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Metabolic Effects of Ethanol on the
Rabbit Heart
By T. Kikuchi, M.D., and K. J. Kako, M.D.
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ABSTRACT
Ethanol in saline solution (15!b, v / v ) was infused into anesthetized rabbits
at a rate of 0.494 ml/min for the first 12 minutes and then at 0.247 ml/min for
108 minutes. Three hours after the infusion, heart triglyceride and lipoprotein
lipase were assayed. Oxidation and esterification of fatty acids (palmitate-14C
as an indicator) were assessed by using either tissue homogenates or perfused
hearts taken from the rabbits. Oxidation-reduction states of the perfused hearts
were examined by measuring the tissue levels of dehydrogenase-linked
substrates. The infusion of ethanol resulted in 180% increase in heart
triglyceride content, but the infusion of norepinephrine (3 /Ltg/kg/min) did not
change the content. No change in plasma free fatty acids and triglyceride or
heart lipoprotein lipase activity was detected. Addition of ethanol had little
effect on the distribution of palmitate-14C in the lipids of tissue slices and
homogenates. On the other hand, prior infusion of ethanol resulted in
depression of 14 COj production (70 and 50%) and enhanced fatty acid
esterification into triglyceride (270 and 170$) both in homogenates and
perfused hearts. Mitochondrial and cytoplasmic redox states were shifted to
more oxidized states by ethanol infusion. It is postulated from these results that
an accumulation of triglyceride in the rabbit heart in response to ethanol
administration is a result of decreased fatty acid oxidation rather than of increased tnglyceride uptake or increased fatty acid synthesis.
ADDITIONAL KEY WORDS
palmitate oxidation
lipoprotein lipase
fatty acid esterification
heart triglyceride
rabbit heart perfusion
mitochondrial redox state
cytoplasmic redox state
plasma lipids
• Chronic ingestion of alcohol affects cardiac
function and metabolism causing cardiomyopathy (1, 2). Histological and histochemical
changes observed in this syndrome include
large amounts of neutral lipid deposit (1).
However, accumulation of triglyceride in the
heart was found even after an acute, moderate
intoxication of ethanol in experimental animals (3, 4). Furthermore, previous reports
from other laboratories describe a decreased
From the Department of Physiology, Faculty of
Medicine, University of Ottawa, Ottawa, Ontario, Canada.
This investigation was supported by grants from the
Medical Research Council of Canada (MA-2219) and
Ontario Heart Foundation (5-8) and by the J. P.
Bickell Foundation and Licensed Beverage Industries,
Inc. Dr. Kako is a Medical Research Council
Associate.
The present address of Dr. Kikuchi is IV
Department of Internal Medicine, Tokyo University
Branch Hospital, Tokyo, Japan.
Received December 19, 1969. Accepted for
publication March 5, 1970.
CiraUtio* Rtsetrcb, Vol. XXVI, M.y 1970
uptake of free fatty acids (FFA) and a release
of zinc potassium, phosphate and enzymes
from the heart (3-6). These results, together
with the morphological findings, suggest that
ethanol may alter intermediary metabolism in
myocardial cells, resulting in an accumulation
of triglyceride.
The present study was made to observe the
biochemical changes in rabbit hearts under
the influence of a moderate dose of ethanol.
The esterification of labeled palmitate was
quantified by using tissue slices, homogenates
and perfused hearts. Three hours after a 2hour infusion of ethanol, a decreased oxidation and an increased esterification of exogenous fatty acid was recorded, but there was
no change in lipoprotein lipase activity.
Indirect evidence further indicates that triglyceride accumulation is due neither to enhanced fatty acid synthesis nor to an increased
triglyceride uptake.
625
626
KIKUCHI, KAKO
Materials and Methods
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Albino male rabbits weighing 1.9 kg to 2.7 kg
(average 2.5 kg) were anesthetized with 2.5
ml/kg of 2% chloralose in 20% urethane. The
jugular vein was cannulated, and 15% (v/v)
ethanol in physiologic saline solution was infused
at a rate of 0.494 ml/min for the first 12 minutes
and then at a rate of 0.247 ml/min for 108
minutes (total 1.95 g/kg). This infusion plan (3)
was adapted to produce moderate intoxication
(i.e. blood alcohol level of approximately 200
mg/ml, ref. 7, 8). Three hours after stopping the
infusion, the heart was excised and used for the
following experiments: (1) triglyceride determination, (2) lipoprotein lipase assay, (3) tissue
slice experiments, (4) tissue homogenate experiments, and (5) heart perfusion experiments.
Triglyceride
Determination.—Tissue
lipids
were extracted with chloroform-methanol (2:1,
v/v) according to the method of Folch et al. (9).
The chloroform extract was washed twice with a
mixture of chloroform-methanol-salt solution
(3:47:48, v/v/v, "pure solvent upper phase").
Silicic acid was then added to the extract to
remove phospholipids. Triglyceride was determined by a modification of the method based on
chromotropic acid formation (10). The extraction
procedure resulted in recovery of 83% of the
tripalmitin, 89% of the phospholipid phosphorus
(11) and 75% of the palmitic acid (12). Tissue
lipids were also measured in the heart of rabbits
which received, instead of ethanol, norepinephrine infusion (2 to 3 jxg base/kg/min for 2 hours)
or compound F (20 mg hydrocortisone succinate
infused in 1 hour) to exclude the possibility of
ethanol infusion causing intravascular release of
these substances and secondarily changing tissue
triglyceride. These doses were based on studies of
catecholamine secretion (13) and corticoid release (14, 15) during ethanol infusion. Three
hours after completion of the infusion the chest
was opened and the heart lipid was analyzed.
Lipoprotein Lipase Activity.—This
activity
was assayed by a method similar to that
described by Alousi and Mallov (16). Each
incubating flask contained 0.5 ml of 1:5
(w/ v) homogenates of the left ventricle
in Krebs' phosphate buffer (pH 8.5), 210 fj.g
heparin, 0.6 ml of 5% Ediol, 1 2.7 ml serum and
135 mg of bovine serum albumin IN a final volume
of 7.0 ml. After 1 hour's incubation at 37°C, free
fatty acids (FFA) were extracted with isopropanol-heptane-lN H 2 SO 4 (20:5:1, v/v/v) and
titrated with sodium ethoxide (17).
^
Ediol: 50% emulsion of coconut oil containing
12.5% sucrose, 1.5% glycerylmonos tea rate, and 2%
polyoxyethylene sorbitan monostearate, Calbiochem.
Tissue Slice Experiments.—Slices2 about 0.6
mm thick, each weighing 200 to 300 mg, were
prepared from heart tissue in a cold room. This
was placed in a flask containing 0.15 fjuc
palmitate-14C, 1 /xmole palmitate, 30 mg albumin
and Krebs' phosphate buffer (pH 7.4; calcium
was omitted, and the final volume was 3.0 ml)
(18). Incubation proceeded for 3 hours in air at
37°C. A time course was followed and the rate of
incorporation into tissue lipids was essentially
constant between 1 and 4 hours. At the end of the
incubation period, chloroform-methanol (2:1,
v/v) was added to the flask. The contents were
homogenized and lipids extracted. To measure
radioactivity, the volume of chloroform extract
was reduced and applied to a thin-layer plate for
chromatography. The solvent fracn'onation method of Borgstrbm (19) was attempted but
abandoned because of poor recovery (70 to 77%).
The chromatographic plate was coated by silica
gel G to 0.25 mm thick. The chromatogram was
developed by petroleum ether-ether-acetic acid
(80:20:2, v/v/v) (20). The spots were identified
by iodine vapor, then scraped off and collected in
scintillation vials and counted in 0.4% Omnifluor3
in toluene containing 4% Cab-O-Sil ( w / v ) . 4 FFA
in the medium were quantified (12) and their
specific activity calculated.
Tissue Homogenate Experiments.—These experiments were carried out by using 700 mg of
the left ventricle in 7 ml of Krebs' phosphate
buffer (pH 7.4), followed by homogenizing with
a Potter-type glass and Teflon homogenizer (21).
Each incubation employed 1.5 ml of this
homogenate per flask. Before tissue addition,
Erlenmeyer flasks with attached center wells
(22), each containing 0.06 ^ic of potassium
palmitate-14C (specific activity; 7 to 8 X 1 0 "
cpm/fiEq, i.e. 0.14 fiEq palmitate per flask) in
0.2 ml of water were flushed with a mixture of
95% oxygen and 5% CO 2 for approximately 5
minutes. Without this aeration, a high blank value
of 14 CO 2 was invariably observed. The tissue
homogenate (about 150 mg) in buffer solution
was added, and the incubation at 37°C was
started in a Dubnoff metabolic shaker. In some
experiments, ethanol was added to a final
concentration of 12 to 95 niM. After 30 minutes,
the reaction was stopped by adding 1 ml of
methanol, and 0.1 ml of 10% KOH was injected
for CO2 collection through a rubber stopper into
the center well to which a sheet of filter paper
S
The tissue slicer was purchased from Harvard
Apparatus Co.
8Omnifluor: 98% PPO and 2% bis-MSB, New
England Nuclear Corp.
4Cab-O-Sil: thixotropic gel suspension powder,
New England Nuclear Corp.
CircuUtion Riitirch, Vol XXVI, Mtf 1970
627
METABOLIC EFFECTS OF ET HA NO L
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had been added. The reaction mixture was then
brought to pH 4.5 by adding 1 ml of 1M
potassium phosphate buffer (22). CO2 was
liberated by shaking the flask at 37°C for 30
minutes. Radioactivity was counted by adding the
filter paper to 0.4% Omnifluor in a toluene-ethanol
(2:1, v/v) mixture. In this experiment, distilled
water was boiled to expel CO2. An average of 05%
of the label was recovered from NaH 1 4CO s after
completion of the entire procedure. The rate of
14
CO 2 production was constant between 10 and
40 minutes of incubation under these conditions.
Incorporation of palmitate-14C into tissue triglyceride and phospholipid was quantified by
applying the techniques of thin-layer chromatography and liquid scintillation counting as previously described above. Protein was measured by
the method of Lowry et al. (23). The specific
activity of the precursor was calculated in
individual experiments by using measured values
(dpm and /xEq) of FFA in the incubation mixture containing tissue. Quantitative distribution of
fatty acids was expressed in juEq/g protein per
30 minutes by assuming that the specific activity
remained constant. Initially, other incubation
media containing ATP, coA, a-glyeerophosphate,
reducing agents, etc. (24, 25) were studied and
found not to influence the result greatly.
Perfusion Experiments.—-The rabbit heart perfusion technique was previously described (26).
In this experiment, 20 ml of blood was taken from
a rabbit just before perfusion and mixed with the
same volume of Krebs' bicarbonate buffer (pH
7.4) to which albumin-bound palmitate-14C (2
fic:S.A. — 28 ^ic/umole) was added. The perfusing apparatus, which is similar to that used by
Morgan et al. (27), was filled with this perfusing
solution. The'heart was excised from the animal,
which also served as blood donor, and attached to
the perfusing system. Coronary perfusing pressure
was 60 to 80 mm Hg. Perfusate samples were
taken at 0 minutes and after 30 minutes of
perfusion, at which time the heart was cooled and
used for analysis. The perfusate was analyzed for
FFA (12) and " C , and the analytical methods
for heart lipids were as described above.
Incorporation of FFA was expressed in /LtEq/g
wet weight by using the specific activity of FFA
found in the perfusing solution, although there
might be some objection in using palmitate alone
to represent fatty acids of various chain lengths
and unsaturation since the myocardium may
preferentially utilize some fatty acids (28). For
assessment of redox states, the heart was frozen
with tongs cooled in liquid nitrogen (26). The
frozen pellet was pulverized by a percussion and
a porcelain mortar, then weighed in 6% HC1O4
and mixed (VirTis homogenizer). The extract
was neutralized (pH meter) with K.,COa.
CircuUiio* Ktsemcb, Vol. XXVI, MTJ 1970
Dihydroxy ace tone
phosphate,
a glycerophosphate, malate, pyruvate, glucose-6-phosphate and
a-ketoglutarate were assayed by enzymatic,
fluorometric methods (29) using a metabolite
fluorimeter.5 Glutamate and lactate were similarly
assayed, using an Eppendorf photometer (29).
NH 4 was determined eolorimetrically by applying
the indophenol reaction of ammonia (30). In a
few experiments, 14 CO 2 was collected as follows:
The recirculating perfusion system, which was
constantly oxygenated by a CO 2 -O 2 mixture, was
closed during the last 10 minutes of a 30-minute
perfusion period. At the end of this period, 4N
H o SO 4 was injected into the system to release
CO2 into the gaseous phase. The accumulated
CO 2 was flushed through the system by nitrogen
gas into 15 ml of 10* KOH. The washing with
'Metabolite fluorimeter was made in a work shop of
the Johnson Foundation, University of Pennsylvania.
30
2O
rh
IO .
o
CONT. EtOH
NE. COMRF
Change in triglyceride level in heart in situ influenced
by ethanol. Triglyceride concentration was expressed
in fiEq/g wet weight of the left ventricle. The mean,
standard error, and number of experiments (in the
columns) are shown. See text for detail of the technique
of infusion of ethanol (EtOH), norepinephrine (NE)
and hydrocortisone succinate (COMP.F).
KIKUCHI, KAKO
628
', of Ethanol Infusion on the Plasma Free Fatty
Acid and Triglyceride Levels
Control
Free fatty acids
Triglyceride
1.32
0.94
0.19 (4)
0.14 (4)
Ethanol infusion
1.59 ± 0.14 (4)
1.25 ± 0.29 (4)
The concentration is expressed as jxEq/ml of plasma
(means =* =SB). The number in parentheses is the number of determinations.
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nitrogen was continued for 1 hour. An aliquot of
KOH solution was counted in Bray's solution
(31) with about 55% efficiency.
Miscellaneous.—In some experiments, plasma
levels of triglyceride and FFA were measured 3
hours after the end of ethanol infusion. Plasma
was extracted by using chloroform containing
Florisil (activated magnesium silicate) for the
triglyceride determinations (10) or by chloroform-heptane-methanol (200:150:7, v/v/v) containing silicic acid for the FFA determinations
(12). All lipid solvents (ethanol, methanol,
chloroform, heptane) were redistilled. This was
found to be particularly important for sensitive colorimetry of the FFA. Bovine serum
albumin, fraction V (Sigma) contained approximately 0.01 fiEq FFA/mg. Enzymes were
purchased from Boehringer Corp., New York. The
efficiency of counting of the radioactivity was
calculated by the channels ratio method.
case. The plasma level of these lipids was not
significantly changed. These values were
measured at the time of heart extirpation, 3
hours after the end of infusion. However, in
two cases the FFA and triglyceride levels in
plasma were determined 30 and 120 minutes
after start of the infusion and were found to
be unchanged. This finding differs again from
that reported as the effect of catecholamines
(3,6,32,33,34).
Although the question regarding the penetration of triglyceride molecules across the
myocardial cell membrane is still open, the
most likely mode of triglyceride transport is
through prior hydrolysis by lipoprotein lipase
(34). To test this, the enzyme activity was
measured. The results indicated that an increase in activity under the influence of ethanol infusion is unlikely, namely, the activity
Results
Triglyceride content in the heart under the
influence of various interventions in our
experiment is shown in Figure 1. There was a
significant increase in triglyceride 3 hours
after the infusion of ethanol. In contrast, the
infusion of norepinephrine at a rate of 2 to 3
;u.g/kg/min or that of 20 mg of compound F
did not influence the myocardial triglyceride
level (Fig. 1). None of these interventions
influenced myocardial FFA and phospholipid
levels. The prior administration of reserpine
(10 or 20 mg, 20 hours before) did not
prevent triglyceride accumulation by ethanol
administration (32.7 jw.Eq/g, n = 2). Since
FFA uptake by the heart depends on the
plasma FFA concentration (28), it could have
been possible that FFA or triglyceride concentrations were increased by the infusion of
ethanol, resulting in an increase of the uptake.
However, Table 1 shows that this is not the
O.I
.
O
18
36
FIGURE 1
Effects of adding various concentrations of ethanol on
palmitate distribution in lipids of tissue slices of
normal rabbit hearts. Tissue slices were incubated in
the presence of ethanol (0-72 VIM), palmitate-l-'iC,
albumin and Krebs' phosphate buffer for 3 hours at
37°C. Incorporation in nEq/g wet weight into triglyceride (TG), phospholipid (PhL) and free fatty
acids (FFA) is shown. The number of experiments was
3 in each case.
CircuUim Rtiuicb, yd. XXVI, Ma) 1970
629
METABOLIC EFFECTS OF ETHANOL
Effect of Elhanol on Faily Acid Oxidation and Esleri ficaiion by Heart Hoviogenales
Control
Fattyacids-» "COi
Fatty acids -> triglyceride
Fatty acids -> phospholipid
1.42 ± 0.20
0.20 ± 0.05
0.84*0.07
Ethanol infusion
1.46 ± 0.19
0.23 ± 0.07
0.84±0.16
0.93 ± 0.11 (P < 0.05)
0.48 ± 0.10 (P < 0.05)
0.89±0.10
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Approximately 150 mg of 1:10 homogenates of rabbit left ventricle were incubated at 37°C
for 30 minutes in 1.5 ml of the medium containing 0.14 ^Eq (0.06 iiv) of potassium palmitate
and Krebs' phosphate buffer (pH 7.4). The concentration of ethanol added was 24 ma. The
infusion rate was 74 mg/min for the first 12 minutes, and then 37 mg/min for 102 minutes. The
rabbit was killed 3 hours after cessation of the infusion. "COj was collected by absorbing it with
a filter paper soaked in 0.1 ml of 10% KOH after the medium pH was brought down to 4.5. The
homogenates were treated with chloroform-methanol and the lipid extract was separated by
thin-layer chromatography. Specific activities of the precursor in individual experiments were
calculated by using the results of chemical and radiochemical determinations of fatty acids in
the incubation mixture. All the above lesults are means of six experiments expressed as /
protein ± SE.
was 90.5 ± 21.9$ (mean ± SE) of the control in
seven paired cases (140 ± 24.4 ^tEq/hour/g of
the control, vs. 103.3 ± 15.2 /xEq/hour/g of
the ethanol-treated).
The effect of ethanol on the distribution of
labeled fatty acids in the tissue slice obtained
from the normal rabbit is shown in Figure 2.
The addition of increasing concentrations of
ethanol from 0 to 72 MM resulted in a
decreasing esterification; concomitantly, an
increasing proportion of the tissue label was
found in the fatty acid fraction as the
concentration of ethanol was raised. Since the
tissue slice is a broken cell preparation and
since the cellular transport of FFA is governed
by the concentration, chain length and unsaturation of the fatty acids and FFA-albumin
ratio (28, 34), the fatty acid uptake was
presumably uninfluenced by the above experimental conditions. Therefore, these results
suggest less esterification and probably less
beta oxidation of palmitate as the medium
ethanol concentration becomes higher. In
another experiment in which 144 mM ethanol
was added, 92% of the total tissue label was
found in the FFA fraction as compared to
82.7 ± 2.6$ at 72 mM and 62.5 ± 2.9$ at 0 mM
(data shown in Fig. 1). Addition of ethanol
did not change the chemically determined
content of heart lipids.
More isotope was incorporated into tissue
lipids when homogenates were used for the
OrcaUiion Rtusrcb, Vol. XXVI. May 1970
experiment than when slices were used (Table
2), probably because of different methods of
calculation of specific activity of the precursor
and because of the presence of albumin in the
slice experiment. Although the addition of
ethanol, 12 to 95 mM (24 MM in Table 2), did
not influence the oxidation and esterification
of FFA significantly, homogenates prepared
from the animal that had received the ethanol
infusion incorporated more (270%) fatty acids
into the triglyceride fraction than did homogenates from the normal rabbit heart. The
14
CO2 production from fatty acids, on the
other hand, was depressed to approximately
70$ of the control level. Interestingly, the
incorporation into phospholipid was uninfluenced by prior treatment with ethanol (table
2).
In the perfused heart, the incorporation of
labeled FA was even greater than that of the
homogenates (Table 3). In particular, incorporation into triglyceride was more than 50fold greater (1.96 /^Eq/g vs. 0.027 /iEq/g).
Although the medium fatty acid concentration
was also greater by 13-fold (1.02 vs. 0.08
/LtEq/ml), the incorporation into phospholipid
in the homogenate and perfusion experiments
was of a similar magnitude. The labeling
found in FFA and phospholipid of the heart
was again not influenced by the addition of 2
mg/ml (44.5 MM) of ethanol to the perfusing
fluid or by prior infusion of ethanol into the
630
KIKUCHI, KAKO
TABLE 3
Effect of Elhanol on Folly Acid Esterificalion
by Perfused Rabbit Hearts
Control
(n = l6)
Incorporation into triglyceride (
Incorporation into phospholipid (»iEq/g)
Incorporation into free fatty acid (FFA) (/i
Concentration of FFA in perfusate (MEq/ml)
Specific activity of FFA in perfusate (cpm/VEq)
1.96 ±
0.37 ±
0.16 ±0.
10.2 ±0
61 000
ollanol aadditil
(n = 3)
2,36 ±
0.48 ±
0.21 =
1.28 ±
21 000
0.18
0.07
0.04
0.06
Ethanol intus
(n=6)
3.42 ±
0.36 ±
0.10 ±
1.18 =<=
51 000
0.46
0.11
0.03
0.20
0.54 (P < 0.01)
0.08
0.02
0.14
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The perfusion was performed by using the heart and blood of the same rabbit. The blood was diluted 1:1 with
Krebs' bicarbonate buffer. The perfusion pressure was 60 to 80 mm Hg and the duration 30 minutes. Heart lipids
were extracted by chloroform-methanol and analyzed by thin-layer chromatography. Free fatty acids in the medium
were determined according to Laurell and Tibbling (12). The concentration of ethanol added was 44.5
I
Ethanol was infused as described in the legend of Table 2. g indicates gram of wet weight. Values are
means ± SE.
rabbit from which the blood and heart were
obtained for perfusion (Table 3).
In contrast, esterification to triglyceride was
significantly increased by prior infusion of
ethanol to 174% of the value found in the
control heart (Table 3). This relative increase
was comparable in magnitude to that observed in in-vivo experiments in which triglyceride concentration was determined (Fig. 1).
Incorporation into triglyceride was somewhat
increased in the heart perfused in the presence
of ethanol, although the change was statistically insignificant (Table 3). The production
of 14CO2 was semiquantitatively compared,
since the work level and the heart rate were
not rigidly controlled in these perfusion
experiments. Nevertheless, a 10-minute CO?
collection indicated that ethanol suppressed
fatty acid oxidation (from 0.122 ±0.010,
n = 3, to 0.058±0.005, yu.Eq/g/10 min, n =
2).
The ratios of redox pairs of substrates,
a-glycerophosphate/dihydroxyacetone phosphate, lactate/pyruvate and glutamate/ (aketoglutarate) X (NH 4 ) were measured in
hearts perfused under conditions similar to the
above study (Fig. 3). All of the ratios tended
to decrease, indicating that both cytoplasmic
and mitochondrial redox states shifted to a
more oxidized state (35). In other words, free
pyridine nucleotides of the two compartments
became more oxidized by the action of
ethanol. These results again suggest that the
mitochondrial fatty acid oxidation decreased,
together with a decreased glycolytic flux in the
cytoplasm. Simultaneously, an increase in the
malate level (93 ±13, n = 9, to 126 ± 14,
n = 5, /u,moles/g wet weight) and in the
glucose-6-phosphate level (108 ± 18 to 200
2O
r-
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COMT. E t O H
15
bl
60
IO
4O
2O
ft
,O6
T .04
.02
i
•'tJHAP
GLUT
GTKGXN
-KG)(NH4)
Effect of ethanol on heart redox states. Hearts were
perfused as described in text. Ethanol (EtOH) was
infused between 5 and 3 hours before perfusion. The
heart was freeze-quenched and the substrates determined by enzymatic-fluoronwtric methods. The ratio
of a-glycerophosphate (A-GP) to dihydroxyacetone
phosphate (DHAP), that of lactate (LACT) to pyruvate (PYR) and that of glutamate (GLUT) to the
product of a-ketoglutarate (a-KG) and NH^ are illustrated. The number of experiments was 9 in control
and 5 in ethanol-treated.
Circulation Research, Vol. XXVI,
May 1970
METABOLIC EFFECTS OF ETHANOL
± 33 /JLtnoles/g wet weight) were observed in
the hearts of alcohol-infused rabbits.
Discussion
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This study demonstrates that a moderate
dose of ethanol infused into rabbits induces
concurrently a decrease in fatty acid oxidation
and an increase in fatty acid esterification in
the myocardium. These findings were observed both by using heart homogenates and
hearts perfused with diluted autologous blood.
The results showing a decrease in CO2
production from fatty acids were supported
by the finding that the mitochondrial redox
state shifted to a more oxidized state under
the influence of ethanol. Esterification data
was in agreement with the increase in
triglyceride content of the heart in vivo caused
by the infusion of ethanol.
Although it was shown that oxidation in
the tricarboxylic acid cycle in the liver was
inhibited by ethanol (36, 37), such an
inhibition in heart mitochondria seems unlikely. This is because (1) the inhibition of the
Krebs cycle in the liver is related to the
production of reducing equivalents during
ethanol metabolism (37), but in the heart, the
redox state was not reduced (our results), and
(2) the ventricles demand a continuous
supply of energy for contraction and the
oxygen consumption does not decrease (3,
38). Furthermore, it was recently reported
that the glucose, lactate, and pyruvate metabolism of the perfused rat heart was unchanged in the presence of a toxic concentration of ethanol (38). Thus, the myocardial
usage of lactate, acetate and acetoacetate may
even be increased as a consequence of their
increased plasma concentration (3, 6, 39-41).
This alone could cause the suppression of
oxidation of palmitate- ^ C and enhance esterification, as shown in the isolated rat heart
(42). This cannot, however, be the sole mechanism, since our experiment with tissue homogenates, in which the medium contained exogenous and perhaps endogenous fatty acids
as the sole substrate, produced a change in esterification, which suggested the existence of
some control mechanisms other than that mediOrcuUtim Krtc-ch. Vol. XXVI, May 1970
631
ated by acetate. Thus a mode of action of ethanol in directing the flow of fatty acyl moiety
from oxidation to esterification in the heart
cell remains unsolved.
Under the experimental conditions adopted
in this study, the myocardial level of aglycerophosphate tended to decrease (206 ±
37 to 146 ±20 /imoles/g). This is in agreement with the finding of a shift to oxidation of
cytoplasmic pyridine nucleotides (Fig. 3),
which suggests that glycolytic flux is decreasing (43), and hence the supply of aglycerophosphate should also be limited. The
results thus indicate that the level of aglycerophosphate does not primarily control
fatty acid esterification in the heart. An
increased glucose-6-phosphate level implies
that the tissue citrate may be increased, as was
observed when acetate availability was raised
in the isolated perfused rat heart (43).
The measured dehydrogenase system indicated that the mitochondrial state was reduced. This is in contrast to the findings
obtained from the hepatic actions of ethanol
(36, 37). In the latter case, presumably
because of the presence of powerful alcohol
dehydrogenase and aldehyde dehydrogenase,
both mitochondrial and cytoplasmic redox
states changed dramatically to a more reduced
state (37), with its numerous biochemical
consequences (36, 37, 40). A portion of pyridine nucleotides is bound to cellular proteins
and does not necessarily participate in the
oxidation-reduction reactions (35, 43). It
would be feasible, then, to observe a reduction
of the total NAD/NADH2 of the heart by
large doses of ethanol (44), whereas the
determination of dehydrogenase-linked substrates, as carried out in this study, shows the
oxidation of free nucleotides in the cell
compartment. Since reduced states in cellular
compartments are prerequisite for fatty acid
synthesis to take place (34, 36), these results
suggest that the increased synthesis by the
action of ethanol is unlikely. Nevertheless, the
possibility exists that an additional mechanism
is responsible for the change in heart lipid
metabolism caused by ethanol, particularly in
fed rabbits, since the change in the triglyc-
632
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eride level was more dramatic in fed rabbits,
which showed a low preinfusion value of heart
triglyceride (45) compared to fasted rabbits
(unpublished observations), and since the
plasma concentrations of FFA is low in fed
animals (28,46).
Our study provides only an indirect answer
to the question whether an increase in
myocardial triglyceride is the result of an
increase in uptake of FFA or triglyceride. The
uptake of FFA depends on their arterial level
at a given concentration of albumin, a carrier
of FFA (28). Since plasma FFA levels
remained unchanged (Table 1), the FFA
uptake presumably did not change. Rather,
FFA uptake by hearts in vivo was decreased
in previous studies (3, 6, 39).
Factors governing myocardial triglyceride
uptake, on the other hand, have not been
studied in much detail. Most studies favor
hydrolysis of triglyceride prior to its entry into
the myocardial cell (34). If this assumption is
valid, increased uptake of triglyceride-fatty
acids is the result of an increased plasma
triglyceride level, increased heart lipoprotein
lipase activity, or both. Neither of these
predictions was substantiated in the experiments reported here, although higher doses of
ethanol may cause such changes (47, 48).
Regan et al. (3, 6) suggested an increased
permeability of myocardial membrane to
triglyceride as a possible mechanism. However, our work provides two pieces of
circumstantial evidence against the hypothesis
that an increase in triglvceride penetration
may be a cause of ethanol-inducsd lipid
accumulation; namely, (1) an increase in fatty
acid esterification was observed in the experiment in which homogenates were used in the
absence of exogenous triglyceride, and
(2) an even greater degree of esterification
was observed by using the perfused heart
instead of the homogenate. This would not
have been the case if an appreciable amount
of triglyceride uptake had occurred, since the
triglyceride-fatty acids would then have diluted the palmitate-C, resulting in an apparently low incorporation.
The heart does not appear to metabolize
KIKUCHI, KAKO
ethanol (38), and its addition to the incubating medium or to the perfusing fluid has been
shown by our studies to be without effect.
Therefore, the changes in intermediary metabolism reported here are probably not a
result of direct action of ethanol but more
likely mediated by the action of acetaldehyde,
an alteration in membranous components of
the cell (1, 3, 6), or a change in palmitoylcarnitine acyltransferase (49), for example. The
influence of secondarily released catecholamines is negligible (3, 6, 32, 33, 50, and our
results). Further studies are required to
elucidate the functional significance of the
fatty infiltration and depression of fatty acid
oxidation which follow a moderate dose of
alcohol.
Acknowledgment
For help in setting up the fluorometric methods, the
authors are greatly indebted to Dr. J. R. Williamson,
J. T. Veasy, and D. Meyer. C. Huang, M. Sholdice, L.
Silverman, and S. Yates gave valuable help during
various phases of these experiments.
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Metabolic Effects of Ethanol on the Rabbit Heart
T. KIKUCHI and K. J. KAKO
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Circ Res. 1970;26:625-634
doi: 10.1161/01.RES.26.5.625
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