A comparison between different methods for the determination of reduced and oxidized glutathione in mammalian tissues.
In this study, three rapid assay techniques for the determination of glutathione, one enzymatic, one fluorometric and one newly patented colorimetric method, were compared by measuring reduced (GSH) and oxidized (GSSG) glutathione in guinea-pig heart and liver. The HPLC technique was used as a standard, since it is considered the most reliable assay method. In heart, all methods measured the same levels of GSH (about 1 mumole/g wet tissue), whereas in liver the fluorometric assay gave GSH levels about half as high as those measured by the other methods (about 3 vs. 7 mumoles/g wet tissue). Conversely, the fluorometric assay grossly overestimated GSSG concentration (by 5 to 8 times) in both heart and liver. These results confirm previous doubts about the use of the fluorometric technique for GSSG determination in mammalian tissues and also raise some questions about its use for the measurement of GSH in liver. In this tissue, the GSH concentration determined by the fluorometric method was shown to be inversely correlated with the size of the sample, suggesting the presence of some quenching material.
Floreani
M
M
Department of Pharmacology, University of Padova, Italy. floreani@dfem.unipd.it
Petrone
M
M
Debetto
P
P
Palatini
P
P
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000032:analysis; D004228:Dithionitrobenzoic Acid / Q000032:analysis; D005470:Fluorometry; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry*; D019803:Glutathione Disulfide; D006168:Guinea Pigs; D008099:Liver / Q000737:chemistry*; D008297:Male; D009206:Myocardium / Q000737:chemistry*; D013053:Spectrophotometry
D004396:Coloring Agents; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D019803:Glutathione Disulfide