CONTINUOUS ENZYME-LINKED FLUOROMETRIC DETECTION OF L-(-LACTATE RELEASED FROM RAT-BRAIN VESICLES UNDER ANOXIC CONDITIONS())

A method is described for the on-line detection of L-(+)-lactate relea sed from brain vesicles under physiological conditions. The principle of L-lactate detection is based on the reversible oxidation of L-lacta te catalysed by L-lactate dehydrogenase (LDH, EC 1.1.1.27) employing 3 -acetylpyridine-adenine-dinucleotide (APAD) as analogue of NAD accordi ng to the reaction: L-lactate + APAD = pyruvate + APADH. In practical terms, L-lactate synthesis of vesicles incubated in the presence of LD H and APAD was continuously followed by the fluorescence (490 nm) of A PADH excited at 410 nm. Addition of a L-lactate standard (10 mu mol/l) enhanced APADH fluorescence with a half-life of 6.0 +/- 0.6 s allowin g us to uncover a short-term alteration of L-lactate synthesis. This m ethod was applied to evaluate a prospective change of L-lactate genera tion caused by the anoxia-induced increase in intravesicular Na+ and C a+ concentration ([Na+](i), [Ca2+](i)), both fluorometrically determin ed 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-lact ate 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 mu mol/l tetrodotoxin (TTX) suppress ed the increase in [Na+](i) and [Ca2+](i) but failed to influence L-la ctate 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 .