Research articleThe kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes☆
Introduction
Flavonoids are dietary polyphenols found ubiquitously in plant foods. Prospective cohort studies have associated generous flavonoid intake with reduced risk of cardiovascular disease, stroke and some forms of cancer [1], [2], [3]. Flavonoids may confer protection against chronic diseases through an array of putative actions, including antioxidation, detoxification, anti-inflammation and anti-proliferation. Any health benefits derived from dietary flavonoid intake must depend upon their bioavailability, metabolism and distribution [4], parameters that may be affected by age-related changes in physiology. Limited flavonoid bioavailability and extensive metabolism, particularly when combined with a low intake of these phytochemicals [5], may decrease their putative contribution to health promotion and disease prevention in older adults.
Upon consumption, flavonoids are extensively transformed via Phase 2 enzymes through conjugation with glucuronide, sulfate and/or methyl moieties, particularly in small intestine and liver [6]. This pre-systemic biotransformation of flavonoids within liver and small intestine affects their biological half-life and may decrease their efficacy. For example, glucuronidation of flavonoids limit their putative actions to protect cells against oxidative injury and inhibit lipoxygenase and xanthine oxidase activity [7], [8]. Due to the diversity of the Phase 2 enzymes, metabolites of flavonoids produced through this pathway also depend on tissue type. For example, in rats fed quercetin, the liver contained a majority of sulfated monoglucuronide derivatives, whereas intestinal tissue had the greatest proportion of monoglucuronides [6].
Physiological and environmental factors such as age, genetics and dietary behavior affect the bioavailability of nutrients as well as drugs. However, information on the extent to which flavonoid metabolism is altered by these parameters is scarce. The Phase 2 detoxification pathway is essential to flavonoid metabolism and is partly influenced by aging [9]. van Bezooijen [10] has described how age-related changes in Phase 2 metabolism are dependent upon factors such as enzyme isoform, substrate, gender and diet. However, the effect of age-related changes in flavonoid metabolism mediated by Phase 2 enzymes responsible for glucuronidation, sulfation and methylation remains to be established. Characterizing this relationship is important to elucidating the impact of dietary flavonoids on the health of older adults.
Previously, we observed that fasted senescent rats fed genistein for 4 weeks had a lower steady-state concentration of this isoflavone in liver and plasma than adult rats [11]. The mechanisms underlying this difference remain to be determined. We hypothesized that UDP-glucuronosyltransferase (UGT) flavonoid activity changes with age. Therefore, the aim of this study was to determine whether changes in hepatic glucuronidation of selected flavonoids account for the previously observed age-related decline in their bioavailability in rats. Our objective was to determine the variation in glucuronidation kinetics of isoflavone genistein and the flavonol quercetin using pooled hepatic microsomes collected from 4-, 18- and 28-month-old rats. Acetaminophen glucuronidation was also determined in the same samples to validate enzyme kinetic modeling and as a negative control for age-related changes in glucuronidation, as hepatic microsomal rates of acetaminophen glucuronidation are reportedly unchanged with advanced age in rats and mice [12], [13].
Section snippets
Chemicals and supplies
HPLC-grade acetonitrile and methanol were obtained from Fisher Scientific (Thermo Fisher Scientific, Rockford, IL). Genistein was purchased from Cayman Chemical (Ann Arbor, MI), and rutin was obtained from Extrasynthese (Genay, France). Quercetin, UDP-glucuronic acid, alamethicin and all other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO).
Tissues and preparation
Whole rat liver of tumor-free male Fischer F344 rats aged 4, 18 and 28 months (n=5/group) was obtained from the Aging Rodent Tissue
Acetaminophen glucuronidation
The enzyme kinetics of acetaminophen glucuronidation was best fit using the Michaelis–Menten model. Although Vmax values of pooled microsomes increased with advanced age, Km and intrinsic clearance (Vmax/Km) did not follow this trend (Table 1). Age did not affect individual rates of acetaminophen glucuronidation measured at substrate concentrations of 0.5 and 10 mM (Table 2).
Genistein glucuronidation
Genistein 7-O-glucuronidation kinetics was best modeled by the Michaelis–Menten equation (Fig. 1). Age did not affect the
Discussion
A wide variation in flavonoid bioavailability has been observed in clinical trials [19], which can be attributed to environmental, physiological, dietary and genetic factors. Age may be a contributing factor to this variation of flavonoid bioavailability, since we have previously observed that older rats had lower steady state of genistein disposition than younger rats [11]. Although the mechanisms responsible for this difference have not been explored, physiological changes associated with
Acknowledgments
This work is funded through the USDA ARS Cooperative Agreement #58-1950-7-707. Dr. Court was supported by Grant R01GM061834 from the National Institute of General Medical Sciences, National Institutes of Health (NIH; Bethesda, MD). Dr. Bolling was supported by NIH IRACDA Training Grant K12 GM074869.
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