Research ReportAccurate measurement of reduced glutathione in gamma-glutamyltransferase-rich brain microvessel fractions

Investigation of the redox status in the cerebral circulation is of great importance in order to evaluate intensity of oxidative stress-related diseases and the corresponding therapeutic effects. Changes in levels of reduced glutathione (GSH) are a major indicator of oxidative stress conditions. However, an important limitation for measurement of GSH as a biomarker is the possible presence in samples of gamma-glutamyltransferase (GGT) activity, i.e., the enzyme catalysing GSH breakdown.An accurate assay for the measurement of GSH in rat brain microvessels was developed, taking into account the high GGT activity expressed in this tissue compartment. Based on a sensitive fluorescence-based microtiter plate method using 2,3-naphthalenedicarboxyaldehyde as GSH-selective fluorogenic probe, the assay was applied to brain microvessels isolated from individual male Wistar rats. Pooling of microvessel fractions from several animals, as required by other procedures, could thus be avoided. In order to prevent GSH consumption via GGT activity, serine-boric acid complex (SBC) was added as inhibitor all along the microvessels isolation process. In the absence of GGT inhibition GSH in isolated brain microvessels was below the limit of quantification. Addition of SBC almost completely suppressed GGT activity, thus allowing GSH quantification (4.4 ± 1.6 nmol.mg− 1 protein, n = 3). Following the administration of a GSH depletor (diethyl maleate, 1 g.kg− 1, i.p.), decreased GSH levels were measured in liver, brain tissue and brain microvessels as well, thus confirming the reliability of the method for safe GSH measurements in small-sized, individual samples presenting high GGT activity.

Research Highlights►GSH in brain microvessels is below limit of quantification due to consumption by GGT ►Inhibition of GGT by serine-boric acid complex preserves microvessel GSH content ►Fluorimetric microplate method allows quantifying GSH in small size individual sample ►Our method is validated in liver, brain and microvessels from GSH-depleted rats