Blood preservation 35. Red cell 2,3-DPG and ATP maintained by DHA- ascorbate-phosphate

DHA (dihydroxyacetone, 60 mM) with ascorbic acid (d-ascorbate, 10 mM) kept 2,3-DPG concentrations above normal for six weeks. Levels of 2,3- DPG were below normal after four weeks with DHA alone and after two weeks with DHA-ascorbate-phosphate. As in previous studies, high phosphate concentrations decreased 2,3-DPG maintenance. ATP maintenance was best achieved with the following (in order of performance): DHA- phosphate (20 mM); DHA-phosphate (10 mM); the control, CPD-adenine preservative; Phosphate 20 mM; and DHA. DHA with ascorbate provides normal 2,3-DPG for six weeks. The adverse effects of DHA and DHA with ascorbate on ATP levels are modified by 10 mM phosphate.     

Blood preservation. XXIX. Pyruvate maintains normal red cell 2,3-DPG for six weeks of storage in CPD-adenine

Pyruvate was placed in experimental CPD-adenine (0.25 mM) blood preservative mixtures in four concentrations ranging from 40 to 320 mM. In the 320 mM pyruvate preservative, 2,3-DPG levels were elevated above normal for six weeks of whole blood storage at 4 C. The lower pyruvate concentrations maintained elevated or normal 2,3-DPG levels for less time: four weeks with 160 mM, two weeks with 80 mM, and one week or less with 40 mM or the control. ATP values were best maintained in the control. The higher pyruvate concentrations resulted in the most rapid decreases at ATP. However, even the 320 mM pyruvate did not cause ATP to fall below 2 microM/gm of Hb. The higher pyruvate concentrations produced and maintained a higher pH during storage. On the other hand, 2,3-DPG levels increased with pyruvate during the first week of storage when the pH was decreasing rapidly. This could be the result of its oxidation of NADH to NAD. The high pyruvate concentration which maintained elevated 2,3-DPG levels throughout the six weeks might be simulating the effect reported in pyruvate kinase-deficient red blood cells, in which blockage of glycolysis at that step is preventing 2,3- DPG catabolism.     

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.     

Blood storage XXV: Ascorbic acid (vitamin C) and dihydroxyacetone (DHA) maintenance of 2,3-DPG for six weeks in CPD-adenine

In experiments in which ascorbate was included in CPD-adenine preservatives, 2,3-DPG levels were maintained above normal for 28 days with an ascorbate concentration of 10 mM or higher and concentrations of 20 to 80 had no greater effect on 2,3-DPG maintenance. Less ascorbate (5 mM) provided better 2,3-DPG maintenance than was obtained with no ascorbate throughout six weeks of storage but was not as good as the higher concentrations after the third week. ATP concentrations were adversely affected by the presence of ascorbate. The highest ATP concentrations were without ascorbate, next highest with 5 mM, and the worst ATP was with 80 mM. The pH values did not differ from one preservative to another.     

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 XXIII: 2,3-DPG maintenance for six weeks in a CPD-adenine- inosine preservative with and without methylene blue

In a pilot study the optimal concentration of inosine for 2,3-DPG maintenance in a CPD-adenine (0.25 mM) preservative was confirmed to be at lease 10 mM. In these experiments, 2,3-DPG maintenance was nearly normal for six weeks of storage in CPD-adenine-inosine (10 mM) preservative with or without methylene blue. The control preservative lacking inosine showed a statistically significant decrease in 2,3-DPG concentrations after the 3rd week. Finally, 2,3-DPG levels were significantly better maintained in CPD-adenine preservatives that contained 15 mM concentrations of inosine, whether methylene blue was present or not (10(-6)M), compared to CPD-adenine-inosine preservatives that contained 5 mM inosine, with or without methylene blue. The methylene blue effect, while it can be demonstrated in most experiments to help the red blood cell maintain 2,3-DPG during prolonged blood storage, is judges to be a slight value. However, inosine is of great value in maintaining 2,3-DPG for prolonged (five to six weeks) liquid storage.     

Blood preservation. XXXIV. DHA maintains 2,3-DPG and its adverse effect on ATP is reversed with extra phosphate

We have found that the addition of 10 mM inorganic phosphate to DHA in CPD-adenine maintains ATP levels at normal or higher than normal values for six weeks of storage. 2,3-DPG values are slightly lowered by the extra phosphate, but are still maintained at approximately half normal for four weeks by the DHA. The addition of a higher phosphate concentration, 20 mM, to DHA produced lower levels of ATP and 2,3-DPG than those observed with 10 mM phosphate, although both levels were better than in the CPD-adenine control. pH values in this experiment were lowest in the three preservatives containing DHA, probably indicating increased lactate production due to metabolism of this triose sugar, in addition to dextrose present in CPD.     

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.     

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.     

Blood preservation using metabolic regulators and nutrients: XXI. Further studies on pyruvate and DHA (dihydroxyacetone)

CPD-adenine is being adopted in Europe for five weeks for regular blood bank storage and six weeks for emergency use storage. There may be a need to maintain normal levels of 2,3-DPG during this prolonged storage time. In a pilot study from this laboratory, improved 2,3-DPG maintenance was noted with DHA and pyruvate during the fifth and sixth weeks of storage. DHA and pyruvate are relatively unstable in aqucous solutions and in the present study extra care was taken with their experimental use. The additive effect of using DHA and pyruvate together in maintaining 2,3-DPG was confirmed in this study in which significant improvements were seen as early as the seventh day of storage.     

Blood preservation 42: improvement of ascorbate's ability to maintain 2,3-DPG with inosine

Inosine and ascorbate have been shown to maintain normal 2,3-DPG levels during three to four weeks of blood storage. With the introduction of CPD-adenine, which allows five weeks of storage, the desire for 2,3-DPG maintenance may receive new emphasis. Red blood cell 2,3-DPG remained at normal or higher levels for six weeks whenever 10 or 15 mM inosine and 10 mM vitamin C (L-ascorbate) or D-ascorbate were present in the CPD-adenine preservative. Provision by inosine of a five-carbon sugar for 2,3-DPG synthesis, bypassing the rate-limiting phosphofructokinase reaction, may allow NADH oxidation by ascorbate to provide an increased supply of substrate for the Rappoport-Luebering shunt, thus affecting the net increase and maintenance of 2,3-DPG.     

Blood preservation. XLIII. Studies on the ascorbate mechanisms of maintaining red cell 2,3-DPG

Our previous experiments on the mechanisms of ascorbate's effect on the red blood cell failed to show an effect of iodoacetate (IA), a sulfydryl inhibitor. In this study, in contrast to the previous, iodoacetate (85 micromolar) was seen to prevent continued red blood cell metabolism. During the first weeks there was an absence of a continual fall in pH; ATP levels were depressed below half normal; and 2,3-DPG levels fell to very low values within the first week. ATP was best maintained in the control preservative and next best maintained, at adequate levels, with ascorbate, 5 mM, with and without glutathione, 5 mM. 2,3-DPG levels were well maintained with ascorbate and ascorbate with glutathione. Poor ATP maintenance and rapid decreases in 2,3-DPG were observed with iodoacetate, IA plus ascorbate, and IA plus ascorbate and glutathione.     

Dihydroxyacetone, pyruvate, and phosphate effects on 2,3 DPG and ATP in citrate-phosphate-dextrose-adenine blood preservation

Blood was stored with various combinations of 60 mM dihydroxyacetone (DHA), 80 mM pyruvate and 10 mM phosphate in citrate-phosphate-dextrose- adenine-one (CPDA-1) preservative for 42 days. During that time, DHA was found to have a beneficial effect on 2,3 diphosphoglycerate (2,3 DPG), although a deleterious effect on adenosine triphosphate (ATP). Preservatives containing pyruvate gave similar results. However, DHA and phosphate together in CPDA-1 improved both 2,3 DPG and ATP. The adverse effect on 2,3 DPG levels that phosphate normally shows was reversed by the addition of DHA. These results indicate that the addition of phosphate alone, rather than pyruvate, or pyruvate and phosphate, to DHA in CPDA-1 would be preferable to the presently licensed CPDA-1 preservative.     

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.     

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. XIX. Inosine maintenance of 2,3- DPG for 35 days in a CPD-adenine preservative

This study establishes that 10 mM inosine is a sufficient additive to maintain 2,3-DPG levels in blood for five weeks of storage in CPD- adenine. No previous experiments were done with CPD-adenine (0.25 mM) using a design which would give statistical proof of the optimal concentration of inosine needed for maintenance of normal hemoglobin function (2,3-DPG) for five weeks of blood bank storage.     

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.     

Blood preservation. XXVIII. Galactose and maltose maintain red blood cell 2,3-DPG and ATP

Because there may be inadequate dextrose in the newly licensed CPD- adenine for five or six weeks storage of high hematocrit red blood cells, this laboratory has examined some alternate sugars for their ability to maintain red blood cell metabolism during storage. In the current study, dextrose and fructose were studied as model or prototype nutrients. A third six carbon monosacharide, galactose, three dissacharides, lactose, maltose, and sucrose were studied in the same experiment. Of these, fructose best maintained ATP and 2,3-DPG during the fourth to sixth week of whole blood storage at 4 C. Dextrose was next best during this time and was nearly equivalent to fructose in the first three weeks of storage. Galactose and maltose both maintained ATP and 2,3-DPG, but not nearly so well as did fructose and dextrose. Sucrose and lactose were associated with the most rapid deterioration of ATP and DPG levels and they failed to maintain the progressive fall in pH which is usually associated with continuing, useful metabolism.     

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.     

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.