Application of Phosphoenolpyruvate Into Canine Red Blood Cell Cryopreservation with Hydroxyethyl Starch

CryoLetters 26 (1), 1-6 (2005) © Cryoletters, c/o Royal Veterinary College, London NW1 0TU, UK

APPLICATION OF PHOSPHOENOLPYRUVATE INTO CANINE RED BLOOD CELL CRYOPRESERVATION WITH HYDROXYETHYL STARCH

Heejaung Kim1,3, Kazuhito Itamoto2, Satoshi Une3, Munekazu Nakaichi3, Yasuho Taura3, and Sajio Sumida4

1 The United Graduate School of Veterinary Sciences, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan. E-mail:[email protected] 2 Department of Veterinary Internal Medicine and 3 Departments of Veterinary Surgery, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi, 753-8515, and 4 Sumida Laboratory of Cryomedicine and Blood Transfusion, Kenketsu-Kyokyu Bldg. 1F, Tateishi 5-11-16, Katsushika, Tokyo, 124-0012, Japan.

Abstract

Phosphoenolpyruvate (PEP) is a phosphorylated glycolytic intermediate that can penetrate the RBC membrane and be metabolized to 2,3-DPG and ATP. In this study, we evaluated the effects of PEP treatment on canine red blood cells (RBCs) cryopreserved with 12.5% (w/v) HES. RBCs were incubated for 30, 60, and 90 min at 37°C with PEP solution containing 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, 1 mM adenine and 50 mM PEP (340 m osm/kg), pH 6.0 and then cryopreserved in liquid nitrogen with 12.5% (w/v) HES for 2 weeks. 2,3-DPG and saline stabilities of the PEP treated groups were increased and osmotic fragility indices were significantly decreased compared to the untreated control group. There were no differences in 2,3-DPG levels within the PEP treated groups with different PEP incubation times. These results suggest that PEP treatment may be beneficial for the cryopreservation of canine RBCs with HES. Keywords: canine red blood cells, cryopreservation, hydroxyethyl starch, phosphoenolpyruvate

INTRODUCTION

Blood cryopreservation has several distinct advantages over traditional refrigerated storage (36). There are many reports on human red blood cells (RBCs) cryopreservation using glycerol. Unfortunately, this approach has been labor- and cost-prohibitive for animal transfusions. Knorpp et al. (17) reported the successful cryopreservation of human RBCs with hydroxyethyl starch (HES). HES is a non-penetrating cryoprotectant and removal of HES prior to transfusion is not necessary, since it is a substituted polysaccharide slowly metabolized in vivo to a non-utilizable carbohydrate by α-amylases, and then eliminated in the kidney. We reported the comparison of HES and glycerol in canine RBCs cryopreservation (in press). In the study, 12.5% (w/v) HES showed the same value of thaw hemolysis but more cell deformities than glycerol. In reports about the influence of HES on ATP and 2,3-DPG in

1 human RBC cryopreservation, ATP and 2,3-DPG levels dropped after cryopreservation (3,19,25). A study showed that ATP and 2,3-DPG are capable of affecting the stability of the RBC membrane and through the regulation of metabolite levels, the cell could control membrane skeleton organization and those cell properties affected by the skeleton (28). Hamasaki et al. (10,11) reported that phosphoenolpyruvate (PEP) could penetrate the RBC membrane and be metabolized to 2,3-DPG and ATP. Unlike other additives, PEP effectively increases both ATP and 2,3-DPG in RBCs, and under suitable conditions, the 2,3- DPG concentration can be raised several-fold beyond its physiological level. Some experiments on animals (7,18,26,37), humans (11,13,14) and clinical trials in human medicine (21) support the beneficial effect of PEP treated RBCs. However, to the best of our knowledge, there are only a few papers on PEP treated RBCs cryopreservation using glycerol (24,29) and no papers using HES. In this study, different incubation times with PEP solution were used for the treatment of canine RBCs, following which the treated cells were cryopreserved with 25% (w/v) HES (final concentration 12.5% (w/v) HES) for 2 weeks. We determined the optimum PEP incubation time for canine RBCs by analyzing 2,3-DPG levels, saline stability and osmotic fragility. Furthermore, we evaluated that the PEP treated RBCs are superior to untreated RBCs, for cryopreservation with HES.

MATERIALS AND METHODS

Six mature beagle dogs were used in this study. They were housed indoors and maintained according to the Yamaguchi University Animal Care and Use Committee regulations. Dogs were 3 to 5 years of age and weighed 10 to 14 kg. Each dog was subjected to a physical examination and blood tests prior to blood collection. Blood was collected in CPDA-1 (Terumo Blood Bag CPDA, Terumo Co., Tokyo, Japan) and stored up to 24 hr at 4°C until cryopreserved. Plasma and buffy coat were removed by centrifugation (1,500 ×g, 5 min, 4°C). The plasma was frozen and then used as the so-called “autologous fresh frozen plasma (AFFP)” in the post-thaw resuspension experiment conducted later. The RBCs were washed three times with phosphate-buffered saline solution. After the final wash, RBCs were packed by centrifugation to a hematocrit value of about 80%. In the control group, 15 ml of packed RBCs was mixed in the same volume of 25% (w/v) HES (mean molecular weight 200,000 g/M, molar substitution 0.6, Ajinomoto Co. Inc., Tokyo) solution manually. The mixture was transferred to a freezing bag (froze bag, Hutaba Medical Inc., Tokyo). This bag was then placed in a closed aluminum container, submerged in liquid nitrogen, and stored for 2 weeks. The frozen RBCs were thawed by manual agitation in a water bath maintained at 48°C (33,34). In the PEP treated groups, packed RBCs were mixed with an equal volume of PEP solution (29) that consisted of 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, 1 mM adenine and 50 mM PEP (Sigma-Aldrich Co., ST. Louis, MO., USA.), (340 m osm/kg), pH 6.0 and incubated for 30, 60, and 90 min (PEP30, PEP60, PEP90) at 37°C. After incubation and centrifugation, 15 ml of incubated packed RBCs was mixed with HES solution. Then, it was frozen, stored, and thawed as described above. Thawed RBCs of each group were evaluated by the 2,3-diphosphoglycerate (2,3-DPG) UV test (Roche, Mannheim, Germany), saline stability test (36) and osmotic fragility test (16). In the post-thaw resuspension experiments, 10 ml of thawed RBCs of each group were incubated for 30 min at 37°C in the same volume of thawed AFFP immediately after thawing. At the end of incubation, the plasma-HES-RBCs mixture was centrifuged and packed RBCs were evaluated using the osmotic fragility test.

2 All results are given as means ± SD. Student’s t-test was used to identify significant differences between saline stabilities at 30 min and 120 min, and between osmotic fragility tests before and after AFFP incubation. Differences among groups were tested for significance using the ANOVA followed by Fischer’s multiple comparison.

RESULTS

The results of 2,3-DPG, osmotic fragility index, and saline stability test are summarized in Table 1. The 2,3-DPG level (3.45 ± 0.65 µmole/ml RBC) before cryopreservation was decreased in the control group after thawing (P<0.05). The PEP incubation groups were higher than the control group (P<0.05), but there were no significant differences among the PEP incubation times. Saline stabilities increased significantly with PEP incubation except for the PEP90 group incubated for 30 min and there were no significant differences between the 30 min and 120 min incubation times of each group. All the osmotic fragility curves of the PEP incubation groups shifted to the left compared with the control group (Fig. 1).

A A 120 B 120 Control 100 100 PEP30 PEP60 80 80

s s PEP90 ysi l ysi l o

60 m 60

mo e e

H H % % 40 40

20 20

0 0 0 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 0.2 0.3 0.4 0.5 0.6 0.7 0.8

% NaCl concentraion % NaCl concentraion

Figure 1. Osmotic fragility curves (A) Representative osmotic fragility curves of thawed RBCs and (B) resuspended RBCs with plasma. Osmotic fragility curves of PEP treated RBCs groups shifted to left compared with control group.

Using these osmotic fragility curves, the fragility index (Table 1) was quantified by determining the saline percentage at which 50% hemolysis was observed. PEP treated groups of the thawed and post-thaw resuspended RBCs had significantly decreased osmotic fragility indices compared to the control groups and post-thaw resuspension caused a significant decrease in osmotic fragility indices compared to that prior to plasma incubation.

3 Table 1. 2,3-DPG, saline stability, and osmotic fragility index of canine red blood cells frozen-thawed in the presence of phosphoenolpyruvate.

2,3-DPG Saline Stability (%) Osmotic Fragility Index (% NaCl) (µmole/ml RBC) 30 min 120 min Wholea AFFPb ¶ Control 3.18 ± 0.40 77.4 ± 6.3 72.4 ± 6.7 0.434 ± 0.031 0.391 ± 0.024 PEP30 4.77 ± 0.93* 82.4 ± 1.7* 78.9 ± 1.6† 0.407 ± 0.015* 0.354 ± 0.015†§ PEP60 4.84 ± 1.50* 82.2 ± 2.3* 78.7 ± 2.4† 0.401 ± 0.017* 0.346 ± 0.015†§ PEP90 4.54 ± 1.25* 81.5 ± 2.7 77.9 ± 2.4* 0.385 ± 0.021† 0.354 ± 0.020†¶ a: Osmotic fragility index of thawed RBCs. b: Osmotic fragility index of resuspended RBCs (thawed RBCs incubated in autologous fresh frozen plasma (AFFP) for 30 min in 37°C and centrifuged). *Denotes a significant difference (P<0.05) with respect to control group. †Denotes a significant difference (P<0.01) with respect to control group. ¶Denotes a significant difference (P<0.05) with respect to coupled whole group. §Denotes a significant difference (P<0.01) with respect to coupled whole group.

DISCUSSION

Many mammalian erythrocytes contain 2,3-DPG in higher concentrations than other tissue cells. In the RBC, 2,3-DPG not only acts as a potential regulator of energy metabolism through the anaerobic glycolysis pathway, but it also has an important function in regulating the release of oxygen from hemoglobin (4,6). The latter property is that oxygen affinity of red blood cells decreases as their 2,3-DPG content increases, and oxygen affinity increases as 2,3- DPG content decreases. 2,3-DPG can be increased by incubation of preserved red blood cells in a solution containing inosine, pyruvic acid, inorganic phosphate and mannitol, but ATP cannot be increased (22,23,27,35). Rejuvenation solutions containing inosine have not been widely used, because a product of the in vivo catabolism of inosine is uric acid, which can be dangerous to the point of kidney failure in some patients (2). PEP is transported across the cell membrane by band 3 protein, an anion exchanger that mediates the ‘chloride shift’, and is metabolized to 3-phosphoglyceroyl phosphate (1,3-DPG), 2,3-DPG and ATP by a glycolytic enzyme. In cells treated with PEP, the concentration of 1,2- DPG, the precursor of 2,3-DPG, was more than 100 times its physiological concentration and as a consequence, the concentration of 2,3-DPG increased. Both ATP and 2,3-DPG in preserved cells can be increased by short-term incubation with PEP. Moreover, PEP is nontoxic (LD50 more than 2 g/kg in the dog) (15). We incubated RBCs in PEP solution, pH 6.0 at 37°C for 30, 60, and 90 min before cryopreservation with HES. PEP treated RBCs showed increased 2,3-DPG levels compared to untreated RBCs, after thawing. There have been several studies conducted to determine the optimum incubation conditions of RBCs in PEP (10,12,20,26,31). Some studies showed that by incubation at 37°C for 30 min at the end of various storage periods, the levels of ATP and 2,3-DPG in the cells were raised (12) and that physiologic temperature (37°C) and a pH of less than 6.5 were required for transport and metabolism of PEP (26). These data resemble our results even though a slightly different PEP solution was used here, that consisted of 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, 1 mM adenine and 50 mM PEP. Sputtek et al. (32) reported that the 2,3-DPG level was changed from 5.77 ± 1.19 (before cryopreservation) to 5.34 ± 0.20 µmole/ml RBC (after thawing) in canine RBC cryopreservation using HES. Their 2,3-DPG level before cryopreservation was higher than ours, therefore their value after thawing appears higher. However some papers showed that 2,3-DPG level could be increased by PEP in fresh RBCs (31,37).

4 PEP treated cells showed increased saline stability and decreased osmotic fragility indices. In a report about PEP treatment in old human RBCs (stored for 21 days at 4°C) cryopreserved using glycerol, RBC recovery as frozen and thawed RBCs after deglycerolization was increased to 80 ± 4% compared to 43 ± 9% in units without rejuvenation with PEP and the recovery percentage of PEP-treated frozen and thawed RBCs was similar to the percentage of frozen and thawed RBCs recovered from fresh RBCs within 5 days after donation. Incubation of RBCs with PEP solution restored ATP and 2,3-DPG to levels seen in fresh RBCs, and also facilitated transformation of crenated RBCs to discocytes (29). One study reported that the morphological changes after thawing using HES recovered rapidly in fresh autologous plasma (34). The post-thaw resuspended RBCs showed decreased osmotic fragility indices and more stable osmotic fragility curves in this study. Singbartl et al. (30) reported that HES cryopreservation may create changes in protein-protein association in RBC membranes, but these changes can be reversed after incubation in autologous plasma. Osmotic fragility curves of PEP treated RBCs shifted to the left compared with the control group in plasma resuspension. These results indicated that though plasma incubation after thawing improves overall RBC condition, initial high levels of 2,3-DPG and ATP can further enhance RBC recovery. The concentration of ATP and 2,3-DPG in preserved RBC related very closely to the efficacy of transfusion. The viability of RBC after transfusion is dependent on their ATP concentration (1,8), although the ATP level is by no means the only limiting factor in determining their posttransfusion survival (5). The oxygen transport function of hemoglobin is affected by RBC 2,3-DPG level (9). In a report where RBCs treated with PEP in vitro were reinfused into the donor dogs, it was seen that the increases in 2,3-DPG and P50 induced with PEP were maintained for longer than 3 days following transfusion. All of the dogs that received cells treated with PEP were healthy and no elevations in ALT and AST values were observed. PEP treatment had no influence on the viability of red cells in vivo. The 24 h survival, as well as the half-life of cells treated with PEP, was quite similar to those of fresh cells (37). PEP treatment is not only a protectant of cell membranes against physical stress in frozen and thawed red cells processing, but also a modifier of intracellular biochemical microenviroment intended for transformation of deteriorated cells to intact cells (29). In conclusion, PEP is beneficial in improving the 2,3-DPG level, saline stability, and osmotic fragility index in canine RBCs cryopreservation in HES. Considering these data, PEP treatment may be applied to canine RBCs cryopreservation with 12.5% (w/v) HES and a PEP incubation time of 30 min. This is the first paper reporting PEP-treated RBCs cryopreservation using HES.

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Accepted for publication 22/12/04