The origin of hexahydrohippurate (cyclohexanoylglycine) in the urine of herbivores.

Hexahydrohippuric acid was &st described as a metabolite in the urine of cattle at pasture by Suemitsu et al. (1971). It was present to the extent of 50mg/litre; the biochemical origin of this component remained unknown. Schogt & Haverkamp Begemann (1965) had identsed 1 1-cyclohexylundecanoic acid in butter and confirmed it by synthesis. Hansen & Gerson (1967) isolated it from the depot fat of ruminants and bovine rumen bacteria (Hansen, 1967). Subsequently, De Rosa et al. (1972) proved that the alicyclic ring of w-cyclohexyl fatty acids present in the cell lipids of an acidophilic thermophilic bacillus (Bacillus acidocaldarius) was derived from shikimate. In view of our demonstration [see the following paper (Balba & Evans 1977)] that cyclohexanecarboxylate is an intermediate in the methanogenic fermentation of benzoate by an adapted consortium of rumen bacteria, there remained the possibility that, if this occurs in the intact animal, some might be absorbed and conjugated with glycine in the liver or kidney. These alternative hypotheses as to the origin of hexahydrohippurate in the urine have now been tested. A Welsh Mountain wether (castrated ram) (35kg, 3 years old), fistulated to the rumen, was placed in a metabolism crate fitted with a continuous urine-collection attachment and fed on a normal daily diet of hay (400g) and sheep pellets (250g, BOCM Silcock); water was offered ad libitum. The tight seal of the rumen cannula had a polythene tube passing through it directly into the rumen; externally this polythenetubingwas attached toaperistalticpumpfortheinfusion of a measuredvolume of fluid (250ml) over a predetermined time-span (48h). An initial urinary collection over this period was made by using water as the drip-infusion fluid. In separate experiments, non-radioactively labelled substrates followed by the same compounds labelled with I4C were administered in the above volume of water, and the individual urine samples collected, as follows. The substrates used were: (I) [ring-U-14C]benzoate (The Radiochemical Centre, Amersham, Bucks., U.K.), 02pCi/ml (47719c.p.m./ml; Philips liquid-scintillation counter); [ring-U-14C]phenylalanine (The Radiochemical Centre), 0.1 pCi/ml (62226 c.p.m./ml); [U-14C]shikimic acid (New England Nuclear Corp., Boston, MA, USA.), 0.1 pCi/ml(59334c.p.m./ml). Urine samples were acidified to pH3 and extracted with ethyl acetate; acidic components were taken into NaHC03 solution from this solvent, and, after acidification, the diethyl ether-soluble acids (e.g. benzoic acid and cyclohexanecarboxylic acid) separated from those that remained in the aqueous phase (e.g. hippuric acid and hexahydrohippuric acid:). This fractionation was suitable for the identification of some of the components by t.1.c. and g.l.c., but the conjugated acids were not easily separable by these techniques. The metabolites (from the urine) in the ethyl acetate extract were sharply separated by gel filtration by using Sephadex G-10 (Pharmacia, Uppsala, Sweden) (cf. Sinha & Gabrieli, 1968); the extract (lOmg), dissolved in sodium phosphate buffer (1 ml, 0.1 M, pH7), was placed on a column (91 cmx 1.4cm) of Sephadex G-10 (bed vol. 130ml), developed with a similar buffer with a constant-flow-rate peristaltic pump and fractions (5ml/tube) were collected (20ml/h) (Combi Cold Cabinet at 4°C and LKB automatic fraction collector). The Azso of the effluent was automatically recorded to locate the