Kramer J K, Blais L, Fouchard R C, Melnyk R A, Kallury K M
Center for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada.
Lipids. 1997 Mar;32(3):323-30. doi: 10.1007/s11745-997-0040-1.
This paper describes a simple method for the analysis of tocopherols in tissues by which frozen tissues-70 degrees C were pulverized at dry ice temperatures (-70 degrees C) and immediately extracted with hexane. There was no need to remove the coeluting lipids from tissues by saponification, since at that level of neutral lipids in the sample, there was no reduction in fluorescence response. For the analysis of oil, in which large amounts of neutral lipids were coextracted, a 20% reduction of fluorescence response was observed, but the response was equal for all tocopherol forms, and was appropriately corrected. Saponification was used only when tocopherol esters were present, and only after an initial hexane extraction to remove the free tocopherols in order to avoid their loss by saponification, particularly non alpha-tocopherol and tocotrienols. All the tocopherols and tocotrienols were separated on a normal-phase diol (epoxide) column that gave consistent and reproducible results, without the disadvantages of nonreproducibility with silica columns, or the lack of separation with reversed-phase columns. The tocopherols were quantitated by using a tocopherol form not present in the sample as an internal tocopherol standard, or using an external tocopherol standard if all forms were present, or when the sample was saponified. Piglet heart and liver samples showed the presence of mainly alpha-tocopherol, with minor amounts of beta- and gamma-tocopherol and alpha-tocotrienol, but no delta-tocopherol. Only small amounts of tocopherol esters were present in the liver but not in the heart.
本文描述了一种分析组织中生育酚的简单方法，即将冷冻至-70℃的组织在干冰温度（-70℃）下研磨，然后立即用己烷提取。由于样品中中性脂质处于该水平时荧光响应没有降低，因此无需通过皂化从组织中去除共洗脱的脂质。对于共提取大量中性脂质的油样分析，观察到荧光响应降低了20%，但所有生育酚形式的响应相同，并进行了适当校正。仅当存在生育酚酯时才使用皂化，并且仅在最初的己烷提取以去除游离生育酚之后进行，以避免其因皂化而损失，特别是非α-生育酚和生育三烯酚。所有生育酚和生育三烯酚在正相二醇（环氧化物）柱上分离，结果一致且可重复，没有硅胶柱的不可重复性缺点，也没有反相柱缺乏分离效果的问题。生育酚的定量分析是通过使用样品中不存在的一种生育酚形式作为内部生育酚标准，或者如果所有形式都存在，或者当样品进行皂化时使用外部生育酚标准。仔猪心脏和肝脏样品显示主要存在α-生育酚，少量β-和γ-生育酚以及α-生育三烯酚，但没有δ-生育酚。肝脏中仅存在少量生育酚酯，而心脏中没有。
