# Defining NAD(P)(H) catabolism
## 2 NAD(P)(H) Metabolism  
NAD(P)(H) biosynthesis: It has been observed by many that a decrease in NAD\({}^{+}\) levels can negatively affect mitochondrial and cellular function [14; 15]. Mounting evidence also supports that NAD\({}^{+}\) levels decrease with aging and the timeline of such decrease appears to be gender-dependent [7; 16]. Ever since the discovery of the connection between NAD\({}^{+}\) maintenance and the metabolic consequences of its decline in age-related and metabolic diseases such as obesity, diabetes, cardiovascular, Parkinson's, and Alzheimer's disease [17; 18; 19; 20; 21; 22], approaches to increasing NAD\({}^{+}\) levels have become therapeutic pursuits [23; 24]. Some pre-clinical and clinical studies have sought to restore declining NAD\({}^{+}\) levels by interference with NAD\({}^{+}\)-consuming processes (e.g., CD38 inhibitors [25; 26] or PARP inhibitors [27]), by preventing the loss of NAD\({}^{+}\) biosynthetic intermediates [28], by boosting nicotinamide conversion to NAD\({}^{+}\)[29; 30; 31], or by supplementation with NAD\({}^{+}\) precursors [32; 33]. NAD\({}^{+}\) precursors include nicotinamide [19], niacin [34],  
Figure 1: Schematic representation of the formation of intracellular nucleotides and dinucleotides derived from extracellular ribosyllated precursors. niacin derivatives [35; 36], nicotinamide riboside (NR) [37; 38; 39], nicotinamide mononucleotide (NMN) [40; 41; 42; 43], and the reduced forms of NR (NRH) and NMN (NMNH) [44; 45; 46; 47]. From these precursors, NAD\({}^{+}\) can be synthesized by three major biosynthetic pathways: (1) the de novo pathway or kynure pathway from tryptophan; (2) the Preiss-Handler pathway from niacin; and (3) the salvage pathway from nicotinamide. Two additional pathways to NAD\({}^{+}\) uncovered more recently employ ribosylation intermediate-ates. The first employs an NR kinase to generate NMN from NR [48; 49; 50], while the other pathway employs the reduced form of NR, NRH, and adenosine kinase for its phosphorylation to NMNH [13; 44; 51; 52; 53] (Figure 1). NMN and NMNH have also been shown to increase NAD\({}^{+}\) levels in both cells and animal models although some controversy remains as to the feasibility of nucleotide transport across cellular membranes [54; 55; 56]. In addition to the presence of numerous phosphatases that convert nucleotides to nucleosides [57], the equilibrative nucleoside transporters (Figure 1) regulate the levels of nucleoside supplements that make their way to the intracellular space [58], while expression of kinases and hydrolysis of NR by purine nucleoside phosphorylase [59] and BST-1 [60] control the pathway by which NAD\({}^{+}\) precursors truly affect NAD\({}^{+}\) and NADH intracellular levels of a cell [61]. Challenges of measuring levels of precursors and NAD(P)(H) in biospecimens: It should be noted that NRH is readily oxidized in the presence of riboflavin, and free riboflavin cofactors and other oxidants are readily present in cells and tissue extracts, via processes that do not require enzymatic catalysis [62]. This oxidation process also occurs readily upon storage, even at low-temperature, and can also convert NMNH to NMN, NADH to NAD\({}^{+}\), and NADPH to NADP\({}^{+}\) (Figure 2). This oxidative process is often overlooked in the measurements of the NAD\({}^{+}\) metabolome. Yet, on many occasions, only the NAD\({}^{+}\) levels are reported, and it is unclear whether NAD\({}^{+}\) from NADH oxidation contributes to this measurement. Similarly, acidic conditions applied during sample processing of biospecimens affect the levels of NADH and NADPH detected. Under acidic conditions, the reduced forms can degrade and thus does not contribute to the overall NAD(P)(H) measurements [63]. Figure 2: Schematic representation of NRH, NMNH, NADH and NADPH degradation pathways. Considering the importance of total NAD(H) to cellular homeostasis (e.g., [64]), (NAD\({}^{+}\) + NADH) levels should be measured simultaneously under conditions that conserve both NAD\({}^{+}\) and NADH.