The metabolism and disposition of 3-methylindole in goats

The metabolism and disposition of 3-methylindole (3MI), a ruminal metabolite of L-tryptophan which causes acute pulmonary edema and emphysema (APE) when administered to cattle, goats, and sheep was investigated. Goats given jugular infusions of [¹⁴C] 3MI excreted 87 to 92% of the infused radioactivity in the urine, 0.4 to 0.9% in expired air, and negligible amounts in the feces over a period of three days. Composite urine samples were fractionated into 10 peaks by ion exchange chromatography, which represented 80% of the urinary radioactivity. A major route of metabolism involved formation of 3-methyloxindole and suggests that a mixed function oxidase (MFO), pyrrolooxygenase, may be the major metabolic system involved. A minor route of metabolism involved oxidation of the methyl carbon of 3MI.     

Identification of 3-hydroxy-3-ethylglutaric acid in urine of patients with propionic acidaemia

By gas chromatographic mass spectrometric analysis, 3-hydroxy-3-ethylglutaric acid was identified in the urine of patients with propionyl CoA carboxylase deficiency. Simultaneously, large amounts of 3-hydroxyvaleric acid and 3-oxovaleric acid, as well as a number of metabolites previously well known in this disorder, were also found in the urine of the patients. It is suggested that formation of 3-oxovaleric acid and 3-hydroxyvaleric acid proceeds via 3-hydroxy-3-ethylglutaryl CoA as an intermediate by a mechanism similar to that of ketone body formation.     

Skatole metabolites in urine as a biological marker of pigs with enhanced hepatic metabolism

Boar taint is a quality defect in meat, related to accumulation of skatole and androstenone in male pigs. The levels of skatole and its main metabolites in plasma and urine samples were measured with a validated liquid chromatography-MS method and related to activity of hepatic cytochrome P450 (CYP450) in order to identify ‘fast metabolizing’ pigs. Urine (n=46), blood (n=12), liver (n=25) and adipose tissue (n=46) were sampled from a total of 46 entire male pigs. Skatole levels in fat were negatively correlated to CYP2E1 activity and positively to 3-hydroxy-3-methyloxindole (HMOI), indole-3-carboxylic acid (ICA) and 2-aminoacetophenone in urine. HMOI and ICA levels in urine were the best predictors of high skatole levels in fat. In summary, the present study provided further evidence for the key role of CYP2E1 in skatole metabolism and suggested that measurement of HMOI and/or ICA in urine might provide information about skatole levels in live pigs.     

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.     

Gender-related differences in the formation of skatole metabolites by specific CYP450 in porcine hepatic S9 fractions

Higher accumulation of skatole in the fat of male pigs compared with female pigs might be due to gender-related differences in the rate of skatole degradation. In the present study, skatole metabolites and cytochrome P450 (CYP450) isoforms involved in skatole metabolism were for the first time investigated in hepatic S9 fractions from six male and four female pigs (crossbred Landrace×Yorkshire dams and Duroc boar). Surprisingly, the rates of production of major skatole metabolites were similar in male and female pigs. The most abundant metabolite of skatole was 3-hydroxy-3-methyloxindole (HMOI) followed by 3-methyloxindole and indole-3-carbinol in both male and female S9 fractions. Concentrations of formed HMOI and 3-methyloxindole did not differ between the genders (P=0.124 for HMOI, and P=0.575 for 3-methyloxindole). Indole-3-carbinol formation was higher in S9 fractions from the females compared with male pigs (P=0.0001). Enzyme kinetic parameters were similar for both genders (P>0.05). In both male and female pigs, ellipticine, diallyl sulphide (DAS) and quercetin inhibited HMOI formation, confirming the involvement of CYP1A1 and CYP2E1. The formation of 3-methyloxindole was reduced in the presence of the CYP2E1 inhibitor DAS, and formation of indole-3-carbinol was reduced in the presence of CYP1A1 and CYP2A19 inhibitors. We found only minor differences in skatole metabolism between male and female pigs, particularly the involvement of CYP2C and CYP3A in indole-3-carbinol formation in female but not in male pigs. This is a very essential finding, suggesting the involvement of larger number of CYP450 isoforms in female pigs. On the other hand, indole-3-carbinol is a minor skatole metabolite, and the physiological significance of CYP2C and CYP3A involvement in its formation in female pigs, but not in male pigs, needs to be elucidated. Our results, however, should be interpreted with caution because of the low number of animals and possibility of breed and age effects on skatole metabolism.     

Interaction of P-glycoprotein with a hydrophobic component of rat urine.

The presence of an endogenous P-glycoprotein substrate in rat urine was examined by testing the ability of a hydrophobic extract to reverse multidrug resistance in CHO cells and to inhibit [3H]azidopine photolabelling. The accumulation of several hydrophobic drugs and dyes, known to be transported by P-glycoprotein, was dramatically enhanced in multidrug-resistant CHO cells (CHRC5) by a component contained in a hydrophobic extract prepared from rat urine by octadecyl (C18) reverse phase chromatography. The biological action of this urinary component involves a direct interaction with P-glycoprotein since it blocked photolabelling of the protein with [3H]azidopine. The effective concentration of the substance required to enhance drug accumulation and inhibit photolabelling was similar and within the range of its urinary content. These results suggest that a hydrophobic substance in urine may be an endogenous substrate of kidney P-glycoprotein.     

Metabolism of 4-hydroxyanisole: identification of major urinary excretory products

The human metabolism of 4-hydroxyanisole was investigated by the analysis of urine samples from melanoma patients treated with this substance. The samples were hydrolyzed with glucoronidase and/or arylsulphatase, extracted with ethyl acetate, and, after derivatization with pentafluoropropionylanhydride, analyzed by gas chromatography-mass spectrometry. We were able to identify peaks by their retention times and mass spectra corresponding to 4-hydroxyanisole, 3,4-dihydroxyanisole, and two of its o-methyl derivatives, namely, 3-hydroxy-4-methoxy- and 4-hydroxy-3-methoxyanisole. We also detected a peak of hydroquinone, which may have originated (at least partly) from 4-hydroxyanisole. All the above-mentioned compounds were excreted predominantly as sulphates and glucuronides. Only a small proportion of the substances was present in urine in an unconjugated form. Our results demonstrate that 3,4-dihydroxyanisole is the most important metabolite of 4-hydroxyanisole.     

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.     

Bovine adrenal cytochrome P-450(11 beta)-mediated conversion of 11-deoxycortisol to 18- and 19-hydroxy derivatives; structural analysis by 1H-NMR.

Incubation of 11-deoxycortisol with a cytochrome P-450(11β)-reconstituted system yielded, in addition to cortisol, several new steroid products. In this study, the structures of the three steroid products were elucidated. Retention time of the first product (Peak 2 substance) coincided with that of authentic 18-hydrocortisol on reverse phase HPLC. To further confirm the chemical identity of this product, the purified sample was subjected to ¹H-NMR analysis. The spectrum was essentially identical to that of 18-hydrocortisol. The retention time of the second product (Peak 3 substance) did not coincide with those of commonly occuring steroids. The one- and two-dimension ¹H-NMR spectra provided strong evidence for its structure of 19-hydroxy-11-deoxycortisol. The retention time of the third product (Peak 4 substance) did not coincide with those of commonly occuring steroids. The ¹H-NMR spectrum showed the presence of signals of 19-CH₃ and 18-CH₂ protons. There was also evidence that this product is not hydroxylated at the 11-position. Further analysis of the COSY spectra identified its structure as 18-hydroxy-11-depxycortisol. From these results, we conclude that bovine P-450(11β)deoxycortisol can catalyze the hydroxylation of 11-deoxycortisol at 11β-18- and 19-positions and produce cortisol, 18-hydroxy-11-deoxycortisol, 18-hydroxycortisol and 19-hydroxy-11-deoxycortisol.     

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.     

Metabolism and toxicological detection of the designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) in rat urine using gas chromatography-mass spectrometry

The amphetamine-derived designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) is an upcoming substance on the illicit drug market. In the current study, the identification of its metabolites in rat urine and their toxicological detection in the authors' systematic toxicological analysis (STA) procedure were examined. DOI is extensively metabolized by O-demethylation and beside small amounts of parent compound it was found to be excreted mainly in form of metabolites. The STA procedure using full-scan GC-MS allowed proving an intake of a common drug users' dose of DOI by detection of the two O-demethyl metabolite isomers in rat urine. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of DOI in human urine.     

An enzyme-bound intermediate in the biosynthesis of 3-hydroxy-3-methylglutaryl-coenzyme A

1. Purified 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast (free from acetoacetyl-CoA thiolase activity) catalysed an exchange of acetyl moiety between 3'-dephospho-CoA and CoA. The exchange rate was comparable with the overall velocity of synthesis of 3-hydroxy-3-methylglutaryl-CoA. 2. Acetyl-CoA reacted with the synthase, giving a rapid ;burst' release of CoA proportional in amount to the quantity of enzyme present. The ;burst' of CoA was released from acetyl-CoA, propionyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA) but not from acetoacetyl-CoA, hexanoyl-CoA, dl-3-hydroxy-3-methylglutaryl-CoA, or other derivatives of glutaryl-CoA. 3. Incubation of 3-hydroxy-3-methylglutaryl-CoA synthase with [1-(14)C]acetyl-CoA yielded protein-bound acetyl groups. The K(eq.) for the acetylation was 1.2 at pH7.0 and 4 degrees C. Acetyl-labelled synthase was isolated free from [1-(14)C]acetyl-CoA by rapid gel filtration at pH6.1. The [1-(14)C]acetyl group was removed from the protein by treatment with hydroxylamine, CoA or acetoacetyl-CoA but not by acid. When CoA or acetoacetyl-CoA was present the radioactive product was [1-(14)C]acetyl-CoA or 3-hydroxy-3-methyl-[(14)C]glutaryl-CoA respectively. 4. The isolated [1-(14)C]acetyl-enzyme was slowly hydrolysed at pH6.1 and 4 degrees C with a first-order rate constant of 0.005min(-1). This rate could be stimulated either by raising the pH to 7.0 or by the addition of desulpho-CoA. 5. These properties are interpreted in terms of a mechanism in which 3-hydroxy-3-methyl-glutaryl-CoA synthase is acetylated by acetyl-CoA to give a stable acetyl-enzyme, which then condenses with acetoacetyl-CoA yielding a covalent derivative between 3-hydroxy-3-methylglutaryl-CoA and the enzyme which is then rapidly hydrolysed to free enzyme and product.     

Metabolism of Indole-3-Acetic Acid: II. Oxindole Pathway in Parthenocissus tricuspidata Crown-Gall Tissue Cultures

An indoleacetic acid oxidase preparation from an acetone powder of Parthenocissus tricuspidata crown-gall tissue has been examined. An intermediate in the reaction is 3-hydroxymethyloxindole and nonenzymic conversion of it to 3-methyleneoxindole was observed. Neither reaction mixtures nor 3-methyleneoxindole have any auxin-like activity in Avena or wheat coleoptile bioassays. In vivo studies show that although 53% decarboxylation of indoleacetic acid was observed in 48 hours, only a small amount of 3-methyloxindole could be recovered from the medium. The other decarboxylated products remain to be identified but are not 3-hydroxymethyloxindole or 3-methyleneoxindole.     

The identification of 17 -hydroxy-17-methyl-1,4-androstadien-3-one as a metabolite of the anabolic steroid drug 17 -hydroxy-17-methyl-1,4-androstadien-3-one in man

The more polar of the two major urinary metabolites of methandrostenolone, 17β-hydroxy-17-methyl-1,4-androstadien-3-one, in man has already been identified as 6β-hydroxymethandrostenolone, 6β, 17β-dihydroxy-17-methyl-1,4-androstadien-3-one. The other metabolite has now been identified as the 17-epimer of methandrostenolone, 17α-hydroxy-17-methyl-1,4-androstadien-3-one. The compound was isolated from the freely extractable neutral fraction of urine following the administration of 5 mg of the drug to normal men. The relevant chromatographic fractions from thin layer and gas liquid systems were identified by carbon skeleton chromatography. The 17-epimer has been synthesised, details of which are included, and the previously unidentified metabolite was found to be identical with the synthetic compound.     

Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.     

Degradation of 3-phenylbutyric acid by Pseudomonas sp.

Pseudomonas sp. isolated by selective culture with 3-phenylbutyrate (3-PB) as the sole carbon source metabolized the compound through two different pathways by initial oxidation of the benzene ring and by initial oxidation of the side chain. During early exponential growth, a catechol substance identified as 3-(2,3-dihydroxyphenyl)butyrate (2,3-DHPB) and its meta-cleavage product 2-hydroxy-7-methyl-6-oxononadioic-2,4-dienoic acid were produced. These products disappeared during late exponential growth, and considerable amounts of 2,3-DHPB reacted to form brownish polymeric substances. The catechol intermediate 2,3-DHPB could not be isolated, but cell-free extracts were able only to oxidize 3-(2,3-dihydroxyphenyl)propionate of all dihydroxy aromatic acids tested. Moreover, a reaction product caused by dehydration of 2,3-DHPB on silica gel was isolated and identified by spectral analysis as (--)-8-hydroxy-4-methyl-3,4-dihydrocoumarin. 3-Phenylpropionate and a hydroxycinnamate were found in supernatants of cultures grown on 3-PB; phenylacetate and benzoate were found in supernatants of cultures grown on 3-phenylpropionate; and phenylacetate was found in cultures grown on cinnamate. Cells grown on 3-PB rapidly oxidized 3-phenylpropionate, cinnamate, catechol, and 3-(2,3-dihydroxyphenyl)propionate, whereas 2-phenylpropionate, 2,3-dihydroxycinnamate, benzoate, phenylacetate, and salicylate were oxidized at much slower rates. Phenylsuccinate was not utilized for growth nor was it oxidized by washed cell suspensions grown on 3-PB. However, dual axenic cultures of Pseudomonas acidovorans and Klebsiella pneumoniae, which could not grow on phenylsuccinate alone, could grow syntrophically and produced the same metabolites found during catabolism of 3-PB by Pseudomonas sp. Washed cell suspensions of dual axenic cultures also immediately oxidized phenylsuccinate, 3-phenylpropionate, cinnamate, phenylacetate, and benzoate.     

Characteristics of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

A procedure for the preparation of rat liver microsomal fractions essentially devoid of contaminating lysosomes is described. When this preparation was examined by immunoblotting with a rabbit antiserum to rat 3-hydroxy-3-methylglutaryl-CoA reductase, a single band corresponding to an Mr of 100000 was observed. No evidence was found for glycosylation of rat liver-3-hydroxy-3-methylglutaryl-CoA reductase. Native rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase differs from the purified proteolytically modified species in that it displays allosteric kinetics towards NADPH.     

Identification of a unique sertoli cell steroid as 3α-hydroxy-4-pregnen-20-one (3α-dihyroprogesterone: 3α-DHP)

An allylic steroid produced from progesterone by rat Sertoli cells and which does not appear to have been described previously as a product of gonadal or adrenal tissues has been isolated and identified as 3α-hydroxy-4-pregnen-20-one (3α-dihydroprogesterone; 3α-DHP). 3α-DHP appears to be a reactive molecule which is easily oxidized or dehydrated and its identification was possible by a combination of microchemical procedures, derivative formation, specific enzyme reaction, TLC, GC, HPLC, spectrophotometry and mass spectrometry. The biological functions of this Sertoli cell steroid are not known, but it is suggested that 3α-DHP is more than a metabolic waste product because (a) its production rate varies with age and is highest at the onset of meiosis, and (b) there appear to be specific receptors for it in the testis.     

Mitochondrial metabolism of valproic acid

The beta-oxidation of valproic acid (2-propylpentanoic acid), an anticonvulsant drug with hepatotoxic side effects, was studied with subcellular fractions of rat liver and with purified enzymes of beta-oxidation. 2-Propyl-2-pentenoyl-CoA, a presumed intermediate in the beta-oxidation of valproic acid, was chemically synthesized and used to demonstrate that enoyl-CoA hydratase or crotonase catalyzes its hydration to 3-hydroxy-2-propylpentanoyl-CoA. The latter compound was not acted upon by soluble L-3-hydroxyacyl-CoA dehydrogenases from mitochondria or peroxisomes but was dehydrogenated by an NAD(+)-dependent dehydrogenase associated with a mitochondrial membrane fraction. The product of the dehydrogenation, presumably 3-keto-2-propylpentanoyl-CoA, was further characterized by fast bombardment mass spectrometry. 3-Keto-2-propylpentanoyl-CoA was not cleaved thiolytically by 3-ketoacyl-CoA thiolase or a mitochondrial extract but was slowly degraded, most likely by hydrolysis. The availability of 2-propylpentanoyl-CoA (valproyl-CoA) and its beta-oxidation metabolites facilitated a study of valproate metabolism in coupled rat liver mitochondria. Mitochondrial metabolites identified by high-performance liquid chromatography were 2-propylpentanoyl-CoA, 3-keto-2-propylpentanoyl-CoA, 2-propyl-2-pentenoyl- CoA, and trace amounts of 3-hydroxy-2-propylpentanoyl-CoA. It is concluded that valproic acid enters mitochondria where it is converted to 2-propylpentanoyl-CoA, dehydrogenated to 2-propyl-2-pentenoyl-CoA by 2-methyl-branched chain acyl-CoA dehydrogenase, and hydrated by enoyl-CoA hydratase to 3-hydroxy-2-propylpentanoyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one in the rabbit

An acidic metabolite, 2α-carboxy-5α-androstane-3α, 16α, 17αtriol and two neutral metabolites, 2α-hydroxymethyl-5α-androstane-3α, 17α-diol, and 2α-hydroxymethyl-5α-androstane-3α, 16α, 17α-triol have been identified in the urine of rabbits orally dosed with 17β-hydroxy-2-hydroxymethylene-5α-androstan-3-one. 2α-Hydroxymethyl-5α-androstane-3α, 16α, 17α-triol was previously obtained from the urine of rabbits dosed with 17β-hydroxy-2α-methyl-5α-androstan-3-one. The acidic metabolite was the major urinary excretion product.