A Novel Method for Measuring the ATP-Related Compounds in Human Erythrocytes
A Novel Method for Measuring the ATP-Related Compounds in Human Erythrocytes
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DOI：
10.1620/tjem.233.205
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发表时间：
2014-07-01
影响因子：
2.2
通讯作者：
Martinez-Morales, Flavio
中科院分区：
文献类型：
--
作者：
Hugo Aragon-Martinez, Othoniel;Galicia, Othir;Martinez-Morales, Flavio
全血或红细胞中的 ATP 相关化合物已用于评估红细胞的能量状态以及磷酸化化合物在各种条件下的降解水平，例如慢性肾功能衰竭、药物监测、癌症、环境毒物暴露和器官保存。对红细胞能量稳态的完整解释只能使用参与腺嘌呤核苷酸降解途径的化合物以及尿酸值来进行。我们首次报告了一种使用二极管阵列检测器的液相色谱方法，该方法可在 20 分钟的可接受时间内测量少量人类全血样本 (125 μL) 中的所有这些化合物。对所有化合物的稳定性进行了评估，与零日值相比，稳定性范围为 96.3% 至 105.1%。该测量对 ATP 相关化合物具有足够的灵敏度（检测限为 0.001 至 0.097 μmol/L，定量限为 0.004 至 0.294 μmol/L）。该方法特别适用于测量肌苷单磷酸、肌苷、次黄嘌呤和尿酸。此外，该测定具有可接受的线性（r > 0.990）、精密度（变异系数范围为 0.1 至 2.0%）、特异性（所有样品中的保留时间和光谱相似）和回收率（范围为 89.2 至 104.9%）。新开发的方法对于评估红细胞在不同条件下（例如体外实验和临床环境）的能量稳态具有不可估量的价值。
The ATP-related compounds in whole blood or red blood cells have been used to evaluate the energy status of erythrocytes and the degradation level of the phosphorylated compounds under various conditions, such as chronic renal failure, drug monitoring, cancer, exposure to environmental toxics, and organ preservation. The complete interpretation of the energetic homeostasis of erythrocytes is only performed using the compounds involved in the degradation pathway for adenine nucleotides alongside the uric acid value. For the first time, we report a liquid chromatographic method using a diode array detector that measures all of these compounds in a small human whole blood sample (125 mu L) within an acceptable time of 20 min. The stability was evaluated for all of the compounds and ranged from 96.3 to 105.1% versus the day zero values. The measurement had an adequate sensitivity for the ATP-related compounds (detection limits from 0.001 to 0.097 mu mol/L and quantification limits from 0.004 to 0.294 mu mol/L). This method is particularly useful for measuring inosine monophosphate, inosine, hypoxanthine, and uric acid. Moreover, this assay had acceptable linearity (r > 0.990), precision (coefficients of variation ranged from 0.1 to 2.0%), specificity (similar retention times and spectra in all samples) and recoveries (ranged from 89.2 to 104.9%). The newly developed method is invaluable for assessing the energetic homeostasis of red blood cells under diverse conditions, such as in vitro experiments and clinical settings.