Croat Med J. 2013;54:33-41
Aim To determine the circadian rhythm alteration of cor-
tisol excretion and the level of corticosteroids in children
with different grades of autism severity.
Methods The study included 45 children with different
grades of autism severity (low [LFA], medium [MFA], and
high functioning autism [HFA]), 15 in each group, and 45
age/sex-matched children with typical development. The
urinary levels of free cortisol (at three phases of 24-hour cy-
cle), corticosteroids, vanilylmandelic acid, and 5-hydroxyin-
dole acetic acid were determined.
Results Alteration in the pattern of cortisol excretion (Phas-
es I, II, and III) was observed in children with LFA (Phase I:
43.8 ± 4.43 vs 74.30±8.62, P = 0.000; Phase II: 21.1±2.87 vs
62±7.68, P < 0.001; Phase III: 9.9 ± 1.20 vs 40 ± 5.73, P < 0.001)
and MFA (Phase I: 43.8 ± 4.43 vs 52.6±7.90, P < 0.001; Phase
II: 21.1±2.87 vs 27.4±4.05, P < 0.001; Phase III: 9.9 ± 1.20 vs
19 ± 2.50, P < 0.001) compared to the control group. The
corticosteroids excretion levels were higher in all the
groups of children with autism than in the control group.
The level of 5-hydroxyindole acetic acid was significantly
higher in children with LFA (8.2±1.48 vs 6.8±0.85, P < 0.001)
and MFA (8.2±1.48 vs 7.4± 0.89, P = 0.001) and not sig-
nificantly higher in children with HFA than in the control
group. The changes were correlated with degrees of sever-
ity of the disorder.
Conclusion These data suggest that altered cortisol excre-
tion pattern and high level of corticosteroids in urine may
probably be a consequence of altered hypothalamic-pitu-
itary-adrenal axis function, which may contribute to the
pathogenesis and affect the severity of autism.
Received: April 9, 2012
Accepted: February 2, 2013
Department of Biochemistry
Bharathi Women’s College
(Affiliated to University of Madras)
North Chennai – 600 108, Tamil
Lakshmi Priya1, Arumugam
1Department of Biochemistry,
Bharathi Women’s College,
University of Madras, Chennai,
Tamil Nadu, India
2Madras Medical College and
Pediatrician Institute of Child
Health and Hospital for Children,
Chennai, Tamil Nadu, India
Abnormal circadian rhythm and
cortisol excretion in autistic
children: a clinical study
Croat Med J. 2013;54:33-41
Autistic disorder is a behaviorally defined neurodevelop-
mental disorder characterized by deficits in social interac-
tion, language, and communication, and by repetitive be-
haviors, which manifest in early postnatal life (1). It belongs
to the spectrum of closely related conditions also referred
to as autism spectrum disorders (ASDs). The incidence of
ASDs has increased significantly over the last decades and
is currently 1 in 150, affecting boys four times more often
than girls (2). Autism remains one of the very few condi-
tions classified as a syndrome, defined only in terms of
observable symptoms (3), largely because no accepted
biochemical diagnostic markers are currently available. Re-
search into the pathophysiology and etiology of autistic
disorder has been ongoing for nearly half a century and
still the cause remains unknown.
Circadian rhythm is controlled by a “biological clock,” a
time keeping system (24 hours) in the organism. Biologi-
cal clock also provides internal temporal organization and
ensures that internal changes take place in coordination
with one another. Although circadian rhythm is synchro-
nized with light and dark cycle, it is also affected by psy-
chological (defective adaptive capacity to environmental
demand) and physical factors (hypoglycemia, infection), as
well as stress. In people lacking a proper circadian rhythm,
biological clock ceases to function normally resulting in
defective physiological ability to respond to the demands
of the environment (4). Experimental studies have investi-
gated different types of stress and their effects on the hy-
pothalamic-pituitary-adrenal (HPA) axis (5). HPA axis is al-
tered significantly in mood disorders and functional illness,
including anxiety disorders, bipolar disorder, insomnia,
posttraumatic stress disorder, border line personality dis-
order, and attention deficit hyperactivity disorder (ADHD)
(6). Dysregulation of biological systems, including the HPA
axis, has been also observed in autism (7). Autism has of-
ten been characterized as a disorder accompanied by in-
creased arousal, stress, and sensory sensitivity. In research
on autism, HPA axis deserves special attention, since it is
the basis for emotions and social interactions (8,9).
The HPA axis has a well-characterized circadian pattern.
Under the influence of stress, this pattern is altered and
homeostasis of stress-related neuroendocrine function is
disrupted, with adverse impact on health. Essential to this
stress response is the activation of the HPA system, result-
ing in the release of glucocorticoid hormones from the
adrenal cortex. The primary glucocorticoid in humans is
cortisol (hydrocortisone), which exhibits diurnal varia-
tions peaking in the early morning hours (about 30
minutes after waking), declining rapidly in the morning,
decreasing slowly in the afternoon, and reaching its low-
est level in the evening. This circadian rhythmic release of
cortisol can be well studied by the level of its excretion in
urine in different time intervals during the day. This is a well
developed pattern already in the third month of infancy
Impaired immune functions and disturbed circadian
rhythm in ASDs may be due to aberrations in fatty acid me-
tabolism, particularly the eicosanoid production (11,12),
which plays a major role in neuronal development and be-
havior, learning ability, and memory (13).
Prostaglandin E2 (dinoprostone) is an important arachi-
donic acid metabolite formed by the action of cyclooxy-
genase-1. Prostaglandin E2 is an important signaling mol-
ecule involved in pain or synaptic plasticity in the nervous
system. Along with free radicals, prostaglandin metabo-
lites have been shown to influence the pathological dis-
turbance in nervous system (14). The level of cortisol and
its catabolic excretable compounds in urine are a measure
of their metabolic turnover and used to evaluate the neu-
Therefore, the present study aims to evaluate the altera-
tion in the circadian rhythmic pattern of cortisol by mea-
suring its excretion in different time intervals during the
day and also to determine the level of excretion of adre-
nal cortex hormones (11- hydroxy corticosteroids, 17- oxo-
genic steroids, and total 17- hydroxycorticosteroids), along
with vanilyl mandelic acid, 5-hydroxyindoleacetic acid, and
prostaglandin E2, which could also shed more light on the
pathogenesis of autism and its severity.
StuDy DeSiGn AnD MethoDS
The study included 45 children with autism attending the
school for children with special needs Aikya, Maruti Seva
at Chennai, Tamil Nadu, India from 2010-2012. The institu-
tion used Check of Autism in Toddlers (15) to assess autism.
Autistic children were classified according to the method
adopted from Childhood Autism Rating Scale (CARS) (16)
as those with low functioning autism (LFA), medium func-
tioning autism (MFA), and high functioning autism (HFA).
Each group comprised 15 children. Forty-five age- and
sex-matched healthy children were used as controls. They
were attending regular school at the same location and
Lakshmi Priya: Circadian rhythm and corticosteroids in autism
had no history of neurodevelopmental complications. The
boys and girls ratio was 4:1, and they were 4-12 years old
CARS classification is a 15-item scale that identifies chil-
dren with autism and distinguishes them from other chil-
dren with compromised development but without autism
(16). It also differentiates mild to moderate from severe au-
tism (17). It is brief and appropriate for children older than
2 years. The scale evaluates behavior in 14 domains that are
generally affected in autism, plus a single category for gen-
eral impression of autism (18). These 15 items are as follows:
relating to people, imitation, emotional response, body use,
object use, adaptation to change, visual response, listening
response, taste, smell, and touch response and use, fear or
nervousness, verbal communication, nonverbal communi-
cation, activity level, level and consistency of intellectual
response, and general impressions. The scores assigned to
each domain vary from 1 (within the limits of normality) to
4 (severe autistic symptoms). The total score varies from 15
to 60 and the cutoff point for autism is 30 (16).
Collection of urine sample
Parents collected urine samples from children at 3 different
time points as follows: 9.00 pm to 7.00 am (morning sample –
tAbLe 1. Clinical history of children with autism and children with typical development*
Number of children
CARS value (15-60)
Age in years, range
Children with gluten sensitivity
Economic status of the parents
High = 5;
Medium = 38;
Low = 2
45 (15 in each group)
LFA = 46-60; MFA = 31-45; HFA = 15-30
LFA = 12/15; MFA = 10/15; HFA = 5/15
High (above Rs. 4 lakhs)
Medium (- Rs. 2.5 lakhs to Rs. 4 lakhs) LFA = 13; MFA = 12; HFA = 13
Low (Rs. 60 000 to 2.5 lakhs)
LFA = 3/15; MFA = 7/15; HFA = 11/15
LFA = 2; MFA = 1; HFA = 2
LFA = 0; MFA = 2; HFA = NIL
LFA = 0; MFA = 0; HFA = 2
LFA = 2; MFA = 4; HFA = 2
LFA = 13; MFA = 11; HFA = 11
Nutritional status of the children†
Good = 45
Children with special talents (dancing, humming,
drawing, assembling puzzles)‡
No. of children with low muscle tone§
No. of children with ear infection during the study
No. of children with sleep disturbanceII
LFA = 14/15; MFA = 12/15; HFA = 11/15
LFA = 2/15; MFA = 0; HFA = 0
LFA = 13/15; MFA = 11/15; HFA = 6/15 0
No. of children with mood disorder¶
No. of children on antibiotic treatment
No. of children with gastrointestinal problems**
No. of parents given their cooperation††
No. of parents appreciated the study‡‡
*Abbreviations: LFA – low functioning autism; MFA – moderately functioning autism; hFA – high functioning autism, CARS – Childhood Autism Rat-
†Assessed in terms physical examination including weight, stature, head circumference, and arm measurements. these data were obtained from
the children’s clinical histories kept at the school.
‡the children had talents in general but a few autistic children had a very unique and exemplary way of displaying their talents.
§Muscle tone was determined by squeezing the muscle to feel resistance to compression and lifting up and moving the limbs and feeling the resis-
tance to the movement. these data were obtained from the children’s clinical histories kept at the school.
iiSleep disturbance mainly refers the discontinuous sleep pattern, due to frequent bed-wetting or sudden waking up in fear, as observed by the
¶Mood swings in school, including non cooperation with the care takers and sudden screaming.
**Frequent diarrhea, stomach ache, or gut dysbiosis.
††the parents volunteered in giving the samples of their children. they also helped us in follow up studies.
‡‡the parents found the subject interesting and were satisfied with the outcome of the study.
LFA = 11/15; MFA = 9/15; HFA = 6/15
LFA = 9/15; MFA = 6/15; HFA = 0
Croat Med J. 2013;54:33-41
Phase I), 7.00 am to 2.00 pm (noon sample – Phase II), and 2.00
pm to 9.00 pm (evening sample – Phase III) in 3 different ster-
ilized containers labeled accordingly. After taking 25 µL-50
µL for determination of cortisol, the samples were poured
and collected in a 24-hour container for further assays. On
receipt of the specimen at the laboratory, the volume was
noted, and after vigorous shaking, stored at -4°C with 3-4
drops/100 mL of formalin as preservative until subsequent
analysis. Urine samples were collected on three consecu-
tive days and analyzed separately, and the average values
for each sample was calculated. The study protocol was ap-
proved by the Institutional Ethics Committee, Madras Medi-
cal College, Chennai – 3, EC No. 22072012.
The fluorimetric method developed by Mattingly et al (19)
was used for the determination of urinary 11-hydroxycorti-
costeroids against cortisol as the standard. Neutral 17- oxo-
steroids level was determined by the method of Norym-
berski et al (20), which used ethylene dichloride to extract
the steroids, and tetramethyl ammonium hydroxide (25%
w/v aqueous solution) was used as alkali in the color de-
velopment and was read colorimetrically at 520 nm using
a green filter. For the determination of total 17-oxosteroids
(20), sodium bismuthate was used for oxidation and 12%
sodium metabisulphite was used to reduce the bismuth-
ate. The color was developed by Zimmerman reaction sim-
ilar to 17-oxosteroids and the color developed was read
colorimetrically at 520 nm using a green filter. 17-oxogenic
steroids level was determined using the formula:
17-oxogenic steroids = total 17-oxosteroids - neutral 17-
Determination of total 17-hydroxycorticosteroids was
done using metaperiodate by the method of Few (21). The
level of urinary free cortisol was determined by EIAgen cor-
tisol kit (ADALTIS, Rome, Italy) using Elisa strip reader cata-
log no: LI4003K.
Vanilyl mandelic acid was measured using UV-spectrom-
eter by the method of Pisano et al (22). We used a more
rigorous method by Udenfriend et al, which however, only
determines 5-HIAA (23). The urine was first treated with di-
nitrophenyl hydrazine to react with ketoacids, which may
interfere later. Any indole acetic acid present was extract-
ed into chloroform. After saturation with sodium chlo-
ride, the hydroxyindole acetic acid was extracted into
ether and then returned to phosphate buffer pH 7.0
for colorimetric assay. Prostaglandin E2 was determined us-
ing a kit purchased from Arbor Assays (www.arborassays.
com), catalog no: K018-H1.
The results are presented as mean ± standard deviations.
The groups were compared with one-way ANOVA with
post-hoc Bonferroni test, and the P value <0.05 was con-
sidered as significant. The parameters were also analyzed
by Spearman rank test, and rs values were calculated to
find the significance of correlation.
The level of excretion of free cortisol in the LFA (Phase I:
43.8 ± 4.43 vs 74.30±8.62, 95% confidence interval [CI]
69.94-78.66; P < 0.000; Phase II: 21.1±2.87 vs 62±7.68, 95% CI
58.11-65.89, P < 0.001; Phase III: 9.9 ± 1.20 vs 40 ± 5.73, 95%
CI 37.1-42.9, P < 0.001, post-hoc Bonferroni test) and MFA
group (Phase I: 43.8 ± 4.43 vs 52.6±7.90, 95% CI 48.6-56.6,
P < 0.001; Phase II: 21.1±2.87 vs 27.4±4.05, 95% CI 25.35-
29.45, P < 0.001; Phase III: 9.9 ± 1.20 vs 19 ± 2.50, 95% CI
17.73-20.27, P < 0.001, post-hoc Bonferroni test) was signifi-
cantly higher than in the control group (Figure 1). Excretion
of cortisol in HFA group showed no significant alteration
from the control group (Phase I: 43.8 ± 4.43 vs 46.5 ± 6.60,
95% CI 43.16-49.84 P = 0.179; Phase II: 21.1 ± 2.87 vs
FiGuRe 1. Level of urinary free cortisol (mean±standard devia-
tion) collected in three different time intervals in children with
low functioning autism (LFA), medium functioning autism
(MFA), and high functioning autism (hFA) and age and sex-
matched children with typical development. P<0.001 (control
vs LFA); P<0.01 (control vs MFA); P<0.05 (control vs MFA and
hFA); non-significant (control vs hFA); P<0.001 (LFA vs hFA).
Lakshmi Priya: Circadian rhythm and corticosteroids in autism
22.5 ± 2.52, 95% CI 21.22-23.78 P = 0.352; Phase III: 9.9 ± 1.20
vs 9.0 ± 0.97, 95% CI 8.51-9.49 P = 0.177, post-hoc Bonfer-
roni test) but showed a highly significant alteration at the
phases I (74.3 ± 8.62 vs 46.5 ± 6.60, P < 0.001), II (62 ± 7.68 vs
22.5 ± 2.52, P < 0.001), and III (40 ± 5.73 vs 9 ± 0.97, P < 0.001)
when compared to LFA group. The cortisol excretion in the
autistic groups had a significant positive correlation with
the severity of autism (Table 2).
11-hydroxycorticosteroids, neutral 17-oxosteroids, total 17-
oxosteroids, 17-oxogenic steroids, and total 17-hydroxy-
corticosteroids showed a significantly greater excretion in
all groups of autistic children (Table 3). The level of excre-
tion of vanilyl mandelic acid was also significantly higher
(P < 0.001) in autistic children than in the control group.
The level of excretion of 5-hydroxyindole acetic acid was
significantly higher in LFA (6.8 ± 0.85, P < 0.001) and in MFA
(7.4 ± 0.89, P = 0.001) and non-significantly higher in HFA
(7.80 ± 0.98, P = 0.277) when compared to the control group
(8.2 ± 1.48). The level of prostaglandin E2 in autistic children
was higher (LFA = 11.41 ± 1.65; P < 0.001, MFA = 8.02 ± 1.12;
P < 0.001, HFA = 6.86 ± 0.99; P < 0.001) than in the control
group (3.62 ± 0.80). Also, the level of prostaglandin E2 was
higher in LFA than in HFA (6.86 ± 0.99; P < 0.001).
Interestingly, Spearman’s rank correlation test showed that
higher levels of corticosteroids (11-hydroxycorticosteroids
vs CARS (rs: +0.795), neutral 17-oxosteroids vs CARS (rs:
+0.852), total 17-oxosteroids vs CARS (rs: +0.902), 17-oxo-
genic steroids vs CARS (rs: +0.856), and total 17-hydroxy-
corticosteroids vs CARS (rs: +0.799), free cortisol vs CARS
Phase I (rs: +0.899), Phase II (rs: +0.801), Phase III (rs: +0.787),
vanilylmandelic acid vs CARS (rs: +0.834), and prostaglan-
din E2 vs CARS (rs: +0.876) were significantly positively cor-
related with the severity of autism (Table 2).
The present study found higher level of cortisol excretion
in autistic children (LFA and MFA) than in the control group.
Children with typical development showed a significantly
lower cortisol excretion at noon time than LFA group.
The HPA axis, like most biological systems, is highly regu-
lated and dependent on the ability of the system to main-
tAbLe 2. Rank correlation between CARS and corticosteroids,
free cortisol, vanilyl mandelic acid, and prostaglandin e2 in dif-
ferent groups of autistic children (n = 45)*
11- Hydroxycorticosteroids vs CARS
Neutral 17- oxosteroids vs CARS
Total 17- oxosteroids vs CARS
17- Oxogenic steroids vs CARS
Total 17- hydroxycorticosteroids vs CARS
Free cortisol vs CARS
Free cortisol vs CARS
Free cortisol vs CARS
Vanilyl mandelic acid vs CARS
Prostaglandin E2 vs CARS
*CARS – Childhood Autism Rating Scale.
†based on the critical values of the rank correlation (Spearman rho’s),
null hypothesis of no correlation was rejected and it was concluded
that the level of corticosteroids, cortisol, vanilyl mandelic acid, and
prostaglandin e2 correlated with the severity of autism.
tAbLe 3. Level of corticosteroids, vanilyl mandelic acid, 5-hydroxyindoleacetic acid, and prostaglandin e2 in the urine of autistic
children compared with age and sex matched children with typical development, shown as mean and standard deviation*†
11- Hydroxycorticosteroids (μg cortisol/24 h)326.00 ± 63.57
Neutral 17- oxosteroids (mg/day) 3.44 ± 0.74
Total 17- oxosteroids (mg/day) 21.45 ± 4.08
17- Oxogenic steroids (mg/day) 18.01 ± 3.60
Total 17- Hydroxycorticosteroids (mg/day) 15.39 ± 2.85
Vanilyl mandelic acid (mg/day) 5.8 ± 1.28
5- Hydroxyindoleacetic acid (mg/day) 8.2 ± 1.48
Prostaglandin E2 (nmol/day)
*Abbreviations: LFA – low functioning autism; MFA– moderately functioning autism; hFA– high functioning autism; nS – non-significant.
†Variables were examined using one-way AnoVA and symbols represent significant differences from post-hoc bonferroni test.
‡P < 0.000 (control vs LFA, MFA and hFA).
§P = 0.001 (control vs MFA).
iiP = 0.006 (control vs hFA).
¶P < 0.001 (LFA vs hFA).
**P = 0.001 (LFA vs hFA).
649.15 ± 110.36‡
10.68 ± 1.34‡
56.72 ± 9.36‡
46.04 ± 5.99‡
42.73 ± 6.62‡
38.99 ± 4.68‡
6.8 ± 0.85‡
11.41 ± 1.65‡
450.61 ± 56.33‡
8.73 ± 1.48‡
48.01 ± 6.96‡
39.28 ± 6.09‡
33.82 ± 5.41‡
13.35 ± 1.74‡
7.4 ± 0.89§
8.02 ± 1.12‡
377.76 ± 49.10‡¶
4.07 ± 0.67II¶
29.06 ± 4.07‡¶
24.99 ± 3.87‡¶
24.56 ± 3.81‡¶
7.56 ± 1.06‡¶
7.8 ± 0.98NS**
6.86 ± 0.99‡¶
3.62 ± 0.80
Croat Med J. 2013;54:33-41
tain, respond to, and reset itself for homeostasis. Dys-
regulation of the HPA axis may manifest as disruptions in
circadian rhythms, which in turn are represented by the
pulsatile release of cortisol (24). A study in children with
autism showed alterations in the normal circadian pat-
terns of cortisol (25). The value of urinary free cortisol in
assessing of adrenocortical function was first pointed out
by Cope (26,27), who found that it detected increased ad-
renocortical function. Other studies also confirmed that an
increase in the plasma concentration of free cortisol was
accompanied by a linear increase in cortisol excretion in
the urine (28). Urinary free cortisol in 24-hour samples has
been widely used to assess basal cortisol secretion and has
the theoretical advantage of being unaffected by possible
cortisol circadian rhythm differences.
Normal physiological rhythms are responsible for all be-
havioral variables, including sleep organization and pro-
pensity, subjective alertness, and cognitive performance,
which are disturbed in autistic children. There is increasing
evidence to support the role of the sleep-wake cycle and
the endogenous circadian system in the pathogenesis of
major psychiatric disorders (29), and disturbed nocturnal
sleep is a common observation among autistic children.
Our study also found a positive correlation between the
elevated level of cortisol and the severity of autism. Corti-
sol secretion has earlier been shown to also markedly in-
crease in response to stress, and autism has often been
characterized as a disorder accompanied by increased
arousal, stress, and sensory sensitivity (30-32). It was also
shown that more severe autism led to more abnormal di-
urnal rhythm (33), which is in accordance with the pres-
Studies on major neurotransmitter systems (serotonin,
catecholamine) strongly suggest that a major role in au-
tism could be played by neurochemical factors (34). In the
present study, urinary vanilyl mandelic acid, a marker of
catecholamine metabolic status, was significantly higher
in autistic children than in control group, which may be
related to frequent stressful situations to which autistic
children are subjected. The increased response to stres-
sors could be due to worse handling of stress, over-elicita-
tion of the physiological response, or dysfunctional stress
response systems (35).
In this study, 5-hydroxyindoleacetic acid, a major metab-
olite of serotonin was significantly higher in LFA and
MFA group and not significantly in HFA group than
in the control group. Individuals with autism have been
reported to have significantly higher serotonin (5-hy-
droxytryptamine) levels in whole blood and platelets (36).
Though a higher level of serotonin has been reported in
the blood of autistic children (37), the present study found
significantly lower excretion level of 5-hydroxyindoleacetic
acid in LFA and MFA group than in control group, which
may be due to altered activity of monoamine oxidase, the
enzyme responsible for oxidative deamination of sero-
tonin to form the corresponding aldehyde, which is fur-
ther oxidized to 5- hydroxyindoleacetic acid.
In the recent years, there has been a spate of research
into the role of serotonin in neuropsychiatric conditions
in childhood. Interestingly, Cohen et al (38) have reported
lower concentrations of the serotonin metabolite 5-hy-
droxyindoleacetic acid in the cerebrospinal fluid in autis-
tic than in non-autistic psychotic children. The interpreta-
tion of this finding is far from clear, however, neither group
differed significantly from controls, whose concentration
was between the autistic and psychotic group. Boullin et
al (39) reported increased serotonin efflux as a finding spe-
cific to autism, but this was not confirmed by Yuwiler et al
(40). Hence, the serotonin findings may well be important,
but their meaning remains obscure. So far, attempts to re-
late serotonin concentration to clinical differences in au-
tistic groups or groups with mental retardation have been
rather disappointing (41).
Along with metabolic and rhythmic disturbances, there
is emerging evidence of the contributing role of abnor-
mal fatty acid metabolism in the pathology of autism (42).
The present study showed a higher level of excretion of
prostaglandin E2 in autistic children than in control group.
There is evidence for increased prostaglandin metabolism
in individuals with autism spectrum disorders (43). Eico-
sanoids, particularly prostaglandin D2 and E2, are known to
have sedative properties and to be involved in the control
of the sleep-wake cycle (44), which was disturbed in autis-
tic children in the present study.
There are reports (42) suggesting that fatty acid homeo-
stasis may be altered in autism as a result of insufficient di-
etary supplementation, genetic defects, functional altera-
tion of enzymes involved in their metabolism, or various
environmental agents such as infections, inflammation,
and drugs. Eicosanoids, derived from highly unsaturated
fatty acids released from cell membranes by phospholi-
pases and produced by cyclooxygenases, are required for
normal functioning of synaptic junctions (45). Thus, any
Lakshmi Priya: Circadian rhythm and corticosteroids in autism
abnormality in phospholipase activity could result in al-
teration in neuronal structure and functions. Docosa-hex-
enoic acid and other free fatty acids can modulate abnor-
mal electrical discharges from neurons (46) and a deficit
of these fatty acids could increase susceptibility to epilep-
tic seizures, which occurs in many patients with ASDs. In
addition, the involvement of prostaglandin E2 signaling in
early developmental process, including formation of den-
dritic spines and neuronal plasticity, is also emerging (47).
A major limitation of our study is that we did not deter-
mine the blood and saliva levels of corticosteroids, free
cortisol, vanilyl mandelic acid, 5- hydroxyindole acetic acid,
and prostaglandin E2, which still remains to be done, pref-
erably in a study involving a greater number of children.
Abnormal physiological rhythm found in autistic children
should also be further investigated.
This study found that abnormal function of the HPA axis,
evidenced by abnormal pattern of cortisol excretion in au-
tistic children, could be strongly correlated with the sever-
ity of the disorder. Further studies on the factors responsi-
ble for abnormal circadian rhythm are needed, be it a gene,
environmental stimuli, or an enzyme defect.
Acknowledgment The authors thank AIKYA and MARUTI SEVA schools for
children with special needs, Chennai, Tamil Nadu, India and the parents of
children with autism for their cooperation.
Funding Provided by the Indian Council of Medical Research, New Delhi.
ethical approval received from the Institutional Ethics Committee, Madras
Medical College, Chennai – 3, EC No. 22072012.
Declaration of authorship MDLP planned the study, analyzed the samples,
and contributed to manuscript preparation. AG designed the work and
contributed to manuscript preparation. VS contributed to data analysis. SS
screened and selected the patients for sample collection.
Competing interests All authors have completed the Unified Competing
Interest form at www.icmje.org/coi_disclosure.pdf (available on request
from the corresponding author) and declare: no support from any organi-
zation for the submitted work; no financial relationships with any organiza-
tions that might have an interest in the submitted work in the previous 3
years; no other relationships or activities that could appear to have influ-
enced the submitted work.
1 Folstein Se, Rosen-Sheidley b. Genetics of autism: complex
aetiology for a heterogeneous disorder. nat Rev Genet. 2001;2:943-
55. Medline:11733747 doi:10.1038/35103559
2 inglese MD, elder Jh. Caring for children with autism spectrum
disorder. Prevalence, etiology and core features. J Pediatr nurs.
2009;24:41-8. Medline:19159834 doi:10.1016/j.pedn.2007.12.006
3 American Psychiatric Association. Diagnostic and statistical manual
of mental disorders, 3rd edition. Washington (DC): APA; 1987.
4 takahash SJJS, hong hK, Ko Ch, McDearmon eL. the genetics
of mammalian circadian order and disorder: implications
for physiology and disease. nat Rev Genet. 2008;9:764-75.
5 Douglas AJ. Central noradrenergic mechanisms underlying acute
stress responses of the hypothalamo- pituitary- adrenal axis:
adaptations through pregnancy and lactation. Stress. 2005;8:5-18.
6 Spencer RL, hutchinson Ke. Alcohol, aging and the stress response.
Alcohol Res health. 1999;23:272-83. Medline:10890824
7 Corbett bA, Mendoza S, Abdullah S, Wegelin JA, Levine S. Cortisol
circadian rhythms and response to stress in children with autism.
Psychoneuroendocrinology. 2006;31:59-68. Medline:16005570
8 Palmen SJ, Van england h, hof PR, Shmitz C. neuropathological
findings in autism. brain. 2004;127:2572-83. Medline:15329353
9 hamza Rt, hewedi Dh, ismail MA. basal and adrenocorticotropic
hormone stimulated plasma cortisol levels among egyptian
autistic children: Relation to disease severity. ital J Pediatr.
2010;36:71. Medline:21034507 doi:10.1186/1824-7288-36-71
10 Mantagos S, Moustogiannis A, Vagenakis AG. Diurnalvariation
of plasma-cortisol levels in infancy. J Pediatr endocrinol Metab.
1998;11:549-53. Medline:9777576 doi:10.1515/JPeM.1918.104.22.1689
11 Gillberg C. Autism and related behaviors. J intellect Disabil Res.
1993;37:343-72. Medline:8400719 doi:10.1111/j.1365-2788.1993.
12 Samuelsson b, Ramwell PW, Paoletti R, Folco G, Granstrom e.
Advances in prostaglandin, thromboxane, and leukotriene
research, vol 21b. new york (ny): Raven Press; 1991.
13 tassoni D, Kaur G, Weisinger RS, Sincliar AJ. Review article- the role
of eicosanoids in the brain. Asia Pac J Clin nutr. 2008;17:220-8.
14 tamiji J, Crawford DA. Prostaglandin e2 and misoprostol induce
neurite retraction in neuro – 2a cells. biochem biophys Res
Commun. 2010;398:450-6. Medline:20599704 doi:10.1016/j.
15 baron-Cohen S, Wheelwright S, Cox A, baird G, Charman t,
Swettenham J, et al. early identification of autism by the checklist
for autism in toddlers (ChAt). J R Soc Med. 2000;93:521-5.
16 Schopler e, Reichler R, Renner bR. the Childhood Autism Rating
Scale (CARS), 10th ed. Los Angeles (CA): Western Psychological
17 Magyar Ci, Pandolfi V. Factor structure evaluation of the childhood
autism rating scale. J Autism Dev Disord. 2007;37:1787-94.
18 Rellini e, tortolani D, trillo S, Carbone S, Montecchi F. Childhood
Autism Rating Scale (CARS) and Autism behavior Checklist (AbC)
correspondence and conflicts with DSMiV criteria in diagnosis of
autism. J Autism Dev Disord. 2004;34:703-8. Medline:15679189
Croat Med J. 2013;54:33-41
19 Mattingly D, Dennis PM, Pearson J, Cope CL. Rapid screening
test for adrenal cortical function. Lancet. 1964;2:1046-9.
20 norymberski JK, Stubbs RD, West hF. Assessment of adrenocortical
activity by assay of 17-ketogenic steroids in urine. Lancet.
1953;1:1276-81. Medline:13053749 doi:10.1016/S0140-
21 Few JD. A method for the analysis of urinary 17-hydroxy
corticosteroids. J endocrinol. 1961;22:31-46. Medline:13699253
22 Pisano JJ, Crout JR, Abraham D. Determination of 3-methoxy
4-hydroxymandelic acid in urine. Clin Chim Acta. 1962;7:285-91.
23 udenfriend S, titus e, Weissbach h. the identification of 5-hydroxy-
3-indoleacetic acid in normal urine and a method for its assay. J
biol Chem. 1955;216:499-505. Medline:13271330
24 Gunnar MR, Vazquez DM. Low cortisol and a flattening of expected
daytime rhythm: potential indices of risk in human development.
Dev Psychopathol. 2001;13:515-38. Medline:11523846 doi:10.1017/
25 Richdale AL, Prior MR. urinary cortisol circadian rhythm in a group
of high-functioning children with autism. J Autism Dev Disord.
1992;22:433-47. Medline:1400105 doi:10.1007/bF01048245
26 Cope CL, hurlock b. Some aspects of adrenal cortical metabolism.
Clin Sci. 1954;13:69-83. Medline:13141425
27 Cope CL, harrison RJ. effect of 9α- fluorohydrocortisone on
adrenal hyperfusion in Cushing’s syndrome. bMJ. 1955;2:457-60.
28 beisel WR, Cos JJ, horton R, Chao Py, Forsham Ph. Physiology of
urinary cortisol excretion. J Clin endocrinol Metab. 1964a;24:887-
93. Medline:14216479 doi:10.1210/jcem-24-9-887
29 boivin Db. influence of sleep-wake and circadian rhythm
disturbances in psychiatric disorders. J Psychiatry neurosci.
30 Garde Ah, hansen AM. Long- term stability of salivary cortisol.
Scand J Clin Lab invest. 2005;65:433-6. Medline:16081365
31 eek FC, Garde Ah, hansen AM, Pearson R, orbaek P, Karlson b. the
cortisol awakening response- an exploration of intra individual
stability and negative responses. Scand J environ health Suppl.
32 hansen AM, Garde Ah, Persson R. Sources of biological
and methodological variation in salivary cortisol and their
impact on measurement among healthy adults: A review.
Scand J Clin Lab invest. 2008;68:448-58. Medline:18609093
33 Corbett bA, Mendoza S, Wegelin JA, Carmean V, Levine S. Variable
cortisol circadian rhythms in children with autism and anticipatory
stress. J Psychiatry neurosci. 2008;33:227-34. Medline:18592041
34 Chugani DC, Muzik o, behen M, Rothermel R, Janisse JJ, Lee J, et
al. Developmental changes in brain serotonin synthesis capacity
in autistic and non-autistic children. Ann neurol. 1999;45:287-95.
35 Schultz Rt, Anderson GM. the neurobiology of autism and
pervasive developmental disorders. in: Charney DS, nestler eJ. the
neurobiology of mental illness, 2nd edition. oxford (uK): oxford
university Press; 2003. p. 1-19.
36 Anderson GM, Gutknecht L, Cohen DJ, brailly-tabard S, Cohen Jh,
Ferrari P, et al. Serotonin transporter promoter variants in autism:
functional effects and relationship to platelet hyperserotonemia.
Mol Psychiatry. 2002;7:831-6. Medline:12232775 doi:10.1038/
37 naffah-Mazzacoratti MG, Rosenberg R, Fernandes MJ, Draque
CM, Silvestrini W, Calderazzo L, et al. Serum serotonin levels of
normal and autism children. braz J Med biol Res. 1993;26:309-17.
38 Cohen DJ, Caparulo bK, Shaywitz bA, bowers Mb. Dopamine and
serotonin metabolism in neuropsychiatrically disturbed children.
CSF homovanillic acid and 5-hydroxyindoleacetic acid. Arch
Gen Psychiatry. 1977;34:545-50. Medline:860891 doi:10.1001/
39 oullin DJ, Coleman M, o’brien RA, Rimland b. Laboratory
predictions of infantile autism based on 5-hydroxytryptamine
efflux from blood platelets and their correlation with the Rimland
e-2 score. J Autism Child Schizophr. 1971;1:63-71. Medline:5172440
40 yuwiler A, Ritvo e, Geller e, Glousman R, Schneiderman G, Matsuno
D. uptake and efflux of serotonin from platelets of autistic and
non-autistic children. J Autism Child Schizophr. 1975;5:83-98.
41 Partington MW, tu Jb, Wong Cy. blood serotonin levels in severe
mental retardation. Dev Med Child neurol. 1973;15:616-27.
42 Ming X, Stein tP, brimacombe M, Johnson WG, Lambert Gh,
Wagner GC. increased excretion of lipid peroxidation biomarkers in
autism. Prostaglandins Leukot essent Fatty Acids. 2005;73:379-84.
43 Lozinsky S. Misoprostol elevates intracellular calcium levels and
increases glutamate release in type-i astrocytes: implications in
autism. Master’s thesis. toronto (Canada): york university; 2010.
44 hayaishi o, Matsumura h, onoe h, Koyama y, Waranabe y. Sleep-
wake regulation by PGD2 and e2. in: Samuelsson, b; Ramwell, P.W;
Paoletti, R; Folco, G; Granstrom e. (eds.), Advances in prostaglandin,
thromboxane and leukotriene research, vol, 21b. new york (ny):
Raven Press; 1991. p. 723-6.
45 Smalheiser nR, Dissernayake S, Kapil A. Rapid regulation of
neurite outgrowth and retraction by phospholipase A2- derived
arachidonic acid and its metabolites. brain Res. 1996;721:39-48.
Lakshmi Priya: Circadian rhythm and corticosteroids in autism
46 Vreugdenhil M, bruehl C, Voskuyl RA, Kang JX, Leaf A, Wadman WJ.
Polyunsaturated fatty acid modulate sodium and calcium currents
in CA1 neurons. Proc natl Sci uSA. 1996;93:12559-63. doi:10.1073/
47 Chen C, Magee JC, bazan nG. Cyclooxygenase-2-regulated
prostaglandin e2 signaling in hippocampal long-term synaptic
plasticity. J neurophysiol. 2002;87:2851-7. Medline:12037188