Comparison of Three Urine Normalization Strategies for Mass Spectrometric Analysis in a Pediatric Asthma Model.

Abstract

INTRODUCTION: Metabolomics studies the products of metabolism within a biological system to determine biomarkers for disease diagnosis. Metabolite values found in urine analysis require a method to normalize the data to account for urine concentration. Creatinine (CRN) is a commonly used method of LC-MS/MS data normalization; however, CRN is affected by muscle mass and height (HT) in children. This could be a concern when comparing children's samples across various age groups. Referencing CRN to HT (CRN/HT) is a possible strategy to correct for such a concern. Another approach is to reference each metabolite to the total concentration of the metabolites, a strategy termed the total metabolome (TM). METHODS: We evaluated age/HT as a confounder in pediatric metabolomic data analysis by comparing CRN, CRN/HT, or TM normalization in children aged 3-13 with or without asthma (n = 152). LC-MS/MS measured 41 metabolites in urine samples, which were normalized to the three methods. These metabolites were then used to differentiate children with asthma versus healthy children. RESULTS: CRN was affected by age, unlike CRN/HT or TM. The metabolites selected to differentiate healthy and asthmatic children differed based on the normalization technique; 10 metabolites were shared across models. Diagnostic accuracy was affected by the normalization strategy. CONCLUSION: Using CRN/HT or TM limits the variation seen across ages and was better than CRN alone. The normalization strategy for hydration affects model accuracy in pediatric research. There is a need for better diagnosis of asthma in children. Metabolomics studies the products of metabolism within a biological system to determine biomarkers for disease diagnosis. Urine contains many potential diagnostic biomarkers, but their quantity is impacted by the urine concentration of the child. To correct for this, urine data are normalized to creatinine. While studying different ages of children, we became concerned that creatinine was not adequate because it was affected by age and height. This article describes the issue and two possible solutions to address creatinine shortcomings within pediatric populations.

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