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Biomedical subjects

H Nohl

Publications and source records attributed to H Nohl.

At least 55 records · Page 3Linked to original sources
PubMed1996

Hydroxylamine and phenol-induced formation of methemoglobin and free radical intermediates in erythrocytes.

As previously shown with isolated oxyhemoglobin, methemoglobin formation can also be induced in intact erythrocytes by hydroxylamine compounds and substituted phenols such as butylated hydroxyanisole (BHA). Electron spin resonance investigations revealed that, accordingly, free radical intermediates were formed in erythrocytes from hydroxylamine, N,N-dimethylhydroxylamine, and N-hydroxyurea. Due to the low stability of the dihydronitroxyl radicals, their detection required the use of a continuous flow system and relatively high amounts of the reactants. As has already been demonstrated with the solubilized hemoglobin system, hemoglobin of intact erythrocytes also reacts with the more hydrophilic xenobiotics such as hydroxylamine. However, the reaction rate was slightly reduced, indicating the existence of an incomplete permeability barrier for these compounds. The limited solubility of phenolic compounds in the aqueous buffer of suspended erythrocytes (in combination with the strict requirement of osmolarity in order to prevent hemolysis) impeded the direct detection of the respective phenoxyl radicals previously reported in hemoglobin solutions. However, in accordance with earlier findings in homogeneous reaction systems, chemiluminescence was observed as well, indicating the existence of a further reaction intermediate, which was also obtained in pure hemoglobin solutions when mixed with the respective reactants. As has recently been demonstrated, this light emission is indicative of the existence of highly prooxidative compound I intermediates during methemoglobin formation. Prooxidant formation in erythrocytes is reflected by a significant decrease in thiol levels even with those compounds where free radical formation was not directly detectable by ESR spectroscopy. The use of the spin-labeling technique revealed membrane effects as a result of oxidative stress. Oxidative metabolism of hemoglobin with hydroxylamine caused a release of low molecular weight iron. The marked hemolysis observed in the presence of BHA results from a direct membrane effect of this compound rather than a consequence of free radical-induced oxidative stress. A correlation of the different results is discussed in terms of possible toxicological consequences.

Animals↗
PubMed1996

The effect of the exogenous NADH dehydrogenase of heart mitochondria on the transmembranous proton movement.

Heart mitochondria can be made to oxidize extramitochondrial NADH via the exogenous NADH dehydrogenase. Oxidation of extramitochondrial NADH was found to be associated with the disappearance of H+ from the suspension medium. Our studies on the possible pathway through which H+ may disappear from the extramitochondrial space were focused on (i) an unspecific transmembranous H+ leakage along the electrochemical H+ gradient following peroxidative membrane alteration, (ii) stimulation of a controlled H+ reconduction through the H+ channel of the ATP synthase, and (iii) stimulation of the Na+/H+ counterporter by Ca2+ release. Our experiments revealed that none of these H+ pathways was involved in the observed alkalinization of the extramitochondrial space during respiration of external NADH. The latter effect was inhibited when oxidation of external NADH via the respiratory chain was blocked and could be turned into the opposite when artificial e- acceptors of the exogenous NADH dehydrogenase were used to reactivate NADH consumption. Stoichiometric analysis of H+ disappearance and O2 consumption revealed that reducing equivalents of external NADH were transferred to oxygen via cytochrome oxidase and H+ from the suspension was used to release water.

Animals↗
PubMed1996

Oxidation of cytosolic NADH via complex I of heart mitochondria.

The exogenous NADH dehydrogenase of heart mitochondria is increasingly reported to mediate cardiomyopathies following adriamycin treatment or reperfusion of ischemic hearts. A great number of studies on the biochemistry and pathobiology exists which indirectly support the existence of this dehydrogenase. Our studies exclude both the rotenone-insensitive NADH dehydrogenase of the outer membrane and the endogenous NADH dehydrogenase of damaged mitochondria as being responsible for external NADH consumption. Reducing equivalents from external NADH were demonstrated to enter complex I of the respiratory chain from the cytosolic phase. Our data support earlier reports on the physical association of the exogenous NADH dehydrogenase with the inner mitochondrial membrane excluding oxidation of external NADH via an enzyme of artefactual origin.

Animals↗
PubMed1996

Conditions allowing redox-cycling ubisemiquinone in mitochondria to establish a direct redox couple with molecular oxygen.

The present investigation seeks to elucidate the molecular mechanism responsible of the transformation of redox-cycling ubiquinone (UQ) from a save electron carrier to an O2.- generator as observed in toluene-treated mitochondria as well as in mitochondria exposed to conditions of organ ischemia/reperfusion. Starting from the earlier finding that for thermodynamic grounds autoxidation of ubisemiquinone (SQ.-) requires the accessibility of protons, two possibilities were considered: a) protons from the aqueous phase may penetrate into the phospholipid bilayer and react with SQ.- due to a decreased hydrophobicity of the membrane, b) the physical state of the membrane remains unchanged while the binding of redox-cycling UQ is changed such that SQ.- will come into contact with the aqueous phase in the polar head group section. Spin probes were used to follow changes of the physical order of phospholipids of the inner mitochondrial membrane. Binding changes of mitochondrial SQ.- were assessed from power saturation experiments and spin-spin interactions with a Cr3+ salt of the aqueous phase were studied to recognize orientation changes via the polar head group section of the membrane. Our results show that autoxidation of SQ.- occurs in two different ways. In the case of membrane insertion of toluene, the physical property of the membrane was affected such that protons could penetrate and allow SQ.- to undergo autoxidation. In contrast, mitochondrial respiration of cytosolic NADH accumulating during ischemia involves a low saturating SQ.- species that readily autoxidizes due to its spatial orientation close to the aqueous face of the membrane. We conclude from these observations that in line with thermodynamics autoxidation of SQ.- in mitochondria requires protons that normally have no access.

Animals↗
PubMed1996

[Feasibility and safety of dobutamine stress echocardiography: experiences with 1,000 studies].

The use of dobutamine stress echocardiography for the evaluation of coronary artery disease is rapidly expanding. Despite its widespread use, the feasibility and safety of dobutamine stress echocardiography has not been sufficiently documented. Between November 1992 and June 1995, we performed 1000 dobutamine stress echocardiographies. There were 744 men and 256 women with a mean age of 59 +/- 11 years. Anti anginal medication was not routinely withdrawn before the test. The mean maximal dobutamine dose was 41,4 +/- 10 mu g/kg center dot min(-1). Atropine was given additionally in 440 patients, with a mean dose of 0.5 mg. In patients receiving beta-blockers additional atropine was more often necessary as compared to those not receiving beta-blockers (278/457 = 61% versus 162/543 = 30 %, p < 0.0001). Reasons for discontinuing dobutamine infusion were achievement of target heart rate (64 % of cases) and maximal dose (12 % of cases). In 791 (79,1 %) patients no side-effects of dobutamine stress echocardiography were noticed. Termination of the study because of adverse side-effects occurred in 6.6 %. A total of 103 (10,3 %) noncardiac side-effects were observed: dizziness or nausea 6.4 %, headache 1.7 %. In one patient a focal cerebral seizure occurred. 156 cardiac side-effects occurred: blood pressure decrease of more than 20 mm Hg in 25 patients, extreme palpitations in 16 patients and pulmonary edema in one case. Most common cardiac side-effects consisted of arrhythmias (11.4 %): 9.1 % ventricular and 2.3 % supraventricular arrhythmias. Most ventricular arrhythmias were less severe (uniform and multiform premature ventricular beats, ventricular bigeminy or couplets in 71 patients). Nonsustained ventricular tachycardia, with a maximum duration of 20 s, occurred in 18 patients. In one patient sustained ventricular tachycardia developed and progressed towards ventricular fibrillation. This patient could be successfully defibrillated. Supraventricular arrhythmias presented as new atrial fibrillation in 10 patients, supraventricular tachycardia in three patients, junctional rhythm with a short decline in heart rate in nine patients and a second-degree AV block in another case. Dobutamine stress echocardiography has proven to be a safe and feasible method in the diagnosis of coronary heart disease. Minor side-effects are common and sometimes unpleasant for the patient, but do not often require termination of the study. Severe side-effects are seldom (< 1 %), but nevertheless, adequate medical and technical (defibrillator) support should be rapidly available.

Adult↗
PubMed1995

Effect of alpha-lipoic acid and dihydrolipoic acid on ischemia/reperfusion injury of the heart and heart mitochondria.

The aim of the present study was to evaluate a possible interference of alpha-lipoic acid (LA) or its reduced form (dithiol dihydrolipoic acid = DHLA) in the cardiac ischemia/reperfusion injury both at the level of the intact organ and at the subcellular level of mitochondria. In order to follow the effect of LA on the ischemia/reperfusion injury of the heart the isolated perfused organ was subjected to total global ischemia and reperfusion in the presence and absence of different concentrations of LA. Treatment with 0.5 microM LA improved the recovery of hemodynamic parameters; electrophysiological parameters were not influenced. However, application of 10 microM LA to rat hearts further impaired the recovery of hemodynamic functions and prolonged the duration of severe rhythm disturbances in comparison to reperfusion of control hearts. Treatment of isolated mitochondria with any concentration of DHLA could not prevent the impairment of respiratory-linked energy conservation caused by the exposure of mitochondria to 'reperfusion' conditions. However, DHLA was effective in decreasing the formation and the existence of mitochondrial superoxide radicals (O2.-). Apart from its direct O(2.-)-scavenging activities DHLA was also found to control mitochondrial O2.- formation indirectly by regulating redox-cycling ubiquinone. It is suggested that impairment of this mitochondrial O2.- generator mitigates postischemic oxidative stress which in turn reduces damage to hemodynamic heart function.

Animals↗
PubMed1995

Reactions of reducing xenobiotics with oxymyoglobin. Formation of metmyoglobin, ferryl myoglobin and free radicals: an electron spin resonance and chemiluminescence study.

The oxygen-haem centre of oxymyoglobin reacts with reducing xenobiotics such as hydroxylamines and phenols with the concomitant formation of metmyoglobin and oxidation of the respective xenobiotic. Metmyoglobin formation rates were measured by visible spectroscopy with xenobiotic concentrations ranging from 100 microM to 30 mM. Analogous to previous results obtained with oxyhaemoglobin, the first step in the reaction of hydroxylamines with oxymyoglobin leads to the formation of the one-electron oxidation product of hydroxylamine, a nitroxyl radical detectable by electron spin resonance. A variety of paramagnetic secondary products were also found. The terminal oxidation product of hydroxylamine and hydroxyurea was the myoglobin-nitric oxide complex, one showing similar spectral characteristics to the analogous haemoglobin-nitric oxide adduct found in our previous experiments. On the other hand, the amount of low-spin ferric complexes obtained from metmyoglobin and an excess of the respective hydroxylamine was considerably lower than the corresponding results with methaemoglobin. A second important reaction intermediate was the compound I-type ferryl haem-species detected by a recently-published chemiluminescence assay. Partial spectral resolution of the emitted light using a set of cut-off filters indicated that maximum light emission occurred above 600 nm, most probably involving excited porphyrin states. The intensity of oxymyoglobin-related light emission was considerably higher than that reported earlier with oxyhaemoglobin. This indicates a difference in the excitation mechanism which leads to the formation of the compound I-type ferry haem species.

Electron Spin Resonance Spectroscopy↗
PubMed1995

Chemiluminescence and EPR studies on the excitation site of ferric-heme-oxo complexes of natural and model heme systems.

Chemiluminescence was detected both in the reaction system of H2O2 plus heme proteins such as methemo- and metmyoglobin and ferric-protoheme complexes used as a model system. The intensity of chemiluminescence was found to be mediated by ligand binding to the sixth coordination site of the ferric-protoheme compounds, e.g. chemiluminescence was not observed with the bisimidazole ferric-protoheme complex. On the other hand the pentacoordinated histidine ferric-protoheme complex exhibited strong light emission. Comparative studies with various ligand-heme compounds elucidated that light emission was inversely correlated with the binding strength of the respective ligand at the sixth coordination site. The basic reaction mechanism causing the establishment of an excited state was studied by monitoring chemiluminescence and EPR signal formation of ligand-modified heme proteins in the presence of different electron donors. External electron donors such as Trolox C, TMPD and ascorbic acid affected a strong reduction in the development of chemiluminescence suggesting the essential involvement of an inner-molecular electron transfer process. Our results allow the conclusion that chemiluminescence is generated from the decay of an excited state of oxo-heme compounds established as a result of a one electron transfer step from a ligand group to heme iron.

Binding Sites↗
PubMed1994

Generation of superoxide radicals as byproduct of cellular respiration.

Cell respiration is associated with the risk of formation of oxygen radicals. Although various conditions of respiration have been described under which O2-radicals are generated it is not clear whether oxygen radical generation is an inevitable side effect of respiration. The answer is necessarily linked to an understanding of the mechanism and molecular site of oxygen radical generation. Redox-cycling ubiquinones of the mitochondrial respiratory chain have often been suggested to account for cellular O2-radical formation. However, there is an increasing body of evidence which refutes this assumption on thermodynamic grounds. The discovery of a novel respiratory enzyme of heart mitochondria, exogenous NADH-dehydrogenase, some years ago, has considerably aided understanding of mitochondrial O2-radical generation and the role of ubiquinones therein. This mitochondrial enzyme can be directly activated by cytosolic NADH. It has been shown that NADH consumption via this enzyme not only stimulates electron flow along components of the respiratory chain but that its activity is also linked to the release O2-. or the single electron reduction of adequate non-physiological oxidants. Anthraquinones which are increasingly used as antitumor drugs can enter this redox-shuttle and initiate radical chain reactions which may be partially responsible for the selective cardiotoxicity of these compounds. Metabolic conditions, causing abnormally high NADH levels in the cytosol, such as ischemia have been found to irreversibly transform intact mitochondria to active radical generators. The present review elucidates the finding of a general phenomenon which gives more insight into the mechanism and the site of O2-radical formation during normal cell respiration.(ABSTRACT TRUNCATED AT 250 WORDS)

Free Radicals↗
PubMed1993

Chemiluminescence from activated heme compounds detected in the reaction of various xenobiotics with oxyhemoglobin: comparison with several heme/hydrogen peroxide systems.

Chemiluminescence was detected in the reaction of oxyhemoglobin with various hydroxylamines and phenols, which have previously been shown to produce free radicals. The emitted light intensity correlated roughly with the methemoglobin formation rate, indicating the involvement of a photoemissive species as a reaction intermediate. In our previous work, we postulated the involvement of a catalase-insensitive, heme-bound hydrogen peroxide species in the methemoglobin formation reaction. In a series of experiments, we showed that intensive chemiluminescence occurred when hydrogen peroxide was mixed with either methemoglobin or metmyoglobin but not with hematin, which lacks the globin moiety. This suggests the involvement of the globin moiety in the light-emitting reaction sequence. The detection of paramagnetic globin species exhibiting similar kinetics as the corresponding light-emitting compound demonstrated that the assumed H2O2-heme compound has strong oxidizing properties. Accordingly, addition of bovine serum albumin to the hematin-hydrogen peroxide system also resulted in a strong chemiluminescence due to the formation of a paramagnetic transient species which could be detected by electron spin resonance (ESR). Several other heme compounds, such as cytochrome c or cytochrome c oxidase which have no vacant ligand site, did not show any light emission under similar conditions. This means that hydrogen peroxide must have access to a free-binding position on the heme. Chemiluminescence most probably stems from the transition of the initially formed heme-H2O2 adduct to the compound II type species. Due to their oxidizing nature, these species might be responsible for deleterious toxic effects such as lipid peroxidation and protein degradation.

Animals↗
PubMed1993

Involvement of free radicals in ageing: a consequence or cause of senescence.

Free oxygen radicals are increasingly discussed as important factors involved in the phenomenon of biological ageing. Higher formation rates of free radicals from senescent animals observed in isolated biological materials (mainly in mitochondria), accumulation of free radical damage and changes of antioxidant capacities appear to prove the correctness of this assumption. In the present review these findings are critically examined in order to evaluate whether free radicals do contribute to the initiation and/or propagation of ageing. It is concluded that data available so far do not allow a definite answer to this question although, free radicals are very likely to contribute considerably to the development of stochastic disorders observed during the progress of ageing.

Aging↗
PubMed1993

Ischemia/reperfusion impairs mitochondrial energy conservation and triggers O2.- release as a byproduct of respiration.

The aim of the present study was to elucidate the role of mitochondria in the development of heart failure following ischemia/reperfusion. Although mitochondria were increasingly assumed to be responsible for the establishment of an oxidative stress situation the lack of suitable methods to prove it required new concepts for an evaluation of the validity of this hypothesis. The principal idea was to expose isolated mitochondria to metabolic conditions which are developed during ischemia/reperfusion in the cell (anoxia, lactogenesis) and study how they respond. Heart mitochondria treated in that way responded with an incomplete collapse of the transmembraneous proton gradient, thereby impairing respiration-linked ATP generation. The membrane effect affected also the proper control of e- transfer through redox-cycling ubisemiquinone. Electrons were found to leak at this site from its normal pathway to O2 suggesting that ubisemiquinone becomes an active O2.- generator. It was concluded from these observations that mitochondria are likely to play a pathogenetic role in the reperfusion injury of the heart both, by an impairment of energy conservation and their transition to a potent O2.(-)-radical generator. Furthermore, there is considerable evidence that the exogenous NADH-dehydrogenase of heart mitochondria is mainly responsible for functional changes of these organelles during ischemia/reperfusion.

Animals↗
PubMed1992

Methemoglobin formation from butylated hydroxyanisole and oxyhemoglobin. Comparison with butylated hydroxytoluene and p-hydroxyanisole.

The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) react with oxyhemoglobin, thereby forming methemoglobin. The reaction rates were measured using visible spectroscopy, and second order rate constants were established for BHA and compared with p-hydroxyanisole. Using ESR we investigated the involvement of free radical reaction intermediates. The expected one-electron oxidation product of BHA and BHT, the phenoxyl radical, could only be detected with pure 3-t-butyl-4-hydroxyanisole and oxyhemoglobin. With the commercial mixture of 2- and 3-t-butyl-4-hydroxyanisole a very strong ESR signal of a secondary free radical species was observed, similar to the one observed earlier with p-hydroxyanisole and dependent on the presence of free thiol groups, so that we assumed the intermediate existence of a perferryl species, the MetHb-H2O2 adduct. In a second series of experiments we investigated the reactivity of this postulated intermediate with BHA and BHT, starting with a pure MetHb/H2O2-phenol mixture in a stopped-flow apparatus linked to the ESR spectrometer, detecting the expected phenoxyl radicals from BHA and p-hydroxyanisole. Due to the low solubility and decreased reactivity of BHT only traces of phenoxyl type radical were found together with a high concentration of unreacted perferryl species. The reactivity of BHA, BHT and p-hydroxyanisole with free thiol groups is demonstrated by an increased reaction rate in the presence of the thiol group blocking substance NEM.

Anisoles↗
PubMed1992

Ubisemiquinones of the mitochondrial respiratory chain do not interact with molecular oxygen.

The present investigation deals with the suggested role of redox-cycling ubisemiquinones in mitochondrial O2-. generation. Due to the functional complexity of electron-transferring ubiquinones in the respiratory chain, model experiments were designed to study whether ubisemiquinones will directly react with oxygen, thereby generating O2-. radicals. Based on the fact that mitochondrial ubiquinone was reported to operate in an aprotic surrounding of the inner mitochondrial membrane, the reactivity of ubisemiquinones with oxygen was tested in water-free acetonitrile. Our results prove that autoxidation of ubisemiquinones requires the addition of protons to the non-polar reaction system. An experimental evaluation of the validity of this finding with respect to mitochondrial ubiquinones is impeded by the biochemical role that oxygen plays in the establishment of ubisemiquinone populations. To differentiate between a possible direct interaction of oxygen on redox-cycling ubisemiquinones and this indirect biochemical O2 effect, we have successfully introduced ferricyanide instead of oxygen to establish mitochondrial ubisemiquinone pools. Ubisemiquinones in this reaction system were not susceptible to oxygen and no O2-. radicals were released unless the inner mitochondrial membrane was protonated by toluene pretreatment. Since the inner mitochondrial membrane is normally not permeable to protons (which is a prerequisite of the chemiosmotic theory of energy conservation) based on our experiments we can exclude the involvement of redox-cycling ubisemiquinones in mitochondrial O2-. generation.

Coenzymes↗
PubMed1991

Is oxidative stress primarily involved in reperfusion injury of the ischemic heart?

Reperfusion injury of ischemic organs is suggested to result from metabolic derangements initiating an imbalanced formation of free oxygen radicals. Most investigators in this field have used the spin-trap 5,5'-dimethyl-N-pyrroline-N-oxide (DMPO) to stabilize these short-lived radicals and make them visible by means of the electron spin resonance (ESR) technique. ESR signals obtained from intravascular DMPO were reported to indicate the formation of free OH. radicals and, in some cases, also carbon-centered radicals. We were unable to confirm these findings. Carbon-centered radicals were not obtained irrespectively of conditions studied, while oxygen-centered DMPO-adducts could only be detected in minor amounts. Instead, we observed an ascorbyl-related ESR signal. The addition of ethylenediaminetetraacetic acid (EDTA), which was used by many investigators in this field, was found to greatly influence ESR-spectra of the reperfusion fluid. The ascorbyl radical concentration was clearly reduced and the DMPO-OH. adduct became more prominent. The addition of iron further stimulated this change eliciting a Fenton-type reaction responsible for DMPO-OH.-related ESR spectra in the perfusate after ischemia. Accordingly, we observed the release of iron and ascorbic acid into the perfusate as a consequence of ischemia. We could demonstrate that iron in the presence of ascorbate and EDTA causes both types of radicals detected in the perfusate. DMPO-OH. generation in the presence of EDTA was found to result from free OH. radicals that were not generated in the absence of EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Formation of methemoglobin and phenoxyl radicals from p-hydroxyanisole and oxyhemoglobin.

The reaction of p-hydroxyanisole with oxyhemoglobin was investigated using electron spin resonance spectroscopy (ESR) and visible spectroscopy. As a reactive reaction intermediate we found the p-methoxyphenoxyl radical, the one-electron oxidation product of p-hydroxyanisole. Detection of this species required the rapid flow device elucidating the instability of this radical intermediate. The second reaction product formed is methemoglobin. Catalase or SOD had no effect upon the reaction kinetics. Accordingly, reactive oxygen species such as hydroxyl radicals or superoxide could not be observed although the spin trapping agent DMPO was used to make these short-lived species detectable. When the sulfhydryl blocking agents N-ethylmaleimide or mersalyl acid were used, an increase of the methemoglobin formation rate and of the phenoxyl radical concentration were observed. We have interpreted this observation in terms of a side reaction of free radical intermediates with thiol groups.

Anisoles↗