Accurate 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, 1g.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.
EA 3452 Cibles Thérapeutiques, Formulation et Expertise Préclinique du Médicament, Faculty of Pharmacy, Nancy-University, BP 80403, 54001 Nancy Cedex, France.
Katy
K
Maguin Gaté
Isabelle
I
Lartaud
Philippe
P
Giummelly
Romain
R
Legrand
Alfonso
A
Pompella
Pierre
P
Leroy
D000818:Animals; D001921:Brain / Q000098:blood supply* / Q000378:metabolism; D019411:Clinical Laboratory Techniques; D005978:Glutathione / Q000032:analysis*; D008297:Male; D055806:Microvessels / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D005723:gamma-Glutamyltransferase / Q000378:metabolism
D005723:gamma-Glutamyltransferase; D005978:Glutathione