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Carbohydrate beverages attenuate bone resorption markers in elite runners
ME TAB O L IS M CL I N ICA L A N D EX PE R IM EN T AL 6 3 ( 2 0 14 ) 15 3 6 – 15 4 1
Available online at www.sciencedirect.com
Metabolism www.metabolismjournal.com
Carbohydrate beverages attenuate bone resorption markers in elite runners☆,☆☆ Maysa Vieira de Sousa a,⁎, Rosa Maria R. Pereira b , Rosa Fukui a , Valéria Falco Caparbo b , Maria Elizabeth Rossi da Silva a a b
Laboratory of Medical Investigation 18 - LIM-18, Endocrinology Division, Medical School, University of São Paulo, SP, Brazil Bone Metabolism Laboratory, Rheumatology Division, Medical School, University of São Paulo, SP, Brazil
A R T I C LE I N FO Article history: Received 5 November 2013 Accepted 22 August 2014
AB S T R A C T Objective. We evaluated the effects of carbohydrate (CHO) supplementation on markers of bone turnover in elite runners. Design. Twenty-four male runners were randomly assigned to two groups – a CHO and a control (CON) group – using a double-blind design. The participants were submitted to an
Keywords:
overload training program (days 1–8), followed by a high-intensity intermittent running
Bone turnover markers
protocol (10 × 800 m) on day 9. They received a maltodextrin solution (CHO group) or a
CTX
placebo solution as the CON equivalent, before, during, and after these protocols. Results. After 8 days of intensive training, baseline levels of osteocalcin (OC) decreased in
PTH Exercise
both CHO and CON groups (before: 28.8 ± 3.6 and 26.6 ± 2.4 ng/ml, after: 24.8 ± 3.0 and 21.9 ±
Carbohydrate supplementation
1.6 ng/ml, respectively, p < 0.01). On day 9, at 80 min of the recovery period, carboxy-terminal of telopeptide type I collagen (CTX) serum concentration was suppressed in the CHO group (0.3 ± 0.1 ng/ml) vs. 0.6 ± 0.0 ng/ml for the CON group (p < 0.01). CHO supplementation was effective in decreasing CTX levels from baseline to recovery (0.5 ± 0.1 ng/mL to 0.3 ± 0.1 ng/mL, p < 0.001), while an increase from 0.4 ± 0.0 ng/mL to 0.6 ± 0.0 ng/mL (p < 0.001) was observed in the CON group. Conclusion. CHO beverage ingestion attenuated the exercise-induced increase in CTX concentration, suggesting that CHO supplementation is a potential strategy to prevent bone damage in athletes. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Athletes have higher bone turnover than sedentary individuals [1]. Intensive training combined with an inadequate diet can lead to overtraining syndrome, which may reduce athletic performance and play a role in muscle, joint, and bone stresses and injuries, besides several other physiological,
biochemical, and psychological complications affecting athletes’ professional career [2–4]. Bone mass can be viewed as the net product of two metabolic processes, bone formation and bone resorption, coupled in a basic multicellular unit [5]. During a training program, runners expose their lower extremity bones and joints to large repetitive axial loads [6,7]. Such extremes of
Abbreviations: CHO, carbohydrate; CON, control; OC, osteocalcin; CTX, carboxy-terminal of telopeptide type I collagen; P1NP, procollagen type 1N propeptide; ALP, bone alkaline phosphatase; PTH, parathyroid hormone; HPA, hypothalamic–pituitary–adrenal; DXA, dual energy X-ray absorptiometry. ☆ Grant: The project was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: n. 07/08747-7). ☆☆ Disclosure: The authors have nothing to declare. ⁎ Corresponding author at: Av. Dr. Arnaldo, 455-3° andar-Sala 3324, São Paulo, SP, Brazil, 01246-000. Tel.: + 55 11 3061 7259. E-mail address: [email protected] (M.V. de Sousa). http://dx.doi.org/10.1016/j.metabol.2014.08.011 0026-0495/© 2014 Elsevier Inc. All rights reserved.
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chronic loading influence the turnover rate of bone and cartilage; the changes in turnover rate are usually detectable through changes in bone turnover biomarkers [5]. Osteocalcin (OC) is a marker of bone formation that has an important association with energy metabolism and plays a role in fat and glucose metabolism, insulin secretion, and pancreatic β-cell proliferation [8]. Levels of carboxy-terminal of telopeptide type I collagen (CTX), a bone resorption marker, are altered with exercise intensity and may also influence glucose metabolism [9,10]. CTX levels have been reported to increase post-exercise [5,11], whereas no changes were observed in procollagen type 1N propeptide (P1NP) and bone alkaline phosphatase (ALP) serum concentrations, both related to bone formation. On the other hand, in the study by Herman [12], OC decreased with moderate exercise at 75% and increased with intense exercise at 95% in athletes, besides an increase in CTX levels. These data suggest that strenuous exercise may induce osteoclastic activity that is not necessarily accompanied by a compensatory increase in osteoblastic activity. In addition to mechanical loading, bone turnover is regulated by several hormones. It is known that parathyroid hormone (PTH) and cortisol inhibit osteoblastic activity [13,14], gonadal steroids and IGF-1 inhibit the osteoclasts, and GH increases the osteoblastic activity during energy deficit. Moreover, activation of the hypothalamic–pituitary–adrenal (HPA) axis and the resulting hypercortisolemia are some of the main factors suppressing the hypothalamic–pituitary– gonadal (HPG) axis during exercise stress and inducing bone fragility [15]. All these changes are observed in athletes, increasing the risk for developing osteoporosis and stress fractures, which are common disorders in this group [16]. Diet and nutritional status have traditionally been the most relevant factors in the management of skeletal health and have helped to mitigate some losses [6,17]. However, the importance of CHO supplementation in bone markers during intense exercise has not been investigated in athletes. If CHO beverages attenuate post-exercise bone resorption, we suggest that this nutritional strategy is needed for athletes to prevent bone damage while engaged in intensive training. We hypothesized that athletes receiving CHO supplementation during a protocol consisting of 8-day intensive training followed by a session of intermittent high-intensity running have less bone stress afterwards. This study aimed to investigate the impact of intensive training and CHO beverages on bone biochemical markers in elite runners.
2.
Methods
2.1.
Subjects, carbohydrate (CHO) supplementation, and diet
Twenty-four elite male endurance runners (28.0 ± 1.2 years) training for the last 8.6 ± 1.1 years participated in the study. The protocol was approved by the Local Committee on Ethics of Human Research, and written informed consent was obtained from all subjects. These runners were randomly assigned to the CHO group or control (CON) group using a double-blind design. The two groups were matched for
maximal oxygen consumption (VO2max), body weight, and age. Both groups received isocaloric diet, with greater intake of CHO in the CHO group versus the CON group. The CHO group consumed 1 g maltodextrin/kg body weight per hour of running as a supplement during 8 days of training in the morning, corresponding to a daily dietary CHO of 61%, whereas the CON group was given a placebo solution. The diet consumed by the CON group provided a daily CHO of 54%. Data regarding the physical profiles of the athletes are shown in Table 1. The CHO and CON groups consisted of 12 subjects each, presenting the following mean physical profile, respectively: VO2max: 69.8 ± 2.2 and 68.5 ± 1.9 mL · kg−1 · min−1; body weight: 60.2 ± 1.4 and 62.3 ± 1.6 kg; and height: 169.5 ± 2.0 and 170.2 ± 2.5 cm (Table 1). There were no differences in these parameters between the two groups.
2.2. Overload training program and intermittent running protocol After determination of the physical profile, the participants underwent 13 training sessions over a period of 8 days. Eight sessions were held in the morning (days 1–8) and five in the afternoon (days 1–5). The training protocol has been described previously [2]. After the overload training program (days 1–8), the athletes arrived at the running track on day 9 after a 12-h fast and blood samples were collected. Next, the athletes ingested a standard breakfast 140 min before the beginning of the intermittent running protocol. The protocol was performed in the morning on a synthetic surface running track. The running session consisted of 10 series of 800 m (10 × 800 m) performed at a speed corresponding to the 3-km time trial (Vm3km) performed previously, with resting intervals of 1 min and 30 s, and two maximum performance tests of 1000 m. The first maximum performance test (1st 1000 m) was performed 20 min before the beginning of the intermittent exercise session and the second test (2nd 1000 m) was performed 20 min after the end of the intermittent exercise session. The CHO group received a 7% maltodextrin solution and the CON group received water artificially sweetened with aspartame before (− 30 min), during (after the 1st 1000 m, after every 2 series of 10 × 800 m), and immediately after the intermittent running
Table 1 – Mean (± SEM) biometric and physical profile of the participants (n = 24). Carbohydrate Control Age (years) 29.1 ± 1.6 Height (cm) 169.5 ± 2.0 Weight (kg) 60.2 ± 1.4 8.3 ± 0.3 Percent body fat a HRmax (bpm) 175.9 ± 1.4 VO2 max (ml/kg/min) 69.8 ± 2.2 Duration of training 9.5 ± 1.6 (years) a
26.9 170.2 62.3 9.9 177.5 68.5 7.4
± ± ± ± ± ± ±
1.9 2.5 1.6 0.5 2.4 1.9 1.2
P value (CHO vs. Control) 0.40 0.84 0.34 0.0043 0.60 0.67 0.35
Evaluated by bone densitometry (DXA; Hologic QDR, 2000 W).
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Osteocalcin (ng/mL)
c
Carbohydrate Control
c
20
10
0
m in
X8 0
80
10 po st
in e el
re c
m
m in ) (-1
(-9
40
da y
s)
0
in e ba s
The body composition of the athletes was determined pre- and post-training after breakfast. Whole-body composition was measured by DXA using Hologic 4500 QDR densitometry equipment (Bedford, MA) at the Bone Metabolism Laboratory, Rheumatology Division, Medical School, University of São Paulo.
c
b c
30
el
2.3. Body composition by dual-energy X-ray absorptiometry (DXA)
40
ba s
protocol. After the 2nd 1000 m and at 60 min of recovery, the athletes received supplemental CHO at a concentration of 1.2 g/kg body weight (CHO) or the CON equivalent.
Time point
Blood sample collection and analysis
P1NP (ng/mL)
100
c
80 60 40 20
Results
3.1.
Overload training program (days 1–8)
Osteocalcin levels decreased after 8 days of intense training in both CHO and CON groups (before: 28.8 ± 3.6 and 26.6 ± 2.4 ng/mL, after: 24.8 ± 3.0 and 21.9 ± 1.6 ng/mL; p < 0.01) (Fig. 1). Baseline values of P1NP, CTX, and PTH pre-training (day − 9) and post-training (baseline − 140 min) were similar between the CHO and CON groups (Figs. 1 and 2).
3.2.
Intermittent running protocol (day 9)
The osteocalcin concentration increased after the intermittent running protocol (post 10 × 800 m) in the two groups (p < 0.001) and it remained higher at 80 min of the recovery
c in
00
m
X8
80
10 st po
se ba
Time point 50
PTH (pg/mL)
Carbohydrate Control
c
40
3.
re
m
) in lin
e
lin
e
(-1
(-9
40
da
m
ys
)
0
Statistical analysis
30
b
20 10
c m
x8 10 po
st
in
00
re
m
) in e
se lin ba
se
lin
e
(-1
(-9
40
da
m
ys
)
0
ba
Data are presented as mean (±) and standard error of the mean (SEM). After assessing normality of the data through the Shapiro–Wilk test, a two-way analysis of variance with repeated measures was performed to evaluate differences in the blood variables between the CHO and CON groups (treatment by time). If the differences were significant, the post-hoc Student–Newman–Keuls test was applied. The physical profile and body composition results were compared by the t test for unpaired samples. A p-value < 0.05 was considered to indicate statistical significance and all analyses were carried out using the Statistical Package Sigma Plot 11.0.
Carbohydrate Control
c
80
2.5.
120
se
Blood samples were collected at four different time points [pre-training (baseline –9 days), post-training (baseline – 140 min), post 10 × 800 m, and 80 min of recovery] for assessing serum concentration of PTH, osteocalcin, P1NP, and CTX. Serum samples were centrifuged at 4 °C in a Sorvall RC3 Plus (H-2000b rotor) at 2000 rpm for 15 min. Serum aliquots were stored frozen at − 20 °C. Serum levels of CTX, P1NP, and PTH were determined by an automated Roche electrochemiluminescence system (E411, Roche diagnostics, Mannheim, Germany). The intra-assay and interassay coefficients of variation were 2.5% and 3.4% for CTX, 2.2% and 1.8% for P1NP, and 4.3% and 5.3% for PTH.
140
ba
2.4.
Time point
Fig. 1 – Carbohydrate supplementation on serum PTH and bone formation marker (osteocalcin and P1NP) concentrations during intensive training followed by a session of intermittent running (10 × 800 m) in elite runners. (b) Significant difference compared to the baseline (−140 min) (p < 0.01). (c) Significant difference compared to the baseline (−140 min) (p < 0.001).
period in the CON group (p < 0.001) when compared to the post-training program levels (baseline − 140 min; Fig. 1). PTH levels increased after running in both CHO (p < 0.01) and CON (p < 0.001) groups, but these concentrations decreased rapidly at 80 min of the recovery period and were not significantly different from baseline − 140 min (Fig. 1). A similar response was observed for P1NP levels in both groups (p < 0.001; Fig. 1). The CON group showed an increase in CTX at 80 min of the recovery period (from 0.4 ± 0.0 ng/ml to 0.6 ± 0.0 ng/ml,
ME TAB O L IS M CL I N ICA L A N D EX P ER IM EN T AL 6 3 ( 2 0 14 ) 15 3 6 – 1 54 1
0.8
CTX (ng/mL)
0.7
Carbohydrate Control
**
0.6
c
0.5 0.4 0.3
c
0.2 0.1 re c 80
m
in
m st 10 X8 00
in )
po
m (-1 40
(-9 ba se lin e
ba se lin e
da ys )
0.0
Time point
Fig. 2 – Carbohydrate supplementation on serum CTX concentration during intensive training followed by a session of intermittent running (10 × 800 m) in elite runners. (c) Significant difference compared to the baseline (−140 min) (p < 0.001). **Significant difference between the carbohydrate and control groups (p < 0.01).
p < 0.001). On the other hand, CHO supplementation suppressed the acute exercise-induced increase in CTX levels throughout the study, being significant at 80 min of the recovery period (0.3 ± 0.1 ng/ml) when compared to the post-training concentrations (− 140 min: 0.5 ± 0.1 ng/ml, p < 0.001). CTX levels was significant lower in the CHO group (0.3 ± 0.1 ng/ml) vs. 0.6 ± 0.0 ng/ml for the CON group at 80 min of the recovery period (Fig. 2).
3.3.
Body composition evaluated by DXA
There were no significant differences in body composition parameters measured by DXA pre- and post-training between CHO and CON groups (Table 2).
4.
Discussion
Our results suggest that CHO supplementation reduces the exercise-induced bone resorption markers, as evidenced by a decrease in CTX levels after acute exercise. This nutritional strategy seems particularly interesting when we consider our previous study, in which CHO supplementation during overload training resulted in higher energy store [18] and testosterone levels and reduced exercise-induced stress hormone levels and tissue damage, as demonstrated by lower cortisol and circulating free plasma DNA and superior athletic performance [2,3]. Similar to what has been observed in stress hormone levels, there seems to be an exercise intensity relationship with CTX levels [9], as running at 75% VO2max caused significantly higher CTX levels when compared to the intensities of 55% and 65% VO2max. In the present study, the effect of exercise on bone markers was even higher due to the training load intensity, which reaches above 100% of VO2max. However, the expected increase of CTX levels during
1539
exercise was suppressed with CHO supplementation, significant at 80 min of the recovery period, in contrast to the increase observed in the CON group. Studies have reported the effects of dietary manipulation on bone resorption markers such as on CTX suppression [10,11]. In the study by Guillemant et al. [11], a high-calcium mineral water provided before and during exercise may have contributed to the suppression of CTX levels. A similar decrease in CTX levels was also observed in postmenopausal women with varying degrees of glucose tolerance after an oral glucose tolerance test (OGTT), contributing to the amelioration of the impairment of bone quality in type 2 diabetic patients [10]. Furthermore, in the study by Scott et al. [19], a standard meal provided 3 h before exercise reduced CTX levels pre-exercise. However, to our knowledge, this is the first study reporting the impact of CHO supplementation during intensive training exercise on bone resorption. The mechanism underlying bone turnover suppression with CHO supplementation remains unknown. However, it is known that the enteric hormones, such as gastric inhibitory polypeptide (GIP), glucagon-like peptide 1 (GLP1), and glucagon-like peptide 2 (GLP2), which are involved in glucose control, have been reported to influence bone metabolism [10,20]. Our findings suggest that the decrease of CTX levels during exercise and recovery due to CHO supplementation may be involved in the mechanisms underlying lower bone resorption, probably by reducing osteoclastic activity. Moreover, osteoclastic activity may be enhanced by cortisol [13,21] and suppression of hypothalamaic–pituitary function [22]. This may occur in long distance runners undergoing intensive training sessions, as a consequence of a marked catabolic state. Our previous results have shown that CHO supplementation attenuated the suppression of testosterone/cortisol ratio in the CHO group which may have caused a number of benefits to the body such as better postexercise muscle injury recovery, better modulation of immune response, improved bone health and attenuation of the endocrine system’s stress [2]. Both PTH and P1NP increased only transitorily in the exercise session and returned to basal levels at recovery. Although PTH concentrations were lower in the CHO group post 10 × 800 m, this difference did not reach statistical significance. PTH increased before CTX, suggesting that changes in PTH are usually related to greater bone resorption and CTX levels, increasing calcium and phosphorus availability, and to a compensatory increase in OC and P1NP [5]. This compensatory increase of OC may have occurred in the CON group. Therefore, CHO supplementation acutely altered the exercise-induced changes in CTX and OC, but not in P1NP levels, perhaps less related to energetic substrate. Proper nutritional counseling with CHO supplementation while exercising should be recommended during intensive training program to attenuate bone resorption, as demonstrated in the present study, helping to prevent stress fractures in athletes. However, the effect of acute exercise on bone remodeling seems to be short-lived. Except for OC, bone turnover markers did not change significantly after the 8-day overload training program when measured after 12-h fasting, pointing to no sustained effect of exercise on CTX, P1NP, and PTH levels in highly trained athletes, despite greater CHO ingestion in the
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ME TAB O L IS M CL I N ICA L A N D EX PE R IM EN T AL 6 3 ( 2 0 14 ) 15 3 6 – 15 4 1
Table 2 – Bone densitometry (% increment) in pre- and post-training in elite runners with carbohydrate supplementation. Carbohydrate
Total Mass (kg) BMC a (g) Fat (kg) Lean (kg) Lean_BMC (kg) a
Control
Pre-training
Post-training (% increment)
Pre-training
Post-training (% increment)
60.5 2511.0 4.9 52.9 55.5
−0.2 0.3 −2.7 −0.0 0.0
62.5 2528.0 6.2 53.7 56.3
0.1 1.6 −2.0 0.4 0.3
± ± ± ± ±
1.4 90.1 0.2 1.2 1.3
± ± ± ± ±
0.5 0.3 2.7 0.5 0.5
± ± ± ± ±
1.6 96.4 0.3 1.4 1.5
± ± ± ± ±
0.4 0.6 1.4 0.4 0.3
Bone mineral content. NS: No significant difference on % increment was observed between groups.
CHO group. These short-lived alterations suggest that markers of bone turnover not related to recent exercise are not useful in predicting the likelihood of stress fractures in athletes. They probably do not reflect the changes in bone biomechanics or the skeletal fragility and bone quality induced by exercise. Several studies showed that after an acute exercise bout, osteocalcin levels either remained unchanged or decreased immediately post-exercise protocols or a marathon race [9,23,24]. In a recent study by Scott et al. [9], independently of exercise intensity, an increase in P1NP levels was also observed after running in all three groups (55%, 65%, and 85% of VO2max). Conversely, Pomerants et al. [25] reported no changes in P1NP concentrations immediately after an acute exercise bout. In this study, the high-intensity running was associated with an important increase in bone turnover markers, as assessed by PTH (~ 145%), osteocalcin (~32%), and P1NP (~ 77%). Although the physiological functions and clinical relevance of these hormones are well established, there are still controversies regarding the impact of exercise on bone remodeling, probably because of the differences in preanalytical conditions (exercise protocols, intensity, duration, sample size, and diet control) [8,16]. Furthermore, recent findings have demonstrated undercarboxylated OC (ucOC) as a new homeostatic mechanism that alters CHO metabolism [26]. This aspect will be considered in a future study. Although the present study displays originality, especially with regard to the decrease in bone resorption with CHO supplementation, it has limitations such as the long-term effect of this nutritional practice on bone health and sample size. We suggest that the cumulative effect of CHO supplementation during training sessions may prevent long-term bone impairment in highly trained athletes. Furthermore, the sample size of the study was relatively small and the results are limited to male subjects that practice a specific kind of activity. As strength of this study we suggest the intake of carbohydrate supplementation as part of a balanced diet during short term overload training followed by a session of high-intensity intermittent running (<1 h duration each training session) in highly trained runners. The present study showed that the administration of a CHO beverage resulted in significant reduction in bone resorption markers post-exercise, suggesting that CHO supplementation can be a potential strategy to prevent bone damage in athletes. The findings of this study emphasize the need for supplementation with CHO in high performance athletes
during high intensity interval and distance training, both lasting less than 1 h and representing typical stages of overload training programs. Moreover, the impact of this nutritional strategy suppressing bone resorption and helping to prevent stress fractures in athletes is the translational potential message of the present study.
5.
Author contributions
MVS designed the research. MVS, RF, and VFC performed research and/or data analysis. MVS wrote the first version of the paper, which was reviewed by MRS and RRP.
Acknowledgments We acknowledge Aritania Santos and Greci Paula from Laboratory of Medical Investigation 18 (LIM-18) and Valéria Samuel Lando from LIM-42 for their technical assistance and Liliam Takayama from LIM-17 for the DXA analysis. We thank Dr. Pedro Henrique Silveira Correa from Endocrinology Division, Medical School, University of Sao Paulo for encouragement in publishing this manuscript. This study was supported by grants from the State of São Paulo Research Foundation–Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP).
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Metabolism www.metabolismjournal.com
Carbohydrate beverages attenuate bone resorption markers in elite runners☆,☆☆ Maysa Vieira de Sousa a,⁎, Rosa Maria R. Pereira b , Rosa Fukui a , Valéria Falco Caparbo b , Maria Elizabeth Rossi da Silva a a b
Laboratory of Medical Investigation 18 - LIM-18, Endocrinology Division, Medical School, University of São Paulo, SP, Brazil Bone Metabolism Laboratory, Rheumatology Division, Medical School, University of São Paulo, SP, Brazil
A R T I C LE I N FO Article history: Received 5 November 2013 Accepted 22 August 2014
AB S T R A C T Objective. We evaluated the effects of carbohydrate (CHO) supplementation on markers of bone turnover in elite runners. Design. Twenty-four male runners were randomly assigned to two groups – a CHO and a control (CON) group – using a double-blind design. The participants were submitted to an
Keywords:
overload training program (days 1–8), followed by a high-intensity intermittent running
Bone turnover markers
protocol (10 × 800 m) on day 9. They received a maltodextrin solution (CHO group) or a
CTX
placebo solution as the CON equivalent, before, during, and after these protocols. Results. After 8 days of intensive training, baseline levels of osteocalcin (OC) decreased in
PTH Exercise
both CHO and CON groups (before: 28.8 ± 3.6 and 26.6 ± 2.4 ng/ml, after: 24.8 ± 3.0 and 21.9 ±
Carbohydrate supplementation
1.6 ng/ml, respectively, p < 0.01). On day 9, at 80 min of the recovery period, carboxy-terminal of telopeptide type I collagen (CTX) serum concentration was suppressed in the CHO group (0.3 ± 0.1 ng/ml) vs. 0.6 ± 0.0 ng/ml for the CON group (p < 0.01). CHO supplementation was effective in decreasing CTX levels from baseline to recovery (0.5 ± 0.1 ng/mL to 0.3 ± 0.1 ng/mL, p < 0.001), while an increase from 0.4 ± 0.0 ng/mL to 0.6 ± 0.0 ng/mL (p < 0.001) was observed in the CON group. Conclusion. CHO beverage ingestion attenuated the exercise-induced increase in CTX concentration, suggesting that CHO supplementation is a potential strategy to prevent bone damage in athletes. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
Athletes have higher bone turnover than sedentary individuals [1]. Intensive training combined with an inadequate diet can lead to overtraining syndrome, which may reduce athletic performance and play a role in muscle, joint, and bone stresses and injuries, besides several other physiological,
biochemical, and psychological complications affecting athletes’ professional career [2–4]. Bone mass can be viewed as the net product of two metabolic processes, bone formation and bone resorption, coupled in a basic multicellular unit [5]. During a training program, runners expose their lower extremity bones and joints to large repetitive axial loads [6,7]. Such extremes of
Abbreviations: CHO, carbohydrate; CON, control; OC, osteocalcin; CTX, carboxy-terminal of telopeptide type I collagen; P1NP, procollagen type 1N propeptide; ALP, bone alkaline phosphatase; PTH, parathyroid hormone; HPA, hypothalamic–pituitary–adrenal; DXA, dual energy X-ray absorptiometry. ☆ Grant: The project was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP: n. 07/08747-7). ☆☆ Disclosure: The authors have nothing to declare. ⁎ Corresponding author at: Av. Dr. Arnaldo, 455-3° andar-Sala 3324, São Paulo, SP, Brazil, 01246-000. Tel.: + 55 11 3061 7259. E-mail address: [email protected] (M.V. de Sousa). http://dx.doi.org/10.1016/j.metabol.2014.08.011 0026-0495/© 2014 Elsevier Inc. All rights reserved.
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chronic loading influence the turnover rate of bone and cartilage; the changes in turnover rate are usually detectable through changes in bone turnover biomarkers [5]. Osteocalcin (OC) is a marker of bone formation that has an important association with energy metabolism and plays a role in fat and glucose metabolism, insulin secretion, and pancreatic β-cell proliferation [8]. Levels of carboxy-terminal of telopeptide type I collagen (CTX), a bone resorption marker, are altered with exercise intensity and may also influence glucose metabolism [9,10]. CTX levels have been reported to increase post-exercise [5,11], whereas no changes were observed in procollagen type 1N propeptide (P1NP) and bone alkaline phosphatase (ALP) serum concentrations, both related to bone formation. On the other hand, in the study by Herman [12], OC decreased with moderate exercise at 75% and increased with intense exercise at 95% in athletes, besides an increase in CTX levels. These data suggest that strenuous exercise may induce osteoclastic activity that is not necessarily accompanied by a compensatory increase in osteoblastic activity. In addition to mechanical loading, bone turnover is regulated by several hormones. It is known that parathyroid hormone (PTH) and cortisol inhibit osteoblastic activity [13,14], gonadal steroids and IGF-1 inhibit the osteoclasts, and GH increases the osteoblastic activity during energy deficit. Moreover, activation of the hypothalamic–pituitary–adrenal (HPA) axis and the resulting hypercortisolemia are some of the main factors suppressing the hypothalamic–pituitary– gonadal (HPG) axis during exercise stress and inducing bone fragility [15]. All these changes are observed in athletes, increasing the risk for developing osteoporosis and stress fractures, which are common disorders in this group [16]. Diet and nutritional status have traditionally been the most relevant factors in the management of skeletal health and have helped to mitigate some losses [6,17]. However, the importance of CHO supplementation in bone markers during intense exercise has not been investigated in athletes. If CHO beverages attenuate post-exercise bone resorption, we suggest that this nutritional strategy is needed for athletes to prevent bone damage while engaged in intensive training. We hypothesized that athletes receiving CHO supplementation during a protocol consisting of 8-day intensive training followed by a session of intermittent high-intensity running have less bone stress afterwards. This study aimed to investigate the impact of intensive training and CHO beverages on bone biochemical markers in elite runners.
2.
Methods
2.1.
Subjects, carbohydrate (CHO) supplementation, and diet
Twenty-four elite male endurance runners (28.0 ± 1.2 years) training for the last 8.6 ± 1.1 years participated in the study. The protocol was approved by the Local Committee on Ethics of Human Research, and written informed consent was obtained from all subjects. These runners were randomly assigned to the CHO group or control (CON) group using a double-blind design. The two groups were matched for
maximal oxygen consumption (VO2max), body weight, and age. Both groups received isocaloric diet, with greater intake of CHO in the CHO group versus the CON group. The CHO group consumed 1 g maltodextrin/kg body weight per hour of running as a supplement during 8 days of training in the morning, corresponding to a daily dietary CHO of 61%, whereas the CON group was given a placebo solution. The diet consumed by the CON group provided a daily CHO of 54%. Data regarding the physical profiles of the athletes are shown in Table 1. The CHO and CON groups consisted of 12 subjects each, presenting the following mean physical profile, respectively: VO2max: 69.8 ± 2.2 and 68.5 ± 1.9 mL · kg−1 · min−1; body weight: 60.2 ± 1.4 and 62.3 ± 1.6 kg; and height: 169.5 ± 2.0 and 170.2 ± 2.5 cm (Table 1). There were no differences in these parameters between the two groups.
2.2. Overload training program and intermittent running protocol After determination of the physical profile, the participants underwent 13 training sessions over a period of 8 days. Eight sessions were held in the morning (days 1–8) and five in the afternoon (days 1–5). The training protocol has been described previously [2]. After the overload training program (days 1–8), the athletes arrived at the running track on day 9 after a 12-h fast and blood samples were collected. Next, the athletes ingested a standard breakfast 140 min before the beginning of the intermittent running protocol. The protocol was performed in the morning on a synthetic surface running track. The running session consisted of 10 series of 800 m (10 × 800 m) performed at a speed corresponding to the 3-km time trial (Vm3km) performed previously, with resting intervals of 1 min and 30 s, and two maximum performance tests of 1000 m. The first maximum performance test (1st 1000 m) was performed 20 min before the beginning of the intermittent exercise session and the second test (2nd 1000 m) was performed 20 min after the end of the intermittent exercise session. The CHO group received a 7% maltodextrin solution and the CON group received water artificially sweetened with aspartame before (− 30 min), during (after the 1st 1000 m, after every 2 series of 10 × 800 m), and immediately after the intermittent running
Table 1 – Mean (± SEM) biometric and physical profile of the participants (n = 24). Carbohydrate Control Age (years) 29.1 ± 1.6 Height (cm) 169.5 ± 2.0 Weight (kg) 60.2 ± 1.4 8.3 ± 0.3 Percent body fat a HRmax (bpm) 175.9 ± 1.4 VO2 max (ml/kg/min) 69.8 ± 2.2 Duration of training 9.5 ± 1.6 (years) a
26.9 170.2 62.3 9.9 177.5 68.5 7.4
± ± ± ± ± ± ±
1.9 2.5 1.6 0.5 2.4 1.9 1.2
P value (CHO vs. Control) 0.40 0.84 0.34 0.0043 0.60 0.67 0.35
Evaluated by bone densitometry (DXA; Hologic QDR, 2000 W).
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Osteocalcin (ng/mL)
c
Carbohydrate Control
c
20
10
0
m in
X8 0
80
10 po st
in e el
re c
m
m in ) (-1
(-9
40
da y
s)
0
in e ba s
The body composition of the athletes was determined pre- and post-training after breakfast. Whole-body composition was measured by DXA using Hologic 4500 QDR densitometry equipment (Bedford, MA) at the Bone Metabolism Laboratory, Rheumatology Division, Medical School, University of São Paulo.
c
b c
30
el
2.3. Body composition by dual-energy X-ray absorptiometry (DXA)
40
ba s
protocol. After the 2nd 1000 m and at 60 min of recovery, the athletes received supplemental CHO at a concentration of 1.2 g/kg body weight (CHO) or the CON equivalent.
Time point
Blood sample collection and analysis
P1NP (ng/mL)
100
c
80 60 40 20
Results
3.1.
Overload training program (days 1–8)
Osteocalcin levels decreased after 8 days of intense training in both CHO and CON groups (before: 28.8 ± 3.6 and 26.6 ± 2.4 ng/mL, after: 24.8 ± 3.0 and 21.9 ± 1.6 ng/mL; p < 0.01) (Fig. 1). Baseline values of P1NP, CTX, and PTH pre-training (day − 9) and post-training (baseline − 140 min) were similar between the CHO and CON groups (Figs. 1 and 2).
3.2.
Intermittent running protocol (day 9)
The osteocalcin concentration increased after the intermittent running protocol (post 10 × 800 m) in the two groups (p < 0.001) and it remained higher at 80 min of the recovery
c in
00
m
X8
80
10 st po
se ba
Time point 50
PTH (pg/mL)
Carbohydrate Control
c
40
3.
re
m
) in lin
e
lin
e
(-1
(-9
40
da
m
ys
)
0
Statistical analysis
30
b
20 10
c m
x8 10 po
st
in
00
re
m
) in e
se lin ba
se
lin
e
(-1
(-9
40
da
m
ys
)
0
ba
Data are presented as mean (±) and standard error of the mean (SEM). After assessing normality of the data through the Shapiro–Wilk test, a two-way analysis of variance with repeated measures was performed to evaluate differences in the blood variables between the CHO and CON groups (treatment by time). If the differences were significant, the post-hoc Student–Newman–Keuls test was applied. The physical profile and body composition results were compared by the t test for unpaired samples. A p-value < 0.05 was considered to indicate statistical significance and all analyses were carried out using the Statistical Package Sigma Plot 11.0.
Carbohydrate Control
c
80
2.5.
120
se
Blood samples were collected at four different time points [pre-training (baseline –9 days), post-training (baseline – 140 min), post 10 × 800 m, and 80 min of recovery] for assessing serum concentration of PTH, osteocalcin, P1NP, and CTX. Serum samples were centrifuged at 4 °C in a Sorvall RC3 Plus (H-2000b rotor) at 2000 rpm for 15 min. Serum aliquots were stored frozen at − 20 °C. Serum levels of CTX, P1NP, and PTH were determined by an automated Roche electrochemiluminescence system (E411, Roche diagnostics, Mannheim, Germany). The intra-assay and interassay coefficients of variation were 2.5% and 3.4% for CTX, 2.2% and 1.8% for P1NP, and 4.3% and 5.3% for PTH.
140
ba
2.4.
Time point
Fig. 1 – Carbohydrate supplementation on serum PTH and bone formation marker (osteocalcin and P1NP) concentrations during intensive training followed by a session of intermittent running (10 × 800 m) in elite runners. (b) Significant difference compared to the baseline (−140 min) (p < 0.01). (c) Significant difference compared to the baseline (−140 min) (p < 0.001).
period in the CON group (p < 0.001) when compared to the post-training program levels (baseline − 140 min; Fig. 1). PTH levels increased after running in both CHO (p < 0.01) and CON (p < 0.001) groups, but these concentrations decreased rapidly at 80 min of the recovery period and were not significantly different from baseline − 140 min (Fig. 1). A similar response was observed for P1NP levels in both groups (p < 0.001; Fig. 1). The CON group showed an increase in CTX at 80 min of the recovery period (from 0.4 ± 0.0 ng/ml to 0.6 ± 0.0 ng/ml,
ME TAB O L IS M CL I N ICA L A N D EX P ER IM EN T AL 6 3 ( 2 0 14 ) 15 3 6 – 1 54 1
0.8
CTX (ng/mL)
0.7
Carbohydrate Control
**
0.6
c
0.5 0.4 0.3
c
0.2 0.1 re c 80
m
in
m st 10 X8 00
in )
po
m (-1 40
(-9 ba se lin e
ba se lin e
da ys )
0.0
Time point
Fig. 2 – Carbohydrate supplementation on serum CTX concentration during intensive training followed by a session of intermittent running (10 × 800 m) in elite runners. (c) Significant difference compared to the baseline (−140 min) (p < 0.001). **Significant difference between the carbohydrate and control groups (p < 0.01).
p < 0.001). On the other hand, CHO supplementation suppressed the acute exercise-induced increase in CTX levels throughout the study, being significant at 80 min of the recovery period (0.3 ± 0.1 ng/ml) when compared to the post-training concentrations (− 140 min: 0.5 ± 0.1 ng/ml, p < 0.001). CTX levels was significant lower in the CHO group (0.3 ± 0.1 ng/ml) vs. 0.6 ± 0.0 ng/ml for the CON group at 80 min of the recovery period (Fig. 2).
3.3.
Body composition evaluated by DXA
There were no significant differences in body composition parameters measured by DXA pre- and post-training between CHO and CON groups (Table 2).
4.
Discussion
Our results suggest that CHO supplementation reduces the exercise-induced bone resorption markers, as evidenced by a decrease in CTX levels after acute exercise. This nutritional strategy seems particularly interesting when we consider our previous study, in which CHO supplementation during overload training resulted in higher energy store [18] and testosterone levels and reduced exercise-induced stress hormone levels and tissue damage, as demonstrated by lower cortisol and circulating free plasma DNA and superior athletic performance [2,3]. Similar to what has been observed in stress hormone levels, there seems to be an exercise intensity relationship with CTX levels [9], as running at 75% VO2max caused significantly higher CTX levels when compared to the intensities of 55% and 65% VO2max. In the present study, the effect of exercise on bone markers was even higher due to the training load intensity, which reaches above 100% of VO2max. However, the expected increase of CTX levels during
1539
exercise was suppressed with CHO supplementation, significant at 80 min of the recovery period, in contrast to the increase observed in the CON group. Studies have reported the effects of dietary manipulation on bone resorption markers such as on CTX suppression [10,11]. In the study by Guillemant et al. [11], a high-calcium mineral water provided before and during exercise may have contributed to the suppression of CTX levels. A similar decrease in CTX levels was also observed in postmenopausal women with varying degrees of glucose tolerance after an oral glucose tolerance test (OGTT), contributing to the amelioration of the impairment of bone quality in type 2 diabetic patients [10]. Furthermore, in the study by Scott et al. [19], a standard meal provided 3 h before exercise reduced CTX levels pre-exercise. However, to our knowledge, this is the first study reporting the impact of CHO supplementation during intensive training exercise on bone resorption. The mechanism underlying bone turnover suppression with CHO supplementation remains unknown. However, it is known that the enteric hormones, such as gastric inhibitory polypeptide (GIP), glucagon-like peptide 1 (GLP1), and glucagon-like peptide 2 (GLP2), which are involved in glucose control, have been reported to influence bone metabolism [10,20]. Our findings suggest that the decrease of CTX levels during exercise and recovery due to CHO supplementation may be involved in the mechanisms underlying lower bone resorption, probably by reducing osteoclastic activity. Moreover, osteoclastic activity may be enhanced by cortisol [13,21] and suppression of hypothalamaic–pituitary function [22]. This may occur in long distance runners undergoing intensive training sessions, as a consequence of a marked catabolic state. Our previous results have shown that CHO supplementation attenuated the suppression of testosterone/cortisol ratio in the CHO group which may have caused a number of benefits to the body such as better postexercise muscle injury recovery, better modulation of immune response, improved bone health and attenuation of the endocrine system’s stress [2]. Both PTH and P1NP increased only transitorily in the exercise session and returned to basal levels at recovery. Although PTH concentrations were lower in the CHO group post 10 × 800 m, this difference did not reach statistical significance. PTH increased before CTX, suggesting that changes in PTH are usually related to greater bone resorption and CTX levels, increasing calcium and phosphorus availability, and to a compensatory increase in OC and P1NP [5]. This compensatory increase of OC may have occurred in the CON group. Therefore, CHO supplementation acutely altered the exercise-induced changes in CTX and OC, but not in P1NP levels, perhaps less related to energetic substrate. Proper nutritional counseling with CHO supplementation while exercising should be recommended during intensive training program to attenuate bone resorption, as demonstrated in the present study, helping to prevent stress fractures in athletes. However, the effect of acute exercise on bone remodeling seems to be short-lived. Except for OC, bone turnover markers did not change significantly after the 8-day overload training program when measured after 12-h fasting, pointing to no sustained effect of exercise on CTX, P1NP, and PTH levels in highly trained athletes, despite greater CHO ingestion in the
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Table 2 – Bone densitometry (% increment) in pre- and post-training in elite runners with carbohydrate supplementation. Carbohydrate
Total Mass (kg) BMC a (g) Fat (kg) Lean (kg) Lean_BMC (kg) a
Control
Pre-training
Post-training (% increment)
Pre-training
Post-training (% increment)
60.5 2511.0 4.9 52.9 55.5
−0.2 0.3 −2.7 −0.0 0.0
62.5 2528.0 6.2 53.7 56.3
0.1 1.6 −2.0 0.4 0.3
± ± ± ± ±
1.4 90.1 0.2 1.2 1.3
± ± ± ± ±
0.5 0.3 2.7 0.5 0.5
± ± ± ± ±
1.6 96.4 0.3 1.4 1.5
± ± ± ± ±
0.4 0.6 1.4 0.4 0.3
Bone mineral content. NS: No significant difference on % increment was observed between groups.
CHO group. These short-lived alterations suggest that markers of bone turnover not related to recent exercise are not useful in predicting the likelihood of stress fractures in athletes. They probably do not reflect the changes in bone biomechanics or the skeletal fragility and bone quality induced by exercise. Several studies showed that after an acute exercise bout, osteocalcin levels either remained unchanged or decreased immediately post-exercise protocols or a marathon race [9,23,24]. In a recent study by Scott et al. [9], independently of exercise intensity, an increase in P1NP levels was also observed after running in all three groups (55%, 65%, and 85% of VO2max). Conversely, Pomerants et al. [25] reported no changes in P1NP concentrations immediately after an acute exercise bout. In this study, the high-intensity running was associated with an important increase in bone turnover markers, as assessed by PTH (~ 145%), osteocalcin (~32%), and P1NP (~ 77%). Although the physiological functions and clinical relevance of these hormones are well established, there are still controversies regarding the impact of exercise on bone remodeling, probably because of the differences in preanalytical conditions (exercise protocols, intensity, duration, sample size, and diet control) [8,16]. Furthermore, recent findings have demonstrated undercarboxylated OC (ucOC) as a new homeostatic mechanism that alters CHO metabolism [26]. This aspect will be considered in a future study. Although the present study displays originality, especially with regard to the decrease in bone resorption with CHO supplementation, it has limitations such as the long-term effect of this nutritional practice on bone health and sample size. We suggest that the cumulative effect of CHO supplementation during training sessions may prevent long-term bone impairment in highly trained athletes. Furthermore, the sample size of the study was relatively small and the results are limited to male subjects that practice a specific kind of activity. As strength of this study we suggest the intake of carbohydrate supplementation as part of a balanced diet during short term overload training followed by a session of high-intensity intermittent running (<1 h duration each training session) in highly trained runners. The present study showed that the administration of a CHO beverage resulted in significant reduction in bone resorption markers post-exercise, suggesting that CHO supplementation can be a potential strategy to prevent bone damage in athletes. The findings of this study emphasize the need for supplementation with CHO in high performance athletes
during high intensity interval and distance training, both lasting less than 1 h and representing typical stages of overload training programs. Moreover, the impact of this nutritional strategy suppressing bone resorption and helping to prevent stress fractures in athletes is the translational potential message of the present study.
5.
Author contributions
MVS designed the research. MVS, RF, and VFC performed research and/or data analysis. MVS wrote the first version of the paper, which was reviewed by MRS and RRP.
Acknowledgments We acknowledge Aritania Santos and Greci Paula from Laboratory of Medical Investigation 18 (LIM-18) and Valéria Samuel Lando from LIM-42 for their technical assistance and Liliam Takayama from LIM-17 for the DXA analysis. We thank Dr. Pedro Henrique Silveira Correa from Endocrinology Division, Medical School, University of Sao Paulo for encouragement in publishing this manuscript. This study was supported by grants from the State of São Paulo Research Foundation–Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP).
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