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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of the present
study was to examine whether parameters of isolated mitochondria could
account for the in vivo maximum oxygen uptake
(O2 max) of human skeletal muscle.
O2 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
O2 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
O2 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
O2 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
(O2 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
O2 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
O2 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 O2 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
O2 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
O2 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
O2 max.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).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).
|
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
O2 max.
|
|
|
|
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 O2 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
O2 max and is able to sustain this rate
also at nonsaturating oxygen concentrations.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study showed strong correlations between mitochondrial
parameters and O2 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
O2 max. The balance between PDH activity and
O2 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
O2 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
-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
O2 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
(G) of ATP hydrolysis, and the efficiency, i.e., work
done/free energy lost, of the actomyosin-ATPase reaction. This means
that
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
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
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
O2 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
O2 max. Two possible explanations for
this result will be discussed.
1) The measurements were influenced by systematic errors.
The measurement of O2 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
O2 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
O2 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 O2 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
O2 max, and the individual enzymes
measured in vitro showed activities that could account for
O2 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
O2 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
O2 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
O2 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
O2 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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
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
14.
Hoppeler, H.
The different relationship of O2 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 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
35.
Wikström, M,
Krab K,
and
Saraste M.
Cytochrome Oxidase, a Synthesis. London, UK: Academic, 1983.