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A metabolite profiling approach to identify biomarkers of flavonoid intake in humans.

The Journal of nutrition (2009-10-09)
Wai Mun Loke, Andrew M Jenner, Julie M Proudfoot, Allan J McKinley, Jonathan M Hodgson, Barry Halliwell, Kevin D Croft
ABSTRACT

Flavonoids are phytochemicals that are widespread in the human diet. Despite limitations in their bioavailability, experimental and epidemiological data suggest health benefits of flavonoid consumption. Valid biomarkers of flavonoid intake may be useful for estimating exposure in a range of settings. However, to date, few useful flavonoid biomarkers have been identified. In this study, we used a metabolite profiling approach to examine the aromatic and phenolic profile of plasma and urine of healthy men after oral consumption of 200 mg of the pure flavonoids, quercetin, (-)-epicatechin, and epigallocatechin gallate, which represent major flavonoid constituents in the diet. Following enzymatic hydrolysis, 71 aromatic compounds were quantified in plasma and urine at 2 and 5 h, respectively, after flavonoid ingestion. Plasma concentrations of different aromatic compounds ranged widely, from 0.01 to 10 micromol/L, with variation among volunteers. None of the aromatic compounds was significantly elevated in plasma 2 h after consumption of either flavonoid compared with water placebo. This indicates that flavonoid-derived aromatic compounds are not responsible for the acute physiological effects reported within 2 h in previous human intervention studies involving flavonoids or flavonoid-rich food consumption. These effects are more likely due to absorption of the intact flavonoid. Our urine analysis suggested that urinary 4-ethylphenol, benzoic acid, and 4-ethylbenzoic acid may be potential biomarkers of quercetin intake and 1,3,5-trimethoxybenzene, 4-O-methylgallic acid, 3-O-methylgallic acid, and gallic acid may be potential markers of epigallocatechin gallate intake. Potential biomarkers of (-)-epicatechin were not identified. These urinary biomarkers may provide an accurate indication of flavonoid exposure.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Benzoic acid, ACS reagent, ≥99.5%
Sigma-Aldrich
Gallic acid, 97.5-102.5% (titration)
Sigma-Aldrich
(−)-Epigallocatechin gallate, ≥95%
Supelco
Benzoic acid, Pharmaceutical Secondary Standard; Certified Reference Material
Supelco
Melting point standard 121-123°C, analytical standard
Supelco
4-Hydroxybenzoic acid, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Phenylacetic acid, 99%
Supelco
Mettler-Toledo Calibration substance ME 18555, Benzoic acid, analytical standard, for the calibration of the thermosystem 900, traceable to primary standards (LGC)
Sigma-Aldrich
trans-Ferulic acid, 99%
Sigma-Aldrich
1,2-Dihydroxybenzene, ReagentPlus®, ≥99%
Supelco
Gallic acid, certified reference material, TraceCERT®
Sigma-Aldrich
4-Hydroxybenzoic acid, ReagentPlus®, ≥99%
Sigma-Aldrich
(−)-Epicatechin, ≥90% (HPLC)
Sigma-Aldrich
4-Hydroxybenzoic acid, ReagentPlus®, 99%
Sigma-Aldrich
Pyrocatechol, ≥99%
Sigma-Aldrich
3,4-Dihydroxybenzoic acid, ≥97.0% (T)
Sigma-Aldrich
Benzoic acid, ≥99.5%, FCC, FG
Sigma-Aldrich
Shikimic acid, ≥99%
Sigma-Aldrich
3-(4-Hydroxyphenyl)propionic acid, 98%
Sigma-Aldrich
Benzoic acid, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99.9% (alkalimetric)
Sigma-Aldrich
Vanillic acid, purum, ≥97.0% (HPLC)
Sigma-Aldrich
4-Hydroxyphenylacetic acid, 98%
Sigma-Aldrich
(−)-Epigallocatechin gallate, ≥80% (HPLC), from green tea
Sigma-Aldrich
Vanillic acid, 97%
Sigma-Aldrich
(−)-Epicatechin, ≥98% (HPLC), from green tea
Protocatechuic acid, primary reference standard
Sigma-Aldrich
Hydrocinnamic acid, 99%
Supelco
Benzoic acid, certified reference material for titrimetry, certified by BAM, ≥99.5%
Sigma-Aldrich
3,4-Dihydroxyphenylacetic acid, 98%
Sigma-Aldrich
3,4-Dihydroxyhydrocinnamic acid, 98%