Ubiquinol-10 and ubiquinone-10 levels in umbilical cord blood of healthy foetuses and the venous blood of their mothers

Abstract

Despite their being good markers of oxidative stress for clinical use, little is known about ubiquinol-10 (reduced coenzyme Q₁₀) and ubiquinone-10 (oxidized coenzyme Q₁₀) levels in foetuses and their mothers. This study investigates oxidative stress in 10 healthy maternal venous, umbilical arterial and venous bloods after vaginal delivery by measuring ubiquinol-10 and ubiquinone-10 levels. Serum ubiquinol-10 and ubiquinone-10 levels were measured by HPLC with a highly sensitive electrochemical detector. Maternal venous ubiquinol-10 and ubiquinone-10 levels were significantly higher than umbilical arterial and venous levels (all p < 0.001). However, the ubiquinone-10/total coenzyme Q₁₀ (CoQ₁₀) ratio, which reflects the redox status, was significantly higher in umbilical arterial and umbilical venous blood compared to maternal venous blood (all p < 0.001). The ubiquinone-10/total CoQ₁₀ ratio was higher in umbilical arterial than in umbilical venous blood (p < 0.01). The present study demonstrated that foetuses were under higher oxidative stress than their mothers.     

Cytochrome b558 monitors the steady state redox state of the ubiquinone pool in the aerobic respiratory chain of Escherichia coli

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases, the cytochrome o complex and the cytochrome d complex. These both function as ubiquinol-8 oxidases and reduce molecular oxygen to water. Electron flux is funneled from a variety of dehydrogenases, such as succinate dehydrogenase, through ubiquinone-8, to either of the terminal oxidases. A strain was examined which lacks the intact cytochrome d complex, but which overproduces one of the two subunits of this complex, cytochrome b558. This cytochrome, in the absence of the other subunit of the oxidase complex, does not possess catalytic activity. It is shown that the extent of reduction of cytochrome b558 in the E. coli membrane monitors the extent of reduction of the quinone pool in the membrane. The activity of each purified oxidase was examined in phospholipid vesicles as a function of the amount of ubiquinone-8 incorporated in the bilayer. A ratio of ubiquinol-8:phospholipid as low as 1:200 is sufficient to saturate each oxidase. The maximal turnover of the oxidases in the reconstituted system is considerably faster than observed in E. coli membranes, demonstrating that the rate-limiting step in the E. coli respiratory chain is at the dehydrogenases which feed electrons into the system.     

Ubiquinol-10 ameliorates mitochondrial encephalopathy associated with CoQ deficiency

Coenzyme Q10 (CoQ₁₀) deficiency (MIM 607426) causes a mitochondrial syndrome with variability in the clinical presentations. Patients with CoQ₁₀ deficiency show inconsistent responses to oral ubiquinone-10 supplementation, with the highest percentage of unsuccessful results in patients with neurological symptoms (encephalopathy, cerebellar ataxia or multisystemic disease). Failure in the ubiquinone-10 treatment may be the result of its poor absorption and bioavailability, which may be improved by using different pharmacological formulations. In a mouse model (Coq9^X/X) of mitochondrial encephalopathy due to CoQ deficiency, we have evaluated oral supplementation with water-soluble formulations of reduced (ubiquinol-10) and oxidized (ubiquinone-10) forms of CoQ₁₀. Our results show that CoQ₁₀ was increased in all tissues after supplementation with ubiquinone-10 or ubiquinol-10, with the tissue levels of CoQ₁₀ with ubiquinol-10 being higher than with ubiquinone-10. Moreover, only ubiquinol-10 was able to increase the levels of CoQ₁₀ in mitochondria from cerebrum of Coq9^X/X mice. Consequently, ubiquinol-10 was more efficient than ubiquinone-10 in increasing the animal body weight and CoQ-dependent respiratory chain complex activities, and reducing the vacuolization, astrogliosis and oxidative damage in diencephalon, septum–striatum and, to a lesser extent, in brainstem. These results suggest that water-soluble formulations of ubiquinol-10 may improve the efficacy of CoQ₁₀ therapy in primary and secondary CoQ₁₀ deficiencies, other mitochondrial diseases and neurodegenerative diseases.     

Coenzyme Q10 enrichment decreases oxidative DNA damage in human lymphocytes

Ubiquinol-10, the reduced form of coenzyme Q10, 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 Q10 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 H2O2, we supplemented in vitro human lymphocytes with both forms of coenzyme Q10 and evaluated the DNA strand breaks by Comet assay. The exposure of lymphocytes to 100 microM H2O2 resulted in rapid decrease of cellular ubiquinol-10 content both in ubiquinol-10-enriched and in control cells, whereas alpha-tocopherol and beta-carotene concentration were unchanged. After 30 min from H2O2 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 Q10 in vitro supplementation enhances DNA resistance towards H2O2-induced oxidation, but it doesn't inhibit directly DNA strand break formation.     

Proton magnetic resonance spectroscopic studies of the interaction of ubiquinone-10 with phospholipid model membranes

Proton magnetic resonance spectra of ubiquinone-10 and ubiquinone-10 dispersed with dipalmitoylglycerophosphocholine or egg phosphatidylcholine in aqueous medium have been obtained. The dispersions are in the form of multilamellar liposomes as judged by 31P-NMR spectra and the thermal history of the samples have ensured that ubiquinone not incorporated into the phospholipid structure only gives rise to a broad-line NMR proton spectrum. A high-resolution proton spectrum of ubiquinone is observed with upfield shifts of the O-methyl protons of the benzoquinone rings, indicating close proximity of the molecules but with an arrangement different from the pure liquid ubiquinone. Spectra obtained in the presence of the lanthanide shift reagents, dysprosium fluorooctanedionate and Dy(NO3)3, which have a preferred location in the hydrophobic and hydrophilic domains, respectively, of ubiquinone/phospholipid codispersions, are consistent with the partitioning of ubiquinone into a hydrophobic phospholipid environment remote from the aqueous phase. The type of arrangements of ubiquinone that could be accommodated within bilayers of phospholipid are discussed.     

Magic angle spinning 13C-NMR spin-lattice relaxation study of the location and effects of α-tocopherol,ubiquinone-10 and ubiquinol-10 in unsonicated model membranes

-Tocopherol, ubiquinone-10 and ubiquinol-10 have been studied by high resolution magic angle samples spinning ¹³C-nuclear magnetic resonance in egg yolk phosphatidylcholine multilamellar vesicles model membranes in order to assess their location and the induced perturbations on this model system. -Tocopherol is placed in such a position that it is in close contact with the head group of the phospholipid and exposed to the solvent. In this position it significantly perturbs the phospholipid head group, the 5a-CH₃ and the 7a-CH₃ groups being the closest parts to the membrane surface. On the other hand, ubiquinol-10 perturbs the membrane surface more than ubiquinone-10, but neither compound significantly changed the phospholipid head group conformation.     

Solvation properties of ubiquinone-10 in solvents of different polarity

The solvation properties of ubiquinone-10 and ubiquinol-10 in a wide variety of solvents of polarity varying from alkanes to water are reported. Greatest solubility is observed in solvents of intermediate polarity and particularly where low polarity is combined with a pronounced tendency to interact with the benzoquinone substituent of the ubiquinone molecule. This includes solvents like chloroform and benzene. Ubiquinone-10 is somewhat less polar than ubiquinol-10 as judged by comparative solubilities of the two molecules. Proton-NMR chemical shift measurements and aggregation studies in selected solvents indicate that in ubiquinone-10 in the liquid phase and in solution in hydrocarbons like dodecane the molecules have a preferred association possibly involving stacking of the benzoquinone rings. Surface balance studies indicated that the surface-active character of ubiquinone-10 is relatively weak and only in a comparatively polar and highly structured solvent, formamide, was there evidence of an effect on surface tension of the solvent. The critical micelle concentratiom in this solvent was estimated to be about 5 M on the basis of surface tension measurements. Ubiquinone-10 is well known to form virtually insoluble monolayers at the air/water interface. Studies of the partition of ubiquinone-10 in binary mixtures of solvents suggest that the interaction of the benzoquinone ring substituent with structured polar solvents is considerably weaker than the internal cohesion between molecules of the solvent. No evidence on the basis of wide-angle X-ray diffraction measurements was obtained to indicate that solvent molecules were a component of the crystal lattice of ubiquinone-10 that had precipitated from solvent mixtures.     

Plasma ubiquinol-10 as a marker for disease: is the assay worthwhile?

Ubiquinol-10 and ubiquinone-10 were measured in plasma of patients with several pathologies known to be associated with increased oxidative stress. Plasma ubiquinol-10, expressed as a percentage of total ubiquinol-10 + ubiquinone-10, was found to be significantly lower in hyperlipidaemic patients and in patients with liver diseases than in age-matched control subjects. In contrast, no decrease in ubiquinol-10 was detected in plasma of patients with coronary heart disease and Alzheimer's disease. Except for ubiquinol-10, no other lipophilic antioxidant was found to be decreased in patients with liver diseases. These data suggest that the level of ubiquinol-10 in human plasma may serve as a marker for liver dysfunction, reflecting its diminished reduction by the liver rather than increased consumption by oxidants.     

Anisotropy measurements of intrinsic fluorescence of prenyllipids reveal much higher mobility of plastoquinol than alpha-tocopherol in model membranes

As an alternative to a fluorescent probe approach, the intrinsic fluorescence of reduced forms of prenylquinones has been exploited, which offers a convenient means of determining directly motional properties of these molecules. The steady-state fluorescence anisotropy measurements of plastoquinols (PQH(2)) and alpha-tocopherol (alpha-Toc) incorporated into phospholipid liposomes have been performed. The effect of prenyllipid concentration, PQH(2) side chain length and the composition of the membranes has been studied. For the data interpretation, the fundamental anisotropy of alpha-Toc, PQH(2), ubiquinol-10 and alpha-tocopherolquinol, as well as the angles between the absorption and emission transition moments have been also determined. It was concluded that alpha-Toc shows very low mobility in the lipid bilayer, whereas PQH(2)-9 displays significant motional freedom in dipalmitoylphosphatidylcholine vesicles and even higher in egg yolk lecithin membranes.     

Neutral Magnesium-Dependent Sphingomyelinase from Liver Plasma Membrane: Purification and Inhibition by Ubiquinol

Plasma membranes isolated from pig liver contained almost no acid sphingomyelinase but significant neutral magnesium-dependent sphingomyelinase that was activated by phosphatidylserine. We report here the purification to apparent homogeneity of neutral sphingomyelinase of about 87 kDa from liver plasma membranes. The purified enzyme strictly required magnesium and had a neutral optimal pH. In contrast with neutral sphingomyelinase purified from other sources (such as brain), the enzyme purified from from liver plasma membrane was not inhibited by GSH and, strikingly, it was not activated by phosphatidylserine. Liver sphingomyelinase was inhibited by several lipophilic antioxidants in a dose-dependent way. Ubiquinol-10 was more effective than alpha-tocopherol, alpha-tocopherylquinone, alpha-tocopherylquinone, and ubiquinone-10, and inhibition was noncompetitive. Differential inhibition of neutral sphingomyelinase by antioxidants did not correlate with different levels of protection against lipid peroxidation. The purified sphingomyelinase was not inhibited significantly by ubiquinone-10 and ubiquinol- 10, but ubiquinol-0 and ubiquinone-0 inhibited by 30 and 60% respectively. Our results demonstrate a direct inhibitory effect of ubiquinol on the plasma membrane n-SMase and support the participation of this molecule in the regulation of ceramide-mediated signaling.     

Increased oxidative stress and coenzyme Q10 deficiency in juvenile fibromyalgia: amelioration of hypercholesterolemia and fatigue by ubiquinol-10 supplementation

Abstract

Fibromyalgia (FM) is characterized by generalized pain and chronic fatigue of unknown etiology. To evaluate the role of oxidative stress in this disorder, we measured plasma levels of ubiquinone-10, ubiquinol-10, free cholesterol (FC), cholesterol esters (CE), and free fatty acids (FFA) in patients with juvenile FM (n = 10) and in healthy control subjects (n = 67). Levels of FC and CE were significantly increased in juvenile FM as compared with controls, suggesting the presence of hypercholesterolemia in this disease. However, plasma level of ubiquinol-10 was significantly decreased and the ratio of ubiquinone-10 to total coenzyme Q10 (%CoQ10) was significantly increased in juvenile FM relative to healthy controls, suggesting that FM is associated with coenzyme Q10 deficiency and increased oxidative stress. Moreover, plasma level of FFA was significantly higher and the content of polyunsaturated fatty acids (PUFA) in total FFA was significantly lower in FM than in controls, suggesting increased tissue oxidative damage in juvenile FM. Interestingly, the content of monoenoic acids, such as oleic and palmitoleic acids, was significantly increased in FM relative to controls, probably to compensate for the loss of PUFA. Next, we examined the effect of ubiquinol-10 supplementation (100 mg/day for 12 weeks) in FM patients. This resulted in an increase in coenzyme Q10 levels and a decrease in %CoQ10. No changes were observed in FFA levels or their composition. However, plasma levels of FC and CE significantly decreased and the ratio of FC to CE also significantly decreased, suggesting that ubiquinol-10 supplementation improved cholesterol metabolism. Ubiquinol-10 supplementation also improved chronic fatigue scores as measured by the Chalder Fatigue Scale.     

Modulation of (Na+,K+)-ATPase activity by the lipid bilayer examined with dansylated phosphatidylserine

The fluorescent probe 8-(dimethylamino)naphthalene-1-sulfonylphosphatidylserine (Dns-PS) was incorporated into purified lamb kidney Na+- and K+-stimulated adenosinetriphosphatase (EC 3.6.1.3) [(Na+,K+)-ATPase] by using a purified phospholipid exchange protein. Phospholipase C was used to reduce phospholipid content. Up to 40% of the phospholipid could be hydrolyzed with only 10% inhibition of the (Na+,K+)-ATPase, but when 67% of the phospholipid was hydrolyzed, the enzyme was inhibited 53%. To examine the effect of protein on the phospholipid bilayer, the fluorescent parameters of the probe incorporated into the enzyme preparation were contrasted with the same parameters for the probe incorporated into the total lipid extract of the preparation. The polarization of fluorescence of the probe in the lipid extract was 0.118 while in the enzyme preparation it was 0.218. This reflected a decrease in fluidity of the glycerol region of the phospholipid bilayer which was mediated by the protein. This effect increased as the phospholipid content of the (Na+,K+)-ATPase preparation was reduced so that with maximal phospholipid reduction the polarization of fluorescence was 0.262. The protein caused a decrease in the transition temperature from gel to fluid states of the bilayer detected by polarization of the probe. The midpoint temperature transition of the enzyme preparation decreased from 33 degrees C when all phospholipids were present to 20 degrees C when 67% of the phospholipids were hydrolyzed. This decrease was not observed for the lipid extract of these samples. A direct correlation between the (Na+,K+)-ATPase specific activity and the polarization of fluorescence of Dns-PS was found. The reduction in phospholipid content did not affect the steady-state level of phosphorylation of the enzyme by ATP but did affect the rate of dephosphorylation which would require conformational changes of the enzymes. The data showed that the fluidity of the phospholipid bilayer can modulate the activity of the (Na+,K+)-ATPase.     

Ubiquinones have surface-active properties suited to transport electrons and protons across membranes

Surface-active properties of ubiquinones and ubiquinols have been investigated by monomolecular-film techniques. Stable monolayers are formed at an air/water interface by the fully oxidized and reduced forms of the coenzyme; collapse pressures and hence stability of the films tend to increase with decreasing length of the isoprenoid side chain and films of the reduced coenzymes are more stable than those of their oxidized counterparts. Ubiquinone with a side chain of two isoprenoid units does not form stable monolayers at the air/water interface. Mixed monolayers of ubiquinol-10 or ubiquinone-10 with 1,2-dimyristoyl phosphatidylcholine, soya phosphatidylcholine and diphosphatidylglycerol do not exhibit ideal mixing characteristics. At surface pressures less than the collapse pressure of pure ubiquinone-10 monolayers (approx. 12mN.m(-1)) the isoprenoid chain is located substantially within the region occupied by the fatty acyl residues of the phospholipids. With increasing surface pressure the ubiquinones and their fully reduced equivalents are progressively squeezed out from between the phospholipid molecules until, at a pressure of about 35mN.m(-1), the film has surface properties consistent with that of the pure phospholipid monolayer. This suggests that the ubiquinone(ol) forms a separate phase overlying the phospholipid monolayer. The implications of this energetically poised situation, where the quinone(ol) is just able to penetrate the phospholipid film, are considered in terms of the function of ubiquinone(ol) as electron and proton carriers of energy-transducing membranes.     

Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase.

The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.     

The interaction of coenzyme Q with phosphatidylethanolamine membranes.

Coenzyme Q (CoQ) is a component of the mitochondrial respiratory chain which carries out additional membrane functions, such as acting as an antioxidant. The location of CoQ in the membrane and the interaction with the phospholipid bilayer is still a subject of debate. The interaction of CoQ in the oxidized (ubiquinone-10) and reduced (ubiquinol-10) state with membrane model systems of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (Ela2Gro-P-Etn) has been studied by means of differential scanning calorimetry (DSC), 31P-nuclear magnetic resonance (31P-NMR) and small angle X-ray diffraction (SAXD). Ubiquinone-10 did not visibly affect the lamellar gel to lamellar liquid-crystalline phase transition of Ela2Gro-P-Etn, but it clearly perturbed the multicomponent lamellar liquid-crystalline to lamellar gel phase transition of the phospholipid. The perturbation of both transitions was more effective in the presence of ubiquinol-10. A location of CoQ forming head to head aggregates in the center of the Ela2Gro-P-Etn bilayer with the polar rings protruding toward the phospholipid acyl chains is suggested. The formation of such aggregates are compatible with the strong hexagonal HII phase promotion ability found for CoQ. This ability was evidenced by the shifting of the lamellar to hexagonal HII phase transition to lower temperatures and by the appearance of the characteristic hexagonal HII 31P-NMR resonance and SAXD pattern at temperatures at which the pure Ela2Gro-P-Etn is still organized in extended bilayer structures. The influence of CoQ on the thermotropic properties and phase behavior of Ela2Gro-P-Etn is discussed in relation to the role of CoQ in the membrane.     

Advantages and limitations of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3- sn-phosphatidylcholine as a fluorescent membrane probe

We have investigated the behavior of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn -phosphatidylcholine (DPHpPC) in synthetic, multilamellar phosphatidylcholine vesicles. This fluorescent phospholipid has photophysical properties similar to its parent fluorophore, diphenylhexatriene (DPH). DPHpPC preferentially partitioned into fluid phase lipid (Kf/s = 3.3) and reported a lower phase transition temperature as detected by fluorescence anisotropy than that observed by differential scanning calorimetry. Calorimetric measurements of the bilayer phase transition in samples having different phospholipid to probe ratios demonstrated very slight changes in membrane phase transition temperature (0.1-0.2 degree C) and showed no measurable change in transition width. Nonetheless, measurements of probe fluorescence properties suggested that DPHpPC disrupts its local environment in the membrane and may even induce perturbed probe-rich local domains below the phospholipid phase transition. Temperature profiles of steady-state fluorescence anisotropy, limiting anisotropy, differential tangent, and rotational rate were similar to those of DPH below the main lipid phase transition but indicated more restricted rotational motion above the lipid phase transition temperature. As for DPH, the fluorescence decay of DPHpPC could be described by either a single or double exponential both above and below the DPPC phase transition. The choice seemed dependent on the treatment of the sample. The intensity-weighted average lifetime of DPHpPC was roughly 1.5 ns shorter than that of DPH. In summary, the measured properties of DPHpPC and its lipid-like structure make it a powerful probe of membrane structure and dynamics.     

Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen.

To probe the functional role of a bound ubiquinone-8 in cytochrome bo-type ubiquinol oxidase from Escherichia coli, we examined reactions with ubiquinol-1 and dioxygen. Stopped-flow studies showed that anaerobic reduction of the wild-type and the bound ubiquinone-free (DeltaUbiA) enzymes with ubiquinol-1 immediately takes place with four kinetic phases. Replacement of the bound ubiquinone with 2,6-dibromo-4-cyanophenol (PC32) suppressed the anaerobic reduction of the hemes with ubiquinol-1 by eliminating the fast phase. Flow-flash studies in the reaction of the fully reduced enzyme with dioxygen showed that the heme b-to-heme o electron transfer occurs with a rate constant of approximately 1x10(4) s(-1) in all three preparations. These results support our previous proposal that the bound ubiquinone is involved in facile oxidation of substrates in subunit II and subsequent intramolecular electron transfer to low-spin heme b in subunit I.     

Fluorescence quenching in model membranes An analysis of the local phospholipid environments of diphenylhexatriene and gramicidin A′

Interactions between the fluorophors diphenylhexatriene or gramicidin A′ and lipids are examined using a spin-labeled phosphatidylcholine as a fluorescence quenching probe. It is found that in phospholipid vesicles of mixed lipid composition at temperatures where phospholipids are completely in the liquid crystal phase, several different species of phosphatidylcholines are randomly distributed around the fluorophors. In vesicles of mixed lipid composition which can undergo thermally induced phase separations, the fluorescence quenching observed at lower temperatures reflects a non-random distribution of lipids around each fluorophor. This observation is explained in terms of the partition of fluorophor between a spin-labeled lipid-rich liquid crystal phase, and a spin-labeled lipiddepleted gel phase. Gramicidin A′ strongly favors partition into the liquid crystal phase, whereas diphenylhexatriene partitions about equally between the two lipid phases. A method is described utilizing fluorescence quenching for the calculation of the partition coefficient for a fluorophor. The partition coefficients so calculated are shown to be in good agreement with previously reported values derived from other methods. It is also shown that Ca²⁺-induced lipid phase separations can be monitored by fluorescence quenching.