The effects of lipophilic substances on the shape of erythrocytes demonstrated by a new in vitro-method

Abstract

Low aqueous solubility of lipophilic agents, such as free fatty acids, hampers proper in vitro demonstration of biological effects, yielding an ambiguous in vitro–in vivo correlation. We have therefore developed a method for evaluating the acute effects of lipophilic substances on the shape of erythrocytes and estimated EC 50 and Hill coefficient according to the sigmoidal E max model. The test substance dissolved in medium-chain triglyceride is coated on a polycarbonate slide which serves as a cover sheet of a Bürker chamber. Freshly collected finger-tip blood is diluted with autologous EDTA–plasma and introduced into the chamber. After 10 min at 37 °C, the cells are photographed under microscope and the fractions of normal and defect cells are evaluated. No staining is needed and the cells are kept viable during the test period. With increasing chain length, fatty acids, aliphatic amines and alcohols all increased the fraction of defect erythrocytes in a concentration-dependent manner. The results indicate that several fatty acids are very potent in their acute actions on erythrocytes, and that this effect is due to chain length rather than conformation. Conclusion The technique offers a screening method for testing the harmful effects of small amounts of lipophilic substances on erythrocytes.