Abstract
A rapid and covenient screening method using high-performance chromatography(HPLC) for the simultanous detection of deficiencies of adenine phosphoribosyltransf erase (APUT, EC 2.4.2.7) and hypoxanthine guanine phosphoribosyltransferase (HGPRT, EC 2.4.2.8) is presented.
The enzyme reactions were started by the addition of 0.1 ml of the red cell lysates (2∼5g Hb/dl) treated with charcoal-dextran into 0.5 ml of the reaction mixture A(2 mM PRPP, 6 mM MgCl2, 1 mM HX and 0.2 mM adenine in 50 raM Tris-HCl pH7.4). After incubation at 37°C, 30 min, 0.5 ml of reagent B (20 U/ml ALP, 4 mM MgCl2 in 1 M Tris) were added and incubated for 30 min. at 37°C for the conversion of AMP and IMP into adenosine and inosine. ALP reaction was stopped by adding 1.0 ml of 0.5 M HCIO4. The mixture centrifuged at 3,000 x g for 10 min. The supernatants were used as samples for HPLC analysis. Normal ranges of APRT and HGPRT activities in erythrocytes obtained 28 healthy subjects were 0.40 ± 0.06 and 1.97 μ 0.19 μ mol/min/ g Hb. respectively. The method could detect down to 1 % of normal APRT activity and 0.3 % of normal HGPRT activity.
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Nishina, T., Sakuma, R., Kojima, T. et al. 97 High-Performance Liquid Chromatographic method for Simultaneous Screening of the Deficiencies of APRT and HGPRT. Pediatr Res 24, 127 (1988). https://doi.org/10.1203/00006450-198807000-00121
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DOI: https://doi.org/10.1203/00006450-198807000-00121