Reduced serum total reductive capacity in lethal severe trauma.

BACKGROUND: Risk assessment is a prerequisite for effective treatment and triage in severe injury. A novel substrate-based assay to measure total reductive capacity (TORC) in serum was used to stratify risk of lethal outcome in severe trauma in a clinical trial. METHODS: Serum of patients with severe trauma (Injury Severity Score > 19) was obtained at the accident site, at admission, and at regular intervals thereafter. TORC was determined and correlated to outcome. The TORC assay uses thiol-labeled arachidonic acid as substrate from which free thiols are released by reductive amino acids and the specific activity of phospholipase A2. Free thiols are coupled to monochrombimone, and the resulting fluorescence is proportional to TORC. RESULTS: Eighteen patients with lethal severe trauma and 16 patients who survived were studied. Injury Severity Scores (lethal, 33 (29--43); survival, 31 (25--42); p = NS) and Polytrauma Scores (lethal, 25 [18--32]; survival, 26 [23--31], p = NS) were not significantly different. At the accident site, patients with a lethal course had significantly lower TORC than nonlethal cases (59.2 +/- 5.1 ng/mL vs. 89.5 +/- 6.7 ng/mL; p < 0.001). Values at admission were similar (lethal, 51.2 +/- 7 ng/mL; survival, 73.8 +/- 9 ng/mL; p < 0.01). At the accident site and at admission, TORC < 82.3 ng/mL was prognostic of lethal outcome (sensitivity, 88%; specificity, 65%/73% and 69%, respectively, for admission). CONCLUSION: Serum reductive potential at the site of accident or at admission allows the stratification of trauma patients with respect to lethal outcome in severe trauma when severity scores fail to do so.

pH-dependent changes of nitric oxide, peroxynitrite, and reactive oxygen species in hepatocellular damage.

Low arterial blood pH and sustained nitric oxide (NO) production are critical parameters in inflammatory events such as sepsis, and appropriate treatment is still under debate. Because the stability of nitrogen and oxygen intermediates is dependent on the surrounding pH, we investigated whether the relationship among NO, peroxynitrite (ONOO-), and reactive oxygen species production also depends on the pH value, particularly with respect to their effects on hepatocellular damage. Our studies demonstrate that the extracellular pH influences NO and hydroxyl radical (OH) production in hepatocytes. Acidification (pH 7.0) of the medium revealed a significant increase (P < 0.05) of OH-like radicals, enhanced hepatocellular damage, and a sharp drop in cellular glutathione (GSH) content compared with levels measured at physiological or alkaline pH conditions. Furthermore, inhibition of NO synthesis at all pH conditions resulted in decreased NO production and cellular GSH levels but a simultaneous increase of OH-like radicals and hepatocellular damage with a maximum seen at pH 7.0. Our results suggest that hepatocellular damage is in part regulated by the surrounding pH and that inhibition of NO synthesis at acidic conditions (e.g., in sepsis) leads to increased reactive oxygen-mediated cell injury.

Effects of hydroxyethyl starch-deferoxamine on arachidonic acid metabolism and small bowel wall perfusion in early sepsis.

The effects of hydroxyethyl starch-conjugated deferoxamine (HES-DFO), a macromolecular iron chelator, were investigated on eicosanoid release and bowel wall perfusion following cecal ligation puncture (CLP) in rats. Animals were randomly given an intravenous dose of 3.0 ml of HES-DFO or either vehicle (HES) or 9.0 ml saline immediately following completion of the CLP procedure. At 30, 60, 120, and 240 min after sepsis induction, blood pressure and bowel perfusion were measured. The animals were sacrificed and blood was collected for subsequent analysis of thromboxane, prostacyclin, and prostaglandin F2 alpha. The tissue content of energy-rich phosphates was determined in small-bowel samples at each time point. The antioxidative HES-DFO therapy did not diminish the eicosanoid release after CLP when compared with either HES-treated or saline-infused rats. However, treatment with the polymeric iron chelator resulted in an impaired bowel wall perfusion that was not reflected in alterations in total adenine nucleotide content or in energy charge. Considering hemodynamic and biochemical endpoints, these results are contradictory to the hypothesis that iron-driven oxygen radicals are major determinants of the eicosanoid release that is elevated following CLP-induced sepsis.

Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols.

The aim of this study was to compare and improve standard methods to determine nitrite (NO2-), nitrate (NO3-) and S-nitrosothiol (RSNO) levels in cell culture supernatants, sera, and urine. We modified the conventional Griess reaction by replacing sulfanilamide with dapsone (4,4'-diamino-diphenylsulfone) and compared the NO2- levels in our study samples with a commercially available NO2- assay kit. Our modification, along with ultrafiltration of the samples, resulted in an enhanced sensitivity to measure NO2- down to 0.2 microM. The detection limit was further improved to 0.02 microM when NO2- was identified by the fluorochrome 2,3-diaminonaphthalene (DAN). To measure the stable end product NO3- by the Griess reaction or the DAN method, this anion must be reduced to NO2-. We compared the capacity of bacterial nitrate reductase with the reducing metal cadmium to convert NO3- to NO2-. After reduction, NO2- levels were determined either by the DAN method or by our modified Griess reaction. We found that there was a high correlation (r2 = 0.998) in total NO2- concentrations in the study samples using both methods for reducing NO3- to NO2-. The simultaneous determination of NO2- and NO3- was achieved by using anion-exchange chromatography (HPLC; Polyspher IC AN-1 column). The detection limit of this assay for each anion is 0.5 microM, and it can be applied equally well to sera, urine, and culture media. We also adapted the DAN method to determine RSNO levels in our study samples. Using this approach, we were able to measure RSNO levels down to 0.15 microM. As result we discovered that RSNO levels were markedly increased in urine from septic patients and in supernatants from cytokine-stimulated human tumor cell lines. L-Citrulline, a coproduct of NO biosynthesis, was measured using a colorimetric assay with a sensitivity limit of 3.0 microM. Increased L-citrulline levels in media from cultured cells, but not in sera or urine, correlated with increased NO production. Although all methods studied were suitable for quantifying end products of NO in biological fluids and media, the use of bacterial reductase and the modified Griess reaction proved successful to provide the greatest sensitivity and linear range for routine measurements of NO2- and NO3-.

[Systemic release of prostanoids after surgically-induced injury of lung tissue]. / Systemische Freisetzung von Prostanoiden nach chirurgisch induzierter Verletzung des Lungengewebes.

In a prospective study, the systemic inflammatory consequences of surgery-induced lung tissue injury were evaluated using biochemical markers. The aim was to examine whether this type of injury produces a specific pattern of prostanoid plasma levels (prostacyclin, thromboxane, PGE2, PGF2 alpha, and PGM). We, therefore, compared 18 patients (group 1) who underwent thoracotomy without injury to the lung with 26 patients (group 2) that had a resection of pulmonary tissue due to benign diseases. Group 2 patients clearly revealed increased plasma levels of C-reactive protein as well as of the granulocyte-specific PMN-elastase. In particular, there was a pronounced release of prostacyclin and its antagonist thromboxane A2 following lung tissue resection. In contrast to group 1 patients, lung tissue damage resulted in immediately elevated plasma levels of PGF2 alpha and PGE2. When, however, taking into account the time course of PGM, the stable cleavage product of PGF2 alpha, there was no hint of an altered pulmonary metabolic capacity. Presumably, this pattern of elevated prostanoid levels in group 2 is the result of the surgical damage to the lung tissue. Therefore, it can be suggested to be specific for that type of injury. Thus, the release of prostanoids following surgery-induced lung tissue damage may indicate the importance of these mediators, particularly in thoracic injuries associated with lung damage since those may lead to post-traumatic pulmonary dysfunction. These substances may also be useful in evaluating both the severity and the extent of lung tissue damage following major trauma.

Effect of acadesine treatment on postischemic damage to small intestine.

Hemorrhagic mucosal lesions are produced during intestinal ischemia and after reperfusion due at least in part to the accumulation and activation of polymorphonuclear leukocytes in the tissue. It has been shown in vitro that adenosine is instrumental in attenuating this pathophysiological process. Acadesine [5-amino-4-imidazolecarboxamide (AICA) riboside], a purine nucleoside analogue, increases the availability of adenosine in the tissue. The aim of the study was therefore to assess the influence of acadesine treatment on neutrophil accumulation, purine metabolism, and mucosal damage after intestinal ischemia and reperfusion. Intestinal ischemia was induced in cats by partial occlusion of the superior mesenteric artery for 2 h. Samples of the small intestine were exercised before and at the end of the hypotensive period as well as 10 and 60 min after reperfusion. Conjugated dienes, myeloperoxidase, and reduced and oxidized glutathione, as well as the purine metabolites, were determined in the tissue samples. The tissue was also examined histologically. Six cats received saline, and six cats were treated initially before ischemia with acadesine (2.5 mg/kg body wt i.v.) over 5 min as a bolus. Thereafter, acadesine (0.5 mg.kg-1.min- i.v.) was given continuously during ischemia and 30 min after reperfusion. Acadesine treatment significantly attenuated the mucosal lesions seen during reperfusion. This improvement was due at least in part to the inhibition of neutrophil accumulation, as judged by low myeloperoxidase levels. The prevention of neutrophil activation resulted most likely from increased adenosine concentrations in the intestinal tissue in the early reperfusion period.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid peroxidation and glutathione metabolism in chronic pancreatitis.

In experimental models of pancreatitis lipid peroxidation products are increased possibly because of an enhanced generation of oxygen radicals. The purpose of this study was to determine whether lipid peroxidation products are increased in pancreatic tissue and serum of patients suffering from chronic or acute pancreatitis. In 20 patients undergoing operative treatment for chronic (n = 11) and acute pancreatitis (n = 9) the levels of malondialdehyde, conjugated dienes, and reduced and oxidized glutathione were determined in resected tissue samples. The excised tissue was examined and evaluated by light microscopy. Shortly before operation the serum concentrations of malondialdehyde, alpha-amylase, and lipase were measured. Pancreatic tissue from eight organ donors who had no abdominal trauma or pancreatic disease served as control. In chronic pancreatitis, conjugated dienes as well as malondialdehyde concentrations in the tissue were significantly elevated. Reduced glutathione was significantly decreased, suggesting glutathione depletion due to oxidative stress. In acute pancreatitis only the tissue and serum malondialdehyde levels were significantly high, whereas conjugated dienes remained within the normal range. Serum malondialdehyde levels correlated significantly with tissue concentrations (r = 0.76; p < 0.05) but not with the clinical course or the enzyme levels. In chronic pancreatitis, the increased tissue levels of lipid peroxidation products and the changes in glutathione metabolism suggest ongoing peroxidation of lipids due to an enhanced generation of oxygen radicals. In hemorrhagic necrotizing pancreatitis, however, oxygen radical-induced lipid peroxidation cannot be proven. Apparently, other pathomechanisms are involved in the development of the severe tissue damage.

[Ischemia/reperfusion damage of the liver caused by free radicals--direct radical detection using electron spin resonance (ESR)]. / Ischämie/Reperfusionsschaden der Leber durch freie Radikale--direkter Radikalnachweis mittels Elektronenspinresonanz (ESR).

The generation of free oxygen radicals is presumed to be a pathogenetic principle in various conditions, primarily in postischemic reperfusion injury. Their assessment is difficult. ESR is an excellent tool to assess free radicals directly. In an experimental model of rat liver ischemia and reperfusion the increased generation of free radicals during reperfusion in liver tissue could be demonstrated after 60 min of liver ischemia. Elevated production rates of radicals could be detected after 5 min of reperfusion for at least 45 min with a maximum after 15 min of reperfusion. Scavenging of these radicals has to start in the very beginning of reperfusion.

Quantitative endotoxin determination in blood--chromogenic modification of the limulus amebocyte lysate test.

A newly developed modification of the limulus amebocyte lysate test for quantification of endotoxin levels in blood is described. The chromogenic peptide carbobenzoxy-Gly-Gly-Arg-4-methyl-cumarinyl-7-amid proved to be most suitable. The liberated fluorescent dye is diazotized with N(1-naphtyl-)-ethylen-diamin-dihydrochloride. Using this statistically proved reliable and sensitive test, endotoxin serum levels of healthy persons and patients undergoing major surgical treatment were compared. In the postoperative phase endotoxin serum levels up to 0.5 ng/ml can be detected without clinical signs of septicemia. Healthy persons show endotoxin serum levels up to 0.08 ng/ml. In rats no difference of endotoxin serum levels was detected in the portal vein, and in arterial and venous blood. So a physiological endotoxin resorption from the intestine followed by a clearance during the liver passage seems to be doubtful in this species.