Osmotic tolerance of avian erythrocytes to complete hemolysis in solute free water

Osmotic behavior of erythrocytes is not only important clinically, but is also significant in understanding of material transport across biological membranes. It is most commonly studied through fragiligrams - plots of the degree of hemolysis as a function of extracellular osmolarity. A fundamental assumption in experimental and theoretical studies on osmolarity driven transport of water across the plasma membranes of all cells is the sigmoidal nature of their osmotic behavior. Sigmoidal data is mathematically monotonic showing either a decreasing only or an increasing only trend, but not both, within certain thresholds; beyond these thresholds the data is asymptotic or flat. Fragiligrams of erythrocytes are usually sigmoidal, with maximal hemolysis in plain solute-free water and often up to a certain extracellular hypotonic environment. In this work, we report a new discovery of non-monotonic osmotic behavior of avian erythrocytes. In contrast to the expected monotonic fragiligrams obtained for mammalian erythrocytes, fragiligrams of avian erythrocytes show non-monotonic curves. Maximal hemolysis of avian erythrocytes was not observed at the most hypotonic conditions - instead, maximal hemolysis was observed at mild hypotonic conditions. Hemolysis of avian erythrocytes first increases then decreases with increasing extracellular osmolarity. We also report that the non-monotonic fragiligrams of chicken erythrocytes are converted to the expected monotonic sigmoids subsequent to controlled extracellular trypsinization. While possibly having profound evolutionary implications for vertebrates, the findings reported in this work have a direct impact on understanding of avian physiology. Our results also compel revisiting of experimental and theoretical models for understanding material transport across biological membranes under different osmotic conditions.