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PubMed1991

Quality control of hemoglobin solutions. I. The purity of hemoglobin before modification.

It is not yet clear whether the toxicity of hemoglobin solutions is due to hemoglobin per se, contaminants, or both. To elucidate the problem, it is first necessary to prepare "pure hemoglobin," i.e., hemoglobin completely free of (a) bacterial endotoxins, (b) phospholipids of the red cell membrane, and (c) other proteins and/or peptides. The methods found most sensitive in the determination of such purity are here presented. Consensus on the adoption of these methods and on the meaning of the term "purity" would help to achieve (a) a better understanding of the toxicity problem, (b) a more meaningful exchange of information among investigators and (c) the ultimate development of nontoxic solutions.

Chromatography, High Pressure Liquid↗
PubMed1991

[Treatment of gut-derived endotoxemia with lactulose. An experimental study].

Endotoxemia plays an important role in the origination and development of many dangerous clinical diseases, for which there has been little medication so far. To determine whether lactulose has an effect on endotoxemia, we treated experimental, gut-derived endotoxemia using lactulose in vitro and in vivo. The results showed that 55 mg lactulose inactivated the activity of 0.01 mg endotoxin on limulus lysate in vitro, suggesting that lactulose may have a direct anti-endotoxin effect. In vivo study in rats showed that blood endotoxin level was significantly decreased from 78.61 +/- 6.54 pg/ml to 20.26 +/- 2.38 pg/ml (P less than 0.01), and liver damage significantly reduced after lactulose treatment. It is suggested that lactulose can prevent absorbtion of endotoxin from gut and may have an effect on gut-derived endotoxemia. The mechanism of lactulose for treating endotoxemia is discussed.

Animals↗
PubMed1991

[Effects of different functional groups of phenylurea compounds on indices of their toxicity and MAC in the air of work areas].

Free-Wilson technique with the use of computer correlation-regression and dispersion analysis program made it possible to determine the importance of certain radicals and functional groups for the characteristics of the toxicity of phenylurea compounds. Specific contribution of different functional groups depending on their position in the molecule to the values of toxicometric indices (LD50, Limac, Limch) and MAC of those compounds in the air of the working area were determined. Summary contributions of radicals with their functional groups to the values of toxicity indices and MACs for individual substances of the phenylurea group was calculated. A possibility was shown to use the Free-Wilson technique for the rapid prediction of toxicity and hygienic standards for new phenylurea compounds in the air of the working area.

Air Pollutants, Occupational↗
PubMed1990

[Chemical composition and biological activities of lipopolysaccharides extracted from Treponema denticola and Treponema vincentii].

Lipopolysaccharides (LPSs) were isolated from Treponema denticola (T. denticola) and Treponema vincentii (T. vincentii) by the phenol/water (PW) and the phenol/chloroform/petroleum-ether (PCP) procedures. 1) T. denticola PW-LPS (LPS isolated by the PW procedure), PCP-sup-LPS (LPS isolated by the PCP procedure in the supernates of ultracentrifugation), PCP-ppt-LPS (precipitated LPS isolated by the PCP procedure, obtained after ultracentrifugation), and T. vincentii PCP-ppt-LPS were composed of carbohydrate, hexosamine, protein, fatty acid, and phosphorus. T. vincentii PW-LPS was contained major amount of carbohydrates and small amount of fatty acids. 2-keto-3-deoxyoctonic acid (KDD) was not detected in these LPSs. 2) The major fatty acids of T. denticola PW-LPS and PCP-sup-LPS were palmitic, stearic, oleic, and linoleic acids. The major fatty acids of T. vincentii PCP-ppt-LPS were palmitic, stearic, myristic, oleic, and linoleic acids. Hydroxy fatty acids were not detected. 3) Glucose, galactose, and mannose were comprised in T. denticola PW-LPS. Glucose, galactose, and arabinose were comprised in T. denticola PCP-sup-LPS. Glucose and galactose were comprised in T. vincentii PCP-ppt-LPS. 4) The Limulus amoebocyte lysate (LAL) clotting activity of T. denticola PW-LPS was 1/10, as compared with that of Escherichia coli (E. coli) UKT-B LPS standard. The LAL clotting activities of T. denticola PCP-sup-LPS, T. vincentii PW-LPS, and T. vincentii PCP-ppt-LPS were 1/100, as compared with that of E. coli LPS standard. 5) Five hundred micrograms/kg of T. denticola PCP-sup-LPS was pyrogenic in rabbits. Two thousand micrograms/kg of T. vincentii PCP-ppt-LPS was pyrogenic in rabbits. 6) T. denticola PCP-sup-LPS and T. vincentii PCP-ppt-LPS were capable of increasing or decreasing the release of lysosomal enzymes from human polymorphonuclear leukocytes.

Endotoxins↗
PubMed1990

Bactericidal/permeability-increasing protein has endotoxin-neutralizing activity.

Neutrophil granules contain proteins important in host defense against bacterial pathogens. Granule proteins released from activated neutrophils facilitate opsonization, phagocytosis, tissue digestion, and antimicrobial activity. Three similar, if not identical, neutrophil proteins, bactericidal/permeability-increasing protein (BPI), 57,000 m.w. cationic antimicrobial protein, and bactericidal protein have been described that specifically kill gram negative bacteria. Since LPS is a structure common to all gram-negative bacteria, we investigated whether the microbicidal protein BPI affects biologic activity of LPS in vitro. Human neutrophils can be activated both in vitro and in vivo by LPS. Upon stimulation, surface expression of CR1 and CR3 increases markedly. Using flow microfluorimetry, we analyzed surface expression of CR1 and CR3 as a measure of neutrophil stimulation in response to LPS. CR up-regulation on neutrophils was TNF independent, suggesting direct LPS stimulation of neutrophils in this system. Purified BPI completely inhibited CR up-regulation on neutrophils stimulated with both rough and smooth LPS chemotypes at 1.8 to 3.6 nM (100 to 200 ng/ml). By comparison, the polypeptide antibiotic polymyxin B completely inhibited the same dose of LPS at 0.4 nM. The inhibitory activity of BPI appeared to be specific for LPS because neutrophil stimulation by formylated peptide or TNF was unaffected. The specificity of BPI for LPS was further demonstrated by inhibition of LPS activity in the limulus amebocyte lysate assay. Therefore, the role of BPI in infection may not be limited to its microbicidal activity, but it may also regulate the neutrophil response to LPS.

Antimicrobial Cationic Peptides↗
PubMed1978

The contribution of intestinal endotoxin to mortality in hosts with compromised resistance: a review.

Sepsis, particularly with endotoxin-containing Gram-negative bacilli, is a serious complication in hosts whose defenses are compromised. This review examines work from our laboratory and others concerning infectious processes which may be critical to the survival of compromised individuals. Several avenues for control of sepsis are proposed. Gram-negative bacteria and their endotoxins can escape from the intestines of compromised animals to contaminate normally sterile host tissues. Endotoxins are especially toxic to compromised hosts because essential components of their inflammatory responses are missing (i.e., leukocytes and platelets in irradiated animals). Therefore, regulation of host responses to endotoxin is no longer possible. It is recommended that sepsis be controlled in compromised individuals through elimination of endogenous microbial agents. Should infection occur in these individuals, they should be transfused with blood cells necessary for clearance of bacteria and endotoxin and restoration of homeostasis.

Animals↗
PubMed1986

Properties of free and bound Citrobacter freundii lipopolysaccharides.

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.

Animals↗
PubMed1989

Carcinoscorpius rotunda cauda amoebocyte lysate for detection of endotoxins--its preparation, stability, sensitivity and comparison with Limulus amoebocyte lysate.

Carcinoscorpius amoebocyte lysate (CAL) was prepared from C. rotunda cauda by a modification of the method described by Mahalanabis et al. [Indian J Med Res, 70 (1979) 35]. Seasonal variation as well as batch variation was observed in the yield of haemolymph and the total lysate protein. In the presence of E. coli lipopolysaccharide (pure, free endotoxin) and E. coli and Salmonella cell suspensions (bound endotoxin), the CAL formed a gel after incubation at 37 degrees C. The gelling time varied from 10-90 min depending on the concentration of endotoxin used; higher concentrations formed gel more rapidly. The endotoxin detection capacity (sensitivity) of the lysate preparations was influenced by the season in which prepared, but not by the total protein content. Ten fold increase in the sensitivity was achieved by a purification step using chloroform. Although subsequent frozen storage with or without lyophilization did not alter the initial sensitivity, it was either decreased considerably or lost totally when the lysate was stored for 4 months at 4 degrees C or for 2 months at 30 degrees C. Under the same conditions, Limulus lysate was more stable. The lost sensitivity could not be regained by the incorporation of divalent cations (Ca2+ and Mg2+). The CAL preparations in general were able to detect as little as 10-100 pg of endotoxin or as few as 10(3) cells of E. coli or 10(4) cells of Salmonella and were comparable to LAL. CAL could be used successfully in lieu of Limulus amoebocyte lysate in the detection and assay of endotoxins.

Animals↗
PubMedDate not supplied

Limulus amebocyte lysate reactivity, complement activation, and patients' symptoms. Comparison of dialyzer membranes.

This study was undertaken to clarify the role of Limulus amebocyte lysate reactive material (LAL-RM) in the activation of complement in patients during hemodialysis with cuprophan membranes. The authors measured LAL-RM and complement C3a levels predialysis and at 15 and 60 minutes of dialysis in 16 patients dialyzed for 8 weeks on cuprophan membranes and then for a succeeding 8 weeks on cellulose acetate membranes. Patients' symptoms on dialysis with each membrane were assessed. The LAL-RM levels of these patients were compared to those of six patients dialyzed on cellulose acetate membranes only. The LAL-RM and C3a levels rose significantly in the 16 patients while on dialysis with cuprophan membranes, but there was no correlation between the LAL-RM and C3a levels. C3a levels rose significantly in the 16 patients while on dialysis with cellulose acetate membranes, but there was no rise in LAL-RM levels. C3a levels were significantly lower while the patients were on dialysis with the cellulose acetate membranes. The six patients on dialysis with the cellulose acetate membranes only had no detectable LAL-RM in their blood. Patients' symptoms were less while on dialysis with the cellulose acetate membrane, but the difference did not achieve statistical significance. We conclude that LAL-RM plays a very minor role, if any, in the complement activation caused by cuprophan membranes and that cellulose acetate membranes do not release LAL-RM.

Adult↗
PubMedDate not supplied

Plasma levels of endotoxin and anaphylatoxin in patients with multiple organ failure.

Plasma concentrations of endotoxin and anaphylatoxins were studied inpatients with multiple organ failure (MOF). Thirty one blood samples from nine patients with MOF and twenty samples from preoperative patients as control were determined. The average plasma concentration of endotoxin was 188.7 +/- 37.0 pg-ml-1 (Mean + S.E.) in MOF patients and 12.4 +/- 3.3 pg.ml-1 in control patients (p less than 0.05). Plasma anaphylatoxin (C3a, C5a) levels increased significantly and C4 level decreased in MOF patients as compared with those in control patients. However, no significant correlation was found between plasma endotoxin and anaphylatoxin levels. Endotoxin does not seem to be a major factor in activating of the complement system in patients with MOF.

Adult↗
PubMed1989

The permeability of dialytic membranes to endotoxins: clinical and experimental findings.

This study of 20 endotoxemic patients submitted to 70 hemodialyses (HD) found a reduction of the pre-HD limulus amebocyte lysate (LAL) positivity in 50 HD (71%), without appreciable differences in terms of effectiveness between cuprophan and AN 69 membranes. To define the mechanisms responsible for the reduction in LAL positivity during HD, the membranes were used in two in vitro studies, the first of which showed that the LAL positivity of blood containing lipopolysaccharide (LPS), submitted to hemofiltration (HF) for 300 min, remained unchanged and the ultrafiltrate remained constantly LAL negative. These results suggest that the reduction in LAL positivity observed in HD in vivo, an expression of reduced endotoxemia, cannot be attributed either to the filtration of the LPS as such or to its fragmentation following blood-membrane interaction into theoretically less filtrable molecules or to mechanisms of LPS adsorption on the membrane. The in vivo reduction of LAL positivity is more likely due to removal of the filtrable endotoxin fragments already released in the body, like lipid A, the biologically active component of LPS, known to react to LAL. This hypothesis was borne out by the second in vitro study, where the LAL positivity of blood containing lipid A, treated by HF for 80 min, gradually decreased, and dialytic permeability to lipid A was confirmed by the appearance of LAL positivity in the ultrafiltrate.

Clinical Trials as Topic↗
PubMed1989

Heparin inhibition of endotoxin-dependent Limulus amebocyte lysate coagulation.

Addition of commercial heparin to preparations of the coagulation system obtained by hypotonic lysis of Limulus amebocytes, prior to the addition of endotoxin from gram-negative bacteria, inhibited gelation. Activation of the cell-lysate proclotting enzyme, utilizing a diluted lysate preparation and synthetic substrates, was inhibited to a comparable extent with either anticoagulantly active or inactive heparin. Other glycosaminoglycans, including heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate, did not inhibit the generation of clotting enzyme activity. Experiments conducted with preparations of proactivator(s) and proclotting enzyme, obtained by fractionation of lysate with heparin-Sepharose, revealed that inhibition of endotoxin-dependent activation of amebocyte lysate by heparin is mediated by precipitation of the proactivator(s) and can be prevented by increasing the salt concentration.

Animals↗
PubMedDate not supplied

Expert system computer program for troubleshooting the gel-clot Limulus amebocyte lysate (LAL) assay.

This article describes a knowledge-based or expert system computer program that can assist in troubleshooting the gel-clot Limulus Amebocyte Lysate (LAL) method for the assay of endotoxin in parenteral products and in-process materials. The program may also be used to train new personnel and/or to assist in the development of an LAL method for a new product or material. The program includes LAL assay information for 31 parenteral products and materials. The program uses rules-of-thumb and intuition to diagnose 155 possible LAL assay problems and logically reduce them to 37 recommended problem solving processes. The program also provides references to support its recommendation.

Endotoxins↗