Injuries Of Human Erythrocytes By Sevoflurane In Vitro

Objective Some studies has demonstrated that the cell number, size and functions ofred blood cells are independent risk factor for cardiovascular disease. Patients redblood cell count decreased after administered with sevoflurane in operations, and wespeculate that is because of the damage to red blood cells derived from the inhalationanesthetic—sevoflurane. There will be two typical kinds of pathologicchanges—eryptosis and necrosis after red blood cells were impaired. Eryptosis redblood cells can be recognized and phagocytized by macrophages, which result inshortened life span, or even anemia. Hemolyze and necrosis of red blood cells willrelease free hemoglobin, which can eliminate nitric oxide （NO） then impair bloodvessel system function, harmfulness for all organs. It is known that red blood cellscontain endothelial nitric oxide synthase （eNOS）, which are the main source of NO,and catalyzes the transformation between arginine and citrulline. The structure andfunction of red blood cells can be impaired easily by endo-and exogenous oxygencontent changes, which is always the ideal and reliable model for free radical oxidativedamage. In this study, we use this experiment model to evaluate the influence andmechanism of sevofluran to red blood cells from erythrocytes anti-oxidation ability,NO metabolism and cells eryptosis.Method: Part1: Influence of different concentration sevoflurane on people red bloodcells hematocytolysis ratio and antioxidase. Blood samples from healthy volunteerswere prepared to2%red cell suspension （RCS）, and the suspension contained sodiumchloride145mmol/l, potassium chloride4mmol/l, calcium chloride2mmol/l, glucose 5mmol/l, and the pH was7.4. RCS samples were divided to8groups （n=4for each）:air group （A）,1%sevoflurane group （S1）,3%sevoflurane group （S3）,5%sevofluranegroup （S5）, air plus H₂O₂group （A+H）,1%sevoflurane plus H₂O₂group （S1+H）,3%sevoflurane plus H₂O₂group （S3+H）,5%sevoflurane plus H₂O₂group （S5+H）.Administered with sevoflurane for30minutes according to the groups settings, thenincubated15hours. After that, medicated200μmol/L hydrogen dioxide to A+H,S1+H, S3+H and S5+H groups, as control, Ringer’s solution was used in other groups.All samples were incubated for another3hours to response. Red blood cellshematocytolysis ratio was detected by elisa reader, while catalase （CAT）,malonaldehyde （MDA）, glutathione （GSH）, glutathione peroxidase （GSH-PX） contentwere determined by Beijing Huaying Biotechnology Research Company. Part2:Change of red blood cells NO, NO₂^-, NO₃^-, iNOS and eNOS content after differentconcentration sevoflurane administration. The group settings and administrationstrategies were the same as Part1. Red blood cells NO, NO₂^-, NO₃^-, iNOS and eNOScontent were detected by Griess method, also by Beijing Huaying BiotechnologyResearch Company. Part3: Human red blood cells eryptosis induced by differentconcentration sevoflurane administration. Besides8groups settings the same as Part1,the study added other8groups, including air plus high glucose group （A+G）,1%sevoflurane plus high glucose group （S1+G）,3%sevoflurane plus high glucose group（S3+G）,5%sevoflurane plus high glucose group （S5+G）, air plus CaCl₂（A+C）,1%sevoflurane plus CaCl₂group （S1+C）,3%sevoflurane plus CaCl₂group （S3+C）,5%sevoflurane plus CaCl₂group （S5+C）. After administered with sevoflurane for30minutes, samples were incubated in37℃thermostat incubator for5hours,50rpm.Then added200μmol/L hydrogen dioxide,600mmol/L glucose solution,5mmol/Lcalcium chloride, incubated for another3hours. Using flow cytometry to determineAnnexin Ⅴ fluorescence labeled red blood cells PS labeling rate and forward scatter（FSC）. Result: Part1: Hematocytolysis ratio: S3+H （17.384±1.976）> A+H（10.848±0.274）group, P=0.007; S3+H> S3（12.417±0.716）, P=0.027. CAT content: A （17.301±4.222）> S3（5.431±0.531）group， P=0.009；A> S5（5.175±0.658）， P=0.009； S1+H（4.319±0.235）, S3+H （8.257±1.389）, S5+H （9.156±0.742）all significantly lower thanA+H （13.689±2.003） group,（P<0.05）. MDA content: S1+H （3.547±0.898）> A+H（2.654±0.509） group，P=0.031； S3+H （4.474±0.653）> A+H group，P=0.021. GSHcontent: S3（6.266±0.254）> S3+H （6.097±0.322） group, P=0.018; A （7.335±0.106）>S3group, P=0.023. there was no significant differences in GSH-PX groups. Part2:NO content: A （3.199±0.208）<A+H （3.622±0.262） group, P=0.001; S1（3.371±0.236）> S1+H （2.801±0.12） group, P=0.003; S3（2.808±0.244）<S3+H （3.372±0.261） group,P<0.001; S5（3.172±0.22）>S5+H（3.015±0.308）, P=0.039; A+H> SI+H, P=0.002. NO₂^-:There was no significant differences between A （16.731±0.434） and A+H（17.334±0.969）group, S1（16.778±0.263） and S1+H （17.709±1.547）group, S5（16.539±0.411） and S5+H （16.397±0.35）group; but S3（16.861±0.315）<S3+H（17.173±0.294）group， P=0.016. NO₃^-content: A （49.837±6.09）<A+H（65.000±4.173）group, P=0.002; S1（55.128±3.676）<S1+H （64.736±2.326）group,P=0.002; S5（47.233±4.886）<S5+H （57.526±2.148） group, P=0.005; There was nosignificant differences between S3（55.779±11.342） and S3+H （58.161±2.36）group,P=0.642. eNOS content: A （1.403±0.103）<A+H （1.62±0.117）group, P<0.001; S1（1.503±0.107）<S1+H （1.23±0.075）group, P=0.002; S3（1.256±0.112）> S3+H（1.508±0.122）group, P<0.001; S5（1.414±0.097）> S5+H （1.349±0.133）group,P=0.037; A+H> S1+H, P=0.002. iNOS content: A （2.818±0.239）<A+H（3.215±0.224）group， P=0.001; S1（3.002±0.208）> S1+H （2.495±0.106）group,P=0.003; S3（2.495±0.209）<S3+H （2.984±0.216） group, P<0.001; S5（2.818±0.203）> S5+H （2.659±0.284）group, P=0.043; A+H> S1+H group, P=0.002. Part3: PSlabeling rate: A （1.28±0.62）<S3（2.803±0.516） group，A <S5（3.1775±0.474）group， S1（1.5925±0.167）<S3group，S1<S5group，P<0.05；A+H （1.47±0.337）<S3+H（3.1325±0.223）group，A+H <S5+H （3.605±0.622）group，S1+H （2.225±0.357）<S3+Hgroup， S1+H <S5+H group， P<0.05. FSC: A （1239.27±59.701）> S3（1065.3125±14.607）group，A> S5（1060.385±26.493）group，S1（1244.3±88.409）> S3group，S1> S5group，S3> S5group, P<0.05； A+H （1185.868±51.738）> S5+H（1059.733±12.06） group，P=0.025.Conclusion:1. Sevoflurane could decrease human red blood cells anti-oxidation ability bydecreasing resistance to active oxygen （H₂O₂）, increasing hematocytolysis ratio andMDA content. The mechanism may be the decreased red blood cells CAT activityand GSH content.2. Sevoflurane could inhibit red blood cells NO production by decreasing NO contentand the cells tolerance to H₂O₂stress injury. The reason may because the eNOS andiNOS activity were suppressed.3. There exists activity iNOS and eNOS in red blood cells,and the content of iNOS ishigher than that of eNOS. In normal oxygen content circumstances, NO mainlycomes from the two kinds of enzyme’s activity.4. High concentration sevoflurane could induce human red blood cells eryptosis, itshowed that exposed PS, reduced cells size, and further, impaired red blood cellsresistance to harmful environment factors.5. It exists big gap between in vivo and in vitro environment, these in vitro findingsneed further evaluation in clinical patients to know the potential hazards ofsevoflurane.