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.     

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.     

Oxygen Equilibria and Biochemical Changes of Whole Blood Stored in Different Preservation Media

Human blood was stored in its oxygenated and in its deoxygenated forms for a period of 14 to 56 days at 4 C in six different preservation media, namely, ACD and CPD, ACD and CPD supplemented with adenine, ACD and CPD supplemented with adenine and with tris (hydroxymethyl) amino-methane at pH 7.2. At regular intervals determinations were made of the pH and the oxygen affinities of the total blood, of the intracellular concentrations of ATP, 2,3-DPG and inorganic phosphate, of plasma electrolytes and lactic add concentrations, and of the minor hemoglobin components. The investigations have shown: 1) Storage of blood in the deoxygenated form results in a rapid decrease of the blood pH, an increased lactic acid production, and a less rapid change in the oxygen affinities and 2,3-DPG levels; 2) Storage of blood at a more neutral pH prevents the changes in 2,3-DPG level and oxygen affinity; 3) Supplementation of the media with adenine may increase to some extent the ATP level of the red blood cells; 4) No significant changes are observed in the percentages of the minor hemoglobins. A striking correlation was observed between the oxygen affinity of the blood and the intracellular 2,3-DPG level; no such correlation was present between the ATP level of the red blood cells and the oxygen affinity of the blood. The 2,3-DPG level (and the oxygen affinity) was also dependent on the pH of the blood-preservation media. A rapid disappearance of 2,3-DPG was noted below rather fixed pH values; these pH values were consistently lower when blood was stored in its deoxygenated form than in its oxygenated form.     

Effect of initial storage at room temperature on human red blood cell ATP, 2,3-DPG, and viability

The concentrations of ATP and 2,3-DPG and post-transfusion viability were measured in human red blood cells exposed for one, four and seven hours to room temperature before refrigerated storage for 21 days. No effect of room temperature storage was observed on ATP or viability. Decrease in 2,3-DPG was accelerated by room temperature exposure but the differences in 2.3-DPG were small and unlikely to have a significant adverse effect on red blood cell oxygen delivery. Delays of up to seven hours in refrigeration of blood do not appear to have serious adverse effects on red blood cell viability or function.     

The potential use of dihydroxyacetone for improved 2,3-DPG maintenance in red blood cell storage: solution stability and use in packed cell storage

Dihydroxyacetone (DHA) is effective in maintaining 2,3- diphosphoglycerate (2,3-DPG) concentrations in stored red blood cells. One limitation to the use of DHA is its instability when added to anticoagulant solutions during blood bag manufacture. The stability of DHA solutions have been evaluated. Solutions of DHA are stable at 25 C in water or isotonic saline, with or without the addition of glucose or adenine. DHA is stable to autoclaving; 99 + per cent surviving at 150 mM, and 89 per cent surviving at 1.9 M concentrations. DHA can be incorporated into a satellite addition pouch attached to the main blood drawing bag, and be added to the blood-anticoagulant mixture after phlebotomy or the preparation of red blood cells. Addition of the DHA solution, containing adenine and extra glucose, to packed cells causes significantly improved maintenance of 2,3-DPG during 42 days of 4 C storage, while maintaining adequate concentrations of red blood cell ATP. The use of DHA, adenine, and glucose in extended storage of packed cells, using either zero or seven day addition of the nutrient solution, produces similar efficacious results.     

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.     

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.     

Prolonged maintenance of 2,3-DPG in liquid blood storage: use of an internal CO2 trap to stabilize pH.

A close relationship exists between the decrease in concentration of 2,3-diphosphoglycerate (2,3-DPG) and a fall in the pH of stored blood. Buffering the stored red cells with bicarbonate is one solution to the problem of maintaining pH during storage. The effectiveness of this buffer depends upon loss from the stored blood of carbonic acid in the form of CO2. We describe a system in which the CO2 is trapped in a small internal package which contains calcium hydroxide, or calcium hydroxide embedded in Silastic. A medium containing bicarbonate, adenine, glucose, phosphate and mannitol (BAGPM) is added after initial packing of the erythrocytes. With this approach, it has been possible to maintain 2,3-DPG at 92 percent of original, and ATP was approximately 62 percent of initial levels at the end of 42 days of storage if an internal Silastic bag containing calcium was used in bags agitated once weekly. More frequent agitation (five times weekly) produced acceptable maintenance of both 2,3-DPG (78 percent of original) and ATP (44 percent of original) after 42 days of storage when a Silastic block impregnated with calcium hydroxide was utilized to absorb CO2.     

Blood preservation 33. Phosphate enhancement of ribose maintenance of 2,3-DPG and ATP

Blood storage in CPD-adenine supplemented with 25 mM inosine and 10 mM phosphate gave 2,3-DPG levels as high as 140 per cent of normal for six weeks of blood storage at 4 C. Lower but normal 2,3-DPG levels were maintained throughout six weeks with inosine or inosine plus ribose. Ribose alone provided marginally increased DPG maintenance over the control, but ribose with phosphate maintained 2,3-DPG levels above 70 per cent of normal for five weeks of storage and two weeks longer than the control preservative. ATP levels were maintained at normal or above for six weeks with phosphate plus ribose or inosine. 2,3-DPG maintenance has previously been shown to be impaired by phosphate, unless inosine is also present. The ribose and inosine effects on 2,3-DPG maintenance are not additive. Phosphate also has an enhancement effect on ATP maintenance in the presence of either ribose or inosine.     

Blood storage XXII. Improvement in red blood cell 2,3-DPG levels at six weeks by 20 mM PO4 in CPD-adenine-inosine

Inorganic phosphate has been known to assist red blood cell maintenance of ATP and in the presence of inosine to assist in the maintenance of 2,3-DPG. High concentrations of phosphate, while helping ATP maintenance, were found to be deleterious to 2,3-DPG maintenance in CPD- adenine preservatives. However, in the presence of inosine, concentrations of phosphate as high as 10 mM were advantageous to 2,3- DPG maintenance. The present study extends the observations on ATP and 2,3-DPG maintenance in CPD-adenine-inosine preservatives from the previous 10 mM to 20 mM phosphate. A high phosphate (20 mM) effect has been seen as improved maintenance of 2,3-DPG levels during the fifth and sixth weeks of storage of whole blood at 4C. This supports the previously reported observation of improved maintenance of 2,3-DPG in a 10 mM phosphate preservative. This is ten times the 2 mM phosphate concentration in CPD-adenine. In the low phosphate preservative (2 mM), 2,3-DPG maintenance is less than that in all of the higher phosphate preservatives after the second week of storage. ATP concentrations in this experiment show good maintenance throughout six weeks of storage.     

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.     

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.     

The Effect of Ascorbate and Dihydroxyacetone on the 2,3-Diphosphoglycerate and ATP Levels of Stored Human Red Cells

Fifty ml aliquots of blood were stored in modified CPD-adenine preservative solutions at pH 4.8, 5.6, and 7.0 containing either dihydroxyacetone alone, ascorbic acid alone, or a combination of both. Red blood cell ATP and 2,3-DPG determinations showed that the effect of dihydroxyacetone and ascorbic acid were synergistic at all pH levels, and that even at the lowest pH levels excellent 23-DPG maintenance was observed. A reciprocal relationship existed between 2,3-DPG and ATP maintenance. Studies in 500 ml units of blood containing both dihydroxyacetone and ascorbate gave similar results to those in 50 ml aliquots. There was excellent maintenance of 2,3-DPG levels throughout the 28-day storage period.     

Lactic acidemia in baboons after transfusion of red blood cells with improved oxygen transport function and exposure to severe arterial hypoxemia

Baboons were bled one-third of their blood volume and then transfused with an equivalent volume of compatible donor red blood cells with 160 per cent of normal 2,3-diphosphoglycerate (2,3-DPG) levels and improved capacity to release oxygen to tissue. The mixture of baboon donor- recipient red blood cells in the circulation had a 2,3-DPG level of 130 per cent of normal. After transfusion, the baboon's inspired oxygen was first lowered from 21 to 10 per cent to produce severe arterial hypoxemia with a PO2 tension of less than 40 mm Hg for two hours and then restored to 21 per cent. Lactic acidemia occurred when the alveolar oxygen tension was reduced so as to produce an arterial oxygen tension of less than 40 mm Hg, even though oxygen consumption was maintained. The data suggest that when red blood cells with normal or improved oxygen delivering capacity are transfused to patients, the alveolar oxygen tension should be sufficient to maintain an arterial oxygen tension of greater than 40 mm Hg.     

Blood preservation XLIV. 2,3-DPG maintenance by dehydroascorbate better than D-ascorbic acid

A study was designed to compare the effects of D-ascorbate and dehydroascorbate on red blood cell metabolism during blood storage. Dehydroascorbate increased red blood cell concentrations of 2,3-DPG such that the levels are above normal for four weeks and normal at six weeks of storage. In contrast, there is a gradual decrease in 2,3-DPG levels with D-ascorbate such that the levels are approximately 80 per cent of normal after six weeks. ATP levels were adversely effected such that the worst levels were produced by 10 and 5 mM dehydroascorbate, with 10 mM having a more adversive effect than 5 mM. Intermediate levels of ATP were produced by D-ascorbate, with the 10 mM concentration. The control CPD-adenine preservative maintained near normal ATP levels for the entire six-week storage period. pH values were initially slightly lower with dehydroascorbate compared to the other preservatives early in storage, the difference being slightly over 0.1 pH units.     

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. 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.     

Viability and function of outdated human red blood cells after biochemical modification to improve oxygen transport function, freezing, thawing, washing, postthaw storage at 4 C, perfusion in vitro through a bubble oxygenator, and autotransfusion

The quality of transfused blood is especially important during cardiac surgery, and red blood cell viability and function may be adversely affected during perfusion through the artificial blood oxygenator used during extracorporeal bypass. In this study, we administered 10 ml aliquot autotransfusions of rejuvenated red blood cells to 13 healthy volunteers after perfusion through an infant bubble oxygenator for one to three hours. Twenty-three other volunteers received rejuvenated red blood cells that had not been perfused. The red blood cells were biochemically modified after they had reached their outdating period, a process used to increase 2,3 DPG and ATP levels and improve oxygen transport function. The rejuvenated red blood cells were frozen with 40% W/V glycerol, stored frozen at -80 C for about 3 months, thawed, washed, and stored in a sodium chloride-glucose-phosphate solution at 4 C for as long as three days. Freeze-thaw recovery was about 97 per cent, and freeze-thaw-wash recovery about 90 per cent. Twenty-three units were transfused after 1 to 3 days of post-wash storage, and 13 units were perfused through an infant bubble oxygenator for as long as three hours before transfusion. The 24-hour posttransfusion survival values were about 80 per cent and oxygen transport function was either normal or improved whether or not the units were perfused before transfusion.     

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.     

Blood storage XXIV: red blood cell 2,3-DPG and ATP maintenance for six weeks in CPD-adenine with higher phosphate, pyruvate, and dihydroxyacetone

The individual and collective effects of various phosphate, pyruvate and dihydroxyacetone concentrations on 2,3-DPG and ATP maintenance during blood storage with CPD-adenine (0.25 mM), were studied. Phosphate concentrations ranged from 2 to 100 mM. Low concentations were best for 2,3-DPG maintenance during the first three weeks, after which there was no difference. ATP concentrations were better maintained by the highest phosphate concentrations in the first week. After the second week the lower concentrations of phosphate were better. With pyruvate 40 and 60 mM were the best for 2,3-DPG levels through six weeks of storage. ATP concentrations were poorest with high pyruvate. Maintenance of 2,3-DPG was above half normal for six weeks of storage in the 60, 80 and 100 mM DHA preservatives. ATP concentrations were best maintained in the preservative lacking DHA. Combinations of phosphate, pyruvate and DHA in concentrations which had been found to be effective when used individually were studied. Best maintenance of 2,3-DPG (above half normal levels) for six weeks was afforded by pyruvate, phosphate and DHA, and by pyruvate and DHA. ATP maintenance was best afforded by CPD-adenine alone and CPD-adenine with pyruvate and phosphate. Pyruvate alone maintained ATP less well and the pyruvate- DHA was worst. Intermediate in maintenance of ATP was the preservative containing pyruvate, phosphate and DHA.