1Department of Musculoskeletal Science, 2Magnetic Resonance and Image Analysis Research Centre and 3Department of Medical Imaging, University of Liverpool, Liverpool, 4Renal Unit, Royal Liverpool University Hospital, Liverpool and 5Countess of Chester Hospital NHS Trust, Chester, UK
Correspondence and offprint requests to: G. J. Kemp, Department of Musculoskeletal Science, University of Liverpool, Liverpool L69 3GA, UK. Email: gkemp{at}liv.ac.uk.
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Abstract |
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Methods. We performed 31P-MRS/NIRS/MRI studies on the lateral gastrocnemius during isometric plantarflexion and recovery in 23 male patients on haemodialysis (age 2471 years; haemoglobin 9.914.2 g/dl; bicarbonate 1730 mmol/l; urea reduction ratio 5377%; parathyroid hormone 195 U/l) and 15 male controls (age 2971 years). To understand the relationships between calf CSA and body mass we also performed MRI only in a further six male patients and 18 male controls.
Results. In patients, exercise duration was 30±11% lower than in controls. Muscle CSA was lower by 26±5%, but contractile efficiency (force/CSA/ATP turnover) was normal. Slowing of post-exercise phosphocreatine (PCr) recovery implied a 22±5% defect in effective mitochondrial capacity. That PCr recovery was slow relative to NIRS recovery suggests that this is largely an intrinsic mitochondrial problem (not the result of impaired O2 supply), one which, furthermore, correlated with CSA. Urea reduction ratio showed a negative correlation with body mass and CSA, but none with PCr rate constant.
Conclusions. The relationships to urea reduction ratio reflect the effect of muscle mass on dialysis efficiency, rather than direct effects on muscle CSA or metabolism. The relationship between PCr recovery and calf CSA suggests a role for the mitochondrial defect, whatever its cause, in the development of muscle wasting, although a common cause (e.g. physical inactivity) for both abnormalities cannot be ruled out.
Keywords: haemodialysis; magnetic resonance spectroscopy; mitochondria; near-infrared spectroscopy
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Introduction |
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Simultaneous measurement of blood flow, muscle/capillary PO2, muscle O2 consumption and energy metabolism is not possible. The non-invasive technique of 31P-magnetic resonance spectroscopy (31P-MRS) has shown abnormalities in energy metabolism [36], but published interpretations have not always distinguished some possible implications. When muscle contracts, ATP use increases and temporary failure of (in this context, largely mitochondrial) ATP production to match this results in a fall in phosphocreatine (PCr) concentration [7]. An increased PCr fall during exercise is a commonly reported 31P-MRS abnormality in dialysed patients [36,8] and this has two possible mechanisms, which have never been separated clearly. One is increased ATP demand, which implies an increased initial rate of PCr fall [9]. This could be due to decreased contractile efficiency, perhaps resulting from altered fibre-type proportions or, in some experimental designs [5,6], simply from muscle atrophy [10]. The other cause of increased PCr depletion during exercise is defective mitochondrial ATP synthesis, which would not affect the initial rate of fall of PCr, but which would be expected to increase the (steady state) fall in PCr [9]. The importance of this distinction lies in the therapeutic implications: mitochondrial therapies, such as carnitine and coenzyme Q, may not help if normal mitochondrial function is overstressed by increased ATP demand. Evidence for a mitochondrial defect is the slow post-exercise PCr recovery seen in some [3,5,6,8], but not all [4,8], studies of dialysed patients. PCr recovery depends both on intrinsic mitochondrial function and, contrary to what is sometimes assumed [4], on vascular O2 supply [9]. Both of these may be abnormal in dialysed patients: invasive methods have shown that intramuscular O2 transport is impaired [2] and possible causes of intrinsic mitochondrial dysfunction include detraining, nutritional deficiencies and damage by reactive oxygen species (ROS).To distinguish mitochondrial lesions (i.e. in O2 usage) and vascular lesions (O2 supply) we use the non-invasive technique of near-infrared spectroscopy (NIRS): an O2-supply defect would be favoured by observation of slow post-exercise reoxygenation, e.g. in peripheral vascular disease (PVD) [11]. The only previous study in haemodialysed patients showed no obvious abnormality [12]. However, there certainly are O2-supply problems, e.g. reduced capillary O2 conductance [2,8] and, of course, anaemia, which is now better treated than at the time of earlier 31P-MRS studies [36]. Only when these distinctions are made can we establish the relationships between muscle wasting and mitochondrial metabolic defects that are expected if, for example, both result from detraining [13].
We combined 31P-MRS, magnetic resonance imaging (MRI) and NIRS to examine this in more detail. Our aims were to define the muscle metabolic abnormalities in contemporary dialysed patients, to establish the possible contributions of reduced muscle O2 supply and intrinsic mitochondrial dysfunction (i.e. ATP supply) and alterations in contractile efficiency (i.e. ATP demand), and to define the relationship to muscle wasting.
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Subjects and methods |
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Methods
These have been described elsewhere [9,11]. 31P-MRS was performed in a SIGNA Advantage 1.5 tesla whole body MR system (General Electric, Milwaukee, WI, USA) using a 10±6 cm elliptical surface coil over the lateral gastrocnemius, with simultaneous NIRS using a RunMan CWS 2000 (NIM Inc., Philadelphia, PA, USA). An axial T1-weighted spin echo MR image (10 mm slices with 5 mm gaps) was obtained for measuring CSA. Muscle was studied at rest and during 35 min voluntary 0.5 Hz isometric plantarflexion at 50% and then at 75% maximum voluntary force (MVC), each followed by 5 min recovery. If exercise ended early because of fatigue, recovery collection started early, but the next exercise started at the normal time. The CSA of the posterior calf (soleus + gastrocnemius) was measured in the slice at which it was maximal by point-counting using ANALYZE software (Mayo Foundation, Rochester MN, USA). 31P-MRS data were analysed using Magnetic Resonance User Interface software (MRUI 97.1). Cell pH was obtained from the chemical shift of inorganic phosphate Pi relative to PCr. Changes in PCr and derived quantities were expressed relative to resting PCr concentration, which did not differ significantly between the groups.
In this protocol there is little pH change and glycolytic ATP production can usually be ignored. The key 31P-MRS measurements are of PCr kinetics, which are fitted to exponential functions. The analysis is described in detail elsewhere [7,9,11] and will be summarized briefly here. In general (see Figure 2), ATP turnover = glycolytic ATP synthesis rate + oxidative ATP synthesis rate + rate of PCr splitting. Very early in exercise the first two are negligible, so the initial rate of PCr splitting measures ATP turnover rate (F). This is proportional to force/CSA, the slope being the contractile efficiency. Subsequently, PCr splitting slows as oxidative and/or glycolytic ATP synthesis increase and a steady state may be reached. In the largely aerobic constant-power exercise used here, PCr kinetics are monoexponential. The change in PCr ([PCr]) is both an overall measure of how far ATP synthesis has failed to meet ATP demand and also a correlate of the changes (in e.g. ADP) that are important in the feedback stimulation of oxidative ATP synthesis; the extrapolated steady-state PCr change (
[PCr]SS) is related to ATP turnover rate and mitochondrial function. The initial post-exercise PCr resynthesis rate (V) measures the end-exercise oxidative ATP synthesis rate and the rate constant of PCr recovery (= 0.693/halftime = V/
[PCr]) is proportional to the capacity for oxidative ATP synthesis (i.e. the notional rate at complete PCr depletion). Initial post-exercise PCr resynthesis rate = end-exercise oxidative ATP synthesis rate and this declines with the same halftime as PCr. Making a small correction for end-exercise PCr splitting, total ATP turnover rate minus end-exercise oxidative ATP synthesis rate (i.e. F V) gives end-exercise glycolytic ATP synthesis rate, which is always small here. The NIRS signal represents the difference in absorption at two wavelengths, proportional to deoxygenated haemoglobin + myoglobin content [14], normalized for instrument gain. The key measurement is the rate constant of post-exercise NIRS recovery (= 0.693/halftime), which reflects the extent to which O2 supply exceeds demand in recovering muscle [11]. To simplify the presentation, results from the two exercise intensities (50% and 75% MVC) were combined: recovery rate constants did not differ significantly and are presented as means; initial PCr depletion rates were constrained in the ratio 75:50 and a single 50% MVC value presented; exercise duration and all-exercise changes in PCr and NIRS were combined as a mean weighted 75:50 [9,11].
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Results |
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Muscle metabolism: fluxes
Neither ATP turnover rate nor the contractile cost was significantly abnormal in patients (Figure 1B). The end-exercise oxidative fraction at 50% and at 75% MVC was not significantly different from unity in controls, implying that glycolytic ATP production was negligible. In patients it was significantly less than unity at both 50% MVC and 75% MVC (Table 1 gives a mean). Figure 2B shows how estimated oxidative ATP synthesis rate was reduced in patients throughout exercise at 50% MVC, the shortfall being made up by extra PCr depletion and glycolytic ATP production. Glycolysis, however, was not enough to produce significant acidification: pH changes during exercise were <0.15 units in controls and not significantly abnormal in patients (data not shown). PCr recovery was slowed in patients, the rate constant being reduced (Table 1). The end-exercise oxidative fraction correlated (P = 0.007) with the PCr recovery rate constant, as expected if the reduced oxidative contribution during exercise is mainly due to the defect in mitochondrial function, which is measurable during recovery, rather than to increased ATP demand.
Muscle metabolism and muscle deoxygenation
NIRS showed the expected pattern of deoxygenation during exercise followed by recovery: the exercise changes were not significantly abnormal in patients. The NIRS recovery rate constant was not significantly abnormal overall. However, there was a correlation between the PCr and NIRS rate constants in both groups (Figure 3A): the slopes not being significantly different, the significant vertical difference between the two lines measures the amount by which PCr recovery was slower than expected for given a NIRS recovery rate constant. Figure 3B shows a relationship in patients (not control subjects) between the PCr rate constant and calf CSA expressed relative to height squared. A similar correlation is seen with a number of other measures of CSA, as well as CSA itself (P = 0.008).
Relationships to plasma biochemistry and haematology
URR correlated inversely with body mass (Figure 4A) and with weight/height (both P<0.007), but not with either PCr rate constant or excess rate constant (Figure 4B). Furthermore, when the patient group is divided into two on the basis of URR <65% and 65%, the abnormality in the PCr rate constant is not significantly different between these two groups; the reduction compared with control being 24±6% and 33±7%, respectively (both significant, P<0.002). URR showed a negative correlation with both calf CSA and body mass (Figure 4A) and with CSA/height2 (P = 0.02; data not shown), but not with CSA/weight2/3 (P = 0.2; data not shown). Plasma Pi correlated positively with body mass (P = 0.04) and calf CSA (P = 0.03) and negatively with URR (P = 0.03). There were no other significant correlations with biochemical plasma measurements or with haemoglobin.
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Discussion |
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Abnormalities in muscle
First, we wished to establish the muscle abnormalities in our patients. We found, as expected, slow PCr recovery, which suggests a functional defect of mitochondrial ATP synthesis and, consistent with this [7], a significant increase in the steady-state PCr change. Unlike in previous studies [36,8], this analysis is not complicated by a significant glycolytic contribution and the consequent pH change [8]. ATP demand was normal and, accordingly, the reduced oxidative contribution during exercise is due to the mitochondrial defect measurable during recovery. We also found evidence of muscle wasting, assuming the controls represent the patients pre-morbid state.
It is of interest to compare our results with those of invasive studies of maximal muscle O2 consumption VO2max [2,15]. To a first approximation, the PCr recovery rate constant is proportional to maximum O2 consumption [7,16]. Direct arteriovenous-differences measurements of muscle VO2max are usually expressed per leg, or exercising muscle, and so the appropriate comparison is with the product of PCr recovery rate constant and muscle CSA: this is significantly reduced, by 50%, a similar abnormality to that seen in VO2max in quadriceps in young patients in [2] and larger than in [15].
Contributions to mitochondrial dysfunction
Second, we wished to estimate the contributions to abnormalities in exercising muscle of reduced muscle O2 supply, intrinsic mitochondrial dysfunction (i.e. ATP supply) and alterations in contractile efficiency (i.e. ATP demand). Compared with controls, the patients exert less force because they have less muscle; contractile cost is normal and so is the ATP demand. There is a relative failure of oxidative ATP production during exercise, due to the reduced capacity for oxidative synthesis inferred from the slow PCr recovery [7]. This depends on actual mitochondrial content, intrinsic mitochondrial function and vascular O2 delivery, any of which might be abnormal.
A candidate cause for impaired O2 delivery is anaemia. The results of a comparison with the literature are suggestive: the mitochondrial abnormality of 30% seen in the present study (Table 1) and others where mean haemoglobin was 1012 g/dl [12,15] is smaller than abnormalities of 4050% seen in earlier studies [3,5,6] where mean haemoglobin was 78 g/dl (calculations based [7,9] on published data). Against this, pre- and post-Epo studies conducted over periods of a few months showed little improvement in mitochondrial function measured by 31P-MRS [6] or invasively [8], while haemoglobin increased from 78 to 12 g/dl. Also, Epo treatment lowers peak blood flow and does not normalize the reduced O2 conductance [2]. Recent work has shown that elevating haemoglobin from 10 to 14 g/dl does improve various measures of exercise performance [1]. Nevertheless, in the present work there was no correlation between mitochondrial function and haemoglobin. It is, therefore, likely that there is no direct influence on muscle mitochondrial function over this range.
Another possible cause of impaired O2 delivery is the reduced muscle O2 conductance in patients on haemodialysis [2]. We tested this using NIRS to report cellular/capillary deoxygenation. Unlike in PVD, where the lesion is straightforwardly in blood supply [11], the absolute change in NIRS was normal. This currently cannot be interpreted in any more detail because of the current debate about the source of the NIRS signal: intracellular deoxymyoglobin [17] or intracapillary deoxyhaemoglobin [14]. However, the recovery kinetics of NIRS should be largely independent of this. The overall normal NIRS recovery shows that, unlike in PVD [11], there is no gross defect of O2 supply. NIRS recovers because vascular O2 supply, which we do not measure non-invasively, exceeds O2 use, which is declining during recovery (Figure 2B). Given that PCr resynthesis can be limited by O2 supply [16], theory and simulation predict the observed correlation between the rate constants of PCr and NIRS recovery (Figure 3A), which allows us to control for the O2 supply by means of a joint linear regression. That PCr recovery is slow for a given NIRS recovery suggests an oxidative defect independent of O2 supply.
The implication of the recent finding that O2 supplementation normalizes VO2max [15] is that the entire mitochondrial defect is secondary to impaired O2 supply. However, there must be a difference between the patients in our studies and those patients, because O2 supplementation could normalize whole-leg O2 consumption in the presence of reduced muscle mass. Comparison is made difficult by the differences in the muscle studied and because that protocol gives highly pH-dependent PCr recovery rate constants [8] (see above).
The older literature on muscle content and activity of mitochondria in dialysed patients is confused [18,19]. A recent finding of normal mitochondrial content and activity [20] has been taken as evidence that the oxidative defect is all in muscle O2 supply, although it might also be related to a difference in the studied populations, or a defect in mitochondrial control in vivo. A mitochondrial defect could be related to fibre-type change; however, the relative type 1 (fast) fibre atrophy sometimes reported [10] would tend to have the opposite effect. Furthermore, in quadriceps muscle of 10 patients on haemodialysis and 10 control subjects we have found no difference in the proportion of type 1 vs type 2 fibres, but there is a significant decrease relative to control in the mean diameter of both fibre types (A.V.Crowe, unpublished data).
Muscle metabolism and muscle atrophy
Thirdly, we wished to establish the relationship of metabolic abnormalities to muscle atrophy. Muscle wasting is a known feature of chronic renal failure [10] and many mechanisms have been proposed. As muscle makes substantial contribution to body weight, an appropriate comparison is not entirely straightforward, but several methods agree in finding a reduced calf muscle CSA in these patients, while the ability to generate force was unchanged. Figure 3B shows that the metabolic component of the PCr recovery defect (i.e., as we argue, the intrinsic mitochondrial defect) correlates with the abnormality in CSA. This could, of course, mean that both are consequences of some other factor. Age can be provisionally ruled out, as neither quantity shows a significant age-dependence in this group. Detraining is a possible cause, as experimental disuse decreases both muscle CSA and mitochondrial content [13] and it is known that haemodialysed patients are less active than even sedentary controls [21]. Another possible link is damage by ROS: mitochondria [22] are damaged by ROS in dialysed patients and are themselves a source of ROS, and this might be important in disuse atrophy.
Even though these may be, at least in part, rather general features of chronic disease, defining their contributions may be useful. For example, the persistence of mitochondrial dysfunction might suggest that it is still worth testing mitochondrial therapies, such as carnitine and coenzyme Q. One would expect this to be one of the benefits of endurance-training.
Muscle metabolism, muscle atrophy and plasma biochemistry
Some other findings are of pathophysiological relevance. The range of URR seen here is fairly wide, with about half of the patients falling below the currently recommended minimum of 65% [23]. Although URR is not an entirely satisfactory measure of dialysis dose, in the present study it shows some interesting relationships. First, URR correlated inversely with body mass (Figure 4A) and with weight/height, as in large patient series [24], presumably because, for a given dialysis dose, the smaller the body mass the greater the reduction in urea [24]. We cannot be sure that more complete dialysis would not have resulted in smaller muscle metabolic abnormalities, although the lack of correlation between URR and mitochondrial function (Figure 4B) argues against this. Second, the negative correlation between URR and muscle CSA (Figure 4A) no doubt reflects the negative correlation between URR and body mass (Figure 4A) and remains even after correcting CSA for height2. However, the lack of correlation with CSA/weight2/3, which should control for the contribution of CSA to body mass, suggests that URR has no causal relationship to muscle wasting. URR also correlates negatively with plasma Pi, as one might expect [25], and this no doubt explains the negative correlations between plasma Pi and both calf CSA and body mass: all result from the influence of body mass on dialysis effectiveness [24].
In summary, we observed slow PCr recovery relative to NIRS recovery, suggesting an intrinsic mitochondrial defect rather than a result of impaired O2 supply. We also observed relationships to URR that reflect the effect of muscle mass on dialysis efficiency, but not direct effects of URR on muscle CSA or metabolism. We noted a relationship between PCr recovery and calf CSA, which suggests a role for the mitochondrial defect in the development of muscle wasting, although a common cause (e.g. physical inactivity) for both abnormalities cannot be ruled out.
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Acknowledgments |
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Conflict of interest statement. None declared.
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References |
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