Dynamics of blood NAD and glutathione in health, disease, aging, and under NAD+-booster treatment.

Nicotinamide adenine dinucleotides (NAD+, NADH, NADP+ and NADPH) and glutathione (GSH and GSSG) metabolites are vital regulators of redox state, enzyme functions, and metabolic flux in hundreds of cellular metabolic reactions. NAD+/NADH ratio increases during fasting and has been associated with health outcomes in model systems. A low ratio occurs in specific diseases, treated with high-dose B3-vitamin forms, so-called NAD+-boosters. Still, neither healthy human values nor NAD+ booster-treated or disease-related levels of NAD(P)(H) or glutathione have been established in humans. Here, we report a standardized workflow of enzymatic cycling assays to quantitatively measure NAD+, NADH, NADP+, NADPH, GSH, and GSSG from a single whole-blood sample. In the blood of a healthy population aged 18-70 years, these metabolites follow a normal distribution and remain unchanged during aging. High-dose nicotinic acid, a B3 vitamin, resulted in 4-6-fold increase of blood NAD+ in healthy individuals, suggesting the need for personalized dosing and follow-up in treatment and drug trials. Our data also indicate disease-dependent "redox fingerprints" of NAD and glutathione forms in different degenerative diseases, including cancers. The evidence highlights the potential of redox profiling as an indicator of probable pathology and as a measure of treatment response.