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.     

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 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.     

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.     

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 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 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.     

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.     

Adaptation to hypoxia in liver cirrhosis: the role of 2,3-DPG (author's transl)

By observing a group of 20 patients with liver cirrhosis, we have clarified some features concerning the tissue hypoxia, which is often present in such a disease. By determining the levels of the haemoglobin and of the intraerythrocytic 2,3-DPG, and by evaluating the acid-base state of such patients, we have emphasized the increased output of the 2,3-DPG as mechanism of adaptation to hypoxia associated with hepatic cirrhosis, both in subjects with anemia and alkalosis and in subjects without anemia but with alkalosis.     

Prolonged maintenance of 2,3-diphosphoglycerate acid and adenosine triphosphate in red blood cells during storage

BACKGROUND: Current additive solutions (ASs) for red cells (RBCs) do not maintain a constant level of critical metabolites such as adenosine triphosphate (ATP) and 2,3-diphosphoglycerate acid (2,3-DPG) during cold storage. From the literature it is known that the intracellular pH is an important determinant of RBC metabolism. Therefore, a new, alkaline, AS was developed with the aim to allow cold storage of RBCs with stable product characteristics. STUDY DESIGN AND METHODS: Whole blood-derived RBCs (leukoreduced) were resuspended in experimental medium phosphate-adenine-guanosine-glucose-gluconate-mannitol (PAGGG-M; pH 8.2) with and without washing in the same medium. During cold storage several in vitro variables, such as intracellular pH, 2,3-DPG, ATP, and hemolysis, were analyzed. RESULTS: During cold storage, RBCs resuspended in PAGGG-M showed a constant ATP level (approx. 6 mumol/g Hb) and a very limited hemolysis (<0.2%). The 2,3-DPG content showed an increase until Day 21 (150% of initial level), followed by a slow decrease, with at Day 35 still 100 percent of the initial level. RBCs washed in PAGGG-M even showed a continuous increase of 2,3-DPG during 35 days, with a maximum level of 200 percent of the initial value. The effect of PAGGG-M appears to be related to long-lasting effects of the initial intracellular pH shortly after production. CONCLUSION: Resuspension of RBCs in our alkaline medium PAGGG-M resulted in a RBC unit of high quality during storage for up to at least 35 days, with 2,3-DPG levels of higher than 10 mumol per g Hb, hemolysis of less than 0.2 percent, and ATP levels of higher than 5 mumol per g Hb.     

Pediatric reference intervals for lymphocyte vitamin C (ascorbic acid)

ObjectiveTo establish pediatric reference intervals for lymphocyte vitamin C.Design and methodsThis was a prospective study of 194 well children aged 0–7 years old of mixed ethnicity who had blood drawn for the purpose of this study. Blood was collected during elective surgery under general anesthesia and lymphocytes isolated and stored as frozen ascorbic acid lymphocyte lysates for later HPLC analysis by previously described methodology. Reference intervals were established according to the Clinical and Laboratory Standards Institute (CLSI) and the International Federation of Clinical Chemistry (IFCC) guidelines (C28-A3). Horn–Pesce robust method was used to estimate the 95% confidence interval and 95% reference interval.ResultsReference intervals were independent of age or gender and shown to be 12.9–52.8 μg/10⁸ cells (lymphocytes).ConclusionWe have defined pediatric reference ranges for lymphocyte vitamin C in healthy, fasted children at a relevant age group (0–7 years). The new reference interval can now be used to more reliably explore possible implications of variation of vitamin C levels on bleeding and other clinical signs.     

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.     

Viability and function of platelet concentrates stored in CPD-adenine (CPDA-1)

The effective use of CPDA-1 as an anticoagulant in routine blood banking practice requires demonstration that platelet concentrates prepared in this solution meet both in vitro quality control standards and maintain posttransfusion viability and function after storage. In this study of 138 units of CPDA-1 platelet concentrates, the average platelet count was 8.0 +/− 0.2 × 10(10) with 81 per cent of the units having greater than 5.5 × 10(10) platelets. The mean poststorage pH was 6.68 +/− 0.03 and only four of the units had a pH of less than 6.0 (3%). Residual plasma volume averaged 75 +/− 1 ml. Platelet viability was determined in 16 normal volunteers by measuring survival of 51Cr- labeled autologous platelets after storage for 72 hours at 22 +/− 2 C. Platelet recovery averaged 50 +/− 4 per cent, while survival was 7.3 +/− 0.4 days for the 15 units with a pH above 6.0. Measurements of posttransfusion platelet viability and function were made in 12 paients with thrombocytopenia secondary to marrow failure. Their mean pretransfusion platelet count was 17,000 +/− 2,000/microliter, and their standardized template bleeding times were all greater than 30 minutes. Platelet recovery averaged 44 +/− 5 per cent and survival 3.3 +/− 0.5 days. In seven of the patients with the best posttransfusion increments, bleeding time was improved. Five patients with poor posttranfusion platelet increments showed no improvement in bleeding time with CPDA-1; two of these patients were also transfused with CPD platelets and had no response. Our studies indicate that platelet concentrates prepared in CPDA-1 meet in vitro quality control standards and after transfusion, maintain viability and function comparable to that of CPD collected platelets.     

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.     

Inhibitory effect of ascorbic acid (vitamin C) on cortisol secretion following adrenal stimulation in children.

Measurements of plasma cortisol values before and at 4 and 6 hours after intramuscular administration of a depot preparation of synthetic b1-24 corticotrophin were carried out in 12 healthy children (group B) before, as well as on the 5th day of continuous ascorbic acid (AA) administration (1 g t.i.d. orally). Comparison of the results in group B with those of 8 healthy children similarly treated with corticotrophin but not given AA (group A) showed that, on the 5th day of AA administration the mean cortisol values after ACTH were significantly lower than the corresponding values in group A (p less than 0.02), or the post-ACTH values in group B observed on the 1st experimental day, i.e., before AA administration (p less than 0.001). On the other hand, AA administration had no significant effect on the fasting plasma cortisol values. These data suggest that AA excess following adrenal stimulation with ACTH exerts an inhibitory effect on cortisol secretion and consequently it may be of no benefit in conditions of stress.