A high-sensitivity fluorometric high-performance liquid chromatographic method for determination of glutathione and other thiols in cultured melanoma cells, microdialysis samples from melanoma tissue, and blood plasma.
A high-performance liquid chromatographic method with fluorometric detection is described which is suitable for determination of glutathione in small samples. Reduced glutathione (GSH) and total glutathione obtained as GSH after reduction with glutathione reductase is derivatized with N-(7-dimethylamino-4-methyl-3-coumarinyl) maleimide (DACM) and subjected to chromatography. The detection limit for the GSH-DACM derivative was 5-10 fmol/injection, and analytical recovery was quantitative. The method is suitable for determination of both reduced and total glutathione in samples from microdialysis of melanoma tumours, and cysteine can be quantified in the same chromatogram. Application is shown also for glutathione determinations in cultured melanoma cells, melanoma homogenates and plasma.
Dizdar
N
N
Department of Clinical Chemistry, University Hospital, Linköping, Sweden.
Kågedal
B
B
Smeds
S
S
Arstrand
K
K
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000032:analysis; D003956:Dialysis; D005470:Fluorometry / Q000379:methods*; D005978:Glutathione / Q000032:analysis*; D006801:Humans; D008301:Maleimides; D008545:Melanoma / Q000097:blood / Q000737:chemistry* / Q000473:pathology; D008832:Microchemistry; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000032:analysis*; D014407:Tumor Cells, Cultured / Q000737:chemistry
D008301:Maleimides; D013438:Sulfhydryl Compounds; C019347:N-(7-dimethylamino-4-methylcoumarinyl)maleimide; D005978:Glutathione; D003545:Cysteine