American Zoologist

American Zoologist 2001 41(2):229-246; doi:10.1093/icb/41.2.229
© 2001 by The Society for Integrative and Comparative Biology

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Gas Exchange, MRS and NIRS Assessment of Metabolic Transients in Skeletal Muscle¹

Paolo Cerretelli¹ and Bruno Grassi^1
1 I.T.B.A.—National Research Council, Via F.lli Cervi 93, I-20090 Segrate (MI), Italy

	SYNOPSIS

TOP SYNOPSIS INTRODUCTION "EARLY" STUDIES CARDIOVASCULAR AND GAS EXCHANGE... RECENT STUDIES ON SKELETAL... METABOLIC STUDIES OF PCR... THE POSSIBLE ROLE OF... CONCLUSIONS References

 
The study of the kinetics of O₂ consumption (o₂) at the onset and offset of constant-load submaximal exercise (o₂ on- and off-kinetics) is useful from a practical point of view (a faster adjustment of oxidative metabolism following an increased metabolic demand reduces the need for substrate level phosphorylation, with implications on exercise tolerance and muscle fatigue) and can give valuable insights into the regulation of oxidative metabolism in skeletal muscle. Measurements have been carried out both in man and in animals, at the tissue and at the whole body level. At the tissue level, the o₂ on- and off-kinetics were determined: a) Directly, by dynamic solution of the Fick equation throughout the transients; attempts were also made to obtain similar informations by near-infrared spectroscopy. b) Indirectly, from the kinetics of phosphocreatine hydrolysis and resynthesis, by chemical methods or by ³¹P magnetic resonance spectroscopy. At the whole body level, o₂ on- and off-kinetics are determined from breath-by-breath measurements of pulmonary gas exchange. The o₂ = f(t) function is a complex one, particularly during the on-transient. The so-called "phase 2" of the o₂ on-response, as well as the o₂ off-response, yield relevant metabolic informations. In muscle the o₂ on- and off-kinetics are characterized by half-times (t) of 15–20 sec. At the whole-body level, t of the o₂ on-kinetics show a wider variability, related to the experimental protocol and to other factors. The o₂ off-phase is more constant, and its kinetic parameters appear closer to those obtained at the tissue level. The study of the o₂ kinetics is valuable for a functional evaluation of skeletal muscle oxidative metabolism. In ordinary conditions muscle o₂ kinetics appears mainly imposed by intrinsic (metabolic) rather than extrinsic (O₂ delivery) factors.

	INTRODUCTION

TOP SYNOPSIS INTRODUCTION "EARLY" STUDIES CARDIOVASCULAR AND GAS EXCHANGE... RECENT STUDIES ON SKELETAL... METABOLIC STUDIES OF PCR... THE POSSIBLE ROLE OF... CONCLUSIONS References

 
Skeletal muscle may be considered a biological engine. Its main characteristic is that of being able to carry out almost instantaneous changes of mechanical performance dictated by the requirements of any of its activated masses, mainly within its locomotor structures. Sudden changes in work output are frequently encountered in sports as well as during many activities of everyday life. The changes of mechanical power are supported by a sequence of exergonic processes that over the last decades have been the subject of extensive research in biochemistry and biophysics. As is well known, the energy sources of the muscle, both anaerobic (alactic and lactic) and oxidative, are characterized by variable capacities and different maximal rates of production (power) and time courses. A faster adjustment of skeletal muscle oxidative metabolism during increases in work rate reduces the need for substrate level phosphorylation, with lesser disturbance for cellular and organ homeostasis, and has obvious implications for exercise capacity and muscle fatigue. Thus, besides providing valuable insights into basic mechanisms of metabolic control during muscular contractions, the study of metabolic adjustments during shifts of power output can have practical implications related to exercise tolerance, in sports, work, health and disease.

	"EARLY" STUDIES

TOP SYNOPSIS INTRODUCTION "EARLY" STUDIES CARDIOVASCULAR AND GAS EXCHANGE... RECENT STUDIES ON SKELETAL... METABOLIC STUDIES OF PCR... THE POSSIBLE ROLE OF... CONCLUSIONS References

 
The initial approach to the study of muscle performance was the analysis of its mechanical properties, which was mainly carried out in isolated, perifused frog limb preparations. These studies were followed in the late twenties and early thirties by the identification of the fundamental energy-yielding anaerobic events, i.e., anaerobic glycolysis with lactate accumulation and the hydrolysis of high energy phosphates, mainly phosphocreatine (PCr). The time course of the latter after stimulation was determined much later in isolated perifused anoxic frog muscle by chemical methods (Danforth, 1965; Cerretelli et al., 1972). The main conclusion from these studies was that, at the onset of constant-load exercise, lactate begins to accumulate in the muscle to a variable extent depending on the load but with a detectable delay compared to PCr hydrolysis. According to Cerretelli et al. (1972) anaerobic glycolysis becomes quantitatively relevant only when PCr concentration drops to 40% of its initial resting level. More recently, a similar analysis was carried out in the dog gracilis preparation (Connett et al., 1985). These authors demonstrated that, in their experimental model: 1) Lactate can be produced, under fully aerobic conditions, in two distinct bouts during the rest-to-contraction transition. The first bout occurs at the very onset on contractions (within the first 5 sec), whereas the second (much greater) bout follows after about 25 sec, and lasts until about 180 sec during the transition. According to Connett et al. (1985) the accumulation of lactate within the muscle deriving from these glycolytic "bouts" (particularly from the first one) has a regulatory role, as a "buffer of cytosolic redox status and/or substrate supply in support to oxidative phosphorylation." 2) PCr concentration decreases sharply at the very onset of contractions, representing the major fraction of cumulative energy contribution during the transition. In order to analyze the rate of the P regenerative process which is carried out by oxidative metabolism, an ideal experimental model would be the isolated-perfused muscle preparation, whereby the various stages of anaerobic energy yielding processes can be assessed simultaneously with O₂ uptake. To our knowledge, this was done for the first time only at the end of the sixties (Piiper et al., 1968) (see below) and later by Connett et al. (1985).

With regard to the kinetics of the adjustment of oxidative reactions, starting from the beginning of the century several investigations were conducted in humans, ever since Krogh and Lindhard (1913) and subsequently Hill and Lupton (1923) had noticed that at the onset of constant-load exercise, the O₂ uptake by the whole organism lags behind the mechanical work and the release of anaerobic energy. This phase necessarily gives rise to an O₂ deficit during the rest-to-work transient which is paid (O₂ debt payment) after the end of the performance as o₂ subsides gradually to the pre-exercise level. The analysis of the o₂ on- and o₂off-kinetics, particularly of the latter (Margaria et al., 1933) led to the identification of the metabolic significance of the various components of the O₂ debt payment, particularly of their relationship to the resynthesis of high energy phosphates (alactic fraction of the O₂ debt) and to the lactic component. These conclusions are in part still valuable with the exception of those concerning the fate of lactate in blood and muscle during recovery.

Whereas the interest of such an integrated approach is undeniable, the quality of the analysis of the mechanisms underlying the adaptations of gas exchanges, mainly o₂, occurring in the muscle during the rest to work transient is certainly affected by at least two variables: 1) the "distance" between the tissues where the primary changes occur and the upper airways of the subject where the o₂ on-response is assessed, and 2) the possible limitations induced during the o₂ on-phase by O₂ convective and diffusive delivery to the mitochondria of the active muscles.

Despite these methodological drawbacks, several studies on gas exchange kinetics were conducted in man over the years and a number of interesting data were obtained before 1975 which could be compared with the results of the few available studies on isolated-perfused muscles. The main results were:

1. The rate of adjustment of o₂ upon exercise onset in moderately active adults is described by a single exponential function with half time (t) of 25 to 30 sec (Hill and Lupton, 1923).
   
2. The o₂ off-phase is made up of at least four components, two of which are related to the payment of the O₂ debt (fast alactic and slow lactic) (Margaria et al., 1933).
   
3. The kinetics of o₂ off- is faster in young than in middle aged adults (Berg, 1947).
   
4. Training reduces both t of the o₂ on- and o₂ off-responses (Berg, 1947).
   
5. The velocity constant of the fast component (alactic) of the O₂ debt payment is independent of work load (Henry and DeMoor, 1950; Henry, 1951).
   
6. The rate of the oxidative processes in muscle is "dictated by the concentration at a given time of the high energy phosphate compounds when split" (Margaria et al., 1965).
   

The link between the above preliminary results on exercising man and those on isolated, unperfused amphibian muscles was provided by the study of Piiper et al. (1968) whereby combined physiological and biochemical analytical procedures allowed the simultaneous assessment of O₂ uptake during the rest to work transient and vice-versa together with the determination of muscle lactate accumulation and of the kinetics of the depletion/repletion of muscle PCr. The results obtained from an intact isolated perfused dog gastrocnemius showed a monoexponential rise of o₂ with a half time of 16.5 sec (i.e., faster than that found in man) at work loads requiring up to 100 ml O₂·kg^–1·min^–1 (Fig. 1). The t of at work onset was also quite fast (10 sec) whereas the maximal blood flow level attained at steady state was extremely high (800 ml·kg^–1·min^–1) allowing an average venous O₂ saturation of about 40%. The t of the o₂ off curve was, on the average, 20 sec and that of 21 sec. These figures suggest that the O₂ convective transport to the muscle was in all cases in excess of the requirements and that the kinetics of the aerobic machinery was likely regulated by "metabolic inertia" and not by lack of O₂ delivery. A fast o₂ on-response was also described by Connett et al., (1985), with a t of 15 sec.

View larger version (27K): [in this window] [in a new window]   FIG. 1. Time course of O2 uptake (o2), blood flow (), and mechanical work (W) of isolated in situ dog gastrocnemius muscle at the onset of work and during the recovery (schematic). At the beginning of the stimulation period o2 and increase, approaching the steady-state net values exponentially (half-times 16.5 and 9.0 sec, respectively). In the recovery phase o2 and reach 50% of the steady-state net value after 20 and 21 sec, respectively. After about 2 min in the recovery o2 has declined to 10% of the steady-state net value. In the upper part of the figure the ratio O2 debt paid/O2 debt built are indicated: they amount to 0.57 and 1.37 after 20 and 120 sec, respectively, from the end of the stimulation period (from Piiper et al., 1968)

 
Following this study, with the exception of the work by Mahler (1978, 1985) on perifused frog sartorious in which the time constant () of the o₂ off-kinetics was estimated at 150 sec (or the t at 120 sec) at 20°C, the gas exchange as well as the readjustment of both central and peripheral circulation were determined mainly in humans.

	CARDIOVASCULAR AND GAS EXCHANGE ADJUSTMENTS DURING METABOLIC TRANSITIONS

TOP SYNOPSIS INTRODUCTION "EARLY" STUDIES CARDIOVASCULAR AND GAS EXCHANGE... RECENT STUDIES ON SKELETAL... METABOLIC STUDIES OF PCR... THE POSSIBLE ROLE OF... CONCLUSIONS References

 
The number of studies dealing with the problem of gas exchange and, to a lesser extent, of circulation kinetics at the onset and/or offset of exercise over the last 25 yr is impressive (see e.g., the review by Tschakovsky and Hughson, 1999). Apart from several confirmatory results of earlier studies, the main indications deriving from these studies can be summarized as follows:

1. The kinetics of o₂ on-, both in dogs (Cerretelli et al., 1964) and in humans (Cerretelli et al., 1966; Pendergast et al., 1980), is consistently slower than that of cardiac output. This finding represents indirect evidence that O₂ convective transport to the limbs during the rest to work transient, by and large, is not a limiting factor to the o₂ kinetics. This statement is also applicable to changes of work load from light to heavy (Davies et al., 1972).
   
2. The kinetics of adjustment of tissue blood flow (_tissue) defined as the time required after constant-load exercise onset to attain steady state at a higher level (i.e., 100% of the blood flow response from rest to any steady state level within 80% of the maximum aerobic power) is, in most cases, below 30 sec (corresponding to t of about 5 sec) both in exercising arms and legs (see e.g., Fig. 2). The adjustment of local circulation appears therefore 3 to 4 times faster than that of o₂ on- (Pendergast et al., 1980). This finding, together with the constantly very high _tissue absolute levels attained by the contracting muscles would imply that even at the tissue level O₂ convective transport in most cases is adequate.
   
3. The t of the o₂ on-response for supine arm exercise is longer than for any similar (absolute and relative) work level carried out by the legs in the same posture (Cerretelli et al., 1977; Pendergast et al., 1979). Since gravitational factors are the same in the supine posture for all limbs, local mechanical factors associated with the grip would seem to be a likely mechanism to impair blood flow early in arm work.
   
4. With the exception of very mild exercise, the O₂ deficit values are consistently smaller than the corresponding O₂ debts, independent of work load and posture. This is evident particularly for arm exercise in the supine position (see point 3 above). The relatively slow o₂ on-kinetics during arm exercise is associated with a transient release of lactate even at relatively low work loads. In these conditions, "early" anaerobic glycolysis (Cerretelli et al., 1977, 1979), besides its hypothesized regulatory role (see Connett et al., 1985, as discussed above) must supply part of the energy for ATP resynthesis that (during arm exercise) cannot be provided by oxidations because of inadequate convective O₂ transport, and/or activation of greater fractions of type II muscle fibres with limited aerobic potential. Despite experimental evidence of fast blood flow readjustment rates also in the arms of subjects exercising in the supine posture (see point 2 above), several studies (Hughson and Morrissey, 1982; Tschakovsky and Hughson, 1999) seem to indicate that, in particular postural conditions, circulatory factors may become responsible for part of the delay of the o₂ readjustment during the on-phase. In the latter case the increase of the t of o₂ on- must be associated, due to a deficit of O₂ convective transport, with a corresponding accumulation of lactate in the contracting muscle and in blood ("early lactate," Cerretelli et al., 1977, 1979). The finding of "early lactate" (Cerretelli et al., 1977, 1979) appears compatible with the occurrence of a "second burst" of anaerobic glycolysis at the onset of aerobic exercise, as described by Connett et al. (1985). In man, during the first minutes of the rest-to-work transient, the accumulation of lactate in muscles and blood can be quite ramarkable. After the initial phase, lactate concentrations may "level off," indicating an equilibrium between lactate production and disposal. Independently on the level reached, an unchanged lactate concentration in muscle and blood indicates fully aerobic conditions (see also the recent review by di Prampero and Ferretti, 1999). The "early" accumulation of lactate depends on the work load intensity, on the muscles involved in the exercise, on the training status of the subjects (Cerretelli et al., 1977, 1979). At a given rate of ATP resynthesis, which is obviously constant for a given "rectangular" submaximal workload, the extent of ATP resynthesis provided by anaerobic glycolisys during the transition is greater with higher "early lactate" accumulation. It follows that the rate of o₂ increase, i.e., the rate of ATP supply by oxidative phosphorylation, may be lower in proportion with the accumulation of "early lactate."
   
5. Overall, for whole body exercise in humans, the measured t of the O₂ off- responses are shorter than those of the corresponding on-responses, independent of posture and type of exercise, and rather constant, ranging among different subjects with variable training conditions, between 20 and 30 sec (see e.g., Cerretelli et al., 1977; di Prampero et al., 1989). Besides, their t are very close to the values reported originally for the fast component of the O₂ debt payment by Margaria et al. (1933). Moreover, average t o₂ off- are similar to the estimated minimum o₂ on- levels calculated after subtraction of the part of the O₂ deficit attributed to early lactate formation in muscle. The t of both these processes approaches that of the isolated-perfused dog gastrocnemius muscle (Piiper et al., 1968) and that of intact running dogs (Marconi et al., 1982) in which the accumulation of early lactate is absent or negligible.
   
6. In some experimental conditions, the observation has been made that o₂ on-kinetics is enhanced when given work loads are superimposed on preexisting exercise baselines carried out by the same or by a different muscle mass (di Prampero et al., 1970, 1989; Davies et al., 1972; Paganelli et al., 1989). This would be further evidence of "metabolic inertia" that may reach a minimum level corresponding to a minimum accumulation of "early lactate" and with the depletion of the O₂ stores of the body. In these conditions t o₂ on-approaches t o₂ off- and both values might become very close to the o₂ on-kinetics of the dog isolated perfused gastrocnemius. However, it must be pointed out that various authors (Diamond et al., 1977; Casaburi et al., 1977; Hughson and Morrissey, 1982) were unable to confirm the above findings, even though in different experimental conditions. At least in part these apparently conflicting results can be reconciled by taking into account the effects of the type of the adopted exercise protocol (cycling, stepping, running) on the size of body O₂ stores depletion and "early lactate" (di Prampero et al., 1989).
   
7. The analysis of the o₂ on-response curves and of their superimposed repetitions obtained with the most sophisticated breath-by-breath analytical devices has made it possible to identify various components of the response and to make interesting inferences regarding their functional significance. At a closer inspection, the o₂ on-kinetics is not a single exponential function but is a function characterized by at least three "phases" (Whipp et al., 1982): a) Phase 1, following work onset and lasting for about 15–20 sec, reflects mainly increased pulmonary blood flow (Linnarson, 1974; Wasserman et al., 1974; Weissman et al., 1982). This part of the o₂ on-curve, which is considered devoid of metabolic implications, is often excluded from kinetics analysis. b) Phase 2, very close to a monoexponential function, reflects the metabolic events occurring in the muscle upon step changes of activity, i.e., the building of the O₂ deficit which appears mainly dictated by inertia of the oxidative machinery and possible differences and/or perturbations of the kinetics of the anaerobic reactions (bursts of anaerobic glycolysis or "early lactate"). This is the part of the o₂ on-curve utilized for comparison with the curves obtained in isolated-perfused muscle preparations and/or with those describing PCr hydrolysis in man by MRS. c) Phase 3, a "slow component" that is detectable with work loads exceeding the so-called ventilatory anaerobic threshold, and whose significance is still under investigation (Gaesser and Poole, 1996). The o₂ slow component has indeed been associated with physical (increase of temperature), and metabolic factors (e.g., some glucose resynthesis from lactate) and, particularly, with preferential recruitment of fast-twitch fibers (Gaesser and Poole, 1996). On theoretical grounds, the kinetics of the phase 2 of the o₂ on-response (except for early lactate accumulation) should be identical with that of the o₂ off-phase. In fact, the latter, following an instantaneous arrest of the "engine," is not affected (in the opposite direction) by the blood-gas exchange associated with phase 1 of the o₂ on- response and/or by release of early lactate during the transient. Moreover, the influence of increased metabolic activity of the cardiac and of the respiratory pumps as well as the depletion/replenishment of the body O₂ stores during recovery should be the mirror image of events occurring also during work onset.
   
8. Physical training or athletic conditioning affects favourably the kinetics of the o₂ on-response since it reduces the accumulation of the early lactate (Cerretelli et al., 1979). By contrast, the "metabolic inertia" of the system which is imposed by the known sequence of biochemical reactions, appears to be less affected, as indicated by the narrow variability of the o₂ off-response (Figs. 3A and B). On the other hand, it is known that bed rest deconditioning determines slower than normal pulmonary o₂ on-kinetics (Convertino et al., 1984).
   
9. Also age seems to affect the kinetics of the o₂ on-response as originally described by Berg (1947). Indeed, children appear to be characterized by shorter t o₂ on-responses, a conclusion which has been recently challenged by Heberstreit et al. (1998) but that is supported by the authors of the present manuscript in association with a lower lactate accumulation during the transient. On the other hand, pulmonary o₂ on-kinetics is slower in old subjects compared to young controls (Babcock et al., 1994a) during cycle exercise, but not during exercises conducted with muscles commonly utilized during everyday life (Chilibeck et al., 1996). It is also known that a 6-mo endurance training program in aged subjects (70 yr-old) determines a faster pulmonary o₂ on-kinetics during cycling exercise (Babcock et al., 1994b). Moreover, the effects of exercise training on pulmonary o₂ on-kinetics in old subjects appear much earlier than those described on o₂ max (Fukuoka et al., 1999).
   
10. With regards to work load, there appears to be substantial consensus on the concept of constancy of t or of phase 2 of the o₂ on-response below the so-called ventilatory threshold. The recent finding of shorter time constants in the rest to work transitions to supramaximal work loads (130% o₂ max) by Heberstreit et al. (1998) are in conflict with the work of Margaria et al. (1965) and needs confirmation.
    
11. Lowering muscle temperature from 35.5°C to 28.0°C does not influence the o₂ on-kinetics at submaximal work loads. By contrast, t _tissue on- as well as steady state muscle blood flow are sharply reduced (Ishii et al., 1992). This is a further proof that the velocity of readjustment as well as the steady state blood flow level in cycle exercise, within broad limits, are not the determinants of the o₂ on-kinetics. By contrast, at maximum exercise, the t of the o₂ on-response rises significantly as expected, due to the drop of temperature and the consequent effects on the rate of chemical reactions involved in the ATP regenerative process (Ferretti et al., 1995).
    
12. The kinetics of the o₂ on-response may be significantly decreased by impairments of the oxidative machinery induced by disease and/or pharmacological agents. This is the case for heart transplant recipients whose t o₂ on- and off-responses are significantly longer than expected on the basis of the t of the on-response (Cerretelli et al., 1992; Grassi et al., 1997). The slow o₂ on-kinetics found in these patients as compared to control subjects, appears at least in part attributable to detraining and, most likely, to the negative effects of the pharmacological treatment (corticosteroids, cyclosporin A) on skeletal muscle metabolism. Very recently, Bauer et al. (1999) have shown that patients affected by peripheral arterial disease are characterized by a slow o₂ on-response which could be attributed to peripheral blood flow limitation and/or to changes in the regulation of skeletal muscle oxidative function (as suggested by the observation, in these patients, of mitochondrial DNA injury [Bhat et al., 1999]).
    

View larger version (20K): [in this window] [in a new window]   FIG. 2. Individual 133Xe washout curves for a trained (kayaker) and untrained subject during arm exercise (biceps) at the indicated loads (75 and 37.5 W, respectively). The log of the accumulated counts for 6 sec periods are plotted as a function of time during rest and exercise. Tissue blood flow can be calculated from the slope of the regression lines drawn through the experimental points

 

View larger version (24K): [in this window] [in a new window]   FIG. 3. A. Breath-by-breath o2on- and off-responses of a sedentary subject and a trained kayaker to square-wave arm work load of 125 W. Note longer t of o2on-response in sedentary subject while t of the o2off-response is the same. B. Changes in blood lactate (Lab) as function of t of the o2on-response of leg-trained (LT) subjects performing leg exercise and arm-trained (AT) subjects performing arm exercise (from Cerretelli et al., 1979)

 
The conclusions that can be drawn from the above reported experimental results are mainly in favour of the hypothesis that, in ordinary working conditions, "metabolic inertia" is the main determinant of the rate of readjustment of o₂ on- in normal subjects, particularly when work load does not exceed 70–80% of o₂ max. However, it goes without saying that there are conditions imposed by posture, gravitational or environmental (e.g., hypoxia) factors compatible with a limitation attributable to convective O₂ transport (Tschakovsky and Hughson, 1999).

Be that as it may, it is the authors' opinion that the debate between those who claim the priority of the circulatory factors and those who maintain that the control of the velocity of readjustment of the oxidative machinery is metabolism-dependent could be settled, provided that the experimental conditions leading to the above divergent conclusions are known. Further steps towards the solution of the problem were taken in the early and mid nineties by extending to humans and animals the experimental techniques used earlier (Piiper et al., 1968) with isolated-perfused muscle preparations, and by adopting magnetic resonance spectroscopy for the study of the metabolic adjustment of human muscle (Binzoni et al., 1992; Blei et al., 1993; McCann et al., 1995).

	RECENT STUDIES ON SKELETAL MUSCLE

TOP SYNOPSIS INTRODUCTION "EARLY" STUDIES CARDIOVASCULAR AND GAS EXCHANGE... RECENT STUDIES ON SKELETAL... METABOLIC STUDIES OF PCR... THE POSSIBLE ROLE OF... CONCLUSIONS References

 
After showing that during the first 10 sec of an unloaded-to-loaded pedaling transition the adjustment rate of O₂ delivery to exercising human muscle was faster than the adjustment of O₂ utilization (Grassi et al., 1996), Grassi and coworkers performed further experiments, by utilizing an isolated in situ dog gastrocnemius preparation, aimed at evaluating the role of convective O₂ delivery as a limiting factor to muscle O₂ on-kinetics (Grassi et al., 1998a). The preparation permitted to keep muscle blood flow (m) constantly elevated, during rest-to-contraction transitions (up to 60% of O₂peak), at a level corresponding to that obtained at steady-state during preliminary trials conducted with "spontaneous" adjustment of m. A vasodilatory drug (adenosine) was also infused to prevent vasoconstriction. This allowed elimination of all delays in convective O₂ delivery during the transition. The main results of the study are presented in Figure 4, in which average values of O₂ delivery (m·Cao₂) and o₂m in the two experimental conditions ("Control" and "Fast O₂ Delivery") are shown: a complete dissociation of the time-courses of the independent variable ( m·CaO₂) between the two conditions did not affect the dependent variable, i.e., o₂m, whose time-courses in the two conditions were remarkably similar. Parenthetically, the authors observed that the t for m was significantly lower (12 sec) than that for o₂m (18 sec) confirming previous results by Piiper et al. (1968). These results provide further evidence that, for the adopted experimental model and within power transients up to 60% of o₂peak, convective O₂ delivery to muscle is not a factor limiting the o₂m on-kinetics. This conclusion might not fully apply to transitions involving contractions (or exercises) of higher metabolic intensity. Indeed, data obtained by the same group in the same dog preparation, during transitions from rest to contractions corresponding to 100% of o₂peak, showed that elimination of delays in convective O₂ delivery to muscle determined a slightly but significantly faster o₂m on-kinetics, a lower O₂ deficit and less muscle fatigue (Grassi et al., 2000a). This would be in agreement with more indirect evidence obtained by Gerbino et al. (1996) and by MacDonald et al. (1997) in humans. According to these authors, enhancing O₂ delivery to muscle (by a warm-up exercise and/or by hyperoxic breathing) determines a slightly faster pulmonary o₂ on-kinetics during exercises above the ventilatory threshold (VT), but not during exercises below VT. According to a recent study by Burnley et al. (2000), the speeding of the "overall" (no distinction between the phase 2 and the slow component) pulmonary o₂ kinetics obtained by a preceeding "warm up" exercise is attributable to a reduced amplitude of the slow component, whereas the of the "primary" (i.e., phase 2") component would be substantially unaffected. Thus, in humans, VT could discriminate between work intensities at which O₂ delivery is not (i.e., those below VT) or is (those above VT) one of the limiting factors for the pulmonary o₂ on-kinetics.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Average values (±SE) of O2 delivery to muscle (·CaO2) and O2 uptake by muscle (o2), during a rest to submaximal contraction transition, in the two experimental conditions: "Control" (upper panels) and "Fast O2 Delivery" (lower panels). Vertical hatched lines indicate contractions onset. Faster adjustment of O2 delivery does not affect o2 on-kinetics in isolated in situ canine muscle. (from Grassi et al., 1998a, used with permission of the American Physiological Society)

 
The above data (Grassi et al., 1998a) showing that bulk convective O₂ delivery to muscle is not a major limiting factor to the o₂m on-kinetics, do not allow inferences on convective O₂ delivery within the muscle. The latter could be affected by intramuscular uneven distribution not only of m (Piiper et al., 1985; Marconi et al., 1988) but also of m/o₂m, which, in theory, could affect the o₂m on- kinetics by inducing areas of anaerobiosis within the muscle. The model by Grassi et al. (1998a) could not directly test the issue of m/o₂m maldistribution. In fact, all muscle fibers were synchronously activated by electrical stimulation, so that o₂m heterogeneity was eliminated or significantly reduced compared with the situation of asynchronous and heterogeneous fiber activation in physiologically contracting muscle. Some degree of intramuscular m/o₂m maldistribution in physiologically contracting muscle, during the early phase of the transition, appears likely, considering the different patterns of distribution and/or activation of motor units and "microvascular units" (Segal and Kurjiaka, 1995) within the muscle. At present it is not known whether a m/o₂m maldistribution is indeed a limiting factor for o₂m on-kinetics. Clarification of this issue awaits the development of methods allowing to effectively determine /o₂ distribution in tissues.

Another determinant of the o₂ on- kinetics could be represented by the finite delivery of O₂ to mitochondria by diffusion, a process which is known to represent one of the limiting factors to o₂max (Wagner, 1995). The approach adopted earlier by Linnarson (1974) and more recently by MacDonald et al. (1997), based on the study of the effects on o₂ on-kinetics during hyperoxia, has generated controversial results (either no effect or a faster o₂ on- above the ventilatory threshold). This approach, moreover, is characterized by an intrinsic limitation represented by the fact that the hyperoxia-induced enhancement of peripheral O₂ diffusion, secondary to increased capillary PO₂, is likely counterbalanced by peripheral vasoconstriction and intramuscular maldistribution, which are known to be associated with hyperoxia. The net results of these contrasting effects on O₂ delivery to muscle fibers are difficult to establish. Grassi et al. (1998b) were able to overcome these limitations, unavoidable when dealing with human subjects, by utilizing again the isolated "in situ" dog gastrocnemius model. In their study, the constantly elevated m and adenosine administration prevented the negative effects of hyperoxia on convective O₂ delivery. The authors enhanced peripheral O₂ diffusion (during transitions from rest to 60% of o₂peak), by a) hyperoxic breathing (FIO₂ = 1.00), and b) hyperoxic breathing combined with the administration of a drug (RSR13, Allos Therapeutics) which, as an allosteric inhibitor of Hb-O₂ binding, induced a significant rightward shift of the Hb-O₂ dissociation curve, and therefore a significant increase in mean capillary PO₂ (as calculated by numerical integration). Enhancement of peripheral O₂ diffusion obtained by these maneuvers did not affect o₂m on-kinetics. Thus, after eliminating possible influences on convective O₂ delivery (in all conditions m was constantly elevated and adenosine was given), peripheral O₂ diffusion was not a limiting factor for o₂m on-kinetics. The conclusion that can be drawn from the above studies (Grassi et al., 1998a, b; Grassi, 2000a) is that up to 60% of o₂peak muscle o₂ on-kinetics is mainly determined by an intrinsic "inertia" of oxidative metabolism. The latter could be imposed by the level of cellular metabolic controllers and/or enzyme activation, among which the activity of pyruvate dehydrogenase might play a significant role (Timmons et al., 1998).

The essentially mono-exponential increase in skeletal muscle o₂ during the rest to work transition observed in humans (Grassi et al., 1996) as well as in canine muscle (Grassi et al., 1998a, b) is in agreement with the metabolic models of muscle respiratory control during contraction (Margaria et al., 1965; di Prampero and Margaria, 1968; Mahler, 1985; Meyer, 1988; Binzoni and Cerretelli, 1994; Meyer and Foley, 1996) according to which a single reaction with first order kinetics in conditions of O₂ excess controls muscle o₂. Such reaction can be identified with the ATP regenerative process, whose rate is directly proportional to phosphocreatine (PCr) concentration. A monoexponential decrease of the latter during the o₂ on- transition has indeed been described by several authors both in isolated muscle (Piiper and Spiller, 1970) and in man by magnetic resonance spectroscopy (Taylor et al., 1983; Molé et al., 1985; Binzoni et al., 1992). More recently Rossiter et al. (1999) determined simultaneously pulmonary o₂ on- kinetics and PCr kinetics by ³¹P-NMR, during moderate constant-load quadriceps exercise, and confirmed that phase 2 of the pulmonary o₂ on-kinetics is almost identical to the kinetics of PCr hydrolysis.

	METABOLIC STUDIES OF PCR KINETICS BY MRS

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Magnetic resonance spectroscopy (MRS), as is well known, has proven to be a powerful tool for investigating muscle function in vivo. Phosphorus MRS (³¹P-MRS) can be used in man to determine, non-invasively, the concentration of ATP, PCr, Pi and hexose phosphates as well as the cytosolic pH in resting and exercising muscles. The high time resolution of the measurements, which, for practical purposes, is on the order of 10 sec, allows the assessment not only at steady state but also during metabolic changes, particularly in the rest-to-work transition and during recovery when the artifacts related to motion are eliminated. The information that may be obtained from ³¹P-MRS is extremely valuable and complementary to that from muscle biopsies. ³¹P-MRS in muscle was pioneered by the Oxford group, developed by Wilkie and coworkers and then applied to exercising human limbs.

Binzoni et al. (1992) determined the PCr kinetics at the onset of constant-load exercise and recovery by high resolution ³¹P-MRS in the human plantar flexors. This was assumed to be the mirror image of the ATP oxidative regenerative process and therefore the quantitative counterpart of the latter (o₂ on-response). In Figure 5 (A) examples are shown of relative PCr concentration as a function of the number of spectra (obtained at 10.8 sec intervals) at three different work loads. The two upper curves are compatible with the criteria defining aerobic exercise (i.e., no change of intracellular pH compared to rest, and the attainment of a steady [PCr]). By contrast, the lower curve, in which [PCr] values are as low as 20% of the initial resting level, is an example of an exercise at load exceeding the so-called anaerobic threshold whereby the muscle is steadily accumulating lactic acid. In Figure 5 (B) the average kinetics is shown for 13 curves from 5 subjects performing aerobic loads. The relation between normalized [PCr] and time, on the basis of an assumed monoexponential fitting, yields a value of 23.3 sec (t = 16.3 sec). This value is very similar to those found for the o₂ on-response of the isolated-perfused dog gastrocnemius by Piiper et al. (1968) and by Grassi et al. (1998 a, b) and not very different from the values of the t of phase 2 of the o₂ on-response of human muscles (after taking into account the influence of "early lactate") and of the o₂ off-response when allowance is made for the replenishment of tissue O₂ stores.

View larger version (25K): [in this window] [in a new window]   FIG. 5. A. PCr concentration in arbitrary units (a.u.) as function of time (or number of spectra obtained at 10.8 sec intervals) at the onset and offset (see arrows) of three different constant work loads by the plantar flexors. B. Normalized PCr content as a function of time of exercise. Average values (±SD) of 13 experiments for 5 sedentary subjects. Line: monoexponential function calculated by a fitting procedure (from Binzoni et al., 1992)

 
Steady state normalized phosphocreatine concentration values ([PCr]) are a linear function of work load (Binzoni et al., 1992) (w, in Watt per square cm of cross sectional area of the plantar flexors). Based on the above results, a different approach for the description of muscle metabolic control was proposed by Binzoni and Cerretelli (1994). The flux of aerobic energy is described by a transfer function analogous to that of Chance et al. (1985) but constructed without knowing, as was the case for the previous models, the characteristics of the reactions regulating the ATP regenerative process in the tissue. The experimental protocol on which this model is based is the instantaneous rest-to-work metabolic transition occurring in a muscle mass contracting "in situ." According to the system theory, this protocol corresponds to the study of the output of a system after a given input step function. The definition of the model has been made elsewhere (see Binzoni and Cerrerelli, 1994; Cerretelli and Binzoni, 1997). Shortly, starting from the general energy balance equation for the muscle:

(1)

where:, ß, are the moles of ATP resynthesized per mole of hydrolysed phosphocreatine (PCr), O₂ consumed and lactic acid (La) produced, respectively, and the over dots signify first time derivatives. Equation [1] describes the energy flux in a given muscle mass at time t, when a mechanical load w is added. Imposing a number of constraints, a series of expressions may be obtained that satisfy the conditions imposed by the model and are coherent with most experimental data and with other models. The following equation:

represents the proposed transfer function which, upon replacement of PCr by the creatine-kinase equilibrium reaction, is analogous to Chance's original formulation (Chance et al., 1985).

The model is based on the assumption that Lohmann's reaction describes the transient depletion of the only available energy buffer (PCr) in the neighbourhood of time zero (onset of the rest to work transient) when both aerobic metabolism and anaerobic glycolysis are not fast enough to keep up with the imposed load.

Should the kinetics of the ATP regenerative process from both aerobic and anaerobic glycolysis in a given identical activated muscle mass vary among subjects, defined as the velocity constant of PCr hydrolysis during the rest to work transient which is shown to be independent of work load, would change accordingly (see Eq. [2]). For example, let us assume that the value of measured in a limb muscle of an endurance athlete is shorter than that of a sedentary subject. This is tantamount to saying that the kinetics of the ATP regenerative reactions of the athlete is faster. Such kinetics, as is well known, depends on the enzymatic activity of both cytosolic and mitochondrial exergonic reactions. A shorter value implies a lesser PCr depletion of the muscle stores for a given constant workload. The faster response could be the consequence of either of the two following functional conditions of the muscle: a greater enzymatic activity (whence a shorter ) of a given identical mitochondrial mass and/or a greater concentration of functionally identical mitochondria. In the latter case, to sustain a given submaximal power w, the mitochondrial mass needs to be activated to a lesser extent, which would imply a shorter time to reach steady state. On the basis of the above hypothesis, it is reasonable to expect that an endurance athlete, whose muscle mitochondrial concentration is greater, would be characterized by a shorter and, consequently, by higher [PCr] steady-state levels. This would also be in agreement with data obtained by Wegener et al. (1991) on locusts. These authors showed that the phosphoarginine (insect equivalent of PCr) concentration of locusts reaches a steady state after only about 2 sec of flight. Assuming that this also reflects the kinetics of o₂ in the insect muscle, it would appear that the "double-packed" mitochondria of insect muscle leads to a remarkably fast o₂ on-kinetics.

The above concepts are illustrated schematically in Figure 6. It appears from the top panel that muscles characterized by shorter values are those incurring at steady state a smaller PCr deficit. These are muscles with higher mitochondrial density and/or greater activity of oxidative enzymes, like the myocardium (Katz et al., 1989). Muscles such as the diaphragm, are expected to display intermediate levels whereas skeletal muscles are characterized by a wide range of values depending on their fiber composition and training level. The slower levels are to be found in patients affected by mitochondrial diseases, particularly those due to defects of the respiratory proteins.

View larger version (16K): [in this window] [in a new window]   FIG. 6. A. Expected relative PCr concentration (initial = 100) vs. time for three hypothetical muscles characterized by decreasing oxidative potential (from = 2 to = 30), when performing an identical constant work load requiring o2 = 1.9 arbitrary units. B. PCr relative concentration vs. o2. The slope of the lines decreases for tissue with increasing oxidative metabolic potentials. The symbols indicate the expected steady state PCr levels for the three curves appearing in the A panel

 
³¹P-MRS was used by Mizumo et al. (1994) to study the involvement of various wrist flexor muscles together with same metabolic characteristics of the different muscle components (e.g., fiber type distribution). Based on pH and Pi determinations, these authors found different patterns of fiber recruitment between subjects who were also characterized by different kinetics of Pi and PCr recovery. Takahashi et al. (1995) have examined in the human quadriceps the effects of differences in exercise intensity on the time constant () of phosphocreatine resynthesis after exercise and the relationship between and maximal oxygen consumption (o₂ max) in endurance runners and control subjects. The runners, as expected, were characterized by shorter at the same PCr and pH, than control subjects. There was a significant negative correlation between o₂ max and at all levels of exercise. As indicated earlier, very recently, Rossiter et al. (1999) have determined simultaneously in man the kinetics of PCr hydrolysis and that of phase 2 of the o₂ on-response. The values of obtained were very close to each other (35 and 36 sec, respectively).

	THE POSSIBLE ROLE OF NEAR-INFRARED SPECTROSCOPY FOR THE ASSESSMENT OF O2 KINETICS

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Whereas MRS has been widely used to study muscle metabolism in normal (Kemp and Radda, 1994) and pathological conditions (Kent-Braun et al., 1995), no MRS technique is available to directly monitor O₂ consumption (o₂). Near-infrared spectroscopy (NIRS) appears to be an emerging technique for monitoring oxidative metabolism in muscle (Mancini et al., 1994; Cerretelli and Binzoni, 1997; Ferrari et al., 1997). This non-invasive method allows the monitoring of "tissue" oxygenation on the principle that the near-infrared light absorption characteristics of hemoglobin (and myoglobin) depend on their O₂ saturation. NIRS allows measurements, at the tissue level, of oxyhemoglobin ([O₂Hb]) and deoxyhemoglobin concentration changes ([HHb]), thereby allowing an evaluation of the balance between O₂ delivery and O₂ utilization in the region of interest of the tissue under consideration. At steady state, in the absence of blood inflow and outflow to and from the tissue, [O₂Hb] decrease and [HHb] increase can be considered an index of o₂. [HHb] is, in fact, the mirror image of the disappearance of O₂ stored in the tissue before ischemia is induced (increase in [HHb] = decrease in [O₂Hb]). O₂ uptake could be measured in human arm (De Blasi et al., 1993; Hampson and Piantadosi, 1988) and calf muscles (Binzoni et al., 1998) by NIRS at rest and during steady isometric contractions in ischemic conditions (Colier et al., 1995). By contrast, the o₂ on- transient from the onset of isotonic exercise cannot be easily assessed by NIRS. Only recently a mathematical model was devised allowing determination of o₂ at steady state as well as of the kinetics of o₂ readjustment during the rest to work transient (Binzoni et al., 1999) based on the analysis of [HHb] or [O₂Hb] vs. time curves of ischemic muscles. The experimental approach is rather complicated and further work is required to make it possible to assess routinely o₂ on-kinetics by NIRS.

The kinetics of muscle oxygenation has been followed also during recovery from aerobic exercise by Chance et al. (1992), and it was subsequently found to be closely correlated to that of PCr resynthesis (McCully et al., 1994). More recently, Grassi et al. (2000b) followed by NIRS the on-kinetics of human muscle oxygenation, and observed a "biphasic" time course (unchanged for 6.5 sec, then a monoexponential decrease to a new steady-state with a of 8.5 sec), that is remarkably similar to that obtained invasively for C(a-v)O₂ increase across human (Grassi et al., 1996) and isolated in situ canine (Grassi et al., 1998a) muscle; the observed muscle oxygenation time-course appears compatible with the well known rapid increase of m at the transition.

Although several issues still need to be clarified (such as the role of myoglobin in determining the NIRS signal, or the influence of changes in blood volume on the oxygenation indices) NIRS appears a promising method to investigate non-invasively oxidative metabolism (both during steady states and transitions) at the skeletal muscle level. The method offers the additional advantage to allow to selectively investigate specific muscle groups.

	CONCLUSIONS

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It has taken about 80 years after the pioneering measurements of Krogh and Lindhard (1913) and A. V. Hill and co-workers (Hill and Lupton, 1923) before a clear-cut description of the sequence of circulatory and metabolic events occurring in the muscle upon rest to work transition and vice-versa could be made available. As for most of the progress in physiology, the "quantum leap" was made possible mainly by the availability of high technology, in the present case fast-response gas analysers and MRS.

The conclusions that can be drawn from the latest results may be summarized as follows:

1. With an excess of convective O₂ delivery to the active muscle mass, the kinetics of the o₂on-response (during the metabolically relevant "phase 2") recorded at the mouth after the onset of a constant-load exercise may reflect the time lag of the oxidative machinery ("metabolic inertia").
   
2. Enhancing convective and diffusive O₂ delivery in isolated-perfused dog gastrocnemius during transitions to 60 of o₂ max does not affect o₂ on-kinetics.
   
3. The kinetics of the o₂ on-response (phase 2) may be affected by anaerobic glycolysis. This is not the case for the o₂ off-response which, therefore, may be a better predictor of the o₂ kinetics at the muscle level.
   
4. The kinetics of the PCr hydrolysis assessed by MRS in both animal and human muscles attain values that are very close to those of the o₂ on- and off-responses recorded at the muscle level by the Fick method.