Proc. Nati. Acad. Sci. USAVol. 83, pp. 9458-9462, December 1986Biochemistry

Multiple controls of oxidative metabolism in living tissues asstudied by phosphorus magnetic resonance

(metabolic control/adaptation to exercise/oxygen delivery/instability of cardiac performance)

B. CHANCE*tt, J. S. LEIGH, Jr.*, J. KENT*§, K. MCCULLY*, S. NIOKA$, B. J. CLARKII, J. M. MARIS*,AND T. GRAHAM**Departments of Biochemistry and Biophysics, and TPhysiology, The University of Pennsylvania, tThe Institute for Structural and Functional Studies, andI"Department of Cardiology Childrens Hospital of Philadelphia, Philadelphia, PA 19104; and §The University of Southern California, Los Angeles, CA 90089

Contributed by B. Chance, September 12, 1986

ABSTRACT Three types of metabolic control of oxidativemetabolism are observed in the various tissues that have beenstudied by phosphorous magnetic resonance spectroscopy. Theprincipal control of oxidative metabolism in skeletal muscle isby ADP (or P1/phosphocreatine). This conclusion is basedupon studies of arm muscles of humans during steady-stateexercise. A work-cost (Vm vs. Pi/phosphocreatine) relation-ship follows a Michaelis-Menten rectangular hyperbola,where Km values from 0.5 to 0.6 and Vm.. values from 50 to200 (at nearly constant pH) are found in linearized plots of theequation V/V.AX = 1/(1 + 0.6 phosphocreatine/Pi) where V iswork level (which is equal to the velocity of the enzymatic reac-tion) and V.n,. is the maximal work capacity that is a measureof the enzyme activity (E) of oxidative metabolism. Adaptationto exercise enhances the slope of the work-cost relationshipand causes large changes in V.. or E. A second metaboliccontrol may enhance the slope of the work-cost relationshipbut not V.. For example, the initiation of exercise can lead toan improved characteristic that can be explained by 2-fold in-creased substrate delivery, for example, increased oxygen de-livery by microcirculatory control. Cardiac tissue of the adultdog affords an example of optimal endurance performance ad-aptation and exhibits the steepest work-cost relationship ob-served and is attributed to a coordinated control of substratedelivery that may involve Ca2' and inorganic phosphate con-trol of NADH; control of 02 delivery may also be involved.The calculated work-cost relationship is similar to that ob-served in the beagle heart. The theoretical curve illustratesthat the liability of multiple controls is a sharp break point inmetabolic control at the end of the multiple control range-apossible cause of instability of cardiac performance at highV/Vmax.

Ideas and experiments on oxidative metabolism have devel-oped along the lines of pathways and stoichiometries. War-burg (1), Meyerhoff (2), Cori and Cori (3), and Krebs (4) de-lineated important pathways while Kalckar (5), Ochoa (6),Loomis and Lipman (7), Slater (8), Lardy and Wellman (9),and Kennedy and Lehninger (10) studied and contributed tostoichiometries particularly the P/O and H+/O ratio of oxi-dative phosphorylation. Metabolic control emerged as a top-ic of great importance in the works of Krebs (4), Bucher andKlingenberg (11), Lardy and Wellman (9), Lowry et al. (12),Chance and Hess (13), and others. Fundamental principlesof negative feedback control of metabolism elegantly illus-trated by the ADP control of ATP synthesis in mitochondria(9, 14) of which many examples are described in the 1955Ciba Symposium on Metabolic Control (15). Analytical bio-chemistry of tissues gave >200-400 AM ADP while NADHfluorometry of stimulated muscle gave

Proc. NatL Acad ScL USA 83 (1986) 9459

col was repeated if the initial "transfer function" was sigmoi-dal, which eliminated the sigmoidal shape (see Fig. 3).NMR spectra were acquired just before each contraction,

using a 1.9-T, 30-MHz phosphorus spectrometer and a 2-cmsurface coil. No "baseline conditioning" was used, and theprecision of the peaks (or areas) was better than ±5.0% for a3-min average.ADP Control of Exercise Performance. The role of ADP

(and Pi) in the control of oxidative metabolism has beenshown (20) to lead to a hyperbolic relationship between work(the velocity of the enzymatic reaction is equal to work) andthe biochemical cost [a velocity (V) vs. substrate (S) con-centration profile]. This is termed the work-cost relation-ship or transfer function. We have shown how the creatinekinase equilibrium can be used to convert from S = ADP toS = P1/PCr with the conversion of Km from 20 juM to itsequivalent of 0.6:

V 1 1Vmax (s1( + 0.6 PCr [2]

This formulation has been used for the evaluation of exercisetraining adaptations, particularly for triathlon and yachtingevents (a 12-m winch man) requiring elite endurance per-formance (21).

Control by the Phosphate Potential [ATP/(ADP x P1)]. Un-der steady-state conditions of mitochondrial respiratory ac-tivity, ATP is maintained constant to within ±20 AM or 0.4%from V/Vmax = 0 to V/Vmax = 0.5 and thus ATP is not regu-latory. In the resting muscle, Pi is 2 to 3 mM as comparedwith its in vitro Km of 1 mM. In exercise at V/Vmax = 0.5,PCr/Pi = 0.6 and Pi 20 mM (PCr + Pi = 30 mM) and thusPi is not appreciably regulatory. The fact that Pi appears ex-plicitly in Eq. 2 is not that Pi (or PCr for that matter) is regu-lating the rate of oxidative metabolism, but that Pi reflectsthe ADP concentration that is the major regulator. The fits toADP(Pi/PCr) control for the athletes of Fig. 1 (below) sug-gests that the simple hypothesis of ADP control of Michae-lis-Menten kinetics seems more useful in these studies thancontrol by the phosphate potential or the energy charge.

Verification by Linearized Graphics. An experimental testis obtained in steady-state exercise from 10-40% of Vmax,emphasizing the low values of V where negligible lactic aci-

2.0¢H\0.

o \o2z .0 4

-t 4. / ^ = 4 S VC = -\go/X~~~~~~~~~~~V

0.5S, Pi/PCr

1.0

dosis and minimal stress of structural tissue occurs. In thiscase a linearized transformation of the equation gives betterweighting of the data as discussed (22).

5= V (Km + S) or1P/P = 1 (m + P/PCr) [3V Vmax V Vmax K + r

In this case, Km is the intercept and 1/Vmax the slope of theHanes plot (22). Fig. 1 illustrates this plot for an elite yachts-man (12 m) and an oarsman. The best fit Vmax is 8.2 W forcurve RY and 34 W for curve RX, and the corresponding Kmvalues are 0.48 and 0.51 for the yachtsman and oarsman, re-spectively. The rowing training apparently requires a greaterendurance performance than the winch operator. We havepointed out that scaling for muscle mass is important (20),and in these cases, the limb circumferences are similar. Thefit requires an assignment of an apparent V to the Pi/PCrvalue observed at rest and values of :'15% Vmax are em-ployed. These are significantly larger than the values expect-ed from studies or isolated mitochondria (14).

Acidosis in Exercise Stress. While steady-state exercise atlow V/Vmax need not involve an acidosis in the enduranceperformance (highly oxidative fibers), many nonsteady-stateprotocols involve exercise to exhaustion** tt and pH1 < 6.5.Similarly congestive heart failure (23) and deficiency of cyto-chrome b (24) leads to peripheral hypoxia and acidosis. H'is a direct participant in the oxidative phosphorylation reac-tion and in the creatine kinase equilibrium. Experimentally,the H' in oxidative phosphorylation is not significantly op-erating in the range pH 6.5-7.5 (25), while the H' in the cre-atine kinase equilibrium appears to decrease PCr/Pj on aone-to-one basis, for example, in response to CO2 inhalation(26, 35, 14). Thus metabolic control by ADP is largely pHindependent while the value of Pi/PCr decreases with pH[log(Pi/PCr) pH].Correction of Pi/PCr for the effect of pH is readily incor-

porated in Eq. 2.

VVmax

1

1+ (6 x 106 [H+] \k Pi/PCr )

[4]

Measurements of ADP in acidosis involve errors of bothP,/PCr and pHi determinations and thus are less accuratethan the determinations of ADP based on Pi/PCr alone.Also, acidosis in muscles of mixed fiber types is difficult toassign, although an equation for calculating P1/PCr has beenproposed (27). In these studies, we have exercised the volun-teers at low V/Vmax and nearly constant pH. Under theseconditions simplified formulations are also possible.

Alterations of the Work-Cost Characteristic: The Effect ofEnzyme Biosynthesis in Human Muscle. The Vmax obtained inthe exercise performance represents the enzyme concentra-tion, E, in Eq. 1 and affords a measure of the effects of exer-cise stress adaptation. In a particular case of the four-weekexercise training of a leg that had been in a cast and thusatrophied (Fig. 2), the increase of Vmax orE in Eq. 2 is calcu-

FIG. 1. Plots of Eq. 2 to illustrate the determination ofKm to Vmand PCr from steady-state graded muscle exercise performance of a12-m winch man (RY) and an elite oarsman (RX). A Hanes plot isused for the equation,

S 1V Vmax (S + Ki)

Thus the intercept is Km, and the slope is 1/V,,,. Vi,, is given inJ/min. VR is the rest work.

**Boska, M., Moussavi, R., Miller, R., Layzer, R. & Weiner,M. W., Society for Magnetic Resonance in Medicine, Fifth Annu-al Meeting, August 19-22, 1986, Montreal, Vol. 2, p. 466 (abstr.).

ttMassie, B., Conway, M., Yonge, R., Rajagopalan, B., Frostick,S., Ledingham, J., Sleight, P. & Radda, G. K., Society for Mag-netic Resonance in Medicine, Fifth Annual Meeting, August 19-22, 1986, Montreal, Vol. 2, pp. 341-342 (abstr.).

#lNioka, S., Chance, B., Smith, D., Subramanian, H. V., Alter, C.,Richardson, M., Gianitsos, A. & Butler, S., Society for MagneticResonance in Medicine, Fourth Annual Meeting, August 19-22,1985, Montreal, pp. 286-287 (abstr.).

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Proc. NatL Acad Sci USA 83 (1986)

In the resting muscle the combined effect of substrate con-trol in addition to that provided by'ADP (Pi/PCr) and Pi con-trol of the phosphorylation rate is given by Eq. 7:

*-* 5/24/83

A-A 4/24/83

Muscle tested: Right gastrocTraining program: Running (a

0 0.2 0.4Pi/PCr

FIG. 2. The effect of exercise adaptation upothe work-cost relationship. Here we plot work rbiochemical costs (Pi/PCr).

lated according to the steady-state versionMenten equation as follows (20). The ratiofor the "trained" (T) to the untrained (U) cthe increased values of Vma,, or E (ET, leveing; Eu, level of enzyme prior to training).For work at equal V and KM for Pi/PCr

ET 1 + 0.6 (PCr/Pi)TEu 1 + 0.6 (PCr/Pi)u'

V 1

Vmax 1 + + K2 + K3'ADP Pi S

where S is 02 or NADH and K3 is the appropriate Km. Prod-:nemius uct terms for interacting controls may also be included (31)aerobic regimen) but will not alter the basic principles of multiple metabolic

controls illustrated here.0.6 0.8 A change of S may account for the shift from the sigmoidalto hyperbolic transfer characteristic observed in the exercise

in the initial slope of protocol of Fig. 3 that represents a sequence of exercise per-ate in J/min against formances starting from rest (test 1) and proceeding in se-

quence to test 10. The "warm-up" phenomenon observedhere may be due to stimulation by exercise of substrate de-livery. Since Pi exceeds its Km in exercising muscle, the al-

of the Michaelis- tered performance in tests 6-10 as compared to tests 1-5 canof Pi/PCr values be analyzed on the basis of two substrate controls, S, =Organ is related to ADP and S2 either NADH or 02. Since the values of V/Vm.Xl of E after train- are similar for tests 5-9, 4-8, and 3-7, we can equate the

expressions for the V/Vmax in the two profiles where theprimed values of S refer to the tests 6-10 and the unprimedvalues refer to tests 1-5:

[5]

At 0.6 PCr/P»>> 1, (low V/Vma,), the relationship is simpli-fied.

V 1

Vmax 1 + K1 K2S S2

Simplifying,ET (PCr/Pi)TEu (PCr/Pi)u [6]

Thus the slope of PCr/Pj values as measured by phospho-rous magnetic resonance spectroscopy is equal to the rela-tive enzyme biosynthesis and a 3.1-fold increase of E is cal-culated for Fig. 2. This simple equation may also be useful inquantifying changes of enzyme level in exercise perform-ance, in dystrophies, and in genetic disease.

Effects of Substrate Delivery in Skeletal Muscle. Evidencefor additional complexities in muscle exercise suggested bydeviations from the hyperbolic form of the transfer functionare observed, and a sigmoidal form is obtained. As a test ofthe applicability of this analysis, we postulate that there is aphysiological parsimony of 02 or NADH delivery in the rest-ing state of muscle. If the concentration of these substratesat rest is near their Km values for respiration and ATP syn-thesis, their concentration changes can participate in meta-bolic control. Control by 02 delivery in resting muscle hassupport from other studies, for example, the phosphorousmagnetic resonance spectroscopy studies of Idstrom et al.(28) and the microspectropic studies of Pittman et al.§§. Den-ton and McCormack (29) and Hansford (30) suggest sub-strate (NADH) control in heart tissue. The analysis is com-patible with control by either substrate.An approximate fit to the sigmoid transfer characteristic

was obtained by assuming that the 02 delivery was propor-tional to Pi/PCr that in turn altered Vmax, shifting the valueof Pi/PCr accordingly (20). However, the enzyme concentra-tion E can only be increased by biosynthesis, a slow process.Rapid changes of Pi/PCr can occur with the Michaelis-Men-ten control.

S - Si K2 S2 - S2Si x Si K1 52 XS2

We can relate these values to changes of concentration;for example, at points 3-7 Pi/PCr decreases from Si = 0.35to S' = 0.21. If K2 = K1 then S2 = 0.21 and S = 0.35. Thechanges of the additional substrate can be numerically equaland opposite to those of ADP to provide the left-shiftedtransfer characteristic.

Cardiac Control Characteristics. The transfer function orwork-cost relationship for cardiac tissue is very steep andpossible explanations are discussed. The change of Pi/PCrwith the rate pressure product is very small in the heart of an

16

14

12

.C 10

.c o-E0

4

2

a!~~~~~~~'-10mA' "*5 A'

/// /// 7

I/

/7A/6 ./2

0 0.1 'I 0.2 0.3 0.4 0.5Pi/PCr

0.6 0.7 0.8 0.9

FIG. 3. Altered work-cost performance due to supplementarycontrols of oxidative metabolism. The performance test is consecu-tive from 1 through 10. The greater efficiency of tests 6-10 is attrib-uted to increased substrate delivery.

a)

'M

~c

0)

4-

Y o)

I.

M

[7]

1

K1 K2.1 +

St St51 S2

[81

§§Pittman, R. N., Ellsworth, M. L. & Swain, D. P., Proceedings ofthe IEEE Seventh Annual Conference ofEngineering in Medicineand Biology, September 27-30, 1985, Chicago, pp. 536-540.

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Proc. NatL Acad Sci. USA 83 (1986) 9461

6.0-

5.0-

5 4.0

x

L- 3.0mx

T 2.0

PCr

ATP

,m1.0 ,'

I,1,///

0 0.5 1.0

Rest Pj/PCr(State 4)

15 10 5 0 5 10 15 20PPm

FIG. 5. Illustrating the low inorganic phosphate content of theworking heart in vivo. The small peak labeled Pi may in part be con-tributed by phosphate compounds in the erythrocytes seen throughthe heart wall bv the NMR probe.

FIG. 4. The work-cost characteristic for an endurance perform-ance heart (beagle, A) and a neonate heart (lamb, *). In the beagleheart, there is an effective homeostasis of the phosphate potential(ATP/ADP x Pi). HR, heart rate; BP, blood pressure.

adult endurance performance dog (Fig. 4). Measurementswith a piezoelectric crystal attached to the myocardium veri-fy the increased work of the isoproteronol-stimulated myo-cardium. As a control on the method we have also studiedlamb heart where the feedback loop is less tight and wherethe transfer (work-cost) characteristic is similar to that ofskeletal muscle, suggestive of a smaller degree of enduranceperformance adaptation.

Multiple Controls of Cardiac Bioenergetics. An explanationfor the heart work-cost anomaly can be found in the under-lying biochemistry and physiology. In this case we considerthe control of oxidative metabolism by the enzyme concen-tration E and by all the parameters of Eq. 1. The relativevelocity V/Vmax can be expressed by an extended steady-state Michaelis-Menten equation for multiple noninteractingcontrols (for variable pH see Eq. 4 above):

V 1Vmax + + K2 + K3 + K4

ADP Pi 02 NADH

The actual value of Pi in the beating heart spectrum shownin Fig. 5 is uncertain due to its small value and to possiblecontributions due to 2,3-bisphosphoglycerate of the erythro-cytes. Nevertheless, we can set the maximum value of Pi < 1mM. Thus PCr/P1 > 5. Evidence for NADH control is basedupon the observation in perfused heart that glucose is a less-effective substrate than infused pyruvate (32), which we in-terpret to indicate that the fraction of Vmax available withglucose as a substrate is less than that with infused pyruvate,a consequence of NADH control of substrate delivery. Inview of the low free Pi values of the heart in vivo, we proposethat Pi is regulating the glycolytic rate and, hence, the deliv-ery of NADH to the mitochondria. For example, regulationof the flux through the glyceraldehyde 3-phosphate dehydro-genase by Pi may control the pyruvate level and, hence, theNADH level. It is also possible that Ca2+ regulates the activ-ity of pyruvate dehydrogenase making regulation of NADHan important factor in minimizing the variation of ADP andPi with changes of work of the heart (29). Oxygen deliveryitself is regulated by the flow through the microvasculatureof the heart in accordance with the work of the heart andthus may exert a further control of the transfer characteris-tic.

An essential feature of multiple controls is that the con-centration of the control chemical lies within the range ofKm, i.e.,

9462 Biochemistry: Chance etalP

SCHEMATIC DIAGRAM OF ADP, OXYGEN, ANDSUBSTRATE CONTROL OF TISSUE METABOLISM

( triple control )

ATP-ase

FIG. 7. A block diagram indicating multiple feedback pathways that can be operative in the heart to create a homeostasis of phosphatepotential.

genase activity (pyruvate and ketoglutarate) and thereby in-creases substrate delivery to a substrate limited oxidativemetabolism (state 2). Denton and McCormack (29) illustratea number of transfer characteristics for Ca' control of dehy-drogenase activity that suggest a very high gain for the over-all system from muscle twitch to ATP formation.Comparison of ADP, Oxygen, and NADH Control. ADP is

remarkable in the simplicity and efficiency of its control ofelectron transport and ATP production; a single chemical ina very low range of concentration activates electron flowthrough over a dozen steps of electron transport includingthe multisubunit translocase and ATPase in a time of 70msec (33). Changes of [ADP] (and in indeed Ca2+) galvanizethe function of the respiratory chain in response to energyneeds in a minimum of time and with a minimum of concen-tration.

Control of electron flow by oxygen or NADH is less effec-tive than ADP or Ca21 control because it is countered by theremarkable property of the respiratory chain to compensatefor any deficiency of oxygen or substrate by alterations ofthe redox state of electron carriers, a "cushioning effect."This cushion acts so effectively that the titration of cyto-chrome a3 by oxygen differs from that ofNADH by a factorof 10 (34).

This work has been supported by Grants HL 31934, RR 02305,HL 18708, HL 06817, NS 22881, HL 36010, and AA 05662 from theNational Institutes of Health; the James S. McDonnell Foundation;and by the Benjamin Franklin Partnership's Advanced TechnologyCenter of Southeastern Pennsylvania.

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17. Jacobus, W. E. (1985) Annu. Rev. Physiol. 47, 700-725.18. Meyer, R. A., Brown, T. R., Krilowicz, B. L. & Kushmerick,

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28. Idstrom, J.-P., Harihara Subramanian, V., Chance, B., Scher-sten, T. & Bylund-Fellenius, A.-C. (1985) Am. J. Physiol. 17,H40-H48.

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Leigh, J. S., Jr., Veech, R. L. & Forster, R. (1987) Am. J.Physiol., in press.

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