Metabolism of d-glucuronolactone in mammalian systems. Identification of d-glucaric acid as a normal constituent of urine.

The inhibition of P-glucuronidase by mamResearch Unit, Aberdeen Royal Infirmary, to whom the malian urine has been noted by a number of author is indebted. Fresh samples of cat urine were workers. This complicates assay of the enzyme in supplied by Dr A. J. Carr, Pathology Department, Aberurine, and the use of added *-e to deen University; 24 hr. urines of rat and guinea pig, and urine, and the us of -glucuronidase 1 samples of sheep, pig and calf urines, were collected from lhberate excretedl phenols and alcohols' includm' let xo s, i ng animals maintained on normal diets in metabolism cages. steroids, from the fl-D-glucosiduronic acid conThe rat and guinea-pig exereta were separated by filtration, jugates. P-Glucuronidase assay is normally conand the faeces washed with water, before the combined ducted with a chromogenic substrate, the enzymic urine and washings were tested. Urines were stored at 0' if hydrolysis of which may be diminished in the examined within 24 hr. of collection, or otherwise at 20°. presence of urine which contains competing subTreatment of urine. The following standard procedure strates of different affinities (see Levvy & Marsh, was used in testing the inhibitory powers of mammalian 1954). In addition to natural substrates for p urine. Crude urine (pH 4-5-7-0) was treated (a) at 1000 glucuronidase, however, mammnalian urine confor 40 min. after adjustment with 3N-HCl to pH 2-0-2-2, tains trueenzymeinhibitrs Abul-Fadl(1957) followed by readjustment with NaOH to pH 4-0-45, or tamns true enzyme inhibitors. Abul-Fadl (1957) (b) at 100° for 15 min. after adjustment with 0-1 N-NaOH to found both dialysable and non-dialysable material pH 7-5-8-0, follQwed by readjustment with HCI to pH 60in human urine which was inhibitory to /-gluc6-5. The inhibitory power was maximal after treatment uronidase; the dialysable inhibitor was stable to (a) and minimal after treatment (b). Either treatment was treatment with hot acid. Similar inhibitory fracsufficient to rid the urine of ,B-glucuronidase activity, and tions were present in rabbit urine (Conzelman & after the readjustments there was no interference with the Crout, 1961). pH of the subsequent enzyme assay. Heating at a pH Mammalian /3-glucuronidase is inhibited by above 2-5 was inadequate for development of maximum heavy-metal ions (Levvy & Marsh, 1957a, b; inhibitory power of acidified urines, for there was then a