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W3

Novel method for determination of oxidative status of tissues and body fluids as routine clinical parameter

Dr Matthias Schittmayer-Schantl, Medical University of Graz; Prof. Ruth Birner-Gruenberger, Medical University of Graz; Tamara Tomin, Medical University of Graz

Medical University of Graz


Challenge

Determination of the ratio of glutathione (GSH) to glutathione disulfide (GSSG) is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter because of the analytically challenging interconversion of glutathione to glutathione disulfide, which starts directly after sampling.


Technology

This is a methodology to stabilize glutathione and glutathione disulfide status aiding its incorporation as routine clinical parameter. A simple derivatization route that yields different isotopologues of N-ethylmaleimide alkylated glutathione from glutathione and glutathione disulfide after chemical reduction for mass spectrometric analysis has been demonstrated. A third isotopologue is employed as isotopic standard for simultaneous absolute quantification. Using customized blood vacuum containers the common pre-analytical errors associated with redox state measurement are avoided. As all isotopologues have similar chromatographic properties, matrix effects arising from different sample origin can only influence method sensitivity but not glutathione to glutathione disulfide ratio or absolute quantification.


Commercial Opportunity

The technology is of interest for companies developing and marketing venipuncture equipment, such as blood collection tubes and additives. It could also be of interest for diagnostic companies providing assays for the measurement of oxidative stress. Reagents applied in this method could be supplied in the form of a convenient kit. It is possible to provide blood collection tubes with dried reagents ready for use in clinical routine.


Development Status

Complete analytical method including sample preparation from tissues and body fluids. Clinical validation has to be performed.


Patent Situation

European Patent Application filed (EP18191196) Priority: 28/08/2018


Further Reading

1. McGill, M. R. & Jaeschke, H. A direct comparison of methods used to measure oxidized glutathione in biological samples: 2-vinylpyridine and N-ethylmaleimide. Toxicol. Mech. Methods 25, 589-595, doi:10.3109/15376516.2015.1094844 (2015).

2. Giustarini, D. et al. Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room. J Chromatogr B Analyt Technol Biomed Life Sci 1019, 21-28, doi:10.1016/j.jchromb.2016.02.015 (2016).

3. Giustarini, D., Dalle-Donne, I., Milzani, A., Fanti, P. & Rossi, R. Analysis of GSH and GSSG after derivatization with N-ethylmaleimide. Nat. Protoc. 8, 1660-1669, doi:10.1038/nprot.2013.095 (2013).

4. Danielson, S.R. et al. Quantitative mapping of reversible mitochondrial Complex I cysteine oxidation in a Parkinson disease mouse model. J. Biol. Chem. 286, 7601-7608 (2011).


 

Novel method for determination of oxidative status of tissues and body fluids as routine clinical parameter