Highly sensitive simultaneous detection of cultured cellular thiols by laser induced fluorescence-capillary electrophoresis

Abstract

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.

Protein‐bound glutathione measurement in cultured cells by CZE with LIF detection

“…The LOQ evaluated as S/N of 10 and calculated by a 44-nL injection of sample, was about 5 nmol/L. This value is higher than those obtained by our previous work [30] in which LOQ value was 1 nmol/L. The lost sensitivity is probably due to the combination of several factors such as different instrumentation (MDQ system with a vs. P/ACE 5500), different run buffer (low-concentrated in this work) and different injection mode (in the previous work we utilized a water plug to increase sample staking and sensitivity).…”

“…Moreover, as well-known, in CE a higher sensitivity is obtained when migration time of analytes is retarded, therefore the little decrease in the LOQ of the present method was preferred in order to obtain a higher rapidity. The accuracy of our new CE method was assessed by comparing the GSH levels from 27 different cultured cells obtained by the new method and by our previous CE assay [30]. The Bland-Altman test shown in Fig.…”

“…However, when the measurement is restricted to cultured cells the number of assays able to perform GSH determination decreases drastically for the elevated sensitivity required. We have recently carried out some works for the evaluation of GSH and other thiols in biological fluids and cultured cells by using 5-IAF as thiol specific fluorogenic reagent [27][28][29][30]. In our previous method for the evaluation of intracellular thiols [30], analysis was accomplished in about 15 min.…”

“…We have recently carried out some works for the evaluation of GSH and other thiols in biological fluids and cultured cells by using 5-IAF as thiol specific fluorogenic reagent [27][28][29][30]. In our previous method for the evaluation of intracellular thiols [30], analysis was accomplished in about 15 min. Although cysteinyglycine, Cys glutamylcysteine and in some cases homocysteine were also detected, the GSH peak migrated in about 12 min.…”

Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection

“…Previous studies employed capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) to measure GSH in red blood cells [10], plasma [11], serum [12], bacteria [13] and various cell lines [14]. However, these methods were designed to determine only the concentrations of GSH and (or) other thiols.…”