Extraction and Analysis of Cortisol from Human and Monkey Hair

Summary

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.

Abstract

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.

Introduction

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 excreti....

Protocol

1. Sample Collection and Storage

1. Human hair
   1. Secure the entire length of the hair to be sampled (up to a pencil-width in diameter) with a rubber band or clip. Cut hair as close to the scalp as possible (taking care not to nick the skin) with a clean scissors. Note: The standard sampling area is the posterior vertex of the skull.
   2. Some investigators have reported a decline in human hair CORT levels  with distance from the scalp⁶, which may be due to washout from repeated exposure to water and shampoo⁷ (though see Manenschijn et al.⁸ and Thomson et al.⁹ for contrary resul....

Results

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........

Discussion

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 inste.......

Materials

Name	Company	Catalog Number	Comments
HPLC-grade isopropanol	Fisher	A451
HPLC-grade methanol	Fisher	A452
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
Name of Equipment	Company	Catalog Number	Comments/Description
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
Mini-Beadbeater 16	BioSpec	607
Savant DNA Speedvac	Fisher	DNA120-115
Savant refrigerated vapor trap	Fisher	RVT400-115
Savant chemical trap	Fisher	SCT120	Alternative to refrigerated vapor trap
Microplate reader
Microplate washer
Microplate mixer
Multichannel pipetter
Analytical balance