Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies

Javier I Ottaviani, Redmond Fong, Jennifer Kimball, Jodi L Ensunsa, Abigail Britten, Debora Lucarelli, Robert Luben, Philip B Grace, Deborah H Mawson, Amy Tym, Antonia Wierzbicki, Kay-Tee Khaw, Hagen Schroeter, Gunter G C Kuhnle, Javier I Ottaviani, Redmond Fong, Jennifer Kimball, Jodi L Ensunsa, Abigail Britten, Debora Lucarelli, Robert Luben, Philip B Grace, Deborah H Mawson, Amy Tym, Antonia Wierzbicki, Kay-Tee Khaw, Hagen Schroeter, Gunter G C Kuhnle

Abstract

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk.

Conflict of interest statement

H.S. and J.I.O. are employed by Mars, Inc., a company engaged in flavanol research and flavanol-related commercial activities. G.G.C.K. has received an unrestricted research grant from Mars Inc.

Figures

Figure 1
Figure 1
Typical chromatogram of EPIC Norfolk spot urine sample, showing γ-valerolactone-3′/4′-O-glucuronide (top, 3.9 minutes) and γ-valerolactone-3′-sulphate (bottom, 3.8 minutes) in urine.
Figure 2
Figure 2
Distribution of gVLM (sum of γ-valerolactone-3′/4′-sulfate and γ-valerolactone-3′/4′-O-glucuronide) concentrations in 5000 random samples of EPIC Norfolk. The red bar indicates samples with concentration below the lower limit of quantification (0.1 µmol/L).
Figure 3
Figure 3
Urinary excretion of flavan-3-ol biomarker (gVLM, sum of γ-valerolactone-3′/4′-sulphate and O–glucuronide) following the consumption of different flavan-3-ols (error bars show standard error; n = 12–8). *indicate statistically significant differences (p 

Figure 4

Flavan-3-ol precursors of the microbial…

Figure 4

Flavan-3-ol precursors of the microbial metabolite 5-(3′/4′-dihydroxyphenyl)-γ-valerolactone (gVL). Only compounds with intact (epi)catechin…

Figure 4
Flavan-3-ol precursors of the microbial metabolite 5-(3′/4′-dihydroxyphenyl)-γ-valerolactone (gVL). Only compounds with intact (epi)catechin moiety result in the formation of γVL by the intestinal microbiome. ECG, (−)-epicatechin-3-O-gallate; EGCG, (−)-epigallocatechin-3-O-gallate; EGC, (−)-epigallocatechin.

Figure 5

Association between flavan-3-ol intake and…

Figure 5

Association between flavan-3-ol intake and gVLM (sum of 5-(3′/4′-dihydroxyphenyl)-γ-valerolactone-3′/4′-sulphate and O–glucuronide metabolites). Results…

Figure 5
Association between flavan-3-ol intake and gVLM (sum of 5-(3′/4′-dihydroxyphenyl)-γ-valerolactone-3′/4′-sulphate and O–glucuronide metabolites). Results from the intake-amount escalation study and regression analysis (R2 = 0.66; 95% CI 0.49; 0.80). The intake amount used is comparable with the estimated habitual intake in EPIC Norfolk. For comparison, Pearson and Spearman correlation coefficients for other biomarkers are shown (†; ‡).
Figure 4
Figure 4
Flavan-3-ol precursors of the microbial metabolite 5-(3′/4′-dihydroxyphenyl)-γ-valerolactone (gVL). Only compounds with intact (epi)catechin moiety result in the formation of γVL by the intestinal microbiome. ECG, (−)-epicatechin-3-O-gallate; EGCG, (−)-epigallocatechin-3-O-gallate; EGC, (−)-epigallocatechin.
Figure 5
Figure 5
Association between flavan-3-ol intake and gVLM (sum of 5-(3′/4′-dihydroxyphenyl)-γ-valerolactone-3′/4′-sulphate and O–glucuronide metabolites). Results from the intake-amount escalation study and regression analysis (R2 = 0.66; 95% CI 0.49; 0.80). The intake amount used is comparable with the estimated habitual intake in EPIC Norfolk. For comparison, Pearson and Spearman correlation coefficients for other biomarkers are shown (†; ‡).

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