Background

Oxidized Nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NAD⁺ and NADH) are important biological cofactors that donate and accept electrons in several anabolic and catabolic functions. They participate in reactions such as glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. In addition, it serves as a substrate for several enzymes involved in DNA damage repair, such as the sirtuins and poly (ADP-ribose) polymerases (PARPs) (Imai and Guarente, 2014; Verdin, 2015; Yoshino et al., 2018).

NAD⁺ levels decrease during aging and are involved in age-related metabolic decline. It has been shown that the cellular NAD pool is determined by a balance between the activity of NAD-synthesizing and NAD-consuming enzymes (Aksoy et al., 2006; Barbosa et al., 2007; Yang et al., 2007; Nahimana et al., 2009; Bai et al., 2011; Yoshino et al., 2011). In previous publications, our laboratory has demonstrated that expression and activity of the NADase CD38 increases with age and that CD38 is required for the age-related NAD decline and mitochondrial dysfunction via a pathway mediated at least in part by regulation of SIRT3 activity (Camacho-Pereira et al., 2016). We also identified CD38 as the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo. That indicates that CD38 has a key role in the modulation of NAD-replacement therapy for aging and metabolic diseases (Camacho-Pereira et al., 2016). CD38 was originally identified as a cell-surface enzyme that plays a key role in several physiological processes such as immune response, inflammation, cancer, and metabolic disease (Frasca et al., 2006; Barbosa et al., 2007; Guedes et al., 2008; Malavasi et al., 2008).

Several different assays and methods have been described due to the great importance of monitoring NAD⁺ and NADH cellular levels under various physiological conditions. Specifically two of them, ultra-performance liquid chromatography (UPLC)-mass spectroscopy assay and cycling assay have finality and both are equally sensitive and specific (Camacho-Pereira et al., 2016).

Our laboratory also further optimized and validated the cycling assay. We determined NAD⁺ and NADH specific and does not detect any of the other nucleotides or NAD derivatives tested, including nicotinamide adenine dinucleotide phosphate (NADP), nicotinic acid adenine dinucleotide (NAAD), nicotinic acid adenine dinucleotide phosphate (NAADP), cyclic-adenine diphosphate ribose (cADPR), adenine triphosphate (ATP), ADP, and others. The results obtained with both methods confirm that there is indeed a decrease in levels of both NAD⁺ and NADH in murine tissues during chronological aging (Camacho-Pereira et al., 2016). Furthermore, both techniques correlated well for both nucleotides (correlation coefficient of r = 0.95 for NAD⁺ and 0.97 for NADH) (Camacho-Pereira et al., 2016).

In regards to the cycling assay (pathway in which the main reaction happens), it takes NAD⁺ and NADH present in the samples and they are coupled to both enzymes alcohol dehydrogenase (ADH) and diaphorase in a cycling assay. Every time NAD⁺ or NADH cycles, it produces a molecule of resorufin, which is highly fluorescent. This fluorescence is directly captured by the multi-well fluorescence plate reader therefore indirectly the NAD⁺ levels can be measured. Most studies have relied on separated extractions for NAD⁺ and reduced nicotinamide adenine dinucleotide (NADH) determination: a basic extraction for the reduced species and a separate acidic extraction for the oxidized species (Ashrafi et al., 2000; Smith et al., 2000; Lin et al., 2001; Anderson et al., 2002; Lin et al., 2004). The extraction conditions are specific for the stabilization of either oxidized compounds, which are more stable in acid or reduced compounds, which are more stable in base.

Conversely, the ultra-performance liquid chromatography (UPLC)-mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides identity of the individual components with high molecular specificity and detection sensitivity. This tandem technique can be used to analyze very accurate components such as NAD⁺ and NADH (Dass, 2007).