Development of LC-MS/MS Method for 2,3-Dpg Measurement in Human RBCs

Background: 2,3 Diphosphoglycerate- (2,3DPG) is a metabolic intermediate and a key regulator of hemoglobin affinity for oxygen in RBCs. Reductions in 2,3DPG concentrations cause increased affinity of hemoglobin (Hgb) for O₂, lowering P50, thus decreasing O₂ release in tissues and potentially worsening tissue hypoxia. Prior studies have identified higher pre-dialysis serum phosphate as an important risk factor of intradialytic hypotension, myocardial stunning and cardiovascular mortality. Each dialysis session is associated with an acute decline in serum phosphate, and our preliminary studies show that they may lead to greater % reduction in RBC 2,3DPG - a critical modulator of tissue hypoxia via P50. Unfortunately, the commercial assay for 2,3DPG used in several previous studies is no longer available. To fully elucidate the complex pathophysiological interplay between intra-dialytic phosphate, intra-dialytic 2,3DPG and cardiac complications, the development of a reliable method to quantify 2,3DPG is essential. Given its role as an allosteric effector to enhance oxygen release, 2,3DPG assay is also relevant in other important fields such as maternal-fetal oxygenation, hyperthyroidism, chronic anemia and chronic respiratory disease.

Methods: Whole blood samples were collected from healthy volunteers and dialysis patients in ice-cold K2EDTA tubes and centrifuged within 15 minutes followed by RBC collection. RBCs were aliquoted and then snap frozen/stored in -80C. At time of analysis, QCs and study samples were thawed on wet ice and extracted with ice-cold 80% MeOH. A standard curve was generated by spiking 2,3DPG into aged blood bank aliquots. After overnight protein precipitation at -80C, the extracts were clarified and then further diluted for LC-MS analysis using an ion exchange column and an Ion Chromatography System coupled with an Q Exactive mass spectrometer. 2,3DPG peaks were identified and quantified using Maven software.

Results: The working range of the assay was 0.125 – 8mg/ml. The fit-for-purpose method demonstrated excellent accuracy and precision. Two independent analyses on samples collected during one blood draw from the same subjects but processed and quantified separately, indicated a strong correlation coefficient of 0.84 (P<0.001). Importantly, the assay was able to reflect clear physiological differences in concentrations between groups anticipated to have low 2,3DPG (RBC samples stored for 21 days), normal 2,3DPG (fresh RBCs from healthy volunteers) and high 2,3DPG (fresh RBCs from dialysis patients; 0.11 ± 0.04 vs 1.95 ± 0.29 vs 2.56 ± 0.54 mg/ml; p<0.05).

Conclusions: 2,3DPG was found to be stable and quantifiable in human K2EDTA packed RBCs for at least 90 days when stored at −80 °C, quantification of 2,3DPG on separate LC:MS/MS runs strongly correlated, and this assay was able to distinguish between known low, normal and high 2,3DPG states. Development of a robust and reproducible assay will make it possible to study the role of erythrocyte phosphate metabolism, 2,3DPG in intradialytic hypotension and other physiological and disease conditions affecting the erythrocyte glycolytic pathway.