Anphen and energy status of the animal body

Energy status of rats was altered after administration of anphen [2,4-(hydroxy-3,5-ditretbutyl phenyl)-2-aminomalonic acid] at a dose of 40 mg/kg. Within 30 min after administration, maximal rates of NAD-dependent substrates and succinate oxidation were detected in liver mitochondria, which appears to occur due to activation of the mitochondrogenesis. The rate of electron transport in respiratory chain of mitochondria was decreased 1.3-1.6-fold within 1-1.5 hr, whereas within 3 hrs the patterns of oxidation and energetic coupling in liver mitochondria were reduced to control values. The similar alterations were observed in activities of lymphocyte alpha-glycerophosphate- and succinate dehydrogenases. Shifts in the pool of lipid peroxidation products in biological membranes was apparently responsible for alterations in activity of the energy metabolizing enzymes in lymphocytes and liver mitochondria.     

Hypertension in a patient with acrodermatitis enteropathica

3'3'-diaminobenzidine (DAB) oxidation was studied in the human and rat brain by electron cytochemistry at different periods after death. The product of DAB oxidation localized in mitochondria, lipofucsin pigment granules and erythrocytes. DAB oxidation by rat and human brain mitochondria proved to decrease but slightly even two days after death.     

Evaluation of birth cohort patterns in population disease rates

1. The effects of piroxicam, a nonsteroidal anti-inflammatory drug, on rat liver mitochondria were investigated in order to obtain direct evidence about a possible uncoupling effect, as suggested by a previous work with the perfused rat liver. 2. Piroxicam increased respiration in the absence of exogenous ADP and decreased respiration in the presence of exogenous ADP, the ADP/O ratios and the respiratory control ratios. 3. The ATPase activity of intact mitochondria was increased by piroxicam. With 2,4-dinitrophenol uncoupled mitochondria, inhibition was observed. The ATPase activity of freeze-thawing disrupted mitochondria was insensitive to piroxicam. 4. Swelling driven by the oxidation of several substrates and safranine uptake induced by succinate oxidation were inhibited. 5. The results of this work represent a direct evidence that piroxicam acts as an uncoupler, thus, decreasing mitochondrial ATP generation.     

Fragmented mitochondrial DNA is the predominant carrier of oxidized DNA bases

Previous analyses indicated a high level of oxidative base modification in mitochondrial DNA, the extent of which raised questions about the methodological validity and biological implications. In the present study DNA was isolated from rat liver mitochondria under carefully controlled conditions, and the extent of base oxidation, DNA fragmentation, and nuclear DNA contamination were analyzed. DNA isolated from intact mitochondria treated with DNase consisted of 16.3 kilobase pairs, mostly circular, mitochondrial DNA molecules and a mixture of nuclear and mitochondrial DNA fragments, as identified by agarose gel electrophoresis and hybridization. High-performance liquid chromatography in combination with electrochemical detection confirmed that the overall level of 8-hydroxy-2'-deoxyguanosine, a marker commonly used in the analysis of base oxidation, is higher in mitochondrial than in nuclear DNA. Importantly, 8-hydroxy-2'-deoxyguanosine is relatively scarce in the 16.3 kilobase pair mitochondrial DNA molecules (0.051 pmol/microgram) but is present in high levels in mitochondrial DNA fragments (0.741 pmol/microgram). The fragments constitute about 18% of total mitochondrial DNA. The antitumor agent bleomycin, which binds to DNA, forms an iron complex capable of transferring electrons from Fe2+ to molecular oxygen. Exposure of mitochondria to bleomycin and iron resulted in nicking but not in a significant increase in base oxidation of 16.3 kilobase pair mitochondrial DNA, whereas the amount and the oxidation level of fragmented mitochondrial DNA significantly increased. These findings are relevant for a better understanding of the role of mitochondria in aging and various diseases and are consistent with the notion that despite the overall high DNA oxidation level, mitochondria can faithfully proliferate.     

Effect of bioresmethrin on mitochondrial ultrastructure and oxidative phosphorylation in the flight muscles of Chrysomya albiceps (Wied.) (Diptera: Calliphoridae)

The sublethal effect of bioresmethrin on the ultrastructure, oxidation and phosphorylation of flight muscle mitochondria in the blow fly Chrysomya albiceps which survived treatment of newly formed pupae (0-day old) with 10 micrograms/pupa (LD30) was studied. The ultrastructure of the flight muscle mitochondria was severely deteriorated by treatment with bioresmethrin. The deterioration included disalignment and sometimes destruction of the cristae and vacuolation of mitochondria. The oxidation rate was decreased in treated flies, although the P/O ratio was increased. The present study may indicate that bioresmethrin is neither an uncoupler nor a direct inhibitor of oxidative phosphorylation but may have functioned as an inhibitor of the oxidation rate.     

Inhibition of pyruvate oxidation by skeletal muscle mitochondria by phenylpyruvate

1. Phenylpyruvate inhibits pyruvate plus malate oxidation in human and rat skeletal muscle mitochondria in state 3 and in the uncoupled state, it has, however, no effect in state 4. 2. Inhibition by phenylpyruvate of pyruvate oxidation by intact uncoupled rat muscle mitochondria was competitive, with the Ki value about 0.18 mM. 3. It is suggested that the inhibition of pyruvate oxidation is due to the action of phenylpyruvate on muscle pyruvate dehydrogenase, and is the principal cause of the elevated concentration of pyruvate and lactate in blood plasma of phenylketonuric patients.     

Features of noradrenaline stimulation of rat liver mitochondria respiration by ADP and calcium ions

A single administration of a physiological dose of noradrenaline to animals. in contrast to adrenaline, stimulates the respiration of mitochondria not only under oxidation of FAD-dependent Krebbs cycle substrate of the succinase but also HAD-dependent substrate of alpha-ketoglutarate. In the both cases the phosphorylation rate increases, since the action of noradrenaline, separating the respiration and oxidative phosphorylation, was not found. Noradrenaline increases the capacity of mitochondria to more actively absorb calcium ions under oxidation of succinate than under that of alpha-ketoglutarate.     

Energization of mitochondrial inner membranes caused by L-malate

It was found that 0.06 mug antimycin A/mg mitochondrial protein, an amount sufficient to inhibit electron transfer between cytochromes b and c1 completely, fully reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. The effect of L-malate on cytochrome a was insensitive to oligomycin, but all the uncouplers and detergents tested reversed the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria. It was also found that addition of L-malate to anaerobic mitochondria, like addition of ATP, decreased the fluorescence of 1-anilinonaphthalene-8-sulphonate, and that subsequent addition of uncouplers reversed this effect. The effect of L-malate on the fluorescence of the dye was insensitive to oligomycin. The present findings suggest that addition of L-malate may cause energization of the mitochondrial inner membranes and that the oxidation of cytochrome a caused by L-malate in anaerobic mitochondria may result from an L-malate-induced, energy-linked reversal of electron transfer in site II.     

Absence of relationship between the level of electron transport chain activities and aging in human skeletal muscle

We investigated the relationship between age and respiratory chain function of skeletal muscle mitochondria in 132 control individuals (15 to 95 years old). Muscle mitochondria were studied polarographically and spectrophotometrically. By regression analysis, we found a significant inverse correlation between age and oxygen uptake linked to substrate oxidation (succinate, glutamate-malate or ascorbate-TMPD). However, this significance disappeared after including physical activity and tobacco consumption as confounding variables in a multivariate statistical analysis. Similarly, the activity of respiratory chain complexes individually measured did not decline with age. It therefore appears that respiratory chain activity in human skeletal muscle mitochondria is substantially undamaged during the aging process.     

Preparation and properties of mitochondria derived from synaptosomes

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats

We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.     

Genetic-demographic characteristics of farming regions and small cities of the Tomsk district

The effect of the herbicide 4,6-dinitro-o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10-50 microM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium, in the presence of valinomycin. At higher concentrations (> 50 microM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca(2+)-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca(2+)-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.     

Loop-L bulbar ventricular malformation.II. Surgical treatment

In vitro Mn2+ decreases respiration at metabolic state III and at the uncoupled state. Pretreatment with Mn2+ decreased also ADP to oxygen ratio in rat liver and brain mitochondria. The mechanism of manganese toxicity involving suppression of substrate oxidation and decrease of oxidative phosphorylation efficiency in brain mitochondria is discussed.     

Membrane potential generation coupled to oxidation of external NADH in liver mitochondria

Oxidation of added NADH by rat liver mitochondria has been studied. It is found that exogenous NADH, when oxidized by rat liver mitochondria in sucrose hypotonic medium supplemented with Mg2+ and EGTA, generates a membrane potential (delta psi) even in the absence of added cytochrome c. ADP and phosphate decrease delta psi, the effect being reversed by oligomycin. Rotenone and myxothiazol do not inhibit delta psi generated by oxidation of exogenous NADH. Added cytochrome c increases the rate of the exogenous NADH oxidation and coupled delta psi formation. In sucrose isotonic medium, or in hypotonic medium without Mg2+, exogenous NADH fails to stimulate respiration and to form a membrane potential. In the presence of Mg2+, exogenous NADH appears to be effective in delta psi generation in isotonic sucrose medium if mitochondria were treated with digitonin. In isotonic KCl without Mg2+, oxidation of exogenous NADH is coupled to the delta psi formation and MgCl2 addition before mitochondria prevents this effect. In hypotonic (but not in isotonic) sucrose medium, Mg2+ makes a portion of the cytochrome c pool reducible by exogenous NADH or ascorbate. It is assumed that (i) hypotonic treatment or digitonin causes disruption of the outer mitochondrial membrane, and, as a consequence, desorption of the membrane-bound cytochrome c in a Mg2+-dependent fashion; (ii) incubation in isotonic KCI without Mg2+ results in swelling of mitochondrial matrix, disruption of the outer membrane and cytochrome c desorption whereas Mg2+ lowers the K+ permeability of the inner membrane and, hence, prevents swelling; (iii) desorbed cytochrome c is reduced by added NADH via NADH-cytochrome b5 reductase and cytochrome b5 or by ascorbate and is oxidized by cytochrome oxidase. The role of desorbed cytochrome c in oxidation of superoxide and cytoplasmic NADH as well as possible relations of these events to apoptosis are discussed.     

Comparison of side chain oxidation of potential C27-bile acid intermediates between mitochondria and peroxisomes of the rat liver: presence of beta-oxidation activity for bile acid biosynthesis in mitochondria

The oxidation of the side chains of two potential bile acid intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestanoic acid (THCA) and 3 alpha,7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA), were investigated in rat liver mitochondria and peroxisomes. Both THCA and DHCA were efficiently oxidized to yield cholic acid and chenodeoxycholic acid, along with 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-enoic acid and 3 alpha,7 alpha-dihydroxy-5 beta-cholest-24-enoic acid, respectively, in both the mitochondria and peroxisomes. However, the spectrum of the metabolites in the mitochondria differed greatly from those in the peroxisomes. The major products from THCA and DHCA in the mitochondria were 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-chol-22-enoic acid and 3 alpha,7 alpha-trihydroxy-5 beta-chol-22-enoic acid, respectively, which were tentatively identified from the mass spectral data. However, the formation of these C24-unsaturated bile acids was not observed in the peroxisomes. These results strongly suggest that the cleavage of the side chain of the C27-intermediates for bile acid biosynthesis also occurs independently in the mitochondria, not due to the contamination of peroxisomes.     

The effect of hypoxic hypoxia on energy metabolism in the liver mitochondria and the acetylcholine content of different tissues

It has been found, the 1 or 2 days exposure of animals to hypoxic hypoxia (during 4 hours every day; 32 mm Hg) leads to the decrease of acetylcholine level in rat heart, liver and pancreas tissues and to activation of succinate oxidation in rat liver mitochondria. Beginning from the 7th day of hypoxia treatment the level of acetylcholine in the tissues was increasing and it was connected with the activation of NAD-dependent substrate alpha-ketoglutarate oxidation in the rat liver mitochondria and with the increase of the efficiency of oxidative phosphorylation. The effect increases to the 12-16-th day of animal adaptation. The important role of acetylcholine in formation of nonspecific adaptation reactions of organism to hypoxia has been shown.     

In vitro inhibition of carnitine acyltransferase activity in mitochondria from rat and mouse liver by a diethylhexylphthalate metabolite

The effects of mono(2-ethyl-5-oxohexyl)phthalate [ME(O)HP], a di(2-ethylhexyl)phthalate (DEHP) metabolite and a potent peroxisomal inducer, on the mitochondrial beta-oxidation were investigated. In isolated rat hepatocytes, ME(O)HP inhibited long chain fatty acid oxidation and had no effect on the ketogenesis of short chain fatty acids, suggesting that the inhibition occurred at the site of carnitine-dependent transport across the mitochondrial inner membrane. In rat liver mitochondria, ME(O)HP inhibited carnitine acyltransferase I (CAT I; EC 2.3.1.21) competitively with the substrates palmitoyl-CoA and octanoyl-CoA. An analogous treatment of mouse mitochondria produced a similar competitive inhibition of palmitoyl-CoA transport whereas ME(O)HP exposure with guinea pig and human liver mitochondria revealed little or no effect. The addition of clofibric acid, nafenopin or methylclofenopate revealed no direct effects upon CAT I activity. Inhibition of transferase activity by ME(O)HP was reversed in mitochondria which had been solubilized with octyl glucoside to expose the latent form of carnitine acyltransferase (CAT II), suggesting that the inhibition was specific for CAT I. Our results demonstrate that in vitro ME(O)HP inhibits fatty acid oxidation in rat liver at the site of transport across the mitochondrial inner membrane with a marked species difference and support the idea that induction of peroxisome proliferation could be due to an initial biochemical lesion of the fatty acid metabolism.     

Relations between tocopherol depletion and coenzyme Q during lipid peroxidation in rat liver mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe++/ascorbate and NADPH/ADP/Fe++ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

Effect of restoration of light flashes on energy processes in the cerebral visual system of animals developing under conditions of light deprivation

The changes in the rate and the character of energy processes in the mitochondria of the visual system of the brain caused by prolonged dark-rearing of the animals (for 2 1/2 months from the time of birth) proved to be reversible. The extent of normalization differed in the case of mitochondria of the optic cortex and the superior colliculus. After the animals were placed under normal light conditions the rate of the mentioned processes (when glutamic acid was used as a substrate) increased in the course of a two-week restorative period, and reached the control level or even exceeded it. The rate of the electron transport in the area of cytochrome c -- cytochromoxidase succinic toxidase oxidation chain decreased at the period of restoration and approached the control level. The role of specific impulsation in the age formation of the energy processes of the brain mitochondria is discussed.     

Effects of rapeseed oil on fatty acid oxidation and lipid levels in rat heart and liver

The comparative rates of oxidation of erucic and oleic acids and of their CoA esters were studied in heart and liver mitochondria of rats fed a standard diet or semisynthetic diets containing 25% of the calories as either rapeseed oil (46.6% erucic and 10.4% eicosenoic acid) or olive oil, for a period of 5 months. The long exposure to the diet containing 25% rapeseed oil did not alter the oxidative activity of mitochondria and did not induce morphological changes in the heart. It is confirmed that erucic acid is oxidized in mitochondria at lower rates than other long chain fatty acids and that its activation as CoA derivative may be one of the rate limiting steps of the overall oxidationprocess. Total lipids and triglycerides do not significantly change in the heart whereas they increase in the liver of rats fed the diet containing rapeseed oil.