Highly sensitive simultaneous detection of cultured cellular thiols by laser induced fluorescence-capillary electrophoresis.
We have recently described a new method to determine physiological thiols, in which the quantification of plasma homocysteine, cysteine, cysteinylglycine, glutathione, and glutamylcysteine was achieved after derivatization with 5-iodoacetamidofluorescein. Samples were separated and measured by capillary electrophoresis with laser-induced fluorescence in an uncoated fused-silica capillary, using a phosphate/borate run buffer and the organic base N-Methyl-D-glucamine as effective electrolyte addictive to obtain a baseline peak separation. In this paper, we propose an improvement of our method useful for the analysis of the intracellular thiols in different cultured cells. In particular, we studied run buffer and injection conditions in order to increase the sensitivity of the assay and we found that, by incrementing two times the injected volume and using the water plug before the sample injection, the sensitivity of our previous method was increased by about ten times. To maintain a good resolution between peaks, particularly between homocysteine and the internal standard d-penicillamine, we lengthened the run time by incrementing the concentration of the electrolyte buffer and the organic base d-glucamine and by decreasing the cartridge temperature from 40 to 25 degrees C. After these changes in electrophoretical parameters, cellular thiols were baseline-resolved in less than 14 min instead of 9 min as in our previous method, but the limit of quantification is increased from 50 to 1 nmol/L. This new procedure allows also to measure the intracellular thiols commonly found at low concentration, such as cysteinylglycine, glutamylcysteine, and homocysteine. The new analytical method performance was assessed by measuring the intracellular thiols in three different cell lines, i.e., HUVEC, ECV304, and R1 stem cells.
Zinellu
Angelo
A
Chair of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, 1-07100 Sassari, Italy.
Sotgia
Salvatore
S
Posadino
Anna M
AM
Pasciu
Valeria
V
Perino
Maria G
MG
Tadolini
Bruna
B
Deiana
Luca
L
Carru
Ciriaco
C
D000818:Animals; D002478:Cells, Cultured; D003545:Cysteine / Q000032:analysis; D004151:Dipeptides / Q000032:analysis; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology; D005452:Fluoresceins; D005978:Glutathione / Q000032:analysis; D006710:Homocysteine / Q000032:analysis; D006801:Humans; D007834:Lasers; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013234:Stem Cells; D013438:Sulfhydryl Compounds / Q000032:analysis*
D004151:Dipeptides; D005452:Fluoresceins; D013438:Sulfhydryl Compounds; D006710:Homocysteine; C028505:cysteinylglycine; C015296:5-iodoacetamidofluorescein; D005978:Glutathione; D003545:Cysteine; C017341:gamma-glutamylcysteine