Biochemistry & Pharmacology: Open Access
Open Access

Abstract

Benzoic Acid and its Derivatives Increase Membrane Resistance to Osmotic Pressure in Isolated Sheep Erythrocytes

Hitoshi Mineo, Chinami Matsuda, Yukiko Suzuki, Saeri Takahashi, Akina Chiba and Rikako Kasai

There have been many reports that osmotic fragility (OF) in red blood cells (RBCs) is a valuable tool for assessing the actions of various chemicals on the cell membrane in vitro. We determined the effects of benzoic acid and its derivatives on OF in sheep RBCs in vitro. Isolated sheep RBCs were exposed to these substances at 0-100 mM in a buffer solution for 1 h, and the 50% hemolysis was then determined by soaking in 0.1-0.8% NaCl solution. OF was determined as the NaCl concentration inducing 50% hemolysis which was colorimetrically measured by the released hemoglobin concentration. Benzoic acid decreased OF in a dose-dependent manner. Heptanoic acid and cyclohexane carboxylic acid, both of which have 6 carbons in their saturated hydrocarbon structure, did not change OF. Replacement of the COOH bound to the benzene ring with PO(OH)2 or SO2OH abolished the OF response obtained by benzoic acid. Replacement with OH did not affect OF up to 25 mM, but did induce hemolysis at 50 and 100 mM. Replacement with CONH2 decreased OF, with the degree of the OF-lowering effect being greater than that of benzoic acid. Most derivatives possessing other groups (OH, CH3 or NH3) or halogens (Cl or Br) decreased or tended to decreased OF, with the degree of change in OF dependent on the position of the group introduced onto the benzene ring. 4-methylbenzoic acid, and 3-,4- and 4-, 5-dichlorobenzoic acids demonstrated a biphasic effect on OF in sheep RBCs; lowering OF up to 50 mM followed by a lytic effect at 100 mM. The results of a regression analysis using the values of all substances tested revealed no significant correlations between the partition coefficient of the substances and their effects on OF response. However, some groups of substances at concentrations of 10-50 mM showed a negative and statistically significant correlation. For substances sharing very close chemical structures, partition coefficients can probably be used as an indicator for evaluating the effect on OF within an appropriate range of concentrations.