Erythrocyte Preservation. VIII. Metabolic Degradation of Nucleosides In Vitro and In Vivo1

Abstract

It has been shown previously that the addition of inosine to ACD preservative 2 prolongs the effective period of in vitro storage of erythrocytes (1). Inosine is utilized by the red cell after a phosphorolytic cleavage to ribose-l-phosphate and hypoxanthine, mediated by a nucleoside phosphorylase (2, 3). Ribose-l-phosphate enters the "aerobic shunt pathway" of glucose metabolism after conversion to ribose-5-phosphate, and a subsequent effect is a generation of ATP 8 and the resultant maintenance of the energy reserve of the red cell (4). Inasmuch as the red cell lacks the enzyme, xanthine oxidase (5), it is evident that the other cleavage product, hypoxanthine, is not metabolized further but remains in the red cell and the plasma during storage.The present investigation is concerned with the rate of conversion of inosine to hypoxanthine by the red cells and the ratio of these substances in the plasma of blood stored in ACDI, as well as the in vivo metabolism of inosine after infusion. Related studies on the nucleosides, adenosine and guanosine, will be presented also. METHODSHuman blood was collected in ACD, and the nucleoside, dissolved in 0.9 per cent NaCi, was added with sterile precautions.Inosine and adenosine were obtained from Schwarz Laboratories; hypoxanthine and guanosine from Nutritional Biochemicals Corporation. adjusted to pH 10 with NH4OH, according to the methods described previously (1). After identification of the purine-containing compounds with the use of an ultraviolet light (Mineralite), the materials were eluted from the paper with water and measured spectrophotometrically. These substances were estimated also by quantitative densitometry4 of the paper chromatograms. The hypoxanthine content of the plasma and red cells was determined enzymatically, using xanthine oxidase (7), except that the assays were performed on neutralized. PCA filtrates of the various fractions.Acid filtrates of plasma were prepared in the following way: 2 ml. of plasma were added to 2 ml. of cold 0.6 N PCA, mixed well, and centrifuged at 40 C for 5 minutes at 15,200 X g.5 The precipitate was washed once, with 2 ml. of cold 0.3 N PCA, and the centrifugation was repeated. The supernatant fluid and the washing were, combined and neutralized with cold 20 per cent KOH, followed by centrifugation at 4°C for 10 minutes at 15,200 X g. The KCIO4 precipitate was washed once with 2 ml. of cold distilled water and centrifugation repeated. This second supernatant fluid and the washing were combined for analysis. Acid filtrates of the red cells were prepared in essentially the same way. Approximately 4 ml. of cells were washed twice with an equal volume of cold 0.9 per cent NaCl each time with centrifugation for 20 minutes at 1,700 X g.6 The washings were discarded, since they contained only negligible amounts of ultraviolet absorbing materials.A 50 per cent cell suspension was prepared with 0.9 per cent NaCl. Hematocrit determinations were made on this cell suspension. One ml. of cell suspension was added to 3 ml. of cold 0.6 N P...

Adenine in Red Cell Preservation*

“…To date, cleavage of adenosine to adenine has not been demonstrated with human red cells (3,(17)(18)(19). While significant direct phosphorylation of adenosine to form adenine nucleotides (51) does not appear to occur in rabbit erythrocytes (52,53), such data for human red cells have not been reported.…”

The Effects of Nucleosides On the Resistance of Normal Human Erythrocytes to Osmotic lysis12

“…One may speculate that an effective purine riboside must have a specific structure which enables it to permeate or to be bound to the cell membrane in such fashion that the deaminase or nucleoside phosphorylase can readily react with it. Once the ribose has been cleaved phosphorolytically from the purine, the purine, in large part and perhaps wholly, is released to the environment of the cell (23,26).…”