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Antioxidants Research Laboratory and * Mass Spectrometry Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA
4 To whom correspondence should be addressed: E-mail: jeffrey.blumberg{at}tufts.edu.
ABSTRACT
Quercetin and quercetin glycosides from food or dietary supplements appear in body tissues almost exclusively as glucuronated, sulfated, and methylated quercetin conjugates, suggesting that the in vivo bioactivity of quercetin may be due to its metabolites. In this study, pre- and postabsorptive metabolism of orally ingested quercetin was examined by comparing the metabolite pattern in gastrointestinal (GI) tissues, contents, and internal tissues. F344 rats (n = 6) were fed for 6 wk a diet containing 0.45% quercetin and the metabolite patterns were determined in the tissues and contents of stomach, small intestine, cecum, and colon and in liver, kidney, and plasma using LC-MS/MS. GI contents contained predominantly unmetabolized quercetin at 94–100%, whereas quercetin in GI tissues was present as 11 different sulfated, glucuronated, and methylated metabolites at 32% in stomach, 88% in small intestine, 27% in cecum, and 46% in colon. Quercetin was further metabolized postabsorption and found in liver, kidney, and plasma almost exclusively as sulfated methyl-quercetin glucuronide. The unique pattern of quercetin metabolites in each GI tissue indicates extensive biotransformation before absorption and distribution in rats.
KEY WORDS: • quercetin • metabolism • gastrointestinal tract • rats • LC-MS/MS
The flavonol quercetin is ubiquitous in plant foods; it is particularly abundant in apples, broccoli, and onions at 4.4, 3.2, and 13.3 mg/100 g, respectively (1). Consumption of fruit and vegetables containing quercetin has been associated with several health benefits, including reduced risk of cardiovascular disease and some forms of cancer (2). However, our understanding of the bioavailability, metabolism, and mechanisms of action of quercetin is limited (3). Orally ingested quercetin is metabolized extensively before its entry into blood and internal organs, suggesting biotransformation in gastrointestinal (GI)5 tissues and/or liver (4,5). Although the acidic environment in the stomach does not facilitate the release of quercetin aglycone from glycoside complexes, this process can be mediated via GI enzymes such as lactase phlorizin hydrolase and cytosolic ß-glycosidases. Phase II metabolizing enzymes such as UDP-glucuronyl transferases (UGT), catechol-O-methyltransferases, and phenol sulfotransferases form glucuronated, sulfated, and methylated quercetin conjugates that are found in blood and urine (6). The unabsorbed majority of flavonoid glycosides is subject to deconjugation by microbial ß-glycosidases, 
-rhamnosidases, and ß-glucuronidases in the colon. In addition, hydrolytic cleavage of the ether bond at the 1,2 position of the C ring converts quercetin into phenolic acids, including phenyl acetic and phenyl propionic acids, that can be absorbed and are present in plasma, mostly as phase II conjugates (7). Biotransformation of quercetin may affect its function and activity because its metabolites are generally more hydrophilic and have a negative charge at physiological pH (8).
Although most in vitro studies of quercetin have tested its aglycone or glycoside forms, only a small number of physiologically relevant metabolites were investigated. For example, in vitro, quercetin-mono-glucuronides (Fig. 1) possess antioxidant-scavenging activity (9,10), delay lipid peroxidation of cell membranes (11), and reduce Cu2+-induced LDL oxidation (12). Further, quercetin-mono-glucuronides inhibit the expression and activity of cyclooxygenase, lipoxygenase, and xanthine oxidase, enzymes implicated in atherosclerosis and inflammatory reactions (13,14). Recently, Saito et al. (15) reported that quercetin-3-sulfate reduced H2O2-induced chromosomal damage in cultured human lymphocytes, a model of carcinogenesis.
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MATERIALS AND METHODS
Chemicals. Authentic flavonoid standards isorhamnetin, tamarixetin, quercetin-4'-glucoside, and isorhamnetin-3-glucoside were purchased from Extrasynthese. Quercetin was purchased from Sigma Chemical. All other chemicals were of analytical grade; solvents were of HPLC grade and were purchased from Fisher Scientific.
Animals and sample collection.
Male weanling (3 wk old) F-344 rats were obtained from Harlan and housed individually in wire cages at the animal care facility of the Jean Mayer USDA Human Nutrition Research Center on Aging (HNRCA) at Tufts University. The animal room was maintained at 25°C with a 12-h light:dark cycle throughout the study. After a 1-wk acclimation period, the rats were weight matched and divided into a control group (n = 6) fed the AIN-93G basal diet (Dyets) (20) and a supplemented group (n = 6) fed a diet containing 4.5 g quercetin dihydrate (98% purity from Alfa Aesar)/kg diet for 6 wk. The diets were stored at –20°C under nitrogen gas during the study and the quercetin content was monitored by HPLC analysis. During the last week of the study, the rats fed the supplemented diet consumed 
13 g/d diet, which provided 58.5 mg/d of quercetin. After 6 wk of consuming the respective diets, the rats were food deprived for 2–3 h and anesthetized with AerraneTM (Barter) before being killed by CO2 asphyxiation. Blood was collected from the orbital plexus into heparinized tubes (Becton-Dickenson) and plasma was prepared by centrifugation at 1000 x g for 10 min at 4°C and stored at –80°C before analyses. The GI tract, liver, and kidney were quickly harvested, snap-frozen in liquid nitrogen, and stored at –80°C until analyses. The protocol complied with NIH guidelines and was approved by the HNRCA Institutional Animal Care and Use Committee.
Metabolite extraction. The GI tract was thawed, divided into stomach, small intestine, cecum, and colon; their contents were separated from the tissue by washing 5 times with PBS. Tissues from 6 rats were pooled and homogenized in extraction buffer (50% methanol in 0.1 mol/L phosphate buffer, pH 7.0) containing 20 mmol/L sodium diethyldithiocarbamate (as an antioxidant) using a SDT-1810 homogenizer (Tekmar). The tissue homogenate was divided in half for extraction and analysis in duplicate as described by Mullen et al. (21). Briefly, the homogenized tissue was extracted 3 times under constant shaking with extraction buffer. After centrifugation (160 x g; 30 min), the 3 methanolic supernatants were combined and the methanol removed in vacuo using a SC110 Speed Vac (Savant Instruments). The remaining aqueous phase was adjusted to pH 3 and partitioned 3 times with an equal volume of ethyl acetate. Metabolites present in the aqueous phase were further purified using a C18 Sep Pak cartridge (Waters), which was eluted with methanol. The eluent was combined with the ethyl acetate extracts and reduced to volumes <500 µL in vacuo and stored at –80 °C. To precipitate proteins, plasma was treated twice for 10 min with 2.5 volumes of acetone. After centrifugation (160 x g; 15 min), the 2 acetone extracts were combined, 20 mL extraction buffer was added, and the solvents removed in vacuo. The ethyl acetate and aqueous extracts were obtained as described above.
Metabolite identification by LC-MS/MS.
Quercetin metabolites were separated and identified on an Agilent HPLC workstation fitted with a Synergy RP-Max column (4 µm, 250 x 4.6 mm, Phenomenex) at 40°C using a gradient from 5 to 40% methanol in 1% aqueous formic acid over 120 min at a flow rate of 0.2 mL/min. The column eluate was directed through an Agilent UV G1315A diode array detector monitoring 250–700 nm (Agilent Technology) and then into an Esquire ion trap MS/MS (Bruker, Daltonic GmbH) fitted with an electrospray interface operating in negative ion mode. The instrument was set to scan ions from 150 to 1000 Da, employing alternating MS and MS/MS scanning mode. Metabolite identification was based on the appropriate mass to charge ratio (m/z) of molecular ions and MS/MS fragmentation patterns, UV absorption at 365 nm (with a 
-max between 355 and 370 nm), and conversion into the aglycones quercetin, tamarixetin, and isorhamnetin upon treatment with ß-glucuronidase and sulfatase.
Enzyme hydrolysis. To confirm that the detected compounds were quercetin conjugates, tissue extracts were treated with 7350 units ß-glucuronidase containing 0.18 units sulfatase (Sigma Chemical) and 5 volumes hydrolysis buffer (1.1 mol/L ascorbate, 3.42 mmol/L EDTA, 0.4 mol/L NaH2PO4 adjusted with NaOH to pH 5.5). After 2 h of incubation at 37°C, samples were extracted 3 times with methanol. The supernatant was removed after centrifugation (8000 x g; 10 min), concentrated under nitrogen to the original volume of the extract, and analyzed using LC-MS/MS as described above.
Metabolite Quantification.
All samples were extracted and analyzed in duplicate. The metabolite profile in the duplicate extracts (prepared 6 mo after the first extracts) was almost identical to that of the fresh extracts; however, partial degradation of some of the more complex metabolites (diglucuronides, glucuronated sulfates) was observed. Therefore, data obtained from the fresh extract were used for quantitative purposes. The quercetin and isorhamnetin aglycones were quantified based on their UV absorption at 365 nm using standard curves of authentic compounds. The absorption spectra and -max values of quercetin metabolites and quercetin-4'-glucoside and isorhamnetin-3-glucoside were similar; thus, without authentic standards, quercetin metabolites were quantified as quercetin-4'-glucoside equivalents. The use of these "equivalent values" may result in potential under- or overestimates of actual amounts; thus these values listed in the tables should be considered as approximate rather than precise concentrations.
RESULTS
Distribution of quercetin metabolites
Extracts of the tissues, gut contents, and plasma from control rats did not contain quercetin or quercetin metabolites. After 6 wk of supplementation with 0.45% quercetin, 16 different phase II metabolites were detected in the GI tissues and contents as well as in liver, kidney, and plasma (Table 1, Fig. 2). GI tissue weight (mean ± SD) was as follows: stomach, 0.74 ± 0.07 g; intestine, 2.45 ± 0.48 g; cecum, 0.52 ± 0.06 g; colon, 0.65 ± 0.12 g and contained 3.0 nmol quercetin metabolites and 2.7 nmol quercetin per rat, whereas liver (7.05 ± 0.89 g), kidneys (1.68 ± 0.18 g), and plasma (6.03 ± 0.75 mL) contained 17.9, 5.2, and 14 µmol/L quercetin and metabolites, 0.009, 0.003, and 0.04% of the daily ingested dose (194 µmol, 58.5 mg), respectively (Table 2).
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GI contents. GI contents contained predominantly unmetabolized quercetin (Table 2, peak 12). Compared with the luminal contents of the stomach, cecum and colon, the luminal content of the small intestine held the highest quantity of quercetin metabolites (6%) (Table 3). The luminal contents of the stomach contained no metabolites, and 0.4 and 1.2% of the quercetin in the cecum and colon was metabolized, respectively.
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Liver, kidney and plasma. In liver and kidney, 92 and 96% of the quercetin was metabolized; in plasma, all quercetin was metabolized. A methylated glucuronated quercetin sulfate (peak 6) was the predominant metabolite in all 3 samples, representing 55, 65, and 78% of all quercetin and quercetin metabolites, respectively.
Stability of quercetin metabolites
Degradation of quercetin metabolites with a molecular weight >550 Da was observed in tissue homogenates and extracts after 6 mo of storage at –80°C. Diglucuronides (peaks 1 and 3) completely degraded in GI tissue homogenates and extracts. Partial loss of the sulfuryl moiety of sulfated quercetin glucuronide (peak 4) and sulfated methyl-quercetin glucuronide (peak 6) was observed in tissue homogenates and extracts as well as plasma, but not in plasma extracts. All other metabolites were stable under our storage and extraction conditions.
DISCUSSION
Quercetin bioavailability and metabolism were examined previously in rats only after an acute administration; thus, we characterized these variables after rats underwent chronic feeding of a diet rich in quercetin for 6 wk, and determined the identity and quantity of quercetin metabolites in GI tissues, liver, kidney, and plasma. Metabolite profiles in plasma, liver, and kidney differed from those obtained in GI tissues. Each GI tissue displayed a unique metabolite pattern, suggesting different pathways of biotransformation in stomach, intestine, cecum, and colon. In contrast, the metabolite profiles of quercetin in plasma, liver, and kidney did not differ. All GI tissues contained both free quercetin and quercetin metabolites, suggesting absorption of free quercetin followed by metabolic transformation. Active involvement of intestinal tissue in quercetin metabolism was reported in a number of recent reports (4,19,22,23). When rat intestine was perfused in situ with quercetin and intestinal eluent and bile were collected separately (5), bile contained only 10% of the quercetin conjugates, whereas the intestinal eluent held 90% of the formed quercetin conjugates, thus indicating that the majority of metabolism had occurred in the gut and not in the liver. After oral ingestion of quercetin by pigs, Cermak et al. (24) found that their portal blood contained only quercetin metabolites and suggested that quercetin was biotransformed in the GI tract before reaching the liver. Our results in rats are consistent with these findings. The distinct metabolite patterns along the descending segments of the GI tract, as well as in the liver and kidney, may reflect the different activity and expression of phase II metabolizing enzymes in these tissues. Tissue-specific expression of individual enzymes of the UGT1A superfamily was reported for rat esophagus, duodenum, jejunum, ileum, cecum, colon, rectum, liver, and kidney (25). Similarly, sulfotransferases have different activities in human stomach, intestine, and colon (26). Five individual sulfotransferases (SULT-1A1, SULT-1A3, SULT-1B2, SULT-2A1, SULT-1E1) with different tissue locations and kinetic parameters were reported to conjugate flavonoids in vitro at 4 different positions (27,28).
The metabolite profile of quercetin in liver and kidney contrasted with that of the GI tract and suggests a second stage of biotransformation in internal tissues with the addition of subsequent methyl, glucuronyl, or sulfuryl moieties or deconjugation and immediate reconjugation, possibly at different positions. Such a pathway is consistent with the in vitro observations of O'Leary et al. (29) who found that HepG2 cells hydrolyzed quercetin-7-glucuronide and quercetin-3-glucuronide followed by sulfation of the resulting aglycone to quercetin-3'-sulfate. The metabolite profiles in plasma, liver, and kidney are sufficiently similar to suggest a common origin of the metabolites present. If flavonoid metabolites are distributed from the enterocyte to chylomicrons in lymph, as recently suggested by Murota and Terao (30), the lung could also prove a potential location for postabsorption metabolism of quercetin; thus, further investigation of the metabolic contribution of this tissue is warranted. Some reports suggest substantial excretion of quercetin metabolites via the bile (31,32); however, different metabolite profiles in liver and gut indicate that enterohepatic circulation is only a minor pathway in our rat model or that metabolites are hydrolyzed and reconjugated as described earlier.
The stomach and small and large intestines appear to absorb and actively metabolize quercetin because the tissues contain both free quercetin and a complex and differing metabolite profile depending on the region of the GI tract. Of all GI tissues, stomach tissue had the highest concentration of metabolites and quercetin (1.2 µg/g or 3.19 nmol/g fresh weight), supporting previous findings that quercetin and other flavonoids may be absorbed from the stomach (33,34).
Human plasma and urine contain similar types (i.e., diglucuronides, monoglucuronides, sulfates, glucuronated sulfates) and numbers (i.e., 12–21) of phase II metabolites (35–37) compared with rats (4,38). The differences in the number and quantity of individual metabolites reported in human and animal plasma may be due to species-dependent variation of phase II metabolism as well as experimental variables such as the dose, duration, route of administration, and form of quercetin fed.
Validating recovery rates of individual quercetin metabolites extracted from biological matrices requires a research approach such as feeding radiolabeled material. Graf et al. (4) fed rats [2-14C]quercetin-4'-glucoside and found on average that 50% of the radiolabeled compounds were recovered from plasma and tissues in the purified extracts. However, they did not determine whether any metabolites were preferentially recovered during the extraction process. We employed the same extraction method in this study; thus, a preferential extraction of some metabolites is a potential confounding factor.
The chemical instability of some quercetin metabolites during storage, extraction, or analysis, as well as the potential for enzymatic hydrolysis and conversion of metabolites, also present confounding factors in the effort to identify bioactive forms of quercetin in vivo. For example, the presence of quercetin aglycone in liver and kidney (<8%) may be due to ex vivo hydrolysis of quercetin metabolites. Appropriate enzyme inhibitors should be considered in future studies to control for ex vivo conversion of metabolites. The substantial amount of one predominant methylated, sulfated, and glucuronated metabolite found in liver, kidney, and plasma after chronic consumption of quercetin suggests that the physiochemistry of quercetin or the specific metabolic activity of tissue(s) channels its biotransformation toward this single compound in rats. Thus, studies of the bioactivity of this particular quercetin metabolite are warranted.
ACKNOWLEDGMENTS
The authors thank Ting Li and Jennifer O'Leary for their excellent technical assistance and Dr. Donald Smith for his help with the animal study.
FOOTNOTES
1 Presented in part at Experimental Biology 04, April 2004, Washington, DC [Graf AB, Milbury PE, Ameho C, Li T, Dolnikowski GG, Blumberg J. Gastrointestinal tissues of the rat metabolize dietary quercetin (abstract). FASEB J. 2004;18:A516]. 
2 Supported by the U.S. Department of Agriculture Agricultural Research Service under Cooperative Agreement 58-1950-4-401 and the Yamanouchi USA Foundation. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture nor does mention of trade names, commercial products or organizations imply endorsement by the U.S. government.
3 Current address: Department of Animal Science, University of Kentucky, Lexington, KY 40536-0215.
5 Abbreviations used: GI, gastrointestinal; m/z, mass to charge ratio; SULT, sulfotransferase; UGT, UDP-glucuronyl transferases.
Manuscript received 12 July 2005. Initial review completed 6 August 2005. Revision accepted 30 September 2005.
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