Scavengers Of Oxygen-derived Free Radicals Research Articles

Free Radical Scavengers Prevent Intestinal Ischemia-Reperfusion-Mediated Cardiac Dysfunction

Our previous studies show that intestinal ischemia impairs cardiac function. This present study examined the contribution of oxygen-derived free radicals to cardiac dysfunction after intestinal ischemia-reperfusion in a rat model of superior mesenteric artery (SMA) occlusion (atraumatic clip for 20 min) and ligation of collateral arcades from the right colic and jejunal arteries. Controls were sham operated (Group 1, n = 10); in Group 2, 20 rats with SMA occlusion were sacrificed 2-5 hr after reperfusion without treatment. Superoxide dismutase (SOD) and catalase, scavengers of oxygen-derived free radicals which have been shown to effectively reduce ischemic injury in several models of traumatic injury, were given as 6000 units/350 g body wt either 1 min after SMA occlusion (Group 3, n = 11) or 2 min after reperfusion (Group 4, n = 10). To examine the contribution of neutrophils as a source of free radicals, additional groups of animals were treated with pentoxifylline (PTX, a methylxanthine derivative which has been shown to decrease neutrophil adherence and aggregation as well as to decrease superoxide production) either 1 min after SMA occlusion (Group 5, n = 10) or 2 min after reperfusion (Group 6, n = 10). Cardiac contractile depression occurred in the untreated ischemic group as indicated by a fall in left ventricular pressure (from 77 ± 3 to 63 ± 4 mm Hg, P < 0.01) and + dP/dt max (from 1827 ± 60 to 1558 ± 98 mm Hg/sec, P < 0.03) and - dP/dt max (from 1267 ± 57 to 953 ± 68 mm Hg/sec, P < 0.02). Contractile depression after untreated ischemia-reperfusion was confirmed by a rightward shift in left ventricular function curves and decreased responsiveness to perfusate calcium. Both free radical scavengers (SOD and catalase) and neutrophil stabilization (PTX) prevented ischemia-reperfusion-mediated cardiac dysfunction, and left ventricular function curves for these treated groups were identical to those generated by control hearts. We conclude that oxygen-derived free radicals from activated neutrophils contribute, in part, to intestinal ischemia-reperfusion-mediated cardiac dysfunction.

Scavengers of Free Oxygen Radicals in Combination with Magnesium Ameliorate Perinatal Hypoxic-Ischemic Brain Damage in the Rat

The effect of oxygen radical scavengers in combination with magnesium administered after a hypoxic-ischemic insult was evaluated in a model of perinatal brain damage. A mixture of scavengers of oxygen-derived free radicals (L-methionine, 0.2 g; mannitol, 0.5 g) and magnesium sulfate (0.3 g) per kg body weight was given to 34 1-wk-old rat pups immediately after a session of unilateral carotid artery ligation and 2 h of hypoxia (8% O2 in N2). Thirty-four littermates served as controls; they received a placebo. At 3 wk of age, there was a significantly smaller reduction of hemisphere weight ipsilateral to the ligation in the treated animals compared with the controls (0.7 versus 8.8% of contralateral hemisphere weight median values, p < 0.01). The difference was especially marked for the most severe degrees of brain damage. Only one of the 34 treated animals, compared with 13 of 34 control animals, had a reduction of ipsilateral hemisphere weight > 25%. The protection offered by the mixture used was larger than in previously published studies using this model and treatment after the hypoxic exposure with only one protective agent. It is concluded that a combination of oxygen radical scavengers and magnesium administered in the phase of resuscitation mitigates perinatal postasphyxial brain damage in the rat. An additive protective effect of different therapeutic strategies on the brain damage may be present in this situation.

Improved recovery of heart transplants by combined use of oxygen-derived free radical scavengers and energy enhancement

Oxygen free radical injury during reperfusion of ischemically stored heart transplants may further impair the ability of the transplanted heart to reuse substrate for recovery. We compared the effects of oxygen free radical scavengers, superoxide dismutase and catalase, either alone or combined with glucose-insulin-potassium, in an improved model of the heterotopically transplanted rat heart. Group 1 hearts (n = 8) received no preservation before transplantation and were transplanted immediately. Hearts in four other groups (n = 8 for each group) underwent cold storage (4 degrees to 6 degrees C) for 3 1/2 hours before transplantation. Five minutes before reperfusion of the transplanted hearts, recipient rats received one of the following intravenous treatments: saline (group 2), glucose-insulin-potassium (group 3), superoxide dismutase/catalase (group 4), and superoxide dismutase/catalase plus glucose-insulin-potassium (group 5). Left ventricular end-diastolic pressure, rate of rise of left ventricular pressure, myocardial blood flow, coronary resistance, and tissue adenosine triphosphate content of the heart transplants were assessed during or at the end of 2 hours of reperfusion. Hearts treated with superoxide dismutase/catalase alone showed improvement of end-diastolic pressure and myocardial blood flow. The use of glucose-insulin-potassium alone did not facilitate the recovery of transplanted hearts. In contrast, the combined use of superoxide dismutase/catalase plus glucose-insulin-potassium resulted in a superior recovery of all functional and hemodynamic parameters. These results indicate that free radical scavengers in the presence of glucose-insulin-potassium significantly improve functional recovery in the setting of heart transplantation.

Role of oxygen-derived free radicals on rat splanchnic eicosanoid production during acute hemorrhage

The effect of superoxide dismutase (SOD), an oxygen-derived free radical scavenger, on rat splanchnic eicosanoid synthesis was examined following hemorrhagic shock. Anesthetized male rats were hemorrhaged to 30 mm Hg for 30 minutes (Shock), killed, or treated with the shed blood (Shock plus reperfusion). The Shock plus reperfusion group was treated with saline vehicle or SOD (2500, 5000, 7500, 10,000 or 15,000 U/Kg, i.v.) 15 minutes prior to the reperfusion of the shed blood. The superior mesenteric artery was removed in continuity with the end organ intestine (SV+SI) and perfused in vitro with oxygenated Krebs-Henseleit buffer (3 ml/min at 37°C). Venous effluent was measured for basal release of 6-keto- PGF 1 α , PGE 2 and thromboxane B 2 at 15, 30, 60 and 90 minutes of perfusion. The (SV+SI) compensated for acute shock by increased release of 6-keto-PGF 1α (300%) (and not PGE 2 or thromboxane B 2) which was abolished by reperfusion of the shed blood following shock. Prior treatment of the Shock plus reperfusion group with 7500 U/Kg or more of SOD restored the increased release of SV+ SI 6-keto- PGF 1 α found following shock alone ( p<0.05). These data provided indirect evidence that ODFRs contributed to endogenous SV+ SI regulation of PGI 2 synthesis and release during hemorrhagic shock and reperfusion of shed blood.

Preventive actions of N-(3-aminopropionyl)-L-histidinato zinc (Z-103) through increases in the activities of oxygen-derived free radical scavenging enzymes in the gastric mucosa on ethanol-induced gastric mucosal damage in rats.

Z-103 at 1 to 25 mg/kg, p.o. prevented 100% ethanol-induced gastric mucosal lesions in a dose-dependent manner. Z-103 at 3 to 25 mg/kg, p.o. significantly elevated gastric mucosal superoxide dismutase (SOD)-like activity 1 hr after its administration to normal rats. In addition, Z-103 at doses (10 and 25 mg/kg, p.o.) which prevented 100% ethanol-induced gastric lesion further increased gastric mucosal SOD-like and glutathione peroxidase (GSH-px) activities elevated by 60% ethanol. Z-103 (10 and 25 mg/kg) significantly inhibited the increase in thiobarbituric acid-reactive substances in gastric mucosa injured by 60% ethanol. The combination with cycloheximide, a protein synthesis inhibitor, completely abolished the prevention of 60% ethanol-induced gastric mucosal lesions and the elevation of both free radical scavenging enzyme activities in the mucosa by Z-103 (10 mg/kg, p.o.). These results suggest that Z-103 may partly protect rat gastric mucosa against ethanol-induced damage by scavenging oxygen-derived free radicals via increases in the synthesis of SOD-like and GSH-px enzymes in the mucosa.

Preventive Actions of N-(3-Aminopropionyl)-L-Histidinato Zinc (Z-103) through Increases in the Activities of Oxygen-Derived Free Radical Scavenging Enzymes in the Gastric Mucosa on Ethanol-Induced Gastric Mucosal Damage in Rats

Z-103 at 1 to 25 mg/kg, p.o. prevented 100% ethanol-induced gastric mucosal lesions in a dose-dependent manner. Z-103 at 3 to 25 mg/kg, p.o. significantly elevated gastric mucosal superoxide dismutase (SOD)-like activity 1 hr after its administration to normal rats. In addition, Z-103 at doses (10 and 25 mg/kg, p.o.) which prevented 100% ethanol-induced gastric lesion further increased gastric mucosal SOD-like and glutathione peroxidase (GSH-px) activities elevated by 60% ethanol. Z-103 (10 and 25 mg/kg) significantly inhibited the increase in thiobarbituric acid-reactive substances in gastric mucosa injured by 60% ethanol. The combination with cycloheximide, a protein synthesis inhibitor, completely abolished the prevention of 60% ethanol-induced gastric mucosal lesions and the elevation of both free radical scavenging enzyme activities in the mucosa by Z-103 (10 mg/kg, p.o.). These results suggest that Z-103 may partly protect rat gastric mucosa against ethanol-induced damage by scavenging oxygen-derived free radicals via increases in the synthesis of SOD-like and GSH-px enzymes in the mucosa.

Mechanism of liver injury following ischemia

To clarify whether ischemic liver injury is due to ischemia itself or reperfusion, histopathological and functional changes in the liver were examined before and after liver ischemia in rats with porto-systemic collateral channels. Effects of oxygen-derived free radical scavengers or an inhibitor of platelet aggregation on development of ischemic liver injury were also examined. Liver ischemia was produced by ligation of the portal vein and hepatic artery at liver hilum for 1 hr. The primary lesion of ischemic liver injury was cloudy swelling of liver cells in the periportal and midzonal regions; it developed during ischemia. The cloudy swelling of liver cells induced uneven distribution of sinusoidal blood flow after reperfusion, and consequently individual liver cell necrosis and focal hepatocellular necrosis in the midzonal regions developed later. Elevation of cytoplasmic enzyme activities in the serum after reperfusion was due to leakage across the damaged plasma membrane of liver cells. The treatment with superoxide dismutase, catalase, or heparin had not altered the liver injury that was attributed to ischemia, biochemically and histologically. These results suggest that ischemic liver injury is due to liver cell damage developed during ischemia, and that the ischemic liver injury is not alleviated or prevented by superoxide dismutase, catalase, or heparin.

Role of Oxygen-Derived Free Radical Scavengers in the Treatment of Recurrent Pain Produced by Chronic Pancreatitis

This randomized double-blind trial examined the influence of the radical scavengers allopurinol (50 mg per rectum, four times per day) and dimethyl sulfoxide (500 mg per rectum, four times per day) on pancreatic pain treated with intramuscular pethidine hydrochloride (100 mg followed by 50 mg every 4 hours until complete pain relief) in patients given nothing orally and intravenously hydrated. Addition of allopurinol or dimethyl sulfoxide to the analgesic regimen significantly enhanced its efficacy, enabling at least 57% (13 patients receiving allopurinol and 12 patients receiving dimethyl sulfoxide) of 43 patients to be free of pain within 12 hours after admission compared with only four (17%) of 23 controls achieving the same effect. This advantage extended to all patients within 24 hours after admission, leaving 11 controls (48%) still in pain. Consequently, all patients given allopurinol or dimethyl sulfoxide were discharged 3 days after admission, a result realized in only five (22%) of the assessable controls who were discharged after 5 days of hospitalization. The results suggest that oxygen-derived free radicals are implicated in the mechanism of abdominal pain caused by alcohol-induced chronic pancreatitis and that removing them results in a beneficial therapeutic effect.

Metabolism of ethanol by rat hepatocytes results in generation of a lipid chemotactic factor: Studies using a cell-free system and role of oxygen-derived free radicals

We have reported that liver cells metabolizing ethanol release a lipid that has chemotactic activity for human polymorphonuclear leukocytes. The purpose of the present study was to investigate the role of ethanol and of oxygen-derived free radicals in generation of this activity. A cell-free, ethanol-metabolizing system consisting of rat liver cytosol was found to generate a polar lipid which possessed a chemotactic activity identical to the activity from intact cells. The importance of acetaldehyde in this process was established by the findings that (i) an inhibitor of the metabolism of ethanol to acetaldehyde, 4-methylpyrazole, blocked production of the chemotactic lipid; (ii) acetaldehyde could substitute for ethanol; and (iii) cytosol alone incubated with ethanol in the absence of mitochondrial acetaldehyde metabolism generated the activity. Acetaldehyde can serve as a substrate for cytosolic oxidases in reactions that generate radicals (e.g., superoxide anion) which have been shown to attack unsaturated lipids to yield lipid hydroperoxides. When scavengers of oxygen-derived free radicals (superoxide dismutase, catalase, and dimethylsulfoxide) were added to the ethanol-metabolizing system they prevented generation of the activity. The data are consistent with a mechanism of chemotactic factor generation by ethanolmetabolizing liver cytosol in which acetaldehyde causes formation of oxygen-derived free radicals that attack unsaturated cytosolic lipid to generate a polar chemotactic compound.

Angiotensin converting enzyme inhibitors improve contractile function of stunned myocardium by different mechanisms of action

Angiotensin converting enzyme (ACE) inhibitors enhance contractile function of myocardium “stunned” by a brief episode of coronary artery occlusion, yet their mechanism(s) of action remain unresolved. In addition to possible hemodynamic effects, ACE inhibitors may stimulate the synthesis of cardioprotective prostaglandins. Furthermore, the beneficial effects of ACE inhibitors that contain a sulfhydryl group may be due in part to the ability of thiol compounds to act as nonspecific antioxidants or direct scavengers of cytotoxic oxygen-derived free radicals. To investigate this question we compared the effects of (1) the sulfhydryl-containing ACE inhibitor zofenopril, (2) the sulfhydryl-containing stereoisomer of captopril (SQ 14,534) with essentially no ACE inhibitor properties, (3) the nonsulfhydryl-containing ACE inhibitor enalaprilat, and (4) solvent alone, given at the time of reperfusion, on recovery of contractile function after 15 minutes of coronary occlusion in the anesthetized open-chest dog. Segment shortening in control animals remained depressed or “stunned” after reperfusion, recovering to only −5 ± 12% of baseline preocclusion values at 3 hours after reperfusion. In contrast, all three treatment agents attenuated postischemic dysfunction: segment shortening was restored to 33 ± 12%, 54 ± 6%, and 83 ± 5% of baseline values at 3 hours after reflow in dogs treated with SQ 14,534 ( p < 0.05), zofenopril ( p < 0.01), and enalaprilat ( p < 0.01), respectively (all vs control value). These improvements in segment shortening did not appear to be the result of altered oxygen supply or demand after reperfusion, inasmuch as no significant differences in systemic hemodynamic parameters or myocardial blood flow were observed among the groups. In the second phase of the study, we found that the improved contractile function associated with enalaprilat treatment could largely be reversed by infusion of the potent cyclooxygenase inhibitor indomethacin: segment shortening was reduced from 69 ± 12% at 2 hours after treatment/reperfusion to 38 ± 12% at 2 hours after indomethacin infusion ( p < 0.01 vs 2 hours after reperfusion). Infusion of indomethacin had no effect, however, on the improved contractile function associated with zofenopril treatment. We therefore conclude that sulfhydryl- versus nonsulfhydryl-containing agents enhance contractile function of stunned myocardium by different mechanisms of action: enalaprilat attenuates postischemic dysfunction at least in part by a prostaglandin-mediated mechanism, whereas the salutary effects of zofenopril and SQ 14,534 may be due in part to the antioxidant properties of the sulfhydryl moiety.

Protection Against Postischemic Myocardial Dysfunction in Anesthetized Rabbits with Scavengers of Oxygen-Derived Free Radicals

Postischemic myocardial dysfunction in canine myocardium has been reported to be reduced by scavengers of oxygen-derived free radicals. One potential source of oxygen-derived free radicals in canine myocardium is xanthine oxidase, but human and rabbit myocardium either lack or possess very low levels of this enzyme. Therefore, the effects of scavengers of oxygen-derived free radicals on postischemic myocardial dysfunction produced by 15 min of ischemia and 3 h of reperfusion were evaluated in vivo in the rabbit. Superoxide dismutase (SOD) (45,000 U/kg) and catalase (55,000 U/kg) were given into the left atrium 10 min before ischemia, and followed by an additional 45,000 U/kg of SOD and 55,000 U/kg of catalase given over 85 min. This treatment reduced postischemic myocardial dysfunction, as did sulfhydryl-containing free radical scavengers N-2-mercaptopropionyl glycine (4 mg/kg, i.v.) and captopril (3 mg/kg, i.v.) given 5 min before and 60 min after reperfusion. SOD given alone at the same dose was ineffective, as was enalaprilat (0.3 mg/kg, i.v.), an angiotensin-converting enzyme inhibitor that does not scavenge oxygen-derived free radicals. Thus, postischemic myocardial dysfunction was reduced by scavengers of oxygen-derived free radicals in vivo in a species that is deficient in myocardial xanthine oxidase. This suggests that oxygen-derived free radicals derived from a source other than xanthine oxidase play a role in postischemic myocardial dysfunction.

A Water-Soluble Quaternary Ammonium Analog of α-Tocopherol, that Scavenges Lipoperoxyl, Superoxyl and Hydroxyl Radicals

The new water-soluble ammonium-analog of alpha-tocopherol (vitamin E) (compound 1: 3,4-dihydro-6-hydroxy-N,N, N-2,5,7,8-heptamethyl-2H-1-benzopyran-2-ethanaminium 4-methylbenzenesulfonate) and its tertiary amine derivative (compound 2: 3,4-dihydro-2-(2-dimethylaminoethyl)-2,5,7,8-tetramethyl-2H-1-benzopyran -6-ol hydrochloride) were investigated as scavengers of oxygen-derived free radicals. Compounds 1 and 2 were at least 40 times more potent inhibitors of Fe-driven heart microsomal lipid peroxidation than Trolox. While the alpha-tocopherol analogs had the same potency as scavengers of xanthine/xanthine oxidase-generated superoxyl radicals, the thiol compounds D,L-penicillamine and N-2-mercaptopropionyl glycine reacted at a much slower rate. The O-acetyl derivatives of compounds 1 and 2 were not scavengers of superoxyl radicals. Considerable differences between the alpha-tocopherol analogs were observed in their competition with 2-deoxyribose for hydroxyl radicals (OH.). Compound 2 was equipotent with Trolox and thiourea, whereas the reactivity of these substances was diminished by more than 30% as compared to compound 1. Although showing lower reactivity, the O-acetyl derivatives of compounds 1 and 2 were active nevertheless as OH.-scavengers. The previously reported high potency of compound 1 in reducing infarct size during myocardial ischemia/reperfusion appears to be due to its radical-scavenging properties, likely to be enhanced by its previously described cardioselectivity.

The Role of Endogenous Free Radical Scavengers on Tissue Recovery in the Experimental Ulcer Model

The role of lipid peroxidation and endogenous oxygen-derived free radical scavengers on ischemia-reperfusion injury and tissue recovery in rat ulcer model corresponding to the gastric histopathology was investigated. Male Wistar rats weighting 200-250 g were heparinized before occlusion of the celiac axis for 1.5 h. Endogenous CuZn-superoxide dismutase (SOD), Mn-SOD, glutathione peroxidase, fumarase, cytochrome c oxidase, and thiobarbituric acid-reactive compounds as lipid peroxidation products were measured in the gastric tissue at 3 h, and 1, 2, 4, and 7 days after release and in the controls (no occlusion). At 3 h after release, erosion of the gastric mucosa was observed, and gastric ulcers beyond the muscularis mucosae were present in the gastric body 2 days later. Seven days after release, gastric ulcers had disappeared. Activity levels for all five enzymes (CuZn-SOD, Mn-SOD, glutathione peroxidase, fumarase, and cytochrome c oxidase) were low for days 1-4 after release and did not return to control levels by the seventh day. It was observed that the ulcer formation, as evidenced by the histopathology, was significantly related to the levels of endogenous CuZn-SOD, Mn-SOD, glutathione peroxidase, fumarase, and cytochrome c oxidase activities. Thiobarbituric acid-reactive compounds were also low through the entire course of ulcer formation. The study concludes that decreases in the levels of these oxygen-derived free radical scavengers may result in the formation of gastric ulcers; however, endogenous free-radical scavengers may not correspond with tissue recovery. Lipid peroxidation may not be related to ulcer formation.

Pulmonary reperfusion injury: evidence for oxygen-derived free radical mediated damage and effects of different free radical scavengers

Blood granulocyte-mediated reactions involving generation of oxygen-derived free radicals have recently been shown to be capable of causing injury to the lungs. These findings suggest a similar mechanism also to be involved in the development of pulmonary ischemia/reperfusion injury. In the present study, therefore, the effects of three oxygen-derived free radical scavengers, superoxide dismutase (SOD; 1 mg/kg), catalase (20,000 IU/kg) and allopurinol (45 mg/kg), were evaluated during reperfusion in a rabbit model after 2 h normothermic ischemia of the lung. During reperfusion, ischemic lungs were found to have an elevated pulmonary vascular resistance, increased total and extravascular lung water content, and decreased arterial oxygen tension (PaO2) compared to control animals. SOD and catalase, but not allopurinol, were able to reduce pulmonary injury by lowering the pulmonary vascular resistance, but could not prevent pulmonary damage as shown by total lung water (TLW) or PaO2. It is concluded that oxygen-derived free radicals such as hydrogen peroxide and the superoxide anion may play an important role in precipitating pulmonary injury after ischemia. The failure of xanthine oxidase inhibition (allopurinol) to exert protective effects may suggest that oxygen-derived free radical generation following pulmonary ischemia occurs predominantly via leukocyte-mediated reactions.

Mechanisms Underlying the Inhibitory Interaction Between the Nitrovasodilator SIN-1 and the Endothelium

Experiments were designed to determine the influence of the endothelium on the relaxing potency of the nitrovasodilator SIN-1. Rings of coronary arteries were suspended for isometric-tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution, aerated with 95% O2-5% CO2 and warmed to 37 degrees C. The experiments were performed in the presence of indomethacin and propranolol in order to inhibit cyclooxygenase and beta-adrenoceptors, respectively. In rings contracted with prostaglandin F2 alpha, SIN-1 caused concentration-dependent relaxations that were increased following endothelium removal. In rings denuded of endothelium, the relaxations evoked by SIN-1 were not affected by NG-monomethyl-L-arginine (L-NMMA, which inhibits the production of endothelium-derived relaxing factor), or by superoxide dismutase and catalase (scavengers of oxygen-derived free radicals), or by L-NMMA plus superoxide dismutase and catalase. In rings with endothelium, relaxations evoked by SIN-1 were increased significantly by L-NMMA or by superoxide dismutase and catalase, and were increased further by the combination of L-NMMA plus superoxide dismutase and catalase. The difference in potency of SIN-1 between arterial rings with and without endothelium was reduced by either L-NMMA or by superoxide dismutase and catalase, and was abolished by the combination of L-NMMA plus superoxide dismutase and catalase. Therefore, the inhibitory interaction between SIN-1 and the endothelium may result from an endothelium-dependent production of oxygen-derived free radicals that may inactivate the nitric oxide generated by SIN-1, and from an inhibitory interaction between SIN-1 and endothelium-derived relaxing factor, released under basal conditions.

Mechanisms Underlying the Inhibitory Interaction Between the Nitrovasodilator SIN-1 and the Endothelium

SUMMARY Experiments were designed to determine the influence of the endothelium on the relaxing potency of the nitrovasodilator SIN-1. Rings of coronary arteries were suspended for isometric-tension recording in organ chambers filled with modified Krebs-Ringer bicarbonate solution, aerated with 95% O2-5% CO2 and warmed to 37°C. The experiments were performed in the presence of indomethacin and propranolol in order to inhibit cyclooxygenase and β-adrenoceptors, respectively. In rings contracted with prostaglandin F2α. SIN-1 caused concentration-dependent relaxations that were increased following endothelium removal. In rings denuded of endothelium, the relaxations evoked by SIN-1 were not affected by NG-monomethyl-L-arginine (L-NMMA, which inhibits the production of endothelium-derived relaxing factor), or by superoxide dismutase and catalase (scavengers of oxygen-derived free radicals), or by L-NMMA plus superoxide dismutase and catalase. In rings with endothelium, relaxations evoked by SIN-1 were increased significantly by L-NMMA or by superoxide dismutase and catalase, and were increased further by the combination of L-NMMA plus superoxide dismutase and catalase. The difference in potency of SIN-1 between arterial rings with and without endothelium was reduced by either L-NMMA or by superoxide dismutase and catalase, and was abolished by the combination of L-NMMA plus superoxide dismutase and catalase. Therefore, the inhibitory interaction between SIN-1 and the endothelium may result from an endothelium-dependent production of oxygen-derived free radicals that may inactivate the nitric oxide generated by SIN-1, and from an inhibitory interaction between SIN-1 and endothelium-derived relaxing factor, released under basal conditions.

Generation of chemotactic activity for neutrophils by liver cells metabolizing ethanol

The mechanism of polymorphonuclear leukocyte (PMN) infiltration of the liver in acute alcohol-related liver injury is unknown. We have reported that ethanol metabolism by hepatocytes incubated with moderate concentrations of ethanol (2–50 mM) results in the release into the medium of a chemoattractant for human PMN. This response to ethanol is time- and concentration-dependent with peak activity at 10 mM ethanol. Generation of the factor is specific for hepatocytes and is blocked by inhibiting ethanol metabolism with 4-methylpyrazole. It does not appear to be due to cell death. The activity has been partially characterized: it behaves as a polar lipid, possibly an arachidonic acid metabolite distinct from leukotriene B4. Preliminary studies indicate that a cell-free system derived from liver generates a similar activity. In that system scavengers of oxygen-derived free radicals block production of the factor.

Long-term neonatal heart preservation

Donor availability is a major limiting factor in neonatal heart transplantation. Prolonging donor heart preservation would facilitate distant heart procurement. Forty-two neonatal (1 to 5 days) piglet hearts in seven groups were arrested with cold cardioplegic solutions, stored for 12 hours at 4 degrees C in storage solutions, and reperfused with blood from an adult support pig. The cardioplegic solutions used were a crystalloid solution with potassium chloride 30 mEq/L and bicarbonate (Stanford), the Stanford cardioplegic solution with the addition of calcium (1.2 mmol/L), or an intracellular solution (Sacks) with added glucose. Storage solutions were normal saline, Sacks II, or Sacks II with glucose 20 gm/L. Reperfusion was done with normal blood or modified blood for 20 minutes with superoxide dismutase, catalase, aspartate, glutamate, citrate-phosphate-dextrose, potassium, tromethamine, and 50% dextrose followed by normal blood. Evaluation of stroke work index after 60 minutes of recovery (as percent of control) was performed using the isolated, blood perfused, working heart preparation in all groups: Group I (Stanford cardioplegia, saline storage, normal blood reperfusion) had a recovery of 11%; group II (Stanford + calcium, saline, normal blood) 8%; group III (Stanford + calcium, saline, modified blood, superoxide dismutase 35,000 U/L, catalase 35,000 U/L) 37%; group IV (Stanford + calcium, Sacks II, modified blood, superoxide dismutase 35,000 U/L, catalase 35,000 U/L), 47%; group V (Stanford + calcium, Sacks + glucose, modified blood, superoxide dismutase 35,000 U/L, catalase 105,000 U/L) 89%; group VI (Stanford + calcium, Sacks + glucose, modified blood, superoxide dismutase 150,000 U/L, catalase 150,000 U/L) 107%; group VII (Sacks + glucose, Sacks + glucose, modified blood, superoxide dismutase 35,000 U/L, catalase 105,000 U/L) 115%. The neonatal heart stored hypothermically for 12 hours tolerates normal blood reperfusion poorly. Modified blood reperfusion markedly improves the recovery. Complete functional recovery was achieved by the intracellular Sacks plus glucose storage solution and modified blood reperfusion with oxygen-derived free radical scavengers (high catalase). Extended preservation of the neonatal heart is feasible.

Oxygen-derived free radical scavengers for amelioration of reperfusion damage in heart transplantation

In this study we tried to define the possible benefits of the oxygen-derived free radical scavengers after 3 hours of cold myocardial global ischemia, as required in the setting of cardiac transplantation. Twenty-one pig hearts were harvested after preservation with a cold cardioplegic solution (St. Thomas' Hospital solution) and topical cooling. Normothermic reperfusion with blood was achieved with a special heart-lung machine preparation, which allows the heart to beat in a working or nonworking mode. Twelve hearts served as control hearts (group I), and nine (group II) were subjected to superoxide dismutase and catalase. Superoxide dismutase was applied at a dose of 40 U/ml of cardioplegic solution and 1500 U/kg body weight with the start of reperfusion. Catalase was added to the cardioplegic solution in a dose of 100 U/kg and 3500 U/kg body weight with the start of reperfusion. After 15 minutes of retrograde reperfusion, both left ventricular developed pressure and its first derivative were significantly higher in group II (137 +/- 7.6 mm Hg, 2467 +/- 162 mm Hg/sec) than in group I (105 +/- 6 mm Hg, 1676 +/- 231 mm Hg/sec, p less than 0.05 for each). In addition, a considerably higher coronary blood flow was observed in group II throughout the 180-minute period of reperfusion (p = 0.047). We therefore conclude that the combined administration of superoxide dismutase and catalase during the initial period of cardioplegic arrest and during early reperfusion of donor hearts submitted to 3 hours of cold ischemia has a beneficial effect on myocardial performance.

The mechanism of neuronal resistance and adaptation to hypoxia

In this work we provide a theoretical explanation for the observations that: (i) young animals are more resistant to hypoxia than adult ones and (ii) repeated exposure to a hypoxic insult increases the tolerance of young animals and isolated brain tissue to that insult. Considered here is the role of taurine, a putative Ca 2+ transport modulator, in attenuating Ca 2+ influx and overload in brain tissue upon hypoxia. It is proposed that the higher resistance of young animals to hypoxia stems from their higher brain content of taurine as compared with adults. The increased resistance to lack of oxygen upon re-exposure to hypoxia may occur as a result of protein and coenzyme A (CoA) breakdown which leads to the accumulation of products like cystine, cysteine, cysteamine and other sulfur-containing compounds. Upon reoxygenation, these compounds are oxidized to form taurine, which in turn attenuates neuronal Ca 2+ accumulation. The sulfur-containing compounds are considered to be natural scavengers of oxygen-derived free radicals which are formed upon reoxygenation and have been implicated as a major component in the process leading to ischemic/hypoxic brain damage. Repeated hypoxic insults bring about the formation of higher levels of taurine and hence the observed adaptation to oxygen lack. The hypothesis presented here is supported by experimental observations in our laboratory and those of others.