Glucocorticoid sensitivity in health and disease

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Glucocorticoid Sensitivity in Health and Disease

Christopher Barton University of Adelaide

Departments of Medicine and Psychiatry

Submitted 2411211999

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Table of Contents

Declaration

Acknowledgements Thesis Summary

Chapter One:

- Introduction

and Review of the

literature

Glucocorticoids in the normal subject

Glucocorticoids and disease Setting the Scene - what is stress?

The Stress Systems:

The Hypothalamic Pituitary Adrenal axis (HPA axis) Dehydroepiandrosterone (DHEA) and adrenal androgens HPA-axis dysregulation in Chronic Stress

Posttraumatic Stress Disorder (PTSD) The Biological Basis of PTSD:

Functioning of the hippocampus in chronic stess and PTSD Measures of HPA activity in PTSD and

MDD

Aims Hypotheses

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18

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Chapter

2:

Methods Subjects

Radioimmunoassay

(RIA)

for cortisol

A

fluorescent polarisation immunoassay for cortisol Gas Chromatography

Determination of Glucocorticoid Receptor-B by

flow

cytometry Determination of Glucocoricoid Receptor-cr by

flow

cytometry

Chapter 3:

Overnight administration of dexamethasone: Effect of time of administration of

dexamethasone and dose.

Summary Introduction Subjects Protocols Results Discussion

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Chapter

4

-

Regulation

of GlucocorticoÍd

Receptor-cr and

Glucocorticoid

Receptor-B expression

in

human lymphocytes:

Diurnal variation

and effect of dexamethasone.

Summary Introduction Subjects Protocol

Results(I): Response to dexamethasone

Results(Il):

Diurnal variation Discussion

Chapter 5 - Endocrinological and psychological

responses

to a motor

vehicle

accident: Identification of factors important in the

development

of

posttraumatic stress disorder.

Summary Introduction Subjects Protocol

Results (I): Demoraphic information

Results(Il): Psychological response to trauma

Results(lll):

Biological responses to trauma Discussion

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118 84 85 86 87 88

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135

Chapter 6 - Conclusions

References

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148

5

List of

Tables

Table 1:

Table 2:

Table 3 Table 4a Table 4b Table 4c Table 5

Table 6

TableT

Table 8

Summary of findings of HPA-axis alterations in PTSD and

MDD

diagnosis groups compared to normal healthy controls.

Details of Motor Vehicle Accident Victims Employment details

Time that motor vehicle accidents occurred How motor vehicle accidents occurred

Position of subjects during the motor vehicle accident Admission diagnoses for motor vehicle accident victims Psychiatric history of subjects and their families

Alcohol use of motor vehicle accident victims prior to,

one month after and six months after a motor vehicle accident Mean and standard error scores for the Clinician Administered PTSD Scale, Beck Depression Inventory, Impact of Events Scale, Stanford Acute Stress Questionnaire and Dissociative Experiences Scale.

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115

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Líst of Figures

Figure 1

Figure ² Figure 3 Figure 4 Figure 5

Figure 6a Figure 6b

Figure 7 Figure 8

Figure 9 Figure 10

Figure

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Figure 12

The Hypothalamic-Pituitary-Adrenal-axis (HPA-axis) Afferent control of the HPA-axis.

Steroid pathways

Anti

- Glucocorticoid Receptor-B antibody titration.

Representative histograms of GRB autofluorescent control, isotype control and bound receptor.

Freeze storage of lymphocytes for GRB determination.

Paraforrnaldehyde storage of lymphocytes for GRp determination.

Anti

-

Glucocorticoid Receptor-cr antibody titration.

Representative histograms of GRcr autofluorescent control, isotype control and bound receptor.

Dexamethasone Suppression Testing

in

normal subjects Glucocorticoid Receptor-cr response to

0.5mg and 1.Omg doses of dexamethasone.

Glucocorticoid Receptor-p response to 0.5mg and 1.Omg doses of dexamethasone.

Diurnal variation in the expression of Glucocorticoid Receptor-ø in human lymphocytes.

Diurnal variation in the expression

of

Glucocorticoid Receptor-p in human lymphocytes.

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75 81

90

90

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Figure 13

Figure

14

^Plasma cortisol levels within four hours of a

MVA,

and at 08.00hrs and at 16.00hrs two days after the accident.

Figure

15

Glucocorticoid Receptor-p responses to

0.5mg Dexamethasone in motor vehicle accident victims with PTSD.

Figure

16

^{24-hour urinary free} cortisol concentration one day and one

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Figure ¹⁷ Figure 18a

Figure 18b

Figure 19

Figure 20

Figure

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month after of motor vehicle accident

in

subjects who developed PTSD and those who did not.

Total cortisol fraction in urine

Cortisol metabolites in urine including THE, THF and allo-THF the day after a motor vehicle accident.

Cortisol metabolites in urine including THE, THF and allo-THF one month after a motor vehicle accident.

24-hour Dehydroepiandrosterone

(DHEA)

in urine the day after and one month after ^a motor vehicle accident

in subjects who developed PTSD and those who did not Total androgen fraction in urine the one day after and one month after ^a motor vehicle accident.

Androgen metabolites

in

a24-hour urine collection one day after, and one month afrcr a motor vehicle accident

in

subjects who developed PTSD and those who did not.

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Declaration

The work presented

in

this thesis has been submitted to the University

of

Adelaide for the degree

of Master of Medical Science. This work

contains

no

material

that

has been accepted

for

the award

of

any other degree or diploma

in

any University or other tertiary

institution.

Some of the results of this study have been published as scientific papers.

The material contained

within

this thesis may be photocopied and loaned

in

compliance with the rules and regulations of the University library.

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9

Acknowledgments

The research undertaken as part

of

this degree was multidisciplinary

in

the broadest sense of the

word.

Foremost,

I

must acknowledge Susan March, who coordinated the project and performed the

clinical

interviewing

of

subjects. Not

only

a fantastic professional, but also a great person to work with.

A

special thanks is extended to my supervisors Dr. Gary'Wittert, and Professor McFarlane, who have provided so much assistance to me over the last two ^years.

I

must also thank

the staff of

the various laboratories

who

allowed me

to

perform the assays at the core

of

this

project. In

particular the Endocrine and Metabolism

unit

at the Queen Elizabeth Hospital where the urinary steroid and cortisol assays were performed.

Special thanks

to Dr.

George

Phillipov

without whose help these tests would have been impossible. Thanks are also extended to Bob Farrant,

for

his technical assistance

in

both the before mentioned procedures.

A

thank you is also extended to

Allan

Bishop director

of

the

flow

cytometry laboratory at the

IMVS,

and co-author of one of the papers arising from this study.

Two

special people also deserve

mention. Firstly

my fiancée Elsa, who has provided me

with

so much support and encouragement during a

very trying period. And finally

the wonderful

lady who

supported me throughout

my life, my

Grandma,

who

sadly passed away before seeing the completion of my studies.

Summary

It

has been shown previously that chronic PTSD

is

associated

with

alterations

in

the activity of the hypothalamic-pituitary-adrenal axis (HPA

-axis).

^These include diminished cortisol excretion, increased numbers

of

glucocorticoid receptors and increased sensitivity

of the

axis

to low

doses

of

dexamethasone. However,

it is not known

whether these alterations are fundamental to the development

of

the disorder, or whether they arise once the condition

is

established.

Initially

a study was conducted

with

six healthy controls to determine suitable dosage and administration time

for

dexamethasone.

A

second series

of

studies

in

healthy subjects

was

used

to

develop

a

receptor

binding protocol for flow

cytometry to enable the analysis of both isoforms of the glucocorticoid receptor (GR-cr and GR-P). Finally, a study of HPA-axis activity in motor vehicle accident victims admitted to the Royal Adelaide Hospital was conducted

to

determine

if

aberrant HPA-axis activation

in

the

first

month

following

a motor vehicle accident

is

related

to

adverse psychological experiences in some individuals

following

^a traumatic event and

if

the activity of the HPA- axis

is

related

to

the development

of

posttraumatic stress disorder

in a

sub-set

of

these

individuals.

Twenty male (mean age

3I + 3

years) and seven female (mean age 45

+

6 years)

victims of

a motor vehicle accident were recruited

into

the

study.

Subjects were studied within two days of the accident, one month after the accident and again six months after the accident. Female victims were more

likely

to develop PTSD then male victims

of

a motor vehicle accident

(p = 0.OZ). Within two

days

of

the accident,

high

scores on measures

of the 'Impact of Events

Scale

(IES-R)', and the

Stanford

Acute

Stress Questionnaire

(SASQ)' were strongly

related

to the

development

of PTSD.

^Plasma

tl

cortisol measured

with 4

hours

of

the accident, and again

two

days after the accident at 08.00hrs and 16.00hrs was not different between subjects who developed PTSD and those

who did not.

^Plasma

cortisol was not

related

to

subjects

self

reported

level of

pain.

Additionally,

plasma cortisol at the time

of

the accident was not related

to

scores on the IES or

SASQ. 24-hou

Urinary Free Cortisol the day after the accident was not related to the development of PTSD, but at one month was related to the symptoms of avoidance (p =

0.01)

and SASQ

total

score

(p = 0.046).

^Cortisol metabolites were measured

by

Gas Chromatography, but 24-hour excretion

of

cortisol metabolites THF, allo-THF, and THE

did not differ between subjects who

developed

PTSD and those who did

not.

Dehydroepiandrosterone

(DHEA) is not normally

excreted

into urine,

however

it

was found

in

the urine

of all

subjects who developed PTSD the day after the accident, and at

one month after

the

accident,

but

was

not

significantly higher than

DHEA in

urine

of

subjects who did not develop

PTSD. DHEA

^was positively related to the total Dissociative Experiences Scale score

(p = 0.025). A

small cohort, and the large

variability in

the release

of

adrenal steroids

following

trauma meant

that

differences between diagnostic groups in the activation of the HPA-axis could not be detected. However, one month after the trauma strong relationships were found between symptoms of PTSD such as avoidance

of

reminders

of the

event, and

intrusive

thoughts

that

were related

to

24-hour urinary cortisol excretion. Additionally,

DHEA

in urine was related to synmptoms of dissociation,

which is

a common symptom

of PTSD.

The results suggest that different neuorsteroids

may

^mediate

different

components

of

the psychological response

to a

traumatic event.

Dysregulation

of

these neuroendocrine systems may be important

in

the consolidation

of

adverse psychological reactions

to

the trauma that take place

in

the

first

month after the event and may lead to the development of PTSD.

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