Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk

Abstract

Background: Nicotinamide riboside (NR) is a recently discovered NAD(+) precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD(+) precursors was unknown.

Objective: We aimed to determine NAD(+) precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk.

Methods: LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD(+) precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk.

Results: Cow milk typically contained ∼12 μmol NAD(+) precursor vitamins/L, of which 60% was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD(+) metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = -0.58, P = 0.014), and NR was degraded by S. aureus Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk.

Conclusions: NR is a major NAD(+) precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD(+) precursor vitamin concentration of milk.

Keywords: LC-MS; metabolomics; milk; nicotinamide adenine dinucleotide; pellagra-preventive factor.

© 2016 American Society for Nutrition.

Figures

FIGURE 1

NR is stable in milk and is degraded by S. aureus. (A) Stability of [18O]-NR in 4 store-bought brands of skim milk and in water at pH 5, 7, and 11 (n = 3) was assessed with LC-MS/MS. NR metabolism by S. aureus was determined with [13C1, D1]-NR and LC-MS (B–H). (B–F) Extracellular concentration of [13C1, D1]-NR, nonlabeled Nam, [13C1]-Nam, nonlabeled NA, and [13C1]-NA in Todd-Hewitt + 50 mmol/L Bis-Tris (pH 6.7) media containing 10 μmol/L [13C1, D1]-NR incubated at 37°C without or with S. aureus RN3170 inoculation (n = 3 for inoculated media). In each panel, the concentration of the metabolite is shown in the noninoculated media (n = 1). (G) Intracellular concentration of endogenous or 13C1 enriched Nam. (H) Intracellular concentration of endogenous 13C1-, or 13C1, D1–enriched NAD+ from extracts of S. aureus RN3170 cell pellets (n = 3/time point). For intracellular measurements, a Holm-Sidak multiple-comparisons post hoc test was performed to test for statistical significance compared with time point zero for each metabolite (B and G). For extracellular measurements, a Holm-Sidak multiple-comparisons test was performed to test for statistical significance compared with noninoculated medium within each time point (C–F and H). Data are represented as means ± SEMs. *P < 0.05, **P < 0.01, and ***P < 0.001. NA, nicotinic acid; Nam, nicotinamide; NR, nicotinamide riboside.

FIGURE 2

NR binding to milk demonstrated by NMR. Organic and conventional skim milk was separated into soluble and resuspended particulate fractions. NR was added to the nonfractionated milk and to the fractions. NR binding was analyzed with NMR as described. (A, E) Normal 1-dimensional 1H NMR spectra of NR. (B–D, F–H) WaterLOGSY spectra of NR in the presence of milk. (A) NR alone. (B) NR + total conventional milk. (C) NR + conventional milk (soluble fraction). (D) NR + conventional milk (particulate fraction). (E) NR alone. (F) NR + total organic milk. (G) NR + organic milk (soluble fraction). (H) NR + organic milk (particulate fraction). The assigned 1H resonances of the nicotinamide ring aromatic protons are labeled. 1H, proton; NR, nicotinamide riboside; ppm, parts per million; WaterLOGSY, water-ligand observed via gradient spectroscopy.