以对乙酰基苯基磷酸二钠盐为底物的碱性磷酸酶连续监测法

An assay for the determination of alkaline phosphatase activity using disodium p-acetylphenylphosphate as a substrate

目的合成对乙酰基苯基磷酸二钠盐(PAP-PNa2),并建立以PAP-PNa2为底物的碱性磷酸酶(ALP)测定方法。方法在半自动生化分析仪上采用连续监测法,建立实验参数。结果本法测定对乙酰基酚(PAP)最大吸收峰为325nm;ALP的Km=0.376mmol/L;340nm时PAP的ε为23390L·mol-1·cm-1,AMP缓冲液浓度为0.438mol/L时,最适底物浓度5.0mmol/L,最适pH值为10.4,延迟期60s,线性反应期15min;线性范围为0～1110U/L。Hb<115.62mg/L及胆红素浓度达171.0μmol/L对测定结果无影响。本法总ALP的参考范围为:男63.1～118.3U/L,女52.5～89.0U/L。结论该方法重复性好,线性反应期长,干扰因素少,适用于自动生化分析。

Objective To establish a new method to measure alkaline phosphatase （ALP） activity using disodium p-acetylphenylphosphate （ PAP-PNa2 ） as substrate. Methods Experimental parameters were set up with continuous-monitoring procedures on a semiautomatic analyzer. Results The wavelength of maximum absorption of PAP was 325 nm, and the Km of ALP was O. 376 mmol/L. The molecular extinction coefficient of PAP at 340 nm was 23 390 L · mol^-1· cm^-1 and the concentration of citrate buffer was 0. 438 mol/L. In the measurement, the optimum pH was 10.4, and the optimum concentration of substrate was 5.0 mmol/L. The delay time was 60 see and the time keeping the linear reaction was 15 min,and the linear range was 0 - 1 110 U/L. The measurement was not interfered by the hemoglobin 〈 115.62 mg/L and bilirubin. Total serum ALP was 63.1 - 118.3 U/L in male and 52.5 - 89.0 U/L in female. Conclusions The method developed in this study has wide range for linear reaction and good reproducibility. The method is less interfered by other factors and is suitable for automatic biochemical analysis.