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: savasguzel@yahoo.com

G. Andican (&) � A. Seven � M. Aslan

Department of Biochemistry, Cerrahpasa Medical Faculty,

Istanbul University, Istanbul, Turkey

e-mail: gulnurandican@gmail.com

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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