Validation of high-performance liquid chromatography-boron-doped diamond detection for assessing hepatic glutathione redox status.
Glutathione redox status is a commonly used oxidative stress biomarker. High-performance liquid chromatography-ultraviolet (HPLC-UV) and HPLC-electrochemical detection (HPLC-ECD) have been used to assess glutathione status but have potential limitations due to challenging sample preparation procedures or electrochemical signal degradation. Thus, this study aimed to validate an HPLC-ECD approach using boron-doped diamond (BDD), a novel electrode material exhibiting excellent electrochemical stability. Liver homogenates from obese (ob/ob) mice and their lean littermates (n=4/genotype) as well as from rats fed high- or low-fat diets (n=8/treatment) were analyzed in parallel by HPLC-BDD and -UV. HPLC-BDD responses for reduced glutathione (GSH) and oxidized glutathione (GSSG) were linear over more than four orders of magnitude at 1475 mV, the optimal oxidation potential. Within- and between-day precision values of GSH, GSSG, and GSH/GSSG were 2.1% to 7.9%, and accuracy values of GSH and GSSG were 96% and 105%, respectively. Electrochemical responses were stable up to 48 h of continuous system use. Using HPLC-BDD and -UV, hepatic GSH, GSSG, and GSH/GSSG from mice (r=0.64-0.94) and rats (r=0.79-0.92) were well correlated (P<0.05), and no significant differences in thiol levels were observed between detection methods. Collectively, our findings support HPLC-BDD as a relatively simple, accurate, and validated approach for evaluating hepatic glutathione redox status.
Park
Hea Jin
HJ
Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269, USA.
Mah
Eunice
E
Bruno
Richard S
RS
D000818:Animals; D001895:Boron / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018130:Diamond / Q000737:chemistry*; D004041:Dietary Fats; D055664:Electrochemical Techniques / Q000379:methods*; D004566:Electrodes; D005978:Glutathione / Q000737:chemistry*; D008099:Liver / Q000378:metabolism*; D051379:Mice; D008820:Mice, Obese; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D051381:Rats; D013696:Temperature; D013997:Time Factors
D004041:Dietary Fats; D018130:Diamond; D005978:Glutathione; D001895:Boron