Endotoxin

Endotoxins are potentially toxic, natural compounds found inside pathogens such as 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. 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" was in fact due to lipopolysaccharide.

There are, however, endotoxins other than LPS: (hA study was carried out using GC-MS in addition to LAL test on a number of gram-neg & gram-pos (including Listeria) by Maitra et al (1986) Appl & Env Microbiology 52:510 (http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=203564) The GC-MS results found a good correlation with LAL test in that all the gram-neg bacteria screened. Moreover, Listeria did not show any positive results with either GC-MS or LAL.
 * For example, delta endotoxin of Bacillus thuringiensis makes crystal-like inclusion bodies next to the endospore inside the bacteria. It is toxic to larvae of insects feeding on plants, but is harmless to humans (as we do not possess the enzymes and receptors necessary for its processing followed by toxicity).
 * The only gram positive bacteria that produces endotoxin is Listeria monocytogenes, however to be precise, they are best describled as "endotoxin-like" AND SHOULD NOT BE CONFUSED WITH THAT OF THOSE FOUND IN THE CELL WALL OF GRAM NEGATIVE BACTERIA, coming from the paper by Wexler & Oppenheim (1979) Infect. Immunol. 23:845. Another study was carried out using GC-MS in addition to LAL test on a number of gram-neg & gram-pos (including Listeria) by Maitra et al (1986) Appl & Env Microbiology 52:510

LPS consist of a polysaccharide (sugar) chain and a lipid moiety, known as lipid A, which is responsible for the toxic effects. The polysaccharide chain is highly variable amongst different bacteria. Endotoxins are approximately 10 kDa in size but can form large aggregates up to 1000 kDa. Humans are able to produce antibodies to endotoxins after exposure but these are generally directed at the polysaccharide chain and do not protect against a wide variety of endotoxins. Injection of a small amount of endotoxin in human volunteers produced fever, a lowering of the blood pressure, and activation of inflammation and coagulation. Endotoxins are in large part responsible for the dramatic clinical manifestations of infections with pathogenic Gram-negative bacteria, such as Neisseria meningitidis, the pathogen that causes fulminant meningitis.

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 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.

Endotoxin contamination
Endotoxins are frequent contaminants in plasmid DNA prepared from bacteria, and must be removed from the DNA to avoid unwanted inflammatory responses prior to in vivo applications such as gene therapy.

In pharmaceutical production, it is necessary to remove all traces of endotoxin from drug product containers as even small amounts of endotoxin will cause illness, but not disease, in humans. A depyrogenation oven is used for this purpose. Temperatures in excess of 300 degrees celsius are required to break down this substance. A defined endotoxin reduction rate is a correlation between time and temperature. Based on primary packaging material as syringes or vials a glass temperature of 250°C and a holding time of 30min is typical to achieve 3log reduction on endotoxin levels.

A very sensitive assay for detecting presence of endotoxin is the Limulus Amebocyte Lysate assay, utilizing blood from the Horseshoe crab. Very low levels of LPS can cause coagulation of the limulus lysate due to a powerful amplification through an enzymatic cascade.