A method is described for the on-line detection of L-(+)-
lactate released from brain vesicles under physiological conditions. The principle of L-
lactate detection is based on the reversible oxidation of L-
lactate catalysed by
L-lactate dehydrogenase (LDH, EC 1.1.1.27) employing 3-acetylpyridine-adenine-dinucleotide (
APAD) as analogue of
NAD according to the reaction: L-
lactate +
APAD reversible
pyruvate +
APADH. In practical terms, L-
lactate synthesis of vesicles incubated in the presence of LDH and
APAD was continuously followed by the fluorescence (490 nm) of
APADH excited at 410 nm. Addition of a L-
lactate standard (10 mumol/l) enhanced
APADH fluorescence with a half-life of 6.0 +/- 0.6 s allowing us to uncover a short-term alteration of L-
lactate synthesis. This method was applied to evaluate a prospective change of L-
lactate generation caused by the
anoxia-induced increase in intravesicular Na+ and Ca2+ concentration ([Na+]i, [Ca2+]i), both fluorometrically determined by
SBFI and Fura, respectively. Upon
anoxia, [Na+]i and [Ca2+]i increased continuously up to 40 mmol/l Na+ and 900 nmol/l Ca2+ within 400 s. Concurrently, intravesicular
NADH ([
NADH]i) and basal L-
lactate synthesis were enhanced within a few seconds, the latter from 4.2 +/- 1.5 to 15.8 +/- 1.5 nmol L-
lactate/min per mg
protein. Incubation of vesicles in the presence of 10 mumol/l
tetrodotoxin (TTX) suppressed the increase in [Na+]i and [Ca2+]i but failed to influence L-
lactate synthesis. The data indicate a continuous Na+ influx via voltage-dependent Na+ channels accompanied by an increase in [Ca2+]i during
anoxia which did not affect anaerobic L-
lactate synthesis. The method of fluorometric L-
lactate determination was confirmed to be suitable for the detection of L-
lactate released under physiological conditions from brain vesicles and seems to be applicable to various cell models.