NADH Redox State Research Articles

Effect of Ca2+-homopantothenate and mild hypoxia on some enzyme activities evaluated in subcellular fractions from different rat brain regions

The effect of Ca2+-homopantothenate (HOPA) treatment (250 mg/kg for 5 d) has been studied by evaluating the specific activity of enzymes related to: glycolytic pathway (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), tricarboxylic acid cycle (citrate synthase, malate dehydrogenase), mitochondrial electron transfer chain (succinate dehydrogenase, cytochrome oxidase), NADH redox state (NADH cytochrome c reductase), acetylcholine metabolism (acetylcholinesterase), and glutamate metabolism (glutamate dehydrogenase). The enzymatic activity assays were performed on homogenate in toto, nonsynaptic mitochondria and synaptosomes isolated from: cerebral cortex, hippocampus, striatum, hypothalamus, medulla oblongata, and cerebellum of normoxic rats and rats submitted to intermittent normobaric hypoxia (90:10, N2:O2). In normoxic rats, HOPA was unable to induce any modification. Hypoxia per se induced a decrease in the activity of synaptosomal cytochrome oxidase in cerebral cortex, hippocampus, and cerebellum.

Effects of acetazolamide on cerebrocortical NADH and blood volume.

Acetazolamide (AZ), a potent carbonic anhydrase inhibitor in human and animal tissues, increases cerebral blood flow (CBF) by acidifying cerebral extracellular fluids. To demonstrate the relationship of increased CBF to brain O2 availability after AZ administration, a compensated fluorometer was used to study changes in the cerebrocortical redox balance in rabbits. Seven rabbits were anesthetized with pentobarbital sodium. Excitation light (366 nm) was conducted to the cerebrocortical surface of each animal by a 4-mm-diam fiberoptic light guide. Fluorescence emissions from cerebrocortical NADH (450 nm) were compared at different inspired O2 (FIO2) tensions. Reflected light (366 nm), which was used to determine a correction to the fluorescence signal, was separately quantitated and interpreted as an index of cerebrocortical blood volume. Reductions in FIO2 from 1.0 to 0.21, 0.14, 0.10, and 0.07 resulted in increases in both tissue blood volume and [NADH]. Intravenous AZ (25 mg/kg) increased cerebrocortical blood volume and reduced the [NADH], even during ventilation with 100% O2. The changes in brain redox balance caused by vasodilation with AZ were compared with those caused by vasodilatation with CO2. The NAD+/NADH redox state was a continuous function of FIO2 at all levels of arterial PCO2 (PaCO2), both before and after AZ administration. The improvement in cerebral O2 delivery caused by AZ-induced vasodilation was comparable to that caused by the vasodilatation that results from a PaCO2 elevation approximately equal to 12-15 Torr above normal. The slope of the relationship between [NADH] and FIO2 was similar at normal, low, and high levels of PaCO2. We conclude that AZ administration and PaCO2 elevation improve cerebral oxygenation by similar mechanisms.

Brain oxidative metabolism of the newborn dog: correlation between 31P NMR spectroscopy and pyridine nucleotide redox state.

The effects of both anoxia and short- and long-term hypoxia on brain oxidative metabolism were studied in newborn dogs. Oxidative metabolism was evaluated by two independent measures: in vivo continuous monitoring of mitochondrial NADH redox state and energy stores as calculated from the phosphocreatine (PCr)/Pi levels measured by 31P nuclear magnetic resonance (NMR) spectroscopy. The hemodynamic response to low oxygen supply was further evaluated by measuring the changes in the reflected light intensity at 366 nm (the excitation wavelength for NADH). The animal underwent surgery and was prepared for monitoring of the two signals (NADH and PCr/Pi). It was then placed inside a Phosphoenergetics 260-80 NMR spectrometer magnet with a 31-cm bore. Each animal (1-21 days old) was exposed to short-term anoxia or hypoxia as well as to long-term hypoxia (1-2 h). The results can be summarized as follow: (a) In the normoxic brain, the ratio between PCr and Pi was greater than 1 (1.2-1.4), while under hypoxia or asphyxia a significant decrease that was correlated to the FiO2 levels was recorded. (b) A clear correlation was found between the decrease in PCr/Pi values and the increased NADH redox state developed under decreased O2 supply to the brain. (c) Exposing the animal to moderately long-term hypoxia led to a stabilized low-energy state of the brain with a good recovery after rebreathing normal air. (d) Under long-term and severe hypoxia, the microcirculatory autoregulatory mechanism was damaged and massive vasoconstriction was optically recorded simultaneously with a significant decrease in PCr/Pi values.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic substrate utilization by rabbit proximal tubule. An NADH fluorescence study

The effects of various short-chain fatty acids, carboxylic acids, and amino acids on NADH fluorescence and oxygen consumption (QO2) of rabbit proximal tubule suspensions were determined. The short-chain fatty acids were the most effective substrates in increasing NADH fluorescence and QO2, followed by the carboxylic acids and amino acids. All of the substrates tested that increased NADH fluorescence proportionally increased QO2. This implies that the primary effect of these substrates was to increase QO2 by increasing the delivery of reducing equivalents to NAD and not by stimulating ATP hydrolysis directly. The relative affinity of several substrates to increase NADH fluorescence was also determined. The short-chain fatty acids had the highest affinity (10 microM range) followed by the carboxylic acids (100 microM range). These data demonstrate that the metabolic rate and NADH redox state of the renal cortical cell is very sensitive to the type of metabolic substrate available.

Respiratory control in the glucose perfused heart: A 31P NMR and NADH fluorescence study

The phosphate metabolises, adenosine diphosphate (ADP), inorganic phosphate (P i), and adenosine triphosphate (ATP), are potentially important regulators of mitochondrial respiration in vivo. However, previous studies on the heart in vivo and in vitro have not consistently demonstrated an appropriate correlation between the concentration of these phosphate metabolites and moderate changes in work and respiration. Recently, mitochondrial NAD(P)H levels have been proposed as a potential regulator of cardiac respiration during alterations in work output. In order to understand better the mechanism of respiratory control under these conditions, we investigated the relationship between the phosphate metabolites, the NAD(P)H levels, and oxygen consumption (Q 0 2 ) in the isovolumic perfused rat heart during alterations in work output with pacing. ATP, creatine phosphate (CrP), P i and intracellular pH were measured using 31P NMR. Mitochondrial NAD(P)H levels were monitored using spectrofluorometric techniques. Utilizing glucose as the sole substrate, an increase in paced heart rate led to an increase in Q 0 2 from 1.73 ± 0.09 to 2.29 ± 0.12 mmol Q 2/h per g dry wt. No significant changes in the levels of P i, PCr, ATP, or the calculated ADP levels were detected. Under identical conditions, an increase in heart rate was associated with a 23±3% increase in NAD(P)H fluorescence. Thus, under the conditions of these studies, an increase in Q 0 2 was not associated with an increase in ADP or P i. In contrast, increases in Q 0 2 were associated with an increase in NAD(P)H. These data are consistent with the notion that increases in the mitochondrial NADH redox state regulate steady-state levels of respiration when myocardial work is increased.

Metabolic, ionic and electrical activities during and after incomplete or complete cerebral ischaemia in the Mongolian gerbil.

During the last 10 years the Mongolian gerbil (Meriones unguiculatus) has been adopted as an animal model for cerebral ischaemia because of the incompleteness of its Circle of Willis (Levy and Brierley, 1974; Levy, Brierley and Plum, 1975; Kobayashi, Lust and Passonneau, 1977; Levy and Duffy, 1977). The posterior connection between the vertebro-basilar and the carotid arteries is missing in all animals hence complete cerebral ischaemia develops after bilateral carotid artery occlusion. The degree of connection between the two anterior cerebral arteries varies in different individuals, so with unilateral carotid artery occlusion, the level of ischaemia developing in the ipsilateral hemisphere can vary between 0 and 100%. Using fibre optic surface fluorometry we monitored the intramitochondrial NADH redox state under partial or complete ischaemia in the gerbil brain (Mayevsky, Lebourdais and Chance, 1980; Mayevsky, Zarchin and Friedli, 1982; Mayevsky and Chance, 1983). The purpose of the present study was: 1) to correlate the degree of ischaemia developed under unilateral and bilateral carotid artery occlusion with the impairment of ionic homeostasis developed under these conditions; 2) to test the recovery rate of the extracellular K+ level (K +e ) under the same conditions.KeywordsAnterior Cerebral ArteryLight GuideMongolian GerbilCarotid Artery OcclusionIschaemic EpisodeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

O2 uptake in periportal and pericentral regions of liver lobule in perfused liver.

O2 uptake by the perfused liver decreased at O2 concentrations considerably higher than levels that caused NADH reduction when the input O2 concentration was varied. The maximal rate of O2 uptake was two- to threefold higher in periportal (137 +/- 8 mumol . g-1 . h-1; O2 concentration = 478 +/- 37 microM) than pericentral regions (59 +/- 5 mumol . g-1 . h-1; O2 concentration = 263 +/- 21 microM); however, the O2 concentration required for half-maximal O2 uptake was similar (approximately 20 microM) in the two areas. The infusion of atractyloside, antimycin A, or KCN inhibited O2 uptake in both zones by 50-85%, indicating that O2 uptake in both regions was largely dependent on mitochondrial electron transport. The content of ATP and ADP and ATP:ADP were similar in microdissected samples from periportal and pericentral areas. In contrast, when livers were perfused in the retrograde direction, O2 uptake was two- to threefold greater in pericentral than in periportal regions. Maximal rates of O2 uptake correlated with the local O2 concentration irrespective of the direction of flow when the electrode was moved across the liver lobule with a micromanipulator. Lower rates of O2 uptake in pericentral areas were not altered appreciably by infusion of agents known to uncouple oxidative phosphorylation (DNP), increase ADP supply (fructose), or increase the NADH redox state (ethanol or octanoate). These data are consistent with the hypothesis that maximal rates of O2 uptake are regulated, in part, in the perfused liver by O2 concentrations far above the Km of cytochrome oxidase for O2.

Metabolic, ionic, and electrical responses of gerbil brain to ischemia

The effects of short- or long-term complete cerebral ischemia were studied in the gerbil brain using a multi-parameter monitoring system. Metabolic (NADH redox state) and hemodynamic responses were monitored by surface fluorometry-reflectometry. Ionic activities (K+ and pH) were measured by surface macroelectrodes. Electrical activity was evaluated by monitoring the general electrocorticogram (ECoG) as well as local DC steady potential (two sites). Two groups of gerbils were studied to compare the effects of 4-5 min occlusions with those of 30 min complete ischemia. During bilateral carotid artery occlusion the cortex is exposed to complete ischemia resulting in the complete depletion of O2 with attendant maximal reduction of NADH. Extracellular K+ began to increase as soon as energy reserves were decreased with a time course suggesting two different kinetic areas. Surface pH decreased very shortly after the occlusion. During the recovery phase, NADH was reoxidized soon after recirculation, whereas the pH and K+ recovery showed a short delay. ECoG did not recover even when all other parameters reached base-line levels. The recovery of all the measured parameters was correlated to the duration of the ischemic insult; i.e., the recovery from 30 min of ischemia took significantly longer than after 5 min of ischemia. We conclude that pH recovery depends on recirculation and adequate O2 supply to the tissue, whereas K+ recovery required not only an adequate O2 supply but also the integrity of the adenosine triphosphatase system.

Human liver cytosolic malate dehydrogenase: Purification, kinetic properties, and role in ethanol metabolism

Cytosolic malate dehydrogenase from human liver was isolated and its physical and kinetic properties were determined. The enzyme had a molecular weight of 72,000 ± 2000 and an amino acid composition similar to those of malate dehydrogenases from other species. The kinetic behaviour of the enzyme was consistent with an Ordered Bi Bi mechanism. The following values (μ m) of the kinetic parameters were obtained at pH 7.4 and 37 °C: K a , 17; K ia , 3.6; K b , 51; K ib , 68; K p , 770; K ip , 10,700; K q , 42; K iq , 500, where a, b, p, and q refer to NADH, oxalacetate, malate, and NAD +, respectively. The maximum velocity of the enzyme in human liver homogenates was 102 μmol/min/g wet wt of liver for oxalacetate reduction and 11.2 μmol/min/g liver for malate oxidation at pH 7.4 and 37 °C. Calculations using these parameters showed that, under conditions in vivo, the rate of NADH oxidation by the enzyme would be much less than the maximum velocity and could be comparable to the rate of NADH production during ethanol oxidation in human liver. The rate of NADH oxidation would be sensitive to the concentrations of NADH and oxalacetate; this sensitivity can explain the change in cytosolic NAD + NADH redox state during ethanol metabolism in human liver.

Correlation of brain NADH redox state, K+, PO2 and electrical activity during hypoxia, ischemia and spreading depression.

The normal functioning brain requires a continuous supply of blood in order to obtain glucose as well as oxygen. Any change in the oxygenation of the tissue will result in an impairment of the normal function. Most of the oxygen taken up by the brain is consumed by the mitochondria in order to supply the large amount of ATP needed. A major part of the ATP is used by the ATPase system in order to keep the neurons in the polarized state. Figure 1 shows in a schematic way the interrelation between the energy production system and the pumping activity going on in the membranes. In order to understand the connection between various events occurring in the brain in vivo, one has to measure as many parameters as possible from the same site.

Multisite measurements of NADH redox state from cerebral cortex of the awake animal.

There are few non-destructive non-invasive approaches to the study of cortical oxidative metabolism. Nevertheless, the great necessity for the development and application of such approaches arises from the inadequacy of cell and brain slice models on the one hand and the need for interpretive monitoring of brain metabolism in humans, or if possible, under non-operative conditions. Two techniques can be used to study metabolism of the brain without the necessity of an operation, 31P NMR which is totally non-invasive and positron emission tomography which requries injection and delivery of the radio isotope. Neither of these methods affords an adequately sharp localization to provide better than regional localization (lam) under current conditions of development and application, on the other hand, when the subject is sacrificed and autoradiography of tritium labelled deoxyglucose is employed, a high degree of metabolic resolution can be obtained albeit the method averages events over times as long as 45 minutes. The need for a non-destructive continuous read out method for brain metabolism providing a high degree of localization, both spacially and within appropriate metabolic compartments is obtained with the fluorescence of mitochondrial pigments, NADH or flavoprotein. Furthermore, this method is applicable as well to frozen tissue surfaces affording high resolution 3D spacial resolution. The discovery that mitochondrial NADH is fluorescent and that the fluorescence is enhanced 15 or more times over that of the pigment in solution afforded a unique “look” at metabolic events in the matrix base of mitochondria; the NADPH therein was found not to respond to variations of electron transport in the respiratory chain (1–3). Furthermore, comparisons of changes of NADH fluorescence could be well correlated with actual tissue assays of NADH in heart and liver (4,5).KeywordsLight GuideSpreading DepressionCarotid Artery OcclusionAwake AnimalGerbil BrainThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Factors affecting the oxygen balance in the awake cerebral cortex exposed to spreading depression

Oxygen balance was evaluated in the cerebral cortex, using the surface fluorometry technique of the intramitochondrial NADH redox state, exposed to various physiological and pathological situations. Using flexible fiber optic light guide, connected to the brain surface via a cemented holder, the measurements were done continuously from the awake rat and gerbil. In few experiments the NADH redox state was correlated with the electrical and ionic activity (measured by surface K + and DC electrodes). Three different animal models were used in the study: the adult rat, the very young rat (20 g) and the adult gerbil. Those 3 models were used in studying the effect of hypoxia, partial ischemia, and anesthesia on the metabolic and ionic activities measured from the awake brain. Spreading cortical depression (elicited by topical KCl solution) was used as a standard stimulation of the ionic and metabolic activities of the cerebral cortex. Two typical metabolic responses to spreading depression (SD) were recorded, namely ‘oxidation cycle’ and ‘reduction cycle’ depending upon the ability of the tissue to compensate for the extra amount of oxygen needed for the higher mitochondrial activity. It was found that the adult rat brain showed oxidation cycles in most conditions (besides partial ischemia), while the young rat and the gerbil brains were much more sensitive to the various perturbations of the brain and exhibited reduction cycle (as a response to SD) under all pathological situations tested. We conclude from our detailed studies that the type of response to SD, as measured by NADH surface fluorometry, represents the oxygen balance which exists in the tissue under various conditions.

Altered redox states in corneal epithelium and endothelium: NADH fluorescence in rat and rabbit ocular tissue

NADH fluorescence signals can be obtained from the freeze-trapped cornea and are assignable to the epithelial and endothelial layers. Alteration of the NADH redox state is obtained by systemic hypoxia or histotoxic anoxia (hydrosulfide inhibition). In a series of studies using paired corneas from the same animal, sulfide treatment resulted in 26 and 15% increases in epithelial NADH fluorescence in rabbits and rats, respectively. Paired controls showed no significant alteration (<6%). The corresponding change for the sulfide treated endothelium was 16%.

Redox transitions in mitochondria of cat cerebral cortex with seizures and hemorrhagic hypotension.

Fluorometry and dual-wave-length spectrophotometry were used to detect transitory shifts in the redox state of mitochondrial NADH and cytochrome aa3 in the exposed cerebral cortex of anesthetized paralyzed cats as seizures were induced with pentylenetetrazol. In normotensive animals, NADH and cytochrome aa3 oxidation accompany the seizures, but when the mean arterial pressure (MAP) is reduced to 40.2 +/- 1.1% of the base line by hemorrhaging, the NADH fluorescence response converts to a biphasic oxidation-reduction sequence. In extreme hypotension (MAP lowered to an average of 28%), only NAD reduction transients are observed with seizures, and cytochrome aa3 is oxidized irrespective of the low MAP. Our data show that a reversible perfusion impairment, perhaps inhomogeneous in its distribution, appears in the cortex at the 40% MAP level and modifies electron flux in the respiratory chain between NADH and cytochrome aa3, and uniform oxygen insufficiency is an unlikely cause for the reversal of NADH oxidation toward reduction during seizures under hypovolemic conditions.

Pyridine nucleotide redox state and blood flow of the cerebral cortex following middle cerebral artery occlusion in the cat.

Acute changes in the redox state of NADH in the cerebral cortex of cats were investigated following occlusion of the middle cerebral cortex (MCA) and were correlated with alterations of regional cerebral blood flow in the ischemic cortex determined autoradiographically. Arterial occlusion was accomplished via the transorbital approach. Cortical fluorescence and reflected light signals were recorded from the central MCA territory by means of a beam-splitting fluorometer, and a fluorescence signal corrected for alterations in intravascular hemoglobin was derived. Following arterial occlusion, there was a rapid increase in cortical NADH fluorescence, peaking within 30 to 70 seconds at 20% to 40% of full scale. This was followed by a slow linear decline in fluorescence over the next several minutes. The behavior of cortical NADH fluorescence was unaffected by replacement of the ambient air over the cortical surface with nitrogen. Mean regional blood flow values in the most ischemic gyri two to 15 minutes following arterial occlusion were 21% to 23% of the corresponding values in the opposite, nonischemic hemisphere. In individual animals, peak NADH fluorescence values following arterial occlusion correlated with the extent of blood flow reduction in the affected ischemic gyri (P less than 0.05).

Intracellular redox changes in functioning cerebral cortex. II. Effects of direct cortical stimulation.

Intracellular redox changes in functioning cerebral cortex. II. Effects of direct cortical stimulation.

Control of citrate and acetoacetate synthesis in rat liver

The mechanisms by which ethanol (EtOH) inhibits the human chorionic gonado-tropin (hCG)-stimulated testosterone synthesis was studied in isolated rat Leydig cells in vitro. EtOH inhibited steroidogenesis, but this inhibition was reversed by l-glutamate (Glu) and an uncoupler of the oxidative phosphorylation, 2,4-dinitrophenol (DNP). The mechanism of EtOH-induced inhibition was studied by measuring steroidogenic precursors and comparing them with the cytosolic and mitochondrial NADH redox states during uncoupling or in the presence of Glu. DNP had a dual effect. Low concentrations abolished the EtOH-induced inhibition of progesterone to testosterone formation suggesting that the inhibitory step was at or before progesterone formation. A large concentration led to an overall decrease in steroidogenesis indicating toxic effects on steroidogenesis. The mitochondrial NADH/NAD+ ratio, measured as the 3-hydroxybutyrate/acetoacetate ratio, decreased simultaneously when steroidogenesis was stimulated, either during uncoupling or in the presence of Glu, whereas cytosolic NADH/NAD+ ratio, measured as lactate/pyruvate ratio showed no response. These results demonstrate that the rise in the mitochondrial NADH/NAD+ ratio rather than in the cytosolic one is connected with the inhibition of testosterone synthesis by EtOH in isolated Leydig cells. The EtOH-induced high mitochondrial NADH/NAD+ ratio may deplete mitochondrial oxalacetate concentrations. This can decrease the activity of several transport shuttles and interrupt the flow of mitochondrial citrate into the smooth endoplasmic reticulum, which then reflects to decreased rate of steroidogenesis in the presence of ethanol.