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

Abstract

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.

A LC-MS/MS Method for Quantifying Adenosine, Guanosine and Inosine Nucleotides in Human Cells

“…More RBCs could be analyzed to increase sensitivity, but results would then be above the limit of quantification for ATP and GTP when assaying more than 10×10 6 cells. However, for all validation runs the adjusted LLOQ was still relatively sensitive at 0.625 pmol/sample; a lower amount than other methods published for the measurement of inosine or its IMP metabolite (17, 34). Additionally, because ITP is not incorporated into RNA or DNA, previous methods have not, to our knowledge, attempted to quantify this nucleotide.…”

“…As mentioned earlier, DBS has been used for the measurement of many endogenous biomarkers including nucleotides, but stability of this matrix has not been thoroughly evaluated (22, 31, 32). Other studies that have measured nucleotide pools in RBC samples have reported that immediate processing is a necessity for accurate results (17, 33). Both the RBCs and DBS samples reported here were stored at −70°C for 815–929 days prior to this analysis, but DBS appeared to have a breakdown of TP to the MP compared to RBCs.…”

“…Several methods were previously developed to specifically measure adenosine and guanosine related nucleotides in red blood cells (RBCs) with HPLC coupled to ultraviolet-visible and/or diode array detection (10, 12–17). The range of concentrations detected specifically for ATP and GTP was similar for all of these methods (~114 to 213 and ~3.3 to 8.6 pmol/10 6 cells (10, 13, 16, 17)). Methods that assayed other cell types, like peripheral blood mononuclear cells (PBMCs), found adenosine and guanosine nucleotide concentrations that were roughly 10 fold higher than RBCs (10, 13).…”

Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells

“…Previously, Aragon-Martinez et al developed a liquid chromatographic method for quantification of ATP-related compounds in erythrocytes using a diode array detector [18]. In this method, the authors reported sensitivity for ATP-compounds with limits of detection from 0.001 to 0.097 µmol/L and quantification limits from 0.004 to 0.294 µmol/L [18].…”

“…Previously, Aragon-Martinez et al developed a liquid chromatographic method for quantification of ATP-related compounds in erythrocytes using a diode array detector [18]. In this method, the authors reported sensitivity for ATP-compounds with limits of detection from 0.001 to 0.097 µmol/L and quantification limits from 0.004 to 0.294 µmol/L [18]. Similarly, McFarland and coworkers developed a combined liquid chromatography UV-Vis assay for detection of adenosine, inosine, and hypoxanthine and detected urate and xanthine via coulometric detection at 150 and 450 mV [19].…”