Fluorimetric determination of monobromobimane and o-phthalaldehyde adducts of gamma-glutamylcysteine and glutathione: application to assay of gamma-glutamylcysteinyl synthetase activity and glutathione concentration in liver

Journal of Chromatography. B, Biomedical Applications
C C Yan, R J Huxtable

Abstract

The reversed-phase HPLC separation of fluorescent o-phthalaldehyde (OPA) derivatives has been applied to the assay of hepatic gamma-glutamylcysteine and glutathione (GSH) levels and the enzymes producing these peptides. The method has been compared to the assay using monobromobimane (MB) as the derivatizing agent. The OPA method has the advantage of faster derivatization, the lack of need to adjust the pH, isocratic separation and selectivity for GSH and gamma-glutamylcysteine. The MB method requires pH adjustment following derivatization and gradient elution chromatography. MB is also non-selective, yielding fluorescent derivatives of all biological thiols and more interfering peaks on the chromatogram. MB-based analyses are also approximately sixty times more expensive per sample. MB yields fluorescent degradation products on exposure to light. OPA adducts are stable for up to ten days when stored at -20 degrees C. OPA detection is sensitive to 12.5 pmol in the sample, at a signal-to-noise ratio of 2.5. The two methods correlate well. Hepatic gamma-glutamylcysteine synthetase in the same liver preparation was found to be 4.85 +/- 0.47 nmol min-1 mg-1 protein by the OPA method and 4.42 +/- 0.52 nmol min-1 mg-1 protein by the M...Continue Reading

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Citations

May 15, 2013·Chemico-biological Interactions·Young Suk JungSang Kyum Kim
Nov 27, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Emanuela Camera, Mauro Picardo
Feb 9, 1996·Biochemical Pharmacology·C C Yan, R J Huxtable
Sep 6, 2012·Journal of Pharmaceutical and Biomedical Analysis·Isabella SquellerioViviana Cavalca
Feb 3, 2007·Journal of Controlled Release : Official Journal of the Controlled Release Society·Amnon C Sintov, Uri Wormser
Jun 28, 2005·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·N MaesoC Barbas
Feb 9, 2006·Journal of Neurochemistry·Mary Latha RathinamGeorge I Henderson
Jul 28, 2015·Rapid Communications in Mass Spectrometry : RCM·Jia YuJiang Zheng
Feb 23, 2000·Archives of Biochemistry and Biophysics·J ChoiH J Forman
Jun 5, 1998·Journal of Chromatography. B, Biomedical Sciences and Applications·C MuscariC Guarnieri
Jul 31, 1998·Journal of Pharmaceutical and Biomedical Analysis·M StachowiczD Pradeau
Jun 20, 2003·Analytical Biochemistry·Collin C WhiteTerrance J Kavanagh
Feb 16, 2008·Biochemical and Biophysical Research Communications·Young S JungYoung C Kim
Mar 13, 2009·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Dakshine ShanmugarajahFrederick H Hausheer
Jun 18, 2009·Archives of Biochemistry and Biophysics·Daniela GiustariniRanieri Rossi

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