DETERMINATION OF THE INVIVO REDOX STATUS OF CYSTEINE, CYSTEINYLGLYCINE, HOMOCYSTEINE, AND GLUTATHIONE IN HUMAN PLASMA
DETERMINATION OF THE INVIVO REDOX STATUS OF CYSTEINE, CYSTEINYLGLYCINE, HOMOCYSTEINE, AND GLUTATHIONE IN HUMAN PLASMA
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DOI：
10.1016/0003-2697(92)90456-h
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发表时间：
1992-02-01
影响因子：
2.9
通讯作者：
UELAND, PM
中科院分区：
文献类型：
--
作者：
MANSOOR, MA;SVARDAL, AM;UELAND, PM
描述了一种测定人类血浆中半胱氨酸、半胱氨酸甘氨酸、同型半胱氨酸和谷胱甘肽的还原、氧化和蛋白结合形式的方法。利用NaBH4将氧化的和蛋白质结合的硫醇转化为还原的硫醇，然后与单溴双烷（mBrB）衍生，通过反相离子对液相色谱和荧光检测对硫醇双烷加合物进行定量。在很低的硫醇浓度下，50 μm二硫赤藓糖醇的存在提供了标准曲线的线性。氧化形式的选择性测定是通过n -乙基马来酰亚胺（NEM）阻断游离巯基来完成的，多余的NEM通过随后添加NaBH4来失活。血浆中硫醇的还原形式在采集时通过衍生化血液样品以最小的氧化被捕获。这是通过将血液直接吸入含有mBrB或NEM等渗溶液的管中来实现的。该试验具有足够的灵敏度（<2 pmol），可检测人体血浆中四种硫醇化合物的各种形式。等离子体。半胱氨酸、半胱氨酸甘氨酸、同型半胱氨酸和谷胱甘肽的分析回收率接近100%，日内精密度分别对应于7、8、6和7%的变异系数。该试验已被用于测定人体血浆中四种硫醇化合物的各种形式。
An assay that measures the reduced, oxidized, and protein-bound forms of cysteine, cysteinylglycine, homocysteine, and glutathione in human plasma is described. Oxidized and protein-bound thiols are converted to their reduced counterparts by the use of NaBH4, and, following derivatization with monobromobimane (mBrB), the thiol-bimane adducts are quantified by reversed-phase ion-pair liquid chromatography and fluorescence detection. The presence of 50 μm dithioerythritol provides linearity of the standard curves at very low thiol concentrations. Selective determination of the oxidized forms was accomplished by blocking free sulfhydryl groups with N-ethylmaleimide (NEM) and excess NEM is inactivated by the subsequent addition of NaBH4. The reduced forms of the thiols in plasma were trapped with minimal oxidation by derivatizing blood samples at the time of collection. This was attained by drawing blood directly into tubes containing isotonic solutions of mBrB or NEM. The assay is sufficiently sensitive (<2 pmol) to detect the various forms of the four thiol compounds in human plasma. plasma. The analytical recovery of cysteine, cysteinylglycine, homocysteine, and glutathione was close to 100%, and the within-day precision corresponded to a coefficient of variation of 7, 8, 6, and 7%, respectively. The assay has been used to determine the various forms of the four thiol compounds in human plasma.