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PubMed1976

[Endotoxinemia in liver cirrhosis].

Endotoxins of gram-negative bacteria and of intestinal origin, insufficiently cleared by the hepatic reticulo-endothelial system are of an increasing interest within the pathogenesis of liver diseases. With purpose to obtain data concerning incidence and course of endotoxaemia in patients with liver cirrhosis an unselected group of these patients, sequentially admitted, was investigated by means of the Limulus-gelation test, regarded as most sensitive to endotoxins. At the admittance, 65% of the patients had endotoxaemia, further 14% developed endotoxaemia later. In total 79% of the patients investigated had endotoxaemia.---Bleeding from oesophageal varices was associated with endotoxaemia in 78%, functional renal impairment in 75%, consumption coagulopathy in 81%, encephalopathy in 77% and a pyrogen reaction in 82% of the patients. Regarding the Limulus assay, the dilution technique was more sensitive in detection of free endotoxaemia as opposed to the chloroform extract. It is concluded from the results that endotoxaemia in patients with liver cirrhosis is frequent and has to be viewed as relevant within the pathogeneses of chronic liver diseases.

Blood Coagulation Disorders↗
PubMed2001

Endotoxin release and endotoxin neutralizing capacity during colonoscopy.

In 38 patients who underwent elective colonoscopy, endotoxin and endotoxin neutralizing capacity (ENC) were determined by use of the limulus--amebocyte--lysate test. A control group of 10 patients, prepared for colonoscopy, were sampled in the same manner as the study group prior to endoscopy. Elevated endotoxin plasma levels were only found when comparing the plasma levels before endoscopy with the highest levels available during endoscopy. The timed endotoxin plasma levels did not change significantly by use of the conventional limulus amebocyte test. However, ENC was found to decrease significantly 5 min after the onset of endoscopy. Maximal values were reached at the end of colonoscopy which recovered completely 24 h later. These results, obtained in a population which did not receive any infusions, demonstrate that the half life of endotoxin in the circulation seems to be very short and therefore endotoxin cannot itself be detected. On the other hand, small amounts of endotoxin reaching the blood stream are able to reduce ENC which can be analyzed by a modified limulus--amebocyte--lysate test. With the use of ENC and plasma endotoxin determinations, we are able to show significant endotoxemia during a minimal invasive procedure such as colonoscopy.

Adult↗
PubMed1989

[Colony count and LPS content of gram-negative bacteria in cold storage foods and water].

In storage trials, the multiplication of gram negative bacteria was monitored by means of both colony counts and LPS-formation as determined by the three LAL tests methods (the Capillary test, the "Mini" Endotoxin test and the Coatest endotoxin method). The detection limits of the colony forming units which could be determined by the three LAL tests were initially set in model experiments in which beef (M. cleidooccipitalis) was variously inoculated with stationary-phase cells of Pseudomonas sp. previously grown at 30 degrees C. In all three methods, measurable amounts of LPS were possible only at colony counts above 10(3)/ml, g or cm2. The detection limits for colony counts of vacuum packed, aerobically stored beef muscle (Caput long. of M. triceps brachii) were found to lie between 1.0 x 10(3) and 1.0 x 10(4)/g for the Capillary test and the Coatest endotoxin method, and 5.0 x 10(4)/g for the "Mini" Endotoxin test. In the case of poultry carcasses the detection limits lay between 2.0 x 10(1) and 7.0 x 10(2)/cm2 and were thus considerably lower than for beef. With very low levels of bacterial loads, substrate interference in the LPS-LAL reaction must be taken into account. Linear regression analysis gave satisfactory correlation between the concentration of LPS and colony forming units for beef, poultry carcasses, ground beef and mixed salad. An acceptable proportionality was established only for beef and poultry carcasses. Potable water, stagnant for a fortnight in an experimental piping system and sampled at five different points, showed significant regrowth of oligocarbotolerant aquatic bacteria. However, only very low levels of LPS could be determined. All three LAL test methods can be recommended for rapid determination of the load of gram negative bacteria in meat and meat products.

Animals↗
PubMed2000

The role of polar and facial amphipathic character in determining lipopolysaccharide-binding properties in synthetic cationic peptides.

Two series of peptides, designated K and NK were synthesized and tested for lipid A binding and neutralizing properties. K2, which has an 11-residue amphiphilic core, and a branched N-terminus bearing two branched lysinyl residues does not bind lipid A, while NK2, also with an 11-residue amphiphilic core comprised entirely of non-ionizable residues, and a similarly branched, cationic N-terminus, binds lipid A very weakly. Both peptides do not inhibit lipopolysaccharide (LPS) activity in the Limulus assay, nor do they inhibit LPS-induced TNF-alpha and NO production in J774 cells. These results are entirely unlike a homologous peptide with an exclusively hydrophobic core whose LPS-binding and neutralizing properties are very similar to that of polymyxin B [David SA, Awasthi SK, Wiese A et al. Characterization of the interactions of a polycationic, amphiphilic, terminally branched oligopeptide with lipid A and lipopolysaccharide from the deep rough mutant of Salmonella minnesota. J Endotoxin Res 1996; 3: 369-379]. These data suggest that a clear segregation of charged and apolar domains is crucial in molecules designed for purposes of LPS sequestration and that head-tail (polar) orientation of the cationic/hydrophobic regions is preferable to molecules with mixed or facial cationic/amphipathic character.

Amino Acid Sequence↗
PubMedDate not supplied

[Testing for Pseudomonas aeruginosa: an additional parameter for assessing microbiological quality of dialysis water].

In 1995 the regional council of the Lazio region passed Decree N.1650 which regulates the maintenance of dialysis equipment and sets limiting values for microbiological and chemical parameters used to assess the quality of water used for dialysis fluids. As regards the microbiological parameters, limiting values were set for total bacterial count and endotoxins. However, in recent literature, serious health effects following dialysis have been described; these suggest the advisability of including testing for more specific microorganisms and of testing greater samples of dialysis water. In this study, conducted over 2 years, in addition to normal microbiological parameters, dialysis fluids were tested for the presence of bacteria of the Pseudomonas species. Results show that samples which met the standards for dialysis water set by legislation were actually found to be contaminated by Pseudomonas species.

Colony Count, Microbial↗
PubMedDate not supplied

An approach to the determination of endotoxin in anesthetics and antibiotics: use of an ultrafiltration system and enzymatic LAL reaction.

An ultrafiltration system is used to obtain an endotoxin-free buffer for the LAL test. The procedure is combined with a kinetic LAL reaction of high sensitivity. This approach allows for the easy determination of endotoxin levels in parenterals with LAL-interfering substances, reducing the maximum valid dilution necessary for use in product development.

Anesthetics↗
PubMed1987

Quantitation of endotoxin in products using the LAL kinetic turbidimetric assay.

The data presented here show the kinetic turbidimetric LAL assay to be a highly quantitative and effective method for determining endotoxin concentrations in products. The assay allows for the accurate assessment of inhibiting or enhancing effects in products when related to a LRW standard curve. However, designating some products as inhibitors or enhancers can be both misleading and erroneous unless qualified as to the dilution and/or endotoxin concentration. Our results demonstrate that some products can yield both inhibiting and enhancing results when related to water. Due to the enhanced resolution of the kinetic turbidimetric assay, these complicating inhibition/enhancing effects can usually be avoided by diluting to the WED. Alternatively, products could be related to a PSC in which the endotoxin response is defined and quantified within the product itself. The practicality of a PSC, however, depends upon the pass/fail limit established, the "cleanliness" of the product used as a standard and the degree of product "lot to lot" variability. Current FDA Guidelines consider a PSC valid providing the value of the "unspiked" product extrapolated from the regression line of the PSC is less than 10% of lambda, the lowest endotoxin concentration used to construct the standard. All products in which LAL kinetics have not been previously analyzed will require a characterization similar to that used with the four products described. From these data, the optimal methodology for kinetically testing the product (dilution to a WED or generation of a PSC) can be determined. Although analysis of endotoxin in a product will always require a characterization of the kinetics of the LAL-endotoxin-product reaction, subsequent testing should be rapid and straightforward. More importantly, the kinetic turbidimetric assay allows the user to quantitatively assess product endotoxin levels with a degree of precision greater than that of any other methodology currently in use.

Endotoxins↗
PubMed1989

An inhibition enzyme-linked immunosorbent assay technique for the detection of endotoxins in proteins extracted from Escherichia coli K12 recombinant DNA.

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed for the specific quantitation of rough (R) mutant E. coli K12 lipopolysaccharide (LPS). Since R-LPS binds poorly to polystyrene microplates, an LPS-BSA covalent complex was prepared following glutaraldehyde activation and used as a coating surface antigen. A 100-fold higher signal was observed using the LPS-BSA complex as solid-phase antigen instead of free LPS. The LPS detection limit obtained was 0.5 ng/ml. This test was applied to hGH extracts produced genetically engineered E. coli K12 and a good correlation was found with the LAL test. This new LPS titration technique will be useful for detecting LPS in complex mixtures and the antigen-antibody reaction will ensure the specificity of the detection.

Antigens, Bacterial↗
PubMedDate not supplied

Aseptic peritonitis due to peptidoglycan contamination of pharmacopoeia standard dialysis solution.

BACKGROUND: Manufacturers of parenteral solutions adhere to European and US Pharmacopoeia standards to define safety and sterility. In response to excess cases of aseptic peritonitis in peritoneal dialysis patients using icodextrin-containing dialysate that met all pharmacopoeia standards, a global recall was issued in May, 2002. We aimed to establish the cause of aseptic peritonitis. METHODS: We analysed 186 reports of aseptic peritonitis between September, 2001, and January, 2003. Extensive physical, chemical, and microbiological investigations of recalled dialysate were done. We calculated dose-response curves for peptidoglycan-induced interleukin 6 elaboration in peripheral blood mononuclear cells (PBMCs) from healthy donors and for sterile peritonitis in rats. FINDINGS: Although its chemical constituents and concentrations of endotoxin were within pharmacopoeia specifications, the dialysis solution elicited an interlukin 6 response in vivo and in vitro. We identified peptidoglycan from thermophilic acidophilic bacteria (Alicyclobacillus acidocaldarius) as the contaminating proinflammatory substance. In the PBMC assay, strong dose-response relations were noted between peptidoglycan concentrations and interleukin 6. In rats injected with peptidoglycan, dose-dependent increases of intraperitoneal neutrophils and pyrogenic cytokines were recorded. We measured a positive relation between peptidoglycan concentrations in recalled dialysate and reports of aseptic peritonitis. After implementation of corrective actions, the rate of peritonitis returned to baseline. INTERPRETATION: Excess cases of aseptic peritonitis in peritoneal dialysis patients were due to peptidoglycan contamination of dialysate by Alicyclobacillus. This outbreak serves as an example of how contemporary parenteral products with microbial contaminants can be considered safe under current pharmacopoeia tests, but provoke adverse clinical effects.

Animals↗
PubMed1987

CSF evaluation in neurologic disease.

Contraindications to performing a lumbar puncture include local infection, intracranial hypertension and complete spinal block. Routine parameters include pressure, appearance, glucose, protein and cytology. Specific studies such as antigen-antibody tests may identify infectious agents. The color of the CSF may indicate infection, previous hemorrhage or, rarely, metastatic melanoma. The IgG-albumin index is useful when there is inflammation, as in multiple sclerosis.

Central Nervous System Diseases↗
PubMed1977

An improved in vitro pyrogen test: to detect picograms of endotoxin contamination in intravenous fluids using limulus amoebocyte lysate.

A method for in vitro pyrogen testing using Limulus amoebocyte lysate (LAL) has been described. The method is based upon the measurement of endotoxin-precipitable protein and can be used to measure picogram quantities equivalent to E. coli endotoxin in unknown solutions. When increasing concentrations of E. coli endotoxin are added to a constant amount of LAL and the reaction is allowed to proceed to completion, there is a proportional increase in the protein precipitated by endotoxin. Therefore, by measuring the amount of protein precipitated from LAL, it is possible to determine the equivalent E. coli endotoxin concentration in unknown solutions, when samples of the unknowns are run simultaneously with E. coli endotoxin standards and negative controls. The endotoxin proportional precipitation of protein occurs in reaction mixture showing gelation as well as in reaction mixture where the levels of endotoxin are lower than required for gelation. Determination of precipitated protein provides greater sensitivity for endotoxin detection than the gelation methods currently in use.

Analysis of Variance↗
PubMed1995

Pharmacokinetics of endotoxin in a rhesus macaque septic shock model.

Using a prospective, randomized, controlled study, we tested the hypothesis that the initial administered dose of endotoxin determines its pharmacokinetics in a rhesus macaque septic shock model. Twelve adult male rhesus macaques, weighing 6 to 10 kg, were equally divided into two groups. The first group received a 20 mg/kg intravenous bolus of the gram-negative endotoxin. The second group received a bolus comparable to the concentration of endotoxin found in the plasma of the first group, 12 hr postendotoxin injection. Both groups were monitored for 12 hr and sacrificed. Plasma endotoxin concentrations were measured using the limulus amebocyte lysate assay and a pharmacokinetic model was applied to the concentration curves. Results of the pharmacokinetic evaluation revealed differences in half-life, clearance, and total apparent volume of distribution between the two groups of animals, suggesting that the changes in these parameters may have a biphasic pattern and may be related to the initial dose of endotoxin injected.

Animals↗
PubMed2004

Beta-D-glucan as a diagnostic adjunct for invasive fungal infections: validation, cutoff development, and performance in patients with acute myelogenous leukemia and myelodysplastic syndrome.

The Glucatell (1-->3)- beta-D-glucan (BG) detection assay (Associates of Cape Cod) was studied as a diagnostic adjunct for invasive fungal infections (IFIs). On the basis of findings from a preliminary study of 30 candidemic subjects and 30 healthy adults, a serum BG level of >or=60 pg/mL was chosen as the cutoff. Testing was performed with serial serum samples obtained from 283 subjects with acute myeloid leukemia or myelodysplastic syndrome who were receiving antifungal prophylaxis. At least 1 serum sample was positive for BG at a median of 10 days before the clinical diagnosis in 100% of subjects with a proven or probable IFI. IFIs included candidiasis, fusariosis, trichosporonosis, and aspergillosis. Absence of a positive BG finding had a 100% negative predictive value, and the specificity of the test was 90% for a single positive test result and >or=96% for >or=2 sequential positive results. The Glucatell serum BG detection assay is highly sensitive and specific as a diagnostic adjunct for IFI.

Adult↗
PubMed1993

Role of the physical state of Salmonella lipopolysaccharide in expression of biological and endotoxic properties.

Lipopolysaccharide (LPS) extracted from three strains of Salmonella typhimurium, i.e., the rough Re mutant SL1102, the rough Ra mutant TV119, and the smooth strain SH4809, was first electrodialyzed (eLPS) and then divalent cation deprived by EDTA treatment and finally made monomeric by deoxycholate solubilization. The removal of excess detergent by extensive dialysis in the absence of mineral cations resulted in the reassociation of LPS subunits into monodisperse micelles of reduced aggregation number (dLPS) as estimated by electron microscopy and gel filtration chromatography. For all LPS chemotypes tested, the developed procedure reproducibly results in stable and clear solutions of dLPS in concentrations of up to 100 mg/ml. The dLPS and eLPS preparations possessed the same reactivity with monoclonal antibodies (MAbs) raised against different LPS domains. The 100% lethal dose in galactosamine-sensitized mice of 0.01 microgram for the smooth eLPS was from 10- to 100-fold lower than that of dLPS at 0.1 to 1.0 microgram. dLPS from both the smooth strain and the Ra mutant had a significantly reduced capacity to activate the proenzyme cascade in the Limulus amoebocyte lysate assay in comparison with the slightly reduced activity of dLPS from the Re mutant. In contrast, dLPS as well as the deoxycholate-dispersed and then diluted eLPS from the smooth strain had a higher mitogenic activity on splenocytes than eLPS. The results indicate that the biological and endotoxic properties of LPS are significantly influenced by the physical state of its aggregates in aqueous solutions. The approach developed for production of a stable and dispersed form of LPS should further assist in investigation of LPS properties and interpretation of the data of endotoxic research.

Animals↗
PubMed1979

Characteristics of lipid A-protein complex from endotoxin of Shigella dysenteriae type 1 (S and R strains).

Mild acetic acid hydrolysis of endotoxin (lipopolysaccharide-protein complex) of Shigella dysenteriae type 1 (S and R forms) yielded a lipid A-protein complex that consisted of amino acids, fatty acids, and sugar and, in terms of chemical composition, displayed no marked differences between the S and R forms. Its protein portion (53 to 56%) consisted of at least 16 amino acids. In the fatty acid portion (14 to 18%), myristic, 3-hydroxymyristic, palmitic, and stearic acids accounted for 50%. The sugar portion (10 to 12%) consisted solely of glucosamine. The remainder was unidentified substances, most of which contained phosphorus. Lipid A-protein complexes derived from both S and R forms were not toxic for mice in doses up to 1,000 microgram/mouse, but their Linulus test activity had increased considerably as compared with the starting lipopolysaccharide-protein complex material: from 10(-6) to 10(-10--10(-12) mg/ml. The lipid A-protein complexes were readily soluble in a water solution of triethylamine, in dimethyl sulfoxide, and in pyridine.

Amino Acids↗
PubMed1992

Pyrogenic reactions in patients receiving conventional, high-efficiency, or high-flux hemodialysis treatments with bicarbonate dialysate containing high concentrations of bacteria and endotoxin.

High-efficiency (HE) and high-flux (HF) hemodialysis are becoming increasingly popular methods for treating patients with chronic renal failure because they reduce the time required for dialysis treatment. HF and HE dialyzers require bicarbonate dialysate, often prepared from concentrates that can support bacterial growth with endotoxin production. There is a concern that endotoxins or bacteria may cross or interact at the membranes of these dialyzers, triggering the release of endogenous pyrogens (cytokines) by peripheral blood mononuclear cells to cause pyrogenic reactions (PR). To determine the incidence of PR and to examine the association between PR and levels of bacteria and endotoxin in dialysate, a cohort of patients receiving conventional, HE, or HF hemodialysis with bicarbonate dialysate and reprocessed dialyzers at three dialysis centers during a 12-month period was studied prospectively. All dialyzers underwent a test of membrane integrity before use. A total of 19 PR were identified among 18 patients in 26,877 hemodialysis treatments (0.7 PR/1,000 treatments). There was no significant difference in PR rates by treatment modality: conventional, 0.5 per 1,000 (7 PR/13,123 treatments) versus HE, 0.9 per 1,000 (9 PR/11,345) versus HF, 1.2 per 1,000 (3 PR/2,409) (P = 0.21; chi 2 test). Throughout the study period, bacterial counts for dialysate at each center significantly exceeded the Association for the Advancement of Medical Instrumentation's (AAMI) microbiologic standards for dialysate of less than 2,000 CFU/mL (mean, 19,000 CFU/mL), but water used in the reuse of dialyzers tested less than 200 CFU/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1996

[LAL test used for detection of undesirable biologically active substances in biopreparations].

Detection of bacterial endotoxin was performed in four groups of biopreparations: IVIG, virus and bacterial vaccines and antibiotics. The 44 samples of biopreparations were tested by the qualitative LAL-test (gel-clot) and some of them (22 samples) by the quantitative, LAL-test (chromogenic end-point). The concentration of endotoxin in 10 samples of IVIG was in the range from 0.457 EU/ml to 19.46 EU/ml. Only 4 of them did not exceed the limit recommended by FDA for human globulins (5 EU/ml). In the 9 samples out of 10 samples of virus vaccines the presence of endotoxin was in the range from 0.06 to 0.15 EU/ml. The concentration of endotoxin in 10 samples of bacterial vaccines determined by gel- clot method was below sensitivity of test (2 EU/ml). In antibiotics we did not find the presence of endotoxin in the range recommended by Ph. Eur. 1995 limits (0.1-0.2 EU/ml). The presented data show the necessity for requirements elaboration for each type of biopreparations.

Anti-Bacterial Agents↗
PubMed2005

The screening and isolation of an effective anti-endotoxin monomer from Radix Paeoniae Rubra using affinity biosensor technology.

Lipopolysaccharide (LPS) is a known trigger in the pathogenesis of sepsis, lipid A being the toxic component. One of several adjuvant therapeutic approaches for severe sepsis is currently focusing on the neutralization of LPS. In order to obtain the components from traditional Chinese herbs that can neutralize the endotoxin, aqueous extractions were tested using affinity biosensor technology. From amongst 42 herbs, eight were found to possess lipid A-binding abilities. Radix Paeoniae Rubras had the highest lipid A-binding ability; therefore an aqueous extraction from this plant was investigated further. After preparation using standard methods, including silica gel chromatography and HPLC, we obtained 1, 2, 3, 4, 6-beta-d-pentagalloylglucose (PGG), with lipid A-binding ability. It was found that in vitro, PGG directly bound to lipid A, with a Kd of 32 microM, and that it neutralized the endotoxin both in the Limulus Amebocyte Lysate (LAL) assay and in a TNF-alpha release experiment, in a dose-dependent manner. In in vivo experiments, PGG was found to protect mice from a lethal challenge by LPS, and significantly decreased the plasma endotoxin level both in endotoxemic mice and rats, the reduction of the endotoxin level in rats being tightly associated with the TNF-alpha level. In conclusion, we demonstrate the effectiveness of affinity biosensor technology in discovering useful agents amongst traditional Chinese herbs and using this approach we found a new anti-endotoxin agent.

Animals↗