Some Pitfalls in the Measurement of Blood Glutathione

Abstract

1. We report some pitfalls in the measurement of whole blood and plasma glutathione in man. 2. Using a simple tourniquet to the forearm and a 21-gauge needle, blood samples were collected by brachial vein puncture from healthy subjects. Whole blood and plasma were analysed for total glutathione, including the reduced and oxidized forms, by a spectrophotometric recycling method involving the glutathione reductase/NADPH couple. 3. The concentration of oxidized glutathione was determined after treatment of aliquots with either 2-vinylpyridine or N-ethylmaleimide to trap reduced glutathione. Reduced glutathione in the native samples could then be obtained by subtraction. 4. When the reagents were added to separated plasma, 2-vinylpyridine yielded values for oxidized glutathione that were twice as high as with N-ethylmaleimide. In whole blood studies the discrepancy was even greater, and the problem was not resolved by deproteinization of samples with 5-sulphosalicylic acid. Using N-ethylmaleimide, levels of oxidized glutathione were less than 1% of total glutathione in whole blood. 5. Despite attempts to minimize haemolysis, lysed erythrocytes contributed on average 25% to the 'plasma' glutathione concentration.

Pitfalls in the analysis of the physiological antioxidant glutathione (GSH) and its disulfide (GSSG) in biological samples: An elephant in the room

“…Additionally, only in samples treated with NEM before acid deproteinization we could obtain a good recovery of GSSG added to hemolysate [8]. These data together with those obtained for 2-VP [14,15] let us to select NEM as the most suitable alkylating agent to protect the oxidation of thiol groups, according to what suggested previously [Butler, Tietze]. NEM was found to be the best alkylating agent because, in addition to penetrate cell membranes and to rapidly block all available GSH within seconds, it is also a strong rapid irreversible inhibitor of GR [8].…”

“…Because of this, if applied to intact cells or tissues, this method requires cell lysis which is usually carried out by acidification with consequent onset of large amounts of GSSG as an artifact. Indeed, in comparative analyses, where GSSG was determined after treatment of blood aliquots with either 2-VP or NEM, the levels of GSSG were at least 12 times higher in 2VP-treated samples with a corresponding decrease of the GSH/GSSG ratio [14]. A direct comparison between the two methods for analysis of GSH and GSSG in several mouse tissues has been performed also recently [15].…”

“…about 40 mM in whole blood) was shown to be able to block any oxidation of thiols to disulfides and to represent the reference procedure to measure the intracellular GSH and GSSG concentration in a reliable way. The acidification step of samples for deproteinization must follow the reaction with NEM [8,10,14,20,34,35]. In fact, that addition of NEM together with acid or after acid deproteinization is not correct since it does not avoid artificial oxidation of GSH.…”

Glutathione homeostasis is disturbed in CD4‐positive lymphocytes of HIV‐seropositive individuals

“…In plasma, a rapid redistribution of thiols occurs, whereas under influence of air the protein-bound glutathione fraction increases at the expense of the free acid-soluble [15,17] and total glutathione concentration [17]. Blood samples should be collected in evacuated tubes containing EDTA, preferably without using a tourniquet, and processed immediately [18]. Fasting also affects leucocyte and plasma glutathione concentrations.…”

Effect of Glutathione on Lung Activator Protein-1 Activation and Heme Oxygenase-1 Induction in the Immature Rat

“…Because our exposures were longer than in other studies where GSH content or GSSG/GSH ratios were measured (72 h versus 2-54 h) (25), this could be an important factor in the decreased lung GSSG/ GSH ratio we observed. Another consideration is that the method we used for GSH content measurement could have also artificially increased GSSG in air exposed adults because the vinylpyridine may cause higher value of GSSG (26). We would have expected this to affect all samples if this were the case.…”