An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases

A method for measuring nicotinamide-adenine dinucleotide by enzymatic cycling is described which uses malic and alcohol dehydrogenases (EC 1.1.1.37, and EC 1.1.1.1) for the enzyme couple. After cycling, malate is measured with either malic dehydrogenase or malic enzyme (EC 1.1.1.40). The method has a number of advantages compared to those previously described. The cycling rate is high (greater than 30 000/hr); blank values are low; the reaction is linear over a wide range of NAD concentrations; and the terminal indicator reaction requires only one step. In addition the system is well suited for double cycling. This was shown by measurements of NAD in nuclei and cytoplasm from single dorsal root ganglion cells (rabbit). The overall amplification in this case was about 1 000 000.