Quinone-responsive multiple respiratory-chain dysfunction due to widespread coenzyme Q10 deficiency.

BACKGROUND: The respiratory-chain deficiencies are a broad group of largely untreatable diseases. Among them, coenzyme Q10 (ubiquinone) deficiency constitutes a subclass that deserves early and accurate diagnosis. METHODS: We assessed respiratory-chain function in two siblings with severe encephalomyopathy and renal failure. We used high-performance liquid chromatography analyses, combined with radiolabelling experiments, to quantify cellular coenzyme Q10 content. Clinical follow-up and detailed biochemical investigations of respiratory chain activity were carried out over the 3 years of oral quinone administration. FINDINGS: Deficiency of coenzyme Q10-dependent respiratory-chain activities was identified in muscle biopsy, circulating lymphocytes, and cultured skin fibroblasts. Undetectable coenzyme Q10 and results of radiolabelling experiments in cultured fibroblasts supported the diagnosis of widespread coenzyme Q10 deficiency. Stimulation of respiration and fibroblast enzyme activities by exogenous quinones in vitro prompted us to treat the patients with oral ubidecarenone (5 mg/kg daily), which resulted in a substantial improvement of their condition over 3 years of therapy. INTERPRETATION: Particular attention should be paid to multiple quinone-responsive respiratory-chain enzyme deficiency because this rare disorder can be successfully treated by oral ubidecarenone.

Reduction of brain antioxidant defense upon treatment with butylated hydroxyanisole (BHA) and Sudan III in Syrian golden hamster.

Treatment with the antioxidant butylated hydroxyanisole (BHA) or the azo dye Sudan III during two weeks led to changes in the brain enzymatic antioxidant defense of Syrian golden hamsters. BHA was able to induce liver superoxide dismutase (SOD) 2-fold but had no effect on the brain SOD activity, whereas SOD activity was reduced to 50% in brain and remained unchanged in liver with Sudan III. These two substances are known inducers of DT-diaphorase and in fact this enzymatic activity was induced 4- and 6-fold in liver with BHA and Sudan III, respectively. However, BHA promoted a significant 40% reduction, whereas no change was observed with Sudan III in brain DT-diaphorase activity. Glutathione(GSH)-related enzymatic activities were also assayed in brain and liver. No induction was observed with BHA or Sudan III for any of the activities tested in hamster brain: GSH S-transferase (GST), GSH peroxidase (GSH-Px) and glutathione disulfide (GSSG) reductase (GR). Only 1.3- and 1.4-fold increases of GST and GR activities were observed in liver and no change in any of these enzymatic activities in brain with BHA; a partial limitation of permeability to BHA of the blood-brain barrier may explain this results. Furthermore, Sudan III promoted reductions in all these GSH-related enzymatic activities in brain and liver. The possible explanations for these results are discussed.

The protective role of plasmalogens in iron-induced lipid peroxidation.

The role of plasmalogens in iron-induced lipid peroxidation was investigated in two liposomal systems. The first consisted of total brain phospholipids with and without plasmalogens, and the second of phosphatidylethanolamine/phosphatidylcholine liposomes with either diacyl- or alkenylacyl-phosphatidylethanolamine. By measuring thiobarbituric acid reactive substances, oxygen consumption, fatty acids and aldehydes, we show that plasmalogens effectively protect polyunsaturated fatty acids from oxidative damage, and that the vinyl ether function of plasmalogens is consumed simultaneously. Furthermore, the lack of lag phase, the increased antioxidant efficiency with time, and the experiments with lipid- and water-soluble azo compounds, indicate that plasmalogens probably interfere with the propagation rather than the initiation of lipid peroxidation, and that the antioxidative effect cannot be related to iron chelation.

Lipid peroxidation and changes in the ubiquinone content and the respiratory chain enzymes of submitochondrial particles.

The relationship between, lipid peroxidation induced by ascorbate and adenosine ADP/Fe3+, and its effect on the respiratory chain activities of beef heart submitochondrial particles has been investigated. Lipid peroxidation, measured as thiobarbituric acid reactive substance formation, resulted in an inhibition of the NADH and succinate oxidase activities. Examination of several partial reactions of the respiratory chain revealed inactivation primarily of those involving endogenous ubiquinone, i.e., NADH- and succinate-ubiquinone1 and cytochrome c reductases. Ubiquinol-cytochrome c reductase, measured with reduced ubiquinone2 as electron donor, was unaffected. The amount of NADH- or succinate-reducible cytochrome b in the presence of cyanide was strongly decreased, but could be recovered by the addition of antimycin. There occurred a substantial decrease of the ubiquinone content in the course of lipid peroxidation, with a linear relationship between this decrease and the NADH and succinate oxidase activities. The results are consistent with the conclusion that the ubiquinone pool undergoes an oxidative modification during lipid peroxidation, to a form that can no longer function as a component of the respiratory chain. Lipid peroxidation also led to a partial inhibition of the succinate dehydrogenase and cytochrome c oxidase activities and a minor decrease of the cytochrome c and cytochrome a contents. Reduction of endogenous ubiquinone prevented lipid peroxidation as well as the concomitant modification of ubiquinone and inactivation of the respiratory chain. These observations suggest that the destruction of ubiquinone through lipid peroxidation is the primary cause of inactivation of the respiratory chain, and emphasize the antioxidant role of ubiquinol in preventing these effects. The possible implications of these findings for regulation of the cellular turnover of ubiquinone by the prevailing oxidative stress are discussed.

Oxidative modification of nicotinamide nucleotide transhydrogenase in submitochondrial particles: effect of endogenous ubiquinol.

The present paper describes the sensitivity of the mitochondrial nicotinamide nucleotide transhydrogenase (EC 1.6.1.1) to oxidative modification, and the effects of endogenous ubiquinol on this modification. A comparison is made between the effects of treatment with ADP-Fe3+ and ascorbate and with peroxynitrite, using kinetic, electrophoretic, and immunological analyses, together with lipid peroxidation measurements. The transhydrogenase was inactivated by both types of oxidative modification, but apparently through different mechanisms. Ubiquinol protected the enzyme against inactivation only when the modification was caused by ADP-Fe3+ and ascorbate treatment. Kinetic measurements revealed a threefold increase of the Km value of the enzyme for NADPH after exposure to ADP-Fe3+ and ascorbate, and a twofold increase of the Km values for both NADH and NADPH after exposure to peroxynitrite. NAD(H) exerted a protection against trans-hydrogenase inactivation when added to the preincubation in the case of peroxynitrite, but neither NAD(H) or NADP(H) protected in the case of ADP-Fe3+ and ascorbate. Using immunoblotting it was shown that the enzyme became both aggregated and fragmented, although to different extents, depending on the oxidative system used. Again, ubiquinol prevented these effects only in the case of ADP-Fe3+ and ascorbate treatment. Furthermore, there occurred a striking decrease in the 66-kDa trypsin fragment after exposure of the enzyme to ADP-Fe3+ and ascorbate, and of the 48-kDa trypsin fragment after exposure to peroxynitrite. It is concluded that the mitochondrial nicotinamide nucleotide transhydrogenase is sensitive to oxidative stress and that the mechanism underlying this can vary according to the challenge to which the enzyme is exposed. Endogenous ubiquinol may play a role in protecting the enzyme against agents perturbing the lipid phase of the membrane.

P/O ratios reassessed: mitochondrial P/O ratios consistently exceed 1.5 with succinate and 2.5 with NAD-linked substrates.

The efficiency of ATP synthesis coupled to cell respiration, commonly referred to as the P/O ratio, has been the subject of extensive studies for more that 50 years. The general conclusion from these studies is that respiring mitochondria can convert external ADP to ATP at a maximal P/O ratio of 3 for NAD-linked substrates and 2 for succinate. However, in recent years the validity of these "integral" values has been questioned on both mechanistic and thermodynamic grounds, and a mechanistic P/O ratio of 2.5 for NAD-linked substrates and 1.5 for succinate have been concluded on the basis of experiments with isolated mitochondria. These values have been widely adopted in the scientific literature, including several recent textbooks. In this paper we report that under optimal conditions with respect to preparation and assay procedures, the P/O ratios obtained with isolated rat liver mitochondria consistently exceed 2.5 with NAD-linked substrates and 1.5 with succinate. These results, although not excluding "nonintegral" P/O ratios due to various energy-dissipating side reactions, warrant caution in accepting the reported lower values and, in general, in referring to mechanistic considerations unless the underlying molecular mechanisms are understood.

Suspended animation for delayed resuscitation.

Suspended animation is defined as the therapeutic induction of a state of tolerance to temporary complete systemic ischemia, i.w., protection-preservation of the whole organism during prolonged circulatory arrest ( > or = 1 hr), followed by resuscitation to survival without brain damage. The objectives of suspended animation include: a) helping to save victims of temporarily uncontrollable (internal) traumatic (e.g., combat casualties) or nontraumatic (e.g., ruptured aortic aneurysm) exsanguination, without severe brain trauma, by enabling evacuation and resuscitative surgery during circulatory arrest, followed by delayed resuscitation; b) helping to save some nontraumatic cases of sudden death, seemingly unresuscitable before definite repair; and c) enabling selected (elective) surgical procedures to be performed which are only feasible during a state of no blood flow. In the discussion session, investigators with suspended animation-relevant research interests brainstorm on present knowledge, future research potentials, and the advisability of a major research effort concerning this subject. The following topics are addressed: the epidemiologic facts of sudden death in combat casualties, which require a totally new resuscitative approach; the limits and potentials of reanimation research; complete reversibility of circulatory arrest of 1 hr in dogs under profound hypothermia ( < 10 degrees C), induced and reversed by portable cardiopulmonary bypass; the need for a still elusive pharmacologic or chemical induction of suspended animation in the field; asanguinous profound hypothermic low-flow with cardiopulmonary bypass; electric anesthesia; opiate therapy; lessons learned by hypoxia tolerant vertebrate animals, hibernators, and freeze-tolerant animals (cryobiology); myocardial preservation during open-heart surgery; organ preservation for transplantation; and reperfusion-reoxygenation injury in vital organs, including the roles of nitric oxide and free radicals; and how cells (particularly cerebral neurons) die after transient prolonged ischemia and reperfusion. The majority of authors believe that seeking a breakthrough in suspended animation is not utopian, that ongoing communication between relevant research groups is indicated, and that a coordinated multicenter research effort, basic and applied, on suspended animation is justified.

Coenzymes Q9 and Q10 in skeletal and cardiac muscle in tumour-bearing exercising rats.

Physical exercise increases metabolic rate, and induces both adaptational biogenesis of mitochondria in skeletal muscle and an increase in antioxidant capacity. The onset of experimental anorexia and cachexia can be delayed by voluntary exercise. As skeletal muscle is the main target for cancer cachexia, we determined the levels of coenzymes Q9 and Q10 in skeletal muscle from tumour-bearing exercising rats, and compared them to those of sedentary tumour-bearers and controls. Both tumour-bearing groups had increased levels of coenzymes Q9 and Q10 in the anterior tibial muscle (P < 0.05 for exercised animals). In the soleus muscle, only the tumour-bearing exercising animals demonstrated an increase in the levels of both coenzymes (P < 0.05). In cardiac muscle, the presence of tumour and exercise reduced the levels of coenzymes below that of sedentary controls. Exercise counteracted the anaemia in the tumour-bearing host (P < 0.05). In conclusion, the increase in antioxidant capacity in skeletal muscle indicates a defence mechanism in the tumour-bearing hosts which is augmented by physical exercise.

Activity and immunohistochemistry of DT-diaphorase in hamster and human kidney tumours.

We have studied the biochemical and immunohistochemical changes of DT-diaphorase in diethylstilbestrol (DES)-induced hamster kidney tumours and human biopsies from normal kidneys and renal clear cell carcinoma. The activities of primary and secondary antioxidants in these hamster and human tissues are also reported. DT-diaphorase is decreased in the different subcellular fractions of hamster and human tissues. In hamster kidney the activities of the one-electron quinone reductases show a nearly two-fold increase. Immunohistochemical findings confirm the decrease in DT-diaphorase in hamster and human tissues. This image is of special interest in the case of nephroblastoma (Wilms' tumour), since it has been proposed that the DES-induced tumour is a 'nephroblastoma-like' one. Primary anti oxidant enzymatic activities, i.e. superoxide dismutase and glutathione peroxidase, are increased in hamster kidney bearing DES-induced tumours and decreased in human renal clear cell carcinoma. Glutathione disulphide reductase is decreased in hamster and human tumours. The role of these enzymatic activities in the carcinogenic process is also discussed.

Occurrence of prenylated proteins in plant cells.

In this paper evidence is presented for the occurrence of prenylated proteins in plants. When spinach leaves were incubated in the presence of [3H]mevalonate non-extractable lipids were found in the protein fraction after extraction with organic solvents. Alkaline hydrolysis liberated phytol, polyprenyl phosphates-11-15 and also, in contrast to animal cells, polyprenols-11-15. Complete removal of farnesol and geranylgeraniol required the cleavage of thioether linkages by iodomethane. The results indicate that several polyisoprenoid lipids in plant cells are covalently bound to proteins. So far a protein fraction dominated by one or more proteins in the 23 kDa region has been identified.

Biosynthesis of ubiquinone and plastoquinone in the endoplasmic reticulum-Golgi membranes of spinach leaves.

The localization of ubiquinone (UQ) and plastoquinone (PQ) biosynthesis in subfractions isolated from spinach leaves has been studied. UQ-9 and UQ-10 were found mainly in mitochondria, whereas PQ was enriched in chloroplasts, but also found in Golgi membranes. alpha-Unsaturated polyprenol-11 was also present at a low concentration in chloroplasts. Autoradiography revealed the presence of nonaprenyl-4-hydroxybenzoate (NPHB) and nonaprenyl-2-methylquinol (NPMQ) transferase activities involved in quinone biosynthesis in all subfractions, but the specific activities involved in quinone biosynthesis in the total microsomal fraction were 20 times higher than those in mitochondria and chloroplasts. The isolated Golgi vesicles were particularly enriched in both activities. When the incubation medium containing total microsomes or Golgi membranes was supplemented with NADH, NADPH, S-adenosylmethionine, and an ATP-generating system, NPHB and NPMQ were transferred to UQ-9 and PQ, respectively. trans-Prenyltransferase, which synthesizes the side chain of UQ and PQ, was present in the total microsomal fraction. With farnesyl-PP as substrate, no product was formed, but with geranyl-PP, solanesyl-PP was synthesized and transferred to 4-hydroxybenzoate present in the total microsomal fraction. The results show that these membranes from spinach contain farnesyl-PP synthetase. It is concluded that the plant leaf Golgi membranes contain the enzymes for both UQ and PQ biosynthesis and that a specific transport and targeting system is required for selective transfer of UQ to the mitochondria and of PQ to the chloroplast.

Initiation of lipid peroxidation in submitochondrial particles: effect of respiratory inhibitors.

Initiation of lipid peroxidation in the inner mitochondrial membrane was investigated using respiratory substrates and inhibitors and various iron chelates. An iron chelate was required for initiation of lipid peroxidation in the presence of either NADH or NADPH. The two nicotinamide nucleotides exhibited different activities in initiating lipid peroxidation with regard to concentration and to the effects of rotenone and rhein. Succinate and both nicotinamide nucleotides supported lipid peroxidation in the presence of thenoyl trifluoroacetone (TTFA), without a requirement for exogenously added iron. ADP stimulated lipid peroxidation in the case of NAD(P)H and TTFA, but inhibited it in the case of succinate and TTFA. Lipid peroxidation is thought to be enzymatically induced in both the NADH and the succinate dehydrogenase regions of the respiratory chain, and evidence is presented for a novel pathway of NADPH oxidation that may also be involved. Possible initiation mechanisms are discussed.

Inhibition of lipid peroxidation by ubiquinol in submitochondrial particles in the absence of vitamin E.

The relationship between the antioxidant effects of reduced coenzyme Q10 (ubiquinol, UQH2) and vitamin E (alpha-tocopherol) was investigated in beef heart submitochondrial particles in which lipid peroxidation was initiated by incubation with ascorbate + ADP-Fe3+. These effects were examined after extraction of coenzyme Q10 (UQ-10) and vitamin E from the particles and reincorporation of the same components alone or in combination. The results show that UQH2 efficiently inhibits lipid peroxidation even when vitamin E is absent. It is concluded that UQH2 can inhibit lipid peroxidation directly, without the mediation of vitamin E.

The levels of quinone reductases, superoxide dismutase and glutathione-related enzymatic activities in diethylstilbestrol-induced carcinogenesis in the kidney of male Syrian golden hamsters.

The level of quinone oxidoreductases (microsomal and cytosolic DT-diaphorase, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase), superoxide dismutase and glutathione-related enzymatic activities in diethylstilbestrol (DES)-induced carcinogenesis in kidney from Syrian golden hamsters are presented. Animals that exhibited two different stages of DES-induced carcinogenesis in kidney--pre- and neoplastic lesions and tumorous lesions (after 6 and 8 months of continuous exposure to DES respectively)--were studied in comparison to kidneys from control animals. A dramatic decrease in microsomal and cytosolic DT-diaphorase activities (13.6 and 37.8% of controls), as well as in glutathione disulphide reductase (39.5%), and less marked in superoxide dismutase (45.6%), NADH cytochrome b5 reductase (61.9%) glutathione transferase (GST) towards 1-chloro-2,4-dinitrobenzene (CDNB) (66.2%) and glutathione peroxidase (GSH-Px) (80%) activities, were observed in kidneys with pre- and neoplastic lesions. NADPH-cytochrome P450 reductase and GST activity towards 4-hydroxy-2,3-trans-nonenal (4-HNE) showed no statistically significant variation at this stage of carcinogenesis. In kidney from animals with tumorous lesions, all the enzymatic activities mentioned above decreased, except for superoxide dismutase, which was increased to 186% of the control activity. GST activity towards 4-HNE again showed no statistically significant variation. These results suggest that if one-electron reduction of diethylstilbestrol-4',4''-quinone (DESQ) occurs, it may play a very important role in the development of DES carcinogenesis (pre- and neoplastic lesions), since at this stage of carcinogenesis the primary defense mechanisms against the oxygen free radicals generated in this way, i.e. SOD activity, is reduced to less than a half of control values. Both cytosolic and microsomal DT-diaphorase activities are unable at this stage of carcinogenesis to promote effectively the two-electron reduction of DESQ, which would avoid the initial formation of superoxide anion. The consequences of these decreases may be an increased steady-state concentration of superoxide anion and hydrogen peroxide, which in the presence of iron might lead to lipid peroxidation. GST activity towards 4-HNE could be responsible for the possible higher steady-state concentration of this lipid peroxidation product during DES treatment. The induction of DT-diaphorase and its protective role in the prevention of the development of pre- and neoplastic lesions in kidney from Syrian golden hamster during DES treatment is also discussed.

Hydrogen peroxide production by monoamine oxidase in isolated rat-brain mitochondria: its effect on glutathione levels and Ca2+ efflux.

H2O2 production and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H2O2 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial NADPH to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H2O2 generated by monoamine oxidase. The H2O2 accumulation was also inhibited by externally added reduced glutathione or NADPH but not NADH. External NADPH was oxidized by added oxidized glutathione but not alpha-ketoglutarate + NH4+. These results suggest that the removal of H2O2 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and NADPH, possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H2O2 generated by monoamine oxidase enhances Ca2+ release from mitochondria and may thus function as a regulator of mitochondrial Ca2+ efflux.

The oxidative inactivation of mitochondrial electron transport chain components and ATPase.

Bovine heart submitochondrial particles (SMP) were exposed to continuous fluxes of hydroxyl radical (.OH) alone, superoxide anion radical (O2-) alone, or mixtures of .OH and O2-, by gamma radiolysis in the presence of 100% N2O (.OH exposure), 100% O2 + formate (O2- exposure), or 100% O2 alone (.OH + O2- exposure). Hydrogen peroxide effects were studied by addition of pure H2O2. NADH dehydrogenase, NADH oxidase, succinate dehydrogenase, succinate oxidase, and ATPase activities (Vmax) were rapidly inactivated by .OH (10% inactivation at 15-40 nmol of .OH/mg of SMP protein, 50-90% inactivation at 600 nmol of .OH/mg of SMP protein) and by .OH + O2- (10% inactivation at 20-80 nmol of .OH + O2-/mg of SMP protein, 45-75% inactivation at 600 nmol of .OH + O2-/mg of SMP protein). Importantly, O2- was a highly efficient inactivator of NADH dehydrogenase, NADH oxidase, and ATPase (10% inactivation at 20-50 nmol of O2-/mg of SMP protein, 40% inactivation at 600 nmol of O2-/mg of SMP protein), a mildly efficient inactivator of succinate dehydrogenase (10% inactivation at 150 nmol of O2-/mg of SMP protein, 30% inactivation at 600 nmol of O2-/mg of SMP protein), and a poor inactivator of succinate oxidase (less than 10% inactivation at 600 nmol of O2-/mg of SMP protein). H2O2 partially inactivated NADH dehydrogenase, NADH oxidase, and cytochrome oxidase, but even 10% loss of these activities required at least 500-600 nmol of H2O2/mg of SMP protein. Cytochrome oxidase activity (oxygen consumption supported by ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine) was remarkably resistant to oxidative inactivation, with less than 20% loss of activity evident even at .OH, O2-, OH + O2-, or H2O2 concentrations of 600 nmol/mg of SMP protein. Cytochrome c oxidase activity, however (oxidation of, added, ferrocytochrome c), exhibited more than a 40% inactivation at 600 nmol of .OH/mg of SMP protein. The .OH-dependent inactivations reported above were largely inhibitable by the .OH scavenger mannitol. In contrast, the O2(-)-dependent inactivations were inhibited by active superoxide dismutase, but not by denatured superoxide dismutase or catalase. Membrane lipid peroxidation was evident with .OH exposure but could be prevented by various lipid-soluble antioxidants which did not protect enzymatic activities at all.(ABSTRACT TRUNCATED AT 400 WORDS)