http://informahealthcare.com/arp ISSN: 1381-3455 (print), 1744-4160 (electronic) Arch Physiol Biochem, 2014; 120(2): 80–85 ! 2014 Informa UK Ltd. DOI: 10.3109/13813455.2014.884141
The effect of zinc deficiency and zinc supplementation on element levels in the bone tissue of ovariectomized rats: Histopathologic changes Abdulkerim Kasim Baltaci1 Fusun Sunar1, Rasim Mogulkoc1, Musa Acar2, and Hatice Toy3 1
Selcuk University, Medical Faculty, Department of Physiology, Konya, Turkey, 2Mevlana University, High School of Health, Department of Physiotherapy and Rehabilitation, Konya, Turkey, and 3Necmettin Erbakan University Meram Medical School Department of Pathology, Konya, Turkey Abstract
Keywords
Objective: Study aimed to determine the effects of zinc supplementation/deficiency on the histological structure and elements levels in bone tissue in ovariectomized rats. Methods: The study included 40 Sprague-Dawley type adult female rats, divided as follows: Control, ovariectomized, ovariectomized + zinc supplemented, ovariectomized + zinc deficient groups. At the end of the study bone tissues (femur) were collected to determine the levels of calcium, phosphorus, magnesium, zinc, iron, aluminium, chrome, lithium, lead, nickel, and manganese. The bone tissue was examined for histopathology. Results: Ovariectomy leaded to significant decrease in magnesium. Zinc supplementation to ovariectomized rats restored the reduced calcium, phosphorus, zinc. However, zinc deficiency in ovariectomized rats further reduced calcium, phosphorus, zinc, and manganese levels. Zinc deficiency in ovariectomized significantly increased Al, Cr, Li, Pb, and Ni levels. Tissue integrity was impaired due to ovariectomy and zinc deficiency. Conclusion: Ovariectomy and zinc deficiency leads significant decreases elements of the bone.
Bone, elements, ovariectomy, zinc supplementation and deficiency
Introduction Oestrogen is a required element for the preservation of bone mineral balance (Avila et al., 2009) and its deficiency resulting from ovariectomy leads to osteoporosis (Arslan et al., 2011). Being a major pathological bone condition, osteoporosis causes a serious decline in the quality of life. Decreases that occur in oestrogen levels with ageing constitute the main risk factor for osteoporosis (Yamada et al., 1998). It was established that oestrogen deficiency caused re-structuring of the bone tissue and altered the tissue element distribution (Ames et al., 2010). Likewise, it was reported in a study exploring the effects of ovariectomy on the bone metabolism that ovariectomy increased bone turnover by affecting the levels of osteocalcin and alkaline phosphatase, which are sensitive markers of bone formation, and type I collagen C-terminal (CTX), a bone resorption parameter (Yoon et al., 2012). Zinc is a trace element essential for different metabolic events and cellular signalling pathways that are involved in growth, development and sustenance of life (Oh et al., 2012). Zinc deficiency impairs the bone tissue (Oh et al., 2012). Correspondence: Dr. Abdulkerim Kasim Baltaci, Selcuk University, Medical School, Department of Physiology, Selcuk University, Medical Faculty, Department of Physiology, Konya, 42075 Turkey. E-mail: [email protected]
History Received 12 December 2013 Revised 9 January 2014 Accepted 10 January 2014 Published online 5 February 2014
Zinc is required for the differentiation and growth of osteoblastic cells (Yusa et al., 2011). Similarly, zinc was reported to inhibit osteoclastic bone resorption and stimulate osteoblastic bone formation (Hie et al., 2011). It was seen in a study of male rats that zinc supplementation had a protective effect against femoral fracture in rats exposed to cadmium (Brzo´ska et al., 2007). There has been increasing emphasis on the significance of trace elements in bone formation, development, and structure (Arikan et al., 2011). It was reported in a study involving pregnant rats that zinc deficiency in the mothers impaired bone mineralization and matrix formation in the offspring (Nagata et al., 2011). A review of the relevant literature shows that both oestrogen which originates from the ovary and zinc, an essential element, affect the bone metabolism. The purpose of the present study is to determine the effect of ovariectomy and 6-week zinc deficiency and zinc supplementation on the element levels and histological structure of bone tissue in rats.
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ORIGINAL ARTICLE
Methods The study was conducted upon the approval of the ethics committee of Selcuk University Experimental Medicine and Application Center. The study included 40 Wistar-Albino type female rats whose mean weight was 250 to 260 g. Kept in
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DOI: 10.3109/13813455.2014.884141
Zinc deficiency and supplementation, bone tissue and ovariectomy
rooms with controlled temperature and lighting conditions, all the rats were supplied with water and feed. Group 1 (n ¼ 10): The control group that was not subjected to any procedure. Group 2 (n ¼ 10): The group fed on a normal diet after being ovariectomized under general anaesthesia. Group 3 (n ¼ 10): The group supplemented with intraperitoneal zinc (3 mg/kg/day) for 6 weeks after being ovariectomized under general anaesthesia. Group 4 (n ¼ 10): The group fed on a zinc-deficient diet (0.65 ppm zinc/g diet) for 6 weeks after being ovariectomized under general anaesthesia. In order to minimize zinc contamination, the experimental animals were fed in special steel cages which were cleaned daily by washing. The feed were given in special steel bowls and water in glass feeding bottles. Both zinc-deficient and normal forms of the animal feed were prepared in Korkuteli Feed Supplement Industry Factory. Experimental procedures Zinc sulphate administration The appropriate amount of zinc sulphate was dissolved in distilled water and supplemented by intraperitoneally to the animals. The dose was 3 mg zinc/kg and was given daily at 9 am for 6 weeks. Ovariectomy The rats were injected with 60 mg/kg ketamine and 5 mg/kg rompun to induce general anaesthesia. After the hair on the back of the rats was shaved, proper asepsis and antisepsis were ensured using betadine. After the rats were put into ventral position, the skin was incised at the 1/3 upper point of the distance between the tail and mid-dorsal area. The subcutaneous tissues were released and the spinal muscle was reached. Peritoneal cavity was entered through the back wall muscles of the abdomen. The ovaries were removed together with the fatty tissue. The ovaries were first cleaned off the fatty tissue, and then clamped, tied, and cut. After checking for bleeding, the other organs were put back into the peritoneal cavity. Lastly, the muscle was sutured using a 2/0 chrome catgut and the skin with 2/0 silk (Waynfort & Flecnell, 1994). After the animals were decapitated at the end of the procedures, bone tissue samples were taken to determine the levels of some elements. Analyses of calcium, phosphorus, magnesium, zinc, iron, aluminium, chrome, lithium, lead, nickel, and manganese levels in the bone tissue Samples of bone tissue were put into capped polyethylene tubes washed with NHO3 and de-ionized water to prevent contamination. The tubes were stored at 35 C until the day of analysis. Bone tissue was ground into powder using a mortar. Wet weights of the bone tissue were recorded. Concentrated H2SO4 and HNO3 were added to the samples (gram tissue/ml H2SO4/ml HNO3 ¼ 1/1/10). The samples were then left to wait in a closed system microwave oven (CEM – Marsx5) at 170 psa pressure and 200 C for
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20 minutes. Then, they were added deionized water to obtain the final volume of 25 ml. After a maximum waiting time of half an hour, samples were read. The analyses were conducted using the Atomic Emission (ICP-AES) device found in the Department of Soil Science of SU Faculty of Agriculture (Saygı et al., 1991). Magnesium values in the bone tissue were calculated as mg/g/wet tissue, while the other elements were determined as mg/g/wet tissue. Histological examination Bone tissue samples taken from the animals at the end of the study and were decalcified by formic acid and the tissues were followed autotechnicon. Following autotechnicon processing, samples were buried into paraffin and 5 mm crosssections were obtained using a microtome. The sections were placed on a microscope slide and stained with PAS. Coverslip on a slide and viewed by light microscopy under 40 magnification. Histological results including calcification, inflammation, and sclerotic changes in the bone tissue were examined. When evaluating bone density and morphology, ovariectomized rat bone tissue was based and other groups were compared with this. Osteoclast activity was evaluated by surrounding bone tissue examined (trabecular thinning, disfigurement). Counting the number of osteoclasts in bone adjacent to the five areas were averaged. Statistical evaluations The statistical evaluation of the results was carried out using computer software. Arithmetic means and standard deviations of all parameters were calculated. Variance analyses were used to identify the differences between groups. The least significant difference (LSD) test was employed to compare group means in the statistically significant analysis results. Differences for which p50.05 were accepted significant. The Mann-Whitney U test was used in the statistical analysis of histology results, which were evaluated by comparing median values. Level of significance was set at p50.05.
Results Of the parameters presented in Table 1, calcium and phosphorus values in the bone tissue were found higher in the control group which was not subjected to any procedure (group 1) and zinc-supplemented ovariectomized group (group 3), when compared with groups 2 and 4 (p50.05), with the ovariectomized control group (group 2) having higher calcium and phosphorus values than the ovariectomized group fed on a zinc-deficient diet (p50.05). There was no significant difference among the groups in terms of their bone tissue magnesium levels. Bone tissue zinc levels in groups 1 and 3 were not different relative to each other, but higher than the levels in groups 2 and 4 (p50.05). The lowest bone zinc level was found in group 4 (p50.05). When aluminium values in the bone tissue were examined, this parameter was seen to fall especially after ovariectomy, relative to the control group (p50.05). Zinc supplementation after ovariectomy did not affect aluminium levels in the bone tissue. However, zinc deficiency induced after ovariectomy
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Table 1. Calcium, phosphorus, magnesium, zinc, and iron levels in the bone tissue of study groups.
Groups C Ovx Ovx-Zn S Ovx-Zn D p
Calcium (mg/g/wet tissue)
Phosphorus (mg/g/wet tissue)
Magnesium (mg/g/wet tissue)
Zinc (mg/g/wet tissue)
Iron (mg/g/wet tissue)
92.18 ± 27.87a 62.35 ± 33.69b 91.57 ± 29.60a 58.05 ± 28.18c 0.05
56.07 ± 16.80a 44.74 ± 14.05b 53.42 ± 13.25a 32.27 ± 16.43c 0.05
25.32 ± 13.02 25.94 ± 12.04 25.97 ± 15.19 25.41 ± 13.99
0.11 ± 0.01a 0.07 ± 0.02b 0.11 ± 0.01a 0.05 ± 0.02c 0.05
73.75 ± 32.22a 52.03 ± 11.44b 41.90 ± 14.67b 48.69 ± 14.04b 0.05
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C, control; Ovx-C, ovariectomy; Ovx-Zn S, ovariectomy + zinc supplementation; Ovx-Zn D, ovariectomy + zinc deficiency. a,b,c Means with different superscripted letters in the same column are statistically significant (p50.05).
Table 2. Aluminium, chrome, lithium, lead, nickel, and manganese levels in the bone tissue.
Groups C Ovx Ovx-Zn S Ovx-Zn D p
Aluminium (mg/g/wet tissue)
Chrome (mg/g/wet tissue)
Lithium (mg/g/wet tissue)
Lead (mg/g/wet tissue)
Nickel (mg/g/wet tissue)
Manganese (mg/g/wet tissue)
6.40 ± 3.80a 3.82 ± 1.76b 3.87 ± 1.37b 4.20 ± 2.38b 0.05
2.94 ± 2.76b 1.96 ± 0.75c 1.96 ± 0.75c 3.46 ± 1.29a 0.05
4.82 ± 2.64b 3.83 ± 1.90b 4.26 ± 2.99b 9.10 ± 5.10a 0.05
0.86 ± 0.31b 0.78 ± 0.36b 0.83 ± 0.38b 1.96 ± 0.29a 0.05
0.22 ± 0.13b 0.09 ± 0.10b 0.11 ± 0.08b 0.36 ± 0.13a 0.05
0.24 ± 0.15a 0.12 ± 0.10b 0.14 ± 0.11b 0.07 ± 0.02c 0.05
C, control; Ovx-C, ovariectomy; Ovx-Zn S, ovariectomy + zinc supplementation; Ovx-Zn D, ovariectomy + zinc deficiency. a,b,c Means with different superscripted letters in the same column have statistical significance (p50.05).
elevated aluminium levels in this group, in comparison to the aluminium levels in the ovariectomized and ovariectomized and zinc supplemented groups (p50.05) (Table 2). Bone tissue levels of iron and manganese decreased after ovariectomy. Secondly, manganese decreases in the ovariectomized plus zinc-deficient group (Tables 1 and 2, p50.05). An examination of chrome, lithium, lead, and nickel levels in the bone tissue demonstrated that the levels of these elements dropped, though statistically insignificantly, in ovariectomized and ovariectomized and zinc-supplemented groups, compared with the control group (p50.05). However, these parameters were found markedly higher than the control levels especially in the ovariectomized group in which zinc deficiency was induced (p50.05). Histological results including loss of trabecular mass and altered osteoclastic activity in the bone tissue were evaluated. There was no significant difference between the control group which was not subjected to any procedure (group 1) and ovariectomized and zinc-supplemented group (group 3) in terms of trabecular mass loss, osteoclastic activity, and bone density (Figures 1, 3). However, these values in the ovariectomized control group, group 2 (Figure 2), were higher than their counterparts in groups 1 and 3 (p50.05), and lower than the values in group 4 (Figure 4) (p50.05). Loss of trabecular mass, and changes in osteoclastic activity and bone density were higher in group 4, the group fed on a zinc-deficient diet, than in other groups (p50.05) (Table 3).
Figure 1. Normal rat bone tissue.
Discussion It was reported in a similar study that Ca levels in the serum, though not in the bone tissue, decreased after ovariectomy, and this result is supportive of ours (El-Shitany et al., 2010; Sherman et al., 1989; Ulas & Cay, 2011). Decreased bone tissue calcium levels resulting from zinc deficiency show that zinc deficiency has negatively affected the tissue level of this
Figure 2. Bone tissue of ovariectomized rat (loss of trabecular mass and changes in osteoclastic activity and bone density were higher than their counterparts in groups 1 and 3).
Zinc deficiency and supplementation, bone tissue and ovariectomy
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DOI: 10.3109/13813455.2014.884141
Figure 3. Bone tissue of ovariectomized and zinc-supplemented rat (not different from Figure 1).
Figure 4. Bone tissue of ovariectomized rat fed on a zinc-deficient diet (increased loss of trabecular mass and changes in osteoclastic activity and bone density).
Table 3. Histological changes in the bone tissue of study groups.
Group Control Ovariectomy control Ovariectomy + zinc supplementation Ovariectomy + zinc deficiency p
Osteoclastic Trabecular activity Bone density mass loss (median value) (median value) (median value) 1.000c 3.000b 1.000c
1.000c 3.000b 1.000c
1.000c 3.000b 1.000c
5.000a
5.000a
5.000a
0.05
0.05
0.05
a,b,c
Means with different superscripted letters in the same column are statistically significant (p50.05).
element. In fact, a similar decrease in serum calcium levels was already reported in a previous study (Sunar et al., 2009). The drop in tissue calcium may have resulted from increased urinary calcium excretion. Actually, there are previous studies reporting increased urinary calcium excretion due to Ovx (Zhao et al., 2009). As oophorectomy enhances bone loss, bone mineral loss results from increased calcium excretion from the gastrointestinal tract and kidney (Cai et al., 2005; O’Loughlin & Morris, 2003). The results also suggest that
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6-week zinc supplementation can reinstate the bone tissue calcium loss resulting from ovariectomy and zinc deficiency. When phosphorus levels in the bone tissue were examined, it was seen that this element was negatively affected by ovariectomy and zinc deficiency. However, these diminished bone tissue phosphorus levels were significantly restored by 6-week zinc supplementation and reached control levels. In line with these results, Avila et al., (2009) reported a decline in phosphorus levels due to ovariectomy. Along the same line, Nagata et al. (2011) found that the offspring of rats fed on a zinc-deficient diet suffered from impaired bone mineralization and highlighted the importance of adequate zinc intake. Re-absorption of phosphorus from the kidney is regulated by the effect of the parathyroid hormone on the sodium-phosphorus co-transporter mechanism, and within this mechanism, oestrogen was reported to be involved in the renal phosphorus retention (Dick & Prince, 2001). In line with our study, there are those stating that ovariectomy lowered phosphorus levels (Sunar et al., 2009; Ulas & Cay, 2011). However, the decrease caused by ovariectomy in phosphorus levels was reversed by zinc supplementation which restored the levels of this element to normal, parallel to the results of previous studies (Sunar et al., 2009). Previous studies show that ovariectomy affected Mg levels in a different way. For instance, Avila et al., (2009) noted that ovariectomy lowered Mg levels. In another study, Mg loss through urine was reported to increase as a result of ovariectomy (Zhao et al., 2009). However, there are also studies demonstrating that there was no ovariectomyassociated change in the Mg levels and our results are in consistency with the results of these latter studies (Sunar et al., 2009; Ulas & Cay, 2011). Thus, in our study neither ovariectomy nor zinc supplementation or deficiency had any effect on magnesium levels in the bone tissue. Concerning the Zn values in our study, it was seen that both ovariectomy and zinc deficiency had a negative impact on the level of this element. Previous studies also reported decreased zinc values after ovariectomy (Ulas & Cay, 2011; Yazici et al., 2011; Zhao et al., 2009). Thus, our results are parallel to the results of previous studies. It was reported in these studies that zinc had a critical effect on the bone metabolism. It was established that zinc altered serum osteocalcin and tissue osteoblastic and osteoclastic activities, all of which are parameters related to the bone metabolism (Hie et al., 2011). In a study involving women with osteoporosis, a positive correlation was found between zinc and lumbar vertebra in the postmenopausal period, and this result emphasizes the importance of zinc in maintaining the normal bone structure (Arikan et al., 2011). Being a critical factor stimulating the differentiation and growth of osteoblasts, zinc is an essential element for protecting the bone structure and sustaining normal bone function. In fact, it was demonstrated in an experimental study that zinc-releasing implants favourably influenced bone fixation (Yusa et al., 2011). In our study, 6-week zinc supplementation significantly restored the zinc reduction caused by both ovariectomy and zinc deficiency. When iron levels in the bone tissue were investigated in our study, it was seen that the level of this element dropped relative to control values after ovariectomy. However, iron
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levels were not significantly affected by zinc supplementation or deficiency. In a previous study, it was shown that zinc supplementation and deficiency together with Fe supplementation affected the bone metabolism and iron supplementation increased the tibia size (Abrisham et al., 2010). However, dietary calcium intake was also demonstrated to affect the iron levels in the bone tissue (tibia) (Behling & Greger, 1998). Besides, the report showing that iron accumulation caused by the cessation of menstruation led to resorption in the bone tissue indicates a crucial relation between bone tissue and iron (Yang et al., 2011). In the present study, the increase brought about by zinc supplementation in the reduced bone tissue iron levels may be considered a positive effect on bone tissue formation. Previous studies showed a relation between Oestrogen and aluminium (Zhao et al., 2009). In fact, the combination of ovariectomy and aluminium toxicity was seen to alter the tissue element distribution in the concerned study. Oestrogen replacement to ovariectomized rats increased aluminium levels in the tibia and urinary aluminium loss. Furthermore, the protective effect of Oestrogen is attested by the study which showed that 3-month aluminium chloride administration to ovariectomized rats caused neurodegeneration, which was restored by Oestrogen treatment (Mohamd et al., 2011). Similar results were also reported by Contini et al. (2011). Interestingly in our study, zinc deficiency induced after ovariectomy caused a certain amount of aluminium accumulation in the bone tissue, and it was thought that the presence of this element might cause a certain degree of toxicity in the bone tissue, which is consistent with the cited studies. An examination of chrome levels in the bone tissue revealed that the level of this element in the tissue dropped after ovariectomy (group 2), but zinc supplementation following ovariectomy did not bring about a further change. However, a significant increase was observed in the bone tissue chrome levels of the group which was fed on a zincdeficient diet after ovariectomy (group 4). In a study including postmenopausal women, it was established that hormone replacement therapy increased chrome levels, while decreasing urinary chrome loss (Bureau et al., 2002). In a similar study, untreated postmenopausal women were shown to have lower chrome levels (Roussel et al., 2002), and this is consistent with our results. In our study, zinc deficiency together with ovariectomy was found to increase bone tissue chrome levels. These results suggest that decreased zinc levels changed the levels of chrome in the concerned tissue. In fact, several studies already demonstrated that zinc had a regulatory effect on the distribution of elements in the serum and tissues (Bicer et al., 2011; Sivrikaya et al., 2012). When the studies exploring ovarian hormones and lithium are considered, it is seen that this element acts upon the decreased Oestrogen receptors in the uterus tissues (Gunin et al., 2004). Elevated lithium levels we found in this study may be important in terms of the toxicity this element causes in the relevant tissue. As a matter of fact, previous studies reported that lithium treatment was associated with impairment in pubertal growth and changes in sex hormone levels (Allagui et al., 2006; Samuel et al., 2012). When the effect of ovariectomy together with zinc supplementation and deficiency on manganese levels in the
Arch Physiol Biochem, 2014; 120(2): 80–85
bone tissue was explored, it was established that ovariectomy lowered the levels of this element and zinc supplementation brought about a partial improvement, while zinc deficiency rendered the decrease more marked. It was already suggested that manganese and MnSOD activity were affected by ovariectomy (Pajovic et al., 1993; Rico et al., 2000). It was found in this study that manganese levels in the bone tissue dropped significantly after ovariectomy. Thus, these results are parallel to the results of previous studies (Ulas & Cay, 2011). The reason for this decrease may be increased MnSOD activity. Actually, increased MnSOD activity was reported after ovariectomy (Pajovic et al., 1993). However, the further decrease observed in manganese levels after zinc deficiency suggests that zinc has a regulatory effect on this element as well. With regard to the lead and nickel levels in the bone tissue, these two elements were seen to significantly increase especially in zinc deficiency. Having a toxic character, both of these elements can alter physiological functions. The toxic effects of these metals had already been reported (Levesque et al., 2003; Valko et al., 2005; Ebrahimi & Taherianfard, 2010; Ebrahimi et al., 2014). Thus, accumulation of these metals in the bone tissue due to zinc deficiency indicates an increase in tissue toxicity in the case of zinc deficiency. Bone tissue was also histopathologically assessed in the present study. Results of the histopathologic assessment revealed that ovariectomy resulted in bone tissue loss, which deteriorated with zinc deficiency and was restored by zinc supplementation. Previous studies showed that oral zinc treatment had a positive effect on osteogenesis (Abrisham et al., 2010). It was seen that zinc treatment reduced the risk for fractures due to toxic metals like cadmium in the bone tissue (Brzo´ska et al., 2007). Zinc deficiency, on the other hand, was found to have a negative effect on the biomechanical quality of bone tissue (Scrimgeour et al., 2007). Impairment in the bone tissue due to zinc deficiency and the restoration of this impairment with zinc supplementation in our study are consistent with the results of previous literature studies. An overall assessment of study results indicates that ovariectomy and zinc deficiency in rats have a negative effect on the elements involved in the bone structure and damage the tissue integrity. The second important result of this study is that zinc deficiency can cause toxicity in the tissue by increasing the amounts of some elements such as lead, nickel, aluminium, and chrome.
Declaration of interest Authors declare no conflict of interest.
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O’Loughlin PD, Morris HA. (2003). Oophorectomy acutely increases calcium excretion in adult rats. J Nutr, 133:2277–80. Pajovic´ S, Nikezic´ G, Martinovic´ JV. (1993). Effects of ovarian steroids on superoxide dismutase activity in the rat brain. Experientia, 49: 73–5. Rico H, Go´mez-Raso N, Revilla M, et al. (2000). Effects on bone loss of manganese alone or with copper supplement in ovariectomized rats. A morphometric and densitomeric study. Eur J Obstet Gynecol Reprod Biol, 90:97–101. Roussel AM, Bureau I, Favier M, et al. (2002). Beneficial effects of hormonal replacement therapy on chromium status and glucose and lipid metabolism in postmenopausal women. Maturitas, 42:63–9. Samuel JB, Stanley JA, Sekar P, et al. (2012). Persistent hexavalent chromium exposure impaired the pubertal development and ovarian histoarchitecture in wistar rat offspring. Environ Toxicol, doi: 10.1002/ tox.21810. Saygi S, Deniz G, Kutsal O, Vural N. (1991). Chronic effects of cadmium on kidney, liver, testis, and fertility of male rats. Biol Trace Elem Res, 31:209–14. Scrimgeour AG, Stahl CH, McClung JP, et al. (2007). Moderate zinc deficiency negatively affects biomechanical properties of rat tibiae independently of body composition. J Nutr Biochem, 18:813–19. Sherman SS, Smith Jr JC, Tobin JD, Soares Jr JH. (1989). Ovariectomy, dietary zinc, and bone metabolism in retired breeder rats. Am J Clin Nutr, 49:1184–91. Sivrikaya A, Bicer M, Akil M, et al. (2012). Effects of zinc supplementation on the element distribution in kidney tissue of diabetic rats subjected to acute swimming. Biol Trace Elem Res, 147: 195–9. Sunar F, Baltaci AK, Ergene N, Mogulkoc R. (2009). Zinc deficiency and supplementation in ovariectomized rats, their effect on serum estrogen and progesterone levels and their relation to calcium and phosphorus. Pak J Pharm Sci, 22:150–4. Ulas M, Cay M. (2011). Effects of 17b-estradiol and vitamin E treatments on blood trace element and antioxidant enzyme levels in ovariectomized rats. Biol Trace Elem Res, 139:347–55. Valko M, Morris H, Cronin MT. (2005). Metals, toxicity and oxidative stress. Curr Med Chem, 12:1161–208. Waynfort HB, Flecnell PA. (1994). Ovariectomy in experimental and surgical technique in the rat, 2nd ed. London: Academic Press, 76–8. Yamada G, Nakamura S, Fukuzaki K, et al. (1998). Dysregulation of trace element composition in ovariectomized cynomolgus monkey bones. Cell Mol Biol (Noisy-le-grand), 44:1205–13. Yang Q, Jian J, Abramson SB, Huang X. (2011). Inhibitory effects of iron on bone morphogenetic protein 2-induced osteoblastogenesis. J Bone Miner Res, 26:1188–96. Yazici Z, Baltaci AK, Mogulkoc R, et al. (2011). Effect of boron supplementation on plasma element distribution in ovariectomized rats subjected to acute swimming exercise. Bratisl Lek Listy, 112: 323–6. Yoon KH, Cho DC, Yu SH, et al. (2012). The change of bone metabolism in ovariectomized rats: Analyses of MicroCT scan and biochemical markers of bone turnover. J Korean Neurosurg Soc, 51: 323–7. Yusa K, Yamamoto O, Fukuda M, et al. (2011). In vitro prominent bone regeneration by release zinc ion from Zn-modified implant. Biochem Biophys Res Commun, 412:273–8. Zhao C, Xu N, Zhang W, Zhao C. (2009). Changes of some elements in rat’s tissues except nerve centre with both ovariectomy and chronic aluminium toxication and the effects of estrogen supplement. Wei Sheng Yan Jiu, 38:99–103.
The effect of zinc deficiency and zinc supplementation on element levels in the bone tissue of ovariectomized rats: Histopathologic changes Abdulkerim Kasim Baltaci1 Fusun Sunar1, Rasim Mogulkoc1, Musa Acar2, and Hatice Toy3 1
Selcuk University, Medical Faculty, Department of Physiology, Konya, Turkey, 2Mevlana University, High School of Health, Department of Physiotherapy and Rehabilitation, Konya, Turkey, and 3Necmettin Erbakan University Meram Medical School Department of Pathology, Konya, Turkey Abstract
Keywords
Objective: Study aimed to determine the effects of zinc supplementation/deficiency on the histological structure and elements levels in bone tissue in ovariectomized rats. Methods: The study included 40 Sprague-Dawley type adult female rats, divided as follows: Control, ovariectomized, ovariectomized + zinc supplemented, ovariectomized + zinc deficient groups. At the end of the study bone tissues (femur) were collected to determine the levels of calcium, phosphorus, magnesium, zinc, iron, aluminium, chrome, lithium, lead, nickel, and manganese. The bone tissue was examined for histopathology. Results: Ovariectomy leaded to significant decrease in magnesium. Zinc supplementation to ovariectomized rats restored the reduced calcium, phosphorus, zinc. However, zinc deficiency in ovariectomized rats further reduced calcium, phosphorus, zinc, and manganese levels. Zinc deficiency in ovariectomized significantly increased Al, Cr, Li, Pb, and Ni levels. Tissue integrity was impaired due to ovariectomy and zinc deficiency. Conclusion: Ovariectomy and zinc deficiency leads significant decreases elements of the bone.
Bone, elements, ovariectomy, zinc supplementation and deficiency
Introduction Oestrogen is a required element for the preservation of bone mineral balance (Avila et al., 2009) and its deficiency resulting from ovariectomy leads to osteoporosis (Arslan et al., 2011). Being a major pathological bone condition, osteoporosis causes a serious decline in the quality of life. Decreases that occur in oestrogen levels with ageing constitute the main risk factor for osteoporosis (Yamada et al., 1998). It was established that oestrogen deficiency caused re-structuring of the bone tissue and altered the tissue element distribution (Ames et al., 2010). Likewise, it was reported in a study exploring the effects of ovariectomy on the bone metabolism that ovariectomy increased bone turnover by affecting the levels of osteocalcin and alkaline phosphatase, which are sensitive markers of bone formation, and type I collagen C-terminal (CTX), a bone resorption parameter (Yoon et al., 2012). Zinc is a trace element essential for different metabolic events and cellular signalling pathways that are involved in growth, development and sustenance of life (Oh et al., 2012). Zinc deficiency impairs the bone tissue (Oh et al., 2012). Correspondence: Dr. Abdulkerim Kasim Baltaci, Selcuk University, Medical School, Department of Physiology, Selcuk University, Medical Faculty, Department of Physiology, Konya, 42075 Turkey. E-mail: [email protected]
History Received 12 December 2013 Revised 9 January 2014 Accepted 10 January 2014 Published online 5 February 2014
Zinc is required for the differentiation and growth of osteoblastic cells (Yusa et al., 2011). Similarly, zinc was reported to inhibit osteoclastic bone resorption and stimulate osteoblastic bone formation (Hie et al., 2011). It was seen in a study of male rats that zinc supplementation had a protective effect against femoral fracture in rats exposed to cadmium (Brzo´ska et al., 2007). There has been increasing emphasis on the significance of trace elements in bone formation, development, and structure (Arikan et al., 2011). It was reported in a study involving pregnant rats that zinc deficiency in the mothers impaired bone mineralization and matrix formation in the offspring (Nagata et al., 2011). A review of the relevant literature shows that both oestrogen which originates from the ovary and zinc, an essential element, affect the bone metabolism. The purpose of the present study is to determine the effect of ovariectomy and 6-week zinc deficiency and zinc supplementation on the element levels and histological structure of bone tissue in rats.
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ORIGINAL ARTICLE
Methods The study was conducted upon the approval of the ethics committee of Selcuk University Experimental Medicine and Application Center. The study included 40 Wistar-Albino type female rats whose mean weight was 250 to 260 g. Kept in
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DOI: 10.3109/13813455.2014.884141
Zinc deficiency and supplementation, bone tissue and ovariectomy
rooms with controlled temperature and lighting conditions, all the rats were supplied with water and feed. Group 1 (n ¼ 10): The control group that was not subjected to any procedure. Group 2 (n ¼ 10): The group fed on a normal diet after being ovariectomized under general anaesthesia. Group 3 (n ¼ 10): The group supplemented with intraperitoneal zinc (3 mg/kg/day) for 6 weeks after being ovariectomized under general anaesthesia. Group 4 (n ¼ 10): The group fed on a zinc-deficient diet (0.65 ppm zinc/g diet) for 6 weeks after being ovariectomized under general anaesthesia. In order to minimize zinc contamination, the experimental animals were fed in special steel cages which were cleaned daily by washing. The feed were given in special steel bowls and water in glass feeding bottles. Both zinc-deficient and normal forms of the animal feed were prepared in Korkuteli Feed Supplement Industry Factory. Experimental procedures Zinc sulphate administration The appropriate amount of zinc sulphate was dissolved in distilled water and supplemented by intraperitoneally to the animals. The dose was 3 mg zinc/kg and was given daily at 9 am for 6 weeks. Ovariectomy The rats were injected with 60 mg/kg ketamine and 5 mg/kg rompun to induce general anaesthesia. After the hair on the back of the rats was shaved, proper asepsis and antisepsis were ensured using betadine. After the rats were put into ventral position, the skin was incised at the 1/3 upper point of the distance between the tail and mid-dorsal area. The subcutaneous tissues were released and the spinal muscle was reached. Peritoneal cavity was entered through the back wall muscles of the abdomen. The ovaries were removed together with the fatty tissue. The ovaries were first cleaned off the fatty tissue, and then clamped, tied, and cut. After checking for bleeding, the other organs were put back into the peritoneal cavity. Lastly, the muscle was sutured using a 2/0 chrome catgut and the skin with 2/0 silk (Waynfort & Flecnell, 1994). After the animals were decapitated at the end of the procedures, bone tissue samples were taken to determine the levels of some elements. Analyses of calcium, phosphorus, magnesium, zinc, iron, aluminium, chrome, lithium, lead, nickel, and manganese levels in the bone tissue Samples of bone tissue were put into capped polyethylene tubes washed with NHO3 and de-ionized water to prevent contamination. The tubes were stored at 35 C until the day of analysis. Bone tissue was ground into powder using a mortar. Wet weights of the bone tissue were recorded. Concentrated H2SO4 and HNO3 were added to the samples (gram tissue/ml H2SO4/ml HNO3 ¼ 1/1/10). The samples were then left to wait in a closed system microwave oven (CEM – Marsx5) at 170 psa pressure and 200 C for
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20 minutes. Then, they were added deionized water to obtain the final volume of 25 ml. After a maximum waiting time of half an hour, samples were read. The analyses were conducted using the Atomic Emission (ICP-AES) device found in the Department of Soil Science of SU Faculty of Agriculture (Saygı et al., 1991). Magnesium values in the bone tissue were calculated as mg/g/wet tissue, while the other elements were determined as mg/g/wet tissue. Histological examination Bone tissue samples taken from the animals at the end of the study and were decalcified by formic acid and the tissues were followed autotechnicon. Following autotechnicon processing, samples were buried into paraffin and 5 mm crosssections were obtained using a microtome. The sections were placed on a microscope slide and stained with PAS. Coverslip on a slide and viewed by light microscopy under 40 magnification. Histological results including calcification, inflammation, and sclerotic changes in the bone tissue were examined. When evaluating bone density and morphology, ovariectomized rat bone tissue was based and other groups were compared with this. Osteoclast activity was evaluated by surrounding bone tissue examined (trabecular thinning, disfigurement). Counting the number of osteoclasts in bone adjacent to the five areas were averaged. Statistical evaluations The statistical evaluation of the results was carried out using computer software. Arithmetic means and standard deviations of all parameters were calculated. Variance analyses were used to identify the differences between groups. The least significant difference (LSD) test was employed to compare group means in the statistically significant analysis results. Differences for which p50.05 were accepted significant. The Mann-Whitney U test was used in the statistical analysis of histology results, which were evaluated by comparing median values. Level of significance was set at p50.05.
Results Of the parameters presented in Table 1, calcium and phosphorus values in the bone tissue were found higher in the control group which was not subjected to any procedure (group 1) and zinc-supplemented ovariectomized group (group 3), when compared with groups 2 and 4 (p50.05), with the ovariectomized control group (group 2) having higher calcium and phosphorus values than the ovariectomized group fed on a zinc-deficient diet (p50.05). There was no significant difference among the groups in terms of their bone tissue magnesium levels. Bone tissue zinc levels in groups 1 and 3 were not different relative to each other, but higher than the levels in groups 2 and 4 (p50.05). The lowest bone zinc level was found in group 4 (p50.05). When aluminium values in the bone tissue were examined, this parameter was seen to fall especially after ovariectomy, relative to the control group (p50.05). Zinc supplementation after ovariectomy did not affect aluminium levels in the bone tissue. However, zinc deficiency induced after ovariectomy
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Table 1. Calcium, phosphorus, magnesium, zinc, and iron levels in the bone tissue of study groups.
Groups C Ovx Ovx-Zn S Ovx-Zn D p
Calcium (mg/g/wet tissue)
Phosphorus (mg/g/wet tissue)
Magnesium (mg/g/wet tissue)
Zinc (mg/g/wet tissue)
Iron (mg/g/wet tissue)
92.18 ± 27.87a 62.35 ± 33.69b 91.57 ± 29.60a 58.05 ± 28.18c 0.05
56.07 ± 16.80a 44.74 ± 14.05b 53.42 ± 13.25a 32.27 ± 16.43c 0.05
25.32 ± 13.02 25.94 ± 12.04 25.97 ± 15.19 25.41 ± 13.99
0.11 ± 0.01a 0.07 ± 0.02b 0.11 ± 0.01a 0.05 ± 0.02c 0.05
73.75 ± 32.22a 52.03 ± 11.44b 41.90 ± 14.67b 48.69 ± 14.04b 0.05
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C, control; Ovx-C, ovariectomy; Ovx-Zn S, ovariectomy + zinc supplementation; Ovx-Zn D, ovariectomy + zinc deficiency. a,b,c Means with different superscripted letters in the same column are statistically significant (p50.05).
Table 2. Aluminium, chrome, lithium, lead, nickel, and manganese levels in the bone tissue.
Groups C Ovx Ovx-Zn S Ovx-Zn D p
Aluminium (mg/g/wet tissue)
Chrome (mg/g/wet tissue)
Lithium (mg/g/wet tissue)
Lead (mg/g/wet tissue)
Nickel (mg/g/wet tissue)
Manganese (mg/g/wet tissue)
6.40 ± 3.80a 3.82 ± 1.76b 3.87 ± 1.37b 4.20 ± 2.38b 0.05
2.94 ± 2.76b 1.96 ± 0.75c 1.96 ± 0.75c 3.46 ± 1.29a 0.05
4.82 ± 2.64b 3.83 ± 1.90b 4.26 ± 2.99b 9.10 ± 5.10a 0.05
0.86 ± 0.31b 0.78 ± 0.36b 0.83 ± 0.38b 1.96 ± 0.29a 0.05
0.22 ± 0.13b 0.09 ± 0.10b 0.11 ± 0.08b 0.36 ± 0.13a 0.05
0.24 ± 0.15a 0.12 ± 0.10b 0.14 ± 0.11b 0.07 ± 0.02c 0.05
C, control; Ovx-C, ovariectomy; Ovx-Zn S, ovariectomy + zinc supplementation; Ovx-Zn D, ovariectomy + zinc deficiency. a,b,c Means with different superscripted letters in the same column have statistical significance (p50.05).
elevated aluminium levels in this group, in comparison to the aluminium levels in the ovariectomized and ovariectomized and zinc supplemented groups (p50.05) (Table 2). Bone tissue levels of iron and manganese decreased after ovariectomy. Secondly, manganese decreases in the ovariectomized plus zinc-deficient group (Tables 1 and 2, p50.05). An examination of chrome, lithium, lead, and nickel levels in the bone tissue demonstrated that the levels of these elements dropped, though statistically insignificantly, in ovariectomized and ovariectomized and zinc-supplemented groups, compared with the control group (p50.05). However, these parameters were found markedly higher than the control levels especially in the ovariectomized group in which zinc deficiency was induced (p50.05). Histological results including loss of trabecular mass and altered osteoclastic activity in the bone tissue were evaluated. There was no significant difference between the control group which was not subjected to any procedure (group 1) and ovariectomized and zinc-supplemented group (group 3) in terms of trabecular mass loss, osteoclastic activity, and bone density (Figures 1, 3). However, these values in the ovariectomized control group, group 2 (Figure 2), were higher than their counterparts in groups 1 and 3 (p50.05), and lower than the values in group 4 (Figure 4) (p50.05). Loss of trabecular mass, and changes in osteoclastic activity and bone density were higher in group 4, the group fed on a zinc-deficient diet, than in other groups (p50.05) (Table 3).
Figure 1. Normal rat bone tissue.
Discussion It was reported in a similar study that Ca levels in the serum, though not in the bone tissue, decreased after ovariectomy, and this result is supportive of ours (El-Shitany et al., 2010; Sherman et al., 1989; Ulas & Cay, 2011). Decreased bone tissue calcium levels resulting from zinc deficiency show that zinc deficiency has negatively affected the tissue level of this
Figure 2. Bone tissue of ovariectomized rat (loss of trabecular mass and changes in osteoclastic activity and bone density were higher than their counterparts in groups 1 and 3).
Zinc deficiency and supplementation, bone tissue and ovariectomy
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Figure 3. Bone tissue of ovariectomized and zinc-supplemented rat (not different from Figure 1).
Figure 4. Bone tissue of ovariectomized rat fed on a zinc-deficient diet (increased loss of trabecular mass and changes in osteoclastic activity and bone density).
Table 3. Histological changes in the bone tissue of study groups.
Group Control Ovariectomy control Ovariectomy + zinc supplementation Ovariectomy + zinc deficiency p
Osteoclastic Trabecular activity Bone density mass loss (median value) (median value) (median value) 1.000c 3.000b 1.000c
1.000c 3.000b 1.000c
1.000c 3.000b 1.000c
5.000a
5.000a
5.000a
0.05
0.05
0.05
a,b,c
Means with different superscripted letters in the same column are statistically significant (p50.05).
element. In fact, a similar decrease in serum calcium levels was already reported in a previous study (Sunar et al., 2009). The drop in tissue calcium may have resulted from increased urinary calcium excretion. Actually, there are previous studies reporting increased urinary calcium excretion due to Ovx (Zhao et al., 2009). As oophorectomy enhances bone loss, bone mineral loss results from increased calcium excretion from the gastrointestinal tract and kidney (Cai et al., 2005; O’Loughlin & Morris, 2003). The results also suggest that
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6-week zinc supplementation can reinstate the bone tissue calcium loss resulting from ovariectomy and zinc deficiency. When phosphorus levels in the bone tissue were examined, it was seen that this element was negatively affected by ovariectomy and zinc deficiency. However, these diminished bone tissue phosphorus levels were significantly restored by 6-week zinc supplementation and reached control levels. In line with these results, Avila et al., (2009) reported a decline in phosphorus levels due to ovariectomy. Along the same line, Nagata et al. (2011) found that the offspring of rats fed on a zinc-deficient diet suffered from impaired bone mineralization and highlighted the importance of adequate zinc intake. Re-absorption of phosphorus from the kidney is regulated by the effect of the parathyroid hormone on the sodium-phosphorus co-transporter mechanism, and within this mechanism, oestrogen was reported to be involved in the renal phosphorus retention (Dick & Prince, 2001). In line with our study, there are those stating that ovariectomy lowered phosphorus levels (Sunar et al., 2009; Ulas & Cay, 2011). However, the decrease caused by ovariectomy in phosphorus levels was reversed by zinc supplementation which restored the levels of this element to normal, parallel to the results of previous studies (Sunar et al., 2009). Previous studies show that ovariectomy affected Mg levels in a different way. For instance, Avila et al., (2009) noted that ovariectomy lowered Mg levels. In another study, Mg loss through urine was reported to increase as a result of ovariectomy (Zhao et al., 2009). However, there are also studies demonstrating that there was no ovariectomyassociated change in the Mg levels and our results are in consistency with the results of these latter studies (Sunar et al., 2009; Ulas & Cay, 2011). Thus, in our study neither ovariectomy nor zinc supplementation or deficiency had any effect on magnesium levels in the bone tissue. Concerning the Zn values in our study, it was seen that both ovariectomy and zinc deficiency had a negative impact on the level of this element. Previous studies also reported decreased zinc values after ovariectomy (Ulas & Cay, 2011; Yazici et al., 2011; Zhao et al., 2009). Thus, our results are parallel to the results of previous studies. It was reported in these studies that zinc had a critical effect on the bone metabolism. It was established that zinc altered serum osteocalcin and tissue osteoblastic and osteoclastic activities, all of which are parameters related to the bone metabolism (Hie et al., 2011). In a study involving women with osteoporosis, a positive correlation was found between zinc and lumbar vertebra in the postmenopausal period, and this result emphasizes the importance of zinc in maintaining the normal bone structure (Arikan et al., 2011). Being a critical factor stimulating the differentiation and growth of osteoblasts, zinc is an essential element for protecting the bone structure and sustaining normal bone function. In fact, it was demonstrated in an experimental study that zinc-releasing implants favourably influenced bone fixation (Yusa et al., 2011). In our study, 6-week zinc supplementation significantly restored the zinc reduction caused by both ovariectomy and zinc deficiency. When iron levels in the bone tissue were investigated in our study, it was seen that the level of this element dropped relative to control values after ovariectomy. However, iron
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levels were not significantly affected by zinc supplementation or deficiency. In a previous study, it was shown that zinc supplementation and deficiency together with Fe supplementation affected the bone metabolism and iron supplementation increased the tibia size (Abrisham et al., 2010). However, dietary calcium intake was also demonstrated to affect the iron levels in the bone tissue (tibia) (Behling & Greger, 1998). Besides, the report showing that iron accumulation caused by the cessation of menstruation led to resorption in the bone tissue indicates a crucial relation between bone tissue and iron (Yang et al., 2011). In the present study, the increase brought about by zinc supplementation in the reduced bone tissue iron levels may be considered a positive effect on bone tissue formation. Previous studies showed a relation between Oestrogen and aluminium (Zhao et al., 2009). In fact, the combination of ovariectomy and aluminium toxicity was seen to alter the tissue element distribution in the concerned study. Oestrogen replacement to ovariectomized rats increased aluminium levels in the tibia and urinary aluminium loss. Furthermore, the protective effect of Oestrogen is attested by the study which showed that 3-month aluminium chloride administration to ovariectomized rats caused neurodegeneration, which was restored by Oestrogen treatment (Mohamd et al., 2011). Similar results were also reported by Contini et al. (2011). Interestingly in our study, zinc deficiency induced after ovariectomy caused a certain amount of aluminium accumulation in the bone tissue, and it was thought that the presence of this element might cause a certain degree of toxicity in the bone tissue, which is consistent with the cited studies. An examination of chrome levels in the bone tissue revealed that the level of this element in the tissue dropped after ovariectomy (group 2), but zinc supplementation following ovariectomy did not bring about a further change. However, a significant increase was observed in the bone tissue chrome levels of the group which was fed on a zincdeficient diet after ovariectomy (group 4). In a study including postmenopausal women, it was established that hormone replacement therapy increased chrome levels, while decreasing urinary chrome loss (Bureau et al., 2002). In a similar study, untreated postmenopausal women were shown to have lower chrome levels (Roussel et al., 2002), and this is consistent with our results. In our study, zinc deficiency together with ovariectomy was found to increase bone tissue chrome levels. These results suggest that decreased zinc levels changed the levels of chrome in the concerned tissue. In fact, several studies already demonstrated that zinc had a regulatory effect on the distribution of elements in the serum and tissues (Bicer et al., 2011; Sivrikaya et al., 2012). When the studies exploring ovarian hormones and lithium are considered, it is seen that this element acts upon the decreased Oestrogen receptors in the uterus tissues (Gunin et al., 2004). Elevated lithium levels we found in this study may be important in terms of the toxicity this element causes in the relevant tissue. As a matter of fact, previous studies reported that lithium treatment was associated with impairment in pubertal growth and changes in sex hormone levels (Allagui et al., 2006; Samuel et al., 2012). When the effect of ovariectomy together with zinc supplementation and deficiency on manganese levels in the
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bone tissue was explored, it was established that ovariectomy lowered the levels of this element and zinc supplementation brought about a partial improvement, while zinc deficiency rendered the decrease more marked. It was already suggested that manganese and MnSOD activity were affected by ovariectomy (Pajovic et al., 1993; Rico et al., 2000). It was found in this study that manganese levels in the bone tissue dropped significantly after ovariectomy. Thus, these results are parallel to the results of previous studies (Ulas & Cay, 2011). The reason for this decrease may be increased MnSOD activity. Actually, increased MnSOD activity was reported after ovariectomy (Pajovic et al., 1993). However, the further decrease observed in manganese levels after zinc deficiency suggests that zinc has a regulatory effect on this element as well. With regard to the lead and nickel levels in the bone tissue, these two elements were seen to significantly increase especially in zinc deficiency. Having a toxic character, both of these elements can alter physiological functions. The toxic effects of these metals had already been reported (Levesque et al., 2003; Valko et al., 2005; Ebrahimi & Taherianfard, 2010; Ebrahimi et al., 2014). Thus, accumulation of these metals in the bone tissue due to zinc deficiency indicates an increase in tissue toxicity in the case of zinc deficiency. Bone tissue was also histopathologically assessed in the present study. Results of the histopathologic assessment revealed that ovariectomy resulted in bone tissue loss, which deteriorated with zinc deficiency and was restored by zinc supplementation. Previous studies showed that oral zinc treatment had a positive effect on osteogenesis (Abrisham et al., 2010). It was seen that zinc treatment reduced the risk for fractures due to toxic metals like cadmium in the bone tissue (Brzo´ska et al., 2007). Zinc deficiency, on the other hand, was found to have a negative effect on the biomechanical quality of bone tissue (Scrimgeour et al., 2007). Impairment in the bone tissue due to zinc deficiency and the restoration of this impairment with zinc supplementation in our study are consistent with the results of previous literature studies. An overall assessment of study results indicates that ovariectomy and zinc deficiency in rats have a negative effect on the elements involved in the bone structure and damage the tissue integrity. The second important result of this study is that zinc deficiency can cause toxicity in the tissue by increasing the amounts of some elements such as lead, nickel, aluminium, and chrome.
Declaration of interest Authors declare no conflict of interest.
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DOI: 10.3109/13813455.2014.884141
Zinc deficiency and supplementation, bone tissue and ovariectomy
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