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

TitleDetermination of NAD(+) and NADH in a Single Cell under Hydrogen Peroxide Stress by Capillary Electrophoresis
Publication TypeJournal Article
Year of Publication2009
AuthorsXie WJ, Xu AS, Yeung ES
Journal TitleAnalytical Chemistry
Volume81
Pages1280-1284
Date Published02/01
ISBN Number0003-2700
Accession NumberISI:000262915100057
Keywordsastrocytes, dehydrogenase, erythrocytes, extraction, inhibition, injury, metabolism, nicotinamide adenine-dinucleotide, pyridine-nucleotides, synthetase
Abstract

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.

URL<Go to ISI>://000262915100057
DOI10.1021/Ac802249m