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*
Discipline of Nutrition and Dietetics, Faculty of Medicine & Health Sciences, University of Newcastle, Callaghan, NSW 2308, Australia,
Department of Cardiology, John Hunter Hospital, New Lambton, NSW 2299, Australia and
**
Discipline of Reproductive Medicine, Faculty of Medicine & Health Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
2To whom correspondence and reprint requests should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • nonesterified fatty acids • (n-3) polyunsaturated fatty acids • ischemia • myocardium • pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Dietary supplementation with marine (n-3)
polyunsaturated fatty acids
(PUFA)3
were shown to prevent ventricular arrhythmias in experimental animals,
such as rats (McLennan et al. 1988
), marmoset monkeys
(McLennan et al. 1993
) and dogs (Billman et al. 1994 and 1997
). Mechanisms responsible for the
anti-arrhythmic properties of (n-3) PUFA are not clear at present.
It is believed that (n-3) PUFA exhibit their beneficial effects via
incorporation into sarcolemmal phospholipids, changing membrane
properties and altering the eicosanoid metabolism (Abeywardena et al. 1991
, Charnock et al. 1992
). Hallaq et al. (1990)
demonstrated that the protective effect of (n-3)
PUFA results from the prevention of high toxic levels of free cytosolic
calcium within the myocytes induced by ouabain. Eicosapentaenoic acid
(EPA) and DHA, the two (n-3) PUFA predominantly present in the marine
oils, modulate the calcium current through L-type calcium
channels in the sarcolemma of the rat myocytes (Pepe et al. 1994
). In a study by Billman et al. (1994)
, they
demonstrated that ischemia-induced cardiac arrhythmia can be
prevented by infusion of a fish oil emulsion just 50–60 min prior to
the test. These experiments ruled out the necessity for (n-3) PUFA to
be incorporated into sarcoplasmic membrane phospholipids for them to
exhibit their anti-arrhythmic effects. More recently, studies by
the same group (Billman et al. 1997
) showed that (n-3)
PUFA have to be in the nonesterified form for the prevention of
ventricular fibrillations.
Our working hypothesis is that, following the consumption of a diet
rich in (n-3) PUFA, the myocyte membranes are enriched with these fatty
acids that are preferentially released from the myocyte phospholipids
in the nonesterified form during occlusion of the coronary artery and
thus prevent arrhythmias (Nair et al. 1997
). Therefore,
in the present study, we have examined the content and composition of
nonesterified fatty acids (NEFA) in the myocardium of pigs fed fish
oil- or beef tallow-supplemented diets following occlusion of the
left anterior descending coronary artery. To delineate the origin of
the NEFA, the phospholipid and triglyceride fractions of myocardium was
also analyzed for fatty acid content and composition. Pigs was chosen
as our model because of their similar cardiac physiology and fatty acid
metabolism to humans (Chapman 1980
, Harris 1997
).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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All chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia), unless stated otherwise in the text. For lipid analysis, fatty acid methyl ester (FAME) standards were purchased from NuCheck Prep (Elysian, MN) and silica gel G plates from Alltech (Deerfield, IL). All solvents were of a HPLC grade.
Animals and experimental diets.
Specific, pathogen-free, large white female pigs, 6-wk-old, with an average body weight of 6.1 kg (±1.2), were purchased from the University of Sydney animal farm. They were fed a 3.5 g fat/kg basal diet (Grower pig feed, Y. S. Feeds, Young, NSW, Australia) on arrival at the Central Animal House of the University of Newcastle. Proximate composition of the basal diet were protein, 16%; crude fat, 3%; crude fiber, 10%; and NaCl, 0.4%. One group (n = 7) had their diet supplemented with 50 gMaxEPA fish oil (FO)/kg (R. P. Scherer, Melbourne), while the other half consumed diets containing an iso-energetic amount of fat in the form of Allowrie prime beef dripping (Bonlac Foods, Melbourne). It was not economical to use a purified diet because the pigs consumed up to 1 kg of feed a day. The dietary intervention was maintained for 6 wk. Animals in both diet groups were fed similar amounts of diet each day. At the end of the 6-wk dietary supplementation, the pigs weighed 35.5 kg (±1.3). The surgical protocol was reviewed and approved by the Animal Care and Ethics Committee of the University of Newcastle.
Surgical protocol.
The animals were sedated with 3–5 mL Stresnil (Janssen, Belgium) then intubated and artificially respirated with O2 saturation at >90%. Anesthesia was induced with 3% halothane and 1% nitrous oxide and maintained with 1% halothane and hourly infusions of 0. 5 mg fentanyl citrate (David Bull Laboratories, Victoria). The arterial pH of each animal was maintained between 7.38 and 7.44. Once anesthetized, a cannula was inserted into the animal's femoral vein, and a 10 mL baseline blood sample was collected. The chest cavity was exposed via thoracotomy, and the heart positioned by creating a pericardial cradle. Ischemia was induced by the occlusion of the left anterior descending coronary artery 1 cm below the origin of the first diagonal by using a silk ligature. Normoxic and hypoxic tissue samples were collected 35–45 min after occlusion. Following electrophysiological measurements, the myocardial tissue sections from the normoxic and hypoxic areas were rapidly excised and quickly frozen in liquid nitrogen and stored at -80°C before NEFA analysis. Prior to lipid extraction, the tissue was minced finely on ice and rinsed with ice-cold saline to remove any contaminating blood.
Colored microspheres (Triton Technology, Australia) were used to measure, by using different colored spheres, blood flow to the heart wall before and after occlusion of the coronary artery. Before the injection of microspheres (10 s), a reference sample of blood was collected, via catheter placed in the femoral artery and advanced into the descending aorta, into a heparinized, weighed syringe. Flow in the reference sample was calculated from the time of withdrawal (75–90 s) and the weight of blood. Blood was withdrawn for 5 s before injection and for 60 s after the injection of the microspheres was complete. A total of 50–100 million microspheres were injected directly into the left ventricle through the ventricle wall over 10–15 s. Heart tissue was collected from the wall of the left ventricle in the center of the region seen to be ischemic during coronary artery occlusion. Ischemia was confirmed by blood flow determination in the surrounding tissues.
Quantitative fatty acid analysis.
Frozen sections of normoxic and hypoxic myocardium were thawed on ice
and blotted well to remove excess moisture. Lipids were extracted from
the tissues by the method of Folch et al. (1957)
. Briefly, the sections
were minced, rinsed in ice-cold saline to remove contaminating
blood, weighed and homogenized in a volume of chloroform:methanol
mixture (2:1, v/v) containing butylated hydroxy toluene (0.05 g/L) to
prevent oxidation of fatty acids during extraction. Samples were
vortexed; KCl was added; and the mixture allowed to separate. The lower
organic phase was collected and dried down using N2. The
lipid extract was weighed and redissolved in a small volume of hexane.
NEFA fractions were separated by TLC (Christie 1982
).
Heptadecanoic acid was added to the lipid extracts as an internal
standard, and separation was carried out on silica gel plates by using
hexane:diethyl ether:acetic acid (85:15:1) as the solvent system. After
the plates were dried and fractions identified, the NEFA fractions were
scraped from the plates and redissolved in hexane. The NEFA fractions
were methylated by using BF3-methanol at 100°C for 1 h (Metcalfe and Schmitz 1961
) and analyzed by gas
chromatography.
Gas chromatographic analysis.
Using a 30 m x 0.25 mm (DB-225) fused carbon-silica column,
coated with cyanopropylphenyl (J & W Scientific, Folsom, CA) and
employing a method modified from Garg and Blake (1997)
the derivatized
fatty acids were analyzed. The injector and the detector port
temperatures were set at 250°C. The oven temperature was initially
held at 170°C for 2 min, increased 10°C/min to 190°C, held for 1
min, then increased 3°C/min up to 220°C and maintained for 15 min
to give a total run time of 30 min. A split ratio of 10:1 and an
injection volume of 5 µL was used. The chromatograph was equipped
with a flame ionization detector, autosampler and autodetector. Sample
FAME peaks were identified by comparing their retention times to those
of an authentic standard mixture of FAME and quantified using a Hewlett
Packard (NSW, Australia) 6890 Series Gas Chromatograph with
Chemstations Version A.04.02 software for gas chromatographic analysis.
Statistical analysis.
Where indicated, all values were expressed as means ± SEM. The two-tailed, unpaired Student's t-test was used to assess differences between dietary groups, and the one-tailed, paired Student's t-test for differences within groups. P-values of < 0. 05 were regarded as significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The beef tallow (BT) diet was rich in saturated and monounsaturated
fatty acids and contained sufficient amounts of 18:2(n-6) to prevent
essential fatty acid (EFA) deficiency. The MaxEPA (FO) diet contained a
similar amount of 18:2(n-6) to the beef tallow diet, but was enriched
with EPA and DHA (Table 1
).
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There was no significant difference in the content of heart lipids
between beef tallow- and fish oil-fed pigs in normoxic myocardium
(Table 2
). Total phospholipids (PL), total NEFA and total triglyceride (TG)
concentrations in normoxic myocardium in the two groups of pigs were
not significantly different.
|
Fatty acid concentration in the phospholipid and triglyceride fraction.
In the PL fraction (Table 3
) of the beef tallow-fed pigs, linoleic acid and arachidonic acid
(AA) make up ~50% of the total fatty acids. In fish
oil-fed pigs, the lower levels of the (n-6) fatty acids (AA and
linoleic acid) in both normoxic and hypoxic myocardium were accompanied
by higher levels of (n-3) fatty acids, EPA and DHA compared to beef
tallow-fed pigs (P < 0.05). However, no
significant effects of the diet treatment or hypoxia on the total PL
were found (data not shown).
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NEFA concentration and composition.
Total NEFA concentration did not differ between the diet groups in
either the normoxic or the hypoxic regions of the myocardium
(Fig. 1
). Despite large differences in the proportions of saturated fatty acids
(14:0 and 18:0) in the two diets (Table 1)
, there was little or no
difference in the saturated fatty acid concentration of the NEFA
fractions (Fig. 1)
.
|
), which is a reflection of the dietary fats. The major monounsaturated
fatty acid (oleic acid) concentration was unaffected by fat treatment
(Table 4)
. Pigs fed fish oil had significantly lower levels of
arachidonic acid (P < 0.05) compared to the beef
tallow diet in both the normoxic and hypoxic areas of the heart.
Arachidonic acid concentration was significantly greater (P
< 0.05) after occlusion of the left anterior descending artery in
the hypoxic myocardium in both the fish oil- and the beef
tallow-fed pigs.
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. After occlusion, the hypoxic
myocardium of fish oil-fed pigs contained significantly higher
levels of total (n-3) NEFA (P < 0.05) compared to the
normoxic myocardium (Fig. 1)
. Total saturated and monounsaturated fatty
acid concentrations were unaffected by diet or occlusion (Fig. 1)
.
The percentage change in NEFA content caused by hypoxia was calculated
to illustrate the release of the different fatty acids after occlusion
(Fig. 2
) in both diet groups. (n-3) NEFA release in the fish oil-fed pigs
was significantly higher (P < 0.001) than in the beef
tallow-fed pigs. In the fish oil-fed pigs, release of (n-3)
NEFA was markedly greater than that of saturated, monounsaturated and
(n-6) NEFA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) that can develop within hours or days of its onset.
Prevention of arrhythmia in these patients is of vital importance
because this would allow them to recover and live many healthy and
productive years. Recent in vitro studies (Leaf and Kang 1997
) have shown that nonesterified, very long chain PUFA, such
as AA, EPA and DHA, can prevent induced arrhythmias in cardiac
myocytes. In the present study we demonstrated that such a pool of
nonesterified, very long chain PUFA exists in the ventricular
myocardium, that this pool is increased by supplementing the diet with
(n-3) PUFA from fish oil and that it increases further when the left
anterior descending artery is occluded in an in vivo pig model.
Regional ischemia produced by coronary artery occlusion in experimental
animals will eventually develop into myocardial infarction. The
severity of ischemia is determined by the extent to which collateral
circulation, which varies among species, penetrates the ischemic zone
from the nonischemic tissue. In the ischemic zone, a number of cells
are still surviving by continuing normal oxidative metabolism while the
zone as a whole is dying (Opie 1991
). Within the heart,
ischemic injury does not evolve in a uniform manner. The endocardium is
most vulnerable to injury. Tissue necrosis originates here and with
time, migrates as a wave front of cell death towards the epicardial
surface (Hearse 1998
). In a study examining the uptake
and tissue content of fatty acids in normoxic and ischemic dog
myocardium (van der Vusse et al. 1982
), the accumulation
of NEFA was highest in the subendocardium of the ischemic tissue. This
study showed that the different layers of normoxic tissue had lower
triacylglycerol content, and the authors speculated that this could be
due to an increased need for more combustible substrates for energy
production in the normoxic area to compensate for loss of work by the
ischemic region. Therefore, alterations in the composition of the
ischemic myocardium could influence the functioning of the nonischemic
myocardium.
During restriction of flow (ischemia), fatty acid homeostasis is
severely disturbed because of oxygen deprivation. Accumulation of
nonesterified fatty acids and their metabolites occur because of
diminishing mitochondrial oxidation and respiratory chain activity. The
precise origin of the fatty acid that accumulates in the
flow-deprived cardiac tissue is not completely known. Speculations
about the origin of these fatty acids include 1) residual
uptake of fatty acids from extracellular sources, such as the blood and
adipose tissue and their accumulation caused by reduced use (van der Vusse et al. 1980
) and 2) release of fatty acids from
intracellular lipid pools, such as the phospholipids and
triacylglycerol caused by enhanced phospholipase
A2 (PLA2) activity.
In our study, although we did not perfuse the heart tissue with saline
before freezing it, before we extracted the lipids, the tissue was
finely minced on ice and rinsed with ice-cold saline to remove any
contaminating blood. Therefore, it is likely that the NEFA fatty acids
represented that of the heart and not the blood. Victor et al. (1984)
critically evaluated different steps in the extraction
of lipid from the myocardium. They found that there was no significant
difference in NEFA contents of hearts that were freeze-clamped
immediately and those that were perfused to remove the blood present in
the heart and coronary system.
The accumulation of NEFA in the ischemic zone of the fish oil-fed
pigs in our study appeared to be from the uptake of circulating plasma
NEFA as well as from the release of fatty acids from phospholipids and
triglyceride breakdown. In fish oil-fed pigs, plasma TG
concentration was lower than that in beef tallow-fed pigs, although
this difference was not reflected in myocardial tissue. The absence of
any difference in the TG concentration with the rise in NEFA may be
explained by the fact that the amount of accumulating NEFA corresponded
to a very small proportion of the total fatty acids. Incomplete TG
recycling in the myocardium could also be another source for the
accumulation of NEFA in the myocardium of fish oil-fed pigs, as was
suggested by van Bilsen et al. (1989)
. Plasma total PL levels
in fish oil-fed pigs was significantly reduced (P
< 0.05) compared to beef tallow fed-pigs, suggesting an
increase in the activity of PLA2. A similar trend
in TG was observed in the plasma with FO-fed pigs having
significantly lower (P < 0.005) levels compared to
those fed BT.
A significant elevation of arachidonic acid in the ischemic zone occurs
within 20–45 min of ischemia (van der Vusse et al. 1980
). The
accumulation of nonesterified arachidonic acid signals the beginning of
a chain of events that include eicosanoid synthesis. Prostaglandins and
leukotrienes derived from arachidonic acid may influence
arrhythmogenesis and the electrophysiological properties of ischemic
tissue (Corr et al. 1987
). Arrhythmia can occur
when certain ion (sodium and calcium) channels in the myocyte become
hyperactive and fatty acids are well positioned to act as messenger
molecules that regulate ion channels (Ordway et al. 1991
).
In this study, following occlusion, the ventricular fibrillation
threshold was measured in both fish oil- and beef tallow-fed pigs.
After occlusion, the fibrillation threshold fell in the beef
tallow-fed group, but increased or remained unchanged in the fish
oil-fed group (P < 0.05) (unpublished
observations). This between-group difference in fibrillation
threshold in response to occlusion can be related to the differences
observed in myocardial NEFA, especially (n-3) NEFA. Nonesterified (n-3)
fatty acids have been shown to act as sodium and calcium channel
blockers (Leaf and Kang 1997
). The electrophysiological
effect of the nonesterified form of PUFA has been demonstrated by Kang
and Leaf (1996a
and 1996b
) in isolated, neonatal, rat cardiac
myocytes. By adding EPA and DHA (2–10 µmol/L) to the fluid bathing
the myocytes, the beating rate of cardiac myocytes exposed to
arrhythmogenic agents was decreased. This led to the observation that
the effective form of (n-3) PUFA is the free acid itself, not a
metabolite or covalent incorporation into any membrane component. The
free (n-3) fatty acids inhibit ion channels by direct interaction of
both the unsaturated hydrophobic acyl chain and the free carboxyl group
with the channel protein. Similarly, Huang et al. (1992)
reported that concentrations of 3–30 µmol/L of long chain PUFA
directly activate calcium channels in cardiac myocytes, whereas the
esterified fatty acids had no effect. In a study by Conquer and Holub (1998)
, conducted in people of Asian Indian descent who are known to
exhibit high rates of coronary heart disease, serum NEFA was measured
after supplementation of 1. 5 g/d DHA for 6 wk. After 6 wk, DHA in
serum NEFA increased from 1–12 µmol/L. The authors concluded that
7.7–12.7 µmol/L DHA in NEFA may offer anti-arrhythmic effects
under physiological conditions.
Therefore, ready availability of (n-3) PUFA in the NEFA pool following the consumption of fish oil supplemented diet, as demonstrated in the present study, may play an important role in the prevention of ischemia-induced arrhythmias. This also lends support to the observations of Billman et al. (1994 and 1997) who demonstrated that ischemia-induced cardiac arrhythmia can be prevented by infusion of a fish oil emulsion just 50–60 min prior to the test and that it was not necessary for (n-3) PUFA to be incorporated into sarcoplasmic membrane phospholipids for them to exhibit anti-arrhythmic effects. These studies showed that (n-3) PUFA have to be in the nonesterified form for the prevention of ventricular fibrillations and that the esterified fatty acids do not have such properties.
In our study, after the occlusion of the left anterior descending coronary artery, the (n-3) PUFA and arachidonic acid in the NEFA fraction of the ischemic area was further increased, which is consistent with previous studies. It is likely that following ischemia, phospholipase A2 is stimulated in the myocardial tissue that is devoid of oxygen, resulting in the hydrolysis of phospholipids and accumulation of (n-3) PUFA and arachidonic acid in the NEFA fraction.
A recent study has shown that lipoprotein lipase is highly active
following the consumption of (n-3) fatty acids supplementation
(Harris et al. 1997
). The effect of fish oil on plasma
levels of circulating apolipoproteins, which are cofactors for
lipoprotein lipase, has been studied in animals and humans. In fish
oil-fed swine, a decrease in LPL activity was accompanied by
accelerated clearance of VLDL apolipoprotein B-100 (Huff and
Telford 1993
), whereas in humans, fish oil inhibited
the synthesis of apolipoprotein B (Connor and Connor 1997
). In that study, a group of seven mildly
hypertriglyceridemic subjects were fed a high carbohydrate,
fish-oil diet. The levels of apo A-I and apo C-III
decreased, while concentrations of apo B and E did not change. Whether
myocardial ischemia is accompanied by further up regulation of the
lipoprotein lipase activity to release fatty acids from the sn-2
position is not known and deserves further investigation.
The increase in (n-3) NEFA in the ischemic area of the myocardium
observed in our study could possibly alter the course of myocardial
injury in two possible ways. First the release of NEFA from PL by
PLA2 in the ischemic area may be an adaptive
mechanism that occurs when oxygen supply to that region is abolished
and the oxidation of fatty acids provides fuel to the nonischemic
region. In addition the fatty acid pool could also be involved in
various cell signalling processes (Nunez 1998
).
Secondly, the accumulation of the (n-3) NEFA in the ischemic area
inhibits the rapid metabolism of arachidonic acid into
pro-arrhythmogenic metabolites and modulates calcium channels and
calcium homeostasis in cardiac myocytes.
In this study, tissue was collected 35–45 min after occlusion. Studies
to determine NEFA accumulation at different time points in
oxygen-deprived myocardium have been carried out by van Bilsen et al. (1989)
in rats and by Shaikh and Downar (1981)
in
pigs. In the former study, samples were taken at various time points
(10–90 min) of ischemia. NEFA started to accumulate after 45 min, and
the concentrations of different fatty acids varied at the different
time points. In the same study, tissue content of myocardial PL did not
change during 90 min of ischemia, while tissue content of TG also was
also not significantly different. In the second study (Shaikh and Downar 1981
), myocardial accumulation of NEFA
started after 20–30 min of ischemia and ischemia of up to 40 min did
not produce any change in either total PL or PL fractions. These
studies suggest that the time point of collection in our study was
appropriate.
We have demonstrated the quantitative accumulation of (n-3) PUFA, particularly in the NEFA fraction, of the cardiac lipids after occlusion in an experimental model that has a cardiac physiology similar to humans. The increase in the (n-3) PUFA seen in the ischemic myocardium suggests that supplementation of diet with these fatty acids ensures that they replace the accumulation of arrhythmogenic arachidonic acid and thus prevent lethal ventricular arrhythmias after myocardial ischemia. The source of the accumulated nonesterified (n-3) PUFA , whether caused by increased myocardial lipid hydrolysis or selective uptake from plasma, remains to be elucidated.
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FOOTNOTES |
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3 Abbreviations used: AA, arachidonic acid; BT,
beef tallow; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid;
EFA, essential fatty acid; FAME, fatty acid methyl ester; FO, fish oil;
NEFA, nonesterified fatty acids; PLA2, phospholipase
A2; PL, phospholipids; PUFA, polyunsaturated fatty acids;
TG, Triglyceride.
Manuscript received February 8, 1999. Initial review completed March 5, 1999. Revision accepted April 22, 1999.
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