Simultaneous Determination for Adenine-Phosphoribosyltransferase and Hypoxanthine-Phosphoribosyltransferase Activities in Human Erythrocytes by High-Performance Liquid Chromatography

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A simple and rapid nonradiochemical method for the simultaneous determination of adenine phosphoribosyltransferase (APRT, EC 2. 4. 2. 7) and hypoxanthine phosphoribosyltransferase (HPRT, EC 2. 4. 2. 8) activities in human erythrocytes by high-performance liquid chromatography is described. APRT and HPRT activities were simultaneously assayed with adenine and hypoxanthine as substrates. adenosine-5'-monophosphate (AMP) and inosine-5'-monophosphate (IMP) produced by both enzyme reactions were converted to adenosine and inosine with alkaline phosphatase (EC 3. 1. 3. 1), respectively, and the adenosine and inosine separated from adenine and hypoxanthinelby a reversedphase silica column were determined by the absorbance at 270nm. The activities of APRT in erythrocytes of 36 healthy subjects were 0.42±0.17μmol/min/gHb and 2.00±0.39μmol/min/gHb (mean±2SD), respectively The method is suitable for screening erythrocytes for APRT and HPRT deficiencies.
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