Gorman Mark W, Feigl Eric O, Buffington Charles W
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195-7290, USA.
Clin Chem. 2007 Feb;53(2):318-25. doi: 10.1373/clinchem.2006.076364. Epub 2006 Dec 21.
Human plasma ATP concentration is reported in many studies as roughly 1000 nmol/L. The present study tested the hypothesis that the measured plasma ATP concentration is lower if ATP release from formed blood elements is inhibited during blood sample processing. A second hypothesis was that pretreatment with aspirin to inhibit platelets would reduce the measured plasma concentration of ATP.
Blood was sampled from the antecubital vein in 20 healthy individuals 30 and 60 min after ingestion of aspirin (325 mg) or placebo. Aliquots of each blood sample were added to the usual EDTA/saline solution to inhibit ATP catabolism, or to a new stabilizing solution designed to both stop ATP catabolism and inhibit ATP release from blood elements. The stabilizing solution contained NaCl, EDTA, tricine buffer, KCl, nitrobenzylthioinosine, forskolin, and isobutylmethylxanthine. Plasma ATP was measured with the luciferin-luciferase assay with standard additions in each sample to determine ATP content. Hemoglobin concentration was used as an index of sample hemolysis, and the plasma ATP concentration was corrected for the hemolysis component.
Aspirin pretreatment had no effect on plasma ATP concentrations. However, use of the stabilizing solution resulted in mean (SD) ATP concentrations 8-fold lower than the use of EDTA alone [28 (16) vs 236 (201) nmol/L; P <0.001].
When precautions are taken to inhibit ATP release from blood elements during sample preparation, human venous plasma ATP concentration is much lower than previously reported.
许多研究报道人类血浆ATP浓度约为1000 nmol/L。本研究检验了以下假设:如果在血样处理过程中抑制血液有形成分释放ATP,所测得的血浆ATP浓度会更低。第二个假设是,用阿司匹林预处理以抑制血小板会降低所测得的血浆ATP浓度。
在20名健康个体摄入阿司匹林(325 mg)或安慰剂30分钟和60分钟后,从前臂肘静脉采血。将每份血样的等分试样加入到常用的EDTA/盐溶液中以抑制ATP分解代谢,或加入到一种新的稳定溶液中,该溶液既能阻止ATP分解代谢,又能抑制血液有形成分释放ATP。该稳定溶液含有氯化钠、EDTA、三羟甲基甘氨酸缓冲液、氯化钾、硝基苄基硫代肌苷、福斯可林和异丁基甲基黄嘌呤。用荧光素-荧光素酶测定法测定血浆ATP,并在每个样品中加入标准品以确定ATP含量。血红蛋白浓度用作样品溶血的指标,并对血浆ATP浓度进行溶血成分校正。
阿司匹林预处理对血浆ATP浓度没有影响。然而,使用稳定溶液导致的平均(标准差)ATP浓度比单独使用EDTA低8倍[28(16)对236(201)nmol/L;P<0.001]。
当在样品制备过程中采取措施抑制血液有形成分释放ATP时,人静脉血浆ATP浓度比先前报道的要低得多。