Analysis of Glutathione, Glutathione Disulfide, Cysteine, Homocysteine, and Other Biological Thiols by High-performance Liquid Chromatography Following Derivatization by N-(1-pyrenyl)maleimide

Abstract

The compound N-(1-pyrenyl)maleimide (NPM) reacts with free sulfhydryl groups to form fluorescent derivatives. A new method for measurement of glutathione and other biological thiols utilizing reverse-phase high-performance liquid chromatography to separate and quantify these derivatives is described. Separation and quantification of glutathione, cysteine, homocysteine, cysteinylglycine, and γ-glutamylcysteine derivatives are achieved. The method allows for the measurement of glutathione disulfide by masking free glutathione with 2-vinylpyridine, reducing glutathione disulfide with glutathione reductase, and measuring the resulting glutathione. Coefficient of variations for the various thiols measured by the NPM method range from 1.5 to 8.8%. The lower detection limit is around 50 fmol of glutathione. NPM derivatives are shown to be stable for 2 months at 4°C. Between 94.2 and 97.2% of glutathione and/or glutathione disulfide added to a sample is recovered using the NPM method. The NPM method is compared to the monobromobimane high-performance liquid chromatography method and the Tietze assay by measuring glutathione in homogenates from five different cell lines. The newly developed method offers some advantages over the currently accepted techniques, including specificity, speed, sensitivity, and ease of use.

Department(s)

Chemistry

International Standard Serial Number (ISSN)

0003-2697

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 1995 Elsevier, All rights reserved.

Publication Date

01 May 1995

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