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Print version ISSN 0482-5004
Rev. Bras. Reumatol. vol.52 no.3 São Paulo May/June 2012
Christiano Robles Rodrigues AlvesI; Igor Hisashi MuraiI; Pamella RamonaII; Humberto NicastroIII; Lilian TakayamaIV; Fabiana GuimarãesV; Antonio Herbert Lancha JuniorVI; Maria Claudia IrigoyenVII; Rosa Maria Rodrigues PereiraVIII; Bruno GualanoIX
IGraduation student of Physical Education, Escola de Educação Física e Esporte, Universidade de São Paulo - EEFE/USP
IIMaster's degree candidate in Experimental Pathophysiology, Instituto do Coração, Hospital das Clínicas, Medical School, Universidade de São Paulo - InCor-HCFM/USP
IIIPhD candidate in Physical Education, EEFE/USP
IVSpecialist of the Bone Metabolism Laboratory, FMUSP
VMaster's degree candidate in Sciences, EEFE/USP
VIPhD in Experimental Nutrition, Pharmaceutical Sciences School - FCM/USP; Full Professor, EEFE/USP
VIIPhD in Cardiovascular Physiology, USP; Professor of the Department of Cardiopneumology FMUSP
VIIIPhD in Rheumatology, FMUSP; Post-PhD in Cellular and Molecular Biology Applied to the Bone Cell, Connecticut University; Associate Professor of the Division of Rheumatology, FMUSP
IXPhD, Professor of the Human Movement Biodynamics Department, EEFE/USP
INTRODUCTION: Recent evidence has suggested that creatine supplementation (Cr) can increase the bone mineral density (BMD) of the femur in healthy growing rats. Nevertheless, studies assessing the efficacy of the Cr supplementation in conditions characterized by bone mass loss are scarce.
OBJECTIVE: To investigate the effect of Cr supplementation on BMD and bone mineral content (BMC) in spontaneously hypertensive rats (SHRs), an experimental model of osteoporosis.
MATERIALS AND METHODS: Sixteen 8-month-old male SHRs were randomly allocated into two groups matched by body weight: 1) Pl group: SHRs treated with placebo (distilled water; n = 8); and 2) Cr group: SHRs treated with Cr (n = 8). After nine weeks of supplementation, the animals were euthanized and their femur and spine (L1-L4) were analyzed by use of densitometry (Dual Energy X-Ray Absorptiometry).
RESULTS: No significant difference was observed between the groups regarding either the spine or the total femur measures as follows: spine - BMD (Pl = 0.249 ± 0.003 g/cm2 vs. Cr = 0.249 ± 0.004 g/cm2; P = 0.95) and BMC (Pl = 0.509 ± 0.150 g vs. Cr = 0.509 ± 0.017 g; P > 0.99); and total femur - BMD (Pl = 0.210 ± 0.004 g/cm2 vs. Cr = 0.206 ± 0.004 g/cm2; P > 0.49) and BMC (Pl = 0.407 ± 0.021 g vs. Cr = 0.385 ± 0.021 g; P > 0.46).
CONCLUSION: In this study, using the experimental model of osteoporosis, Cr supplementation had no effect on bone mass.
Keywords: osteoporosis, creatine, bone mineral density.
The spontaneously hypertensive rat (SHR) has been considered the arterial hypertension genetic model most similar to the primary hypertension observed in humans.1,2 There is evidence of a reduction in bone mineral density (BMD) in that model, resulting from changes in calcium metabolism, which cause an increase in bone resorption.3-5 While the bone mass of healthy Sprague Dawley rats peaks around the age of 30-36 weeks, SHRs stabilize their bone growth at the age of only 18 weeks. In addition, the peak bone mass in SHRs is approximately 40 mg/cm2 lower than that in healthy rats.6 Thus, SHRs are considered an experimental model for the study of osteoporosis.7
The energy need of bone cells to survive, proliferate, differentiate, and synthesize extracellular matrix is known to be high.8 Evidence has shown that part of the energy required for those processes originates from creatine (Cr; α-methyl guanidine-acetic acid), which plays a central role in maintaining ATP and ADP levels in several tissues, such as skeletal muscle, brain, testicles, cartilage, and bone (for a recent and comprehensive review, see Wallimann et al.9).
The hypothesis that Cr could play an important role in bone metabolism was first suggested based on the identification of creatine kinase isoforms (CK), enzyme responsible for the reversible reaction as follow: phosphocreatine + ADP + H+ creatine + ATP in the bone.9,10 In addition, in vitro assays have shown that stimuli that can induce the development of bone mass (i.e., insulin growth factor-1 and parathyroid hormone) concomitantly increase CK activity, suggesting that the Cr/CK system is associated with the process of bone remodeling.11,12 In fact, a study has shown that incubating Cr in a culture medium with primary osteoblasts has stimulating effects on the differentiation, metabolic activity, and bone mineralization, elevating the phosphorylcreatine/Cr ratio and preserving the ultrastructure and mitochondrial function of osteoblasts.8
In vivo evidence13,14 has corroborated those findings. Supplementation with Cr can increase the BMD and cause beneficial biomechanical adaptations in the femur of healthy rats.15 In humans, there is preliminary evidence that the Cr supplementation can prevent bone mass loss in patients with Duchenne dystrophy14 and in the elderly undergoing physical training.16
However, the role of the dietary Cr supplementation remains little explored. Aiming at increasing the understanding about the effects of Cr on possible bone mass loss conditions, the present study assessed the effect of that nutrient on the BMD and bone mineral content (BMC) of SHRs.
MATERIALS AND METHODS
The sample comprised 16 8-month-old male SHRs, which were maintained at the animal facility of the Laboratory of Nutrition and Metabolism Applied to Motor Activity (Escola de Educação Física e Esporte of the Universidade de São Paulo - EEFE/USP) in plastic cages (three to four animals per cage), at room temperature of 22.0 ºC-24.0 ºC and 12-hour cycle (inverted light and dark). The rats were fed a normal protein diet (12% of protein) and water ad libitum.
The animals were randomized into two experimental groups matched by body weight, as follows: 1) Pl group: SHRs treated with placebo (n = 8); and 2) Cr group: SHRs treated with Cr (n = 8). After nine weeks of intervention the animals were
The procedures were approved by the Ethics Committee in Research of the EEFE/USP (protocol 2011/10).
The Cr group received, through gavage, Cr supplement daily (Ethika, Ribeirão Preto, SP, Brazil) for nine weeks. Creatine powder was diluted in water (room temperature) at the proportion of 200 g for each liter of water, and the dosage used was 5 g/kg weight/day.13 The animals were weighted daily for the required corrections. The Pl group received distilled water through gavage to simulate the stress imposed to the Cr group.
Bone densitometry (Dual Energy X-Ray Absorptiometry; DXA) was used to assess the BMD and BMC of the spine (L1-L4) and total femur (total femur length, including diaphysis and epiphyses). The device Discovery-A SN: 80999 Hologic (Bedford, MA, USA) in the high resolution mode was used, with the aid of the small animal software, provided by the same manufacturer. The accuracy of the DXA for assessing BMD was previously analyzed by measuring the coefficient of variation, expressed as a percentage of the mean.17,18 The coefficient of variation was 1.9% for the spine and 0.6% for the total femur. Together, those data indicate high accuracy of measures.
Data were expressed as mean ± standard deviation. The nonpaired t test was used to compare BMD and BMC of the groups and two-way ANOVA to assess body weight every week. The significance level adopted to reject the null hypothesis was P < 0.05.
Only one rat (Pl) died during follow-up. The body weight did not significantly differ between both groups during the study (P = 0.48; Figure 1).
No significant difference was observed between the groups regarding either the spine or the total femur measures as follows: spine - BMD (Pl = 0.249 ± 0.003 g/cm2 vs. Cr = 0.249 ± 0.004 g/cm2; P = 0.95; Figure 2A), and BMC (Pl = 0.509 ± 0.150 g vs. Cr = 0.509 ± 0.017 g; P > 0.99; Figure 2B); and total femur - BMD (Pl = 0.210 ± 0.004 g/cm2 vs. Cr = 0.206 ± 0.004 g/cm2; P > 0.49; Figure 3A), and BMC (Pl = 0.407 ± 0.021 g vs. Cr = 0.385 ± 0.021 g; P > 0.46; Figure 3B).
This study aimed at assessing the influence of Cr supplementation in the bone mass of SHRs, a well-described experimental model to study low bone mass.3-5 Our results are not in accordance with those of Antolic et al.,15 who have reported beneficial effects of Cr supplementation on the bone mass of Sprague Dawley rats. Some methodological differences can explain the contradictory results. Antolic et al.15 have reported benefits with Cr supplementation to growing rats, while the present study used adult rats. The process of bone growth and development is characterized by high bone turnover, a period more susceptible to environmental influences on bone mass.19 The gains with Cr supplementation might have been intensified in that phase. In addition, it is worth emphasizing that the SHR model is known to have high bone resorption, and, thus, low bone mass. On the other hand, the Sprague Dawley model studied by Antolic et al.15 has no change in bone metabolism. Based on the differences of the experimental models, one can speculate that Cr supplementation might be more effective in potentiating bone mass gain in healthy growing rats than in attenuating bone mass loss in rats undergoing bone mass loss.
This study has some limitations. The tissue capture of Cr, to guarantee the success of supplementation, was not assessed. However, the dosage used in this study (5 g/kg weight/day) has been considered high in the literature20,21 and effective to increase the musculoskeletal content of Cr in Wistar rats.22 Future studies have to assess whether the Cr supplementation can also increase Cr concentrations in bone tissue. Finally, it is worth emphasizing that our study assessed only male rats. Knowing that gender is a factor that influences directly the bone mass response,22 and, considering the impossibility of generalizing those data for both genders, further studies should also assess the therapeutic potential of Cr in bone mass remodeling in females.
Despite the evidence that Cr supplementation can promote important therapeutic effects, such as bone mass increase,23 our findings indicate that SHRs supplemented with Cr do not experience such gains. Considering the evident difficulty in carrying out large longitudinal clinical assays, other models characterized by bone mass loss (for example, polycystic rats or rats treated with corticoids) should be investigated to more deeply assess the therapeutic potential of Cr on the preservation of BMD in low bone mass conditions. However, it is worth emphasizing that the Cr metabolism seems to differ substantially between species, and, thus, studies in humans should be conducted to confirm all preclinical findings.
1. Trippodo NC, Frohlich ED. Similarities of genetic (spontaneous) hypertension. Man and rat. Circ Res 1981;48(3):309-19. [ Links ]
2. Bing OH, Brooks WW, Robinson KG, Slawsky MT, Hayes JA, Litwin SE et al. The spontaneously hypertensive rat as a model of the transition from compensated left ventricular hypertrophy to failure. J Mol Cell Cardiol 1995;27(1):383-96. [ Links ]
3. Bastos MF, Brilhante FV, Bezerra JP, Silva CA, Duarte PM. Trabecular bone area and bone healing in spontaneously hypertensive rats: a histometric study. Braz Oral Res 2010;24(2):170-6. [ Links ]
4. Wright GL, DeMoss D. Evidence for dramatically increased bone turnover in spontaneously hypertensive rats. Metabolism 2000; 49(9):1130-3. [ Links ]
5. Metz JA, Karanja N, Young EW, Morris CD, McCarron DA. Bone mineral density in spontaneous hypertension: differential effects of dietary calcium and sodium. Am J Med Sci 1990;300(4):225-30. [ Links ]
6. Inoue T, Moriya A, Goto K, Tanaka T, Inazu M. What is the difference of bone growth in SHR and SD rats? Clin Exp Pharmacol Physiol Suppl 1995;22(1):S242-3. [ Links ]
7. Yamori Y, Fukuda S, Tsuchikura S, Ikeda K, Nara Y, Horie R. Stroke-prone SHR (SHRSP) as a model for osteoporosis. Clin Exp Hypertens A 1991;13(5):755-62. [ Links ]
8. Gerber I, ap Gwynn I, Alini M, Wallimann T. Stimulatory effects of creatine on metabolic activity, differentiation and mineralization of primary osteoblast-like cells in monolayer and micromass cell cultures. Eur Cell Mat 2005;10:8-22. [ Links ]
9. Wallimann T, Tokarska-Schlattner M, Schlattner U. The creatine kinase system and pleiotropic effects of creatine. Amino Acids 2011;40(5):1271-96. [ Links ]
10. Wallimann T, Hemmer W. Creatine kinase in non-muscle tissues and cells. Mol Cell Biochem 1994; 133-134:193-220. [ Links ]
11. Sömjen D, Kaye AM, Rodan GA, Binderman I. Regulation of creatine kinase activity in rat osteogenic sarcoma cell clones by parathyroid hormone, prostaglandin E2, and vitamin D metabolites. Calcif Tissue Int 1985;37(6):635-8. [ Links ]
12. Sömjen D, Kaye AM. Stimulation by insulin-like growth factor-I of creatine kinase activity in skeletal-derived cells and tissues of male and female rats. J Endocrinol 1994;143(2):251-9. [ Links ]
13. Chilibeck PD, Chrusch MJ, Chad KE, Shawn Davison K, Burke DG. Creatine monohydrate and resistance training increase bone mineral content and density in older men. J Nutr Health Aging 2005;9(5):352-3. [ Links ]
14. Louis M, Lebacq J, Poortmans JR, Belpaire-Dethiou MC, Devogelaer JP, Van Hecke P et al. Beneficial effects of creatine supplementation in dystrophic patients. Muscle Nerve 2003;27(5):604-10. [ Links ]
15. Antolic A, Roy BD, Tarnopolsky MA, Zernicke RF, Wohl GR, Shaughnessy SG et al. Creatine monohydrate increases bone mineral density in young Sprague-Dawley rats. Med Sci Sports Exerc 2007;39(5):816-20. [ Links ]
16. Candow DG, Chilibeck PD. Potential of creatine supplementation for improving aging bone health. J Nutr Health Aging 2010;14(2):149-53. [ Links ]
17. Gala Paniagua J, Díaz-Curiel M, de la Piedra Gordo C, Castilla Reparaz C, Torralbo García M. Bone mass assessment in rats by dual energy X-ray absorptiometry. Br J Radiol 1998;71(847):754-8. [ Links ]
18. Patullo IM, Takayama L, Patullo RF, Jorgetti V, Pereira RM. Influence of ovariectomy and masticatory hypofunction on mandibular bone remodeling. Oral Dis 2009;15(8):580-6. [ Links ]
19. Guadalupe-Grau A, Fuentes T, Guerra B, Calbet JA. Exercise and bone mass in adults. Sports Med 2009; 39(6):439-68. [ Links ]
20. Nicastro H, Gualano B, de Moraes WM, de Salles Painelli V, da Luz CR, Dos Santos Costa A et al. Effects of creatine supplementation on muscle wasting and glucose homeostasis in rats treated with dexamethasone. Amino Acids 2011 Mar 5 [Epub ahead of print].
21. Aoki MS, Lima WP, Miyabara EH, Gouveia CH, Moriscot AS. Deleteriuos effects of immobilization upon rat skeletal muscle: role of creatine supplementation. Clin Nutr 2004;23(5):1176-83. [ Links ]
22. Felson DT, Zhang Y, Hannan MT, Anderson JJ. Effects of weight and body mass index on bone mineral density in men and women: the Framingham study. J Bone Miner Res 1993;8(5):567-73. [ Links ]
23. Gualano B, Artioli GG, Poortmans JR, Lancha Junior AH. Exploring the therapeutic role of creatine supplementation. Amino Acids 2010;38(1):31-44. [ Links ]