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Things I’m Glad I Don’t Do: Isolating Ciguatoxin

The natural products topic (which I’ll return to in a couple of days) has me starting up a companion to the “Things I Won’t Work With” category. This is the first in the new category of “Things I’m Glad I Don’t Do”.
I mentioned ciguatoxin, and I notice that one of the comments to that post was from someone who’d had a brush with the stuff. My sympathies – it’s supposed to be really awful. A number of warm-water fish species can have dangerous concentrations of the compound in them, and it’s probably one of the most common non-bacterial sources of food poisoning. The thing is, the fish themselves don’t make the stuff. They concentrate it from marine algae, who produce a lot of extravagantly crazy molecules.
So if you want some ciguatoxin yourself, you fool, you, a good source is an organism near the top of the food chain. Moray eels turn out to be a good bet. But you don’t just turn one of them upside down over a beaker and squeeze his tail. No, the isolation is a bit more involved:
The moray eels (ca. 4000 kg) were collected from the Tuamotu Archipelago and from the Island of Tahiti in French Polynesia. The viscera (125 kg) were homogenized and extracted with two volumes of acetone twice. After filtration, the extract was left at -20 “C for 1 day to precipitate oily residue. The supernatant was evaporated to dryness and partitioned between diethyl ether and water. The ether layer was condensed and suspended in aqueous 80% MeOH, followed by defatting with hexane. The methanolic layer was condensed, dissolved in acetone. . .
The prep goes on in this vein, through six different columns, one after the other. Now, imagine joining this research group (which was Yasumoto’s, in Japan). It’s your first day in the lab, and here comes one of the post-docs carrying a couple of blenders in his arms. Behind him, another one is wheeling in the bags of frozen eel guts. It’s moray margarita time, and will be for some time to come.
One other aspect of this isolation deserves comment, because I don’t think you could do it like this today. In the final column or two, the paper outlines a brutal but effective method for cutting fractions to get the ciguatoxin: take a sample from each cut of the column and inject it into a mouse. If it doesn’t die immediately, that fraction doesn’t have any ciguatoxin in it. Gloves recommended.

16 comments on “Things I’m Glad I Don’t Do: Isolating Ciguatoxin”

  1. Harry says:

    That reminds me of a book I read a few years back, called “Anatomy of a Scientific Discovery”. It was about the isolation of the first endorphins (then called endogenous morphines).
    The fellow that was working on this project got to make a trip to the slaughterhouse every day that they killed pigs to collect the heads. He then cracked open the skulls and removed the brains (at the slaughterhouse). He trundled back to the lab with the brains and froze them, crushed thm up in a stainless beaker with lots of acetone (no wimpy blenders for this guy). He then followed the same general lines outlined above. And he was the Principal (indeed, the ONLY) Investigator.
    Imagine the PI in a modern group cracking open pigs heads and crushing frozen brains!
    I’m glad I don’t do that sort of stuff too (and I religiously avoid column chromatography).
    My $0.02

  2. Harry says:

    Oh yeah- the method for testing for endorphins is cool too. It involved removing the ileum from a Guinea Pig and timing the twitches, then the ileum was treated with the test compound. If the twitching decreased, then the ileum was treated with naloxone (a morphine antagonist) to see if it reversed the effect.
    Nice line for the ol’ resume’: One year as assistant in charge of Guinea Pig ileum removal and testing.
    The book is an interesting read- not sure if its still in print, I picked it up in the bargain bin. Has some interesting insights on the jockeying around for the Nobel Prize, too.

  3. Grubbs the cat says:

    The last paragraph reads like the isolation of psilocybin from magic mushrooms. If I remember correctly, the isolation team tested the fractions orally. If they felt slightly weird, it contained the right stuff (I hope they made sure about the correct dose…).
    Pretty efficient – I can’t think of an easier method.

  4. daen says:

    I read the description of John Thomson freezing and blending kilograms of calf thymus at Vertex in Barry Werth’s excellent book “The Billion-Dollar Molecule” and thinking “that’s disgusting”, but at least it wouldn’t kill you. Not unless you ate too much of it, I guess.

  5. Derek Lowe says:

    Harry’s comments recall the pre-cloned-receptor days, when tissue assays like that were the only way to assay compounds. When I started doing muscarinic receptor work in 1989, we had the cloned receptors in cell lines, but not everyone did. The literature was still full of assays against things like rat vas deferens and possum trachea.
    Those assays have their good and bad points. The big problems are the trouble with setting things up in volume, reproducibility, and the big issue: the fact that there are no tissues that are totally clean sources of any one receptor type.
    On the plus side, you were testing in something that much more closely approximates a living system, so if you got the effect you were looking for, you had a little more confidence that your compound was something real. It was like going straight to the cell assay under modern conditions, but in this case the cells were much more realistic than some of the cultured lines that we use now.
    But I am not advocating a return to possum tracheas.

  6. Mark Reviea says:

    Wow, that sure brings back memories! In the early years of my career, I worked on a project to isolate gonadotropins from equine pituitary glands. The method was pretty much as described by Derek; however, we used abs. alc. for the extraction solvent. Unwilling to pay the exorbitant duties to a chem. distributor for that reagent, we just went down to the liquor store and bought a case of Everclear. This was a nice modification, as when the slimy task was completed, we treated ourselves to shots (don’t try this at home, kids) out of griffin beakers!
    Cheers.

  7. Harry’s comments recall the pre-cloned-receptor days, when tissue assays like that were the only way to assay compounds. When I started doing muscarinic receptor work in 1989, we had the cloned receptors in cell lines, but not everyone did. The literature was still full of assays against things like rat vas deferens and possum trachea.

    Ah, now you’re talking about things I can relate to. Cutting bits out of rodents and suspending them from potentiometers is (was) a staple of pharmacology. During my PhD (not that many years ago) we were working on EDHF, and my boss, who trained under John Vane, had an idea to see if the response we were seeing was due to the release of a mediator.
    Cue several hours setting up bits of glassware on stands, helical cut vessels and half the department crowded into the lab to see an actual cascade bioassay! It raised a lot of laughs, and didn’t give us any decent results, but it did make me appreciate the advances made since the 1960s and 1970s…

  8. Jason says:

    Derek, why can’t you use mice to test ciguatoxin fractions? They’re just mice. My kid feeds them to his pet snake.

  9. Milo says:

    Is that 4000 kilo-grams of eel?!?
    Man, this is like “Fear Factor” for chemists!

  10. Derek Lowe says:

    I don’t think that any company’s Animal Care and Use Committee would sign off on that sort of thing. You’re supposed to use animals when there aren’t any reasonable substitutes, which covers all the in vivo models and PK (blood level) tests.
    But in this case, these guys were basically using mice as a substitute for analysis. Run an NMR, run a mass spec. The mouse detector has a good signal-to-noise, especially if there are a lot of similar nontoxic compounds coming out around the same time, but that’s not going to be enough to convince the care-and-use folks.

  11. NJBiologist says:

    Milo: It’s also Fear Factor for biologists. In quantity, you’ll have a hard time finding Morays over 10 kg. So that’s several hundred congenitally surly critters to be caught. Can you say “multiple puncture wounds”?
    Harry et al: Don’t forget, NIGMS is trying to bring back organ pharmacology through training programs at four universities. They’re on the same page as Derek regarding realism of the assay system.
    Derek: Possum trachea? I’ve never heard of that one.

  12. Jason says:

    At what animal cuteness level do drug companies draw the line? Animal Care and Use Committees must be aware of mosquito assays for ciguatera toxin.

  13. Tot. Syn. says:

    And now they can make it synthetically; http://dx.doi.org/10.1021/ja063041p

  14. Do mosquitos even count as animals? I tend to think of them as evil in corporeal form, with wings. I’d have more qualms about killing bacteria than mosquitos.
    On the other hand, I’d rather not have them in the lab.

  15. Fabius says:

    Horrible, horrible toxin. I encountered it during a fishing trip in the Pacific. We captured three wahoos and two yellowfin tunas, which were preserved in an ice chest. That night, we pan fried it (beautiful, juicy fish steaks, freshest fish I had eaten) and happily ate it with patacones (fried plantain slices). I woke up that night with the horrible feeling that someone was arranging heavy furniture on my chest. I managed to get to the porcelain altar where both ends of my GI tract competed to unravel from my body. About 10 persons affected with mild to severe symptoms (a cousin and his wife had to be hospitalized). We ended up discarding about 100 lbs of perfectly edible fish. Is there a good test to detect ciguatoxin?

  16. Rachel says:

    haha! Reminds me of some trophic transfer research I once did where we had to make agar pellets dosed with toxic metals palatable to crayfish. Suspicious creatures crayfish…. the only way we could do it was by blending raw fish in with the agar prior to autoclaving it. Lets just say it was fragrant.

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