|Year : 2013 | Volume
| Issue : 4 | Page : 344-347
Effect of methylprednisolone on bone mineral density in rats with ovariectomy-induced bone loss and suppressed endogenous adrenaline levels by metyrosine
Mehmet Yilmaz1, Unal Isaoglu2, Turan Uslu3, Kadir Yildirim4, Bedri Seven5, Fatih Akcay6, Ahmet Hacimuftuoglu7
1 Department of Obstetrics and Gynecology, Ataturk University, Faculty of Medicine, Erzurum, Turkey
2 Department of Obstetrics and Gynecology, Nenehatun Obstetrics and Gynecology Hospital, Ministry of Health, Erzurum, Turkey
3 Department of Physical Medicine and Rehabilitation, Fatih Sultan Mehmet Education and Research Hospital, Istanbul, Turkey
4 Department of Physical Medicine and Rehabilitation, Ataturk University, Faculty of Medicine, Erzurum, Turkey
5 Department of Nuclear Medicine, Ataturk University, Faculty of Medicine, Erzurum, Turkey
6 Department of Biochemistry, Ataturk University, Faculty of Medicine, Erzurum, Turkey
7 Department of Pharmacology, Ataturk University, Faculty of Medicine, Erzurum, Turkey
|Date of Submission||08-Jul-2011|
|Date of Decision||16-Aug-2011|
|Date of Acceptance||23-Apr-2013|
|Date of Web Publication||15-Jul-2013|
Department of Obstetrics and Gynecology, Ataturk University, Faculty of Medicine, Erzurum
Source of Support: None, Conflict of Interest: None
Objectives: In this study, effect of methylprednisolone on bone mineral density (BMD) was investigated in rats with overiectomy induced bone lose and suppressed endogenous adrenalin levels, and compared to alendronate.
Materials and Methods: Severity of bone loss in the examined material (femur bones) was evaluated by BMD measurement.
Results: The group with the highest BMD value was metyrosinemetyrosine + methylprednisolone combination (0.151 g/cm 2 ), while that with the lowest BMD was methylprednisolone (0.123 g/cm 2 ). Alendronate was effective only when used alone in ovariectomized rats (0.144 g/cm 2 ), but not when used in combination with methylprednisolone (0.124 g/cm 2 ). In the ovariectomized rat group which received only metyrosine, BMD value was statistically indifferent from ovariectomized control group.
Conclusions: Methylprednisolone protected bone loss in rats with suppressed adrenaline levels because of metyrosinemetyrosine.
Keywords: Bone mineral density, bone loss, metirosine, methylprednisolone, ovariectomy, osteoporosis
|How to cite this article:|
Yilmaz M, Isaoglu U, Uslu T, Yildirim K, Seven B, Akcay F, Hacimuftuoglu A. Effect of methylprednisolone on bone mineral density in rats with ovariectomy-induced bone loss and suppressed endogenous adrenaline levels by metyrosine. Indian J Pharmacol 2013;45:344-7
|How to cite this URL:|
Yilmaz M, Isaoglu U, Uslu T, Yildirim K, Seven B, Akcay F, Hacimuftuoglu A. Effect of methylprednisolone on bone mineral density in rats with ovariectomy-induced bone loss and suppressed endogenous adrenaline levels by metyrosine. Indian J Pharmacol [serial online] 2013 [cited 2020 Jul 6];45:344-7. Available from: http://www.ijp-online.com/text.asp?2013/45/4/344/115008
| » Introduction|| |
Osteoporosis is a chronic bone disease, which is defined by decrease of bone mass per bone volume.  Bone consists of about 70% minerals, 5–8% water, and the rest as matrix. About 95% of the minerals are hydroxyapatite. Bone matrix is largely made up of type 1 collagen and non-collagen proteins.  Hydroxyapatite can contain phosphate group, carbonate or hydroxyl groups, and chlorine and fluorine.  Importance of hydroxyapatite crystals resides in their providing bone hardness and endurance, together with collagens. Inorganic matter (minerals) makes up about 50% of bone dry weight. Despite its high inorganic content, bone is an active tissue.  Besides hydroxyapatite, which composes crystalline structure of bone, there are minerals like sodium, magnesium, calcium, and phosphate, which make up the amorphous (non-crystalline) structure of bone. 
Osteoporosis is defined as a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fracture. Bone strength reflects the integration of two main features, bone density, and bone quality. Bone density is expressed as grams of mineral per area or volume and in any given individual it is determined by peak bone mass and amount of bone loss. Bone mineral density (BMD) measurement is a widely used parameter.  Decrease of BMD in osteoporosis increases the risk of bone fractures. Low BMD is the most important risk factor for bone fracture. 
On the other hand, among other contributing factors to osteoporosis, estrogen deficiency and corticosteroid usage are also important. , Ovariectomy or administration of glucocorticoids is frequently employed to develop a bone loss model in animals. Effect of glucocorticoids decreases the bone production and BMD, and thus increases the bone destruction.  Glucocorticoid usage, therefore, leads to osteoporosis and related fractures.  However, there are studies, which show that toxic effects of glucocorticoids turn into positive effects in animals, whose endogenous adrenaline levels are lowered or completely removed. , However, it is not known if the toxic effects of glucocorticoids on the bone tissue will translate into osteoprotective effects in animals with reduced amount of adrenaline. Metyrosine has been shown to suppress the endogenous adrenaline production by 30-80%. , Therefore, our study aimed to probe the effect of methylprednisolone, a glucocorticoid, on ovariectomy-induced bone loss in rats with the metyrosine-reduced amount of endogenous adrenaline.
| » Materials and Methods|| |
Animals: Fifty-six female albino Wistar rats were obtained from Atatürk University Medical Experimental Application and Research Center. Animals weighed between 220 and 230 g and were kept under normal laboratory conditions in groups (22°C). Twelve-week-old animals were used.
Chemicals: Metyrosine (Demser, Aton Pharma Inc. Lawrenceville, NJ, USA), depomedrol (Eczacýbasi Ýlac San. Istanbul, Turkey), alendronate (Fosamax, Merck-Sharp and Dohme, Whitehouse Station, NJ, USA), and thiopental sodium (Pentotal-Abbott, Illinois, USA) were used.
Fifty-six female, 12-hour starved rats were ovariectomized with Kelly and Robert method. Animals were anesthetized with 25 mg/kg thiopental sodium. Ovariectomized rats were kept and fed under normal laboratory conditions (22°C) for 3 months. Then first group of rats received 20 mg/kg metyrosine, second group 2 mg/kg alendronate per orally (p.o.). Third group received a combination of 20 mg/kg metyrosine (p.o.) and 10 mg/kg methylprednisolone (i.m.). Fourth group received a combination of 2 mg/kg alendronate and 10 mg/kg methylprednisolone (i.m.). Fifth group received 10 mg/kg methylprednisolone (i.m.). Sixth group of ovariectomized rats received equal volume of distilled water. Seventh group of intact animals served as control. Metyrosine and alendronate were administered daily, while methylprednisolone was administered once every 10 days, for 3 months. At the end of 3 months, animals were sacrificed with high-dose anesthesia. Hip bones were removed and sent to Nuclear Medicine Department for BMD determination. Blood samples taken from animals were sent to Biochemistry Department for adrenaline measurements.
Dual Energy X-Ray Absorptiometry (DEXA) Measurements
Bone mineral density (BMD, g/cm 2 ) and bone mineral content (BMC, g) were determined with DEXA method using a Hologic QDR 4500 (Hologic Inc., Waltham, MA, USA) machine and an experimental animal assessment software. Femur bones were placed on the imaging positioning tray and scanned three times. All specimens were placed in a similar orientation for comparison. Total femur bone BMD and BMC was evaluated by inclusion of the whole femur in the region of interest (ROI). All measurements were performed by the same investigator. The coefficients of variations for repeated measurements on the same bone was <1.0%. To ensure DEXA functionality, phantom calibration and quality assurance checks were also conducted prior to specimen scans.
We collected blood samples from the hearts of rats in 5-ml vacutainer tubes and 2-ml EDTA vacuum glass tubes for determination of adrenaline and noradrenaline. The EDTA samples for the adrenaline measurement were immediately placed on ice and centrifuged at 3500 g for 5 min within 15 min of venesection. After centrifugation, the plasma samples were stored for up to 2 h at room temperature. The plasma adrenaline concentration was measured by an isocratic system using a high-performance liquid chromatography (HPLC) pump (model HP1100, Hewlett Packard) (flow rate: 1 ml/min; injection volume: 40 μl; analytical run time: 20 min) and electrochemical detector. We used a reagent kit for HPLC analysis of catecholamines in plasma serum (Chromsystems, Munich, Germany). Intra-assay and inter-assay coefficients of variation were 5 and 6%, respectively, with 70% recovery.
All results were shown as mean ± standard error (SE). One-way analysis of variance was used to evaluate the results. A value of P < 0.05 was considered significant.
| » Results|| |
DEXA Results (BMD Measurement)
As seen in [Table 1], BMD value in ovariectomized control group was 0.133 g/cm 2 , while it was 0.139, 0.144, and 0.123 g/cm 2 in metyrosine, alendronate, and methylprednisolone receiving ovariectomized rat groups, respectively. In ovariectomized groups receiving metyrosine/methylprednisolone and alendronate/methylprednisolone combinations, BMD values were 0.151 and 0.135 g/cm 2 , respectively. BMD value of intact control group was 0.156 g/cm 2.
|Table 1: Bone area (BA), bone mineral content (BMC) and bone mineral density (BMD) in various experimental groups|
Click here to view
Biochemical Results (Adrenaline Levels)
Results are summarized in [Figure 1]. Mean endogenous adrenaline level in healthy intact control group was found to be 665 ± 13.8 ng/dl (P < 0.05). In ovariectomized control group it was 691 ± 19.3 ng/dl. Mean endogenous adrenaline levels were 786 ± 21 (P < 0.05), 448 ± 16.5 (P < 0.001), 323 ± 16.3 (P < 0.0001) ng/dl in ovariectomized methylprednisolone receiving, ovariectomized metyrosine + methylprednisolone receiving, and ovariectomized metyrosine receiving rat group, respectively. Mean adrenaline level was 677 ± 12.7 (P e" 0.05), and 712 ± 14.5 ng/dl (P < 0.05) in ovariectomized alendronate receiving and ovariectomized alendronate + methylprednisolone receiving rat group, respectively.
|Figure 1: Endogenous adrenaline levels (ng/dl) in various experimental groups. Vertical bars represent standard error of mean (*P < 0.05, **P< 0.001, ***P < 0.0001)|
Click here to view
| » Discussion|| |
In this study, we aim to investigate the effects of methylprednisolone, a glucocorticoid, on ovariectomy-induced bone loss in rats with a metyrosine-reduced amount of endogenous adrenaline and compare them with alendronate.
Severity of bone loss in the examined femur bone tissues were evaluated with BMD measurements. Results show that highest BMD value was measured in metyrosine/methylprednisolone combination group, while the lowest BMD was measured in methylprednisolone receiving ovariectomized rats. Alendronate was found to be effective only when used alone in ovariectomized rats, and it could not increase BMD value compared to ovariectomized control group, when used in combination with methylprednisolone. There was a slight, statistically insignificant increase in BMD values of metyrosine receiving ovariectomized rat group.
In a recent study, we showed that gastrotoxic effect of glucocorticoids (prednisolone) was turned into gastroprotective effect in rats with suppressed endogenous adrenaline levels by metyrosine. To concretize whether gastroprotective effect of prednisolone was dependent on inhibition of endogenous adrenaline, we showed that prednisolone completely removed indomethacin-induced stomach ulcer in adrenalectomized rats. In addition, we showed that prednisolone could attach to alpha-2 adrenergic receptors when endogenous adrenaline level was lowered, and produce protective effect through these receptors.  Based on this information, it can be interpreted that methylprednisolone produced a protective effect on BMD via stimulation of alpha-2 adrenergic receptors in rats with metyrosine-induced suppression of endogenous adrenaline (methylprednisolone/metyrosine combination receiving ovariectomized rats group). In our study, we found a significant reduction in the amount of BMD in the femoral bone tissue of rats that underwent ovariectomy. It is known that reduced serum estrogen level is associated with low BMD.  Low levels of BMD in rats with ovariectomy might have increased by estrogen over the alpha-2 adrenergic receptor. This is because it has been reported that estrogen stimulates alpha-2 adrenergic receptors and creates a protective effect around these receptors.  However, it is not clear that stimulation of alpha-2 receptors in which tissue produces protective effect. On the other hand, the estrogen receptors are said to be of two types, alpha (ESRα) and beta (ESRβ). ,, The polymorphism of the ESRα genes is found to have a significant association with BMD.  The results of our study and literature data show that osteoporosis consists of a polyetiologic and complex mechanism. Our study demonstrates that alpha-2 adregenic receptors as well as estrogen receptors may have role in the osteoprotective effect mechanism of methylprednisolone in animals with reduced endogenous adrenaline.
As endogenous adrenaline levels are lowered in metyrosine administered rat group, endogenous cortisol might, at least slightly, stimulate alpha-2 adrenergic receptors and produce this protective effect. Metyrosine is a tyrosine hydroxylase enzyme inhibitor. Inhibition of this enzyme leads to inhibition of adrenaline and other catecholamines.  While metyrosine given to patients with hypercalcemia causes a fall in the amount of catecholamines, it does not prevent hypercalcemia.  It is known that hyperparathyroidism-induced hypercalcemia may result in osteoporosis.  In our study too, metyrosine failed to significantly prevent bone loss. No data is available about the effect of metyrosine alone or in combination with glucocorticoids on the bones. It is postulated that the protective effect of metyrosine on tissues may be due to inhibition of the COX-2 enzyme without affecting COX-1.  It has been reported that the prostaglandin E2 (PGE2) production increased by COX-2 activation, results in the destruction of the bone tissue  and that PGE2s stimulates the osteoclasts responsible for bone destruction. 
It has been reported that prednisolone, a glucocorticoid, significantly inhibits the COX-1 enzymes responsible for cytoprotection and COX-2 enzymes responsible for inflammation in intact rats, it increases COX-1 activity in rats with adrenalectomy (endogenous adrenaline source removed), and strongly suppressed COX-2; it was found that prednisolone produces such effects through the alpha-2 adrenergic receptors.  This suggests that glucocorticoids have protective effect in rats with suppressed production of endogenous adrenaline and that the inhibition of COX-2 is a part of the osteoprotective mechanism.
It has been experimentally demonstrated that different doses of alendronate give rise to positive effects on bone content. ,, In our study, 2 mg/kg dose of alendronate significantly increased BMD in animals with ovariectomy. But, when alendronate was administered with methylprednisolone, no positive effect on BMD was observed. This could be explained as alendronate could not prevent bone loss when osteoporosis was aggravated by methylprednisolone. The mean endogenous adrenaline levels in ovariectomized alendronate receiving rat group and ovariectomized control group were almost the same. Mean endogenous adrenaline level in ovariectomized alendronate + methylprednisolone receiving group was significantly higher than ovariectomized control group. This also indicates that alendronate uses a different mechanism to produce effect, from methylprednisolone + metyrosine combination. Alendronate is reported to increase BMD in osteoporosis patients.  It is a bisphosphonate derivative. Bisphosphonates are known to produce effect by binding to hydroxyapatite and by decreasing the activity and number of osteoclasts. The difference between alendronate and other bisphosphonates is that, alendronate inhibits mevalonic acid pathway, which is necessary for osteoclast survival. 
In conclusion, BMD levels were decreased in ovariectomized rats and this decrease was aggravated by administration of methylprednisolone. Alendronate could only prevent ovariectomy-induced bone loss and not the severe BMD loss observed in methylprednisolone-administered ovariectomized rats. Methylprednisolone prevented the BMD decrease in ovariectomized rats where endogenous adrenaline levels were also suppressed by metyrosine. This finding could be useful in prevention of bone loss in patients who have to be treated with glucocorticoids for a long time and also in treatment of severe bone loss.
| » References|| |
|1.||Ambrus JL, Hoffman M, Ambrus CM, Hreshchyshyn MM, Moore D, Munschauer FE. Prevention and treatment of osteoporosis. One of the most frequent disorders in American women: A review. J Med 1992;23:369-88. |
|2.||Marcus R, Feldman D, Kelsey J. California, San Diego, CA, USA: Osteoporosis California Academic Press; 2001. |
|3.||Cassandra A, Thomas AE. The bone organ system: Form and Function. In: Marcus R, Feldman D, Kelsey J, editors. Osteoporosis: Academic Press; 2001. p. 3-20. |
|4.||Downey PA, Siegel MI. Bone biology and the clinical implications for osteoporosis. Phys Ther 2006;86:77-91. |
|5.||Robinson RA. An electron-microscopic study of the crystalline inorganic component of bone and its relationship to the organic matrix. J Bone Joint Surg Am 1952;34-A:389-435. |
|6.||Compston JF, Rosen C. Bone densitometry in clinical practise. Osteoporosis: Oxford Health Press; 2002. p. 35-41. |
|7.||Andreassen H, Rungby J, Dahlerup JF, Mosekilde L. Inflammatory bowel disease and osteoporosis. Scand J Gastroenterol 1997;32:1247-55. |
|8.||Delmas PD. Treatment of postmenopausal osteoporosis. Lancet 2002;359: 2018-26. |
|9.||Reid DM. Corticosteroid-induced osteoporosis: Guidelines for prevention-are they useful? Br J Rheumatol 1997;36:1035-7. |
|10.||Adachi JD, Olszynski WP, Hanley DA, Hodsman AB, Kendler DL, Siminoski KG, et al. Management of corticosteroid-induced osteoporosis. Semin Arthritis Rheum 2000;29:228-51. |
|11.||Suleyman H, Halici Z, Cadirci E, Hacimuftuoglu A, Keles S, Gocer F. Indirect role of alpha2-adrenoreceptors in anti-ulcer effect mechanism of nimesulide in rats. Naunyn Schmiedebergs Arch Pharmacol 2007;375:189-98. |
|12.||Takeuchi K, Nishiwaki H, Okada M, Niida H, Okabe S. Bilateral adrenalectomy worsens gastric mucosal lesions induced by indomethacin in the rat. Role of enhanced gastric motility. Gastroenterology 1989;97:284-93. |
|13.||Hoffman BB. Therapy of hipertension. Goodman and Gilman's The Pharmacological Basis of Therapeutics. New York: McGraw-Hill; 2006. p. 845-97. |
|14.||Ettinger B, Pressman A, Sklarin P, Bauer DC, Cauley JA, Cummings SR. Associations between low levels of serum estradiol, bone density, and fractures among elderly women: The study of osteoporotic fractures. J Clin Endocrinol Metab 1998;83:2239-43. |
|15.||Borekci B, Kumtepe Y, Karaca M, Halici Z, Cadirci E, Albayrak F, et al. Role of alpha-2 adrenergic receptors in anti-ulcer effect mechanism of estrogen and luteinising hormone on rats. Gynecol Endocrinol 2009;25:264-8. |
|16.||Enmark E, Gustafsson JA. Oestrogen receptors-An overview. J Intern Med 1999;246:133-8. |
|17.||Gruber CJ, Wieser F, Gruber IM, Ferlitsch K, Gruber DM, Huber JC. Current concepts in aesthetic endocrinology. Gynecol Endocrinol 2002;16:431-41. |
|18.||Lunt M, Masaryk P, Scheidt-Nave C, Nijs J, Poor G, Pols H, et al. The effects of lifestyle, dietary dairy intake and diabetes on bone density and vertebral deformity prevalence: The EVOS study. Osteoporos Int 2001;12:688-98. |
|19.||Rizzoli R, Bonjour JP, Ferrari SL. Osteoporosis, genetics and hormones. J Mol Endocrinol 2001;26:79-94. |
|20.||Perretti M, Mugridge KG, Wallace JL, Parente L. Reduction of aspirin-induced gastric damage in rats by interleukin-1 beta: Possible involvement of endogenous corticosteroids. J Pharmacol Exp Ther 1992;261:1238-47. |
|21.||Stewart AF, Hoecker JL, Mallette LE, Segre GV, Amatruda TT Jr, Vignery A. Hypercalcemia in pheochromocytoma. Evidence for a novel mechanism. Ann Intern Med 1985;102:776-9. |
|22.||Mosekilde L. Primary hyperparathyroidism and the skeleton. Clin Endocrinol (Oxf) 2008;69:1-19. |
|23.||Albayrak A, Polat B, Cadirci E, Hacimuftuoglu A, Halici Z, Gulapoglu M, et al. Gastric anti-ulcerative and anti-inflammatory activity of metyrosine in rats. Pharmacol Rep 2010;62:113-9. |
|24.||Morton RS, Dongari-Bagtzoglou AI. Cyclooxygenase-2 is upregulated in inflamed gingival tissues. J Periodontol 2001;72:461-9. |
|25.||Bezerra MM, de Lima V, Alencar VB, Vieira IB, Brito GA, Ribeiro RA, et al. Selective cyclooxygenase-2 inhibition prevents alveolar bone loss in experimental periodontitis in rats. J Periodontol 2000;71:1009-14. |
|26.||Suleyman H, Dursun H, Bilici M, Cadirci E, Halici Z, Gulaboglu M, et al. Relation of adrenergic receptors, which have roles in gastroprotective and anti-inflammatory effect of adrenal gland hormones, with cyclooxygenase enzyme levels in rats. J Physiol Pharmacol 2009;60:129-34. |
|27.||Gasser JA, Ingold P, Venturiere A, Shen V, Green JR. Long-term protective effects of zoledronic acid on cancellous and cortical bone in the ovariectomized rat. J Bone Miner Res 2008;23:544-51. |
|28.||Sliwinski L, Janiec W, Pytlik M, Folwarczna J, Kaczmarczyk-Sedlak I, Pytlik W, et al. Effect of administration of alendronate sodium and retinol on the mechanical properties of the femur in ovariectomized rats. Pol J Pharmacol 2004;56:817-24. |
|29.||Wimalawansa SJ, Simmons DJ. Prevention of corticosteroid-induced bone loss with alendronate. Proc Soc Exp Biol Med 1998;217:162-7. |
|30.||Tiras MB, Noyan V, Yildiz A, Yildirim M, Daya S. Effects of alendronate and hormone replacement therapy, alone or in combination, on bone mass in postmenopausal women with osteoporosis: A prospective, randomized study. Hum Reprod 2000;15:2087-92. |
|31.||Fleisch H. Biphosphonates. In: Marcus R, Friedman D, Kelsey J, editors. Osteoporosis. San Diego: Academic Press; 2001. p. 449-67. |
|This article has been cited by|
||Protective effects of resveratrol on postmenopausal osteoporosis: regulation of SIRT1-NF- B signaling pathway
| ||J. Feng,S. Liu,S. Ma,J. Zhao,W. Zhang,W. Qi,P. Cao,Z. Wang,W. Lei |
| ||Acta Biochimica et Biophysica Sinica. 2014; 46(12): 1024 |
|[Pubmed] | [DOI]|