Hepatic drug metabolizing enzyme activity in the channel catfish, Ictalurus punctatus

1. Several pathways of drug metabolizing enzymic activity were measured in hepatic fractions of the channel catfish and rat using model substrates. The pathways examined included the O-demethylation of p-nitroanisole, microsomal ester hydrolysis of procaine and glucuronidation of p-nitrophenol, and the cytosolic acetylation of sulfamethazine and sulfation of 2-naphthol. Catfish liver preparations were incubated at both 25 degrees C and 37 degrees C. 2. The oxidative metabolism of p-nitrophenol was only one-eighth that of the rat at 37 degrees C and one-twelfth that of the rat at 25 degrees C. 3. Procaine ester hydrolysis was negligible in catfish microsomal preparations. 4. At 37 degrees C, p-nitrophenol glucuronidation was equivalent in catfish and rat microsomes. 5. Catfish cytosolic preparations exhibited N-acetyltransferase and arylsulfotransferase nearly comparable to those of the rat. 6. Rates of glucuronidation and sulfation were higher at 37 degrees C than at 25 degrees C in hepatic fractions of catfish.     

Characterization of rat liver bile acid acyl glucuronosyltransferase

Recent studies have suggested that bile acid acyl glucuronides form covalently bound protein adducts which may cause hypersensitivity reactions and increased morbidity in patients. Although the preferential biosynthesis of the acyl glucuronides has been known, the characterization of hepatic bile acid acyl glucuronosyltransferase has not yet been clearly elucidated. We have investigated the substrate specificity of the hepatic bile acid acyl glucuronosyltransferase using five common bile acids as substrates. The glucuronidation rate was dependent on the number of the hydroxy group on the steroid nucleus and mono-hydroxylated lithocholic acid, the more lipophilic common bile acid, was most effectively metabolized into its acyl glucuronide. The tri-hydroxylated cholic acid, the more water-soluble common bile acid, barely transformed into its glucuronide. Results showed decreasing of the initial velocity of the acyl glucuronidation with increasing of the concentration of substrate, lithocholic acid, owing to the substrate inhibition of the hepatic bile acid acyl glucuronosyltransferase. The substrate analogues, glycine and taurine conjugated bile acids, which exist in the body fluids in high concentrations, also inhibited the enzyme's activity. In addition, enzymatic reaction products, bile acid acyl glucuronides, also inhibited the activity. These inhibitory mechanisms may be responsible for the low concentration of bile acid acyl glucuronides in urine and may be an important detoxification system in the body.     

Bile acid sulfates. II. Formation, metabolism, and excretion of lithocholic acid sulfates in the rat

Sulfate esterification has been shown previously to be a prominent feature of lithocholate metabolism in man. These studies were undertaken to ascertain whether this metabolic pathway is also present in rats, and to investigate the physiological significance of bile acid sulfate formation. Lithocholic acid-24-(14)C was administered to bile fistula rats, and sulfated metabolites were identified in bile by chromatographic and appropriate degradative procedures. They constituted only a small fraction (2-9%) of the total metabolites but a more significant fraction (about 20%) of the secreted monohydroxy bile acids, most of the lithocholate having been hydroxylated by the rat liver. When sulfated glycolithocholate was administered orally, it was absorbed from the intestine without loss of the sulfate, presumably by active transport, and secreted intact into the bile. In comparison with non-sulfated lithocholate, an unusually large fraction (24%) of the sulfated bile acid was excreted in the urine, and fecal excretion took place more rapidly. Both the amino acid and sulfate moieties were extensively removed prior to excretion in the feces. Hydroxylation of bile acid sulfates or sulfation of polyhydroxylated bile acids did not occur to any great extent, if at all.     

Identification of hyperoside metabolites in rat using ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry

In this paper, ultra performance liquid chromatography (UPLC)/quadrupole-time-of-flight mass spectrometry (QTOF) with automated data analysis software (Metabolynx?) were applied for fast analysis of hyperoside metabolites in rat after intravenous administration. MS(E) was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of 12 metabolites in rat plasma, urine and bile. The results indicated that methylation, sulfation and glucuronidation were the major metabolic pathways of hyperoside in vivo, and among them, 3'-O-methyl-hyperoside was confirmed by matching its fragmentation patterns with standard compound. The present study provided important information about the metabolism of hyperoside which will be helpful for fully understanding the mechanism of this compound's action. Furthermore, this work demonstrated the potential of the UPLC/QTOFMS approach using Metabolynx for fast and automated identification of metabolites of natural product.     

Factors influencing sulfation in isolated rat hepatocytes

Sulfation of harmol by isolated hepatocytes was dependent on an exogenous source of sulfate. Inorganic sulfate ion stimulated sulfation by over ten fold. Analysis of the stimulation of harmol sulfation by sulfate indicated a K_m of 239 μM and a V_max of 1.1 μmoles harmol sulfate/min/10⁶ cells. Cysteine also stimulated the rate of harmol sulfation but was less effective than sulfate ion. Lithium chloride inhibited harmol sulfation. Sulfation was unaffected by several metabolic alterations which inhibited harmol glucuronidation. Fasting for 24 hours, and incubation with ethanol or linoleic acid, did not influence the rate of sulfation but inhibited glucuronidation by 50 percent.     

Release of mutagenic metabolites of benzo[a]pyrene from the perfused rat liver after inhibition of glucuronidation and sulfation by salicylamide

The role of glucuronide and sulfate conjugation in presystemic inactivation of benzo[a]pyrene (BP) metabolites was investigated with rat livers perfused with BP (12 mumol). Comparisons were made between metabolite profiles and mutagenicity of medium from perfusions with and without salicylamide, a selective inhibitor of glucuronide and sulfate conjugation. After 4 h perfusion in the presence of salicylamide, certain BP metabolites (diols, quinones, phenols, and metabolites more polar than BP-9,10-diol) were significantly increased at the expense of quinones and phenols in the glucuronide fraction. Mutagenicity of medium (detected by the Ames test, using tester strains TA98 and TA100) was low in perfusion without salicylamide. Mutagenicity detected with tester strain TA98 was significantly increased in perfusions with salicylamide. Involvement of glucuronidation in BP inactivation was also observed at the subcellular level; when cofactors of glucuronidation were added to liver homogenates along with the NADPH regenerating system in the Ames test, BP mutagenicity was markedly decreased. Both the activation of BP to mutagenic metabolites and the inactivation of BP metabolites by glucuronidation was much more pronounced with liver homogenates from 3-methylcholanthrene-treated rats than with those from phenobarbital-treated animals or untreated controls. The results suggest an important role for glucuronidation and sulfation in the inactivation and elimination of polycyclic aromatic hydrocarbons.     

Hepatocyte metabolism of coenzyme Q1 (ubiquinone-5) to its sulfate conjugate decreases its antioxidant activity

Previous studies on the metabolism of coenyzme Q (CoQ) have focused on products found in the urine, bile or feces. However, the metabolites found in these samples were end products from a multitude of catabolic processes which did not necessarily reflect CoQ intracellular metabolism (e.g. in the liver, the major site of CoQ synthesis or metabolism). Using isolated rat hepatocytes, we have found that the sulfation of coenzyme Q1 (CoQ1) was the initial and dominant step following its reduction to the hydroquinone. This metabolic process is important as conjugation may occur on the hydroquinone metabolites of any coenzyme10 scission product retaining the quinone ring. By using rat liver cytosol, we were able to identify the monosulfated metabolite of CoQ1. The CoQ1 sulfate conjugate was identified by mass spectrometry followed by tandem mass spectrometry. The rate of formation of the CoQ1 sulfate conjugate was markedly increased by the addition of NADH and was prevented by dicumarol, a DT-diaphorase (NQO1) inhibitor. CoQ1 sulfate conjugate formation catalysed by cytosol was inhibited by the sulfotransferase 1A (SULT1A) inhibitor, pentachlorophenol (PCP) suggesting that sulfation was carried out by the SULT 1A isoform. CoQ1 sulfation in isolated hepatocytes and inversely CoQ1 hydroquinone formation were dependent on the concentration of inorganic sulfate in the media. Intracellular sulfation also decreased CoQ1 antioxidant and cytoprotective activity towards cumene hydroperoxide (CHP) induced cell death. Sulfotransferases may therefore play a significant role in endogenous CoQ metabolism following its degradation to short chain products.     

Localization of the sulfate/anion exchanger in the rat liver

Although the sulfate/anion transporter (sat-1; SLC26A1) was isolated from a rat liver cDNA library by expression cloning, localization of sat-1 within the liver and its contribution to the transport of sulfate and organo sulfates have remained unresolved. In situ hybridization and immunohistochemical studies were undertaken to demonstrate the localization of sat-1 in liver tissue. RT-PCR studies on isolated hepatocytes and liver endothelial and stellate cells in culture were performed to test for the presence of sat-1 in these cells. In sulfate uptake and efflux experiments, the substrate specificity of sat-1 was evaluated. Sat-1 mRNA was found in hepatocytes and endothelial cells. Sat-1 protein was localized in sinusoidal membranes and along the borders of hepatocytes. The canalicular region and bile capillaries were not stained. Sulfate uptake was only slightly affected by sulfamoyl diuretics or organo sulfates. Sulfate efflux from sat-1-expressing oocytes was enhanced in the presence of bicarbonate, indicating sulfate/bicarbonate exchange. Estrone sulfate was not transported by sat-1. Sat-1 may be responsible for the uptake of inorganic sulfate from the blood into hepatocytes to enable sulfation reactions. In hepatocytes and endothelial cells, sat-1 may also supply sulfate for proteoglycan synthesis.     

In Vitro and In Vivo Metabolism and Inhibitory Activities of Vasicine,a Potent Acetylcholinesterase and Butyrylcholinesterase Inhibitor

Vasicine (VAS), a potential natural cholinesterase inhibitor, exhibited promising anticholinesterase activity in preclinical models and has been in development for treatment of Alzheimer’s disease. This study systematically investigated the in vitro and in vivo metabolism of VAS in rat using ultra performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry. A total of 72 metabolites were found based on a detailed analysis of their ¹H- NMR and ¹³C NMR data. Six key metabolites were isolated from rat urine and elucidated as vasicinone, vasicinol, vasicinolone, 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-yl hydrogen sulfate, 9-oxo-1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-yl hydrogen sulfate, and 1,2,3,9-tetrahydropyrrolo [2,1-b] quinazolin-3-β-D-glucuronide. The metabolic pathway of VAS in vivo and in vitro mainly involved monohydroxylation, dihydroxylation, trihydroxylation, oxidation, desaturation, sulfation, and glucuronidation. The main metabolic soft spots in the chemical structure of VAS were the 3-hydroxyl group and the C-9 site. All 72 metabolites were found in the urine sample, and 15, 25, 45, 18, and 11 metabolites were identified from rat feces, plasma, bile, rat liver microsomes, and rat primary hepatocyte incubations, respectively. Results indicated that renal clearance was the major excretion pathway of VAS. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of VAS and its main metabolites were also evaluated. The results indicated that although most metabolites maintained potential inhibitory activity against AChE and BChE, but weaker than that of VAS. VAS undergoes metabolic inactivation process in vivo in respect to cholinesterase inhibitory activity.     

Functional relationship between initial oxidation of 7-ethoxycoumarin and subsequent conjugation of 7-hydroxycoumarin in isolated perfused rat livers

Functional relationship between the initial mixed function oxidation of 7-ethoxycoumarin (EC) to 7-hydroxycoumarin (HC) and the subsequent conjugation of this metabolite to sulfate ester and glucuronide has been studied using isolated perfused rat livers. When increasing concentrations of EC (from 25 to 200 microM) were infused, perfused liver can oxidize only up to about 60 nmol of the infused EC to HC per min/g liver tissue. Most of this HC metabolite was released as sulfate ester, but there was a dose dependent shift to a more significant glucuronidation at the expense of the sulfate form. The dose dependent shift observed upon infusions with increasing dose of EC was not extensive so that the major portion of metabolite released was always the sulfate ester. However, the shift observed with HC was extensive and the major portion released was the glucuronide conjugate. Upon infusions with increasing concentrations of HC, the maximal rates of sulfation and glucuronidation were found to be 60 nmol and 120 nmol of HC conjugated per min/g liver tissue, respectively. Furthermore, the ranges in the rates of conjugation for the infused HC were divided into a sulfate ester 'zone' (less than 20 nmol), a dose-dependent shift 'zone' (between 20 and 180 nmol) with the 'cross-over' occurring at 80 nmol/min/g liver, and reaching the maximal conjugation 'capacity' rate (180 nmol), above which the unconjugated free form of HC was released. Under conditions when EC was infused into normal rat livers, the calculated maximal oxidation rate was only 60 nmol of HC produced/min/g liver. Consequently, under such a condition, the oxidation rate may never reach the 'cross-over' rate and this explains the lack of extensive dose-dependent shift and further indicates that there remained a large reserve conjugation capacity (120 nmol/min/g).     

Evidence for an ordered reaction mechanism for bile salt: 3'phosphoadenosine-5'-phosphosulfate: sulfotransferase from rhesus monkey liver that catalyzes the sulfation of the hepatotoxin glycolithocholate

The in vivo formation of the sulfate ester of glycolithocholate is a critical step in the elimination of this hepatotoxic bile salt. Rhesus monkeys fed chenodeoxycholate or ursodeoxycholate, the precursors of lithocholate, develop frank cirrhosis in association with accumulation of nonsulfated glycolithocholate in bile. An enzyme catalyzing the formation of glycolithocholate-3-sulfate has been isolated from hepatic cytosol of adult female rhesus monkeys and has been purified 146-fold. When reduced it appears as a 30 kD band on an SDS-polyacrylamide gradient gel. It has a pH optimum of 7.0 and is stimulated by low concentrations of Mg2+ (up to 2 mM), but does not have an absolute requirement for this metal ion. The kinetics of this enzyme have been investigated to ascertain whether its reaction mechanism can account for the poor in vivo rate of glycolithocholate sulfation. Inhibitor studies with an oxidized metabolite of lithocholate, 3-keto-5 beta-cholanoate, showed that the latter is a competitive inhibitor of glycolithocholate and is noncompetitive with the active form of sulfate, 3'phosphoadenosine-5'-phosphosulfate. The monophosphonucleotide 3'-AMP is a competitive inhibitor of 3'phosphoadenosine-5'-phosphosulfate, and is noncompetitive with glycolithocholate. These observations are consistent with a sequentially ordered Bi Bi reaction mechanism in which the bile salt is the first substrate to bind to the enzyme. Such a reaction mechanism for bile salt:3'phosphoadenosine-5'-phosphosulfate:sulfotransferase would be, therefore, the first time in which the sulfate acceptor (the bile salt) is the initial substrate to bind to a sulfotransferase. These studies have shown that although rhesus monkeys have a liver enzyme capable of forming the sulfate ester of glycolithocholate, its reaction mechanism and the potent inhibition caused by simple metabolites, such as 3-keto-5 beta-cholanoate, may serve to under-express the activity of the enzyme in vivo.     

6 alpha-glucuronidation of hyodeoxycholic acid by human liver, kidney and small bowel microsomes

Kinetic constants for the glucuronidation of hyodeoxycholic acid in man were determined using microsomal preparations of liver, kidney and small bowel. The affinity of hyodeoxycholic acid for the microsomal hepatic and extrahepatic enzymes was in the same range as previously observed for the monohydroxy bile acid lithocholic acid and about 3-14-times the affinity for the dihydroxy bile acids chenodeoxycholic, deoxycholic and ursodeoxycholic acids. The Vmax values for glucuronidation of hyodeoxycholic acid with hepatic microsomes were 10-30-times higher and with kidney microsomes 50-110-times higher than for the bile acids lacking a 6 alpha-hydroxy group. The site of glucuronidation was determined by gas chromatographic-mass spectrometric analysis of derivatives of products formed after periodate and chromic acid oxidation. Hyodeoxycholic acid glucuronides synthesized with microsomal preparations from the three organs were all found to be conjugated at the 6 alpha position. This has previously been shown to be the site of glucuronidation of endogenous hyodeoxycholic acid glucuronide excreted in urine.     

Species differences in metabolism of ketotifen in rat, rabbit and man: demonstration of similar pathways in vivo and in cultured hepatocytes

In vitro drug metabolism by cultured rat, rabbit and human adult hepatocytes has been studied, using ketotifen (ZADITEN) as a model substrate because it is biotransformed in vivo by various metabolic pathways in man and animals. The major in vivo pathways were demonstrated in vitro, namely oxidation in rat hepatocytes, oxidation, glucuronidation and sulfation in rabbit hepatocytes, reduction and glucuronidation in human hepatocytes. Human hepatocytes were the most stable in culture, displaying ketotifen biotransformation for at least one week. These results clearly demonstrated that cultured hepatocytes retain their in vivo specific drug metabolizing activities, including inter-species polymorphism, for a few days. Therefore, pure hepatocyte cultures represent a useful system suitable for drug metabolism studies.     

Synthesis of the 3-sulfates of S-acyl glutathione conjugated bile acids and their biotransformation by a rat liver cytosolic fraction

The 3-sulfates of the S-acyl glutathione (GSH) conjugates of five natural bile acids (cholic, chenodeoxycholic, deoxycholic, ursodeoxycholic, and lithocholic) were synthesized as reference standards in order to investigate their possible formation by a rat liver cytosolic fraction. Their structures were confirmed by proton nuclear magnetic resonance, as well as by means of electrospray ionization-linear ion-trap mass spectrometry with negative-ion detection. Upon collision-induced dissociation, structurally informative product ions were observed. Using a triple-stage quadrupole instrument, selected reaction monitoring analyses by monitoring characteristic transition ions allowed the achievement of a highly sensitive and specific assay. This method was used to determine whether the 3-sulfates of the bile acid-GSH conjugates (BA-GSH) were formed when BA-GSH were incubated with a rat liver cytosolic fraction to which 3'-phosphoadenosine 5'-phosphosulfate had been added. The S-acyl linkage was rapidly hydrolyzed to form the unconjugated bile acid. A little sulfation of the GSH conjugates occurred, but greater sulfation at C-3 of the liberated bile acid occurred. Sulfation was proportional to the hydrophobicity of the unconjugated bile acid. Thus GSH conjugates of bile acids as well as their C-3 sulfates if formed in vivo are rapidly hydrolyzed by cytosolic enzymes.     

Sulfation and glucuronidation of acetaminophen by cultured hepatocytes reproducing in vivo sex-differences in conjugation on matrigel and type 1 collagen

Summary The sulfate and glucuronide conjugation of acetaminophen (APAP) by hepatocytes cultured on Matrigel or type 1 collagen was compared to APAP metabolism in vivo. The metabolic fate of low (15 mg/kg), medium (125 mg/kg), and high (300 mg/kg) doses of APAP injected intraperitoneally were determined in male and female rats. Males excreted more APAP as the sulfate conjugate than females, which correlated with the twofold greater APAP sulfotransferase activity in the male vs. females (301±24 vs. 156±18 pmol · mg⁻¹ protein · min⁻¹). Also, as sulfate conjugation became saturated, there was a dose-related shift in APAP metabolism from sulfate to glucuronide conjugation in both sexes. After death, the livers of the same animals were perfused with collagenase and the hepatocytes cultured in modified Waymouth’s medium on either Matrigel or rat-tail collagen, with various doses of APAP (0, 0.125, 0.25, 0.5, and 1.0 mM). Sex differences in APAP sulfation and glucuronidation persisted in culture for up to 4 days, with sulfation predominating in the male similar to in vivo. With increasing APAP concentration (dose), there was a saturation of sulfate conjugation and a shift to glucuronidation as observed in vivo. Sex differences in APAP sulfation and glucuronidation were no longer significant by Day 4 in culture. Sulfation, and to a lesser extent, glucuronidation, were more stable on Matrigel than collagen. We concluded that APAP metabolism of freshly isolated hepatocytes could replicate in vivo sex differences in conjugation, and that Matrigel was superior to collagen as substrate.     

Determination of 3 beta-hydroxy-5-cholenoic acid in serum of hepatobiliary diseases--its glucuronidated and sulfated conjugates

3 beta-Hydroxy-5-cholenoic acid in the serum of control subjects and 62 patients with various hepatobiliary diseases was quantitated by mass fragmentography after separation into nonglucuronidated-nonsulfated, glucuronidated, and sulfated fractions. Deuterium-labeled deoxycholic acid and its glucuronide and sulfate were used as internal standards. Mean concentrations of total 3 beta-hydroxy-5-cholenoic acid in serum (mumole/liter) were as follows: Control subjects (14), 0.184; obstructive jaundice (15), 6.783; liver cirrhosis, compensated (12), 0.433, and decompensated (12), 1.636; chronic hepatitis (12), 0.241; and acute hepatitis (11), 2.364. Most of the 3 beta-hydroxy-5-cholenoic acid was glucuronidated or sulfated. Only in patients with obstructive jaundice did glucuronidation (60 +/- 14%) exceed sulfation (31 +/- 14%), sulfation exceeding glucuronidation in the others. The UDP-glucuronyltransferase might have different substrate specificities for 3 beta-hydroxy-5-cholenoic acid and other common bile acids, especially in the cholestatic state.     

Hepatic microsomal glucuronidation of bilirubin is modulated by the lipid microenvironment of membrane-bound substrate

Hepatocyte intracellular membranes may facilitate the directed movement of bilirubin and other hydrophobic substrates to the active site of UDP-glucuronyltransferase in the endoplasmic reticulum. We postulated that the lipid composition and physical properties of membranes that transport substrate may modulate bilirubin glucuronidation. To examine this hypothesis, we incorporated [14C]bilirubin substrate into the membrane bilayer of small unilamellar liposomes composed of native phospholipid purified from rat hepatic microsomes. The initial velocity of bilirubin glucuronide formation in rat liver microsomes, measured by radiochemical assay, was considerably more rapid than for bilirubin in liposomes of egg phosphatidylcholine (p less than 0.001). Moreover, the ratio of bilirubin diglucuronide to monoglucuronides synthesized was markedly increased (p less than 0.01), approaching that observed in normal rat bile. Although the rates of bilirubin glucuronidation did not correlate with fluidity of the liposomal membrane core region, specific phospholipid head groups were associated with an increase, and cholesterol a decrease, in rates of glucuronidation. Movement of [3H]bilirubin from dual-labeled liposomes to microsomes occurred without concomitant [14C]phospholipid transfer. Thus, the lipid composition of membranes incorporating bilirubin appears to modulate the rate of glucuronidation and the relative rates of bilirubin mono- and diglucuronide formation. Phospholipid head groups on the surface of the bilayer, not the hydrocarbon core regions, may be implicated in the rapid process of membrane transport, which is likely to involve membrane-membrane collisions or diffusion of free substrate rather than membrane fusion.     

Stereoselective sulfation of terbutaline by the rat liver cytosol: evaluation of experimental approaches

Little is known about the stereochemistry of sulfation of chiral phenolic drugs. In this study we examined several in vitro approaches to this question, using (+)-, (-)-, or (+/-)-terbutaline as the substrate and the rat liver cytosol as the phenolsulfotransferase enzyme source. The cosubstrate PAPS was either generated by the cytosol from inorganic sulfate and ATP or added to the cytosol. The intact sulfate conjugates formed were determined by HPLC. Using the PAPS generating system, which is best suited for the production of relatively large quantities of sulfate conjugates, with the individual enantiomers as substrates, (T)-terbutaline was conjugated to a much greater extent than (-)-terbutaline; the (+)/(-)-enantiomer ratio was 7.3 +/- 0.3 (mean +/- SE). When (+/-)-terbutaline was the substrate and chiral derivatization was employed to separate the sulfate enantiomers formed, a similar (+)/(-)-enantiomer ratio of 7.9 +/- 0.2 was obtained. With PAP35S added to the cytosol, an approach best suited for kinetic studies, the substrate concentration dependence of sulfation could be determined. The Km app for this reaction was identical for (+)- and (-)-terbutaline. However, the Vmax app was 8.1 +/- 0.4 times greater for (+)-terbutaline. This study for the first time shows enantioselectivity in sulfation of a chiral phenolic drug. The experimental approaches used should be valuable for human studies of stereoselective sulfation of terbutaline and other chiral drugs.     

Inhibition of sulfation of phenols in vivo by 2,6-dichloro-4-nitrophenol: selectivity of its action in relation to other conjugations in the rat in vivo

The effect of 2,6-dichloro-4-nitrophenol, an inhibitor of the sulfation of the phenolic compound harmol in vivo, on the sulfation of other phenolic substances and on various conjugation reactions has been studied in the rat in vivo. Compounds chemically related to 2,6-dichloro-4-nitrophenol were also tested as sulfation inhibitors. 2,6-Dichloro-4-nitrophenol inhibited the sulfation of phenol while it had no effect on biliary excretion of dibromosulphthalein, glucuronidation of phenolphthalein, acetylation of procainamide ethobromide or glutathione conjugation of ethacrynic acid. It is concluded that of these conjugation reactions sulfation is inhibited selectively at the dose level used. Some phenols with chloro- or nitro-substituents effectively inhibited the sulfation of harmol but to a lesser extent than 2,6-dichloro-4-nitrophenol. Many other phenols did not affect the conjugation of harmol, which is both glucuronidated and sulfated.