Determination of NAD⁺and NADH in a Single Cell under Hydrogen Peroxide Stress by Capillary Electrophoresis

A capillary electrophoresis (CE) method based on an enzymatic cycling reaction is developed to determine both NAD(+) and NADH in a single cell in a single run. The detection limit can reach down to 0.2 amol of NAD(+) and 1 amol of NADH with a homemade capillary electrophoresis laser-induced fluorescence (CE-LIF) setup. This method shows good reproducibility and specificity. After an intact cell is injected into the capillary and lysed using a Tesla coil, intracellular NAD(+) and NADH were separated, incubated with the cycling buffer, and quantified by recording the amount of fluorescent product generated. Cellular NAD(+) and NADH levels of a rat myoblast cell line were determined using this method. Both NAD(+) and NADH levels decreased when the cells were exposed to oxidative stress induced by H(2)O(2). This may be due to the activation of the DNA repair enzyme, poly(ADP-ribose) polymerase, in response to the oxidative damage imposed on DNA, since pretreatment of the cells with an inhibitor of these enzymes prevented the reduction of cellular NAD(+) and NADH levels.