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Definitions for sepsis, septic shock, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS) were developed by consensus conferences with the goal of achieving standardization of terminology and improved homogeneity of patient populations in clinical studies. Although such definitions have been useful in epidemiologic investigations, the criteria specified by the consensus conferences are broad and insufficiently specific to address the problem of heterogeneous mechanisms leading to clinical syndromes. An important challenge is to progress from clinical syndromes, as presently defined, to more specific entities that are delineated by alterations in specific immunologic or biochemical pathways. Such mechanistic definitions will provide more homogeneous groups of patients who can be identified at early stages of their clinical course. This approach encourages focused investigation of pathways leading to organ system dysfunction and death and, also, provides an efficient framework for the development of new therapies useful in critically ill patients.

Hearts isolated from rats pretreated 24 hr before with endotoxin had increased myocardial catalase activity, but the same superoxide dismutase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase activities, as hearts from untreated rats. Hearts isolated from rats pretreated with endotoxin 24 hr before also had increased myocardial function (decreased injury) after ischemia and reperfusion (Langendorff apparatus, 3rC), as assessed by measurement of ventricular developed pressure, contractility (+dP/dt), and relaxation rate (-dP/dt), compared to control hearts. In contrast, hearts isolated from rats pretreated with endotoxin 1 hr before isolation or hearts perfused with endotoxin did not have increased catalase activity or decreased injury following ischemia and reperfusion. Aminotriazole pretreatment prevented increases in myocardial catalase activity and myocardial function after ischemiareperfusion in hearts from endotoxin-pretreated rats. The results suggest that endotoxin pretreatment decreases cardiac ischemia-reperfusion injury and that increases in endogenous myocardial catalase activity contribute to protection.Hydrogen peroxide generated by xanthine oxidase appears to contribute to reperfusion injury of isolated ischemic rat hearts (1-4). This impression is based on observations that inhibition of xanthine oxidase or scavenging of H202 by addition of exogenous 02-metabolite scavengers decreases reperfusion injury of isolated ischemic hearts (1-4). Because endotoxin pretreatment increases endogenous lung antioxidant activities and decreases lung injury from hyperoxia (5), we hypothesized that endotoxin pretreatment would also increase endogenous myocardial antioxidant activities and, as a result, decrease myocardial susceptibility to ischemiareperfusion injury. Using a standard Langendorff isolated "blood-free" perfused rat heart model, we found that hearts from endotoxin-pretreated rats had increased catalase activity and decreased susceptibility to ischemia-reperfusion injury. MATERIALS AND METHODSPretreatment with Endotoxin and/or Aminotriazole. Quarantined male Sprague-Dawley rats (300-325 g) were treated with saline (0.9% NaCI) or endotoxin (500 ,ug/kg of body weight, i.p., Salmonella typhimurium phenol extract, Sigma) in 5 mM potassium phosphate buffer (pH 7.4) 24 hr or 1 hr before heart isolation. Some rats were pretreated with endotoxin 24 hr before and low-dose aminotriazole (50 mg/kg, i.p. in potassium phosphate buffer, Sigma) 12 hr before or with high-dose aminotriazole (500 mg/kg, i.p. in potassium phosphate buffer) alone 12 hr before isolation.Assay (7), and glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase (8) activities. Preparation of Isolated Rat Hearts. Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and heparinized (500 units, via the right atrium). Hearts were rapidly excised and then perfused (70 mmHg; 1 mmHg = 133 pascals) in retrograde fashion at the aortic root with a Krebs-Henseleit so...

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Exposure to decreasing oxygen tensions progressively increased xanthine dehydrogenase (XD) and xanthIne oxidase (XO) activities over 48 hr in cultured pulmonary artery endothelial cells (EC) without altering XD/XO ratios. Increases in XD and XO activity in EC induced by hypoxia were associated upon reoxygenation with increased (P < 0.05) extraceflular superoxide anion (O2 ) levels that were inhibited by treatment with XO inhibitors (tungsten, allopurinol) or an anion-channel blocker (4,4'-diisothiocyanatoslbene-2,2'-disulfonic acid). EC monolayers subjected to hypoxia/reoxygenation also leaked more preioaded 51Cr, were more adherent to neutrophils, and permitted greater albumin transit than control monolayers.Treatment with tungsten, aflopurinol, and/or superoxide dismutase decreased (P < 0.05) 5'Cr release, neutrophil adherence, and albumin transit in EC monolayers exposed to hypoxia/reoxygenation. We conclude that prolonged hypoxia increases both XO and XD activity in EC and may predispose the endothelium to oxidative and inflammatory damage. Recently, we found a marked suppression of XD and XO activity in EC exposed to high oxygen tensions (6). Accordingly, we examined the effects of prolonged hypoxia on EC XD and XO activities. We found that prolonged hypoxia increased both XD and XO activities in cultured EC and, further, that increases in XD and XO activities contributed to 0j -mediated injury and neutrophil adherence to EC. Measurement of XD and XO Activity. EC were grown in 75-cm2 flasks (Falcon, Becton Dickinson). Medium was removed from confluent EC monolayers and replaced with Hanks' balanced salt solution/5% fetal calf serum; then flasks were placed in sealed chambers, flushed with 5% C02/0-95% 02 and 95-0o N2, and incubated at 370C. 02 tensions of parallel saline samples measured with a Clark electrode were 15, 33, 66, 120, and 660 torr (1 torr = 133.3 Pa) after 48-hr incubations in 0, 3, 10, 21, and 95% 02, respectively, at Denver altitude. In some experiments, EC were grown for three-passages in sodium tungstate (10 ppm final) before use; or allopurinol (100 AM), 4,4'-diisocyanatostilbene-2,2'-disulfonic acid (DIDS, 100 ,AM), or SOD (10 jug/ml) was added to EC before incubation. Monolayers of EC were triply washed with phosphate-buffered saline (20 mM, pH 7.4), mechanically harvested with a plastic policeman in 50 mM phosphate buffer, pH 7.8/1 mM EDTA/1 mM phenylmethylsulfonyl fluoride/1 mM dithioerythritol/SOD at 20 ,ug/ml/catalase at 20 ,ug/ml and sonicated on ice. Nuclei were removed by centrifugation, and supernatants were desalted by using a Centricon filter system. Aliquots (0.25 ml) were incubated aerobically at 370C for 3 hr with xanthine (200 AuM) to measure XO activity or xanthine plus NAD' (300 AM) to measure XO plus XD activity in a final volume of 0.5 ml. Lactic dehydrogenase (70 units per ml) and pyruvate (1.75 mM) were added to tubes containing NAD' to prevent inhibition of XD by NADH (9). Baseline values were obAbbreviations: XO, xanthine oxidase; O-., superoxide anion; XD,...

A B S T R A C T Methane (CH4) production from the anti-inflammatory agent, dimethyl sulfoxide (DMSO), was used to measure *OH from chemical reactions or human phagocytes. Reactions producing * OH (xanthine/xanthine oxidase or Fe++/EDTA/H202) generated CH4 from DMSO, whereas reactions yielding primarily 0r or H202 failed to produce CH4. Neutrophils (PMN), monocytes, and alveolar macrophages also produced CH4 from DMSO. Mass spectroscopy using cJ-DMSO showed formnation of d3-CH, indicating that CH4 was derived from DMSO. Methane generation by normal but not chronic granulomatous disease or heat-killed phagocytes increased after stimulation with opsonized zymosan particles or the chemical, phorbol myristate acetate. Methane production from DMSO increased as the number of stimulated PMN was increased and the kinetics of CH4 production approximated other metabolic activities of stimulated PMN. Methane production from stimulated phagocytes and DMSO was markedly decreased by purportedly potent -OH scavengers (thiourea or tryptophane) and diminished to lesser degrees by weaker -OH scavengers (mannitol, ethanol, or Indeed, stimulated neutrophils (PMN), monocytes (MN) or alveolar marcophages (AM) do produce C2H4 from methional or KMB (6-10). However, the strict dependence of C2H4 production upon -OH is in doubt (8,(10)(11)(12)(13)(14). Methional spontaneously forms C2H4, especially during prolonged incubations with tissues (8), and can also react with H202 to form C2H4 (10). The specificity of KMB as a detector of -OH is also in question because C2H4 production from KMB may reflect, at least in part, reactions with 02 or hypochlorous acid (10).In Ca++ and Mg++ (26). Contaminating erythrocytes were lysed by adding 6 ml of ice-cold sterile distilled water and mixing gently for 35 s. Tonicity was rapidly restored with 2 ml of hypertonic (4x) Ca++ and Mg++ free HBSS. The mixture was then centrifuged at 170 g for 10 min. Cells in the pellet were then washed once more, resuspended in HBSS with Ca++ and Mg++, counted, and used immediately. PMN preparations contained >98% PMN with only a few lymphocytes and rare erythrocytes, platelets or MN. Suspensions of MN were similarly prepared. The percentages of MN were determined by differential counting of 600 cells on Wright's stained smears and cytochemically confirmed by esterase stain. MN preparations contained -30% MN, <1% PMN, and the remainder lymphocytes. AM were obtained by bronchoscopic sterile saline lavage of the unaffected subsegments of the lungs of patients undergoing evaluations for localized pulmonary disease or from healthy volunteers (9). AM were recovered from lavage fluids, washed once, and counted. The final preparations contained >90% AM and <2% PMN. More than 85% of the AM excluded trypan blue. Concentrations of lymphocytes or platelets that were comparable to those remaining in phagocyte preparations did not produce significant amounts of CH4 from DMSO.Preparation of pooled human serum, opsonized zymosan, or PMA. Pooled human serum was prepared from clotted ...

We isolated cDNAs encoding xanthine dehydrogenase (XD; xanthine:NAD+ oxidoreductase, EC 1.1.1.204) from a human liver cDNA library. The complete nucleotide sequence of human XD was determined; the deduced amino acid sequence encoded a protein of 1336 amino acid residues of M(r) 147,782. Human XD possessed many of the signature sequences typical of XDs from flies and rodents, including an unusual cysteine distribution, a potential 2Fe/2S binding site, and a putative molybdopterin cofactor binding domain. Analysis of potential NAD binding sites suggested a simple hypothesis for the conversion of human XD into the oxygen metabolite forming xanthine oxidase (XO; xanthine:oxygen oxidoreductase, EC 1.1.3.22). Using a human XD complementary RNA hybridization probe, we found a 5100-base RNA in human liver by RNA blot-hybridization analysis. This RNA exhibited tissue-specific distribution that may be pertinent to XD- and XO-mediated oxygen radical injury in ischemia/reperfusion and inflammation. A second 4500-base RNA was detected in some tissues and may arise through differential transcription termination.