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 the urine concentration. Creatinine (CRN) is a commonly used method of LC-MS-MS data normalization; however, CRN is affected by muscle mass and height in children. This could be a concern when comparing children’s samples across various age groups. Referencing creatinine-to-height (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/height as a confounder in pediatric metabolomic data analysis, by comparing CRN, CRN/HT, or TM normalization in children between the ages of 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 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.