The dietary vitamin B3, which encompasses nicotinamide, nicotinic acid and nicotinamide riboside, is precursor to the coenzyme nicotinamide adenine dinucleotide (NAD+), its phosphorylated parent (NADP+) and their respective reduced forms (NADH and NADPH). Once converted intracellularly to NAD(P)+, it is used as a co-substrate in two types of intracellular modifications, which control numerous essential signalling events (adenosine diphosphate ribosylation and deacetylation), and is a cofactor for over 400 redox enzymes, thus controlling metabolism. Critically, the NAD(P)(H)-cofactor family can promote mitochondrial dysfunction and cellular impairment if present in sub-optimal intracellular concentrations. Vitamin B3, and other B-vitamins such as thiamine (vitamin B1), riboflavin (vitamin B2) and pyridoxine (vitamin B6) are extracted in their coenzyme forms from food stuff. During digestion, the coenzymes are catabolised to the free circulating vitamins, which are then passively or actively transported across membranes, and salvaged intracellularly to their respective cofactors. Mammals are entirely reliant on a dietary source of vitamin B1 and heavily dependent on the dietary supply of vitamin B3, B2, and B6. Of note, acute deficiencies in vitamin B1 and vitamin B3 effect identical organs, with identical outcomes if left untreated: dementia and death. Conditions such as diabetes and obesity, alcoholism, high fat diet and conditions where therapy impacts nutrition can compromise suitable absorption of these vitamins. The bulk of intracellular NAD+ is regenerated via the effective salvage of nicotinic acid and nicotinamide (vitamin B3), while de novo NAD+ is obtained from tryptophan. Crucially, these salvage and de novo pathways depend on the functional forms of vitamin B1, B2 and B6 to generate NAD+ via a phosphoriboside pyrophosphate intermediate. Nicotinamide Riboside (NR) is the only form of vitamin B3 from which NAD+ can be generated in a vitamin B1, B2 and B6 independent manner and even though NR is a minor component of vitamin B3, the salvage pathway using NR for the production of NAD+ is expressed in most eukaryotes. While major strides have been made in the field of NAD+ biology and NAD+ metabolism, the role of this later pathway and the importance of the interplay between the bioavailability of vitamin B1, B2 and B6 and the pool of NAD(P)(H)-cofactors remain poorly explored. Using our synthetic expertise in nucleotide and stable isotope labelling chemistry, we will generate isotopically labelled vitamin B1 and B3 derivatives. These entities will be used to determine the profile of the vitamin B3 metabolome quantified by mass spectroscopy, under vitamin B1, B2 and B6 depletion conditions, in genetically engineered yeast strains and mammalian (murine and human) hepatocytes. In mammalian cells, these metabolic profiles will be correlated to mitochondrial functions. With this information, we will be able to prioritise the mechanisms cells use to best maintain the NAD(P)(H) pool in time of shortage of vitamin B1, B2 or B6. We predict that the pathway, by which NR is converted to NAD+, provides the means to rapidly yet transiently elevate mitochondrial and cytosolic NAD(P)(H) levels to kick start mitochondrial functions. If demonstrated, this knowledge will help identify new, physiologically relevant, vitamin-B combinations that could better restore mitochondrial function through enhanced bioavailability, in cells and organs where metabolism has been compromised by imbalanced micronutrition. This knowledge will be particularly important in terms of understanding the impacts of a global or partial vitamin B deficiency and vitamin B supplementation on organ functions in relation to malnutrition and over-nutrition.