Scavengers Of Oxygen-derived Free Radicals Research Articles

The Pleiotropic Effects of Oxygen-Derived Free-Radical Scavengers on the Graft During Normothermic Ex Situ Heart Perfusion

<h3>Purpose</h3> The management of oxidative stress is pivotal during ESHP. The lack of sufficient antioxidant systems allow the accumulation of reactive oxygen species (ROS) which can negatively affect myocardial function. We aim to evaluate the use of oxygen-derived free-radical (OFR) scavengers on cardiac functional preservation during ESHP. <h3>Methods</h3> Using a porcine heart perfusion model, we compared the effect of different OFR scavengers. Hearts from juvenile Yorkshire pigs were perfused in working mode for 6 hours either in the control group (n=6) or adding polyethylene glycol (PEG)-catalase (10,000U/L, n=6) and PEG-superoxide dismutase (SOD) (550U/L, n=6) at the initiation of perfusion. Another group (n=6) of freshly procured heart were obtained as tissue control. Contractile function, myocardial injury and endothelial integrity were evaluated. The oxidative modification and antioxidant activity were determined in the myocardium and coronary vasculature. <h3>Results</h3> Cardiac function was better preserved in the catalase group (p<0.05, Figure A). Catalase and SOD both decreased perfusate cTnI, indicating less myocardial injury. The catalase group had lower perfusate vWF than the other two groups, suggesting less endothelial injury (p<0.05, Figure B). Compared to SOD, catalase reduced ROS production and oxidatively modified protein, indicated by protein carbonyl content in the myocardium and nitrotyrosine in the coronary vasculature (p<0.05, Figure C). The catalase and glutathione peroxidase activity decreased in all groups. However, adding catalase partially restored enzyme activity (Figure D). <h3>Conclusion</h3> The antioxidant enzyme activity decreased during <i>ex situ</i> heart perfusion. Catalase better preserved cardiac function and coronary endothelium than SOD, likely resulting from distinct oxidative stress states in the graft. Catalase and SOD have opposing effects on hydrogen peroxide detoxification, which may explain the differences observed in this study.

Defective Ag-In-S/ZnS quantum dots: an oxygen-derived free radical scavenger for mitigating macrophage inflammation.

Oxidative stress plays an important role in the development of inflammatory diseases including allergy, heart disease, diabetes and cancer. Nanomaterial-mediated antioxidant therapy is regarded as a promising strategy to treat oxidative stress-mediated inflammation. Herein, defective Ag-In-S/ZnS quantum dots (AIS/ZnS QDs) with oxygen-derived radical-scavenging capabilities are developed. Owing to their intrinsic defects and abundant surface functional groups, these quantum dots exhibit excellent oxygen-derived free radical removal efficiency in vitro. In macrophages, AIS/ZnS QDs can eliminate intracellular excessive ROS stimulated by either H2O2 or lipopolysaccharide (LPS), thus can effectively protect macrophages against ROS-induced oxidative injury. Moreover, in the model of LPS-triggered macrophage inflammation, they exhibit benign anti-inflammatory ability by inhibiting the expression of related proinflammatory cytokines (e.g., TNF-α and IL-6). These findings indicate that AIS/ZnS QDs hold great potential for the treatment of ROS-related inflammatory disorders.

Rational Design and Synthesis of New, High Efficiency, Multipotent Schiff Base-1,2,4-triazole Antioxidants Bearing Butylated Hydroxytoluene Moieties.

A new series of multipotent antioxidants (MPAOs), namely Schiff base-1,2,4-triazoles attached to the oxygen-derived free radical scavenging moiety butylated hydroxytoluene (BHT) were designed and subsequently synthesized. The structure-activity relationship (SAR) of the designed antioxidants was established alongside the prediction of activity spectra for substances (PASS). The antioxidant activities of the synthesized compounds 4–10 were tested by the DPPH bioassay. The synthesized compounds 4–10 inhibited stable DPPH free radicals at a level that is 10−4 M more than the well-known standard antioxidant BHT. Compounds 8–10 with para-substituents were less active than compounds 4 and 5 with trimethoxy substituents compared to those with a second BHT moiety (compounds 6 and 7). With an IC50 of 46.13 ± 0.31 µM, compound 6 exhibited the most promising in vitro inhibition at 89%. Therefore, novel MPAOs containing active triazole rings, thioethers, Schiff bases, and BHT moieties are suggested as potential antioxidants for inhibiting oxidative stress processes and scavenging free radicals, hence, this combination of functions is anticipated to play a vital role in repairing cellular damage, preventing various human diseases and in medical therapeutic applications.

Pyridine induction of cytochrome P450 1A1, iNOS and metallothionein in Syrian hamsters and protective effects of silymarin

An in vivo assessment for the protective effects of silymarin for pyridine toxicity was investigated through cytochrome P450 isoform CYP1A1 and inducible nitric oxide synthase (iNOS) activity prevention. Moreover, the effect of pyridine-induced oxidative stress on metallothionein I-II (MT), a scavenger of oxygen-derived free radicals, was investigated. Forty Syrian hamsters were allocated into 4 groups. Syrian hamsters were dosed with pyridine (400 mg/kg) intraperitoneally with and without silymarin (200 mg/kg daily by gavage) for 4 days. Pyridine induced diffuse degeneration and necrosis of the proximal and distal renal tubular cells; cloudy swelling, necrosis and hepatocellular atypia of the liver; and degenerative changes in the myocardium. The degree of pathological alterations was less severe with simultaneous silymarin application. CYP1A1, iNOS and MT expression levels were elevated in liver, kidney and heart in response to acute pyridine toxicity. Silymarin application abolished or significantly suppressed the induction of CYP1A1, iNOS and MT expressions in liver, kidney and heart of the pyridine-treated Syrian hamsters. Enhanced synthesis of MT by pyridine possibly implies a purposive cellular response to prevent damage caused by oxygen radicals. However, silymarin significantly reduced the oxidative-stress-inducing effect of pyridine as reflected by decreased synthesis of MT. These results suggest that through oxidant generation, pyridine may cause alteration of the metabolic ways, including nitric oxide-mediated CYP1A1 activity.

Significance of ERK nitration in portal hypertensive gastropathy and its therapeutic implications

Portal hypertensive (PHT) gastric mucosa increases susceptibility to injury and delayed mucosal healing. It is possible that nitration of ERK by peroxynitrite might alter MAPK (ERK) signaling in PHT gastric mucosa, leading to delayed mucosal healing, since excessive nitric oxide production is implicated in PHT gastric mucosa and MAPK (ERK) signaling induces cell proliferation and leads to gastric mucosal healing in response to injury. Portal hypertension was produced by staged portal vein ligation, and sham-operation (SO) rats served as controls. Lipid peroxide (LPO) and nitrotyrosine increased significantly in PHT gastric mucosa compared with SO rats. ERK activation was impaired in PHT gastric mucosa in response to ethanol injury, whereas no significant difference in the phosphorylation of MEK, an upstream molecule of ERK, was seen between the two groups. The nitration of ERK by peroxynitrite, as detected by the coimmunoprecipitation of ERK and nitrotyrosine, was significantly enhanced in PHT gastric mucosa. Administration of rebamipide, a gastroprotective drug that acts as an oxygen-derived free radical scavenger, significantly decreased LPO and nitrotyrosine as well as the nitration of ERK by peroxynitrite in PHT gastric mucosa, therefore normalizing ERK activation and restoring the gastric mucosal healing response to ethanol injury. Enhanced nitration of ERK by peroxynitrite is involved in the impaired MAPK (ERK) signaling in PHT gastric mucosa. These findings demonstrate a new molecular mechanism in which PHT gastric mucosa is predisposed to injury and impaired healing.

Iodinated contrast induced renal vasoconstriction is due in part to the downregulation of renal cortical and medullary nitric oxide synthesis

The loss of renal function continues to be a frequent complication of the iodinated contrast agents used to perform diagnostic angiography and endovascular procedures. This study examined the hypothesis that contrast-induced renal injury is partly due to a decrease in cortical and medullary microvascular blood flow after the downregulation of endogenous renal cortical and medullary nitric oxide (NO) synthesis. Anesthetized male Sprague-Dawley rats (300 g) had microdialysis probes or laser Doppler fibers inserted into the renal cortex to a depth of 2 mm and into the renal medulla to a depth of 4 mm. Laser Doppler blood flow was continuously monitored, and the microdialysis probes were connected to a syringe pump and perfused in vivo at 3 muL/min with lactated Ringer's solution. Dialysate fluid was collected at time zero (basal) and 60 minutes after infusion of either saline or Conray 400 (6 mL/kg). Both groups were treated with saline carrier, N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, 30 mg/kg), L-arginine (400 mg/kg), or superoxide dismutase (10,000 U/kg), an oxygen-derived free radical scavenger. Dialysate was analyzed for total NO and eicosanoid synthesis. The renal cortex and medulla were analyzed for inducible NO synthase (iNOS), cyclooxygenase-2 (COX2), prostacyclin synthase, and prostaglandin E(2) (PGE(2)) synthase content by Western blot analysis. Conray caused a marked decrease in cortical and medullary blood flow with a concomitant decrease in endogenous cortical NO, PGE(2), and medullary NO synthesis. The addition of L-NAME to the Conray further decreased cortical and medullary blood flow and NO synthesis, which were restored toward control by L-arginine. Neither L-NAME nor L-arginine (added to the Conray) altered cortical or medullary eicosanoids release. Medullary PGE(2) synthesis decreased when superoxide dismutase was added to the Conray treatment, suggesting that oxygen-derived free radicals had a protective role in maintaining endogenous medullary PGE(2) synthesis after Conray treatment. Conray did not significantly alter iNOS, COX-2, prostacyclin synthase, or PGE(2) synthase content. These findings suggest that the downregulation of renal cortical and medullary NO synthesis contributes to the contrast-induced loss of renal cortical and medullary microvascular blood flow. Preservation of normal levels of renal cortical and medullary NO synthesis may help prevent or lessen contrast-induced renal vasoconstriction and lessen contrast-induced renal injury found after diagnostic and therapeutic endovascular procedures.

Oxygen-radical regulation of renal blood flow following suprarenal aortic clamping

Renal insufficiency continues to be complication that can affect patients after treatment for suprarenal aneurysms and renal artery occlusive disease. One proposed mechanism of renal injury after suprarenal aortic clamping (above the superior mesenteric artery) and reperfusion (SMA-SRACR) is the loss of microvascular renal blood flow with subsequent loss of renal function. This study examines the hypothesis that the loss of medullary and cortical microvascular blood flow following SMA-SRACR is due to oxygen-derived free radical down-regulation of endogenous medullary and cortical nitric oxide synthesis. Anesthetized male Sprague-Dawley rats (about 350 g) either had microdialysis probes or laser Doppler fibers inserted into the renal cortex (depth of 2 mm) and into the renal medulla (depth of 4 mm). Laser Doppler blood flow was continuously monitored. The microdialysis probes were connected to a syringe pump and perfused in vivo at 3 microL/min with lactated Ringer's solution. The animals were subjected to SMA-SRACR (or sham) for 30 minutes, followed by 60 minutes of reperfusion. Laser Doppler blood flow after the 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion was compared with the time zero (basal) and with the corresponding sham group and reported as percent change compared with the time zero baseline. The microdialysis fluid was collected at time zero (basal) and compared with the dialysis fluid collected after 30 minutes of SMA-SRACR followed by 60 minutes of reperfusion as well as the corresponding sham group. The microdialysis dialysate was analyzed for total nitric oxide (microM) and prostaglandin E2 (PGE2), 6-keto-PGF(1alpha) (PGI2 metabolite), and thromboxane B2 synthesis. The data are reported as percent change compared with the baseline time zero. The laser Doppler blood flow and microdialysis groups were treated with either saline carrier, N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME) (30 mg/kg, nitric oxide synthesis inhibitor), L-arginine (400 mg/kg, nitric oxide precursor), superoxide dismutase (SOD, 10,000 U/kg, oxygen-derived free radical scavenger), L-NAME + SOD, or L-arginine + SOD. SOD was given 30 minutes before the reperfusion, and the other drugs were given 15 minutes before reperfusion. The renal cortex and medulla were separated and analyzed for inducible nitric oxide synthase (iNOS), cyclooxygenase-2, prostacyclin synthase, and PGE2 synthase content by Western blot. Superior mesenteric artery-SRACR caused a marked decrease in medullary and cortical blood flow with a concomitant decrease in endogenous medullary and cortical nitric oxide synthesis. These changes were further accentuated by L-NAME treatment but restored toward sham levels by L-arginine treatment after SMA-SRACR. The kidney appeared to compensate for these changes by increasing cortical and medullary PGE2 synthesis and release. SOD treatment restored renal cortical and medullary nitric oxide synthesis and blood flow in the ischemia-reperfusion group and in the ischemia-reperfusion group treated with L-NAME. These data show that nitric oxide is important in maintaining renal cortical and medullary blood flow and nitric oxide synthesis. These data also support the hypothesis that the loss of medullary and cortical microvascular blood flow following SRACR is due in part to oxygen-derived free radical downregulation of endogenous medullary and cortical nitric oxide synthesis.

Effect of 5-aminosalicylic acid on radiation-induced micronuclei in mouse bone marrow

5-Aminosalicylic acid (5ASA), a prescribed drug for ulcerative colitis, is a potent scavenger of oxygen-derived free radicals. The present study was undertaken to ascertain its ability to protect against radiation-induced damage. The drug dose-dependent effect, optimum time of drug administration and radiation dose-dependent effect (0–4 Gy) on in vivo radiation protection against micronuclei induction in polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were studied in the bone marrow of mice. Intraperitoneal injection of 10–125 mg/kg of the drug 30 min before whole body irradiation with 3 Gy produced a significant reduction in the frequency of micronucleated erythrocytes at 24 h after exposure. The optimum dose for protection without drug toxicity was 25 mg/kg body weight. Injection of 25 mg/kg of the drug 60 or 30 min before or within 15 min after 3 Gy whole body γ-irradiation resulted in a significant decrease in the radiation-induced PCE and NCE with micronuclei (MPCE and MNCE) and an increase in the ratio of PCE to NCE (P/N), at 24 h post-irradiation. Maximum effect was seen when the drug was administered 30 min before irradiation. Therefore, to study the radiation dose–response, mice were pre-treated with 25 mg/kg of 5ASA 30 min before 1–4 Gy of γ-irradiation. Radiation increased the MN frequency linearly ( r 2=0.99) with dose. Pre-treatment with 5ASA significantly reduced the MN counts to 40–50% of the radiation (RT) alone values, giving a dose modification factor (DMF) of 2.02 (MPCE) and 2.53 (MNCE). Irradiation resulted in a dose-dependent decline in the P/N ratio at all the doses of radiation studied. 5ASA produced a significant increase in the P/N ratio from that of irradiated controls, at all doses of radiations tested. These results show that 5ASA protect mice against radiation-induced MN formation and mitotic arrest.

Allopurinol as Prophylaxis Against Pouchitis Following Ileal Pouch-Anal Anastomosis for Ulcerative Colitis: A Randomized Placebo-controlled Double-blind Study

Background: Pouchitis is the major long-term complication of restorative proctocolectomy for ulcerative colitis (UC). Allopurinol is a scavenger of oxygen-derived free radicals, which it is suggested play a role in the development of UC and pouchitis.The first aim was to test the hypothesis that the incidence of pouchitis can be reduced by prophylactic Allopurinol, and secondly to evaluate if Allopurinol influences the overall pouch function. Methods: 273 patients with UC who were planned for proctocolectomy and ileal pouch-anal anastomosis at 12 centres in Sweden between October 1994 and June 1997 were offered the opportunity to participate. 184 patients (67%) were randomized to receive postoperative prophylactic Allopurinol 100 mg twice daily or placebo. All 273 patients had clinical and endoscopic follow-up at 1, 3, 6, 12, 18, 24 months after surgery. Results: Of the 184 randomized patients, 94 were randomized to Allopurinol and 90 to placebo; 116 patients (63%) completed follow-up and the crude incidence of pouchitis among those patients fullfilling the protocol was 31% in the Allopurinol group and 28% in the placebo group (ns). The cumulative risk for a first attack of pouchitis was 30% and 26% after 24 months (ns). The overall pouch function improved over time and did not differ significantly between the two groups. Conclusions: Prophylactic Allopurinol did not reduce the risk of a first attack of pouchitis.

Antioxidant vitamins and enzymatic and synthetic oxygen-derived free radical scavengers in the prevention and treatment of cardiovascular disease.

Oxygen-derived free radical formation can lead to cellular injury and death. Under normal situations, the human body has a free radical scavenger system (catalase, superoxide dismutase) that can detoxify free radicals. Antioxidant vitamins and enzymatic and synthetic oxygen-derived free radical scavengers have been used clinically to prevent the formation of oxidized LDL and to prevent reperfusion injury, which is often caused by free radicals. In this article, the pathogenesis of free radical production and cell injury are discussed, and therapeutic approaches for disease prevention are presented.

Tetrahydrobiopterin Improves Endothelial Function in Patients with Coronary Artery Disease

Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase (NOS) and a scavenger of oxygen-derived free radicals. Decreased availability of BH4 leads, under in vitro conditions, to reduced nitric oxide (NO) production and increased superoxide formation. We studied the effect of exogenous BH4 on endothelial function of angiographically normal vessel segments in patients with coronary artery disease. Nineteen patients with coronary artery disease underwent quantitative coronary angiography with simultaneous coronary flow velocity measurements (Cardiometrics FloWire). Data were obtained in angiographically normal segments of the left coronary artery at baseline, after intracoronary (i.c.) administration of acetylcholine (Ach; 10(-4) M), after infusion of BH4 (10(-2) M), and after co-infusion of ACh and BH4. At the end of the study, 300 microg nitroglycerin (NTG) i.c. was administered to obtain maximal vasodilation. At each step, flow velocity was determined before and after 18 microg adenosine i.c. to assess coronary flow velocity reserve. In 15 patients, ACh induced coronary vasoconstriction of -18 +/- 3% (endothelial dysfunction; p < 0.0001 vs. baseline), and in four patients, vasodilation of +39 +/- 20%. In the 15 patients with endothelial dysfunction, BH4 alone did not influence vessel area but prevented vasoconstriction to ACh (+2 +/- 3%, NS, vs. baseline). Correspondingly, calculated volume flow showed the highest value after co-infusion of ACh and BH4. Coronary flow velocity reserve was comparable during the various infusion steps. BH4 prevents ACh-induced vasoconstriction of angiographically normal vessels in patients with coronary artery disease. Thus substitution of this cofactor of NOS may represent a new approach for the treatment of endothelial dysfunction.

Metallothionein-I and -II knock-out mice are not more sensitive than control mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity

Oxidative stress, resulting from either excess generation or reduced scavenging of free radicals, has been proposed to play a role in damaging striatal neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity. Metallothionein (MT) is a scavenger of oxygen-derived free radicals and it has been suggested that MT protects against oxidative damage in the central nervous system. In the present study we compared MT-I and –II null mice (MT-null) and the parental strain with intact metallothionein genes (control mice) to determine whether the absence of MT protein alters MPTP neurotoxicity. Control and MT-null mice were treated with MPTP at different doses (30, 40 and 50 mg/kg, i.p.) for 11 days. Seven days after the last dose, brains were removed and dopamine (DA) and homovanillic acid (HVA) contents were analyzed in striatum and midbrain by high pressure liquid chromatography (HPLC). Striatal DA content in MPTP-treated control mice was reduced by 53, 74 and 84% after 30, 40 and 50 mg/kg, respectively. Similar decreases were observed in MT-null mice treated with MPTP. MT-null mice were not more sensitive than control mice to MPTP neurotoxicity. The data suggest that MT-I and -II proteins do not play a role in protecting against MPTP neurotoxicity.

Evening primrose meal: a source of natural antioxidants and scavenger of hydrogen peroxide and oxygen-derived free radicals.

Evening primrose meal (EPM: 1% and 2%, w/w) reduced (p </= 0.05) the formation of 2-thiobarbituric acid-reactive substances (TBARS), hexanal, and total volatiles in cooked comminuted pork by 43.6-72.6%. Phenolic compounds in the EPM were extracted under optimum conditions (with 56% acetone at 71 degrees C for 47 min) predicted by a multivariate analysis. The resulting evening primrose crude extract (EPCE) inhibited the bleaching of beta-carotene in a model system. After 2 h of assay, the system containing 200 ppm EPCE [as (+)catechin equivalents] retained 86% of the initial beta-carotene whereas the control retained only 11%. Inhibition of the formation of TBARS, hexanal, and total volatiles in the cooked comminuted pork containing 200 ppm EPCE [as (+)catechin equivalents] ranged from 67. 3% to 97.5%. The EPCE inhibited the formation of conjugated dienes, hexanal, and total volatiles in stripped-bulk corn oil (18.5-63.6% inhibition) and stripped-corn oil-in-water emulsion systems (31.7-65. 6% inhibition). Hydrogen peroxide (H(2)O(2)), hydroxyl radical ((*)OH), and superoxide radical (O(2)(*)(-)) scavenging properties of the EPCE were comparable to those observed for authentic (+)catechin. At 200 ppm of the EPCE [as (+)catechin equivalents], a 100% quenching of (*)OH and O(2)(*)(-) was evident. The EPCE scavenged 44-91% of H(2)O(2) in the assay medium after 10 min as compared to 7% reduction in the control.

Impairment of nitric oxide-mediated relaxations in anaesthetized autoperfused streptozotocin-induced diabetic rats.

This work was designed to determine in vivo the influence of the metabolic control of streptozotocin-induced diabetic rats, measured by the levels of haemoglobin glycosylation in blood (HbA1c), on developing vascular endothelial dysfunction. For this, the vasoactive responses to basal and stimulated endothelial nitric oxide (NO) were studied using the technique of the anaesthetized autoperfused rat, analyzing the responses to acetylcholine (ACh) and N(G)-nitro-L-arginine methyl ester (L-NAME) in non-diabetic and diabetic rats with different degrees of metabolic control (four groups with HbA1c levels of 5.5-7.4%, 7.5-9.4%, 9.5-12%, and >12%, respectively). When administered over a noradrenaline-induced vasopressor tone, ACh (0.25, 0.75, 2.5, 7.5 and 25 microg kg(-1)) induced dose-dependent vasodilatatory responses in all rat groups, reducing both mean arterial pressure and perfusion pressure of the left hindlimb. These responses were similar in non-diabetic and in diabetic rats with good metabolic control (HbA1c 5.5-7.4%), while diabetic rats with levels of HbA1c higher than 7.5% showed significantly lower vasodilatatory responses to ACh. In untreated diabetic rats, the relaxant responses evoked by the NO donor sodium nitroprusside were also impaired. On the other hand, increasing doses of L-NAME (0.1 to 10 mg kg(-1)) enhanced both mean arterial pressure and left hindlimb perfusion pressure in diabetic and non-diabetic rats. As with ACh, the responses to L-NAME were significantly reduced in diabetic rats with HbA1c levels higher than 7.5%. To determine the mechanism underlying the NO-mediated endothelial dysfunction, the responses to ACh in untreated diabetic rats (HbA1c >12%) were studied in the presence of the NO substrate L-arginine, in the presence of the oxygen-derived free radical scavenger superoxide dismutase (SOD), or in the presence of both compounds. Both L-arginine and SOD produced a partial improvement of the ACh-induced vasodilatatory responses, but the effects of these agents were not additive. In this group of animals, SOD also induced a partial recovery of the L-NAME-evoked vasoconstrictions. In non-diabetic and untreated diabetic rats, the plasma levels of NO derivatives and arginine were measured. No significant differences were obtained in the amount of nitrites plus nitrates, while plasma levels of arginine were markedly reduced in the untreated diabetic animals. The results indicate that the endothelial dysfunction associated to diabetes is closely related to the level of metabolic control of the disease. Therefore, it is possible to establish a threshold for developing endothelium impairment from percentages of HbA1c higher than 7.5%. As the responses to the NO synthase blocker L-NAME were analogously impaired, it is reasonable to suggest that diabetic endothelial dysfunction is related to the interference with mechanisms linked both to stimulated and basal production of NO. We suggest that this interference is partially due to a deficit in the substrate availability for NO and to an increased generation of superoxide anions.

U74389G prevents vasospasm after subarachnoid hemorrhage in dogs.

Oxygen-derived free radicals may contribute to vasospasm after the rupture of an intracranial aneurysm through direct vasoconstricting effects occurring within the arterial wall or, secondarily, by causing lipid peroxidation in the subarachnoid erythrocytes with secondary induction of vasoconstriction. U74389G is a potent inhibitor of lipid peroxidation and a scavenger of oxygen-derived free radicals. This study determined the relative contributions of oxygen-derived free radicals and lipid peroxidation to vasospasm in the double-hemorrhage dog model. Sixteen dogs underwent baseline (Day 0) cerebral angiography and induction of subarachnoid hemorrhage by two injections of blood into the cisterna magna 2 days apart. They were randomized to receive drug vehicle (n=8) or U74389G (n=8, 3 mg/kg of body weight/d) intravenously. Drug administration and end point analysis were blinded. The end points were angiographic vasospasm, as assessed by comparison of angiograms obtained before and 7 days after subarachnoid hemorrhage, and the levels of malondialdehyde and salicylate hydroxylation products (dihydroxybenzoic acids) in cerebrospinal fluid and of malondialdehyde in subarachnoid blood clots and basilar arteries 7 days after hemorrhage. Comparisons within groups of Day 0 and Day 7 angiograms and between groups of angiograms obtained at Day 7, showed significant vasospasm in animals in the vehicle group (mean+/-standard error, 51%+/-4) but not in the U74389G group (25%+/-11, P < 0.05, unpaired t test). High-pressure liquid chromatographic assays of malondialdehyde and dihydroxybenzoic acids in cerebrospinal fluid, subarachnoid blood clots, and basilar arteries showed no significant differences between groups. The significant prevention of vasospasm by U74389G without change in levels of indicators of free radical reactions suggests that the effect of the drug is related to other processes occurring in the arterial wall and that cerebrospinal fluid levels of oxygen radicals and lipid peroxides are not useful markers of vasospasm.

Effect of α-tocopherol, taurine and selenium on the attenuation of ischemia/reperfusion injury of splanchnic organs

Background: Splanchnic artery occlusion shock is caused by increased capillary permeability and cellular injury precipitated by oxygen derived free radicals following ischemia and reperfusion of splanchnic organs. The purpose of this study was to assess the role of several wellknown oxygen-derived free radical scavengers in ameliorating or preventing this syndrome. Study design: Anesthetized rats were subjected to periods of occlusion of the visceral arteries and reperfusion. Tocopherol, taurine, selenium or a ‘cocktail’ of these three agents was injected subcutaneously for 4 consecutive days prior to operation. Mean arterial blood pressure was measured throughout the experimental period. Fluorometry and technetium-99m pyrophosphate counting of the visceral organs were performed as well as a histologic grading system for intestinal viability. Results: Final mean arterial blood pressure associated with the ‘cocktail’ and selenium groups was 79.1 ± 27.4 mmHg and 83.6 ± 17.8 mmHg, respectively. These values were significantly higher than the control group, 40.8 ± 11.4 mmHg ( P < 0.05). Similar patterns of the benefit of selenium in contrast with the other groups were obtained with fluorescein perfusion, radioisotopic activity and histologie analysis. Conclusion: Pretreatment with selenium of splanchnic ischemia and reperfusion in the rat improves mean arterial blood pressure and microcirculatory visceral perfusion. Further analysis of the precise protective mechanism of selenium for reperfusion injury will enable visceral organs to withstand the consequences of increased capillary leakage and oxidant injury.

Effect of α-Tocopherol, Taurine and Selenium on the Attenuation of Ischemia/Reperfusion Injury of Splanchnic Organs

Background: Splanchnic artery occlusion shock is caused by increased capillary permeability and cellular injury precipitated by oxygen derived free radicals following ischemia and reperfusion of splanchnic organs. The purpose of this study was to assess the role of several well-known oxygen-derived free radical scavengers in ameliorating or preventing this syndrome. Study design: Anesthetized rats were subjected to periods of occlusion of the visceral arteries and reperfusion. Tocopherol, taurine, selenium or a ‘cocktail’ of these three agents was injected subcutaneously for 4 consecutive days prior to operation. Mean arterial blood pressure was measured throughout the experimental period. Fluorometry and technetium-99m pyrophosphate counting of the visceral organs were performed as well as a histologic grading system for intestinal viability. Results: Final mean arterial blood pressure associated with the ‘cocktail’ and selenium groups was 79.1 ± 27.4 mmHg and 83.6 ± 17.8 mmHg, respectively. These values were significantly higher than the control group, 40.8 ± 11.4 mmHg ( P &lt; 0.05). Similar patterns of the benefit of selenium in contrast with the other groups were obtained with fluorescein perfusion, radioisotopic activity and histologic analysis. Conclusion: Pretreatment with selenium of splanchnic ischemia and reperfusion in the rat improves mean arterial blood pressure and microcirculatory visceral perfusion. Further analysis of the precise protective mechanism of selenium for reperfusion injury will enable visceral organs to withstand the consequences of increased capillary leakage and oxidant injury.

Tetrahydrobiopterin improves endothelial function in patients with coronary artery disease

Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase (NOS) and a scavenger of oxygen-derived free radicals. Decreased availability of BH4 leads, under in vitro conditions, to reduced nitric oxide (NO) production and increased superoxide formation. We studied the effect of exogenous BH4 on endothelial function of angiographically normal vessel segments in patients with coronary artery disease. Nineteen patients with coronary artery disease underwent quantitative coronary angiography with simultaneous coronary flow velocity measurements (Cardiometrics FloWire). Data were obtained in angiographically normal segments of the left coronary artery at baseline, after intracoronary (i.c.) administration of acetylcholine (Ach; 10(-4) M), after infusion of BH4 (10(-2) M), and after co-infusion of ACh and BH4. At the end of the study, 300 microg nitroglycerin (NTG) i.c. was administered to obtain maximal vasodilation. At each step, flow velocity was determined before and after 18 microg adenosine i.c. to assess coronary flow velocity reserve. In 15 patients, ACh induced coronary vasoconstriction of -18 +/- 3% (endothelial dysfunction; p < 0.0001 vs. baseline), and in four patients, vasodilation of +39 +/- 20%. In the 15 patients with endothelial dysfunction, BH4 alone did not influence vessel area but prevented vasoconstriction to ACh (+2 +/- 3%, NS, vs. baseline). Correspondingly, calculated volume flow showed the highest value after co-infusion of ACh and BH4. Coronary flow velocity reserve was comparable during the various infusion steps. BH4 prevents ACh-induced vasoconstriction of angiographically normal vessels in patients with coronary artery disease. Thus substitution of this cofactor of NOS may represent a new approach for the treatment of endothelial dysfunction.

Direct visualization of leukocyte/endothelial cell interaction during extracorporeal circulation (ECC) in a new animal model

The clinical complications of extracorporeal circulation (ECC) have been linked to a systemic activation of cellular and humoral components and to a dysregulation of the microcirculatory compartment. Since to date only in vitro methods exist for evaluation, we developed an animal model to study the effects of ECC on the microcirculation. To establish the model, we assessed whether these effects are dependent on the duration of ECC. Intravital fluorescence microscopy was used on the dorsal skinfold chamber preparation in chronically instrumented, awake Syrian golden hamsters. ECC was realized using a micro-rollerpump and a silicon tube shunting blood between the carotid artery and the jugular vein. ECC was performed in three groups for various times (2, 10 and 20 min) after application of heparin at 300 IU/kg body wt. In hamsters, the application of high-dose heparin releases endothelial bound superoxide dismutase (SOD), a natural scavenger of oxygen-derived free radicals. Protocol II assigned two groups receiving heparin at different doses of 50 and 2000 IU/kg body wt. ECC for 2 min served as control to exclude effects from hemodilution and resulted in a minimal induction of leukocyte/endothelial cell interaction. Isovolemic ECC for 20 min resulted in an increase in rolling (from 11 +/- 3 to 38 +/- 20%, mean +/- S.D., P < 0.05) and adherent leukocytes (from 19 +/- 16 to 215 +/- 145 cells/mm2, mean +/- S.D., P < 0.05) in postcapillary venules. Microhemodynamic parameters and functional capillary density were not significantly affected. Arterial blood pressure and heart rate were stable. Heparin at 2000 IU/kg inhibited post-ECC leukocyte adhesion following ECC, whereas 50 IU/kg showed no protective effects. Leukocyte/endothelial cell interaction, induced by blood contact with synthetic surfaces, was directly visualized in vivo. The number of adherent leukocytes was dependent on the duration of ECC. The application of high-dose heparin followed by release of SOD almost prevented leukocyte activation, suggesting a formation of oxygen free radicals during ECC. The new application of the hamster model may allow to study the underlying pathomechanisms and to develop therapeutic/prophylactic strategies to avert problems associated with ECC.

Neutrophil function and glutathione-peroxidase (GSH-px) activity in healthy individuals after treatment with N-acetyl-L-cysteine

The objective of this study was to evaluate the effect of N-acetyl-L-cysteine (NAC) on neutrophilic functions and as an antioxidant. NAC, 600 mg daily, given orally to healthy individuals for a period of 2 weeks, affected some functions of human neutrophilic granulocytes when tested in vitro. NAC treatment caused a decrease in the production of superoxide anions by stimulated neutrophils and the improvement of their phagocytic capacity although it did not affect their random or chemotactic migration. The level of glutathione peroxidase (GSH-px) in thrombocytes of the NAC-treated individuals was increased in comparison with the activity before treatment. These results suggest that NAC might act as a scavenger of oxygen-derived free radicals released by stimulated neutrophils and thereby protect the tissue against the radical caused injury as well as optimize phagocytosis.