Cardiac #{149} Robert

Myocardial Metabolism in Patients

High-Energy Phosphate and Allograft Rejection with Heart Transplants’

To determine high-energy is altered

whether phosphate in cardiac

patients

undergoing rejection, 14 patients with heart transplants were exammed with image-guided, one-dimensional, phase-encoded surface-coil phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy on 19 occasions 39-2,021 days after transplantation. On average, patients underwent mild rejection (detected with endomyocardial biopsy) and had a reduced ratio of anterior myocardial phosphocreatine (PCr) to adenosine triphosphate (AlT) (1.57 ± 0.50 [standard deviation] vs 1.93 ± 0.2; P < .01) compared with that of 17 healthy control subjects. Ratios of PCr to inorganic phosphate also appeared lower whenever detectable. However, P-31 NMR spectroscopy did not permit reliable identification of patients who required augmented therapy for rejection detected with biopsy either on the day of the P-31 NMR spectroscopic study or at the next scheduled biopsy 10-140 days thereafter (sensitivity, 50%, and specificity, 73% with use of cardiac-averaged PCr/ATP values for each heart; sensitivity, 88%, and specificity, 55% with use of the lowest myocardial PCr/ATP ratios measured in each heart). Index terms: Heart, radionucide 51.1299 #{149} Heart, transplantation, netic resonance (MR), phosphorus 51.1214 #{149}Magnetic resonance copy, 51.1214 Radiology

1991;

181:67-75

G. Weiss,

#{149} Christopher

PhD

MD

myocardial metabolism allograft

MD

J. Hardy,

Paul A. Bottomley, PhD William A. Baumgartner,

studies, 51.459 #{149} Magstudies, (MR), spectros-

C

ARDIAC

allograft

a major morbidity

cause

in patients

rejection

remains

of mortality with

and

heart

transplants, and its early detection and treatment are crucial for optimal graft function and survival (1-4). Unfortunately, few reliable techniques exist for diagnosing rejection. Currently, histologic evidence of myocyte necrosis from serial transvenous endomyocardial biopsy is the only universally accepted diagnostic criterion (1). This procedure is invasive and has a small but inherent risk. Moreover, because early rejection can be focal, multiple tissue samples are required, rendering

sampling Noninvasive

biopsy

and

results

interpretation techniques

subject

to

error. that use elec-

trocardiographic, echocardiographic, and/or blood measurements appear to lack sensitivity and specfficity in

phocreatine (PCr) to adenosine triphosphate (ATP), and PCr to inorgaic phosphate (Pi), or ATP/Pi at, or prior to, the occurrence of prominent histologic evidence for rejection and prior to the occurrence of LV dysfunction (6), compared with ratios measured soon after surgery (6,9) or in animals

with

isografts

(7) or in ani-

mals receiving immunotherapy (8). Some increase in the ratio of myocardial phosphodiesters (PDs) to ATP has also been observed in heterotopic canine allografts undergoing rejection (9). New spatial localization techniques have now been developed for surface-coil P-31 NMR spectroscopy that permit noninvasive detection and monitoring of phosphate metabolites in the human preliminary

heart

(10,11),

and

three

conference reports have various degrees of correla-

detection of early rejection, and abnormal left ventricular (LV) function and other clinical symptoms of rejection are not manifested until late in the process, when reversal is difficult

suggested

or impossible

metabolic abnormalities in normal human cardiac allografts and those undergoing rejection and to assess their potential value for the noninvasive diagnosis of rejection, we used localized surface-coil P-31 NMR spectroscopy to study patients with transplants in whom various degrees of allograft rejection were determined by means of endomyocardial biopsy over a wide range of periods after transplantation.

(4-7).

development reliable, and for assessing therefore treatment Recently,

The

successful

of new, noninvasive, reproducible techniques allograft rejection would

be of major benefit in the of transplant recipients. in vivo phosphorus-31

nuclear magnetic resonance (NMR) spectroscopy with surface coils was used to monitor high-energy phosphate metabolism in heterotopic cardiac allografts implanted near the surface of the neck or groin of nonimmunosuppressed rats (7,8) and dogs (6,9). The results showed significant reductions in the ratios of phos-

the GE Research and Development Center, P0 Box 8, Schenectady, NY 12301 (P.A.B., and the Departments of Medicine, Division of Cardiology (R.G.W.) and Surgery, Division of Cardiac Surgery (W.A.B.), The Johns Hopkins Hospital, Baltimore. Receivedjanuary 18, 1991; revision requested March 27; revision received April 22; accepted May 16. Supported in part by research funds from the GE Research and Development Center; R.G.W. supported by National Institutes of Health grant HL-17655-16 and a Clinician Scientist Award of the American Heart Association. Address reprint requests to P.A.B. I

Radiology

tion

between

tive

intensities

human (12-14).

rejection

and

of PCr,

heart transplant To investigate

Fourteen men age, 50 years ±

aged

rela-

ATP

recipients the range

AND

SUBJECTS

the

Pi, and

in of

METHODS 33-61

7 [standard

years

(mean

deviation

From

C.J.H.);

C RSNA,

1991

Abbreviations: phate, DPG

=

ATP = adenosine 2,3-bisphosphoglycerate,

triphosLV

=

left ventricular, PCr = phosphocreatine, PD = phosphodiester, Pi = inorganic phosphate, PM = phosphomonoester, SD = standard deviation, S/N = signal-to-noise ratio.

67

Table

1

Summary

in 14 Male

of Data

Patients Biopsy

Case

Age at

No.

Exam (y)

Time (d)*

Ia

51 52 52

711 739 1,087

53

58

53

87

lb ic 2a 2b 2c

of Prior Episodes

Exam

No.

Medication at Examt

Time

Score at Exam

Treat or Not Treat$

Pred None Pred Pred Ratg

5 5 6 1 1

5 2 4 5 5

T N 1 T T

54

412

None

2

2

N

44 33

48 2,021

Okt3 None

1 6

5 1

T N

52 44

1,521 478

None

2

2

N

6 7

41

8a 8b

44 44

864 875

None None

None

3 3 6

2 2 2

N N N

I’red

7

4

T

None None

1 0 1 2 2 5

2 1 4 0 4 1

N N T N T N

3 4 5

9 10 11

995

43 57 60 61 59 54

12 13 14

39 40 479

Pied

229

None None None

135

3%

Treat

Mean Myocardial

Lowest Myocardial

to Next Biopsy

Next Biopsy

PCr/ATP

PCr/ATP

(d)

Score

Next?*

2 0 1 5 5

N N N T T

None None None None None

4

T

None

NA 0 1 0 1 4 3 5 2 2 4 5 0

NA N N N N T N T N N T T N

0.90 ± 0.04 1.87±0.12 2.2 ± 0.6 0.87 ± 0.28 1.68±0.6 1.72 ± 0.35 1.21 ± 0.07 1.82±0.13 1.58±0.59 1.04±0.05 0.59±0.13 1.14±0.55 2.11 ± 0.63 1.96±0.49 1.64 ± 0.40 1.89±0.29 1.60 1.50±0.42 2.4±0.15

0.87 1.71 1.25

28 95 42

0.79

28

0.8 1.38 1.16 1.62 1.03 1.01 0.45 0.44 1.24 1.46 1.19 1.61 1.6 1.21

28 105 (Died) 119 119 87 140 119 13 10 10 33 89 28 112

2.3

or Not Treat

Echocardiographic Abnormalities

LVH RVdi1 MIIdLVH,mIIdRVdII SHK,mildLVHandLVdys SHK

None Mild LVH, RV dii SHK SHK, mild LV dys SHK None None None

Note-Exam examination, LV dys = global LV dysfunction, LVH = LV hypertrophy, N - no change, NA - not applicable, Okt3 - suppressor T-cell therapy, Pred - prednisone, Ratg - rat thymic globulin, RV dii - dilated right ventride, SHIC - septal hypokinesia, T - treat. * Examination time is the numberofdays at which P-31 NMR spectroscopy was performed after transplantation t Inaeed doses of medications that were taken at the time ofP-31 NMRspectroscopy to manage rejection detected at the precedingbiopsy examination. $ The decision to augment therapy wasbased on results ofbiopsy performed on the day of P-31 NMR spectroecopy or the next scheduled biopsy thereafter Next). I Errors In PCr/ATP are plus or minus SD from two to five measurements

fsD}])

underwent

NMR

spectroscopy

scheduled

examination

visits

with

during for

P-31

rejection

regularly

endomyocardial

bi-

opsy 39-2,021 days (mean, 590 days ± 547) after cardiac allograft transplantation (Table 1). Three men (patients 1, 2, and 8) underwent examination with P-31 NMR spectroscopy on two occasions (a,b) or three

occasions

(a,b,c)

at intervals

of 28-

large-vessel

myocardial

coronary

infarction,

catheterization performed

and within

spectroscopy.

artery

disease

as shown

or

by cardiac

of P-3i

Echocardiography

perivascular

NMR

per-

formed within a week of P-3i NMR spectroscopy indicated normal LV function in all but two patients (patients 6 and 10), who had mild global LV dysfunction. Four

and

and

myocyte

interstitial

necrosis

scores of 4 and higher indicative of rejection

GE

changes

immunosuppres-

whether

might

metabolic

precede

or follow

logic changes,

biopsy

scores

appointments

10-140

days

after

evaluations

copy

were

number cessitated

of P-31

also reviewed,

of episodes augmented

(6). Biopsy

were considered of sufficient severity

augmented

sive therapy. To determine

electrocardiography 6 months

ifitration,

to warrant

348 days. Data were obtained from a total of 19 examinations. None of the patients had

and

mononuclear cell inifitration; 4-6, moderate rejection, mononudear cell infiltration, and myocyte necrosis; and 7-8, severe rejection, polymorphonuclear leukocyte in-

histo-

from the first

prior to and NMR spectros-

along

of rejection treatment

with

the

that nefrom the of P-31 NMR

Signa

1.5-1

imaging/spectroscopy

sys-

tem (GE Medical Systems, Milwaukee) with a custom-built, coplanar, coaxial surface-coil set comprising a P-31 NMR transmitter coil 40 cm x 40 cm square (16), a P-31

NMR

receiver

coil 6.5 cm in diameter,

and an 8 x 13-cm figure-eight-shaped proton (H-i) receiver coil (17). Detection coils were turned off during excitation, and excitation coils were turned off during detection with passive crossed-diode circuits. This coil set enabled both H-i imaging and P-31 spectroscopy to be performed without moving the patient, with minimal mutual interactions between coils, and with a uniform P-3i NMR excitation field over the sensitive volume of the detection coils (16,17). A vial contain-

time of surgery until the time spectroscopy (Table 1). The timing of prior and subsequent biopsies was dependent on a biopsy schedule determined by routine clinical practice for this group of pa-

ing 0.9 mL of a 0.5 mol/L solution of phosphonitrilic chloride trimer in xy!ene doped with chromium acetylacetonate was embedded in the 6.5-cm P-3i coil to be a localization fiduciary and a check on

medications (cyclosporine, prednisone, and, in some patients, azathioprine) at the time of P-3i NMR spectroscopy. In addition, in seven cases (cases ia, ic, 2a, 2b, 3,

tients, NMR

the spectral

8b, and ii), medication creased during the 2-4 spectroscopy to treat a detected with the prior

no clinical history of heart disease, were control subjects; they underwent 19 examinations with P-31 NMR spectroscopy. Control subjects older than age 40 years

patients (patients 3, 5, 6, and 8 [case 8b]) had evidence of mild LV hypertrophy. All patients were taking immunosuppressive

biopsy.

No medications

had been inweeks preceding rejection episode endomyocardial were

prior to P-3i NMR spectroscopy. Biopsy samples were taken right ventricular endomyocardium tients with transplants on the P-31 NMR spectroscopy, and score was assigned according ham’s criteria (15). The scoring were

68

as follows:

Radiology

0, no rejection;

without

Seventeen 24-83

interference

spectroscopic healthy

years

(mean

the

P-31

Subjects

volunteers,

age,

39 years

face-coil

16) with

showed

from the in pasame day as a rejection to Billingcriteria

trocardiograms or thaffium radionucide scans obtained during separate treadmill testing. All subjects gave informed consent for the study, which was approved by the Johns Hopkins Joint Committee on Clinical Investigation.

mild

All NMR

studies

of ischemia

aged ±

withheld

1-3,

no evidence

from

study.

were

performed

on elec-

on a

net,

but

signal-to-noise

were set

oriented in the

rotated

ratio

prone

center

of

(S/N).

on the surthe NMR mag-

on the left side

to bring

the P-3i receiver coil closest to the anterior LV wall and the septum of the heart. The location of the coils relative to the heart was confirmed by means of conventional

cardiac-gated

H-i

NMR

imaging

with the standard body H-i transmitter coil and the figure-eight receiver coil. The magnetic field homogeneity was shimmed in the region of the heart by maximizing the H-i water resonance detected with the figure-eight coil. The P-31 coils were then connected, and the system was switched to P-3i

NMR

spectroscopy.

October

1991

P-31

spectra

were

acquired

from

nances nances

1-cm-

thick sections parallel to the surface by use of a 32-step, one-dimensional,

coils

per

gradient, spectroscopic sequence (18), with a 1-msec delay and 24-48 acquisitions (8-i6 minutes imaging time). The

step

acquisitions

were

synchronized phase in which

with the LV mo-

diastolic cardiac tion is reduced, at one acquisition per cle, with an optical peripheral gating transducer. The NMR pulse amplitude was

adjusted

to maximize

PCr

which

was

in

muscle have

and similar

heart phosphate relative spin-lattice

metaborelax-

derive

PCr.

from its [3-phosphate 16.3

Overlapping

assumption

ppm

peaks

that

the

relative

were

line

to

fitted

shape

on the

of the

re-

solved portion reflects that of the entire peak, so that overlap principally contributes to, and is included as, random error. Metabolite ratios were measured in one to

five sections

through

the heart,

extending

from the anterior LV wall through the left ventricle and septum. We addressed the possibility that abnormalities might

in PCr/ATP levels be due to systematic

the volumes lions This

of blood

intersecting

the

possibility

in

from

ventricular

arises

sec-

cavity.

because

blood

con-

tains ATP but no PCr. Human blood shows two characteristic resonances phosphomonoester

(PM)

bisphosphoglycerate

about with

DPG ATP

region

(DPG)

0.3 ± 0.02 (20,21). ratios were corrected

tamination

by

from

subtraction

2,3at

spectra, ATP/

Myocardial for blood

=

in the

resonances

5.4 and 6.2 ppm in P-3i a ratio of concentrations,

PCr/ con-

of a blood

ATP

signal corresponding to 15% of the total integrated DPG signal from [3-ATP signal.

In addition

to PCr, AT?, and PD levels,

the quantification tempted in light

of Pi levels was atof the prior observations

of altered despite

Pi levels in allograft rejection, the recognized difficulties of re-

solving

the

Volume

resonance

181

#{149} Number

of Pi from

1

ppm,

the reso-

and

and PD/ATP ratios because Pi and PM were unresolvable or undetectable in unlocalized

ATP

spectra. ratios were

Thus PCr/Pi not corrected

and PM/ for satura-

tion.

used

only

NMR spectroallograft rejection

surface-coil

ratios

for each

were

averaged

subject.

an

analysis of data from single sections showing the largest metabolic changes in each heart was also performed. Analysis of data from all patients with transplants was blinded to results of both the biopsy and P-3i NMR spectroscopy. Statistical significance was tested with the Student unpaired t test.

RESULTS Endomyocardial biopsy samples acquired from patients on the day of the P-31 NMR examinations scored 05 for rejection (mean, 2.8 ± 1.6), corresponding on average to mild rejection (Table 1). The mean score did not change significantly in the biopsy samples acquired at the first sched-

67 days

±

46) after

(mean scores, 2.4 ± 2.0, respectively).

P-31

NMR

2.2 ± 2.2 From the

ter the ble 1).

spectroscopic

Figures

of P-31 sections

1 and

spectra through

in a healthy undergoing

(Ta-

typical

series

from

coronal

and

satu-

myocardial

PCr/ATP ratios were i.64 ± 0.20 and 0.74 ± 0.li in the healthy subject and the patient, respectively.

Overall, the mean correction factors for saturation of myocardial PCr/ATP and PD/ATP ratios did not differ significantly between patients and healthy subjects (i.34 ± 0.27 vs 1.42 ± for

PCr/ATP,

and

1.04

0.56

±

vs

compared

with

69.6

min1

i2

±

in healthy subjects (P < .001). The mean correction factors for blood contamination of the myocardial PCr/ ATP ratios were essentially the same at i.il ± 0.i4 for healthy subjects and 1.i4 ± 0.23 for patients (P > .i), a finding that is consistent with absence of significant bias ferences in blood tween these study

from systematic contamination groups. The

tion does, however, measured PCr/ATP mately 10%. Table 2 summarizes spectroscopic

data

aminations.

Overall,

difbecorrec-

increase the ratios by approxithe

P-3i NMR all of the ex-

from

PCr/ATP

was

significantly reduced (P < .01) by approximately 20% in patients relative to that in healthy subjects. The statis-

tical

error

in the ratios

normal

myocardial

is consistent

with

the

tients, The

ratios

subjects P < .01).

were

1.76

vs 1.38

measurement

± ±

0.22

0.51

of Pi was

in

in papossi-

ity to detect resonances teria for Pi assignment

2 show

subject moderate

find-

for

at the af-

4)

acquired

and

earlier

correction

mean

the 16 and

NMR

intervention

chest

with

After

the

from

ble in heart spectra from 17 of the 19 patient examinations, but from only seven of the 19 examinations in healthy subjects because of the inabil-

examination

the

ration,

signal

intersecting (sections

consistent

(10,ii).

healthy

distinct epi(mean 3.1 ± 2.2). Mod-

was detected with biopsy (scores in eight cases at the time of P-31 spectroscopy and in seven cases next biopsy procedure performed

P-3i

in sections chamber

i7), both ings

PCr/ATP

to seven rejection

number of episodes, erate rejection requiring

corresponding

spectral S/N: The scatter is larger in patients, a fact that suggests other confounding physiologic variables. These findings were independent of the blood correction (the uncorrected

and time of allograft transplantation until and including the date of P-31 NMR spectroscopy, patients underwent treatment for up sodes of allograft

with

increased

DPG

PCr/ATP

uled visit either 13-125 days (mean, 67 days ± 39) before or 10-140 days

(mean,

and

i3.9,

over

However,

along

1.09 ± 0.31 for PD/ATP; P > .i), although heart rates were significantly higher in patients at 88.2 min’ ±

resolution (6-9), the multiand blood-

the myocardium saturation-corrected

13),

blood

0.33

localiza-

tion with little or no spatial within section

11),

annotated H-i NMR images. Note the decrease in the PCr/ATP ratio in spectra from the heart (sections 14 or 15 and deeper), compared with those from chest muscle (sections 12 and

ventricular

indud-

of DPG, phosphorylcholine,

spectroscopy

in patients differences

in spectra

of 5.5-6.8

phosphorylethanolamine, are documented as phosphomonoesters (PMs). In many subjects, saturation factors for PCr/Pi and PM/ATP ratios were unobtainable with the method used for PCr/ATP

the heart

analyzed (after application of a 12-Hz linebroadening ifiter) by fitting peaks to gaussian lines to within the spectral noise level, integrating the curves, and applying the saturation correction for PCr/ATP, PD/ATP, and PD/PCr ratios. All ATP meaat about

peaks

DPG-corrected

constraints. The total patient examination time was 40-60 minutes. P-3i spectra from all sections identified from H-i images as myocardium and exhibiting useful S/Ns ( > 6 for ATP) were

surements

with

in animals

ation times (Ti) (19). Spatially localized saturation factors or metabolite Ti values could not be measured in each 1-cm-thick section in each patient because of time

resonance

The average lower limit of detectability for the PCr/Pi ratio consistent with the experimental S/N was about 10. Integrated reso-

Because all prior P-31 scopic studies of cardiac

to pro-

vide a fully relaxed spectrum (16). This correction was used on the assumption that lites

detectable, the PCr/Pi data were omitted for that subject to prevent statistical bias.

ing those

intensity

assumed

tient

relative to PCr were assigned to or defined as Pi. When myocardial Pi was not

nances

cy-

unlocalized surface-coil spectra acquired in a few seconds. Because heart rates varied, PCr/ATP, PD/ATP, and PD/PCr ratios from all subjects were corrected for spectral distortion caused by nuclear magnetic resonance saturation by acquisition of unlocalized surface-coil spectra at the heart rate and at a sequence repetition period of 15 seconds,

resochemical

shift

phase-encoding localization acquisition

of DPG (22,23). Resolvable in the range 4.0-5.1 ppm

heart

in a patient rejection (pa-

fitting the and obfusca-

cri-

tion by overlapping portions of the neighboring PM and PD resonances. Mean PCr/Pi levels were about 40% lower in patients, was not significant,

the

low

subjects.

rate

but

the mainly

of Pi detection

(If the

detection

difference because

of

in control limit

Radiology

of PCr/ #{149} 69

Pi

10 were

=

assumed

which

Pi was

PCr/Pi

becomes

subjects

8.4

vs 4.2

nificant We nificant ratios

in all cases

undetectable, ±

decrease.) were also differences in patients

but two of the highest scores

in

normal

± 3.9 in control

4.6 in patients,

a sig-

unable to resolve sigin the PD/ATP and healthy subjects,

three patients for rejection

with the individu-

ally showed much higher myocardial PD/ATP ratios than normal (Fig 3). The corrections for blood ATP contamination in all three of these patients-less than 10%-were comparable to those of the other subjects. Thus the elevated PD/ATP in two of

them

is unlikely

attributable

PD unless their were anomalously

Ia.

2a

to blood

blood PD/DPG levels high compared

with the levels in all other subjects (n = 36). Like the DPG findings, PM! ATP levels were not ferent among study Although PCr!ATP

significantly difgroups (Table 2). was lower in

patients, we were unable to detect significant correlations between any of the metabolite ratios and the absolute ples

numeric taken

after

P-3i

scores from before, at the

NMR

biopsy samtime of, or

spectroscopy,

or be-

tween the metabolite ratios and patient age, time after transplantation, number of prior rejection episodes, heart rate during P-31 NMR spectros-

copy,

medications,

or echocardio-

graphic results (r < .6 throughout). For example, Figure 4 shows one of the better correlations observed: that between PD/ATP and biopsy scores recorded at the time of P-31 NMR spectroscopy (r = .58). In patients classified as having mild or moderate rejection on the basis of the findings from the biopsy sample obtained on the day of P-31 NMR spectroscopy, myocardial PCr!ATP

ratios

were

significantly

lower

than

normal (P < .05), whereas patients not undergoing rejection had normal PCr!ATP ratios (Fig 5). In patients

scored

as having

on the tamed

basis of biopsy samples obon the first visit after P-31

NMR

moderate

spectroscopy,

rejection

myocardial

PCr!

ATP ratios were also significantly lower than normal (P < .01), whereas the ratios in patients with mild or no rejection at that visit were not significantly different. The metabolite ratios in mildly and moderately rejecting hearts were not statistically signifi-

cantly different None of the PD/ATP, grouped

rejection, ate 70

from each other ratios

PM/ATP, by severity

mild

rejection) #{149} Radiology

and PD/PCr of rejection

rejection, at the

other. of PCr/Pi,

time

and

(no

moder-

of P-31

NMR

oppm

20 2b.

lb. Figures

1, 2.

(1) Typical P-31 NMR spectra (b) from 1-cm-thick coronal sections as a function the chest and anterior myocardium of a healthy volunteer, and a corresurface-coil H-i NMR image (a) annotated to show the location of the numLV (LV), right ventricle (RV), and septum (SE). Sections 9 and 10 intersect the phosphate reference (Ref.) located off-center in the plane of the surface coil. Chemical shift scale is relative to PCr at 0 ppm. The amplitudes of spectra 14-16 and i2-i3 are respectively magnified 20 times (x20) and 10 times (xlO) those of the reference spectra 9-10. The S/Ns of ATP and PCr in spectra 14 and 15 are approximately 11 and 20, respectively. (2) Typical P-3i NMR spectra (b) from i-cm-thick coronal sections as a function of depth through the chest

of depth through sponding axial bered sections,

and anterior myocardium of a patient with a transplant jection, detected by means of an endomyocardial biopsy axial surface-coil H-i NMR image (a) shows the location tudes of spectra 13-17 are magnified 20 times in spectrum 16 are about 15 and 19; in spectrum RV = right ventricle, SE = septum.

spectroscopy

or the

ter exhibited

significant

In patients immunosuppressive

who

manage

rejection

first

visit

thereaf-

abnormalities.

required increased medication to

detected

in biopsy

(patient ii) undergoing score of 4. Corresponding

of numbered those of spectra 9-li. 17, they are about

samples

P-31

obtained

NMR

moderate reannotated

sections. The ampliThe S/N of ATP and PCr ii and 10, respectively.

at either

spectroscopy

the

or the

time

of

next

scheduled appointment thereafter, myocardial PCr!ATP ratio was sigificantly lower than normal (Table 2).

October

1991

ATP ratios in patients were 1.22 ± 0.46 and 1.86 ± 0.65, respectively. Figure 7 shows scatter plots of the lowest myocardial PCr!ATP ratio observed in each patient classified according to severity of rejection.

DISCUSSION Previous work on animal models of cardiac allograft rejection has demonstrated (a) a correlation between dedining high-energy phosphate ratios and

graft

rejection

as indexed

by the

time interval after transplantation in nonimmunosuppressed animals and (b) significant reductions in high-energy phosphate ratios in grafts classifled by means of histologic examination as undergoing rejection (6-9). Our results showing reduced PCr/ ATP overall in patients with mild rejection on average, in mildly and moderately rejecting allografts treated

Again,

however,

icant

no statistically

difference

signif-

in myocardial

positive quiring analysis.

metab-

olite ratios existed between patients who required intervention and those who did not (P > .1). When values of PCr/ATP between 1.51 and 1.58 were chosen as a threshold below which intervention for rejection might be instigated, P-31 NMR spectroscopy

come

true

yielded

the

as the biopsy positives

same

scores

+ true

out-

specificity

([number

of

negatives]!total)

false

+

positives]),

the predictive value (true positives![true

73%;

of a positive positives

patients who on multiple

separately

underwent occasions,

6a and

of

troscopy

but

PCr!ATP

who

ratios

had

and

nearly

in whom

of

with treatspecnormal

the

re-

at 55% and 58%, respeca new PCr/ATP threshold 1.26 and 1.38. P-31 NMR based on the lowest ob-

a basis

for

standard

sults,

only

ative

detection

P-31

NMR

scores,

dial

therefore

2) with

PCr/ATP

Volume

181

considered

much-reduced

that #{149} Number

resulted 1

myocar-

in a false-

of eight

one

negative

would

as well. Of these false-positive re-

(case

8a)

had

positive

results of the examination for rejection requiring augmented treatment at the next scheduled biopsy after P-31 NMR spectroscopy. However, in the single patient in whom false-neg-

negative. Also shown are heart spectra in three patients (cases 6, 7, and 8a) who had mild rejection (biopsy

were

seven

were

undergo treatment five patients with

false

sults

treatment,

patients who required augmented treatment on the basis of the biopsy examination would qualify, but an additional five patients (cases 5-7, 8a, and 10) in whom results of the examination for treatment according to the

examitreated

4 indicating moderate rejection myocyte necrosis that required ment at the time of P-31 NMR

We

or are un-

PCr/ATP ratio would then the same outcome as endomyocardial biopsy about 68% of the time. If the lowest PCr/ATP ratio were used as

+

was

spectroscopy

indicated

(case

sults of the next scheduled were negative for rejection augmented treatment. The

the lowest

and

highest

cardial PD/ATP in most of the patients with the highest endomyocardial biopsy score of 5 for rejection are also in agreement with canine studies that show significant PD/ATP elevations with biopsy scores of 5.5 ± 1.1, but no significant elevations at scores of 2.8 ± 0.9 (9).

predictive

worse,

served

and

myocardial spectra from patients (cases ic and 11) with biopsy scores

overall

a little

yield

test

examples

with

spectroscopy

biopsy

6b shows

and

fare

between

or together.

Figure

value changed, tively,

false positives]), 57%. As is evident from Table 1, these results did not change significantly in the limited group of

three nation

re-

If we allow for heterogeneity in the rejection process and consider only the sections that show the minimum myocardial PCr!ATP ratio in each patient, the sensitivity improves to 88% at the time of the biopsy, although

obtained either at the same time or following P-31 NMR spectroscopy in 12 of 19 cases (63%). The overall sensitivity (number of true positives! [true positives + false negatives]) was 50%; the specificity (true negatives/[true negatives

classification for rejection therapy in the blinded

separately, and in allografts requiring intervention agree with these findings. Observations of elevated myo-

not,

however,

see

a good

appears creases

to be due solely in mild through

jection, apparent

because no change between patient

classified

with

mild,

to Pi inmoderate

in PCr groups

re-

is

mild-to-moder-

with

ate, and moderate rejection. To the extent that the reliability of our Pi measurements is compromised by the low intensity of Pi and by overlapping PD and PM resonances, and that

11), re-

we

biopsy requiring means of

corrected

did

direct correlation between metabolite ratios and biopsy scores of rejection in the patients in this study, nor could we reliably distinguish patients with mild rejection from those with moderate rejection, or patients requiring intervention from those not requiring intervention on the basis of myocardial PCr/ATP, PCr/Pi, PD/ATP, or PD/PCr alone. Although a correlation between the severity of rejection and myocardial PCr!Pi reductions was reported in a previous conference report on human transplant recipients in whom the same spatial localization method was used (12), this reduction

PCr!

were

also

unable

to distinguish

PCr changes between mild rejection and moderate rejection, our findings are not inconsistent with these results. Radiology

#{149} 71

Published data in canine studies have shown decreases in PCr/Pi and PCr/ATP in the first 2 or 3 days after transplantation

to levels

essentially this latter

static period

lite

endomyocardial

ratios,

that

thereafter of stasis

remain

(6). During in metabo-

of rejection episodes (6), but it is also consistent with our findings of poor discrimination between PCr/ATP or PCr/Pi ratios and biopsy scores of 2 and higher. The value of the PCr/Pi ratio in P-31 NMR spectroscopy for prediction of rejection corresponding to myocyte necrosis (biopsy score 4) in immupressed vestigated findings

and

70%; specificity, predictive value of at the time of P-31 63% (24). These

are comparable to our of 50%, 73%, and 57% and 58% if the minimum

ratios

in each

patient

average values (or 88%, 55%, metabolite

are used),

re-

spectively, for the sensitivity, specificity, and predictive value of PCr! ATP measurements for identification of biopsy scores greater than or equal to 4 on the same day as the P-3i NMR

spectroscopic opsy scores

examination; form the basis

tion

of augmented

sive

treatment

immunosuppres-

to counter

However,

the

dogs

much

were

prediction

biopsy

P-3i

day

better

in

in enabling detected

after

P-31

spectroscopy,

in which diction values improved for all three parameters

with

NMR

case the preto 90%-92% (24). Daily

biopsy

data

patients,

but the predictability of reat the subsequent scheduled

jection

biopsy

were

rejection.

measurements

of rejection

on the

such bifor initia-

not

available

in our

procedure 67 days ± 46 after NMR spectroscopy did not im-

P-31 prove over that on the day of P-31 NMR spectroscopy. One-dimensional localization sequences in conjunction with surface-

coil detection have enabled successful detection of regional myocardial ischemia (22,25) and were used in the conference report on patients with transplants discussed previously (12). Although

unlikely

agreement

cardial those

our

#{149} Radiology

that

our

spectra

from

adjacent

and

3 (c) in patients

undergoing

the

severest

rejection (endomyocardial at the time of P-3i NMR

biopsy scores of 5) spectroscopy shows PD resonances in two patients. The ratios were 7.3, (a), 5.7 (1,), and 2.2

PD/ATP (c). The

PD/ATP

changed

ratio

of patient

at 5.2 at repeat

P-3i

copy 29 days later, when was unchanged

2b). The highest from the deepest

2 was unspectros-

NMR

the biopsy

at 5 (Table numbered

sections

score

i, examination spectra derive

in each subject.

compromised by some contamination from chest muscle. If present, skeletal muscle contamination would tend to increase PCr/ATP ratios toward those in muscle,

ber

of false-negative

our

study

plain

the

which (three

increasing

the

num-

detections.

such

effects

could

false-positive

not

ex-

detections

in

myocardial PCr/ATP was cases in which cardiac-aver-

aged

values

were

used;

low

five cases

in

which In any

PCr/ATP minima were used). case, when minimum PCr/ATP

values

were

selected

only

one

false-

negative detection in 19 examinations was observed. The possible lack of discrimination between metabolite ratios in mild and in more severe cases of rejection is a

potentially major problem in the application of localized P-31 NMR spectroscopy to the noinvasive detection of allograft rejection. To be useful in present clinical practice, P-31 NMR spectroscopy would have to enable reliable identification of patients with rejecting hearts that require intervention, not just differentiate (mild) rejection from the absence of rejection. While the scatter in the patient meta-

bolic data observed in our study is in fact comparable to that observed in several animal studies (6,7,9,24), it is currently too large to provide a reliable basis for clinical decisions vidual patients, assuming that

myocardial reference. Because

biopsy

is the

transplant

heterogeneous detection with a problem with

standard

rejection

of

data

from

single

sections

as tis-

scores

indicating

for augmented therapy some. Moreover, because metabolic changes with appear

in the

myocardial

did improve the largest rejection may PCr!Pi

ratio

(6-9),

show-

and

biopsy

-20

C.

is a

process, erroneous biopsy is recognized the small (1-mm)

sue sampling used, whereas P-31 NMR spectroscopic analysis does average larger volumes of tissue.

When

ppmo

in mdiendo-

are

myo-

-20

a.

In

and it is not

normal

results

NMR

coronal

ing the largest metabolic abnormalities were selected from the P-31 NMR spectroscopic study, the correlation between altered PCr!ATP ratios

of the

PCr/ATP measurements of prior animal studies,

inconceivable

72

between

in view

P-3i

sections through the antenor myocardial wall (LV [spectrum 17, 18], septum [spectrum i9]) in cases ia (a), 2a (1,),

found

nonimmunosup-

canine myocardium was inmore recently (24). The in 17 animals were as fol-

lows: sensitivity, 70%; and overall positive findings NMR spectroscopy,

3.

i-cm-thick

elevated

biopsy

scores of rejection changed from 2 to 7, signifying mild through severe rejection. This was interpreted as suggesting that the results of P-31 NMR spectroscopy might enable prediction

nosuppressed

Figure

a need

newly developed H-1--decoupled P-31 NMR spectroscopic techniques that enhance the spectral resolution of PM, DPG, and PD resonances from Pi (26) might significantly improve measurements,

the reliability and hence the

of Pi potential

October

1991

0 0

3

3

0

I-

0 0

0

0

0

F

0

0

0

+

C) 0

0

>.

0)

Ct

0

!

Ct

0 0 >

C

I

Ct

0

0)

0123456 Biopsy Figure

4. Mean anterior ATP ratio in each patient endomyocardial biopsy P-31 NMR spectroscopy. least squares fit (r = .58;

clinical

utility

0

Score

myocardial PD! as a function of the score at the time of The straight line is a P

of P-31

>

.05).

NMR

I

I

normal

spectros-

copy for detection of transplant rejection. As in the studies of rejection in animals, the primary changes in highenergy phosphate metabolism in cardiac allograft rejection in humans (ie, reduced myocardial PCr!ATP and possibly PCr,IPi ratios where measur-

able) are those normally considered characteristic of ischemia (27). Although no histologic evidence of mi-

0-1

2-3

Figure 5. Mean anterior myocardial PCr!ATP ratios in healthy (0) classified as having no rejection (scores, 0-1), mild rejection rejection (scores, 4-6) on the basis of findings in endomyocardial the day of P-31 NMR spectroscopy (a) and obtained at the first values (#{149}) have error bars at ±1 SD. Asterisks denote statistical subjects (*, P < .05; **, P < .01).

like those seen in our study would result if disturbances in high-energy phosphate metabolism that accompany the rejection process were transient and asynchronous with respect to histologic observations. The PD resonance in myocardial P-31 NMR spectra has been shown, at least

in the

posed

rabbit

(9,27).

necrosis as manifested the magnitude of the

bolic

change

proportional involved

would

be expected

to be

to the fraction of cells in the disease process, as

well

as to the

bolic ATP cells,

derangement. Because PCr and are completely depleted in dead however, tissue comprised of a

intensity

of any

mixture of metabolically and necrotic cells alone show

altered

meta-

normal cells would not

metabolite

ratios

but

could show histologic evidence of moderate rejection. Therefore, given the limited S/N of P-31 NMR spectroscopy

(27),

the

utility

of altered

phosphate metabolite ratios as robust predictors of histologic rejection would depend on the existence of a significant fraction of diseased but nonnecrotic myocytes persisting in this

ischemialike

state

of reduced

PCr/ATP and/or PCr/Pi and contributing to the observed myocardial spectra nation.

at the time of the NMR Note also that false-positive

and false-negative findings of altered Volume

181

#{149} Number

exami-

NMR spectroscopic PCr!ATP ratios 1

rylcholine

our

(31),

Several study

studies

to be com-

of glycerophospho-

the

in

heart,

principally

myocyte biopsies,

in meta-

normal

4-6

none

mild

moderate

0-1

2-3

4-6

b.

monitor

result

moderate

a.

brane phospholipid elevation in more might thus reflect

that

I

mild

crovascular occlusion exists, vascular endothelial injury resulting in reduced coronary blood flow is possible (28), albeit open to further question (6,29,30). If the metabolic changes

processes

I

none

a by-product

of mem-

degradation. Its severe rejection membrane damage

other differences in humans and in animals

between previous

deserve

type, usually nonworking cases

placed location,

(6-8),

consider-

in a heterotopic

untreated

and,

in many

with

prior

or

ongoing immunosuppressive medications. The range of severity of rejection represented by those studies is therefore much greater; indeed, the magnitude of metabolic change observed

than ences

in those

that

found

in the

studies

appears

in our

magnitude

study.

larger

Differ-

of the

meta-

bolic response to rejection may exist between hearts in the acute phase immediately after transplantation and the chronically treated hearts that we studied an average of 20 months after transplantation. The number of patients studied by means of P-31 NMR spectroscopy

weeks

within

of transplantation

the

first

few

in our

group

is currently too limited to asthis possibility. Second, biopsies were performed daily or every few days in animal sess

studies in which the entire process-from no rejection

study

rejection to the

most severe rejection-usually occurred within 7 days of surgery (6,7,9). By contrast, the average biopsy frequency of approximately 67 days in our study of humans does not permit resolution of such short-term changes or reliable evaluation of the suggestion that P-31 metabolic changes with rejection precede changes

ation: First, all of the animal studies were performed within the first 2 weeks of transplantation in acutely rejecting hearts that were unmatched for tissue

subjects (0) and in patients (scores, 2-3), and moderate biopsy samples obtained on biopsy thereafter (b). Mean significance relative to control

indexed

by

means

of endo-

myocardial biopsy should these changes occur over periods of a few days (6,7). It is clear that rejection is largely controlled, at least during the much longer biopsy intervals of the human studies, with long-term administration of immunosuppressive medication. Transient bouts of rejection, which may show early changes in cardiac metabolism, may also disappear as a result of the medication prior to the next biopsy. Although the schedule of P-3i NMR

spectroscopic

examinations

in

our study of humans does not match that of the previous studies in animals, it does nevertheless correspond to the accepted clinical practice in scheduling endomyocardial biopsies for monitoring and managing rejection in heart transplant recipients. While preliminary observations correlating metabolic data with acute rejection appear more promising for the prediction of rejection in patients studied in the first weeks after transplantation (14), it should be noted Radiology

#{149} 73

Figure 6. Examples of P-31 heart spectra from spectral data considered false-negative (a, b) or false-positive (c) for moderate rejeclion

requiring

intervention

(biopsy

scores

4). (a) Adjacent spectra from 1-cm-thick sections in the anterior wall (endocardial [endo] and septal [sept], and epicardial [epi] and endocardial) in case ic, with mean cor-

rected

PCr!ATP

4. (b) Heart

ATP,

of 2.2 and

spectra

1.9; biopsy

from

score,

a biopsy case

score

11 (mean

4). (c) Heart

of PCr!

spectra

from cases 6 (top), 7 (middle), and 8a (bottom) (mean corrected PCr!ATP values, 1.0, 0.6, and 1.1, respectively; biopsy scores, 2).

that most patients with transplants undergo some degree of rejection during this period. For example, in 12 of the 14 subjects in our study, mild or worse rejection was indicated by biopsy within the first 2 weeks (mean time to first detection, 18 days ± 22; n = 14), and the first rejection episode that necessitated intervention occurred within the first 90 days after transplantation (mean interval, 73 days ± 122; n = 14). Because of the high prevalence of rejection in the acute phase, the sample population can be biased to favor rejection as a correlate of any abnormal parameter observed in these patients. Therefore caution is needed in attributing metabolic abnormalities to rejection per se in this immediate postoperative phase. Third, it is possible that prior episodes

of rejection

in which

treatment

after

immunosuppressive

also

prone

ischemic ease that PCr/Pi

and hypertensive could alter the ratios

of the

in our

study

group

of large-vessel

#{149} Radiology

0

0

0 0

the

showed coronary

*

2

2

o

1.

Ct C)

0 >

1

.-5 Ct

O**

01

0> C)

8

o**

0

©

0

1

0

C,)

U)

**

1

a)

0

0

0

u

Figure rejection

0

0

-J

i

I

none

mild

0-1

2-3

0’

I

moderate

i

miid

none 0-1

4-6

a.

disease at catheterization, some LV hypertrophy was present in four patients (Table 1). Of these, only two patients had abnormally low mean myocardial PCr/ATP ratios (1.0, and 1.2), and one of the two was undergoing moderate rejection at the time. Therefore, hypertrophy could explain at most a single false-positive detection of rejection. 74

-20

3

0

re-

Although

evidence

0

C.

3

disand

in transplant no

10 b.

-J

These condisymptoms

recipients.

I

Ct

jection status (25,33,34). tions often occur without patients

-

independent

-20

I

I

a.

C-

accelerated

heart PCr/ATP

,

-I

0

ther-

to develop

‘

-I

I-

apy showed a recovery of the mean myocardial PCr/Pi ratio to only 74% of the initial value after therapy; this difference was not statistically significant but is also not inconsistent with such a possibility (9,32). Fourth, patients with transplants are

J

0

was successful may exhibit histologic recovery but delayed or incomplete metabolic recovery. Experiments monitoring canine allografts before and

ppm

mod’erate 4-6

2-3

b. 7.

Lowest (scores,

anterior

0-i),

the basis of findings

mild

myocardial

rejection

in endomyocardial

PCr/ATP

(scores,

biopsy

ratios 2-3),

and

samples

spectroscopy (a) and during the first biopsy thereafter ± i SD. Asterisks denote statistical significance relative (*,P < .02;**,P < .001).

Fifth, it should be noted that patient numbers are limited, particularly for the more severe cases of rejection, and a better metabolic-histologic correlation for transplant rejection may emerge with larger numbers of pa-

in patients

moderate

obtained (b).

Mean

to average

(0)

classified

as having

no

rejection (scores, 4-6) on on the day of P-3i NMR values

values

(U)

have

error

in control

bars

at

subjects

tients and as the technology continues to improve. Nonetheless, these results suggest that use of localized P-31 NMR spectroscopy does not enable precise prediction of significant histologic rejecOctober

1991

tion

in many

patients

with

allograft

heart

transplants. Because the primary cause of rejection is a heterogeneous

immunologic altered rently

response

energy understood,

metabolism the role

spectroscopy of patients with unclear.

undergo

Because

regular

such

help

10.

re-

patients

for rejec-

data

from earlier examinations serve as controls

which metabolic change measured. Further serial elucidate

the

to

is not curof P-31

monitoring

tion, their own NMR spectroscopic the heart might

thus

link

in the treatment heart transplants

NMR

mains

9.

whose

P-31 of with

could be studies may causes

12.

of the

observed

abnormalities

phosphate how such serve this

metabolism and indicate information might best group of patients. U

spectroscopy

in cardiac

Acknowledgments: We thank V.P. Chacko, PhD, P. Barker, PhD, S. Augustine, MS. and Kahlil, MD, for assisting with NMR examina-

tions and patient

11.

handling,

R. Giaquinto

13.

R.

for fab-

rication of coil hardware, and C. Gerstenblith, MD, J. Glickson, PhD, and W. Brody, MD, PhD, for their helpful comments and support.

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Radiology

#{149} 75