Challenges of fecal hormone analyses (Round 2): finally in Seattle!

By Leila Lemos, Ph.D. Student, Department of Fisheries and Wildlife, OSU

In a previous blog of mine, you could read about the challenges I have been facing while I am learning to analyze the hormone content in fecal samples of gray whales (Eschrichtius robustus). New challenges appeared along the way over the last month, while I was doing my training at the Seattle Aquarium (Fig. 1).

Figure 1: View of the Seattle Aquarium.


My training lasted a week and I am truly grateful to the energy and time our collaborators Shawn Larson (research coordinator), Amy Green and Angela Smith (laboratory technicians) contributed. They accompanied me throughout my training to ensure I would be able to conduct hormonal analysis in the future, and to handle possible problems along the way.

The first step was weighing all of the fecal samples (Fig. 2A). Subsequently, the samples were transferred to appropriate glass tubes (Figs. 2B & 2C) for the next laboratorial step.

Figure 2: Analytical processes: (A) Sample weighing; (B) Transference of the sample to a glass tube; (C) Result from the performed steps.


The second conducted step was the hormone extraction. The extraction began with the addition of an organic solvent, called methanol (CH3OH), to the sample tubes (Fig. 3A & 3B). Hormones leach out from the samples and dissolve in the methanol, due to their affinity for this polar solvent.

Tubes were then placed on a plate shaker (Fig. 3C) for 30 minutes, which is used to mix the substances, in order extract the hormones from the fecal samples. The next step was to place the tubes in a centrifuge (Fig. 3D) for 20 minutes. The centrifuge uses the sedimentation principle, causing denser substances or particles to settle to the bottom of the tube, while the less dense substances rise to the top.

Figure 3: Analytical processes: (A) Methanol addition; (B) Sample + methanol; (C) Plate shaker; (D) Centrifuge.


After this process, the two different densities were separated: the high-density particles of the feces were in the bottom of the tube, while the methanol containing the extracted hormones was at the top. The top phase (methanol + hormones) was then pipetted into a different tube (Fig. 4A). The solvent was then evaporated, by using an air dryer apparatus (Fig. 4B), with only the hormones remaining in the tube.

The third performed step was dilution. A specific amount of water, measured in correlation with sample weight and to the amount of the methanol mixed with each sample, was added to each tube (Fig. 4C). Since the hormones were concentrated in the methanol, the readings would exceed the measurement limits of the equipment (plate reader). Thus, in order to prepare the extracts for the immunoassays, different dilutions were made.

Figure 4: Analytical processes: (A) Methanol transference; (B) Methanol drying; (C) Water addition.


The fourth and final step was to finally conduct the assays. Each assay kit is specific to the hormone to be analyzed with specified instructions for each kit. Since we were analyzing four different hormones (cortisol, testosterone, progesterone, and triiodothyronine – T3) we followed four different processes accordingly.

First, a table was filled with the identification numbers of the samples to be analyzed in that specific kit (Fig. 5A). The kit (Fig. 5B) includes the plate reader and several solutions that are used in the process to prepare standard curves, to initiate or stop chemical reactions, among other functions.

A standard curve, also known as calibration curve, is a common procedure in laboratory analysis for determining the concentration of an element in an unknown sample. The concentration of the element is determined by comparison with a set of standard samples of known concentration.

The plate contains several wells (Fig. 5C & 5D), which are filled with the samples and/or these other solutions. When the plate is ready, (Fig.5D) it is carried to the microplate reader that measures the intensity of the color of each of the wells. The intensity of the color is inversely proportional to the concentration of the hormone in both the standards and the samples.

Figure 5: (A) Filling the assay table with the samples to be analyzed; (B) Assay kit to be used; (C) Preparation of the plate; (D) Plate ready to be read.


Since this is the first fecal hormone analysis being performed in gray whales, a validation process of the method is required. Two different tests (parallelism and accuracy) were performed with a pool of three different samples. Parallelism tests that the assay is measuring the antigen (hormone) of interest and also identifies the most appropriate dilution factor to be used for the samples. Accuracy tests that the assay measurement of hormone concentration corresponds to the true concentration of the sample (Brown et al. 2005).

This validation process only needs to be done once. Once good parallelism and accuracy results are obtained, and we have identified the correct dilution factor and approximate concentration of the samples, the samples are ready to be analyzed. Below you can see examples of a good parallelism test (parallel displacement; Fig. 6) and bad parallelism tests (Fig. 7) that indicate no displacement, low concentration or non-parallel displacement; and a good accuracy test (Fig. 8).

Figure 6: Example of a good parallelism test. The dark blue line indicates the standard curve; the pink line indicates a good parallelism test, showing a parallel displacement; and the ratios in black indicate the dilution factors.
Source: Brown et al. (2005)


Figure 7: Examples of bad parallelism tests. The dark blue line indicates the standard curve; the light blue line is an example of no displacement; the pink line is an example of low concentration of the sample; and the green line is an example of non-parallel displacement.
Source: Brown et al. (2005)


Figure 8: Example of a good accuracy test while analyzing hormone levels of pregnanediol glucuronide (Pdg) in elephant urine. The graph shows good linearity (R2 of 0.9986) and would allow for accurate concentration calculations.
Source: Brown et al. (2005)


After the validation tests returned reliable results, the samples were also analyzed. However, many complications were encountered during the assay preparations and important lessons were learned that I know will allow this work to proceed more smoothly and quickly in the future. For instance, I now know to try to buy assay kits of the same brand, and to be extremely careful while reading the manual of the process to be performed with the assay kit. With practice over the coming years, my goal is to master these assay preparations.

Now, the next step will be to analyze all of the results obtained in these analyses and start linking the multiple variables we have from each individual, such as age, sex and body condition. The results of this analysis will lead to a better understanding of how reproductive and stress hormones vary in gray whales, and also link these hormone variations to nutritional status and noise events, one of my PhD research goals.


Cited Literature:

Brown J, Walker S and Steinman K. 2005. Endocrine manual for reproductive assessment of domestic and non-domestic species. Smithsonian’s National Zoological Park, Conservation and Research Center, Virginia 1-69.