- Email: [email protected]
The effect of olpadronate in ovariectomized thyroxine-treated rats
Bone Vol. 21, No. 4 October 1997:329-333
ELSEVIER
The Effect of Olpadronate in Ovariectomized Thyroxine-Treated Rats S. Z E N I , C. G O M E Z - A C O T r O ,
and C. M A U T A L E N
Seccifn Osteopatfas Mddicas, Hospital de Cllnicas, "Jos~ de San Martin," Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
findings may have clinical relevance in estrogen-depleted patients to whom medical management other than the reduction of T 4 administration would be desirable. (Bone 21: 329-333; 1997) © 1997 by Elsevier Science Inc. All rights reserved.
Hyperthyroidism increases bone turnover and induces bone loss. This study examines the effect of thyroid hormone excess on two biocbemical markers of bone turnover (hydroxyproline and bone alkaline phosphatase) as well as on bone mineral content (BMC) and bone mineral density (BMD). The possible protective role of dimethyl-APD (olpadronate, OLP), on both suppression of bone turnover and bone mineral loss in ovariectomized (ovx) rats, was also studied. Female Sprague-Dawley rats, were assigned to five groups of eight rats each: sham, ovx, ovx OLP treated (0.3 mg/kg per week), ovx T 4 treated (250 Ixg/kg per day), and ovx T4-OLP rats. Rats were killed after 5 weeks of treatment. At the end of the study, blood samples were analyzed for serum calcium, phosphorus, '1"4,total and bone alkaline phosphatase (ALP and b-ALP), .'rod urinary samples for hydroxyproline/ creatinine ratio (HOProl/creat). Moreover, total BMC, BMD, and scanned area were determined by DXA. Ovx T4-OLPtreated rats presented higher values of b-ALP than ovx T4-treated , ovx, and sham rats (p < 0.05). Ovx increased HOProl/creat excretion compared with sham (p < 0.05), but it was similar cortlpared with ovx T4-treated rats. OLP treatment reduced HOProl/creat excretion in both ovx T 4. treated (p < 0.05) and ovx rats (p < 0.05). The final BMC in ovx was lower than iin the sham group, but the difference was not statistically significant (p < 0.08). The lowest BMC was observed in o v x T 4 rats (p < 0.05). When final BMC was expressed per body weight (BMC/W), ovx rats presented a significantly lower BMC/W than sham rats (p < 0.05). Ovx OLP rats had BMC/W levels higher than ovx (p < 0.005), ovx T 4 (p < 0.01), and ovx T4-OLP rats (p < 0.01). The ovx group had a final BMD lower than sham animals (p < 0.05), but not significantly different than the ovx T 4 rats. BMC and BMD of OLP ovx rats, whether they received T 4 or not, was similar to the sham group. The highest final BMD was observed in the ovx T4-OLP group. In summary, the prevention of an increase in HOProl excretion accompanied by the fact that final BMD and BMC in OLP-treated animals were comparable to sham control rats may reflect that OLP administration could inhibit bone resorption in both TI-treated or -untreated rats. Although further studies are necessary, these
Key Words: BMC; BMD; Bone turnover; Olpadronate; Ovariectomized rats; Thyroxine. Introduction Since 1891 when von Recklinghausen 36 recognized the association of thyrotoxicosis and bone loss, several studies have documented the adverse effects of prolonged untreated hyperthyroidism on bone and mineral metabolism. The influence of thyroid hormone excess on bone is multifactorial. Bone histomorphometry in hyperthyroidism reveals activation of both osteoblast and osteoclast activities 14"23 resulting finally in a decrease of bone mass. 8'2°-22 An important clinical problem could be the association of both hyperthyroidism and estrogen deficiency, because the latter is the main cause of bone mass reduction in women during the early stages of the postmenopausal period. 3 Ovariectomized (ovx) rats have been widely used as an animal model for postmenopausal osteoporosis in humans to study the mechanism of bone loss, as well as to evaluate new potential therapies before their possible use in humans. 4° However, there are very few studies on skeletal changes following thyroid hormone administration in ovx rats, assessed densitometrically and biochemically. 43 On this basis, it seems relevant to examine the effect of bone resorption inhibitors in ovx animals receiving long-term thyroid hormones. Bisphosphonates are potent inhibitors of bone resorption, ll'le'2° although their effects on bone formation are complex and not well established. 15 They have been used widely to treat various disorders characterized by an increase in bone resorption both in animals 42 and humans. 4"12'13A9'35 Furthermore, several animal studies have also shown the efficacy of bisphosphonates in the prevention of experimentally induced bone Ioss. 31'33'34 The aim of this study was to clarify the effect of excess thyroid hormone in estrogen deficiency on two biochemical markers of bone turnover (hydroxyproline and bone alkaline phosphatase) and on bone mass as well as to evaluate the possible protective role of 3-dimethylamino-l-hydroxypropylidene-l,1 bisphosphonate, or dimethyl-APD (olpadronate, or OLP), a new potent bisphosphonate, on both suppression of bone turnover and on bone mineral density (BMD).
Address for correspondence and reprints: Susana N. Zeni, Secci6n Osteopatfas Mfdicas, Hospital de Clfnicas, Cordoba 2351, piso 8, 1120Buenos Aires, Argentina. E-mail: [email protected] This research was presented in part at the annual meeting of the American Society of Bone and Mineral Research, Baltimore, Maryland, 1995.
© 1997 by Elsevier ScienceInc. All rights reserved.
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S. Zeni et al. Effect of olpadronate in ovx T4-treated rats
Bone Vol. 21, No. 4 October 1997:329-333
Table 1. Weight measurements and serum biochemical analysis (mean ÷ SEM)
Sham Ovx Ovx-T4 treated Ovx-OLP treated Ovx-T4-OLP treated
Weight gain (g)
Serum T4 (mg %)
41.8 81.1 58.8 66.5 71.1
5.2 5.2 15.1 4.9 13.4
_+ 4.5 ± 5.8 a ± 4.1 a'b - 5.0a _+ 4.2 a'c
± ± ± ± ±
0.3 0.4 2.4a 0.6 2.1 a
Serum Ca (mg %) 10.1 10.2 9.7 9.9 9.6
± 0.1 + 0.1 ± 0.1 a'b -+ 0.1 +_ 0.2 a'b
Serum P (mg %) 6.7 6.9 7.3 7.1 7.2
± 0.1 _+ 0.2 + 0.1 a't' _+ 0.1 ± 0.2
ap < 0.05, compared to sham; bp < 0.05, compared to ovx; Cp < 0.05, compared to ovx-T4 treated.
Materials and Methods
Materials L - T h y r o x i n e (T4) was kindly provided by Glaxo (Buenos Aires)
and olpadronate (OLP) by Gador SA (Buenos Aires). The doses of L-thyroxine and olpadronate were based on previous experimental dose-response studies. 2'28'42 Rats were purchased from IBYME (Buenos Aires) and acclimated to conditions for 2 weeks before experimentation.
Experimental Design A total of 40 female Sprague-Dawley virgin rats, approximately 7 months of age, with an average body weight of 220 -+ 2 g, were assigned to five groups of eight rats each with similar average weights. Rats were housed at the room temperature of 21 -+ 1°C with a humidity of 55 +- 10% and 12 h light/dark cycles. They were fed a commercial formula (Purina, Buenos Aires) and allowed access to water ad libitum. Rats were maintained according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Bilateral ovariectomies were performed in 32 rats by a dorsal approach (ovx groups), and the remaining 8 rats were subjected to a sham operation (sham group). Rats were assigned to the experimental groups with the following four treatment regimes:
Vehicle (sham) and vehicle (ovx): These rats were injected intraperitoneally (ip) 6 days/week with vehicle (0.001N NaOH/0.9% NaC1). c-Thyroxine (ovx T4 treated): Each ovx rat in this group was injected ip with 250 jxg of T4/kg rat/day, 6 days per week. Olpadronate (ovx OLP treated): Each ovx rat in this group was injected ip with 0.30 mg of olpadronate/kg rat, once per week. L-Thyroxine-olpadronate (ovx T4-OLP treated): Each ovx rat in this group was injected ip with both T 4 and olpadronate, as described previously in the L-thyroxine and olpadronate groups. All treatments were initiated 2 days after surgery and performed for a 5 week period. Body weight was recorded once per week. All rats were killed by exsanguination under anesthesia. Blood was obtained by cardiac puncture and serum was kept frozen at - 2 0 ° C for biochemical analysis. The day before killing, rats were kept in metabolic cages to collect urine samples. Urine was kept frozen at - 2 0 ° C until analysis was performed. The effectiveness of ovx was confirmed at the end of the experimental period by visualizing the atrophy of the uterine horns and the absence of ovarian tissue.
CA) using commercial kits with an intraassay coefficient of variation (CV) of 3.3%. Serum calcium (Ca) was measured by atomic absorptiometry using LaC13 as an interference suppressor. Serum phosphorus (P) was measured by the colorimetric assay. The methods used in our laboratory have been described previously. 26 Total alkaline phosphatase (ALP) was measured using a commercial kit (Boehringer, Mannheim, Germany). Bone alkaline phosphatase isoenzyme (b-ALP) was assayed after a selective wheat germ lectin precipitation. 9 Urinary hydroxyproline (HOProl/creat) was determined by the colorimetric method of Firscbein and Shill. 1° Urinary creatinine was measured by the colorimetric method of Jaffe.
DXAMeasuremen~ At the end of the experiment, body BMC and body BMD were determined with a total body scanner using specifically designed software for small animals (DPX Alpha 8034, Small Animal Software, Lunar Radiation Corp., Madison, WI). This scanner uses a constant potential X-ray source at 76 kVp, and the effective energies are 38 and 70 keV. The collimation (size of X-ray beam at the source) used was 0.84 mm. The size of each sample point during the scan was 0.6 × 1.2 mm. With the DXA scanner, a series of transverse scans were made from the top of the nose to the end of the tail at 1.2 m m intervals with a set scan area of 8 x 25 cm for each rat. Scans were performed at a transverse scan speed of 9.6 mm/sec, giving a scan time of 50 min for the total body determination. All rats were scanned using the identical scan procedure. Precision of the software on total body was assessed by measuring five times with repositioning between scans on the same and different days. The CV was expressed as a percentage standard deviation (SD) of the mean. The CV was 3.0% for total body BMC, 0.9% for total body BMD, and 1.5% for body weight.
Statistical Analysis Data are expressed as mean _+ standard error of the mean (mean _+ SEM). Comparison between pairs of variables was assessed by Student's t-test when appropriate. Differences between treatment groups were tested by one-way analysis of variance (ANOVA) using the STATVIEwT M (Macintosh) statistical package. If significant differences were indicated by ANOVA, comparisons between group means were tested by Fisher's test (95% significance level). Results
Body Weight Biochemical Determinations Serum T 4 w a s measured by radioimmunoassay (RIA total T 4 Ab-coated tube; DPC, Diagnostic Products Corp., Los Angeles,
At baseline the average weight of each group was similar (220 -+ 2 g). As expected, ovx animals gained more weight during the study period than sham rats (19 < 0.05). Among ovx animals,
Bone Vol. 21, No. 4 October 1997:329-333
S. Zeni et al. Effect of olpadronate in ovx T4-treated rats
Table 2. Biochemical markers of bone formation ALP (U/L) Sham Ovx Ovx T 4 treated Ovx OLP treated Ovx T4-OLP treated
52.3 ± 49.6 ± 56.5 ± 79.0 ± fi9.0±
B-ALP (U/L)
6.3 4.8 5.2 5.1 ~'bx 5.9
25.4 27.0 26.9 29.2 33.0
-± 2.8 ± 0.8 ± 1.2 ± 2.9 --- 2.4~'bx
HOProl/creat (mg/mg) 0.13 0.28 0.23 0.12 0.14
± 0.02 ± 0.01 a ± 0.02 a - 0.00 b ___0.02 b'c
(ALP: total alkaline phosphatase, B-ALP: bone alkaline phosphatase) and bone resorption (HOProl/Creat: total hydroxyproline-creatinine ratio) (mean - SEM). ap < 0.05, compared to sham; bp < 0.05, compared to ovx; ~p < 0.05, compared to ovx T 4 treated.
neither T 4 nor olpa&onate treatment impaired weight gain (Table 1). However, ovx Ta-treated animals gained less weight than ovx rats (p < 0.05) and than ovx Ta-OLP rats (p < 0.05).
Biochemical Analysis Serum T 4 concentrations were 2.5-3.0 times higher in the T 4t r e a t e d rats than in T4-untreated rats (Table 1).
Ovx rats presented the highest serum Ca level (10.2 ± 0.1 mg%), with no significant differences from sham rats (10.1 ± 0.1 mg%) (Table 1). Ovx T 4 rats (with or without OLP) had a serum Ca level significantly lower than ovx animals (p < 0.05). Sham and ovx rar.s had similar serum P levels that, in tum, were lower than those of ovx T 4 animals (p < 0.05) (Table 1). Olpadronate treatment increased total ALP in both T4-treated and -untreated anim~ds (Table 2). Ovx OLP animals presented the highest values and were statistically different than ovx (p < 0.05) and o v x T 4 rats (p < 0.05). Olpadronate also increased b-ALP (Table 2). Ovx Ta-OLP animals presented higher values than o v x T 4 (p < 0.05), than OVX (p < 0.05), and than sham rats (p < 0.05). As shown in Table 2, ovx produced a significant increase in the HOProl/creat excretion compared to sham control animals (p < 0.05). HOProl/creat was similar in ovx Ta-treated animals and ovx rats. OLP 'Lreatment reduced the mean HOProl/creat excretion in o v x T 4 treated (p < 0.05) and ovx rats (p < 0.05) to sham levels (Table 2).
331
they were treated with T 4. Furthermore, the ovx T4-OLP group had a greater BMC than ovx T 4 rats (p < 0.05). When final BMC was expressed per body weight (BMC/W), ovx rats presented a lower value than the sham group (p < 0.05). Ovx T 4 showed a lower BMC/W than sham rats (p < 0.01), but not statistically different from the ovx group (Table 3). Ovx OLP had a higher BMC/W than ovx (p < 0.05), than ovx T 4 (p < 0.01), and than ovx T4-OLP (p < 0.01), respectively, but comparable with the sham group. Ovx OLP-T 4 rats had a lower BMC/W than sham animals (p < 0.05), but not different than ovx or ovx T 4 rats. Ovx rats presented a similar scanned area than sham control animals (23.6 ± 1.2 vs. 23.0 ± 1.2 cm2). The ovx T 4 group had the lowest area (p < 0.05), and presented significant differences when compared with sham, ovx, and ovx OLP rats. Ovx OLP-T 4 rats had a scanned area comparable to all other groups studied, but it was lower when compared with ovx OLP rats (p < 0.05).
Bone Mineral Density Ovx rats had significantly lower BMD than sham rats (p < 0.05). T 4 t r e a t m e n t did not impair BMD in ovx rats, and an unexpectedly similar BMD between ovx T 4 rats and ovx rats was observed (Table 3). Ovx OLP and ovx Ta-OLP rats had a significantly higher mean BMD than ovx rats (p < 0.05) and o v x T 4 rats (p < 0.05), respectively. On the other hand, the highest BMD was observed in ovx T4-OLP rats. The BMD of this group was even significantly higher than that observed in the sham control group (p < 0.05).
Discussion The slight excesses of thyroid hormone that result in a marked imbalance between resorption and formation appear to be more evident in postmenopausal women. 5 Ovariectomized rats are the most frequently used animal model of postmenopausal bone loss, 16 because the skeletal effect of estrogen depletion as well as estrogen replacement are very similar in both rats and women. 7"17'18"27'32'37"38 On these bases we studied the thyroid-hormone-induced biochemical and densitometric bone changes in ovx rats treated or not with thyroxine, OLP, or both.
Ovariectomy Bone Mineral Content and Scanned Area At the end of the study, ovx rats presented a lower BMC than sham animals, but the difference was not statistically significant (673 --- 38 vs. 607 -+ 37 mg, p < 0.08). Among all groups studied, ovx T 4 rats showed the lowest BMC levels (480 ± 44 mg) (Table 3). It is important to point out that there were no significant differences between the OLP groups, whether or not
As expected, in the present study, the body weight of our ad libitum-fed ovx rats increased significantly vs. sham animals. Although serum Ca, P, and ALP were unchanged, ovx increased bone resorption. Urinary HOProl excretion was increased at least twofold following ovariectomy, whereas the increase of b-ALP did not achieve statistical significance. Similarly, Kalu et al. 17 found that urinary HOProl excretion increased following ovx.
Table 3. DXA measurements Body weight (g) Sham Ovx OVX T 4 treated Ovx OLP treated Ovx T 4 0 L P treated
289 324 268 302 324
± 14 ± 11 -+ 12b -- 10c ± 16c
BMC (mg) 673 607 480 703 636
+ 38 _+ 37 _+ 44 a'b ± 36c ± 57c
BMC/W (mg/g) 23.6 ± 18.6 ± 17.6 ± 23.3 ± 20.0 ±
2.2 0.8a 1.4a 1.0b'c'd 1.l a
Area (cm 2)
23.6 ± 1.2 23.0 ± 1.2 18.3 ± 1.8a'b 25.6 ± 1.3c'd 20.0 +- 1.7
BMD (mg/cm2) 279 264 260 277 319
± ± ± ± ±
3 3a 2~ 2 b'c'd
13a'b'c
Bone mineral content (BMC), BMC per body weight (BMC/W), area and bone mineral density (BMD) (mean ± SEM). ap < 0.05, compared to sham; bp < 0.05, compared to ovx; Cp < 0.05, compared to ovx T4 treated; d p < 0.05, compared to ovx T4-OLP treated.
332
S. Zeni et al. Effect of olpadronate in ovx T4-treated rats
Bone loss associated with increased bone turnover is a wellknown consequence of ovx. 4° In the present study, ovx rats had final BMD and BMC normalized by body weight (BMC/W) significantly lower than control sham rats. Bone growth can be assessed by measuring the scanned area. According to our findings, and in agreement with previous studies by Amman et al.,1 ovariectomy would not affect scanned area. T4 Treatment The high serum T 4 levels and the diminished body weight gain of the ovx Ta-treated rats confirm they were significantly hyperthyroid. Although within the normal range, serum Ca was significantly lower and serum P was significantly higher in o v x T 4 rats than sham and ovx rats. Previous clinical studies by Mosekilde and Christensen2t and Charles et al. 6 showed high serum phosphorus levels in hyperthyroid. Moreover, there are several experimental studies showing that thyroid hormones can increase serum P concentration in animals. 24 It has been reported that serum P is much more variable than serum Ca concentration in hyperthyroidism due to an increase in tubular resorption of p.24 Thyroxine administration, in the dose employed in this study, did not cause further increase in HOProl or b-ALP compared with ovx rats. Our findings agree with a previous histomorphometric study by Yamanra et al. 43 who showed that thyroxine administration did not modify the increased osteoclast surface and osteoid surface showed after ovx. Furthermore, it seems reasonable that the greatly increased bone tumover that occurs during the first 5 weeks after ovx may mask any effects of T 4 treatment on biochemical measurements. The effects of T 4 may become more evident when turnover decreases. It is important to point out that BMD behaved in much the same way as HOProl excretion: it was lower in the ovx group when compared to sham control rats, but T 4 administration to ovx rats did not cause any further change in the final BMD of the total skeleton. However, we have recently found that T 4 t r e a t m e n t caused a greater decrease in tibia, femora, and spine BMD when compared to ovx-untreated animals (Zeni et al., unpublished). A substantially lower final BMC and scanned area was verified in ovx T4-treated rats when compared with the control sham group. The simultaneous change of BMC and area in ovx T 4 rats might explain the nonsignificant variation of BMD (BMC/area) when compared with ovx rats (Table 3). OLP Treatment Previous experimental studies in female rats showed that etidronate and risedronate prevented bone loss induced by ovx. 39'41 In the present report, we examined the protective effect of a new potent bisphosphonate, olpadronate, in estrogen-deficient rats treated or not with T 4. Olpadronate treatment in ovx rats did not impair body weight gain and did not modify serum Ca and P levels. Although the total ALP levels were increased, the levels remained within the normal range for the age of our studied group. However, the possibility of an increase in the liver or intestinal fraction of ALP should be investigated because b-ALP did not rise significantly. There was a decrease in the HOProl excretion to sham control levels. These results are consistent with a decrease in bone resorption after olpadronate treatment. DXA measurements in ovx OLP rats revealed BMD, BMC, and area values comparable to sham control animals, suggesting that olpadronate could prevent the bone loss caused by ovariectomy. It is not yet fully known whether cotherapy with antiresorptive agents would prevent thyroxine-induced bone loss, especially in postmenopausal women. Recently, Rosen et al. 29 found
Bone Vol. 21, No. 4 October 1997:329-333 that HOProl excretion rose after patients received thyroid hormone and fell after treatment with APD. In experimental studies, bone loss induced in male rats by thyroid hormone excess was also prevented by pamidronate, 3° etidronate, 24 or alendronate. 2 In agreement with findings in our hyperthyroid estrogen-depleted rats, olpadronate treatment also could prevent bone resorption, according to the final BMD and BMC values of the ovx Ta-OLP group when compared with control sham rats. Moreover, surprisingly, the highest BMD was observed in this ovx Tn-OLPtreated group and was significantly higher than in sham and ovx OLP groups. This finding could be explained by taking into account that specific inhibitors of bone resorption do not immediately depress bone formation, and continue filling the existing remodeling sites for some time. This phenomenon of apparent temporal uncoupling between formation and resorption results in a temporary gain in bone mass, depending on the initial rate of bone turnover. 11 Recognizing differences in thyroid-hormone-treated humans and the rat model of hyperthyroidism, further clinical studies are necessary to determine if olpadronate could ameliorate the adverse effect of excess thyroid hormone on bone mass in estrogendepleted women. In summary, the prevention of the increase in HOProl excretion accompanied by the fact that final BMD and BMC in OLP-treated animals were comparable to sham control rats may reflect that OLP administration could inhibit bone resorption in both T4-treated or -untreated rats. Although further studies are necessary, these findings may have clinical relevance in estrogen-depleted patients to whom medical management other than the reduction of T 4 administration would be desirable.
Acknowledgments: The authors are grateful to Cristina Arakelian and Maria del Carmen Degrandi for their technical assistance and to Diana Gonzalez for her review and helpful comments. This research was supported in part by the Fundaci6n Argentina de Osteologia.
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cross-linked N-telopeptides of type I collagen as a marker of bone turnover. Calcif Tissue Int 54:26-29; 1994. Rosen, H., Sullivan, E., Middlebrooks, V., Zeind, A., Gundberg, C., DresnerPollak, R., Maitland, L., Hock, J., Moses, A., and Greenspan, S. Parenteral pamidronate prevents thyroid hormone-induce bone loss in rats. J Bone Miner Res 8:1255-1261; 1993. Seedor, J. G., Quartuccio, H., and Thompson, D. D. The bisphosphonate alendronate (MK-217) inhibits bone loss due to ovariectomy in rats. J Bone Miner Res 6:339-346; 1991. Stepan, J. J., Pospichal, J., Presl, J., and Pacovsky, V. Bone loss and biochemical indices of bone remodeling in surgically-induced postmenopausal women. Bone 8:279-284; 1987. Thompson, D. D., Seedor, J. G., Quartuccio, H., Solomon, H., Fiolavanti, C., Davidson, J., Klein, H., Clair, J., Franskenfield, D., Brown, E., Simmons, H. A., and Rodan, G. A. The bisphosphonate, alendronate, prevents bone loss in ovariectomized baboons. J Bone Miner Res 7:951-960; 1992. Thompson, D. D., Seedor, J. G., Weinreb, M., Rosini, S., and Redan, G. A. Aminohydroxybutane bisphosphonate inhibits bone loss due to immobilization in rats. J Bone Miner Res 5:279-286; 1990. Valkena, R., Vismans, F., Papapoulos, S. E., Pauwels, E. K., and Bijvoet, O. L. Maintained improvement in calcium balance and bone mineral content in patients with osteoporosis treated with the bisphosphonate APD. Bone Miner 5:183-192; 1989. von Recklinghausen, F. Die fibrose oder deformierende Ostitis, die Osteomalazie and die Osteoplastiche Carzinose, in ihren gegenseitigen Biziehungen. In: Festschrift Rudolph Virchow. Berlin: Reiner; 1891; 20-89. Wronski, T. J., Cintron, M., and Dann, L. M. Temporal relationship between bone loss and increased bone turnover in ovariectomized rats. Calcif Tissue Int 43:179-183; 1988. Wronski, T. J., Cintron, M., Doherty, A. L., and Dann, L. M. Estrogen treatment prevents osteopenia and depresses bone turnover in ovariectomized rats. Endocrinology 123:681-686; 1988. Wronski, T. J., Dann, L. M., Qi, H., and Yen, C.-F. Skeletal effects of withdrawal of estrogen and disphosphonate treatment in ovariectomized rats. Calcif Tissue Int 53:210-216; 1993. Wronski, T. J., Lowry, P. L., Walsh, C. C., and Ignaszewki, L. A. Skeletal alterations in ovariectomized rats. Calcif Tissue Int 37:324-328; 1985. Wronski, T. J., Yen, C. F., and Scott, K. S. Estrogen and diphosphonate treatment provide long-term protection against osteopenia in ovariectomized rats. J Bone Miner Res 6:387-394; 1991. Yamamoto, M., Markatos, A., Seedor, J. G., Masarachia, P., Gentile, M., Rodan, G., and Balena, R. The effects of the aminobisphosphonate Alendronate on thyroid hormone-induced osteopenia in rats. Calcif Tissue Int 53:278-282; 1993. Yamaura, M., Nakamura, T., Kanou, A., Miura, T., Ohara, H., and Suzuki, K. The effect of 1713-estradiol treatment on the mass and the turnover of bone in ovariectomized rats taking a mild dose of thyroxin. Bone Miner 24:33-42; 1994.
Date Received: May 14, 1996 Date Revised: June 13, 1997 Date Accepted: June 13, 1997
ELSEVIER
The Effect of Olpadronate in Ovariectomized Thyroxine-Treated Rats S. Z E N I , C. G O M E Z - A C O T r O ,
and C. M A U T A L E N
Seccifn Osteopatfas Mddicas, Hospital de Cllnicas, "Jos~ de San Martin," Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
findings may have clinical relevance in estrogen-depleted patients to whom medical management other than the reduction of T 4 administration would be desirable. (Bone 21: 329-333; 1997) © 1997 by Elsevier Science Inc. All rights reserved.
Hyperthyroidism increases bone turnover and induces bone loss. This study examines the effect of thyroid hormone excess on two biocbemical markers of bone turnover (hydroxyproline and bone alkaline phosphatase) as well as on bone mineral content (BMC) and bone mineral density (BMD). The possible protective role of dimethyl-APD (olpadronate, OLP), on both suppression of bone turnover and bone mineral loss in ovariectomized (ovx) rats, was also studied. Female Sprague-Dawley rats, were assigned to five groups of eight rats each: sham, ovx, ovx OLP treated (0.3 mg/kg per week), ovx T 4 treated (250 Ixg/kg per day), and ovx T4-OLP rats. Rats were killed after 5 weeks of treatment. At the end of the study, blood samples were analyzed for serum calcium, phosphorus, '1"4,total and bone alkaline phosphatase (ALP and b-ALP), .'rod urinary samples for hydroxyproline/ creatinine ratio (HOProl/creat). Moreover, total BMC, BMD, and scanned area were determined by DXA. Ovx T4-OLPtreated rats presented higher values of b-ALP than ovx T4-treated , ovx, and sham rats (p < 0.05). Ovx increased HOProl/creat excretion compared with sham (p < 0.05), but it was similar cortlpared with ovx T4-treated rats. OLP treatment reduced HOProl/creat excretion in both ovx T 4. treated (p < 0.05) and ovx rats (p < 0.05). The final BMC in ovx was lower than iin the sham group, but the difference was not statistically significant (p < 0.08). The lowest BMC was observed in o v x T 4 rats (p < 0.05). When final BMC was expressed per body weight (BMC/W), ovx rats presented a significantly lower BMC/W than sham rats (p < 0.05). Ovx OLP rats had BMC/W levels higher than ovx (p < 0.005), ovx T 4 (p < 0.01), and ovx T4-OLP rats (p < 0.01). The ovx group had a final BMD lower than sham animals (p < 0.05), but not significantly different than the ovx T 4 rats. BMC and BMD of OLP ovx rats, whether they received T 4 or not, was similar to the sham group. The highest final BMD was observed in the ovx T4-OLP group. In summary, the prevention of an increase in HOProl excretion accompanied by the fact that final BMD and BMC in OLP-treated animals were comparable to sham control rats may reflect that OLP administration could inhibit bone resorption in both TI-treated or -untreated rats. Although further studies are necessary, these
Key Words: BMC; BMD; Bone turnover; Olpadronate; Ovariectomized rats; Thyroxine. Introduction Since 1891 when von Recklinghausen 36 recognized the association of thyrotoxicosis and bone loss, several studies have documented the adverse effects of prolonged untreated hyperthyroidism on bone and mineral metabolism. The influence of thyroid hormone excess on bone is multifactorial. Bone histomorphometry in hyperthyroidism reveals activation of both osteoblast and osteoclast activities 14"23 resulting finally in a decrease of bone mass. 8'2°-22 An important clinical problem could be the association of both hyperthyroidism and estrogen deficiency, because the latter is the main cause of bone mass reduction in women during the early stages of the postmenopausal period. 3 Ovariectomized (ovx) rats have been widely used as an animal model for postmenopausal osteoporosis in humans to study the mechanism of bone loss, as well as to evaluate new potential therapies before their possible use in humans. 4° However, there are very few studies on skeletal changes following thyroid hormone administration in ovx rats, assessed densitometrically and biochemically. 43 On this basis, it seems relevant to examine the effect of bone resorption inhibitors in ovx animals receiving long-term thyroid hormones. Bisphosphonates are potent inhibitors of bone resorption, ll'le'2° although their effects on bone formation are complex and not well established. 15 They have been used widely to treat various disorders characterized by an increase in bone resorption both in animals 42 and humans. 4"12'13A9'35 Furthermore, several animal studies have also shown the efficacy of bisphosphonates in the prevention of experimentally induced bone Ioss. 31'33'34 The aim of this study was to clarify the effect of excess thyroid hormone in estrogen deficiency on two biochemical markers of bone turnover (hydroxyproline and bone alkaline phosphatase) and on bone mass as well as to evaluate the possible protective role of 3-dimethylamino-l-hydroxypropylidene-l,1 bisphosphonate, or dimethyl-APD (olpadronate, or OLP), a new potent bisphosphonate, on both suppression of bone turnover and on bone mineral density (BMD).
Address for correspondence and reprints: Susana N. Zeni, Secci6n Osteopatfas Mfdicas, Hospital de Clfnicas, Cordoba 2351, piso 8, 1120Buenos Aires, Argentina. E-mail: [email protected] This research was presented in part at the annual meeting of the American Society of Bone and Mineral Research, Baltimore, Maryland, 1995.
© 1997 by Elsevier ScienceInc. All rights reserved.
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S. Zeni et al. Effect of olpadronate in ovx T4-treated rats
Bone Vol. 21, No. 4 October 1997:329-333
Table 1. Weight measurements and serum biochemical analysis (mean ÷ SEM)
Sham Ovx Ovx-T4 treated Ovx-OLP treated Ovx-T4-OLP treated
Weight gain (g)
Serum T4 (mg %)
41.8 81.1 58.8 66.5 71.1
5.2 5.2 15.1 4.9 13.4
_+ 4.5 ± 5.8 a ± 4.1 a'b - 5.0a _+ 4.2 a'c
± ± ± ± ±
0.3 0.4 2.4a 0.6 2.1 a
Serum Ca (mg %) 10.1 10.2 9.7 9.9 9.6
± 0.1 + 0.1 ± 0.1 a'b -+ 0.1 +_ 0.2 a'b
Serum P (mg %) 6.7 6.9 7.3 7.1 7.2
± 0.1 _+ 0.2 + 0.1 a't' _+ 0.1 ± 0.2
ap < 0.05, compared to sham; bp < 0.05, compared to ovx; Cp < 0.05, compared to ovx-T4 treated.
Materials and Methods
Materials L - T h y r o x i n e (T4) was kindly provided by Glaxo (Buenos Aires)
and olpadronate (OLP) by Gador SA (Buenos Aires). The doses of L-thyroxine and olpadronate were based on previous experimental dose-response studies. 2'28'42 Rats were purchased from IBYME (Buenos Aires) and acclimated to conditions for 2 weeks before experimentation.
Experimental Design A total of 40 female Sprague-Dawley virgin rats, approximately 7 months of age, with an average body weight of 220 -+ 2 g, were assigned to five groups of eight rats each with similar average weights. Rats were housed at the room temperature of 21 -+ 1°C with a humidity of 55 +- 10% and 12 h light/dark cycles. They were fed a commercial formula (Purina, Buenos Aires) and allowed access to water ad libitum. Rats were maintained according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Bilateral ovariectomies were performed in 32 rats by a dorsal approach (ovx groups), and the remaining 8 rats were subjected to a sham operation (sham group). Rats were assigned to the experimental groups with the following four treatment regimes:
Vehicle (sham) and vehicle (ovx): These rats were injected intraperitoneally (ip) 6 days/week with vehicle (0.001N NaOH/0.9% NaC1). c-Thyroxine (ovx T4 treated): Each ovx rat in this group was injected ip with 250 jxg of T4/kg rat/day, 6 days per week. Olpadronate (ovx OLP treated): Each ovx rat in this group was injected ip with 0.30 mg of olpadronate/kg rat, once per week. L-Thyroxine-olpadronate (ovx T4-OLP treated): Each ovx rat in this group was injected ip with both T 4 and olpadronate, as described previously in the L-thyroxine and olpadronate groups. All treatments were initiated 2 days after surgery and performed for a 5 week period. Body weight was recorded once per week. All rats were killed by exsanguination under anesthesia. Blood was obtained by cardiac puncture and serum was kept frozen at - 2 0 ° C for biochemical analysis. The day before killing, rats were kept in metabolic cages to collect urine samples. Urine was kept frozen at - 2 0 ° C until analysis was performed. The effectiveness of ovx was confirmed at the end of the experimental period by visualizing the atrophy of the uterine horns and the absence of ovarian tissue.
CA) using commercial kits with an intraassay coefficient of variation (CV) of 3.3%. Serum calcium (Ca) was measured by atomic absorptiometry using LaC13 as an interference suppressor. Serum phosphorus (P) was measured by the colorimetric assay. The methods used in our laboratory have been described previously. 26 Total alkaline phosphatase (ALP) was measured using a commercial kit (Boehringer, Mannheim, Germany). Bone alkaline phosphatase isoenzyme (b-ALP) was assayed after a selective wheat germ lectin precipitation. 9 Urinary hydroxyproline (HOProl/creat) was determined by the colorimetric method of Firscbein and Shill. 1° Urinary creatinine was measured by the colorimetric method of Jaffe.
DXAMeasuremen~ At the end of the experiment, body BMC and body BMD were determined with a total body scanner using specifically designed software for small animals (DPX Alpha 8034, Small Animal Software, Lunar Radiation Corp., Madison, WI). This scanner uses a constant potential X-ray source at 76 kVp, and the effective energies are 38 and 70 keV. The collimation (size of X-ray beam at the source) used was 0.84 mm. The size of each sample point during the scan was 0.6 × 1.2 mm. With the DXA scanner, a series of transverse scans were made from the top of the nose to the end of the tail at 1.2 m m intervals with a set scan area of 8 x 25 cm for each rat. Scans were performed at a transverse scan speed of 9.6 mm/sec, giving a scan time of 50 min for the total body determination. All rats were scanned using the identical scan procedure. Precision of the software on total body was assessed by measuring five times with repositioning between scans on the same and different days. The CV was expressed as a percentage standard deviation (SD) of the mean. The CV was 3.0% for total body BMC, 0.9% for total body BMD, and 1.5% for body weight.
Statistical Analysis Data are expressed as mean _+ standard error of the mean (mean _+ SEM). Comparison between pairs of variables was assessed by Student's t-test when appropriate. Differences between treatment groups were tested by one-way analysis of variance (ANOVA) using the STATVIEwT M (Macintosh) statistical package. If significant differences were indicated by ANOVA, comparisons between group means were tested by Fisher's test (95% significance level). Results
Body Weight Biochemical Determinations Serum T 4 w a s measured by radioimmunoassay (RIA total T 4 Ab-coated tube; DPC, Diagnostic Products Corp., Los Angeles,
At baseline the average weight of each group was similar (220 -+ 2 g). As expected, ovx animals gained more weight during the study period than sham rats (19 < 0.05). Among ovx animals,
Bone Vol. 21, No. 4 October 1997:329-333
S. Zeni et al. Effect of olpadronate in ovx T4-treated rats
Table 2. Biochemical markers of bone formation ALP (U/L) Sham Ovx Ovx T 4 treated Ovx OLP treated Ovx T4-OLP treated
52.3 ± 49.6 ± 56.5 ± 79.0 ± fi9.0±
B-ALP (U/L)
6.3 4.8 5.2 5.1 ~'bx 5.9
25.4 27.0 26.9 29.2 33.0
-± 2.8 ± 0.8 ± 1.2 ± 2.9 --- 2.4~'bx
HOProl/creat (mg/mg) 0.13 0.28 0.23 0.12 0.14
± 0.02 ± 0.01 a ± 0.02 a - 0.00 b ___0.02 b'c
(ALP: total alkaline phosphatase, B-ALP: bone alkaline phosphatase) and bone resorption (HOProl/Creat: total hydroxyproline-creatinine ratio) (mean - SEM). ap < 0.05, compared to sham; bp < 0.05, compared to ovx; ~p < 0.05, compared to ovx T 4 treated.
neither T 4 nor olpa&onate treatment impaired weight gain (Table 1). However, ovx Ta-treated animals gained less weight than ovx rats (p < 0.05) and than ovx Ta-OLP rats (p < 0.05).
Biochemical Analysis Serum T 4 concentrations were 2.5-3.0 times higher in the T 4t r e a t e d rats than in T4-untreated rats (Table 1).
Ovx rats presented the highest serum Ca level (10.2 ± 0.1 mg%), with no significant differences from sham rats (10.1 ± 0.1 mg%) (Table 1). Ovx T 4 rats (with or without OLP) had a serum Ca level significantly lower than ovx animals (p < 0.05). Sham and ovx rar.s had similar serum P levels that, in tum, were lower than those of ovx T 4 animals (p < 0.05) (Table 1). Olpadronate treatment increased total ALP in both T4-treated and -untreated anim~ds (Table 2). Ovx OLP animals presented the highest values and were statistically different than ovx (p < 0.05) and o v x T 4 rats (p < 0.05). Olpadronate also increased b-ALP (Table 2). Ovx Ta-OLP animals presented higher values than o v x T 4 (p < 0.05), than OVX (p < 0.05), and than sham rats (p < 0.05). As shown in Table 2, ovx produced a significant increase in the HOProl/creat excretion compared to sham control animals (p < 0.05). HOProl/creat was similar in ovx Ta-treated animals and ovx rats. OLP 'Lreatment reduced the mean HOProl/creat excretion in o v x T 4 treated (p < 0.05) and ovx rats (p < 0.05) to sham levels (Table 2).
331
they were treated with T 4. Furthermore, the ovx T4-OLP group had a greater BMC than ovx T 4 rats (p < 0.05). When final BMC was expressed per body weight (BMC/W), ovx rats presented a lower value than the sham group (p < 0.05). Ovx T 4 showed a lower BMC/W than sham rats (p < 0.01), but not statistically different from the ovx group (Table 3). Ovx OLP had a higher BMC/W than ovx (p < 0.05), than ovx T 4 (p < 0.01), and than ovx T4-OLP (p < 0.01), respectively, but comparable with the sham group. Ovx OLP-T 4 rats had a lower BMC/W than sham animals (p < 0.05), but not different than ovx or ovx T 4 rats. Ovx rats presented a similar scanned area than sham control animals (23.6 ± 1.2 vs. 23.0 ± 1.2 cm2). The ovx T 4 group had the lowest area (p < 0.05), and presented significant differences when compared with sham, ovx, and ovx OLP rats. Ovx OLP-T 4 rats had a scanned area comparable to all other groups studied, but it was lower when compared with ovx OLP rats (p < 0.05).
Bone Mineral Density Ovx rats had significantly lower BMD than sham rats (p < 0.05). T 4 t r e a t m e n t did not impair BMD in ovx rats, and an unexpectedly similar BMD between ovx T 4 rats and ovx rats was observed (Table 3). Ovx OLP and ovx Ta-OLP rats had a significantly higher mean BMD than ovx rats (p < 0.05) and o v x T 4 rats (p < 0.05), respectively. On the other hand, the highest BMD was observed in ovx T4-OLP rats. The BMD of this group was even significantly higher than that observed in the sham control group (p < 0.05).
Discussion The slight excesses of thyroid hormone that result in a marked imbalance between resorption and formation appear to be more evident in postmenopausal women. 5 Ovariectomized rats are the most frequently used animal model of postmenopausal bone loss, 16 because the skeletal effect of estrogen depletion as well as estrogen replacement are very similar in both rats and women. 7"17'18"27'32'37"38 On these bases we studied the thyroid-hormone-induced biochemical and densitometric bone changes in ovx rats treated or not with thyroxine, OLP, or both.
Ovariectomy Bone Mineral Content and Scanned Area At the end of the study, ovx rats presented a lower BMC than sham animals, but the difference was not statistically significant (673 --- 38 vs. 607 -+ 37 mg, p < 0.08). Among all groups studied, ovx T 4 rats showed the lowest BMC levels (480 ± 44 mg) (Table 3). It is important to point out that there were no significant differences between the OLP groups, whether or not
As expected, in the present study, the body weight of our ad libitum-fed ovx rats increased significantly vs. sham animals. Although serum Ca, P, and ALP were unchanged, ovx increased bone resorption. Urinary HOProl excretion was increased at least twofold following ovariectomy, whereas the increase of b-ALP did not achieve statistical significance. Similarly, Kalu et al. 17 found that urinary HOProl excretion increased following ovx.
Table 3. DXA measurements Body weight (g) Sham Ovx OVX T 4 treated Ovx OLP treated Ovx T 4 0 L P treated
289 324 268 302 324
± 14 ± 11 -+ 12b -- 10c ± 16c
BMC (mg) 673 607 480 703 636
+ 38 _+ 37 _+ 44 a'b ± 36c ± 57c
BMC/W (mg/g) 23.6 ± 18.6 ± 17.6 ± 23.3 ± 20.0 ±
2.2 0.8a 1.4a 1.0b'c'd 1.l a
Area (cm 2)
23.6 ± 1.2 23.0 ± 1.2 18.3 ± 1.8a'b 25.6 ± 1.3c'd 20.0 +- 1.7
BMD (mg/cm2) 279 264 260 277 319
± ± ± ± ±
3 3a 2~ 2 b'c'd
13a'b'c
Bone mineral content (BMC), BMC per body weight (BMC/W), area and bone mineral density (BMD) (mean ± SEM). ap < 0.05, compared to sham; bp < 0.05, compared to ovx; Cp < 0.05, compared to ovx T4 treated; d p < 0.05, compared to ovx T4-OLP treated.
332
S. Zeni et al. Effect of olpadronate in ovx T4-treated rats
Bone loss associated with increased bone turnover is a wellknown consequence of ovx. 4° In the present study, ovx rats had final BMD and BMC normalized by body weight (BMC/W) significantly lower than control sham rats. Bone growth can be assessed by measuring the scanned area. According to our findings, and in agreement with previous studies by Amman et al.,1 ovariectomy would not affect scanned area. T4 Treatment The high serum T 4 levels and the diminished body weight gain of the ovx Ta-treated rats confirm they were significantly hyperthyroid. Although within the normal range, serum Ca was significantly lower and serum P was significantly higher in o v x T 4 rats than sham and ovx rats. Previous clinical studies by Mosekilde and Christensen2t and Charles et al. 6 showed high serum phosphorus levels in hyperthyroid. Moreover, there are several experimental studies showing that thyroid hormones can increase serum P concentration in animals. 24 It has been reported that serum P is much more variable than serum Ca concentration in hyperthyroidism due to an increase in tubular resorption of p.24 Thyroxine administration, in the dose employed in this study, did not cause further increase in HOProl or b-ALP compared with ovx rats. Our findings agree with a previous histomorphometric study by Yamanra et al. 43 who showed that thyroxine administration did not modify the increased osteoclast surface and osteoid surface showed after ovx. Furthermore, it seems reasonable that the greatly increased bone tumover that occurs during the first 5 weeks after ovx may mask any effects of T 4 treatment on biochemical measurements. The effects of T 4 may become more evident when turnover decreases. It is important to point out that BMD behaved in much the same way as HOProl excretion: it was lower in the ovx group when compared to sham control rats, but T 4 administration to ovx rats did not cause any further change in the final BMD of the total skeleton. However, we have recently found that T 4 t r e a t m e n t caused a greater decrease in tibia, femora, and spine BMD when compared to ovx-untreated animals (Zeni et al., unpublished). A substantially lower final BMC and scanned area was verified in ovx T4-treated rats when compared with the control sham group. The simultaneous change of BMC and area in ovx T 4 rats might explain the nonsignificant variation of BMD (BMC/area) when compared with ovx rats (Table 3). OLP Treatment Previous experimental studies in female rats showed that etidronate and risedronate prevented bone loss induced by ovx. 39'41 In the present report, we examined the protective effect of a new potent bisphosphonate, olpadronate, in estrogen-deficient rats treated or not with T 4. Olpadronate treatment in ovx rats did not impair body weight gain and did not modify serum Ca and P levels. Although the total ALP levels were increased, the levels remained within the normal range for the age of our studied group. However, the possibility of an increase in the liver or intestinal fraction of ALP should be investigated because b-ALP did not rise significantly. There was a decrease in the HOProl excretion to sham control levels. These results are consistent with a decrease in bone resorption after olpadronate treatment. DXA measurements in ovx OLP rats revealed BMD, BMC, and area values comparable to sham control animals, suggesting that olpadronate could prevent the bone loss caused by ovariectomy. It is not yet fully known whether cotherapy with antiresorptive agents would prevent thyroxine-induced bone loss, especially in postmenopausal women. Recently, Rosen et al. 29 found
Bone Vol. 21, No. 4 October 1997:329-333 that HOProl excretion rose after patients received thyroid hormone and fell after treatment with APD. In experimental studies, bone loss induced in male rats by thyroid hormone excess was also prevented by pamidronate, 3° etidronate, 24 or alendronate. 2 In agreement with findings in our hyperthyroid estrogen-depleted rats, olpadronate treatment also could prevent bone resorption, according to the final BMD and BMC values of the ovx Ta-OLP group when compared with control sham rats. Moreover, surprisingly, the highest BMD was observed in this ovx Tn-OLPtreated group and was significantly higher than in sham and ovx OLP groups. This finding could be explained by taking into account that specific inhibitors of bone resorption do not immediately depress bone formation, and continue filling the existing remodeling sites for some time. This phenomenon of apparent temporal uncoupling between formation and resorption results in a temporary gain in bone mass, depending on the initial rate of bone turnover. 11 Recognizing differences in thyroid-hormone-treated humans and the rat model of hyperthyroidism, further clinical studies are necessary to determine if olpadronate could ameliorate the adverse effect of excess thyroid hormone on bone mass in estrogendepleted women. In summary, the prevention of the increase in HOProl excretion accompanied by the fact that final BMD and BMC in OLP-treated animals were comparable to sham control rats may reflect that OLP administration could inhibit bone resorption in both T4-treated or -untreated rats. Although further studies are necessary, these findings may have clinical relevance in estrogen-depleted patients to whom medical management other than the reduction of T 4 administration would be desirable.
Acknowledgments: The authors are grateful to Cristina Arakelian and Maria del Carmen Degrandi for their technical assistance and to Diana Gonzalez for her review and helpful comments. This research was supported in part by the Fundaci6n Argentina de Osteologia.
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Date Received: May 14, 1996 Date Revised: June 13, 1997 Date Accepted: June 13, 1997