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

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.