Vox Sang. 37: 229-234 (1979)
Changes in 2,3-DPG Content and Oxygen Affinity in Erythrocytes Stored at 4°C Kenji Honda, Masaki Miyamoto and Shigeru Sasakawa Department of Research, Central Blood Center, Japanese Red Cross Society, Tokyo
Abstract. The 2,3-DPG content of red blood cells increased within the first 24 h when fresh erythrocytes or whole blood were stored at 4°C. This phenomenon was strongly pH dependent. The temporary increase in 2,3-DPG was scarcely observed below pH 7.4 or above pH 7.8. In the case of whole blood, the increase was observed in CPD blood but not in ACD blood. Similar results were obtained with erythrocytes suspended in saline, when its pH was adjusted to approximately 7.6. Plasma proteins were not essential for the increase in 2,3-DPG content. Extracellular oxygen levels were continually measured in erythrocyte suspensions in order to check the changes in oxygen affinity of hemoglobin without damaging the cells. Both extracellular oxygen levels and 2,3-DPG contents were simultaneously increased by keeping fresh erythrocytes at 4°C. Inhibition of glycolysis with sodium fluoride and monoiodoacetic acid indicated that the in vivo steady state of glycolysis in erythrocytes might be altered by chilling to make the rate of 2,3-DPG synthesis faster than that of 2,3-DPG decomposition.
Introduction
Materials and Methods
vious papers [4,5] that the oxygen affinity (Terumo c0.9 Tokyo, Japan) containing ACD O r CPD as anticoagulant (30ml of ACD or CPD to Of hemolysates Of 200 ml of blood). ACD and CPD blood was stored decreased and their 273-DPG ‘Ontent in- at 4 OC without separating plasma, or erythrocytes creased at an early Stage Of preservation. were isolated from ACD blood by centrifugation This temporary increase in 2,3-DPG content (3,000 rpm x 10 min) immediately after collection has an important influence upon the func- of blood, and then washed three times with saline. The washed erythrocytes were resuspended in 30 mM changes Of e@hrocytes’ In this glucose at 50% hematocrit, and stored at 4 OC. paper, such changes Of g1Yco1YSis and Metabolic inhibiton such sodium fluoride WaF) function of eVthrocYtes have been Cmnhed or monoiodoacetic acid (MIA) were added to ,a final concentration of 1OmM. The pH of saline in more detail.
230
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media was adjusted with 0.2 N NaOH as required. Samples of the erythrocyte suspensions were taken at several intervals of time during storage and the cells were washed three times with 8aline before measurement of their 2,3-DPG content. A sealed flask equipped with an oxygen electrode was prepared as an incubation vessel for the measurement of the oxygen level of erythrocyte suspensions (fig. 1). The suspension was placed in the incubation vessel and aerated for 10min. The vessel was immersed in a constant-temperature bath at 4 OC and carefully sealed with wax. The oxygen electrode used in this experiment was of a sensitive type; its cathode was a platinum wire 0.2mm in diameter that was sealed into a capillary outlet of a softglass tube 2 mm in diameter. A silverhiher chloride half-cell in 1M KCl was used as a reference electrode. The polarizing voltage was 0.6V. Coating of the platinum electrode was carried out by dipping the end of the electrode 3-5mm into 7% acetylcellulose dissolved in a mixture of acetonemethanol (1:l) [l]. Oxygen levels were continuously recorded on a Hitachi 056 recorder (Hitachi Ltd., Tokyo, Japan) and calculated with the assumption that 1ml of air-equilibrated water contains 183 nmol of molecular oxygen at 4 OC. Erythrocyte 2,3-DPG content was enzymatically determined according to Michal [3] at 23OC, using a 2,3-DPG teat kit (lot No. 148334; Boehrhger, Mannheim; Yamanouchi Co.).
Fig. 1. Oxygen electrode and reaction vessel. A = Electrode assembly with a closed reaction vessel, 50 ml in volume; a = constanttemperature water; b = stirrer; c =thermometer; Bi= silver eleotrode (Ag/AgCl in 1M KCl, anode); C = platinum electrode (cathode). The numbers in this figure show size in millimeters.
Table I. Reproducibility of the temporary increase in 2,3-DPG content of erythrocytes at an early stage of preservation (0,7 and 24 h) in 100 mM PBS at 50% hematocrit
Sample No.
1 2
3
4 5
6 7 8
2,3-DPG content, %
Oh
7h
24 h
100 (7.62)
99 (7.65) 105 (7.67) 98 (7.64) 95 (7.71)
104 (7.65) 97 (7.68) 102 (7.64) 94 (7.70) 113 (7.63) 103 (7.65)
100
(7.64) 100 (7.65) 100 (7.68) 100 (7.61) 100 (7.64) 100 (7.68) 100 (7.58)
111
(7.63) 100
(7.65) 101 (7.72) 111 (7.64)
100
(7.80) 107 (7.67)
The figures in parentheses are the extracellular pH of erythrocyte suspensions.
Low Temperature Effects on Erythrocyte Storage
23 1
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Fig.2. Changes in extracellular pH and 2,3DPG contents of citrated whole blood during 24hour storage at 4%. Open symbols and closed ones indicate CPD and ACD blood, respectively. Each sample was from a different individual.
Results and Discussion Figure 2 shows the changes during storage of extracellular pH and 2,3-DPG content of erythrocytes which were collected from healthy volunteers as citrated whole blood (ACD or CPD blood) and stored at 4 "C. The pH of CPD and ACD blood were kept almost constant at approximately 7.6 and 7.3, respectively, during 24 h storage at 4 OC. 2,3-DPG contents at arbitrary periods of storage are indicated as percentages of the 2,3-DPG content of fresh erythrocytes determined immediately after collection of blood. The 2,3-DPG content of ACD blood decreased with storage while that of CPD
0
2
4
6
8
24
Incubation time, h
Fig. 3. Changes in extracellular pH and 2,3-DPG contents of erythrocyte-suspending saline during 24hour storage at 4 O C . Open and closed circles indicate a saline suspension and a PBS suspension, respectively. Each medium contained 30 mM glucose. The hematocrit of the erythrocyte suspensions was 50%.
blood increased or was constant within at least 24 h of storage. Figure 3 shows findings for stored erythrocytes which were separated from plasma immediately after collection of ACD blood and then resuspended in saline or phosphate-buffered saline (PBS) at 50% hematocrit. The 2,3-DPG content was also increased without plasma, as shown in this figure but only when the pH of the erythrocyte suspension was near 7.6. A temporary increase of 2,3-DPG content was not observed above pH7.8 or below7.4.This finding was reproducible although there was some difference in the degree of 2,3-DPG increase among the individual samples examined (table I).
232
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Fig. 4. Changes in oxygen levels in themedium of erythrocyte suspensions stored at different pH in saline at 4 OC.
Fig.5. pH dependency of the maximum increments in oxygen level in the medium of chilled erythrocyte suspensionsin saline at 50%hematocrit. The maximum increments were determined from figure 4.
These data also indicate that it becomes more difficult to increase 2,3-DPG contents as the extracellular pH deviates from 7.6. An increase in 2,3-DPG content of erythrocytes should decrease the oxygen affinity of hemoglobin and release molecular oxygen through the membranes of erythrocytes. This effect was continuously measured by an oxygen electrode. Figure 4 shows the changes of oxygen levels in erythrocyte suspensions at various pH levels during 24 h storage at 4 "C. During the first 5-6 h of storage the oxygen levels were scarcely changed, then they suddenly increased after 10 h of storage. The change in the oxygen level in the erythrocyte suspensions is linked to a change
of P,, [partial pressure of oxygen required for hemoglobin to produce 50% saturation of oxygen; 21. The maximum amount of released oxygen was dependent on the pH of the medium used as shown in figure 5. This pH profile of oxygen affinity change seems to be linked to the pH-dependent change in 2,3-DPG content described above. The temporary increase in 2,3-DPG content in chilled fresh erythrocytes might result from the different temperature sensitivity of the enzymes participating in glycolysis. The low temperature (4 "C) might disrupt the steady state of erythrocyte metabolism, so that 2,3-DPG synthesis might be more active than 2,3-DPG decomposition at an
Low Temperature Effects on Erythrocyte Storage
233
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Incubation time, days
Fig.6. Changes in extracellular pH and 2,3DPG contents of erythrocyte suspensions in saline with glycolytic inhibitors. The final concentration of the inhibitors was 10 mM. A = NaF; 0 = MIA; = contral.
Fig.7. Changes in extracellular pH and 2,3DPG contents of erythrocyte suspensions in PBS with glycolytic inhibitors. The final concentration of the inhibitors was 10 mM. A = NaF; 0 = MIA; = control.
early stage of low temperature preservation of blood. To verify this speculation, the effect of glycolytic inhibitors on the changes in 2,3-DPG content were examined. One of the inhibitors used was MIA which inhibits the 2,3-DPG-synthesizing enzyme, glyceraldehyde 3-phosphate dehydrogenase, and the other was NaF which inhibits the 2,3-DPGdecomposing enzyme, enolase. Figure 6 shows the changes in extracellular pH and 2,3-DPG content of erythrocytes which were stored at 4 "C with these glycolytic inhibitors. Decrease in extracellular pH with storage time was less in the inhibited systems than in the controls since lactate production was suppressed due to inhibition of a part of
the glycolytic pathway. Erythrocyte 2,3-DPG contents were increased in the presence of NaF but decreased in the presence of MIA. However, as shown in figure 7,addition of a relatively high concentration (50 mM) of inorganic phosphate led to a great increase in 2,3-DPG content in the absence of glycolytic inhibitors and to a slight decrease of 2,3DPG content in their presence. The activation of glycolysis by inorganic phosphate seems to overcome inhibition by NaF and MIA. In conclusion, the 2,3-DPG contents of erythrocytes were temporarily increased at an early stage of low-temperature liquid preservation of blood, with a decrease in the
234
oxygen affinity of hemoglobin. This phenomenon has a strong pH dependency with an optimum pH of about 7.6, which is the pH of CPD blood at 4 "C.
References 1 Hagihara, B.; Ishibashi, F.; Sasaki, K., and Kamigawara, Y.: Cellulose acetate coating for the polarographic oxygen electrode. Analyt. Biochem. 86: 417-431 (1978). 2 Honda, K. et al.: unpublished data. 3 Michal, G.:in Bergmeyer, Methods of enzymatic analysis, pp. 1433-1438 (Verlag Chemie, Weinheim/Academic Press, New York 1974).
Honda/Miyamto/Sasakawa
4 Sasakawa, S. and Tokunaga, E.: Physical and chemical changes of ACD-preserved blood: a comparison of blood in glass bottles and plastic bags. VOXSang. 31: 199-210 (1976). 5 Sasakawa, S.; Honda, K.; Miyamoto, M., and Tokunaga, E.: Change of oxygen affinity of hemoglobin in different conditions of blood preservation. Vox Sang. 34: 164-170 (1978). Received: April 13, 1979 Accepted: April 23,1979 Dr. K. Honda Department of Research Central Blood Center Japanese Red Cross Society Hiroo 4, Shibuya-ku, Tokyo 150 (Japan)
Changes in 2,3-DPG Content and Oxygen Affinity in Erythrocytes Stored at 4°C Kenji Honda, Masaki Miyamoto and Shigeru Sasakawa Department of Research, Central Blood Center, Japanese Red Cross Society, Tokyo
Abstract. The 2,3-DPG content of red blood cells increased within the first 24 h when fresh erythrocytes or whole blood were stored at 4°C. This phenomenon was strongly pH dependent. The temporary increase in 2,3-DPG was scarcely observed below pH 7.4 or above pH 7.8. In the case of whole blood, the increase was observed in CPD blood but not in ACD blood. Similar results were obtained with erythrocytes suspended in saline, when its pH was adjusted to approximately 7.6. Plasma proteins were not essential for the increase in 2,3-DPG content. Extracellular oxygen levels were continually measured in erythrocyte suspensions in order to check the changes in oxygen affinity of hemoglobin without damaging the cells. Both extracellular oxygen levels and 2,3-DPG contents were simultaneously increased by keeping fresh erythrocytes at 4°C. Inhibition of glycolysis with sodium fluoride and monoiodoacetic acid indicated that the in vivo steady state of glycolysis in erythrocytes might be altered by chilling to make the rate of 2,3-DPG synthesis faster than that of 2,3-DPG decomposition.
Introduction
Materials and Methods
vious papers [4,5] that the oxygen affinity (Terumo c0.9 Tokyo, Japan) containing ACD O r CPD as anticoagulant (30ml of ACD or CPD to Of hemolysates Of 200 ml of blood). ACD and CPD blood was stored decreased and their 273-DPG ‘Ontent in- at 4 OC without separating plasma, or erythrocytes creased at an early Stage Of preservation. were isolated from ACD blood by centrifugation This temporary increase in 2,3-DPG content (3,000 rpm x 10 min) immediately after collection has an important influence upon the func- of blood, and then washed three times with saline. The washed erythrocytes were resuspended in 30 mM changes Of e@hrocytes’ In this glucose at 50% hematocrit, and stored at 4 OC. paper, such changes Of g1Yco1YSis and Metabolic inhibiton such sodium fluoride WaF) function of eVthrocYtes have been Cmnhed or monoiodoacetic acid (MIA) were added to ,a final concentration of 1OmM. The pH of saline in more detail.
230
Honda/Miyamto/Sasakawa
C
T
v)
I 44c-
media was adjusted with 0.2 N NaOH as required. Samples of the erythrocyte suspensions were taken at several intervals of time during storage and the cells were washed three times with 8aline before measurement of their 2,3-DPG content. A sealed flask equipped with an oxygen electrode was prepared as an incubation vessel for the measurement of the oxygen level of erythrocyte suspensions (fig. 1). The suspension was placed in the incubation vessel and aerated for 10min. The vessel was immersed in a constant-temperature bath at 4 OC and carefully sealed with wax. The oxygen electrode used in this experiment was of a sensitive type; its cathode was a platinum wire 0.2mm in diameter that was sealed into a capillary outlet of a softglass tube 2 mm in diameter. A silverhiher chloride half-cell in 1M KCl was used as a reference electrode. The polarizing voltage was 0.6V. Coating of the platinum electrode was carried out by dipping the end of the electrode 3-5mm into 7% acetylcellulose dissolved in a mixture of acetonemethanol (1:l) [l]. Oxygen levels were continuously recorded on a Hitachi 056 recorder (Hitachi Ltd., Tokyo, Japan) and calculated with the assumption that 1ml of air-equilibrated water contains 183 nmol of molecular oxygen at 4 OC. Erythrocyte 2,3-DPG content was enzymatically determined according to Michal [3] at 23OC, using a 2,3-DPG teat kit (lot No. 148334; Boehrhger, Mannheim; Yamanouchi Co.).
Fig. 1. Oxygen electrode and reaction vessel. A = Electrode assembly with a closed reaction vessel, 50 ml in volume; a = constanttemperature water; b = stirrer; c =thermometer; Bi= silver eleotrode (Ag/AgCl in 1M KCl, anode); C = platinum electrode (cathode). The numbers in this figure show size in millimeters.
Table I. Reproducibility of the temporary increase in 2,3-DPG content of erythrocytes at an early stage of preservation (0,7 and 24 h) in 100 mM PBS at 50% hematocrit
Sample No.
1 2
3
4 5
6 7 8
2,3-DPG content, %
Oh
7h
24 h
100 (7.62)
99 (7.65) 105 (7.67) 98 (7.64) 95 (7.71)
104 (7.65) 97 (7.68) 102 (7.64) 94 (7.70) 113 (7.63) 103 (7.65)
100
(7.64) 100 (7.65) 100 (7.68) 100 (7.61) 100 (7.64) 100 (7.68) 100 (7.58)
111
(7.63) 100
(7.65) 101 (7.72) 111 (7.64)
100
(7.80) 107 (7.67)
The figures in parentheses are the extracellular pH of erythrocyte suspensions.
Low Temperature Effects on Erythrocyte Storage
23 1
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7.8
7.6
7.6
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Fig.2. Changes in extracellular pH and 2,3DPG contents of citrated whole blood during 24hour storage at 4%. Open symbols and closed ones indicate CPD and ACD blood, respectively. Each sample was from a different individual.
Results and Discussion Figure 2 shows the changes during storage of extracellular pH and 2,3-DPG content of erythrocytes which were collected from healthy volunteers as citrated whole blood (ACD or CPD blood) and stored at 4 "C. The pH of CPD and ACD blood were kept almost constant at approximately 7.6 and 7.3, respectively, during 24 h storage at 4 OC. 2,3-DPG contents at arbitrary periods of storage are indicated as percentages of the 2,3-DPG content of fresh erythrocytes determined immediately after collection of blood. The 2,3-DPG content of ACD blood decreased with storage while that of CPD
0
2
4
6
8
24
Incubation time, h
Fig. 3. Changes in extracellular pH and 2,3-DPG contents of erythrocyte-suspending saline during 24hour storage at 4 O C . Open and closed circles indicate a saline suspension and a PBS suspension, respectively. Each medium contained 30 mM glucose. The hematocrit of the erythrocyte suspensions was 50%.
blood increased or was constant within at least 24 h of storage. Figure 3 shows findings for stored erythrocytes which were separated from plasma immediately after collection of ACD blood and then resuspended in saline or phosphate-buffered saline (PBS) at 50% hematocrit. The 2,3-DPG content was also increased without plasma, as shown in this figure but only when the pH of the erythrocyte suspension was near 7.6. A temporary increase of 2,3-DPG content was not observed above pH7.8 or below7.4.This finding was reproducible although there was some difference in the degree of 2,3-DPG increase among the individual samples examined (table I).
232
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Fig. 4. Changes in oxygen levels in themedium of erythrocyte suspensions stored at different pH in saline at 4 OC.
Fig.5. pH dependency of the maximum increments in oxygen level in the medium of chilled erythrocyte suspensionsin saline at 50%hematocrit. The maximum increments were determined from figure 4.
These data also indicate that it becomes more difficult to increase 2,3-DPG contents as the extracellular pH deviates from 7.6. An increase in 2,3-DPG content of erythrocytes should decrease the oxygen affinity of hemoglobin and release molecular oxygen through the membranes of erythrocytes. This effect was continuously measured by an oxygen electrode. Figure 4 shows the changes of oxygen levels in erythrocyte suspensions at various pH levels during 24 h storage at 4 "C. During the first 5-6 h of storage the oxygen levels were scarcely changed, then they suddenly increased after 10 h of storage. The change in the oxygen level in the erythrocyte suspensions is linked to a change
of P,, [partial pressure of oxygen required for hemoglobin to produce 50% saturation of oxygen; 21. The maximum amount of released oxygen was dependent on the pH of the medium used as shown in figure 5. This pH profile of oxygen affinity change seems to be linked to the pH-dependent change in 2,3-DPG content described above. The temporary increase in 2,3-DPG content in chilled fresh erythrocytes might result from the different temperature sensitivity of the enzymes participating in glycolysis. The low temperature (4 "C) might disrupt the steady state of erythrocyte metabolism, so that 2,3-DPG synthesis might be more active than 2,3-DPG decomposition at an
Low Temperature Effects on Erythrocyte Storage
233
::k
7.6
7.4
ra 7.2 7.0
I a 7.4 7.2 0
6.8
0
4
8
12
16
Incubation time, days
4
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16
Incubation time, days
120
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100
160
80
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Incubation time, days
Incubation time, days
Fig.6. Changes in extracellular pH and 2,3DPG contents of erythrocyte suspensions in saline with glycolytic inhibitors. The final concentration of the inhibitors was 10 mM. A = NaF; 0 = MIA; = contral.
Fig.7. Changes in extracellular pH and 2,3DPG contents of erythrocyte suspensions in PBS with glycolytic inhibitors. The final concentration of the inhibitors was 10 mM. A = NaF; 0 = MIA; = control.
early stage of low temperature preservation of blood. To verify this speculation, the effect of glycolytic inhibitors on the changes in 2,3-DPG content were examined. One of the inhibitors used was MIA which inhibits the 2,3-DPG-synthesizing enzyme, glyceraldehyde 3-phosphate dehydrogenase, and the other was NaF which inhibits the 2,3-DPGdecomposing enzyme, enolase. Figure 6 shows the changes in extracellular pH and 2,3-DPG content of erythrocytes which were stored at 4 "C with these glycolytic inhibitors. Decrease in extracellular pH with storage time was less in the inhibited systems than in the controls since lactate production was suppressed due to inhibition of a part of
the glycolytic pathway. Erythrocyte 2,3-DPG contents were increased in the presence of NaF but decreased in the presence of MIA. However, as shown in figure 7,addition of a relatively high concentration (50 mM) of inorganic phosphate led to a great increase in 2,3-DPG content in the absence of glycolytic inhibitors and to a slight decrease of 2,3DPG content in their presence. The activation of glycolysis by inorganic phosphate seems to overcome inhibition by NaF and MIA. In conclusion, the 2,3-DPG contents of erythrocytes were temporarily increased at an early stage of low-temperature liquid preservation of blood, with a decrease in the
234
oxygen affinity of hemoglobin. This phenomenon has a strong pH dependency with an optimum pH of about 7.6, which is the pH of CPD blood at 4 "C.
References 1 Hagihara, B.; Ishibashi, F.; Sasaki, K., and Kamigawara, Y.: Cellulose acetate coating for the polarographic oxygen electrode. Analyt. Biochem. 86: 417-431 (1978). 2 Honda, K. et al.: unpublished data. 3 Michal, G.:in Bergmeyer, Methods of enzymatic analysis, pp. 1433-1438 (Verlag Chemie, Weinheim/Academic Press, New York 1974).
Honda/Miyamto/Sasakawa
4 Sasakawa, S. and Tokunaga, E.: Physical and chemical changes of ACD-preserved blood: a comparison of blood in glass bottles and plastic bags. VOXSang. 31: 199-210 (1976). 5 Sasakawa, S.; Honda, K.; Miyamoto, M., and Tokunaga, E.: Change of oxygen affinity of hemoglobin in different conditions of blood preservation. Vox Sang. 34: 164-170 (1978). Received: April 13, 1979 Accepted: April 23,1979 Dr. K. Honda Department of Research Central Blood Center Japanese Red Cross Society Hiroo 4, Shibuya-ku, Tokyo 150 (Japan)
