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Psychoneuroendocrinology (2009) 34, 163171

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n

Salivary cortisol as a biomarker in stress research


Dirk H. Hellhammer a,*, Stefan Wust a,b, Brigitte M. Kudielka a,c

a
Psychology, Department of Clinical and Physiological Psychology, University of Trier, Johanniterufer 15, 54290 Trier, Germany
b
Department of Genetic Epidemiology in Psychiatry, Central Institute of Mental Health, J5, 68159 Mannheim, Germany
c
Jacobs Center on Lifelong Learning and Institutional Development, Jacobs University Bremen,
Campus Ring 1, 28759 Bremen, Germany

Received 15 July 2008; received in revised form 30 October 2008; accepted 31 October 2008

KEYWORDS Summary Salivary cortisol is frequently used as a biomarker of psychological stress. However,
Stress response; psychobiological mechanisms, which trigger the hypothalamuspituitaryadrenal axis (HPAA)
Cortisol; can only indirectly be assessed by salivary cortisol measures. The different instances that control
CRF; HPAA reactivity (hippocampus, hypothalamus, pituitary, adrenals) and their respective
AVP; modulators, receptors, or binding proteins, may all affect salivary cortisol measures. Thus, a
ACTH linear relationship with measures of plasma ACTH and cortisol in blood or urine does not
necessarily exist. This is particularly true under response conditions. The present paper addresses
several psychological and biological variables, which may account for such dissociations, and aims
to help researchers to rate the validity and psychobiological significance of salivary cortisol as an
HPAA biomarker of stress in their experiments.
# 2008 Elsevier Ltd. All rights reserved.

1. Introduction observed. This paper describes such dissociations and puta-


tive mechanisms. We aim to illustrate the physiological
Today, salivary cortisol is routinely used as a biomarker of significance of salivary cortisol as a biomarker of HPAA
psychological stress and related mental or physical diseases. responsivity by addressing several sources of variance con-
Most studies consider salivary cortisol levels a reliable mea- tributing to such dissociations.
sure of hypothalamuspituitaryadrenal axis (HPAA) adapta-
tion to stress. However, the stress response of the HPAA is 2. Dissociations with CRF/AVP
rather complex and modulated by numerous factors. Cortisol
levels in saliva are partly dissociated from levels of para- The process between the initiation of an HPAA response in the
ventricular corticotrophin releasing factor (CRF), arginine central nervous system and salivary cortisol variations as an
vasopressin (AVP), adrenocorticotropic hormone (ACTH), and outcome measure is modulated by numerous psychological
cortisol in blood or urine. Under several circumstances a and biological events. Psychological events initiate an HPAA
partial but significant dissociation between salivary cortisol response by predominantly activating CRF/AVP neurons in
levels and other HPAA related endocrine signals can be the paraventricular nucleus (PVN) of the hypothalamus (see
Chrousos and Kino, 2007 for a review). The degree of activa-
* Corresponding author. Tel.: +49 651 2012928; tion will vary, depending on the psychological components
fax: +49 651 2012934. of the situation, such as unpredictability, uncontrollability,
E-mail address: albertz@uni-trier.de (D.H. Hellhammer). novelty, anticipation, ego-involvement, habituation, etc.

0306-4530/$ see front matter # 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.psyneuen.2008.10.026
164 D.H. Hellhammer et al.

(Mason, 1968; Kudielka et al., 2007). Furthermore, classically HPAA hyporeactivity in chronically stressed individuals seems
conditioned stimuli may also elicit CRF mediated HPAA to be likely. For instance, aspects of chronic work stress were
responses (Kreutz et al., 1992). In addition, whenever the associated with a dampening of the HPAA response to the
brain needs energy to fulfill its function it needs to initiate TSST (Bellingrath and Kudielka, 2008), while perceived
HPAA activity in order to allocate glucose to the brain. This is chronic stress was related to an elevation of salivary cortisol
particularly true under stressful conditions (Peters et al., after awakening (Schulz et al., 1998; Wust et al., 2000b;
2004). The interplay of these factors already accounts for Pruessner et al., 2003; Watts, 2005). In a study by Bellingrath
some variability. et al. (2008) enhanced cortisol suppression to a low dose of
Interestingly, it is generally assumed that endocrine and dexamethasone in a much larger study sample was also
psychological stress responses represent indicators of the observed. Dexamethasone mainly affects glucocorticoid
same construct and thus a high psycho-endocrine covariance receptors in the pituitary. However, both parvocellular CRF
should be expected. On a neuroanatomical level this hypoth- and AVP are also under inhibitory control of glucocorticoids
esis is corroborated by close links between the HPAA and (Erkut et al., 1998) and the glucocorticoid receptor may thus
cortical and limbic structures, which are important media- primarily modulate the dynamic response to acute and
tors of subjective-psychological stress responses (Feldman chronic psychological stress.
et al., 1995; Lopez et al., 1999; Buijs and Van Eden, 2000; Under stress conditions, the interplay between the PVN
Heckmann et al., 2005; Herman et al., 2005; Wang et al., and the noradrenergic neurons arising from the locus coer-
2005). However, the analyses of psycho-endocrine covariance uleus (LC) is most crucial (Chrousos and Kino, 2007). Liu et al.
in studies using a variety of stressors and subjects have (1994) provided evidence that CRF/AVP neurons are acti-
yielded inconsistent and largely negative results (alAbsi vated by noradrenergic or neuropeptide Y neurons, resulting
et al., 1997; Buchanan et al., 1999; Cohen et al., 2000; in an increased AVP/CRH ratio, and their data suggest a major
Oswald et al., 2004). Considering the time lag between involvement of this interplay in depression and stress related
psychological and endocrine stress responses probably disorders. Under these circumstances, paraventricular CRF/
enlarges the detectable covariance (Smyth et al., 1998; AVP neurons activate sympathetic preganglionic neurons in
Schlotz et al., 2008), but the proportion of explained var- the spinal cord, which control cortisol release from the
iance is nevertheless small (Hellhammer and Hellhammer, adrenals and may mainly drive the circadian rhythm (Enge-
2008). Although beyond the scope of this manuscript, it land and Arnhold, 2005). In turn, ACTH does not only stimu-
should be noted that not only neuroendocrine factors late the release of cortisol from the adrenals, but, in
account for the limited association between cortisol and addition, regulates gene expression of Norepinephrine (NE)
subjective-psychological stress responses, but also factors biosynthetic enzymes in the superior cervical ganglia and
related to the assessment of perceived stress by self-report locus coeruleus (LC) by an adrenal independent mechanism
methods. Amongst others, stress questionnaires differ sub- (Serova et al., 2008). In sum, the interplay between CRF/AVP
stantially in underlying theoretical stress concepts and foci, neurons, ACTH and other peptides, as well as noradrenergic
and several personality traits (e.g. neuroticism, locus of and sympathetic activation contributes to the missing covar-
control, general affectivity) as well as gender are known iance between psychological measures of stress and salivary
to impact on self-reported stress levels. cortisol levels.
Hypothetically, it should be expected that a stress- Sex steroids are another important modulator of HPAA
induced activation of CRF/AVP neurons in the PVN results measures. The influence of sex steroids on central HPAA
in a subsequent linear release of ACTH from the pituitary and activity is rather complex, only partially understood and
of cortisol from the adrenals, as finally represented by has predominantly been studied in rodents. For instance,
salivary cortisol levels. However, research investigating the the affinity of hippocampal mineralocorticoid receptors (MR)
linearity between CRF and ACTH release suggests that only for glucocorticoids is significantly higher in male than in
part of the variance of ACTH and glucocorticoid release can female rats (Turner, 1997). Progesterone seems to decrease
be explained by CRF release under stimulated (Carlson et al., the affinity of hippocampal MRs while estrogens decrease MR
2007) and unstimulated conditions (Watts, 2005). The role of binding capacity (Carey et al., 1995). Testosterone was
AVP remains to be fully elucidated. In animals, both peptides shown to enhance glucocorticoid receptor (GR) binding in
seem to act synergistically, with CRF playing a permissive, the preoptic area of the hypothalamus (Viau and Meaney,
and AVP a dynamic role (Plotsky, 1991). Again, it is not yet 1996) and the progesterone receptor binds to hormone
fully clear, how acute or chronic psychological stress differ- responsive elements on the DNA with the same affinity as
entially affects these peptides, and which role each of them the GR and the MR (von der Ahe et al., 1985). Estrogens and
plays in ACTH release under stress conditions in humans. dihydrotestosterone inhibit GR and MR expression in several
However, the multiplicity of central factors regulating the tissues (Patchev and Almeida, 1996). Progesterone was found
HPAA response may well account for response differences to modulate the inhibitory estrogen effect on the GR and
among individuals and occasions. For instance, women in the enhances MR mRNA transcription (Castren et al., 1995;
follicular phase of the menstrual cycle show smaller ACTH Patchev and Almeida, 1996). Recent studies provided further
and salivary cortisol responses to the TSST than men, while convincing evidence for a significant impact of gonadal ster-
women (irrespective of cycle phase) show higher salivary oids on HPAA regulation. Male rats that were prenatally and
cortisol levels 4560 min after awakening (Kirschbaum et al., at an early postnatal stage treated with either an antiandro-
1996, 1999; Wust et al., 2000a). In animal studies, chronic gen or a drug, which inhibits aromatization of testosterone to
stress results in a shift towards an increased AVP/CRF ratio in estradiol, showed a feminized HPAA including enhanced
parvocellular neurons (for review see Dinan and Scott, 2005). corticosterone levels (basal and in response to challenge)
Findings in humans are currently not consistent but a certain as well as elevated CRF mRNA levels in the PVN and proo-
Salivary cortisol as a biomarker in stress research 165

piomelanocortin mRNA levels in the adenohypophysis (Seale Interestingly, physical challenge and psychological stress
et al., 2005b). An analogous HPAA masculinization was seem to exert different effects: physically demanding,
observed in female rats that were exposed to a testosterone life-threatening stressors result in an augmented adrenal
propionate (TP) injection early after birth. Remarkably, in response, whereas psychological, anxiety-producing stres-
adult TP rats these effects on HPAA regulation could be sors result in a diminished response. These findings suggest
reversed by estradiol treatment (Seale et al., 2005a). How- that an extra-ACTH mechanism shapes the adrenal response
ever, it is important to note that sex steroid effects on in exercising animals, providing elevated corticosteroids
processes related to HPAA regulation often depend on the when needed to withstand physical insults, but reduced
experimental design, the scrutinized brain region and the sex corticosteroid when exercise alone is sufficient to offset
of the studied animal. In a recent review Federman (2006) psychological challenges (Bornstein et al., 2008).
concludes that due to the pronounced specificity of present Studies assessing changes of the circadian slope as a
receptors, coactivators, corepressors and enzymes, each measure of stress load may consider that light induces the
target tissue can have its own androgenic or estrogenic expression of clock genes in the adrenal gland independent of
identity. Moreover, it has been shown that cortisol binding ACTH release. In this context also the cortisol awakening
globuline (CBG) levels are influenced by circulating gonadal response (CAR) should be mentioned. Pruessner et al. (1997)
steroids (Kajantie and Phillips, 2006) and that CBG synthesis were the first who proposed that the repeated assessment of
is regulated by glucocorticoids (Cole et al., 1999). In humans this cortisol increase after awakening in saliva might repre-
the use of ethinyl-estradiol containing oral contraceptives sent a useful and easy to measure index of cortisol regulation.
(OC) stimulates CBG synthesis (Wiegratz et al., 2003) result- Later the term CAR was coined (Federenko et al., 2004) and
ing in higher total cortisol levels. The observation of a in most studies the CAR was observed to be an increase in
blunted salivary cortisol response following psychosocial salivary cortisol levels of about 5075% within 3045 min
and pharmacological stimulation was related to the CBG after awakening. The CAR is increasingly used in psychoneur-
enhancing effect of ethinyl-estradiol (Kirschbaum et al., oendocrinology as an indicator of HPAA activity and a com-
1999). This assumption was recently confirmed by the obser- prehensive overview of findings related to the CAR is well
vation that CBG levels were negatively associated with ACTH beyond the scope of the present report (for current reviews
and salivary cortisol responses to the TSST but positively with see Clow et al., 2004; Chida and Steptoe, in press; Fries
total cortisol levels (Kumsta et al., 2007a). Diminished ACTH et al., in press). Interestingly, a certain dissociation between
responses might be explained by changes in pituitary CRF ACTH and cortisol seems to become detectable at the end of
receptors. In the course of OC intake and the concomitant the sleep phase. We could recently show that within a 30 min
and unphysiological increase of CBG, a larger fraction of free interval after awakening plasma ACTH and serum cortisol
cortisol will be bound, resulting in a permanently enhanced levels rose steeper than in the last hours before morning
activation of CRF neurons in the PVN to maintain comparable awakening (Wilhelm et al., 2007). Our data indicate conclu-
free basal cortisol levels. An increased CRF drive could sively that the increased secretory activity of the HPA axis
eventually result in a down-regulation of CRF receptors on after morning awakening is at least in part caused by the
pituitary corticotrophs. This assumption is consistent with process of awakening itself, i.e., represents a genuine
the observed blunted ACTH responses to CRF administration response to awakening. However, we also found that in
in OC users compared to non-users (Jacobs et al., 1989). the last hour before awakening ACTH levels showed a trend
Remarkably, an impact of CBG as a regulatory element of towards a steeper rise as compared to cortisol levels. This
HPAA responses is not limited to women with exogenously up- finding might be suggestive of a partly dissociated regulation
regulated CBG levels. In men, positive associations were before awakening and it is in line with a study by Born et al.
observed between CBG and ACTH as well as total cortisol (1999) who reported a significant ACTH but not cortisol
levels following the TSST (Kumsta et al., 2007a). increase in the last hour of sleep in subjects being woken
rather early. All this information is important in order to
3. Dissociations with ACTH understand that ACTH responses to stress explain only part of
the variance of salivary cortisol response measures. In 212
In a recent review, Bornstein et al. (2008) summarized dis- young healthy males and females (all using oral contracep-
sociations of ACTH and glucocorticoids in critical illness, tives; Kumsta et al., 2007b) the correlation between ACTH
inflammation, and mental disorders. They name numerous and salivary cortisol responses to the TSST was r = .54 for the
factors, such as neuropeptides, neurotransmitters, opioids, area under the curve (AUC) and r = .66 for the response peak.
growth factors, cytokines, adipokines, and bacterial ligands, After premedication with 0.25 mg dexamethasone early
which modulate adrenal glucocorticoid release indepen- morning ACTH levels showed a correlation of r = .35 (AUC)
dently of pituitary ACTH. Thus, it should be noted that the and r = .28 (peak) to the salivary cortisol awakening
salivary cortisol response to stress can be confounded by a response. All these correlations were statistically significant
broad array of intervening variables. but when the proportion of explained variance is taken into
In this review, Bornstein et al. (2008) showed that the account (between 8 and 44%) it becomes obvious that ACTH
adrenal release of cortisol during the circadian slope and the and salivary cortisol are not interchangeable measures.
stress response are under dual control of both ACTH and
preganglionic sympathetic neurons. Other studies suggest 4. Dissociations with blood cortisol levels
that reward can dampen adrenal glucocorticoid release
independent of ACTH release and that several factors mod- There is a high correlation between salivary cortisol levels
ulate ACTH sensitivity of the adrenals. Among those factors and unbound free cortisol levels in plasma and serum, which
are chronic stress, long-term exercise, and gonadal steroids. remains high during the circadian cycle and under different
166 D.H. Hellhammer et al.

dynamic tests, such as dexamethasone suppression and ACTH


stimulation (Vining et al., 1983b; Umeda et al., 1981; Wede-
kind et al., 2000; Levine et al., 2007). Since free cortisol
represents the biologically active hormone fraction, salivary
cortisol measures have early been considered to be a better
measure of adrenocortical function than serum cortisol (Vin-
ing et al., 1983a). Recently, the free hormone hypothesis has
been challenged, and it has been suggested that CBG-bound
cortisol may also have physiological effects on target tissues,
as recently reviewed by Levine et al. (2007).
The proportion of salivary cortisol to total cortisol is about
12% in the lower range, but about 89% in the upper range.
Thus, salivary cortisol levels have to be treated with caution,
since they will not behave linear to serum levels in response
to a challenge or under conditions which affect CBG levels,
such as oral contraceptives, menstrual cycle or pregnancy.
The salivary cortisol assay used by Vining and co-workers
showed such aberrations already for cortisol levels above
10 nmol/l. This is an important issue, which is often
neglected in psychobiological research.
However, the relation between total and free cortisol
levels in blood changes once CBG is saturated (Vining
et al., 1983a). Fig. 1 shows simultaneous measures for sali-
vary and serum cortisol in men, women, and women taking
oral contraceptives. In men (Fig. 1a), CBG seems to be
saturated when serum cortisol levels range between 450
and 500 nmol/l. Sex steroids enhance CBG levels (see above),
which may result in a broader variability of cortisol levels in
women (Fig. 1b), and in a far higher proportion of serum
cortisol to salivary cortisol levels in women taking oral con-
traceptives (Fig. 1c).
This situation has practical implications, which can be
illustrated by the following example: if an HPAA response is
assessed by cortisol in saliva, the magnitude of this response
will differ with respect to CBG saturation. For instance, an
increase of salivary cortisol of only 5 nmol/l would be
detected if total cortisol levels increased from 200 to
400 nmol/l in response to a challenge. But the same total
cortisol increase of 200 nmol/l from 500 to 700 nmol/l would
result in a threefold higher increase of about 15 nmol/l. In
the salivary cortisol assay we are employing in Trier, this
problem of non-linearity affects probably only relatively high
salivary cortisol levels (Fig. 1a and b); for example, mean
TSST responses or mean daytime cortisol peak levels are
usually below the mean CBG saturation range. Surely, salivary
cortisol levels have to be interpreted with caution (a) under
conditions affecting CBG levels, such as oral contraceptives,
menstrual cycle or pregnancy and (b) in response to pharma-
cological or strong psychological challenges.
Salivary cortisol levels do not just reflect a linear adre-
nocortical response to ACTH or adrenergic stimulation, but
vary with different determinants, which have been shown to
be relevant in psychobiological research. For example, pre-
and early postnatal adversity have been reported to affect Figure 1 Simultaneous measures for salivary and serum corti-
cortisol availability by two different mechanisms. First, the sol in (a) men (744 measures), (b) women (100 measures), and (c)
adrenal capacity to mount a cortisol response may be women taking oral contraceptives (920 measures).
impaired, and secondly, the degree of cortisol metabolism
in the liver and visceral fat may be affected (Buffington, et al., 2006). In sum, total cortisol levels cannot be consid-
2004; Yehuda et al., 2006; Klingmann and Hellhammer, 2008; ered a passive bridge between the ACTH and salivary
Klingmann et al., in press). In addition, the hair follicle is able cortisol response to stress, but rather a considerable source
to synthesize CRF, ACTH, and cortisol, and it is not yet known, of variance, particularly under hyper- and hypocortisolemic
how much this source contributes to total cortisol levels (Paus conditions (Fries, 2008; Putz, 2008).
Salivary cortisol as a biomarker in stress research 167

Despite the conditions mentioned above, salivary cortisol sol (THF), 5beta-tetrahydrocortisone (THE), alpha-cortol,
levels can be expected to correlate reasonably well with beta-cortol, alpha-cortolone, and beta-cortolone, encom-
total cortisol levels in the upper or lower range, as defined by pass almost 80% of the cortisol secreted by the adrenal gland
CBG binding. For instance, in the above mentioned cohort of (Stewart and Krozowski, 1999).
212 healthy males and females the correlation between
serum cortisol and salivary cortisol responses to the TSST
was r = .47 (AUC) and r = .58 (peak), respectively. However, 7. Dissociations with effects on target
both ranges need to be treated separately. tissues

5. Dissociations with free cortisol levels in The measurement and interpretation of (salivary) cortisol
blood levels in stress research is often based on the implicit
assumption that, firstly, a given cortisol level reflects a
certain effect across different GC target tissues within one
Although most studies report a high correlation between free
subject and that, secondly, a given level of cortisol elicits
cortisol levels in blood and saliva, some studies call for
comparable target tissue effects in different subjects. Both
caution. Levine et al. (2007) summarize such studies, report-
aspects of this assumption are not necessarily correct. It is
ing dissociation between both measures during the circadian
well documented that within the normal population, a con-
circle, under challenge conditions, and with respect to the
siderable variability in the sensitivity to glucocorticoids
magnitude of free cortisol levels. Referring to data which
across individuals exists (Baxter and Rousseau, 1979; Hui-
suggest that about 14% of salivary cortisol is bound to CBG in
zenga et al., 1998). Furthermore, it has been shown that GC
saliva, they assume that this may be one important reason
sensitivity of one target tissue does often not reflect GC
behind such dissociations. In addition, about 30% of free
sensitivity of other organs in both patients receiving GC
cortisol is enzymatically converted to cortisone in saliva,
treatment (Corrigan et al., 1991, 1996; Sher et al., 1994;
thus resulting in relatively lower levels of free cortisol in
Caldji et al., 1998) and in healthy individuals (Ebrecht et al.,
saliva as compared to plasma.
2000; Vasiliadi et al., 2002). It is well beyond the scope of this
However, given the numerous advantages in the stress-
manuscript to summarize all putative molecular sources of
free collection and processing of salivary cortisol and the
this variability. However, one important determinant of
reasonable overall correlation between free cortisol in saliva
magnitude and efficacy of GC action are characteristics of
and blood, we feel that salivary measures are the method of
the GR. Rare clinical abnormalities in GC sensitivity, such as
choice in stress research, at least for the assessment of free
the generalized inherited GC resistance syndrome, have been
cortisol levels.
linked to mutations of the GR (Charmandari et al., 2004).
More common polymorphisms of the GR gene have been
6. Dissociations with urinary cortisol levels associated with variability in sensitivity to exogenous GCs
in the general population (Koper et al., 1997; Huizenga et al.,
Free cortisol is metabolized in the liver and about 70% of these 1998; Panarelli et al., 1998; DeRijk et al., 2001; van Rossum
biologically inactive metabolites are excreted in urine, about et al., 2002, 2003; Fleury et al., 2003; Wust et al., 2004).
20% (via bile) in stool, and about 8% in the skin. In urine, only Moreover, GR gene variants (and probably also polymorph-
about 1% of free blood cortisol is excreted (Hatz, 1998). In the isms in other genes) cannot only partly explain differences
early stress literature, cortisol conjugates and metabolites between two individuals, they can also mediate GC effect
(17-hydroxycorticosteroids and 17-ketosteroids) were com- differences between different target tissues within one indi-
monly used as HPAA measures, before new assays were avail- vidual. For instance, we have recently observed an associa-
able allowing precise assessment of cortisol in blood and tion between a certain GR genotype (BclI GG) and measures
saliva. Overnight or 24 h urine measures have the advantage of GC sensitivity of the pituitary and subdermal blood vessels,
to provide integrative HPAA measures over larger time periods, but we did not detect an association with the GC sensitivity of
although the compliance is poor, particularly for 24 h mea- peripheral leukocytes (Kumsta et al., 2008). The molecular
sures. Furthermore, renal conditions may specifically affect basis for the observed variability in GC responsiveness can be
urinary cortisol levels. Thus, creatinine clearance is usually partially attributed to existence of isoforms of the GR result-
assessed for the adjustment of urinary cortisol levels. More ing from alternative splicing, alternative translation initia-
than 95% of cortisol is metabolized before excretion. Free tion and posttranslational modifications (Lu and Cidlowski,
cortisol can thus not only be considered a consequence of 2006). Different cellular environments are likely to result in
cortisol production, but, in addition, of cortisol clearance in differential expression of GR isoforms, all possessing unique
the liver. In patients with chronic fatigue, for example, low transcriptional regulatory profiles. Tissue specific GR isoform
salivary cortisol levels have been discussed to be associated compositions could determine the cell specific response to
with a faster clearance of cortisol (Jerjes et al., 2006). glucocorticoids thus accounting for the diverse and specific
However, it should be noted that (if the subjects com- effects of GCs (Lu and Cidlowski, 2006). The molecular
pliance is ensured) urine analyses can be a useful and valid mechanisms underlying GR isoform generation have been
approach to assess glucocorticoid secretion if not only urinary identified (Lu and Cidlowski, 2005). However, how tissues
cortisol but also cortisol metabolites are assessed, ideally direct this expression to achieve their unique cell specific
with a modern gas chromatography/mass spectrometry isoform composition is largely unknown. It has been demon-
method (Heckmann et al., 2005; Remer et al., 2008). It strated that presence of GR gene variants can impact on the
was shown that the seven major urinary cortisol metabolites regulation of isoform expression and thus alter the cellular
5alpha-tetrahydrocortisol (5a-THF), 5beta-tetrahydrocorti- composition and proportion of these isoforms.
168 D.H. Hellhammer et al.

Another presumably very important mechanism contribut- The assessment of salivary cortisol offers the opportunity to
ing to target tissue specificity of glucocorticoid action is the collect the samples stress-free, without medical personnel,
presence or absence of the enzyme 11b-hydroxysteroid dehy- and in many different environments. These advantages are of
drogenase type 1 (11b-HSD1) (Seckl and Walker, 2001). This particular relevance for ambulatory assessments, studies in
enzyme can be found in peripheral tissues (liver, lung, adi- large cohorts and for studies in children (Jessop and Turner-
pose tissue, vasculature, ovary) but it is also expressed in Cobb, 2008).
several brain regions including hippocampus, hypothalamus However, researchers who decide to assess salivary corti-
and pituitary. 11b-HSD1 catalyzes the conversion from inert sol have to consider that variables such as estrogens (gender,
11-keto forms (cortisone, 11-dehydrocorticosterone), which menstrual cycle, oral contraceptives) or medical conditions
are sufficiently present in the blood stream, into active could affect cortisol binding and HPAA responsivity. In addi-
glucocorticoids (cortisol, corticosterone). This results in a tion, the expected range of cortisol measures should be
local and specific amplification of glucocorticoid action in considered as levels are elevated once CBG binding is satu-
11b-HSD1 expressing tissues. rated.
Since alterations in GC sensitivity are implicated in the If the research focus is on free cortisol effects on target
onset and course of stress related diseases and due to the tissue, salivary cortisol could be the measure of choice.
inaccessibility of brain tissue, reliable peripheral markers However, as described above, a linear doseresponse rela-
reflecting changes in GC signaling are needed. Given the tion in terms of target cell effects should not be expected,
highly tissue specific effects, it is a clear limitation that but salivary cortisol will be the most valid parameter of HPAA
markers of GC sensitivity in peripheral tissue do not neces- activation in such studies.
sarily reflect functional changes in central GC signaling. In sum, the complexity of factors accounting for varia-
However, at least in depressed patients, there are consistent bility of stress induced HPAA activation and effects demands
findings of non-suppression in the dexamethasone suppres- a careful a priori analysis to select the right HPAA parameters
sion test and a reduced in vitro inhibition of lymphocyte and to control for the most relevant intervening variables.
proliferation in response to dexamethasone in the same Such an approach will help to design adequate experimental
patients (Pariante, 2004). Recently, Carvalho et al. (2008) designs and to create a data set, which allows for ideal
have shown that patients resistant to antidepressant treat- testing of the research hypotheses.
ment in vivo were also resistant to the antidepressant clo-
mipramine in vitro. Conflict of interest
Taken together, different GR gene variants can probably
have tissue specific effects on GC sensitivity, and polymorph- None declared.
ism of the GR gene might constitute a vulnerability or
protection factor for stress related disorders and altered
GC sensitivity. Thus, variability of effects on target tissues References
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