Thomas D W, van Kuijk F J, Dratz E A, Stephens R J
Life Sciences Division, SRI International, Menlo Park, California 94025.
Anal Biochem. 1991 Oct;198(1):104-11. doi: 10.1016/0003-2697(91)90513-s.
A new method has been developed for the quantitation of lipid peroxidation products by gas chromatography-mass spectrometry. An important advantage over existing gas chromatography-mass spectrometry methods is the elimination of autoxidation during sample preparation. The sensitivity is sufficient to permit measurement of lipid peroxidation products under normal physiological conditions on as little as 1 mg of tissue. Lipids from whole tissue samples or cell preparations are reduced by catalytic hydrogenation during extraction. The hydrogenation stabilizes the compounds by saturating the double bonds and reducing the hydroperoxides to hydroxy derivatives. The saturated lipids are then saponified and the resulting fatty acids are converted to pentafluorobenzyl esters. Hydroxy fatty acids are further converted to trimethylsilyl ether derivatives. Quantitation is accomplished by negative ion chemical ionization gas chromatography-mass spectrometry, using deuterated internal standards. Specific products from polyunsaturated fatty acids can be quantitated, and the method differentiates between products produced by free-radical and photooxidation mechanisms. Increased levels of lipid peroxidation products, above normal physiological levels, that result from prooxidant conditions, such as exposure of animals to carbon tetrachloride, can be measured.
已开发出一种通过气相色谱 - 质谱法对脂质过氧化产物进行定量的新方法。与现有气相色谱 - 质谱法相比，一个重要优势是在样品制备过程中消除了自氧化。其灵敏度足以在正常生理条件下对低至1毫克组织中的脂质过氧化产物进行测量。在提取过程中，全组织样品或细胞制剂中的脂质通过催化氢化进行还原。氢化通过使双键饱和并将氢过氧化物还原为羟基衍生物来稳定化合物。然后将饱和脂质皂化，所得脂肪酸转化为五氟苄酯。羟基脂肪酸进一步转化为三甲基硅醚衍生物。使用氘代内标，通过负离子化学电离气相色谱 - 质谱法进行定量。可以对多不饱和脂肪酸的特定产物进行定量，并且该方法能够区分自由基和光氧化机制产生的产物。可以测量由促氧化条件（如动物接触四氯化碳）导致的高于正常生理水平的脂质过氧化产物水平升高。
