Rabbit and human liver contain a novel pentacyclic triterpene ester with acyl-CoA: cholesterol acyltransferase inhibitory activity

Acyl-coenzyme A (CoA):cholesterol acyltransferase (ACAT) catalyzes the intracellular fatty acid esterification of cholesterol and is thought to play a key role in lipoprotein metabolism and atherogenesis. Herein we describe the purification and characterization of a novel pentacyclic triterpene ester from rabbit liver that has ACAT inhibitory activity. The inhibitor was purified by a combination of silicic acid chromatography and preparative thin layer chromatography. The compound inhibited both rabbit and rat liver microsomal ACAT activity with an IC50 = 20 microM. The lipid did not inhibit fatty acid incorporation into triglycerides, diglycerides, monoglycerides, or phospholipids nor did it inhibit plasma lecithin:cholesterol acyltransferase activity. However, rat liver microsomal acyl-CoA:retinol acyltransferase activity was inhibited by the terpene ester. Kinetic data are consistent with a mechanism in which ACAT is inhibited by the compound in an irreversible manner. The subcellular fractionation pattern of both ACAT activity and the ACAT inhibitor were similar in rabbit liver (both were approximately equally distributed in membranes that pelleted at 10,000 X g and 100,000 X g). A lipid with similar properties to the rabbit liver inhibitor was found in many other rabbit tissues, including adrenal and spleen, as well as in human liver. Rat liver did not contain this lipid. Structural analysis by NMR, mass spectrometry, and x-ray crystallography indicated that the rabbit liver inhibitor was a fatty acid ester (mostly stearate) of a pentacyclic triterpene acid. The carbon skeleton of the triterpene moiety is a new member of the olean-12-ene triterpene family. Both the negatively charged carboxylic acid group of the triterpene moiety and the esterified fatty acid group were necessary for the ACAT-inhibitory activity of the triterpene ester. Lastly, we present preliminary data which, together with the structural homology of the rabbit triterpene with known plant compounds, suggest the hypothesis that the triterpene moiety of the rabbit ACAT inhibitor arises from dietary absorption of a plant triterpene.     

Hepatic ethanol metabolism: Respective roles of alcohol dehydrogenase,the microsomal ethanol-oxidizing system,and catalase

The respective role of alcohol dehydrogenase, of the microsomal ethanol-oxidizing system, and of catalase in ethanol metabolism was assessed quantitatively in liver slices using various inhibitors and ethanol at a final concentration of 50 mm. Pyrazole (2 mm) virtually abolished cytosolic alcohol dehydrogenase activity but inhibited ethanol metabolism in liver slices by only 50–60%. The residual pyrazole-insensitive ethanol oxidation in liver slices remained unaffected by in vitro addition of the catalase inhibitor sodium azide (1 mm). At this concentration, sodium azide completely abolished catalatic activity of catalase in liver homogenate as well as peroxidatic activity of catalase in liver slices in the presence of dl-alanine. Similarly, in vivo administration of 3-amino-1,2,4-triazole, a compound which inhibits the activity of catalase but not that of the microsomal ethanol-oxidizing system, failed to decrease both the overall rates of ethanol oxidation and the activity of the pyrazole-insensitive pathway. Finally, butanol, a substrate and inhibitor of the microsomal ethanol-oxidizing system but not of catalase-H₂O₂, significantly decreased the pyrazole-insensitive ethanol metabolism in liver slices. These results indicate that alcohol dehydrogenase is responsible for half or more of ethanol metabolism by liver slices and that the microsomal ethanol-oxidizing system rather than catalase-H₂O₂ accounts for most if not all of the alcohol dehydrogenase-independent pathway.     

Rabbit liver microsomal lipid peroxidation. The effect of lipid on the rate of peroxidation

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.     

The role of lipid peroxidation in the N-oxidation of 4-chloroaniline.

Irradiation with u.v. light of aerobic aqueous media containing both rabbit liver microsomal fraction and 4-chloroaniline results in N-oxidation of the arylamine. The reaction is severely blocked by exhaustive extraction with organic solvents of the microsomal membranes to remove lipids. Further, scavengers of OH. and O2.-impair the photochemical process. These findings suggest that the observed phenomenon may be closely associated with light-induced lipid peroxidation. Indeed, N-oxidation of 4-chloroaniline is fully preserved when either phospholipid liposomes or dispersed linoleic acid substitute for intact microsomal fraction. Co-oxidation of the amine substrate occurs during iron/ascorbate-promoted lipid peroxidation also, but H2O2 or free OH. radicals do not appear to be involved. Cumene hydroperoxide-sustained rabbit liver microsomal turnover of the amine generates N-oxy product via O2-dependent and -independent pathways; propagation of lipid peroxidation is presumed to govern the former route. Lipid hydroperoxides, either exogenously added to rabbit liver microsomal suspensions or enzymically formed from arachidonic acid in ram seminal-vesicle microsomal preparations, support N-oxidation of 4-chloroaniline. The significance, in arylamine activation, of lipid peroxidation in certain extrahepatic tissues exhibiting but low mono-oxygenase activity is discussed.     

Synthesis, hydrolysis and anti-EBV activity of a series of 3'-modified cycloSal-BVDUMP pronucleotides

A series of cycloSal-BVDUMP phosphate triesters has been prepared. The prototype compound was 3-methyl-cycloSal-BVDUMP 2. Furthermore, a series of 3'-O-acyl-modified derivatives having carboxylic acids with different lipophilicity or a L-configurated alpha-amino acid (phenylalanine) was prepared. The hydrolysis properties in phosphate buffer PBS as well as in PBS containing pig liver esterase (PLE) will be described. Finally, the biological activity against EBV has been determined.     

Lipid alterations in liver and kidney induced by normobaric hyperoxia: correlations with changes in microsomal membrane fluidity

The effects of normobaric hyperoxia on both microsomal membrane fluidity and mechanism of phospholipid synthesis in rabbit liver and kidney have been studied. Hyperoxia induces in both organs an impairment of de novo synthesis of glycerolipids which could be due to an inactivation of acyltransferase activities involved in the initial formation of phosphatidic acid. The ability to replace phospholipid fatty acids by reacylation mechanism decreases slightly in the hyperoxic kidney, while it does not change in the hyperoxic liver. Concerning the effect of high arterial pO2 on microsomal membrane fluidity, the hyperoxic liver shows a more fluid environment within the membrane core of microsomes; however, no difference was shown in that of microsomal membrane core of hyperoxic kidney. An insight into the lipid composition of microsomes indicates that liver microsomal membranes have lower cholesterol content and higher unsaturation degree of phospholipid fatty acids, whereas hyperoxic kidney microsomes become more saturated and did not show any difference in their cholesterol content. In both hyperoxic liver and kidney microsomes, phospholipid content decreases in agreement with the depression of phosphatidic acid biosynthesis. These results are discussed in relation to the values of microsomal membrane microviscosity obtained.     

Oxidation of arylcyclopropanes by rabbit liver cytochrome P-450. Mechanistic implications of a multiple oxidation.

The arylcyclopropanes (cyclopropylarenes) cyclopropylbenzene and diphenylcyclopropane are oxidized by rabbit liver microsomal cytochrome P-450, both by the microsomal fraction and by the purified cytochrome in a reconstituted system. The products formed, principally benzoic acid, are due to an unusual triple oxidation of the substrate, which probably remains attached to the active site during the several steps of the oxidation. Both substrates were found to be inhibitors of the cytochrome P-450-dependent O-de-ethylation of 7-ethoxycoumarin. Model oxidation studies with cumene hydroperoxide as oxidizing agent and rabbit liver microsomal fraction as source of enzyme gave similar products to the microsomal and reconstituted systems. The significance of these results in the mechanism of oxidation catalysed by cytochrome P-450 is discussed.     

Some observations on the chemical and stereochemical specificity of the de-alkylation of organophosphorus esters by a hepatic glutathione transferase

Hepatic glutathione (GSH) S-methyl transferase from rabbit, pig and dog demethylates dimethyl phosphate triesters. No stereospecificity towards racemic ethyl methyl 2-chloro-1-(2,4-dichlorophenyl)vinyl phosphate could be demonstrated but the enzyme exhibited some selectivity towards the (+) and (?) forms of O-methyl S-methyl 1-naphthyl phosphorothiolate. Pig liver enzyme, purified 30-fold, demethylated the prochiral substrate dimethyl 1-naphthyl phosphorothionate with 90% stereo-selectivity.     

Inhibition of lipid-linked oligosaccharide synthesis by aryl phosphates.

Our previous work has shown that phenyl phosphate acts as an exogenous substrate for GDP-mannose:dolichyl phosphate mannosyltransferase in rat liver microsomal fractions to give rise to phenyl phosphate beta-D-mannose, a compound which, unlike Dol-P-Man (dolichyl phosphate beta-D-mannose), cannot act as mannose donor for further mannose-adding reactions in microsomal fractions. The study has now been extended to the action of various aryl phosphates and structurally related compounds on several other glycosyltransferase systems in the microsomal fractions. (1) Examination of the ability of these compounds to accept sugars from various sugar nucleotides indicated that the individual compounds have specificity as sugar acceptors. Thus phenyl phosphate acted as an effective acceptor for both mannose and glucose, whereas benzenephosphonic acid was active only in accepting mannose. p-Nitrophenyl phosphate was a more effective glucose acceptor than phenyl phosphate, but had only 8% of the mannose-accepting activity of phenyl phosphate. (2) Phenyl phosphate had an inhibitory effect on the transfer of mannose form GDP-mannose to lipid-linked oligosaccharides and to glycoproteins in rat liver microsomal fractions. The inhibition depended on the concentration of phenyl phosphate and on the extent of inhibition of Dol-P-Man synthesis. It is proposed that phenyl phosphate has a direct effect on the synthesis of Dol-P-Man and that its inhibition of synthesis of lipid-linked oligosaccharides and glycoproteins could be a consequence of this effect.     

The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.     

Glutathione S-aryl transferase in the metabolism of parathion and its analogs

A soluble enzyme (glutathione S-aryl transferase) which converts parathion and related insecticidal organophosphorus triesters to S-$\text{p}$-nitrophenylglutathione and the corresponding dialkyl phosphorothioic or phosphoric acid has been identified and assayed in vertebrate liver. The activity of this enzyme can be differentiated from that of the analogous glutathione S-alkyl transferase also present in rat tissues. Its relationship to other known glutathione S-aryl transferases remains to be established but considerable differences in optimum pH have been observed.     

The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate

1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.     

A Spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes

1. The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH₂ and oxygen. Activity with NADH₂ was one-third of that with NADPH₂. 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at −12°. Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2′-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.     

Inhibition by gamma-hexachlorocyclohexane of acetylcholine-stimulated phosphatidylinositol synthesis in cerebral cortex slices and of phosphatidic acid-inositol transferase in cerebral cortex particulate fractions

• 1 γ-Hexachlorocyclohexane inhibits the ACh-stimulated synthesis of phosphatidylinositol in guinea pig cerebral cortex slices, as measured either by the incorporation of [2-³H]inositol or of ³²P. Phosphatidylinositol synthesis in the control slices is not inhibited.
• 2 The synthesis of phosphatidylinositol from CDP-diglyceride in cerebral cortex microsomal preparations is inhibited by γ-hexachlorocyclohexane. The incorporation of [2-³H]inositol into lipid in the absence of added cytidine nucleotide in these preparations is not inhibited.
• 3 δ-Hexachlorocyclohexane profoundly inhibits phosphatide synthesis and phosphate metabolism in cerebral cortex slices both in the presence and absence of ACh. This isomer also inhibits the exchange reaction for the incorporation of [2-³H]inositol into lipid in the microsomal preparations.
• 4 α-, and β-Hexachlorocyclohexanes do not inhibit either ACh-stimulated or control synthesis of phosphatidylinositol in cerebral cortex slices; nor do they inhibit the exchange reaction for [2-³H]inositol incorporation into lipid in the microsomal preparations.
• 5 The specific effects of γ-hexachlorocyclohexane are taken as providing evidence that ACh-stimulated phosphatidylinositol synthesis in cerebral cortex slices probably involves the CDP-diglyceride pathway. The possibility is discussed that the primary action of ACh in this system is to cause an increased activity of diglyceride kinase to provide phosphatidic acid for this pathway.

     

Bis-ketol nucleoside triesters as prodrugs of the antiviral nucleoside triphosphate analogues of 3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine

Derivatives of 3'-deoxythymidine (ddT) and 3'-deoxy-2',3'-didehydrothymidine (d4T) were prepared in which the 5'-hydroxyl group of the nucleoside was esterified to a bis-ketol phosphate. The resulting phosphate triesters are postulated to be prodrugs of the corresponding 5'-mononucleotides, which are formed intracellularly by the hydrolysis of the two ketol ester groups. The triesters were tested for anti-HIV activity with the result that those derived from ddT showed enhanced antiviral activity when compared to the parent nucleoside.     

Inter-tissue and inter-species comparison of butyrylcholinesterases

Butyrylcholinesterase (BuChE) occurs in a multiple molecular forms whose catalytic activity depends on tissue distribution and species. The hypothesis led us to the study of BuChE catalytic properties focused on the inter-tissue and inter-species level with benzoylcholine and N-alkyl derivates of benzoylcholine (BCHn) as substrates. These compounds are soft disinfectants easily biodegradable to biologically inactive hydrolytic products, substituted choline and benzoic acid. Different sources of BuChE were used: rabbit and rat liver microsomal fraction (membrane-anchored enzyme) and serum (soluble form). Hydrolytic activity of both these BuChE forms was compared to rat recombinant BuChE (rBuChE). Hydrolytic product (benzoic acid) formation was recorded as function of time, and hydrolytic rate was determined. Tissue distribution of BuChE plays an important role in hydrolysis of BCHn. High BuChE activity was observed in a serum of both studied species rat and rabbit and was significantly dependent on a structure of substrates. Activity of soluble serum forms was the same as that for the rBuChE. Significant change of BuChE activity was recorded on the inter-species level in the microsomal fractions. It is because the rabbit microsomal BuChE activity had absolutely different course for all substrates as compared to rat microsomes. Inhibitory studies of BCHn enzymatic hydrolysis of all BuChE forms were performed to determine the level of BuChE participation in BCHn hydrolysis. It can be concluded that short-chain BCHn substrates are exclusively hydrolyzed by BuChE from all studied sources except for the rabbit liver microsomal fraction. Rabbit seems to have different enzymes involved in the hydrolysis of all studied BCHn compounds.     

A trypsin-sensitive, heat-labile, N-ethylmaleimide-sensitive factor in adipocyte post-microsomal supernatant which affects the assay of adipocyte glycerol phosphate acyltransferase activities.

Addition of adipocyte 100 000 g post-microsomal supernatant to assays of glycerol phosphate acyltransferase in isolated mitochondria or microsomal fractions decreased activity at lower concentrations of palmitoyl-CoA. At higher concentrations of palmitoyl-CoA, activation was observed on addition of post-microsomal supernatant. The effect of post-microsomal supernatant to decrease activity at lower [palmitoyl-CoA] was abolished by heating or by trypsin treatment, and was also abolished by addition of N-ethylmaleimide to assays or by pretreatment of post-microsomal supernatant with N-ethylmaleimide. The stimulatory effect seen at higher [palmitoyl-CoA] was not sensitive to heat or trypsin treatment. The effect of post-microsomal supernatant at lower [palmitoyl-CoA] cannot be attributed to palmitoyl-CoA hydrolase activity. It was found that brief treatment of adipocyte mitochondria with low concentrations of trypsin was an effective way to remove contaminating microsomal glycerol phosphate acyltransferase activity. Adipocyte post-microsomal supernatant was more effective than an equivalent quantity of liver post-microsomal supernatant protein in decreasing adipocyte microsomal glycerol phosphate acyltransferase activity. The effects of the supernatants from both tissues were decreased by flavaspidic acid. Semi-purified Z-protein fraction from rat liver did not mimic the effect of adipocyte post-microsomal supernatant to decrease glycerol phosphate acyltransferase at lower [palmitoyl-CoA]. Post-microsomal supernatants obtained from noradrenaline-treated adipocytes were less effective than those from control cells in decreasing glycerol phosphate acyltransferase activity in microsomal fractions at lower [palmitoyl-CoA]. It is suggested that adipocyte cytosol may contain an acyl-CoA-binding protein or proteins differing from Z-protein in some respects. The physiological significance of the findings is briefly discussed.     

Induction of cytosolic and microsomal epoxide hydrolases in mouse liver by peroxisome proliferators, with special emphasis on structural analogues of 2-ethylhexanoic acid

Using dietary administration, mice were exposed to eight substances known to cause peroxisome proliferation (i.e. clofibrate clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, ICI-55.897, S-8527 and Wy-14.643) or the related substance p-chlorophenoxyacetic acid (group A). Other animals received di(2-ethylhexyl)phthalate, mono(2-ethylhexyl)phthalate, 2-ethylhexanoic acid, or one of 12 other metabolically and/or structurally related compounds (group B). The effects of these treatments on liver cytosolic and microsomal epoxide hydrolases, microsomal cytochrome P-450, cytosolic glutathione transferase activity, the liver-somatic index and the protein contents of the microsomal and cytosolic fractions prepared from liver were subsequently monitored. In general, peroxisome proliferation was accompanied by increases in cytosolic epoxide hydrolase activity. Many peroxisome proliferators also caused increases in microsomal epoxide hydrolase activity, although the correlation was poorer in this case. Immunochemical quantitation by radial immunodiffusion demonstrated that the increases observed in both of these enzyme activities reflected equivalent increases in enzyme protein, i.e. that induction truly occurred. Induction of total microsomal cytochrome P-450 was obtained after dietary exposure to clofibrate, clofibric acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, nafenopin, Wy-14.643, di(2-ethylhexyl)phthalate and di(2-ethylhexyl)phosphate. The most pronounced effects on cytosolic glutathione transferase activity were the decreases obtained after treatment with clofibrate, clofibric acid and Wy-14.643. Our results, together with those reported by others, suggest that the processes of peroxisome proliferation and induction of cytosolic epoxide hydrolase are intimately related. One possible explanation for this is presented.     

Microheterogeneity in the major phenobarbital-inducible forms of rabbit liver microsomal cytochrome P-450 as revealed by nucleotide sequencing of cloned cDNAs

We have isolated one full-length cDNA clone, termed pHP1, and a number of clones of shorter insert lengths, tentatively called b14, b46, etc., all encoding phenobarbital- (PB-) inducible forms of rabbit liver microsomal cytochrome P-450, and determined their nucleotide sequences. The polypeptides encoded by these cDNAs can be classified into five types, represented by HP1, b14, b46, b52, and b54, the deduced amino acid sequences of which are more than 95% similar to one another. Amino acid differences among them total 24 positions, which are distributed over the entire sequence, in contrast to the microheterogeneity observed in two PB-inducible rat liver microsomal cytochromes P-450 (P-450b and P-450e). The primary structure deduced for the HP1 protein is 97% similar to that determined for rabbit P-450 LM2 (form 2), which has been purified by Coon and co-workers [van der Hoeven, T. A., Haugen, D. A., & Coon, M. J. (1974) Biochem. Biophys. Res. Commun. 60, 569-675; Haugen, D. A., & Coon, M. J. (1976) J. Biol. Chem. 251, 7929-7939] as the major PB-inducible form of rabbit liver microsomal cytochrome P-450. The amino acid sequence of P-450(1), which we have purified as the major PB-inducible rabbit liver cytochrome P-450, was partially determined with the sequence reported for P-450 LM2 as a reference. The two sequences are closely similar to each other, but at least two amino acid differences can be detected between them.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of a glutathione conjugate of 2-hydroxyoestradiol by rat liver and kidney preparations in vitro

Adult male rat liver and kidney preparations were incubated with (2-hydroxyoestradiol-1-yl)[(35)S]glutathione. The glutamic acid and glycine residues were removed by enzymes present in the kidney microsomal fraction; the liver preparations had no effect. The resulting 2-hydroxyoestradiol-cysteine conjugate was acetylated at the alpha-amino group by both liver and kidney homogenates fortified with acetyl-coenzyme A, but not significantly in the absence of this coenzyme, or by liver or kidney slices. These results suggest that an oestrogen-glutathione conjugate, if formed in vivo, would be converted into the corresponding mercapturic acid before excretion.