M. S. Moss

Metabolic conjugation of some carboxylic acids in the horse

1. 14C-Labelled benzoic acid, salicylic acid and 2-naphthylacetic acid were administered orally to horses, and urinary metabolites investigated by chromatographic and mass spectral techniques. 2. [14C]Benzoic acid (5 mg/kg) was eliminated rapidly in the urine, and quantitatively recovered in 24 h. The major urinary metabolite was hippuric acid (95% of dose) with much smaller amounts of benzoic acid, benzoyl glucuronide and 3-hydroxy-3-phenylpropionic acid. Administration of [ring-D5]benzoic acid together with [14C]benzoic acid to a pony permitted the mass spectral determination of metabolites of the exogenous benzoic acid metabolites in the presence of the same endogenous compounds. 3. [14C]Salicylic acid (35 mg/kg) was eliminated rapidly in the urine, 98% of the 14C dose being excreted in 24 h. The major excretion product was unchanged salicylate (94% of dose). Gentisic acid, salicyluric acid and the ester and ether glucuronides of salicylic acid were very minor metabolites. 4. 2-Naphthyl[14C]acetic acid (2 mg/kg) was excreted very slowly in the urine, with 53 and 77% of the 14C dose being recovered in six days. 2-Naphthylacetylglycine was the major metabolite (26 and 38% dose) and in addition, the glucuronic acid and taurine conjugates were excreted together with unchanged 2-naphthylacetic acid. 5. The study has shown that the horse can utilize glycine, taurine and glucuronic acid for conjugation of xenobiotic carboxylic acids, and that the relative extents of these pathways are governed by the structure of the carboxylic acid.

3-hydroxy- and 3-keto-3-phenylpropionic acids: Novel metabolites of benzoic acid in horse urine

The metabolism of benzoic acid has been examined in the horse, using 14C- and deuterium-labelled compounds. Chromatographic analysis of the urine showed the presence of hippuric acid, benzoyl glucuronide and benzoic acid and a discrete band which accounted for 2% of the dose administered. This material was isolated by solvent extraction and HPLC and, following treatment with diazomethane, examined by GC/MS. The major component of this fraction was 3-hydroxy-3-phenylpropionic acid methyl ester, which was accompanied by very much smaller amounts of cinnamic acid methyl ester and acetophenone. The two latter minor components have been shown to be artefacts produced during workup and analysis. Cinnamic acid methyl ester arises by the thermal decomposition of 3-hydroxy-3-phenylpropionic acid methyl ester on the GC column. It is proposed that acetophenone has formed, during workup, by decarboxylation of 3-keto-3-phenylpropionic acid. It is suggested that 3-hydroxy and 3-keto-3-phenylpropionic acids, which are also endogenous in horse urine, have arisen by an addition of a 2 carbon fragment to benzoyl CoA, in a sequence analogous to the reactions of fatty acid biosynthesis. Some implications of the metabolic interrelationships between xenobiotic acids and fatty acids are discussed.

The bioavailability of phenylbutazone in the horse

[phenyl-14C]-Phenylbutazone was administered to 2 horses p.o. and i.v. on separate occasions. Plasma levels and urinary and faecal elimination of 14C were monitored for up to 7 days after dosing. Phenylbutazone was rapidly and extensively absorbed after oral administration, and its bioavailability was 91% assessed by comparison of plasma AUCs of unchanged drug after p.o. and i.v. administration. The plasma elimination half-life of phenylbutazone was 9.7 h and this was independent of the route of administration. The pattern of elimination of phenylbutazone was independent of the route of administration, with 55% of the dose being found in the urine in 3 days and a further 39% in the faeces in 7 days. These data, which are the first reports of the absolute bioavailability and excretion pathways of phenylbutazone in the horse, are discussed in terms of their significance for the gastrointestinal toxicity of this drug.

The biotransformation and urinary excretion of dexamethasone in equine male castrates

The pro-drugs of dexamethasone, a potent glucocorticoid, are frequently used as anti-inflammatory steroids in equine veterinary practice. In the present study the biotransformation and urinary excretion of tritium labelled dexamethasone were investigated in cross-bred castrated male horses after therapeutic doses. Between 40-50% of the administered radioactivity was excreted in the urine within 24 h; a further 10% being excreted over the next 3 days. The urinary radioactivity was largely excreted in the unconjugated steroid fraction. In the first 24 h urine sample, 26-36% of the total dose was recovered in the unconjugated fraction, 8-13% in the conjugated fraction and about 5% was unextractable from the urine. The metabolites identified by microchemical transformations and thin-layer chromatography were unchanged dexamethasone, 17-oxodexamethasone, 11-dehydrodexamethasone, 20-dihydrodexamethasone, 6-hydroxydexamethasone and 6-hydroxy-17-oxodexamethasone together accounting for approx 60% of the urinary activity. About 25% of the urinary radioactivity associated with polar metabolites still remains unidentified.

Evidence for the occurrence of a novel pathway of benzoic acid metabolism involving the addition of a two carbon fragment.

Abstract The metabolism of benzoic acid has been investigated in the horse as part of a study on the fate of carboxylic acids in this species (1). The greater part of an orally administered dose of the acid is excreted in the urine in the form of the glycine conjugate, hippuric acid, together with small quantities of benzoyl glucuronide and the free acid. In addition to these the urine samples were found to contain a further metabolite, accounting for some 2% of the dose which exhibited a chromatographic mobility intermediate between that of the parent acid and hippuric acid. This communication provides evidence that this unknown metabolite arises from the addition of a two carbon fragment to the carboxyl group of benzoic acid, leading to the excretion of β-hydroxyphenylpropionic acid and the corresponding β-keto acid in the urine.

The salivary secretion and clearance in the horse of chloral hydrate and its metabolites

Abstract The salivary secretion of chloral hydrate and its metabolites in the horse have been studied. The concentration of chloral hydrate and trichlorethanol in saliva approximated to that in the plasma, but saliva levels of trichloracetic acid were much lower than plasma levels. Chloral hydrate and trichlorethanol were quickly absorbed after oral administration. About 60 per cent of the dose of chloral hydrate was excreted in the urine as the glucuronide of trichlorethanol ; traces of free trichlorethanol and trichloracetie acid were also detected. After chloral hydrate administration the main plasma constituents were trichlorethanol, trichloracetic acid, and chloral hydrate. Trichloracetic acid persisted longest in the plasma. Chloral hydrate could only be detected in the plasma for four or five hours after its administration whereas the other two metabolites persisted for much longer. Methods for the determination of trichlorethanol and chloral hydrate by gas-liquid chromatography using electron capture have been described. Trichloracetic acid was measured using the Fujiwara reaction.6