How should we assess the effects of exposure to dietary polyphenols in vitro?

Commentary How should we assess the effects of exposure to dietary polyphenols in vitro?1–3 Paul A Kroon, Michael N Clifford, Alan Crozier, Andrea J Day, Jennifer L Donovan, Claudine Manach, and Gary Williamson ABSTRACT Human intervention studies have provided clear evidence that dietary polyphenols (eg, flavonoids— eg, flavonols—and isoflavones) are at least partly absorbed and that they have the potential to exert biological effects. Biological activity of polyphenols is often assessed by using cultured cells as tissue models; in almost all such studies, cells are treated with aglycones or polyphenol-rich extracts (derived from plants and foods), and data are reported at concentrations that elicited a response. There are 2 inherent flaws in such an approach. First, plasma and tissues are not exposed in vivo to polyphenols in these forms. Several human studies have identified the nature of polyphenol conjugates in vivo and have shown that dietary polyphenols undergo extensive modification during first-pass metabolism so that the forms reaching the blood and tissues are, in general, neither aglycones (except for green tea catechins) nor the same as the dietary source. Polyphenols are present as conjugates of glucuronate or sulfate, with or without methylation of the catechol functional group. As a consequence, the polyphenol conjugates are likely to possess different biological properties and distribution patterns within tissues and cells than do polyphenol aglycones. Although deconjugation can potentially occur in vivo to produce aglycone, it occurs only at certain sites. Second, the polyphenol concentrations tested should be of the same order as the maximum plasma concentrations attained after a polyphenol-rich meal, which are in the range of 0.1–10 ␮mol/L. For correct interpretation of results, future efforts to define biological activities of polyphenols must make use of the available data concerning bioavailability and metabolism in humans. Am J Clin Nutr 2004;80:15–21. KEY WORDS Polyphenols, flavonoids, isoflavones, phytochemicals, plant bioactives, antioxidants, human metabolism, firstpass metabolism, conjugation, quercetin INTRODUCTION Polyphenols have been shown, in both in vitro test systems and small animal models, to induce responses consistent with the protective effects of diets rich in fruit and vegetables against degenerative conditions such as cardiovascular disease (CVD) and cancer (1, 2). In fact many polyphenols, particularly flavonoids (eg, flavonols) and isoflavones, showed potent bioactivity when tested in vitro, which led to clinical trials assessing them with respect to a variety of effects (3, 4). However, because clinical studies are expensive and time-consuming, it is also necessary to optimize the use and interpretation of in vitro experiments. Human tissues are exposed to polyphenols via the blood, which is the only route through which dietary polyphenols can reach tissues and their cells, except for the cells lining the intestinal tract. Understanding that polyphenols are substantially modified during absorption and identifying the physiologically relevant conjugates are essential to the planning of meaningful in vitro studies of polyphenol activity. Some controversy has attended hypotheses about the nature of circulating conjugates for particular polyphenols, but recent improvements in the analytic methods have resolved many of these questions. In the past few years, studies using physiologic concentrations of polyphenol conjugates helped clarify their specific mechanisms of action in vivo and advanced the field of understanding polyphenols in relation to health. In this article, we briefly discuss the arguments for using physiologic polyphenol conjugates to assess biological responses in vitro, and we define both what is known about polyphenol conjugates in vivo and where the gaps are in our knowledge of this subject. HOW ARE POLYPHENOLS METABOLIZED? The metabolism of several common polyphenols is now reasonably well understood. An important fact is that polyphenols are extensively altered during first-pass metabolism so that, typically, the molecular forms reaching the peripheral circulation and tissues are different from those present in foods (5–10). The term metabolism is used here to describe the typical modifications that occur during or after absorption. In general, the resulting metabolites are conjugates (eg, sulfates and glucuronates) of the parent aglycone or conjugates of methylated parent aglycones. Catabolism of polyphenols in humans usually occurs only as a result of microbial activity in the (large) intestine. 1 From the Nutrition Division, Institute of Food Research, Norwich, United Kingdom (PAK); the Centre for Nutrition and Food Safety, School of Biomedical and Molecular Sciences, University of Surrey, Guildford, United Kingdom (MNC); the Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, United Kingdom (AC); the Procter Department of Food Science, University of Leeds, United Kingdom (AJD); the Laboratory of Drug Disposition and Pharmacogenetics, Medical University of South Carolina, Charleston, SC (JLD); the Unité des Maladies Metaboliques et Micronutriments, INRA de Clermont-Ferand/Theix, St Genes-Champanelle, France (CM); and the Nestlé Research Center, Lausanne, Switzerland (GW). 2 Supported by the Biotechnology and Biological Sciences Research Council, United Kingdom. 3 Address reprint requests to PA Kroon, Institute of Food Research, Colney Lane, Norwich NR4 7UA, United Kingdom. E-mail: . Received October 24, 2003. Accepted for publication January 16, 2004. Am J Clin Nutr 2004;80:15–21. Printed in USA. © 2004 American Society for Clinical Nutrition 15 16 KROON ET AL numerous sites of possible conjugation, and recent efforts have focused on identifying specific structures that exist in vivo. IDENTIFYING STRUCTURES OF PLASMA POLYPHENOLS IS KEY TO DEFINING THEIR BIOLOGICAL ACTIVITIES IN HUMANS FIGURE 1. The structure of quercetin (3,3⬘,4,5,7-pentahydroxyflavone). Quercetin contains 5 hydroxyl functional groups that have the potential to be conjugated and that differ in their inherent chemical reactivities (3 쏜 7 쏜 3⬘ 쏜 4⬘ ⬎⬎ 5). Quercetin in human plasma is found as sulfate and glucuronate conjugates, and conjugation occurs at positions 3, 3⬘, and 4⬘, but not at position 5 or 7. Methylation of the catechol function (3⬘/4⬘-dihydroxy in the B-ring) also occurs, which gives rise to methylated conjugates. Most polyphenol glucosides are deglycosylated by ␤-glucosidases in the small intestine, namely, the broadspecificity cytosolic ␤-glucosidase and lactase phlorizin hydrolase; this step is requisite for the absorption of many of these polyphenols (see, for example, 11, 12). After absorption, flavonoids are metabolized by the phase II drug–metabolizing enzymes, the uridine-5⬘-diphosphate glucuronosyl-transferases, sulfotransferases, and catech (...truncated)