Density Gradient Separation of Inositol Hexaphosphate Loaded Red Blood Cells in Various Preparation Conditions

Abstract

A process for large scale incorporation of drugs into red blood cells (RBC) has been developed by Ropars et al^l, using reversed osmotic lysis inducing transient holes in the RBC membrane. Internalization of inositol hexaphosphate (IHP) in RBC induces a decrease of haemoglobin oxygen affinity, leading to rightward shift of the dissociation curve. This decrease of affinity improves the oxygen extraction and is potentially important for therapeutic applications such as ischemic pathologies. The separation of RBC and IHP-RBC into classes of progressively increasing density has been obtained on discontinuous percoll-albumin buffered density gradients. Eight layers of erythrocytes have been isolated, washed and analysed for RBC numerations, mean cell volume (MCV), mean cell haemoglobin (MCH), and IHP concentration. The modification of RBC profiles according to their P50 reflected the effects of reversed osmotic lysis and the distribution of IHP concentration. The degree of modification of IHP-RBC fractionation on gradient could be an indication of the in vivo survival.