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Measuring cellular Glutathione concentration

One of the impediments to knowing ones GSH status is the lack of standardized analytical methods for determining GSH is blood, plasma, tissue and cellular material.  Unlike most other biochemical blood markers (e.g. cholesterol or glucose) GSH analysis it is not part of the regular battery of tests performed when a blood sample is taken. This is due mostly to the labile nature of GSH in biological systems.  GSH is considered a high turnover dynamic molecule where it can both be readily oxidized to its disulphide form GSSG or also broken down to its constituent amino acids by the action of endogenous enzymes.  This means that samples require immediate chilling and treatment. Sample pre-treatment usually begins with the addition of an acid which not only lyses the cells but also brings about the precipitation of many contaminating proteins. The resulting lower sample pH also prevents GSH autoxidation to GSSG and inhibits most of the enzymes involved in GSH catabolism and oxidation [1].

The resulting treated samples are usually then centrifuged to remove solids and the supernatant stored frozen prior to analysis.  There are many methods available in the scientific literature for the analysis of GSH but none, however, are particularly reliable or simple. This is reflected in the literature values of blood GSH analysis which often vary greatly between laboratories using what seem to be similar methods [2]. Methods include HPLC, HPLC-MS /MS and capillary electrophoresis [3]. One of the most commonly used is asspectroscopic method based on the GSH reductase enzyme recycling assay first described by Tietze at al in 1969 [4]. 

Sampling blood and measuring GSH in either red blood cells (RBC) or plasma would seem like an acceptable method of determining GSH status, however, both present unique technical difficulties that are open to artefacts.  In the case of RBC, their metabolism, physiology and structure are not considered representative of most cell types in the body as they lack a nucleus and most organelles. In addition, the acid pre-treatment of the RBC sample releases a large amount of iron that can oxidize GSH even under acidic conditions.  In the case of blood plasma, there exists an approximate thousand-fold difference between intracellular GSH (mM range) to extracellular or plasma concentration (µM range).  Thus, a small amount of RBC lysis in the sample that could occur simply by using too small a needle to draw blood can result in erroneously high plasma GSH level.  In the Glyteine bioavailability human study, these problems were averted by focussing on assaying the lymphocyte fraction of blood [5].  These nucleated cells were considered more representative of the body’s cells and could be easily isolated by high speed fluorescence activated cell sorting which allowed the collection of a million lymphocytes in less than 5 minutes.  The resulting million cells were acid treated and analysed for GSH content using the GSH reductase enzyme recycling assay.

References

  1. Monostori, P., et al., Determination of glutathione and glutathione disulfide in biological samples: An in-depth review Journal of Chromatography B, 2009. 877: p. 3331.
  2. Rossi, R., et al., Blood glutathione disulfide: In vivo factor or in vitro artifact? Clinical Chemistry, 2002. 48(5): p. 742-753.
  3. Iwasaki, Y., et al., Chromatographic and mass spectrometric analysis of glutathione in biological samples Journal of Chromatography B, 2009. 877(28): p. 3309.
  4. Tietze, F., Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Analytical Biochemistry, 1969. 27(3): p. 502-522.
  5. Zarka, M.H. and W.J. Bridge, Oral administration of γ-glutamylcysteine increases intracellular glutathione levels above homeostasis in a randomised human trial pilot study. Redox Biology, 2017. 11: p. 631-636.
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