Samuel A.J. Trammell
University of Iowa
Copyright 2016 Samuel AJ Trammell
is dissertation is available at Iowa Research Online: http://ir.uiowa.edu/etd/3203
ACKNOWLEDGEMENTS
I must give the greatest thanks to current and former members of the Brenner laboratory for being a constant supportive yet critical force in my thesis work. My mentor, Dr. Charles Brenner, was, is, and always will be a voice of optimism and encouragement that propelled my work forward. Dr. Brenner allowed me to work independently but was always there as a much needed critical, centering voice during the whole of my thesis.
Recounting the contributions of all members of the Brenner laboratory is impossible due to my inability to properly measure the extreme aid and friendship each person provided. However, I would like to specifically thank Dr. Rebecca Fagan for the light, absurd, and humorous conversations had as co-workers and more importantly as friends. I would like to acknowledge former laboratory mates Dr. Szu-Chieh Mei, her husband Dr. Bokuan Wu, and Dr. Jennifer Bolyston for making the laboratory a fun and interesting place in which to work and for their insightful commentary into my work. To my current co-workers, thank you for continuing to contribute to the special milieu that is the Brenner laboratory.
I could never properly thank Dr. Lynn Teesch and Mr. Vic Parcell enough. Dr. Teesch quickly became an unofficial advisor throughout my time working in the High Resolution Mass Spectrometry Facility on all things related to the operation and use of mass spectrometry. Conversations with Mr. Parcell varied from the deeply technical to the most mirthful. Both provided constant expertise and support that continually reinvigorated my passion for science and undoubtedly helped me through the more difficult portions of my time here at the University of Iowa.
I would like to thank Drs. Diane Slusarski, Marry Wilson, Michael Wright, and Rob Piper for first agreeing to serve on my committee and then for the contributions they have made to my growth as a scientist and to my thesis.
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Finally, I would like to thank the many friends outside of my field and the University that I have met in Iowa City. I cannot imagine the person I would be today without their company.
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ABSTRACT
Nicotinamide adenine dinucleotide (NAD+) is a cofactor in hydride transfer reactions and consumed substrate of several classes of glycohydrolyitc enzymes, including sirtuins. NAD+, its biosynthetic intermediates, breakdown products, and related nucleotides (the NAD metabolome) is altered in many metabolic disorders, such as aging and obesity. Supplementation with the novel NAD+ precursor, nicotinamide riboside (NR), ameliorates these alterations and opposes systemic metabolic dysfunctions in rodent models. Based on the hypothesis that perturbations of the NAD metabolome are both a symptom and cause of metabolic disease, accurate assessment of the abundance of these metabolites is expected to provide insight into the biology of diseases and the mechanism of action of NR in promoting metabolic health. Current quantitative methods, such as HPLC, lack specificity and sensitivity to detect distinct alterations to the NAD metabolome. In this thesis, I developed novel sensitive, accurate, robust liquid chromatography mass spectrometry methodologies to quantify the NAD metabolome and applied these methods to determine the effects of disease states and NR supplementation on NAD+ metabolism. My investigations indicate that NR robustly increases the NAD metabolome, especially NAD+ in a manner kinetically different than any other NAD+ precursor. I provide the first evidence of effective NAD+ supplementation from NR in a healthy, 52 year old human male, suggesting the metabolic promoting qualities of NR uncovered in rodent studies are translatable to humans. During my investigation of NR supplementation, my work establishes an unexpected robust, dramatic increase in deamino–NAD+, NAAD, directly from NR, which I argue could serve as an accessible biomarker for efficacious NAD+ supplementation and the effect of disease upon the NAD metabolome. Lastly, I further establish NR as a general therapeutic against metabolic disorder by detailing its ability to oppose aspects of chronic alcoholism and diabetes mellitus.
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PUBLIC ABSTRACT
A century ago in the United States, a disease known as Pellagra ravaged areas mainly subsisting on maize. This disease was detrimental to quality of life and in some instances proved fatal. At the time, this disease was considered a major public health problem and many grant initiatives were announced to identify the cause of the disease and develop an effective treatment. Through these efforts, Pellagra was shown to be a non-infectious disease caused by a diet of maize and lard. It was cured by drinking milk and eating more animal meat. These efforts essentially eliminated the disease from high income nations. Further investigation identified the B3 vitamins commonly referred to as niacin as the anti-Pellagra components of milk and animal meat. Today, obesity, diabetes, and heart disease are prevalent in the US and areas around the world. These diseases are a new public health crisis resulting in the loss of billions of dollars and a decreased quality of life and lifespan. As it was a hundred years ago, public funds are now directed to identify effective treatments to counter these prevalent and devastating diseases. Work generously funded by the public has identified the most recently discovered B3 vitamin, nicotinamide riboside, as a health promoting compound that could treat these diseases. The goal of my thesis was to develop improved tools to answer how this vitamin works in times of health and disease. In so doing, my work further establishes this novel B3 vitamin as a health promoting compound and describes clinically relevant technologies to assess its effectiveness in future human trials.
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TABLE OF CONTENTS
LIST OF TABLES………………………………………………………………………………………………………..xi LIST OF FIGURES …………………………………………………………………………………………………… xiii LIST OF ABBREVIATIONS………………………………………………………………………………………….xv CHAPTER 1………………………………………………………………………………………………………………. 1
INTRODUCTION…………………………………………………………………………………………………….. 1
1.1 Significance of NAD+ and Description of the Need for Improved Technologies for Its Measurement………………………………………………………………………………………………………. 1
1.2 NAD+ Transactions …………………………………………………………………………………………. 3 1.3 Thesis Goals………………………………………………………………………………………………….. 8 1.4 Figure …………………………………………………………………………………………………………..11
CHAPTER 2………………………………………………………………………………………………………………12
NAD METABOLOME ANALYSIS VIA LIQUID CHROMATOGRAPHY MASS SPECTROMETRY…………………………………………………………………………………………………..12
2.1 Quantitative NAD+ Metabolomics ………………………………………………………………………12 Optimized Extraction …………………………………………………………………………………………12 Optimized Internal Standards ……………………………………………………………………………..14 Optimized Liquid Chromatography ………………………………………………………………………17 Mass Spectrometry Optimization…………………………………………………………………………18 Metabolite Measurement Challenges …………………………………………………………………..19 Results in Mammalian Cell Line ………………………………………………………………………….19 Conclusions……………………………………………………………………………………………………..20 Acknowledgements …………………………………………………………………………………………..20
2.2 Continued Method Development Post-Initial Publication ……………………………………….20 ATP and ADP: The Other Problem Metabolites ……………………………………………………..20 Addition of MeNam, Me2PY, and Me4PY to the NAD Metabolomic Assay …………………20 Considerations of Quantitative NAD Metabolomics in Mammalian Tissues ………………..22 Quantification of the Oxidized NAD Metabolome in Liver…………………………………………24 Quantification of NAD(P)H and Extraction from Liver………………………………………………25 Quantification of the Oxidized NAD Metabolome in Skeletal Muscle………………………….29
2.3 Tables and Figures …………………………………………………………………………………………31 CHAPTER 3………………………………………………………………………………………………………………40
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NICOTINAMIDE RIBOSIDE IS A MAJOR NAD+ PRECURSOR VITAMIN IN BOVINE MILK……………………………………………………………………………………………………………………..40
3.1 Distribution of Work…………………………………………………………………………………………40 3.2 Abstract ………………………………………………………………………………………………………..40 3.3 Introduction……………………………………………………………………………………………………41 3.4 Methods………………………………………………………………………………………………………..42
Milk Quality and Herd Health Measurements…………………………………………………………42 Bovine Milk Sample Acquisition and Preparations ………………………………………………….43 NMR Spectroscopy …………………………………………………………………………………………..43 NR Stability Assays …………………………………………………………………………………………..44 Staph a Growth Experiments………………………………………………………………………………44 LC-MS and LC-MS/MS………………………………………………………………………………………45 Statistical Analysis…………………………………………………………………………………………….46
3.5 Results………………………………………………………………………………………………………….47 NR is a Major Component of the B3 Vitamin Content in Bovine Milk ………………………….47 Staph a Depletes NR and Nam …………………………………………………………………………..48 NR Content as a Function of Organic Certification …………………………………………………49 NR is a Bound Metabolite in Bovine Milk ………………………………………………………………50
3.6 Discussion …………………………………………………………………………………………………….50 3.7 Tables and Figures …………………………………………………………………………………………52 3.8 Supplemental Tables ………………………………………………………………………………………55
CHAPTER 4………………………………………………………………………………………………………………57 EFFICACY OF NMN AND NR AS EXTRACELLULAR NAD+ PRECURSORS…………………..57 4.1 Distribution of Work…………………………………………………………………………………………57 4.2 Abstract ………………………………………………………………………………………………………..57 4.3 Introduction……………………………………………………………………………………………………57 4.4 Materials and Methods…………………………………………………………………………………….59 Compounds ……………………………………………………………………………………………………..59 Cell Culture Conditions………………………………………………………………………………………59 Extraction ………………………………………………………………………………………………………..59 LC-MS/MS……………………………………………………………………………………………………….60 4.5 Results………………………………………………………………………………………………………….60
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NMN is Dephosphorylated Extracellularly and Contributes to the Intracellular NAD+
Pool slower than NR………………………………………………………………………………………….60
4.6 Discussion …………………………………………………………………………………………………….61
4.7 Figures………………………………………………………………………………………………………….63 CHAPTER 5………………………………………………………………………………………………………………65 NICOTINAMIDE RIBOSIDE IS UNIQUELY BIOAVAILABLE IN MOUSE AND MAN ………….65 5.1 Distribution of Work…………………………………………………………………………………………65 5.2 Abstract ………………………………………………………………………………………………………..65 5.3 Introduction……………………………………………………………………………………………………66 5.4 Methods………………………………………………………………………………………………………..69 Materials and Reagents……………………………………………………………………………………..69 Mice ……………………………………………………………………………………………………………….69 N of 1 Human Experiment ………………………………………………………………………………….70 Clinical Trial……………………………………………………………………………………………………..70 Sample Preparation and LC-MS………………………………………………………………………….71 Statistical Analyses …………………………………………………………………………………………..71 5.5 Results………………………………………………………………………………………………………….72 Oral NR Increases the Blood NAD Metabolome in a Healthy Adult Male ……………………72 Oral NR, Nam and NA Elevate Hepatic NAD+ with Distinctive Kinetics ………………………73 NR Directly Contributes to Murine Liver NAAD ………………………………………………………77 NR Increases Blood Cell NAD+ Metabolism in Human Subjects ……………………………….79 5.6. Discussion ……………………………………………………………………………………………………81 5.7 Figures and Table for Chapter 5.5-5.6 ……………………………………………………………….84 5.8 Supplemental Materials……………………………………………………………………………………90 Clinical Trial……………………………………………………………………………………………………..90 Sample Preparation and LC-MS………………………………………………………………………….91 5.9 Supplemental Tables and Figures for Chapter 5.5-5.6 ………………………………………….95 5.10 Perspective on Chapter 5……………………………………………………………………………….97 Introduction ……………………………………………………………………………………………………..97 Results and Discussion ……………………………………………………………………………………..97 Methods ………………………………………………………………………………………………………..102 5.11 Figures for 5.10…………………………………………………………………………………………..103 CHAPTER 6…………………………………………………………………………………………………………….107
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NICOTINAMIDE RIBOSIDE PREVENTS ALCOHOL INDUCED FATTY LIVER ………………107 6.1 Distribution of Work……………………………………………………………………………………….107 6.2 Abstract ………………………………………………………………………………………………………107 6.3 Introduction………………………………………………………………………………………………….108 6.4 Materials and Methods…………………………………………………………………………………..110
Animal Husbandry and Experimental Design……………………………………………………….110 Mitochondrial Isolation……………………………………………………………………………………..111 Western Blotting ……………………………………………………………………………………………..112 Microscopy …………………………………………………………………………………………………….112 NAD Metabolomics………………………………………………………………………………………….112 Acetylomics ……………………………………………………………………………………………………113 Statistical Analysis…………………………………………………………………………………………..117
6.5 Results and Discussion………………………………………………………………………………….117
6.6 Tables and Figures ……………………………………………………………………………………….123 CHAPTER 7…………………………………………………………………………………………………………….130
NICOTINAMIDE RIBOSIDE OPPOSES TYPE 2 DIABETES AND NEUROPATHY IN
MICE …………………………………………………………………………………………………………………..130
7.1 Distribution of Work……………………………………………………………………………………….130 7.2 Abstract ………………………………………………………………………………………………………130 7.3 Introduction………………………………………………………………………………………………….131 7.4 Methods………………………………………………………………………………………………………133
Mouse Models………………………………………………………………………………………………..133 NAD Metabolomics………………………………………………………………………………………….133 Statistics………………………………………………………………………………………………………..133 Study Approval ……………………………………………………………………………………………….133
7.5 Results and Discussion………………………………………………………………………………….133 7.6 Acknowledgments…………………………………………………………………………………………138 7.7 Figures and Tables for Sections 3-5…………………………………………………………………139 7.8 Supplemental Materials………………………………………………………………………………….143
Sample Extraction for NAD+ Metabolomics………………………………………………………….143
LC-MS/MS Analysis for NAD Metabolomics ………………………………………………………..143 7.9 Supplemental Figures ……………………………………………………………………………………145
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7.10 Perspective on Chapter 7……………………………………………………………………………..147 7.11 Results and Discussion………………………………………………………………………………..147 Type 1 Diabetes in Rat Compared to Type 2 Diabetes in Mouse…………………………….147 NADH and NADPH Measurement in T2D Murine Liver …………………………………………151 7.12 Methods…………………………………………………………………………………………………….152 7.13 Tables and Figures ……………………………………………………………………………………..154 CHAPTER 8…………………………………………………………………………………………………………….158 GENERAL SUMMARY AND FUTURE DIRECTIONS………………………………………………….158 8.1 General Summary…………………………………………………………………………………………158 8.2 Regulation of the NAD+ Metabolome: a Future Avenue of Inquiry …………………………163 8.3 Future Investigations of NR as a Health Promoting Agent……………………………………164 APPENDIX A …………………………………………………………………………………………………………..166 APPENDIX B …………………………………………………………………………………………………………..172 REFERENCES ………………………………………………………………………………………………………..174
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LIST OF TABLES
Table 2.1 Alkaline separation gradient. ………………………………………………………………………….31 Table 2.2 Acidic separation gradient. …………………………………………………………………………….31 Table 2.3 LCMS/MS SRM parameters, sensitivity, and robustness for each metabolite…………32 Table 2.4 NAD+/nucleotides metabolome of LN428/MPG cell line………………………………………34 Table 2.5 LCMS/MS SRM parameters, sensitivity, and robustness for methylated
nicotinamide metabolites……………………………………………………………………………………….35
Table 2.6 Recovery of murine hepatic oxidized NAD metabolome. …………………………………….36
Table 2.7 Gradient for analysis of NAD(P)H……………………………………………………………………39
Table 2.8 Recovery of the oxidized NAD metabolome in murine quadriceps………………………..39
Table 3.1 Mean NAD+ metabolomes of 18 raw bovine milk samples…………………………………..52
Table 3.2. Correlation coefficients between B3 vitamin concentrations and milk quality………….52
Table 3.3. Vitamin B3 content in store bought bovine milk. ………………………………………………..54
Table 3.4 NAD+ precursor concentrations in 19 individual milk samples………………………………55
Table 3.5. Individual milk quality assessments and breed. ………………………………………………..56
Table 5.1 PBMC NAD+ metabolites (μm) in a 52 year-old male who orally ingested 1000 mg
NR Cl for 7 consecutive days. ………………………………………………………………………………..85
Table 5.2 Plasma NAD+ metabolites (μm) in a 52 year-old male who orally ingested 1000 mg NR Cl for 7 consecutive days. ………………………………………………………………………………..95
Table 5.3 Urinary NAD+ metabolites (μmol/mmol creatinine) in a 52 year-old male who orally ingested 1000 mg NR Cl for 7 consecutive days. ………………………………………………………95
Table 6.1 Ethanol induced NAD metabolome alterations are opposed by NR. ……………………123
Table 6.2 Pathways affected by ethanol-induced acetylation. ………………………………………….125
Table 6.3 Pathways affected by ethanol-induced protein acetylation and responsive to NR treatment…………………………………………………………………………………………………………..126
Table 7.1 The hepatic pool of NADP+ and NADPH is depressed by PD and T2D and is
partially restored by NR……………………………………………………………………………………….142
Table 7.2 Glycemic control, dyslipidemia, and overall health were not improved by NR in T1D………………………………………………………………………………………………………………….154
Table 7.3 NR opposes T1D neuropathy. ………………………………………………………………………154
Table 7.4 NR tends to improve STZ induced NAD metabolome defects in sciatic nerve homogenate. ……………………………………………………………………………………………………..155
Table 7.5 B3 vitamins were ineffective in improving glycemic control and overall health……….155 Table 7.6 Among the B3 vitamins, NR consistently opposed aspects of T1D neuropathy……..156
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Table 7.7 All three B3 vitamins tended to alter the NAD metabolome in sciatic nerve…………..156
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LIST OF FIGURES
Figure 1.1 NAD+ biosynthesis in yeast and vertebrates…………………………………………………….11
Figure 2.1 Chromatograms of all 19 metabolites generated from injection of complex
standard solutions using MRM LC-MS. ……………………………………………………………………33
Figure 2.2 Chromatograms of the methylated nicotinamide species generated from injection
of complex standard solutions using MRM LC-MS. ……………………………………………………35
Figure 2.3 NADH and its [13C10] isotopologue in murine liver sample…………………………………..36
Figure 2.4 Hydrophobic ammonium salts cause intense ion suppression…………………………….37
Figure 2.5 Carryover of NADPH when TBA is used in the mobile phase……………………………..37
Figure 2.6 Extracted ion currents for NAD(P)H on a C18 column with TEA in the mobile phase…………………………………………………………………………………………………………………38
Figure 3.1. NR is stable in milk and is degraded by Staph a………………………………………………53
Figure 3.2. NR-binding to milk demonstrated by NMR………………………………………………………54
Figure 4.1 Proposed model for NMN utilization. ………………………………………………………………63
Figure 4.2. Extracellular NMN is dephosphorylated extracellular and incorporated into the intracellular NAD+ pool at a slower rate compared to extracellular NR. …………………………64
Figure 5.1 The NAD metabolome………………………………………………………………………………….84 Figure 5.2. NR elevates hepatic NAD+ metabolism distinctly with respect to other vitamins. …..86 Figure 5.3 NR contributes directly to hepatic NAAD. ………………………………………………………..87 Figure 5.4 Dose-dependent effects of NR on the NAD Metabolome of human subjects…………88 Figure 5.5 Hepatic NR, NAR, and Me2PY concentrations after gavage of NR, Nam and NA. …96 Figure 5.6 IP administration of NR, Nam, and NA produce similar effects on murine liver
NAD metabolome. ……………………………………………………………………………………………..103 Figure 5.7 NR directly contributes to hepatic NAAD after IP injection………………………………..104 Figure 5.8 NR contributes to muscle NAAD following gavage. …………………………………………105 Figure 5.9 NR contributes to muscle NAAD following IP. ………………………………………………..106 Figure 6.1 NR tends to oppose ethanol induced hepatic lipid deposition……………………………123 Figure 6.2 NR does not oppose ethanol induced global hyperacetylation…………………………..124 Figure 6.3 Proteins acetylated by ethanol and sensitive to NR…………………………………………127 Figure 6.4 NR opposed increased mortality experienced by ethanol fed mice…………………….128 Figure 6.5 NR increased diet consumption in both control and ethanol animals. …………………128 Figure 7.1 NR improves metabolic parameters in PD and T2D. ……………………………………….139 Figure 7.2 NR opposes PDPN and T2DPN. ………………………………………………………………….140
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Figure 7.3 Activity of NR in DPN can be monitored by corneal confocal microscopy (CCM)….141 Figure 7.4 Experimental design and weight gain. …………………………………………………………..145 Figure 7.5 GTT primary data used for Figure 7.1 i and j. …………………………………………………146 Figure 7.6 NADP+ and NADPH were equally depressed by PD and T2D and improved by
NR. ………………………………………………………………………………………………………………….157
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LIST OF ABBREVIATIONS
AMP Adenosine Monophosphate ADP Adenosine Diphosphate
ATP Adenosine Triphosphate ADPR Adenosine diphosphate ribose CMP Cytosine Monophosphate
IMP Inosine monophosphate
LC-MS Liquid Chromatography Mass Spectrometry
LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry Me2PY N1-Methyl-2-pyridone-5-carboxamide
Me4PY N1-Methyl-4-pyridone-5-carboxamide
MeNam N1-Methyl-Nicotinamide
NA Nicotinic Acid
NAAD Nicotinamide Adenine Dinucleotide
NAD+ Nicotinamide Adenine Dinucleotide
NADH Nicotinamide Adenine Dinucleotide, reduced
NADP+ Nicotinamide Adenine Dinucleotide Phosphate
NADPH Nicotinamide Adenine Dinucleotide Phosphate, reduced NAMN Nicotinic Acid Mononucleotide
NAR Nicotinic acid Riboside
NMN Nicotinamide Mononucleotide
NR Nicotinamide Riboside
Trp Tryptophan
UMP Uridine Monophosphate
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CHAPTER 1
INTRODUCTION
“Targeted, LCMS-based Metabolomics for Quantitative Measurement of NAD+ Metabolites”*
Samuel A.J. Trammell1,2 and Charles Brenner1,2
1Department of Biochemistry, 2 Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
*Sections 1.1 and 1.2 and Figure 1.1 of the following chapter are reprinted from a published article in Computational and Structural Biotechnology Journal volume 4 (1). The copyright of the article belongs to the authors. The manuscript was written by Samuel Trammell with guidance from Charles Brenner, PhD.
1.1 Significance of NAD+ and Description of the Need for Improved Technologies for Its Measurement
The essentiality of NAD+-dependent processes in fuel utilization, gene regulation, DNA repair, protein modification, and cell signaling events makes the analysis of NAD+ metabolites central to an understanding of what a tissue is doing. NAD+ is the key hydride transfer coenzyme for a wide variety of oxidoreductases and is also the consumed substrate of sirtuins, poly(adenosine diphosphate ribose (ADPr)) polymerase, mono ADPr transferases, and cyclic ADPr synthases (2, 3). Measurement of NAD+ and related metabolites including several nucleosides and nucleotides (hereafter, the NAD+ metabolome) serves as a powerful indicator of the ability of a cell or tissue to perform processes such as glycolysis, gluconeogenesis, fatty acid oxidation, reactive oxygen species detoxification, among others. Moreover, the state of the NAD+ metabolome can serve as an indication of nutrition, health and disease.
Because NAD+ and related metabolites vary in cellular concentration from ~1 μM to ~1 mM, the analytical procedure must be robust, reproducible, and rapid. Liquid chromatography (LC)-based assays afford the ability to measure multiple metabolites in a timely fashion with the
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duration of each run ranging from 10 minutes to an hour. However, quantification through HPLC-UV-Vis methods is severely compromised based on the complexity of samples. In complex mixtures, a single peak may contain the metabolite of interest in addition to many other metabolites of identical retention time. In addition, peak shapes are rarely unaffected by complexity. Some investigators use a UV-vis signal at a retention time as the primary means for identification of a metabolite of interest—collected fractions are then subjected to mass spectrometry to confirm (nonquantitatively) the presence of the metabolite. This process leaves a great deal of data in the dark. Because every NAD+ metabolite can be converted to one or more other metabolites, snapshots of the levels of NAD+ , nicotinamide (Nam) or any other NAD+ metabolite without assessment of the NAD+ metabolome on a common scale has the potential to be misleading.
Because of its specificity and sensitivity, LC coupled to mass spectrometry (LC-MS) is a leading analytical method in the measurement of small molecules in complex samples. As with HPLC-UV-vis methods, LC serves to separate compounds of interest and must be optimized in the same way as any HPLC method. Because all LC-MS data contain at least two dimensions of data (retention time plus the mass:charge ratio, termed m/z), LC-MS increases specificity with respect to LC-UV-vis methods that report complex absorbance spectra as a function of retention time or matrix-assisted laser desorption ionization (MALDI)-based methods that report complex m/z data without retention times. Multidimensional MS, i.e., LC-MSn, provides further information because a particular analyte breaks down to component ions at a particular ionization energy. An ideal LC-MS method identifies an optimal extraction and separation method for all molecules of interest, detects the compounds in either negative or positive ion mode MS, and has sufficient LC separation to subject each molecule of interest to MSn analysis. The method is then a series
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of selective reaction monitoring (SRM)1 protocols in which analytes are identified and quantified by MS as they come off the LC.
Whereas metabolomic discovery projects require high mass resolution instruments, targeted quantitative LC-MS assays can make use of lower resolution tandem mass spectrometers such as triple quadrupoles (QQQ). Here, the multidimensional data (retention time, m/z, and MS2 transitions) are used to distinguish closely related metabolites, such as NAD+ from NADH. Limits of quantification in optimized targeted, quantitative LC-MS assays are in the femtomole range.
Though mass spectrometers offer great analytical power for measuring the NAD+ metabolome, they also present technical challenges not encountered in other analytical techniques. These challenges include development of optimal mass spectrometry conditions, proper separation of metabolites, and best choice of internal standards. Here we discuss NAD+ metabolism and describe an optimized LC-MS2 assay of the NAD+ metabolome.
1.2 NAD+ Transactions
In fungi and vertebrates, NAD+ concentration is maintained by either de novo synthesis from tryptophan (4) or through salvage of nicotinic acid (NA) (5), nicotinamide (Nam) (6), and the recently identified NAD+ precursor vitamin nicotinamide riboside (NR) (7) (Figure 1.1). Some organisms, such as Candida glabrata, lack de novo synthesis (8). Many vertebrate cell types turn this pathway off (3). De novo synthesis proceeds from tryptophan in six steps to produce nicotinic acid mononucleotide (NAMN) and in two additional steps to produce NAD+. When NAD+ is the substrate of an enzyme such as glyceraldehyde phosphate dehydrogenase (GAPDH), fuel oxidation reactions will reduce NAD+ to NADH. In the case of GAPDH, the reaction is reversible, such that NADH is reoxidized to NAD+ in the gluconeogenic direction. NAD+ and NADH can be phosphorylated to NADP+ and NADPH. NADP+ is required for the
1 Multiple SRMs are referred to as multiple reaction monitoring (MRM). 3
pentose phosphate pathway (PPP), which produces NADPH. NADPH is required for detoxification of reactive oxygen species and reductive biosynthesis of lipids and steroids. Just as glucose-6-phosphate oxidation by the PPP produces NADPH, glutathione reactivation and reductive biosynthesis reoxidizes NADPH to NADP+.
Beyond serving as a coenzyme in hydride reactions, NAD+ is a consumed substrate for enzymes such as sirtuins, PARPs, and other ADPr transfer enzymes (2, 3, 9, 10). Though CD38 has an activity on NADP+, at least in vitro (11), the typical activity of an NAD+-consuming enzyme involves NAD+ as the substrate, and products that include Nam and an NAD+-derived ADPr moiety. Thus, to sustain intracellular NAD+ levels, actions of NAD+-consuming enzymes must be accompanied by Nam salvage (2, 3). Nam salvage differs between fungi and vertebrates. In fungi, Nam is hydrolyzed by the PNC1-encoded nicotinamidase to NA (6). NA is then converted by the first enzyme of the Preiss-Handler pathway, the NPT1-encoded NA phosphoribosyltransferase, to form NAMN. The second and third steps of Preiss-Handler salvage correspond to the final two steps of de novo synthesis, whose last step is glutamine- dependent NAD+ synthetase (12). In vertebrates, Nam produced as a product of NAD+- consuming enzymes cannot be salvaged as NA intracellularly. However, if Nam goes through the gut, bacterial nicotinamidases produce NA (13), which circulates and is used via Preiss- Handler salvage.
Intracellular Nam salvage in vertebrates depends on a Nam phosphoribosyltranferase, which entered the scientific literature with the names pre-B cell colony enhancing factor (PBEF) (14) and Visfatin (15). Now termed Nampt, this protein is widely expressed as an intracellular enzyme and also circulates as an active extracellular molecule (16, 17). First predicted to be part of a partially extracellular NAD+ biosynthetic cycle (2) along with CD73, a homolog of bacterial NMN 5’-nucleotidase, extracellular Nampt clearly has enzymatic activity (17). However, extracellular NMN remains controversial in part due to deficiencies in NAD+ metabolite quantification. As a phosphoribosyltransferase, Nampt activity depends on PRPP, an
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extracellular source of which has not been demonstrated (18). By an HPLC-UV method, which may have been distorted by co-eluting analytes, the abundance of extracellular NMN was reported to be 80 μM (17). However, using LC- MSn, it was reported that PRPP and NMN are virtually absent and, moreover, are unstable in mouse plasma (18). It stands to reason that extracellular Nampt may have activity in local environments and developmental/nutritional conditions in which the substrates, Nam and PRPP, and the ATP activator are at substantial levels. Systemic NMN at 80 μM appears to be implausible, however.
Nam and NA can also be methylated, which would be predicted to block salvage. In plants, NA N-methyltransferase produces a compound known as trigonelline by transfer of the methyl group from S-adenosyl-methionine (19, 20). The corresponding Nam N- methyltransferase (NNMT) has been well characterized in vertebrates (21). Increased NNMT expression has been observed in Parkinson’s Disease (22) with a potential role in disease etiology (23, 24). NNMT is also increased in malignancy (25) and plays an apparent role in cell migration (26). Despite the reported roles in disease, N-methyl Nam (NMNam2) is a natural metabolite in healthy individuals with reported antithrombotic (27) and vasorelaxant (28) activities that is increased in plasma and urine after endurance exercise (29). NMNam is ultimately converted to N1-Methyl-2-pyridone-5-carboxamide and N1-Methyl-4-pyridone-5- carboxamide.
Though the primary breakdown product of NAD+ is Nam and the complete bacterially digested product is NA, nicotinamide riboside (NR) is an additional salvageable precursor that exists intracellularly and in milk (7, 30, 31). The unique NR salvage pathway is via nicotinamide riboside kinases (7). In addition, NR can be split into a Nam moiety and resynthesized to NAD+ via Nam salvage enzymes (32). Nicotinic acid riboside (NAR) is an alternate substrate of
2 In the future, this metabolite is abbreviated MeNam. 5
nicotinamide riboside kinases (33) and purine nucleoside phosphorylase (13) that has been shown to be an intracellular NAD+ precursor (30) but has not been reported to circulate.
Whereas NA is the salvageable precursor of NAD+ that has been exposed to the most digestive enzymes and Nam is the salvageable precursor that is produced by every cell with NAD+-consuming enzymes, the main source of dietary NR is probably partial digestion of NAD+. Depending on one’s nutrition and potentially one’s microbiome, the three vitamin precursors of NAD+ (NA, Nam and NR) and trp should be in circulation (3). The existence of extracellular enzymes with the potential to produce and consume NMN, and which consume NAD+, suggests the circulation of pyridine nucleotides (2). Moreover, NMN supplementation of mice on high fat diet (HFD) increases insulin sensitivity, glucose tolerance, and intracellular NAD+ compared to non-treated mice on the same diet (34). Though extracellular NMN was interpreted to function via direct incorporation of the nucleotide into cells (34), careful examination indicates that extracellular NA, Nam, and NR increase intracellular NAD+ in yeast and vertebrate cells, whereas NMN requires dephosphorylation to NR (35). Consistent with the prediction that the ectoenzyme CD73 has NMN 5’-nucleotidase activity (2), CD73 has the requisite biochemical activity to catalyze NMN dephosphorylation (36). In the yeast system, NR extends replicative longevity in a manner that depends on conversion to NAD+ (32). In mice on high fat diet, NR improves glucose control and insulin sensitivity, while moderating the observed increase in adiposity (37).
In addition to the major difference in Nam salvage between vertebrate and yeast systems, there is a mitochondrial compartmentalization problem in vertebrates. In yeast, transporters Ndt1 and Ndt2 carry NAD+ across the mitochondrial inner membrane (38) and the only mitochondrial NAD+ biosynthetic enzyme is NADH kinase, Pos5 (39). However, in vertebrate cells, the nucleocytoplasm and the mitochondrial matrix constitute distinct pools of NAD+, NADH, NADP+ and NADPH owing to impermeability of the mitochondrial inner membrane to these compounds. Though systems such as the malate-aspartate shuttle and
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nicotinamide nucleotide transhydrogenase transfer reducing equivalents across mitochondrial membranes, vertebrate mitochondria require a system to import an NAD+ precursor into the matrix for conversion to NAD+. On the basis of localization of NAD+ biosynthetic enzymes, that precursor is NMN (35) 3. Nmnat3, which converts NMN to NAD+, is localized to the mitochondrial matrix. Nmnat3 is one of three vertebrate NAMN/NMN adenylyltranferases—the other two are localized in the nucleus and on the cytosolic face of Golgi. Though one could argue that the ability of Nmnat3 to convert NAMN to NAAD suggests that NAMN or NMN could be the mitochondrial NAD+ precursor, the NAAD product of the NAMN reaction requires glutamine- dependent NAD+ synthetase for conversion to NAD+. Glutamine-dependent NAD+ synthetase is not mitochondrially localized (35).
As shown in Figure 1.1, the implication of NMN as the limiting precursor for vertebrate mitochondrial NAD+ biosynthesis is profound. De novo synthesis and NA-dependent Preiss- Handler synthesis can only supply mitochondria with NAD+ by nucleocytosolic conversion to NAD+ followed by the pyrophosphate-dependent conversion of NAD+ to NMN in a back reaction of Nmnat first demonstrated by Arthur Kornberg in 1948 (44) or by conversion of NAD+ to Nam and subsequent conversion of Nam to NMN. In contrast, Nam and NR can be converted directly to NMN by Nampt and NR kinases, respectively.
In mitochondria that are burning fuel, the redox reactions are largely directional because fuel oxidation converts NAD+ to NADH and complex I of the electron transfer chain reoxidizes NADH to NAD+. Three vertebrate sirtuins, Sirt3-5, reside in mitochondria, where they consume NAD+ in reactions that either modify proteins or relieve protein modifications (45). For the
3 The origin of mammalian mitochondrial NAD+ is controversial. Later investigations revealed Nmnat3 is expressed in erythrocytes, which do not contain mitochondria (40). Further, Nmnat3 deficiency does not alter mitochondrial NAD+ (41) and Nmnat3 knockout animals are viable and capable of maintaining in NAD+ both fractions (42), suggesting the nucleocytoplasmic and mitochondrial pool are continuous. So far, no mammalian mitochondrial NAD+ importer has been identified (43) and the partitioning or lack thereof of NAD+ requires further investigation.
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mitochondrial sirtuins to work and avoid robbing redox enzymes of NAD+, NMN must be imported from the cytosol.
Completing the major NAD+ transactions, yeast possess two cytosolic NAD+/NADH kinases and the mitochondrial NADH kinase, Pos5 (39). Vertebrate cytosolic NAD+/NADH kinase is related to the yeast enzymes (46), whereas the vertebrate mitochondrial NAD+/NADH kinase was recently identified as a homolog of A. thaliana Nadk3, which can use ATP or polyphosphate as the phosphate donor (47).
1.3 Thesis Goals
The main focus of my thesis research was to develop LC-MS/MS technologies for the quantitation of NAD+ and related metabolites to further our understanding of NR interventions in healthy and diseased states. Previous members of my thesis laboratory focused upon the enzymes related to NAD+ and its biosynthesis from NAR and NR (7, 30, 32, 33, 48). In their investigations, the first NAD metabolome assay was developed and included substrates and products of these enzymes and other enzymes related to NAD+ metabolism (31). From their work, NR was established as a bona fide salvageable NAD+ precursor that could extend life- span of yeast (32) and work from other groups revealed NR extends life-span in C. elegans (49) and acts to oppose metabolic (37, 50-54) and neurodegenerative disorders (55, 56) in rodents. Translating these health promoting effects of NR in the clinic required the improvement and development of novel technologies for accurate and robust quantitation of NAD+ and related metabolites.
In my thesis work, I improved upon the previous assay by including internal standards, adding additional NAD+ related metabolites, and further optimizing extraction procedures for cell and tissues. With these improvements, we were able to produce a detailed metabolic image of the fate of NAD+ metabolism in a variety of biological contexts with and without NR interventions. The technologies described herein allowed for both the confirmation and
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generation of hypotheses regarding the effect of NR on NAD+ metabolism in the normal and abnormal function of a cell or organism.
In chapter 2, I include a reprint of the remaining publication used to introduce my dissertation in this chapter. Sections are added after section 2.2 Conclusions to describe further method development as necessitated by my thesis work.
Chapters 3, 4, 5, 6, and 7 are demonstrations of the technologies described in chapter 2. Our work in chapter 3 establishes the true B3 vitamin content of bovine milk, uncovers that farming practices may influence the vitamin quality of milk, and reveals that B3 vitamin fortification of bovine milk may be a future route of delivery of NR to populations at risk for developing neurological and metabolic disorders. Specifically, we show that NR represents 40% of the B3 vitamin content of bovine milk from a herd of Bos taurus and in store procured cow’s milk. We uncover that organic milk tends to contain less NR than conventional milk and suggest that, in part, Staphylococcus aureus infection may be responsible. We then uncover that NR is stable in and binds to milk. This chapter illustrates how the same technology utilized to merely elucidate the composition of a food product can be utilized to generate and test hypotheses for how the vitamin content of a food.
In chapter 4, we establish that NR is a superior NAD+ precursor compared to NMN using stable isotope labeling technologies. Work in chapter 3 and 4 were crucial for later work described in chapter 5 where more complex stable isotope labeled experiments were performed.
The work described in chapter 5 is the culmination of my thesis work. This chapter is my perspective and narration of a work that was written by my advisor Dr. Charles Brenner and includes data generated using methods pioneered by me but performed by both myself and Dr. Mark Schmidt, a current staff member of the Brenner laboratory. In this work, we quantified the NAD metabolome in a healthy middle-aged human subject after initial and subsequent supplementation of NR and uncover that NAAD is a potential, non-obvious, accessible
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biomarker for NR supplementation. NAAD is a non-obvious product of NR since there is currently no known mammalian deamidating NAD+ pathway (Figure 1.1). We then compare the effect of NA, Nam, and NR on the murine hepatic NAD metabolome. All three precursors increase NAD+ as expected. However, both Nam and NR increase NAAD. Additionally, we report for the first time that NR is a far superior effector in NAD+ metabolism, increasing both hepatic NAD+ and NAAD to a greater extent compared to NA and Nam. Further, we tested whether and confirmed that NR directly contributes to NAAD using stable isotope technologies. In Dr. Schmidt’s work, we confirm that NAAD positively correlates with NR dosage in a group of healthy human subjects. Together, these works performed in human and murine systems prove NR is superior to other B3 vitamins effecting the NAD metabolome and increasing NAD+ in particular and uncover that NAAD may be a future, clinical biomarker for the effect of NR on NAD+ metabolism.
Chapters 6 and 7 are essentially the phenotypic effects of NR supplementation on metabolic syndromes. In chapter 6, we tested the hypothesis that NR supplementation would prevent alcohol-induced fatty liver disease by increasing hepatic NAD+ and consequently reversing the metabolic damage of alcohol on mitochondrial metabolism. In Chapter 7, we tested the hypothesis that the alteration of the NAD metabolome in diabetic animals is involved in the etiology of diabetic peripheral neuropathy and that supplementation with NR could prevent this devastating complication of diabetes. In this chapter, I present our work with a Type I diabetic animal model and NR and my perspective on my work with a Type II diabetic animal model and its context alongside the work performed by Dr. Mark Yorek and coworkers detailed in Chapter 7.1-4 written by Dr. Charles Brenner, thesis advisor.
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1.4 Figure
Figure 1.1 NAD+ biosynthesis in yeast and vertebrates.
Intracellular NAD+ is derived from either de novo synthesis from tryptophan or from salvage of NA, Nam, or NR. In yeast, Nam is converted to NA by nicotinamidase Pnc1p (dotted line). In yeast and vertebrates, NA is phosphoribosylated to NAMN, an intermediate in de novo synthesis, and converted to NAD+ by way of NAAD in a step catalyzed by glutamine-dependent NAD+ synthetase (12). In vertebrates, Nam conversion to NMN is catalyzed by Nampt (16). The other source of NMN in yeast and vertebrates is phosphorylation of NR by NR kinases. NR and NAR can be split to the corresponding pyridine bases. NAR phosphorylation yields NAMN. NMN is converted to NAD+ by NMN adenylyltransferase activity, which is reversible. As shown, in vertebrates, NMN must be imported into mitochondria for conversion to NAD+. Enzymatic NAD+ and NADP+ consumption releases the Nam moiety and produces ADPr products. Finally, Nam and NA can be converted to non-salvageable products.
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CHAPTER 2
NAD METABOLOME ANALYSIS VIA LIQUID CHROMATOGRAPHY MASS SPECTROMETRY
“Targeted, LCMS-based Metabolomics for Quantitative Measurement of NAD+ Metabolites”*
Samuel A.J. Trammell1,2 and Charles Brenner1,2
1Department of Biochemistry, 2 Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
*The following chapter is a description of the general liquid chromatography and mass spectrometry methodologies employed in all subsequent chapters. Sections 1 – 2 are reprints of the rest of the publication included in Chapter 1.1-1.2 (1) which was written by myself with guidance and editing by CB.
2.1 Quantitative NAD+ Metabolomics
The NAD+ metabolome4, as defined here, includes dinucleotides, nucleotides, nucleosides, nucleobases and related compounds (Table 2.3). The masses of many of the analytes differ by a single Dalton, necessitating optimal separation and careful MS. The current method is an improvement over methods, which measured only select metabolites (17, 57), and more recent methods, which embraced a more complete set of metabolites, but which lacked resolution of several compounds (31, 58). Here we review optimization of all parameters and a solution to the ionization suppression problem that plagued previous methods.
Optimized Extraction
Methods that do not inactivate enzymatic activities upon cell lysis (58) are clearly flawed
and, based upon the amount of time of sample autolysis, cellular NAD+ can be degraded to ~1% of expected values (~10 μM) with elevation of apparent NR concentration to ~100 times
4 After publication of this document, we have referred to the NAD+ metabolome interchangeably with the NAD metabolome.
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expected values (1 mM) (59). The preferred method of extraction is to use boiled, buffered ethanol (60), which is well validated for NAD+ metabolites (30, 31).
For yeast samples, an ideal cell number is 2 to 4.5 x 107, the midrange of which can be obtained by harvesting 25 ml of cells at an OD600 nm of 0.7. For mammalian cell culture, we typically use 4 to 20 x 106 cells, depending upon the cell type. Yeast cell pellets are extracted directly. Mammalian cell pellets are washed once in ice-cold potassium buffered saline. Cells are resuspended in 300 μL of a 75% ethanol/25% 10 mM HEPES, pH 7.1 v/v (buffered ethanol) solution, preheated to 80 °C. Samples are shaken at 1000 rpm in an 80 °C block for three minutes. Soluble metabolites are separated from particulate by refrigerated microcentrifugation (10 min, 16kg). Though the ethanol-soluble extract contains all the metabolites of interest, the weight of the particulate can be used to determine the optimized resuspension volume for dried metabolites. Thus, both the particulate and soluble metabolites are dried by speed vacuum at 40 °C.
Empirically, we determined that 3.6 mg of yeast or mammalian cell-derived particulate corresponds to a metabolite pellet, which can be resuspended in a 100 μl volume and produce the desired absorbance and LC-MS signals. Thus, the dry weight of each pellet is recorded, divided by 3.6 mg, and multiplied by 100 μl to obtain the initial resuspension volume. Extracts are resuspended in 1% (v/v) acetic acid adjusted to pH 9 with ammonium hydroxide (ammonium acetate buffer). These conditions were chosen to preserve NADH and NADPH prior to analysis5 (61).
Resuspended metabolites (2 μl) are checked in a Nanodrop (ThermoFisher) to determine the OD260 nm, which is typically greater than or equal to 14. The remaining volume is diluted to an OD260 nm of 14 in ammonium acetate buffer to obtain the final resuspension volume.
5 In Chapter 2.4, I describe problems in analysis uncovered after publication which required alterations to the re-suspension solvent and to the metabolites included in the metabolomic assay and the way with which they were dealt.
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For LC-MS, this material is diluted two-fold into two different metabolite standards and 2.5 μl of the resulting material is injected and analyzed. Because these dilutions convert the total intracellular volume into a known volume of which an effective volume of 1.25 μl is analyzed, it is straightforward to calculate the intracellular volume of cells under analysis.
The calculation of intracellular volume is as follows. For yeast cells, the intracellular volume of a single cell is taken as 70 fl (62). Thus, the calculated intracellular volume is obtained as 70 fl times the cell number. For mammalian cells, we use 2.5 pl as volume of a HeLa cell (63) and calculate the total extracted intracellular volume in the same way as for yeast cells. For example, an extraction of 3 x 107 yeast cells has a calculated intracellular volume of 2.1 μl. If this sample were resuspended into 100 μl and require no further adjustment after checking on the Nanodrop, the 1.25 μl of cell extract in a 2.5 μl injection would represent 1.25% of 2.1 μl = 26 nl. Because the internal standards permit metabolites to be quantified on a mol scale, intracellular metabolite concentrations are determined, in this example, as mol of metabolite divided by 2.6 x 10-8 l.
Optimized Internal Standards
Ionization suppression is the tendency for sample components to dampen the ionization
and detectabilit