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.     

The rate of phosphocreatine hydrolysis and resynthesis in exercising muscle in humans using 31P-MRS

Time-resolved 31-phosphorus nuclear magnetic resonance spectroscopy (31P-MRS) of the biceps femoris muscles was performed during exercise and recovery in six healthy sedentary male subjects (maximal oxygen uptake; 46.6 +/- 1.7 (SEM) ml.kg-1.min-1), 5 male sprinters (56.2 +/- 2.5), and 5 male long-distance runners (73.6 +/- 2.2). Each performed 4 min of knee flexion exercises at absolute values of 1.63 W and 4.90 W, followed by 5 min of recovery in a prone position in a 2.1 T superconducting magnet with a 67 cm bore. 31P-MRS spectra were recorded every 12.8 s during the rest-exercise-recovery sequence. Computer-aided contour analysis and pixel imaging of phosphocreatine peaks (PCr) and inorganic phosphate (Pi) were performed. The work loads in the present study were selected as mild exercise (1.63 W) and heavy exercise (4.90 W), corresponding to 18-23% and 54-70% of maximal exercise intensity. Long-distance runners showed a significantly smaller decrement in PCr and less acidification at a given exercise intensity compared to those shown by sedentary subjects. The transient responses of PCr and Pi during recovery were characterized by first-order kinetics. After exercise, the recovery rates of PCr and Pi were significantly faster in long-distance runners than in sedentary subjects (P < 0.05). Since it is postulated that PCr resynthesis is controlled by aerobic metabolism and mitochondrial creatine kinase, it is suggested that the faster PCr and Pi recovery rates and decreased acidification seen in long-distance runners during and after exercise might be attributed to their greater capacity for aerobic metabolism.     

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

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.     

Effect of circulatory occlusion on human muscle metabolism during exercise and recovery

To assess muscle metabolism and inorganic phosphate (P_i) peak splitting during exercise, 31-phosphorus nuclear magnetic resonance spectroscopy was performed during ramp incremental and submaximal step exercise with and without circulatory occlusion. Seven healthy men performed calf flexion in a superconducting magnet. There was no P_i splitting during ramp incremental exercise with the circulation present and phosphocreatine (PCr) decreased linearly by 0.07 (SEM 0.01) mmol · l⁻¹ · s⁻¹, while exercise with the circulation occluded caused the P_i peak to split into a high and a low pH peak. The rate of PCr decrease during exercise with the circulation occluded was 0.15 (SEM 0.03) mmol · l⁻¹ · s⁻¹ which with the efficiency of the adenosine 5′-triphosphate (ATP) hydrolysis reaction corresponded well to the mechanical energy. Both with and without occlusion of the circulation PCr decreased with some time lag which may reflect the consumption of residual oxygen. In submaximal step exercise PCr decreased exponentially at the onset of exercise with the circulation open whereas it decreased linearly by 0.15␣mmol · l⁻¹ · s⁻¹ when the circulation was occluded. After exercise, occlusion of the circulation was maintained for 1 min more and there was no PCr resynthesis. It is suggested that ATP synthesis was limited by the availability of oxygen. Accepted: 14 August 1996     

Control of the rate of phosphocreatine resynthesis after exercise in trained and untrained human quadriceps muscles

We examined the effect of differences in exercise intensity on the time constant (t _c) of phosphocreatine (PCr) resynthesis after exercise and the relationships betweent _c and maximal oxygen uptake (VO_2max) in endurance-trained runners (n = 5) and untrained controls (n = 7) (average VO_2max = 66.2 and 52.0 ml · min^–1 · kg^–1, respectively). To measure the metabolism of the quadriceps muscle using phosphorus nuclear magnetic resonance spectroscopy, we developed a device which allowed knee extension exercise inside a magnet. All the subjects performed four types of exercise: light, moderate, severe and exhausting. The end-exercise PCr: [PCr + inorganic phosphate (P_i)] ratio decreased significantly with the increase in the exercise intensity (P < 0.01). Although there was little difference in the end-exercise pH, adenosine diphosphate concentration ([ADP]) and the lowest intracellular pH during recovery between light and moderate exercise, significant changes were found at the two higher intensities (P < 0.01). These changes for runners were smaller than those for the controls (P < 0.05). The _c remained constant after light and moderate exercise and then lengthened in proportion to the increase in intensity (P < 0.05). The runners had a lowert _c at the same PCr and pH than the controls, particularly at the higher intensity (P < 0.05). There was a significant correlation betweent _c and [ADP] in light exercise and betweent _c and both end-exercise PCr and pH in severe and exhausting exercise (P < 0.05). The threshold of changes in pH andt _c was a PCr: (PCr + P_i) ratio of 0.5. There was a significant negative correlation between the VO_2max andt _c after all levels of exercise (P<0.05).However, in the controls a significant correlation was found in only light and moderate exercise (P < 0.05). These findings suggest the validity of the use oft _c at an end-exercise PCr:(PCr + P_i) ratio of more than 0.5 as a stable index of muscle oxidative capacity and the correlation between local and general aerobic capacity. Moreover, endurance-trained runners are characterized by the faster PCr resynthesis at the same PCr and intracellular pH.     

Evaluation of muscle oxidative potential by 31P-MRS during incremental exercise in old and young humans

The purpose of this study was to compare muscle oxidative capacity between moderately active young and old humans by measuring intracellular threshold (IT) during exercise with ³¹P-magnetic resonance spectroscopy (³¹P-MRS). Changes in phosphocreatine, inorganic phosphate, and intracellular pH were measured by ³¹P-MRS during a progressive unilateral ankle plantar flexion exercise protocol in groups of moderately active old (n=12, mean age 66.7 years) and young (n=13, mean age 26.2 years) individuals. From muscle biopsy samples of the lateral gastrocnemius, citrate synthase (CS) activity was determined in six subjects from each group, and fibre type composition was determined in nine old and ten young subjects. The old group had a lower IT for pH, as a percentage of peak work rate (P<0.05), despite a similar CS activity compared to the young. IT was significantly correlated with CS activity (R=0.59; P<0.05), but not with fibre type composition. It was concluded that metabolic responses to exercise are affected by ageing, as indicated by a lower IT in old compared to young individuals. Accepted: 7 May 1998     

31P nuclear magnetic resonance study on changes in phosphocreatine and the intracellular pH in rat skeletal muscle during exercise at various inspired oxygen contents

We measured ATP, phosphocreatine (PCr), inorganic phosphate (P_i), and the intracellular pH in rat hindlimb muscles during submaximal isometric exercise with various O₂ deliveries using³¹P nuclear magnetic resonance spectroscopy (³¹P NMR) to evaluate changes in energy metabolism in relation to O₂ availability. Delivery of O₂ to muscles was altered by controlling the fractional concentration of inspired oxygen (F _IO₂) at 0.50, 0.28, 0.21, 0.11 and 0.08 with monitoring partial pressure of oxygen and carbon dioxide, and bicarbonate at the femoral artery. The steady-state ratio of PCr : (PCr + P_i) during exercise decreased as a function ofF _IO₂ even at 0.21. Significant acidification of the intracellular pH during exercise occurred at 0.08F _IO₂. Change in the PCr : (PCr + P_i) ratio demonstrated that the oxidative capacity, i.e. the maximal rate of the oxidative phosphorylation reaction, in muscle was not limited by O₂ delivery at 0.50F _IO₂, but was significantly limited at 0.21F _IO₂ or below. Change in the intracellular pH at 0.08F _IO₂ could be interpreted as an increase in lactate, suggesting activation of glycolysis. Correlation between the PCr : (PCr + P_i) ratio and the intracellular pH revealed the existence of a critical PCr : (PCr + P_i) ratio and pH for glycolysis activation at around 0.4 and 6.7, respectively.     

Muscle metabolism in track athletes, using 31P magnetic resonance spectroscopy.

We tested whether preferred running event in track athletes would correlate with the initial rate of phosphocreatine (PCr) resynthesis following submaximal exercise. PCr recovery was measured in the calf muscles of 16 male track athletes and 7 male control subjects following 5 min of repeated plantar flexion against resistance. Pi, PCr, and pH were measured using phosphorus magnetic resonance spectroscopy (31P MRS) with an 8-cm surface coil in a 1.8-T magnet. During exercise, work levels were gradually increased to deplete PCr to 50-60% of the initial value. No drop in pH was seen in any of the subjects during this exercise. The areas of the PCr peaks following exercise were fit to monoexponential curves. Two or three tests were performed on each subject and the results averaged. Athletes were divided into three groups based on their primary event: sprinters running 400 m or less, middle-distance athletes running 400-1500 m, and long-distance athletes running farther than 1500 m. The maximal rates of PCr resynthesis (mmol.min-1.kg-1 muscle weight) were 64.8 +/- 8.6, for long-distance runners; 41.4 +/- 11, for middle-distance runners; 32.0 +/- 7.0, for sprinters; and 38.6 +/- 10, for controls (mean +/- SE). The faster PCr recovery rates seen in long-distance runners compared with sprinters indicate greater oxidative capacity, which is consistent with the known differences between athletes in these events.     

Peak blood ammonia and lactate after submaximal, maximal and supramaximal exercise in sprinters and long-distance runners

The purpose of this study was to elucidate the difference in peak blood ammonia concentration between sprinters and long-distance runners in submaximal, maximal and supramaximal exercise. Five sprinters and six long-distance runners performed cycle ergometer exercise at 50% maximal, 75% maximal, maximal and supramaximal heart rates. Blood ammonia and lactate were measured at 2.5, 5, 7.5, 10 and 12.5 min after each exercise. Peak blood ammonia concentration at an exercise intensity producing 50% maximal heart rate was found to be significantly higher compared to the basal level in sprinters (P less than 0.01) and in long-distance runners (P less than 0.01). The peak blood ammonia concentration of sprinters was greater in supra-maximal exercise than in maximal exercise (P less than 0.05), while there was no significant difference in long-distance runners. The peak blood ammonia content after supramaximal exercise was higher in sprinters compared with long-distance runners (P less than 0.01). There was a significant relationship between peak blood ammonia and lactate after exercise in sprinters and in long-distance runners. These results suggest that peak blood ammonia concentration after supramaximal exercise may be increased by the recruitment of fast-twitch muscle fibres and/or by anaerobic training, and that the processes of blood ammonia and lactate production during exercise may be strongly linked in sprinters and long-distance runners.     

Purification and properties of a lectin from the seeds of Croton tiglium with hemolytic activity toward rabbit red cells

The pH dependence of the chemical shift of the inorganic phosphate (P_i) inside mitochondria observable by ³¹P nmr has been examined and used for the measurement of the internal pH. The pH gradient and the P_i concentration gradient were in the simple relation expected for the neutral exchange process of H₂PO₄^? and OH^?. This P_i distribution across the mitochondrial membrane was not influenced by the cross-membrane electrical potential. Both the P_i, distribution and the pH titration curve of the internal P_i indicate that the activity of the internal P_i can be well represented by the concentration of P_i measured by ³¹P nmr peak intensity. The present results give a sound base for applying ³¹P nmr to study bioenergetics and cell metabolism.     

Work-Related Pain in Extrinsic Finger Extensor Musculature of Instrumentalists Is Associated with Intracellular pH Compartmentation during Exercise

Background

Although non-specific pain in the upper limb muscles of workers engaged in mild repetitive tasks is a common occupational health problem, much is unknown about the associated structural and biochemical changes. In this study, we compared the muscle energy metabolism of the extrinsic finger extensor musculature in instrumentalists suffering from work-related pain with that of healthy control instrumentalists using non-invasive phosphorus magnetic resonance spectroscopy (³¹P-MRS). We hypothesize that the affected muscles will show alterations related with an impaired energy metabolism.

Methodology/Principal Findings

We studied 19 volunteer instrumentalists (11 subjects with work-related pain affecting the extrinsic finger extensor musculature and 8 healthy controls). We used ³¹P-MRS to find deviations from the expected metabolic response to exercise in phosphocreatine (PCr), inorganic phosphate (Pi), Pi/PCr ratio and intracellular pH kinetics. We observed a reduced finger extensor exercise tolerance in instrumentalists with myalgia, an intracellular pH compartmentation in the form of neutral and acid compartments, as detected by Pi peak splitting in ³¹P-MRS spectra, predominantly in myalgic muscles, and a strong association of this pattern with the condition.

Conclusions/Significance

Work-related pain in the finger extrinsic extensor muscles is associated with intracellular pH compartmentation during exercise, non-invasively detectable by ³¹P-MRS and consistent with the simultaneous energy production by oxidative metabolism and glycolysis. We speculate that a deficit in energy production by oxidative pathways may exist in the affected muscles. Two possible explanations for this would be the partial and/or local reduction of blood supply and the reduction of the muscle oxidative capacity itself.     

Measurement of an intracellular pH rise after fertilization in crab eggs using 31P-NMR

The effect of fertilization upon the intracellular pH, pH_i, in crab ovulated eggs was examined by ³¹P-NMR. The pH_i values were obtained from the chemical shift differences between the phosphoarginine PA resonance and the inorganic phosphate P_i resonance. The detection of the P_i peak was accomplished by Hahn spin-echo experiments in order to cancel the broad signal arising from phosphoproteins which overlaps the P_i signal. The average pH_i of the unfertilized unactivated eggs was 6.55 and a rise of 0.12 pH unit occurred after fertilization.     

A 31P NMR study of the internal pH of yeast peroxisomes

The internal pH of peroxisomes in the yeasts Hansenula polymorpha, Candida utilis and Trichosporon cutaneum X4 was estimated by ³¹P nuclear magnetic resonance (NMR) spectroscopy. ³¹P NMR spectra of suspensions of intact cells of these yeasts, grown under conditions of extensive peroxisomal proliferation, displayed two prominent P_i-peaks at different chemical shift positions. In control cells grown on glucose, which contain very few peroxisomes, only a single peak was observed. This latter peak, which was detected under all growth conditions, was assigned to cytosolic P_i at pH 7.1. The additional peak present in spectra of peroxisome-containing cells, reflected P_i at a considerably lower pH of approximately 5.8–6.0. Experiments with the protonophore carbonyl cyanide m-chlorophenylhydrazon (CCCP) and the ionophores valinomycin and nigericin revealed that separation of the two P_i-peaks was caused by a pH-gradient across a membrane separating the two pools. Experiments with chloroquine confirmed the acidic nature of one of these pools. In a number of transfer experiments with the yeast H. polymorpha it was shown that the relative intensity of the P_i-signal at the low pH-position was correlated to the peroxisomal volume fraction. These results strongly suggest that this peak has to be assigned to P_i in peroxisomes, which therefore are acidic in nature. The presence of peroxisome-associated P_i was confirmed cytochemically.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazon - DCCD N,N-dicyclohexylcarbodiimide     

Changes in phosphometabolites and intracellular pH in the tail muscle of the prawnPalaemon serratus as shown by in vivo31P-NMR

Summary ³¹P NMR spectra were recorded from tail muscles of the prawnPalaemon serratus, at rest, after exhaustive work and during subsequent recovery. At rest, the spectra indicated concentrations of phosphoarginine and ATP in good agreement with those obtained from resting fast skeletal muscles in mammals, which are characterized by a high phosphocreatine/P_i ratio. Following exhaustive work, phosphoarginine dropped by ca. 60% and ATP by 20%, while inorganic phosphate increased by 160%. The increase in inorganic phosphate immediately after contractions and in the first minutes of recovery corresponded partially to the changes in phosphoarginine and ATP. During recovery, the decrease of inorganic phosphate balanced the resynthesized phosphoarginine which was fully replenished within 30–40 min. The position of the inorganic phosphate resonance peak was used to monitor changes in intracellular pH (pH_i). The average pH_i in resting tail muscles was 7.20. After stimulation it was observed to decrease by 0.22 units. The return to pre-stimulation value was not achieved within 45 min. A NMR index (ATP+Arg-P)/(ATP+Arg-P+P_i) was calculated to characterize the extent of energetic changes caused by exercise.     

A 31P-NMR study of the cross-membrane pH gradient induced by ATP hydrolysis in mitochondria

³¹P-NMR has been used to study the increase of ΔpH in mitochondria by externally added ATP. Freshly prepared mitochondria was treated with N-ethylmaleimide to inhibit the exchange between internal and external P_i. Upon addition of ATP, phosphocreatine (30 mM) and creatine kinase to a NMR sample of mitochondria suspension (approx. 120 mg protein/ml) at 0°C, an increase of ΔpH by approx. 0.5 pH unit was observed. However the increased ΔpH could not be maintained, but slowly decayed along with the increase of external ADP/ATP ratio. Further addition of valinomycin to the suspension induced a larger ΔpH (approx. 1) which was maintained by the increased rate of internal ATP hydrolysis as seen in the growth of the internal P_i peak intensity in NMR spectra and the concomitant decrease of the external phosphocreatine peak. The external P_i and ATP peaks stayed virtually constant. When carboxyatractyloside was added to inhibit the ATP/ADP translocase, the internal P_i increase was stopped and the ΔpH decayed. These observations in conjunction with those made earlier in respiring mitochondria clearly show the reversible nature of the ATPase function in which the internal ATP hydrolysis is associated with outward pumping of protons.     

pH Tolerance in Freshwater Bacterioplankton: Trait Variation of the Community as Measured by Leucine Incorporation

pH is an important factor determining bacterial community composition in soil and water. We have directly determined the community tolerance (trait variation) to pH in communities from 22 lakes and streams ranging in pH from 4 to 9 using a growth-based method not relying on distinguishing between individual populations. The pH in the water samples was altered to up to 16 pH values, covering in situ pH ± 2.5 U, and the tolerance was assessed by measuring bacterial growth (Leu incorporation) instantaneously after pH adjustment. The resulting unimodal response curves, reflecting community tolerance to pH, were well modeled with a double logistic equation (mean R² = 0.97). The optimal pH for growth (pH_opt) among the bacterial communities was closely correlated with in situ pH, with a slope (0.89 ± 0.099) close to unity. The pH interval, in which growth was ≥90% of that at pH_opt, was 1.1 to 3 pH units wide (mean 2.0 pH units). Tolerance response curves of communities originating from circum-neutral pH were symmetrical, whereas in high-pH (8.9) and especially in low-pH (<5.5) waters, asymmetric tolerance curves were found. In low-pH waters, decreasing pH was more detrimental for bacterial growth than increasing pH, with a tendency for the opposite for high-pH waters. A pH tolerance index, using the ratio of growth at only two pH values (pH 4 and 8), was closely related to pH_opt (R² = 0.83), allowing for easy determination of pH tolerance during rapid changes in pH.     

Relaxation of solvent protons by cobalt bovine carbonic anhydrase.

In a recent report, Bertini et al. (Biochem. Biophys. Res. Comm.78, 158–160 (1977)) argued that the low-pH form of Co²⁺-substituted bovine carbonic anhydrase contains a rapidly exchanging water molecule at the cobalt site. The basis for this was the observation of a pH-independent contribution to the solvent water proton relaxation rate; it was suggested that the result was unobserved by previous workers because of the presence of sulfate in the sample buffer. We have repeated the experiments of Bertini et al. and find that the results can be attributed to an ionic strength-induced shift of the pK of the group responsible for the relaxation enhancement. The amount of high-pH form of the enzyme present (determined spectrophotometrically) at every pH correlates with the relaxation rate, whereas the fraction of high-pH form present at a given pH depends on ionic strength. These results are in agreement with earlier data indicating that the low-pH form of the enzyme does not contribute to solvent water proton relaxation.     

Inorganic Phosphate Compartmentation in the Normal Isolated Canine Brain

Abstract: In vivo ³¹P magnetic resonance spectra of 16 isolated dog brains were studied by using a 9.4-T wide-bore superconducting magnet. The observed P_i peak had an irregular shape, which implied that it represented more than one single homogeneous pool of P_i. To evaluate our ability to discriminate between single and multiple peaks and determine peak areas, we designed studies of simulated ³¹P_i spectra with the signal-to-noise (S/N) ratios ranging from ∞ to 4.4 with reference to the simulated P_i peak. For the analysis we used computer programs with a linear prediction algorithm (NMR-Fit) and a Marquardt–Levenberg nonlinear curve-fit algorithm (Peak-Fit). When the simulated data had very high S/N levels, both methods located the peak centers precisely; however, the Marquardt-Levenberg algorithm (M-L algorithm) was the more reliable at low S/N levels. The linear prediction method was poor at determining peak areas; at comparable S/N levels, the M-L algorithm determined all peak areas relatively accurately. Application of the M-L algorithm to the individual experimental in vivo dog brain data resolved the P_i peak into seven or more separate components. A composite spectrum obtained by averaging all spectral data from six of the brains with normal O₂ utilization was fitted using the M-L algorithm. The results suggested that there were eight significant peaks with the following chemical shifts: 4.07, 4.29, 4.45, 4.62, 4.75, 4.84, 4.99, and 5.17 parts per million (ppm). Although linear prediction demonstrated the presence of only three peaks, all corresponded to values obtained using the M-L algorithm. The peak indicating a compartment at 5.17 ppm (pH 7.34) was assigned to venous pH on the basis of direct simultaneous electrode-based measurements. On the basis of earlier electrode studies of brain compartmental pH, the peaks at 4.99 ppm (pH 7.16) and 4.84 ppm (pH 7.04) were thought to represent interstitial fluid and the astrocyte cytoplasm, respectively.     

The influence of maximal aerobic power on recovery of skeletal muscle following anaerobic exercise

Phosphorus magnetic resonance spectroscopy (³¹P-MRS) was used to investigate the influence of maximal aerobic power (˙VO _2max) on the recovery of human calf muscle from high-intensity exercise. The (˙VOO_2max) of 21 males was measured during treadmill exercise and subjects were assigned to either a low-aerobic-power (LAP) group (n = 10) or a high-aerobic-power (HAP) group (n = 11). Mean (SE) ˙VO _2max of the groups were 46.6 (1.1) and 64.4 (1.4) ml · kg⁻¹ · min⁻¹, respectively. A calf ergometry work capacity test was used to assign the same relative exercise intensity to each subject for the MRS protocol. At least 48 h later, subjects performed the rest (4 min), exercise (2 min) and recovery (10 min) protocol in a 1.5 T MRS scanner. The relative concentration of phosphocreatine (PCr) was measured throughout the protocol and intracellular pH (pH_i) was determined from the chemical shift between inorganic phospate (P_i) and PCr. End-exercise PCr levels were 27 (3.4) and 25 (3.5)% of resting levels for LAP and HAP respectively. Mean resting pH_i was 7.07 for both groups, and following exercise it fell to 6.45 (0.04) for HAP and 6.38 (0.04) for LAP. Analysis of data using non-linear regression models showed no differences in the rate of either PCr or pH_i recovery. The results suggest that ˙VO_2max is a poor predictor of metabolic recovery rate from high-intensity exercise. Differences in recovery rate observed between individuals with similar ˙VO_2max imply that other factors influence recovery. Accepted: 17 December 1996     

NMR Studies of Pi-Containing Extracellular and Cytoplasmic Compartments in Brain

Abstract: The inorganic phosphate (P_i) NMR peak in brain has an irregular shape, which suggests that it represents more than a single homogeneous pool of P_i. To test the ability of the Marquardt-Levenberg (M-L) nonlinear curve fit algorithm software (Peak-Fit) to separate multiple peaks, locate peak centers, and estimate peak heights, we studied simulated P_i spectra with defined peak centers, areas, and signal-to-noise (S/N) ratios ranging from ∞ to 5.8. As the S/N ratio decreased below 15, the M-L algorithm located peak centers accurately when they were detected; however, small peaks tended to grow smaller and disappear, whereas the amplitudes of larger peaks increased. We developed an in vitro three-compartment model containing a mixture of P_i buffer, phosphocreatine, phosphate diester, and phosphate monoester (PME), portions of which were adjusted to three different pHs before addition of agar. Weighed samples of each buffered gel together with phospholipid extract and bone chips were placed in an NMR tube and covered with mineral oil. Following baseline correction, it was possible to separate the P_i peaks arising from the three compartments with different pH values if each peak made up 10–35% of total P_i area. In vivo, we identified the plasma compartment by intraarterial infusion of P_i. It was assumed that intracellular compartments contained high-energy phosphates and took up glucose. Based on these assumptions we subjected the brains to complete ischemia and observed that P_i compartments at pH 6.82, 6.92, 7.03, and 7.13 increased markedly in amplitude. If the brain cells took up and phosphorylated 2-deoxyglucose (2-DG), 2-DG-6-phosphate (2-DG-6-P) would appear in the PME portion of the spectrum ionized according to pH_i. Four 2-DG-6-P peaks with calculated pH values of 6.86, 6.94, 7.04, and 7.15 did appear in the spectrum, thereby confirming that the four larger P_i peaks represented intracellular spaces.