Why Studies Need Multiple Experiments, An Illustration Using Manic Rats And Beef Jerky

When we start out with a question in science, we need to break it down to its most fundamental components.

Let’s say we want to know the answer to “Do non-human animals have language?”.  First we need to specify what is meant by language.  Do we just mean verbal language, or are we including gestural languages like sign language?  Do we want to know about all animals, or just the most intelligent ones like chimps or dolphins?  Does the communication need grammar?  And so on, and so forth.

For every question we answer, a thousand smaller questions crop up.

For this reason, scientific research tends to be very niche.  A single study might attempt to answer one facet of one section of a broader question.

By doing this, we can learn more about that question in order to ask better questions.  One day, when enough of the question has been answered, we find ourselves with a theory.

Theories are the collections of observations we have made, that have been put together into a coherent structure.  We can use these to generate new questions and, more importantly, to make predictions about what the answers to those questions will be.

If the answer is what we predicted, we keep probing.  If it isn’t, then we have to adjust the theory.

That’s the basic premise of science.  Keep asking questions.

But not every study has to answer just one part of a broader question.  It is very common for a study to answer multiple parts.  To proceed down the chain of questions to some logical endpoint, then report how they got there.

This is what happened in a recent study about the link between nitrates and mania.

Mania is one half of bipolar disorder.  You may know bipolar disorder as the condition where a person’s mood swings rapidly from grand highs to deep lows.  Mania is the grand highs.  People with bipolar disorder who are manic experience great feelings of motivation and self-belief.  But they also experience delusions and in some cases aggression.

Nitrates on the other hand are a chemical that is added to dried cured meats and fish as part of the preservative process.  If you have ever eaten salami, prosciutto, or beef jerky, you’ve eaten nitrates.  This is generally seen to be a safe substance to add to food.

So why look at it?  And why look at mania?

One risk for bipolar disorder is the environment in which you grow up and live.  Part of that environment is the food you eat.

The question that the researchers were trying to answer was Is bipolar disorder linked to what foods are eaten?

To do this they conducted a first study.

Part 1 – Is food intake related to bipolar disorder?

The researchers interviewed a group of people at hospitals and health care facilities around the Baltimore area.  They were specifically looking for people who had recently been admitted for a manic episode.

Those people were asked a long series of questions about their diet.

“Have you ever eaten undercooked fish such as raw tuna?”

“… undercooked meat?”

“… dried meats?”

Here they found an association.  Among all those questions asked, as well as ones about their age, sex, race, and so on, dried meat was singled out.

Those people who were admitted for manic episodes ate more dried meats than the control group, a group of similar people who did not have manic episodes.

So the researchers had an answer.  They found an association between eating dried meats and experiencing mania.  They could have stopped there.  That’s an interesting finding after all.

But answering questions is addictive, once you’ve got one answer you need the next one.  Not to mention that journals are not very likely to just publish one finding by itself.

The researchers now knew that eating dried meats was linked with manic episodes.  But correlation does not equal causation.  It does imply it though.  To test whether eating dried meats did indeed increase the risk of mania, the researchers would have to do more experiments.

So they kept going.

Part 2 – Do rats show mania after eating dried meats?

Anyone who works in showbusiness will tell you – never work with children or animals.  In science that principle is often ignored.  Animal research is a cornerstone of scientific progress.  It allows researchers to do experiments that would be unethical or impractical on human participants.

Any discussion about animal research will of course encounter controversy, but that is a topic for another day.  For more information visit Understanding Animal Research, an organisation that provides information about animal research.

They separated two groups of normal rats.  One they gave normal rat chow, and the other had rat chow with rat-friendly beef jerky added into the mix.  The amount they were given was proportionally the same as a human eating one stick of jerky or one hot dog a day.

This was carried out for two weeks, with the rats living normal rat lives together.  The only difference between the two groups was whether or not they had the added jerky.

Then testing began.  The rats were taken from their home cage and put into a featureless room.  They were filmed, and the researchers observed how much they moved around.

This locomotor agitation is an important clue as to whether the rats are experiencing mania.  Agitation is a very common symptom during manic episodes in humans.

This part of the researcher’s question was answered.  Rats who had beef jerky added to their diet were more agitated in the testing room than control rats.  It appeared that dried meats did cause an increased risk of mania, or at least mania-like symptoms.

The researchers could have stopped there too.  They had found an association in humans, and had seen that this was a real relationship by studying it in rats.  Great.

But there were still more questions.

Part 3 – Are the nitrates in dried meats responsible for increased symptoms of mania in rats?

Imagine for a second you are a rat.  You have spent your whole life eating rat chow.  Dry, bland, same-textured rat chow.  And one day you are given beef jerky.  For two whole weeks you get beef jerky with your normal boring food.

Is it any wonder that these rats may be more excited?  Perhaps the increased agitation the researchers found in rats who ate beef jerky was just because their diet changed.

This is an important question.  Was it the beef jerky?  Or was it something in the beef jerky?

The best way to find out is to test it.

So the researchers commissioned the creation of a new beef jerky from a local company.  A jerky which had no nitrates.  In every other respect these two jerkies were identical.

Why nitrates?  In short, they have been implicated in many different disorders and diseases from cancer to asthma.  In long, you’ll have to read the paper.  For our purposes, it doesn’t terribly matter.

If the rats were just agitated because of the jerky, it should make no difference whether there were nitrates or not.  But if the nitrates were to blame, then only the rats who ate the nitrated beef jerky should show agitation.

To examine this, the researchers used the same behaviour observation method as before, with new rats.  What they found was that rats who ate nitrate-free beef jerky were no more agitated than the control rats who ate no jerky at all.  The rats with the nitrated beef jerky were still more agitated than the other two groups.

This was strong evidence that the nitrates in the jerky were responsible for increasing the manic symptoms being displayed by the rats.  By extension, nitrates in the dried meats eaten by the human participants in the hospitals are likely to have played a role in their own manic episodes.

This seems like a foregone conclusion now.  The researchers’ question about diet has been answered surely?  It looks like nitrates in dried meats cause an increase in manic symptoms in both humans in rats.

Well not quite.

There is a concept called double dissociability that is quite important when trying to show that two things are related.  Double dissociability refers to the ability to tell apart what effects are caused by what part of an experimental manipulation, like beef jerky and nitrates.

The researchers had successfully shown that beef jerky without nitrates did not cause increased agitation.  However, they had not shown that nitrates could have the same effect without beef jerky.

Although it seems very unlikely, it is possible that nitrates only cause increased agitation if eaten in dried meat products.  Perhaps if nitrates were eaten with cured fish or fresh meat, then there would be no effect.

To test this and round off the paper, the researchers would have to do one last bit of research.

Part 4 – Are nitrates alone causing increased manic symptoms in rats?

For this last part, the researchers gathered a new sample of rats who had not been exposed to any of the previous conditions.

These rats either ate the control diet of just rat chow, or one of two diets with chow and beef protein.  One of these diets had purified nitrate added to it, and the other did not.  By isolating the nitrate and not including dried meat, it becomes possible to see whether the nitrate alone is the cause or if there is something in dried meat that is also increasing agitation.

The researchers this time found that rats fed the nitrate diet showed increased agitation compared to the other, nitrate-free, diets.  However the increase was not as strong and did not last for as long either.

This suggested to them that nitrates did play a role, but that there was something else happening with nitrated dried meats that also increased the level of agitation.

This something else was having an interaction effect with the nitrates, making the effect of the nitrate stronger.  By itself it did not seem to have an effect, as the nitrate-free dried meat diet did not cause increased agitation.  But, combined with the nitrate, mania was stronger than if nitrate alone was added.

The researchers’ original question was now mostly answered.  Not completely, but mostly.

Diet does, it seems, have an effect on mania.  Dried meats appear to increase agitation, which is a major symptom of mania.  Nitrates in these dried meats were found to be the likely cause of this, but they did not explain the whole increase.


This experiment wasn’t perfect, but no experiment ever is.  And the researchers did look at other things like gut bioflora and neural firing changes.

What it does do is nicely illustrate why it is that scientific studies tend to do more than one experiment.  Every question leads to more questions.  It’s a matter of seeing how deep the rabbit hole goes, and finding a sensible place to stop.

After all, one paper doesn’t have to answer every question out there.  Provided it gives a good answer to the important question it started with, that’s good enough.  Other people can take up the questions that follow.  Often those other people are the researchers themselves mind you, but the questions are there.

And if you’re worrying about eating dried meats, I wouldn’t get too concerned.  It’s still early days on that research front.  I’d maybe think twice about a dodgy hot dog though.

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