Biochimica et Biophysica Acta (BBA) - General Subjects
Mini reviewQuantitation of NAD+: Why do we need to measure it?
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.
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