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1Department of Psychology, University of Massachusetts, Amherst, 2Neuroscience and Behavior Program, University of Massachusetts, Amherst
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Cortisol (CORT) accumulates in the growing hair shaft of humans and nonhuman primates. We describe methods for extracting and analyzing hair CORT with high precision and sensitivity. Measurement of hair CORT is particularly well-suited for assessing chronic stress over periods of weeks to months.
Meyer, J., Novak, M., Hamel, A., Rosenberg, K. Extraction and Analysis of Cortisol from Human and Monkey Hair. J. Vis. Exp. (83), e50882, doi:10.3791/50882 (2014).
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.
Measurement of CORT in plasma, saliva, or occasionally in urine or feces has been used as an index of physiological stress since Selye's discovery of the role of the HPA axis in stress1. Although numerous papers have been published relating HPA activity to acutely stressful situations, the field has been hampered by the lack of a simple and reliable index of chronic physiological stress. This problem arises because plasma and saliva both yield "point" estimates of HPA activity that are subject to circadian variation and can be confounded by environmental disturbances. Urinary and fecal samples yield measurements of CORT and/or metabolite excretion that span a number of hours up to a full day in some cases. Collection of multiple samples using any of these matrices may provide a rough composite index of CORT levels over time; however, none of these approaches provides a truly long-term index of HPA activity and the responsiveness of this system to chronic stressors.
Measuring CORT in hair has begun to fill this important need in the stress literature. Initial studies by several laboratories demonstrated the presence of CORT in human hair but did not investigate whether hair CORT levels changed as a function of stress2. As our laboratory has been interested for many years in the regulation of the rhesus monkey HPA axis by various social and behavioral factors3, we set out to establish and validate methods for extraction and analysis of rhesus monkey hair4. Based on the premise that blood-borne CORT is slowly and continuously incorporated into growing hair, the purpose of this new method was to use levels of hair-derived CORT as an integrated index of HPA activity over periods of weeks to months.
Several methodological challenges were encountered in developing the present protocol. First, previous studies had shown that small amounts of circulating CORT are excreted in sweat and sebum and therefore could coat the outside of the hair shaft2. In order to eliminate this potential confound, we developed a mild wash procedure that appears to remove external CORT while having a minimal effect on CORT present within the growing hair shaft. Thus, monkey hair subjected to this procedure (i.e. two 3-min washes with isopropanol) lost approximately 7-8% of the total hair CORT content, and a third wash removed less than 1% more steroid from the sample4. There appears to be more external CORT in human hair, since the same procedure removed an average of 27% total CORT content from the samples (K. Rosenberg and J. Meyer, unpublished). Like monkey hair, however, an additional wash contained much less CORT (about 7%) than the first two washes. Therefore, results from both monkey and human hair support the contention that most (if not all) external CORT can be removed while maintaining a major fraction of CORT within the internal hair matrix. Second, our pilot studies also showed that grinding the hair prior to extraction significantly increased CORT recovery from the sample, presumably by breaking open the complex proteinaceous matrix of the hair shaft as well as increasing the surface area available for solvent penetration. Two different grinding methods were developed, each with advantages and disadvantages. Method 1, which uses a ball mill, has the advantage of producing the finest powder. However, a ball mill is a relatively expensive equipment item and, if used with standard grinding jars and balls, it is capable of grinding only two samples at a time. Small samples are also difficult to process using a ball mill with standard grinding jars. Method 2, which uses a beadbeater, is less effective in its grinding ability. As a result, average CORT recovery is approximately 10% lower using this method compared to the ball mill (unpublished data). On the other hand, a beadbeater is considerably less expensive than a ball mill, 16-24 samples can be ground at once depending on the model, and the method is well suited for small samples. Because of the above mentioned differential recovery, it is advisable to use the same grinding method for all samples within a particular study.
Once the hair samples have been processed, they are extracted with methanol and CORT in the extracts is analyzed by means of a sensitive and specific commercial EIA kit originally designed to measure salivary CORT. The extraction and assay procedures were validated in part by demonstrating that serial dilutions of extracts from monkey hair samples yielded EIA readings that closely paralleled the readings obtained from authentic CORT standards. We then showed that hair CORT (in addition to plasma and salivary CORT) was sensitive to the major life stressor of an administratively mandated relocation of the monkeys to new housing quarters4,5. The present paper provides a detailed account of the methods used routinely in our laboratory to process human and monkey hair samples and to extract and analyze CORT from such samples.
1. Sample Collection and Storage
2. Sample Washing and Drying
3. Sample Grinding and CORT Extraction - Method 1 for Large Samples
4. Sample Grinding and CORT Extraction - Method 2 for Small Samples
5. Solvent Evaporation and Sample Reconstitution
6. CORT Assay and Data Conversion
Figure 1 shows the printout from a representative set of human hair samples (adult male and female human subjects) processed using method 2 grinding and extraction. Computer software was used to generate the data output and to fit a 4-parameter sigmoidal curve to the CORT standards (Figure 2). The between-well CVs from this plate ranged from 0.01-5.73% with an average intra-assay CV of 1.34%. The inter-assay CV determined using the QC values from nine recent human hair assays was 4.41%. The 37 samples analyzed on this plate yielded a range of hair CORT values from 3.1-650 pg/mg (median = 10.2 pg/mg; mean±SD = 36.0±110 pg/mg).
Method 1 is used in our laboratory to process hair samples from nonhuman primates and other large animals. A representative assay of adult rhesus monkey hair (mostly from females) yielded a range of CORT values from 50.1-102 pg/mg (median = 75.0; mean±SD = 75.8±14.0 pg/mg). The average intra-assay CV for this assay was 2.08%, and the inter-assay CV determined using the QC values from nine recent monkey hair assays was 4.63%.
Figure 1. Software output for a representative human hair CORT assay. The duplicate wells are as follows: S1-S6 - CORT standards ranging from 0.012 μg/dl (S6) to 3.0 μg/dl (S1); S7 - 0 CORT wells; B1 - nonspecific binding (NSB) wells that lack anti-CORT antibody (the software automatically subtracts the average NSB optical density [OD] value from all other OD readings); C1 and C2 — high and low CORT calibrators provided by the manufacturer; T1 - QC;
T2-T38 - test samples. From C1 to T38, the upper value in each cell of the array is the measured OD value from the corresponding well, and the lower value in the left-hand cell of the pair is the CORT value in μg/dl calculated from the mean OD value. Note that samples T9 and T31 yielded readings above the highest CORT standard. As a result, both samples were later diluted 4-fold in assay diluent and reanalyzed. The reanalyzed values were used after correction for the dilution factor. Click here to view larger image.
Figure 2. Standard curve of OD values versus log CORT concentration for the same human hair assay. The R-squared value shown in panel A is the calculated goodness of fit of the curve to the standards. Click here to view larger image.
The hair CORT procedure described above is simple to perform, is relatively inexpensive, makes use of readily available chemicals, reagents, and supplies, and requires equipment that, with one exception, is likely to be present in a typical analytical laboratory. The exception is a grinding apparatus such as a ball mill or mini-beadbeater. We note that some research groups mince hair samples into small fragments roughly 1 mm in length12, but based on our observations we recommend performing grinding instead of mincing if at all possible. Another area of methodological variation in the literature involves whether or not to wash the hair prior to CORT extraction13, and if so, what wash conditions to use. If washing is not performed, then one risks the possibility that the methanol extract will contain not only CORT incorporated slowly over the weeks or months of hair growth but also sweat- and/or sebum-derived CORT deposited recently on the surface of the hair. Studies on polar bear samples support the existence of two CORT fractions in hair: a loosely-bound fraction removable by brief isopropanol washing of intact hair that presumably represents mainly surface contamination, and a more tightly-bound fraction that is accessible by extensive methanol extraction of powdered hair samples14. The same conclusion can be drawn from the diminishing amounts of CORT extracted from monkey and human hair by repeated isopropanol washing (see Introduction). On the other hand, if washing is performed, then the choice of solvent and wash conditions can have a significant impact on the resulting CORT values. The Society of Hair Testing has published guidelines for selecting an appropriate solvent to minimize hair swelling and potential solute elution from the interior of the hair matrix during the washing process15. Although these recommendations were developed for application to drug testing in hair, they are relevant for steroid analysis as well.
Measuring CORT in hair rather than plasma or saliva offers a number of advantages2. Most importantly, this approach provides a biomarker of integrated CORT levels over periods of weeks to months that is uninfluenced by the time of day when samples are collected or by brief stress exposure prior to collection. Hair collection is noninvasive, although sampling of some animal species such as rhesus monkeys or bears may require anesthetization of the subject for safety reasons. Another advantage is that CORT is extremely stable in hair compared with other sample matrices, which permits analysis of historical or archival samples even if they have been stored at ambient temperature for a long period of time. Finally, CORT levels in human hair segments cut at successively greater distances from the scalp have sometimes been used to create a retrospective calendar of HPA activity over time. In such cases it is especially important to be aware of the previously mentioned “washout” effect produced by repeated hair washing. Although some studies have failed to replicate this effect8,9, it is worth noting that in those studies the samples were not washed prior to methanol extraction. This important methodological difference may help explain why hair CORT levels did not decline with distance from the scalp.
Some limitations of the hair CORT approach should also be mentioned. First, this approach cannot detect changes in the circadian rhythmicity of HPA activity (as seen in some depressed patients) or the awakening CORT response. Hair CORT levels also might not detect the impact of relatively brief stressors that occurred during the period of hormone incorporation. Hence, this approach should be thought of as complementary to measurements of salivary and/or plasma CORT, not as a replacement for such measurements. Second, whereas the use of hair CORT will likely be of particular value to researchers interested in psychosocial and environmental stressors, it is important to keep in mind that elevated HPA activity can occur under a variety of conditions, including physical exercise, metabolic abnormalities, and infectious disease. Third, whereas data exist from both humans and monkeys supporting the hypothesis that hair CORT is derived mainly from the bloodstream2, this hypothesis has not yet been proven. Indeed, Ito and coworkers16 have demonstrated the existence of a functional HPA-like system in microdissected human hair follicles maintained in organ culture. The degree to which hair follicles contribute to the CORT measured in the hair shaft remains unknown at this time.
In just a few years since its inception as a new biomarker of HPA axis activity, hair CORT has been used in a wide variety of applications across numerous species. Many of these applications fall within several major themes aimed at determining how long-term HPA activity is related to chronic stress, endocrine disorders such as Cushing’s disease, or neuropsychiatric disorders such as post-traumatic stress disorder2,17-19. Other studies have used hair CORT to examine HPA axis function in relation to behavioral temperament, normal development, the influence of developmental factors such as early childhood experiences in humans or different rearing conditions in monkeys, environmental conservation of wild-living animals, and retrospective investigation of historical or archival samples. Species studied to date with respect to hair CORT include humans, several species of nonhuman primates (macaques, vervet monkeys, and baboons), dogs, cats, cattle, horses, and several species of bears. It is likely that the use of hair CORT to assess long-term HPA activity, whether to investigate the physiological response to chronic stress or to address other experimental questions, will continue to expand and to be applied to an even greater range of species.
The authors have no conflicts of interest to declare.
We thank Kymberlee O'Brien, Celia Moore, and Edward Tronick (Department of Psychology, University of Massachusetts, Boston) for providing the human hair samples analyzed in this study, and Stephen Suomi and Amanda Dettmer (Laboratory of Comparative Ethology, NICHD) for providing the rhesus monkey hair samples. Initial development and continued use of this method has been supported by NIH RR11122 to M.A.N.
|Salivary cortisol assay kits||Salimetrics||1-3002||See manufacturer's kit insert for information on assay sensitivity and specificity|
|15 ml Polypropylene screw-cap centrifuge tubes||Max Scientific||10-9151|
|1.5 ml Safe-Lock microcentrifuge tubes||Fisher||05-402-25|
|2.0 ml Safe-Lock microcentrifuge tubes||Fisher||05-402-7|
|2.0 ml XXTuff reinforced microvials||BioSpec||330TX||Use with mini-beadbeater|
|3.2 mm chrome-steel beads||BioSpec||11079132c||Use with mini-beadbeater|
|10 ml stainless steel grinding jars||Retsch||02.462.0061||Use with mixer mill|
|12 mm stainless steel grinding balls||Retsch||05.368.0037||Use with mixer mill|
|Savant activated carbon cartridge||Fisher||DTK120R||Use with Savant chemical trap|
|Rotator for 15 ml centrifuge tubes||Fisher||S02135|
|Rotator for microcentrifuge tubes||Fisher||NC9854190|
|Benchtop centrifuge for microcentrifuge tubes||Fisher||13-100-675|
|MM 200 mixer mill||Retsch||20.746.0001|
|Savant DNA Speedvac||Fisher||DNA120-115|
|Savant refrigerated vapor trap||Fisher||RVT400-115|
|Savant chemical trap||Fisher||SCT120||Alternative to refrigerated vapor trap|
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