ANALYSIS OF KEY SUGAR PHOSPHATE INTERMEDIATES IN GLYCOLYSIS BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY TO ASSES AND EVALUATE POSSIBLE ALTERATIONS IN METABOLITE POOL OF ERYTHROCYTES IN TYPE 2 DIABETES

In previous studies, it has been shown that the activities of the glycolysis cycle enzyme are significantly altered with changes in kinetic attributes of hexokinase, phosphorfructokinase and lactate dehydrogenase. Moreover, the kinetic parameters of the terminal enzyme lactate dehydrogenase also changed significantly. In view of these observed changes, it was of interest to know if and how the flux of intermediates of the glycolytic pathway was also affected. With this objective, a simple, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for quantification of five important glycolysis intermediates of rate-limiting steps [viz. glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P), fructose-1,6-bis-phosphate (FBP), 3-phosphoglyceric acid (3PGA), and pyruvate in human erythrocytes. Liquid chromatography was performed using Supelco Ascentis Express RP amide (250 mm × 4.6 mm, 5µm) column with mobile phase, 10mM ammonium acetate in water and acetonitrile. The detection of metabolites was carried out by tandem mass spectrometry. In erythrocytes from Type 2 diabetic subjects, there was a significant increase in intracellular concentrations of G-6-P and 3-PGA, with a marked decrease in FBP and pyruvate. The results suggest that the metabolic flux of glycolysis is altered significantly in the erythrocytes from Type 2 diabetic patients. The significance of these findings in relation to adenosine triphosphate (ATP) synthesis and loss of membrane deformability, which lead to diabetic complications, is discussed.