Sattler W, Reicher H, Ramos P, Panzenboeck U, Hayn M, Esterbauer H, Malle E, Kostner G M
Department of Medical Biochemistry, University Graz, Austria.
Lipids. 1996 Dec;31(12):1302-10. doi: 10.1007/BF02587917.
A simple, accurate, and fast procedure for quantitative analysis of fatty acids (FA) in simple lipid subclasses from different biological specimens is presented. Lipid extracts of isolated plasma lipoproteins (very low, low, and high density lipoproteins; VLDL, LDL, and HDL, respectively) and permanent J774 mouse macrophages were fractionated into lipid subclasses by thin-layer chromatography (TLC) on silica gel 60 plates. Bands comigrating with authentic lipid standards were scraped off under argon and subjected to direct, in situ transesterification with BF3/MeOH in the presence of the TLC adsorbent. Fatty acid methyl esters were subsequently quantitated by capillary gas chromatography. A comparison of the FA content present in total lipid extracts and in lipid subclasses separated by TLC revealed recoveries ranging from 93 (J774 cell extracts) to 99.7% (LDL). The method described is applicable for the measurement of FA in individual lipid subclasses and was successfully applied to quantitatively analyze the FA composition of the phospholipid, triacylglycerol, and cholesteryl ester fraction derived from VLDL, LDL, and HDL. In J774 lipid extracts, the FA composition of the phospholipid-, monoacylglycerol-, diacylglycerol-, free fatty acid-, triacylglycerol-, and cholesteryl ester fraction was quantitated. In addition we have analyzed the time-dependent loss of the major HDL polyunsaturated fatty acids (18:2, 20:4) in the phospholipid and cholesteryl ester fraction during copper-dependent peroxidation of HDL. We have not encountered analytical problems concerning low FA recoveries from CE-rich lipid extracts as indicated by almost quantitative recoveries of FA in LDL, HDL, and J774 extracts.
本文介绍了一种简单、准确且快速的方法，用于对来自不同生物样本的简单脂质亚类中的脂肪酸（FA）进行定量分析。将分离的血浆脂蛋白（分别为极低密度、低密度和高密度脂蛋白；VLDL、LDL和HDL）以及永久性J774小鼠巨噬细胞的脂质提取物，通过在硅胶60板上进行薄层色谱（TLC），分离为脂质亚类。与真实脂质标准品共迁移的条带在氩气保护下刮下，并在TLC吸附剂存在的情况下，用BF3/MeOH进行直接原位酯交换反应。随后通过毛细管气相色谱法对脂肪酸甲酯进行定量分析。对总脂质提取物和通过TLC分离的脂质亚类中FA含量的比较显示，回收率范围为93%（J774细胞提取物）至99.7%（LDL）。所描述的方法适用于测量单个脂质亚类中的FA，并成功应用于定量分析源自VLDL、LDL和HDL的磷脂、三酰甘油和胆固醇酯组分的FA组成。在J774脂质提取物中，对磷脂、单酰甘油、二酰甘油、游离脂肪酸、三酰甘油和胆固醇酯组分的FA组成进行了定量分析。此外，我们还分析了在HDL的铜依赖性过氧化过程中，磷脂和胆固醇酯组分中主要HDL多不饱和脂肪酸（18:2、20:4）随时间的损失情况。正如LDL、HDL和J774提取物中FA几乎定量的回收率所示，我们未遇到富含CE的脂质提取物中FA回收率低的分析问题。
