Brain Mitochondrial Enzymes Research Articles

Effects of Allanblackia floribunda stem-bark Extracts on Oxidative stress and Mitochondrial Dysfunction in Rats Exposed to Crude Oil

The development of degenerative diseases has been linked to oxidative stress and mitochondrial dysfunction. Additionally, it has been demonstrated that the changes caused by crude oil exposure contribute to the onset of degenerative diseases. There is little or no information on the anti-degeneration properties of Allanblackia floribunda. This study was carried out to investigate brain mitochondrial tissues exposed to crude oil and the effects of A. floribunda as a preventive and therapeutic intervention against oxidative stress and mitochondrial dysfunction. Twenty-five Wistar rats were divided into five groups: the control group received distilled water, the second group received ethanol extracts of A. floribunda, the third group received crude oil (5 mL/kg), the fourth and fifth groups received crude oil and A. floribunda extract concurrently (200 and 400 mg/kg, respectively). The results showed that crude oil treatment caused a significant increase in brain mitochondrial MDA levels and induced significant alterations in brain mitochondrial antioxidant enzyme activities compared with control. Treatment with the extract alone revealed a significant increase in protein carbonyl, conjugated dienes, and ATPases compared with crude oil-treated rats. This indicates that A. floribunda inhibited crude oil-induced mitochondrial lipid peroxidation and improved ATPase and antioxidant status in the rat brain mitochondria, which further suggests that A. floribunda possesses potential and may efficiently inhibit crude oil-induced oxidative damage and improve mitochondrial functions.

A Look into Liver Mitochondrial Dysfunction as a Hallmark in Progression of Brain Energy Crisis and Development of Neurologic Symptoms in Hepatic Encephalopathy.

Background: The relationship between liver disease and neuropathology in hepatic encephalopathy is well known, but the genesis of encephalopathy in liver failure is yet to be elucidated. Conceptually, the main cause of hepatic encephalopathy is the accumulation of brain ammonia due to impaired liver detoxification function or occurrence of portosystemic shunt. Yet, as well as taking up toxic ammonia, the liver also produces vital metabolites that ensure normal cerebral function. Given this, for insight into how perturbations in the metabolic capacity of the liver may be related to brain pathology, it is crucial to understand the extent of ammonia-related changes in the hepatic metabolism that provides respiratory fuel for the brain, a deficiency of which can give rise to encephalopathy. Methods: Hepatic encephalopathy was induced in starved rats by injection of ammonium acetate. Ammonia-induced toxicity was evaluated by plasma and freeze-clamped liver and brain energy metabolites, and mitochondrial, cytoplasmic, and microsomal gluconeogenic enzymes, including mitochondrial ketogenic enzymes. Parameters of oxidative phosphorylation were recorded polarographically with a Clark-type electrode, while other measures were determined with standard fluorometric enzymatic methods. Results: Progressive impairment of liver mitochondrial respiration in the initial stage of ammonia-induced hepatotoxicity and the subsequent energy crisis due to decreased ATP synthesis lead to cessation of gluconeogenesis and ketogenesis. Reduction in glucose and ketone body supply to the brain is a terminal event in liver toxicity, preceding the development of coma. Conclusions: Our study provides a framework to further explore the relationship between hepatic dysfunction and progression of brain energy crisis in hepatic encephalopathy.

Trigonella foenum-graecum seeds extract plays a beneficial role on brain antioxidant and oxidative status in alloxan-induced Wistar rats

AbstractContextTrigonella foenum-graecum (TriFG) exhibits increased scavenger enzymatic activities and reduces the production of reactive oxygen species in diabetic rats.ObjectiveThe present study was aimed to investigate the effect of TriFG on lipid peroxidation levels and antioxidant status in brain tissue of rats exposed to alloxan.Materials and MethodsHealthy male rats (180 ± 10 g) were allocated into five groups. Animals in group 1 maintained on normal tap water served as controls and rats in groups 2, 3, 4, and 5 were treated as experimental groups. Rats in group 2 were intraperitoneally injected with alloxan (120 mg/kg BW) and treated as diabetic rats, whereas rats in groups 3 and 4 were maintained on same experimental regimen as that of rats in groups 1 and 2, respectively, and in addition, they were orally gavaged with herbal extracts of TriFG (0.25 g/kg BW). Diabetic rats treated with glibenclamide in group 5 were used as positive controls.Results and DiscussionSignificant (P < 0.001) increase in the antioxidant enzymes with a significant (P < 0.001) decrease in the lipid peroxidation levels were observed in the brain tissue of diabetic rats treated with TriFG extract as compared to diabetic and glibenclamide-treated rats. No significant changes were observed in pro- and antioxidant levels in brain tissue of rats treated with TriFG extract alone when compared to normal rats. In diabetic rats, brain mitochondrial and cytosolic enzymes like succinate dehydrogenase, glutamate dehydrogenase, and glucose-6-phosphate dehydrogenase activity levels were significantly (P < 0.05) decreased with reversely increased was observed in lactate dehydrogenase activity (P < 0.05).ConclusionsThe findings of the present study suggested that TriFG, through its antioxidant properties, protects brain tissue by mitigating oxidative stress induced by alloxan-exposed rats. TriFG extract significantly increased the antioxidant and oxidative properties in diabetic rats when compared with the control group rats.

Nicorandil attenuates neuronal mitochondrial dysfunction and oxidative stress associated with murine model of vascular calcification.

Evidences suggest that the presence of chronic kidney disease (CKD) is associated with cerebrovascular diseases related cognitive decline in dialysis patients. As mitochondrial dysfunction is implicated in neurodegenerative disorders, we hypothesized that changes in brain mitochondria occur due to vascular calcification induced by renal failure and the opening of the mitochondrial potassium channel using nicorandil may prevent its dysfunction. Brain tissues from rats with vascular calcification were studied. Nicorandil (7.5 mg/kg b.wt.) was given either concomitantly or after the induction of calcification. The brain tissues were evaluated for antioxidant capacity, mitochondrial enzymes and oxidative phosphorylation efficiency along with the progression of calcification. The results suggested that renal failure, elevated the calcium, phosphorus product in the brain. The brain cytoplasm and mitochondrial fractions showed an elevated TBARS and a corresponding decline in the antioxidant enzymes, indicating a sev ere oxidative stress. The elevated brain mitochondrial enzymes like NADH dehydrogenase, and succinate dehydrogenase in the disease control groups, reversed to the near control level after nicorandil treatment. We observed that nicorandil was more effective when given after calcification. It reduced the biochemical alterations associated with calcium and phosphorous toxicity in the brain, by preserving mitochondria, the key target for treating neurodegenerative diseases.

Combination therapy of normobaric oxygen with hypothermia or ethanol modulates pyruvate dehydrogenase complex in thromboembolic cerebral ischemia

Pyruvate dehydrogenase complex (PDH) is a brain mitochondrial matrix enzyme. PDH impairment after stroke is particularly devastating given PDH's critical role in the link between anaerobic and aerobic metabolism. This study evaluates the restoration of oxidative metabolism and energy regulation with a therapeutic combination of normobaric oxygen (NBO) plus either therapeutic hypothermia (TH) or ethanol. Sprague-Dawley rats were subjected to middle cerebral artery occlusion with an autologous embolus. One hour after occlusion, tissue-type plasminogen activator (t-PA) was administered alone or with NBO (60%), EtOH (1.0 g/kg), or TH (33°C), either singly or in combination. Neurological deficit score and infarct volume were assessed 24 hr after t-PA-induced reperfusion. PDH activity and reactive oxygen species (ROS) levels were measured 3 and 24 hr after t-PA. Western blotting was used to detect PDH and pyruvate dehydrogenase kinase (PDK) protein expression. After t-PA in ischemic rats, NBO combined with TH or EtOH most effectively decreased infarct volume and neurological deficit. The combined therapies produced greater increases in PDH activity and protein expression as well as greater decreases in PDK expression. Compared with the monotherapeutic approaches, the combined therapies provided the most significant declines in ROS generation. Reperfusion with t-PA followed by 60% NBO improves the efficacy of EtOH or TH in neuroprotection by ameliorating oxidative injury and improving PDH regulation. Comparable neuroprotective effects were found when treating with either EtOH or TH, suggesting a similar mechanism of neuroprotection and the possibility of substituting EtOH for TH in clinical settings. © 2016 Wiley Periodicals, Inc.

Abstract TP268: Combination Therapy of Normobaric Oxygen With Hypothermia or Ethanol Modulates Pyruvate Dehydrogenase Complex in Thromboembolic Cerebral Ischemia

Background and Hypothesis: Pyruvate Dehydrogenase Complex (PDH) is a brain mitochondrial matrix enzyme that is inactivated during stroke injury. PDH impairment after stroke can be particularly devastating given PDH’s critical role in the conversion from anaerobic to aerobic energy metabolism. In this study, we evaluated the restoration of oxidative metabolism by measuring reactive oxygen species (ROS) levels and energy regulation by characterizing modulation of PDH and its inhibitor, pyruvate dehydrogenase kinase (PDK), with therapeutic combination of normobaric oxygen (NBO) plus either hypothermia (Hypo) or ethanol (EtOH). Methods: Sprague-Dawley rats were subjected to middle cerebral artery (MCA) occlusion induced with an autologous embolus, the more clinically relevant stroke model. One hour after occlusion, tissue-type plasminogen activator (t-PA) was administered alone or with NBO (60%), EtOH (1.0g/kg) or Hypo (33°C), either singly or in combination. PDH activity and ROS levels were measured at 3 and 24 hours after t-PA administration. Western blotting was used to detect PDH and PDK protein expression levels. Results: Administration of 60% NBO alone after reperfusion by t-PA treatment did not affect PDH activity. Under t-PA, compared to EtOH or Hypo alone, combined administration of NBO plus either EtOH or Hypo produced the greatest increases in PDH activity and protein expression levels, as well as the greatest decrease in PDK expression. Combination therapy also provided the most significant decline in ROS generation compared to any monotherapeutic approach. Conclusions: Reperfusion with t-PA followed by 60% NBO improves the efficacy of EtOH or Hypo in neuroprotection by ameliorating oxidative injury and improving metabolic regulation with PDH. Comparable neuroprotective effects were found when treating with either EtOH or Hypo, suggesting a similar mechanism and the possibility of substituting EtOH for Hypo in the clinical setting.

Hesperidin potentiates the neuroprotective effects of diazepam and gabapentin against pentylenetetrazole-induced convulsions in mice: Possible behavioral, biochemical and mitochondrial alterations.

Aim:Epilepsy is a chronic neurological disorder with complex pathophysiology. Several evidences suggest a role of oxidative stress and mitochondrial dysfunction in pathophysiology of epilepsy. Hesperidin (Hesp) acts as a powerful anti-oxidant agent against superoxide, singlet oxygen, and hydroxyl radicals. Thus, this study was undertaken to evaluate the possible neuroprotective mechanism of Hesp against pentylenetetrazole (PTZ)-induced convulsions in mice.Materials and Methods:Sixty males Laca mice (20-25 g) were randomly divided into 10 treatment groups (n = 6). Seven days pretreatment of Hesp (100, 200 mg/kg, p.o.) was carried out before PTZ (80 mg/kg, intraperitoneal [i.p.]) challenge, whereas diazepam (DZP) (0.2, 0.5 mg/kg) and gabapentin (Gbp) (10, 20 mg/kg) were administered i.p. 30 min before PTZ administration, that is, on 7th day. Following PTZ challenge, severity of convulsions (onset of jerks, myoclonic seizures, extensor phase and death), brain anti-oxidant enzyme levels and mitochondrial complex enzymes activities were estimated.Results:Single i.p. PTZ (80 mg/kg) challenge demonstrated severe convulsions, oxidative damage (raised lipid peroxidation [LPO], nitrite concentration as well as depleted reduced glutathione, superoxide dismutase and catalase levels), and depletion of mitochondrial enzyme Complex (I, II, IV) activities. Hesp (200 mg/kg), DZP (0.5 mg/kg) and Gbp (20 mg/kg) pretreatments attenuated PTZ induced behavioral, biochemical and mitochondrial alterations. However, administration of Hesp (100 mg/kg) in combination with DZP (0.2 mg/kg) or Gbp (10 mg/kg) potentiated their neuroprotective effect, which was significant as compared to their effects in PTZ treated animals.Conclusion:Hesp possesses potent anticonvulsant activity which might be mediated through modulation of gamma-amino butyric acid/benzodiazepine receptor action.

Age-related changes in the rat brain mitochondrial antioxidative enzyme ratios: Modulation by melatonin

Oxidative stress is an important factor for aging. The antioxidative enzymes glutathione peroxidase (GPx), glutathione reductase (GRd) and superoxide dismutase (SOD) play a crucial role protecting the organism against the age-dependent oxidative stress. Glutathione (GSH) is present in nearly all living cells. GSH is one of the main antioxidants in the cell and it serves several physiological functions. Our purpose was to evaluate the age-related changes in mitochondrial GPx, GRd and SOD activities, and mitochondrial GSH pool in the brains of young (3months) and aged rats (24months). We also investigated whether melatonin administration influences these brain mitochondrial enzyme activities and GSH levels in young and aged rats. The results showed that GPx activity increased with age, whereas melatonin treatment decreased GPx activity in the aged rats at levels similar to those in young and young+melatonin groups. The activities of GRd and SOD, however, did not change with age. But, melatonin treatment increased SOD activity in the aged rats. GSH levels, which also increased with age, were not modified by melatonin treatment. The reduction in the SOD/GPx and GR/GPx ratios with age was prevented by melatonin administration. Together, our results suggest that the age-related oxidative stress in rat brain mitochondria is more apparent when the antioxidant enzyme ratios are analyzed instead of their absolute values. The antioxidative effects of melatonin were also supported by the recovery of the enzyme ratios during aging.

Brain mitochondrial dysfunction in aging

Aging of mammalian brain is associated with a continuous decrease of the capacity to produce ATP by oxidative phosphorylation. The impairment of mitochondrial function is mainly due to diminished electron transfer by complexes I and IV, whereas inner membrane H+ impermeability and F1-ATP synthase activity are only slightly affected. Dysfunctional mitochondria in aged rodents show decreased rates of respiration and of electron transfer, decreased membrane potential, increased content of the oxidation products of phospholipids and proteins, and increased size and fragility. In aging mice, the activities of brain mitochondrial enzymes (complexes I and IV and mtNOS) are linearly correlated with neurological performance (tightrope and T-maze tests) and with median life span and negatively correlated with the mitochondrial content of lipid and protein oxidation products. Conditions that increased mice median life span, such as moderate exercise, vitamin E supplementation, caloric restriction, and high spontaneous neurological activity; also improved neurological performance and mitochondrial function in aged brain. The diffusion of mitochondrial NO and H2O2 to the cytosol is decreased in the aged brain and may be a factor for reduced mitochondrial biogenesis.

Association of reduced extracellular brain ammonia, lactate, and intracranial pressure in pigs with acute liver failure

We previously demonstrated in pigs with acute liver failure (ALF) that albumin dialysis using the molecular adsorbents recirculating system (MARS) attenuated a rise in intracranial pressure (ICP). This was independent of changes in arterial ammonia, cerebral blood flow and inflammation, allowing alternative hypotheses to be tested. The aims of the present study were to determine whether changes in cerebral extracellular ammonia, lactate, glutamine, glutamate, and energy metabolites were associated with the beneficial effects of MARS on ICP. Three randomized groups [sham, ALF (induced by portacaval anastomosis and hepatic artery ligation), and ALF+MARS] were studied over a 6-hour period with a 4-hour MARS treatment given beginning 2 hours after devascularization. Using cerebral microdialysis, the ALF-induced increase in extracellular brain ammonia, lactate, and glutamate was significantly attenuated in the ALF+MARS group as well as the increases in extracellular lactate/pyruvate and lactate/glucose ratios. The percent change in extracellular brain ammonia correlated with the percent change in ICP (r(2) = 0.511). Increases in brain lactate dehydrogenase activity and mitochondrial complex activity for complex IV were found in ALF compared with those in the sham, which was unaffected by MARS treatment. Brain oxygen consumption did not differ among the study groups. The observation that brain oxygen consumption and mitochondrial complex enzyme activity changed in parallel in both ALF- and MARS-treated animals indicates that the attenuation of increased extracellular brain ammonia (and extracellular brain glutamate) in the MARS-treated animals reduces energy demand and increases supply, resulting in attenuation of increased extracellular brain lactate. The mechanism of how MARS reduces extracellular brain ammonia requires further investigation.

Dietary Thioproline Decreases Spontaneous Food Intake and Increases Survival and Neurological Function in Mice

Male mice on a diet supplemented with thioproline (l-thiazolidine-4-carboxylic acid), a physiological metabolite of 5-hydroxytryptamine, at 2.0 g/kg of food from 28 weeks of age and for their entire life, showed a 23-29% increased median and maximal life span. These survival increases were associated with improved neurological functions. Compared to control mice, thioproline-supplemented mice had a 20% lower integral spontaneous food intake, and 10% lower body weight at 100 weeks of age. Body weight showed a statistically significant inverse relationship with survival and neurological performances. Thioproline-supplemented mice exhibited a 58-70% decrease of the age-dependent oxidative damage in brain and liver mitochondria at 52 weeks (old mice) and 78 weeks (senescent mice) of age, respectively. The age-associated decrease of brain mitochondrial enzyme activities, NADH-dehydrogenase, cytochrome c oxidase, and mitochondrial nitric oxide synthase (mtNOS), in old and senescent mice were markedly prevented (51-74%) by thioproline. In vitro, thioproline neither exhibited direct antioxidant activity nor had any effect on the electron transfer or mtNOS functional activities of brain and liver mitochondria. It is surmised that thioproline induces an anorexic effect associated with improved survival and neurological function through a decreased oxidative damage and regulation that may involve hypothalamic appetite centers.

Effect of Ethanol or Thiamine Administration to Cadmium-Intoxicated Rats on some Essential Metals in Brain Tissue and Serum and some Brain Mitochondrial Enzymes

Effect of Ethanol or Thiamine Administration to Cadmium-Intoxicated Rats on some Essential Metals in Brain Tissue and Serum and some Brain Mitochondrial Enzymes

Protective role of Bacopa monniera on morphine-induced brain mitochondrial enzyme activity in rats

Alcoholic extract of Bacopa monniera was tested for its protective role on morphine-induced brain mitochondrial enzyme status in rats. The level of the brain mitochondrial enzymes was significantly lower in the morphine-treated group when compared with control animals. These enzymes were maintained at normal level when Bacopa extract was administered orally at a dose of 40 mg/kg, 2 h before the administration of morphine.

Purification and properties of cytosolic and mitochondrial malic enzyme isolated from human brain

Three isoforms of malic enzyme have been described in mammalian tissues: a cytosolic NADP +-dependent enzyme, a NADP +-dependent mitochondrial isoform and a mitochondrial isozyme which can use both NAD + and NADP + but is more effective with NAD +. We purified mitochondrial and cytosolic malic enzyme from human brain extract to apparent homogeneity in order to compare properties of these isozymes and to verify whether mitochondria containe one or two malic enzyme. Specific activities of both isoforms are approx. 90 μmol/min/mg of protein, which corresponds to about 1900-fold purification. The two isozymes have identical native molecular mass (257 kDa) and are presumably tetramers composed of four identical subunits (M r = 64 kDa). The isoelectric point of cytosolic isozyme is 5.65, and that of mitochondrial one is 7.0. The isozymes show a substantial difference in their capability to catalyse the reductive carboxylation of pyruvate to malate: the maximal carboxylation rate approaches 80% that of decarboxylation velocity for the cytosolic enzyme, and only 17% for the mitochondrial isozyme. The coenzyme specificity of both isozymes is not stringent; NADP + is the preferred and NAD + can substitute it, although with much lower efficiency. The homogenous cytosolic malic enzyme catalysed decarboxylation of oxaloacetate and NADPH-dependent reduction of pyruvate at about 24 and 0.5% of the maximum rate of NADP-dependent oxidative decarboxylation of malate respectively. Decarboxylation of oxaloacetate catalysed by mitochondrial malic enzyme has not been detectable, while NADP-linked reduction of pyruvate approaches only 0.15% of the maximum rate of NADP-linked oxidative decarboxylation of malate. Both isozymes differ also in their affinity to malate, NADP +, Mn 2+ and Mg 2+. The results indicate that human brain mitochondrial and cytosolic malic enzymes are NADP-dependent [EC 1.1.1.40; l-Malate:NADP + oxidoreductase (oxaloacetate decarboxylating)] and display similar properties to the enzyme from animal brain.

L-BOAA induces selective inhibition of brain mitochondrial enzyme, NADH-dehydrogenase

Lathyrism, a human neurological disorder has been linked to the excessive consumption of a plant toxin, β-oxalylamino- l-alanine ( l-BOAA) present in Lathyrus sativus. The present study was carried out to elucidate the biochemical mechanisms underlying l-BOAA-induced toxic insult. Incubation of sagittal slices of mouse brain with l-BOAA resulted in dose and time-dependent inhibition of mitochondrial NADH-dehydrogenase (NADH-DH). Significant inhibition of NADH-DH was seen following incubation of brain slices with very low concentration of l-BOAA (0.1 pM). l-BOAA also induced lactate dehydrogenase (LDH) leakage from the slice into the medium in dose-dependent manner. The inhibition of NADH-DH preceded LDH leakage from the slices into the medium. l-BOAA had no effect on other mitochondrial enzymes, namely, isocitrate dehydrogenase or cytochrome c oxidase. Incubation of isolated mouse brain mitochondria with l-BOAA also resulted in inhibition of NADH-DH. l-BOAA-induced inhibition of NADH-DH was prevented by non- N-methyl- d-aspartate (non-NMDA) glutamate receptor antagonists in general and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor antagonist (NBQX) in particular. Other glutamate agonists examined namely, N-methyl- d-aspartate, β- N-methylamino- l-alanine ( l-BMAA), l-glutamic acid, N-acetylaspartylglutamate (NAAG), quisqualic acid, kainic acid or AMPA did not have any effect on NADH-DH activity in slices although they induced LDH leakage from the slice into the medium. Incubation of brain slices with l-BOAA did not induce lipid peroxidation or changes in glutathione levels. Prior incubation of slices with glutathione (GSH) or GSH-isopropyl ester did not prevent l-BOAA-induced inhibition of NADH-DH. However, incubation of isolated mitochondria with l-BOAA in the presence of GSH-isopropyl ester prevented l-BOAA-induced inhibition of NADH-DH, indicating the protective effect of mitochondrial glutathione in the prevention of l-BOAA-induced toxicity.

D-Penicillamine-induced copper deficiency in suckling mice: neurological abnormalities and brain mitochondrial enzyme activities

Brain mitochondrial enzyme activities were examined in 15-day-old suckling mice which were daily injected with d-penicillamine (DP), a chelating agent of copper. Newborn mice treated with DP (1 g/kg/day) showed retarded weight gain, hyperelasticity of skin, and a bizarre forelimb posture with subcutaneous edema on experimental day (ED) 7. Paraparesis or dragging of the hindlimbs was observed by ED 15. Brain copper contents of DP-treated mice decreased to 34% of the controls on ED 15. Cytochrome c oxidase activity (complex IV) in the brain showed 51% decrease of the controls, on the contrary, rotenone-sensitive NADH cytochrome c reductase (complex I + III) and succinate cytochrome c reductase (complex II + III) were normal. Histochemistry of cytochrome c oxidase in the cerebellum of DP-treated mice disclosed diffuse reduction of staining, especially in Purkinje cells. These data show that DP-induced copper deficiency in the brain subsequently disturbs mitochondrial electron transport system, selectively cytochrome c oxidase activity. This seems to be a useful animal model not only for Menkes' kinky hair disease but also for mitochondrial encephalomyopathy.

Purification and characterization of aldehyde dehydrogenase from human brain

Aldehyde dehydrogenase (EC 1.2.1.3) has been purified from human brain; this constitutes the first purification to homogeneity from the brain of any mammalian species. Of the three isozymes purified two are mitochondrial in origin (Peak I and Peak II) and one is cytoplasmic (Peak III). By comparison of properties, the cytoplasmic Peak III enzyme could be identified as the same as the liver cytoplasmic E1 isozyme ( N. J. Greenfield and R. Pietruszko (1977) Biochim. Biophys. Acta 483, 35–45 ). The Peak I and Peak II enzymes resemble the liver mitochondrial E2 isozyme, but both have properties that differ from those of the liver enzyme. The Peak I enzyme is extremely sensitive to disulfiram while the Peak II enzyme is totally insensitive; liver mitochondrial E2 isozyme is partially sensitive to disulfiram. The specific activity is 0.3 μmol/mg/min for the Peak I and 3.0 μmol/mg/min for the Peak II enzyme; the specific activity of the liver mitochondrial E2 isozyme is 1.6 μmol/min/mg under the same conditions. The Peak I enzyme is also inhibited by acetaldehyde at low concentrations, while the Peak II enzyme and the liver mitochondrial E2 isozyme are not inhibited under the same conditions. The precise relationship of brain Peak I and II enzymes to the liver E2 isozyme is not clear but it cannot be excluded at the present time that the two brain mitochondrial enzymes are brain specific.

Brain alpha-ketoglutarate dehydrogenase complex: kinetic properties, regional distribution, and effects of inhibitors.

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.

Glycemia, ketonemia, and brain enzymes of ketone body utilization in suckling and adult rats undernourished from intrauterine life.

The effect of undernutrition from the 16th day of pregnancy up to 70th day of life on blood glucose and ketone bodies and on several brain mitochondrial enzymes related to energy metabolism or biosynthetic function was investigated. Undernutrition in perinatal period was established by means of a food restriction to pregnant rats and, later, to the lactating mother; undernourished postweaned rats received half the diet consumed by the controls. Body and brain weight from undernourished rats was less than controls throughout the entire period studied. Glycemia and ketonemia were also always lower than controls. Cytochrome c oxidase, citrate synthase, 3-hydroxybutyrate dehydrogenase, 3-oxoacid coenzyme A transferase, and acetoacetyl-coenzyme A thiolase activities during the suckling period were in most stages lower than controls; subsequently, activities in undernourished rats reached or surpassed the control values. These results could explain the "catch up" phenomenon in several ultrastructural parameters found by other authors in undernourished postweaned rats.

Comparative effects of thyroid hormone analogs on the activities of brain and liver mitochondria and nuclei in thyroidectomized rats

Several thyroid hormone analogs have been tested for thyromimetic activity on rat brain and liver subcellular organelles. The compounds were administered immediately after thyroidectomy to 90 g male S-D rats for 10 days, by daily s.c. injection. In cerebral cortex and liver we measured the activities of mitochondrial succinate cytochrome c reductase and a-GPD, and nuclear RNA polymerase I. Brain mitochondrial enzymes were unchanged in thyroidectomized (Tx) and in Tx-treated rats, whereas the activities of these enzymes in liver mitochondria were partially restored by the treatments. RNA polymerase I activity in brain and liver dropped significantly 10 days after thyroidectomy and daily injection of thyroid hormones or analogs maintained the nuclear activity at a normal level. Correlation between the structure of thyroid hormone analogs and their subcellular effects is in good agreement with previous binding and in vivo studies. Enzyme activities stimulated by T 3 were lowered by replacing the T 3 side-chain by an acetic acid group or by substituting the bridged oxygen atom by atom by CO. In contrast, the activity was enhanced by substituting iodine with a 3' isopropyl group. Although less active than iodine, the 3,5-dimethyl substituents may be introduced without a complete loss of nuclear activity.