Phosphorylated Carbohydrate Intermediates of the Human Erythrocyte During Storage in Acid Citrate Dextrose. I. Effect of the Addition of Inosine at the Beginning of Storage*

Abstract

The post-transfusion survival of stored human or rabbit erythrocytes can be influenced favorably by the addition of certain purine nucleosides to the preserving solution (1-5). It had been noted earlier that the water-soluble organic phosphates of the erythrocyte disappeared to a large extent during storage (6), but nucleosides have been found to retard this change and, if added late in storage followed by a short incubation at 370 C, they bring about appreciable resynthesis of the organic phosphate (7-10).Early studies on the nature and changes of the phosphate compounds of the erythrocyte during storage were performed for the most part by using hydrolysis rates and paper chromatography (1, 2, 7-9). Recently improved technics for the isolation and identification of the metabolic intermediates, which use columns of ion exchange resin and more specific chemical and enzyme analytical methods, have greatly extended our knowledge of the normal carbohydrate intermediates of the human erythrocyte and make possible a more detailed analysis of changes occurring during storage (11-18).We have previously reported on the phosphorylated carbohydrate intermediates of the rabbit erythrocyte during storage with and without inosine ( 19). The present paper describes a similar study in which the glycolytic intermediates and nucleotides of human erythrocytes were analyzed at biweekly intervals during 8 weeks of storage at 40 C in acid citrate dextrose (ACD) and in ACD plus inosine (ACDI).* Supported by the US Army Medical Research and Development Command, Office of the Surgeon General, and by the National Heart Institute. METHODSFrom each of 2 healthy male donors who had normal blood counts, 480 ml of blood was drawn by vacuum into standard blood storage bottles containing 120 ml of ACD (NIH formula B). Inosine which had been autoclaved in normal saline in a concentration of 40 mg per ml was added to one sample in the amount of 4 mg (15 ,gmoles) per ml of blood; 100 ml of the ACD-blood mixture was removed aseptically from each sample for assay immediately and after 2, 4, 6, and 8 weeks of storage at 40 C.The blood samples were centrifuged, plasma and buffy coat removed, and the erythrocytes washed once with normal saline. The erythrocytes were mixed with 2 vol of 10 per cent trichloroacetic acid (TCA), the mixture centrifuged, and the precipitate re-extracted with 5 per cent TCA. The above procedures were carried out in the cold. The TCA was removed with ether.The erythrocyte extracts were neutralized, diluted 4-fold, and run through 1 X 15 cm columns of Dowex 1 X 8-chloride (60 to 80 wet mesh). The columns were eluted at a rate of 2.5 ml per minute with solutions of HCl and NH4C1, as shown in Figures 1 and 2

The Mechanism of Action of Adenine in Red Cell Preservation*

“…Finally, incubation of human red cells with adenosine-8-C14 failed to produce more labeling in the adenine portion of ATP than did inosine-8-C14 (22). Nevertheless, red cell viability is better maintained with adenosine than with inosine (3), and adenosine appears to be more effective than inosine, inosine plus adenine, or adenine alone in maintaining and regenerating ATP in stored or nucleotide-depleted cells (2,(31)(32)(33)(34)(35)(36)(37)(38). Since adenosine, through deamination, may raise pH and would thus be expected to maintain hexokinase activity (39), possibly some of the aforementioned effects of adenosine on ATP maintenance and viability may be explained by this means.…”

Hemoglobin function in stored blood

“…Furthermore, 2,3-DPG levels were much better maintained in the presence of inosine. Others have reported similar data on the changes in ATP and 2,3-DPG levels in blood stored with these preservatives (21)(22)(23)(24).…”

Phosphorylated Carbohydrate Intermediates of the Human Erythrocyte During Storage in Acid Citrate Dextrose. Ii. Effect of the Addition of Inosine Late in Storage*

“…Formate rechromatography was also carried out on the combined 0.003 and 0.01 N HCl elution sections to obtain better resolution of the sugar monophosphates ( Figure 3). The results are summarized in Figure 4, which for comparison includes data from the zero-day control and 4-week inosine experiments from the previous study (1).…”

“…The aliquots treated with inosine were incubated for 1 and 3 hours at 370 C. Trichloroacetic acid extracts of the four samples were prepared, the metabolic intermediates were separated on columns of ion-exchange resin, and the compounds were analyzed as described in the preceding paper (1).…”