Journal of Nutritional Science and Vitaminology
Online ISSN : 1881-7742
Print ISSN : 0301-4800
ISSN-L : 0301-4800
Regular Paper
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
ジャーナル フリー

2014 年 60 巻 1 号 p. 17-21


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.

© 2014 by the Center for Academic Publications Japan
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