CD38 is a multifunctional enzyme involved in calcium signaling and Nicotinamide Adenine Dinucleotide (NAD⁺) metabolism. Through its major activity, the hydrolysis of NAD⁺, CD38 helps maintain the appropriate levels of this molecule for all NAD⁺-dependent metabolic processes to occur. Due to current advances and studies relating NAD⁺ decline and the development of multiple age-related conditions and diseases, CD38 gained importance in both basic science and clinical settings. The discovery and development of strategies to modulate its function and, possibly, treat diseases and improve health span put CD38 under the spotlights. Therefore, a consistent and reliable method to measure its activity and explore its use in medicine is required. We describe here the methods how our group measures both the hydrolase and cyclase activity of CD38, utilizing a fluorescence-based enzymatic assay performed in a plate reader using 1,N⁶-Ethenonicotinamide Adenine Dinucleotide (ε-NAD) and Nicotinamide Guanine Dinucleotide (NGD) as substrates, respectively.

Current studies on the age-related development of metabolic dysfunction and frailty are each day in more evidence. It is known, as aging progresses, nicotinamide adenine dinucleotide (NAD⁺) levels decrease in an expected physiological process. Recent studies have shown that a reduction in NAD⁺ is a key factor for the development of age-associated metabolic decline. Increased NAD⁺ levels in vivo results in activation of pro-longevity and health span-related factors. Also, it improves several physiological and metabolic parameters of aging, including muscle function, exercise capacity, glucose tolerance, and cardiac function in mouse models of natural and accelerated aging.

Given the importance of monitoring cellular NAD⁺ and NADH levels, it is crucial to have a trustful method to do so. This protocol has the purpose of describing the NAD⁺ and NADH extraction from tissues and cells in an efficient and widely applicable assay as well as its graphic and quantitative analysis.