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 phosphoenolpyruvate
red
blood
cells,
cryopreservation,
hydroxyethyl
starch,
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 2,3DPG 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.
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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
