Simultaneous separation of intracellular and extracellular lactate NMR signals of human erythrocytes

Intracellular/extracellular lactate (Lac) distribution has been determined before in human and animal erythrocytes (red blood cells [RBCs]) with various methods. However, all previous methods determine intra- and extracellular Lac separately or indirectly. Now, (13)C-NMR spectroscopy has been used to monitor intra- and extracellular Lac simultaneously in intact RBCs. Isolated human RBCs were incubated with [3-(13)C]-Lac, [3-(13)C]-pyruvate (Pyr), and [1-(13)C]-glucose (Gluc). A distortionless enhancement by polarization transfer (DEPT) sequence was used (TR = 3.3 s, N = 128) to monitor the (13)C-NMR resonances in both compartments. The intra- and extracellular methyl group resonances of Lac and Pyr were clearly separated by 9.6 Hz and 7.0 Hz, respectively, under normoxic conditions due to the RBC chemical-shift effect. The results show that the chemical-shift effect of RBCs is convenient to monitor intra- and extracellular Lac simultaneously in intact RBCs under normoxic conditions.