The colorimetric determination of NAD (NAD^+ NADH) as well as NADP (NADP^+ + NADPH), which has been developed by Shibata et al., has been successfully improved to estimate very small amount of NAD. The method is highly reproducible, very straightforward, and can treat many samples at a time. In the present method, micro plate reader was used and the sample volume needed were only 10 μl for NAD determination and the minimum detection limit was 5 pmol/10μl in the sample solution. For NADP determination, the sample volume needed were 20μl and the minimum detection limit was 2 pmol/20μl. This method was applied to estimate NAD (or NADP) in many biological samples such as cultured cells, blood, tissues and so on.
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