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

作者：Xie Wenjun; Xu Aoshuang; Yeung Edward S^*

来源：Analytical Chemistry, 2009, 81(3): 1280-1284.

DOI：10.1021/ac802249m

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 H2O2. 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.

• 出版日期2009-2-1