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Endokrynologia Polska
DOI: 10.5603/EP.a2020.0058
Volume/Tom 71; Number/Numer 6/2020
ISSN 0423–104X
ORIGINAL PAPER
Department of Endocrinology and Internal Diseases, Medical University of Gdansk, Gdansk, Poland
Abstract
Introduction: Cortisol concentration is measured in blood, urine, and saliva samples. It has been recently proven that cortisol could also
be detected in hair samples. Cortisol measurements in different samples have their own individual characteristics and clinical utility.
We aimed to investigate the correlation between hair cortisol concentration and standard cortisol measurements used in clinical practice.
Material and methods: Fifty adult volunteers with a negative history of endocrine disorders were enrolled in the study. Morning serum
cortisol (MSC), evening serum cortisol (ESC), evening free salivary cortisol (EFSC), urine free cortisol (UFC), and hair cortisol concentra-
tion (HCC) were analysed in all participants. Eventually, 41 volunteers were included into the study, whose cortisol concentration in the
1 mg overnight dexamethasone suppression test (1 mg ONDST) were < 50 nmol/L, and cortisol levels in serum, saliva, and urine were
within reference ranges. Hair cortisol concentration test was performed for 20 mg of hair strands of the proximal 1 cm hair segments.
Results: Hair cortisol concentration ranged from 0.3036 to 2.65 nmol/mg, and the average value was 0.8125 ± 0.4834 nmol/mg. No signifi-
cant correlations were found between HCC and MSC (rho = 0.04419, p = 0.7838), HCC and ESC (rho = –0.2071, p = 0.1938), HCC and
EFSC (rho = 0.1005, p = 0.532), or HCC and UFC (rho = 0.1793, p = 0.262).
Conclusions: This work is another step in the discussion on the application of HCC determinations in clinical practice. Our results have
showed no correlations between HCC and single point cortisol assessment in blood, saliva, and urine in patients with reference cortisol
levels. (Endokrynol Pol 2020; 71 (6): 539–544)
Key words: hair cortisol; serum cortisol; salivary cortisol; urine cortisol; cortisol assessment; cortisol correlation
Łukasz Cieszyński, Department of Endocrinology and Internal Medicine, Medical University of Gdansk,
Debinki 7, 80–952 Gdansk, Poland; tel/fax: (+48) 58 349 28 40; e-mail: lukaszdoc@wp.pl
539
Hair cortisol correlation Łukasz Cieszyński et al.
The determination of cortisol in saliva reflects its and sebum production by androgens), age (slower hair
free fraction (free salivary cortisol, FSC) because its growth with age), dyeing hair (possible leaching of
presence in saliva is the result of passive diffusion of cortisol by chemical agents, interference in the assay),
SFC from the blood into saliva. The same amount of cor- physical activity, hygiene habits (frequency of hair
tisol is converted to cortisone in the salivary glands by washing), or exposure to sunlight [19–25]. One study
11b-hydroxysteroid dehydrogenase type 2 (11bHSD2). demonstrated the possibility of cortisol production by
Therefore, FSC concentrations are lower than serum hair bulb cells [26]. HCC determinations are performed
cortisol. It is assumed that FSC constitutes about 50–70% by liquid chromatography tandem-mass spectrometry
of SFC [10]. A saliva sample collection is a stress free (LC-MS/MA) or immunoenzymatic assays [27, 28]. As-
non-invasive procedure, and it does not require the sessment of HCC in healthy volunteers versus standard
ORIGINAL PAPER
involvement of medical personnel; hence, it is cost blood cortisol measurement methods was presented
effective and minimises the impact of stress-induced in our previous work [29]. Here we present analysis of
hypercortisolaemia related to hospitalisation and inva- HCC correlation with cortisol concentrations in blood,
sive derivation of blood samples. Furthermore, cortisol saliva, and urine in individuals with no HPA disorders.
in saliva is stable at room temperature for up to seven
days, which allows a sample to be taken at any time Material and methods
of the day of night. This in turn allows easy analysis
of the circadian rhythm of cortisol. All this makes the All participants gave written informed consent. The study was
approved by the Bioethics Committee at the Medical University
assessment of cortisol in saliva a very good screening
of Gdansk.
test for cortisol level deviation [11, 12]. Fifty adult volunteers with a negative history of endocrine disor-
In the past, urine free cortisol (UFC) was recognised ders were enrolled into the study. Subjects treated with GCS over
as the main test (gold standard) in cortisol secretion the past year prior to the enrolment were excluded from the study.
Whole blood (3 mL) was collected at 8.00 a.m. and 8:00 p.m. on
disorder assessment. Currently it is used as a comple- the same day to assess serum total cortisol concentration in the
mentary assay. Similar to salivary cortisol, UFC con- morning (morning serum cortisol, MSC) and evening (evening
centrations are also significantly reduced due to the serum cortisol, ESC), respectively. In order to assess the daily
excretion of UFC, 24 h urine collection under medical supervi-
renal activity of 11bHSD2 [13]. Difficulties in assessing
sion was done, and 5 ml of urine sample was immediately sent
UFC include frequent interference of determinations for analysis. Saliva collection was carried out using a dedicated
with drugs or GCS metabolites, such as prednisolone, kit at 8 p.m. (evening free salivary cortisol, EFSC). The obtained
methylprednisolone, spironolactone, dexamethasone, material was frozen at –32°C and stored for further analysis. After
collecting samples for MSC, ESC, UFC, and EFSC, 1 mg overnight
cortisone, 17-hydroxyprogesterone, or carbamazepine. dexamethasone suppression test (1mg ONDST) was performed.
In addition, patients’ self-urine collection is not always Eventually, 41 volunteers were included in the study, whose cor-
properly performed, which in turn might affect UFC re- tisol concentration in the 1 mg ONDST were < 50 nmol/L, and
cortisol levels in serum, saliva, and urine were within reference
sults. In patients with renal failure (glomerular filtration
ranges (Tab. 1).
rate [GFR] < 30 mL/min), it is possible to obtain false The hair sampling technique consisted of cutting off approximately
negative results. Due to these disadvantages, instead of 100 hair strands from the posterior vertex area of the head with
UFC assessment it has been suggested to measure the a sterile scalpel close to the scalp. Next, 1 cm hair fragments were
cut away close to their follicles, and after weighing 20 mg samples
cortisol:creatinine ratio in a single urine sample [14]. they were sealed in paper envelopes until further analysis. In
Hair cortisol concentration (HCC) assessment is not HCC assessment, the hair samples were placed into a 10 ml plastic
routinely performed due to a number of pre-laboratory tube and flushed with methanol. Next, they were transferred into
5 ml tubes and incubated at 50°C in methanol for 24 hours. The
impairing factors. The first determinations of HCC in extract was transferred into a 3 mL tube for methanol evaporation.
humans were described in 2004 [15]. Considering the Subsequently, the residue was dissolved in 250 µL of phosphate
average hair growth of 1 cm per month, a hair sample, buffered saline (PBS) by incubation for 1 hour and sent for cortisol
depending on its length, might reflect cortisol levels
over the last days, weeks, or months [16]. An additional
Table 1. Reference ranges of the laboratory assay
benefit of the HCC assessment is the non-invasive
sample collection, and the ability to store it at room Assay Reference ranges
temperature for an extended period of time without 1 mg ONDST < 50 nmol/L
any preparation [17]. The presence of cortisol in hair, Morning serum cortisol 138–690 nmol/L
like other endogenous substances and xenobiotics, is the
Evening serum cortisol 55–331 nmol/L
result of the passive diffusion of free cortisol from the
Evening free salivary cortisol 1.10–11.32 nmol/L
blood to the hair matrix during its formation in the bulb
[18]. In addition, a certain amount of cortisol may come Urine free cortisol 138–524 nmol/24 hr
from sweat and sebum. Therefore, HCC may depend on Hair cortisol concentration Not validated
additional factors, such as gender (stimulation of sweat 1 mg ONDST — 1 mg overnight dexamethasone suppression test
540
Endokrynologia Polska 2020; 71 (6)
Table 2. Characteristics of the population. Data presented as mean ± standard deviation (SD) and median ± interquartile
range (IQR)
ORIGINAL PAPER
Evening free salivary cortisol 2.09 ± 1.18 2.07 ± 1.21 2.16 ± 1.18
0.99
[nmol/L] (1.78 ± 0.99) (1.83 ± 0.91) (1.71 ± 1.55)
Hair cortisol concentration 0.81 ± 0.48 0.70 ± 0.28 1.08 ± 0.73
0.19
[nmol/mg] (0.65 ± 0.3) (0.65 ± 0.26) (0.78 ± 0.75)
1 mg ONDST — 1 mg overnight dexamethasone suppression test
measurement. Finally, HCC obtained in the samples was calculated nmol/mg, and the average value was 0.8125 ± 0.4834
for 1 mg hair specimens (nmol/mg).
nmol/mg. The characteristics of the studied group are
All cortisol assessments were performed in the Central Clinical
Laboratory of the Medical University of Gdansk, Poland. Com- presented in Table 2.
mercial ELISA kit (IBL International GmbH, Hamburg, Germany, No significant correlations were found between
catalogue number RE 52611) was used for the MSC, ESC, EFSC, HCC and EFSC (rho = 0.1005, p = 0.532), HCC and
and HCC measurements. According to the manufacturer’s data,
cross-reactivity was determined to be 30% for prednisolone, 7% for
MSC (rho = 0.04419, p = 0.7838), HCC and ESC
11-desoxycortisol, 4.2% for cortisone, 2.5% for prednisone, 1.4% for (rho = –0.2071, p = 0.1938), and HCC and UFC
corticosterone, and < 1% for other test substances. The detection (rho = 0.1793, p = 0.262) (Fig. 1).
threshold was set at 0.138 nmol/L, with functional sensitivity set at The general linear model was fitted to perform
0.828 nmol/L. The intra and interassay coefficient of variation was
determined at 7.3% for concentrations 7.452 nmol/L and 8.8% for multivariate analysis. The dependent variable was the
concentrations 14.904 nmol/L (saliva), and 9.9% for concentrations concentration of cortisol in the hair. Independent vari-
49.68 nmol/L and 20% for concentrations 41.4 nmol/L (serum). ables for the model were selected by the step method
Urine free cortisol determinations were made with the set from the
based on the AIC (Akaike Information Criterion) val-
same manufacturer (IBL International GmbH, Hamburg, Germany,
catalogue number RE52241). In UFC measurements, cross-reactivity ues. However, a statistically significant linear regression
was determined at 18.7% for 11-alpha-desoxycortisol, 10.8% for model could not be fitted.
cortisone, 2.4% for corticosterone, and < 0.1% for the other sub-
stances. The declared intra assay variation was 7%, and the inter
assay variation was < 9%. Cortisol measurements in saliva and Discussion
hair were done twice, and arithmetic average values were used
in statistical analysis. Several publications have indicated a strong relation-
ship between serum cortisol and salivary cortisol [6,
Results 30–34]. It is a result of the simultaneous cortisol secretion
into the blood by the adrenal glands and subsequent
The assessment of variable distribution was performed cortisol diffusion from blood into saliva. Hence, salivary
by using the quantile-quantile plots. The t-test and cortisol correlates with cortisol concentrations in blood
the Mann-Whitney U test were used for comparisons at a given time point. On the other hand, correlations
between subgroups. Correlations were analysed by the of HCC with cortisol concentrations in blood, urine,
Spearman test. The general linear model was fitted to or saliva have been assessed as moderate or weak
perform multivariate analysis. Some variables were [35]. Our results have shown no correlation between
transformed before inclusion into the linear model. HCC and cortisol concentrations in saliva, urine, and
Values of p < 0.05 were considered to be statistically sig- blood. The passive diffusion of free cortisol from the
nificant. R Studio software (RStudio Inc., Boston, USA, blood into the hair bulb is the main mechanism of
ver. 1.2.1335) was employed for the statistical analysis. the presence of cortisol in hair. Thus, HCC reflects the
The values had a normal distribution for: age average concentration of serum cortisol over a given
(p = 0.5999), MSC (p = 0.3008), ESC (p = 0.3725), and period of time depending on the length of hair used
UFC (p = 0.6905). A non-normal distribution was de- for analysis. Indeed, statistically significant correlations
tected for: 1mgONDST (p = 0.2741), EFSC (p = 0.9886), between HCC and multi-point measurements of urine
and HCC (p = 0.1873). HCC ranged from 0.3036 to 2.65 and salivary cortisol have been reported [35–38]. Our
541
Hair cortisol correlation Łukasz Cieszyński et al.
500 6
400
EFSC [nmol/L]
4
300
200
2
100
400 200
ESC [nmol/L]
MSC [nmol/L]
300 150
200 100
50
100
–1.0 –0.5 0.0 0.5 –1.0 –0.5 0.0 0.5
HCC [nmol/mg] HCC [nmol/mg]
Figure 1. Correlation analysis between hair cortisol concentration (HCC) vs. urinary free cortisol (UFC), evening free salivary cortisol
(EFSC), morning serum cortisol (MSC), and evening serum cortisol (ESC). rho — Spearman’s rank correlation coefficient
results are in turn consistent with a number of studies fecting the HPA axis were eliminated, such as social
assessing the relationship between HCC and single status, anthropometric parameters, or emotional state
point cortisol measurement in saliva and serum where [41–44]. The impact of these factors is prominent [22].
such correlations were weak or absent [36, 38, 39]. In Assessed cortisol levels reflected various time inter-
our opinion, despite progress in laboratory diagnostics, vals: minutes and hours (blood, saliva), hours and
including cortisol assays, it is still challenging to transfer days (urine), and days and months (hair). Such a wide
complex regulation of cortisol production in vivo to time slot of determination of cortisol concentrations
relatively simple statistical models based on laboratory may better reflect the variability of its concentrations
tests. Cortisol is secreted in a pulsating manner that is in analysed individuals. Additionally, we assessed
subject to circadian rhythms, and negative and posi- the patients’ clinical status during the year preced-
tive feedback may depend on exogenous factors and ing enrolment in the study. The studied group was
numerous genetic variants determining the sensitivity not large; however, it was homogeneous in terms of
of the cortisol receptor, which in turn, might alter its hormonal status.
level and impact on clinical manifestations [6]. This The study has one major limitation, which is that
may lead to misinterpretation of laboratory results a relatively small population was studied. This was
and incorrect diagnosis [8, 9]. Another problem raised due to the protocol of the study, which included strict
by many other authors is the lack of sufficient valida- exclusion criteria. Any potential factors that might
tion of studies assessing the correlation of HCC with have impacted the HPA axis excluded individuals
other biological samples [20, 36, 40]. Additionally, the from the study. Hence, we tested only individuals
possibility of pre-analytical errors, especially for more with no HPA axis impairment. The enrolment was
complex determinations such as HCC, should be taken performed in a hospital where many patients are
into account. given to steroid treatment or suffer from diseases
For instance, the impact of substances used in hair impacting glucocorticosteroids levels. However, the
care has not been fully evaluated. Interestingly, we studied population was small, it was very carefully
did not see any difference in HCC between males and selected, and represented individuals with no disor-
females despite the fact that the latter population prob- ders of the HPA axis, which is a major advantage of
ably use substances such as hair bleach more frequently. our study. We believe that if the correlation across
Unlike other studies, our enrolment protocol was saliva, blood, and hair cortisol was strong, it would
based on a preliminary assessment of cortisol levels be displayed even in a relatively small population.
in routine laboratory determinations of saliva, blood, Indeed, our data are consistent with other studies
and urine. Therefore, a number of external factors af- showing no such correlations.
542
Endokrynologia Polska 2020; 71 (6)
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