Fructose-2,6-bisphosphate, a potent stimulator of phosphofructokinase, is increased by high exogenous glucose perfusion.

BACKGROUND: We have previously demonstrated that perfusion of isolated hearts with high concentrations of glucose results in increased glycolysis during ischemia, diminished ischemic injury, and improved functional recovery with reperfusion. OBJECTIVE: To evaluate a possible mechanism by which glucose conferred this protection. We examined the hypothesis that increased exogenous glucose concentrations results in increased concentrations of fructose-2,6-bisphosphate, a potent activator of phosphofructokinase-1, and thus increases glycolysis. METHODS: Perfused rabbit hearts were subjected to 60 min of low-flow ischemia. Control hearts were perfused with buffer containing 0.4 mmol/l palmitate, 5 mmol/l glucose, and 70 mU/l insulin, and treated hearts were perfused with buffer containing 0.4 mmol/l palmitate, 15 mmol/l glucose and 210 mU/l insulin. RESULTS: Ischemic contracture was attenuated by perfusion of high concentrations of glucose (high glucose) (P < 0.05 compared with control). Glucose uptake and lactate production were greater in hearts perfused with high glucose, as was the ATP concentration at the end of ischemia (P < 0.05 compared with controls). Exogenous glucose uptake and lactate production correlated well with fructose-2,6-bisphosphate content (P = 0.007). CONCLUSIONS: Enhancement of glycolysis in hearts perfused with high glucose may be the result of stimulation of phosphofructokinase-1 by fructose-2,6-bisphosphate. Accordingly, this may serve as an important mechanism by which cardioprotection may be achieved.

Traumatic versus postischemic induction of oxidative stress in rat liver.

A series of experiments was performed to characterize the effects of tissue trauma, extracellular calcium concentration, and prior ischemia on oxidative stress, measured by the accumulation of malondialdehyde-like materials (MDA-LM) in slices of rat liver. Liver tissue was rendered ischemic for 1 hour at 37 degrees C, either minced (to create traumatized fragments) or cleanly cut and washed (to create nontraumatized fragments), and then reoxygenated for 30 minutes in flasks of buffered salt solution. Nonischemic tissue was incubated similarly but without the 60-minute prior ischemia. The production of MDA-LM in the tissues was used as an indicator of lipid peroxidation. Production of MDA-LM in the tissues was used as an indicator of lipid peroxidation. Production of MDA-LM was always enhanced by prior ischemia and reoxygenation. However, trauma also increased the production of MDA-LM both in nonischemic liver slices in vitro and in those subjected to ischemia and reoxygenation. Furthermore, the elimination of calcium from incubation buffer significantly reduced MDA-LM production both in nontraumatized, ischemic, and reoxygenated tissues and in traumatized, nonischemic tissues; while the addition of the calcium ionophore A23187 (10 mumol/L) increased MDA-LM production in nontraumatized tissues independently of ischemia and reoxygenation. In nonischemic, traumatized tissues, the iron chelators deferoxamine and CGP-46,700A (1,2-diethyl-3-hydroxypyrid-4-one) quenched MDA-LM production. These data indicate that either ischemia or mechanical trauma may predispose liver tissue to calcium-dependent and iron-dependent oxidative stress.

Potential use of simple manganese salts as antioxidant drugs in horses.

The scavenging of superoxide radicals by endogenous and therapeutically administered superoxide dismutases may prevent superoxide-mediated oxidative stress leading to lipid peroxidation, membrane lysis, and cell death in a wide variety of normal and pathologic states. Simple inorganic manganous salts such as MnCl2 also have superoxide dismutase-like activity and are extremely inexpensive, compared with enzymatic superoxide dismutase preparations. In this study, we explored the use of Mn salts as antioxidant drugs. We used the percentage of inhibition of nitroblue tetrazolium reduction by superoxide as a measure of the amount of superoxide dismutase-like activity. We found concentration-related increases in superoxide scavenging activity in simple buffer solutions upon addition of 1.25, 2.5, and 5.0 microM MnSO4. To determine whether Mn salts can inhibit oxidative damage in tissues, we used an in vitro model of lipid peroxidation in ischemic and reoxygenated rat liver slices. Concentrations of 10, 100, and 1000 mumoles MnCl2/L of buffer significantly decreased indicators of lipid peroxidation believed to be initiated by intracellular superoxide. We then determined the effectiveness of MnCl2 as a superoxide scavenger in conscious horses by measuring the superoxide scavenging ability of equine plasma before and during intravenous infusions of 1.0 L volumes of 0.9% saline solution containing 0, 12.5, or 25 mM MnCl2. Plasma Mn concentrations, which were determined by atomic absorption spectrophotometry, increased as a function of time and dose. Intravenously administered MnCl2 concomitantly produced dose-related increases in superoxide scavenging ability of equine plasma at 15, 30, 45, and 60 minutes after the onset of infusion, compared with preinfusion control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Methylene blue as an inhibitor of superoxide generation by xanthine oxidase. A potential new drug for the attenuation of ischemia/reperfusion injury.

Tissue oxidases, especially xanthine oxidase, have been proposed as primary sources of toxic oxygen radicals in many experimental models of disease states. Among these, ischemia-reperfusion injury may be of the greatest clinical interest. In this paper we propose the use of methylene blue as a means of suppressing the production of superoxide radicals O2- by acting as an alternative electron acceptor for xanthine oxidase. Previous work has indicated that methylene blue accepts electrons from xanthine oxidase at the iron-sulfur center. Initial experiments in our laboratory demonstrated that (1) pairs of electrons from each enzymatic oxidation are transferred to methylene blue, (2) the reduction of methylene blue can be achieved by model iron-sulfur centers, similar to the iron-sulfur center of xanthine oxidase, (3) reduced methylene blue auto-oxidizes to produce H2O2 directly, rather than O2-, and (4) methylene blue is effective at non-toxic levels (2-5 mg/kg) in preventing free radical damage to liver and kidney tissues in an in vitro model of ischemia and reoxygenation. Accordingly, we propose that methylene blue may represent a new class of antioxidant drugs that competitively inhibit reduction of molecular oxygen to superoxide by acting as alternative electron acceptors for tissue oxidases. We have termed these agents "parasitic" electron acceptors.

Cytochemical studies of hydrogen peroxide generation in postischemic hepatocytes.

Reoxygenation injury that occurs when blood circulation is restored to previously ischemic tissues is currently discussed as a pathophysiological entity distinct from the primary anoxic injury that develops during ischemia per se. To test the hypothesis that reoxygenation injury in hepatocytes is caused by a postischemic burst of reactive oxygen species (ROS), including superoxide radicals, O2-., and hydrogen peroxide, H2O2, we performed a cytochemical study exploiting the peroxidase activity within peroxisomes as a sensitive ultrastructural detector of intracellular H2O2 generation. The osmiophilic polymer formed when tissue peroxidase is incubated with 3,3'-diaminobenzidine (DAB) and H2O2 was used as a marker for endogenous H2O2 in rat liver slices in short-term organ culture subjected to a cycle of 60-min ischemic anoxia and 30-min reoxygenation in the presence of DAB without exogenous H2O2. Peroxisomal reaction product was quantitatively evaluated in transmission electron micrographs of systematically sampled hepatocytes. Mean densities of positive peroxisomes per 1,000 micron2 (+/- SE) in liver slices subjected to various treatments were as follows: continuous anoxia (negative control) 0 +/- 0; normoxia + exogenous H2O2 (positive control) 45 +/- 12; normoxia only 26 +/- 2; ischemia-reoxygenation 13 +/- 6; ischemia-reoxygenation + xanthine oxidase inhibitor, oxypurinol 5 +/- 3; ischemia-reoxygenation + peroxidase inhibitor, aminotriazole 7 +/- 3. Endogenous H2O2 can be detected in hepatocytes by electron microscopic cytochemistry and may in part derive from xanthine oxidase, but it is not substantially increased in the postischemic state. We conclude that hepatocytes do not exhibit a postischemic burst of reactive oxygen species that could cause reoxygenation injury.

Effect of oxygen concentration on the formation of malondialdehyde-like material in a model of tissue ischemia and reoxygenation.

This study was conducted to explore the functional relationship between oxygen concentration during tissue reoxygenation after ischemia and the extent of postischemic lipid peroxidation, an indicator of reoxygenation injury. Excised rat liver or kidney tissue was rendered ischemic for 1 h at 37 degrees C, minced into 1 mm3 fragments, and then reoxygenated for 1 h in flasks of buffered salt solution containing various amounts of oxygen. Production of malondialdehyde-like material (MDA) was measured to indicate lipid peroxidation. MDA production was minimal at oxygen tensions less than 10 mmHg, increased sharply from 10 to 50 mmHg, and plateaued at approximately 100 mmHg. A similar functional relationship was produced by a simple mathematical model of free radical mediated lipid peroxidation in biological membranes, suggesting that MDA production is indeed caused by free radical oxidation of membrane phospholipids and that the oxygen effect is governed by simple competition between chain propagation and chain termination reactions within the membrane. These experimental and analytical results confirm that relatively low concentrations of oxygen are sufficient to produce oxidative damage in post-ischemic tissues.

A rapid, widely applicable screen for drugs that suppress free radical formation in ischemia/reperfusion.

Substantial injury can occur during reoxygenation of previously ischemic tissue in many experimental models, as the result of the generation of oxygen-derived free radicals. To test the antiradical activity of potentially protective compounds in this setting, we developed a simple screening system, applicable to fresh biopsy specimens, in which warm ischemia and reoxygenation of excised tissue are performed in vitro. Tissue production of malondialdehyde (MDA) equivalents is used as a nonspecific-but-sensitive marker of oxygen radical damage. Test compounds with putative antiradical activity are added prior to the reoxygenation phase, and their ability to suppress MDA production is an index of activity in preventing reoxygenation injury. Comparison with ischemic but not reoxygenated controls confirms the oxygen-dependent nature of the effect. Standard positive controls of known effective agents, such as butylated hydroxytoluene or deferoxamine, provide a reference for the activity of the test compound. The method is applicable to surgical biopsy specimens in veterinary and human medicine.