等度洗脱HPLC分析冻存与新鲜心肌线粒体心磷脂含量的比较  

Isocratic elution system for the determination of cardiolipin in frozen mitochondria using HPLC

作　　者：魏稼黎 段忠心[2] 喻田[3] 李李[1] 刘镭[1] 

机构地区：[1]中南大学湘雅二医院麻醉科,湖南长沙410011 [2]南华大学附属第二医院麻醉科,湖南衡阳421000 [3]遵义医学院附属医院麻醉科,贵州遵义563000

出　　处：《贵州医药》2015年第12期1065-1067,共3页Guizhou Medical Journal

摘　　要：目的建立稳定、快速检测心磷脂(cardiolipin,CL)的等度洗脱(HPLC)检测法,并检测线粒体冻存前后CL含量变化。方法雄性SD大鼠心脏30个,分成C、I、IR 3组,每组10个。C组于Langendorff装置灌注80min;I组灌注20min后,缺血30min;IR组灌注20min,缺血30min,再灌注30min。提取其线粒体,均分成2份,1份即可提取总磷脂,1份冻存3个月后提取总磷脂。采用等度洗脱正相HPLC分析心磷脂。结果 CL保留时间为为9.697min,最小检测限为0.156 25μg(S/N≥3),线性范围0.31~40μg,回归方程为CL(μg)=0.003 582×峰面积-0.728 0(r=0.988)。线粒体CL含量缺血后显著减少,但再灌注未进一步加重CL丢失。冻存线粒体CL含量无明显变化。结论正相等度洗脱分析大鼠心肌线粒体CL快速、有效,线粒体冻存不影响CL检测。Objective To establish a stable and fast detection of CL by HPLC isocratic elution and detect the change of mitochondrial Cardiolipin before and after cryopreserved.Method Thirty hearts of male SD rats were randomly divided three groups.Each group contained 10 hearts.The control group was subjected to perfusion for 80 min in Langendorff device.Ischemia group was subjected to perfusion for 20 min and 30 min of no-flow ischemia.Ischemia-reperfusion group was subjected to perfusion for 20 min and 30 min of no-flow ischemia followed by reperfusion for a 30 min period.After this,Mitochondria were extracted and divided into 2aliquots.Total phosphatide of the first aliquot was extracted right now and another extracted after three months＇ cryopreservation.Cardioplipin was qualitatively and quantitatively analyzed by NP-HPLC isocratic elution.Result Under the condion of HPLC,the retention time of cardiolipin was 9.697min;the lowest quality of detection was 0.15625ug（S/N≥3）.The regression curve was Cardiolipin（ug）=0.003582×Peak Area-0.7280（r=0.988）with a validated quantitation range of 0.31ug^40ug.Myocardial ischemia injury can significantly reduce cardiolipin content,while reperfusion injury won＇t cause more lose of cardiolipin.Cryopreserved mitochondrial cardiolipin content had no obvious change.Conclusion NPHPLC isocratic elution was shown to be sensitive,suitable and accurate for determination of cardiolipin located in mitochondria from perfused hearts of rats.Mitochondrial cryopreservation will not affect the detection of cardiolipin.

关 键 词：心磷脂 高效液相色谱 等度洗脱 线粒体 

分 类 号：R33[医药卫生—人体生理学] R313[医药卫生—基础医学]