Andrew J. Hutt

The metabolism of aspirin in man: a population study

The metabolism of a 900 mg oral dose of aspirin has been investigated in 129 healthy volunteers. For this purpose, the 0-12 h urine was collected and analysed for the following excretion products: salicylic acid, its acyl and phenolic glucuronides, salicyluric acid, its phenolic glucuronide and gentisic acid. The total excretion of salicylate and metabolites was normally distributed within the population group studied, showing a 2.5-fold variation: a mean of 68.1% of the dose was recovered in 12 h. The excretion of salicylic acid was found to be highly variable within the study panel (1.3-31% of dose in 12 h), and was related to both urine volume and pH. Salicyluric acid was the major metabolite in the majority of the volunteers and its excretion was normally distributed amongst the study panel. The elimination of this metabolite ranged from 19.8 to 65% of the dose and was related to the total recovery of salicylate. The excretion of the two salicyl glucuronides was highly variable, ranging from 0.8 to 42% of the dose. The elimination of the glucuronides was inversely related to that of salicyluric acid. Gentisic acid and salicyluric acid phenolic glucuronide were minor metabolites of salicylate, accounting for 1 and 3% of the dose, respectively. The recovery of gentisic acid was statistically significantly greater in female subjects than in males, whilst the opposite was found for salicyluric acid and total salicylate. However, these differences were small in magnitude.

Application of a radial compression column to the high-performance liquid chromatographic separation of the enantiomers of some 2-arylpropionic acids as their diastereoisomeric S-()-1-(naphthen-1-YL)ethylamides

The enantiomers of 2-phenylpropionic acid and four congeneric anti-inflammatory drugs were separated as their diastereoisomeric amides with S-(-)-1-(naphthen-1-yl)ethylamine by high-performance liquid chromatography using a silica-packed radial compression cartridge. The order of elution of the diastereoisomeric amides was always R, S or -, S before S,S or +,S. The conditions for the derivatization, using 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide as coupling agent, were optimized, and it was found that the addition of 1-hydroxybenzotriazole rendered the reaction quantitative. Good calibration curves were obtained for the quantitation and determination of the enantiomeric composition of 2-phenylpropionic acid in urine, and the application of the method to the study of the metabolism of this acid in vivo is described.

The metabolism of [carboxyl-14C]aspirin in man

1. [carboxyl-14C]Aspirin has been orally administered to four male volunteers and the urinary metabolites examined by paper chromatography, t.l.c., h.p.l.c. and mass spectrometry. 2. 14C Radioactivity was eliminated rapidly in the urine, 94 to 98% of the dose in the first 24 h and approx. 1% in 24-48 h. 3. The major urinary metabolite was salicyluric acid (56-68% dose). In addition, free salicylic and gentisic acids were also detected as were both the acyl and phenolic glucuronides of salicylate. 4. A phenolic glucuronide of salicyluric acid has also been identified. The importance of this metabolite in relation to analytical methods for salicylphenolic glucuronide determination is discussed. 5. The presence of other di- and tri-hydroxybenzoic acids or gentisuric acid could not be demonstrated.

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.

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.

Metabolism of imipramine in vitro: synthesis and characterization of N-hydroxydesmethylimipramine

The synthesis of N-hydroxydesmethylimipramine via the corresponding primary hydroxylamine and oxime is described. The N-oxygenated products are unstable to g.l.c. analysis without prior derivatization; the decomposition products are identified by g.l.c.-mass spectrometry. N-Hydroxydesmethylimipramine is shown to be a metabolite of imipramine and desmethylimipramine on incubation of either with fortified 9000 g liver homogenates of male New Zealand white rabbits. The metabolic product is characterized by mass spectrometry and n.m.r. Didesmethylimipramine is shown to undergo metabolic alpha-C-oxidation, to yield the carboxylic acid, 3-(10,11-dihydro-5H-dibenz[b, f]azepin-5-yl)propionic acid, but not N-oxidation. N-Hydroxydesmethylimipramine is metabolically reduced to desmethylimipramine and metabolized further to 10-hydroxydesmethylimipramine, 2-hydroxydesmethylimipramine and the carboxylic acid. The possible role of N-hydroxydesmethylimipramine and 3-(10,11-dihydro-5H-dibenz[b,f]azepin-5-yl)propionic acid in the formation of iminodibenzyl is discussed.

Isolation and Characterization of Amino Acid and Sugar Conjugates of Xenobiotic Carboxylic Acids

The carboxyl group in xenobiotics such as certain drugs and pesticides may be conjugated with glucuronic acid, glycine, glutamine or taurine. A logical system, developed for excreta but of general applicability, is now presented for isolating and characterizing such conjugates. The choice of TLC or HPLC system is determined by the mobility of the parent acid. It cannot be taken for granted that all alkali-labile conjugates are ester glucuronides. Problems in splitting off and identifying different conjugate moieties are considered. Chemical synthesis of amino acid conjugates is facile and aids identification.

Metabolic oxidation of desmethylchlorimipramine in vitro by 9000 g rabbit-liver preparations

1. The metabolism of desmethylchlorimipramine (I) has been investigated in vitro using fortified 9000 g liver homogenates of male rabbits. 2. Four metabolic products: N-hydroxydesmethylchlorimipramine (II), 3-(3-chloro-10, 11-dihydro-5H-dibenz[b,f]azepin-5-yl)propanoic acid (III), 10/11-hydroxydesmethylchlorimipramine (IV) and 2/8-hydroxydesmethylchlorimipramine (V) were isolated and identified by electron-impact mass spectrometry and n.m.r. spectroscopy. 3. Desmethylchlorimipramine (I) undergoes both N- and alpha-carbon oxidation in addition to aromatic and alicyclic carbon oxidation.

PITFALLS IN THE ENANTIOSELECTIVE ANALYSIS OF CHIRAL DRUGS

There is currently considerable interest in the stereochemical aspects of biochemical pharmacology, largely originating from analytical developments permitting the determination of the enantiomeric composition of chiral drugs in biological media. These new methods allow new types of experiments to be carried out, but there is a need for them to be used critically: a number of pitfalls await those who use them inappropriately. These particularly present themselves in metabolic studies where authentic standards are not available for method validation. Both of the major methods for determining enantiomeric composition can give rise to errors. The formation of diastereoisomers may exhibit stereoselectivity, while the use of chiral columns is fraught with difficulties. In both cases, calibration lines for the two enantiomers are frequently different. Additionally, enantiodifferentiation may occur during sample work-up, as a consequence of achiral processes such as solvent extraction. The importance of proper method validation, with each of a pair of enantiomers, cannot be stressed too highly. If standards of appropriate enantiomeric purity are not available, individual methods cannot be acceptably validated, and data can be accepted only when the same result is obtained with two methods relying on different principles, e.g. a chiral column plus a diastereomeric derivatization, or a Chromatographic method plus NMR.⊗