Academic nonsense! (scientific info on thiamine/thiaminase)

[DrKate]

OK, since there's at least mild interest out there, here's a thread specifically for scientific information and debate on The Thiaminase Issue.

The rules are that anything posted here has to be real scientific information, with a reference. I think it's fair to say that accounts of your own personal experiences are OK too, but not "I know this guy who said..." stories.

As far as an overall goal for the thread, I still think that there are two critical unanswered questions of practical importance:

1) What is the garter snake's dietary thiamine requirement - how much is necessary to prevent thiamine deficiency symptoms? And also for completeness, at what level does thiamine supplementation become toxic?

2) How much thiamine does a garter snake actually obtain (that is, amount initially present minus amount destroyed by thiaminase on the way from the mouth to the intestine) from a goldfish/rosy red/shiner fed live? And how much variability is there in the thiamine/thiaminase content of commercial feeder species?

[DrKate]

This is a copy of an e-mail I wrote a couple weeks ago to someone who wanted a thiaminase primer. It breaks the rules a little because it's not actually fully referenced, but I hope you'll forgive that.

Hi Chris, I can certainly give you some more information on thiaminase in general if you're interested. There are reference books that contain general information (CRC Handbook of Marine Mammal Medicine, Nutritional Biochemistry of the Vitamins) that you could find in a university library. You can also find a lot of info online because this is a big issue for captive and commercially farmed piscivores - one very general reference is Feeding Captive Piscivorous Animals (http://www.nagonline.net/Technical%2...02MODIFIED.pdf).

Thiamine is a vitamin (vitamin B1), meaning it's something that animals must get in their diet; the body can't make it. Thiaminase is an enzyme that breaks down thiamine, and is part of some animals' normal metabolism during life. But the enzyme remains active after the animal dies, and breaks down whatever thiamine was present in the body at death. Thiaminase itself is a protein, and it is broken down in the digestive tract of the predator just like all the other proteins in the prey animal's body. But the issue for our purposes is how much thiamine gets broken down between when the prey animal dies and when the enzyme itself is destroyed. If the level of thiaminase is high or if the prey animal died long before being consumed, most of the thiamine is broken down and very little is available to the predator. If the predator is heavily dependent on that food source then its overall diet may be thiamine deficient, and it can develop potentially fatal neurologic and sometimes reproductive problems.

From what I've read, there are two main ways that thiaminase causes problems for predator species. The first is when a particular prey species makes for a thiamine-deficient diet even if freshly killed. Enough thiamine is lost between the mouth (where the prey animal dies) and the intestine (where the thiamine would have been absorbed) that the predator can't obtain sufficient thiamine from that prey. There was a big problem with salmonid fish in the Great Lakes some years ago feeding on an introduced/invasive fish species called the alewife. There are a lot of scientific publications studying this particular episode because it was such a big commercial issue, but there are also other reports where a predator population has become dependent on a high-thiaminase prey species and suffered thiamine deficiency.

The second (and much more common) issue with thiaminase is that it remains active (though less so) even in frozen fish. This is particularly a problem with captive or commercially farmed animals fed pre-killed fish. In this case a prey species that might be safe to eat when freshly killed becomes thiamine deficient during storage. The longer the storage, the more depletion of thiamine. And if storage conditions are not ideal (stored unfrozen or not adequately frozen) then the depletion happens faster. Thiaminase is inactivated by heating, which is why most commercial prepared fish meal diets are shelf stable - but they're also heavily supplemented with vitamins including thiamine to make up for what's missing in the source fish or lost during processing. Again most of the scientific publications in this area are for commercially important farmed species, or those commonly kept in zoos.

You'll notice that in both of the above scenarios, the predator is not consuming its natural diet. In one case, high-thiaminase invasive species have displaced natural low-thiaminase prey of a wild population, and in the other, captive animals eat whatever they're fed, which is usually whatever can be obtained cheaply. I have to assume that the natural predators of high-thiaminase prey (like tuna, which eat herring/sardines/anchovy) have evolved ways of dealing with the problem - perhaps just a varied diet that also includes low-thiaminase (or high-thiamine) prey species. Predators eating live fish in the wild also escape the additional depletion of thiamine during storage, of course.

The most thorough single reference I know of is Nutrient Requirements of Mink and Foxes, which has a 2-page table of thiaminase content in various fish (pages 64 and 65): Nutrient Requirements of Mink and Foxes, Second Revised Edition, 1982. However, this table is not comprehensive (just because it's not listed doesn't mean it's safe). Both goldfish and fathead minnows (a.k.a. rosy red minnows) are listed in this table. You can also find a lot of scientific publications, including surveys of particular geographic regions, by searching Google Scholar. (You'll need to go to a university library for free access to the full text of most of these articles, but can usually read the abstract for free at home.)

In the aquarium and reptile hobbies, there are other prey which are generally considered free of thiaminase, though I haven't found specific scientific references for them. These are live fish of the guppy/molly/platy family, and frozen true silversides (genus Menidia).

[DrKate]

Here's a re-post from another thread addressing the common misconception that thiaminase blocks thiamine absorption, or somehow stays in the intestine of the fish and affects later meals, so there's no use in giving supplemental thiamine...

http://www.thamnophis.com/forum/husb...tml#post112680

...thiaminase doesn't so much destroy the thiamine, as that it blocks it's absorption, and when the absorption is blocked, it doesn't matter how much extra you throw at it

I've seen that elsewhere too, but never with an actual reference for the information. So, I tried looking it up. Here's the most directly relevant thing I could find on the mechanism of thiaminase:

Nutritional Biochemistry of the Vitamins
David A. Bender
2003
"There are two classes of thiaminase. Thiaminase I catalyzes a base-exchange reaction [...chemistry stuff we don't care about...]. Thiaminase I is relatively widespread in a variety of microorganisms, plants, and fish. In addition to depleting thiamin, the products of base exchange by thiaminase I are structural analogs of the vitamin and may have antagonistic effects (Edwin and Jackman, 1970). [...] Thiaminase II catalyzes a simple hydrolysis [...]. It is relatively rare and is restricted to a small number of microorganisms."

That bold part there may be the source of the idea thrown around among hobbyists that thiaminase "blocks absorption or something." Quite simply, it doesn't - it just breaks down thiamine. BUT, those breakdown products may compete with any surviving thiamine for absorption into and use by the body, which would make the deficiency appear even more severe.

But again, if the supplement level is high enough then this competition should be overcome, too. For example:

Experimental thiamine deficiency in captive harp seals, Phoca groenlandica, induced by eating herring, Clupea harengus, and smelts, Osmerus mordax. Geraci, JR
Can. J. Zool. Vol. 50, no. 2, pp. 179-195. 1972.
Freshwater smelts, and Atlantic herring, both shown to contain thiaminase, were fed to harp seals under a variety of experimental conditions. When thiamine was not administered, the seals developed thiamine deficiency which, in some cases, was fatal. [...medical stuff we don't care about...] Thiamine administered either i. m. or orally, during the course of deficiency, effected prompt recovery. Thereafter, seals maintained on herring required 25-33 mg thiamine/kg of ingesta, if the vitamin was consumed in the diet; if administered 2 hr before feeding, 35 mg/day was sufficient for normal maintenance.

So, it is possible to feed extra thiamine along with thiaminase-containing food and have the seals be OK, but you need more than if you give the thiamine supplement on an empty stomach (no thiaminase present in the gut).

[DrKate]

Last post for now, I promise! But I think this one's important.

Thiamine Content and Thiaminase Activity of ten Freshwater Stocks and One Marine Stock of Alewives
Fitzsimons, JD, et al.
Journal of Aquatic Animal Health vol. 17, no. 1, pp. 26-35. Mar 2005.
"We sampled alewives of uniform size (60-120 mm) during the summer of 1998 from the Gulf of St. Lawrence, seven of New York's Finger Lakes, one inland lake in Ontario, and two Great Lakes [...]. Thiaminase activity varied significantly among the 11 locations but was unrelated to thiamine concentration, which did not vary significantly. [...] Variation in alewife thiaminase activity has the potential to affect the extent of a thiamine deficiency associated with salmonines who feed on alewives as well as the viability of their offspring."

So all of the different populations had essentially the same amount of thiamine to start with, but there was wide variation in the amount of thiaminase they contained (as much as a four-fold difference as reported in the results). So all in all, there would be a big difference in how quickly thiamine was broken down after the alewives were killed, and therefore how thiamine-deficient they were as food.

We're not considering alewives as a feeder fish for garter snakes, but I think the important point is that different populations of the same species of fish can vary widely in the amount of thiaminase they contain. I think this is probably how we get situations like Jimyd (the "I have fed my garter goldfish for 13 years" thread) and Shannon having VERY different personal experiences with goldfish-fed garter snakes - they likely had feeder goldfish coming from different farms, with different diets and other environmental factors...

[Zephyr]

So, is the solution to the myths surrounding thiaminase-"filled" fish to feed the fish themselves a varied diet and increase the quality of their surroundings?
Seeing as the environmental and dietary conditions surrounding would be monitored by a concerned and caring hobbyist, wouldn't it therefore increase the quality of the fish and make them of edible quality to the snakes?

Also, something else I'd like to bring up on supposed "thiaminase-seizures."
Feeder fish from pet stores are, for the most part, kept in bait house (inadequate) conditions. It can be concluded, therefore, that they've also got a higher-than-normal parasite content. Could a parasite be the cause of these seizures, as opposed to the fish's thiaminase content?

[DrKate]

Well, I lied... One more. But this one is more directly relevant to feeding garters, since shiners do get sold as feeders:

Thiamine and Thiaminase Status in Forage Fish of Salmonines from Lake Michigan
Tillitt, DE, et al.
Journal of Aquatic Animal Health vol. 17, no. 1, pp. 26-35. Mar 2005.
"Thiaminase activity varied significantly among the forage species collected in Lake Michigan. The lowest thiaminase activities (12–35 pmol · g−1 · min−1) were measured in yellow perch, round goby, and bloater; there were no significant differences observed among these three species (Duncan's multiple-range test). The mean amount of thiaminase activity in these three species was near the limit of detection for the thiaminase assay (approximately 5 pmol · g−1 · min−1). Slightly greater then these three species was the mean thiaminase activity of ninespine stickleback at 85 pmol · g−1 · min−1 and deepwater sculpin at 172 pmol · g−1 · min−1. The amount of thiaminase activity observed in the deepwater sculpin and ninespine stickleback is still considered low, even though the concentrations were detectable. Thiaminase activity in alewife (mean = 4,280 pmol · g−1 · min−1) and rainbow smelt (mean = 2,640 pmol · g−1 · min−1) were significantly greater than the aforementioned species, but not significantly different from one another. Thiaminase activity in alewife was consistently greater than that observed in rainbow smelt at a given location or collection season, but the amount of variation in thiaminase measurements and among individuals within a location made these differences statistically not significant. The greatest amount of thiaminase activity detected in Lake Michigan forage fish species was measured in gizzard shad (31,800 pmol · g−1 · min−1) and spottail shiner (32,700 pmol · g−1 · min−1). These values were not different from one another but were significantly greater than the thiaminase activity of all of the other species examined."

That's a paragraph from the results. You don't even need to know what pmol x g-1 x min-1 means to see that spottail shiners have a whole lot more thiaminase than even the alewives - especially given that the lowest detectable amount is still way lower than even the alewife number. So... I wouldn't plan on relying on shiners as a staple diet item, anyway.

Anybody know if spottails specifically are the "shiners" sold as feeders and bait?

[DrKate]

So, is the solution to the myths surrounding thiaminase-"filled" fish to feed the fish themselves a varied diet and increase the quality of their surroundings?

Well, that one study says that the amount of thiaminase in alewives is unrelated to the amount of thiamine they contain. That last study I posted, on the forage species in the Great Lakes, found that thiaminase levels in some fish go UP with the amount of thiamine they contain (I didn't post that part). So I don't think that changing the husbandry and feeding of feeder fish is a 100% solution - they'd still contain thiaminase, but they may also contain more thiamine and might therefore be less deficient by the time they're digested. But you couldn't really know (unless you've got a thiamine/thiaminase test kit in your basement!) whether you had a safe feeder at the end of all that.

Could a parasite be the cause of these seizures, as opposed to the fish's thiaminase content?

Good point. Very generally, I know there are parasites that get into the brain, especially in non-adapted host species. But I don't know if there are any that could be transmitted from fish (or other feeders) to snakes. I'm not terribly hopeful that this has even been studied, but I'd love to know if anyone finds anything on it...

[Stefan-A]

(unless you've got a thiamine/thiaminase test kit in your basement!)

By the way, how is it actually tested?

[Zephyr]

Very generally, I know there are parasites that get into the brain, especially in non-adapted host species.

Generally the "rosy-reds" sold are albinos; I know it's not a major difference like two different species, however I'm sure there are some things that their genetics make them more susceptible to than their dark-pigmented brethren.
Goldfish definitely aren't native fish, so I'd assume that they'd have the highest likelihood of carrying pathogens.
Coincidentally... I don't think too many wild garters are consuming platies or guppies. Definitely mosquitofish, but not other species of livebearers.

[DrKate]

By the way, how is it actually tested?

Hee hee... Well, I just happened to see this when I looked up those other studies...

A Method for Measuring Total Thiaminase Activity in Fish Tissues
Zajicek, JL, et al.
Journal of Aquatic Animal Health vol. 17, no. 1, pp. 82-94. Mar 2005.
(Paraphrasing here, of course...)
1. Pulverize the fish you want to test and make an extract in buffer solution.
2. Add a known amount of radioactive thiamine and let stand 10 min.
3. Add ethyl acetate. Thiazole, one of the breakdown products of thiamine, is soluble in the organic solvent ethyl acetate while whole thiamine is not.
4. Measure the amount of radioactivity in the ethyl acetate. This tells you how much radioactive thiazole is there, from which you can calculate how much radioactive thiamine must have been broken down in 10 minutes, from which you can calculate how much thiaminase there must have been in your fish.

It seems that older assays were variants of this same general method (find a way to measure the amount of breakdown product created in a known time period). They just made some technical improvements that should give more accurate results.

EDIT: This is why if you look at any of these studies, they all talk about "thiaminase activity" - none of them is measuring the literal number of thiaminase enzyme molecules in the fish. What matters practically speaking is how much thiamine gets broken down between the time the fish dies and the time it gets to the predator's intestine, not how many enzyme molecules it takes to do that.