The Effect of the Addition of Adenine and Nucleosides at the Beginning of Storage on the Concentrations of Phosphates of Human Erythrocytes during Storage in Acid-Citrate-Dextrose and Citrate-Phosphate-Dextrose

Bloods collected in ACD and CPD preservatives, supplemented with varying amounts of adenine, inosine, adenosine, and guanosine, were stored anaerobically and aerobically at 4 C for six weeks. At two-week intervals, phosphorylated carbohydrate intermediates of washed red cells were assayed by ion-exchange chromatography. The 2,3-diphospho-glycerate values were higher in the CPD or CPD-supplemented than in the ACD bloods which were at lower pH levels. Generally, the ATP and the sum of adenylate (ΣAd) concentrations were maintained at higher levels for longer periods when bloods were collected in more acidic solutions. The highest ATP and ΣAd levels were observed in ACD-adenine (.075, 0.10 and 0.25 mmole/100 ml blood) and in CPD-adenine (0.5 mmole) anaerobically-stored bloods. Relatively high ATP and ΣAd levels were maintained in blood collected in ACD-inosine -+ adenine + guanosine and in inosine-adenine preservatives. Lower values were obtained with ACD-inosine, ACD-adenosine and ACD-adeno-sine + adenine, in the order listed. The ADP values remained relatively constant during storage. An appreciable increase in AMP concentration was observed only in the adenine-supplemented bloods. The IMP concentration increased in all stored bloods and was particularly marked in the presence of adenosine and least in the adenine-supplemented bloods.     

Rejuvenation capacity of red blood cells in additive solutions over long-term storage

BACKGROUND: Red blood cells (RBCs) are Food and Drug Administration (FDA)‐approved for 42‐day storage with the use of additive solutions (ASs). However, adenosine triphosphate (ATP) and 2,3‐diphosphoglycerate (2,3‐DPG) levels in the RBCs decline over this time. These constituents may be restored by treatment with rejuvenation (REJ) solutions. This study was done to assess the response capability of RBCs from 30 to 120 days of storage in three FDA‐licensed RBC storage solutions after incubation with a rejuvenating solution of pyruvate, inosine, phosphate, and adenine. STUDY DESIGN AND METHODS: Three units each of RBCs in approved AS (AS‐1 [Adsol, Fenwal, Inc.], AS‐3 [Nutricel, Medsep Corp.], and AS‐5 [Optisol, Terumo Corp.]) were stored under standard conditions at 1 to 6°C for up to 120 days. Aliquots (4 mL) on Days 30, 42, 60, 80, 100, and 120 (±2 days) were REJ by incubating with Rejuvesol (Encyte Corp.). Control untreated and REJ aliquots were extracted using perchloric acid and stored at ?80°C until assayed for 2,3‐DPG and ATP. RESULTS: RBCs responded to REJ by increasing DPG and ATP contents. The response declined linearly at 0.070 ± 0.008 µmol DPG/g hemoglobin (Hb)/day and 0.035 ± 0.004 µmol ATP/g Hb/day with no differences between ASs. CONCLUSION: We conclude that Rejuvesol is able to restore ATP and 2,3‐DPG levels in RBCs stored up to 120 days in AS. The response diminishes as storage time increases. This rejuvenation (REJ) capability does not seem useful for routine assessment of RBC anabolic capacity in research programs, but may be useful to the investigator when studying unique and novel treatment methods.     

An improved red blood cell additive solution maintains 2,3‐diphosphoglycerate and adenosine triphosphate levels by an enhancing effect on phosphofructokinase activity during cold storage

BACKGROUND: Current additive solutions (ASs) for red blood cells (RBCs) do not maintain constant 2,3‐diphosphoglycerate (DPG) and adenosine triphosphate (ATP) levels during cold storage. We have previously shown that with a new AS called phosphate‐adenine‐glucose‐guanosine‐gluconate‐mannitol (PAGGGM), both 2,3‐DPG and ATP could be maintained throughout storage for 35 days. STUDY DESIGN AND METHODS: In this study, the mechanism underlying the effect of PAGGGM on RBC storage was studied in more detail. By using double‐erythrocytapheresis units (leukoreduced), a direct comparison could be made between the current AS saline‐adenine‐glucose‐mannitol (SAGM) and the experimental solution PAGGGM. During cold storage, several in vitro characteristics were analyzed. RESULTS: In agreement with our previous findings with single RBCs, PAGGGM maintained 2,3‐DPG and ATP levels for 35 days of cold storage. Furthermore, glucose consumption and lactate production were higher in PAGGGM units during the first 21 days of cold storage. Fructose‐1,6‐diphophate and dihydroxyacetone phosphate levels were also increased during the first 21 days of storage in PAGGGM units. CONCLUSION: These results indicate that it is likely that phosphofructokinase (PFK) activity is enhanced in PAGGGM units relative to SAGM units. After 21 days, PFK activity also decreases in PAGGGM units, but sufficient metabolic reserve in these units prevents depletion of 2,3‐DPG and ATP.     

Storage of Erythrocytes in Artificial Media

The storage of red blood cells (RBC) for extended periods in artificial media without plasma has been studied. Blood was collected in either heparin or ACD solution; the plasma was removed; and one or two volumes of a solution containing 2 to 3 mM adenine, 5 to 60 mM Na₂HPO₄ 55 mM glucose, and 120 to 140 mM NaCl was added to the packed RBC. Control samples were stored in ACD plasma containing an equivalent concentration of adenine (ACD-ad). Viability studies were done on three consecutive days in each of 22 subjects, using the subject's own blood stored in various preservatives for 41 to 57 days. After this period of storage, the mean viability of ACD-ad stored blood was 73.5 per cent and of erythrocytes stored in artificial media, 74.4 per cent. Cells stored for longer periods had diminished viability, but the viability of cells stored in artificial media was equivalent to or superior to that of controls. After 42 days' storage, 2,3 DPG levels of RBC stored in artificial media were higher than those of controls, and in some instances, the 2,3 DPG content was one-third to two-thirds that of fresh blood. Some of the potential advantages of this system for blood preservation are:
1. The suspending medium is discarded prior to infusion so that less potentially toxic substances are administered.
2. Plasma is removed at the beginning of storage so that labile factors are available for fractionation.
3. 2,3 DPG levels are higher, so that, theoretically, the oxygen-delivering capacity of the transfused cells is greater.     

Development of an optimized additive solution containing ascorbate-2- phosphate for the preservation of red cells with retention of 2,3 diphosphoglycerate

An additive solution containing adenine, ascorbate-2-phosphate, sodium phosphate, dextrose, and saline was developed for packed red cell preservation. The combination of all components was simultaneously optimized so that the resulting solution produced the maximum retention of both red cell adenosine triphosphate (ATP) and 2,3 diphosphoglycerate (2,3 DPG) concentrations. Fourteen nutrient combinations were tested; each combination was evaluated for 42 days of storage using cells from three donors. The nutrient combinations were chosen with the aid of a computerized experimental design process. Results of the experiments were modeled by regression analysis, and the model was optimized to produce the "best" formulation for simultaneous maintenance of ATP and 2,3 DPG. The resulting mathematically optimal formulation was tested in the laboratory using 10 units of red cells. With this solution, it was possible to store red cells for 42 days with retention of 45 to 55 percent of the initial ATP and 85 to 150 percent of the initial 2,3 DPG. Red cell lysis was low (0.8 percent), and most of the cells were biconcave discs (by scanning electron microscopy) at the end of storage. The studies were carried out in an efficient manner by using computer-optimized experimental design techniques coupled with multiple regression modeling and subsequent computer optimization of the models. This experimental approach has potential application to many current blood banking procedures. This additive solution should maintain viable red cells for 42 days. In addition, the solution will maintain red cell 2,3 DPG throughout storage.     

Refrigerated storage of lyophilized and rehydrated, lyophilized human red cells

Human red cells (RBCs) were collected in CPDA-1 and then freeze-dried in lyoprotective solution. The lyophilized RBCs were then stored at -20 degrees C for 7 days. At the end of the storage period, the lyophilized RBCs were rehydrated and washed in dextrose saline. The washed, reconstituted, lyophilized RBCs were resuspended in final wash solutions of ADSOL, CPDA-1, or a special additive solution containing glucose, citrate, phosphate, adenine, and mannitol, and then they were stored at 4 degrees C for an additional 7 days. The main purpose of this study was to determine whether human RBCs can be lyophilized in such a manner that normal metabolic, rheologic, and cellular properties are maintained during rehydration and subsequent storage in standard blood bank preservative solutions. Our results show that reconstituted, lyophilized RBCs maintained levels of ATP, 2,3 DPG, lactate, and cellular properties that are equal to or better than those in control nonlyophilized RBCs stored for a comparable period in CPDA-1. Reconstituted, lyophilized RBCs stored at 4 degrees C after rehydration also show better maintenance of ATP, 2,3 DPG, and lactate than do control RBCs stored in the same preservative solutions for comparable periods.     

Preservation of Red Blood Cell 2,3‐Diphosphoglycerate in Stored Blood Containing Dihydroxyacetone

In a search for red blood cell metabolites which would preserve 2,3‐DPG during storage of blood, it was discovered that dihydroxyacetone (DHA) prolonged the maintenance of 2,3‐DPG levels for up to four weeks of storage, compared to about one week for presently used preservatives such as CPD. Four‐week preservation of 2,3‐DPG at normal levels was desired. CPD‐adenine was used as the starting point and a formulation having a pH of 7.0 and a DHA concentration of 20 millimoles per liter of blood was developed. The 2,3‐DPG level at four weeks of storage was proportional to DHA concentration in the 5 to 20 mM range. The osmotic fragility, red blood cell ATP levels, and plasma sodium, potassium, and hemoglobin during four weeks of storage in CPD‐adenine‐DHA were similar to those in blood stored in CPD‐adenine.     

Five-week red cell storage with preservation of 2,3 DPG

The 2,3 diphosphoglycerate (2,3 DPG) content of red cells stored in current anticoagulant-preservative products decreases rapidly after the first few days of storage, and by 3 weeks the red cells are essentially depleted of 2,3 DPG. Because ascorbic acid and ascorbate-2-phosphate (A-2-P) are effective in maintaining erythrocyte 2,3 DPG during liquid preservation, ascorbate was stabilized through autoclaving and subsequent storage by adding it as the trisodium salt of A-2-P to a phosphate-adenine-saline solution at a pH of 8.5 to 9.0. Red cell concentrates prepared from blood drawn into citrate-phosphate-double-dextrose were supplemented with the A-2-P additive solution (AS-4) and studied in vitro and in vivo. Mean 2,3 DPG values for 22 units were 147.6, 113.5, and 82.3 percent of initial value after storage for 3, 4, and 5 weeks, respectively. Maintenance of 2,3 DPG was at the expense of adenosine triphosphate (ATP), which fell to as low as 22.2 percent of initial value after 5 weeks. Despite the low ATP values, the 24 hour 51Cr-labeled red cell recoveries averaged 80.8 and 74.1 percent after 4 and 5 weeks of storage, respectively. The AS-4 system provides a red cell product with acceptable viability and improved oxygen off-loading function.     

Hemoglobin Function of Blood Stored at 4 C in ACD and CPD with Adenine and Inosine

Normal hemoglobin function depends on adequate erythrocyte levels of 2,3‐diphosphoglycerate (2,3‐DPG), a compound that is poorly maintained during blood bank storage in acid‐citrate‐dextrose (ACD). Since 2,3‐DPG is better maintained at the higher pH afforded by citrate‐phosphate‐dextrose (CPD), hemoglobin function was compared during storage in CPD and ACD. Further, hemoglobin function was studied in CPD blood containing adenine and inosine, compounds that provide metabolic energy and thus prolong the shelf‐life of blood, because they also effect the levels of 2,3‐DPG during storage. Hemoglobin function, expressed as the P₅₀ (the P₀₂ at 50 per cent oxygenation, an inverse but direct measure of oxygen affinity) is considerably better maintained during storage in CPD than in ACD. The hemoglobin function or P₅₀ of blood stored in CPD‐adenine is not maintained as well as blood stored in CPD without adenine, but the oxyhemoglobin dissociation curves show only a small difference when compared to the difference between ACD and CPD. Blood stored in CPD‐adenine with inosine, present initially or added at day 25, allows higher P₅₀ values late in storage, thus providing better hemoglobin function for more of the storage period.     

Steroid hormones in the preservation of human blood

Units of blood were stored for 42 days under different conditions: (1) packed cells with addition of progesterone, testosterone or androsterone; (2) erythrocytes suspended in Krebs-Ringer phosphate buffer with addition of adenine or progesterone plus adenine; and (3) whole blood with addition of progesterone. At selected intervals during storage, determinations were made of ATP levels, spontaneous lysis, and osmotic fragility. By these criteria, progesterone was effective in protecting the red blood cells during storage by reducing spontaneous lysis, changes in osmotic resistance, and loss of ATP. The greatest effect in this respect was obtained by addition of progesterone plus adenine to erythrocytes suspended in Krebs-Ringer phosphate buffer. Testosterone and androsterone did not show any effect. The use of the “osmotic fragility index” to represent the osmotic resistance of erythrocytes is proposed.     

Manufacture of red cells in additive solution from whole blood refrigerated for 5 days or remanufactured from red cells stored in plasma

Background and Objectives: To investigate methods for the production of red cell concentrates (RCC) in saline, adenine, glucose and mannitol (SAG‐M), from whole blood or red cells stored in plasma for 5 or 6 days and to provide evidence that exchange transfusion RCC in citrate phosphate dextrose (CPD) plasma or citrate, phosphate, dextrose, adenine (CPDA‐1) plasma are of comparable quality. Methods and Materials: Ten RCC in SAG‐M were produced following the remanufacture of red cells in CPD plasma on day 5/6 or after 5 days hold as leucodepleted CPD whole blood. In addition, 10 RCC in CPD plasma and 9 in CPDA‐1 plasma were stored without further processing. Units were assessed for red cell parameters including haemolysis, adenosine triphosphate (ATP), 2,3‐diphosphoglycerate (2,3‐DPG) and extracellular potassium. Results: Units in SAG‐M produced by remanufacture of RCC in plasma or by delayed manufacture of whole blood had comparable levels of haemolysis, ATP and 2,3‐DPG. Furthermore, these units underwent biochemical changes similar to reference SAG‐M units, with the exception of haemolysis which was greater at the end of shelf life and supernatant potassium which was lower following remanufacture. As expected, the decline in ATP was greater in red cells stored in CPD plasma compared with CPDA‐1 plasma. In general, units in CPD plasma were of similar quality at day 28 compared to those in CPDA‐1 plasma at day 35. Conclusions: RCC produced following the remanufacture of RCC in plasma or the delayed manufacture of whole blood are of acceptable in vitro quality and should be assigned the same shelf life as standard RCC in SAG‐M.     

Acid-citrate-dextrose-phosphoenolpyruvate medium as a rejuvenant for blood storage

Stored, depleted RBC were rejuvenated with respect to their levels of adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), and P50 by acid-citrate-dextrose perservatives containing phosphoenolpyruvate (PEP) without sucrose. The restorations of P50 and 2,3-DPG were dependent on the phosphoenolpyruvate concentration. Erythrocyte P50 and 2,3-DPG, even after treatment with these preservatives, decreased with increasing storage period, but the P50 and 2,3-DPG of five-week-old blood were still higher than the corresponding values of fresh blood. ATP concentration was also increased by treating stored blood with preservatives containing phosphoenolpyruvate, but the elevated ATP of five-week-old blood was only about 50 percent of fresh blood. The ATP level could not be raised further by increasing phosphoenolpyruvate concentration but was improved by supplementation with adenine and nucleosides. Incubation of stored blood with 15 mM phosphoenolpyruvate was sufficient to restore ATP, 2,3-DPG and P50 of three-week-old blood to nearly normal. The results of these studies indicate that sucrose is not necessary for PEP to be effective as a preservative additive.     

Rejuvenation of irradiated AS-1 red cells

BACKGROUND: Gamma irradiation of blood components is used to prevent transfusion-associated graft-versus-host disease. The demand for irradiated blood components is increasing because of the increase in directed donation by family members. Irradiated units currently have a recommended maximum storage life of 28 days. Since in vivo recovery is related to red cell ATP levels, rejuvenation of stored irradiated units using a pyruvate-inosine phosphate-adenine additive was explored. STUDY DESIGN AND METHODS: Units of AS-1 red cells from 16 volunteer donors were divided into two equal volumes and one split unit from each was irradiated with 25 Gy. Ten units were irradiated on Day 5, 6, or 7 of 4 degrees C storage and 6 units were irradiated on Day 1 of 4 degrees C storage. All units were rejuvenated for 1 hour at 37 degrees C using a pyruvate-inosine-phosphate-adenine additive on Day 42 of 4 degrees C storage. Units were assayed for ATP, 2, 3 DPG and supernatant sodium, potassium, and glucose. RESULTS: ATP and 2, 3 DPG levels were restored equally well in irradiated and non-irradiated units. The previously reported irradiation-induced red cell potassium-sodium shift was demonstrated. Supernatant potassium and sodium levels did not reverse 1 hour after rejuvenation was completed. There was no significant difference in results between units irradiated on Day 1 or Day 5, 6, or 7. CONCLUSION: Red cell ATP and 2, 3 DPG levels were restored in irradiated AS-1 units stored at 4 degrees C for 42 days using a pyruvate-inosine-phosphate-adenine rejuvenation additive.     

A comparison of biochemical and functional alterations of rat and human erythrocytes stored in CPDA-1 for 29 days: implications for animal models of transfusion

Animal models of transfusion are employed in many research areas yet little is known about the storage-related changes occurring in the blood used in these studies. This study assessed storage-related changes in red blood cell (RBC) biochemistry, function and membrane deformability in rat and human packed RBCs when stored in CPDA-1 at 4 degrees C over a 4-week period. Human blood from five volunteers and five bags of rat RBC concentrates (five donor rats per bag) were collected and stored at 4 degrees C. RBC function was assessed by post-transfusion viability and the ability to regenerate adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) when treated with a rejuvenation solution. Membrane deformability was determined by a micropipette aspiration technique. ATP in rat RBCs declined more rapidly than human RBCs; after 1 week rat ATP fell to the same level as human cells after 4 weeks of storage (rat, 2.2 +/- 0.2 micromol g(-1) Hb; human, 2.5 +/- 0.3 micromol g(-1) Hb). Baseline DPG concentrations were similar in rat and human RBCs (16.2 +/- 2.3 micromol g(-1) Hb and 13.7 +/- 2.4 micromol g(-1) Hb) and declined very rapidly in both species. Human RBCs fully regenerated ATP and DPG when treated with a rejuvenation solution after 4 weeks of storage. Rat RBCs regenerated ATP but not DPG. Post-transfusion viability in rat cells was 79%, 26% and 5% after 1, 2 and 4 weeks of storage, respectively. In rats, decreased membrane deformability became significant (- 54%) after 7 days. Human RBC deformability decreased significantly by 34% after 4 weeks of storage. The rejuvenation solution restored RBC deformability to control levels in both species. Our results indicate that rat RBCs stored for 1 week in CPDA-1 develop a storage lesion similar to that of human RBCs stored for 4 weeks and underscores significant species-specific differences in the structure and metabolism of these cells.     

The effect of storing whole blood at 22 degrees C for up to 24 hours with and without rapid cooling on the quality of red cell concentrates and fresh-frozen plasma

BACKGROUND: Storage of whole blood (WB) for less than 24 hours at ambient temperature is permitted in Europe, but data directly comparing storage with and without active cooling are lacking, which was investigated and compared to current standard methods. STUDY DESIGN AND METHODS: WB was stored in one of four different ways for 24 hours after donation before processing on Day 1 to red cell concentrates (RCCs) in saline‐adenine‐glucose‐mannitol and fresh‐frozen plasma (FFP; n = 20 each): 1) at 22°C in plastic trays, 2) in cooling devices (Compocool II, NPBI), 3) at 4°C, or 4) processed from WB without storage less than 8 hours from donation (Day 0). RESULTS: 2,3‐Diphosphoglycerate (2,3‐DPG) in RCCs were lower after ambient storage compared with those processed on Day 0 or after 4°C storage. Rapid cooling slowed the loss of 2,3‐DPG but levels were undetectable by Day 21 with any method. On Day 42 of RCC storage, there was no significant difference between storage methods in levels of adenosine triphosphate or hemolysis. Potassium levels were lower in RCCs from WB stored at ambient compared with those produced on Day 0, regardless of the use of cooling plates. FFP produced from WB on Day 0 or after storage at ambient with or without active cooling met UK specifications (>75% of units >0.70 IU/mL Factor VIII). CONCLUSION: These data suggest that RCCs and FFP produced from WB that has been stored at ambient temperature with or without active cooling are of acceptable quality compared with those produced using current standard methods in the United Kingdom.     

Adenine in Blood Preservation: Posttransfusion Viability and Biochemical Changes

Posttransfusion viability was studied in red blood cells stored for 21 days in ACD solution and for 35 days in ACD solution supplemented with adenine to a final concentration of 0.5 mM. The survival of radio-chromium-labeled red cells was determined after transfusion of 10 ml. of autologous blood and 350–400 ml. homologous blood. The viability values were about the same for the two transfusion procedures. The mean posttransfusion viability was 80 per cent for erythrocytes stored for 35 days in the medium containing adenine and 79 per cent for cells preserved in ACD solution for 21 days.
The concentration in the erythrocytes of ATP, ADP, AMP, reduced and oxidized glutathione, 2,3-diphosphoglycerate, and four glycolytic enzymes was measured before and after storage in the two media. The ATP and the total adenine nucleotide concentrations were much higher in the red cells stored in the adenine-containing solution. Of the enzymes tested, only phosphofructokinase decreased in activity during 35 days of storage. The decrease was about 50 per cent and was not dependent on the storage solution.
This study supports the theory that decreased adenine supply is an important cause of damage to erythrocytes in ACD solution.     

Adenine nucleotide metabolism in human blood – important roles for leukocytes and erythrocytes

Adenosine diphosphate (ADP) released into blood induces platelet aggregation and contributes to hemostasis and thrombosis. Released ATP can also induce platelet aggregation and there is evidence that blood leukocytes and also erythrocytes play important roles in this. Rapid metabolism of ADP and ATP by endothelial cells is important in protecting platelets from their effects. Here we have performed a systematic investigation of adenine nucleotide metabolism in human blood and the involvement of blood cells. Conversion of ATP to ADP in blood was due almost exclusively to the presence of leukocytes; plasma, platelets and erythrocytes made little or no contribution. Mononuclear leukocytes (MNLs) and polymorphonuclear leukocytes (PMNLs) were equally effective. Conversion of ADP to AMP was also promoted by leukocytes, with no involvement of platelets or erythrocytes. Some ADP was also converted to ATP in blood, apparently via an enzyme present in plasma, but ATP was then rapidly removed by the leukocytes. Conversion of AMP to adenosine occurred via a plasma enzyme with little or no contribution from any cellular element. As expected, in blood the adenosine produced was removed very rapidly by erythrocytes and then converted to inosine and then hypoxanthine. In the absence of erythrocytes plasma supported only a slow conversion of adenosine to inosine and hypoxanthine, which was not influenced by platelets or leukocytes. This study has demonstrated that leukocytes and erythrocytes play a major role in adenine nucleotide metabolism in blood and that these cells, as well as endothelial cells, may be important determinants of the effects of ATP and ADP on platelets.     

Studies of the metabolic integrity of human red blood cells after cryopreservation. II. Effects of low-glycerol-rapid-freeze preservation on glycolysis

The effects of freeze-preservation on the metabolic integrity of human erythrocytes are shown to be largely dependent on the length of time they are stored in ACD at 4 C prior to being frozen. In vitro evaluation involving incubation for 12 hours at 37 C with adenine, inosine and buffering to pH 7.3 showed that when stored for ten days, red blood cells could be preserved by the low-glycerol-rapid-freeze process with no deleterious effects on their capability to produce ATP and 2,3 DPG and to restore the oxygen dissociation curve and sodium -potassium gradient to normal. Storage for 20 days, however, resulted in changes in the metabolic response of red blood cells to the incubation so that ATP production was increased while that of 2,3 DPG was decreased and freeze-preservation lowered production of both compounds. The findings indicate that red blood cells can be stored in ACD at 4 C for up to ten days prior to being freeze-preserved and still be capable of resuming normal metabolic function. This, in view of previous evidence showing that the oxygen affinity and metabolic status of red blood cells remain unaffected by the low-glycerol-rapid-freeze process, indicates that if the cells are frozen immediately after being drawn, preservation of their respiratory function will be optimal.     

The distribution and utilization of adenine in red blood cells during 42 days of 4 C storage

The fate of adenine in CPD whole blood (17.3 mg/500 ml, or 0.25 mM) was evaluated during 42 days of 4 C storage. In whole blood, 95 per cent of the adenine was removed from the plasma by 42 days while the cellular adenosine triphosphate (ATP) levels remained above 60 per cent of the initial concentration. Packed red blood cells (concentrates) were stored with the same relative quantity of adenine (0.1 mg/ml red blood cells) used in whole blood units by the addition of adenine after packing and were shown to take up adenine in a similar manner. Calculations of the initial adenine distribution indicated higher intracellular adenine concentrations than predicted from distribution equilibrium based on volume considerations. The presence of inorganic phosphate has marginal effects on adenine incorporation but does elevate ATP levels, while contributing to the reduction of 2,3-diphosphoglycerate (2,3-DPG) content. The free adenine equilibrium between plasma and red blood cells favors the red blood cells, suggesting adenine binding by red blood cell membranes as shown by initial distribution studies with ¹⁴C-adenine and equilibrium dialysis.     

Phosphoenolpyruvate in the rejuvenation of stored red cells in SAGM medium: optimal conditions and the indirect effect of methemoglobin formation

Red cells stored in saline-adenine-glucose-mannitol (SAGM) medium were rejuvenated by incubation with phosphoenolpyruvate (PEP) under conditions that can be achieved easily in ordinary blood banking. Regeneration of 2,3 diphosphoglycerate (2,3 DPG) and adenine nucleotides of stored red cells was dependent on the pH of the incubation medium and the incubation time. In red cells stored for 3 and 5 weeks, the optimal pH and incubation time for regeneration of 2,3 DPG and adenine nucleotides were 5.8 and 90 minutes and 6.1 and 60 minutes, respectively. During the incubation of red cells with PEP, methemoglobin was formed; it increased when the medium pH was below 6.0 and the incubation time exceeded 60 minutes. We conclude that incubation at a medium pH of 6.1 for 60 minutes is optimal for the rejuvenation of stored red cells with PEP. Under such incubation conditions, the concentrations of 2,3 DPG and adenine nucleotides in red cells stored for 5 weeks were restored to normal without methemoglobin formation.