Internalization and distribution of inositol hexakisphosphate in red blood cells

Inositol hexakisphosphate (InsP6), an allosteric effector of haemoglobin, is able to modify the oxyhaemoglobin dissociation curve. The rightwards shift of the curve increases the in vivo oxygen delivery to tissues. Such an exogenous substance may be internalized into red blood cells (RBC) using a reversed lysis-resealing process following a hypoosmotic shock, resulting in InsP6-RBC with modified oxygen transport capacity. The efficacy of the process depends on various physicochemical parameters which can be fixed during the experimental protocol. The variability of InsP6 internalization from one sample to another appeared to be mainly due to the natural variation in osmotic fragility of RBC. This factor was also modified during the storage of RBC units before the lysis-resealing process. The separation of InsP6-RBC on a density gradient revealed a wide heterogeneity of internalized InsP6 concentration, varying with the degree of osmotic shock. The control of these various parameters will result in resealed InsP6-RBC in reproducible conditions suitable for in vivo use.