Measurement of glycolysis with heavy water labeling

We introduce a method to calculate the contribution of glycolysis to lactate and the sources of cytosolic NADH, using heavy water labeling, mass spectrometric measurement, and combinatorial analysis (mass isotopomer distribution analysis). The number (n) of deuterium incorporation sites into C-H bonds during flux through glycolysis versus the oxaloacetate/phosphoenolpyruvate-carboxykinase pathway is calculated. The stereospecificity of lactate dehydrogenase reveals the metabolic source of cytosolic NADH used because tricarboxylic acid cycle dehydrogenases have a different spatial orientation (reduce pyruvate from 4R position) than glycolytic GAPDH (4S position, which derives intramolecularly, not from cellular water). We calculate n in lactate and validate the model in vivo in mouse liver and skeletal muscle and in HepG2 cells. We also compare lactate measurement of glycolysis from [U-¹³C₆]-glucose to M₃-lactate. In summary, this heavy water method is operationally simple, provides information about both carbon fluxes and sources of NADH, and is translatable into humans.