Differential Isotope Labelling Of 38 Dietary Polyphenols To Measure Exposure To Dietary Polyphenols In Epidemiological Studies

David ACHAINTRE, International Agency for Research on Cancer, France
RINALDI S. 1 , CREN C. 2 , BULETÉ A. 2 , LI L. 3 , SCALBERT A. 1

1 International Agency for Research on Cancer (IARC), Nutrition and Metabolism Section, Biomarkers Group, F-69372 Lyon, France
2 University Lyon 1, ENS Lyon, Institut des Sciences Analytiques, Departement Service Central d’Analyses, UMR5280, CNRS, Equipe TRACES, F-69100 Villeurbanne, France
3 Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 Canada

Purpose: A large number of studies support a role of polyphenols in the prevention of chronic diseases such as cardiovascular diseases, diabetes or cancers, however epidemiological evidence is still limited. Robust methods are needed to reliably assess exposure to a large variety of dietary polyphenols which can be easily applied to epidemiological studies.
Methods: We report here the development of a method to quantify dietary polyphenols in urine and in plasma, based on differential 12C-/13C-isotope labelling of polyphenols through derivatization with isotopic dansyl chloride reagents and on the analysis of the labelled polyphenols by tandem mass spectrometry. Urine or plasma samples are first hydrolysed with a β-glucuronidase/sulfatase enzyme mixture and the resulting polyphenol aglycones extracted twice with ethyl acetate. Samples containing the polyphenols tagged with 13C-dansyl groups are mixed with a reference urine or plasma sample containing polyphenols tagged with 12C-dansyl groups and analyzed by tandem mass spectrometry. Polyphenol concentrations are estimated through the calculation of ratios of labelled over non-labelled polyphenols
Results: The method was successfully applied to the measurement of 38 different polyphenols, either native compounds representative of the main classes and subclasses found in the diet, or metabolites formed by the microbiota or human tissues. It was applied to the analysis of 475 urine samples of the European Prospective Investigation on Cancer and nutrition (EPIC) cross-sectional study, and 1170 plasma samples of a case-control study on colorectal cancer nested in EPIC.
Conclusions: The method provides a simple and robust mean for measuring polyphenols in urine and in plasma and overcomes the need of having expensive labelled standards for each compound.

Funding sources: Institut National du Cancer (INCa 2011-105) and Wereld Kanker Onderzoek Fonds (WCRF NL 2012/604).