1. Incubation of isolated rat hepatocytes with nicotinamide or nicotinic acid showed that while both vitamers were taken up from the incubation medium, neither was utilized to any significant extent as a precursor of the nicotinamide nucleotide coenzymes, NAD and NADP, and neither was capable of preventing the loss of nucleotides that occurs on incubating the cells.2. Incubation of hepatocytes with tryptophan showed that de novo synthesis from tryptophan permitted replacement of the nucleotides lost during incubation ; at high concentrations of tryptophan there was an increase above the initial intracellular concentration of NAD(P). Incubation of hepatocytes with tryptophan also resulted in the formation and release from the cells of a considerable amount of niacin, as well as the two principal metabolities of NAD(P), "-methyl nicotinamide and methyl pyridone carboxamide.3. It is suggested that, in the liver, preformed niacin is not utilized for nucleotide synthesis, and indeed the function of the liver appears to be synthesis of niacin from tryptophan, and its release for use by extrahepatic tissues that lack the pathway for de novo synthesis of nicotinamide nucleotides from tryptophan.Previous studies from this laboratory have suggested that the de novo synthesis of nicotinamide nucleotides (NAD and NADP) from tryptophan may be a more important source of these coenzymes than is the utilization of dietary nicotinamide or nicotinic acid. The enzyme kinetic variables are such that the three enzymes involved in the incorporation of preformed niacin into nucleotides, nicotinamide phosphoribosyltransferase (EC 2.4.2.12), arylformamidase (nicotinamide deamidase; EC 3.5.1 -19) and nicotinate phosphoribosyltransferase (EC 2.4.2.11) (see Fig. l), all act at or near their maximum rates at the normal intracellular concentrations of nicotinamide and nicotinic acid (Bender et al. 1982). This suggests that there would be little or no utilization of any additional niacin which might be available from the diet or arising from the catabolism of NAD(P).Feeding rats on diets providing high intakes of either nicotinic acid or nicotinamide leads to a relatively small increase in the concentration of NAD(P) in the liver. A tenfold increase in dietary niacin resulted in only a 50 % increase in liver NAD(P). By contrast, a threefold increase in dietary tryptophan resulted in a 2.6-fold increase in liver NAD(P), as well as a very considerable increase in the urinary excretion of "-methyl nicotinamide and methyl pyridone carboxyamide, the two principal metabolites of nicotinamide, resulting from hydrolysis of NAD(P) (McCreanor & Bender, 1986).The present study was undertaken in order to investigate further the relative importance of utilization of preformed niacin and de novo synthesis from tryptophan in the synthesis of nicotinamide nucleotide coenzymes in isolated rat hepatocytes.
METHODS
Cell preparation and incubationsMale Wistar rats, weighing 200 g, fed from weaning on animal house stock diet (Diet 86;