Bacterial endotoxins are pyrogens originating from gram-negative bacteria; these endotoxins are found within the exterior membranes containing lipopolysacchrides (LPS). A pyrogen when in contact with blood or cerebrospinal fluid can cause an increase in body temperature, septic shock and in some cases death. These endotoxins excreted by gram-negative bacteria are a major cause of pharmaceutical products contamination. Endotoxins are exceptionally potent, heat stable, able to transcend sterilizing membrane filters and are existent where bacteria are or have been. Sterility tests do not accurately identify endotoxins due to their chemical nature and because they are only produced by gram-negative bacteria. Therefore Bacterial Endotoxin Test (BET) is performed on sterile products (officially in the Pharmacopoeia) and medical devices to be implanted or injected into the body to avoid antagonists effects. These effects include; fever, activation of cytokine system, destruction of endothelial cells, permeability of blood vessels causing low blood pressure,among others.
BET detects/quantifies dangerous levels of microbial cell wall fragments from live or dead gram-negative bacteria, using amoebocyte lysate from the horseshoe crab. The horseshoe crab is immune against invasion of gram-negative bacteria. This lysate is used in the Limulus Amoebocyte Lysate (LAL) test, which is an analysis that quantifies endotoxin in-vitro. The manner in which the test is carried out avoids endotoxin contamination, within the limits set by Pharmacopoeia. There are 3 variant methods for the LAL test:
1. Gel-clot, combination of LAL and endotoxin (reaction mixture) resulting in clotting;
2. Turbidimetric, where rate of turbidity development after cleavage of an endogenous substrate is equivalent to the concentration of substrate;
3. Chromogenic, centered on development of yellow color after cleavage of a synthetic peptide – chromogen complex which is compared to reaction mixture on a standard curve.
Before carrying out the test for endotoxins it’s important to verify; sensitivity of lysate (gel-clot method), linearity of standard curve (quantitative methods), and absence of interfering factors. Initial rate of reaction is determined by concentration of endotoxin present, pH and temperature. The reaction requires certain bivalent cations, a clotting protein and clotting-cascade enzyme system, all of which are provided by the lysate.
The 3 methods are used to evaluate end-product injectable drugs, medical devices and raw materials. In the gel-clot method, endotoxin catalyzes activation of proenzyme (found in LAL) to produce coagulase. The activated coagulase hydrolyzes specific bonds in coagulogen (a clotting protein found in LAL) to form coagulin. Coagulin forms a gelatinous clot through self-association. A positive results forms a solid gel that remains after inversion, a negative result is void of a solid clot after inversion. Results should be compared against a reference sample, control standard endotoxin (CSE). All glassware has to depyrogenized and test run in quadruples.
The turbidimetric and chromogenic methods are both photometric assays wherein the concentration of endotoxin is calculated from a standard curve. The turbidimetric method analyzes either the time needed to reach a predetermined absorbance of reaction mixture or rate of turbidity development. The chromogenic assay measures either; rate of color development of chromophore released by chromogenic substrate of the reaction mixture, or the time needed to reach a predetermined absorbance of the reaction mixture. Both assays should be performed in duplicates.
The gel-clot method is the more sensitive and accurate procedure, it has fewer false positives/negatives. Despite its accuracy, it’s not automated, time consuming and is subject to; chemical and physical inhibitors, protein denaturation and pH disruption. The quantitative methods can be automated and results can be easily calculated. Although user friendly these methods can be altered by testing blood, plasma, albumin, serum and similar materials. Both methods are sensitive to excess turbidity. The turbidimetric method is associated with numerous false positives, whereas many compounds interact with the chromogenic method rendering it ineffective in most cases.