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a new method for evaluation of
myocardial energy metabolism
Institute of Experimental Surgery, Heinrich Heine University, 40225 Düsseldorf; and Institute of Geology, Ruhr University Bochum, 44801 Bochum, Germany
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Schwanke, Uwe, Harald Strauss, Gunther Arnold, and Jochen D. Schipke. Analysis of respiratory water
a new method for evaluation of myocardial energy metabolism. J. Appl.
Physiol. 81(5): 2115-2122, 1996.Aerobic ATP
synthesis via oxidative phosphorylation causes a proportional
production of respiratory water. Thus the amount of respiratory water
produced at a given time should be a reliable measure of the current
ATP demand of the mammalian myocardium. Respiratory water from isolated
rabbit hearts was labeled by using the stable oxygen isotope
18O. The hearts were perfused
according to the method of Langendorff (O. Langendorff.
Pfluegers Arch. 61: 291-332,
1895
) with
18O2-equilibrated
Krebs-Henseleit solution. Control hearts were exclusively perfused with
carbogen-equilibrated Krebs-Henseleit solution. Myocardial tissue was
then lyophilized; the extracted water and samples from the coronary
venous effluent were converted to
CO2 by using the guanidine
hydrochloride technique. The
18O values within the
CO2 samples were determined by
mass spectrometry and related to the standard mean ocean water
(SMOW) scale. Compared with control
hearts, the 18O-labeled hearts
exhibited a significant increase of
18O values from tissue water
(
47.50 ± 0.64 vs. 40.35 ± 2.05
SMOW; P < 0.05). The values were also
significantly increased in the coronary venous effluent after a
perfusion time of only 50 s (47.50 ± 0.64 vs. 43.66 ± 0.91 SMOW;
P < 0.05). Thus this first
adaptation of the guanidine hydrochloride technique on microliter
samples of myocardial tissue water and coronary venous effluent
demonstrates that this method can be used to evaluate both respiratory
activity and the kinetics of cardiac metabolic processes.
isolated rabbit hearts; myocardial energy metabolism; stable oxygen
isotope 18O; guanidine
hydrochloride technique; mass spectrometry
INTRODUCTION THE CORONARY CIRCULATION of the mammalian myocardium
has a significant inhomogeneous distribution that is stable for several hours (18). Such inhomogeneity not only exists transmurally but also
within each myocardial layer (8). Associated with this phenomenon is
the regional inhomogeneity of coronary reserve. The term "coronary
reserve" characterizes the property of coronary vessels to vary
their diameter owing to changing oxygen and/or metabolic
demands; it is defined as the ratio of maximal coronary blood flow to
flow at rest. On the assumption of a global coronary blood flow of
100% in the entire heart, the relative regional coronary blood flow
can change between 20 and 200% (3). Given such immense differences in
coronary blood flow and the resulting inhomogeneous distribution of
oxygen and substrates to the myocardial tissue, regional differences in
the energy demand of cardiomyocytes are very likely.
The energy demand must be met in terms of ATP production. Due to the
almost exclusively aerobic metabolism of the myocardium, ATP production
predominantly takes place within the mitochondria via oxidative
phosphorylation (16). Under aerobic conditions, the respiratory chain
is the last metabolic pathway leading electrons and protons from the
decomposing substrates to their terminal acceptor molecular oxygen
(Fig. 1).
Most of the electrons are transferred to the respiratory chain via the
coenzyme NAD (NADH + H+), a
negligible quantity via the coenzyme FAD
(FADH2). The transfer of two
electrons supplied by one molecule of NADH + H+ or
FADH2 results in formation of
three or two molecules of ATP, respectively, and one molecule of
respiratory water (16)
Fig. 1.
Schematic representation of ATP synthesis linked to respiratory chain
(top). Oxidative phosphorylation
occurs when electrons carried by reduced coenzymes NADH + H+ and
FADH2 are transferred to molecular
oxygen. Offering
18O2
to aerobically metabolizing cells such as cardiomyocytes results in
formation of 18O-labeled
respiratory water
(H218O;
bottom).
[View Larger Version of this Image (60K GIF file)]
The above reaction equations show that the amount of respiratory water is expected to be a reliable measure for the cellular ATP demand. For example, the decomposition of 1 mol of glucose during glycolysis and the citrate cycle generates 10 mol of NADH + H+ and 2 mol of FADH2 to transfer electrons and protons to the respiratory chain. Consequently, during oxidative phosphorylation, 34 mol of ATP and 12 mol of respiratory water are formed while 6 mol of molecular oxygen are consumed (16).
Many techniques are available for investigating coronary circulation and myocardial metabolic processes in both isolated and in situ hearts. Regional coronary blood flow can be assessed by using radiolabeled (1, 7) or colored (13, 19) microspheres. The blood flow within defined tissue samples can be calculated by either measuring the incorporated radioactivity and/or the amount of X-ray fluorescence (1, 7, 23, 27) or after alkaline digestion of the tissue and subsequent photometric analysis (13, 19).
For the measurement of myocardial metabolism, radiolabeled substrates or intermediates of cellular energy metabolism may be employed. The accumulation of such tracers within the tissue and/or the coronary venous appearance of catabolic fragments can be assessed by using autoradiography and scintigraphy (5, 12, 20, 26). Positron emission tomography, on the other hand, permits the assessment of both coronary blood flow and metabolism (6, 14).
By using the conventional biochemical techniques, only an indirect
assessment of myocardial energy demand is possible. Measurement of the
regional accumulation of radiolabeled carbohydrates, fatty acids, amino
acids, or their metabolic intermediates additionally contains the
anabolic performance of the cardiomyocytes, e.g., the synthesis of
fatty and/or amino acids to regenerate cellular components, as
well as glycogen synthesis via gluconeogenesis to maintain essential
substrate stores (Fig. 2).
The purpose of this study was to adapt a new technique for evaluating the regional performance of oxidative phosphorylation within the mammalian myocardium. Experiments were performed on isolated rabbit hearts that were perfused with a Krebs-Henseleit solution equilibrated with 18O2. Myocardial tissue samples and samples taken from the coronary venous effluent were then analyzed for 18O-labeled respiratory water (H218O).
METHODS
The experiments on 14 isolated hearts from adult New Zealand White rabbits (age 6-8 mo, weight 3.5-4.1 kg) were performed in accordance with the animal welfare regulations of the German federal authorities. The hearts were prepared according to the method of Langendorff (20a) and perfused with carbogen (95% O2-5% CO2)-equilibrated Krebs-Henseleit solution by using a roller pump; the perfusate was maintained at 37°C, and the coronary perfusion pressure was adjusted to 90 mmHg. The composition of the perfusate was as follows: (in mM) 90 NaCl, 30 NaHCO3, 4 KCl,1 Na2HPO4, 0.5 MgSO4, 2.5 CaCl2, 2.2 pyruvate, and 11.1 glucose.
In a second perfusion system, 500 ml of Krebs-Henseleit solution were
equilibrated with 80% enriched recirculating
18O2.
This solution had been equilibrated previously with 95%
N2-5% CO2 to remove the residual oxygen.
Our experimental setup permitted switching between the perfusion media
within the experiment (Fig. 3).
A latex balloon was inserted into the left ventricle via the mitral valve; the balloon was connected to an artificial systemic circuit (Fig. 3, right) that was separated from the perfusion circuit (Fig. 3, left). To monitor the stability of the preparation, the coronary perfusion pressure, the coronary flow, and the coronary venous oxygen partial pressure were measured in the perfusion circuit, and the left ventricular pressure, the aortic pressure, and the aortic flow were measured in the artificial systemic circuit.
For the assessment of global myocardial oxygen consumption
(M
O2), the coronary
arterial oxygen partial pressure was measured at the beginning, in the
middle, and at the end of an experiment. The global oxygen consumption
was calculated according to the Fick principle
|
is the absorption coefficient (= 0.024),
PaO2 is the arterial
PO2,
PvO2 is the venous
PO2, and CF is the coronary flow.
Experimental Procedures
Langendorff heart preparation. Control hearts (n = 5) were perfused exclusively with carbogen-equilibrated Krebs-Henseleit solution for 15 min and then immediately shock-frozen by using liquid nitrogen to instantly stop myocardial metabolism and, moreover, to trap the respiratory water within the cardiomyocytes where it had been formed. To label respiratory water, nine hearts were first perfused with carbogen-equilibrated solution. After an initial 15-min stabilization period, they were perfused with 18O2-enriched solution for a maximum of 4 min. During this period, sequential samples of coronary venous effluent were taken. Finally, these hearts were also shock-frozen. Basal, median, and apical components of the frozen hearts were separated by using a belt saw. From these tissue slices, samples of right ventricular wall, septum, and left ventricular free wall were taken by using a scalpel. The basal, median, and apical portions of the left ventricular free wall were further cut into subendocardial and subepicardial layers. Each tissue sample weighed ~100 mg.Water-Extraction Procedure
The water was extracted from each tissue sample over an ethanol-dry ice trap within a high-vacuum line (
10
2 mb) during 2 h of
lyophilization. The oxygen isotope ratios in these water samples had to
be determined by mass spectrometry. Direct mass spectrometric analysis
of fluid compounds is very unusual. In general, the compounds are
converted into suitable gases, usually
CO2 in the case of oxygen.
Therefore, 7.5-µl aliquots from the tissue water samples as well as
from the samples taken from the coronary venous effluent were converted
quantitatively to CO2 by using the
guanidine hydrochloride technique (Fig. 4). This technique has been described in detail previously (10, 29).
In brief, incubation of a 7.5-µl water sample together with 50 mg
guanidine hydrochloride within an evacuated
(<103 mb) sealed Pyrex
glass tube at 260°C for 16 h results in the formation of ammonia,
CO2, and ammonium chloride. At a
temperature below 70°C, ammonia and
CO2 form ammonium carbamate, which
sublimates to the wall of the sample tube; the ammonium chloride does
not take part in this reaction. In a second step, the sample tube is
transferred into a reaction assembly containing an acid reservoir of
100% phosphoric acid. The reaction assembly is evacuated to <103 mb and then sealed,
after which the sample tube inside the assembly is broken. The entire
assembly is placed in an oven at 80°C for 1 h. Under these
conditions, ammonium carbamate decomposes and ammonia is trapped by the
phosphoric acid, producing ammonium phosphate. The
CO2 is purified by passage through
an ethanol-dry ice trap and cryogenically transferred to an evacuated
(<103 mb) sample tube for
isotope ratio measurement. These glass tubes containing the
CO2 may be stored indefinitely for
later analysis (10).
Oxygen Isotope Ratios
Naturally occurring 18O accounts for ~0.2% of total oxygen. When water evaporates from the sea, primarily H216O is selected, leading to the enrichment of sea water with H218O over time; conversely, H218O condenses first within the atmosphere. These fractionation effects are due to the different molecular masses, and it is obvious that geography and climate have an important influence on the 18O content of ground water and surface water and therefore of inorganic and organic oxygen compounds (24).In 1957, Craig (9) defined a reference for the determination of oxygen
isotope ratios in water samples and termed it standard mean ocean water
(SMOW). The
SMOW standard defines the
H218O/(H218O + H216O) or
18O/(18O + 16O) ratio of an arbitrary ocean
water sample. The proportion of 18O in
SMOW
[18O/(18O + 16O)] is 1,989.5 ± 2.5 × 106 (2). This
isotopic composition was assigned a value of 0 on the
SMOW scale (Fig.
5); the unit of the scale is per thousand (). Water samples containing less
18O than the
SMOW standard are assigned negative
SMOW values; samples containing more
18O receive positive values.
); compared with SMOW
standard, oxygen isotope ratio of any water sample can be expressed as
18O ( SMOW).
18O enrichment results in an
increased SMOW value. Our myocardial water samples were located in the range between about 39.00 and 49.00 SMOW, indicating
that 18O content was lower
compared with SMOW standard.
Determination of the oxygen isotope ratios
18O/(18O + 16O) within the
CO2 samples was performed by using
a mass spectrometer (Finnigan, MAT 251) equipped with a
multicollector. Results are expressed as
18O values related to the
SMOW scale. The
18O values are defined as
|
The Krebs-Henseleit solution used in our study was prepared by
using double-distilled water. The distillation procedure caused a
depletion of
H218O from
the distilled water fractionthe
H216O content
increased, i.e., the SMOW value
decreased. The mean 18O value
equaled 47.50 SMOW.
The significance of this unfamiliar unit can be remembered as follows:
18O values express the content
of 18O atoms within a water sample
compared with the absolute 18O
content of the arbitrary SMOW water
sample; consequently, 47.50 SMOW means 47.50 of 1,989.5 ± 2.5 × 106
[18O/(18O + 16O)] less compared with
the SMOW standard.
Data Processing
Data were processed by using an IBM-compatible personal computer and the statistics program SYSTAT (28). Data are presented as means ± SE. The significance of changes was determined with a one-way analysis of variance for repeated measures. If significant differences were detected, comparisons were performed with a t-test for paired data with the Bonferroni correction. A P value <0.05 was taken as statistically significant.RESULTS
In our isolated rabbit heart preparations, the PaO2 in the Krebs-Henseleit solution was ~560 ± 30 Torr, and the venous PO2 was ~190 ± 25 Torr. The mean coronary flow was 50 ± 7 ml/min, and the mean heart rate was 200 ± 26 beats/min. The wet weight of the 14 hearts ranged from 8.2 to 10.8 g (mean 9.5 g).
Applying these data to the Fick principle resulted in an
average MO2 of 60 µl · g1 · min1
(
2.7 µmol · g1 · min1).
Control Series
The control series (n = 5) was performed to determine a18O
reference value in the cellular (respiratory) water of isolated rabbit
hearts perfused exclusively with carbogen-equilibrated Krebs-Henseleit
solution. In all experiments, this solution was prepared by using water
from the same still. Figure 6 shows the 18O values in water extracted
from five different myocardial areas. The results show low scatter
among both different hearts (maximum SD: 0.90
SMOW) and different myocardial areas
(maximum SD: 0.53 SMOW).
For comparison with 18O-labeled
hearts, the 18O reference value
was taken as the mean of all measured oxygen isotope ratios in the
control hearts: 47.50 ± 0.64
SMOW.
18O values of
respiratory water extracted from 5 different myocardial areas
[right ventricular free wall (RV), septum, and left ventricular
free wall (basal, median, and apical)] of control hearts that
were perfused exclusively with carbogen-equilibrated Krebs-Henseleit
solution. Far right open bar shows
18O value ( SMOW) taken as mean ± SE of all
measured oxygen isosope ratios within control series that was used as
reference for later analysis of
18O-labeled hearts.
18O-Labeled Series
18O values were significantly
increased in all myocardial areas of the nine hearts labeled with
18O2
(Fig. 7,
right). Again, scatter among the
hearts (2.45 SMOW) and
among different myocardial areas (0.94
SMOW) was low. The 18O values were 40.68 ± 1.74 SMOW in the right
ventricular free wall and with 39.82 ± 1.79
SMOW slightly higher in the apical left ventricular subendocardium; significant differences among myocardial areas could not be detected. The mean
18O value determined from all
measured oxygen isotope ratios in this series was 40.35 ± 2.05 SMOW. The difference
between control and 18O-labeled
hearts averaged 7.15 SMOW (=
15.05%).
18O mean values
of respiratory water from control series [far
left open bar,
18O reference value = 47.50 ( SMOW)]
and 18O-labeled series
(right). Values are means ± SE.
Perfusion of isolated rabbit hearts with
18O2-equilibrated
perfusate resulted in significantly increased 18O values within all
myocardial areas (* P < 0.05).
The kinetics of
H218O were
investigated from the coronary venous effluent of five isolated rabbit
hearts. After only 10 s of perfusion,
H218O began
to appear in the coronary venous effluent, increasing the
18O value (47.50 ± 0.64 vs. 45.73 ± 1.12
SMOW). This increase became
statistically significant after a perfusion time of 50 s (47.50 ± 0.64 vs. 43.66 ± 0.90
SMOW). Over time, the slope of the
curve flattened, indicating that
H218O
synthesis and
H218O
disposal had reached a steady state. Because of this behavior, the
obtained values were fitted by using a logarithmic function (y = a × ln (x) b; a = 1.44, b = 49.60); the
coefficient of determination
(r2) was 0.92 (Fig. 8).
18O values were detectable in
all hearts. After a perfusion time of 50 s, changes reached statistical
significance. * P < 0.05 compared with onset of perfusion.
DISCUSSION
Inhomogeneities in coronary blood flow, even within the same myocardial layer, have recently been reported in the literature (1, 4, 8, 17, 23, 27). The background of this intriguing finding is still unclear because no obvious histological differences between high- and low-flow areas are apparent (15, 25). This inhomogeneous blood flow probably results in inhomogeneous substrate and oxygen supply to the myocardial tissue (5, 6, 12, 20, 26) and thus is expected to reflect inhomogeneous metabolic demand in different regions of the heart.
The purpose of our study was to establish a new methodology for the assessment of the degree of oxidative phosphorylation in various regions of the myocardium. Because we wanted to clearly separate catabolism, the breakdown of substrates for aerobic ATP production, from anabolism, the conversion of substrates for the regeneration of cellular components and the maintainance of substrate stores, we labeled respiratory water, the final product of aerobic ATP synthesis via oxidative phosphorylation, by using the stable oxygen isotope 18O. By analyzing the regional 18O enrichment in cardiac tissue water and within sequential samples from the coronary venous effluent, we tried to assess myocardial energy demand. In this first step, we have not attempted to quantify the regional myocardial ATP production precisely but rather sought to detect regional differences in oxidative phosphorylation.
The experiments were performed on well-oxygenated buffer-perfused
rabbit hearts. To ensure the adequate oxygenation, the global oxygen
consumption of the hearts was determined. The average value of 60 µl
O2 · g1 · min1
is sufficient to rule out hypoxia and the concomitant accumulation of
negatively charged free radicals in the mitochondria of the isolated
hearts (16, 21, 22).
Our major finding is that
H218O can be
detected in microliter samples of myocardial tissue water. These
samples and samples from the coronary venous effluent were
quantitatively converted to CO2 by
using the guanidine hydrochloride technique (10, 29). The oxygen
isotope composition of the CO2
samples was then determined by using mass spectrometry, and the results
were related to the SMOW scale (2, 9).
Hence a technique is available to evaluate both regional myocardial
energy metabolism (sample size 100 mg) and the kinetics of global
myocardial energy metabolism (effluent aliquots 7.5 µl).
The guanidine hydrochloride technique was first used by Wong et al.
(29) for determinating oxygen isotope ratios in microliter quantities
of biological fluids like saliva, urine, plasma, and milk. The results
exhibited reproducible 18O
values even without prior purification of the fluids, except milk
samples, from which the fat was removed before analysis. The authors
made the assumption that organic compounds present in the fluids may
undergo dehydratation at 260 °C, and thus only slightly affect the
oxygen isotope composition of the free water.
Most of the water samples of this study were obtained by lyophilization of myocardial tissue. Thus cellular and respiratory water without any contamination of myocardial metabolites/intermediates was later analyzed. The samples taken from the coronary venous effluent were microliter aliquots of Krebs-Henseleit solution containing glucose (11.1 mM), pyruvate (2.2 mM), and probably small amounts of cardiac excretions such as taurine, alanine, and glutamate. According to the results of Wong et al. (29), dehydratation of these compounds probably had only a negligible effect on the oxygen isotope composition of the free water.
In earlier experiments, Fleckenstein et al. (11) assessed the rate of intracellular phosphate turnover and hence, in part, energy metabolism in isolated skeletal muscle fibers by labeling ATP and other organic phosphate compounds with H218O. At first sight, these experiments appear to be very similar to ours, but there are major differences. In contrast to those studies, our experiments were performed on entire isolated hearts and 18O2 was used that was incorporated into respiratory water during oxidative phosphorylation. Hence we could clearly separate mitochondrial ATP synthesis via oxidative phosphorylation, i.e., myocardial energy demand, from the immense pool of intracellular phosphate compounds.
The results from our control series demonstrate the applicability of
the technique to small samples of myocardial tissue. This series
provided results in a narrow range: the SD of our 18O reference value
(47.50 SMOW) was
only 0.64 SMOW, equal to only
1.35% of the mean, and maximal scatter among different hearts and
maximal difference between myocardial areas were only 0.90 and
0.53 SMOW (1.89 and 1.12% of
the mean), respectively.
This high reproducibility was confirmed in measurements on
18O-labeled hearts. In that
series, the mean value of
H218O was
significantly increased to 40.35 ± 2.05
SMOW, an average difference of
7.15 SMOW (15.05%) compared
with control hearts. Again, the SD among the various myocardial areas
was small and averaged 0.94
SMOW (2.33%). The maximal scatter
among the hearts in this group averaged 2.45
SMOW (6.07%).
Because of the significant differences between unlabeled and labeled hearts, we conclude that the method provides a means for investigating cardiac metabolism. Thus we present for the first time the successful adaptation of the guanidine hydrochloride technique to the evaluation of aerobic metabolism in small myocardial tissue samples.
Analysis of the coronary venous effluent of
18O-labeled hearts demonstrates
that the technique can possibly provide valuable information about the
kinetics of cardiac metabolism. Because mean
18O values in the coronary
venous effluent showed a tendency to saturate over time, we conclude
that an equilibrium had been reached between
H218O
production in the cardiomyocytes and
H218O
diffusion out of them.
Critique of Methods
In the present study, we detected only insignificant inhomogeneities in myocardial respiratory activity. Except for the condition that no significant inhomogeneities in the myocardium exist, there are three probable methodological reasons for this shortcoming.In the first place, the time-dependent analysis of the 18O enrichment of the coronary venous effluent shows that due to the unphysiologically high coronary flow in our buffer-perfused hearts (50 ± 7 ml/min), respiratory water is rapidly eliminated from the myocardium. Due to the relatively long perfusion times that were used and the free diffusion of water throughout the myocardial tissue, the differences among areas with differing activity of oxidative phosphorylation were obliterated. This effect led to a rather uniform distribution of H218O to the entire myocardium. Thus even samples from the right ventricle, which can be expected to have a lower energy metabolism compared with the left ventricle, only exhibited insignificantly decreased energy metabolism.
In the second place, water is not only a solvent within the cellular metabolism but also takes part in a multitude of hydrolytic reactions, such as the hydrolysis of ATP. The myocardial respiratory water consumed in such reactions cannot be detected by our method. However, on the assumption that the regional amount of metabolized water is proportional to the regional metabolic performance of the cardiomyocytes, this applies to all regions and should not affect in principle the applicability of our method.
Furthermore, buffer-perfused hearts tend to become edematous; this
circumstance is due to the reduced oncotic pressure of the chosen
perfusate. The dry-to-wet ratio in the 14 hearts was 12%, whereas in
normal blood-perfused hearts it averages 21%. Within edematous
myocardial areas, the amount of diffusion is higher, so that
respiratory water is more or less diluted, which, in turn, decreases
the 18O values. For this
reason, it would have been inaccurate to convert the regional
alterations of 18O values into
precise values of regional oxygen consumption.
18O.
Because the interpretation of
18O values and their conversion
to MO2 may be unfamiliar to
most readers, we describe the procedure by using some explicit data
from our experiments.
The absolute
H218O content
of SMOW averages 1,989.5 ± 2.5 H218O × 106
(H218O + H216O) 2 H218O × 103
(H218O + H216O) 2 (2). Shifts from this
18O content within the water
samples analyzed in our study were expressed as
18O values. The absolute
H218O content
of any water sample is given by
|
18O value from our
control series averaged 47.50 ± 0.64
SMOW. Thus the absolute
H218O content
averaged 1,895 ± 2.0 H2180 × 106
(H218O + H216O).
Labeling respiratory water with
18O2
resulted in significantly increased
18O values; the mean
18O of all measured oxygen
isotope ratios was 40.35 ± 2.05
SMOW. The absolute difference of
H218O content
between two water samples is approximated by
|
1 and
2 are the measured
18O
SMOW values. Thus the difference of
absolute
H218O content
between control hearts and
18O-labeled hearts averaged 14.22 H218O × 106
(H218O + H216O).
To convert absolute
H218O
contents into common SI-units, the following expression may be employed
|
C1 is the absolute difference of
H218O
contents between the control and the
18O-labeled sample.
For example, the global amount of
H218O in the
myocardium could be determined as follows.
The dry-to-wet ratio of our isolated hearts was ~12%. The wet weight
averaged 9.5 g; thus the H2O
content was ~8.36 ml. Hence
|
|
|
|
|
O2 in this series (= 60 µl
O2 · min1 · g1)
and from the recent literature (= 100 µl
O2 · min1 · g1)
are considerably higher. This result is certainly due to our experimental procedure, in which major quantities of
H218O were
washed out of the myocardium. Reducing the perfusion time, employing
higher enriched
18O2
(
95%), and reducing coronary flow by choosing blood as a more physiological perfusate in the future will very likely warrant detection of metabolic inhomogeneities and thus enable us to calculate actual regional concentrations of synthetized
H218O from
the substrate
18O2.
Conclusion
Due to the prolonged perfusion with 18O2-enriched saline perfusate, we were not successful in demonstrating regional metabolic inhomogeneities. Nevertheless, we conclude from our results on isolated hearts that this technique is well suited to investigate both regional metabolism in small myocardial tissue samples and the kinetics of cardiac metabolism from coronary venous effluent.ACKNOWLEDGEMENTS
We greatly appreciate reading and correcting of the manuscript by Drs. Brian Guth and Sinclair Cleveland. We are grateful to Elke Vasilescu for excellent secretarial help.
FOOTNOTES
Address for reprint requests: U. Schwanke, Institut für
Experimentelle Chirurgie, Heinrich-Heine-Universität,
Universitätsstra
e 1, D-40225 Düsseldorf, Germany.
Received 10 September 1995; accepted in final form 14 June 1996.
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