Klaus Stolze - Independent Researcher (original) (raw)
Papers by Klaus Stolze
FEMS Yeast Research, 2004
We show that the dominant activated allele of the yeast RAS gene, RAS2 ala18,val19 , led to redox... more We show that the dominant activated allele of the yeast RAS gene, RAS2 ala18,val19 , led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cellspecific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2 + rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2 + rho-zero strain to that of the wild-type cells. The double mutant RAS2 ala18,val19 rhozero cells showed the same lifespan as the RAS2 ala18,val19 cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2 ala18,val19 mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.
A new flat cell for flow-orientation ESR experiments
Journal of Magnetic Resonance (1969), 1987
Acta Biochimica Polonica, Feb 1, 2000
Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especiall... more Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe 2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe 2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid rearrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe 2+ /LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were simi-Vol. 47 No. 4/2000 923-930 QUARTERLY * Presented at the 5th Symposium on "Free Radicals in Biology and Medicine", £ódŸ, 2000, Poland. . The present investigation was supported by the Oesterreichische Fonds zur Förderung der wissenschaftlichen Forschung.
Free Radical Intermediates in the Oxidation of N-Methylhydroxylamine and N,N-Dimethylhydroxylamine by Oxyhemoglobin
Free Radical Research Communications, Feb 1, 1990
ABSTRACT Nitroxide radicals have been detected in the methemoglobin formation reaction between ox... more ABSTRACT Nitroxide radicals have been detected in the methemoglobin formation reaction between oxyhemoglobin and the substituted hydroxylamine compounds, N-methylhydroxylamine and N,N-dimethylhydroxylamine, by ESR spectroscopy. The stability of these nitroxide radicals was considerably higher than that of the NH2O. radical derived from unsubstituted hydroxylamine. Only in the case of N-methylhydroxylamine the detection of the nitroxide radical required the use of a flow system, because the radical was found to undergo a rapid degradation with the concomitant formation of a secondary product, the beta-aminonitroxide CH3NO.CH2NH2. The nitroxide radical derived from N,N-dimethylhydroxylamine and oxyhemoglobin was stable for more than 1 hour. In addition, formation of low-spin iron-(III)-complexes from methemoglobin and excess substituted hydroxylamine was observed in both cases. Neither N-methylhydroxylamine nor N,N-dimethyldroxylamine formed the hemoglobin-nitric oxide complex found with unsubstituted hydroxylamine. Parallels and differences in the reaction path of un-, mono- and disubstituted hydroxylamines are discussed.
[16] Noninvasive measurement of thiol levels in cellsand isolated organs
Meth Enzymology, 1995
Changes in thiol levels of tissues are sensitive indicators of the metabolic challenges of intoxi... more Changes in thiol levels of tissues are sensitive indicators of the metabolic challenges of intoxication with heavy metals and arsenic compounds and of an imbalance between pro- and antioxidants known as “oxidative stress.” A noninvasive analysis of low- and high-molecular-weight thiols in a particular organ or in distinct biological systems cannot be performed with the existing methods that use optical or chromatographic methods, which require the preparation of tissue samples after the destruction of intact functional systems by homogenization, centrifugation, freeze clamping, or other procedures. The biradical method can be used to determine the changes in steady state levels of thiol pools without the need to disrupt the biological system. Thiol–disulfide exchange reactions between low-molecular-weight thiols (GSH) and high-molecular-weight disulfides are important biological processes for maintaining stable tissue thiol redox states. The biradical method is based on a reaction mechanism in which the reactant used is a symmetrical paramagnetic disulfide that rapidly undergoes thiol–disulfide exchange reactions with tissue thiols. The use of this biradical for a noninvasive assessment of thiol levels in any biological system requires its penetration through the phospholipid membranes.
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 mitoc... more 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.
Investigations of the Light-Emitting Species in the Reaction of Metmyoglobin and Methemoglobin With Hydrogen Peroxide
Photochemistry and Photobiology, 1994
Methemoglobin Formation from Butylated Hydroxyanisole and Oxyhemoglobin. Comparison with Butylated Hydroxytoluene and P-Hydroxyanisole
Free Radical Research Communications, Feb 1, 1992
The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) ... more 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.
Biochemical Pharmacology, Sep 15, 1989
Four distinct paramagnetic intermediates could be observed in the reaction between oxyhemoglobin ... more Four distinct paramagnetic intermediates could be observed in the reaction between oxyhemoglobin and hydroxyIamine using ESR spectroscopy. The radical species exhibited different stability properties thus different techniques were required for their detection. Two of them were identified as the hydronitroxide radical (NH,O') and the hemoglobin-nitric oxide complex (Hb'+-NO). The third one is a low-spin iron-(III)-complex, possibly the methemoglobin-hydroxylamine adduct. A fourth paramagnetic species was detected only in the absence of the iron chelator DETAPAC thus indicating that free iron ions were responsible for the formation of this intermediate. The same species was observed when a Fenton system was used to generate the radicals. This species was identified as being the Fe(N0)2X, complex described in the literature (X = inorganic anions such as OH-or PO:-). The identification of the radical intermediates detected in the hydroxyfamine-induced methemoglobin formation contributes to a more detailed understanding of the reaction sequence.
[16] Noninvasive measurement of thiol levels in cellsand isolated organs
Methods in Enzymology, 1995
ABSTRACT
The Effects of Xenobiotics on Erythrocytes
General Pharmacology: The Vascular System, 1998
... Butterfield, DA, Sun, B., Bellary, S., Arden, WA and Anderson, KW, 1994. Effect of endotoxin ... more ... Butterfield, DA, Sun, B., Bellary, S., Arden, WA and Anderson, KW, 1994. Effect of endotoxin on lipid order and motion in erythrocyte membranes. Biochim. Biophys. Acta 1225, pp. 231234. Abstract | PDF (328 K) | View Record in Scopus | Cited By in Scopus (14). Carter, P., 1971 ...
Effect of Copper on Fatty Acid Profiles in Non- and Semifermented Teas Analyzed by LC-MS-Based Nontargeted Screening
Journal of agricultural and food chemistry, Jan 30, 2015
Unsaturated fatty acids are well-known precursors of aroma compounds, which are considered import... more Unsaturated fatty acids are well-known precursors of aroma compounds, which are considered important for green tea quality. Due to the known copper-induced oxidation of unsaturated fatty acids and the broad variability of the amount of copper present in tea infusions, this paper investigates the influence of copper, added at a nontoxic concentration (300 μM) to non- and semifermented teas, on the degradation of fatty acids and fatty acid hydroperoxides thereof. The abundance of fatty acids in green and oolong tea was determined by means of a nontargeted approach applying high-resolution MS/MS. As a result, most of the fatty acids in green and oolong tea were already oxidized prior to copper addition. Addition of 300 μM CuSO4 to the oolong tea sample resulted in a decrease of 13-hydroperoxy-9Z,11E-octadecadienoic acid, an important flavor precursor, from 0.12 ± 0.02 to 0.05 ± 0.01 μM (p = 0.035), and other oxidized fatty acids decreased as well. However, copper-induced degradation of...
The British journal of cancer. Supplement
The Influence of Oxidation Conditions on Free Radical Formation in Kaempferol
Acta biochimica Polonica, 2000
Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especiall... more Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl r...
Noninvasive measurement of thiol levels in cells and isolated organs
Methods in enzymology, 1995
Free Radical Reactions with DNA and Its Nucleotides
Antimutagenesis and Anticarcinogenesis Mechanisms II, 1990
The effects of the xenobiotics atrazine, benzene, butylated hydroxyanisole (BHA), butylated hydro... more The effects of the xenobiotics atrazine, benzene, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lindane, toluene, and xylenol on the respiration of isolated rat heart mitochondria were studied. Bioenergetic parameters such as respiratory control (RC) and ATPIoxygen (P/O) values decreased considerably in the presence of these substances, and a concomitant increase of superoxide radical (0, ~ ) formation was observed. These observations were discussed in terms of partial damage of the inner mitochondrial membrane, impairing the maintenance of the proton gradient across this membrane.
Agricultural Sciences, 2013
Coffees and teas are beverages that are both exceptionally rich in antioxidant molecules, and are... more Coffees and teas are beverages that are both exceptionally rich in antioxidant molecules, and are also both associated with beneficial health effects. Thus although the quality characteristics of these beverages are conventionally assessed on the basis of their sensory properties, their antioxidant contents represent an additional and increasingly valued attribute of quality based on their contributions to healthy diets. Both beverages are prepared by hot water extraction of a pure plant-derived product, and thus their compositions can potentially change quite rapidly as a result of oxidation in contact with air. Oxidative processes often proceed via free radical intermediates, and sometimes also result in the formation of stable radical endproducts; thus EPR spectroscopy is a convenient technique for investigating some of the various free radical reactions that occur in these beverages. This paper reviews progress that has been made in elucidating free radical processes that occur during the preparation and storage of coffees and teas, and the results are discussed in terms of quality criteria of the beverages.
Photochemistry and Photobiology, 1997
Oxidation of low-density lipoprotein (LDL) by low amounts of cupric ions resulted in the formatio... more Oxidation of low-density lipoprotein (LDL) by low amounts of cupric ions resulted in the formation of singlet oxygen (lo,, lAa) when hydroxylamine (NH,OH) was added. Direct evidence on this excited species came from partial spectral resolution of the emitted light in the red spectral region (634 nm and 703 nm), which can be attributed to the dimol decay of singlet oxygen. Additional evidence for the existence of singlet oxygen came from the enhancing effect of deuterium oxide buffer (D20) on chemiluminescence intensity and the quenching effect of sodium azide. A linear correlation between NH,OH-dependent chemiluminescence intensity and the amount of diene conjugates (DC) formed in this reaction was observed. Removal of adventitious transition metals b y adequate chelators prevented chemiluminescence in this system; NH20H was also found to efficiently decrease metabolites of lipid peroxidation (LPO). Our findings are consistent with a sequence of reactions in which NH,OH first converts transition metals to their reduced state, thereby stimulating the formation of alkoxy-and peroxyradicals. Peroxyradicals decompose in a bimolecular Russel reaction to hydroxyl compounds and singlet oxygen while the majority of alkoxy radicals are eliminated by a secondary reaction with NH20H. Identical effects were observed when reducing antioxidants such as ascorbic acid or trolox C were used instead of hydroxylamine.
FEMS Yeast Research, 2004
We show that the dominant activated allele of the yeast RAS gene, RAS2 ala18,val19 , led to redox... more We show that the dominant activated allele of the yeast RAS gene, RAS2 ala18,val19 , led to redox imbalance in exponential-phase cells and to excretion of almost all of the cellular glutathione into the medium when the cells reached early-stationary phase. The mitochondria of the mutant stained strongly with dihydrorhodamine 123 (DHR) and the cells displayed a very short mother cellspecific lifespan. Adding 1 mM reduced glutathione (GSH) to the medium partly restored the lifespan. The corresponding RAS2 + rho-zero strain also displayed a short lifespan, excreted nearly all of its GSH, and stained positively with DHR. Adding 1 mM GSH completely restored the lifespan of the RAS2 + rho-zero strain to that of the wild-type cells. The double mutant RAS2 ala18,val19 rhozero cells showed the same lifespan as the RAS2 ala18,val19 cells, and the effect of glutathione in restoring the lifespan was the same, indicating that both mutations shorten lifespan through a similar mechanism. In the RAS2 ala18,val19 mutant strain and its rho-zero derivative we observed for the first time a strong electron spin resonance (ESR) signal characteristic of the superoxide radical anion. The mutant cells were, therefore, producing superoxide in the absence of a complete mitochondrial electron transport chain, pointing to the existence of a possible non-mitochondrial source for ROS generation. Our results indicate that oxidative stress resulting from a disturbance of redox balance can play a major role in mother cell-specific lifespan determination of yeast cells.
A new flat cell for flow-orientation ESR experiments
Journal of Magnetic Resonance (1969), 1987
Acta Biochimica Polonica, Feb 1, 2000
Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especiall... more Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe 2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe 2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid rearrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl radical. We also synthesized lipophilic derivatives of the spin trap DEPMPO in order to detect lipid radical species generated in the lipid phase. With all spin traps studied a lipid-derived carbon-centered radical was obtained in the anaerobic incubation system Fe 2+ /LOOH indicating the trapping of a lipid radical, possibly generated as a secondary reaction product of the primary lipid alkoxyl radical formed. Under aerobic conditions an SOD-insensitive oxygen-centered radical adduct was formed with DEPMPO and its lipophilic derivatives. The observed ESR parameters were simi-Vol. 47 No. 4/2000 923-930 QUARTERLY * Presented at the 5th Symposium on "Free Radicals in Biology and Medicine", £ódŸ, 2000, Poland. . The present investigation was supported by the Oesterreichische Fonds zur Förderung der wissenschaftlichen Forschung.
Free Radical Intermediates in the Oxidation of N-Methylhydroxylamine and N,N-Dimethylhydroxylamine by Oxyhemoglobin
Free Radical Research Communications, Feb 1, 1990
ABSTRACT Nitroxide radicals have been detected in the methemoglobin formation reaction between ox... more ABSTRACT Nitroxide radicals have been detected in the methemoglobin formation reaction between oxyhemoglobin and the substituted hydroxylamine compounds, N-methylhydroxylamine and N,N-dimethylhydroxylamine, by ESR spectroscopy. The stability of these nitroxide radicals was considerably higher than that of the NH2O. radical derived from unsubstituted hydroxylamine. Only in the case of N-methylhydroxylamine the detection of the nitroxide radical required the use of a flow system, because the radical was found to undergo a rapid degradation with the concomitant formation of a secondary product, the beta-aminonitroxide CH3NO.CH2NH2. The nitroxide radical derived from N,N-dimethylhydroxylamine and oxyhemoglobin was stable for more than 1 hour. In addition, formation of low-spin iron-(III)-complexes from methemoglobin and excess substituted hydroxylamine was observed in both cases. Neither N-methylhydroxylamine nor N,N-dimethyldroxylamine formed the hemoglobin-nitric oxide complex found with unsubstituted hydroxylamine. Parallels and differences in the reaction path of un-, mono- and disubstituted hydroxylamines are discussed.
[16] Noninvasive measurement of thiol levels in cellsand isolated organs
Meth Enzymology, 1995
Changes in thiol levels of tissues are sensitive indicators of the metabolic challenges of intoxi... more Changes in thiol levels of tissues are sensitive indicators of the metabolic challenges of intoxication with heavy metals and arsenic compounds and of an imbalance between pro- and antioxidants known as “oxidative stress.” A noninvasive analysis of low- and high-molecular-weight thiols in a particular organ or in distinct biological systems cannot be performed with the existing methods that use optical or chromatographic methods, which require the preparation of tissue samples after the destruction of intact functional systems by homogenization, centrifugation, freeze clamping, or other procedures. The biradical method can be used to determine the changes in steady state levels of thiol pools without the need to disrupt the biological system. Thiol–disulfide exchange reactions between low-molecular-weight thiols (GSH) and high-molecular-weight disulfides are important biological processes for maintaining stable tissue thiol redox states. The biradical method is based on a reaction mechanism in which the reactant used is a symmetrical paramagnetic disulfide that rapidly undergoes thiol–disulfide exchange reactions with tissue thiols. The use of this biradical for a noninvasive assessment of thiol levels in any biological system requires its penetration through the phospholipid membranes.
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 mitoc... more 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.
Investigations of the Light-Emitting Species in the Reaction of Metmyoglobin and Methemoglobin With Hydrogen Peroxide
Photochemistry and Photobiology, 1994
Methemoglobin Formation from Butylated Hydroxyanisole and Oxyhemoglobin. Comparison with Butylated Hydroxytoluene and P-Hydroxyanisole
Free Radical Research Communications, Feb 1, 1992
The widely used food additives butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) ... more 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.
Biochemical Pharmacology, Sep 15, 1989
Four distinct paramagnetic intermediates could be observed in the reaction between oxyhemoglobin ... more Four distinct paramagnetic intermediates could be observed in the reaction between oxyhemoglobin and hydroxyIamine using ESR spectroscopy. The radical species exhibited different stability properties thus different techniques were required for their detection. Two of them were identified as the hydronitroxide radical (NH,O') and the hemoglobin-nitric oxide complex (Hb'+-NO). The third one is a low-spin iron-(III)-complex, possibly the methemoglobin-hydroxylamine adduct. A fourth paramagnetic species was detected only in the absence of the iron chelator DETAPAC thus indicating that free iron ions were responsible for the formation of this intermediate. The same species was observed when a Fenton system was used to generate the radicals. This species was identified as being the Fe(N0)2X, complex described in the literature (X = inorganic anions such as OH-or PO:-). The identification of the radical intermediates detected in the hydroxyfamine-induced methemoglobin formation contributes to a more detailed understanding of the reaction sequence.
[16] Noninvasive measurement of thiol levels in cellsand isolated organs
Methods in Enzymology, 1995
ABSTRACT
The Effects of Xenobiotics on Erythrocytes
General Pharmacology: The Vascular System, 1998
... Butterfield, DA, Sun, B., Bellary, S., Arden, WA and Anderson, KW, 1994. Effect of endotoxin ... more ... Butterfield, DA, Sun, B., Bellary, S., Arden, WA and Anderson, KW, 1994. Effect of endotoxin on lipid order and motion in erythrocyte membranes. Biochim. Biophys. Acta 1225, pp. 231234. Abstract | PDF (328 K) | View Record in Scopus | Cited By in Scopus (14). Carter, P., 1971 ...
Effect of Copper on Fatty Acid Profiles in Non- and Semifermented Teas Analyzed by LC-MS-Based Nontargeted Screening
Journal of agricultural and food chemistry, Jan 30, 2015
Unsaturated fatty acids are well-known precursors of aroma compounds, which are considered import... more Unsaturated fatty acids are well-known precursors of aroma compounds, which are considered important for green tea quality. Due to the known copper-induced oxidation of unsaturated fatty acids and the broad variability of the amount of copper present in tea infusions, this paper investigates the influence of copper, added at a nontoxic concentration (300 μM) to non- and semifermented teas, on the degradation of fatty acids and fatty acid hydroperoxides thereof. The abundance of fatty acids in green and oolong tea was determined by means of a nontargeted approach applying high-resolution MS/MS. As a result, most of the fatty acids in green and oolong tea were already oxidized prior to copper addition. Addition of 300 μM CuSO4 to the oolong tea sample resulted in a decrease of 13-hydroperoxy-9Z,11E-octadecadienoic acid, an important flavor precursor, from 0.12 ± 0.02 to 0.05 ± 0.01 μM (p = 0.035), and other oxidized fatty acids decreased as well. However, copper-induced degradation of...
The British journal of cancer. Supplement
The Influence of Oxidation Conditions on Free Radical Formation in Kaempferol
Acta biochimica Polonica, 2000
Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especiall... more Unsaturated lipids are rapidly oxidized to toxic products such as lipid hydroperoxides, especially when transition metals such as iron or copper are present. In a Fenton-type reaction Fe2+ converts lipid hydroperoxides to the very short-lived lipid alkoxyl radicals. The reaction was started upon the addition of Fe2+ to an aqueous linoleic acid hydroperoxide (LOOH) emulsion and the spin trap in the absence of oxygen. Even when high concentrations of spin traps were added to the incubation mixture, only secondary radical adducts were detected, probably due to the rapid re-arrangement of the primary alkoxyl radicals. With the commercially available nitroso spin trap MNP we observed a slightly immobilized ESR spectrum with only one hydrogen splitting, indicating the trapping of a methinyl fragment of a lipid radical. With DMPO or 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) adducts were detected with carbon-centered lipid radical, with acyl radical, and with the hydroxyl r...
Noninvasive measurement of thiol levels in cells and isolated organs
Methods in enzymology, 1995
Free Radical Reactions with DNA and Its Nucleotides
Antimutagenesis and Anticarcinogenesis Mechanisms II, 1990
The effects of the xenobiotics atrazine, benzene, butylated hydroxyanisole (BHA), butylated hydro... more The effects of the xenobiotics atrazine, benzene, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lindane, toluene, and xylenol on the respiration of isolated rat heart mitochondria were studied. Bioenergetic parameters such as respiratory control (RC) and ATPIoxygen (P/O) values decreased considerably in the presence of these substances, and a concomitant increase of superoxide radical (0, ~ ) formation was observed. These observations were discussed in terms of partial damage of the inner mitochondrial membrane, impairing the maintenance of the proton gradient across this membrane.
Agricultural Sciences, 2013
Coffees and teas are beverages that are both exceptionally rich in antioxidant molecules, and are... more Coffees and teas are beverages that are both exceptionally rich in antioxidant molecules, and are also both associated with beneficial health effects. Thus although the quality characteristics of these beverages are conventionally assessed on the basis of their sensory properties, their antioxidant contents represent an additional and increasingly valued attribute of quality based on their contributions to healthy diets. Both beverages are prepared by hot water extraction of a pure plant-derived product, and thus their compositions can potentially change quite rapidly as a result of oxidation in contact with air. Oxidative processes often proceed via free radical intermediates, and sometimes also result in the formation of stable radical endproducts; thus EPR spectroscopy is a convenient technique for investigating some of the various free radical reactions that occur in these beverages. This paper reviews progress that has been made in elucidating free radical processes that occur during the preparation and storage of coffees and teas, and the results are discussed in terms of quality criteria of the beverages.
Photochemistry and Photobiology, 1997
Oxidation of low-density lipoprotein (LDL) by low amounts of cupric ions resulted in the formatio... more Oxidation of low-density lipoprotein (LDL) by low amounts of cupric ions resulted in the formation of singlet oxygen (lo,, lAa) when hydroxylamine (NH,OH) was added. Direct evidence on this excited species came from partial spectral resolution of the emitted light in the red spectral region (634 nm and 703 nm), which can be attributed to the dimol decay of singlet oxygen. Additional evidence for the existence of singlet oxygen came from the enhancing effect of deuterium oxide buffer (D20) on chemiluminescence intensity and the quenching effect of sodium azide. A linear correlation between NH,OH-dependent chemiluminescence intensity and the amount of diene conjugates (DC) formed in this reaction was observed. Removal of adventitious transition metals b y adequate chelators prevented chemiluminescence in this system; NH20H was also found to efficiently decrease metabolites of lipid peroxidation (LPO). Our findings are consistent with a sequence of reactions in which NH,OH first converts transition metals to their reduced state, thereby stimulating the formation of alkoxy-and peroxyradicals. Peroxyradicals decompose in a bimolecular Russel reaction to hydroxyl compounds and singlet oxygen while the majority of alkoxy radicals are eliminated by a secondary reaction with NH20H. Identical effects were observed when reducing antioxidants such as ascorbic acid or trolox C were used instead of hydroxylamine.