Progress in Nutrition 2018; Vol. 20, N. 1: 86-93 DOI: 10.23751/pn.v20i1.5223 © Mattioli 1885 Original article Biological effect of calcium and vitamin D dietary supplements against osteoporosis in ovariectomized rats Reham A. Mustafa1, Nehal A. A. Alfky1, Haifa H. Hijazi1, Eslam A. Header1,2, Firas S. Azzeh1 1 Clinical Nutrition Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, KSA. - E-mail: [email protected]; 2Department of Nutrition and Food Science Faculty of Home Economics Minufiya University, Egypt. Summary. There is a direct association between the lack of estrogen and the progress of osteoporosis. This study was done to evaluate the biological effect of diet supplementation with calcium (Ca), and vitamin D (VD) on osteoporosis in ovariectomized (OVX) rats and to examine the possible potential mechanisms. Twenty-eight rats were randomly divided into 4 equal groups (n=7). To induce estrogen deficiency in rats, bilateral ovariectomy and sham (SHAM; negative control) surgery were done. In the basal diet, Ca and VD was supplemented with 210 mg/kg and 600 IU/kg, respectively, for 6 weeks. Alendronate as a standard anti- osteoporotic drug was used in a single weekly dose (3 mg/kg) for 6 weeks. After six weeks, serum markers of osteoporosis and bone femur status were evaluated. The results exposed that Ca and VD supplementation increased the body weight gain and diminished the uterine weight as a result of ovariectomy operation. These supplements significantly raised the serum Ca, bone-specific alkaline phosphatase, free thyroxin, and osteo- calcin in OVX-rats, while the serum interleukin-1beta, interleukin-6, parathormone, and pyridinoline levels were significantly dropped. There were also significantly improved in femur bone mineral density and bone ash contents, mainly Ca and phosphorous. In conclusion, feeding of Ca and VD dietary supplements have an anti-osteoporotic activity in OVX rats due to improvement of bone formation and abolition of bone loss. The study recommends that intake of Ca and VD together may be beneficial for the inhibition of osteoporosis in postmenopausal women due to estrogen deficiency. Key words: calcium, vitamin D, osteoporosis, bone mineral density Introduction elderly women and younger postmenopausal women. Moreover, menopause drastically increases the risk of Osteoporosis is a silently progressing disease of osteoporosis (3). Postmenopausal osteoporosis occurs bones characterized by low bone mass and decreased due to imbalance between osteoblastic bone formation bone mineral density (BMD) leading to high inci- and osteoclastic bone resorption as a result of estrogen dence of bone fragility and fractures (1). The mass of loss (4). Estrogen deficiency is the most potent initiator skeletal bone is controlled by a combination of some of osteoclastic bone loss and has been associated with endogenous (genes, metabolic hormones) and exoge- osteoporosis (5). In addition to maintaining adequate nous (nutrition, exercise) factors (2). Osteoporosis rep- Ca and VD intake and practicing exercise, the preven- resents a serious health problem that prevails among tive measures against osteoporosis include avoiding Biological effect of calcium and vitamin D dietary supplements against osteoporosis in ovariectomized rats 87 of smoking, excessive alcohol and caffeine intake (6). obtained in the form of tablets each contains 70 mg Estrogen, Ca, VD, calcitonin and several antioxidants Alendronate sodium. The dose of Alendronate 3 mg/ help in the prevention of postmenopausal osteoporosis kg body weight (b.wt)/week was orally given to rats (7, 8). Estrogen replacement therapy (ERT) has been according to Maria et al. (17). established as a regimen for prevention of postmeno- pausal bone loss, but long term ERT may be accom- Rats panied with severe adverse effects and increased risk of Twenty-eight mature female Sprague Dawley rats ovarian and endometrial cancers (9, 10). (235-245 g b.wt and 10-12 weeks old) were used in Nutrition plays an important role in bone health, this study. The rats were purchased from Laboratory and there is an increasing interest in dietary nutrients Animal Colony, Helwan, Egypt. The animals were which influence bone metabolism and health such as housed under hygienic conditions at room temperature Ca and VD. Reduced dietary intake of Ca is associ- of 24°C, relative humidity of 50% and 12 hr light/12 ated with reduced bone mass and leads to osteoporosis. hr dark cycles. The rats were fed on either basal or ex- Chronic VD deficiency leads to osteomalacia (11). On perimental diets and water was provided as required. the other side, adequate intake of Ca and VD is essen- The experiment on rats was carried out according to tial for bone health (2, 7, 11). the National regulations on animal welfare and Insti- Oxidative stress, resulting from excessive forma- tutional Animal Ethical Committee. tion of reactive oxygen species (ROS) or lowering of body antioxidant defense system, represents a main Basal and experimental diets cause of postmenopausal bone loss (12). ROS are in- The dietary supply of protein, fat, carbohydrates, volved in bone resorption because of superoxide free vitamins and minerals was equivalent to the recom- radicals generate osteoclastic bone loss (13). Oxidative mended dietary allowances for rats according to stress increased differentiation and function of osteo- Reeves et al. (18). Basal diet consisted of 20% protein, clasts, so increasing bone loss (14). 10% sucrose, 5% corn oil, 2% choline chloride, 1% vi- Therefore, the present study was undertaken to tamin mixture, 3.5% salt mixture and 5% fibers. The evaluate the effect of Ca and VD micronutrients on remainder was corn starch up to 100%. Experimental serum and bone biomarkers of osteoporosis in ovari- diets were basal diet supplemented with Ca (210 mg/ ectomized rat model, and to examine the potential kg) and VD (600 IU/kg). mechanisms. Ovariectomy procedure Under ether anesthesia, the bilateral ovariectomy Materials and methods was performed in rats by making two dorsolateral in- cisions using sharp dissecting scissors. The skin and Dietary supplements dorsal muscles were then cut and the peritoneal cav- Calcium carbonate was procured from El- ity was thus reached. The uterine horn was picked out Gomhoryia Company, Egypt, in the form of fine and the fatty tissue around the ovary was removed. powder. Calcium carbonate is widely used as an The connection between the Fallopian tube and the inexpensive dietary Ca supplement. It was added uterine horn was clamped by artery forceps and cut to basal diet at 210 mg/kg according to Chen et al. was made under the clamped area to remove the ovary. (15). Vitamin D (Cholecalciferol, vitamin D3) was Skin was closed bilaterally with one simple catgut su- obtained in the form of capsules and added to basal ture. Tincture iodine solution (antiseptic) was applied diet at 600 IU/kg according to Ghanizadeh et al. (16). locally on the skin at both sites of the operation. This technique was described by Lasota and Danowska- Alendronate drug Klonowska (19). Similarly, sham (SHAM) operation Alendronate (Fosamax®, Merck Sharp and was performed where the ovaries were exposed but not Dohme Company, USA) is class of bisphosphonates removed. that widely used for treatment of osteoporosis. It was 88 R. A. Mustafa, N.A. A. Alfky, H. H. Hijazi, et al. Experimental design using Vernier caliper. Femur bone volume and BMD Twenty-eight rats were randomized into to 4 were calculated according to the principle of Archi- equal groups. Group 1 was sham-operated (SHAM) medes (25). In brief, the femur was cut out at the mid and fed on basal diet and the other 3 groups were diaphyses and bone marrow washed out. Each femur ovariectomized (OVX) and left for 3 weeks post-op- bone was placed in a vial filled with deionized wa- eration to ensure almost complete clearance of their ter and the vial was placed in vacuum desiccator for bodies from sex hormone residues. Group 2 was kept 90 minutes. The femurs were removed from the vial, OVX (positive control) and fed on basal diet. Group 3 dried by blotted paper, weighed, and placed again in was fed on experimental diets supplemented with Ca other vial containing deionized water. The bone was + VD for 6 weeks. Group 4 was orally given Alen- reweighed and bone volume was measured. Femur dronate (standard anti-osteoporotic drug) in a single BMD was calculated using this formula: BMD = fe- weekly dose (3 mg/kg) for 6 weeks. The initial and fi- mur weight/femur volume. To obtain the ash, femur nal body weights of rats were recorded and changes bones were dehydrated and defatted in acetone and in weight gains were calculated. Blood samples were anhydrous ether, dried for 6 hr in an oven at 700°C. collected for biochemical analyses. The rats were then The remaining ash was weighed, solubilized with 0.1 euthanized by prolonged exposure to ether anesthetic Mol/L HCl, transferred into volumetric flask and and uterine horns were dissected out and weighed. Fe- completed to 100 ml with 0.1Mol/L HCl according mur bones were dissected out and prepared for bone to Yang et al. (26). The final solution was used for es- analysis. timation of calcium and phosphorus in the ash using colorimetric methods. Biochemical analyses Blood samples were withdrawn by cardiac punc- Statistical analysis ture, left standing for 10 minutes to clot and centri- Data were presented as means ± standard error fuged at 12000 rpm for 15 minutes to separate the (SE). The statistical analysis was performed using com- serum which kept frozen at – 80°C till biochemical puterized statistical package of social sciences (SPSS, analyses. Serum concentrations of Ca (20) and phos- version 20) program with one-way analysis of variance phorus (21) were colorimetrically determined using (ANOVA) followed by Duncan›s multiple range tests specific diagnostic reagent kits (BioMérieux, France) according to Snedecor and Cochran (27).