Abstract
We report a novel (^13^C, ^2^H) nuclear magnetic resonance (NMR) procedure to investigate lactate recycling through the monocarboxylate transporter of the plasma membrane of cells in culture. C6 glioma cells were incubated with [3‐^13^C]lactate in Krebs‐Henseleit Buffer containing 50% ^2^H~2~O (vol/vol) for up to 30 hr. ^13^C NMR analysis of aliquots progressively taken from the medium, showed: (1) a linearly decreasing singlet at ∼20.85 parts per million (ppm; −0.119 μmol/mg protein/hr) derived from the methyl carbon of [3‐^13^C]lactate; and (2) an exponentially increasing shifted singlet at ∼20.74 ppm (0.227 μmol/ mg protein/hr) from the methyl carbon of [3‐^13^C, 2‐^2^H]lactate. The shifted singlet appears because during its transit through the cytosol, [3‐^13^C]lactate generates [3‐^13^C, 2‐^2^H]lactate in the lactate dehydrogenase (LDH) equilibrium, which may return to the incubation medium through the reversible monocarboxylate carrier. The methyl group of [3‐^13^C, 2‐^2^H]lactate is shifted −0.11 ppm with respect to that of [3‐^13^C]lactate, making it possible to distinguish between both molecules by ^13^C NMR. During incubations with 2.5 mM [1‐^13^C]glucose and 3.98 mM [U‐^13^C~3~]lactate or with 2.5 mM [1‐^13^C]glucose and 3.93 mM [2‐^13^C]pyruvate, C2‐deuterated lactate was produced only from [1‐^13^C]glucose or [U‐^13^C~3~]lactate, revealing that this deuteration process is redox sensitive. When [1‐^13^C]glucose and [U‐^13^C~3~]lactate were used as substrates, no significant [3‐^13^C]lactate production from [1‐^13^C]glucose was detected, suggesting that glycolytic lactate production may be stopped under the high lactate concentrations prevailing under mild hypoxic or ischemic episodes or during cerebral activation. © 2004 Wiley‐Liss, Inc.