Factors Affecting the in Vitro Stability of ¹⁴C-Urea in the Urine of Rats

Abstract

To assess possible factors affecting the degradative reaction of urea (ureolysis) catalyzed by urease, the in vitro stability of urea in rat urine was investigated by changing the reaction temperature and pH or amount of added urease or by masking the reaction mixture from the atmosphere. The fraction of the radioactivity spiked as ¹⁴C-urea which was decomposed to ¹⁴CO₂ in rat urine at 15 and 25°C for 24 h was only about 1.5 and 3.3%, respectively, while that at 35°C was as much as 42%. The activation energy for this reaction was estimated to be approximately 25 kcal/mol. Added urease enhanced the degradation of ¹⁴C-urea, the initial apparent rate constant was proportional to the amount of the added enzyme (0.01 to 5.0 U/ml) to yield an approximate catalytic constant of 1.7×10^-2 ml/U/h. When urease was added at 5.0 U/ml, the highest degree of ureolysis was observed at neutral to very slightly alkaline pH. Covering the surface of the reaction mixture of rat urine with a layer of liquid paraffin appeared to depress almost completely the capture of radioactivity as ¹⁴CO₂. It is, therefore, suggested that two precautions, i.e., keeping the urine isolated from the atmosphere and keeping the metabolic cage at a temperature below 25°C, should be taken in disposition experiments with urea which involve the measurement of expiratory excretion of radioactivity.