The Quantitation of Metabolites of Quercetin Flavonols in Human Urine"2

The flavonoid quercetin, or its metabolites, inhibit chemical carcinogenesis in rodents and may have a role in the prevention of human cancers. Quercetin exposure in human populations results from the dietary intake of various plant foods; high concentrations of quercetin are found in apples, onions, tea, and red wine. Determination of the relationship between dietary intake and cancer risk depends on the characterization of quercetin intake. The development and use of biomarkers for quercetin intake may provide a basis for the objective classification of this exposure. One possible biomarker is metabolic products of quercetin. We report the development of a highperformance liquid chromatography (HPLC)-based assay for quantitation of quercetin metabolites in human urine. The metabolites include 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid), metahydroxyphenylacetic acid, and 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid). The assay has only two major steps, ether extraction and HPLC analysis, and is suitable for analysis of large sample numbers. Analytical characteristics of the assay include a sensitivity of less than 1 ?tg, precision with coefficients of variation < 10%, and metabolite recoveries >90%. The mean concentrations of 3,4-dihydroxyphenylacetic acid, metahydroxyphenylacetic acid, and homovanillic acid in two human urine samples are approximately 0.7, 4.8, and 2.8/xg/ml, respectively. The identification of each metabolite is confirmed by HPLC, UV absorbance scans, and gas chromatography-mass spectrometry analysis. These results verify the occurrence of quercetin metabolites in human urine and the feasibility of quercetin metabolite quantitation, by the assay described herein, for epidemiological studies. Development of the Received 5/5/96; revised 6/11/96; accepted 6/13/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Grant P01 CA50305 (a Colon Cancer Prevention Research Unit) from the National Cancer Institute. 2 A portion of the results reported herein were presented at the annual meeting of the American Association for Cancer Research in 1994 in San Francisco. 3 To whom requests for reprints should be addressed, at Division of Epidemiology, School of Public Health, University of Minnesota, 1300 South Second Street, Suite 300, Minneapolis, MN 55454-1015. analytical procedure is an essential first step for validation of the metabolites as biomarkers of quercetin intake. Introduction Human diets contain a wide range of compounds with possible "semi-essential" functions. One group of such compounds is the flavonoids. Flavonoids have multiple chemical and biological actions, including antioxidant (1-4), chelation (5, 6), anticarcinogenic (7, 8), bacteriostatic (9), and secretory activities (10, 11). Several enzymes, including trypsin, leucine aminotransferase, phenosulfotransferase, DNA topoisomerase, phosphoinositol kinase, NADPH diaphorase, H+,K~+)-ATPase, and xanthine oxidase, are modulated by flavonoids (12-18). Furthermore, some flavonoids may act as antiviral, antitumor, anti-inflammatory and antiallergenic agents (19-21). Flavonoids have a widespread distribution among food plants. In general, fruits and vegetables are good dietary sources of flavonoids (22-28). The flavonoids are found primarily in the outer layers of fruits and vegetables; the peel and outer leaves have the highest concentrations (22, 23, 29-32). Human consumption of flavonoids is estimated at 1 g or more per day for the average American diet (33). One flavonoid, quercetin, is of particular interest because of its anticarcinogenic activities (7) and a significant quantitative presence in human foods. Quercetin is found in numerous fruits and vegetables (22-24), the highest concentrations occurring in apples, onions, and tea (34-36). Biological activities of quercetin include the inhibition of cell proliferation in animals and cell cultures (37) and prevention of chemically induced tumors. Early reports of carcinogenic activity by quercetin are based on bacterial mutagenicity tests (38). Extrapolation of the results to humans, in this case, led to an erroneous conclusion. Tests of the carcinogenicity of quercetin in mammalian species, with few exceptions (39, 40), have proven to be negative (41, 42), and epidemiological data do not implicate carcinogenic activity for quercetin in humans (43). Interpretations of more recent data from in vitro experiments, suggesting carcinogenic activity for quercetin, do not consider the absence of quercetin absorption (44) and almost complete metabolism of quercetin in the human gastrointestinal tract (45). Thus, the evidence to date does not support carcinogenic activities for quercetin in humans (46, 47) but rather a chemopreventive effect (48). The chemopreventive effect may be mediated by metabolites of quercetin. Also, quercetin or its metabolites may have cytotoxic activities (49). Because diets associated with a reduction in cancer risk generally contain numerous potential anticarcinogens (43, 50, 51), biomarkers of dietary quercetin would be useful in studies of diet-cancer relationships. Studies on the metabolism of quercetin suggest degradation by intestinal microbes and relatively low absorption of quercetin (52, 53), limiting the use of quercetin as a biomarker. However, metabolites of quercetin have on June 16, 2017. © 1996 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from 712 Measurement of Quercetin Metabolites