A new method for the quantification of β-glucan in plasma and its application in the diagnosis of postoperative infection

In order to correctly diagnose and treat severe postoperative infections, it may be critical to detect and differentiate between endotoxin derived from Gram-negative bacteria and/or β-glucan derived from fungi. In addition to the chromogenic assay, the turbidimetric kinetic assay has been performed for the quantification of endotoxin in plasma usingLimulus amebocyte lysate as previously reported. However, it is also known that β-glucan triggers the coagulation ofLimulus amebocyte lysate. In the present study, the differentiation of β-glucan from endotoxin and its clinical application were studied. Endotoxin was able to be inactivated in plasma using one-tenth dilution by 10 per cent ethanol or distilled water, followed by heating at 100°C for 120 min, without affecting the activity of coexisting β-glucan. The treated sample was then subjected to the turbidimetric kinetic assay using Toxinometer ET-201^®. Using this method, as little as 30 pg/ml of β-glucan in the plasma may be assayed separately, with the amount of circulating β-glucan in the plasma of normal subjects being less than 50 pg/ml. On the other hand, in patients with a fungal infection, the amount of β-glucan in their plasma was elevated significantly. Clinically, β-glucanemia may often occur in severe postoperative infection even if fungi are not detected.     

A study on limulus amebocyte lysate (LAL) reactive material derived from dialyzers

The amebocytes of horseshoe crab (Limulus) hemolymph contain a coagulation system highly sensitive to bacterial endotoxins. Limulus amebocyte lysate (LAL) reactive material derived from cuproammonium rayon membranes, however, is not an endotoxin and acts upon the alternative pathway in the coagulation cascade found in Limulus amebocyte lysate. This study confirmed these facts by using the coagulation system of Limulus without factor G, which is a substrate of the alternative pathway. LAL reactive material lingered in the circulation for a relatively long time. In acute hemodialysis, its plasma concentration increased by an average of 100 pg/ml with each dialysis and eventually reached a plateau of approximately 300 pg/ml. In patients with chronic renal failure under regular hemodialysis, the mean level of LAL reactive material was 330.0 +/- 8.0 pg/ml before hemodialysis which increased by 70.6 +/- 20.7 pg/ml after four hours of hemodialysis.     

A study on limulus amebocyte lysate (LAL) reactive material derived from dialyzers

The amebocytes of horseshoe crab (Limulus) hemolymph contain a coagulation system highly sensitive to bacterial endotoxins. Limulus amebocyte lysate (LAL) reactive material derived from cuproammonium rayon membranes, however, is not an endotoxin and acts upon the alternative pathway in the coagulation cascade found in Limulus amebocyte lysate. This study confirmed these facts by using the coagulation system of Limulus without factor G, which is a substrate of the alternative pathway. LAL reactive material lingered in the circulation for a relatively long time. In acute hemodialysis, its plasma concentration increased by an average of 100 pg/ml with each dialysis and eventually reached a plateau of approximately 300 pg/ml. In patients with chronic renal failure under regular hemodialysis, the mean level of LAL reactive material was 330.0±8.0 pg/ml before hemodialysis which increased by 70.6±20.7 pg/ml after four hours of hemodialysis.     

Beta-glucan reflects liver injury after preservation and transplantation in dogs.

Graft failure and extrahepatic organ complications, which frequently develop after transplantation, may be related to inflammatory mediators stimulated by endotoxin (ET). The role of endotoxemia after liver transplantation is controversial and may depend upon differences in the ET assay method used in the various contradicting studies. While the standard Limulus amebocyte lysate (LAL) is reactive for ET and beta-glucan, a novel turbidimetric assay method enables separate determinations of ET and beta-glucan. Beagle dogs undergoing orthotopic liver transplantation were divided into two groups. In Group I (n = 6) the grafts were transplanted immediately and in Group II (n = 6) grafts were preserved for 48 h in University of Wisconsin (UW) solution. Animals received cyclosporine immunosuppression and were followed for 14 days. Daily measurements of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were performed. Samples for ET and beta-glucan measurement were collected serially and processed using the turbidimetric assay method. While no graft failure was seen in Group I, three of six Group II animals died from graft failure within 1 day after transplantation. Preservation and reperfusion injury was much more severe in the Group II grafts than in Group I grafts. While endotoxemia could not be detected, postoperative beta-glucan levels (undetectable pretransplant) were seen in both groups. Beta-glucan levels were much higher in Group II grafts than in Group I grafts, and correlated with the severity of liver damage. In conclusion, this study shows that beta-glucan, instead of ET, appears during the early posttransplant period. We believe that posttransplant elevation of beta-glucan is related to liver damage, especially endothelial damage by preservation and reperfusion.     

Endotoxemia is masked in fungal infection due to enhanced endotoxin clearance by beta-glucan.

Employing a novel turbidimetric assay, the amount of endotoxin and beta-glucan in plasma was monitored in postoperative patients suffering from both bacterial and fungal infections. The patients, whose beta-glucan level was markedly elevated, were always negative for endotoxin, suggesting a possible role of beta-glucan in the clearance of circulating endotoxin. In order to test the above hypothesis, effects of beta-glucan (carboxy methylated curdlan or lentinan) on endotoxin clearance were studied in rabbits. beta-glucan (4 micrograms/kg or 40 micrograms/kg) was intravenously administered to rabbits which received 4 micrograms/kg endotoxin injection, simultaneously or 120 min before the injection. Either of the beta-glucans increased the endotoxin clearance in a dose-dependent manner, and the preinjection of beta-glucan was more effective than the coinjection. The increased amount of plasma beta-glucan results in the unexpectedly low level of plasma endotoxin, which may be removed by the reticuloendothelial system activated by beta-glucan, in cases with both bacterial and fungal infections. Thus, it is important to recognize that endotoxemia may be masked by the coexisting fungal infection.     

Application of a quantitative spectrophotometric endotoxin assay on lymph and plasma from the rat

A quantitative spectrophotometric assay for endotoxin, utilizing Limulus amebocyte lysate and a chromogenic peptide substrate, was standardized for the application on lymph and plasma from the rat. The influence of inhibitors and unspecific amidolytic activity, the optimal incubation times, the adhesion of endotoxin to platelets and glass surface, the variation in endotoxin standard as well as the effects of cold storage on endotoxin activity were settled. In vitro added as well as endogenously derived endotoxin was studied. The method was found to be reliable when in practical use on these media.     

Endotoxemia and enhanced generation of oxygen radicals by neutrophils from patients undergoing cardiopulmonary bypass

Plasma endotoxin concentrations and oxidative burst response of peripheral blood polymorphonuclear leukocytes were examined in 12 patients undergoing coronary artery bypass. The measurements were made just before the operation, 5 minutes after removal of the aortic crossclamp, and 24 hours after the operation. Endotoxin was quantitated by a combination of a sensitive Limulus amebocyte lysate assay and rocket immunoelectrophoresis measuring picogram amounts of endotoxin. Peripheral blood neutrophils were purified by a two-step dextran sedimentation and metrizoate sodium Ficoll (Lymphoprep., Nyegaard, Oslo, Norway) centrifugation. The oxidative burst response of these cells was measured for their ability to generate superoxide anion and was determined by a cytochrome c reduction assay. Preoperatively, all the plasma samples except one were free of endotoxin. The endotoxin levels reached 100 pg/ml 5 minutes after removal of the aortic crossclamp, and except in one sample they had decreased 24 hours after the operation. Studies on the generation of superoxide by neutrophils showed a decline in the response 5 minutes after removal of the aortic crossclamp and an enhancement of the response to f-Met-Leu-Phe by cells obtained from 11 of 12 patients 24 hours postoperatively. In vitro addition of bacterial lipopolysaccharide to blood from healthy individuals also enhanced the superoxide response of the neutrophils. We conclude that during cardiopulmonary bypass the circulating blood is contaminated by endotoxin and the neutrophils are primed for enhanced generation of oxygen radicals. The released oxygen radicals may be involved in the tissue damage observed in these patients.     

Presence of circulating endotoxins during cardiac operations

Ten patients having coronary artery bypass grafting were intraoperatively and postoperatively analyzed for endotoxins with the Limulus amoebocyte lysate test. A new highly sensitive rocket immunoelectrophoretic assay for reading the reactions of endotoxins with Limulus amoebocyte lysate was used. Preoperatively, all blood samples from the patients had negative Limulus amoebocyte lysate tests, negative blood cultures, normal total white cell counts, and were clinically without signs of infection. Intraoperatively, a substantial amount of endotoxins were found in samples from the extracorporeal circuit, the pulmonary artery, and the cardiac suction lines, which persisted during the cardiopulmonary bypass. The endotoxin content decreased significantly (p less than 0.05) 6 hours after cardiopulmonary bypass and further decreased within the seventh postoperative day (p less than 0.01). A positive Limulus amoebocyte lysate test was also found in some of the fluids administered during the operation, that is, the cardioplegic fluids, the priming fluids for the extracorporeal circuit, the blood transfusions, and the ice for local cooling. Postoperatively, all patients had rectal temperatures below 38.5 degrees C, but no correlation was found between the magnitude of endotoxin content and the degree of fever. Only one of the patients had positive blood cultures. Despite the measured endotoxin content, no intraoperative or postoperative complications were found.     

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.     

Measurement of operative plasma endotoxin levels in jaundiced and non-jaundiced patients

A study of portal plasma endotoxin levels was performed using a chromogenic limulus amoebocyte lysate (LAL) assay. The assay proved sensitive and reproducible. In only 1 of 25 healthy subjects was the systemic plasma endotoxin level above 100 pg/ml (equivalent Escherichia coli 0111B4). In 30 non-jaundiced patients undergoing surgery the mean (+SEM) portal plasma endotoxin level (60 + 9 pg/ml) was significantly higher (p less than 0.05) than the mean level in the systemic blood (46 + 6 pg/ml), supporting the concept of endotoxin absorption from the intestine into the portal blood. In 20 patients with obstructive jaundice undergoing surgery 42% of portal, 45% of inferior mesenteric and 35% of systemic venous plasma endotoxin levels were above 100 pg/ml. There were significantly higher levels in the portal (p less than 0.05) and inferior mesenteric (p less than 0.05) compared with the systemic blood. Neither the presence of malignancy nor the duration of surgery appeared to influence endotoxin absorption. The significance of raised plasma endotoxin levels in obstructive jaundice is discussed.     

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.     

Bacterial Challenge of NISSHO Ultrafilter ETF 609: Results of In Vitro Testing

Abstract: In hemodialysis, a certain degree of bacterial contamination on the dialysate side is a regular finding. Concern has been growing that this contamination may lead to a chronic inflammatory response in the patient. Ultrafiltration of dialysate can be used to reduce bacterial content and levels of cytokine-inducing substances upstream of the patient's dialyzer. The aim of this study was to test in vitro the rejection capacity of a polysulfone hollow-fiber ultrafilter (ETF 609, NISSHO Co., Osaka, Japan) challenged with bacterial filtrates derived from Pseudomonus aeruginosa PA 103. Results showed a reduction of interleukin-Iβ-inducing activity (measured on peripheral blood mononuclear cells) from 5,035 ± 394 pg/ml prefilter to nondetectable levels postfilter and endotoxin levels (limulus amebocyte lysate assay) of 4,167 ± 1,079 versus 12 ± 2 pg/ml, respectively. In conclusion, ultrafiltration of dialysate with the polysulfone ultrafilter ETF 609 leads to a potent reduction of cytokine-inducing activity.     

Endotoxaemia in severe burns

In an attempt to detect endotoxaemia the authors carried out the Limulus test in burned patients' plasma. The test was positive mainly in lethal cases, yet it was negative in many other cases where endotoxaemia could be presumed.The authors therefore carried out a series of experiments in rabbits, in which they proved that endotoxin entering the circulation is being accumulated in leucocytes and can be detected in platelet-leucocyte lysate, using Limulus test, even at low concentrations, when Limulus test in plasma is negative.It appears that endotoxin detection by Limulus test in plasma does not offer decisive results and that it should be supplemented by endotoxin detection in platelet-leucocyte lysate.     

Portal and systemic endotoxemia in abdominal operations: the significance of acute abdomen

Little evidence is available for the implication of bacterial translocation in cases of acute abdomen. Intraoperative endotoxemia in both portal and systemic circulation was studied in 20 surgical patients with acute abdomen and in 36 controls undergoing elective abdominal surgery. Blood was sampled simultaneously from a mesenteric vein immediately after opening the peritoneum and from a peripheral vein. Endotoxin was measured by a colorimetric Limulus amebocyte lysate assay and malondialdehyde (MDA) was measured by the thiobarbiturate assay and passage through a high-performance liquid chromatography (HPLC) system as a marker of the oxidative status. LPS concentrations (mean +/- SE) in portal vein blood from patients with acute abdomen was 5.69 +/- 1.58 and from patients with chronic diseases 1.05 +/- 0.07 EU/ml (P < 0.0001). Respective values for the systemic circulation were 4.98 +/- 1.47 and 1.36 +/- 0.31 EU/ml (P < 0.0001). Concentrations of MDA (mean +/- SE) in portal vein blood from patients with acute abdomen was 11.16 +/- 4.00 and from patients with chronic diseases was 10.56 +/- 2.39 mum (P NS). Positive correlations were observed between endotoxin and MDA in both portal and systemic circulation. These results indicate increased levels of endotoxin in acute abdominal conditions pointing to the gut as the site of origin of the bacterial products.     

In vitro assessment of dialysis membrane as an endotoxin transfer barrier: geometry, morphology, and permeability

High-flux dialysis membranes used with bicarbonate dialysis fluid increase the risk of back diffusion of bacterial endotoxin into the blood during hemodialysis. Endotoxin transfer of various synthetic fiber membranes was tested with bacterial culture filtrates using an in vitro system testing both diffusive and convective conditions. Membranes were tested in a simulated dialysis mode with endotoxin challenge material (approximately 420 EU/mL) added to the dialysis fluid, with saline used to model both blood and dialysis fluid. Samples were taken of both blood and dialysis fluid, and analyzed using a kinetic turbidimetric Limulus amoebocyte lysate assay. Endotoxin was found in all of the blood circuit samples, except for the Fresenius Optiflux F200NR(e) and thick-wall membranes. All membranes tested removed approximately 95% of the endotoxin from solution, with the residual approximately 5% recirculating within the dialysis fluid compartment. Endotoxin distribution through the fiber membrane was examined using a fluorescent-labeled endotoxin conjugate. Fluorescence images indicate that adsorption occurs throughout the membrane wall, with the greatest concentration of endotoxin located at the inner lumen. Contact angle analysis was able to show that all membranes exhibit a more hydrophilic lumen and a more hydrophobic outer surface except for the polyethersulfone membranes, which were of equal hydrophobicity. Resulting data indicate that fiber geometry plays an important role in the ability of the membrane to inhibit endotoxin transfer, and that both adsorption and filtration are methods by which endotoxin is retained and removed from the dialysis fluid circuit.     

Gel clot LAL assay in the initial management of peritoneal dialysis patients with peritonitis: a retrospective study.

BACKGROUND: Indiscriminate use of broad-spectrum antibiotic treatment of peritonitis in peritoneal dialysis patients may have either unwanted side-effects or contribute to the development of antibiotic resistance. This may be avoided by improved diagnosis at presentation. The Limulus amoebocyte lysate assay is a convenient test detecting bacterial endotoxins or fungal beta glucans. This study evaluates a qualitative Limulus amoebocyte lysate test as a diagnostic tool used at presentation of a peritoneal dialysis patient with peritonitis. METHODS: One-hundred and eleven episodes of peritonitis in peritoneal dialysis patients have been analysed retrospectively. Limulus amoebocyte lysate results at presentation were compared with culture results. A Limulus amoebocyte lysate assay was performed using a commercial kit by incubating a mixture of dialysate effluent and Limulus amoebocyte lysate reagent at 37 degrees C. The development of a stable solid clot was considered positive. The specificity and sensitivity of the test were calculated. RESULTS: The specificity of the Limulus amoebocyte lysate assay was found to be 98% and the sensitivity 74%. Limulus amoebocyte lysate assay was false-negative in 13 cases of Gram-negative peritonitis (22%). Limulus amoebocyte lysate was positive in three of seven cases of fungal peritonitis. The study included one case each with false-positive Limulus amoebocyte lysate and with culture-negative peritonitis. CONCLUSIONS: The Limulus amoebocyte lysate assay is a convenient and valuable diagnostic tool for excluding Gram-positive peritonitis in peritoneal dialysis patients. This allows more specific antibiotic treatment at presentation and may avoid the development of bacterial resistance. A negative Limulus amoebocyte lysate test is not reliable for the exclusion of Gram-negative peritonitis. In the absence of a positive culture result 48 h after presentation, accompanied by a delayed response to treatment, a positive Limulus amoebocyte lysate assay may indicate the presence of fungus. This justifies early empiric antifungal treatment before definitive culture results are made available. Routine Limulus amoebocyte lysate assay of dialysate effluent from continuous ambulatory peritoneal dialysis patients presenting with peritonitis is recommended.     

Correlativity of plasma endotoxin, plasma fibronectin and common bile duct pressure in severe acute cholangitis]

On the basis of common bile duct pressure measurement (CBDP) in 18 patients of severe acute cholangitis, plasma endotoxin (ET) was determined by modified synthetic chromogenic limulus amebocyte lysate assay and plasma fibronectin (FN) was detected with Laurell's rocket immunoelectrophoresis. CBDP was 2.23 +/- 0.49 KPa in patient group. There was striking positive correlation between ET and CBDP. Preoperative ET was 202.73 +/- 88.57 ng/L in patient group which was much higher than 15.47 +/- 7.38 ng/L in the control (P less than 0.001). Preoperative FN was 141.77 +/- 82.37 mg/L in the patient group which was lower than 317.21 +/- 12.57 mg/L in the control (p less than 0.001). Statistical differences could be noticed in postoperative ET and FN between the survivor and the dead. The study suggested that plasma ET levels are greatly influenced by pressure gradient of bile duct, dynamic observations of ET and FN levels are helpful to monitor disease course and predict prognosis.     

Are plasma endotoxin levels related to burn size and prognosis?

Plasma endotoxin concentrations were determined in 42 patients with burns covering more than 20 per cent of the body surface area, using the endotoxin-specific Endospecy assay and treatment of plasma by a new method developed by ourselves. The normal endotoxin level was 9.8 pg/ml or less. In the early period after injury when no infection was present, very few patients had an endotoxin level above 9.8 pg/ml and endotoxin levels did not correlate with the area of the burns or with prognosis. However, later in the clinical course, endotoxin levels were correlated significantly with the burned area and with the prognosis.     

Endotoxin-neutralizing capacity of serum from cardiac surgical patients

OBJECTIVE: To determine if endotoxin core antibody (EndoCAb) from the serum of cardiac surgical patients neutralizes endotoxin in an ex vivo biologic assay. DESIGN: Prospective blinded cohort study. SETTING: Academic medical center. PARTICIPANTS: Patients (n = 203) undergoing cardiac surgery. INTERVENTIONS: Sera were obtained from patients preoperatively. MEASUREMENTS AND MAIN RESULTS: EndoCAb levels were determined by enzyme-linked immunosorbent assay. Sera were incubated for 15 minutes at 37 degrees C with varying concentrations of endotoxin from a clinically relevant bacterium (Escherichia coli serotype O18), then tested for the presence of endotoxin activity using the validated Limulus amebocyte lysate assay. Median (interquartile range) IgM and IgG EndoCAb levels were 118 median units (range, 31 to 259 median units) and 208 median units (range, 108 to 401 medium units). Increasing levels of IgM EndoCAb were associated with increased neutralization of endotoxin (p < 0.0001). Increasing levels of IgG EndoCAb were associated with increased neutralization of endotoxin (p < 0.0001). An additive effect of IgM and IgG EndoCAb levels on endotoxin neutralization was observed without evidence of synergistic or plateau effects. EndoCAb levels did not completely predict serum neutralization capacity. CONCLUSION: Anti-EndoCAbs of both classes (IgM and IgG) were able to neutralize lipopolysaccharide from a clinically relevant bacterium in an ex vivo model. Neither Igm nor IgG appeared to be more capable of neutralization in this model. These antibodies did not completely predict neutralization capacity; other endogenous factors in human serum must be capable of lipopolysaccharide neutralization.     

Comparison of endotoxin antagonism of linear and cyclized peptides derived from limulus anti-lipopolysaccharide factor

BACKGROUND: Limulus anti-lipopolysaccharide (LPS) factor (LALF) is a 102-amino acid LPS-binding protein from the horseshoe crab, Limulus polyphemus. The peptide includes the LPS-binding domain of holoLALF, yet it lacks the loop structure stabilized by disulfide or other covalent bonds that is a common motif in the LPS-binding regions of holo- LALF and several other LPS-binding proteins. Although it neutralizes LPS and is bactericidal against Pseudomonas aeruginosa, the LALF 28-54 portion of LALF is not protective in a murine model of intraperitoneal sepsis compared with holoLALF. We examined the effects of cyclizing this linear peptide to determine if this action would recapitulate the stable loop-type structure and enhance its LPS-neutralization and bactericidal activity in vitro. METHODS: Cyclic LALF 28-54 was produced by oxidizing linear LALF 28-54. Each peptide, along with appropriate controls, was assayed for LPS neutralization using the chromogenic Limulus amebocyte lysate (LAL) assay and a bioassay to measure inhibition of LPS-stimulated production of tumor necrosis factor-alpha (TNF-alpha) by murine macrophages. Bactericidal activity against Pseudomonas aeruginosa was also assessed. Data were analyzed using a two-tailed Student t-test. RESULTS: Polymyxin B and holoLALF exhibited potent endotoxin antagonism in both assays, as well as bactericidal activity. Concordant with prior studies, the linear form of LALF 28-54 exhibited either similar or a slightly lesser degree of activity in all assays. However, cyclization was associated with significantly diminished endotoxin neutralization in both assays (p < 0.05) and decreased bactericidal activity (p < 0.05) compared with linear LALF 28-54. CONCLUSIONS: Whereas a synthetic linear peptide based on the endotoxin-binding region of holoLALF retained activity, cyclization was associated with a diminution in potency in vitro. We postulate that cyclization does not constrain the peptide in a manner that recreates the loop structure necessary for potent endotoxin antagonism.