Measurement of reduced and oxidized coenzyme Q9 and coenzyme Q10 levels in mouse tissues by HPLC with coulometric detection

Abstract

Background: Ubiquinone-responsive multiple respiratory chain dysfunction due to coenzyme Q(10) (CoQ(10)) deficiency has been previously identified in muscle biopsies. However, previous methods are unreliable for estimating CoQ(10) redox status in tissue. We developed an accurate method for measuring tissue concentrations of reduced and oxidized coenzyme Q (CoQ).

Methods: Mouse tissues were weighed in the frozen state and homogenized with cold 1-propanol on ice. After solvent extraction, centrifugation and filtration, the filtrate was subsequently analyzed by reversed-phase HPLC with coulometric detection.

Results: Reference calibration curves were used to determine reduced and oxidized coenzyme Q(9) (CoQ(9)) and CoQ(10) concentrations in tissues. The method is sensitive ( approximately 15 microg/l), reproducible (6% CV) for CoQ(9) and CoQ(10), and linear up to 20 mg/l for CoQ(9) and CoQ(10). Analytical recoveries were 90-104%. In mouse tissues the amounts of total CoQ (TQ) ranged from 261 to 1737 nmol/g of protein. Total CoQ(9) levels are comparable with the values of those previously reported. CoQ is found to be mostly in the reduced form in mouse liver ( approximately 87%), heart ( approximately 60%), and muscle tissues ( approximately 58%); in the brain, most of the CoQ is in the oxidized state ( approximately 65%).

Conclusion: This procedure provides a precise, sensitive, and direct assay method for the determination of reduced and oxidized CoQ(9) and CoQ(10) in mouse hindleg muscle, heart, brain, and liver tissues.