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The metabolism of some xenobiotic compounds when incubated with strains of intestinal bacteria has been studied. The reactions included: 1) glucuronide hydrolysis, 2) glucoside hydrolysis, 3) sulphate ester hydrolysis, 4) glycine conjugate hydrolysis, 5) N-acetyl derivative hydrolysis, 6) amide hydrolysis, 7) ester hydrolysis, 8) dehydroxylation, 9) decarboxylation, 10) double bond reduction, 11) nitro reduction, 12) azo reduction and 13) heterocyclic ring scission. The results showed that strains of the following test microorganisms were able to carry out the indicated reactions to varying degrees: Str. faecalis (4, 11, 12), E. coli (1, 11, 12), Lactobacillus (2, 10, 11, 12), Bacillus (9, 11, 12), Ps. aeruginosa (12), Pr. vulgaris (2, 10, 11, 12), A. aerogenes (2, 3, 4, 5, 9, 11, 12, 13), Clostridium (10, 11, 12) and Bacteroides (1, 11, 12). These results are discussed in relation to earlier findings on the metabolism of xenobiotics by members of the intestinal microflora.

The metabolism of the 3,4-methylenedioxy derivatives of mandelic acid (1), phenylacetic acid (2), benzoic acid (3), 3-phenylpropionic acid (4) and cinnamic acid (5) was studied in rats. Following intragastric dosage (1 mmol/kg) the compounds and their metabolites were excreted in the urine within 24 hrs. Recoveries of roughly 85% were obtained. Except for compound (1) which was excreted to a large extent unchanged, glycine conjugates were the major urinary metabolites. Compound (2) formed 3,4-methylenedioxyphenylacetylglycine whereas compounds (3), (4) and (5) were converted to 3,4-methylenedioxybenzoylglycine. No evidence was found with any of the compounds for demethylenation and subsequent excretion of catecholic metabolites.

The metabolism of p-tert.-butyltoluene (TBT) was studied in the rat and guinea pig. Both the methyl and the tert.-butyl group were oxidized to alcohol and carboxylic acid derivatives in these species. The major urinary metabolites in rats were p-tert.-butylbenzoic acid and its alcohol derivative 2-(p-carboxyphenyl)-2-methylpropan-1-ol whereas p-tert.-butylbenzoylglycine was the most prominent metabolite in guinea pig urine. No significant differences in metabolism were found when TBT was given intragastrically or by inhalation. The intragastric administration of 14C-TBT to rats showed that the bulk of the excretion of radioactivity occurred within three days. A recovery of 83% was achieved and the ratio of urinary/faecal radioactivity was roughly 3.5:1.

The urinary metabolites of safrole (4-allyl-1,2-methylenedioxybenzene) in the rat were identified using gas chromatographic - mass spectrometric methods. The amounts of the individual metabolites excreted were determined gas chromatographically. Metabolite excretion was 93% in 72 hrs and most of this material (86%) consisted of metabolites formed via demethylenation of the methylenedioxy moiety. The other metabolic routes observed were allylic hydroxylation and the epoxide-diol pathway.

1. The metabolites of 3,4-dimethoxyallylbenzene (eugenol methyl ether) and 3,4-dimethoxypropenylbenzene (isoeugenol methyl ether) in the rat were identified and quantitatively determined by g.l.c. and g.l.c.-mass spectrometry. 2. The major metabolic reactions of 3,4-dimethoxyallylbenzene were oxidation of the allylic side chain to 2-hydroxy-3-(3,4-dimethoxyphenyl)-propionic acid, 3,4-dimethoxybenzoic acid and 3,4-dimethoxycinnamic acid, the two latter being largely excreted as their glycine conjugates. The formation of the hydroxy acid presumably involved epoxidation of the double bond and subsequent hydration to the diol whereas the formation of 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid involved migration of the double bond and the formation of cinnamoyl intermediates. Other reactions were O-demethylation to 4-hydroxy-3-methoxyallylbenzene (eugenol) and 3-hydroxy-4-methoxyallylbenzene in equal amounts, oxidation to 1-(3,4-dimethoxyphenyl)-2-propen-1-ol, hydroxylation of the benzene ring to a hydroxy-3,4-dimethoxyallylbenzene and oxidation to 3,4-dimethoxyphenylacetic acid. The formation of 1-(3,4-dihydroxyphenyl)propane was found to be carried out by the rat intestinal micro-organisms. A total of at least 63% but as much as 95% dose was accounted for. 3. The major metabolic pathway of 3,4-dimethoxypropenylbenzene was via the cinnamoyl derivatives, leading to the formation of 4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3,4-dimethoxycinnamic acid and 3,4-dimethoxybenzoic acid, the two latter being excreted largely as their glycine conjugates. Other reactions were O-demethylation to 4-hydroxy-3-methoxypropenylbenzene (isoeugenol) and 3-hydroxy-4-methoxypropenylbenzene in equal amounts, and oxidation to 3,4-dimethoxyphenylacetic acid and 4-hydroxy-3-methoxyphenylacetone. Epoxidation of the side chain appeared to be a minor metabolic reaction with the propenyl derivative. 4. The biliary metabolites of 3,4-dimethoxyallylbenzene and 3,4-dimethoxypropenylbenzene were identified and most of the urinary metabolites were also found in the bile.

1. The metabolism of 4-(4-hydroxy-3-methoxyphenyl)butan-2-one (zingerone), a pungent principle of ginger, has been investigated in rats. 2. Oral or intraperitoneal dosage (100mg/kg) of zingerone resulted in the urinary excretion of most metabolites within 24 h, mainly as glucuronide and/or sulphate conjugates. While zingerone itself accounted for roughly 50-55% of the dose, reduction to the corresponding carbinol (11-13%) also occurred. Side chain oxidation took place at all three available sites and oxidation at the 3-position, giving rise to C6-C2 metabolites, predominated. About 95-97% of the dose was accounted for. 3. Appreciable (40% in 12 h) biliary excretion occurred. Biliary studies and studies in vitro using caecal micro-organisms indicated that several O-demethylated metabolites found in the urine are of bacterial origin.

The urinary metabolites of isosafrole (1,2-methylenedioxy-4-propenylbenzene) and dihydrosafrole (1,2-methylenedioxy-4-propylbenzene) in the rat were identified using gas chromatographic mass spectrometric methods. Additionally, the amounts of the individual metabolites excreted were determined gas chromatographically. Metabolite excretion was 89% (isosafrole) and 97% (dihydrosafrole) of the dose in 72 h. Although isosafrole was metabolized by allylic hydroxylation and via the epoxide-diol pathway, demethylenation leading mainly to 1,2-dihydroxy-4-propenylbenzene was by far the most prominent reaction. This was similarly true with dihydrosafrole which was metabolized mainly to 1,2-dihydroxy-4-(1-propyl) benzene. The total amount of demethylenated metabolites formed were 92% (isosafrole) and 95% (dihydrosafrole) of the identified material.

The urinary metabolites of flavanone in the rate were investigated using a gas chromatographic mass spectrometric method employing an OV-1 capillary column. Forty-three metabolites were detected and the most prominent of these were identified. The most common metabolic reactions were reduction of the keto group and hydroxylation at the 3- or 6-positions. Little hydroxylation occurred in ring B. The major metabolites were flavan-4 alpha-ol, trans-3-hydroxyflavan-4 beta-ol, 6-hydroxyflavanone and 6-hydroxyflavan-4 beta-ol. Gas chromatography analysis of underivatized flavanone metabolites, especially with stainless steel columns, results in the formation of dehydrated products including flavone, flav-3-ene and flavanone itself.

The conversion of cyclohexanecarboxyl-CoA to hippuric acid in submitochondrial fractions from guinea pig liver was studied using a gas chromatographic-mass spectrometric method employing selected ion monitoring. Comparison of the activities of the cyclohexanecarboxyl-CoA to hippuric acid converting system (CCoAHC-system) and marker enzymes in the various submitochondrial fractions showed that the CCoAHC-system is localized in the mitochondrial matrix. Partial separation of the inner and outer membranes has been accomplished by treating mitochondria with digitonin in isotonic medium and fractionating the treated mitochondria by differential centrifugation. A digitonin-protein ratio of 2.6 mg of digitonin/10 mg of protein must be used in order to release significant amounts of amine oxidase activity (outer membrane marker) from low speed mitochondrial pellets. This pellet still contained most of the glutamate dehydrogenase activity and was insignificantly contaminated with adenylate kinase. Moderate concentrations of phenazine methosulfate (PMS) greatly stimulated the activity of the CCoAHC-system, even in intact mitochondria (optimal concentration of PMS: 1 mM) whilst higher concentrations (greater than 1 mM) decreased the activity. The formation of hippuric acid in these mitochondrial preparations was linear with time for at least 40 min and linear with respect to protein concentration up to approximately 2.0 mg mitochondrial protein X ml-1.

The ability to convert cyclohexanecarboxylic acid to hippuric acid has been studied in liver from guinea pigs, rabbits, rats and mice using a gas chromatographic - mass spectrometric method employing selected ion monitoring. Guinea pig liver showed the highest activity, giving values double of those found in rabbit liver and five times those in rat liver. Only very weak activity was found in mouse liver. (Hydroxymethyl)cyclohexane, cyclohexanealdehyde and alpha-hydroxyethylcyclohexane, which are structurally related to cyclohexanecarboxylic acid but lack the carboxyl group, were not aromatized by guinea pig liver mitochondria. This finding indicates that the carboxyl group is essential for aromatization. Absence of aromatization was also found with the homologs cyclohexaneacetic acid and cyclohexanepropionic acid and with the di-acids trans-1,2- and trans-1,4-cyclohexanedicarboxylic acid. The effect of a methyl group in cyclohexanecarboxylic acid depended on its position. 2-Methyl-1-cyclohexanecarboxylic acid was not aromatized, however the 3- and 4-methyl derivatives underwent aromatization and subsequent conjugation with glycine. The rates of formation of m-methyl- and p-methylhippuric acid were 16% and 9%, respectively, of that found for hippuric acid from cyclohexanecarboxylic acid (8.0 nmol/min/mg protein).

1. The metabolites of elemicin (3,4,5-trimethoxyallylbenzene) and isoelemicin (3,4,5-trimethoxypropenylbenzene) in the rat were identified by g.l.c.-mass spectrometry. 2. The major metabolic reactions of elemicin follow the cinnamoyl pathway or the epoxide-diol pathway. The former route gives 3-(3,4,5-trimethoxyphenyl)propionic acid and its glycine conjugate as major urinary metabolites, whereas 3-(3,4,5-trimethoxyphenyl)propane-1,2-diol is the most prominent metabolite of the latter route. Small amounts of the epoxide of the 3-O-demethylated derivative of elemicin were identified in the urine. 3. Isoelemicin was metabolized by both aforementioned pathways; the cinnamoyl pathway predominated and 3-(3,4,5-trimethoxyphenyl)propionic acid was the major urinary metabolite. 4. All of the acidic metabolites detected were C6--C3 derivatives and further oxidation to benzoic acid derivatives did not occur. 5. Most of the urinary metabolites were also found in the bile, but in different relative amounts.

1. The metabolism of p-cymene was studied in rats and guinea-pigs.2. Following intragastric or inhalation dosage (100mg/kg) urinary metabolite excretion was nearly complete within 48h, amounting to 60–80% dose. The inhalation experiments gave the lowest values.3. 18 urinary metabolites were detected and identified. Of these, rats did not excrete two and guinea-pigs did not excrete a third.4. No ring-hydroxylation of p-cymene was detected in rats, but guinea-pigs formed small amounts of carvacrol and hydroxycarvacrol.5. Oxidation of both the methyl and isopropyl groups of p-cymene occurred extensively in both species. The following types of metabolites were formed: monohydric alcohols, diols, mono- and di-carboxylic acids and hydroxyacids. Conjugation with glycine of the cumic acid formed was extensive in guinea-pigs.

The metabolism of carvacrol and thymol in rats was studied using gas chromatographic-mass spectrometric methods. The urinary excretion of metabolites was rapid. Only very small amounts were excreted after 24 hrs. Although large quantities of carvacrol and, especially, thymol were excreted unchanged (or as their glucuronide and sulphate conjugates), extensive oxidation of the methyl and isopropyl groups also occurred. This resulted in the formation of derivatives of benzyl alcohol and 2-phenylpropanol and their corresponding carboxylic acids. In contrast, ring hydroxylation of the two phenols was a minor reaction.

1. The metabolism of o-methoxycinnamaldehyde (1·3 mmol/kg, intragastrically) was studied in rats.2. Identification of the urinary metabolites by g.l.c.-mass spectrometry and quantification by h.p.l.c. showed that the major metabolic pathway (approx. two-thirds of the dose) was oxidation to the corresponding cinnamic and phenylpropionic acids (C6-C3 acids) which were largely excreted as glycine conjugates.3. Intermediate amounts (approx. 10% of the dose) of the O-demethylated C6-C3 acids were excreted.4. Relatively large amounts of the β-hydroxylated phenylpropionic acid derivative were found, however only traces of the further products of β-oxidation (2-methoxylated derivatives of benzoic and hippuric acid) were excreted.5. No evidence was obtained for conjugation of o-methoxycinnamaldehyde with glutathione.6. Urinary excretion of metabolites was rapid (91% in 24h and 98% in 48 h).

1. The metabolism of 3,4,5-trimethoxycinnamic acid and some related compounds in the rat has been investigated.2. The metabolic reactions observed were hydrogenation of the double bond, O-demethylation, p-dehydroxylation, dehydrogenation and O-methylation. In addition, the urinary metabolites were partly excreted as glucuronide and/or sulphate conjugates.3. Experiments with antibiotic-treated and germ-free rats showed that O-demethylation in the tissues occurred only at one of the meta-positions. Further O-demethylation and p-dehydroxylation to give the ultimate metabolite, 3,5-dihydroxyphenylpropionic acid, were dependent upon the activities of the intestinal micro-organisms.4. Interconversion of cinnamic and phenylpropionic acids has been demonstrated. Hydrogenation was mainly carried out by the intestinal microorganisms whereas dehydrogenation occurred only in the tissues.5. O-Methylation of 3,4,5-trihydroxyphenylpropionic acid occurred at both the para- and meta-positions. In the latter case further O...

Methods for conjugate hydrolysis and ether extraction of simple phenols in urine have been investigated. The losses of monohydric phenols could be compensated for by adding an internal standard and p - or m -methoxyphenol is suggested for this use. However, recoveries of some dihydroxybenzenes were considerably reduced and these losses were shown to occur during the removal of phenolic acids. Both acid and enzymic hydrolysis are recommended, as they do not yield the same quantities of all the phenols. Formation of simple phenols from phenolic acids was shown to occur during treatment with acid but was not significant with the quantities of naturally occurring phenolic acids encountered in the present experiments.

1. Hippuric acid (12·3% of the dose) and catechol (1·0% of the dose) were found as urinary metabolites when (-)-quinic acid (100 mg) was administered orally to rats. Neither protocatechuic acid nor vanillic acid was detected.2. No significant differences in the extent of urinary excretion of hippuric acid and catechol were found when the quinic acid was given at the end of two successive 24-day periods during which the rats were fed a purified diet containing 1% quinic acid.3. Incubation of quinic acid with rat caecal microorganisms resulted in the formation of benzoic acid under aerobic and anaerobic conditions. Catechol was not found in these incubates but small amounts of protocatechuic acid were detected when aerobic conditions were employed.

The hydroxylation of benzene, toluene, ethylbenzene, styrene, cumene, o -xylene, m -xylene, p -xylene, p -cymene, pseudocumene, and mesitylene was studied following their oral administration to rats. The animals were fed a purified diet containing neomycin in order to reduce the levels of normally occurring simple urinary phenols. Phenolic metabolites were quantitatively estimated in hydrolyzed urine samples by gas chromatography. The aromatic hydrocarbons administered at a dose of 100 mg/kg were metabolized to the following monohydric phenols (% of dose): benzene to phenol (3.7), toluene to o -cresol (0.04–0.11) and p -cresol (0.4–1.0), ethylbenzene to p -ethylphenol (0.3), styrene to 4-vinylphenol (0.1), o -xylene to 3,4-dimethylphenol (0.1) and very small amounts of 2,3-dimethylphenol, m -xylene to 2,4-dimethylphenol (0.9), p -xylene to 2,5-dimethylphenol (1.0), pseudocumene to 2,4,5-trimethylphenol (0.05), and mesitylene to 2,4,6-trimethylphenol (0.4). Cumene and p -cymene were not metabolized to phenols. The analytical methods allowed the detection of several alcoholic metabolites. The following compounds were shown to be urinary metabolites: benzyl alcohol from toluene, 1- and 2-phenylethanol from ethylbenzene and styrene, 2-phenyl-1-propyl alcohol and 2-phenyl-2-propyl alcohol from cumene, 2-methylbenzyl alcohol from o -xylene, and p-isopropylbenzyl alcohol from p -cymene. The possible significance of the formation of phenolic metabolites to the toxicity of aromatic hydrocarbons is briefly discussed.

1. The metabolism of some substituted cinnamic and phenylpropionic acids by mixed cultures of rat caecal micro-organisms has been investigated. The compounds studied were: 3,4,5-trimethoxy-, 4-hydroxy-3,5-dimethoxy- and 3,5-dimethoxycinnamic acid, and 3,4,5-trimethoxy-, 3-hydroxy-4,5-dimethoxy-, 4-hydroxy-3,5-dimethoxy-, 3,4-dihydroxy-5-methoxy-, 3,5-dihydroxy-4-methoxy-, 3,4,5-trihydroxy-, 3,5-dimethoxy- and 3-hydroxy-5-methoxy-phenyl-propionic acid.2. The metabolic reactions found to occur were hydrogenation of the double bond, O-demethylation and p-dehydroxylation. No decarboxylation was detected.3. The ultimate metabolic product was 3,5-dihydroxyphenylpropionic acid which was formed from all of the trihydric derivatives.4. The metabolic sequence from the trimethoxy derivatives to 3,5-dihydroxyphenylpropionic acid was not found to proceed along a single pathway. However, the major pathway appears to involve an initial meta-O-demethylation followed by para-O-demethylation and, then after the ultimate O-...

1. The metabolism by rat caecal micro-organisms of several benzaldehyde and benzyl alcohol derivatives containing hydroxyl and/or methoxyl groups has been investigated.2. The benzaldehyde derivatives were found to be reduced to the corresponding alcohols and oxidized to the corresponding acids. The former reaction was predominant and the oxidative pathway could be selectively reduced by using aged cultures or antibiotic treatment.3. Several of the benzaldehyde derivatives were metabolized to compounds suggested to be 1,1-diols (aldehyde hydrates).4. The benzyl alcohol derivatives containing a r-hydroxyl group or those capable of obtaining such a group through O-demethylation were reduced to the corresponding toluene derivatives.

1. The metabolism of 2′,4′,4-, 2′,6′,3- and 2′,6′,4-trihydroxydihydrochalcone was studied in rats2. Approx. 25–35% of the oral-dose (0.75 mmol/kg) was excreted in the urine during a five to six day period.3. The unchanged compounds were the most prominent urinary excretion products and were also present in large amounts in the faeces.4. Other metabolites of the three dihydrochalcones included minor amounts of hydroxylated products and subsequent O-methylated derivatives as well as minor amounts of degradation products (resorcinol and hydroxyphenylpropionic acids) arising from scission of the compounds by the normal gut microflora.5. The present results support the view that the degradative metabolism of dihydrochalcones involves scission leading to equal amounts of products derived from the A- and B-ring regions of these flavonoids.

Metabolites present in the urine and bile of coalfish (Pollachius virens) 48 hrs after the intragastric administration of phenanthrene (75 mg/kg) were studied. Using gas chromatographic-mass spectrometric methods small amounts of unchanged compound, all five of the possible monohydroxy derivatives and the 1,2- and 9,10-dihydrodiols of phenanthrene were identified inthese samples. The major metabolite detected was phenanthrene-1,2-dihydrodiol which was excreted mainly as glucuronide and/or sulfate conjugates.

The metabolism of phloretin (2',4',6',4-tetrahydroxydihydrochalcone) was studied in rats. Approx. half of the intragastric dose (0.75 mmol/kg) was excreted in the urine, mainly within two days. Small initial amounts of phloretin were found, however most of the metabolites were degradation products. The latter included phloroglucinol and, in larger amounts, phloretic acid and related metabolites formed by its dehydrogenation, beta-oxidation and glycine conjugation. Phloroglucinol, administered in similar experiments, was rapidly (90% within 24 h) excreted in the urine, either unchanged or as conjugates (glucuronide/sulphate). Incubation of phloretin and its glucoside phloridzin with rat-caecal micro-organisms resulted in the formation of phloroglucinol and phloretic acid. The degradative pathways of metabolism of dihydrochalcones and other flavonoids are discussed.

1. The metabolism of 4-(4-hydroxyphenyl)butan-2-one(raspberry ketone) was studied in rats, guinea-pigs and rabbits. 2. Following intragastric dosage (1 mmol/kg) urinary metabolite excretion was nearly complete within 24 h, amounting to roughly 90% of the dose in all species. 3. The most prominent urinary metabolites were raspberry ketone and its corresponding carbinol, both largely conjugated with glucuronic acid and/or sulphate. The extent of ketone reduction was greatest in rabbits. 4. Oxidative metabolism included ring hydroxylation and side-chain oxidation. The latter pathway led to 1,2- and 2,3-diol derivatives. It is proposed that the latter undergo cleavage to furnish the C6-C3 and C6-C2 derivatives detected.

1. The metabolism in rats of several kava pyrones from Piper methysticum Forst. was studied. The compounds were the 5,6-dihydro-alpha-pyrones, dihydrokawain, kawain and methysticin, and the alpha-pyrones, 7,8-dihyroyangonin and yangonin. 2. Approx. half the dose (400 mg/kg, p.o.) of dihydrokawain was found in the urine in 48 h. About two-thirds of this was hydroxylated metabolites (three mono- and three di-hydroxylated derivatives), of which p-hydroxydihydrokawain was the most abundant. The remaining third consisted of metabolites formed by scission of the 5,6-dihydro-alpha-pyrone ring and included hippuric acid (9--13% dose). 3. Lower amounts of urinary metabolites were excreted when kawain was given, but both hydroxylated and ring-opened products were formed. 4. Methysticin gave rise to only small amounts of two urinary metabolites formed by demethylenation of the methylenedioxyphenyl moiety. 5. Urinary metabolites of the alpha-pyrones, 7,8-dihydroxyangonin and yangonin, were formed via omicron-demethylation. No ring-opened products were detected. 6. These lipophilic kava pyrones have extremely low solubility in water, which would be expected to reduce their absorption rates and appears to be responsible for the variable and low extent of metabolism observed.

The metabolic disposition of 14C–biphenyl in the rat was studied by liquid scintillation counting. The rats were given an oral dose of 14C–biphenyl (100 mg/kg, 0.7–1.0 μci) and the total excretion of radioactivity after 96 hrs was 92.2 % of the dose. Urinary excretion accounted for 84.8 % and faecal excretion for 7.3% of the dose. Most of this radioactivity, 75.8 % and 5.8% respectively, was excreted within 24 hrs. Only trace amounts of 14CO2 were detected in the expired air and 0.6% of the dose was found to be still present in the rats 96 hrs after biphenyl administration. Extraction and fractionation of the 24 hrs urine samples showed that the largest fraction (nearly 30% of the dose) consisted of conjugated phenolic metabolites. Acidic metabolites accounted for a quarter of the dose and the low levels of expired 14CO2 indicated that these were not products resulting from extensive degradation and decarboxylation.

1. The metabolism of several alkyl- and alkoxy-substituted pyrazines in the rat has been investigated. 2. Alkyl substituted compounds were oxidized to the corresponding acids which were excreted in the urine as such or as their glycine cojugates. The extent of oxidation was reduced when two adjacent alkyl groups were present. In the latter case ring hydroxylation also occurred. Methoxy-substituted pyrazines underwent O-demethylation and ring hydroxylation. 3. Little or no biliary excretion of the pyrazines or their metabolites occurred. 4. Some preliminary results on the metabolism of 2-isobutyl-3-methoxy-pyrazine (the major characteristic flavour component of bell pepper) have been obtained. 5. For comparative purposes the metabolism of some similarly substituted pyridines was investigated.

The degradation of Fast Red E, Amaranth, Ponceau 4R and Ponceau 6R in anaerobic incubates of rat caecal extract was studied by spectrophotometric scanning. The compounds underwent reduction of the azo linkage and thus loss of colour. The reaction rates seem to depend on the number rather than position of the sulphonate groups in the molecule.

1. The metabolism of the plant ketones acetovanillone (4-hydroxy-3-methoxyacetophenone) and paeonol (2-hydroxy-4-methoxyacetophenone), was studied in rats. Identification and quantification of metabolites was carried out by g.l.c.-mass spectometry and g.l.c., respectively, following intragastric doses of 1 mmol/kg. 2. Acetovanillone was rapidly excreted in the urine, mainly unchanged but also as the demethylated compound and three ring-hydroxylated metabolites. Minor additional metabolic pathways produced the para-methoxy derivative, acetoisovanillone, a dimethoxy-hydroxy derivative and two 1-phenylethanol derivatives formed by ketone reduction of acetovanillone and 3,4-dihydroxyacetophenone. 3. Paeonol was metabolized more extensively and larger amounts of the demethylated (resacetophenone) and hydroxylated (mainly 2,5-dihydroxy-4-methoxyacetophenone and a trihydroxyacetophenone) metabolites were excreted. Paeonol was not found to undergo ketone reduction, however, small amounts of the hydroxymethyl derivative formed by omega-oxidation were detected. 4. The metabolites were excreted mainly as glucuronide and/or sulphate conjugates. Faecal recoveries of metabolites were very small and the urinary excretion (48 h) was 97% (acetovanillone) and 61% (paeonol).

The phenolic metabolites of biphenyl in both male and female pigs were qualitatively and quantitatively analysed as trimethylsilyl (TMS) ethers by combined gas chromatography/mass spectrometry and gas chromatography, respectively. This xenobiotic was metabolized mainly to mono–hydroxylated biphenyl, and in small amounts to di– and tri–hydroxylated biphenyls. Urinary excretion was the only route of elimination of these compounds from the body. The total urinary recovery in male pigs was 44.8 % of the dose 96 hrs after administration of the drug, and the corresponding value in female pigs was 27.6%. The main part of the urinary metabolites was excreted during the first 24 hrs after dosing in both sexes.

A method for the assay of uridine diphosphate (UDP)-glucuronosyltransferase activities toward some phenolic compounds and monoterpenoid alcohols is described. The method is based on the disappearance of the free substrate after incubation with microsomes and UDP-glucuronate. This disappearance is recorded using a gas chromatographic process. This method has been used, for example, to characterize the glucuronidation process of eugenol (4-allyl-2-methoxyphenol). The method could be extended to other substrates. Analytical conditions are given for some of them, especially monoterpenoid alcohols since the studies of their conjugations are a growing field of interest in evaluation of heterogeneity of UDP-glucuronosyltransferase. The method could also be used with other biological materials including cell suspension and crude liver biopsies.

1. The metabolism of vanillin, isovanillin and the corresponding alcohols and acids in rats was investigated using t.l.c., g.l.c. and combined g.l.c.-mass spectrometry. 2. Oral dosage (100 mg/kg) of the aldehyde resulted in urinary excretion of most metabolites within 24 h, mainly as glucuronide and/or sulphate conjugates although the acids formed were also excreted free and as their glycine conjugates. In 48 h 94% of the dose of vanillin was accounted for as follows (%) : vanillin (7), vanillyl alcohol (19), vanillic acid (47), vanilloylglycine (10), catechol (8), 4-methylcatechol (2), guaiacol (0-5) and 4-methylguaiacol (0-6). Similarly, 89% of the dose of isovanillin was accounted for as follows: isovanillin (19), isovanillyl alcohol (10), isovanillic acid (22), vanillic acid (11), isovanilloylglycine (19), catechol(7) and 4-methylcatechol (1). Protocatechuic acid was also formed from both aldehydes. 3. By means of (a) investigation of biliary metabolites, (b) prevention of biliary excretion, (c) suppression of intestinal bacteria with neomycin sulphate and (d) inhibition of intestinal beta-glucuronidase with saccharo-1,4-lactone, it was found that glucuronides of the aldehydes and their respective alcohol and acid derivatives are excreted in the bile and that the conjugates are metabolized by the intestinal bacteria to toluene derivatives and decarboxylated products.

The conversion of cyclohexanecarboxylic acid to hippuric acid in subcellular fractions from guinea pig liver was studied using a gas chromatographic-mass spectrometric method employing selected ion monitoring. Comparison of the activities of the cyclohexanecarboxylic acid to hippuric acid converting system (CHC-system) and marker enzymes in the various subcellular fractions showed that the CHC-system is localized exclusively in the mitochondria. No contribution to the total activity of the system was made by microsomal enzymes. The activity of the CHC-system in whole liver homogenate and in isolated mitochondria was similar when the latter were supplemented with ATP, alpha-ketoglutaric acid, glycine, ethylenediaminetetraacetate, PO4(3-) and Mg2+. The formation of hippuric acid in these mitochondrial preparations was linear with respect to time over a period of at least 60 min. Studies designed to optimize the incubation conditions showed that the activity of the CHC-system was reduced by PO4(3-) concentrations greater than approximately 70 mM. Conversely, both ATP and alpha-ketoglutaric acid stimulated the system. It is possible that two different types of acyl-CoA synthetases, one which is ATP-specific and one which is GTP-specific, may operate in the activation of cyclohexanecarboxylic acid.

1. The metabolism of umbelliferone (7-hydroxycoumarin) and herniarin (7-methoxycoumarin) was studied in rats after oral and intraperitoneal administration.2. Umbelliferone was excreted in the urine partly unchanged but mainly as a glucuronide and/or sulphate conjugate. It was also metabolized to 2,4-dihydroxyphenylpropionic acid and, following intraperitoneal dosage, to a very small amount of 3,7-dihydroxycoumarin.3. Herniarin was metabolized to the following unconjugated urinary metabolites: 2-hydroxy-4-methoxyphenylacetic acid (major metabolite), 2-hydroxy-4-methoxyphenylpropionic acid, 2,4-dihydroxyphenylpropionic acid, 2-hydroxy-4-methoxyphenyllactic acid and umbelliferone (trace). A trace of unchanged compound was excreted. The conjugated metabolites excreted were umbelliferone, 3,7-dihydroxycoumarin, 3-hydroxy-7-methoxycoumarin, isoscopoletin and two unidentified metabolites.4. The metabolism of umbelliferone and herniarin and a few related compounds by the rat caecal microflora was investigated. Um...

Rat caecal contents decarboxylate phenolic benzoic acid derivatives when a free hydroxyl group is in the para position but the presence of substituents adjacent to this group or the carboxyl group reduce or abolish the reaction. Compounds containing a hydroxyl group in the ortho or meta position but lacking one in the para position are not decarboxylated. Some methoxy-derivatives are demethylated. The possible relationship between these findings and urinary phenols is discussed.

Summary Streptococcus faecalis , isolated from rat faeces, has been shown to reduce the azo dye, acid yellow, to its component amines, and the properties of the enzyme system effecting this reduction have now been studied. Acid yellow was anaerobically incubated, under various conditions, with the supernatant fraction from sonically disrupted cells of S. faecalis . Reduction was slight when the dye was incubated with the supernatant fraction alone, but was extensive in the presence of NADPH- or, particularly NADH-generating systems. Riboflavin, FMN and FAD each markedly stimulated the reducing activity of the supernatant fraction, while the reduction was considerably depressed by the flavin inhibitor, quinacrine. Flavoproteins are thus implicated in the reduction of acid yellow by S. faecalis extracts, but the study suggests that more than one system may be involved.