胆固醇脱氢酶测定血清胆固醇方法的建立

Determination of cholesterol in serum with cholesterol dehydrogenase

目的 评价胆固醇脱氢酶测定法在临床应用的可行性。方法 胆固醇酯在胆固醇酯酶的作用下 ,水解为游离胆固醇 ,后者被胆固醇脱氢酶还原为胆烷 4 烯 3 酮 ,同时 β NAD+ 还原为 β NADH +H+ 。联胺能清除其反应产物胆烷 4 烯 3 酮 ,使反应完全。结果 回收率为 99.9%和 10 0 .1% ,在 1.1～ 2 0 .2 8mmol/L内为线性 ;批内CV为 0 .2 9%～ 0 .39% ,批间CV为 0 .4 2 %～ 0 .4 5 % ;乳酸脱氢酶无影响 ;与化学比色法 (X1)和胆固醇氧化酶法 (X2 )比较 ,回归方程分别为Y =0 .992X1- 0 .0 0 6 ,r =0 .997;Y =0 .989X2 - 0 .0 4 8,r =0 .999。结论 本法结果准确、可靠、易自动化。

Objective To evaluate cholesterol dehydrogenases(CDH) assay for measurement of cholesterol. Methods Cholesterol esters were first hydrolyzed to free cholesterol by cholesterol ester hydrolase(CEH). The free cholesterol was then reduced by CDH to cholest-4-ene-3-one with the simultaneous production of β-NADH+H + from β-NAD +. We added hydrazine to the reaction mixture to remove cholest-4-ene-3-one,which allowed the reaction to proceed to completion. Results Recoveries were 99.9% to 100.1%. The reaction was linear up to 20.28 mmol/L. The mean within-day and between day imprecision(CV) was 0.29% to 0.39% and 0.42% to 0.45% respectively. No interference by LDH was observed. The equation obtained in comparison with the modified Abell-Levy-Bredie-Kendall method(X_1) and CEH-CO-POD method(X_2) were Y=0.992X_1-0.0068,r=0.997;Y=0.989X_2- 0.048,r= 0.999 ,respectively. Conclusions This method is accurate,reliable for serum cholesterol analysis and is amenable to automation.