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17 Jan 2019

Auto Oxidation of Glutathione and its Relevance to IV Use

Dr. Paul S. Anderson

This is an incredibly common issue in the assay of GSH-GSSG in lab testing so the mechanisms are well described. In-vitro (i.e. in the IV syringe or bag) this phenomenon happens even faster than in plasma or tissue samples when additives are introduced due to lack of in-vivo enzymes. So protecting the GSH from the pharmacy until infusion is important. (Yes, one can infuse GSSG but then glutathione reductase has to work even harder).

Below are some of the many discussions with clinical chemists regarding the procedures they have to go through to diminish auto oxidation of GSH to GSSG (there are hundreds as it is a common technical issue).

So when preparing an IV it is best to dilute it with sterile water or saline as neither has an oxidizing component. Trace elements, most B-Vitamins etc. will auto-oxidize GSH on contact. If infusing it in a water or saline diluent it remains mainly GSH and then can enter the plasma where it has the best chance of having a higher GSH to GSSG ratio until delivery to target tissue.

One of the many papers on “auto-oxidation of GSH” and an attempt to mitigate this laboratory finding comes from Curello et.al. Reiterating the perpetual lab issue of “reduced glutathione in plasma undergoes spontaneous autoxidation, with mixed disulfide formation” and then describing the lengths one must go to not have this happen. [Curello, et.al CLINICAL CHEMISTRY, Vol. 33, No. 8, 1987]

CLINICAL IMPORTANCE: So if it is this difficult for clinical chemists why in IV preparation would one purposefully auto-oxidize that costly GSH you are about to infuse?

More lab nerd information below for flavor:

From Prof. Alan Slusarenko: RWTH Aachen University

We use Ellman’s reagent in a glutathione reductase based recycling assay to measure glutathione. Here is an extract from Gruhlke et al. FRBM 49:1916-1924 (2010): Glutathione levels were determined using a standard enzymatic recycling assay based on glutathione reductase (GR) [54, modified by 55]. Glutathione (GSH) was sequentially oxidized to glutathione disulfide (GSSG) by 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB, Merck) and reduced by NADPH (AppliChem) in the presence of GR. The rate of 2-nitro-5-thiobenzoic acid formation was monitored at 412 nm and the total glutathione present read off from a standard curve prepared using GSH (AppliChem). In any given sample the assay can be rendered to reflect only GSSG levels by utilising 2-vinylpyridine to remove GSH from the GSSG /2GSH couple [54]. The system was calibrated using standard curves prepared with GSH and GSSG purchased from AppliChem and GR from Sigma (E 3664). Under the test conditions used the detection limit for GSH was 1.25 µg mL-1.

[54] Griffith, O. W. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106:207-212; 1980.

[55] Anderson, M. E. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol. 113:548-555; 1985.

There are many methods to estimate glutathione but we like this one because its specific and sensitive and since it is an enzyme-based assay, you get a good feeling for what is going on by seeing that the enzyme kinetics are behaving as they should.