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

• Published in: The Tohoku Journal of Experimental Medicine Vol. 233; no. 3; pp. 205 - 214
• Main Authors: Aragon-Martinez, Othoniel Hugo, Galicia, Othir, Isiordia-Espinoza, Mario Alberto, Martinez-Morales, Flavio
• Format: Journal Article
• Language: English
• Published: Japan Tohoku University Medical Press 2014
• Subjects: Adenosine - analysis; Adenosine - metabolism; adenosine triphosphate; Adenosine Triphosphate - metabolism; Chromatography, High Pressure Liquid - methods; energetic homeostasis; Erythrocyte Count; erythrocytes; Erythrocytes - chemistry; Erythrocytes - physiology; Homeostasis - physiology; Humans; Hydrogen-Ion Concentration; Hypoxanthine - analysis; Hypoxanthine - metabolism; Inosine - analysis; Inosine - metabolism; Inosine Monophosphate - analysis; Inosine Monophosphate - metabolism; liquid chromatography; Sensitivity and Specificity; Uric Acid - analysis; Uric Acid - metabolism; whole blood sample
• ISSN: 0040-8727; 1349-3329
• DOI: 10.1620/tjem.233.205

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.