Studies of glutathione metabolism in sheep erythrocytes

The tripeptide, reduced glutathione (GSH), is present in high concentration in mammalian erythrocytes, where its major role is the protection of the cell against oxidative damage. Sheep exhibit two distinct types of erythrocyte GSH deficiency. In Finnish Landrace sheep (Finns) low GSH is inherited as an autosomal recessive trait, and is associated with a markedly diminished erythrocyte life-span, and the presence of unusually high concentrations of some erythrocyte amino acids, notably ornithine and lysine. In contrast, in Tasmanian Merino sheep (Merinos) the low GSH characteristic is inherited in an autosomal dominant manner and both the erythrocyte life-span and amino acid concentrations are normal. This Thesis describes an investigation of the biochemical mechanisms responsible for the Finn and Merino erythrocyte GSH deficiencies. The Finn and Merino sheep investigated were maintained by the ARC Animal Breeding Research Organisation, Edinburgh. Both breeds contained substantial numbers of GSH-deficient animals. In agreement with other studies, GSH-deficient Finn erythrocytes were found to contain very high concentrations of ornithine and lysine, a phenomenon not encountered in Merinos. Sheep were allotted their GSH type on the basis of their erythrocyte total non-protein reduced thiol concentration as determined by the non-specific thiol reagent 5, 5'-dithiobis-(-2-nitrobenzoate) (DTNB). The validity of equating total DTNB reactive thiol with GSH was established by estimating GSH using novel automated versions of methods employing DTNB and alloxan as chromogens. GSH-deficient erythrocytes of both breeds were also found to have a diminished GS3G concentration so that they had a diminished total glutathione content (GSH + 2GSSG). The alloxan GSH and GSSG estimates were used to calculate the redox potential of the GSH:G3SG couple in the various cell types. In both breeds the difference in redox potential between normal and GSH-deficient cells was small. It is suggested that the diminished life-span of GSH-deficient Finn cells may not be a direct consequence of their GSH status