Abstract
Cortisol is a hydrophobic molecule that is largely bound to corticosteroid-binding globulin (CBG) in the circulation. In the assessment of adrenal insufficiency, many clinicians measure a total serum cortisol level, which assumes that CBG is present in normal concentrations and with a normal binding affinity for cortisol. CBG concentration and affinity are affected by a number of common factors including oral contraceptive pills (OCPs), fever and infection, as well as rare mutations in the serine protease inhibitor A6 (SERPINA6) gene, and as such, total cortisol levels might not be the ideal way to assess adrenal function in all clinical circumstances. This paper reviews the limitations of immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the measurement of total cortisol, the challenges of measuring free serum cortisol directly as well as the difficulties in calculating an estimated free cortisol from total cortisol, CBG and albumin concentrations. Newer approaches to the evaluation of adrenal insufficiency, including the measurement of cortisol and cortisone in the saliva, are discussed and a possible future role for these tests is proposed.
Introduction
In pediatric endocrinology, clinical evaluation of the hypothalamic-pituitary-adrenal (HPA) axis relies heavily on the measurement of a total cortisol level [1]. In this paper we describe the challenges of evaluating the HPA axis, including the common use of total serum cortisol rather than the measurement or calculation of the biologically active free cortisol. We argue that total serum cortisol may not accurately reflect the complex balance between the different elements of the HPA axis and, as such, might not be the ideal way to assess adrenal function in all clinical circumstances.
Cortisol physiology
Only a small fraction of total serum cortisol is unbound and free to enter cells, where it interacts with glucocorticoid receptors, provides feedback inhibition in the hypothalamus and pituitary gland and is ultimately responsible for crucial functions such as controlling inflammation and ensuring normotension and euglycemia [2]. This delicate system is influenced by the intrinsic pulsatility of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH)-secreting cells, diurnal variation, physical and psychological stress, inflammatory cytokines, medications and genetic factors affecting receptors, binding proteins and adrenal enzymes [2], [3]. Intricate local fluctuations in cortisol concentration are essential for maintaining homeostasis in the body, through mechanisms that are still not entirely understood.
Cortisol physiology in the systemic circulation
Cortisol is a hydrophobic molecule that binds to protein transport molecules in the circulation [1]. Eighty to 90% of circulating cortisol is bound to corticosteroid-binding globulin (CBG), a highly conserved 50–60 kDa glycoprotein encoded on the serine protease inhibitor A6 (SERPINA6) gene on chromosome 14q32.1 [4]. CBG binds cortisol with high affinity and low capacity; there is only one cortisol binding site on each CBG molecule [3]. CBG is saturated when the total cortisol concentration in serum is 400–500 nmol/L [3]. Ten to 15% of cortisol is bound, with low affinity, to albumin which is abundant in the circulation and essentially non-saturable. In the steady state, approximately 5% of cortisol is unbound [3]. Recent work has emphasized the “dual role” of CBG as both a reservoir for cortisol and a modulator of cortisol release [5]. CBG binds cortisol when it is synthesized by the adrenal glands and acts as a buffer to prevent large fluctuations in free cortisol levels [1]. It directs local free cortisol concentration through changes in CBG-cortisol binding affinity [6]. The presence of neutrophil elastase, produced by neutrophils and macrophages at sites of inflammation, causes CBG to undergo a conformational change resulting in a much lower affinity for cortisol [3], [4]. In the steady state, there is a balance between the two forms, with 30%–35% of CBG in the low-affinity form [7].
Table 1 lists many of the biological factors that affect CBG levels in the human body. The half-life of CBG in the blood is approximately 5 days [4]; however this varies widely depending on factors such as temperature and glycosylation. The relatively long half-life means that factors that affect the synthesis and secretion of CBG are only significant if they are prolonged [1], while factors affecting cortisol affinity have an immediate effect. In addition to the presence of inflammation, increases in temperature result in a significant decrease in CBG’s affinity for cortisol [3], leading to an increased proportion of free cortisol. Glycosylation, a form of post-translational modification in which carbohydrate molecules are attached to specific sites on protein molecules in an enzyme-dependent fashion, has been shown, in vitro, to be influenced by the presence of certain hormones [8]. Several glycoforms of CBG have been identified [8], [13]. Glycosylation has been demonstrated to affect CBG secretion, its affinity for cortisol, its half-life in the circulation and direction to specific tissues [7]. The degree of glycosylation of CBG has been found to interact with the variables of temperature and conformation to determine CBG’s affinity for cortisol in a given situation [6].
Biological factors that affect CBG levels and affinity for cortisol [1], [6], [8], [9], [10], [11], [12].
| Factors that increase CBG concentration | Factors that increase CBG’s affinity for cortisol | Factors that decrease CBG | Factors that decrease CBG’s affinity for cortisol |
|---|---|---|---|
| Oral estrogen/OCP | Glycosylation | Cirrhosis | Inflammation |
| Pregnancy | Ethanol | High temperature | |
| SERMs | IL-6 | Ethanol | |
| Mitotane | Insulin | SERPINA6 mutations | |
| Evening | IGF-1 | ||
| Hyperthyroidism | |||
| Exogenous glucocorticoids | |||
| Cushing syndrome | |||
| Nephrotic syndrome | |||
| SERPINA6 mutations | |||
| Neutrophil elastase | |||
| Morning |
SERM, selective estrogen receptor modulator; IGF-1, insulin-like growth factor 1.
The free hormone hypothesis [14] states that the biological activity of a hormone is affected by its unbound (free) rather than protein-bound concentration in the plasma. There is more recent evidence that CBG itself has a biologically active role by binding to CBG receptors, resulting in both targeted delivery of cortisol to specific tissues and possibly second-messenger effects as well [13], [15]. It is hypothesized that some CBG receptors might bind only specific glycoforms of CBG [8], [13], [16]. The exact role of CBG receptors is not completely clear; the most recent literature suggests that free cortisol is the main determinant of cortisol effects, with CBG itself playing a more minor role [7]. The fact that null mutations in the SERPINA6 gene are not lethal demonstrates that these interactions are not as important as the effects of free cortisol, however they represent a poorly understood aspect of glucocorticoid physiology [15].
Mutations in SERPINA6
Nine SERPINA6 mutations have been identified in humans [7], [17], with varying effects depending on the exact genetic locus involved. Some mutations result in low or absent circulating CBG, and some mutations affect only the cortisol-binding site, resulting in normal CBG concentrations but poor cortisol carrying capacity [1]. Some SERPINA6 polymorphisms have no clinical significance but can complicate the biochemical picture; Simard et al. [17] reported a CBG variant which was not identified by the CBG immunoassay, but was otherwise functional. Approximately 1 in 35 individuals in the Han Chinese population have a SERPINA6 variant leading to a 50% reduction in CBG levels [7]. These individuals have normal free cortisol levels and normal ACTH, but a low total cortisol concentration. In other ethnic groups, these mutations are rare [17].
Free cortisol fractions reported in unstressed adults are typically close to 5% [1], [7]. Lewis et al. [9] reported a free cortisol fraction of 25% in an unstressed individual with a null SERPINA6 mutation; this was the same free cortisol fraction noted in heat-inactivated plasma of normal volunteers [9]. Animal studies suggest that individuals with SERPINA6 mutations may also respond differently to critical illness. CBG knockout mice that are injected with lipopolysaccharide [5] (an animal model of septic shock) or tumor necrosis factor-alpha (TNF-α [17] have considerably lower survival than wild-type mice. It seems that the larger free fraction of cortisol does not fully compensate for the lower cortisol pool.
Some individuals with SERPINA6 mutations present with hypotension or nonspecific symptoms such as fatigue, nausea or chronic pain, but symptomatology varies widely, even between family members with identical mutations [10], [15]. Proposed explanations for the symptoms experienced by some of these individuals include dysregulation of cortisol pulsatility leading to changes in transcription of glucocorticoid-responsive genes [1] or epigenetic changes [17], and loss of the tissue-specific targeting and second-messenger effects that occur when CBG binds its receptors [15].
Methods for measuring cortisol
The two main methods for measuring cortisol, both total and free, are immunoassays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Immunoassay is currently the most popular method, given its speed, lower cost and ease of use [18]. There are important limitations of this technology, however, that must be understood to ensure appropriate interpretation of results [1], [19], [20], [21]. The main issue, affecting the measurement of both total and free cortisol, is imperfect antibody specificity. The antibodies used in these assays may cross-react with cortisol precursors or metabolites, as well as synthetic steroids. Because of this cross-reactivity, cortisol levels by immunoassay are usually higher than those obtained by other methods [1]. These molecules are easily distinguished by mass spectrometry [22]. The second issue, which affects the measurement of total cortisol, involves the step in which the cortisol molecule is removed from CBG. This process can involve a change in temperature or pH or the addition of a reagent [21]. If a reagent is used, then it must be added in sufficient quantities to allow for high-CBG states, such as pregnancy or oral contraceptive use, or cortisol levels will be underestimated [21], [23]. The third and very significant issue is that both the antibodies and the methods for removing cortisol from CBG in immunoassays are proprietary and can change over time [24], so even a patient followed at the same institution may have multiple results that are not directly comparable. These inconsistencies complicate the application of the literature to clinical practice [18], [22], [25] as the methods used to derive clinical guidelines may be different than those available at the institution attempting to implement the guidelines.
Many authors [1], [19], [22], [24], [26] have argued that LC-MS/MS should be the gold standard for quantifying all steroid hormones. If proper quality control measures are followed, mass spectrometry allows steroid hormones to be measured accurately and reproducibly, even in tiny concentrations, without the issue of antibody specificity [27]. A clear advantage of mass spectrometry is the ability to measure a concurrent steroid profile, which is useful in conditions such as polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH) which involve aberrations in multiple steroids [22]. LC-MS/MS can screen for exogenous steroids (prescribed or not) [22], which can sometimes confuse the clinical picture. Hawley et al. [21] compared the performance of five commercially available cortisol immunoassays with an LC-MS/MS candidate reference management procedure [27] in five patient populations. They found considerable variation between immunoassay results and LC-MS/MS results with a greater discrepancy in both pregnant and non-pregnant women compared to men [21]. They found that all immunoassays they tested (though less on the newer assays) overestimated cortisol levels in patients taking metyrapone and prednisolone [21]. While LC-MS/MS is a superior technique to immunoassay in many ways, a clear limitation is the expense and the special training required for laboratory personnel to maintain quality control [20]. Certainly in the short term, it is realistic that many clinics will continue to use immunoassays as the main method for measuring serum cortisol levels. It is therefore crucial that individuals using this method understand the assumptions and limitations of their assay.
Methods for measuring serum free cortisol directly
The direct measurement of serum free cortisol involves first separating the cortisol that is bound to CBG or albumin from the plasma containing free cortisol, followed by quantification of free cortisol by immunoassay or LC-MS/MS as already described. The most common techniques used to separate bound cortisol from free cortisol in the serum are ultrafiltration and equilibrium dialysis [28]. Equilibrium dialysis involves incubating the plasma in a cell with a membrane that CBG is not able to cross, often overnight, while ultrafiltration involves centrifugation of plasma samples in a tube fitted with a filter with pores smaller than CBG molecules. In all of the techniques, controlling temperature is extremely important as small fluctuations cause the total:free cortisol ratio to change dramatically. Several authors have noted that equilibrium dialysis provides slightly higher results than ultrafiltration, with inferior reproducibility and a longer incubation time [28], [29], [30].
Measuring serum free cortisol indirectly
Several formulae for estimating free cortisol concentrations from the more easily measured values of total cortisol, CBG and albumin have been published. The most straightforward is the free cortisol index, a ratio of total cortisol to CBG [31], [32], [33]. While simple and somewhat useful, this method does not consider the saturability of CBG in high-cortisol states [11] and the changes in binding affinity due to fever, infection, pregnancy and some SERPINA6 mutations [17]. Concerns with this approach led Coolens et al. [11] to develop a more complicated formula to derive free cortisol concentration when the concentrations of total cortisol and CBG are known. Compared to the free cortisol index, Coolens’ formula is a more accurate representation of free cortisol levels when CBG is saturated [11], but it does not account for changes in albumin concentration or CBG affinity [8] and has been demonstrated to have low precision in patients with sepsis [30].
Dorin et al. [34] attempted to improve the accuracy of Coolens’ method by including albumin concentration and the equilibrium dissociation constant for cortisol binding to albumin as independent variables, rather than incorporating them into a constant. They tested their formula on three populations: healthy controls, patients with sepsis and patients with septic shock. They found that their formula was less biased than Coolens’ formula, and it was better able to model the interaction effect observed when both CBG and albumin were low [34], as observed in profound sepsis. Dorin et al. [34] noted that there was no correlation between albumin and CBG levels, indicating that these proteins change in response to different factors. The Dorin formula oversimplifies some aspects of cortisol dynamics, including the inaccurate assumption that an albumin molecule can only bind one cortisol molecule, and the use of a single dissociation constant for CBG, while physiologically the affinity of CBG to cortisol is significantly lower in sepsis [34]. Nguyen et al. [35] developed a formula that addressed some of the limitations of the Dorin formula; however, the tests required (separate immunoassays for the high-and low-affinity forms of CBG) were less practical than measuring free cortisol directly.
The limitations of indirect methods in general are the oversimplification of a highly complex system as well as the incorporation of multiple sources of error, both from the methods used to measure cortisol, CBG and albumin in the patient but also the methods used to derive the formulae [29]. For a calculated free cortisol value to be useful clinically, it must be accurate, reproducible, and easier and less expensive than measuring free cortisol directly. To maximize precision, the formula and its constants should be derived from free cortisol values measured by ultrafiltration followed by LC-MS/MS. For accuracy in sepsis, a measure of albumin should be included. Consideration of different dissociation constants for different clinical situations is worth investigating.
Measuring free cortisol and cortisone in the saliva
Given the technical challenges of separating bound and unbound cortisol in serum, many researchers have considered measuring free cortisol in parts of the body where CBG and albumin are not present – in the saliva and urine. Salivary cortisol is widely used in psychological research [36], [37]. As with serum cortisol, salivary cortisol can be measured by immunoassay or LC-MS/MS [38]. The antibody specificity of immunoassays may be problematic in saliva due to the presence of other steroids and steroid metabolites [38]. As with serum measurements, LC-MS/MS reference ranges for cortisol in saliva are lower than for immunoassays, reflecting the superior specificity of LC-MS/MS [36], [39]. Measuring free cortisol in saliva instead of in serum offers many potential advantages, particularly in children. The stress associated with phlebotomy is removed, and samples can be collected at home at any time of day, allowing the early-morning cortisol peak and late-night nadir to be captured. Samples are stable at room temperature for 1–2 days [38] and longer if refrigerated. The main challenge is ensuring standardization of collection, particularly if the test is done at home. The kit must be used as directed and avoidance of eating, drinking, smoking and tooth brushing prior to the test is key. Patients with xerostomia, intraoral bleeding or recent dental work will have inaccurate results [37]. However, similar to serum cortisol levels, non-stimulated salivary cortisol levels may only be useful as a screening tool [40] and are less accurate in individuals without an established circadian rhythm [23], such as infants and shift workers, and during intercurrent illness.
Lipophilic unbound cortisol in the serum diffuses passively into the parotid glands and across the epithelial membrane into saliva [1]. Salivary cortisol reflects levels of serum free cortisol and shows the same circadian rhythm as serum free cortisol [38]; cortisol in saliva increases within minutes of a rise in serum free cortisol [23]. The parotid glands produce 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which converts cortisol to cortisone such that levels of cortisone in the saliva are 4 to 6 times higher than in salivary cortisol [1], [41]. Salivary cortisone measured by LC-MS/MS may be a more accurate proxy for serum free cortisol than salivary cortisol as it is detectable even when serum cortisol levels are low and avoids potential contamination with oral hydrocortisone [42], [43], [44].
Measuring free cortisol in urine
Urinary free cortisol (UFC) has long been used in the diagnosis of Cushing syndrome in adults. UFC correlates with free serum cortisol and, like salivary cortisol and cortisone, is more closely associated with cortisol production than total serum cortisol levels [38]. While CBG is not a confounding factor in the measurement of UFC, there are several limitations of this test including day-to-day variation, several common conditions that affect UFC (obesity, pregnancy and stress) and higher levels of cross-reactive metabolites in urine relative to serum [38], [41], [45]. Despite the limitations of the test, UFC continues to be a useful tool in the evaluation of cortisol excess; however, it has not been established as a test for the evaluation of adrenal insufficiency.
Free cortisol in the diagnosis of adrenal insufficiency
Free cortisol in the diagnosis of primary, secondary and tertiary adrenal insufficiency
The limitations of measurement of total cortisol are typically less important in the evaluation of primary adrenal insufficiency when ACTH levels are elevated. However, the diagnosis of secondary or tertiary adrenal insufficiency depends on a low stimulated cortisol measurement in the context of a low or inappropriately normal ACTH level. Low total cortisol and normal ACTH are also seen in individuals with low CBG levels, due to SERPINA6 polymorphisms or concurrent medical conditions that lower CBG (Table 1). If free cortisol levels are not measured, secondary or tertiary adrenal insufficiency may be overdiagnosed when factors that decrease CBG concentrations are present (i.e. critical illness or SERPINA6 polymorphisms), and may be missed if factors that elevate CBG are present (i.e. oral contraceptive pills [OCPs]). Symptoms of adrenal insufficiency are nonspecific; therefore, if there is poor clinical response to glucocorticoid therapy in an individual with presumed central adrenal insufficiency, the possibility of a false-positive test secondary to low CBG levels should be considered.
Clinical guidelines on the diagnosis of Cushing syndrome indicate that salivary cortisol levels measured during the late-night nadir may be a useful diagnostic tool in the evaluation of hypercortisolism [23], [45], [46], and recent research has suggested that stimulated salivary cortisol and cortisone levels may be used as an alternative to serum cortisol in the evaluation of adrenal insufficiency as well [42], [47]. Similar to serum cortisol, early-morning basal salivary cortisol levels may only be useful as a screening tool for adrenal insufficiency in patients with a diurnal rhythm [40]. While there is emerging research evaluating the use of basal and stimulated salivary cortisol levels as diagnostic tools, to date, there are no widely supported reference ranges for these tests and more recent evidence suggests that stimulated salivary cortisone may be a superior measure [40]. For use in an endocrinology clinic, reference ranges following ACTH stimulation testing would be of greater use. Table 2 summarizes the advantages and disadvantages of dynamic tests that could be used in the evaluation of adrenal insufficiency.
Summary of dynamic tests that could be used for the evaluation of adrenal insufficiency.
| Testa | Laboratory requirements | Indications and advantages | Contraindications and disadvantages |
|---|---|---|---|
| Stimulated total serum cortisol | LC-MS/MS or immunoassay |
|
|
| Stimulated free serum cortisol (direct measurement) | Ultrafiltration or equilibrium dialysis followed by LC-MS/MS or immunoassay |
|
|
| Stimulated free serum cortisol (calculated) | Measurement of total cortisol, CBG and albumin |
|
|
| Stimulated salivary free cortisol | LC-MS/MS |
|
|
| Stimulated salivary free cortisone | LC-MS/MS |
|
|
aOf note, sex, age and Tanner-stage-specific reference ranges based on LC-MS/MS values have not been established for these tests and this remains a major limitation for all.
Establishing a pediatric reference range for free cortisol
Establishing reference ranges for cortisol is more complicated than for other hormones, due to diurnal variation and the association with stress, including the stress of phlebotomy. To overcome this variability, the diagnosis of adrenal insufficiency relies on dynamic testing. Eyal et al. [48] provide an important first step in establishing reference ranges for stimulated free cortisol levels in children. They measured free cortisol levels by equilibrium dialysis, followed by chemiluminescence assay, in 28 girls and 57 boys aged 0.6–17.7 years before and after a 250 µg/m2 ACTH stimulation test [48]. The mean basal free cortisol level was 11.1 (±8.3) nmol/L and the mean peak free cortisol level was 50.0 (±16.7) nmol/L [48]. At baseline, they noted a mean free fraction of 3.95% (95% confidence interval [CI] 3.57%–4.33%) and after stimulation testing they found a mean free fraction of 6.69% (95% CI 6.23%–7.13%). The increase in free cortisol levels after stimulation testing was greater than the increase in total cortisol levels, consistent with saturation of CBG [48]. The authors concluded that an appropriate cutoff for peak free cortisol following a standard-dose ACTH stimulation test in children is >25 nmol/L [48]. This was the same cutoff that was determined by similar studies in adults [49], [50]. Eyal et al. [48] noted significant differences in peak free cortisol levels by Tanner stage for both genders, but no difference between genders. Although the sample size in each age/Tanner stage group was small and the study did not use LC-MS/MS to measure cortisol, this study provides a useful starting point and is, to our knowledge, the only study that attempts to determine reference ranges for serum free cortisol in children.
The use of stimulated salivary cortisone levels by LC-MS/MS in the evaluation of cortisol deficiency, especially in states of altered cortisol binding, may address many of the challenges with testing of the HPA axis; however, further study is needed to establish pediatric reference ranges. Several studies have attempted to develop age, sex and time of day-specific reference ranges for salivary free cortisol levels, notably the CIRCORT database [36], a meta-dataset made up of 15 studies that included 104,623 saliva samples from 18,698 participants. Though salivary cortisol was measured on two types of immunoassay in this study, the results were calibrated to LC-MS/MS in order to increase generalizability [36]. These results did not take into account the conversion to cortisone. Two recent studies [47], [51] published stimulated salivary cortisol and cortisone thresholds for the diagnosis of adrenal insufficiency in adults using LC-MS/MS after low- [51] and standard-dose [47] ACTH stimulation tests. Attempts to establish pediatric norms for stimulated salivary cortisol levels have also been made in smaller studies [52], [53].
Free cortisol in the diagnosis of critical illness-related corticosteroid insufficiency (CIRCI)
Levels of free cortisol are significantly affected by critical illness through two main mechanisms: activation of the HPA axis and changes in CBG binding affinity due to elevated temperature and cleavage by neutrophil elastase. Free serum cortisol assays performed at 37 °C may underestimate levels in febrile patients [1]. Interleukin-6 (IL-6) inhibits transcription of SERPINA6 and CBG secretion by hepatocytes [1], leading to higher free cortisol in situations of extreme inflammation. While albumin is less affected by temperature changes compared to CBG, it is highly sensitive to acidosis [54]; therefore in the context of septic shock, cortisol is released from both of its binding proteins. The low CBG and resultant free cortisol levels in sepsis mimic a SERPINA6 polymorphism, with high baseline free cortisol levels and increased pulsatility due to lack of buffering [1].
It is an unresolved debate in critical care medicine as to how and when corticosteroids should be given to patients in septic shock [55], [56]. This discussion is beyond the scope of this paper, other than to highlight the importance of considering (and ideally measuring) free cortisol in studies evaluating the role of glucocorticoids in critical illness, as nearly all aspects of cortisol physiology, including ACTH, CBG cleavage, CBG transcription, CBG binding affinity and albumin binding affinity, are profoundly affected by sepsis. This is the patient group in whom indirect measures of free cortisol are most likely to be inaccurate; however, direct measurements of free cortisol would have to be available very quickly to be useful clinically. Measuring salivary cortisol in critically ill patients has been explored in the literature and research on this subject is ongoing [57].
Conclusions
In this paper, we argue that direct measurement of serum free cortisol, by ultrafiltration followed by LC-MS/MS, is the most accurate way to quantify the biologically active free fraction of cortisol and address many of the challenges that we face in the evaluation of the HPA axis. Although significantly more research would be required, the measurement of salivary cortisol and/or cortisone could also become a useful and convenient approach. We argue that reference ranges should be established using LC-MS/MS, and should be post-stimulation test and specific for sex and age (early childhood) or Tanner stage (from middle childhood to maturity) [48], [53], [58]. Currently, many of us rely on immunoassays, total serum cortisol levels and non-standardized reference ranges for the purpose of clinical decision-making. We hope to raise awareness of the limitations of our current approaches and to highlight areas where further research would be helpful.
Total cortisol might still be the most cost-effective measurement for evaluating the HPA axis in many clinical situations, however, consideration of free cortisol measurement, either directly in serum or saliva, or indirectly using calculated values, is useful in more complicated situations when cortisol levels do not explain the patient’s clinical presentation. Further investigation may also be helpful when faced with a patient with borderline results after ACTH stimulation testing or in those with underlying conditions or medications that affect binding proteins, including critical illness or use of oral contraceptives. The possibility of abnormal CBG binding must also be considered if results are unexpected, as calculated free cortisol formulae do not allow for changes in CBG’s affinity for cortisol. As Vogeser et al. [29] suggest, perhaps providing context for the cortisol level, such as a concurrent C-reactive protein level to diagnose an acute-phase response, could help decide if cortisol-CBG binding is likely to be altered. While the current literature suggests that free cortisol is the main determinant of cortisol effects, the role of CBG on cortisol action and the degree of correlation between serum free cortisol levels and clinical outcomes require further study [7], [9]. Unfortunately, the currently available reference ranges for measured and calculated free cortisol levels are not sufficient to make specific recommendations, but at minimum an understanding of the importance of considering free vs. total cortisol is important for the clinician.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: KCV was supported by the Canadian Pediatric Endocrine Group fellowship program and the Children’s Hospital Academic Medical Organization.
Honorarium: Neither author received an honorarium for the preparation of this manuscript.
Competing interests: The funding organization played no role in the writing of this report or in the decision to submit the report for publication.
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