twenty old female wistar albino rats raised in the same environment and weighing 175215 g were used in this study ( 14 ) . they were maintained in a well - ventilated controlled room at 2022c on a 12-h light/12-h dark schedule . the rats were randomly divided into three groups : control group ( c , n=6 ) , ovariectomy group ( o , n=7 ) , and ovariectomy+vitamin c group ( ov , n=7 ) . the rats had access to food and water until 2 h prior to the anesthesia . after anesthesia ( intraperitoneal ketamine , 50 mg / kg ) , bilateral ovariectomy was performed with a ventral approach . after surgical procedure , vitamin c ( l - ascorbic acid ) was added to the drinking water of the ov group at a concentration of 1 g/500 ml water . a period of 100 days was allowed for the development of op ( 3 , 4 ) . bone mineral densities of all rats were measured 100 days after ovariectomy prior to euthanasia , using dual - energy x - ray absorptiometry ( norland xr-36 ; norland inc . , fort atkinson , wi ) with small subjects program ( 11 mm resolution , 60 mm / s sweep speed ) . bmd was determined as the amount of mineral per cubic centimeters of bone ( g / cm ) . anesthesia was performed with intraperitoneal injection of ketamine ( 50 mg / kg ) , and 5 ml blood sample was collected from intra - abdominal region prior to euthanasia . blood samples with ethylenediaminetetraacetic acid were centrifuged and the plasma was stored at 80c together with bone samples until analyzed . the homogenates were centrifuged and the supernatant was used for analysis . to assess os , the levels of cat , sod , gpx , mda , no , and t - sh were measured both in plasma and bone homogenates . cayman ( ann arbor , mi ) kits were used in cat analysis and randox ( crumlin , uk ) kits in sod and gpx analysis . statistical package for the social sciences ( chicago , il ) v12.0 program was used for statistical analysis . variations in bone and plasma oxidative state parameters between study groups were assessed by using kruskal whitney u test was used after significant kruskal  wallis to determine which group differs from the other . spearman 's rank correlation test was used to determine the relation between bmd and oxidative state parameters . results were expressed as meanstandard deviation ( meansd ) and minimum  maximum ( min  max ) . statistical significance was set at p<0.05 for all analysis and p<0.033 ( 0.1/3 ) for bonferroni - adjusted mann bone mineral densities of all rats were measured 100 days after ovariectomy prior to euthanasia , using dual - energy x - ray absorptiometry ( norland xr-36 ; norland inc . , fort atkinson , wi ) with small subjects program ( 11 mm resolution , 60 mm / s sweep speed ) . bmd was determined as the amount of mineral per cubic centimeters of bone ( g / cm ) . anesthesia was performed with intraperitoneal injection of ketamine ( 50 mg / kg ) , and 5 ml blood sample was collected from intra - abdominal region prior to euthanasia . blood samples with ethylenediaminetetraacetic acid were centrifuged and the plasma was stored at 80c together with bone samples until analyzed . the homogenates were centrifuged and the supernatant was used for analysis . to assess os , the levels of cat , sod , gpx , mda , no , and t - sh were measured both in plasma and bone homogenates . cayman ( ann arbor , mi ) kits were used in cat analysis and randox ( crumlin , uk ) kits in sod and gpx analysis . statistical package for the social sciences ( chicago , il ) v12.0 program was used for statistical analysis . variations in bone and plasma oxidative state parameters between study groups were assessed by using kruskal whitney u test was used after significant kruskal  wallis to determine which group differs from the other . spearman 's rank correlation test was used to determine the relation between bmd and oxidative state parameters . results were expressed as meanstandard deviation ( meansd ) and minimum  maximum ( min  max ) . statistical significance was set at p<0.05 for all analysis and p<0.033 ( 0.1/3 ) for bonferroni - adjusted mann there was no statistically significant difference observed in body weights of rats at onset and at conclusion of the study within and between groups ( table 1 ) . a significant decrease in bmd was observed in o group compared with c group ( p=0.015 ) and a significant increase was observed in ov compared with o group ( p=0.003 ) . comparison of body weights at the beginning and end of treatment period c : control , o : ovariectomy , ov : ovariectomy + vitamin c meansd : mean standard deviation , min  max : minimum  maximum statistical significance was set at p < 0.05 with kruskal  wallis test statistical significance was set at p < 0.033 with bonferroni - adjusted mann whitney u test comparison of bone mineral densities c : control , o : ovariectomy , ov : ovariectomy + vitamin c meansd : mean standard deviation , min  max : minimum  maximum statistical significance was set at p < 0.05 with kruskal  wallis test statistical significance was set at p < 0.033 with bonferroni - adjusted mann whitney u test when groups were compared regarding oxidant and antioxidant parameters , mda levels in bone tissue were significantly higher in group o than in c and ov ( p=0.032 and p=0.025 , respectively ) . no levels were significantly higher in group o than in either group c or group ov ( p=0.022 , p=0.018 , respectively ) . sod activity was significantly higher in group o than in group c ( p=0.032 ) . there were no statistically significant differences in bone t - sh , cat , and gpx levels ( table 3 ) oxidative stress parameters in femur bone tissue c : control , o : ovariectomy , ov : ovariectomy + vitamin c meansd : mean standard deviation , min  max : minimum  maximum statistical significance was set at p<0.05 with kruskal whitney u test the plasma mda activity was significantly higher in o than in c ( p=0.022 ) ; no level was significantly higher in o than in c or ov ( p=0.017 , p=0.018 , respectively ) . there was no statistically significant difference in plasma t - sh , sod , cat , and gpx levels ( table 4 ) . plasma oxidant stress parameters c : control , o : ovariectomy , ov : ovariectomy + vitamin c meansd : mean standard deviation , min  max : minimum  maximum statistical significance was set at p<0.05 with kruskal this study revealed that bmd was significantly reduced in ovariectomized rats and that vitamin c supplement restored bmd to control levels . os was induced in bone and plasma with ovariectomy and levels of antioxidants raised in response to decrease in os . it is suggested that by reducing or eliminating os , the loss of bmd and op could be prevented ( 10 ) . there is an increase in superoxide formation by the osteoclasts that accompanies active bone resorption and loss of bone density ( 14 ) . it is considered that oxidative state is important in op and coherent with the relation between diseases like op and arteriosclerosis in which os plays a major role . in addition , it is stated that os and bmd are correlated in women ( urine 8-iso - p6f2 levels indicates os ) ( 7 , 15 ) . recently , in vitro or animal studies showed that os has an important role in osteoclast differentiation and functions . on the other hand , it was indicated that ros plays an important role in osteoblastic functions ( 16 ) . in vivo and in vitro studies proved that no ( no is a free radical that causes os ) plays a role in osteoblastic and osteoclastic activities ( 8 , 15 ) . animal and human studies showed that no is an important regulator in bone metabolism ( 17 , 18 ) . but the outcomes of these experimental results are debated and the roles of the mechanisms are unclear . it is known that bone cell produces no as a response of estrogens , proinflammatory cytokines , and mechanic stress , during this process different nos isoforms take part ( 17 , 19 ) . van't hof et al . ( 19 ) noticed that neuronal nos ( nnos ) in knockout rats plays a role on increased bone mass regulation and bone circle . ( 20 ) reported that , in early postmenopause , l - arginine treatment ( natural no percussor ) is not effective in bone resorption or in bone formation . aguirre ( 21 ) advocated that resection of no gene in guinea pigs decreased bone volume , bone formation rate , and mineral density , whereas wimalawansa ( 22 ) found that no donors are protected from postmenopause bone loss . ( 8) found a significant relationship between erythrocyte no and proximal femur bmd rats , but not with lumbar bmd . in addition , compared with non - porotic controls , females with postmenopausal op have an increased no level , but they found no difference in plasma no levels . same investigators in another study found increased no levels in women with op compared with non - porotic controls . on comparison between calcitonin , risedronat , and raloksifen treatment in osteoporotic patients , no level is decreased only in the risedronat group ( 23 ) . yalin et al . ( 24 ) investigated the effect of antioxidants and free radicals on postmenopausal op and found that no levels in op patients were higher . in our study , we found that in the ovariectomized group no level in femur bone tissue and plasma was higher than the ovariectomized+vitamin c group . although it has been reported that there was a negative correlation between no levels and bmd , we could not find a correlation . we believe that a correlation could be found if we increase the number of subjects . studies showed that the effect of no on bone formation is related to other stimuli , which regulates the bone formation cycle . in situations with high stress , like increased osteoblastic activity , no levels increase in order to eliminate o2 . 
 . onoo is a very potent oxidant , which can affect deoxyribonucleic acid ( dna ) and fatty acids and initiate lipid peroxidation . many ros , which are made in cells and tissues , occur in very low levels . ros is a very reactive molecule when it is very high and it affects the defensive systems , causes tissue damage , and starts lipid peroxidation ( 25 ) . lipid peroxidation is a well - known tissue damage mechanism in humans and is used in tissue and cells as an os indicator . lipid peroxides are made of polyunsaturated fatty acids ; they are unstable and they decompose to make complex compounds . mda is the biggest group of this compound and for this reason measuring the level of mda is the criteria to understand the lipid peroxidation ( 26 ) . recently it is shown that oxide lipids affect osteoclastic and osteoblastic bone cells ( 27 ) . levels of the femur hydrogen peroxide ( h2o2 ) and lipid peroxidation in rats with overectomy indicate the deficiency of estrogen levels and activate ros , which leads to bone loss . it is well known that osteoclasts express nadph and produce high levels of ros during bone resorption ( 28 ) . ( 8) showed that increase in mda levels ( in plasma and erythrocyte ) was a result of lipid peroxidation in osteoporotic females compared with the non - porotic control group . in another study , ozgocmen et al . ( 23 ) notified an increase in mda levels in osteoporotic females and a decrease in mda levels in the group which was treated with calcitonin , risedronat , and raloksifen . ( 29 ) marked the increase in mda levels in osteoporotic females , but there was no change in alkaline phosphatase level . although there are studies that investigated the role of free radicals on bone resorption , muthusami et al . ( 6 ) noted that the data of the bone loss in ovariectomized rats are limited and they also found that the levels of mda increase , which is induced by lipid per oxidation . increased production of h2o2 in femur may cause an increase in the lipid per oxidation . mda , which has high reactivation against the amino groups , inhibits the protein synthesis and deactivates the enzymes . for this reason , it is reported that decreased antioxidant enzymes of the femur corresponded to increased lipid peroxidation . ( 10 ) investigated the lipid peroxidation levels by assessing the urine levels of the 8-iso - pgf(2 ) in osteoporotic patients ; they indicated that there is a correlation between lipid peroxidation and bmd . experimental and clinical studies about op point different results in other findings , except maggio et al . ( 7 ) , besides increased lipid peroxidation and as a result of this increased mda activity is indicated ( 14 , 30 ) . in our study , we found increased mda activity in femur compared with other groups and increased mda activity in plasma group compared with the control group . the results of the earlier studies on animals showed that osteoclastic functions and differentiations are stimulated by the ros , especially h2o2 and o2 
  ( 6 , 15 , 16 , 28 , 31 ) . reaction of the radicals with the potential substrates and formation of hydroxyl , which is a kind of toxic product , inhibits sod ( 32 ) . osteoclasts produce high levels of o2 
  and calciotropic hormones which stimulate or inhibit the osteoclasts have also stimulating or inhibiting effect on cell 's production of anion . h2o2 functions both as intra- and inter - cellular pulses and has the most appropriate characteristic besides the other reactive oxygen types . from this point of view , ros , which is responsible for bone loss due to estrogen deficiency , increased os is related with the risk factors of op , like smoking , hypertension ( ht ) , and diabetes mellitus ( dm ) . estrogen deficiency decreases the defense of the thiol antioxidants and induces tnf- expressions , which result in bone loss ( 6 ) . ( 6 ) indicated increased lipid peroxidation and h2o2 levels and decreased enzymatic antioxidants like sod , gpx , and glutathione - s - transferase in preovariectomized rats compared with the control group . they claimed that h2o2 is necessary for osteoclast differentiation . in another study , the major intracellular enzyme for h2o2 gpx and its expression comparing macrophages is amplified and the levels of enzymes increased by 17--eustradiol are shown ( 31 ) . sontakke and tare ( 14 ) in a group of pmo , renal osteo dystrophy , and patients with fractured bone have found decreased gpx and sod activity compared with the control group , and this indicates the potential role of ros in bone metabolism . ( 30 ) found similar erythrocyte sod results in women with both premenopausal and postmenopausal op . ( 8) found that in patients with os , sod enzyme activity was almost same compared with the control group . but on the other hand , osteoporotic women have high sod enzyme activity in plasma and that there was a correlation between erythrocyte sod enzyme activity and femur bmd . ( 24 ) , in their study about the role of antioxidants and free radicals in postmenopausal op , reported that sod activity in patients with op was similar compared with the control group . on the other hand , chavan et al . ( 29 ) showed that serum sod activity is notably higher than the control group . maggio ( 7 ) and sontakke ( 14)and tare found sod and gpx levels to be low in the osteoporotic patients and they believed that as a result of this the antioxidant defense mechanisms were reduced in op . in our study , ( 29 ) , we found increased sod activity in the femur bone tissue in ovariectomized group compared with the control group . plasma sod activities increased in the ovariectomized group , but it was not statistically significant . among various studies about the evaluation of gpx , which is one of the enzymatic antioxidant enzymes , maggio et al . ( 7 ) reported that plasma gpx activity is significantly low in osteoporotic patients . ( 6 ) showed that levels of enzymatic antioxidants decreased like gpx and glutathione - s - transferase in ovariectomized rats compared with the control group and pronounced that h2o2 is necessary for osteoclast differentiation . bednarek - tupikowska et al . ( 30 ) compared premenopausal and postmenopausal women and showed a significant decrease in erythrocyte gpx activity in postmenopausal women . similarly , sontakke and tare ( 14 ) , in a group of pmo , found decreased gpx activity in patients with renal dystrophy and bone fracture compared with the control group . ( 8) found similar results in plasma gpx enzyme activities in op patients and the control group . the same investigators ( 9 ) showed that male op patients have significantly low gpx activities , but the difference is not statistically significant . but in osteoporotic patients , compared with the control group , sod / gpx was higher . differences in sod / gpx ratio are very important in cellular resistance over cell death and damage caused by oxidants and also cause increased ros formation . in our study , ( 9 ) , we found that , in femur bone tissue and in plasma , gpx activity was similar in all the three groups . causes of increase in sod activity and unchanged gpx activity in ovariectomized group had higher sod / gpx ratio . ( 8) reported that erythrocyte cat activity is meaningly low in patients with postmenopausal op and also reported that they found a correlation between erythrocyte cat enzyme activity and proximal femur bmd . in our study , we found that sod activity was increased and on the contrary cat activity was the same in femur antioxidant defenses . catalase , which speeds up the h2o2 formation with a catalytic reaction transforms h2o2 to two molecules of water and in this way removes it from the environment . in our study , the formation speed of h2o2 might not be so high and for that reason we might have found cat activities alike . plasma thiols are cleaners of the free radicals and in some instances they can be antioxidant . in pathological and physiological situations , to evaluate the overproduction of free radicals , t - sh is measured ( 33 ) . protein thiols in some mechanisms can protect their antioxidant function ; in the beginning of peroxidation they can remove the antioxidants in a preemptive way ( 33 ) . we could not find any study in the literature that evaluated thiol in plasma , but there are some studies in which the t - sh is measured ( especially in illnesses which are related with os ) ( 33 , 34 ) . in our study , we found t - sh levels were almost same in each group . although femur bone tissue t - sh level in ovariectomized group decreased from 113.33 + 57.14 ( mm / g prt ) to 76.0 + 42.16 ( mm / g prt ) , this is probably due to increased standard deviation and low number of subjects in the study . a study has shown that vitamin c therapy increases the t - sh levels ( 34 ) . in our study , vitamin c supplementation has increased the t - sh levels in femur bone tissue and in plasma , but this increase is not statistically significant and further studies will make us understand the role of t - sh in op . other mechanisms in which vitamin c assists bmd are still not clear , but it is known that it takes place in collagen formation growth of osteoblasts or increase in ca absorption ( 35 ) . in various studies , it was shown that , in postmenopausal females there is a relationship between dietary vitamin c intake and bmd ( 36 ) . hall and greendale ( 36 ) showed a consistent positive association of vitamin c with bmd in postmenopausal women with dietary calcium intake of at least 500 mg . ( 37 ) in a cross - sectional study showed significant relationship between bmd and several critical nutrients such as : energy , protein , calcium , magnesium , zinc , and vitamin c. it was shown that people who smoke and take low doses of vitamin c and e in their diets have a higher incidence of hip fracture . it was shown that vitamin c and niacin intake in postmenopausal females and iron and zinc intake in premenopausal females are related closely with the forearm bone mineral content . ( 12 ) did not find any correlation between vitamin c and bmd , but with hormone therapy they found a relation . they also found that in females with high total vitamin c concentration it plays a good role . vitamin c deficiency is known in rats and pigs ( 38 ) , and when they are feeded with low levels of ascorbate many deformities have been pointed out : shortness in the legs , multiple fractures , op , growth retardation , and tendencies to haemoragies . ( 39 ) investigated the potential role of antioxidating vitamin on the hip fractures in people who smoke . they reported that if vitamin c and e are taken in low quantities , besides other factors such as age , weight , and other op risk factors , they cause as much as five times more hip fractures . they concluded that sufficient vitamins intake could protect people from the side effects of smoking . ( 13 ) studied 994 postmenopausal women , in which 277 of them were regularly taking vitamin c support . daily intake of vitamin c was 1005,000 ( average 745 mg ) and the average of the year of intake was 12.4 ; 85% of them reported that they were taking vitamin c support for more than 3 years . as a result we can say that vitamin c intake has beneficial effects in bmd , especially if they are combined with hormone therapy . in this study , but best results ( high bmd level ) were by obtained/ taking 1,000 mg / day or higher . simon and hudes ( 40 ) showed a linear relationship between ascorbic acid intake with diet and bmd in premenopausal women . a relationship was found between decreased spontaneous fractures and ascorbic acid levels in females who took estrogen and smoke , but surprisingly they reported a correlation in females who do not use estrogen or smoke . maggio et al . ( 7 ) pronounced that in elderly osteoporotic patients , there is a significant decrease in vitamin levels , sod , and gpx activity . they also reported that taking vitamin c and e with diet is protective against hip fractures . ( 12 ) in a cohort study indicated a correlation between dietary antioxidant intake , to total intake serum concentrations and bmd . with all these results , decreasing on bone resorption and on bmd , vitamin c plays an important role . , we added 1 g vitamin c ( l - ascorbic acid ) to 500 ml of water . we changed the water every day and vitamin c was administered ( as explained earlier ) as rats consumed 1012 ml ( ml/100 g / day ) water every day ( 41 ) . in this case , our rats took 5075 g of vitamin c every day . in our study , bmd was totally restored and os was prevented in female rats . in our study the other hand , li and schellhorn ( 42 ) indicated that understanding the effect of vitamin c on various illnesses , serum vitamin c level along with serum glucose level must be measured . it is pointed that hormones and glucose levels affect the process of measuring serum vitamin c level . for the aforementioned reasons , vitamin c level ( 3 ) characterized the bone deficiencies in rats with postmenopausal or ovarian deficiency in 12 control and 12 ovariectomized rats ( 90 days old ) in a 18-month period . they observed that in 6-month - old rats cancellous bone volume were almost stable and reduced later . in contrast to this , in ovariectomized rats the proximal tibia bone volume was dramatically lost in 2 weeks and in 100 days callose bone volume was 57% and on day 270 bone volume was 12% . in ovariectomized rats , osteoblast level , osteoclast level , and bone formation were higher compared with control group ( especially in 100 days in ovariectomized rats ) as a result , they said ovariectomy in mature rats causes a rapid bone loss in conjunction with maximum increase in bone circle . for that reason we used mature rats ( 100 days old ) in our study . after this period we also found that there was a statically significant reduction in bmd . if the study was done with large number of animals , we could have been more successful . on the other hand , vitamin c level intake and output liquid were not measured in our study , and these were our limitations . to conclude , our results suggest that ovariectomy may produce op and os in females , and vitamin c supplementation may provide alterations regarding improvement in os and bmd values . we assume that studies including more subjects are needed to make a decisive conclusion about os  bmd relation . the authors have not received any funding or benefits from industry or elsewhere to conduct this study .
objectivesthe aim of this study is to investigate the effects of ovariectomy on bone mineral density ( bmd ) and oxidative state in rats , and the alterations in these effects that vitamin c supplementation may produce.materials and methodstwenty female wistar albino rats were randomly divided into three groups : control ( c , n=6 ) ; ovariectomy ( o , n=7 ) ; and ovariectomy+vitamin c supplement ( ov , n=7 ) . oxidative stress ( os ) was assessed 100 days postovariectomy by measuring the activity of several enzymes , including catalase ( cat ) , superoxide dismutase ( sod ) , and glutathione peroxidase , as well as the concentrations of malondialdehyde ( mda ) , nitric oxide ( no ) , and total sulfhydryl groups in plasma and bone homogenates.resultsa significant decrease in bmd was observed in o group compared with c group ( p=0.015 ) , and a significant increase was observed in ov compared with o group ( p=0.003 ) . 
 when groups were compared with respect to parameters of os , mda and no levels in bone tissue were significantly higher in o than in c ( p=0.032 , p=0.022 ) and were significantly lower in ov than in o ( p=0.025 , p=0.018 ) . 
 sod activity was significantly higher in o than in c ( p=0.032 ) . in plasma , 
 mda activity was significantly higher in o than in c ( p=0.022 ) and no level was significantly higher in o than in c and ov ( p=0.017 , p=0.018).conclusionsour results suggest that ovariectomy may produce osteoporosis and os in females , and vitamin c supplementation may provide alterations regarding improvement in os and bmd values . 
 we assume that studies including more subjects are needed to make a decisive conclusion about os 
 bmd relation .