31P-Nuclear magnetic resonance spectroscopy study of the time course of energy metabolism during exercise and recovery

Summary This study evaluated the time courses of intracellular pH and the metabolism of phosphocreatine (PCr) and inorganic phosphate (P) at the onset of four exercise intensities and recoveries. Non-invasive evaluation of continuous changes in phosphorus metabolites has become possible using³¹P-nuclear magnetic resonance spectroscopy (³¹P-MRS). After measurements at rest, six healthy male subjects performed 4 min of femoral flexion exercise at intensities of 0 (loadless), 10, 20 and 30 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore. Measurements were continuously made during 5 min of recovery. During a series of rest-exercise-recovery procedures,³¹P-MRS were accumulated using 32 scans · spectrum^–1 requiring 12.8 s each. At the onset of exercise, PCr decreased exponentially with a time constant of 27–32 s regardless of the exercise intensity. The time constant PCr resynthesis during recovery was about 27–40 s. The PCr kinetics were independent of exercise intensity. There were similar Pi kinetics at the onset of all types of exercise, while those of Pi recovery became significantly longer at the higher exercise intensities (P < 0.05). Furthermore, the intracellular pH indicated temporary alkalosis just at the onset of exercise, probably due to absorption of hydrogen ions by PCr hydrolysis, and then decrease at a point about 40%–50% of the preexercise PCr. The pH recovery time was longer than that for the P_i or PCr kinetics. By using a more efficient resolution system it was possible to obtain the phosphorus kinetics during exercise and to follow PCr resynthesis within the first few minutes of recovery. From our results it was concluded that in general the time course of PCr and Pi metabolism were unaffected by the exercise intensity, both at the onset of exercise and during recovery, with the exception of P_i recovery.     

Fatigue and recovery of phosphorus metabolites and pH during stimulation of rat skeletal muscle: an evoked electromyography and in vivo31P-nuclear magnetic resonance spectroscopy study

³¹P-nuclear magnetic resonance spectroscopy and evoked electromyography were applied to rat skeletal muscle to examine the mechanism of muscle fatigue and the recovery of muscle phosphorus metabolites and pH during fatigue. When the sciatic nerve was electrically stimulated at 1 Hz, the contraction force of the gastrocnemius muscle decreased gradually to 46% of the maximal force, accompanied by a decrease in phosphocreatine (PCr) and a corresponding increase in inorganic phosphate (P_i) and diprotonated inorganic phosphate (H₂PO₄ ^–). Neither the amplitudes of compound muscle action potentials (CMAP) nor muscle pH changed significantly. At 10-Hz stimulation, contraction force rapidly decreased to 26% of maximal force, accompanied by a decrease in PCr and increases in P_i and H₂PO₄ ^–. Muscle pH decreased for a few minutes, then gradually recovered during continued stimulation. The amplitude of the CMAP also decreased for a few minutes and then reached steady values. At 100-Hz stimulation, the contraction force decreased to 6% of the maximal force and there was a decrease in the amplitude of the CMAP. However, the changes in the phosphorus metabolites and pH were transient and recovered to the control value during the stimulation. These results indicated that fatigue at 1 and 100-Hz stimulation was mainly caused by the change in phosphorus metabolite concentrations and electrical failure, respectively, and that fatigue at 10-Hz stimulation might have been due to both of the these factors. These results also indicated that electrical failure might have been the cause of the recovery of the phosphorus metabolites and pH during 100-Hz stimulation and of pH during 10-Hz stimulation.     

Changes in intracellular pH during repeated exercise

Summary The rates of change in intracellular pH during repeated exercise sessions with rest periods was determined by 31 phosphorus-nuclear magnetic resonance spectroscopy (³¹P-MRS). Five long-distance runners and six healthy male subjects as controls performed a 2-min femoral flexion at 20 kg · m · min^–1 in a 2.1 T superconducting magnet with a 67-cm bore and repeated this exercise four times with 2-min rest periods intervening. In all cases during exercise the inorganic phosphate (P_i) peak split into two, the earlier increased rapidly (high-pH P_i) and the later (low-pH P_i) increased more slowly. The P_i peaks were separated by a fitting procedure using the least square mean method. The high-pH P_i area during exercise decreased as the number of repeated exercise periods increased, while the low-pH P_i area gradually increased. Although the total P_i area decreased exponentially during the recovery period, the high-pH P_i area decreased first and then the low-pH P_i area reduced gradually. The pH values were estimated from the chemical shift between the phosphocreatine peak and each split peak in the P_i. The high-pH in pooled data ranged from 6.6 to 7.0 during exercise and recovery, while the low pH decreased to 6.2 during exercise. As the number of exercise periods increased, each pH value gradually became less acidic, although there was a tendency to more acidity in the control subjects than in the long-distance runners. In conclusion, it was possible to obtain by non-invasive, continuous³¹P-MRS, a split pattern of P_i peaks during exercise and there were at least tow different intracellular pH values during exercise, suggesting that each P_i peak might be attributed to the types of muscle fibre recruited.     

Assessment of muscular metabolism in peripheral arterial occlusive disease using31P nuclear magnetic resonance spectroscopy

Summary The behaviour of muscular metabolism was investigated in 10 patients with peripheral arterial occlusive disease stage II at rest and after maximum erometric calf exercise. The intracellular concentrations of phosphocreatine, inorganic phosphate and adenosine triphosphate as well as muscle pH were measured by means of³¹P magnetic resonance spectroscopy and compared with those from a control group. In addition, arteriovenous differences in concentrations of lactate, pyruvate, ammonia, hypoxanthine and alanine in the femoral blood were determined. The fall in intracellular phosphocreatine concentration during exercise was significantly greater in the calf muscles of patients with arterial occlusion than in controls and correlated linearly with the increase in femoral arteriovenous differences in lactate, ammonia and alanine. A significant fall in intracellular pH occurred during muscular activity only in the patient group, but not in the identically exercised control group. The fall in pH correlated closely with the rise in arteriovenous lactate difference in the femoral blood. The intramuscular ATP concentration remained constant throughout the exercise procedure. The behaviour of both the directly and indirectly measured metabolites permits the deduction of activation of the creatine kinase reaction, glycolysis, myokinase reaction and the purine nucleotide cycle during exercise-induced hypoxia in the presence of arterial occlusive disease. The anaerobic production of energy is sufficient to maintain the ATP concentration even during claudication pain. Magnetic resonance spectroscopy proved to be a useful tool for non-invasive assessment of the metabolic changes in peripheral arterial occlusive disease.Abbreviations ADP adenosine diphosphate - AMP adenosine monophosphate - ATP adenosine triphosphate - AVD arteriovenous difference - (P)AOD (peripheral) arterial occlusive disease - IMP inosine monophosphate - NAD⁺ nicotinamide adenine dinucleotide - PCr phosphocreatine - P₁ inorganic phosphate - ³¹P-MRS ³¹phosphorus magnetic resonance spectroscopy - NMR nuclear magnetic resonance - PNC purine nucleotide cycle     

High-energy phosphate metabolism during two bouts of progressive calf exercise in humans measured by phosphorus-31 magnetic resonance spectroscopy

According to the literature the steady-state level of phosphocreatine (PCr) has a linear relationship to the workload during muscle exercise intensities below the lactate threshold, whereas this linearity is impaired during exercise intensities above the lactate threshold. The purpose of this study was to investigate the linearity between PCr kinetics and workload during two bouts of isotonic incremental calf exercise with transitions from moderate- to high-intensity as well as from high- to moderate-intensity work rates. Using a whole-body 1.5 T MR scanner and a self-built exercise bench, we performed serial phosphorus-31 magnetic resonance spectroscopy (³¹P-MRS) with a time resolution of 30 s in nine healthy male volunteers. Changes in PCr, inorganic phosphate (Pi) and pH were statistically evaluated in comparison to the baseline. The exercise protocol started with a 4.5 W interval of 6 min followed by two bouts of 1.5 W increments. The workload was increased in 2-min intervals up to 9 W during the first bout and up to 7.5 W during the second bout. The second bout was preceded by a 4.5 W interval of 2 min and followed by a 4.5 W interval of 4 min. PCr hydrolysis achieved a steady state during each increment and was highly linear to the work rate (r ², –0.796; P <0.001). Pi accumulated during each bout, whereas the pH decreased continuously during the first bout and did not exhibit any substantial decrease during the second bout. The metabolite levels and pH were expressed as the median value and the range. Our study confirms that steady-state PCr levels also have a linear relationship to work intensities above the lactate threshold, while pH changes do not have any impact on PCr degradation. The lack of substantial changes in pH during the second exercise bout indicates that prior high-intensity exercise leads to an activation of oxidative phosphorylation.     

Blood flow and muscle bio-energetics by31P-nuclear magnetic resonance after local cold acclimation

Summary To clarify the origin of local cold adaptation and to define precisely its influence on muscle bio-energetics during local exercise, five subjects were subjected to repeated 5°C cold water immersion of the right hand and forearm. The first aim of our investigation was therefore carried out by measuring local skin temperatures and peripheral blood flow during a cold hand test (5°C, 5 min) followed by a 10-min recovery period. The³¹P by nuclear magnetic resonance (³¹PNMR) muscle bio-energetic changes, indicating possible heat production changes, were measured during the recovery period. The second aim of our investigation was carried out by measuring³¹PNMR muscle bioenergetics during handgrip exercise (10% of the maximal voluntary contraction for 5 min followed by a 10-min recovery period) performed both at a comfortable ambient temperature (22°C; E) and after a cold hand test (EC), before and after local cold adaptation. Local cold adaptation, confirmed by warmer skin temperatures of the extremities (+30%,P<0.05), was related more to an increased peripheral blood flow, as shown by the smaller decrease in systolic peak [–245 (SEM 30) Hz vs –382 (SEM 95) Hz,P<0.05] than to a change in local heat production, because muscle bioenergetics did not vary. Acute local cold immersion decreased the inorganic phosphate: phosphocreatine (PC) ratio during EC compared to E [+0.006 (SEM 0.010) vs +0.078 (SEM 0.002) before acclimation and +0.029 (SEM 0.002) vs +0.090 (SEM 0.002) after acclimation respectively, P<0.05] without significant change in the PC:-adenosine triphosphate ratio and pH. Local adaptation did not modify these results statistically. The recovery of PC during E increased after acclimation [9.0 (SEM 0.2) min vs 3.0 (SEM 0.4) min,P<0.05]. These results suggested that local cold adaptation is related more to peripheral blood flow changes than to increased metabolic heat production in the muscle.     

Electromyogram spectrum changes during sustained contraction related to proton and diprotonated inorganic phosphate accumulation: a 31P nuclear magnetic resonance study on human calf muscles

Summary The calf muscles of five clinically healthy men were submitted to isometric exercise and examined by ³¹P nuclear magnetic resonance (NMR) spectroscopy and electromyography (EMG) to evaluate the influence of proton (H⁺) and diprotonated forms of inorganic phosphate (H₂PO ₄ ^– ) accumulation on EMG spectrum changes. The experiments were performed in a supraconducting magnet (2.35 Tesla, 35-cm effective diameter) using a surface coil (7-cm diameter) positioned against the calf muscles. The EMG surface electrodes were applied on the gastrocnemius medialis muscle and acquisition of both NMR and EMG signals was synchronized. The exercise consisted of a sustained isometric contraction at 70% of the maximal voluntary contraction until exhaustion. A continuous decrease in phosphocreatine content and a large concomitant increase in H₂PO ₄ ^– was observed in the calf muscles of each subject. A significant increase in H⁺ concentration was also found when considering the whole population but intracellular acidosis was low for two subjects. Moreover, a quasilinear decrease in mean power frequency (MPF) was found during the test. Changes in MPF were correlated with variations in H⁺ and H₂PO ₄ ^– concentration but a more significant relationship was found when MPF changes were correlated with H₂PO ₄ ^– concentration. An interpretation of EMG spectrum changes in terms of an accumulation of by-products of anaerobic metabolism and an increase in the relative number of activated slow fibres is proposed.     

Phosphorus-31 nuclear magnetic resonance study on the effects of endurance training in rat skeletal muscle

Summary To evaluate changes in muscle energetics following endurance training, we measured phosphorus-31 nuclear magnetic resonance (³¹P NMR) spectra on rat muscle in vivo before and after training in the same animals. The endurance training lasted for 3 months. The³¹P NMR spectra were obtained serially at rest, during exercise by electrical stimulation, and during recovery. Intramuscular phosphocreatine (PCr), inorganic phosphate (P_i, adenosine 5-triphosphate (ATP) and pH were determined from the NMR spectra. The ratio of PCr : (PCr + P_i) at rest showed no difference between the trained and control groups even after 3 months of training. During exercise, however, this ratio was significantly higher in the trained group than in the control group. The ratio also recovered more rapidly after exercise in the trained group. The intramuscular pH decreased slightly by approximately 0.1 pH unit during exercise but did not show a significant difference between the groups. These results indicated that endurance training of 3 months duration improved the ATP supply system in the muscle. They also demonstrated that³¹P NMR is a potent method for evaluating the effects of training in the same individuals.     

Parameter estimation in modeling phosphocreatine recovery in human skeletal muscle

Commonly, muscle phosphocreatine (PCr) recovery from exercise has been fitted to a monoexponential function. However, a number of experiments indicate that at low muscle pH, a monoexponential fit is not suitable. We have performed in vivo ³¹P-MRS measurements of PCr during recovery from high-intensity intermittent exercise where muscle pH dropped below 6.5 (35 out of 40 cases). Results of a statistical analysis showed that monoexponentiality should be rejected in 32 out of 40 cases. Therefore, a Monte Carlo simulation was carried out to test the quality of competing models used in the literature at low pH: monoexponential, biexponential and changing rate utilization resource (CRUR). For each model, random Gaussian-distributed errors were imposed on simulated PCr recovery before performing the fits. A monoexponential function (three estimated parameters) provided a correct estimation of parameters (unbiased, normally distributed, poorly correlated estimates) and, therefore, should be preferred. When alternative functions are required, as in 32 cases out of 40 in the present study, it is demonstrated that a biexponential function (five estimated parameters) is not well suited (estimates were correlated), whereas a CRUR function (four estimated parameters) provides correct estimation of the parameters. It is concluded that a biexponential fit to PCr recovery is too sensitive to experimental errors to be practicable. Statistical and physiological relevance of CRUR are discussed.     

Muscle metabolism during exercise using phosphorus-31 nuclear magnetic resonance spectroscopy in adolescents

Very little has been reported on muscle energetics during exercise in adolescents. This is attributable to the difficulty of subjecting children to muscle biopsy. The purpose of this study was to investigate the characteristics of muscle metabolism during exercisein vivo in adolescents by comparing firstly, with adults and secondly, the differences resulting from physical activity using phosphorus-31 nuclear magnetic resonance (³¹PNMR) spectroscopy. The subjects were boys aged 12 to 15 years, comprising 21 trained boys and 23 control boys, and 6 adults controls. The ratio of phosphocreatine (PCr):(PCr + P_i), where P_i is inorganic phosphate intracellular pH at exhaustion and the time constant of PCr during recovery were measured in all the subjects using³¹PNMR. Both groups of children showed higher values of PCr:(PCr + P_i) and intracellular pH at exhaustion than did the adult control group (P < 0.01 orP < 0.05). However, no significant differences were found between the trained boys and the control boys with respect to PCr:(PCr + P_i) and intracellular pH at exhaustion. On the other hand, we found the same values for PCr time constant in all groups. This result suggested no differences of the muscle oxidative capacity between children and adults. We concluded that the adolescents, aged 12 to 15 years in both the trained and control groups, had less glycolytic ability during exercise than the adults.     

Mitochondrial coupling in humans: assessment of the P/O<Subscript>2</Subscript> ratio at the onset of calf exercise

Coupling of oxidation to ATP synthesis (P/O₂ ratio) is a critical step in the conversion of carbon substrates to fuel (ATP) for cellular activity. The ability to quantitatively assess mitochondrial coupling in vivo can be a valuable tool for basic research and clinical purposes. At the onset of a square wave moderate exercise, the ratio between absolute amount of phosphocreatine split and O₂ deficit (corrected for the amount of O₂ released from the body O₂ stores and in the absence of lactate production), is the mirror image of the P/O₂ ratio. To calculate this value, cardiac output whole body O₂ uptake O₂ deficit and high-energy phosphates concentration (by ³¹P-NMR spectroscopy) in the calf muscles were measured on nine healthy volunteers at rest and during moderate intensity plantar flexion exercise (3.44 ± 0.73 W per unit active muscle mass). and increased (from 4.68 ± 1.56 to 5.83 ± 1.59 l min⁻¹ and from 0.28 ± 0.05 to 0.48 ± 0.09 l min⁻¹, respectively), while phosphocreatine (PCr) concentration decreased significantly (22 ± 6%) from rest to steady-state exercise. For each volunteer, “gross” was corrected for the individual changes in the venous blood O₂ stores yielding the “net” . Resting PCr concentration was estimated from the appropriate spectroscopy data. The so calculated P/O₂ ratio amounted on average to 4.24 ± 0.13 and was, in all nine subjects, very close to the literature values obtained directly on intact skeletal muscle. This unfolds the prospect of a non-invasive tool to quantitatively study mitochondrial coupling in vivo.     

Exercise-induced splitting of the inorganic phosphate peak: investigation by time-resolved 31P-nuclear magnetic resonance spectroscopy

To investigate the splitting of the inorganic phosphate (Pi) peak during exercise and recovery, a time-resolved ³¹phosphorus nuclear magnetic resonance spectroscopy (³¹P-MRS) technique was used. Seven healthy young sedentary male subjects performed knee flexion exercise in the prone position inside a 2.1-T magnet, with the surface coil for ³¹P-MRS being placed on the biceps femoris muscle. After a 1-min warm-up without loading, the exercise intensity was increased by 0.41 W at 15-s intervals until exhaustion, followed by a 5-min recovery period. The ³¹P-MRS were recorded every 5 s during the rest-exercise-recovery sequence. Computer-aided contour analysis and pixel imaging of the Pi and phosphocreatine peaks were performed. Five of the seven subjects showed two distinct Pi peaks during exercise, suggesting two different pH distributions in exercising muscle (high pH and low pH region). In these five subjects, the high-pH increased rapidly just after the onset of exercise, while the low-pH peak increased gradually approximately 60 s after the onset of exercise. During recovery, the disappearance of the high-pH peak was more rapid than that of the low-pH peak. These findings suggest that our method ³¹P-MRS provides a simple approach for studying the kinetics of the Pi peak and intramuscular pH during exercise and recovery.     

Intracellular pH during secretion in the perfused rabbit mandibular salivary gland measured by31P NMR spectroscopy

Intracellular pH (pH_i) was measured in the isolated, perfused rabbit mandibular salivary gland by³¹P NMR spectroscopy. In the unstimulated gland perfused with HCO ₃ ^– /CO₂-buffered Ringer's solution, pH_i was 7.27±0.01. Continuous stimulation with acetylcholine elicited dose- and time-dependent changes in pH_i. 10^–6 mol/l acetylcholine caused a brief intracellular acidosis (–0.19±0.06 pH units) followed by an increase in pH_i to a more alkaline steady-state value (7.33±0.02). In the absence of perfusate HCO ₃ ^– or in the presence of 10^–4 mol/l DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonic acid), the transient acidosis was abolished and pH_i increased rapidly to give a sustained alkalosis (7.49±0.03 and 7.44±0.03 respectively). In the presence of 10^–3 mol/l amiloride, the response to acetylcholine was a rapid decrease in pH_i to 7.02±0.02. The data suggest that, during perfusion with HCO ₃ ^– /CO₂-buffered solutions, stimulation with acetylcholine results in a transient loss of HCO ₃ ^– from the acinar cells (causing a transient acidosis), and, independently, the activation of Na⁺–H⁺ exchange (causing a sustained alkalosis). In the unstimulated gland, DIDS and the HCO ₃ ^– -free perfusate caused decreases in pH_i to 7.12±0.02 and 7.04±0.01 respectively. In contrast, amiloride had little effect. The relatively high value of pH_i maintained by the unstimulated gland is therefore probably not due to Na⁺–H⁺ exchange.     

Quantification of skeletal muscle mitochondrial function by 31P magnetic resonance spectroscopy techniques: a quantitative review

Magnetic resonance spectroscopy (MRS) can give information about cellular metabolism in vivo which is difficult to obtain in other ways. In skeletal muscle, non‐invasive ³¹P MRS measurements of the post‐exercise recovery kinetics of pH, [PCr], [Pi] and [ADP] contain valuable information about muscle mitochondrial function and cellular pH homeostasis in vivo, but quantitative interpretation depends on understanding the underlying physiology. Here, by giving examples of the analysis of ³¹P MRS recovery data, by some simple computational simulation, and by extensively comparing data from published studies using both ³¹P MRS and invasive direct measurements of muscle O₂ consumption in a common analytical framework, we consider what can be learnt quantitatively about mitochondrial metabolism in skeletal muscle using MRS‐based methodology. We explore some technical and conceptual limitations of current methods, and point out some aspects of the physiology which are still incompletely understood.     

Phasic changes in intracellular pH during action potentials of sheep Purkinje fibres

Regulation of intracellular pH (pH_i) and the relationship between H⁺ and Ca²⁺ may vary during activity. Ion-selective microelectrodes were used to record pH_i during action potentials of sheep Purkinje fibres prolonged by low temperature (21°C) and elevated CO₂ content. Intracellular pH also was measured during changes in extracellular calcium concentration, [Ca²⁺]_o. Cytosolic alkalinization (peak pH_i change, 0.03–0.05) was observed during the long action-potential plateau and transient acidification (0.01–0.02 units) upon repolarization. Potassium-induced depolarization to plateau potentials (i.e. to –15±2 mV) simulated the peak magnitude of the alkalinization. However, compensation for the alkalinization occurred at a faster rate during the action potential (8.9±4.3 nM/min) than during K⁺ depolarization (1.2±0.5 nM/min). In comparison, the cytoplasm acidified in resting fibres (0.06–0.07 log units) during changes of [Ca²⁺]_o thought to increase intracellular calcium concentration. Alterations of pH_i were translated into changes of proton concentration ([H⁺]_i). Ten-to twenty-fold elevation of [Ca²⁺]_o evoked a comparable change in [H⁺]_i (mean increase, 5.7 nM) but oppositely directed from that during the plateau (mean decrease, 8.8 nM). The findings in resting fibres seem consistent with displacement of bound protons by Ca²⁺. In contrast, the initial change in pH_i during the plateau is proposed to be consequent to Ca²⁺-release from sarcoplasmic reticulum and/or phosphocreatine hydrolysis coupled to ATP regeneration.     

Dependence of intracellular free calcium and tension on membrane potential and intracellular pH in single crayfish muscle fibres

The dependence of intracellular free calcium ([Ca²⁺]_i) and tension on membrane potential and intracellular pH (pH_i) was studied in single isolated fibres of the crayfish claw-opener muscle using ion-selective microelectrodes. Tension (T) was quantified as a percentage of the maximum force, or as force per cross-sectional area (N/cm²). In resting fibres, pH_i had a mean value of 7.06. Contractions evoked by an increase extracellular potassium ([K⁺]₀) produced a fall in pH_i of 0.01–0.05 units. The lowest measured levels of resting [Ca²⁺]_i corresponded to a pCa_i (= –log [Ca²⁺]_i) of 6.8. Intracellular Ca²⁺ transients recorded during K⁺-induced contractions did not reveal any distinct threshold for force development. Both the resting [Ca²⁺]_i and resting tension were decreased by an intracellular alkalosis and increased by an acidosis. The sensitivity of resting tension to a change in pH_i (quantified as –dT/ dpH_i) showed a progressive increase during a fall in pH_i within the range examined (pH_i 6.2–7.5). The pH_i/[Ca²⁺]_i and pH_i/tension relationships were monotonic throughout the multiphasic pH_i change caused by NH₄Cl. A fall of 0.5–0.6 units in pH_i did not produce a detectable shift in the pCa_i/tension relationship at low levels of force development. The results indicate that resting [Ca²⁺]_i is slightly higher than the level required for contractile activation. They also show that the dependence of tension on pH_i in crayfish muscle fibres is attributable to a direct H⁺ and Ca²⁺ interaction at the level of Ca²⁺ sequestration and/or transport. Finally, the results suggest that in situ, the effect of pH on the Ca²⁺ sensitivity of the myofibrillar system is not as large as could be expected on the basis of previous work on skinned crustacean muscle fibres.     

Muscle phosphocreatine and pulmonary oxygen uptake kinetics in children at the onset and offset of moderate intensity exercise

To further understand the mechanism(s) explaining the faster pulmonary oxygen uptake kinetics found in children compared to adults, this study examined whether the phase II kinetics in children 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 ³¹P-magnetic resonance spectroscopy, and (2) breath by breath changes in during upright cycle ergometry. Only subjects (n = 12) with 95% confidence intervals ≤±7 s for all estimated time constants were considered for analysis. No differences were found between the PCr and phase II time constants at the onset (PCr 23 ± 5 vs. or offset (PCr 28 ± 5 vs. of exercise. The average difference between the PCr and phase II time constants was 4 ± 4 s for the onset and offset responses. Pooling of the exercise onset and offset responses revealed a significant correlation between the PCr and time constants (r = 0.459, P = 0.024). The close kinetic coupling between the and PCr responses at 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 phosphorylation may explain the faster kinetics found in children compared to adults.     

Continuous fluorometric measurement of intracellular pH and Ca2+ in perfused salivary gland and pancreas

Intracellular pH (pH_i) has been measured in intact, perfused rat mandibular salivary glands loaded with the fluorescent pH indicator BCECF [2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]. Glands mounted in the cuvette of a conventional bench-top spectrofluorometer were perfused for 5 min with the acetoxymethyl ester of BCECF and fluorescence was measured ratiometrically at 6-s intervals. The mean value of pH_i in glands perfused with a HCO ₃ ^– -free, N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES)-buffered solution at 37°C was 7.36±0.01 (n=52) which is comparable with values obtained by ³¹P nuclear magnetic resonance (NMR) spectroscopy. NMR data confirmed that the BCECF loading period was accompanied by a transient acidification of the cells, but there was no significant change in the content of the major phosphorus metabolites. Changes in pH in response to NH₄Cl pulses and acetylcholine stimulation were comparable with results reported previously for isolated acini. Additional, preliminary experiments show that the method can also be used to monitor intracellular Ca²⁺ (using fura-2) in perfused salivary glands, and can be adapted for studies of the isolated, perfused pancreas.     

In vivo human myocardial metabolism during aerobic exercise by phosphorus-31 nuclear magnetic resonance spectroscopy

A few studies have been made in vivo on human myocardial energy metabolism. Hence, no discussion has taken place on metabolism during exercise or of training effects on metabolism. We examined human myocardial energy metabolism at rest and during exercise, and also training effects on the metabolism by phosphorus-31 nuclear magnetic resonance (³¹P NMR)-spectroscopy. Six sedentary male students (Cont) and six male long distance runners (Tr) were the subjects. Energy metabolism data were obtained from myocardium during rest and exercise by the region selection method using ³¹P NMR. Rotation of the legs while riding a bicycle, which was fitted with an ergometer we had made ourselves for NMR, imposed given exercise intensities. The heart rate was measured in a stationary phase during exercise. Although the heart rate at rest in the Tr group was significantly lower [Tr, 52.5 (SD 3.1) beat · min^–1; Cont, 67.1 (SD 2.9) beat · min^–1], no significant difference was observed in myocardial energy metabolism using the ³¹P NMR method [Tr, phosphocreatine/-adenosine 5-triphosphate (PCr/-ATP); 1.51 (SD 0.02); Cont, 1.51 (SD 0.01)]. When NMR measurements were investigated at two different intensities of exercise, heart rates in the Cont group were significantly higher by about 20 beat · min ^–1 than those in the Tr group at both exercise intensities, while no difference in energy metabolism was observed between the groups or between rest and exercise [Tr, 75.9 (SD 3.6), 88.3 (SD 3.7) beat · min^–; PCr/-ATP 1.51 (SD 0.03), 1.51 (SD 0.03); Cont, 95.9 (SD 2.4), 115.1 (SD 3.5) beat · min^–1 PCr/-ATP 1.51 (SD 0.01), 1.51 (SD 0.04)]. Thus, during submaximal exercise as employed in this study, it would seem that the high energy phosphate level normally observed during rest may still be maintained. From these results, the absence of change in the myocardial PCr: ATP ratio suggested that adenosine 5-diphosphate was not the primary regular of the increased metabolism needed to meet the higher cardiac workload during aerobic exercise in either group.     

Effects of exercise‐induced intracellular acidosis on the phosphocreatine recovery kinetics: a 31P MRS study in three muscle groups in humans

Little is known about the metabolic differences that exist among different muscle groups within the same subjects. Therefore, we used ³¹P‐magnetic resonance spectroscopy (³¹P‐MRS) to investigate muscle oxidative capacity and the potential effects of pH on PCr recovery kinetics between muscles of different phenotypes (quadriceps (Q), finger (FF) and plantar flexors (PF)) in the same cohort of 16 untrained adults. The estimated muscle oxidative capacity was lower in Q (29 ± 12 mM min^‐1, CV_{inter‐subject} = 42%) as compared with PF (46 ± 20 mM min^‐1, CV_{inter‐subject} = 44%) and tended to be higher in FF (43 ± 35 mM min^‐1, CV_{inter‐subject} = 80%). The coefficient of variation (CV) of oxidative capacity between muscles within the group was 59 ± 24%. PCr recovery time constant was correlated with end‐exercise pH in Q (p < 0.01), FF (p < 0.05) and PF (p <0.05) as well as proton efflux rate in FF (p < 0.01), PF (p < 0.01) and Q (p = 0.12). We also observed a steeper slope of the relationship between end‐exercise acidosis and PCr recovery kinetics in FF compared with either PF or Q muscles. Overall, this study supports the concept of skeletal muscle heterogeneity by revealing a comparable inter‐ and intra‐individual variability in oxidative capacity across three skeletal muscles in untrained individuals. These findings also indicate that the sensitivity of mitochondrial respiration to the inhibition associated with cytosolic acidosis is greater in the finger flexor muscles compared with locomotor muscles, which might be related to differences in permeability in the mitochondrial membrane and, to some extent, to proton efflux rates. Copyright © 2013 John Wiley & Sons, Ltd.