Myocardial flavin reductase and riboflavin: a potential role in decreasing reoxygenation injury.

Ferrylmyoglobin has been implicated in cardiac reoxygenation damage. Flavin reductase, an enzyme previously isolated from erythrocytes, can reduce ferrylmyoglobin in the presence of sufficient flavin concentrations. Flavin reductase mRNA signals were detected in rabbit heart, lung, liver, kidney, and isolated cardiomyocytes. It was hypothesized that increasing flavin reductase catalysis by administering flavins exogenously could decrease cardiac reoxygenation damage in isolated rabbit hearts. Riboflavin (150 microM) inhibited reoxygenation-induced lactate dehydrogenase release by 57%, an effect prevented by hematoporphyrin, a flavin reductase inhibitor. The results suggest that riboflavin supplementation has cardioprotective effects during reoxygenation and that these effects are mediated by flavin reductase.

The effect of pass/fail grading and weekly quizzes on first-year students' performances and satisfaction.

BACKGROUND: In 1992-93 the University of Michigan Medical School revised its first-year curriculum. An evaluation system using honors, high-pass, pass, and fail grading and only two examinations (a midterm and a final) was replaced with a system using pass/fail grading and weekly quizzes in addition to the two examinations. The objective was to increase students' satisfaction while maintaining a high level of achievement. METHOD: Students' performance scores and survey data from the final year of the former system (1991-92, 222 students) and the first year of the new system (1992-93, 195 students) were used to investigate whether overall performance decreased and whether the students liked the new approach to grading. Statistical methods used were one-sample t-tests, Student's t-test, and Fisher's Z-test. RESULTS: Under the new system, the average scores for courses remained well above passing, and no evidence was found that the students achieved at lower levels than had their predecessors with the former, more traditional grading system. Also, higher cumulative pre-final scores (i.e., scores on the weekly quizzes as well as the midterm) did not predict lower, "just passing" achievement on final examinations. The students' responses to the surveys included comments that pass/fail grading eased anxiety and reduced competition while encouraging the students' co-operation. CONCLUSION: Despite concerns that implementing pass/fail grading for all first-year courses would result in lower overall performance and decreased motivation among students, during the first year of implementation these fears proved to be unfounded as the students continued to perform well and reported greater satisfaction with the new system.

Pyrroloquinoline quinone acts with flavin reductase to reduce ferryl myoglobin in vitro and protects isolated heart from re-oxygenation injury.

Pyrroloquinoline quinone has been isolated from bacteria and recently has been detected in mammalian tissues and fluids. We report in vitro studies which show that pyrroloquinoline quinone serves as a high-affinity substrate for an erythrocyte "flavin reductase" and that the pyrroloquinoline quinol generated by this catalysis reacts rapidly with ferryl myoglobin radical. Western blot analysis of rat and rabbit heart homogenates detects a cross-reactive protein which has a molecular weight identical to the erythrocyte reductase from the same species. Low concentrations of pyrroloquinoline quinone protect isolated rabbit heart from re-oxygenation injury, serving as an effective tissue-protective agent in this model for cellular oxidative damage. We propose that this tissue protection is due to a pyrroloquinoline quinol-mediated reduction of reactive oxygen species.

Ultrastructural demonstration of peroxidative activity and peroxidation in ischaemic and ischaemic-reperfused rabbit hearts.

OBJECTIVE: The aim was to characterise subcellular histochemical evidence of the involvement of peroxidation and peroxidases in myocardial reperfusion injury. The histochemical technique involved the use of 3,3'-diaminobenzidine (DAB), which reacts with peroxides and proteins with peroxidase activity to form an electron dense polymer. METHODS: Isolated rabbit hearts were perfused (Langendorff method) for 30 min with oxygenated physiological saline solution. Some were subjected to 30 min of normothermic global ischaemia, with or without 30 min reperfusion. Non-ischaemic control hearts were perfused continuously for 90 min. Hearts were fixed with glutaraldehyde and cut into 100-150 microns sections that were incubated for 1 h in buffered DAB (1 mg.ml-1) with or without added KCN or H2O2. They were processed further for transmission electron microscopy. Planimetry was done on micrographs taken from random fields (approximately 500 photos). RESULTS: The total amount of DAB polymer in non-ischaemic control heart sections incubated with DAB alone occupied 1.19(SEM 0.44) micron 2 x 1000 micron-2 total cell area. For ischaemic-nonreperfused hearts, the value was 2.32(0.90) micron 2 x 1000 micron-2 (p = 0.223 v control); DAB occupied 7.49(1.42) micron 2 x 1000 micron-2 in ischaemic-reperfused hearts (p = 0.001 v control). DAB positive staining of mitochondria and lipid droplets, but not of peroxisomes, was significantly increased in reperfused hearts compared with non-ischaemic controls. CONCLUSIONS: Reperfusion, but not ischaemia, was associated with increased DAB staining. This suggests a reperfusion induced increase in myocyte peroxidation. Increased staining may be due to the actions of haem proteins with peroxidase activity on peroxidized lipid.

Evidence that NADPH-dependent methemoglobin reductase and administered riboflavin protect tissues from oxidative injury.

NADPH-dependent methemoglobin reductase, first detected in erythrocytes sixty years ago, has subsequently been purified and characterized as a methylene blue reductase and a flavin reductase. The reductase plays no role in methemoglobin reduction under normal conditions, but its activity serves as the basis for the treatment of methemoglobinemia with methylene blue or flavin. On-going studies demonstrate that this cytosolic protein is also present in liver and that its primary structure distinguishes it from other known proteins. The bovine erythrocyte reductase tightly binds hemes, porphyrins, and fatty acids with resulting loss of activity. Pyrroloquinoline quinone serves as a high-affinity substrate of the reductase, suggesting that this naturally-occurring compound may be a physiological substrate. The ability of the reductase to catalyze the intracellular reduction of administered riboflavin to dihydroriboflavin suggested that this system might be exploited to protect tissues from oxidative damage. This hypothesis was supported by our finding that dihydroriboflavin reacts rapidly with Fe(IV)O and Fe(V)O oxidation states of hemeproteins, states that have been implicated in tissue damage associated with ischemia and reperfusion. Preliminary studies demonstrate that, as predicted, administration of low concentrations of riboflavin protects isolated rabbit heart from reoxygenation injury, rat lung from injury resulting from systemic activation of complement, and rat brain from damage caused by four hours of ischemia. Data from these animal studies suggest that flavin therapy holds promise in protecting tissue from the oxidative injuries of myocardial infarction, acute lung injury, stroke, and a number of other clinical conditions.

Human neutrophil hydrogen peroxide generation following physical exercise.

The effects of prolonged, submaximal exercise by seven healthy, untrained individuals on the generation of H2O2 by neutrophils was studied. Hydrogen peroxide generation by neutrophils isolated from pre-exercise (control) and post-exercise blood samples was measured 10, 15 and 20 minutes following stimulation with phorbol myristate acetate (PMA). Exercise was associated with a significant elevation in the number of circulating neutrophils and a diminished capacity for neutrophil H2O2 generation following PMA stimulation. Addition of post-exercise plasma to neutrophils isolated from pre-exercise blood caused a small reduction in H2O2 generation, suggesting the presence of an inhibitory factor(s) in the plasma during physical exercise. These results support the concept that exercise may contribute to an attenuation of oxygen-dependent neutrophil killing.

Hemodynamic effects of intraatrial administration of deferoxamine or deferoxamine-pentafraction conjugate to conscious dogs.

Deferoxamine (DFX) is a specific Fe3+ chelator that is used to manage iron overload, and is being evaluated as an agent to reduce ischemic organ damage that involves iron-mediated OH formation. However, high intravascular doses cause significant hemodynamic changes that may limit or counteract beneficial effects. We used conscious, closed-chest dogs to test the hypothesis that conjugating DFX to pentafraction, a high molecular weight fraction of pentastarch, could reduce such hemodynamic changes. We infused 50 mg/kg of body weight of native DFX, or an equivalent dose as DFX-pentafraction, intraatrially over 15 min. Within 10 min of starting the infusion. DFX increased heart rate from pre-drug values of 105 +/- 11 (mean +/- SEM; N = 9) to 158 +/- 13 beats/min, and reduced left ventricular (LV) systolic pressure from 131 +/- 3 to 99 +/- 16 mm Hg, LV end-diastolic pressure from 12 +/- 3 to 3 +/- 3 mm Hg, and mean arterial pressure (MABP) from 101 +/- 5 to 74 +/- 13 mm Hg. In two dogs, MABP decreased to less than or equal to 35 mm Hg. These parameters returned to predrug values by 60 min after infusion. All of these changes were statistically significant (p less than 0.05). In contrast, infusing DFX-pentafraction (N = 9) caused no significant cardiac or hemodynamic changes other than a transient and slight (approximately 7%) increase in systolic arterial pressures. This conjugate, which prolongs the plasma half-life and does not alter the iron-chelating activity of native DFX, eliminates many undesirable hemodynamic actions. It may be a useful therapeutic alternative to native DFX in some settings.

Hydrogen peroxide generation by mitochondria isolated from regionally ischemic and nonischemic dog myocardium.

We occluded the left anterior descending coronary artery of anesthetized, open-chest dogs, for 1 or 2 h. Some hearts were reperfused for 1 h after 1 h of ischemia. We isolated mitochondria from the central ischemic zone (CIZ) and a surrounding nonischemic zone (NIZ) of the left ventricle, and assayed H2O2 production using a horseradish peroxidase-dual wavelength spectrophotometric technique. Mitochondria, studied in the absence of exogenous respiratory chain inhibitors, generated H2O2 during State 4 respiration with succinate as the substrate. NIZ mitochondria in all groups produced ca. 1.5 nmols H2O2/min/mg protein (no significant differences between groups). The State 4 O2 consumption rates of NIZ mitochondria from hearts subjected to 1 h ischemia plus reperfusion, or 2 h of ischemia (ca. 30 nmols/min/mg) were significantly higher than that of NIZ mitochondria of hearts subjected to only 1 h of ischemia (23 nmols/min/mg). Thus, the ratio between H2O2 produced and State 4 O2 consumption fell from 6.5% to 5%. Mitochondria from all CIZ samples had State 4 O2 consumption rates that were not different from corresponding NIZ values. However CIZ mitochondria of hearts subjected to 1 h ischemia without reperfusion produced less H2O2 (1.1 +/- 0.1 nmols/min/mg), and had a slightly reduced H2O2/O2 ratio (4.4 +/- 0.7%), compared with their NIZ samples (1.5 +/- 0.1 nmols/min/mg; 5.3%). Reperfusion after 1 h of ischemia abolished these regional differences. The CIZ mitochondria from hearts subjected to 2 h ischemia produced only 0.75 +/- 0.22 nmols H2O2/min/mg (2.5% of State 4 O2 consumption). These values were 50% of corresponding NIZ values, and were significantly less than for any other group or tissue region. If similar phenomena occur in conscious animals subjected to incomplete regional ischemia, especially of relatively brief duration or if accompanied by reduced intracellular defenses against oxidants such as H2O2, they suggest that mitochondria persist as H2O2 sources and so may contribute to the oxidant load and myocardial dysfunction.

The oxygen consumption paradox of "stunned myocardium" in dogs.

The contractile state of the heart is a major determinant of myocardial oxygen consumption. Since regional myocardial contractility can be severely impaired following a transient coronary occlusion, post-ischemic myocardium is frequently assumed to consume less oxygen. To test this assumption, regional myocardial function and oxygen consumption were studied in anesthetized dogs during 2 h of myocardial reperfusion following either a 15-min (Group I) or 4-h (Group II) left anterior descending coronary artery occlusion. Both groups developed similar post-ischemic regional dysfunction characterized by paradoxical motion (negative shortening). Measured as a percent of baseline segment shortening, anterior wall function in Group I (n = 8) and Group II (n = 5) at 30 min of reperfusion was -33 +/- 11% and -34 +/- 16% (p = NS) and at 120 min was -23 +/- 9% and -40 +/- 16% (p = NS). However, the two groups showed a marked difference in regional myocardial oxygen consumption during reperfusion. Despite the abnormal wall motion, regional oxygen consumption in Group I at 30 and 120 min of reperfusion was unchanged from pre-ischemic levels as measured as a percent of baseline: 104 +/- 20% (p = NS) and 111 +/- 21% (p = NS). In contrast, regional oxygen consumption in Group II was markedly depressed from baseline at 30 and 120 min of reperfusion: 42 +/- 7% (p less than .01) and 40 +/- 8% (p less than .01). To determine whether the dissociation between regional myocardial oxygen consumption and function in Group I was related to mitochondrial uncoupling, six additional dogs were studied. Tissue samples were obtained from post-ischemic myocardium after 120 min of reperfusion following a 15-min coronary artery occlusion, and compared to non-ischemic myocardium. There were no differences in the in vitro mitochondrial respiratory rates or oxidative phosphorylation capacity between the post-ischemic and non-ischemic myocardium. Therefore, in the post-ischemic myocardium, significant depressions in regional contractility may not be associated with falls in oxygen consumption. Following a 15-min coronary artery occlusion, the injured myocardium maintains a paradoxically high oxygen consumption with normal mitochondrial function despite decreased contractility and abnormal wall motion.

Cerium chloride as a histochemical marker of hydrogen peroxide in reperfused ischemic hearts.

Hydrogen peroxide (H2O2) has been implicated in cardiac damage due to ischemia and reperfusion. We adapted an electron microscopic, histochemical method for demonstrating H2O2 produced by isolated cells to isolated, buffer-perfused rabbit hearts. The method involves formation of an electron-dense precipitate when H2O2 reacts with cerium chloride (CeCl3). We perfused hearts retrograde via the aorta with well-oxygenated bicarbonate-buffered solution, followed by one in which bicarbonate was replaced with imidazole (IPSS) to prevent precipitation of bicarbonate and CeCl3. Some hearts were made globally ischemic (30 min, 37 degrees C), reperfused 5 min with well-oxygenated IPSS containing 1 mM CeCl3, then processed for electron microscopy. Others were perfused with IPSS containing catalase (300 U/ml) or albumin before ischemia and upon reperfusion, followed by CeCl3 administration. Nonischemic control hearts perfused with IPSS (+/- catalase) were also studied. Electron micrographs were assessed visually and by computer for precipitate localization and amount. There was abundant precipitate on the luminal face of the coronary vascular endothelium in ischemic-reperfused, cerium-treated hearts, including those treated with albumin. There was significantly less in reperfused catalase-treated or nonischemic control hearts. X-ray microbeam analysis of the endothelial precipitate indicated the presence of Ce. This appears to be the first visual demonstration of a CeCl3-H2O2-dependent reaction product in intact isolated ischemic hearts. The data indicate that at the time of reperfusion some H2O2 is accessible to the vascular space, and that its amount can be reduced by perfused catalase. Further modifications this technique may be useful for assessing the sites and pathways by which H2O2 is generated by hearts or other buffer-perfused organs subjected to stresses such as ischemia or hypoxia.

Beta-hydroxybutyrate and response to hypoxia in the ground squirrel, Spermophilus tridecimlineatus.

1. Previous studies have suggested that elevated ketone levels are associated with increased survival time in rodents exposed to hypoxia. In this study the association between whole blood BHB (beta-hydroxybutyrate) and hypoxic survival time was investigated in hibernating and non-hibernating ground squirrels and in rats. 2. Non-hibernating ground squirrels and rats were exposed to hypoxia (4.5% O2). One hundred per cent of ground squirrels survived 1 hr of hypoxia vs 20% of rats. 3. Ketone levels were significantly higher in ground squirrels than rats during hypoxia, and rats surviving the longest had the highest ketone levels. 4. When hibernation was induced in ground squirrels there was a significant increase in beta-hydroxy-butyrate from 0.45 to 1.6 mM (P = 0.0005). 5. Ground squirrel heart mitochondrial respiratory control ratios and ATP synthesis rates indicated no preferential ketone utilization which might suggest a possible extramitochondrial role of BHB during hypoxia. 6. We conclude that elevated blood BHB levels are associated with increased hypoxic survival and they may have evolved in response to life-threatening hypoxia as experienced during hibernation.

The effect of ischemia-reperfusion derived oxygen free radicals on skeletal muscle calcium metabolism.

This study investigated the contribution of cytotoxic oxygen-derived free radicals to the skeletal muscle injury seen in a rat hindlimb tourniquet model after ischemia and reperfusion. The free radical scavengers superoxide dismutase (SOD) and catalase (CAT) were used as biologic probes to detect free radical activity, while Ca2+ uptake by sarcoplasmic reticulum (SR) was used to measure subcellular muscle function. Anesthetized rats received SOD (2 mg/kg IV) plus CAT (3.5 mg/kg IV, n = 6 treated group) or saline alone (4 ml/kg, n = 6 control group) 5 min before unilateral hindlimb tourniquet ischemia of 3 hr duration. SOD and CAT were conjugated to polyethylene glycol to increase their plasma half-life. After 19 hr reperfusion, muscle from ischemic and non-ischemic lower legs of each rat was excised and homogenized. Skeletal muscle SR was isolated by differential centrifugation and ATP-dependent Ca2+ uptake by SR was then measured with dual wavelength spectrophotometry and a calcium-sensitive dye. In control rats, Ca2+ uptake velocity by SR from ischemic muscle was reduced by 48% compared with contralateral non-ischemic muscle (p less than .001). Rats pretreated with SOD + CAT showed a less severe (27%) reduction in Ca2+ uptake velocity by SR from ischemic muscle. Thus, SOD + CAT significantly (p less than .01) reduced the dysfunction of SR Ca2+ transport seen in this tourniquet ischemia model. These results strongly implicate the involvement of oxygen-derived free radicals in abnormal Ca2+ transport observed in skeletal muscle after ischemia and reperfusion.

Absence of detectable xanthine oxidase in human myocardium.

The enzyme xanthine oxidase has been implicated as a generator of toxic oxygen metabolites that contribute to ischemic injury. Because substantial species variability has been demonstrated and because there are minimal human data available, the relevance of xanthine oxidase to human heart damage has been in doubt. We report the absence of xanthine oxidase activity in nine human heart biopsy specimens obtained during cardiac surgery, and in two larger samples obtained during heart transplantation. A sensitive radiochemical assay was used to assess enzyme activity. Our findings imply that oxygen free radicals generated by xanthine oxidase are not relevant in terms of human myocardial injury.

Spatial and temporal characteristics of circumferential flow-function relations during acute myocardial ischemia in the conscious dog.

In the anesthetized open-chest dog the ischemic area produced by coronary occlusion is surrounded by an area of nonischemic contractile dysfunction, identified as the functional border zone. To establish whether a similar functional border zone exists in the conscious animal during acute regional ischemia and to determine its spatial dimensions and temporal changes, we performed simultaneous two-dimensional echocardiography and radioactive microsphere studies in nine chronically instrumented dogs. We produced circumferential flow-function maps at 22.5-degree intervals over the full circumference of the left ventricle at the midpapillary muscle level during control conditions, 5 minutes after left circumflex occlusion, and 2.5 hours after left circumflex occlusion. After occlusion there was no change in left ventricular end-diastolic area, an increase in left ventricular end-systolic area (p less than 0.01), and a decrease in left ventricular area ejection fraction (p less than 0.01). The circumferential extent of left ventricular dysfunction was 197 +/- 11 degrees (mean +/- SEM) at 5 minutes of left circumflex occlusion, whereas the extent of subendocardial hypoperfusion was 144 +/- 6 degrees (p less than 0.0005). This produced a functional border zone measuring 54 +/- 8 degrees, or 25% of the nonischemic myocardium, which did not change over the 2.5-hour occlusion period. Despite a modest but significant decrease in wall thickening (70 +/- 6% to 43 +/- 6%; p less than 0.01) in the functional border zone, there was no difference in subendocardial blood flow between the functional border zone and the control nonischemic area. We conclude that a discrete functional border zone exists in the conscious dog during acute regional ischemia produced by circumflex coronary occlusion, which does not change during the early evolution of myocardial infarction. The functional border zone likely contributes to minor overestimation of infarct size in the early hours after myocardial infarction if extent of left ventricular dysfunction is used as an index of infarction in humans.

The effect of hypothermic ischemia on recovery of left ventricular function and preload reserve in the neonatal heart.

Neonatal and adult myocardium respond differently to ischemia. In addition, the neonatal heart possesses a limited preload reserve. The effect of uninterrupted hypothermic ischemia on recovery of left ventricular function and preload reserve was studied in two groups of isolated rabbit hearts: group 1 (neonates, n = 8), 7 to 10 days old; group 2 (adults, n = 15), 6 to 12 months old. Peak left ventricular systolic pressure, the first derivative of left ventricular systolic pressure, and heart rate were measured at left ventricular pressures of 0, 5, 10, and 15 mm Hg before and after 120 minutes of global ischemia at 27 degrees C. Before ischemia, left ventricular systolic pressure increased significantly at each increment of left ventricular end-diastolic pressure for both groups of hearts. After hypothermic ischemia, recovery of left ventricular systolic pressure was significantly reduced at each level of left ventricular end-diastolic pressure among neonatal hearts (range 75% to 79% of control values). The postischemic recovery of left ventricular systolic pressure in the adult hearts was markedly reduced from baseline values (range 43% to 53% of control values) and was significantly worse than that of neonatal hearts at each level of left ventricular end-diastolic pressure (p less than 0.001). Both groups were able to respond to increasing preload after ischemia. The slope of the curve describing the relationship between left ventricular end-diastolic pressure and percent recovery of left ventricular systolic pressure was not different from zero for neonatal hearts but was significantly greater than zero among the adults (0.22 +/- 0.21 versus 0.73 +/- 0.07, p = 0.0056). After ischemia, the first derivative of left ventricular systolic pressure fell significantly from control values among neonatal hearts (71% of control values). The reduction was considerably greater, however, among the adult hearts (54% of control values). These data indicate that the neonatal heart recovers systolic function better than the adult heart after global ischemia with moderate hypothermia.

Mitochondrial hydrogen peroxide generation and activities of glutathione peroxidase and superoxide dismutase following global ischemia.

We used isolated, buffer-perfused rabbit hearts to evaluate whether global, normothermic ischemia altered mitochondrial hydrogen peroxide (H2O2) generation and mitochondrial activities of the major enzymes responsible for degrading H2O2 and superoxide anion (O2-.): glutathione peroxidase (GPD) and superoxide dismutase (SOD), respectively. This preparation lacks exogenous neutrophils and endogenous xanthine oxidase, which are other potential sources of oxygen metabolites. Ischemia depressed mitochondrial oxidative phosphorylation parameters, State 4 succinate-supported H2O2 generation rates, and the relative flux of State 4 oxygen consumption that was diverted to H2O2 formation. The production of H2O2 was not abolished. Ischemia and reperfusion significantly reduced the activities of SOD (by 43%) and GPD (by 39%) in the mitochondrial fraction. Cytosolic GPD activity was also depressed. The results suggest that the myocardial cell's ability to enzymatically degrade H2O2 and O2-. is compromised, particularly in the mitochondrion. Although mitochondrial H2O2 production is decreased, the mitochondria may persist as a source of this oxygen metabolite following ischemia. Collectively, the data may help explain why mitochondria are vulnerable targets of free radical-mediated damage due to ischemia.

The functional border zone in conscious dogs.

Studies focusing on the functional border zone have been performed largely with anesthetized, open-chest preparations. Therefore, we instrumented 14 dogs at sterile surgery with sonomicrometers arrayed to measure systolic wall thickening across the perfusion boundary produced by circumflex coronary occlusion. We fitted sigmoid curves to the data to model the distribution of wall thickening impairment as a function of distance from the perfusion boundary, which was delineated with myocardial blood flow (15 micron diameter microspheres) maps. Using this approach, we defined the functional border zone as the distance from the perfusion boundary to 97.5% of the sigmoid curve's nonischemic asymptote. The lateral extent of the functional border zone, measured 10 min and 3 hr after occlusion, was 32 and 28 degrees of circumference, respectively. To evaluate the severity of nonischemic dysfunction, we measured average systolic wall thickening within the functional border zone. It was reduced from 3.69 +/- 1.10 (mean +/- SD) mm to 2.98 +/- 1.07 mm (p less than .01) and 2.74 +/- 1.12 mm (p less than .01) early and late after coronary occlusion. Thus, a narrow functional border zone was evident during circumflex coronary occlusion in conscious dogs. Its lateral extent was limited to approximately 30 degrees (similar to findings in open-chest, anesthetized dogs), severe dysfunction was restricted to the immediate vicinity of the perfusion boundary, and the average severity of nonischemic dysfunction within the functional border zone was mild.

Purine efflux after cardiac ischemia: relevance to allopurinol cardioprotection.

Allopurinol is thought to protect hearts against damage due to hypoxia or ischemia by inhibiting xanthine oxidase and oxygen radical generation. We subjected isolated rabbit hearts, equilibrated by perfusion at 37 degrees C, to 1 h of global ischemia at 27 or 37 degrees C with or without brief pretreatment with 100 microM allopurinol. The total absence of xanthine or uric acid in the coronary effluent following ischemia, the presence of hypoxanthine (25 +/- 4 microM peak concentration), and the failure of allopurinol to alter purine washout profiles or postischemic cardiac function suggest that rabbit myocardium lacks xanthine oxidase or dehydrogenase. Data obtained with a similar rat heart preparation showed appreciable formation of xanthine (12 +/- 2 microM peak) and uric acid (10 +/- 3 microM). Allopurinol pretreatment inhibited xanthine and uric acid formation and significantly improved key indicators of postischemic left ventricular function. We conclude that there is species dependency in the myocardial activity of xanthine oxidase or dehydrogenase, that when present it can be inhibited by acute allopurinol pretreatment, and that xanthine oxidase activity and its ability to generate oxygen radicals are not universal contributors to cardiac ischemic damage.

Effect of superoxide dismutase plus catalase on Ca2+ transport in ischemic and reperfused skeletal muscle.

Cytotoxic oxygen metabolites may contribute to skeletal muscle damage associated with ischemia and reperfusion. This study utilized a rat hindlimb ischemia model to investigate the effect of pretreatment with oxygen free radical scavengers superoxide dismutase (SOD) and catalase (CAT) on skeletal muscle Ca2+ uptake by sarcoplasmic reticulum (SR) in limbs subjected to periods of ischemia and reperfusion. SOD and CAT were conjugated to polyethylene glycol to prolong their half lives. Anesthetized rats (ca. 350 g) received an iv injection of either conjugated SOD (2 mg/kg) plus CAT (3.5 mg/kg) (n = 6, Treated Group) or 0.9 saline (4 ml/kg) (n = 6, Control Group) 5 min before unilateral hindlimb tourniquet ischemia of 3 hr duration. After 19 hr of reperfusion, muscle from each lower leg was excised and homogenized. Skeletal muscle SR was isolated by differential centrifugation. ATP-dependent Ca2+ uptake by the SR was then measured with dual wavelength spectrophotometry and used as an index of muscle function. Pretreatment with SOD and CAT maintained higher rates of Ca2+ uptake by SR of skeletal muscle from postischemic reperfused limbs (Treated Group 2.29 +/- 0.21 vs Control Group, 1.61 +/- 0.06 mumole Ca2+/mg protein/min). These results implicate cytotoxic oxygen metabolites in the pathogenesis of ischemic reperfusion skeletal muscle injury.

Changes in cardiac muscle function and biochemistry produced by long-term amiodarone and amiodarone + triiodothyronine administration in the rabbit.

Cardiac muscle function and biochemistry were examined after long-term amiodarone administration in the rabbit (20 mg/kg/day for 28 days). Isolated cardiac muscle preparations were obtained from control and amiodarone-treated rabbits, and were studied in vitro. Amiodarone treatment did not alter the magnitude of force development in isolated atrial and papillary muscle preparations, but depressed the rate of force development (dF/dt). The muscle preparations responded similarly to inotropic and chronotropic stimulation with isoproterenol, histamine, and tyramine, although the intrinsic rate of right atrial preparations from the drug-treated animals was reduced. Na+-K+ ATPase activity in crude ventricular homogenates was increased in the amiodarone-treated group. Mitochondrial respiratory function in amiodarone-treated left ventricular tissue was depressed for glutamate, malate, and glutamate + malate. The reduction in respiratory function occurred without uncoupling oxidative phosphorylation or altering respiratory function for succinate. The pharmacologic effects of amiodarone observed in the present study were not observed with the simultaneous administration of triiodothyronine (5 micrograms/day). No difference in ATP-dependent calcium uptake or in calcium-dependent ATPase activity were observed in sarcoplasmic reticulum preparations from control, amiodarone, and amiodarone + T3 groups. The pharmacologic effects of amiodarone in rabbit hearts resemble those previously reported with hypothyroidism and are not observed after triiodothyronine administration.