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

Abstract

NAD1 plays critical roles in various biological processes, such as metabolism of carbohydrates (1) and fat (2), the circadian cycle (3,4), and aging (5, 6) through the function of SIRT1. Cellular NAD 1 concentration, which is regulated through the synthesis from nicotinamide (Nam) and nicotinic acid (NA) and breakdown to Nam (Fig. 1A) (7), would thus have a significant impact on mammal physiology, including human. Because the NAD 1 concentrations are determined by the balance of its synthesis and breakdown, measurement of the rates of the synthesis and breakdown are required to understand the mechanisms regulating the concentrations. For this purpose, incorporation of stable isotope-labeled precursors into NAD 1 should be quantified. We have analyzed NAD 1 formed from 2 H (D)-labeled precursors in mammalian cells using a liquid chromatographytandem mass spectrometry (LC/MS/MS) and found the cellular conversion of [2,4,5,6-D4]Nam (Fig. 1B) and [2,4,5,6-D4]NA (Fig. 1B) 1 has not directly been demonstrated. Furthermore, the mechanism for the 1-Da mass decrease in NAD 1 formed with the D4-labeled precursors in the cells remains unknown.In the cells, synthesized NAD 1 is reduced to NADH through the acceptance of an additional hydrogen (H) at the redox site (C-4 carbon of the pyridine ring), whereas the removal of either of the two H atoms at the redox site of NADH reforms NAD 1 (9, 10). The reversible transfer of H to and from either the pro-R or pro-S side at the redox site of NAD 1 /NADH is catalyzed by oxidoreductases exhibiting the opposite stereospecificity for the coenzymes (9, 10). We postulated that after the synthesis of [Nam-2,4,5,6-D4]NAD 1 by NAD 1 biosynthetic enzymes in the cells, D at the C-4 carbon in its pyridine ring is exchanged with H through the cellular redox reactions of NAD 1 /NADH involving oxidoreductases, resulting in [Nam-2,5,6-D3]NAD 1 (Fig. 1C)

NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion

“…Labeled (heavy) or non-labeled (light) NAD + was quantified as described below with LC-MS/MS. For heavy-labeled NAD + , D3-NAD + as well as D4-NAD + was observed as previously reported ( Hara et al, 2014 ). This is due to the replacement of deuterium at C4 position with non-labeled proton during NAD + d this combined value-NADH cycling.…”

Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry

“…To measure R S and R B , cells pre-incubated with unlabeled Nam (d0-Nam) are then incubated with d4-Nam (Fig 1B). Based on the continual synthesis and breakdown of NAD + [15, 16], d3-NAD + is produced in the cell [17] while unlabeled NAD + (d0-NAD + ) is degraded, depending on the incubation time (Fig 1C). We here defined R S and R B as a / t and b / t , respectively, where t , a , and b are respectively the incubation time, the amount of d3-NAD + newly appeared, and that of d0-NAD + decreased during the incubation, as schematically shown in Fig 1C.…”

“…[2, 4, 5, 6-D 4 ]Nam (d4-Nam) was purchased from CDN isotope (Quebec, Canada) and was further HPLC-purified to remove contaminating a significant level of d4-nicotinic acid (NA) (5%) [17]. Nam was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).…”

“…NAD + and 5-phosphoribosyl 1-pyrophosphate (PRPP) were purchased from Oriental Yeast (Tokyo, Japan) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Calibration standards d3-NAD + and d3-Nam were prepared with recombinant NAD + biosynthetic enzymes, as described previously [17]. The concentrations of the labeled standards were determined based on the integrated peak areas of the absorbance at 254 nm compared to those of unlabeled ones on HPLC.…”

“…(A) HepG2N cells [17] were cultured in d0-Nam-free MEM supplemented with 10 μM d4- ( squares ) or d3-Nam ( triangles ). After the incubation for the indicated times, cellular contents of d3-NAD + were quantified.…”