Dealing with Variation in Hormone Metabolite Measurements: A Tale of Poop

Post provided by EVE DAVIDIAN and SARAH BENHAIEM (DEPARTMENT OF EVOLUTIONARY ECOLOGY, IZW, BERLIN)

On the Art of Collecting Faeces

Sarah Benhaiem waiting for a faecal sample from a spotted hyena in the Serengeti National Park.©Sarah Benhaiem

Sarah Benhaiem waiting for a faecal sample from a spotted hyena in the Serengeti National Park.©Sarah Benhaiem

Whether you are a laboratory or a field scientist, you have to be willing to get your hands dirty from time to time for the good of science. Sarah and I took that literally and spent a large part of our respective PhD projects handling faeces of free-ranging spotted hyenas from the Serengeti National Park and the Ngorongoro Crater, Tanzania.

Though faeces often are underrated, they are highly valuable material to work with because they conceal the most secret details about an animal’s social and sexual life. But having the privilege of holding a still-steaming poop is something you have to earn!

Becoming a professional poop hunter first requires patience; sometimes waiting long hours in the hope that an animal eventually gets up and delivers the precious goods. It also requires getting familiar with the species’ body language to recognize the signs of an animal that is about to ‘do it’ and be able to react fast. When dealing with shy, wild or potentially dangerous animals, you further have to be discrete and cautious when collecting a sample. This can be quite challenging with spotted hyenas because they’re curious little rascals. You can never completely rule out the possibility of a fuzzy snout popping out from behind the car, wondering what kind of goodie you found on the ground.

People too, can often be confused by what you’re doing. Once, a car full of tourists stopped next to me as I was crouching to collect a fresh sample on the side of a road in the Ngorongoro Crater. The passengers seemed rather puzzled and one of them threw a sarcastic: “Nice job!”

“Well, somebody has to clean,” I replied.

While many would agree that it is rather natural to get attached to your study animal, you may be considered a bit of a weirdo if you also adore what they leave behind. But faeces, urine, and other excreta contain hormone metabolites (the by-product of hormone degradation) that can provide very valuable information on the health, level of stress, reproductive state or behaviour of an animal without interfering with its life.

The Art of Reading an Animal’s Past (or Future) from its Faeces

Biochemical reactions involved in an enzyme immunoassay and quantification of hormone metabolite concentrations. © Eve Davidian & Sarah Benhaiem

Biochemical reactions involved in an enzyme immunoassay and quantification of hormone metabolite concentrations. © Eve Davidian & Sarah Benhaiem (click to enlarge)

Enzyme immunoassays constitute a widespread method to quantify the concentration of hormone metabolites contained in excrement. The principle of an enzyme immunoassay is straightforward: the hormone metabolites contained in a sample compete with a known amount of tracer – the mother-hormone conjugated with an enzyme – for the binding sites of an antibody specific to the hormone of interest. The tracer generates a visible response that is proportional to the amount of tracer bound to the antibody. The metabolite concentration in a sample is then estimated by relating the measured response to a calibrated dose–response curve generated by standards of known hormone concentration.

The Downside of Enzyme Immunoassays

Well, we did not get that one! Good old male "Lampone" doing it in a pond. © Eve Davidian

Well, we did not get that one! Good old male “Lampone” doing it in a pond.
© Eve Davidian

No method is perfect though and dealing with faecal samples and enzyme immunoassays also has its downside. Because the chemical structure of hormone metabolites is usually unknown, working with excrements requires conducting additional validation experiments (unlike studies based on plasma hormones). You need to test whether the immunoassay detects the metabolites of the hormone of interest (‘physiological’ validation), and verify that it is sensitive enough to detect natural fluctuations in concentration of hormone metabolites (‘biological’ validation).

Even when your immunoassay passes these validation steps with honours, you have to keep an eye on its performance to make sure it provides repeatable, precise and comparable measurements. This is the tricky bit. The binding reactions involved in immunoassays are sensitive to variations in the laboratory environment, like room temperature or light exposure. Any variation can affect your measurements and their comparability if samples were analyzed during different periods of time. Previously, the only solution in cases like this was to discard your measurements and re-analyze all your samples. This is very costly and time-consuming when dealing with several hundreds of samples though, and may not even be possible if some samples are no longer available.

A New Method to Standardize Measurements from Large Data Sets

Eve Davidian collecting a faecal sample from a spotted hyena in the Ngorongoro Crater. © Oliver Höner

Eve Davidian collecting a faecal sample from a spotted hyena in the Ngorongoro Crater.
© Oliver Höner

In our paper published in Methods in Ecology and Evolution, we provided an alternative to the re-analysis of complete sample sets. With our colleagues from the Leibniz Institute for Zoo and Wildlife Research in Berlin, we developed a handy method to standardize hormone metabolite concentrations based on the re-analysis of only a small subset of samples.

Using over 450 faecal samples from spotted hyenas collected and analyzed (for cortisol metabolites) over several years, we illustrate the potential problems associated with variation in metabolite measurements. Our article provides simple and flexible tools to identify groups of measurements that require standardization and to estimate the minimum number of samples to re-analyze to obtain a reliable standardization.

This method should be particularly useful for long-term studies that deal with large data sets and international collaborative projects that share the laboratory workload between different facilities and are likely to experience some variation in enzyme immunoassay accuracy.

Our method is simple, cheap and reliable and enables the standardization of measurements from samples that are no longer available. Plus it can be applied to any species and sample type, too! Think about it next time you have to clean after your beloved pet!

To find out more about our standardization method, read Determining hormone metabolite concentrations when enzyme immunoassay accuracy varies over time. Our paper contains detailed R scripts to apply each step of the method.

If you are curious to know more about spotted hyenas and our project, visit the Hyena Project – Ngorongoro Crater website.

Advertisements

One thought on “Dealing with Variation in Hormone Metabolite Measurements: A Tale of Poop

  1. Pingback: Hormone assays in the lab this Fall | The Lizard Log

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s