Respiratory health of children is a health priority. Club cell protein (CC16) is an interesting biomarker of lung diseases and adverse effects towards the airway epithelium integrity. Osteopontin (OPN) and nuclear factor-kappa B (NF-κB) also play a role in respiratory health. The use of urine as biomarker source is useful in studies involving children but necessitates proper adjustment for physiological confounders influencing the urinary excretion, potentially characterized with beta-2-microglobulin (β2M), retinol binding protein 4 (RBP4) or myoglobin (MYO), as well as adjustment for possible renal dysfunction, characterized by human serum albumin (HSA). The simultaneous quantification of all these proteins in urine could facilitate children’s health monitoring. A multiple reaction monitoring method (MRM) was developed and validated for the relative quantification of the seven mentioned urinary proteins. A total of nine proteotypic peptides were selected and used for the relative quantification of the seven proteins. The MRM method was completely validated for all proteins and partially for OPN. LOQ’s ranged from 0.3 to 42.8 ng/ml, a good reproducibility and a good linearity were obtained across the analytical measurement range (r2 > 0.98). The method yielded varying correlations (r2 of 0.78, 0.71, 0.34 and 0.15 for CC16, β2M, RBP4 and HSA respectively) with available immunoassay data. It also allowed the identification and successful quantification of β2M and RBP4 as a protein candidate for adjustment of renal handling and dysfunction. All proteins were detected in the urine samples except for MYO and NF-κB. Our validated MRM-method is able to simultaneously quantify in urine biomarkers of airway epithelium integrity and biomarkers of variation in renal function and urinary dilution. This will allow to investigate further in future studies if urine can be used as a good surrogate source for biomarkers of airway epithelium integrity, and to understand the complex relationship between cause and effect in children’s respiratory health monitoring.
Background
Studies that investigated the association between the CC16 A38G polymorphism and the risk of asthma yielded conflicting results. The aim of this study among schoolchildren was to assess the relationships of CC16 A38G polymorphism with aeroallergen sensitization and fractional exhaled nitric oxide (FeNO), two outcomes predicting asthma later in life.
Methods
The study included 139 children (72 boys), median age of 7.7. Information on each child's health, lifestyle, and environment was collected through a questionnaire completed by their parents. CC16 genotypes were determined using urinary DNA. We measured FeNO, the CC16 protein in urine and nasal lavage fluid and aeroallergen‐specific immunoglobulin E in nasal mucosa fluid.
Results
Children with the homozygous mutant CC16 38AA genotype had higher odds of increased FeNO (>30 ppb) compared with their peers with the wild‐type genotype 38GG (OR, 9.85; 95% CI, 2.09‐46.4; P = .004). This association was female gender specific (P = .002) not being observed in boys (P = .40). It was also independent of allergic sensitization, which yet emerged as the strongest predictor of FeNO along with the use of bleach for house cleaning. Children with the CC16 38AA genotype had lower covariates‐adjusted urinary CC16 levels than those with 38GG (median, μg/L, 1.17 vs 2.08, P = .02).
Conclusion
Our study suggests that the CC16 38AA allele promotes airway inflammation as measured by FeNO through a gender‐dependent association. Deficient levels of CC16 in the deep lung, measured noninvasively in urine, as a possible proxy for serum CC16, might underlie this promoting effect.
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