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PubMed1988

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.

Adult↗
PubMed1987

[Clinical value of endotoxin determination in infection. Comparison of the Limulus amebocyte lysate test with detection of bacterial pathogens].

To evaluate usefulness of Limulus amoebocyte lysate test and blood culture in the diagnosis of septicemia both tests were performed in 27 intensive care patients. Test results were compared with a clinical sepsis score. Ten (62%) out of 16 patients with clinical diagnosis of septicemia showed a positive endotoxin test and 11 (69%) a positive blood culture. In 14 patients (87%) either endotoxin test or blood culture revealed a positive result. Two out of 11 patients (20%) classified by the sepsis score as non-septic showed positive blood cultures as well as positive endotoxin tests. 4 patients with gram-positive bacteria in the blood cultures showed a positive endotoxin test. Due to lack of sensitivity and specificity the Limulus amoebocyte lysate test is of rather low value in the diagnosis of septicemia. Simultaneous performance of Limulus amoebocyte lysate test and blood culture is able to improve the sensitivity, which then over-rules the one obtained when only blood cultures are performed.

Bacteria↗
PubMed1977

Activation of protein mediators of inflammation and evidence for endotoxemia in Borrelia recurrentis infection.

Fifteen patients with Borrelia recurrentis infection were studied to evaluate the role of certain plasma proteins and endotoxin in the pathophysiology of both the acute illness and the Jarisch-Herxheimer-like reaction. The causative spirochetes disappeared from the blood during the Jarisch-Herxheimer-like reaction, which occurred about 2 hours after antibiotic therapy. The mean titers of Hageman factor, plasma prekallikrein and serum hemolytic complement activity were decreased at the time of admission and 2 hours after treatment, and rose to normal values during convalescence. Serum properdin titers were decreased in 14 patients at the time of admission, in 12 patients 2 hours after treatment, and in none during convalescence. The frequency of elevated levels of fibrinogen-related antigens increased from three patients at the time of admission to 12 patients 2 hours after treatment. Results of plasma limulus tests for endotoxin-like material were positive in 11 patients at the time of admission and in 13 patients 2 hours after treatment. These findings demonstrated that Hageman factor, prekallikrein and proteins of the complement system are activated in B. recurrentis infection and that endotoxin may play a role in both the acute illness and in the development of the Jarisch-Herxheimer-like reaction after treatment.

Adolescent↗
PubMedDate not supplied

Plastics, endotoxins, and the Limulus amebocyte lysate test.

A variety of polypropylene and polystyrene tubes have been tested for use with the Limulus amebocyte lysate (LAL) test. Polypropylene tubes tended to be more contaminated with endotoxin than polystyrene. One brand of polypropylene tube contained a water extractable inhibitor of the LAL test. Polystyrene tubes from some manufacturers caused enhancement of the LAL test. Other polystyrene tubes were not significantly different from glass for storage of endotoxin or dilution water. Results of these studies indicate that while some tubes are well suited for use with the LAL test, others are not.

Biocompatible Materials↗
PubMed1983

Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa.

Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3-deoxy-D-manno-octulosonic acid, glucose, 3-deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram-negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assays than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi.

Animals↗
PubMed1978

Dithiols simulate endotoxin in the Limulus reaction.

Dithiothreitol, dithioerythritol and bacterial lipopolysaccharides increase optical absorbance and clot Limulus lysate. Purification of dithiothreitol from possible endotoxin contamination by vacuum sublimation or chromatography does not abolish the reaction with lysate. The dithiols reported active here represent the smallest molecules capable of simulating endotoxin in the Limulus test.

Bacterial Toxins↗
PubMed1983

[Do latent bacteremia and endotoxinemia exist in cirrhotics? A study of 48 patients].

Forty-eight patients with alcoholic cirrhosis and absence of patent infection were assessed for asymptomatic bacteriemia and endotoxemia. 280 blood cultures have been performed and 190 serum samples collected for study by two different methods of the limulus test (LT). Bacteriemia was found in 7 blood cultures from 3 patients. In these patients, occult infection was demonstrated in each case (cholecystitis, ulcerated rectal adenocarcinoma, cutaneous infection). Sixteen LT from 14 patients were positive initially. Only one LT remained positive after control by two different methods. These results suggest that in patients with alcoholic cirrhosis and in the absence of patent infection: 1) bacteriemia is an infrequent feature and, if present, explained by other causes than cirrhosis. 2) LT is negative in systemic blood when rigorous controls are performed, and endotoxemia cannot be substantiated by this test.

Adult↗
PubMed1978

Typhoid fever. Studies of blood coagulation, bacteremia, and endotoxemia.

Patients with typhoid fever were studied to determine whether disseminated intravascular coagulation (DIC), circulating bacteria, and endotoxemia were responsible for the signs and symptoms of their illnesses. Coagulation tests in 28 patients detected thrombocytopenia in 17, hypofibrinogenemia in nine, and elevated titers of fibrinogen-related antigens in 20. Repeated testing during convalescence showed a return toward normal values. Intestinal bleeding, however, did not correlate with abnormalities of coagulation tests. Thus, DIC occurred commonly but appeared to be a subclinical event in these patients. In 25 patients with positive blood cultures for Salmonella typhi, quantitative cultures detected from less than 10 to 9 x 10(2) bacteria/ml. Limulus tests for endotoxin in plasma were negative in all 21 patients tested. These results indicated that the concentrations of circulating bacteria and endotoxin in typhoid fever are lower than in other Gram-negative bacterial infections and suggested that circulating bacteria and endotoxin do not play a major role in the pathogenesis of typhoid fever.

Antigens↗
PubMed1992

G test, a new direct method for diagnosis of Candida infection: comparison with assays for beta-glucan and mannan antigen in a rabbit model of systemic candidiasis.

An indirect method to measure beta-glucan, a major structural component of yeast cell walls, is available, but has the disadvantage of requiring the combined use of two assays. Recent reports describe the fungal index, which measures the difference between the conventional limulus test, in which factors C and G react with endotoxin and beta-glucan, and a new endotoxin-specific test, in which only factor C reacts with endotoxin. The G test was developed as a direct method to measure beta-glucan, and contains only factor G reacting with beta-glucan alone. In this study, the G test was examined in sera of rabbits with experimental systemic candidiasis, and compared with the fungal index and mannan assay. The G test showed positive in all rabbits with systemic candidiasis faster and with higher titers than with the fungal index. Three rabbits with fulminant systemic candidiasis showed higher levels of reactivity with the G test and the fungal index than two rabbits with mild reactions. Mannan was positive by at least one serum in four of five rabbits by the latex agglutination test, and there was a good correlation between these assays. The G test is a good serodiagnostic method for the detection of candidiasis.

Animals↗
PubMed1991

New trends in the use of Al(OH)3-conjugated endotoxins and their subunits from the S- and R-forms of Shigella dysenteriae serovar 1 for model vaccination purposes.

The lipopolysaccharides, lipopolysaccharide-protein complexes and their lipids A, isolated from Shigella dysenteriae 1, exhibited lethal toxicity (LD50 300-400 micrograms per mouse), pyrogenicity (0.01-1.0 microgram), activity in the Limulus test (10(-3)-10(-12) mg ml-1) and produced a positive local Shwartzman reaction. It was found that these substances bound to Al(OH)3 were capable of successfully protecting mice from challenge with both homologous and heterologous infections (up to 90% of mice survived a challenge with LD100), whereas immunization with substances with no biological carrier exhibited very low or zero protective effects. Furthermore, the positive cross-reactivity of lipids A in the agar immunodiffusion test confirms the wide applicability for vaccination purposes.

Aluminum Hydroxide↗
PubMed1980

Pathogenesis of experimental Leptospira interrogans serotype icterohaemorrhagiae infection in the guinea pigs: possible role of endotoxin of intestinal bacteria in the development of lesions.

Germ-free guinea pigs were infected with a virulent strain of Leptospira interrogans serotype icterohaemorrhagiae to evaluate the possible role of endotoxin of intestinal bacteria in the development of hemorrhages and hepatic and renal failure. Clinical manifestations, necropsy findings and histological lesions were similar to those seen in control animals. In the second part of the experiment, endotoxin was not detected by the Limulus test in the blood of ten guinea pigs during the five day course of leptospirosis.

Animals↗
PubMed1988

Endotoxemia in patients who underwent ultrasonic lithotripsy and extracorporeal shock wave lithotripsy.

The authors conducted limulus tests of assay blood endotoxin in order to study the fever which often develops after ultrasonic lithotripsy (USL) and extracorporeal shock wave lithotripsy (ESWL). Assays of endotoxin were also conducted after transurethral resection (TUR) and other operations as a basis for comparison. Endotoxemia was observed in 18 among 28 patients (64.3%) who underwent USL, in 8 among 34 patients (23.5%) who underwent ESWL, in 1 among 14 patients (7.1%) who underwent TUR and in 1 among 11 patients (9.1%) who underwent other operations. The incidence of endotoxemia was significantly higher (p less than 0.01 by chi 2 test) in patients who had undergone USL than in those who had undergone ESWL, TUR or other operations.

Endotoxins↗
PubMed1977

Effect of transient intestinal ischaemia on the thoracic duct lymph absorption of endotoxin.

One-hour intestinal ischaemia by clamping the superior mesenteric artery and interrupting the intramural collaterals was produced in 8 dogs. Thoracic duct lymph, mesenteric venous blood and arterial blood were examined before and during intestinal ischaemia and after revascularization of the small bowel. Venous blood was the major route of transport of lactic acid and lactate dehydrogenase from the ischaemic bowel. Endotoxin activity determined by the limulus test was found in the lymph before the induction of intestinal ischaemia in 4 of 6 dogs while it was absent in arterial blood. After one hour lasting revascularization endotoxin was present in the lymph of all dogs and in 3 and 4 dogs in arterial and mesenteric venous blood respectively. It was demonstrated that limulus-positive substances escape from the intestine both via the lymph and blood.

Animals↗
PubMed1981

[Endotoxins in the plasma and ascitic fluid of the cirrhotic patient. Their clinical significance].

The endotoxin presence was tested by Limulus Lysate Test (LLT) in plasma and ascites of 29 patients with cirrhosis, all having demonstrable portal hypertension and ascite. It was positive in plasma in 7 cases (24.1%) and in ascite in 12 cases (73.12%). All the cases with endotoxin in the plasma were positive in ascite too. 21 patients presented porto-sistemic encephalopathy from 1 to 4 grade, 1 case only had the clinical features of hepatorenal syndrome. The CHE and protrombin test values were significantly different in LLT positive patients in plasma and ascite respect to the values of negative ones.

Ascitic Fluid↗
PubMed1985

A new chromogenic endotoxin-specific assay using recombined limulus coagulation enzymes and its clinical applications.

A conventional limulus test is not specific to endotoxin because of the presence in amebocyte lysate of a (1----3)-beta-D-glucan-sensitive factor. By fractionating coagulation enzymes in the lysate and recombining only those factors involved in endotoxin-induced coagulation, we have developed a new test specific to endotoxin. The recombined enzymes reacted only with endotoxin, and not with fungal polysaccharides. Conventional amebocyte lysate, on the other hand, reacted with both of them. A good linearity was obtained with this method between endotoxin concentration and absorbance with a sensitivity of 1 pg/ml of Escherichia coli 0111:B4 endotoxin. The regression lines for different types of endotoxins were parallel to one another. For the correct diagnosis of endotoxemia, this new test has a definite advantage over the one using whole amebocyte lysate.

Arthropod Proteins↗
PubMed2001

Biophysical characterization of lipopolysaccharide and lipid A inactivation by lactoferrin.

The interaction of bacterial endotoxins (LPS Re and lipid A, the 'endotoxic principle' of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fourier-transform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein - as monitored by the amide I band - is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a [LF]:[LPS] ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. X-ray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharide-binding protein (LBP)-induced intercalation of LPS. The LPS-induced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentration-dependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed.

Animals↗
PubMed2000

Release of membrane vesicles containing endotoxic lipopolysaccharide in Escherichia coli O157:H7 clinical isolates.

Membrane vesicles released by E. coli O157:H7 strains were investigated by immuno-electron microscopy using anti-O157 antibody. Anti-O157 antibody enhanced the negative-staining of vesicles and we found numerous small vesicles clearly formed around bacterial cells. An immunogold-electron microscopic examination confirmed that lipopolysaccharide (LPS) including the O-side chain is present on the surface of the vesicles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified vesicles showed that the vesicles contained LPS consisting of a lipid-A and an O polysaccharide. In addition, the endotoxic activity of the vesicle was confirmed by a limulus test. These results suggest that the vesicles may play an important role in the pathogenesis of Escherichia coli O157:H7.

Animals↗
PubMed1990

[Interference in endotoxin and fungal polysaccharide assays from blood products and antimicrobial agents].

A conventional chromogenic limulus test (CCLT, normal plasma value: less than 10 pg/ml) detects both of endotoxin and beta-1,3-glucan, while a chromogenic endotoxin-specific test (EST: less than 3 pg/ml) detects only endotoxin. Two sources of pseudo-positive result in CCLT and/or EST were studied; (1) factor G-activating material found in blood products, which is indicative of fungal infections, and (2) interaction in diazo-coupling process of the tests by antibiotics or sulfonamides. CCLT and EST values obtained from 13 vials of blood products including human albumin, antithrombin III, and antihemophilic globulin were 284.6 +/- 284.6 pg/ml and 20.2 +/- 26.4 pg/ml (mean +/- SD), respectively. High CCLT values accompanied by low EST values indicate factor G-activating material in the blood products rather than endotoxin. Sample blanks of 42 plasma specimens obtained from 19 patients were 23.7 +/- 34.7 pg/ml by CCLT and 19.2 +/- 31.5 pg/ml by EST. All the patients were administered Sulfamethoxazole/Trimethoprim (SMX/TMP), Aztreonam (AZT), Ceftazidime (CAZ), Imipenem/Cilastatin (IPM/CS), or Silver sulfadiazine. SMX/TMP, AZT, CAZ, IPM/CS, and Cefmenoxime were demonstrated to react with the diazo-coupling reagents used in CCLT and EST, and to cause false-positive result in the tests. Thus the investigation of blood product and sample blank is required for the assay of endotoxins or fungal polysaccharides using CCLT and EST.

Albumins↗