Relative Pyrogenicity

Endotoxin achieves greater leverage in eliciting deleterious host effects than any other microbial pyrogen as is seen in the relative amount of endotoxin needed to provoke a response, which is in the nanogram per kilogram range. If endotoxin is considered to be one of many alarm markers for hosts in recognizing microbial invasion (16), then it elicits the loudest and most variable response of the group. The great leverage of endotoxin can be seen in the wide variety of endogenous mediators elicited, which are active in the picogram (even femtogram) per kilogram range. Therefore, a small amount of endotoxin generates a very large host response in terms of both severity and variety. The complexity of the host response has frustrated efforts to devise treatments against it. The complexity arises from the interplay of the various mediators produced that may have proinflammatory and anti-inflammatory host effects as well as synergistic effects on their own kind. A few nanograms of endotoxin translate into the production of a myriad of manufactured endogenous pyrogens that are extremely bioactive. Arguably, the closest exogenous pyrogenic mediator analogous to endotoxin is prostaglandin (PG), a significant constituent of the cell wall of gram-positive bacteria (and present in lesser amounts in gram-negative bacteria). The pyrogenicity and endotoxin-like characteristics of PG were demonstrated by Roberson and Schwab in 1961 by using Streptococcus pyogenes cellular walls to induce fever in rabbits. Importantly though, the relative pyrogenicity of PG was shown to be on the order of 50,000 times less than the minimum pyrogenic dose of endotoxin (3).

In the early use of the pyrogen assay, no attempt was made to quantitate the amount of endotoxin needed to produce a pyrogenic response in rabbits. Because they are part of one of the most endotoxic families (Enterobacteriaceae) of bacteria, Escherichia coli and Salmonella were later chosen to determine and quantify the amount of endotoxin by weight considered to be pyrogenic. In 1969, Greisman and Hornick (17) performed a study using healthy male inmates (volunteers) and found the threshold pyrogenic response (TPR) level to be about 1 ng/kgb for E. coli and Salmonella typhosa (approximately 0.1-1.0) and 50 to 70 ng/kg for Pseudomonas. The same study revealed that the rabbit and human TPRs are approximately the same. Therefore, the amount of purified E. coli needed to initiate pyrogenicity in both man and rabbits is approximately 1 ng/kg, which represents about 25,000 E. coli bacterial cells (18). In terms of whole cells, the injection of an estimated 1000 organisms per milliliter (10,000/kg) of E. coli causes a pyrogenic reaction in rabbits, as compared to 107 to 108 organisms per kilogram of gram-positive or fungal organisms (19). The pyrogenic dose response curve in man is much steeper than it is in rabbits, although the minimum pyrogenic dose on a weight basis is about the same. Man is the most sensitive creature studied in his response to endotoxin exposure. It requires 10 times as much S. typhosa endotoxin in the rabbit as in man (0.005 mg/kg) to elicit the same degree of relative febrile response (20). Due to the steep response curve in man and the associated dangers, many of the effects of endotoxin exposure at higher doses cannot be studied. It has also been observed that certain illnesses (typhoid fever, hepatic cirrhosis) can increase the host's toxic responsiveness to endotoxin just as other diseases can decrease it (malaria) (17).

Pearson (21) has described that environmental endotoxins are notably less pyrogenic than purified LPS. His study indicated that a given test may fail the bSee Chapter 9 for a discussion of the relationship of the units: EU versus nanogram.

5.0 EU/kg FDA cutoff [established by the Endotoxin Unit designation as 0.2 ng of FDA RSE EC-2 (3)], but may exhibit a low order of actual pyrogenicity. Thus, the routine use of purified LPS as a control standard serves as a "significant safety factor unwittingly built into LAL endotoxin limits when real-world endotoxins are assayed by LAL" and "the Limulus test 'overpredicts' pyrogenicity." This is due to the high correlation observed between the rabbit pyrogen test and the LAL test using purified LPS, but a much less predictable correlation (biased conservatively) between the pyrogen assay and endogenous endotoxin.c Nevertheless, the routine use of worst-case validation philosophy has been borne out as a viable scientific approach for demonstrating the absence of contaminants (in a manner analogous to the use of Bacillus stearothermophilis in sterilization validation) and is incorporated into official regulatory requirements the world over.

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