The overall goal of the following procedure is to extract and measure the stress hormone cortisol from human or monkey hair. This is accomplished by first washing the sample in isopropanol to remove any excreted cortisol from the external surface of the hair. In the second step, the sample is ground into a fine powder to break up the hair matrix and to increase the surface area for extraction.
The cortisol present within the interior of the hair shaft is then extracted with methanol and the same cortisol content is quantified using an enzyme immunoassay. Ultimately, the level of cortisol deposition over time within the hair sample can be evaluated. The main advantage of this technique over existing methods like point measurements of cortisol in blood or saliva, is that hair cortisol provides a measure of chronic activity of the hypothalamic pituitary adrenal cortical system.
This method could help answer key questions in the stress field, such as what is the relationship between stress and psychopathology in humans, or what are the effects of different rearing conditions on stress physiology in non-human primates? The implications of this technique extend toward the therapy of disorders such as post-traumatic stress disorder as hair. Cortisol can help determine whether endocrine dysfunction associated with this disorder has been ameliorated by the treatment.
Begin by placing each hair sample into individual 15 milliliter screw cap polypropylene centrifuge tubes. Then wash the samples two times in five milliliters of HPLC grade isopropanol by repeated inversion on a rotator for three minutes. Each time decant the isopropanol into a waste container after each wash, taking care not to lose any of the sample to grind.
Large samples. Place up to 250 milligrams of dried hair into a 10 milliliter stainless steel grinding jar, along with a 12 millimeter stainless steel grinding ball. Use a ball mill to grind the sample at 25 hertz.
After six minutes, weigh up to 50 milligrams of powdered hair on an analytical balance. Then transfer the sample to a clean two milliliter polypropylene micro centrifuge tube for subsequent cortisol extraction. For cortisol extraction from large samples, add one milliliter of HPLC grade methanol to the micro centrifuge tube containing the powdered sample.
Then cap the tube and incubate the sample for 18 to 24 hours at room temperature with constant inversion on a rotator. Next, spin down the powdered hair for one minute at 13, 000 gs, and then taking care not to disturb the pellet transfer. Point six milliliters of the supernatant into a clean 1.5 milliliter micro centrifuge tube to grind small samples.
Place up to 60 milligrams of hair into a pre weigh two milliliter micro centrifuge tube reinforced for bead beating. Reigh the vial to obtain the sample weight. Then add three 3.2 millimeter chrome steel beads to each vial and grind the sample in a bead beater after two minutes.
If visual inspection reveals that the sample is insufficiently pulverized, perform additional grinding for 30 to 60 more seconds to extract the cortisol. Next, add 1.5 milliliters of HPLC grade methanol to the micro centrifuge tube containing the powdered sample. Then cap the tube and incubate the sample at room temperature with constant inversion on a rotator.
After 18 to 24 hours, centrifuge the tubes for five minutes at 6, 500 Gs at room temperature. Then taking care not to disturb the pellet of powdered hair. Transfer one milliliter of the supernatant into a clean 1.5 milliliter micro centrifuge tube to remove the rest of the solvent from the hair samples dry down the methanol using a vacuum evaporator, the methanol vapor is captured using a cold trap.
Finally, reconstitute the cortisol extract in an appropriate volume of enzyme immunoassay buffer, and then assay the sample immediately using a high sensitivity enzyme immunoassay kit. According to the manufacturer's recommendations for saliva samples, this first figure shows the printout from a representative set of adult male and female human hair samples processed using the small sample grinding and extraction method. The S one to S six wells contain the cortisol standards ranging from 0.0 12 micrograms per deciliter to 3.0 micrograms per deciliter.
With the S seven well containing the zero cortisol control. The B one wells are the non-specific binding wells that lack anti cortisol antibody as the software automatically subtracts the average non-specific binding optical density value from all the other optical density readings in the C one and C two wells. The high and low cortisol calibrators provided by the manufacturer are shown respectively.
The T one wells contain the quality control sample. Wells T two to T 38 contain the test samples from C one to T 38. The upper value in each cell of the array is the measured optical density value from the corresponding well, and the lower value in the left hand cell of the pair is the cortisol value in micrograms per deciliter.
Calculate from the mean optical density value. Note that in this experiment, samples T nine and T 31 yielded readings above the highest cortisol standard. As a result, both samples were later diluted fourfold in assay diluent and reanalyzed.
The reanalyzed values were used after correction for the dilution factor. Computer software can then be used to generate the data output and to fit a four parameter sigmoidal curve to the cortisol standards. For example, in this graph, the output of the cortisol assay with the standards from the enzyme immunoassay kit displayed as the individual data points is shown Following this procedure, other methods like repeated measurement of cortisol and bladder, saliva can be performed to answer additional questions such as whether differences in hair cortisol are related to changes in the circadian rhythm of cortisol Secretion after its development.
This technique paved the way for researchers in the field of wildlife conservation to explore the influence of factors such as climate change and pollution burden in polar bears.
