Chemical instability of [2-14C]uric acid in alkaline solution: The effect on observed kinetics of urate transport in human erythrocytes
✍ Scribed by B.A. Brooks; A.F. Lant
- Publisher
- Elsevier Science
- Year
- 1976
- Tongue
- English
- Weight
- 313 KB
- Volume
- 75
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
2-"C]Uric acid. in strongly alkaline solution, is chemically unstable when stored at +4"C or -20°C. Two major degradation products occur, which cochromatograph with allantoin and allantoic acid. The nonenzymic decomposition of [2-'T]uric acid markedly alters the observed kinetics of uric acid efflux from human erythrocytes.
Studies of the transport of uric acid in biological systems, both in riro and in vitro. involve use of alkaline solutions. This is due to poor solubility of the free acid form of the urate molecule at neutral pH (1). In tissues lacking uricase, the oxidative decomposition of uric acid by peroxidising haemoproteins at physiological pH, has been well characterised (2,3). By contrast, although there are indications that aqueous solutions of uric acid are unstable (4,.5), little attention has been paid to the nature and extent of nonenzymic uricolysis in such solutions. During a study of uric acid transport across red blood cell membranes, we have made use of [2-14C]uric acid obtained from The Radiochemical Centre, Amersham, England. This preparation is stated to be 98% pure chromatographically (data sheet CFA 221) and has served as a source material for studies of urate transport by various workers in different parts of the world (5-7). Quantitative discrepancies which appeared in some kinetic parameters of the transport system we were investigating led us to examine, chromatographically, samples of [2-W]uric acid which had been stored in aqueous solution at -2o"C, for varying periods of time. On finding substantial amounts of radioactive impurities, we proceeded to study the effect of storage of [2-lAC]uric acid on the observed kinetic behaviour of the transport system in human erythrocyte membranes. Variations in chemical environment, pH, and temperature of storage were assessed for their influence on the stability of [2-ldC]uric acid in solution.
Samples of [2-l"C]uric acid (50 &i), prepared from the same batch, were dissolved in 1 ml of 100 mM NaOH, 1 mM NaOH, or 100 mM Na,HPO,.
Each sample was further divided into two aliquots which were stored at 4°C and -20°C. The [2J4C]uric acid solutions were used immediately, and also after varying lengths of storage, for the determination of rate constants for [2-14C]uric acid efflux from preloaded red blood cells (8). Freshly drawn heparinised venous blood from healthy volunteers was spun down at 3000g. The plasma and buffy layer were 640 Copyright