一种新型肌酸激酶检测方法的研究

Study on a novel determination for creatine kinase in urine

目的根据肌酸激酶(CK)的检测原理,以肌酸为对象,快速检测CK的活性。方法根据CK可催化磷酸肌酸和二磷酸腺苷(ADP)产生等摩尔肌酸和三磷酸腺苷(ATP)的机理,通过肌酸与碱性甲萘酚和丁二酮作用显色(525 nm),用比色法测定尿肌酸的含量。经HPLC(验证集)的验证和经典酶偶联法(校正集)的校正,由MedCal统计软件计算尿肌酸含量与尿ATP含量的关联性,以此说明肌酸值对CK的活性表达。结果1.287~51.47μg·mL^-1肌酸与峰面积的线性关系良好,回归方程为:Y=0.023X-0.0288(r^2=0.9992),检测限为0.4290μg·mL^-1,平均加样回收率为100.3%,平均日内和日间精密度的RSD分别为2.22%、8.63%。尿肌酸的测定结果与验证集比较,具有良好的相关性;与校正集比较,具有较好的相关性。SD大鼠在高强度运动后,其尿肌酸的水平发生明显变化,48 h达峰,具有性别差异。其中,雄鼠达峰后下降缓慢,而雌鼠出现快速下降趋势。结论尿肌酸与尿ATP的含量均能反映CK活性,且检测步骤更简捷、成本更低,可成为快速检测CK的较好方法。

OBJECTIVE To rapidly detect creatine kinase(CK)activity based on the principle of detection of CK.METHODS According to the mechanism that CK can catalyze the production of equimolar creatine and adenosine triphosphate(ATP)by creatine phosphate and adenosine diphosphate(ADP),a color-developing method was established through the interaction of creatine with basic menadione and butadione(525 nm),and urine creatine content was determined by colorimetry.After HPLC(validation set)verification and classic enzyme coupling method(calibration set)correction,MedCal statistical software calculated the correlation between urinary creatine content and urinary ATP content,thereby proving that creatine value had an effect on CK activity expression.RESULTS Creatine concentration in the range of 1.287-51.47μg·mL^-1 showed good linear relationship Y=0.023X-0.0288(r^2=0.9992),the detection limit was 0.4290μg·mL^-1,and the average sample recovery was 100.3%.The intra-day and inter-day precision RSD were 2.22%and 8.63%,respectively.The measurement results of urine creatine had a good correlation with the validation set,and a good correlation with the calibration set.SD rats experienced significant changes in urine creatine levels after high-intensity exercise,reaching peaks at 48 hours,with gender differences.Male rats declined slowly after reaching peaks.Female rats showed a rapid downward trend.CONCLUSION The results show that both the creatine and ATP levels of urine can reflect the activity of CK,and the detection steps are simpler and cheaper,which can be a better method for rapid detection of CK.