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 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. 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 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 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.     

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.     

不同保养液对血2,3-二磷酸甘油酸与ATP的保存效果

目的　研究不同保养液对血中 2 ,3 二磷酸甘油酸 ( 2 ,3 DPG)和三磷酸腺苷 (ATP)的保存能力。寻找适宜 2 ,3 DPG和ATP保存的保养液。方法　同一血源于采血的当日及 4d、7d、1 0d、1 4d、2 1d、2 8d、35d、4 2d分别测定 2 ,3 DPG、ATP、Hb和 pH值 ,观察不同保养液对血中 2 ,3 DPG和ATP的保存效果。 结果　ACD保养液保存血第 1 4d、CPD及CPD AI保存血第2 1d就检测不到 2 ,3 DPG ,CPD AI DHA类保养液中 2 ,3 DPG到 4 2d维持在初始水平的 6 0 % ,CPD AI DHA Vit .C保养液中2 ,3 DPG的含量至 4 2d仍然保持在初始水平。CPD AI类保存血对ATP的保存效果较好 ,ACD与CPD保养液对ATP保存效果较差。结论　CPD AI DHA Vit .C保养液对血中 2 ,3 DPG和ATP的保存效果最好     

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.     

Regeneration of 2,3-bisphosphoglycerate and ATP in stored erythrocytes by phosphoenolpyruvate: a new preservative for blood storage

Phosphoenolpyruvate is transported through erythrocyte membrane in acid citrate or sucrose solution and metabolized to 2,3-bisphosphoglycerate and pyruvate. The levels of ATP and 2,3-bisphosphoglycerate of depleted, stored erythrocytes were increased effectively by incubating the cells in acid-citrate-dextrose-sucrose solution with phosphoenolpyruvate. The concentration of 2,3-bisphosphoglycerate exceeded by three times its physlological level under suitable conditions. When fresh blood was stored at 4 C in acid-citrate-dextrose-sucrose solution with phosphoenolpyruvate, the levels of ATP and 2,3-bisphosphoglycerate of erythrocytes decreased during storage in essentially the same extent as those of erythrocytes preserved in acid-citrate-dextrose solution. However, the levels of ATP and 2,3-bisphosphoglycerate of the cells increased when preserved erythrocytes with phosphoenolpyruvate were incubated at 37 C for 30 minutes. Especially noteworthy was the ATP concentration of erythrocytes preserved with phosphoenolpyruvate at 4 C for 14 days. It was kept at the same level as in fresh blood by the short incubation time.     

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.     

Use of methylene blue in sepsis: a systematic review

A systematic review of the literature was conducted to determine if the administration of methylene blue in humans improves hemodynamic status and/or outcome in patients with septic shock. Studies were identified from MEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials database. The review included human studies of patients with septic shock treated with methylene blue in which hemodynamic variables or mortality rates were reported. An electronic form was used to extract items including study design, population characteristics, intervention details, and outcomes. No meta-analysis was performed. Methylene blue administration in patients with septic shock increases mean arterial pressure and systemic vascular resistance while decreasing vasopressor requirements. Increased pulmonary vascular resistance has been reported with bolus administration but might be avoided by continuous infusion. No other ill effects were reported. Effects on mortality have not been adequately evaluated in the literature.     

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.     

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.     

Mammalian genotoxicity assessment of methylene blue in plasma: implications for virus inactivation

BACKGROUND: The risk of adverse consequences of virus-inactivation procedures for plasma and cellular blood components must be less than the risk of transfusion-associated viral disease. Previous studies demonstrated that methylene blue, which is currently used in Europe for virus inactivation in fresh-frozen plasma, can elicit mutations in bacterial test systems. This study investigates the potential for methylene blue genotoxicity in two mammalian test systems. STUDY DESIGN AND METHODS: Different concentrations of methylene blue were prepared in plasma (heat-treated at 56 degrees C for 1 hour to reduce cytotoxicity) and used, without illumination, in an in vitro mouse lymphoma cell assay designed to detect forward mutations in the gene encoding thymidine kinase. The assay was performed in the presence or absence of rat liver S9 microsomal fraction. Similarly prepared samples of methylene blue in heat-treated plasma were used in an in vivo mouse micronucleus assay. Each system included a negative vehicle control (heat-treated plasma without methylene blue) and a positive control consisting of a known genotoxic agent. RESULTS: Intravenous administration to mice of 62 mg per kg of methylene blue did not increase the frequency of micronuclei in polychromatic red cells harvested from bone marrow. However, methylene blue concentrations of 10 micrograms per mL (with S9 activation) and 30 micrograms per mL (without S9 activation) significantly increased the thymidine kinase mutation frequency of mouse lymphoma cells to approximately 110 × 10(- 6), from a spontaneous frequency of 28 × 10(-6). CONCLUSION: Methylene blue is mutagenic in cultured mammalian cells. In contrast, results from the mouse micronucleus assay suggest that the genotoxicity is not expressed in vivo. Considerably more investigation will be required to assess the genotoxic potential of intravenously administered methylene blue used in virus-inactivation procedures, because of the likelihood of the formation of methylene blue photoproducts or the impact of metabolic conversion of methylene blue to leukomethylene blue in vivo.     

Successful storage of RBCs for 10 weeks in a new additive solution

BACKGROUND: The effect of storing packed RBCs suspended in 300 mL of an alkaline, experimental additive solution (EAS 64) was explored. STUDY DESIGN AND METHODS: RBC units prepared from blood collected from healthy donors into CPD were WBC reduced and stored for 10 weeks under blood bank conditions after the addition of 300 mL of EAS 64 (adenine, 2 mM:; dextrose, 50 mM:; mannitol, 20 mM:; NaCl, 75 mM:; Na(2)HPO(4), 9 mM:). For comparison, non-WBC-reduced units from the same donors were stored in a different additive solution (AS-1, Baxter Healthcare) for 6 weeks. Standard methods were used for the in vitro assays. The 24-hour in vivo recoveries were measured by using (51)Cr- and (99m)Tc-labeled RBCs. RESULTS: Mean recovery in the EAS 64 units after 10 weeks was 84 +/- 8 percent, the same as in the AS-1 units stored for 6 weeks. For EAS 64 and AS-1 units, respectively, the ATP of the RBCs was 85 percent and 64 percent of the initial value, hemolysis was 0.43 percent and 0.63 percent, supernatant potassium was 24 mEq per L and 44 mEq per L, and the morphologic index was 98 and 71. CONCLUSION: RBCs suspended in 300 mL of EAS 64 can be stored satisfactorily for 10 weeks. Longer RBC storage should reduce outdating, increase availability of transfusions in remote locations, and improve the efficiency of autologous donor programs.     

Study protocol: stability of morphine injected without preservative, delivered with a disposable infusion device

Background and objective : Morphine hydrochloride, a major analgesic drug, is being increasingly administered using portable disposable infusion devices. The objective of this study was to investigate the stability of morphine in such a system at two concentrations (2·50 and 5·00 mg/ml) over a 30‐day period. Method : High‐performance liquid chromatography of stored morphine solutions. Results : The best stability was observed with disposable infusion devices filled with a morphine solution containing sodium metabisulphite as a preservative. No breakdown products were detected after 1 month of storage at room temperature, in light or darkness.
On the other hand, 2·50 and 5·00 mg/ml morphine solutions without sodium metabisulphite, stored in the infusion device led to the formation of 0·205% and 0·235% of pseudomorphine, respectively, after 6 days of storage in the light, and 1·50% and 0·94% after 30 days storage. Conclusion : Morphine hydrochloride solutions stored in disposable infusion devices degraded very slowly, particularly when preserved with sodium metabisulphite. The solutions are stable over 5 days, the maximum period of storage normally required when using disposable infusers.