Proton NMR study of water permeability of hepatocyte membrane

It is known that Nuclear Magnetic Resonance (NMR) relaxation methods may provide useful information regarding the permeability of the cell membrane. In the case of erythrocyte membrane, the NMR relaxation methods put in evidence the permeability changes due to different diseases or to chronic internal irradiation. The aim of this work was to investigate the water transport across the normal and oncological stressed hepatocyte membrane and to evaluate in which extend the T2 - NMR may reveal permeability changes due to the oncological factor. Isolated hepatocytes were obtained from Wistar male rat liver using a two phase perfusion technique with collagenase. The oncogenic stress was induced by acute administration of 2 - acetaminofluorene (2-AAF), 20 mg per body weight, twice in 48 hours before liver extraction. The cell viability after isolation was assessed by trypan blue exclusion. The NMR measurements were carried out on hepatocytes suspension doped with a relaxation agent (8 mM Mn Cl2). The transversal relaxation functions were determined for temperatures within the range 7 - 40 deg. C, using the standard CPMG pulse sequence. For all investigated temperatures the relaxation curves have a biexponential behavior corresponding to the water exchange between inner (A) and outer (B) cell compartments. The apparent relaxation times, T2A, T2B were obtained from experimental data via Singular Value Decomposition fitting and were used to determine experimentally the mean lifetime value of water molecules inside the liver cells. Since T may be directly related to the permeability of the membrane by P = (V/A)/T, it results that the oncogenic stress induces an increase of membrane water permeability accompanied by the change of activation energy. Therefore one may conclude that the acute administration of 2 - AAF induces conformation changes of the membrane structure which significantly affects the water transport process across the membrane