**2. Laboratory determination of hair cortisol concentration (HCC)**

Different laboratories use similar methods to measure HCC with minor variations in experimental procedures. Usually, the proximal 3 cm of hair which represents the last 3 months of cortisol production, is collected [8, 9]. Using the most proximal segments reduces the potential for a 'wash-out effect' which relates to a decline in cortisol levels in distal segments due to ultraviolet radiation and hair care practices [8]. The hair sample is carefully sectioned into segment lengths that approximate the period of interest, for example, cut into 1 cm fragments, or whole hair samples are used. Hair grows approximately 1 cm per month, so this is a convenient approximation of changes in HCC each month [9].

The hair is then either finely minced with scissors or pulverized with a ball mill. The mass of hair used for analysis varies between studies, with a range of 2.5–50 mg for ELISA and 1.25–20 mg for LCMS [10]. Next, the samples are incubated in a solvent such as methanol for a set period to extract the cortisol. The extraction medium is evaporated to dryness and the extracted cortisol is reconstituted in a solution such as phosphate-buffered saline or distilled water. HCC is typically measured using two types of analyses: immunoassays or mass spectrometry [9, 10]. Immunoassays used for the analysis include enzyme-linked immunosorbent assay (ELISA), immunoassay with chemiluminescence detection (CLIA), and radioimmunoassay (RIA).

*The Association of Hair Cortisol and Cardiometabolic Risk Factors in Cardiovascular… DOI: http://dx.doi.org/10.5772/intechopen.108356*

Alternatively, liquid chromatography-mass spectrometry (LCMS) is used to determine HCC. Typically, the inter- and intra-assay coefficients of variability (cvs) are reported to indicate variability between measurements using different plates or tests and between duplicate samples on the same plate, respectively [10].

As the clinical utilization of HCC increases, it is important to establish an international benchmark [8, 9, 11]. Russell *et al.* conducted a study comparing the cortisol testing protocols of four leading laboratories, which include four ELISA methods and two LCMS methods, by analyzing the same hair samples representing the low, intermediate, and high ranges of hair cortisol concentrations (HCC) [9]. This study showed significant positive correlations between the four different immunoassay methods, while the results for the LCMS methods were almost identical. Further, the study concluded that laboratories using immunoassays could use a correction factor to convert results into standard LCMS equivalents.

## **3. The role of long-term hair cortisol levels in CVD risk**

Stress can be defined as any stimuli that can alter homeostasis and causes physical, emotional, or psychological strain. It impairs both physical and physiological health [6], resulting in a higher risk for obesity, type 2 diabetes mellitus, and cardiovascular diseases [5, 6]. Stress can be the result of psychosocial factors like anxiety, social isolation, and traumatic life events. Although some stress is beneficial and helps the body cope (e.g., with inflammation), excessive/prolonged stress can be harmful [12–15]. The response to stress is mediated by the "stress hormone" cortisol, which is released in higher doses under an excessive amount of stress [12]. Cortisol is a lipid-soluble glucocorticoid hormone that regulates a wide range of basal processes throughout the body, including metabolism, and immune response, and, most importantly, it helps the body to respond to stress and to maintain homeostasis. Cortisol does this by increasing blood sugar through gluconeogenesis, suppressing the immune system, and increasing the metabolism of fat, glucose, protein, and carbohydrates [13]. The production of cortisol is housed in the cortex of the adrenal glands and then released into the bloodstream, which transports it throughout the body [12]. Cortisol is an end product of the Hypothalamic-Pituitary-Adrenal (HPA) axis; its secretion in response to biochemical stress may influence health and cognitive events [12, 13].

The hypothalamic-pituitary-adrenal (HPA) axis is a crucial stress response system in the human body [12]. The primary function of the HPA axis is to maintain homeostasis and facilitate successful adaption to the surrounding environment through an intricate cascade of hormonal reactions. The stress response initiates when a stressor triggers the hypothalamus, releasing corticotropin-releasing factor (CRF) and vasopressin (AVP). These hormones stimulate the production of adrenocorticotropic hormone (ACTH) from the pituitary gland, eliciting the release of glucocorticoids, most notably cortisol, from the adrenal glands (**Figure 2**). Chronic stress has been well-documented as increasing the risk of cardiovascular disease through sustained exposure to increased levels of endogenous cortisol [6, 13]. The elevation of plasma cortisol levels is one of the best-studied components of the stress response, and this hormone is one of the best indicators of acute stress in humans.

Elevated cortisol leads to downregulation of the glucocorticoid receptor and potently affects lipid and carbohydrate metabolism [6]. Repeated or chronic stress can result in HPA axis dysfunction and cortisol remaining at high levels [12]. Clinical and population studies have shown that excessive cortisol levels are associated with

#### **Figure 2.**

*The HPA axis response to stress resulting in elevated cortisol, increased cardiometabolic risk (CMR), and cardiovascular disease (CVD).*

*The Association of Hair Cortisol and Cardiometabolic Risk Factors in Cardiovascular… DOI: http://dx.doi.org/10.5772/intechopen.108356*

developing central adiposity, insulin resistance and hyperglycemia, hypertension, and dyslipidemia [14, 15]. Traditionally these studies measured cortisol levels in body fluids, including saliva, blood, and urine [16]. While blood and saliva samples provide a cortisol measurement of a single timepoint, urine reflects the total exposure to bioactive cortisol over 12 or 24 hours. In contrast, hair allows for retrospective assessment of long-term cortisol concentrations over weeks, months, and even years [11, 17], potentially providing a better measure of an individual's long-term exposure to stress. HCC is increasingly important in establishing and providing significant insight into cardiovascular disease prognosis, diagnosis, and management [13]. Elevated cortisol has been shown to be associated with an increased risk of cardiovascular diseases (9). Also, several cross-sectional studies found positive associations of hair cortisol with adverse cardiometabolic outcomes, including higher systolic blood pressure [18], diabetes, metabolic syndrome [18], and adiposity [18, 19].
