They need replenishing only to the extent that they are accidentally lost or degraded. We have lost what ability we once had to synthesize nicotinic acid and riboflavin biologically, and are forced to obtain the raw materials for NAD⁺ and FAD from our diet. Substances such as these, which are needed only in minute quantities but nevertheless are absolutely essential in these quantities, are termed vitamins. Vitamin A, the precursor of retinal, is one example. Riboflavin is vitamin B₂, and nicotinic acid is niacin. Niacin deficiency in humans causes penagra, a disease once common in the American rural South but which now has been largely eradicated.

When NAD⁺ is reduced, one H binds to the ring, the electron from the second H cancels the positive charge, and the proton goes into solution, as shown above. When FAD is reduced, two hydrogen atoms are attached to the flavin ring at two points, as at the left. Energy is stored in both of these reduced molecules, to be released again when the carrier molecule is reoxidized. Just as the amount of energy obtained in an oxidation depends on what is used as the oxidizing agent, so the energy that we can think of as stored in reduced NADH or FADH₂ varies with the substances used to reoxidize them.

In normal 02 respiration, reoxidization of NADH takes place with a liberation of 52.7 kcal mole-' of free energy: (next page)
NADH + H⁺ + ½O₂ --> NAD⁺ + H₂0 D` = 52.7 kcal mole^-'

(The prime indicates a free energy change under the physiological conditions of pH 7, or [H+] = 1(-7 mole litre^-1, rather than 1 mole litre^-l.) Under these conditions we can think of each mole of NADH as "carrying" 52.7 kcal of free energy from the place where it was reduced to the place where it will be reoxidized. A mole of FADH₂ carries somewhat less energy:
FADH₂ + ½0₂ --> FAD + H₂0 D` = -36.2 kcal mole^-'

The dinucleotides NAD⁺ and FAD, and the nucleotide ATP, cooperate as "big buckets" and "little buckets" for energy in the energy extracting processes of living cells. When foodstuffs are broken down, 53-kcal quantities of energy are stored by reducing NAD⁺ to NADH, or smaller amounts by reducing FAD to FADH₂. These reduced dinucleotides, no matter what their source, then can funnel into a common respiratory machinery that reoxidizes them and transfers their energy in smaller packages to ATP: three ATP per NADH molecule reoxidized, or two ATP per FADH₂. In the banking analogy for energy storage in Chapter 21, NADH molecules are the nickels of the energy coinage and ATP molecules are the pennies.