Citrate metabolism in red blood cells stored in additive solution-3

Red blood cells (RBCs) are thought to have a relatively simple metabolic network compared to other human cell types. Recent proteomics reports challenge the notion that RBCs are mere hemoglobin carriers with limited metabolic activity. Expanding our understanding of RBC metabolism has key implications in many biomedical areas, including transfusion medicine.

In-gel digestion coupled with mass spectrometric analysis proteomics approaches were combined with state-of-the-art tracing experiments by incubating leukofiltered RBCs in additive solution-3 for up to 42 days under blood bank conditions, in presence of ¹³ C_1,2,3 -glucose, 2,2,4,4-d-citrate, and ¹³ C,¹⁵ N-glutamine.

Results indicate that the pentose phosphate pathway/glycolysis ratio increases during storage in additive solution-3. While the majority of supernatant glucose is consumed to fuel glycolysis, incorporation of glucose-derived pentose phosphate moieties was observed in nucleoside monophosphates. Incubation with deuterated citrate indicated that citrate uptake and metabolism contribute to explain the origin of up to approximately 20% to 30% lactate that could not be explained by glucose oxidation and 2,3-diphosphoglycerate consumption alone. Incubation with ¹³ C,¹⁵ N-glutamine showed that glutaminolysis fuels transamination reactions and accumulation of millimolar levels of 5-oxoproline, while de novo glutathione synthesis was not significantly active during refrigerated storage.

Quantitative tracing metabolic experiments revealed that mature RBCs can metabolize other substrates than glucose, such as citrate, an observation relevant to transfusion medicine (i.e., formulation of novel additives), and other research endeavors where metabolic modulation of RBCs opens potential avenues for therapeutic interventions, such as in sickle cell disease.