Assessment of reproducibility and biological variability of fasting and postprandial plasma metabolite concentrations using 1H NMR spectroscopy

Abstract

Introduction: It is crucial to understand the factors that introduce variability before applying metabolomics to clinical and biomarker research.

Objectives: We quantified technical and biological variability of both fasting and postprandial metabolite concentrations measured using 1H NMR spectroscopy in plasma samples.

Methods: In the Netherlands Epidemiology of Obesity study (n = 6,671), 148 metabolite concentrations (101 metabolites belonging to lipoprotein subclasses) were measured under fasting and postprandial states (150 minutes after a mixed liquid meal). Technical variability was evaluated among 265 fasting and 851 postprandial samples, with the identical blood plasma sample being measured twice by the same laboratory protocol. Biological reproducibility was assessed by measuring 165 individuals twice across time for evaluation of short- (<6 months) and long-term (>3 years) biological variability. Intra-class coefficients (ICCs) were used to assess variability. The ICCs of the fasting metabolites were compared with the postprandial metabolites using two-sided paired Wilcoxon test separately for short- and long-term measurements.

Results: Both fasting and postprandial metabolite concentrations showed high technical reproducibility using 1H NMR spectroscopy (median ICC = 0.99). Postprandial metabolite concentrations revealed slightly higher ICC scores than fasting ones in short-term repeat measures (median ICC in postprandial and fasting metabolite concentrations 0.72 versus 0.67, Wilcoxon p-value = 8.0×10-14). Variability did not increase further in a long-term repeat measure, with median ICC in postprandial of 0.64 and in fasting metabolite concentrations 0.66.

Conclusion: Technical reproducibility is excellent. Biological reproducibility of postprandial metabolite concentrations showed a less or equal variability than fasting metabolite concentrations over time.

Fig 1. Workflow of study design and statistical analyses for biological replication.

Fig 2. The ICC score distributions on biological reproducibility by (A) comparisons between fasting and postprandial, and (B) short- and long-term visits.

Metabolites are ranked ascendingly by fasting (top) and short-term ICC scores (bottom). The lower corner represents the density plot of ICC score distributions from fasting and postprandial samples (top) or short- and long-term samples (bottom) separately, with the p-value derived from Wilcoxon two-sided test. The dash lines correspond to the median ICC scores among fasting (blue) and postprandial (green) samples (top) or short- (blue) and long-term (green) samples (bottom).

Fig 3. The ICC score distributions for biological reproducibility by comparisons between fasting/postprandial state, stratified on time interval between visits (short-/long-term).

Metabolites are ranked ascendingly by fasting short-term (blue dots). The lower corner contains a density plot of ICC score distributions from fasting short-term (blue), fasting long-term (green), postprandial short-term (purple) and postprandial long-term (orange) separately. The dash lines correspond to the median ICC scores among four scenarios.

Fig 4. Decomposition of variance for each metabolite.

Metabolites are ordered by commercial clusters. Fasting/postprandial state (F/P) corresponds to fasting (F) and postprandial; Time interval (S/L) stands for short- (S) and long-term.