Thiol redox status evaluation in red blood cells by capillary electrophoresis-laser induced fluorescence detection

Abstract

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 degrees 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 x 75 microm 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 gamma-glutamylcysteine (GluCys) were baseline-resolved in approximately 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.