Identification of Glutathione-Related Quercetin Metabolites in Humans

In humans, several glutathione-related metabolites of quercetin were identified in urine as mercapturic acids of common hydroxyphenylacetic acids gene...
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Chem. Res. Toxicol. 2006, 19, 1525-1532

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Identification of Glutathione-Related Quercetin Metabolites in Humans Yun-Jeong Hong and Alyson E. Mitchell* Department of Food Science and Technology, UniVersity of California DaVis, DaVis, California 95616 ReceiVed July 28, 2006

The glutathionylation of quercetin was investigated in murine hepatic suspensions, in the absence of chemically or enzymatically induced oxidative stress, and in human urine after the consumption of 200 g of cooked onions (∼74 mg of quercetin). In murine hepatic suspensions, 22 metabolites, including glucuronide, sulfate, and glutathione conjugates of quercetin, were identified by LC/ESI-MS/MS. In total, eight glutathione conjugates were identified in these suspension, including three isomeric forms of monoglutathionyl quercetin, two isomers of monoglutathionyl quercetin glucuronide, and three isomers of glutathionyl methyl quercetin. Quinone forms of glutathionyl quercetin and glutathionyl methyl quercetin were also apparent in mass spectra. In humans, several glutathione-related metabolites of quercetin were identified in urine as mercapturic acids of common hydroxyphenylacetic acids generated by the microbial degredation of quercetin in the gut. These include mercaptic acids of dihydroxytoluene, dihydroxybenzaldehyde, dihydroxyphenylacetic acid, dihydroxycinnamic acid, and dihydroxyphenylpropionic acid. Our results suggest that glutathionylation of quercetin occurs in both murine hepatic suspensions and humans and indicate that under certain conditions, quercetin intermediates require inactivation through conjugation with glutathione. Introduction Flavonoids are potent antioxidants that occur in most plant species and can account for a significant percentage of the chemical constituents in vegetables, fruits, and beverages such as tea and red wine. Contemporary interest in flavonoids focuses on their potent antioxidant properties and the epidemiological association between flavonol-rich diets and a lower incidence of cardiovascular disease (1) and to a lesser extent certain cancers (2, 3). Flavonoids are diphenylpropanes with different oxidation levels that are generally subclassified on the basis of the oxidation state of the central C ring (e.g., flavones, flavonols, flavanones, flavanols, anthocyanidins, and isoflavones). The major flavonols found in the Western diet are glucosides of quercetin (Figure 1). Major dietary sources of quercetin glycosides are onions (∼200-650 mg/kg), apples (∼36 mg/kg), and broccoli (∼6-30 mg/kg) (4-6). Although quercetin is found primarily as glycosides in plants, the majority of in Vitro investigations have focused on the aglycone form of quercetin and have given conflicting results. For example, several studies indicate that quercetin aglycone acts as a potent antioxidant in Vitro (7-9), whereas other studies demonstrate pro-oxidant activites for quercetin (10, 11). The pro-oxidant effects, such as the induction of apoptosis (12, 13), cell cycle arrest (14), and anti-proliferative effects (15), are difficult to interpret because the metabolism of quercetin by phase II enzymes was not considered in these studies. Given that quercetin undergoes extensive metabolism in ViVo, the biological activities associated with quercetin aglycone, including the pro-oxidative behavior and alkylating properties, have to be reassessed using likely phase II metabolites of quercetin rather than the aglycone. The bioavailability and metabolism of flavonols such as quercetin is complex. In general, about 75-99% of ingested * Corresponding author. Phone: 530-752-7926. Fax: 530-752-4759. E-mail: [email protected].

Figure 1. The structure of (a) quercetin and (b) rutin (quercetin-3-Oβ-rutinoside).

quercetin glycosides are not recovered in urine, and levels of quercetin aglycone rarely exceed 1 µM in human plasma when the quantities ingested do not exceed those common in the diet (16). The small intestine is the primary site of absorption of quercetin glycosides (16-19), and it appears that the sugar moiety of the glycoside is an important determinant in the absorption process (20, 21). The absorption of quercetin glucosides is thought to occur via interactions with epithelial brush border membrane transporters such as the sodiumdependent glucose transporter-1 (SGLT-1) and subsequently

10.1021/tx0601758 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/06/2006

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Figure 2. The structures derived from the ring fission of quercetin by colonic bacteria. The structure on top is quercetin, (a) hydroxybenzoic acid, (b) hydroxyphenylacetic acid, and (c) phydroxyphenylpropionic acid. (The arrows indicate the site of ring cleavage.)

deglycosylated (20, 22, 23). Additional studies indicate that flavonoids can be absorbed after deglycosylation by hydrolases (e.g., lactose-phlorizin hydrolase or beta-glucosidase) located at the intestinal brush border membrane (24-26). In either case, once the flavonoid is absorbed, it is further metabolized by UDPglucuronyl transferases, sulfotransferases, and methyl transferase, forming numerous glucuronidated, sulfated, and methylated conjugates prior to reaching systemic circulation (2731). Because the majority of quercetin remains in the gut, it is a substrate for the intestinal microflora. Intestinal degredation of quercetin leads to the formation of the aglycone and phenolic acids that result from ring fission reactions (32). Two studies (33, 34) report that bacteria of the colon can cleave the heterocyclic ring of flavonols to form phenyl-C3 and phenylC2 metabolites, such as hydroxyphenyl acetic acid, hydroxyphenyl propionic acid, and hydroxycinnamic acid (Figure 2). These metabolites are reabsorbed and subsequently metabolized by enzymes located mainly in the liver (35) Several studies utilizing LC/MS/MS have undertaken the task of profiling the range of quercetin metabolites circulating in plasma and urine in humans (30, 36). Studies from our lab indicate that that 21 quercetin metabolites are present in human urine after the ingestion of 200 g of cooked onion (∼74 mg of quercetin) and include quercetin monoglucuronides, methyl quercetin monoglucuronides, quercetin monoglucuronide sulfate, quercetin diglucuronides, methyl quercetin diglucuronide, quercetin-glucoside sulfates, methyl quercetin, and quercetin aglycone (36). In these studies of the metabolism of quercetin, the formation of conjugates with the tripeptide glutathione (GSH) were not considered. However, if the basic mechanism by which flavonoids provided their protective effect is by neutralizing reactive oxygen species (37), then at some point, they will be converted into quinoidal products that could potentially react with cellular nucleophiles such as GSH. GSH is present in significant levels in most tissues (e.g., the average cellular GSH concentrate is 5 mM) and is easily oxidized to GSSG in the presence of quinones. The ratio of GSH/GSSG is a critical factor in assessing the oxidative stress burden of a tissue. The reaction of GSH with electrophiles occurs spontaneously or can be catalyzed by the phase II enzymes gluthione S-transerfases (GSTs). Several in Vitro studies demonstrate that quercetin aglycone readily forms a glutathione adduct in the presence of peroxidases or tyrosinases as these enzymes oxidize quercetin to electrophlic

Hong and Mitchell

quinoidal products that react with GSH, forming mono and diglutathione adducts (38-40). In unpublished studies, our lab has determined that the rate of the reaction between GSH and oxidized quercetin is rapid and not accelerated by the presence of the major mammalian isozymes of GST (alpha, pi, and mu). It has been suggested that the oxidation of quercetin has the potential to deplete intracellular levels of GSH, rendering cells more sensitive to oxidative stress (41) as well as generating pro-oxidant quinoid products. Previous investigations have shown that other catechol-containing flavonoids, such as catechin (42), taxifolin, luteolin (43), and fisetin, are capable of reacting with GSH to generate mono and diglutathionyl adducts in the presence of chemically induced oxidative stress. To date, there are no in ViVo studies demonstrating a reaction between quercetin and GSH. However, Spencer et al. (44) have shown that glutathione-related quercetin conjugates are formed in cultured fibroblasts, suggesting that the oxidative metabolism of quercetin in fibroblasts occurs in the intracellular environment. More recently, Hernandez-Montes et al. (2006) demonstrated that genestein but not dadzein protects against oxidative stress-induced injury in endothelial cells in culture through the mediation of nuclear factor E2-related factor 1 (Nrf1) activation of glutathione peroxidase (45). Genistein produced decreases in intracellular GSH levels and the formation of a 2′-glutathionyl conjugate of the oxidized genistein metabolite (5,7,3′,4′tertrahydroxyisoflavone). Conversely, daidzein did not form a glutathione adduct or invoke the nuclear translocation of Nrf1 and activation of glutathione peroxidase activity (45). This suggests that the oxidative activation and accumulation of nontoxic levels of the reactive intermediate may trigger the upregulation of phase II detoxification enzymes via Nrf1 activation of the antioxidant response element (ARE)/electrophile response element (EpRE) pathway. Furthermore, Van der Woude et al. (2005) demonstrated that the intracellular formation of prooxidant metabolites of quercetin is independent of tyrosinase and/or peroxidase-type oxidative enzyme activities and that these reactive intermediates lead to the formation of quercetin-DNA adducts that are transient in nature (46). The transient nature of these adducts may explain the lack of in ViVo toxicity associated with quercetin (46), whereas the upregulation of phase II enzymes might explain antioxidant and anticarcinogenic effects. This article describes two independent studies examining the metabolism of quercetin. In the first, the metabolism of quercetin was investigated in murine hepatic suspensions in the absence of any pro-oxidant using LC/ESI-MS/MS. The second study describes the LC/ESI-MS/MS identification of several intestinal microflora degredation products of quercetin in humane urine after the consumption of 200 g of onion. In both studies, several glutathione conjugates of quercetin were identified. To our knowledge this is the first description of the in ViVo formation of glutathione conjugates of quercetin in humans.

Materials and Methods Chemicals. Quercetin, UDPGA (uridine 5′-diphosphoglucuronic acid), PAPS (adenosine 3′-phosphate-5′-phosphosulfate), SAM (sadenosyl-L-methionine chloride) were purchased from Sigma Chemical Co. All HPLC solvents were purchased from Fisher (Pittsburgh, PA). Other reagents and chemicals were purchased from either Fisher or EM Science (Gibbstown, NJ) as analytical grades. Murine Hepatic Suspensions. Quercetin metabolites were generated using a modified method from Morand et al. (47). Male Swiss Webster mice were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Males (2 per group) were housed in wire-bottom cages and fed ad libitum on a purified diet. Liver homogenate was prepared by homogenizing 2 g of liver in

Glutathione-Related Quercetin Metabolites 10 mL of ice-cold Krebs-Henseleit buffer (48). Quercetin (750 µM) was added to 1 mL of liver homogenate and incubated at 37 °C for 3 h in the presence of 4 mM UDPGA, 400 µM PAPS, and 800 µM SAM. The reaction was stopped by the addition of 750 µM ice-cold methanol. The reaction mixture was centrifuged at 4000 rpm for 20 min. The supernatant was filtered through a 0.45 µL PTFE membrane filter. Twenty milliliter aliquots were analyzed directly by LC/ESI-/MS/MS. Quercetin metabolite generation by hepatic incubation were done in triplicate, and the analysis by LCESI/MS/MS were duplicated per sample. Collection and Preparation of Urine Samples. Subjects (five women, one men, 23-40 ages) consumed 200 g of cooked onion fried with oil (∼74 mg of quercetin) after 24 h of low-flavonoid diet. Urine was collected at 0 h as a control and also over 12 h (between 80 and 100 mL) in a sterile container before and after onion consumption, respectively. Urine samples were purified by SPE. The urine sample (about 100 mL) was loaded onto Strada (Phenomenax, Torrence, CA) C18 sep-pak cartridge (10 g/60 mL) pretreated with 60 mL of methanol followed by 60 mL of water. The column was rinsed with 5 column volumes of water (300 mL) and eluted with 60 mL of aqueous methanol (1:1, v/v). The extract was freeze-dried after methanol evaporation by nitrogen flushing and measured as 25 mg aliquot of freeze-dried powder. This sample was then reconstituted in 100 µL of water and filtered through 0.45 µM PTFE and HPLC membrane filter prior to LC/ESI-MS/MS analysis. The injection volume was 10 µL. LC/ESI-MS/MS Analysis. Metabolites generated using the murine hepatocyte mixture were separated by a reversed-phase HPLC system (Shimadzu Scientific, Columbia, MD) using a 5 micron Prodigy (Phenomenex, Torrance, CA), ODS column (250 × 2 mm), equipped with a SIL-10A auto injector, binary LC 10AD pumps, and a SPD-10A UV/vis detector monitoring at 370 nm. The mobile phase consisted of 1% formic acid in water (solvent A) and 1% formic acid in acetonitrile (solvent B). Separation was effected using a series of linear gradients at a flow rate of 0.2 mL/ min as follows: elution starting with 5% B, 0-10 min; 5-20% B, 10-30 min; 20% B, 30-50 min; 20-21% B, 50-70 min; 21% B, 70-80 min; 21-60% B, 80-90 min; 60% B, 90-95 min; and 60100% B, 95-100 min. For the separation of mercapturic acid conjugates, the same HPLC system was used except for the gradient applied as follows: 5%- 60% in B, 0-60 min. A Quattro LC triplequadrupole mass spectrometer (Micromass, Altrincham, UK) equipped with a dual orthogonal (ZSPRAY) ion source was used for all analyses. Samples were run in positive ion mode using a capillary voltage of 3.2 kV. The cone and extractor voltages were set to 20 V and 2 V, respectively. The source temperature and a desolvation gas temperature were 150 and 300 °C, respectively. Optimum nebulization was achieved using a nitrogen flow rate of 600 L/hour. Total ion chromatograms (TIC) were recorded over a mass range of m/z 50-800. The m/z value for each peak in the TIC was first determined in order to identify possible quercetin metabolites. This approach allows for the preliminary identification of metabolites that may not have been previously realized (e.g., glutathione conjugates) and is critical to the success of selected ion monitoring (49). Peaks showing m/z values corresponding to possible quercetin metabolites were further investigated using electrospray ionization-tandem mass spectrometry (LC/ESI-MS/ MS). Parent ions were detected by selected ion monitoring, and daughter ions were generated using data-dependent scanning techniques. Data were collected and processed using MassLynx software (v3.5). Argon gas (2.3 × 10-3 bar) was applied as the collision-induced dissociation (CID). Collision voltage was varied from 10 to 35 eV depending on different metabolites.

Results and Discussion In order to better understand the metabolism of quercetin and determine if the murine hepatic suspension was an appropriate model for the generation of querceitn metabolites, the metabolites of quercetin were compared in murine hepatic suspension

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Figure 3. UV chromatogram (370 nm) of quercetin extracted from the murine hepatic suspension. Numbered peaks represent quercetin metabolites identified by LC/ESI-MS/MS in positive ion mode.

and human urine taken after the consumption of 200 g of onions. Figure 3 shows the UV chromatogram at 370 nm of hepatic incubation mixture. All peaks appearing in this chromatogram were examined by full scanning mode MS prior to MS/MS experiments to identify all possible metabolites derived from quercetin biotransformation (e.g., glucuronidation, sulfation, methylation, and also glutathionylation) on the basis of calculated masses. Table 1 summarizes the 18 quercetin metabolites identified in murine hepatic suspensions. Our results indicate that four isomers of quercetin monoglucuronides are formed in murine hepatic suspensions (peaks 9, 11, 12, and 15 in Figure 3). The identification of monoglucuronidated metabolites of quercetin has been reported in previous studies. Day et al. (50) also demonstrated the presence of four quercetin monoglucuronides isomers in human liver cell-free extracts, which agrees with our results. This finding also agrees with other studies, showing four isomers of quercetin monoglucuronides generated in rat liver microsomes (50-53). In addition, the conjugational positions of these four isomers have been described and confirmed by UV absorption spectral shift assays (50) and more unequivocally by LC/MS/MS and 1H NMR (51). A representative product ion scan (PIS) chromatogram and ESI-MS/MS spectrum of peaks 9, 11, 12, and 15 of Figure 3 are given in Figure 4 a. By comparing the elution order of these four isomers with the previous study using the similar LC conditions (51), it appears that peak 9, 11, 12, and 15 are the 3, 7, 4′, and 3′ quercetin monoglucuronides, respectively. Very recently, Mullen et al. (31) identified three major quercetin monoglucuronides including 3, 4′, and 3′ monoglucuronides in human urine and plasma after the consumption of red onions. Peaks 2, 5, and 7 in Figure 3 were identified as quercetin diglucuronides, showing an [M + H]+ ion at m/z 655 and generating a product ion at m/z 479 (the loss of glucuronic acid) and one at m/z 303 (quercetin aglycone). A representative PIS chromatogram and ESI-MS/MS spectrum is given in Figure 4b. Using the same approach described above, peaks 13, 16, and 18 in Figure 3 were identified as three isomers of methyl quercetin monoglucuronides, whereas peak 10 in Figure 3 was identified as quercetin sulfate monoglucuronide. The three methyl quercetin monoglucuronides generated product ions at m/z 317 (methyl quercetin) and 303 (quercetin). A representative PIS chromatogram and ESI-MS/MS spectrum for these isomers are given in Figure 4c. Van der Woude (51) reported the presence of 2 isomers of quercetin monosulfate in an in Vitro model system using the rat liver S9 fraction incubated with the sulfate transferase cofactor PAPS. They were also found in human urine in the experiments with the red onion consumption (31). Our results show that a sulfated form of quercetin occurs as a mixed conjugate with the glucuronic acid (Peak 10 in Figure

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Table 1. Qurcetin Metabolites Identified by Lc/ESI-MS/MS in Murine Hepatic Suspensions peaka 1 2 3

glutathionyl quercetin monoglucuronide quercetin diglucuronide

[M + H]+ (m/z) 784 655

4

glutathionyl quercetin monoglucuronide glutathionyl quercetin

608

5

quercetin diglucuronide

655

6

glutathionyl methyl quercetin quercetin diglucuronide

622

7 8 9 10 11 12 13 14 15 16/17

18 a

compound

glutathionyl methyl quercetin quercetin monoglucuronide quercetin sulfate monoglucuronide quercetin monoglucuronide quercetin monoglucuronide methyl quercetin monoglucuronide glutathionyl quercetin (quinone) quercetin monoglucuronide methyl quercetin monoglucuronide/glutathionyl methyl quercetin methyl quercetin monoglucuronid

784

655 622 479 559 479 479 493 606 479 493/622

493

fragment ions (m/z) 707, 681, 608, 590, 534, 479, 335 (Scheme 1) 479 ([M + H]+- GlucUA), 303 ([M + H]+- GlucUA - GlucUA) 707, 681, 608, 590, 534, 479, 335 (Scheme 1) 534, 479, 461, 376, 335, 303 (Scheme 1) 479 ([M + H]+- GlucUA), 303 ([M + H]+- GlucUA GlucUA) 547, 479, 347, 317 (Scheme 1) 479 ([M + H]+- GlucUA), 303 ([M + H]+GlucUA - GlucUA) 547, 479, 347, 317 (Scheme 1) 303 ([M + H]+- GlucUA) 383 ([M + H]+- GlucUA), 303 ([M + H]+GlucUA- SO3) 303 ([M + H]+- GlucUA) 303 ([M + H]+- GlucUA) 317 ([M + H]+- GlucUA) 532, 477, 459, 374, 333, 301 (Scheme 1) 303 ([M + H]+- GlucUA) 317 ([M + H]+- GlucUA), 303 ([M + H]+GlucUA- CH3)/ 547, 479, 347, 317 (Scheme 1) 317 ([M + H]+- GlucUA)

Peak numbers refer to Figure 1. GlucUA, glucuronyl unit; Glc, glucosyl unit; [M + H]+, molecular ion.

Figure 4. PIS (product ion scanning) chromatogram and MS/MS spectra of (a) quercetin monoglucuronide, (b) quercetin diglucuronide, (c) methyl quercetin monoglucuronide, and (d) queretin monoglucuronide sulfate

4) in the murine hepatic incubation mixture. The PIS chromatogram and ESI-MS/MS spectrum for peak 10 is given in Figure 4d. More interestingly, we found several glutathione-related quercetin metabolites. These were present as glutionylated quercetin and as mixed conjugates with glucuronides and

methylated forms of quercetin. As a catechol, quercetin can potentially be converted into quercetin-o-semiquinone and quercetin-o-quinone and subsequently conjugated with glutathione (GSH). Previous reports have indicated that GSH conjugates of quercetin (e.g., 6-glutathionyl and 8-glutathionyl conjugates) are formed in Vitro (39, 44, 54). However, in these

Glutathione-Related Quercetin Metabolites

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Figure 5. PIS (product ion scanning) chromatogram and MS/MS spectra of (a) glutathionyl queretin, (b) glutathionyl quercetin (quinone), (c) glutathionyl quercetin monoglucuronide, and (d) glutathionyl methylquercetin.

Scheme 1. CID Fragmemtation of Glutathione Adducts of Quercetin and the Description of the Major Fragment Ions Formed (a-f)

experiments, the formation of GSH adducts was expected due to the addition of tyrosinase or peroxidase in these cells, which catalyze quercetin quinone/quinone methide formation prior to their reaction with GSH (55-57). Using the murine hepatic suspensions, seven glutathione-related conjugates of quercetin and/or quercetin monoglucuronide or methyl quercetin were identified (peaks 1, 3, 4, 6, 8, 14, and 17 in Figure 3). Peak 4 and 14 in Figure 3 were identified as glutathionyl quercetin and glutathionyl quercetin quinone, respectively, and PIS and M/MS spectra are given Figure 5 a and 5b. CID fragmentation patterns for these conjugates are described in Scheme 1. Peak 4 demonstrates the precursor ion ([M + H]+) at m/z 608 (peak 4), which corresponds to a glutathione conjugate of quercetin, whereas the precursor ion for peak 14 was observed at m/z 606; 2 mass units lower, indicating peak 14 is the quinoid form of glutathionyl quercetin. The longer retention time for peak 14 compared to peak 4 would be expected of the quinoidal structure as this would result in a relatively higher hydrophobicity. Spencer et al. (44) also found two novel cellular metabolites,

2′-glutathionyl quercetin conjugate and another product with similar spectral characteristics and, one mass unit lower, putatively a quinine/quinine methide. Peaks 1 and 3 in Figure 3 were identified as 2 isomers of the glutathionyl quercetin monoglucuronides. A respresentative PIS and MS/MS spectrum are given in Figure 5c demonstrating an [M + H]+ at m/z 784 corresponding to the glutathionyl quercetin monoglucuronides and characteristic fragment ions relating to the loss of the glucuronic acid and fragmentation of glutathione. Peaks 6, 8, and 17 in Figure 3 were identified as methylated forms of glutathionylated quercetin with the [M + H]+ appearing at m/z 622 and characteristic fragments relating to the fragmentation of glutathione as represented in Figure 5d. A description of the fragmentation of glutathione-related quercetin metabolites is given in Scheme 1. It appears that the methylation of the catechol group has no effect on the ability of quercetin to form a thiol-adduct with GSH. Methylation of quercetin, which is facilitated by catechol O-methyl transferase, occurs at either the 3′ or 4′ position. Methylation at either of these positions would prohibit quercetin from forming a quinone structure in the B ring. It is possible that glutathionylation occurs prior to the methylation of quercetin, indicating that gluthationylated quercetin is still a susbstrate for catechol O-methyl transferase. However, Van der Woude et al. recently (2006) demonstrated that the formation of 6′- and 8′-glutathionyl quercetin conjugates as well as formation of quercetin DNA adducts was still possible with methylation of the catechol group, and a mechanism to explain the formation of the quinine methides without the intermediate orthoquinone at the B ring catechol formation was suggested (58). Taking this into account, it seems more likely that glutathione conjugation occurs with the methylated quercetin because binding of the glutathione conjugate to the catechol O-methyl transferase seems less likely. Taken together, the data obtained in the murine hepatic suspensions revealed that glutathione adducts of quercetin form in Vitro in the absence of chemically or enzymatically generated oxidative stress. The identification of glutathione-related quercetin adducts may help explain some of the variable results obtained in earlier cell culture assays (53, 59).

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Scheme 2. CID Fragmentation of Mercapuric Acid Conjugates of Colonic Degradation Products of Quercetin and Suggested Mercapturic Acid Metabolites

In a previous study, we identified the profile of quercetinrelated phase II metabolites in human urine taken after the consumption of 200 g of cooked onion using LC/ESI-MS/MS (36). In this study, we were able to identify 21 phase II metabolites of quercetin as conjugates, or mixed conjugates, of glucuronic acid, sulfate or a methyl group; however, no glutathione-related conjugates (mercapturic acids) were observed in this study. On the basis of results obtained in the murine hepatic suspension, these urine samples were reanalyzed, emphasizing peaks that gave characteristic CID fragments of mercapturic acids. Interestingly, early on in the total ion chromatogram, we identified several peaks that gave CID fragment ions corresponding to mercapturic acids (60) (Scheme 2). Upon further MS/MS investigation, it was recognized that these peaks corresponded to mercapturic acid derivatives of several colonic microbial degradation products of quercetin, including dihydroxyacetic acid, dihydroxypropionic acid, hydroxycinnamic acid, dihydroxytoluene, and dihydroxybenzaldehyde. The structures and masses of the identified degradation products of quercetin are illustrated in Figure 6. It was previously reported that quercetin is metabolized in the gut by microflora, forming hydroxyphenylacetic acids, including 3,4dihydroxylphenyl acetic acid, 3-hydroxyphenyl-acetic acid, and 4-hydroxy-3-methoxyphenyl-acetic acid (homovanillic acid). These products were considered as possible biomarkers for microbial and/or hepatic metabolism of quercetin (61). There have also been a number of studies in Vitro showing the bacterial

Figure 6. Structures of colonic degradation products of quercetin (a) dihydroxytoluene, (b) dihydroxybenzaldehyde, (c) dihydroxyphenylacetic acid, (d) dihydroxycinnamic acid, and (e) dihydroxyphenylpropionic acid.

degradation of rutin and quercetin-3-glucoside to 4-hydroxyphenyl-acetic acid (32, 62), 3,4-dihydroxyphenyl-acetic acid (32, 62), 3,4-dihydroxytoluene (34), and 3,4-dihydroxy-benzaldehyde (63). In order to improve the resolution of these relatively more hydrophilic compounds eluted out in the beginning of the chromatogram, a different gradient and mobile phase were applied during LC separation, monitoring 280 nm instead of 370 nm as described in the Materials and Methods section.

Figure 7. PIS (product ion scanning) chromatogram and MS/MS spectra of mercapturic acid conjugates of (a) dihydroxytoluene, (b) dihydroxybenzaldehyde, (c) dihydroxyphenylacetic acid, (d) dihydroxycinnamic acid, and (e) dihydroxyphenylpropionic acid. The arrows indicate isomers.

Glutathione-Related Quercetin Metabolites

Figure 7 demonstrates the PIS chromatograms and the MS/ MS spectra for the mercapturates of the hydroxyphenylacetic acids. Fragmentation of these mercapturates results in three primary ions that arise from the loss of the mercapturate, the loss of carboxylic acid and the methyl group and one more ion produced by the attachment of one water molecule. One example of this fragmentation during MS/MS is given in Scheme 2 with the description of suggested all other metabolites showing the same type of fragmentation pattern. Mass spectra demonstrate that the mercapturates of dihydroxyphenylacetic acid and dihydroxycinnamic acid are present as isomers. This can be explained by the observation that glutathionylation of flavonoid containing catechol structures like quercetin are formed through quinone intermediates (11, 53, 55), which result in multiple active conjugation sites on the B ring under physiological conditions. Further identification of these products by NMR is required for the unambiguous structural identification of these metabolites.

Conclusion This present study indicates that glutathione conjugates of quercetin can form in murine hepatic suspension in the absence of chemically or enzymatically induced oxidative stress. They appear as glutathione adducts of quercetin, methyl quercetin, and quercetin glucuronides. More importantly, our studies of quercetin metabolites in human urine also reveal evidence for the glutathionylation of quercetin as supported by the observation that mercapturic acids of common hydroxyphenylacetic acids generated by the microbial degredation of quercetin exist in human urine after the consumption of 200 g of cooked onions (∼74 mg of quercetin). It remains unclear as to whether the GSH adduct of quercetin is formed in the liver during first pass metabolism prior to excretion into bile or whether the GSH adduct of quercetin forms in the gut, is metabolized by gut microflora, and then reabsorbed. Further research to answer this question is currently underway in our lab.

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