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

Abstract

The concentrations of total oxidized pyridine nucleotides and of DPN did not decrease significantly in normal human erythrocytes during storage at 4C in acid‐citrate‐dextrose solution for periods up to 47 days. The capacity of washed erythrocytes which had survived storage to incorporate radioactive nicotinic acid and nico‐tinamide into DPN and TPN during a four‐hour period of incubation at 37 C declined progressively with aging in vitro. The net synthesis of total oxidized pyridine nucleotides and of DPN from nicotinic acid and of total oxidized pyridine nucleotides from nicotinamide also was decreased in these erythrocytes. Addition of inosine to the incubation medium inhibited both incorporation and synthesis by erythrocytes stored for less than three to four weeks. The purine nudeoside enhanced incorporation and synthesis by cells stored for more than about 30 days. It was suggested that the availability of substrates, such as ATP or phosphoribosylpyrophosphate, influenced the extent of pyridine nudeotide synthesis.

Pyridine nucleotides in erythrocyte metabolism.

“…The present evidence indicates, however, that pyridine nucleotides and pyridine nucleotide-dependent enzymes are maintained at approximately their steady state levels throughout storage with or without additives. These findings are of interest in light of the evidence of Jaffe and Neumann that stored erythrocytes have a diminished capacity to synthesize DPN from nicotinic acid as well as incorporate nicotinic acid or nicotinamide into DPN and TPN in comparison with fresh cells (38). Further, Jaffe and Neumann found that although inosine is inhibitory to these processes in fresh blood, the nucleoside could effect, in part, a restoration of the ability of stored cells to carry out these reactions (38).…”

“…These findings are of interest in light of the evidence of Jaffe and Neumann that stored erythrocytes have a diminished capacity to synthesize DPN from nicotinic acid as well as incorporate nicotinic acid or nicotinamide into DPN and TPN in comparison with fresh cells (38). Further, Jaffe and Neumann found that although inosine is inhibitory to these processes in fresh blood, the nucleoside could effect, in part, a restoration of the ability of stored cells to carry out these reactions (38). Since the pyridine nucleotide levels in the present study remained stable throughout storage despite the decline in the cell's pertinent synthetic capacity, it might be concluded that the enzymes that hydrolyze pyridine nucleotides in erythrocytes of adults are inactive during storage at 40 C. Quite different results in the levels of these nucleotides and enzymes can be obtained if the temperature of storage is increased to 200 C. LUhr and Waller (4) found that DPN and TPN declined to about 20% of their original levels and PGD, LDH, G-6-PD, and Hb8R to about 10 to 40%o of their original activities after 7 days of storage at 20 C. The latter data serve to emphasize that experiments performed on blood stored at elevated temperatures are not comparable to those carried out at the standard 40 C.…”

Reduced nicotinamide adenine dinucleotide and the reduction of oxidized glutathione in human erythrocytes

“…The activity of G-6-PD in hemolysates was determined by the method of Kornberg and Horecker as modified by Marks (11). The concentrations of NAD were measured by a fluorometric method with alcohol dehydrogenase in filtrates prepared after the addition of aliquots of the erythrocyte suspensions to 2 volumes of cold 7% trichloroacetic acid (9,12). Concentrations of hemoglobin and packed erythrocyte volumes were determined by routine hematologic methods.…”

“…Preparation of erythrocytes with increased concentrations of NAD Erythrocytes with increased concentrations of NAD were prepared by incubating suspensions of washed cells (packed cell volume 66-68%) with 5.8 X 10' M glucose, 9.2 X10-3 M L-glutamine, 2.3 X 10' M inorganic phosphate, 1.38 X 10-' M nicotinic acid, penicillin, 0.6 mg/ml, and streptomycin, 0.6 mg/ml, as described previously (9). We prepared control suspensions by incubating erythrocytes in a medium in which the nicotinic acid was replaced by an equimolar amount of nicotinamide.…”

“…Since regeneration of GSH in hemolysates was dependent upon the addition of NAD, as well as on lactate, we prepared erythrocytes containing increased concentrations of NAD by incubating cells with nicotinic acid and the mediumh described above (9,13). Both normal erythrocytes and glucose and inorganic phosphate alone, elevated NAD concentrations were not observed.…”