Therapeutic effect of vegetable oils and ubiquinone-9 on radiation injuries

The comparison was made of the protective (the administration 3 h before irradiation with a dose of 7.3 Gy) and therapeutic (the administration immediately and later after exposure) effects of soya oil (150 mg/kg) and oil solution of ubiquinone-9 (100-200 mg/kg) on survival of exposed rats. It was shown that soya oil and ubiquinone-9 increased the survival rate of rats when administered before and, to a lesser extent, immediately after irradiation. Corn oil administered immediately after exposure increased the survival rate as well. DMF for the therapeutic effect of soya oil solution of ubiquinone-9 was 1.08.     

The concentration and bisynthesis of ubiquinone-9 in the liver of white rats adapted to altitude hypoxia]

The content and biosynthesis of ubiquinone-9 in the thin slices of the liver of rats was studied during altitude adaptation. There was a three-fold acceleration of ubiquinone biosynthesis during the first period of altitude adaptation. Acceleration of biosynthesis of ubiquinone-9 in rat liver was insignificant after two weeks of adaptation. The content of ubiquinone-9 in rat liver changed but insignificantly in the course of one month of altitude adaptation.     

Ubiquinone content of eight plant species in cell culture

Crystals of ubiquinone-10 were isolated from soyabean, peanut and Ruta cell cultures, while crystals of ubiquinone-9 were obtained from rice and wheat cell cultures. These crystals also contained lesser amounts of lower and higher homologues (ubiquinone-7 to 10). The ubiquinone content of eight higher plants in cell culture was determined. Ubiquinone-9 content of rice was 680 μg per g dry wt, and this was 3–6 times higher than that of the other plants.     

Untersuchungen zur Anreicherung von Ergosterol und Ubichinonen aus Lipid-Kohlenwasserstoff-Fraktionen

Adsorption on Silicagel was used for the enrichment of ergosterol and ubiquinone-9 from a lipid-hydrocarbon extract. The main components of the lipid-hydrocarbon extract were hydrocarbons of gas oil (B. p. 513 to 653 K)phosphatides, glycerides, and fatty acids. By adsorption on silicagel (1 part silicagel to 2–3 parts of lipid-hydrocarbon extract dissolved in hexane) were ergosterol and ubiquinone-9 enriched and nearly completely separarated from hydrocarbons and phosphatides, partly from glycerides and fatty acids. Ergosterol and ubiquinone-9 can be obtained by a simplified separation from the enriched fraction.     

Production of ubiquinone-10 using bacteria

Among the bacterial strains known to contain ubiquinone-10, three strains, Agrobacterium tumefaciens KY-3085 (ATCC4452), Paracoccus denitrificans KY-3940 (ATCC19367) and Rhodobacter sphaeroides KY-4113 (FERM-P4675), were selected as excellent producers of this ubiquinone. The ubiquinone-10 production by the Agrobacterium and Rhodobacter strains was affected by aeration. An ethionine-resistant mutant (M-37) derived from A. tumefaciens KY-3085 promoted increased production of ubiquinone-10 (20% higher than the parent). Another Agrobacterium mutant (AU-55), which was induced by the successive addition of four genetic markers, showed a tolerance to the suppression of ubiquinone-10 production caused by aeration, and the fermentation time for production was remarkably shortened. The amount of ubiquinone-10 produced by this Agrobacterium mutant reached 180 mg/l in a 58 h culture. A green mutant (carotenoid-deficient mutant, Co-22-11) derived from R. sphaeroides KY-4113 produced 350 mg/l of ubiquinone-10 under culturing conditions with a limited supply of air, the ubiquinone-10 content being 8.7 mg/g-dry cell. In this case, the amount and content corresponded to 2.8 and 3.6 times larger than those given by the wild-type strain, respectively. A multiple-layer structure of cell membrane was observed in the highly ubiquinone-10 accumulating cell of the green mutant by electron microscopy. The amount of ubiquinone-10 produced by P. denitrificans was much lower than those of the other two strains.     

Ubiquinone-10 Protects Neurons from Virus-Induced Degeneration

Abstract: Cultured neurons from rat dorsal root ganglia and cerebral cortex were infected with Sendai virus, which gives a productive replication with lysis of most neurons, and with the RW strain of mumps virus, which undergoes defective replication causing degeneration of only 30–40% of the neurons within 5 days after initial infection. In Sendai virus-infected cells the amount of polyisoprenoid lipids was enhanced. In mumps virus-infected cultures there were transient reductions in the contents of cholesterol, dolichol, and ubiquinone-9 in the cultures, whereas the reduction in the ubiquinone-10 level was progressive, reaching 20% of its original value 21 days after infection. Treatment of mumps virus-infected cultures with ubiquinone-10 protected the neurons from degeneration, whereas no effects were observed on exposure to ubiquinone-9. Linolenic acid (18:3) and arachidonic acid (20:4), but not myristic acid (14:0) and palmitic acid (16:0), also had significant neuroprotective effects.     

Nonaprenyl-4-hydroxybenzoate transferase, an enzyme involved in ubiquinone biosynthesis, in the endoplasmic reticulum-Golgi system of rat liver

The properties and distribution of nonaprenyl-4-hydroxybenzoate transferase in rat liver were investigated with subcellular fractions, liver perfusion, and in vivo labeling with [3H]solanesyl-PP. In addition to some ubiquinone-9, only one labeled intermediate, i.e. nonaprenyl-4-hydroxybenzoate, was obtained. In the total microsomal fraction, the enzyme had a pH optimum of 7.5 and was completely inhibited by Triton X-100 and deoxycholate, but not by taurodeoxycholate and beta-octyl glucoside. Liver, kidney, and spleen demonstrated the highest activities of nonaprenyl-4-hydroxybenzoate transferase. Upon subcellular fractionation, high specific activities were found in smooth II microsomes and Golgi III vesicles. The enzyme was also found in lysosomes and plasma membranes, but only at low levels in rough and smooth I microsomes and mitochondria and not at all in peroxisomes and cytosol. When the product of the transferase reaction was used as a substrate in vitro and in a perfusion system, the only product obtained was end product ubiquinone-9. Although the transferase reaction was associated with the inner, luminal surface of microsomal vesicles, the terminal reaction(s) for ubiquinone-9 synthesis are found at the outer cytoplasmic surface. The results suggest that the major site for ubiquinone synthesis is the endoplasmic reticulum-Golgi system, which also participates in the distribution of ubiquinone-9 to other cellular membranes.     

Untersuchungen zur Anreicherung von Ubichinon-9 aus Lipid-Kohlenwasserstoff-Fraktionen mittels Kurzwegdestillation

Short path distillation was used for the enrichment of ubiquinone-9 from a lipid hydrocarbon extract. The main components of the lipid hydrocarbon extract were hydrocarbons of gas oil (b. p. 513 to 653 K), phosphatides, glycerides, and fatty acids. Phosphatides were isolated by extraction with acetone before distillation. The acetone soluble fraction was distilled by a pressure of ≦ 0.5 kPa and a temperature of 433 to 453 K. The concentration of the ubiquinone-9 in the residue of distillation was three times higher than in the acetone soluble fraction. Ubiquinone-9 can be obtained by a simplified separation from the enriched fraction.     

Identification of ubiquinone-50 as the major methylated nonpolar lipid in human monocytes. Regulation of its biosynthesis via methionine-dependent pathways and relationship to superoxide production

Human blood monocytes incorporated the methyl group from methionine into their neutral lipids. The major methylated product was identified as ubiquinone-50 in monocytes, lymphocytes, and a variety of human tumor cell lines by several analytical procedures including TLC or high performance liquid chromatography and as ubiquinone-45 in a mouse tumor cell line. Up to three methyl groups were shown to be derived from methionine by mass spectrometry. The rate of synthesis of ubiquinone-50 by monocytes as assessed by measuring labeled methyl group incorporation was shown to be linear over a 3-h period. Degradation of ubiquinone proceeded slowly; 80% of the labeled compound persisted after 18 h. The dependence of ubiquinone-50 synthesis upon methionine concentration was established in monocytes, with an estimated apparent Km for methionine of about 20 microM. The tumor promoter, tetradecanoate phorbol acetate, a potent stimulator of superoxide anion (O2-) production in phagocytic cells, inhibited ubiquinone-50 synthesis at nanomolar concentrations in monocytes, but not in lymphocytes, under conditions where oxidation of methionine takes place. Degradation of the labeled ubiquinone was unaffected. Formylmethionylleucyl-phenylalanine, a chemoattractant peptide which stimulates O2- production in phagocytic cells, also inhibited ubiquinone-50 synthesis. The degree of inhibition by either stimulus was increased when the methionine concentration in the medium was low. These findings demonstrate that in human monocytes ubiquinone-50 biosynthesis is regulable and that methionine concentration modulates both its rate of synthesis and the inhibitory effects of two stimuli of O2- production.     

Participation of chlorobiumquinone in the transplasma membrane electron transport system of Leishmania donovani promastigote: effect of near-ultraviolet light on the redox reaction of plasma membrane

The respiratory quinone composition of the parasitic protozoa Leishmania donovani promastigote was investigated. 1'-oxomenaquinone-7, a chlorobiumquinone was found to be the major isoprenoid quinone. Substantial level of ubiquinone-9 was also present. Isolation and identification of the quinone from the purified plasma membrane yielded mainly 1'-oxomenaquinone-7 and ubiquinone-9; menaquinone was not detected. Membrane bound 1'-oxomenaquinone-7 could be destroyed by near-ultraviolet irradiation, with a concomitant loss or stimulation of plasma membrane electron transport activities. The abilities of different quinones to restore alpha-lipoic acid and ferricyanide reductase activity in near UV-irradiated cell preparations were compared. The order was; conjugate of chlorobiumquinone and sphingosine base approximately conjugate of 2-methyl-3-(1'-oxooctadecyl)-1,4-napthoquinone and octadecylamine > chlorobiumquinone approximately 2-methyl-3-(1'-oxooctadecyl)-1,4-napthoquinone > menaquinone-4 approximately ubiquinone-10. After irradiation with near-UV light, transmembrane alpha-lipoic acid reduction was inhibited, while transmembrane ferricyanide reduction was stimulated. The result obtained indicates that chlorobiumquinone mediates the plasma membrane electron transport between cytosolic reductant and oxygen as well as alpha-lipoic acid. UV-inactivation of chlorobiumquinone shuts down the plasma membrane oxygen uptake and diverts the electron flux towards ferricyanide reduction via ubiquinone-9. Chlorobiumquinone is the only example of a polyisoprenoid quinone containing a side chain carbonyl group from photosynthetic green-sulphur bacteria. Recent work has revealed numerous genes of trypanosomatid sharing common ancestry with plants and/or bacteria. These observations pose some fascinating questions about the evolutionary biology of this important group of parasitic protozoa.     

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.     

Untersuchungen zur Anreicherung von Ubichinon-9 aus Lipid-Kohlenwasserstoff-Franktionen mittels Sepahdex LH-20

Sephadex LH-20 was tested for a potential application in the enrichment of ubiquinone-9 from a lipid-hydrocarbon-extract. The lipid-hydrocarbon-extract was isolated from yeast grown on gas oil (b. p. 240 to 380°C) as carbon-source. The main components of the lipid-hydrocarbon extract were phosphatides, glycerides, fatty acids, and hydrocarbons of gas oil. By use of Sephadex LH-20 and CHCl₃/CH₃OH (2:1)as eluent it is possible to concentrate the whole ubiquinone-9 in a special fraction both directly from the lipid-hydrocarbon-extract and from the aceton-soluble part of the extract The concentration of ubiquinone in the special fraction was ten times higher than in the raw-material and the hydrocarbons were separated completely. Ubiquinone-9 can be obtained by a simplified separation from the enriched fraction.     

Identification of 5-desmethylubiquinone-9 as an intermediate in the biosynthesis of ubiquinone-9 by rat liver mitochondria

Radioactive 5-desmethylubiquinone-9 has been isolated from mitochondria synthesizing ubiquinone-9-¹⁴C from p-hydroxybenzoate-U-¹⁴C. By mass spectrometry, the natural 5-desmethylubiquinone-9 has been shown to be identical with that chemically synthesized from fumigatol and solanesol. Synthetic 5-desmethylubiquinone-9-³H can be methylated to ubiquinone-9-³H by S-adenosyl-L-methionine in submitochondrial particles.     

Participation of chlorobiumquinone in the transplasma membrane electron transport system of Leishmania donovani promastigote: Effect of near-ultraviolet light on the redox reaction of plasma membrane

The respiratory quinone composition of the parasitic protozoa Leishmania donovani promastigote was investigated. 1′-oxomenaquinone-7, a chlorobiumquinone was found to be the major isoprenoid quinone. Substantial level of ubiquinone-9 was also present. Isolation and identification of the quinone from the purified plasma membrane yielded mainly 1′-oxomenaquinone-7 and ubiquinone-9; menaquinone was not detected. Membrane bound 1′-oxomenaquinone-7 could be destroyed by near-ultraviolet irradiation, with a concomitant loss or stimulation of plasma membrane electron transport activities. The abilities of different quinones to restore α-lipoic acid and ferricyanide reductase activity in near UV-irradiated cell preparations were compared. The order was; conjugate of chlorobiumquinone and sphingosine base ? conjugate of 2-methyl-3-(1′-oxooctadecyl)-1,4-napthoquinone and octadecylamine >> chlorobiumquinone ? 2-methyl-3-(1′-oxooctadecyl)-1,4-napthoquinone > menaquinone-4 ? ubiquinone-10. After irradiation with near-UV light, transmembrane α-lipoic acid reduction was inhibited, while transmembrane ferricyanide reduction was stimulated. The result obtained indicates that chlorobiumquinone mediates the plasma membrane electron transport between cytosolic reductant and oxygen as well as α-lipoic acid. UV-inactivation of chlorobiumquinone shuts down the plasma membrane oxygen uptake and diverts the electron flux towards ferricyanide reduction via ubiquinone-9. Chlorobiumquinone is the only example of a polyisoprenoid quinone containing a side chain carbonyl group from photosynthetic green-sulphur bacteria. Recent work has revealed numerous genes of trypanosomatid sharing common ancestry with plants and/or bacteria. These observations pose some fascinating questions about the evolutionary biology of this important group of parasitic protozoa.     

Role of ubiquinone-10 in electron transport system of chromatophores from Rhodospirillum rubrum.

The role of ubiquinone-10 in the activities for the reduction of free cytochrome c2 and bound cytochrome cc' by succinate was studied with chromatophores from a blue-green mutant (G-9) of Rhodospirillum rubrum. 1. By a single extraction with isooctane, approximately 90% of ubiquinone-10 was easily removed from the chromatophores. In the extracted chromatophores, the activity for succinate-cytochrome c2 reduction decreased to 5-10% of the original activity. This depressed activity was mostly restored by adding ubiquinone-10. The remaining quinone was hardly extractable, even by repeated extractions. With repeatedly extracted chromatophores, the activity for succinate-cytochrome c2 reduction was mostly restored to the same extent as with once-extracted chromatophores, whereas the extent of inhibtion of the activity by antimycin A gradually fell. 2. In isooctane-extracted chromatophores, the activity for the reduction of bound cytochrome cc' by succinate under anaerobic conditions decreased to 35 to 95% of the original level. With chromatophores in which the remaining activity was as low as 40% of the original level, the activity was partially restored by adding ubiquinone-10, but this was not the case with chromatophores in which the remaining activity was higher than approximately 50% of the original level.     

Free radicals and metal paramagnetic ions of the tissues of rats exposed to gamma irradiation and hormones

It was shown that gamma-radiation (8-16 Gy) did not influence the number of paramagnetic centers of hemal (g = 2.42, g = 2.25) and nonhemal (g = 1.94) iron of rat tissues: free radicals were significantly reduced 5 min after 16 Gy irradiation. The combined effect of gamma-radiation (8 Gy) and ubiquinone-9 decreased the number of the above-mentioned ESR signals after 48 h. The number of free radicals in the rat liver, kidneys and spleen was almost twice reduced 28 days following castration. The administration of methyl testosterone increased the free radical amount in the heart and was ineffective in other rat organs.     

Free-living nematodes Caenorhabditis elegans possess in their mitochondria an additional rhodoquinone, an essential component of the eukaryotic fumarate reductase system.

The respiratory chain of Caenorhabditis elegans was characterized in mitochondria isolated from aerobically grown nematodes. Nematode mitochondria contain ubiquinone-9 as a major component and rhodoquinone-9 as a minor component. The ratio of ubiquinone-9/rhodoquinone-9 is higher in C. elegans mitochondria than in mitochondria from second-stage larvae of Ascaris suum, the free-living stage of porcine gut-dwelling nematode. The individual oxidoreductase activities comprising succinate oxidase and the amount of substrate-reducible cytochromes are comparable to those of mitochondria from second-stage larvae of A. suum. The specific activity of fumarate reductase is lower in C. elegans mitochondria than in mitochondria from second-stage larvae of A. suum, but still higher than in mammalian mitochondria. These results indicate that the free-living nematode C. elegans is capable of synthesizing rhodoquinone, as distinguished from aerobic mammalian species, although its mitochondria appear more aerobic than A. suum larval mitochondria.     

Analytical method for ubiquinone-9 and ubiquinone-10 in rat tissues by liquid chromatography/turbo ion spray tandem mass spectrometry with 1-alkylamine as an additive to the mobile phase

We investigated the application of 1-alkylamines, as additives to the mobile phase, to a quantification method for ubiquinone-9 (CoQ9) and ubiquinone-10 (CoQ10) in rat thigh muscle and heart using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the optimization of the analytical method, we found that 1-alkylamines mixed with CoQ9 and CoQ10 in the turbo ion sprayed solution formed the 1-alkylammonium adduct molecules of these compounds during the ionization process and that the intensity of the adduct ions was considerably higher than that of the protonated molecules ([M+H]+) of these compounds. Furthermore, we investigated a variety of 1-alkylamines in the mobile phase for LC-MS/MS analysis to select the most appropriate 1-alkylamine for higher sensitivities of CoQ9 and CoQ10. After these examinations, we found that methylamine was the most suitable additive for the mobile phase, allowing a 12.5-fold gain in signal intensity in the full ion mass spectrum compared with that without methylamine. The internal standard (IS) used was ubiquinone-11 (CoQ11) for each analyte. The analytes and IS were extracted with methanol from the tissue homogenates at neutral pH and were injected into an LC-MS/MS with a turbo ion spray interface. The calibration curves for CoQ9 (5-500 microg/g in thigh muscle and 50-10,000 microg/g in heart) and CoQ10 (1-500 microg/g in thigh muscle and 10-10,000 microg/g in heart) showed good linearity. The method was precise; the relative standard deviations of the method for rat thigh muscle were not more than 13.5 and 9.0% for CoQ9 and CoQ10, respectively, and those for rat heart were not more than 6.7 and 5.4% for CoQ9 and CoQ10, respectively. The accuracies of the method for both rat thigh muscle and heart were good, with the deviations between the nominal concentration and calculated concentration of CoQ9 and CoQ10 typically being within 12.3 and 4.3%, respectively. This method provided reliable concentration levels for CoQ9 and CoQ10 in rat thigh muscle and heart.     

Polyprenols of Aspergillus niger. Their characterization, biosynthesis and subcellular distribution

A polyprenol complex of Aspergillus niger was shown, by using spectrometric methods, to consist of a family of exo-methylene-hexahydroprenols that contain between 18 and 24 isoprene residues per molecule. Each prenol contains two trans residues, three saturated residues (alpha, omega and psi) and an exo-methylene substituent on the carbon atom beta to the isopropyl group in each omega-residue. The ubiquinone complex consisted of 90% ubiquinone-9, 9% ubiquinone-8 and 1% ubiquinone-10. The amount of polyprenol complex present reached a maximum of 1.7mg/culture bottle after 9-10 days of growth, coincident with the maximum weight of mycelium. The amount of ergosterol (10mg/culture bottle) and ubiquinone (1mg/culture bottle) reached a peak at 8 days. By the 13th day of growth the yield of ergosterol had fallen by 20% and that of ubiquinone by 85%. A study of the incorporation of [2-(14)C]mevalonate over different time-intervals confirmed that there was a slow turnover of prenol, a more rapid turnover of ergosterol and a very rapid turnover of ubiquinone. At any one time each member of the prenol complex had essentially the same specific radioactivity as other members of the complex. A similar conclusion was made about the ubiquinone mixture. Just over half of the polyprenol present was esterified to fatty acids. Subcellular fractionation studies indicated that the unesterified prenol is associated primarily with a mitochondrial fraction, whereas the ester is more widely distributed.     

Distribution and redox state of ubiquinones in rat and human tissues.

The distribution and redox state of ubiquinone in rat and human tissues have been investigated. A rapid extraction procedure and direct injection onto HPLC were employed. It was found in model experiments that in postmortem tissue neither oxidation nor reduction of ubiquinone occurs. In rat the highest concentrations of ubiquinone-9 were found in the heart, kidney, and liver (130-200 micrograms/g). In brain, spleen, and intestine one-third and in other tissues 10-20% of the total ubiquinone contained 10 isoprene units. In human tissues ubiquinone-10 was also present at highest concentrations in heart, kidney, and liver (60-110 micrograms/g), and in all tissues 2-5% of the total ubiquinone contained 9 isoprene units. High levels of reduction, 70-100%, could be observed in human tissues, with the exception of brain and lung. The extent of reduction displayed a similar pattern in rat, but was generally lower.