血清中腺苷脱氨酶同工酶活性的自动化酶法测定

Automated enzymatic measurement of adenosine deaminase isoenzyme activities in serum.

作者信息

Muraoka T, Katsuramaki T, Shiraishi H, Yokoyama M M

机构信息

Oyodo Research Laboratories, Maruho Company, Ltd., Shiga, Japan.

出版信息

Anal Biochem. 1990 Jun;187(2):268-72. doi: 10.1016/0003-2697(90)90455-i.

DOI:10.1016/0003-2697(90)90455-i

PMID:2382828

Abstract

We developed a simple, rapid, and automated method for simultaneous measurement of adenosine deaminase (ADA, EC 3.5.4.4) isoenzymes in human serum, based on their apparent difference in Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) as inhibitor. Serum ADA was partially purified by CM-Sephadex, gel-filtration, and affinity chromatography into two types of isoenzymes, designated ADA1 (300 kDa) and ADA2 (120 kDa). Because ADA2 has a higher Km for adenosine and higher Ki values for EHNA than does ADA1, the activity of ADA1 is almost completely inhibited by EHNA at 0.1 mM (analytical recovery 4.1%), whereas ADA2 is practically unaffected (analytical recovery 94.8%) by that concentration of EHNA. We measured the activities of ADA2 and total ADA in the presence and absence of 0.1 mM EHNA. ADA1 activities were calculated by subtracting the activity of ADA2 from that of total ADA. The mean within-assay CV was 5.7% for ADA1 and 2.7% for ADA2. The interassay CV was 2.8% for ADA1 and 3.1% for ADA2. Results of the present method correlated well (r = 0.9026 for ADA1, 0.9438 for ADA2) with those of the ion-exchange chromatography method. The upper limits of the reference intervals, as calculated from data for 320 healthy donors, are 7.2 U/liter for ADA1, and 14.6 U/liter for ADA2. This method is suitable for analysis of large numbers of samples in clinical laboratories for routine monitoring of the activities of ADA isoenzymes in serum.

摘要

基于人血清中腺苷脱氨酶（ADA，EC 3.5.4.4）同工酶对红细胞 -9-(2-羟基 -3-壬基)腺嘌呤（EHNA）作为抑制剂的表观 Ki 值差异，我们开发了一种简单、快速且自动化的同时检测人血清中 ADA 同工酶的方法。血清 ADA 通过 CM - 葡聚糖凝胶、凝胶过滤和亲和层析部分纯化，分为两种同工酶类型，分别命名为 ADA1（300 kDa）和 ADA2（120 kDa）。由于 ADA2 对腺苷的 Km 值高于 ADA1，且对 EHNA 的 Ki 值也高于 ADA1，所以在 0.1 mM 的 EHNA 浓度下，ADA1 的活性几乎被完全抑制（分析回收率为 4.1%），而该浓度的 EHNA 对 ADA2 实际上没有影响（分析回收率为 94.8%）。我们在有和没有 0.1 mM EHNA 的情况下测量了 ADA2 和总 ADA 的活性。ADA1 的活性通过从总 ADA 的活性中减去 ADA2 的活性来计算。ADA1 的批内变异系数均值为 5.7%，ADA2 为 2.7%。ADA1 的批间变异系数为 2.8%，ADA2 为 3.1%。本方法的结果与离子交换色谱法的结果相关性良好（ADA1 的 r = 0.9026，ADA2 的 r = 0.9438）。根据 320 名健康供体的数据计算得出的参考区间上限，ADA1 为 7.2 U/升，ADA2 为 14.6 U/升。该方法适用于临床实验室对大量样本进行分析，以常规监测血清中 ADA 同工酶的活性。