Effects of Calcitonin, Risedronate, and Raloxifene on Erythrocyte Antioxidant Enzyme Activity, Lipid Peroxidation, and Nitric Oxide in Postmenopausal Osteoporosis

Archives of Medical Research 38 (2007) 196e205

ORIGINAL ARTICLE

Effects of Calcitonin, Risedronate, and Raloxifene on Erythrocyte Antioxidant Enzyme Activity, Lipid Peroxidation, and Nitric Oxide in Postmenopausal Osteoporosis Salih Ozgocmen,a Huseyin Kaya,a Ersin Fadillioglu,b and Zumrut Yilmazc a

Division of Rheumatology, Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Firat University, Elazig, Turkey b Department of Physiology, Faculty of Medicine, Hacettepe University, Ankara, Turkey c Department of Physiology, Faculty of Medicine, Inonu University, Malatya, Turkey Received for publication July 12, 2006; accepted September 18, 2006 (ARCMED-D-06-00284).

Background. The aims of this study were to compare erythrocyte antioxidant enzyme activities, lipid peroxidation, and nitric oxide levels (NO) in women with postmenopausal osteoporosis (PMO) and non-porotic postmenopausal healthy controls and to assess the relationship between bone mineral density and these oxidant/antioxidant parameters. Additionally, in vivo effects of three different anti-osteoporotic drugs, calcitonin, risedronate and raloxifene, on the erythrocyte oxidanteantioxidant status in women with PMO were also assessed. Methods. Postmenopausal women aged 40e65 years and without previous diagnosis or treatment for osteoporosis and independent in activities of daily living were included. Bone mineral density was measured at the lumbar spine and proximal femur using DXA. Erythrocyte enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and lipid peroxidation end-product malondialdehyde (MDA) and nitrite/nitrate levels, by product of NO, were assessed. Fifty-nine women with PMO were included (mean age 56.7 years), 44 completed course of therapy and were analyzed. Twenty-two non-porotic healthy women (mean age 55.8 years) were included as controls. Results. Patients had significantly lower CAT and GSH-Px enzyme activity and higher levels of MDA and NO than non-porotic healthy controls. Proximal femur BMD measurements significantly correlated with NO levels. QUALEFFO scores improved in different levels with these short-term treatments. In all treatment groups, erythrocyte MDA levels significantly decreased; moreover, risedronate reduced NO levels and raloxifene enhanced CAT enzyme activity. Conclusions. Oxidative stress plays an important role in the pathogenesis of PMO. Studied drugs had ultimate effects on reducing lipid peroxidation. Raloxifene also had potent effects in the enhancement of antioxidant defense system. Ó 2007 IMSS. Published by Elsevier Inc. Key Words: Postmenopausal osteoporosis, Calcitonin, Risedronate, Raloxifene, Antioxidant enzyme, Nitric oxide.

Introduction Bone is a complex tissue composed of several types of cells like osteoclasts and osteoblasts, the primary actors of the

Address reprint requests to: Dr. Salih Ozgocmen, Firat Tip Merkezi, FTR AD, Romatoloji BD, 23119, Elazig, Turkey; E-mail: sozgocmen@ hotmail.com

continuous process of renewal and repair termed as ‘‘bone remodeling.’’ In this process, there is a balance in the activities of these two types of cells that is carefully coordinated by several hormones and cytokines. During the course of opening up tiny holes by osteoclasts, which will be further refilled by osteoblasts, the important chisel molecule is superoxide radical (O2.), which is one of the highly reactive oxygen species (ROS).

0188-4409/07 $esee front matter. Copyright Ó 2007 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2006.09.010

Antioxidants and Nitric Oxide in Osteoporosis

A limited number of previous reports have shown the association of bone mineral density (BMD) and oxidative stress and postulated that oxidative stress and antioxidant systems play important roles in the development of osteoporosis (1e6). Nitric oxide (NO) is a type of short-lived free radical involved in several biological processes. The production of NO from L-arginine is catalyzed by a dioxygenase, nitric oxide synthase (NOS) that has three isoforms: neuronal (nNOS), inducible (iNOS), and endothelial (eNOS). Nitric oxide has been implicated in diverse processes as vasodilatation or vascular regulation, modulation of nociception, immune function, neurotransmission, and excitationcontraction coupling (7,8). Previous studies conducted in animal models and in humans have shown that NO is an important regulator of bone metabolism (9e14). However, results of these studies are controversial and data on in vivo effect of widely used anti-osteoporotic drugs are limited. In this study, we first assessed erythrocyte antioxidant enzyme activities, lipid peroxidation and nitric oxide levels in postmenopausal osteoporotic (PMO) women compared to non-osteoporotic postmenopausal healthy controls. Additionally, relationships between BMD and these parameters were also assessed. We secondly investigated in vivo effects of three different anti-osteoporotic drugs, calcitonin, risedronate and raloxifene, on erythrocyte oxidanteantioxidant status in women with postmenopausal osteoporosis. Materials and Methods Subjects were consecutively recruited among 90 women referred to the out-patient clinic of our department for osteoporosis screening. Inclusion criteria for the study group (PMO and healthy controls) were female gender, age between 40 and 65 years and postmenopausal status, non-smoking, completely independent in activities of daily living. Exclusion criteria were previous diagnosis of osteoporosis or history of treatment for metabolic bone disorders [including hormone replacement therapy, bisphosphonates, selective estrogen receptor modulators (SERMs) or calcitonins], having self-reported fracture history or diseases known to affect bone metabolism and oxidanteantioxidant status (including diabetes, thyroid diseases, neurological and inflammatory diseases, hepatic and renal diseases), use of antioxidant supplementations (antioxidant vitamins, omega-3 or -6 fatty acids, special diets or potent herbal medicines) within 6 months prior to enrollment. Lateral radiographs of the thoracic and lumbar spine were taken and assessed for vertebral deformities (T4-L4) using Genant’s method (15). The study protocol was approved by the local ethics committee of our institution and all participants gave written informed consent. Bone mineral density was measured at the lumbar spine and proximal femur, using dual X-ray absorptiometry

197

(DXA), on a Lunar DPX densitometer (Lunar, Madison, WI). Values for results of DXA measurements were expressed as BMD (g/cm2) and T scores as supplied by the manufacturer. Short-term precision for spine and proximal femur measurements had a coefficient of variation (CV) of 1e2%. Women with a T score #2.5 on L24 or femur neck or total femur were accepted as having PMO. The control group consisted of women with a T-score $1.5 at the same sites. A trained interviewer applied a questionnaire to each subject. This questionnaire consisted of all the study variables such as age, body mass index (BMI), self-reported fracture history, daily energy expenditure, and daily calorie and calcium intake. Quality of Life Questionnaire of the European Foundation for Osteoporosis-41 item (QUALEFFO) was applied at baseline and at the end of the study (16). Basal energy expenditure (BEE) was calculated using Harris Benedict Equation for females; BEE (in calories) 5 655 þ [9.6  (weight in kg)] þ [1.8  (height in cm)]  [4.7  (age in years)]. Daily calorie expenditure was obtained by summing BEE with daily calories expended, which was calculated from the subjects’ daily living activities. Dietary calcium intake was calculated using a modified food frequency questionnaire, which was in agreement with a traditional diet. In the second examination, 600 mg elementary calcium taken during treatment period was not added to the dietary calcium intake calculation. All of the participants’ routine biochemical analyses (including glucose, urea, creatine, liver function tests, calcium, and phosphorus), complete blood count, thyroid function tests, parathyroid hormone, sex hormones, urinary calcium, erythrocyte sedimentation rate and C-reactive protein were assessed on the same day of admission. Urinary levels of collagen type-I cross-linked C-telopeptide (CTx) was measured using Osteosal kits (Provalis Diagnostics Ltd, Flintshire, UK). Vitamin D metabolite 25-hydroxyvitamin D [25(OH)D] was measured by Chromosystems Instruments and Chemicals (Munich, Germany) using high-performance liquid chromatography (HPLC) method. Serum 25(OH)D concentration O20 ng/mL was considered sufficient. Fasting venous blood was collected in heparinized tubes. After centrifugation of the blood, plasma and erythrocyte were separated. Erythrocytes were washed three times with 0.9% NaCl solution and packed. Erythrocytes were then stored at 80 C. The following determinations were made on the samples using commercial chemicals supplied by Sigma (St. Louis, MO). Determination of Catalase Activity in Erythrocytes Catalase (CAT, EC 1.11.1.6) activity was determined according to Aebi’s method (17). The principle of the assay is based on the determination of the rate constant (s-1, k) or the H2O2 decomposition rate at 240 nm.

198

Ozgocmen et al./Archives of Medical Research 38 (2007) 196e205

Determination of Glutathione Peroxidase Activity in Erythrocytes Glutathione peroxidase (GSH-Px, EC 1.6.4.2) activity was measured by the method of Paglia and Valentine (18). The enzymatic reaction in the tube, which contained the following items: NADPH, reduced glutathione (GSH), sodium azide, and glutathione reductase, was initiated by addition of H2O2, and the change in absorbance at 340 nm was monitored by a spectrophotometer. Determination of Malondialdehyde Level in Erythrocytes Malondialdehyde (MDA) level was determined by a method (19) based on the reaction with thiobarbituric acid (TBA) at 90e100 C. In the TBA test reaction, MDA or MDA-like substances and TBA react with the production of a pink pigment having an absorption maximum at 532 nm. The reaction was performed at pH 2e3 at 90 C for 15 min. The sample was mixed with two volumes of cold 10% (w/v) trichloroacetic acid to precipitate protein. The precipitate was pelleted by centrifugation, and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm. The results were expressed according to a standard graphic prepared from a standard solution (1,1,3,3-tetramethoxypropane).

morning. Subjects who received risedronate 5 mg/day were instructed to take the drug once daily on an empty stomach 30e60 min before breakfast with water and to remain in an upright position for at least 1 h after taking the drug. Raloxifene 60 mg/day was instructed to be taken with water in the morning. All participants in the medication groups received 1500 mg daily calcium carbonate supplement (equivalent to 600 mg elemental calcium) with the evening meal. All measurements (except BMD measurements) in the treated groups were made at baseline and at the end of the third month. Statistical Analysis Statistics Package for Social Sciences (SPSS Inc., Chicago, IL) was used for statistical analyses. Results are expressed as mean  SD. Normality of distribution was assessed by Kolmogorov-Smirnov test. Differences between groups at baseline were assessed using t test (for normally distributing variables) and Mann-Whitney U test (for non-normal distribution variables, i.e., QUALEFFO scores, daily coffee intake, urinary calcium). Pre- and post-treatment differences were assessed using Wilcoxon test. Spearman rank correlation (for categorical variables) and Pearson correlation coefficients were used to analyze the relationship between parameters. Two-tailed p value of !0.05 was considered statistically significant.

Determination of SOD Activity in Erythrocytes Total (CueZn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al. (20). The principle of the method is based on the inhibition of NBT reduction by the xanthineexanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the sample after 1.0 mL ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate.

Results The Consolidated Standards for Reporting Trials (CONSORT) diagram is shown in Figure 1. Demographic data and clinical variables of the study population were shown in Table 1. Seven vertebra fractures are found in five patients on thoraco-lumbar radiographs (four grade 1 and three grade 2). All of the osteoporotic women and controls had sufficient levels of 25(OH)D (48.66  21.18 vs. 48.97  17.66, respectively, p 5 0.84).

Determination of Erythrocyte Nitric Oxide Levels Nitric oxide has a half-life of only a few seconds because it is readily oxidized to nitrite (NO 2 ) and subsequently to nitrate (NO ), which serves as index parameters of NO production. 3 The method for erythrocyte nitrite and nitrate levels was based on the Griess reaction (21). Samples were initially deproteinized with Somogyi reagent. Total nitrite (nitrite þ nitrate) was measured by spectrophotometry at 545 nm after conversion of nitrate to nitrite by copperized cadmium granules. A standard curve was established with a set of serial dilutions of sodium nitrite. The resulting equation was used to calculate the unknown sample concentrations. Subjects were randomly allocated into three study arms and received calcitonin, risedronate or raloxifene during a 3-month course of treatment. Subjects who received calcitonin 200 IU/day were instructed to use nasal spray every

Comparison of Osteoporotic Women with Non-porotic Healthy Controls Women with osteoporosis had significantly lower erythrocyte CAT and GSH-Px enzyme activity and higher MDA and NO levels than non-porotic healthy controls, whereas SOD enzyme activity was similar (Table 1). Relationship between Oxidative Stress Parameters with Bone Mass and Other Clinical Variables There was no significant correlation between oxidative stress parameters and baseline clinical variables such as age, body mass index, waist and pelvic circumference, 100-m walking time, gestation, duration of lactation, age at menarche, years since menopause, daily tea intake, daily

Antioxidants and Nitric Oxide in Osteoporosis

199

Assessed for eligibility (n = 90)

Excluded (total n = 31) Not meeting inclusion criteria (n = 25) Refused to participate (n = 3) Other reasons (n = 3) Cross-sectional study

Included (n = 59)

Longitudinal study

3-months

Baseline

End

Randomized (n = 44)

Calcitonin 200 IU/day (n = 16) Received (n = 16)

Risedronate 5 mg/day (n = 14) Received (n = 14)

Raloxifene 60 mg/day (n = 14) Received (n = 14)

Analyzed (n = 16)

Analyzed (n = 14)

Analyzed (n = 14)

Figure 1. The Consolidated Standards for Reporting Trials (CONSORT) diagram showing the flow of participants through each stage of randomized trial.

coffee intake, dietary calcium intake, 25(OH)D or daily calorie expenditure in women with PMO. A significant negative correlation was found between alkaline phosphatase and NOe (r 5 0.28, p 5 0.037) (Figure 2), urinary CTx and GSH-Pxe (r 5 0.34, p 5 0.01) (Figure 3), urinary calcium and CATe (r 5 0.30, p 5 0.025) (Figure 4). There was no significant correlation between oxidative stress parameters and L2-4 and proximal femur BMD measurements except for NO levels which were correlated significantly with femur neck and total femur BMD (r 5 0.34, p 5 0.01 and r 5 0.31, p 5 0.019, respectively). Significant relationship was found between QUALEFFO scores and oxidative stress parameters such as social function with MDAe (r 5 0.26, p 5 0.05) and GSH-Pxe (r 5 0.27, p 5 0.048); mental function with SODe (r 5 0.30, p 5 0.026) and MDAe (r 5 0.26, p 5 0.05); total score with MDAe (r 5 0.29, p 5 0.033). There was no significant correlation between clinical variables and oxidative stress markers in control group. Changes in Oxidative Stress Parameters and Clinical Variables with Anti-osteoporotic Treatments Tables 2e4 show changes in clinical and laboratory parameters with a 3-month course of treatment in calcitonin,

risedronate, and raloxifene groups. In a relatively short treatment period for an anti-osteoporotic drug, calcitonin achieved significant improvement in some of the QUALEFFO scores. Risedronate and raloxifene also achieved improvement in some scores of this questionnaire. The common effect of these three medications was the reduction in lipid peroxidation ensuing declined erythrocyte MDA levels. Furthermore, risedronate reduced erythrocyte NO levels, and raloxifene enhanced erythrocyte CAT enzyme activity.

Discussion Our study revealed that osteoporotic women had significantly reduced erythrocyte antioxidant enzyme activity (CAT, GSH-Px), increased NO and lipid peroxidation end-product MDA. Used drugs significantly reduced lipid peroxidation as evident by reduced MDA levels after a 3month course of therapy. In addition, raloxifene increased erythrocyte CAT enzyme activity, and risedronate reduced NO levels significantly. Some of the anthropometric variables in osteoporotic women were different from those of non-porotic healthy controls. Although BMI was similar, non-porotic healthy

200

Ozgocmen et al./Archives of Medical Research 38 (2007) 196e205

Table 1. Demographic data and clinical and laboratory variables in the study and control group Study group (n 5 59)

Age, years Body mass index, kg/m2 Waist circumference, cm Pelvic circumference, cm 100-m walking time, min Gestation Duration of lactation, weeks Age of menarche Years since menopause Daily tea intake, cups/day Daily coffee intake, cups/day Dietary calcium intake, mg Daily calorie expenditure, Kcal/day Alkaline phosphatase, U/L Parathormone, pg/mL Urinary CTx, mg/L Urinary calcium, mg/dL L2e4 BMD, g/cm2 Femur neck BMD, g/cm2 Total proximal femur BMD, g/cm2 L2e4 T score Femur neck T score Total proximal femur T score QUALEFFO-Pain QUALEFFO-Physical function QUALEFFO-Social function QUALEFFO-General health perception QUALEFFO-Mental function QUALEFFO-Total score CATe, k/gHb SODe, U/gHb MDAe, nmol/gHb GSH-Pxe, U/gHb NOe, mmol/gHb

Controls (n 5 22)

Mean

SD

Minimum

Maximum

Mean

SD

Minimum

Maximum

p

56.75 27.87 93.54 105.25 2.09 5.90 77.83 13.83 9.88 4.31 0.07 571.53 1317.01 107.02 70.51 2.58 12.21 0.86 0.80 0.82 2.81 1.49 1.39 40.95 34.56 51.97 63.27 53.59 44.64 421.77 1814.49 47.21 2.21 0.30

5.38 4.44 10.32 6.96 0.42 3.26 58.99 1.15 7.29 2.46 0.25 91.20 125.40 37.57 28.46 1.10 13.89 0.08 0.11 0.12 0.63 0.90 0.91 17.72 9.76 10.88 12.89 13.93 8.11 134.14 554.80 20.65 0.83 0.19

41.00 19.37 66.00 88.00 1.25 0 0 12.00 1.00 0 0 400.0 1016.0 2.20 8.03 0.10 1.00 0.66 0.60 0.59 4.50 3.20 3.00 20.00 20.00 25.71 33.33 22.22 28.73 202.55 943.23 22.61 0.36 0.05

65.00 38.95 119.00 120.00 3.05 15.00 256.00 18.00 34.00 10.00 1.00 730.00 1905.50 229.00 155.00 4.80 107.00 1.08 1.07 1.09 1.00 0.80 1.00 76.00 57.64 74.28 93.33 91.11 66.14 795.61 3672.02 120.40 3.93 0.99

55.86 29.12 102.64 114.14 2.42 5.55 78.23 13.68 6.27 4.41 0.27 528.15 1792.01 108.60 72.68 ND 14.16 1.14 0.93 0.94 0.76 0.53 0.41 44.91 34.76 51.01 57.57 65.04 47.59 658.02 1735.74 34.21 2.65 0.22

6.01 4.55 12.47 9.92 0.54 3.35 69.33 1.13 4.84 2.82 0.63 267.60 184.77 39.29 34.01 ND 6.46 0.18 0.17 0.08 0.92 0.75 0.65 21.66 11.06 11.27 21.44 8.00 6.02 115.71 245.00 8.35 0.74 0.13

45.00 22.15 81.00 100.00 1.40 0 0 12.00 1.00 0 0 181.20 1547.57 8.00 2.55 ND 4.80 0.97 0.77 0.82 1.50 1.50 1.40 20.00 20.00 25.88 12.30 55.55 38.04 393.08 1293.79 19.01 1.56 0.10

65.00 38.28 120.00 131.00 3.50 12.00 240.00 17.00 15.00 10.00 2.00 1293.70 2173.0 180.00 143.00 ND 23.50 1.80 1.60 1.09 1.90 0.70 0.80 92.00 54.11 71.42 100.00 82.22 62.43 900.78 2216.64 46.33 4.79 0.51

NS NS 0.005 0.001 0.019 NS NS NS 0.013 NS NS NS 0.0001 NS NS NS 0.0001 0.002 0.0001 0.0001 0.0001 0.0001 NS NS NS NS 0.0001 NS 0.0001 NS 0.0001 0.028 0.043

Abbreviations: CTx, collagen type I cross-linked C-telopeptide; CATe, erythrocyte catalase; SODe, erythrocyte superoxide dismutase; MDAe, erythrocyte malondialdehyde; GSH-Pxe, erythrocyte glutathione peroxidase; NOe, erythrocyte nitric oxide; BMD, bone mineral density; QUALEFFO, Quality of Life Questionnaire of the European Foundation for Osteoporosis; ND, not done; NS, not significant.

controls had significantly higher waist and pelvic circumference than osteoporotic females. This evidence supports the notion that females with endomorphic somatotype had higher BMD. Previous reports have shown that ROS, like H2O2 and superoxide anion, is involved in the pathogenesis of bone loss by stimulating osteoclasts differentiation and bone resorption (22e29). Hydrogen peroxide has important properties like acting both as intra- and intercellular signal, having a long half-life and being membrane permeable and essential for osteoclastic differentiation and functions (26). Lane and colleagues found that ovariectomy caused a significant decrease in the level of glutathione and thioredoxin and activities of the enzymes glutathione and thioredoxin reductases in rodents (25). Furthermore, they showed that these antioxidants and their regenerative enzymes were rapidly normalized by a single dose of 17b-estradiol and

antioxidants (N-acetyl cysteine-NAC and ascorbate), whereas L-buthionine-(S,R)-sulfoximine (BSO), which depletes thiol antioxidants, induced bone loss (25). In a recent study, the same group demonstrated that expression of GSH-Px, a major intracellular scavenging enzyme for hydrogen peroxide, was amplified in osteoclasts compared to macrophages and was further increased by administration of 17b-estradiol. They underscored that restraint formation of osteoclasts was associated with suppression of NFkB-activation by RANKL and TNF-a and by enhanced resistance to oxidation (hydrogen peroxide induced) of dihydrodichlorofluorescein in GSH-Px transfected cells. Additionally, administration of pegylated CAT to mice prevented ovariectomy-induced bone loss (26). Bai and co-workers tried to explain the role of ROS in RANKL expression and signaling mechanisms (24). The authors demonstrated that ROS stimulated RANKL expression by

Antioxidants and Nitric Oxide in Osteoporosis

201

250.00

Urinary Ca mg/dL

Alkaline phosphatase U/L

30.00 200.00

150.00

100.00

20.00

10.00

50.00

0.00 0.00 0.00

0.20

0.40

0.60

0.80

1.00

NOe umol/gHb

200.00 300.00 400.00 500.00 600.00 700.00 800.00

Figure 2. Relationship between alkaline phosphatase and NOe (r 5 0.28, p 5 0.037).

different signaling pathways in mouse osteoblasts and human osteoblast-like MG63 cells and highlighted important stimulator effect of ROS in osteoclastogenesis (24). Despite many empirical studies on ROS and bone metabolism in in vivo or in in vitro animal models, in vivo human studies are scanty. Basu et al. investigated a possible relationship between 8-iso-PGF2a (F2 isoprostane is a marker of oxidative stress and reflects nonenzymatic process of lipid peroxidation) with BMD in 48 women and 53 men. They concluded existence of a link between lipid peroxidation and BMD in the studied population (1). Sontakke and Tare showed elevated levels of MDA and reduced

5.00

Urinary CTx mcg/L

4.00

3.00

2.00

1.00

0.00 0.00

1.00

2.00

3.00

4.00

GSH-PxeU/gHb Figure 3. Relationship between urinary CTx and GSH-Pxe (r 5 0.34, p 5 0.01).

CATe k/gHb Figure 4. Relationship between urinary calcium and CATe (r 5 0.30, p 5 0.025).

activities of SOD and GSH-Px in osteoporotic groups (PMO, renal osteodystrophy and bone fractures) in comparison to healthy younger controls, indicating the potential role of ROS in bone metabolism (2). In another controlled study, Maggio et al. investigated a possible link between plasma antioxidants and BMD in elderly osteoporotic women (3). The authors studied plasma vitamin C, E, A, uric acid and MDA levels and activities of SOD (erythrocyte and plasma) and GSH-Px (plasma) in 75 osteoporotic elderly (femoral neck T score !3.5) and 75 non-porotic controls. Osteoporotic women have been reported to have significantly reduced vitamin levels, SOD and GSH-Px activity but similar MDA levels. Vitamin A, C and GSH-Px activity has been found significantly related to femur neck BMD in osteoporotic elderly women (3). Albeit conflicting results in some parameters, the existence of a link between oxidative stress and altered bone metabolism is the conclusive result in these in vivo studies (1e3). There are conflicting results in the literature on the possible relationship between antioxidant vitamin intake and bone mineral density (3,30e33). In a recently published large population study, no association was found between BMD and dietary intake, total intake or serum concentrations of antioxidants (33). In our study, we excluded subjects taking vitamin supplementations and estimated nutritional details using a self-reported questionnaire. Regarding the effects of anti-osteoporotic drugs, some investigations revealed antioxidant activities of risedronate and raloxifene. In a recent study, Dombrecht et al. showed that risedronate together with some other bisphosphonates revealed radical scavenging activity and lipid peroxidation reducing capacity in vitro (34). Ozgonul et al. showed that MDA levels in brain tissue returned to normal by

Ozgocmen et al./Archives of Medical Research 38 (2007) 196e205

202

Table 2. Clinical and laboratory measurements at baseline and at end of a 3-month course of treatment in calcitonin-treated group Baseline (n 5 16)

2

Body mass index, kg/m Waist circumference, cm Pelvic circumference, cm 100-m walking time, min Daily tea intake, cups/day Daily coffee intake, cups/day Dietary calcium intake, mg Daily calorie expenditure, Kcal/day Urinary CTx, mg/L Urinary calcium, mg/dL QUALEFFO-Pain QUALEFFO-Physical function QUALEFFO-Social function QUALEFFO-General health perception QUALEFFO-Mental function QUALEFFO-Total score CATe, k/gHb SODe, U/gHb MDAe, nmol/gHb GSH-Pxe, U/gHb NOe, mmol/gHb

3 months (n 5 16)

Mean

SD

Minimum

Maximum

Mean

SD

Minimum

Maximum

p

27.28 93.81 106.38 1.95 3.81 0.06 541.88 1285.76 2.18 10.12 41.50 32.13 46.20 60.41 49.86 41.62 423.56 1946.44 45.43 2.14 0.33

4.72 8.95 7.17 0.42 2.34 0.25 87.19 90.84 1.17 6.20 16.58 8.88 9.57 8.93 14.61 7.13 162.04 619.96 16.14 0.72 0.19

20.96 73.00 94.00 1.25 1.00 0.00 400.00 1123.20 0.20 2.50 20.00 20.00 25.71 46.66 22.22 28.73 238.00 1174.27 22.61 0.36 0.14

38.95 107.00 120.00 2.45 9.00 1.00 710.00 1515.80 3.90 25.60 76.00 49.40 60.40 80.00 80.00 50.24 795.61 3672.02 78.64 3.35 0.92

27.43 94.00 106.38 2.06 4.25 0.06 560.00 1280.88 3.23 9.23 32.75 30.50 47.31 51.87 46.93 38.98 505.05 1668.64 31.46 2.60 0.19

4.60 9.21 7.08 0.31 2.11 0.25 58.65 101.93 4.64 6.93 10.55 7.44 9.38 11.48 11.96 4.70 223.83 479.42 9.83 0.59 0.21

20.96 73.00 94.00 1.40 1.00 0.00 450.00 1128.50 0.40 1.50 20.00 21.17 29.42 23.33 28.80 29.26 206.08 965.58 15.37 1.65 0.03

38.09 111.00 120.00 2.45 9.00 1.00 650.00 1545.40 20.00 28.00 48.00 47.00 62.85 73.33 73.33 45.85 1229.23 2494.81 48.26 3.46 0.68

0.197 0.670 1.000 0.108 0.210 1.000 0.351 0.281 0.898 0.245 0.008 0.535 0.975 0.038 0.530 0.163 0.278 0.121 0.003 0.125 0.074

Abbreviations: CTx, collagen type I cross-linked C-telopeptide; CATe, erythrocyte catalase; SODe, erythrocyte superoxide dismutase; MDAe, erythrocyte malondialdehyde; GSH-Pxe, erythrocyte glutathione peroxidase; NOe, erythrocyte nitric oxide; BMD, bone mineral density; QUALEFFO, Quality of Life Questionnaire of the European Foundation for Osteoporosis.

Table 3. Clinical and laboratory measurements at baseline and at end of a 3-month course of treatment in risedronate-treated group Baseline (n 5 14)

2

Body mass index, kg/m Waist circumference, cm Pelvic circumference, cm 100-m walking time, min Daily tea intake, cups/day Daily coffee intake, cups/day Dietary calcium intake, mg Daily calorie expenditure, Kcal/day Urinary CTx, mg/L Urinary calcium, mg/dL QUALEFFO-Pain QUALEFFO-Physical function QUALEFFO-Social function QUALEFFO-General health perception QUALEFFO-Mental function QUALEFFO-Total score CATe, k/gHb SODe, U/gHb MDAe, nmol/gHb GSH-Pxe, U/gHb NOe, mmol/gHb

3 months (n 5 14)

Mean

SD

Minimum

Maximum

Mean

SD

Minimum

Maximm

p

28.84 96.29 106.43 2.07 4.29 0.07 601.43 1368.18 2.59 9.55 41.14 32.54 51.45 62.38 53.17 43.66 447.37 1796.14 55.19 2.24 0.30

4.66 13.21 8.64 0.42 2.76 0.27 86.19 174.49 1.14 5.10 15.96 10.04 7.59 14.93 13.29 7.76 145.57 458.43 28.35 0.90 0.13

19.37 76.00 91.00 1.50 0.00 0.00 400.00 1181.40 0.40 3.90 20.00 20.00 38.85 33.33 28.88 29.75 202.55 1098.10 22.72 0.41 0.14

35.50 119.00 117.00 3.00 10.00 1.00 730.00 1905.50 4.40 18.90 72.00 49.41 62.85 86.66 77.77 57.21 694.89 2541.98 120.40 3.66 0.65

29.13 96.71 105.14 2.01 3.29 0.07 599.29 1369.83 2.51 8.90 37.43 33.27 50.74 58.09 44.61 335.97 503.32 1496.04 28.65 2.46 0.19

5.15 12.89 7.45 0.35 2.70 0.27 73.14 175.98 0.85 3.97 17.09 10.00 8.54 16.00 13.68 1104.99 150.62 525.96 7.48 0.80 0.16

19.37 76.00 91.00 1.50 0.00 0.00 420.00 1200.40 1.60 2.80 20.00 20.00 31.14 33.33 24.44 25.31 293.44 577.50 19.69 1.53 0.01

36.89 117.00 117.00 2.50 10.00 1.00 690.00 1905.50 4.30 16.10 64.00 48.23 68.23 80.00 68.88 4175.00 828.80 2918.36 39.27 3.92 0.50

0.314 0.399 0.175 0.801 0.259 1.000 0.950 0.530 0.509 0.552 0.305 0.724 0.753 0.165 0.079 0.510 0.433 0.109 0.005 0.363 0.041

Abbreviations: CTx, collagen type I cross-linked C-telopeptide; CATe, erythrocyte catalase; SODe, erythrocyte superoxide dismutase; MDAe, erythrocyte malondialdehyde; GSH-Pxe, erythrocyte glutathione peroxidase; NOe, erythrocyte nitric oxide; BMD, bone mineral density; QUALEFFO, Quality of Life Questionnaire of the European Foundation for Osteoporosis.

Antioxidants and Nitric Oxide in Osteoporosis

203

Table 4. Clinical and laboratory measurements at baseline and at end of a 3-month course of treatment in raloxifene-treated group Baseline (n 5 14)

2

Body mass index, kg/m Waist circumference, cm Pelvic circumference, cm 100-m walking time, min Daily tea intake, cups/day Daily coffee intake, cups/day Dietary calcium intake, mg Daily calorie expenditure, Kcal/day Urinary CTx, mg/L Urinary calcium, mg/dL QUALEFFO-Pain QUALEFFO-Physical function QUALEFFO-Social function QUALEFFO-General health perception QUALEFFO-Mental function QUALEFFO-Total score CATe, k/gHb SODe, U/gHb MDAe, nmol/gHb GSH-Pxe, U/gHb NOe, mmol/gHb

3 months (n 5 14)

Mean

SD

Minimum

Maximum

Mean

SD

Minimum

Maximum

p

27.42 89.71 104.14 2.00 4.57 0.07 572.86 1306.55 2.49 12.61 42.86 35.87 53.79 62.85 54.90 45.94 395.51 1578.90 40.92 2.41 0.25

4.46 9.79 7.56 0.30 2.17 0.27 99.49 73.78 1.11 8.33 19.69 7.77 12.94 12.19 15.94 9.26 126.10 428.70 14.81 0.59 0.22

19.91 67.00 87.00 1.40 0.00 0.00 460.00 1033.20 0.90 2.20 20.00 21.17 38.85 40.00 26.66 29.56 292.42 1107.01 18.36 1.89 0.01

37.57 106.00 111.00 2.50 7.00 1.00 680.00 1445.70 3.80 18.20 76.00 49.41 68.57 80.00 77.77 60.48 1035.12 3594.74 45.83 3.43 0.58

27.53 89.07 103.43 2.06 3.79 0.14 557.14 1310.46 2.40 9.31 36.57 35.46 52.47 61.90 46.66 42.92 580.63 2050.71 31.05 2.60 0.21

4.90 9.70 7.79 0.37 2.15 0.36 59.15 106.19 0.82 5.08 16.59 8.74 9.12 12.92 15.74 8.55 221.24 714.64 7.35 0.48 0.19

20.34 66.00 88.00 1.40 2.00 0.00 400.00 1125.70 0.10 1.00 20.00 21.17 37.14 53.33 33.33 29.26 236.58 943.23 23.81 1.53 0.05

36.15 104.00 114.00 2.30 10.00 1.00 700.00 1409.90 4.10 31.00 76.00 48.23 74.00 93.33 91.11 66.14 660.67 2585.17 68.18 3.51 0.92

0.844 0.629 0.427 0.109 0.109 0.317 0.450 0.470 0.196 0.124 0.167 0.906 0.826 0.944 0.008 0.060 0.004 0.140 0.02 0.397 0.778

Abbreviations: CTx, collagen type I cross-linked C-telopeptide; CATe, erythrocyte catalase; SODe, erythrocyte superoxide dismutase; MDAe, erythrocyte malondialdehyde; GSH-Pxe, erythrocyte glutathione peroxidase; NOe, erythrocyte nitric oxide; BMD, bone mineral density; QUALEFFO, Quality of Life Questionnaire of the European Foundation for Osteoporosis.

administration of raloxifene in ovariectomized rats (35). Antiinflammatory activity of raloxifene has also been demonstrated by others in ovariectomized rat model of carrageenan-induced acute inflammation (36). Furthermore, raloxifene has been shown to protect osteoblasts from apoptosis induced by sodium nitroprusside by inhibition of C22 and C24 ceramide generation (37). Additionally, Gur et al. showed that calcitonin treatment regulates cytokine levels in women with PMO (38). An enormous amount of research has been conducted on the effect of NO on bone cell functions. These investigations showed that bone cells produce NO in response to various stimuli including estrogens, pro-inflammatory cytokines and mechanical stress (9,13,14,39), and during these processes different NOS isoforms take place. It is suggested that NO has biphasic effects on osteoclastic bone resorption and osteoblastic functions. In this field, human studies and animal models have revealed inconclusive and sometimes conflicting results. Caballero-Alias et al. found that human iliac (transiliac biopsies from female osteoporotic patients) and femoral neck (female postmortem femoral neck biopsies) bone was rich from nNOS and eNOS isoforms, and the percentage was in favor of nNOS isoform (40). In another recently published study, van’t Hof and colleagues showed that nNOS knockout mice had increased bone mass and decreased bone turnover, indicating that nNOS isoforms play an important role in the regulation

of bone mass and bone turnover in mice (14). On the other hand, different doses of NO donors (medium and low doses of nitroglycerin) have been shown to prevent osteoporosis in ovariectomized rats (13). Similar results in rats have been previously reported by others (41,42). Jamal and coworkers showed that women taking daily nitrates had significantly higher hip BMD compared to nonusers, whereas heel BMD were similar. However, women who used nitrates intermittently had considerably high hip and heel BMD (43). In a pilot study, Wimalawansa suggested that nitroglycerin ointment (applied to the skin once a day within 4 weeks of undergoing oophorectomy) was as effective as estrogen in preventing bone loss in women with oophorectomy-induced menopause (44). In contrast, Baecker et al. showed that Larginine—the natural precursor of NO—supplementation was found ineffective in reducing bone resorption or increasing bone formation in early postmenopausal women (45). In this present study, we found a significant relationship between erythrocyte NO and hip BMD but not with lumbar BMD and higher erythrocyte NO levels in women with PMO compared to non-porotic controls. Anti-osteoporotic drugs, calcitonin and risedronate, reduced erythrocyte NO levels; however, this reduction reached statistical significance in only risedronate-treated group ( p 5 0.074 vs. p 5 0.041, respectively). On the other hand, NO levels remained nearly unchanged in the raloxifene group ( p 5 0.77).

204

Ozgocmen et al./Archives of Medical Research 38 (2007) 196e205

It is necessary to address that net effects of NO on bone depend on various interactions with other stimuli that regulate bone turnover like estrogen, proinflammatory cytokines, mechanical stress and/or free radical generation and related oxidative stress. In highly stressed conditions such as increased osteoclastic activity, NO levels tend to be high in order to scavenge O2.; however, they produced a potent oxidant species peroxynitrite (ONOO) that can interact with protein, DNA and fatty acids and induces lipid peroxidation. This mechanism is explanatory for increased NO and MDA levels in osteoporotic women and reduction after anti-osteoporotic therapy. There are some important limitations in the present study. First, we measured drug effects after 3 months, a threshold time or beginning time for drugs to take effect on bone turnover. Secondly, we could not assess other important bone resorption and formation markers, which might be of value for interpreting the results. Consequently, oxidative stress may play an important role in postmenopausal bone loss, and anti-osteoporotic drugs have an effect on oxidative stress. Further research assessing the oxidative stress markers and nitric oxide in bone tissue and changes with anti-osteoporotic drugs may be of value to better understand the role of oxidants, antioxidants and nitric oxide in the regulation of bone mass.

References 1. Basu S, Michaelsson K, Olofsson H, Johansson S, Melhus H. Association between oxidative stress and bone mineral density. Biochem Biophys Res Commun 2001;288:275e279. 2. Sontakke AN, Tare RS. A duality in the roles of reactive oxygen species with respect to bone metabolism. Clin Chim Acta 2002;318:145e148. 3. Maggio D, Barabani B, Pierandrei M, Polidori MC, Catani M, Mecocci P, et al. Marked decrease in plasma antioxidants in aged osteoporotic women. Results of a cross-sectional study. JCEM 2003;88: 1523e1527. 4. Melhus H, Michaelsson K, Holmberg L, Wolk A, Ljunghall S. Smoking, antioxidant vitamins and risk of hip fracture. J Bone Miner Res 1999;14:129e135. 5. Yalin S, Bagis S, Polat G, Dogruer N, Aksit SC, Hatungil R, et al. Is there a role of free oxygen radicals in primary male osteoporosis. Clin Exp Rheumatol 2005;23:689e692. 6. Varanasi SS, Francis RM, Berger CEM, Papiha SS, Datta HK. Mitochondrial DNA deletion associated oxidative stress and severe male osteoporosis. Osteoporos Int 1999;10:143e149. 7. Kigwell BA. Nitric oxide-mediated metabolic regulation during exercise: effect of training in health and cardiovascular disease. FASEB J 2000;14:1685e1696. 8. Akyol O, Zoroglu SS, Armutcu F, Sahin S, Gurel A. Nitric oxide as a physiopathological factor in neuropsychiatric disorders. In Vivo 2004;18:377e390. 9. van’t Hof RJ, Ralston SH. Nitric oxide and bone. Immunology 2001; 103:255e261. 10. Aguirre J, Buttery L, O’Shaughnessy M, Afzal F, Fernandez Marticorena I, Hukkanen M, et al. Endothelial nitric oxide synthase gene-deficient mice demonstrate marked retardation in postnatal bone formation, reduced bone volume, and defects in osteoblast maturation and activity. Am J Pathol 2001;158:247e257.

11. van’t Hof RJ, Armour KJ, Smith LM, Armour KE, Wei XQ, Liew FY, et al. Requirement of the inducible nitric oxide synthase pathway for IL-1-induced osteoclastic bone resorption. Proc Natl Acad Sci USA 2000;97:7993e7998. 12. Armour KE, Van’T Hof RJ, Grabowski PS, Reid DM, Ralston SH. Evidence for a pathogenic role of nitric oxide in inflammation-induced osteoporosis. J Bone Miner Res 1999;14:2137e2142. 13. Hao YJ, Tang Y, Chen FB, Pei FX. Different doses of nitric oxide donor prevent osteoporosis in ovariectomized rats. Clin Orthop 2005; 435:226e231. 14. van’t Hof RJ, Macphee J, Libouban H, Helfrich MH, Ralston SH. Regulation of bone mass and bone turnover by neuronal nitric oxide synthase. Endocrinology 2004;145:5068e5074. 15. Leidig-Bruckner G, Genant HK, Minne HW, Storm T, Thamsborg G, Bruckner T, et al. Comparison of a semiquantitative and a quantitative method for assessing vertebral fractures in osteoporosis. Osteoporos Int 1994;4:154e161. 16. Lips P, Cooper C, Agnusdei D, Caulin F, Egger P, Johnell O, et al. Quality of life in patients with vertebral fractures: validation of the Quality of Life Questionnaire of the European Foundation for Osteoporosis (QUALEFFO). Working Party for Quality of Life of the European Foundation for Osteoporosis. Osteoporos Int 1999;10: 150e160. 17. Aebi H. Catalase. In: Bergmeyer HU, ed. Methods of Enzymatic Analysis. New York: Academic Press;1974. pp. 673e677. 18. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158e170. 19. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. In: Packer L, Glazer AN, eds. Methods in Enzymology, vol. 186, Oxygen Radicals in Biological Systems. San Diego: Academic Press;1990. pp. 186, 407e421. 20. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497e500. 21. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990; 36:1440e1443. 22. Bax BE, Alam ASMT, Banerji B, Bax CMR, Bevis PJR, Stevens CR, et al. Stimulation of osteoclastic bone resorption by hydrogen peroxide. Biochem Biophys Res Commun 1992;183:1153e1158. 23. Garrett IR, Boyce BF, Oreffo ROC, Bonewald L, Poser J, Mundy GR. Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo. J Clin Invest 1990;85:632e639. 24. Bai XC, Lu D, Liu AL, Zhang ZM, Li XM, Zou ZP, et al. Reactive oxygen species stimulates receptor activator of NF-kB ligand expression in osteoblasts. J Biol Chem 2005;280:17497e17506. 25. Lean JM, Davies JT, Fuller K, Jagger CJ, Kirstein B, Partington GA, et al. A crucial role for thiol antioxidants in estrogen-deficiency bone loss. J Clin Invest 2003;112:915e923. 26. Lean JM, Jagger CJ, Kirstein B, Fuller K, Chambers TJ. Hydrogen peroxide is essential for estrogen-deficiency bone loss and osteoclasts formation. Endocrinology 2005;146:728e735. 27. Bai XC, Lu D, Bai J, Zheng H, Ke ZY, Li XM, et al. Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kB. Biochem Biophys Res Commun 2004;314:197e207. 28. Hall TJ, Schaueblin M, Jeker H, Fuller K, Chambers TJ. The role of reactive oxygen intermediates in osteoclastic bone resorption. Biochem Biophys Res Commun 1995;207:280e287. 29. Steinbeck MJ, Appel WH Jr, Verhoeven AJ, Karnovsky MJ. NADPH oxidase expression and in situ production of superoxide by osteoclasts actively resorbing bone. J Cell Biol 1994;126:765e772. 30. Hall SL, Greendale GA. The relation of dietary vitamin C intake to bone mineral density: results from the PEPI Study. Calcif Tissue Int 1998;63:183e189.

Antioxidants and Nitric Oxide in Osteoporosis 31. Morton DJ, Barret-Connor EL, Schneider DL. Vitamin C supplement use and bone mineral density in postmenopausal women. J Bone Miner Res 2001;16:140e145. 32. Simon JA, Hudes ES. Relation of ascorbic acid to bone mineral density and self-reported fractures among US adults. Am J Epidemiol 2001;154:427e433. 33. Wolf RL, Cauley JA, Pettinger M, Jackson R, Lacroix A, Leboff MS, et al. Lack of a relation between vitamin and mineral antioxidants and bone mineral density: results from the Women’s Health Initiative. Am J Clin Nutr 2005;82:581e588. 34. Dombrecht EJ, Cos P, Van den Berghe D, Van Offel JF, Schuerwegh AJ, Bridts CH, et al. Selective in vitro antioxidant properties of bisphosphonates. Biochem Biophys Res Commun 2004;314: 675e680. 35. Ozgonul M, Oge A, Sezer ED, Bayraktar F, Sozmen EY. The effects of estrogen and raloxifene treatment on antioxidant enzymes in brain and liver of ovarectomized female rats. Endocr Res 2003;29:183e189. 36. Esposito E, Iacono A, Raso GM, Pacilio M, Coppola A, Di Carlo R, et al. Raloxifene, a selective estrogen receptor modulator, reduces carrageenan-induced acute inflammation in normal and ovariectomized rats. Endocrinology 2005;146:3301e3308. 37. Oliver S, Fillet M, Malaise M, Piette J, Bours V, Merville MP, et al. Sodium nitroprusside-induced osteoblast apoptosis is mediated by long chain ceramide and is decreased by raloxifene. Biochem Pharm 2005;69:891e901.

205

38. Gur A, Denli A, Nas K, Cevik R, Karakoc M, Sarac AJ, et al. Possible pathogenetic role of new cytokines in postmenopausal osteoporosis and changes during calcitonin plus calcium therapy. Rheumatol Int 2002;22:194e198. 39. Das UN. Nitric oxide as the mediators of the antiosteoporotic actions of estrogens, statins, and essential fatty acids. Exp Biol Med 2002;227: 88e93. 40. Caballero-Alias AM, Loveridge N, Lyon A, Das-Gupta V, Pitsillides A, Reeve J. NOS isoforms in adult human osteocytes: multiple pathways of NO regulation? Calcif Tissue Int 2004;75:78e84. 41. Wimalawansa SJ. Restoration of ovariectomy-induced osteopenia by nitroglycerine. Calcif Tissue Int 2000;66:56e60. 42. Wimalawansa SJ, De Marco G, Gangula P, Yallampalli C. Nitric oxide donor alleviates ovariectomy-induced bone loss. Bone 1996;18: 301e304. 43. Jamal SA, Browner WS, Bauer DC, Cummings SR. Intermittent use of nitrates increases bone mineral density: the study of osteoporotic fractures. J Bone Miner Res 1998;13:1755e1759. 44. Wimalawansa SJ. Nitroglycerin therapy is as efficacious as standard estrogen replacement therapy (premarin) in prevention of oophorectomy-induced bone loss: a human pilot clinical study. J Bone Miner Res 2000;15:2240e2244. 45. Baecker N, Boese A, Schoenau E, Gerzer R, Heer M. L-arginine, the natural precursor of NO, is not effective for preventing bone loss in postmenopausal women. J Bone Miner Res 2005;20:471e479.