Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography
Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography
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DOI：
10.1007/s004030100212
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发表时间：
2001-04
影响因子：
3
通讯作者：
A. Weerheim;M. Ponec
中科院分区：
文献类型：
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作者：
A. Weerheim;M. Ponec
摘要角质层细胞间脂质是哺乳动物皮肤屏障功能的重要组成部分。SC脂质的主要成分是神经酰胺，胆固醇和游离脂肪酸，建立从人类和哺乳动物SC提取的脂质的薄层色谱分析。到目前为止，脂质分析的提取物的整个SC已被使用和信息的脂质组合物是否随深度在SC的变化是稀缺的。胶带剥离是一种使用粘合膜逐步去除角质细胞层的技术。使用该技术进行脂质分析受到脂质提取物与化合物的污染的阻碍，所述化合物是用用于提取SC脂质的有机溶剂从胶带中共提取的。本研究的目的是建立一个合适的分析方法，用于测定当地SC脂质组成。为此，在5名健康志愿者中通过Leukoplex胶带连续剥离收集SC样品。脂质用乙酸乙酯：甲醇混合物（20：80）萃取，并通过HPTLC分离。本研究的结果显示，在最上层SC层（约4次剥离）中，游离脂肪酸水平最高，胆固醇和神经酰胺水平最低。在底层SC层中的水平保持不变。在这些层中，神经酰胺水平为约60重量%，游离脂肪酸和胆固醇水平各为约20重量%。神经酰胺可以被分离成七个不同的馏分和个别神经酰胺馏分的相对量没有显着改变与SC深度。硫酸胆固醇水平约为总胆固醇的5%，并且不随SC深度变化，除了第一条的水平约为1%。所开发的方法使得有可能研究在健康和患病的人皮肤中的SC脂质分布的差异与SC脂质组织和体内皮肤屏障功能。
AbstractIntercellular lipids in the stratum corneum (SC) are responsible for the barrier function of mammalian skin. The main components of the SC lipids are ceramides, cholesterol, and free fatty acids, as established by thin-layer chromatographic analysis of lipids extracted from the human and mammalian SC. Up to now, for lipid analysis the extracts of the entire SC has been used and information on whether the lipid composition changes with the depth in the SC is scarce. Tape stripping is a technique which removes corneocyte layers step by step with an adhesive film. The use of this technique for lipid analysis was hampered by the contamination of lipid extracts with compounds co-extracted from the tape with organic solvents used for the extraction of SC lipids. The aim of the present study was to establish a suitable analytical method for the determination of the local SC lipid composition. For this purpose, the SC samples were collected by sequential stripping with Leukoplex tape in five healthy volunteers. The lipids were extracted with ethyl acetate:methanol mixture (20:80) and separated by means of HPTLC. The results of this study revealed that the free fatty acid level is highest and the cholesterol and ceramide levels lowest in the uppermost SC layers (about 4 strippings). The levels remained unchanged in the underlying SC layers. In these layers, the ceramide level was about 60 wt% and the free fatty acid and cholesterol levels were about 20 wt% each. Ceramides could be separated into seven different fractions and the relative amounts of individual ceramide fractions did not significantly change with the SC depth. Cholesterol sulfate levels were about 5% of total cholesterol and did not change with the SC depth, except for the for the first strip where the level was about 1%. The method developed makes it possible to study the differences in the SC lipid profile in healthy and diseased human skin with relation to the SC lipid organization and to the skin barrier function in vivo.