`
`
`
`flV’THEjOURNALOFNUTRITION
`
`The Journal of Nutrition. First published ahead of print April 6, 2016 as doi: 10.3945Ijn.116.230078.
`
`line Journal of Nutrition F
`Genomics, Proteomics, and Metabolomias ASN
`
`Nicotinamide Riboside Is a Major NAD+
`Precursor Vitamin in Cow Milk"3
`
`Samuel AI Trammell,"5 Liping 3m:-6 Philip Redmt 7 Marie E Migaud,” and Charles Brenner“5‘
`
`‘Department of Biochemistry. Carver College of Medicine, slnlerdisciplinary Graduate Program in Genetics, and 6Nuclear Magnetic
`Resonance Facility, Carver College of Medicine, University of Iowa. Iowa City, IA; and 7Queen‘s University Belfast, School of Phamlacy,
`Belfast. Northem Ireland, United Kingdom
`
`Abstract
`
`Background: Nicotinamide rboside (NR) is a recently discovered NAD+ precursor vitamin with a unique biosynthetic
`pathway. Although the presence of NH 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 NH binding to substances in milk.
`Results: Cow mik typically contained ~12 pmol W precursor vitamins/L, of which 60% was presmt as nicotinamide and
`40% was presert as NR. Nicotinic acid and other NAD* metabolites were below the limits of detection. Mlk from samples
`testing positive for Smphylococcus aureus contained lower concentrations of NR (Spearman p = —0.58, P: 0.014), and NR
`was degraded by S. aileus. Convertional milk contained more NR tlnn milk sold as organic. Nonetheless, NR was stable in
`orgnic milk and exhbited an NMR spectnm 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. J Nutr doi: 10.3945/1n.116.230078.
`
`Keywords:
`
`LC MS, metabolomics, milk, nicotinamide adenine dinudeotide, pellagra preventive factor
`
`Introduction
`
`One hundred years ago, pellagra was common in the rural
`American South. One of the early treatments for pellagra was
`consumption of a 1.5 2 pints of cow milk (1). In 1937,
`nicotinamide and nicotinic acid (NA)8 were identified as
`pellagra preventive (PP) factors (2, 3), and tryptophan was
`subsequently discovered as a molecule with PP activity (4).
`Nicotinamide and NA, which are collectively termed niacin,
`contain a pyridine ring that can be salvaged to form NAD’ in
`2 or 3 enzymatic steps, whereas tryptophan is the de novo
`
`' Supported by the Roy J. Carver Trust.
`2 Author dsdosues: C Brenner is an inventor of intellectual property enabling
`commercial development of nicotinamide riboside. He is also a member of the
`Scientific Advisory Board anda stockholderot ChromaDex, Inc., which develops
`aid sells nicotinamide riboside. SNTrarrmell, LYu, P Redpath, and ME Migaud,
`no conflicts of interest.
`3 Supplemental Tables 1 and 2 are avalable from the "Onl‘ne Supporting
`Material" Ink in the mine posting of the article and from the same link in the
`online table of contents at htth/innnritionorg.
`3 Abbreviations used: NA, nicotinic and; NR, ricotinarride riboside; PP, pellaga
`preventive; WaterLOGSY, water ligand observed via gadient spectroscopy.
`'To whom correspondence should be addreswd. E ma‘l: Charles brennerO
`uiowa.edu.
`
`precursor of NAD’, requiring 7 enzymatic steps (5). largely
`because tryptophan can be incorporated into protein, oxidized
`as a fuel, and convened to many other metabolites such as
`serotonin, 50 60 mg tryptophan is considered the niacin
`equivalent of 1 mg nicotinamide or NA. In addition, much of
`the niacin equivalent in food is, in fact, NAD’ (6).
`NAD‘ and its phosphorylated and oxidized derivatives,
`NAD(P)", NAD(H), and NAD(P)H, are essential hydride
`transfer cofactors in hundreds of oxidoreductase reactions and
`
`consumed substrates of several classes of enzymes with activities
`required for DNA repair, gene expression, regulation of energy
`metabolism, and calcium mobilization (7). NAD‘ is one of the
`most abundant metabolines in the human body and is turned
`over at a rapid pace (8), requiring near constant replenishment.
`Hence, although pellagra is described as niacin deficiency, at a
`cellular level, pellagra is a disease of NAD" depletion as a result
`of diets deficient in NAD’ precursors.
`It has long been known that the NAD‘ precursors in milk
`include nicotinamide (9) and tryptophan (10). More recently, it
`has been discovered that milk also contains nicotinamide riboside
`
`(NR), another salvageable NAD’ precursor vitamin (11). Boost
`ing NAD+ concentrations with NR extends life span in yeast (12)
`
`© 2016 American Sodety for Nutrition.
`Manusa'pt received Jammy 14, 2016. Initial review oornpleted February 12, 2016. Revision accepted Mardt 1, 2016.
`doi: 10.3945lin.11623m)78.
`
`Copyright (C) 2016 by the American Society for Nutrition
`
`1 of 7
`
`THORNE - EXHIBIT 1030
`
`
`
`
`
`9lOZ'SlllldV“0SEIHWEI'IVMOI:lOMISHSAINn13610'uomiznu‘uluroaPWBOIUMDG
`
`
`
`
`
`
`
`THORNE - EXHIBIT 1030
`
`

`

`Downloaded from
`
`jn.nutrition.org
`
` at UNIVERSITY OF IOWA LIBRARIES on April 13, 2016
`
`and has been shown to prevent and treat metabolic (13 16) and
`neurodegenerative (17, 18) conditions in mouse models. Although
`these studies suggest that dairy products as a source of NR could
`be beneficial to many aspects of human health, the amount of NR
`in milk has not been established. In this work, we determine the
`complete NAD+ precursor vitamin concentration in individual
`and pooled commercial samples of cow milk with the use of an LC
`tandem MS (LC MS/MS) based method (19). Our results show
`that milk from Staphylococcus aureus positive samples contained
`lower concentrations of NR and nicotinamide, and that milk
`sold as organic milk contained lower concentrations of NR than
`conventionally sourced milk. Moreover, we show that NR is
`stable in milk and bound by substances in milk and that ~40% of
`the NAD+ precursor vitamin concentration of cow milk is present
`as NR.
`
`Methods
`
`Milk quality and herd health measurements. Milk flows and
`representative samples were obtained from 19 conventionally raised
`cows with a Dairy Herd Improvement Association testing meter. Samples
`were dispensed into 2 oz snap cap Dairy Herd Improvement Association
`vials containing liquid bronopol for analysis by Dairy Lab Services of
`fat, protein, lactose, other solids, milk urea nitrogen, and somatic cells
`(FOSS). Additional aseptic individual milk samples were obtained for
`bacterial analysis after teats were sterilized with 70% ethanol before
`collecting 3 mL milk from each teat into 12 3 75 mm culture tubes. All
`samples were frozen before further analysis. Blood agar culture plates
`were inoculated with sample and then incubated at 37°C and evaluated
`for bacterial growth at 24 and 48 h. Bacterial growth was characterized
`by morphology, and samples were subjected to confirmatory tests to
`identify genus and species.
`
`Milk sample acquisition and preparations. Nineteen milk samples
`from individual cows plus 8 skim milk samples (4 organic and 4
`conventional) purchased in the Iowa City area were analyzed by
`LC MS/MS. Two 50 mL aliquots were extracted from each milk
`sample. Each aliquot was dosed with either solution A [18.75 pmol
`[18O1] NR, 18.75 pmol [18O1] nicotinamide, 18.75 pmol [D3, 18O1]
`1 methyl nicotinamide, and 150 pmol [D4] NA] or solution B, a
`13C labeled yeast extract at 1:50 dilution. Aliquots were extracted with
`0.5 mL of 1.5% formic acid at room temperature. Each aliquot was
`vortexed for 10 s and then sonicated for 10 min in a bath sonicator.
`Aliquots were then centrifuged at a speed of 16,100 3 g for 10 min at
`room temperature. Extracts were transferred to fresh 1.5 mL centrifuge
`tubes and dried overnight via speed vacuum. Recovery was >90% for
`all metabolites of interest.
`
`NMR spectroscopy. NR 1H resonances were assigned with 1H/13C
`2 dimensional heteronuclear multiple quantum coherence and hetero
`nuclear multiple bond coherence experiments. NR binding to skim
`milk was analyzed with the use of water ligand observed via gradient
`spectroscopy (WaterLOGSY) (20, 21). To analyze fractions of milk for
`NR binding activity, 2 mL total milk was centrifuged for 1 h at 4°C at
`16,100 3 g. The supernatant was termed the soluble fraction, whereas
`the pellet resuspended in 2 mL 50 mmol/L sodium phosphate (pH 7) was
`termed the particulate fraction. NMR samples were prepared by adding
`150 mL skim milk, skim milk soluble fraction, or resuspended skim
`milk particulate fraction to 352.2 mL buffer that contained 300 mL
`50 mmol/L sodium phosphate (pH 7), 50 mL D2O, and 2.2 mL NR
`stock, giving a final NR concentration in the NMR samples of
`0.3 mmol/L. For the WaterLOGSY experiment, a T2 relaxation filter of
`100 ms was used just before data acquisition to suppress signals derived
`from macromolecules, and a water nuclear Overhauser effect mixing
`time of 1 s was used in the experiment. All NMR data were acquired
`with a Bruker Avance II 800 MHz NMR spectrometer equipped with a
`sensitive cryoprobe and recorded at 25°C. The 1H chemical shifts were
`referenced to 2,2 dimethyl 2 silapentane 5 sulfonate. NMR spectra
`
`2 of 7 Trammell et al.
`
`were processed with the NMRPipe package software (22) and analyzed
`with NMRView (23).
`
`NR stability assays. [18O] labeled and [13C1, D1] NR were synthesized
`as described (24) (S Trammell, M Schmidt, B Weidemann, P Redpath,
`F Jaksch, R Dellinger, M Migaud, C Brenner, unpublished results, 2016).
`[18O] NR was suspended in conventional and organic milk samples or in
`water at pH 5, 7, or 11 at a concentration of 10 mmol/L and allowed to
`sit at room temperature. In total, 20 mL aliquots were collected at 0, 0.5,
`1, 2, 4, and 8 h and extracted as described above.
`
`S. aureus growth experiments. Strain RN3170 was a kind gift from
`Patrick Schlievert (University of Iowa) (25). Bacteria were streaked onto
`Todd Hewitt (Becton & Dickinson) 2% agar plates, incubated overnight
`at 37°C, and then stored at 4°C. S. aureus was then inoculated into Todd
`Hewitt media containing 50 mmol/L Bis Tris (pH 6.7) and 10 mmol/L
`[13C1, D1] NR at a starting OD600 nm of 0.1. Noninoculated medium
`was used as control for NR stability. All cultures were incubated at 37°C
`with constant shaking at 220 rpm, and 15 mL aliquots were collected at
`0, 1, 2, 4, 6, and 8 h. OD600 nm and pH values were recorded at each time
`point. Aliquots were centrifuged at 2060 3 g for 30 min at 4°C, at which
`time 1 mL culture medium was collected and snap frozen in liquid
`nitrogen. The remainder of culture medium was aspirated and cell pellets
`were washed with 1 mL ice cold PBS and centrifuged at 16,100 3 g for
`10 min at 4°C. PBS was aspirated and pellets were flash frozen. Then,
`50 mL media was analyzed with LC MS/MS as described below. Cells
`were extracted with buffered ethanol (3 parts ethanol to 1 part 10 mmol/L
`HEPES, pH 7.1) heated to 80°C for 3 min with constant shaking at
`1050 rpm. Extracts were clarified by centrifugation (16,100 3 g, 10 min,
`4°C). Pellets were extracted again following the same procedure as
`above. Supernatants from both rounds of extraction were combined
`and dried via speed vacuum. Extracts were analyzed with LC MS as
`described below.
`
`LC MS and LC MS/MS. Media samples were diluted 1:1 with double
`distilled H2O. Standard solutions in double distilled H2O were diluted
`1:1 with noninoculated Todd Hewitt media containing 50 mmol/L Bis
`Tris and [13C1, D1] NR, producing a standard curve with the final
`concentrations of 0, 0.1, 0.3, 0.5, 1, 3, 5, and 10 mmol/L. Quality control
`samples at a final concentration of 0.75 and 7.5 mmol/L were also
`prepared by diluting standard 1:1 with media. Then, 10 mL of media
`samples, quality controls, and standards was injected and quantified
`with a Waters TQD mass spectrometer with the use of the acid
`separation chromatographic conditions described previously (19). Me
`dia were quantified with raw peak areas and converted to micromoles
`per liter with background subtracted standard curves.
`For cow milk, standards (final concentrations of 0.08, 0.24, 0.8,
`2.4, 8, 24, 80, and 120 mmol/L) and 2 quality control samples (final
`concentration of 2.5 and 25 mmol/L) were treated in the same manner as
`the samples and as described above. In total, 5 mL of samples, quality
`controls, and standards containing solution A or 10 mL of those
`containing solution B was loaded onto the column and quantified with a
`Waters TQD mass spectrometer according to the procedures previ
`ously described (19). Newly quantified metabolites in the acidic separa
`tion, N methyl nicotinamide, N methyl 2 pyridone 5 carboxamide, and
`N methyl 4 pyridone 5 carboxamide, were assayed with the following
`transitions: 1 methyl nicotinamide (m/z = 137 > 94), N methyl 2
`pyridone 5 carboxamide (m/z = 153 > 107), and N methyl 4 pyridone
`5 carboxamide (m/z = 153 > 136). Analyte peak areas were normalized to
`internal standard peak areas and converted to mmol/L with the standard
`curve. S. aureus cell pellets were suspended in 50 mL of 10 mmol/L
`ammonium acetate in LC MS grade water. A260 nm values for each sample
`were measured with a Thermo Scientific 2000c Nanodrop spectropho
`tometer operated in nucleic acid mode. Samples at 0 and 1 h time points
`were diluted 1:1 with either solution A or solution B. Samples at all
`remaining time points were diluted to a final A260 nm value of 14 and then
`diluted 1:1 with solution A or B. All samples were analyzed according to
`the chromatography protocols previously described (19) and detected and
`quantified with a Waters Q Tof Premier mass spectrometer operated in
`positive full scan mode. The alkaline separation was altered by increasing
`
`
`
`

`

`Downloaded from
`
`jn.nutrition.org
`
` at UNIVERSITY OF IOWA LIBRARIES on April 13, 2016
`
`the flow rate to 0.55 mL/min and shortening the runtime to 11.6 min.
`Separation was performed with the use of a modified gradient with initial
`equilibration at 3% B; a 0.9 min hold; a gradient to 50% B over 6.3 min,
`followed by a 1 min wash at 90% B; and a 3 min reequilibration at 3% B.
`When performing the alkaline separation, the scanning window was set to
`m/z = 120 800 with a scan rate of 0.1 and an interscan rate of 0.01. When
`performing the acid separation, the scanning window was set to m/z =
`120 600 with a scan rate of 0.5 and an interscan delay of 0.05. In both
`cases, leucine enkephalin was infused and used for mass accuracy
`correction. Analyte peak areas were normalized to internal standard
`peak areas and converted to mmol/L with the standard curve. Nicotin
`amide concentrations were corrected for the contribution of [12C1]
`nicotinamide and [13C1] nicotinamide to the [18O1] nicotinamide internal
`standard area counts. Enrichment for all metabolites was corrected for the
`natural abundance of the analyte, 13C abundance, and the purity of the
`doubly labeled NR (95/5% [13C1, D1] NR/[13C1] NR). The corrected
`concentrations of each analyte were converted to intracellular concentra
`tions by calculating the total intracellular volume of S. aureus with an
`intracellular volume of 0.28 fL (26) and an assumption of 1 3 109 cells/mL
`per OD600 nm (27).
`
`Statistical analysis. Unless otherwise stated, all values are expressed as
`means 6 SDs. Two tailed, unpaired t tests were performed on all com
`parisons involving 2 groups. Outliers were identified with the robust
`regression and outlier removal method (28). Two factor, repeated
`measures ANOVA followed by Holm SidakÕs multiple comparisons test
`was performed on experiments involving S. aureus. Media samples were
`compared with noninoculated medium within time points. Intracellular
`samples were compared with initial concentrations within condition.
`Spearman rank correlation coefficient was calculated for the concentra
`tion of nicotinamide and NR compared with the milk quality, herd
`health metrics, and breed. P < 0.05 was considered significant. Statisti
`cal analyses were performed with GraphPad Prism version 6.00 for
`Windows (GraphPad Software).
`
`Results
`
`NR is a major component of the NAD+ precursor vitamin
`concentration in cow milk. The NAD+ metabolome of 19
`individual cow milk samples was determined with LC MS/MS
`and isotope dilution techniques. We define the NAD+ precursor
`vitamin concentration as the concentrations of salvageable
`NAD+ precursor (nicotinamide, NA, and NR) plus concentra
`tions of the higher molecular weight species [nicotinic acid
`riboside, nicotinic acid mononucleotide, NAD+, nicotinic acid
`adenine dinucleotide, and NAD(P)+] from which a vitamin can
`be released by enzymatic or chemical decomposition. NAD(H)
`and NAD(P)H are oxidized in extraction, such that these
`
`TABLE 1 Mean NAD+ metabolomes of 18 raw cow milk
`samples1
`
`Metabolome, mmol/L
`
`Total (n = 18)
`
`S. aureus
`negative (n = 12)
`
`S. aureus
`positive (n = 6)
`
`Nicotinamide
`NR
`NA
`NMN
`NAD+
`NAR
`NAD(P)+
`NAAD
`
`7.3 6 1.52
`4.3 6 2.6
`,1.0
`,0.4
`,0.08
`,0.04
`,0.02
`,0.01
`
`7.7 6 1.2
`5.1 6 2.6
`,1.0
`,0.4
`,0.08
`,0.04
`,0.02
`,0.01
`
`6.4 6 1.7
`2.7 6 1.9
`,1.0
`,0.4
`,0.08
`,0.04
`,0.02
`,0.01
`
`metabolites, if present, would contribute to the peaks of NAD+
`and NAD(P)+.
`As shown in Table 1, in all 19 samples, nicotinamide and NR
`and no other NAD+ metabolite were quantifiable. Thus, neither
`NAD+ nor NA is a PP factor in milk. Excluding one unusual milk
`sample that contained 24 mmol nicotinamide/L and 27 mmol
`NR/L (Supplemental Table 1), milk samples contained 7.3 6
`1.5 mmol nicotinamide/L and 4.3 6 2.6 mmol NR/L. To
`determine whether other parameters correlate with NAD+
`precursor vitamin concentrations in the 18 remaining samples,
`breed was recorded and metrics of the health and milk quality
`of each cow were measured (Supplemental Table 2). As shown
`in Table 2, concentrations of NR positively correlated with
`concentrations of lactose (P = 0.013) and milk urea nitrogen
`(P = 0.018), whereas nicotinamide negatively correlated
`with somatic cell count (P = 0.029) and positively correlated with
`NR (P = 0.011). NR concentration negatively correlated
`with S. aureus infection (P = 0.014). Nicotinamide concentra
`tion also negatively correlated with S. aureus infection, but the
`correlation was not significant (P = 0.09). When we recalculated
`concentrations of NR and nicotinamide in the 12 samples
`without S. aureus infection or extremely high concentrations of
`NAD+ precursor vitamins, nicotinamide rose to 7.7 6 1.2 mmol/L
`and NR rose to 5.1 6 2.6 mmol/L. Thus, although it was clear
`from previous work that there is no NA in cow milk (9), there has
`been a substantial underreporting of NAD+ precursor vitamin on
`account of lack of an assay for NR.
`
`S. aureus depletes NR and nicotinamide. Because the
`presence of S. aureus was associated with lower concentrations
`of NR and nicotinamide, we tested whether S. aureus growth
`might directly alter the concentrations of these metabolites in
`rich media. Before testing stability in the presence of S. aureus,
`we investigated the stability of [18O] NR in pasteurized cow
`milk or in water adjusted to pH values of 5.0, 7.0, and 11.0. As
`shown in Figure 1A, [18O] NR was stable in pasteurized milk and
`in water at neutral pH but exhibited lesser stability at pH 5.0 and
`pH 11.0, with pH 11.0 producing nearly complete hydrolysis
`within 1 h. We measured the pH of cow milk in 4 store bought
`milk brands and determined the pH to be 6.72 6 0.01.
`Bacteria might alter concentrations of NR found in milk by
`incorporating NR intact into NAD+ and/or by converting NR
`into nicotinamide or NA, either of which could be subsequently
`incorporated into NAD+. To distinguish between these possibil
`ities, we synthesized a double labeled NR containing a 13C in the
`
`TABLE 2 Correlations of cow milk NR and nicotinamide
`concentrations with breed and indicators of milk quality1
`
`NR, mmol/L
`
`Nicotinamide, mmol/L
`
`Breed
`Fat, %
`Protein, %
`Lactose, %
`Nonfat solids, %
`Total solids, %
`Somatic cell count, 3103 cells/mL
`Urea nitrogen, mg/dL
`S. aureus
`NR, μmol/L
`
`0.28
`0.14
`0.21
`0.58*
`0.13
`0.08
`0.38
`0.55*
`0.57*
`
`0.21
`0.39
`0.13
`0.5
`0.07
`0.3
`0.52*
`0.27
`0.41
`0.58*
`
`1 NA, nicotinic acid; NAAD, nicotinic acid adenine dinucleotide; NAR, nicotinic acid
`riboside; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside.
`2 Mean 6 SD (all such values).
`
`1 Spearman correlation coefficients were determined between NAD+ precursor
`vitamins and milk quality and cow breed with data from Supplemental Table 1 (except
`cow 3) and Supplemental Table 2. *P , 0.05. NR, nicotinamide riboside.
`
`Nicotinamide riboside concentrations in cow milk
`
`3 of 7
`
`
`
`

`

`Downloaded from
`
`jn.nutrition.org
`
` at UNIVERSITY OF IOWA LIBRARIES on April 13, 2016
`
`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 con
`centration 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 mmol/L [13C1, D1] NR incubated
`at 37°C without or with S. aureus
`RN3170 inoculation (n = 3 for inocu
`lated media). In each panel, the con
`centration of the metabolite is shown
`in the noninoculated media (n = 1). (G)
`Intracellular concentration of endoge
`nous or 13C1 enriched Nam. (H) Intra
`cellular concentration of endogenous
`13C1 , or 13C1, D1 enriched NAD+ from
`extracts of S. aureus RN3170 cell
`pellets (n = 3/time point). For intra
`cellular 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 signif
`icance compared with noninoculated me
`dium within each time point (C F and H).
`Data are represented as means 6 SEMs.
`*P , 0.05, **P , 0.01, and ***P ,
`0.001. NA, nicotinic acid; Nam, nicotin
`amide; NR, nicotinamide riboside.
`
`nicotinamide moiety and a D1 in the ribose. Incorporation of NR
`into the bacterial NAD+ pool would be accompanied by a 2 Da
`mass shift (m/z 664 / 666), whereas breakdown of NR to
`nicotinamide or NA would be accompanied by appearance of
`1 Da shifts in the peaks of these metabolites and a 1 Da mass
`shift in bacterial NAD+.
`Three individual colonies of S. aureus strain RN3170 were
`cultured separately in Todd Hewitt media supplemented with
`10 mmol [13C1, D1] NR/L and buffered at pH 6.7 with 50 mmol
`Bis Tris/L. The inoculated media and a noninoculated medium
`control were incubated at 37°C with constant shaking over an
`8 h period. Clarified media and cell pellets were collected and
`analyzed by LC MS/MS and LC MS. The pH of the clarified
`media was also recorded at each time point. The pH consistently
`remained between 6.5 and 6.7 until between the 6 and 8 h
`time points, at which time the pH rose to 7.8. As shown in Figure
`1B, [13C1, D1] NR was stable in noninoculated medium over the
`time course of the experiment. However, S. aureus inoculation
`significantly decreased the concentration of extracellular NR
`
`within 1 h and eliminated the presence of NR as an extracellular
`metabolite within 4 h. As shown in Figure 1C, singly labeled
`nicotinamide appeared in growth media within 1 h. As shown in
`Figure 1D F, at 4 h, there was a simultaneous rise in singly
`labeled cellular NAD+, singly labeled cellular nicotinamide, and
`extracellular NA.
`Todd Hewitt media contain beef heart extract, nicotina
`mide, and NA (29). As shown in Figure 1G, H, nonlabeled
`nicotinamide was exhausted within 4 h, whereas the non
`labeled NA slowly declined. Thus, S. aureus principally uses
`NR as an extracellular source of nicotinamide. Consistent with
`nicotinamide deamination (30 32), S. aureus can also degrade
`NR and nicotinamide to NA. Because NR was eliminated by
`4 h and the rise in pH occurred after 6 h, pH mediated
`mechanisms cannot be responsible for S. aureus mediated NR
`instability.
`
`NR concentration as a function of organic certification.
`Milk with organic certification requires avoidance of synthetic
`
`4 of 7 Trammell et al.
`
`
`
`

`

`Downloaded from
`
`jn.nutrition.org
`
` at UNIVERSITY OF IOWA LIBRARIES on April 13, 2016
`
`TABLE 3 NAD+ precursor concentrations in commercial cow milk1
`
`Organic
`
`Conventional
`
`Brand A Brand B Brand C Brand D
`
`All
`
`Brand A Brand B Brand C Brand D
`
`All
`
`Nicotinamide, μmol/L
`NR, μmol/L
`
`2.4
`3.1
`
`7.1
`0.84
`
`5.0
`2.2
`
`7.9
`1.4
`
`5.6 6 2.5
`1.9 6 1.0
`
`5.6
`2.5
`
`0.67
`1.7
`
`8.9
`5.4
`
`5.4
`2.7
`
`5.2 6 3.4
`3.1 6 1.6
`
`1 Values are expressed as means 6 SDs, n
`
`4. NR, nicotinamide riboside.
`
`chemical inputs, irradiation, genetically modified seed, and ad
`herence to certain standards of feed, housing, and breeding (33).
`Because one or more of these variables could affect NAD+
`precursor vitamin concentrations in milk, we purchased 4 brands
`of conventional and 4 brands of organic milk and quantified the
`NAD+ metabolome. As observed in milk samples from individual
`cows, only nicotinamide and NR were above the limit of
`quantification (Table 3). In 3 of 4 conventional samples and 3 of
`4 organic samples, the concentration of nicotinamide exceeded that
`of NR. Moreover, the concentration of nicotinamide was similar in
`conventional (5.2 6 3.4 mmol/L) and organic (5.6 6 2.5 mmol/L)
`milk. In the samples we obtained, NR tended to have a higher
`concentration in conventional (3.1 6 1.6 mmol/L) compared with
`organic (1.9 6 1.0 mmol/L) milk. We note that only one brand of
`store bought milk had a combined NAD+ precursor vitamin
`concentration (8.9 mmol nicotinamide/L plus 5.4 mmol NR/L)
`that was higher than the mean of S. aureus negative individual
`cow samples (7.7 mmol nicotinamide/L plus 5.1 mmol NR/L). NR
`concentration may be lower as a function of organic dairy practices,
`but sample sizes were too small for adequate assessment. Future
`work following NR concentration before and after processing in
`conventional and organic dairies will be necessary to assess NR
`concentration as a function of dairy practices.
`
`NR is a bound metabolite in cow milk. Although a higher
`level of S. aureus infection in organic milk production (32)
`
`could potentially account for lower concentrations of NR in
`organic milk, there was no change in nicotinamide and no
`appearance of NA that would be consistent with bacterial
`exposure. As shown in Figure 1, NR is more stable in milk than
`in water, suggesting that the metabolite might be complexed to
`a protective factor.
`Organic dairies frequently ultrapasteurize milk at 135°C for
`2 s, whereas most conventional dairies pasteurize at 72°C for
`15 s (34). Although ultrapasteurization is employed to kill
`bacterial spores, it might damage a macromolecule responsible
`for the stabilization of NR. WaterLOGSY NMR measurements
`were used to detect and map protons in NR (Figure 2A, E) that
`are potentially bound by slower rotating macromolecules in
`milk (20, 21). As shown in Figure 2B and 2F, when NR was
`added to conventional or organic skim milk, 4 aromatic protons
`(H2, H4, H5, and H6) from the nicotinamide moiety of NR
`produced positive WaterLOGSY signals consistent with protein
`binding from both sources of milk. Interestingly, when conven
`tional and organic milk were separated into soluble and
`resuspended particulate fractions, the conventional soluble
`fraction retained more NR binding activity than did the or
`ganic soluble fraction (Figure 2C and 2G). Consistent with
`denaturation of an NR binding protein by heat, the solubilized
`organic particulate fraction produced stronger NR Water
`LOGSY signals than did the solubilized conventional particulate
`fraction (Figure 2D and 2H).
`
`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.
`
`Nicotinamide riboside concentrations in cow milk
`
`5 of 7
`
`
`
`

`

`Downloaded from
`
`jn.nutrition.org
`
` at UNIVERSITY OF IOWA LIBRARIES on April 13, 2016
`
`10. Sutton TS, Esh GC. The nutrition of the newborn dairy calf: I. Changes
`in the tryptophan content of the blood plasma following birth and the
`ingestion of colostrum. J Dairy Sci 1947;31:183–7.
`11. Bieganowski P, Brenner C. Discoveries of nicotinamide riboside as a
`nutrient and conserved NRK genes establish a Preiss-Handler inde-
`pendent route to NAD+ in fungi and humans. Cell 2004;117:495–
`502.
`12. Belenky P, Racette FG, Bogan KL, McClure JM, Smith JS, Brenner
`C. Nicotinamide riboside promotes Sir2 silencing and extends
`lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+. Cell
`2007;129:473–84.
`13. Cant ´o C, Houtkooper RH, Pirinen E, Youn DY, Oosterveer MH, Cen Y,
`Fernandez-Marcos PJ, Yamamoto H, Andreux PA, Cettour-Rose P,
`et al. The NAD(+) precursor nicotinamide riboside enhances oxidative
`metabolism and protects against high-fat diet-induced obesity. Cell
`Metab 2012;15:838–47.
`14. Khan NA, Auranen M, Paetau I, Pirinen E, Euro L, Forsstrom S, Pasila
`L, Velagapudi V, Carroll CJ, Auwerx J, et al. Effective treatment
`of mitochondrial myopathy by nicotinamide riboside, a vitamin B3.
`EMBO Mol Med 2014;6:721–31.
`15. Gariani K, Menzies KJ, Ryu D, Wegner CJ, Wang X, Ropelle ER, Moullan
`N, Zhang H, Perino A, Lemos V, et al. Eliciting the mitochondrial
`unfolded protein response via NAD+ repletion reverses fatty liver disease.
`Hepatology 2015 Sept 24 (Epub ahead of print; DOI: ).
`16. Xu W, Barrientos T, Mao L, Rockman Howard A, Sauve Anthony A,
`Andrews Nancy C. Lethal cardiomyopathy in mice lacking transferrin
`receptor in the heart. Cell Rep 2015;13:533–45.
`17. Gong B, Pan Y, Vempati P, Zhao W, Knable L, Ho L, Wang J, Sastre M,
`Ono K, Sauve AA, et al. Nicotinamide riboside restores cognition
`through an upregulation of proliferator-activated receptor-gamma
`coactivator 1alpha regulated beta-secretase 1 degradation and mito-
`chondrial gene expression in AlzheimerÕs mouse models. Neurobiol
`Aging 2013;34:1581–8.
`18. Brown KD, Maqsood S, Huang JY, Pan Y, Harkcom W, Li W, Sauve A,
`Verdin E, Jaffrey SR. Activation of SIRT3 by the NAD(+) precursor
`nicotinamide riboside protects from noise-induced hearing loss. Cell
`Metab 2014;20:1059–68.
`19. Trammell SA, Brenner C. Targeted, LCMS-based metabolomics for
`quantitative measurement of NAD+ metabolites. Comput Struct
`Biotechnol J 2013;4:e201301012.
`20. Dalvit C, Fogliatto G, Stewart A, Veronesi M, Stockman B. Water-
`LOGSY as a method for primary NMR screening: practical aspects and
`range of applicability. J Biomol NMR 2001;21:349–59.
`21. Dalvit C, Pevarello P, Tato` M, Veronesi M, Vulpetti A, Sundstro¨ m M.
`Identification of compounds with binding affinity to proteins via
`magnetization transfer from bulk water. J Biomol NMR 2000;18:65–8.
`22. Delaglio F, Grzesiek S, Vuister G, Zhu G, Pfeifer J, Bax A. NMRPipe: a
`multidimensional spectral processing system based on UNIX pipes.
`J Biomol NMR 1995;6:277–93.
`23. Johnson BA, Blevins R. NMR View: a computer program for the
`visualization and analysis of NMR data. J Biomol NMR 1994;4:603–14.
`24. Yang T, Chan NY, Sauve AA. Syntheses of nicotinamide riboside and
`derivatives: effective agents for increasing nicotinamide adenine dinu-
`cleotide concentrations in mammalian cells. J Med Chem 2007;50:6458–
`61.
`25. Thurmond MC, Tyler JW, Luiz DM, Holmberg CA, Picanso JP. The
`effect of pre-enrichment on recovery of Streptococcus agalactiae,
`Staphylococcus aureus and mycoplasma from bovine milk. Epidemiol
`Infect 1989;103:465–74.
`26. Maass S, Sievers S, Zuhlke D, Kuzinski J, Sappa PK, Muntel J, Hessling
`B, Bernhardt J, Sietmann R, Volker U, et al. Efficient, global-scale
`quantification of absolute protein amounts by integration of targeted
`mass spectrometry and two-dimensional gel-based proteomics. Anal
`Chem 2011;83:2677–84.
`27. Rasigade JP, Moulay A, Lhoste Y, Tristan A, Bes M, Vandenesch F,
`Etienne J, Lina G, Laurent F, Dumitrescu O.