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Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis

Carru, Ciriaco and Deiana, Luca and Sotgia, Salvatore and Pes, Giovanni Mario and Zinellu, Angelo (2004) Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis. Electrophoresis, Vol. 25 (6), p. 882-889. eISSN 1522-2683. Article.

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DOI: 10.1002/elps.200305768

Abstract

High concentrations of total plasma thiols such as cysteine and homocysteine are important risk factors for atherosclerosis and cardiovascular diseases. We have recently described a new laser-induced fluorescence capillary electrophoresis (CE-LIF) method to measure total plasma thiols, in which the baseline separation of cysteinylglycine, homocysteine, cysteine, and glutathione was achieved by adding the organic base N-methyl-D-glucamine to the run buffer. However, because the active fractions of homocysteine and cysteine responsible for vascular injuries are still unknown, research calls for a set up of methods able to analyze different forms of plasma thiols. In this paper, we present an improvement of our previous method that allows the measurement of different thiol forms. Total, reduced, and free thiols were measured by varying the order of disulfide reduction with tributylphosphine and proteins precipitation with 5-sulfosalicylic acid. After derivatization with 5-iodoacetamidofluorescein, samples were separated and measured by CE-LIF using a phosphate/borate buffer in the presence of 75 mmol/L N-methyl-D-glucamine. Oxidized thiols and protein bound thiols were calculated by difference, free minus reduced and total minus free form, respectively. Linearity, reproducibility, analytical recovery, and sensitivity were evaluated. The assay was used to measure the thiols redox status in 15 plasma samples from healthy volunteers.

Item Type:Article
ID Code:2529
Status:Published
Refereed:Yes
Uncontrolled Keywords:Atherosclerosis, capillary electrophoresis, 5-iodoacetamidofluorescein, N-methyl-D-glucamine, oxidative stress, redor status, 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 10:08

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