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Papers Please – Biological Activity of Masked Endotoxin

As written previously (A Paper a Day Keeps the Doctorate Away: Saturated Fatty Acids Do Not Directly Stimulate Toll-Like Receptor Signaling), research suggesting that saturated fatty acids are inflammatory via activation on toll-like receptor 4 (TLR4) has been questioned. This 2017 Nature Scientific Report expands greatly on the subject (1).

The primary question: can endotoxin, in commercial and research preparations, be masked by reagents used in buffer from detection by the standard limulus amebocyte (LAL) assay. This masking is called low endotoxin recovery (LER). Depending on the extent of LER in research preparations, Erridge and Samani’s argument that so-called saturated fatty acid activation of TLR4 is really caused by latent endotoxin contamination could be strongly supported.

Schwarz et al. begin by explaining how the LAL assay works and how it may be vulnerable to false negatives by the masking phenomenon (Figure 1). A protein named Factor C binds aggregate lipopolysaccharide (LPS), initiating an enzymatic cascade ending in the cleavage of a colorimetric substrate. This enzyme system is extracted from red blood cells of the Atlantic horseshoe crab Limulus polyphemus. Importantly, monomeric LPS does not work to activate Factor C.

Monomeric LPS, however, is what is produced when contaminated preparations are exposed to chelating buffers (such as sodium citrate or phosphate) and detergents (such as polysorbate 20 aka Tween-20 or octoxinol 9 aka Triton X-100). The chelator removes stabilizing cations from endotoxin micelles, and detergents bind and isolate LPS monomers.

The resulting LPS monomer/detergent complexes cannot activate Factor C based assays. There is disagreement in the field on whether naturally occurring endotoxin is susceptible to micelle weakening and intercalation, or if this only occurs in purified LPS. A series of experiments was performed in pursuance of this question.

In Figure 2, two Factor C based assays detected purified LPS but not endotoxin in a protein preparation from E. coli or LPS-null Clear Coli cells.

A monocyte-based assay of cytokine production, however, was able to detect endotoxin in the E. coli-expressed proteins (Figure 3). Both the Clear Coli preparation, and the E. coli preparation treated with TLR4 antibodies, produced negative results, making a false positive unlikely.

Further results are reported to round out the paper, but the primary question has already been answered. Naturally occurring endotoxin, at concentrations sufficient to cause inflammatory cytokine production in monocytes, can indeed be masked from LAL detection by common buffer reagents. This endotoxin-contaminated material may go on to cause pyrogenic symptoms (when used in medical preparations) or confound experimental results (when used in research preparations). A follow-up experiment on how fatty acids, saturated and unsaturated, affect the detection ability of Factor C and monocyte-based endotoxin assays would be interesting. The results would serve as further evidence on whether research showing TLR4 activation from saturated fatty acids is describing the direct effects of these fatty acids on these receptors or their effects on the delivery mechanism to these receptors of masked endotoxin.


  1. Schwarz H, Gornicec J, Neuper T, Parigiani MA, Wallner M, Duschl A, et al. Biological Activity of Masked Endotoxin. Sci Rep. Nature Publishing Group; 2017;7(44750):1–11.

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