ORIGINAL ARTICLE
Acute phase response and oxidative stress status in familialMediterranean fever (FMF)
Savas Guzel • Gulnur Andican • Arzu Seven •
Mahmure Aslan • Murat Bolayirli •
Eda Celik Guzel • Vedat Hamuryudan
Received: 25 May 2011 / Accepted: 16 August 2011 / Published online: 23 September 2011
� Japan College of Rheumatology 2011
Abstract We aimed to determine acute phase response
(APR) and oxidative stress in patients with familial Medi-
terranean fever (FMF) and compare these characteristics
with those in healthy controls; 20 patients with FMF and 15
healthy controls were enrolled in the study. The erythrocyte
sedimentation rate (ESR), and C-reactive protein (CRP),
fibrinogen, and leukocyte levels were determined as markers
of APR. Thiobarbituric acid reactive substances (TBARS),
conjugated diene, and lipid hydroperoxide levels were
measured as markers of lipid peroxidation. Carbonyl group
and thiol (T-SH) levels were analyzed to determine the
oxidative damage to proteins, and 8-hydroxy-2-deoxygua-
nosine (8-OHdG) was measured to reflect DNA oxidation.
The erythrocyte glutathione (GSH) level, and glutathione
peroxidase (GSH-Px), CuZn superoxide dismutase (CuZn
SOD), and catalase activities were measured as markers of
antioxidant status. Conjugated diene (p \ 0.001) and car-
bonyl group (p \ 0.05) levels were significantly higher and
GSH-Px activity (p \ 0.01) was significantly lower in FMF
patients compared with controls. FMF patients in the attack
period (n = 8) had significantly higher CRP, ESR, fibrino-
gen, and leukocyte levels (p \ 0.001) than patients in the
attack-free period (n = 12). The T-SH level (p \ 0.05) was
significantly higher and CuZn SOD activity was signifi-
cantly lower (p \ 0.05) in FMF patients in the attack period.
The findings revealed upregulated APR during the attack
period in FMF patients and enhanced oxidative stress in the
FMF patients as compared to controls.
Keywords Familial Mediterranean fever � Acute phase
response � Oxidative stress � Antioxidant status
Introduction
Familial Mediterranean fever (FMF) is an autosomal
recessively inherited disorder, characterized by fever and
recurrent aseptic inflammation of serosal spaces, joints, and
skin. FMF is a chronic autoinflammatory disease, and
persistent respiratory burst caused by activated neutrophils
may generate reactive oxygen species (ROS) in patients
with this disease [1–3].
Reactive oxygen species are formed during oxidative
processes that normally occur at relatively low levels in all
cells and tissues. Circulating human erythrocytes possess
the ability to scavenge O2- and H2O2 by CuZn superoxide
dismutase (SOD)-, catalase (CAT)-, and glutathione per-
oxidase (GSH-Px)-dependent mechanisms. If ROS are not
effectively scavenged, these species may lead to wide-
spread lipid, protein, and DNA damage [4–6].
S. Guzel (&)
Department of Biochemistry, Medical Faculty,
Namik Kemal University, Tekirdag, Turkey
e-mail: [email protected]
G. Andican (&) � A. Seven � M. Aslan
Department of Biochemistry, Cerrahpasa Medical Faculty,
Istanbul University, Istanbul, Turkey
e-mail: [email protected]
M. Bolayirli
Fikret Biyal Central Research Laboratory,
Cerrahpasa Medical Faculty, Istanbul University,
Istanbul, Turkey
E. C. Guzel
Department of Family Medicine, Medical Faculty,
Namik Kemal University, Tekirdag, Turkey
V. Hamuryudan
Department of Internal Medicine, Cerrahpasa Medical Faculty,
Istanbul University, Istanbul, Turkey
123
Mod Rheumatol (2012) 22:431–437
DOI 10.1007/s10165-011-0517-5
In the present study we aimed to determine acute phase
response (APR) and lipid, protein, and DNA oxidation and
antioxidant status in newly diagnosed FMF patients.
Thiobarbituric acid reactive substances (TBARS) and
conjugated diene and lipid hydroperoxide levels were
analyzed as markers of lipid peroxidation. To evaluate the
extent of oxidative damage to proteins, carbonyl group and
thiol (T-SH) levels were measured. As a DNA oxidation
marker; 8-hydroxy-2-deoxyguanosine (8-OHdG) was ana-
lyzed. To reflect antioxidant status, the erythrocyte gluta-
thione (GSH) level and GSH-Px, CuZn SOD, and CAT
activities were measured. Oxidative stress and antioxidant
status indexes in FMF patients were compared with those
in controls. We also evaluated the analyzed parameters in
FMF patients during attack and remission periods.
Patients, materials, and methods
Patients
This study was conducted in 20 patients attending the
Rheumatology Department of Internal Medicine at Cer-
rahpasa Medical Faculty, Istanbul University. The clinical
diagnosis of FMF was based on the Tel-Hashomer criteria
[7]. The study protocol was approved by the Ethics Com-
mittee of Cerrahpasa Medical Faculty. Written informed
consent was obtained from all patients and controls. Newly
diagnosed FMF patients (11 females, 9 males) were
enrolled in the study. The mean age of the FMF patients
was 35.15 ± 10.41 years (range 19–59). Fifteen healthy
age-matched individuals (8 females, 7 males) aged between
24 and 55 years (mean 37.06 ± 9.73) formed the control
group.
Twelve of the newly diagnosed FMF patients were in
remission, and 8 of them were in an attack period. The
attack period is determined according to the presence of
clinical findings of fever, abdominal pain, and arthritis, and
according to laboratory measurements of fibrinogen,
leukocytes, and the erythrocyte sedimentation rate (ESR).
An attack-free period (remission) is defined as being free of
attacks for at least 3 weeks. The type of attack was
abdominal in 8, articular in 6, and protracted febrile in 8.
Exclusion criteria were as follows: presence of systemic
diseases, including chronic renal failure, diabetes mellitus,
ischemic heart disease, and malignancy; trauma; heavy
exercise; and use of drugs with potential effects on bio-
chemical parameters.
Blood samples
Blood samples from patients and controls (heparinized and
not) were drawn by venipuncture into precooled tubes. The
ESR, and C-reactive protein (CRP), fibrinogen, and leu-
kocyte levels were measured immediately. The blood
samples to be examined for other measurements were
centrifuged at 15009g at 4�C for 10 min. Serum and
plasma samples were stored at -70�C until the analysis.
Measurements
Determination of TBARS levels
The level of TBARS was determined spectrophotometri-
cally by the method of Buege and Aust [8]. One volume of
plasma was mixed thoroughly with 2 volumes of stock
solution of 15% w/v trichloroacetic acid, 0.375% w/v
thiobarbituric acid, and 0.25 N hydrochloric acid. The
mixture was heated for 30 min in a boiling water bath.
After cooling, the flocculent precipitate was removed by
centrifugation at 10009g for 10 min. The light absorbance
of the sample was determined at 535 nm, and the TBARS
concentration was calculated using 1.56 9 105 M-1 cm-1
as the molar extinction coefficient.
Determination of lipid hydroperoxide levels
Lipid hydroperoxides were measured spectrophotometri-
cally by an iodometric method [9]. The assay was per-
formed by adding 1 ml color reagent (consisting of
0.2 M potassium phosphate; pH 6.2, 0.12 M potassium
iodide, 0.15 mM sodium azide, 2 g/l Triton X-100, 0.1 g/
l benzalkonium chloride, and 10 lM ammonium molyb-
date) to 100 ll sample and to 100 ll saline as the blank.
The samples were placed in the dark for 30 min. After
centrifugation at 12,0009g for 3 min, the absorbance of
the supernatant was measured at 365 nm against the
blank. Lipid hydroperoxide content of the plasma was
calculated by using the molar absorbance of measured
triiodine.
Determination of conjugated diene levels
Conjugated dienes were determined according to the
method of Slater [10]. Lipids were extracted with chloro-
form–methanol (2:1), followed by centrifugation at
10009g for 5 min. The chloroform layer was evaporated to
dryness under a stream of nitrogen. The lipid residue was
dissolved in 1.5 ml cyclohexane and the absorbance read at
233 nm measured the amount of hydroperoxide formed.
Determination of thiol (T-SH) levels
A spectrophotometric assay based on the thiol/disulfide
reaction of thiol and Ellman’s reagent (5,50-dithiobis-2
432 Mod Rheumatol (2012) 22:431–437
123
nitrobenzoic acid) was used for the thiol assay [11]. The
concentrations were calculated using a molar extinction
coefficient of 13.600 M-1 cm-1. Thiol levels were
expressed as lmol/l.
Determination of carbonyl levels
Protein carbonyl levels were determined, using 2,4-dini-
trophenyl hydrazine (DNPH), from the difference in
absorbance at 365 nm between DNPH-treated samples and
HCl-treated controls, with e370 = 22.000 M-1 cm-1. Car-
bonyl levels were expressed as nmol DNPH incorporated/
mg of protein [12].
Determination of 8-OHdG levels
The 8-OHdG level in serum samples, filtered by ultra-fil-
tration (cut-off molecular weight 10,000), was determined
with a competitive enzyme-linked immunosorbent assay
(ELISA) (Bioxytech 8-OHdG-EIA Kit; Oxis Health Prod-
ucts, Inc., Portland, OR, USA).
Determination of GSH levels
Erythrocyte GSH concentration was determined according
to the method of Beutler et al. [13], using 5,50dithiobis
2-nitrobenzoic acid as a disulfide chromogen that is easily
reduced by sulfhydryl compounds to an intensely yellow
compound. GSH concentration was expressed as mg per g
hemoglobin.
Determination of CuZn SOD activity
CuZn SOD activity was determined according to the
method of Sun et al. [14], based on the inhibition of
nitroblue tetrazolium (NBT) reduction, with xanthine–
xanthine oxidase used as a superoxide generator. One unit
of SOD was defined as the amount of protein that inhibits
the rate of NBT reduction by 50%. SOD activity was
expressed as ng/l.
Determination of GSH-Px activity
The activity of GSH-Px was quantified by a colorimetric
assay, as described by Paglia and Valentine [15], based
upon the oxidation of nicotinamide adenine dinucleotide
phosphate, reduced (NADPH) at 340 nm in the presence of
H2O2 used as a substrate. Erythrocyte GSH-Px activity was
expressed as U/g hemoglobin (Hb). The enzyme unit of
GSH-Px (U) is defined as the number of micromoles of
reduced NADPH oxidized per minute at 37�C by 1 g of Hb
under standard assay conditions.
Determination of catalase activity (CAT)
The spectrophotometric method of Goth [16] was used for
the determination of CAT activity in erythrocytes. One unit
of CAT decomposes 1 lmol of H2O2/l min; results are
expressed as kU/gHb.
Serum levels of CRP were measured by standard
nephelometry (Behring Latex Enhanced on the Behring
Nephelometer BN-100; Behring Diagnostic, Westwood,
MA, USA.) with a sensitivity of 0.1 mg/l. The ESR was
determined by the Westergren method with an established
normal range of 0–20 mm/1 h, fibrinogen was determined
by the clotting time method (Biopool, USA), and leuko-
cytes were determined with an automatic hematology
analyzer (Beckman Coulter, Inc., Fullerton, CA, USA).
Statistical analysis
Statistical analyses were performed with a Statistical
Package for the Social Sciences (SPSS) 10.0 Package
(SPSS Inc., Chicago, IL, USA). Values were expressed as
means ± SD. The significance of the mean differences
between groups was assessed by Dunnett’s t-test and Stu-
dent’s t-test. Relationships between variables were tested
using Pearson’s correlation analysis. p values of less than
0.05 were regarded as significant.
Results
No statistically significant difference was observed with
respect to age distribution between the FMF and control
groups. Dermatologic and/or amyloid accumulation was
not observed in any of the patients (Table 1).
In the FMF patients; conjugated diene (p \ 0.001) and
carbonyl group (p \ 0.05) levels were found to be signif-
icantly higher than those in the control group. As to anti-
oxidant status parameters, erythrocyte GSH-Px activity
was significantly lower (p \ 0.01) in FMF patients with
respect to the control group (Table 2).
Comparison of FMF patients in the attack period with
those in the attack-free period revealed significantly higher
values for the inflammatory markers–CRP (p \ 0.001),
ESR (p \ 0.001), fibrinogen (p \ 0.001), and leukocytes
(p \ 0.001) in the attack period. Thiol (T-SH) was found to
be significantly (p \ 0.05) higher and erythrocyte CuZn
SOD activity significantly (p \ 0.05) lower in the attack
period (Table 3).
Significant positive correlations were observed between
CRP and ESR, leukocyte, and fibrinogen levels (r = 0.494,
p \ 0.05, r = 0.571, p \ 0.01, and r = 0.483, p \ 0.05,
respectively). Fibrinogen was observed to be positively
correlated with both ESR and the leukocyte level
Mod Rheumatol (2012) 22:431–437 433
123
(r = 0.821, p \ 0.001 and r = 0.829, p \ 0.001, respec-
tively). A positive correlation was found between ESR and
the leukocyte level (r = 0.832, p \ 0.001). The T-SH level
was found to be positively correlated with both leukocyte
and fibrinogen levels (r = 0.466, p \ 0.05 and r = 0.453,
p \ 0.05, respectively). A positive correlation at the
p \ 0.05 level (r = 0.497) was observed between CuZn
SOD activity and the lipid hydroperoxide level.
Discussion
Familial Mediterranean fever is a recessively inherited
periodic inflammatory disease that stimulates a very
intense APR which manifests as increased oxidative stress.
Under normal conditions a delicate balance exists between
oxidative processes and antioxidant activity at all levels of
organization in complex organisms. Oxidative stress occurs
when antioxidant activity is unable to preserve the equi-
librium, and results in tissue damage [3–5].
The predominant prooxidant chemicals are singlet
oxygen molecules, superoxide anions, hydrogen peroxide,
and hydroxyl radicals: molecules collectively referred to as
ROS. Some of the consequences of increased ROS include
depletion of ATP and nicotinamide dinucleotide, DNA
damage, alterations in protein stability, the destruction of
membranes via lipid peroxidation, and the release of pro-
inflammatory cytokines [17, 18].
In recent years, increasing attention has been focused on
the role of reactive oxygen metabolites in the pathogenesis
of inflammatory diseases such as rheumatoid arthritis.
Increased activity of free radicals, the unstable molecules
associated with cell damage, is theorized to underlie the
Table 1 Demographic data and inflammatory markers in FMF
patients and control group
Control group
(n = 15)
FMF group
(n = 20)
Age (years) 37.06 ± 9.73 35.15 ± 10.41
Sex (F/M) 8/7 11/9
Fever, ?/- – 8/12
Myalgia, ?/- – 15/5
Abdominal pain, ?/- – 8/12
Joint pain, ?/- – 6/14
CRP (mg/dl) NA 8.16 ± 7.38
ESR NA 29.35 ± 15.84
Leukocytes NA 9012.50 ± 3180.20
Fibrinogen (mg/l) NA 419.05 ± 68.58
FMF familial Mediterranean fever, NA not assessed, CRP C-reactive
protein, ESR erythrocyte sedimentation rate
Table 2 Values (mean ± SD) of the analyzed parameters in FMF
patients and control group, and statistical significance
Control group
(n = 15)
FMF group
(n = 20)
TBARS (lM) 3.52 ± 0.69 3.93 ± 0.79
Lipid hydroperoxide (lM) 33.11 ± 8.90 37.87 ± 11.17
Conjugated diene (lM) 82.04 ± 18.16 100.29 ± 22.47***
Carbonyl (nmol/mg protein) 0.85 ± 0.11 1.02 ± 0.19*
T-SH (lM) 448.49 ± 65.64 388.90 ± 107.30
8-OHdG (ng/ml) 8.24 ± 4.80 10.26 ± 4.63
GSH (mg/gHb) 3.57 ± 0.63 3.59 ± 0.70
GSH-Px (U/gHb) 19.94 ± 4.42 14.89 ± 5.47**
CuZn SOD (ng/l) 304.64 ± 66.21 331.31 ± 73.40
Catalase (kU/l) 47.42 ± 11.03 43.91 ± 7.16
TBARS thiobarbituric acid reactive substances, T-SH thiol, 8-OHdG8-hydroxy-2-deoxyguanosine, GSH glutathione, GSH-Px glutathione
peroxidase, CuZn SOD CuZn superoxide dismutase, Hb hemoglobin
* p \ 0.05, ** p \ 0.01, *** p < 0.001
Table 3 Comparison of demographic data and analyzed parameters
in FMF patients in attack and attack-free periods
Intra-group comparison of FMF patients
Attack period
(n = 8)
Attack-free period
(n = 12)
Age (years) 37.50 ± 9.13 33.58 ± 11.29
Sex (F/M) 4/4 7/5
Fever, ?/- 8/8 7/5
Myalgia, ?/- 5/3 1/11
Abdominal pain, ?/- 8/8 8/4
Joint pain, ?/- 7/1 1/11
CRP (mg/dl) 14.56 ± 7.95*** 3.89 ± 2.03
ESR 44.50 ± 14.54*** 19.25 ± 4.56
Fibrinogen (mg/l) 479.75 ± 48.04*** 378.58 ± 46.80
Leukocytes 12275 ± 1982.60*** 6837.50 ± 1437.39
TBARS (lM) 3.79 ± 0.78 4.03 ± 0.82
Lipid hydroperoxide
(lM)
35.09 ± 11.69 39.73 ± 10.92
Conjugated diene (lM) 95.14 ± 25.87 95.41 ± 33.39
Carbonyl (nmol/mg
protein)
1.07 ± 0.21 1.00 ± 0.19
T-SH (lM) 457.99 ± 90.76* 342.85 ± 94.14
8-OHdG (ng/ml) 10.51 ± 4.30 10.10 ± 3.74
GSH (mg/gHb) 3.36 ± 0.60 3.75 ± 0.75
GSH-Px (U/gHb) 15.02 ± 7.07 14.82 ± 4.46
CuZnSOD (ng/l) 297.23 ± 58.71* 351.53 ± 79.23
Catalase (kU/l) 44.19 ± 8.45 43.72 ± 6.57
CRP C-reactive protein, ESR erythrocyte sedimentation rate, TBARSthiobarbituric acid reactive substances, T-SH thiol, 8-OHdG8-hydroxy-2-deoxyguanosine, GSH glutathione, GSH-Px glutathione
peroxidase, CuZn SOD CuZn superoxide dismutase
* p \ 0.05, *** p < 0.001
434 Mod Rheumatol (2012) 22:431–437
123
mucosal injury commonly seen in the various inflammatory
diseases [19, 20].
In the literature there is evidence of increased oxidative
stress in FMF patients both in remission and attack periods.
In FMF patients, the spontaneous production of superoxide
radicals by neutrophils has been shown to be greater than
that in healthy people, with a positive correlation between
superoxide production and clastogenic factors (CFs) [21,
22]. CFs are reported to stimulate superoxide production
and thus create a prooxidant state [23].
Hydrogen peroxides, formed from the dismutation of
superoxide radicals, can react with metal ions and lead to
the formation of highly reactive hydroxyl radicals that can
cause lipid, protein, and DNA damage. The typical clinical
course of FMF is that of exacerbations and remissions, and
the increased APR observed during attacks usually returns
to normal in attack-free periods. In this respect the change
in the magnitude of APR between attack and attack-free
periods has been used as a means of diagnosis of FMF.
CRP and ESR are reported to be the most frequently used
indicators to monitor APR [24, 25].
In the present study we observed significantly higher
CRP, ESR, fibrinogen, and leukocyte levels in FMF
patients during the attack period compared to levels during
the attack-free period. Similar to our findings, Baykal et al.
[26] and Bagcı et al. [24] reported that FMF patients had
significantly higher ESR, CRP, and fibrinogen levels in the
acute attack period compared to the findings in the quies-
cent period.
As a ligand-binding protein, CRP contributes to innate
resistance to pneumococcal and possibly other bacterial
infections and may thus have important scavenging prop-
erties with respect to autologous ligands generated by tis-
sue damage and inflammation [27]. In this respect,
upregulation of the APR during the attack period may
contribute to better resistance to infection [28]. However, a
high ESR level, reported in some FMF patients during
remission, has been attributed to the presence of continuing
subclinical inflammation in 25% of FMF patients during
attack-free periods [29, 30].
As to our findings of oxidative stress markers, of the
lipid peroxidation markers, only conjugated diene levels
appeared to be significantly higher in FMF patients than in
controls. Diene conjugation resulting from lipid oxidation
is now commonly used as an end-point for determining the
antioxidant activity of a sample. Measurement of the for-
mation of diene conjugation has an advantage in that it
measures an early stage in the oxidation process. The
persistent activation of neutrophils may be the cause of the
accumulation of conjugated dienes due to excess produc-
tion of ROS.
The specificity of the thiobarbituric acid (TBA) method
has been a subject of controversy due to the presence in
plasma of many substances, including bilirubin, DNA,
sucrose, and aldehydes that interfere with the assay. The
TBA test has been found to underestimate the extent of
lipid peroxidation [31]. These factors may explain the
nonsignificant difference in TBARS values between the
FMF patients and control group in the present study.
The iodometric procedure used for the lipid hydroper-
oxide analysis in our study could possibly have underes-
timated the hydroperoxide amount, due to interference by
plasma phospholipids and other amphipathic molecules,
and this could have led to the nonsignificant difference
observed between FMF patients and controls in this study.
In vitro studies have shown that the action of ROS on
proteins results in the formation of carbonyl groups with a
relatively longer half-life than lipid peroxidation products.
Evaluation of the carbonyl group content in plasma pro-
teins is the most generally respected indicator of both
protein oxidation and free radical reaction intensity [19,
32], and our finding of a significantly high carbonyl group
level in FMF patients appears to be compatible with pre-
vious findings.
The persistent respiratory burst caused by activated
neutrophils in FMF patients may generate ROS, leading to
oxidative DNA damage. However, our findings revealed no
significant difference in 8-OHdG levels between FMF
patients and controls and no significant difference in
8-OHdG levels between attack and remission periods in
FMF patients. In contrast to our reported findings; Kirkali
et al. [33] reported the accumulation of statistically sig-
nificant levels of mutagenic and cytotoxic DNA lesions in
the polymorphonuclear leukocytes of FMF patients.
Antioxidants and antioxidant enzymes work indepen-
dently and in concert to protect against oxygen toxicity.
Our finding of decreased GSH-Px activity reflects the
exhaustion of the antioxidant system and seems to be
responsible for the increased conjugated diene levels in
FMF patients. The findings of Gurbuz et al. [34] related to
malondialdehyde levels and GSH-Px activity are in line
with our data.
Comparison of findings in FMF patients during attack
and remission periods revealed no significant differences in
lipid, protein, and DNA markers. As to antioxidant status,
the T-SH level was found to be significantly increased and
the CuZn SOD activity significantly decreased in attack
periods. The positive correlation observed between thiol
group and leukocyte and fibrinogen levels indicates acti-
vation of the antioxidant system in enhanced APR.
In our previous study [35] in rheumatoid arthritis (RA)
patients, we found ESR and leukocyte levels to be signifi-
cantly increased in patients with moderate disease activity
compared to those with low disease activity. Furthermore,
the percentages of RA patients who were positive for
rheumatoid factor and anti-nuclear antibodies were higher
Mod Rheumatol (2012) 22:431–437 435
123
in those with moderate disease activity. In our other study,
published in Clinical Biochemistry [20], we observed sig-
nificantly increased levels of lipid peroxidation markers
(TBARS, conjugated dienes, lipid hydroperoxides) and a
DNA oxidation marker (8-OHdG) in RA patients. In that
study, reflecting protein oxidation, the protein carbonyl
group was found to be significantly increased and the T-SH
level was found to be significantly decreased in RA patients.
As antioxidant status markers, GSH, GSH-Px, and CuZn
SOD levels were observed to be significantly decreased in
RA patients. Comparison of RA patients grouped according
to disease activity revealed enhanced lipid, protein, and
DNA oxidation status and exhaustion of the antioxidation
defense system in those with greater disease activity.
In conclusion, our findings reveal upregulated APR dur-
ing attack periods in FMF patients, and enhanced oxidative
stress, as indicated by significantly increased lipid peroxi-
dation and protein oxidation markers and compromised
GSH-Px activity, in FMF patients compared with controls.
Acknowledgments This study was supported by Istanbul Univer-
sity Research Foundation, Turkey.
Conflict of interest None.
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