The Half Decent Pharmaceutical Chemistry Blog

It’s July and I have begun to receive emails from worried, nerd undergraduates who ask me what they could do now that they are going to be kicked out of the lab.

I’m happy to announce, my nerdy friends, that I’ve got a perfect solution for you, which, believe it or not, will take you to sunny beaches so that you’ll all finally lose your ugly paleness, while giving you the opportunity of doing science.

It’s time to go crab hunting! In particular, this is the last month when you could hunt for Limulus Polyphemus!

This lovely animal is better known as horseshoe crab: these crabs evolved from sea scorpions in the Paleozoic era (fossils were found in Germany). They depose eggs in the sand of estuaries in April: there, trilobate larvae appear until, when the newborn is ready for living in the sea, high tide gently takes them out of the sand.

You shouldn’t bother about the offspring: concentrate on the parents. We need their blood! No, it’s not for a gory sacrifice or a delicious, Spanish plate: it’s all about saving lives, dear.

Let’s begin with a bit of a history lesson. In the 1940, the Brits set a first official test to assay the content of pyrogens in parentally-administered drugs: they injected in three rabbits the product and, if the sum of their temperature rise was less than 1.15°C, there wasn’t any problem. Otherwise, whether the sum reached 2.65°c, that product had to be destroyed. Anything in between would require the assay to be repeated employing 6 rabbits (with different values, of course).

In 1960s, however, Dr. Frederik Bang, at the Marine Biological Laboratory in Woods Hole, Massachusetts, discovered the properties of horseshoe crabs.

You see, this lovely creatures present a granular amebocyte in their blood, where Cu replaces Fe. So you can really say this is a blue-blooded crab!
From May to July a third of hemolymph of Limulus Polyphemus is collected. The animal is therefore marked so that it won’t be used for the next two years or so and is released in the sea where, it has been proved, it goes on with its normal life as always. Perhaps it’ll look like an undergraduate preparing for exams among its peers…

However, this hemolymph is rather precious: in the presence of endotoxin G (typical of Gram- bacteria), it coagulates. That’s because endotoxins trigger the activation of factor C. This protein then acts on factor B, which in turn activates a proclotting enzyme, that converts coagulogen to coagulin. As a result a gel-clot is yielded.
Interestingly, this test works for yeasts and moulds too, which present β-1, 3 glucans on the surface, because this alternative substance activates factor G whose role is exactly the same as that of factor B.
It is interesting to highlight that the last 8 amino acids of the peptide C of coagulogen are the same of fibrinogen.

Briefly, the LAL test (Limulus Amebocyte Lysate) works as follows: physiologic coagulogen is substituted with a derivative which bears p-nitro aniline bound to an arginyl group, so that an aromatic amine is yielded. This is a great advantage as the concentration of this molecule can be easily detected through a colorimetric titration. To sum up, the concentration of endotoxins is determined using standard solutions whose pyrogen levels are known.

For what concerns the limits of endotoxins parentally administered, they are calculated through the K/M ratio, where K stands for the quantity of endotoxins that an average human being can receive without showing any pathological consequence (intravenous = 5.0 endotoxin units/kg/hour; intratechal = 0.2 EU/kg/h) and M is the maximum recommended human dose of that drug.

For more information, log on to this website (I’ve always wanted to say this phrase!). Hey, you! What are you waiting for?! You’re running out of time: go crab hunting!