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MwGNiiK R6UN.nr( IN Mem(l\E 7. 143-ISS (1988) 

Acute Volume Loading Studied in Cat Myocardium with 31P Nuclear 

, Magnetic Resonance• 

MARY OSBAKKEN . MARIE YOUNG. JUDY HUDDELL. JEFFERYROSTER. 

MANFRED PRAMMER, AND BRITTON CHA\IC[ 

Drtnnmaus nl.inerrAma and Brmkemrun/&orArnn, l'anwaor!fPrnnsytrome . 

PAiladtlphm. Penmlitnnsa 19100 

Rettitrzd Aupul $. 1987; rerited Nonembn 19.1997 

Tu nudy the nfrets of acute tolume irodine on myorardW meubotic and mcchanid 

funnion- se.m ens ..rt .ernrne loadrd vla anauorrmsis of the aDdomioal aom r9 Iht 

.tnap+a(AVShunIL hleuboliceRecauxnnalumr .d oiN"PnudarmaTneurnaonanre 

(NMR) . MecMniral funnion -arrr .lumN•im 1mn rate x I.ywac nbod peasuepadmi 

(HR x SOP) . Shunts rete opened (or 1-2 h durin8 .hich timt pnuNhorreanne (PCr) . 

Wenosinr rripbosphan (ATP1 . Inor,amc phosplutt (P,L and HR x SOP a,rae monitomt . 

Hirh~erg,v phosphate enerteues asdeeennined bf P,flY'rand P(WATP onmrerr com . 

re6tcd with HR X SBP.Openin8 of ine AV aaunu wm arsocialed .ith an inrrcue (four 

ntsl or a dernase (thrce nn1 ao HR x SBP . P,/PCr ratim increased and PCNATP rnins 

demeased in r.n ..ilb an innesu in HR x SOP. In nt. .itb a dernuse in HR x SBP . 

P,/PCr and PCr/ATP genenlp- drd nm clranet siani6o .ntly. In summary . amu rMume 

leadilra could be aROrlnled elih aE illnraR or derleaM in m)'neardial MICIaa1 xVrk ar 

evaluated bf HR X SOP. acroc .rdid 

meehanial Ioadin8 can bt naluated .ita "P Nh1R technlpun and may pra.ide insiaht 

into in Wro meutnlic eontml mrrhenums.

144 oSaAKKEV FT .V .. 

are these changes^ It is postdlated that tin understanding of thest mechanisms in 

animal models mav lead to a better understanding of similar processes in a clinical 

setting. 

)n the following studies. s'P nuclear magoetic resonance (NMR)was used togenemte 

data concerning phosphate rrdetabo)ism (IK'r . phosphocreatine: ATP. adenosine ariphosphata 

P„ inorganic phosphate). Ratios of P,/PZ-r and PCr/A'iP were used to 

estimate phosphorvlation potential (PP) ahd adenosine diphosphate (ADP) coneentration, 

both potential regulators ofoxidatna phosphorylation (24) . The P,/PCr ratio 

would be a better estimator of oxidative pho-cphoq-(ation based on the equation 

PP' J~ADPT]P) . l P~l x KcK jCrj ' [I) 

where KcK is the equilibrium constant ol' creatine kinase . H' is the hydrogen ion 

concentration. and Cr is cteatine (2f. =t) . Howe.rr• at times it is impoisible to interpret 

information concerning the P. peaks because of its contamination by 2 .3-diphosphoglycerie 

aeid (2J-DPG) . Therefore . the second ratio. PCr/ATP, can be used to provide 

similar. if not as sensitive. informaion concerning bioenergetic control mechanisms . 

Aafrnal Preparoriwr 

METHdDS 

Nine cats were anesthetized with nembutal (20-30 mg/kg. ip) and intubated for 

respiratory maintenance. (Onl% data from seven cats are presented because the shunts 

were found to be paniallp Ihrombosed in ~tw•o cats at the end of the experiment .) 

Anesthesia was maintained b% intravenous nembuWl (10-20 nrg/kg, iv) . Intravenous 

and intraarterial lines were placed for fluid and dmg administmtion, and blood preswte 

and arterial blood gas monitoring . nespatisrly. Median stcrnotomy was done to expose 

the hean for surface coil placement . A pericardial cradle was created for support and 

movement of the hran within the magnetic frcld (to help position the hean in the 

sensitive volume of the magnet) . A Iwo-turn . 1-cm-diameter surface coil, tuned to 

33 .8 MHz for phosphnrus and 8$A MHz for protons• wasattaehed to the left rentricle 

with cyanoaccrylate. Creation of an abdominal aorta to vena cava shunt was done 

using 3-mm (id) pol.rthylene tubing which was inserted between the two vessels (/? . 

7f). This shunt w-as kept closed wirh a plastic roller clamp until volume overload was 

required . Each animal was maintained under stable physiological conditions by monitoring 

and maintaining anerial Wood gases within physiologic range and by mainte• 

nance of volume and blood pressure status with fluid administration as required . 

X.t fR JYudres 


Cats were placed in a plexigla$s holder and introduced into a 2 .1-T . 23-em-bore. 

Oxford magnet . The surface coil used for transmission and reception of radiofrequency 

signal was tuned to 35.8 MHi (phosphorus) and 88 .4 MHz (protons). The animalsurface 

coil-magnet combination was made as homogeneous as possible with the magnet 

shim coils (shimming was dorfe using proton signals . 'H NMR). Pulse width (P W) 

optimization was performed for each animal-coil interface . For this procedure, a 

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VOLUME LOADING EiTECrS ON THE HEART USING "P NMR 145 

series of pulses of diH'erent dumsion'(15-50 us) was applied and spectra were acquired 

with a delay time of 4 s. The pulse width which provided the best signal/noise for PCr 

and ATP was used for radiofiepuency stimulation for each expnimmt . Opttlmat pulses 

were between 35 and 30 µs . 

The schematic repraenlation of pla,ccment of the 1-cm-diameter surface cail on 

the heart is shown in Frg 1 . Practically a)1 the NMR signal cantributing to the spectrum 

was generated from the indicated sensitive region . which extends from the heae sullhtt 

by 4 mm into the myocardium and is roughly ellipsoidally shaped with a diameter 

varying from 12 to 13 mm . These data were generated by integrating over the threedimensional 

modulation profile of a suPface coil, BI x sie(ryBJ . 8ased on this infor 

mation, we do not expect an appreciabfe amount of contamination of the muscle 

spectrum from blood within the ventricle. 

t1P NMR spectra were obtained using respiratory and electrocardiographic gating . 

To do this , the following procedure was used . The respirator was outfened with a 

switch which was turned on at end expiration . Respiratory informarion along with 

ECG signal was fed into a uiggering lrox . (built in our lab) . The first QRS. on the ECG, 

observed by the sensing device in the triggering box. after end expimtion was used to 

trigger the mdiofrequertcy pulse for NMR spectra acquisition . Each spectrum consisted 

of 100 seans obtained with a delay tiine of 4 s(based on preset respiration rate main• 

tained by the ventilator) and an NMR acquisition time of 200 ms per wan, for a total 

time per spectrum of approximarely 6 min . 

Since the TI's for PCr. d-ATP, and P, are approximately 4. 1 .5 . and 1 . S s. respectively 

(26), saturation e&cts wnuid result from using a delay time of 4 s . However. using 

longer delay times would make data collection times inordinately long . Therefore. 

MY[e aqe 

qe~ 

C~CM Yt~Yn r, NrO .~ 

fuYm M 

.aep.M1 n,rpr . 

F•lo . 1 . Cwn•stcuond diatram of tulfatt eui7 placement an the heart weh a pmale urUhe eendti .

® 


146 oSBAKKEN ET AL . 

sereral sets of experiments were performed to determine the etfects of saturation . 

Delay times (TR time) of 4, 8 . 12 . 16. and 20 s were used . No change in signal/noise 

occurred for ATP or P, with a TR time of 4 s or greater . The PCr siplal increased 

progressively up to a TR time of 16 s(16% incpease from a 4 f TR time), with no 

funher increase in PCr signal with longer TR times . Since only one p:ak, the PCr 

peak, would experience saturation effecu and because we were osing taYeo data and 

not pertonning absolute quantification, we chose not to apply a correction /atctor for 

saturation to our data . 

The Pmblinn uf glotd ?.3J)ipposphughar~dre 

Samples otblood from three cats were taken and placed immediately in NMR tubes 

and studied at room remperature with 1aP NMR using a 12 .4-T magnet to determine 

the quantity of 2 .3-diphosphoglrceric acid in the btood which might contaminate the 

heart spectm under in rnn co:n3itions . Spectra wers obtained using melhylene diphosphonic 

acid (Meth Phos) as a standard. as wasdodie in trro. Inaddition. NaH2P0s 

was used as a P, reference . and 2 .3-DPG. PCr. and ATP were added sequentially to 

the blood to determine the relation or the chemical shi/t of blood peaks to those of 

known standards (Fig. 2). There was no evidence of 2•3•DPG in the blood spectra. 

B n. S~sbo•n 

.n. .+w 

C n~em n~.r N1 

froneem btt•^t . K' e// ~ 

rw.r t .e¢ota 

~ tla. 

II I a 

Fq. 2. B1ood fm

VOLUME LOADI.'G EFfEC13ON THE HEARt USING "P NMR 147 

This inditates Nat 2 .3-DPG is present in too small a qudntity to cause a problem with 

contamination of in vira hean spectm. There were twro blood peaks (peak 1 . peak 2) 

in the s'P NMR spectra of cat blood . Peak one had a chemical shift consistent with 

dtat of P, . Peak two had a chemical shift consistent with that of a phosphodiester . 

Thus.any increase in the P, peak ofin viraspectm would most likeiy be that generated 

fmnt intracellular metabolism . 

Pln:ciologira/ lmrnrntirnr 

~ After control spectra were acquired . volume o.Yrload (high output state) was produced 

by opening the previously constructed abdominal aorta-vena cava shunts . a'P 

spectra were obtained sequentially at 6-min intervals o .er I to 2 h or acute volume 

loading. Upon opening of the AVS . all cats had the typical increase in pulse pressure 

which is a good indication that the shunt has been opened . 

N.71R Data Anahsis 

Each free induction decay (FlD) was Fourier tmnsformed and filtered with 10 Hz 

line bmadening Spectra .verc phased and curve fi0ed(Lorcnt8an) using a least-squarex 

fit algorithm. Areas under each peak were integrated . Each spcctrvm contained the 

following peaks from left to right : inorganic phosphate . phosphocrcatine. and adenosine 

triphosphate (ATP: T . a . tT). Figs. 3 and 4. Ratios of P,/PCr and PCr/ATP were used 

to estimate the slatc ofoaidati•r phosphopiation . The chemical shift of P, with respect 

to PCr was used to determine the pH of myocardial cells during physiological intervention 

. 

To determine the reproducibility of"P NhfR data, the following was done . Under 

• contrd conditions (no physiological intervention) . five sets of spectra were obtained 

at the beginning of each esperfinent and at the end of each experiment for each cat . 

P,IPCr and PCr/ATP data were averaged and the standard deviation was determined . 

TTere was ±6% variability for P,/PCr ratios from acquisition of one slxctnnn m the 

next and t 108% variability for PCr/ATP ratios. Thus, there is mme random variability 

in "P NMR data which must be acknowledged when responses to physiological in• 

tervention areevalualed . A change in the P~/PCr mtio of greater than 61, and a ch ange 

in the PCr/ATP ratio of greater than i0't, must ocrur for a responsc to be considered 

due to physiological intervention and not due to random variability . 

Bioenergetir data as determined by P,/PCr and PCr/ATP ratios were corrclated 

with mechanical function as determined by hean m1e x systolic blood pressureprodun 

lHR x SBP is an estimate of oxygcn consumption (27)) with a linear regression algorithm 

. In addition, in order to follow changes in the above-mentioned parameters 

• with time and intervention (i .e . . to observe the effects of continuous modulation of 

feedback control ofchanges in wark load on metabolism and vice versa). percentage 

changes of P,/PCr, PCr/ATP. and HR x SBP from control were plotted with respect 

• to time and intervention . 

• 


RESULTS 

Although all animals were prepared and treated in a similar manner concerning 

maintenance of stable physiological conditions (maintenance of arterial blood gases . 

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149 OSBAKKEN ET AL 

a,,,. ena 

w M e 

^ . t : hta . .ric' 

flG . 3 . tompaile gaure of syeetri from a ar who ItapaMed to arule ancnrl-•enoYf Shumina rAVSr 

aith an increase in HR x SBP. Note that openina af ine AVS wat avonned ith an inneam in the as. 

under the P, pesk and a dennne in the uea under the Pt7 penk- flownr of the shanl %w associated %ith 

the relurn of P, and PCG peaks to neal avnuol lerclx Vme that the ehe/ni Wshia di8nenre bnuten p and 

PCe wu connant indicating Ihat PH did nol change durins •nlume loading Melhyienr diphovyhonic acid 

tMeah Phmr was used as a standard todelemmne that Ihe mqmerie Re1d/Bs1 rHnained tonstam Ihrouthout 

the eapenment P, inorganic Phosphure PCr . phosphocrtatinr: and ATT, adenodne tnphotphte . 

blood pressure. and volume status in physiological mngesl . metabolic and HR x SBP 

responses to acute volume loading nere .ariaMe for the different cats . Cats 1 . 3. 5 . 

and 6 had an increase and cats 2 . 4 . and 7 had a decrease in HR x SBP during acute 

volume loading. This may he due to intrinsicdiFerences in baseline physical condition 

or due to variable refle% responses secondary to imposition of volume load . Because 

these phenomena are not uncommon in clinical situations, ur think it is valid to 

preamt our data as such . 

Acute volume loading was not associated with a change in intmcdlular pH . as the 

chemical shiB betazen the P, and PC7peaks did not change during orafter completion 

of acute volume loading (as can be seen in representali .r spectra in Figs. 3 and 4) . 

After exlensive analysis . we determined that data woubd be most meoningfull,v rcporud 

by using a graphic presentmion ofpercentage chanibe from control ofinetabolic 

and mechanical fbncrional pammeurs. and a graphic presentation of the correla• 

tion analysis of raw data and percentage change Itom control data parameters 

(Fgs. 5-10). 

There were t.vo general tppes of HR x SBP responses resulting from imposition of 

the acute volume load of an AV shunt on the hean : krut cats (1, 3. 5. and 6) had an 

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VOLUh1E LOADIVG ER.r(TS ON THE HEART USING "P NMR 149 

~ . Rn A . n1b' 

~\.. .. ../w' V 1I~`M^b 

Fla. 4 . Comlwute 6purc of spmn fmro a aaw .tno mpondrC to .rnle anesial-.xnous sEuntinr IA VS/ 

nilh a dntease in HR x SBP . Nole aat ,q'reirl opening of thr,huni aau asaonaed uith a $111111 innease 

in P, and slighl derreasP ; PC, peaL. . Mowtn. ahhourh sAen am 0unwnons In both peakt dnh 

minimal ehanm in both I . .aks ottVnM airouthnm rhe semmndnofmaimrnm•e ohhe nnmd-anour 

shunt . Spergra obtained aRer Hmure of ine shunt sho.d rhar P, irc¢ned and PCr d[deased slighti) . The 

legrnd u the Rme as that for Fig . I . 

increase in HR x SBP. and three cats (1, . 4_ 7) had a decrease in HR x SBP . Reprcsenmtice 

spectra throughout snlume loading for croswith ahe t ..-o di/ferent typrs of 

HR x SBP responses are presented in Figs . 3 and 4.IVotc that in cat 6 (Fig. 31• the P, 

peak increased and PCr peak decreased during volume load . while in cat 7(Fig 4) . 

there were minimal changes in these parameters. As can be seen from group dam for 

all cats presented in Fig . 5 . the bioenergetic responses to volume loading teqd to 

correspond to changes in mechanical work estimated by HR x SBP. Although not all 

of the P,/PCr or PCr/ATP responses w•ere gmater or less than 6 and 1011 . mspectisrly, 

which was considered necessan' for a response to he considered due to a ph)siologir 

intervention (determined via error analysis) . them wxreclear trends. When HR x SBP 

increased. P,/PCr increased and PCr/ATP decreased . When HR X SBP decreased . 

the R/PCr and PCr/ATP rrtios generally did not change significantly . 

Cortelation of P,/PCr ratios and PCr/ATP ratios viih HR x SBP was done for all 

cats and data are presented in Figs . 6 and 7 . For tsll cats, excepl cat 3 . the P,/PCr vs 

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150 OSB{KKEx ET AL 

r 

w 

Cot 1 Cot 3 

aul R ^ p"^ " " 4eRRSgCRRSPGg ^_ 

~rcggcgY°- v ~_ t 

~ s ti r L r ` t Y 

1 .] 1 

Co12 

••4qy.1 : 6~~CR^n$a[ir$ 9 


Cot 4 

An 

-/L 

Cot 5 ~a Cot 6 

e P./PCr , 

• PCr/0.TP 

Cot7 nleRwSBP 

- ~ccccxsrcas =f - F .^ asa stffc } 

FlG. 5 . Metabolic and meAunrral respnmrs to araje anenal-trnout shunts L41'SI in sr .en pn. Data 

tle pesented as pr+KnLigt changt from cnntrul (Su Y, J. 3- and 6 genenllv had an mctrase ,n HR x SBP 

during rmpoutlon of thunr tondmonf Cars 2 . l and 7 genrmlh had a dernaac m HR x SBP dunng 

impovuon nf .hunt eondniom The meut+obc pnameten IP,/PCr and PL-r/ATPI Parallel rhanges on mt . 

duniul panmeten tHR x SRPI: that is, am mivease in HR N SBr,canmmed .nlh am mmase in P{PCr 

end d.ertase in PCr/ATP nuos A derrease in HR x SBP is generally auwnaud ..iM a sl,ght ,nneau . 

drnrate. on rw change tn O,/P('r arM a 9rgln derrtau, mrrease, or no change on PCR!ATP ranos . P, . 

Inorgamr phosphate: PCr. phouphoc¢aone: ATP, denoune ttiphmPhn2 HR. hean ratr. and SBP-slxobr 

blood pnssure . 

HR x SBP relationship appeared to be quite stable : i.e. . there was minimal change in 

P/PCr for any change in the HR x SOP (Fig . 6) . The PCr/ATP vs HR x SOP r41ationship 

also appeared to be quite stable . with very little change in PCr/ATP with a 

change in HR x SBP (Fig. 7/. 

Evaluation of comMned cat data . for the entire volume loading time . demonslrated 

that PCr/ATP almosl always decreased (even if the decrease oas sometimes small) 

after opening of the AV shums. regardless of the HR x SBP /esponse (Fig . 8). In 

addition. P,/PCr generally increased in conjunction with a decrease in PCr/ATP for 

all points of time throughout acu7e volume loading (Rg . 9) . When changes in P,/f°Cr 

were eorrelated with changes in HR x SBP for all points in time for the entire grolap . 

there appeared to be a trend towald linearity (Fig . 10) t+'ith P,/PCr increasing as 11R 

x SOP increased . 

DISiL351O.`t 

There are a number of hypotheses concerning the tissue events which may lead In 

myroeardial eell damage and/or death (-'8--i1): ischemia . hypoxia . acidemia. oxygen 

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VOLUME LOADING EFFFCfS ON THE HEART USING 'P Nh/R 

a .,cm L-r 

> • i 

cnr S cm h ,Cm s 

• ~r t 

a .~ 

` a ~:;~ 

• 

s T 

.~~~•rrs 

• • or 

. a e w r• a ~o r• a s 

w ..e . 

frcc mdicals. abnomlal calcium metabolism, lactate accumulation . pusrinsult Vascular 

changea, and possibly bioencrgetic changes. There have been many inveatigations tonmrning 

the eBccts of ischemia and/or h,vpoxia on energv metabolism of dte hean . h 

~ is now known that both of thesr conditions are associated aith changes in oxidative 

phosphorylation (dd-33). Although much is understood about rhe ellects of these 

stresses on energy metabolism. less is known about the'etTecl of other types of stress . 

. 


~ a . •• e m r a m • e eo ~ r . r e ~o 

o s Cor 2 6ar n Cnr 7 

Flc. 6. forrelaron of HR X SBP rirh P,IPCr fou se.en sau durins acme aruriol-nnom shunung . Note 

auu t


I 

--Srt-EO 

SWn1 . MNT 

e1a. S. RcLtiomhip /aRer AV snum opeoed) of 1he .1>


t 

VOLUME LOADING EFFEe'fS ON THE HEART USING "P NMR 

00 

wn•» 

ss 

, 1 •ea.a .np 

• 1 • Co~6_n .q 

•[ .nqe n .nt. 

FiG . 10. Relatiomhiptaher AV alunl openedl d,htpenYn4yee chynPe frum eunlm7 ofP,/P(Y to pefeenteae 

ehang from eontrol of HR a SBP for all points,n time dur,np olume loading for all u .tn alt, Nme thn 

then appears to be a Iinear tetannn .hip 4vuxn tAerc pnropcd vrfaMet narch wat not ew{hm w-hen 

individual data utrc anaretd :,.e .. as HR x SBP increased P,/P(T intremed Thn indican tnn as rn& 

load mcro0.s. meaboOa mdummi, Iregulnionl of oudun .e phorpnmllmion is prubabl) appmpuate for 

the incrnsad merhamcal orL loads . 

Acute volume loaditly produced by creation of abdominal aona .•vena cava shunts 

(12. ?1 . 36) was associated with variable changes in NR x SBP . some cats with an 

increase and some wilh a decrease in HR x SBP . These responses were probxbly 

closely relaed to each animal's baseline homcostalic condition and/or ability ofregex 

recruitmem secondarc to volume loading . Metabolic responses to .nlume overload 

as reflected by changes in P./PCr and PCr/ATP generally paralleled mechanical re• 

sponses : i.e. . an increase in HR x SRP was associated with an increase in P,/PCr and 

a decrease in PCr/ATP and a decrease in HR x SBP was associated with a slight 

inerease . decrease . or no change in P,/PCr and a slighl decrease. inerease. or no change 

in PCr/ATP. Hoariern when group data were analyzed 

. the P,/PCr ratio wasgenemlhincreased during volume loading and appeared to be Iinearl,v related to HR X SBP• 

even though this relationship was not linear in each individual animal . This indicates 

that the P,/PCr ratio may be a go,x1 measure of metabolic induction and stability 

during myocardial loading conditions (when _^,3•DPG does not obscure the P, peak) . 

The P,/PCr response to volume loading generally appeared to be greater than the 

PCr/ATP response. This is reasonable because both parameters (P, and PCrI of the 

P,/PCr ratio change during intenenlion . whereas only one parameter of the PCr/ATP 

catio ostensibly changes. These changes in P,/PCr and/or PCr/ATP ratios may regulate 

the increase in oxidame phosphor)1ation whice may result from increasing the work 

of the hean: i .e. . phosphorylation potential as eslimated by P,/PCr and ADP cmncen• 

nation as estimated b. PCr/ATP appear to be regulators of myocardial metabolism 

during acute volume loading . 

In conclusion . acute .rolumc loading in cats uus associated with hioenergetic changes 

as measured by "P NhlR . Oxidative phospor.lafion . asestimated by P,/PCr and PCr/ 

d,TP ratios. eas changed during acute snlume overload as dem,mstrated Man increase 

and/la decrease in P,/PCr and PCr/ATP. The changes in P,IPCr and PCr/ATP may 

ah as mesxngers for enryme induction to ultimately increase oxidative phosphory- 

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154 OSBAKKEN ET AL 

Idtion (state 4 to state 3 : inactive to aetire state . although since hean is always working, 

it is always in some sense in slate 3) and protein synthesis, which are compensatory 

mechanisms associated with more ehornic volume loading conditions If the work 

Iqad increased (} HR X SBP). there would be greater activation of oxidative phos . 

phorylation ; if work load did not change signigcantly. there would be no need for 

flhnher activation of oxidative phosphorylotion . It may be possible m use measurement 

of these phenomena in the following way to estimate the noetabolic reserve and/or 

viability of the hean under clinical conditions where changes in myocardial loading 

occur. Small changes in P,/PCr and/ar PCr/ATP ratios may indicate that there is 

considerable metabolic reserve and thus myocardial function 6s stable . Larger changes 

in these ratios may indicate that myocardial metabolism is strverdy tased and there 

is minimal metabolic rcsen•e, thus making myocardial function relatively tess stable. 

Fa,nher studies using J' P NMR techniques will be needed to rnore speciflplly suppon 

tlv.ese hypotheses . 

ACKNOWLEDGMENTS 

The wthnD7lunkluhn HaH18rDte and laek L


VOLUME WADING EFfECFS ON THE HEART IISING "P NMR 155 

23 . B. CIe+N[E . J. S. LEIDH . J. KENr. K. MCCVLLY . S. NIOIU, B J . CwDN . J. MARn, AND T. GIUHAM. 

Pmr. NaN. Aaed. Sci LSU 113, 9438 f 19161 

26. H . L KANroR . R . W. BlaD6i K . MeTi AND R S BuAUN, Amn. J. Cardrot 231 (lJrort Cin 

PAI+IaJ. 20). H171 (19861 

27. S, 1- Swtwnrs, E BuUNWwLD . G. R WEICH, 1N.. R- R CASE. W. K STnINCeV. AND R . MAC71vi. 

Amm^ J. PAISioA 19L 146 (1959) . 

28. 1. KooANw A. WIInE . M. l. lAem D. DAODER, AND K. YAreADa. l. d/oL Cef/ CardiaL 16. 247 

(1984 

19. B. Hw1aNU.ro AND M. L Hcs.IACC 6I1 L 215 (19851 

30 M. SN~. . P. F. KAat . U. V . WwDt.'s, AND M. M. KIasH . C~mfanar 66(1). 485 (1962) . 

31. M . D. SfuN. A. 1.1. CHiEN, M . C. CArAOnOSA . D.1. PnTa AND E . C. Lucor7A. Cnr Bn. l6161. 

699 (19851 . 

32. T. NA~uaism H. H . Yot1ND . T . SxIN¢o. K . ND~NNxE. AND l. G1 . IAID~utwm. J A/d Ca•droJ 16 . 

519419941 - 

!l. R. Aww. M . TANUnA. AND 1. TAMAZnN1. (1rc. 8e. 51448 (19AJ1. 

JI. I:. H . HDEEnMD. B. . 0 EmsaD~.l. KIEwnE/L R. MAvND,AND K.O . R06FN.Ana IkJSId. SnrtQ 

11). 111981). 

37. E G . Kwa[ AND A. WmacN11exDEA. Adr. Crr NvW. Bn. 12, y911980) . 

36. P. V . HAn . K. RnN~N, P. Gasmm..U, M . LArut4, AND F. L7UilwuD. Barvr Rn Co.dlol 7S, 

103 (1980). 

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