Metabolism of aromatic plant ketones in rats: acetovanillone and paeonol.

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).

Metabolism in rats of p-cymene derivatives: carvacrol and thymol.

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.

Metabolism of the cinnamon constituent o-methoxycinnamaldehyde in the rat.

The metabolism of o-methoxycinnamaldehyde (1.3 mmol/kg, intragastrically) was studied in rats. 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. Intermediate amounts (approx. 10% of the dose) of the O-demethylated C6-C3 acids were excreted. Relatively large amounts of the beta-hydroxylated phenylpropionic acid derivative were found, however only traces of the further products of beta-oxidation (2-methoxylated derivatives of benzoic and hippuric acid) were excreted. No evidence was obtained for conjugation of o-methoxycinnamaldehyde with glutathione. Urinary excretion of metabolites was rapid (91% in 24 h and 98% in 48 h).

Dihydrochalcone metabolism in the rat: trihydroxylated derivatives related to phloretin.

The metabolism of 2',4',4-, 2',6',3- and 2',6',4-trihydroxydihydrochalcone was studied in rats. Approx. 25-35% of the oral dose (0.75 mmol/kg) was excreted in the urine during a five to six day period. The unchanged compounds were the most prominent urinary excretion products and were also present in large amounts in the faeces. 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. 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.

The aromatization of cyclohexanecarboxylic acid to hippuric acid: substrate specificity and species differences.

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).

The aromatization of cyclohexanecarboxyl-CoA to hippuric acid by guinea pig liver mitochondria: submitochondrial localization.

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.

Intracellular localization and some properties of the system in guinea pig liver responsible for the aromatization of cyclohexanecarboxylic acid to hippuric acid.

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.

Dihydrochalcone metabolism in the rat: phloretin.

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.

Studies of UDP-glucuronosyltransferase activity toward eugenol, using a gas chromatographic method of measurement.

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.

p-Cymene metabolism in rats and guinea-pigs.

The metabolism of p-cymene was studied in rats and guinea-pigs. Following intragastric or inhalation dosage (100 mg/kg) urinary metabolite excretion was nearly complete within 48 h, amounting to 60-80% dose. The inhalation experiments gave the lowest values. 18 urinary metabolites were detected and identified. Of these, rats did not excrete two and guinea-pigs did not excrete a third. No ring-hydroxylation of p-cymene was detected in rats, but guinea-pigs formed small amounts of carvacrol and hydroxycarvacrol. 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.

Metabolism of p-tert.-butyltoluene in the rat and guinea pig.

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.

Metabolism of safrole in the rat.

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.

Metabolism of isosafrole and dihydrosafrole in the rat.

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 metabolism of 4-(4-hydroxyphenyl)butan-2-one (raspberry ketone) in rats, guinea-pigs and rabbits.

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.

Metabolism in rats of several carboxylic acid derivatives containing the 3,4-methylenedioxyphenyl group.

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.

Metabolism of alkenebenzene derivatives in the rat. III. Elemicin and isoelemicin.

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.

Urinary and biliary metabolites of phenanthrene in the coalfish (Pollachius virens).

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.