Identification of Novel Electrophilic Metabolites of Piper methysticum

Chem. Res. Toxicol. 2003, 16, 733-740

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Identification of Novel Electrophilic Metabolites of Piper methysticum Forst. (Kava) Benjamin M. Johnson,†,‡ Sheng-Xiang Qiu,§ Shide Zhang,§ Fagen Zhang,† Joanna E. Burdette,†,‡ Linning Yu,† Judy L. Bolton,†,‡ and Richard B. van Breemen*,†,‡ Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231, and Herbstandard, Inc., 1743 Canyon View Court, Chesterfield, Missouri 63017 Received December 16, 2002

Dietary supplements containing Piper methysticum Forst. (kava) have been implicated in multiple cases of liver injury in humans, including 10 recently reviewed cases in which patients required liver transplantation following the usage of kava-containing products (Centers for Disease Control and Prevention, reprinted. (2003) J. Am. Med. Assoc. 289, 36-37). To investigate a possible mechanism(s) of kava-induced hepatotoxicity, an extract of kava was incubated in vitro with hepatic microsomes, NADPH, and GSH. Electrophilic intermediates that were generated via metabolic activation were trapped as GSH conjugates and removed from the protein mixture using ultrafiltration. Positive ion electrospray LC-MS/MS with precursor ion scanning was used for the selective detection of GSH conjugates, and LC-MSn product ion scanning was used to elucidate their structures. Using this in vitro MS-based screening assay, two novel electrophilic metabolites of kava, 11,12-dihydroxy-7,8-dihydrokavaino-quinone and 11,12-dihydroxykavain-o-quinone, were identified. Mercapturic acids of these quinoid species were not detected in the urine of a human volunteer following ingestion of a dietary supplement that contained kava; instead, the corresponding catechols were metabolized extensively to glucuronic acid and sulfate conjugates. These observations indicate that quinoid metabolites, under most circumstances, are probably not formed in substantial quantities following the ingestion of moderate doses of kava. However, the formation of electrophilic quinoid metabolites by hepatic microsomes in vitro suggests that such metabolites might contribute to hepatotoxicity in humans when metabolic pathways are altered (e.g., because of a drug interaction, genetic difference in enzyme expression, etc.) or if conjugation pathways become saturated.

Introduction In March 2002, a consumer advisory was issued by the United States Food and Drug Administration warning that severe liver injury might be caused by the consumption of dietary supplements containing kava (1). According to the advisory, more than 25 cases of liver injury associated with kava including hepatitis, cirrhosis, and liver failure have been reported in other countries. The Centers for Disease Control and Prevention recently reviewed 10 such case reports (two in the United States, six in Germany, and two in Switzerland) in which liver transplants were necessary following hepatic failure that was associated with the use of kava-containing supplements (2). Adverse neurological and dermatological reactions to kava have also been reported (3-8). These cases have prompted several European regulatory agencies to issue warnings about the safety of supplements contain* To whom correspondence should be addressed. Tel: (312)996-9353. Fax: (312)996-7107. E-mail: [email protected]. † Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago. ‡ UIC/NIH Center for Botanical Dietary Supplements Research, College of Pharmacy, University of Illinois at Chicago. § Herbstandard, Inc..

ing kava or remove them from the marketplace altogether. Regulatory action by the Food and Drug Administration concerning the legal sale of such supplements in the United States might be necessary as more details about kava toxicity become available (1). Mechanisms that have been proposed to explain adverse reactions to kava include an immunoallergic response (9) and the inhibition of cytochrome P450 isoforms by various kava constituents (10). Elevated levels of γ-glutamyltransferase have also been reported among heavy kava users (11). The chemical compounds that are unique to kava and appear to be responsible for its anxiolytic activity (12) include the 5,6-dihydro-R-pyrones KV,1 MT, and their 7,8-dihydro derivatives (Figure 1). Several stable metabolites of these compounds were identified in urine and feces of rats following administration via gavage (13); however, reactive metabolites resulting from their bioactivation have not been reported previously. 1 Abbreviations: CID, collision-induced dissociation; DDKV, 11,12dihydroxy-7,8-dihydrokavain; DHKV, 11,12-dihydroxykavain; DKV, 7,8-dihydrokavain; DMT, 7,8-dihydromethysticin; KV, kavain; MT, methysticin.

10.1021/tx020113r CCC: $25.00 © 2003 American Chemical Society Published on Web 05/15/2003

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Figure 1. Six compounds isolated previously from kava and associated with anxiolytic activity (12).

Quinones are a class of electrophilic phase I metabolites that can cause toxicity via the covalent modification of biological nucleophiles and/or through redox cycling leading to the formation of reactive oxygen species (14). Ultrafiltration LC-MS/MS methods for the selective detection of such metabolites in complex mixtures have been developed recently in our laboratory (15, 16). These methods entail the in vitro incubation of a combinatorial library or botanical extract with hepatic microsomes, NADPH, and GSH. Quinoid and epoxide metabolites that are generated by metabolic activation are trapped as GSH conjugates and removed from the protein mixture using ultrafiltration. Positive ion electrospray LC-MS/ MS, using precursor ion scanning or constant neutral loss scanning and CID, is used for the selective detection of ions that fragment to form the ion of m/z 130 or eliminate a neutral fragment weighing 129 u corresponding to the γ-glutamyl moiety of GSH. The output from this analysis is a chromatogram in which every peak corresponds to a GSH conjugate. After such conjugates are detected, additional LC-MSn analyses using product-ion scanning may be carried out to provide information about their structures. During the analysis of botanical extracts, the NAPRALERT natural products database may be used to help identify unknown metabolites by providing a list of compounds previously isolated from a particular plant (17). Hence, this assay facilitates the rapid identification of electrophilic species that are formed during the metabolism of complex mixtures. The purpose of the present study was to identify novel reactive metabolites that might result from the bioactivation of compounds present in an extract of kava and to determine whether such metabolites are formed in vivo. Evidence for the formation of reactive metabolites of kava constituents by hepatic enzymes would provide a possible mechanism for kava-induced hepatotoxicity in humans.

Materials and Methods All reagents and cofactors were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted, and all

Johnson et al. solvents were from Fisher Scientific (Pittsburgh, PA) and were HPLC grade or better. Isolation and Identification of Kavalactones. Dried kava roots (2 kg, Frontier Botannicals, Norway, IA) were extracted three times with 4 L of methanol/water (1:1, v/v). The extracts were filtered and dried under vacuum to afford 250 g of residue. The composition of the residue was determined using HPLCPDA with a YMC (Wilmington, NC) Basic 5 µm (4.6 mm × 150 mm) column and absorbance detection at 220 nm. The mobile phase consisted of methanol/acetonitrile/0.1% acetic acid (20: 20:60, v/v/v) at 1 mL/min. For the isolation of kava lactones, the residue (10 g) was separated on a silica gel column, eluting stepwise with hexane, CHCl3, CHCl3/acetone (1:1), and finally acetone. The CHCl3 eluate was subjected to additional chromatographic separations using a Fuji Silysia (Durham, NC) Chromatorex ODS column eluted with 80% methanol and then a silica gel column eluated with hexane-CHCl3 (1:2), hexane-CHCl3-EtOAc (3:2:1), and then hexane-acetone (2:1) to give MT (300 mg), DMT (120 mg), KV (280 mg), DKV (160 mg), yangonin (295 mg), and desmethoxyyangonin (50 mg). The abundance of each lactone in the extract was determined using standards that were provided by Herbstandard (Chesterfield, MO). Structures were verified by comparison of the 1H NMR, 13C NMR, and mass spectra with those reported in the literature (18, 19). Screening Assay for GSH Conjugates of Electrophilic Metabolites. Dexamethasone-induced Sprague-Dawley rat liver microsomes were prepared as previously reported (20), and pooled human microsomes were purchased from In Vitro Technologies (Baltimore, MD). Microsomes were stored at -80 °C until use. Stock solutions of 10 mM GSH and 10 mM NADPH in deoxygenated water were prepared ahead of time, separated into 100 µL aliquots, and stored at -20 °C for use within 1 week. A sample containing kava extract (1 mg), microsomes (50 µL, 20 mg protein/mL), and 100 µL aliquots of GSH and NADPH were dissolved in 400 µL of 50 mM phosphate buffer (pH 7.4). A negative control was prepared in which additional phosphate buffer was substituted for the microsomes. The samples were incubated at 37 °C for 30 min, subjected to centrifugal ultrafiltration at 10 000g using Microcon (Millipore Corp., Bedford, MA) regenerated cellulose 30 000 MWCO filters, and stored at 4 °C for analysis within 24 h. The samples were then analyzed using LC-MS/MS with a ThermoFinnigan (San Jose, CA) Surveyor HPLC system, equipped with a Waters (Milford, MA) Xterra MS C18 (2.1 mm × 150 mm) column and a ThermoFinnigan TSQ Quantum triple quadrupole mass spectrometer. The HPLC solvent system consisted of 0.1% formic acid and 5% acetonitrile for 5 min (eluent diverted to waste), followed by a 15 min linear gradient from 5 to 20% acetonitrile, isocratic 20% acetonitrile for 10 min, a gradient from 20 to 90% acetonitrile over 10 min, and finally 90% isocratic acetonitrile for 15 min. Positive ion electrospray tandem mass spectra were acquired using precursor ion scanning (for precursors of m/z 130) with a collision energy of 26 V and a scan time of 2 s over the range m/z 406-906. GSH conjugates that were detected during screening were reanalyzed for structural characterization using positive ion electrospray LC-MSn on a ThermoFinnigan LCQ Deca ion trap mass spectrometer. (Although triple quadrupole mass spectrometers are ideal for precursor ion scanning, ion traps are more sensitive for product ion scanning and have the added capability of multiple stages of tandem mass spectrometry for structure elucidation and confirmation.) The NAPRALERT database was queried for compounds previously isolated from kava, and the structures of these compounds were retrieved from the SciFinder database. The output from these databases and the product ion MSn spectra were used to infer probable identities for the compounds detected during screening. To confirm these identities, 10 mM (25 µL) solutions of the authentic standards of MT and DMT were substituted for the kava extract, and the ultrafiltration LC-MS/MS product ion analyses were repeated. Incubations using the MT and DMT standards were also carried out in the absence of GSH so that

Metabolites of Piper methysticum Forst.

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Figure 2. Positive ion electrospray ultrafiltration LC-MS/MS screen for precursors of the b1 GSH fragment ion in a metabolite preparation of kava. (A) Control without microsomes. (B) Experiment with microsomes, GSH, P. methysticum, and NADPH. negative ion electrospray LC-MS/MS product ion spectra could be recorded for selected phase I metabolites of these compounds. The solvent system for this negative ion LC-MS/MS analysis consisted of 50 mM ammonium acetate and acetonitrile as follows: isocratic 5% acetonitrile for 5 min (eluent diverted to waste), 5-90% acetonitrile over 40 min, and isocratic 90% acetonitrile for 10 min. Investigation of Electrophilic Metabolites In Vivo. A healthy human male, 27 years of age, who periodically takes dietary supplements that contain kava, volunteered to participate in this study. This subject took a single dose of a dietary supplement containing a kava root extract (lot MA11167; Nature’s Resource Products, Mission Hills, CA). Per the product label, this dose contained 300 mg of extract and was standardized to contain 90 mg of kava lactones. Urine samples were collected immediately before and 8 h after ingestion of the product and were immediately cooled to 4 °C. Within 12 h, a 10 mL aliquot of each urine sample was acidified with 50 µL of concentrated formic acid and extracted using a RESPREP Drug Prep I solid phase extraction cartridge (Restek, Bellefonte, PA). Each cartridge was washed with 1 mL of 0.5% formic acid and eluted with 2 mL of formic acid/water/methanol (0.5:10:90, v/v/ v). The eluates were then analyzed using negative ion electrospray LC-MS on the LCQ Deca ion trap mass spectrometer. The solvent system consisted of 50 mM ammonium formate and acetonitrile as follows: isocratic 5% acetonitrile for 5 min (eluent diverted to waste), 5-90% acetonitrile over 35 min, and isocratic 90% acetonitrile for 10 min. Negative ion electrospray LC-MSn with multiple stage product ion scanning was used to record mass spectra for the metabolites of interest.

Results and Discussion An incubation containing GSH and rat liver microsomal metabolites of a kava extract was screened for the presence of reactive metabolites such as quinoids and epoxides that might have been trapped as GSH conju-

Figure 3. Positive ion electrospray LC-MS/MS product ion analysis of the bis-glutathionyl-DDKV conjugate eluting at 26.4 min in Figure 2. The peaks of m/z 365.1, 373.5, and 400.5 represent doubly charged z2, y2, and b2 product ions, respectively, which were formed via the dissociation of a single GSH moiety. The peak of m/z 617.1 corresponds to a monoprotonated product ion formed by the removal of γ-glutamyl groups from both GSH moieties.

gates. The results of this screening assay for GSH conjugates are shown in Figure 2. As compared to the negative control incubation that did not contain microsomes, the incubation that contained active microsomes exhibited new peaks of m/z 438.6, 570.5, and 568.2 at retention times of 26.4, 28.8, and 29.0 min, respectively. Subsequent analysis of these species using positive ion electrospray LC-MS/MS with product ion scanning confirmed that they were GSH conjugates. For example, the MS/MS product ion spectrum (Figure 3) of the GSH conjugate of m/z 438.6 eluting at 26.4 min indicated

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Figure 4. Positive ion electrospray LC-MS/MS product ion analysis of m/z 570.3 corresponding to the GSH conjugate of DDKV eluting at 28.8 min in Figure 2. Note that the MS/MS spectrum is dominated by peptide fragment ions corresponding to cleavage of the GSH moiety.

that it was a doubly charged conjugate [M + 2H]2+ in which two GSH moieties had become attached to an activated phase I metabolite. Following a search of the NAPRALERT database for compounds previously isolated from kava, as well as a review of the pertinent literature, it was inferred that the doubly charged ion of m/z 438.6 corresponded to a di-GSH adduct of DDKV. This structural assignment was subsequently confirmed by comparing the HPLC retention time and tandem mass spectrum of the kava metabolite to those of an authentic standard produced by incubating DMT with rat liver microsomes, NADPH, and GSH. Another GSH adduct of m/z 570.5 was detected at a retention time of 28.8 min in the LC-MS/MS chromatogram shown in Figure 2. This peak corresponded to a mono-GSH adduct of DDKV and was also formed during the incubation of a DMT standard with microsomes, NADPH, and GSH. The LC-MS/MS product ion mass spectrum of this GSH adduct of m/z 570.5 is shown in Figure 4 and contains b2, y2, and z2 product ions that are characteristic of GSH conjugates (16). However, little information regarding the structure of the phase I kava metabolite is evident from this spectrum since the only intense peaks are due to fragmentation within the GSH moiety. To facilitate the formation of more structurally significant fragment ions, a positive ion electrospray LC-MS4 analysis was carried out on the same conjugate. The abundant y2 product ion from MS/MS was fragmented during CID to produce the [RS + H]+ product ion of m/z 295 (MS3), which was selected as a precursor ion for CID and a fourth stage of MS. The MS4 product ion mass spectrum of the thiolated phase I metabolite and a proposed fragmentation scheme are shown in Figure 5. Because only the sulfur from the GSH moiety remained on this fragment ion of m/z 295, the abundant product ions in the MS4 mass spectrum reveal structural information about the natural product part of the molecule. For example, cleavage on either side of a methylene group to produce ions of m/z 141 and m/z 155 confirms that this is a DKV derivative and localizes the sites of attachment of the sulfur and hydroxyl groups to one particular ring. Therefore, this MS4 mass spectrum is consistent with that of a GSH conjugate of DDKV.

Johnson et al.

Figure 5. Positive ion electrospray LC-MS4 analysis of the mono-GSH conjugate of DDKV. The CID sequence was [M + H]+ of m/z 570.3 f y2 of m/z 441.1 f [RS + H]+ of m/z 294.9 f product ion mass spectrum.

Figure 6. Positive ion electrospray LC-MS/MS product ion analysis of m/z 568 from an ultrafiltrate of an incubation containing kava extract, NADPH, hepatic microsomes, and GSH. The peaks at 28.6, 29.1, and 29.7 min correspond to GSH adducts of DHKV.

Although the peak of m/z 568.2 at 29.0 min corresponding to the GSH conjugate of DHKV is only barely visible on the chromatogram shown in Figure 2, an MS/MS product ion scan of the same botanical ultrafiltrate revealed the presence of three distinct but closely eluting species of m/z 568.2 (Figure 6). Positive ion electrospray product ion mass spectra of these three compounds indicated that they were regioisomers of the DHKV-GSH conjugate (Figure 7), and these assignments were verified by comparison to the LC-MS/MS product ion analyses of an identical incubation in which an authentic MT standard was substituted for the kava extract. In all cases, the retention times and product ion scans of the standard GSH conjugates matched those of the conjugates in the botanical incubation. Dexamethasone-induced rat liver microsomes are used frequently in our laboratory to carry out metabolism studies because they can be produced in-house and are significantly less expensive than commercially available



Figure 7. Positive ion electrospray LC-MS/MS product ion analysis of three isomeric GSH conjugates of DHKV. The retention times of these isomers are (A) 28.6, (B) 29.1, and (C) 29.9 min. The normalized collision energy is 28% for all three mass spectra.

pooled human microsomes. However, to confirm that the results of the studies using dexamethasone-induced rat liver microsomes were relevant to human metabolism, the incubations of the kava extract and MT and DMT standards were repeated using noninduced pooled human microsomes. Positive ion electrospray LC-MS and LC-MS/MS analyses of the metabolites that were formed using human microsomes again revealed the presence of mono-GSH conjugates corresponding to DHKV and DDKV. The di-GSH conjugate of DDKV was detected in trace amounts. These data are consistent with a bioactivation mechanism (Figure 8) involving oxidation of the methylenedioxy ring to give catechols followed by oxidation to o-quinones and reaction with GSH. Previously, Rasmussen et al. (13) investigated the urinary metabolites in rats of some of the most abundant kava pyrones, including KV, DKV, and MT, but not DMT, and noted that dihydroxylated derivatives (catechols) were formed following the oral administration of DKV and MT. In particular, they identified DHKV as a metabolite of MT and DDKV as a phase I metabolite of MT or DKV. However, Rasmussen et al. did not report the detection of any mercapturic acids following administration of the kava pyrones (400 mg/kg). On the basis of our data indicating that the catechols DDKV and DHKV are subject to enzymatic oxidation to form oquinones followed by conjugation with GSH, the urine of rats administered DKV and MT would have been expected to contain mercaputuric acids. However, mercapturic acid derivatives of these catechols might have been too polar or thermally labile to be detected using the GC-FID method of Rasmussen et al. On the basis of the work of Rasmussen et al. and our present studies, cytochrome P450-catalyzed O-demethylenation of the methylenedioxyphenyl compounds is

Figure 8. Cytochrome P450-catalyzed bioactivation of MT and DMT.

probably the most important source of catechol formation from kava. It should be noted that hepatotoxicity of compounds containing the methylenedioxyphenyl moiety is not without precedent. For example, the hepatotoxicant safrole [1-allyl-3,4-(methylenedioxy)benzene] contains this structural feature and is metabolized extensively to catechols via demethylenation by cytochrome P450 (21). Safrole may be bioactivated to multiple electrophilic metabolites including carbonium ions (22), o-quinones, and p-quinone methides (23).

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Although quantitative analyses were not carried out, it should be noted that the relative levels of the GSHtrapped quinones detected in the LC-MS/MS chromatogram in Figure 2 were unequal. For example, the GSH conjugates of DDKV produced LC-MS/MS peaks at 26.4 and 28.8 min that were more intense than the peak corresponding to the GSH adducts of DHKV at 29.0 min. This is in contrast to the approximately equal levels of MT, DMT, and DKV in the kava extract. The observation by Rasmussen et al. (13) that DDKV can be formed via the hydrogenation of MT might help to explain this result, although other factors including o-quinone reactivity could have also affected the relative amounts of GSH conjugates that were produced. To investigate whether the quinoid species that were generated during the ultrafiltration LC-MS/MS screening assay might also be formed in humans, a commercially available dietary supplement containing a kava root extract was administered to a human volunteer. Urine samples from the volunteer were analyzed using LC-MSn for the detection of possible urinary mercapturates and other metabolites of the kavalactones. A sample of the kava root preparation from the same vendor was analyzed for kavalactone composition using HPLC. The total kavalactone content was 29.4 wt %, which included 4.4% KV, 7.5% DKV, 2.9% MT, 0.75% DMT, 8.3% yangonin, and 4.0% desmethoxyyangonin. A positive control for the detection of mercapturic acids in human urine using ultrafiltration LC-MS/MS was carried out previously, in which a mercapturic acid conjugate was detected in the urine of a subject who ingested a 500 mg dose of acetaminophen.2 However, mercapturic acid conjugates of DHKV and DDKV were not detected in human urine during this study, indicating that quinoid metabolites of kava pyrones are not formed in significant quantities following ingestion of moderate doses of kava. This is consistent with the conclusion of Stevinson et al. that kava is usually well-tolerated by humans (8). However, we detected glucuronic acid and sulfate conjugates of both catechols in urine that was collected following kava administration. As a control, none of these conjugates were detected in urine from the same individual, which was collected immediately before ingestion of kava. For example, Figure 9 shows the LC-MS3 analysis of the sulfate conjugate of DHKV. Additional LC-MS3 data regarding the sulfate conjugate of DDKV were as follows: retention time, 20.4 min; MS3 sequence, m/z 343.1 f m/z 263.1; product ions: m/z 263.1 [M-H-SO3], 35%; m/z 263.2, 20%; m/z 219.1, 100%; m/z 204.1, 15%; m/z 187.2, 10%; m/z 177.0, 15%; m/z 161.1, 8%; m/z 146.9, 8%; m/z 140.9, 10%; m/z 121.0, 8%; and m/z 83.0, 18%. Glucuronic acid conjugates of DHKV and DDKV were detected eluting at 19.8 and 19.7 min, respectively. These ions were analyzed using MS3 with product ion scanning, in which the glucuronic acid moiety was eliminated via CID, and then, the ion containing the natural product moiety was subjected to additional fragmentation and a third stage of MS. The MS3 spectrum of each glucuronic acid conjugate was essentially identical to the MS3 spectrum of the corresponding sulfate conjugate in which the sulfate moiety had been removed via CID. (These spectra were also essentially identical to the MS/MS 2 Johnson, B. M., and van Breemen, R. B. Formation of electrophilic metabolites of the dietary supplement Actea racemosa (Cimicifuga racemosa; black cohosh): investigation of corresponding mercapturates in women. Unpublished results.

Johnson et al.

Figure 9. Negative ion electrospray LC-MS3 analysis of a urine sample from a human volunteer who took a supplement containing kava. (A) Elution at 20.6 min of a sulfate conjugate of DHKV. (B) MS3 product ion scan of the conjugate eluting at 20.5 min in part A. The product ion of m/z 261, formed via loss of the sulfate group from the precursor ion (m/z 341) during MS/ MS, was selected as a precursor for CID for a third stage of MS. The third stage product ion of m/z 217 corresponds to removal of CO2 from the lactone ring, and the ion of m/z 185 is formed via the additional loss of the methoxyl group. The ion of m/z 109 represents the deprotonated catechol ring.

spectra of unconjugated DHKV and DDKV that were recorded following the in vitro incubation of MT and DMT, respectively, with NADPH and hepatic microsomes in the absence of GSH.) Although Duffield et al. (24) did not detect these catechol metabolites in human urine following ingestion of a beverage containing an aqueous extract of kava, their GC-MS method did not include steps to hydrolyze glucuronic acid and sulfate conjugates of these compounds. In vitro incubations of KV and DKV with rat and human liver microsomes showed that metabolic products of these compounds included the catechols DHKV and DDKV, respectively, and that oxidation to the corresponding quinones could lead to the subsequent formation of GSH adducts. If DHKV and DDKV were already present in the kava extract or the commercial kava formulation, then metabolic O-demethyleneation of KV and DKV would not be required for the formation of the electrophilic quinones. Therefore, negative ion electrospray LC-MS analyses were carried out to determine whether DHKV and DDKV were already present in the kava extract studied in vitro or in the commercial kava formulation used for the in vivo study. Even though DHKV and DDKV have not been reported to be constituents of Piper methysticum Forst., both of these compounds were detected in these preparations (data not shown). Whether these compounds are biosynthetic products that occur naturally in kava or are formed as degradation products during sample processing is unknown. However, the biotransformation in vitro of MT and DMT standards to GSH conjugates of DHKV and DDKV, respectively (described above), demonstrates the formation of additional catechols during kava metabolism. The formation of sulfate and glucuronic acid conjugates of DHKV and DDKV represents a detoxication step that


precludes the oxidation of these catechols to o-quinones, and the absence of corresponding mercapturic acids indicates that such quinoid metabolites were not formed in substantial quantities during the metabolism of kavalactones in this particular human volunteer. However, there is circumstantial evidence that electrophilic metabolites might be formed during kava metabolism in vivo in other individuals or under other circumstances. First of all, the immunoallergic response reported by Stoller (9) is consistent with the covalent modification of hepatic proteins by quinoid metabolites; this adverse reaction to kava would be analogous to the immunologic response induced by the reactive quinone imine metabolite of sulfanilamide in patients who are allergic to sulfa drugs (25). Second, the elevation of plasma levels of γ-glutamyltransferase in heavy kava users (11) suggests that GSH is involved in kava metabolism since γ-glutamyltransferase facilitates the disposition of GSH conjugates and plays an important role in the maintenance of high intracellular GSH concentrations (26). Third, the formation of GSH conjugates of DHKV and DDKV in our ultrafiltration LC-MS/MS screening analysis demonstrates that kavalactones can be converted to o-quinones by enzymes in human and rat liver microsomes. These observations suggest that the formation of electrophilic metabolites could lead to kava-induced hepatotoxicty in select cases where the conjugation pathways that help detoxify kavalactones have become saturated or modified. Because sulfation pathways can be saturated via the depletion of the 3′-phosphoadenosine-5′-phosphosulfate cofactor (27), the existence of alternative metabolic pathways involving quinone formation might become relevant at high doses. Alternatively, certain individuals with unusual metabolic profiles, or for whom exposure to other xenobiotics has induced, down-regulated, or inhibited certain metabolic enzymes, might be especially susceptible to kava-induced hepatotoxicity. In conclusion, two electrophilic mammalian metabolites of the botanical dietary supplement kava, DHKVo-quinone and DDKV-o-quinone, were identified as GSH conjugates using an in vitro ultrafiltration LC-MS/MS screening assay. Mercapturic acids corresponding to these metabolites were not found in the urine of a human subject who took a single, moderate dose of kava. However, glucuronic acid and sulfate conjugates of the catechols DHKV and DDKV were detected. Although kava is usually well-tolerated by humans, several cases of kava-related liver injury have been reported. The results of this study suggest a possible mechanism of kava-induced hepatotoxicity for cases in which the detoxification pathways for certain kava pyrone catechol metabolites have become altered or saturated.

Acknowledgment. We thank ThermoFinnigan for providing the ion trap LC-MSn instrument used in this study. This project was funded by Grant P50 AT00155 provided to the UIC/NIH Center for Botanical Dietary Supplements Research by the Office of Dietary Supplements, the National Institute of General Medical Sciences, the Office for Research on Women’s Health, and the National Center for Complementary and Alternative Medicine. Its contents are the responsibility of the authors and do not necessarily represent the official views of the sponsors.


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Identification of Novel Electrophilic Metabolites of Piper methysticum

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