Serum eicosanoid analysis in Chinese pediatric patients with asthma
Asthma is a reversible obstructive lung disease, caused by increased reaction of the airways as various stimuli, and is diagnosed by the medical measurement of the airflow in and out of the lungs. However, children who are pre‐school age or younger may be unable to complete the airflow test, which requires blowing very hard into a tube.1 Therefore, it is vital to develop a simple and fast method for the determination of pediatric asthma.2 Eicosanoids such as cysteinyl leukotrienes (Cys‐LTs), prostaglandins (PGs), and thromboxanes (TXs) play important roles in the pathophysiology of asthma.3 So far, several independent studies have indicated that elevated levels of eicosanoid metabolites are associated with adult asthma. However, the correlation of eicosanoids and pediatric asthma is still unknown.
In this study, thirty‐nine children with persistent asthma and twenty‐seven healthy children with no history of atopy or respiratory diseases were recruited in Shanghai PuDong Women and Children Hospital. The ethics committee of this hospital approved this study, and written informed consent was obtained from the child's parents. According to international guidelines,4 the diagnosis of asthma was based on clinical history and medical examination, spirometry, and forced expiratory volume in 1 second (FEV1) reversibility ≥12% demonstrated at least once in the previous 6 months. Asthmatic symptoms in patients were well controlled with β2 short‐acting agonist as need. All children have no significant allergen exposure and do not take medicines in the 3 weeks before the recruitment.
There are no significant differences in age, gender, and body mass index (BMI) z‐scores between asthmatics and healthy controls. Fraction exhaled nitric oxide (FeNO) measurement and lung function tests were performed 15 minutes after blood collection. Meanwhile, a complete blood count test was carried out using a standard automated cell counter.5 The asthmatics showed a significantly higher absolute peripheral blood eosinophil count, serum total immunoglobulin E (IgE), and fractional exhaled nitric oxide (FENO) levels (Table S1).
Serum eicosanoid levels were measured based our earlier method.6 In brief, 20 μL serum was mixed with 180 μL phosphate‐buffered saline (PBS) containing internal standards (listed in Table S2). The mixture was extracted using Strata‐X 33u Polymeric SPE cartridge (100 mg 3 mL). After loading, cartridges were washed with 1 mL 10% methanol in water to remove impurities, and eluted with 1 mL MeOH to collect eicosanoids. All elutes were stored in −80°C to prevent metabolite degradation. Prior to analysis, the eluents were dried with N2 and dissolved in 50 μL methanol for liquid chromatography mass spectrometry (LC‐MS) analysis. The LC‐MS parameters were provided in Tables S2 and S3.
A total of 16 eicosanoids were consistently detected in all samples. Their concentrations were shown in Table S4. The eicosanoid levels in two groups were compared using student's test, and values of P‐value <.05 were considered statistically significant. Statistical analysis indicated that prostaglandin D2 (PGD2), 6keto‐prostaglandin F1α (6keto‐PGF1α), thromboxane B2 (TXB2), leukotriene E4 (LTE4), 5‐Hydroxyeicosatetraenoic acid (5‐HETE), and 15‐HETE in serum are markedly elevated in the asthmatic group (Figure 1).
Among these eicosanoids, PGD2, 6keo‐PGF1α, and TXB2 that are formed from arachidonic acid by cyclooxygenase (COX), showed significant increases in the asthma group, and these data agreed with earlier findings.7 We also noted higher levels of LTE4 in the serum of asthmatic children when compared with healthy control subjects, which is consistent with a previous study in adult urine sample.8 5‐HETE that is the intermediate metabolite of 5‐lipoxygenase (5‐LO) pathway has also been found to be considerably elevated in asthmatic children as compared with healthy controls. 5‐HETE has been reported to be significantly enhanced in exhaled breath condensate (EBC) in adult asthmatics following bronchial aspirin challenge.