Determination of lactate dehydrogenase isoenzymes in single rat glioma cells by capillary electrophoresis with electrochemical detection.

A method for determination of lactate dehydrogenase (LDH) isoenzymes in single rat glioma cells (C6) was developed. In this method, a whole cell was electrokinetically injected into the front end of the separation capillary. After that, the cell was lysed by ultrasonication and the isoenzymes in the cell were pre-separated at 20 kV for 5 min and then incubated for 2 min with the enzyme substrates nicotinamide adenine dinucleotide (NAD(+)) and lactate in the capillary electrophoresis running buffer. The electroactive product NADH generated by the isoenzymes through on-capillary enzyme-catalyzed reaction was detected at the outlet of capillary by using the end-capillary amperometric detection with a constant potential mode at a carbon fiber bundle microdisk electrode. Since the amplification of signal via the enzyme reaction, the concentration of nicotinamide adenine dinucleotide (NADH) is much higher than that of LDH. The external standardization was used to quantify isoenzymes in individual cells. Three LDH isoenzymes in single rat glioma cells (C6) were determined and quantified.