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The chain-breaking antioxidant potential of caeruloplasmin and bovine serum albumin (BSA) has been investigated in comparison with other well-established antioxidants. Their Oxygen Radical Absorbing Capacity (ORAC), was measured by using beta-phycocyanin (beta-PC) as a fluorescent indicator protein, 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) as a peroxyl radical generator and the water soluble vitamin E analogue, Trolox, as a reference standard. The relative peroxyl absorbing capacities/mole for Trolox, caeruloplasmin, heat-denatured caeruloplasmin (hCP), catalase, bovine serum albumin (BSA), superoxide dismutase (SOD), and deferoxamine were 1; 2.6; 3.3; 3.7; 1.2; 0.1; 0.2, respectively. Caeruloplasmin was far more effective as a peroxyl radical scavenger than SOD, deferoxamine and BSA, but slightly less effective than catalase. The peroxyl radical absorbing capacity of caeruloplasmin was enhanced by heat-denaturation of the protein. Electron paramagnetic resonance (EPR) spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trap, was applied in order to measure the scavenger abilities of caeruloplasmin on superoxide radical and hydroxyl radical production and the concentration required to inhibit by 50% oxygen free radical formation (IC50) was determined. The IC50 values of caeruloplasmin, hCP, and BSA for the superoxide radical were 12, 2, 260 microM and for the hydroxyl radical 15, 2, 200 microM. These results show that caeruloplasmin is an effective chain-breaking antioxidant for a variety of radicals, independently of its catalytic ferroxidase activity.

Nitric oxide (NO) has been shown to inhibit the proliferation of lymphocytes. However, in tumour-bearing rats treated with the immunomodulator OM 163, the regressing nodules were heavily infiltrated by T lymphocytes, although they contained high levels of NO. We show here that NO, while inhibiting the proliferation of lymphocytes, increased their life-span, pointing to the ambivalence of this molecule in the course of tumour growth and regression.

The aim of these experiments was to investigate the radical scavenging properties of three diuretics: indapamide (IND) and its major metabolite, 5-OH indapamide (5-OH IND), compared to a reference diuretic, hydrochlorothiazide (HTZ). Electron Paramagnetic Resonance (EPR) was used to determine the scavenging abilities of these compounds on enzymatically produced superoxide radical anion, with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) used as a spin-trap. These experiments revealed that IND and specially 5-OH IND were effective superoxide radical anion scavengers at 0.2 mg/ml. In the second part of these studies, allophycocyanin was used as an indicator of free radical mediated protein damage. In the assay, 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) was used as a peroxyl radical generator, Trolox (a water-soluble analogue of vitamin E) as a control standard, and the loss of allophycocyanin fluorescence was monitored. The antioxidant effects of the diuretics were expressed in oxygen-radical absorbing capacity (ORAC), where one ORAC unit equals the net protection produced by 1 microM Trolox. HTZ showed no protection up to 100 microM final concentration, whereas IND and 5-OH IND showed linear correlation with respect to concentration when expressed in ORAC units: 5-OH IND induced the highest protection against peroxyl radical. The above observations suggested that IND and 5-OH IND are potent radical scavengers, with the metabolite 5-OH IND having a superior antioxidant potency than IND. By contrast, HTZ had no effect. These radical scavenging properties of 5-OH IND may be of clinical interest for vascular protection and may help to protect the heart from oxidative injury.

Nitric oxide (NO.) is a free radical characterized by a high spontaneous chemical reactivity with many other molecules including the superoxide radical (O2.-). This complex interaction may generate a peroxynitrite anion (ONOO-), which behaves as an important mediator of oxidative stress in many pathological states. In the present study, in vitro experiments were performed to assess directly the O2.- and hydroxyl (.OH) radical scavenging effects of various NO. donor drugs, i.e. sodium nitroprusside (SNP), sodium nitrite (NaNO2), molsidomine and SIN 1, at pH 7.4, 7 or 6. Concentrations of NO. in the incubation medium containing the different NO. donor drugs were measured by the assay based on the reaction of Fe-N-methyl-D-glucamine dithiocarbamate (MGD) with NO. that yields a stable spin-adduct measured by electron paramagnetic resonance (EPR). O2.- and .OH generation was characterized by EPR spin trapping techniques, using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). These free radicals were generated from the enzymatic system xanthine-xanthine oxidase, in phosphate buffer adjusted at pH 7.4, 7 and 6. Under these experimental conditions, SNP exhibited the strongest superoxide scavenging properties, characterized by IC50 values expressed in the micromolar range, which decreased at low pH. Addition of SNP (800 microM) to solution containing MGD and Fe2+ (5:1) at pH 7 4 produced a three line EPR spectrum which is identified to [(MGD)2-Fe2+-NO]. In control experiments no EPR signal was observed. We obtained the same results with NaNO2 and an augmentation of the spin-adduct level was noted with the prolongation of the incubation period. In return, molsidomine (2 mM) did not produce, in our conditions, a detectable production of NO.. NaNO2 displayed a significant superoxide scavenging effect only at pH 6, whilst neither molsidomine nor SIN 1 had any effect. Therefore, the superoxide scavenging properties of SNP, NaNO2, and molsidomine appeared to be closely related to their potential for NO release, which partially depends on the pH conditions. The behaviour of SIN 1 is more complicated, the speed of oxygen diffusion probably acting as a limiting factor in NO. formation in our conditions. The production of NO. was detected in presence of SIN 1. The intensity of the complex is comparable with the signal founded with NaNO2. By contrast, all molecules exhibited hydroxyl radical scavenging properties, highlighting the capacity of .OH to react with a wide range of molecules. In conclusion, considering the poor chemical reactivity of O2.-, the NO. donor drugs/O2.- interactions suggest a special relationship between these two radical species, which, in certain pathological states, could lead to the generation of cytotoxic end-products with strong oxidizing properties.

Post-ischemic reperfusion phenomenon has been studied in two methods of myocardial protection: a crystalloid cardioplegia (St Thomas no.2) and a cold blood cardioplegia (Buckberg) during cardiopulmonary bypass for myocardial revascularisation in patients. Myocardial protection has been assessed from the evolution of hemodynamic parameters, reperfusion arrhythmias and biochemical analysis of the coronary flow after cross-clamp removal: creatinine phosphokinase (CPK_MB) and nucleotide adenine metabolites (adenosine, inosine, hypoxanthine, xanthine and uric acid). The study was performed in two groups of 14 patients. Hemodynamic conditions were similar in both groups during reperfusion in order to avoid different coronary flow. In those conditions, myocardial protection by cold blood cardioplegia reduced reperfusion arrhythmias, and resulted in a loss of CPK-MB release. Furthermore, the reduction of metabolites release, purine bases and oxypurine bases into coronary sinus after cold blood cardioplegia suggest a better protection of myocardial high energy phosphates in this group than after crystalloid cardioplegia. Our results also show that hypoxanthine is probably the final product of ATP degradation in human myocardial tissue.

Post-ischemic reperfusion phenomena were studied in two methods of myocardial protection: crystalloid cardioplegia (St Thomas n(o) 2) and cold blood cardioplegia (Buckherg) during cardiopulmonary bypass for human myocardial revascularisation. Myocardial protection was assessed on the course of hemodynamic parameters, reperfusion arrhythmias and biochemical analysis of the coronary flow after cross-clamp removal: creatine phosphokinase (CPK-MB) and nucleotide adenine metabolites (adenosine, inosine, hypoxanthine, xanthine and uric acid). The study was performed in two groups of 14 patients. Hemodynamic conditions were similar in both groups during reperfusion in order to avoid different coronary flow. Under these conditions, myocardial protection by cold blood cardioplegia reduced reperfusion arrhythmias, and resulted in a loss of CPK-MB release. Furthermore, cold blood cardioplegia provided protection of myocardial energy metabolism by reducing the loss of metabolites, purine bases and oxypurine bases into the coronary sinus. Our results also show that hypoxanthine is probably the final product of ATP degradation in human myocardial tissue.

During radiotherapy of thoracic tumors, the heart is often included in the primary treatment volume, and chronic impairment of myocardial function occurs. The cellular biomolecules are altered directly by radiation or damaged indirectly by free radical production. The purpose of this investigation was to evaluate the biochemical and functional responses of the rat heart to a single high dose of radiation. The effect of 20 Gy local X irradiation was determined in the heart of Wistar rats under general anesthesia. Mechanical performances were measured in vitro using an isolated perfused working heart model, and cardiac antioxidant defenses were also evaluated. Hearts were studied at 1 and 4 months after irradiation. This single dose of radiation induced a marked drop in the mechanical activity of the rat heart: aortic output was significantly reduced (18% less than control values) at 1 month postirradiation and remained depressed for the rest of the experimental period (21% less than control 4 months after treatment). This suggests the development of myocardial failure after irradiation. The decline of functional parameters was associated with changes in antioxidant defenses. The decrease in cardiac levels of vitamin E (-30%) was associated with an increase in the levels of Mn-SOD and glutathione peroxidase (+45.5% and +32%, respectively, at 4 months postirradiation). However, cardiac vitamin C and catalase levels remained constant. Since these antioxidant defenses were activated relatively long after irradiation, it is suggested that this was probably due to the production of free radical species associated with the development of inflammation.

Purpose: To determine the variations of vitamin E in rat retinas after transient ischemia and reperfusion., Methods: Male Wistar rats 11 to 12 weeks of age were used in all experiments. Ischemia was induced by ligation of the optic nerve and vessels, then reperfusion was obtained by removing the ligature. Retinas were dissected, and vitamin E was analyzed by a modified high-performance liquid chromatography assay., Results: There was an insignificant rise in vitamin E during ischemia and a dramatic decrease after reperfusion. No changes were dependent on the period of ischemia (30, 60, or 90 minutes) or reperfusion (15 or 120 minutes)., Conclusion: Vitamin E decreased only in the reperfused groups, demonstrating a depletion of defenses against free radical production occurring during reperfusion. Vitamin E changes may represent an indirect measure of lipoperoxidation during ischemia-reperfusion and, thus, a potential parameter to evaluate drugs in vitro.

The purpose of this study was to evaluate changes in the proton nuclear magnetic resonance relaxation times (T1 and T2) after chronic infarction in the rat. Ligation of the left coronary artery was followed by various degrees of reduction in myocardial blood flow. The ligation induced infarction in the left ventricle and compensatory hypertrophy in the right ventricle, as evaluated by the ratio of right ventricle to body weight. The interventricular septum and the right ventricle did not become ischemic in this model and served as control areas. In the infarcted left ventricle our results showed an increase in the T1 and T2 relaxation times after 15 and 30 days of ligation and a slight decrease after 60 days. A similar change in the T1 values was observed in the right ventricle. In contrast, a persistent increase in the T2 relaxation times was observed in the right ventricle and correlated with the ratio of right ventricle to body weight (r = 0.54, p < 0.01). The observation that the magnetic resonance relaxation times in vitro are modified in the hypertrophic right ventricle after myocardial infarction could be important in interpreting magnetic resonance imaging in vivo. There was no relation between the changes in the relaxation times and the degree of myocardial ischemia.

It is well established that early reperfusion of ischemic myocardium limits or even prevents necrosis. However, sometimes restitution of coronary flow to the ischemic myocardium may precipitate a sequence of events that would have occurred either later or not at all. To prove the existence of reperfusion-induced injury, it is necessary to show that cells, potentially viable before reperfusion, are killed by the onset of reperfusion. The consequences of reperfusion are reperfusion-induced arrhythmias, myocardial stunning, accelerated necrosis and lethal reperfusion injury. The two main theories explaining the development of reperfusion injury are formation of free radicals and cellular calcium overload. Free radicals produced during early reperfusion may cause oxidant stress to membrane lipids and proteins associated with a potassium loss and a temporary cytosolic calcium overload. Results of experiments with free radical scavengers are contradictory for unknown reasons suggesting that reperfusion injury is a complex event innowing endothelial damage, leukocyte-plugged vessels, microvascular damage and production of heat-shock proteins.

Three groups of sixteen male rats each were fed semi-purified diets containing 15% by weight of lipid for a period of 4 wk. The diets contained the same amount of polyunsaturated fatty acids (PUFA) (20% of total fatty acids) and saturated fatty acids (19% of total fatty acids). Dietary PUFA were represented exclusively by linoleic acid (18:2 diet), or 10% linoleic acid and 10% linolenic acid (18:3 diet), or 10% linoleic acid and 10% long-chain n-3 fatty acids (LCn-3 diet). The overall amount of vitamin E was similar in the three diets, i.e., 140, 133 and 129 mg/kg diet, respectively. Following appropriate extraction, tocopherol levels in heart, liver, brain, adipose tissue (AT) and plasma were measured by high-performance liquid chromatography. The level of vitamin E in the heart decreased with n-3 PUFA diets, most markedly with LCn-3 PUFA. Liver and AT vitamin E contents also decreased with n-3 PUFA diets when expressed as micrograms/mg total lipids and micrograms/mg phospholipids, respectively. Total plasma vitamin E was lower in rats fed the LCn-3 diet, but there was no significant difference when expressed as microgram/mg total lipids. Brain vitamin E was not affected by the various diets. In vitro cardiac lipid peroxidation was quantified by the thiobarbituric acid reactive substances (TBARS) test. Heart homogenates were incubated at 37 degrees C for 15 and 30 min in both the absence (uninduced) or presence (induced) of a free radical generating system (1 mM xanthine, 0.1 IU per mL xanthine oxidase, 0.2 mM/0.4 mM Fe/ethylenediaminetetraacetic acid). TBARS release was time-independent but significantly higher when LCn-3 fatty acids were fed to rats in either the uninduced or induced system. The study demonstrated that n-3 PUFA diets can influence vitamin E status of rats even in short-term experiments and can change the susceptibility of the heart to in vitro lipid peroxidation.

The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because of the relative resistance of this tissue to normothermic ischemia. Under normal conditions small quantities of oxygen free radicals are produced but they are quenched by intracellular free radical scavenging enzymes (superoxide dismutase, catalase and glutathione peroxidase) or alpha-tocopherol. The increase in malondialdehyde suggests increased lipid peroxidation initiated by free radical reactions. Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. The results of recent studies indicate that: a) oxygen free-radicals mediates, at least in part, the increased microvascular permeability produced by reoxygenation, b) free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged ischemia. Additional evidence support the hypothesis of the interrelationship between ischemic tissue and inflammatory cells. So capillary plugging by granulocytes and oxygen free radical formation may contribute to the ischemic injury.

Free radicals have been implicated in several aspects of cellular injury, both during ischemia and reperfusion of the myocardium. In this study, formation of free radicals in the isolated rat heart was measured a) directly using electron paramagnetic resonance (EPR) spectroscopy and b) indirectly using the generation of thiobarbituric acid reactants as an index of lipid peroxidation. EPR spectra of frozen heart powder recorded at 100 degrees K show several lines and consist of different components separated by temperature studies: signal C disappears after warming the sample 1 minute at 190 degrees K and is suggestive of a triplet signal g = 2.001, aN = 25 Gauss; signal B g parallel = 2.034, g perpendicular = 2.007, disappears after 1 min at 240 degrees K, and is similar to those previously reported for oxygen alkylperoxyl free radical; the remaining signal, signal A with g = 2.004 is identical to that of a carbon-centered ubiquinone free radical. The total free radical concentration in isolated rat heart perfused at a constant flow rate of 12 ml/min was increased by 44% compared with control (p less than 0.05) after 10 minutes of normothermic global ischemia with a 10% residual flow, and by only 31% compared with control after 20 seconds of reflow with oxygenated perfusate (p less than 0.05). Compared with the reperfused group, trimetazidine 10(-5) M administered 15 minutes before the ischemic period decreased the free radical concentration (-20%). However, this free radical generation in heart was not associated with a concomitant increase of lipid peroxides.

Free radical species have been implicated as important agents involved in myocardial ischemic and reperfusion injuries. In our study, formation of free radicals was measured directly with electron paramagnetic resonance spectroscopy before ischemia, during 10 minutes of global ischemia, and 20 seconds after reperfusion in the rat heart. We also investigated the formation of thiobarbituric acid-reactive material as index of lipoperoxidation induced by free radicals and measured arrhythmias. Production of free radicals takes place during ischemia since the signal intensity with a g value of 2.004 attributed to free radical species was increased by 50% after 10 minutes of global ischemia. In hearts reperfused with oxygenated perfusate for 20 seconds, the signal doubled. These experiments supply evidence that free radicals are generated in isolated rat heart during a short period of global ischemia and reperfusion. However, this increase was not associated with a concomitant increase of lipid peroxides in the myocardium nor with the development of reperfusion arrhythmias.