Why do other measurements of stress suck worse than cortisol?

After an overwhelming number of requests (2) for a sequel to my post Why cortisol sucks as a measurement of stress, I am obliging. The fact that I am in the middle of writing this particular section of my thesis and need some high-level perspective on it might also have something to do with it. So: why do other measurements of stress suck worse than cortisol?

When I left you, you were trying to design a study of stress in hospitalized dogs using cortisol as your marker of psychological distress. You were confounded by the fact that cortisol measures both psychological and physiological distress, and that it varies a lot between individuals. I haven’t been around to keep an eye on you lately, so you have started investigating other approaches to measuring stress other than cortisol.

Cortisol is a messenger used by the HPA (hypothalamic-pituitary-adrenal) axis, for the brain to send a message about stress levels out to the body, for the body to pass that message along to the organs that need to change their operations as a result, and for the body to then report back to the brain that the message has been received, so the brain can stop yelling about it. There are multiple levels in this axis; cortisol comes from the bottom-most level, the adrenals. Why not go up one level, to the pituitary? It is actually in the brain, so it is closer to the source of the message and might be less distorted by the game of telephone.

The hormone that the pituitary gland releases as part of the HPA axis is ACTH (adrenocorticotropic hormone, or “the hormone that makes the adrenal cortex change”). ACTH causes cortisol release. Why don’t you measure ACTH release directly? Unfortunately, ACTH can only be measured in the blood; it doesn’t get into the saliva. (Or urine, hair, or feces, three other places you can go to get an estimate of cortisol levels.) The owners of your hospitalized dogs aren’t going to be happy if you tell them you need to draw blood from their dogs for your study. And remember, you’d have to draw the blood pretty quickly in order to get it before the brain mounted a stress response as a result of having a needle stuck into the body. Cortisol levels change in under three minutes. I don’t actually know how long it takes ACTH levels to change, but I will hazard a guess that since they are farther up the telephone chain, they change faster.

What about farther down the chain? CBG (corticosteroid binding globulin, a.k.a. transcortin) is a protein that carries cortisol around in the blood. The body uses CBG as a way of regulating the stress response. When there is less CBG, cortisol is more able to jump inside cells and do its work. OK, no one actually uses CBG to measure stress levels, because we have no real idea how it works. But it is a very cool system that I’m really curious about. And stress researchers would do well to remember that it is there. If the dogs you are studying are very sick, they might not be able to make as much CBG as a healthy dog would, and that would affect their cortisol levels.

That pretty much exhausts using the HPA. Luckily there is an entire second axis for you to mine: the SAM (sympatho-adrenomedullary) axis. This is the series of chemicals that regulate the well-known “fight or flight” response. This particular game of telephone includes adrenaline (epinephrine), the effects of which which many people enjoy abusing when they go on roller coasters. This axis works much more quickly than the HPA. If you hear a sudden loud noise, you will get an adrenaline rush within a second. So you can try to measure adrenaline levels in the blood, but there is just no way you will be able to get the blood out fast enough to not have the stress of the needle (damn needle) affecting them. If you had a very controlled population of animals, with catheters already placed that they were used to, so that you could draw out blood without stressing them, that might work, assuming you could catch the animals without stress. (Catch a mouse without stressing it: difficult. Catch a dog without stressing it: actually, when I went into the runs with the hospitalized dogs I was studying, they definitely experienced eustress, or happy stress.)

You can also measure adrenaline levels in pee! This turns out not to be useful, though. Adrenaline levels go up and down, as we’ve said, very quickly, in response to individual stressors. Pee collects all those changes and averages them out over however many hours (say six). So this approach is definitely not good for measuring responses to specific stressors, like a sudden loud noise. It might be better at measuring something longer term (hey, like the response to being in a hospital!) but initial studies haven’t shown it to work very well at that, either. Adrenaline is just the most interesting when you can map it as it goes up and down, not when you have to look at an average and guess about what was smoothed out.

What about the other end of the SAM? When you get an adrenaline rush, you have some physical changes. Among many other things, your heart rate gets faster. Can you measure that? Well, again, good luck measuring that in a dog without having the excitement of interacting with a human confound your measurement! And heart rate is very sensitive to physical changes; you might be measuring whether the dog is standing up versus lying down, rather than its level of distress.

It turns out that what is a better way to measure physiologic changes from SAM activation is heart rate variability. Your heart rate normally speeds up a little when you breathe in, and slows down a little when you breathe out. (I actually did notice this in a dog once, in a lab where I was supposed to be learning how to find abnormal heart rhythms, and I had to call a vet over to ask if it was actually normal, because it sounded so weird once I noticed it.) When you are stressed (physically or psychologically), this variability goes away. This is not a bad way to measure stress, but you can’t measure it with a stethoscope; you have to hook up equipment to the dog in the form of a little vest with a monitor attached. This is expensive (too expensive for you to use, because your project is on a shoestring budget!). You would also have to get the dog used to the vest, so that you were sure you wouldn’t be measuring stress from having clothing on when the dog is used to being naked. It is therefore not a good measurement for hospitalized dogs on their first day in the hospital, but it is a good measurement for some studies. It’s best when used in conjunction with cortisol, so that the two measurements can catch each other’s mistakes.

That uses up the SAM, but there is a system that is the opposite of the SAM. When your body is not in “fight or flight” mode, it is in “rest and digest” mode. This mode is regulated by the parasympathetic branch of the ANS (autonomic nervous system). (The SAM is the sympathetic branch of the ANS.) Can you measure parasympathetic activity? It should increase when stress decreases, and vice versa. It turns out that when your body is thinking it’s time to rest and digest, it releases a digestive protein into your saliva, known as α-amylase. This protein is useful for pre-digesting carbohydrates. More α-amylase suggests less stress. And it’s even in the saliva, so it can be measured non-invasively! You are very excited until you find a paper from the 1950s (I am not kidding) which is the last time anyone bothered to look for α-amylase in dog saliva. Dogs don’t make it. Because they are not meant to eat lots of carbs? Oh wait, this isn’t a post about nutrition.

(For those of you who say “OK, but what about measuring stress via α-amylase in humans?” — I didn’t delve any deeper into this one after I learned it wasn’t useful in dogs. My guess is that it suffers from similar problems to measuring cortisol: it measures more than just [lack of] distress. It also has been less widely used than cortisol, so we understand its pitfalls less. This would be another good measurement to use as a complement to measuring cortisol. If you want to use it in humans, read lots studies that have used it before you commit.)

So much for the ANS. But you know that increases in stress cause decreases in parts of the immune system. In fact, that’s partly why we care about stress in hospitalized dogs — stressed dogs may not heal as quickly or as well. Can we measure the immune system?

We can. Your saliva normally contains a kind of antibody called IgA. This presumably provides a first line of defense against the bugs on your food. When you are stressed, you make less of it. (At a guess, this is because when you’re running from a lion, you’re not likely to be eating. You’re more likely to be getting bitten, so your immune system needs to focus on defenses against open wounds instead of microbes in food.) Salivary IgA is known as “sIgA.” Can you measure that in dogs? You can, and it is being fairly widely used in humans, in fact. Only some initial work has been done on it in dogs, though. It seems to be prey to some of the same issues cortisol is — varying regularly throughout the day, varying irregularly between individuals — so it’s not yet clear if it’s really a better option. It might be a good way to go for a long term project. For something short, though, it might be better to stick with what is well-understood.

Are there any other ways to measure immune system function as it relates to stress? As I said, your immune system reorients when it thinks you’re running from a lion, to protect against open wounds. It does this in part by packing the blood full of a kind of white blood cell called a neutrophil. Neuts are the first line of defense against microbes coming in through open wounds. You can measure their ratio to another kind of white blood cell, a lymphocyte, to measure stress. A greater N : L (neutrophil : lymphocyte) ratio implies greater stress levels. In some ways, this is a really great measure of stress, because it takes a little while — an hour or so — for the N : L ratio to change after a stressor. So when dogs first come in to the hospital, if you can get blood right away, you could actually measure their unstressed baseline. A later blood sample could provide a comparison. Then you could ignore all that annoying individual variability, because you would be measuring the difference pre- and post-stressor in the same individual. I would have loved to have use this measurement.

But, as always, good luck getting an owner to consent to not one but two unnecessary blood draws. I am not sure I would have felt good about adding that much stress to an already stressed dog’s hospital visit, either. For a different kind of study, this might be a really good option, though as always, it measures the effects of multiple systems, so there is going to be some extra variability to account for.

And that is why, though cortisol is a really appalling way to try to measure stress (looking at my salivary cortisol data right now, I keep saying “why does anyone use this hormone?!”), it is still the most widely used approach. As we learn more about how all these systems interact, it is possible that some day we will develop a method of taking multiple kinds of measurements and basically triangulating distress. Or maybe we’ll develop hand-held fMRI scanners and be able to directly measure activation of specific parts of the brain. For now, we are stuck with spit.