Removing and monitoring bacterial endotoxins in biological samples

The removal of bacterial endotoxins from biological samples is a routine practice in many research laboratories. Gram-negative bacteria, where bacterial endotoxins come from, are characterised by their ability to grow in almost all conditions and types of samples where tests are performed in laboratories. Since the pyrogenicity of bacterial endotoxins and the consequences on the health of human and animal exposure to them was discovered, scientists have been studying the different methods of separating endotoxins when they are found to be contaminating any of the samples in laboratories.

The most common method for removing endotoxins from solutions of parenteral drugs is filtration. Bacterial endotoxins are lipopolysaccharides found in different states of aggregation as contaminants: in the form of vesicles, micelles or detergents that are soluble. All these forms in which the lipopolysaccharide molecules may be associated are removed using filters with different pore sizes. In the case that the solute and the aggregates of endotoxins have a similar size, supramolecular interactions between lipopolysaccharide molecules can be overcome using methods such as the extraction of cations. It has been shown that cations stabilize the bilayers formed by endotoxin molecules, where the hydrophilic part is exposed to the environment and the hydrophobic chains on the inside of the miscella or vesicle. These aggregates are stabilised by divalent cations, such as calcium or magnesium cations present in aqueous solutions. When removing cations from the solutions, we can remove endotoxins using filters with a pore size allowing the solute to pass and preventing the passage of contaminating endotoxins.

A useful method for removing endotoxins from biological samples is affinity chromatography, which is commonly used for protein samples. This technique consists of making the solutions pass through porous adsorbent material with immobilised histidine. Polymyxin B can also be immobilised. Endotoxins have a great affinity for histidine in a wide range of pH and temperatures. Cases have been reported where, using this chromatographic method, the concentration of endotoxins derived from various types of Gram-negative bacteria has been reduced from 1000 to less than 0.01 ng/mL of aqueous solution. This type of separation can be carried out, for example, in samples containing factor of tumour necrosis and lysozymes with good results. Other authors have compared chromatographic methods for removing endotoxins from samples containing proteins with separation by phase with the detergent Triton X-114. Through this procedure, a greater recovery of endotoxins in the phase containing the detergent is achieved compared to affinity chromatography. This technique is most useful in large scale processes, such as the purification of recombinant proteins.

A biological substance that has attracted the attention of researchers for replacing synthetic polymers in plastics with a molecule allowing to obtain biodegradable and/or biocompatible materials is the poly (3-hydroxybutyrate) or PHB. PHB is produced by Gram-negative bacteria, among which we can list the Ralstonia eutropha, Alcaligenes latus, and recombinant Escherichia coli. Following any of the procedures conducted to isolate the PHB synthesised by the bacteria, samples are found to be contaminated with endotoxins. To reduce the amount of endotoxins in extractions, the chloroform extraction method is replaced by digestion with sodium hydroxide. In this particular case, the separation of endotoxins is indispensable to use the PHB in any biomedical application, such as packaging design for controlled drug delivery, the manufacture of threads for sutures and the replacement of bone tissue.

In all cases where it is necessary to remove endotoxins from a specific medium, researchers perform the LAL test as a method of monitoring the separation. The LAL or Limulus Amebocyte Lysate test is based on the gelation that occurs in the hemolymph of the horseshoe crab in the presence of bacterial endotoxins. The gelation process can be measured qualitatively and quantitatively by turbidimetry as the gel causes turbidity to appear in the environment. The brand Wako, through the brand PYROSTAR™, offers a series of reagents and accessories for conducting the LAL test.

These kits can be purchased as single tests or as multi-tests, based on the needs of the researchers. For example, the simple Limulus PS is a unique test where, through the absorption of endotoxins in Pyrosep™, resin with an affinity and selectivity for these types of substances can be measured by the content of these substances in a sample. Unlike other kits for the analysis of LAL (Limulus Amebocyte Lysate), Limulus PS can dissolve samples that are non-soluble in water in organic solvents for performing the test. This feature is an advantage of the product given that in the past, in drug research, there was often a need to know the presence of endotoxins in non-aqueous solutions of the analyte since the molecules were insoluble in water. Fat-soluble samples are an example of the samples where the Limulus PS is useful.

Bibliography:

1) K J Sweadner, M Forte and L L Nelsen, Appl. Environ. Microbiol. October 1977 vol. 34 no. 4 382-385.

2) Matsumae, H., et al., Biotechnology and Applied Biochemistry, Volume 12, Issue 2, pages 129–140, (1990).

3) Shigui Liu, Rowel Tobias, Shannon McClure, Garth Styba, Qinwei Shi, George Jackowski, Clinical Biochemistry, 30, 6, 455-463, 1997.

4) Sang Yup Lee1, Jong-il Choi1, Kyuboem Han, Ji Yong Song, Appl. Environ. Microbiol., 65, 6, 2762-2764, (1999).

LAL REAGENTS AND RELATED ACCESSORIES:

Guardar