Tohoku J. Exp. Med., 2014 July, 233(3)

A Novel Method for Measuring the ATP-Related Compounds in Human Erythrocytes

OTHONIEL HUGO ARAGON-MARTINEZ,¹ OTHIR GALICIA,¹ MARIO ALBERTO ISIORDIA-ESPINOZA² and FLAVIO MARTINEZ-MORALES¹

¹Department of Pharmacology, School of Medicine, University of San Luis Potosi, San Luis Potosi, S.L.P., México
²Laboratory of Basic Sciences, School of Stomatology, University of San Luis Potosi, San Luis Potosi, S.L.P., México

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 μL) 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 μmol/L and quantification limits from 0.004 to 0.294 μmol/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.

keywords —— adenosine triphosphate; energetic homeostasis; erythrocytes; liquid chromatography; whole blood sample

© 2014 Tohoku University Medical Press

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Tohoku J. Exp. Med., 2014, 233, 205-214

Correspondence: Flavio Martinez-Morales, M.D., Ph.D., Department of Pharmacology, School of Medicine, University of San Luis Potosi, 2405 Carranza Avenue, 78210-Los Filtros, San Luis Potosi, S.L.P., México.

e-mail: martinej@uaslp.mx