Comparison of metabolomic steady state phenotypes and metabolic fluxes in response to oxidant stress in fresh and matched deglycerolized RBCs.

(A) Tracing experiments were performed by incubating RBCs with [1,2,3-13C3]glucose, a heavy substrate that can be metabolized in RBCs through (B) the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (generating isotopologue M+3) or through the pentose phosphate pathway (PPP), generating isotopologue M+2 of lactate, (C) owing to the release of the first carbon atom of heavy glucose in the form of 13CO2 at the oxidative phase reactions of the PPP. (F) Schematic overview of glucose uptake and consumption in RBC supernatants and cells, in fresh, or deglycerolized RBCs, either untreated, or treated with the superoxide generator (SOTS-1), (D) demonstrating in both RBCs and media similar glucose consumption rates. (E) Total lactate, i.e., sum of lactate isotopologues (unlabeled M+0, M+3 generated via glycolysis, or M+2 generated via the PPP), in fresh and DG RBCs, either untreated or treated with the superoxide generator (SOTS-1), demonstrating in both RBCs and media similar lactate accumulation. (G and H) Schematic overview of expected isotopologues and relative ratios of lactate, from glycolysis (EMP, M+3 isotopologue) or PPP (M+2) and relative ratios in fresh and DG RBCs, either untreated, or treated with the supoxide generator SOTS-1. (H) Fresh RBCs were characterized by significantly higher levels of supernatant–but not intracellular—M+2 lactate. Conversely, deglycerolized RBCs were characterized by faster glycolysis than fresh RBCs. Lower lactate M+2 export in deglycerolized RBCs following SOTS-1 was observed, while intracellular glycolysis to pentose phosphate pathway ratios were comparable between fresh and deglycerolized RBCs following treatment with SOTS-1. Continuous lines indicate median, while dashed lighter lines are representative of interquartile ranges, according to the color legend left. Symbols indicate p+ = fresh vs DG; & = fresh + SOTS vs DG + SOTS; * = fresh vs fresh + SOTS; # = DG vs DG + SOTS.