Studies on the biosynthesis of nad by a direct method of tracing metabolism in vivo

Abstract

In order to investigate the biosynthetic process of NAD in the intact organism, the pulse-labeling experiments in vivo were carried out, which involves the direct injection of the radioactive substrates into the portal vein, followed by the analyses of the isotopically labeled metabolites in the liver. When administered in small doses (78 mμmoles per mouse) into the portal vein, nicotinic acid was a much better precursor of liver NAD than nicotinamide. The incorporation of nicotinic acid- 14C into NAD proceeded almost linearly up to 10 min, thereafter NAD- 14C gradually decreasing to about one-half by 10 hr. Nicotinic acid ribonucleotide- 14C and deamido-NAD- 14C, the presumed intermediates in this conversion, were also detected during several minutes after the injection. In contrast, nicotinamide- 14C injected in this way was not utilized efficiently for the de novo synthesis of NAD during the first hour after the injection. Quinolinic acid- 14C hardly penetrated into the liver cells. On the contrary, when administered in large doses (75 μmoles per mouse), nicotinamide was a much better precursor of liver NAD than nicotinic acid. With a large dose of nicotinamide- 14C as substrate, the total radioactivity in the liver decreased rapidly during the first hour and then increased up to almost 8 hr after the injection. During the initial phase, the incorporation of 14C into NAD was almost insignificant but NAD- 14C in the liver began to increase about 1 hr after the injection with the concomitant increase of the total radioactivity in the liver. Analyses of the distribution of 14C in various organs and tissues indicated that a large portion of nicotinamide- 14C was first excreted from liver, accumulated in the gastrointestinal tract, deamidated to nicotinic acid, reabsorbed into the liver and served as precursor to NAD over a prolonged period of time.