Muscle phosphocreatine and pulmonary oxygen ... - Springer Link

Eur J Appl Physiol (2008) 102:727–738 DOI 10.1007/s00421-007-0650-1

ORIGINAL ARTICLE

Muscle phosphocreatine and pulmonary oxygen uptake kinetics in children at the onset and offset of moderate intensity exercise Alan R. Barker Æ Joanne R. Welsman Æ Jonathan Fulford Æ Deborah Welford Æ Craig A. Williams Æ Neil Armstrong

Accepted: 4 December 2007 / Published online: 3 January 2008 Ó Springer-Verlag 2007

Abstract To further understand the mechanism(s) _ 2Þ explaining the faster pulmonary oxygen uptake ðpVO kinetics found in children compared to adults, this study _ 2 kinetics in children examined whether the phase II pVO are mechanistically linked to the dynamics of intramuscular PCr, which is known to play a principal role in controlling mitochondrial oxidative phosphorylation during metabolic transitions. On separate days, 18 children completed repeated bouts of moderate intensity constant work-rate exercise for determination of (1) PCr changes every 6 s during prone quadriceps exercise using 31Pmagnetic resonance spectroscopy, and (2) breath by _ 2 during upright cycle ergometry. breath changes in pVO Only subjects (n = 12) with 95% confidence intervals B±7 s for all estimated time constants were considered for analysis. No differences were found between the PCr _ 2 time constants at the onset (PCr 23 ± and phase II pVO _ 5 vs. pVO2 23 4 s; P ¼ 1:000Þ or offset (PCr 28 ± 5 _ 2 29 5 s; P ¼ 1:000Þ of exercise. The average vs. pVO _ 2 time difference between the PCr and phase II pVO constants was 4 ± 4 s for the onset and offset responses. Pooling of the exercise onset and offset responses

A. R. Barker J. R. Welsman C. A. Williams N. Armstrong (&) Children’s Health and Exercise Research Centre, University of Exeter, St Luke’s Campus, Exeter EX1 2LU, UK e-mail: [email protected] J. Fulford Peninsula Medical School, University of Exeter, Exeter, UK D. Welford Cardiff School of Sport, University of Wales Institute, Cardiff, UK

revealed a significant correlation between the PCr and _ 2 time constants (r = 0.459, P = 0.024). The close pVO _ 2 and PCr responses at kinetic coupling between the pVO the onset and offset of exercise in children is consistent with our current understanding of metabolic control and suggests that an age-related modulation of the putative phosphate linked controller(s) of mitochondrial oxidative _ 2 kinetics phosphorylation may explain the faster pVO found in children compared to adults. Key words 31P-MRS Developmental Metabolism Muscle oxygen consumption

Introduction At the onset of a ‘‘step’’ transition from rest to a higher _ 2Þ metabolic rate, the rise in muscle O2 utilization ðmVO exhibits considerable delay before the rate of oxidative ATP supply and ATP hydrolysis in the myocyte are suitably matched. Debate exists as to whether this _ 2 is limited by the provision of delayed rise in mVO oxygen to the contracting muscle, or through an intrinsic ability of the muscle to utilize O2 (Grassi 2005; Hughson 2005). While an interaction between the above factors is _ 2 (Tschakovsky likely to determine the adaptation of mVO and Hughson 1999), there is strong evidence supporting _ 2 is principally controlled the theory that the rise in mVO by one (or a combination) of the reactant(s) and/or product(s) involved in the creatine kinase splitting of muscle PCr (Grassi 2005; Meyer 1988; Rossiter et al. 2005). Using 31P-magnetic resonance spectroscopy (31P-MRS), Rossiter et al. (1999) established a close kinetic coupling between the respective fall and rise in PCr and phase II

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_ 2 ; which provides a close pulmonary O2 uptake ðpVO _ 2 ; Grassi et al. 1996) at the onset of reflection of mVO prone knee-extensor exercise. Moreover, the acute inhibition of the creatine kinase reaction resulted in a pronounced _ 2 speeding (*50%) in the fall in PO2 (which reflects mVO in this model) at the onset of isometric contractions in frog muscle (Kindig et al. 2005). Therefore, following a ‘‘step’’ change in external work-rate, muscle PCr provides an immediate temporal buffer for ATP, thereby delaying the rise in the putative metabolic feedback controllers to signal an increased rate of mitochondrial oxidative phosphorylation. Specifically, changes in cellular PCr/Cr may modulate the sensitivity of the mitochondria to ADP via creatine kinase isoforms in the cytoplasmic and mitochondrial spaces (Walsh et al. 2001). Investigations designed to quantify the adjustment of _ 2 at the onset of exercise in children have provided pVO interesting insights into the kinetic response parameters. A consistent finding is that younger children display a _ 2 at the onset of moderate and faster phase II rise in pVO heavy intensity exercise compared to older children or adults (Fawkner and Armstrong 2004; Fawkner et al. 2002b; Williams et al. 2001). In a longitudinal study, _ 2 Fawkner and Armstrong (2004) found the phase II pVO time constant during heavy intensity cycling exercise increased (i.e. slower kinetics) by 25 and 29% in 11–13 year-old girls and boys, respectively. In combination with the results obtained from an earlier study for moderate cycling exercise (Fawkner et al. 2002b), the authors proposed an age-related decline in the muscles’ potential to utilize O2, perhaps via a slower activation of metabolic enzymes and/or the build-up of putative metabolic controllers may account for these findings. This, however, has yet to be investigated. Considering the prominent role muscle PCr plays in _ 2 during metabolic controlling the adaptation of mVO transitions, we were interested in examining the kinetic _ 2 and muscle PCr association between the phase II pVO responses in children in an attempt to further understand _ 2 kinetics the mechanisms underlying the faster pVO found in children compared to adults. In contrast to experimental work conducted in adults whereby the _ 2 were determined kinetics of muscle PCr and pVO simultaneously during knee-extensor exercise (Rossiter _ 2 signal arising from the quadriceps et al. 1999), the pVO muscle in children is insufficient to accurately quantify the response characteristics. We therefore based our experimental model upon the work by Barstow et al. _ 2 were measured during (1994). The kinetics of pVO upright cycle ergometry and compared to the kinetics of muscle PCr determined during prone quadriceps exercise using 31P-MRS. Although cycling in the supine position _ 2 kinetics results in a slowing of the exercise onset pVO

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Eur J Appl Physiol (2008) 102:727–738

compared to the upright position (Hughson et al. 1993), exercise modalities that recruit less muscle mass, i.e. plantar-flexors or knee-extensors, in the supine/prone body position demonstrate no differences in the kinetics _ 2 _ 2 when compared to upright cycling pVO of PCr or pVO kinetics (Barstow et al. 1994; Rossiter 2000). Therefore, _ 2 kinetics determined during upright in comparison to pVO cycling, these data indicate that body position does not exert a limiting influence on the kinetics of muscle PCr _ 2 providing that the exercise modality in the and pVO prone/supine body position is restricted to a small muscle mass. Therefore, the purpose of the present study was to test _ 2 kinetics in children the hypothesis that the phase II pVO are mechanistically linked to the putative metabolic controller muscle PCr, both at the onset and offset of moderate intensity exercise.

Materials and methods Subjects Eighteen 9–11 year-old children (eight boys and ten girls) recruited from a local primary school were included in the current study. After written and verbal explanation of the study’s aims, risks and procedures, all children and their parent(s)/guardian(s) provided informed consent to partake in the project that was approved by the institutional ethics committee. Preexperimental questionnaires identified that all subjects were healthy and showed no contraindications to exercising inside the MR scanner. Each subject made between six and ten visits to the Research Centre. The purpose of the first visit was to ensure that the subjects were fully familiarised with the laboratory setting and exercise procedures. In particular, all children were habituated to exercising on a knee extension/ flexion ergometer at the required cadence inside a purpose built, to scale model of the MR scanner. All subjects completed a number of repeat trials on the ergometer at a range of work intensities, identical to the actual protocols employed in the study. All subjects were well habituated to the test procedures.

Descriptive characteristics Subjects’ body mass and stature were measured using a calibrated balance beam scale (Avery, Birmingham, UK) and a stadiometer (Holtain, Crymych, Dyfed, UK), respectively. Subjects’ age was calculated as the difference between the date of birth and the date of the first visit.


729

Quadriceps exercise inside the MR scanner

Constant work-rate exercise

Ergometer description

Subsequently, each subject completed repeated constantload exercise transitions inside the MR scanner with the work rate set to 80% of ITPi/PCr. The exercise protocol consisted of a 2 min rest period for baseline measures, then 6 min constant work-rate quadriceps exercise followed by 6 min rest for assessment of the recovery dynamics. Between two to four repeat constant work-rate exercise transitions were performed on a given day, with at least 15 min rest given between each test. In total between three and nine repeat transitions were performed by each subject.

The quadriceps exercise consisted of performing dynamic knee extensions and flexions with the right leg on a nonmagnetic ergometer whilst lying prone inside the MR scanner. The right foot was fastened to a padded foot brace, which was connected to the ergometer load basket using a rope and pulley system. This provided resistance against which continuous concentric and eccentric quadriceps contractions could be performed inside the MR scanner over a distance of *0.22 m. To ensure interrogation of the quadriceps muscles for metabolite changes occurred in the same volume of interest (VOI) and to standardise the exercise protocol both within and between subjects, the quadriceps exercise was performed at a cadence set in unison with the magnetic pulse sequence (40 repetitions min-1). Alignment of the knee extensions/flexions with the pulse sequence was guided using a projected image of a vertical moving cursor set to the frequency of 40 pulses min-1. The subjects were required to follow the metronomic cursor using a second vertical cursor under voluntary control of the subject. To prevent displacement of the quadriceps VOI relative to the surface coil and minimise adjacent muscles contributing to the exercise task, nylon straps were fastened over the subject’s legs, hips and lower back. Power output (W) was calculated continuously during the quadriceps exercise as described previously (Barker et al. 2006).

Step-incremental test Each subject completed an incremental test to exhaustion inside the MR scanner for determination of the intracellular threshold (IT) between the ratio of Pi and PCr (ITPi/PCr). Following a 2 min baseline measurement period and starting with an initial basket load of 0.5 kg, an incremental test was undertaken whereby the basket load was increased in ‘‘steps’’ of 0.5 kg min-1 until subject exhaustion occurred. This was typically within 7–12 min. Using a plot of Pi/PCr versus power output at a sample resolution of 30 s, each subject’s ITPi/PCr was identified by two investigators as previously described (Barker et al. 2006). The ITPi/PCr was defined as the power output at which the first sudden and sustained increase in Pi/PCr from the initial linear slope occurred (Marsh et al. 1991). Using this technique with children, the ITPi/PCr typical error over three repeat tests within our laboratory resulted in a coefficient of variation of 10% (Barker et al. 2006).

31

P-MRS measurement and quantification

A 1.5 T whole body MR scanner (Philips Gyroscan Intera) was used to monitor the changes in quadriceps muscle energetics. A 6 cm 31P transmit/receive surface coil was fastened securely to the scanner bed and positioned under the subject’s right quadriceps muscle at the midpoint between the hip and knee joints. Gradient echo images were initially acquired to ensure the quadriceps muscle was positioned correctly relative to the coil. An automatic shimming protocol using the 1H signal was undertaken within a volume that defined the quadriceps muscle in order to optimise the homogeneity of the local magnetic field (with a typical line width of *14 Hz at FWHM), thereby leading to maximum signal collection. Tuning and matching of the coil was then performed to maximise energy transfer between the coil and the muscle. 31P spectra were obtained using an adiabatic pulse every 1.5 s, with a spectral width of 1,500 Hz and 512 data points. Phase cycling with four phase cycles was employed and four measurements were performed, leading to spectra acquired every 6 s to improve the signal to noise of the profile yet provide a high PCr sampling resolution for kinetic analysis. As the signal intensities for 31P spectra were significantly saturated during the test protocol, T1 correction factors were determined during the rest phase using a pulse interval of 20 s and applied to all peak intensities. The assumption that the T1 relaxation time for muscle PCr does not change from rest to steady-state exercise has recently been confirmed during calf exercise below the intracellular threshold (Cettolo et al. 2006). The 31P spectra areas were quantified using a non-linear least squares peak fitting software package (jMRUI Software, version 2.0) employing the AMARES fitting algorithm (Naressi et al. 2001; Vanhamme et al. 1997). Spectral areas were fitted assuming prior knowledge of the following peaks: Pi, phosphodiester, PCr, a-ATP (two peaks, amplitude ratio 1:1), c-ATP (two peaks, amplitude ratio 1:1) and b-ATP (three peaks, amplitude ratio 1:2:1).

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Changes in PCr were expressed as a percentage change from baseline, set to 100%, using the mean PCr concentration obtained during the 2 min rest period. Intracellular pH was determined using the chemical shift of the Pi spectral peak relative to the PCr peak: pH ¼ 6:75 þ logðr 3:27Þ=ð5:96 rÞ where r represents the chemical shift in parts per million between the Pi and PCr resonance peaks (Taylor et al. 1983).

Upright cycling exercise Peak oxygen uptake Each child exercised to volitional exhaustion on a mechanically braked cycle ergometer (Lode, Groningen, Netherlands) in a temperature controlled laboratory (1922°C) for determination of the ventilatory threshold (VT). Appropriate adjustments were made to the ergometer seat, _ 2 handlebar and pedal cranks for each subject. Peak pVO was determined using a ramp incremental test initiated from baseline pedalling with work rate increasing 10 W min-1 until subject exhaustion occurred (typically within 8–12 min). Cycle cadence was maintained between 70 ± 5 rpm during the test. Heart rate was measured continuously during the test using telemetry (Polar, Accurex Plus, Kemple, Finland). In addition to subjective signs of sweating, hyperpnea and intense effort, the attainment of a _ 2 was supported using the following criteria: (1) a peak pVO respiratory exchange ratio (RER) C 1.00, and/or (2) attainment of a maximum heart rate C 90% of the age predicted maximum. In all cases, each subject satisfied at _ 2 was recorded as the least one of these criteria. Peak pVO highest 10 s stationary average attained during the test. The _ 2 at which the VT occurred was established using a pVO combination of methods: (1) a plot of carbon dioxide _ _ production ðVCO 2 Þ against pVO2 and (2) plots of the _ _ 2 against time. ventilatory equivalents for pVO2 and VCO The reliability of these techniques with children in our laboratory has previously being reported (Fawkner et al. 2002a).

Constant work-rate exercise On subsequent visits, each child completed moderate intensity constant work-rate exercise transitions on the cycle ergometer. The protocol consisted of 6 min baseline pedalling (*15 W) followed by 6 min constant-work exercise at the subject’s target intensity (80% VT), and then 6 min baseline pedalling for recovery measurements.

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Between two to three repeat transitions were performed on a given day, with at least 30 min rest given between each test. In total the children repeated between three and ten exercise _ 2 kinetic responses. transitions for determination of the pVO

Measurement of breath by breath gas exchange Breath by breath changes in gas exchange and ventilation were measured using a standard algorithm (Beaver et al. 1973) and displayed using an on-line computer system. Gas fractions of oxygen and carbon dioxide were drawn continuously from a low dead space (90 mL) mouthpieceturbine assembly and determined by mass spectroscopy following calibration with gases of known concentration (EX671, Morgan Medical, Kent, UK). Expired volume was measured by a turbine flow meter (VMM-401, Interface Associates, CA, USA), which was manually calibrated over a range of flow rates using a 3-L syringe (Hans Rudolph, Kansas City, MO, USA). Appropriate adjustments for the capillary line gas transport and analyser response time were made before gas concentrations and volumes were aligned for calculation of breath by breath _ 2 and VCO _ 2 : All calibration procedures changes in pVO were performed prior to each exercise test.

Kinetic parameter estimation _ 2 kinetic parameters were estimated using All PCr and VO an iterative least-squares non-linear regression procedure along with their corresponding 95% confidence intervals (95% CIs) to establish the precision of the point estimate (GraphPad Prism, GraphPad Software, San Diego, CA, USA).

Muscle PCr kinetic analysis Each separate constant-work exercise transition was initially checked for PCr sample fluctuations that were greater than ±4 standard deviations (SD) from a moving local mean (Whipp and Rossiter 2005). Such large PCr fluctuations are considered unrelated to the underlying physiological profile and are likely to arise due to the low signal to noise properties of the 31P-MRS technique and/or the acute mistiming of a quadriceps contraction relative to the pulse sequence during exercise. To enhance the underlying features of the PCr response profile for kinetic parameter estimation, each subject’s repeat constant workrate exercise transitions were time aligned to the onset of exercise (t = 0 s) and averaged, yielding a single PCr response profile with a 6 s sample resolution (Rossiter et al.


2000). The resultant averaged PCr response profile for each subject was normalised relative to the previous steady-state baseline, using the average PCr value during the 2 min rest or exercise period for determination of the onset and offset kinetics, respectively. The PCr exercise onset and offset responses were initially modelled using a single-exponential function including a delay term: PCrðtÞ ¼ DPCrss ð1 eðtTDÞ=s Þ where PCrðtÞ ; DPCrss ; TD and s are the value of PCr at a given time (t), the amplitude change in PCr from the control baseline to a new steady-state at the onset or offset of exercise, time delay, and the time constant of the response, respectively. However, preliminary analyses revealed the model delay term was not significantly (P [ 0.05) greater than 0 s both at the onset and offset of exercise. This indicates that the breakdown and resynthesis of muscle PCr occurred with no detectable delay at both the onset and offset of exercise. A single exponential model with no delay term was therefore employed for all analyses: PCrðtÞ ¼ DPCrss ð1 et=s Þ _ 2 kinetic analysis Pulmonary VO Each breath by breath constant work-rate profile was interpolated on a second by second basis to provide a _ 2 sample resolution. Following time alignuniform pVO ment to the onset of exercise (t = 0 s), subjects’ repeat constant work-rate exercise transitions were superimposed and averaged to enhance the signal to noise ratio and _ 2 response thereby the underlying features of the pVO _ (Lamarra et al. 1987). The averaged pVO2 profile for each subject was subsequently normalised relative to the previous steady-state baseline as described above for the PCr _ 2 data analysis. As the accurate identification of the pVO phase I-II transition using the RER, and end tidal (PCO2 and PO2) dynamics was difficult due to the noise present in the children’s response profile, the duration of phase I was taken as 15 s for all subjects both at the onset and offset of exercise (Hebestreit et al. 1998; Ozyener et al. 2001). _ 2 exercise onset and Characterisation of the phase II pVO offset responses was achieved by fitting a single-exponential model including a delay term following the initial 15 s (to exclude phase I) of the exercise or recovery regions (Whipp et al. 1982): _ 2ðtÞ ¼ DpVO _ 2ss ð1 eðtTDÞ=s Þ pVO _ 2ss ; TD and s represent the value of _ 2ðtÞ ; DpVO where pVO _ _ 2 pVO2 at a given time (t), the amplitude change in pVO

731

from baseline to a new steady-state amplitude, time delay and the time constant of the response, respectively.

Statistical analyses To enhance the statistical confidence and sensitivity in _ 2 kinetic time constants comparing the PCr and phase II pVO either within or between subjects, only subjects with 95% CIs equal to or less than ±7 s for all estimated time constants were considered for analysis. Potential mean differences in the estimated time constants were examined using a two-way repeated measures ANOVA with response _ 2 Þ and exercise transition (onset vs. variable (PCr vs. pVO offset) as the model factors. Changes in pH during the rest, exercise and recovery regions of the constant work-rate exercise tests were examined using a one-way repeated measures ANOVA. The assumption of sphericity was verified using Mauchly’s test for all repeated measures ANOVA models. Significant differences were followed-up using planned pairwise comparisons employing the Bonferroni alpha adjustment procedure. Single linear regression was employed to investigate the association between the _ 2 estimated time constants. Results PCr and phase II pVO are presented as mean ± SD, with rejection of the null hypothesis accepted at an alpha level of P = 0.05. Analyses were performed using SPSS (version 11.0). Results Descriptive and exhaustive test responses Out of the 18 subjects recruited, 12 children (six boys and six girls) satisfied the time constant 95% confidence interval criterion. The descriptive characteristics of these 12 subjects’ were: age (9.9 ± 0.3 years), body mass (33.8 ± 6.7 kg) and stature (1.38 ± 0.06 m). The subjects’ average exercise responses during the incremental tests to exhaustion for determination of the ITPi/PCr and VT are presented in Table 1.

Constant work-rate exercise tests The average work rate and pH responses during the quadriceps constant work-rate exercise are presented in Fig. 1. It is clear from the power profile in Fig. 1a that work rate increased and decreased instantaneously at the onset and offset of exercise, thus highlighting the subjects’ high compliance with the quadriceps ergometer. This resulted in the attainment of an average power output of 7 ± 1 W, which corresponds to 83 ± 13% of the ITPi/PCr. The

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a

Table 1 Subjects’ incremental test responses Variable

10

Group (n = 12) 8

Peak power (W) ITPi/PCr (W) ITPi/PCr (% PP) Cycle ergometry _ 2 (l min-1) Peak pVO _ 2 at VT (l min-1) pVO _ 2 (%) VT peak pVO

15 ± 3 9±2 59 ± 9

Work rate (W)

Quadriceps ergometry

1.81 ± 0.23

6

4

2

1.05 ± 0.19 58 ± 8

0

Data are presented as mean ± SD W, watts; IT, intracellular threshold; % PP, IT work rate expressed as _ 2 ; pulmonary oxygen uptake; VT, ventilatory a % peak power; pVO threshold

b

7.15

averaged pH response in Fig. 1b demonstrates that the quadriceps constant work-rate exercise did not result in a metabolic acidosis during the exercise tests. Compared to rest (7.05 ± 0.03) a small but significant increase in quadriceps pH was noted during the last 2 min of constant-work exercise (7.08 ±0.02, P = 0.033). pH during the final 2 min of recovery (7.02 ± 0.02) was significantly lower than both rest (P = 0.018) and exercise (P = 0.000). During the cycle ergometry constant work-rate exercise, the children _ 2 amplitude of 0.90 ± 0.13 l min-1 achieved a pVO _ 2 at VT). In combi(equivalent to 87 ± 11% of the pVO nation, these observations indicate that the imposed work rates for both exercise modalities were below the metabolic ITs (ITPi/PCr and VT) and considered to reflect ‘‘moderate’’ exercise. On average, the children completed 6 ± 2 and 7 ± 2 repeat constant work-rate exercise transitions for determi_ 2 kinetics responses nation of the PCr and pVO _ 2 response profile for a respectively. A typical PCr and pVO child subject at the onset and offset of exercise is shown in Fig. 2, along with the fitted non-linear regression model. In all cases the single-exponential model provided an appro_ 2 dynamics, as indicated by priate fit of the PCr and pVO the unsystematic and random residual profiles. The estimated time constants and 95% CIs for the _ 2 responses at the onset subjects’ PCr and phase II pVO and offset of exercise are shown in Table 2. The twoway repeated measures ANOVA revealed a significant main effect for exercise transition (P = 0.001), with the _ 2 offset time constant (28 average PCr and phase II pVO ± 5 s) being slower than those at the onset (23 ± 4 s). However, the follow-up pairwise comparisons revealed no significant difference between the PCr onset and offset kinetics (23 ± 5 vs. 28 ± 5 s, P = 0.064), _ 2 onset kinetics were signifiwhereas the phase II pVO cantly faster than those at the offset (23 ± 4 vs. 29

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pHi

7.10

7.05

7.00

Exercise

Rest

Recovery

6.95 -120

0

120

240

360

480

600

720

Time (s)

Fig. 1 Average work-rate (a) and pH (b) responses during quadriceps constant work-rate exercise. Dotted lines signify the onset and offset of exercise. The solid horizontal line in a represents the group average power output at the ITPi/PCr

±5 s, P = 0.015). Comparison of the kinetic responses _ 2 time constants between the PCr and phase II pVO revealed similar values at the onset (PCr 23 ± 5 vs. _ 2 23 4 s; P ¼ 1:000Þ and offset (PCr 28 ± 6 vs. pVO _ pVO2 29 7 s; P ¼ 1:000) of exercise. The 95% CIs for the time constants at the onset and offset of exercise were: PCr 5 ± 1 s (range 3–7 s), 5 ± 2 s (range 2–7 s); _ 2 5 1 s (range 3–7 s), 5 ± 2 s (range 2–7 s), pVO respectively. Within a given child’s response, the average difference _ 2 time constants was 4 ± between the PCr and phase II pVO 4 s for the onset and offset kinetics profiles. Out of the 24 kinetic response profiles, the 95% CIs spanning the esti_ 2 time constants failed to mated PCr and phase II pVO overlap in only two subjects (see footnote a in Table 2). A line of identity plot showing the agreement between the _ 2 time constants at the onset and PCr and phase II pVO offset of exercise is presented in Fig. 3. Linear regression analysis revealed no significant relationship between the _ 2 time constants at the onset PCr and phase II pVO (r = 0.225, P = 0.482) or offset (r = 0.298, P = 0.347) of exercise. However, after pooling of the exercise onset and

Eur J Appl Physiol (2008) 102:727–738 VO 2 onset kinetics

PCr onset kinetics

0.2

Residuals

R e s id u a ls

10 5 0 -5

0.0 -0.1

0.8 0.6

VO 2 (l/min)

0

-10

-20

-30 -120 -60

0.1

-0.2

-10 10

% PCr change

_ 2 kinetics Fig. 2 PCr and pVO at the onset and offset of exercise in a child subject. An _ 2 taken example PCr and pVO from subject number 3. The kinetic parameters are shown in Table 2. The data presented are the result of time-aligning and averaging six repeat exercise transitions for determination of the PCr kinetics, and ten for the _ 2 kinetics. The continuous pVO line represents the fitted singleexponential function, with the resulting residuals displayed above. Vertical dotted lines signify either the onset or offset of exercise

733

0.4 0.2 0.0

0

60

120 180 240

-0.2 -120 -60

300 360

0

60

300 360

VO 2 offset kinetics

0.2

5

0.1

Residuals

10

0 -5

0.0 -0.1

-10 30

-0.2

20

-0.0

0.2

VO 2 (l/m in)

Residuals

PCr offset kinetics

% PCr change

120 180 240

Time (s)

Time (s)

10

-0.2 -0.4

0 -0.6 -10 -120 -60

0

60

120 180 240 300 360

Time (s)

offset responses to form a single data set, a significant _ 2 time relationship between the PCr and phase II pVO constants was observed (r = 0.459, P = 0.024). Moreover, when the two responses that failed to demonstrate an _ 2 time overlap in the 95% CIs for the PCr and phase II pVO constants were removed from the analysis (see asterisk in Fig. 3), a stronger correlation was observed (r = 0.711, P = 0.000).

Discussion This is the first study to investigate the kinetic association between muscle PCr, a putative metabolic feedback con_ 2 ; and the phase II pVO _ 2 response at the troller of mVO onset and offset of moderate intensity exercise in children. The main finding of this study is that a close kinetic

-0.8 -120 -60

0

60

120 180 240 300 360

Time (s)

coupling was evident between the quadriceps PCr and _ 2 responses both at the onset and cycling phase II pVO offset of exercise. This finding is supported by three lines of reasoning. Firstly, at both the onset and offset of exer_ 2 time cise, the group average PCr and phase II pVO constants maintained a striking equivalence despite the recovery kinetics demonstrating a longer time constant compared to the onset responses. Secondly, we observed an _ 2 time overlapping between the PCr and phase II pVO constants 95% CIs in *92% of the kinetic responses, suggesting that within the sensitivity of the experimental measures employed, the physiological responses are mechanistically linked. Thirdly, a significant correlation _ 2 time was observed between the PCr and phase II pVO constants. Collectively, these results are consistent with the control _ 2 being functionally linked to the kinetics of PCr in of mVO

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_ 2 kinetic responses at the onset and Table 2 Subjects’ PCr and VO offset of exercise Subject

Onset kinetics sPCr

CIs

Offset kinetics _ 2 spVO

sPCr

CIs

CIs

_ 2 spVO

CIs

2

25

6

21

5

24

7

26

3

3

32

6

27

5

33

6

37

4

4 5

15a 22

5 5

28a 19

5 4

31 22

7 4

33 22

7 5

9

22

5

24

5

35a

7

20a

2

10

28

5

25

7

26

4

27

6

13

27

6

25

5

25

5

31

5

14

19

5

24

5

31

6

31

6

15

25

5

19

4

31

6

33

7

16

19

4

17

4

25

5

33

5

17

22

3

27

3

28

2

28

3

18

14

3

20

5

21

5

25

4

Mean

23

5

23

5

28

5

29

5

5

1

4

1

5

2

5

2

SD

The time constant (s) and 95% confidence intervals (CIs, ±s) values are reported in seconds. Only subjects with 95 % confidence intervals equal to or less than ±7s are shown _ 2 ; pulmonary oxygen uptake PCr, phosphocreatine; pVO _ 2 profile where the 95% CIs around the time Subject’s PCr and pVO constant failed to overlap a

20%

10%

0%

50

r=0.459,

P=0.024

10% 20%

PCr tim e constant (s)

40

* 30

20

* 10

0 0

10

20

30

40

50

VO2 time constant (s)

_ 2 line of identity plot. PCr and pVO _ 2 time Fig. 3 PCr and pVO constants at the onset (open circle) and offset (closed circle) of moderate intensity exercise for all 12 subjects. When the two _ 2 time constant’s confidence responses where the PCr and pVO intervals failed to overlap were removed from the analysis (asterisk), the Pearson correlation increased to r = 0.711, P = 0.000

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children, as previously demonstrated in adults (Barstow et al. 1994; Rossiter et al. 1999), and implies that an agerelated modulation of the dynamics of the creatine kinase reaction and/or build-up of metabolic feedback controllers, _ 2 found in children may explain the faster phase II pVO compared to adults (Fawkner and Armstrong 2004; Fawkner et al. 2002b; Williams et al. 2001). At the onset of moderate intensity exercise the rise of _ 2 occurs immediately, i.e. without delay, and is well mVO characterised by a single exponential function (Behnke et al. 2002). The modelling simulations by Barstow et al. (1990) predict that for a variety of muscle blood flow and _ 2 response venous volume parameters, the underlying mVO _ 2 is expressed to within ±10% through the phase II pVO time constant at the onset of moderate exercise. This association has been experimentally confirmed using the direct _ 2 across the contracting Fick technique to determine mVO thigh muscle during upright cycle ergometry in humans (Grassi et al. 1996). In contrast, a recent computerised simulation found a significant dissociation between the _ 2 and pVO _ 2 at the onset of mean response time for mVO moderate (13 vs. 65 s), heavy (13 vs. 100 s) and very heavy (15 vs. 82 s) cycling exercise in adolescent boys (Lai et al. 2006). However, these results must be interpreted with caution as no attempt was made to characterise the phase II _ 2 response profile, which clearly kinetics from the pVO _ 2 slow component resulted in the development of the pVO during heavy and very heavy exercise. In addition, no slow _ 2 response component was evident for the simulated mVO during heavy or very heavy exercise, which is in contradiction with human studies (Koga et al. 2005). The first-order model of metabolic control predicts an inverse proportional relationship between the dynamics of _ 2 at the onset and offset of submaximuscle PCr and mVO mal exercise (Mahler 1985; Meyer 1988). One would therefore expect a similar kinetic coupling (±10%) between _ 2 to be evident if the kinetics of PCr PCr and phase II pVO _ 2 : In support of this was the principal controller of mVO model, Rossiter et al. (1999, 2002) established that the fall _ 2 were in agreement in muscle PCr and rise in phase II pVO to within ±10% at the onset and offset of moderate intensity knee-extensor exercise in adult men. However, the associ_ 2 kinetic ation between the muscle PCr and phase II pVO responses at the onset and offset of exercise in the present study demonstrates a lower level of agreement in children (±18%, see Fig. 3). This, however, appears to be a consequence of the inherently ‘fast’ PCr kinetics in children (*25 s) compared to adults (*35 s, Rossiter et al. 1999), as the PCr time constant for a given subject was on average _ 2 response both at to within ±4 s to that of the phase II pVO the onset and offset of exercise. Such an association is in direct agreement with the results obtained by Rossiter et al. (1999, 2002). In contrast, McCreary et al. (1996) found an


average dissociation of approximately ±16 s (±40%) _ 2 and PCr time constants during between the phase II pVO the onset and offset of submaximal plantar-flexor exercise. However, a limitation of this study was the poor level of _ 2 time constant was confidence in which the phase II pVO obtained (95% CIs ±10 s), which confines interpretation of these data with respect to the role muscle PCr may have in _ 2: controlling mVO Collectively, the results from the present study and those previously published in adults (Barstow et al. 1994; Rossiter et al. 1999, 2002) highlight the prominent role _ 2 muscle PCr plays in modulating the dynamics of mVO during a ‘‘step’’ change in metabolic rate. These results are consistent with the first-order model of metabolic control proposed by Meyer (1988), an appreciation of which, may provide greater insight into the mechanisms underlying the _ 2 kinetics found in children compared to adults faster pVO (Fawkner and Armstrong 2004; Fawkner et al. 2002b; Williams et al. 2001). The model predicts that the kinetics of muscle PCr will be faster in muscle with a higher mitochondrial density and/or content of mitochondrial enzymes (‘‘resistor’’), and slower in muscle with a higher PCr content (‘‘capacitance’’, Meyer 1988). There is some evidence supporting a decrease in the activity of oxidative enzymes in 13–15 year-old children through to adulthood (Haralambie 1982), although the data available are equivocal (Berg et al. 1986; Kaczor et al. 2005). Moreover, the relative density of mitochondria in 6year old boys’ and girls’ vastus lateralis muscle is not appreciably different from that recorded in sedentary adults (Bell et al. 1980). There are limited data showing a progressive increase in the rectus femoris muscle PCr stores (equivalent to the model ‘‘capacitance’’) in a small group of boys between the ages of 11 and 16 years (Eriksson and Saltin 1974), which may account for the faster phase II _ 2 kinetics found in children compared to adults. pVO However, adding further complexity are studies showing children to have faster (Taylor et al. 1997) or similar (Kuno et al. 1995) recovery of muscle PCr compared to adults following a progressive maximal exercise test. Clearly, further research is needed to examine the muscle PCr kinetic responses between children and adults in various exercise intensity domains before any firm conclusions can be drawn with regard to the mechanisms underlying the _ 2 kinetics. child–adult differences in pVO An interesting finding in the present study was the lack of symmetry between the kinetic responses at the onset and offset of exercise, which was consistent across both exercise modalities and response variables. While the muscle _ 2 dynamics retained single exponential PCr and pVO properties, the recovery time constants for both variables were *25% longer than the onset kinetics. Interestingly, Rossiter et al. (2002) also demonstrated a close identity

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_ 2 kinetics but an between the muscle PCr and phase II pVO even greater slowing of the time constants during the offset (*50 s) compared to the onset (*35 s) of moderate intensity knee-extensor exercise. The presence of asymmetrical time constants implies that the mechanism(s) _ 2 differ at the onset and controlling the dynamics of mVO offset of exercise. Candidate mechanisms may include a greater sensitivity of metabolic control to O2 delivery during recovery compared to the onset of exercise (Haseler et al. 1999, 2004), a pH dependent effect on the creatine kinase equilibrium favouring PCr breakdown thereby slowing the recovery of PCr (McMahon and Jenkins 2002), and/or a direct effect of acidosis limiting the rate of mitochondrial oxidative phosphorylation and thereby the resynthesis of muscle PCr (Jubrias et al. 2003). In contrast, using a theoretical model of energy balance, Kushermick (1998) attributed the asymmetric behaviour of the muscle PCr kinetics to changes in the flux of creatine kinase at the onset (forward flux) and offset (backward flux) of exercise. However, the magnitude of difference between the onset and offset time constants in the present study (*5 s) lies within the boundaries of confidence surrounding the time constants (95% CIs *±5 s). This reduces the certainty _ 2 kinetic with which the muscle PCr and phase II pVO responses can be described as asymmetrical in children and warrants further investigation.

Methodological considerations The results of the current study provide support for the hypothesis that muscle PCr, or some related function, _ 2 at the onset controls the respective rise and fall in mVO and offset of exercise in children. However, a number of methodological considerations require discussion. While it _ 2 response prohas been established that the phase II pVO _ 2 during vides a close reflection of the rise in mVO moderate intensity work-rates in adults (Grassi et al. 1996), this association has yet to be verified in children, largely due to ethical and methodological constraints. In particular, factors such as the cardiac output response dynamics, muscle-lung transit time and the utilisation of body O2 stores during the non-steady state, all have the potential to _ 2 response from the kinetics of dissociate the phase II pVO _ 2 during the non steady-state (Barstow et al. 1990; mVO Francescato et al. 2003). However, given the close kinetic _ 2 and muscle PCr in *92% coupling between phase II pVO of the responses in the present study, the latter of which is _ 2 (e.g. Barstow et al. routinely used as surrogate of mVO 1994; McCreary et al. 1996), the assumption that phase II _ 2 provides a reflection of the dynamics of mVO _ 2 pVO appears appropriate in children and is within acceptable limits.

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_ 2 in the present study were The kinetics of phase II pVO determined on a breath by breath basis using a conventional algorithm which has been criticized for its failure to account for changes in alveolar O2 stores (Cautero et al. 2002). However, despite intense investigation, the most _ 2 appropriate algorithm to determine ‘true’ alveolar pVO during exercise remains controversial, largely due to the various assumptions imposed by each technique. Indeed, a recent study highlighted the considerable variability (*10 s or *25%) across the proposed algorithms for determination of the phase II time constant within a group of adults (Cautero et al. 2002). Although a technique that _ 2 at the alveolar level has measures breath-by-breath pVO been developed (Aliverti et al. 2004), its comparison against the traditionally used algorithms in humans, and in particular children, has yet to be investigated. Therefore, _ 2 time constant in the the degree to which the phase II pVO _ 2 present study represents that of ‘‘true’’ alveolar pVO dynamics is unknown. The use of different exercise modalities to investigate _ 2 and muscle PCr is a the kinetic association between pVO limitation of the present study, as inter-modality differences in body posture, muscle recruitment patterns and muscle contraction type, and muscle mass may result in _ 2 (and presumably muscle fundamentally different pVO PCr) kinetics (Jones and Burnley 2005). Although the _ 2 alongside PCr during simultaneous measurement of pVO the quadriceps exercise would allay such concerns (Whipp et al. 1999), this technique has limited application in _ 2 amplitude during singlechildren. In particular, the pVO legged quadriceps exercise is insufficient to achieve an _ 2 adequate signal to noise ratio to determine the pVO kinetic response parameters to within an acceptable level of precision in children. As a consequence, the measurement _ 2 during upright cycling was necessary in order to of pVO _ 2 time constant to within an determine the phase II pVO acceptable level of confidence in order to infer control mechanisms in relation to the PCr dynamics, i.e. 95% CIs ±5 s on average. At the onset of moderate intensity upright cycling, the _ 2 kinetics have been demonstrated to be phase II pVO similar to that determined during moderate knee-extensor exercise both in the upright (Koga et al. 2005) and prone body positions (Rossiter et al. 2000). As highlighted in the introduction, this latter finding is in conflict with the slower _ 2 kinetics found during moderate intensity cycling in pVO the supine compared to the upright body position (Hughson et al. 1993). Moreover, Barstow et al. (1994) found no difference between the kinetics of muscle PCr determined during plantar-flexor exercise when compared to the _ 2 measured during upright cycling. Colleckinetics of VO tively, these data therefore support the notion that body _ 2 position is not modulating the kinetics of PCr or VO

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determined during prone quadriceps exercise in the current study. As upright cycling predominantly involves concentric muscle contractions, an important consideration is the degree to which the concentric and eccentric components of the quadriceps exercise may have influenced the kinetics of muscle PCr in the present study. While the steady-state PCr cost of muscle contraction is twofold higher during 5 min of concentric exercise at 30% maximum voluntary contraction when compared to eccentric exercise (Ryschon et al. 1997), the recovery of muscle PCr has been shown to be independent of muscle contraction type (Combs et al. 1999). Whether this is also the case for PCr kinetics during concentric and eccentric exercise at the onset of exercise is currently unknown. However, Perrey et al. (2001) observed _ 2 time conno significant differences in the phase II pVO stant between high intensity eccentric and moderate intensity concentric cycling exercise. This indirectly suggests that the kinetics of muscle PCr may be similar between concentric and eccentric exercise, although this requires clarification from future studies. In addition, the extrapolation of results obtained from studies using ‘‘isolated’’ concentric and eccentric muscle contraction regimes to the ‘‘mixed’’ concentric and eccentric exercise in the current study is difficult. Given the reasoning above, it appears that the PCr and _ 2 kinetics during prone quadriceps exercise phase II pVO and upright cycling are similar regardless of inter-modality differences in body posture, muscle recruitment and contraction regimes, and muscle mass. For these reasons, the current experimental design appears appropriate to investigate the kinetic association between quadriceps muscle _ 2 dynamics during moderate PCr and upright cycling pVO exercise and importantly, with high statistical confidence in the kinetic parameters.

Practical implications The recovery kinetics of muscle PCr (determined using P-MRS) is routinely employed as a non-invasive and valid measure of the muscle oxidative capacity (McCully et al. 1993; Paganini et al. 1997). However, this technique is very expensive, time consuming and often inaccessible to paediatric physiology research groups. The close kinetic _ 2 and PCr kinetic profiles at the coupling between the pVO onset and offset of exercise in the present study show that in majority of cases (*92%), similar information can be _ 2 recovery provided by characterising the phase II pVO time constant following moderate upright cycling exercise in young people. These results therefore support the use of _ 2 time constant as a proxy measure of the the phase II pVO muscle PCr kinetics, which can for example, be used to 31


investigate the influence of maturity and physical training on the muscles’ O2 capacity in young people.

Conclusion This is the first study to quantify the kinetics of quadri_ 2 during moderate ceps muscle PCr and cycling pVO intensity exercise in children. The kinetic changes in muscle PCr were closely coupled (to within ±4 s) with _ 2 response both at the onset and offset of the phase II pVO _ 2 being exercise and are therefore consistent with mVO controlled either directly or indirectly by the dynamics of muscle PCr during the non-steady-state in children. These data support the theory that an age-related modulation of _ 2 may the putative phosphate linked controller(s) of mVO _ explain the faster VO2 kinetics found in children compared to adults. Acknowledgments We would like to express our gratitude to the children and staff from Wynstream Primary School for participation in this project. The technical expertise provided by David Childs in designing the quadriceps ergometer and analysis software was most appreciated. Grants: this project was funded by the Darlington Trust.

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