Plasma thiols redox status by laser-induced fluorescence capillary electrophoresis.
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.
Carru
Ciriaco
C
Chair of Clinical Biochemistry, University of Sassari, Viale San Pietro 43/B, I-07100 Sassari, Italy. carru@uniss.it
Deiana
Luca
L
Sotgia
Salvatore
S
Pes
Giovanni Mario
GM
Zinellu
Angelo
A
D019075:Electrophoresis, Capillary / Q000379:methods*; D005453:Fluorescence; D007834:Lasers; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000097:blood* / Q000737:chemistry
D013438:Sulfhydryl Compounds