标题：Formation of [Nicotinamide-2H3]NAD+ from [2H4]Nicotinamide and [2H4]Nicotinic Acid in Human HepG2N Cells and Involvement of 2H/1H Exchange at the Redox Site of NAD+/NADH
期刊名称：Journal of Nutritional Science and Vitaminology
印刷版ISSN：0301-4800
电子版ISSN：1881-7742
出版年度：2014
卷号：60
期号：1
页码：17-21
DOI：10.3177/jnsv.60.17
出版社：Center for Academic Publications Japan
摘要：To determine the rates of cellular NAD+ synthesis and breakdown, incorporation of stable isotope-labeled precursors into NAD+ should be quantified. Although with 2H (D)-labeled precursors [2,4,5,6-D4]nicotinamide ([D4]Nam) and [2,4,5,6-D4]nicotinic acid ([D4]NA), [D3]NAD+ is formed in human cells, why only three of four D atoms from [D4]Nam and [D4]NA are present in NAD+ remains unknown. Using a liquid chromatography-tandem mass spectrometry, we tested the involvement of D/1H (H) exchange at the redox site of NAD+/NADH (C-4 carbon of the pyridine ring) by oxidoreductases exhibiting opposite stereospecificity for the coenzymes in the 1-Da mass decrease in the cellular NAD+ formation. In all cells examined, [ Nam -D3]NAD+, but not [ Nam -D4]NAD+, was obtained after the incubation with the D4-labeled precursors, whereas [ Nam -D4]NAD+, but not [ Nam -D3]NAD+, was synthesized from the same precursors with purified recombinant NAD+ biosynthetic enzymes. [D4]Nam group of [ Nam -D4]NAD+ was converted to [D3]Nam group via [D4]NADH by in vitro sequential reduction and oxidation with oxidoreductases exhibiting opposite stereospecificity for the coenzymes. Furthermore, using [2,5,6-D3]Nam, which has H instead of D at the C-4 carbon, as a precursor of NAD+ in the cells, the 1-Da mass decrease in the nucleotide was not observed. Based on these observations, we conclude that following the synthesis of [ Nam -2,4,5,6-D4]NAD+, cellular redox reactions of NAD+/NADH convert [ Nam -2,4,5,6-D4]NAD+ to [ Nam -2,5,6-D3]NAD+. Quantification of [ Nam -2,5,6-D3]NAD+ and [2,5,6-D3]Nam would successfully determine the rate of the NAD+ turnover and provide clues to understand regulatory mechanisms of cellular NAD+ concentrations.
