Endotoxins

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Endotoxins (not to be confused with enterotoxins) are toxins associated with certain bacteria. Classically, an "endotoxin" is a toxin which, unlike an "exotoxin", is not secreted in soluble form by live bacteria, but is a structural component in the bacteria which is released mainly when bacteria are lysed.

Lipopolysaccharide and other endotoxins

The prototypical examples of endotoxin are lipopolysaccharide (LPS) or lipo-oligo-saccharide (LOS) found in the outer membrane of various Gram-negative bacteria and is an important cause of their ability to cause disease. The term LPS is often used exchangeably with endotoxin, owing to its historical discovery. In the 1800s it became understood that bacteria could secrete toxins into their environment, which became broadly known as "exotoxin". The term endotoxin came from the discovery that portions of Gram-negative bacteria itself can cause toxicity, hence the name endotoxin. Studies of endotoxin over the next 50 years revealed that the effects of "endotoxin" were, in fact, due to lipopolysaccharide.

The only gram positive bacteria that produces endotoxin is Listeria monocytogenes; however these toxins are more accurately described as "endotoxin-like" and should not be confused with that of those found in the cell wall of gram negative bacteria.

Mechanism

In humans, LPS binds to the lipid binding protein (LBP) in the serum which transfers it to CD14 on the cell membrane, which in turn transfers it to another non-anchored protein, MD2, which associates with Toll-like receptor-4 (TLR4).

CD14 and TLR4 are present in several immune system cells (including macrophages and dendritic cells), triggering the signaling cascade for macrophage/endothelial cells to secrete and pro-inflammatory cytokines and Nitric oxide that lead to "endotoxic shock".

Other than TLR4, components of gram negative cell wall may also activate other pathways which may contribute to the overall endotoxic effect.

Assay for detecting presence of endotoxin

A very sensitive assay for detecting presence of endotoxin is the Limulus Amebocyte Lysate assay, utilizing blood from the Horseshoe crab (The horseshoe crab or Atlantic horseshoe crab (Limulus polyphemus) is a marine chelicerate arthropod. Despite its name, it is more closely related to spiders, ticks, and scorpions than to crabs. Horseshoe crabs are most commonly found in the Gulf of Mexico and along the northern Atlantic coast of North America. A main area of annual migration is Delaware Bay, although stray individuals are occasionally found in Europe).

Very low levels of LPS can cause coagulation of the limulus lysate due to a powerful amplification through an enzymatic cascade.

Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amoebocytes) from the horseshoe crab, Limulus polyphemus. LAL reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. This reaction is the basis of the LAL test, which is used for the detection and quantification of bacterial endotoxins.

There are three basic LAL test methodologies:
-gel-clot
-turbidimetric
-chromogenic
The primary application for LAL is the testing of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid. In the United States, the FDA has published a guideline for validation of the LAL test as an endotoxin test for such products .

The LAL cascade is also triggered by (1,3)-β-D-glucan. Both bacterial endotoxins and (1,3)-β-D-glucan are considered "Pathogen-Associated Molecular Patterns", or PAMPS, substances which elicit inflammatory responses in mammals.