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Thiol redox status evaluation in red blood cells by capillary electrophoresis‐laser induced fluorescence detection
Author(s) -
Zinellu Angelo,
Sotgia Salvatore,
Usai Maria Franca,
Chessa Roberto,
Deiana Luca,
Carru Ciriaco
Publication year - 2005
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200400042
Subject(s) - capillary electrophoresis , thiol , laser induced fluorescence , fluorescence , chemistry , electrophoresis , chromatography , redox , analytical chemistry (journal) , biochemistry , optics , inorganic chemistry , physics
Thiols and in particular glutathione (GSH) play a central role in human metabolism, including the detoxification of xenobiotics, cell homeostasis, radioprotection, and antioxidant defence. Here, a new method is provided for the measurement of reduced and total forms of thiols in red blood cells. In order to minimize oxidation of reduced thiols, a water erythrocyte lysis (15 min at 4°C) was performed followed by a protein precipitation step with acetonitrile. The supernatant was rapidly derivatized with 5‐iodoacetoamidefluorescein that trapped thiol groups, thus minimizing auto‐oxidation. Derivatized samples were separated in a 57 cm × 75 μm ID capillary by using 5 mmol/L sodium phosphate, 4 mmol/L boric acid as electrolyte solution with 75 mmol/L N ‐methyl‐ D ‐glucamine at pH 11.0. Under these conditions, cysteinylglycine (CysGly), cysteine (Cys), glutathione, and γ‐glutamylcysteine (GluCys) were baseline‐resolved in ˜ 4 min. Precision tests showed a good repeatability of our method both for migration times (coefficient of variation CV < 0.8%) and areas (CV < 3.3%). Furthermore, a good reproducibility of intrassay and interassay tests was obtained (CV < 5% and CV < 8%, respectively). The method was employed to investigate the effect of acidic precipitation on intracellular thiol concentration. Our data suggest that sample acidification causes a modification of the measured redox thiol status due to the development of a pro‐oxidant environment; moreover, the thiol redox status of red blood cells was evaluated in 22 healthy volunteers.