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.

Pro- and anti-oxidant activities of the mitochondrial respiratory chain: factors influencing NAD(P)H-induced lipid peroxidation.

This paper is a study of factors influencing the rate of lipid peroxidation in beef heart submitochondrial particles induced by NAD(P)H via the NADH-ubiquinone oxidoreductase (Complex I) of the respiratory chain. In accordance with earlier observations, both NADH and NADPH initiated lipid peroxidation in the presence of ADP-Fe3+. The rate of the reaction, measured as oxygen consumption and formation of thiobarbituric acid reactive substances, was biphasic as a function of NADH concentration, reaching a maximum at low NADH concentrations and then declining. In contrast, the NADPH-initiated lipid peroxidation showed a monophasic concentration profile of hyperbolic character. Rotenone did not eliminate the biphasicity of the NADH-induced reaction, indicating that this was not due to an antioxidant effect of reduced ubiquinone at high NADH concentrations. This conclusion was further supported by the demonstration that extraction of ubiquinone from the particles did not relieve the inhibition of lipid peroxidation by high NADH concentrations. However rhein, another inhibitor of Complex I, eliminated the biphasicity, and even caused a substantial stimulation of the NADH-induced lipid peroxidation in the particles upon extraction of ubiquinone by pentane. No similar effect occurred in the case of NADPH-induced lipid peroxidation. Furthermore, rhein facilitated both NADH- and NADPH-induced lipid peroxidation even in the absence of added ADP-Fe3+, in a fashion similar to that earlier reported with succinate in the presence of theonyltrifluoroacetone. Based on these findings and measurements of the redox states of ubiquinone and cytochromes in the presence of KCN and NADH or NADPH, it is concluded that Complex I may distinguish between electron input from NADH and NADPH by differences in the site(s) of substrate binding and in the pathways and rates of NADH and NADPH oxidation.

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.

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.

Cerebral resuscitation from cardiac arrest: pathophysiologic mechanisms.

Both the period of total circulatory arrest to the brain and postischemic-anoxic encephalopathy (cerebral postresuscitation syndrome or disease), after normothermic cardiac arrests of between 5 and 20 mins (no-flow), contribute to complex physiologic and chemical derangements. The best documented derangements include the delayed protracted inhomogeneous cerebral hypoperfusion (despite controlled normotension), excitotoxicity as an explanation for selectively vulnerable brain regions and neurons, and free radical-triggered chemical cascades to lipid peroxidation of membranes. Protracted hypoxemia without cardiac arrest (e.g., very high altitude) can cause angiogenesis; the trigger of it, which lyses basement membranes, might be a factor in post-cardiac arrest encephalopathy. Questions to be explored include: What are the changes and effects on outcome of neurotransmitters (other than glutamate), of catecholamines, of vascular changes (microinfarcts seen after asphyxia), osmotic gradients, free-radical reactions, DNA cleavage, and transient extracerebral organ malfunction? For future mechanism-oriented studies of the brain after cardiac arrest and innovative cardiopulmonary-cerebral resuscitation, increasingly reproducible outcome models of temporary global brain ischemia in rats and dogs are now available. Disagreements exist between experienced investigative groups on the most informative method for quantitative evaluation of morphologic brain damage. There is agreement on the desirability of using not only functional deficit and chemical changes, but also morphologic damage as end points.

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.

Antioxidant activity of reduced plastoquinone in chloroplast thylakoid membranes.

The antioxidant effect of reduced plastoquinone was studied in chloroplast membranes. Isolated spinach thylakoid membranes were subjected to strong illumination followed by analysis of pigment bleaching and lipid peroxidation. The plastoquinone pool was kept in the reduced or oxidized state during the light stress by the addition of the electron transport inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and o-phenanthroline, respectively. In the absence of inhibitors there occurred a bleaching of carotenoids and chlorophyll a, while chlorophyll b was unchanged. Formation of thiobarbituric acid reactive substances, used as a measure of lipid peroxidation, was negligible during the first hour of strong illumination, but during the second hour there was a marked increase in the rate of lipid peroxidation. Reduction of the plastoquinone pool resulted in a virtually complete inhibition of lipid peroxidation and pigment bleaching. In contrast, conditions of an oxidized plastoquinone pool markedly enhanced lipid peroxidation and pigment bleaching. It is argued that the reduced form of plastoquinone can act as a scavenger of toxic oxygen species generated in the thylakoid membranes during strong illumination.

Endogenous ubiquinol prevents protein modification accompanying lipid peroxidation in beef heart submitochondrial particles.

This article is a study of the relationship between lipid peroxidation and protein modification in beef heart submitochondrial particles, and the protective effect of endogenous ubiquinol (reduced coenzyme Q) against these effects. ADP-Fe3+ and ascorbate were used to initiate lipid peroxidation and protein modification, which were monitored by measuring TBARS and protein carbonylation, respectively. Endogenous ubiquinone was reduced by the addition of succinate and antimycin. The parameters investigated included extraction and reincorporation of ubiquinone, and comparison of the effect of ubiquinol with those of various antioxidant compounds and enzymes, as well as the iron chelator EDTA. Under all conditions employed there was a close correlation between lipid peroxidation and protein carbonylation, and the inhibition of these effects by endogenous ubiquinol. SDS-PAGE analysis revealed a differential effect on individual protein components and its prevention by ubiquinol. Conceivable mechanisms behind the observed oxidative modifications of membrane phospholipids and proteins and of the role of ubiquinol in preventing these effects are considered.

Biochemical, physiological and medical aspects of ubiquinone function.

This presentation is a brief review of current knowledge concerning some biochemical, physiological and medical aspects of the function of ubiquinone (coenzyme Q) in mammalian organisms. In addition to its well-established function as a component of the mitochondrial respiratory chain, ubiquinone has in recent years acquired increasing attention with regard to its function in the reduced form (ubiquinol) as an antioxidant. Ubiquinone, partly in the reduced form, occurs in all cellular membranes as well as in blood serum and in serum lipoproteins. Ubiquinol efficiently protects membrane phospholipids and serum low-density lipoprotein from lipid peroxidation, and, as recent data indicate, also mitochondrial membrane proteins and DNA from free-radical induced oxidative damage. These effects of ubiquinol are independent of those of exogenous antioxidants, such as vitamin E, although ubiquinol can also potentiate the effect of vitamin E by regenerating it from its oxidized form. Tissue ubiquinone levels are regulated through the mevalonate pathway, increasing upon various forms of oxidative stress, and decreasing during aging. Drugs inhibiting cholesterol biosynthesis via the mevalonate pathway may inhibit or stimulate ubiquinone biosynthesis, depending on their site of action. Administration of ubiquinone as a dietary supplement seems to lead primarily to increased serum levels, which may account for most of the reported beneficial effects of ubiquinone intake in various instances of experimental and clinical medicine.

Uptake of dietary coenzyme Q supplement is limited in rats.

Coenzyme Q is an important mitochondrial redox component and the only endogenously produced lipid-soluble antioxidant. Its tissue concentration decreases with aging and in a number of diseases; dietary supplementation of this lipid would fulfill important functions by counteracting coenzyme Q depletion. To investigate possible uptake, rats were administered 12 mumol coenzyme Q10/100 g body wt once daily by gastric intubation. The appearance of coenzyme Q10 in various tissues and blood after 6 h, 4 d or 8 d was studied. The control group of rats received rapeseed-soybean oil (the vehicle in the experimental group). Lipids were extracted with petroleum ethermethanol, and the reduced and oxidized forms of coenzyme Q9 and Q10 were separated and quantified by reversed-phase HPLC. In the plasma, the total coenzyme Q concentration was doubled after 4 d of treatment. Coenzyme Q10 was also recovered in liver homogenates, where, as in the plasma, it was largely in the reduced form. Uptake into the spleen could be to a large extent accounted for by the blood content of this organ. No dietary coenzyme Q10 was recovered in the heart or kidney. The uptake in the whole body was 2-3% of the total dose. Coenzyme Q10 found in the liver was located mainly in the lysosomes. Dietary coenzyme Q10 did not influence the endogenous biosynthesis of coenzyme Q9. This is in contrast to dietary cholesterol, which down-regulates cholesterol biosynthesis. The dietary coenzyme Q10 level in the plasma decreased to approximately 50% after 4 d. These results suggest that dietary coenzyme Q10 may play a role primarily in the blood and that no appreciable uptake occurs into tissues.

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.