Clinical and Medical Research
Beta Glucans and Endotoxin Testing
The presence of bacterial endotoxins in different media, solutions and laboratory materials can be determined by utilising the limulus amebocyte lysate (LAL) test. This assay, which is the compendia test for the examination of bacterial endotoxin in pharmaceutical products, has become an essential tool for both industry and research over the last few decades, and the most reliable test for the detection of pyrogens. The biochemical basis of the LAL test involves the unique clotting mechanism initiated by amoebocytes from the blood of the Atlantic horseshoe crab (Limulus polyphemus) when exposed to gramnegative bacterial endotoxin. There is a specific cascade associated with LAL that makes it possible to observe either qualitative or quantitative effects when endotoxin is present. The enzymatic reactions that occur along this cascade result in the conversion of amoebocyte coagulogen into a fibrinogen-like clotting protein, which forms a coagulin gel.1 A more detailed review of these reactions shows that the endotoxin interaction initiates Factor C (first serine protease precursor) from its inactivated form, which in turn activates Factor B (second serine protease precursor). The activated Factor B stimulates the clotting enzyme, converting it from a proclotting enzyme.
The clotting enzyme then cleaves peptide bonds within coagulogen to yield coagulin, the insoluble gel-forming protein produced in the gel clot assay. There are, however, other compounds which also cause the gelation in the amoebocyte lysate from the horseshoe crab, leading to an interference in the test of bacterial endotoxins. Some of these compounds are known as (1,3)-β-Dglucans, which can lead to false positives in the LAL test due to initiation of the clotting cascade when the glucans trigger the protease enzyme Factor G. To overcome this interference, the assay can be modified to include a wide range of glucan-blocking reagents. The (1,3)-β-D-glucans are polysaccharide compounds consisting of glucose monomers, particularly the monomers of D-glucose, linked via β-glycosidic bonds, which are produced by many prokaryotic and eukaryotic organisms. One of the main sources of glucan contamination in laboratories comes from paper cellulose as these compounds are found in the cellular walls of tree cells. As filtering operations are a common process in most laboratories and are conducted on many occasions using cellulose filter paper, it is not unusual to find that samples being tested for the presence of bacterial endotoxins contain (1,3)-β-Dglucans introduced by the fibres of these same filters. In many cases, an examination of the filters fails to be completed prior to testing, allowing for the glucans to be
leached from certain types that ultimately end up in the filtrate. Often, this problem is only detected through routine testing performed on the finished product using the LAL assay. The samples can also be contaminated upon contact with other organisms which produce glucans. The contaminant glucans can come from different sources, such as the zymosan of yeast, the laminarin from algae, lentinan from the shiitake mushrooms (often used as an antitumoral drug) and curdlan that is produced by bacteria. β-glucans have also been regarded as pathogens in mammals and thus a serious concern of possible contamination in both bioprocessing and in blood products. High amounts of β-glucans in pharmaceutical end-products can illicit an immune response and so it is necessary to control their levels to prevent any associated reactions from occurring within the general public. The β-D-glucans, which have various effects on mammals, mediate the immune response by activating different receptors involved in proinflammatory reactions, such as those found on lymphocytes, macrophages and within the complement cascade. Generally, they are not digested in the intestine. They are substances that serve as an energy source for different microorganisms that live in the
68 INTERNATIONAL PHARMACEUTICAL INDUSTRY
Spring 2021 Volume 13 Issue 1
