| OBJECTIVE: to determine the effectiveness of each concentration ( 1.0μmol/L, 5.0μmol/L, 10.0μmol/L ) of methylene blue that are subjected to irradiation intensity of 40000LUX within0, 20, 40, and 60 minutes on the inactivation of Hepatitis B virus in blood plasma using FQ-PCR assay; to observe the inactivation effects and choose the proper concentration, to determine the effects on compositions of plasma. to evaluate the effect of 5.0μmol/L methylene blue concentration that is subjected to irradiation intensity of 40000LUX within 0, 20, 40and 60 minutes on the erythrocyte suspension using photochemical treatment method; to observe the effects on structure and biological function of erythrocyte treated with 5.0μmol/L methylene blue concentration that is subjected to irradiation intensity of 40000LUX within 0, 20, 40and 60 minutes, and; to determine the feasibility of applying methylene Blue to RBC suspension.METHODS: Methylene blue(MB) concentrations of 1.0μmol/L, 5.0μmol/L, 10.0μmol/L were added to Hepatitis B Virus (HBV) DNA-positive human blood plasma. Each concentration solutions was then subjected to irradiation intensity of 40000LUX within0,20, 40 and 60 minutes. DNA concentration of each concentration solutions that was subjected to irradiation intensity of 40000LUX within 0 , 20, 40, and 60 minutes was then determined by extracting genome DNA using FQ-PCR assay. The effects of the different concentration of MB and length of time in subjecting to light on the HBV DNA-positive human blood plasma were evaluated. The variations on the plasma fraction, the biochemical indicator, the enzymology indicator, and the coagulation factor FⅧ:C activity were determined. The variations of sub-unit were observed using SDS-PAGE while the variations of human blood plasma antibodies were also observed using western-blot technique.Methylene blue concentration of 5.0μmol/L was also added to erythrocyte suspension and was subjected to 40000LUX illuminance within 0, 20, 40, and 60 minutes. Erythrocyte membranes were then extracted to detect Na+-K+ ATP activity, Acetylcholinesterase (AchE) activity and to observe the variations of erythrocytes'shape and permeability.RESULTS: The experiment showed that the most effective concentration of MB and length of irradiation time was 10.0μmol/L and 60 minutes, respectively. The result showed that methylene blue cannot inactivate high copy viruses completely but it can lower the viruses' concentration. As shown in the inactivation table, the irradiation time and concentration are positively correlated to the inactivation effect. There was no notable change on the plasma principle constitution using biochemical indicator when irradiated for 60 minutes. Also, there was nearly no influence to blood coagulation factor FⅧ:C activity,but some enzymes decreased notably (P<0.05). The subunit content and moving speed had no conspicuous change, while, antibodies had normal biological activity. On the other hand, Methylene blue caused serious damage to erythrocyte membrane as suggested by the result of photochemical treatment made on erythrocyte suspension. As the irradiation time was prolonged, the structure of erythrocyte showed irregularities and accretions, as well as the increase of lesions. Hematocytolysis degree increased notably (P<0.05), nearly approaching linear. Na+-K+ ATP enzyme didnot slow down notably (P>0.05) as the irradiation time was prolonged. AchE decreased significantly by 65% (P<0.05) after 40 minutes of irradiation time. Osmotic fragility dramatically increased after irradiating for 20 minutes (P<0.05), then significantly increased with longer irradiation time. The primary mechanism to membrane damage may due to production of singlet oxygen and free radicals.CONCLUSION: The HBV concentration in human blood plasma decreased when treated with 10μmol/L methylene blue and visible light irradiating for 60 min, but could not be completely inactivated. Slight changes to the activities of blood plasma were observed , but overall activities remained normal. The damages to erythrocyte membranes induced by MB photochemical method were mainly induced by the production of singlet oxygen and free radicals.
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