Metabolic dependence of the critical hemolytic volume of human erythrocytes: relationship to osmotic fragility and autohemolysis in hereditary spherocytosis and normal red cells.

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Abstract
This investigation was undertaken to relate in vitro autohemo-lysis and osmotic fragility to previous observations. In vitro loss of membrane lipid from hereditary spherocytes [HS] is much greater than from normal red cells. Blood from 5 splenectomized patients with HS and 10 normals was incubated in-vitro with or without glucose, as well as with or without ouabain for 12-48 hr. Observations were made on cell volume in plasma, cation content, and critical hemolytic volume (CHV) of the cells, determined with a Model-B Coulter Counter. Metabolically depleted HC cells over 24 hr. and normal cells at 36-48 hr. undergo the disc-sphere transformation with a decrease in critical hemolytic volume. These changes were associated with fragmentation loss of pieces of membrane illustrated by phase and electron micrographs. The addition of glucose protected against this decrease in CHV and loss of membrane lipid whether ouabain was present or not. Thus, although ouabain produced Na+ accumulation, glucose enabled the cells to maintain their CHV above the volume resulting from the increased cation content. Metabolically depleted HS cells did not show increased cation content at 24 hr. These studies indicated: the existence of a ouabain-insensitive, energy-dependent mechanism for the maintenance of membrane in-tegrity. Osmotic fragility is an expression of the relationship be-tween osmotically-active cell contents and the surface area of the cell. The CHV is a reflection of the surface area of the cell which is decreased by loss of membrane material. The in vitro hemolysis and osmotic fragility of substrate-depleted HS cells are related to a decrease in critical hemolytic volume sufficient to produce lysis at the tonicity of plasma.