Takeo Kishi

Determination of reduced and total ubiquinones in biological materials by liquid chromatography with electrochemical detection

A convenient and reliable liquid chromatographic (LC) method with electrochemical detection (ED) was developed for the determination of reduced (ubiquinol) and total ubiquinones in biological materials. After extraction of samples with n-hexane, ubiquinol was separated on a reversed-phase column and assayed directly by ED. In order to determine the total amount of a ubiquinone in biological samples, the unbiquinone was converted into the corresponding reduced form by treatment with sodium borohydride. No significant interfering peak (plastoquinol-9, ubichromenol-9, etc.) was observed in the elution areas of ubiquinol-7 to -11. This LC-ED method was about 70 times more sensitive than the previous LC-UV method and was able to detect 150 pg of ubiquinol-10. The method was applied satisfactorily to the determination of the contents of ubiquinol homologues in biological materials. The content of ubiquinols is a major component of the total ubiquinones in human plasma and urine and rat plasma and liver, but a minor component in rat heart and kidney.

High-performance liquid chromatography of coenzyme Q-related compounds and its application to biological materials

A convenient and precise method for the separation and determination of coenzyme Q (CoQ)-related compounds (CoQ homologues, plastoquinone-9, ubichromenol-9, etc.) was developed using high-performance liquid chromatography (HPLC). All compounds tested were separated using a reverse-phase column with a suitable mobile phase and detected at a wavelength of 275 nm. CoQ extracts in plasma and erythrocytes were purified by thin-layer chromatography prior to HPLC analysis, but such purification was not necessary when determining CoQ in urine and tissues. Hydroquinone forms of CoQ existing in animal tissues were oxidized to the corresponding quinone forms with potassium hexacyanoferrate(III). This HPLC method was applied satisfactorily to the determination of the contents of CoQ homologues in human and animal samples. CoQ10 was the only homologue detected in human samples, and CoQ8, CoQ9 and CoQ10 were native homologues of CoQ in rat tissues. Ubichromenol-9 and plastoquinone-9 were not detected in these samples.

Distribution of ubiquinone and ubiquinol homologues in rat tissues and subcellular fractions

The oxidized (UQox) and reduced (UQred) forms of ubiquinone (UQ) homologues in rat tissues and subcellular fractions were analyzed to elucidate their distribution and physiological role. UQ-9 and UQ-10 were detected in all tissues studied, and UQ-9 was the predominant homologue. The total amount of UQox-10 and UQred-10 was 20–50% that of UQox-9 and UQred-9. The levels of these homologues were highest in heart with lesser amounts occurring in kidney, liver and other organs. In liver and blood plasma, the UQred homologue amounted to 70–80% of the total UQ (UQox+UQred=t-UQ). UQred was less than 30% of t-UQ in other tissues and blood cells. t-UQ was much higher in leukocytes and platelets in blood than in erythrocytes. In erythrocytes, t-UQ was exclusively located in the cell membranes. UQox and UQred were also found in all subcellular fractions isolated from liver and kidney in about the same ratio as UQred/t-UQ was present in the whole organ. The levels of UQox and UQred per mg protein in subcellular fractions from liver were highest in mitochondria, with lesser amounts present in plasma membranes, lysosomes, Golgi complex, nuclei, microsomes and cytosol. In the mitochondria, the outer membranes were richer in t-UQ than the inner membranes. In the Golgi complex, the light and intermediate fractions were rich in t-UQ when compared to the heavy fraction. The possible physiological role of UQox and UQred in tissues and subcellular fractions is discussed.

Protective Effects of Coenzyme Q10 on Decreased Oxidative Stress Resistance Induced by Simvastatin

The effects of simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), on oxidative stress resistance and the protective effects of coenzyme Q (CoQ) were investigated. When simvastatin was administered orally to mice, the levels of oxidized and reduced CoQ9 and CoQ10 in serum, liver, and heart, decreased significantly when compared to those of control. The levels of thiobarbituric acid reactive substances induced by Fe2+-ascorbate in liver and heart mitochondria also increased significantly with simvastatin. Furthermore, cultured cardiac myocytes treated with simvastatin exhibited less resistance to oxidative stress, decreased time to the cessation of spontaneous beating in response to H2O2 addition, and decreased responsiveness to electrical field stimulation. These results suggested that oral administration of simvastatin suppresses the biosynthesis of CoQ, which shares the same biosynthesis pathway as cholesterol up to farnesyl pyrophosphate, thus compromising the physiological function of reduced CoQ, which possesses antioxidant activity. However, these undesirable effects induced by simvastatin were alleviated by coadministering CoQ10 with simvastatin to mice. Simvastatin also reduced the activity of NADPH-CoQ reductase, a biological enzyme that converts oxidized CoQ to the corresponding reduced CoQ, while CoQ10 administration improved it. These findings may also support the efficacy of coadministering CoQ10 with statins.

Quantitative determination of coenzyme Q10 in human blood for clinical studies

A quantitative method for the determination of coenzyme Q10 (CoQ10) in human blood has been devised which allows recovery of essentially 100% of the CoQ10. The use of whole blood rather than plasma includes the CoQ10 in white cells. The method utilizes TLC instead of saponification to fractionate lipid impurities, because CoQ10 is sensitive to saponification, and utilizes CoQ11 as an internal standard which is advantageous over CoQ9 and a synthetic quinone. The final step of HPLC frequently reveals a peak with a retention time like that of CoQ9 which, being less than that of CoQ10, can be near other peaks of impurities.

A novel ubiquinone reductase activity in rat cytosol

Ubiquinone (UQ) reductase activity which reduces UQ to ubiquinol (UQH2) in rat tissues was roughly proportional to the UQH2/total UQ ratio in respective tissues. The honest activity was found in the liver, showing the highest UQH2/total UQ ratio. A greater part of liver UQ reductase activity was located in the cytosol. Within a week, the liver UQ reductase activity decreased by 80% even at −20°C. The DT‐diaphorase activity was stable. UQ reductase required NADPH as the hydrogen donor and was not inhibited by a less than 1μM concentration of dicoumarol. There was no stimulation of UQ reductase in the presence of bovine serum albumin nor in Triton X‐100. Yet, both stimulated DT‐diaphorase. As a result, UQ reductase appeared to be a novel NADPH‐UQ oxidoreductase and responsible for the UQ redox state in liver.

Effect of dicumarol, a Nad(P)h: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes.

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 w M dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro, was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 w M dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.

The Quality Control Assessment of Commercially Available Coenzyme Q10-Containing Dietary and Health Supplements in Japan

Coenzyme Q10 (CoQ10) has been widely commercially available in Japan as a dietary and health supplement since 2001 and is used for the prevention of lifestyle-related diseases induced by free radicals and aging. We evaluated CoQ10 supplements to ensure that these supplements can be used effectively and safely. Commercially available products were selected and assessed by the quality control tests specified in the Japanese Pharmacopoeia XV. When the disintegration time of CoQ10 supplements was measured, a few tested supplements did not completely disintegrate even after incubation in water for an hour at 37°C. In the content test, many samples were well controlled. However, a few supplements showed low recovery rates of CoQ10 as compared to manufacturer’s indicated contents. Among soft capsule and liquid supplements, the reduced form of CoQ10 (H2CoQ10), as well as the oxidized form, was detected by HPLC with electrochemical detector. The results for experimental formulated CoQ10 supplements demonstrated that H2CoQ10 was produced by the interaction of CoQ10 with vitamins E and/or C. From these results, we concluded that quality varied considerably among the many supplement brands containing CoQ10. Additionally, we also demonstrated that H2CoQ10 can be detected in some foods as well as in CoQ10 supplements.

Protective effect of γ-glutamylcysteinylethyl ester on dysfunction of the selenium-deficient rat heart

We investigated the protective effect of intracellular GSH against cardiac dysfunction in selenium (Se)-deficient neonatal rats and cultured fetal rat myocytes. A Se-deficient diet with or without daily subcutaneous injections of gamma-glutamylcysteinylethyl ester (gammay-GCE) (a membrane-permeating GSH precursor) was given to rats from gestation day 4 via the dam to postnatal day 14. Se deficiency induced a 62% incidence of electrocardiographic abnormalities such as sinus arrhythmias or extrasystole, a 63% reduction in dP/dt in the left ventricle, and an increase in thiobarbituric acid reacting substances (TBARS), but no ultrastructural cardiac lesions were observed. Administration of gamma-GCE increased the intracellular GSH concentration ([GSH]i) of both neonatal rat hearts and cultured fetal rat cardiac myocytes. gamma-GCE-like sodium selenite prevented the cardiac dysfunction and the TBARS increment. gamma-GCE also prevented H2O2 toxicity in the cultured myocytes. The Vmax, but not the Km, for GSH of Se-dependent GSH peroxidase (Se-Gpx) activity in Se-deficient rat heart homogenates was one-third that of normal rat heart homogenates. Although gamma-GCE did not affect the Se-Gpx Vmax and Km for GSH, it did induce a substantial and significant increase in [GSH]i, which was postulated to increase the velocity of H2O2 decomposition by Se-Gpx activity 1.6-fold. These data suggest that the increase in [GSH]i may have played a role in preventing the TBARS increase and cardiac dysfunction in Se-deficient rats.

Cardiostimulatory action of coenzyme Q homologues on cultured myocardial cells and their biochemical mechanisms.

SummaryThe effect of coenzyme Q (CoQ) homologues on the beating of myocardial cells was investigated in cultured cell sheets from mouse fetuses and quail embryos. Myocardial cell sheets grown in Eagles minimum essential medium with fetal bovine serum showed very weak and irregular beating when this serum was removed from the medium. However, the depressed beating rate and amplitude recovered almost completely within a few minutes by adding CoQ10 to the medium, and the effect of Co10 continued over 1 h. CoQ9 showed a cardiostimulatory effect similar to that of CoQ10 but CoQ8 and COQ7 showed almost no effect. Short homologues (less than CoQ4) inhibited the beating of cell sheets. The cardiostimulatory effect of CoQ10 was not blocked by atenolol, a selective β-blocker. In addition, CoQ10 stimulated the formation of ATP, not cAMP. CoQ0 and COQ3 inhibited beating rates by inhibiting ATP formation. In conclusion, only native CoQ homologues having a nona- or decaprenyl group showed a cardiostimulatory effect on cultured myocardial cells, probably by stimulating mitochondrial ATP formation.