Aerobic metabolism of human quadriceps muscle: in vivo data parallel measurements on isolated mitochondria

Ulla F. Rasmussen1, Hans N. Rasmussen1, Peter Krustrup2, Bjørn Quistorff3, Bengt Saltin4, and Jens Bangsbo2

1 Department of Biochemistry, August Krogh Institute, University of Copenhagen; 2 Copenhagen Muscle Research Center, Institute of Exercise and Sport Sciences, University of Copenhagen; 3 Nuclear Magnetic Resonance Center, Panum Institute, University of Copenhagen; and 4 Copenhagen Muscle Research Center, Rigshospitalet, Copenhagen DK-2100, Denmark


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to examine whether parameters of isolated mitochondria could account for the in vivo maximum oxygen uptake (VO2 max) of human skeletal muscle. VO2 max and work performance of the quadriceps muscle of six volunteers were measured in the knee extensor model (range 10-18 mmol O2 · min-1 · kg-1 at work rates of 22-32 W/kg). Mitochondria were isolated from the same muscle at rest. Strong correlations were obtained between VO2 max and a number of mitochondrial parameters (mitochondrial protein, cytochrome aa3, citrate synthase, and respiratory activities). The activities of citrate synthase, succinate dehydrogenase, and pyruvate dehydrogenase, measured in isolated mitochondria, corresponded to, respectively, 15, 3, and 1.1 times the rates calculated from VO2 max. The respiratory chain activity also appeared sufficient. Fully coupled in vitro respiration, which is limited by the rate of ATP synthesis, could account for, at most, 60% of the VO2 max. This might be due to systematic errors or to loose coupling of the mitochondrial respiration under intense exercise.

skeletal muscles; maximal oxygen uptake; work rate; respiration; adenosine triphosphate synthesis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

DURING STEADY-STATE WORK, most energy formation and oxygen consumption of the working muscle occur in the mitochondria. It is therefore of interest whether in vitro measurements on isolated mitochondria can account for the rates of oxygen uptake and work output measured in vivo. If so, isolated mitochondria may constitute an important model in the study of physiological and pathophysiological phenomena of human skeletal muscle energy metabolism.

Skeletal muscle is particularly well suited for studying energy metabolism, because the metabolic rate can be increased close to one hundred times between rest and work (19). Humans are well suited for such studies, because they are motivated and a considerable variation exists with respect to performance capability (28). The maximal oxygen uptake (VO2 max) of the human body is limited by the circulation and will be reached with exercise of only a limited part of the total muscle mass (14, 27). The one-legged knee extensor exercise model, in which exercise is confined to the quadriceps muscle, was developed to cope with the limitation in oxygen delivery to the working muscle (1, 2). In this model, the VO2 max of the active muscle has been measured and related to marker enzymes determined in biopsies taken from the same muscle (5). Estimation of in vivo rates of energy metabolism in a single muscle have also been made from nuclear magnetic resonance (NMR) measurements of creatine phosphate kinetics, e.g., on gastrocnemius muscle (16, 17). The in vivo measurements of VO2 max and rate of phosphocreatine formation are much higher than can be accounted for by previously determined respiratory rates of mitochondria isolated from human skeletal muscle (see surveys in Refs. 24, 33, 34). An explanation for this discrepancy might be loss of regulatory mechanisms in the mitochondria upon isolation, and models assuming multistep activation of the mitochondrial systems have been developed (Ref. 15 and references therein).

Isolation of intact mitochondria from skeletal muscle presents some problems, because the mitochondria are scarce and partially integrated in the myofibrillar structure and because the amount of tissue is limited when healthy volunteers are studied. A recently developed small-scale technique provides mitochondria with the highest specific activities reported and in yields close to 50% (21, 23-26).

The VO2 max determined in the knee extensor model is the highest value reported for an "isolated" muscle in vivo under defined work rate (1, 2, 5). It was, consequently, of interest to test whether the in vitro measured activities of the mitochondrial preparation could account for the in vivo measured VO2 max and work rate of the quadriceps muscle in the knee extensor model.

The present study shows strong correlations between in vitro and in vivo data. When extrapolated to in vivo metabolism from glycogen and cyclic operation of the tricarboxylic acid cycle, pyruvate dehydrogenase (PDH) and the other enzyme activities assayed in the isolated mitochondria will all account for the VO2 max. But the fully coupled in vitro respiration, which was observed to be limited by the rate of ATP synthesis, might account for, at most, 60% of the VO2 max.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In vivo measurements. Six healthy young men with different backgrounds of physical activity were tested in the one-legged knee extensor model, the technical details of which have been described previously (1, 2). In this model, power is produced by the knee extensor muscles, and the oxygen supply is not limited by cardiac output (2, 27). The subjects were familiarized with the experimental setup on separate occasions before the experiment. Furthermore, a number of preexperiments served to select the work intensity for the exercise so that the subject would be exhausted within 4 min (range 56-76 W). This criterion was shown in previous studies to lead to the maximal oxygen consumption of the contracting muscles (4, 13).

The knee extensor exercise was performed in two 3-min periods separated by 6 min of rest. Toward the end of each exercise period, leg blood flow was measured frequently, and blood was drawn three times from the femoral artery and vein. Contribution of anaerobic work was minimized by performing the measurements at the end of the 3-min exercise periods. In a similar protocol, the anaerobic energy turnover in the last phase of exercise could account for 10-20% of the work (13).

VO2 max was obtained as the product of the rate of blood flow obtained immediately before blood sampling and the femoral arteriovenous difference of oxygen concentration. Muscle mass was estimated by magnetic resonance imaging. These methods were essentially as described by Blomstrand et al. (5).

The study was approved by the ethics committees of the municipalities of Copenhagen and Frederiksberg. The volunteers were informed in accordance with the rules.

Mitochondrial preparation and yield estimation. The preparation method has been described in detail (21). A part of a single biopsy obtained at rest from vastus lateralis muscle was used, and the preparation was started within 30 s after the sampling. The major features of the preparation procedure were: 1) no dissection of the biopsy, 2) a brief proteinase treatment followed by dilution and removal of the proteinase, 3) homogenization in a very large volume, and 4) one low- and two high-speed centrifugations. The preparation media were conventional salt media: KCl (100 mM), Tris (50 mM), MgSO4 (5 mM), and EDTA (1 mM), pH 7.40, supplemented with ATP (1 mM) and bovine serum albumin (0.5% wt/vol) in the initial steps of the preparation, and further supplemented with Subtilisin A (0.2% wt/vol) for the proteinase treatment. The final pellet, which appeared completely homogeneous, was suspended in mannitol + sucrose (225 + 75 mM) to a protein concentration of ~5 mg/ml. Subtilisin A (~30 Anson units/g) was obtained from Novo Nordisk, Copenhagen, Denmark.

The yield of mitochondria was measured as citrate synthase (CS) activity in the mitochondrial suspension relative to that in the actual biopsy used for the preparation. The tissue homogenate was not sufficiently homogeneous to allow representative sampling for measurement of the tissue activity. This activity was distributed among the proteinase wash (a few percent) and the pellet plus supernatant of the first centrifugation. Because all transfer and sampling operations were quantitative, the tissue activity could be obtained by summing the activities of these fractions. An aliquot of the homogeneous supernatant was withdrawn and supplemented with Triton X-100 (0.1%) to effect liberation of the enzyme. Triton X-100 was also added to the entire pellet, which was then vigorously homogenized. Addition of Triton X-100 to the entire protease wash usually produced a homogeneous solution without homogenization. Aliquots of these solutions were assayed for CS at 25°C, as described by Shepherd and Garland (31).

Respiratory experiments and functional enzyme assays. Mitochondrial respiration was measured at 25°C in a miniature vessel of 36.5 µl volume (22). The assay medium was mannitol (225 mM), sucrose (75 mM), Tris (20 mM), phosphate (10 mM), and EDTA (0.5 mM) at pH 7.35. The oxygen concentration of this medium was measured to 234 µM O2 by two independent methods (25°C, 101.3 kPa, 100% humidity).

Respiration with different substrates was measured in separate experiments under nonphosphorylating and phosphorylating conditions (classical state 4-3-4 experiments) and under uncoupled conditions, obtained with 3,5-di-tert- butyl-4-hydroxybenzylidenemalononitrile (SF-6847, 28 nM). State 3 respiration was measured with ADP (175-350 µM) and optimized MgCl2 (0-1.3 mM). The substrates were used in the following concentrations: glutamate (4 mM), malate + glutamate (4 + 8 mM), palmitoyl-carnitine + malate (10 µM + 1 mM), pyruvate + malate (9 + 4 mM), succinate (12 mM + 1 µM rotenone), and succinate + glutamate (8 + 4 mM).

In some cases, the respiratory experiments could be used as so-called functional assays of particular enzymes, namely if the rate of respiration was limited by the enzyme activity in question and the oxygen-substrate stoichiometry was known. The essential features of a functional assay are that the reaction is realized in the mitochondria and that it occurs between physiological substrates. It may thus be expected that the activity measured in a functional assay applies more readily to the in vivo situation than that measured in an assay involving extraction and reaction in an entirely artificial system. Some examples may illustrate the concept (see Ref. 24 for more details).

Succinate dehydrogenase (SDH) was assayed by the respiratory rate with succinate (+rotenone), either in state 3 or uncoupled. Under these conditions, only the SDH reaction caused oxygen consumption, and this reaction exerted major flux control over respiration.

The functional assay of PDH was based on the respiratory rate with pyruvate and malate, in state 3 or uncoupled. Malate added alone was not respired by the mitochondria, because oxaloacetate could not be further metabolized. When pyruvate was also added, the rate of malate oxidation was controlled by the rate of formation of acetyl-CoA from pyruvate, i.e., one-half the oxygen consumption was used for malate oxidation and the other one-half for pyruvate oxidation. The balance was maintained, because the mitochondrial activities of malate dehydrogenase and CS were high, the content of CoA was low, and continuous recycling of this compound was necessary. The equilibrium constant at aconitase predicts that only ~4% of the citrate formed will be converted to isocitrate, i.e., the isocitrate causes virtually no respiration, as is also substantiated by the observation that the rate of pyruvate + malate respiration is constant until anaerobiosis. The PDH activity could therefore be calculated as one-half of the respiratory rate.

The total respiratory chain activity was measured as the rate of the rotenone-sensitive NADH oxidase in freeze-permeabilized mitochondria (650 µM NADH + 2 µM cytochrome c; 2 µM rotenone). The assay requires optimal permeabilization without damage to the system. Two freezing cycles at 77K at different protein concentrations were used in the present study. This procedure had worked with other kinds of skeletal muscle mitochondria (23, 26) but turned out to be problematic with quadriceps mitochondria. Comparison of the rates per mitochondrial protein in the present study with those obtained later with an improved permeabilization technique point to an underestimation of the mean value by as much as 30% (24, 25).

The rate of ATP synthesis was calculated from the state 3 rate of respiration and the P/O ratio, i.e., moles ATP produced per O-atom consumed, in the same experiment. It might be used as a functional assay of the phosphorylating system as described in RESULTS.

Tissue activities. Tissue activities were calculated from the rates of mitochondrial respiration or ATP synthesis, a dilution factor, the yield of mitochondria, the biopsy mass, and a temperature factor.

The temperature factor adjusted the rates measured at 25°C to the rate at the temperature of the working muscle (38°C). The factor used, 2.44, was calculated by the Arrhenius equation from data for human skeletal muscle mitochondria indicating a twofold increase of rate between 25 and 35°C (8). Data for horse muscle mitochondria would give a factor 1.23 times the one used (9), whereas data for rat muscle mitochondria might be interpreted as supporting the factor used (7). Reliable measurement of the temperature factor presents some problems, such as calibration of the oxygen electrode and lack of conformity with the Arrhenius equation. Our preliminary experiments with a novel method of measurement indicate that the factor used probably should be multiplied by 1.25.

Other in vitro measurements. Cytochrome aa3 (cyt.aa3) was determined by low-temperature spectroscopy with a modification (23) of the glycerol technique invented by Keilin and Hartree (12). Protein was analyzed with bovine serum albumin as standard according to Schaffner and Weissmann (30).

Statistics. The tables give mean values ± SD (n = 6). The correlation curves in Fig. 1 were calculated by regressions forced through origin, because the assumptions of proportionality were not rejected in normal regression (P = 0.05). The significance of correlation coefficients was tested with the t-test (18).


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Fig. 1.   Comparison of in vitro and in vivo parameters of the quadriceps muscle. For each subject, the in vitro parameters are plotted against the mean value of the 2 estimates of maximal O2 uptake (VO2 max). All data are expressed per kg tissue mass (wet wt). Dashed lines are calculated by regression, and solid lines indicate the theoretical correlations in case of balance between in vitro activities and VO2 max. The 6 in vitro parameters are (with values of r2 and P for the correlations) as follows. A: mitochondrial protein content (r2 = 0.94, P < 0.002). B: cytochrome aa3 (cyt.aa3) content (r2 = 0.87, P < 0.01). C: citrate synthase (CS) activity (slope = 4.9, r2 = 0.87, P < 0.01). D: succinate dehydrogenase (SDH) activity (slope = 1.0, r2 = 0.96, P < 0.001). E: pyruvate dehydrogenase (PDH) activity (slope = 0.38, r2 = 0.92, P < 0.01). F: state 3 rate of succinate + glutamate respiration (slope = 0.60, r2 = 0.92, P < 0.01).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects and mitochondrial preparations. In Tables 1-4, the subjects are ranked according to declining mitochondrial protein content in the muscle tissue. This ranking matched their physical activity, which ranged from long-distance running (subject C-2) to no regular activities (subjects B-4 and K-3). The numbers in the codes indicate the experimental chronology. Table 1 gives the personal data for the volunteers and the results from both determinations of VO2 max.

                              
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Table 1.   Anthropometric data and in vivo measurements on the six male subjects


                              
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Table 2.   Data for mitochondrial preparations and markers


                              
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Table 3.   In vitro respiratory activities in mmol O2 · min-1 · kg-1 at 38°C, referred to tissue mass


                              
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Table 4.   In vitro rates of ATP synthesis in mmol ATP · min-1 · kg-1 (38°C), referred to tissue mass

Table 2 shows data for the mitochondrial preparations and some markers. The yield amounted to as much as one-half of the tissue content, and it is unlikely that only a subpopulation of the mitochondria was studied. The tissue content of mitochondrial protein varied by a factor of 1.5 among the subjects. The contents of cyt.aa3 and CS varied in a similar fashion, the former being a marker of the inner mitochondrial membrane and the latter a marker of the mitochondrial matrix.

Respiratory capacity of isolated mitochondria referred to tissue. Table 3 shows the tissue activities calculated from the in vitro data. Except for the last two substrates, NADH and succinate + glutamate + SF-6847, all rates are state 3.

The state 3 rate of respiration with succinate + glutamate represented the highest rate that could be measured under phosphorylating conditions. The substrate reactions involved are catalyzed by SDH, fumarase, malate dehydrogenase, glutamate-oxaloacetate transaminase, and probably also glutamate dehydrogenase. Uncoupling caused a marked increase of the rate of respiration (Table 3). This indicates that it is the activity of the system for ATP synthesis that limits the coupled succinate + glutamate respiration. Neither the activities of the substrate reactions nor the activity of the respiratory chain limits the respiratory rate in this system.

In other cases, the rate of respiration was limited by the activity of the enzymes catalyzing the substrate reaction. This applies to, for instance, the succinate respiration, which therefore might be used as a functional assay of SDH as described in METHODS.

The suboptimal freeze-permeabilization in the NADH oxidase assay (see METHODS) is obvious from the data in Table 3. Two preparations (subjects B-4 and K-3) showed rates of uncoupled succinate + glutamate respiration that were higher than the NADH oxidase rates. Probably only the preparation from subject J-5 was optimally permeabilized, and the most likely mean activity would have been ~15 mmol O2 · min-1 · kg-1 (cf. 25). This value is slightly above the mean value of VO2 max (Table 1).

The mitochondrial capacity of ATP synthesis referred to tissue. For each subject, the substrate combinations succinate + glutamate, malate + glutamate, and pyruvate + malate showed different rates of phosphorylating (state 3) respiration (Table 3) but similar rates of ATP synthesis (Table 4). This supports the aforementioned notion that the major flux-controlling step of the phosphorylating succinate + glutamate respiration is the ATP synthesis, i.e., the ATP synthase and/or the involved translocases. The rate of ATP synthesis with succinate + glutamate may therefore be used as a functional assay of the phosphorylating system.

SDH, as a single enzyme, was able to sustain a high rate of ATP synthesis but not to saturate the phosphorylating system. Palmitoyl-carnitine + malate respiration showed only about one-half of the maximal rate of ATP synthesis.

Comparison of in vitro and in vivo data. In Fig. 1, the content of mitochondrial protein and five other in vitro parameters are correlated with VO2 max of the quadriceps muscle. All correlations appear to be significant at least at the P < 0.01 level. In the case of cyt.aa3 (Fig. 1B), the slope of the regression line corresponds to an activity of cyt.aa3 of ~30 mol O2 · s-1 · mol-1, at 38°C. Isolated cytochrome oxidase has been reported to possess an activity three times higher at 25°C (35). Consequently, cyt.aa3 is present in a considerable excess relative to VO2 max and is able to sustain this rate also at nonsaturating oxygen concentrations.

During in vivo respiration, three oxygen molecules are reduced per molecule of pyruvate formed by glycolysis and are subsequently oxidized in the tricarboxylic acid cycle. They are needed for reoxidation of SDH and five NADH molecules (from glycolysis, PDH, and the tricarboxylic acid cycle dehydrogenases). The minimal tissue activity of the single enzymes involved should be one-third of VO2 max, and this limit is marked in Fig. 1, C-E, by solid lines. As seen from the figures, the CS activity represents a considerable excess (~15 times), the SDH activity a moderate excess (~3 times), and the PDH activity only a slight excess (~1.1 times). CS was assayed in a conventional enzyme assay after extraction, whereas SDH and PDH were assayed by functional assays based on the respiratory rates of intact mitochondria.

Figure 1F shows the correlation between the highest rate of phosphorylating in vitro respiration and VO2 max. The slope indicates that this in vitro respiration can account for 60% of the VO2 max.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study showed strong correlations between mitochondrial parameters and VO2 max. With this protocol, the energy was probably obtained only from glycogenolysis and pyruvate oxidation via PDH and the tricarboxylic acid cycle (4). Based on this reaction scheme, the in vitro activities of CS, SDH, and PDH could account for VO2 max. The balance between PDH activity and VO2 max is interesting, because this enzyme has been considered rate limiting for pyruvate oxidation in skeletal muscle (for a review, see Ref. 3). The mitochondria were apparently isolated in a condition that required no further activation of PDH by chemical modification. This notion is supported by the observation that the maximal state 3 rate was attained immediately upon addition of ADP.

The in vitro measurements of mitochondrial respiration of palmitoyl-carnitine + malate may be used to evaluate whether VO2 max can be obtained from oxidation of fatty acids. Mitochondrial respiration of these compounds most likely has an acetyl-O2 stoichiometry of 2:3, owing to beta -oxidation and malate oxidation. The data in Table 3 indicate that the reactions of fatty acid oxidation, coupled to the tricarboxylic acid cycle, can account for <50% of VO2 max.

The in vitro measurements also provide an estimate of the activity of the phosphorylating system, and comparison of this with the in vivo work rate is relevant, even though it necessarily involves some assumptions. Data from Tables 1 and 4 indicate that the ratio between the work rate and the in vitro rate of ATP synthesis is 57 ± 4 kJ/mol. Ideally, the work rate equals the product of three quantities, namely the rate of ATP synthesis, the in vivo Gibbs energy change (-Delta G) of ATP hydrolysis, and the efficiency, i.e., work done/free energy lost, of the actomyosin-ATPase reaction. This means that -Delta G times the efficiency should be larger than 57 kJ/mol if the in vitro rate of ATP synthesis could account for the in vivo work rate. It is possible to obtain some estimate of -Delta G for ATP hydrolysis in the working muscle. An ATP-to-ADP ratio of 50:100 (16), a total phosphate concentration of 10 mM, and an apparent equilibrium constant of 105 M (20) give a range of 52-54 kJ/mol. These values of -Delta G leave no room for an efficiency of the actomyosin reaction that is <100%, i.e., the measured rate of mitochondrial ATP synthesis does not appear to account fully for the work rate. But within the cell, glycolytic ATP formation also contributes to the overall balance, and, with likely assumptions regarding the efficiencies, 80-85% of the ATP needed can be accounted for (25).

The tricarboxylic acid cycle cannot be established in optimal, cyclic operation with isolated mitochondria, but parts of the in vivo reaction scheme may be realized in experiments with substrate combinations. The activity of the respiratory chain does not limit phosphorylating respiration, and it is possible to obtain conditions in which the combined activities of the involved dehydrogenases saturate the activity of the phosphorylating system. This occurred with succinate + glutamate (see RESULTS), malate + glutamate, and probably also with pyruvate + malate. In these cases, addition of supplementary substrates would not lead to a higher rate of ATP synthesis. The complete pyruvate oxidation, with six simultaneous oxygen-consuming steps, will also be limited by the activity of the phosphophorylating system. It will attain a rate of respiration that equals the phosphorylating activity divided by the P/O value. This applies only to fully coupled respiration; completely uncoupled mitochondria will respire at rates limited by the activities of the substrate reaction enzymes or by the activity of the respiratory chain.

The highest state 3 rate of respiration in vitro is the rate with succinate + glutamate. This rate is compared with VO2 max in Fig. 1F, and the slope of the curve indicates that fully coupled in vitro respiration was able to account for, at most, 60% of the in vivo VO2 max. Two possible explanations for this result will be discussed.

1) The measurements were influenced by systematic errors. The measurement of VO2 max involved sampling of blood from the femoral vein, which also drains the hamstrings/adductor muscles. Any involvement of these muscles to produce the power would lead to an overestimation of the oxygen uptake of the quadriceps muscle. However, force recordings in the flexion phase showed that such contributions caused <10% overestimation. When performing knee extensor exercise, there is no limitation of the oxygen supply to the active muscle, and all portions of the muscle are engaged in the exercise (1, 2, 27). The exercise intensity was selected to cause exhaustion within 4 min, and the finding of a plateau in oxygen uptake in the last minute of this exercise indicates that the true VO2 max of the active muscle was reached (4, 13). This is further supported by the observation of a similar oxygen uptake when exercise was repeated. Thus it is likely that the measured oxygen uptake equals VO2 max of the quadriceps muscle within a <10% range.

The temperature factor used to adjust the in vitro rates is critical, and an underestimated factor might have been used. But the alternative estimates of the factor would increase the calculated rates by at most ~25% (see METHODS). Overestimation of the yield of mitochondria would also result in underestimation of the tissue activities, but any large error in the yield determination seems unlikely. Our tissue activity of CS was close to the value in Blomstrand et al. (5), and assays of all fractions in the preparation scheme indicated that only 7% of the total activity was not accounted for (24).

The in vitro assay conditions were carefully optimized, and all test parameters suggested high integrity of the isolated mitochondria. The rates of coupled respiration are very sensitive to the integrity, and the present preparation showed specific activities that were higher than those previously obtained with isolated human skeletal muscle mitochondria and skinned muscle fibers (24). This does not exclude the possibility that the present mitochondria exhibit less than optimal characteristics, but it probably does exclude the possibility that they exhibit only about one-half of their in situ activity.

To summarize, no single systematic error appears to be of a magnitude that can account for the difference between in vitro coupled respiratory rate and VO2 max. On the other hand, the possibility cannot be excluded that a combination of systematic errors is responsible for the difference.

2) The mitochondria were loosely coupled in vivo. The respiratory chain activity most likely exceeded the VO2 max, and the individual enzymes measured in vitro showed activities that could account for VO2 max assuming glycogenolysis and pyruvate oxidation followed by the tricarboxylic acid cycle reactions. But the in vitro rate of coupled respiration, which was controlled by the activity of the ATP synthesis reactions, did not balance VO2 max. A higher rate of respiration would accompany the rate of ATP synthesis if the mitochondria were loosely coupled under in vivo conditions. The formation of ATP need not be altered in such a state; only the overall P/O value is lowered. It is possible to establish this situation under in vitro conditions. The possibility of loose coupling of the mitochondria in a muscle performing severe work is not improbable. It might, for instance, be related to increased mitochondrial permeability due to increased Ca2+ concentration and oxidative stress (10, 11). The uncoupling protein UCP-3, which is present in skeletal muscle (see Ref. 6 for a review), might also be responsible for uncoupling under the conditions of very high work rates. Mild uncoupling has even been considered a beneficial mechanism by counteracting formation of reactive oxygen compounds (32).

In conclusion, the present study shows strong correlations between parameters measured in vitro and VO2 max, and the mitochondrial capacity seems limiting for the performance of the muscle. The in vitro activities of CS, SDH, and PDH can account for the in vivo VO2 max without further activation. The fully coupled in vitro respiration, which is controlled by the rate of mitochondrial ATP synthesis, may account for, at most, 60% of the VO2 max. The possibility cannot be excluded that systematic errors cause this difference, but it is also possible that the mitochondria are loosely coupled under the conditions of intense exercise.


    ACKNOWLEDGEMENTS

We thank Dr. José González-Alonso for careful evaluation of the tissue scannings. We also wish to thank the enthusiastic students who volunteered in this study. The expert technical assistance of I.-L. Føhns and H. Lauritzen is gratefully acknowledged.


    FOOTNOTES

This work was supported by grants from the Danish Natural Research Foundation (504).

Address for reprint requests and other correspondence: U. F. Rasmussen, Dept. of Biochemistry, The August Krogh Institute, Universitetsparken 13, DK-2100 Copenhagen, Denmark

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.

Received 24 March 2000; accepted in final form 27 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andersen, P, Adams RP, Sjøgaard G, Thorboe A, and Saltin B. Dynamic knee extension as model for study of isolated exercising muscle in humans. J Appl Physiol 59: 1647-1653, 1985[Abstract/Free Full Text].

2.   Andersen, P, and Saltin B. Maximal perfusion of skeletal muscle in man. J Physiol (Lond) 366: 233-249, 1985[Abstract].

3.   Bangsbo, J. Muscle oxygen uptake in humans at onset of and during intense exercise. Acta Physiol Scand 168: 457-464, 2000[ISI][Medline].

4.   Bangsbo, J, Gollnick PD, Graham TE, Juel C, Kiens B, Mizuno M, and Saltin B. Anaerobic energy production and O2 deficit-debt relationship during exhaustive exercise in humans. J Physiol (Lond) 422: 539-559, 1990[Abstract].

5.   Blomstrand, E, Rådegran G, and Saltin B. Maximum rate of oxygen uptake by human skeletal muscle in relation to maximal activities of enzymes in the Krebs cycle. J Physiol (Lond) 501: 455-460, 1997[Abstract].

6.   Boss, O, Hagen T, and Lowell BB. Uncoupling proteins 2 and 3. Potential regulators of mitochondrial energy metabolism. Diabetes 40: 143-156, 2000.

7.   Brooks, GA, Hittelman KJ, Faulkner JA, and Beyer RE. Temperature, skeletal muscle mitochondrial functions, and oxygen debt. Am J Physiol 220: 1053-1059, 1971[ISI][Medline].

8.   Byrne, E, and Trounce I. Oxygen electrode studies with human skeletal muscle mitochondria in vitro. J Neurol Sci 69: 319-333, 1985[ISI][Medline].

9.   Gollnick, PD, Bertocci LA, Kelso TB, Witt EH, and Hodgson DR. The effect of high-intensity exercise on the respiratory capacity of sceletal muscle. Pflügers Arch 415: 407-413, 1990[ISI][Medline].

10.   Gunter, TE, and Pfeiffer DR. Mechanisms by which mitochondria transport calcium. Am J Physiol Cell Physiol 258: C755-C786, 1990[Abstract/Free Full Text].

11.   Halestrap, AP, Griffiths EJ, and Connern CP. Mitochondrial calcium handling and oxidative stress. Biochem Soc Trans 21: 353-358, 1993[ISI][Medline].

12.   Hartree, EF. Haematin compounds. In: Modern Methods in Plant Analysis, edited by Paech K, and Tracey MW. Berlin, Germany: Springer, 1955, vol. 4, p. 208-210.

13.   Hellsten, Y, Richter EA, Kiens B, and Bangsbo J. AMP deamination and purine exchange in human skeletal muscle during and after intense exercise. J Physiol (Lond) 520: 909-919, 1999[Abstract/Free Full Text].

14.   Hoppeler, H. The different relationship of VO2 max to muscle mitochondria in humans and quadrupedal animals. Respir Physiol 80: 137-146, 1990[ISI][Medline].

15.   Korzeniewski, B. Regulation of ATP supply in mammalian skeletal muscle during resting state right-arrow intensive work transition. Biophys Chem 83: 19-34, 2000[ISI][Medline].

16.   Lodi, R, Taylor DJ, Tabrizi SJ, Kumar S, Sweeney M, Wood NW, Styles P, Radda GK, and Schapira AHV In vivo skeletal muscle mitochondrial function in Leber's hereditary optic neuropathy assessed by 31P magnetic resonance spectroscopy. Ann Neurol 42: 573-579, 1997[ISI][Medline].

17.   McCully, KK, Fielding RA, Evans WJ, Leigh JS, and Posner JD. Relationships between in vivo and in vitro measurements of metabolism in young and old human calf muscles. J Appl Physiol 75: 813-819, 1993[Abstract].

18.   Miller, JC, and Miller JN. Statistics for Analytical Chemistry (3rd ed.). Chichester, UK: Ellis Horwood, 1993, p. 108.

19.   Newsholme, EA, and Leech AR. Biochemistry for the Medical Sciences. Chichester, UK: Whiley, 1983.

20.   Nicholls, DG, and Ferguson SJ. Bioenergetics 2. London: Academic, 1992, p. 58-59.

21.   Rasmussen, HN, Andersen AJ, and Rasmussen UF. Optimimization of preparation of mitochondria from 25-100 mg skeletal muscle. Anal Biochem 252: 153-159, 1997[ISI][Medline].

22.   Rasmussen, HN, and Rasmussen UF. Respiration measurements in small scale. Anal Biochem 208: 244-248, 1993[ISI][Medline].

23.   Rasmussen, HN, and Rasmussen UF. Small scale preparation of skeletal muscle mitochondria, criteria of integrity, and assays with reference to tissue function. Mol Cell Biochem 174: 55-60, 1997[ISI][Medline].

24.   Rasmussen, UF, and Rasmussen HN. Human quadriceps muscle mitochondria: a functional characterization. Mol Cell Biochem 208: 37-44, 2000[ISI][Medline].

25.   Rasmussen, UF, and Rasmussen HN. Human skeletal muscle mitochondrial capacity. Acta Physiol Scand 168: 473-480, 2000[ISI][Medline].

26.   Rasmussen, UF, Rasmussen HN, Andersen AJ, Fogd Jørgensen P, and Quistorff B. Characterization of mitochondria from pig muscle: higher activity of exo-NADH oxidase in animals suffering from malignant hyperthermia. Biochem J 315: 659-663, 1996[ISI][Medline].

27.   Richardson, RS, Frank LR, and Haseler LJ. Dynamic knee-extensor and cycle exercise: functional MRI of muscular activity. Int J Sports Med 19: 182-187, 1998[ISI][Medline].

28.   Saltin, B, and Gollnick PD. Skeletal muscle adaptability: significance for metabolism and performance. In: Handbook of Physiology, Skeletal Muscle, edited by Peachey LD, and Adrian RH. Bethesda, MD: Am Physiol Soc, 1983, p. 555-631.

29.   Saltin, B, and Rowell LB. Functional adaptations to physical activity and inactivity. Fed Proc 39: 1506-1513, 1980[ISI][Medline].

30.   Schaffner, W, and Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem 56: 502-514, 1973[ISI][Medline].

31.   Shepherd, D, and Garland PG. Citrate synthase from rat liver. Methods Enzymol 13: 11-16, 1969.

32.   Starkov, AA. "Mild" uncoupling of mitochondria. Bioscience Reports F17F: 273-279, 1997[ISI][Medline].

33.   Tonkonogi, M, and Sahlin K. Rate of oxidative phosphorylation in isolated mitochondria from human skeletal muscle: effect of training status. Acta Physiol Scand 161: 345-353, 1997[ISI][Medline].

34.   Wibom, R, and Hultman E. ATP production rate in mitochondria isolated from microsamples of human muscle. Am J Physiol Endocrinol Metab 259: E204-E209, 1990[Abstract/Free Full Text].

35.   Wikström, M, Krab K, and Saraste M. Cytochrome Oxidase, a Synthesis. London, UK: Academic, 1983.


Am J Physiol Endocrinol Metab 280(2):E301-E307
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