|Year : 2014 | Volume
| Issue : 1 | Page : 21-26
Effects of angiotensin converting enzyme inhibition on adiponectin levels and lipid profile in the ovariectomized-aged rats
Turhan Dost1, Samet Kafkas2, Filiz Gokalp1, Aslihan Karul3, Mustafa Birincioglu1
1 Department of Pharmacology, Adnan Menderes University, Medical School, Aydin, Turkey
2 Department of Obstetrics and Gynecology, Adnan Menderes University, Medical School, Aydin, Turkey
3 Department of Biochemistry, Adnan Menderes University, Medical School, Aydin, Turkey
|Date of Web Publication||7-Jan-2014|
Department of Medical Pharmacology, Adnan Menderes University, Medical School, Aydin 09010
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: To investigate the relationship between angiotensin converting enzyme (ACE) and adiponectin and lipid profile in the ovariectomized-aged rats. Materials and Methods: Wistar albino rats were first divided into two groups; control (C) and ovariectomized (OVX). Bilateral ovariectomy were carried out on rats (n = 30) except control group (n = 10). After 6 weeks from ovariectomy, ovariectomized rats were subdivided into three groups; one group received no treatment (OVX), two groups received low dose (OVX + Cap5; 5 mg/kg/day) and high dose (OVX + Cap20; 20 mg/kg/day) captopril (Cap). Body weights were monitored weekly. Adiponectin, triglyceride, cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and very low density lipoprotein cholesterol (VLDL-C) levels were measured at the end of the 6 weeks. Results: In the OVX group, body weights increased (P < 0.001). In the OVX + Cap20 group, body weights significantly decreased compared with the OVX group during weeks 5 and 6 (P < 0.05). While adiponectin levels increased in the OVX + Cap5 group (P = 0.014), triglyceride and cholesterol levels decreased in the OVX + Cap20 group (P = 0.016 and P < 0.001, respectively) compared to the OVX group. HDL-C and VLDL-C levels decreased only in OVX + Cap20 group (P < 0.005). Conclusions: ACE inhibitors may be decreasing the ovariectomy-induced weight gain by increasing adiponectin levels, and by affecting lipid profiles. The adipose tissue renin-angiotensin system (RAS) may be playing an important role in the development of adiposity.
Keywords: Adiponectin, angiotensin converting enzyme, cholesterol, ovariectomy, triglyceride
|How to cite this article:|
Dost T, Kafkas S, Gokalp F, Karul A, Birincioglu M. Effects of angiotensin converting enzyme inhibition on adiponectin levels and lipid profile in the ovariectomized-aged rats. J Pharmacol Pharmacother 2014;5:21-6
|How to cite this URL:|
Dost T, Kafkas S, Gokalp F, Karul A, Birincioglu M. Effects of angiotensin converting enzyme inhibition on adiponectin levels and lipid profile in the ovariectomized-aged rats. J Pharmacol Pharmacother [serial online] 2014 [cited 2020 Aug 15];5:21-6. Available from: http://www.jpharmacol.com/text.asp?2014/5/1/21/124413
| Introduction|| |
During menopause, with the dramatic fall in serum estrogen levels, together with relative hyperandrogenism which contribute to weight gain and changes in adipose tissue distribution, women tend to gain body fat. , It was shown that estrogen reduced adiposity, at least in part, in ovariectomized-mice by promoting the use of lipid as fuel. Estrogen was reported to cause this effect via enhancement of pathways that promote fat oxidation in muscle, by inhibiting fat storage (lipogenesis) in adipose tissue, liver and muscle, and increasing rates of adipocyte lipolysis.  The lack of estrogen causes obesity and metabolic syndrome after menopause. It is also one of the reasons for insulin resistance, dyslipidemia (elevated triglycerides, small dense low density lipoprotein cholesterol (LDL-C) particle, and reduced high density lipoprotein cholesterol (HDL-C)), elevated blood pressure, and increased hypercoagulability and proinflammatory state in blood.  It is known that estrogen prevents the progression of atherosclerosis by decreasing LDL-C, and increasing HDL-C plasma levels. 
Adiponectin, derived from adipose tissue, has roles in both carbohydrate and lipid metabolisms. Plasma adiponectin levels are affected by various factors and metabolic parameters. Adiponectin levels were reported to decrease in conditions such as obesity, type II diabetes, and atherosclerosis; and were shown to positively correlate with HDL-C levels. ,, Adiponectin levels are sex-dependent and were shown to be two- to three-fold higher in women than men. 
Angiotensin II type 1 (AT1) receptor which mediates most biological effects of angiotensin II (Ang II); such as vasoconstriction, aldosterone release, sodium and water retention, and cellular growth is one of the major components of the renin-angiotensin system (RAS). Estrogen induces down regulation of AT1 receptor expression, and shows protective effect against hypertension and atherosclerosis. Risks of cardiovascular disease and atherosclerosis increase in the menopause, because activation of AT1 receptor plays a key role in the regulation of blood pressure and fluid homeostasis. 
Several studies show that angiotensin converting enzyme (ACE) inhibitors change plasma adiponectin levels by affecting the hormonal function of adipose tissue. ,, Thus, ACE inhibitors which are frequently used for the treatment of hypertension and atherosclerosis in the postmenopause might lead to change of circulating adiponectin levels. Therefore, we investigated the effects of ACE inhibition on adiponectin levels and lipid profile in ovariectomized-aged rats.
| Materials and Methods|| |
Six-month-old adult female Wistar albino rats (220 ± 25 g; n = 40) were obtained from the Experimental Animal Center, Adnan Menderes University, Aydin, Turkey. The protocol for the experiment was approved by the Animal Experimentation Ethics Committee of Medical School (2007/0056). Adult female Wistar rats were kept in conventional room with controlled light (12:12, dark:light), temperature (22 ± 1°C), relative humidity (40-50%), and ventilation (15 air changes per hour). Food and water were given ad libitum. They were allowed to adapt to their environment for one week prior to the experiments.
Rats were first divided into two groups; control (C) and ovariectomized (OVX). Rats in control group (n = 10) were not ovariectomized and did not receive any treatment. In the study group, ovariectomies were carried out intraperitoneally with ketamine hydrochloride (50 mg/kg) and xylazine (5 mg/kg). The skin area was shaved from a hip to the lowest rib. Bilateral incisions were performed, and ovaries and the fat tissue around them were removed. Incision on the muscles and skin was sutured. 
After at least 6 months of recovery from the operation, ovariectomized rats were divided into three subgroups of ten animals each. First group (OVX) was given only water (n = 10). Second group of OVX rats received captopril (5 mg/kg/day) (OVX + Cap5; n = 10). Third group of OVX rats received captopril (20 mg/kg/day) (OVX + Cap20; n = 10). Captopril (C4042, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in water and given orally (p.o.) for 6 weeks. Body weights were monitored weekly.
Animals were decapitated under ketamine and xylazine anesthesia after 6 weeks. Blood was taken from the heart by midline laparotomy. Adiponectin, triglyceride, total cholesterol, HDL-C, LDL-C, and very low density lipoprotein cholesterol (VLDL-C) levels in blood were measured.
Blood samples were centrifuged at 4,000 rpm for 10 min at room temperature. Sera were removed and stored at -80°C for later studies.
Serum total cholesterol, HDL cholesterol, and triglyceride levels were determined with autoanalyzer (Architect C 8000, Abbott, Abbott Park, IL, USA). LDL-C and VLDL-C levels were calculated using "Friedewald" and "triglyceride/5" formulas, respectively. Rat adiponectin concentrations were determined with an ELISA kit (Linco Research Inc., St Charles, MO, USA) using a nonradioactive detection method.
Experimental values are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) followed by "Student-Newman-Keuls" multiple comparison test was used to compare the study groups. P values of less than 0.05 were considered statistically significant.
| Results|| |
Weekly body weight changes
Changes in body weights were assessed within each group. Weights in OVX group increased throughout 6 weeks and changes were statistically significant in the later weeks compared to the first two weeks (P < 0.001). Weight gain was statistically significant in the OVX + Cap5 and OVX + Cap20 groups only in the 6 th week when compared to week one (P < 0.001). Body weight changes in captopril-intake groups were not statistically significant from 3 rd week onwards [Figure 1].
|Figure 1: The comparison of the weekly changes in body weights within each group. Body weights increased throughout 6 weeks. Data were expressed as mean ± SEM. *P < 0.001 vs. 1st week, #P < 0.001 vs. 1st and 2nd week|
Click here to view
When changes in the body weights among groups were compared, the differences in the body weight between the groups OVX and C were statistically significant from 3 rd week on (P < 0.05), due to an increase in body weights in the OVX group. The increase was statistically significant in the OVX + Cap5 group compared to the control group in the 4 th and 5 th weeks (P < 0.05). In the OVX + Cap20 group, there was a statistically significant decrease in body weights compared to the OVX group (P < 0.05) [Figure 2].
|Figure 2: The comparison of weekly changes in body weights between groups. Data were expressed as mean ± SEM. *P < 0.05 vs. C, #P < 0.05 vs. OVX|
Click here to view
Plasma adiponectin levels increased in OVX group without any statistical significance. Adiponectin levels were higher in OVX + Cap5 group compared to the other three, being statistically significant between OVX + Cap5 group and controls. Adiponectin levels significantly decreased in the OVX + Cap20 group compared with the OVX + Cap5 group (P = 0.014) [Figure 3].
|Figure 3: Changes in adiponectin levels among groups at the end of 6th week. Data were expressed as mean ± SEM. *P = 0.014 vs. C and OVX + Cap20|
Click here to view
Triglyceride levels were higher in OVX group than other groups, but not statistically significantly. They decreased in OVX + Cap5 and OVX + Cap20 groups, being statistically significant in the OVX + Cap20 group compared to the OVX group (P = 0.016) [Figure 4].
|Figure 4: Changes in triglyceride and cholesterol levels. Data were expressed as mean ± SEM. *P = 0.016 vs. OVX, # P < 0.001 vs. C, OVX and OVX + Cap5|
Click here to view
Similar to the triglyceride levels, cholesterol levels were higher in the OVX group. In the OVX + Cap20 group, cholesterol levels were low with statistical significances between OVX + Cap20 group and all the others (P < 0.001) [Figure 5].
|Figure 5: Changes in HDL-C, LDL-C, and VLDL-C levels. Data were expressed as mean ± SEM. *P = 0.001 vs. C, #P < 0.005 vs. OVX,+ P = 0.005 vs. OVX + Cap5|
Click here to view
HDL-C, LDL-C, and VLDL-C
The HDL-C levels were higher in OVX group than the controls (P = 0.001). Low HDL-C levels in the OVX + Cap20 group were statistically significant compared to the OVX group (P < 0.005). The LDL-C did not show any significant changes among groups. The VLDL-C levels were lower in OVX + Cap20 group than the others. There were statistical significance between OVX + Cap20 group and groups of control, OVX and OVX + Cap5 (P < 0.005) [Figure 5].
| Discussion|| |
One of the aims of the current work was to evaluate body weight changes after ovariectomy. We observed an increase in body weight after ovariectomy in association with lack of estrogen. We previously showed that weight gain due to increased adipose tissue in menopausal period might be prevented by high-dose estrogen treatment (20 μg/kg).  Weight gain and obesity largely drives the increased prevalence of several disorders in postmenopausal women. These include increased central distribution of body fat, insulin resistance, dyslipidemia (elevated triglycerides, small dense LDL-C particle, and reduced HDL-C), elevated blood pressure, and increased hypercoagulability and proinflammatory state in blood.  Increased adiposity is associated with systemic loss of estrogen at menopause. Estrogen replacement alone or in combination with progesterone can prevent menopause-induced gains in adipose tissue mass. Reduction in adiposity is higher in intra-abdominal fat depots than subcutaneous, consistent with the observation in menopausal women that estrogen therapy primarily acts on intra-abdominal depots, thereby preventing central adiposity. 
Another aim of this study was to observe the effects of inhibition of ACE on body weight, lipid profile, and plasma adiponectin levels; and therefore captopril was used. Captopril treatment prevented body weight gain. While the classical RAS is known for its role in body fluid and cardiovascular homeostasis, there is an analogous RAS located in adipose tissue. Plasma angiotensinogen, plasma renin activity, plasma ACE activity, and adipose tissue angiotensinogen expression are positively correlated with adiposity in humans.  The adipose tissue RAS is thought to play a major role in the development of adiposity. Weisinger et al., reported that treatment with perindopril, an ACE inhibitor, from birth, reduced the body fat of rats maintained on a 7% fat diet.  In our study, body weight did not change in captopril-treated animals, and this may be related to its functions within adipose tissue. The RAS including Mas receptor is present in adipose tissue. It has been shown that captopril-induced weight loss may be related to activation of Mas receptor in the adipose tissue.  The decreased body weight is also associated with decreased body fat and plasma leptin and importantly, with increased adiponectin. Many studies have shown a relationship between plasma adiponectin levels and variety of diseases. First of all, it was demonstrated that plasma adiponectin levels were inversely correlated with body mass index. Reduction of body weight increases plasma adiponectin levels. 
Adipocytes secrete factors that play central roles in energy balance regulation, insulin sensitivity, immunological responses, and vascular diseases. , Adiponectin, released at high levels from differentiated adipocytes, is found at high levels in blood. Its expression is especially higher in subcutaneous adipose tissue. Plasma adiponectin levels decline before the onset of obesity in nonhuman primates, suggesting that hypoadiponectinemia contributes to the pathogenesis of this condition.  Plasma concentrations of adiponectin are significantly lower in obese subjects than in lean controls,  indicating a role for this adipocyte-secreted factor in the energy balance regulation. Adiponectin effects on energy homeostasis are mediated, at least in part, by increased free fatty acid oxidation in muscle. It was suggested that increasing free fatty acid oxidation pharmacologically in muscle might provide a new way to control body weight without interfering with food intake.  Kohlstedt et al., showed that ACE inhibitors, such as enalaprilat or ramiprilat, significantly increased soluble adiponectin production. In ACE-deficient 3T3 L1 cells, inhibitors of the enzyme did not affect the release of adiponectin. ACE inhibitors were shown to significantly increase the release of adiponectin from preadipocytes.  In the present study, we also observed that treatment with ACE inhibitor, captopril (5 mg/kg/day), significantly enhanced circulating adiponectin levels, whereas high-dose captopril did not have any effect.
The mechanism by which ACE inhibitors affect adiponectin levels is unknown, but it was indicated that ACE inhibitors modulated adipocyte gene expression via the induction of cellular retinol-binding protein 1 (CRBP1).  The effects of ACE inhibitor therapy are related to both changes in enzyme activity and ACE signaling cascade. Binding ACE inhibitors to the cytoplasmic site of the enzyme causes phosphorylation, activating specific signaling pathways affecting expression of several genes such as endothelial genes. Adipose tissue expresses all the components necessary for the renin-angiotensin system.  It has been reported that renin, angiotensinogen, aldosterone, and ACE levels were higher in obese women compared to slim women and low angiotensinogen gene expression in their adipose tissues, suggesting a correlation between RAS components and body weight. Weight reduction (≅5%) was shown to reduce angiotensinogen, renin, and aldosterone levels; and decrease ACE expression. 
Some of the ACE inhibitors such as perindopril and ramipril, exhibit a strong affinity for target tissues, while the others (enalapril and captopril) are characterized by relatively weak tissue affinity. Effects of ACE inhibitors on plasma adiponectin levels may show these differences. Although both enalapril and perindopril increased plasma adiponectin level, perindopril was shown to have much stronger action than enalapril.  Here, we showed that adiponectin levels were higher in the low dose captopril group compared to the high dose one, indicating possible differences in pharmacokinetics and pharmacodynamics between various ACE inhibitors.
It has been generally accepted that HDL-C level decreased in women with menopause.  However, recent reports showed different results. Kim et al., showed that menopause might associate with the elevating of HDL-C. They also showed that cholesterol, triglyceride, LDL-C, and VLDL-C levels were higher in the postmenopausal women than the premenopausal and perimenopausal women.  In our study, HDL-C levels were significantly higher in ovariectomy group than the controls. Triglyceride and cholesterol levels were also increased in the OVX group, but LDL-C and VLDL-C levels did not change. ACE inhibitor, captopril changed the lipid profile. Increased triglyceride, cholesterol, HDL-C, and VLDL-C levels after ovariectomy were decreased by especially high dose of captopril. But, LDL-C level was not affected by captopril. These results showed that the adipose tissue RAS might play a major role in the development of adiposity.
It was well documented that adiponectin is associated with dyslipidemia. Plasma adiponectin levels correlate positively with HDL-cholesterol and negatively with triglyceride. , A study by Matsubara et al., on nondiabetic women reported that plasma adiponectin values correlated negatively with triglyceride, apolipoprotein B and E levels, and atherogenic index (total/HDL cholesterol); but positively with serum HDL-C and apoprotein A1 levels.  These results are compatible with our results. While low dose of captopril caused an increase in adiponectin levels, high-dose of the ACE inhibitor decreased triglyceride, cholesterol, HDL-C, and VLDL-C levels. These findings suggest that the hypoadiponectinemia observed in dyslipidemia may accelerate the atherosclerotic changes seen in the metabolic syndrome in postmenopausal period.
| Conclusion|| |
Ovariectomy-induced weight gain was decreased by ACE inhibitors. This effect may explain at least in part, increased adiponectin levels. Triglyceride, cholesterol, HDL-C, and VLDL-C levels were also decreased by ACE inhibitors. Our results demonstrate that the adipose tissue RAS may play a major role in the development of adiposity.
| Acknowledgment|| |
We thank Leyla Didem Kozaci MD, PhD for her invaluable contribution in the linguistic revision of the final manuscript.
| References|| |
|1.||D'Eon TM, Souza SC, Aronovitz M, Obin MS, Fried SK, Greenberg AS. Estrogen regulation of adiposity and fuel partitioning. Evidence of genomic and non-genomic regulation of lipogenic and oxidative pathways. J Biol Chem 2005;280:35983-91. |
|2.||Rachon D, Teede H. Ovarian function and obesity--interrelationship, impact on women's reproductive lifespan and treatment options. Mol Cell Endocrinol 2010;316:172-9. |
|3.||Lobo RA. Metabolic syndrome after menopause and the role of hormones. Maturitas 2008;60:10-8. |
|4.||Zandberg P, Peters JL, Demacker PN, de Reeder EG, Smit MJ, Meuleman DG. Comparison of the antiatherosclerotic effect of tibolone with that of estradiol and ethinyl estradiol in cholesterol-fed, ovariectomized rabbits. Menopause 2001;8:96-105. |
|5.||Kazumi T, Kawaguchi A, Hirano T, Yoshino G. Serum adiponectin is associated with high-density lipoprotein cholesterol, triglycerides, and low-density lipoprotein particle size in young healthy men. Metabolism 2004;53:589-93. |
|6.||Kleiblova P, Springer D, Haluzik M. The influence of hormonal changes during menstrual cycle on serum adiponectin concentrations in healthy women. Physiol Res 2006;55:661-6. |
|7.||Shetty GK, Economides PA, Horton ES, Mantzoros CS, Veves A. Circulating adiponectin and resistin levels in relation to metabolic factors, inflammatory markers, and vascular reactivity in diabetic patients and subjects at risk for diabetes. Diabetes Care 2004;27:2450-7. |
|8.||Bottner A, Kratzsch J, Muller G, Kapellen TM, Bluher S, Keller E, et al. Gender differences of adiponectin levels develop during the progression of puberty and are related to serum androgen levels. J Clin Endocrinol Metab 2004;89:4053-61. |
|9.||Nickenig G, Baumer AT, Grohe C, Kahlert S, Strehlow K, Rosenkranz S, et al. Estrogen modulates AT1 receptor gene expression in vitro and in vivo. Circulation 1998;97:2197-201. |
|10.||Kohlstedt K, Gershome C, Trouvain C, Hofmann WK, Fichtlscherer S, Fleming I. Angiotensin-converting enzyme inhibitors modulate cellular retinol-binding protein 1 and adiponectin expression in adipocytes via the ACE-dependent signaling cascade. Mol Pharmacol 2009;75:685-92. |
|11.||Krysiak R, Sierant M, Marek B, Okopien B. The effect of perindopril and enalapril on plasma resistin levels in normotensive patients with coronary heart disease. Endokrynol Pol 2010;61:683-90. |
|12.||Nakamura T, Kawachi K, Saito Y, Saito T, Morishita K, Hoshino J, et al. Effects of ARB or ACE-inhibitor administration on plasma levels of aldosterone and adiponectin in hypertension. Int Heart J 2009;50:501-12. |
|13.||Feng Z, Zhang JT. Long-term melatonin or 17beta-estradiol supplementation alleviates oxidative stress in ovariectomized adult rats. Free Radic Biol Med 2005;39:195-204. |
|14.||Kafkas S, Dost T, Ozkayran H, Yenisey C, Tuncyurek P, Birincioglu M. Effect of estrogen therapy on adipocytokines in ovariectomized-aged rats. J Obstet Gynaecol Res 2012;38:231-8. |
|15.||Kershaw EE, Flier JS. Adipose tissue as an endocrine organ. J Clin Endocrinol Metab 2004;89:2548-56. |
|16.||Weisinger RS, Stanley TK, Begg DP, Weisinger HS, Spark KJ, Jois M. Angiotensin converting enzyme inhibition lowers body weight and improves glucose tolerance in C57BL/6J mice maintained on a high fat diet. Physiol Behav 2009;98:192-7. |
|17.||Oh YB, Kim JH, Park BM, Park BH, Kim SH. Captopril intake decreases body weight gain via angiotensin-(1-7). Peptides 2012;37:79-85. |
|18.||Nishida M, Funahashi T, Shimomura I. Pathophysiological significance of adiponectin. Med Mol Morphol 2007;40:55-67. |
|19.||Kim S, Moustaid-Moussa N. Secretory, endocrine and autocrine/paracrine function of the adipocyte. J Nutr 2000;130:3110-5S. |
|20.||Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun 1999;257:79-83. |
|21.||Gregoire FM. Adipocyte differentiation: From fibroblast to endocrine cell. Exp Biol Med (Maywood) 2001;226:997-1002. |
|22.||Fleming I. Signaling by the angiotensin-converting enzyme. Circ Res 2006;98:887-96. |
|23.||Engeli S, Bohnke J, Gorzelniak K, Janke J, Schling P, Bader M, et al. Weight loss and the renin-angiotensin-aldosterone system. Hypertension 2005;45:356-62. |
|24.||Li Z, McNamara JR, Fruchart JC, Luc G, Bard JM, Ordovas JM, et al. Effects of gender and menopausal status on plasma lipoprotein subspecies and particle sizes. J Lipid Res 1996;37:1886-96. |
|25.||Kim CJ, Kim TH, Ryu WS, Ryoo UH. Influence of menopause on high density lipoprotein-cholesterol and lipids. J Korean Med Sci 2000;15:380-6. |
|26.||Chandran M, Phillips SA, Ciaraldi T, Henry RR. Adiponectin: More than just another fat cell hormone? Diabetes Care 2003;26:2442-50. |
|27.||Matsubara M, Maruoka S, Katayose S. Decreased plasma adiponectin concentrations in women with dyslipidemia. J Clin Endocrinol Metab 2002;87:2764-9. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
|This article has been cited by|
||Reduction in nitric oxide bioavailability shifts serum lipid content towards atherogenic lipoprotein in rats
| ||Esther Oluwasola Aluko,Temidayo Olutayo Omobowale,Ademola Adetokunbo Oyagbemi,Olumuyiwa Abiola Adejumobi,Temitayo Olabisi Ajibade,Adesoji Adedipe Fasanmade |
| ||Biomedicine & Pharmacotherapy. 2018; 101: 792 |
|[Pubmed] | [DOI]|
||Chronic treatment with angiotensin-converting enzyme inhibitor increases cardiac fibrosis in young rats submitted to early ovarian failure
| ||Sabrina Graziani Veloso Dutra,Ana Carolina Sanches Felix,Ada Clarice Gastaldi,Tábata De Paula Facioli,Suenimeire Vieira,Hugo Celso Dutra De Souza |
| ||Autonomic Neuroscience. 2017; |
|[Pubmed] | [DOI]|
||Renin-angiotensin system blockers regulate the metabolism of isolated fat cells in vitro
| ||R de O. Caminhotto,R.A.L. Sertié,S. Andreotti,A.B. Campaăa,F.B. Lima |
| ||Brazilian Journal of Medical and Biological Research. 2016; 49(8) |
|[Pubmed] | [DOI]|