Physiological biomarkers of chronic stress: A systematic review
Posted May 11, 2022
By Shamoon Noushad, et al.
Stress is a usual psychophysiological response generated by the body due to the undesirable, challenging, and difficult circumstances or stressors.[1-3] The stress effects on the nervous system are known for more than 50 years; and literature confirms the adverse effects of stress on the human brain. The structural changes, like that in brain atrophy due to chronic stress, lead to differential response and its impact on cognition and memory is also reported.
The intensity of effects varies as per the duration of stress, and it causes long-term structural defects to the brain, leading to psychological alterations.[5,7] Any traumatic event can serve as the trigger, for instance, being part of a road accident and getting a lifetime disability after it. Sometimes even witnessing any horrible event can cast a lifelong impact on mental health and well-being.
Furthermore, maternal separation is also determined as a powerful stressor that affects the individual in the postnatal period, ultimately extending till adulthood. Acute stress refers to a short term and adaptive state. In contrast, chronic stress is a long-lasting condition known to be related to maladaptive response, implying harmful effects on bodily mechanisms.[3,8]
Persistent or prolonged stress results in the secretion of certain hormones or chemicals from the body, which indicates the body’s constant stressful condition and affects the vital organs such as the brain, heart, or liver, in various aspects that might not favor the subject’s health. There are many systems in the body that either individually or collectively regulate the level of stress,[9,10] including the hypothalamic–pituitary–adrenal axis (HPA-Axis), Autonomic Nervous System (ANS), and immune system.[11-14]
HPA axis reacts to stressors by secreting cortisol, a chemical that prepares the body for fight and flight conditions and is a significant biomarker for stress. Cortisol is mostly concerned with psychological stress making HPA axis a system that mainly responds to psychosocial stress and interacts with ANS and the immune system.[9,10] Due to this interaction, the HPA axis is considered the mediating system and used to determine the effects of stress on disease processes, playing an essential role in cognition, metabolism, behavior, and immune reactivity.[9,15]
Although usually, the cortisol level remains high in the body at specific times of the day, for instance, in the morning, if it is persistent throughout the day, it is an alarming situation. Cortisol levels are detectable through various biological media such as saliva, blood, urine, and hair sample.
As mentioned, ANS also contributes to acute and chronic stress generation and regulation. It regulates bodily functions by autonomic reflexes in response to external (environmental, vision, smell, touch, etc.) and internal stimuli (maintenance of body homeostasis including temperature, blood sugar, removal of excess water, obesity etc.).[12,16-19] Epinephrine, norepinephrine, and acetylcholine (ACh) are the prime neurotransmitters released by the sympathetic and parasympathetic ANS.
During a stressful condition, catecholamines level increases while acetylcholine decreases, regulating the fight, and flight response.[13,20] The HPA-axis and ANS regulate a series of nonimmunological biomarkers, for example, arginine vasopressin (AVP) and dehydroepiandrosterone that are not involved in an immune response. Still, a few of the immunological biomarkers regulated by these are accountable for the immune system’s response to stress.
The body’s immune system has fought against these stressors; initiation of such stressful events, for instance, any injury or infection, promotes immune cells’ release into the bloodstream, which then induce fight and flight response. Increase inflammatory responses due to the immune system dysregulation among acute and chronic stress subjects, as the level of pro-inflammatory cytokines rapidly excels under both conditions increasing the likelihood of chronic diseases and associated frailty.[22-24]
Moreover, chronic stress’s health consequences are severe compared to acute and lead to latent viral activation, ultimately affecting the immune system. These responses and their intensity may vary from person to person, as an exaggerated immune response to stress might be generated among some individuals.[26,27]
These immunological biomarkers are mainly cytokines, that is, interleukin 6 (IL-6),[9,28,29] IL1- beta, C-Reactive Protein (CRP),[9,30] tumor necrosis factor-alpha (TNF-alpha)[9,28] also termed as pro-inflammatory cytokines, and natural killer cells (NK).[31-33] These immunological indicators and mediators, in particular cytokines and CRP, not only prepare the body against pathogens but also play an essential role in stimulating either acute or chronic stress concerning psychological stimuli or social interactions.
Along with these specified mediators, there are also other metabolic biomarkers such as Fasting glucose, glucose tolerance,[35,36] glycosylated hemoglobin (HbA1c), triglycerides, and cholesterol levels,[38,39] for examining the association of chronic stress with prolonged diseases. Other than these stress hormones, endocrine hormones such as prolactin,[40,41] estradiol, oxytocin, Growth Factor (GF), and Dehydroepiandrosterone Sulfate (DHEA-S)[43,44] are also studied to evaluate the level of chronic stress through blood samples.
Hence, these neural endocrine-immune system interactions play an essential role in shaping an individual’s reactivity to persistent stress. In addition to these hormones, the human body also has a complex array of enzymatic and nonenzymatic antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase (GTPx), malondialdehyde (MDA) and Ascorbic Acid, which are involved in antioxidant defense system, that is one of the natural defense systems against the harmful effects of reactive oxygen species (ROS) inducing oxidative stress.[45-47]
Therefore, it provides a useful link to understand the association between stress and diseases also specifies the significance of testing these biomarkers among stressed subjects in routine clinical practices.
Study design and search strategy
A systematic search of articles was conducted using PubMed, Embase, and Google scholar, using the combinations of search terms such as chronic stress together with the keywords HPA-Axis, ANS, immune system, metabolic biomarkers, cortisol, hair cortisol, salivary cortisol, urinary cortisol, epinephrine, norepinephrine, Adrenocorticotropic hormone (ACTH), BDNF, metabolic biomarkers, antioxidants, glucose, hemoglobin, CRP, cytokines, Pro-inflammatory cytokines, anti-inflammatory cytokines, and TNF.
The study population was human subjects. Since the aim of the study is to identify the potential biomarkers for chronic stress. Hence, all similar studies with the selected biomarkers, without restricting age and gender of the participants, were studied.
Data sources, studies selection, and data extraction
Studies of the last 40 years were included in the systematic review. All the articles fulfilling the eligibility criteria were read thoroughly for relevancy. The data were extracted, screened, and scrutinized for the robustness of the article and errors in the inclusion, to confirm the authenticity of the review. The primary information sources were the published research articles, while the reference lists of eligible articles were also searched and scrutinized to obtain all potential data.
Quality of studies
The quality of the included studies was assessed on the basis of selection bias, study design, confounders, and data collection methods.
Inclusion and exclusion criteria
Eligible studies reporting empirical findings on the association between physiological biomarkers and chronic stress were included in the study. Studies of the last 40 years were included in the systematic review with no age restrictions, only human studies published in English were included in the study. While all studies on non-human subjects, outside specified time duration, review articles, and studies with biomarkers other than those studied were excluded from this review.
A systematic summary was established through data synthesis including extraction, and management of the studies. The results were then screened, and combined according to the PRISMA guidelines.
The systematic search and review yielded 628 studies from electronic database searching and 43 additional studies identified through other sources. A total of 616 non-human subject studies were excluded and remaining 55 full-text experimental studies were assessed for eligibility. Finally, 37 studies were included in the quantitative synthesis [Figure 1].
Biomarkers of HPA-axis
Among the biomarkers of HPA-Axis in relation to chronic stress, cortisol, ACTH, and BDNF were studied. A total of nine studies supported cortisol measurement as a chronic stress biomarker through hair samples. HPA-axis starts from the hypothalamus by secreting the corticotropin-releasing hormone (CRH) is secreted that further induces the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary stimulating the secretion of cortisol.
Cortisol is mainly synthesized from cholesterol as the main glucocorticoid in zona fasciculata of the human adrenal cortex secreted by the influence of biochemical stress.[15,48,49] Cortisol is present as bound and unbound form; the unbound cortisol is of low molecular weight and is lipophilic. It enters the cell through passive diffusion, thus, easily determined in body fluids.
The blood cortisol measurement timings have significant importance as it is increased in blood cortisol level in the early morning and decreases in the evening time and initial phase of sleep. Herein, we focused on those studies that helped us identify cortisol as a biomarker of chronic stress and cortisol quantification methods through hair samples, saliva, and urine. Rauel et al. were the first who examined cortisol through hair samples.
According to Table 1, hair cortisol is demonstrated as a potential clinical biomarker for infants to address neonatal chronic stress better using a baseline measure comparison in which hair cortisol is influenced by day of ventilation in the neonatal intensive care unit (NICU). Moreover, another study assessed stress among mothers and their infants, using intra stability of hair, cortisol suggests its potential biomarker in detecting chronic stress experiences during the first year of life and the postpartum period.
Furthermore, hair cortisol can be a good source for measuring chronic stress in pregnant women. This long-term biomarker provides a vital implication in clinical practices and research. Moreover, another study examined the role of cortisol in pregnant women’s hair. This study suggested that hair cortisol is a potential biomarker of chronic stress. Hence, this longterm biological marker has a key association in clinical research practices.[54-57]
The quantification of scalp hair cortisol in children seems to be a biomarker for long-term cortisol measurement and thereby a biomarker of stress. A study examined HCC in children in the early phase of elementary school. It proposed a high-stress hormone level in fearful children at that stage. Furthermore, another study demonstrated that the chronic stress evaluated by increased cortisol levels in hair were positively associated with obesity measures among children with disabilities.
Besides, Quellete et al. examined the validity of hair cortisol concentration (HCC) in mother and daughter characterized by higher and lower maternal chronic stress, and overall findings indicated HCC as a useful biomarker of cortisol in response to chronic stress.
Moreover, chronic stress is associated with low socioeconomic status (SES) in children of young age and adolescent population by activating HPA-axis and releasing a high level of the stress hormone cortisol. According to this study, chronically high hair cortisol levels were found in children of young age and adolescents in low SES. However, older people with increasing age have more chances for a stress-related disease that correlates with chronically elevated cortisol secretion.
HCC showed a positive relation with age, female sex, alcohol consumption, and other factors such as smoking, contributing to significantly elevated cortisol levels in older people. Moreover, increased hair cortisol suggested a high level of chronic stress among the Walpole island first nation volunteers compared to volunteers of the non-first nation. Thus, the most reliable biomarker of chronic stress and the most readily used validation exhibited hair cortisol measurements.
There were a total of seven studies for salivary cortisol as a biomarker of chronic stress (Table 2). The use of salivary cortisol as a stress biomarker increases and provides useful results when estimating cortisol through saliva samples.[64-70] However, when investigated the role of the physiological stress response and basal physiological values in health and stress (burnout), individual study suggested that stress (burnout) patients showed higher heart rate and high cortisol level during the 1st h after awakening from the sleep when compared with healthy subjects. 
Furthermore, this study also suggested the association between chronic stress and cortisol changes during the 1st h of post-sleep in the morning. The salivary cortisol was found to be elevated in chronically stressed subjects compared to unstressed subjects. According to Soderstrom et al., 2006, burnout (stress), patients showed a high awakening cortisol response during the workday than during the weakened.
However, when examined the role of free cortisol response after awakening among both genders, the study suggested high cortisol response in females with high (stress) burnout than to females with low burnout (stress) at awakening and +15 min, +30 min, and +60 min after awakening whereas, male participants with moderate burnout (stress) showed high cortisol level at +60 min, after awakening than to low burnout (stress) male participants.
Therefore, the measurement of free cortisol in response to awakening should be considered a possible biomarker for chronic stress. Moreover, this study examined whether chronic stress (burnout) is associated with somatic and physiological arousal, intensified somatic arousals and elevated salivary cortisol levels. In addition, the study for determining the physiological biomarker of chronic stress through a salivary sample on middle-aged women revealed that stressed women have high evening (9 pm) salivary cortisol level and they had high urinary testosterone from the first-morning void whereas no association found between overnight urinary cortisol and catecholamines Table 6.
Free cortisol was found in chronically stressed (burnout) patients when compared to control healthy subjects. Hormonal and biochemical changes remained indifferent in these patients versus control subjects. These results indicated hypocortisolism in the stressed patient Tables 3 and 6.
Adrenocorticotropic hormone (ACTH)
Corticotrophin releasing hormone (CRH) is a crucial mediator of endocrine, autonomic, and immune responses to stress. There are two studies in which ACTH was used as a measure to evaluate chronic stress (Table 4).[71,72]
Brain-derived neurotropic factor (BDNF)
BDNF is considered necessary in the regulation, development, and survival of neurons and the maintenance of their physiological activity. However, the studies suggested that the stress (burnout) subjects have significantly lowered the level of BDNF and symptoms such as altered mood and cognition factor when compared with healthy control subjects (Table 5).
Biomarkers of Autonomic Nervous System (ANS)
Two physiological systems commonly; HPA-axis and sympathetic adrenergic medullary-axis (SAM) are involved in biomarkers of the autonomic nervous system. HPA-axis has a slow response toward a stressor whereas the SAM axis activates instantaneously and shows a very adaptive response to a stressor.[9,10] Moreover, SAM deals with immediate sympathetic activation. Hence, it stimulates the ability of an individual to cope with the conditions such as increased heart rate, blood pressure, and the release of catecholamines which include epinephrine and norepinephrine.
Catecholamine is one of the major biomarkers for the ANS activity. Epinephrine and norepinephrine are mainly synthesized by the central noradrenergic neurons and are released mainly from the adrenal medulla in the bloodstream.
Biomarkers of Metabolic Processes
Like HPA-axis and SAM-axis, several metabolic biomarkers play a major role in the physiology of stress, entailing a group of biomarkers such as glucose, glycosylated hemoglobin (HbA1c), cholesterols, and insulin. Thus, categorizing this group as metabolic biomarkers can be an effective tool for a biological measure of chronic stress level in the body.
Glucose, HbA1c, triglycerides, and cholesterol
The first study in Table 7 shows that the association of chronic stress with fasting glucose, glucose tolerance, HbA1c also involves the indirect effect of stress through inflammatory marker CRP.[73,74] However, it was found that high chronic stress leads to elevated levels of fasting glucose, post loaded glucose, and HbA1c level that there was no indirect effect of stress with CRP, this result suggested that high chronic stress is associated with poor glucose regulation in Hispanics subjects before onset of clinical diabetes diagnosis.
Prolactin and oxytocin
Many other hormones such as prolactin, oxytocin, Dehydroepiandrosterone Sulfate (DHEA-S), and growth hormone are studied in various stress groups to determine the effects of these endocrine hormones as a physiological biomarker (Table 8).[75,76]
Dehydroepiandrosterone sulfate (DHEA-S)
It is a steroid hormone with a capability of immunomodulatory function opposite to cortisol, and it is produced by the zona – reticularis in the adrenal cortex in response to ACTH Table 9.[43,45,77] It has a significant role as a regeneration and protection function that’s leads to maintenance of health status, whereas increased and decreased levels of DHEA-S are associated with the diagnosis of health issues and outcomes.
Several enzymes such as SOD, catalase, and MOD are involved in the body’s natural defense system against reactive oxygen species (ROS), contributing to aging, and disease-related problems.
SOD and catalase
The role of two antioxidant enzymes, SOD and catalase, in stress in healthy workers has been studied,[78,79] it was shown that SOD was higher in workers of evening and night shift Table 10.
The stressful condition leads to free radicals along with MDA as an end product of lipid peroxidation. The relationship between MDA and burnout levels in workers, when identified at night and evening show increased level of MDA and high-stress level. In contrast, MDA increases with age. No changes in MDA levels are found for sex; however, MDA is considered a biomarker of lipid peroxidation, occupational, and oxidative stress rather than chronic stress biomarker Table 11.[29,82]
Biomarkers of Immune System
The immunological biomarkers are an effective tool in creating a link between pathways between the immune and neuroendocrine systems in chronic stress-related outcomes. However, few studies determine the role of immunological biomarkers and the association of different cytokines measures in plasma in the blood samples and it involves individual cytokine anti-inflammatory and pro-inflammatory or it showed the ratio of anti-inflammatory and pro-inflammatory in several studies to detect the role of cytokines as a biomarker of chronic stress.
There are certain groups of cytokines such as growth factors, interleukins, and interferon. There are certain immune biomarkers such as pro-inflammatory (IL-6, IL-1β) cytokines, anti-inflammatory (IL-10), TNF-alpha, T, B cell, and NK cells and some of them are discussed below. All of these play a major role in immune responses by promoting the cell to cell communication and thus stimulates its role at the side of inflammation, infection, and trauma.
Anti-inflammatory and pro-inflammatory cytokines
As discussed earlier (Table 12), few studies demonstrated cytokines’ role in stress physiology.[32,85-88] A previous study has presented the association of stress (burnout) with circulating anti-inflammatory (IL-4, IL-10) and pro-inflammatory (TNFalpha) cytokines, where burnout stress teachers possessed higher levels of TNF-alpha/IL-4 ratio, suggests burnout stress subjects are related to increased systemic inflammation.
Natural killer cells (NK) and leukocytes
Few studies have found the association between different types of white blood cells (leukocytes) and the levels of stress (burnout) state (Table 13).[28,89] The present study demonstrated the relationship between burnout (stress) state and immune variables indicating that high scores for stress lead toward an increased leukocyte level and several T cell counts above the normal range. Several studies do not involve the increased number of lymphocytes subsets due to burnout stress state.[28,90] Hence, to summarize, burnout (stress) does not usually associate with a large number of leukocyte changes in the blood.
C-reactive protein (CRP) and fibrinogen
A study in support showed a significant relationship between chronic stress, inflammatory markers, and daily stressors (Table 14). It suggested that among family dementia caregivers, the increased daily stressors experience contributes to an elevation in the IL-6 and CRP levels. However, CRP showed more contrasting results with such variations, that either showing no relationship between burnout stress state and nonburnout state,[42,74] while in some studies it showed increased levels of CRP for burnout (stress) state.
All of the included 37 studies were original researches from the past 40 years and included any of the specific biomarkers that we have studied in this review. Among the biomarkers of HPA-Axis in relation to chronic stress, cortisol, ACTH, and BDNF were studied. A total of nine studies supported cortisol measurement as a chronic stress biomarker through hair samples. The useful cortisol parameters are urine, saliva, and blood; however, these parameters have some limitations.
The best and recent innovation for cortisol estimation is from hair. According to Kimberly et al, potential biomarker during maternal stress is cortisol; they proposed that hair cortisol as a valid biomarker rather than samples from urine or salivary samples. Hair cortisol concentration (HCC) is a useful tool in evaluating long-term cortisol levels. It provides a reliable measurement of HPA-axis activity throughout pregnancy and the reflecting total cortisol release.
However, the working population depicted the association between chronic stress and hair cortisol concentration and suggested that hair cortisol concentration’s limited applicability is an essential biomarker in work-related stress. Besides, it may be useful for the early detection of depression. Compared to salivary cortisol, hair cortisol appears to be a significant biomarker for evaluating stress and is a better indicator of stress system.
Furthermore, the role of ACTH as a biological measure of chronic stress in the study of non-Hispanic black and white pregnant women suggested that black Hispanic pregnant women had elevated mean levels of ACTH and C – reactive protein (CRP) in the 2nd and 3rd trimester of pregnancy. However, another study did not show any ACTH association with biological measures of chronic stress in blood samples. Similarly, for BDNF studies suggest that the stress subjects have significantly low BDNF levels.[35,36]
Among the ANS biomarkers, Catecholamines were studied while among those for the metabolic processes were glucose, HbA1c, triglycerides, and cholesterol. The included study suggested that there is no association found between ANS mediated hormones and chronic stress. The previous studies did not identify changes in stressed subjects versus healthy subjects,[70,72] thus, it cannot be used as a valid biomarker for chronic stress detection.
A study determined the role of the stressor on the individual, through metabolic endocrine, and immunological biomarker without any significant changes found through endocrine and immunological markers; however, statistical changes were found in glucose and HbA1c in two different caregivers group.
However, another study determined metabolic, endocrine, and immune correlates of burnout (stress) state among women, and high HbA1c and TNF-alpha levels in women with burnout and suggested these women had enhanced inflammatory responses and oxidative stress. Moreover, the association of tensed burnout with cardiovascular disease (CVD) risk factors results showed reduced health state and elevated levels of glucose, cholesterols, triglycerides, and uric acid.
Prolactin, oxytocin, and Dehydroepiandrosterone Sulfate (DHEA-S) were the endocrine hormones studied. Three articles compared DHEA-S levels through blood samples and showed the association of DHEA-S levels between stress (burnout) subjects and controlled healthy subjects. The low dose dexamethasone and DHEA-S levels were identified; however, higher DHEA-S levels were found in burnout, and no difference was found in cortisol level.
Furthermore, Pro-inflammatory cytokine IL-10 was found to be increased in stress subjects. When DHEA-S levels were compared between stress subjects and healthy subjects within each three 10 years interval age group, the youngest age group (25-34 years) showed lower DHEA-S levels more in females than in males of stress subjects. In comparison, no change in stress subjects for DHEA-S levels was found in the age group (35–44 years) and (45–54 years) when compared with control subjects.
Moreover, changes in DHEA-S levels in stress patients during the 1st year of their treatment leads led to the development in health, hence changes in DHEA-S sulfate associated with the prognosis of outcome in stress patients. Furthermore, we studied SOD and catalase among the antioxidants, only one study analyzed the role of two antioxidant enzymes, SOD, MDA, and catalase, in stress in healthy workers, showed activity SOD was higher in workers of evening and night shift.
Biomarkers of immune system included cytokines, antiinflammatory, pro-inflammatory cytokines, NK cells, leukocytes, CRP, and fibrinogen. A reviewed study showed high levels of IL-6 due to chronic stress that explains the changes due to chronic stress that produces adaptations in the immunological system reflected in the certain number of cytokines and immune cells, which do not return to normal baseline even after removal of stressors.
That justify decreased immune function in people with chronic stress provides a possibility to identify a personal vulnerability with specific biomarkers.[31,86] Furthermore, the presented study that shows showed the association of IL-6 and chronic stress; hence, it was found that IL-6 was four times greater in caregivers than noncaregivers, and this provides an evidenced mechanism through which chronic stressors may be related to age-associated disease and ageing of the immune system.
However, this present study indicates that chronic stress and its responses are widely associated with oxidative stress and inflammation, which may further lead to the risk of type 2 diabetes. The anti-inflammatory cytokines (IL-10) found to be increased in patients with burnout (stress) state. However, another study shows that interleukin-8 is a robust biomarker of chronic stress, and elevated levels of IL-8 are associated with cardiovascular disease and negative psychological consequences.
While, changes in NK cell activity and its number due to chronic stress were found in a few studies. One study shows that due to high burnout, depersonalization scores indicate decreased NK cell activity in male office workers and no changes in NK cell activity due to other burnout scores were found to in these male workers. The above studies summarized that the exact role of NK cell activity and variation in leukocyte number and activity due to chronic stress response is not clear. Hence, NK cells and leukocytes cannot be such valid biomarkers as compared to that of others.
Furthermore, CRP is one of the significant biomarkers of inflammation, and studies have presented its enhanced levels in chronic stress conditions.[92,93] A previous study has reported a positive relation of burnout with hs-CRP and fibrinogen levels in women, but not in men.
Summary of Main Findings and Study Limitations
Each biomarker plays an important role in stress, and each portray different outcomes. Moreover, this review helps to identify the relevance for biomarkers in chronic stress that can be helpful as the biological predecessor of disease, prognosticator of disease progression, and a potential target for behavioral interferences in chronic stress. Among the major limitation of this review was a few of the studies had a fragmented outcomes; however, conclusions are drawn based on this descriptive systematic analysis. However, we recommend a more extensive and yet specific literature search for each of these included biomarkers.
This review highlights the physiological biomarkers of HPA axis, ANS, immune, endocrine, metabolic, and defense system for how each of these play a major role in chronic stress. Hair and salivary cortisol, HPA axis biomarkers are considered as a major source of evaluating chronic stress levels in the targeted individuals. As the salivary and urinary biomarkers provide convenient measurement methods.
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