组织脂质抽提方法评估与应用

Evaluation and application of lipid extraction methods from tissues

目的比较3种常用的组织三酰甘油(TG)、总胆固醇(TC)抽提方法。方法脂质提取分别采用3种方法。A:氯仿-甲醇抽提,B:庚烷-异丙醇-聚山梨酯20抽提,C:叔醇-Triton X-100-甲基乙醇抽提。采用氧化酶法检测混合人血清、普食或高脂饲养大鼠肝组织TG和TC水平,自动生化仪或手工比色测定。结果在3种方法中,A法较其他方法耗时长;C法需手工测定。尽管3种方法的批内和批间差异无明显不同,方法A、B提取的TG、TC回收率随抽提溶液体积增大而增加,而C法回收率随抽提液体积增大而呈下降趋势。采用B法作组织抽提,普食大鼠肝脏中TG、TC的抽提量不随抽提液体积的改变而变化,高脂大鼠肝脏TG、TC含量随抽提液体积增加而升高。进一步选用5 mL抽提液提取普食和高脂大鼠肝脏,高脂大鼠TG、TC含量均明显高于普食组(P<0.01)。结论 3种脂质抽提方法中,B法回收率高,自动化程度高、省时,适合肥胖大鼠肝脏脂质的抽提。用B法提取50 mg高脂肝脏脂质时,抽提溶液体积以5或7 mL为宜。

AIM To compare three common lipid extraction methods for triglycerides （TG） and total cholesterol （TC）. METHODS Lipids were extracted using the following 3 methods： method A （chloroformmethanol extraction）, method B （heptane-isopropanol-polysorbate extraction） and method C （tert-butyl alcohol- Triton X-100-ymethyl alcohol extraction）. The concentrations of TG and TC were determined using enzymatic commercial kits by an automatic biochemical analyzer （method A and B） or manual operation （method C）. RESULTS Among three methods, method A was the most time-consuming one and method C was the least automated one due to its manual operation. Recoveries of method A and B were both dose-dependently associated with volume of extraction solution, while a negative relationship existed between recovery and extraction liquid volume in method C. There were no significant differences in within-run CV and day-to-day CV among the three methods. When method B was used for lipid extraction, we found TG and TC content in liver from high fat diet fed rats were increased with the extraction volume, whereas those in liver from chow fed rats were not. More TG and TC were found in liver from high fat diet fed rats than those in liver from chow fed rats （P 〈 0.01 ）. CONCLUSION Method B has the characteristics of higher precision, higher degree of automatization and less time-consuming, and is suitable for lipid extraction in livers from obese rats. A volume of 5 or 7 mL extraction solution is suitable for as much as 50 mg liver tissue.