Benzoic acid is widely used as a preservative in food products and is detoxified in humans through glycine conjugation. Different viewpoints prevail on the physiological significance of the glycine conjugation reaction and concerns have been raised on potential public health consequences following uncontrolled benzoic acid ingestion. We performed a metabolomics study which used commercial benzoic acid containing flavored water as vehicle for designed interventions, and report here on the controlled consumption of the benzoic acid by 21 cases across 6 time points for a total of 126 time points. Metabolomics data from urinary samples analyzed by nuclear magnetic resonance spectroscopy were generated in a time-dependent cross-over study. We used ANOVA-simultaneous component analysis (ASCA), repeated measures analysis of variance (RM-ANOVA) and unfolded principal component analysis (unfolded PCA) to supplement conventional statistical methods to uncover fully the metabolic perturbations due to the xenobiotic intervention, encapsulated in the metabolomics tensor (three-dimensional matrices having cases, spectral areas and time as axes). Identification of the biologically important metabolites by the novel combination of statistical methods proved the power of this approach for metabolomics studies having complex data structures in general. The study disclosed a high degree of inter-individual variation in detoxification of the xenobiotic and revealed metabolic information, indicating that detoxification of benzoic acid through glycine conjugation to hippuric acid does not indicate glycine depletion, but is supplemented by ample glycine regeneration. The observations lend support to the view of maintenance of glycine homeostasis during detoxification. The study indicates also that time-dependent metabolomics investigations, using designed interventions, provide a way of interpreting the variation induced by the different factors of a designed experiment–an approach with potential to advance significantly our understanding of normal and pathophysiological perturbations of endogenous or exogenous origin.

Metabolomics studies of disease conditions related to chronic alcohol consumption provide compelling evidence of several perturbed metabolic pathways underlying the pathophysiology of alcoholism. The objective of the present study was to utilize proton nuclear magnetic resonance (1H-NMR) spectroscopy metabolomics to study the holistic metabolic consequences of acute alcohol consumption in humans. The experimental design was a cross-over intervention study which included a number of substances to be consumed—alcohol, a nicotinamide adenine dinucleotide (NAD) supplement, and a benzoic acid-containing flavoured water vehicle. The experimental subjects—24 healthy, moderate-drinking young men—each provided six hourly-collected urine samples for analysis. Complete data sets were obtained from 20 of the subjects and used for data generation, analysis and interpretation. The results from the NMR approach produced complex spectral data, which could be resolved sufficiently through the application of a combination of univariate and multivariate methods of statistical analysis. The metabolite profiles resulting from acute alcohol consumption indicated that alcohol-induced NAD+ depletion, and the production of an excessive amount of reducing equivalents, greatly perturbed the hepatocyte redox homeostasis, resulting in essentially three major metabolic disturbances—up-regulated lactic acid metabolism, down-regulated purine catabolism and osmoregulation. Of these, the urinary excretion of the osmolyte sorbitol proved to be novel, and suggests hepatocyte swelling due to ethanol influx following acute alcohol consumption. Time-dependent metabolomics investigations, using designed interventions, provide a way of interpreting the variation induced by the different factors of a designed experiment, thereby also giving methodological significance to this study. The outcomes of this approach have the potential to significantly advance our understanding of the serious impact of the pathophysiological perturbations which arise from the consumption of a single, large dose of alcohol—a simulation of a widespread, and mostly naive, social practice.

Metabolomics studies of diseases associated with chronic alcohol consumption provide compelling evidence of several perturbed metabolic pathways. Moreover, the holistic approach of such studies gives insights into the pathophysiological risk factors associated with chronic alcohol-induced disability, morbidity and mortality. Here, we report on a GC–MS-based organic acid profiling study on acute alcohol consumption. Our investigation — involving 12 healthy, moderate-drinking young men — simulated a single binge drinking event, and indicated its metabolic consequences. We generated time-dependent data that predicted the metabolic pathophysiology of the alcohol intervention. Multivariate statistical modelling was applied to the longitudinal data of 120 biologically relevant organic acids, of which 13 provided statistical evidence of the alcohol effect. The known alcohol-induced increased NADH:NAD+ ratio in the cytosol of hepatocytes contributed to the global dysregulation of several metabolic reactions of glycolysis, ketogenesis, the Krebs cycle and gluconeogenesis. The significant presence of 2-hydroxyisobutyric acid supports the emerging paradigm that this compound is an important endogenous metabolite. Its metabolic origin remains elusive, but recent evidence indicated 2-hydroxyisobutyrylation as a novel regulatory modifier of histones. Metabolomics has thus opened an avenue for further research on the reprogramming of metabolic pathways and epigenetic networks in relation to the severe effects of alcohol consumption.