Chemiluminescent Assay of NAD (P)H and its Application to Clinical Chemistry

建立了1-甲氧基-5-甲基吩嗪甲基硫酸盐（1-MPMS）/异鲁米诺（IL）/微过氧化物酶（m-POD）体系荧光法测定NAD（P）H的方法。在10 ~ 10 μ mol/l范围内，化学发光强度与NAD（P）H浓度（log/log）呈线性关系<-9><-12>。该荧光测定已与葡萄糖-6-磷酸脱氢酶（G6 PDH）、β-D-半乳糖苷酶（β-Gal）和碱性磷酸酶（ALP）的测定相结合。G6 PDH、β-D-Gal和ALP的检测限分别为10 μ mol/次<-18>、10 μ mol/次<-20>和10 μ <-18>mol/次。将这些酶的荧光分析应用于17 α-羟孕酮的荧光酶免疫分析和DNA杂交分析，并将酶循环系统与NADH荧光分析联用，以提高该方法的灵敏度。在3 x 10 μ mol/l<-14>至5 x 10 μ <-12>mol/l范围内获得标准曲线。NADH的检测限为30 fmol/assay，与细菌荧光素酶生物发光法相当，同时还建立了己糖激酶/G6 PDH和i-MPMS/ IL/m-POD体系测定ATP的荧光分析法。利用ATP与己糖激酶/丙酮酸激酶的酶循环反应，建立了ATP的增强荧光分析法。该方法的灵敏度是前一种方法的1000倍。ATP的检测限为10 fmol/次。

We have developed a chemiluminescent method for the assay of NAD (P)H using the 1-methoxy- 5-methylphenazinium methylsulphate (1-MPMS) / isoluminol (IL) / microperoxidase (m-POD) system. A linear relationship between chemiluminescence intensity and NAD (P)H concentration (log/log) was obtained ranged from 10^<-9> mol/l to 10^<-12> mol/l. This chemiluminescent assay has been coupled to the assay of glucose-6-phosphate dehydrogenase (G6PDH), beta-D-glactosidase (beta-Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, beta-D-Gal and ALP were 10^<-18>, 10^<-20> and 10^<-18> molper assay, respectively. The chemiluminescent assay of these enzymes were appplied to chemiluminescent enzyme immunoassay for 17alpha-hydroxyprogesterone and DNA hybridization assay.In order to increase the sensitivity of this method, enzyme cycling system was coupled to the chemiluminescent assay of NADH. The standard curve was obtained in the range from 3 x 10^<-14> to 5 x 10^<-12> mol/l. The detection limit of NADH was 30 fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase.The chemiluminescent assay of ATP was also developed using hexokinase/G6PDH and i-MPMS/ IL/m-POD system. The enhanced chemiluminescent assay of ATP was developed by using enzyme cycling reaction of ATP with hexokinase/pyruvate kinase. This method is 1000 fold more sensitive than the former method. The detection limit of ATP was 10 fmol/assay.