Satoru Ikenoya

Simultaneous determination of reduced and oxidized ubiquinones

Publisher Summary Quantitative analyses of individual ubiquinones (Q) homologs in biological samples have been performed by high-performance liquid chromatography (HPLC) combined with an ultraviolet spectrometric detector (UVD) or by mass spectrometry (MS). An electrochemical detector (ECD) for HPLC was confirmed to be simple and sensitive for the determination of Q. However, only Q was determined by these methods. For the determination of QH 2 (ubiquinol) and Q in mitochondria, submitochondrial particle and cell-free bacterial homogenates, the dual-wavelength spectrometric method has been generally used. The method, however, cannot simultaneously measure the amounts of QH 2 and Q in whole tissues owing to the presence of vitamin A and other interfering compounds, which have an absorbance in the same spectral region as Q and undergo an absorption change by chemical reduction. The dual-wavelength spectrometric method cannot separately determine individual Q homologs. The analytical procedure described was developed to provide a rapid, sensitive, and direct assay method for QH 2 and Q in biological samples. This method is based on extraction from tissues, mitochondria, microsomal fractions, or plasma with organic solvents, followed by quantitation by means of reversed-phase chromatography with UVD and ECD.

Studies on reduced and oxidized coenzyme Q (ubiquinones). II. The determination of oxidation-reduction levels of coenzyme Q in mitochondria, microsomes and plasma by high-performance liquid chromatography.

Reduced and oxidized coenzyme Q10 (Q10H2 and Q10) in guinea-pig liver mitochondria were rapidly extracted and determined by high-performance liquid chromatography (HPLC). The percentages of Q10H2 as compared to the total (sum of Q10 and Q10H2) were increased by the addition of respiratory substrates such as succinate, malate and beta-hydroxybutyrate (State 4). The levels of Q10H2 in State 4 were increased more extensively with electron-transport inhibitors such as KCN, NaN3 and antimycin A. These results indicate that the method for determining Q10H2 and Q10 by HPLC is quite useful for investigation of the physiological function of coenzyme Q in mitochondria and other organelles. The reduced and oxidized coenzyme Q levels of rat liver mitochondria, which contain both coenzyme Q9 and coenzyme Q10, were measured simultaneously. The results suggest that coenzymes Q9 and Q10 play a similar role as an electron carriers. The liver microsomes of guinea-pig contained approx. 133 nmol total coenzyme Q10 per g protein. The Q10H2 levels of microsomes were increased from 46.5 to 67.5 and 64.8% with NADH and NADPH, respectively. The plasma levels of total coenzyme Q were 0.92 microgram/ml for man, 0.35 microgram/ml for guinea-pig and 0.27 microgram/ml for rat. The reduced coenzyme Q were also present in those plasma samples. The levels of reduced coenzyme Q were 51.1, 48.9 and 65.3%, respectively.

Simultaneous determination of ubiquinone-10 and ubiquinol-10 in tissues and mitochondria by high performance liquid chromatography

Abstract A method of high performance liquid chromatography with both of a UV detector and an electrochemical detector for the simultaneous determination of ubiquinone and ubiquinol was established. This method could sensitively and specifically measure the redox state of ubiquinone in mitochondria and tissues.