Table 1.
A summary of the experimental designs, data analysis approaches and main metabolic conclusions of some NMR-based ethanol administration studies.
Fig 1.
Representative 1H-NMR spectrum of urine collected one hour following the “alcohol plus vehicle” intervention.
EtOH = ethanol (1.18 t, 3.64 q); LA = lactic acid (1.33 d, 4.12 q); SA = succinic acid (2.41 s); TMAO = trimethylamine-N-oxide (3.27 s); CA = citric acid (2.61 AB); MA = methylamine (2.61 s); TMA = trimethylamine (2.90 s); DMG = N,N-dimethylglycine (2.93 s); DMF = N,N-dimethylformamide (2.87 s, 3.02 s); CT = creatine (3.04 s, 3.93 s); GLY = glycine (3.57 s); SO = sorbitol (3.60–3.69 m, 3.73 d, 3.74–3.80 m, 3.82 d, 3.85 m); IS = indoxyl sulphate (7.51 d, 7.70 d); HX = hypoxanthine (8.20 d); HA = hippuric acid (3.97 d, 7.56 tt, 7.64 tt, 7.84 dd); Cr = creatinine (3.05 s, 4.06 s). [Not observed in the present spectrum: fumaric acid (6.52 s) and 3-hydroxybutyric acid (1.20 d, 2.36 m, 4.15 m)].
Fig 2.
Confirmation of sorbitol annotation.
(a) 1D 1H-NMR of a representative urine sample collected one hour after alcohol consumption, zoomed into the 3.50–3.90 ppm region (black), compared to the pure compound spectrum of sorbitol (blue). (b) Correlating 2D 1H-NMR COSY, confirming the sorbitol annotation based upon proton correlation.
Fig 3.
Group separation between participants, based on equidistant binned spectral data from the “vehicle only” and the “alcohol plus vehicle” interventions, illustrated as dendrograms, ML–PCA plots and Volcano plots.
The respective analyses were constructed on subsets of the data representing the same three time points—time 0 (a, d and g), 2 hours (b, e and h) and 4 hours (c, f and i) following the two interventions. Data from the 21 participants in the dendrograms and ML–PCA plots are shown as blue dots/areas for the “vehicle only” intervention and pink dots/areas for the “alcohol plus vehicle” intervention. The single outlier is shown as a red square in the dendrograms. All data from this participant were excluded from further analyses, resulting in the analysis of the data from a total of 20 participants.
Table 2.
Quantified data of important metabolites following alcohol consumption.
Fig 4.
Changes in the concentrations of the six up-regulated metabolites from time 0 to time 4 following alcohol consumption.
Each subplot displays a set of lines (coloured solid lines for 19 of the individuals and a red dotted line for one highlighted individual) representing the observations from the 20 individuals across 5 time points for a given metabolite: (a) ethanol; (b) sorbitol; (c) TMAO; (d) lactic acid; (e) hypoxanthine; and (f) uric acid. The black dashed lines represent one potential LOWESS regression for each metabolite against time.
Fig 5.
Indications of differences in the average levels of hypoxanthine and sorbitol across the four interventions and six time points.
(a) Differences in the average levels of hypoxanthine from time –1 to time 4 between the four interventions. (b) Differences in the average change in hypoxanthine levels, measured from time 0, following the four interventions. (c and d) The comparative results for sorbitol. Significant differences were based on the Greenhouse–Geisser-corrected p-values from the RM ANOVA model or the Wilcoxon signed-rank tests, assessing differences between the sets of means, and are indicated by the arrows.
Fig 6.
Model of the metabolite profile based on the important metabolites up-regulated following alcohol consumption.
The main organs involved are the gut and the liver. Ethanol absorption is indicated in the upper region of the gut, and benzoic acid (from the vehicle and microbiome metabolism) in the lower gut. The main metabolism of ethanol, and the associated consequences of the disturbed NAD+:NADH ratio, occur in the liver. The structural formulas and names of the six important metabolites are shown in red (uric acid is indicated in brackets because it was not measured by NMR). Blue arrows are not related to enzyme kinetic reactions, but are used as indicators of the proposed flow directions following alcohol consumption. Osmoregulation is proposed as efflux in hepatocytes in the early phases following alcohol consumption, and potential influx (shown in brackets) in the later phases. Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; IMP, inosine monophosphate; G-3-P, glyceraldehyde-3-phosphate; GLYAT, glycine-N-acyltransferase; SD, sorbitol dehydrogenase; AR, aldose reductase; OXPHOS, oxidative phosphorylation.