The effect of dietary flavonoids and phenolics on the formation of nitrotyrosine by acidic nitrite
Ceri Oldreive, Catherine Rice-Evans, and George Paganga
International Antioxidant Research Centre, UMDS-Guyķs Hospital, London, SE1 9RT, United Kingdom
The purpose of this study was to investigate the peroxynitrite independent formation of 3nitroLtyrosine and assess the inhibitory effect of various dietary antioxidants.
Nitrotyrosine has been detected in a number of disease states, implicating peroxynitrite in their pathogenesis. In vitro studies, from this laboratory, have demonstrated the inhibitory effects of the dietary consituents catechin / gallate compounds (constituents of teas and wines) and hydroxycinnamates in inhibiting peroxynitrite ń induced tyrosine nitration.
This study shows the formation of nitrotyrosine through the interaction between acidic nitrite and tyrosine, in vitro, in conditions similar to those found in the human stomach. The process is inhibited by dietary antioxidants in the order epicatechin epigallocatechin > ferulic acid > rutin, whereas ascorbic acid appeared to have no effect. They exert their effect by removing or reducing the formation of the nitrating species, being nitrated or oxidised themselves in the process. Their direct reaction with acidic nitrite is in the order catechin > ascorbic acid > ferulic acid > rutin > tyrosine.
These results suggest that along with peroxynitrite and the product of the reaction between HOCl and nitrite (Whiteman and Halliwell, 1996) tyrosine nitration may also occur in acidic nitrite. Thus nitro tyrosine may be formed in the acidic conditions of the gastric juice in the stomach after ingestion of nitrate and tyrosine which may be prevented by the simultaneous ingestion of dietary antioxidants.
We acknowledge the Ministry of Agriculture, Fisheries and Food for financial support.
Stereospecific effects of lipoic acids on mammalian pyruvate dehydrogenase complex
M.S. Patel, Y.S. Hong, S.J. Jacobia, and L. Packer
Department of Biochemistry, State University of New York, Buffalo, NY 14214 and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
Mammalian pyruvate dehydrogenase complex (PDC) plays a pivotal role in the oxidation of pyruvate derived from hexoses and some aminoacids. R-Alpha-lipoic acid (natural enantiomer) serves as a covalently linked prosthetic group for two components of PDC, namely the dihydrolipoamide acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3)-binding protein (E3BP). Additionally, of the three catalytic sites of the pyruvate dehydrogenase (E1), E2 and E3 components, two active sites (E2 and E3) also recognize free lipoic acid. Since R-lipoic acid is increasingly used as an anti-oxidant, we investigated the stereospecific effects of lipoic compounds (R-lipoic acid, R-LA; S-lipoic acid, S-LA; diseleno-lipoic acid, Se-LA) on PDC catalytic activities as well as on pyruvate oxidation by cultured HepG2 cells. Both R-LA and S-LA inhibited overall PDC activity at a concentration of 1 mM; whereas Se-LA displayed less inhibition than either enantiomer. Examination of the effects of LAs on the individual catalytic components of PDC indicated that Se LA significantly inhibited E1 activity; whereas both enantiomers of LA inhibited E2 activity. These lipoic compounds reduced E3 activity (forward reaction) by about 30 to 45% due most likely to the reoxidation of NADH by its reverse reaction. Kinetic analyses of E3 showed that both R-LA and Se-LA are used as substrates for the reverse reaction. Decarboxylation of [1-14C]pyruvate by intact HepG2 cells was significantly increased with R-LA, and decreased by either S LA or Se-LA. These findings indicate that (i) PDC and its catalytic components are affected by lipoic compounds based on their stereospecificity, and (ii) the oxidation of pyruvate by HepG2 cells is not inhibited by R-LA, suggesting that R-LA does not accumulate in mitochondria at a sufficient level toexert any inhibitory effect on PDC activity. Since R-LA enhances the decarboxylation of pyruvate by cultured HepG2 cells, it would enhance the flux of glucose-carbon via PDC reaction.
This study was supported in part by USPHS Grants DK42885 and DK50430
D. Pendergrass, S. Pinckney, Y. Mukin, M. Garnovskoya, J.R. Raymond*, and E.L. Greene*
Medical University of South Carolina, Charleston, South Carolina
Many mitogens cause cellular proliferation by activating ERK family kinases. We have recently shown that the type 1A serotonin (5-HT) receptor (5-HT1A receptor) expressed in fibroblasts activates ERKs through Gi protein - subunits, nonreceptor tyrosine kinase Src, adaptor protein Grb2, docking platform Shc, PI3¹-kinase and sequential actions of Ras, Raf, and MEK. These studies tested the potential role for receptor-stimulated reactive oxygen species in activating ERK through G-b-g. Our major readout assay was a phospho-ERK immunoblot. Studies were confirmed with an immunoprecipitation kinase assay. Activation of ERK by 5-HT was blocked by thiol- reactive antioxidants (a-lipoic acid and N-acetylcysteine). The effect of a-lipoic acid was reversed by depletion of intracellular glutathione with buthionine sulfoximine. ERK was also strongly activated by direct application of several oxidant chemicals (arsenite, diamide, H2O2), but not t-butyl-hydroperoxide, suggesting that oxidation of a critical cysteine (but not methionine) residue is required for fibroblast ERK activation. We tested a potential role for NAD(P)H oxidase in generating the oxidant signal with several inhibitors having distinct modes of action (apocyanin competes with NAD(P)H; diphenylene iodinium blocks FAD binding to the oxidase; phenylarsine oxide blocks distal electron transport; AEBSF blocks assembly of the oxidase subunits). All of the compounds blocked the ERK activation by 5-HT. When possible, closely related compounds without known effects on NAD(P)H activity were also used and in all cases were inactive. The block by phenylarsine oxide was specifically reversed by British anti-Lewisite, confirming the importance of a critical vicinal thiol in the activity of phenylarsine oxide. Mapping studies with transfected constitutively activated Raf and MEK were consistent with a location of the NAD(P)H oxidase enzyme upstream of Raf, in that ERK activation with those constructs was not attenuated by N-acetylcysteine or diphenylene iodinium. We conclude that NAD(P)H oxidase regulates the activation of ERK by 5-HT and G-b-g in fibroblasts and does so at a point upstream of Raf.
J. Pincemail, R. Larbuisson, F. Blaffart, B. Abdallaah, S. Simon, R. Limet, and J.O. Defraigne
Universtiy of Ličge - CHU, Dept of Cardiovascular Surgery, CREDEC Sart Tilman 4000 Ličge, Belgium
Introduction. During cardiac operations in patients operated with cardiopulmonary bypass (CPB), the contact between the blood and the artificial surface of the oxygenator and tubing leads to complement activation that is associated with significant granulocytes activation. Heparin immobilisation on the surfaces seems to be able to reduce complement activation but contradictory results have been observed with granulocytes activation.
Material and methods. A number of 100 patients were randomly assigned to control (n = 50, uncoated group) or a study group (n = 50, entire blood-contact surface coated with the Duraflo II heparin surface). Heparin (3 mg/kg body weigth) was administered before onset of CPB, to all patients in addition to 2875 IU added to the priming solution. Blood samples were obtained before start of CPB (T1), 5 min after aorta declamping (T2), after protamine administration (T3) and one day after the operation (T4). There was no difference in age distribution, cross-clamp time and lowest hematoctrit between the two groups. Infammatory response was assessed with the determination of 1) myeloperoxidase (MPO) and elastase (Elast) in plasma as markers of granulocytes activation and 2) of antioxidants protein-SH (PSH) and vitamin E (Vit E) consumption as indirect index of free radical production.
Results
Table I. Control group (values corrected for hemodilution* p < 0.0001 vs T1)
| MPO (ng/ml) | 6.7 +/- 0.6 | 186.1 +/- 6.5* | 53.2 +/- 6.5* | 25.5 +/- 3.1* |
| Elast (ng/ml) | 46.4 +/- 3.6 | 346.6 +/- 24.9* | 326.3 +/- 23.4* | 144.6 +/- 16.0* |
| PSH (µM) | 206.1 +/- 7.3 | 160.3 +/- 7.2* | 154.5 +/- 7.5* | 241.3 +/- 12.4* |
| Vit E (µg/ml) | 12.9 +/- 0.4 | 9.8 +/- 0.3* | 9.2 +/- 0.3* | 7.2 +/- 0.2* |
Table II. Study group (values corrected for hemodilution* p < 0.0001 vs T1)
| MPO (ng/ml) | 6.6 +/- 0.6 | 188.3 +/- 16.6* | 40.5 +/- 3.7* | 33.5 +/- 7.0* |
| Elast (ng/ml) | 59.4 +/- 6.6 | 316.7 +/- 31.9* | 279.9 +/- 25.8* | 126.8 +/- 7.3* |
| PSH (µM) | 203.4 +/- 7.0 | 165.7 +/- 7.8* | 162.2 +/- 9.1* | 251.8 +/- 9.9* |
| Vit E (µg/ml) | 13.7 +/- 0.4 | 10.1 +/- 0.3* | 8.8 +/- 0.3* | 7.1 +/- 0.2* |
Conclusion. The CPB procedure results in a significant activation of granulocytes that remains present even the day after the operation (table I). It is associated with a free radical production as evidenced with the decrease of antioxidants PSH and Vitamin E. Table II indicates that coating the circuits with Heparin by Duraflo II method fails to reduce this inflammatory response.
The peroxisome proliferator-activated receptor a regulates cellular redox state in aging
Matthew E. Poynter and Raymond A. Daynes§
Department of Pathology, University of Utah, Salt Lake City, Utah 84132 and §Geriatric Research, Education and Clinical Center, Veterans Affairs Medical Center, Salt Lake City, Utah 84112
The peroxisome proliferator-activated receptors (PPAR) are ligand-inducible transcription factors that function to transduce environmental, nutritional, and inflammatory signals into cellular responses. The alpha isoform of PPAR (PPARa) modulates the expression of genes encoding a number of metabolic, catabolic, and antioxidant enzymes following its activation by natural molecules like dehydro epiandrosterone sulfate (DHEAS), leukotriene B4, certain polyunsaturated fatty acids, or xenobiotic agents such as WY-14,643 and eico satetrayonic acid (ETYA). In aged mice, the redox-regulated transcription factor NF-kB is constitutively active in many tissues, including cells of the hematopoietic system. This activity promotes the spontaneous production of a number of pro-inflammatory cytokines which contribute to the pathology of many disease states and aging. The administration of vitamin E, DHEAS, or WY-14,643 to aged PPARa +/+ mice was able to restore the cellular redox balance, evidenced by an elimination of constitutively active NF-kB and a loss in spontaneous inflammatory cytokine production. Only vitamin E treatment was able to correct the dysregulated expression of NF-kB and pro-inflammatory cytokines seen in aged PPARa knockout mice. It was also determined that normal aged mice express reduced transcript levels of PPARa and the peroxisome-associated genes acyl-CoA oxidase and catalase. Dietary supplementation of aged mice with vitamin E or DHEAS restored normal levels of these transcripts. Our results demonstrate that the administration of modest amounts of PPARa-specific activators to redox-imbalanced aged mice reestablishes redox balance through mechanisms that require a functional PPARa.
Impairment of mitochondrial function by transient hypoxia and/or calcium
L. Schild, I. Wiswedel, F. Plumeyer, T. Reinheckel, and W. Augustin
Institute of Clinical Chemistryf, Department of Pathological Biochemistry, Otto-von-Guericke University, Magdeburg, Germany
The aim of the present study was to elucidate the role of mitochondria in liver impairment after ischemia/reperfusion. It is commonly assumed that mitochondria are in part responsible for tissue damage by impaired oxidative phosphorylation as a consequence of high calcium levels and the attack of reactive oxygen species generated within the mitochondria. The principal support of this hypothesis was provided by exposing isolated mitochondria to temporary hypoxia in combination with elevated calcium concentrations and alterations of the substrate supply. Rat liver mitochondria exhibited a decreased rate of oxidative phosphorylation of about 40% of the initial value after 5 minutes anoxia and 10 minutes reoxygenation in calcium-free incubations. The decline of the activity of the NADH-cytochrome c oxidoreductase complex found under these conditions is likely due to a drop of ADP stimulated respiration. Moreover, oxidative modifications of mitochondrial proteins, indicated by carbonyl formation, were found. Likewise, products of lipid peroxidation, such as lipid peroxides and malodialdehyde were also formed.f These data and the beneficial effects of water-soluble antioxidants suggest a reactive oxygen species mediated mechanism of the injury of mitochondrial function. Extramitochondrial calcium concentrations of about 3 µM and higher amplified the damage of mitochondrial function caused by transient hypoxia. Free calcium concentrations between 1 µM and 2 µM, however, exerted a protective effect. As under those conditions less carbonyls were formed, calcium seems to suppress oxidative stress by a still unknown mechanism.
Beth Schomer and David Epel
Hopkins Marine Station, Stanford University, Pacific Grove CA 93950
Fertilization of the sea urchin egg initiates three biochemical changes within the first minute: (1) a transient increase in calcium, (2) a permanent rise in pH, and (3) a permanent shift in the redox ratio of NADPH:NADP from 1:1 to 3-6:1. These early changes could be related to the later increases in the rate of protein translation, movement of the nucleus to the center of the egg, activation of DNA synthesis, and re-initiation of the cell cycle. The pH change might be critical since incubation in ammonia, which raises the pHi, activates all of these phenomena in unfertilized eggs. However, ammonia also causes a small redox shift (but without the initial calcium wave which occurs during fertilization), and its effects might also arise from weak base effects on acidic compartments.
We dissected the relative contribution of pH and NAD(P)H to the activation of the egg nucleus by microinjecting zwitterionic bases to increase pHi and by injecting NADPH to mimic the redox shift. The advantage of using zwitterionic bases instead of ammonia is that they will not diffuse into lysosomes and disrupt lysosomal function. We find that increasing the NAD(P)H of the egg alone does not activate DNA synthesis or other changes of the nucleus. Injection of zwitterionic bases directly increases pHi and activates nuclear changes in the egg, similarly to the activation by ammonia, suggesting the pHi change is critical.
Role of the alcoholic and non alcoholic fraction in the antioxidant effect of wine
M. Serafini, G. Maiani, and A. Ferro-Luzzi
Human Nutrition Unit, National Institute of Nutrition, Rome, Italy
Moderate wine consumption is reputed to exert a protective effect against coronary heart disease. It is still unclear whether the non-alcoholic fraction (mainly represented by phenolic compounds) and/or the alcoholic fraction is the main factor responsible for this protective effect. We set out to establish whether the non-alcoholic fraction of wine increases plasma antioxidant potential (TRAP), and whether such effect is associated to the presence in plasma of phenolic compounds (PC). The effect of alcoholic fraction on protein- phenolics interaction (reducing PC bioavaila-bility) and on in vitro TRAP of wine was also investigated.
Results showed that alcohol-free wines possessed an in vitro dose- dependent peroxyl-radical scavenger activity, but red wine with a PC concentration of 3630 g/mL Quercetin Equivalent (QE) was twenty times more active (40 mmol/L) than white wine (1.9 mmol/L) which has a PC concentration of 31 g/mL QE. The TRAP and plasma levels of PC were measured in 10 healthy subjects after the ingestion of alcohol-free red and white wine (113 mL). Alcohol-free red wine caused a significant increase in TRAP (p < 0.004) as well as PC levels (p < 0.01). Alcohol-free white wine had no effect on either plasma values.
Wine¹s phenolic fraction precipitated by proteins account for by the 78% of in vitro TRAP value. Scalar amounts of ethanol proportionally reduce the extent of protein-phenolics interaction with respect to alcohol- free red wine (p < 0.05). This effect is parallel to and is highly correlated (r = 0.978) with the TRAP increase in wine fractions, partially restoring the antioxidant activity lost as a consequence of the interactions with proteins. Alcohol had no direct effect on in vitro antioxidant capacity of wine.
The parallel and prompt increase of antioxidant status and of circulating levels of PC in fasting subjects after bolus ingestion of a moderate amount of alcohol-free red wine suggest that PC are absorbed and might be directly involved in the dietary modulation of oxidative stress. Alcoholic fraction of wine, reducing the chemical interactions between proteins and red wine PC, could to contribute indirectly to the antioxidant capacity of wine by increasing the bioavailability of its PC.
Oxidative stress in patients with obstructive lymphoedema
Werner G. Siems, Rainer Brenke, Alexander Beier, Eberhard Conradi, and Tilman Grune
Herzog-Julius Hospital of Rheumatology & Orthopaedics, D-38667 Bad Harzburg; Hospital Simbach, D-84359 Simbach; Clinics for Physical Therapy & Rehabilitation, Medical Faculty, Humboldt University, D-10098 Berlin; Germany
Obstructive lymphoedema is one of the most frequent and debilitating complications after surgical and radiological tumor treatment. It arises in approximately 40% of women with breast cancer who have had radical mastectomies followed by radiotherapy. Prevention and therapy of lymphoedema is an important problem of cancer rehabilitation.
The hypothesis on accelerated lipid peroxidation and increased oxidative stress in lymphoedema of surgically treated tumor patients was studied. The patients were at the time of the study without any tumor recidiv and without detectable metastases.
The blood of 38 patients with chronic lymphedema contains diminished concentrations of GSH and increased levels of GSSG and of cytotoxic lipid peroxidation products such as MDA and HNE as compared with a control group of 90 healthy persons. Erythrocytic GSH was 2.09 ± 0.68 mM compared to 2.70 ± 0.51 mM (p<0.0001), and erythrocytic GSSG was 101 ± 14µM compared to 50 ± 20 µM (p=0.0002). Serum MDA was 1.24 ± 0.69 µM compared to 0.44 ± 0.18 µM (p<0.0001), and HNE was 0.185 ± 0.027 µM compared to 0.088 ± 0.010 µM (p<0.0001), respectively. That demonstrates the enhanced formation of oxygen free radicals and accelerated lipid peroxidation processes in chronic lymphoedema of patients after cancer treatment. As higher the MDA concentration as lower were the levels of important serum antioxidants such as GSH or uric acid. From the significant detection of oxidative stress even in the circulating blood a tremendous generation of oxygen radicals in the lymph edematous tissue may be concluded.
The accelerated lipid peroxidation processes were additionally demonstrated by the liberation of MDA and HNE into the blood serum after the manual lymph drainage and compression bandaging of the edematous extremity. The lymph drainage led to considerable increases of serum MDA and HNE levels. The HNE level increased up to 0.3 µM.
It is suggested, that the formation of oxygen radicals contributes significantly to local lymphedematous tissue damage.
O.Sommerburg1, K.Sostmann2, T.Grune3, G.Filler2, and J.H.H.Ehrich4
1University Children¹s Hospital of Heidelberg; 2Dept. of Pediatric Nephrology, 3Dept. of Physiotherapy & Rehabilitation, University Hospital Charite, Berlin; Dept. of Pediatrics, Medical School Hannover, Germany
Background: MDA, a major product of LPO, was shown to be increased in plasma of patients with end-stage renal failure (ESRF) undergoing hemodialysis (HD). Elevated oxidative stress in ESRF patients is a result of multiple pathogenetic factors. HD treatment has been shown to be one important cause of accelerated radical generation. The aim of our study is to examine whether treatment with a dialysis membrane which has alpha-tocopherol hydrophobically bonded to its surface (Excebrane by Terumo, Japan) can reduce oxidative stress due to HD. Methods: 10 ESRF patients undergoing HD three times weekly were examined. First analysis was done when patients were still dialyzed with the membranes they used before the study. Thereafter, samples were taken when patients were dialyzed first time with the Excebrane membrane, and 6 weeks after Excebrane treatment. Samples were collected always before and after HD session. A method with HPLC-separation and flourimetric detection was used to measure plasma concentration of MDA. Results: Data are given as median and interquartile ranges. After HD with the regularly used membranes MDA was found significantly increased (1.92 [1.81-2.02] µM before vs. 2.26 [2.60-2.02] µM after HD, p<0.05). Using Excebrane first time MDA was decreased significantly after HD (2.04 [1.95-2.88] µM before vs. 1.35 [1.19-1.92] µM after HD, p<0.05). After 6 weeks of Excebrane treatment plasma MDA did not change significantly (2.01 [1.69-2.62] µM before vs. 1.95 [1.42-2.20] µM after HD). Conclusion: Oxidative stress due to HD might be decreased by the Excebrane membrane. After 6 weeks of treatment with Excebrane no effect was seen on the initial plasma concentration of MDA compared to the time before. More work is necessary to analyze long term effects of the Excebrane dialysis membrane.