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 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 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 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. 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.

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. 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 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.

Preservation of erythrocytes using metabolic regulators and mutrients. V. Inosine and methylene blue.

Methylene blue and inosine have been shown to stimulate glycolytic metabolism in the erythrocytes, increasing the concentration of 2.3-diphosphoglycerate (2,3-DPG), which is necessary for hemoglobin function, by regulating oxidative metabolism and providing a five-carbon nutrient for glycolysis, respectively. However, a recent study suggested that the methylene blue effect was dependent on the presence of inosine. This study was designed to establish, if possible, the existence of a methylene blue effect and to confirm the usefulness of inosine. The optimal concentration of inosine for increasing 2,3-DPG synthesis in a CPD-adenine preservative is confirmed to be 10--15 mM. Concentrations of 2,3-DPG were maintained in the erythrocytes at normal or higher levels for 21 days of storage with 10 or 15 mM inosine, whether the methylene blue was present or not. However, when methylene blue was present, 2,3-DPG concentrations were significantly better maintained.

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 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 XVI packed red cell storage in CPD-adenine.

Interest has been renewed in CPD-adenine as a long-term liquid blood preservative. The question of whether the metabolic product of adenine, 2,8-dioxyadenine was toxic to humans has apparently been resolved by extensive animal and human studies in favor of there being no potential toxicity in the amounts used in blood preservation. Sweden is adopting CPD-adenine (0.25 mM) as its national blood preservative after ten years of clinical experience in trials. They have shown that each additional week of storage time beyond the current three weeks with CPD results in a 50 per cent reduction of wasteage caused by outdating. They are adopting the 35-day time for regular use with 42 days for an emergency reserve supply. However, many units of blood in the U.S. are stored as packed red blood cells and the question has been raised as to whether there is sufficient glucose in the preservative to maintain red blood cell metabolism in the packed cell unit. The present investigation indicates that there is sufficient glucose for 35 days of packed cell storage in CPD-adenine (0.25 mM) but in some units this might be marginal at 42 days of storage.