CIRCADIAN DYSFUNCTION IN FIBROMYALGIA

1. “CIRCADIAN DYSFUNCTION IN FIBROMYALGIA
RHEUMATICA”.
Dr. Ghizal Fatima
Assistant Professor,
Department of Biotechnology,
Era University, Lucknow

2. Circadian rhythm regulates the amount of hormones and neurotransmitters
the body produces, and by this it creates circadian rhythm in balance. In FMS
there is alteration in hormones and neurotransmitters production. The study
is conducted to study the relationship of FMS & circadian pattern of release
of hormones and cytokines.

3. • 50 patents and 50 controls were enrolled for the study, all the
clinical and biochemical assessments of both patients and
controls have been completed.
• Samples were collected by admitting the patients and
controls in the Department of Rheumatology.
1- General Assessment Questionnaire (self designed)
2- Fibromyalgia impact Questionnaire Revised (Bennett et al,
2009)
3- Circadian rhythm Phillip questionnaire
(www.golite.philip.com)
4- Circadian rhythm symptoms of FMS questionnaire (self
designed)
All the data analysis has been completed.
Clinical assessment of Patients and Controls

4. Clinical and biochemical characteristics among Study
and Control groups:
Parameters FMS=50
[mean ± SD]
Controls=50
[mean ± SD]
P-value
Age [years] 36.7±9.8 32.8±10.5 >0.05
BMI (Kg/m2) 25.7±3.9 23.9±3.3 <0.05
ESR 27.2±9.7 24.9±8.2 >0.05
ALT 39.8±14.1 37.6±14.4 >0.05
FIQR 91.9±8.0 5.0±8.3 <0.01
Tender Points 16.8±1.9 1.9±2.4 <0.01
CRA 40.80±16.16 14.56±24.41 <0.01

5. Variables FMS patients n=50 (%) Control n=50) (%)
Residence
Rural 17 (34) 11 (22)
Urban 33 (66) 39 (78)
Tobacco chewing
Yes 2 (4) 4 (8)
No 48 (96) 46 (92)
Widespread pain history
>3 months 14 (28) Nil
>6 months 10 (20) Nil
>1 year 26 (52) Nil

6. Clinical characteristics of women with FMS and control women:
Variables FMS patients n=50 (%) Control n=50 (%) p-value
Weight loss
Yes 16 (32) 7 (14)
<0.05No 34 (68 43 (86)
Family history
Yes 7 (14) 0 (0)
<0.01No 43 (86) 50 (100)
Muscles twitching
Yes 50 (100) 6 (12) <0.01
No 0 (0) 44 (88)
Disequilibrium in Climbing stairs
Yes 38 (76) 3 (6) <0.01
No 12 (24) 47 (94)
Frequent awakening
Yes 42 (84) 4 (8) <0.01

7. Sleep Status
Yes (Sound sleep) 3 (6) 46 (92) <0.01
No (Disturbed sleep) 47 (94) 4 (8)
Morning Stiffness
Yes 47 (94) 3 (6) <0.01
No 3 (6) 47 (94)
Morning fatigue
Yes 48 (96) 5 (10) <0.01
No 2 (4) 45 (90)
Headache
Yes 43 (86) 24 (48) <0.01
No 7 (14) 26 (52)
Abdominal pain
Yes 28 (56) 20 (40) >0.05
No 22 (44) 30 (60)
Appetite change
Yes 18 (36) 0 (0) <0.01
No 32 (64) 50 (100)
Lack of energy
Yes 49 (98) 8 (16) <0.01
No 1 (2) 42 (84)
Jaw pain
Yes 11 (22) 46 (92) >0.05
No 39 (32) 4 (8)

8. Circadian Rhythm Test (Phillip online
questionnaire)
Circadian rhythm
Condition
Patients FMS N=50
(%)
Controls N=50 (%) P-value
Misaligned body clock 39 (78) 4 (8)
<0.01Non body-clock related 9 (18) 0 (0)
Body clock amplitude
disorder
1 (2) 10 (20)
Normal body clock 1 (2) 36 (72)
Circadian Rhythm
Severity
Mild 32 (64) 5 (10)
<0.01Moderate 17 (34) 9 (18)
Severe 0 (0) 0 (0)
N.A 1 (2) 36 (72)
Circadian Rhythm
Score
40.80±16.16 14.56±24.41 <0.01

9. Sampling design
Blood samples were collected at the 4 designated times of the day.
However, 6 or 7 control Samples were collected at 11-11:30 pm and at 6 -6:30 am.
The mid-night samples of patients and Controls were drawn And kept for 30 min.
for serum Separation and then stored at 2-8 C. for 6 hr.
and centrifuged In the morning and stored at -40 C.

10. Objectives: 1
Assessment of the circadian changes in cortisol, melatonin and serotonin
levels in patients with FMS.

11. A. Grouping of experiment subjects
Control (n=50) FMS (n=50)
Cortisol analysis by Elisa Kit
Collection of Blood samples
B. Experimental Design
Melatonin analysis by Elisa Kit Serotonin analysis by Elisa Kit
All the Patients and Controls
have been analyzed
All the Patients and Controls
have been analyzed
FLOW CHART OF THE STUDY DESIGN FOR OBJECTIVE-1
All the Patients and Controls
have been analyzed
Data analysis completed

12. Circadian Rhythm of serum cortisol level in study and control group.
Time of Day Study (n=50)
(Mean ± SD)
Control (n=50)
(Mean ± SD)
P-Value
Morning (6 AM)
27.91±12.81 28.30±13.63
>0.05
Afternoon (12 Noon)
14.82±5.91 13.68±6.21
>0.05
Evening (6 PM)
10.68±5.58 8.60±3.61
<0.05
Night (12 Midnight)
15.45±9.80 6.06±3.03
<0.01

14. Circadian Rhythm of serum Melatonin level in study and control group
Time of Day Study (n=50)
(Mean ± SD)
Control (n=50)
(Mean ± SD)
P-Value
Morning (6 AM)
35.18±12.75 19.73±9.98
<0.01
Afternoon (12 Noon)
14.91±6.40 13.48±5.14
>0.05
Evening (6 PM)
17.84±11.57 22.74±16.83
>0.05
Night (12 Midnight)
38.39±18.51 63.68±13.64
<0.01

16. Serum serotonin level was found high in patients but no significant circadian rhythm was
found in serum Serotonin level in study and control group.
Time of Day Study (n=50)
(Mean ± SD)
Control (n=50)
(Mean ± SD)
P-Value
Morning (6 AM)
76.53±34.02 115.54±38.68
<0.01
Afternoon (12 Noon)
86.76±47.46 113.69±28.10
<0.01
Evening (6 PM)
88.06±47.49 108.10±27.31
<0.05
Night (12 Midnight)
89.78±51.27 108.44±26.38
<0.05

18. Objective: 2
• Analysis of circadian pattern of symptoms of fibromyalgia and its possible relationship
with cortisol, melatonin and serotonin.

19. Symptoms Morning N=50
(%)
Afternoon N=50
(%)
Evening N=50 (%) Mid night N=50
(%)
Control Max. pain 0 (0) 0 (0) 10 4 (8)
Least pain 11 (22) 2 (4) 0 (0) 1 (2)
Patients Max. pain 19 (38) 1 (2) 12 (24) 17 (34)
Least pain 12 (24) 28 (56) 8 (16) 1 (2)
Control Max. stiffness 1 (2) 0 (0) 9 (18) 4 (8)
Least stiffness 5 (10) 7 (14) 2 (4) 0 (0)
Patients Max. stiffness 18 (36) 1 (2) 13 (26) 16 (32)
Least stiffness 11 (22) 26 (52) 9 (18) 1 (2)
Control Max. anxiety 0 (0) 13 (26) 15 (30) 5 (10)
Least anxiety 19 (38) 3 (6) 9 (18) 2 (4)
Patients Max. anxiety 15 (30) 1 (2) 15 (30) 13 (26)
Least anxiety 9 (18) 29 (58) 5 (10) 0 (0)
Control Max. numbness 1 (2) 0 (0) 0 (0) 2 (4)
Least numbness 1 (2) 2 (4) 0 (0) 0 (0)
Patients Max. numbness 7 (14) 0 (0) 7 (14) 35 (70)
Least numbness 8 (16) 37 (74) 4 (8) 0 (0)
Control Feel worst 0 (0) 1 (2) 0 (0) 1 (2)
Feel best 42 (84) 0 (0) 6 (12) 2 (4)
Circadian pattern of Fibromyalgia symptoms in patients and control

20. Patients Feel worst 20 (40) 0 (0) 10 (20) 20 (40)
Feel best 3 (6) 32 (64) 9 (18) 6 (12)
Control Max. stress 0 (0) 17 (34) 17 (34) 3 (6)
Min. stress 24 (48) 0 (0) 2 (4) 11 (22)
Patients Max. stress 10 (20) 0 (0) 19 (38) 20 (40)
Min. stress 10 (20) 34 (68) 3 (6) 2 (4)
Control Diff. conc. 2 (4) 14 (28) 20 (40) 4 (8)
No diff. conc. 23 (46) 3 (6) 14 (28) 1 (2)
Patients Diff. conc. 18 (16) 3 (6) 22 (44) 7 (14)
No diff. conc. 9 (18) 30 (60) 5 (10) 6 (12)
Control Max. Headache 2 (4) 9 (18) 8 (16) 6 (12)
Min. Headache 14 (28) 3 (6) 5 (10) 3 (6)
Patients Max. Headache 3 (6) 0 (0) 19 (38) 18 (36)
Min. Headache 14 (28) 26 (52) 0 (0) 0 (0)
Control Max. bowl 30 (60) 6 (12) 13 (26) 1 (2)
Suppress bowl 6 (12) 23 (46) 8 (16) 13 (26)
Patients Max. bowl 11 (22) 22 (44) 15 (30) 1 (2)
Suppress bowl 30 (60) 6 (12) 6 (12) 7 (14)

21. Decreased
Melatonin Level
Maximum Stress
Elevated Cortisol Level
Impaired sleep
Maximum
Numbness
Worst Stiffness
Fatigue
Least stiffness,
Anxiety and
Pain
Worst
Fibromyalgia Syndrome : Circadian
Rhythm of Symptoms
Feel good time
Day time
Sleepiness
Immense Fatigue
minimally Alert
Difficulty in
Concentration.Dizziness
12
Mid night
12
Noon
6 PM
6 AM
Headache onset
Maximum Pain,
stiffness
and Anxiety
Low Cortisol
level, High
stress
12:Midnight
High alertness
12
Noon

22. 3-Determination of circadian changes in cytokines level (IL-6 and TNF-
alpha) and its possible relationship with melatonin, serotonin, cortisol
and symptoms of FMS.
Objective: 3

23. A. Grouping of experimental subjects
Control (n=50) FMS (n=50)
Estimation of Circadian
Rhythm
IL-6
Collection of Blood samples
B. Experimental Design
Biochemical Estimation
TNF-alpha
Data analysis completed
FLOW CHART OF THE STUDY DESIGN FOR OBJECTIVE-3
Estimation of Circadian
Rhythm

24. Time of Day Study (n=50)
(Mean ± SD)
Control (n=50)
(Mean ± SD)
P-Value
Morning (6 AM)
3.04±2.41 2.36±1.75
>0.05
Afternoon (12 Noon)
2.91±2.29 2.19±1.69
>0.05
Evening (6 PM)
2.67±2.17 2.24±1.45
>0.05
Night (12 Midnight)
2.99±2.48 2.15±1.47
>0.05
Serum IL-6 level was not found significant in patients and control group and hence there
was no significant differences observed in the circadian Rhythm of serum IL-6 level in study
and control group.

26. Time of Day Study (n=50)
(Mean ± SD)
Control (n=50)
(Mean ± SD)
P-Value
Morning (6 AM)
5.85±3.63 2.99±2.05
<0.01
Afternoon (12 Noon)
5.86±3.49 3.11±2.02
<0.01
Evening (6 PM)
5.90±3.51 2.96±2.11
<0.01
Night (12 Midnight)
5.88±3.44 2.78±1.99
<0.01
Serum TNF-alpha levels were found significant in patients group but there was no
significant circadian changes observed in serum TNF-alpha level in study and control group.

28. Simple correlation analysis in cytokines and hormonal profiles
Parameters Groups
Cortisol Melatonin 5-HT
r r r
IL-6 Control
-0.127 -0.124 0.136
Patients
0.301* 0.137 -0.347*
TNF- α Control
-0.053 0.082 0.150
Patients
-0.182 -0.145 0.040
Simple correlation analysis of Melatonin with cortisol and 5-HT
Parameters Groups
Cortisol 5-HT
r r
Melatonin
Control
-0.318* 0.044
Patients
0.147 -0.025

29. Pearson correlation analysis in between IL-6, TNF-α, and age, TPC, BMI and FIQR.
Parameters Groups Age TPC BMI FIQR
r r r r
IL-6 Level
(Morning)
Control
0.022 0.075 -0.113 0.080
Patients
0.152 0.079 0.144 0.167
IL-6 Level
(After Noon)
Control
0.060 0.049 -0.203 0.116
Patients
0.162 0.168 0.107 -0.115
IL-6 Level
(Evening)
Control
0.001 0.004 -0.173 0.048
Patients
0.120 0.088 0.154 0.208
IL-6 Level
(Mid Night)
Control
0.191 0.127 -0.184 0.176
Patients
0.235 0.054 0.088 0.237
TNF-α Level (Morning) Control
-0.078 -0.093 -0.044 0.006
Patients
-0.232 0.051 -0.182 0.093
TNF-α Level (After Noon) Control
-0.138 0.008 -0.102 0.096
Patients
-0.246 0.117 -0.250 0.126
TNF-α Level (Evening) Control
-0.130 0.049 -0.120 0.097
Patients
-0.189 0.038 -0.134 0.086
TNF-α Level (Mid Night) Control
-0.148 0.022 -0.184 0.116
Patients
-0.235 0.054 -0.176 0.094

30. Pearson correlation analysis in between Serotonin, Melatonin and Cortisol, and age, TPC, BMI and FIQR.
Parameters Groups Age TPC BMI FIQR
r r r r
Melatonin Level
(Morning)
Control 0.084 -0.043 0.068 0.263
Patients 0.043 0.065 0.021 0.551**
Melatonin Level
(After Noon)
Control 0.038 0.082 -0.017 0.087
Patients -0.064 -0.190 -0.242 0.242
Melatonin Level
(Evening)
Control -0.063 -0.048 -0.083 0.107
Patients 0.022 0.197 0.194 0.053
Melatonin Level (Mid
Night)
Control 0.100 0.124 0.012 -0.126
Patients -0.029 0.209 0.010 -0.648**
Cortisol Level
(Morning)
Control -0.049 0.019 -0.009 -0.041
Patients 0.031 -0.122 0.122 -0.020
Cortisol Level (After
Noon)
Control -0.177 -0.251 -0.182 -0.151
Patients 0.097 0.178 0.036 0.009
Cortisol Level
(Evening)
Control -0.182 -0.262 -0.138 -0.247
Patients 0.010 0.141 -0.056 0.045
Cortisol Level (Mid
Night)
Control 0.220 -0.041 0.250 -0.129
Patients 0.125 -0.296* -0.165 -0.417**

31. Relevance of the Study:
1)-The observations of abnormalities in melatonin and cortisol which are highly regulated by circadian pacemaker
raise the possibility that there is an abnormality of circadian rhythm in FMS patients.
2)- Our results suggests that circadian rhythm of symptoms in FMS patients varies substantially with the time of the
day.
3)-This study resulted in better understanding of the circadian rhythm in FMS patients. The Chronobiological theory
has important implications with regard to the treatment or management of patients with FMS. Moreover, the
diurnal variation of symptoms suggests that the optimal midday time for the least pain and fatigue provides
opportunities to promote and to monitor the response to treatment programmes and has important implications
in the assessment of the patients. Therefore, timing may be crucial in treating disease like FMS and this has
important implications for scheduling activities of daily living and possibly for timing the administration of
medications.

33. Conclusions
• Our results suggest that the disturbance in the circadian pattern of cortisol is found
in FMS patients. Interestingly, this increase in nocturnal serum cortisol in patients
group suggests deregulated circadian patterns which may explain in part the
patient complaint of unrefreshing sleep. In sum, our results showing lower cortisol
values in the morning, support the hypothesis of a circadian dysfunction of the
cortisol among patients group.
• Further, our results suggest that there was a weak circadian pattern of 5-HT in
patients with FMS. And this decrease in 5-HT levels, might contribute to the
pathogenesis of the disease. Interestingly, the decrease in 5-HT in patients group
throughout the day is important because it may explain in part the patients
complaint of unrefreshing sleep.
• Low melatonin level during the hours of darkness may cause disturbed sleep among
patients group. Due to which patients may complain of increased lethargy,
emotional distress, and cognitive and performance difficulties that accompany the
diffuse pain, fatigue and stiffness in the morning.
• Furthermore, our results suggest that there were no perturbations in the circadian
pattern of serum levels of IL-6 and TNF-alpha in patients with FMS.

34. • The sleep disturbance in FMS patients and that the pain is increased during night-time imply
that the ideal treatment at night hours may promote restful sleep.
• For treatment to be effective, the Chronobiological model implies that there must be a
reduction in the sleep -arousal disturbance.
• Moreover, the diurnal variation of symptoms suggests that the optimal midday time for the
least pain and fatigue provides opportunities to promote and to monitor the response to
treatment programmes. For example, excessive physical exertion or energy-consuming tasks
should be discouraged in the early morning or evening.
• On the other hand, any interventional programme would be more acceptable by the patient
during the midday time. Therefore, timing may be crucial in treating FMS and this has
important implications for scheduling activities of daily living and possibly for timing the
administration of medications in FMS patients, and, therefore, these findings may lead to
novel interventions in the treatment of Fibromyalgia Syndrome.
• Future studies are important to validate the results presented here and especially to further
clarify the interplay of circadian rhythm symptoms with hormonal profiles and cytokines.

35. Circadian rhythm assessment by Phillip
Questionnaire
 Circadian rhythm test was done using Philip Questionnaire.
(www.golite.philip.com)
 We have recorded the data of patients and controls
 The data of both patients and controls was entered on Philip Questionnaire
and it gave the result online.
 We do not know how the data got calculated.

36. Our bodies are in harmony with the 24-hour light dark cycle
Secretion of hormones that are sensitive to light and dark orchestrate
rhythms around the 24hour cycle and thus ensure optimal functioning of
the body and disturbances in secretion of these hormones results in
abnormal functioning of the body.
• A rhythm with a period of 24hours is termed circadian.
• It is the scientific study of how biological rhythms or 24 hr. cycle impact both on health and diseases.
In other words, circadian rhythm teaches that human biological and physiological processes
demonstrate a predictable-in time cyclic variability.

37. Diagnosis of patients: (FMS)
• Diagnosis is based on the standardized criteria developed by the
American College of Rheumatology (1990). The criteria is-
1)-Widespread musculoskeletal pain for at least 3 months.
2)-Tenderness is found in at least 11 out of 18 anatomical sites in
making a fibromyalgia diagnosis with the application of 4 kg pressure by
palpation through first three fingers.

38. Circadian rhythm of Control
This graph depicts Normal circadian rhythm of healthy control.

39. Abnormal Circadian rhythm of FMS patient
showing slow rhythm.
This graph depicts that circadian rhythm of FMS patients is running slower
than a normal circadian rhythm.

40. Abnormal Circadian rhythm of FMS patient
showing Faster rhythm.
This graph depicts that circadian rhythm of FMS patients is running Faster
than a normal circadian rhythm.