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Department of Legal Medicine, Kobe University Graduate School of Medicine and Departments of
Clinical Biochemistry and
Nutrition and Dietetics, King’s College London, London, U.K.
*
2To whom correspondence should be addressed. E-mail: adachi{at}med.kobe-u.ac.jp
ABSTRACT
Recently, cholesterol hydroperoxides have been shown to be sensitive
pathogenic markers of reactive oxygen species (ROS)-mediated damage
though they have never been measured in heart tissue. We hypothesized
that cholesterol hydroperoxides and oxysterols, putative cardiotoxic
products of cholesterol oxidation, are elevated in the hearts of
alcoholics as a consequence of ROS-mediated reactions. To test
this, we measured 7
- and 7ß-hydroperoxycholest-5-en-3ß-ol
(7-OOH and 7ß-OOH) by HPLC with postcolumn chemiluminescence as
well as 7- and 7ß-hydroxycholesterol (7-OH and 7ß-OH) and
3ß-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto)
by HPLC-UV in cardiac muscle of alcohol-fed rats. Alcohol
feeding was carried out using a pair-feeding protocol with 35% of
total dietary energy as ethanol; controls were pair-fed isocaloric
glucose. After 6–7 wk treatment with alcohol, heart 7-OOH, 7ß-OOH
and 7ß-OH were significantly greater than in controls. Levels of
heart phospholipid 16:0 and 18:1 were lower than in controls, while
18:0 and 18:2 were greater. This is the first report of the presence of
7-OOH, 7ß-OOH and 7-OH in cardiac tissue. The elevations in
7-OOH and 7ß-OOH as well as 7ß-OH are evidence of increased
oxidative stress and possible membrane changes. Alterations in the
proportions of 16:0, 18:1, 18:2 and 18:0 in heart phospholipids provide
further evidence of an altered membrane domain.
KEY WORDS: • 7-hydroperoxycholesterols • oxysterols • ethanol • rats • heart
Heart muscle damage occurs as a consequence of excessive alcohol
ingestion and is manifested as a variety of metabolic and function
abnormalities, including left ventricular hypertrophy, cardiomegaly,
diastolic dysfunction, atrial fibrillation and reduced ejection
fractions (1
, 2
). The pathogenic mechanisms responsible for
alcoholic cardiomyopathy may involve injury by reactive oxygen species
(ROS)3.Thus, elevated myocardial lipopigments are found in myocardial tissues
of subjects with a previous history of chronic alcohol consumption and
who had died from acute ethanol consumption (3
). In animal
models of alcohol toxicity, increased cardiac lipid peroxidation is
observed (4
). There is also a shift in the cardiac fatty
acid profile in alcohol-fed rats, which is inhibited by the dietary
anti-oxidant -tocopherol (5
). A decrease in the
activity of myocardial creatine kinase activity has also been reported
in rats administered chronic doses of alcohol and ascribed to
free-radical mediated injury (6
).
However, in that study, a pair-feeding regimen was not used
(6
). This introduces two points of contention. The first
is that alcohol-injury may be mediated by nutritional impairment;
for example, by thiamine deficiency. However, the use of
pair-feeding regimens with nutritionally adequate diets containing
ethanol with micronutrient and vitamin supplementation has circumvented
the possibility that malnutrition is involved in the pathogenesis of
alcohol induced heart muscle damage [reviewed in (7
)].
The second point of contention pertains to the most suitable indices of
oxidative stress. There are a variety of markers that can used as
markers of oxidative stress [see for example (8
)].
Recently the applicability of using cholesterol hydroperoxides has been
demonstrated (9
–11
). These hydroperoxides include 7-
hydroperoxycholest-5-en-3ß-ol (7-OOH) and
7ß-hydroperoxycholest-5-en-3ß-ol (7ß-OOH) and have been shown to
be particularly sensitive markers of metabolic stress induced by ROS
(10
, 11
). Nevertheless, there are also other pathways by
which cholesterol can be modified including the oxidative formation of
7- and 7ß-hydroxycholesterol (7- and 7ß-OH) and
3ß-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto)
collectively termed oxysterols (Fig. 1
) (12
). Both 7ß-OH and 7-keto are major oxysterols in
oxidized LDL and are reported to reflect increased lipid peroxidation
(13
).
|
-OOH, 7ß-OOH, 7-OH, 7ß-OH and 7-keto in heart
muscle of rats exposed chronically to alcohol. We used a
pair-feeding protocol in which both control and ethanol-fed
rats were fed the same diet, albeit with differences in the proportion
of energy provided by ethanol (treated) or glucose (controls).
MATERIALS AND METHODS
Materials.
3,5-Di-tert-butyl-4-hydroxytoluene, luminol
(3-aminophthaloyl-hydrazine) and cytochrome c (from horse
heart, type VI) were purchased from Wako Pure Chemical (Osaka, Japan).
Hydrogen chloride (50 g/L) in methanol was obtained from Nacalai
Tesque, (Kyoto, Japan). Cholesterol hydroperoxides
5-hydroperoxycholest-6-en-3ß-ol (5-OOH), 7-OOH, 7ß-OOH and
ß-sitosterol 5-hydroperoxide as an internal standard (IS) for HPLC
with postcolumn chemiluminescence (HPLC-CL) were prepared as described
previously (14
). 7-Keto, 7-OH, 7ß-OH and
ß-sitosterol as IS for HPLC-UV were purchased from Steraloids
(Wilton, NH). Fresubin, a nutritionally complete diet with added
vitamins and minerals, was obtained from the Department of Dietetics,
King’s College Hospital, and ’Orovite 7', a vitamin supplement
(Beecham Group, Brentford, U.K.), was purchased from the high street
retailers Boots Chemists (U.K.).
Animal treatments.
Male Wistar rats were obtained from Charles River (U.K.) at
60g body wt. They were maintained in a temperature- (19–23°C) and
humidity-controlled (45–65%) animal house for 1 wk until they
weighed 0.1 kg. They were then ranked and divided into two groups of
equal mean body weight and subjected to a pair-feeding
alcohol-dosing regimen in which treated rats were given a nutritionally
complete liquid diet containing 35% of total energy as ethanol
(7
) (see below). Controls were pair-fed the same diet
having ethanol replaced by isocaloric glucose. The pair-feeding
entailed measuring the volume of diet consumed by the alcohol-fed
rats. The next day, the exact amount of glucose-containing diet was
fed to its individually matched control. After 6–7 wk, rats were
killed by decapitation and hearts were dissected free of atrial tissue.
The work was carried out under institutional supervision that ensured
humane treatment of the animals.
Liquid diets.
Fresh liquid diets used for the 6-wk chronic ethanol-feeding
experiment were prepared on a daily basis (Table 1
). A food blender was used to thoroughly mix the ingredients. To prevent
the possibility of ethanol precipitating the protein in the alcohol
diet, absolute ethanol was the last ingredient to be added carefully,
and contents were then thoroughly stirred during the addition. The
diets (Table 2
) were freshly prepared each day and presented to the rats between
09:00–12:00 h. Control and alcohol-containing diets were
isolipidic, isonitrogenous and isoenergetic.
|
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Total lipid was extracted by adding 4 mL of ice-cold
chloroform/methanol (3:1, v/v), containing 0.005% (v/v) butylated
hydroxytoluene (as antioxidant) and 500 pmol ß-sitosterol
5-hydroperoxide as an IS for HPLC-CL, and 60 nmol ß-sitosterol
as an IS for HPLC-UV to 0.1 g of heart and was then homogenized
under ice-cold conditions. The homogenate was mixed with 4 mL of
chloroform/methanol (3:1, v/v) and 1 mL of distilled water, vortexed
vigorously and centrifuged at 800g and 4°C for 20 min.
The chloroform layer was aspirated, concentrated in a rotary evaporator
and dried under nitrogen. A cholesterol-rich fraction was isolated
from the total lipid by solid-phase extraction. A silica column
(Sep-Pak, Waters, Milford, MA) packed with
aminopropyl-derivatized silica (-NH2) was initially
conditioned by washing with 5 mL of acetone and 10 mL of n-hexane.
The total lipid sample, dissolved in a small amount of chloroform, was
added to the column, which was flushed with a mixture of 2 mL
chloroform and 1 mL iso-propanol, giving an eluate that mainly
consisted of cholesterol. This was concentrated in a rotary evaporator
and dried under a nitrogen stream. The residue was dissolved in
methanol and stored until analysis.
Phospholipids were eluted with methanol and evaporated under a nitrogen
stream, and the residue was placed in a screw cap–sealed reaction vial
with hydrogen chloride in methanol and heated at 110°C for 1 h
to convert the esterified fatty acids into methyl esters. The methanol
was evaporated under a nitrogen stream, and the lipid residue was
redissolved in chloroform for gas chromatography analysis, as described
previously (11
).
HPLC-CL analysis of cholesterol hydroperoxides.
Cholesterol hydroperoxides were quantified by HPLC-CL as
previously described (9
).
HPLC-UV analysis of oxysterols.
Oxysterols were determined by HPLC with an L-7100 pump (Hitachi, Tokyo, Japan), an SPD-10Avp UV detector (Shimadzu, Kyoto, Japan) set at 210 and 245 nm, and a Chromatopac C-R8A integrator (Shimadzu). An Inertsil ODS-2 column (GL Sciences) was used (5 µm, 150 x 4.6 mm internal diameter). Acetonitrile/methanol/water (46:45:9) was used as the mobile phase at the flow rate of 0.7 mL/min. All oxysterols were detected at 210 nm, while 7-keto was detected at 245 and 210 nm. The area of absorbance at 245 nm was 2.6 times as large as that at 210 nm (hence the determination of 7-keto at 245 nm).
Standard curves were prepared by the analyses of 25–200 ng of 7-OH,
50–200 ng of 7ß-OH and 7-keto using 250 ng of IS (ß-sitosterol),
7-keto.
Statistical analysis.
All data are expressed as mean ± SEM Differences between groups were assessed by Student’s t test.
RESULTS
Heart and body weights.
Heart weights of the control rats at the end of the study were 706 ± 31 and 698 ± 47 mg in ethanol-fed rats (P > 0.05). Body weights were 0.215 ± 0.004 and 0.199 ± 0.004 kg, respectively (P = 0.02). The relative heart weights of control rats were 0.328 ± 0.012 g/100 g body vs. 0.348 ± 0.016 g/100 g body in ethanol-fed rats (P = 0.34). Body weight differences were small and likely reflect differences in gut contents.
Heart cholesterol hydroperoxides and oxysterols in control rats.
A mixture of standard cholesterol hydroperoxides (5-OOH, 7-OOH,
and 7ß-OOH), the IS ß-sitosterol 5-hydroperoxide were
successfully separated with a TSK gel Octyl-80Ts column (Fig. 2
). Figure 2
also shows HPLC-CL chromatograms for a heart sample.
Peaks 1 and 2 of the heart sample, at the respective retention times of
6.9 and 7.4 min, corresponded with those of the respective standards
7ß-OOH and 7-OOH. Lipid extracts from heart contained 7-OOH and
7ß-OOH (Table 3
) but not 5-OOH as previously described (11
). The
concentration of 7ß-OOH was more than twice that of 7-OOH. This is
the first reported identification of 7-OOH and 7ß-OOH in heart
tissue.
|
|
shows the HPLC chromatograms of a mixture of standard 7-OH, 7ß-OH,
7-keto, cholesterol and the IS (ß-sitosterol) when an Inertsil ODS-2
column was used. The retention times of standard 7-OH, 7ß-OH,
7-keto, cholesterol and the IS, respectively, on the chromatograms were
9.7, 10.1, 10.8, 34.3 and 45.6 min. There was successful separation of
oxysterols. Figure 4
shows chromatograms of oxysterols in a heart sample. The retention
times of peaks 1, 2, 3 and 4 of the heart sample corresponded with
those of standards 7-OH, 7ß-OH, 7-keto, and cholesterol. The
concentration of 7-keto (166 ± 21 nmol/g) was more than three
times that of 7ß-OH (50.08 ± 6.04 nmol/g) and 7-OH (48.21
± 6.20 nmol/g). 7-Keto concentration was 14 times as large as the
sum of 7-OOH and 7ß-OOH. The cholesterol concentration was 2,052
± 210 nmol/g heart.
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In control rats there were large amounts of 16:0 (25%), 18:0 (37%)
and 18:1 (19%) but low levels of 18:2, 20:4, 22:0, 22:6 and 24:1 (each
<10%) (Table 4
).
|
After chronic treatment with alcohol, cardiac 7-OOH, 7ß-OOH and
7ß-OH were 28, 34 and 33% greater, respectively, than in controls
(Table 3)
. A trend for elevations of 7-OH (26%, P = 0.1) and 7-keto (22%, P = 0.3) also was observed in
heart after chronic alcohol. Because the heart cholesterol
concentration was not significantly affected by chronic alcohol (data
not shown), we did not correct oxysterol concentrations for
cholesterol. In alcohol-fed rats, heart phospholipids 16:0 and 18:1
were significantly lower than in controls, while 18:0 and 18:2 were
elevated. Heart phospholipids 20:4, 22:0, 22:6 and 24:1 were not
affected by chronic ethanol treatment (Table 4)
.
DISCUSSION
We used a paired-feeding protocol using liquid diets with
additional supplementation of nutrients and vitamins (see Tables 1
and 2
). Thus, both controls and ethanol-treated rats ingested the same
amounts of protein, lipids, carbohydrates, vitamins and minerals.
Although no overt changes in heart weights are usually seen after 6–8
wk, there are significant reductions in the contractile protein
contents, a condition which is also seen clinically (15
).
We believe that our 6- to 7-wk feeding model represents a transitional
or preclinical phase in the development of the full spectrum of
cardiomyopathic lesions (2
).
In these studies we sought to test the hypothesis that alcohol feeding
increases ROS or sufficiently reduces antioxidant defenses to alter the
biochemistry of cardiac membranes as reflected by changes in
cholesterol moieties and phospholipid composition. Assays of
malondialdehyde-protein adducts in hearts of chronically (6 wk)
ethanol-fed rats have been unsuccessful (16
), and we
have previously shown that protein carbonyl concentration in heart
muscle is unaffected by alcohol feeding (Preedy, V. R. & Mantle,
D., unpublished observations).
Our study is the first in which cholesterol hydroperoxides have been
identified in the rat heart and are increased in response to alcohol
exposure, indicative of oxidative stress and/or enhanced lipid
peroxidation. Both 7-OOH and 7ß-OOH are formed as a consequence of
ROS-mediated stress per se rather than other routes of metabolism
(9
). There is also other evidence to show increased
cardiac lipid peroxidation or ROS-damage in response to alcohol
(17
). However, there are also various other mechanisms
whereby tissue lipids may be affected by alcohol including the
nonoxidative formation of ethyl adducts and inhibition of fatty acid
synthesis [for example see (18
)].
Additionally, we measured the oxysterols 7-OH, 7ß-OH, and 7-keto.
7ß-OH was increased in hearts of alcohol-fed rats, which may
reflect constitutive oxidative processes. Oxysterols are particularly
important in disease because they have cytotoxic properties. For
example, 7ß-OH and 7-keto increase radiolabeled calcium incorporation
in cultured endothelial cells (19
). However, there is only
one investigation in which 7ß-OH or 7-keto have been measured in
heart muscle per se (20
). In the aforementioned study,
7ß-OH and 7-keto were increased in the hearts of diabetic rats and
assigned a role in the pathogenesis of the related cardiomyopathy
(20
). In contrast, 7-OH has not been measured in heart
tissue apart from this investigation.
We believe that our data has important implications based on the possible structural and/or functional alterations in the cardiac membrane and the putative cytotoxicity of the oxysterols.
In terms of structural and/or functional changes, including the altered
membrane lipid composition, previous work has shown that acute or
chronic ethanol administration in rats alters the fatty acid/membrane
lipid components in noncardiac tissue [for example, increased 18:2 and
decreased 20:4 in liver and platelets (21
)]. In hearts,
20:0 and 22:0 fatty acids are increased (5
). In regard to
the latter study, ethanol feeding for 7 wk did not alter either 18:0 or
18:2 heart phospholipids (5
). This contrasts with our data
showing increases in 18:0 and 18:2. We are unable to explain these
differences, but we used younger rats at the commencement of alcohol
feeding to obtain earlier alcohol-induced metabolic lesions.
Nevertheless, altered fatty acid composition will have functional
consequences, such as perturbing membrane fluidity (22
).
Oxysterols have putative toxic effects in general (23
) as
well as in alcoholism (24
). They inhibit mitosis, increase
apoptosis and have pro-oxidant effects (25
). How these
might affect the heart in cases of alcohol exposure remain speculative
at present. Most of the cholesterol is associated with the membrane and
increased ROS-products imply compositional and/or functional
changes.
In conclusion, we have found for the first time evidence of both altered cardiac membrane fatty acid content and increased lipid peroxidation as a result of ROS. These perturbations in membrane lipids may have important implications for the pathogenesis of alcohol-induced cardiac abnormalities.
FOOTNOTES
1 Supported by The Royal Society for funding
travel between the U.K. and Japan and supported by a Grant-in-Aid for
Scientific Research from Japan Society for Promotion of Science. 
3 Abbreviations used: HPLC-CL, HPLC with
chemiluminescence detection; IS, internal standard; 7-keto,
3ß-hydroxycholest-5-en-7-one or 7-ketocholesterol; 7-OH,
7-hydroxycholest-5-en-3ß-ol; 7ß-OH,
7ß-hydroxycholest-5-en-3ß-ol; 5-OOH,
5-hydroperoxycholest-6-en-3ß-ol; 7-OOH,
7-hydroperoxycholest-5-en-3ß-ol; 7ß-OOH,
7ß-hydroperoxycholest-5-en-3ß-ol; ROS, reactive oxygen species.
Manuscript received 4 June 2001. Initial review completed 30 June 2001. Revision accepted 16 August 2001.
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