抄録
Establishment of new reaction systems for rapid and precise analysis are urgently desired in clinical chemistry. In this report, the conditions of reaction rate analysis of uric acid and creatinine were studied so as to establish the rapid analysis systems.
In reaction rate analysis with enzyme systems, the first order velocity must be proportional to the substance to be measured (substrate). Thus, the dynamic range of calibration depends upon the Michaelis constant of the enzyme for substrate.
Uricase and uricase-catalase systems were examined for analysis of uric acid in biological fluids. The Michaelis constant of uricase for uric acid was small (9.4×10-6M) so that the dynamic range of the uricase system is narrow in calibration. Thus, it is required that the sample size of a specimen must be decreased to as small volume as possible to be suitable for sensitivity of the detector and precision of pipette sampling. Assay condition was as follows: 10 microliters of sample was incubated at 37°C with 2.9ml of borate buffer pH 8.5. After 2minutes incubation, 0.1ml of uricase solution was added and decrease of optical density at 293nm was measured by a Hitachi 124 double beam spectrophotometer equipped with a scale expanded recorder.
On the other hand, in case of reaction rate analysis without enzyme system, when concentrations of the reagents were high enough for the substances to be measured, the reaction velocity at zero time could be proportional to the concentration of measured substances.
Reaction rate analysis of picrate formation of creatinine was investigated. Picrate and sodium hydroxide concentration was determined to give a linear time course within setted reaction time (one or two minutes). Creatinine in biological fluids was measured with the following conditions: 0.2ml of serum sample was incubated at 37°C with 2.0ml of picric acid solution (0.13g/dl). After 2 minutes incubation, 0.1ml of 1N-NaOH was added and increase in absorbance at 510nm was measured by a Hitachi 124 spectrophotometer. In this case, the final concentration of picric acid and sodium hydroxide were 4.93mM and 0.044M, respectively.
Under these conditions, the linear calibration curve of creatinine was obtained up to concentration of 20mg/dl. Each serum examined gave the same affinity for picric acid and recovery of creatinine was about 100% in this system. This condition was easily applied to an LKB 8,600 Reaction Rate Analyzer without modification.
