Effect of sprint interval training on circulatory function ... - Springer Link

Eur J Appl Physiol (2011) 111:1591–1597 DOI 10.1007/s00421-010-1777-z

ORIGINAL ARTICLE

Effect of sprint interval training on circulatory function during exercise in sedentary, overweight/obese women Jennifer L. Trilk • Arpit Singhal • Kevin A. Bigelman Kirk J. Cureton

•

Accepted: 7 December 2010 / Published online: 29 December 2010 Ó Springer-Verlag 2010

Abstract Very high-intensity, low-volume, sprint interval training (SIT) increases muscle oxidative capacity and _ 2max ), but whemay increase maximal oxygen uptake (VO ther circulatory function is improved, and whether SIT is feasible in overweight/obese women is unknown. To _ 2max and circulatory examine the effects of SIT on VO function in sedentary, overweight/obese women. Twentyeight women with BMI [ 25 were randomly assigned to SIT or control (CON) groups. One week before pre-testing, _ 2max testing and the subjects were familarized to VO _ workload that elicited 50% VO2max was calculated. Preand post-intervention, circulatory function was measured at _ 2max , and a GXT was per50% of the pre-intervention VO _ formed to determine VO2max . During the intervention, SIT training was given for 3 days/week for 4 weeks. Training consisted of 4–7, 30-s sprints on a stationary cycle (5% body mass as resistance) with 4 min active recovery between sprints. CON maintained baseline physical activity. Post-intervention, heart rate (HR) was significantly lower and stroke volume (SV) significantly higher in SIT (-8.1 and 11.4%, respectively; P \ 0.05) during cycling at _ 2max ; changes in CON were not significant (3 and 50% VO _ and -4%, respectively). Changes in cardiac output (Q)

Communicated by Niels Secher. J. L. Trilk A. Singhal K. A. Bigelman K. J. Cureton Department of Kinesiology, University of Georgia, Athens, GA, USA J. L. Trilk (&) Department of Exercise Science, Arnold School of Public Health, University of South Carolina, 921 Assembly St., Suite 212, Columbia, SC 29208, USA e-mail: [email protected]

arteriovenous oxygen content difference [(a - v)O2 diff] were not significantly different for SIT or CON. The _ 2max by SIT was significantly greater than by increase in VO CON (12 vs. -1%). Changes by SIT and CON in HRmax (-1 vs. -1%) were not significantly different. Four weeks of SIT improve circulatory function during submaximal _ 2max in sedentary, overweight/ exercise and increases VO obese women. Keywords Aerobic capacity Cardiac output Stroke volume Physical conditioning Training intensity

Introduction _ 2max ) is frequently used as an Maximal aerobic power (VO indicator of cardiorespiratory fitness and is inversely related to all-cause mortality, primarily due to lowered risk of cardiovascular disease (Blair et al. 1989; Taylor et al. _ 2max , physical inactivity, and being over1955). Low VO weight/obese are all risk factors for developing cardiovascular disease, hypertension, and diabetes, and individuals with these diseases have increased morbidity and mortality (Katzmarzyk et al. 2004). Therefore, _ 2max is essential for reducing risk of cardioimproving VO vascular and other chronic diseases in overweight/obese individuals. Repeated bouts of very-intense cycling ([100% _ VO2max ) of short-duration (30 s), termed sprint interval training (SIT), increase skeletal muscle oxidative capacity _ 2max in healthy and has been shown by some to increase VO men and women (Bailey et al. 2009; Burgomaster et al. 2005, 2007, 2008; Gibala et al. 2006; MacDougall et al. 1998). However, studies examining whether circulatory

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function is altered after SIT are scarce. Investigators exam_ 2max ) ining the effect of moderate-intensity (*55–65% VO _ continuous training have found improvements in VO2max , as well as a reduction in heart rate (HR) and an increase in stroke volume (SV) during submaximal exercise (Cunningham and Hill 1975; Ekblom et al. 1968; Pollock et al. 1998; Saltin et al. 1968). High-intensity interval training (85–90% _ 2max ) improves VO _ 2max more than moderate-intensity VO continuous training of equal volume (Daussin et al. 2007, 2008; Warburton et al. 2004). Therefore, intensity can play a _ 2max improvement. Although SIT role in the magnitude of VO _ 2max in healthy, normalhas been shown to increase VO weight college-aged individuals (Bailey et al. 2009; Burgomaster et al. 2008; MacDougall et al. 1998), it has not been established whether SIT is a suitable training modality for _ 2max in individuals improving circulatory function and VO who are sedentary and overweight/obese. The purpose of our study was to examine the effect of SIT on circulatory function during submaximal exercise _ 2max in sedentary, overweight/obese women. We and on VO hypothesized that SIT would improve circulatory function during submaximal exercise at the same power output by decreasing HR and increasing SV, and that SIT would _ 2max in sedentary, overweight/obese women. increase VO

Eur J Appl Physiol (2011) 111:1591–1597

Research design _ 2max and circulaTo determine whether SIT improves VO tory function, a randomized, pretest–posttest control group design was used. Participants were randomly assigned to either a sprint interval training group (SIT, n = 14) or control (CON, n = 14) group. Outcome variables were measured on both groups before and after the intervention period. Familiarization One week prior to pre-testing, the participants came to the laboratory to practice testing procedures. Anthropometric data were collected, and then participants cycled on an electronically braked ergometer (Lode, Groningen, Netherlands) for 10 min at a power output estimated to elicit _ 2max to practice the CO2-rebreathing method used 50% VO to measure cardiac output (Q). After a brief (10 min) rest period, participants performed a graded cycling exercise test (GXT) to become familiarized to give a maximal effort _ 2max test and to determine the workload that for a VO _ 2max . Following a 20-min recovery, parelicited 50% VO ticipants performed two practice sprints on a mechanically braked cycle ergometer (Monark model 874E, Varberg, Sweden) to simulate a partial training session.

Methods Participants

Experimental trials

Twenty-eight women participated in the study. Only women were studied due to lack of information of the effects of SIT in this population, as well as to reduce the heterogeneity of the outcome variables and to increase statistical power. Using a mixed-model, repeated-measures ANOVA, a sample size of 14 per group is sufficient to detect a moderate (Cohen’s d = 0.57) Group 9 Time _ 2max (Burgomaster et al. 2008) interaction effect for VO with an experiment-wise a level of 0.05 and a power of 0.8, assuming a correlation between repeated trials of 0.9 (Park and Schutz 1999). Inclusion criteria included women who were sedentary (exercise B1 day per week) and had a BMI of [25 kg/m2. Women who had been clinically diagnosed with type 1 or type 2 diabetes, had a history of smoking (B6 months), hypertension, or who were on antidepressant, antianxiety, thyroid, or hypertension medication were excluded. The experimental protocol was approved by the University’s Institutional Review Board and all participants provided written informed consent.

All testings were performed in thermoneutral conditions (*25°C, *40% RH). Body mass was measured on an electronic scale (A & D Company, Ltd., Tokyo, Japan) and body composition was measured by dual-energy X-ray absorptiometry (iDXA, GE Healthcare-Lunar, Madison, WI, USA). Then, a sterile, flexible catheter (AngiocathTM, Becton–Dickinson, Franklin Lakes, NJ, USA) was inserted into a forearm vein and kept patent with 0.5 mL of 10 USP units/mL heparin lock flush. Participants sat upright for 20 min in order for plasma volume to stabilize. A resting 2-mL blood sample was drawn into a Vacutainer blood collection tube containing EDTA (Becton–Dickinson, Franklin Lakes, NJ, USA) and analyzed for hemoglobin ([Hb]) in duplicate (HemoCue, Inc., Lake Forest, CA, USA), and hematocrit (Hct) in triplicate using the microhematocrit method. Resting plasma volume (PV) was estimated from fat-free mass (Sawka et al. 1992), and pre- to postintervention change in PV was estimated from changes in resting [Hb] and Hct (Dill and Costill 1974).

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Circulatory measures After the resting measures, participants cycled for 20 min _ 2max so that at a power output estimated to elicit 50% VO circulatory measures could be obtained. From 15 to 17 min, expired air was analyzed by open-circuit spirom_ 2 and VCO _ 2 using a Parvo Medics etry to measure VO TrueOne 2400 Metabolic Measurement System (Parvo Medics, Inc., Sandy, UT, USA). HR was also recorded (Polar Electro, Inc, Woodbury, NY, model 145900). From 17 to 20 min, the CO2-rebreathing procedure was per_ Two rebreathing trials, separated by formed to obtain Q. approximately 1 min, were completed and averaged. Q_ was calculated using the indirect Fick CO2-rebreathing method described by Heigenhauser and Jones (1989). The reliability of Q_ from the two trials was high (ICC for Q_ = 0.93). SV was determined by dividing Q_ by HR. _ 2max VO _ 2max was measured using a continuous, progressive, VO load-incremented cycle ergometer protocol. The graded exercise test (GXT) protocol was adapted from Lafortuna et al. (2006), taking into account the higher energy cost of cycling in obese women. Participants began at 40 W and the power output increased 20 W every 2 min until participants could no longer continue. Metabolic measurements and HR were obtained continuously and averaged over 1-min intervals. Maximal HR, respiratory exchange ratio (RER), and rating of perceived exertion (RPE) using the Borg 15-point scale (Borg and Shephard 1971) were obtained at exhaustion. A fingerstick blood sample was obtained 3 min after the GXT to determine end-test lactate concentration (Lactate Pro, Quesnel, BC, Canada). _ 2 was attained, an addiTo ensure that a plateau in VO tional bout of cycling was performed following a 20-min recovery. Participants cycled to exhaustion at a power output equivalent to the last workload performed during the graded test (if \1 min was completed during the last stage of the graded test) or at a power output 20 W higher than the last workload performed during the graded test (if C1 min was completed during the last stage of the GXT). _ 2max was based on evidence of a plaAttainment of VO _ 2 with increasing power output customized for teau in VO this population (Lafortuna et al. 2006). Lafortuna et al. _ 2 (L/min) (2006) found that the mean (±SD) increase in VO for obese women undergoing a 20-W incremental graded exercise test was 0.264 ± 0.0267 L/min. Using these data, _ 2 increase accepted as the maximum allowed to the VO provide evidence of a plateau was [2 standard deviations _ 2 increase associated with the less than the expected VO

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increase in work (i.e. \0.210 L/min) (Taylor et al. 1955). Using this approach, all participants met the plateau criterion on both the pre- and posttests. In addition, all participants met or exceeded proxy criteria that indicated maximal effort (RPEmax C 18, Lactatemax C 7 mmol/l, RERmax C 1.1, and HRmax C 95% of age-predicted maximum HR). Diet Participants were instructed to maintain their usual diet throughout the intervention period. Participants recorded their dietary intake detailing their daily food consumption for 2 days prior to pretesting and did not consume caffeine or alcohol for 24 h before the test day. The food diaries were collected, photocopied, and returned to the participants after pretesting. Two days prior to posttesting, participants were instructed to replicate their pretest diet to decrease any confounding effects of food intake on their performance. Interventions Participants in SIT followed a training protocol modified from Burgomaster et al. (2005). Training sessions consisted of repeated cycling sprints (4–7 bouts/session) on a Monark ergometer. Participants warmed up for 4 min with no resistance, and then began pedaling at maximal cadence *5 s before a fixed resistance of 0.05 kg/kg (5%) body mass was applied. Participants continued pedaling as fast as possible against the resistance for 30 s. After the sprint, the resistance was removed and they continued cycling for 4 min of active recovery (low RPMs at 0% body mass). These intervals were repeated until the prescribed number was performed. On the first training day, participants performed four sprints with 4 min of active recovery between bouts. The subsequent training sessions increased by one sprint bout every week, finishing with cycling 7 sprints/ session in the last week. Participants were trained 3 days/ week (1–2 days rest between sessions) for 4 weeks and were instructed to maintain their pre-study physical activity outside of the training intervention. Work performed by SIT during the 4-week training regimen was recorded to quantify the training volume. An optical sensor (Sports Medicine Industries, Inc., St. Cloud, MN, USA) was secured to the ergometer frame and 16 reflective markers were placed on the flywheel to record flywheel revolutions. The sensor was interfaced with a computer and SMI Power software (version 1.02) recorded pedal rate (rpm) and calculated work each second (W s-1). The work for the 30 s during the sprint was summed and converted to Joules (J) to obtain work performed for each sprint. The work for each session over the 4 weeks was

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calculated to obtain weekly and total work of the intervention. Finally, work done on the 4th sprint of each session was used to assess any changes in power output during the course of training. The 4th sprint bout was chosen because it would allow for any changes in effort or power to be observed over the training period (vs. using the 1st sprint bout), and it was the minimum number of sprints performed in a session. Participants in CON were instructed to maintain their baseline physical activity during the intervention period and to neither increase nor decrease their activity level. After completion of the posttests, participants were offered the opportunity to participate in SIT training for 4 weeks.

Work performed The weekly average total work performed during the 30-s sprints for SIT increased significantly each week by an average of 35 ± 2 kJ (mean ± SEM) (139 ± 8, 177 ± 10, 210 ± 13, 245 ± 10 kJ for weeks 1, 2, 3, and 4, respectively), due to the addition of one more sprint bout every week. Work performed on the 4th sprint of each session was not statistically significantly different across weeks, indicating that the progression in work performed across weeks was primarily due to the greater number of intervals performed. Cardiovascular function during submaximal exercise

Statistical analysis Data were analyzed using SPSS v. 15.0, (SPSS, Inc., Chicago, IL, USA). Differences between groups at baseline for all physical characteristics and outcome variables were analyzed using a t test for independent samples. Differences in work performed by SIT across 4 weeks of training were analyzed using a one-way, repeated measures ANOVA. Bonferroni post hoc tests were performed to test for differences between weeks. To test the significance of the effects of the intervention, a two-way (Group 9 Time), mixed-model ANOVA was used. If an interaction existed, tests for simple effects were performed to examine the effect of the treatment on each group separately. Differences were considered statistically significant if P \ 0.05.

Results Participant characteristics No statistically significant differences existed between groups at the pretest for age, BMI, or percentage body fat (Table 1). Pretest-to-posttest changes in body mass, BMI, and percent body fat in SIT were not significantly different from changes in CON.

_ 2 at 50% of the pretest VO _ 2max was Power output and VO not significantly different between SIT and CON at the pretest and did not change significantly with training in _ 2max , either group (Table 2). During cycling at 50% VO HR was significantly lower and SV was significantly higher (P \ 0.001) in SIT (-11 beats min-1, -8.1%, 9.7 mL beat-1, 11.4%, respectively), whereas changes in CON were not statistically significant (4 beats min-1, 3%, P = 0.165; -3.2 mL beat-1, -4%, Table 2). Changes by SIT and CON in Q_ and (a - v)O2 diff (P = 0.180) were not significantly different. _ 2max VO _ 2max (L min-1) was not significantly different between VO _ 2max SIT and CON at the pretest. The change in VO -1 (L min ) by SIT was significantly different (Fig. 1) than _ 2max increased 0.245 L min-1 (12%) in SIT CON. VO while CON did not change (-1%). Because there were no significant changes in body mass during the intervention _ 2max expressed relative to body mass period, VO -1 (mL kg min-1) also increased significantly for SIT, but not for CON (Table 3). Indicators of maximal effort on the GXT were similar in the two groups before and after training (Table 3). HRmax

Table 1 Participant physical characteristics (mean ± SD) Variable

SIT

CON

Age (years)

30.1 ± 6.8

31.4 ± 5.5

Mass (kg)

96.8 ± 15.2

96.6 ± 22.7

Height (cm)

164.5 ± 6.2

166.4 ± 7.7

Body mass index (kg m-2)

35.7 ± 6.3

34.6 ± 5.9

Fat mass (%)

48.0 ± 5.7

46.9 ± 5.2

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HRmax was not different between groups at the pretest. The changes in HRmax in the two groups were not significantly different (Table 3). Mean changes in SIT and CON were -2 and -2 beats min-1, respectively. Estimated resting plasma volume Estimated PV at rest was not significantly different between groups at the pretest. The changes in PV were not


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Table 2 Metabolic and circulatory measures at 50% VO2max (mean ± SEM) Variable

SIT Pre

CON Post

60 ± 17a

Power output (W) -1

Pre

Post

55 ± 9a

VO2 (L min )

1.17 ± 0.05

1.16 ± 0.05

1.13 ± 0.05

1.11 ± 0.05

Heart rate (beats min-1)

135 ± 4

124 ± 4*

133 ± 4

137 ± 4 81.3 ± 5.2

-1

Stroke volume (mL beat )

86.9 ± 5.0

96.6 ± 6.1*

84.5 ± 5.6

Cardiac output (L min-1)

11.5 ± 0.5

11.8 ± 0.6

11.3 ± 0.7

11.2 ± 0.8

(a - v)O2 diff (mL 100 mL-1)

10.2 ± 0.3

9.9 ± 0.3

10.0 ± 0.5

10.2 ± 0.4

* Significant change from pre- to post-treatment (P \ 0.05) a

Power output was the same for pretest and posttest

_ 2max measured before and after the intervention. Values Fig. 1 VO are mean ± S.E.M. *Significant Group 9 Treatment interaction (P \ 0.001)

significantly different, although PV in SIT tended to increase (4%, 86 mL) compared to no change increase for CON.

Discussion We examined whether SIT improves circulatory function _ 2max in sedentary, during submaximal exercise and VO overweight/obese women. Our primary finding was that

SIT improved cardiovascular function during submaximal exercise by reducing HR and increasing SV, and increased _ 2max . These results indicate that very high-intensity, VO low-volume cycling training is an effective and time-efficient method for improving circulatory function and _ 2max , thereby increasing functional capacity and VO decreasing risk for all-cause mortality in sedentary, overweight/obese women. _ 2max . It is generally SIT elicited a 12% increase in VO _ accepted that improvements in VO2max are related to total amount of work (volume) completed, as determined by the intensity, duration, and frequency of training (Pollock et al. 1998). However, Gormley et al. (2008) found that adap_ 2max to training was directly related to intensity tation of VO and independent of volume up to an intensity of 95% _ 2max . Similarly, Bailey et al. (2009) observed a 7.5% VO _ 2peak after 2 weeks of SIT in healthy, normal increase in VO weight adults but no change after 2 weeks of work-matched, moderate-intensity endurance training. The training used in our study involved a much-higher intensity and about one-half the volume compared to previous studies _ 2max with prolonged continuous or showing improved VO interval exercise training. Based on the power outputs elicited during training and extrapolation of the oxygen

Table 3 VO2max and associated measures obtained during the GXT (mean ± SEM) Variable

SIT

CON

Pre

Post

Pre

Post

VO2max (mL/kg min-1)

21.6 ± 1.1

24.5 ± 1.1*

20.5 ± 0.9

20.4 ± 0.8

VO2max (mL/kg FFM min-1)

43.1 ± 1.6

47.7 ± 3.6*

40.6 ± 1.3

40.0 ± 1.0

O2 Pulsemax (mL beat-1)

11.1 ± 0.5

12.7 ± 0.5*

10.7 ± 0.4

10.7 ± 0.4

Heart ratemax (beats min-1)

184 ± 3

182 ± 3

182 ± 3

180 ± 3

RERmax

1.16 ± 0.01

1.16 ± 0.01

1.17 ± 0.01

1.16 ± 0.02

Lactatemax (mmol L-1) RPEmax

9.8 ± 0.5 19.4 ± 0.2

10.3 ± 0.4 19.0 ± 0.2

9.2 ± 0.4 19.4 ± 0.2

8.5 ± 0.3* 19.2 ± 0.1

* Significant change from pre- to post-treatment (P \ 0.05)

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uptake required by overweight/obese women during submaximal intensities, we estimated participants trained at _ 2max during intensities that would elicit 243 to 284% of VO the sprint intervals. However, total volume of work performed during sprinting for the 4-week intervention was 772 ± 37 kJ (mean ± SEM), and total duration was 33 min. In contrast, if the women in our study would have _ 2max for 30 min, 3 times/week for exercised at 65% VO 4 weeks, the minimum recommended quantity and quality of exercise for developing and maintaining cardiorespiratory fitness (Pollock et al. 1998), they would have expended *1,469 kJ and cycled a total of 6 h. Our study and others (Bailey et al. 2009; Burgomaster et al. 2008; MacDougall et al. 1998) have shown that substantial increases _ 2max can be obtained when the intensity of training is in VO _ 2max ) and the volume is relatively very high ([100% VO low. This finding reinforces the conclusion that intensity is the most important element of the physical activity stim_ 2max . Very high ulus (Hickson et al. 1981) for increasing VO intensity apparently can negate the need for high volume, a clear advantage for those who need to improve their aerobic power, but are unaccustomed to long-duration exercise of moderate-to-high intensities. _ 2max observed in our The greater improvement in VO 4-week study (12%) compared to other studies of SIT lasting 6–7 weeks (7%) (Burgomaster et al. 2008; MacDougall et al. 1998) may have been due to the very low _ 2max (average VO _ 2max 21.6 mL kg initial level of VO -1 -1 BW min at baseline) in the sedentary, overweight/ obese women we studied. MacDougall et al. (1998) trained healthy, young, normal weight, physically active men _ 2max 50.8 mL kg BW-1 min-1 at baseline), (average VO whereas Burgomaster et al. (2008) trained healthy young, normal weight individuals not engaged in a regular exer_ 2max 40.6 mL kg BW-1 cise training program (average VO -1 min at baseline). The rapid, relatively large improvement _ 2max in sedentary, overweight/obese women suggests in VO SIT should be considered as an intervention when rapid changes in cardiorespiratory fitness and reduced risk of chronic disease are desired in this population. Our study is the first to demonstrate that SIT improves circulatory function during submaximal exercise in sedentary, overweight/obese women. The mechanisms underlying increased SV typically include a training-induced enlargement of left ventricular chamber size (Moore 2006; Saltin et al. 1968), a slight increase in blood volume (Warburton et al. 2004), and cardiac muscle hypertrophy with enhanced contractility during systole (Moore 2006). Mechanisms underlying reduced HR include increased parasympathetic and reduced sympathetic stimulation of the sinoatrial node, and altered intrinsic firing rate of the

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sinoatrial node (Moore 2006). Submaximal (a - v)O2 diff did not change after SIT. Although Warburton et al. (2004) examined circulatory function at maximal exercise, they also observed an increase in SV (due to increased enddiastolic volume and blood volume with no change in contractility), with no change in (a - v)O2 diff following 12 weeks of high-intensity interval training (2-min exercise _ 2max ). Our study extends the previous intervals at 90% VO research on high-intensity interval training by showing that interval training involving much higher intensities ([240% _ 2max ) and considerably lower volumes also favorably VO alters circulatory function during submaximal exercise. Therefore, SIT may be a feasible training modality to improve circulatory function in sedentary, overweight/ obese women. Increased PV can be a significant contributor to increased SV. Whether an increase in PV is an acute or chronic response to exercise can be debated. Gillen et al. (1991) observed a 10% increase in PV 24 h after a single session of intermittent aerobic exercise (4-min bouts at _ 2max with 5-min rests). However, even after 85% VO _ 2max ), as longer-duration continuous (30-min at 64% VO _ 2max ) well as high-intensity intermittent (2 min at 90% VO aerobic exercise training (12 weeks), has been shown to increase PV * 11% (Warburton et al. 2004). This increase is due in part to an increase in plasma albumin content (Nagashima et al. 1999, 2000). After 3 days of SIT (1-min _ 2max ), Green et al. (1984) exercise, 4-min rest; 120% VO found an 11.3% increase in PV; however, their training protocol consisted of 24 sprints/session, whereas ours was, at maximum, 7 sprints/session in the final week of training. The relatively small, non-significant 4% increase in resting PV after 4 weeks of SIT was considerably less than that reported in these studies. It is possible that the shorter duration of the work intervals (30 s) and lower volume of the SIT training was inadequate to stimulate PV expansion. In our study, the increased PV apparently contributed little to the increase in SV following SIT. However, our data are limited because PV was estimated and not directly measured. Changes in hydration status of the subjects also could have confounded any changes that may have been observed pre- versus post-testing. In summary, we found that 4 weeks of very highintensity, low-volume SIT improved cardiovascular function during submaximal exercise by increasing SV and _ 2max in sedentary, overreducing HR, and increased VO weight/obese women. Very high intensity apparently can negate the need for high volume, a clear advantage for those who need to improve their aerobic power and cardiovascular capacity, but are unaccustomed to long-duration exercise of moderate-to-high intensities. Despite very


low fitness at the onset, the sedentary, overweight/obese women in our study completed the training program without undue duress or mishap, indicating that SIT can be an effective training modality for increasing cardiorespiratory fitness and decreasing risk of all-cause mortality in this population subgroup at risk for developing multiple chronic diseases. Acknowledgments The authors thank Miley Duvall, Mai Nguyen, Hemal Patel, Anna Gelbrich, Diana Kim and Sahir Ahsan for their technical assistance with the study. Conflict of interest

The authors declare no conflict of interest.

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