Reactive oxygen metabolites and reperfusion injury: aberrant triggering of reticuloendothelial function.
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Animals↗
Biomedical subjects
G B Bulkley
Publications and source records attributed to G B Bulkley.
Antioxidants modulate induction of programmed endothelial cell death (apoptosis) by endotoxin.
OBJECTIVE: To evaluate the potential role of reactive oxygen metabolites as signals for endothelial cell apoptosis. DESIGN: A series of antioxidants were evaluated for their ability to block apoptosis in cultured porcine aortic endothelial cells in vitro. RESULTS: Scavenging of the hydroxyl radical with the membrane-permeable scavenger dimethyl sulfoxide or blocking its generation via the Fenton reaction by the chelation of iron with o-phenanthroline blocked apoptosis, whereas the cell membrane-impermeable scavengers superoxide dismutase and catalase did not block apoptosis. Inhibition of xanthine oxidase with enzyme-inhibitory levels of allopurinol also failed to block apoptosis, whereas high levels of allopurinol, which also scavenge the hydroxyl radical in vitro, conferred protection. In each case (dimethyl sulfoxide, o-phenanthroline, and high-dose allopurinol), hydroxyl radical ablation was only effective when administered before the priming step (lipopolysaccharide) and was ineffective when administered later, prior to the activation step (heat shock). CONCLUSIONS: These findings suggest a novel role for the hydroxyl radical as a nonlethal intracellular signal in endothelial cell apoptosis. Moreover, the results support a role for programmed cell death in the pathogenesis of multiple organ dysfunction syndrome and suggest novel strategies for prophylaxis and therapy of the most common cause of death in surgical intensive care units.
Allopurinol↗
Shock and multiple organ failure.
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Animals↗
Proposed toxic oxidant inhibitors fail to reduce brain edema.
Toxic oxidants (oxygen free radicals) have been implicated in the formation of brain edema from ischemia-reperfusion injury or tumor growth. We investigated the ability of an iron chelator, a calcium channel blocker, and a xanthine oxidase inhibitor to reduce formation of brain edema following a cold lesion in cats. The agents were given independently of each other in an attempt to inhibit the Haber-Weiss reaction, prevent Ca++ modulated uncoupling of oxidative phosphorylation, and inhibit the generation of toxic oxidants via xanthine oxidase, respectively. Pentastarch-deferoxamine conjugate at a dose of 50 mg/kg was given 15 minutes before and 60 minutes after the cold lesion. Nimodipine was given at a dose of 1 mg/kg 1 hour before and 2 hours after the cold lesion. Allopurinol was given at a dose of 50 mg/kg 24 hours before, at the time of the lesion and, 24 and 48 hours after the lesion. Gravimetric measurements of multiple brain areas were performed at 24 hours post-lesion in the pentastarch-deferoxamine and nimodipine groups and at 72 hours post-lesion in the allopurinol group. None of these agents led to significant reduction in brain edema formation as measured with a gravimetric column of kerosene and bromobenzene. Pentastarch-deferoxamine conjugate was utilized to avoid the confounding effects of arterial hypotension which is seen with intravenous deferoxamine. There was even a suggestion of increased edema in the periventricular white matter in animals treated with nimodipine. Taken together, independent inhibition of the Haber-Weiss reaction, of calcium channels, or of xanthine oxidase does not reduce formation of brain edema in the cold lesion model.
Allopurinol↗
Induction of heat-shock gene expression in postischemic pig liver depends on superoxide generation.
BACKGROUND/AIMS: Both hemorrhagic and cardiogenic shock are associated with hepatic shock gene expression at resuscitation. This study investigated the potential role of intravascular superoxide anion as a proximal trigger of heat shock protein gene expression. METHODS: Preanesthetized pigs were subjected to 120 m of total warm hepatic ischemia. The survival model consisted of warm, total hepatic ischemia and reperfusion (with active portal-systemic bypass) followed by reperfusion and survival for 3 days. Serial hepatic biopsy samples were evaluated for the expression of heat shock protein 72 (HSP-72) messenger RNA (mRNA) by Northern and Western analysis and by in situ RNA hybridization. The possible role of intravascular O2- as a mediator of heat shock response was evaluated by its specific inhibition by the intravenous infusion of recombinant human superoxide dismutase (SOD). RESULTS: Ischemia for 120 minutes followed by 60 minutes of reperfusion caused accumulation of HSP-72 mRNA. Transcripts were localized to hepatocytes. HSP-72 mRNA was detected neither following ischemia alone nor when SOD was infused for 15 minutes at reperfusion. Three days later, transcripts were not detectable, but HSP-72 protein accumulated irrespective of SOD administration. CONCLUSIONS: Warm hepatic ischemia induces the hepatocyte expression of HSP-72 at reperfusion by a mechanism that is dependent upon the superoxide anion, probably generated intravascularly. However, the transient dismutation of superoxide is insufficient to suppress subsequent accumulation of HSP-72.
Animals↗
Visceral lipid peroxidation occurs at reperfusion after supraceliac aortic cross-clamping.
PURPOSE: Recently, we have reported that lipid peroxidation specific to oxygen free radical-mediated injury increased immediately after reperfusion of human liver allografts. However, in the human liver transplantation setting it was impossible to disassociate the contributions to lipid peroxidation caused by the warm and cold ischemic phases from those caused by reperfusion. Therefore we now have studied lipid peroxidation at reperfusion after supraceliac aortic cross-clamping in patients with normal livers. METHODS: Ethane, a noninvasive biomaker of lipid peroxidation, was measured in exhaled breath of patients before and during cross-clamping of the thoracic aorta and at sequential time intervals after visceral reperfusion. RESULTS: Approximately a two-fold transient increase in the ethane level was observed at around 15 minutes after reperfusion in those patients whose aortas were cross-clamped for more than 18 minutes. CONCLUSIONS: These results indicate that free radical-mediated lipid peroxidation occurs at reperfusion of warm ischemic viscera in the clinical setting of aortic repair. This observation supports the hypothesis that substantial lipid peroxidation occurs when tissues are subjected to cold or warm ischemia followed by reperfusion.
Aged↗
Evaluating oxidant or antioxidant status.
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Animals↗
Evidence for free radical-mediated lipid peroxidation at reperfusion of human orthotopic liver transplants.
BACKGROUND: Generation of toxic oxygen metabolites at reperfusion may contribute to the injury sustained as a consequence of harvest and ischemic preservation of organ allografts. Because there is a paucity of evidence that this mechanism is operative in human beings, we measured the generation of ethane into the exhaled breath as a biomarker of free radical-mediated lipid peroxidation in human liver transplantation. METHODS: A novel technique that increased the previous standard of sensitivity 100-fold was used to measure picomole quantities of ethane in exhaled breath of eight recipients undergoing human orthotopic liver transplantation. RESULTS: Ethane production correlated closely with the specific events of liver transplantation including the initial reperfusion of the allografts. In every case a twofold to threefold increase in ethane production was superimposed on a stable baseline immediately after reestablishment of portal vein blood flow through the donor liver. CONCLUSIONS: Ethane production was interpreted as evidence of hepatic lipid peroxidation, presumably mediated by toxic metabolites of oxygen occurring at reperfusion. This noninvasive approach allowed localization of the time point at which lipid peroxidation occurred and may facilitate quantification of lipid peroxidation mediated by free radicals and other toxic oxygen metabolites during operation.
Adult↗
Endothelial xanthine oxidase: a radical transducer of inflammatory signals for reticuloendothelial activation.
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Endothelium↗
Prospects for treatment of free radical-mediated tissue injury.
Toxic metabolites of oxygen are generated normally by aerobic metabolism in cells and this generation can significantly increase in certain pathologic conditions. When endogenous antioxidant defense capabilities are exceeded by this oxidant flux, tissue injury occurs. This process can be intercepted pharmacologically at different levels with agents that scavenge reactive oxygen metabolites, block their generation, or enhance endogenous antioxidant capabilities. In many situations, such as the treatment of post-ischaemic reperfusion injury, efficacy of antioxidant is related primarily to the proportionate magnitude of the total injury sustained that is due to an antioxidant mechanism. This approach has shown great promise in animal models of clinical problems and has been tested successfully in early, controlled clinical trials.
Animals↗
Vasoactive mediators and splanchnic perfusion.
OBJECTIVE: To provide an overview of the splanchnic hemodynamic response to circulatory shock. DATA SOURCES: Previous studies performed in our own laboratory, as well as a computer-assisted search of the English language literature (MEDLINE, 1966 to 1991), followed by a selective review of pertinent articles. STUDY SELECTION: Studies were selected that demonstrated relevance to the splanchnic hemodynamic response to circulatory shock, either by investigating the pathophysiology or documenting the sequelae. Article selection included clinical studies as well as studies in appropriate animal models. DATA EXTRACTION: Pertinent data were abstracted from the cited articles. RESULTS OF DATA SYNTHESIS: The splanchnic hemodynamic response to circulatory shock is characterized by a selective vasoconstriction of the mesenteric vasculature mediated largely by the renin-angiotensin axis. This vasospasm, while providing a natural selective advantage to the organism in mild-to-moderate shock (preserving relative perfusion of the heart, kidneys, and brain), may, in more severe shock, cause consequent loss of the gut epithelial barrier, or even hemorrhagic gastritis, ischemic colitis, or ischemic hepatitis. From a physiologic standpoint, nonpulsatile cardiopulmonary bypass, a controlled form of circulatory shock, has been found experimentally to significantly increase circulating levels of angiotensin II, the hormone responsible for this selective splanchnic vasoconstriction. CONCLUSIONS: While angiotensin II has been viewed primarily as the mediator responsible for the increased total vascular resistance seen during (and after) cardiopulmonary bypass, it may also cause the disproportionate decrease in mesenteric perfusion, as measured in human subjects by intraluminal gastric tonometry and galactose clearance by the liver, as well as the consequent development of the multiple organ failure syndrome seen in 1% to 5% of patients after cardiac surgery.
Angiotensin II↗
Induction of heat shock response leads to apoptosis in endothelial cells previously exposed to endotoxin.
The homeostatic response of complex eukaryotes to the challenge of environmental stress includes the induction of several programs of gene expression; among them are those for the acute phase genes and those for the heat shock genes. In some systems, the heat shock response, which is often elicited by more severe stimuli, preempts the acute phase response, which is seen in response to less severe challenges, as well as constitutive gene expression. Nevertheless, each response appears to provide a natural selective advantage for survival of the organism in a toxic environment. However, when cultured porcine endothelial cells were exposed first to a nonlethal level of bacterial endotoxin lipopolysaccharide (LPS), an inducer of the acute phase response, and then, simultaneously to standard stimuli, which normally elicit a salutary heat shock response, the cells died manifesting a pattern of DNA fragmentation (nucleolysis) characteristic of programmed cell death (apoptosis). The treatment of LPS-exposed cells with cycloheximide to block protein synthesis reproduced the lethal apoptosis that had been elicited by the induction of heat shock gene expression. Therefore, the preemption of other programs of stress gene expression by the prioritized expression of heat shock genes is associated with apoptosis.
Animals↗
Tissue perfusion in critical illnesses. Antioxidant therapy.
OBJECTIVES: To provide an overview of the mechanisms of free radical-mediated reperfusion injury and to review the available antioxidant agents and strategies available to prevent or ameliorate this injury. DATA SOURCES: Previous studies performed in our own laboratory, as well as a computer-assisted search of the English language literature (MEDLINE, 1966 to 1991), followed by a selective review of pertinent articles. STUDY SELECTION: Articles representing the mechanisms of free-radical generation, free-radical-mediated tissue injury, and antioxidant function. DATA EXTRACTION: Pertinent data were abstracted from the cited articles. RESULTS OF DATA SYNTHESIS: The small intestine is disproportionately susceptible to ischemia during circulatory shock. Ischemia activates xanthine oxidase, which then generates a massive burst of superoxide free radicals when oxygen is reintroduced at reperfusion. These radicals and their toxic oxygen metabolites, both by direct action and by the secondary activation of circulating neutrophils, then generate much of the mucosal injury that had been previously attributed to anoxia itself. CONCLUSIONS: Recent evidence indicates that although the mucosal epithelial cell is rich in xanthine oxidase, it is probably endothelial cell xanthine oxidase that triggers this mucosal reperfusion injury, not only in the intestine, but in other organs as well. Antioxidant therapies directed toward the ablation of free-radical generation at reperfusion offer a potential therapy for these injuries and their sequelae.
Animals↗
Free radicals and other reactive oxygen metabolites: clinical relevance and the therapeutic efficacy of antioxidant therapy.
As in any new field, justifiable enthusiasm for the potential for antioxidant therapy has led to hyperbole, hastily designed, poorly conceived clinical trials, and premature reporting of uncontrolled, anecdotal indicators of efficacy that have not held up when subjected to close scrutiny or more careful, controlled trial design. This tendency has been augmented by strong pressure for early positive results from a few, but not most, members of the pharmaceutical industry and by a few clinicians in highly competitive fields who were anxious not to be left behind. The sobering reality of negative or, even worse, indeterminate clinical trials has culled the field and educated those that remain. As a result, we are beginning to see the publication of quite promising results from large, well-controlled, carefully designed clinical studies, many, but not all, of which are quite promising. This has been associated with a much better understanding of the basic mechanism of free radical-mediated human disease, without which further substantial progress would be quite limited. Because the manipulation of oxidant-mediated tissue injury represents treating disease at its most basic level, the therapeutic potential of this approach remains not only promising but exciting.
Antioxidants↗
Mesenteric vasoconstriction in cardiogenic shock in pigs.
The quantitative impact of mesenteric vasoconstriction on the systemic hemodynamic response to cardiogenic shock induced by pericardial tamponade was evaluated. Graded increases in pericardial pressure produced corresponding decreases in cardiac output to 44% +/- 2% and arterial pressure to 64% +/- 3% of baseline and increases in total peripheral vascular resistance to 131% +/- 4% of baseline. Total mesenteric blood flow decreased disproportionately, to 28% +/- 3% of baseline, because of a disproportionate increase in mesenteric vascular resistance to 223% +/- 6% of baseline. Nonmesenteric vascular resistance increased only to 119% +/- 4% of baseline. Thus mesenteric vasoconstriction accounted for 42% of the increase in total peripheral resistance. Prior blockade of the renin-angiotensin axis ablated this response and eliminated the mesenteric contribution to systemic vascular resistance, while confirmed blockade of the alpha-adrenergic system or vasopressin system had no effect. Without shock, central intravenous infusions of angiotensin II (but not norepinephrine or vasopressin) closely mimicked this selective vasoconstriction. Angiotensin-mediated selective mesenteric vasoconstriction accounts for more than 40% of the overall increase in systemic vascular resistance in cardiogenic shock.
Animals↗
Free radicals and other reactive oxygen metabolites in inflammatory bowel disease: cause, consequence or epiphenomenon?
Oxygen-derived free radicals and other reactive oxygen metabolites have emerged as a common pathway of tissue injury in a wide variety of otherwise disparate disease processes. This has given rise to the hope that efforts directed towards the pharmacologic control of free radical-mediated tissue injury (Reilly, P.M., Schiller, H. J. and Bulkley, G. B. (1991) Pharmacologic approach to tissue injury mediated by free radicals and other reactive oxygen metabolites. Am. J. Surg. 161: 488-503) may have particular application to patients suffering from Crohn's disease and/or ulcerative colitis. However, because tissue injury by any mechanism, even direct mechanical trauma, can elicit an inflammatory response which entails the secondary generation of toxic oxidants by neutrophils and tissue macrophages, it is important that the evidence for this association be examined critically, so as to discriminate the possibility of an etiologic role for these toxic compounds from their presence as a reflection of injury caused primarily by other agents. Similarly, in considering the therapeutic potential of free radical ablation for the treatment of patients with IBD it is important to distinguish between interventions that might specifically block the fundamental injury mechanism from those which would act in a more nonspecific, anti-inflammatory role.
Animals↗
Breath ethane: a specific indicator of free-radical-mediated lipid peroxidation following reperfusion of the ischemic liver.
A major component of the organ injury mediated by toxic oxidants, such as seen following reperfusion of the ischemic liver, is due to the peroxidation of polyunsaturated fatty acids, especially of cell membranes. We utilized the measurement of exhaled breath ethane, a metabolic product unique to oxidant-mediated lipid peroxidation, as a noninvasive indicator of this process in swine liver subjected to warm ischemia/reperfusion. Under rigorously controlled anesthesia conditions, pig livers were subjected to 2 h of warm total ischemia, followed by reperfusion in situ. Expired air was collected and its ethane content quantitated by a novel gas chromatographic technique. The time course of breath ethane generation correlated closely with the appearance of hepatocellular injury as measured by impairment of Factor VII generation and other measures of liver integrity. Moreover, the administration of the specific superoxide free radical scavenger, superoxide dismutase (SOD), significantly attenuated both the elaboration of ethane and the hepatocellular injury. These findings not only provide confirmation of the previously reported link between hepatocellular injury by free radicals generated at reperfusion, but also establish the use of expired breath ethane analysis as a sensitive, specific, and noninvasive indicator of the injury process in real time.
Alanine Transaminase↗
Heat-shock gene expression excludes hepatic acute-phase gene expression after resuscitation from hemorrhagic shock.
BACKGROUND: To determine whether the gene expression of both acute-phase reactants (APR) and the major heat-shock protein (hsp-72) can occur simultaneously, transcriptional rates were measured during shock and resuscitation. METHODS: A nuclear runoff technique was applied to hepatic biopsy specimens obtained from pigs before shock, during 40% blood volume hemorrhagic shock (1 and 2 hours), and after resuscitation (4 and 6 hours). RESULTS: Shock-induced transcription of hsp-72 was elevated elevenfold over sham operation at 2 hours (p less than 0.02, Mann-Whitney rank test). Individually shocked animals did not transcribe both classes of stress genes but segregated into two groups: (1) strong APR transcriptional responders and (2) hsp-72 transcriptional responders. In group 2, APR transcription was significantly suppressed. Antichymotrypsin transcription was an average of eighteenfold lower in group 2 versus group 1 (p less than 0.05 at 1,2, and 6 hours). CONCLUSIONS: Different classes of stress protein genes are not transcribed simultaneously. We infer that their increased accumulation at the mRNA level is the result of sequential transcription. Hsp-72 transcription excludes that of the APR genes that may be critical to survival after stress.
Acute-Phase Proteins↗