Alternative explanation for the effect of myo-Inositol trispyrophosphate on normal human erythrocytes: 31P NMR studies

Abstract myo -Inositol trispyrophosphate (ITPP) is an allosteric effector of haemoglobin in vitro and has been proposed as a therapeutic agent for diseases associated with tissue hypoxia. Its surmised action on erythrocytes (red blood cells; RBCs) requires transmembrane passage and intracellular binding to haemoglobin; however, direct evidence, of the kind provided by 31 P NMR spectroscopy, for entry of ITPP into RBCs has been absent despite proposed clinical use of the compound. We tested transport by monitoring fresh human RBC suspensions exposed to ITPP using non-invasive 31 P NMR spectroscopy over several hours. No intracellular ITPP was detected within the sensitivity limits of the method (~ 30 µM), and no evidence of significant accumulation was observed. Neither was there evidence of its hydrolysis in haemolysates. We conclude that ITPP does not permeate the normal human RBC membrane in measurable quantities under the conditions examined (and realistic in vivo ones). Consequently, the reported effects of ITPP when added to suspensions of RBCs, on haemoglobin oxygenation are unlikely to result from direct intracellular interaction with haemoglobin and are more plausibly ascribed to indirect effects arising from perturbation of ionic equilibria (Donnan effects) that affect intracellular pH and membrane potential.