Analysis of Cortisol

Biomarkers of acute stress, particularly the measurement of glucocorticoids, are well established, with the most common being cortisol. Traditionally, cortisol has been measured using liquid chromatography-mass spectrometry (LC-MS) or enzyme- linked immunoassays (ELISA) in serum, saliva or urine as a biomarker of stress (45). However, these matrices have major disadvantages for measuring stress over longer periods of time, as they represent only a single point in time. Cortisol levels in these matrices are also subject to major physiological-based daily fluctuations and thus, in order to characterize long-term stress, multiple samples must be taken which is labour intensive for both participants and researchers (45). These limitations make it difficult to measure chronic stress in large populations using the traditional matrices.

In serum, cortisol is found both as a bioactive, free molecule, as well as bound to proteins. Thus, when there are increases in protein bound cortisol, such as during pregnancy or when taking birth control tablets, an increase in cortisol occurs when there is no concomitant increase in stress (45). The actual act of having blood taken can also be a source of stress and thus, could acutely increase serum cortisol levels (46). Although salivary levels of cortisol are not bound to proteins and the procedure is less invasive, levels still fluctuate widely throughout the day decreasing its value as a matrix to measure systemic stress. Collecting urine over 24 h can be used to overcome daily fluctuations, however it is labour intensive (47).

Hair cortisol content is increasingly being accepted as a validated biomarker of chronic stress (32, 48-58). Circulating, lipophilic compounds such as cortisol are incorporated into the hair shaft following diffusion from the capillaries that nourish the growing hair follicle and can remain trapped inside the hair shaft for hundreds of years (59-62). Hair grows on average one cm per month and thus one cm of hair represents one month of cortisol exposure. This mitigates the issue of inter- and intra-daily cortisol fluctuations, and of great importance to studies such as ours,

cortisol production can be documented retrospectively (11-13). Hair samples are also easy to collect, transport and store making this an especially attractive matrix.

Historically, hair analysis has been used extensively to monitor exposure to exogenous compounds, particularly drugs of abuse (63). Interest in monitoring endogenous compounds, such as cortisol, in hair is more recent. The first study to investigate whether glucocorticoids, including cortisol, could be measured in hair was conducted by Cirimele in 2000 using high performance liquid chromatography– ion spray mass spectrometry (HPLC-IS/MS) (64). The first study to utilize the enzyme-linked immunosorbent assay (ELISA) method on human hair, showed that hair cortisol concentrations were positively correlated with 24h urine sampling, but not salivary or serum cortisol, reflective of daily fluctuations (65). Sauvé et al. (2007) determined that the optimal region to take the hair sample from is the posterior vertex region of the scalp as it has the lowest intra-individual coefficient of variation for cortisol levels (65). Current findings also demonstrate that there is intra-individual stability in hair cortisol levels (66). These facts highlight the utility of hair cortisol as a biomarker of chronic stress. Evidence from several studies shows a direct correspondence between hair cortisol levels and physiological conditions that exhibit well-defined changes in classical HPA axis activity (e.g. Cushing’s syndrome and Addison syndrome) both in animals and humans (55, 67, 68). These data also provide strong support for the concept that hair cortisol levels do indeed reflect systemic cortisol levels. There are several studies wherein hair cortisol content correlates positively with other more traditional matrices used to measure cortisol (urine, serum, saliva and feces) (69-71). These and earlier studies demonstrate that hair cortisol reflects systemic cortisol, the major reason it is accepted as a validated biomarker of chronic stress.

There is limited research on the effects of the external environment on hair cortisol levels including frequency of hair washing, cosmetic treatments, use of hair products, and exposure to ultraviolet (UV) radiation. Repeated hair washing in hot water and shampoo was reported to decrease cortisol content in hair of rhesus monkeys and of humans (65, 72, 73). Hair cortisol content in the rhesus monkeys was similar after 20 washes with only water when compared to 20 shampoo washes, indicating the decrease on hair cortisol content is likely due to water exposure.

Conversely in human hair, there was a significant cortisol decrease in hair washed with shampoo compared to hair washed with just water. However, the washing procedures in these two studies were equivalent to 3-month and 12-month washing equivalents in monkeys and humans, respectively. Considering the most proximal 1 cm segment is traditionally used for measuring hair cortisol content, the hair would have been exposed to a maximum of 1-month washing equivalent and thus the effects of hair washing seem not be as dramatic as is presented in these few studies. Immersion in water is reported to damage hair structure, which could be contributing to the decreases seen in hair cortisol content (74). The study by Li et al. (2012) also found that exposure to UV irradiation decreased hair cortisol content (73).

A study by Sauvé et al. (2007) showed that chemically treated hair (i.e. hair that has been dyed) had significantly lower hair cortisol concentrations than did untreated hair (65). Conversely, Manenschijn et al. (2011) found no significant differences in hair that had been treated (i.e. dyed, bleached or permanently waved/straightened) or in use of hair products on the day of sample collection (e.g. hair spray, mousse, gel or wax) or in frequency of hair washing (68).

In a study by Keckeis et al. (2012), a small amount of radiolabelled cortisol was administered to guinea pigs and its disposition followed (75). It was found that most of the administered radioactive cortisol was secreted through feces and that only small amounts were incorporated into hair. In fact, most hair cortisol was not radioactive. The authors concluded that central cortisol thus did not enter the hair shaft. However, this finding is not surprising, as the short in vivo half-life of radioactive cortisol used in this study does not allow sufficient time for its disposition in hair and so, this study does not prove the source of deposition of cortisol into hair of humans.

Two groups have shown that human hair follicles possess their own HPA axis, also known as a “peripheral” HPA axis and that cortisol levels in hair are the result of the localized HPA axis activity and not reflective of central responses to stress (76-79). While further corroboration on the contribution of the ”peripheral” HPA axis activity to hair cortisol is needed, it can be argued that local cortisol production by human hair follicles will only marginally influence hair cortisol levels, if

at all. First, these studies have used arm and leg hair, structurally different from scalp hair. The hair that was shaved off the leg and wrist was not washed prior to analyses and thus could have been contaminated by cortisol in sweat or sebum. This is reflected in the fact that levels of cortisol detected were particularly, and unusually high, reaching levels up to 9,000 ng/g (wheras normal levels in human hair range from 46.1 – 225 ng/g (65, 80-83)).

However, as demonstrated in Ito et al. in 2005, exogenous ACTH influences follicular hair cortisol levels, and thus, if follicular-produced cortisol is a dominant contributor to hair cortisol content, hair cortisol levels should reflect systemic ACTH activity (76). On the other hand, if systemic cortisol is the dominant contributor, hair cortisol levels should reflect systemic cortisol activity. Some Cushing’s and Addison disease patients can present with a contradictory alteration of ACTH and cortisol levels, such as the former condition having undetectable ACTH levels and elevated cortisol levels and vice versa in the latter condition. From the studies on Cushing’s and Addison disease patients (55, 68), it was found that hair cortisol levels indeed follow the aberrant pattern of cortisol and not ACTH, supporting the notion that systemic cortisol is the major contributor to hair cortisol content. Also, elevated hair cortisol levels are seen during the third trimester of pregnancy, another physiological condition associated with increased levels of cortisol in plasma (51, 84).

There are data that indicate that cortisol concentrations in human hair are short-term to the period of a pain stressor (rather than cumulative), and localized (i.e. vary according to the stressor localization) (85, 86). These studies support the argument that cortisol in hair is a function of localized, temporary, peripheral HPA- axis activity rather than a cumulative record of relatively static, central HPA axis activity. However, a rapid incorporation of cortisol into the hair shaft, as shown in these studies, seems inconsistent with known structures of hair such as its barriers to diffusion due to its low water and lipid content. Several previous studies utilizing segmental analyses of hair have demonstrated that cortisol remains relatively static in the hair shaft after deposition. Both of these studies (85, 86) used a very small number of participants and are thus not robust enough to constitute conclusive findings.

numerous health conditions; thus, this biomarker may allow better insight into pathogenesis and disease progression. A Canadian study reported that mean hair cortisol concentrations were higher in patients with major chronic pain compared to non-obese controls (82). Another study by this group found that median hair cortisol content in patients with a confirmed acute myocardial infarction was significantly higher than in patients whose chest pains were attributed to other causes (81). These studies lend strong support to the use of hair cortisol as a biomarker of chronic stress, particularly in relation to a health effect.