Research Article: Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study

Date Published: January 2, 2014

Publisher: Springer

Author(s): Simone Schaumberger, Andrea Ladinig, Nicole Reisinger, Mathias Ritzmann, Gerd Schatzmayr.


Endotoxins are part of the cell wall of Gram-negative bacteria. They are potent immune stimulators and can lead to death if present in high concentrations. Feed additives, which bind endotoxins in the gastrointestinal tract of animals, could help to prevent their negative impact. The objective of our study was to determine the potential of a bentonite (Bentonite 1), a sodium bentonite (Bentonite 2), a chemically treated smectite (Organoclay 1) and a modified attapulgite (Organoclay 2) to bind endotoxins in vitro. Polymyxin B served as positive control. The kinetic chromogenic Limulus Amebocyte lysate test was adapted to measure endotoxin activity. Firstly, a single sorption experiment (10 endotoxin units/mL (EU/mL)) was performed. Polymyxin B and organoclays showed 100% binding efficiency. Secondly, the adsorption efficiency of sorbents in aqueous solution with increasing endotoxin concentrations (2,450 – 51,700 EU/mL) was investigated. Organoclay 1 (0.1%) showed a good binding efficiency in aqueous solution (average 81%), whereas Bentonite 1 (0.1%) obtained a lower binding efficiency (21-54%). The following absorbent capacities were calculated in highest endotoxin concentration: 5.59 mg/g (Organoclay 1) > 3.97 mg/g (Polymyxin B) > 2.58mg/g (Organoclay 2) > 1.55 mg/g (Bentonite 1) > 1.23 mg/g (Bentonite 2). Thirdly, a sorption experiment in artificial intestinal fluid was conducted. Especially for organoclays, which are known to be unspecific adsorbents, the endotoxin binding capacity was significantly reduced. In contrast, Bentonite 1 showed comparable results in artificial intestinal fluid and aqueous solution. Based on the results of this in vitro study, the effect of promising clay minerals will be investigated in in vivo trials.

Partial Text

Endotoxins are toxins that are kept in the cell wall of Gram-negative bacteria. In pure chemical form endotoxins are so called Lipopolysaccharides (LPS). LPS consist of a lipid part (lipid A) and a polysaccharide part with an inner core and O-specific side chains. They are characterised as amphiphilic (hydrophilic and lipophilic) molecules (Hodgson
2006), which are heat resistant and very pH stable. Endotoxin activity is indicated as endotoxin units (EU). In general, around 10 EU are equivalent to 1 ng endotoxin.

The use of clay minerals as a feed additive for farm animals to prevent the effects of different mycotoxins (e.g. aflatoxin and ergotamine), bacteria and other toxic compounds is widespread and has been discussed for years (Slamova et al.2011; Tateo and Summa
2007; Trckova et al.2004). Therefore, the hypothesis is that binding endotoxins in the gut lumen will reduce the number of endotoxins entering the organism. As animal studies are expensive, and the effect of the additive has to be assured, the in vitro screening of sorbents is important for estimating their adsorptive potential. Although, results of in vitro studies may not be reflected in in vivo studies, conclusions regarding the mode of action under controlled conditions can be gained (Ganner and Schatzmayr
2012). Approaches for screening binding materials in vitro and collecting information on the underlying functional mechanisms, are of great interest.

The authors declare that they have no competing interests.




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