R. T. Hershey

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.

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 stored 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 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 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 XXVI. CPD-Adenine Packed Cells: Benefits of Increasing the Glucose

In searching for the optimal glucose concentration, this lab has monitored ATP, 2,3‐DPG, pH, and glucose levels of samples taken from full blood units stored for 6 weeks at 4 C. The blood was collected into CPD‐adenine containing 100, 125, 150, 175, and 200 per cent of the glucose present in CPD. The units were stored as whole blood, soft packed (50 to 70% Hct), or hard packed units (80 to 95% Hct). ATP values in general did not decrease very greatly in whole blood units and only moderately in soft packed units. However, in hard packed units a steady progressive decrease in the ATP values was seen to begin at day 14. In these hard‐packed units the only improvement with extra glucose was seen beginning at day 14 when ATP maintenance was better with 200 per cent glucose, but the improvement was not significant until day 42. However, at 35 days the ATP values for 200 and 175 per cent glucose were noticeably better than for the other preservatives. Therefore, it appears from this study that the glucose concentration in CPD‐adenine for hard‐packed cells should be at least 175 per cent of that in regularly formulated CPD. Also, there would appear to be an advantage of having 200 per cent glucose in those units of blood that may be stored beyond 35 days for emergency blood shortage times.

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 μM/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 23‐DPG catabolism.

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

Our previous experiments on the mechanisms of ascorbates 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.

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.