Erythrocyte Preservation. Vi. The Storage of Blood With Purine Nucleosides 1

Red blood cells of the rabbit appear to react about the same as those of the human to storage in acid citrate dextrose (ACD) preservative in the cold; the cells of both species show a posttransfusion survival of approximately 70 per cent at three weeks and less than 50 per cent at six weeks. The fall in survival is accompanied by a reduction in the glycolytic rate and in the organic phosphate content and these changes are retarded in the presence of certain nucleosides (1-3). These facts suggest that rabbit blood may be substituted for human blood in the study of certain problems related to the maintenance of red cell viability during storage.The mechanism responsible for the loss of red cell viability during storage is not known, but one reasonable hypothesis would implicate a disturbance in the cell's carbohydrate metabolism.To learn more about the state of the metabolic machinery, which is predominantly glycolytic, rabbit blood was kept in ACD at 40 C. with and without inosine and the phosphorlylated metabolic intermediates of the red cells were analyzed by ionexchange chromatography at the beginning and after three and six weeks of storage. METHODSBlood was collected by heart puncture with heparinized syringes from female New Zealand strain rabbits, 2 to 3 Kg. in weight, and was mixed with Formula B (National Institutes of Health) ACD solution. Four mg. of inosine-which had been autoclaved in normal salineper ml. of blood, was added to half of the pooled blood mixture. One hundred ml. aliquots of the blood mixture were removed for analysis initially and after three and six weeks of storage at 40 C. Aseptic precautions were followed throughout the experimental manipulations and there was no evidence of bacterial contamination at the end of the storage period. * Supported in part by the U. S. Army Medical Research and Development Command, Office of the Surgeon General, and the San Diego Heart Association.The aliquot of blood was centrifuged, the plasma and buffy coat withdrawn, and the packed red cells were suspended in two volumes of normal saline and recentrifuged. The washed red cells were weighed, the small amount of saline present being measured by a hematocrit determination, and then were mixed thoroughly with two volumes of 10 per cent trichloroacetic acid and the suspension centrifuged. The residue was extracted with the same volume of 5 per cent trichloroacetic acid. All operations to this point were performed at 00 to 50 C.The acid was removed by extracting with two volumes of ether four times.The red cell extract was neutralized with 1 N ammonium hydroxide, diluted fourfold and passed through a 1 X 15 cm. column of Dowex 1-X8 chloride (60 to 80 wet mesh) which was eluted at 2.5 ml. per minute with solutions of hydrochloric acid and ammonium chloride, as shown in Figures 1 and 2. Fractions of 20 ml. volume were collected and each was assayed for optical density at 260 m/A and for total phosphorus. The HCO elution fractions were neutralized on the day of collection. The compounds were identified from ...

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confidence: 99%

Phosphate Compounds of the Rabbit Red Blood Cell During Storage in Acid Citrate Dextrose (Acd) and Acd-Inosine*

Bartlett¹,

Shafer²

1960

“…Procedures for the determination of phosphate partition, glucose, and osmotic fragility of erythrocytes have been described elsewhere (3,6).…”

Section: Methodsmentioning

confidence: 99%

“…Previous investigations on erythrocyte preservation (1)(2)(3)(4) have demonstrated a marked effect of purine nucleosides on the metabolism of the red cell throughout storage. Attention has been focused upon the effect of adenosine in the maintenance of glucose metabolism and of high energy phosphate compounds, such as adenosine triphosphate (ATP).…”

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confidence: 99%

Erythrocyte Preservation. Vii. Acid-Citrate-Dextrose-Inosine (Acdi) as a Preservative for Blood During Storage at 4° C1

Gabrio¹,

Donohue²,

Huennekens³

et al. 1956

Self Cite

Previous investigations on erythrocyte preservation (1-4) have demonstrated a marked effect of purine nucleosides on the metabolism of the red cell throughout storage. Attention has been focused upon the effect of adenosine in the maintenance of glucose metabolism and of high energy phosphate compounds, such as adenosine triphosphate (ATP). As a result, not only are the physical and chemical characteristics of the erythrocyte preserved, but the storage period, during which satisfactory post-transfusion viability is maintained, is extended to approximately twice that observed in acid-citrate-dextrose (ACD) preservative alone. It was postulated that the effect of adenosine was the result of an initial enzymatic conversion to inosine by a deaminase and the subsequent splitting of inosine to yield hypoxanthine and ribose-l-phosphate, mediated by a nucleoside phosphorylase (4, 5). Thus, it appeared that inosine might be the actual substrate for the initial enzymic reaction, and hence the preferred substance for viability studies. In order to evaluate a new preservative, it is desirable to correlate 1) the chemical characteristics of the stored red cells, 2) the ability of the cells to survive after transfusion, and 3) possible toxic effects of the preservative. Studies employing acid-citrate-dextroseinosine (ACDI) as a blood preservative are presented in this paper, as well as further investigations on the mechanism of action of purine nucleosides. METHODSProcedures for the determination of phosphate partition, glucose, and osmotic fragility of erythrocytes have been described elsewhere (3,6).Inosine, adenosine, deoxyadenosine, and adenine were obtained from Schwarz Laboratories; guanine, xanthine,

“…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)(2)(3)(4)(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)(8)(9)(10).…”

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confidence: 99%

“…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)(8)(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)(12)(13)(14)(15)(16)(17)(18).…”

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confidence: 99%

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*

Shafer¹,

Bartlett²

1961

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