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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 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.

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 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.

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.

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.

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.