An improved HPLC measurement for GSH and GSSG in human blood

The pathophysiological sequelae of oxidative/nitrosative stress are notoriously difficult to quantify. Despite these impediments, the medical significance of oxidative/nitrosative stress has become increasingly recognized to the point that it is now considered to be a component of virtually every disease. The level of oxidative stress can be quantified in blood by the measurement of the increase in glutathione disulfide (GSSG) and the decrease in the GSH/GSSG ratio, which has been shown to be altered in a variety of human diseases such as lung inflammation, amyotrophic lateral sclerosis, chronic renal failure, malignant disorders, and diabetes. Among the proposed methods for GSH/GSSG detection, the amino group derivatization with 2,4-dinitrofluorobenzene followed by HPLC separation has the advantage of allowing evaluation of both parameters within a single run contemporaneously. However, it has been shown that the application of this method on blood samples is not reproducible. In this report, we offer an explanation for these experimental limits and suggest some modifications that allow the application of this method to blood samples. The modified method has a low detection limit (0.5 microM, i.e., 1.4 pmoles) and a high reproducibility with a within-run imprecision of less than 2%. It could have a wide application as it is simple, virtually artifact-free, and not time-consuming, especially for large-scale screening studies.