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Research Notes: Sepsis-like Conditions and Mitochondrial Function in PWSRelated topics: Scattered throughout the medical literature about Prader-Willi Syndrome (PWS) are a disturbing number of reports of the occurrence of rapid deterioration during seemingly minor illnesses, as well as during or following surgery, including the development of sepsis-like conditions and sudden unexpected death (SED). Although the literature is mostly silent on the point, it appears that mitochondrial disorders, including significant Kreb's cycle and respiratory chain transport impairments, are common in PWS. Interestingly, rapid deterioration during minor illness and the development of sepsis-like conditions, including SED, are also relatively common in other genetic syndromes where mitochondrial function is impaired, such as Leigh syndrome, Kearns-Sayre syndrome, etc. Recent research into mitochondrial function during sepsis and sepsis-like conditions may help explain why those with PWS and other genetic syndromes characterized by impaired mitochondrial function are so vulnerable to minor illnesses and perisurgically, as it turns out that the body's proinflammatory responses to infection and injury can set off a whole cascade of biological events that results in the inhibition of pyruvate dehydrogenase and other mitochondrial enzymes, and a subsequent sharp reduction in mitochondrial respiratory chain activity, a phenomenon that has been termed "cytopathic hypoxia." That's obviously not good in someone who may already have impaired mitochondrial function and may help explain why those with PWS can have such potentially catastrophic reactions during even minor surgery or mild respiratory infections. Interestingly, research has found that vitamin C, melatonin, N-acetylcysteine and other antioxidants have remarkable protective effects against the collapse of mitochondrial function in sepsis and sepsis-like conditions. Shock. 2007 May 3. Mitochondrial dysfunction is thought to play an important role in the pathogenesis of many different disease states. It has been proposed that an acquired defect in oxidative phosphorylation prevents cells from using molecular oxygen for adenosine triphosphate production and potentially causes sepsis-induced organ dysfunction. This concept, termed cytopathic hypoxia, however, has been difficult to prove because impaired oxidative phosphorylation has never been shown to cause sepsis-induced organ failure or to be a reversible phenomenon. Presented here is a review of oxidative phosphorylation, evidence of defective electron-transport-chain function in the heart and other organ systems during sepsis, and support for a link between mitochondrial dysfunction and pathologic metabolic down-regulation. Ann Fr Anesth Reanim. 2007 Apr. OBJECTIVE: Mitochondria play a key role in energy metabolism within the cell through the oxidative phosphorylation. They are also involved in many cellular processes like apoptosis, calcium signaling or reactive oxygen species production. The objectives of this review are to understand the interactions between mitochondrial metabolism and anaesthetics or different stress situations observed in ICU and to know the clinical implications. DATA SOURCES: References were obtained from PubMed data bank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi) using the following keywords: mitochondria, anaesthesia, anaesthetics, sepsis, preconditioning, ischaemia, hypoxia. DATA SYNTHESIS: Mitochondria act as a pharmacological target for the anaesthetic agents. The effects can be toxic like in the case of the local anaesthetics-induced myotoxicity. On the other hand, beneficial effects are observed in the anaesthetic-induced myocardial preconditioning. Mitochondrial metabolism could be disturbed in many critical situations (sepsis, chronic hypoxia, ischaemia-reperfusion injury). The study of the underlying mechanisms should allow to propose in the future new specific therapeutics. J Appl Physiol. 2007 Jan 11. Loss of functional capacity of skeletal muscle is a major cause of morbidity in patients with a number of acute and chronic clinical disorders, including sepsis, COPD, heart failure, uremia and cancer. Weakness in these patients can manifest as either severe limb muscle weakness (even to the point of virtual paralysis), respiratory muscle weakness requiring mechanical ventilatory support and/or some combination of these phenomena. While factors such as nutritional deficiency and disuse may contribute to the development of muscle weakness in these conditions, systemic inflammation may be the major factor producing skeletal muscle dysfunction in these disorders. Importantly, studies conducted over the past 15 years indicate that free radical species (superoxide, hydroxyl radicals, nitric oxide, peroxynitrite, and the free radical derived product hydrogen peroxide) play an key role in modulating inflammation/infection induced alterations in skeletal muscle function. Substantial evidence exists indicating that several free radical species can directly alter contractile protein function, and evidence suggests that free radicals also have important effects on sarcoplasmic reticulum function, on mitochondrial function, and on sarcolemmal integrity. Free radicals also modulate activation of several proteolytic pathways, including proteosomally mediated protein degradation and, at least theoretically, may also influence pathways of protein synthesis. As a result, free radicals appear to play an important role in regulating a number of downstream processes that collectively act to impair muscle function and lead to reductions in muscle strength and mass in inflammatory conditions. J Appl Physiol. 2007 Jan. Previous studies indicate that ATP formation by the electron transport chain is impaired in sepsis. However, it is not known whether sepsis affects the mitochondrial ATP transport system. We hypothesized that sepsis inactivates the mitochondrial creatine kinase (MtCK)-high energy phosphate transport system. To examine this issue, we assessed the effects of endotoxin administration on mitochondrial membrane-bound creatine kinase, an important trans-mitochondrial ATP transport system. Diaphragms and hearts were isolated from control (n = 12) and endotoxin-treated (8 mg.kg(-1).day(-1); n = 13) rats after pentobarbital anesthesia. We isolated mitochondria using techniques that allow evaluation of the functional coupling of mitochondrial creatine kinase MtCK activity to oxidative phosphorylation. MtCK functional activity was established by 1) determining ATP/creatine-stimulated oxygen consumption and 2) assessing total creatine kinase activity in mitochondria using an enzyme-linked assay. We examined MtCK protein content using Western blots. Endotoxin markedly reduced diaphragm and cardiac MtCK activity, as determined both by ATP/creatine-stimulated oxygen consumption and by the enzyme-linked assay (e.g., ATP/creatine-stimulated mitochondrial respiration was 173.8 +/- 7.3, 60.5 +/- 9.3, 210.7 +/- 18.9, was 67.9 +/- 7.3 natoms O.min(-1).mg(-1) in diaphragm control, diaphragm septic, cardiac control, and cardiac septic samples, respectively; P < 0.001 for each tissue comparison). Endotoxin also reduced diaphragm and cardiac MtCK protein levels (e.g., protein levels declined by 39.5% in diaphragm mitochondria and by 44.2% in cardiac mitochondria; P < 0.001 and P = 0.009, respectively, comparing sepsis to control conditions). Our data indicate that endotoxin markedly impairs the MtCK-ATP transporter system; this phenomenon may have significant effects on diaphragm and cardiac function. Novartis Found Symp. 2007. Growing evidence suggests that mitochondrial inhibition plays a major role in the development of multiple organ failure during sepsis. Early correction of tissue hypoxia, strict control of glycaemia and modulation of oxidative and nitrosative stress may protect mitochondria during the acute inflammatory response. Once mitochondrial dysfunction has developed, the regulated induction of a hypometabolic state, analogous to hibernation, may protect the cells from severe bioenergetic failure and a critical fall in ATP. Though this is clinically manifest as organ dysfunction, it may actually represent an adaptive response to a prolonged, severe inflammatory stress. Repair of damaged organelles, stimulation of mitochondrial biogenesis and re-activation of cellular metabolism may accelerate the recovery phase and thus improve clinical outcomes. The aim of this review is to discuss putative interventions aimed at preventing or reversing mitochondrial dysfunction that may have possible clinical relevance, and to stress the importance of the correct timing of intervention. Contrib Nephrol. 2007. Background/Aims: Sepsis and multiple organ failure are complex processes that result from dysregulation of the immune response and its associated hematological, hemodynamic and metabolic disturbances. Methods: Review of the pathophysiological basis for sepsis and a review of the literature on its mechanisms of expression. Results: Sepsis is the host response to an injury, often infectious in origin, that creates both pro- and anti-inflammatory immune responses. The level and duration of this response roughly correlates with outcome. Subcellular injury characterized by increased oxidative stress defines the central mitochondrial component of this process. Treatments which minimize the amplification of this response are usually more effective at reducing tissue injury than are measures aimed at suppressing the inflammatory response. Conclusions: Sepsis is a complex process whose expression and treatment are just now being defined. Treatments that minimize the overall host response still represent the most effective strategies. Surg Infect (Larchmt). 2007 Feb. BACKGROUND AND PURPOSE: Studies in sepsis suggest that mitochondria mediate multiple organ dysfunction, including cardiac failure; however, the underlying molecular mechanisms remain elusive. This study examined changes in mitochondrial membrane integrity, antioxidant activities, and oxidative stress in the heart after infectious challenge (intratracheal Streptococcus pneumoniae, 4 x 10(6) colony-forming units). Inflammation responses also were examined. METHODS: Cardiac tissues were harvested from Sprague-Dawley rats 4, 8, 12, and 24 h after bacterial challenge (or intratracheal vehicle for sham-treated animals) and homogenized, followed by preparation of subcellular fractions (mitochondrial, cytosol, and nuclei) or whole-tissue lysate. We examined mitochondrial outer membrane damage and cytochrome C translocation to evaluate mitochondrial integrity, mitochondrial lipid and protein oxidation to assess oxidative stress, and mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities to estimate antioxidant defense. In addition, we measured nuclear factor-kappa B (NF-kappaB) activation in myocardium and cytokine production to investigate inflammatory responses to septic challenge. RESULTS: Oxidation of mitochondrial protein and lipid was evident 4 h through 24 h after bacterial challenge. Mitochondrial outer membrane damage and cytochrome C release were accompanied by down-regulation of mitochondrial SOD and GPx activity. After bacterial challenge, systemic and myocardial cytokine production increased progressively, and NF-kappaB was activated gradually. CONCLUSION: Sepsis impaired cardiac mitochondria by damaging membrane integrity, increasing oxidative stress, and altering defenses against reactive oxygen species. These alterations occur earlier than or simultaneously with inflammatory responses in myocardium after infectious challenge, suggesting that mitochondria play a role in modulating inflammation in sepsis. Am J Physiol Endocrinol Metab. 2006 Nov. Critically ill patients treated for multiple organ failure often develop muscle dysfunction. Here we test the hypothesis that mitochondrial and energy metabolism are deranged in leg and intercostal muscle of critically ill patients with sepsis-induced multiple organ failure. Ten critically ill patients suffering from sepsis-induced multiple organ failure and requiring mechanical ventilation were included in the study. A group (n = 10) of metabolically healthy age- and sex-matched patients undergoing elective surgery were used as controls. Muscle biopsies were obtained from the vastus lateralis (leg) and intercostal muscle. The activities of citrate synthase and mitochondrial respiratory chain complexes I and IV and concentrations of ATP, creatine phosphate, and lactate were analyzed. Morphological evaluation of mitochondria was performed by electron microscopy. Activities of citrate synthase and complex I were 53 and 60% lower, respectively, in intercostal muscle of the patients but not in leg muscle compared with controls. The activity of complex IV was 30% lower in leg muscle but not in intercostal muscle. Concentrations of ATP and creatine phosphate were, respectively, 40 and 34% lower, and lactate concentrations were 43% higher in leg muscle but not in intercostal muscle. We conclude that both leg and intercostal muscle show a twofold decrease in mitochondrial content in intensive care unit patients with multiple organ failure, which is associated with lower concentrations of energy-rich phosphates and an increased anaerobic energy production in leg muscle but not in intercostal muscle. Anesthesiology. 2006 Oct. Mitochondria produce metabolic energy, serve as biosensors for oxidative stress, and eventually become effector organelles for cell death through apoptosis. The extent to which these manifold mitochondrial functions are altered by previously unrecognized actions of anesthetic agents seems to explain and link a wide variety of perioperative phenomena that are currently of interest to anesthesiologists from both a clinical and a scientific perspective. In addition, many surgical patients may be at increased perioperative risk because of inherited or acquired mitochondrial dysfunction leading to increased oxidative stress. This review summarizes the essential aspects of the bioenergetic process, presents current knowledge regarding the effects of anesthetics on mitochondrial function and the extent to which mitochondrial state determines anesthetic requirement and potential anesthetic toxicity, and considers some of the many implications that our knowledge of mitochondrial dysfunction poses for anesthetic management and perioperative medicine. Neuro Endocrinol Lett. 2006 Oct. Objectives: To evaluate the changes in the mitochondrial ATP production during sepsis and the participation of iNOS in these changes. We also assessed the effect of melatonin administration in this experimental paradigm. Methods: The activity of ATPase, the level of adenine nucleotides, and the ATP production were measured in mitochondria of diaphragm and hind leg skeletal muscle of wild type (iNOS+/+) and knockout iNOS (iNOS-/-) mice. Three experimental groups were done: control group; group of septic mice induced by cecal ligation and puncture (CLP), and group of septic mice treated with melatonin. Mice were killed 24 hours after CLP. Melatonin was administrated in four doses (30 mg/kg b.w.) as follows: 30 min before CLP (i.p.) and 30 min, 4 h and 8 h after CLP (s.c.). Results: Mitochondrial production of ATP decreased in iNOS+/+ but not in iNOS-/- mice after sepsis. No changes in the ATPase activity were found in any group. Melatonin treatment normalized the production of ATP in iNOS+/+ mice, without affecting iNOS-/- animals. Conclusions: The reduction of the ATP production in iNOS+/+ but not in iNOS-/- mice suggest the participation of iNOS in the impairment of mitochondrial function in the former. Because ATPase was unaffected by sepsis, it is suggested the ATP deficit depended on the sepsis-induced respiratory chain damage. The normalization of the production of ATP with melatonin may explain the reduction of the mortality reported elsewhere in experimental and clinical sepsis after treatment with the indoleamine. J Am Coll Cardiol. 2006 Jul 18. OBJECTIVES: The purpose of this study was to test whether mitochondrial dysfunction is causative of sepsis sequelae, a mouse model of peritonitis sepsis induced by cecal ligation and perforation. Inhibition of mitochondrial permeability transition was achieved by means of pharmacological drugs and overexpression of the antiapoptotic protein B-cell leukemia (Bcl)-2. BACKGROUND: Sepsis is the leading cause of death in critically ill patients and the predominant cause of multiple organ failure. Although precise mechanisms by which sepsis leads to multiple organ dysfunction are unknown, growing evidence suggests that perturbations of key mitochondrial functions, including adenosine triphosphate production, Ca2+ homeostasis, oxygen-derived free radical production, and permeability transition, might be involved in sepsis pathophysiology. METHODS: Heart and lung functions were evaluated respectively by means of isolated heart preparation, bronchoalveolar lavage fluid protein concentration, lung wet/dry weight ratio, lung homogenate myeloperoxidase activity, and histopathologic grading. Respiratory fluxes, calcium uptake, and membrane potential were evaluated in isolated heart mitochondria. RESULTS: Peritonitis sepsis induced multiple organ dysfunction, mitochondrial abnormalities, and increased mortality rate, which were reduced by pharmacological inhibition of mitochondrial transition by cyclosporine derivatives and mitochondrial Bcl-2 overexpression. CONCLUSIONS: Our study provides strong evidence that mitochondrial permeability transition plays a critical role in septic organ dysfunction. These studies demonstrate that mitochondrial dysfunction in sepsis is causative rather than epiphenomenal and relevant in terms of vital organ function and outcome. Regarding the critical role of heart failure in the pathophysiology of septic shock, our study also indicates a potentially new therapeutic approach for treatment of sepsis syndrome. Am J Respir Crit Care Med. 2006 Jun 1. RATIONALE: Sepsis produces significant mitochondrial and contractile dysfunction in the heart, but the role of superoxide-derived free radicals in the genesis of these abnormalities is not completely understood. OBJECTIVES: The study was designed to test the hypothesis that superoxide scavenger administration prevents endotoxin-induced cardiac mitochondrial and contractile dysfunction. METHODS: Four groups of rats were studied, and animals were injected with either saline, endotoxin, endotoxin plus polyethylene glycol-adsorbed-superoxide dismutase (PEG-SOD; a free-radical scavenger), or PEG-SOD alone. Animals were killed 48 h after injections. We then measured cardiac mitochondrial generation of reactive oxygen species (ROS), formation of free-radical reaction products (protein carbonyls, lipid aldehydes, nitrotyrosine), mitochondrial function, and cardiac contractile function. MEASUREMENTS AND MAIN RESULTS: Endotoxin elicited increases in cardiac mitochondrial ROS formation (p < 0.001), increases in cardiac levels of free-radical reaction products, reductions in mitochondrial ATP generation (p < 0.001), and decrements in cardiac pressure-generating capacity (p < 0.01). Administration of PEG-SOD blocked formation of free-radical reaction products, prevented mitochondrial dysfunction, and preserved cardiac contractility. For example, mitochondrial ATP generation was 923 +/- 50, 392 +/- 32, 753 +/- 25, and 763 +/- 36 nmol/min/mg, respectively, for control, endotoxin, endotoxin + PEG-SOD, and PEG-SOD groups (p < 0.001). In addition, cardiac systolic pressure generation at a diastolic pressure of 15 mm Hg averaged 110 +/- 11, 66 +/- 7, 129 +/- 10 and 124 +/- 5 mm Hg, respectively, for control, endotoxin, endotoxin + PEG-SOD, and PEG-SOD groups (p < 0.01). CONCLUSION: These data indicate that superoxide-derived oxidants play a critical role in the development of cardiac mitochondrial and contractile dysfunction in endotoxin-induced sepsis. Pol Merkuriusz Lek. 2006 Jun. The aim of this study was to investigate the effect of lipoic acid (LA) on lipid peroxidation, hydrogen peroxide concentration (H2O2), sulphydryl group (-SH) contents and total capacity protein a few hours after administration lipopolysaccharide (LPS)-induced oxidative stress in rats. Materials and methods: Male albino Wistar rats were used in this study. The animals were divided into four groups, each group consisting of ten male animals. Control group received physiological saline alone i.v. The second group was given LA (60 mg/kg b.w., i.v.). The third second group received LPS (30 mg/kg b.w., i.v.). The fourth group received LPS (30 mg/kg b.w., i.v.) and after 0.5h received LA (60 mg/kg b.w., i.v.). In the lung homogenates were measured thiobarbituric acid reactants (TBARS), hydrogen peroxide concentration, -SH groups and protein contents. Results: The administration of LA after to LPS-induced oxidative stress caused a significant decrease in TBARS and H2O2 concentrations (appropriate 3.65 +/- 0.2 microM and 0.014 +/- 0.008 microM) compared with the group treated with LPS (appropriate 6.030 +/- 0.16 microM and 0.189 +/- 0.03 microM). Treatment of LPS-injected rats with LA caused increase in -SH groups and protein concentration (p < 0.05). Conclusion: The early administration of LA [caused] a significant decrease [in] symptoms of oxidative stress-induced LPS, [including a] decrease in lipid peroxidation process [and] H2O2 concentration and [an] increase in -SH groups and protein contents in rat's lung homogenates. Pharmacol Rep. 2006 May-Jun. Lipopolysaccharide (LPS), called endotoxin, is a major component of Gram-negative bacteria cell wall. LPS stimulates the synthesis and release of several metabolites from mammalian phagocytes which leads to fulminant systemic inflammation (endotoxic shock). Among LPS-induced metabolites, reactive oxygen species are considered to play crucial role in the pathogenesis of endotoxic shock via oxidative stress generation. In this study, the effect of early administration of antioxidant alpha-lipoic acid (LA) on plasma lipid peroxidation and total antioxidant blood capacity was evaluated in endotoxic shock in rats. Lipid peroxidation was measured as plasma thiobarbituric acid reactive substances (TBARS) levels, while total blood antioxidant capacity was assessed as ferric reducing ability of plasma (FRAP). The endotoxic shock was induced by administration of LPS (Escherichia coli 026:B6, 30 mg/kg, iv) in anesthetized rats. Then, 30 min later, animals were treated intravenously (iv) with LA at 60 mg/kg. After 5 h observation animals were killed and blood from heart was taken for TBARS and FRAP measurements. LPS injected to saline-pretreated animals resulted in development of oxidative stress indicated by significant increases in plasma TBARS and significant decrease in total antioxidant capacity of plasma. Conversely, LA injected to saline pretreated animals caused an increase in FRAP values and the decrease in TBARS levels. The administration of LA0.5 h after LPS challenge resulted in an increase in FRAP values and decrease in plasma lipid peroxidation as compared to LPS group. Moreover, the levels of TBARS and FRAP in LPS + LAgroup were similar to those observed in LA group. In conclusion, our present study demonstrates that early treatment with LA significantly protects against endotoxin-induced oxidative stress in rats. Respir Physiol Neurobiol. 2006 Apr 28. For the last half century, scientists have studied the biological importance of free radicals in respiratory and limb muscles. We now know that muscle fibers continually produce both reactive oxygen species (ROS) and nitric oxide (NO) and that both cascades play critical roles in contractile regulation. Under basal conditions, muscle-derived ROS and NO exert opposing effects. Low-level ROS activity is an essential part of the homeostatic milieu and is required for normal force production whereas NO derivatives function as a brake on the system, limiting force. The modulatory effects of ROS and NO are disrupted by conditions that exaggerate production including mechanical unloading, inflammatory disease, and strenuous exercise. Marked increases in ROS or NO levels cause contractile dysfunction, resulting in muscle weakness and fatigue. These principles provide a foundation for ongoing research to identify the mechanisms of ROS and NO action and develop interventions that protect muscle function. Int J Biochem Cell Biol. 2006 Feb. Sepsis provokes an induction of inducible nitric oxide synthase (iNOS) and melatonin down-regulates its expression and activity. Looking for an inducible mtNOS isoform, we induced sepsis by cecal ligation and puncture in both normal and iNOS knockout mice and studied the changes in mtNOS activity. We also studied the effects of mtNOS induction in mitochondrial function, and the role of melatonin against induced mtNOS and mitochondrial dysfunction. The activity of mtNOS and nitrite levels significantly increased after sepsis in iNOS+/+ mice. These animals showed a significant inhibition of the respiratory chain activity and an increase in mitochondrial oxidative stress, reflected in the disulfide/glutathione ratio, glutathione redox cycling enzymes activity and lipid peroxidation levels. Interestingly, mtNOS activity remained unchanged in iNOS-/- septic mice, and mitochondria of these animals were unaffected by sepsis. Melatonin administration to iNOS+/+ mice counteracted mtNOS induction and respiratory chain failure, restoring the redox status. The results support the existence of an inducible mtNOS that is likely coded by the same gene as iNOS. The results also suggest that sepsis-induced mtNOS is responsible for the increase of mitochondrial impairment due to oxidative stress in sepsis, perhaps due to the high production of NO. Melatonin treatment prevents mitochondrial failure at the same extent as the lack of iNOS gene. J Pineal Res. 2006 Jan. Mitochondrial nitric oxide synthase (mtNOS) produces nitric oxide (NO) to modulate mitochondrial respiration. Besides a constitutive mtNOS isoform it was recently suggested that mitochondria express an inducible isoform of the enzyme during sepsis. Thus, the mitochondrial respiratory inhibition and energy failure underlying skeletal muscle contractility failure observed in sepsis may reflect the high levels of NO produced by inducible mtNOS. The fact that mtNOS is induced during sepsis suggests its relation to inducible nitric oxide synthase (iNOS). Thus, we examined the changes in mtNOS activity and mitochondrial function in skeletal muscle of wild-type (iNOS(+/+)) and iNOS knockout (iNOS(-/-)) mice after sepsis. We also studied the effects of melatonin administration on mitochondrial damage in this experimental paradigm. After sepsis, iNOS(+/+) but no iNOS(-/-) mice showed an increase in mtNOS activity and NO production and a reduction in electron transport chain activity. These changes were accompanied by a pronounced oxidative stress reflected in changes in lipid peroxidation levels, oxidized glutathione/reduced glutathione ratio, and glutathione peroxidase and reductase activities. Melatonin treatment counteracted both the changes in mtNOS activity and rises in oxidative stress; the indole also restored mitochondrial respiratory chain in septic iNOS(+/+) mice. Mitochondria from iNOS(-/-) mice were unaffected by either sepsis or melatonin treatment. The data suggest that inducible mtNOS, which is coded by the same gene as that for iNOS, is responsible for mitochondrial dysfunction during sepsis. The results also suggest the use of melatonin for the protection against mtNOS-mediated mitochondrial failure. Free Radic Biol Med. 2006 Jan 1. Free radical-mediated mitochondrial dysfunction may play a role in the genesis of sepsis-induced multiorgan failure. Several cellular defenses protect against free radicals, including heme oxygenase. No previous study has determined if measures that increase heme oxygenase levels reduce mitochondrial dysfunction following endotoxin. The purpose of the present study was to determine if mitochondrial dysfunction following endotoxin (LPS) administration can be attenuated by administration of hemin, a pharmacological inducer of heme oxygenase. Blood pressure, heart rate, cardiac and diaphragm mitochondrial function, plasma nitrite/nitrate levels, and tissue markers of free radical generation were compared among rats given saline, LPS, hemin, or a combination of hemin and LPS. Endotoxin (LPS) administration produced large reductions in mitochondrial function (e.g., ATP production rate decreased in both tissues, P < 0.001). Administration of hemin increased tissue heme oxygenase levels, ablated LPS-induced alterations in mitochondrial function, attenuated LPS-induced increases in plasma nitrite/nitrate levels, and prevented LPS-mediated increases in tissue markers of free radical generation. These data indicate that tissue heme oxygenase levels modulate the degree of LPS-induced mitochondrial dysfunction. Measures that increase heme oxygenase levels may provide a means of reducing sepsis-induced mitochondrial dysfunction and tissue injury. Crit Care. 2006. The pathogenesis of sepsis-induced multiple organ failure may crucially depend on the development of mitochondrial dysfunction and consequent cellular energetic failure. According to this hypothesis, interventions aimed at preventing or reversing mitochondrial damage may have major clinical relevance, although the timing of such interventions will be critical to both ensuring benefit and avoiding harm. Early correction of tissue hypoxia, strict control of glycaemia, and modulation of oxidative and nitrosative stress may afford protection during the initial, acute systemic inflammatory response. The regulated induction of a hypometabolic state resembling hibernation may protect the cells from dying once energy failure has developed, allowing the possibility of functional recovery. Repair of damaged organelles through stimulation of mitochondrial biogenesis and reactivation of cellular metabolism may accelerate resolution of the multiple organ failure syndrome. Arzneimittelforschung. 2006. Sepsis impairs diaphragmatic contractility and endurance capacity and increases diaphragmatic fatigability. Several investigations have shown that administration of a number of free radical scavengers, such as N-acetylcysteine (NAC), protects the diaphragm from the development of endotoxin-mediated diaphragmatic dysfunction. The aim of this study was to evaluate the effects of melatonin (CAS 73-31-4), a naturally occurring potent antioxidant, on diaphragmatic contractility and lipid peroxidation as a marker of oxidative stress in endotoxemic rats. Rats were randomly divided into four groups: control group, endotoxemic group, melatonin group and endotoxemic plus melatonin group. Melatonin was administered by intraperitoneal injection 30 min before endotoxin inoculation to animals. Diaphragmatic function and malondialdehyde (MDA) level analysis as an indicator of lipid peroxidation were assessed 17 h after endotoxin or saline inoculation. Endotoxemia decreased the development of diaphragm fatigue and diaphragmatic MDA levels. The effects of endotoxemia on diaphragmatic contractions and fatigability were reversed and returned to control levels by melatonin administration. However, melatonin did not prevent the increase in muscle MDA content. In conclusion, the present study demonstrated that melatonin attenuated the endotoxin-induced impairment of diaphragm function. This effect of melatonin does not seem to be related to its antioxidant properties. Am J Respir Crit Care Med. 2005 Oct 1. RATIONALE: Sepsis significantly alters skeletal muscle mitochondrial function, but the mechanisms responsible for this abnormality are unknown. OBJECTIVES: We postulated that endotoxin elicits specific changes in electron transport chain proteins that produce derangements in mitochondrial function. To examine this issue, we compared the effects of endotoxin-induced sepsis on mitochondrial ATP (adenosine triphosphate) formation and electron transport chain protein composition. METHODS AND MEASUREMENTS: Diaphragm mitochondrial oxygen consumption and mitochondrial nicotinamide adenine dinucleotide, reduced form, oxidase assays were measured in control rats (n=13) and rats given endotoxin (8 mg/kg/d) for 12 (n=14), 24 (n=14), 36 (n=14), and 48 h (n=13). Electron transport chain subunits from Complexes I, III, IV, and V were isolated using Blue Native polyacrylamide gel electrophoresis techniques. MAIN RESULTS: Endotoxin administration: 1) elicited large reductions in mitochondrial oxygen consumption (e.g., 201+/-3.9 SE natoms O/min/mg for controls and 101+/-4.5 SE natoms O/minutes/mg after 48 h endotoxin, p<0.001), in nicotinamide adenine dinucleotide, reduced form, oxidase activity (p<0.002), and in uncoupled respiration (p<0.001) and 2) induced selective reductions in two subunits of Complex I, three subunits of Complex III, one subunit of Complex IV, and one subunit of Complex V. The time course of depletion of protein subunits mirrored alterations in oxygen consumption. CONCLUSIONS: Our data indicate that endotoxin selectively depletes critical components of the electron transport chain that diminishes electron flow, reduces proton pumping and decreases ATP formation. Pediatr Int. 2005 Oct. Background: Thermoregulation problems, resulting in hypo- or hyperthermia, have been infrequently reported in children with Prader Willi syndrome (PWS), yet their clinical details remained unknown. Methods: The clinical characteristics of three infants with PWS are reported. Results: Etiologies of high fever could not be identified in three children with PWS. One of these children was also admitted to the intensive care unit with extremely high body temperature in a life-threatening condition, similar to septic shock, without a plausible explanation. Conclusion: Hyperthermia may be a part of the clinical spectrum in young infants with PWS and should be carefully monitored, since it may cause life-threatening complications. J Appl Physiol. 2005 Sep. Cellular energy metabolism is altered in sepsis as a consequence of dysfunction of mitochondrial electron transport and glycolytic pathways. The purpose of the present study was to determine whether sepsis is associated with compensatory increases in gene expression of electron transport chain and glycolytic pathway proteins or, alternatively, whether gene expression decreases in sepsis, contributing to abnormalities in energy metabolism. Studies were performed using diaphragms from control and endotoxin-treated (8 mg x kg(-1) x day(-1)) rats; at 48 h after endotoxin administration, animals were killed. Microarrays and RNAse protection assays were used to assess the expression of several electron transport chain components (cytochrome-c oxidase subunits Cox 5A, Cox 5B, and Cox 6A, ATP synthase, and ATP synthase subunit 5B) and of the rate-limiting enzyme for glycolysis, phosphofructokinase (PFK). Western blotting was used to assess protein levels for these electron transport chain subunits and PFK. Activity assays were used to assess electron transport chain and phosphofructokinase function. We found that sepsis evoked 1) a downregulation of genes encoding all examined electron transport chain components (e.g., cytochrome-c oxidase 5A decreased 45 + 7%, P < 0.01) and PFK (P < 0.001), 2) reductions in protein levels for these electron transport chain subunits and PFK (P < 0.05 for each), and 3) decreases in mitochondrial state 3 respiration rates and phosphofructokinase enzyme activity (P < 0.01 for each comparison). We speculate that these sepsis-induced reductions in the expression of genes encoding critical electron transport and glycolytic proteins contribute to the development and persistence of sepsis-induced abnormalities in cellular energy metabolism. Am J Respir Crit Care Med. 2005 Jun 15. Rationale: Long-term intermittent hypoxia (LTIH) exposure in adult mice, modeling oxygenation patterns of moderate-severe obstructive sleep apnea, results in lasting hypersomnolence and is associated with nitration and oxidation injuries in many brain regions, including wake-active regions. Objectives: We sought to determine if LTIH activates inducible nitric oxide synthase (iNOS) in sleep/wake regions, and if this source of NO contributes to the LTIH-induced proinflammatory gene response, oxidative injury, and wake impairments. Methods: Mice with genetic absence of iNOS activity and wild-type control animals were exposed to 6 weeks of long-term hypoxia/reoxygenation before behavioral state recordings, molecular and biochemical assays, and a pharmacologic intervention. Measurements and main results: Two weeks after recovery from hypoxia/reoxygenation exposures, wild-type mice showed increased iNOS activity in representative wake-active regions, increased sleep times, and shortened sleep latencies. Mutant mice, with higher baseline sleep times, showed no effect of long-term hypoxia/reoxygenation on sleep time latencies and were resistant to hypoxia/reoxygenation increases in lipid peroxidation and proinflammatory gene responses (tumor necrosis factor alpha and cyclooxygenase 2). Inhibition of iNOS after long-term hypoxia/reoxygenation in wild-type mice was effective in reversing the proinflammatory gene response. Conclusions: These data support a critical role for iNOS activity in the development of LTIH wake impairments, lipid peroxidation, and proinflammatory responses in wake-active brain regions, and suggest a potential role for inducible NO inhibition in protection from proinflammatory responses, oxidative injury, and residual hypersomnolence in obstructive sleep apnea. [Note: Interesting study that suggests that those with PWS with untreated sleep-related breathing disturbances may be at increased risk for sepsis and sepsis-like conditions due to increased iNOS activity as a result of long-term intermittent hypoxia.]] Toxicology. 2005 Mar 15. Mitochondria play a central role in the life and death of cells. These organelles serve as the major energy-producing power-house, whereby the generation of ATP is associated with the utilization of molecular oxygen. A significant fraction (2-3%) of molecular oxygen consumed by mitochondria may be reduced in a one-electron fashion to yield a series of reactive oxygen species (ROS) such as superoxide anion radical, hydrogen peroxide, and hydroxyl radical. ROS are capable of damaging components of the electron transport apparatus and can, in turn, disrupt mitochondrial functioning, limiting cellular ATP levels and ultimately resulting in cell death. ROS-induced disruption of electron transport can perpetuate production of deleterious ROS and propagate mitochondrial damage. Consequently, mitochondria are highly enriched with water-soluble and lipid-soluble antioxidants (glutathione, ascorbate, Vitamin E, and coenzyme Q) and antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, catalase, thioredoxins, and peroxiredoxin. Another important antioxidant acting as a very effective scavenger of reactive lipid (peroxyl, alkoxyl) radicals is nitric oxide (NO) generated by mitochondrial nitric oxide synthase. However, NO can also be very disruptive to mitochondria function, a process facilitated by its high reactivity with superoxide. This interaction results in the formation of peroxynitrite, an oxidant capable of causing oxidative/nitrosative stress, further aggravating mitochondrial dysfunction, causing ATP depletion and damage to cells. Thus, in the most general sense, the effects of NO in mitochondria may be either protective or deleterious depending on specific conditions of local redox environment (redox potential, ratio of oxidized to reduced glutathione, transition metals, and the presence of other oxygen- and nitrogen-centered radicals). J Neurosci Res. 2005 Feb 15. Efficient functioning of maintenance and repair processes seem to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so-called longevity assurance processes, under control of several genes termed vitagenes. These include members of the heat shock protein system, and there is now evidence that the heat shock response contributes to establishing a cytoprotective state in a wide variety of human conditions, including inflammation, neurodegenerative disorders, and aging. Among the various heat shock proteins, heme oxygenase-1 has received considerable attention; it has been recently demonstrated that heme oxygenase-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Acetyl-L-carnitine is proposed as a therapeutic agent for several neurodegenerative disorders. Accordingly, we report here that treatment of astrocytes with acetyl-L-carnitine induces heme oxygenase-1 in a dose- and time-dependent manner and that this effect was associated with up-regulation of heat shock protein 60 as well as high expression of the redox-sensitive transcription factor Nrf2 in the nuclear fraction of treated cells. In addition, we show that addition of acetyl-L-carnitine to astrocytes, prior to proinflammatory lipopolysaccharide- and interferon-gamma-induced nitrosative stress, prevents changes in mitochondrial respiratory chain complex activity, protein nitrosation and antioxidant status induced by inflammatory cytokine insult. Given the broad cytoprotective properties of the heat shock response, molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, manipulation of endogenous cellular defense mechanisms via acetyl-L-carnitine may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. We hypothesize that maintenance or recovery of the activity of vitagenes may delay the aging process and decrease the risk of age-related diseases. Shock. 2005 Feb. Gram-negative bacterial infection predisposes to the development of shock and acute lung injury with multiple organ dysfunction in the critically ill. Although overexpression of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta, IL-6, IL-8, and other mediators is causally implicated in the pathogenesis of shock and lung injury, the underlying mechanisms following cellular exposure to gram-negative endotoxin remain unclear. De novo generation of reactive oxygen species (ROS) by monocytes/macrophages in particular has been proposed as a pivotal regulatory mechanism by which enhanced transactivation of redox-sensitive genes culminates in augmented cytokine expression within the lower respiratory tract. Here we sought to characterize the mechanism of action of a synthetic, nonpeptide, low-molecular-weight, Mn-containing superoxide dismutase mimetic (SODm), M40403, in modulating E. coli lipopolysaccharide serotype 0111:B4 (LPS)-induced cytokine production by cultured rat alveolar macrophages. Intracellular superoxide (O2) ion generation was measured using hydroethidine (HE) dye, and the dose-dependent effects of M40403 on TNF-alpha and IL-6 biosynthesis by ELISAs. Upstream redox-sensitive signaling events involving the pleiotropic transcription factor NF-kappaB were determined in nuclear extracts by electrophoretic mobility shift assays (EMSAs) and p65 subunit Western blot. The levels of the cytosolic inhibitory protein IkappaB-alpha were also assessed by Western analysis. We found that M40403 potently suppressed the production of superoxide, TNF-alpha, and IL-6 in LPS-stimulated alveolar macrophages, suggesting a key role for superoxide in endotoxin-induced cytokine production in the distal air spaces. In addition, M40403 decreased E. coli LPS-induced activation of NF-kappaB, and this effect was associated with modest suppression of cytoplasmic IkappaB-alpha degradation. Together, these results suggest that removal of superoxide by M40403 inhibits endotoxin-induced production of TNF-alpha and IL-6 in alveolar macrophages by a mechanism involving suppression of redox-sensitive NF-kappaB transactivation or signaling. Curr Pharm Des. 2005. Severe sepsis leading to shock is the principal cause of death in intensive care units. It is a systemic inflammatory response caused by excessive secretion of pro-inflammatory mediators, such as tumor necrosis factor-alpha (TNFalpha) and reactive oxygen species (ROS), mainly induced by endotoxin (a major component of the Gram-negative bacterial outer membrane). Immune cells use ROS in order to support their functions and need adequate levels of antioxidant defenses to avoid harmful effects of an excessive ROS production. In addition, nitric oxide (NO) is thought to play a key role in the pathogenesis of sepsis and in the development of multiple organ failure. This article discusses the toxic effects of endotoxin, paying particular attention to cardiovascular damage. It continues by analysing the mechanism by which endotoxin is recognized by specific cells of the immune system, and the pathway leading to nuclear factor-kappaB (NF-kappaB) activation and pro-inflammatory gene transcription. In relation to this process, this review focuses on the involvement of reactive oxygen and nitrogen species. Finally, the protective role of antioxidants against homeostatic disturbances such as those caused by endotoxin toxicity, their potential clinical use and the effects on the redox state of the immune cells is discussed. Ann N Y Acad Sci. 2004 Nov. Carnitine and its congeners may regulate the immune networks, and their influence on functions of immune cells predominantly or exclusively relies on carnitine-dependent energy production from fatty acids. A reduced pool of carnitines has been demonstrated in either serum or tissues, or both, from patients with a wide spectrum of disorders characterized by unregulated or impaired immune responses ranging from sepsis syndrome to systemic sclerosis, infection with human immunodeficiency virus, and chronic fatigue syndrome. Furthermore, experimental studies have consistently reported that the deranged immune responses and the less efficient inflammation towards infectious organisms associated with aging may be enhanced or modulated by treatment with carnitines. There is also evidence that carnitine deprivation could adversely affect the course of the sepsis syndrome, at least in experimental models, and preliminary studies suggest that carnitine deficiency is ultimately implicated in the pathophysiology of endotoxin-mediated multiple organ failure. Several data indicate that carnitine deficiency is a contributing factor to the progression of infection with human immunodeficiency virus, and carnitine therapy in those patients could counteract the unregulated process of lymphocyte apoptosis and improve CD4 counts. Some case reports have suggested the use of carnitine for the treatment of the severe lactic acidosis that complicates in some patients the use of reverse transcriptase inhibitors. Free Radic Biol Med. 2004 Oct 15. Compromised microvascular responsiveness is one of the key factors associated with mortality of septic patients. The present study addresses the mechanism of protection by ascorbate against impaired vasoconstriction in septic mice. Sepsis (i.e., cecal ligation and puncture (CLP) model) elevated both plasma protein carbonyl (i.e., an index of oxidative stress) and plasma nitrite/nitrate (NOx) levels, reduced baseline mean arterial blood pressure (MABP), and inhibited the MABP pressor response to angiotensin II (Ang II) at 6 h post-CLP. At the microvascular level, sepsis increased the inducible nitric oxide synthase (iNOS) mRNA level in cremaster muscle arterioles (18-25 microm diameter) at 3 h post-CLP, and impaired vasoconstriction to Ang II in these arterioles at 6 h post-CLP. At 24 h post-CLP, sepsis resulted in 9% survival. An intravenous bolus of ascorbate (200 mg/kg body wt) given 30 min prior to CLP prevented the protein carbonyl and NOx increases, partially restored the baseline arterial pressure, and completely protected against all arteriolar iNOS mRNA increases, arteriolar constriction hyporesponsiveness, and pressor response impairment. Survival increased to 65%. In septic mice, iNOS gene knockout resulted in protection of arteriolar constriction and pressor responses identical to that provided by ascorbate. Ascorbate bolus given 3 h post-CLP protected against the increase in plasma NOx concentration and against the pressor response impairment. We conclude that ascorbate may protect arteriolar vasoconstrictor responsiveness in sepsis by inhibiting excessive NO production. Lancet. 2004 Aug 7-13. Sepsis and other critical illnesses produce a biphasic inflammatory, immune, hormonal, and metabolic response. The acute phase is marked by an abrupt rise in the secretion of so-called stress hormones with an associated increase in mitochondrial and metabolic activity. The combination of severe inflammation and secondary changes in endocrine profile diminish energy production, metabolic rate, and normal cellular processes, leading to multiple organ dysfunction. This perceived failure of organs might instead be a potentially protective mechanism, because reduced cellular metabolism could increase the chances of survival of cells, and thus organs, in the face of an overwhelming insult. We propose that, first, multiple organ failure induced by critical illness is primarily a functional, rather than structural, abnormality. Indeed, it may not be failure as such, but a potentially protective, reactive mechanism. Second, the decline in organ function is triggered by a decrease in mitochondrial activity and oxidative phosphorylation, leading to reduced cellular metabolism. Third, this effect on mitochondria might be the consequence of acute-phase changes in hormones and inflammatory mediators. J Lab Clin Med. 2004 Jun. Energy-metabolism disturbances during sepsis are characterized by enhanced glycolytic fluxes and reduced mitochondrial respiration. However, it is not known whether these abnormalities are the result of a specific mitochondrial alteration, decreased pyruvate dehydrogenase (PDH) complex activity, depletion of ubiquinone (CoQ(10); electron donor for the mitochondrial complex III), or all 3. In this study we sought to specify metabolism disturbances in a murine model of sepsis, using either a PDH-activator infusion (dichloroacetate, DCA) or CoQ(10) supplementation. After anesthesia, Sprague-Dawley rats received intravenous saline solution (control; n = 5), DCA (n = 5; 20 mg/100 g), or CoQ(10) (n = 5; 1 mg/100 g), before the induction of sepsis. Increased plasma lactate levels and increased muscle glucose content were observed after 4 hours in the control group. In the DCA group, a decrease in the muscle content of lactate (P <.05) and an increase in muscle glucose content (P <.05) were observed at 4 hours, but no lactatemia variation was noted. In the CoQ(10) group, only increased plasma lactate levels were observed. Increased muscle glycolysis fluxes were observed after 4 hours in the control group, but to a slighter degree in both the DCA and CoQ(10) groups. Only DCA restored a normal temperature sensitivity in the hyperthermia range, but we noted no differences in survival time. In conclusion, only DCA infusion restores normal glycolysis function. Int Immunopharmacol. 2004 Mar. The excessive production of reactive oxygen species (ROS), associated with inflammation, leads to a condition of oxidative stress. Oxidative stress is a major contributing factor to the high mortality rates associated with several diseases such as endotoxic shock. This condition can be controlled to a certain degree by antioxidant therapies. Immune cells use ROS in order to support their functions and therefore need adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive production of ROS. This review discusses the toxic effects of endotoxin, paying particular attention to immune function. It continues by analyzing the mechanism to which specific cells of the immune system recognize endotoxin, and the resulting pathways leading to nuclear factor-kappaB activation and proinflammatory gene transcription. We also focus on the involvement of reactive oxygen and nitric oxide (NO) and the protective role of antioxidants. The potential clinical use of antioxidants in the treatment of sepsis and the effects on the redox state of the immune cells are discussed. Curr Med Chem. 2004 Feb. The pathogenetic sequence of reactions mediated by endotoxin (LPS) leading to the production of sepsis involves the oxygen radicals or reactive oxygen species, which has been evaluated in the present review. Among reactive oxygen species hydroxyl radical either singly or in combination with peroxynitrite, produces tissue damage often observed during septic injury. Inactivation of these damaging radicals by antioxidants or nitric oxide inhibitor(s) may be helpful for protecting sepsis mediated derangements but the application of these agents as drugs in humans has not been fully successful. Transcription factor NF-kappa B is reported to be the oxygen sensor in LPS induced endotoxemia. Polyphenols, especially the catechin group of compounds, are important therapeutic agents, which may be used for the treatment of endotoxin mediated sepsis. J Mol Cell Cardiol. 2004 Jan. Our previous studies indicate that hearts from septic rats have decreased work with oxygen wasting. The present studies test if there is energy deficit, changes in cardiac mitochondrial content and caspase activation during sepsis. Anesthetized, male Sprague-Dawley rats received no surgical treatment (control), laparotomy (sham), or laparotomy with cecal ligation and puncture (CLP) to induce polymicrobial septic shock. Hearts were isolated 12-14 h later. Cardiac work, oxygen consumption, substrate oxidation and energy stores were measured in perfused hearts. Normalized density of mitochondria was determined in ventricles without perfusion by morphometric analysis with electron microscopy. Citrate synthase activity was assessed in homogenates and isolated mitochondria. Cardiac work decreased significantly in CLP (47%), while oxygen consumption and glucose oxidation were unchanged compared with control or sham hearts (oxygen and substrate wasting). Tissue adenosine triphosphate, creatine phosphate and glycogen were lower in CLP hearts (energy deficit). Mitochondrial grid intersects decreased significantly from 151 +/- 8 sham to 130 +/- 4 CLP out of 361 possible intersects and autophagy was observed in CLP hearts. Total activity of citrate synthase decreased in homogenates (99 +/- 8 micromol/min/g wet weight sham vs. 62 +/- 7 CLP, P < 0.05) and in the mitochondrial fraction (27 +/- 1 micromol/min/g wet weight sham to 22 +/- 1 CLP, P < 0.05). Calculated mitochondrial content decreased from 63 +/- 4 mg protein/g wet weight sham to 46 +/- 5 CLP, P < 0.05 (mitochondrial depletion). Caspase-3 activity doubled and tumor necrosis factor alpha content tripled in CLP hearts. CONCLUSIONS. - Oxygen and substrate wasting in CLP occurs with fewer mitochondria and energy deficit, processes that are coincident with caspase-3 activation. Curr Infect Dis Rep. 2003 Oct. Sepsis is an increasingly common problem, particularly among critically ill patients. Mechanisms by which sepsis induces organ dysfunction have not been elucidated. The coexisting findings (unique to sepsis) of metabolic acidosis yet increased tissue oxygen tensions suggest cellular availability but decreased use of oxygen (tissue dysoxia). Because mitochondria use more than 90% of total body oxygen consumption for adenosine triphosphate (ATP) generation, a bioenergetic abnormality is implied. Cell and animal data have shown that nitric oxide (and its metabolites), produced in considerable excess in patients with sepsis, can affect oxidative phosphorylation by inhibiting several of its component respiratory enzymes. Human data are scarce. However, in skeletal muscle biopsies taken from patients with sepsis, we have recently demonstrated a relationship between increased nitric oxide production, antioxidant depletion, reduced respiratory chain complex I activity, and low ATP levels. These findings correlated with severity of disease and outcome and support the notion that mitochondrial dysfunction resulting in bioenergetic failure may be an important factor in the pathophysiology of sepsis-associated multiorgan failure. However, a reasonable argument can be made that the reduction in energy supply could represent a last-ditch adaptive response to ongoing inflammation, resulting in a cellular shutdown analogous to hibernation that allows eventual restoration of organ function and long-term survival in patients fit enough to survive the acute phase. Free Radic Res. 2003 Sep. The excessive production of reactive oxygen species (ROS) associated with inflammation leads to oxidative stress, which is involved with the high mortality from several diseases such as endotoxic shock and can be controlled to a certain degree by antioxidants. The immune cells use ROS in order to support their functions and, therefore, need adequate levels of antioxidant defenses in order to avoid the harmful effect of an excessive ROS production. In the present work, the effect of the administration of the antioxidant N-acetylcysteine (NAC) on the redox state of peritoneal macrophages and lymphocytes from mice with lethal endotoxic shock (100 mg/kg i.p. of lipopolysaccharide, LPS), was studied. In both types of immune cells at 0, 2, 4, 12 and 24 h after LPS injection, an increase of ROS, of the proinflammatory cytokine tumor necrosis factor alpha (TNFalpha), the lipid peroxidation (malonaldehyde levels, MDA), inducible nitric oxide synthase (iNOS) expression and the oxidized/reduced glutathione (GSSG/GSH) ratio, as well as a decrease of enzymatic antioxidant defenses, such as superoxide dismutase (SOD) and catalase (CAT) activity, was observed. The injection of NAC (150 mg/kg i.p. at 30 min after LPS injection) decreased the ROS, the TNFalpha the MDA levels, iNOS expression and the GSSG/GSH ratio, and increased the antioxidant defenses in both macrophages and lymphocytes. Moreover, the NAC treatment prevented the activation of nuclear translocation of the nuclear factor kappaB (NF-kappaB), which regulates ROS, inflammatory cytokines and antioxidant levels. Our present results provide evidence that both cell types have a relevant role in the pathogenesis of endotoxic shock, and that NAC, by improving the redox state of these immune cells, could increase mouse survival. Thus, antioxidants could offer an alternative treatment of human endotoxic shock. Am J Physiol Regul Integr Comp Physiol. 2003 Jul. Inducible nitric oxide synthase (iNOS) expression in blood vessels contributes to the vascular hyporeactivity characteristic of sepsis. Our previous work demonstrated in vitro that ascorbate inhibits iNOS expression in lipopolysaccharide- and interferon-gamma-stimulated skeletal muscle endothelial cells (ECs) through an antioxidant mechanism. The present study evaluated in vivo the hypothesis that administration of ascorbate decreases oxidative stress, prevents endothelial iNOS expression, and improves vascular reactivity in septic skeletal muscle. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Plasma nitrite and nitrate (NOx) levels were elevated by 6 h after CLP. Prior ascorbate bolus injection (200 mg/kg body wt iv) blocked the elevation of plasma NOx and abolished the expression of iNOS protein and activity in the septic skeletal muscle. We also demonstrated that iNOS mRNA determined by RT-PCR was induced in the microvascular ECs of the muscle at 3 h after CLP. This induction was attenuated by prior ascorbate administration. Ascorbate inhibition of iNOS expression was associated with decreased oxidant levels in the septic muscle. Moreover, ascorbate administration restored partially the baseline arterial pressure and preserved completely the microvascular constriction and arterial pressure responses to norepinephrine in CLP mice. These results suggest that early administration of ascorbate may be a valuable adjunct treatment of sepsis. FASEB J. 2003 May. Mitochondrial nitric oxide synthase (mtNOS) is expressed constitutively, although it might be induced. Nitric oxide (NO) is a physiological regulator of mitochondrial respiration. Melatonin prevents mitochondrial oxidative damage and inhibits iNOS expression induced by bacterial lipopolysaccharide (LPS). The loss of melatonin with age may be related to the age-dependent mitochondrial damage. Thus, we examined the protective role of melatonin against the effects of LPS on mtNOS and on respiratory complexes activity in liver and lung mitochondria from young and old rats. The activity of mtNOS in control lung was low and did not change with age. LPS administration (10 mg/kg, i.v.) significantly increased mtNOS expression and activity and NO production in lung mitochondria, and the effect was greater in old rats. LPS administration also reduced the age-dependent decrease of the respiratory complexes I and IV. Melatonin administration (60 mg/kg, i.p.) prevented the LPS toxicity, decreasing mitochondrial NOS activity and NO production. Melatonin also counteracted LPS-induced inhibition of complexes I and IV. In general, the actions of melatonin were stronger in older animals than in younger ones. The results suggest that an inducible component of mtNOS, together with mitochondrial damage, occurs during sepsis, and melatonin prevents the mitochondrial failure that occurs during endotoxemia. Am J Physiol Renal Physiol. 2003 Mar. Acute renal failure (ARF) during sepsis is associated with increased nitric oxide (NO) and oxygen radicals, including superoxide (O(2)(-)). Because O(2)(-) reacts with NO in a rapid manner, it plays an important role in modulating NO levels. Therefore, scavenging of O(2)(-) by superoxide dismutase (SOD) may be critical for preserving NO bioavailability. In mice, substantial renal extracellular SOD (EC-SOD) expression implies its important role in scavenging O(2)(-) in the kidney. We hypothesized that during endotoxemic ARF, EC-SOD is decreased in the kidney, resulting in increased O(2)(-) and thus decreased vascular NO bioavailability with resultant renal vasoconstriction and ARF. In the present study, normotensive endotoxemic ARF was induced in mice using lipopolysaccharide (LPS; 5 mg/kg ip). Sixteen hours after LPS, glomerular filtration rate (GFR; 50 +/- 16 vs. 229 +/- 21 microl/min, n = 8, P < 0.01) and renal blood flow (RBF; 0.61 +/- 0.10 vs. 0.86 +/- 0.05 ml/min, n = 8, P < 0.05) were subsequently decreased. EC-SOD mRNA and protein expression in endotoxemic kidneys were decreased at 16 h compared with controls. A catalytic antioxidant, metalloporphyrin, reversed the deleterious effects of endotoxemia on renal function as GFR (182 +/- 40 vs. 50 +/- 16 microl/min, n = 6, P < 0.01) and RBF (1.08 +/- 0.10 vs. 0.61 +/- 0.10 ml/min, n = 6, P < 0.05) were preserved. Similar results were obtained with tempol, a chemically dissimilar antioxidant. Specific inhibition of inducible nitric oxide synthase (iNOS), l-N(6)-(1-iminoethyl)-lysine, reversed the renal protective effect on GFR and RBF observed with antioxidant treatment during endotoxemia. In summary, renal EC-SOD expression is decreased during endotoxemia. Antioxidant therapy preserved GFR and RBF during endotoxemia. The reversal of this protective effect by inhibition of iNOS suggests the importance of the bioavailability of NO for preservation of renal function during early endotoxemia. Int Immunopharmacol. 2003 Jan. Changes in several functions of peritoneal macrophages from mice with oxidative stress caused by intraperitoneal injection of endotoxin (Escherichia coli lipopolysaccharide, LPS) (100 mg/kg), and associated with a high production of reactive oxygen species (ROS), have been observed in our previous studies. Antioxidants such as N-acetylcysteine (NAC) are free radical scavengers that improve and modulate the immune response, especially in oxidative stress situations. Therefore, in the present work, we have studied the effects of the administration of NAC (150 mg/kg i.p.) on different functions of peritoneal macrophages from Swiss mice suffering that oxidative stress, caused by LPS (100 mg/kg). NAC was injected 30 min after LPS injection, and the peritoneal macrophages were obtained at 2, 4, 12, and 24 h after endotoxin injection. The following functions, key stages of the phagocytic process, were studied: adherence to substrate, chemotaxis, ingestion of particles, and production of ROS (reactive oxygen species), as well as tumor necrosis factor (TNFalpha) release. The decrease in chemotaxis and the increase in adherence, ingestion, superoxide anion production, and TNFalpha release shown by macrophages from animals with oxidative stress were counteracted by NAC injection. These data suggest that NAC administration may be useful for the treatment of oxidative stress-linked endotoxic shock, modulating the function of macrophages, specifically in decreasing the production of ROS and of inflammatory cytokines such as TNFalpha. Free Radic Res. 2003 Jan. The immune cells, such as phagocytes and lymphocytes, which use reactive oxygen species (ROS) for carrying out many of their functions, need appropriate levels of intracellular antioxidants to avoid the harmful effect of oxidative stress. In previous studies, we have observed changes in several functions of those leukocytes from female BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of Escherichia coli 055:B5 lipopolysaccharide (LPS) (100 mg/kg), which were associated with high ROS production. In the present study, we have investigated the redox state of the above mentioned immune cells in that lethal endotoxic shock model measuring the oxidant/antioxidant balance through the following parameters: production of ROS, proinflammatory cytokine TNFalpha reduced glutathione (GSH), oxidized glutathione (GSSG), superoxide dismutase (SOD) and catalase (CAT) activities, malonaldehyde (MDA) and transcription factor NF-kappaB expression at different times after LPS injection. The results show an increase in ROS, TNFalpha and MDA production in both cell types, being higher in macrophages than in lymphocytes. GSSG/GSH ratio was increased in both macrophages and lymphocytes after LPS injection. With respect to the activity of the antioxidant enzymes SOD and CAT were decreased in both macrophages and lymphocytes. The activation of the transcription factor NF-kappaB was stimulated in macrophages and lymphocytes. These results point out that both lymphocytes and macrophages, which are able to play an important role in host response to endotoxin, show an oxidative stress thus contributing to the pathogenesis of this septic shock. Physiol Res. 2003. Free radicals and proinflammatory cytokines from phagocytes have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin. In the present study, male BALB/c and Swiss mice received intraperitoneally lipopolysaccharide (LPS) at 100 mg/kg and 150 mg/kg, respectively, that led to a lethal endotoxic shock (100 % of mortality before 30 h). Swiss mice injected with 100 mg/kg, that did not show lethal endotoxic shock, were also studied. Peritoneal macrophages were obtained from animals at 2, 4, 12 or 24 h after injection of LPS or saline (control) solutions. Superoxide anion and tumor necrosis factor (TNFalpha) production were determined in these cells as well as other functions such as adherence capacity, chemotaxis and phagocytosis. The increase in superoxide anion production after endotoxin injection was higher in cells from mice with lethal shock than in those with non-lethal shock. However, the enhancement of TNFalpha production was similar in all cases, although in Swiss mice the highest levels of TNFalpha were observed at 1.5 h after endotoxin injection, while in BALB/c mice they occurred at 2 h after LPS injection. This oxidative stress was also revealed by the other functions analyzed, since adherence to substrate and phagocytosis were stimulated and chemotaxis was decreased after endotoxin injection as compared to controls, the differences being even more significant in animals with lethal shock. These data suggest that these changes, mainly the increased production of free radicals even more than the TNFalpha release, could be involved in mouse mortality caused by LPS. Crit Care. 2002 Dec. The rate of oxygen consumption by certain tissues is impaired when mice or rats are injected with lipopolysaccharide. A similar change in the rate of oxygen consumption is observed when Caco-2 human enterocyte-like cells are incubated in vitro with cytomix, a cocktail of cytokines containing tumor necrosis factor, IL-1beta, and IFN-gamma. The decrease in the rate of oxygen consumption is not due to a change in oxygen delivery (e.g. on the basis of diminished microvascular perfusion), but rather to an acquired intrinsic defect in cellular respiration, a phenomenon that we have termed 'cytopathic hypoxia'. A number of different biochemical mechanisms have been postulated to account for cytopathic hypoxia in sepsis, including reversible inhibition of cytochrome a,a3 by nitric oxide, and irreversible inhibition of one or more mitochondrial respiratory complexes by peroxynitrite. Recently, however, our laboratory has obtained data to suggest that the most important mechanism underlying the development of cytopathic hypoxia is depletion of cellular stores of nicotinamide adenine dinucleotide (NAD+/NADH) as a result of activation of the enzyme, poly(ADP-ribose) polymerase-1. If cytopathic hypoxia is important in the pathophysiology of established sepsis and multiorgan dysfunction syndrome, then efforts in the future will need to focus on pharmacological interventions designed to preserve normal mitochondrial function and energy production in sepsis. Toxicology. 2002 Oct 30. Septic shock is a serious problem in critically ill and surgical patients throughout the world. It is a systemic inflammatory response caused by excessive secretion of proinflammatory mediators, such as tumor necrosis factor-alpha, mainly induced by endotoxin, a major component of the Gram-negative bacterial outer membrane. Experimental evidence suggests that reactive oxygen species (ROS) may be important mediators of cellular injury during endotoxemia, either as a result of macromolecular damage or by interfering with extracellular and intracellular regulatory processes. In addition, nitric oxide is thought to play a key role in the pathogenesis of sepsis. This review begins with a brief overview of the toxic effects of endotoxin at organism level, paying particular attention to cardiovascular damage. It continues by analysing the mechanism by which endotoxin is recognized by specific cells of the immune system, which then respond to bacterial infection and the pathway leading to nuclear factor-kappaB activation and proinflammatory gene transcription. With regard to this process, the review focuses on the involvement of reactive oxygen and nitrogen species. Lastly, the protective role of antioxidants against endotoxin toxicity and their potential clinical use is discussed. Rev Mal Respir. 2002 Oct. In sepsis contractile weakness of the diaphragm is a major cause of the onset of respiratory failure. This muscular weakness is the result of haemodynamic and metabolic disorders secondary to sepsis and also the damaging effects of inflammatory mediators, among which oxygen free radicals play a crucial role. This role is demonstrated by the protective effect of various exogenous anti-oxidants on diaphragmatic contraction. Early in the course of sepsis there is, in animal models and in man, an increased production of oxygen free radicals and nitric oxide (NO) in the diaphragm, principally within the mitochondria. The formation of peroxinitrite as the result of the action of NO on superoxide anions impairs mitochondrial respiration and consequently the energy production necessary for diaphragmatic contraction. Among the endogenous anti-oxidant systems haem oxygenase, which splits haemoglobin into bilirubin, iron and carbon monoxide, is an effective system for the protection of diaphragmatic function by limiting the damage of oxidant stress. Nevertheless a transient deficiency of local anti-oxidant defences during the early stages of sepsis, when the production of oxygen free radicals is intense, encourages the onset of contractile weakness. Biochem Biophys Res Commun. 2002 Aug 30. Reduction-oxidation (redox) state constitutes such a potential signaling mechanism for the regulation of an inflammatory signal associated with oxidative stress. Exposure of alveolar epithelial cells to ascending DeltapO(2) regimen+/-reactive oxygen species (ROS)-generating systems induced a dose-dependent release of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha. Similarly, the Escherichia coli-derived lipopolysaccharide-endotoxin (LPS) up-regulated cytokine biosynthesis in a dose- and time-dependent manner. Irreversible inhibition of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of glutathione (GSH), by L-buthionine-(S,R)-sulfoximine (BSO), induced the accumulation of ROS and augmented DeltapO(2) and LPS-mediated release of cytokines. Analysis of the molecular mechanism implicated revealed an inhibitory-kappaB (IkappaB-alpha)/nuclear factor-kappaB (NF-kappaB)-independent pathway in mediating redox-dependent regulation of inflammatory cytokines. BSO stabilized cytosolic IkappaB-alpha and down-regulated its phosphorylation, thereby blockading NF-kappaB activation, yet it augmented cytokine secretion. Glutathione depletion is associated with the augmentation of oxidative stress-mediated inflammatory state in a ROS-dependent mechanism and the IkappaB-alpha/NF-kappaB pathway is redox-sensitive but differentially involved in regulating redox-dependent regulation of cytokines. Lancet. 2002 Jul 20. BACKGROUND: Sepsis-induced multiple organ failure is the major cause of mortality and morbidity in critically ill patients. However, the precise mechanisms by which this dysfunction is caused remain to be elucidated. We and others have shown raised tissue oxygen tensions in septic animals and human beings, suggesting reduced ability of the organs to use oxygen. Because ATP production by mitochondrial oxidative phosphorylation accounts for more than 90% of total oxygen consumption, we postulated that mitochondrial dysfunction results in organ failure, possibly due to nitric oxide, which is known to inhibit mitochondrial respiration in vitro and is produced in excess in sepsis. METHODS: We did skeletal muscle biopsies on 28 critically ill septic patients within 24 h of admission to intensive care, and on nine control patients undergoing elective hip surgery. The biopsy samples were analysed for respiratory-chain activity (complexes I-IV), ATP concentration, reduced glutathione (an intracellular antioxidant) concentration, and nitrite/nitrate concentrations (a marker of nitric oxide production). FINDINGS: Skeletal muscle ATP concentrations were significantly lower in the 12 patients with sepsis who subsequently died than in the 16 septic patients who survived (p=0.0003) and in controls (p=0.05). Complex I activity had a significant inverse correlation with norepinephrine requirements (a proxy for shock severity, p=0.0003) and nitrite/nitrate concentrations (p=0.0004), and a significant positive correlation with concentrations of reduced glutathione (p=0.006) and ATP (p=0.03). INTERPRETATION: In septic patients, we found an association between nitric oxide overproduction, antioxidant depletion, mitochondrial dysfunction, and decreased ATP concentrations that relate to organ failure and eventual outcome. These data implicate bioenergetic failure as an important pathophysiological mechanism underlying multiorgan dysfunction. Crit Care Med. 2002 Feb. OBJECTIVE: This study was designed to determine whether mitochondrial function in a systemic organ is acutely impaired in a resuscitated model of sepsis (endotoxemia, lipopolysaccharide) and the relationship, if any, between this impairment and the extent of mitochondrial ultrastructural damage that occurs. DESIGN: Perspective, controlled laboratory investigation. SETTING: Animal laboratory in a university research institute. SUBJECTS: Adult male cats. INTERVENTIONS: A well-established feline model of acute endotoxemia was used wherein measures were taken to minimize tissue hypoxia. After lipopolysaccharide (3 mg/kg intravenously, n = 9) or isotonic saline vehicle (control, n = 5) administration, liver samples were obtained at 4 hrs posttreatment, and mitochondrial ultrastructure and respiratory function were assessed. Mitochondrial ultrastructural injury was graded on a scale of 0 (no injury) to 5 (severe injury), and mitochondrial respiration was evaluated by using standard techniques. MEASUREMENTS AND MAIN RESULTS: Significant mitochondrial injury was apparent by 4 hrs, but only in the lipopolysaccharide-treated group (2.5 +/- 0.2 vs. 1.3 +/- 0.2, p <.001) and despite maintenance of tissue oxygen availability. In addition, lipopolysaccharide treatment reduced the rate of state 3 (adenosine 5'-diphosphate-dependent) respiration, especially at complex IV (40% inhibition), and increased the rate of state 4 (adenosine 5'-diphosphate-independent) respiration, reflecting partial uncoupling of mitochondrial oxidative phosphorylation. Finally, a significant correlation was demonstrated between the severity of ultrastructural injury and the magnitude of mitochondrial respiratory dysfunction after lipopolysaccharide treatment and despite resuscitation efforts. CONCLUSION: Mitochondrial function is significantly impaired during acute sepsis, and this impairment is strongly associated with the extent of mitochondrial ultrastructural abnormalities present in the tissues. These findings in conjunction with those previously shown suggest that mitochondrial functional impairment may contribute to the pathogenesis of altered oxygen metabolism in systemic organs during sepsis. Crit Care Clin. 2002 Jan. Several lines of evidence indicate that cellular energetics are deranged in sepsis, not by inadequate tissue perfusion but rather by impaired mitochondrial respiration; that is, organ dysfunction in sepsis may result from cytopathic hypoxia. If this concept is correct, the therapeutic implications are enormous. Efforts to improve outcome in septic patients by monitoring and manipulating cardiac output, systemic oxygen (DO2), and regional blood flow are doomed to failure. Instead, the focus should be on developing pharmacologic strategies (e.g., isoform-selective iNOS or PARP inhibitors) to restore normal mitochondrial function and cellular energetics. Free Radic Res. 2002 Jan. Reactive oxygen species (ROS) and proinflammatory cytokines produced by immune cells cause the oxidative stress involved in septic shock induced by endotoxin. This oxidative stress can be controlled to a certain degree by antioxidants, which is specially important for a type of immune cell, i.e. the phagocyte, that uses ROS to kill microorganisms and needs antioxidants in order to support its functions. In a previous study we have observed changes in several functions of peritoneal macrophages from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg), which were associated with a high production of superoxide anion. N-acetylcysteine (NAC) is a thiolic antioxidant that improves the immune response, and we have observed that when administered intraperitoneally (150 mg/kg) at 30 min after LPS injection it counteracts the effects of LPS on macrophages and lymphocytes. In the present work, we have studied the in vitro effect of several concentrations of NAC (0.001, 0.01, 0.1, 1 and 2.5 mM) on the following functions: adherence to substrate, chemotaxis, ingestion of particles, ROS production and the release of tumor necrosis factor (TNFalpha) of peritoneal macrophages from BALB/c mice at 2, 4,12 and 24 h after LPS injection. The results show that the administration of NAC (especially at 0.1 mM) decreases raised adherence, ingestion, ROS production and TNFalpha levels in macrophages from animals injected with LPS, bringing these functions to values near those of control animals. These effects which seem to be linked to a modulation of NF-kappaB, suggest that the improvement of immune functions observed in previous work after injection of NAC to animals with endotoxic shock could be due to a direct action of this thiol antioxidant on immune cells. Mol Cell Biochem. 2002 Jan. Oxidative stress, associated with a high production of reactive oxygen species (ROS) by immune cells, is involved in the endotoxic shock caused by endotoxin. This oxidative stress is linked to the inability of the immune cells to maintain adequate levels of antioxidants with free radical-scavenging action. Glutathione (GSH) and ascorbic acid (AA) are intracellular and extracellular antioxidants (ROS scavengers) that improve the leukocyte functions. Therefore, in the present work we have determined the reduced GSH and AA content in axillary nodes, spleen, thymus and peritoneal mononuclear leukocytes from BALB/c mice subjected to lethal endotoxic shock produced by intraperitoneal injection of E. coli lipopolysaccharide (LPS, 100 mg/kg), at several times (0, 2, 4, 12 and 24 h) after LPS injection. Endotoxic shock decreased the levels of AA in the leukocytes from the three organs as well as the levels of GSH in axillary nodes and spleen cells while it increased the GSH levels in thymus and peritoneum. These results are in agreement with the oxidative stress and the altered function previously observed in those leukocytes, and they suggest that antioxidant administration may be useful for the treatment of endotoxic shock and other oxidative stress situations with altered immunological responses. Free Radic Res. 2001 Dec. Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30 h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24 h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24 h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30-60 min of incubation in cells from controls and at 10 min in cells from treated mice 12-24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it. J Neuroimmunol. 2001 Nov 1. We determined innate and adaptive immune responses in children with developmental regression and autism spectrum disorders (ASD, N=71), developmentally normal siblings (N=23), and controls (N=17). With lipopolysaccharide (LPS), a stimulant for innate immunity, peripheral blood mononuclear cells (PBMCs) from 59/71 (83.1%) ASD patients produced >2 SD above the control mean (CM) values of TNF-alpha, IL-1beta, and/or IL-6 produced by control PBMCs. ASD PBMCs produced higher levels of proinflammatory/counter-regulatory cytokines without stimuli than controls. With stimulants of phytohemagglutinin (PHA), tetanus, IL-12p70, and IL-18, PBMCs from 47.9% to 60% of ASD patients produced >2 SD above the CM values of TNF-alpha depending on stimulants. Our results indicate excessive innate immune responses in a number of ASD children that may be most evident in TNF-alpha production. Free Radic Res. 2001 Jul. Oxidative stress associated with reactive oxygen species (ROS) and cytokines produced by immune cells, which is involved in septic shock caused by endotoxin, can be controlled to a certain degree by antioxidants with free radical scavenging action. N-acetylcysteine (NAC) and ascorbic acid (AA) are ROS scavengers that improve the immune response, and modulate macrophage function in mice with endotoxin-caused oxidative stress. Therefore, we have investigated the in vitro effects of these antioxidants on the functions of lymphocytes from BALB/c mice with lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg). Adherence to tissues and chemotaxis (the earliest two functions of lymphocytes in the immune response), as well as ROS levels and TNF alpha production were determined in the presence or absence of NAC or AA (0.001, 0.01, 0.1, 1 and 2.5 mM) in lymphocytes from peritoneum, axillary nodes, spleen and thymus obtained at several times (2, 4, 12 and 24 hours) after LPS injection. Endotoxic shock decreases the chemotaxis of lymphocytes from all the above localizations and increases their adherence, TNF alpha and ROS production. These changes in lymphocyte function were counteracted by NAC and AA, bringing these functions to values near those of control animals. Our data suggest that lymphocytes are important targets of endotoxins contributing to oxidative stress by septic shock, and that antioxidants can preserve the function of lymphocytes, preventing the homeostatic disturbances caused by endotoxin. Am J Respir Cell Mol Biol. 2001 Feb. Recent studies have indicated that sepsis is associated with enhanced generation of several free-radical species (nitric oxide [NO], superoxide, hydrogen peroxide) in skeletal muscle. It is also known that this enhanced free-radical generation results in reductions in skeletal muscle force-generating capacity, but the precise mechanism(s) by which free radicals exert this effect in sepsis has not been determined. We postulated that free radicals might react directly with the contractile proteins in this condition, altering contractile protein force-generating capacity. To test this theory, we compared the force generation of single Triton-skinned diaphragmatic fibers (Triton skinning exposes the contractile apparatus, permitting direct assessment of contractile protein function) from the following groups of rats: (1) control animals; (2) endotoxin-treated animal; (3) animals given endotoxin plus polyethylene glycol- superoxide dismutase (PEG-SOD), a superoxide scavenger; (4) animals given endotoxin plus N(omega)-nitro-L-arginine methylester (L-NAME), a NO synthase inhibitor; (5 ) animals given only PEG-SOD or L-NAME; and (6 ) animals given endotoxin plus denatured PEG-SOD. We found that endotoxin administration produced both a reduction in the maximum force-generating capacity (Fmax) (i.e., a decrease in Fmax) of muscle fibers and a reduction in fiber calcium sensitivity (i.e., an increase in the Ca2+ concentration required to produce half-maximal activation [Ca50]). L-NAME and PEG-SOD administration preserved Fmax and Ca50 in endotoxin-treated animals; neither drug affected these parameters in non-endotoxin treated animals. Denatured PEG-SOD failed to inhibit endotoxin-related alterations in contractile protein function. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of skinned fibers from endotoxin-treated animals revealed a selective depletion of several proteins; administration of L-NAME or PEG-SOD to endotoxin-treated animals prevented this protein depletion, paralleling the effect of these two agents to prevent a reduction in contractile protein force-generating capacity. These data indicate that free radicals (superoxide, NO, or daughter species of these radicals) play a central role in altering skeletal muscle contractile protein force-generating capacity in endotoxin-induced sepsis. Crit Care Clin. 2001 Jan. Several lines of evidence support the notion that cellular energetics are deranged in sepsis, not on the basis of inadequate tissue perfusion, but rather on the basis of impaired mitochondrial respiration and/or coupling; that is, organ dysfunction in sepsis may occur on the basis of cytopathic hypoxia. If this concept is correct, then the therapeutic implications are enormous. Efforts to improve outcome in patients with sepsis by monitoring and manipulating cardiac output, systemic Do2, and regional blood flow are doomed to failure. Instead, the focus should be on developing pharmacologic strategies to restore normal mitochondrial function and cellular energetics. Free Radic Biol Med. 2001 Jan 1. Recent studies indicate that sepsis is associated with enhanced generation of several free radical species (nitric oxide, superoxide, hydrogen peroxide) in skeletal muscle. While studies suggest that free radical generation causes uncoupling of oxidative phosphorylation in sepsis, no previous report has examined the role of free radicals in modulating skeletal muscle oxygen consumption during State 3 respiration or inhibiting the electron transport chain in sepsis. The purpose of the present study was to examine the effects of endotoxin-induced sepsis on State 3 diaphragm mitochondrial oxygen utilization and to determine if inhibitors/scavengers of various free radical species would protect against these effects. We also examined mitochondrial protein electrophoretic patterns to determine if observed endotoxin-related physiological derangements were accompanied by overt alterations in protein composition. Studies were performed on: (a) control animals, (b) endotoxin-treated animals, (c) animals given endotoxin plus PEG-SOD, a superoxide scavenger, (d) animals given endotoxin plus L-NAME, a nitric oxide synthase inhibitor, (e) animals given only PEG-SOD or L-NAME, (f) animals given endotoxin plus D-NAME, and (g) animals given endotoxin plus denatured PEG-SOD. We found: (a) no alteration in maximal State 3 mitochondrial oxygen consumption rate at 24 h after endotoxin administration, but (b) a significant reduction in oxygen consumption rate at 48 h after endotoxin, (c) no effect of endotoxin to induce uncoupling of oxidative phosphorylation, (d) either PEG-SOD or L-NAME (but neither denatured PEG-SOD nor D-NAME) prevented endotoxin-mediated reductions in State 3 respiration rates, (e) some mitochondrial proteins underwent tyrosine nitrosylation at 24 h after endotoxin administration, and (f) SDS-page electrophoresis of mitochondria from endotoxin-treated animals revealed a selective depletion of several proteins at 48 h after endotoxin administration (but not at 24 h); (g) administration of L-NAME or PEG-SOD prevented this protein depletion. These data provide the first evidence that endotoxin-induced reductions in State 3 mitochondrial oxygen consumption are free radical-mediated. Cytokines Cell Mol Ther. 2000 Dec. Chemioxyexcitation [deltapO2/reactive oxygen species (ROS)] constitutes a potential signaling mechanism for regulating an inflammatory signal associated with oxidative stress. Exposure of fetal alveolar type II epithelial cells to an ascending deltaPO2 regimen with or without the hydroxyl radical (OH) or the superoxide radical anion (O2*-) induces a dose-dependent release of pro-inflammatory cytokines. Similarly, the Escherichia coli-derived lipopolysaccharide (LPS) upregulates cytokine biosynthesis in a dose- and time-dependent manner. Irreversible inhibition by L-buthionine-(S,R)-sulfoximine (BSO) of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of glutathione (GSH), induces intracellular accumulation of ROS and augments chemioxyexcitation and LPS-mediated release of interleukin (IL)-1beta, IL-6, and tumor necrosis factor alpha (TNF-alpha). Analysis of the molecular mechanism implicated reveals an inhibitory kappaB (IkappaB-alpha)/nuclear factor kappaB (NF-kappaB)-independent pathway mediating the redox-dependent regulation of inflammatory cytokines. Although BSO stabilizes cytosolic IkappaB-alpha and downregulates its phosphorylation, thereby blockading NF-kappaB activation, it augments cytokine biosynthesis in a dose-dependent manner. These results indicate that glutathione depletion is associated with augmentation of an oxidative stress-mediated pro-inflammatory state in an ROS-dependent mechanism and that the IkappaB-alpha/NF-kappaB pathway is otherwise not necessarily indispensable for redox-mediated regulation of cytokines. Am J Respir Crit Care Med. 2000 Dec. We investigated the effects of polyethylene glycol-adsorbed superoxide dismutase (PEG-SOD), polyethylene glycol-adsorbed catalase (PEG-CAT), and DMSO on diaphragmatic contractility and malondialdehyde (MDA) concentrations in septic peritonitis in vitro. One hundred eighty-six rats were divided into two groups. One group (CLP group) was treated with cecal ligation and perforation (CLP), and the other (sham group) was treated with laparotomy. PEG-SOD, PEG-CAT, and DMSO were administered intraperitoneally 30 min before and 12 h after CLP. The left hemidiaphragm was removed at 10 h or 16 h after the operation. We assessed the diaphragmatic contractility by twitch characteristics and force-frequency curves in vitro. We measured MDA concentrations, as an index of oxygen-derived free radical-mediated lipid peroxidation, and the activities of two main antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (GPx), as an index of antioxidant defenses, after CLP. Diaphragmatic force generation capacity was significantly reduced after CLP. Diaphragmatic MDA levels were significantly elevated after CLP. PEG-SOD, PEG-CAT, and DMSO significantly improved diaphragmatic contractility and prevented the elevation in diaphragmatic MDA concentrations after CLP. Diaphragmatic SOD activities were significantly increased after CLP. These results suggest that several types of oxygen-derived free radicals play a role in the reduction in diaphragmatic contractility after CLP. Minerva Anestesiol. 2000 May. The most common cause of death in patients with sepsis is the multiple organ dysfunction syndrome (MODS). One important factor underlying the pathogenesis of MODS may be sepsis-induced alterations in cellular energy metabolism due to acquired intrinsic derangements in cellular respiration, a phenomenon that might be called "cytopathic hypoxia". A number of different biochemical mechanisms have been postulated to account for cytopathic hypoxia in sepsis, including reversible inhibition of cytochrome oxidase by nitric oxide, irreversible inhibition of one or more mitochondrial respiratory complexes by peroxynitrite, and activation of the nuclear enzyme, poly-(ADP-ribosyl)-polymerase. Immunopharmacology. 2000 Jan. The toxic effects of oxygen radicals produced by immune cells can be controlled to certain degree by endogenous antioxidants because of their scavenger action. This control is specially important in a type of immune cell, i.e., the phagocyte, which produces oxygen-free radicals and uses antioxidants in order to support its functions. Antioxidants, such as ascorbic acid (AA), are free radical scavengers and improve the immune response. In the pathogenesis of endotoxic shock, a disease with high mortality caused by gram-negative bacterial endotoxin, the reactive oxygen species (ROS) produced by phagocytes have been implicated. In a previous study, we observed in peritoneal macrophages from BALB/c mice suffering lethal endotoxic shock caused by intraperitoneal (i.p.) injection of Escherichia coli lipopolysaccharide (LPS; 100 mg/kg) a high production of superoxide anion. Therefore, in the present work, we have studied the in vitro effect of AA, at different concentrations (0.001, 0.01, 0.1, 1 and 2.5 mM), on the various steps of the phagocytic process, i.e., adherence to substrate, chemotaxis, ingestion of particles and superoxide anion production of murine peritoneal macrophages obtained from BALB/c mice with that of endotoxic shock, at 2, 4, 12 and 24 h after LPS injection. The increased adherence, ingestion and superoxide anion production by macrophages from animals with endotoxic shock were lower in the presence of AA, reaching similar values to those of the control animals. The most effective AA concentration in cells from mice with endotoxic shock was 0.01 mM. These data suggest that AA can regulate the phagocytic process in endotoxic shock, principally decreasing free radical production and thus it could reduce endotoxic shock severity. Shock. 1999 Feb. Sepsis and hypoxia are important stressors for the neonate. Newborn infants receiving total parenteral nutrition are routinely deprived of carnitine and develop low carnitine plasma and tissue levels. Because of its high metabolic rate and dependence on fatty acids for energy, the newborn heart may be particularly vulnerable to stress in the face of an inadequate carnitine supply. To investigate whether carnitine deprivation affects cardiac performance under stress, 23 neonatal piglets received parenteral nutrition for 2-3 weeks that was either carnitine free (CARN -) or supplemented (CARN +) with L-carnitine (400 mg/L). Bacterial endotoxin (lipopolysaccharide (LPS), 250 microg/kg intravenous bolus) or saline vehicle was administered to anesthetized piglets 3 h prior to study of isolated perfused hearts. Left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure, and left ventricular developed pressure (LVDP) were measured in vitro under aerobic, hypoxic, and reoxygenation conditions in all animals. Plasma and tissue carnitine values were lower in CARN - than in CARN + piglets. In hearts from LPS-treated animals prior to hypoxia, there was no difference in ventricular compliance between CARN - and CARN + groups. LVSP and LVDP were lower in CARN - than CARN + hearts. During hypoxia, LVSP and LVDP fell, but left ventricular end diastolic pressure increased in hearts from both LPS- and saline- treated piglets. Reoxygenation led to poorer recovery in CARN - than CARN + hearts from LPS-treated animals, but not from saline controls. During hypoxia/reoxygenation, lactate efflux initially rose and then fell, while carnitine efflux increased continually. Acetyl- and medium-chain acylcarnitines were detected in the coronary effluent. Our findings suggest that carnitine deprivation diminishes heart carnitine concentrations and impairs cardiac recovery from combined endotoxic and hypoxic stress. Possible mechanisms include reduced acyl buffering and/or impaired transport of fatty acyl groups into mitochondria. Biochem Soc Symp. 1999. The current mainstream view of organ failure induced by sepsis revolves around inflammation and loss of vascular control. However, there has been a resurgence in interest in bioenergetic failure due to mitochondrial dysfunction. This concept is not new--studies date back 30 years; however, the data have been highly conflicting with findings of either decreased, increased or unchanged mitochondrial activity and/or nucleotide levels. These studies are virtually all based on non-human cells, isolated perfused organs or in vivo animal models that have received a variety of insults ranging from mild to severe, and monitored for different durations ranging from minutes to weeks. As a generalization, there does appear to be depression of mitochondrial function with longer-duration models of greater severity. This is confirmed by the scanty human data currently available. This chapter provides an overview, and attempts to relate the biochemical changes to the clinical condition. The potential roles of nitric oxide, intracellular calcium and reactive oxygen species are highlighted. Shock. 1998 Nov. Sepsis and endotoxemia are important stressors for the neonate. Newborn infants receiving total parenteral nutrition are routinely deprived of carnitine. To investigate whether carnitine deprivation affects the neonate's ability to respond to endotoxin, 19 newborn piglets received parenteral nutrition for 2-3 weeks that was either carnitine free (CARN-) or supplemented (CARN+) with L-carnitine (400 mg/L). Cardiovascular performance, i.e., heart rate; blood pressure (BP); cardiac output (CO); systemic vascular resistance (SVR), and metabolic response, i.e., plasma glucose; lactate; tumor necrosis factor alpha; tissue nitric oxide; and urinary nitrites, were studied serially in anesthetized piglets for 3 h after endotoxin (lipopolysaccharide (LPS), 250 microg/kg intravenous bolus) or vehicle administration. Plasma and tissue carnitine values were lower in CARN- than in CARN+ piglets. Prior to LPS, no differences were found for most parameters (excepting lower diastolic BP and SVR in CARN- animals). Systolic, diastolic, and mean BP fell after LPS but recovered by the end of the experiment. Nadirs were lower in CARN- than in CARN+ piglets. CO tended to be higher in CARN- than in CARN+ animals and fell after LPS. SVR fell after LPS and was lower in CARN- than in CARN+ piglets. LPS-treated animals transiently increased urinary flow. By all measures (plasma tumor necrosis factor alpha, glucose and lactate, tissue nitric oxide, and urinary nitrite excretion), LPS provocation was similar for both groups. Chronologically, BP changes were more closely related to SVR than to CO. Our findings suggest that carnitine deprivation diminishes tissue carnitine concentrations and adversely affects cardiovascular response to LPS, in part mediated by the peripheral vasculature. Mol Cell Biochem. 1998 Feb. It is now recognized that respiratory muscle fatigue contributes to the development of respiratory failure in some patients with lung disease. This observation has prompted an examination into the mechanisms of development of muscle fatigue, with the understanding that an elucidation of these processes may lead to new therapeutic approaches to the treatment of these patients. A series of recent studies examining this issue have, moreover, discovered that oxygen-derived free radicals generated during strenuous contraction may modulate respiratory muscle contractile function and contribute to the development of muscle fatigue. The data supporting this concept include: (a) direct (e.g. EPR, ESR studies) and indirect (evidence of lipid peroxidation, protein carbonyl formation, glutathione oxidation) evidence that there is heightened free radical production in contracting muscle, (b) evidence that pharmacologic depletion of muscle antioxidant stores increases degree of muscle fatigue present after a period of exercise, and (c) evidence that administration of agents that act as free radical scavengers retard the development muscle fatigue. Free radicals may produce these changes in muscle force generating capacity by interacting with and altering the function of a number of intracellular-biophysical processes (i.e. sarcolemmal action potential propagation, sarcoplasmic reticulum calcium handling, mitochondrial function, contractile protein interactions). Am J Respir Crit Care Med. 1997 Feb. Recent studies have suggested that free radical scavenger administration reduces the rate of development of diaphragm fatigue. Much of this work has been done, however, using in vitro muscle preparations; the purpose of the present study was to assess the effect of scavengers on in vivo diaphragm contractile function. To accomplish this, we compared the rate of development of fatigue of the electrically stimulated diaphragm in four groups of dogs: (1) animals given intravenous polyethylene glycol adsorbed superoxide dismutase (PEG-SOD, 2,000 units/kg) 1 h before a fatigue trial; (2) a group given intravenous dimethylsulfoxide (DMSO, 0.5 ml/kg of a 50% solution) before fatigue; (3) a group given saline before fatigue; and (4) a group treated with denatured PEG-SOD (2,000 units/kg) before fatigue. We measured diaphragmatic concentrations of thiobarbituric acid reactive substances (TBAR), a marker of free radical-mediated lipid peroxidation, on muscle samples taken at the conclusion of fatigue trials. As a control, we also measured TBAR concentrations for muscle samples taken from nonfatigued diaphragm. We found that the rate of development of diaphragm fatigue was much greater in saline and denatured PEG-SOD-treated groups than for animals pretreated with either PEG-SOD or DMSO, with force falling to 23 +/- 4, 21 +/- 4, 50 +/- 7, and 47 +/- 6% of its initial value, respectively, over a 2-h period of electrophrenic stimulation in these four groups of animals (p < 0.01). TBAR concentrations in fatigued diaphragm from saline and denatured PEG-SOD-treated animals were significantly higher than levels for either nonfatigued fresh diaphragm or fatigued diaphragm taken from PEG-SOD- or DMSO-treated animals (p < 0.01). These data suggest that diaphragm fatigue resulting from repetitive low-frequency stimulation is associated with lipid peroxidation within this muscle and that pretreatment with free radical scavengers prevents lipid peroxidation and reduces the rate of development of fatigue. Acta Anaesthesiol Scand Suppl. 1997. Diminished availability of oxygen at the cellular level might account for organ dysfunction in sepsis. Although the classical forms of tissue hypoxia due to hypoxemia, anemia, or inadequate perfusion all might be important under some conditions, it seems increasingly likely that a fourth mechanism, namely cytopathic hypoxia, might play a role as well. The term cytopathic hypoxia is used to denote diminished production of adenosine triphosphate (ATP) despite normal (or even supranormal) PO2 values in the vicinity of mitochondria within cells. At least in theory, cytopathic hypoxia could be a consequence of several different (but mutually compatible) pathogenic mechanisms, including diminished delivery of a key substrate (e.g., pyruvate) into the mitochondrial tricarboxylic acid (TCA) cycle, inhibition of key mitochondrial enzymes involved in either the TCA cycle or the electron transport chain, activation of the enzyme, poly-(ADP)-ribosylpolymerase (PARP), or collapse of the protonic gradient across the inner mitochondrial membrane leading to uncoupling of oxidation (of NADH and FADH) from phosphorylation of ADP to form ATP. Tantalizing, but limited, data support the view that cytopathic hypoxia occurs in both animals and patients with sepsis or endotoxemia. Biol Pharm Bull. 1996 Jan. Blood concentrations of branched chain amino acids (BCAA; leucine, isoleucine, and valine) and glutamine (Gln) decrease markedly in sepsis. We investigated the effect of carnitine on serum concentrations of BCAA and Gln in fasted septic rats. Rats were made septic by cecal ligation and puncture. They developed extremely high blood concentrations of endotoxin, and serum concentrations of BCAA and Gln were markedly decreased 2 d after the operation. When L-carnitine was administered subcutaneously to the rats at 500 mg/kg body weight every 12 h for 2 d starting at the operation, no decrease in the serum concentrations of BCAA and Gln was observed. This indicates that the administration of carnitine can prevent the decrease of serum concentrations of BCAA and Gln in septic animals. Br J Cancer. 1995 Nov. Inappropriate hepatic lipogenesis, hypertriglyceridaemia, decreased fatty acid oxidation and muscle protein wasting are common in patients with sepsis, cancer or AIDS. Given carnitine's role in the oxidation of fatty acids (FAs), we anticipated that carnitine might promote FA oxidation, thus ameliorating metabolic disturbances in lipopolysaccharide (LPS)- and methylcholanthrene-induced sarcoma models of wasting in rats. In the LPS model, rats were injected with LPS (24 mg kg-1 i.p.), and treated with carnitine (100 mg kg-1 i.p.) at -16, -8, 0 and 8 h post LPS. Rat health was observed, and plasma inflammatory cytokines and triglycerides (TG) were measured before and 3 h post LPS. In the sarcoma model, rats were implanted subcutaneously with tumour, and treated continuously with carnitine (200 mg kg-1 day-1 i.p.) via implanted osmotic pumps. Tumour burden, TG and cytokines were measured weekly for 4 weeks. Carnitine treatment significantly lowered the tumour-induced rise in TG (% rise) in the sarcoma model (700 +/- 204 vs 251 +/- 51, P < 0.03) in control and carnitine groups respectively. Levels of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) (pg ml-1) were also lowered by carnitine in both LPS (IL-1 beta: 536 +/- 65 vs 378 +/- 44: IL-6: 271 +/- 29 vs 222 +/- 32; TNF-alpha: 618 +/- 86 vs 367 +/- 54, P < or = 0.02) and sarcoma models (IL-1 beta: 423 +/- 33 vs 221 +/- 60; IL-6: 222 +/- 18 vs 139 +/- 38; TNF-alpha: 617 +/- 69 vs 280 +/- 77, P < or = 0.05) for control and carnitine groups respectively. We conclude that carnitine has a therapeutic effect on morbidity and lipid metabolism in these disease models, and that these effects could be the result of down-regulation of cytokine production and/or increased clearance of cytokines. J Pediatr Surg. 1995 Jul. Increased oxidation of fat is an important host response to sepsis, and carnitine is essential for long-chain fatty acid oxidation. Because neonates have low levels of carnitine, their ability to respond to a septic insult may be impaired. The purpose of this study was to compare fatty acid and carnitine metabolism in septic weanling (60 to 85 g) and septic adult (285 to 310 g) rats. Sepsis was induced in weanling and adult male Sprague-Dawley rats by cecal ligation and puncture (CLP). The rats were killed 16 hours after CLP or sham operation, and serum glucose, lactate, beta-hydroxybutyrate, fatty acid, carnitine, liver fatty acid, and tissue carnitine levels were measured. The data suggest that during sepsis weanling rats may be more dependent on fatty acid oxidation than adult rats are, as evidenced by their elevated serum fatty acid and acylcarnitine levels, and relative hypoglycemia and hyperketonemia. In addition, although total serum carnitine levels were increased in both adult and weanling septic rats, tissue carnitine levels of weanling rats became significantly depleted during sepsis, unlike in adult rats. This study supports further investigation regarding the role of exogenous carnitine in newborn sepsis. Am Rev Respir Dis. 1993 Nov. Recent studies have suggested that free radicals contribute to the diaphragmatic dysfunction observed in sepsis. However, previous work has not determined which species of free radicals are responsible for producing these effects or whether the intercostal muscles are affected similarly during sepsis. The purpose of this study was to examine these issues using a hamster model of endotoxin-mediated sepsis in which diaphragm and intercostal muscle function was assessed on muscle strips excised from these animals after killing. Several groups of animals were studied, including animals injected with (1) saline, (2) endotoxin, (3) endotoxin plus active PEG-SOD, a superoxide scavenger, (4) endotoxin plus active PEG-catalase, a hydrogen peroxide scavenger, (5) endotoxin plus DMSO, a hydroxyl scavenger, and (6) endotoxin plus denatured PEG-SOD. We found that endotoxin administration elicited significant reductions in diaphragm and intercostal muscle contractility. In each of the three groups of animals to which active free radical scavengers were administered, the effects of endotoxin were attenuated. Denatured PEG-SOD did not protect the respiratory muscles from endotoxin-mediated dysfunction, however. These data indicate that both the diaphragm and intercostal muscles are affected similarly by sepsis; moreover, several free radical species (superoxide ions, hydrogen peroxide, and hydroxyl ions) play a role in mediating this type of injury. Am Rev Respir Dis. 1992 Jun. Although it is known that endotoxin can induce diaphragmatic dysfunction, the mechanism of this effect is not fully understood. However, because the effects of endotoxin on other tissues appear to be mediated in part by free radicals, the present study sought to determine if free radicals may also contribute to the diaphragmatic dysfunction induced by endotoxin administration. Studies were performed on four groups of hamsters. One group of animals received intraperitoneal injections of endotoxin on the first and second days of study (i.e., 10 and 20 mg/kg, respectively). The second group received saline rather than endotoxin, the third group received both endotoxin and a free radical scavenger, PEG-SOD (2,000 U/kg given intraperitoneally every 12 h on Days 1 and 2), and the fourth group received PEG-SOD alone. All groups were killed on the third study day (i.e., 48 h after the initial injections). Diaphragmatic contractile function was assessed in vitro using muscle strips excised from the costal diaphragms of freshly killed animals; diaphragm samples were also assayed for malondialdehyde (MDA), a commonly used index of free-radical-mediated lipid peroxidation. MDA levels were higher in diaphragms from endotoxin-treated animals than from saline-treated control animals, and the contractility of diaphragm strips from endotoxin-treated animals was reduced when compared with strips from saline-treated control animals. Administration of PEG-SOD prevented MDA formation and contractile dysfunction in endotoxin-treated animals. Diaphragm contractility and MDA levels for animals given PEG-SOD alone were similar to those for saline-treated control animals. Crit Care Med. 1991 May. OBJECTIVE: To quantitate the contribution of lactate, phosphate, urate, total serum proteins, and unidentified anions to the anion gap in patients with severe sepsis. DESIGN: Thirty critically ill patients with evidence of severe sepsis and systemic hypoperfusion were prospectively studied. MEASUREMENTS: The anion gap was calculated as [Na+] + [K+] - [Cl-] - [HCO3]. A corrected anion gap was calculated as the anion gap minus the anionic contribution of lactate, phosphate, urate, and total serum proteins. The corrected anion gap is a marker of unmeasured anion less unmeasured cation concentration. RESULTS: The mean anion gap was 21.8 +/- 1.4 mmol/L and the corrected anion gap was 3.7 +/- 0.8 mmol/L. The mean arterial blood lactate concentration was 5.9 +/- 0.8 mmol/L. The magnitude of the lactate concentration correlated linearly with the anion gap (r2 = .61, lactate = 0.4 anion gap - 3.9, n = 30, p less than .01). The corrected anion gap was greater than 0 in 24 (80%) of 30 patients. The magnitude of the corrected anion gap correlated linearly with the anion gap (r2 = .66, corrected anion gap = 0.5 anion gap - 6.3, n = 30, p less than .01). Since the slope of the regression line for estimating corrected anion gap from anion gap was 0.5, the contribution of unmeasured anions was as important as lactate in determining the anion gap. CONCLUSION: These data indicate that lactic acidosis does not entirely account for the metabolic acidosis during severe sepsis. Furthermore, the increased corrected anion gap suggests the presence of an unidentified anion (or anions) that is (or are) responsible, in large part, for the development of metabolic acidosis in patients with sepsis. Int J Clin Pharmacol Res. 1991. The clinical use of acetyl carnitine in circulatory shock has its theoretical basis in the ability of this molecule to restore enzyme activity inhibited by hypoxia, acting as an acetyl donor. Moreover the action of carnitine on an injured myocardium encouraged us to examine the clinical effect of this drug during heart failure. A double-blind clinical study was performed in ten Italian intensive care units on 115 patients with septic, cardiac or traumatic shock, by using acetyl-L-carnitine infusion for 12 hours, with a previous single bolus intravenously. The results showed a good response to the drug in terms of blood oxygenation during the course of sepsis and heart failure. The heart rate as well as right atrial pressure decreased significantly in patients with cardiogenic shock. In septic patients systolic and mean arterial pressures increased also. The present data suggests the use of acetyl-L-carnitine as an adjuvant to the commonly used therapy in hypoxic conditions. Surgery. 1989 Feb. Gram-negative bacterial endotoxin (lipopolysaccharide) induces hepatic damage in mice caused by lipid peroxidation, and administration of antioxidants (coenzyme Q10 and alpha-tocopherol) suppresses this lipid peroxidation, preserves energy metabolism, and enhances the survival of endotoxin-administered mice. Therefore experiments were done to determine whether experimental endotoxemia in mice affected the levels of endogenous antioxidants and whether treatment with antioxidants altered these levels. Endotoxin produced decreases in hepatic endogenous antioxidants with time: reduced coenzyme Q9, alpha-tocopherol, and reduced glutathione were lowered to 29.9%, 27.1%, and 45.4% of the control levels, respectively, 24 hours after administration. Exogenously administered coenzyme Q10 (oxidized) accumulated in the liver and showed a maximal plateau between 8 and 16 hours after injection when 82% of coenzyme Q10 was converted to the reduced form. Coenzyme Q10 administered with endotoxin totally or partially prevented the decreases in these endogenous antioxidants, and furthermore, total coenzyme Q10 and the reduced form, both at levels of approximately 30 mg protein, were consumed during the period of elevated lipid peroxidation, 16 hours after endotoxin injection. These results indicate that coenzyme Q10 acts in vivo as an antioxidant after it has been converted to the reduced form. alpha-Tocopherol administered also showed an 84-fold accumulation in the liver 8 hours after injection, completely preventing any decrease in endogenous reduced coenzyme Q9 and partially preventing reduction of glutathione, which indicated an in vivo antioxidant action of alpha-tocopherol. These results support the assumption that administered coenzyme Q10 or alpha-tocopherol functions cooperatively with endogenous antioxidants to prevent tissue damage caused by lipid peroxidation in endotoxemia. Am J Physiol. 1989 Jan. The activities of palmitoyl-coenzyme A (CoA) synthetase, carnitine acetyltransferase (CAT), and carnitine palmitoyltransferase (CPT) and the levels of ketone bodies, reduced coenzyme A (CoASH), carnitine, and their esters, which are involved in fatty acid metabolism, in rat liver and plasma were measured after the administration of Escherichia coli lipopolysaccharide (LPS). We also studied the effect of L-carnitine treatment before LPS administration on survival and on hepatic fatty acid metabolism. The activities of CAT and CPT and the concentrations of ketone bodies, CoA, and carnitine derivatives (except for malonyl-CoA) declined in the liver after LPS administration. The activity of palmitoyl-CoA synthetase was changed little after LPS administration, and the level of hepatic malonyl-CoA increased significantly, suggesting that LPS causes activated fatty acids to undergo esterification and lipogenesis rather than oxidation. Treatment of rats with L-carnitine before LPS greatly increased the survival rate, but did not affect enzymes that metabolize fatty acids, CoA, or carnitine derivatives in the liver. Further studies are necessary to elucidate the mechanism of the effect of carnitine on post-LPS survival. JPEN J Parenter Enteral Nutr. 1988 Jan-Feb. Endogenous and exogenous supplies of carnitine are decreased in septic patients under total parenteral nutrition, while carnitine urinary elimination is increased. But the increase of lipid role in the energetic cover requires a greater intervening role of tissue carnitine. So one may hope that in septic patients additional supply of L-carnitine would increase the catabolism of infused lipids. Twenty-eight septic patients, admitted in an intensive care unit were given parenteral nutrition (200 g of glucose, 12.5 g of N/24 hr). On the day of the study, 250 ml of Intralipid 20% (Kabi Vitrum) were administered in 4 hr. During the same period 13 patients were infused with 2 g of L-carnitine (Sigma-Tau). The remaining 15 patients constituted the control group. Basic plasma levels of triglycerides, nonesterified fatty acids, free glycerol, phospholipids, and ketone bodies remained within physiological limits. They increased during the lipid infusion and returned to initial values, 4 hr after the end of the infusion. Free and total carnitine levels and free/total carnitine ratio were comparable to healthy subjects' reference values. These parameters increased during L-carnitine infusion. This infusion had no effect on exogenous lipid clearance. However, it seemed to increase the uptake and the hepatic oxidation of circulating fatty acids. It invalidated the increase of lactate and pyruvate that had been noticed when lipids were solely infused. |