I thought I'd change gears here and post another informative post, actually it'll be a couple of them under the "stress & spit" title. So what am I talking about when I say "stress & spit"? Well, specifically I'll be talking cortisol in this post. Other posts will also include other steroids/enzymes involved as salivary biomarkers of HPA axis reactivity. Cortisol has been used for a long time and has been the big boy on the block but it isn't necessarily the best marker but I'll get to others later on. You'll either find it fascinating or it will bore you to death...
Cortisol is a glucocorticoid hormone which occurs naturally in the human body. It is produced by the zona fasciculata portion of the adrenal cortex. It is heavily active in the stress response and is secreted within minutes following stimulation by a stressor. It circulates in the blood bound to plasma proteins and has a plasma half-life of approximately 70-90 minutes. It has a predictable diurnal variability in which it is at its lowest levels during deep sleep (Findling, Aron and Tyrrell, 1997). The hormone has been implicated in several roles – learning, memory, emotion and, most importantly for this study, the stress response (Miller, Chen and Zhou, 2007). Cortisol becomes elevated in the stress response in animals. While it can have a local anti-inflammatory consequence, prolonged cortisol secretion can be damaging to tissues as well as acting as an immunosuppressor (Selye, 1978; Rice, 1999). Chronic stimulation of the HPA axis by stressors and the subsequent release of cortisol impairs the normal negative feedback mechanisms to retain homeostasis and may also result in diseases or disorders such as Cushing’s syndrome, hypoglycemia, truncal obesity, insulin resistance and dyslipidemia (Chrousos and Gold, 1998; Whitworth, Williamson, Mangos and Kelly, 2005). As cortisol is intimately tied with the stress response, it is reasoned that measurement of its levels in the body will provide an accurate physiological assessment tool of stress level. Use of salivary cortisol as a measure of stress has much support in the literature.
Many researchers have examined the viability of utilizing salivary cortisol as a measure of the functionality of the HPA axis. For example, Laudat et al. in 1988 assessed this usage of salivary cortisol as a measure of HPA functioning. Their study included 101 healthy participants, 18 Cushing’s patients and 21 with primary or secondary adrenal deficiency. From 20 normal adults and 10 Cushing’s patients, saliva was collected every four hours over a 24 hour period. Samples were also collected from 58 normal adults, 4 Cushing’s patients and 8 adrenal deficiency participants at 8 in the morning and 10 at night. 58 normal participants and 21 adrenal deficiency participants had saliva and serum collected simultaneously before and one hour after administration of 240 µg of βACTH. For 23 normal and 8 Cushing’s participants, serum and salivary cortisol were assessed 8 hours after administration of 1 mg of dexamethasone. Salivary samples were between 2 and 3 mL each. Assessment and comparison of salivary and blood cortisol levels revealed eight discrepancies but all belonged to the participants with adrenal deficiency. Amongst the normal participants, no such discrepancies were found. The authors found that the patterns amongst the groups were as predicted with Cushing’s participants showing higher levels than the normal group and the adrenal deficient showing not only lower levels but a gap between salivary and blood levels. The DST and βACTH tests established upper and lower bounds for each group thereby allowing a range to be established. The authors conclude that salivary cortisol is indeed a reliable indicator of HPA function in normal as well as pathological participants.
Coste, Strauch, Letrait, and Bertagna (1994) also examined the reliability of hormonal levels to assess the HPA axis function. They utilized a sample of 20 healthy male volunteers in a double-blind randomized placebo crossover design experiment. Saliva collection was conducted at a baseline at 8 in the morning and a nocturnal measure between midnight and 8 a.m. Collection then followed administration of .25mg of tetracosactrin (synthetic ACTH) and 100mg of corticotrophin releasing hormone (CRH). The collections occurred on three separate occasions with intervals of 1 and 5 weeks. It was found that salivary cortisol for the peak following tetracosactrin administration had a satisfactory reliability. The baseline, however, was assessed to have a poor reliability. The authors conclude this is due to great individual variability among the participants (individual differences) and suggest that it can be controlled for by utilizing more measurements (approximately 18). This would aid in providing a greater intraclass reliability coefficient of the desired 0.8. The authors recommend conducting collections at a greater number of intervals per participant throughout the day to increase the reliability of the salivary cortisol analysis. This is in concordance with findings from other literature which show a higher reliability when utilizing a greater number of collections as well as larger sample groups (Young, Abelson and Lightman, 2004) and controlling for confounding variables which may affect cortisol levels such as caffeine intake, menstruation and medication amongst others (Kirschbaum et al., 1997; Lovallo et al., 2005)
Furthermore, Chiapelli et al. (2006) reviewed the use of saliva in the analysis of neuro-endocrine responses. The obvious benefits of saliva collection, the authors point out, are it being non-invasive and painless as opposed to other techniques such as sampling blood serum. The ease of the procedure has also made the technique very attractive to researchers as well. Collection of samples can be done several times throughout the day easily which allows for monitoring of diurnal patterns. Cortisol is acknowledged as a measure of HPA functioning. Saliva is utilized for collection and analysis of cortisol as from the time of stimulation it is then filtered by the salivary glands and is found in its free, unbound form in saliva.
Measurement of cortisol by means of saliva collection has become a simple, non-invasive method often utilized in research involving assessment of HPA axis activity (Kirschbaum and Hellhammer, 1989). Collection requires small amounts of saliva ranging from 0.025 – 2 mL (de Weerth, Graat, Buitelaar and Thijssen, 2003). Examination of cortisol levels in saliva is often performed by enzyme-linked immunosorbent assay, a non-radioactive assay which has become more widely utilized over the past three decades (Hausmann, Vleck and Farrar, 2007; Lequin, 2005). Another popular measurement method of cortisol examines the hormone’s levels in blood serum. Many studies have focused on the the question of whether salivary measurements are as realiable as those done through blood withdrawl. A quick analysis of the available literature shows that it is just as reliable a method.
In a review of the technique of utilizing salivary cortisol as a stress measure, Kirschbaum and Hellhammer (1994) stress the reliability of utilizing the measure. Not only do they cite numerous references to the reliability of the technique to indicate a level of plasma cortisol concentrations but also reference support for it as a better indicator of free cortisol in plasma. It is suggested by the authors that a choice to utilize salivary measurements are less costly, less invasive and easier to perform than analysis of blood serum. This supports earlier work by the authors in reviewing experimental assessment of salivary cortisol as a reliable source of cortisol for assessment of neuro-endocrine function (Kirschbaum and Hellhammer, 1989). This work is echoed by a review of the literature from Lewis (2006). Lewis indicates that “salivary cortisol levels parallel those plasma following ACTH and CRH stimulation, and following exercise induced stress” (pp. 141) and he further notes the technique is also useful in detection of pathological states such as those found in Cushing’s and metabolic disturbances.
For many years, it was thought that only measurement of hormones from blood samples would reflect accurate levels (Kirschbaum and Hellhammer, 1989). Subsequent research correlating salivary measures with blood samples has shown that salivary cortisol measures are as accurate as blood serum level measurements. Tunn et al. (1992) specifically examined cortisol levels in saliva and blood serum from samples collected simultaneously after administration of cortisol orally, intravenously or rectally. Endogenous production of cortisol was suppressed by administration of 4 mg of dexamethasone the night before the day of the study. The researchers found the measures of serum and salivary cortisol to correlated well. In the situation of elimination kinetics, the serum and salivary cortisol were nearly identical and the general time course of the hormone in both situations was close. The researchers also substantiated what many previous and subsequent studies have found in that salivary cortisol is a good indicator of unbound hormone.
Another study, by Gozansky et al. (2005), studied the correlation between salivary cortisol measures and that found in blood serum. Participants included 10 healthy women and 2 men. Salivary collection was done first late in the date when cortisol levels should be low and declining. The participants where then asked to engage in 10 minutes of exercise at 90% their maximum heart rate. They were monitored for the following two hours as cortisol levels decreased. Simultaneously with the saliva collection, blood samples were also taken. Samples were taken at the onset, midpoint and conclusion of the exercise. To calculate the lower value binding capacity of cortisol, participants were administered 1mg of dexamethasone at night and returned the next morning without eating for specimen collection. This procedure is called the dexamethasone suppression test or DST. Of the saliva, each participant provided at least 1 mL. As was noted earlier, that amount is within the standard range of collection for accurate analysis. Analysis of cortisol was performed utilizing an EIA (enzyme immunoassay) and analysis of blood serum performed utilizing an RIA (radioimmunoassay). Comparison of the blood serum and salivary cortisol showed similar patterns for baseline as well as exercise stimulation. Linear correlations obtained significance between the blood serum and salivary cortisol levels indicating one may act as a predictor of the other.
Salivary measurements of cortisol are not only applicable to adults. Research has shown that the procedure is also applicable to children and adolescents as well. Silva et al. (2007) studied the use of salivary cortisol as a measurement of HPA functionality and showed that the technique can also be utilized as a measure in children as well as adults. Nintey-one children ranging in age from 45 days to 36 months were examined using this method. Forty-six children were male and 45 were female. They were subdivided into groups of 6 month age intervals. Comparisons were made of collections done in the morning and evening for all age groups. The typical circadian rhythm of cortisol levels was found in all groups even present in those children at 45 days of age. The circadian rhythm of cortisol levels is important in the assessment of HPA function to determine normal from pathological ranges. The authors conclude that salivary cortisol measurement is a reliable measure of HPA functionality in children under three years of age.
Next Stress & Spit post I'll talk a bit about dehydroepiandrosterone and its sulfated ester.
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