Synthesis of reduced collagen crosslinks.

A new synthetic route to reduced collagen crosslinks (LNL and HLNL) is described in this report. It enables an enantioselective synthesis of LNL. HLNL was obtained as a mixture of two diastereoisomers. This method also provides the possibility to introduce radio-labels during the synthesis.

Inhibition of glutathione S-transferase 3-3 by glutathione derivatives that bind covalently to the active site.

In all, 13 GSH derivatives have been synthesized and tested for their potency to inhibit glutathione S-transferase (GST) 3-3. All of these derivatives contained a reactive group that could potentially react with the enzyme active site. Best results were obtained with the phenylthiosulphonate derivative of GSH, GSSO2Ph. Preincubation of GST 3-3 with a 100 microM concentration of this inhibitor resulted in a time-dependent loss of activity: after 30 min at pH 6.5 and 25 degrees C, 51% of the activity was lost. At more alkaline pH, the activity is more rapidly inhibited: at pH 8.0 the 90%-inhibition level is already reached after 10 min preincubation. Separation of enzyme and excess unbound GSSO2Ph after preincubation by gel-filtration chromatography did not result in a reappearance of enzyme activity. If 100 microM-GSH was added to the preincubation mixture at pH 7.4, inhibition was almost completely prevented. Addition of S-(hexyl)glutathione (20 microM) could delay the inhibition but, ultimately, not prevent it. The inhibited enzyme could be re-activated by addition of 10 mM-2-mercaptoethanol: 60 min after this thiol was added, the inhibited GST-3- activity was bacxk to the control level. GSH at the same concentration could not re-activate the enzyme. On the basis of these results, on the known reactivity of thiosulphonate compounds, and on current knowledge about the amino acid residues involved in GST catalysis, a covalent modification of an active-site cysteine residue by mixed-disulphide formation between enzyme and the cosubstrate GSH is postulated. Information on the synthesis and characterization of the GSH derivatives is given in Supplementary Publication SUP 50166 (5 pages) which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1991) 273, 5.

Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues.

The substrate specificity of rat liver microsomal glutathione transferase toward glutathione has been examined in a systematic manner. Out of a glycyl-modified and eight gamma-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, alpha-L-Glu-L-Cys-Gly, alpha-D-Glu-L-Cys-Gly, and gamma-L-Glu-L-Cys-beta-Ala) function as substrates. The kinetic parameters for three of these substrates (the alpha-D-Glu-L-Cys-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the N-ethylmaleimide-activated microsomal glutathione transferase. The alpha-L-Glu-L-Cys-Gly analogue is similar to GSH in that it has a higher kcat (6.9 versus 0.6 s-1) value with the activated enzyme compared with the unactivated enzyme but displays a high Km (6 versus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kcat (8.9 versus 6.7 s-1) with the N-ethylmaleimide-treated enzyme but retains a higher Km value (50 versus 15 mM). Thus, the alpha-amino group of the glutamyl residue in GSH is important for the activity of the activated microsomal glutathione transferase. These observations were quantitated by analyzing the changes in the Gibbs free energy of binding calculated from the changes in kcat/Km values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the alpha-amino group of the glutamyl residue in GSH contributes 9.7 kJ/mol to catalysis by the activated enzyme, whereas the corresponding value for the unactivated enzyme is 3.2 kJ/mol. The importance of the acidic functions in glutathione is also evident as shown by the lack of activity with 4-aminobutyric acid-L-Cys-Gly and the low kcat/Km values with gamma-L-Glu-L-Cys-beta-Ala (0.03 and 0.01 mM-1s-1 for unactivated and activated enzyme, respectively). Utilization of binding energy from a correctly positioned carboxyl group in the glycine residue (10 and 17 kJ/mol for unactivated and activated enzyme, respectively) therefore also appears to be required for optimal activity and activation. A conformational change in the microsomal glutathione transferase upon treatment with N-ethylmaleimide or trypsin, which allows utilization of binding energy from the alpha-amino group of GSH as well as the glycine carboxyl in catalysis, is suggested to account for at least part of the activation of the enzyme.

Inhibition of rat liver glutathione S-transferase isoenzymes by peptides stabilized against degradation by gamma-glutamyl transpeptidase.

Inhibitors for glutathione S-transferase (GST) iso-enzymes from rat liver with high affinity for the glutathione-binding site (G-site) have been developed. In previous studies, a model was described for the G-site of GST (Adang, A. E. P., Brussee, J., van der Gen, A., and Mulder, G. J. (1990) Biochem. J. 269, 47-54) in terms of essential and nonessential interactions between groups in glutathione (GSH) and the G-site. Based on this model, compounds were designed that have high affinity for the G-site but cannot be conjugated. In the dipeptide gamma-L-glutamyl-D-aminoadipic acid (gamma-L-Glu-D-Aad), the L-cysteinylglycine moiety is replaced by D-aminoadipic acid. This dipeptide is an efficient competitive inhibitor (toward GSH) of mu class GST isoenzymes with Ki values of 34 microM for GST isoenzyme 3-3 and 8 microM for GST isoenzyme 4-4. Other GSH-dependent enzymes, such as gamma-glutamyl transpeptidase (gamma-GT), glutathione reductase, and glutathione peroxidase, were not inhibited by 1 mM of gamma-L-Glu-D-Aad. Inhibition is also highly stereospecific since gamma-L-Glu-L-Aad is only a poor inhibitor (Ki = 430 microM for GST 3-3). Gamma-L-Glutamyl-D-norleucine also had a much higher Ki value for GST 3-3. Thus, the presence of a delta-carboxylate group in D-Aad appears to be essential for a high affinity inhibitor. An additional hydrophobic group did not result in increased inhibitory potency. In a different approach, the gamma-L-glutamyl moiety in GSH was replaced by delta-L-aminoadipic acid; delta-L-Aad-L-Cys-Gly is an efficient cosubstrate analogue for GSTs with Km values comparable to GSH and Vmax values ranging from 0.24 to 57 mumol/min/mg for the different GSTs. The structures of the efficient inhibitor and the cosubstrate analogue were combined in delta-L-Aad-D-Aad, which had a Ki value of 68 microM with GST 3-3. In order to investigate their possible use in vivo studies, the degradation of gamma-L-Glu-D-Aad and delta-L-Aad-L-Cys-Gly by gamma-GT was investigated. The peptides showed no measurable hydrolysis rates under conditions where GSH was rapidly hydrolyzed. Thus, an efficient, mu class-specific GST inhibitor and a gamma-glutamyl-modified cosubstrate analogue of GSH were developed. Their gamma-GT stability offers the possibility to use these peptides in in vivo experiments.

The glutathione-binding site in glutathione S-transferases. Investigation of the cysteinyl, glycyl and gamma-glutamyl domains.

The GSH-binding site of glutathione S-transferase (GST) isoenzymes was studied by investigating their substrate-specificity for three series of GSH analogues; further, a model of the interactions of GSH with the G-site is proposed. Twelve glycyl-modified GSH analogues, four ester derivatives of GSH and three cysteinyl-modified GSH analogues were synthesized and tested with purified forms of rat liver GST (1-1, 2-2, 3-3 and 4-4). The glycyl analogues exhibited spontaneous chemical reaction rates with 1-chloro-2,4-dinitrobenzene comparable with the GSH rate. In contrast, the enzymic rates (Vmax.) differed greatly, from less than 1 up to 140 mumol/min per mg; apparently, a reaction mechanism is followed that is very sensitive to substitutions at the glycyl domain. No correlation exists between the chemical rates and Vmax. values for the analogues. Analogues of GSH in which L-cysteine was replaced by D-cysteine, L-homocysteine or L-penicillamine showed little or no capacity to replace GSH as co-substrate for the GSTs. GSH monomethyl and monoethyl esters showed Vmax. values greater than the Vmax. measured with GSH: the Vmax. for the monoethyl ester of GSH and GST 3-3 was 5-fold that for GSH. The data obtained in this and previous studies [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724; Adang, Meyer, Brussee, van der Gen, Ketterer & Mulder (1989) Biochem. J. 264, 759-764] allow a model of the interactions of GSH in the G-site in GSTs to be postulated. The gamma-glutamyl site is the main binding determinant: the alpha-carboxylate group is obligatory, whereas shifting of the amino group and shortening of the peptide backbone only decreased kcat./Km. Furthermore, the GSTs appear to be very critical with respect to a correct orientation of the thiol group of the GSH analogue. The glycyl site is the least restrictive domain in the G-site of GSTs: amino acid analogues all showed Km values between 0.2 and 0.6 mM (that for GSH is 0.2-0.3 mM), but large differences in Vmax. exist. The glycyl carboxylate group is not essential for substrate recognition, since decarboxy analogues and ester derivatives showed high activities. The possible mechanisms for an increased Vmax. in some analogues are briefly discussed.

Inhibition of UDP-glucuronosyltransferase activity by possible transition-state analogues in rat-liver microsomes.

A series of possible transition state analogues of the glucuronidation reaction catalyzed by UDP-glucuronosyltransferase were tested for their inhibitory effect on glucuronidation of various substrates in a rat liver microsomal fraction. In general 4-nitrophenol glucuronidation was more effectively inhibited than that of 1-naphthol, bilirubin or testosterone. 2-(1-Naphthyl)ethyl-UDP and 2,2,2-(triphenyl)ethyl-UDP were the most effective inhibitors. Their inhibitory effect was competitive towards both UDP-glucuronic acid and 4-nitrophenol. These compounds were much more effective inhibitors than UDP; therefore addition of a lipophilic group enhances the inhibitory potency of UDP. The various UDP derivatives showed differences in their abilities to inhibit the glucuronidation of the four acceptor substrates, supporting the concept that the different forms of UDP-glucuronosyl transferase have different active sites.

Quantitative HPLC analysis of the level of fecapentaenes and their precursors in human feces by a chemical conversion method.

A fast and reliable hplc method for the quantitative analysis of total fecapentaene-12 (FP-12 and its precursors) and total fecapentaene-14 (FP-14 and its precursors) in human feces is described. The analysis is based on the rapid chemical conversion of fecapentaenes and their precursors to more stable methoxytetraenols and the use of synthetic, not naturally occurring, fecapentaene-13 (FP-13) as an internal standard. The synthesis and physical properties of this internal standard are described. The convenience and reproducibility of the method were illustrated by applying the procedure to stool samples obtained from twelve individuals on 3 consecutive days. Levels of total FP's were in the range of 0.1-25.4 micrograms for total FP-12 and 0.1-8.5 micrograms for total FP-14 per g of wet feces. Appreciable fluctuations were observed between levels in samples from the same individual on different days. Reproducibility and recovery were shown to be good.

Interaction of rat glutathione S-transferases 7-7 and 8-8 with gamma-glutamyl- or glycyl-modified glutathione analogues.

Analogues of GSH in which either the gamma-glutamyl or the glycyl moiety is modified were synthesized and tested as both substrates for and inhibitors of glutathione S-transferases (GSTs) 7-7 and 8-8. Acceptor substrates for GST 7-7 were 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid (ETA) and for GST 8-8 CDNB, ETA and 4-hydroxynon-trans-2-enal (HNE). The relative ability of each combination of enzyme and GSH analogue to catalyse the conjugation of all acceptor substrates was similar with the exception of the combination of GST 7-7 and gamma-L-Glu-L-Cys-L-Asp, which used CDNB but not ETA as acceptor substrate. In general, GST 7-7 was better than GST 8-8 in utilizing these analogues as substrates, and glycyl analogues were better than gamma-glutamyl analogues as both substrates and inhibitors. These results are compared with those obtained earlier with GSH analogues and GST isoenzymes 1-1, 2-2, 3-3 and 4-4 [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] and the implications with respect to the nature of their active sites are discussed.

Importance of multiple hydroxylated metabolites in hexamethylphosphoramide (HMPA)-mediated mutagenesis in Drosophila melanogaster.

The mutagenic profiles in Drosophila and the influence of inhibition of metabolism on genotoxic activity were determined for hexamethylphosphoric triamide (HMPA), some synthetically prepared presumed metabolites and ethylated analogs. Demethylated HMPA metabolites are considerably less mutagenic than HMPA, dependent on the degree of demethylation. The mutagenicity of the presumptive primary metabolite, hydroxymethyl pentamethylphosphoramide (HM-Me5-PA), is comparable to HMPA and can be decreased considerably by inhibition of the metabolism by 1-phenylimidazole or iproniazid. This suggests that further oxidative metabolism is required for mutagenic activity. The mutagenicity of the doubly hydroxylated HMPA metabolite, N,N'-bis(hydroxymethyl)-tetramethylphosphoramide (N,N'-(HM)2-Me4-PA) can also be decreased by inhibition of metabolism, whereas the 3-fold hydroxylated N,N',-N"-(HM)3-Me3-PA is not affected by pretreatment with enzyme inhibitors, indicating that no further oxidative metabolism is required for its activation. A second hydroxylation on 1 dimethylamino group, forming N,N-(HM)2-Me4-PA, results in a drastic loss of mutagenic activity. Further oxidation of HM-Me5-PA to formyl pentamethylphosphoramide (formyl-Me5-PA) also leads to a strong reduction of the genotoxic activity. The rearrangement product of N-oxidation, N-[bis(dimethylamino)phosphinyl)-oxy)dimethylamine (HMPOA) is not mutagenic in Drosophila. The very low mutagenicity of hexaethylphosphoramide (Et6-PA) allowed us to study the mutagenicity of some ethyl-hydroxymethyl hybrid compounds. For the ethylated phosphoramides also the presence of only 1 hydroxymethyl group is insufficient for mutagenic activity, whereas the introduction of 2 or 3 hydroxymethyl groups resulted in considerable genotoxicity in the sex-linked recessive lethal (SLRL) test as well as in the ring-X loss test. It is concluded that the bioactivation of HMPA in Drosophila proceeds via multiple metabolic hydroxylations to form multifunctional, cross-linking agents. The presence of an oxygen atom on the phosphorus appears to be a prerequisite for the genotoxic activity of HMPA as hexamethylphosphorus triamide (HMPT), a derivative lacking this oxygen, is only weakly mutagenic in Drosophila. The results presented in this paper do not support the theory that formaldehyde is the active principle of activated HMPA.

Synthesis and nucleophilic reactivity of a series of glutathione analogues, modified at the gamma-glutamyl moiety.

A series of GSH analogues with modifications at the gamma-glutamyl moiety was synthesized and purified by following peptide chemistry methodology. Benzyl, benzyloxycarbonyl and t-butyloxycarbonyl protective groups were used to protect individual amino acid functional groups. The formation of peptide bonds was accomplished through coupling of free amino groups with active esters, generated by reaction of the carboxylate functions with dicyclohexylcarbodi-imide and 1-hydroxybenzotriazole. The protecting groups in the tripeptides were removed in a single step by using Na in liquid NH3. Precautions were taken in order to prevent oxidation of the thiol function in the cysteine residue. Thus GSH analogues containing both L- and D-glutamic acid and L- and D-aspartic acid, coupled to cysteinylglycine through both the alpha- and the omega-carboxylate group, were synthesized. Also, decarboxy-GSH and deamino-GSH, lacking one functional group in the glutamate moiety, were prepared. The spontaneous non-enzyme-catalysed nucleophilic reaction of these GSH analogues with the electrophilic model substrate 1-chloro-2,4-dinitrobenzene showed appreciable rate differences, indicating the importance of intramolecular interactions in determining the nucleophilic reactivity of the thiol function in the cysteine residue. In particular, the free amino group in the gamma-L-glutamic acid residue appears to play a crucial role in activating the thiol group in GSH. In an adjacent paper [Adang, Brussee, Meyer, Coles, Ketterer, van der Gen & Mulder (1988) Biochem. J. 255, 721-724] these results are compared with those obtained in a study on the ability of these GSH analogues to act as a co-substrate in the glutathione S-transferase-catalysed conjugation reaction with 1-chloro-2,4-dinitrobenzene.

Substrate specificity of rat liver glutathione S-transferase isoenzymes for a series of glutathione analogues, modified at the gamma-glutamyl moiety.

The substrate specificity of purified rat liver glutathione S-transferases (GSTs) for a series of gamma-glutamyl-modified GSH analogues was investigated. GST isoenzyme 3-3 catalysed the conjugation of 1-chloro-2,4-dinitrobenzene with six out of the nine analogues. alpha-L-Glu-L-Cys-Gly and alpha-D-Glu-L-Cys-Gly showed catalytic efficiencies of 40% and 130% that of GSH respectively. The GSH analogue with an alpha-D-glutamyl moiety appeared to be a highly isoenzyme-3-3-specific co-substrate: kcat./Km with GST isoenzyme 4-4 was only about 5% that with GST isoenzyme 3-3, and no enzymic activity was detectable with GST isoenzymes 1-1 and 2-2. GST isoenzyme 4-4 showed some resemblance to GST 3-3: five out of nine co-substrate analogues were accepted by this second isoenzyme of the Mu multigene family. Isoenzymes 1-1 and 2-2, of the Alpha multigene family, accepted only two alternative co-substrates, which indicates that their GSH-binding site is much more specific.

Stereoselective glutathione conjugation of the separate alpha-bromoisovalerylurea and alpha-bromoisovaleric acid enantiomers in the rat in vivo and in rat liver subcellular fractions.

Glutathione (GSH) conjugation of the separate alpha-bromoisovalerylurea (BIU) enantiomers was studied in the rat. Administration of (R)-BIU resulted in excretion of a single glutathione conjugate in bile (IU-S-G/I) and a single mercapturate in urine (IU-S-MA/B). The other enantiomer, (S)-BIU, was exclusively metabolized to the other diastereomeric conjugates, IU-S-G/II and IU-S-MA/A. Thus, the conjugation of BIU with glutathione was completely stereospecific. Both the GSH conjugate and mercapturate derived from (R)-BIU were excreted two to three times more rapidly than their diastereomeric (S)-BIU counterparts. The enantiomers did not influence each others metabolism as reflected by identical metabolite excretion rates when the BIU enantiomers were administered either separately or as the racemic mixture. A similar rate difference for GSH conjugation of the separate BIU enantiomers was observed in incubations with rat liver cytosol as source of GSH transferases, suggesting that the stereoselectivity in vivo was due to glutathione conjugation properly. Similar results were obtained with a rat liver microsomal fraction, indicating that microsomal GSH transferases are active towards BIU and have a similar stereoselectivity as the cytosolic enzymes. Comparison of the GSH conjugation of BIU with that of its analogue alpha-bromoisovaleric acid (BI, which lacks the amide-linked urea group) revealed an opposite stereoselectivity: while (R)-BIU was conjugated faster than (S)-BIU, the (R) enantiomer of the acid was conjugated more slowly than (S)-BI. The alpha-bromocarbonyl compounds BI and BIU present a new type of substrate for the GSH transferases and allow studies of these enzymes in intact organisms as well as investigations on the stereoselectivity of GSH conjugation.

1,2-Dibromo compounds. Their mutagenicity in Salmonella strains differing in glutathione content and their alkylating potential.

The mutagenic activities of several structurally related dibromo compounds were compared in Salmonella strains sensitive to base substitution mutagenesis (TA1535 and/or TA100) and in the glutathione (GSH)-deficient derivative TA100/NG-57, using a preincubation procedure. The compounds tested were 1,2-dibromoethane (DBE), 1,2-dibromopropane (DBP), 1,2-dibromo-1-phenylethane (DBPE) and model compounds for the half-mustards resulting from their conjugation with GSH, i.e. the N-acetyl-S-2-bromoalkyl-L-cysteine methyl esters SBE, SBP, and SBPE, respectively. The alkylating potential of all compounds was assayed with the 4-(p-nitrobenzyl)pyridine (NBP) alkylation test. Five of the compounds showed a good correlation between relative mutagenic activity in TA100 and electrophilic reactivity in the NBP-test, the order of decreasing potency being SBE greater than SBP greater than DBPE greater than DBP. SBPE displayed the highest reactivity in the NBP-test, but was devoid of mutagenic activity. The mutagenic activity of DBE was substantially decreased in the GSH-deficient strain TA100/NG-57 and could be restored by pretreating the cells with GSH. None of the other chemicals showed different mutagenic activities in TA100 and TA100/NG-57. From the results it can be concluded that 2-bromothioethers possess higher alkylating activities than the 1,2-dibromo compounds. Methyl substitution has a deactivating effect on the mutagenic activity. The results with the phenyl-substituted analogue, DBPE, show that a higher alkylating activity does not always lead to a higher mutagenic activity.

Glutathione conjugation of 1,2-dibromo-1-phenylethane in rats in vivo.

The metabolism of 1,2-dibromo-1-phenylethane (DBPE) was studied in rats. Administration of DBPE orally, in doses of 0.25-1.25 mmol/kg (66-330 mg/kg), to male Wistar rats resulted in the excretion of a single mercapturic acid in urine. The methyl esters of three potential mercapturic acid metabolites were synthesized: N-acetyl-S-(2-oxo-2-phenylethyl)-L-cysteine methyl ester (O),N-acetyl-S-(2-hydroxy-1-phenylethyl)-L-cysteine methyl ester (I), and N-acetyl-S-(2-hydroxy-2-phenylethyl)-L-cysteine methyl ester (II). GC/MS analysis showed that the methyl ester of the excreted mercapturic acid was identical with II. Quantitative measurement of II in urine by GLC showed that, after 24 hr, excretion of the mercapturic acid was almost complete and amounted to 41% of the administered dose. At doses higher than 1.00 mmol/kg, the excretion no longer increased. Inhibition of the oxidative pathways by ip injection of 1-phenylimidazole resulted in an excretion decrease of about 40%. (Pre)treatment with diethyl maleate lowered the excretion of mercapturic acid by 30-60%. Glutathione conjugates synthesized from DBPE and styrene oxide were separated by HPLC. Both compounds can produce the same two pairs of diastereomers, viz. (R)- and (S)-(2-hydroxy-1-phenyl-ethyl)glutathione ((R)-1 and (S)-1), and (R)- and (S)-(2-hydroxy-2-phenylethyl)glutathione ((R)-2 and (S)-2). These could be separated in the order (R)-2, (R)-1, (S)-1, and (S)-2 within 20 min. This method was also applied to examine glutathione conjugates excreted in bile after DBPE administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological activity of analogs of fecapentaene-12.

Analogs of the potent fecal mutagen fecapentaene-12 have been prepared and tested both for mutagenicity and for their ability to serve as biological precursors of 1. It was found that mutagenicity in three different Salmonella tester strains TA96, TA100, and TA104, decreased rapidly as the number of conjugated double bonds was reduced. The aldehyde 8, analogous to the hydrolysis product of 1, showed only low mutagenicity, even in the aldehyde-sensitive strain TA104. None of the polyenes prepared was able to function as a direct biological precursor of 1 under the conditions employed.

alpha-Bromoisovalerylurea as model substrate for studies on pharmacokinetics of glutathione conjugation in the rat. I. (Bio-) synthesis, analysis and identification of diastereomeric glutathione conjugates and mercapturates.

In order to find a model substrate for kinetic characterization of glutathione conjugation in vivo alpha-bromoisovalerylurea (BIU) was studied. After administration of racemic [14C]urea BIU to rats, two radioactive metabolites were found in bile by high-performance liquid chromatography. The identity of these metabolites was established by various methods. Based on the hydrolytic activity of gamma-glutamyltranspeptidase (presence of the gamma-glutamyl moiety), high resolution nuclear magnetic resonance (isovaleryl and glutathionyl moieties) and fast atom bombardment mass spectrometry (molecular weight and fragmentation pattern), they were identified as glutathione conjugates of BIU. Because both conjugates in bile had these characteristics in common they must be diastereomers. Incubation of BIU with glutathione in the presence of rat liver cytosol resulted in formation of the same diastereomeric glutathione conjugates. Chemical synthesis of the diastereomers confirmed their identity. The major urinary excretion products of [14C]urea BIU in the rat were identified as diastereomeric mercapturates. A convenient chromatographic separation of the diastereomeric glutathione conjugates and the mercapturic acids is described. Electrochemical detection was used to determine the presence of the thioethers in both urine and bile. Pharmacokinetic results on BIU conjugation are described in the accompanying paper.