Marshal Shlafer

Involvement of hydrogen peroxide and hydroxyl radical in the ‘oxygen paradox’: Reduction of creatine kinase release by catalase, allopurinol or deferoxamine, but not by superoxide dismutase **

The objective of this study was to test the hypothesis that cytotoxic oxygen metabolites participate in lytic cardiac cell damage, detected as creatine kinase release, upon reoxygenation of hypoxic, isolated buffer-perfused hearts (oxygen paradox). Perfusate additives included: superoxide dismutase (30 mg/l); catalase (2 mg/l); deferoxamine (0.5 mM); and allopurinol (1 mM). Creatine kinase release upon reoxygenation was reduced, to levels not significantly different from nonhypoxic controls, by adding either catalase, allopurinol or deferoxamine to the buffer during hypoxia. Reduced creatine kinase leakage was not accompanied by parallel preservation of ventricular function or coronary vascular resistance. Administration of catalase during hypoxia was superior to administering it only during reoxygenation. Treatment with catalase during both hypoxia and reoxygenation provided no more protection than administration only during hypoxia. The data suggest that an important component of hypoxia-induced cardiac cell damage is due primarily to hydrogen peroxide, which may then form hydroxyl radical. Superoxide anion plays an important role as a precursor of these species, but added superoxide dismutase alone did not significantly reduce creatine kinase loss. The data also suggest that damage resulting in creatine kinase release upon reoxygenation occurs during oxygen deprivation, and it is mediated in part by cytotoxic oxygen metabolites.

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 cells 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.

Absence of xanthine oxidase or xanthine dehydrogenase in the rabbit myocardium

We directly measured the activity of the enzymes xanthine oxidase and xanthine dehydrogenase in rabbit and rat hearts, using a sensitive radiochemical assay. Neither xanthine oxidase activity nor xanthine dehydrogenase activity was detected in the rabbit heart. In the rat heart, xanthine oxidase activity was 9.1 +/- 0.5 mIU per gram wet weight and xanthine dehydrogenase activity was 53.0 +/- 1.9 mIU per gram wet weight. These results argue against the involvement of the xanthine oxidase/xanthine dehydrogenase system as a mechanism of tissue injury in the rabbit heart, and suggest that the ability of allopurinol to protect the rabbit heart against hypoxic or ischemic damage must be due to a mechanism other than inhibition of these enzymes.

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.

The oxygen consumption paradox of “stunned myocardium” in dogs

SummaryThe 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 ancsthetized 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 bascline: 104±20% (p=NS) and 111±21% (p=NS). In contrast, regional oxygen consumption in Group II was markedly depressed from bascline at 30 and 120 min of reperfusion: 42±7% (p<.01) and 40±8% (p<.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.

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.

A method to reduce interference by sucrose in the detection of thiobarbituric acid-reactive substances☆

A thiobarbituric acid (TBA) reaction for measuring lipid peroxidation products was evaluated for interference by several ingredients commonly used in solutions to prepare or analyze tissue homogenates or subcellular organelles. These included sucrose (up to 100 mM final concentration in the assay medium), Tris-maleate (up to 40 mM), imidazole (up to 20 mM), inorganic phosphate (up to 10 mM), and 4- morpholinepropanesulfonic acid (up to 20 mM). When the samples were heated at 95 degrees C as recommended in some procedures, only sucrose significantly affected color development. Sucrose concentrations as low as 10 mM significantly increased absorbance at 532 nm of aqueous tetramethoxypropane (TMP) standards, and so the assay could not be applied reliably to tissue samples prepared in sucrose. Sucrose interference was only partially reduced by subsequent organic extraction (n-butanol plus pyridine), with measured absorbances remaining significantly greater (50-100%) than sucrose-free controls at sucrose concentrations of 20 mM or more. Modifying the assay to include sucrose in blanks and TMP standards failed to adequately correct for interference when the absorbance of unextracted (aqueous) solutions was measured. Further modification by adding sucrose to blanks and TMP standards, followed by butanol-pyridine extraction, gave standard curves that were linear, through the origin, and had slopes equivalent to those of sucrose-free standards. This modification enabled almost complete recovery (average 2% error) of known amounts of TMP added to aliquots of tissue homogenates containing amounts of sucrose that otherwise significantly interfered. Also, with the modified method the content of TBA-reactive substances in tissues homogenized in sucrose was found to be not significantly different from that measured in tissues homogenized in a noninterfering substance, KCl.

β-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.

Time-dependent changes in canine cardiac mitochondrial function and ultrastructure resulting from coronary occlusion and reperfusion

SummaryTime-dependent changes in mitochondrial function and structure resulting from 1 hr of left circumflex coronary artery occlusion followed by 2 to 24 hrs of reperfusion were examined. These changes were correlated with changes in myocardial ultrastructure, tissue water content, infarct size and mitochondrial calcium content. The heart was removed after different periods of reperfusion, and mitochondria were isolated from ischemic and nonischemic regions of the left ventricle. Tissue samples from ischemic and nonischemic myocardium also were taken for electron microscopy and tissue water content determinations. Infarct size was measured by the nitroblue tetrazolium staining method. Oxygen consumption by mitochondria isolated from ischemic and nonischemic myocardium was measuredin vitro. Mitochondria from ischemic myocardium showed time-dependent decreases in rates of oxygen consumption and tightness of coupling. Electron microscopy revealed progressive ultrastructural deterioration in ischemic myocardium, including accumulation of calcium deposits within mitochondria, a finding corroborated by elevated concentrations of calcium in mitochondria isolated from the same area. Tissue wet-to-dry weight ratios were increased significantly in ischemic myocardium. A small, but significant, decrease in respiratory function was observed in mitochondria isolated from nonischemic myocardium several hrs after reperfusion; however, nomal respiration was observed 24 hrs after release of occlusion. This latter observation indicates that the nonischemic zone also is affected by regional ischemia. The results obtained indicate that temporary left circumflex artery occlusion and reperfusion result in progressively decreasing mitochondrial function and structure within the ischemic myocardium, and that these changes are accompanied by cellular electrolyte alterations.ZusammenfassungUntersucht wurden zeitabhängige Veränderungen in Struktur und Funktion der Mitochondrien, die durch einstündigen Verschluß und 2- bis 24stündige Reperfusion des Ramus circumflexus der linken Koronararterie erzeugt wurden. Diese Veränderungen wurden mit Veränderungen der myokardialen Ultrastruktur, dem Wassergehalt des Gewebes, der Infarktgröße und dem mitochondrialen Calciumgehalt korreliert. Das Herz wurde nach verschiedenen Reperfusionszeiten entnommen und die Mitochondrien aus ischämischen und nichtischämischen Gebieten des linken Ventrikels isoliert. Ebenso wurden Gewebeproben von ischämischem und nichtischämischem Myokard für Elektronenmikroskopie und Bestimmung des Wassergehaltes des Gewebes entnommen. Die Infarktgröße wurde durch die Anfärbung mit Nitroblau-Tetrazolium bestimmt. Der Sauerstoffverbrauch der Mitochondrien aus ischämischem und nichtischämischem Myokard wurdein vitro gemessen. Mitochondrien aus ischämischem Myokard zeigten eine zeitabhängige Abnahme des Sauerstoffverbrauchs und seiner Bindung an die Phosphorylierung von ADP. Die Elektronenmikroskopie zeigte eine fortschreitende Zerstörung der Ultrastruktur im ischämischen Myokard, einschließlich einer Zunahme der Calciumablagerungen in Mitochondrien, was mit erhöhten Calciumkonzentrationen in Mitochondrien aus dem gleichen Gebiet übereinstimmte. Im ischämischen Myokard war die Relation Feuchtgewicht/Trockengewicht signifikant erhöht. Eine geringe, aber signifikante Abnahme der Atmung wurde in Mitochondrien, die nach einigen Stunden Reperfusion aus nichtischämischem Myokard isoliert worden waren, beobachtet; aber nach 24 h Reperfusion fand sich normale Atmung. Letzteres weist darauf hin, daß auch das nichtischämische Gebiet von der regionalen Ischämie betroffen ist. Die Ergebnisse zeigen, daß vorübergehender Verschluß des Ramus circumflexus der linken Koronararterie und Reperfusion zu fortschreitender Zerstörung mitochondrialer Funktion und Struktur führen und daß diese Veränderungen von Änderungen des Electrolytstatus der Zelle begleitet werden.