Kather H, Wieland E, Waas W
Anal Biochem. 1987 May 15;163(1):45-51. doi: 10.1016/0003-2697(87)90091-1.
A fully automatic method for analysis of adenosine, inosine, and hypoxanthine/xanthine which combines the specificity of enzymatic catalysis and sensitivity of chemiluminescence is presented. The hydrogen peroxide formed by sequential catabolism of purines to uric acid is detected by the oxidation of luminol in the presence of peroxidase. The method takes advantage of the fact that light output in the H2O2/luminol system is transient. By adopting a two-step procedure this feature enables selective determination of adenosine, inosine, and hypoxanthine/xanthine. In step 1 any purines lower in the catabolic sequence than the analyte under study are converted to uric acid. Light emission is allowed to decay to baseline levels. During step 2 the analyte is selectively degraded. The H2O2 formed leads to a new light emission which is proportional to the square of analyte concentration. The method can be performed with commercially available reagents and enzymes and requires minimal processing of biological samples. Excellent agreement has been obtained with HPLC analysis. Sensitivity is in the range of 5-10 nmol/liter in as little as 0.1 ml. More than 200 samples per day can be analyzed by a single operator.
本文介绍了一种结合酶催化特异性和化学发光灵敏度的全自动分析腺苷、肌苷和次黄嘌呤/黄嘌呤的方法。嘌呤依次分解代谢为尿酸所产生的过氧化氢，在过氧化物酶存在的情况下，通过鲁米诺的氧化来检测。该方法利用了H2O2/鲁米诺系统中光输出是瞬态的这一事实。通过采用两步法，这一特性能够选择性地测定腺苷、肌苷和次黄嘌呤/黄嘌呤。在步骤1中，将分解代谢序列中比所研究分析物更低的任何嘌呤转化为尿酸。使发光衰减至基线水平。在步骤2中，选择性地降解分析物。所形成的H2O2导致新的发光，其与分析物浓度的平方成正比。该方法可以使用市售试剂和酶来进行，并且对生物样品的处理要求最低。与HPLC分析结果具有良好的一致性。灵敏度在低至0.1 ml时为5 - 10 nmol/升。单个操作人员每天可分析200多个样品。
