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GH responses to two consecutive bouts of whole body vibration, maximal voluntary contractions or vibration alternated with maximal voluntary contractions administered at 2-h intervals in healthy adults
Growth Hormone & IGF Research 20 (2010) 416–421
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Growth Hormone & IGF Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g h i r
GH responses to two consecutive bouts of whole body vibration, maximal voluntary contractions or vibration alternated with maximal voluntary contractions administered at 2-h intervals in healthy adults A. Sartorio a,b,⁎, C.L. Lafortuna c, N.A. Maffiuletti d, F. Agosti a, N. Marazzi a, F. Rastelli e, A.E. Rigamonti f, E.E. Muller f a
Istituto Auxologico Italiano, Laboratorio Sperimentale di Ricerche Auxo-endocrinologiche, IRCCS, Milan, Italy Istituto Auxologico Italiano, Divisione Malattie Metaboliche, IRCCS, Piancavallo (VB), Italy c Istituto di Bioimmagini e Fisiologia Molecolare, CNR, Segrate, Milan, Italy d Neuromuscular Research Laboratory, Schulthess Clinic, Zurich, Switzerland e Facoltà di Scienze motorie, Università degli Studi dell'Aquila, L'Aquila, Italy f Università degli Studi di Milano, Dipartimento di Farmacologia Medica, Milan, Italy b
a r t i c l e
i n f o
Article history: Received 14 June 2010 Received in revised form 7 September 2010 Accepted 19 September 2010 Available online 12 October 2010 Keywords: GH Whole body vibration Isometric contraction Lactate Repeated stimuli
a b s t r a c t Background: Pharmacological or exercise stimuli repeated at a short interval (but not electrical muscle stimulation) are associated with a blunting of GH responsiveness. Aim: To compare GH responses to repeated bout of three different GH-releasing stimuli. Methods: The effects of two consecutive bouts (with a 2-h interval) of whole body vibrations (WBV), maximal voluntary contractions alone (MVC), or alternated with WBV (MVC–WBV) on blood GH and lactate (LA) were assessed in nine young males. Results: Baseline levels of both GH and LA increased significantly after the first bout of all the tested stimuli, and were significantly lower after WBV than after MVC or MVC alternated with WBV, no difference being detected between these last. The administration of a second bout resulted in significantly lower GH increases than those elicited in the first bout in the three different tests; significantly lower LA responses were recorded after the second bout of MVC and MVC–WBV when compared with those obtained after the first bout, while no significant differences were observed after the two WBV bouts for LA. All responses after the second bout of MVC and MVC–WBV were significantly higher than those observed after WBV alone. GH concentrations were significantly correlated with LA after all stimuli, although LA concentrations after the second bout were associated with markedly lower GH levels. Conclusions: A significant blunting of GH responsiveness ensues after a second bout of different GH-releasing stimuli, independent from the amount of GH released after the first bout. This is a pattern also observed for other pharmacological stimuli and exercise modalities, and suggests a common mechanism underlying different GH-releasing stimuli. © 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.
1. Introduction The secretion of growth hormone (GH) is mainly under the influence of hypothalamic GH-releasing hormone (GHRH) and somatostatin, and GH regulates its own secretion via a negative auto-feedback mechanism, which operates at both at the pituitary and hypothalamic levels [1]. Increased GH concentrations evoke prompt hypothalamic somatostatin release, which in turn inhibits GHRH secretion, blocks pituitary GH exocytosis GH and sensitizes somatotrophs to the next GHRH stimulus ⁎ Corresponding author. Laboratorio Sperimentale di Ricerche Auxo-Endocrinologiche, Istituto Auxologico Italiano, IRCCS, Via Ariosto 13, 20145 Milan, Italy. Tel.: +39 02 619112426; fax: +39 02 619112435. E-mail address: [email protected] (A. Sartorio).
[1]. In healthy adults, repeated GHRH stimuli administered at 2-h intervals are associated with blunting of GH responsiveness to the second stimulus [2–5], with restoration of GH responsiveness occurring when the time elapsed between the two pharmacological stimuli is longer [6]. Similar results have been reported with repeated bouts of highintensity physical exercise, an attenuation of GH response being present when the exercise bouts are repeated within 1–2 h [6–8]. Reciprocal to the pattern recorded with repeated GHRH administration, normal GH responsiveness reappears progressively as the time interval between the two consecutive stimuli increases. When the interval between the two stimuli was 6 h, the GH response after the second exercise bout was comparable with that observed after the first bout [6]. By contrast, we have recently demonstrated that nonvoluntary physical exercise (electrical stimulation of lower limb
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A. Sartorio et al. / Growth Hormone & IGF Research 20 (2010) 416–421
muscles) was able to maintain GH responsiveness also after a second bout repeated at a 2-h interval [9], probably due to the low GH response to the first bout (mean GH peak of 4.5 μg/l) in comparison to the greater response elicited by pharmacological and high-intensity voluntary exercise (N25 μg/l). Among the different tools for strength training exercise, vibrating platforms have become increasingly popular and used in sport settings and rehabilitation institutes [10,11]. Whole body vibration (WBV), i.e., standing in different static positions or exercising on a vibrating platform, is being commercially promoted as an attractive and efficient complement or as an alternative to strength training for the enhancement of muscle function and athletic performance [12–18]. Recent evidence suggests that acute vibration exercise elicits a specific warm-up effect and that vibration improves muscle power, although the potential benefits over traditional forms of resistive exercise are yet to be completely understood [18]. Hormonal evaluation of healthy young and old subjects after acute sessions of WBV has yielded conflicting results. Particularly, Bosco et al. [19] have shown a sharp rise in circulating GH levels, a finding which could not be replicated by other authors [20,21]. We have recently detected a significant rise in plasma GH concentration after acute WBV in young male volunteers, although it was much less marked than that observed after isometric muscle contractions [22]. The aim of the present study was to compare changes in GH and lactate concentrations between three bouts of different GH-releasing stimuli: WBV, maximal voluntary contractions (MVC) and MVC alternated with WBV (MVC–WBV), administered at a 2-h interval in healthy adults. We have hypothesised that, due to the relatively small response of GH secretion to WBV [22], a second bout of the vibrating stimulus could not blunt the hormonal response, similar to what was observed after electrical muscle stimulation (9). 2. Materials and methods 2.1. Subjects Nine healthy male adults, recruited among friends and colleagues (mean age ± S.D.: 23 ± 2 years, range: 21–26 years; weight: 68 ± 9 kg, range: 58–81 kg; body mass index (BMI): 22.2 ± 0.8 kg/m2, range: 21.0–23.8 kg/m2), volunteered to participate in this investigation after giving their written informed consent. The study protocol was approved by the Ethical Committee of the Italian Institute for Auxology. All the subjects were habitually active but were not involved in strength or endurance training on a regular basis, and none of them had any signs of musculoskeletal disorders. Subjects were asked not to perform any strenuous exercise for at least 48 h prior to the experiment. None of them reported ingestion of drugs or nutritional supplements known to interfere with GH and/or cortisol secretion. The subjects were admitted to the laboratory 1 h before the start of the tests, after an overnight fast (10–14 h); all tests started between 8 and 8.30 AM. 2.1.1. Testing All the subjects admitted to the study performed two consecutive and identical bouts of exercise (separated by 2 h) under the following conditions: WBV, MVC, and MVC alternated with WBV (MVC–WBV). The three different protocols were randomly performed in separate days with an interval of at least two days in between. After a standardized warm-up (5 min on a cycloergometer; power: 50 W, cadence: 60 rpm), the different protocols were completed as follows. In WBV, subjects initially seated in a semi-recumbent position on a horizontal leg press machine (Technogym, Gambettola, Italy) for 30 s, with the trunk–thigh and thigh–shank angles at 80°, without making any efforts. Then a 30-s WBV bout was delivered while the subject stood on a vibrating platform (Nevisys H1©, RME, Ferrara, Italy) with the knees at 110°. The vibration platform produced vertical
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sinusoidal vibrations at a frequency of 35 Hz. At a peak-to-peak amplitude of vibration of 5 mm the acceleration of the platform was 2.85 g, as assessed with the standing subject by means of a magnetic monoaxial accelerometer (Vibration Meter, Lutron VB-8200). These two rest–WBV cycles were repeated 15 times, for a total duration of 15 min. In MVC, subjects were initially placed on the leg press machine in the same supine position as in the WBV protocol, and performed three 5-s MVC, separated by 5-s resting periods in between. Then, 30 s of rest was respected in the same static position as WBV, with the knees at 110° but without WBV. These two MVC– rest cycles were repeated 15 times, for a total duration of 15 min. In MVC–WBV, subjects initially performed the three 5-s MVC like in the MVC condition, and then received the 30-s WBV bout, as in the WBV condition. These two MVC–WBV cycles were repeated 15 times, for a total duration of 15 min. The temporal pattern of the different protocols is summarised in Fig. 1. The force generated during isometric efforts in tests MVC and MVC–WBV was measured by a strain gauge (Globus, Codognè, Italy) properly mounted on the leg press machine with chains attached to the frame of the machine and the sliding axis of the leg press seat. The signal from the strain gauge was sampled at 100 Hz and stored on a computer for later analysis with a commercially available software (TCS-SUITE 400, Globus, Codognè, Italy). The average isometric force developed during MVC and MVC–WBV was comparable, both for the first bout (MVC: 196 ± 42 kg; MVC–WBV: 186 ± 46 kg, p = 0.164), and for the second one (MVC: 180 ± 44 kg; MVC–WBV: 172 ± 43 kg, p = 0.503). 2.1.2. Blood sampling and measurements Blood samples for glucose and GH measurements (5 ml at each time point) were collected before the start (baseline) and immediately after each 15-min exercise bout at 0, 10, 20 and 30 min postexercise. While the baseline blood sample was obtained by syringe venipuncture, the remaining ones were drawn through an indwelling cannula inserted into an ante-cubital vein kept patent via a continuous infusion of isotonic saline. This approach was used both for the first and the second bouts of exercise. Between the end of the last blood sampling, ensuing the first bout and the baseline sampling of the second bout, the subjects were asked to stay relaxed in a semirecumbent position. All blood samples were allowed to clot, centrifuged for 5 min, and immediately stored at −20 °C for the next analysis. Blood glucose levels were determined by an oxidase enzymatic method (Roche Diagnostics GmbH, Mannheim, Germany) and GH concentrations by a commercially available immunometric kit (Immulite 2000, DPC, Los Angeles, CA, USA). In addition to peak GH values, integrated GH concentration [net incremental area under the curve (nAUC)] over 45 min, including all time points, was calculated by using the trapezoidal method [23]. All samples were run in the same assay to minimize inter-assay variability. Intra- and inter-assay coefficients of variation were 2.5% and 6%, respectively. Before the standardized warm-up, a small blood sample (5 μl) was obtained from the earlobe for the determination of basal lactate concentration. The next blood samples were obtained immediately after the end of exercise and at 2-min intervals until the detection of the peak value. Blood lactate was measured by a portable analyzer (Lactate Pro, Akray, Japan). Intra- and inter-assay coefficients of variation were 3% and 7%, respectively. 2.1.3. Muscle soreness Muscle soreness was assessed 24 and 48 h after exercise using an 11-point visual analogue scale (VAS), starting from “no pain” (level 0) up to “extremely painful” (level 10). Subjects were asked to mark their pain level on the VAS under supervision of the examiner, and the marked point provided a numeric measure of soreness.
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WBV on platform
MVC on leg press
On platform without WBV
On leg press without MVC
WBV
MVC
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10
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30
40
50
60
Time (s) Fig. 1. Schemes of protocols used in the study: vibrations only (WBV), maximal isometric voluntary contractions only (MVC), and maximal isometric voluntary contractions alternated with vibrations (MVC–WBV). See the text for further details.
2.1.4. Statistical analysis GH and blood lactate responses at different time intervals after both the first and second bouts of the three tests were evaluated by means of ANOVA for repeated measures. Differences in peak GH values after the three different tests were assessed by means of ANOVA. Differences in GH peaks and GH nAUCs between the first and second bouts were evaluated with a Student's t-test. Standardized mean difference was evaluated as Cohen's d effect size. The statistical significance was set at P b 0.05 for all analyses. 3. Results In the three types of exercise, baseline GH levels significantly increased after the first bout (WBV: p b 0.05, + 560%, d = 1.38; MVC: p b 0.001, + 2379%, d = 2.83; MVC–WBV: p b 0.01, + 2593%, d = 2.07, respectively) with a complete normalization at 120 min (Fig. 2). Mean GH peaks and AUC after WBV were significantly lower than after MVC (p b 0.01) and MVC–WBV (p = 0.01), whereas no significant difference was detected between MVC and MVC–WBV (Fig. 3). In protocols, the administration of the second bout resulted in a GH increase (WBV: NS, + 415%, d = 1.08; MVC: p b 0.01, + 555%, d = 2.98; MVC–WBV: p b 0.05, + 576%, d = 1.45, respectively) significantly lower than that observed after the first bouts (WBV: p = 0.05; MVC: p b 0.01; and MVC–
WBV: p b 0.05). GH responses after the second bout of MVC and MVC–WBV were comparable, being significantly higher than those observed after WBV alone. Baseline lactate levels (WBV: 1.1±0.2 mmol/l; MVC: 1.3±0.3 mmol/l; MVC–WBV: 1.4±0.4 mmol/l) significantly increased after the first bout for the three different stimuli (WBV: 1.9±0.5 mmol/l, pb 0.01,+ 71%, d=2.23; MVC: 6.9±2.3 mmol/l, pb 0.001,+ 424%, d=3.62; MVC-WBV: 7.6 ± 1.0 mmol/l, p b 0.001, + 461%, d = 9.08). Mean blood lactate concentrations after WBV were significantly lower than after MVC and MVC–WBV, whereas no difference was present between MVC and MVC–WBV (p= 0.533). Peak lactate values in response to the second exercise bouts were 1.8± 0.5 mmol/l for WBV (vs. baseline p b 0.05, + 43%, d = 1.61), 5.5± 1.5 mmol/l for MVC (pb 0.001, + 205%, d = 3.41) and 5.9± 1.6 mmol/l for WBV–MVC (pb 0.001,+ 211%, d = 3.61). Mean lactate peaks after the second bout were significantly lower than those attained after the first bout for both MVC (pb 0.05) and MVC–WBV (pb 0.01), but not for WBV (p= 0.49). Individual peak GH values after the different tests were plotted as a function of the respective lactate values (Fig. 4). A significant correlation was observed, both for the first (R2 = 0.24, p = 0.015, n = 24) and for the second bout (R2 = 0.21, p = 0.023, n = 24), but a comparison of the linear regression equations revealed a significant difference between the two bouts (t = 3.61, p b 0.001, total df = 23), thus indicating a non-univocal relation between lactate and GH.
25 WBV MVC MVC-WBV
GH (µg/L)
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15
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0 0
30
60
90
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Time (min) Fig. 2. GH responses to the two consecutive bouts of the three different tests: vibrations only (WBV), maximal isometric voluntary contractions only (MVC), and maximal isometric voluntary contractions alternated with vibrations (MVC–WBV). Values are expressed as mean + SD. The black box represents the time spent to perform the exercise.
A. Sartorio et al. / Growth Hormone & IGF Research 20 (2010) 416–421
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4. Discussion 1st Bout 2nd Bout
800
GH nAUC (µg/L*45min)
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600
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0 WBV
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Fig. 3. Mean GH nAUC values (μg/ml × 45 min) after the two bouts of the three tests. Vibrations only (WBV), maximal isometric voluntary contractions only (MVC), and maximal isometric voluntary contractions alternated with vibrations (MVC– WBV). Vertical bars represent SD.
Mean baseline glucose concentrations were not significantly different before starting the three types of exercise (first and second bouts), they did not significantly change during the two recovery periods and the patterns were comparable in the three experimental conditions (data not shown). The average score for muscle soreness determined after repeated MVC (24 h: 4.4 ± 2.3, 48 h: 3.5 ± 2.3) was not significantly different from that obtained after MVC–WBV (24 h: 4.1 ± 2.6, 48 h: 3.9 ± 3.5). By contrast, muscle soreness determined after WBV alone (24 h: 0.4 ± 0.5, 48 h: 0.1 ± 0.4) was significantly lower (p b 0.01) than those recorded after MVC and MVC–WBW.
45 First Bout Second Bout
40 35
GH (µg/L)
30 y = 1.851x + 3.227 R² = 0.241
25 20 15
y = 0.684x + 0.972 R² = 0.212
10 5 0 0
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2
4
6
8
10
12
Lactate (mmol/L) Fig. 4. Correlations between blood lactate and GH levels observed after the first and second bouts of the three different tests.
It is well established that in healthy adults repeated GHRH stimuli [2–5] or bouts of high-intensity physical exercise [6–8,24,25] administered at 2-h intervals are associated with a blunted GH responsiveness to the second stimulus in healthy adults, a restoration of which is taking place when the time elapsed between the two stimuli is more prolonged (6 h) [6,26]. In contrast, we have recently shown that GH responses to non-voluntary exercise (electrical muscle stimulation) were persistent when the second stimulus was administered at a 2-h interval, thus suggesting different underlying mechanisms exerted by different GH-releasing stimuli [9]. In the present study, we have tested the effects on GH secretion of WBV exercise, which elicits small but rapid changes in muscle length producing reflex muscle activity, named “tonic vibration reflex”. Baseline GH levels significantly increased after the first exercise bout for the three different stimuli, with a complete normalization after 120 min. Mean GH peaks and nAUC after WBV were significantly lower than both those recorded after isometric MVC and MVC alternated with WBV, whereas no significant difference was detected between these two stimuli after the first bout of stimulation. WBV exercise has been shown to acutely increase GH in young healthy individuals after a single bout of 10 min [19]. In contrast, both Di Loreto et al. [20] and Cardinale et al. [21] did not find any acute changes in GH (former study only) and insulin-like growth factor 1 (both studies) titers in subjects undergoing 5 and 20 min of WBV exercise, albeit with relatively small amplitude and frequencies of 27 and 30 Hz, respectively. Furthermore, in a recent study, Kvorning et al. [27] reported an increase in the GH-releasing effect of WBV when combined with squatting at a load equal to 10 repetitions maximum. One plausible explanation of these conflicting results is the diversity of the WBV protocols used in these different studies. The administration of the second bout of exercise resulted in GH levels significantly lower than those observed after the first bout for all the protocols, independent from the amount of GH produced following the first bout (range between 4.4 and 18.9 μg/l). GH nAUC after the first and second bouts were also significantly different, indicating that the overall pattern of GH secretion following the two consecutive bouts was characterized by a proportional blunting of GH responsiveness in the three experimental conditions. The blunting of GH responsiveness to repeated bouts of exercise at 2-h intervals detected after maximal voluntary isometric contractions, alone or in combination with WBV, strongly mimics the GH response to GHRH stimulation, suggesting therefore a common underlying mechanism, possibly resting on a relative somatotroph refractoriness and/or reactive increase of the somatostatinergic tone [1]. Blunting of GH responsiveness after the second bout of exercise presently observed was comparable to those occurring after repeated pharmacological stimuli [2–5] or high-intensity voluntary exercise of different types [6–8,24,25]. Further studies of voluntary exercise performed at different intensities and WBV are therefore mandatory to establish the appropriate interpretation of this finding. The results of the blunted GH response to different exercise and pharmacological stimuli contrast with the persistent GH responsiveness after electrically evoked muscle contractions [9]. The persistence of GH responsiveness to repeated electrical stimuli was hypothesised to depend from the relatively scarce GH-releasing ability of electrical muscle stimulation. However, based on the present results, this interpretation appears actually unlikely. In fact, the amount of GH released following the first bout of WBV was comparable to that observed after electrically induced muscle activity (4.4 vs. 4.2 μg/l), whereas the patterns of GH responsiveness to the second bouts were clearly different (blunting of GH responsiveness after WBV vs. its maintenance after electrical stimulation). Although Giustina and Veldhuis [28] proposed that the final common pathway underlying exercise-induced GH release likely involves either increase in GHRH
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function and/or decrease in somatostatin tone, this different GH response to a second equal stimulus in the presence of comparable acute GH-releasing potency in the first bout bespeaks the involvement of different neuroendocrine mechanisms, which remain to be fully evaluated. The present results may elicit a further consideration concerning the role of blood lactate (taken per se or as a marker of blood hydrogen ion concentration) as a regulatory mechanism in GH exercise-related secretion [25,29,30]. In fact, there was a significant correlation between blood lactate and GH concentrations in response to the tested stimuli within either of the two exercise bouts; lactate concentrations after the second bout were similar to those observed after the first one, but they were associated with markedly lower GH levels. This finding suggests some caution in considering lactate as an univocal determinant of GH secretion. In keeping with this view, Stokes et al. [31] reported a divergent pattern between the increased blood lactate concentrations and the attenuated serum GH responses following exercise. Goto and Takamatsu [32], who examined the effects of WBV on GH levels and lipolytic response, observed a significant increase of free fatty acid during the recovery period after the WBV session (150– 210 min). Since in our study protocol the second bouts of WBV, WBV– MVC and MVC occurs 105 mi after the end of the first exercise bouts, the blunted GH response to the second bouts might be actually amenable to a rise of free fatty acids following the first bouts, which might have blocked GH release to the second bouts directly acting at the pituitary level [33]. Although plasma concentrations of free fatty acids were not measured in the present study, however this hypothesis is not completely tenable in these experimental conditions since the blunting of GH responsiveness to the second bouts was similar in the three experimental tests, in spite of markedly different GH peaks after the first bouts. Alternatively, blunting of GH responses to the second bouts might have occurred via changes in blood glucose concentrations, which could be increased for the effects of cathecolamines on hepatic glucose production and/or pancreatic glucagon release [34]. However, this mechanism does not seem working in these experimental conditions, since no significant changes of blood glucose were observed. The lack of changes in glucose concentrations was also observed in our previous studies evaluating repeated bouts (at a 2-h interval) of aerobic exercise in healthy young volunteers [6]. In conclusion, the present study reveals that both MVC and WBV induce a significant response in terms of GH secretion, although with different potencies, and that the response to both stimuli delivered after the short interval of 2 h is blunted in similar proportion, irrespective from the extent of GH rise initially induced by each stimulus. Further studies are needed to identify the complex mechanisms underlying the blunted GH responsiveness to different repeated stimuli and, in addition, the training modalities (even combining different GH-releasing stimuli) capable to increase the endogenous GH production through different physiological processes and mechanisms. The persistence of GH responsiveness after repeated stimuli, which probably requires intervals longer than 2 h (with the exception of electrical muscle stimulation), could tentatively be useful to athletes, GH-deficient patients and sarcopenic elderly subjects for producing physiologically greater amounts of GH-IGF-I, able to positively influence muscle mass and function. Acknowledgements The authors acknowledge dr. S. Chiavaroli, dr. P. Gola, dr. A De Col, dr. R. Galli, dr. C. Busti, Mrs. F. Pera, head nurse at the Division of Metabolic Diseases, and Mr. F Villano for their qualified assistance during the experiments and all the volunteers for their kind collaboration. The Authors are also indebted with Mr. C. Cotelli, past
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Contents lists available at ScienceDirect
Growth Hormone & IGF Research j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g h i r
GH responses to two consecutive bouts of whole body vibration, maximal voluntary contractions or vibration alternated with maximal voluntary contractions administered at 2-h intervals in healthy adults A. Sartorio a,b,⁎, C.L. Lafortuna c, N.A. Maffiuletti d, F. Agosti a, N. Marazzi a, F. Rastelli e, A.E. Rigamonti f, E.E. Muller f a
Istituto Auxologico Italiano, Laboratorio Sperimentale di Ricerche Auxo-endocrinologiche, IRCCS, Milan, Italy Istituto Auxologico Italiano, Divisione Malattie Metaboliche, IRCCS, Piancavallo (VB), Italy c Istituto di Bioimmagini e Fisiologia Molecolare, CNR, Segrate, Milan, Italy d Neuromuscular Research Laboratory, Schulthess Clinic, Zurich, Switzerland e Facoltà di Scienze motorie, Università degli Studi dell'Aquila, L'Aquila, Italy f Università degli Studi di Milano, Dipartimento di Farmacologia Medica, Milan, Italy b
a r t i c l e
i n f o
Article history: Received 14 June 2010 Received in revised form 7 September 2010 Accepted 19 September 2010 Available online 12 October 2010 Keywords: GH Whole body vibration Isometric contraction Lactate Repeated stimuli
a b s t r a c t Background: Pharmacological or exercise stimuli repeated at a short interval (but not electrical muscle stimulation) are associated with a blunting of GH responsiveness. Aim: To compare GH responses to repeated bout of three different GH-releasing stimuli. Methods: The effects of two consecutive bouts (with a 2-h interval) of whole body vibrations (WBV), maximal voluntary contractions alone (MVC), or alternated with WBV (MVC–WBV) on blood GH and lactate (LA) were assessed in nine young males. Results: Baseline levels of both GH and LA increased significantly after the first bout of all the tested stimuli, and were significantly lower after WBV than after MVC or MVC alternated with WBV, no difference being detected between these last. The administration of a second bout resulted in significantly lower GH increases than those elicited in the first bout in the three different tests; significantly lower LA responses were recorded after the second bout of MVC and MVC–WBV when compared with those obtained after the first bout, while no significant differences were observed after the two WBV bouts for LA. All responses after the second bout of MVC and MVC–WBV were significantly higher than those observed after WBV alone. GH concentrations were significantly correlated with LA after all stimuli, although LA concentrations after the second bout were associated with markedly lower GH levels. Conclusions: A significant blunting of GH responsiveness ensues after a second bout of different GH-releasing stimuli, independent from the amount of GH released after the first bout. This is a pattern also observed for other pharmacological stimuli and exercise modalities, and suggests a common mechanism underlying different GH-releasing stimuli. © 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.
1. Introduction The secretion of growth hormone (GH) is mainly under the influence of hypothalamic GH-releasing hormone (GHRH) and somatostatin, and GH regulates its own secretion via a negative auto-feedback mechanism, which operates at both at the pituitary and hypothalamic levels [1]. Increased GH concentrations evoke prompt hypothalamic somatostatin release, which in turn inhibits GHRH secretion, blocks pituitary GH exocytosis GH and sensitizes somatotrophs to the next GHRH stimulus ⁎ Corresponding author. Laboratorio Sperimentale di Ricerche Auxo-Endocrinologiche, Istituto Auxologico Italiano, IRCCS, Via Ariosto 13, 20145 Milan, Italy. Tel.: +39 02 619112426; fax: +39 02 619112435. E-mail address: [email protected] (A. Sartorio).
[1]. In healthy adults, repeated GHRH stimuli administered at 2-h intervals are associated with blunting of GH responsiveness to the second stimulus [2–5], with restoration of GH responsiveness occurring when the time elapsed between the two pharmacological stimuli is longer [6]. Similar results have been reported with repeated bouts of highintensity physical exercise, an attenuation of GH response being present when the exercise bouts are repeated within 1–2 h [6–8]. Reciprocal to the pattern recorded with repeated GHRH administration, normal GH responsiveness reappears progressively as the time interval between the two consecutive stimuli increases. When the interval between the two stimuli was 6 h, the GH response after the second exercise bout was comparable with that observed after the first bout [6]. By contrast, we have recently demonstrated that nonvoluntary physical exercise (electrical stimulation of lower limb
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A. Sartorio et al. / Growth Hormone & IGF Research 20 (2010) 416–421
muscles) was able to maintain GH responsiveness also after a second bout repeated at a 2-h interval [9], probably due to the low GH response to the first bout (mean GH peak of 4.5 μg/l) in comparison to the greater response elicited by pharmacological and high-intensity voluntary exercise (N25 μg/l). Among the different tools for strength training exercise, vibrating platforms have become increasingly popular and used in sport settings and rehabilitation institutes [10,11]. Whole body vibration (WBV), i.e., standing in different static positions or exercising on a vibrating platform, is being commercially promoted as an attractive and efficient complement or as an alternative to strength training for the enhancement of muscle function and athletic performance [12–18]. Recent evidence suggests that acute vibration exercise elicits a specific warm-up effect and that vibration improves muscle power, although the potential benefits over traditional forms of resistive exercise are yet to be completely understood [18]. Hormonal evaluation of healthy young and old subjects after acute sessions of WBV has yielded conflicting results. Particularly, Bosco et al. [19] have shown a sharp rise in circulating GH levels, a finding which could not be replicated by other authors [20,21]. We have recently detected a significant rise in plasma GH concentration after acute WBV in young male volunteers, although it was much less marked than that observed after isometric muscle contractions [22]. The aim of the present study was to compare changes in GH and lactate concentrations between three bouts of different GH-releasing stimuli: WBV, maximal voluntary contractions (MVC) and MVC alternated with WBV (MVC–WBV), administered at a 2-h interval in healthy adults. We have hypothesised that, due to the relatively small response of GH secretion to WBV [22], a second bout of the vibrating stimulus could not blunt the hormonal response, similar to what was observed after electrical muscle stimulation (9). 2. Materials and methods 2.1. Subjects Nine healthy male adults, recruited among friends and colleagues (mean age ± S.D.: 23 ± 2 years, range: 21–26 years; weight: 68 ± 9 kg, range: 58–81 kg; body mass index (BMI): 22.2 ± 0.8 kg/m2, range: 21.0–23.8 kg/m2), volunteered to participate in this investigation after giving their written informed consent. The study protocol was approved by the Ethical Committee of the Italian Institute for Auxology. All the subjects were habitually active but were not involved in strength or endurance training on a regular basis, and none of them had any signs of musculoskeletal disorders. Subjects were asked not to perform any strenuous exercise for at least 48 h prior to the experiment. None of them reported ingestion of drugs or nutritional supplements known to interfere with GH and/or cortisol secretion. The subjects were admitted to the laboratory 1 h before the start of the tests, after an overnight fast (10–14 h); all tests started between 8 and 8.30 AM. 2.1.1. Testing All the subjects admitted to the study performed two consecutive and identical bouts of exercise (separated by 2 h) under the following conditions: WBV, MVC, and MVC alternated with WBV (MVC–WBV). The three different protocols were randomly performed in separate days with an interval of at least two days in between. After a standardized warm-up (5 min on a cycloergometer; power: 50 W, cadence: 60 rpm), the different protocols were completed as follows. In WBV, subjects initially seated in a semi-recumbent position on a horizontal leg press machine (Technogym, Gambettola, Italy) for 30 s, with the trunk–thigh and thigh–shank angles at 80°, without making any efforts. Then a 30-s WBV bout was delivered while the subject stood on a vibrating platform (Nevisys H1©, RME, Ferrara, Italy) with the knees at 110°. The vibration platform produced vertical
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sinusoidal vibrations at a frequency of 35 Hz. At a peak-to-peak amplitude of vibration of 5 mm the acceleration of the platform was 2.85 g, as assessed with the standing subject by means of a magnetic monoaxial accelerometer (Vibration Meter, Lutron VB-8200). These two rest–WBV cycles were repeated 15 times, for a total duration of 15 min. In MVC, subjects were initially placed on the leg press machine in the same supine position as in the WBV protocol, and performed three 5-s MVC, separated by 5-s resting periods in between. Then, 30 s of rest was respected in the same static position as WBV, with the knees at 110° but without WBV. These two MVC– rest cycles were repeated 15 times, for a total duration of 15 min. In MVC–WBV, subjects initially performed the three 5-s MVC like in the MVC condition, and then received the 30-s WBV bout, as in the WBV condition. These two MVC–WBV cycles were repeated 15 times, for a total duration of 15 min. The temporal pattern of the different protocols is summarised in Fig. 1. The force generated during isometric efforts in tests MVC and MVC–WBV was measured by a strain gauge (Globus, Codognè, Italy) properly mounted on the leg press machine with chains attached to the frame of the machine and the sliding axis of the leg press seat. The signal from the strain gauge was sampled at 100 Hz and stored on a computer for later analysis with a commercially available software (TCS-SUITE 400, Globus, Codognè, Italy). The average isometric force developed during MVC and MVC–WBV was comparable, both for the first bout (MVC: 196 ± 42 kg; MVC–WBV: 186 ± 46 kg, p = 0.164), and for the second one (MVC: 180 ± 44 kg; MVC–WBV: 172 ± 43 kg, p = 0.503). 2.1.2. Blood sampling and measurements Blood samples for glucose and GH measurements (5 ml at each time point) were collected before the start (baseline) and immediately after each 15-min exercise bout at 0, 10, 20 and 30 min postexercise. While the baseline blood sample was obtained by syringe venipuncture, the remaining ones were drawn through an indwelling cannula inserted into an ante-cubital vein kept patent via a continuous infusion of isotonic saline. This approach was used both for the first and the second bouts of exercise. Between the end of the last blood sampling, ensuing the first bout and the baseline sampling of the second bout, the subjects were asked to stay relaxed in a semirecumbent position. All blood samples were allowed to clot, centrifuged for 5 min, and immediately stored at −20 °C for the next analysis. Blood glucose levels were determined by an oxidase enzymatic method (Roche Diagnostics GmbH, Mannheim, Germany) and GH concentrations by a commercially available immunometric kit (Immulite 2000, DPC, Los Angeles, CA, USA). In addition to peak GH values, integrated GH concentration [net incremental area under the curve (nAUC)] over 45 min, including all time points, was calculated by using the trapezoidal method [23]. All samples were run in the same assay to minimize inter-assay variability. Intra- and inter-assay coefficients of variation were 2.5% and 6%, respectively. Before the standardized warm-up, a small blood sample (5 μl) was obtained from the earlobe for the determination of basal lactate concentration. The next blood samples were obtained immediately after the end of exercise and at 2-min intervals until the detection of the peak value. Blood lactate was measured by a portable analyzer (Lactate Pro, Akray, Japan). Intra- and inter-assay coefficients of variation were 3% and 7%, respectively. 2.1.3. Muscle soreness Muscle soreness was assessed 24 and 48 h after exercise using an 11-point visual analogue scale (VAS), starting from “no pain” (level 0) up to “extremely painful” (level 10). Subjects were asked to mark their pain level on the VAS under supervision of the examiner, and the marked point provided a numeric measure of soreness.
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WBV on platform
MVC on leg press
On platform without WBV
On leg press without MVC
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10
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40
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Time (s) Fig. 1. Schemes of protocols used in the study: vibrations only (WBV), maximal isometric voluntary contractions only (MVC), and maximal isometric voluntary contractions alternated with vibrations (MVC–WBV). See the text for further details.
2.1.4. Statistical analysis GH and blood lactate responses at different time intervals after both the first and second bouts of the three tests were evaluated by means of ANOVA for repeated measures. Differences in peak GH values after the three different tests were assessed by means of ANOVA. Differences in GH peaks and GH nAUCs between the first and second bouts were evaluated with a Student's t-test. Standardized mean difference was evaluated as Cohen's d effect size. The statistical significance was set at P b 0.05 for all analyses. 3. Results In the three types of exercise, baseline GH levels significantly increased after the first bout (WBV: p b 0.05, + 560%, d = 1.38; MVC: p b 0.001, + 2379%, d = 2.83; MVC–WBV: p b 0.01, + 2593%, d = 2.07, respectively) with a complete normalization at 120 min (Fig. 2). Mean GH peaks and AUC after WBV were significantly lower than after MVC (p b 0.01) and MVC–WBV (p = 0.01), whereas no significant difference was detected between MVC and MVC–WBV (Fig. 3). In protocols, the administration of the second bout resulted in a GH increase (WBV: NS, + 415%, d = 1.08; MVC: p b 0.01, + 555%, d = 2.98; MVC–WBV: p b 0.05, + 576%, d = 1.45, respectively) significantly lower than that observed after the first bouts (WBV: p = 0.05; MVC: p b 0.01; and MVC–
WBV: p b 0.05). GH responses after the second bout of MVC and MVC–WBV were comparable, being significantly higher than those observed after WBV alone. Baseline lactate levels (WBV: 1.1±0.2 mmol/l; MVC: 1.3±0.3 mmol/l; MVC–WBV: 1.4±0.4 mmol/l) significantly increased after the first bout for the three different stimuli (WBV: 1.9±0.5 mmol/l, pb 0.01,+ 71%, d=2.23; MVC: 6.9±2.3 mmol/l, pb 0.001,+ 424%, d=3.62; MVC-WBV: 7.6 ± 1.0 mmol/l, p b 0.001, + 461%, d = 9.08). Mean blood lactate concentrations after WBV were significantly lower than after MVC and MVC–WBV, whereas no difference was present between MVC and MVC–WBV (p= 0.533). Peak lactate values in response to the second exercise bouts were 1.8± 0.5 mmol/l for WBV (vs. baseline p b 0.05, + 43%, d = 1.61), 5.5± 1.5 mmol/l for MVC (pb 0.001, + 205%, d = 3.41) and 5.9± 1.6 mmol/l for WBV–MVC (pb 0.001,+ 211%, d = 3.61). Mean lactate peaks after the second bout were significantly lower than those attained after the first bout for both MVC (pb 0.05) and MVC–WBV (pb 0.01), but not for WBV (p= 0.49). Individual peak GH values after the different tests were plotted as a function of the respective lactate values (Fig. 4). A significant correlation was observed, both for the first (R2 = 0.24, p = 0.015, n = 24) and for the second bout (R2 = 0.21, p = 0.023, n = 24), but a comparison of the linear regression equations revealed a significant difference between the two bouts (t = 3.61, p b 0.001, total df = 23), thus indicating a non-univocal relation between lactate and GH.
25 WBV MVC MVC-WBV
GH (µg/L)
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0 0
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60
90
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Time (min) Fig. 2. GH responses to the two consecutive bouts of the three different tests: vibrations only (WBV), maximal isometric voluntary contractions only (MVC), and maximal isometric voluntary contractions alternated with vibrations (MVC–WBV). Values are expressed as mean + SD. The black box represents the time spent to perform the exercise.
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4. Discussion 1st Bout 2nd Bout
800
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Fig. 3. Mean GH nAUC values (μg/ml × 45 min) after the two bouts of the three tests. Vibrations only (WBV), maximal isometric voluntary contractions only (MVC), and maximal isometric voluntary contractions alternated with vibrations (MVC– WBV). Vertical bars represent SD.
Mean baseline glucose concentrations were not significantly different before starting the three types of exercise (first and second bouts), they did not significantly change during the two recovery periods and the patterns were comparable in the three experimental conditions (data not shown). The average score for muscle soreness determined after repeated MVC (24 h: 4.4 ± 2.3, 48 h: 3.5 ± 2.3) was not significantly different from that obtained after MVC–WBV (24 h: 4.1 ± 2.6, 48 h: 3.9 ± 3.5). By contrast, muscle soreness determined after WBV alone (24 h: 0.4 ± 0.5, 48 h: 0.1 ± 0.4) was significantly lower (p b 0.01) than those recorded after MVC and MVC–WBW.
45 First Bout Second Bout
40 35
GH (µg/L)
30 y = 1.851x + 3.227 R² = 0.241
25 20 15
y = 0.684x + 0.972 R² = 0.212
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2
4
6
8
10
12
Lactate (mmol/L) Fig. 4. Correlations between blood lactate and GH levels observed after the first and second bouts of the three different tests.
It is well established that in healthy adults repeated GHRH stimuli [2–5] or bouts of high-intensity physical exercise [6–8,24,25] administered at 2-h intervals are associated with a blunted GH responsiveness to the second stimulus in healthy adults, a restoration of which is taking place when the time elapsed between the two stimuli is more prolonged (6 h) [6,26]. In contrast, we have recently shown that GH responses to non-voluntary exercise (electrical muscle stimulation) were persistent when the second stimulus was administered at a 2-h interval, thus suggesting different underlying mechanisms exerted by different GH-releasing stimuli [9]. In the present study, we have tested the effects on GH secretion of WBV exercise, which elicits small but rapid changes in muscle length producing reflex muscle activity, named “tonic vibration reflex”. Baseline GH levels significantly increased after the first exercise bout for the three different stimuli, with a complete normalization after 120 min. Mean GH peaks and nAUC after WBV were significantly lower than both those recorded after isometric MVC and MVC alternated with WBV, whereas no significant difference was detected between these two stimuli after the first bout of stimulation. WBV exercise has been shown to acutely increase GH in young healthy individuals after a single bout of 10 min [19]. In contrast, both Di Loreto et al. [20] and Cardinale et al. [21] did not find any acute changes in GH (former study only) and insulin-like growth factor 1 (both studies) titers in subjects undergoing 5 and 20 min of WBV exercise, albeit with relatively small amplitude and frequencies of 27 and 30 Hz, respectively. Furthermore, in a recent study, Kvorning et al. [27] reported an increase in the GH-releasing effect of WBV when combined with squatting at a load equal to 10 repetitions maximum. One plausible explanation of these conflicting results is the diversity of the WBV protocols used in these different studies. The administration of the second bout of exercise resulted in GH levels significantly lower than those observed after the first bout for all the protocols, independent from the amount of GH produced following the first bout (range between 4.4 and 18.9 μg/l). GH nAUC after the first and second bouts were also significantly different, indicating that the overall pattern of GH secretion following the two consecutive bouts was characterized by a proportional blunting of GH responsiveness in the three experimental conditions. The blunting of GH responsiveness to repeated bouts of exercise at 2-h intervals detected after maximal voluntary isometric contractions, alone or in combination with WBV, strongly mimics the GH response to GHRH stimulation, suggesting therefore a common underlying mechanism, possibly resting on a relative somatotroph refractoriness and/or reactive increase of the somatostatinergic tone [1]. Blunting of GH responsiveness after the second bout of exercise presently observed was comparable to those occurring after repeated pharmacological stimuli [2–5] or high-intensity voluntary exercise of different types [6–8,24,25]. Further studies of voluntary exercise performed at different intensities and WBV are therefore mandatory to establish the appropriate interpretation of this finding. The results of the blunted GH response to different exercise and pharmacological stimuli contrast with the persistent GH responsiveness after electrically evoked muscle contractions [9]. The persistence of GH responsiveness to repeated electrical stimuli was hypothesised to depend from the relatively scarce GH-releasing ability of electrical muscle stimulation. However, based on the present results, this interpretation appears actually unlikely. In fact, the amount of GH released following the first bout of WBV was comparable to that observed after electrically induced muscle activity (4.4 vs. 4.2 μg/l), whereas the patterns of GH responsiveness to the second bouts were clearly different (blunting of GH responsiveness after WBV vs. its maintenance after electrical stimulation). Although Giustina and Veldhuis [28] proposed that the final common pathway underlying exercise-induced GH release likely involves either increase in GHRH
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function and/or decrease in somatostatin tone, this different GH response to a second equal stimulus in the presence of comparable acute GH-releasing potency in the first bout bespeaks the involvement of different neuroendocrine mechanisms, which remain to be fully evaluated. The present results may elicit a further consideration concerning the role of blood lactate (taken per se or as a marker of blood hydrogen ion concentration) as a regulatory mechanism in GH exercise-related secretion [25,29,30]. In fact, there was a significant correlation between blood lactate and GH concentrations in response to the tested stimuli within either of the two exercise bouts; lactate concentrations after the second bout were similar to those observed after the first one, but they were associated with markedly lower GH levels. This finding suggests some caution in considering lactate as an univocal determinant of GH secretion. In keeping with this view, Stokes et al. [31] reported a divergent pattern between the increased blood lactate concentrations and the attenuated serum GH responses following exercise. Goto and Takamatsu [32], who examined the effects of WBV on GH levels and lipolytic response, observed a significant increase of free fatty acid during the recovery period after the WBV session (150– 210 min). Since in our study protocol the second bouts of WBV, WBV– MVC and MVC occurs 105 mi after the end of the first exercise bouts, the blunted GH response to the second bouts might be actually amenable to a rise of free fatty acids following the first bouts, which might have blocked GH release to the second bouts directly acting at the pituitary level [33]. Although plasma concentrations of free fatty acids were not measured in the present study, however this hypothesis is not completely tenable in these experimental conditions since the blunting of GH responsiveness to the second bouts was similar in the three experimental tests, in spite of markedly different GH peaks after the first bouts. Alternatively, blunting of GH responses to the second bouts might have occurred via changes in blood glucose concentrations, which could be increased for the effects of cathecolamines on hepatic glucose production and/or pancreatic glucagon release [34]. However, this mechanism does not seem working in these experimental conditions, since no significant changes of blood glucose were observed. The lack of changes in glucose concentrations was also observed in our previous studies evaluating repeated bouts (at a 2-h interval) of aerobic exercise in healthy young volunteers [6]. In conclusion, the present study reveals that both MVC and WBV induce a significant response in terms of GH secretion, although with different potencies, and that the response to both stimuli delivered after the short interval of 2 h is blunted in similar proportion, irrespective from the extent of GH rise initially induced by each stimulus. Further studies are needed to identify the complex mechanisms underlying the blunted GH responsiveness to different repeated stimuli and, in addition, the training modalities (even combining different GH-releasing stimuli) capable to increase the endogenous GH production through different physiological processes and mechanisms. The persistence of GH responsiveness after repeated stimuli, which probably requires intervals longer than 2 h (with the exception of electrical muscle stimulation), could tentatively be useful to athletes, GH-deficient patients and sarcopenic elderly subjects for producing physiologically greater amounts of GH-IGF-I, able to positively influence muscle mass and function. Acknowledgements The authors acknowledge dr. S. Chiavaroli, dr. P. Gola, dr. A De Col, dr. R. Galli, dr. C. Busti, Mrs. F. Pera, head nurse at the Division of Metabolic Diseases, and Mr. F Villano for their qualified assistance during the experiments and all the volunteers for their kind collaboration. The Authors are also indebted with Mr. C. Cotelli, past
scientific responsible of the Italian Ski Federation, for the discussion concerning WBV data. The study was partially supported by Progetti di Ricerca Corrente, Istituto Auxologico Italiano, IRCCS, Milan and by Dipartimento di Scienze e Tecnologie Biomediche, Università dell'Aquila, Italy. The research was carried out as a collaborative project among members of the study group “Ottimizzazione della performance motoria: ruolo dei fattori endocrini” of the Italian Society for Endocrinology, Rome, Italy. References [1] E.E. Muller, V. Locatelli, D. Cocchi, Neuroendocrine control of growth hormone secretion, Physiol. Rev. 79 (1999) 511–607. [2] A. Sartorio, A. Spada, F. Morabito, G. Faglia, Different GH responsiveness to repeated GHRH administration in normal children and adults, J. Endocrinol. Invest. 11 (1988) 727–729. [3] E. Ghigo, S. Goffi, E. Mazza, E. Arvat, M. Procopio, J. Bellone, E.E. Muller, F. 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