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Activity of the hypothalamic-pituitary-adrenocortical (HPA) axis is commonly assessed by measuring glucocorticoids such as cortisol (CORT). For many years, CORT was obtained primarily from blood plasma or urine, whereas later approaches added saliva and feces for noninvasive monitoring of HPA functioning. Despite the value of all these sample matrices for answering many research questions, they remain limited in the temporal range of assessment. Plasma and saliva are point samples that vary as a function of circadian rhythmicity and are susceptible to confounding by environmental disturbances. Even urine and feces generally assess HPA activity over a period of only 24 h or less. We and others have recently developed and validated methods for measuring the concentration of CORT in the body hair of animals (e.g. rhesus monkeys) and scalp hair of humans. CORT is constantly deposited in the growing hair shaft, as a consequence of which such deposition can serve as a biomarker of integrated HPA activity over weeks and months instead of minutes or hours. Since the advent of this methodological advance, hair CORT has already been used as an index of chronic HPA activity and stress in human clinical and nonclinical populations, in a variety of laboratory-housed and wild-living animal species, and in archival specimens that are many decades or even centuries old. Moreover, because human hair is known to grow at an average rate of about 1 cm/month, several studies suggest that CORT levels in hair segments that differ in proximity to the scalp can, under certain conditions, be used as a retrospective calendar of HPA activity during specific time periods preceding sample collection.

The stress hormone cortisol (CORT) is slowly incorporated into the growing hair shaft of humans, nonhuman primates, and other mammals. We developed and validated a method for CORT extraction and analysis from rhesus monkey hair and subsequently adapted this method for use with human scalp hair. In contrast to CORT "point samples" obtained from plasma or saliva, hair CORT provides an integrated measure of hypothalamic-pituitary-adrenocortical (HPA) system activity, and thus physiological stress, during the period of hormone incorporation. Because human scalp hair grows at an average rate of 1 cm/month, CORT levels obtained from hair segments several cm in length can potentially serve as a biomarker of stress experienced over a number of months.In our method, each hair sample is first washed twice in isopropanol to remove any CORT from the outside of the hair shaft that has been deposited from sweat or sebum. After drying, the sample is ground to a fine powder to break up the hair's protein matrix and increase the surface area for extraction. CORT from the interior of the hair shaft is extracted into methanol, the methanol is evaporated, and the extract is reconstituted in assay buffer. Extracted CORT, along with standards and quality controls, is then analyzed by means of a sensitive and specific commercially available enzyme immunoassay (EIA) kit. Readout from the EIA is converted to pg CORT per mg powdered hair weight. This method has been used in our laboratory to analyze hair CORT in humans, several species of macaque monkeys, marmosets, dogs, and polar bears. Many studies both from our lab and from other research groups have demonstrated the broad applicability of hair CORT for assessing chronic stress exposure in natural as well as laboratory settings. Video LinkThe video component of this article can be found at

Our results indicate that relocation is a significant stressor for rhesus macaques and that this stressor triggers an increase in self-biting behavior as well as sleep disturbance in monkeys previously identified as suffering from SIB. These findings suggest that life stresses may similarly exacerbate SIB in humans with this disorder. The HPA axis results underscore the potential role of CBG in regulating long-term neuroendocrine responses to major stressors.

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