First, the pyrogen (progon)
In the clinical use of drug injections in hospitals, there are often cold sensations, chills, fever, headache, nausea, vomiting, gray complexion, shock, and serious death. This symptom is called pyrogen reaction.
In order to improve the quality of medicines and the safety of medicines, extensive research has been carried out on pyrogens. Until 1923, Seibert proposed a method for detecting pyrogens in rabbits. In 1942, the United States Pharmacopoeia first became the statutory method for the income of the rabbit pyrogen check item. The Chinese Pharmacopoeia began to include the method in the 1953 edition. The subsequent pharmacopoeia of the world used the animal pyrogen test method as one of the methods for drug quality monitoring.
The advantage of the rabbit pyrogen test method is that the body temperature change of the rabbit can be observed within the prescribed time, which correspondingly reflects the complex body temperature reaction process of the mammal caused by the pyrogen. Therefore, the pyrogen inspection method has played an important role in ensuring the quality of medicines and the safety of medicines for more than half a century.
However, with the development of the pharmaceutical industry and the requirements of clinical drugs, the limitations of this method are becoming more and more obvious. This pyrogen test method is limited to whether a certain drug enters the body (blood circulation) can cause a change in body temperature or a pyrogen reaction as a method for judging whether a drug contaminates a pyrogen, and cannot meet the needs of the development of the pharmaceutical industry. Its disadvantages:
1 The degree of standardization is low, and it is impossible to judge what kind of substance or which substance is present in the test sample.
2 Because the test animals rabbits are in an environment contaminated by bacteria, they are immunized by inhalation or skin infection with bacterial endotoxin, resulting in a large individual difference in animals.
3 Test animals are interfered by the pharmacological activity of the drug, which affects changes in body temperature (such as radiopharmaceuticals, antibiotics, biological products, etc.), and the experimental results are difficult to judge.
4 equipment and experiment costs are expensive (such as the construction of animal rooms, water and electricity, animal feed, etc.), to do a drug requires 280 yuan / time, while the sputum reagent is only 28 yuan / time.
In summary, the sputum test method can avoid the deficiencies of the above animal pyrogen test method. The success and application of this technology can be described as a revolution in drug quality monitoring.
What is a pyrogen? At present, there is still no unified understanding at home and abroad, but from the domestic and foreign literature reports, a common opinion is generally believed: it refers to the lipopolysaccharide of bacterial endotoxin.
J. Van Noordwijk, Vice-Chairman of the European Pharmacopoeia Commission, said: "Strictly speaking, not every pyrogen has a lipopolysaccharide structure, but all known bacterial endotoxin lipopolysaccharides have pyrogen activity." Under the conditions of Good Manufacturing Practice (GMP), the quality control of pharmaceutical production is generally acceptable: the absence of bacterial endotoxin means the absence of pyrogens.
Second, bacterial endotoxin (Endotoxin)
Bacterial endotoxin is a complex of a lipopolysaccharide (Lipoply Saccharide) and a microprotein (Protein) on the cell wall of Gram-negative bacteria. Its specificity is not a bacterial or bacterial metabolite, but a bacterial death or disintegration. A substance with endotoxin biological activity released. Its chemical composition is widely distributed in the cell wall layer of Gram-negative bacteria (such as Escherichia coli, Brucella, Salmonella typhi, Proteus, Salmonella, etc.) and other microorganisms (such as Chlamydia, Rickettsia, spirochete, etc.) The chemical composition of polysaccharides is mainly composed of O-specific chain, core polysaccharide and lipid A.
<1>, O-specific chain: The polysaccharide chain located at the outermost layer of the lipopolysaccharide molecule is composed of 3 to 5 monosaccharides (generally not more than 25) connected to form a polysaccharide chain. The monosaccharide includes pentose, aminopentose, hexose, aminohexose, deoxyhexose, etc. The type, position, arrangement order and spatial configuration of the monosaccharide vary depending on the species. Therefore, it determines the specificity of the bacterial pyrogen.
<2> Core polysaccharide: The core polysaccharide has less variability, and is located between the lipid A and the 0-specific chain (inner layer), and is structurally divided into an inner core and an outer core. The outer core contains several hexoses, including glucose, galactose, and acetylglucosamine. The inner core contains heptose and a special ketose (3-deoxy-D-mannose-octulose KDO). This part of the structure is basically similar to the LPS of different strains, and KDO is linked to the glucosamine of the lipid A by the acid-tolerant ketose chain, which is the core part of the endotoxin lipopolysaccharide.
<3> Lipid A: Located in the outer layer of the molecular structure of LPS, it is composed of glucosamine, phosphoric acid and fatty acid (10-18C), so it is called glycophospholipid, which is also a kind of bacterial outer membrane, forming monomeric polymer. . Has a hydrophobic (strong) and hydrophilic (weak) duplex. However, the lipid A can be separated from the O-specific chain and the core polysaccharide, and the free lipid A can itself agglomerate into a complex of macromolecules, is hardly soluble in water, and is biologically active. Therefore, Lipida is the major group of various biological activities or toxic reactions of endotoxin. This group is not species-specific, so the lipid A structure of each genus is similar, and its toxicity is similar. Such as fever, hemodynamic changes, diffuse intravascular coagulation, and lead to shock and so on. (Figure 2)
Since the lipid A has four main chains and two branched fatty acids linked to the lactam, the purified endotoxin LPS is extremely unstable. This requires endotoxin to be stored under low temperature conditions. Endotoxin dilution should be as short as possible during work and should be used now.
Third, the correlation between the biological activity of endotoxin and disease
According to the literature, in the very early days (about the end of the 19th century), the Italian scholar Centanne extracted a substance similar to toxin from Gram-negative bacilli by the method of autolysis of the genus, because this substance produces heat to the animal. At the same time as the activity, a pathological reaction is also produced, which is named as pyrotoxin (Pyrotoxina). At the same time, Buchner from Germany also extracted similar thermotoxins from various bacteria, and confirmed that this toxin can increase the immunity of the body to bacterial infection while causing changes in the number of white blood cells. Therefore, "fever therapy" was established.
In New York, a clinician in New York, William B. Coley used a heat method to kill the bacillus and Streptococcus pyogenes, and used the supernatant filtrate for the treatment of various malignant tumors (especially sarcoma). He named the bacterial supernatant as Coley's toxin. MurrayJ.Shear then confirmed that the anti-tumor substance in Coley's toxin is endotoxin.
Until 1933, when Boivin and other scholars studied the pathogenesis of Salmonella typhimurium, endotoxin was extracted from Salmonella typhimurium. After the 1950s, research on the chemical composition and chemical structure of endotoxin was rapidly developed. A large number of experiments have shown that endotoxin has extremely strong biological activity, especially when Gram-negative bacteria infection and intravenous injection of endotoxin solution can cause endotoxin shock and death in animals.
The pathogenesis of endotoxin is mainly due to Gram-negative bacilli (such as Escherichia coli, Salmonella, Salmonella typhi, Brucella, Proteus aureus, etc.) and other microorganisms (virus, rickettsia) , Chlamydia trachomatis, etc.), when the genus enters the blood circulation with the exudate of the lesion, and spreads to various tissues and organs and body fluids to reproduce. After the bacteria die and disintegrate in the body, only a large number of bacteria are diluted. Endotoxin lipopolysaccharide (LPS), according to preliminary experiments, can induce endogenous pyrogens such as tumor necrosis factor, interleukin and β2-interferon when the toxin concentration in the body is > 0.005 ng/ml. . These factors stimulate the body's thermoregulatory center to cause fever. When bacterial endotoxin acts directly or indirectly on the liver and pancreas, it can damage liver cells and make glycogenogenic enzymes (such as glucose-6-6 phosphatase and glycogen synthase). The activity is reduced, inhibiting the xenobiotics and decomposition of glycogen. At the same time, endotoxin acts on the pancreas to cause pancreatic dysfunction, and forms insulin resistance, resulting in a series of hyperglycemia symptoms caused by elevated blood sugar and complicated myocarditis and myocardial enlargement. Therefore, Gram-negative bacteria infection or bacteria in the lesions enter the body fluid cells to proliferate, when the endotoxin produced after its death or disintegration, can enter the blood multiple times, causing recurrent attacks, and its pathological changes are extremely extensive, almost all Organs and tissues can be violated, causing dysfunction of various organs. Among them, the reticuloendothelial system is the most common. Lymphocytes, spleen, liver, kidney, and bone marrow all have epithelial cell hyperplasia, which forms granulomas. In addition to liver granuloma, hemorrhage, edema, and hepatocyte necrosis can occur. The occurrence of cirrhosis. Other organs have similar toxic reactions.
Therefore, strengthening the monitoring of bacterial endotoxin in medicines and human body fluids is of great significance for ensuring human health. The quantitative bismuth reagent developed by Zhanjiang Bokang Marine Biological Co., Ltd. and the bacterial endotoxin detection and determination system developed by Tianjin University Radio Factory provide convenience for the majority of medical science workers.
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