Capillary electrophoretic analysis of hydroxyl radicals produced by respiring mitochondria

• Published in: Analytical and bioanalytical chemistry 2013-07, Vol.405 (18), p.6053-6060
• Main Authors: Donoghue, Margaret A., Xu, Xin, Bernlohr, David A., Arriaga, Edgar A.
• Format: Article
• Language: English
• Subjects: Adipose tissue; Adipose Tissue, Brown - metabolism; Analytical Chemistry; Animals; Biochemistry; Body fat; Capillarity; Capillary electrophoresis; Carbonyl compounds; Cell Respiration; Cells, Cultured; Characterization and Evaluation of Materials; Chemical properties; Chemistry; Chemistry and Materials Science; Chromatography; Composition; Diet, High-Fat - adverse effects; Dimethyl Sulfoxide - chemistry; Electrophoresis; Electrophoresis, Capillary - methods; Fluorescein; Fluoresceins - analysis; Fluoresceins - chemistry; Fluorescence; Food Science; Hydrazones - chemistry; Hydrazones - pharmacology; Hydrogen peroxide; Hydroxyl Radical - analysis; Hydroxyl radicals; Identification and classification; Laboratory Medicine; Limit of Detection; Liquid chromatography; Mass spectrometry; Mathematical analysis; Methods; Mice; Mice, Inbred C57BL; Mice, Obese; Microscopy; Mitochondria; Mitochondria - chemistry; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondrial DNA; Monitoring/Environmental Analysis; Myoblasts - metabolism; Obesity; Oxidative stress; Physiological aspects; Purification; Radicals (Chemistry); Rats; Raw materials; Research Paper
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-013-7022-y

Here, we report the use of a capillary electrophoretic method with laser-induced fluorescence detection to evaluate hydroxyl radicals produced by respiring mitochondria. The probe, hydroxyphenylfluorescein (HPF), is separated from the product, fluorescein, in under 5 min with zeptomole and attomole limits of detection for fluorescein and HPF, respectively. Purification of the probe with a C-18 SPE column is necessary to reduce the fluorescein impurity in the probe stock solution from 0.4 % to less than 0.001 %. HPF was responsive to hydroxyl radicals produced by isolated mitochondria from L6 cells, and this signal was blunted when DMSO was added to scavenge hydroxyl radicals and when carbonyl cyanide m -chlorophenylhydrazone was added to depolarize the mitochondria. The method was used to compare hydroxyl radical levels in mitochondria isolated from brown adipose tissue of lean and obese mice. Mitochondria from obese mice produced significantly more hydroxyl radicals than those from lean mice. Figure Caption for figure abstract: Mitochondria are the main source of cellular reactive oxygen species. While all are of interest, the specific detection of hydroxyl radicals can be achieved with the fluorescent probe HPF. MEKC-LIF is used to separate the probe HPF from its product, fluorescein. This was demonstrated by treating enriched mitochondria fractions from L6 cells with HPF. Electropherograms show an increase in fluorescein peak area when mitochondria are stimulated with 100 μM Fe2+.