LFS-14025; No of Pages 4 Life Sciences xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
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Article history: Received 2 November 2013 Accepted 2 May 2014 Available online xxxx
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Keywords: ADMA Aerobic exercise capacity Cardiovascular disease Aging Endothelin
Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
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Aims: Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase, an enzyme responsible for the generation of NO. Plasma concentrations of ADMA increase in the elderly and in postmenopausal women. In fact, an elevated ADMA level is a risk factor of cardiovascular disease. Aerobic exercise has a beneficial effect on cardiovascular disease. However, the relationship between ADMA and aerobic fitness is unknown. The aim of this study was to determine whether plasma ADMA concentrations correlate with aerobic fitness levels in postmenopausal women. Main methods: Thirty healthy postmenopausal women aged 50–76 years participated in this study. We measured plasma concentrations of ADMA and oxygen consumption at the ventilatory threshold (VO2VT) as an index of aerobic fitness. Subjects were divided into the low aerobic fitness (Low fitness) and high aerobic fitness (High fitness) groups, and the dividing line was set at the median VO2VT value. Key findings: VO2VT was significantly higher in the High fitness group than in the Low fitness group (P b 0.01). The plasma ADMA concentrations in the High fitness group were significantly lower than those in the Low fitness group (P b 0.05). There was a negative correlation between plasma ADMA concentrations and VO2VT (r = −0.532, P b 0.01). Significance: We found that plasma ADMA concentrations were associated with aerobic fitness in postmenopausal women. The results of this study suggest that habitual aerobic exercise may decrease plasma ADMA concentrations. © 2014 Published by Elsevier Inc.
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Koichiro Tanahashi a, Nobuhiko Akazawa a, Asako Miyaki a, Youngju Choi b, Song-Gyu Ra a, Tomoko Matsubara a, Hiroshi Kumagai a, Satoshi Oikawa a, Takashi Miyauchi c, Seiji Maeda b,⁎
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Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women☆
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Introduction
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Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase, an enzyme responsible for NO generation. Increased ADMA levels are known to decrease NO synthesis, leading to endothelial dysfunction (Cooke, 2004). Elevated ADMA levels are associated with increased risks of cardiovascular disease (Böger et al., 2009; Leong et al., 2008), and relationships between increased circulating ADMA levels and several traditional cardiovascular risk factors, such as hypertension, hypercholesterolemia, insulin resistance, obesity, and smoking, have been reported (Kielstein et al., 2003;
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☆ This work was presented at the Thirteenth International Conference on Endothelin (held at the University of Tsukuba, Tokyo Campus: September 8–11, 2013), and was published as an abstract form in the Program and Abstract Book (Cross Border Session PC-8: 2013) of this Meeting. ⁎ Corresponding author at: Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8574, Japan. Tel.: +81 29 853 2683; fax: +81 29 853 2986. E-mail address: [email protected] (S. Maeda).
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Böger et al., 1998; Sydow et al., 2005; Eid et al., 2004). Therefore, it is important to attenuate increased circulating ADMA concentrations. ADMA is a naturally occurring amino acid that is produced by methylation of arginine residues in intracellular proteins via protein arginine methyltransferases (PRMTs) (Leiper and Vallance, 1999). ADMA is eliminated by renal clearance (Vallance et al., 1992). However, N80% of ADMA is hydrolyzed into citrulline and dimethylamine by dimethylarginine dimethylamonohydrolase (DDAH) (Achan et al., 2003). The elevation of plasma ADMA that occurs with vascular disease and risk factors is largely due to enhanced PRMT activity or impaired DDAH activity (Cooke, 2004). Aerobic exercise has been recommended for preventing cardiovascular disease (Thompson et al., 2003). Regular aerobic exercise is an effective strategy for reducing the risk of cardiovascular disease in middle-aged and elderly individuals (Tanaka et al., 2000). McMurray et al. reported that increased aerobic fitness is associated with reduced cholesterol and blood pressure levels, risk factors of cardiovascular disease (McMurray et al., 1998). It is well known that physical activity and aerobic fitness decrease with age (Hawkins and Wiswell, 2003). Aging increases plasma ADMA concentrations (Miyazaki et al., 1999), and
http://dx.doi.org/10.1016/j.lfs.2014.05.003 0024-3205/© 2014 Published by Elsevier Inc.
Please cite this article as: Tanahashi K, et al, Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.05.003
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Materials and methods
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A total of 30 healthy postmenopausal women (50–76 years) participated in the present study. The subjects were non-smokers, non-obese, and free of overt cardiovascular disease as assessed by medical records. All subjects were at least 2 years postmenopause and none was taking medications. All potential risks were explained to the study participants, and each provided written informed consent to participate in the study. All of the procedures were reviewed and approved by the Ethics Committee of the University of Tsukuba.
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All experiments, excluding the cycle exercise test, were performed in the morning after a 12-h overnight fast. Subjects abstained from alcohol and caffeine for at least 12 h and did not exercise for at least 24 h before beginning the experiment to avoid any potential acute effects of exercise. Measurements were obtained in a quiet, temperaturecontrolled room (24–26 °C). After a resting period of at least 20 min, arterial blood pressure was measured and a blood sample was drawn to determine the plasma ADMA concentrations and blood chemistry. After these procedures, aerobic fitness was measured during an incremental cycle ergometer exercise.
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Body weight was measured to the nearest 0.1 kg with a digital scale. Height was measured to the nearest 0.1 cm by using a wall-mounted stadiometer. Body mass index (BMI) was calculated as the participant's weight (in kilograms) divided by their height (in meters squared).
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Each blood sample was placed in a chilled tube containing ethylenediaminetetraacetic acid (2 mg/mL) and centrifuged at 2000 ×g for 15 min at 4 °C. The plasma was stored at −80 °C until analysis. Plasma ADMA concentrations were determined using a commercial enzymelinked immunosorbent assay kit (Immundiagnostik AG, Bensheim, Germany). The ADMA assay was performed according to the manufacturer's instructions. The intra-assay coefficients of variation were 5.8–7.9%. The standards and controls provided with the kits were used to take the measurements.
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Blood biochemistry
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Serum concentrations of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and fasting plasma concentrations of glucose were determined using standard enzymatic techniques. Total cholesterol and triglyceride concentrations were determined using the cholesterol dehydrogenase and glycerol kinase methods, respectively (Allain et al., 1974; Kohlmeier, 1986). Low-density lipoprotein cholesterol and high-density lipoprotein cholesterol concentrations were measured directly (Finley et al.,
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Aerobic fitness
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Oxygen uptake at the ventilatory threshold (VO 2VT) was measured during the incremental cycle ergometer exercise using an online computer-assisted circuit spirometer (AE300S; Minato Medical Science, Osaka, Japan). All subjects underwent an incremental cycle exercise test (2 min at 20 W, followed by 10-W increases every 1 min) until they felt exhausted or reached 85% of their agepredicted maximal heart rate (HRmax = 220 − age) (Fox et al., 1971). Each individual VO2VT value was calculated using regression analysis of the slopes of CO2 production, O2 uptake, and the minuteventilation plot. The subjects (9.2–18.2 mL/min/kg) were divided into the low aerobic fitness (Low fitness) and high aerobic fitness (High fitness) groups according to the median VO2VT value (11.8 mL/min/kg).
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1978; Yamashita et al., 2008). The glucose concentration was assayed 124 using the hexokinase and glucose-6-phosphate dehydrogenase methods 125 (Slein, 1963). 126
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Table 1 shows the characteristics of the study participants by subgroup according to VO2VT results (Low fitness and High fitness). There were no significant differences in age, height, systolic blood pressure, diastolic blood pressure, triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, or glucose between the groups. Body weight and BMI were significantly lower in the High fitness group than in the Low fitness group (P b 0.01). VO2VT values in the High fitness group were significantly higher than those in the Low fitness group (P b 0.01). Fig. 1 shows the plasma ADMA
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Unpaired Student's t tests were used to evaluate differences between the Low fitness and High fitness groups. Relationships between plasma ADMA concentrations and aerobic fitness were analyzed by using a Pearson correlation coefficient. All data are expressed as the means ± SD. Differences were considered significant at values of P b 0.05.
All n = 30 Age, years Height, cm Body weight, kg BMI, kg/m2 SBP, mm Hg DBP, mm Hg TG, mol/L TC, mol/L HDL-C, mol/L LDL-C, mol/L Glucose, mol/L VO2VT, mL/min/kg
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Table 1 Subject characteristics.
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Supine systolic and diastolic arterial blood pressures were recorded 141 from the right arm using a semi-automated device (form PWV/ABI; 142 Colin Medical Technology, Komaki, Japan). 143 Statistical analyses
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postmenopausal women have higher plasma ADMA concentrations than premenopausal women (Schulze et al., 2005). However, the relationship between aerobic fitness and ADMA in postmenopausal women is unclear. The purpose of this study was to examine the relationship between aerobic fitness and plasma ADMA concentrations in postmenopausal women. We hypothesized that plasma ADMA concentrations are lower in postmenopausal women with high aerobic fitness levels than in their low aerobic fitness peers. To investigate this hypothesis, we measured aerobic exercise capacity and plasma ADMA concentrations in postmenopausal women.
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60.7 154.3 54.6 22.9 120 73 1.3 5.9 1.7 3.5 5.1 12.5
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Subjects divided according to VO2VT
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61.2 153.7 59.7 25.2 122 75 1.4 6.0 1.6 3.7 5.1 10.7
60.1 155.0 49.4 20.6 117 71 1.1 5.8 1.8 3.4 5.1 14.3
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Data are means ± SD. BMI; body mass index, SBP; systolic blood pressure, DBP; diastolic blood pressure, TG; triglycerides, TC; total cholesterol, HDL-C; high-density lipoprotein cholesterol, LDL-C; low-density lipoprotein cholesterol, VO2VT; oxygen uptake at the ventilatory threshold. ⁎⁎ P b 0.01 vs. the Low fitness group.
Please cite this article as: Tanahashi K, et al, Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.05.003
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In the present study, we demonstrated that plasma ADMA concentrations were lower in individuals with high aerobic fitness levels compared to their peers with low aerobic fitness levels. Furthermore, we showed that plasma ADMA concentrations were negatively correlated with aerobic fitness in postmenopausal women. Our findings suggest that regular exercise or increased physical activity may decrease plasma ADMA concentrations.
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Conflict of interest statement
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cardiovascular disease (Cooke, 2004). Elevated ADMA is a cause of reduction in NO synthesis and activation of renin–angiotensin system, which results in endothelial dysfunction and arteriosclerosis (Cooke, 2004; Hasegawa et al., 2007; Furuki et al., 2008). Furthermore, ADMA levels are increased in patients with hypertension (Kielstein et al., 2003), coronary artery disease (Valkonen et al., 2001) and chronic heart failure (Usui et al., 1998). In the present study, we demonstrated that plasma ADMA concentrations were lower in the High fitness group than in the Low fitness group. These results suggest that a high fitness-related ADMA reduction may help inhibit cardiovascular disease. In the present study, plasma ADMA concentrations were negatively correlated with VO2VT. This is the first study to show the relationship between plasma ADMA concentrations and aerobic fitness in healthy postmenopausal women. However, it should be noted that we could not determine the causal relationship between plasma ADMA concentrations and aerobic fitness. One earlier study reported that aginginduced increases in oxidative stress induce increases in plasma ADMA concentrations (Fabian et al., 2012). Oxidative stress increases ADMA levels by enhancing PRMT activity and/or decreasing DDAH activity (Sydow and Münzel, 2003). In contrast, Bełtowski and Kedra reported that antioxidant treatment, such as intake medications or supplements, decreases plasma ADMA concentrations (Bełtowski and Kedra, 2006). Aerobic exercise capacity is reportedly associated with the antioxidant defense system (Pialoux et al., 2009). In addition, a recent study reported that aerobic exercise training increased DDAH mRNA expression in obese men (Hanssen et al., 2011). Therefore, the decrease in oxidative stress, PRMT, and DDAH levels may contribute to exercise-related ADMA level decreases. In this study, the mean BMI of the Low fitness group was higher than that of the High fitness group. One study reported that circulating levels of ADMA increased in obesity (Eid et al., 2004). However, we did not observe a correlation between plasma ADMA concentrations and BMI in this study. This inconsistent result is likely explained by the focus on only normal weight healthy postmenopausal women in the present study. After adjustment for BMI, the association between plasma ADMA concentrations and VO2VT was confirmed. Therefore, our results suggest that the correlation between plasma ADMA concentrations and physical fitness is independent of BMI in normal weight postmenopausal women. There are several limitations of this study that should be emphasized. First, the present study had a relatively small sample size due to the rigorous screening criteria of healthy postmenopausal women who were non-smokers, non-obese, free of overt cardiovascular disease, and taking no medications or supplements. Accordingly, further studies should include larger sample sizes. Second, although it has been reported that VO2VT is associated with daily physical activity (Malatesta et al., 2004), we did not measure the individual exercise type, frequency, intensity, or duration. We also did not measure DDAH or NO levels or track any indices of endothelial function. Further studies are needed to investigate the effects of individual exercise training protocols on endothelial function.
In the present study, we investigated the relationship between plasma ADMA concentrations and aerobic fitness in postmenopausal women. We demonstrated that the plasma ADMA concentration was significantly lower in the High fitness group than in the Low fitness group. Moreover, plasma ADMA concentrations were negatively correlated to VO2VT, and this relationship was independent of age and BMI. These findings suggest that improved cardiovascular fitness may decrease plasma ADMA concentrations in postmenopausal women. ADMA is an endogenous competitive inhibitor of NO synthase. Plasma concentrations of ADMA increase in elderly individuals and postmenopausal women (Miyazaki et al., 1999; Schulze et al., 2005). An increased ADMA level has been reported as a risk factor of
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concentrations in the Low fitness and High fitness groups. The plasma ADMA concentrations were significantly lower in the High fitness group than in the Low fitness group (Low, 10.7 ± 0.7 mL/min/kg vs. High, 14.3 ± 1.6 mL/min/kg; P b 0.05). Moreover, there was a significant negative correlation between plasma ADMA concentrations and VO2VT (r = − 0.531, P b 0.01; Fig. 2). After adjustment for age and BMI, the association between plasma ADMA concentrations and VO2VT was confirmed (β = − 0.435, P b 0.05). There was no relationship between plasma ADMA concentrations and postmenopausal periods (r = 0.05, P = 0.77).
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Fig. 1. Plasma ADMA concentrations in the Low fitness and High fitness groups. Data are expressed as the mean ± SD.
Fig. 2. Relationship of oxygen uptake at the ventilatory threshold (VO2VT) and plasma ADMA concentrations.
The authors report no conflicts of interest with respect to this manuscript.
Please cite this article as: Tanahashi K, et al, Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.05.003
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Achan V, Broadhead M, Malaki M, Whitley G, Leiper J, MacAllister R, et al. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 2003;23:1455–9. Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974;20:470–5. Bełtowski J, Kedra A. Asymmetric dimethylarginine (ADMA) as a target for pharmacotherapy. Pharmacol Rep 2006;58:159–78. Böger RH, Bode-Böger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, et al. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation 1998;98:1842–7. Böger RH, Sullivan LM, Schwedhelm E, Wang TJ, Maas R, Benjamin EJ, et al. Plasma asymmetric dimethylarginine and incidence of cardiovascular disease and death in the community. Circulation 2009;119:1592–600. Cooke JP. Asymmetrical dimethylarginine: the Uber marker? Circulation 2004;109:1813–8. Eid HM, Arnesen H, Hjerkinn EM, Lyberg T, Seljeflot I. Relationship between obesity, smoking, and the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine. Metabolism 2004;53:1574–9. Fabian E, Bogner M, Elmadfa I. Age-related modification of antioxidant enzyme activities in relation to cardiovascular risk factors. Eur J Clin Invest 2012;42:42–8. Finley PR, Schifman RB, Williams RJ, Lichti DA. Cholesterol in high-density lipoprotein: use of Mg2+/dextran sulfate in its enzymic measurement. Clin Chem 1978;24:931–3. Fox SM, Naughton JP, Haskell WL. Physical activity and the prevention of coronary heart disease. Ann Clin Res 1971;3:404–32. Furuki K, Adachi H, Enomoto M, Otsuka M, Fukami A, Kumagae S, et al. Plasma level of asymmetric dimethylarginine (ADMA) as a predictor of carotid intima-media thickness progression: six-year prospective study using carotid ultrasonography. Hypertens Res 2008;31:1185–9. Hanssen H, Nickel T, Drexel V, Hertel G, Emslander I, Sisic Z, et al. Exercise-induced alterations of retinal vessel diameters and cardiovascular risk reduction in obesity. Atherosclerosis 2011;216:433–9. Hasegawa K, Wakino S, Tatematsu S, Yoshioka K, Homma K, Sugano N, et al. Role of asymmetric dimethylarginine in vascular injury in transgenic mice overexpressing dimethylarginie dimethylaminohydrolase 2. Circ Res 2007;101:e2-10. Hawkins S, Wiswell R. Rate and mechanism of maximal oxygen consumption decline with aging: implications for exercise training. Sports Med 2003;33:877–88. Kielstein JT, Bode-Böger SM, Frölich JC, Ritz E, Haller H, Fliser D. Asymmetric dimethylarginine, blood pressure, and renal perfusion in elderly subjects. Circulation 2003;107:1891–5.
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This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24590654). We thank Dr. Ryuichi Ajisaka, University of Tsukuba, for his advices and Mr. Ryota Higashino, University of Tsukuba, for his technical assistance.
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Kohlmeier M. Direct enzymic measurement of glycerides in serum and in lipoprotein fractions. Clin Chem 1986;32:63–6. Leiper J, Vallance P. Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 1999;43:542–8. Leong T, Zylberstein D, Graham I, Lissner L, Ward D, Fogarty J, et al. Asymmetric dimethylarginine independently predicts fatal and nonfatal myocardial infarction and stroke in women: 24-year follow-up of the population study of women in Gothenburg. Arterioscler Thromb Vasc Biol 2008;28:961–7. Malatesta D, Simar D, Dauvilliers Y, Candau R, Ben Saad H, Préfaut C, et al. Aerobic determinants of the decline in preferred walking speed in healthy, active 65- and 80-yearolds. Pflugers Arch 2004;447:915–21. McMurray RG, Ainsworth BE, Harrell JS, Griggs TR, Williams OD. Is physical activity or aerobic power more influential on reducing cardiovascular disease risk factors? Med Sci Sports Exerc 1998;30:1521–9. Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, et al. Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Circulation 1999; 99:1141–6. Pialoux V, Brown AD, Leigh R, Friedenreich CM, Poulin MJ. Effect of cardiorespiratory fitness on vascular regulation and oxidative stress in postmenopausal women. Hypertension 2009;54:1014–20. Schulze F, Maas R, Freese R, Schwedhelm E, Silberhorn E, Böger RH. Determination of a reference value for N(G), N(G)-dimethyl-L-arginine in 500 subjects. Eur J Clin Invest 2005;35:622–6. Slein MW. D-Glucose: determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Bergmeyer HU, editor. Methods of enzymatic analysisNew York, N.Y., USA: Academic Press; 1963. p. 117. Sydow K, Münzel T. ADMA and oxidative stress. Atheroscler Suppl 2003;4:41–51. Sydow K, Mondon CE, Cooke JP. Insulin resistance: potential role of the endogenous nitric oxide synthase inhibitor ADMA. Vasc Med 2005;10:S35–43. Tanaka H, Dinenno FA, Monahan KD, Clevenger CM, DeSouza CA, Seals DR. Aging, habitual exercise, and dynamic arterial compliance. Circulation 2000;102:1270–5. Thompson PD, Buchner D, Pina IL, Balady GJ, Williams MA, Marcus BH, et al. American Heart Association Council on Clinical Cardiology Subcommittee on Exercise, Rehabilitation, and Prevention; American Heart Association Council on Nutrition, Physical Activity, and Metabolism Subcommittee on Physical Activity. Exercise and physical activity in the prevention and treatment of atherosclerotic cardiovascular disease: a statement from the Council on Clinical Cardiology (Subcommittee on Exercise, Rehabilitation, and Prevention) and the Council on Nutrition, Physical Activity, and Metabolism (Subcommittee on Physical Activity). Circulation 2003;107: 3109–16. Usui M, Matsuoka H, Miyazaki H, Ueda S, Okuda S, Imaizumi T. Increased endogenous nitric oxide synthase inhibitor in patients with congestive heart failure. Life Sci 1998;62:2425–30. Valkonen VP, Päivä H, Salonen JT, Lakka TA, Lehtimäki T, Laakso J, et al. Risk of acute coronary events and serum concentration of asymmetrical dimethylarginine. Lancet 2001;358:2127–8. Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992;339: 572–5. Yamashita S, Nakamura M, Koizumi H, Oku H, Sandoval JC, Tsubakio-Yamamoto K, et al. Evaluation of a homogeneous assay for measuring LDL-cholesterol in hyperlipidemic serum specimens. J Atheroscler Thromb 2008;15:82–6.
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Please cite this article as: Tanahashi K, et al, Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.05.003
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Contents lists available at ScienceDirect
Life Sciences journal homepage: www.elsevier.com/locate/lifescie
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Article history: Received 2 November 2013 Accepted 2 May 2014 Available online xxxx
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Keywords: ADMA Aerobic exercise capacity Cardiovascular disease Aging Endothelin
Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
i n f o
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Aims: Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase, an enzyme responsible for the generation of NO. Plasma concentrations of ADMA increase in the elderly and in postmenopausal women. In fact, an elevated ADMA level is a risk factor of cardiovascular disease. Aerobic exercise has a beneficial effect on cardiovascular disease. However, the relationship between ADMA and aerobic fitness is unknown. The aim of this study was to determine whether plasma ADMA concentrations correlate with aerobic fitness levels in postmenopausal women. Main methods: Thirty healthy postmenopausal women aged 50–76 years participated in this study. We measured plasma concentrations of ADMA and oxygen consumption at the ventilatory threshold (VO2VT) as an index of aerobic fitness. Subjects were divided into the low aerobic fitness (Low fitness) and high aerobic fitness (High fitness) groups, and the dividing line was set at the median VO2VT value. Key findings: VO2VT was significantly higher in the High fitness group than in the Low fitness group (P b 0.01). The plasma ADMA concentrations in the High fitness group were significantly lower than those in the Low fitness group (P b 0.05). There was a negative correlation between plasma ADMA concentrations and VO2VT (r = −0.532, P b 0.01). Significance: We found that plasma ADMA concentrations were associated with aerobic fitness in postmenopausal women. The results of this study suggest that habitual aerobic exercise may decrease plasma ADMA concentrations. © 2014 Published by Elsevier Inc.
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Koichiro Tanahashi a, Nobuhiko Akazawa a, Asako Miyaki a, Youngju Choi b, Song-Gyu Ra a, Tomoko Matsubara a, Hiroshi Kumagai a, Satoshi Oikawa a, Takashi Miyauchi c, Seiji Maeda b,⁎
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Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women☆
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Introduction
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Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of nitric oxide (NO) synthase, an enzyme responsible for NO generation. Increased ADMA levels are known to decrease NO synthesis, leading to endothelial dysfunction (Cooke, 2004). Elevated ADMA levels are associated with increased risks of cardiovascular disease (Böger et al., 2009; Leong et al., 2008), and relationships between increased circulating ADMA levels and several traditional cardiovascular risk factors, such as hypertension, hypercholesterolemia, insulin resistance, obesity, and smoking, have been reported (Kielstein et al., 2003;
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☆ This work was presented at the Thirteenth International Conference on Endothelin (held at the University of Tsukuba, Tokyo Campus: September 8–11, 2013), and was published as an abstract form in the Program and Abstract Book (Cross Border Session PC-8: 2013) of this Meeting. ⁎ Corresponding author at: Faculty of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8574, Japan. Tel.: +81 29 853 2683; fax: +81 29 853 2986. E-mail address: [email protected] (S. Maeda).
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Böger et al., 1998; Sydow et al., 2005; Eid et al., 2004). Therefore, it is important to attenuate increased circulating ADMA concentrations. ADMA is a naturally occurring amino acid that is produced by methylation of arginine residues in intracellular proteins via protein arginine methyltransferases (PRMTs) (Leiper and Vallance, 1999). ADMA is eliminated by renal clearance (Vallance et al., 1992). However, N80% of ADMA is hydrolyzed into citrulline and dimethylamine by dimethylarginine dimethylamonohydrolase (DDAH) (Achan et al., 2003). The elevation of plasma ADMA that occurs with vascular disease and risk factors is largely due to enhanced PRMT activity or impaired DDAH activity (Cooke, 2004). Aerobic exercise has been recommended for preventing cardiovascular disease (Thompson et al., 2003). Regular aerobic exercise is an effective strategy for reducing the risk of cardiovascular disease in middle-aged and elderly individuals (Tanaka et al., 2000). McMurray et al. reported that increased aerobic fitness is associated with reduced cholesterol and blood pressure levels, risk factors of cardiovascular disease (McMurray et al., 1998). It is well known that physical activity and aerobic fitness decrease with age (Hawkins and Wiswell, 2003). Aging increases plasma ADMA concentrations (Miyazaki et al., 1999), and
http://dx.doi.org/10.1016/j.lfs.2014.05.003 0024-3205/© 2014 Published by Elsevier Inc.
Please cite this article as: Tanahashi K, et al, Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.05.003
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Materials and methods
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A total of 30 healthy postmenopausal women (50–76 years) participated in the present study. The subjects were non-smokers, non-obese, and free of overt cardiovascular disease as assessed by medical records. All subjects were at least 2 years postmenopause and none was taking medications. All potential risks were explained to the study participants, and each provided written informed consent to participate in the study. All of the procedures were reviewed and approved by the Ethics Committee of the University of Tsukuba.
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Experimental protocol
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All experiments, excluding the cycle exercise test, were performed in the morning after a 12-h overnight fast. Subjects abstained from alcohol and caffeine for at least 12 h and did not exercise for at least 24 h before beginning the experiment to avoid any potential acute effects of exercise. Measurements were obtained in a quiet, temperaturecontrolled room (24–26 °C). After a resting period of at least 20 min, arterial blood pressure was measured and a blood sample was drawn to determine the plasma ADMA concentrations and blood chemistry. After these procedures, aerobic fitness was measured during an incremental cycle ergometer exercise.
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Anthropometric variables
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Body weight was measured to the nearest 0.1 kg with a digital scale. Height was measured to the nearest 0.1 cm by using a wall-mounted stadiometer. Body mass index (BMI) was calculated as the participant's weight (in kilograms) divided by their height (in meters squared).
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Plasma ADMA concentrations
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Each blood sample was placed in a chilled tube containing ethylenediaminetetraacetic acid (2 mg/mL) and centrifuged at 2000 ×g for 15 min at 4 °C. The plasma was stored at −80 °C until analysis. Plasma ADMA concentrations were determined using a commercial enzymelinked immunosorbent assay kit (Immundiagnostik AG, Bensheim, Germany). The ADMA assay was performed according to the manufacturer's instructions. The intra-assay coefficients of variation were 5.8–7.9%. The standards and controls provided with the kits were used to take the measurements.
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Blood biochemistry
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Serum concentrations of total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and fasting plasma concentrations of glucose were determined using standard enzymatic techniques. Total cholesterol and triglyceride concentrations were determined using the cholesterol dehydrogenase and glycerol kinase methods, respectively (Allain et al., 1974; Kohlmeier, 1986). Low-density lipoprotein cholesterol and high-density lipoprotein cholesterol concentrations were measured directly (Finley et al.,
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Aerobic fitness
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Oxygen uptake at the ventilatory threshold (VO 2VT) was measured during the incremental cycle ergometer exercise using an online computer-assisted circuit spirometer (AE300S; Minato Medical Science, Osaka, Japan). All subjects underwent an incremental cycle exercise test (2 min at 20 W, followed by 10-W increases every 1 min) until they felt exhausted or reached 85% of their agepredicted maximal heart rate (HRmax = 220 − age) (Fox et al., 1971). Each individual VO2VT value was calculated using regression analysis of the slopes of CO2 production, O2 uptake, and the minuteventilation plot. The subjects (9.2–18.2 mL/min/kg) were divided into the low aerobic fitness (Low fitness) and high aerobic fitness (High fitness) groups according to the median VO2VT value (11.8 mL/min/kg).
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1978; Yamashita et al., 2008). The glucose concentration was assayed 124 using the hexokinase and glucose-6-phosphate dehydrogenase methods 125 (Slein, 1963). 126
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Results
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Table 1 shows the characteristics of the study participants by subgroup according to VO2VT results (Low fitness and High fitness). There were no significant differences in age, height, systolic blood pressure, diastolic blood pressure, triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, or glucose between the groups. Body weight and BMI were significantly lower in the High fitness group than in the Low fitness group (P b 0.01). VO2VT values in the High fitness group were significantly higher than those in the Low fitness group (P b 0.01). Fig. 1 shows the plasma ADMA
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Unpaired Student's t tests were used to evaluate differences between the Low fitness and High fitness groups. Relationships between plasma ADMA concentrations and aerobic fitness were analyzed by using a Pearson correlation coefficient. All data are expressed as the means ± SD. Differences were considered significant at values of P b 0.05.
All n = 30 Age, years Height, cm Body weight, kg BMI, kg/m2 SBP, mm Hg DBP, mm Hg TG, mol/L TC, mol/L HDL-C, mol/L LDL-C, mol/L Glucose, mol/L VO2VT, mL/min/kg
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t1:1 t1:2
Table 1 Subject characteristics.
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Supine systolic and diastolic arterial blood pressures were recorded 141 from the right arm using a semi-automated device (form PWV/ABI; 142 Colin Medical Technology, Komaki, Japan). 143 Statistical analyses
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Blood pressure and heart rate
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postmenopausal women have higher plasma ADMA concentrations than premenopausal women (Schulze et al., 2005). However, the relationship between aerobic fitness and ADMA in postmenopausal women is unclear. The purpose of this study was to examine the relationship between aerobic fitness and plasma ADMA concentrations in postmenopausal women. We hypothesized that plasma ADMA concentrations are lower in postmenopausal women with high aerobic fitness levels than in their low aerobic fitness peers. To investigate this hypothesis, we measured aerobic exercise capacity and plasma ADMA concentrations in postmenopausal women.
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60.7 154.3 54.6 22.9 120 73 1.3 5.9 1.7 3.5 5.1 12.5
± ± ± ± ± ± ± ± ± ± ± ±
6.0 4.3 8.4 3.5 17 11 0.8 0.7 0.5 0.7 0.4 2.2
Subjects divided according to VO2VT
t1:3
Low fitness (n = 15)
High fitness (n = 15)
t1:4
61.2 153.7 59.7 25.2 122 75 1.4 6.0 1.6 3.7 5.1 10.7
60.1 155.0 49.4 20.6 117 71 1.1 5.8 1.8 3.4 5.1 14.3
t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16
± ± ± ± ± ± ± ± ± ± ± ±
6.9 4.7 8.2 3.1 15 10 1.1 0.6 0.4 0.6 0.4 0.7
± ± ± ± ± ± ± ± ± ± ± ±
5.1 3.9 4.6⁎⁎ 2.0⁎⁎ 20 12 0.4 0.8 0.6 0.7 0.4 1.6⁎⁎
Data are means ± SD. BMI; body mass index, SBP; systolic blood pressure, DBP; diastolic blood pressure, TG; triglycerides, TC; total cholesterol, HDL-C; high-density lipoprotein cholesterol, LDL-C; low-density lipoprotein cholesterol, VO2VT; oxygen uptake at the ventilatory threshold. ⁎⁎ P b 0.01 vs. the Low fitness group.
Please cite this article as: Tanahashi K, et al, Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.05.003
t1:17 t1:18 t1:19 t1:20 t1:21
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Conclusion
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In the present study, we demonstrated that plasma ADMA concentrations were lower in individuals with high aerobic fitness levels compared to their peers with low aerobic fitness levels. Furthermore, we showed that plasma ADMA concentrations were negatively correlated with aerobic fitness in postmenopausal women. Our findings suggest that regular exercise or increased physical activity may decrease plasma ADMA concentrations.
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Conflict of interest statement
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cardiovascular disease (Cooke, 2004). Elevated ADMA is a cause of reduction in NO synthesis and activation of renin–angiotensin system, which results in endothelial dysfunction and arteriosclerosis (Cooke, 2004; Hasegawa et al., 2007; Furuki et al., 2008). Furthermore, ADMA levels are increased in patients with hypertension (Kielstein et al., 2003), coronary artery disease (Valkonen et al., 2001) and chronic heart failure (Usui et al., 1998). In the present study, we demonstrated that plasma ADMA concentrations were lower in the High fitness group than in the Low fitness group. These results suggest that a high fitness-related ADMA reduction may help inhibit cardiovascular disease. In the present study, plasma ADMA concentrations were negatively correlated with VO2VT. This is the first study to show the relationship between plasma ADMA concentrations and aerobic fitness in healthy postmenopausal women. However, it should be noted that we could not determine the causal relationship between plasma ADMA concentrations and aerobic fitness. One earlier study reported that aginginduced increases in oxidative stress induce increases in plasma ADMA concentrations (Fabian et al., 2012). Oxidative stress increases ADMA levels by enhancing PRMT activity and/or decreasing DDAH activity (Sydow and Münzel, 2003). In contrast, Bełtowski and Kedra reported that antioxidant treatment, such as intake medications or supplements, decreases plasma ADMA concentrations (Bełtowski and Kedra, 2006). Aerobic exercise capacity is reportedly associated with the antioxidant defense system (Pialoux et al., 2009). In addition, a recent study reported that aerobic exercise training increased DDAH mRNA expression in obese men (Hanssen et al., 2011). Therefore, the decrease in oxidative stress, PRMT, and DDAH levels may contribute to exercise-related ADMA level decreases. In this study, the mean BMI of the Low fitness group was higher than that of the High fitness group. One study reported that circulating levels of ADMA increased in obesity (Eid et al., 2004). However, we did not observe a correlation between plasma ADMA concentrations and BMI in this study. This inconsistent result is likely explained by the focus on only normal weight healthy postmenopausal women in the present study. After adjustment for BMI, the association between plasma ADMA concentrations and VO2VT was confirmed. Therefore, our results suggest that the correlation between plasma ADMA concentrations and physical fitness is independent of BMI in normal weight postmenopausal women. There are several limitations of this study that should be emphasized. First, the present study had a relatively small sample size due to the rigorous screening criteria of healthy postmenopausal women who were non-smokers, non-obese, free of overt cardiovascular disease, and taking no medications or supplements. Accordingly, further studies should include larger sample sizes. Second, although it has been reported that VO2VT is associated with daily physical activity (Malatesta et al., 2004), we did not measure the individual exercise type, frequency, intensity, or duration. We also did not measure DDAH or NO levels or track any indices of endothelial function. Further studies are needed to investigate the effects of individual exercise training protocols on endothelial function.
In the present study, we investigated the relationship between plasma ADMA concentrations and aerobic fitness in postmenopausal women. We demonstrated that the plasma ADMA concentration was significantly lower in the High fitness group than in the Low fitness group. Moreover, plasma ADMA concentrations were negatively correlated to VO2VT, and this relationship was independent of age and BMI. These findings suggest that improved cardiovascular fitness may decrease plasma ADMA concentrations in postmenopausal women. ADMA is an endogenous competitive inhibitor of NO synthase. Plasma concentrations of ADMA increase in elderly individuals and postmenopausal women (Miyazaki et al., 1999; Schulze et al., 2005). An increased ADMA level has been reported as a risk factor of
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Discussion
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concentrations in the Low fitness and High fitness groups. The plasma ADMA concentrations were significantly lower in the High fitness group than in the Low fitness group (Low, 10.7 ± 0.7 mL/min/kg vs. High, 14.3 ± 1.6 mL/min/kg; P b 0.05). Moreover, there was a significant negative correlation between plasma ADMA concentrations and VO2VT (r = − 0.531, P b 0.01; Fig. 2). After adjustment for age and BMI, the association between plasma ADMA concentrations and VO2VT was confirmed (β = − 0.435, P b 0.05). There was no relationship between plasma ADMA concentrations and postmenopausal periods (r = 0.05, P = 0.77).
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Fig. 1. Plasma ADMA concentrations in the Low fitness and High fitness groups. Data are expressed as the mean ± SD.
Fig. 2. Relationship of oxygen uptake at the ventilatory threshold (VO2VT) and plasma ADMA concentrations.
The authors report no conflicts of interest with respect to this manuscript.
Please cite this article as: Tanahashi K, et al, Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.05.003
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This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24590654). We thank Dr. Ryuichi Ajisaka, University of Tsukuba, for his advices and Mr. Ryota Higashino, University of Tsukuba, for his technical assistance.
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Please cite this article as: Tanahashi K, et al, Plasma ADMA concentrations associate with aerobic fitness in postmenopausal women, Life Sci (2014), http://dx.doi.org/10.1016/j.lfs.2014.05.003
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