Johannson M, Lenngren S
Department of Research and Development, ACO Läkemedel AB, Solna, Sweden.
J Chromatogr. 1988 Nov 18;432:65-74. doi: 10.1016/s0378-4347(00)80634-9.
The thiol and oxidized forms of cysteine, glutathione and N-acetylcysteine in plasma were determined by ion-pair liquid chromatography and post-column derivatization. The thiol forms were measured after direct injection of deproteinized plasma. The oxidized forms, present either as a dimer or oxidized with other small thiols, were assayed in deproteinized plasma after reduction with dithiothreitol. The total amounts, including the fraction bound to plasma proteins via disulphide bonds, were determined after reductive cleavage in plasma with dithiothreitol. The compounds were separated by ion-pair liquid chromatography on a reversed-phase column (C18) and were detected by fluorimetry after post-column derivatization. The endogenous plasma levels of all forms of cysteine, glutathione and N-acetylcysteine, except for the thiol form of N-acetylcysteine, were above the quantification limits. The quantification limit of N-acetylcysteine as a thiol, was 0.15 microM. The precision was better than 12% for the endogenous concentrations.
采用离子对液相色谱法和柱后衍生法测定血浆中半胱氨酸、谷胱甘肽和N-乙酰半胱氨酸的硫醇形式和氧化形式。直接进样脱蛋白血浆后测定硫醇形式。以二硫苏糖醇还原脱蛋白血浆后，测定以二聚体形式存在或与其他小硫醇氧化的氧化形式。用二硫苏糖醇对血浆进行还原裂解后，测定包括通过二硫键与血浆蛋白结合部分在内的总量。这些化合物在反相柱（C18）上通过离子对液相色谱法分离，并在柱后衍生后用荧光法检测。除N-乙酰半胱氨酸的硫醇形式外，所有形式的半胱氨酸、谷胱甘肽和N-乙酰半胱氨酸的内源性血浆水平均高于定量限。N-乙酰半胱氨酸硫醇形式的定量限为0.15 microM。内源性浓度的精密度优于12%。
