Depletion and Regeneration of 2,3-diphosphoglyceric Acid in Stored Red Blood Cells

2,3-diphosphoglyceric acid appears to be an important regulator of the oxygen dissociation curve of hemoglobin in intact red blood cells. The rate of loss of 2,3-DPG under various storage conditions therefore was investigated. 2,3-DPG disappeared rapidly from conventional preservative media, CPD, and ACD solutions. After only two weeks' storage, 65 per cent to 85 per cent of erythrocyte 2,3-DPG had been lost from ACD-stored blood and slightly less from CPD-stored blood. Although the addition of adenine to ACD solution aided in the maintenance of ATP levels, it hastened the rate of loss of 2,3-DPG. The rate of 2,3-DPG depletion was strongly dependent on pH. In more alkaline storage media, levels of this compound were relatively well maintained for as long as two, or even three weeks. However, under these circumstances ATP maintenance was less satisfactory. The levels of 2,3-DPG and ATP in red blood cells incubated in fresh plasma at 37 C, pH 7.4, to simulate the conditions after reinfusion of stored cells also was investigated. ATP levels remained relatively stable under these circumstances and 2,3-DPG levels were restored gradually. However, the repletion of 2,3-DPG was sufficiently slow so that even after eight hours only approximately one third of the 2,3-DPG which had been lost was regenerated. Thus, stored blood may fail to transport oxygen efficiently for many hours after reinfusion.     

Changes of blood oxygen affinity in different CPD solutions during liquid storage

Soft and hard-packed red blood cells in four different CPD anticoagulant-preservative solutions were stored with and without added glucose, adenine, and electrolytes. The hemoglobin-oxygen affinity of the red blood cell concentrates was tested over a six-week storage period. No single solution conferred better protection than any other against an expected increase in oxygen affinity due to loss of 2,3-DPG during storage. In all solutions, P50 at pH 7.4 decreased linearly when measured in a physiological system using CO2. After six weeks' storage at 4 C, the normal oxygen-binding properties of red blood cells could be restored in all instances following incubation for one hour in a rejuvenation solution. By contrast, red blood cell ATP levels were highest when resuspending solutions contained adenine and added glucose, but did not significantly compensate the allosteric role of 2,3-DPG in regulating oxygen affinity when the latter became depleted.     

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.     

The effect of agitation on in vitro metabolism of erythrocytes stored in CPD-adenine

Agitation of blood stored in plastic containers has been reported to lead to improved posttransfusion survival and it has been found that, in some media, agitation has improved erythrocyte 2,3- diphosphoglycerate (2,3-DPG) levels. Using CPD II media (CPD with 277.5 mM glucose and 2.04 mM adenine), we were not able to identify any improvement in levels of adenosine triphosphate, 2,3-DPG or glucose in whole blood under various agitation conditions when compared with nonagitated control. The 2,3-DPG level was moderately improved through 28 days in the 90 per cent hematocrit packed erythrocytes but the results were not considered to be significantly beneficial to warrant agitation. Thus, the application of agitation to the CPD II blood storage system was of no great benefit in improving metabolic intermediate levels.     

Hemoglobin function in stored blood

Serial oxygen dissociation curves were performed on blood units preserved in acid-citrate-dextrose (ACD), ACD-adenine, and ACD-adenine-inosine. Dividing blood from a single donor into two or more bags allowed direct comparison between preservatives. During the 1st wk of storage in ACD, a progressive increase in oxygen affinity was observed. Thereafter, little further change was noted. Oxygen affinity increased even more rapidly during initial storage in ACD-adenine. However, with the inclusion of inosine as a preservative, oxygen affinity remained unaltered during the first 2 wk. Increases in oxygen affinity correlated well with falling levels of red cell 2,3-diphosphoglycerate (2,3-DPG) during storage. No significant changes in glutathione, reduced form (GSH), or A3 (A(I)) hemoglobin levels were noted during the first 3 wk of storage. No significant accumulation of ferrihemoglobin was detected. When blood stored 20 days in ACD or ACD-adenine was incubated with inosine for 60 min at 37 degrees C, 2,3-DPG and adenosine triphosphate (ATP) were resynthesized, and oxygen affinity was decreased. The distribution of 2,3-DPG in fresh and stored red cells appeared to influence experimental values for Hill's n, a measure of heme-heme interaction.     

Storage of red blood cells with improved maintenance of 2,3-bisphosphoglycerate

BACKGROUND: During storage, red blood cells (RBCs) rapidly lose 2,3-bisphosphoglycerate (2,3-DPG) leading to an increase in the affinity for O(2) and a temporary impairment of O(2) transport. Recent clinical evaluations indicate that the quality of transfused RBCs may be more important for patient survival than previously recognized. STUDY DESIGN AND METHODS: Glucose-free additive solutions (ASs) were prepared with sodium citrate, sodium gluconate, adenine, mannitol, and phosphates at high pH, a solution that can be heat-sterilized. CP2D was used as an anticoagulant. Additional CP2D was added to the AS to supply glucose. RBCs were stored at 4 degrees C and assayed periodically for intracellular pH (pHi), extracellular pH, glucose, lactate, phosphate, ATP, 2,3-DPG, hemolysis, and morphology. RESULTS: Storage in 175 mL of the chloride-free, hypotonic medium at a hematocrit (Hct) level of 59 to 60 percent resulted in an elevated pHi and the maintenance of 2,3-DPG at or above the initial value for 2 weeks without loss of ATP. The addition of 400 mL of storage solution followed by centrifugation and removal of 300 mL of excess solution to a Hct level of 60 to 66 percent further reduced the chloride concentration, resulting in the maintenance of 2,3-DPG for 4 weeks. Hemolysis was at 0.1 percent at 6 weeks. CONCLUSION: Improvements in the maintenance of 2,3-DPG were achieved with 175 mL of a chloride-free storage solution with familiar additives at nontoxic concentrations to increase pHi. Adding, instead, 400 mL of storage solution followed by the removal of 300 mL reduced the chloride concentration, increasing the pHi and extending the maintenance of 2,3-DPG to 4 weeks.     

Alkaline CPD and the preservation of RBC 2,3-DPG

BACKGROUND: Concentrations of 2,3-DPG decline rapidly in the first week of RBC storage because of the low pH of conventional storage solutions. Alkaline additive solutions, which can preserve RBCs for up to 11 weeks, still do not preserve 2,3-DPG because the starting pH is below 7.2. STUDY DESIGN AND METHODS: Alkaline CPD (pH=8.7) was made with trisodium citrate, dextrose, and disodium phosphate. Twelve units of whole blood were collected into heparin and pooled in groups of four units. Each pool was then aliquoted into four units; 63 mL of CPD with pH 5.7, 6.5, 7.5, or 8.7 was added to one unit of each pool, and 300 mL of the alkaline experimental additive solution-76 was added. In Study 2, 12 units were collected into alkaline CPD, pooled in groups of four, aliquoted as described, and stored in four variants of experimental additive solution-76 containing 0, 9, 18, and 27 mM of disodium phosphate. RBC ATP and 2,3-DPG concentrations, intracellular and extracellular pH and phosphate concentrations, hemolysis, and other measures of RBC metabolism and function were measured weekly. RESULTS: RBCs stored in more alkaline conditions made 2,3-DPG, but at the expense of ATP. Concentrations of 2,3-DPG decreased after 2 weeks storage, but ATP concentrations never fully recovered. Providing more phosphate both increased the duration of 2,3-DPG persistence and raised ATP concentrations in the later stages of storage. CONCLUSIONS: Maintaining both 2,3-DPG and ATP requires both high pH and high concentrations of phosphate.     

Hemoglobin function in stored blood. IX. Preservative with pH to maintain red blood cell 2,3-DPG (function) and ATP (viability)

An optimal pH was sought to maintain hemoglobin function, ATP, and red blood cell viability during liquid storage under blood banking conditions. Ten units of blood from normal volunteers were subjected to an automated analytical system for determining concentrations of 2,3-DPG and ATP. Each unit was split during donation into five parts containing citrate—dextrose solutions of pH 5.0, 5.5, 6.0, 6.5, and 7.0. Significant differences at the 95 per cent level were based on the paired t-test. In addition, osmotic fragility and methylene blue uptake were determined to assess their possible usefulness as indicators of either red blood cell viability or ATP. With pH 5.0 preservative 2,3-DPG fell from day 0 to day 3, with pH 5.0 and 5.5 preservatives from day 3 to day 7, and from day 7 to day 14 in all pH groups. A plot of 2,3-DPG versus hydrogen ion concentration showed that in excess of 1 × 10^?7hydrogen ion, corresponding to pH 7.0, 2,3-DPG concentration falls at a rapid rate. From 2,3-DPG and ATP data, a preservative with pH higher than 5.5 would seem to be optimal for maintaining hemoglobin function and red blood cell viability, but adenine may be needed to maintain adequate ATP levels.     

Reversal of the storage lesion of CPD bank blood: a problem in clinical medicine

The effect of phosphate buffer on the course of pH, ATP, and 2,3-PDG of CPD red blood cells stored at three temperatures was observed. Basic phosphate at an equilibrated level of 10 mM (as iP) maintained pH above 7.00 and ATP and 2,3-DPG above 70 per cent of initial value in cells stored at 37 C for 24 hours. In contrast however, at 25 and 4 C no buffering was obtained with basic phosphate concentrations up to 50 mM, but values for both ATP and 2,3-DPG were higher in phosphate treated aliquots than in controls throughout storage. When the pH of blood stored at 4 C was adjusted into the range 7.15 to 7.25 with tromethamine and the level of iP raised to 10 mM by addition of Na2HPO4 on day seven, it was found that ATP and 2,3-DPG levels were maintained at 90 and 120 per cent, while control levels fell to 60 and 12 per cent, respectively at 21 days. The process described parallels the normal repair of damaged red blood cells of bank blood that occurs in vivo following transfusion.     

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 effect of cardiac disease on hemoglobin-oxygen binding

The relation between degree of cardiac functional impairment and changes in hemoglobin-oxygen affinity and 2,3-diphosphoglycerate (2,3-DPG) has been studied in 39 patients with noncyanotic heart disease. A progressive decline in hemoglobin-oxygen affinity was found with worsening cardiac function as assessed by cardiac index, arteriovenous oxygen (A-V O(2)) difference, and cardiac symptoms; this alteration in hemoglobin-oxygen binding represents a significant mechanism for adaptation to the limited oxygen supply imposed by the cardiac lesion. The highly significant correlation of mixed venous blood oxygen saturation (S[unk]V(VO2)) with 2,3-DPG and the position of the oxygen dissociation curve suggests that the level of deoxygenated hemoglobin is an important in vivo regulator of hemoglobin-oxygen affinity.     

The effect of fructose infusions on the oxygen transport system of human blood.

In 9 healthy subjects the erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentration, which modifies the oxygen affinity of haemoglobin, decreased by more than 25 per cent within 60 minutes of the beginning of a fructose infusion (0.5 g.kg body weight-1.h-1). In contrast erythrocyte adenosine triphosphate (ATP) concentration was unchanged. In spite of the diminished 2,3-DPG concentrations the oxygen affinity of whole blood actually measured remained unaltered. However, at the same time both the arterial and the venous blood pH had fallen by 0.05 or more. In vitro experiments indicated that this fall of erythrocyte 2,3-DPG was not due to a direct effect of fructose on the intra-erythrocytic regulation of 2,3-DPG or to changes indirectly related to the intravenous administration of fructose in vivo, i.e. an increase of the blood lactate/pyruvate ratio or a decrease of plasma inorganic phosphate. It is suggested that two opposing effects on the oxygen transport system of blood are induced by fructose infusions: 1) a displacement of the oxygen dissociation curve to the right due to the Bohr effect 2) a virtually counterbalancing shift of the oxygen dissociation curve to the left due to decreased erythrocyte 2,3-DPG concentrations.     

Storage effects on the Cole-Cole parameters of erythrocyte suspensions

Chemical alterations of red blood cells (RBCs) during storage eventually affect the electrical properties of blood. In this study, the physiological parameters such as extracellular (SAGM + CPD + residual plasma) Na(+), K(+), Cl(-), pH, 2,3-DPG and ATP together with the Cole-Cole parameters were measured using erythrocyte suspensions from 51 male donors (31 donors form the training set and 20 donors are used for testing), on the 0th, 10th, 21st, 35th and 42nd days of storage. During storage, while the surrounding fluid resistance (R(e)) and the effective cell membrane capacitance (C(m)) increased progressively with time, the intracellular fluid resistance (R(i)) has decreased. Storage of RBCs resulted in a rise in K(+) and a fall in Na(+), Cl(-), pH, 2,3-DPG and ATP. Accordingly, electrical parameters were all correlated with Na(+), K(+), Cl(-), pH and ATP at varying levels. By applying multi-regression analysis, it is concluded that R(i), R(e) and C(m) are appropriate for modeling Na(+), K(+), Cl(-), pH and ATP during storage.     

Blood preservation XXVII. Fructose and mannose maintain ATP and 2,3-DPG

Mannose and fructose as well as glucose have been shown to be effective for maintaining ATP and thus viability of stored red blood cells. Normal 2,3-DPG levels are desirable in stored red blood cells to provide the needed oxygen transport upon transfusion. ATP levels in sotred concentrated red blood cells in the new preservative, CPD- adenine (citrate-phosphate-dextrose-adenine) become critically low in the 5th week. In this study two hexoses and two pentoses are compared with dextrose in their ability to maintain ATP and 2,3-DPG. ATP levels were best maintained by fructose, then dextrose and mannose. ATP levels had fallen to critically low levels by four weeks with ribose and xylose. Red blood cell 2,3-DPG concentrations were also maintained by hexoses, with mannose being best, dextrose and fructose being similar. When ribose was used in addition to dextrose in CPD-adenine, ATP maintenance was improved and under the same conditions xylose improved 2,3-DPG maintenance. Fructose and mannose may be as useful as dextrose in citrate-phosphate preservatives for maintaining ATP and 2,3-DPG levels. Also, ribose and xylose may help the maintenance of ATP and 2,3- DPG, respectively, in CPD-adenine.     

Hemoglobin Function and 2,3‐DPG Levels of Blood Stored at 4 C in ACD and CPD pH Effect

Normal hemoglobin function depends on adequate erythrocyte levels of 2,3‐diphosphoglycerate (2,3‐DPG), a compound which is poorly maintained in acid‐citrate‐dextrose (ACD). Since 2,3‐DPG is better maintained in citrate‐phosphate‐dextrose (CPD) and this preservative has a higher pH (5.5) than ACD (pH = 5.0), these preservatives were prepared at each pH and studied. The CPD preservatives (pH 5.0, 5.5) had similar amounts of phosphate so the differences between them, obtained by altering the buffer ratio, should relate to pH. The ACD solutions (pH 5.0, 5.5) contained no phosphate. Hemoglobin function, expressed as P₅₀ (the Po₂ at 50 per cent oxygenation, an inverse but direct measure of oxygen affinity), and 2,3‐DPG were better maintained in ACD and CPD of pH 5.5. The lower pH (5.0) preservatives, whether ACD or CPD, showed rapidly declining hemoglobin function and 2,3‐DPG levels. The values at the higher pH remained close to normal for two weeks and above those of the lower pH preservatives for most of the four‐week storage period.     

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.     

Certain Aspects of the Thrombotest Method for the Control of Anticoagulant Therapy

The effect of exposure to simulated high altitude (4,500 m) on the concentration of 2,3-diphosphoglycerate (2,3-DPG) and adenosine triphosphate (ATP) in the red cell and the oxygen affinity of hemoglobin (P50) (measured at PCO₂=40 mm Hg and corrected to plasma pH=7.40) was studied. It was found that a moderate physical activity is a condition for the previously reported decrease in the oxygen affinity during acclimatization to high altitude, and that this decrease is exclusively due to an increase in the concentration of 2,3-DPG in the red cell. A rapid and pronounced decrease in the extracellular concentration of inorganic phosphate was found in resting as well as exercising individuals.     

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.     

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.     

Improved red blood cell storage using optional additive systems (OAS) containing adenine, glucose and ascorbate-2-phosphate

Red blood cells were treated with optional additive system (OAS) solutions to provide component-specific metabolic enhancement for improved storage. Red blood cell viability, as monitored by ATP concentrations, was maintained by use of adenine and extra glucose. Red blood cell oxygen offloading characteristics were improved by maintenance of red blood cell 2,3-DPG concentrations with ascorbate-2- phosphate (AsP). The use of CPD-collected red blood cells with an OAS containing adenine, glucose, and AsP, or CPD-adenine collected red blood cells with an OAS containing AsP demonstrates the potential to store red blood cells at least 42 days and to maintain red blood cell 2,3-DPG.