Further improvement of the ACD-AG protective solution for blood. II. Behavior of the oxygen affinity of preserved blood in ACD-AG medium and in a protective solution with addition of pyruvate and xylitol and elevated Ph values]

The present paper deals with the behaviour of the oxygen transport function as well as the 2.3 DPG and ATP levels of erythrocytes during the storage in an ACD-AG solution. In the ACD-AG blood in P 50 fell from 20mm of Hg to values of 12 mm of Hg within 4 weeks of storage. The 2.3 DPG content had already fallen to values below 10% within a fortnight. Additions of xylitol (10 mM in the blood) and pyruvate (0.3 mM in the blood) will delay the decrease of P 50 and the 2.3 DPG content. Concerning the ATP behaviour there was no significant difference between ACD-AG blood and that with additions of xylitol and pyruvate. Up to a storage of a fortnight stored blood in the ACD-AG solution of xylitol and pyruvate will be equal to ACD-AG fresh blood as far as the parameter of oxygen transport function is concerned.     

Autologous transfusion of erythrocytes with high or low concentration of 2,3-diphosphoglycerate. Survival rate and time and hemoglobin-oxygen affinity]

1. In human erythrocytes the 2.3 DPG concentration was increased three to fourfold of the norm as IPP re-suspension by an incubation time of four hours at 37 degrees C or as ACD-AG blood was lowered below 20% of the norm respectively. After an autologous transfusion the 24 hours' surviving rate and the apparent half survival time of cells as well as the affinity of haemoglobin to oxygen in the total blood were measured. 2. The 24 hours' surviving rate for fresh erythrocytes with increased 2.3 DPG and ATP concentration amounts to 73% and the apparent half survival time amounts to 6 days. If erythrocytes are stored for four weeks as IPP resuspension at 4 degrees C, the 24 hours' surviving rate is 59%. Erythrocytes from fresh ACD-AG blood with lowered 2.3 DPG and a normal ATP concentration have a 24 hours' surviving time of 85% and an apparent half survival time of 24 days. 3. After autologous transfusion of 400 ml of erythrocytes with increased 2.3 DPG concentration the P50 value of the total blood will increase by 3 mm of Hg, after administering 400 ml of erythrocytes with lowered 2.3 DPG concentration it will fall by 1.8 mm of Hg. 4. The findings are discussed in connection with the significance of the changes of affinity of haemoglobin to oxygen produced by 2.3 DPG for the oxygen supply of tissues and under the aspect of using stored blood with increased 2.3 DPG concentration for practical purposes.     

Viability, ATP and 2,3-DPG content of resuspended erythrocytes during several-week storage in cold

Leukocyte-and thrombocyte-poor packed red cells obtained from ACD or. ACD-AG blood were resuspended to a hematocrit of about 55% and stored at 4 degrees C. The resuspension solution consisted of xylitol, inorganic phosphate, bicarbonate, adenine (A) and guanosine (G) solved in water. In one case glucose, citrate and sucrose were also added, in another one, sorbitol. The 2,3-DPG and the ATP level remained for a longer period in the sorbitol-xylitol-medium than in the glucose-xylitol-medium. The ATP content in the red cell suspension was higher than in packed cells. Higher ATP values were obtained in red blood cells from whole blood with adenine and guanosine. The survival rate of resuspended red blood cells in glucose-AG-citrate-sucrose medium was about 80--85% after 3 weeks of storage and 77% after 6 weeks with a higher range.     

The effect of inosine, inorganic phosphates and pyruvate on erythrocyte glycolysis metabolites under conditions of preservation]

Human erythrocytes were stored as resuspensions in solutions containing citrate (Z), inosine + citrate (I), inosine + phosphate (IP), and inosine + phosphate + pyruvate (IPP). The storage was made at + 4 degrees C for 6 weeks; the initial pH-value amounted to 7.4 at + 4 degrees C. The cellular concentrations of 2.3 DPG, ATP, G6P, FDP and DOAP + GAP were determined. The following results were obtained: 1. During the storage in stored Z-blood the 2.3 DPG concentration will fall below 10% of its initial value; it will remain nearly unchanged in stored I-blood and will increase to 170% in stored IP-blood, to 270% of its initial value in stored IPP-blood. 2. The ATP concentration of cells will fall to about 50% of its initial value at the beginning of the storage of all stored blood. After that it will only increase to about 80% of its initial value in stored IP- and IPP-blood. 3. During the storage the G6P concentration will increase to the highest degree in stored IPP-blood and if high pyruvate concentrations are not present, it will have a reciprocal behaviour towards the FDP and triosephosphate level. The results were discussed in view of the regulation of glycolysis under storage conditions.     

ATP-content and shape of erythrocytes during preservation with adenine and nucleosides]

ACD blood with additions of adenine (A, 0.5 mM in blood), ademine + guanosine ((AG, 0.5 mM each) and adenine + guanosine + inosine (IAG, 0.5: 0.5: 18 mM) was stored for 6 weeks at 4 degrees C and the morphological changes in connection with the ATP content were observed. After a storage of 6 weeks 2--3% of the cells were present as diskocytes, 60% as echinocytes, and 40% as spherocytes. The delayed morphological alterations in the ACD-AG blood in comparison with ACD-A blood were also reflected by a higher ATP content of the ACD-AG blood during its storage. The alterations in the form of erythrocytes recorded in the morphological index Im (a subdivision was made according to 6 different stages of form) correlated with the ATP content. The coefficient of correlation amounted to r = 0.85. Thus, Im is a reliable criterium for evaluating possible storage damages of stored erythrocytes.     

Erythrocyte concentrates, low in buffy coat: production, preservation and evaluation of its quality

Buffy coat-poor packed red cells were prepared from fresh ACD-, ACD-AG- and EDTA-blood, than resuspended with a preservation solution, containing glucose, adenine, guanosine, sucrose, citric acid and sodium citrate and stored at 4 degrees C for 6 weeks. The survival rate of resuspended red cells from ACD-AG-blood amounted to 77% after 6 weeks of storage. The ATP content of resuspended red cells was approximately 25% lower than in ACD-AG whole blood during storage caused probably by increased ATP consuming reactions at the red cell membrane. The P2G-content of resuspended red cells from ACD- and ACD-AG-blood decreased above 50% of the normal level during the first week, as fast as in ACD- and ACD-AG whole blood. The P2G-breakdown in red cells from EDTA-blood was delayed for a week due to the higher pH as in CPD blood. Additions of xylitol, inorganic phosphate, and bicarbonate in 6, 5 and 20 mM final concentrations in the red cell suspensions and an increased pH at the same time delayed the breakdown of ATP and P2G. Packed red cells can be administered fast enough at hematocrits to 0.60 that will be achieved by adding 50 to 100 ml preservation solution. Leukocytes and thrombocytes were reduceds to 70 to 80%. With increasing rate of reduction a higher loss of red cells occured. Buffy coat-poor red cell concentrate contains only few microaggregates. It diminishes the risc of febrile transfusion reactions and delays the appearance of alloimmunisation. The circulatory overload of patients is less frequent than after transfusions of red cell resuspensions containing a large resuspension volume.     

An incubation medium for the elevation of adenosine triphosphate and 2,3-diphosphoglycerate in fresh and long-preserved human erythrocytes.

The levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in freshly drawn human erythrocytes can be tripled by a 2 h incubation at 37 degrees C in a medium containing 21 mM glucose, 1.8 mM adenine, 5 mM pyruvate, 10 mM inosine, and 96 mM phosphate. Similar incubation conditions will restore the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood levels preserved for 12 and 15 weeks, respectively, to those of fresh cells. Omission of pyruvate from the incubation medium further increases the level of ATP slightly, but there is little elevation of 2,3-diphosphoglycerate. Under these conditions labelled pyruvate and lactate production from [14-C]glucose or [14-C]inosine is not diminished, but labelled fructose 1,6-diphosphate, rather than 2,3-diphosphoglycerate, accumulates. In addition, omission of pyruvate from the incubation medium, with a concomitant decrease in accumulation of 2,3-diphosphoglycerate, diminishes the concentration of inorganic phosphate required for optimal ATP elevation. A 5 h incubation in the glucose-adenine-pyruvate-inosine-phosphate medium elevates the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood preserved in the cold for 15 weeks to twice that of fresh cells, indicating that the cells retain their metabolic potential even after prolonged storage at 2 degrees C. The medium may provide a method of rejuvenating 10-12 week cold-preserved erythrocytes for transfusion purposes, by a 1 h incubation at 37 degrees C.     

Effect of dihydroxyacetone on metabolic parameters and survival of resuspended erythrocytes after storage at 25 degrees and 4 degrees]

Erythrocyte concentrates from CPD blood were resuspended after separating the leukocyte-thrombocyte layer and stored at 4 degrees C and 25 degrees C. The solutions for resuspension differed in the content of substrate, pH, condition of sterilization and the resuspension volume used, 1 or 4 volumes of cell concentrate to 1 volume of resuspension solution. At 4 degrees C, there was no effect of DHA on the the 2.3 bisphosphoglycerate content (P2G content). The decreased activity of triokinase at low temperature, phosphate deficiency and a partial disintegration of DHA during the autoclave as well as the difference of the commercial DHA preparations are discussed as underlying causes. At 25 degrees C DHA delayed the P2G decrease during storage. The enhanced triokinase activity above 15 degrees C is principally considered to be the cause for it. High rates of haemolysis, rapid ATP decrease, survival rates of 56% after a storage of three weeks for erythrocyte resuspensions with DHA in used compositions render a clinical application impossible at present. It could be well suitable as a substrate for resynthesizing P2G. An addition of ascorbate + nicotinamid + adenin at a storage temperature of 4 degrees C had no influence on the P2G content. A resuspension solution on the basis of xylitol with additions of sorbitol, bicarbonate, inorganic phosphate, pyruvate, adenine and guanosine showed the best properties in the content of P2G and ATP, lactate formation and survival rate during a storage at 4 degrees C.     

Hemoglobin function in stored blood, XVII. Maintenance of red cell 2,3 DPG (function) and ATP (viability) for six weeks in ACD or CPD-adenine-inosine-methylene blue.

Blood preservatives containing adenine for six week storage have been prepared with inosine and methylene blue at various pH levels in order to maintain, 23-DPG levels for immediate oxygen transport upon transfusion. In one experiment, the adverse effect of a high pH on ATP maintenance was demonstrated in the presence of methylene blue and inosine. In this and other experiments it was clear that ATP was better maintained in low pH preservatives and DPG better maintained in higher pH preservatives. However, 2,3-DPG levels were kept from falling with CPD-adenine-inosine over a wide range of pH values. A CPD-adenine-inosine preservative at a pH 5.8 maintained normal DPG levels for three weeks of storage. A similar preservative but with a pH of 6.6 maintained normal DPG levels for 35 days of storage. It is suggested that if all blood bank units are going to have normal DPG levels for optimal oxygen transport at the time of transfusion then a CPD preservative with a higher pH and/or metabolic nutrients and regulators such as inosine or methylene blue would be required.     

Biochemical parameters to assess viability of blood stored for transfusional use

Blood collected in citrate-phosphate-dextrose-adenine (CPDA-1) containing transfusional bags, was weekly tested throughout a 35 day period. Biochemical assays included plasmatic glucose, electrolytes, free Hb, acid-base balance and hemogasanalysis. Intraerythrocytic ATP and 2,3 DPG were also determined. Results show that an almost total 2,3 DPG depletion occurs during the first three weeks, whereas intracellular ATP are about 50% of the initial values, at the same time. Lowering of pH is also maximal at the third week. pCO2 variation pattern is biphasic: an early increase due to HCO3- titration by lactic acid arising from red cells glycolysis, followed by a decrease probably due to plastic bag permeability to CO2 itself. The percentage of O2Hb also rises during blood storage: this might be the combined result of increase in pO2 and decrease in 2,3 DPG content. A rise of free Hb was obtained; extracellular K+ levels underwent a sixfold increase in 35 days. The mechanism of relative variations of these parameters and the gas transport are discussed. Some of these parameters might be used as routine controls to asses viability and functional status of stored red cells for transfusional use.     

Evaluation of internationally used blood stabilizers for effective erythrocyte substitution]

Haemotherapy developed in the direction of transfusing pure blood cell preparations, if possible and enlarging the therapy with plasma fraction considerably. Quality losses of haemotherapeutics will already occur when they are prepared. This is mainly due to the conditions of blood collecting, blood stabiliser, duration and storage temperature from the blood collecting to further processing as well as biological variability of the composition of the donor's blood. The amount of substrate available to erythrocytes differs in various blood stabilisers. Deplasmatized erythrocyte concentrates can be used after several weeks of storage, if a high glucose concentration is present in the blood stabiliser. In CPD media the function of erythrocytes, the oxygen supply of the tissue, will remain intact a week longer than in ACD media. This effect will be increased by xylitol and pyruvate as well as by adding bicarbonate simultaneously. In future a primary importance will have to be attached to an improved storage of erythrocytes in the form of resuspended buffy coat-free erythrocyte concentrates.     

The effect of thyroxine on the 2,3-diphosphoglycerate content of erythrocytes in vivo and in vitro]

After ending a continous treatment with thyroxine the average dropping of the 2,3 DPG level was 0.4 mumol/ml. T4 decreased on the average by 7.6 microgram/ml. One time application of 1 mg thyroxine p.o. led within 24 hours to an increase of the 2,3 DPG level of -chi = 0.2 mumol/ml, the pH in the erythrocytes increased by 0.02 on the average. Blood incubation with thyroxine added in a concentration of -chi = 24 microgram/100 ml showed no increase of 2,3 DPG, pH and phosphate, while there was a significant acidosis and increase of phosphate in the control blood. The lactate production was significantly lower and glucose consumption was significantly higher in the blood with thyroxine.     

Hormonal effects and the control of gluconeogenesis from sorbitol, xylitol and glycerol in perfused chicken liver

Addition of sorbitol or xylitol to perfused chicken liver caused a biphasic increase in the rate of glucose production. The second increase correlated with a decrease in the lactate to pyruvate ratio. Increased glucose production in response to the addition of glycerol was not biphasic. Aminooxyacetate inhibited both the inherent second increase in glucose production and stimulatory effects of alanine and pyruvate. The stimulatory effects of norepinephrine and glucagon on gluconeogenesis from sorbitol decreased in the presence of methylene blue. Only the stimulatory effect of norepinephrine was inhibited by aminooxyacetate.     

Xylitol metabolism in the isolated perfused rat liver

1. Loading the isolated perfused liver from well-fed rats with xylitol (20mm) caused a depletion of adenine nucleotides and P_i and an accumulation of α-glycerophosphate. The ATP content fell to 66% of the control value after 10min and to 32% after 80min. The ADP and AMP contents also fell. After 80min 63% of the total adenine nucleotides and 59% of the P_i had been lost. 2. The α-glycerophosphate content rose from 0.13 to 4.74μmol/g at 10min and reached 8.02μmol/g at 40min. 3. Xylitol was rapidly metabolized, the main products being glucose, lactate and pyruvate. 4. The [lactate]/[pyruvate] ratio in the presence of xylitol rose to 30–40. 5. On perfusion of livers from starved animals the main product of xylitol metabolism was glucose and the mean ratio xylitol removed/glucose formed was 1.29 (corrected for endogenous glucose and lactate production). This is close to the predicted value of 1.2. 6. Evidence is presented indicating that the loss of adenine nucleotides caused by xylitol is not due to the increased ATP consumption but to the accumulation of α-glycerophosphate and depletion of P_i. 7. The loss of adenine nucleotides accounts for the hyperuricaemia which can occur after xylitol infusion in man. 8. The relevance of the findings to the clinical use of xylitol as an energy source is discussed.     

Hypoxic incubation leads to concerted changes of carbonic anhydrase activity and 2.3 DPG concentration of chick embryo red cells

Red cell carbonic anhydrase activity, 2.3 DPG concentration, and activities of three key enzymes controlling DPG metabolism (PK, PFK and DPGM) were measured in normoxic and hypoxic (incubation in 13.5% O₂) chick embroys. In normoxia 2.3 DPG concentration and carbonic anhydrase activity begin to increase by the third week of incubation. Hypoxia induces a rise of 2.3 DPG concentration and carbonic anhydrase activity by Day 8 of development, i.e., 1 week earlier. Since during normal development chick embryos become hypoxic in the third week of incubation, the results suggest that PO₂ has a controlling influence on the timing of differentiation events of definitive embryonic red cells.     

Gluconeogenesis in isolated chicken (Gallus domesticus) liver cells.

1. Gluconeogenesis was studied in isolated avian hepatocytes. The highest rate of glucose production obtained was from lactate, followed by dihydroxyacetone, glyceraldehyde, and fructose. Alanine was converted to glucose at only about 4% the rate of lactate. 2. Addition of 10 mM sorbitol, xylitol, or ethanol to the hepatocytes increased glucose production from pyruvate 25-40%, while glycerol addition increased it only 9%. 3. Addition of beta-hydroxybutyrate had no effect on glucose production from lactate or pyruvate. 4. Addition of octanoate had no effect on glucose production from pyruvate, but depressed it from lactate at 5 mM. 5. Differences in the formation of glucose from various substrates suggest some basic differences in the mode of glucose production between the chick and the rat and guinea-pig.     

Mathematical analysis of multienzyme systems. I. Modelling of the glycolysis of human erythrocytes.

A mathematical model for the glycolysis of human erythrocytes is presented which takes into account ATP-synthesis and -consumption. A set of three differential equations describes the steady states and the time-dependent changes of the metabolite concentrations under blood storage conditions. For a given parameter combination there are in general three stationary points of the system, one of which is unstable. At a low ATP-need the ATP-level is relatively constant for variations in the rate constant of the ATP-consuming processes. Above a critical level of the energy consumption the system breaks down. An important role of the 2.3P2G-bypass of the erythrocytes is its action as an "energy buffer", wasting ATP in case of ATP-overproduction and producing ATP in case of underproduction. A parameter combination consistent with the data on the isolated enzymes was found which gives a good agreement of theoretical predictions with the measured metabolite concentrations. Under blood preservation conditions the difference of the rates of ATP-production and -consumption is the most important factor for a high ATP-level over long periods.     

An in vitro study of the pH-lowering potential of salivary lactobacilli associated with dental caries

AIMS: Lactobacilli are known to produce acids from sucrose or glucose. This acid production can cause a drop in pH which is sufficiently significant to demineralize the hard tissues of the teeth. Some authors have demonstrated the benefits of substituting sorbitol or xylitol for sucrose. The aim of this work was to study the acid production of salivary lactobacilli with one test sugar (glucose) and two polyols (sorbitol and xylitol). METHODS AND RESULTS: The pH-lowering potential of three strains of oral lactobacilli was recorded with glucose or one of the polyols at three different concentrations. The results showed that polyols were broken down by certain strains of lactobacilli. When this degradation took place, the pH dropped to values sufficiently low to demineralize the hard tissues of the teeth. CONCLUSIONS: Further studies must be carried out on the metabolism of polyols before encouraging their widespread substitution for sucrose.     

In vitro evaluation and study of the survival of erythrocytes preserved in a saline-adenine-glucose-mannitol medium for 35 days

The saline-adenine-glucose-mannitol (SAGM) solution for resuspension of red cells was evaluated on 30 blood units tested over 42 days and compared to 5 red cell concentrates collected on the conventional CPD medium. Total and extra-cellular hemoglobin, potassium, pH, ATP and DPG concentrations, osmotic fragility, schizocyte formation, and red cell antigenicity were studied through the storage period. Chromium survival studies of autologous donated red cells were performed in 10 donors. Red cell concentrates resuspended in SAGM solution showed at the 35th day of conservation at 4 degrees C, a mean storage hemolysis of only 0.66%, an ATP concentration of 67% of the initial value, a schizocyte proportion of less than 1.5%, a mean 24 hour posttransfusion viability of 88.33% and a mean red cell T 1/2 survival of 25 days 10 hours. No alteration of common blood group antigens could be found after storage of red cells for 42 days.     

Correction of Metabolic Indicators of Erythrocytes and Myocardium Structure with Ozonized Red Blood-Cell Mass

The early posttransfusion period after acute blood loss using erythromass without ozone was characterized by decreased electrophoretic mobility of erythrocytes (EPME), ATP, 2,3-diphosphoglycerate (2,3- DPG) concentration, Na⁺-K⁺-ATPase activity, and increased concentration of malonic dialdehyde (MDA) and catalase activity in erythrocytes. Most parameters gradually were restored over 5 days, but a reduced concentration of 2,3-DPG was observed throughout the entire experiment with transfusion of erythromass without ozone. Transfusion of the ozonized erythrocyte mass 1 h after its administration caused an increase in 2,3- DPG concentration in erythrocytes circulating in the vascular bed. Twenty-four hours later, increased catalase activity, EPPE, and ATP concentration were registered, and, after 5 days, enhanced Na⁺-K⁺-ATPase activity was registered. Their values were maintained until the end of the experiment at a higher level than in animals that had undergone transfusion of the nonozonized erythromass. Ozonized erythrocyte mass improves the functional metabolic state of erythrocytes, promotes an earlier recovery of the oxygen transport in blood, and limits the damage to the microcirculatory bed of the myocardium and cardiomyocytes during blood loss.