Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage

Abstract

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates.

Heterogeneity of blood processing and storage additives in different centers impacts stored red blood cell metabolism as much as storage time: lessons from REDS‐III—Omics

“…Samples stored in AS-1 were characterized by lower levels of citrate (absolute and relative quant graphs are shown in Figures 4.B and 7, respectively), oxaloacetate, homoaconitate and acyl-carnitine C6-DC (Figure 7). The same samples were instead characterized by higher levels of malate and fumarate – byproducts of reactions preserving NADH homeostasis and purine salvage in the presence of aspartate (an almost completely inactive pathway in RBCs 31 ).…”

Metabolic effect of alkaline additives and guanosine/gluconate in storage solutions for red blood cells

“…However, the observed metabolic benefits do not extend to the prevention of storage‐induced fatty acid release and lipid oxidation. The correlation of metabolites from these pathways with surrogate ex vivo indexes of functional outcomes (e.g., cell morphology and osmotic fragility) are relevant in that they confirm and extend the recent observation on the (correlative) role of purine deamination in RBC recovery after transfusion . However, additional functional in vivo measurements (e.g., posttransfusion recovery) will be instrumental to further expand our understanding of the impact of these storage additives on transfusion efficacy.…”

“…This observation could also be alternatively explained by increased fluxes through the oxidative PPP, resulting in higher nonoxidative/oxidative PPP intermediate ratios (consistent with higher glucose‐6‐phosphate dehydrogenase activity and thus increased antioxidant capacity in alkaline RBCs). Indeed, we recently appreciated the role that oxidative stress plays in activating AMP deaminase 3 to promote purine deamination and thus hypoxanthine accumulation, a phenomenon that negatively correlates with posttransfusion recovery and is prevented by hypoxic storage (which also induces intracellular alkalinization) . However, direct comparison of hypoxanthine levels in alkaline versus nonalkaline additives (SOLX vs. AS‐3) in unpaired studies showed increases in the former group, also accompanied by higher total levels of nondeaminated purines and ATP .…”

“…Activation of purine deaminases such as erythrocyte‐specific adenosine monophosphate (AMP) deaminase 3 in turn results in potentially toxic by‐products such as hypoxanthine. Hypoxanthine has been recently identified as a negative predictor of posttransfusion recovery in stored mouse and human RBCs and a substrate for circulating xanthine oxidase for the generation of urate and hydrogen peroxide . In this view, end‐of‐storage rejuvenation of RBCs has been proposed, which exploits inosine and pyruvate to replenish late glycolysis via phosphoribolysis‐derived pentose phosphate sugars and to promote oxidation of NADH back to NAD+ via lactate dehydrogenase, respectively.…”

Metabolomics evaluation of early‐storage red blood cell rejuvenation at 4°C and 37°C

“…Alterations to energy and redox metabolism may indeed limit RBC capacity to circulate upon transfusion, as historically noted for the storage‐induced depletion of high‐energy phosphate compounds such as adenosine triphosphate (ATP) and, more recently, for the ATP‐breakdown product hypoxanthine . Indeed, the metabolic storage lesion may negatively impact the capacity of transfused RBCs to cope with physiologic challenges such as body temperature, inflammation, and systemic stress present in hypoxic recipients (e.g., patients with sickle cell or critically ill patients).…”