太平洋鳕鱼脑中硫苷脂的分离纯化和分析

The Isolation and Molecular Species Analysis of Sulfatides from Codfish Brain

以太平洋鳕鱼为原料,从鳕鱼脑中分离硫苷脂,确立了提取及纯化的条件,并对硫苷脂的纯度和分子种进行分析。首先,采用氯仿/甲醇(2∶1,v/v)提取总脂。然后,依次采用氯仿/甲醇/水(7∶3∶0.3,v/v/v)洗脱硅胶柱层析,含0.2mol/L乙酸铵的氯仿/甲醇/水(30∶60∶8,v/v/v)洗脱DEAE Sephadex-A25离子交换柱层析,40%甲醇脱盐和100%甲醇洗脱反相C8柱层析,获得硫苷脂纯品。最后,利用500YMC Diol液相色谱柱(3.0mm×250mm,5μm)分离,以正己烷/异丙醇(70∶30,v/v),异丙醇/水/甲酸/氨水(100∶13∶1∶0.14,v/v/v/v)为流动相梯度洗脱,采用负电喷雾电离(ESI)和母离子扫描模式,用液相色谱-串联质谱(LC-MS/MS)法分析了鳕鱼脑硫苷脂的纯度和分子种组成,并比较了其与哺乳动物脑中硫苷脂分子种组成的异同。结果表明,本实验制得的鳕鱼脑硫苷脂纯度为90.74%,与哺乳动物类似,鳕鱼脑硫苷脂的长链碱基以鞘氨醇为主,主要分子种为d18∶1-C24∶1,但脂肪酸的羟基化程度略低,且含少量独特的分子种,如d18∶1-22∶1和d18∶2-25∶2。

The objective of the research is to establish an efficient extraction and isolation workflow for purification of codfish sulfatides as well as procedures to determine the purity and molecular species of the purified sulfatides.The crude lipids extracted with chloroform/methanol（2∶1,v/v）were firstly applied to a normal phase silica column and fractions containing sulfatides were eluted with chloroform/methanol/water（7∶3∶0.3,v/v/v）.The fractions were combined and further applied to a DEAE-Sephadex A25 anion exchange column.Negative charged sulfatides were eluted with solvent chloroform/methanol/water（30∶60∶8,v/v/v）containing 0.2 mol/L ammonium acetate.Subsequent desalting procedure was achieved on a reversed chromatography by cleaning column with 40% methanol in water and recover sulfatides with methanol.The purity and molecular composition of purified sulfatides were determined by a normal phase liquid chromatography coupled to tandem mass spectrometry method.Gradient elution on a500 YMC Diol column（3.0mm × 250 mm,5μm）with solvent hexane/isopropanol（70∶30,v/v）and solvent isopropanol/water（100∶13,v/v）containing 0.8% formic acid and 0.1% aqueous ammonia can separate sulfatides successfully.Mass spectrometer was operated on negative ionization mode and the sulfatide molecules were detected by precursor ion scan.The purity of isolated codfish sulfatides is 90.74%.The most abundant molecule is d18∶1-C24∶1and the major long chain base is sphingosine,both the characteristics are similar to mammalian sulfatides.However,the hydroxylation degree of fatty acids was lower than that of mammalian sulfatides,and unique molecules with structure d18∶1-C22∶1and d18∶2-C25∶2in lower content were also identified.