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Oxidative stress and bone markers in plasma of patients with long-bone fixative surgery: Role of antioxidants

Human and Experimental Toxicology 30(6) 435–442 ª The Author(s) 2010 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327110374203 het.sagepub.com

A Sandukji1, H Al-Sawaf2, A Mohamadin2, Y Alrashidi1 and SA Sheweita2

Abstract It is well known that bone markers (e.g. osteocalcin and alkaline phosphatase) play a significant role in healing of bone fractures, whereas oxidative stress delay such healing. The present study aimed to investigate the effect of a mixture of antioxidants (vitamins A, C, E, and selenium) on oxidative stress parameters, and the levels of bone healing markers in the plasma of male patients following fixative surgery of long bones. Antioxidant tablets (300 mg vitamin A, 10 mg vitamin E, 60 mg vitamin C, and 75 mg selenium) were administered to groups 3 and 4 (10 patients in each) for 1 and 2 weeks, respectively, in addition to the regular postoperative treatment. Groups 1 (25 patients) and 2 (10 patients) received the regular post-operative treatment consisting of intravenous (I.V.) second generation of cephalosporin 1000 mg/day for 3 days, oral diclofenac 50 mg, and paracetamol 500 mg twice daily for 15 days. Osteocalcin level and alkaline phosphatase activity as well as antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR), as well as glutathione (GSH), and thiobarbituric acid reactive substances (TBARS) as indices of oxidative stress, were determined in the plasma of all patients after 1 or 2 weeks of long-bone fixative surgery. The results revealed that osteocalcin level and the activity of alkaline phosphatase were markedly increased in the plasma of patients who received antioxidants for 2 weeks. In addition, after 1 and/or 2 weeks, the levels of TBARS were significantly lower in the antioxidant-treated patients compared with those who did not receive antioxidants. On the other hand, the activities of SOD and GR were markedly elevated in plasma of patients who received antioxidants after 1 or 2 weeks compared with patients who received regular therapy. Moreover, the level of plasma GSH was markedly increased only after 2 weeks in patients who received antioxidants. It is concluded that administration of antioxidant vitamins A, E, and C in addition to selenium could accelerate bone healing after long-bone fixative surgery. Therefore, antioxidants should be considered in designing therapeutic protocols in post-operative bone surgery. Keywords Bone healing, osteocalcin, antioxidants, free radicals

Introduction Osteoblasts (bone-forming cells) and osteoclasts (bone-resorption cells) are involved in bone remodeling. Therefore, any loss of osteoblastic activity or an increase in osteoclastic activity could lead to a decrease in bone-mineral densities (BMD), bone mass, and make the bones more likely to osteoporosis, and ultimately to fractures.1,2 In addition, high levels of reactive oxygen species (ROS) and many other factors such as genetic race, hormonal, mechanical, and nutritional statues are involved in bone weakness and fractures. ROS shift cells into a state of oxidative

stress,3 which contributes to the etiology of various degenerative diseases that cause tissue injury.2,3 Studies have demonstrated that the ischemia-reperfusion processes that occur after a fracture are associated 1

Department of Orthopedic Surgery, Taibah University, KSA Department of Clinical Biochemistry, College of Medicine, Taibah University, KSA 2

Corresponding author: Salah A Sheweita, College of Medicine, Department of Clinical Biochemistry, Tabiah University, Al-Madinah, 30001, Saudi Arabia Email: [email protected]

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with oxidative stress development.2,4 There is no oxidative stress injury during the first 3 days of fracture healing compared to the ischemic stage. However, after this period, the inflammatory response that occurred during the callus formation may be accompanied by an overproduction of free radicals.5,6 In this condition, lipid peroxidation induced by attack of free radical-initiated oxidation leads to lipid hydroperoxides formation, which can be measured indirectly as malondialdehyde (MDA) or thiobarbituric acid reactive substances (TBARS).5,7 In addition to oxidative stress, it has been found that cytokines such as tumor necrosis factor and interleukins are involved in osteoporosis.8 Previous studies showed that persons with osteoporosis have an increased oxidative stress and no changes in transforming growth factor (TGF)-b1 levels.9 Due to the presence of oxidative stress, there is a defense system to alleviate the harmful effects of these oxidants including enzymatic (e.g., superoxide dismutase and glutathione reductase [GR]) and nonenzymatic antioxidants (such as glutathione [GSH] and vitamins C and E). Vitamin C is a key element in the healing of fractures through hydroxylation of proline and lysine needed to form the triple helix of collagen and the formation of immature callus in bone healing.10,11 It also has other important functions in the formation of non-collagenous proteins during bone healing, development of mesenchymal, chondroblast, and osteogenic phenotypes.12-15 Also, it has been found that vitamin C deficiency leads to delaying in healing of bone fractures.16-18 Although some authors have reported beneficial effects of vitamin C, others warn that in the presence of metals such as iron with vitamin C could generate more free radicals via the Fenton reaction.19-22 Of note, there are conclusive lines of evidence showing that the lower of vitamin C levels found in elderly, alcoholics, and smokers are related to osteoporosis process.23,24 In line with this, several epidemiological studies have found a positive correlation between dietary vitamin C intake and BMD.25,26 Indeed, it has been shown that lower dietary intake of antioxidants as vitamins C and E substantially increases the risk of hip fracture.27 Marked decrease in plasma antioxidants was found in aged osteoporotic women28 and reduced BMD in aged men and women,29 and the intakes of antioxidants have beneficial effects on BMD.30-32 GSH is an antioxidant and maintains vitamin C in a reduced and functional form and enhances metabolic clearance of dietary lipid peroxides.33 Both

Human and Experimental Toxicology 30(6)

oxidation-reduction reactions may eventually result in glutathione oxidation and should be effectively compensated by GR activity.34 In this way, an elegant study related to oxidative stress and osteoporosis showed that osteoporotic patients exhibit higher levels of malondialdehyde (MDA) and nitric oxide (NO) and reduced GR activity. On the other hand, no significant alteration was found in erythrocytes GSH levels and catalase activity of these patients.35 Total femoral BMD measurements were significantly correlated with MDA levels. There was no significant relationship between other antioxidants and lumbar or femoral BMD.35 It is believed that bone markers such as osteocalcin and alkaline phosphatase as well as antioxidant enzymes play a significant role in fracture healing. However, to the best of our knowledge, there are no reports about the use of vitamins A, C, E, and selenium as antioxidant therapy to explore their effects in the levels of bone-healing markers and oxidative stress parameters of osteoporotic patients. Therefore, the present study aimed to investigate the effects of these combined antioxidants on the biochemical markers of bone healing and oxidative stress in the plasma of patients following long-bone fixative surgery.

Patients and methods Patients The local ethics committee of the Deanship of Scientific Research approved the protocol of study, and it conforms to the guidelines of Taibah University. After agreement of all patients, a total of 55 male patients (age 20–40 years) living in Al-Madinah Urban area were selected. All patients were selected among traffic-accident cases admitted to the Department of Orthopedic Surgery of King Fahd General hospital at Al-Madinah Al-Munawarah, Kingdom of Saudi Arabia, during the period from September 2007 to June 2008. All patients have been selected to fulfill the following criteria: 1 – Smokers, diabetic, open fracture, liver, and kidney diseases and female patients were excluded. 2 – Only diaphyseal fractures of the long bones (humerus, tibia, radius, ulna, and femur) of male patients were included in this study. 3 – Operative fracture fixation of patients not exceeding 7 days of the accident was chosen.

Fracture fixation method Internal fixation of the femur and tibia were carried out by closed interlocking intra-medullary nailing and

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for the humerus, radius, and ulna by plate and screw techniques under general anesthesia.

Post-operative therapeutic management All patients who fulfilled the above-mentioned criteria were treated with the regular post-operative therapeutic protocol; second-generation cephalosporin 1000 mg/day intravenous (I.V.) for 3 days. Oral non-steroidal anti-inflammatory drugs, diclofenac tablets (50 mg), and paracetamol tablets (500 mg) were given two times/day for all subjects during the whole period of the study.

Antioxidants administration Group 1 (25 patients) and groups 2–4 (10 patients each) received the regular post-operative therapeutic protocol. In addition, groups 3 and 4, (10 subjects each) received a tablet of an antioxidant mixture composed of vitamin A: 300 mg, vitamin E: 10 mg, ascorbic acid: 60 mg, and selenium: 75 mg/day for 7 and 15 days, respectively, following the bone surgery. The numbers of samples in groups 2–4 were more than 10. However, only 10 cases in each of the groups 2–4 were strictly matched with the exclusion criteria mentioned above. Blood samples were collected in heparinzed tubes from groups 1 and 2 and 3 and 4 at 7 and 15 days, respectively, and centrifuged at 3000 rpm. Plasma was stored at –80 C until assaying of biochemical parameters.

Biochemical parameters Chemicals. Glutathione reduced (GSH), and oxidized form (GSSG), NADPH, bis-(3-carboxy-4-nitophenyl)-disulfide, thiobarbituric acid, and 1-chloro-2,4dinitrobenzene, sulfosalicylic acid, were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). Selenium–ACE tablets were obtained from Wassen International Ltd, UK. Commercial kits for determination of osteocalcin was obtained from DRG International Inc., USA, whereas alkaline phosphatase, and superoxide dismutase were obtained from Cayman Chemical Company, USA.

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Assay of alkaline phosphatase activity. Alkaline phosphatase activity was determined according to the method of Principato et al.,37 using commercial kit (Cayman Chemical Company, Michigan, USA). The liberated para-nitrophenol was measured colorimetrically at 405 nm.

Assay of superoxide dismutase activity. Superoxide dismutase (SOD) activity was assayed by a microwell assay, as described by Ewing and Janero,38 using commercial kit (Cayman Chemical Company). The absorbance at 560 nm was continuously monitored over 5 min in a microplate reader. SOD activity was expressed in units/mL.

Assay of glutathione reductase activity. The GR assay was conducted essentially as that described by James et al.39 The oxidation of NADPH was followed at 340 nm under conditions that yielded zero-order kinetics for at least 5 min. A unit of enzyme activity presented as 1 nmole of NADPH oxidized/min/mg protein.

Determination of TBARS levels. Lipid peroxidation endproduct was determined as thiobarbituric acid-reactive substances (TBARS) according to the method of Tappel and Zalkin.40 The color intensity of the reactants (TBARS) was measured at 532 nm. An extinction coefficient of 156,000 mM1 cm1 was used for calculation.

Glutathione levels. Glutathione levels were estimated in human plasma according to the method of Mitchell et al.,41 using sulfosalicylic acid for protein precipitation and bis-(3-carboxy-4-nitrophenyl)-disulfide for color development. The color intensity was measured at 412 nm.

Statistical analysis Statistical analyses were performed by using SPSS statistical software package version 15.0.1.1. Data are presented as means + SEM. Normality and homogeneity of the data were confirmed before analysis of variance (ANOVA), differences among the experimental groups were assessed by one-way ANOVA followed by Duncan’s multiple range tests.

Osteocalcin determination. Human osteocalcin assay was performed on a multiwell plate,36 using commercial kit (DRG International Inc., New Jersey, USA). The color intensity measured colorimetrically at 450 nm wavelength, which is directly proportional to the concentration of osteocalcin.

Results Bone markers, osteocalcin level, and alkaline phosphatase activity were markedly increased after 2 weeks of antioxidant administration to patients, following

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Figure 1. Effect of antioxidant treatment on osteocalcin levels (A) and alkaline phosphatase activity (B) of patients with bone fractures. *Values are the mean + SEM and significantly different from other groups at p < 0.05.

long-bone fixative surgeries (Figure 1A and B). However, osteocalcin level did not change after one week of antioxidant supplementation when compared to values found in the non-antioxidant supplemented group (Figure 1A). The levels of lipid peroxidation, measured as TBARS, were markedly decreased after administration of antioxidants for 1 and 2 weeks compared with those of non-antioxidants groups (Figure 2B). The data of Figure 2A show that the antioxidant administration caused a significant increase on the levels of GSH of patients. However, this effect was observed only 2 weeks after the treatment (Figure 2A). The activity of antioxidant enzymes, SOD, and GR were markedly increased in plasma of patients after 1 and 2 weeks of antioxidant administration compared with patients who did not receive antioxidant (Figure 3A and B).

Discussion The development of specific and sensitive biochemical markers, such as osteocalcin and alkaline phosphatase,

Two weeks

Figure 2. Effect of antioxidant treatment on thiobarbituric acid reactive substances (TBARS) level (A) and glutathione level (B) of patients with bone fractures. *Values are the mean + SEM and significantly different from other groups at p < 0.05.

has markedly improved the assessment of bone turnover in various metabolic bone diseases.42 These biomarkers are useful in monitoring of treatment efficacy in patients with bone fractures and osteoporoses.43,44 In addition, the biochemical, immunohistochemical, histological, and radiological appearances of callus in the experimental animals showed that high levels of osteocalcin increased the healing of bone fractures.45 In the present study, osteocalcin level significantly increased in the plasma of antioxidanttreated patients for 2 weeks compared with those of non-antioxidant-treated group (Figure 1A). Elevation of osteocalcin levels might increase BMD and consequently could accelerate healing of bone fractures since high levels of osteocalcin and alkaline phosphatase are positively correlated with BMD.43 A positive correlation between vitamin C intake and BMD have been shown by many investigators.46-49 Moreover, vitamin E deficiency was found to impair bone calcification.49 Recently, it has been found that

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Figure 3. Effect of antioxidant treatment on superoxide dismutase activity (A), and glutathione reductase activity (B) of patients with bone fractures. *Values are the mean + SEM and significantly different from other groups at p < 0.05.

selenium is required for regulation of thyroid hormones,50,51 which are necessary for bone formation. Moreover, it has been found that the existence of sodium selenite with vitamins E and C was more effective than single vitamins in restoring of structural alterations of bones52. Some clinical conditions, such as Kashin-Beck osteoarthropathy, have been associated with selenium deficiency.53 On the other hand, no association between vitamin A or retinol intake and the risk of hip or total fractures in postmenopausal women was observed.54 It has been found that a significant decrease in bone formation was associated with a decrease in alkaline phosphatase activity and osteocalcin levels.49 In the present study, the activity of alkaline phosphatase increased after 2 weeks in antioxidant-treated patients compared to non-antioxidant-treated group, whereas such activity did not change after 1 week (Figure 1B). This inhibition might be due to the presence of high levels of oxidative stress, which could inhibit osteogenic cells that provide osteocalcin, alkaline phosphatase, and other proteins.55 This finding could explain the mechanism of induction of osteocalcin

level in the plasma of antioxidant-treated patients since oxidant levels were much lower than those of non-antioxidant-treated groups. Supporting our finding, it has been found that oxidant levels are increased after 1 and 2 weeks of fractures perhaps due to callus formation and angiogenesis, which results in reperfusion at the fracture site.56,57 Levels of free radicals have been reported to be increased in chronic inflammatory diseases,58 aging,59 and osteoporoses.60 Therefore, when bone fractures occurred, a remarkably high yield of free radicals (ROS) could be generated by a number of phagocytes including monocytes, macrophages, and neutrophils.61 One of the most damaging effects of ROS is lipid peroxidation, the end product of which is MDA, which serves as a marker of osteoclastic activity.62 Fortunately, vitamin E suppresses the production of free radicals generated by certain cytokines,63,64 which are linked to increment of bone loss,1 and also could protect bone cells from the damaging effects of lipid peroxidation.65,66 In the present study, the levels of lipid peroxidation (measured as TBARS) derived from oxidative stress are significantly lower in the antioxidant-treated patients than those of non-antioxidant groups (Figure 2B). In agreement with the present study, it has been found that adequate intake of vitamin E could reduce the risk of hip fracture in cases with high levels of lipid peroxidation.27 SOD is a metalloenzyme that catalyzes the dismutation of the superoxide anion to molecular oxygen and forms a crucial part of the cellular antioxidant defense mechanism.63 In the present study, the activity of SOD markedly increased in plasma of antioxidant-treated groups after 1 and 2 weeks compared to non-antioxidant groups (Figure 3A). The low levels of free radicals in antioxidant-treated group might be due to induction of SOD activity, which plays a significant role against superoxide ions produced by the osteoclasts.2 In addition, the level of glutathione, non-enzymatic antioxidant, increased after 2 weeks, whereas such level did not change after 1 week of antioxidant administration (Figure 2A). Induction of GSH level might be due to induction of GR activity, which converts the oxidized form of glutathione (GSSG) into its biologically active form (GSH; Figure 3B). In summary, our results show that antioxidant treatment improves some bone markers and parameters related to oxidative stress in patients submitted to long-bone fixative surgery. Therefore, it could be

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suggested that the use of antioxidants (vitamins A, C, E and selenium), as a part of therapeutic protocols, might be beneficial in the healing of long-bone fractures.


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Acknowledgment The authors thank Mr Wael Barakat and Mr Mohamad Abd el-Samad for their technical assistances.

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Funding This work was funded by the Taibah University under the project # 61/427.

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