Identification and Pharmacokinetics of Novel Alkylresorcinol Metabolites in Human Urine, New Candidate Biomarkers for Whole-Grain Wheat and Rye Intake

Abstract

Biomarkers of dietary intake are prominent tools in nutritional research. The alkylresorcinol metabolites 3,5-dihydroxybenzoic acid (3,5-DHBA) and 3-(3,5-dihydroxyphenyl)propanoic acid (3,5-DHPPA) have been proposed as exposure biomarkers of whole-grain (WG) wheat and rye intake. However, the profile of alkylresorcinol metabolites is not fully understood. The aim of this study was to investigate the metabolism of alkylresorcinols in mice and in humans, while further determining urinary pharmacokinetics of the novel alkylresorcinol metabolites to explore their potential as biomarkers of WG wheat intake. Utilization of the liquid chromatography-mass spectrometry approach resulted in 10 alkylresorcinol metabolites identified in mice and in humans, including 3 phenolic acids and 7 of their phase II conjugates. Among them, 2 novel metabolites were discovered: 5-(3,5-dihydroxyphenyl)pentanoic acid (3,5-DHPPTA) and 2-(3,5-dihydroxybenzamido)acetic acid (3,5-DHBA glycine). The structures of these 2 metabolites were confirmed by comparing with authentic standards synthesized in-house. In the pharmacokinetic study, a group of 12 volunteers consumed a polyphenolic-restricted diet for 4 d before ingesting WG wheat bread containing 61 mg of alkylresorcinols. Urine samples were collected for 32 h, and alkylresorcinol metabolites were quantified with HPLC-coulometric electrode array detection. The mean urinary excretion rates and mean apparent half-life of 3,5-DHPPTA, 3,5-DHBA glycine, 3,5-DHBA, and 3,5-DHPPA at each time point were determined. Our results suggest that 3,5-DHPPTA and 3,5-DHBA glycine may be used in combination with 3,5-DHBA and 3,5-DHPPA as potential biomarkers to increase the accuracy of recording WG wheat and rye intake in epidemiologic studies. Further validation of 3,5-DHPPTA and 3,5-DHBA glycine as potential biomarkers is warranted.

Comparison of plasma alkylresorcinols (AR) and urinary AR metabolites as biomarkers of compliance in a short-term, whole-grain intervention study

“…So far, no study has reported the presence of these novel metabolites in urine after rye consumption, and it is possible that the urinary AR metabolite composition differ after wheat and rye intake. This is supported by the DHPPA/DHBA ratio in urine which is usually >2 after wheat-based AR intake as reported here and elsewhere [9,25], while the ratio in studies where rye is the primary AR source commonly <2 after rye-based AR consumption [16,17,33,34]. Unlike previous studies [16,17], no inverse relationship between AR intake and urinary AR metabolite recovery was observed.…”

“…In addition, two novel AR metabolites [5-(3,5-dihydroxyphenyl)-pentanoic acid and 2-(3,5-dihydroxybenzamido)-acetic acid] were recently detected in human urine after a single dose of WG wheat bread and accounted for ~12 % of all AR metabolites in urine [9]. So far, no study has reported the presence of these novel metabolites in urine after rye consumption, and it is possible that the urinary AR metabolite composition differ after wheat and rye intake.…”

“…Plasma ARs are rapidly eliminated, presumably through extensive hepatic metabolism. Although minor metabolites exist [9], the phase I metabolism of AR mainly results in the formation of two phenolic acids: 3,5-dihydroxybenxoic acid (DHBA) and 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA). DHPPA and DHBA can be measured in plasma and urine either as free molecules or as conjugates (glucuronides and sulfonates) resulting from phase II metabolism [10,11].…”

Determination of alkylresorcinols and their metabolites in biological samples by gas chromatography–mass spectrometry

“…A slightly modified protocol based on a previous method [21] was developed to allow accurate quantification of four novel AR metabolites recently identified (DHPPTA, DHBA-glycine, DHCA and DHCA-amide) along with DHBA and DHPPA in urine samples [17,18]. Enzymatic hydrolysis, extraction and SPE of DHBA and DHPPA have been optimized previously [21], and similar conditions were applied in the present method.…”

“…These metabolites are 3-(3, 5-dihydroxyphenyl)-1-propanoic acid (DHPPA) and 3,5-dihydroxybenzoic acid (DHBA). In a recent study, 5-(3,5-dihydroxyphenyl) pentatonic acid (DHPPTA) and 2-(3,5-dihydroxybenzamido) acetic acid (DHBAglycine) were also described as urinary metabolites derived from AR [17]. Moreover, 3,5-dihydroxycinnamic acid (DHCA) and 3,5-dihydroxycinnamic acid amide (DHCA-amide) have recently been identified in urine and suggested as AR metabolites [18].…”