Kinetic evidence for multiple chemically reactive intermediates in the breakdown of phenacetin N-O-glucuronide

N-hydroxyphenacetin glucuronide has been previously shown to be an unstable compound (half-life 8.7 h) that breaks down to phenacetin, 2-hydroxyphenacetin glucuronide, acetaminophen, acetamide, and a deethylated metabolite that covalently binds to protein. Evidence was presented that the acetamide, acetaminophen and a compound that binds covalently were formed from a common intermediate which was postulated to be N-acetylimidoquinone. In the presence of phosphate buffer, 3-hydroxyphenacetin phosphate is formed at the expense of acetaminophen, acetamide and covalent binding. Phosphate buffer, however, only partially blocks covalent binding to protein suggesting that two deethylated reactive metabolites are formed that can covalently bind to protein. These metabolites also may be converted to acetaminophen but only one of them leads to acetamide. Since the phosphate conjugate contains the ethyl group apparently a third reactive intermediate, which can react with phosphate but not with protein, serves as a precursor of one of the metabolites.     

The metabolism of N-hydroxyphenacetin in vitro and in vivo

1. N-Hydroxyphenacetin (100 mg/kg) injected i.p. into rats rapidly appeared in the blood and disappeared with a t_1/2 of 14 min; phenacetin and 4-acetamidophenol were major metabolites in blood. Ferrihaemoglobin was formed, but 4-nitrosophenetole was not detected in blood.

2. N-Hydroxyphenacetin injected i.p. into rats was excreted in the urine unchanged (partly conjugated 2·1% of the dose, 2% was excreted as phenacetin, 19% as 4-acetamidophenol) and 1·8% as 2-hydroxyphenacetin. In addition, small amounts of 3-hydroxyphenacetin (0·4%) and traces of N-[4-(2-hydroxyethoxy)phenyl]acetamide (β-HAP) (0·05%) were found.

3. Time-course kinetics have shown that N-hydroxyphenacetin is metabolized in vitro to phenacetin, 2- and 3-hydroxyphenacetin, and 4-acetamidophenol by microsomal and cytosolic preparations of rat and rabbit liver. However, after the initial reaction, the formation of phenacetin and 2- and 3-hydroxyphenacetin did not continue with time, indicating that these products were not formed enzymically.

4. N-Hydroxyphenacetin incubated with rat erythrocytes formed ferrihaemoglobin; the relationship between ferrihaemoglobin, phenacetin and 4-nitrosophenetole concn indicated that N-hydroxyphenacetin was oxidized by oxyhaemoglobin to acetyl 4-ethoxyphenyl nitroxide, which yielded phenacetin and 4-nitrosophenetole spontaneously.     

Generation of reactive metabolites of N-hydroxy-phenacetin by glucoronidation and sulfation.

Several xenobiotics exert their toxic effects in mammals through the formation of reactive metabolites that combine with cellular macromolecules. Thus, N-hydroxy-2-acetylaminofluorene becomes covalently bound to various cellular macromolecules after sulfation of the N-hydroxy group. A method is presented for the indirect measurement of the rate of formation of the N,O-sulfate conjugate of this compound, which is too unstable to be measured directly. In this assay 3′, 5′-adenosine diphosphate and p-nitrophenylsulfate were used as a 3′-phosphoadenosine 5′-phosphosulfate-generating system (PAPS-GS); the release of p-nitrophenol (which was measured spectrophotometrically) was used to estimate the sulfation rate of N-hydroxy-2-acetylaminofluorene. The PAPS-GS was also used in studying the role of N-O-sulfate conjugates as intermediates in the formation of reactive metabolites that become covalently bound. Using this method we found that N-hydroxy-phenacetin became rapidly covalently bound after sulfation of the N-hydroxy group. N-hydroxy-2-acetylaminonaphthalene also became covalently bound after sulfation, but the N-O-sulfate derivatives of N-hydroxy-p-chloroacetanilide and N-hydroxy-acetanilide did not bind covalently, although their rates of sulfation were similar to that of N-hydroxy-phenacetin. Glucuronidation of the N-hydroxy group of N-hydroxy-phenacetin resulted in a glucuronide conjugate that was bound covalently to protein at pH 7.4, but at a slower rate than the N-O-sulfate conjugate. The N-O-glucuronides of the other N-hydroxy-N-arylacetamides investigated did not become covalently bound to protein at pH 7.4. Characteristics of the conjugation of N-hydroxy-phenacetin, and of the covalent binding of its conjugates, were determined.     

Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6)

Flavonoids are conjugated by phase II enzymes in humans to form glucuronidated and sulfated metabolites that are excreted in urine via the kidney. In this study, we examined the interaction between metabolites of quercetin and isoflavonoids found in vivo with human organic anion transporters 1 (OAT1) and 3 (OAT3) and their potential in attenuating OAT-induced cytotoxicity of adefovir. Accumulation of flavonoid conjugates was studied in human embryonic kidney 293H cells overexpressing OAT1 or OAT3. OAT1-overexpressing cells exhibited an increased uptake of the sulfated conjugates, genistein-4′-O-sulfate and quercetin-3′-O-sulfate. OAT3-overexpressing cells demonstrated enhanced uptake of glucuronide conjugates, such as daidzein-7-O-glucuronide, genistein-7-O-glucuronide, glycitein-7-O-glucuronide and quercetin-3′-O-glucuronide. Position of conjugation was important since quercetin-3-O-glucuronide and quercetin-7-O-glucuronide were poorly transported. Kinetic analysis revealed high affinity uptake of quercetin-3′-O-sulfate by OAT1 (K_m = 1.73 μM; V_max = 105 pmol/min/mg). OAT3 transported isoflavone glucuronides with lower affinity (K_m = 7.9–19.1 μM) but with higher V_max (171–420 pmol/min/mg). Quercetin-3′-O-sulfate strongly inhibited OAT1-mediated p-aminohippuric acid uptake with an IC₅₀ of 1.22 μM. Transport of 5-carboxyfluorescein by OAT3 was potently inhibited by quercetin-3-O-glucuronide, quercetin-3′-O-glucuronide and quercetin-3′-O-sulfate (IC₅₀ = 0.43–1.31 μM). In addition, quercetin-3′-O-sulfate was shown to effectively reduce OAT1-mediated cytotoxicity of adefovir, an antiviral drug, in a dose-dependent manner. These data suggest that OAT1 and OAT3 are responsible for basolateral uptake of flavonoid conjugates in kidney, and flavonoid conjugates inhibit OAT1 and OAT3 activity at physiologically relevant concentrations. Interaction with OATs limits systemic availability of flavonoids and may be a mechanism of food–drug interaction via inhibition of renal uptake.     

Formation of reactive metabolites of phenacetin in humans and rats

1. The metabolism of phenacetin to reactive intermediates in humans was estimated from the excretion of thio adducts in urine. N-Hydroxyphenacetin, a precursor of reactive metabolites, was also quantified.

2. Following an oral dose of phenacetin (10 mg/kg) to humans, these metabolites in 24 h urine were: paracetamol-3-cysteine, 4·4% dose; paracetamol-3-mercapturate, 3·9%; 3-thiomethylparacetamol, 0·4%; N-hydroxyphenacetin, 0·5%.

3. Rats showed a considerable increase in N-hydroxyphenacetin excretion after chronic dosing with phenacetin at high dosage (500 mg/kg) for one month. Chronic dosing with a low dose (50 mg/kg) did not increase N-hydroxyphenacetin excretion, but a marked increase occurred on concomitant administration of aspirin and caffeine.     

The gas-liquid chromatographic estimation of phenacetin and paracetamol in plasma and urine

Methods are described for the gas-liquid chromatographic estimation of phenacetin and paracetamol in plasma and free and conjugated paracetamol in urine. p-Chloracetanilide and p-bromacetanilide were used as internal standards. The drugs were extracted with ethyl acetate and before chromatography were converted to trimethylsilyl derivatives with N,O-bis(trimethylsilyl)acetamide (simultaneous assay of phenacetin and free paracetamol in plasma), or N-trimethylsilylimidazole (assay of paracetamol alone). Phenacetin was extracted with chloroform and chromatographed directly. Paracetamol glucuronide and sulphate were hydrolysed enzymatically to the parent compound before extraction. Recovery of added phenacetin and paracetamol in plasma at concentrations of 1–100 μg/ml was complete, and the limit of detection of the drugs in plasma was 0.05 μg/ml.     

Studies on the mutagenicity of N-hydroxy-2-acetylaminofluorene in the ames-salmonella mutagenesis test system

N-hydroxy-2-acetylaminofluorene (NOH-2AAF) was deacetylated by Sephadex G-25-chromatographed postmicrosomal supernatant fraction to a substance (presumably N-hydroxy-2-aminofluorene) which was a potent frameshift mutagen in the Ames-SalmoneIla tester strain TA 1538. Preincubation of the supernatant fraction with paraoxon decreased the observed mutagenesis; however, the addition of several reducing agents or nucleophiles to the incubations did not alter the mutagenic response. Sulfation of NOH-2AAF decreased the mutagenesis observed under conditions of supernatant activation and increased the covalent binding of [¹⁴C-acetyl]NOH-2AAF to protein. Under conditions of N-O-sulfate ester generation, ascorbate or reduced pyridine nucleotides greatly increased the number of revertant colonies. Ascorbate did not alter the rate of sulfation of NOH-2AAF, but decreased the protein covalent binding and greatly increased the reduction of the arylating species to 2-acetylaminofluorene (2AAF). Since 2AAF was not mutagenic under these conditions, these data are consistent with the concept of a free radical of 2AAF as the mutagenic intermediate.     

The metabolism of N-hydroxyphenacetin in vitro and in vivo

N-Hydroxyphenacetin (100 mg/kg) injected i.p. into rats rapidly appeared in the blood and disappeared with a t1/2 of 14 min; phenacetin and 4-acetamidophenol were major metabolites in blood. Ferrihaemoglobin was formed, but 4-nitrosophenetole was not detected in blood. N-Hydroxyphenacetin injected i.p. into rats was excreted in the urine unchanged (partly conjugated 2.1% of the dose, 2% was excreted as phenacetin, 19% as 4-acetamidophenol) and 1.8% as 2-hydroxyphenacetin. In addition, small amounts of 3-hydroxyphenacetin (0.4%) and traces of N-[4-(2-hydroxyethoxy)phenyl]acetamide (beta-HAP) (0.05%) were found. Time-course kinetics have shown that N-hydroxyphenacetin is metabolized in vitro to phenacetin, 2- and 3-hydroxyphenacetin, and 4-acetamidophenol by microsomal and cytosolic preparations of rat and rabbit liver. However, after the initial reaction, the formation of phenacetin and 2- and 3-hydroxyphenacetin did not continue with time, indicating that these products were not formed enzymically. N-Hydroxyphenacetin incubated with rat erythrocytes formed ferrihaemoglobin; the relationship between ferrihaemoglobin, phenacetin and 4-nitrosophenetole concn indicated that N-hydroxyphenacetin was oxidized by oxyhaemoglobin to acetyl 4-ethoxyphenyl nitroxide, which yielded phenacetin and 4-nitrosophenetole spontaneously.     

Metabolism of aniline mustard [N,N-di-(2-Chloroethyl)-aniline]

The possible metabolic activation of the antineoplastic agent N,N-di-(2-chloroethyl) aniline (aniline mustard) is discussed. Conversion of aniline mustard into the glucuronide $\text{(p-}\text{di}\text{-2-}\text{chloroethylaminophenyl}\text{-β-}\text{d}\text{-glucopyranosid}\text{)}$uronic acid was mediated by a rat liver homogenate containing the appropriate cofactors. The glucuronide was a major metabolite in the serum and bile after administration of aniline mustard to rats and after isolation and purification it was identified as its methyl ester by mass spectrometry. The use of Amberlite XAD-2 resin facilitated the isolation from serum of the glucuronide and another metabolite, N-(2-chloroethyl)-4-hydroxyaniline. The implication of these findings for the clinical application of aniline mustard is discussed.     

Inhibition of DNA,RNA and protein synthesis and chromatin alteration by N-hydroxyphenacetin

1. The effects of N-hydroxyphenacetin on DNA function and structure were investigated to elucidate the involvement of phenacetin in analgesic nephropathy and transitional cell carcinoma.

2. N-Hydroxyphenacetin or a metabolite inhibited synthesis of DNA, RNA and protein; DNA inhibition was greater at higher pH.

3. No single-strand breaks were detectable in DNA after N-hydroxyphenacetin treatment and no appreciable effect on cell viability was observed at concentrations up to 5 mM.

4. N-Hydroxyphenacetin-induced alteration to chromatin structure was detected using nucleoid sedimentation analysis. Direct binding to plasmid DNA was not observed.

5. These observations are consistent with a role for phenacetin metabolites in renal disease.     

Varying effects of sulfhydryl nucleophiles on acetaminophen oxidation and sulfhydryl adduct formation

The effects of glutathione, cysteine, N-acetylcysteine, cysteamine, α-mercaptopropionylglycine and methionine on the NADPH-dependent metabolism and covalent binding of acetaminophen have been examined in mouse liver microsomal incubations. With the exception of methionine, all of the nucleophiles decreased covalent binding by forming adducts with the electrophilic metabolite of acetaminophen. The adducts were measured quantitatively by high pressure liquid chromatography. In contrast to glutathione, N-acetylcysteine and α-mercaptopropionylglycine, both cysteamine and cysteine in high concentrations also decreased covalent binding of acetaminophen through another mechanism, inhibition of the formation of the reactive acetaminophen metabolite. These results indicate that both inhibition of metabolite formation and detoxification of metabolite by sulfhydryl adduct formation are mechanisms that can be important in reducing acetaminophen toxicity in overdosed patients treated with these nucleophiles.     

In Vitro Studies of Intestinal Permeability and Hepatic and Intestinal Metabolism of 8-Prenylnaringenin, a Potent Phytoestrogen from Hops (Humulus lupulus L.)

Purpose The absorption potential and metabolism of 8-prenylnaringenin (8-PN) from hops (Humulus lupulus L.) were investigated. 8-PN is a potent estrogen with the potential to be used for the relief of menopausal symptoms in women. Methods Monolayers of the human intestinal epithelial cancer cell line Caco-2 and human hepatocytes were incubated with 8-PN to model its intestinal absorption and hepatic metabolism, respectively. Results The apparent permeability coefficients for 8-PN in the apical-to-basolateral and basolateral-to-apical directions of a Caco-2 monolayer were 5.2 ± 0.7 × 10⁻⁵ and 4.9 ± 0.5 × 10⁻⁵ cm/s, respectively, indicating good intestinal absorption via passive diffusion. Both glucuronide and sulfate conjugates of 8-PN were detected in the Caco-2 cell incubations. The 4′-O-glucuronide was the predominant Caco-2 cell metabolite, followed by 7-O-sulfate and 4′-O-sulfate. Both phase I and phase II metabolites of 8-PN were formed by human hepatocytes. The 7-O-glucuronide was the most abundant hepatocyte metabolite, and no sulfate conjugates were detected. Incubations with various cDNA-expressed UDP-glucuronosyltransferases indicated that the isozymes UGT1A1, UGT1A6, UGT1A8, and UGT1A9 were responsible for glucuronidation of 8-PN. Conclusions Although orally administered 8-PN should be readily absorbed from the intestine, its bioavailability should be reduced significantly by intestinal and hepatic metabolism.     

The metabolism and disposition of 3,4,4'-trichlorocarbanilide in the intact and bile duct-cannulated adult and in the newborn rhesus monkey (M. mulatta).

The metabolism and disposition of intravenously infused radioactive 3,4,4′-trichloro[¹⁴C]carbanilide (TCC) in the adult and newborn rhesus monkey have been evaluated. In adult animals the major metabolic reactions were N-glucuronide formation or ring hydroxylation followed by conjugation to glucuronic acid or sulfuric acid. Removal of ¹⁴C from the plasma was biphasic; TCC and the N-glucuronides accounted for the fast phase, and the O-sulfate conjugates accounted for the slow phase. The major urinary metabolites were the N-glucuronides of TCC. The tissue residue of ¹⁴C was low in the monkeys and was limited primarily to tissues that are active in drug metabolism (liver, kidneys, and lungs). The bile was the major route of elimination with glucuronide conjugates as the major radioactive component. Enterohepatic circulation was extensive but did not affect the plasma concentrations or the elimination kinetics of TCC-derived material from the plasma. The newborn monkey also metabolized TCC by ring hydroxylation or N-glucuronidation. The plasma kinetics were similar to those observed in adults as was the tissue distribution. Unlike the adult, there were only very low amounts of O-glucuronides and, instead, high amounts of O-sulfate conjugates. It is concluded that the infant monkey can readily metabolize and eliminate TCC.     

Liver cytosol catalyzed conjugation of reduced glutathione with a reactive metabolite of acetaminophen.

The effect of liver cytosol enzymes on the rate of formation of a glutathione (GSH) conjugate and on the rate of covalent binding of acetaminophen to microsomal proteins was studied in vitro. Mouse liver microsomes were incubated with $\text{[}{}^{14}\text{C}\text{]}\text{acetaminophen}$, GSH, a NADPH-generating system, and Sephadex G-25-treated mouse liver supernatant. The addition of liver cytosol to the microsomal incubation increased the rate of conjugate production, as measured by high pressure liquid chromatography and decreased the rate of covalent binding of the reactive intermediate of acetaminophen to microsomal proteins at all concentrations of GSH and acetaminophen studied. The effect of the liver supernatant enzymes was most pronounced at low GSH concentrations. Although cysteine and N-acetyl-l-cysteine form conjugates with acetaminophen, the cytosol preparations did not facilitate conjugate formation with these nucleophiles. Cytosol enzymes may be important in the detoxification of acetaminophen particularly when the liver concentration of GSH is low and may contribute to the marked “threshold” effect seen with acetaminophen toxicity.     

N-O glucuronidation: a major human metabolic pathway in the elimination of two novel anticonvulsant drug candidates

1. The urinary metabolites of the anti-convulsant compound 4-amino-1-(2,6-difluorobenzyl)-1H-1,2,3-triazolo[4,5-c]-pyridine hydrochloride (GI265080) obtained following a single oral dose to man have been detected and quantified relative to each other using ¹⁹F-NMR spectroscopy. 2. The human urinary metabolites of GI265080 were isolated using semipreparative HPLC and unequivocally characterized using ¹H-NMR spectroscopy, two-dimensional heteronuclear NMR spectroscopy and mass spectrometry. The assignments of the N-(5)oxide and the N-(5)-O-glucuronide metabolites of GI265080 were further confirmed by independent synthesis. The urinary metabolites obtained following single oral doses to dog and rat have also been isolated and characterized. 3. The human urinary metabolites of GI265080 comprise the N-(5)-oxide, the quaternary N⁺-(5)-glucuronide, the 7-hydroxy glucuronide and a glucuronide conjugate of the N-(5)-oxide. The N-(5)-O-glucuronide conjugate is a novel species in human metabolism and is a significant route of elimination of GI265080 in man. 4. The urinary metabolites of the potential anti-convulsant GW273293 (6-amino-3-(2,3,5-trichlorophenyl)pyrazin-2-ylamine) obtained following a single oral dose to man have also been isolated and characterized. The formation of a novel N-O-glucuronide was also observed and was shown to constitute a significant route of elimination of GW273293 in man.     

Metabolism and disposition of [14C]5-amino-o-cresol in female F344 rats and B6C3F1 mice

The metabolism and disposition of [¹⁴C]5-amino-o-cresol (AOC) in female F344 rats following oral, intravenous, and dermal administration and in female B6C3F1 mice following oral administration were studied. Greater than 80% of a single oral dose (4.0–357 mg kg^?1) or intravenous dose (2.7 mg kg^?1) was excreted in urine within 24 h. When the dosing site was protected from grooming, less than 10% of the dermal dose (2.5 and 26 mg kg^?1, rinsed off after 6 h) was absorbed within 24 h, and most of the absorbed radioactivity was excreted in urine. For the unprotected dermal dose, grooming played a major role in the absorption of AOC. Very little AOC-derived radioactivity was present in the surveyed tissues after 24 or 72 h regardless of route, dose level, or species. Five urinary metabolites were identified: 5-acetamido-1,4-dihydroxy-2-methylbenzene glucuronide, AOC O-glucuronide, AOC O-sulfate, N-acetyl-AOC O-glucuronide, and N-acetyl-AOC O-sulfate.     

N-acetyl, 4-O-methyldopa,a major metabolite of L-4-O-methyldopa in man and rat

N-Acetyl, 4-O-methyldopa was identified as a major urinary metabolite of $\text{L}\text{-4-O-}\text{methyldopa}$, both in man and in the rat. The urinary metabolites were examined in man after oral and in rat after intraperitoneal administration of $\text{L}\text{-4-O-}\text{methyldopa}$, $\text{L}\text{-3-O-}\text{methyldopa}$, L-dopa and N-acetyl, 4-O-methyldopa. 4-Mdopa was found to be converted mainly to N-acetyl, 4-Mdopa and iso-HVA and very little to the corresponding pyruvic and lactic acids, whereas 3-Mdopa was metabolized to its pyruvic, lactic and acetic (HVA) derivatives and practically not acetylated. It is suggested therefore that the 3-hydroxy,4-methoxy group (the iso-vanyl structure) prevents transamination and that N-acetylation represents an alternative metabolic pathway. The lack of N-acetyl,4-O-methyldopa after the L-dopa loads shows that L-dopa is not 4-O-methylated and therefore that 4-O-methyldopa is not, or only in a minute amount if any, an in vivo metabolite of L-dopa. N-Acetyl,4-Mdopa administration to rats resulted in excretion of the compound almost unchanged. These results agree with a previous suggestion by the authors of partly distinct metabolic routes for the O-methyl catecholamines according to whether the methyl group is bound on the meta or on the para position and raises the question as to what extent iso-HVA levels in body fluids are representative of a para-O-methylation of dopamine, implicated in neuropsychiatric disorders.     

Factors Affecting the Metabolism of Phenacetin I. Influence of Dose,Chronic Dosage,Route of Administration and Species on the Metabolism of [1-14C-Acetyl]Phenacetin

1. Metabolism and excretion of [acetyl-1-¹⁴C]phenacetin have been studied in relation to dose, chronic dosage, route of administration and species.

2. The drug is largely metabolized in the rat, rabbit, guinea-pig, ferret and man by oxidative de-ethylation and deacetylation as well as by minor pathways of aromatic hydroxylation and cysteine conjugation.

3. Species differences exist in the extent to which the various reactions occur; deacetylation is highest in the rat and ferret (21 and 13% of dose respectively); aromatic hydroxylation of phenacetin to 2-hydroxyphenacetin is highest in the ferret (6% of dose) but low (0·1%) in the other species; the formation of the 3-cysteine conjugate of N-acetyl-p-aminophenol is highest in the rabbit (8%) and least in the guinea-pig (0·3%); the pattern of conjugation of N-acetyl-p-aminophenol varies with species, glucuronide conjugation being dominant in the rabbit, guinea-pig and ferret, whereas sulphate conjugation is the main pathway in the rat.

4. The pattern of elimination in the rat of a large oral dose (2000?mg/kg) of [acetyl-¹⁴C]phenacetin differs from that of a smaller dose (125?mg/kg), with (a) slower overall excretion at the higher dose (b) a marked relative increase in the proportion of the drug undergoing deacetylation and (c) a change in the pattern of conjugation of the N-acetyl-p-aminophenol at the higher dose in favour of glucuronic acid conjugation.

5. The pattern of excretion of [acetyl-¹⁴C]phenacetin in the rat is the same when the drug is given orally or by intraperitoneal injection. However, the elimination of radioactivity is slower in rats treated chronically for 14 days with the drug.

6. The findings are discussed in relation to some of the biological properties of phenacetin.     

Formation and metabolism of [14C]dopamine 3-O-sulfate in dog,rat and guinea pig

Oral administration of [¹⁴C]dopamine to dogs resulted in urinary excretion of predominantly [¹⁴C]dopamine 3-O-sulfate, while after intravenous administration the labeled drug was metabolized largely by O-methylation and deamination pathways. Experiments in vitro pinpointed the small intestinal wall of the dog as the site of dopamine sulfate conjugate formation. The small intestines of rat and guinea pig lack this sulfating ability. When trace amounts of [¹⁴C]dopamine 3-O-sulfate were administered to dog, rat and guinea pig, the compound turned out not to be metabolically inert. In the guinea pig, [¹⁴C]dopamine 3-O-sulfate was almost completely desulfated and metabolized according to the pattern characteristic to orally administered dopamine in this animal species. In rat, about 40 per cent of the administered [¹⁴C]dopamine 3-O-sulfate (19.1 μg/kg) was excreted in urine unchanged, whereas a smaller dose (7.4 μg/kg) was totally metabolized according to the pattern characteristic to rat. In dog urine, more than 80 per cent of the radioactive dose (2.5 μg/kg) emerged in urine as unchanged [¹⁴C]dopamine 3-O-sulfate, the normal metabolism end product of dopamine in dog.     

Isolation and identification of urinary metabolites of tectoridin in rats

Tectoridin is an active isoflavone from the rhizome of Belamcanda chinensis and flower of Pueraria thomsonii Benth, possessing an anti-inflammation action and the protective effect against ethanol-induced intoxication and hepatic injury. The metabolism of tectoridin was investigated in rats. Nine metabolites were isolated by using repeated chromatographic methods and identified by spectroscopic methods including UV, IR, mass spectrometry, and NMR experiments. A new compound was determined as tectorigenin-7-O-β-d-glucuronide (M-1), together with eight known compounds identified as irisolidone-7-O-β-d-glucuronide (M-2), tectorigenin-7-O-sulfate (M-3), tectorigenin-4′-O-sulfate (M-4), tectorigenin (M-5), irisolidone (M-6), isotectorigenin (M-7), genistein (M-8), and daidzein (M-9), respectively. The metabolic pathway of tectoridin was proposed, which is important to understand its metabolic fate and disposition in humans.