(Circulation. 1998;97:2557-2566.)
© 1998 American Heart Association, Inc.
99mTc-HL91
"Hot Spot" Detection of Ischemic Myocardium In Vivo by Gamma Camera Imaging
Robert D. Okada, MD;
Gerald Johnson, III, PhD;
Kiem N. Nguyen, BS;
Zhonglin Liu, MD;
Barbara Edwards, BS;
Colin M. Archer, PhD;
Timothy L. North, BS;
Adam C. King, PhD;
; James D. Kelly, PhD
From the William K. Warren Medical Research Institute of the University
of Oklahoma Health Sciences Center, Tulsa (R.D.O., G.J., K.N.N., Z.L.), and
Amersham International, Bucks, England (B.E., C.M.A., T.L.N., A.C.K., J.D.K.).
Correspondence to Gerald Johnson III, PhD, William K. Warren Medical Research Institute, 6465 S Yale, Suite 1010, Tulsa, OK 74136. E-mail gerald-johnson{at}uokhsc.edu
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Abstract
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Background—99mTc-HL91 is
a new hypoxia imaging agent that demonstrates increased uptake
and retention in globally hypoxic myocardium in vitro. The
purpose of this study was to determine whether 99mTc-HL91
could detect regional ischemia in vivo by gamma camera
imaging.
Methods and Results—Eight open-chest dogs with left circumflex
(LCx) stenoses were studied. Injection of 5 mCi of
99mTc-HL91 and microspheres was followed by imaging
over 4 hours. Heart slices were imaged, then stained with
triphenyltetrazolium chloride (TTC), and
tissues were well-counted. TTC staining demonstrated no injury. Mean
LCx blood flow was 0.32±0.04 mL · min-1 ·
g-1, and mean left anterior descending coronary
artery (LAD) flow was 0.96±0.02 mL · min-1
· g-1 (ratio, 0.33). "Hot spots" were detected in 8
of 8 experiments in vivo within 60 minutes and improved over 4 hours.
Region of interest analysis of LCx/LAD activity ratios
demonstrated significant increases within 30 minutes (final ratio, 3.0;
P<0.05). LCx and LAD washout curves demonstrated
significant differences within 15 minutes. Washout curves were
biexponential over 1 hour, followed by linear retention from 1 to 4
hours. Four-hour fractional retention was 0.12 for LAD and 0.44 for LCx
(P<0.01). Myocardial flow versus tracer uptake
demonstrated 2 phases: phase 1 (flow, 0.05 to 0.7 mL ·
min-1 · g-1) had an inverse linear
correlation (r=-0.80); phase 2, (flow, >0.7 mL
· min-1 · g-1) had no correlation.
Ischemic heart/liver ratios remained near 1.0 for 4 hours.
Conclusions—99mTc-HL91 positively identifies regional
myocardial ischemia in a canine model using 99mTc
imaging. Quantitative techniques allowed identification of
ischemic myocardium within 15 minutes of tracer
administration.
Key Words: imaging • hypoxia • technetium • ischemia • myocardium
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Introduction
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An imaging agent
demonstrating increased uptake and long-term retention in
ischemic myocardium would be an important addition
to currently available agents. Synthesis of a new
99mTc-labeled imaging agent,
99mTc-HL91, has recently been
reported.1 Initial studies demonstrated increased
uptake in tumors with a hypoxic fraction and also low hepatic
uptake.1 Using a reperfused, isolated,
perfused-heart model, our laboratory subsequently described increased
myocardial 99mTc-HL91 uptake and retention in
ischemic and hypoxic hearts.2 We also
found that there was no increased uptake in acutely and irreversibly
injured myocardium early after
reperfusion.3 The purpose of the present
study in an intact canine model with a partial coronary
stenosis at rest was to (1) determine whether these properties
could be used to detect regional ischemic viable
myocardium in vivo with gamma camera imaging, (2) describe
uptake and clearance kinetics and their time course to determine the
optimal imaging period, (3) describe microsphere-determined
flow versus tracer activity relationships, (4) describe
target-to-nontarget ratios over time (lung/liver/gallbladder), and (5)
determine blood clearance over time.
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Methods
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Eight adult mongrel dogs with LCx coronary clamp (mean
weight, 23 kg; range, 20 to 25 kg) were prepared as previously
reported.4 99mTc-HL91 was
prepared and radiochemical purity determined as
reported.2
Gamma Camera Imaging
Our methods for acquiring gamma camera images have been
reported.4 Images were qualitatively assessed
without background subtraction.
Image quality was assessed quantitatively by use of
target-to-background pixel-count ratios. Images were also quantified by
calculation of pixel count ratios in ROIs in the control (LAD) and
ischemic (LCx) myocardial zones. ROIs in the LCx region were
defined by the size of the "hot spot." In the LAD region, ROI size
was equal to the entire septal wall. Average counts per pixel in these
ROIs were then obtained and ratios calculated for each experiment.
Images were background-subtracted and corrected for physical decay of
99mTc. Background subtraction was performed by
placing an ROI on the thoracic wall in an area that avoided overlap
with lung, heart, great vessels, and liver. The size of the ROI varied
slightly between experiments because of anatomic variations (153.5±8.5
pixels, mean±SEM) but was constant within an experiment. The location
of the ROI was unchanged across images within an experiment. Mean
background counts (cpm/pixel) for each image were then subtracted from
the organ counts within that image. LCx/lung and LCx/liver ratios were
calculated for each experiment.
Experimental Protocol
Figure 1
illustrates the
experimental protocol. Baseline hemodynamic
measurements were recorded during a 20-minute period after surgical
preparation and instrumentation. During the baseline period,
microspheres were injected to determine regional myocardial
blood flows as previously described.4 Then, the
occluder on the LCx was partially tightened to provide a 90% reduction
in baseline epicardial resting blood flow. Thirty minutes later, 5 mCi
of 99mTc-HL91 was injected into the left femoral
vein via the Teflon catheter. A microsphere blood flow
determination was made simultaneously. The
microspheres were labeled with either
113Sn or 103Ru. The order
in which the microspheres were injected was randomized. Serial
gamma camera images were acquired every 30 minutes between 30 minutes
and 4 hours after injection. No splanchnic shielding was used.
To measure blood 99mTc activity over the 4-hour
study period, 1.0-mL serial arterial blood samples were
collected at 30-second intervals during the first 2 minutes and then at
2, 4, 6, 8, 10, 20, 30, 60, 120, 180, and 240 minutes after injection.
The dogs were then euthanized.
Postmortem Analysis
After death, the LCx was completely occluded, and hearts were
infused with Evans blue dye to delineate the area at risk. Hearts were
immediately excised, and the precise location of the LCx was marked
with a suture. The entire heart in each experiment was then placed in
plastic wrap on a plastic board and imaged for 5 minutes on the face of
the gamma camera. After the right ventricle and great vessels were
removed, hearts were then sliced into four 1-cm-thick rings from apex
to base. The 4 rings were placed on the face of the gamma camera and
imaged together. In a subset of 4 experiments, the entire heart, liver,
and gallbladder were sectioned and assayed in a dose calibrator for
99mTc activity immediately after the end of the
experiment.
The heart slices were then weighed, and the myocardial rings were
stained in a 2% solution of TTC for 15 minutes at 37°C. Any
unstained areas were measured as a percentage of the total left
ventricle, and infarct size was quantified by computerized digital
planimetry of slice photographs. Forty-eight tissue samples, 24 samples
representing the normally perfused LAD zone and 24
representing the occluded LCx zone, were assayed in the
gamma well counter for 99mTc and
microsphere activity. These samples were taken from the middle
2 slices. The apex and base slices tended not to have an
ischemic zone. Inclusion of these slices would have
disproportionately increased the number of normal pieces.
Ten 1-mL samples were pipetted from different, randomly chosen
locations within the stirred TTC solution after removal of the heart
slices. These samples were subsequently assayed in a gamma well counter
for 99mTc activity. Count data were then
background-subtracted and decay-corrected, and counts were averaged for
the 10 samples. The average value was then multiplied to equal the
final volume of the TTC solution. Activity calculated in this manner
was compared with resident heart activity and was found not to exceed
1% of resident heart activity in any experiment.
Within 12 hours of collection, the 99mTc serial
blood samples and the myocardial tissue samples were counted for 5
minutes each in a gamma well counter as previously
described,4 and myocardial blood flow was
calculated.4
Data Analysis and Statistical Methods
For calculation of 99mTc-HL91 blood
clearance, the first blood sample (t=0) in each experiment was
discarded, because the counts in this sample were lower than that of
the 30-second sample as a result of inadequate time for complete mixing
of 99mTc-HL91 in the blood pool. Then,
background-subtracted and decay-corrected serial blood sample counts
were normalized as a percentage of the counts at 30 seconds (peak
activity), and the individual activity versus time curves were fitted
by a nonlinear regression analysis (Tablecurve 2D for Windows,
Jandel Scientific).
The first minute of myocardial clearance data from images was omitted
from further analysis because of the potential for added
activity from the blood pool. The count data were then normalized to
the percent activity at 2 minutes. The clearance data from both zones
(LCx and LAD) for each experiment were fitted by nonlinear and linear
regression techniques as described above to compare
parameters of myocardial clearance kinetics.
Fractional myocardial clearance from the normal and stenosed zones was
defined as the difference between the initial and final counts divided
by the initial counts. Clearance was calculated beginning 2 minutes
after 99mTc-HL91 injection from the background
and decay-corrected, normalized data.
Percent injected dose calculations were made for heart, liver, and
gallbladder in 4 experiments by dividing the decay-corrected assayed
activity in these organs by the initial injected activity and
expressing this as a percentage.
The myocardial blood flow versus 99mTc-HL91
activity data for each individual experiment were normalized to a flow
of 1.0 mL · min-1 ·
g-1, then normalized to the tissue piece with
the highest activity, which was arbitrarily assigned as 100%. Data
points for flows <0.05 mL · min-1
· g-1 were not included in the regression
analysis, although they are demonstrated in Figure 2. Previous studies using other related
agents have demonstrated markedly reduced tracer uptake at these very
low flows, probably due to poor tracer delivery or irreversible
myocardial injury. The remaining data were then divided into 2 phases.
The first phase ranged from a flow of 0.05 mL ·
min-1 · g-1 to a
flow of <0.7 mL · min-1 ·
g-1. The cutoff value for flow of 0.7 mL/min was
computer-derived. The flat second phase was for flows 
0.7 mL ·
min-1 · g-1. These
individual phases were fitted by linear regression analysis.
The individual experimental results were then combined to demonstrate
mean regression lines.
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Figure 2. Microsphere flow vs 99mTc-HL91
myocardial activity curves for each of 8 individual experiments.
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All results were expressed as mean±SEM. The significance of mean
differences among groups was assessed with a one-way repeated-measures
ANOVA. Post hoc comparisons were made by use of t tests with
correction for multiple comparisons made by the Bonferroni procedure
(Crunch Software Corp). Temporal comparisons were made by a paired
t test. Values of P<0.05 were considered
significant. The goodness of fit for linear and nonlinear regression
analyses was calculated with the fit standard error statistic.
Pearson r correlation coefficients were also reported.
Ethics
All experimental animals were handled in accordance with the
guiding principles of the American Physiological
Society and by the Institutional Animal Care and Use Committee of the
University of Oklahoma Health Sciences Center.
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Results
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Hemodynamic Data
Table 1 lists the
hemodynamic data for the 8 experiments. After the
stenosis was applied, relative LCx epicardial blood flow fell
markedly, from 100% to 10% (P<0.05), and mean
arterial pressure fell slightly, from 114.2±3.0 to
100.7±3.3 mm Hg (P<0.05). During the
stenosis period, mean heart rate fell slightly, from 102.1±9.2
to 80.9±8.8 bpm. Mean left atrial pressure and cardiac output did not
change significantly after stenosis.
Microsphere Flow
Table 2 lists the individual and
mean microsphere-determined blood flows for the 8 experiments.
Before occlusion, the mean LCx flow was 0.94±0.03 mL ·
min-1 · g-1, the
mean LAD flow was 1.00±0.01 mL ·
min-1 · g-1, and
the mean ratio was 0.93 (LCx/LAD zone). After stenosis, the
mean LCx flow was 0.33±0.05 mL ·
min-1 · g-1, the
mean LAD flow was 0.96±0.02 mL ·
min-1 · g-1, and
the mean ratio was 0.33 (LCx/LAD zone).
TTC Data
TTC staining demonstrated no myocardial injury. Furthermore, after
the myocardial tissue was removed from the staining solution, the
solution was found to have no significant activity above background
when counted in a well counter. This analysis indicates that
TTC did not extract 99mTc-HL91 from myocardial
tissue in this study.
99mTc-HL91 Myocardial Uptake Versus Flow
Figure 2
demonstrates the myocardial
99mTc-HL91 uptake versus
microsphere-determined flow curves for the 8 individual
experiments. Each curve was divided into 2 phases and analyzed
(Table 3). Phase 1 consisted of data
points between flows >0.05 and <0.7 mL ·
min-1 · g-1. This
phase had a linear regression equation of
y=116.8-125.2x (r=0.80). Phase 2
consisted of data points for flows 0.7 mL ·
min-1 · g-1. This
phase had a linear regression equation of
y=24.3+0.01x (r=0.06). Because this
phase was flat, the linear correlation was near zero, as expected.
However, the goodness of fit was excellent (fit standard error, 1.5).
The mean regression lines for the 2 phases are demonstrated in Figure 3.
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Figure 3. Mean best-fit linear regression lines for 2 phases
of microsphere flow vs 99mTc-HL91 myocardial
activity data.
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99mTc-HL91 In Vivo Gamma Camera Images
Figure 4 demonstrates
representative 99mTc-HL91 in vivo
gamma camera images. Lung activity was not prominent. Liver and
gallbladder activities were present but did not significantly
affect myocardial image quality. Images demonstrated increased
99mTc-HL91 uptake in the stenosis zone
(LCx) at 60 minutes after tracer administration, although there was
also significant uptake in the nonstenosis zone (LAD). However,
by 120 minutes after tracer administration and thereafter, there was
progressively less activity in the normal zone (LAD) compared with the
stenosis zone (LCx). Qualitative analysis of in vivo
images demonstrated hot spots in the stenosis zone (LCx) in all
8 experiments.
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Figure 4. In vivo serial gamma camera 99mTc-HL91
images from 1 representative dog. Top, Images at 30,
60, 90, and 120 minutes. LCx is to left and LAD to right of cardiac
images, and liver activity is shown below. Bottom, Images at 150, 180,
210, and 240 minutes.
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Quantitative Analysis of In Vivo Myocardial Gamma
Camera Imaging
Figure 5 demonstrates the in vivo
myocardial washout curve from scans. The 240-minute fractional
myocardial 99mTc-HL91 retention was 0.44±0.06
for the stenosis zone (LCx) and 0.12±0.01 for the normal zone
(LAD) (P<0.001). The results of nonlinear and linear
regression fits are listed in Table 4.
The stenosis zone (LCx) washout curve was biexponential during
the first hour of clearance (r2=0.99),
followed by a linear retention from 1 to 4 hours (fit standard error,
0.71). The first exponential phase was rapid, with
t1/2=2.4±0.8 minutes. The second exponential was
slow, with t1/2=161.6±73.6 minutes. The late
linear retention appeared to have a slightly positive slope, but this
was not significant. The control zone (LAD) washout curve was
biexponential during the first hour of clearance
(r2=0.99), followed by a linear retention
from 1 to 4 hours (fit standard error, 3.11). The first exponential
phase for the normal zone (LAD) was rapid and not significantly
different from the stenosis zone (LCx), with
t1/2=2.2 minutes (P=NS versus LCx).
The second exponential phase was faster than the stenosed LCx, with
t1/2=32.8 minutes (P<0.05 versus
LCx). The late linear retention had a slope of 0.01±0.05
(P=NS versus LCx).
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Figure 5. Mean 99mTc-HL91 myocardial
time-activity curves determined by ROI analysis of serial gamma
camera images. LCx indicates LCx myocardial zone; LAD, LAD myocardial
zone.
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Figure 6 demonstrates the mean hot
spot–to–normal myocardial (LCx/LAD), LCx/lung, and LCx/liver activity
ratios over 240 minutes. The LCx/LAD myocardial activity ratio
progressively increased over time (P<0.05). The final
LCx/LAD myocardial activity ratio was 3.0±0.4 at 4 hours. The LCx/lung
and LCx/liver activity ratios did not change significantly over time.
The LCx/lung activity ratio was 3.4±0.7 at the end of the 4-hour
experiment. The LCx/liver activity ratio was 1.0±0.2 at the end of the
4-hour experiment.
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Figure 6. 99mTc-HL91 count ratios over
time determined by ROI analysis of serial gamma camera images.
LCx/LAD indicates LCx/LAD myocardial zones; LCx/lung, LCx myocardial
zone/lung; and LCx/liver, LCx myocardial zone/liver.
*P<0.05 vs time zero.
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Ex Vivo Gamma Camera Imaging
Figure 7 demonstrates ex vivo images
of myocardial slices from a representative dog.
Qualitative analysis of these images demonstrated increased
99mTc-HL91 activity in the stenosed LCx zone in
all 8 experiments.
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Figure 7. Ex vivo gamma camera 99mTc-HL91
images from 4 slices after euthanasia for 1
representative dog. The entire heart is shown in the
left image with LCx to the left. Slices are arranged with base at upper
right, slice 2 upper left, slice 3 lower right, and apex lower left.
LCx zone is to right on all slices and LAD zone to left. Images clearly
demonstrate increased tracer activity in LCx zone vs LAD zone.
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Quantitative analysis of the ex vivo gamma camera images is
demonstrated in Figure 8. For the whole
heart, the mean LCx/LAD activity ratio was 2.8±0.2. The mean LCx/LAD
activity ratio for the individual slices ranged from 2.2±0.2 for the
base to 3.9±0.7 for slice 2.
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Figure 8. Mean 99mTc-HL91 myocardial ex vivo
count ratios for whole heart and individual slices after euthanasia.
Ratio is for LCx zone/LAD zone.
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99mTC-HL91 Biodistribution
The mean percent injected 99mTc-HL91 dose
was 1.1±0.1% in the heart, 5.8±0.7% in the liver, and 0.6±0.1% in
the gallbladder 240 minutes after tracer administration. After
completion of the protocol scans, additional scans of the urinary
bladder indicated that this is a major route of excretion for this
agent.
99mTc-HL91 Blood Clearance
Figure 9 demonstrates the
99mTc-HL91 blood activity clearance curve.
Clearance was triexponential (r2=0.99). The
best-fit equation was y=a exp(-x/b)+c
exp(-x/d)+e exp(-x/f), where a=444.1, b=0.5,
c=43.0, d=9.7, e=9.2, and f=609.5.
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Discussion
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Nitroimidazoles were initially used as markers of tissue
hypoxia in tumor cells.5 Initial cardiac
studies in cultured myocytes, perfused hearts, and dogs detected
hypoxic myocardial tissue by use of 18F-labeled
misonidazole and positron imaging.6 7 This
technology was limited by the need for a cyclotron and the expense of a
positron camera. Studies using an 123I-labeled
analogue were described, thus allowing gamma camera
imaging.8 However, high liver uptake and the
relative unavailability of 123I limited the
usefulness of this agent. More recently, a
99mTc-labeled nitroimidazole has been
reported.9 10 11 12 13 This agent had the advantages of
using a widely available molybdenum-generated radiolabel that could be
detected by standard gamma cameras. However, high hepatic activity on
canine images was described.
More recently, several new nitroimidazole-containing ligands have been
synthesized for labeling with technetium. The
99mTc complex of one of these compounds,
99mTc-HL91M, demonstrated improved hypoxic/normal
count ratios compared with other compounds. Furthermore, when the
2-nitroimidazole moiety was removed from the core ligand of HL91M, the
resulting technetium-labeled compound
(99mTc-HL91) demonstrated significantly improved
hypoxic/normal myocardial uptake and reduced liver activity compared
with other compounds.1 Our laboratory used a
perfused-heart model to demonstrate increased
99mTc-HL91 uptake and retention in
ischemic and hypoxic
myocardium.2 Both models had
increased uptake and retention compared with controls. Similar results
were noted in Krebs-Henseleit–perfused and red blood cell–perfused
hearts. Subsequent studies in our laboratory demonstrated no increased
99mTc-HL91 uptake in reperfused nonviable
myocardium.3 The purpose of the
present study was to determine whether increased uptake and
retention of 99mTc-HL91 could be demonstrated
with gamma camera imaging in an intact canine model. A model of
coronary stenosis–induced regional ischemia
was used to compare ischemic with normal
myocardium.
Hemodynamics
In the present study, epicardial LCx blood flow fell
significantly, as expected, with the placement of the coronary
stenosis. This resulted in a slight fall in mean
arterial blood pressure but no significant change in heart
rate, left atrial pressure, or cardiac output. During the subsequent 4
hours, heart rate fell slightly, but epicardial LCx blood flow, mean
arterial blood pressure, left atrial pressure, and cardiac
output did not change significantly. The fall in heart rate is probably
due to compensatory adaptation to a severe LCx flow reduction. Thus, no
hemodynamic changes could account for the results
reported below. It should be noted that although myocardial blood flow
was reduced and ischemia was assumed to be present, there
was no direct measurement of tissue hypoxia.
Blood Flow as Determined by Microspheres
Mean microsphere myocardial blood flows (mL ·
min-1 · g-1)
before the stenosis were 0.94 for the LCx and 1.00 for the LAD,
indicating no differences at baseline. After the stenosis was
stable, mean microsphere blood flow for the LCx zone was 0.33
versus 0.96 mL · min-1 ·
g-1 for the LAD zone (ratio, 0.33),
consistent with a significant resting flow reduction in the LCx
zone, whereas normal flow was maintained in the LAD zone.
TTC Assay
TTC demonstrated no significant myocardial injury. Furthermore,
after the myocardial tissue was removed from the TTC solution, samples
of the solution were found to have no significant activity above
background in a well counter. This would indicate that
99mTc-HL91 is not leached significantly from
myocardial tissue by the TTC salts, as has been previously described
with sestamibi and thallium.14
In Vivo Gamma Camera Imaging
In the present study, qualitative assessment of in vivo gamma
camera images demonstrated increased LCx zone activity compared with
LAD zone activity at 60 minutes. However, there was still significant
LAD zone activity at 60 minutes. By 90 to 120 minutes, differential
99mTc-HL91 clearance had significantly decreased
LAD zone activity; consequently, the LCx increased activity was easily
discernable as a hot spot. The resolution of this hot spot continued to
improve over the remaining 4-hour imaging period. All 8 experiments
qualitatively demonstrated increased LCx zone activity at 1, 2, 3, and
4 hours. Hot spot identification was possible without splanchnic (ie,
hepatic) shielding, both qualitatively and quantitatively. Thus,
99mTc-HL91 can identify acutely ischemic
viable myocardium on planar scans.
Quantitative analysis demonstrated differences between the LCx
and LAD zones in 2 ways: First, the LCx/LAD activity ratio determined
by ROI analysis of the serial images progressively increased
over 4 hours. This increasing ratio was statistically significant as
early as 30 minutes after tracer administration. By 2 hours after
tracer administration, the LCx/LAD activity ratio was 
90% of the
4-hour value. The final 4-hour in vivo LCx/LAD zone activity ratio was
3.0. Second, LCx and LAD zone time-activity curves determined by ROI
analysis of serial images demonstrated significantly greater
retention in the LCx zone than in the LAD zone. This differential
retention was statistically significant 15 minutes after tracer
administration. At the end of the 4-hour period, LCx zone fractional
retention was 0.44 versus 0.12 for the LAD zone (P<0.001).
Therefore, use of quantitative methods allows early detection of
ischemic territories.
The results of the present study are consistent with
previously published perfused-heart results from our
laboratory.2 The present canine study
demonstrated ischemic zone 1-hour fractional retention of 0.44
and normal zone fractional retention of 0.12. The corresponding
perfused-heart 1-hour fractional retention was 0.31 for the
ischemic zone and 0.13 for the normal zone. It should be noted
that the 4-hour LCx/LAD activity ratio was 3.0 in the present
study, compared with a 1-hour normal/ischemic ratio of 13.6 in
the perfused-heart studies.2 This difference is
probably due to uniformly severe ischemia in the globally
perfused hearts compared with a more heterogeneous pattern
of ischemia in an intact dog heart with a regional
stenosis and with the presence of recirculating blood activity
in the intact-animal study.
In the present study, the normal and ischemic zone
myocardial 99mTc-HL91 clearance curves were
biexponential over the first hour of clearance. This agrees with our
previous results in isolated perfused hearts.2 In
the present study, however, we observed
99mTc-HL91 myocardial clearance for an additional
3 hours after tracer administration. Clearance during this time
demonstrated linear retention with a minimal positive slope for the
ischemic zones and a minimal negative slope for the normal
zones (P=NS). These results indicate a late myocardial
99mTc-HL91 component that is relatively tightly
sequestered. Biexponential clearance implies an early component of
unsequestered fractions related to flow and a later component related
to a slower process perhaps due to reversible binding or enzymatic
reconstitution. Linear retention very late implies a tightly
sequestered fraction. Although the LCx late retention phase appeared to
have a slightly positive slope, this was not significant. A slightly
positive slope could be explained by delayed uptake due to residual
blood activity.
Ex Vivo Gamma Camera Imaging
In the present study, images of 8 of 8 ex vivo hearts and
heart slices demonstrated increased 99mTc-HL91
activity in the LCx zone when assessed qualitatively and
quantitatively. After 4 hours, the LCx/LAD ratios ranged from 2.2 at
the base to 3.9 in the second slice. These values compare well in
magnitude with the LCx/LAD activity ratio of 3.0 obtained at 4 hours in
vivo with gamma camera imaging. This indicates that other organ and
blood activities did not contribute significantly to values obtained
from in vivo scans of the heart.
Regional Myocardial Blood Flow Versus 99mTc-HL91
Tissue Activity
Analysis of the 8 individual graphs of regional myocardial
blood flow versus 99mTc-HL91 myocardial activity
demonstrated 2 phases. During the first phase, decreases in flow from
0.7 to 0.05 mL · min-1 ·
g-1 resulted in marked increases in tracer
activity. This phase was described by an ascending straight line with
r=0.80. Thus, within this phase, increasing severity of
ischemia led to increasing 99mTc-HL91
uptake in a direct negative linear relationship. The second phase
consisted of flat tracer activity for flows >0.7 mL ·
min-1 · g-1. This
phase was described by a flat line with an r value near
zero, as expected (r=0.06). The goodness of fit statistic
(fit standard error, 0.71) was excellent. Thus, it appears that as
blood flow is decreased below normal resting values (<1 mL ·
min-1 · g-1),
there is no increased uptake of 99mTc-HL91. At
70% of normal resting flows, 99mTc-HL91 begins
to accumulate in direct linear proportion to the severity of flow
reduction.
99mTc-HL91 uptake was markedly reduced at flows
<0.05 mL · min-1 ·
g-1. Previous studies using
18F-misonidazole7 and
BMS18132111 also demonstrated reduced tracer
uptake at very low flows. This reduced uptake could be a result of poor
delivery of the tracer due to very low flow. Alternatively, reduced
uptake at very low flows could be due to irreversible myocardial
injury. Perfused-heart studies in our laboratory have demonstrated no
increased uptake of 99mTc-HL91 in irreversibly
injured reperfused myocardium. Although the present
study demonstrated no gross injury by TTC staining, microscopic islands
of injury cannot be excluded.
It should be noted that 99mTc-HL91 tissue
activity at 4 hours was compared with microsphere blood flow at
the time of tracer injection. Epicardial blood flow was kept constant
during this period.
Blood, Lung, Liver, and Gallbladder Activity
In the present study, 99mTc-HL91 blood
activity levels fell rapidly. Clearance was triexponential, and the fit
was excellent (fit standard error, 0.45). Thus, background activity
from blood pools probably did not affect image quality beyond a few
minutes.
The hot spot/lung activity ratio remained relatively constant
throughout the 4-hour experiment, ending at 3.4 after 4 hours. Thus,
lung activity was essentially background and did not degrade myocardial
image quality. Gallbladder activity was observed on the images but did
not significantly affect myocardial image quality.
The hot spot/liver activity ratio remained relatively constant
throughout the 4-hour experiment, ending at 1.0 after 4 hours.
Unshielded images were thought to be of excellent quality. Thus, this
agent is the first to demonstrate excellent ischemic/normal
uptake and retention ratios while maintaining reduced liver uptake
compared with previously described agents.
Nitroimidazole Trapping Mechanism
In normal tissue, it was thought that previously described
nitroimidazoles enter cells by way of their lipophilicity and exit by
back-diffusion without further reaction. When a nitroimidazole
undergoes a single-electron reduction of the nitro group, a free
radical is formed. Free radicals are unstable and highly reactive
species that are unlikely to remain in this state for long.
Nitroimidazoles may undergo 1 reduction reaction and subsequently gain
an electron, restoring the original molecular configuration and
allowing diffusion out of the cell. In this situation, retention is not
prolonged in normal tissue. In hypoxic tissue, nitro free radicals are
less likely to gain an electron, and cellular retention is prolonged.
99mTc-HL91 is actually produced by removal of the
2-nitro- imidazole moiety from the parent nitroimidazole. Thus, in
hypoxic tissue, nitroimidazoles may actually undergo a sequence of
reduction reactions with intracellular enzymes to form more reactive
products that are able to bind to cellular components. Although
this sequence of reactions is not completely understood, further
biochemical modifications to the original molecule are possible through
additional reduction reactions that may result in binding to cellular
elements. The net effect of these structural alterations is to reduce
the membrane permeability of the compound so that cellular retention is
prolonged.
Comparison of 99mTc-HL91 With Other Agents
Initial cardiac studies in vitro and in vivo detected hypoxic
myocardial tissue by use of 18F-labeled
misonidazole and positron imaging.6 7 Shelton and
associates6 studied
18F-misonidazole in a canine model of
coronary occlusion. They reported a 23% retention in the
ischemic zone and a 2% retention in the normal zone. Martin
and associates7 also studied
18F-misonidazole in a canine model of
coronary occlusion. They reported an ischemic/normal
zone activity ratio of 3.0 after 4 hours. This is the same ratio as the
hot spot/LAD myocardial activity ratio determined from gamma camera
images at 4 hours in the present study.
One other 99mTc-labeled hypoxia-avid
imaging agent has been studied extensively to date. This agent is
BMS181321.9 In perfused-heart experiments, serial
increases in BMS181321 myocardial uptake have correlated with both
serial decreases in tissue oxygen and serial decreases in blood
flow.10 12 Stone and
associates13 studied this agent in a swine model
of ischemia with extracorporeal circulation. They demonstrated
increased tracer uptake and retention in ischemic
myocardium. The correlation of flow to tracer uptake was
r=0.68 (compared with r=0.80 in the present
study with 99mTc-HL91). They reported an
ischemic/normal myocardial BMS181321 activity ratio of 1.7
(compared with 3.0 for 99mTc-HL91 in the
present study), a heart/liver ratio of 0.58 (1.0 in the present
study), and a heart/lung ratio of 3.1 (3.4 in the present study).
Shi and associates11 also studied BMS181321 in a
canine model of coronary stenosis with pacing. There
was an inverse correlation of myocardial blood flow to tracer uptake,
with r=0.67 with a second-order polynomial fit. The
ischemic/normal BMS181321 activity ratio was 1.61 at 60
minutes. The hepatic/ischemic myocardium BMS181321
ratio was 4.2 at 60 minutes. Although BMS181321-increased
ischemic zone uptake was found in all ex vivo and in vivo
studies, the authors cautioned that "an unfavorable heart to liver
ratio was observed with in vivo planar imaging which may limit its use
in clinical myocardial imaging." Preliminary reports have described
BMS194796, a modification of BMS181321, which may have reduced hepatic
tracer uptake and faster ischemic tissue tracer
uptake.15
Clinical Implications
The present study using 99mTc-HL91
demonstrates the ability to positively identify regionally
ischemic myocardium by use of
99mTc imaging with standard gamma camera
equipment. By qualitative analysis, identification of regional
ischemia is possible within 60 minutes of tracer
administration. Image quality subsequently improves from 60 to 240
minutes. By use of quantitative analysis of LCx/LAD activity
ratios and LCx and LAD zone washout rates, identification of regional
ischemia may be possible as early as 15 minutes after tracer
administration.
An assessment of the severity of myocardial ischemia may be
theoretically possible with 99mTc-HL91, because
there is a progressive linear increase in activity for flows between
0.7 and 0.05 mL · min-1 ·
g-1. It should be noted that this study used an
open-chest model. Clinically, it may be more difficult to quantify
uptake and clearance. Furthermore, clinically, one may not have a
severe ischemia reference point, although normal tissue uptake
should provide a baseline for comparison. Possible clinical situations
in which the use of a hot spot ischemia–detecting agent could
be useful include (1) the detection of "hibernating"
myocardium before cardiac transplantation or
coronary bypass surgery; (2) the detection of salvageable
myocardium before reperfusion therapy during acute
myocardial infarction. This potential application would be feasible
only if an assessment could be made rapidly by quantitative techniques;
and (3) the detection of exercise-induced transient myocardial
ischemia in the detection of chronic coronary artery
disease. This potential application may be problematic,
given the short period of ischemia.
Conclusions
1. This study demonstrates the ability of
99mTc-HL91 to detect regional myocardial
ischemia in vivo in an intact canine model with a
coronary stenosis by use of gamma camera imaging. Hot
spot detection is possible qualitatively within 60 minutes and improves
until 4 hours after tracer administration. Quantitative ROI
analysis of in vivo LCx/LAD activity ratios demonstrated
significant increases within 30 minutes after tracer administration,
with a final value of 3.0 at 4 hours. Quantitative ROI analysis
of LCx and LAD zone washout curves demonstrated significant differences
within 15 minutes of tracer administration.
2. 99mTc-HL91 normal and ischemic zone
washout curves were biexponential over the first hour of clearance,
followed by a flat linear retention from 1 to 4 hours. The 4-hour
fractional retention was 0.12 for the normal zone and 0.44 for the
ischemic zone.
3. Myocardial blood flow versus 99mTc-HL91
retention curves demonstrated 2 phases. The first phase was gradually
increasing tracer activity for flows between 0.7 and 0.05 mL ·
min-1 · g-1. This
phase was described by an ascending straight line with
r=0.80. The second phase demonstrated no increase in tracer
activity for flows 0.7 mL · min-1
· g-1, indicating no flow dependence at normal
and high flows.
4. Ischemic myocardial/liver activity ratios remained near 1.0
for 4 hours after tracer administration.
5. Blood clearance was relatively rapid and did not interfere with
early myocardial imaging.
|
Selected Abbreviations and Acronyms
|
|---|
| BMS181321 |
= |
propyleneamine oxime-1,2-nitroimidazole |
| HL91 |
= |
4,9-diaza-3,3,10,10-tetramethyldodecan-2,11-dione dioxime |
| LAD |
= |
left anterior descending coronary artery |
| LCx |
= |
left circumflex |
| ROI |
= |
region of interest |
| TTC |
= |
triphenyltetrazolium chloride |
|
Received February 4, 1997;
revision received January 15, 1998;
accepted January 23, 1998.
|
References
|
|---|
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Abstract
Introduction
