Preliminary Study On The Effects Of Glutathione To The Antioxidant Properties Of PolyCHb

Background:Hemoglobin oxygen carriers(HBOCs)are of great value in many application scenarios,such as pre-hospital emergency treatment,blood incompatibility,and sio forth.HBOCs products showed a better therapeutic effect at early time in clinical trials,but there were also many toxic and side effects produced by them.However the oxidation of hemoglobin is one of the main reasons for that.There are two main ways to increase the antioxidant capacity of hemoglobin,one is to crosslink the big molecules such as antioxidant enzymes,the other is to add small molecule antioxidant substances such as ascorbic acid(AA),glutathione,mannitol(GSH),selenium,etc.Tripeptide GSH is one of the body's most important endogenous antioxidants and can remove the free radicals,protect protein sulfhydryl from oxygenization.In this article,we intend to add glutathione as a antioxidant and to research its influence to auto-oxidation of polymerized human cord hemoglobin(PolyCHb)to provide reference and theoretical support for the glutathione as an antioxidant protective agent for HBOCs.Objective:To have a Preliminary study on the effects of GSH to the antioxidant properties of PolyCHb in vitro and the intervention influence on the oxidative stress induced by PolyCHb in vivo.Methods:GSH and AA were added to the PolyCHb solution respectively to detect the concentrations of methemoglobin(MetHb)at 4?and 25?.The secondary structure of protein,the molecular weight distribution and the function of PolyCHb before and after adding GSH,AA were analyzed through circular dichroism(CD)spectra,high performance liquid chromatography(HPLC)and oxygen dissociation curve respectively.For the in vivo experiment,rats were randomly divided into sham operation group,saline injection group,(0.30 g/kg,0.61 g/kg,0.91 g/kg)PolyCHb group,0.91 g/kg PolyCHb +(75 mg/kg,150 mg/kg,225 mg/kg)GSH group,0.91 g/kg PolyCHb +5 mg/kg AA group.MAP and BPM were measured and the femoral artery blood was taken at 1 hour after intravenous infusion to analyze the arterial blood gas and detect several indexes of oxidative stress such as superoxide dismutase(SOD)activity,reduced glutathione(GSH)concentration,total antioxidant capacity(T-AOC),reactive oxygen species(ROS)concentration,malondialdehyde(MDA)concentration and 8-hydroxy deoxyguanosine glycosides(8-OHDG)concentration in plasma.Results:In 72 hours of in vitro conservation experiments,after adding GSH into PolyCHb,the MetHb(%)of solution at 4? and 25? were 24.45%±0.83%,47.32%±1.58%respectively which was significantly lower than the control group(P<0.01).Before and after adding GSH,there was no obvious effect on the molecular weight distribution and the oxygen carrying capacity of PolyCHb,but it caused the protein helix,angle increasing and fold decreasing.For infusion of different doses of PolyCHb groups,MPA increased but BPM and arterial blood gas did not change significantly,OD activity,GSH concentration,T-AOC,ROS concentration,MDA concentration,8-OHDG concentration in plasma significant increased(P<0.01).For adding different concentrations of GSH with PolyCHb groups,MPA,BPM,arterial blood gas,SOD activity,T-AOC,ROS concentration,MDA concentration,8-OHDG concentration had no obvious change(P>0.05),but the concentration of GSH in plasma increased.Conclusions:GSH can effectively inhibit the spontaneous oxidation of PolyCHb in vitro;PolyCHb induced oxidative stress in rats after infusion,and GSH had no significant effect on the oxidative stress induced by PolyCHb.