Measurement of adenine nucleotides in plasma
Measurement of adenine nucleotides in plasma
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DOI：
10.1002/bio.721
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发表时间：
2003-05-01
期刊：
影响因子：
2.9
通讯作者：
Feigl, EO
中科院分区：
文献类型：
--
作者：
Gorman, MW;Marble, DR;Feigl, EO
本研究的目的是确定影响血浆中生物发光ATP，ADP和AMP测量的最重要变量，并开发一种考虑这些变量的测定方法。从清醒的犬中抽取血样。制备了含有EDTA的“终止液”，其通过螯合Mg+2和Ca+2（其是许多ATP酶的辅因子）而大大延缓血浆ATP降解。使用双注射器抽取系统混合终止液和血液。将样品离心两次以除去红细胞，并使用萤火虫荧光素酶测定法测量上清液中的ATP。将样品pH调节至最佳范围（7.75-7.95），并在荧光素酶添加前2秒将Mg 2+（荧光素酶反应所需）添加回光度计内的样品中。为每个血浆样品制备四个测定管，含有0-15 pmol添加ATP的标准添加物，以定量天然血浆ATP含量。在单独的血浆/终止液样品中，通过丙酮酸激酶反应将ADP转化为ATP后测量ADP + ATP，并在加入肌激酶和丙酮酸激酶后测量AMP + ADP + ATP。在终止液中加入毛喉素和异丁基甲基黄嘌呤（IBMX）以抑制血小板，导致ATP浓度降低。测量ATP和血红蛋白从溶解的红细胞显示，溶血产生强烈的影响，血浆ATP浓度，必须考虑。版权所有（C）2003约翰威利父子有限公司。
The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A 'stop solution' containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg+2 and Ca+2 that are co-factors for many ATPases. Stop solution and blood were mixed using a two-syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75-7.95) and Mg2+ (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0-15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account. Copyright (C) 2003 John Wiley Sons, Ltd.