Gorman Mark W, Marble David R, Ogimoto Kayoko, Feigl Eric O
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290, USA.
Luminescence. 2003 May-Jun;18(3):173-81. doi: 10.1002/bio.721.
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 Mg(2+) (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.
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检测方法。从清醒的狗身上采集血样。制备了一种含有EDTA的“终止溶液”，通过螯合作为许多ATP酶辅助因子的Mg(+2)和Ca(+2)，大大延缓了血浆ATP的降解。使用双注射器抽取系统将终止溶液和血液混合。样品离心两次以去除红细胞，并使用萤火虫荧光素酶检测法测量上清液中的ATP。将样品pH值调节至最佳范围(7.75 - 7.95)，并在加入荧光素酶前2秒在发光计内将荧光素酶反应所需的Mg(2+)添加回样品中。为每个血浆样品准备四个检测管，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/终止溶液样品中，通过丙酮酸激酶反应将ADP转化为ATP后测量ADP + ATP，加入肌酸激酶和丙酮酸激酶后测量AMP + ADP + ATP。在终止溶液中添加福斯可林和异丁基甲基黄嘌呤(IBMX)以抑制血小板会导致ATP浓度降低。对裂解红细胞中的ATP和血红蛋白进行测量表明，溶血对血浆ATP浓度有很大影响，必须予以考虑。
