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PubMed1984

Comparison of chemical analyses of hollow-fiber dialyzer extracts.

Hollow-fiber hemodialyzers containing cellulose-based membranes have been shown to produce positive results with the Limulus amebocyte lysate (LAL) test. This study was undertaken to determine whether endotoxin was causing the reaction. Rinses from 45 parallel-plate and hollow-fiber dialyzers from eight different manufacturers were tested using three lysates and four LAL methods. In addition, four in vitro cellular methods--human leukocytic pyrogen, lymphocytic activating factor, peritoneal macrophage, and arginase release--were used to evaluate endotoxin activity. The substance causing the reaction was identified using chromatographic methods. Results indicate that the LAL-reactive material is cellulose derived and is not pyrogenic.

Arginase↗
PubMed2005

In vitro bioassay of endotoxin using fluorescein as a pH indicator in a macrophage cell culture system.

Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH-indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS-detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.

Animals↗
PubMed1984

Enhancement of human MLR by very low concentrations of lipopolysaccharide and blocking of this enhancement by polymyxin B.

Addition of 50 micrograms LPS/ml to as little as 5 pg of LPS/ml to the allogeneic human mixed leukocyte reaction (MLR) enhanced [3H]thymidine incorporation 2-4-fold. The same concentrations of LPS had no effect on thymidine incorporation in cultures without the allogeneic cells. The Limulus amebocyte lysate (LAL) test we used was sensitive only to 15 pg LPS/ml indicating that the MLR is more sensitive to LPS than the LAL test. A commercially available RPMI 1640 culture medium used by many investigators was found to contain concentrations of LPS that would significantly enhance the MLR. Polymyxin B blocked the MLR-enhancing effect of LPS while having no inhibitory or stimulating effect itself. We concluded that low concentrations of LPS are helpful in revealing MLR incompatibilities by enhancing the lymphocyte responses and that the addition of polymyxin B is essential in the study of factors affecting the MLR by eliminating any LPS contribution.

Cells, Cultured↗
PubMed1985

Quantification of endotoxin inhibition in serum and plasma using a turbidimetric LAL assay.

A modified Limulus amebocyte lysate (LAL) test was developed which quantifies the inhibition associated with lipopolysaccharide (LPS) in serum and plasma. The assay utilized the LR50 value to determine relative inhibition. The LR50, measured in ng/ml, represented the concentration of endotoxin needed to elicit a turbidimetric response equal to 50% of the maximum above the control (no added endotoxin). The LR50 concept was used to compare plasma and serum from a normal donor population. Plasma LR50 values ranged from 115 to 5000 ng/ml, while serum samples ranged from 48 to 615 ng/ml. Endotoxin in saline measured in a similar manner yielded values averaging 0.07 ng/ml (LAL sensitivity). Various means to remove inhibition were also tested by this method. Heating, dilution, and chloroform extraction of serum were all found to be efficient in removing inhibition. Although no direct attempt was made to elucidate the biochemical nature of the inhibitor(s) present in serum and plasma, the results presented were consistent with other studies and indicate the involvement of a heat labile component(s). The LR50 may provide a tool for the elaboration of these components. It also was suggested that LR50 may be the best in vitro indicator of the overall ability of serum or plasma to neutralize endotoxin.

Chloroform↗
PubMedDate not supplied

Recent development of rigors during infusion of urokinase: is it related to an endotoxin?

PURPOSE: This study was undertaken to determine the prevalence of rigors associated with the use of urokinase (UK) and to assay for the presence of an endotoxin in the UK solution. PATIENTS AND METHODS: Records of 75 patients who underwent 86 UK infusions between January 1988 and July 1992 were reviewed to evaluate for the development of UK-associated rigors. A modified chromogenic limulus amebocyte lysate (LAL) test was performed to determine the presence of endotoxin in four samples of UK from lots associated with rigors, one sample of UK not associated with rigors, sterile water, nonionic contrast medium, and ionic contrast medium. RESULTS: Between January 1, 1988, and July 10, 1990, 43 patients underwent 46 UK treatments (group 1) with no documented rigors (0% prevalence). In 45 of these 46 treatments, a standard, non-pulse-spray bolus of 75,000-500,000 IU of UK (mean dose, 182,222 IU) was used. Between July 11, 1990, and July 6, 1992, 38 patients underwent 40 UK treatments (group 2). In 33 of these 40 treatments, a standard bolus was given. Five patients received a pulse-spray bolus. The mean bolus was 213,768 IU (range, 100,000-500,000 IU). Eleven group 2 patients developed rigors (28% prevalence; P = .0005 vs group 1). The chromogenic LAL tests demonstrated no endotoxin in sterile water, nonionic contrast media, or ionic contrast media. Endotoxin was detected in small concentrations in the four samples of UK associated with rigors and in the UK sample not associated with rigors. CONCLUSION: The increase in the prevalence of rigors associated with the use of UK does not appear to be related to an endotoxin in UK, since the concentration of endotoxin detected is well below the threshold pyrogenic dose in humans.

Contrast Media↗
PubMed1995

[Application of quantitative assay for endotoxin using immobilized histidine and filtration plate to parenteral drugs].

We improved the Limulus amebocyte lysate (LAL) test for endotoxins in parenteral drugs using immobilized histidine and a filtration plate. In order to deal with many samples at the same time and to apply to a routine assay, we used a filtration plate having 96 wells instead of a filter unit with a working volume of 2 ml. LAL test-affecting substances which are contained in a parenteral drug were separated from endotoxins by adsorbing endotoxins on immobilized histidine in the well of a filtration plate. Then the absorbed endotoxins were allowed to react with LAL reagent in the same well. We defined that this method had the higher precision than conventional methods and was not influenced by the concentrations of endotoxin and parenteral drugs. Hence we examined the recovery of endotoxin spiked to 23 kinds of parenteral drugs by this method, as a result, 100 +/- 25% of recovery was obtained from 17 kinds of them.

Endotoxins↗
PubMed1982

Pyrogens from surgeons' sterile latex gloves.

Pyrogens have been detected in water extracts from surgeons' sterile latex gloves, using both the Limulus Amebocyte Lysate (LAL) test and the rabbit test. After the gloves were rinsed with pyrogen-free water, the amount of pyrogens in extracts was markedly reduced. Pyrogens could also be detected in extracts from an angiographic catheter after it had been handled with latex gloves, whereas an unused catheter was free from pyrogens. The incidence of febrile reactions in our catheterization laboratory has been markedly reduced from 11.6% to 0.6% when rinsing the latex gloves prior to the catheterization was included in our routine procedure. So far the absence of pyrogens in surgeons' sterile gloves has not been demanded. Our observations indicate that such a requirement may be justified.

Animals↗
PubMed1999

Changes in endotoxin-binding proteins during major elective surgery: important role for soluble CD14 in regulation of biological activity of systemic endotoxin.

Assessment of circulating endotoxin during the perioperative period, which is only demonstrated by the Limulus amebocyte lysate (LAL) test, may be modulated by several endotoxin-binding proteins. Endotoxin-neutralizing capacity (ENC) and the plasma levels of soluble CD14 (sCD14), lipopolysaccharide-binding protein, and bactericidal/permeability-increasing protein (BPI) were determined in 40 patients 6 h prior to skin incision for major abdominal surgery. The bioactivity of plasma endotoxin was tested by the polymyxin B-inhibited stimulatory activity of the plasma samples on healthy monocytes as measured by the release of tumor necrosis factor alpha. Plasma endotoxin levels in almost all patients increased from 0.05 +/- 0.01 to 0.23 +/- 0.03 experimental units (EU) per ml (P < 0.001); more specifically, 17 of 40 samples showed endotoxin levels of greater than 0.2 EU per ml and corresponding reductions in ENC. Soluble CD14 plasma levels were decreased from 5. 6 +/- 0.3 to 4.6 +/- 0.3 microg per ml (P < 0.05). ENC was strongly correlated with the sCD14 plasma concentration throughout the period of observation. The addition of sCD14-neutralizing monoclonal anti-sCD14 antibodies reduced ENC both pre- and postoperatively. No correlation could be established between ENC and the plasma levels of BPI, high-density lipoproteins, or low-density lipoproteins determined by measuring the concentrations of apoprotein A and apoprotein B. Biologically active endotoxin was found in only 6 of 17 samples with endotoxin levels greater than 0.2 EU per ml in the LAL test. These samples could be characterized by their perioperative loss of at least 35% of their sCD14. No change in sCD14 was detected in the remaining 11 samples. The perioperative loss of ENC is partly caused by the loss of sCD14 resulting from its consumption by endotoxin reaching the bloodstream. This study demonstrated the role of sCD14 on the bioactivity of circulating endotoxin in a human model of endotoxemia after major abdominal surgery.

Abdomen↗
PubMedDate not supplied

Recovery of endotoxin preparations from the surface of glass capillary tubes.

The gel-clot method of the Limulus Amebocyte Lysate (LAL) test was used to determine the recovery efficiency of four different endotoxin preparations from Type I glass capillary tubes. Each capillary tube was charged with 10,000 endotoxin units (EU) and vacuum-dried at 50 degrees C. Average recovery for the Whittaker M.A. Bioproducts Control Standard Endotoxin (CSE), which contained fillers, was 90% (80-100%), for the Difco Laboratories and Associates of Cape Cod endotoxins 47% (40-80% and 20-100%, respectively), and for the rough lipopolysaccharide 70% (40-100%). The presence of fillers appears to increase the percentage of endotoxin recovered and decrease the inconsistency in recovery results. The overall recovery rates for the pure endotoxin formulations were higher and slightly more consistent than those reported in previous studies.

Endotoxins↗
PubMed1993

Endotoxins and the pathogenesis of Trypanosoma brucei brucei infection in mice.

The involvement of endotoxins in Trypanosoma brucei brucei infection in CD-1 mice was investigated by the Limulus amoebocyte lysate (LAL) test. At 7 days post-infection mean serum endotoxin level was elevated by 2.5 times (36.4 pg/ml cf. control 14.25 pg/ml, P < 0.001) and a similar increase was maintained throughout the infection (survival 28-35 days). Purified disrupted parasites contained significant endotoxin activity (mean value 280 pg/mg protein). The mouse infections were also associated with progressive Gram-negative bacteraemia (present in 4 out of 5 infected animals by day 28 p.i.). The increased endotoxin levels may be due to parasite products, the products of intercurrent bacterial infections, other unidentified sources (e.g. from the gut), or a combination of these. It is concluded that the raised endotoxins may be important contributive factors in the pathogenesis of experimental murine trypanosomiasis.

Animals↗
PubMed2001

Urinary endotoxin excretion and urinary tract infection following kidney transplantation.

Following kidney transplantation, urine endotoxin levels were measured among 44 patients and compared to bacterial cultures. Urine samples were collected either via transurethral catheters or - after removal of the catheter on postoperative day 4 - by midstream void. In a control group of ten healthy volunteers, urine endotoxin levels were measured daily for 10 days. Urinary endotoxin concentration was measured by means of a chromogenically modified Limulus amebocyte lysate (LAL) test. The levels among patients with positive bacteriological findings (n = 21) were always elevated ( > 0.7 EU/ml). Furthermore, there was a marked, statistically significant difference in endotoxin values between samples with bacterial growth and samples with fungal or without any growth (P < 0.001). All 21 of the 44 patients with urinary tract infection (UTI) were endotoxin-positive. Seven more patients who received antibiotics had elevated urinary endotoxin levels, but no bacterial growth in the urine culture. No bacterial infection or significant urinary endotoxin was found in the control group. In summary, the detection of urinary endotoxin in samples obtained by either suprapubic/transurethral catheters or midstream void is an early, sensitive, and specific means of diagnosis that can be carried out even during antibiotic treatment.

Bacteria↗
PubMed1989

[Determination of blood endotoxin in severely burned patients and its clinical significance].

Since October 1985 to June 1987, in 12 severely burned patients with endotoxemia was observed by using a quantitative endotoxin assay of limulus amoebocyte lysate (LAL) test with a chromogenic substrate. Among the 12 patients, 4 died and 8 survived. The average age of dead group was 31.8 years (19-45 years), mean TBSA was 63% (58-80%) and mean I 18.5%. The survival group average age of survival group was 27.5 years (18-39 years), mean TBSA was 58% (18-85%) and mean I 24.4% (6-56%). The plasma endotoxin concentrations of burn patients in dead group were 105-571 pg/ml, significantly higher than that of survival group (30-240 pg/ml) and healthy human (6.44 +/- 1.96 pg/ml). It was found that the increase of endotoxemia was closely related to burn wound sepsis, positive of blood culture, systemic disseminated septicaemia. Systemic use of sensitive antibiotics may increase the level of blood endotoxin in severe gram-negative bacillus infection. Polymixin-B is an exception.

Adolescent↗
PubMed1992

Specific assay for endotoxin using immobilized histidine, Limulus amoebocyte lysate and a chromogenic substrate.

The Limulus amoebocyte lysate (LAL) test is inhibited or enhanced by many substances. In order to overcome this problem, a specific endotoxin assay method using a membrane filter unit, a chromogenic LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins) was developed. Endotoxins are quantitatively adsorbed on immobilized histidine. The adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the membrane filter unit, and their activities are directly assayed with the LAL reagent in a filter cup without any inhibition or enhancement. The reproducibility and the accuracy of this method are high. This new endotoxin assay method using immobilized histidine can be used for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics, as an alternative to the more common gel-clot technique.

Adsorption↗
PubMed1991

The effect of endotoxin-contaminated medium on in vitro fertilization and development of bovine oocytes matured in vitro.

The purpose of this study was to determine whether the presence of bacterial endotoxin in the bovine serum albumin (BSA) used to supplement media utilized for sperm preparation and co-culture of bovine sperm and oocytes affects in vitro penetration and embryonic development of oocytes matured in vitro. The chromogenic limulus amoebocyte lysate (LAL) test was used for quantification of the content of endotoxin. The proportion of penetrated ova was significantly greater (P greater than 0.0005) for the endotoxin-contaminated group (89%) versus the non-contaminated group (61%), but this was probably not due to endotoxin contamination. The presence of endotoxin resulted in a high rate of polyspermy (27% versus 4%, respectively; P greater than 0.0005), while the occurrence of parthenogenetic activation was the same for each group (8%). The proportion of total embryos put into culture that developed to the blastocyst stage by day 8 was similar (30% and 26%) for the contaminated and non-contaminated group, respectively. Fifty-three and 69%, respectively, hatched on day 10. These results suggest that endotoxin induces polyspermy, but has no adverse effect on embryonic development.

Animals↗
PubMedDate not supplied

[Quantitative determination of bacterial endotoxins by the chromogenic limulus method: critical analysis and study of interactions between 3 divalent cations].

The reproducibility of the limulus amoebocyte lysate (LAL) test with the use of a chromogenic substrate was evaluated in the presence of 5 bacterial endotoxins (2 from Escherichia coli, 1 from Salmonella, 1 from Serratia marcescens and 1 from Vibrio cholerae) and the FDA reference endotoxin EC5. Endotoxins from E. coli 055B5, S. marcescens and V. cholerae were significantly more active than the reference endotoxin (p less than 0.01). Addition of Mg2+ activated the LAL chromogenic reaction (optimum = 160 mmol/l added), whereas Ca2+ in concentrations above 5 mmol/l inhibited the reaction. As little as 0.3 mmol/l Zn2+ strongly inhibited the reaction. Inhibition by Zn2+ was partly suppressed by addition of 160 mmol/l Mg2+. These divalent cations modified the LAL chromogenic reaction when added in the first step of the reaction (incubation with endotoxin and LAL reagent). The LAL chromogenic reaction was not modified by divalent cations when these were added in the second step of the reaction (with the chromogenic substrate).

Animals↗
PubMed2005

[Non-specific reactions in four methods measuring (1-->3)-beta-D-glucan levels in plasma].

We detected non-specific reactions in the measurement of (1-->3)-beta-D-glucan levels (beta-glucan) in plasma, and the influences of the non-specific reactions on sensitivity and specificity of measurement methods were examined. In this study, 460 plasma samples from 174 patients at Kawasaki Medical School Hospital were used, and the plasma beta-glucan levels were measured by different four methods. The methods included the dilution-heating-endpoint (DHE), dilution-heating-turbidimetric (DHT), alkaline-kinetic (AK), and alkaline-endpoint method (AE) with and without 4-amidinophenyl benzoate hydrochloride (APB) of a protease inhibitor blocking the Limulus reaction. Non-specific reactions were detected from the calculated value under conditions with APB. Therefore, both of the actual values and the values equivalent to non-specific reactions were calculated. The incidence of non-specific reactions was 2.4% in DHE method, 0% in DHT, 53.3% in AK, and 99.3% in AE. The sensitivity and specificity in the methods were 35.7% and 96.0%, 28.6% and 96.0%, 78.6% and 80.1%, and 57.1% and 84.1%, respectively. When subtracted the non-specific reaction values from the actual values in AK and AE method, the specificity was increased by 91.4% and 94.0%, respectively. In these two methods, the non-specific reaction was considered to be a major cause of the low specificity. Finally, to measure plasma beta-glucan levels accurately, non-specific reactions should be excluded as possible by further improvement of measurement methods.

Alkalies↗
PubMed1994

Use of immobilized histidine in assay for endotoxin in patients with liver disease.

The Limulus amebocyte lysate (LAL) test has the disadvantage of being influenced by various inhibitors and activators. We have developed a method for the LAL reaction that involves the specific adsorption and isolation of endotoxin using a membrane filter unit and immobilized histidine; in this present study we used the method to measure endotoxin in the plasma of patients with acute or chronic liver disease. The adsorbed endotoxins are separated from LAL-inhibitors or -activators by the membrane filter unit, and their activity is directly assayed with the LAL reagent in a filter cup without any inhibition or activation. The study population consisted of 23 subjects, 3 with fulminant hepatitis and 20 with cirrhosis (9 with esophageal varices and 11 without). All 3 (100%) of the samples of plasma from patients with fulminant hepatitis were positive for endotoxin, as were the samples of 7 (78%) of the 9 patients with cirrhosis and esophageal varices, and 2 (18%) of the 11 patients with cirrhosis but without such varices. The results suggested that this method appears to be useful for assaying the concentration of endotoxin in patients with fulminant hepatitis or cirrhosis of the liver.

Acute Disease↗
PubMed2002

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller).

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.

Animals↗