Direct Detection of Biologically Significant Thiols and Disulfides with Manganese(IV) Chemiluminescence

Citations Over TimeTop 10% of 2011 papers

Abstract

The quantification of low-molecular mass thiols and disulfides involved in cellular redox processes is hindered by oxidation or degradation of analytes during conventional sample preparation steps (including deproteinization and derivatization). Researchers therefore seek techniques that minimize sample handling and permit direct detection of thiols and disulfides within a single chromatographic separation. We demonstrate a new HPLC procedure for these biologically important analytes that incorporates direct chemiluminescence detection with a manganese(IV) reagent. A mixture of seven thiols and disulfides (cysteine, N-acetylcysteine, homocysteine, glutathione (GSH), glutathione disulfide (GSSG), cystine, and homocystine) in their native forms were separated using a C18 column within 20 min. Detection limits for these analytes ranged from 5 × 10(-8) to 1 × 10(-7) M, and the precision for retention times and peak areas was excellent, with relative standard deviations of less than 0.3% and 2%, respectively. This approach was employed to determine two key biomarkers of oxidative stress, GSH and GSSG, in whole blood taken from 12 healthy volunteers. Samples were deproteinized, centrifuged, and diluted prior to analysis using a simple procedure that was shown to avoid significant artificial oxidation of GSH.

Related Papers

• → Oxidation of the Glutathione/Glutathione Disulfide Redox State Is Induced by Cysteine Deficiency in Human Colon Carcinoma HT29 Cells(2002)57 cited
• → Thiol-disulfide interchange between cystine and N-2-mercaptoethyl-1,3-diaminopropane as a potential treatment for cystinuria(1984)13 cited
• The effect of thiol compounds on lymphocytes stimulated in culture.(1978)
• → Spectrophotometric determination of cystine by formation of an o-phthalaldehyde/N-acetyl-l-cysteine derivative(1989)4 cited
• → Activation of murine lymphocytes by 2-mercaptoethanol and related thiol compounds and its mechanism. II Effect of thiol compounds on the viability of the lymphocytes under a cystine-deficient culture condition.(1986)2 cited