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Publication type: Doctoral Thesis

Year: 2014

Author(s): Schaumberger, Simone

Title: Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test.

Other title: Evaluierung der Endotoxin Bindungskapazität von Tonmineralien mit Hilfe des Limulus Amebocyten Lysat Tests: Eine in vitro Studie

Source: Dissertation, Vet. Med. Univ. Wien, pp. 38.


Advisor(s):

Ritzmann Mathias

Reviewer(s):
Zebeli Qendrim

Vetmed Research Units:
University Clinic for Swine


Graduation date: 13.03.14


Abstract:
Endotoxins (chem. Lipopolysaccharide, LPS) are located in the cell wall of Gram-negative bacteria. LPS is continuously released into the environment and is known to be a potent immune stimulator. If there is a failure in endotoxin removal in the organism, endotoxins can lead to systemic inflammation, endotoxemia, shock and death. A feed additive which binds endotoxins in the gastrointestinal tract could help to inhibit the negative impact in the animal. We used the kinetic chromogenic Limulus Amebocyte Lysate test to investigate the endotoxins binding efficiency of: A bentonite (smectite, Bentonite 1), a Sodium bentonite (Bentonite 2) and two surface treated clays, one smectite (Organoclay 1) and an attapulgite (Organoclay2). As positive control poylmyxin B (PMB) was used. PMB is a polypeptide antibiotic which binds LPS in a two way binding mechanism. In our experiments included were: a single sorption study (low dose of LPS 10 endotoxin units/mL (EU/mL)) at room temperature and 37 °C, an experiment on the adsorption efficiency in aqueous solutions with increasing LPS concentrations (2,450-51,705 EU/mL) and sorption studies in artificial intestinal fluid (AIF) with LPS concentrations up to 120,000 EU/mL. Both experiments were done at 37 °C and 2 hours of incubation. In the single sorption experiment PMB and Organoclay 1 could bind 100 % of the added LPS at both temperatures. Organoclay 2 showed varying results, suggesting an inhomogeneous material or an unspecific binding. Bentonite 1 and 2 showed a binding efficiency below 14 %. Differences in endotoxins removal efficiency (%) and in binding capacity (mg/g) of organoclays and bentonites were observed in aqueous solution and AIF. Organoclay 1 showed comparable binding efficiencies (81.6 %; P=0.385) in aqueous solutions to PMB (76.4 %). Organoclay 2, Bentonite 1 and 2, whereas, obtained a significantly decreased binding efficiency (P=0.013; P=0.026; P=0.022) compared to PMB in aqueous solution. Binding capacities showed a linear increase and highest binding capacities were reached in highest endotoxins concentrated working solutions (51,704 EU/mL): 5.59 mg/g (Organoclay 1) >3.97 mg/g (Polymyxin B) > 2.58 mg/g (Organoclay 2) > 1.55 mg/g (Bentonite 1) > 1.23 mg/g (Bentonite 2). Experiments in AIF were only performed with PMB, Organoclay 1 and Bentonite 1 to get an idea on the behavior of sorbents in a complex medium. Results in a comparable endotoxin concentration revealed a decreased binding capacity (0.65 mg/g) for Organoclay 1 compared to experiments in aqueous solution (3.03 mg/g). Bentonite 1, whereas, showed an increased binding capacity (0.81 mg/g vs. 0.72 mg/g) in AIF compared to aqueous solution. Media have an influence on binding behavior of LPS on clay minerals, which may be due to the formation of LPS in different solutions or because of the behavior of the clay minerals in complex solution.

Keywords:
Endotoxin / Lipopolysaccharide / Bentonite / Organoclay / Binding / LAL test


Publication(s) resulting from University thesis:

Schaumberger, S; Ladinig, A; Reisinger, N; Ritzmann, M; Schatzmayr, G (2014): Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study. AMB Express. 2014; 4(1):1
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