedure was stopped when FEV(1) declined more than 12.5%. We measured eNO, leucocytes, eosinophils, polymorphonuclear neutrophils (PMNs), C-reactive protein (CrP), lipopolysaccharide binding protein (LBP), eosinophilic cationic protein (ECP), leucotriene B4 (LTB4), thromboxane B2 (TXB2), and body temperature., Results: Initial eNO values were higher in asthmatics (P < 0.01), but only increased in an asthmatic subgroup. Marked differences were observed in the systemic response to LPS inhalation. Significant increases were found for CrP, LBP, and PMNs. There was no correlation between FEV(1) decrease and basal eNO levels., Conclusions: Inhalation of endotoxin was followed by clinical and laboratory signs of systemic inflammation, with asthmatics responding to the challenge similar as healthy subjects. Bronchial eNO increased only temporarily in asthmatics.

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.

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.

The aqueous extract of propolis has been formulated as a nutritional food product and administered, as an adjuvant to therapy, to patients with mild to moderate asthma daily for 2 months in the framework of a comparative clinical study in parallel with a placebo preparation. The diagnosis of asthma was made according to the criteria of patient classification of the National Institutes of Health and Global Initiative for Asthma Management. At inclusion, the pulmonary forced expiratory volume in the first second (FEV1) as a percentage of the forced vital capacity (FVC) was more than 80% in mild persistent cases, and between 60 and 80% in moderate persistent cases, showing an increase in the degree of reversibility of > 15% in FEV1. All patients were on oral theophylline as controller therapy, none was receiving oral or inhaled corticosteroids, none had other comorbidities necessitating medical treatment, and all were from a middle-class community and had suffered from asthma for the last 2-5 years. Twenty-four patients received the placebo, with one drop-out during the study, while 22 received the propolis extract, with no drop-outs. The age range of the patients was 19-52 years; 36 were male and 10 female. The number of nocturnal attacks was recorded on a weekly basis, while pulmonary function tests were performed on all patients at the beginning of the trial, 1 month later and at the termination of the trial. Immunological parameters, including various cytokines and eicosanoids known to play a role in asthma, were measured in all patients at the beginning of the trial and 2 months later. Analysis of the results at the end of the clinical study revealed that patients receiving propolis showed a marked reduction in the incidence and severity of nocturnal attacks and improvement of ventilatory functions. The number of nocturnal attacks dropped from an average of 2.5 attacks per week to only 1. The improvement in pulmonary functions was manifested as a nearly 19% increase in FVC, a 29.5% increase in FEV1, a 30% increase in peak expiratory flow rate (PEFR), and a 41% increase in the forced expiratory flow rate between 25 and 75% of the vital capacity (FEF25-75). The clinical improvement was associated with decreases by 52, 65, 44 and 30%, respectively, of initial values for the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, ICAM-1, interleukin (IL)-6 and IL-8, and a 3-fold increase in the 'protective' cytokine IL-10. The levels of prostaglandins E2 and F2alpha and leukotriene D4 were decreased significantly to 36, 39, and 28%, respectively, of initial values. Patients on the placebo preparation showed no significant improvement in ventilatory functions or in the levels of mediators. The findings suggest that the aqueous propolis extract tested is potentially effective as an adjuvant to therapy in asthmatic patients. The benefits may be related to the presence in the extract of caffeic acid derivatives and other active constituents.

1. Cyclo-oxygenase (COX) and lipoxygenase (LO) share a common substrate, arachidonic acid. Aspirin and related drugs inhibit COX activity. In a subset of patients with asthma aspirin induces clinical symptoms associated with increased levels of certain LO products, a phenomenon known as aspirin-sensitive asthma. The pharmacological pathways regulating such responses are not known. 2. Here COX-1 and LO activity were measured respectively by the formation of thromboxane B(2) (TXB(2)) or leukotrienes (LT) C(4), D(4) and E(4) in whole blood stimulated with A23187. COX-2 activity was measured by the formation of prostaglandin E(2) (PGE(2)) in blood stimulated with lipopolysaccharide (LPS) for 18 h. 3. No differences in the levels of COX-1, COX-2 or LO products or the potency of drugs were found in blood from aspirin sensitive vs aspirin tolerant patients. Aspirin, indomethacin and nimesulide inhibited COX-1 activity, without altering LO activity. Indomethacin, nimesulide and the COX-2 selective inhibitor DFP [5,5-dimethyl-3-(2-isopropoxy)-4-(4-methanesulfonylphenyl)-2(5H)-furanone] inhibited COX-2 activity. NO-aspirin, like aspirin inhibited COX-1 activity in blood from both groups. However, NO-aspirin also reduced LO activity in the blood from both patient groups. Sodium salicylate was an ineffective inhibitor of COX-1, COX-2 or LO activity in blood from both aspirin-sensitive and tolerant patients. 4. Thus, when COX activity in the blood of aspirin-sensitive asthmatics is blocked there is no associated increase in LO products. Moreover, NO-aspirin, unlike other NSAIDs tested, inhibited LO activity in the blood from both aspirin sensitive and aspirin tolerant individuals. This suggests that NO-aspirin may be better tolerated than aspirin by aspirin-sensitive asthmatics.

Inflammation plays a major role in the pathophysiology of asthma. Therefore, monitoring of the disease and its management preferably should include assessment of mediators of airways inflammation. Much interest has been focused on using eosinophil granule proteins in blood and urine as markers of asthma inflammation. The eosinophil granule proteins are important mediators in allergic inflammation. They often function well as inflammatory markers when used in controlled clinical studies, and are therefore useful research tools. With urinary eosinophil-derived protein X (EPX) venous blood sampling is avoided. Disadvantages which limit usefulness in clinical practice are overlap between values in patients and controls, a rather weak correlation to traditional lung function variables, elevation of levels by concurrent allergic disease, and the delay between sampling and test results. Urinary excretion of leukotriene E4 (LTE(4)) reflects the production of cysteinyl leukotrienes, which are major mediators in asthma. The excretion of LTE4 possibly reflects lung function better than serum ECP. In the future, monitoring of cytokines may be used in clinical asthma to monitor control and aid in the prognosis of the disease in the young child. Techniques for simple and rapid monitoring of key type 1 and type 2 cytokines are needed.

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.

The underlying mechanisms of bronchoconstriction in aspirin-intolerant asthmatics (AIAs) are still unknown, but the hypothesis of an altered metabolism of arachidonic acid is generally accepted. So far, no in vitro test for aspirin intolerance is available. The hypothesis that the profile of eicosanoid mediators is changed in AIA-even before aspirin challenge was tested. The release of prostaglandin E2 (PGE2), peptidoleukotrienes and histamine was measured using competitive enzyme immunoassays in 10 asthmatics with a history of aspirin intolerance, 10 controls and eight aspirin-tolerant asthmatics (ATAs) before and after bronchial provocation with lysine-aspirin. Comparing basal release of eicosanoids before challenge, peptidoleukotrienes were significantly elevated and PGE2 was vastly reduced in AIAs, whereas ATAs had elevated basal peptidoleukotrienes but only slightly reduced basal PGE2. The decrease in forced expiratory volume in one second (FEV1) was not associated with changes in histamine release. After aspirin challenge, there was a massive increase of already elevated peptidoleukotrienes in AIAs, but not in ATAs. Arachidonic acid-induced PGE2 release in AIAs was not significantly changed, whereas it was significantly reduced in ATAs and healthy controls. Histamine release was unaffected by aspirin challenge in all three groups. There is a typically altered profile of eicosanoids in aspirin-intolerant asthmatics which could make in vitro diagnosis of aspirin intolerance possible.

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.

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.

To assess the contribution of the leukotrienes, LTC4, D4, E4 and B4 during bronchial asthma attacks, simultaneous determination was made of their levels in venous blood. 25 patients with bronchial asthma (15 atopic types, 10 non-atopic types) participated in this study and 4 normal controls were used. Samples were obtained using heparinized syringe from the patients before treatment. A radioimmunoassay was conducted to measure LTs after purification with a Sep-pak column and separation by HPLC. In normal subjects, the levels were less than the minimal detectable amounts. LTC4, D4, E4 and B4 during asthmatic attacks were 100 +/- 179, 88 +/- 116, 479 +/- 291, and 55 +/- 73 (Mean +/- SD) pg/ml respectively (n = 27). Peptide LTs in remission were below minimal detectable levels. LTD4 in patients with moderate attacks was significantly (p < 0.05) higher than in those with mild attacks. Peptide LTs in moderate attack exceeded those in mild attacks, although not to a statistically significant degree. No significant differences in LT during attacks could be detected in atopic or non-atopic type patients. LTs would thus appear importantly involved in asthmatic attacks in atopic and non-atopic type patients, although other chemical mediators may give rise to airway inflammation.

In order to determine the mechanism of bronchoconstriction induced by the inhalation of adenosine in asthmatics, we measured, by the astograph method, cyclic nucleotides, leukotrienes and thromboxane B2 before and after the inhalation, and we studied the relationship between serum theophylline concentration and Rrs cont, Dmin and S Grs/Grs cont of the astogram obtained by inhaled adenosine. Plasma TXB2 level was significantly raised (p less than 0.05) after inhalation, but cAMP, cGMP, LTB4 and LTC4 did not change. In the astogram, Dmin was significantly elevated (p less than 0.01) when plasma theophylline concentration was raised to the therapeutic level, but Rrs cont and S Grs/Grs cont showed no remarkable change. We confirmed that inhaled adenosine caused bronchoconstriction in asthmatic patients, and that Dmin was elevated after the administration of theophylline. These results suggest that TXB2, a metabolic product of arachidonic acid, may be one of the most important mediators in the bronchoconstriction mechanism of inhaled adenosine.

The generation and metabolism of leukotrienes (LTs) B4, C4, D4, and E4 were studied in vitro in the A23187-stimulated whole blood of normal (N) and atopic asthmatic (AA) human subjects. Using a combination of reversed-phase high performance liquid chromatography and radioimmunoassay, we have demonstrated that the blood cells of atopic asthmatic patients have an enhanced ability to release LTB4 and LTC4 when compared to those of normal subjects. The release of LTB4 and LTC4 in response to ionophore is dose- and time-dependent. Half-maximal doses of ionophore caused the generation of high, sustained levels of LTB4, which are significantly higher in the AA blood than in N blood. Incubations of 3H-LTB4 in ionophore-stimulated N and AA blood revealed a slow metabolism to 20-OH-LTB4 and 20-COOH-LTB4. LTC4 is generated in smaller amounts than LTB4, with an early peak after 10 min which is significantly higher (p less than 0.01) in the AA blood compared to the N blood. Subsequent metabolism of LTC4 elicits significantly greater amounts of LTD4, and consistently higher levels of LTE4, in the AA blood. Parallel incubations of 3H-LTC4 in ionophore-stimulated N and AA blood demonstrated rapid metabolism of LTC4 by the glutathione detoxification pathway. The elevated production of LTB4 and LTC4 in AA blood was not accounted for by differences in leukocyte sub-type counts in the two groups, nor by differences in their rates of catabolism. The novel, selective 5-lipoxygenase inhibitor BW A4C [N-(3-phenoxycinnamyl) acetohydroxamic acid] caused dose-dependent inhibition of LTB4 and LTC4 generation and was equipotent in N and AA blood.

Recently identified Ascaris suum sensitive cynomolgus monkeys were further characterized to determine if a chronologic relationship existed between mediator release and onset of bronchoconstriction. In these anesthetized Ascaris-sensitive monkeys, aerosol antigen challenge of each animal produced rapid and severe bronchoconstriction, as determined by decreases in dynamic lung compliance (-80.2 +/- 4.1%) and airway conductance (-64.5 +/- 13.8%). Maximum changes were achieved within 5 min following exposure and remained substantially altered throughout the 30 min observation period. However, changes in pulmonary function related to duration of onset and maximum change seemed to have some correlation with each animals' sensitivity to the antigen. Comparison of pre- and post-challenge blood gas profiles, showed a progressive formation of respiratory acidosis through decreases in arterial blood pH, partial pressure of O2 (pO2), O2 saturation (sO2) and an increase in partial pressure of CO2 (pCO2). When arterial blood plasma was assayed by RIA for mediators of anaphylaxis, large increases in 5-hydroxyeicostetraenoi acid (5-HETE), leukotriene B4 (LTB4) and histamine were observed. No amount of prostaglandin F2-alpha (PGF2 alpha) or thromboxane A2 were detected. Two of the three monkeys also produced detectable amounts of leukotriene C4 (LTC4). Therefore, in Ascaris-sensitive monkeys, histamine is the predominate mediator released and is probably responsible for at least the early part (5-10 min) of the observed bronchoconstriction. However, mediators from the lipoxygenase pathway may also be playing a role in the antigen-induced bronchoconstriction, especially beyond the 10 min period following anaphylaxis.

Plasma levels of arachidonic acid metabolites, such as thromboxane A2 (TXA2), prostacyclin (PGI2) and leukotrienes (LTS) were measured in patients with exercise-induced asthma (EIA). Asthmatic patients were exercised by treadmill. Before, immediately after and at 5, 15 and 30 minutes after exercise, pulmonary function tests were performed and peripheral venous blood was collected at the indicated times. Plasma TXB2 levels rose after exercise, and this tendency was stronger in EIA negative patients than in EIA positive patients. Plasma 6-keto-PGF1 alpha levels rose slightly after exercise in both EIA positive and negative patients. Plasma LTC4 levels were almost unchanged after exercise in EIA positive patients. Plasma LTC4 levels rose after exercise in EIA negative patients, though the value was always higher in EIA positive patients than in EIA negative patients. Plasma LTB4 levels in both EIA positive and negative patients increased slightly till 5 minutes after exercise, then decreased, and then tended to rise again. Plasma LTB4 levels were higher in EIA positive patients than in EIA negative patients at all times. The ratio of TXB2 to 6-keto-PGF1 alpha after exercise rose more remarkably in EIA negative patients than in EIA positive patients. From these results, we suspected that the metabolites of arachidonic acid may contribute less to chemical mediators of EIA, but the arachidonic acid products of EIA positive patients activated rather towards lipoxygenase cascade than EIA negative patients.