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PubMed2004

Methodological studies on plasma endotoxin level and endotoxin inactivation capacity.

To establish stable methods for detecting plasma endotoxin level and endotoxin inactivation capacity in a normal population and general surgical patients and evaluate their perioperative changes. 50 healthy people and 50 patients receiving gastrointestinal operation were enrolled, their plasma endotoxin levels and plasma endotoxin inactivation capacity were assayed. Our results showed that plasma endotoxin levels were 0.044 +/- 0.009 EU/ml in the normal population and 0.044 +/- 0.023 EU/ml in the preoperative patients. Endotoxin level peaked 3 h after the operation (0.223 +/- 0.041 EU/ml), and then decreased rapidly on the first day after the operation (0.134 +/- 0.164 EU/ml). Endotoxin inactivation capacity also had the same time course as endotoxin level. Systemic inflammatory response syndrome and infection induced another elevation in the time course. It is concluded that establishing the endotoxin standard curve by using pyrogenic free water is better than by using plasma. Plasma endotoxin inactivation capacity can be used as an indirect indicator of postoperative immune depression. Plasma endotoxin level and endotoxin inactivation capacity peaked shortly after operation, indicating surgical stress is closely related with the changes.

Adult↗
PubMed2000

Endotoxaemia in patients with severe sepsis or septic shock.

OBJECTIVE: To examine the incidence and the bacteriological and clinical significance of endotoxaemia in ICU patients with severe sepsis or septic shock. DESIGN: Prospective review. SETTING: A 15-bed general ICU in a university hospital. PATIENTS: One hundred sixteen patients hospitalised in our ICU fulfilling Bone's criteria for severe sepsis or septic shock and with an available early endotoxin assay (chromogenic limulus assay). INTERVENTIONS: None. MEASUREMENTS AND RESULTS: The clinical characteristics of the population were: age 63.6 +/- 11.4 years; SAPS II: 45.4 +/- 15.6; mechanical ventilation: 72.4%; septic shock: 51.7% (n = 60); bacteraemia: 28.4% (n = 33); gram-negative bacteria (GNB) infection 47.4% (n = 55); ICU mortality: 39.6% (n = 46). Detectable endotoxin occurred in 61 patients (51.2%; mean level: 310 +/- 810 pg/ml). There was no relationship between detectable endotoxin and severity of infection at the moment of the assay. Endotoxaemia was associated with a higher incidence of bacteraemia (39.3% vs 16.3%; p = 0.01). There was a trend (p = 0.09) towards an association between positive endotoxin and gram-negative bacteraemia or GNB infection but this was non-significant. This relationship became significant only in the case of bacteraemia associated with GNB infection irrespective of the site of infection. CONCLUSION: Early detection of endotoxaemia appeared to be associated with GNB infection only in cases of bacteraemic GNB infection. Early endotoxaemia correlated neither to occurrence of organ dysfunction nor mortality in patients with severe sepsis or septic shock. This study suggests that the use of endotoxaemia as a diagnostic or a prognostic marker in daily practice remains difficult.

APACHE↗
PubMed2004

Modification of the Limulus amebocyte lysate assay for the analysis of glucan in indoor environments.

beta-1,3- D-Glucan is a biologically active component mainly from fungi that has been shown in several studies to be related to respiratory health outcomes from damp building exposures. Here, we report the development and application of a method for the analysis of the glucan extracted in 0.5 N NaOH solution making use of an available preparation of Limulus amebocyte lysate (LAL). The method yields reproducible beta-1,3- D-glucan measurements from samples of outdoor air, yeast cells, fungal spore preparations and ragweed pollen, and is more sensitive than competing measurements. The LAL-based measurement compared favourably to that based on size-exclusion chromatography using UV and refractive index detection. Growth conditions of the fungi did not materially change the concentrations of glucan in spores indicating that this is a stable property. Glucan content was proportional to spore surface area; however, some species contain higher relative spore glucan contents.

Air↗
PubMed1998

Antibacterial activity of meropenem against Pseudomonas aeruginosa, including antibiotic-induced morphological changes and endotoxin-liberating effects.

The in vitro effects of meropenem on Pseudomonas aeruginosa were examined by studying (i) the inhibitory and bactericidal concentrations of meropenem versus those of imipenem for clinical isolates; (ii) changes in bacterial morphology during in vitro culture; and (iii) release of endotoxin induced by meropenem compared with that induced by other antipseudomonal compounds. Meropenem MIC90 and MBC90 values for 108 clinical isolates were 2 and 4.8 mg/l compared to 4.5 and 9.6 mg/l for imipenem. Morphological studies using phase-contrast and scanning electron microscopy showed that meropenem induced the formation of indeterminate bacterial cell forms at drug concentrations of 1-2.5 mg/l (0.5- to 1.25-fold the MIC), while spheroplasts predominated at drug levels exceeding 5 mg/l (2.5-fold the MIC). Determination of free and EDTA-releasable endotoxin activity by means of the Limulus lysate test showed that both meropenem and imipenem liberated significantly less endotoxin than did ceftazidime. Therefore, although meropenem binds to penicillin-binding proteins (PBPs) 2 and 3 (in contrast to imipenem, which binds to PBP2 only), endotoxin release should not be a cause of concern when treating systemic gram-negative infections with this drug.

Anti-Infective Agents↗
PubMed1980

Gonococcal lipopolysaccharide: a toxin for human fallopian tube mucosa.

Gonococci damaged the mucosa of human fallopian tubes in organ culture (FTOC), producing characteristic pathologic features. Filter-sterilized supernatant fluid from donor gonococcal-infected FTOC damaged recipient FTOC in a similar fashion. Gonococcal lipopolysaccharide (LPS) was detected in these toxic donor fluids in concentrations of 1.2 to 8.3 microgram/ml. Purified gonococcal LPS in concentrations as low as 0.015 microgram/ml produced damage equivalent to that caused by toxic donor fluid and was neutralized by polymyxin B. Such LPS-mediated damage to ciliated cells, if it occurs in gonococcal salpingitis, may impair mucociliary flow and predispose to ectopic pregnancy and recurrent ascending infection.

Animals↗
PubMed1988

Labor and infection. II. Bacterial endotoxin in amniotic fluid and its relationship to the onset of preterm labor.

We have previously reported the detection of endotoxin in the amniotic fluid of patients with gram-negative intraamniotic infection. Endotoxin or lipopolysaccharide is a potent biologic product capable of inducing prostaglandin release from several cell types and, therefore, may be involved in the onset of human parturition in the presence of intraamniotic infection. This article describes a technique for the quantification of endotoxin in amniotic fluid. The method uses a computer-assisted quantification of the turbidimetric reaction between the Limulus amebocyte lysate and endotoxin. Serial dilutions of Escherichia coli endotoxin in culture-negative amniotic fluid were prepared, and the samples were run in the assay. Amniotic fluid was found to enhance the reaction, and a dilution of 1:20 was required for this biologic fluid to behave similarly to pyrogen-free water. The sensitivity of this kinetic turbidimetric technique in the detection of endotoxin in amniotic fluid was 40 pg/ml. This method was applied to the quantification of endotoxin concentration in amniotic fluid in 26 patients with intraamniotic infection and premature rupture of membranes. Patients in active labor had higher concentrations of endotoxin (median = 47,514 pg/ml) than nonlaboring patients (median = 635 pg/ml) (p less than 0.025). Therefore, women with preterm labor had a higher median concentration of endotoxin in amniotic fluid than patients who were not in labor.

Amniotic Fluid↗
PubMed1988

Assay of bacterial endotoxin (lipopolysaccharide) in human amniotic fluid: potential usefulness in diagnosis and management of preterm labor.

The long-range goal of our research is to determine whether the presence of bioactive agents of infection in amniotic fluid may serve as sensitive indexes of the existence of infection as the cause of preterm labor in a given pregnancy. The aim of this study was to explore the possibility that bacterial endotoxin (lipopolysaccharide) could be detected and quantified in amniotic fluid. In particular, we sought to ascertain (1) if amniotic fluid could be collected in a manner to prevent endotoxin contamination, (2) whether there were inhibitors of lipopolysaccharide in amniotic fluid, and (3) if Limulus amebocyte lysate-based assays could be used to identify and quantify lipopolysaccharide in this fluid. We found that the Limulus amebocyte lysate assays (gelation assay and chromophore generation assay) were useful in the qualitative and quantitative analysis of lipopolysaccharide in amniotic fluids. Lipopolysaccharide-mediated generation of chromophore in the presence of amniotic fluid was accelerated strikingly compared with that of lipopolysaccharide in endotoxin-free water. In three amniotic fluid samples obtained during preterm labor, lipopolysaccharide was detectable in aliquots of 1 to 10 microliters by use of the gelation assay and lipopolysaccharide was quantifiable by use of chromophore generation assays. Two of these amniotic fluid samples were sterile as determined by bacteriologic examination; in the third sample, Fusobacterium species was identified. We suggest that these assays may be extremely useful in the identification of lipopolysaccharide in amniotic fluid. Indeed, lipopolysaccharide may serve as one marker of infection useful in establishing the cause of preterm labor.

Amniotic Fluid↗
PubMed1986

Endotoxemia after relief of biliary obstruction by internal and external drainage in rats.

Systemic and portal endotoxemia were studied in rats with biliary obstruction and after relief of the obstruction by internal and external drainage. Endotoxemia was increased after bile duct ligation (p less than 0.001) compared with control values. The incidence of systemic and portal endotoxemia was significantly reduced after internal drainage (p less than 0.001). A significantly higher incidence of portal (86 percent) and systemic (57 percent) endotoxemia, however, was found after external drainage. The persistence of endotoxemia after external drainage, when serum bilirubin levels returned to normal units, indicates that bile flow is important in controlling endotoxemia during preoperative biliary drainage. These results suggest that the systemic endotoxemia observed after relief of obstruction by external drainage may contribute to the increased mortality, as found in previous rat studies. This observation may contribute to an understanding of why patients with preoperative external drainage of biliary obstruction have a higher incidence of septic complications.

Animals↗
PubMed1978

Quantitative Limulus lysate assay for endotoxin activity: aggregation of radioiodinated coagulogen monomers.

This communication describes a modification of the Limulus lysate assay which allows precise quantitation of picograms of bacterial lipopolysaccharide activity. The method measures the incorporation of 125I-labeled coagulogen monomers into the lysate clot as a function of lipopolysaccharide concentration. The method is more precise and requires less lysate than the previously described quantitative assays for endotoxin activity.

Animals↗
PubMed1992

Application of a novel apparatus, the quartz chemical analyzer, to the determination of endotoxin in blood.

A novel apparatus called a quartz chemical analyzer (QCA) has been developed using a quartz crystal resonator. This apparatus measures sample viscosity changes based on resonant frequency changes of the quartz crystal. The apparatus was used to determine bacterial endotoxin concentrations by monitoring the gelation reaction of Limulus amebocyte lysate. The QCA determined endotoxin concentrations with good accuracy and reproducibility in the range of 0.001-3 EU/ml for endotoxin standard (JP XII). For endotoxin determination in human whole blood and plasma samples, the inhibitory reaction was eliminated by pretreatment of a fourfold dilution at 60 degrees C and incubation for 30 min. There are many advantages of the QCA method compared with the turbidimetric and chromogenic methods. For example, QCA can measure sample viscosity changes with high sensitivity and accuracy because QCA detects minor resonant frequency changes and the frequency data give a numerical value for easy quantitation. QCA can examine turbid samples, and the required quantities of samples and reagents are small, since the quartz crystal detects sample viscosity changes directly. The endotoxin determination time may be shortened by raising the reaction temperature, and QCA can detect other types of coagulation reactions.

Antithrombin III↗
PubMed1992

Soybean trypsin inhibitor and beta-amylase induce alveolar macrophages to release nitrogen oxides.

Rat alveolar macrophages incubated with soybean trypsin inhibitor and beta-amylase produced nitrite in a dose- and time-dependent manner. This production depends on the presence of L-arginine (L-arg) in the culture medium. The precursor of this nitrite was demonstrated as being nitric oxide by bleaching ferredoxin at 410 nm when added to the culture medium. NG-Monomethyl-L-arginine and the tetrahydrobiopterin biosynthesis inhibitor 2,4-diamino-6-hydroxypyrimidine inhibited the release of nitrite in a dose-dependent manner. Dexamethasone was able to modulate this release. These data indicate that alveolar macrophages are capable of secreting L-arg-derived nitrogen oxides when stimulated with certain alimentary proteins.

Animals↗