Biomedical subjects

Reiner Sustmann

Publications and source records attributed to Reiner Sustmann.

18 recordsLinked to original sources

PubMed2007

Assessment of chelatable mitochondrial iron by using mitochondrion-selective fluorescent iron indicators with different iron-binding affinities.

Chelatable cellular iron, and chelatable mitochondrial iron in particular, has yet to be well characterized, so the overall strength with which these "loosely bound" iron ions (presumably mainly Fe(II)) are intracellularly/intramitochondrially bound is unclear. We have previously reported the first selective mitochondrial iron indicator: rhodamine B 4-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). With this compound as a model, we have now developed two additional mitochondrial iron indicators with very different iron-binding affinities and have applied these to the study of the chelatable iron pool in the mitochondria of isolated rat liver cells. With the new indicator rhodamine B 4-[(2,2'-bipyridin-4-yl)aminocarbonyl]benzyl ester (RDA), with 2,2'-bipyridine as chelating unit (log beta(3)=17.5), essentially the same iron concentration (16.0+/-1.9 microM) was determined as with RPA (log beta(3)=21.1), despite the four orders of magnitude difference in Fe(II)-binding affinity. This not only demonstrates the reliability of the procedure, but also confirms that iron complexation by these indicators does not induce any significant release of iron from the iron-storage proteins on the timescale of the experiment. In contrast, the indicator rhodamine B 4-[bis(pyridin-2-ylmethyl)aminomethyl]benzyl ester (PIRO), with an N,N-bis(pyridin-2-ylmethyl)amine group as chelating component (log beta(2)=12.2), could not compete against the array of endogenous ligands. The intramitochondrial concentrations of the three indicators were determined to be in the range of 100 microM: that is, about three orders of magnitude lower than the total concentration of endogenous compounds that might chelate iron ions. It is therefore estimated that chelatable mitochondrial iron ions are bound by endogenous ligands with apparent stability constants (log K(app)) of between 9 and 14.

Animals↗

PubMed2006

On the mechanism of the ascorbic acid-induced release of nitric oxide from N-nitrosated tryptophan derivatives: scavenging of NO by ascorbyl radicals.

During the past years, there has been increasing interest in endogenous nitric oxide storage compounds. Recently, we briefly reported on the ascorbate-dependent release of nitric oxide ((.)NO) from N-nitrosotryptophan derivatives. In the present study, the underlying mechanism of (.)NO release is studied in more detail, primarily utilizing N-acetyl-N-nitrosotryptophan (NANT) as a model compound. The initial rate of the ascorbate-induced release of nitric oxide has been found to correspond to the rate of NANT decay. In this process, N-acetyltryptophan (NAT) is produced almost quantitatively. The final yield of nitrite amounted to around 90 % with respect to the applied amount of NANT. However, the total release of nitric oxide was only 60 %, as determined by using an FNOCT-4(fluorescent nitric oxide cheletropic trap number 4) assay. Besides nitric oxide, a second volatile product, dinitrogen oxide (N(2)O), has been identified by using (15)N NMR spectrometry, strongly indicating the intermediacy of nitroxyl (HNO). The formation of intermediate ascorbyl radical anions during the NANT-ascorbate reaction has been monitored by using ESR spectrometry. Unexpectedly, it was found that the primary oxidized product of vitamin C, dehydroascorbic acid (DHA), efficiently consumes nitric oxide. Since ESR spectrometry further revealed that ascorbyl radical anions are also generated during the spontaneous decay of DHA, the DHA-nitric oxide reaction is related to recombination of (.)NO with the thus formed ascorbyl radical anions. A conclusively established mechanism of the NANT-ascorbate reaction is presented, with O-nitrosoascorbate as a key intermediate, as additionally supported by CBS-QB3 calculations. The present study suggests that vitamin C and its oxidation products can chemically counterbalance endogenous nitric oxide levels.

Ascorbic Acid↗

PubMed2006

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes.

Heme catalases are considered to degrade two molecules of H(2)O(2) to two molecules of H(2)O and one molecule of O(2) employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H(2)O(2) (relative to catalase concentration), adjusted by H(2)O(2)-generating systems. At a ratio of a H(2)O(2) flux (given in microM/min(- 1)) to catalase concentration (given in microM) of 10 min(- 1) and above, H(2)O(2) degradation occurred via the catalatic cycle. At lower ratios, however, H(2)O(2) degradation proceeded with increasingly diminished production of O(2). At a ratio of 1 min(- 1), O(2) formation could no longer be observed, although the enzyme still degraded H(2)O(2). These results strongly suggest that at low physiological H(2)O(2) fluxes H(2)O(2) is preferentially metabolised reductively to H(2)O, without release of O(2). The pathways involved in the reductive metabolism of H(2)O(2) are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H(2)O(2) fluxes but kinetically outcompete the reaction of compound I with H(2)O(2) at low H(2)O(2) production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H(2)O(2) are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.

Animals↗

PubMed2004

Protection against iron- and hydrogen peroxide-dependent cell injuries by a novel synthetic iron catalase mimic and its precursor, the iron-free ligand.

Hydrogen peroxide is involved in many types of cell injury and exerts most of its injurious effects in conjunction with chelatable iron. We previously described a synthetic nonporphyrin iron-containing catalase mimic, TAA-1/Fe. Its ligand TAA-1 was designed for application in biological systems in which it is supposed to fulfill a dual task: it should chelate cellular labile iron and thus form the active catalase mimic, thereby decreasing levels of redox-active iron and enhancing the degradation of hydrogen peroxide. Here, we tested these novel compounds in cellular systems, i.e., in cultured hepatocytes and liver endothelial cells. Both the iron complex, i.e., the complete mimic, and the ligand, i.e., the putative precursor of this mimic, provided protection against endothelial cell injury induced by exogenous hydrogen peroxide. Furthermore, the ligand--but not (or less so) the complex--strongly protected both cell types against iron-dependent hypothermic injury and hepatocytes against iron-induced cell injury and against iron-dependent, histidine-induced injury. Together, these results demonstrate that the putative catalase mimic precursor TAA-1 is able to protect cells against iron- and/or hydrogen peroxide-dependent cell injuries and that--in line with our initial concept--it is likely to exert its protection by both iron chelation and hydrogen peroxide degradation.

Animals↗

PubMed2004

The effect of pressure on hydrogen transfer reactions with quinones.

The effect of pressure on the oxidation of hydroarenes 3-9 with 2,3-dichloro-5,6-dicyano-1,4-quinone (DDQ; 1 a) or o-chloranil (10), leading to the corresponding arenes, has been investigated. The activation volumes were determined from the pressure dependence of the rate constants of these reactions monitored by on-line UV/Vis spectroscopic measurements in an optical high-pressure cell (up to 3500 bar). The finding that they are highly negative and only moderately dependent on the solvent polarity (DeltaV( not equal ) = -13 to -25 in MTBE and -15 to -29 cm(3) mol(-1) in MeCN/AcOEt, 1:1) rules out the formation of ionic species in the rate-determining step and is good evidence for a hydrogen atom transfer mechanism leading to a pair of radicals in the rate-determining step, as was also suggested by kinetic measurements, studies of kinetic isotope effects, and spin-trapping experiments. The strong pressure dependence of the kinetic deuterium isotope effect for the reaction of 9,10-dihydroanthracene 5/5-9,9,10,10-D(4) with DDQ (1 a) can be attributed to a tunneling component in the hydrogen transfer. In the case of formal 1,3-dienes and enes possessing two vicinal C--H bonds, which have to be cleaved during the dehydrogenation, a pericyclic hydrogen transfer has to considered as one mechanistic alternative. The comparison of the kinetic deuterium isotope effects determined for the oxidation of tetralin 9/9-1,1,4,4-D(4)/9-2,2,3,3-D(4)/9-D(12) either with DDQ (1 a) or with thymoquinone 1 c indicates that the reaction with DDQ (1 a) proceeds in a stepwise manner through hydrogen atom transfer, analogously to the oxidations of 1,4-dihydroarenes, whereas the reaction with thymoquinone 1 c is concerted, following the course of a pericyclic hydrogen transfer. The difference in the mechanistic courses of these two reactions may be explained by the effect of the CN and Cl substituents in 1 a, which stabilize a radical intermediate better than the alkyl groups in 1 c. The mechanistic conclusions are substantiated by DFT calculations.

Benzoquinones↗

PubMed2004

Chemical and biological investigations of beta-oligoarginines.

In view of the important role arginine plays in living organisms as the free amino acid and, especially, as a residue in peptides and proteins, the homologous beta-homoarginines are central in our investigations of beta-peptides (Fig. 1). The preparation of beta2-homoarginine derivatives suitably protected for solution- or solid-phase peptide syntheses is described with full experimental detail (9 and 12 in Scheme 1). The readily available Fmoc-beta3 hArg(Boc)2-OH is used for manual solid-phase synthesis of beta3-oligoarginines (on Rink amide or Rink amide AM resin) either by single amino acid coupling (Scheme 3) or, much better, by dimer-fragment coupling (Scheme 4). In this way, beta3-oligoarginine amides composed of 4, 6, 7, 8, and 10 residues, both with and without fluorescein labelling, were synthesized (Schemes 2-4), purified by preparative HPLC and identified by high-resolution mass spectrometry. The free amino acids (R)- and (S)-H-beta2 hArg-OH and (S)-H-beta3 hArg-OH were tested for their ability to function as substrates for NO synthase (iNOS); the beta3-oligoarginine amides (5, 6, and 7 residues) were tested for antibacterial (against six pathogens) and hemolytic (against rat and human erythrocytes) activities. All test results were negative: none of the free beta-homoarginines induced NO formation (Fig. 3), and there was no lysis of erythrocytes (concentrations up to 100 microM; Table 1), and no significant antibiotic activity (MIC > or = 64 microg/ml; Table 2). Cell-penetration studies with the fluorescence-labelled, peptidase-resistant beta3-oligoarginine amides were carried out with HeLa cells and human foreskin keratinocytes (HFKs). The results obtained with fluorescence microscopy are: i) the longer-chain beta-oligoarginine amides (8 and 10 residues; Figs. 4-6) enter the cells and end up in the nuclei, especially in the nucleoli, irrespective of temperature (37 degrees and 4 degrees with HFKs) or pretreatment with NaN3 (with HFKs), indicating a non-endocytotic and non-energy-dependent uptake mechanism; ii) the beta-tetraarginine derivative occupies the cell surface but does not enter the cells (with HeLa); iii) the cell-growth rate of the HFKs is not affected by a 1-microM concentration of the fluorescence-labelled beta-octaarginine amide (Fig. 7), i.e., there is no antiproliferative effect. In vivo experiments with mouse skin and the beta-octaarginine derivative show migration of the beta-peptide throughout the epidermis (Fig. 8). As a contribution to understanding the mechanism, we have also studied the behavior of fluorescence-labelled beta-octa- and beta-decaarginine amides (TFA salts) towards giant unilamellar vesicles (GUVs) built of neutral (POPC) or anionic (POPC/POPG mixtures) phospholipids: the beta-oligoarginine amides bind tightly to the surface of anionic GUVs but do not penetrate the lipid bilayer (Fig. 9) as they do with living cells. In contrast, a beta-heptapeptide FL-22, which had been used as a negative control sample for the cell-penetration experiments, entered the GUVs of negative surface charge. Thus, the mechanisms of cell and GUV-model penetration appear to be different. Finally, the possible applications and implications of the 'protein transduction' by beta-oligoarginines are discussed.

Animals↗

PubMed2004

Iron-induced mitochondrial permeability transition in cultured hepatocytes.

BACKGROUND/AIMS: We previously described that the cold-induced apoptosis of cultured hepatocytes and liver endothelial cells is mediated by an increase in the cellular chelatable iron pool-in the absence of any increase in O(2)(.-)/H(2)O(2) formation. As this is an unusual mechanism, we here set out to assess whether an increase in cellular chelatable iron per se is sufficient to trigger cell injury/apoptosis. METHODS: Cultured rat hepatocytes were acutely loaded with iron using the membrane-permeable complex Fe(III)/8-hydroxyquinoline and incubated under otherwise 'physiological' conditions. RESULTS: Incubation with Fe(III)/8-hydroxyquinoline (15 microM/30 microM) increased the cellular chelatable iron and induced strong hepatocellular injury with morphological features of apoptosis, but also of necrosis. The iron-induced cell injury was oxygen-dependent, and although it was not inhibitable by extracellular catalase, it was strongly inhibited by the novel membrane-permeable catalase mimic TAA-1/Fe. The experimentally induced increase in cellular chelatable iron triggered a mitochondrial permeability transition (MPT) as assessed using double-staining with calcein and tetramethylrhodamine methyl ester. The MPT inhibitor cyclosporine A partially and the well-known inhibitor combination trifluoperazine+fructose completely inhibited the iron-induced cell injury/apoptosis. CONCLUSIONS: These results show that iron per se can induce cell injury/apoptosis and that this injury is mediated via an MPT.

Animals↗

PubMed2003

Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron.

We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4 degrees C) incubation in cell culture medium or University of Wisconsin solution, the vast majority of cells rapidly lost mitochondrial membrane potential. This loss was due to MPT as assessed by confocal laser scanning microscopy and as evidenced by the inhibitory effect of the MPT inhibitors trifluoperazine plus fructose. The occurrence of the MPT was iron-dependent: it was strongly inhibited by the iron chelators 2,2'-dipyridyl and deferoxamine. Addition of trifluoperazine plus fructose also strongly inhibited cold-induced apoptosis, suggesting that the MPT constitutes a decisive intermediate event in the pathway leading to cold-induced apoptosis. Further experiments employing the non-site-specific iron indicator Phen Green SK and specifically mitochondrial iron indicators and chelators (rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, RPA, and rhodamine B-[(2,2'-bipyridin-4-yl)aminocarbonyl]benzyl ester, RDA) suggest that it is the cold-induced increase in cytosolic chelatable iron that triggers the MPT and that mitochondrial chelatable iron is not involved in this process.

Animals↗

PubMed2003

A computational study of the cycloaddition of thiobenzophenone S-methylide to thiobenzophenone.

The cycloaddition of thiobenzophenone S-methylide to thiobenzophenone, an experimentally well-known reaction, was studied, using (U)HF/3-21G* for finding stationary points and (U)B3LYP/6-31G*//(U)HF/3-21G* single-point calculations for energies. Some optimizations were performed by (U)B3LYP/ 6-31G* to check the reliability of the calculations. The comparison of the concerted pathways and stepwise reactions via C,C-biradicals and C,S-zwitterions showed that the formation of a tetraphenyl-substituted C,C-biradical and its ring closure to 4,4,5,5-tetraphenyl-1,3-dithiolane constitutes the energetically most probable pathway of product formation, despite the fact that the regioisomeric 2,2,4,4-tetraphenyl-substituted product is more favorable by 17 kcal mol(-1). Model calculations on bond dissociation energies showed that (U)B3LYP with various basis sets overestimates radical stabilization, whereas CBS-QB3 closely reproduced experimental values. Results with the BLYP functional are similar to those with B3LYP. The consequences of the overestimation of radical stability for the cycloaddition mechanism involving biradicals are discussed. Thiobenzophenone S-methylide, if not captured by a dipolarophile, dimerizes to 2,2,3,3-tetraphenyl-1,4-dithiane. Calculation disclosed likewise a tetraphenyl-substituted C,C-biradical as intermediate.

Journal Article↗

PubMed2003

Product formation and kinetic simulations in the pH range 1-14 account for a free-radical mechanism of peroxynitrite decomposition.

The yields of nitrate and nitrite from decomposition of peroxynitrite in phosphate buffer at 37 degrees C were determined in the pH range 1-14. The NO(2)(-)/NO(3)(-) yields showed a stepwise variation with pH, with inflection points at approximately pH 3.1, 5.8, 6.8, 8.0, and 11.9. Nitrite formation increased strongly above pH 7 at the expense of nitrate, but above pH 12 nitrate again became the major product (80% at pH 14). At this pH, the Arrhenius parameters were E(a)=24.1+/-0.2kcal mol(-1) and A=(4.9+/-1.3)x10(12)s(-1). The yields of NO(2)(-), NO(3)(-), and O(2) measured at pH 5.8, 7.4, and 8.5 as a function of the initial peroxynitrite concentration (50-1000 microM) were linear only at pH 5.8. In the presence of carbon dioxide, oxygen production at pH 7.5 and pH 10 was found to be linear on the CO(2) concentration. The experimental observations were satisfactorily reproduced by kinetic simulations including principal component analyses. These data strongly suggest that the chemistry of peroxynitrite is exclusively mediated by z.rad;NO(2) and HO(z.rad;) radicals in the absence, and by z.rad;NO(2) and CO(3)(z.rad;-) radicals in the presence of CO(2).

Carbon Dioxide↗

PubMed2003

Reduction of Fe(III) ions complexed to physiological ligands by lipoyl dehydrogenase and other flavoenzymes in vitro: implications for an enzymatic reduction of Fe(III) ions of the labile iron pool.

Enzymatic reduction of physiological Fe(III) complexes of the "labile iron pool" has not been studied so far. By use of spectrophotometric assays based on the oxidation of NAD(P)H and formation of [Fe(II) (1,10-phenanthroline)3]2+ as well as by utilizing electron paramagnetic resonance spectrometry, it was demonstrated that the NAD(P)H-dependent flavoenzyme lipoyl dehydrogenase (diaphorase, EC 1.8.1.4) effectively catalyzes the one-electron reduction of Fe(III) complexes of citrate, ATP, and ADP at the expense of the co-enzymes NAD(P)H. Deactivated or inhibited lipoyl dehydrogenase did not reduce the Fe(III) complexes. Likewise, in the absence of NAD(P)H or in the presence of NAD(P)+, Fe(III) reduction could not be detected. The fact that reduction also occurred in the absence of molecular oxygen as well as in the presence of superoxide dismutase proved that the Fe(III) reduction was directly linked to the enzymatic activity of lipoyl dehydrogenase and not mediated by O2. Kinetic studies revealed different affinities of lipoyl dehydrogenase for the reduction of the low molecular weight Fe(III) complexes in the relative order Fe(III)-citrate > Fe(III)-ATP > Fe(III)-ADP (half-maximal velocities at 346-485 microm). These Fe(III) complexes were enzymatically reduced also by other flavoenzymes, namely glutathione reductase (EC 1.6.4.2), cytochrome c reductase (EC 1.6.99.3), and cytochrome P450 reductase (EC 1.6.2.4) with somewhat lower efficacy. The present data suggest a (patho)physiological role for lipoyl dehydrogenase and other flavoenzymes in intracellular iron metabolism.

Adenosine Diphosphate↗

PubMed2003

Thioformaldehyde S-methylide and thioacetone S-methylide: an ab initio MO study of structure and cycloaddition reactivity.

The mechanisms of cycloaddition of thioformaldehyde S-methylide and thioacetone S-methylide, as models for an alkyl-substituted ylide, to thioformaldehyde and thioacetone, as well as to ethene as a model for a C=C double bond have been studied by ab initio calculations. Restricted and unrestricted B3LYP/6-31G* calculations were performed for the geometries of ground states, transition structures, and intermediates. Although basis sets with more polarization functions were tested, the 6-31G* basis set was applied throughout. Single-point CASPT2 calculations are reported for analysis of the unsubstituted system. The stabilities of structures with high biradical character seem to be overestimated by DFT methods in comparison to CASPT2. The general trends of the results are independent of the level of theory. Thioformaldehyde adds to thioformaldehyde S-methylide without activation energy, and the activation energies for two-step biradical pathways to 1,3-dithiolane are low. C,S biradicals are more stable than C,C biradicals. The two-step cycloaddition is not competitive with the concerted cycloaddition. Methyl substitution in the 1,3-dipole and the dipolarophile does not change the mechanistic relationships. TSs for the concerted formation of the regioisomeric cycloadducts of thioacetone Smethylide and thioacetone were located. Concerted addition remains the preferred reaction. The reactivity of the C=S double bond is high relative to that of the C=C double bond.

Journal Article↗

PubMed2003

1,3-dithiolanes from cycloadditions of alicyclic and aliphatic thiocarbonyl ylides with thiones: regioselectivity.

The regiochemistry of 1,3-dithiolanes obtained from thiocarbonyl ylides 9 and thiones 10 shows a striking dependence on substituents. Previously and newly performed experiments indicate that sterically hindered cycloalkanethione S-methylides and dialkylthioketone S-methylides react with alicyclic and aliphatic thiones to give the 2,2,4,4-tetrasubstituted 1,3-dithiolanes 11 exclusively. Aryl groups in one or both reactants lead to a preference for, or even complete formation of, 4,4,5,5-tetrasubstituted 1,3-dithiolanes 12. Several mechanisms appear to be involved, but the paucity of experimental criteria is troubling. Quantum-chemical calculations (see preceding paper) on the cycloaddition between thioacetone S-methylide and thioacetone furnish lower activation energies for the concerted process than for the two-step pathways via C,S- or C,C-biradicals; the favoring of the 2,4-substituted 1,3-dithiolanes over the 4,5-substituted type would be expected to increase with growing bulk of substituents. Aryl groups stabilize intermediate biradicals. Experimental criteria for the differentiation of regioisomeric dithiolanes are discussed. Thiocarbonyl ylides 9 are prepared by 1,3-cycloadditions between diazomethane and thioketones and subsequent N(2) elimination from the usually isolable 2,5-dihydro-1,3,4-thiadiazoles 17; different ratios of the two rate constants lead to divergent product formation scenarios.

Journal Article↗

PubMed2003

The autoxidation of tetrahydrobiopterin revisited. Proof of superoxide formation from reaction of tetrahydrobiopterin with molecular oxygen.

It has been known for quite some time that tetrahydrobiopterin (H4B) is prone to autoxidation in the presence of molecular oxygen. Evidence has been presented that in this process superoxide radicals may be released, although their intermediacy never has been directly proven. In the present study, the autoxidation of H4B was reinvestigated with the aim to find direct evidence for superoxide formation. By means of two specific assays, namely elicitation of luminescence from lucigenin and ESR-spectrometric detection of the DEPMPO-OOH radical adduct, the release of free superoxide radicals was unequivocally demonstrated. The production of superoxide radicals was further corroborated by interaction with nitric oxide. The kinetics of the autoxidation process was established. Our data fully confirm earlier conclusions that the direct reaction between H4B and oxygen serves as an initiation reaction for the further, rapid reaction of the thus formed superoxide with H4B, thereby very likely establishing a chain reaction process involving reduction of molecular oxygen by the intermediary tetrahydrobiopterin radical. Conclusively, because H4B can per se induce oxidative stress, an in vivo overproduction of this pterin, as is evident in various diseases, may be responsible for the observed acceleration of pathophysiological pathways.

Biopterins↗

PubMed2002

Selective determination of mitochondrial chelatable iron in viable cells with a new fluorescent sensor.

Mitochondrial chelatable ("redox-active") iron is considered to contribute to several human diseases, but has not yet been characterized in viable cells. In order to determine this iron pool, we synthesized a new fluorescent indicator, rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester (RPA). In a cell-free system, RPA fluorescence was strongly and stoichiometrically quenched by Fe(2+). RPA selectively accumulated in the mitochondria of cultured rat hepatocytes. The intramitochondrial RPA fluorescence was quenched when iron was added to the cells in a membrane-permeant form. It increased when the mitochondrial chelatable iron available to the probe was experimentally decreased by the membrane-permeant transition metal chelators pyridoxal isonicotinoyl hydrazone and 1,10-phenanthroline. The concentration of mitochondrial chelatable iron in cultured rat hepatocytes, quantified from the increase in RPA fluorescence after addition of pyridoxal isonicotinoyl hydrazone, was found to be 12.2 +/- 4.9 microM. Inhibition of haem synthesis with succinylacetone did not alter the signal obtained in hepatocytes, but a rapid increase in the concentration of mitochondrial chelatable iron was observed in human erythroleukaemia K562 cells. In conclusion, RPA enables the selective determination of the highly physiologically and pathophysiologically interesting mitochondrial pool of chelatable iron in intact cells and to record the time course of alterations of this pool.

Animals↗

PubMed1996

Reaction of Phenyl-Substituted o-Quinodimethanes with Nitric Oxide. Are Benzocyclobutenes Suitable Precursors for Nitric Oxide Cheletropic Traps?

In order to elucidate the potential of substituted o-quinodimethanes as reagents for the trapping of nitric oxide (NO) in biological systems, the reaction of alkoxyl- and alkyl-substituted 7,8-diphenyl- and 7,7,8-triphenyl-o-quinodimethanes with nitric oxide in solution was investigated by ESR spectroscopic and UV/vis stopped-flow techniques. Photolytic decarbonylation of 1,3-diphenyl- and 1,1,3-triphenylindan-2-ones gave the corresponding phenyl-substituted benzocyclobutenes as the major products and low photostationary concentrations of o-quinodimethanes. During 266-nm laser flash photolysis (LFP) of 1,3-dimethoxy-1,3-diphenylindan-2-one and 1-methoxy-1,3,3-triphenylindan-2-one in acetonitrile, species absorbing in the 400-600 nm range were produced, which were attributed to configurational isomers of the corresponding 7,7,8,8-substituted o-quinodimethanes. The isomeric o-quinodimethanes decayed at significantly different rates, indicating a strong influence of the relative orientation of the terminal substituents on their stability. Reaction of the raw photolysates of the 2-indanones with NO produced strong ESR spectra of the corresponding cyclic nitroxide radicals, isoindolin-2-oxyls. The nitroxide radicals were generated in a two-phase process, the first, rapid phase being attributed to the reaction of NO with the photolytically formed o-quinodimethanes and the second, slow phase reflecting the reaction with small amounts of o-quinodimethanes, generated by thermal ring opening of the phenyl-substituted benzocyclobutenes and probably a direct reaction of NO with the benzocyclobutenes. The kinetics of both steps, as evaluated by stopped-flow UV/vis and ESR spectroscopy, revealed a strong dependence of the rate constants of the o-quinodimethane + NO reaction on the substitution pattern of the o-quinodimethanes, with rate constants spanning a range of 10-4000 M(-)(1) s(-)(1). The rate constants ((0.4-7.5) x 10(-)(4) s(-)(1)) for the reaction of NO with the 7,7,8,8-tetrasubstituted benzocyclobutenes are much less influenced by the substitution pattern. The utility of phenyl-substituted benzocyclobutenes as "reservoirs" for o-quinodimethane-type nitric oxide traps is discussed.

Journal Article↗

PubMedDate not supplied

The pathobiochemistry of nitrogen dioxide.

Nitrogen dioxide (*NO2) is an oxidizing free radical which can initiate a variety of destructive pathways in living systems, and several diseases are suspected to be connected with both exogenously and endogenously formed *NO2. Peroxynitrite (ONOO-/ONOOH) is believed to be an important endogenous source of *NO2 radicals, but other sources, among them enzymatically ones, have been identified recently. It also became clear during the last few years that in vivo formation of 3-nitrotyrosine strictly depends on the availability of *NO2 radicals. Since nitrogen dioxide is a very toxic compound an arsenal of antioxidants (e.g. vitamin C, glutathione, vitamin E, and beta-carotene) must eliminate this harmful radical in vivo. Here the recently identified superoxide (O2*-)-dependent formation of peroxynitrate (O2NOO-) and the central role of vitamin C are of special importance.

Animals↗

PubMedDate not supplied

The chelatable iron pool in living cells: a methodically defined quantity.

A very small, predominantly cytosolic pool of iron ions plays the central role in the cellular iron metabolism. It links the cellular iron uptake with the insertion of the metal in iron storage proteins and other essential iron-containing molecules. Furthermore, this transit ('labile') pool is essentially involved in the pathogenesis of a number of diseases. Due to its high physiological and pathophysiological significance, numerous methods for its characterization have been developed during the last five decades. Most of these methods, however, influence the size and nature of the transit iron pool artificially, as they are not applicable to viable biological material. Recently, fluorescence spectroscopic methods for measurements within viable cells have become available. Although these methods avoid the artifacts of previous methods, studies using fluorescent iron indicators revealed that the 'intracellular transit iron pool', which is methodically assessed as 'chelatable iron', is substantially defined by the method and/or the iron-chelating indicator applied for its detection, since the iron ions are bound to a large number of different ligands in different metabolic compartments. A more comprehensive characterization of the nature and the role of the thus not uniform cellular transit iron pool therefore requires parallel employment of different indicator molecules, which clearly differ in their intracellular distribution and their physico-chemical characteristics.

Animals↗