Ex vivo 13C6-glucose tracing in red blood cells to study the fate of glucose in hereditary enzymopathies

• Ex vivo 13 C 6 -glucose tracing in RBCs can reveal disease-specific disturbances in glucose metabolism. • Ex vivo 13 C 6 -glucose tracing provides new insights into the molecular consequences of RBC enzymopathies on glucose metabolism. As the maturing red blood cell (RBC) loses its nucleus and organelles including mitochondria to maximize capacity for oxygen transportation, it becomes dependent on glycolysis for the generation of adenosine triphosphate. RBC enzymopathies are a group of hereditary nonspherocytic hemolytic anemias in which impaired cellular energy balance decreases RBC integrity and life span. In this study, we monitored the fate of glucose through glycolysis and the pentose phosphate pathway (PPP) over time in purified RBCs from controls and patients with hexokinase (HK) or pyruvate kinase (PK) deficiency. We combined 13 C 6 -glucose tracing and liquid chromatography-mass spectrometry to quantify incorporation of the 13-labeled carbon atoms from glucose into downstream metabolites. We observed reduced carbon labeling of glycolytic metabolites downstream of HK in RBCs from HK-deficient patients compared to controls, whereas carbon labeling of metabolites of the PPP was similar to controls. In RBCs from PK-deficient patients, we quantified accumulation of carbon-labeled glycolytic metabolites directly upstream of PK compared to controls, but noted decreased carbon labeling of metabolites more upstream in the glycolytic pathway. This result was accompanied by increased carbon labeling of metabolites of the PPP compared to controls. Our findings provide new insights into the consequences of the respective enzyme deficiencies and demonstrate the suitability of ex vivo 13 C 6 -glucose tracing in RBCs to study disease-specific disturbances in glycolysis. This technique can be applied to RBCs from patients with various RBC disorders to generate a better understanding of metabolic disturbances in hereditary anemias.