Mini review
Quantitation of NAD+: Why do we need to measure it?

https://doi.org/10.1016/j.bbagen.2018.07.023Get rights and content

Highlights

  • Nicotinamide adenine dinucleotide (NAD+) is an important target to extend lifespan and health span.

  • Age-related accumulation of oxidative stress can deplete NAD+.

  • NAD+ precursors, exercise and caloric restriction can increase NAD+ levels.

  • NAD+ is also an important factor in tumor biology and some infections and immunological roles.

  • Accurately quantifying NAD+ represents an unexplored yet important component of NAD+ therapy.

Abstract

Background

Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that is currently investigated as an important target to extend lifespan and health span. Age-related NAD+ depletion due to the accumulation of oxidative stress is associated with reduced energy production, impaired DNA repair and genomic instability.

Scope of review

NAD+ levels can be elevated therapeutically using NAD+ precursors or through lifestyle modifications including exercise and caloric restriction. However, high amounts of NAD+ may be detrimental in cancer progression and may have deleterious immunogenic roles.

Major conclusions

Standardized quantitation of NAD+ and related metabolites may therefore represent an important component of NAD+ therapy.

General significance

Quantitation of NAD+ may serve dual roles not only as an ageing biomarker, but also as a diagnostic tool for the prevention of malignant disorders.

Introduction

Nicotinamide adenine dinucleotide (NAD+) is an important coenzyme that is found in all living organisms [1]. Apart from serving as a critical coenzyme for enzymes involved in reduction-oxidation reactions and energy production, NAD+ is also a cosubstrate for other enzymes such as the sirtuins, NAD+ glycohydrolase (CD38), and poly(adenosine diphosphate–ribose) polymerases. Cellular NAD+ levels can be reduced by as much as 80% in old age and among other things, leads to mitochondrial dysfunction, altered gene expression, immune disruption, and the loss of function of sirtuin deacetylation activity and Foxo3A, both of which are associated with healthy longevity [[2], [3], [4]]. Experiments to increase NAD+ in rodents have shown that it can prolong both healthspan and lifespan [5].

In 2011, our group hypothesized that oxidative stress induced NAD+ depletion could play a significant role in the ageing process, by compromising, energy production, DNA repair and genomic surveillance [2]. More recently, it was hypothesized, that NAD+ as not only a cofactor in redox reactions and coenzyme in metabolic processes that has the ultimate role in ageing, but rather the role of NAD+ in cellular signaling when used as substrate for sirtuins (SIRT1-7 in mammals), PARPs (Poly(ADP-ribose) polymerases) and CD38 NAD-dependent glycohydrolases [6].

With these limitations in mind, it has been hypothesized that pharmacological manipulation of the NAD+ synthesis pathway can increase NAD+ levels. Recent studies have shown that oral administration of the NAD+ precursor nicotinamide riboside (NR) can increase whole blood NAD+ levels in humans [[7], [8], [9]]. However, using a direct intravenous (IV) infusion of NAD+ is likely to represent a more efficient way of increasing NAD+ levels than the indirect administration of oral supplements that act as precursors for NAD+ synthesis or spare its consumption. IV NAD+ has been utilized by clinicians for the treatment of alcoholism since the 1950s, as documented by Dr. Paul O'Hollaren, who claimed to have treated >100 acute and chronic alcoholic patients for years, with 1000 mg/day IV, without toxic effects [10].

Building on this principle, several clinics around the United States, and elsewhere, have been using IV NAD therapy over the past several decades to alleviate the symptoms of drug and alcohol withdrawal, increase energy, and improve mood, memory and mental clarity. Thus, while it can be argued that several means are available to increase NAD+ levels, accurate measurement of the endogenous intracellular and extracellular levels of NAD+ and related metabolites (hence forth the NADome) remains unclear. Therefore, establishing the ‘safe’ therapeutic range of the NADome, and standardising NAD+ testing in biological specimens as part of good laboratory practices is warranted and can be applied for the clinical diagnosis of symptoms of metabolic syndrome and addiction disorders.

Section snippets

The NADome – A cellular currency

NAD+ is a highly required coenzyme for over 400 different oxidoreductases, due to its reversible oxidation-reduction potential [6]. When utilized as a substrate for some metabolic enzymes such as glyceraldehyde phosphate dehydrogenase (GAPDH), NAD+ is reduced to NADH, which can be reoxidised to NAD+ in a gluconeogenesis direction. NAD+ and NADH can also undergo phosphorylation to NADP+ and NADPH. NADP+ can be converted to NADPH in the pentose phosphate pathway (PPP). NADPH plays a crucial role

How is NAD+ synthesised and transported into the cell?

In eukaryotic cells, NAD+ levels are regulated by either de novo synthesis from catabolism of the amino acid tryptophan (TRP) via the kynurenine pathway (KP), or through salvage of NAM, nicotinic acid (NA) and nicotinamide riboside (NR) form of vitamin B3 [11] (Fig. 2). The de novo synthesis of NAD+ involves a six-step process that catabolizes TRP to form nicotinic acid mononucleotide (NAMN) which produces NAD+ via two additional steps. To maintain the demand for NAD+, eukaryotic cells have

NADome hormesis: does it exhibit a biphasic dose response?

The beneficial effect of the NADome in the regulation of cellular redox status is well established, and NAD+ decline has been associated with the development and progression of several metabolic and age-related degenerative disorders. An important question is whether too much NAD+ is always desirable. Given the data from in vitro and in vivo studies currently available in the literature, it is likely that promotion of NAD+ levels may induce a hormetic dose response that may influence clinical

How can we measure NAD+?

Given the role of NAD+ in complex biological processes, promotion of NAD+ anabolism may be useful to delay ageing and treat age-related diseases. However, this requires the design of clinical trials to further assess the effect of these compounds on global metabolism. Routine measurement of the NADome may provide an additional means to verify the beneficial effects of NAD+ as a therapeutic strategy to ameliorate metabolic disorders, and counteract the development of malignant cells. However,

Prospects and clinical implications

It is well established that the NADome has several important cellular roles. Alterations in intra- and extracellular levels of NAD+ have been associated with oxidative-stress induced cellular degeneration and cancer. NAD+ is not only an important cofactor for redox reactions, but it is also required as a substrate for optimal PARP, sirtuin, and CD38 activity. While emerging studies are highlighting the beneficial effects of oral administration of NR to increase NAD+ in human, the effects of

Acknowledgements

NB is recipient of the Australian Research Council (DE170100628) DECRA Fellowship.

References (60)

  • V. Kulikova

    Generation, release, and uptake of the NAD precursor nicotinic acid riboside by human cells

    J. Biol. Chem.

    (2015)
  • A. Nikiforov

    Pathways and subcellular compartmentation of NAD biosynthesis in human cells: from entry of extracellular precursors to mitochondrial NAD generation

    J. Biol. Chem.

    (2011)
  • D. Wang

    Hormesis as a mechanistic approach to understanding herbal treatments in traditional Chinese medicine

    Pharmacol. Ther.

    (2018)
  • M. Fukui et al.

    Mechanism for the protective effect of resveratrol against oxidative stress-induced neuronal death

    Free Radic. Biol. Med.

    (2010)
  • A. Mangerich

    Inflammatory and age-related pathologies in mice with ectopic expression of human PARP-1

    Mech. Ageing Dev.

    (2010)
  • B.E. Kennedy

    NAD(+) salvage pathway in cancer metabolism and therapy

    Pharmacol. Res.

    (2016)
  • E. Limagne

    Sirtuin-1 activation controls tumor growth by impeding Th17 differentiation via STAT3 deacetylation

    Cell Rep.

    (2017)
  • P. Bubner et al.

    Structure-guided engineering of the coenzyme specificity of Pseudomonas fluorescens mannitol 2-dehydrogenase to enable efficient utilization of NAD(H) and NADP(H)

    FEBS Lett.

    (2008)
  • C. Bernofsky et al.

    An improved cycling assay for nicotinamide adenine dinucleotide

    Anal. Biochem.

    (1973)
  • J. Evans

    LC/MS analysis of NAD biosynthesis using stable isotope pyridine precursors

    Anal. Biochem.

    (2002)
  • K. Yamada

    The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry

    Anal. Biochem.

    (2006)
  • T.S. Blacker et al.

    Investigating mitochondrial redox state using NADH and NADPH autofluorescence

    Free Radic. Biol. Med.

    (2016)
  • N. Braidy

    Serum nicotinamide adenine dinucleotide levels through disease course in multiple sclerosis

    Brain Res.

    (2013)
  • H. Massudi

    NAD+ metabolism and oxidative stress: the golden nucleotide on a crown of thorns

    Redox Rep.

    (2012)
  • N. Braidy

    Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats

    PLoS One

    (2011)
  • N. Braidy

    Mapping NAD(+) metabolism in the brain of ageing Wistar rats: potential targets for influencing brain senescence

    Biogerontology

    (2014)
  • N. Braidy

    Differential expression of sirtuins in the aging rat brain

    Front. Cell. Neurosci.

    (2015)
  • N. Braidy et al.

    Role of nicotinamide adenine dinucleotide and related precursors as therapeutic targets for age-related degenerative diseases: Rationale, Biochemistry, Pharmacokinetics, and outcomes

    Antioxid. Redox Signal.

    (2018)
  • R.W. Dellinger

    Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD(+) levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study

    NPJ Aging Mech. Dis.

    (2017)
  • W. Shi

    Effects of a wide range of dietary nicotinamide riboside (NR) concentrations on metabolic flexibility and white adipose tissue (WAT) of mice fed a mildly obesogenic diet

    Mol. Nutr. Food Res.

    (2017)
  • Cited by (14)

    • Pharmacology of NAD<sup>+</sup> boosters

      2022, Anti-Aging Pharmacology
    • Simultaneous determination of 49 amino acids, B vitamins, flavonoids, and phenolic acids in commonly consumed vegetables by ultra-performance liquid chromatography–tandem mass spectrometry

      2021, Food Chemistry
      Citation Excerpt :

      The content of free riboflavin in tomatoes was the lowest among the 26 vegetables, while the content of pyridoxine in tomatoes was the highest, at 633.5 ± 23.1 µg/kg FW, which was 77 times higher than that in carrots. Nicotinic acid, derived from plant-based foods, is a precursor of nicotinamide adenine dinucleotide (NAD+) (Liu, Clement, Grant, Sachdev, & Braidy, 2018). Nicotinic acid was not detected in any of the three Compositae vegetables, green lettuce, red lettuce, leaf lettuce, nor in tomato.

    • Improving the production of NAD<sup>+</sup> via multi-strategy metabolic engineering in Escherichia coli

      2021, Metabolic Engineering
      Citation Excerpt :

      Nicotinamide adenine dinucleotide (NAD+) is a classical metabolite that mediates various biological processes, including DNA repair, gene expression, and energy production (Liu et al., 2018; Yaku et al., 2018).

    View all citing articles on Scopus
    View full text