Abstract
The application of metabolic phenotyping in clinical and epidemiological studies is limited by a poor understanding of inter-individual, intra-individual and temporal variability in metabolic phenotypes. Using 1H NMR spectroscopy we characterised short-term variability in urinary metabolites measured from 20 children aged 8–9 years old. Daily spot morning, night-time and pooled (50:50 morning and night-time) urine samples across six days (18 samples per child) were analysed, and 44 metabolites quantified. Intraclass correlation coefficients (ICC) and mixed effect models were applied to assess the reproducibility and biological variance of metabolic phenotypes. Excellent analytical reproducibility and precision was demonstrated for the 1H NMR spectroscopic platform (median CV 7.2%). Pooled samples captured the best inter-individual variability with an ICC of 0.40 (median). Trimethylamine, N-acetyl neuraminic acid, 3-hydroxyisobutyrate, 3-hydroxybutyrate/3-aminoisobutyrate, tyrosine, valine and 3-hydroxyisovalerate exhibited the highest stability with over 50% of variance specific to the child. The pooled sample was shown to capture the most inter-individual variance in the metabolic phenotype, which is of importance for molecular epidemiology study design. A substantial proportion of the variation in the urinary metabolome of children is specific to the individual, underlining the potential of such data to inform clinical and exposome studies conducted early in life.
Highlights
A study with access to repeat sampling under controlled conditions with human subjects showed that 22% of the identified metabolites in 1H NMR urinary spectroscopic profiles exhibited a significant 24 h cosine rhythm[7]
The percentage coefficient of variation (CV%) of the Quality control (QC) sample was calculated for integrals of NMR signals representing individual metabolites and showed an average CV% of 7.2% and median 7.7%
Metabolites with a low signal to noise ratio (S/N) in QCs, such as N-methylpicolinic acid, 3-aminoisobutyrate, N1-methylnicotinamide and acetone, presented a CV% over 10%
Summary
A study with access to repeat sampling under controlled conditions with human subjects showed that 22% of the identified metabolites in 1H NMR urinary spectroscopic profiles exhibited a significant 24 h cosine rhythm[7] This effect is believed to be marked in the morning in comparison to the rest of the day. This reflects a more pronounced influence on homeostasis when food is consumed after an 8 h fast as compared to a shorter inter-meal time interval during the day, which may be due to circadian influences independent of meal consumption[6] It has been shown in in-patient studies with tightly controlled environmental conditions, that diet or day-to-day variability do not account for the largest source of variability in either blood or urine metabolic profiles[8,9]. We confirm the high analytical reproducibility and robustness of NMR-based urinary metabolic phenotyping (as reported elsewhere, ref. 12) and illustrate the benefits of pooling spot urines when seeking the stable component of the metabolome
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