779
Distribution of Carbon Flux Within
Fatty Acid Utilization During Myocardial
Ischemia and Reperfusion
Stephen H. Nellis, A. James Liedtke, and Britta Renstrom
were extracorporeally perfused and divided into two
study groups (16 Aerobic and 13 Ischemic/Reflow hearts). Step function, equilibrium labeling
with ['4Clpalmitate was used to develop uptake and washout curves of radioactive fatty acid
products contained in coronary effluent during either aerobic perfusion or reperfusion after
ischemia (60% reduction in left anterior descending coronary flow for 30 minutes). Left anterior
descending control flows were slightly overperfused in Aerobic hearts (18% higher than in
Ischemic/Reflow hearts); otherwise, circumflex and right coronary flows, left ventricular
pressure, and serum fatty acids and blood sugar levels were comparable between groups. As
expected in Ischemic/Reflow hearts, recovery of regional systolic shortening and myocardial
oxygen consumption in reperfusion was only modestly impaired (-20%o and -19%, respectively,
not significant and p<0.011 compared with preischemic values, not significant from Aerobic
hearts). The only significant metabolized product to be released from labeled fatty acid
utilization in either group was '4CO2. A smaller fatty acid pool also was measured and
accounted for by that contained in the coronary intravascular volume. We could determine no
significant back diffusion of fatty acids from myocardium in either perfusion condition. Uptake
time constants of the early phase of 14C02 production also were virtually identical in both
groups (19.9±3.2 versus 16.7±3.2 minutes in Aerobic and Ischemic/Reflow hearts, respectively)
and strongly correlated with hemodynamics as described by heart rate. In washout studies,
tissue radioactivity in the aqueous soluble and fatty acid pools declined in both study groups,
and counts in complex lipids and cholesterol/cholesteryl esters remained steady, whereas those
in triacylglycerols varied. Washout of 1'4CO2 in both groups never reached background
radioactivity over a 40-minute sampling after cessation of isotope infusion into the perfusate,
suggesting slow release of trapped substrate from intracellular pools, which then proceeded to
fatty acid oxidation. In conclusion, these experiments have demonstrated very similar findings
with respect to fatty acid uptake, storage, and release characteristics between aerobic and
reperfused myocardium. We found no differences in preferred substrate utilization and
oxidation as a result of reversible ischemia followed by reflow. (Circulation Research
1991;69:779-790)
Twenty-nine intact, working pig hearts
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
ontroversy exists as to the level of impairment
in fatty acid metabolism and substrate utilization during early reperfusion after myocardial ischemia. In perfused pig hearts, we have
shown a hierarchical preference for fatty acids with
full return of fatty acid oxidation during acute reflow
accompanied by inhibitions of glucose, pyruvate, and
G
From the University of Wisconsin Hospital and Clinics, Madison, Wis.
Supported in part by US Public Health Service grant HL-41914,
the Rennebohm Foundation of Wisconsin, and the Oscar Mayer
Cardiovascular Research Fund.
Address for correspondence: Stephen H. Nellis, PhD, Director,
Cardiovascular Research, University of Wisconsin Hospital and
Clinics, 600 Highland Avenue, H6/339, Madison, WI 53792.
Received October 17, 1990; accepted May 15, 1991.
lactate oxidations.'-3 Myears et a14 similarly noted
normal rates of oxidative metabolism with fatty acid
substrate during reflow after 1 hour of coronary
occlusion in dog hearts but reduced rates of net fatty
acid uptake from the perfusate. This latter finding
may not be important, because endogenous release
of fatty acids from complex lipid stores is enhanced
severalfold in reperfusion.5 Conversely, Schwaiger
and colleagues6 reported prolonged abnormalities in
fatty acid utilization after a 3-hour coronary occlusion protocol in dogs with delays in clearance of
[1C]palmitate from myocardium, increases in tissue
residual activity, and reduced uptake of palmitic acid
lasting for up to 1 week of reperfusion. However, the
data using positron-emitting isotopes may have prob-
780
Circulation Research Vol 69, No 3 September 1991
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lems with interpretation and subsequently have
been criticized. Fox and coworkers7 described a
fourfold increase in efflux from myocardium of
initially extracted but nonmetabolized fatty acids in
ischemia that confounded estimates of oxidative
metabolism by external detection. Levels of back
diffusion of fatty acids in reperfusion are at present
unknown. Rosamond et a18 argued further that
clearance rates of [1-11C]palmitate could not be
extrapolated to measurements of substrate oxidation not only because of the effilux problem but also
because the positron signal per se nonselectively
records all events simultaneously within the utilization pathway. In addition, it has been hypothesized
that complex lipids within myocytes serve as a large
reservoir for fatty acids with slow turnover kinetics
that cause problems with estimating rates of oxidation, even when [1'4C]palmitate and equilibrium
labeling are used.9 This difficulty would be markedly exaggerated with short-lived [11C]palmitate,
which would not adequately interrogate those distal
portions of the utilization pathway taking up to 30
minutes to come into equilibrium.1101' Thus, the
purpose of the present studies was 1) to review
further the substrate kinetics of long-chain fatty
acids leading to CO2 production, with particular
attention paid to the time constants of incorporation and distribution of substrate; and 2) to
contrast the metabolic behavior between aerobically perfused hearts and those rendered ischemic
and then reperfused.
Materials and Methods
Twenty-nine adolescent swine weighing 48.9±0.6
kg were studied. Sixteen of these animals also had
been used in a separate study (unpublished data) to
develop baseline characteristics of fatty acid utilization kinetics but in this study served as a control
(Aerobic) group to contrast with observations acquired in a separate group (Ischemic/Reflow) during
reperfusion after ischemia. Data for both studies
were obtained contemporaneously. All animals were
anesthetized with pentobarbital (35 mg/kg i.v.) and
underwent establishment of controlled positive pressure ventilation using oxygen-supplemented room
air. The preparation used in this study has been
reported extensively elsewhere.10'12'3 Briefly, the coronary arteries were perfused separately via an arterioarterial shunt connected extracorporeally. Blood
was withdrawn from a femoral artery and returned by
three low flow perfusion pumps to proximally cannulated right, main left, and left anterior descending
(LAD) coronary arteries. Three access ports were
included in the LAD perfusion circuit. One port
allowed infusion of indocyanine green directly into
the perfusion circuit through a 50 -ml mixing chamber. Another port was for infusion of [14C]palmitate
(48±3 ,uCi/animal), which was mixed with the perfusate by passage of the isotope through a segment of
tubing (8-ml volume) contained in series within the
perfusion circuit consisting of alternating sections of
small-bore (0.64-cm i.d.) and large-bore (1.3-cm i.d.)
tubing. A third port was used to sample arterial blood
for gases and metabolites; this blood was compared
with venous blood obtained from a sampling catheter
inserted anteriorly into the great cardiac vein.
Left ventricular pressure and its maximal first
derivative were measured by a high-fidelity, manometer-tipped pressure device (Millar Instruments,
Houston, Tex.) placed into the left ventricular chamber. Regional function was estimated from myocardial shortening as measured by ultrasonic crystals
placed at midmyocardial depth in the LAD perfusion
system. Segment shortening was used to characterize
regional contractility.
Aerobic coronary flows were adjusted so that coronary perfusion pressures approximated arterial
pressures, and the venous oxygen saturations were
estimated at or about 40%.
By prospectively agreed-on entrance criteria, venous saturations in Aerobic hearts were not permitted to decrease below 20% at any time in the
perfusion trial. To set conditions of near constant
myocardial oxygen demand in all hearts, blood volumes were replaced with 6% dextran in saline to
maintain systemic pressures at approximately 100
mm Hg. Serum glucose levels were monitored continuously and maintained at 7.1+0.04 gtmol/ml with
intravenous infusions of dextrose. Indocyanine green
indicator (0.33 mg/ml) was infused into the LAD
perfusate for 5 minutes every 10 minutes at a flow
rate of 1.0 ml/min during the radiolabeled perfusion
period to measure the cross-contamination of any
unlabeled coronary blood entering the venous system
of the LAD circuit. From this, a dilution factor was
calculated to correct the venous counts that then
were used in the calculations.
Metabolic and mechanical data were obtained at
specified intervals throughout the perfusion studies.
Blood samples obtained from the LAD artery and
vein were used to measure blood gases and 1'4CO2
production from labeled palmitate. The former samples were used to calculate regional myocardial oxygen consumption (MVo2 in millimoles per hour per
gram dry weight) by a previously described formula.12
The latter samples were centrifuged at 1,500g at 4°C
for 10 minutes to obtain plasma. Blood from the
femoral artery likewise was centrifuged to acquire a
similar plasma fraction. These plasma samples from
the LAD artery, great cardiac vein, and femoral
artery were counted to measure total radiocarbon in
the blood. 1'4CO2 production from labeled palmitate
also was calculated and expressed in micromoles per
hour per gram dry weight as
,A 4CO2 X QLAD X 60
(1)
ASAxLAD dry wt
where /A4CO2=(V-A)/K in disintegrations per
minute per milliliter. This was the difference in
radioactive counts between venous (V) and arterial
(A) '4CO2 in coronary perfusate. The difference in
~ ~ dt
Nellis et al Flux Through Fatty Acid Metabolism in Ischemia/Reflow
the carbons defined by this integration, for the most
part, do not exist as CO2 or free fatty acid but instead
as intermediates. The term free fatty acid mole
equivalence has been used for these data. A second
index (milliliters per gram dry weight) also was
defined using
Time Activity Curve
.
Ce
CD
._
5
E
10
E
0
I-
l
i
j
T
PERFUSION TIME
)e6n
(min.
)
781
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FIGURE 1. Conceptual time-activity curves of radioactive
uptake analysis using a constant infusion metthod ofsubstrate
delivery with [14C]palmitate. To marks the iinitiation of substrate infusion; Te is the time of equilibriumi or steady-state
uptake of isotope; Tn is the time of isotope tewninatson.C,e s
the concentration of substrate or product at steady-state
delivery, and the hatched area represents the calculated tissue
pool size.
of radioactive
counts was corrected by the indocyani ne green dilution factor, K. QLAD is flow in the LAD bed in
milliliters per minute, and ASA is the a rterial specific
activity of free fatty acids in disint egrations per
minute per micromole, expressed as th4 e ratio of A/B,
where A is the arterial palmitate raidioactivity in
disintegrations per minute per millilite r, and B is the
arterial palmitate concentration in m icromoles per
milliliter. Palmitate was measured by tthe method of
Ho.14
With respect to determining tissue carbon pool
p
size for fatty acid constituents, numeri( .,calinteraion
integration
,al
was used. The pool counts for CO2 a
were calculated by subtracting the arrea under the
time-activity curve for labeled product from the area
beneath the equilibrium concentration level (Ce) (see
Figure 1). For both areas, the range of integration
was from the time at which the labe:1 entered the
mixing tubing (To) until the last tim e sample was
taken (Tn). Area under the time-acti vity curve was
determined by fitting data with a natural cubic
spline.15 The spline then was integratedI from TO to Tn
using a trapezoidal method that allomved successive
approximations to the routine.16 Each estimate further subdivided the interval until the change in the
area was less than or equal to 0.001%. The Ce value
was determined by first integrating th.e spline trom
the point at which equilibrium occurre'A (Ta) until Tn,
then dividing this area by the time intcerval (Tn-Te).
The area under CQ then was calculaited from the
product of Ce and the time interval (Tn-TO). The
units represented on this curve are miicromoles CO2
or fatty acid per minute per gram dry w'eight, and the
value at each time point is calcula ted using an
equation similar to Equation 1. The in tegrated units
are micromoles CO2 or free fatty acid per gram dry
weight, which are an index of the pool size, because
14C02XQLAD
AxLAD dry wt
(2)
where A is arterial concentration of labeled palmitate in disintegrations per minute per milliliter. This
index can be thought of as the volume that would be
occupied by the radioactive carbons if they were
found in the heart at the same concentration as they
occurred in blood. This value has been termed the
volume equivalence and is analogous to the distribution volume described in positron emission tomography (PET) studies.
PET studies also have demonstrated that the timeactivity curves derived from reperfused myocardium
have prolonged clearance times in the early phase of
exponential decay after bolus administration of isotope. To provide a comparison for these studies, the
uptake curves in the present experiments also were
approximated by a single exponential function, and
the time constants were tabulated.
At the end of the studies, tissue samples from the
LAD and adjacent circumflex (LCF) beds were
freeze clamped and stored at -70°C for further
analysis. These samples were obtained before the
infusion of isotope was discontinued. The location of
the tissue perfused by the LAD artery was determined at the conclusion of each study by injection of
india ink into the LAD cannula. The relation of the
previously obtained tissue samples to the perfusion
regions was verified. Then, LAD myocardium was
dissected free from that perfused by the combined
right and LCF arteries, and both perfusion systems
were
weighed.
Frozen tissue samples obtained from the separate
perfusion beds were analyzed for metabolites and
extracted with chloroform/methanol according to
Bligh and Dyer.17 The organic fraction then was
analyzed using a prepacked Sep-Pak silica column
after the method of Hamilton and Comail8 for separation of cholesteryl esters, triacylglycerols, free
fatty acids, cholesterol, and polar lipids.
Protocol
Studies were performed in intact, whole bloodperfused, working swine hearts that were divided into
the Aerobic (n = 16) and Ischemic/Reflow (n = 13)
groups. In all animals of both groups, right coronary
and LCF flows were held at aerobic flows throughout
the experiments. Within each group, separate studies
were conducted to study the kinetics of labeled
palmitate incorporation and washout. In the 13
782
Circulation Research Vol 69, No 3 September 1991
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hearts of the Ischemic/Reflow group, LAD flow was
maintained at aerobic levels for 10 minutes of perfusion, reduced by 60% for the next 30 minutes, and
then returned to aerobic levels for the final 50
minutes. Infusion of [U-14C]palmitate at 2.6 x 106
dpm/min was started 10 minutes after the conclusion
of ischemia and continued for a total of 40 minutes
during reflow. In five of these animals, reflow was
continued for an additional 40 minutes after the
palmitate infusion was terminated to create a washout period. In the 16 Aerobic animals, the LAD flow
was maintained at aerobic flows throughout the
studies. [U-14C]Palmitate was infused (2.4x 106 dpm/
min) at 50 minutes and was continued for the next 40
minutes. In five of these hearts, perfusion also was
continued for an additional 40 minutes after infusion
of labeled palmitate had been discontinued. Venous
and arterial blood samples for blood gas determinations were obtained throughout the studies. Other
blood samples were collected to describe the kinetics
of labeled palmitate infusions. For Ischemic/Reflow
hearts, venous blood samples for 14C02 measurements were taken every 2 minutes for 10 minutes,
then every 5 minutes for the next 30 minutes. Venous
plasma samples were collected every 0.5 minute for 3
minutes, then every 2 minutes for the next 6 minutes,
followed by every 5 minutes for the next 30 minutes.
Coronary arterial blood samples for 14CO2 measurements were obtained every 5 minutes throughout the
studies, and coronary arterial samples together with
femoral arterial samples were collected every 10
minutes to analyze for non-14C02 radioactivity. For
the Aerobic hearts, venous blood samples for CO2
data were obtained every 2 minutes throughout the
40-minute labeling period, and venous plasma samples were collected every 0.5 minute for 3 minutes,
then every 2 minutes for the rest of the 40-minute
labeling period. Arterial CO2 and plasma samples
together with femoral arterial samples were taken
every 10 minutes for 40 minutes. During the washout
period, blood was obtained at the same time intervals
as described above for the Ischemic/Reflow group.
Statistical comparisons of data were made within
each group using paired Student's t tests and between
groups using nonpaired Student's t tests. Statistical
significance was defined by two-tailed probability
values less than 5%.
Results
Figure 2 shows coronary flows and left ventricular
pressures for both Ischemic/Reflow and Aerobic
groups. Venous oxygen saturations in the LAD bed
at aerobic perfusions (0-10 minutes) were comparable between groups, although flow rates were slightly
higher in Aerobic hearts (7.1+0.3 versus 6.0+0.3
ml/min/g dry wt, p<0.034). We do not feel this
difference is physiologically significant. The LAD
flow in Aerobic hearts was purposely maintained
slightly higher than that required to meet the metabolic demands of the heart. This perfusion level was
selected to guarantee that the tissue would not be
exposed to ischemic conditions. In the Ischemic/
Reflow group, LAD flow was reduced to 2.4±0.1
ml/min/g dry wt during ischemia and then acutely
returned to preischemic levels at 40 minutes. Circumflex plus right coronary flows in Aerobic and Ischemic/Reflow hearts were maintained at 11.3 ±0.6 and
10.2±0.6 ml/min/g dry wt, respectively, throughout
the experiments. Left ventricular pressure averaged
102.5± 1.2 mm Hg for both groups throughout the
perfusion trial and was not statistically different
between groups. Serum fatty acids were 0.51±0.04
,umol/ml, and blood sugar concentrations were
7.13±0.04 ,umol/ml (average of all animals in both
groups). Neither substrate level was statistically different between groups.
Figure 3 shows regional mechanical and metabolic
performance in the two groups. Aerobic values for
both systolic shortening and myocardial oxygen consumption were steady over the course of the perfusion trials (not significant in the middle [10-40
minutes] and later [40-90 minutes] portion of the
runs, as compared with the early [0-10 minutes]
intervals by paired Student's t tests). There were the
expected decreases in mechanical and metabolic
functions (p<0.001) in Ischemic/Reflow hearts during ischemia and partial recovery during reflow.
Although MVo2 remained statistically lower during
reperfusion, systolic shortening was only marginally
different from aerobic values, with statistical significance at only the 50- and 70-minute time points.
Specialized studies (Table 1) were conducted to
monitor radioactivity of metabolized and nonmetabolized products of labeled palmitate in coronary
effluent. Blood from Ischemic/Reflow hearts was
compared with blood labeled in vitro with [14C]palmitate. [14C]Palmitate was added to blood that had
no radioactive material present. The amount of radioactivity added was such that the counts were
similar to those of blood taken from the in vivo
studies (approximately 6 x 104 dpm/ml). The distribution of radioactivity in this in vitro labeled blood then
was determined in a manner identical to the approach used with the in vivo samples. These spiked
standard data represent the distribution of "normal"
contamination of radioactivity affected by adding
fatty acids to blood constituents independent of
products accumulating from myocardial metabolism.
These data are compared with the Ischemic/Reflow
blood (Table 1). Distribution of radioactivity in coronary venous effluent was almost totally contained in
the organic or fatty acid compartments, with a small
additional component noted in the aqueous soluble
phase and phospholipids. The distribution was nearly
identical to the pattern in the nonmetabolized spiked
standard data. Indeed, the only significant metabolized product to be released from labeled fatty acid
utilization in hearts was 14C02.
Uptake and washout of fatty acids and 14CO2 are
shown for two representative animals in the Ischemic/Reflow group in Figures 4 and 5 and for both
groups in Table 2. Again, in the uptake studies, there
_. _ ~ ~ ~ ~
Nellis et al Flux Through Fatty Acid Metabolism in Ischemia/Reflow
15 r
A
T
C,
s
0
.i
IL
dr
z
C,
._
&m
0
T
T
1~~
1
1
Io
o
10 1
CD
0 E
0 E
U
Q%
T
I
T
I
I
1
O ~
~
5
T
T
o ±
~
m_
~
~
~
~
m~E~-. .
o
+-
T
T
T
1
1
-L
Aerobi
IcYFi
FIGURE 2. Controlled variables of coro-
nary flow separately displayed as the com-
Reflow
I-11
0
bined circumflex (CIRC) plus right coronary (RCA) flows and left anterior
descending (LAD) flows (panel A) and
left ventricular pressures (LVP) (panel B)
for Aerobic and Ischemic/Reflow study
groups. Aerobic hearts were slightly overperfused in the LAD bed, which met statistical significance (p<0.034).
125 r
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B
z
C
100
h
75
h
T
T
Tr~~91
9
783
T
T-L
~-A
CD
0.
EJ
50
I
0
I
10
I-
I
20
30
40
50
60
70
I
80
90
PERFUSION TIME (min)
were no counts in coronary effluent in either group
over and above normal background (spiked standard
counts as described above) that were not accounted
for in either the labeled CO2 or fatty acid pools. The
latter was the smaller pool in both groups, and this
pool could all be contained in the coronary intravascular compartment as defined by the volume between
the [14C]palmitate infusion port and the coronary
venous sampling port. Back diffusion of nonmetabolized fatty acids was estimated in five animals from
each group using three different approaches. First,
the difference between the free fatty acid pool during
washout and the pool contained in the vascular blood
volume was obtained in each group. The latter was
calculated as 8 ml (the volume of the perfusate
tubing) + 11% xbed weight (the coronary blood volume contained in myocardial tissue weight). This
difference was essentially nil, indicating that all of the
free fatty acid pools in both groups could be accounted for by the combined blood volumes in the
perfusion tubing and the coronary blood volumes.
The level of ischemia selected in this study followed
by reperfusion appeared to have no effect in exacerbating back diffusion above that in aerobic hearts.
Second, the free fatty acid pools were compared
between the Aerobic and Ischemic/Reflow groups
and tended to be slightly smaller for the Ischemic/
Reflow group (see Table 2). Finally, the levels of
radioactive free fatty acid in the 20- and 40-minute
samples of venous effluent during washout were
compared between the two groups. Again, there was
no difference between the two groups (125±60
pmol/g dry wt in Aerobic versus 56+50 pmol/g dry wt
in Ischemic/Reflow hearts at 20 minutes and 42+30
versus 86±+70 pmol/g dry wt, respectively, at 40
minutes). Counts in the venous effluent also were not
statistically different from zero at either of the two
time points for both groups.
The larger pool of radioactivity was that represented by 1'4CO2 (Table 2). In the uptake curve analysis, there was a trend toward reduced pool size in the
Ischemic/Reflow hearts, whereas equilibrium levels of
'4CO2 production were comparable or decreased depending on the units of measure. Interestingly, the
uptake time constant for the radioactive buildup of
'4C02 in coronary effluent was similar for the two
groups (19.9+±3.2 minutes for the Aerobic group and
16.7±+3.2 minutes for the Ischemic/Reflow group).
These were equal to half-times of 13.8 and 11.5
minutes, respectively. Oddly, these time constants
Circulation Research Vol 69, No 3 September 1991
784
n 125
A
i 100 h
.E
75
t
.! 50
h
-0-
Aerobic
h-
-+-
ischemia
Reflow
o
,a
b.
0
25
0
cn
0
a
a
2.0F
a
a
a
a
-
-
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FIGURE 3. Dependent variables for both study
groups describing regional mechanics (systolic shortening [SS] normalized to initial values in the left
anterior descending myocardium) (panel A) and
metabolic function (myocardial oxygen consumption [MV2J) (panel B). Recovery of metabolic
function was impaired in the IschemiclRefiow group,
but systolic shortening almost retumed to that observed in Aerobic hearts.
-
B
I-
0
1.5
h
1.0
h
0.5
h
0-0
9-
L.
.a
I~~~~~~~~~
T
T
-L
+,
E
OC
:E
^
^
1
0
-
1
10
-
1
.
*
a
20
30
40
50
----
9
60
-9
9
70
80
----
--J
90
PERFUSION TIME (min)
were not significantly correlated with either the aqueous or lipid phase fractions of tissue radioactivity or
the myocardial oxygen consumption in either group.
Conversely, the time constants of '4CO2 release in
both groups were inversely correlated with heart rate
(Figure 6) according to the expression Time
Constant=57.1-0.25 x heart rate; r=0.62, p<0.0004
(all animals included from both groups).
In separate studies in 10 animals, washout curve
analysis also was performed (Figure 5, Table 2).
Exponential decay of counts was similar between
groups but different between labeled constituents.
TABLE 1. Distribution of Radioactivity in Coronary Venous Effluent of Ischemic/Reflow Hearts (n=5)
Ischemic/
reflow (%)
Blood
Fractions
7.74± 1.48
Aqueous Soluble
91.62± 1.52
Organic
99.36±0.01
Total of two phases
Total direct (plasma)
100
Lipids species
0.11±0.02
Cholesteryl ester
0.07±0.04
Triacylglycerols
Free fatty acids
83.38±2.87
0.77+0.22
Cholesterol
1.32±0.49
Phospholipids
87.74±2.75
Total in fractions
5.89±1.57
Residuet
Total organic
91.63±1.53
*Nonmetabolized blood spiked with ['4C]palmitate.
tRemaining radioactivity in glass tubes.
Standard*
(%)
p
5.70±2.55
93.90±2.52
99.60±0.04
100
NS
NS
<0.063
0.08+0.02
0.10±0.05
83.48±2.58
0.54±0.04
0.68±0.23
84.88±2.64
9.03±2.20
93.92±2.50
NS
NS
NS
NS
NS
NS
NS
NS
Nellis et al Flux Through Fatty Acid Metabolism in Ischemia/Reflow
785
5.0
so
4.0
3.0
E
EC
2.0
&L
U.
0.0
X
2.0
1.0
FIGURE 4. Labeled fatty acid (FFA)
and 14CO2 uptake measurements in left
anterior descending coronary venous effluent in representative hearts from the Ischemic/Reflow group. Interpolated areas
(gray hatched areas) are the estimated
pool sizes of each constituent. Insert: Expanded time scale to show the small size of
the FFA pooL The configuration of these
two curves was quite similar to thosefound
in Aerobic hearts.
B
am
v
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1.5
CD
r.
1.0
g
0
0.5
I*~~~
a
a
U
10
20
a
30
a
40
PERFUSION TIME (min)
Fatty acids quickly reached background levels of
radioactivity after cessation of ['4C]palmitate infusions, whereas '4CO2 did not. This trend is compatible with the concept that labeled substrate is still
present in intracellular fatty acid pools, probably
complex lipids, and after discontinuance of labeled
infusions is slowly released to enter fatty acid oxidation and '4CO2 production.
In a final comparison, distribution of radioactivity
in tissue was compared between groups in both
uptake and washout protocols (Table 3). In uptake
studies in Aerobic hearts, counts between lipid and
aqueous soluble phases were almost evenly divided
(52% and 48%, respectively), whereas in Ischemic/
Reflow hearts, counts were increased in the lipid
fraction (66%). Generally, there were no major statistical differences between groups among the various
fractions and lipid species in either the uptake or
washout data. Triacylglycerol radioactivity tended to
be higher in the Ischemic/Reflow group, and the
aqueous soluble phase radioactivity was slightly
smaller as measured by the volume equivalency
method. In washout studies, counts in complex lipids
and cholesterol/cholesteryl esters in both groups remained relatively fixed, with more variability noted in
the triacylglycerol compartment, particularly in Aerobic hearts. Conversely, counts in the aqueous soluble fraction and tissue fatty acid compartment dramatically declined with washout in both groups.
These latter two constituents correlated closely with
the '4CO2 uptake pool in both groups (Figure 7) and
probably represent the same material.
Discussion
There has been renewed interest in the mechanical
and metabolic behavior of reperfused myocardium,
in part stemming from the development of clinical
salvaging techniques to reperfuse injured ischemic
myocardium in patients with acute coronary events.
Stunning,19-21 an impairment in mechanical recovery
after reperfusion in the absence of obvious histological damage, has been variously explained to result
from problems with ATP resynthesis or compartmentalization, calcium overload, or free radical injury.22-28 Metabolism in reflow is not completely understood, and there is possible disagreement as to the
rank of substrate preference between fatty acids and
glucose in the reperfused condition.' 4,29 34 With
respect to fatty acid metabolism, differences in interpretation may result from the choice of techniques
used to measure rates of utilization and oxidation.
The PET technique, essentially a pulse labeling
technique, has provided evidence that "C-tracer
clearance and decay from bolus injection of positronlabeled fatty acids is substantially depressed early
after reperfusion35 and recovers only slowly.6 Conversely, other studies using ['4C]palmitate and equi-
786
Circulation Research Vol 69, No 3 September 1991
4.0-
A
3.0
3:
2.01.0 -
FIGURE 5. Labeled fatty acid (FFA)
and 14CO2 washout measurements in left
anterior descending coronary venous effluent in representative hearts from the Ischemic/Reflow group. Interpolated areas
(gray hatched areas) are the estimated
tissue pool sizes of each constituent. Insert: Expanded time scale to show the
small size of the C02 pool. Note that the
14C02 decay, in contrast to that of FFA,
never asymptotically reached background
levels of radioactivity during the 40 minutes we sampled. This is similar to the
pattems observed in Aerobic hearts.
0.0 -1.0
3:
1.0 B
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0.8
U
E
E
0.5-
0.3
9-
0.0 40
50
60
70
80
PERFUSION TIME (min)
librium labeling have shown that fatty acid oxidation
is not depressed.1-4
More than one hypothesis is available to explain
this discrepancy. One possibility, although not shown
in our study, is that the "C-tracer is removed from
the heart by nonmetabolic means, such as back
diffusion, that might be enhanced under some conditions of reflow. If the rates of metabolic and
nonmetabolic turnover are similar, the deconvolution
of the time-activity curve to separate the two would
be difficult. In short, the time-activity curve might be
misinterpreted based on nonmetabolic events. Alternatively, because the time required to dissipate radioactivity in a pool depends not only on the rate of
removal but also on the size of the pool, an increase
in the distribution volume could prolong the timeactivity curve described by PET, and in so doing,
skew the interpretive results. Neither event would
bias the data defined by equilibrium labeling.
On the other hand, some problems in analysis are
common to both techniques. The time-activity curves
acquired by PET represent transmural information
reflecting heterogeneously perfused myocardium.
The venous samples obtained in this study also are
derived transmurally, but the heterogeneity of perfusion is diminished in pig hearts because of the deficit
in preformed collateral circulation.36,37 The consequence of this heterogeneity on the measured time
constants is different for the two techniques. Time-
activity curves from PET represent tissue retention of
radioactivity and thus would over-represent cells with
high retention or slow metabolism. Conversely, myocardial efflux of '4CO2 as determined in this study
primarily reflects behavior in the most metabolically
active cells.
A final problem deals with radioactive labeling of
intracellular lipid pools and its effect on quantitating
fatty acid oxidation. This study has demonstrated by
washout curve analysis a prolonged release of '4CO2
after termination of infused ['4C]palmitate. We interpreted this to reflect the reintroduction of labeled
substrate into the fatty acid utilization pathway from
temporary storage in complex lipids, particularly
triacylglycerols. Because this pool size may be quite
large, the release kinetics of isotope are complex and
prolonged (beyond our 40-minute sampling interval)
and may account for a 20% error in estimating 14C02
production based on equilibrium labeling. PET studies also have recognized a late phase in the timeactivity curves, which has been attributed to tracer
deposition in the endogenous lipid pool but which
has been ignored as an element of metabolic activity.
Analysis by PET would be even more compromised
because of the short half-life of the "C-tracer and the
difficulties in adequately labeling all intracellular
lipid pools homogeneously within the allotted time
constraints of the isotope.
Nellis et al Flux Through Fatty Acid Metabolism in Ischemia/Reflow
787
TABLE 2. Estimated CO2 and Free Fatty Acid Pool Sizes and Equilibrium Values: Uptake and Washout Curve Analysis
CO2
Free fatty acids
Pool
(gmol/g
Equilibrium
(ml/g
dry wt)
(,mmolmin/g
dry wt)
dry wt)
Equilibrium
Pool
(M1/min/g
(gmol/g
diy wt)
(ml/g
(,mmnol/min/g
diy wt)
(mlJminlg
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
diy wt)
Uptake
Aerobic (n=16)
4.77±0.91
17.6+2.85
0.44+0.08
1.58+0.23
Ischemic/
2.77+0.41
9.33+1.34
0.30+0.04
0.99±0.13
Reflow (n=13)
p
<0.075
<0.022
NS
<0.041
Aerobic (n=6)
0.58±0.12
1.72±0.12
1.60±0.24
4.71±0.36
Ischemic/
0.34±0.07
1.11±0.19
1.16±0.34
3.61±0.27
Reflow (n=9)
p
<0.08
<0.033
NS
<0.027
Washout
Aerobic (n=5)
3.92±1.07
11.4±3.15
0.04±0.004
0.12±0.02
Ischemic/
2.84±0.54
9.16±2.28
0.03±0.01
0.09±0.02
Reflow (n=5)
p
NS
NS
NS
NS
Aerobic (n=4)
0.49±0.08
1.24±0.19
0.005±0.004
0.02±0.02
Ischeniic/
0.35±0.10 0.98±0.11
-0.000±0.007
0.01±0.02
Reflow (n=5)
p
NS
NS
NS
NS
p values indicate statistical comparisons of data between Aerobic and Ischemic/Reflow hearts. Units are volume equivalence and mole
equivalence of free fatty acids (see text).
The following data were observed in this study.
Metabolic analysis of the coronary effluent virtually
precludes the existence of significant nonmetabolic
routes of release of radioactivity in reflow after the
moderate levels of ischemia imposed in these experiments. Although back diffusion cannot be categorically eliminated, the magnitude of this release, if
present, must be trivial and would occur only during
capillary transit. This should not obscure the rapid
phase of the uptake curve. There was a modest
80
50
F
40
F
0
r =
p =
0
0.62
0.0004
C
E
0
z
0
i- 30 _
v
z
0
0
c)
W 20 h
F
10
0
* *c*
h
00
00
@tO
I-
0'
so
0
100
150
200
250
HEART RATE (beats/min)
FIGURE 6. Time constants of 14C02 release arrayed against
heart rates in the two study groups (Aerobic hearts, open
circles; IschemiclReflow hearts, closed circles). A significant
inverse relation is noted, which is described for all animals by
the expression Time Constant=57.1-0.25xheart rate.
dry wt)
dry wt)
decrease in the free fatty acid pool in Ischemic/
Reflow hearts. This reduction could represent either
a decline in blood volume or an alteration in fatty
acid uptake by the sarcolemma, perhaps as a residual
consequence of the ischemic stress.
The tissue fraction and pool sizes expressed as
volume equivalents in our results are analogous to
the distribution volume described by PET. Our studies revealed modest changes in these pool sizes. The
pool size measured from 14CO2 efflux decreased, as
did the tissue fraction composed of the aqueous
soluble phase and free fatty acids. This same tissue
fraction was rapidly lost during washout. These findings would not explain the prolonged clearance rates
of the rapid exponential decay portion of the timeactivity curve described by PET in reflow. The PET
data would imply a shift to a larger pool size, not a
smaller one as found in these studies. The triacylglycerol tissue fraction in Ischemic/Reflow hearts did
evince a trend toward larger values, but this pool
manifests slow turnover kinetics and is thought to
influence the late phase of time-activity curves.
These studies provide further evidence that the
oxidation of fatty acid is not depressed during reperfusion. Although there is a trend toward decreased
14CO2 production after equilibrium in the Ischemic/
Reflow hearts, the significant decrease in '4CO2 and
free fatty acid efflux expressed as a volume/flow
equivalent is most likely an indication of the higher
LAD flow in Aerobic hearts. This ratio represents
the venous flow required to contain venous radioactivity if the venous concentration was equal to arterial
concentration. As such, it is a function of perfusion.
Circulation Research Vol 69, No 3 September 1991
788
TABLE 3. Distribution of Radioactivity in Tissue in Aerobic (n=16) and Ischemic/Reflow (n=13) Hearts
Aerobic
Ischemic/Reflow
(,umol/g
(ml/g
(,umol/g
(ml/g
dry wt)
dry wt)
dry wt)
dry wt)
p
p
25.1±4.68
14.3±1.79
0.13±0.03
18.6±4.90
NS
NS
NS
<0.05
NS
NS
NS
NS
<0.07
NS
<0.08
NS
NS
NS
(n= 10)
Uptake
Lipid fraction
Aqueous soluble
Cholesteryl esters
Triacylglycerols
Free fatty acids
Cholesterols
Phospholipids
4.90±1.18
(n=7)
19.5±3.04
18.8+1.45
0.14+0.02
10.9±0.89
1.24±0.38
1.20±0.30
6.25+2.26
4.46+0.61
0.04+0.008
2.59+0.31
0.33±0.12
0.31+0.11
1.70±0.80
7.87±1.99
4.01±0.33
0.04±0.01
6.01±1.90
0.28±0.08
0.34±0.05
1.2±0.13
0.93+0.26
1.15±0.08
4.31+0.68
Washout
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(n=5)
(n=5)
9.14+3.97
23.5+8.21
7.90±2.78
20.5+4.50
NS
NS
Lipid fraction
1.43±0.27*
4.00±0.66*
0.98±0.13*
3.00+0.42*
NS
NS
Aqueous soluble
0.04±0.01
0.11±0.02
0.03±0.01
0.08±0.03
NS
Cholesterol esters
NS
7.04+3.76
17.5+8.01
15.1±4.22
Triacylglycerols
6.07±2.57
NS
NS
0.08±0.02
0.10±0.02
NS
Free fatty acids
NS
0.22+0.06t
0.29±0.08t
0.28+0.11
0.75±0.22
Cholesterols
0.24±0.08
0.62+0.12*
NS
NS
1.70±0.26
4.92±0.84
1.47±0.27
4.52±0.76
Phospholipids
NS
NS
p' values indicate statistical comparisons of free fatty acid equivalent data between Aerobic and Ischemic/Reflow heart; p values indicate
statistical comparisons of volume equivalent data between Aerobic and Ischemic/Reflow heart. Units are volume and mole equivalence of
free fatty acids (see text).
*p<0.05, tp<0.1 between washout and uptake experiments.
The surprising aspect of this study was that the
time constants were insensitive to ischemia and
reperfusion. These time constants should be comparable to those measured from PET time-activity
curves, and their insensitivity to altered perfusion
states was unexpected. Several explanations are possible. The ischemic stress imposed in our studies may
be less severe than that reported with the use of PET.
However, at least one PET study in dogs35 did use
comparable ischemic times but with complete coronary occlusion. Although our experiments were performed with only a partial occlusion, such a restriction would provoke at least moderate insult given the
absence of collateral flow in this heart model. Impairments in both mechanical and metabolic func40
I
tions were noted during the ischemic interval, which
continued to compromise oxygen consumption during reperfusion. Another possibility is that the two
techniques, because they are interrogating cells with
different metabolic activities (see above), are not
measuring the same biological information from the
heterogeneously perfused myocardium. The assumption of comparability of time constants might then be
fallacious. A final concern deals with heart rate and
its relation with the time constants of 14CO2 release.
This relation was not dependent on oxygen consumption and suggested some nonmetabolic interaction
that directly related to hemodynamic performance.
Perhaps this determinant is influencing the two techniques differently, and the time-activity curves de-
r = 0.60f15
p = 0.013
r
0
30
FIGURE 7. Correlation of calculated 14CO2 tissue pool
size estimates from uptake curve analysis in both Aerobic and Ischemic/Reflow hearts, with tissue counts of
radioactivity measured directly from the aqueous soluble fraction and tissue free fatty acids (FFA). The
correlation was significant and linear, suggesting the two
parameters may be measuring the same pool.
0
-J
0E
0.
20
0
L0L
bes
0
10
CA.
0
0
0
0
5
10
15
20
25
30
AQUEOUS SOLUBLE FRACTION AND TISSUE FFA (mulg dry wt)
Nellis et al Flux Through Fatty Acid Metabolism in Ischemia/Reflow
rived from PET should be reexamined with a focus
on heart rate.
In summary, the present data do not support a
major metabolic alteration in fatty acid metabolism
in reflow and are confirmatory of previous data
acquired in this heart model.' Under the conditions
of flow restriction in these studies, fatty acid oxidation was not significantly impaired in reperfusion but
rather closely approximates metabolic behavior defined in aerobic hearts.
Acknowledgments
The authors thank T.M. Lange, S.K. Rasmussen,
D.K. Paulson, E. Scarbrough, and C.R. Kidd for
technical assistance.
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KEY WORDS * fatty acids * reperfusion * coronary effluent
radioactivity * stunned myocardium
.
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Distribution of carbon flux within fatty acid utilization during myocardial ischemia and
reperfusion.
S H Nellis, A J Liedtke and B Renstrom
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Circ Res. 1991;69:779-790
doi: 10.1161/01.RES.69.3.779
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Copyright © 1991 American Heart Association, Inc. All rights reserved.
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