Psychoneuroendocrinology (2013) 38, 1271—1280
Available online at www.sciencedirect.com
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
Analysis of baseline hypothalamic-pituitary-adrenal
activity in late adolescence reveals gender specific
sensitivity of the stress axis
Rebecca M. Reynolds a,*, Hilary L. Hii b, Craig E. Pennell c, Ian W. McKeague d,
E. Ron de Kloet e, Stephen Lye f, Fiona J. Stanley g, Eugen Mattes b,
Jonathan K. Foster b,h,i
a
Endocrinology Unit, University/BHF Centre for Cardiovascular Sciences, Queen’s Medical Research Institute, University of
Edinburgh, Edinburgh, United Kingdom
b
Developmental Neuroscience, Telethon Institute for Child Health Research and Centre for Child Health Research, University of
Western Australia, Perth, Australia
c
The School of Women’s and Infants’ Health, University of Western Australia at King Edward Memorial Hospital, Perth, Australia
d
Department of Biostatistics, Columbia University, New York, NY, United States
e
Department of Medical Pharmacology, The University of Leiden, Leiden, The Netherlands
f
Samuel Lunenfeld Research Institute, University of Toronto, Canada
g
Population Science, Telethon Institute for Child Health Research and Centre for Child Health Research, University of Western
Australia, Perth, Australia
h
School of Psychology and Speech Pathology, Faculty of Health Sciences, Curtin University, Perth, Australia
i
School of Paediatrics & Child Health, University of Western Australia; Neurosciences Unit, Health Department of Western Australia,
Perth, Australia
Received 17 September 2012; received in revised form 7 November 2012; accepted 7 November 2012
KEYWORDS
HPA axis;
Adolescence;
Cortisol;
ACTH;
Corticosteroid binding
globulin;
The Raine Study
Summary Dysfunctional regulation of the hypothalamic-pituitary-adrenal (HPA) axis has been
proposed as an important biological mechanism underlying stress-related diseases; however, a
better understanding of the interlinked neuroendocrine events driving the release of cortisol by
this stress axis is essential for progress in preventing or halting irreversible development of
adverse HPA-function. We aimed to investigate basal HPA-activity in a normal population in late
adolescence, the time of life believed to overlap with HPA-axis maturation and establishment of a
lasting set point level of HPA function. A total of 1258 participants (mean age 16.6 years) recruited
from the Western Australian Pregnancy (Raine) Cohort provided fasting morning blood and saliva
samples for basal HPA activity assessment. Irrespective of gender, linear regression modelling
identified a positive correlation between the main components of the HPA-cascade of events,
ACTH, total cortisol and free cortisol in saliva. Corticosteroid binding globulin (CBG) was inversely
* Corresponding author. Tel.: +44 131 2426762; fax: +44 131 2426779.
E-mail address: [email protected] (R.M. Reynolds).
0306-4530/$ — see front matter # 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.psyneuen.2012.11.010
1272
R.M. Reynolds et al.
associated with free cortisol in saliva, an effect most clearly observed in boys. ACTH levels were
lower, but cortisol levels were higher in girls than in boys. Girls may also be exposed to more
bioactive cortisol, based on higher average free cortisol measured in saliva at awakening. These
relatively higher female free cortisol levels were significantly reduced by oral contraceptive use,
eliminating the gender specific difference in salivary cortisol. Free plasma cortisol, calculated from
total circulating cortisol and CBG concentrations, was also significantly reduced in girls using oral
contraceptives, possibly via an enhancing effect of oral contraceptives on blood CBG content. This
study highlights a clear gender difference in HPA activity under non-stressful natural conditions. This
finding may be relevant for research into sex-specific stress-related diseases with a typical onset in
late adolescence.
# 2012 Elsevier Ltd. All rights reserved.
1. Introduction
The primary biological mechanism underlying stress regulation and adaptation, the hypothalamic-pituitary-adrenal
(HPA) axis, regulates cortisol to maintain homeostasis (McEwen, 2003). Circulating cortisol levels vary between individuals and throughout the course of the day. More pronounced
fluctuations in cortisol are triggered by stress-induced circumstances. A healthy stress response is characterised by a
rapid rise in cortisol within minutes, followed by a return to
baseline levels within 1—2 h following stress exposure (de
Kloet et al., 2008). This phenomenon relies on the autoregulatory ability of cortisol to bind to glucocorticoid (GR) and
mineralocorticoid (MR) receptors in the pituitary and specific
limbic and cortical brain regions which influence HPA-activity
(de Kloet et al., 2008).
Aberrant HPA-function in adulthood has been associated
with adverse physical and psychological health, such as the
metabolic syndrome (Phillips et al., 2006) and depression (de
Kloet et al., 2007; Pariante and Lightman, 2008). Various
association studies in humans suggest that overexposure to
cortisol may contribute to these adverse conditions (Seckl
and Holmes, 2007); however, such studies mostly analyse
cortisol levels in patients with existing disease, making it
difficult to assign cause or consequence to this relationship
(Bremmer et al., 2007; Vreeburg et al., 2009). Moreover, the
‘normal’ range of systemic cortisol and the extent to which
levels outside this range may influence diseases remain to be
clearly defined (Herbert et al., 2006).
Our main understanding of intrinsic activity of the HPA
axis has been derived from detailed animal studies, including measurements of circulating products of the HPA axis
together with abundance of GR and MR in HPA-axis central
negative feedback sites (Van Eekelen et al., 1988, 1995;
Herbert et al., 2006; de Kloet et al., 2008). The full
translation of similar approaches to human cohort studies
is clearly hampered by issues relating to the costs and
logistics of large scale sample collection and analysis, as
well as the burden of appropriate sample collection
imposed on study participants. However, the availability
of complementary information on the activity of the human
HPA axis downstream of the pituitary (investigated by
assaying a combination of ACTH, CBG, total and calculated
free cortisol in the same individual under controlled circumstances) is feasible, and is likely to offer a better
understanding of the interlinked neuroendocrine processes
underpinning HPA function.
The aim of this study was to measure HPA axis activity
under resting conditions in late adolescence, when long-term
levels of HPA activity are established (Goel and Bale, 2009;
McCormick and Mathews, 2010). Notably, development of the
HPA-axis remains sensitive to genetic predisposition, intrinsic
biological development and extrinsic environmental exposures well into adolescence, affecting the baseline state of
HPA-function (Walker et al., 2004; Romeo et al., 2006; de
Kloet et al., 2007). Genetic variance between individuals
characterises associations between genotype and atypical
HPA function (Wust et al., 2004; DeRijk et al., 2006; DeRijk
and de Kloet, 2008). Furthermore, the onset of adolescent
development at puberty is associated with a rise in HPAactivity in the early teenage years (Walker et al., 2001; Oskis
et al., 2009), while environmental influences as early as in
infancy are known to shape HPA-function later in life (Phillips, 2006; Levine et al., 2007; Seckl and Holmes, 2007;
Weinberg et al., 2008). As a consequence, baseline and
stress-induced levels of HPA activity have a significant degree
of individual variability (Hellhammer et al., 2009).
HPA-axis maturation in late adolescence runs parallel to
the overall process of adolescent forebrain maturation
(Spear, 2004; Paus, 2005; Steinberg, 2005; McCormick
and Mathews, 2010). HPA-axis maturation also appears to
overlap with the onset of stress-related disorders (Paus
et al., 2008; Gogtay and Thompson, 2010). Among relevant
clinical conditions, psychopathology associated with
depression and schizophrenia shows significant gender differences (Goodyer et al., 2000; Highley et al., 2003; Malaspina et al., 2008; Goel and Bale, 2009). Animal studies
have also shown sexually dimorphic HPA axis responses,
with females generally showing greater cortisol responsiveness (Weinstock et al., 1992). Although studies in humans
have been less consistent, cortisol levels have generally
been higher in post-pubertal girls or adults compared with
males (Klimes-Dougan et al., 2001; Adam et al., 2010;
Netherton et al., 2004; Reynolds et al., 2005). We therefore
hypothesised in this study that gender differences in HPA
axis activity may be apparent in teenagers.
We aimed to measure a complementary set of plasma ACTH,
total cortisol, CBG, calculated free plasma cortisol and salivary free cortisol levels among more than 1000 boys and girls at
17 years of age in the population based Western Australian
Pregnancy (Raine) cohort (Newnham et al., 1993). The use of
the Raine cohort as an unselected teenage population for
assessment of detailed resting HPA function allowed us to gain
insight into the spectrum of ‘normal’ baseline HPA-activity
Resting HPA function in 17-year-old teenagers
levels among adolescents, and to examine whether there were
gender specific stress-system profiles.
2. Materials and methods
2.1. Participants
All adolescent participants were part of The Western Australian Pregnancy (Raine) cohort, a prospective populationbased pregnancy cohort study of 2868 live births occurring
between 1989 and 1991 (Newnham et al., 1993). The Raine
participants were followed from 16 weeks gestation, with
longitudinal data collection updated at 34 weeks gestation
and 1, 2, 3, 5, 8, 10, 14 and 17 years of age (including
questionnaires, physical assessments and biological sampling). Informed consent was provided by the mother at
enrolment and by one of the parents at each follow-up.
The 17-year follow-up was carried out between 2006 and
2009 and included 1771 Raine participants, representing
61.8% of the original cohort. The study was approved by
the Human Research Ethics Committees at Princess Margaret
Hospital for Children in Perth, Western Australia.
2.2. Sample collection
Biological samples were provided by 1400 subjects. A fasting
morning EDTA-blood sample was collected under non-arousing circumstances during a home visit by a Raine Study
phlebotomist. Participants refrained from eating or drinking
anything other than water from 2200 h the night before blood
collection. The date and time of blood collection before
1000 h in the morning was documented. Saliva was selfcollected by 1121 Raine participants using Salivette saliva
collection tubes (Sarstedt, Germany) 15 min after spontaneous awakening on 3 consecutive weekdays (S1-3). The
majority of participants (75.1%) provided three samples
collected on consecutive days just prior to the day of blood
collection. The participants recorded the date and time of
collection before 10 am in the morning on sample tubes,
which were all kept at room temperature until transport,
processing and storage at 80 8C.
2.3. Hormone analysis
There was sufficient sample suitable for measurement of
cortisol in 1258 subjects, ACTH in 1257 subjects and CBG in
1239 subjects (48% female). Cortisol and CBG were measured
by 125I radioimmunoassay (GammaCoat cortisol RIA — DiaSorin,
MN, USA and CBG-RIA-100 — BioSource Europe S.A., Belgium).
ACTH was measured by 125I immunoradiometric assay (ACTHIRMA; DiaSorin, MN, USA) as per the manufacturer’s instructions. All samples were assayed in duplicate. Outcomes outside
the assay standard range were re-analysed with an adjusted
sample volume. Analysis of sample duplicates which differed
more than 20% was repeated. AssayZap-3 was used for raw data
transformation; assay performance paralleled sample collection over the 3-year study period. Salivary samples S1-3 of each
individual were analysed in the same assay. The intra- and
interassay variations of 23 cortisol RIAs and 20 CBG RIAs were
8% and 14.0% (salivary cortisol), 8.8% and 13.7% (plasma
cortisol) and 3.9% and 5.5% (plasma CBG) respectively. The
1273
intra- and inter-assay variations of 19 ACTH-IRMAs were 5.3%
and 12.1% respectively. Free cortisol was calculated from total
cortisol and CBG concentrations using Coolen’s equation with
the formula U = H(Z2 + 0.0122T) Z, where Z = 0.0167 +
0.182 (G T). U, G and T represent calculated free cortisol,
CBG and total cortisol, respectively (Coolens et al., 1987).
Coolen’s equation assumes normal albumin concentrations;
these were normal in our cohort (mean 45 sd 3.37 g/L). Coolen’s equation is widely used for calculation of free cortisol and
has been shown to predict measured free cortisol in both
normal control subjects as well as in subjects with sepsis or
septic shock (Ho et al., 2006).
2.4. Statistical analysis
Statistical analysis was performed using SPSS Version 19.
Outcome variables portraying basal HPA-activity were plasma
ACTH, total cortisol, CBG, average salivary free cortisol over
consecutive collection days, and calculated free cortisol. The
outcomes of nine pregnant girls at the time of sampling were
not included in the analysis. Gender differences were
assessed using t-tests (two-sided, except for the effect of
oral contraceptive use among female teenagers). Pearson
correlation (two-sided, except for the effect of age and sexspecific adolescent development) was used to study covariate influences of continuous measures of age (years), body
mass index (BMI) (kg/m2); sampling time of day (hours:minutes), and categorical variables representing sex-specific
adolescent development. Sampling time of year was grouped
into one of the four seasons in the southern hemisphere:
summer (November—January), fall (February—April), winter
(May—July) and spring (August—October). Analysis of variance (ANOVA) was used to test whether HPA data differed
according to season. We used available information in the
Raine Study database on the use of oral contraceptive (OC) at
the time of biological sampling (‘yes’ for confirmed indication of OC-use, ‘no’ for all other cases) and the onset of
menarche (i.e. age in years at first menstrual bleeding) in the
girls. The stage of pubic hair and genital development at 17
years of age using the Tanner‘s Stage Scale of Puberty
(Tanner, 1962) was applied in boys.
Association between plasma ACTH, total cortisol and CBG
was analysed using linear regression based on generalised
linear modelling (GLM). A linear mixed model (LMM) with a
random intercept was used to study the effect of plasma ACTH,
total cortisol and CBG on repeated measures of salivary free
cortisol. In both models, we also adjusted for gender, age,
adolescent development, sampling time of the day, season and
female oral contraceptive (OC) use (Walker et al., 1997, 2001;
Kirschbaum et al., 1999; Matchock et al., 2007; Hellhammer
et al., 2009). To exclude an interactive effect of ACTH, total
cortisol and CBG on free cortisol, additional LMM was performed with centred predictor outcomes for comparison.
3. Results
3.1. Gender-specific morning HPA-axis function
in late adolescence
The characteristics of baseline morning HPA-activity in late
adolescence are shown in Table 1. Under non-stressful
1274
Table 1
R.M. Reynolds et al.
Descriptive statistics of baseline morning HPA-activity in late adolescence.
HPA outcome variable
Participants
n
Mean (SD)
Plasma ACTH (pg/ml)
All
Males
Females
Females (no known OC use)
Females (OC use)
1257
651
606
402
204
48.90
53.11
44.37
44.66
43.81
All
1258
22.25 (8.35)
Total plasma
cortisol (mg/dl)
Males
Females
Females (no known OC use)
Females (OC use)
Plasma CBG
(mg/ml)
All
Males
Females
Females (no known OC use)
Females (OC use)
Salivary free
cortisol (mg/dl)
All
Males
Females
Females (no known OC use)
Females (OC use)
Calculated plasma
free cortisol (mg/dl)
All
Males
Females
Females (no known OC use)
Females (OC use)
653
605
401
204
1239
636
603
401
202
1121
573
548
362
186
1235
635
600
400
200
19.98
24.71
21.42
31.17
(26.10)
(28.73)
(22.08)
(20.05)
(25.66)
(5.60)
(9.99)
(7.35)
(11.28)
p
<0.001 a
<0.001 a
<0.001 a
0.657 b
(10.75)
(33.97)
(19.79)
(38.07)
<0.001 a
<0.001 a
<0.001 a
<0.001 b
(0.51)
(0.65)
(0.69)
(0.59)
<0.001 a
<0.001 a
<0.001 a
<0.001 b
(0.77)
(0.86)
(0.90)
(0.73)
21.50—154.50
12.43—217.93
12.42—179.75
23.25—217.93
0.11—5.33
0.003 a
<0.001 a
0.217 a
0.072 b
1.44 (0.81)
1.47
1.40
1.51
1.18
1.33—42.72
1.65—59.97
1.65—54.32
3.17—59.97
12.42—217.93
0.99 (0.59)
0.94
1.04
1.07
0.99
10.88—278.71
10.88—278.71
10.88—243.54
10.88—130.43
11.25—243.54
1.33—59.97
54.67 (26.71)
45.27
64.58
50.23
93.07
Range Min—Max
0.11—3.38
0.12—5.33
0.14—5.37
0.12—3.95
0.05—7.13
0.156 a
0.893 a
<0.001 a
<0.001 b
0.05—7.13
0.05—6.95
0.05—6.95
0.06—5.84
Characteristics of HPA-related outcome variables are presented as mean and standard deviation (SD) in the total group of teenage Raine
participants (All) who provided blood and/or saliva samples for ACTH, total cortisol, CBG and free cortisol analysis (n = number of
participants). Free cortisol was calculated from measured total cortisol and CBG using Coolen’s equation (Coolens et al., 1987). Cortisol
concentrations are presented in mg/dl; to convert to nmol/l multiply by 27.59.
a
Comparative t-test analysis between males and females with probability ( p) values listed behind the specified female category.
b
Comparative t-test analysis between female oral contraceptive (OC) users on the day of sampling and those not having used OC at that
time. The absolute lowest (min) and highest (max) outcome detected across all participants as well as across each gender group reflect the
range of each variable. Identical minimal levels of ACTH in all 3 groups reflect the lowest detectable peptide level in the ACTH-IRMA.
domestic conditions, ACTH levels were lower but total cortisol levels were higher in girls compared to boys. Girls also
showed significantly higher free cortisol in saliva upon awakening; however, this was only statistically significant among
girls not using OCs for birth control or other medically related
reasons and salivary cortisol levels in girls using OCs were
similar to levels in boys. Furthermore, although CBG levels in
girls using OCs were almost twice as high as those not using
OCs, Raine teenage girls had significantly higher levels of CBG
in their circulation than Raine boys, irrespective of OC-use.
No overall gender difference was observed in the levels of
calculated free cortisol in blood, though Raine girls using OCs
had significantly lower concentrations of calculated free
cortisol in their circulation.
3.2. Analysis of covariate influences on baseline
HPA-activity
The mean age on the day of sampling was 16.6 0.5 years
and was not significantly different in boys and girls. Age
correlated positively with ACTH particularly in boys (all:
p = 0.007; boys: p = 0.002), whereas neither total cortisol,
CBG, free salivary nor calculated plasma cortisol were significantly related to age in either boys or girls in late adolescence. Mean BMI was similar in boys and girls (22.99 4.45
vs. 22.90 4.37 kg/m2, p = 0.72) and did not correlate with
any of the HPA axis variables. Within the context of sexspecific development in adolescence, the average female age
at the onset of menarche was 12.76 1.10 years (ranging
Resting HPA function in 17-year-old teenagers
between 9 and 16 years; n = 614) and the average Tanner
stage for male participants in the study was 4.82 0.42
[ranging between Tanner stage 3 (relative early pubic development stage) 5 (final pubic development stage); n = 591].
None of the gender specific adolescent development-related
variables correlated significantly with any of the HPA outcome variables. The average blood collection time was at
7:16 am 1:02 h (n = 1239) and this was significantly earlier
in boys than in girls (7:10 am 1:02 vs 7.23 1:01 h,
p < 0.001). ACTH, total and free cortisol levels were all
higher in samples collected earlier in the morning (all participants: p < 0.001, p < 0.001 and p = 0.01, respectively),
whereas CBG levels were not affected by blood collection
time. Saliva collection time did not differ between boys and
girls but salivary cortisol levels were higher in samples
collected earlier in the morning on each collection day
(average collection time — 8:13 am 1:40; n = 1009;
p < 0.001), S2 (average collection time — 8:05 am 1:38;
n = 945; p = 0.001) and S3 (average collection time —
7:18 am 1:12; n = 745; p < 0.001). Levels of ACTH
( p = 0.031), total and calculated free cortisol ( p = 0.002,
p < 0.001 respectively) and CBG ( p = 0.004) were highest
in winter months and lowest in the summer. Salivary cortisol
levels did not vary according to season ( p = 0.447).
3.3. Linear relationship between the HPAcomponents
Univariate analyses demonstrated a significant positive correlation between total cortisol and ACTH when all participants were grouped together (r = 0.074, p = 0.009); this
finding was also significant in boys (r = 0.124, p = 0.02) but
not in girls (r = 0.023, p = 0.56). These relationships were
similar in the regression model (Table 2A), where the
Table 2A
1275
association in girls became close to significance. Higher
CBG levels were also independently associated with higher
total cortisol levels in all participants grouped together, as
well as in each gender group. Other variables independently
associated with total cortisol included blood sampling time,
season, age, oral contraceptive use in females and stage of
puberty in males (Table 2A).
In univariate analyses, there were significant positive
correlations between free cortisol in saliva blood and total
cortisol in all participants (r = 0.128, p < 0.001) as well as in
boys (r = 0.123, p = 0.007), and in girls (r = 0.135, p = 0.003)
separately. These findings remained significant in the regression analysis (Table 2B). Other variables independently associated with salivary free cortisol included lower CBG levels
and earlier salivary sampling time in all subjects analysed
together, and in boys and girls analysed separately. Salivary
free cortisol was not related to stage of adolescent development or use of OCs.
In univariate analyses, there were significant positive
correlations between CBG and total plasma cortisol in all
participants grouped together (r = 0.586, p < 0.001), in boys
(r = 0.604, p < 0.001) and in girls (r = 0.571, p < 0.001).
These findings remained significant in regression analyses
(Table 2C). Higher CBG levels were also associated with lower
ACTH levels in all participants and teenage boys only, but not
among girls. CBG measurements were higher in boys at later
stages of puberty. There were no associations of CBG with age
at menarche in girls, but levels were higher among those
taking OCs.
4. Discussion
We have completed a detailed evaluation of resting HPAactivity in late adolescence in an unselected population
Correlates of total cortisol in blood.
Variable
Unit
All participants (n = 1207)
Plasma ACTH
Age
Blood sampling time
Plasma CBG
Season
Females (n = 270)
Males (n = 502)
Estimate
p-Value
pg/ml
years
hours:minutes
mg/ml
1.080
29.233
0.004
5.162
20.414
<0.001
0.005
0.008
<0.001
<0.001
Plasma ACTH
Age
Blood sampling time
Plasma CBG
Season
Oral contraceptive use
Age at menarche
pg/ml
years
hours:minutes
mg/ml
1.488
74.927
0.003
3.908
30.230
70.212
10.836
0.054
0.002
0.388
<0.001
0.006
0.030
0.357
Plasma ACTH
Age
Blood sampling time
Plasma CBG
Season
Adolescent development
pg/ml
years
hours:minutes
mg/ml
1.465
29.615
0.007
5.305
13.14
10.882
<0.001
0.053
<0.001
<0.001
0.068
0.023
yes/no
years
Tanner stage
All participants represent the total group of participants who provided the blood and saliva samples and the covariate information required
for inclusion in the respective epidemiological models. The number of participants in each group is represented by n. Probability ( p) values
0.05 were required for statistical significance.
1276
Table 2B
R.M. Reynolds et al.
Correlates of free cortisol in saliva.
Variable
Unit
All participants (n = 836)
Plasma ACTH
Plasma total cortisol
Plasma CBG
Age
Blood sampling time
Saliva sampling time
Females (n = 190)
Males (n = 361)
Estimate
p-Value
pg/ml
mg/dl
mg/ml
years
hours:minutes
hours:minutes
0.014
0.012
0.065
0.763
0.000
0.000
0.536
<0.001
0.009
0.463
0.061
0.002
Plasma ACTH
Plasma total cortisol
Plasma CBG
Age
Blood sampling time
Saliva sampling time
Oral contraceptive use
Age at menarche
pg/ml
mg/dl
mg/ml
years
hours:minutes
hours:minutes
yes/no
years
0.024
0.013
0.023
1.314
0.000
0.001
4.919
0.180
0.744
0.048
0.664
0.611
0.424
0.003
0.189
0.880
Plasma ACTH
Plasma total cortisol
Plasma CBG
Age
Blood sampling time
Saliva sampling time
Adolescent development
pg/ml
mg/dl
mg/ml
years
hours:minutes
hours:minutes
Tanner stage
0.017
0.015
0.117
1.748
0.000
0.000
0.470
0.588
<0.001
0.001
0.200
0.511
0.001
0.304
All participants represent the total group of participants who provided the blood and saliva samples and the covariate information required
for inclusion in the respective epidemiological models. The number of participants in each group is represented by n. Probability ( p) values
0.05 were required for statistical significance.
based cohort. Our aim was to assess exposure levels to
cortisol in a typical 17-year old population around the estimated time of maturation of this stress-regulatory system.
We applied a multifaceted approach to analyse neuroendocrine events downstream of the pituitary as a reflection of
Table 2C
basal HPA-function. This approach permitted the delineation
of gender specific pituitary peptide, adrenal hormone and
blood hormone binding protein outcomes, indicative of different intrinsic HPA-axis activities and free cortisol exposure
levels under non-stressful natural conditions.
Correlates of CBG in blood.
All participants (n = 942)
Females (n = 213)
Males (n = 405)
Variable
Unit
ACTH
Total cortisol
Age
Blood sampling time
Season
pg/ml
mg/dl
years
hours:minutes
ACTH
Total cortisol
Age
Blood sampling time
Season
Oral contraceptive use
Age at menarche
pg/ml
mg/dl
years
hours:minutes
ACTH
Total cortisol
Age
Blood sampling time
Season
Adolescent development
pg/ml
mg/dl
years
hours:minutes
yes/no
years
Tanner stage
Estimate
0.128
0.074
2.058
9.14 10
1.869
p-Value
5
<0.001
<0.001
0.149
0.661
0.005
0.125
0.053
6.800
0.001
1.260
34.379
0.914
0.233
<0.001
0.046
0.265
0.007
<0.001
0.545
0.119
0.075
1.399
0.000
1.314
2.342
0.007
<0.001
0.498
0.499
0.170
<0.001
All participants represent the total group of participants who provided the blood and saliva samples and the covariate information required
for inclusion in the respective epidemiological models. The number of participants in each group is represented by n. Probability ( p) values
0.05 were required for statistical significance.
Resting HPA function in 17-year-old teenagers
An integrated assessment of a combination of key HPAregulatory biomarkers in a group of teenagers of unprecedented size revealed that on average 17-year old Raine girls
had lower ACTH levels but higher plasma cortisol than boys, a
finding suggesting that female adrenals were more sensitive
to ACTH. Girls also showed higher CBG in blood and free
cortisol in saliva upon awakening. Despite their relatively
high CBG, this would imply that teenage girls are still more
likely to be exposed to higher levels of functional cortisol.
Underlying mechanisms are unknown, but are likely due to
complex interactions between the HPA axis and secretion of
sex steroids. Previous studies reporting gender differences in
cortisol responses are inconsistent (reviewed in Clow et al.,
2004; Hellhammer et al., 2009), perhaps related to smaller
study size, and because responses may vary according to age
of the child or stage of puberty (Törnhage, 2002; Netherton
et al., 2004; Matchock et al., 2007). For example, there have
been previous studies reporting higher cortisol levels in girls
(Klimes-Dougan et al., 2001; Adam et al., 2010) particularly
inpost-pubertal (Netherton et al., 2004) or in adult women
(Reynolds et al., 2005), whereas others have reported higher
levels in boys, but only in early puberty (Matchock et al.,
2007) and some other investigations have found no gender
differences (Kelly et al., 2008; Roisman et al., 2009). Our
findings may have arisen because of greater power in our
study, which points to the importance of ensuring that such
investigations are of an adequate size to observe a statistically significant effect.
Our multifaceted approach entailed two different measures of the end product of the HPA axis (total and free
cortisol). Free cortisol represents the functional fraction of
systemic cortisol and reflects the level of cortisol that can
bind to widely distributed GR and MR in discrete non-epithelial peripheral target tissues and the limbic brain, influencing
cortisol sensitive systems and processes. Moreover, the exposure level to morning free cortisol was based on the average
of repeated samples collected on successive days. This specific type of saliva sampling was geared towards minimising
the potentially compromising influence of participants’
anticipation of ‘out of the ordinary’ events on any one of
the collection days. We recognise that this single measure
design cannot address the cortisol awakening response (Clow
et al., 2004; Chida and Steptoe, 2009). In fact, to reduce its
impact on free cortisol levels, sampling of saliva was set at
15 min after waking up, i.e. before the cortisol awakening
response peaks between 30 and 45 min after waking up
(Buckley and Schatzberg, 2005; Oskis et al., 2009). The
influence of circadian fluctuations in HPA-outcomes between
sample days was limited by adjustment of free cortisol for
heterogeneity in the sampling time shortly after awakening
on the 3 collection days. We were unable to correct for the
capacity of salivary gland 11b-hydroxysteroid dehydrogenase
type 2 to inactivate cortisol (Lewis, 2006), the potential
presence of minor amounts of CBG in saliva (Lewis, 2006)
or the pulsatile nature of cortisol output (Lightman et al.,
2008), but we should assume that these influences potentially contributed to the degree of inter-individual variation
in this study. The data collection also did not allow participant stratification into morning versus evening chronotypes
(‘early birds’ vs. ‘night owls’; Kudielka et al., 2006)
to determine the effect of personal preference for timing
of activity on awakening cortisol. Nevertheless, saliva
1277
collection on weekdays ensured sampling following the
‘routine’ time of awakening for each individual.
The collection of a single morning fasting blood sample per
participant may hinder accurate interpretation of the three
biomarker (ACTH, total cortisol and CBG) measures in blood.
In particular, plasma ACTH is known to be more labile and
more rapidly responsive than cortisol to a potential HPA
response to blood collection; however, we attempted to limit
this stress-induced influence by moving away from a ‘clinical’
environment and collecting blood at a home visit under
familiar domestic circumstances. The contribution of differential developmental traits of the study participants to outcome variance in HPA-peptide and hormone levels was also
considered. Puberty-related teenage development may not
be accurately represented by age alone. We therefore
further adjusted statistically in our analyses for existing
differences in the onset of menarche in girls and the stage
of puberty-induced changes to body hair and genital organ
growth at 17 in boys. Finally, one in three Raine girls used oral
contraceptives (OCs), which have been reported to enhance
blood CBG and reduce free cortisol in adults (Kirschbaum
et al., 1999; Wiegratz et al., 2003). We confirmed this effect
of OCs on plasma CBG levels and salivary free cortisol in late
adolescence in girls; its impact was adjusted for in the gender
specific statistical analyses that were undertaken. Lack of
information concerning the phase of the menstrual cycle
during which samples were collected did not permit inclusion
of this potential covariate; however, whilst rodent studies
show corticosterone levels differ according to stage of the
estrous cycle (Atkinson and Waddell, 1997), because of the
action of sex steroids (Carey et al., 1995), the literature in
humans reporting whether cortisol levels change across the
menstrual cycle is inconsistent (Kudielka et al., 2009).
The normative range of baseline HPA-activity in this late
adolescent cohort appeared higher than reference ranges
reported in other teenage studies. Comparative examination
with other studies is limited to free salivary cortisol exposure
ranges, as to our knowledge no normative human data on
blood ACTH, total cortisol or CBG in late adolescence are
available to date. The few reports studying salivary free
cortisol consistently presented a lower average level
(Klimes-Dougan et al., 2001; Walker et al., 2001; Kelly
et al., 2008; Oskis et al., 2009), albeit in early teenagers.
The closest approximation to the current study in size and
sample collection design is offered by Roisman et al. (2009)
and Adam et al. (2010), who reported a mean awakening
salivary cortisol level of 0.36 mg/dl (within a range of 0.015—
1.12 mg/dl) at 15 years of age and of 0.44 mg/dl (11.99 nmol/
l) in 17 year olds, respectively.
Interestingly, comparison of our results with normative
free cortisol levels in adults showed closer resemblance. A
healthy adult study with a mean age of 30 years found free
cortisol averaging approximately 0.87 mg/dl at 15 min after
awakening (Kudielka and Kirschbaum, 2003). Meta-analysis of
12 adult studies indicated an average awakening level of
0.6 mg/dl (Clow et al., 2004), while a recent review on
salivary cortisol as a biomarker for HPA-activity illustrated
variation in adult cortisol comparable to the late adolescent
normative ranges that we report here (Hellhammer et al.,
2009). These observations support the notion that HPA-activity matures in late adolescence, and that baseline HPAactivity at age 17 can be considered as a highly informative
1278
prelude to its adult level of functioning. Concordance
between late adolescent and adult baseline HPA-activity
also suggests a potential role of late teenage HPA-function
as a predictor for stress-related diseases in adulthood. Prospective cohort studies such as the Raine Study appear ideal
to address the issue of risk prediction based on cortisol
deficiency or overload, before disease signs or symptoms
becomes apparent.
The question remains as to which HPA-outcome offers the
best insight into altered HPA-function. World-wide differences in HPA-modulators, such as ethnicity and health status
(Kudielka and Kirschbaum, 2003; DeSantis et al., 2007), argue
against the use of normative ranges of absolute levels of a
single HPA-parameter. Likewise, we demonstrated clear seasonal differences in responses, as has been reported by others
(Walker et al., 1997; Matchock et al., 2007). Recognition that
a linear relationship between ACTH, CBG, total and free
cortisol does not necessarily exist within a normal population
(Hellhammer et al., 2009) may justify the call for more
integrated profiling of HPA-activity. In this study, the positive
correlation between total cortisol and CBG but negative
association of CBG with free cortisol, suggests that total
cortisol influences free cortisol levels via CBG. This is consistent with data in rodents and CBG deficient mice demonstrating that plasma CBG regulates free corticosterone
(Droste et al., 2008; Richard et al., 2010) and access of free
corticosterone to target tissues such as fat (Qian et al., 2011)
or brain (Minni et al., 2012). Interestingly, there was a
discrepancy between free salivary cortisol measurements
and calculated free plasma cortisol levels, derived from
measured total cortisol and CBG in the circulation and presumed to represent bioavailable cortisol, which can freely
diffuse into glucocorticoid target tissues. Free salivary cortisol levels were higher in girls not taking OCs compared to
boys, but there were no gender differences in calculated free
plasma cortisol regardless of OC use. Reasons for this discrepancy are unknown, though the correlation between
calculated free plasma cortisol and measured salivary cortisol was weak (r = 0.14), possibly reflecting assumptions in the
calculation of free plasma cortisol or due to activity of
salivary 11b-hydroxysteroid dehydrogenase type 2. The
importance of CBG measures in studying resting HPA function
is highlighted. Though, in view of the need to extend individual variability in HPA function to genetic variance in the
SerpinA6 gene encoding CBG (Moisan, 2010), a more careful
consideration regarding the role of CBG in corticosteroid
biology and disease vulnerability may be needed.
Our integrated approach to resting HPA-analysis in a
relatively large human cohort allowed distinction of relative
adrenal hypersensitivity and hormone buffering capacity in
late adolescent girls compared to boys. The combined use of
pituitary ACTH, adrenal total and free cortisol and blood CBG
outcomes in individuals and gender groups revealed different
intrinsic patterns of baseline HPA-axis function. The specific
neuroendocrine patterns showed subtle differences in key
events of the HPA cascade at the time of onset of sex-specific
psychopathology (Paus et al., 2008; Gogtay and Thompson,
2010). Depression in adolescence is best known to be associated with an individual’s stress responsiveness (Goodyer
et al., 2000; Goel and Bale, 2009). Our finding supports the
notion that a threefold higher prevalence of adolescent
depression in mid to late teenage girls is related to relatively
R.M. Reynolds et al.
higher stress responsiveness in girls compared to boys (Goel
and Bale, 2009). It has been suggested that boys benefit from
an initial modulating effect of perinatal testosterone on
emotional behaviour followed by a dampening effect of
the pubertal rise in testosterone on adolescent stress responsiveness and the development of depressive behaviour
(Gomez et al., 2004; Romeo et al., 2006). Further investigation into the lowering effect of OC-use on higher free cortisol
in saliva of late adolescent girls upon awakening may shed
light on an alternative protective mechanism in girls to
reduce the risk that higher awakening free salivary cortisol
levels may pose for the increased incidence off depression in
adulthood (Adam et al., 2010).
It is likely that differences in late adolescent stress regulation and behavioural adjustment can be explained by
gender specific sensitivity of adolescent brain maturation
processes to stress and sex steroid hormones (Romeo and
McEwen, 2006; Goel and Bale, 2009; Schulz et al., 2009).
Further delineation of the complex neurobiological mechanisms underpinning stress-related states of health and disease
will advance our understanding of the role that aberrant
cortisol exposure plays in the context of adaptation, resilience and adolescent disease vulnerability and the association of these mechanisms with long term health problems in
adult life (Goodyer et al., 2000; Klimes-Dougan et al., 2001;
Spear, 2004; Compas, 2006).
Role of funding source
The funding source had no role in study design, data collection, analysis or interpretation of the data. The manuscript
was prepared independently from the funding source and the
funding source did not influence the decision to submit the
paper for publication.
Conflict of interest
RMR, HLH, CEP, IWM, ERdK, SL, FJS, JKF and EM all declare
that there is no actual or potential conflict of interest related
to the submitted manuscript.
Acknowledgements
We are most grateful to the study families for their participation in the 17-year Raine Study follow-up. We also thank the
Raine management team, Dr. H. Atkinson and Mrs B. PenovaVeselinovic, for the re-analysis of some blood cortisol levels,
and Dr. D. Blache for use of his radioactive research facility at
the University of Western Australia. The authors further
acknowledge the support of the National Health and Medical
Research Council of Australia (NHMRC Project Grants 458623)
and the Canadian Institutes of Health Research (Grant MOP
82893). Core management of the Raine Study is funded by the
generous support of NHMRC (Program Grant 353514, Stanley
et al.), the University of Western Australia, the Raine Medical
Research Foundation, the Faculty of Medicine, Dentistry and
Health Sciences at UWA, the Telethon Institute for Child
Health Research and the Women and Infants Research Foundation and Curtin University. These funding organizations had
no further role in the following: study design; collection,
Resting HPA function in 17-year-old teenagers
analysis and interpretation of data; writing of the report; or
the decision to submit the paper for publication.
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