Endotoxins are important to me. In addition to impacting the core function of my chosen field – intrathecal drug therapy — they are a singularly fascinating phenomenon. I take them – and Bacterial Endotoxin Testing (BET) — very seriously.
Endotoxins are small impurities present in bacteria. They are actually sections of bacterial walls – more specifically, sections of gram-negative bacterial walls. They are also very lethal and detrimental to the human body. As such, they receive careful consideration in the compounding, manufacture and administering of parenteral preparations. BET is performed on all commercially manufactured parenteral products.
As a college student, I developed a strong interest in parenteral drug therapy and (by association) endotoxin testing. Up until 1982, said testing employed rabbits as the primary test subject – either three or five. The drug was injected into these test rabbits, the resultant body temperature changes (or lack thereof) monitored by a well placed thermometer. If the body temperature varied 0.5 ° or more, the test element was considered pyrogenic – “fire generating” (causing fever) – and indicated the presence of endotoxins.
Allow me to digress here by explaining that in those early days of endotoxin testing, endotoxins were referred to as pyrogens – until it was discovered that not all endoxins are actually pyrogenic. The distinction was made, and the use of “pyrogen” synonymously with endotoxins was discontinued.
Cued by this revelation and the ongoing evolution of available technology, in 1982 an innovative new endotoxin test was developed. I followed its development over the years as I developed my practice, learning more and more about the nature and specifics of this new testing method. In 1998, I contacted a company – one of only three that manufactured the endotoxin test at the time – Massachusetts’ Associates of Cape Cod. (www.acciusa.com )
Key to the testing was a lysate – an enzyme — known as LAL (Limulus Amebocyte Lysate). Derived from a HORSESHOE CRAB [Limulus polyphemus] found only on the eastern seaboard of North America along the various coastal bays of Massachusetts and Connecticut and Japan, the lysate acts to clot and expel bacteria with which the horseshoe crab comes in contact. The crab species has been around for about 250 million years, and for all this time, the lysate has served as the creature’s immune system. Someone researched this phenomenon and found that this lysate has affinity to the lipopolysaccharide section of bacterial cell walls.
Entranced and engaged by this story and its implications for the field of intrathecal drug therapy, in 1999 I pulled the trigger. I bought the equipment that would make it possible for me to conduct my own bacterial endotoxin testing.
This test was very specific and very complicated. We compounded our drugs, took samples and injected the lysate into the test tubes, but there was more. You had to perform a standard curve. The rationale for the standard curve was to ensure the lysate reaction worked with a cross section of endotoxins concentrations. If that standard curve played out correctly — if the mixture clotted — you said “OK, I’m prepared to submit samples of my active drug.”
We tested our samples in duplicates. This discipline of not only running a standard curve, but preparing samples in duplicate – both spiked and non-spiked — took us to a new level of “You’ve gotta be kidding”…and a new level of expertise in analytical chemistry. We explored and learned and grew – and, in time, we mastered this sometimes maddeningly complicated procedure.
We continue to administer this test on our compounded preparations, with new advanced instruments – and it has made all the difference in our finished product. We learned volumes about our preparations and how they interacted with the enzyme – how they actually effect the way the enzyme can enhance or inhibit the outcomes – and we turned that knowledge and know-how into a significantly serious service asset, for our company, our clients and their patients.