In rat liver, the total amount of NAD and NADH is approximately 1 μmole per gram of wet weight, about 10 times the concentration of NADP and NADPH in the same cells. The actual concentration of NAD in cell cytosol is harder to measure, with recent estimates in animal cells ranging around 0.3 mM, and approximately 1.0 to 2.0 mM in yeast. However, more than 80% of NADH fluorescence in mitochondria is from bound form, so the concentration in solution is much lower.
NAD concentrations are highest in the mitochondria, constituting 40% to 70% of the total cellular NAD. NAD in the cytosol is carried into the mitochondrion by a specific membrane transport protein, since the coenzyme cannot diffuse across membranes. The intracellular half-life of NAD was claimed to be between 1–2 hours by one review, whereas another review gave varying estimates based on compartment: intracellular 1–4 hours, cytoplasmic 2 hours, and mitochondrial 4–6 hours.
The balance between the oxidized and reduced forms of nicotinamide adenine dinucleotide is called the NAD/NADH ratio. This ratio is an important component of what is called the redox state of a cell, a measurement that reflects both the metabolic activities and the health of cells. The effects of the NAD/NADH ratio are complex, controlling the activity of several key enzymes, including glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase. In healthy mammalian tissues, estimates of the ratio of free NAD to NADH in the cytoplasm typically lie around 700:1; the ratio is thus favorable for oxidative reactions. The ratio of total NAD/NADH is much lower, with estimates ranging from 3–10 in mammals. In contrast, the NADP/NADPH ratio is normally about 0.005, so NADPH is the dominant form of this coenzyme. These different ratios are key to the different metabolic roles of NADH and NADPH.