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CELLULAR METABOLISM
Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands
Submitted 18 January 2007 ; accepted in final form 21 March 2007
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCr) has been used as a measure of mitochondrial function. However, cytosolic pH has a strong influence on the kinetics of PCr recovery, and it has been suggested that PCr should be normalized for end-exercise pH. A general correction can only be applied if there are no intersubject differences in the pH dependence of PCr. We investigated the pH dependence of PCr on a subject-by-subject basis. Furthermore, we determined the kinetics of proton efflux at the start of recovery. Intracellular acidosis slowed PCr recovery, and the pH dependence of PCr differed among subjects, ranging from –33.0 to –75.3 s/pH unit. The slope of the relation between PCr and end-exercise pH was positively correlated with both the proton efflux rate and the apparent proton efflux rate constant, indicating that subjects with a smaller pH dependence of PCr have a higher proton efflux rate. Our study implies that simply correcting PCr for end-exercise pH is not adequate, in particular when comparing patients and control subjects, because certain disorders are characterized by altered proton efflux from muscle fibers. 31P magnetic resonance spectroscopy; skeletal muscle; oxidative capacity; mitochondrial function; intracellular pH
PCr) provides information about mitochondrial function. This technology was very recently applied to study in vivo mitochondrial function in patients with Type 2 diabetes (46). Several studies have shown that cytosolic pH has a strong influence on the kinetics of PCr recovery (1, 2, 5, 8, 21, 31, 34, 44, 49, 51, 56). The slower PCr recovery in the presence of intracellular acidosis could reflect a decreased mitochondrial respiration at low pH. However, there are conflicting data about the effects of low pH on respiratory rates, ranging from inhibition (17, 22, 54) to a very small or no significant effect (10, 36, 38, 52, 56, 57) to even an increased effectiveness (15, 16). PCr recovery in the presence of intracellular acidosis could also be slowed down because of factors downstream of oxidative phosphorylation, i.e., increased ATP consumption by cellular ion pumps (4, 7, 29, 44) and/or a pH-dependent shift in the creatine kinase (CK) equilibrium (19, 30).
As an alternative to PCr, the time constant of ADP recovery (ADP) can be used to assess oxidative capacity. The concentration of ADP is the principal error signal in the feedback loop controlling mitochondrial oxidation, and therefore ADP recovery is one of the most sensitive MRS indices of mitochondrial function (23). Finally, the maximum aerobic capacity (Qmax), which can be calculated from the 31P MRS recovery data, provides a parameter for mitochondrial function. Both ADP (1–3, 8, 31) and Qmax (31, 44, 56) have been shown to be independent of end-exercise pH. A drawback of the use of these parameters compared with PCr is that they are indirectly derived from the PCr recovery data, with a number of assumptions.
It has been suggested that PCr can be normalized for end-exercise pH (5, 31, 44). However, a general correction for pH can only be applied if there are no intersubject differences in the pH dependence of PCr recovery kinetics. In previous studies, data of different subjects have been grouped to investigate the effect of pH on PCr recovery (1, 2, 5, 8, 21, 31, 34, 44, 49, 51, 56). We investigated the effect of acidosis on PCr in the vastus lateralis muscle on a subject-by-subject basis. To this end, each subject performed 10–13 exercise protocols of different intensity to reach different levels of acidification. Furthermore, we studied the pH dependence of ADP and Qmax and we determined the kinetics of proton efflux at the start of recovery to obtain a measure of the rate of pH recovery.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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31P magnetic resonance spectroscopy. 31P MRS of the vastus lateralis was performed by using a 1.5-T whole body scanner (Gyroscan S15/ACS, Philips Medical Systems, Best, The Netherlands). Subjects were measured in a supine position. After transversal and sagittal scout images were collected, the magnetic field homogeneity was optimized by localized shimming on the proton signal with the body coil. The 31P signals were collected with a 6-cm-diameter surface coil placed over the vastus lateralis. From the dimension of the coil and the size and geometry of a typical upper leg, it was estimated that the majority of the signal in the unlocalized 31P MRS measurements originated from the vastus lateralis, with minimal contaminations from the adjacent rectus femoris and underlying vastus intermedius. Data were acquired after a 90° adiabatic excitation pulse with a sweep width of 2 kHz and 1,024 data points. Spectra were acquired with a repetition time of 3 s during a rest-exercise-recovery protocol (2 scans/spectrum yielding a time resolution of 6 s; total of 150 spectra/15 min). The first 20 spectra (2 min) were measured at rest, after which the subjects started the exercise (see below). The duration of the exercise varied per subject but never exceeded 9 min, so that at least 4 min of recovery was recorded.
Exercise protocol inside magnet. All subjects performed a single leg extension exercise that has been shown to be limited to the four muscles of the quadriceps (41) in the supine position inside the magnet. The exercise was conducted by rhythmically lifting a lever (resting on the lower leg, proximal of the foot) connected to an ergometer. The upper leg was supported with the hip joint in a 30° anteflexed position and immobilized with two 3-cm-wide Velcro straps. One contraction was performed every 1.5 s, acoustically guided by a digital metronome. The initial workload varied per subject and ranged between 7.5 and 12.5 W. This level was maintained for the first minute, and the workload was then increased by 5 W each minute. To achieve different levels of metabolic activation, and hence different degrees of cytosolic acidification, subjects performed exercises of different durations. Each subject performed 10–13 different protocols during 4–9 different sessions in a randomized order, with at least 15-min rest between different protocols within 1 session. The position of the 31P surface coil was marked on the leg during the first session, and the coil was placed at the same location during the next sessions.
The reproducibility of the 31P MRS measurements was determined in one subject. This subject performed the same protocol 10 times during 5 different sessions. In one other subject, we tested whether the position of the 31P surface coil on the vastus lateralis, when varied in the proximal and distal direction, influenced the 31P MRS measurements. For this purpose, the subject performed the same protocol 5 times, with a maximal difference of 15 cm of the position of the 31P surface coil in the proximo-distal direction.
Data analysis. Spectra were fitted in the time domain by using a nonlinear least-squares algorithm (AMARES) in the jMRUI software package (53). PCr, Pi, and ATP signals were fitted to Lorentzian line shapes. The three ATP peaks were fitted as two doublets and one triplet, with equal amplitudes and line widths and prior knowledge for the J-coupling constant (17 Hz). For the time series, the PCr line width during recovery was constrained to the average PCr line width during recovery (excluding the first 10 data points), obtained from a prior, unconstrained fit.
Absolute concentrations of the phosphorylated metabolites were calculated after correction for partial saturation and assuming that the ATP concentration ([ATP]) is 8.2 mM at rest (51). Intracellular pH was calculated from the chemical shift difference between the Pi and PCr resonances (
; measured in parts per million) with the following formula (50):
 | (1) |
 | (2) |
Recoveries of PCr and ADP were fitted to monoexponential functions with Matlab (version 6.1; Mathworks, Natick, MA). Results are expressed as the metabolite's time constant of recovery, i.e., PCr and ADP.
Calculation of the initial rate of PCr recovery (VPCr) was based on the PCr recovery rate (1/PCr) and the difference between the resting and end-exercise [PCr] (
PCr) (26):
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The calculation of the maximum aerobic capacity (Qmax) was based either on the ADP-control model (12), in which VPCr has a hyperbolic dependence on the end-exercise [ADP] ([ADP]end) according to Michaelis-Menten kinetics with a Km of 30 µM (26):
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The proton efflux rate at the start of recovery was calculated as described by Kemp et al. (24, 27) from the changes in [PCr] and pH during the first 12 s of recovery according to
 | (6) |
is the amount of protons consumed per mole of PCr hydrolysis { = 1/[1 + 10(pH–6.75)]; Ref. 58}, m is the number of protons produced per mole of oxidative ATP synthesis {m = 0.16/[1 + 10(6.1–pH)]; Ref. 32}, and 
is the cytosolic buffering capacity [20 slykes, i.e., mmol·l–1·pH–1, plus the calculated contribution of Pi, which is given by 2.3[Pi]·(1 – ) (42)]. The apparent proton efflux rate constant was calculated as 
= E/(–pH), where pH is defined as pH – pHrest (23). Both the proton efflux rate E and the apparent proton efflux rate constant at the start of recovery depend on the end-exercise pH (pHend) (27), and therefore only the data sets with a pHend between 6.6 and 6.8 were used to calculate an average value for E and for each subject. These data sets with rather low pHend values were chosen for two reasons: 1) they had a greater PCr depletion and therefore a larger PCr resynthesis rate and proton production rate at the start of recovery, and 2) they had a greater increase in Pi and therefore a more visible Pi peak at the start of recovery, which is important for an accurate and precise pH determination. Statistics. Data are expressed as means (SD). Reproducibility is reported as the coefficient of variation [CV = (SD/mean) x 100]. Linear regression analyses were performed with the SPSS 14.0 software package (SPSS, Chicago, IL). The level of statistical significance was set at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCr, ADP, VPCr, and Qmax calculated according to the ADP-control model (Qmax-ADP) and calculated by a linear approximation of the ADP-control model (Qmax-lin), was determined in one subject, and the results are shown in Table 1. When all 10 measurements were included, the CV ranged from 6.7% for ADP to 11.3% for PCr. However, even though the exercise protocol was identical for all 10 measurements, there was still some variation in pHend. For 7 of the 10 data sets pHend ranged between 6.91 and 6.96 [mean pHend 6.93 (SD 0.02)], while for 3 data sets pHend was lower than 6.9, i.e., 6.87, 6.84 and 6.81. When only the seven data sets with pHend above 6.9 were considered, the CV for PCr became 6.9%, which is comparable to other studies (29, 56). No systematic differences were observed for the two measurements performed during one session.
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PCr and ADP were 4.5% and 3.0%, respectively. Therefore, it can be concluded that within a certain range the exact positioning of the 31P surface coil does not affect the parameters for mitochondrial function and that regional variations in fiber type composition in the proximo-distal direction of the vastus lateralis are probably small.
End-exercise status.
To achieve different levels of metabolic activation, and hence different degrees of cytosolic acidification, subjects performed 10–13 exercises of different durations. For all measurements, homeostasis of ATP was maintained throughout the exercise protocol. None of the subjects showed a split Pi peak during exercise or recovery, and therefore acidosis was not extremely heterogeneous in the measured muscle tissue. The ranges of pHend, PCr, and [ADP]end reached for each subject are summarized in Table 2. The smallest range in pHend values was obtained for subject 4 and covered 0.3 pH units, while the largest range was obtained for subject 2 and covered 0.6 pH units. For subject 5, one protocol resulted in a rather low [ADP]end of 25 µM. For all other measurements, [ADP]end was well above the accepted Km value of 30 µM for oxidative ATP synthesis. In the top two rows of Fig. 3, PCr and [ADP]end are plotted as a function of pHend, from which it can be seen that PCr was negatively correlated with pHend for all three subjects, whereas [ADP]end was not significantly correlated with pHend.
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PCr, ADP, VPCr, Qmax-ADP, and Qmax-lin for all subjects. For each subject, there was a strong negative linear relationship between PCr and pHend. The third row of Fig. 3 shows the correlation between PCr and pHend for three of the subjects. Around pHend 7, PCr was very similar for these three subjects, but at lower pHend values PCr differed. Therefore, the pH dependence of PCr differed, with subject 1 showing the weakest pH dependence and subject 3 showing the strongest pH dependence. The results of the linear regression analyses for all subjects are shown in Table 4. The slope of the relation between PCr and pHend ranged from –33.0 to –75.3 s/pH unit. For five of the six subjects, PCr at pHend 7 calculated from the linear relation between PCr at pHend was very similar. Only the older subject, subject 6, had a longer PCr at pHend 7.
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ADP was again very similar, while subject 6 had a longer ADP (Table 3). In the fourth row of Fig. 3, ADP is plotted against pHend for three of the subjects. For subjects 2, 3, and 5, ADP did not depend on pHend (Table 4). However, for subjects 1, 4, and 6, ADP was significantly positively correlated with pHend (Table 4).
VPCr is a measure of the actual mitochondrial ATP synthesis rate, and according to the ADP-control model (12) VPCr has a hyperbolic dependence on [ADP]end (Eq. 4). For all but one measurement, [ADP]end was well above the accepted Km value of 30 µM for oxidative ATP synthesis, and therefore differences in [ADP]end would not have a large effect on VPCr. From Fig. 3, it can be seen that PCr and PCr vary in the same direction as a function of pHend. As a consequence, for five of the subjects, VPCr was independent of pHend (Table 4). Only for subject 2 a significant positive correlation between VPCr and pHend was found (Fig. 3, Table 4).
The average values of Qmax-ADP were smaller than the average values of Qmax-lin (Table 3). In accordance with the longer PCr (at pH 7) and ADP, subject 6 also showed smaller values for Qmax-ADP and Qmax-lin compared with the other subjects (Table 3). Qmax-ADP showed significant positive correlations with pHend for three of the six subjects (Fig. 3, Table 4). These are the subjects with the largest correlation coefficients for VPCr vs. pHend. For each subject, there was a strong positive linear relationship between Qmax-lin and pHend (Fig. 3, Table 4).
For subject 5, the proton efflux rate E and the apparent proton efflux rate constant could not be determined. For the other five subjects, the mean total cytosolic buffering capacity at the start of recovery amounted to 35 (SD 1) slykes, the mean proton efflux rate E was 16 (SD 3) mM/min, and the mean apparent proton efflux rate constant was 38 (SD 6) mM·min–1·pH unit–1. In Fig. 4, A and B, the slope of the relation between PCr and pHend is plotted against E and , respectively. The slope of the relation between PCr and pHend was positively correlated with both E (R = 0.91, P = 0.03) and (R = 0.96, P = 0.01).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCr and pHend, one or a few data points of different subjects have generally been grouped. However, this procedure will not reveal intersubject differences in the pH dependence of PCr, and, moreover, the effect of pH on PCr recovery might be exaggerated by a systematic bias. We investigated the effect of acidosis on PCr recovery on a subject-by-subject basis by collecting 10–13 data sets per subject, using protocols of different intensity and duration resulting in different degrees of cytosolic acidification. We showed that for each subject there is a strong negative linear relationship between PCr and pHend but the slopes are different for different subjects, ranging from –33.0 to –75.3 s/pH unit. This implies that no general formula can be applied to correct PCr for differences in pHend. Qualitatively and quantitatively, the results obtained when the data of the different subjects are grouped (Table 4) are in good agreement with data from the literature on the pH dependence of PCr recovery kinetics measured in different muscle types, revealing linear relationships between PCr and pHend (or the minimum pH reached during recovery, pHmin) with slopes ranging from roughly –20 to –90 s/pH unit (1, 8, 21, 31, 44, 49, 56). The slower PCr recovery in the presence of intracellular acidosis could reflect a decreased mitochondrial respiration at low pH. The mechanisms by which protons affect oxidative phosphorylation include 1) a direct effect on the mitochondria, i.e., a decreased oxidative capacity at low pH, or 2) an indirect effect, because a low pH decreases the ADP concentration through the constraints set by the CK equilibrium resulting in a lower signal for mitochondrial ATP supply. There are conflicting data about the effects of low pH on respiratory rates. Hypercapnic acidosis has been found to reduce the aerobic capacity of perfused cat soleus muscle by a factor of 3 (17). However, it is not clear whether this change was caused by acidosis per se or by some other effect of hypercapnic perfusion. Moreover, it has been shown that in skinned fibers from rat soleus muscle the rate of respiration is impaired by lactic acidosis and elevated [Pi] (54). Jubrias et al. (22) showed that intracellular acidosis inhibits oxidative phosphorylation in vivo in hand and lower limb muscle, and their results suggest that pH has a direct effect on mitochondrial function, because oxidative flux did not increase during exercise that generated acidosis despite a significant rise in [ADP]. In contrast, in vitro studies on isolated mitochondria suggest that the effect of acidosis on oxidative phosphorylation is very small (10, 36, 52, 57) and not significant in the range pH 6.5–7.5 (10). Likewise, an in vivo study of electrically stimulated rabbit muscle showed that CO2-induced acidosis (to pH 6.7) did not decrease the maximum aerobic capacity (38). Moreover, in human medial gastrocnemius muscle aerobic ATP synthesis rates were not lowered by acidosis (56). Other reports have suggested that mitochondrial respiration is even more effective at low pH (15, 16).
PCr recovery in the presence of intracellular acidosis could also be slowed down because of factors downstream of oxidative phosphorylation, consistent with the observation that the recovery of oxyhemoglobin saturation measured by near-infrared spectroscopy is not affected by acidosis (34). Ion pumping reactions also require ATP, and therefore not all the ATP that is synthesized oxidatively during recovery is available for the CK reaction. At low pH the amount of ATP that is shuttled to cellular ion pumps might be increased (29, 44) in order to reestablish pH homeostasis. It has been reported that ion pumping reactions can consume 
43% of the total ATP produced (4, 7). The slow PCr recovery at low pH has also been attributed to a pH-dependent shift in the CK equilibrium. The CK Keq depends on proton and metal ion concentrations (30). Iotti et al. (19) showed that at the end of muscular exercise Keq can increase even more than threefold compared with rest, because of a decrease in pH and an increase in the free Mg2+ concentration. Therefore, net PCr resynthesis throughout recovery behaves as a function of both intracellular pH and net ATP flux (2, 56). This was confirmed by a model for ATP production (according to the ADP-control model; see Eq. 4) and pH recovery, which reproduced the main features of recovery from exercise, including the feature that PCr recovery is slowed when the pH is low (48). Finally, with the incremental exercise protocol that we used, part of the pH dependence of PCr recovery could originate from selective fiber type recruitment, i.e., recruitment of mainly oxidative type I fibers (with short PCr) during the low exercise intensities with a high pHend and recruitment of relatively more type II fibers (with long PCr) during the higher exercise intensities with a low pHend.
The observed intersubject differences in the pH dependence of PCr are likely to reflect differences in the rate of pH recovery. Unfortunately, the recovery of pH could not be investigated, because the Pi peak consistently disappeared within the noise after 1 min of recovery (Fig. 1D) and for the exercises at higher intensities was not fully recovered by the end of the time series. This phenomenon has been reported before in the literature (1, 8, 21, 29, 43, 50) and has been attributed to sequestering of Pi inside the mitochondria, where it becomes "NMR invisible" (2, 20), or trapping of Pi into the glycogenolytic pathway during exercise leading to phosphomonoester production (6). The recovery of pH is much slower than PCr recovery (1, 2, 55, 56), and therefore it was attempted to increase the signal-to-noise ratio of the Pi peak by the summation of spectra during the recovery phase. However, even when four spectra were added, yielding a time resolution of 24 s, the position of the Pi peak could not be accurately determined.
The recovery of cytosolic pH to the resting value is a function of net proton efflux (50). Several mechanisms are responsible for proton efflux, such as sodium/proton exchange, sodium-dependent chloride/bicarbonate exchange, efflux of undissociated lactic acid, and outward proton-lactate cotransport. The change in proton concentration in the cell can be calculated from the change in pH multiplied by the cytosolic buffer capacity and equals the proton efflux rate minus the rate of proton generation by PCr resynthesis and aerobic ATP production (24). We calculated proton efflux rates E and apparent proton efflux rate constants at the start of recovery. Both E and are pH dependent (27), and therefore only the data sets with a pHend between 6.6 and 6.8 were used to calculate an average value for E and for each subject. The mean values that we found for the total cytosolic buffering capacity , E, and correspond with values reported by Kemp et al. (27), calculated for a similar pHend range with exactly the same formulas. The slope of the relation between PCr and pHend was positively correlated with both E (R = 0.91, P = 0.03) and (R = 0.96, P = 0.01), indicating that subjects with a smaller pH dependence of PCr have a higher proton efflux rate, most likely as a result of a better blood flow due to, e.g., an increased capillary density possibly related to the subject's fiber type composition. Higher proton efflux rates will lead to faster pH recovery, and therefore the observed correlations support our hypothesis that the intersubject differences in the pH dependence of PCr are caused by differences in the rate of pH recovery.
To overcome the problems of pH determination during recovery associated with the transient loss of Pi signal, Chen et al. (14) modeled the pH recovery based on the CK equilibrium by considering the transition from exercise to recovery as a step function input. The entire pH recovery was characterized by calculating the time required for pH recovery (tpHrec), and a strong linear correlation was observed between tpHrec and the half-time of PCr recovery in normal subjects (average pHend 6.7). This strong correlation corroborates the link between the PCr recovery rate and the overall pH recovery rate. Moreover, the large variation in tpHrec within normal subjects (tpHrec ranged from 2 to 18 min) implies that differences in the pH dependence of PCr can be significant, as we demonstrated in the present study. Certain disorders, e.g., hypertension and mitochondrial myopathy (24) and dermatomyositis and polymyositis (9), are associated with altered proton efflux from muscle fibers, which will affect the rate of pH recovery. Therefore, when comparing PCr recovery measurements between patients and control subjects, differences in proton efflux rates or rates of pH recovery should be considered, as these might lead to systematic changes of PCr, in particular when pHend is low.
As an alternative to PCr, the kinetics of ADP recovery can be used to assess oxidative capacity. Because [ADP] is the principal error signal in the feedback loop controlling mitochondrial oxidation, ADP recovery is one of the most sensitive MRS indices of mitochondrial function (23). It has been shown that, in contrast to PCr, ADP is independent of pHend (1–3, 8, 31), which was confirmed by a theoretical model (48). We investigated the pH dependence of ADP for each subject. For three subjects ADP was not significantly correlated with pHend, while for the other three subjects ADP was positively correlated with pHend, i.e., ADP recovery became faster at low pH. This phenomenon was also observed by Larson-Meyer et al. (29), but we doubt that it has any physiological meaning. It has been reported that the recovery of ADP is not always monoexponential and that [ADP] can decrease below the resting concentration during the second minute of recovery (1). The size and duration of this so-called ADP undershoot were found to correlate with pHmin (1). We also observed an ADP undershoot in some of our data sets. However, it was difficult to quantify this effect, because the undershoot occurs in the period during which the Pi peak becomes invisible, resulting in a less reliable pH estimation. When the ADP recovery data with an undershoot are fitted with a monoexponential function, the time constant will be underestimated (13), and this could explain the positive correlation between ADP and pHend. Furthermore, it was assumed that the CK Keq was not affected by the different metabolic conditions present after exercise, and therefore changes in pH and the free Mg2+ concentration (18), in particular for the exercises at higher intensities, are sources of error for the [ADP] calculation that might lead to deviations in ADP.
VPCr is a measure of the actual mitochondrial ATP synthesis rate and therefore does not represent an absolute measure of oxidative capacity. Still, a number of studies have reported that VPCr is independent of pH (31, 44, 56). This is a consequence of [ADP]end being either similar for different degrees of acidification (44) or well above the accepted Km value of 30 µM for oxidative ATP synthesis (31, 56). The latter was also the case for most of our measurements, and for five of the subjects VPCr was independent of pHend. VPCr can be calculated from the product of 1/PCr and PCr (Eq. 3) (26, 31, 44), as in our study, or VPCr can be measured directly from the first data points (typically 10–14 s) (8, 29, 56). Boska et al. (8) applied both methods and found good correlations between calculated and measured VPCr in control subjects (R = 0.753) and patients with peripheral vascular occlusive disease (R = 0.646). However, Walter et al. (56) observed that at low pH (pHend 6.45) the calculated VPCr was about two times smaller than the measured VPCr. Under conditions in which intracellular pH is decreased, any model that relies on PCr is no longer valid and VPCr should be measured directly from the initial phase of recovery. However, this method is extremely sensitive to sampling rate and signal-to-noise values (56). Moreover, it has been shown that VPCr measured from the first 10 s of recovery is underestimated by up to 56% and that a 1- to 2-s time window is needed for the determination of VPCr, requiring very high-time-resolution 31P MRS data (37). The fact that VPCr calculated from PCr was still independent of pHend in five of our subjects results from the fact that 1/PCr and PCr vary in opposite directions as a function of pHend (Fig. 3). For the sixth subject changes in these two quantities apparently did not compensate each other completely.
In the literature, Qmax-ADP has been found to be independent of pH (31, 44), although in the study of Walter et al. (56) only Qmax-ADP based on the measured VPCr was pH independent. In our study, Qmax-ADP was based on the calculated VPCr and [ADP]end (Eq. 4). Although VPCr and [ADP]end were not significantly correlated with pHend (except for 1 subject), these parameters tend to vary in opposite directions as a function of pHend (Fig. 3), resulting in significant positive correlations between Qmax-ADP and pHend for three of six subjects. For each subject, there was a strong positive linear relationship between Qmax-lin and pHend. Qmax-lin was calculated as the inverse of PCr multiplied by [PCr]rest (Eq. 5). Within one subject, [PCr]rest was more or less constant within the time span of the study and therefore Qmax-lin was equivalent to the inverse of PCr, showing an equally strong relationship with pHend. The slopes of the relation between Qmax-ADP and pHend were much smaller than for Qmax-lin, and thus at low pH the error is smaller for Qmax-ADP. However, in accordance with Walter et al. (56), our data show that none of the models that rely on PCr is reliable to predict Qmax in the presence of intracellular acidosis (56).
In conclusion, intracellular acidosis slowed PCr recovery, and the pH dependence of PCr differed among subjects, ranging from –33.0 to –75.3 s/pH unit. The effect of acidosis on PCr recovery kinetics after exercise correlated with the kinetics of proton efflux at the start of recovery, strongly indicating that the intersubject differences in the pH dependence of PCr reflect differences in the rate of pH recovery. Our study implies that simply correcting PCr for pHend with a general formula is not adequate, in particular when comparing patients and controls, because certain disorders are characterized by altered proton efflux from muscle fibers. Also, matching for pHend is not sufficient when subject groups systematically differ in proton efflux kinetics. Therefore, PCr can only be used as a measure of mitochondrial function when pHend is close to resting values. Avoiding a decrease in intracellular pH along with a sufficient drop in PCr to model PCr recovery may, however, be difficult in untrained subjects or patients (56). An exercise protocol that progressively increases work, like that used in the present study, has been reported to be successful in decreasing PCr without severe acidification as opposed to sustained-load exercise (22, 56). Indeed, we obtained data sets with pHend close to 7 with a drop in PCr of roughly 50%. Alternatively, one could use an exercise protocol of short duration (9 s) with very rapid contractions, which has the advantage that it is believed to simultaneously recruit all fibers (56), or a gated protocol in which the acquisition is gated to contractions of short duration without significant muscle acidification that are repeated in a steady state for as many times as necessary to obtain the desired signal-to-noise ratio (47). The kinetics of ADP recovery is independent of pHend. Disadvantages of using ADP as a measure of mitochondrial function are the complex time-dependent undershoot of ADP during recovery and the assumptions that have to be made to calculate [ADP] (1). Qmax can only be used when based on VPCr directly measured from the initial recovery data points. However, the reproducibility of the latter parameter is much lower than for PCr and ADP (56).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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, 3 phosphate groups of ATP.
