Properties of aqueous-saline human placental extracts and their fractions after storage of placenta at various subzero temperatures

Abstract

Objective: In this study we analyzed the influence of low temperature storage of placental tissue on the effect of obtained aqueous-saline placental extracts and their extract fractions on the state of membranes and cytosol of erythrocytes. Methods: Fractions of human placenta extract (HPE) were obtained with gel-chromatography method. Spontaneous hemolysis of erythrocytes was measured spectrophotometrically by free hemoglobin content in supernatant at 543 nm wavelength. Osmotic fragility (resistance) of erythrocytes was assessed by the level of hemolysis in 0.45% NaCl solution. Low-pH resistance of erythrocytes was evaluated by the time of 50% hemolysis in sodium-phosphate-citrate buffer (pH 3.8), calculating the data from kinetic curve of the change of optical density at 700 nm. Electron paramagnetic resonance (EPR) spin probe method was applied for analysis of cytosol dynamic state. Results: We recorded that low temperature storage of placental tissue allows decreasing membranotropic effect, which was inherent to certain aqueous-saline placental extracts and some of their fractions. At the same time, storage of placental tissue at both 20 С and 196 С does not influence the ability of HPE to decrease erythrocyte osmotic fragility. Erythrocytes exposed with HPE derived from placenta stored for 6 months at 20 С displayed decrease of low-pH resistance. This effect was inherent not only to the whole placental extract, but also to its certain fractions, especially those with molecular weight below 5 kDa. Conclusions: Storage of placental tissue at 196 С seems to be advantageous for preservation of studied properties of placental aqueous-saline extract in comparison to storage at 20 С.