Journal of Exercise Physiologyonline

188 Journal of Exercise Physiologyonline February 2017 Volume 20 Number 1 Editor-in-Chief Official Research Journal of Tommy the American Boone, PhD, Society MBA of Review Exercise Board Physiologists Todd Astorino, PhD Julien Baker, ISSN 1097-9751 PhD Steve Brock, PhD Lance Dalleck, PhD Eric Goulet, PhD Robert Gotshall, PhD Alexander Hutchison, PhD M. Knight-Maloney, PhD Len Kravitz, PhD James Laskin, PhD Yit Aun Lim, PhD Lonnie Lowery, PhD Derek Marks, PhD Cristine Mermier, PhD Robert Robergs, PhD Chantal Vella, PhD Dale Wagner, PhD Frank Wyatt, PhD Ben Zhou, PhD Official Research Journal of the American Society of Exercise Physiologists ISSN 1097-9751 JEPonline Effects of Muay Thai Aerobic Dance on Biochemical Bone Markers and Physical Fitness in Elderly Women Nisakorn Tantiwiboonchai 1, Thanomwong Kritpet 1, Pongsak Yuktanandana 2 1 Faculty of Sports Science, Chulalongkorn University, Bangkok, Thailand, 2 Department of Orthopedics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand ABSTRACT Tantiwiboonchai N, Kritpet T, Yuktanandana P. Effects of Muay Thai Aerobic Dance on Biochemical Bone Markers and Physical Fitness in Elderly Women. JEPonline 2017;20(1):188-199. The purpose of this study was to investigate the effects of Muay Thai aerobic dance on biochemical bone markers and physical fitness in elderly women. Forty elderly women (60 to 77 yrs old) who lived in a nursing home were divided into an experimental group that performed Muay Thai aerobic dance (MAD, N = 20) and a control group (CON, N = 20). Both groups took vitamin D 2 20,000 IU wk -1 and calcium carbonate 1,000 mg d -1 after breakfast. The MAD group worked out at an intensity of 70-75% of maximum heart rate 3 times wk -1, 40 min d -1 for 16 wks. The findings indicate that after 16 wks the level of CTx (cross-linked C-telopeptide of collagen type 1) in both groups significantly decreased from 0.66 ± 0.32 to 0.48 ± 0.19 and 0.66 ± 0.24 to 0.57 ± 0.25 (ng ml -1 ), respectively. The greater percentage reduction and benefit was found in the MAD group (-27.27%) versus the CON group (-13.64%). The results also showed several variables in physical fitness were significantly different between MAD and CON. The elderly women in the MAD group had greater improvement in their lower back flexibility, muscle strength, muscle endurance, cardiorespiratory fitness, static balance, and dynamic balance. The results indicate that the Muay Thai aerobic dance program was effective in reducing the bone resorption process and in increasing physical fitness in elderly women. Key Words: Muay Thai Aerobic Dance, Vitamin D and Calcium, Biochemical Bone Markers, Physical Fitness

189 INTRODUCTION Osteoporosis poses a great challenge in today s aging society. It stems from a continuation of bone loss, and is a major cause of bone fractures in the elderly (1,19). As a result, preventing bone loss is the first step for impeding osteoporosis. Bone loss is correlated closely with the mechanical loading of bones (physical activities), nutrition, and aging. Inadequate intake of calcium and vitamin D leads to secondary hyperparathyroidism and increased bone resorption that results in an increased incidence of osteoporosis related fractures in elderly populations (9,27). Not only are elderly women most prone to bone loss and, therefore, to osteoporosis due to menopause, it is also widely reported that elderly women experienced vitamin D insufficiency and lacked of calcium intake and physical activity (5,18,21). The situation is worse for elderly women who live in nursing homes. Kruavit et al. (15) reported that most nursing home residents had osteoporosis or osteopenia. Moreover, they reported that one-third of the elderly women residing in nursing homes had vitamin D insufficiency with a dietary calcium intake less than 350 mg d -1. To prevent bone loss and osteoporosis, it is recommended that the elderly take their medications while consuming the appropriate nutritional supplements, exercise regularly, and avoid tobacco and alcohol consumption (1). Exercise, especially weight bearing exercise, has been shown to be effective in stimulating an osteogenic response (25,28), improving bone by increasing bone mass, and exerting positive effects on physical fitness (6,22). Aerobic dance is classified as a weight bearing exercise. It is divided into non-impact, low-impact, and high-impact aerobic dance where lowimpact aerobic dance is most appropriate for the middle-aged and elderly women (30). The movement of low-impact aerobic dance requires at least 1 foot on the ground at all-time, thus sharing the same principle of keeping balance in martial art (7). Martial aerobic dance, such as Aero box, Muay Thai aerobic dance, Body combat and Tae Bo, has gained popularity as well (8,10). In particular, the Muay Thai aerobic dance utilizes Muay Thai movements that include punches, elbows, knees, and kicks to create aerobic exercise (23,26). Hence, the dance offers a multidimensional movement exercise that could be regarded as an ideal modality for older people (especially since it has been demonstrated to enhance physical fitness, balance, body composition, muscle strength, muscle endurance, flexibility, and cardiorespiratory fitness) (12,14). The purpose of this paper was to determine the effects of the Muay Thai aerobic dance with vitamin D and calcium supplementation on biochemical bone markers and physical fitness in elderly women. Biochemical bone markers are useful predictor for long-term response of bone mass, while physical fitness is an excellent indicator for overall health in elderly. To our knowledge, no research has addressed the benefits of a combination of aerobic dance, especially the Muay Thai aerobic dance, with vitamin D and calcium supplementation in elderly women, while only a few studies (3,25) found significant improvements in biochemical bone markers after a period of aerobic dance in working women. It is hypothesized that the Muay Thai aerobic dance with vitamin D and calcium supplementation will yield substantial improvements in the biochemical bone markers and physical fitness in elderly women and, therefore, can be used as a means to impede bone loss and osteoporosis.

190 METHODS Subjects Forty elderly women (ages between 60 to 77 yrs) living in public nursing homes in Bangkok and its vicinity (Ban-Bangkhae 1 and Pathum Thani) volunteered to participate in this study. The inclusion criteria were ambulatory. Moreover, the subjects were not allowed to smoke or consume alcoholic beverage, but they were allowed to have no more than 2 cups of tea or black coffee per day. The subjects could not have taken medication for bone metabolism (hormonal replacement therapy, bisphosphonate, calcium, and vitamin D) during the period of 1 yr prior to the study. They could not have renal and/or hepatic disease. The subjects must not be exercising more than twice a week. Also, the subjects would be excluded if they participated in the program fewer than 80% of the training sessions or if they missed the training session for more than 2 consecutive weeks. They were also excluded if they were injured or sick during the experiment. The subjects were separated into 2 groups by the location of nursing homes. The subjects who lived in Ban-Bangkhae 1 were assigned into the experiment group (Muay Thai aerobic dance: MAD) while those who lived in Pathum Thani were assigned to the control group (CON). Both groups engaged in similar routine physical activity arranged by the nursing home, such as recreational activity (singing and dancing) and household chores. Both groups received vitamin D 2 (ergocalciferol) 20,000 IU wk -1 and calcium carbonate 1,000 mg d -1 after a morning meal 16 wks. The only difference between the control and the experimental group was the introduction of Muay Thai aerobic dance to the experimental group. The research design was approved by The Ethics Committee of Health Science group, Chulalongkorn University, and all subjects provided their written informed constant. Procedures Testing Procedures Biochemical bone markers and physical fitness were assessed before training (baseline) and 16 wks after training. The tests were separated into two consecutive days. The subjects were tested for: (a) biochemical bone markers on the first day; and (b) physical fitness on the second day. Measurement of Biochemical Bone Markers The biochemical bone markers included bone formation (procollagen type 1 N-propeptide: P1NP), bone resorption (cross-linked C-telopeptide of collagen type 1: CTx), bone turnover (NMIDosteocalcin: OC), total vitamin D (25(OH)D), and Parathyroid hormone (PTH). All of these biochemical bone markers were analyzed using Electrochemiluminescence immunoassay ECLIA with Cobas e 411 technique. The serum calcium, alkaline phosphatase: ALP (liver function), and serum creatinine: Cr (kidney function) using an automated biochemical analyzer with Cobas Integra 400 plus. The biochemical markers (ALP and Cr) at the baseline indicated that there were no liver or kidney malfunctions in the subjects. All subjects were asked to fast for 8 hrs prior to sampling their blood between 7:00-8:00 a.m. All biochemical bone markers were tested at the clinical laboratory at King Chulalongkron Memorial Hospital.

Measurement of Physical Fitness The subjects physical fitness measurements that were determined included their body composition, flexibility, muscle strength, muscle endurance, cardiorespiratory fitness, and balance. All physical fitness and balance test were measured at baseline and after 16 wks of training. The detailed assessment of each physical fitness measurement is as follows. Body Composition Body mass index was calculated using the BMI equation, while percent fat was analyzed using the bioelectrical-impedance analysis (BC-532 Tanita, Japan). Flexibility Upper body shoulder flexibility was tested using the back scratch test for both right and left sides of the body (24). Lower body flexibility was tested using the sit and reach test. The subjects were asked to sit on the floor and bend their body forward on a sliding ruler that measured from 0-30 centimeters (2). Muscle Strength and Endurance Leg muscle strength and endurance were assessed using the 30-sec chair sit to stand test (CS-30). Arm strength was assessed only in the dominant hand using a hand grip dynamometer (T.K.K.5001, Takei, Japan). Upper body strength and endurance were measured during the arm curl test using a 2 kg dumbbell in which the subjects were asked to performed as many curls as possible in a 30-sec time period (24). Estimated Cardiorespiratory Fitness The Six-Minute Walk Test was used. The subjects distance, blood pressure, and heart rate were recorded immediately after finishing the test. Peak VO 2 was then calculated using the following equation (2): Peak VO 2 = [0.02 distance (m)] - [0.191 age] [0.07 weight (kg)] + [0.09 height (cm)] + [0.26 HR systolic BP (10-3 )] + 2.45 Balance Static balance and dynamic balance were measured using the following procedure. Static balance was measured using the Single Leg Stance Test (SLS) with the eyes open. The timer was started after the subjects raised one foot about 15 to 20 cm off the ground with both hands holding the waist. The test was stopped when the hand moved off the waist or the foot touched the ground. Dynamic balance was evaluated by the 3-m Time Up and Go Test (TUG). Test times were recorded when the subjects got up from a chair with a seat height of 43 cm, and walked 3 m and then returned to the chair and resumed a sitting position (24). Measurements of Bone Mineral Density (BMD) Each subject s BMD was measured with a quantitative ultrasound measurement of the calcaneus (heel bone) (Sahara Clinical bone sonometer, Hologic, USA). The BMD was only used as characteristic of the subject, thus BMD was performed only at baseline. Exercise Program The Muay Thai aerobic dance was derived from Mae Mai Muay Thai, the art of self-defense in Thai boxing (13). It was validated using the index of congruence technique from 5 experts. Then, the pilot study of Muay Thai aerobic dance was conducted with 5 elderly women aged 60 to 75 yrs who lived in Bangkok. The study showed that the average heart rate during the 191

Muay Thai aerobic dance in the first and second pilot studies were 110 and 106 beats min -1, respectively, and there was no statistical difference between the results of the two pilot studies (P = 0.53). During a 40-min session, the Muay Thai aerobic dance consisted of: (a) a 10-min warm-up (that consisted of a 5-min of walking and stretching); (b) a 20-min Muay Thai aerobic dance; and (b) a 10-min cool down (that consisted of stretching exercises). The exercises used for the lower extremities included mambo, heel touch, V-step, step touch, leg curl, side tap, walking forward-backward, straight knee strike, pecking foot-thrust, low round kick, and marching. The exercises used for the upper extremities were the straight punch, hook, uppercut-short, uppercut, elbow, reverse elbow strike, and round elbow strike. The exercise training was 3 times wk -1 for 16 wks at Ban-Bangkhae 1. During the first two wks, the training group performed at an intensity of 60 to 69% of their maximal heart rate. From week 3 to week 16, the subjects worked out at an intensity of 70 to 75% of their maximal heart rate. Heart rate was monitored using a Polar heart rate monitor (M53 system) throughout the exercise sessions. Statistical Analyses The data are shown as mean ± SD. A paired t-test was used for comparing pre- and post-test within groups, while a one-way analysis of covariance (ANCOVA) was used to find difference between groups. All of biochemical bone markers and physical fitness at baseline were used as a covariate in all tests. The SPSS package version 22 (IBM SPSS Statistics, USA) was used to analyze the data. Statistical significance was set at P 0.05. RESULTS Baseline Characteristics Table 1 presents the baseline characteristics of the elderly women aged 60 to 77 yrs. There were no significant differences between the control group and the experimental group. The average serum creatinine and alkaline phosphatase were within the reference range for each (15). 192 Table 1. Subject Characteristics (mean ± SD). Training Group (MAD: N = 20) Control Group (CON: N = 20) P-value Age (yr) 70.8 ± 4.4 71.0 ± 5.2 0.84 Height (cm) 149.8 ± 8.6 148.3 ± 8.0 0.57 T score (BMD of the right heel) -3.2 ± 0.9-3.7 ± 0.9 0.07 T score (BMD of the left heel) -3.2 ± 0.9-3.7 ± 0.8 0.10 Serum creatinine (mg dl -1 ) 0.7 ± 0.2 0.7 ± 0.2 0.92 Alkaline phosphatase (U L -1 ) 75.2 ± 16.7 75.6 ± 22.9 0.96

193 Biochemical Bone Markers The values for the subjects biochemical bone markers and serum calcium are reported in Table 2. The results indicate that only CTx was significantly decreased from the baseline in both the control and the experimental groups. A percentage reduction of 13.64% and 27.27% was observed in the CON and the MAD groups, respectively. In addition, CTx in the MAD group was significantly different from CTx in the CON group after 16 wks of intervention. No statistical significance was found in other biochemical bone markers when compared within groups and between groups. Table 2. Biochemical Bone Markers and Serum Calcium (mean ± SD). Training Group Control Group (MAD) Change (CON) (%) Baseline 16 wks Baseline 16 wks Change (%) CTx (ng ml -1 ) 0.66 ± 0.32 0.48 ± 0.19*# -27.27 0.66 ± 0.24 0.57 ± 0.25* -13.64 P1NP (ng ml -1 ) 58.25 ± 22.64 54.85 ± 20.09-5.84 57.62 ± 22.36 57.61 ± 18.57-0.02 OC (ng ml -1 ) 28.94 ± 11.04 27.10 ± 9.27-6.36 33.31 ± 11.69 30.74 ± 10.17-7.72 25(OH)D (ng ml -1 ) 36.49 ± 6.57 33.88 ± 8.44-7.15 37.22 ± 8.03 34.50 ± 9.52-7.31 PTH (pg ml -1 ) 39.49 ± 13.02 42.87 ± 14.34 8.56 48.30 ± 11.82 47.57 ± 12.09-1.51 S-Ca (mg dl -1 ) 9.01 ± 0.38 9.15 ± 0.47 1.55 8.91 ± 0.34 9.08 ± 0.39 1.91 *P<0.05 vs. baseline, #P<0.05 vs. the control group, CTx = Serum cross-linked C-terminal telopeptides of collagen type 1, P1NP = Serum N-terminal propeptides of collagen type 1, OC = Serum osteocalcin, 25(OH)D = 25-hydroxyvitamin D, PTH = Parathyroid hormone, S-Ca = Serum calcium Physical Fitness The subjects physiological data and physical fitness are shown in Table 3. A comparison within groups showed that no statistical significance was found in all variables in physical fitness of the CON group, but the subjects in the MAD group experienced statistically significant improvements in blood pressure, flexibility, muscle strength, muscle endurance, cardiorespiratory fitness, and balance. When compared between groups after 16 wks of intervention, systolic blood pressure, the sit and reach test, muscle strength and muscle endurance in both the upper and the lower extremities, cardiorespiratory fitness, static balance, and dynamic balance in the MAD group were statistically different from the same measurements in the CON group.

194 Table 3. Physical Fitness (mean ± SD). Training Group (MAD) Physiological Data Control Group (CON) Baseline 16 wks Baseline 16 wks Body Weight (kg) 57.3 ± 14.0 57.6 ± 14.6 52.7 ± 11.8 54.1 ± 12.6 Heart Rate Resting (beats min -1 ) 78.6 ± 8.7 78.7 ± 9.1 75.9 ± 8.8 74.9 ± 10.4 Systolic Blood Pressure (mm Hg) 132.9 ± 18.5 125.8 ± 14.3*# 132.6 ± 19.7 134.4 ± 17.7 Diastolic Blood Pressure (mm Hg) 70.4 ± 11.5 65.1 ± 9.2* 67.4 ± 9.9 67.2 ± 8.3 Health-Related Physical Fitness Body Composition BMI (kg m -2 ) 25.6 ± 5.9 25.7 ± 6.2 24.0 ± 5.0 24.6 ± 5.4 Body Fat (%) 33.3 ± 6.4 34.4 ± 6.1 34.1 ± 7.8 35.0 ± 8.5 Waist-Hip Ratio 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 Flexibility Back Scratch R (cm) -11.6 ± 12.1-9.7 ± 10.9* -16.5 ± 17.3-17.0 ± 15.5 Back Scratch L (cm) -16.7 ± 13.5-15.8 ± 10.3-25.9 ± 16.5-23.3 ± 16.8 Sit and Reach (cm) 8.2 ± 6.9 10.8 ± 8.2*# 5.3 ± 11.3 4.1 ± 10.8 Muscle Strength and Endurance Legs: Chair Sit to Stand-30 S (times) 14.8 ± 4.6 19.7 ± 6.8*# 10.3 ± 5.4 10.4 ± 5.0 Arm Curl-30 S (times) Arms: 18.2 ± 3.6 22.0 ± 5.5*# 14.6 ± 5.1 14.5 ± 5.4 Hand Grip (kg) 17.0 ± 4.9 19.4 ± 5.3*# 16.1 ± 5.8 16.2 ± 5.9 Cardiorespiratory Fitness (6 Min Walk Test) Peak VO 2 (ml kg -1 min -1 ) 10.2 ± 2.8 11.9 ± 3.2*# 7.8 ± 3.6 8.1 ± 3.8 Distance (m) 362.6 ± 103.3 410.0 ± 108.3*# 267.7 ± 128.2 274.6 ± 132.3 Balance Single Leg Stance (sec) 17.7 ± 21.7 30.8 ± 30.0*# 12.0 ± 11.6 11.5 ± 11.0 Time Up & Go-3 m (sec) 10.6 ± 2.0 8.4 ± 2.7*# 17.2 ± 10.2 16.4 ± 10.3 *P<0.05 vs. baseline, #P<0.05 vs. the control group

195 DISCUSSION This study investigated the effects of a combination of Muay Thai aerobic dance with calcium and vitamin D supplementation. The results showed the subjects in the MAD group had positive improvements in both biochemical bone markers and physical fitness. As people experience the bone remodeling process whereby bone loss occurs when the bone resorption rate is greater than the rate of bone formation, it is important to prevent bone loss by promoting the bone formulation process while reducing the bone resorption process. After the intervention, the level of CTx, which is the biochemical bone marker for bone resorption, significantly decreased when compared to the baseline. However, the biochemical bone markers for bone formation (P1NP) and for bone turnover (OC) after 16 wks were not statistically different from the levels before the intervention. As a result, the findings suggest that vitamin D and calcium supplementation significantly reduced the bone resorption process, which is in agreement with the findings by Grados et al. (9) and Kruger et al. (16). Calcium is an important mineral component of bone. Individuals who consume inadequate calcium can cause high bone turnover. The calcium supplementation can offset the calcium requirement of postmenopausal or elderly women that can prevent bone loss or have an antiresorptive effect on bone (17,20). After 16 wks of intervention, the subjects in the MAD group had significantly lower level of CTx than the subject in the CON group. This is a positive benefit of the Muay Thai aerobic dance to bone resorption process. The level of CTx in the MAD group demonstrated a 27.27% reduction, which was twice the response in the CON group. The Muay Thai aerobic dance program required movements in the lower extremities that required the subjects to stand with one leg, thus resulting in an increase in the impact force and muscle contraction, which induced interstitial fluid movement in bone transmitted by biochemical signals (4,11). The mechanical loading of exercise inhibited osteoclast function by mechanoreceptors via osteocyte. The decreased osteoclastogenesis led to decreased bone resorption and the maintenance of bone mass (11). Moreover, the findings in the present study suggest that low-impact aerobic dance with vitamin D and calcium supplementation help to stabilize bone metabolism through reduction of bone resorption process rather than bone formation as observed by the results where the level of CTx was significantly lower but the levels of P1NP and OC were unchanged. The level of a subject s physical fitness is a good indication of overall health. In this study, the analysis of physical fitness included body composition, flexibility, muscle strength, muscle endurance, cardiorespiratory fitness, and balance. While some physical fitness test batteries do not include balance, the present study used balance tests to help assess the risk of falls in the elderly. This information is critical to understanding why falls increase with aging and bone loss. After 16 wks of training, the results showed great benefits of the Muay Thai aerobic dance. The subjects in the MAD group experienced improvement in lower back flexibility, muscle strength and muscle endurance, cardiorespiratory fitness, and balance compared to the subjects in the CON group who received only supplementation. These findings are in agreement with previous studies showing the benefit of exercise training in elderly subjects (6,30). The Muay Thai aerobic dance clearly increased the activity of the both the lower and the upper extremities. The movement of the Muay Thai aerobic dance required the subjects to

punch and elbow strike, to have footwork during dancing, and to stand with one leg while kneeing, pecking, or kicking. Thus, the subjects enjoyed various movements in both upper and lower extremities suitable for increasing muscle strength and balance. Moreover, the subjects in the MAD group continuously trained with a moderate intensity for 20 min that resulted in a significant improvement in cardiorespiratory fitness. This finding was in line with previously published research by Kanjanavanit et al. (12), Kritpet et al. (14), and Suwannarat (26). As a result, it is clear that the Muay Thai aerobic dance intervention reduced bone resorption and improved physical fitness. These findings are important for numerous healthrelated reasons, and they are consistent with other studies (6,19,22,29) that suggest an improvement in muscle strength and cardiorespiratory endurance helps to reduce premature moratlity, disability, and other health complication (related to the subjects neuroadptation, reflex responses and proprioception, bone density, and quality of life). In addition, moderateintensity exercise yielded benefits to blood vessels by helping them to maintain their essential elasticity, thus leading to a decrease in resting blood pressure (1,29). CONCLUSIONS The purpose of this study was to investigate the effects of the Muay Thai aerobic dance with calcium and vitamin D supplementation. The subjects were recruited from the elderly in nursing home residents. This study controlled for physical activity and supplementation of calcium and vitamin D where the subjects in both the control and the experimental groups received vitamin D 2 20,000 IU wk -1 and calcium carbonate 1,000 mg d -1 after breakfast. The only difference between the two groups was the 40-min session of the Muay Thai aerobic dance required of the subjects in the experimental group. The experimental group performed the Muay Thai aerobic dance 3 times wk -1 for 16 wks. Biochemical bone markers and physical fitness were tested at baseline and after 16 wks of training. The results showed that the subjects in the experimental group (MAD) had better bone resorption percentage reduction and improvement in physical fitness. In particular, the subjects in the experimental group had twice the reduction in percentage of bone resorption versus the subjects in the control group. Moreover, the subjects in the experimental group experienced significantly better lower back flexibility, muscle strength and muscle endurance in both the upper and the lower extremities, cardiorespiratory fitness, and both static balance and dynamic balance than the subjects in the control group. Therefore, the findings indicate the suitability of the Muay Thai aerobic dance program as an alternative training program in the ambulatory elderly subjects, particularly in regards to its advantage of multidimensional movements. 196 ACKNOWLEDGMENTS This research was funded by the 90th anniversary of Chulalongkorn University and Faculty of Sports Science, Chulalongkorn University. The authors also appreciate the Faculty of Medicine, Chulalongkorn University for providing the laboratory service and expertise. The authors wish to thanks the subjects who volunteered for the present study.

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