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

Zinellu, Angelo and Sotgia, Salvatore and Posadino, Anna Maria and Pasciu, Valeria and Perino, Maria G. and Tadolini, Bruna and Deiana, Luca and Carru, Ciriaco (2005) Highly sensitive simultaneous detection of cultured cellular thiols by laser induced fluorescence-capillary electrophoresis. Electrophoresis, Vol. 26 (6), p. 1063-1070. eISSN 1522-2683. Article.

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DOI: 10.1002/elps.200406191

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°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.

Item Type:Article
ID Code:2496
Status:Published
Refereed:Yes
Uncontrolled Keywords:Capillary electrophoresis, cultured cells, 5-iodoacetamidofluorescein, N-methyl-D-glucamine, thiols
Subjects:Area 05 - Scienze biologiche > BIO/12 Biochimica clinica e biologia molecolare clinica
Divisions:001 Università di Sassari > 01 Dipartimenti > Scienze biomediche
Publisher:Wiley
eISSN:1522-2683
Deposited On:18 Aug 2009 10:07

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