PAGE 1

University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 1 Relationship between Cortisol Concentrations and Tic Severity Christopher J McDonald Dr. Tanya K. Murphy, and Dr. Darragh P. Devine College of Liberal Arts and Sciences, University of F lorida T yndrome i s a movement disorder characterized by persistence of involuntary, sudden, rapid, recurrent, non rhythmic stereotyped motor movements or vocalization s known as tics for a period of over a year. Often in individuals with s yndrome, the presentat ion of stressful situations or stimuli exacerbate the prevalence of the tics. Cortisol is a stress marker that is released by the adrenal glands in response to stress. We examined the potential relationship between resting serum cortisol concentrations and severity of tics in school age children with severe, mild, and no tics. No significant difference s were found in cortisol concentrations between the three subject groups. This negative finding suggests that basal cortisol does not contribute to tic severi ty but leaves open the possibility that hormonal responses to stressful stimuli may contribute INTRODUCTION A tic as defined by the latest edition of the Diagnostic and Statistical Manual of M ental Disorders 4 th edition (DSM IV) is an involuntary, su dden, rapid, recurrent, non rhythmic stereotyped motor movement or vocalization. The best studied tic disorder s yndrome which is characterized by the presence of both multiple motor tics and one or more vocal tics throughout a period of mor e than one year, during which period there was never a tic free period of more than three consecutive months (Chappell et al., 1994; Hoekstra et al. 2004 ) An s seen during times of emotional distress (Fin dley et al., 2003; Silva et al., 1995) to release various endogenous compounds to alter the However, the question as to whether stress related agents are part of the etiology of T or ar e symptoms still remains. To analyze this relationship we examined the relationship between severity of tics and basal serum cortisol levels in school age children. C ortisol is the principal form of glucocorticoid in humans. Glucocorticoids are hormones produced in the adrenal cortex that mobilize energy stores involved in maintenance of brain and muscle function in a stressful situation and modulate inflammation and other immune responses. They act by forming complexes with receptors that regulate genes involved in inflammatory and immune responses, blocking leukocyte access to sites of inflammation and functionally interfering with other molecules involved in immune response ( for reviews see Boumpas et al. 1993 ; Carrasco, 2002 ). In response to a stressor there is a chain of chemical signaling from the hypothalamus to the pituitary gland and terminating on the adrenal gland, which is called the hypothalamic pituita ry adrenal (HPA) axis (Habib et al., 2001 ) A ctivation of the HPA axis leads to release of Adrenocorticotropic hormone (ACTH) by the pituitary gland which in turn increases circulating levels of glucocorticoids. The glucocorticoids participate in maintena nce of homeostasis and negative feedback of the HPA axis at the hypothalamic and pituitary levels (d e Kloet, 1995). METHODS Subjects and Assessment of Tic Severity We acquired serum samples of 34 school age children from the lab oratory of Dr. Tanya Mur phy from Shands at the University of Florida. To assess the level of tics and group used two methods of evaluation. A choreiform screening was conducted during which the children had to wait in line in groups of five Whi le the children would wait for their turns, they were observed for three minutes at rest, with tics and behaviors noted. The tics noted consisted of six types : facial, shoulder, arms/hands/fingers, legs, vocal, and other. Additional behaviors were also cou nted, including fidgeting in line, twirling hair, excessive touching/tapping, picking skin/nose, cracking joints, hitting/shoving/pulling hair, balance/swaying, and grimacing (non tic facial movements). To assess choreiform movements the rater instructed the children to hold their arms out with palms downward with fingers separated and then palms upward for 20 seconds each, with choreiform movements noted during each interval. The choreiform movements were scored on a scale from 0 3 : 0 = no twitches, 1 = 2 5 isolated twitches, 2 = 6 10 twitches in bursts, or 3 = continuous twitches Choreiform scores of 1 were disregarded to reduce

PAGE 2

C HRISTOPHER J M C D ONALD D R T ANYA K M URPHY & D R D ARRAGH P D EVINE University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 2 potential for false positives (Murphy 2007). Using the observations of tics and choreiform movements, the children were clas sified into groups of no tics, mild tics, and severe tics. Blood samples were then taken from the children, and serum was isolated which was used in our experiment. Serum Samples Because of the natural cycle of cortisol levels throughout the day, the timing of the collection of the samples was critical. Fifteen subjects with severe tics had serum samples collected between 1610 and 1650 hours. Twelve subjects w ith mild tics had serum samples collected between 1610 and 1725 hours. Seven control subje cts had serum samples collected between 1500 and 1800 hours. Samples were collected and placed in 80 o C freezer for storage within 15 minutes to prevent cortisol degradation. Serum Cortisol Immunoassay The serum samples were later thawed and c ortisol w as assayed with an enzyme immuno assay (R&D Systems, Inc., Minneapolis, MN). This assay was performed by an experimenter who was blind to the tic severity scores of the subjects. Goat anti mouse polyclonal antibody was provided in a 96 well polystyrene micr oplate in 12 strips of 8 wells. The c ortisol conjugate (6 mL) was c ortisol conjugated to horseradish peroxidase (HRP c ort) with red dy e and preservatives. The c ortisol s tandard was 100 ng of c ortisol in buffer with preservatives, which was lyophilized pr ior to packaging. Mouse monoclonal antibody to cortisol (6 mL) was provided in buffer with blue dye and preservatives. Two 21 mL vials of buffered protein base with preservatives constituted the c alibrator d iluent RD5 43. 21 mL of 25 fold concentrated solution of buffered surfactant with preservatives se rved as wash buffer concentrate. Color r eagent A consisted of 12.5 mL of stabilized hydrogen peroxide, and c olor r eagent B was 12.5 mL of stabilized chromagen (tetramethylbenzidine). Stop solution was 6 mL of 2 N sulfuric acid ( H 2 SO 4 ). Four adhesive films were used as plate covers to prevent evaporation during incubation of the assay The c ortisol s tandard was reconstituted with 1.0 mL of deionized/distilled water to produce 100 ng/mL stock solution of c ortisol standard and gently shaken on a microplate shaker. The reconstituted c ortisol s tandard was allowed to sit for 15 minutes to allow complete reconstitution. Serum samples were diluted 20 fold by adding 380 L of c alibrator d iluent RD5 43 to 20 L o f each serum sample and a ll tubes were vortexed. Seven test tubes were readied for dilution of c ortisol s tandard. A 100 L aliquot of c ortisol s tandard was pipetted into the first test tube with 900 L of c alibrator d iluent RD5 43 for an initial ten fold dilution of the c ortisol s tandard to a concentration of 10 ng/mL. For each pipetting, the pipette was filled up, discharged back into solution, and then refilled to ensure maximal precision and accuracy during pipetting measures. In the six subsequent tube s serial dilutions of 500 L of c ortisol s tandard were pipetted with 500 L of c alibrator d iluent RD5 43 to form diluted standards of 5, 2.5, 1.25, 0.625, 0.312, and 0.156 ng/mL ) All test tubes were vortexed following addition of c ortisol s tandard and Cal ibrator Diluent RD5 43 to maximize mixing. Calibrator d iluent RD5 43 served as the zero standard (B 0 ). Calibrator diluent RD5 43 was added into NSB (150 L) wells and B 0 (100 L) wells. E ach dilution of c ortisol s tandard added to the appropriate wells (100 L per well) to form the standard curve and serum samples (100 L per well) were added in duplicate to wells according to a pre planned set up of the microplate Horseradish peroxidase conjugated to cortisol (HRP c ort isol ) was added to each well (50 L p er well) Mouse anti human antibody was then added to each well ( 50 L ) except the NSB well. The adhesive film was then placed over the top of the microplate. The m icroplate was then incubated on a orbit) for 2 h ours at 500 rpm. After the microplate finished shaking, the well s were washed using 400 L of wash solution per well. After all the wells were filled, the microplate was flipped and blotted onto a paper towel. The plate was shaken and blotted quite vigoro usly to ensure all unbound solution had been removed. This process was repeated four times. The color reagents A (Hydrogen p eroxide) and B (chromagen) were mixed together to form a substrate solution shortly before the microplate finished shaking. T he sub strate solution w as then added to each well in the microplate (200 L per well) The m icroplate was wrapped in aluminum foil and incubated for 30 minutes to allow for the substrate solution to react with the contents of each well. After 30 minutes 50 L o f Stop solution was added to each well to prevent any further development of the color reaction. The p late was gently tapped to ensure thorough mixing in each well. Optical densities were taken using a microplate reader set at 450 n m with correction set at 570 nm. Data Analysis Once optical density (OD) readings were obtained, the blind on the samples was broken. The OD value of the NSB well was subtracted from the OD of each standard and sample well to correct for non specific binding of cortisol in each microplate well. The m aximum possible binding of c ortisol was obtained from the B 0 well. A s tandard curve was generated using Gra phPad Prism software plotting logit vs. log[ cortisol ] and log[ cortisol ] vs. percent cortisol bound A n a verage OD value was c alculated for each set of duplicate samples. The OD values were divided by B 0 OD value to determine the

PAGE 3

R ELATIONSHIP BETWEEN C ORTISOL C ONCENTRATIONS AND T IC S EVERI TY IN C HILDREN WITH T OURETTE S S YNDROME University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 3 percent of maximum binding of cortisol in the wells and to calculate the l ogit values for each averaged sample The log [cortisol] values were then calc ulated by extrapolation from the linear regression of the plot of logit vs. log [cortisol] values of the standard curve. Finally, antilogs were calculated and the cortisol concentrations were corrected for the 20 fold dilution. Between groups ( i.e. tic s everity ) differences in cortisol concentrations were assessed with a one way ANOVA. RESULTS The linear regression of the standard curve revealed a very strong goodness of fit (r 2 = 0.992; p < 0.001) with a limit of detection less than 1.0 ng/ml The N SB well revealed an ext remely low non specific binding ( OD = 0.013 ; equivalent to 1.23% of the B 0 ) There were no significant differences in cortisol concentrations (F=0.3658, p>0.05) between the group with severe tics those with mild tics and the contro l s ( see Fig ure 1 ). Figure 1 : Cortisol concentrations for the groups with severe tics, mild tics, and no tics DISCUSSION In this study we investigated the relationship between cortisol and severity of tics in school age children with T ourett e's s yndrome Similar studies have been performed in the past on the role s yndrome, but none specifically looked at cortisol levels or at basal functioning of the HPA axis in Tourette's patients. Chappell et al. (1996) inves tigated stress responsiveness of patients with Tourette's s yndrome or obsessive compulsive disorder (OCD) in comparison with normal controls by measuring cerebrospinal fluid (CSF) concentrations of corticotrophin releasing hormone (CRH) obtained through a lumbar puncture. They found significantly elevated levels of CRH in Tourette's patients as compared with the OCD patients and normal controls. Interestingly, no relationship was seen between the CRH concentrations and measures of depression, anxiety, OCD like behaviors, and tics. Our results are consistent with these findings as no correlation was seen between severity of tic presentation and basal cortisol levels. Considering the data from the Chappel et al. (1996) study, in combination with the current d ata, it seems that cortisol is a contributor to the exacerbation of symptoms during times of duress, but it is unrelated to the basal expression of tics and associated behaviors in s yndrome outside of stressful situations. On the other hand, i t is interesting to note the role of cortisol in suppressing the immune response because there appears to be a relationship between early s treptococcus infections and the early s ymptoms in individuals identified as having Pedia tric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus ( PANDAS) (Murphy 2007). Thus, elevated cortisol could potentially play a role in etiology of was no evidence of ongoing differenc es in basal cortisol function in the current study. Future studies should examine the potential that circulating cortisol concentrations may be abnormally elevated during stress exposure in patients with

PAGE 4

C HRISTOPHER J M C D ONALD D R T ANYA K M URPHY & D R D ARRAGH P D EVINE University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 4 s yndrome. This would determine if the dow nstream activation of the HPA axis concurs with the abnormal stress responsive activation of CRH (Chappell et al., 1996) in these patients. It would also be interesting to know if basal CRH concentrations (obtained from post mortem samples) are abnormal in patients with Tourette s yndrome. In summary, s tress has a clear role in the presentation of tics in individuals with Tourette syndrome and these patients have abnormal stress responsive elevations in CSF CRH concentrations ( Chappell et al., 1994 ) ; ho wever, the basal HPA axis tone appears to be unrelated to the diagnosis or severity of Tourette syndrome. T he relationship of the HPA axis response to stress with Tourette is still relatively unexplored REFERENCES Boumpas DT Chro usos GP Wilder RL Cupps TR Balow JE. Glucocorticoid therapy for immune mediated diseases: B asic and clinical correlates Ann Intern Med 1993 ; 119 :1198 208. Carrasco GA., Van de Kar LD. Neuroendocrine pharmacology of stress Eur J Pharmacol 2003 ; 463 : 2 35 272 Chappell P Riddle M Anderson G Scahill L Hardin M Walker D Cohen D Leckman J Enhanced stress responsivity of Tourette syndrome patients undergoing lumbar puncture Biol Psychiatry 1994 ; 36 :35 43. Findley DB, Leckman JF, Katsovich L, Lin H Zhang H Grantz H, Otka J, Lombroso PJ, King, RA Development of the Yale Children's Global Stress Index (YCGSI) and its application in children and adolescents with Tourette syndrome and obsessive compulsive disorder J Am Acad Child Adoles Psychiatry 2003 ; 42 :450 7 Habib KE, Gold PW, Chrousos GP Neuroendocrinology of stress Endocrinol Metab Clin North Am 2001 ; 30 :695 728. Hoekstra PJ, Anderson GM, Limburg PC, Kallenberg CGM, Minderaa RB Neurobiology and Neuroimmu n update Cell Mol Life Sci 2004 ; 61 :886 898 de Kloet ER. Steroids, stability, and stress Front Neuroendocrin 1995 ; 6 :416 25 Murphy TK, Snider LA, Mutch PJ, Harden E, Zaytoun A, Edge PJ, Storch EA, Yang MC, Mann G, Goodman WK, Swedo SE. Relationship of mo vements and behaviors to Group A Streptococcus infections in elementary school children Biological Psychiatry 2007; 61: 279 284. Silva RR, Munoz DM, Barickman J, Friedhoff AJ Environmental factors and related fluctuation of symptoms in children and adol escents with Tourette disorder J Child Psychol Psychiatry 1995 ; 36 :305 12.


Summer Focus on Medical Research : Relationship between Cortisol Concentrations and Tic Severity in Children with Touret...
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Title: Summer Focus on Medical Research : Relationship between Cortisol Concentrations and Tic Severity in Children with Tourette’s Syndrome
Series Title: Journal of Undergraduate Research
Physical Description: Serial
Language: English
Creator: McDonald, Christopher J.
Murphy, Tanya K.
Devine, Darragh P.
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011
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Abstract: Tourette’s syndrome is a movement disorder characterized by persistence of involuntary, sudden, rapid, recurrent, non-rhythmic, stereotyped motor movements, or vocalizations known as tics for a period of over a year. Often in individuals with Tourette’s syndrome, the presentation of stressful situations or stimuli exacerbate the prevalence of the tics. Cortisol is a stress marker that is released by the adrenal glands in response to stress. We examined the potential relationship between resting serum cortisol concentrations and severity of tics in school-age children with severe, mild, and no tics. No significant differences were found in cortisol concentrations between the three subject groups. This negative finding suggests that basal cortisol does not contribute to tic severity but leaves open the possibility that hormonal responses to stressful stimuli may contribute.
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Relationship between Cortisol Concentrations and Tic Severity

in Children with Tourette's Syndrome

Christopher J. McDonald, Dr. Tanya K. Murphy, and Dr. Darragh P. Devine

College of Liberal Arts and Sciences, University of Florida

Tourette's syndrome is a movement disorder characterized by persistence of involuntary, sudden, rapid, recurrent, non-rhythmic,
stereotyped motor movements, or vocalizations known as tics for a period of over a year. Often in individuals with Tourette's
syndrome, the presentation of stressful situations or stimuli exacerbate the prevalence of the tics. Cortisol is a stress marker that is
released by the adrenal glands in response to stress. We examined the potential relationship between resting serum cortisol
concentrations and severity of tics in school-age children with severe, mild, and no tics. No significant differences were found in
cortisol concentrations between the three subject groups. This negative finding suggests that basal cortisol does not contribute to tic
severity but leaves open the possibility that hormonal responses to stressful stimuli may contribute.


INTRODUCTION

A tic as defined by the latest edition of the D,,,,1,i'ic
and Statistical Manual of Mental Disorders, 4th edition
(DSM-IV) is an involuntary, sudden, rapid, recurrent, non-
rhythmic, stereotyped motor movement or vocalization.
The best studied tic disorder is Tourette's syndrome, which
is characterized by the presence of both multiple motor tics
and one or more vocal tics throughout a period of more
than one year, during which period there was never a tic-
free period of more than three consecutive months
(Chappell et al., 1994; Hoekstra et al., 2004).
An exacerbation of the tics in Tourette's patients is seen
during times of emotional distress (Findley et al., 2003;
Silva et al., 1995). Part of the body's response to stress is
to release various endogenous compounds to alter the
body's physiological state to deal with the demand.
However, the question as to whether stress-related agents
are part of the etiology of Tourette's syndrome or are
exclusively intensifiers of Tourette's syndrome symptoms
still remains. To analyze this relationship, we examined the
relationship between severity of tics and basal serum
cortisol levels in school-age children.
Cortisol is the principal form of glucocorticoid in
humans. Glucocorticoids are hormones produced in the
adrenal cortex that mobilize energy stores involved in
maintenance of brain and muscle function in a stressful
situation and modulate inflammation and other immune
responses. They act by forming complexes with receptors
that regulate genes involved in inflammatory and immune
responses, blocking leukocyte access to sites of
inflammation and functionally interfering with other
molecules involved in immune response (for reviews see
Boumpas et al., 1993; Carrasco, 2002). In response to a
stressor, there is a chain of chemical signaling from the
hypothalamus to the pituitary gland and terminating on the


adrenal gland, which is called the hypothalamic-pituitary-
adrenal (HPA) axis (Habib et al., 2001). Activation of the
HPA axis leads to release of Adrenocorticotropic hormone
(ACTH) by the pituitary gland, which in turn increases
circulating levels of glucocorticoids. The glucocorticoids
participate in maintenance of homeostasis and negative
feedback of the HPA axis at the hypothalamic and pituitary
levels (de Kloet, 1995).

METHODS

Subjects and Assessment of Tic Severity

We acquired serum samples of 34 school-age children
from the laboratory of Dr. Tanya Murphy from Shands at
the University of Florida. To assess the level of tics and
choreiform movements, Dr. Murphy's group used two
methods of evaluation. A choreiform screening was
conducted during which the children had to wait in line in
groups of five. While the children would wait for their
turns, they were observed for three minutes at rest, with
tics and behaviors noted. The tics noted consisted of six
types: facial, shoulder, arms/hands/fingers, legs, vocal, and
other. Additional behaviors were also counted, including
fidgeting in line, twirling hair, excessive touching/tapping,
picking skin/nose, cracking joints, hitting/shoving/pulling
hair, balance/swaying, and grimacing (non-tic facial
movements).
To assess choreiform movements, the rater instructed the
children to hold their arms out with palms downward with
fingers separated and then palms upward for 20 seconds
each, with choreiform movements noted during each
interval. The choreiform movements were scored on a
scale from 0-3: 0 = no twitches, 1 = 2-5 isolated twitches,
2 = 6-10 twitches in bursts, or 3 = continuous twitches.
Choreiform scores of 1 were disregarded to reduce


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
1





CHRISTOPHER J. MCDONALD, DR. TANYA K. MURPHY, & DR. DARRAGH P. DEVINE


potential for false positives (Murphy 2007). Using the
observations of tics and choreiform movements, the
children were classified into groups of no tics, mild tics,
and severe tics. Blood samples were then taken from the
children, and serum was isolated, which was used in our
experiment.

Serum Samples

Because of the natural cycle of cortisol levels throughout
the day, the timing of the collection of the samples was
critical. Fifteen subjects with severe tics had serum
samples collected between 1610 and 1650 hours. Twelve
subjects with mild tics had serum samples collected
between 1610 and 1725 hours. Seven control subjects had
serum samples collected between 1500 and 1800 hours.
Samples were collected and placed in -80 oC freezer for
storage within 15 minutes to prevent cortisol degradation.

Serum Cortisol Immunoassay

The serum samples were later thawed, and cortisol was
assayed with an enzyme immunoassay (R&D Systems,
Inc., Minneapolis, MN). This assay was performed by an
experimenter who was blind to the tic severity scores of the
subjects.
Goat anti-mouse polyclonal antibody was provided in a
96 well polystyrene microplate in 12 strips of 8 wells. The
cortisol conjugate (6 mL) was cortisol conjugated to
horseradish peroxidase (HRP-cort) with red dye and
preservatives. The cortisol standard was 100 ng of cortisol
in buffer with preservatives, which was lyophilized prior to
packaging. Mouse monoclonal antibody to cortisol (6 mL)
was provided in buffer with blue dye and preservatives.
Two 21 mL vials of buffered protein base with
preservatives constituted the calibrator diluent RD5-43. 21
mL of 25-fold concentrated solution of buffered surfactant
with preservatives served as wash buffer concentrate. Color
reagent A consisted of 12.5 mL of stabilized hydrogen
peroxide, and color reagent B was 12.5 mL of stabilized
chromagen (tetramethylbenzidine). Stop solution was 6 mL
of 2 N sulfuric acid (HzSO4). Four adhesive films were
used as plate covers to prevent evaporation during
incubation of the assay.
The cortisol standard was reconstituted with 1.0 mL of
deionized/distilled water to produce 100 ng/mL stock
solution of cortisol standard and gently shaken on a
microplate shaker. The reconstituted cortisol standard was
allowed to sit for 15 minutes to allow complete
reconstitution. Serum samples were diluted 20-fold by
adding 380 .iL of calibrator diluent RD5-43 to 20 .iL of
each serum sample, and all tubes were vortexed. Seven test
tubes were readied for dilution of cortisol standard. A 100
iL aliquot of cortisol standard was pipetted into the first
test tube with 900 iL of calibrator diluent RD5-43 for an
initial ten-fold dilution of the cortisol standard to a


concentration of 10 ng/mL. For each pipetting, the pipette
was filled up, discharged back into solution, and then
refilled to ensure maximal precision and accuracy during
pipetting measures. In the six subsequent tubes serial
dilutions of 500 .iL of cortisol standard were pipetted with
500 .iL of calibrator diluent RD5-43 to form diluted
standards of 5, 2.5, 1.25, 0.625, 0.312, and 0.156 ng/mL).
All test tubes were vortexed following addition of cortisol
standard and Calibrator Diluent RD5-43 to maximize
mixing. Calibrator diluent RD5-43 served as the zero
standard (Bo). Calibrator diluent RD5-43 was added into
NSB (150 .iL) wells and Bo (100 .iL) wells. Each dilution
of cortisol standard added to the appropriate wells (100 .iL
per well) to form the standard curve, and serum samples
(100 pL per well) were added in duplicate to wells
according to a pre-planned set-up of the microplate.
Horseradish peroxidase conjugated to cortisol (HRP-
cortisol) was added to each well (50 .iL per well). Mouse
anti-human antibody was then added to each well (50 .iL)
except the NSB well. The adhesive film was then placed
over the top of the microplate. The microplate was then
incubated on a horizontal orbital microplate shaker (0.12"
orbit) for 2 hours at 500 rpm.
After the microplate finished shaking, the wells were
washed using 400 .iL of wash solution per well. After all
the wells were filled, the microplate was flipped and
blotted onto a paper towel. The plate was shaken and
blotted quite vigorously to ensure all unbound solution had
been removed. This process was repeated four times.
The color reagents A (Hydrogen peroxide) and B
(chromagen) were mixed together to form a substrate
solution shortly before the microplate finished shaking.
The substrate solution was then added to each well in the
microplate (200 .iL per well). The microplate was wrapped
in aluminum foil and incubated for 30 minutes to allow for
the substrate solution to react with the contents of each
well. After 30 minutes, 50 .iL of Stop solution was added
to each well to prevent any further development of the
color reaction. The plate was gently tapped to ensure
thorough mixing in each well. Optical densities were taken
using a microplate reader set at 450 nm with correction set
at 570 nm.

Data Analysis

Once optical density (OD) readings were obtained, the
blind on the samples was broken. The OD value of the
NSB well was subtracted from the OD of each standard
and sample well to correct for non-specific binding of
cortisol in each microplate well. The maximum possible
binding of cortisol was obtained from the Bo well.
A standard curve was generated using GraphPad Prism
software, plotting logit vs. log[cortisol] and log[cortisol]
vs. percent cortisol bound. An average OD value was
calculated for each set of duplicate samples. The OD
values were divided by Bo OD value to determine the


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
2





RELATIONSHIP BETWEEN CORTISOL CONCENTRATIONS AND TIC SEVERITY IN CHILDREN WITH TOURETTE'S SYNDROME


percent of maximum binding of cortisol in the wells and to
calculate the logit values for each averaged sample. The
log [cortisol] values were then calculated by extrapolation
from the linear regression of the plot of logit vs. log
[cortisol] values of the standard curve. Finally, antilogs
were calculated and the cortisol concentrations were
corrected for the 20-fold dilution. Between groups (i.e., tic
severity), differences in cortisol concentrations were
assessed with a one-way ANOVA.


RESULTS

The linear regression of the standard curve revealed a
very strong goodness of fit (r2 = 0.992; p < 0.001), with a
limit of detection less than 1.0 ng/ml. The NSB well
revealed an extremely low non-specific binding (OD =
0.013; equivalent to 1.23% of the Bo).
There were no significant differences in cortisol
concentrations (F=0.3658, p>0.05) between the group with
severe tics, those with mild tics, and the controls (see
Figure 1).


Ir


-!-


S Control
Severe
Mild


group


Figure 1: Cortisol concentrations for the groups with severe tics, mild tics, and no tics.


DISCUSSION

In this study, we investigated the relationship between
cortisol and severity of tics in school-age children with
Tourette's syndrome. Similar studies have been performed
in the past on the role of the HPA axis in Tourette's
syndrome, but none specifically looked at cortisol levels or
at basal functioning of the HPA axis in Tourette's patients.
Chappell et al. (1996) investigated stress-responsiveness of
patients with Tourette's syndrome or obsessive-compulsive
disorder (OCD) in comparison with normal controls by
measuring cerebrospinal fluid (CSF) concentrations of
corticotrophin releasing hormone (CRH) obtained through
a lumbar puncture. They found significantly elevated levels
of CRH in Tourette's patients as compared with the OCD
patients and normal controls. Interestingly, no relationship
was seen between the CRH concentrations and measures of
depression, anxiety, OCD-like behaviors, and tics. Our
results are consistent with these findings as no correlation
was seen between severity of tic presentation and basal
cortisol levels.


Considering the data from the Chappel et al. (1996)
study, in combination with the current data, it seems that
cortisol is a contributor to the exacerbation of symptoms
during times of duress, but it is unrelated to the basal
expression of tics and associated behaviors in Tourette's
syndrome outside of stressful situations.
On the other hand, it is interesting to note the role of
cortisol in suppressing the immune response because there
appears to be a relationship between early streptococcus
infections and the early manifestation of Tourette's
symptoms in individuals identified as having Pediatric
Autoimmune Neuropsychiatric Disorders Associated with
Streptococcus (PANDAS) (Murphy 2007). Thus, elevated
cortisol could potentially play a role in etiology of
Tourette's syndrome associated with PANDAS, but there
was no evidence of ongoing differences in basal cortisol
function in the current study.
Future studies should examine the potential that
circulating cortisol concentrations may be abnormally
elevated during stress exposure in patients with Tourette's


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 1 Summer 2011
3





CHRISTOPHER J. MCDONALD, DR. TANYA K. MURPHY, & DR. DARRAGH P. DEVINE


syndrome. This would determine if the downstream
activation of the HPA axis concurs with the abnormal
stress-responsive activation of CRH (Chappell et al., 1996)
in these patients. It would also be interesting to know if
basal CRH concentrations (obtained from post mortem
samples) are abnormal in patients with Tourette's
syndrome. In summary, stress has a clear role in the
presentation of tics in individuals with Tourette's




REFERENCES

Boumpas DT, Chrousos GP, Wilder RL, Cupps TR, Balow JE. Glucocorticoid
therapy for immune-mediated diseases: Basic and clinical correlates. Ann
Intern Med. 1993;119:1198-208.

Carrasco GA., Van de Kar LD. Neuroendocrine pharmacology of stress. Eur J
Pharmacol. 2003;463:235-272

Chappell P, Riddle M, Anderson G, Scahill L, Hardin M, Walker D, Cohen D,
Leckman J. Enhanced stress responsivity of Tourette syndrome patients
undergoing lumbar puncture. Biol/ i. 1994;36:35-43.

Findley DB, Leckman JF, Katsovich L, Lin H, Zhang H, Grantz H, Otka J,
Lombroso PJ, King, RA. Development of the Yale Children's Global Stress
Index (YCGSI) and its application in children and adolescents with Tourette's
syndrome and obsessive-compulsive disorder. J Am Acad Child Adoles
2003;42:450-7


syndrome, and these patients have abnormal stress-
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(Chappell et al., 1994); however, the basal HPA axis tone
appears to be unrelated to the diagnosis or severity of
Tourette's syndrome. The relationship of the HPA axis
response to stress with Tourette's syndrome is still
relatively unexplored.






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University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 | Summer 2011
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