高效液相色谱法研究年龄和缺血对大鼠心肌线粒体心磷脂含量的影响

Effects of age and ischemia on cardiolipin measured by silicic acid high-performance liquid chromatography in isolated rat hearts

目的 建立大鼠心肌线粒体膜心磷脂（CL）定量分析的方法；同时利用该法研究年龄和缺血对大鼠心肌线粒体膜CL含量的影响。方法 不同年龄（4、12、24mon）♂SD大鼠各24只，同一年龄大鼠随机分为3组（n=8）。对照组于Langendorff模型上用Krebs-Henseleit缓冲液（KH液）灌注70min；缺血组于Langendorff模型利用KH液平衡灌注20min，然后停止灌注30min或40min，最后再灌注20min。分别提取心肌细胞肌纤维膜下线粒体（subsarcolemmal mitochondfia,SSM）和肌纤维间线粒体（interfibfillar mitochondria，IFM）脂质。高效液相色谱法（High-performance liquid chromatography，HPLC）采用正相色谱柱梯度洗脱对CL进行分离和定量分析。结果 在所建立的方法学下，CL的保留时间为13．092min；标准曲线为P（μV·s）=20877455X（mmol·L^-1）-352028（r=0．9993），在0．2—3．2mmol·L^-1浓度范围内呈良好的线性关系，最低检测浓度为0．005mmol·L^-1（S／N=3）；在0．2、0．8、3．2mmol·L^-1低、中、高3个浓度下的绝对回收率为0．8505—0．9519，相对回收率为0．9898—1．0429；日内及日间RSD均小于0．11（n=5）。24mon未缺血大鼠心肌SSM膜上CL含量比4mon大鼠低（P〈0．05），也比12mon大鼠低（P〈0．05）；但3种年龄未缺血大鼠IFM膜上CL含量差异无显著性。3种年龄大鼠缺血30min组和缺血40min组心肌SSM膜上CL含量比未缺血组低（P〈0．01），而缺血30min组和缺血40min组间CL含量差异无显著性。3种年龄大鼠正常组、缺血30min组和缺血40min组IFM膜上CL含量之间差异均无显著性。结论 正相梯度洗脱可满足对大鼠心肌线粒体心磷脂的分离和定量的要求。大鼠心肌SSM膜上CL对老龄和缺血较敏感，而老龄和缺血对IFM膜上的CL无明显影响。

Aim To establish a method to quantitatively determine cardiolipin content and investigate effects of age and ischemia on cardiolipin in isolated rat hearts. Methods Male SD rats, 4 months old, 12 months old and 24 months old were used throughout the experiment. Each had 24 ones, which were randomly distributed to control, ischemia 30 min and ischemia 40 rain groups（ n = 8 ）. Control group hearts were perfused for a total of 70 min and ischemia groups hearts were perfused for a 20 min equilibration period, and then underwent 30 or 40 min of no-flow ischemia followed by reperfusion for a 20 min period, respectively. Cardiolipin was quantitatively determined by high-performance liquid chromatography following extraction of lipids from subsarcolemmal mitochondria （SSM） and interfibrillar mitochondria（IFM）. Results Under the condition of HPLC, the retention time of cardiolipin was 13. 092 min. The standard curve was Y （μV· s） = 20 877 455X （ mmol · L^-1 ） - 352 028 （ r = 0. 9993 ） with a validated quantitation range of 0. 2 - 3.2 mmol· L^-1, and the lowest concentration of detection was 0. 005 mmol · L^-1 （ S/N = 3 ）. At the concentration of 0. 2,0. 8, 3.2 mmol· L^-1, absolute recovery rates ranged from 0. 8505 to 0. 9519 and relative recovery rates ranged from 0. 9898 to 1. 0429. The RSD of the precision within-day and between-day was less than 0. 11. Cardiolipin content in SSM of nonischemic group of 24 months old rats was lower than that of both 4 months old and 12 months old, but no differences among them were observed in IFM. In all age rats, compared with control group, cardiolipin contents of SSM from ischemia 30 rain and ischemia 40 min groups were lower, and effects of ischemia 40 min are not pronounced; there were no differences in CL contents of IFM among three groups. Conclusions The assay method was shown to be sensitive, suitable and accurate for determination of cardiolipin located in mitochondria from perfused hearts of rats. Myocardial ischemia and older age decreased cardiolipin content in SSM, and had no remarkable effects on cardiolipin in IFM.