[Endotoxin contents of phytopharmaceuticals: correlation with clinically observed side effects]. / Untersuchungen zum Endotoxingehalt von Phytopharmaka. Korrelation zu klinisch beobachteten Nebenwirkungen.

Four phytopharmaceutics (Carnivora, Pascotox forte-Injectopas, Esberitox N, Iascador M), which sometimes cause side effects after parenteral administration (fever, rigor, nausea), were examined for their endotoxin content by the kinetic turbidometric Limulus-amebocyte-lysate (LAL) microtitre test. Contaminations of over 10(5) EU/ml (endotoxin units; 1 EU = 0.1 ng of the FDA standard EC-5) were found in correlation with the clinical picture. In one preparation (Carnivora) very high endotoxin levels were always found; contaminations were only occasionally found in the others. These endotoxin measurements are supported by tests of endotoxin-dependent parameters in in-vivo experiments (reduction in leukocytes, acute death in hyperreactive mice). These findings underline the urgent need for a widening of the regulations on testing for pyrogens to include those parenteral preparations which now do not have to be tested because of their small volume (less than 15 ml).

Protective effects and role of endotoxin in experimental septicemia.

An experimental model was used in mice in which septicemia develops following invasion of the animals' own intestinal flora after cecal ligation and puncture. Pretreatment with 1 microgram of endotoxin administered 24 hours before surgery significantly reduced the rate of lethality. Bacteria were counted and differentiated in cardiac blood at various times throughout a 48-hour period after induction of septicemia in mice, with and without pretreatment. Endotoxin levels and plasma-related interference of the Limulus-amebocyte-lysate (LAL)-endotoxin reaction also were determined as were hematologic and metabolic parameters. A shift from mixed gram-positive and gram-negative to predominantly gram-negative bacteria occurred in both groups. In pretreated mice, a reduction in aerobic bacterial counts and reduced hyperglycemia were seen in the initial phase; and a decrease in anaerobic and aerobic bacteria and in endotoxin levels were observed at the end of the experiment. This appears to be related to endotoxin-induced increased resistance against the consequences of intraabdominal sepsis. These studies also indicate that the measured amount of circulating endotoxin does not necessarily correlate to the severeness of infection. Individual differences in plasma-related interference with the LAL-endotoxin reaction also emphasize the requirement for sample-internal standardization in order to reliably quantify endotoxin in plasma.

Ability of various adsorbents to bind endotoxins in vitro and to prevent orally induced endotoxemia in mice.

The efficacy of various adsorbents for endotoxin was tested in vitro and in vivo using a murine experimental model of gut-derived endotoxemia. A quantitative limulus amebocyte lysate microtiter test and the limulus amebocyte lysate tube test were used to determine intestinal and circulating levels of endotoxin. Kaopectate, kaolin/pectin mixture, kaolin, pectin, bentonite, charcoal particles, and lactulose were tested for their ability to bind endotoxins both in vitro and in vivo. The most effective material in the prevention of endotoxemia provided to be bentonite followed by Kaopectate and charcoal particles. Kaolin least effectively bound endotoxin at similar concentrations, while lactulose and pectin had minimal effects. Good correlation was shown between the ability of these drugs to bind endotoxin in vitro as compared with in vivo action.

[Quantitative endotoxin determination. Automated kinetic Limulus amebocyte lysate microtiter test with measurement of sample-related interferences]. / Quantitativer Endotoxin-Nachweis. Automatisierter, kinetischer Limulus-Amöbozyten-Lysat-Mikrotiter-Test mit Messung probenabhängiger Interferenzen.

A turbidometric, automated limulus amoebocyte lysate (LAL) microtiter test has been developed based on the evaluation of the LAL-endotoxin reaction kinetics. The maximal increase in optical density of each reaction mixture within 1 min is recorded. With this method an endotoxin standard curve is achieved which is linear over a concentration range of six decades. With presently available LAL methods sample-related inhibition or enhancement of the LAL endotoxin reaction may be overlooked and lead to false results. The quality of interfering factors can be characterized with our methods by spiking serial dilutions of the sample with constant endotoxin concentrations. The additional introduction of an internal standardization in our system allows the determination of endotoxin with simultaneous detection of quality and quantity of sample-induced interference. This procedure is based on a mathematic model which describes interference-caused alterations of the reaction revealed by addition of endotoxin in increasing concentrations. In comparison to the LAL tube test and the turbidometric determination at a given time the advantages of the developed method are demonstrated using three different samples (gelatin solution, adenine-HCl solution and a concentrate of coagulation factors (PPSB)). These are paradigmaticly selected because and enhancement of the LAL endotoxin reaction.