oxidative stress in fibromyalgia syndrome

1. Deciphering the role of oxidative and
antioxidative parameters in women with
Fibromyalgia Syndrome
Dr. Ghizal Fatima
Assistant Professor,
ERA UNIVERSITY,
LUCKNOW, INDIA

2. What is this thing that lurks inside?
It steals my body and my mind,
It leaves me with a fear so strong
I think and wonder, what is wrong?
I cannot walk the way I should,
I cannot run, I wish I could.
What else will it steal in the morning light?
I cannot let it, I must fight.
How can I fight what I cannot see
This monstrous thing inside of me?

3. Introduction: Why we did this study
• Fibromyalgia Syndrome (FMS) is a chronic musculoskeletal syndrome
characterized by diffuse pain, stiffness, and tenderness of specific
anatomic sites which are called tender points.
• FMS has been associated with a wide spectrum of symptoms such as
allodynia, debilitating fatigue, joint stiffness, sleep disturbances,
migraine headaches, irritable bowel syndrome, numbness or tingling of
the extremities, restless legs syndrome,dizziness and balance
problems,cognitive and memory problems, mood disturbance such as
depression and anxiety.
• The prevalence of FMS ranges from 1 to 2% in the general population
and the condition is more common among females than in males with a
ratio of 9:1. The etiology of FMS is still unknown.

4. This condition affects mainly women, with a
female-to-male ratio of 9:1, and its
estimated prevalence in various populations
varies between 2% - 5%.
Epidemiology of FMS

5. • Oxidative stress means an alteration in the delicate balance
between free radicals and the scavenging capacity of
antioxidant enzymes in the body.
• There are several clinical conditions associated with increased
oxidative stress, but novel data suggest a relationship between
oxidative stress and pain perception. Furthermore, there is
little information available in scientific literature about
oxidative stress in FMS patients.
• In the present study we examined the involvement of oxidative
stress in women with FMS and also evaluated its correlation
with the severity of its symptoms.
Introduction: cont.

6. Objectives
• Assessment of the Lipid Peroxides (LPO) nitric oxide (NO) and Protein carbonyl
in plasma of female patients satisfying American College of Rheumatology (ACR)
criteria for FMS and control group.
• Assessment of antioxidative parameters like catalase, Glutathione
peroxidase (GPx) and Glutathione Reductase (GR) in lysate in patients
with FMS and control group.
• Analysis of FIQR in women with FMS and control group.
• To evaluate its relationship with FIQR and oxidative stress parameters in
women with FMS and control group.
• .

7. Materials and Method
• Oxidative stress was determined by measuring the levels of Lipid Peroxides
(LPO) (Ohkawa et al 1979), Protein carbonyl group ((Levine and Williams,
1994) and nitric oxide in plasma and antioxidative parameters in lysate in
60 female patients satisfying American College of Rheumatology criteria
(1990) for FMS and 60 healthy females without FMS.
• Clinical parameters of FMS were evaluated by Fibromyalgia Impact
Questionnaire Revised (FIQR).
• Clinical assessment was done by following questionnaires
1- General Assessment Questionnaire (self designed)
2- Fibromyalgia impact Questionnaire Revised (Bennett et al, 2009)

8. Inclusion and exclusion criteria
Inclusion Exclusion
Patients who fulfilled the criteria
developed by the American
College of Rheumatology (1990)
were included in the study.
Smokers and those using any oral
contraceptives were excluded from the
study as these factors can influence the
oxidative stress parameters.
Informed consent for inclusion in
the study were taken from all the
subjects.
Moreover, subjects with known co-morbid
conditions like diabetes mellitus, psychiatric
and those suffering from Rheumatoid
arthritis or other inflammatory joint disease
were also excluded from the study.

9. 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.

11. • Statistical analysis was done using SPSS statistical software (16.0 versions).
• Quantitative variables of FMS patients and controls were presented as the
mean ± standard deviation, and are compared by independent t-test.
• Pearson correlation was done to find the pattern of associations between the
groups.
• A value of p<0.05 was considered statistically significant and p<0.01 is
considered highly significant.
• .
Statistical analysis

12. Clinical and Biochemical Characteristics among Study and Control groups
Parameters FMS=60
[mean ± SD]
Controls=60
[mean ± SD]
P-value
Age (years) 34.7±9.8 32.8±10.5 N.S
ESR 27.2±9.7 24.9±8.2 N.S
FIQR 87.2±11.4 35.0±8.3 <0.05
Tender Points 16.6±1.9 14.3±2.2 <0.05

13. Clinical characteristics of FMS patients and control group
Variables FMS (n=60)
Mean
Control (n=60)
Mean
p-value
Muscles twitching
Yes 60 4
<0.05No 0 56
Disequilibrium in Climbing stairs
Yes 54 7
<0.05No 6 53
Frequent awakening
Yes 56 9
<0.05
No 4 51
Sleep status
Sound sleep (Yes) 2 49
<0.05
Disturbed sleep (No) 58 11

14. Morning Stiffness
Yes 60 5 <0.05
No 0 55
Morning fatigue
Yes 60 8 <0.05
No 0 52
Headache
Yes 52 10 <0.05
No 8 50
Lack of energy
Yes 60 7 <0.05
No 0 53
Clinical characteristics: cont.

15. Oxidative Parameters Study (n=60)
(Mean ± SD)
Control (n=60)
(Mean ± SD)
P-Value
Lipid Peroxides (LPO)
3.31±0.57 2.24±0.35
p<0.01
Protein carbonyl group
1.90±0.45 1.32±0.26
p<0.01
Nitric Oxide (NO)
2.49±1.26 1.41±0. 49
p<0.01
Oxidative stress parameters among Study and Control groups

16. Antioxidative parameters FMS patients
n=60 Mean ±SD
Control n=60
Mean ±SD
p-value
Catalase 41.0±5.1 57.3±8.6 <0.01
Glutathione peroxidase
(GPx)
28.2±3.7 38.7±5.1 <0.01
Glutathione Reductase
(GR)
24.4±4.2 29.1±6.2 <0.01
Antioxidative parameters among Study and Control groups

17. Pearson correlation analysis in between Lipid Peroxides (LPO), Protein
carbonyl group and TPC and FIQR.
Parameters Groups TPC FIQR
r r
Lipid Peroxides (LPO) FMS patients -0.043 0.533*
Control 0.065 0.129
Protein carbonyl group
FMS patients 0.082
0.434*
Control 0.090 0.115
Nitric Oxide (NO) FMS patients -0.063 0.479*
Control 0.045 0.139

18. Pearson correlation analysis in between antioxidative parameters and TPC
and FIQR
Parameters Groups TPC FIQR
r r
Catalase
FMS patients -0.093 .064
Controls .114 -.179
Glutathione peroxidase (GPx)
FMS patients -0.138 -.116
Controls -.133 .130
Glutathione Reductase (GR)
FMS patients 0.058 -.156
Controls -.101 .064

19. • In conclusion, the present study indicate that women with FMS are exposed
to oxidative stress and this increased oxidative stress may play a role in the
etiopathogenesis of the disease.
• These findings may support the hypothesis of fibromyalgia as an oxidative
disorder.
• Moreover, our results also show that increased oxidative stress parameters are
more strongly associated with FMS symptoms.
• The limitations of the study is the sample size we need good sample size to
draw much better conclusion.
Conclusion

20. • Therefore, an intervention study is recommended to address the question of
whether improved oxidative stress parameters in FMS patients can lead to
decrease in FMS symptomatology.
• Furthermore, supplementation of the regular treatment with
antioxidants may lead to development of new therapeutic
strategies for prevention and treatment of this disease.

21. Relevance of the Study
These findings resulted in better understanding
of oxidative stress parameters in FMS patients
which is found elevated in patients group as
compared to the control group. Therefore,
increased oxidative stress levels may be
responsible for the development of FMS.

23. • Lipid peroxidation (LPO) (Ohkawa et al 1979)
Mix 0.2 ml of blood plasma with 0.5 ml of acetic acid
subsequently 0.5 ml of 8% SDS will be added and shake the
above mixture, after this, add 1.5 ml of 0.8% TBA solution. The
reaction mixture was incubated in a boiling water bath for one
hour. After cooling at room temperature, add 3 ml n-butanol and
this reaction mixture will be centrifuged at 10,000 rpm for 15
minute. A clear supernatant obtained after centrifugation will be
used for measuring the absorbance at 532 mm against reagent
blank.

24. • Protein carbonyl group (Levine and Williams, 1994):
• The experiment will be carried out in two setups. In one set
(experimental), 5 ml DNPH and 1.5 ml blood plasma will be taken
and in the second set (reference) 1.5 ml blood plasma will be added
in the 5 ml of 2.5 M HCl instead of DNPH. Tubes will be left for 1
hour at room temperature (in dark). The samples vortexes for 15
min. then 5ml of 20% TCA will be added in both sets to a final
concentration of TCA by 10% itself. Tubes then are kept to ice water
for 30 min to get protein precipitated and centrifuged. The protein
precipitate collected, washed with 4 ml of 10% TCA and recovered
by centrifugations. Protein pellet washed 3 times with 4 ml mixture
of ethanol: ethyl acetate (1:1 v/v) to remove unreacted DNPH and
lipid components. Finally precipitate of experimental and reference
dissolved in 2ml of 6M guanidine HCl and left for 10 min at 37◦ C
with general vortex mixing and insoluble material will be removed
by additional centrifugation. A clear supernatant obtained. The
absorbance of this will be measured at 365nm (UV) against
guanidine HCl on spectrophotometer. The results expressed as
nmol/mg protein.

25. • Nitric Oxide (Schmit et. al. 1994)
• Procedure:-
• Reagents and standards will be prepared first. Then we will add 85
μL of each standard in duplicate, to respective wells. Depending on
the nitrite concentration in the sample, we will add between 5 μL to
85 μL of each sample to wells in duplicate. If the expected nitrite
concentration of a sample is unknown, it may be helpful to perform
more than one dilution.
• Then we will add sufficient buffer to each sample to bring the total
volume to 85 μL (e.g., 80 μL for 5 μL of sample). 10 μL of
reconstituted Nitrate Reductase to each sample will then be added.
And then 10 μL of 2 mM NADH will be added to each well and the
plate will be shaken for 20 minutes at room temperature. After that
we will add 50 μL of Color Reagent #1 and shake briefly. Then we
will add 50 μL of Color Reagent #2 and shake for 5 minutes at room
temperature. Measure absorbance values at 540 nm in the
microtiter plate reader.
• Lastly we will plot the standard curve and estimate the
concentrations of the samples from the curve.

26. • Catalase (Aebi 1974): (Done in lysate)
2ml of phosphate buffer and 1 ml. of diluted (0.2M) H2O2 will be
taken in a cuvette, in this we will add 0.02 ml enzyme source and
mix thoroughly. The decrease in absorbance at 240nm will be
recorded after every 30 seconds for 3 minutes against reagent
blank. One catalase unit is defined as the amount of enzyme
required to cause a decrease in optical density by 0.100 of substrate
(H2O2).We will also determine Protein content in enzyme source.
The results will be expressed as unit/mg protein.

27. • Super Oxide Dismutase (MC Cord and Fridovich, 1969): (Done in lysate)
• The experiment will be carried out in two setups. In one setup, 1.1 ml pyrophosphate
buffer, 0.2 ml NBT, 0.2 ml PMS, 20µl enzyme source will be taken. The second
setup received all the above reagents minus the enzyme source. We will start the
reaction simultaneously in two sets by the addition of 0.2 ml NADH. After an
interval of 90 seconds, 0.5 ml glacial acidic acid will be added to each tube for
checking the reaction, after this we will add same amount of enzyme source in
reference tubes. The absorbance of both sets tubes will be read at 560 nm against
reagent blank. Difference between reference and experimental OD will give the
inhibition of NBT reduction by enzyme source. Protein will also be estimated in
enzyme source. The unit of SOD enzyme activity will be defined as the amount of
enzyme required to inhibit the optical density at 560 nm of NBT reduction by 50%
in one minute under the assay conditions. The results will be expressed as unit/mg
protein.

28. • Glutathione Reductase (Hazelton and Lang; 1995)
(Done in lysate)
• Procedure: The reaction mixture consist of 0.1 ml NADPH, 0.2 ml GSSG,
0.1 ml EDTA, 2.5 ml buffer and 0.1 ml of enzyme source to a total volume of
3.0 ml. The reaction will be initiated by the addition of enzyme source.
Oxidation of NADPH followed at 340 nm. Blank reaction will be run
simultaneously. The decrease in absorbance at 340 nm is followed at 30
second intervals. We will also determine Protein content in the enzyme
source. Enzyme unit will be defined as nmole of NADPH oxidized per minute
per mg protein. The results will be expressed as unit/min/mg protein.

29. • Glutathione Peroxidase (Pagila and Valentine; 1967)
(Done in lysate)
• Procedure: An incubation mixture containing 0.4 ml buffer, 0.2
ml of GSH, 0.2 ml EDTA, 0.2 ml Sodium azide and 0.2 ml hydrogen peroxide
will be pre-incubated at 37o C for 10 min. 0.1 ml of enzyme source will be
added and incubated at 37oC for 10 min. The reaction will then be
terminated by the addition of the 0.1 ml of 10% TCA. Supernatant taken
and 3 ml of phosphate buffer and 1 ml of DTNB will be added. The color
developed read immediately at 412 nm in a spectrophotometer. We will
also estimate Protein in enzyme source. GPx enzyme activity will be
expressed as µg GSH oxidized per mg protein and the results will be
expressed as unit/mg protein.