NAD and NADH values in rapidly sampled dog heart tissues by two different extraction methods

To clarify the most quantitative extraction method for the determination of NAD and NADH in dog heart tissues, both pyridine dinucleotides were extracted from normal and ischemic heart tissues by the Klingenberg method and the Karp method and determined by bacterial luciferase. Tissues from normal beating hearts were sampled by a specially developed freeze-clamping device in 120 ms to minimize ischemic NADH production during sampling. Samples were obtained from both the subendocardium and the subepicardium of the frozen heart tissues. In the Klingenberg method, NAD and NADH were separately extracted with 0.6 m HClO₄ and 0.5 m KOH in 50% ethanol, respectively. Both pyridine dinucleotides were simultaneously extracted with 70% ethanol in 0.01 m phosphate buffer in the Karp method. The mean values of NAD and NADH in the normal tissues were 5.08 ± 0.84 and 0.18 ± 0.10 nmol/mg protein, respectively, with a NAD NADH ratio of 25-30 by the Klingenberg method. While the values by the Karp method were 4.37 ± 0.68 and 0.09 ± 0.04 nmol/mg protein, with a NAD NADH ratio of 55-65. The efficiency of extraction of both pyridine dinucleotides by the Karp method was lower than that by the Klingenberg method in all tested samples and states of the tissues. These results suggest that the Klingenberg method is preferable for the extraction of both pyridine dinucleotides from dog heart tissues and that the mean NAD NADH ratio in normal dog heart tissues is 25-30.