Elevated eosinophil protein X in urine from healthy neonates precedes development of atopy in the first 6 years of life.

RATIONALE: Biomarkers predicting development of atopic disease are needed for targeted preventive measures and to study if disease pathology may be active before onset of symptoms. OBJECTIVES: To investigate whether eosinophil protein X, leukotriene-C4/D4/E4, and 11ß-prostaglandin (PG) F2α (PGD2 metabolite) assessed in urine from healthy at-risk neonates precede development of atopic disease during the first 6 years of life. METHODS: We measured eosinophil protein X (n = 369), leukotriene-C4/D4/E4 (n = 367), and 11ß-PGF2α (n = 366) in urine from 1-month-old children participating in the Copenhagen Prospective Studies on Asthma in Childhood birth cohort. Clinical data on development of allergic sensitization, allergic rhinitis, nasal eosinophilia, blood eosinophilia, eczema, troublesome lung symptoms (significant cough or wheeze or dyspnea), and asthma were collected prospectively until age 6 years. Associations between urinary biomarkers and development of atopic traits were investigated using general estimating equations, logistic regression, and Cox regression. MEASUREMENTS AND MAIN RESULTS: Eosinophil protein X in the urine of the asymptomatic 1-month-old neonates was significantly associated with development of allergic sensitization (odds ratio, 1.49; 95% confidence interval [CI], 1.08­1.89), nasal eosinophilia (odds ratio, 3.2; 95% CI, 1.2­8.8), and eczema (hazard ratio, 1.4; 95% CI, 1.0­2.0), but not with allergic rhinitis, asthma, or blood eosinophilia. Neither leukotriene-C4/D4/E4 nor 11ß-PGF2α was associated with any of the atopic phenotypes. CONCLUSIONS: Eosinophil protein X in urine from asymptomatic neonates is a biomarker significantly associated with later development of allergic sensitization, nasal eosinophilia, and eczema during the first 6 years of life. These findings suggest activation of eosinophil granulocytes early in life before development of atopy-related symptoms.

Enhanced synthesis of cysteinyl leukotrienes in psoriasis.

Cysteinyl leukotriene synthesis was investigated in patients with psoriasis. A non-invasive test requiring no stimulation was employed by measuring the major index metabolite of LTC4, which appears in urine. The presence of this metabolite, LTE4, was shown unequivocally by gas chromatography-mass spectrometry. Routinely LTE4 was quantitated by specific radio immunoassay after its isolation by reversed-phase high-performance liquid chromatography. Furthermore, in representative samples amounts of LTE4 obtained by radioimmunoassay were validated by gas chromatography-mass spectrometry. We demonstrate a significant (p less than 0.01) more than fourfold increase of urinary LTE4 in psoriasis compared to healthy volunteers. Urinary LTE4 was log normally distributed with geometric mean values (95% confidence intervals) of 11 (9-14) nmol LTE4/mol creatinine in healthy volunteers (n = 11) and 51 (28-95) nmol LTE4/mol creatinine in psoriasis (n = 9). The present study shows that cysteinyl leukotriene synthesis is enhanced in patients with psoriasis and that measurement of urinary LTE4 is a useful parameter to monitor its rate of synthesis.

Urinary leukotriene E4 excretion in exercise-induced asthma.

Recent evidence suggests that the cysteinyl-leukotrienes (LTC4, LTD4, and LTE4) may be important in the pathogenesis of exercise-induced asthma. To evaluate the role of these mediators further, nine asthmatic subjects with exercise-induced bronchoconstriction were studied on two occasions. On visit 1, subjects performed 6 min of treadmill exercise; the mean maximal percent fall in FEV1 was 38.0 +/- 5.3%. On visit 2, maximal bronchoconstriction observed after exercise was matched with aerosolized methacholine. Urine was collected in two 90-min fractions (0-90 and 90-180 min) after challenges and analyzed by high-performance liquid chromatography-radioimmunoassay for LTE4. There were no significant differences in urinary LTE4 excretion between exercise and methacholine challenges for the periods 0-90 min (16.9 +/- 5.4 vs. 20.4 +/- 4.2 ng/mmol urinary creatinine), 90-180 min (24.9 +/- 8.2 vs. 20.1 +/- 5.5), or 0-180 min (21.5 +/- 6.5 vs. 18.8 +/- 4.1). Thus in contrast to allergen-induced bronchoconstriction, there is little evidence for enhanced cysteinyl-leukotriene generation in exercise-induced bronchoconstriction as assessed by urinary LTE4. If local release and subsequent participation of functionally active cysteinyl-leukotrienes in the pathways that ultimately lead to bronchoconstriction after exercise challenge do occur, these are of insufficient magnitude to perturb urinary LTE4 excretion.

Increased leukotriene E4 excretion during antigen-induced bronchoconstriction in allergic sheep.

The metabolism of leukotrienes (LT) in the sheep was investigated to define markers of 5-lipoxygenase involvement in allergic responses, obtainable by noninvasive techniques. Intravenous administration of 14, 15-[3H]LTC4 (0.5 microCi/kg) revealed a rapid clearance from the circulation (half time = 90 s). Circulatory metabolism was apparent, with early formation (within 1 min) of LTD4 and LTE4 shown by reverse-phase high-pressure liquid chromatography (RP-HPLC). Urinary 3H excretion comprised 10% of the original dose. [3H]LTE4 (characterized by coelution with authentic standards during RP-HPLC analysis) was observed in early urine samples. By use of a sensitive and specific RP-HPLC radioimmunoassay analysis, immunoreactive material coeluting with LTE4 was detected in urine from allergic sheep. Excretion of this material was significantly increased during antigen-induced acute bronchoconstriction in eight conscious allergic sheep [preantigen, 65.70 +/- 24.27 (SE) pg; 0-1 h postantigen, 208.00 +/- 71.10 pg, P less than 0.05], but not during late responses. However, total postantigen LTE4 excretion (37.8 - 956.1 pg/8 h) was highly correlated (r = 0.976, P less than 0.001) with the severity of bronchoconstriction (445.3 - 2,409.1% specific pulmonary resistance per hour) assessed by measurement of the area under the curve of pulmonary function plotted against time. These findings represent an important demonstration of in vivo allergen-induced peptide LT generation in a physiologically characterized animal model of prolonged allergic bronchoconstriction and further substantiate an important role for LT in this model of allergic asthma.

Inhaled PAF stimulates leukotriene and thromboxane A2 production in humans.

Platelet-activating factor (PAF) is a potent bronchoconstrictor in humans and has been implicated as an inflammatory mediator in asthma. This study was performed to evaluate whether PAF-induced bronchoconstriction in vivo could be mediated through the release of the bronchoconstrictor eicosanoids, thromboxane (Tx) A2 and the cysteinyl leukotrienes. Ten asthmatic subjects were studied on three occasions after bronchial challenges with aerosolized PAF, methacholine, or isotonic saline. PAF caused bronchoconstriction in all 10 subjects (mean maximal percent fall in specific airway conductance 48.2 +/- 4.6) and was matched by methacholine challenge. Saline caused no changes in specific airway conductance. Urinary leukotriene E4 was significantly elevated after inhaled PAF (366.0 +/- 66.9 ng/mmol creatinine, P less than 0.01) compared with methacholine (41.6 +/- 13.3) and saline (33.6 +/- 4.6). The major urinary TxA2 metabolite 2,3-dinor TxB2 was elevated after inhaled PAF (41.3 +/- 7.1 ng/mmol creatinine, P less than 0.01) compared with methacholine (14.0 +/- 2.7) and saline (17.1 +/- 3.9). Urinary 2,3-dinor 6-oxo-prostaglandin F1 alpha after PAF (22.2 +/- 1.4) was raised with respect to the methacholine challenge (13.9 +/- 1.8, P less than 0.02), although no significant increase was observed compared with the saline control (18.6 +/- 3.3). Inhaled PAF leads to the secondary generation of cysteinyl leukotrienes and TxA2, and it is possible that these mediate some of the acute effects of inhaled PAF in vivo.

Elevated urinary leukotriene E4 excretion in patients with ARDS and severe burns.

Increased synthesis of peptidoleukotrienes may occur in a variety of inflammatory diseases. To test this theory, hospitalized patients with a variety of diseases were studied and urine LTE4 quantitated as an index of total body peptidoleukotriene synthesis. 10 patients with ARDS, 7 of which had additional organ involvement, and 5 patients suffering from severe burn injuries were studied. Patients with uncomplicated ARDS excreted approximately 6-fold higher amounts of LTE4 in urine compared to healthy subjects. When ARDS was complicated by multiple organ failure (MOF), urine LTE4 levels were 2- to 150-fold higher than in healthy volunteers. Patients with severe burn injuries had peak urine LTE4 levels which were approximately 20-fold higher than in healthy volunteers. As additional controls, patients with cardiac arrhythmias (absence of inflammatory disease) and patients with uncomplicated pneumonia (localized inflammation) showed normal or mildly elevated urinary LTE4 levels. The urinary LTE4 levels in ARDS patients did not correlate with serum creatinine, bilirubin, or LDH levels, or with the WBC, nor did renal or liver failure by itself predict extremely elevated urinary LTE4 levels. In conclusion, patients with ARDS or ARDS/MOF and patients with severe injuries and sepsis syndrome excrete higher levels of urinary LTE4 than patients healthy volunteers or patients with limited inflammatory disease. In certain situations, urinary LTE4 levels may be useful as a marker of the degree of inflammation.

[Urinary leukotrienes levels in asthmatic children].

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.

A patient with neurological symptoms and abnormal leukotriene metabolism: a new defect in leukotriene biosynthesis.

A 15-year-old male patient presented with mental retardation, mild motor impairment, and partial deafness. Biochemical investigations showed an abnormal urinary profile of leukotrienes. Concentration of leukotriene D(4) (LTD(4)), which is usually not detectable, was highly increased, whereas LTE(4), the major urinary metabolite in humans, was completely absent. These data suggest membrane-bound dipeptidase deficiency, a new defect in leukotriene biosynthesis on the step of LTE(4) synthesis, as underlying defect.

Stability of leukotrienes C4 and D4 in human urine.

The ex vivo stability of leukotrienes (LT) C4 and D4 in human urine was tested in an experimental model. The various LTs were identified/measured by HPLC, liquid scintillation counting, RIA, UV-absorbance and GC-MS. If fresh urine was incubated under physiological conditions added LTC4 was converted in a time-dependent manner to LTD4 (t1/2 about 0.5 h) and to LTE4 (t1/2 about 6 h), respectively. At the end of the 22 h incubation period less than 10% of the initial LTC4 was detectable. LTB4 was stable under these conditions. Therefore the enzymatic urinary metabolism of LTs should be considered in experimental and clinical studies when urine is collected under different conditions.

Measurement of urinary leukotrienes by reversed-phase liquid chromatography and radioimmunoassay.

Leukotriene (LT) E4, an important LT metabolite appearing in urine, can be rapidly separated from normal and pathological urines by automated reversed-phase HPLC after a simple sample-processing. The recoveries of LTE4 afforded by this system (86.4 +/- 6.5%, mean +/- SEM for 60 ng/L, 85.4 +/- 0.3% for 200 ng/L) are superior to those obtained by a manual extraction method. Consistency of results is similar. Highly reproducible retention times combined with a radioimmunoassay allow one to identify (based on co-elution) and quantify as little as 8 ng/L LTE4 in a 10-mL urine sample. LTE4 concentrations in urine from healthy persons approach this value (17 +/- 5 ng/L), whereas samples from patients with cardiac ischemia show a wider range of concentrations (8 to 388 ng/L), up to 50 times the detection limit. Thus this method is applicable to the noninvasive investigation of leukotriene involvement in a wide range of ischemic, inflammatory, and hypersensitive conditions.

Increased excretion of leukotriene E4 during aspirin-induced asthma.

The etiology of aspirin-sensitive asthma is unknown, but a plausible hypothesis is that the inhibitory effect of aspirin on the cyclooxygenase enzyme increases formation of bronchoconstrictor leukotrienes via "shunting" of unmetabolized arachidonic acid into metabolism by the 5-lipoxygenase enzyme. The severity and rapidity of bronchospasm that is induced by cyclooxygenase-inhibiting drugs in aspirin-sensitive asthmatics is directly related to the dose and to the potency of the drug to inhibit the cyclooxygenase enzyme. Since increased leukotriene synthesis has recently been shown to occur during allergen-induced asthma, we have examined whether altered leukotriene synthesis correlates with the degree of either cyclooxygenase inhibition or bronchospasm during asthma that is induced by doses of aspirin that range from 30 to 365 mg in individual patients. Excretion of leukotriene E4 was increased by a mean of 361% +/- 76% (p less than 0.05) during aspirin-induced asthma episodes, but the degree of increase for individual patients did not correlate with the degree of bronchospasm or inhibition of platelet thromboxane B2 formation. Thus although the endogenous synthesis of potent bronchoconstrictor leukotrienes increases during aspirin-induced bronchospasm, it appears unlikely that a direct "shunting" of unmetabolized arachidonate into leukotriene synthesis represents the mechanism of aspirin-induced asthma.

Tissue distribution, elimination and metabolism of [3H]-leukotriene C4 in the American bullfrog, Rana catesbeiana.

Tissue distribution, elimination, and metabolism of [3H]-leukotriene C4 were studied at 2.5 hours after injection in the conscious and anesthetized American bullfrog, Rana catesbeiana. Conscious frogs were injected via the dorsal lymph sac or the sciatic vein. Anesthetized frogs were injected via the abdominal vein. The organs containing the greatest percent of injected radioactivity at 2.5 hours after injection were liver, small intestine and kidney. Route of injection and anesthesia appears to alter distribution and elimination of leukotrienes. [3H]-leukotrienes were eliminated into bladder water and bile. In addition, 7.8 +/- 2.2 and 5.2 +/- 2.5 percent of the injected radioactivity was found in the pan water bathing the ventral surface of the venously and dorsally injected conscious frogs, respectively, suggesting transfer of radioactivity across the skin. At 2.5 hours, polar metabolites represented 50% of the radioactivity found in liver, bile, and bladder water. These polar metabolites were determined to be 18-carboxy-19,20-dinor-leukotriene E4, 20-carboxy-leukotriene E4, and 20-hydroxy-leukotriene E4. Of the non-oxidized leukotrienes, bile contained mainly LTD4 while bladder water contained primarily LTE4. N-acetyl LTE4 was not detected in any samples. The tissue distribution, elimination and metabolism of leukotrienes in the bullfrog was similar to mammalian studies and suggests evolutionary conservation of leukotriene processing.

In vivo metabolism of leukotriene C4 in man: urinary excretion of leukotriene E4.

Five - 20 nmoles of [5,6,8,9,11,12,14,15-3H8]leukotriene C4 was injected into three male volunteers. Forty-eight percent of the administered 3H was recovered from urine and 8% from feces, within a 72 hr period. Of the total urinary radioactivity 44% was excreted during the first hour after injection. This activity was mainly found in one compound, designated "I". The radioactivity excreted into urine later than one hour after injection, consisted partly of Compound I and two additional components, and partly of polar, non-volatile material. Compound I was identified as leukotriene E4 by UV-spectroscopy and cochromatographies in three high performance liquid chromatography systems with synthetic reference compounds. A total of 13% of administered radioactivity was excreted in urine as leukotriene E4.

Biliary and urinary excretion of peptide leukotrienes in the domestic pig.

The metabolism of leukotriene (LT)C4 and its major routes of elimination in vivo have been studied in four anesthetized domestic pigs administered intravenous [3H]-LTC4 (0.5 microCi/kg). The kinetic profile of LTC4 in the blood was followed for 60 min after administration while the biliary and urinary excretion of LTC4 and its metabolites were determined over a 120 min interval. The total recovery of radioactivity in bile and urine was 45% +/- 1 (n = 3) and 18% (n = 2) respectively. Examination of the radioactive metabolites in bile showed LTD4 (44% of biliary content) and LTE4 (21% of biliary content) as the major identified lipoxygenase products at t 1/2 (27 min). The only identified cysteinyl leukotriene observed in the urine was LTE4 (13% of urinary content). In both bile and urine substantial amounts of radioactivity were detected at the solvent front of the reverse phase chromatographic system indicating the presence of additional unidentified metabolites. We suggest that measurement of metabolites using these sampling methods may be useful for the detection and measurement of peptide leukotriene production in vivo.

The excretion of leukotriene E4 into urine following inhalation of leukotriene D4 by human individuals.

Healthy volunteers underwent bronchial challenge with increasing doses of nebulized leukotriene D4 (0.007 - 200 nmol) at 15 min intervals. Total amounts of 200 nmol (females) and 400 nmol (males) were inhaled, corresponding to approximately 100 nmol and 200 nmol deposited in the lung, respectively. Of the latter amounts 3 +/- 1% (mean +/- S.E.M., n = 5) was found to be excreted as leukotriene E4 into the urine within 12 h. No further excretion after this period was observed. Approximately 50% of the total urinary leukotriene E4 was excreted during the first 2 h. These results suggest that a possible formation of sulfidopeptide leukotrienes in the lung in vivo can be monitored by measuring leukotriene E4 excretion into the urine.

BIO-Fully Automated Sample Treatment high-performance liquid chromatography and radioimmunoassay for leukotriene E4 in human urine from asthmatics.

BIO-Fully Automated Sample Treatment (BIO-FAST) high-performance liquid chromatography (HPLC) is a sophisticated column-switching technique in which a fresh pre-column is used for each sample prior to reversed-phase HPLC. The pre-columns, Varian Advanced Automated Sample Processor (AASP) cartridges, are held and automatically advanced by the Varian AASP. A rapid and efficient extraction and separation for leukotrienes C4 and E4 from human urine has been developed using a C8 cartridge and subsequent C18 analytical HPLC column. Quantitation of leukotriene E4, accomplished by post-column radioimmunoassay, shows significantly increased leukotriene E4 concentrations in urine samples from asthmatics after antigen challenge. This further confirms an active role for leukotrienes in the pathogenesis of bronchial asthma.

Comparison of urinary leukotriene E4 and 16-carboxytetranordihydro leukotriene E4 excretion in allergic asthmatics after inhaled antigen.

Antisera to 16-carboxytetranordihydro leukotriene E4 (tetranor LTE4), a major urinary oxidative metabolite (via omega- and beta-oxidation) of leukotriene E4 (LTE4) in primates, were obtained by immunisation of rabbits with a related, non-naturally occurring synthetic metabolite (16-carboxytetranordihydro leukotriene C4 ester) conjugated to Keyhole Limpit haemocyanin. Material which competed with [11, 12-3H]tetranor LTE4 for binding to this antisera was isolated from urine from allergic asthmatics by reversed-phase HPLC. This material eluted with the retention time of synthetic standards, and its mean urinary excretion was elevated during both the first three hours (6.13 +/- 2.15 ng/h) and 3-6 h (5.87 +/- 1.99 ng/h) after antigen inhalation, compared with baseline values (3.42 +/- 1.49 ng/h), in 5 allergic mild asthmatics. A much greater and statistically significant increase in urinary leukotriene E4 (LTE4) excretion, occurring in all subjects, was seen during acute antigen-induced bronchoconstriction (baseline, 1.62 +/- 0.66 ng/h; 0-3 h, 19.58 +/- 8.79 ng/h; p less than 0.05) in these subjects. These data support the suggestion that endogenous peptide leukotrienes are metabolised by omega- and subsequent beta-oxidation in man, but emphasize the relative importance of urinary LTE4 excretion after allergen elicited leukotriene generation, further substantiating a pathological role for peptide leukotrienes in allergic asthma.

Urinary leukotriene E4 levels during early and late asthmatic responses.

The sulphidopeptide leukotrienes C4 and D4 (LTC4, LTD4) are potent bronchoconstrictor mediators, released from human lung fragments after challenge with specific allergens in vitro. The purpose of this study was to measure urinary LTE4 (metabolite of LTC4 and LTD4) in subjects undergoing inhalation challenges with allergens or occupational sensitizing agents in the laboratory. Eighteen subjects with previously documented isolated early asthmatic responses (EARs), isolated late asthmatic responses (LARs), or dual (both early and late) asthmatic responses were studied. Urinary LTE4 levels increased in subjects who developed either isolated EARs (mean fall in FEV1, 27.98%) or early responses preceding LARs (mean fall in FEV1, 15.01%). The baseline levels of LTE4 were 150.26 (SEM, 49.5) pg/mg of creatinine in the isolated responders and 66.60 (SEM, 13.5) pg/mg of creatinine in the dual responders. These levels increased to 1816 (SEM, 606.1) pg/mg of creatinine (p = 0.041) and 174.80 (SEM, 40.1) pg/mg of creatinine (p = 0.025), respectively, after the EAR. The degree of maximal bronchoconstriction during the EAR correlated with the levels of LTE4 (r = 0.68; p = 0.001). No significant increase in urinary LTE4 levels occurred during the LAR. These results suggest that the LTE4 precursors, LTC4 and LTD4, are important bronchoconstrictor mediators causing EARs after allergen inhalation.