Endotoxin is an important component of the cell wall of Gram-negative bacteria (such as E. coli), the main chemical component is lipopolysaccharide (LPS). LPS is toxic to the host and will only be released when the bacteria die. LPS consists of the O antigen, the core oligosaccharide, and the Lipid A molecule (Figure 1). The main toxic component is lipid A. Different Gram-negative bacteria have different LPS chemical compositions, but all contain lipid A.
When the bacterial cell wall dissolves, the lipid A molecule is released and acts with the immune system, causing inflammation and fever. So the endotoxin is also called "pyrogen". Endotoxin is an exogenous pyrogen that activates neutrophils to release an endogenous pyrogen, which acts on the thermoregulatory center to cause fever.
Fig.1 Diagram of a Gram-negative cell membrane
The specific mechanism by which endotoxin works is as follows. When LPS is released upon bacterial cell lysis, the lipid A component is first bound by serum LPS-Binding Protein (LBP) and then transferred to CD14 (either free CD14 in the serum or bound to the cell surface of macrophages or monocytes). This monomerises the aggregated LPS, as the LPS receptor Toll-like Receptor 4 (TLR4) cannot recognise LPS while aggregated. Monomeric LPS is then transferred to MD-2 pre-complexed with TLR4 on macrophages and monocytes. This leads to release of pro-inflammatory cytokines and nitric oxide, which may lead ultimately to septic shock depending on the strength of response.
Vascular endothelial cells also express TLR4 and MD-2 and so respond to LPS directly, as well as via cytokines and nitric oxide. Bronchial epithelial cells and colonic epithelial cells also express TLR4, but as they do not express MD-2 they rely on LPS precomplexed with serum MD-2 in order to signal to LPS.
Fever is part of the reaction of the innate immune system to invading microbes. Microbial products or so-called pathogen-associated molecular patterns are recognized by Toll-like receptors (TLRs) of phagocytic cells (monocytes/macrophages, neutrophils) and endothelial cells lining the inner surface of blood vessels and capillaries1. TLR-activation leads to secretion of cytokines, predominantly IL-1, IL-6 and TNF (by macrophages) or IL-1 and IL-8 (by neutrophils). The cytokines IL-1, IL-6 and TNF act as endogenous pyrogens on the thermoregulatory center of the hypothalamus to induce prostaglandin E2 production and to raise the hypothalamic thermostatic set point. Depending on the amount of pyrogenic material, the induced response can range from mild increases in body temperature to septic shock.
Endotoxin enters the body through the digestive tract of the body without harm, but when endotoxin enters the blood, it can cause harmful symptoms such as fever, septic shock, endotoxemia, and may even be fatal. Therefore, any drug that enters the body, including parenteral drugs and injection devices, must be tested for endotoxin. The Chinese Pharmacopoeia stipulates that endotoxin can be detected by two methods: rabbit pyrogen test (RPT) and bacterial endotoxin test (BET). The BET is using LAL/TAL, which is divided into Gel-Clot LAL assay and photometric method. Wherein the photometric method is further divided into turbidimetric LAL assay and chromogenic LAL assay.
Endotoxin is highly stable, heated at 180 ℃ for 2 to 4 hours, or boiled with a strong base, strong acid or strong oxidant for 30 minutes to destroy its biological activity. Therefore, the removal of endotoxin is not simple, and different methods need to be selected according to different situations.