Urinary Leukotriene E4 and Prostaglandin D2 Metabolites Increase in Adult and Childhood Severe Asthma Characterized by Type 2 Inflammation. A Clinical Observational Study

Abstract

Rationale: New approaches are needed to guide personalized treatment of asthma.Objectives: To test if urinary eicosanoid metabolites can direct asthma phenotyping.Methods: Urinary metabolites of prostaglandins (PGs), cysteinyl leukotrienes (CysLTs), and isoprostanes were quantified in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study including 86 adults with mild-to-moderate asthma (MMA), 411 with severe asthma (SA), and 100 healthy control participants. Validation was performed internally in 302 participants with SA followed up after 12-18 months and externally in 95 adolescents with asthma.Measurement and Main Results: Metabolite concentrations in healthy control participants were unrelated to age, body mass index, and sex, except for the PGE₂ pathway. Eicosanoid concentrations were generally greater in participants with MMA relative to healthy control participants, with further elevations in participants with SA. However, PGE₂ metabolite concentrations were either the same or lower in male nonsmokers with asthma than in healthy control participants. Metabolite concentrations were unchanged in those with asthma who adhered to oral corticosteroid treatment as documented by urinary prednisolone detection, whereas those with SA treated with omalizumab had lower concentrations of LTE₄ and the PGD₂ metabolite 2,3-dinor-11β-PGF_2α. High concentrations of LTE₄ and PGD₂ metabolites were associated with lower lung function and increased amounts of exhaled nitric oxide and eosinophil markers in blood, sputum, and urine in U-BIOPRED participants and in adolescents with asthma. These type 2 (T2) asthma associations were reproduced in the follow-up visit of the U-BIOPRED study and were found to be as sensitive to detect T2 inflammation as the established biomarkers.Conclusions: Monitoring of urinary eicosanoids can identify T2 asthma and introduces a new noninvasive approach for molecular phenotyping of adult and adolescent asthma.Clinical trial registered with www.clinicaltrials.gov (NCT01976767).

Keywords: U-BIOPRED; mass spectrometry; severe asthma; type 2 inflammation; urinary eicosanoid metabolites.

Figure 1.

Schematic overview of arachidonic acid–derived lipid mediators (eicosanoids) following both enzymatic and nonenzymatic metabolism. Blue text indicates the known or proposed biologic effect of the indicated pathway. Gray boxes highlight eicosanoids quantified in urine from participants in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study by UPLC-MS/MS. 5-LOX = 5-lipoxygenase; COX = cyclooxygenase; cPLA₂ = cytosolic phospholipase A₂; FLAP = five lipoxygenase-activating protein; iPF_2α = isoprostane-F_2α; LT = leukotriene; LTC4S = LTC4-synthase; PG = prostaglandin; PGDS = PGD-synthase; PGES = PGE-synthases; PGFS = PGF-synthase; PGIS = PGI-synthase; tetranorPGDM = tetranor PGD₂ metabolite; tetranorPGEM = tetranor PGE₂ metabolite; TX = thromboxane; TXAS = TXA-synthase; UPLC–MS/MS = ultraperformance liquid chromatography–tandem mass spectrometry.

Figure 2.

Median (interquartile range) urinary concentration of individual eicosanoids in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) adult baseline healthy participant (healthy control) group (n = 100). TetranorPGEM concentrations are stratified by sex. CysLT = cysteinyl LT; iPF_2α = isoprostane-F_2α; LT = leukotriene; PG = prostaglandin; tetranorPGDM = tetranor PGD₂ metabolite; tetranorPGEM = tetranor PGE₂ metabolite; TX = thromboxane.

Figure 3.

Distribution of urinary eicosanoid concentrations in HC (n = 100), MMA (n = 86), SAn (n = 302), and SAs/ex (n = 109) for (A and B) PGD₂ metabolites, (C) PGF_2α, (D) LTE₄, (E and F) thromboxane metabolites, (G–I) isoprostanes, (J) PGE₂, and (K and L) and PGE₂ metabolites. Data are plotted on a log₂ scale. Boxes highlight the interquartile range with the group median; bars display the total distribution range (minimum to maximum). Significant group differences are indicated by P values determined by the Mann-Whitney U test. HC = healthy control participants; iPF_2α = isoprostane-F_2α; LT = leukotriene; MMA = participants with mild-to-moderate asthma; PG = prostaglandin; SAn = nonsmokers with severe asthma; SAs/ex = smokers or ex-smokers with severe asthma; tetranorPGDM = tetranor PGD₂ metabolite; tetranorPGEM = tetranor PGE₂ metabolite; TX = thromboxane.

Figure 4.

Effect of oral corticosteroids (OCS) on observed urinary eicosanoid concentrations. (A) Stratification of adult U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) participants with severe asthma according to reported daily use of OCS and detection of prednisolone or prednisolone metabolites in urine (OCS, n = 90; no OCS, n = 167) (data from Table 4). (B) Median (interquartile range [IQR]) of urinary LTE₄, tetranorPGDM, and 2,3-dinor-11β-PGF_2α concentrations across participants with detectable urinary prednisolone in the adult U-BIOPRED study. Of the 90 individuals in which prednisolone or its metabolites were detected, parent prednisolone was only detected in 68 individuals. Quantified urinary prednisolone is stratified as follows: <500 (n = 17), 500–2,000 (n = 27), and >2,000 ng/ml (n = 24). (C) Median (IQR) of urinary concentration of CysLT and PGD₂ metabolites across the three inhaled corticosteroid budesonide dose groups in adolescent children from the Swedish Search study. Budesonide eq. is stratified as follows: <500 (n = 38), 500–<1,000 (n = 46), and ≥1,000 μg (n = 11). CysLT = cysteinyl LT; eq. = equivalents; iPF_2α = isoprostane-F_2α; LT = leukotriene; PG = prostaglandin; tetranorPGDM = tetranor PGD₂ metabolite; tetranorPGEM = tetranor PGE₂ metabolite; TX = thromboxane.

Figure 5.

Participants with SA from the adult U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study with a history of omalizumab treatment (n = 52) were compared in a case-control design (1:2) to a group (n = 104) matched for similarities in serum IgE. The violin-plot horizontal lines correspond to group median and interquartile-range values for the two PGD₂ metabolites and LTE₄. Group comparisons were evaluated by the Mann-Whitney U test (data from Table E3). LT = leukotriene; PG = prostaglandin; SA = severe asthma; tetranorPGDM = tetranor PGD₂ metabolite.

Figure 6.

Selection of U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) adult participants with mild-to-moderate asthma, nonsmokers with severe asthma, and smokers/ex-smokers with severe asthma at baseline using the 25th and 75th concentration percentile of urinary LTE₄ and calculated composite (log₂-transformed and z-scored) variables for PGD₂ metabolites (c-PGD₂). A complete list of significant associations is provided in Table E6. At the 12- to 18-month follow-up visit (longitudinal), 302 participants with severe asthma were used for internal validation of the type 2 associations. Data are presented as the median and interquartile range and were evaluated by using the Mann-Whitney U test. c-PGD₂ = combined PGD₂ metabolites; Fe_NO = fractional exhaled nitric oxide; LTE₄ = leukotriene E₄; PGD₂ = prostaglandin D₂; ppb = parts per billion.

Figure 7.

Validation of adult type 2 associations in adolescent participants from the Swedish Search study. Participants with severe or controlled persistent asthma were stratified using the 25th and 75th concentration percentile of urinary metabolites of PGD₂ and CysLTs. For each percentile, the corresponding variable group median (interquartile range) differences were evaluated by the Mann-Whitney U test. *Total urinary PGD₂ metabolites were determined by enzyme immunoassay, which has a 9:1 binding ratio toward 2,3-dinor-11β-PGF_2α:11β-PGF_2α as determined by a cross-reactivity test and ultraperformance liquid chromatography–tandem mass spectrometry (9). CysLT = cysteinyl leukotrienes; EDN = eosinophil-derived neurotoxin; Fe_NO = fractional exhaled nitric oxide; PGD₂ = prostaglandin D₂; PGF_2α = prostaglandin F_2α; ppb = parts per billion.

Figure 8.

Relationship between proposed type 2 (T2) markers (A) at baseline and (B) at the 12- to 18-month longitudinal follow-up visit in the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) study. The heat map displays the percentage overlap between high amounts of four common T2 markers and the two urinary eicosanoid markers leukotriene E₄ (LTE₄) and combined prostaglandin D₂ metabolites (c-PGD₂). Established cutoffs (21) for the T2 markers were used (blood eosinophils ≥ 300 counts/μl, Fe_NO ≥ 30 ppb, periostin ≥ 55 ng/ml, and IgE ≥ 150 IU/ml). Cutoff values for the urinary metabolites were calculated from the median + 1 SD in the healthy control group (Table 3). Each cell represents the percentage of participants satisfying both cutoffs for a given comparison. For example, in the first row in A, 62% of the n = 195 participants with blood eosinophils ≥ 300 counts/μl also have urinary LTE₄ ≥ 6.4 ng/mmol creatinine. The total number of participants positive for each row criterion is displayed in the far-right column. Serum IgE data were only available at baseline. c-PGD₂ is a z-scored composite variable consisting of tetranorPGDM and 2,3-dinor-11β-PGF_2α (see Methods). Fe_NO = fractional exhaled nitric oxide; ppb = parts per billion; tetranorPGDM = tetranor PGD₂ metabolite.

Figure 9.

Multivariate correlation analysis between the latent variable consisting of the markers employed to calculate the Refractory Asthma Stratification Program (RASP) type 2 severity score (B-eos, Fe_NO, and serum periostin) (21) and the latent variable representing concentrations of the three urinary eicosanoid metabolites: LTE₄, tetranorPGDM, and 2,3-dinor-11β-PGF_2α. The correlation for participants with a high RASP score with urinary eicosanoid concentrations was significant across all U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) asthma groups at both (A) baseline (n = 112) and (B) the longitudinal follow-up (n = 52). In contrast, the correlation for baseline participants with a (C) intermediate (n = 200) or (D) low (n = 75) RASP score displayed no relevant correlation with the urinary eicosanoid concentrations, as evidenced by the flat slopes (y = 0.04x). PGD₂-metabolites include tetranorPGDM and 2,3-dinor-11β-PGF_2α. B-eos = blood eosinophils; Fe_NO = fractional exhaled nitric oxide; LTE₄ = leukotriene E₄; MMA = participants with mild-to-moderate asthma; PGD₂ = prostaglandin D₂; SAn = nonsmokers with severe asthma; SAs/ex = smokers or ex-smokers with severe asthma; t = scores vector for x-block, calculated from the variables defined in brackets; tetranorPGDM = tetranor PGD₂ metabolite; u = scores vector for y-block, calculated from the variables defined in brackets.