Factors associated with patent foramen ovale-related stroke: SAFAS study.

BACKGROUND: Persistent foramen ovale (PFO) contributes to cryptogenic stroke and is associated with stroke recurrence, although the exact mechanism of ischemic events is not fully understood. Several biomarkers have been developed for the prediction of atrial fibrillation after stroke, but there are currently only limited data on their potential value for the diagnosis of PFO-related stroke. METHODS: This study was a prospective single-center study that included all patients hospitalized between March 31, 2018, and January 18, 2020, in the stroke department of the Dijon University Hospital for ischemic stroke without obvious cause and without a history of atrial fibrillation. PFO was systematically screened by transthoracic echocardiography and images were reviewed by an independent cardiologist blinded from clinical data. PFO was defined according to the CLOSE trial criteria: PFO associated with interatrial septal aneurysm or significant interatrial shunt (> 30 microbubbles in the left atrium within three cardiac cycles after right atrial opacification). The potential association of PFO-related stroke with biomarkers of cardiac fibrosis and inflammation such as galectin-3, GDF-15, ST-2, osteoprotegerin and NT-proBNP was tested using multivariate backward stepwise logistic regression. RESULTS: Of the 240 patients included in the SAFAS study, 229 had complete echocardiographic data, and 23 (10%) had PFO-related stroke. Patients with PFO-related stroke were significantly younger (58±14 vs. 69±14, P<0.001), had less frequent previous arterial hypertension (30 vs. 60%, P=0.008), and more frequent cerebellar territory involvement (26 vs. 9%, P=0.014) compared to the other patients. In addition, they had less frequently left atrial dilatation (left atrial index volume>34mL/m2 [9 vs. 35%, P=0.009]). After ROC curve analysis for definition of thresholds, PFO-related stroke patients more often had galectin-3<9.5ng/mL (59 vs. 27%, P=0.002), ST2<13380pg/ml (23 vs. 50%, P=0.007), GDF-15<1200ng/mL (59 vs. 27%, P=0.002), osteoprotegerin<1133pg/mL (82 vs. 58%, P=0.033) and NT-proBNP<300pg/mL (88 vs. 55%, P=0.009). After multivariate analysis, only galectin-3<9.5ng/mL (OR [95% CI] 3.4 [1.18; 9.8], P=0.024) and osteoprotegerin<1133pg/L (OR [95% CI] 5.0 [1.1; 22.9], P=0.038) were independently associated with PFO-related stroke. CONCLUSION: Patients in whom cryptogenic stroke is attributed to a significant PFO have a specific clinical and biological phenotype. Low levels of galectin-3 and osteoprotegerin may help identify patients with PFO-related strokes.

Transient postnatal over nutrition induces long-term alterations in cardiac NLRP3-inflammasome pathway.

BACKGROUND AND AIMS: The prevalence of obesity is increasing worldwide at an alarming rate. Altered early nutrition, in particular postnatal overfeeding (PNOF), is a risk factor for impaired cardiac function in adulthood. In the understanding of the initiation or progression of heart diseases, NLRP3 inflammasome and non-coding RNAs have been proposed as key players. In this context, the aim of this study was to decipher the role of NLRP3 inflammasome and its post transcriptional control by micro-RNAs in the regulation of cardiac metabolic function induced by PNOF in mice. METHODS AND RESULTS: Based on a model of mice exposed to PNOF through litter size reduction, we observed increased cardiac protein expression levels of NLRP3 and ETS-1 associated with alterations in insulin signaling. Additionally, miR-193b levels were down-regulated in the adult hearts of overfed animals. In a cardiomyocyte cell line, transfection with miR-193b induced down-regulation of ETS-1 and NLRP3 and improved insulin signaling. CONCLUSIONS: These findings suggest that the miR-193b could be involved in cardiac phenotypic changes observed in adulthood induced by PNOF likely through the regulation of ETS-1 and NLRP3 expression, and through this of insulin signaling.

Antioxidant properties of alpha-lipoic acid: effects on red blood membrane permeability and adaptation of isolated rat heart to reversible ischemia.

The aim of our work was to study (1) the antioxidant properties of lipoic acid (LA) and its reduced metabolite dihydrolipoic acid (DHLA) formed by reduction of LA and (2) the effects of treatment with LA and DHLA on (a) K(+) efflux from human red blood cells and (b) post-ischemic recovery and oxidative stress in isolated perfused rat hearts challenged with an ischemia-reperfusion (IR) sequence. In vitro, we used xanthine and xanthine oxidase to generate superoxide anion, which is not directly measurable by electron paramagnetic resonance (EPR), but specifically oxidizes the spin probe CPH into an EPR-detectable long lasting CP(*) nitroxide radical. While 5 mM of LA was ineffective in reducing the kinetics of CP(*) nitroxide formation, DHLA was shown to lessen this rate in a dose-dependent manner and at 30 mM was even more efficient than 300 UI/ml SOD. These results are in agreement with the fact that DHLA is able to directly scavenge superoxide anion. Red cells are a good model to investigate oxidative damage in biological membranes; hence, we used a suspension of erythrocytes incubated with 2,2(')-azobis(2-amidinopropane) hydrochloride (AAPH) which generates in vitro free radicals. DHLA provided more effective protection of red cells membranes than LA; DHLA was comparable to Trolox for its antioxidant potency. In vivo, treatment of rats (50 mg/kg/day i.p. for 7 days) with LA induced a slight increase in coronary flow (CF) in isolated perfused hearts, after 30 min of global total ischemia. This effect was not associated with an improvement in contractile function and reduction of myocardial oxidative stress. In conclusion, because of their ability to scavenge free radicals, LA and to an even greater degree DHLA were able to protect the membranes of red blood cells. This finding suggests that LA and DHLA might be useful in the treatment of diseases associated with oxidative stress such as diabetes.

[Hydrogen sulfide (H2S), an endogenous gas with odor of rotten eggs might be a cardiovascular function regulator]. / Le sulfure d'hydrogène (H2S), un gaz endogène à l'odeur d'oeuf pourri, pourrait être un régulateur des fonctions cardiovasculaires.

A novel concept of "gasotransmitter" arrived recently. They are small molecules of endogenous gases. Hydrogene sulfide (H2S) is qualified as the third gasotransmitter beside nitric oxide (NO) and carbon monoxide (CO). The physiological functions of endogenous H2S are not well-known. The location of the H2S synthetizing enzymes as well as the detector of endogenous levels of H2S in the tissues suggests that the cardiovascular system is a source of H2S generation. This gas relaxes vascular smooth muscle both in vitro and in vivo probably by opening smooth K+ATP channels. Being a reducing agent, H2S may alter cellular redox status. It is able to produce thiyls free radicals: SH degrees and S degrees . The advances in H2S researchs may revolutionize many conventional doctrines in the cardiovascular area.

[An endogenous dithiol with antioxidant properties: alpha-lipoic acid, potential uses in cardiovascular diseases]. / Un dithiol endogène aux propriétés antioxydantes: l'acide alpha-lipoïque, utilisation potentielle dans les pathologies cardiovasculaires.

Alpha-Lipoic acid (ALA) is a natural compound, chemically named 1,2-dithiolane-3-pentanoic acid, also referred to as thioctic acid. In humans, ALA is synthetized by the liver and other tissues with high metabolic activity: heart, kidney. ALA is both water and fat soluble and therefore, is widely distributed in both cellular membranes and cytosol. Recently, a greater deal of attention has been given to antioxidant function for ALA and its reduced formed: dihydrolipoic acid (DHLA). ALA scavenges hydroxyl radicals, hypochlorous acid and singlet oxygen. It may also exert antioxidant effects in biological systems through transitional metal chelation. Dihydrolipoic acid has been shown to have antioxidant but also pro-oxidant properties in systems in which hydroxyl radical was generated. ALA/DHLA ratio has the capacity to recycle endogenous antioxidants such as vitamin E. A number of experimental as well as clinical studies point to the usefulness of ALA as a therapeutic agent for such diverse conditions as diabetes, atherosclerosis, insulin resistance, neuropathy, neurodegenerative diseases and ischemia-reperfusion injury. ALA represents a potential agent on the vascular endothelium, recording to ALA/DHLA redox couple is one of the most powerful biological antioxidant systems.

[Oxidative stress is exacerbated in diabetic patients during cardiopulmonary bypass]. / Exacerbation du stress oxydatif chez les patients diabétiques durant une circulation extracorporelle.

UNLABELLED: Circulation on blood extracorporeally through plastic tubing activates several pathways including systemic inflammation and oxidative stress. These phenomena are suspected to participate to neurological and cardiovascular side effects observed in the patients under cardiopulmonary bypass (CPB). A direct relationship, in diabetic patients, between hyperglycemia and morbidity and mortality has been established. However, it is still unclear whether perioperative hyperglycemia has a direct effect on adverse events in cardiac surgery. The purpose of this study was to determine the influence of hyperglycemia on inflammation and oxidative stress in patients under CPB during cardiac surgery. MATERIAL AND METHODS: Control patients (n=17) and diabetic (type 2) patients (n=13) were included in this study. Blood samples were drawn before, during and after the CPB. Oxidative stress was evaluated in the plasma by direct and indirect approaches. Direct detection of ascorbyl radicals was assessed by electron spin resonance spectroscopy. An index: ascorbyl radical/vitamin C ratio is an indicator of the degree of oxidative stress taking place in the plasma. Oxygen radical absorbing capacity (ORAC) values were used as measurement of antioxidant capacity of the plasma. To determine inflammation profile of patients, we measure the evolution of plasma concentration of interleukin 8 (IL-8). RESULTS: During cross clamping and post-CPB, the index ascorbyl radical/vitamin C is increased; the value of the index is more significant in diabetic patients. Concomitantly, ORAC values decreased in all the patients during cross clamping (p<0.05). Results concerning inflammatory index showed that IL-8 levels increased during the CPB. CONCLUSION: In conclusion, the current study indicates that a systemic oxidative stress occurs during CPB and post-CPB periods and that in patients with type 2 diabetes mellitus, the systemic oxidative stress was increased.

Influence of rosuvastatin on the NAD(P)H oxidase activity in the retina and electroretinographic response of spontaneously hypertensive rats.

BACKGROUND AND PURPOSE: Retinal complications may be encountered during the development of hypertension as a response to oxidative stress. Statins may reduce the risk of developing hypertension and ocular diseases. We evaluate the effects of rosuvastatin (ROSU) on retinal functionality and oxidative stress levels in normotensive and spontaneously hypertensive rats (SHR). EXPERIMENTAL APPROACH: Wistar Kyoto (WKY) and SHR were treated for 3 weeks with rosuvastatin (10 mg kg(-1) day(-1)). Electroretinograms (ERG) were recorded before and after rosuvastatin treatment. Reactive oxygen species (ROS) were determined in the retina with dihydroethidium staining and NAD(P)H oxidase activity was evaluated. KEY RESULTS: Retinal ganglion cell ROS and retinal NAD(P)H oxidase activity were higher in SHR than in WKY rats, respectively (17.1+/-1.1 vs 10.2+/-1.2 AU, P<0.01; 38095+/-8900 vs 14081+/-5820 RLU mg(-1); P<0.05). The ERG b-wave amplitude in SHR was significantly lower than that in WKY rats. Rosuvastatin reduced SBP in SHR but did not change plasma lipid levels. Rosuvastatin treatment in SHR significantly decreased ROS levels (11.2+/-1.3, P<0.01), NAD(P)H activity in retinal ganglion cells (9889+/-4290; P<0.05), and increased retinal plasmalogen content in SHR, but did not modify the ERG response. CONCLUSIONS AND IMPLICATIONS: Rosuvastatin, beyond lowering cholesterol levels, was able to lower ROS in the retina induced by hypertension, but without improving retinal function in SHR. These findings point to a complex relationship between ROS in the pathogenesis of retinal disease and hypertension.

Delayed 24 hours Nomega-nitro-L-arginine methyl ester injection induces pharmacological cardioprotection against reperfusion injury.

Previous studies indicate that adenosine supplementation or nitric oxide synthase (NOS) inhibition during reperfusion exert protective effects against myocardial ischemia-reperfusion (I/R) injury. We wanted to test the hypothesis that NOS inhibition before I/R also protects the myocardium against further injury and aimed to determine the involvement of adenosine receptors in a perfused rat heart model. Rats were injected with 10 mg/kg of L-NAME (N(omega)-nitro-L-arginine methyl ester) or L-NAME + SPT (8-(p-sulfophenyl)-theophylline)--an adenosine antagonist - at 2 x 25 mg/kg or with a saline buffer, 24 hrs prior to heart excision. The hearts, perfused retrogradely were subjected to 60 min of global ischemia followed by 120 min reperfusion. L-NAME decreased NOx (nitrite and nitrate) production (16.2 +/- 3.2 vs. 7.0 +/- 1.8 micromol/L; P<0.05) in vivo and increased the release of troponin I (0.04 +/- 0.01 vs. 0.02 +/- 0.01 microg/L; P<0.05) in the plasma, compared to controls. After 120 min of reperfusion, there was a higher release of adenosine (26.1 +/- 2.2 vs. 2.4 +/- 1.2 nmol/min; P<0.01) and a decrease in troponin I levels (0.19 +/-0.07 vs. 0.59 +/- 0.16 ng/min; P<0.05) in the L-NAME group compared to controls. These results were accompanied by a higher proportion of recovery of left ventricular developed pressure (72.0 +/- 4.0 vs. 60.0 +/- 4.0%; P<0.05) and coronary flow (72.0 +/- 5.0 vs. 51.0 +/- 4.0%; P<0.05) in the L-NAME group. These beneficial effects were not blocked by the adenosine receptor antagonist. The present study reveals that L-NAME protects against I/R injury when the inhibitor is administered 24 hrs before ischemia. The beneficial effects observed in this model appear to be independent of adenosine receptor stimulation.

[Preventing the cardiotoxic effects of anthracyclins. From basic concepts to clinical data]. / Prévention de la cardiotoxicité des anthracyclines: approche fondamentale des mécanismes mis en jeu; relations avec les données cliniques.

The chronic cardiotoxicity of the cytotoxic agents such as anthracyclines is one of the main factors, which limits their prolonged use. Clinically, this cardiotoxicity results in a cardiomyopathy with irreversible congestive heart failure, with high mortality. The molecular mechanisms, which could explain this cardiac toxicity, are complex but it seems distinct from the anticancer mechanism. Several hypotheses were advanced but it appears that the induction of an oxidative stress within myocardial tissue constitutes the common denominator. The prevention of this cardiotoxicity lies on:--a rigorous cardiac monitoring--the use of anthracyclines analogues with lower cardiotoxicity,--modifications of the protocols of administration. The myocardial protection, with cardioprotective agents targeting oxidative stress during chemotherapy would be of great interest for an optimal use of the anthracyclines.

In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative.

N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]- phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) has been selected from a chemical program aimed at identifying an optimized inhibitor of multidrug resistance (MDR). The potency of GF120918 is assessed by dose-dependent sensitization of CHRC5, OV1/DXR and MCF7/ADR cells to the cytotoxicity of doxorubicin and vincristine respectively: GF120918 fully reverses multidrug resistance at 0.05 to 0.1 microM and is half maximally active at 0.02 microM. The spectrum of drugs sensitized by GF120918 coincides with those having the classical MDR phenotype. In CHRC5 cells, 0.01-0.1 microM GF120918 enhances the uptake of [3H]daunorubicin and blocks the efflux from preloaded cells. It is also shown that GF120918 is still active several hours after being taken away from the culture medium showing that it is not, like verapamil, effluxed rapidly by P-glycoprotein. GF120918 effectively competes with [3H]azidopine for binding P-glycoprotein, pointing to this transport membrane protein as its likely site of action. After i.v. administration to mice, GF120918 penetrates thoroughly various organs that have a tissue level/blood level ratio above 10. It is eliminated from organs and blood with a half-time of approximately 2.7 h. It is well absorbed after p.o. administration. In mice implanted i.p. with the MDR P388/Dox tumor, a single i.v. or p.o. dose of GF120918 restores sensitivity of the tumor to a single i.p. dose (5 mg/kg) of doxorubicin administered 1 h later. A statistically significant effect is observed at 1 mg/kg GF120918 i.v. and maximal effect is reached at 5 mg/kg. Similarly, whereas neither drug alone is effective, GF120918 (10 mg/kg i.p.) associated with doxorubicin (5 mg/kg i.p.) inhibits the growth of the moderately MDR C26 tumor implanted s.c. as assessed by tumor size at day 19. GF120918 does not modify significantly the distribution or the elimination of doxorubicin in mice ruling out the possibility that the antitumor effects seen might be explained by pharmacokinetic interactions.

[A treatment with rosuvastatin induced a reduction of arterial pressure and a decrease of oxidative stress in spontaneously hypertensive rats]. / Un traitement par la rosuvastatine induit une réduction de la pression artérielle et une diminution du niveau du stress oxydatif chez le rat spontanément hypertendu.

The aim of this study was to appreciate consequences of rosuvastatin administration on hemodynamic function, vascular oxidative stress and ischemia/reperfusion disorders in normotensive and hypertensive rats. At 10 weeks of age, spontaneously hypertensive rats (SHR, n=20) and normotensive Wistar Kyoto male rats (WKY, n=20) were divided into four groups and given, either vehicle or 10 mg/kg/day of rosuvastatin by gavage for 3 weeks. Systolic blood pressure was assessed every week. At the end of these treatments, vascular NADPH oxidase activity was evaluated by chemiluminescence (lucigenin 0.5 microM). Hearts were isolated and perfused according to the Langendorff method and were subjected to 30 min of global ischemia. Reactive oxygen species (ROS) produced during reperfusion were quantified by electron spin resonance (ESR) spectroscopy using a spin probe (CP-H, 1 mM). After one week of treatment, rosuvastatin reduced the arterial pressure in SHR rats (180.3 +/- 2.1, SHR vs 169.7 +/- 2.3 mmHg, SHR+rosuvastatin; p < 0.01), without lowering plasma cholesterol levels; these effects were not observed in WKY. NADPH activity was 25% higher in control SHR rat aortas compared to control WKY, and was reduced by rosuvastatin in SHR rats. In isolated rat hearts subjected to ischemia/reperfusion sequences, there was a deterioration in functional parameters in control SHR compared to control WKY hearts. Rosuvastatin decreased post-ischemic contracture in WKY hearts by 50% (41.5 +/- 7.5, WKY control vs 18.4 +/- 4.6 mmHg, WKY+rosuvastatin; p < 0.01) and increased left ventricular developed pressure. This beneficial effect was accompanied by a decrease in ROS detected by ESR during reperfusion (312.5 +/- 45.3, WKY control; vs 219.3 +/- 22.9 AUC/mL, WKY+rosuvastatin; p < 0.05). In conclusion, these results are in accordance with the hypothesis that oxidative stress plays a crucial role in the pathogenesis of cardiovascular diseases including hypertension, and demonstrate the beneficial effects of rosuvastatin.

Alteration in plasma antioxidant capacities in chronic renal failure and hemodialysis patients: a possible explanation for the increased cardiovascular risk in these patients.

OBJECTIVE: The high incidence of cardiovascular diseases in chronic renal failure (CRF) and hemodialyzed (HD) patients is now well established and the involvement of oxidative stress has been hypothesized in these phenomena. The aim of our study was to evaluate the level of oxidative stress in healthy controls (CTL) compared with CRF and HD patients before (pre-HD) and after (post-HD) the dialysis session, carried out on a high biocompatible polyacrylonitrile membrane AN69. METHODS: Several indicators of the extracellular redox status were evaluated in plasma. The ascorbyl free radical (AFR) was directly measured using electron spin resonance spectroscopy (ESR) and expressed with respect to the vitamin C level to obtain a direct index of oxidative stress. Indirect plasma parameters such as vitamin E, thiol and uric acid levels were also quantified. The plasma antioxidant status (PAS) was evaluated by the allophycocyanin test. Nitric oxide (NO) stable-end metabolites: nitrites and nitrates (NO(x)), were measured in plasma. RESULTS: In CRF patients, vitamin C and thiol levels were low, and the AFR/vitamin C ratio high compared with the CTL. On the other hand, PAS and uric acid levels were shown to be higher in CRF patients. After the dialysis session, vitamin C level decreased and AFR/vitamin C ratio increased. The thiol levels were shown to be increased, in return PAS and uric acid levels were significantly lower after the dialysis session. NO(x) levels rose during CRF, but were significantly decreased after the dialysis procedure. No differences in vitamin E status were observed between CTL, CRF and HD patients. CONCLUSION: Our study demonstrates that profound disturbances in the extracellular redox system occur during the course of chronic renal failure and hemodialysis, and may provide an explanation for the cardiovascular complications in these patients.

Postischemic myocardial recovery and oxidative stress status of vitamin C deficient rat hearts.

OBJECTIVE: To investigate the role of vitamin C tissue content as a protective agent during myocardial ischemia-reperfusion injury, we have evaluated the postischemic functional recovery and free radical release of osteogenic disorder Shionogi (ODS) inherently scorbutic rat hearts and compared them to healthy Wistar rat hearts. METHODS: Isolated perfused hearts of ODS or Wistar rats underwent 30 min of a global total normothermic ischemia followed by 30 min of reperfusion. The lipid-soluble spin trap alpha-phenyl N-tert-butylnitrone (3 mM) was perfused upstream of the coronary bed. Functional parameters were recorded and samples of coronary effluents were analysed using electron spin resonance spectroscopy to characterise and quantify the amount of radical species released. RESULTS: From the onset of reperfusion, a large and long-lasting release of alkyl/alkoxyl radicals was detected, with a peak value of 29.0+/-3.2 nM obtained after 13 min, which was associated with a persistent contractile dysfunction. However, ODS rat hearts showed a higher myocardial recovery with lower left ventricular end diastolic pressure (44.34+/-1.74 vs. 55.03+/-1.57 mmHg for Wistar), higher recovery of rate pressure product (12.3+/-1.4 vs. 1.9+/-1.7x10(3) mmHg beats/min for Wistar) and shorter duration of contractile abnormalities during reperfusion (3.7+/-1.0 vs. 20.8+/-5.3 min for Wistar). Moreover, free radical release was identical in ODS rat hearts as compared to control Wistar rats. Ascorbic acid tissue content was significantly altered in ODS rats (31.9+/-3.3 vs. 591.0+/-54.9 mmol/g of tissue for Wistar) but superoxide dismutases, glutathion peroxidases and inducible heat shock protein 70 genes were up-regulated. CONCLUSIONS: This study shows that ascorbic-acid-deficient ODS rat hearts are more resistant to an ischemic insult than control Wistar rats, probably through the development of alternative protective defences, like the induction of heat shock proteins. These paradoxical results raise the question of the relative importance of each endogenous antioxidant in the cardiac resistance to ischemia-reperfusion injury.

Influence of the severity of myocardial ischemia on the intensity of ascorbyl free radical release and on postischemic recovery during reperfusion.

Ascorbyl free radical (AFR), can be considered as an atoxic and endogenous indicator of oxidative stress. The purpose of our experiments was to investigate the influence of the severity and length of ischemia on the extent of AFR release during myocardial ischemia and reperfusion. For that purpose, isolated perfused rat hearts were submitted to a global ischemia, either total (residual flow 0%) or low flow (residual flow 5%), of 20 or 60 min length. Coronary effluents were collected at different times of experimentation and analyzed with Electron Spin Resonance (ESR) spectroscopy. AFR ESR doublet (g = 2.0054, aH = 0.188 MT) was not detected in coronary effluents collected during control perfusion periods. Nevertheless, during low-flow ischemia, a weak AFR release was noted. Moreover, a sudden and massive AFR liberation was observed at the time of reperfusion: this AFR release was weaker after low-flow ischemia than after total ischemia and was enhanced when the duration of ischemia increased from 20 min to 60 min. The large liberation of AFR noticed during global total ischemia was associated with a greater depression in myocardial contractile function and a lower recovery in coronary flow. In conclusion, our study demonstrates that AFR production at the time of reperfusion depends on the duration and strength of the ischemia, and is related to free radical injury. According to previously described ascorbate/AFR properties, we can conclude that AFR liberation in coronary effluents could represent a marker of oxidative stress during ischemia and/or reperfusion of hearts. This AFR release could be considered a sign of the severity of the ischemic episode, and could be related to the functional impairment during reperfusion.

In vitro free radical scavenging capacity of thyroid hormones and structural analogues.

It was reported that thyroid hormones decreased Cu(2+)-induced low-density lipoprotein (LDL) oxidation in vitro. Here, we investigated free radical scavenging capacities of thyroid hormones (3,5,3'-tri-iodo-L-thyronine (T(3)), thyroxine (T(4)) and 3,3',5'-tri-iodo-L-thyronine (rT(3))) and structural analogues (L-thyronine (T(0)), 3,5,3'tri-iodothyroacetic acid (TA(3)) and 3,5,3',5'-tetra-iodothyroacetic acid (TA(4))), using three different models of free radical generation. T(0), T(3) and TA(3) slowed down production of conjugated diene and thiobarbituric acid-reactive substances during LDL oxidation by 2,2'-azobis-[2-amidinopropane] (water-soluble), whereas rT(3), T(4) and TA(4) had practically no effect. In this system, T(0) was the more active compound. Using a 1,1-diphenyl-2-picrylhydrazyl (lipid-soluble) test, all compounds also revealed free radical scavenging capacities, but rT(3), T(4) and TA(4) were more active than T(0), T(3) and TA(3). T(3) was able to scavenge superoxide anion and hydroxyl radicals generated in an aqueous phase by a xanthine-xanthine oxidase system, as measured by electron paramagnetic resonance spectroscopy. It may be concluded that: (1) thyroid hormones and analogues with a 4'-hydroxy diphenylether structure have free radical scavenging capacities, (2) this property is influenced by the number of iodines on the phenolic ring, and (3) thyroid hormone scavenging capacity should not be the only mechanism explaining their protective effect on Cu(2+)-induced LDL oxidation. The physiological significance of the findings is discussed.

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: cardiovascular effects of the spin trap alpha-phenyl N-tert-butylnitrone.

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: alpha-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.

In vivo and in vitro antioxidant properties of furosemide.

The aim of this study was to investigate in vivo and in vitro antioxidant properties of furosemide. In vitro, human red blood cells were submitted to oxidative stress (AAPH), in absence or in presence of different concentrations of furosemide. Potassium efflux was measured in order to quantify the oxidative stress after the action of AAPH on red blood cells. Allophycocyanin assay was also used to investigate antioxidant capacities of furosemide. For the in vivo experiment, male Wistar rats were used. A control group (n = 5) was treated by a daily intraperitoneal injection of saline solution (0.2 ml); 2 other groups (J0 and J+) were treated for 7 days by one daily intraperitoneal injection of furosemide (0.10 mg/kg/day). In the J+group, the injection of furosemide was done one hour before the experiment, while in the J0 group the last injection of furosemide was done on the 6th day and an injection of saline was performed one hour before the experiment. On the day of experiment, a laparotomy was performed under general anesthesia and blood was collected from abdominal aorta. Oxidative stress and antioxidant capacities were evaluated on Wistar rat red blood cells and plasma. In vitro results (oxidative challenge with AAPH) showed that oxidative stress was decreased in presence of furosemide. This was due to a potent free radical scavenging effect of furosemide. In vivo studies confirmed that furosemide had antioxidant properties. These data may be of great relevance in clinical practice, considering the use of large doses of furosemide in patients presenting pathology involving the production of free radicals.

Studies by electron paramagnetic resonance of the importance of iron in the hydroxyl scavenging properties of ascorbic acid in plasma: effects of iron chelators.

Ascorbic acid is considered to be the most important antioxidant of plasma. Its oxidation leads to the ascorbyl free radical (AFR), detected by electron paramagnetic resonance (EPR) spectroscopy. The purpose of this study was to investigate by EPR the interaction of plasma AFR levels in different situations of oxidative stress. Our results showed that plasma AFR remains constant after rat feeding with vitamin C (5 mg or 50 mg per 100 g body weight). We also demonstrated that: (1) the ascorbyl free radical (AFR) level was increased after direct addition of iron Fe3+/EDTA to plasma, the optimal level was reached after addition of 8 microM Fe3+/EDTA (1:2); (2) this AFR production was associated with the formation of hydroxyl radicals. Iron chelators (deferrioxamine, a synthetic iron chelator and apotransferrine, a biological iron chelator) added just before the Fe3+/EDTA complex inhibited the increase of AFR signal induced by this complex. The scavenging effect of plasma was significantly correlated with the AFR production. Therefore, AFR, which is naturally present in plasma, could be used as an index of oxidative stress in which free radicals or adverse iron mobilisation are implicated.

Vitamin E analogues reduce the incidence of ventricular fibrillations and scavenge free radicals.

The aim of our study was to analyse the protective effects of different alpha-tocopherol analogues 1) against fibrillations induced by an ischemia-reperfusion sequence, and 2) to further investigate in vitro the radical scavenging properties of these analogues by two sensitive methods. Concerning 1: isolated rat hearts underwent 10 min of coronary ligation followed by reperfusion and the alpha-tocopherol analogues were infused 15 min before occlusion. Functional parameters including heart rate and fibrillations were recorded. Concerning 2: the beta-phycoerythrin assay was utilised to determine the oxygen radical absorbing capacity (ORAC) of these vitamin E analogues against peroxyl radicals. Electron paramagnetic resonance (EPR) was used to measure their scavenger abilities on hydroxyl radical and superoxide anion production. Concerning 1: ventricular fibrillation times were reduced for all analogues treated hearts at concentrations of 1 microM and 5 microM, with Trolox being the most efficacious. Concerning 2: in our experimental conditions of intense production of free radicals, scavenging IC50 values for hydroxyl radical were 1.15, 2.17 and 4.04 mM for Trolox, MDL 74270 and MDL 74366 respectively. Superoxide anion IC50 values were 1.0 and 6.75 mM for Trolox and MDL 74270. Our results show that water-soluble analogues of vitamin E are effective in the prevention of coronary ligation induced reperfusion arrhythmia, under our experimental conditions. Moreover, our data demonstrate that these vitamin E analogues are effective scavengers for a variety of radicals. Our studies support the view that compounds that can either inhibit the formation or scavenge free radicals can protect the heart against arrhythmia associated with ischemia-reperfusion.