- Email: [email protected]
Characterization of Phosphatidylethanolamine N-Modified by Oxidized Lipids
antiserum to study porphyrin-facilitated photooxidation of tryptophan residues of α-crystallin. In vitro experiments show that four porphyrins of biological interest cause aggregation of αcrystallin monomers into higher molecular weight polypeptides as well as accumulation of NFK in α-crystallin monomers and polymers. Confocal microscopy of human lens epithelial cell cultures indicates that while all four porphyrins can photosensitize cellular proteins, they show variable ability to oxidize α-crystallin tryptophan residues to NFK. doi:10.1016/j.freeradbiomed.2010.10.463
452 Increased Heme Degradation and Impaired Deformability of Red Blood Cells in Sickle Cell Disease and Anemia Nagababu Enika1, Barodka M Viachaslau2, Joy G Mohanty1, Daniel Nyhan2, Dan E Berkowitz2, John J Strouse2, and Joseph M Rifkind1 1 2 National Institute on Aging, Johns Hopkins Medical Institutions Hydrogen peroxide generated in association with autoxidation of oxyhemoglobin in red blood cells, reacts with hemoglobin and initiates a cascade of reactions that results in heme degradation with the formation of two fluorescent heme degradation bands and the release of iron. A 4 -7 fold increase in heme degradation has been shown in pathological red blood cells of Sickle, Thalassemia and Hb CC transgenic mice. The increase in heme degradation has also been demonstrated in severe anemic mice. In the present study, we have investigated red cell oxidative stress, as measured by an increase in heme degradation and a decrease in deformability, in human subjects with sickle cell disease, hemoglobin C disease and anemia. For subjects in sickle cell crisis a 2 fold increase in heme degradation and 2 fold decrease in deformability was observed. Comparing controls, subjects with sickle cell trait and sickle cell subjects, both under crisis and not, heme degradation correlated with the deformability of cells as well as elevated levels of methemoglobin, suggesting that oxidative stress is responsible for the increase in heme degradation and decrease in deformability. Red cell heme degradation was also elevated during mild anemia. These studies suggest that heme degradation can be used to identify certain disease states associated with oxidative stress and to assess the severity of the disease. doi:10.1016/j.freeradbiomed.2010.10.464
453 Characterization of Cytochrome C Modifications Promoted by Cholesterol Carboxyaldehyde Thiago Cardoso Genaro-Mattos1, Fernando Valentim Mansano1, Miriam Uemi1, Paolo Di Mascio1, and Sayuri Miyamoto1 1 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil. Cholesterol is a neutral lipid present in eukaryotic cell membranes that regulates their fluidity and stability. It can be oxidized by reactive oxygen species originating several modified and reactive products, such as hydroperoxides and aldehydes. Among them, the cholesterol carboxyaldehyde (ChAld) has a particular interest, once it was detected in atherosclerotic plaques and brain tissue of patients with neurodegenerative diseases. The reaction between aldehydes and proteins has gained much attention, since it could lead to protein malfunction and diseases development. Thus, our goal is to characterize the mechanisms of the reaction between ChAld and cytochrome c (cyt c), a protein present in the mitochondrial inner membrane which participates in the electrontransport chain. MALDI-ToF MS studies showed up to 8 dosedependent covalent additions of ChAld to the cyt c molecule. Moreover, each addition occurred with a loss of one water
molecule, which is consistent to a Schiff base formation with amino groups like Lys and/or Arg residues. In order to study the implications of these additions to the native structure of the protein we performed spectrophotometric analyses. We observed a red shift of γ-Soret absorption band when cyt c was incubated with ChAld, which indicates a perturbation in the heme group region. Tryptophan fluorescence analyses were also performed to evaluate alterations in the protein’s tertiary structure before and after incubation with ChAld. We observed an increase in the fluorescence intensity of the Trp emission peak, which explains that the Trp residue is interacting with different aminoacids in a different environment after the reaction with ChAld. In conclusion, our results demonstrate that ChAld covalently reacts with cyt c modifying its native structure, which could cause a loss of the protein activity and, consequently, an interruption of the electrontransport chain. Supported by: FAPESP, INCT de Processos Redox em Biomedicina-Redoxoma, CNPq and Pró Reitoria de PesquisaUSP. doi:10.1016/j.freeradbiomed.2010.10.465
454 Critical Role of the NAD(P)H Oxidase Subunit p47phox in the Formation of Oxidative DNA Damage Rajaraman Gnana Oli1, Nicole Schupp1, and Helga Stopper1 1 University of Würzburg, Germany. NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species (ROS). A vital knockout mouse model is available with deletion of the p47phox subunit of NAD(P)H oxidase and consequently reduced generation of endogenous ROS. The present study was designed to evaluate -/oxidative stress markers in p47phox mice compared with the relevant background (C57BL/6J) wild type (WT). We analyzed 8oxodG, 8-nitroguanine, 8-isoPGF2alpha, reduced and oxidized glutathione, along with the blood pressure regulating hormones angiotensin II (Ang II) and aldosterone (Aldo) in urine with a newly developed LC-ESI-MS/MS method. The method has the advantage of analyzing all markers in a single run including creatinine by using both the positive and the negative mode. Limits of detection of the analytes were between 1 fmol and 6.2 pmol. The oxidative stress markers 8-oxodG and 8-isoPGF2alpha -/were 2.5- and 1.5-fold higher in WT than p47phox mice. Also DNA damage quantified with the comet assay in kidney and liver -/was higher in WT tissues than in p47phox tissues. Recently we reported that 1nM Ang II causes oxidative DNA damage in the perfused rat kidney model via AT1 receptor activation. In the present study we found a correlation of the level of 8-oxodG and the levels of Ang II and Aldo in the urine samples. Therefore we treated cell isolates of the kidney with 200nM Ang II and showed 2 fold higher DNA damage in WT treated with Ang II than WT -/without Ang II, but no significant difference with the p47phox mice. Most importantly, we report the first evidence that the p47phox subunit of NAD(P)H oxidase plays a critical role in the formation of the oxidative DNA adduct 8-oxodG. doi:10.1016/j.freeradbiomed.2010.10.466
455 Characterization of Phosphatidylethanolamine N Modified by Oxidized Lipids Lilu Guo1, Zhongyi Chen1, Venkataraman Amarnath1, and Sean Davies1 1 Vanderbilt University Lipid peroxidation generates a myriad of lipid aldehydes including 4-hydroxynonenal (HNE), 4-oxononenal (ONE), malondialdehyde (MDA), acrolein, and isoketals (IsoK). Although the modification
SFRBM/SFRRI 2010
S165
of proteins by these aldehydes has been well-studied, the modification of other abundant cellular nucleophiles such as the amine headgroup of phosphatidylethanolamine (PE) has received little attention until recently. A few well-known lipid aldehydes have been reacted with PE and used as authentic standards to show that PE N-modified by oxidized lipids (oxN-PEs) form in vivo. What other species of oxN-PE are formed during lipid peroxidation is unknown. We therefore oxidized arachidonic acid in the presence of dipalmitoyl PE and characterized the resulting products using precursor scanning LC/MS/MS with m/z 255 (palmitate) as the product ion. This method detects only those oxidation products that contain a dipalmitoyl PE moiety. On gradient RP-HPLC, oxN-PE species eluted earlier than unreacted PE or N-acyl-PE (NAPE, used as an internal standard). While some of the major ions detected were previously identified oxNPE products such as IsoK-PE lactam (m/z 1022) and IsoK-PE hydroxylactam (m/z 1038), most were novel species. We tentatively identified three peaks as N-glutaryl-PE, N-hexanoyl-PE and MDA-PE. The elution time of synthesized authentic standards were similar to the putative species derived from arachidonic acid oxidation, supporting these identifications. Interestingly, while IsoK-PE and ONE-PE induced endothelial cell death, N-glutaryl-PE and NAPE did not. Therefore, lipid peroxidation leads to N-modification of PE by a variety of oxidized lipids, at least in vitro, but only some of these oxN-PE are deleterious to the cell. doi:10.1016/j.freeradbiomed.2010.10.467
456 Nterminal Proline of Macrophage Migration Inhibitory Factor as a Biological Target of Isothiocyanates Kristin K Brown1, and Mark B Hampton1 1 University of Otago, Christchurch, New Zealand Isothiocyanates are electrophilic phytochemicals with antioxidant capability. While not direct antioxidants, isothiocyanates can activate the transcription factor Nrf2, which then upregulates endogenous antioxidant systems. At a molecular level, isothiocyanate target proteins are not well characterised. To explore direct biological targets of phenethyl isothiocyanate (PEITC) we used an affinity purification approach. One of the major proteins captured in cell lysates was the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF). Mass spectrometry and site-directed mutagenesis showed that in a complex biological milieu PEITC covalently modified the Nterminal proline residue of MIF. While cysteine is generally considered to be a major redox sensitive target, the local protein environment of MIF promotes the reactivity of the prolyl secondary amine. Other proteins may contain proline residues that are also responsive to biological electrophiles and oxidants. doi:10.1016/j.freeradbiomed.2010.10.468
457
doi:10.1016/j.freeradbiomed.2010.10.469
458 Inactivation of Intracellular Thioldependent Enzymes by HOSCN: Evidence for a Role of Sulfenic Acid Intermediates Tessa J Barrett1, David I Pattison1, Michael J Davies1, and Clare L Hawkins1 1 Heart Research Institute, Sydney, Australia Myeloperoxidase (MPO) released by activated phagocytes at sites of inflammation, forms reactive oxidants including hypothiocyanous acid (HOSCN). Although MPO oxidants have been linked to tissue damage in a range of inflammatory diseases, the role of HOSCN is poorly understood, as HOSCN is a weak oxidant. However, recent studies have shown that this species reacts rapidly and specifically with thiols and hence may induce specific cellular alterations. This is of particular relevance to smokers who have elevated levels of the precursor thiocyanate (SCN ) ion, as HOSCN will account for a significant proportion of the MPO-derived oxidants in these people. In this study, we have utilized a LC-MS peptide mass mapping approach to examine the mechanism of HOSCN-mediated inactivation of glyceraldehyde-3phosphate dehydrogenase (GAPDH) and creatine kinase (CK), which are important thiol-dependent enzymes targeted by this oxidant in J774A.1 murine macrophage-like cells. The loss in enzymatic activity correlates with the oxidation of the active site thiol residue in each case. At low oxidant doses, inactivation occurs in a reversible manner, consistent with the formation of unstable intermediates. Evidence was obtained for the formation of both sulfenyl thiocyanate (RSSCN) and sulfenic acid (RSOH) 14 products using radioactive HOS CN and peptide mass mapping studies with the chemical probe dimedone, respectively. Exposure of GAPDH and CK to high doses of HOSCN, resulted in nonreversible enzyme inactivation and the formation of other oxygenated Cys products, including sulfinic acid (RSO2H) and sulfonic (RSO3H) acids. This is the first evidence for the formation of protein sulfenic acids with HOSCN, and highlights the potential of this oxidant to act as a second messenger in cellular redox signaling processes, which may contribute to the pathogenesis of inflammatory disease. doi:10.1016/j.freeradbiomed.2010.10.470
Influence of Systematically Administered Engineered Ceria Nanomaterial on Rat Brain Redox Homeostasis
459
Sarita S. Hardas1, and Sarita S. Hardas1 1 University of Kentucky Ceria (a.k.a.: CeO2, ceric oxide) engineered nanomaterial (ENM) is extensively used as a diesel fuel additive, an abrasive, and an industrial catalyst. Although useful in industry, unintentional exposure to ceria ENM may have undesirable effects on human health. Interestingly, it has been reported that ceria can display both pro- and anti-oxidant activities and ceria has also been nominated by NIEHS for toxicological consideration. In order to
S166
determine potential oxidative stress effects of ceria on rat brain, 5% dispersion of commercially available (avg. size ~ 31 ± 4nm) and in-house manufactured (avg. size ~ 42 ± 3nm) ceria ENM was administered intravenously to separate rats. Oxidative stress effects were assessed in three brain regions, i.e., hippocampus, cortex and cerebellum separately by analyzing markers of oxidative stress such as, levels of protein oxidation and lipid peroxidation after 1h and 20h of ceria ENM infusion. Furthermore, this study compares oxidative stress generated by commercially available and in-house engineered ceria nanoparticles and the difference in the results was correlated with the individual characterization of engineered nonmaterial to understand the role of shape and surface coating. Support: EPA STAR grant to R.Y., D.A.B., E.G., and M.T.
Validation of Best Detection Methods for Measuring Oxidative Stress Maria Kadiiska1 1 NIEHS/NIH Many techniques exist that potentially allow the measurement of oxidative stress in animal models and humans. The concept of measuring oxidative stress in humans has not been approached as a real possibility in clinical practice because standardized
SFRBM/SFRRI 2010
452 Increased Heme Degradation and Impaired Deformability of Red Blood Cells in Sickle Cell Disease and Anemia Nagababu Enika1, Barodka M Viachaslau2, Joy G Mohanty1, Daniel Nyhan2, Dan E Berkowitz2, John J Strouse2, and Joseph M Rifkind1 1 2 National Institute on Aging, Johns Hopkins Medical Institutions Hydrogen peroxide generated in association with autoxidation of oxyhemoglobin in red blood cells, reacts with hemoglobin and initiates a cascade of reactions that results in heme degradation with the formation of two fluorescent heme degradation bands and the release of iron. A 4 -7 fold increase in heme degradation has been shown in pathological red blood cells of Sickle, Thalassemia and Hb CC transgenic mice. The increase in heme degradation has also been demonstrated in severe anemic mice. In the present study, we have investigated red cell oxidative stress, as measured by an increase in heme degradation and a decrease in deformability, in human subjects with sickle cell disease, hemoglobin C disease and anemia. For subjects in sickle cell crisis a 2 fold increase in heme degradation and 2 fold decrease in deformability was observed. Comparing controls, subjects with sickle cell trait and sickle cell subjects, both under crisis and not, heme degradation correlated with the deformability of cells as well as elevated levels of methemoglobin, suggesting that oxidative stress is responsible for the increase in heme degradation and decrease in deformability. Red cell heme degradation was also elevated during mild anemia. These studies suggest that heme degradation can be used to identify certain disease states associated with oxidative stress and to assess the severity of the disease. doi:10.1016/j.freeradbiomed.2010.10.464
453 Characterization of Cytochrome C Modifications Promoted by Cholesterol Carboxyaldehyde Thiago Cardoso Genaro-Mattos1, Fernando Valentim Mansano1, Miriam Uemi1, Paolo Di Mascio1, and Sayuri Miyamoto1 1 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, SP, Brazil. Cholesterol is a neutral lipid present in eukaryotic cell membranes that regulates their fluidity and stability. It can be oxidized by reactive oxygen species originating several modified and reactive products, such as hydroperoxides and aldehydes. Among them, the cholesterol carboxyaldehyde (ChAld) has a particular interest, once it was detected in atherosclerotic plaques and brain tissue of patients with neurodegenerative diseases. The reaction between aldehydes and proteins has gained much attention, since it could lead to protein malfunction and diseases development. Thus, our goal is to characterize the mechanisms of the reaction between ChAld and cytochrome c (cyt c), a protein present in the mitochondrial inner membrane which participates in the electrontransport chain. MALDI-ToF MS studies showed up to 8 dosedependent covalent additions of ChAld to the cyt c molecule. Moreover, each addition occurred with a loss of one water
molecule, which is consistent to a Schiff base formation with amino groups like Lys and/or Arg residues. In order to study the implications of these additions to the native structure of the protein we performed spectrophotometric analyses. We observed a red shift of γ-Soret absorption band when cyt c was incubated with ChAld, which indicates a perturbation in the heme group region. Tryptophan fluorescence analyses were also performed to evaluate alterations in the protein’s tertiary structure before and after incubation with ChAld. We observed an increase in the fluorescence intensity of the Trp emission peak, which explains that the Trp residue is interacting with different aminoacids in a different environment after the reaction with ChAld. In conclusion, our results demonstrate that ChAld covalently reacts with cyt c modifying its native structure, which could cause a loss of the protein activity and, consequently, an interruption of the electrontransport chain. Supported by: FAPESP, INCT de Processos Redox em Biomedicina-Redoxoma, CNPq and Pró Reitoria de PesquisaUSP. doi:10.1016/j.freeradbiomed.2010.10.465
454 Critical Role of the NAD(P)H Oxidase Subunit p47phox in the Formation of Oxidative DNA Damage Rajaraman Gnana Oli1, Nicole Schupp1, and Helga Stopper1 1 University of Würzburg, Germany. NAD(P)H oxidase is one of the critical enzymes mediating cellular production of reactive oxygen species (ROS). A vital knockout mouse model is available with deletion of the p47phox subunit of NAD(P)H oxidase and consequently reduced generation of endogenous ROS. The present study was designed to evaluate -/oxidative stress markers in p47phox mice compared with the relevant background (C57BL/6J) wild type (WT). We analyzed 8oxodG, 8-nitroguanine, 8-isoPGF2alpha, reduced and oxidized glutathione, along with the blood pressure regulating hormones angiotensin II (Ang II) and aldosterone (Aldo) in urine with a newly developed LC-ESI-MS/MS method. The method has the advantage of analyzing all markers in a single run including creatinine by using both the positive and the negative mode. Limits of detection of the analytes were between 1 fmol and 6.2 pmol. The oxidative stress markers 8-oxodG and 8-isoPGF2alpha -/were 2.5- and 1.5-fold higher in WT than p47phox mice. Also DNA damage quantified with the comet assay in kidney and liver -/was higher in WT tissues than in p47phox tissues. Recently we reported that 1nM Ang II causes oxidative DNA damage in the perfused rat kidney model via AT1 receptor activation. In the present study we found a correlation of the level of 8-oxodG and the levels of Ang II and Aldo in the urine samples. Therefore we treated cell isolates of the kidney with 200nM Ang II and showed 2 fold higher DNA damage in WT treated with Ang II than WT -/without Ang II, but no significant difference with the p47phox mice. Most importantly, we report the first evidence that the p47phox subunit of NAD(P)H oxidase plays a critical role in the formation of the oxidative DNA adduct 8-oxodG. doi:10.1016/j.freeradbiomed.2010.10.466
455 Characterization of Phosphatidylethanolamine N Modified by Oxidized Lipids Lilu Guo1, Zhongyi Chen1, Venkataraman Amarnath1, and Sean Davies1 1 Vanderbilt University Lipid peroxidation generates a myriad of lipid aldehydes including 4-hydroxynonenal (HNE), 4-oxononenal (ONE), malondialdehyde (MDA), acrolein, and isoketals (IsoK). Although the modification
SFRBM/SFRRI 2010
S165
of proteins by these aldehydes has been well-studied, the modification of other abundant cellular nucleophiles such as the amine headgroup of phosphatidylethanolamine (PE) has received little attention until recently. A few well-known lipid aldehydes have been reacted with PE and used as authentic standards to show that PE N-modified by oxidized lipids (oxN-PEs) form in vivo. What other species of oxN-PE are formed during lipid peroxidation is unknown. We therefore oxidized arachidonic acid in the presence of dipalmitoyl PE and characterized the resulting products using precursor scanning LC/MS/MS with m/z 255 (palmitate) as the product ion. This method detects only those oxidation products that contain a dipalmitoyl PE moiety. On gradient RP-HPLC, oxN-PE species eluted earlier than unreacted PE or N-acyl-PE (NAPE, used as an internal standard). While some of the major ions detected were previously identified oxNPE products such as IsoK-PE lactam (m/z 1022) and IsoK-PE hydroxylactam (m/z 1038), most were novel species. We tentatively identified three peaks as N-glutaryl-PE, N-hexanoyl-PE and MDA-PE. The elution time of synthesized authentic standards were similar to the putative species derived from arachidonic acid oxidation, supporting these identifications. Interestingly, while IsoK-PE and ONE-PE induced endothelial cell death, N-glutaryl-PE and NAPE did not. Therefore, lipid peroxidation leads to N-modification of PE by a variety of oxidized lipids, at least in vitro, but only some of these oxN-PE are deleterious to the cell. doi:10.1016/j.freeradbiomed.2010.10.467
456 Nterminal Proline of Macrophage Migration Inhibitory Factor as a Biological Target of Isothiocyanates Kristin K Brown1, and Mark B Hampton1 1 University of Otago, Christchurch, New Zealand Isothiocyanates are electrophilic phytochemicals with antioxidant capability. While not direct antioxidants, isothiocyanates can activate the transcription factor Nrf2, which then upregulates endogenous antioxidant systems. At a molecular level, isothiocyanate target proteins are not well characterised. To explore direct biological targets of phenethyl isothiocyanate (PEITC) we used an affinity purification approach. One of the major proteins captured in cell lysates was the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF). Mass spectrometry and site-directed mutagenesis showed that in a complex biological milieu PEITC covalently modified the Nterminal proline residue of MIF. While cysteine is generally considered to be a major redox sensitive target, the local protein environment of MIF promotes the reactivity of the prolyl secondary amine. Other proteins may contain proline residues that are also responsive to biological electrophiles and oxidants. doi:10.1016/j.freeradbiomed.2010.10.468
457
doi:10.1016/j.freeradbiomed.2010.10.469
458 Inactivation of Intracellular Thioldependent Enzymes by HOSCN: Evidence for a Role of Sulfenic Acid Intermediates Tessa J Barrett1, David I Pattison1, Michael J Davies1, and Clare L Hawkins1 1 Heart Research Institute, Sydney, Australia Myeloperoxidase (MPO) released by activated phagocytes at sites of inflammation, forms reactive oxidants including hypothiocyanous acid (HOSCN). Although MPO oxidants have been linked to tissue damage in a range of inflammatory diseases, the role of HOSCN is poorly understood, as HOSCN is a weak oxidant. However, recent studies have shown that this species reacts rapidly and specifically with thiols and hence may induce specific cellular alterations. This is of particular relevance to smokers who have elevated levels of the precursor thiocyanate (SCN ) ion, as HOSCN will account for a significant proportion of the MPO-derived oxidants in these people. In this study, we have utilized a LC-MS peptide mass mapping approach to examine the mechanism of HOSCN-mediated inactivation of glyceraldehyde-3phosphate dehydrogenase (GAPDH) and creatine kinase (CK), which are important thiol-dependent enzymes targeted by this oxidant in J774A.1 murine macrophage-like cells. The loss in enzymatic activity correlates with the oxidation of the active site thiol residue in each case. At low oxidant doses, inactivation occurs in a reversible manner, consistent with the formation of unstable intermediates. Evidence was obtained for the formation of both sulfenyl thiocyanate (RSSCN) and sulfenic acid (RSOH) 14 products using radioactive HOS CN and peptide mass mapping studies with the chemical probe dimedone, respectively. Exposure of GAPDH and CK to high doses of HOSCN, resulted in nonreversible enzyme inactivation and the formation of other oxygenated Cys products, including sulfinic acid (RSO2H) and sulfonic (RSO3H) acids. This is the first evidence for the formation of protein sulfenic acids with HOSCN, and highlights the potential of this oxidant to act as a second messenger in cellular redox signaling processes, which may contribute to the pathogenesis of inflammatory disease. doi:10.1016/j.freeradbiomed.2010.10.470
Influence of Systematically Administered Engineered Ceria Nanomaterial on Rat Brain Redox Homeostasis
459
Sarita S. Hardas1, and Sarita S. Hardas1 1 University of Kentucky Ceria (a.k.a.: CeO2, ceric oxide) engineered nanomaterial (ENM) is extensively used as a diesel fuel additive, an abrasive, and an industrial catalyst. Although useful in industry, unintentional exposure to ceria ENM may have undesirable effects on human health. Interestingly, it has been reported that ceria can display both pro- and anti-oxidant activities and ceria has also been nominated by NIEHS for toxicological consideration. In order to
S166
determine potential oxidative stress effects of ceria on rat brain, 5% dispersion of commercially available (avg. size ~ 31 ± 4nm) and in-house manufactured (avg. size ~ 42 ± 3nm) ceria ENM was administered intravenously to separate rats. Oxidative stress effects were assessed in three brain regions, i.e., hippocampus, cortex and cerebellum separately by analyzing markers of oxidative stress such as, levels of protein oxidation and lipid peroxidation after 1h and 20h of ceria ENM infusion. Furthermore, this study compares oxidative stress generated by commercially available and in-house engineered ceria nanoparticles and the difference in the results was correlated with the individual characterization of engineered nonmaterial to understand the role of shape and surface coating. Support: EPA STAR grant to R.Y., D.A.B., E.G., and M.T.
Validation of Best Detection Methods for Measuring Oxidative Stress Maria Kadiiska1 1 NIEHS/NIH Many techniques exist that potentially allow the measurement of oxidative stress in animal models and humans. The concept of measuring oxidative stress in humans has not been approached as a real possibility in clinical practice because standardized
SFRBM/SFRRI 2010