科研之友
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摘要
Altered plasma aminothiol concentrations are thought to be a valuable risk indicator and are interestingly utilized for routine clinical diagnosis and for the monitoring of various metabolic disorders and human diseases, and accordingly there is a need for an accurate and reliable assay capable of simultaneously determining aminothiols including glutathione (GSH), N-acetylcysteine (NAC), homocysteine (Hcys), and cysteine (Cys) in human plasma. Herein, a highly sensitive, selective, and very fast HPLC-chemiluminescence (HPLC-CL) coupled method is reported, exploiting for the first time the strong nucleophilicity and high reactivity of aminothiols toward quinones for a CL assay. The unique redox-cycling capability of quinone and/or Michael addition adducts, thioether-quinone conjugates, was utilized to establish a novel analytical method based on the reaction of adducts with dithiothreitol (DTT) to liberate reactive oxygen species (ROS), which are detected by using a luminol-CL assay. Specimen preparation involved the derivatization of aminothiols with menadione (MQ) for 5 minutes at room temperature. A unique green chemistry synthesis of thioether-quinones in HEPES buffer (pH 8.5) was introduced by using our reaction methodology without needing any hazardous organic solvent or catalyst. The aminothiol-MQ adducts were separated using solid-phase extraction followed by isocratic elution on an ODS column. Linearity was observed in the range of 2.5-500, 5-500, 10-1500, and 20-2000 nM with detection limits (S/N of 3) of 3.8, 4.2, 8, and 16 (fmol per injection) for GSH, NAC, Hcys, and Cys, respectively. The method was successfully applied for the selective determination of aminothiols in human plasma from healthy people and patients with rheumatic arthritis and diabetes mellitus. The obtained results postulated the usefulness of our method for investigating the relationship between aminothiol metabolism and related human disorders.
- 出版日期2015
