INTRODUCTION

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THERE IS SOME EVIDENCE that, at similar submaximal work rates, blood and intracellular lactate, intracellular phosphocreatine (PCr), and other proposed regulators of respiration (P_i and ADP) may vary significantly with different fractions of inspired O₂ (FI_O2) (13, 18, 21). However, at submaximal work rates, levels of O₂ uptake (O₂) remained constant while breathing varied FI_O2 (13, 15, 18, 21-23, 30, 31); hence, the causes of these changes in metabolic processes that occur during steady-state submaximal exercise while breathing varied FI_O2 have not been fully elucidated. It has been suggested that these changes at the same O₂ may be a result of differences in O₂ onset kinetics that occur when FI_O2 is varied (16, 17, 23). Additionally, it has recently been proposed that the time required to achieve a steady-state O₂ may set the subsequent levels of cytosolic signals driving mitochondrial recruitment (11), and the time course for O₂ onset has been related to changing PCr concentrations in muscle at the start of exercise (1, 26-28). With respect to FI_O2, exercise in hyperoxia has been shown to have short O₂ onset times (23, 24), which may result in small depletions of PCr, compared with exercise in hypoxic situations in which the onset time is longer, and may result in greater PCr hydrolysis and an increased reliance on anaerobic ATP turnover during the extended transition to steady state (17, 23).

However, even under steady-state conditions, there is evidence that metabolic processes can be affected by alterations in tissue or intracellular oxygenation (4, 12, 37). It has been shown that variations in intracellular PO₂ can alter the ratio of the concentration of ATP to the concentration of ADP ([ATP]/[ADP]) at a given steady-state O₂ to maintain that rate of cellular respiration (35-37). Any alterations in the concentration of O₂ ([O₂]) will change the concentrations of the other reactants of oxidative phosphorylation to maintain kinetic steady state. Thus changes seen in metabolites at a similar O₂ when FI_O2 is varied may be the result of changes in intracellular oxygenation and not of differences in O₂ onset kinetics. However, there is little direct experimental evidence that alterations in tissue oxygenation during steady-state exercise can influence intracellular metabolic processes.

The purpose of this study was to test in vivo the hypotheses that 1) changes in intracellular metabolic processes do occur at constant submaximal O₂ as FI_O2 is changed and 2) these changes are not due to differences in O₂ onset kinetics. Human subjects performed submaximal steady-state plantar flexion exercise, during which FI_O2 was varied (0.1, 0.21, and 1.00) and the levels of PCr were measured by ³¹P-magnetic resonance spectroscopy (MRS). The results demonstrate that there were significant changes in the intracellular metabolic processes due to altered FI_O2 and that these changes were, by experimental design, not the result of O₂ onset kinetics but were likely the result of changes in intracellular oxygenation.

	METHODS

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Subjects. Six healthy men, aged 21-42 yr [32 ± 6.8 (SD) yr], volunteered for this study and gave written informed consent. The study was approved by the Human Subjects Committee of the University of California, San Diego. Subjects were all healthy and active, ranging from recreational to well-trained athletes. The subjects refrained from strenuous exercise for 24 h before data collection.

Exercise protocol. Subjects were familiarized with plantar flexion exercise in the confines of a whole body magnetic resonance imaging system. At this time, ~60% of maximum work rate was determined for each subject. Subjects performed constant-load submaximal plantar flexion at this intensity (7.2 ± 0.6 W) at a frequency of 1 contraction/s (keeping time with an electronic metronome) while lying supine in a superconducting 1.5-T magnet. The exercise was a square-wave protocol with the same work rate maintained for each FI_O2. Throughout each exercise bout, subjects breathed through a low-resistance, two-way breathing valve (2700, Hans-Rudolph, Kansas City, MO), and end-tidal O₂ and CO₂ were sampled continuously, allowing arterial O₂ saturation to be calculated (assuming no alveolar-to-arterial PO₂ gradient and no significant metabolic acidosis). Heart rate was monitored continuously with a finger probe (Omni-Trak, In Vivo Research) throughout the experiment. After a 5-min warm-up period followed by 5 min of rest, each subject performed a control bout of 10 min of exercise under normoxic conditions. After 40 min of rest, subjects performed three separate exercise bouts with the FI_O2 switched after 5 min of the 10-min exercise. MRS data were acquired continuously for 2 min before exercise, 10 min during exercise, and 6 min during recovery. The subjects breathed the first FI_O2 for 4 min before the commencement of MRS acquisition, then for 2 min before exercise with MRS data acquisition, and then for the first 5 min of constant-load, constant-rate exercise (11 min total). After this initial 5 min of exercise, the FI_O2 was switched, and the subjects continued to exercise while breathing the second FI_O2 (without change in work rate) for a further 5 min and for the 6 min of recovery (11 min total). The three FI_O2 switches were 1) 0.1 switched to 0.21 (hypoxia to normoxia; Hypo-Norm), 2) 0.1 switched to 1.00 (hypoxia to hyperoxia; Hypo-Hyper), and 3) 1.00 switched to 0.1 (hyperoxia to hypoxia; Hyper-Hypo). Subjects were allowed 40 min to rest between exercise bouts. The order of the three treatments was varied to allow all six possible orders to be performed once. The subjects were unaware of the treatment order during the exercise.

³¹P-MRS. MRS was performed by using a clinical 1.5-T General Electric Signa system (version 4.8) operating at 25.86 MHz for ³¹P. ³¹P-MRS data were acquired with a transmit/receive surface coil (diameters 20 and 10 cm, respectively) placed under the calf at its maximum diameter. The centering of the leg over the coil was confirmed by T₁-weighted ¹H localizing images obtained in the axial plane. Magnetic field homogeneity was optimized by shimming on the proton signal from tissue water. For ³¹P-MRS, the pulse power was adjusted so that ~72% of the signal acquired was from tissue within 5 cm of the surface coil. The spectral width was 2,500 Hz, and data were acquired continuously for 18 min with a single free induction decay (FID) generated every 4 s. Thus 270 single FIDs were acquired during the 2-min rest period, 10-min exercise period, and 6-min recovery period. As a result, the data can be expressed with a time resolution of 4 s or summed as spectra representing an average over a defined time period.

Data analysis. Data were processed by using SAGE/IDL software on a Silicon Graphics Indigo workstation. Each FID consisted of 1,024 complex points and was processed with 5-Hz exponential line broadening before zero filling and Fourier transformation. Summing 10 spectra provided a sufficient signal-to-noise ratio to allow the determination of PCr levels at 40-s time intervals during the experiment. All spectra were manually phased by using zero and first-order phase corrections. The levels of PCr determined from the intensity of that peak were normalized to 100% by using as a reference the average value obtained for the last 40 s of rest for each subject. Muscle intracellular pH was calculated from the chemical shift difference () of the P_i peak relative to the PCr peak by using the following equation: pH = 6.75 + log[(3.27)/(5.69)] (34). PCr and pH values were pooled and expressed as the means ± SE for the six subjects and plotted as a function of time. The changing PCr levels and pH values in response to varied FI_O2 were statistically assessed by a one-way repeated-measures ANOVA. A 0.05 level of significance was used.

	RESULTS

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The levels of PCr and pH determined with a temporal resolution of 40 s for the rest, exercise, and recovery periods are shown in Figs. 1 and 2, respectively. On commencement of exercise, there is a rapid net hydrolysis of PCr, reaching a steady-state level of 65.7 ± 4.3% of resting levels under control (normoxic) conditions (Fig. 1A). The following values of PCr reported for the three different FI_O2 switch treatments represent the average of the last 40 s of data acquired before the FI_O2 switch and before the recovery period and are expressed as a percentage of resting PCr levels. For Hypo-Norm, PCr levels fell to 55.5 ± 4.8% and after the switch increased to 67.8 ± 4.9% (Fig. 1B). For Hypo-Hyper, PCr levels fell to 59.0 ± 4.3% and increased to 72.3 ± 5.1% after the switch (Fig. 1C). For Hyper-Hypo, PCr levels fell to 72.6 ± 3.1% and decreased further to 64.2 ± 3.4% after the switch (Fig. 1D). For all three treatments, the changes in PCr levels after the FI_O2 switch were significantly different (P < 0.05), compared with the first FI_O2 breathed, even though the work rate remained constant.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Pooled magnetic resonance spectroscopy data for 6 subjects showing percentage of phosphocreatine (%PCr) (means ± SE) during rest, constant-load submaximal exercise, and recovery for control (normoxic) bout (A) and 3 treatments: hypoxia to normoxia (Hypo-Norm; B), hypoxia to hyperoxia (Hypo-Hyper; C), and hyperoxia to hypoxia (Hyper-Hypo; D). When fraction of inspired O2 (FIO2) was varied, PCr levels for last 40 s before switch and for last 40 s before recovery were significantly different for each treatment (P < 0.05). FIO2 = 0.21 (A); 0.1 switched to 0.21 (B); 0.1 switched to 1.00 (C); 1.00 switched to 0.1(D).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Pooled intracellular pH data for control bout and 3 treatments (means ± SE) show that exercise was submaximal. There were no significant differences in pH for last 40 s beforeFIO2 switch and for last 40 s before recovery. Panels are as described in Fig. 1 legend.

The intracellular pH data in Fig. 2 show that, for the control exercise bout and the three FI_O2 switch bouts, there is an increase in pH of ~0.1 at the commencement of exercise. There were no significant differences in pH values for the 40 s before the FI_O2 switch and before the recovery period for any of the treatments, i.e., at the same times that PCr levels were determined. The PCr and pH data, as well as arterial O₂ saturation and heart rate for the 40 s before the switch and before the recovery period, are summarized in Table 1.

The plots for Hypo-Hyper and Hyper-Hypo are overlaid in Fig. 3. This shows that the PCr levels were better maintained when the FI_O2 of 0.1 was second compared with when it was first (64.2 ± 3.4% vs. 59 ± 4.3%). When the second FI_O2 was 1.00, PCr levels increased and matched the levels maintained when 1.00 was the first FI_O2 (72.3 ± 5.1% vs. 72.6 ± 3.1%). In Fig. 3, the numbers in parentheses are the arterial O₂ saturations for the 40 s before the FI_O2 switch and before the recovery period. The arterial O₂ saturation was 70.5 ± 2.9% with the initial 0.1 FI_O2 in Hypo-Hyper, but it was higher, at 77.0 ± 0.5%, when 0.1 FI_O2 was second in Hyper-Hypo. It is interesting to note that the higher saturation when hypoxia was the second treatment did not appear to be a function of time after the FI_O2 switch, as exercising for longer periods while breathing the FI_O2 of 0.1 as the second treatment did not cause a further fall in saturation.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   %PCr for Hypo-Hyper () overlaid with Hyper-Hypo () showing that PCr levels do not fall as low when hypoxia is 2nd FIO2 as when hypoxia is 1st FIO2. PCr levels are same in hyperoxia regardless of order. Arterial O2 saturations are shown in parentheses and reflect the different levels of PCr.

	DISCUSSION

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The major findings of this study were that 1) changes in FI_O2 resulted in altered levels of PCr with exercise at the same constant submaximal intensity and 2) this effect was clearly not the result of onset kinetics.

O₂ onset kinetics. Numerous studies have documented that, at submaximal work intensity, O₂ is independent of the FI_O2 breathed (13, 15, 18, 21-23, 30, 31). The altered cellular energy states at varied FI_O2 observed in this study occurred at the same submaximal work intensity and, therefore, by assumption, the same O₂. Previous investigators (16, 23) have suggested that any metabolic changes with altered FI_O2 may be the result of differences in O₂ onset kinetics, i.e., the time required for the muscle to achieve a steady state of respiration. It has been demonstrated that breathing low FI_O2 results in slower onset times to steady state and that faster kinetics occur with the breathing of higher than normal FI_O2 (23, 24). In addition, it was recently proposed that the time required to achieve a steady-state O₂ may influence the metabolic state of the cell by setting the subsequent levels of the cytosolic signals driving mitochondrial recruitment (11). A longer time to reach steady state with reduced FI_O2 would result in a higher level of substrate-level phosphorylation, resulting in increased lactate production and greater PCr hydrolysis. The reverse would be true with the breathing of a hyperoxic gas mixture. This study tested the hypothesis that any changes in intracellular metabolic processes are due to some direct effect of breathing-varied FI_O2 and not to alterations in the time taken to achieve a new steady-state O₂ after the onset of exercise. The experimental design was such that by "quick switching" the FI_O2 after 5 min of steady-state exercise, the effect of FI_O2 could be studied independent of onset kinetics. Thus, because PCr hydrolysis was significantly different after the FI_O2 switch, these data clearly show that onset kinetics do not explain the altered levels of PCr with varied FI_O2.

FI_O2 and muscle PCr. The data presented in Fig. 1 and summarized in Table 1 show the different levels of PCr reached while the same work rate is maintained in response to altered FI_O2. In Fig. 3, the overlaid plots of Hypo-Hyper and Hyper-Hypo showing the switches between hypoxia and hyperoxia suggest that PCr levels during steady-state exercise may have been influenced by arterial O₂ saturation. When the FI_O2 of 0.1 was second in treatment order, the saturation did not fall to the same extent as it did when the FI_O2 of 0.1 was first, thereby resulting in different levels of PCr at the same FI_O2 (Table 1, Fig. 3). By the end of the exercise period in Hyper-Hypo, the PCr levels appeared to have reached a new steady-state level, reflecting the higher arterial O₂ saturation of 77%. The PCr levels were the same in hyperoxia regardless of whether the FI_O2 of 1.00 was first or second in treatment order. Hyperoxic conditions, such as those encountered in this study (100% O₂), will fully saturate arterial blood, which results in an increase of only ~3% over normoxia but will give rise to arterial PO₂ values of ~650 Torr (an almost 6-fold increase over normoxia). The effects of these differences in arterial PO₂, caused by the varied FI_O2, on myocyte energetics during exercise are shown in Figs. 4 and 5. When the data from Hypo-Norm are overlaid with the control data, it can be seen that the PCr levels rise to similar levels as those obtained in the control setting after the FI_O2 switch from hypoxia to normoxia (Fig. 4, Table 1). In comparison, when the data from Hypo-Hyper are overlayed with the control data, the PCr levels begin below and tend to rise above the control levels after the FI_O2 switch from hypoxia to hyperoxia (Fig. 5, Table 1). Additionally, the PCr level when hyperoxia was the first treatment fell to 72.6 ± 3.1% and was significantly higher compared with the 65.7 ± 4.3% PCr level for the control bout (P  0.01, Table 1). In this situation, arterial O₂ saturations, and thereby arterial [O₂], were similar, but arterial PO₂ was certainly higher. Accordingly, there was less PCr hydrolysis while the same rate of ATP turnover was maintained. This suggests that the higher arterial PO₂ in hyperoxia, even at nearly the same arterial saturation and [O₂], influenced the PCr level required for the same submaximal work rate.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   %PCr for control bout (normoxia; ) overlaid with Hypo-Norm (). After FIO2 switch to normoxia, level of PCr returns to control levels.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   %PCr for control bout (normoxia; ) overlayed with Hypo-Hyper (). After FIO2 switch to hyperoxia, level of PCr tends to rise above control levels.

O₂ tension and myocyte metabolism. At the end of the O₂ cascade, the O₂ required for oxidative phosphorylation diffuses from the capillary to the cytoplasm and finally to the mitochondria. This movement of O₂ from the blood to the mitochondria depends on a PO₂ difference between these two sites. The diffusion-induced pressure difference between the capillary and the cytoplasm is also dependent on the flux of O₂, determined by the rate of mitochondrial respiration. Previous intravascular-to-intracellular investigations of PO₂ have demonstrated that cellular PO₂ rises and falls with an increase or decrease in calculated mean capillary PO₂, which occurs when a hyperoxic or hypoxic gas mixture is breathed (31, 32). Richardson and colleagues (31), using proton MRS to detect myoglobin desaturation and hence estimate intracellular PO₂, have reported muscle intracellular PO₂ values of 3.1 and 2.1 Torr for subjects performing isolated quadriceps exercise while breathing normoxic and hypoxic (12% O₂) gases, respectively. These results were confirmed and extended in a more recent study in which intracellular PO₂ values of 2.6 ± 0.8, 3.2 ± 0.7, and 4.8 ± 2.0 Torr were measured for subjects exercising while breathing 12, 21, and 100% O₂, respectively (32). An interesting observation in those studies was that intracellular PO₂ was unaltered from submaximal to maximal exercise. Because exercise intensity has not been shown to affect intracellular PO₂, we can directly relate those previous findings to the present study, as the subjects were exercising at ~60% of maximum. On the basis of this previous work, it is almost certain that changes in FI_O2 and the subsequent changes in arterial PO₂ altered intracellular PO₂. We postulate that it was these alterations in intracellular PO₂ that resulted in altered levels of PCr with exercising at a steady state. As the oxymyoglobin dissociation curve is extremely steep at low PO₂, a small change in mean intracellular PO₂ at these low PO₂ values will cause large changes in myocyte [O₂], as most of the myocyte O₂ is myoglobin bound (38).

Modulation of respiratory regulation by O₂. It has been demonstrated that PCr hydrolysis is important not only for substrate-level phosphorylation but also as a signal for mitochondrial respiration (7, 25, 29). The rate of ATP production in the mitochondria is tightly coupled to the utilization of ATP by the myofibrils in the cytoplasm. Thus the signal that couples ATP hydrolysis to its production must travel from the point of utilization to the mitochondria, where oxidative phosphorylation occurs. It has been suggested that, rather than ADP and P_i diffusing between the sites of utilization and production, PCr acts as a shuttle of high-energy phosphate (2, 19). Creatine kinase is present in the cytosol close to myosin ATPase and in the mitochondria (2, 19, 20, 33). At the ATPase in the cytosol, PCr serves as the phosphate donor restoring ADP to ATP, locally generating the ATP utilized in contraction. Creatine recycles back to the mitochondria and is phosphorylated back to PCr and thus may influence the control of oxidative phosphorylation. In this way, PCr is a specific carrier of high-energy phosphate, whereas creatine serves as the primary phosphate acceptor. PCr then would serve as a signal linking ATP utilization in the cytosol to production in the mitochondria. The data presented in the present study show PCr levels rising or falling with increased or decreased FI_O2, suggesting that O₂ is modulating one of the proposed regulators of tissue respiration during steady-state exercise, even when the global O₂ availability is well above that considered critical for tissue respiration (8). Whereas at submaximal work rates these metabolic adjustments may have minimal impact on cell function, the differences in PCr hydrolysis resulting from the differences in cell oxygenation may profoundly influence cell function at higher work rates as both glycolysis and contractility are affected by [P_i].