Production of biological drugs: in-process controls with the LAL-assay.
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Recent investigations of several authors on portal venous and systemic endotoxemia in healthy adults have shown that endotoxins absorbed from the intestinal mucosa are found in portal venous blood, cleared by the RES of the liver and usually cannot be determined in peripheral blood. In patients with liver disease, however, there was often a reduced endotoxin clearance with spillover of endotoxin resulting in systemic endotoxemia. Among the complications of systemic endotoxemia, hepatocytotoxicity, pyrogen reaction, disseminated intravascular coagulation, impaired renal function, and endotoxic shock are most hazardous. In addition, O-antibody titers and lipid-A-antibody titers were found to be higher in patients with liver disease and in patients with Crohn's disease than in control groups. The investigations indicate that intestinal endotoxins are of importance in the pathogenesis of liver disease and of Crohn's disease and that reduction of intestinal endotoxins by antibiotics may be of value in the therapy of these diseases.
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To determine if endotoxin liberation occurs after antibiotic administration to patients with syphilis, serial plasma samples were obtained from 15 patients receiving intramuscular penicillin G benzathine for secondary syphilis. The endotoxin content of these plasma samples was determined using a Limulus lysate assay (detection limit, 0.025 ng of reference Escherichia coli endotoxin per milliliter of patient plasma). Although secondary syphilis is the stage of syphilis having the greatest burden of spirochetes and the highest incidence of Jarisch-Herxheimer reactions, no endotoxin was detected in plasma either before or after antibiotic therapy. Despite the absence of detectable endotoxemia, five patients experienced mild Jarisch-Herxheimer-like reactions. These results suggest that endotoxin is not an important factor in either secondary syphilis or the reactions associated with antibiotic therapy of this disease.
A smooth-type lipopolysaccharide (HpS-LPS), a rough-type lipopolysaccharide (HpR-LPS), and a capsular-enriched polysaccharide preparation (HpC-PS) were extracted and purified from Haemophilus pleuropneumoniae serotype 5, strain J45, by the use of phenol-water (HpS-LPS) and phenol-chloroform-petroleum-ether (HpR-LPS) techniques. Chemical analysis of the HpS-LPS and HpR-LPS indicated that they contained 0.7% and 4.4% (w/w) 2-keto-3-deoxyoctonate, 11.8% and 10.4% phosphate, 0.8% and 0.8% nucleic acid, and 0.8% and 1.1% protein, respectively. The HpC-PS contained 0.3% 2-keto-3-deoxyoctonate, 1.4% phosphate, 0.2% nucleic acid, and 0.8% protein. With sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the HpS-LPS banded as a smooth-type LPS and the HpR-LPS banded as a rough-type LPS. Electrophoresis of HpC-PS indicated the presence of a broad high molecular weight band. Gelation of Limulus amoebocyte lysate developed at a minimum concentration of 8 ng/ml of HpS-LPS, 0.3 ng/ml of HpR-LPS, and 35 ng/ml of HpC-PS. The lipopolysaccharide preparations provoked a positive dermal Shwartzman reaction in rabbits and swine, a biphasic febrile response in rabbits, and a monophasic response in swine. Responses were typical of endotoxic activity with swine having greater sensitivity than rabbits. The chick embryo 50% lethal dose was calculated to be 7.3 ng for HpS-LPS, 1.6 ng for HpR-LPS, 5.1 ng for the lipopolysaccharide of Escherichia coli 0111:B4; and the HpC-PS was not toxic. In all assays, HpR-LPS was significantly more toxic than was HpS-LPS. The HpC-PS preparation was not toxic, even at high concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
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Endotoxemia without sepsis was detected with a chromogenic Limulus assay in 36 of 39 (92.3%) cirrhotic patients and was absent in seven healthy volunteers. In 11 patients who underwent elective portasystemic shunt, portal vein endotoxemia was higher than inferior vena caval: p less than 0.05, systemic endotoxin levels did not change, compared to preoperative levels, on the 1st, 2nd, and 3rd postoperative days, attendant to an uneventful recovery. In 21 patients in hepatic encephalopathy after esophagogastric hemorrhage, systemic endotoxemia was higher than in well-compensated cirrhotics: p less than 0.001; it was higher in deep than in light coma: p less than 0.05; it was higher in those who died than in those who survived: p less than 0.001. Endotoxin levels showed a positive correlation with serum bilirubin: r = 0.59, p less than 0.001, and a negative correlation with prothrombin activity: r = -0.59, p less than 0.001. These data show endotoxemia without sepsis is a constant finding in cirrhosis and increasing levels of endotoxemia are associated with hepatic failure, encephalopathy, and death.