Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism

Abstract

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are conserved metabolic cofactors that mediate reduction-oxidation (redox) reactions throughout all domains of life. The diversity of synthetic routes and cellular processes involving the transfer of reducing equivalents to and from these cofactors makes the accurate quantitation and metabolic tracing of NAD(H) and NADP(H) of broad interest. However, current analytical techniques typically rely on standard curves that do not incorporate confounding effects of the sample matrix. We utilized the essential requirement of niacin and tryptophan for NAD synthesis in mammalian cells to devise a stable isotope labeling by essential nutrients in cell culture (SILEC) method for efficient labeling of intracellular NAD(H) and NADP(H) pools. Coupling this approach with detection by liquid chromatography-high resolution mass spectrometry (LC-HRMS), we demonstrate the utility of incorporating a [¹³C₃¹⁵N₁]-nicotinamide moiety into a library of NAD-derived metabolites for use as internal standards in matrixed samples. Using a two-label system incorporating [¹³C₃¹⁵N₁]-nicotinamide and [¹³C₁₁]-tryptophan, we quantify the relative contribution of salvage and de novo NAD synthesis, respectively, in S. cerevisiae and HepG2 human hepatocellular carcinoma cells under basal conditions. As a further proof-of-principle, we demonstrate an improvement in the linear range for quantification of NAD and apply this method to analysis of NAD(H) in mouse liver. This method demonstrates the generalizability of SILEC, and provides a simple method for generating a library of stable isotope labeled internal standards for quantifying and tracing the metabolism of cellular and tissue NAD(H) and NADP(H).

Fig. 1

Biosynthesis and stable isotope labeling of NAD(H)/NADP(H). (A) NAD biosynthetic pathway from tryptophan, niacin, or nicotinamide. (B) Stable isotope labeling using [¹³C₃, ¹⁵N₁]-nicotinamide with the resulting incorporation of isotopes into NAD.

Fig. 2

Chromatograms of NAD analogs. (A) Co-elution of stable isotope analogs with their unlabeled analogs from second generation HepG2 cell extract analyzed by LC-HRMS. There was no appreciable pure [¹³C] labeling observed in HepG2s under conditions tested. (B) Interference in the channel for [¹³C₃]-NAD+/NADP+ (retention time 4.2 and 6.4 min, respectively) was observed from [¹³C₃¹⁵N₁]-NADH/NADPH (retention time 6.2 and 6.6 min, respectively) even at a 5 ppm window with 60 000 resolving power.

Fig. 3

Calibration curves for NAD by LC-HRMS. Calibration curve for quantification of NAD by an untargeted metabolomics method (A) without or (B) with isotope dilution (ID). A targeted NAD method (C) without or (D) with ID. Inserts of the lower points are provided to highlight the non-linearity observed without ID.

Fig. 4

Liver samples from NR versus vehicle treated mice. Liver samples from mice treated with vehicle or NR at 400 mg kg⁻¹ day⁻¹ were analyzed by SID-LC-HRMS for (A) NAD, (B) NADH content.