Determination of oxidized and reduced nicotinamide adenine dinucleotide in cell monolayers using a single extraction procedure and a spectrophotometric assay.
While many investigations measuring oxidized nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADH) have been carried out on several mammalian tissues and blood cells, few reports have dealt with monolayers of cultured cells. Here we show a novel method to measure NAD+ and NADH in monolayers of a neuroblastoma cell line. The method was established by modifying a single extraction procedure originally developed for erythrocytes and an enzymatic cycling assay using a dye that absorbs in visible range. The following modifications were made. (i) Addition of 0.05% of a detergent, Triton X-100, to carbonate-bicarbonate extraction buffer enabled us to accurately measure cellular [NADH]/([NAD+]+[NADH]). (ii) Addition of N-ethyldibenzopyrazine ethyl sulfate salt (phenazine ethosulfate) immediately before the incubation suppressed the gradual decline of the sensitivity of the assay. The procedure presented here provides a simple and inexpensive measurement of NAD+ and NADH in cell monolayers.
Umemura
Ken
K
National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8551, Japan.
Kimura
Hideo
H
D000818:Animals; D002021:Buffers; D006358:Hot Temperature; D051379:Mice; D009243:NAD / Q000032:analysis* / Q000302:isolation & purification; D009447:Neuroblastoma / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010084:Oxidation-Reduction; D010619:Phenazines; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D002021:Buffers; D010619:Phenazines; D009243:NAD; C028122:5-ethylphenazine; D017830:Octoxynol