I wrote a while back about the problem of compounds sticking to labware. That sort of thing happens more often than you’d think, and it can really hose up your assay data in ways that will send you running around in circles. Now there’s a report in Science of something that’s arguably even worse. (Here's a good report on it from Bloomberg, one of the few to appear in the popular press).
The authors were getting odd results in an assay with monoamine oxidase B enzyme, and tracked it down to two compounds leaching out of the disposable plasticware (pipette tips, assay plates, Eppendorf vials, and so on). Oleamide is used as a “slip agent” to keep the plastic units from sticking to each other, but it’s also a MAO-B inhibitor. Another problem was an ammonium salt called DiHEMDA, which is put in as a general biocide – and it appears to be another MAO-B inhibitor.
Neither of them are incredibly potent, but if you’re doing careful kinetic experiments or the like, it’s certainly enough to throw things off. The authors found that just rinsing water through various plastic vessels was enough to turn the solution into an enzyme inhibitor. Adding organic solvents (10% DMSO, methanol) made the problem much worse; presumably these extract more contaminants.
And it’s not just this one enzyme. They also saw effects on a radioligand binding assay to the GABA-A receptor, and they point out that the biocides used are known to show substantial protein and DNA binding. These things could be throwing assay data around all over the place – and as we work in smaller and smaller volumes, with more complex protocols, the chances of running into trouble increase.
What to do about all this? Well, at a minimum, people should be sure to run blank controls for all their assays. That’s good practice, but sometimes it gets skipped over. This effect has probably been noted many times before as some sort of background noise in such controls, and many times you should be able to just subtract it out. But there are still many experiments where you can’t get away from the problem so easily, and it’s going to make your error bars wider no matter what you do about it. There are glass inserts for 96-well plates, and there are different plastics from different manufacturers. But working your way through all that is no fun at all.
As an aside, this sort of thing might still make it into the newspapers, since there have been a lot of concerns about bisphenol A and other plastic contaminants. In this case, I think the problem is far greater for lab assays than it is for human exposures. I’m not so worried about things like oleamide, since these are found in the body anyway, and can easily be metabolized. The biocides might be a different case, but I assume that we’re loaded with all kinds of substances, almost all of them endogenous, that are better inhibitors of enzymes like MAO-B. And at any rate, we’re exposed to all kinds of wild stuff at low levels, just from the natural components of our diet. Our livers are there to deal with just that sort of thing, but that said, it’s always worth checking to make sure that they’re up to the job.
College chemistry, 1983
The 2002 Model
After 10 years of blogging. . .
1. SRC on November 11, 2008 11:49 AM writes...
As a voice crying out in the wilderness, I have on many occasions urged others (unsuccessfully) to run such "unnecessary" controls. "Waste of time" was the response.
Even a three month wild goose chase that ended with the realization that the assay had been buggered all along failed to convince some that there are wastes of time and then there are wastes of time. None so blind, and all that.
Permalink to Comment2. RB Woodweird on November 11, 2008 11:51 AM writes...
That reminds me of a paper I saw in the late 80s-early 90s. Someone was doing radioligand binding assays and first ran a blank series - with radioligand - without receptor. They got a beautiful, reproducible Scatchard plot with saturable sites and a nanomolar binding coefficient. I still remember the plot. It was textbook. The authors concluded that their peers might want to run similar blanks before publishing.
Permalink to Comment3. Smurf on November 11, 2008 2:13 PM writes...
Look out for glue from sticky!
Permalink to Comment4. KC on November 11, 2008 2:52 PM writes...
I got forwarded the brevia from some ex-collegues, with a `you might want to use pyrex when you can` attached to it. We're only slightly effected - the most common work I do is PCR - but the results are concerning enough that I'd basically decided to discontinue autoclaving consumables in plastic. No need to speed up leeching anymore than we have to.
I do wonder about the QT PCR, and whether this means they'll have to go through and re-evaluate old data like they did after they found sterilizing UV light releases inhibitors.
Permalink to Comment5. Green Koala on November 11, 2008 5:21 PM writes...
This reminds me of a project from almost 20 years ago, where a series of inhibitors prepared via a classical Diels-Alder reaction came up as hits in a screen. Hits resynthesized and purified on a flash column confirmed, but those resynthesized and purified by HPLC did not. Nobody worried about this much at the time.
Project went along for six months or so with all compounds showing activity but flat SAR. All were made via a Diels-Alder route and purified by flash. Once the compounds started being prepared via a different synthesis rounte, they were all of a sudden completely inactive.
Turns out that some type of polymeric material was being produced in the Diels-Alder reaction that carried through silica, but not HPLC. It just mucked up the assay in a general way.
It was a good lesson to see early on in one's career.
Permalink to Comment6. Brian Orelli on November 11, 2008 6:10 PM writes...
There was a lab while I was in grad school studying chromosomal non disjunction when all of a sudden the rates in their control mice jumped through the roof. Turned out to be a combination of a new cleaner on the plastic cages was increasing the rate.
Permalink to Comment7. Anonymous BMS Researcher on November 11, 2008 10:03 PM writes...
In some of the experiments I've helped to design, as many as ONE THIRD of the samples were various kinds of controls.
Glass can hold detectable traces of some organic compounds for a very long time. For many years I've used old applesauce jars to keep water in the fridge, and even after an astonishing number of washing-and-refilling cycles I can still detect a little apple taste in the water.
Permalink to Comment8. Lu on November 11, 2008 10:53 PM writes...
to Anonymous BMS Researcher:
...and lysozyme powder from our suppliers still smells like a chicken barn.
Permalink to Comment9. Maks on November 14, 2008 6:18 AM writes...
Control means control. If you store your sample in DMSO in the freezer, then store your blank DMSO in an identical tube in the same freezer, don't just take the DMSO from the glass bottle. One of the compounds in the Science paper is also a known CB1 and TRPV1 agonist, two pharmaceutical targets, this is something you don't want in your assay.
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