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Joint Bone Spine 76 (2009) 514–518

Original article

Antioxidant status in patients with osteoporosis: A controlled study

Omer Faruk Sendur a, Yasemin Turan a,∗, Engin Tastaban a, Mukadder Serter b

a Department of Physical Medicine and Rehabilitation, Adnan Menderes University School of Medicine, Aydın, Turkeyb Department of Clinical Biochemistry, Adnan Menderes University School of Medicine, Aydın, Turkey

Accepted 24 February 2009Available online 21 May 2009

bstract

bjective: We aimed to investigate serum antioxidant enzymes and nitric oxide (NO) levels in postmenopausal women with osteoporosis (OP)nd in healthy controls; and to determine the relationship between these enzymes, NO and clinical parameters in this present study.ethods: Forty-five postmenopausal women fulfilling OP diagnostic criteria of World Health Organization (WHO) and 42 postmenopausal healthyomen without OP were enrolled. Patients in the study population were selected among individuals that were not pre-diagnosed or pre-treated

or OP. Patients with metabolic bone diseases, fracture history, which were smokers, alcohol users and taking antioxidant drug treatment, werexcluded from the study. Dual Energy X-ray Absorptiometry (DXA) results, body mass indices and demographic data were recorded. Erythrocyteatalases (CAT), glutathione reductase (GR) enzyme activities and erythrocyte glutathione (GSH) levels, plasma malondialdehyde (MDA) levelsere measured by spectrophotometer whereas plasma nitrite+nitrate (NOx) levels were measured by ELISA microplate-reader.esults: Patients had significantly lower GR (P < 0.01) enzyme activity and higher levels of MDA (P < 0.01) and NO (P < 0.01) than non osteoporoticealthy controls. There was no significant difference between both groups in erythrocyte GSH levels and CAT activities. Total femoral BMDeasurements significantly correlated with MDA levels (P = 0.001). There was no significant relationship between other antioxidants and lumbar

r femoral BMD.onclusion: Oxidative stress may play an important role in postmenopausal bone loss and therefore it might be considered when pathogenesis ofostmenopausal OP has been investigated.

2009 Société francaise de rhumatologie. Published by Elsevier Masson SAS. All rights reserved.

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eywords: Osteoporosis; Antioxidant; Nitric oxide

. Introduction

Osteoporosis (OP), which is characterized by low bone massnd microarchitectural deterioration of bone tissue leading toncreased bone fragility, can result in an increased risk of frac-ures. The pathogenesis of OP is poorly understood. Agingnd genetic factors are important, moreover, many factors suchs smoking, immobilization, inefficient calcium intake, thyroidnd parathyroid function disorders, gastrointestinal and kidneyiseases cause OP [1,2]. In the literature, limited numbers ofrevious reports have shown the relationship between bone min-

ral density (BMD) and oxidative stress. Antioxidant systemslay important roles in the development of OP [3–10]. On thether hand, some authors as Wolf et al. have noted that serum

∗ Corresponding author. 250 sk No: 109 3/2 D:23, Bornova /35040, Izmir,urkey.

E-mail address: dryaseminturan@gmail.com (Y. Turan).

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297-319X/$ – see front matter © 2009 Société francaise de rhumatologie. Publishedoi:10.1016/j.jbspin.2009.02.005

oncentrations of antioxidants were not related to BMD andupportive intake of antioxidants had no effect on BMD [11].dditionally, role of nitric oxide (NO), which has a biphasic

ffect on bone, in the pathogenesis of OP, is still being debated12–17]. As a result of these studies, catabolic process on boneells is believed to be accelerated by increased serum NO, whichas arisen from serum antioxidant enzyme deficiency.

In order to clarify these conflicting results, we aimed to definend match the levels of serum antioxidants and NO in post-enopausal OP patients and healthy control groups, and make a

ecision about whether there is relationship of these antioxidantsnd NO with clinical parameters or not.

. Methods

Forty-five postmenopausal women fulfilling OP diagnosticriteria of World Health Organization (WHO) [18] and 42 post-enopausal healthy women without OP were enrolled in this

by Elsevier Masson SAS. All rights reserved.

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While there was a significant correlation between plasmaMDA levels and femoral BMD in the study population(r = −0.464, P = 0.001), there was no significant correlation

Table 1Demographic data of both groups.

Patients (n = 45) Controls (n = 42)

Age [year, mean (S.D.)] 55.6 (2.9) 56.6 (2.4)

O.F. Sendur et al. / Joint B

tudy. Patients in the study population were selected amongndividuals that were not pre-diagnosed or pre-treated for OP.atients with metabolic bone diseases, fracture history, and whoere smokers, taking alcohol and antioxidant drug treatmentere excluded from the study. Demographic data of study pop-lation were recorded. The study protocol was approved by theocal ethics committee of our institution and all participants gaveritten informed consents. BMD of patients was measured byual Energy X-ray Absorptiometry (DXA).

.1. BMD measurements

BMD was measured at the lumbar spine (L2-4) and femoralrea by DXA using a LUNAR DPX densitometer (GE Lunarorporation, Madison, WI, USA). The diagnosis of OP wasased on the WHO criteria [18]. So OP is defined as a T scoref −2.5 or less, indicating a BMD that is at least 2.5 S.D.s lesshan the mean for young adults.

In order to investigate the presence of compression fractures,horacic and the lumbar spine radiographs were performed.

.2. Biochemical measurements

For laboratory investigations, following 12 hours of fasting,lood samples of all patients were collected at eight o’clock inhe morning. Blood samples were collected into tubes contain-ng sodium citrate as anticoagulant in the early morning aftern all night fasting and were separated immediately by cen-rifugation at 3000 rpm 10 min at +4 ◦C. The plasma samplesere frozen at −80 ◦C until for nitrite + nitrate (NOx) and MDA

ssays. The buffy coat on the erythrocyte sediment was sepa-ated carefully after the plasma was removed. The erythrocyteediment washed three times with 0.9% NaCl solution to removeeftover leukocytes and plasma components. After each proce-ure, erythrocyte-saline mixture was centrifuged at 3000 rpm0 min at +4 ◦C. Erythrocyte sediments were treated with 4-foldce-cold deionized water to obtain stock hemolysate containing

5 g hb/100 ml for the erythrocyte CAT and Glutathione reduc-ase (GR) activity assays. Hemolysate hemoglobin content wasetermined using Drabkin reagent.

CAT activities were measured in a fresh suspension ofemolysates. 1:1000 dilution of this concentrated homolysateas prepared with phosphate buffer immedialety before the

ssay CAT activity was determined by the method of Aebi. Therinciple of the assay is based on the determination of the rateonstant (s-1,k) of hydrogen peroxide decomposition by catalasenzyme. The rate constant was calculated from the followingormula: k = (2,3/�t)log(A1/A2).

GR detection was performed according to Carlberg and Man-ervik, modified by Kazim Husain [19,20]. GR activity ofamples was calculated by observing absorbance change in firstnd third minutes at 340 nm by Shimadzu UV 160 A spectropho-ometer.

MDA concentration was measured in terms of thiobarbi-uric acid reactive substances, spectrophotometrically by the

ethod of Yoshioka T and the values expressed as �moles ofalondialdehyde (MDA) formed per liter plasma [21]. Direct

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pine 76 (2009) 514–518 515

uantitative measurement of NO level in the biological sam-les is very difficult, because it is a very labile molecule. Inqueous solution, NO reacts with molecular oxygen and accu-ulates in the plasma as nitrite (NO2

−) and nitrate (NO3−)

ons. Therefore, NOx, stable oxidation end products of NO, cane readily measured in biological fluids and have been used initro and in vivo as indicators of tissue NO production. In thistudy, the levels of NO metabolites in serum samples were ana-yzed using a modification of the cadmium-reduction methods described by Navarro-Gonzalves et al. This reaction usingre-treatment of samples to reduce nitrate to nitrite which cane accomplished by catalytic reactions using Cd. The samplesere analyzed spectrophotometrically using a microplate reader

nd quantified automatically against NaNO2 standard curve andhe results were expressed as �mol/L.Blood was collected intoubes containing EDTA as anticoagulant in the early morningfter an all night fasting for total GSH levels were analyzedsing method as described by Teitze [22].

.3. Statistical analyses

Software Statistical Package Sciences for windows ver-ion 14.0 was used for all calculations. For the discrepanciesmong the groups, Student’s t-test was employed. For correla-ion between results, the Pearson’s correlation coefficient waserformed. Chi-square test was used for comparing categoricalariables. The level of significance was set at p value less than.05.

. Results

The mean age of patient group and controls were 55.6S.D. = 2.9) years and 56.6 (S.D. = 2.4) years, respectively. Thereas no significant difference between ages of the both groups

p = 0.085). The demographic characteristics of patients andontrols were shown in Table 1.

No compression deformities were encountered in the thoracicnd lumbar spine of OP patients.

Plasma MDA and NO levels were significantly higher intudy population than those of in the control group (Table 2).owever, erythrocyte GR activity was significantly lower in

tudy group than that of the control group, there was no sig-ificant difference between the both groups in erythrocyte GSH

ex (F/M) 45/0 42/0MI [kg/m2, mean (S.D.)] 29.4 (4.4) 27.9 (4.3)enopause age [year, mean (S.D.)] 49.7 (1.5) 50.1 (1.5)

/M: female/male; BMI: body mass index.

516 O.F. Sendur et al. / Joint Bone S

Table 2Plasma antioxidant and nitric oxide levels in both groups.

Patients (n = 45) Controls (n = 42)

MDA (�mol/L) 4.8 (0.8)* 4.2 (0.8)NO (�mol/L) 42.1 (6.2)* 38.5 (5.7)GSH (mg/gHb) 2.9 (0.9) 3.1 (0.9)GR (�mol/min/gHb) 11.3 (3.8)* 13.2 (3.4)C

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* p < 0.01 versus control.

etween other antioxidants and lumbar or femoral BMDalues.

. Discussion

Oxidative stress has been demonstrated to play a role inathogenesis of many diseases such as cardiovascular disease23,24], cerebrovascular disease [25] and Diabetes Mellitus [26].owadays, studies indicating that formation and resorption ofynamic bone tissue all through life have been affected by anncrease in oxidative stress and/or antioxidant enzyme deficiencyre increasing in number in the literature.

In this present study, there were significant differencesetween patients and control group in plasma MDA, NOx lev-ls and erythrocyte GR activities. In the literature, there were aew studies investigating antioxidant enzyme conditions in OPatients [4,5,9,10]. In one of these studies, it is recently reportedhat serum MDA and NO levels have been increased signifi-antly higher in study group than those of in the control group,hich is in line with the results of our study [9]. Altındag et al.

eported results of their study that increased osteoclastic activ-ty and decreased osteoblastic activity may be associated withn imbalance between oxidant and antioxidant status in post-enopausal OP [10]. Similarly, Maggio et al. demonstrated that

ntioxidant defenses were markedly decreased in osteoporoticemales [5]. Sontakke and Tare showed elevated levels of MDAnd reduced activities of GSH-Px in osteoporotic groups in com-arison to healthy controls [4]. A correlation between oxidativetress and reduced bone density was reported in three differ-nt studies other than these four studies [3,7,8]. Furthermore,n previous studies, in which NO effects on bone were investi-ated, NO was reported to be an important regulator on boneetabolism [12–17].Reactive oxygen species (ROS) are normal by-products of

ellular metabolism. In many joint diseases, proinflammatoryactors such as cytokines and prostaglandins are released at sitesf inflammation, together with ROS [27]. Enhanced osteoclasticctivity observed in bone disorders may have been responsibleor increased production of ROS in superoxide forms, which isvident by increased levels of serum malondialdehyde (MDA)evels. One of the most damaging effects of ROS is lipid per-xidation, the end product of which is MDA [28]. MDA is

ne of the most frequently used indicators of lipid peroxida-ion. MDA is known as a potential biomarker for oxidativetress [29]. Sontakki et al. reported that apart from lipid per-xidation function, MDA had an osteoclastic activity [4]. In

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pine 76 (2009) 514–518

his present study, serum MDA levels were higher in the studyroup than in the control group. Compatible results with ouresults have been reported in the literature [4,7,9]. Besides, inur study, there was a significant relationship between MDAevels and femoral neck BMD values. Contrary to our results,zgocmen et al. reported no significant relationship betweenDA levels and neither lumbar nor femoral BMD values [9].ll of these results support that increased serum MDA levelsay be used as a biochemical marker indicating osteoclastic

ctivity.NO is an important biological mediator produced from L-

rginine through the enzyme nitric oxide synthase (NOS). Itlays an important role in vascular relaxation, regulation oflood flow, immune function, stress, neurotransmission, mod-lation of nociception, induced vasodilatation and in painodulation [17]. Previous studies conducted in animal mod-

ls and in humans have shown that NO is an important regulatorf bone metabolism [12–16]. In our study, serum NO level wasignificantly higher than the values in the control group andhere was no significant relationship between NO levels andeither lumbar nor femoral BMDs. However, Yalin et al. foundignificant correlation between serum NO levels and both lum-ar and femur neck BMD [7]. Additionally, Ozgocmen et al.eported no significant relationship between NO levels and lum-ar BMD, there was a significant relationship between NO levelsnd femoral BMD [9]. According to these results, we may statehat NO levels regulate the bone mass. But more studies inves-igating the relation between NO and lumbar and femoral BMDre required.

Glutathione is a compound classified as a tripeptide madef three amino acids: cysteine, glutamic acid and glycine. Glu-athione is an antioxidant that protects cells from toxins suchs free radicals [30]. Decrease in GSH level is a marker for theegree of oxidative stress [31]. In our study, serum GSH levelas lower in the study group than in the control group. However,

he difference between these groups was not significant.Reduction of oxidized GSH is provided by glutathione reduc-

ase, which is rather important for keeping reduced GSH at highevels within cells [32]. Avitabile et al. reported that there was

strong relationship between decreased bone mineral densitynd decreased GR levels. Authors proposed the hypothesis thatdecrease in antioxidant enzyme activity, like GR, might causearkedly increased bone demineralization and, as a result, may

ncrease destructive free radical levels [33]. Our findings arelso in line with their hypothesis. In contrast to the results ofhis present study, Sontakke et al. reported that there was noignificant difference between GR level in postmenopausal OPatients and individuals in the control group [4]. However, num-ers of patients and healthy subjects enrolled into this studyere fewer than patients and healthy subjects enrolled into our

tudy. Also, in this study mean age of the control group (20–30ears) was younger than our study. We found significant differ-nce between total GSH levels in OP patients compared with

he control group. Low levels of GR were also obtained in OPatients; actually it should not be regarded as an inconsistency.esides the researchers indicating that total GSH is decreasedt oxidative stress, some researchers state an unchanged level of

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SH [34–36]. Annuk et al. reported that oxidative stress occurst chronic renal injury; oxidized glutation is increased, reducedSH is decreased. However, total GSH is found unchanged and

ven had a tendency to decrease in control group [37]. If we hadnalyzed oxidized and reduced GSH, we would expect increasedevels of oxidized GSH and decreased levels of reduced GSH inP patients.Catalase is a common enzyme found in living organisms. Its

unctions include catalyzing the decomposition of hydrogen per-xide to water and dioxygen [38]. Catalase has one of the highesturnover rates of all enzymes; one molecule of catalase can con-ert millions of molecules of hydrogen peroxide to water andxygen per second. Low level of catalase activity could primar-ly damage the endoplasmic reticulum in the cells [35]. Althougherum CAT activity was lower than that of in the control group,he difference was not significant. CAT values in OP patients inzgocmen et al. study were also low, but the difference between

he groups was significant [9].It has been reported in the previous studies that the level of NO

nd other anti-oxidants showed some daily variations [39–43].e prevent these daily variations of the level of anti-oxidants

y collecting the blood samples of patients in both study andontrol groups at the same time of the day [43].

The important limitation of the present study is that we couldot assess any other bone resorption and formation markers.urther studies investigating relationship between antioxidantnzymes, NO levels and these markers should be performed.

Consequently, we believe that oxidative stress may play anmportant role in postmenopausal bone loss. Therefore it mighte considered when pathogenesis of postmenopausal OP haseen investigated.

onflicts of interest

None of the authors has any conflicts of interest to declare.

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