The Incorporation of Nicotinic Acid and of Nicotinamide Into the Pyridine Nucleotides of Erythrocytes and Reticulocytes of Rabbits in Vitro*

Abstract

There is abundant evidence that loss of intracellular structure and alteration in metabolic activity occur as mammalian reticulocytes mature to become nonreticulated erythrocytes (2). Loss of the ability to synthesize heme (3), proteins (3, 4), lipids (5-7), purine nucleotides (8), and pyrimidine nucleotides (9) from small molecule precursors accompanies the disappearance of mitochondria, ribosomes, and ribonucleic acid from reticulocytes. An intact Krebs tricarboxylic acid cycle and a complete cytochrome system are not present in the mature mammalian erythrocyte (10). The concentration of ATP is approximately 2.5 times greater in rabbit reticulocytes than in the mature erythrocytes of rabbits (11). Further changes, including a progressive decrease in the activity of several enzymes of the glycolytic pathway (12) and of the hexose monophosphate shunt pathway (12, 13) and in the concentration of ATP (12,14), are associated with aging of mature mammalian erythrocytes in vivo. The concentrations of DPN and of TPN of avian and human erythrocytes were reported to decrease during storage in vitro (15), and the concentration of DPN in human erythrocytes was found to decrease with aging in vivo (16). It was of interest, therefore, to study in more detail the metabolism of the pyridine nucleotides in mammalian erythrocytes as a function of the age of the cells. The present report provides evidence for a difference in the ability of reticulocytes and of mature erythrocytes of rabbits to incorporate radioactive nicotinic acid and nicotinamide into DPN and TPN in vitro. MATERIALS AND METHODSWhole blood, anticoagulated with heparin, was obtained from normal, adult, white New Zealand rabbits. In order to obtain blood enriched with reticulocytes, rabbits were made severely anemic by the administration of acetylphenylhydrazine, 10 mg per kg daily for 5 days. These rabbits were exsanguinated 2 days after the final injection. The blood was centrifuged immediately, the plasma and buffy coat were removed, and the cells were washed three times with 4 to 5 vol of cold 0.9% sodium chloride solution. Flasks for incubation were prepared to contain 10 ml of erythrocytes or reticulocytes, 1.6 to 1.7 ,emoles and 9.4 to 10.0 ,uc of radioactive nicotinic acid or nicotinamide, 870 ,umoles of glucose, 140 ,umoles of glutamine, 330 jcmoles of inorganic phosphate, 9 mg of penicillin, and 9 mg of streptomycin in a total volume of 15 ml. The specific activity of the nicotinic acid-7-C14 was 5.9 ,c per ,umole and that of the nicotinamide-7-C'4 was 5.6 lc per uemole.1 In experiments in which different concentrations of nicotinic acid were employed, the specific activity was reduced proportionally by the addition -of unlabeled compound.

The Synthesis of Pyridine Nucleotides in Fresh and Stored Human Erythrocytes*

“…The procedures employed for the isolation of nucleotides from cold trichloroacetic acid extracts of washed erythrocytes, for the identification of the pyridine nucleotides and for the determination of radioactivity were described in detail previously. 18 The relative specific activities (MA) of the isolated DPN and TPN were calculated from the formula:…”