EFFECT OF OVARIECTOMY ON BIOCHEMICAL MARKERS OF BONE TURNOVER (ALP, ACP) AND CALCIUM CONTENT IN RAT MANDIBLE AND TEETH

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1 Bull. Vet. Inst. Pulawy 46, , 2002 EFFECT OF OVARIECTOMY ON BIOCHEMICAL MARKERS OF BONE TURNOVER (ALP, ACP) AND CALCIUM CONTENT IN RAT MANDIBLE AND TEETH MANSUR RAHNAMA AND WOJCIECH ŚWIĄTKOWSKI Department and Clinic of Dental and Maxillofacial Surgery, Medical University of Lublin, Lublin, Poland The aim of this study was to determine the correlation between the level of calcium in the mandible and teeth and the level of alkaline and acid phosphatases in ovariectomized rats with or without supplementation of 17β-estradiol. Rats were qualified for the following groups: Cl control, SH sham - operated, OV ovariectomized, OVH ovariectomized and receiving 17β-estradiol in three different doses (1.25, 12.5, 125 μg) during seven weeks. The negative correlation between level of calcium in the examined tissue and the level of both phosphatases was observed. Key words: rat, bones ovariectomy, 17β-estradiol, alkaline phosphatase, acid phosphatase. Bone metabolic processes are mainly regulated by hormonal milieu. It can influence the activity of a group of enzymes. Changes in the bone tissue can be also activated by bone turnover markers. They have been divided into markers of bone resorption and bone formation (15). Among the bone formation markers we can distinguish serum concentration of alkaline phosphatase (ALP), osteocalcin (OC) and type I collagen. Markers of bone resorption are: hydroxyproline (OHP), hydroxylysine (GHYL/GGHYL), pyridinoline (PYD), deoxypyridinoline (DPD), urine calcium level, acid phosphatase (ACP) and C-terminal telopeptide of collagen of type I (11, 12, 15) In healthy organisms, processes of osseous reconstruction (synthesis of matrix by osteoblasts, mineralisation and resorption) are in an equilibrium. In osteoporosis this balance may be disturbed and it can lead to the increasing of resorption processes. First symptom of these disturbances is osteopenia (diminution of osseous mass). When osseous mass is reduced, the correct structures of skeleton are impossible to maintain and it causes breaks and other clinical symptoms on the background of osteoporosis (12). Previous results showed that postmenopausal osteoporosis (experimental or natural) characterized by hypoestrogenism causes characteristic changes in levels of bone turnover markers (9). Also many in vitro and in vivo studies demonstrated the effect of estrogen on osteoblast proliferation and differentiation. Estrogen has been reported to stimulate, inhibit, or have no effect on cell proliferation. Alkaline

2 282 phosphatase, type I collagen, and osteocalcin gene expression have also been shown to be stimulated or inhibited by estrogen, or was unresponsive to the hormone (5). Also calcium metabolism is very important during postmenopausal osteoporosis. It is widely acknowledged that calcium ions are important for mineralization. However, little is known about the modulation by calcium of cartilage and bone alkaline and acid phoshpatases (10). The aim of this study was to evaluate correlation between calcium level in mandible and teeth and the level of alkaline and acid phosphatases in ovariectomized rats with or without 17β-estradiol supplementation. Material and Methods Young, adult female Wistar rats, weighing g were used. The animals were fed a standard chow and housed in the cages with light-dark cycle and allowed free access to water and diet. After two-week adaptation to the diet and new environment the rats were randomly divided into the following seven groups, 10 animals in each: CL - control group; SH - rats sham operated; OV - rats after bilateral ovariectomy; OVO - rats after bilateral ovariectomy receiving oleum pro injectione; OVH 1 - rats after bilateral ovariectomy taking 17β-estradiol in a dose of 1.25 μg per animal, twice a week, during seven weeks; OVH 2 - rats after bilateral ovariectomy taking 17β-estradiol in a dose of 12.5 μg per animal, twice a week, during seven weeks, and OVH 3 - rats after bilateral ovariectomy taking 17β-estradiol in a dose of 125 μg per animal, twice a week, during seven weeks. Sham operated rats (SH group) were used to determine the influence of operation stress on the level of the examined substances in serum and hard tissues. In OV group the ovaries were removed in general anaesthesia. To examine the influence of the oil base of estradiol oleum pro injectione was applied in OVO group. In OVH 1 - OVH 3 groups Oestradiolum benzoicum (Jelfa Jelenia Góra) was administered intramuscularly. After the end of the experiment rats were anaesthetized by the lethal dose of Tiopenthal. The blood samples, drawn with anticoagulant, were centrifuged immediately and the serum was portioned for the determination of ALP and ACP levels. The samples were kept frozen at -30 C until tests. The level of alkaline phosphatase was determined by the spectrophotochemic method with 450 nm wave length (3, 8) and the level of acid phosphatase was determined by the colorimetric method with 405 nm wave length (7). Tooth and mandible bone samples were mineralized in muffle furnace at C (dry method) and the calcium level was then estimated with a Pye Unicam atomic absorption spectrophotometer with nm wave length (13). The calcium level per unit tooth tissue amount (mg/g tissue) was calculated. Rats incisors were used because these teeth are the best in an experiment. They have a wide apical foramen, spacious root canal and big pulp. They are good for experimental conditions and ensure regular mineral metabolism (1). The data obtained were analyzed by calculating mean (M) and standard deviation (SD). The significance between groups were determined on the basis of confidence intervals (NIR), which were determined from variance analysis (ANOVA).

3 283 Correlations between calcium levels in examined tissues and alkaline and acid phosphatases were determined by r-pearson test. Results Table 1 presents the level of the examined phosphatases (ALP, ACP). In the control group the level of ALP was U/L and in SH group U/L, and differences between these groups were statistically significant. The level of ALP was the maximal in OV group U/L. This result was statistically significant in relation to all groups except OVO group ( U/L). The administration of 17βestradiol decreased the level of the examined phosphatase. It was U/L in OVH 1 group, U/L in OVH 2 group and U/L in OVH 3 group. There were not significant differences between these groups. The level of ACP in CL group was 1.36 U/L and in SH group 1.46 U/L and the differences were not statistically significant. After ovariectomy concentration of ACP increased to 2.50 U/L in OV group and to 2.21 U/L in OVO group. After giving the 17β-estradiol in three different doses, ACP level was gradually decreased. Differences between mean results of OVH 1 -OVH 3 groups and remaining groups were statistically significant. Table 2 presents the level of calcium in the mandible and teeth. In the control group the level of mandible calcium level was mg/g and in SH group mg/g. The differences were statistically significant. After ovariectomy Ca concentration in the mandible was the minimal mg/g. Administration of oleum pro injectione did not significantly influence the Ca mandible level in comparison with OV group. Administration of 17β-estradiol caused an increase in the calcium content from mg/g in OVH 1 group to mg/g in OVH 2, and mg/g in OVH 3 group. Differences between these groups were statistically significant. The level of calcium in teeth in the control group was mg/g and this result was statistically significant in relation to all groups. After bilateral ovariectomy calcium concentration in teeth decreased to mg/g. But, after giving 17βestradiol in three different doses calcium level was gradually increased. Differences between these groups (OVH 1, OVH 2, OVH 3 ) were not statistically significant, but in the comparison with OV group statistical significance was observed. Table 3 presents the correlation between the examined markers of bone turnover (ALP, ACP) and calcium in the mandible and teeth. In all groups statistically significant negative correlation was observed. Only correlation between tooth calcium content and serum ALP was not significant. Discussion Bone loss is the primary risk factor for postmenopausal osteoporosis. It was reported that similar in nature disorder occurred also in ovariectomized rats and was prevented by estradiol treatment suppressing bone cell activity (14). That has stimulated the development of biochemical markers to assist in the assessment of osteoporotic risk and in monitoring the efficiency of treatment. Biochemical markers of bone turnover are products released from osteoblasts and osteoclasts or collagen breakdown products (15).

4 Table 1 Serum ALP and ACP activities (U/L) in the examined groups of rats (P 2 ) * Examined group Number of rats ALP (M±SD) (P 1 ) * ACP (M±SD) CL ±13.07 b 1.36±0.16 a SH ±22.16 a 1.46±0.24 a OV ±42.71 c 2.50±0.08 c OVO ±32.94 ac 2.21±0.49 d OVH ±0.41 d 0.70±0.34 b OVH ±4.07 d 0.62±0.15 be OVH ±2.34 d 0.41±0.07 e * Differences between means are significant when means are not designated with the same letter. Examined group Table 2 The level of the mandible and teeth calcium (mg/g) in the examined groups of rats Number of rats Mandible (M±SD) Teeth (P 1 ) * (M±SD) (P 2 ) * CL ±24.04 d ±9.48 e SH ±21.82 c ±14.25 d OV ±5.19 a ±4.67 a OVO ±2.71 a ±6.70 ab OVH ±9.09 a ±6.08 bc OVH ±22.94 c ±11.05 c OVH ±62.79 b ±16.43 bc * Differences between means are significant when means are not designated with the same letter. 284

5 285 Examined traits ALP Ca Mandible Number of rats Table 3 Correlation between the examined traits Correlation coefficient (r) of differences (P) * Regression equation P<0.006 Ca M =-0.42 * ALP ALP Ca Teeth NS NS ACP Ca Mandible P<0.001 Ca M =-6.46 * ACP ACP Ca Teeth P<0.01 Ca T = * ACP * Assigned correlation coefficients are significant at P<

6 286 The obtained results showed that hypoestrogenism after ovariectomy caused characteristic increase in the bone turnover markers (alkaline and acid phosphatases) in serum of experimental animals. Lack of inhibiting activity of estrogen on osteoclasts caused the increase in ACP activity and in consequence increase in bone resorption. Simultaneously with intensification of resorption processes bone formation process was also increased by enhancement of osteoblast activity. It led to growth of ALP activity. In literature it was noticed that ALP measurements correlate with the rates of bone mineralization (3). Deficiency of estrogen in organism after ovariectomy caused also changes in bone and tooth structures during the experiment. Lower level of calcium in the mandible and incisors was observed. Mineralized tissues play an important role in the metabolism in humans. When the level of ions in plasma decreases these ions are mobilized from reserves in bone and teeth. Hypoestrogenism influence the mineral homeostasis in the body (2). It is characteristic for osteoporotic process. Estrogen prevents from excessive escape of calcium from the organism. In treatment of osteoporosis, it seems to be adequate to use estrogen as well as calcium supplementation (4, 5, 6). The obtained data suggest that there was a correlation between calcium level in the examined tissues (mandible, teeth) and activity of bone turnover markers (ALP and ACP). A significant increase in the investigated enzyme activity and simultaneously decrease in calcium levels in each of the examined tissue were observed after ovariectomy. This state was recovered after the administering of 17βestradiol. Bone, which is a metabolically active tissue, undergoes continuous remodelling, involving bone resorption and formation. Biochemical markers could reliably predict imbalance between bone formation and bone resorption, and therefore, predict the rate of bone loss. If it is possible, it will provide an easy and inexpensive method of identification of those at the risk for developing osteoporosis and bone demineralization (loss of calcium). References 1. Belec Cz., Młochowski J.: Wpływ pitnych wód mineralnych Krynicy na zawartość składników mineralnych w zębach zwierząt doświadczalnych. Czas. Stomat., 1967, 48, Bikle, D.D.: Biochemical markers in the assessment of bone disease. Am. J. Med., 1997, 103, Bowers G.N., McComb R.B.: A continuos spectrophotometric method for measuring the activity of serum alkaline phosphatase. Clin. Chem., 1966, 12, Bushinsky D.A., Monk R.D.: Calcium. Lancet, 1998, 325, Chen F.P., Lee N., Wang K.Ch., Soong Y.K., Huang K.E.: Effect of estrogen and 1α,25(OH) 2 vitamin D 3 on activity and growth of human primary osteoblasts-like cells in vitro. Fertility and Sterility, 2002, 77, Davis J.W., Ross P.D., Johnson N.E., Wasnich R.D.: Estrogen and calcium supplement use among Japanese-American women: effects upon bone loss when used singly or in combination. Bone, 1995, 17,

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