The current research shows the possibility of long-term conservation of the erythrocytes an-tioxidant activity in the process of donor packed red blood cells while introducing mexidol to the hemoconservant glugicir composition.
Academic Editor: Jie Yin, Institute of Subtropical Agriculture & University of Chinese Academy of Sciences, China.
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Copyright © 2018 Gavryuchenkov D.V., et al.
The authors have declared that no competing interests exist.
Storage and supply of blood components for possible emergency disaster situation is one of the main tasks of emergency disaster medicine.
While donor blood and its components storage, the viability, morphological and functional values of erythrocytes are gradually decreasing. Alterations in structural and functional organization of cells membranes during donor blood components storage and failure in functioning of membrane connected enzymes and excessive synthesis of free radicals inevitably leads to accelerated blood cells “ageing” 1, 2. Hence, correction of pathophysiological changes in blood transfusion environment at the stage of blood storage is our priority task 3.
We consider creation of new hemopreservatives and further development of the existing ones as the most important trends in transfusiology which allows raising functional value of erythrocytes, medical efficiency of blood transfusions, and lasting blood components storage time 4, 5.
The overall aim of this research is to investigate the capacity of long-termpreservation of the erythrocyte native state in donor packed red blood cells (RBC) by inactivating the processes of lipid peroxygenation (LP) and correction of antioxidant activity (AAC) in erythrocytes with standard RBC storage (at t + 4 0С), by adding 2-ethyl-6-methyl-3-hydroxypyridine succinate antioxidant to composition of the blood conversant “Glugicir”.
Materials and Methods
We prepared several samples of packed RBC on glugicir preservative which contained 0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml 2-ethyl-6-methyl-3-hydroxypyridine succinate antioxidant, respectively. As a control sample we used packed RBC prepared on glugicir with no supplements. We examined the biochemical values of erythrocites in the packed RBC samples immediately after their preparation and in during its storaging at the t = + 4 0С for 6 hours, 3 days, 7 days, 14 days, 21 days and 30 days.
The AAC intensity has been estimated by concentration of malondialdehyde (MDA), which was defined by the reaction with 2-thiobarbituric acid, according to the L.I. Andreeva’s method 6. The intensity of the Free Radical Oxidation (FRO) and General Antioxidant Activity (GAA) were assessed by the chemiluminometer Emilite-1105 (Latvia). The level of catalase was defined according to the M.A. Koroluk’s and Co method) 7. The method based on the ability of hydrogen peroxide to form a stable colored complex with molybdate salts. We applied a modification of this technique, which imply selection the optimum amounts of ammonium molybdate and hydrogen peroxide by titration on the amount of hydrogen peroxide in the solution). 10 ml of 4% solution of ammonium molybdate (0.4 mg per sample volume in a cuvette) was used. The optimal amount of hydrogen peroxide was selected by titration method, the molar concentration of hydrogen peroxide in the solution was calculated: 30 μl of 4% solution of hydrogen peroxide with total content of H2O2 of 1.2 mg was used.
Results and Discussion
The intensification of FRO was observed since the first hours of the packed RBC storage. The greatest increase of FRO by 168 % (p<0.0001) was detected in the sample of the packed RBC stored with 0.25 mg/ml 2-ethyl-6-methyl-3-hydroxypyridine succinate while in samples with higher concentrations of 2-ethyl-6-methyl-3-hydroxypyridine succinate (0.5 mg/ml and 1.0 mg/ml) FRO observed to be decreasing by 16 % and 4 %, respectively (Table 1).Table 1. FRO activity, relative unit
|Hemotransfusion medium||Outcome||6 hours||3 days||7 days||14 days||21 days||30 days|
|Packed RBC (glugicir)||0,664±0,11||0,908±0,17||2,44±0,06||3,14±0,07||3,35±0,07||3,59±0,06||4,11±0,08|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 0.25 mg/ml)||1,871±0,01||1,69±0,04||1,51±0,06||1,41±0,07||1,73±0,03||2,04±0,06|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 0.5 mg/ml)||1,016±0,02||1,18±0,05||1,52±0,04||2,46±0,06||3,19±0,05||2,45±0,04|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 1.0 mg/l)||0,924±0,05||2,09±0,06||2,28±0,08||2,23±0,04||2,48±0,03||2,66±0,02|
By the end of the first week of storage the FRO activity in all test samples was significantly lower than in control samples. Within the group with 1.0 mg/ml concentration of 2-ethyl-6-methyl-3-hydroxypyridine succinate the FRO activation was observed to be increased by 37 % (p<0.001) and 48 % (p<0.001) comparatively to the samples with concentration of 2-ethyl-6-methyl-3-hydroxypyridine succinate in hemostabilizer 0.25 mg/ml and 0.5 mg/ml, respectively.
The antioxidant activity in control samples has decreased by 57 % (p<0.001) compared with initial values by 30 day of storage. GAA into the packed RBCs with added 2-ethyl-6-methyl-3-hydroxypyridine succinate at a dose of 0.25 mg/ml remained on the initial level by the third day. By the end of the first week, it gradually started decreasing and reached the seven-day level of control samples by the 30th day. The packed RBCs samples that containined 2-ethyl-6-methyl-3-hydroxypyridine succinate 0.5 mg/ml during the whole storable life, had the GAA level higher than the control sample, and this level had increased by 166 % (p<0.0001) higher than in the samples with the 2-ethyl-6-methyl-3-hydroxypyridine succinate 0.25 mg/ml concentration by the 30th day of storage. 2-ethyl-6-methyl-3-hydroxypyridine succinate at a dosage of 1.0 mg/ml in the packed RBCs was able to activate GAA during the whole period of storage, while at a dosage of 0.25 mg/ml the efficacy of the antioxidant effect by the 14-th day of storage decreased (Table 2).Table 2. Antioxidant activity, relative unit.
|Hemotransfusion medium||outcome||6 hours||3 days||7 days||14 days||21 days||30 days|
|Packed RBC (glugicir)||2,07±0,048||2,03±0,053||1,78±0,044 p1<0,0001||1,74±0,037 p1<0,0001||1,52±0,081 p1<0,0001||1,26±0,065 p1<0,0001||0,89±0,024 p1<0,0001|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 0.25 mg/ml)||2,08±0,031||2,34±0,030 p1<0,0001 p2<0,0001||2,08±0,018 p2<0,0001||1,90±0,022 p1<0,0001 p2<0,0001||1,67±0,022 p1<0,0001 p2<0,0001||1,44±0,010 p1<0,0001 p2<0,0001|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 0.5 mg/ml)||2,35±0,039 p1<0,0001 p2<0,0001 p3<0,0001||2,42±0,058 p1<0,0001 p2<0,0001||2,15±0,017 p1<0,0001 p2<0,0001 p3<0,05||2,69±0,011 p1<0,0001 p2<0,0001 p3<0,0001||2,81±0,015 p1<0,0001 p2<0,0001 p3<0,0001||2,36±0,023 p1<0,0001 p2<0,0001 p3<0,0001|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 1.0 mg/l)||2,11±0,013 p4<0,0001||2,28±0,025 p1<0,0001 p2<0,0001 p4<0,05||1,97±0,018 p2<0,0001 p3<0,0001 p4<0,0001||2,54±0,015 p1<0,0001 p2<0,0001 p3<0,0001 p4<0,0001||2,79±0,017 p1<0,0001p2<0,0001 p3<0,0001||2,45±0,009 p1<0,0001 p2<0,0001 p3<0,0001 p4<0,001|
The catalase activity in the control samples of the packed red blood cells kept growing for the two weeks of storage and reached 151% (p<0.001), reducing insignificantly by the end of the storage term comparing to the initial values. In the samples of the packed RBCs with 2-ethyl-6-methyl-3-hydroxypyridine succinate we observed the significant growth of catalase activity, increased by the 14th day up to 138 % (p<0.0001) and up to 161 % (p<0.0001) in the packed RBCs with 2-ethyl-6-methyl-3-hydroxypyridine succinate 0.25 mg/ml and 0.5 mg/ml concentration in hemostabillizer, by the 30th day with control samples (Table 3).Table 3. Catalase activity (mcaT/l).
|Hemotransfusion medium||outcome||6 hours||3 days||7 days||14 days||21 days||30 days|
|Packed RBC (glugicir)||12,7±1,53||14,42±1,48||15,5±1,38||25,6±1,21||31,8±3,15||24,50±1,59||26,4±0,22|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 0.25 mg/ml)||21,9±0,32||24,0±0,28||29,2±0,51||30,1±0,60||28,894±0,62||28,5±0,54|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 0.5 mg/ml)||19,5±0,35||22,5±0,18||28,4±0,28||29,4±0,41||28,871±0,59||27,5±0,65|
|Packed RBC (glugicir +2-ethyl-6-methyl-3- hydroxypyridine succinate 1.0 mg/l)||21,5±0,27||23,0±0,34||29,15±0,32||30,8±0,32||28,229±1,16||29,7±1,03|
It was observed that the packed RBCs, when stored, underwent the intensive decrease of the antioxidant activity protection, activating lipid peroxidation. Applying the exogenic antioxidant 2-ethyl-6-methyl-3-hydroxypyridine succinate allows to decrease the lipid peroxidation activity and the “load” on antioxidant erythrocyte systems. Introduction of 2-ethyl-6-methyl-3-hydroxypyridine succinate into hemoconservant causes the evident membrane-protective action and allows adapting blood corpuscles to their lives in the conditions of hypoxia. The minimal level of lipid peroxidation activation has been defined in the erytheromass, while stored with 2-ethyl-6-methyl-3-hydroxypyridine succinate in 0.25 mg/ml glugicir concentration; however, antioxidant activity was lower than in other experimental samples.
Possibly, the activation of lipid peroxidation in the groups with the 2-ethyl-6-methyl-3-hydroxypyridine succinate 0.5 and 1.0 mg/ml glugicir concentration is linked to the inhibition of endogenic antioxidants of RBCs while introducing large dosage of exogenic antioxidants based on principles of biofeedback.
Based on the obtained results, we advise to prepare packed RBCs on glugicir, containing 2-ethyl-6-methyl-3-hydroxypyridine succinate in its minimal concentration — 0.25 mg/ml glugicir.