Arteriosclerosis, Thrombosis, and Vascular

Biology
atvb.ahajournals.org

Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;

17: 2540-2547
doi: 10.1161/01.ATV.17.11.2540

Articles
BeneficialEffectsofAlcoholWithdrawalonLDLParticleSize
DistributionandOxidativeSusceptibilityinSubjectsWithAlcohol
InducedHypertriglyceridemia

Makoto Ayaori, Toshitsugu Ishikawa, Hiroshi Yoshida, Michio Suzukawa,

Masato Nishiwaki, Hideki Shige, Toshimitsu Ito, Kei Nakajima, Kenji Higashi,
Atsushi Yonemura, Haruo Nakamura

+ Author Affiliations

Correspondence to Makoto Ayaori, MD, First Department of

Internal Medicine, National Defense Medical College, 3-2
Namiki, Tokorozawa, Saitama 359, Japan. E-mail
ayaori@ba2.so-net.or.jp

Abstract
Abstract LDL subclass pattern B, reported to have a higher
prevalence in hypertriglyceridemics (HTGs), is considered to
be associated with an increased risk for coronary artery
disease, and the small dense LDL characteristic of this
pattern is susceptible to oxidative modification. Alcohol is
considered one of the most frequent causes of increases in
plasma triglyceride (TG) levels. We investigated the effects of
alcohol withdrawal on LDL subclass distribution and
oxidizability in drinkers with different plasma TG levels.
Thirty-seven male subjects with relatively heavy alcohol-
consumption habits were divided into four groups;
normotriglyceridemic (NTG)/withdrawal (n=11), NTG/control
(n=8), hypertriglyceridemic (HTG)/withdrawal (n=10), and
HTG/control (n=8). Both withdrawal groups abstained form
alcohol for 4 weeks, while the control subjects maintained
their usual intake of alcohol. Peak LDL particle diameter
(PPD) was smaller in the combined HTG groups than in the
combined NTG groups before abstinence, although PPD
increased significantly (P<.01) from 25.5 to 26.1 nm in the
HTG/withdrawal group. Before abstinence, lag times
preceding LDL oxidation in the combined HTG groups were
shorter than in the combined NTG groups; after withdrawal,
lag time was prolonged significantly (P<.01) from 49.9 to
57.3 minutes in the HTG-withdrawal group. No significant
changes in PPD and lag time were observed in the other
three groups. Significant correlations (P<.05) were observed
between the change () in lag time and TG and between
lag time and PPD. We conclude that in alcohol-induced
HTG subjects, alcohol withdrawal has beneficial effects on
the LDL profile by shifting the particle size from smaller to
larger and decreasing its susceptibility to oxidation.

Key Words:
alcohol withdrawal
LDL subclass distribution
LDL particle size
LDL oxidizability
hypertriglyceridemia

Received April 1, 1997.

Accepted August 4, 1997.

Several epidemiological studies have reported a correlation

between a lower incidence of CAD and moderate alcohol
12
intake. In some studies, alcohol intake was positively
34
associated with an elevated concentration of HDL, a factor
that is believed to be negatively correlated with CAD.
Furthermore, the level of LDL, one of the strongest risk
factors for CAD, has been reported to be decreased in
alcohol drinkers. However, other studies have reported a U-
shape trend, showing that a moderate amount of alcohol
intake decreases CAD mortality whereas a high level of
1
alcohol intake increases CAD mortality. It is unclear why a
moderate alcohol intake protects against atherosclerosis
while heavy drinking has an opposite effect. Quantitative
4
changes in HDL and LDL levels with alcohol intake levels
3
and the effects of HDL subfractions have been described.
However, descriptions of the qualitative changes in LDL due
to alcohol ingestion are relatively rare, especially the
relationship between LDL subclass pattern and LDL
oxidizability during alcohol intake.

LDL can be separated into subclasses by size, and individual

LDL patterns are considered to be determined by
5
combinations of different sizes of LDL particles. LDL
subclass pattern B is characterized by a predominance of
small, dense LDL particles and is associated with an
increased risk of CAD, whereas pattern A is characterized by
a predominance of larger LDL particles with a low risk for
67 8
CAD. McNamara et al have previously reported that
pattern B is related to high plasma TG levels, and several
investigators have observed that medication for HTG altered
LDL particle size from small and dense to large and buoyant,
9 10 11 12 13
together with a reduction in plasma TG levels.
14 15
Beard et al reported that the mean particle diameter
and oxidizability of LDL were improved with concomitant
reduction in plasma TG levels after an intensive diet and
exercise program. However, the association between
alcohol-induced HTG and LDL subclass pattern is still
unclear. Furthermore, it has yet to be determined whether or
not a reduction in TG levels after alcohol withdrawal results
in a beneficial change in terms of LDL subclass pattern.

Recently, the possibility of involvement of oxidatively

16
modified LDL in atherogenesis has been suggested.
Oxidized LDL is easily taken up by macrophages via
scavenger receptors and transforms the macrophages to
foam cells, which characteristically appear in the early
16 17
atherosclerotic plaque. The oxidative properties of
18
alcohol and its metabolites have been reported. Lin et al
reported that LDLs separated from the plasma of heavy
alcohol consumers had been oxidatively modified in vivo.
19
Croft et al also described increased oxidizability of LDL
after alcohol ingestion. There may be a correlation between
the amount of alcohol intake and the extent of oxidative
modification of LDL. However, this point has not been
clarified, and the mechanism for oxidative modification of
LDL by alcohol is also unclear.

It has been well documented that the susceptibility to

oxidative stress differs in LDL subfractions. Specifically, it is
considered that the larger, more buoyant LDL particles are
more resistant to oxidation, whereas the smaller, more
20 21 22
dense LDL particles are more susceptible. The
smaller, dense LDL particle may play an important role in
LDL oxidation. Thus, it is important to determine whether or
not alcohol affects LDL oxidizability associated with the
change in LDL subclass pattern. The purpose of this study
was to investigate the effects of alcohol abstinence in terms
of LDL subclass pattern and oxidizability in subjects with
alcohol-induced HTG.

Methods
Subjects

From a pool of potential subjects consisting of soldiers or

employees of the Japan Self-Defense Force who were
examined by blood and physiological examinations during
periodic medical checks, 41 healthy, male volunteers
ranging in age from 36 to 52 years (meanSD, 41.57.2)
were selected to participate in this study. The 41 selected
subjects were free of diabetes mellitus, endocrine disease,
renal dysfunction, or liver dysfunction except for slight
elevations in aspartate aminotransferases (up to 58 IU/L).
Each subject had a history of consuming 40 to 150 g of
alcohol daily in the form of Japanese sake, whiskey, and/or
beer. None had a preference for wine or other spirits brewed
from fruits, and none were taking supplemental vitamins or
routine medications of any kind. All subjects were physically
in good condition throughout the study. The protocol of the
present study was approved by the medical ethics committee
of our institution, and all subjects gave informed consent to
participate in this study.

BloodSamplingandPreparationofLDL

Fasting blood samples were obtained from each subject

upon entry into the study (week 0) and again during week 4.
All subjects stopped drinking alcohol for at least 36 hours
before blood sampling. Blood was collected into Vacutainers
containing disodium EDTA (1 g/L) and placed immediately
on ice, and the plasma was separated by centrifugation.
Plasma samples were subjected to biochemical analysis and
the remainder was stored at 70C for the other assays. LDL
(d=1.019 to 1.063 g/mL) was isolated by sequential
23
ultracentrifugation by using the methods of Havel et al.

ExperimentalDesign

The subjects were separated into two groups according to

TG levels: the first group, the NTG group, included those
individuals whose TG levels were <1.69 mmol/L; the second
group, the HTG group, included those individuals whose TG
levels equaled or exceeded 1.69 mmol/L. The NTG and HTG
groups were further randomly divided into two groups each,
a withdrawal group and a control group. Three subjects in
the NTG/withdrawal group and 1 in the HTG/withdrawal
group subsequently dropped out, so the final number of
subjects who completed the study was 11, 8, 10, and 8 in
the NTG/withdrawal, NTG/control, HTG/withdrawal, and
HTG/control groups, respectively (Table 1). The withdrawal
groups abstained from alcohol for 4 weeks while the control
groups maintained their usual alcohol consumption habits.
Blood samples were obtained at week 0 (baseline) and week
4. The subjects in each group were advised not to change
their usual dietary, smoking, and exercise habits throughout
the study. By analyses of 3-day food records at weeks 1, 0,
2, and 4, daily total caloric intake; percentage contributions
of protein, carbohydrate, and fat; antioxidant intake (-
tocopherol, -carotene, and L-ascorbate); and the type of
fatty acid (saturated/monounsaturated/polyunsaturated, n-
3/n-6) were deemed unchanged throughout the study in all
four groups. The amount of alcohol intake did not change
significantly in the control groups, and all subjects in the
withdrawal groups completely abstained from alcohol
throughout the study.
 Table 1.
View this table:
Clinical In this window
Characteristics of the In a new window
Study Subjects

BiochemicalAnalyses

Plasma TC, LDL-C, TG, FC, and PLs were determined by

enzymatic methods using commercially available enzymatic
reagents (Kyowa Medics Co). Plasma -GTP activity was
24
measured by the method of Szasz. The normal range of -
GTP is <50 IU/L. Apolipoproteins were measured by
25
immunoturbidimetry. The protein content of LDL was
26
measured by the method of Lowry et al. Vitamin E (-
tocopherol) in LDL was determined by high-performance
27
liquid chromatography. In brief, LDL was precipitated with
ethanol and vitamin E was subsequently extracted with
hexane. The hexane phase was evaporated under N2 gas and
the residue dissolved in ethanol. Vitamin E was separated by
reversed-phase high-performance liquid chromatography
on C18 columns (250.46 cm, 5-m particle size; TSK gel
ODS-8OTs, TOSOH) that were eluted with ethanol/distilled
water (92:8, vol/vol) at 1.0 mL/min as the mobile phase and
monitored at 295 nm in a UV detector (UV-8000, TOSOH).
Vitamin A was used as an internal standard. LPO in LDL was
measured by using a commercially available kit (Determiner
LPO, Kyowa) based on a colorimetric method by adopting the
reaction of a leukomethylene blue derivative with lipid
28
peroxides on the presence of heme compounds.

DeterminationofLDLPPD

LDL subfractions were separated at 10C by 2% to 16%

gradient polyacrylamide gel electrophoresis (PAA 2-16,
29
Pharmacia) as reported by Nichols et al. The gels were
prerun in 90 mM Tris, 80 mM boric acid, and 3 mM EDTA,
pH 8.3 (TBE buffer) at 125 V for 20 minutes in a GE-2/4
electrophoretic chamber (Pharmacia). After addition of 40%
sucrose containing 0.02% bromophenol blue to the samples,
5 to 10 L was applied to each line on the gels. The samples
from identical subjects at weeks 0 and 4 were applied to the
same gels. Electrophoresis was initiated by applying voltage
to the chamber in the following sequence: 15 V for 15
minutes, 70 V for 20 minutes, and 125 V for 24 hours. For
fixation prior to protein staining, the gels were exposed to
10% sulfosalicylic acid for 1 hour immediately after
electrophoresis. The gels were stained in 50% methanol10%
acetic acid (vol/vol) containing Coomassie brilliant blue R-
250. After destaining in 20% methanol10% acetic acid
(vol/vol), the gels were scanned at 596 nm on a laser
densitometer (Ultrascan XL, LKB). The calibration curve
determined from the high-molecular-weight standard was
applied to each peak to estimate the LDL particle diameter.
LDL migration distance (Rf) was measured relative to that of
apoferritin. LDL diameters were estimated form the
migration distances of latex beads (38 nm, carboxylated PS
latex, Magsphere), thyroglobulin (17 nm), and apoferritin
(12.2 nm). The estimated diameter for the major peak in
each scan was termed the PPD. A quadratic equation
30
proposed by Williams et al was used to determine the LDL
particle diameters.

DeterminationofOxidativeSusceptibilityofLDL

Oxidative susceptibility of LDL was evaluated by the method

31
of Esterbauer et al with some modification. LDL oxidation
was initiated by incubation of 100 g of LDL protein with 20
mol/L 2,2-azobis(4-methoxy-2,4-dimethylvaleronitrile);
V-70, Wako Pure Chemicals) in 2 mL of PBS at 37C, and
conjugated-diene formation was monitored by the change in
the 234-nm absorbance in a spectrophotometer (Shimazu
160A, Shimazu) equipped with a six-position automatic
changer. The dienes formed during LDL oxidation produced
an absorption spectrum with a distinct peak at 234 nm with
essentially no interindividual variation; the initial absorbance
was taken as the baseline and the changes in absorbance
were recorded every 5 minutes for 4 hours. The absorbance
curve at 234 nm was divided into three phases, ie, a lag
phase, a propagation phase, and a decomposition phase.
The lag time of LDL oxidation was defined as the intercept of
the tangent of the slope during the propagation phase with
that of the lag phase and expressed in minutes.

StatisticalAnalyses

Values were expressed as meanSD. Comparison between

the values at week 0 and week 4 was performed by paired t
tests. Comparison between the values in subgroups was
performed by unpaired t tests. Associations between
different parameters were determined by Pearsons product-
moment correlation coefficients. Values of P<.05 were
considered significant.

Results
There were no significant differences between withdrawal
and control groups with respect to age, body mass index,
prior levels of alcohol consumption, the number of smokers
(Table 1), plasma lipid or -GTP levels (Table 2), fasting
blood glucose, aspartate transaminases, and fructosamine
(data not shown). The mean body mass index at baseline in
the overall HTG group was significantly higher than that in
the overall NTG group (25.22.9 versus 23.44.3, P<.05).
There were no significant changes in body weight in any
group at week 4 (data not shown), but -GTP decreased
significantly in the withdrawal groups while no change was
observed in the control groups (Table 2).

Table 2.
View this table:
Effects of Alcohol In this window
Withdrawal on Plasma In a new window
Lipids,
Apolipoproteins, and
-GTP

Before alcohol withdrawal, the means of initial TC, TG, HDL-

C, and LDL-C levels were 5.05, 1.24, 1.36, and 2.69
mmol/L, respectively, in the overall NTG group and 5.62,
4.02, 1.17, and 2.52 mmol/L, respectively, in the overall
HTG group. After alcohol cessation, HDL-C, apo AI, and apo
AII significantly decreased in the NTG/withdrawal group but
TC, TG, LDL-C, and the other apolipoproteins did not
change significantly (Table 2). In the HTG/withdrawal
group, significant reductions were found in TG and apos AI,
AII, B, CII, CIII, and E but not in TC, HDL-C, and LDL-C (Table
2). Thee were no changes in plasma lipids or
apolipoproteins in the NTG/ or HTG/control groups (Table
2).

Changes in LDL lipid composition as ratios of each lipid

value to that of apo B are shown in Table 3. The mean
ratios of TC/apo B and CE/apo B increased and that of
TG/apo B decreased significantly in the HTG/withdrawal
group after alcohol withdrawal (3.07 versus 3.36, P<.05;
2.57 versus 2.79, P<.01; and 0.42 versus 0.33, P<.05,
respectively; Table 3). FC/apoB and PL/apoB ratios did not
change in the HTG/withdrawal group. No changes in any
parameters were observed in any of the other three groups.

Table 3.
Effects of Alcohol
Withdrawal on LDL
Lipid Composition
View this table:
In this window
In a new window

The mean PPD in the HTG/withdrawal and HTG/control

group before abstinence was 25.5 and 25.3 nm,
respectively, and in the NTG/withdrawal and NTG/control
group 26.8 and 26.4 nm, respectively, indicating that in
terms of LDL subclass pattern, the HTG group had pattern B
and the NTG group had pattern A (Table 4). The mean PPD
increased significantly (P<.01) from 25.5 nm to 26.1 nm in
the HTG/withdrawal group, but there was no change in the
other groups (Table 4). Fig 1 clearly demonstrates that
increased PPD and decreased plasma TG were concomitantly
observed in the HTG/withdrawal group but not in the
NTG/withdrawal group. Before alcohol withdrawal, the mean
lag times of the HTG/withdrawal and HTG/control groups
were relatively low (49.9 minutes and 48.9 minutes,
respectively) compared with those of the NTG/withdrawal
and NTG/control groups (58.9 minutes and 60.0 minutes,
respectively). After cessation of alcohol intake, the mean lag
time was significantly (P<.01) prolonged from 49.9 to 57.3
minutes in the HTG/withdrawal group, although no
significant change was observed in the NTG/withdrawal or
either control group (Table 4). The mean contents of
vitamin E and LPO in LDL did not change significantly in any
group.

Figure 1.

Changes in PPD and

plasma TG levels after
alcohol withdrawal in
the NTG/withdrawal
group (A) and the
HTG/withdrawal
group (B). Open and
View larger version:
closed circles indicate
In this page
values before and
In a new window
after withdrawal,
Download as PowerPoint Slide
respectively.

Table 4.
View this table:
Effects of Alcohol In this window
Withdrawal on In a new window
Oxidizability, PPD, -
Tocopherol Levels,
and LPO Levels of LDL

Coefficients of correlation between the changes () in lag

time and the changes in various parameters in the combined
subjects of both HTG groups are presented in Table 5.
Significant correlations were observed between lag time
and TG (P=.023) and between lag time and PPD
(P=.012) in the overall HTG groups, indicating that
decreased oxidizability of LDL is associated with increased
LDL particle size and with reduced plasma TG levels after
alcohol withdrawal. The changes in LDL oxidizability, PPD,
and plasma TG levels were significantly correlated with each
other in the overall HTG group (Fig 2).

Figure 2.

Correlations among
lag time, plasma
TG, and PPD in
subjects of both HTG
groups. Open and
closed circles indicate
subjects in the
View larger version:
HTG/control group
In this page
(n=8) and the
In a new window
HTG/withdrawal
Download as PowerPoint Slide
(n=10) group,
respectively. A,
Correlation between lag time and plasma TG; B,
correlation between lag time and PPD; C,
correlation between PPD and plasma TG.

Table 5.
View this table:
Correlations Between In this window
the Change () in Lag In a new window
Time and the
Changes in Various
Parameters in Subjects in Both HTG Groups

Discussion
A negative correlation between the incidence of CAD and
moderate alcohol intake has been well documented. With
respect to the effects of alcohol intake on lipid metabolism,
elevated HDL-C levels could play an important role in the
prevention of CAD. However, the relation between
atherogenicity and the effects of alcohol intake on apo B-
containing lipoproteins remains unclear. The report of the
4
Cooperative Lipoprotein Phenotyping Study by Castelli et al
describes a consistent observation that alcohol consumption
is moderately associated with an elevation in plasma TG
levels and moderately to strongly associated with a
depression in LDL-C levels. Several possible mechanisms for
the decreased LDL-C levels in alcohol drinkers have been
postulated. Decreased conversion from VLDL to LDL was
reported in several articles, and acetaldehyde modification
of LDL, resulting in its accelerated catabolism, was reported
32
by Kesaniemi et al. However, although a slight increase in
LDL-C level was observed in both the NTG/ and
HTG/withdrawal groups after alcohol withdrawal in the
current study, these increases were not significant. Thus, an
effect of alcohol intake on LDL-C level was not observed in
this study.

With respect to the effect of alcohol withdrawal on plasma

TG levels, a significant reduction was observed in the
HTG/withdrawal group but not the NTG/withdrawal group.
However, the mean TG level in the HTG/withdrawal group
was still high after abstinence. It may be that other factors
that elevate plasma TG levels (except for alcohol), such as
insulin resistance, might be related to this phenomenon
because of the higher body mass index in the HTG group. In
terms of the responses of plasma TG or TG-rich
lipoproteins, these levels may increase more after alcohol
intake in obese subjects than in lean subjects, as reported
33
by Crouse and Grundy.

It has been documented that individuals with a

predominance of small LDL particles are characterized as
having LDL subclass pattern B and that this pattern is
67
associated with an increased risk for CAD. LDL pattern B
has often been observed in individuals with increased levels
8
of TGs. In the current study, the mean PPD in HTG subjects
before abstinence was smaller than those in NTG groups.
This indicates that in terms of LDL subclass pattern, LDL
pattern B was also observed in alcohol-induced HTG
subjects while NTG subjects who continued drinking alcohol
revealed pattern A. Moreover, the mean PPD in the
HTG/withdrawal group increased significantly in conjunction
with decreased plasma TG levels, but no change was
observed in the other groups. Thus, abstinence from alcohol
did not affect the LDL pattern in drinkers with normal TG
levels but it did have a favorable effect on this pattern in
drinkers who had increased TG levels. The observed change
from B to A in the LDL subclass pattern could be beneficial
for ameliorating atherosclerosis.

It is reasonable to ask why the change in LDL particle size

did not occur in NTG subjects. The precise mechanisms for
the different responses between NTG and HTG subjects are
yet unclear. It is likely that concomitant evaluations of
lipoprotein lipase, hepatic TG lipase, and CETP activities
would be needed. It has been reported that lipoprotein
34 35 36 37
lipase, hepatic TG lipase, and CETP affect LDL
38 39
subclass distribution. Musliner et al and Krauss reported
that large LDL was formed from small VLDL after lipolysis by
lipoprotein lipase and that small LDL was formed from large
VLDL by hepatic TG lipase. Additionally, inactivation of CETP
has been reported to induce TG-rich LDL, which is
hydrolyzed by hepatic TG lipase to form small LDL. With
respect to the effects of alcohol intake on lipid metabolism,
it has been reported that alcohol intake induces activation of
40 41 42
lipoprotein lipase and inhibition of CETP but does not
40
affect hepatic TG lipase activity. These reports are
consistent with the present study, in which the effect of
alcohol was to reduce the size of LDL particles. It could be
postulated that alcohol intake increases lipoprotein lipase
activity and decreases CETP activity without changing
hepatic TG lipase activity and consequently shifts LDL from
larger to smaller particles (pattern A to pattern B). In the
data presented herein, the mean ratios of LDL-C/apo B and
CE/apo B increased and that of TG/apoB decreased
significantly in the HTG/withdrawal group after alcohol
cessation, as has been suggested form recovered CETP
42
activity by alcohol withdrawal. In HTG with an increase in
VLDL production (as often seen in HTG), the effect of
enzymes on VLDL might be emphasized and consequently,
the LDL pattern change might be exaggerated. Therefore, to
make this clear, a study analyzing lipoprotein lipase, hepatic
TG lipase, and CETP activities based on different TG levels
43
would be needed. However, Jansen et al reported in a
cross-sectional multivariate analysis that the major
determinant of LDL size distribution was the plasma TG level
rather than lipoprotein lipase, hepatic TG lipase, and CETP
activity. Moreover, alterations in LDL subclass distribution
from small and dense to large and buoyant have been
observed in conjunction with reduction in plasma TG levels
9
after treatment with lipid-lowering agents in HTG patients
10 11 12 13 14
and after an intensive diet and exercise
15
program in obese subjects. Therefore, the plasma TG level
itself may be the most important factor for changing the LDL
subclass pattern, and a reduction in plasma TG levels could
be a pivotal factor in the shift toward larger, more buoyant
LDL particles after alcohol withdrawal, as well as the
previous studies that used medication for HTG.

Several investigators have proposed possible mechanisms

underlying the link between small, dense LDL particles and
atherogenesis, such as decreased binding to LDL
44 45
receptors, increased binding to arterial wall
46
proteoglycans, enhanced uptake by aortic subendothelial
47
cells, and so on. Recently, the focus has been on the
oxidative susceptibility of small, dense LDL. Increased
21
oxidizability of all LDLs in subjects with pattern B and of

20
the small, dense LDL subfraction in healthy subjects has
 20
the small, dense LDL subfraction in healthy subjects has
been reported. It has also been reported that there are
improvements in LDL oxidizability after reductions in plasma
9
TG levels by medication for HTG and by diet and exercise in
15 48
obese subjects. Furthermore, Regnstrm et al reported
that LDL oxidizability was associated with the severity of
coronary atherosclerosis. Thus, it is important to evaluate
the susceptibility of LDL to oxidation as well as to determine
LDL particle size. As described above in the current study,
LDL particle size in the HTG groups before abstinence was
smaller than that in NTG groups, and after alcohol
withdrawal, the LDL particle size distribution and lipid
composition revealed a shift from smaller and CE-poor, TG-
rich particles toward larger and CE-rich, TG-poor particles,
respectively. Interestingly, mean lag times were shorter in
the HTG groups than in the NTG groups before alcohol
withdrawal, and after the withdrawal the mean lag time
increased significantly in the HTG/withdrawal group,
although no significant change was observed in the
NTG/withdrawal or either control group. These findings
point to a close association between a predominantly small-
particle LDL subclass pattern and LDL oxidizability and
indicate that alcohol withdrawal would have a favorable
effect on the LDL profile in HTG. In addition, among the
combinations between lag time and changes in the various
parameters in the HTG groups, significant correlations were
observed between lag time and TG (P=.023) and between
lag time and PPD (P=.012). This would confirm the above
observation that LDL oxidizability is associated with LDL
particle size. A significant correlation between lag time and
TG might be produced as a secondary result due to the
48
correlation between PPD and TG. Regnstrm et al
reported that LDL oxidizability was positively correlated with
TG contents in LDL, which decreased in the HTG/withdrawal
group after cessation of alcohol intake, but in the present
study, LDL-TG levels or TG/apoB was not associated with
LDL oxidizability (Table 5). With regard to HDL, the
49
enzymes associated with it, such as paraoxonase and
50
platelet-activating factor acetylhydrolase, may play an
important role in protecting LDL against oxidation in the
artery wall and may account in part for the inverse relation
between HDL and the risk of atherosclerotic clinical events.
Therefore, altered HDL levels might affect LDL oxidizability.
Although we did not investigate the effect of HDL on LDL
oxidation in the present study, it may be important to
evaluate LDL oxidizability in relation to HDL. Because HDL
levels did not change in the HTG/withdrawal group, it is
unlikely that HDL affected LDL oxidizability in this study. In
terms of the effect of LDL oxidizability on atherogenicity,
51
Salonen et al reported that lag time during LDL (+VLDL)
 51
Salonen et al reported that lag time during LDL (+VLDL)
oxidation was the strongest predictor of a 3-year increase in
carotid wall thickness as detected by ultrasonography. The
difference between the mean lag time in the progression
group and that in the nonprogression group was only 27%,
even when the range of values was from 55 to 301 minutes.
In our study, the mean lag time in the HTG/withdrawal
group was prolonged by 14.8% from the initial value, even
though our study had a relatively small range of values
compared with those in the study by Salonen et al. Although
we cannot directly compare our results with those of Salonen
et al, it is suggested that this small but significant change in
lag time could affect atherogenesis.

In recent years, there have been reports of an inhibitory

effect of phenolic substances in red wine against LDL
52
oxidation and decreased LDL oxidizability after
53
consumption of red wine. In the present study, none of
the subjects consumed wine and/or other alcoholic
beverages brewed from fruit. Therefore, the presence of
phenolic substances, which could alter LDL oxidizability, was
not considered a problem in the present study. In terms of
the contribution of alcohol itself to LDL oxidation, ethanol
has been considered an exogenous factor that can generate
free radicals in vivo. Oxidative modification of LDL was
18
reported by Lin et al in alcohol abusers who consumed
100 g of alcohol daily. These investigators demonstrated
that rabbit antibodies raised by a protein modified in vitro
by 4-hydroxynonenal as an immunogen reacted more
strongly with the alcoholics LDL than with control LDL,
indicating the presence of oxidatively modified epitopes.
Although Lin et al did not evaluate the oxidative
susceptibility of LDL, it is possible that the oxidative
susceptibility of alcoholics LDL increased because of the
19
lower contents of vitamin E in LDL. Croft et al also
reported that increased oxidizability of LDL was observed
54
after alcohol ingestion. On the other hand, Suzukawa et al
previously reported that LDL oxidizability and vitamin E
levels in LDL did not change after moderate alcohol intake
(30 to 40 g/d) for 4 weeks, although -carotene levels in
LDL decreased. In the present study, no change was
observed in vitamin E levels after alcohol withdrawal.
Additionally, ethanol consumption did not correlate with any
parameters; the baseline values; and the changes after the
study period in plasma lipids, apolipoproteins, LDL lipid
compositions, vitamin E, LPO, PPD, and lag time (data not
shown). We suggest that LDL oxidizability and the
parameters affecting LDL oxidizability were unaffected by
the amount of alcohol intake, at least in the amounts
consumed by the subjects in this study. The decreased
oxidizability observed in this study might be mainly due to
the change in LDL particle size induced by a reduction in
plasma TG levels.

In conclusion, alcohol consumers with alcohol-induced

increases in plasma TG levels have an unfavorable LDL
profile; their LDLs are CE poor and they have TG-rich, small
LDL with increased oxidizability. Alcohol withdrawal
produces beneficial effects on this LDL profile and oxidative
susceptibility.

SelectedAbbreviationsandAcronyms

CAD =coronary artery

disease
CE(TP)=cholesteryl ester
(transfer protein)
FC =free cholesterol
-GTP=-glutamyl
transpeptidase
HDL- =HDL cholesterol
C
HTG =hypertriglyceridemic
LDL- =LDL cholesterol
C
LPO =lipid peroxide
NTG =normotriglyceridemic
PL =phospholipid
PPD =peak particle diameter
TC =total cholesterol
TG =triglyceride

Acknowledgments
We are grateful to Dr C. Maruyama and her colleagues for
analysis of dietary records and to Emiko Miyajima and her
colleagues for technical assistance.

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