Relaxation of arterial smooth muscle: a new function of a water-soluble degradation product of coenzyme Q (ubiquinone)

Treatment of coenzyme Q with ozone yielded a degradation product having unmodified ring that retained its spectral characteristics and a truncated side-chain that made it water-soluble. This derivative, but not the intact lipid-quinone, showed relaxation of phenylephrine-contracted rat arterial rings. This effect offers an explanation for the known hypotensive action of exogenous coenzyme Q regardless of its side-chain length.     

Inhibition by coenzyme Q of ethanol- and carbon tetrachloride-stimulated lipid peroxidation in vivo and catalyzed by microsomal and mitochondrial systems

The ability of coenzyme Q to inhibit lipid peroxidation in intact animals as well as in mitochondrial, submitochondrial, and microsomal systems has been tested. Rats fed coenzyme Q prior to being treated with carbon tetrachloride or while being treated with ethanol excrete less thiobarbituric acid-reacting material in the urine than such rats not fed coenzyme Q. Liver homogenates, mitochondria, and microsomes isolated from rats treated with carbon tetrachloride and ethanol catalyze lipid peroxidation at rates which exceed those from animals also fed coenzyme Q. The rate of lipid peroxidation catalyzed by submitochondrial particles isolated from hearts of young, old, and endurance trained elderly rats was inversely proportional to the coenzyme Q content of the submitochondrial preparation in assays in which succinate was employed to reduce the endogenous coenzyme Q. Reduced, but not oxidized, coenzyme Q inhibited lipid peroxidation catalyzed by rat liver microsomal preparations. These results provide additional evidence in support of an antioxidant role for coenzyme Q.     

Comparison of growth stimulation of HeLa cells,HL-60 cells,and mouse fibroblasts by coenzyme Q10

Summary The addition of coenzyme Q₁₀ to culture media stimulates the serum-free growth of HeLa, HL-60 cells, and mouse fibroblasts (Balb/3T3). With HeLa cells, the stimulation by coenzyme Q₁₀ is additive to the stimulation by ferricyanide, an impermeable electron acceptor for the transplasma membrane electron transport. This combined response to coenzyme Q₁₀ and ferricyanide is enhanced with insulin. -Tocopherylquinone can also stimulate the growth of HeLa cells, but vitamin K₁ is inactive. Specificity of quinone effects is indicated. Serum-free growth of Balb/3T3 and SV 40 transformed BaIb/3T3 (SV/T2) cells is also stimulated by coenzyme Qio with stimulation similar to HeLa cells. However, Balb/3T3 cells are not stimulated by ferricyanide, which does not increase the response to coenzyme Q₁₀. The transformed cells (SV/T2) respond better to ferricyanide alone, but the effects of coenzyme Qio and ferricyanide are not additive. Serum-free growth of HL-60 cells is stimulated dramatically by coenzyme Q₁₀. The extent of growth stimulation on HL-60 cells is almost six-fold that of HeLa or Balb/3T3 cells. The stimulation of NADH-ferricyanide reductase (a transmembrane redox enzyme) by coenzyme Q₁₀ with HL-60 cells is similar to their growth pattern in response to coenzyme Q₁₀. Unlike HL-60, HeLa and Balb/3T3 cells show little stimulation of ferricyanide reduction by coenzyme Q₁₀. The stimulatory effect on both ferricyanide reduction and cell growth by the short side-chain coenzyme Q₂ is much less than that of the long side-chain coenzyme Q₁₀. Ferricyanide reduction by HeLa cells is inhibited by coenzyme Q analogs such as 2,3-dimethoxy-5-chloro-6-naphthyl-mercapto-coenzyme Q and 2-methoxy-3-ethoxyl-5-methyl-6-hexadecyl-mercapto-coenzyme Q. However, these inhibitions are reversed by coenzyme Q₁₀. The growth inhibition of HL-60 cells by other coenzyme Q analogs, such as capsiacin can also be reversed by coenzyme Q₁₀. These data indicate that plasma membrane-based NADH oxidation or modification of the membrane quinone redox balance may be a basis for the growth stimulation.     

A Note on the Separation of Natural Mixtures of Bacterial Ubiquinones using Reverse-phase Partition Thin-layer Chromatography and High Performance Liquid Chromatography

Natural mixtures of bacterial ubiquinones (coenzyme Q) were separated according to the length of the polyisoprenyl side-chain using reverse-phase thin-layer chromatography and high performance liquid chromatography. The chromatographic systems described afford a rapid and sensitive means of ubiquinone characterization.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

Protective effects of coenzyme Q10 on ischemia-induced reperfusion injury in ischemic limb models.

We examined simple cold preservation of rat limbs in Euro-Collins' solution to elucidate the protective effect of coenzyme Q10 (CoQ10) on the ischemia-induced reperfusion injury in an ischemic extremity replant model. A total of 126 Lewis rat limb replants were performed. Limbs were amputated from donor rats and preserved at 4 degrees C in Euro-Collins' solution and were orthotopically grafted to isogeneic rats by microsurgical technique. In the experimental groups (n = 42), coenzyme Q10 (10 mg/kg) was injected intraperitoneally into the recipients about 1 hour before reperfusion. In the control groups (n = 84), the same dose of solvent was given by the same route. We evaluated vascular patency of anastomoses by direct observation or microangiogram and performed histologic examinations 7 days after replantation. In the control groups, the ischemic limit was 96 hours. Ischemic limbs treated with coenzyme Q10 showed a statistically significant (p < 0.05) improvement in vascular patency after 72 and 96 hours of ischemia. Histologically, bone viability with osteoblastic activity was maintained in coenzyme Q10-treated animals of the 72-hour ischemic group. We conclude that the protective effect of coenzyme Q10 on reperfusion injury is suggested in this replant model.     

Effect of dopaminergic neurotoxin MPTP/MPP+ on coenzyme Q content

Coenzyme Q10, an endogenous lipophilic antioxidant, plays an indispensable role in ATP synthesis. The therapeutic value of coenzyme Q10 in Parkinson's disease and other neurodegenerative disorders is still being tested and the preliminary results are promising. The 1-methyl-4-phenyl-1, 2, 3, 6 tetrahydropyridine (MPTP)-treated mouse is a valid and accepted animal model for Parkinson's disease. 1-methyl-4-phenylpyridinium (MPP(+)) is an active toxic metabolite of MPTP. MPP(+) and MPTP are known to induce oxidative stress and mitochondrial dysfunction. However, the effect of MPP(+) and MPTP on coenzyme Q is not clearly understood. The present study investigated the in vitro and in vivo effect of MPP(+) and MPTP on coenzyme Q content. Coenzyme Q content was measured using HPLC-UV detection methods. In the in vitro studies, MPP(+) (0-50 microM) was incubated with SH-SY5Y human neuroblastoma cells and NG-108-15 (mouse/rat, neuroblastomaxglioma hybrid) cells. MPP(+) concentration dependently increased coenzyme Q10 content in SH-SY5Y cells. In NG-108-15 cells, MPP(+) concentration dependently increased both coenzyme Q9 and Q10 content. In the in vivo study, mice were administered with MPTP (30 mg/kg, twice 16 h apart) and sacrificed one week after the last administration. Administration of MPTP to mice significantly increased coenzyme Q9 and coenzyme Q10 levels in the nigrostriatal tract. However, MPTP did not affect the coenzyme Q content in the cerebellum, cortex and pons. This study demonstrated that MPP(+)/MPTP significantly affected the coenzyme Q content in the SH-SY5Y and NG-108 cells and in the mouse nigrostriatal tract.     

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 β-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 μg/ml for man, 0.35 μg/ml for guinea-pig and 0.27 μg/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.     

Sensitivity of Caenorhabditis elegans clk-1 mutants to ubiquinone side-chain length reveals multiple ubiquinone-dependent processes

Ubiquinone (coenzyme Q, or Q) is a membrane constituent, whose head group is capable of accepting and donating electrons and whose lipidic side chain is composed of a variable number of isoprene subunits. A possible role for Q as a dietary antioxidant for treating conditions that involve altered cellular redox states is being intensely studied. Mutations in the clk-1 gene of the nematode Caenorhabditis elegans affect numerous physiological rates including behavioral rates, developmental rates, reproduction, and life span. clk-1 encodes a protein associated with the inner mitochondrial membrane that is necessary for Q biosynthesis in C. elegans. clk-1 mutants do not synthesize Q but accumulate demethoxyubiquinone, a Q synthesis intermediate that is able to partially sustain mitochondrial respiration in worms as well as in mammals. Recently, we and others have found that exogenous Q is necessary for the fertility and development of clk-1 mutants. Here, we take advantage of the clk-1 genetic model to identify structural features of Q that are functionally important in vivo. We show that clk-1 mutants are exquisitely sensitive to the length of the side chain of the Q they consume. We also identified differential sensitivity to Q side-chain length between null alleles of clk-1 (qm30 and qm51) and the weaker allele e2519. This allows us to propose a model where we distinguish several types of Q-dependent processes in vivo: processes that are very sensitive to Q side-chain length and processes that are permissive to Q with shorter chains.     

Protective effect of endogenous coenzyme Q on both lipid peroxidation and respiratory chain inactivation induced by an adriamycin-iron complex

Mitochondria from beef heart have been partially depleted of coenzyme Q by pentane extraction. It has been found that lipid peroxidation induced by an adriamycin-iron complex proceeds at a higher rate in this preparation than in coenzyme Q reincorporated mitochondria. Moreover in coenzyme Q depleted mitochondria both NADH and succinate oxidase activities result more affected. These observations indicate that endogenous coenzyme Q can effectively protect mitochondria from membrane lipid oxidative damage induced by adriamycin-iron and can reduce the inactivation of NADH and succinate oxidases.     

An analysis of the role of coenzyme Q in free radical generation and as an antioxidant.

The vital role of coenzyme Q in mitochondrial electron transfer and its regulation, and in energy conservation, is well established. However, the role of coenzyme Q in free oxyradical formation and as an antioxidant remains controversial. Demonstration of the existence of the semiquinone form of coenzyme Q during electron transport, coupled with recent evidence that hydrogen peroxide (but not molecular oxygen) may act as an oxidant of the semiquinone, suggests that the highly reactive OH. radical may be formed from the semiquinone. On the other hand, data exist implicating the Fe-S species as the source of electron transfer chain, free radical production. Additional data exist suggesting instead that the unpaired electron of the coenzyme Q semiquinone most likely dismutases superoxide radicals. These concepts and those arising from observations at several levels of organization including subcellular systems, intact animals, and human subjects in the clinical setting, supporting the concept of reduced coenzyme Q as an antioxidant, will be presented. The results of recent studies on the interaction between the two-electron quinone reductase--DT diaphorase and coenzyme Q10 will be presented. The possibility that superoxide dismutase may interact with reduced coenzyme Q, in conjunction with DT diaphorase inhibiting its autoxidation, will be described. The regulation of cellular coenzyme Q concentrations during oxidative stress accompanying aerobic exercise, resulting in increased protection from free radical damage, will also be presented.     

Isolation and functional expression of human COQ3, a gene encoding a methyltransferase required for ubiquinone biosynthesis

The COQ3 gene in Saccharomyces cerevisiae encodes an O-methyltransferase required for two steps in the biosynthetic pathway of ubiquinone (coenzyme Q, or Q). This enzyme methylates an early Q intermediate, 3,4-dihydroxy-5-polyprenylbenzoic acid, as well as the final intermediate in the pathway, converting demethyl-Q to Q. This enzyme is also capable of methylating the distinct prokaryotic early intermediate 2-hydroxy-6-polyprenyl phenol. A full-length cDNA encoding the human homologue of COQ3 was isolated from a human heart cDNA library by sequence homology to rat Coq3. The clone contained a 933-base pair open reading frame that encoded a polypeptide with a great deal of sequence identity to a variety of eukaryotic and prokaryotic Coq3 homologues. In the region between amino acids 89 and 255 in the human sequence, the rat and human homologues are 87% identical, whereas human and yeast are 35% identical. When expressed in multicopy, the human construct rescued the growth of a yeast coq3 null mutant on a nonfermentable carbon source and restored coenzyme Q biosynthesis, although at lower levels than that of wild type yeast. In vitro methyltransferase assays using farnesylated analogues of intermediates in the coenzyme Q biosynthetic pathway as substrates showed that the human enzyme is active with all three substrates tested.     

Olive oil supplemented with vitamin E affects mitochondrial coenzyme Q levels in liver of rats after an oxidative stress induced by adriamycin.

In this study we have evaluated the supplementation of olive oil with vitamin E on coenzyme Q concentration and lipid peroxidation in rat liver mitochondrial membranes. Four groups of rats were fed on virgin olive, olive plus 200 mg/kg of vitamin E or sunflower oils as lipid dietary source. To provoke an oxidative stress rats were administered intraperitoneally 10 mg/kg/day of adriamycin the last two days of the experiment. Animals fed on olive oil plus vitamin E had significantly higher coenzyme Q and vitamin E levels but a lower mitochondrial hydroperoxide concentration than rats fed on olive oil. Retinol levels were not affected, by either different diets or adriamycin treatment. In conclusion, an increase in coenzyme Q and alpha-tocopherol in these membranes can be a basis for protection against oxidation and improvement in antioxidant capacity.     

Mechanism of O2- generation in reduction and oxidation cycle of ubiquinones in a model of mitochondrial electron transport systems

O2- generation in mitochondrial electron transport systems, especially the NADPH-coenzyme Q10 oxidoreductase system, was examined using a model system, NADPH-coenzyme Q1-NADPH-dependent cytochrome P-450 reductase. One electron reduction of coenzyme Q1 produces coenzyme Q1-. and O2- during enzyme-catalyzed reduction and O2+ coenzyme Q1-. are in equilibrium with O2- + coenzyme Q1 in the presence of enough O2. The coenzyme Q1-. produced can be completely eliminated by superoxide dismutase, identical to bound coenzyme Q10 radical produced in a succinate/fumarate couple-KCN-submitochondrial system in the presence of O2. Superoxide dismutase promotes electron transfer from reduced enzyme to coenzyme Q1 by the rapid dismutation of O2- generated, thereby preventing the reduction of coenzyme Q1 by O2-. The enzymatic reduction of coenzyme Q1 to coenzyme Q1H2 via coenzyme Q1-. is smoothly achieved under anaerobic conditions. The rate of coenzyme Q1H2 autoxidation is extremely slow, i.e., second-order constant for [O2][coenzyme Q1H2] = 1.5 M-1.s-1 at 258 microM O2, pH 7.5 and 25 degrees C.     

Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition.

Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments. In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study .     

Histochemical demonstration of a circadian rhythm of succinate dehydrogenase in rat pineal gland. Influence of coenzyme Q10 addition

Summary Succinate dehydrogenase activity was investigated histochemically in the rat pineal gland. The influence of fixation on the activity pattern, the possible diffusion of enzyme, the nothing dehydrogenase reaction, and the substantivity of the tetrazolium salts and formazans were investigated in control experiments.In rats maintained on a 17/7 h light/dark schedule a distinct circadian rhythm of the succinate dehydrogenase was demonstrated in the pineal gland. Activity was lowest during the day and highest during the night. The dorsocaudal part of the gland showed the highest activity and within the same part of the gland the activity varied between individual pinealocytes. A relative lack of endogenous coenzyme Q, as well as a circadian rhythm of this coenzyme, highly influenced the activity of succinate dehydrogenase. It is concluded that succinate dehydrogenase activity in the pineal gland of the rat is regulated by changing the concentration of the active enzyme itself as well as the level of the endogenous coenzyme Q. Whether this is caused by a circadian rhythm in the synthesis or in the catabolism of the enzyme and the coenzyme was not revealed by the present study.     

Role of glutamine 148 of human 15-hydroxyprostaglandin dehydrogenase in catalytic oxidation of prostaglandin E2

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase/reductase (SDR) family, catalyzes the first step in the catabolic pathways of prostaglandins and lipoxins. This enzyme oxidizes the C-15 hydroxyl group of prostaglandins and lipoxins to produce 15-keto metabolites which exhibit greatly reduced biological activities. A three-dimensional (3D) structure of 15-PGDH based on the crystal structures of the levodione reductase and tropinone reductase-II was generated and used for docking study with NAD+ coenzyme and PGE2 substrate. Three well-conserved residues among SDR family which correspond to Ser-138, Tyr-151, and Lys-155 of 15-PGDH have been shown to participate in the catalytic reaction. Based on the molecular interactions observed from 3D structure of 15-PGDH, we further propose that Gln-148 in 15-PGDH is important in properly positioning the 15-hydroxyl group of PGE2 by hydrogen bonding with the side-chain oxygen atom of Gln-148. This residue is found to be less conserved and replaceable by glutamyl, histidinyl, and asparaginyl residues in SDR family. Accordingly, site-directed mutagenesis of Gln-148 of 15-PGDH to alanine, glutamic acid, histidine, and asparagine (Q148A, Q148E, Q148H, and Q148N) was carried out. The activity of mutant Q148A was not detectable, whereas those of mutants Q148E, Q148H, and Q148N were comparable to or higher than the wild type. This indicates that the side-chain oxygen or nitrogen atom at position 148 of 15-PGDH plays an important role in anchoring C-15 hydroxyl group of PGE2 through hydrogen bonding for catalytic reaction.     

Effect of Pre‐ and Post–Combined Multidoses of Epigallocatechin Gallate and Coenzyme Q10 on Cisplatin‐Induced Oxidative Stress in Rat Kidney

The nephroprotective effect of coenzyme Q10 and epigallocatechin gallate was investigated in rats with acute renal injury induced by a single nephrotoxic dose of cisplatin. Two days prior to cisplatin administration, epigallocatechin gallate and coenzyme Q10 alone and in four different combinations were given for 6 days. The treatment with antioxidants significantly protected the cisplatin‐induced increase in the levels of blood urea nitrogen and serum creatinine. Both the antioxidants alone or in different combinations significantly compensated the increased malondialdehyde and reduced glutathione levels. Moreover, the decrease in the activities of superoxide dismutase, catalase, and glutathione peroxidase and the concentration of selenium, zinc, and copper ions were significantly attenuated in renal tissue. In conclusion, epigallocatechin gallate and coenzyme Q10 are equally effective against cisplatin‐induced nephrotoxicity, whereas the intervention by combining these two antioxidants was found to be highly effective at low doses in attenuating oxidative stress in rat kidney.     

Effect of atorvastatin withdrawal on circulating coenzyme Q10 concentration in patients with hypercholesterolemia

Statin therapy can reduce the biosynthesis of both cholesterol and coenzyme Q10 by blocking the common upstream mevalonate pathway. Coenzyme Q10 depletion has been speculated to play a potential role in statin-related adverse events, and withdrawal of statin is the choice in patients developing myotoxicity or liver toxicity. However, the effect of statin withdrawal on circulating levels of coenzyme Q10 remains unknown. Twenty-six patients with hypercholesterolemia received atorvastatin at 10 mg/day for 3 months. Serum lipid profiles and coenzyme Q10 were assessed before and immediately after 3 months and were also measured 2 and 3 days after the last day on the statin. After 3 months' atorvastatin therapy, serum levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and coenzyme Q10 (0.43 +/- 0.23 to 0.16 +/- 0.10 microg/mL) were all significantly reduced (all p<0.001). On day 2 after the last atorvastatin, the coenzyme Q10 level was significantly elevated (0.37 +/- 0.16 microg/mL) and maintained the same levels on day 3 (0.39 +/- 0.18 microg/mL) compared with those on month 3 (both p< 0.001), while TC and LDL-C did not significantly change within the same 3 days. These results suggest that statin inhibition of coenzyme Q10 synthesis is less strict than inhibition of cholesterol biosynthesis.     

Elevation of tissue coenzyme Q (ubiquinone) and cytochrome c concentrations by endurance exercise in the rat

Six months of enforced and voluntary endurance training of young female Wistar rats resulted in significant decreases of body weight and gastrocnemius muscle wet weight and protein content, and increases in heart weight and protein content, and liver protein content. The coenzyme Q and cytochrome c concentrations of cardiac, gastrocnemius, and deep red region of the vastus lateralis muscles were increased, while small or nonsignificant trends toward increases in cytochrome c and coenzyme Q were seen in kidney, brain, lung, liver, internal + external oblique muscles, and the superficial white region of the vastus lateralis muscle. These results are discussed with regard to several roles for coenzyme Q in cellular function.