Ex vivo glucose tracing in red blood cells from patients with rare hereditary anemias
Metabolism is affected in many rare hereditary red cell disorders, either directly (i.e. enzymopathies) or indirectly (e.g. sickle cell disease). Red blood cells (RBC) have no mitochondria and rely solely on glycolysis for the generation of adenosine triphosphate (ATP). ATP is needed to maintain RBC ion balance, anti-oxidative defense mechanisms and deformability. Disturbed completion of the glycolytic pathway results in an overall lack of energy, which ultimately decreases RBC life span and manifests as hemolytic anemia. Currently, novel therapies are being developed that target pyruvate kinase (PK), a key glycolytic enzyme, with the aim to increase ATP levels. This new form of therapy is currently under (clinical) investigation for various RBC disorders. We aimed to set up a technique that allows us to observe how RBCs metabolize glucose in normal and pathophysiological conditions.
Whole blood was obtained from four healthy controls (HCs), one transfusion-dependent patient with PK deficiency, one transfusion-dependent patient with hexokinase (HK) deficiency, and four patients with hereditary xerocytosis (HX). Purified RBCs were incubated at 37 ⁰C in glucose-free RPMI medium supplemented with 13C6-carbon labeled glucose. Metabolites were extracted from pelleted RBCs and culture medium after incubation for 0, 0.1, 0.5, 1, 2, 4, and 6 hours with 13C6-carbon labeled glucose. Glycolytic metabolites were measured using ultra high pressure liquid chromatography coupled to high resolution mass spectrometry.
Carbon labeled glucose was taken up and metabolized by the RBCs. Over time, we observed a decrease in the level of unlabeled and an increase in the level of carbon labeled glycolytic metabolites. In HCs, unlabeled glycolytic metabolites were almost completely replaced by carbon labeled metabolites after 6 hours. In HK deficient RBCs, we observed reduced formation of carbon labeled glucose 6-phosphate, the metabolite downstream of HK, and decreased formation of carbon labeled pyruvate and lactate. In PK deficient RBCs, we observed accumulation of carbon labeled glycolytic intermediates upstream of PK, including glucose-6-phosphate and phosphoenolpyruvate, and decreased formation of carbon labeled pyruvate and lactate. In HX RBCs, we observed replacement of unlabeled metabolites with carbon labeled metabolites already after 4 hours, and increased production of carbon labeled glucose-6-phosphate, pyruvate and lactate.
We demonstrate a method to measure glycolytic flux in RBCs upon ex vivo incubation with carbon labeled glucose. Our results show stalled glycolytic flux in HK and PK deficient patients, and strongly suggest glycolytic flux is enhanced in HX patients. The results of this study suggest that glucose tracing is a useful tool to better understand (patho)physiology of RBC disorders and it has the potential to study the effect of PK activation therapy in patients with several hereditary red blood cell disorders.