Background: Phenyl-γ-valerolactones (PVLs) have been proposed as potential biomarkers of dietary flavan-3-ol exposure. Objective: We investigate the performance of a range of PVLs as biomarkers indicative of flavan-3-ol intake. Methods: We report results of two companion studies: a 5-way randomised cross-over trial (RCT) and an observational cross-sectional study. In the RCT, 16 healthy participants were randomly assigned to 1-day flavan-3-ol rich interventions (of either apple, cocoa, black tea, green tea, or water [control]). Participants collected 24-hour urine and first morning urine samples, with diet standardised throughout. For each participant, one of the five intervention periods was randomly extended to two days, to monitor PVL kinetics following repeated days of flavan-3-ol exposure. In the cross-sectional study, 86 healthy participants collected 24-hour urines and concurrent weighed food diaries from which flavan-3-ol consumption was estimated using Phenol-Explorer. A panel of 10 urinary PVLs was quantified using liquid chromatography tandem mass spectrometry. Results: In both studies, two urinary PVLs [5-(3ʹ-hydroxyphenyl)-γ-valerolactone-4ʹ-sulfate and tentatively identified 5-(4ʹ-hydroxyphenyl)-γ-valerolactone-3ʹ-glucuronide] were the principal compounds excreted (>75%). In the RCT, the sum of these PVLs was significantly higher than the water (control) following each intervention; individually, there was a shift from sulfation towards glucuronidation as the total excretion of PVLs increased across the different interventions. In the extended RCT intervention period, after two days of treatment, there was no evidence of accumulation of these compounds in the urine, and following withdrawal of treatment on the third day, there was a return towards negligible PVL excretion. All results were consistent, whether compounds were measured in 24-hour urine or first morning voids. In the observational study, the sum of the principal PVLs correlated dose-dependently (Rs = 0.37, P = 0.0006) with dietary flavan-3-ol intake, with similar associations for each individually. Conclusion: Urinary 5-(3ʹ-hydroxyphenyl)-γ-valerolactone-4ʹ-sulfate and tentatively identified 5-(4ʹ-hydroxyphenyl)-γ-valerolactone-3ʹ-glucuronide are recommended biomarkers for dietary flavan-3-ol exposure.

The authors have declared no competing interest.

ACTRN12619001553167

This project is funded by a 2019 West Family Cardiovascular Grant (SBG 0050/2019) and a 2019 Edith Cowan University Early Career Researcher Grant (G1004660). BHP is supported by an Australian Government Research Training Program Scholarship. NPB is funded by a National Health and Medical Research Council Early Career Fellowship, Australia (APP1159914). The salary of CPB is supported by a Royal Perth Hospital Research Foundation Lawrie Beilin Career Advancement Fellowship (CAF 127/2020).

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

We report two companion studies: a clinical trial and an observational investigation. The clinical trial was approved by the University of Western Australia Human Research Ethics Committee (RA/4/20/5366). The RCT was prospectively registered with the Australian New Zealand Clinical trials registry (ACTRN12619001553167) under the World Health Organisation, Universal Trial Number: U1111-1236-7988. The observational study was approved by the Edith Cowan University, Human Research Ethics Committee (13402). The conduct and reporting of the clinical trial adheres to the Consolidated Reporting Statement for Randomised Controlled Trials (CONSORT). The observational study adheres to the reporting recommendations of the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement. All participants provided written informed consent before inclusion in each study.

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The data that support the findings of this study are available from the corresponding author upon reasonable request in-line with governing ethical considerations.