The influence of ozone on the domain structure of human erythrocyte membranes: an EPR study
Abstract
Introduction. Autotransfusions of ozonised blood or infusions of gaseous ozone into blood vessels andbody cavities are believed to exert therapeutic effects in some pathological states. Investigations on thereaction of ozone with biological molecules and membrane structures are a subject of crucial importance. The present study aimed to yield more precise data about the alterations, which occur in different erythrocytemembrane regions subjected to medical ozone. This could provide some additional informationabout the structural changes in the membrane at a molecular level.
Material and methods. Blood was obtained from 22 healthy volunteers (aged 21 to 63) by vein puncture andmixed with 1/10 volume of 0.13 M trisodium citrate. Erythrocytes were isolated from fresh blood by centrifugationat 4°C, at 1,500 × g and purified by three cycles of resuspension and washing with PBS. Ozone was generatedby passing pure gaseous oxygen at 30 l/h through an apparatus producing silent electric discharges. EPRspectra were obtained at X-band (9.4 GHz), at modulation frequency of 100 kHz. The scan time was 4 min, and the time constant 0.3 s. Since biological membranes are heterogeneous systems composed of severalcoexisting domains, EPR spectra are superimpositions of several spectra with different fluidity parameters.
Results. The effects of ozone at two concentrations (10 and 45 g/m3) on fluidity and phospholipid domainstructure of erythrocyte membranes were investigated by electron paramagnetic resonance (EPR). Atincreased ozone concentration (45 g/m3), the portion of the least ordered domains (WLO) increased, witha corresponding decrease of ordered (WMO, WO), more rigid regions. The order of lipid acyl chains of twoordered (MO and O) as well as the least ordered (LO) domains diminished, as expressed by smaller orderparameters. For ozone concentration of 10 g/m3 values of order parameter were slightly increased, whichindicates a tendency to rigidisation of lipid bilayer at this ozone concentration. This change was, however,statistically insignificant. There were no statistically significant differences in the thermotropic behaviour ofweight factor of ordered domains (WMO, WO) between ozonised and control red cells.
Conclusion. The obtained data shows that ozonation of erythrocytes results in cell membrane fluidisationand an increase in red cell deformability. On the other hand, these results could suggest that ozonationof erythrocytes leads to structural changes in the membranes, especially in cytoskeletal proteins, but thiseffect is probably dose-dependent.