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.
Xie
Wenjun
W
Ames Laboratory, U.S. Department of Energy, Iowa State University, Ames, Iowa 50011, USA.
Xu
Aoshuang
A
Yeung
Edward S
ES
D000818:Animals; D045744:Cell Line, Tumor; D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007834:Lasers; D009243:NAD / Q000032:analysis*; D018384:Oxidative Stress; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D009243:NAD; D006861:Hydrogen Peroxide