Characteristics of Normalization Methods in Quantitative Urinary Metabolomics—Implications for Epidemiological Applications and Interpretations

Abstract

A systematic comparison is presented for the effects of seven different normalization schemes in quantitative urinary metabolomics. Morning spot urine samples were analyzed with nuclear magnetic resonance (NMR) spectroscopy from a population-based group of 994 individuals. Forty-four metabolites were quantified and the metabolite–metabolite associations and the associations of metabolite concentrations with two representative clinical measures, body mass index and mean arterial pressure, were analyzed. Distinct differences were observed when comparing the effects of normalization for the intra-urine metabolite associations with those for the clinical associations. The metabolite–metabolite associations show quite complex patterns of similarities and dissimilarities between the different normalization methods, while the epidemiological association patterns are consistent, leading to the same overall biological interpretations. The results indicate that, in general, the normalization method appears to have only minor influences on standard epidemiological regression analyses with clinical/physiological measures. Multimetabolite normalization schemes showed consistent results with the customary creatinine reference. Nevertheless, interpretations of intra-urine metabolite associations and nuanced understanding of the epidemiological associations call for comparisons with different normalizations and accounting for the physiology, metabolism and kidney function related to the normalization schemes.

Clinical and biochemical associations of urinary metabolites: quantitative epidemiological approach on renal-cardiometabolic biomarkers

“…For the details of the urine sample preparation and the NMR spectroscopy experimentation, we refer to earlier open-access publications. 7 , 17 …”

“…Referencing to creatinine has been a long-term standard choice in urine NMR metabolomics, but we have also recently comprehensively studied the effects of various normalization methods, also supporting the use of creatinine referencing in epidemiological studies. 17 …”

“…The chemical taxonomy of the metabolites can be seen in Table 2 . 17 Replication of the associations is illustrated in Figure 4 . In all, 56 principal components explained over 99% of variation in the 60 creatinine-referenced urinary metabolite concentrations and the 49 clinical and biochemical measures in NFBC1966.…”

“…Towards this far-reaching aim, we have recently developed a basis for an open-access methodology for quantitative high-throughput urinary nuclear magnetic resonance (NMR) metabolomics. 1 , 7 , 17 …”