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Perhaps one of the more astounding scientific discoveries that I have been involved in is the discovery of the salivary fatigue biomarker. 

This story begins 10 years ago when the U.S. Army determined that there was a significant issue at hand concerning fatigue in the Iraq and Afghanistan wars. Too many soldiers had to work for long hours, carry heavy ammunition and armor, be subjected to extreme heat, and yet manage to maintain constant vigilance.

Not surprisingly, Army leadership became worried about their vulnerability to failures caused by fatigue, and they were curious if better analytics could help solve the issue.

The analytics solution had many components, and one part was getting objective measures of fatigue during combat. The idea was that if fatigue could be measured objectively, then commanding officers could distinguish between which soldiers could be safely deployed and which ones needed to stay home and recover.

The Start of a Long Journey

I wrote a grant proposal to the U.S. Army, proposing that we could identify substances in the body that would be associated with the fatigue state. The proposition was not totally outrageous – there are many physiological adjustments made in response to various stresses. For example, when we experience fear, our bodies initiate a response.

If you get scared enough, your body can release large amounts of neurotransmitters into your bloodstream. This causes an almost instantaneous increase in vigilance, strength, and metabolism, and, most importantly, an almost complete reversal of the fatigue state.

This is called the “fight-or-flight” response, and it can be objectively measured in a number of ways. However, measuring fatigue in a purely objective matter has proven to be a difficult challenge.

The Idea to Collect Saliva Samples

Another interesting concept was the idea of collecting saliva samples instead of blood samples for measuring fatigue markers. From a military operations standpoint, this was a great idea. Obtaining blood samples from a soldier during combat is difficult (to say the least), but in comparison, getting saliva samples would be a far more simple task.

There are a couple of problems with saliva, however. First, the medical community has been focused on blood since the beginning of time, so research on saliva is sparse in comparison to the abundance of research on blood.

Second, mice produce only a small amount of saliva—and getting a saliva sample from a mouse presents difficulties. This is important to consider, as so much biomedical research, including behavioral research in the United States, is conducted on mice. If you’re doing an experimental study on mice, you will typically collect and evaluate blood samples.

Vials of Blood. Vials of blood taken in the course of patient care at the National Institutes of Health Clinical Center in Bethesda, Maryland. Test tubes. Blood test. Creator: Daniel SoneVials of blood samples taken from patients. Research on blood is much more robust than the current body of research on saliva, but saliva samples present their own benefits in terms of easier and less invasive collection procedures.

What About Cell Lines?

The third problem is the lack of useful cell lines in salivary glands. You might be wondering, what is a cell line? In short, cell lines are immortal cells that grow forever in a laboratory. In most instances, cell lines come from cancerous tumors in humans. In biosciences, cell line experiments are often done before mouse experiments.

Cell line-based experiments can often provide tremendous insight into how things work in the human body. These experiments allow researchers to easily manipulate the genome, send cells chemical messages, and study exactly how cells respond on a variety of levels. Yet while there are cell lines derived from many different organs, there are no cell lines available that mimic the function of the salivary glands.

In summary, compared to working with blood, researchers face many challenges when working with saliva. In my opinion, these disadvantages are more than offset by the ease with which saliva samples can be taken from people in contexts important to us – in combat, at a sporting/competitive event, at home, etc. The ability to measure fatigue from saliva samples would open up numerous possibilities for fatigue testing applications.

Getting Started with the Fatigue Experiment

So now the question is, how do you do a fatigue experiment? There are several ways to go about it, but we chose to focus on the one that we thought would have the highest relevance to the U.S. Army: endurance exercises.

In collaboration with a university, we asked eight individuals to participate in high levels of physical exertion (treadmill jogging and cycling) in a laboratory for eight hours. We collected saliva samples before, during, and after the experiment and looked for differences.

We assumed that after 8 hours of training, these subjects would be tired; our assumption was correct. The markers of fatigue we were looking for include molecular entities that appear after 8 hours of exercise, but not in an initial sample from before the start of exercise.

A photo of a group of endurance bikers during a race.
A group of bikers during a race. An endurance sport like biking is defined by the ability to sustain a submaximal work rate for an extended period.

The Experimental Procedure

We took various measurements throughout this experiment. We measured blood, saliva, and exhaled breath, additionally taking surveys and muscle biopsies. For the sake of simplicity, I won’t describe the technical details here, but in the end, we decided to pursue two paths when searching for fatigue biomarkers.

Our first step was to examine the “usual suspects” – things people have claimed indicate fatigue (for years and, in some cases, decades). As was expected, many of these changed during this very fatiguing challenge. The problem with the “usual suspects” is that they are poor indicators of fatigue. This is mainly because the extent of change observed is relatively modest compared to the seemingly random variation in levels that can be detected.

This is a very important concept because, ideally, you want to measure something that will unequivocally signal “fatigue”. The bottom line is that the “usual suspects” may tell us something about fatigue, but the message is far from clear. The information just isn’t very useful in helping people understand how they can better manage their own fatigue.

Next, we examined all low-molecular-weight proteins in saliva. We looked for those molecules that were significantly increased or decreased after eight hours of exercise. While this approach may be a bit more complicated than the “usual suspects” method, it has the advantage of potentially discovering something novel.

Compared to blood and urine, saliva contains many small proteins called peptides. We examined 5,000 or so peptides and found that most were unchanged by exercise. However, a few peptides did change. We searched for proteins that both showed the most change and were reasonably abundant in saliva. What we found was truly startling.

So, What Did We Find?

We discovered that the ratio of two small peptides was decreased by about 1,000-fold in fatigue. We also found that the variation associated with this measure was relatively modest, meaning that the uncertainty was very low. By measuring these two peptides, we were able to make a truly objective measurement of fatigue!

Using this approach, we knew the molecular weight and several important chemical properties of the molecule of interest, but we didn’t know much else. And, to succeed in the world of proteins, you need to understand the sequence of amino acids and their modifications.

There was a lot of speculation chatter, and even a few bets were made. The most promising theories were that we had discovered a neuropeptide from the brain or some early inflammation marker.

In order for us to identify the sequence, we had to work for another couple of months to isolate the molecule from saliva. We also collaborated with another laboratory that owned a very expensive machine that could tell us the exact molecular identity.

The Next Steps

To validate our findings, we had to synthesize the molecule and compare it to what we thought we had discovered. This is how good science is done, and I am proud of our team’s work. When we finally got the results, we were very surprised.

We found that both of our proteins of interest turned out to be unique to the salivary gland itself, not neuropeptides or inflammation signals! By sleuthing through databases, it became absolutely certain that both small proteins were part of a larger protein called Proline-Rich Protein (or PRP).

PRP and associated peptides make up about 37% of all the proteins found in saliva. They are only produced in the salivary gland, making them the most abundant proteins found in saliva.

What is PRP & What Does It Do?

Interestingly, nobody seems to know everything that they do. They might be involved in keeping our gums and teeth healthy. More importantly, they don’t closely resemble anything else, though some scientists have speculated that they have a structure similar to wheat gluten.

Despite all of this, it’s still surprising that there are only a few proteins that don’t fit into any of the other categories, essentially standing alone. Some mammals (such as primates and select rodents) have the gene to produce PRP, but it’s not clear what they produce – or if it’s produced in abundance. So… why do we make it?

A photo of a brown mouse outdoors.Some mammals, such as rodents like this mouse, have the gene to produce PRP. However, the scale of production and purpose in other species is not well known.  


Protein In Our Saliva

To make things even stranger, we produce a lot of PRP – our bodies make and swallow about a gallon of saliva each day. Of that, quantities in grams of PRP protein are made and processed in the gastrointestinal tract every single day.

Making protein is a very energy-intensive process. Protein is expensive to make (in terms of energy), and even if you recycle most of it, your body still has to spend a lot of energy doing so. And now, in addition to asking “why do we make it?” the question becomes: why do we make so much?

Peptides & Proteases

As if our story wasn’t interesting enough, the parent protein PRP is huge, weighing in at about 42,000 molecular weight units. Each of the peptides that comprise the fatigue biomarker weighs about 700 molecular weight units (or 8 amino acids). The PRP parent is processed by enzymes, called proteases, that specifically cleave – or split – the larger protein into smaller, individual peptides.

Proteases have two features: 

  1. the ability to recognize, bind to, and fix a protein
  2. functioning as a “cutting machine”

Some proteases are relatively non-specific and will cut up many types of proteins, while others are very specific, targeting a very precise location on a large protein.

With regards to the fatigue biomarker peptides, it seems likely that the proteases are very specific given that they lead to such a dramatic difference in abundance of the two peptides that make up the fatigue biomarker.

Together, these results lead me to conclude that the production of the fatigue peptides is part of a very specific and highly controlled system related to fatigue. In a highly fatigued state, one peptide virtually disappears. But what could any of this have to do with fatigue? Well, the data that we have accumulated over several years all point in one direction: energy.

Diving into Sleep Deprivation

Consider this: sleep deprivation and intense physical exertion can both cause fatigue, but more importantly, they can alter the abundance of fatigue peptides. Anecdotal data suggests that the quality of a diet can also alter this composition. 

All of them have one thing in common: a state of uncertainty and high stress that would most likely benefit from more energy. These peptides are eventually swallowed and then processed through the gastrointestinal tract. This makes it seem possible that they perform a function there that may something having to do with the gut microbiome.

An illustration of a human silhouette adorned with larger-than-life microbes. What does the gut microbiome have to do with fatigue? It is hypothesized to be part of a larger signaling network involving fatigue biomarkers.

The Next Frontier: The Gut, Peptides, & Fatigue

We know that the gut microbiome is composed of bacteria and bacteriophages, as well as human immune cells and other cells that line the gastrointestinal tract. There is a growing awareness that the gut microbiome plays an important role in regulating energy recovery from food.

What if the changing levels of the peptides associated with fatigue biomarkers are a sign that we should be recovering as much energy as possible from the food that’s already in our bodies? This seems to fit pretty well with what we know. However, this is only a hypothesis that is yet unproven.

The content from this article was adapted from a previous series of articles written by John Kalns entitled The Fatigue Biomarker Story, and has been edited for clarity and formatting.

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