Ronald R. Scheline

Hydroxylation of aromatic hydrocarbons in the rat

Abstract 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.

Metabolism of Alkenebenzene Derivatives in the Rat. II. Eugenol and Isoeugenol Methyl Ethers

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.

Decarboxylation and demethylation of some phenolic benzoic acid derivatives by rat caecal contents.

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.

Metabolism of Alkenebenzene Derivatives in the Rat I. p-Methoxyallylbenzene (Estragole) and p-Methoxypropenylbenzene (Anethole)

Abstract1. The metabolism of p-methoxyallylbenzene (estragole) and p-methoxy-propenylbenzene (anethole) in the rat has been investigated. The metabolites were identified and quantitatively determined mainly by g.l.c. and by combined g.l.c.-mass spectrometry.2. The major metabolic reactions of p-methoxyallylbenzene were found to be O-demethylation to p-hydroxyallylbenzene and oxidation of the allylic side chain to p-hydroxy-3-(p-methoxyphenyl)propionic acid or to p-methoxybenzoic acid (anisic acid) which was largely excreted as p-methoxyhippuric acid. The former oxidative pathway involved epoxidation of the double bond and subsequent hydration to the diol whereas the latter pathway involved migration of the double bond and the formation of cinnamoyl intermediates. Other reactions gave rise to 1-(p-methoxyphenyl)allyl alcohol and to p-methoxyphenylacetic acid and its glycine conjugate. A total of at least 60–65% but as much as 90% dose was accounted for.3. A major metabolic reaction of p-methoxypropenylbenz...

The Metabolism of Vanillin and Isovanillin in the Rat

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.

Metabolism in the Rat of Some Pyrazine Derivatives having Flavour Importance in Foods

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.

Enzymatic reduction of the azo dye, acid yellow, by extracts of Streptococcus faecalis isolated from rat intestine

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.

Metabolism of Some Kava Pyrones in the Rat

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 Metabolism of Zingerone, a Pungent Principle of Ginger

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.

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.