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 not available: see fulltext.]
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Acknowledgments This work was supported in part by NIH P30DK050456 and R01AG020866 grants. M.A.D. was supported by NIH Training Grant T32DK007203. The authors thank Rocio Foncea and Wendy Hahn for assistance in obtaining the adipose tissue.
- Capillary electrophoresis
- Hydroxyl radicals
- Micellar electrokinetic chromatography