Reduced Form Of Coenzyme Research Articles

Biologically Mediated RAFT Polymerization for Electrochemical Sensing of Kinase Activity

The assay of kinase activity with ultrahigh sensitivity is important to medical diagnostics and drug discovery. Herein, we report the biologically mediated RAFT polymerization (BMRP) and its potential use as an efficient amplification strategy in the ultrasensitive electrochemical sensing of kinase activity. In BMRP, the reversible addition-fragmentation chain-transfer (RAFT) process is initiated and sustained by the reduced form of coenzyme I (i.e., NADH), which can efficiently mediate the direct fragmentation of thiocarbonylthio (TCT) compounds (or the TCT-capped dormant chains) to produce an initiating/propagating radical under mild conditions. Due to the absence of exogenous radicals, the notorious radical termination in RAFT equilibrium can be greatly suppressed. For the sensing of kinase activity, the recognition peptides, without carboxyl groups, are immobilized via the Au-S self-assembly. After phosphorylation, TCT compounds (as RAFT agents) are tethered to the enzymatically generated phosphate groups via the carboxylate-Zr(IV)-phosphate (CZP) linkage. Subsequently, the BMRP of ferrocenylmethyl methacrylate (FcMMA) results in the labeling of each phosphate group with hundreds to thousands of Fc tags, thereby greatly amplifying the sensing signal. Obviously, the BMRP-based strategy is biologically friendly, highly efficient, uncomplicated, and quite low-cost. The detection limit of 1.85 mU/mL has been achieved toward the selective sensing of the cAMP-dependent protein kinase (PKA). Moreover, the proposed kinase sensor is applicable to inhibitor screening and kinase activity sensing in serum samples. By virtue of its low cost, high sensitivity and selectivity, and uncomplicated operation, the proposed kinase sensor holds great potential in medical diagnostics and drug discovery.

The reduced form of coenzyme Q10 mediates distinct effects on cholesterol metabolism at the transcriptional and metabolite level in SAMP1 mice

Studies in humans and mice indicate a role for coenzyme Q(10) (CoQ(10)) in gene expression. To analyze this function in relation to metabolism, SAMP1 mice were supplemented with the reduced (ubiquinol) or oxidized (ubiquinone) form of CoQ(10) (500 mg/kg BW/d) for 14 months. Microarray analyses in liver tissues of SAMP1 mice identified 946 genes as differentially expressed between ubiquinol-treated and control animals (≥1.5-fold, P < 0.05). Text mining analyses revealed for a part of the ubiquinol-regulated genes, a functional connection in PPARα and LXR/RXR signalling pathways. Because these pathways are involved in cholesterol homeostasis, relevant metabolites were determined by gas chromatography/mass spectrometry (GC/MS). We found a significant increase of desmosterol (2.0-fold, P < 0.001) in the liver of ubiquinol-supplemented SAMP1 mice when related to control animals. In agreement, cholesterol concentrations were also distinctly increased (1.3-fold, P = 0.057). The Q(10)H(2)-induced PPARα and LXR/RXR gene expression signatures and effects on cholesterol metabolism were not apparent for the oxidized form of CoQ(10). In conclusion, the reduced form of CoQ(10) mediates distinct effects on cholesterol metabolism at the transcriptional and metabolite level in SAMP1 mice.

Supplementation with the reduced form of Coenzyme Q10 decelerates phenotypic characteristics of senescence and induces a peroxisome proliferator‐activated receptor‐α gene expression signature in SAMP1 mice

Our present study reveals significant decelerating effects on senescence processes in middle-aged SAMP1 mice supplemented for 6 or 14 months with the reduced form (Q(10)H(2), 500 mg/kg BW/day) of coenzyme Q(10) (CoQ(10)). To unravel molecular mechanisms of these CoQ(10) effects, a genome-wide transcript profiling in liver, heart, brain and kidney of SAMP1 mice supplemented with the reduced (Q(10)H(2)) or oxidized form of CoQ(10) (Q(10)) was performed. Liver seems to be the main target tissue of CoQ(10) intervention, followed by kidney, heart and brain. Stringent evaluation of the resulting data revealed that Q(10)H(2) has a stronger impact on gene expression than Q(10), primarily due to differences in the bioavailability. Indeed, Q(10)H(2) supplementation was more effective than Q(10) to increase levels of CoQ(10) in the liver of SAMP1 mice. To identify functional and regulatory connections of the "top 50" (p<0.05) Q(10)H(2)-sensitive transcripts in liver, text mining analysis was used. Hereby, we identified Q(10)H(2)-sensitive genes which are regulated by peroxisome proliferator-activated receptor-alpha and are primarily involved in cholesterol synthesis (e.g. HMGCS1, HMGCL and HMGCR), fat assimilation (FABP5), lipoprotein metabolism (PLTP) and inflammation (STAT-1). These data may explain, at least in part, the decelerating effects on degenerative processes observed in Q(10)H(2)-supplemented SAMP1 mice.

UBIQUINONE-10 AS AN ANTIOXIDANT

ABSTRACT Ubiquinol, the reduced form of coenzyme Q, is known as a lipid antioxidant. Its fully oxidized form, ubiquinone, would theoretically not have this activity. However, we report that ubiquinone-10, the oxidized form of coenzyme Q, demonstrated antioxidant activity in model studies against a lipid-soluble free radical generator, 2,2′-azobis(2,4-dimethylvaleronitrile). This was demonstrated at both 1:100 and 1:1,000 ratios of ubiquinone-10 to lipid (for both methyl linoleate and methyl linolenate). Ubiquinone-10 should therefore not be discounted as a possible antioxidant in more complex systems such as food. PRACTICAL APPLICATIONS Coenzyme Q (ubiquinol/ubiquinone) is an important component of electron transport in biological tissues and is also classified as a potent antioxidant. It is generally believed that the form that is active as an antioxidant is the reduced ubiquinol. In evaluating the loss of antioxidants in mackerel light and dark muscle tissues, it was observed that the oxidized form, ubiquinone, comprised over 87% of the total coenzyme Q in light muscle and over 93% in dark muscle and this observation did not change under anaerobic conditions. This study was done to determine if the oxidized form of coenzyme Q, ubiquinone, was capable of acting as an antioxidant. In this study, it is shown that it can function in this manner.

Study on safety and bioavailability of ubiquinol (Kaneka QH ™) after single and 4-week multiple oral administration to healthy volunteers

The safety and bioavailability of ubiquinol (the reduced form of coenzyme Q 10), a naturally occurring lipid-soluble nutrient, were evaluated for the first time in single-blind, placebo-controlled studies with healthy subjects after administration of a single oral dose of 150 or 300 mg and after oral administration of 90, 150, or 300 mg for 4 weeks. No clinically relevant changes in results of standard laboratory tests, physical examination, vital signs, or ECG induced by ubiquinol were observed in any dosage groups. The C max and AUC 0–48 h derived from the mean plasma ubiquinol concentration-time curves increased non-linearly with dose from 1.88 to 3.19 μg/ml and from 74.61 to 91.76 μg h/ml, respectively, after single administration. Trough concentrations had nearly plateaued at levels of 2.61 μg/ml for 90 mg, 3.66 μg/ml for 150 mg, and 6.53 μg/ml for 300 mg at day 14, and increased non-linearly with dose in the 4-week study. In conclusion, following single or multiple-doses of ubiquinol in healthy volunteers, significant absorption of ubiquinol from the gastrointestinal tract was observed, and no safety concerns were noted on standard laboratory tests for safety or on assessment of adverse events for doses of up to 300 mg for up to 2 weeks after treatment completion.

UV radiation increases the reduced coenzyme Q ratio in marine bacteria

Oxidative stress is often indicated by an oxidative shift in cellular coenzyme Q (ubiquinol/ubiquinone) redox status. However, exposing two cultures of marine bacteria to intense UVA radiation increased their relative abundance of the reduced form of coenzyme Q, presumably as an adaptive response to photo-oxidative stress. This UV-signalling pathway in marine bacteria may be useful to examine molecular processes that regulate cellular coenzyme Q redox balance.

Coenzyme Q 10 and differences in coronary heart disease risk in Asian Indians and Chinese

Indians or South Asians have been found to be particularly susceptible to coronary heart disease (CHD) in many countries. A novel risk factor for CHD may be coenzyme Q 10 (CoQ 10). In this study, plasma CoQ 10 (including ubiquinol-10, CoQ 10H 2, and total CoQ 10), various lipid parameters, and antioxidant levels were determined in a random sample of Indians and Chinese from the general population of Singapore. The reduced form of coenzyme Q 10, CoQ 10H 2, and total Q 10 concentrations in plasma were significantly lower in Indian males than Chinese males. Although no significant differences were found in plasma concentrations of total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL) between the two ethnic groups, the ratios of ubiquinol and total CoQ 10 to triglycerides, total cholesterol, and LDL were significantly lower in Indian males than Chinese males. There were no significant ethnic differences in other antioxidant levels, including trans-retinol, α-tocopherol, and ascorbic acid. The consistently lower values of coenzyme Q 10, especially its reduced form, in Indian males may contribute to the higher susceptibility of this ethnic group to coronary heart disease.

Coenzyme Q blocks biochemical but not receptor-mediated apoptosis by increasing mitochondrial antioxidant protection

Generation of free radicals is often associated with the induction and progression of apoptosis. Therefore, antioxidants can prove anti-apoptotic, and can help to elucidate specific apoptotic pathways. Here we studied whether coenzyme Q, present in membranes in reduced (ubiquinol) or oxidised (ubiquinone) forms, can affect apoptosis induced by various stimuli. Exposure of Jurkat cells to α-tocopheryl succinate (α-TOS), hydrogen peroxide, anti-Fas IgM or TRAIL led to induction of apoptosis. Cell death due to the chemical agents was suppressed in cells enriched with the reduced form of coenzyme Q. However, coenzyme Q did not block cell death induced by the immunological agents. Ubiquinol-10 inhibited reactive oxygen species (ROS) generation in cells exposed to α-TOS, and a mitochondrially targeted coenzyme Q analogue also blocked apoptosis triggered by α-TOS or hydrogen peroxide. Therefore, it is plausible that ubiquinol-10 protects cells from chemically-induced apoptosis by acting as an antioxidant in mitochondria. Our results also indicate that generation of free radicals may not be a critical step in induction of apoptosis by immunological agents.

Coenzyme Q 10 enrichment decreases oxidative DNA damage in human lymphocytes

Ubiquinol-10, the reduced form of coenzyme Q 10, is a powerful antioxidant in plasma and lipoproteins. It has been suggested that endogenous ubiquinol-10 also exerts a protective role even towards DNA oxidation mediated by lipid peroxidation. Even though the antioxidant activity of coenzyme Q 10 is mainly ascribed to ubiquinol-10, a role for ubiquinone-10 (the oxidized form), has been suggested not only if appropriate reducing systems are present. To investigate whether the concentration of ubiquinol-10 or ubiquinone-10 affects the extent of DNA damage induced by H 2O 2, we supplemented in vitro human lymphocytes with both forms of coenzyme Q 10 and evaluated the DNA strand breaks by Comet assay. The exposure of lymphocytes to100 μM H 2O 2 resulted in rapid decrease of cellular ubiquinol-10 content both in ubiquinol-10-enriched and in control cells, whereas α-tocopherol and β-carotene concentration were unchanged. After 30 min from H 2O 2 exposure, the amount of DNA strand breaks was lower and cells’ viability was significantly higher in ubiquinol-10-enriched cells compared with control cells. A similar trend was observed in ubiquinone-10-enriched lymphocytes when compared with control cells. Our experiments suggest that coenzyme Q 10 in vitro supplementation enhances DNA resistance towards H 2O 2-induced oxidation, but it doesn’t inhibit directly DNA strand break formation.

Expansion of antioxidant function of vitamin E by coenzyme Q.

Coenzyme Q (Q) is an integral redox and proton translocating component of the mitochondrial electron transport chain [5,8]. It is, however, widely distributed in other subcellular membranes [4,6] and even serum lipoproteins [7] where its role in electron transport is not apparent. Moreover, its concentration in some membranes, including those of the Golgi and lysosomes, exceed that found in mitochondria. This raises the question of what functions coenzyme Q may play in the biology of the cell. Coenzyme Q may exist in one of three oxidation states; the fully reduced ubiquinol form (QH2), the fully oxidized ubiquinone form (Q) and the radical semiquinone (Q·) intermediate [15]. The radical form tends to be stabilized by delocalization of the unpaired electron about the fully substituted benzoquinone ring but can undergo a disproportionation reaction to yield equal amounts of fully reduced and fully oxidized products. Another feature of coenzyme Q is the relatively hydrophobic character of the molecule. This is due largely to the long polyisoprene substituent of the benzoquinone ring which, in biological membranes, varies in length from 6 to 10 or more isoprene units. The polarity of the molecule increases in the order oxidized > semiquinone > reduced and this is believed to influence the location of the benzoquinone ring with respect to the aqueous membrane interface [1,21]. The reduced form of coenzyme Q is known to have direct antioxidant action in which radical chain reactions are terminated [2,10,11,16,20,24]. Coenzyme Q is also present in membranes with another lipid antioxidant, vitamin E. The relative amount of each varies from one membrane to another and together they represent a formidable defense against oxidative attack [9,12]. It should be added parenthetically that coenzyme Q in its semiquinone form can generate superoxide from oxygen and this, in turn, can result in oxidation of biomolecules [3,25]. Recent studies in our laboratories have indicated that coenzyme Q and vitamin E, rather than acting independently in protecting biological membranes from oxidative attack, are integrated into a regenerative cycle. In this cycle the more powerful antioxidant, vitamin E, which is converted in the first instance to its phenoxyl radical (tocopheroxyl radical), is reconverted by reduced forms of coenzyme Q to recover

Oxidation of Free Fatty Acids in Low Density Lipoprotein by 15-Lipoxygenase Stimulates Nonenzymic, α-Tocopherol-mediated Peroxidation of Cholesteryl Esters

15-Lipoxygenase has been implicated in the in vivo oxidation of low density lipoprotein (LDL) a process thought to be important in the origin and/or progression of human atherogenesis. We have suggested previously that oxidation of LDL's cholesteryl esters (CE) and phospholipids by soybean (SLO) or human recombinant 15-lipoxygenase (rhLO) can be ascribed largely to alpha-tocopherol (alpha-TOH)-mediated peroxidation (TMP). In this study we demonstrate that addition to LDL of unesterified linoleate (18:2), other free fatty acid (FFA) substrates, or phospholipase A2 (PLA2) significantly enhanced the accumulation of CE hydro(pero)xides (CE-O(O)H) induced by rhLO, whereas the corresponding CE and nonsubstrate FFA were without effect. The enhanced CE-O(O)H accumulation showed a dependence on the concentration of free 18:2 in LDL. In contrast, addition of 18:2 had little effect on LDL oxidation induced by aqueous peroxyl radicals or Cu2+ ions. Analyses of the regio- and stereoisomers of oxidized 18:2 in SLO-treated native LDL demonstrated that the small amounts of 18:2 associated with the lipoprotein were oxidized enzymically and within minutes, whereas cholesteryl linoleate (Ch18:2) was oxidized nonenzymically and continuously over hours. alpha-Tocopheroxyl radical (alpha-TO.) formed in LDL exposed to SLO was enhanced by addition of 18:2 or PLA2. With rhLO and 18:2-supplemented LDL, oxidation of 18:2 was entirely enzymic, whereas that of Ch18:2 was largely, though not completely, nonenzymic. The small extent of enzymic Ch18:2 oxidation increased with increasing enzyme to LDL ratios. Ascorbate and the reduced form of coenzyme Q, ubiquinol-10, which are both capable of reducing alpha-TO. and thereby preventing TMP, inhibited nonenzymic Ch18:2 oxidation induced by rhLO. Trolox and ascorbyl palmitate, which also inhibit TMP, ameliorated both enzymic and nonenzymic oxidation of LDL's lipids, whereas probucol, a radical scavenger not capable of preventing TMP, was ineffective. These results demonstrate that rhLO-induced oxidation of CE is largely nonenzymic and increases with LDL's content of FFA substrates. We propose that conditions which increase LDL's FFA content, such as the presence of lipases, increase 15-LO-induced LDL lipid peroxidation and that this process requires only an initial, transient enzymic activity.

Inhibition of LDL oxidation by ubiquinol-10. A protective mechanism for coenzyme Q in atherogenesis?

The oxidation of low density lipoprotein (LDL) is now commonly regarded as an important early event in atherogenesis. As such there is considerable interest in the ability of antioxidant supplementation to attenuate LDL oxidation and hence atherosclerosis. A majority of studies on LDL antioxidation have focused on α-tocopherol (α-TOH), biologically and chemically the most active form of vitamin E and quantitatively the major lipid-soluble antioxidant in extracts prepared from human LDL. In addition to α-TOH, circulating LDL also contains low levels of ubiquinol-10 (CoQ 10H 2; the reduced form of coenzyme Q). Recent studies have shown that in intact, isolated LDL, α-TOH can act as either an anti- or pro-oxidant for the lipoprotein's lipids. This article reviews the molecular action of α-TOH in LDL undergoing radical-initiated oxidation, and how the presence of CoQ 10H 2 supresses the pro-oxidant or complements the antioxidant activity of the vitamin. We also comment on the plasma and intimal levels of α-TOH and CoQ 10H 2 in patients suffering from coronary artery disease and discuss the potential implications of these results for atherogenesis.

Cosupplementation With Coenzyme Q Prevents the Prooxidant Effect of α-Tocopherol and Increases the Resistance of LDL to Transition Metal–Dependent Oxidation Initiation

There is considerable interest in the ability of antioxidant supplementation, in particular with vitamin E, to attenuate LDL oxidation, a process implicated in atherogenesis. Since vitamin E can also promote LDL lipid peroxidation, we investigated the effects of supplementation with vitamin E alone or in combination with coenzyme Q on the early stages of the oxidation of isolated LDL. Isolated LDL was obtained from healthy subjects before and after in vitro enrichment with vitamin E (D-alpha-tocopherol, alpha-TOH) or dietary supplementation with D-alpha-TOH (1 g/d) and/or coenzyme Q (100 mg/d). LDL oxidation initiation was assessed by measurement of the consumption of alpha-TOH and cholesteryl esters containing polyunsaturated fatty acids and the accumulation of cholesteryl ester hydroperoxides during incubation of LDL in the transition metal-containing Ham's F-10 medium in the absence and presence of human monocyte-derived macrophages (MDMs). Native LDL contained 8.5 +/- 2 molecules of alpha-TOH and 0.5 to 0.8 molecules of ubiquinol-10 (CoQ10H2, the reduced form of coenzyme Q) per lipoprotein particle. Incubation of this LDL in Ham's F-10 medium resulted in a time-dependent loss of alpha-TOH with concomitant stoichiometric conversion of the major cholesteryl esters to their respective hydroperoxides. MDMs enhanced this process. LDL lipid peroxidation occurred via a radical chain reaction in the presence of alpha-TOH, and the rate of this oxidation decreased on alpha-TOH depletion. In vitro enrichment of LDL with alpha-TOH resulted in an LDL particle containing sixfold to sevenfold more alpha-TOH, and such enriched LDL was more readily oxidized in the absence and presence of MDMs compared with native LDL. In vivo alpha-TOH-deficient LDL, isolated from a patient with familial isolated vitamin E deficiency, was highly resistant to Ham's F-10-initiated oxidation, whereas dietary supplementation with vitamin E restored the oxidizability of the patient's LDL. Oral supplementation of healthy individuals for 5 days with either alpha-TOH or coenzyme Q increased the LDL levels of alpha-TOH and CoQ10H2 by two to three or three to four times, respectively. alpha-TOH-supplemented LDL was significantly more prone to oxidation, whereas CoQ10H2-enriched LDL was more resistant to oxidation initiation by Ham's F-10 medium than native LDL. Cosupplementation with both alpha-TOH and coenzyme Q resulted in LDL with increased levels of alpha-TOH and CoQ10H2, and such LDL was markedly more resistant to initiation of oxidation than native or alpha-TOH-enriched LDL. These results demonstrate that oral supplementation with alpha-TOH alone results in LDL that is more prone to oxidation initiation, whereas cosupplementation with coenzyme Q not only prevents this prooxidant activity of vitamin E but also provides the lipoprotein with increased resistance to oxidation.

Effect of lipoic acid on redox state of coenzyme Q in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and diethyldithiocarbamate

We investigated the effects of a combined treatment of male C57Bl/6 mice with diethyldithiocarbamate and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the absence or presence of different forms of lipoic acid (Thioctacid T R; commonly used for treatment of diabetic polyneuropathies) on levels and redox states of α-tocopherol and coenzyme Q in vivo and on activities of various enzymes of energy metabolism ex vivo. Treatment of mice with diethyldithiocarbamate plus MPTP resulted in a decrease in dopamine (67%) and its major metabolites dihydroxyphenylacetic acid (38%) and homovanillic acid (37%) in striatum. α-Tocopherol levels were unaltered in striatum; however, the reduced forms of coenzyme Q were decreased in frontal cortex and hippocampus following diethyldithiocarbamate plus MPTP. In frontal cortex activity of NADH dehydrogenase was significantly inhibited by diethyldithiocarbamate plus MPTP ex vivo, suggesting that the neurotoxic metabolite of MPTP, 1-methyl-4-phenylpyridinium ion, is acting in brain regions other than striatum as well. Lipoic acid, administered 6 times, each at 90 min prior to MPTP, could not restore dopamine in striatum but in contrast maintained a normal ratio of the reduced form to the oxidized form of coenzyme Q, suggesting an interaction of lipoic acid with energy metabolism which seems, however, not only to be due to an activation of pyruvate dehydrogenase.

Free radical production by activated haem proteins: Protective effect of Coenzyme Q

The interaction of hydrogen peroxide with haem proteins leads readily to the formation of myoglobin and/or haemoglobin higher oxidation states (Mb IV and/or Hb IV), which are capable of promoting the oxidation of cellular costituents and are probably to blame for myocardic tissue damage in ischaemia/reperfusion. This study supports the evidence that the reduced form of Coenzyme Q, like other reducing agents, has an antioxidant activity exerted through the progressive reduction of ferryl forms (Mb IV and/or Hb IV) back to met and oxy forms (Mb and/or Hb IIO 2). Furthermore, the strong inactivation afforded by ferryl states of myoglobin on several enzymes, especially creatine kinase (CK), can be prevented by the addition of ubiquinol which protects the enzyme from the oxidative modifications. The ability of ubiquinol to recycle ferryl states of haem proteins provides a novel antioxidant mechanism for Coenzyme Q, besides its direct or indirect antiperoxidative activity, and may represent an important defense mechanism against oxidative tissue injury.

Dietary supplementation with coenzyme Q 10 results in increased levels of ubiquinol-10 within circulating lipoproteins and increased resistance of human low-density lipoprotein to the initiation of lipid peroxidation

Ubiquinol-10 (CoQH 2, the reduced form of coenzyme Q 10) is a potent antioxidant present in human low-density lipoprotein (LDL). Supplementation of humans with ubiquinone-10 (CoQ, the oxidized coenzyme) increased the totaentrations of CoQH 2 in plasma acid and in all of its lipoproteins. Intake of a single oral dose of 100 or 200 mg CoQ increased the total plasma coenzyme content by 80 or 150%, respectively, within 6 h. Long-term supplementation (three times 100 mg CoQ/day) resulted in 4-fold enrichment of CoQH 2 in plasma and LDL with the latter containing 2.8 CoQH 2 molecules per LDL particle (on day 11). Approx. 80% of the coenzyme was present as CoQH 2 and the CoQH 2/CoQ ratio was unaffected by supplementation, indicating that the redox state of coenzyme Q 10 is tightly controlled in the blood. Oxidation of LDL containing various [CoQH 2] by a mild, steady flux of aqueous peroxyl radicals resulted immediately in very slow formation of lipid hydroperoxides. However, in each case the rate of lipid oxidation increased markedly with the disappearance of 80–90% CoQH 2. Moreover, the cumulative radical dose required to reach this ‘break point’ in lipid oxidation was proportional to the amount of CoQH 2 incorporated in vivo into the LDL. Thus, oral supplementation with CoQ increases CoQH 2 in the plasma and all lipoproteins thereby increasing the resistance of LDL to radical oxidation.