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

To determine the rates of cellular NAD(+) synthesis and breakdown, incorporation of stable isotope-labeled precursors into NAD(+) should be quantified. Although with H-2 (D)-labeled precursors [2,4,5, 6-D-4]nicotinamide ([D(4)Nam) and [2,4,5, 6-D-4]nicotinic acid ([D-4]NA), [D-3]NAD(+) is formed in human cells, why only three of four D atoms from [D-4]Nam and [D-4]NA are present in NAD(+) remains unknown. Using a liquid chromatography-tandem mass spectrometry, we tested the involvement of D/H-1 (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-D-3]NAD(+), but not [Nam-D(4)NAD(+), was obtained after the incubation with the D-4-labeled precursors, whereas [Nam-D-4]NAD(+), but not [Nam-D-3]NAD(+), was synthesized from the same precursors with purified recombinant NAD(+) biosynthetic enzymes. [D-4]Nam group of [Nam-D-4]NAD(+) was converted to [D-3]Nam group via [D-4]NADH by in vitro sequential reduction and oxidation with oxidoreductases exhibiting opposite stereospecificity for the coenzymes. Furthermore, using [2,5,6-D-3]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-D-4]NAD(+), cellular redox reactions of NAD(+)/NADH convert [Nam-2,4,5,6-D-4]NAD(+) to [Nam-2,5,6-D-3]NAD(+) Quantification of [Nam-2,5,6-D-3]NAD(+) and [2,5,6-D-3]Nam would successfully determine the rate of the NAD(+) turnover and provide clues to understand regulatory mechanisms of cellular NAD(+) concentrations.