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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2540-2547

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*Triglycerides
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2540-2547.)
© 1997 American Heart Association, Inc.

Articles

Beneficial Effects of Alcohol Withdrawal on LDL Particle Size Distribution and Oxidative Susceptibility in Subjects With Alcohol-Induced Hypertriglyceridemia

Makoto Ayaori; Toshitsugu Ishikawa; Hiroshi Yoshida; Michio Suzukawa; Masato Nishiwaki; Hideki Shige; Toshimitsu Ito; Kei Nakajima; Kenji Higashi; Atsushi Yonemura; ; Haruo Nakamura

From the First Department of Internal Medicine, National Defense Medical College, Saitama, Japan.

Correspondence to Makoto Ayaori, MD, First Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359, Japan. E-mail ayaori{at}ba2.so-net.or.jp


*    Abstract
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 *Abstract
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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 ({Delta}) in lag time and {Delta}TG and between {Delta}lag time and {Delta}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


*    Introduction
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 *Introduction
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Several epidemiological studies have reported a correlation between a lower incidence of CAD and moderate alcohol intake.1 2 In some studies, alcohol intake was positively associated with an elevated concentration of HDL,3 4 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 alcohol intake increases CAD mortality.1 It is unclear why a moderate alcohol intake protects against atherosclerosis while heavy drinking has an opposite effect. Quantitative changes in HDL and LDL levels with alcohol intake levels4 and the effects of HDL subfractions have been described.3 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 combinations of different sizes of LDL particles.5 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 CAD.6 7 McNamara et al8 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, together with a reduction in plasma TG levels.9 10 11 12 13 14 Beard et al15 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 modified LDL in atherogenesis has been suggested.16 Oxidized LDL is easily taken up by macrophages via scavenger receptors and transforms the macrophages to foam cells, which characteristically appear in the early atherosclerotic plaque.16 17 The oxidative properties of alcohol and its metabolites have been reported. Lin et al18 reported that LDLs separated from the plasma of heavy alcohol consumers had been oxidatively modified in vivo. Croft et al19 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 dense LDL particles are more susceptible.20 21 22 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
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 *Methods
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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 (mean±SD, 41.5±7.2) 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.

Blood Sampling and Preparation of LDL
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 -70°C for the other assays. LDL (d=1.019 to 1.063 g/mL) was isolated by sequential ultracentrifugation by using the methods of Havel et al.23

Experimental Design
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 1Down). 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 ({alpha}-tocopherol, ß-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.


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  		Table 1. Clinical Characteristics of the Study Subjects

Biochemical Analyses
Plasma TC, LDL-C, TG, FC, and PLs were determined by enzymatic methods using commercially available enzymatic reagents (Kyowa Medics Co). Plasma {gamma}-GTP activity was measured by the method of Szasz.24 The normal range of {gamma}-GTP is <50 IU/L. Apolipoproteins were measured by immunoturbidimetry.25 The protein content of LDL was measured by the method of Lowry et al.26 Vitamin E ({alpha}-tocopherol) in LDL was determined by high-performance liquid chromatography.27 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 (25x0.46 cm, 5-µm 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 peroxides on the presence of heme compounds.28

Determination of LDL PPD
LDL subfractions were separated at 10°C by 2% to 16% gradient polyacrylamide gel electrophoresis (PAA 2-16, Pharmacia) as reported by Nichols et al.29 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 µL 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% methanol–10% acetic acid (vol/vol) containing Coomassie brilliant blue R-250. After destaining in 20% methanol–10% 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 proposed by Williams et al30 was used to determine the LDL particle diameters.

Determination of Oxidative Susceptibility of LDL
Oxidative susceptibility of LDL was evaluated by the method of Esterbauer et al31 with some modification. LDL oxidation was initiated by incubation of 100 µg of LDL protein with 20 µmol/L 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile); V-70, Wako Pure Chemicals) in 2 mL of PBS at 37°C, 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.

Statistical Analyses
Values were expressed as mean±SD. 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 Pearson's product-moment correlation coefficients. Values of P<.05 were considered significant.


*    Results
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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 1Up), plasma lipid or {gamma}-GTP levels (Table 2Down), 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 (25.2±2.9 versus 23.4±4.3, P<.05). There were no significant changes in body weight in any group at week 4 (data not shown), but {gamma}-GTP decreased significantly in the withdrawal groups while no change was observed in the control groups (Table 2Down).


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  		Table 2. Effects of Alcohol Withdrawal on Plasma Lipids, Apolipoproteins, and {gamma}-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 2Up). 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 2Up). Thee were no changes in plasma lipids or apolipoproteins in the NTG/ or HTG/control groups (Table 2Up).

Changes in LDL lipid composition as ratios of each lipid value to that of apo B are shown in Table 3Down. 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 3Down). 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.


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  		Table 3. Effects of Alcohol Withdrawal on LDL Lipid Composition

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 4Down). 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 4Down). Fig 1Down 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 4Down). The mean contents of vitamin E and LPO in LDL did not change significantly in any group.


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  		Table 4. Effects of Alcohol Withdrawal on Oxidizability, PPD, {alpha}-Tocopherol Levels, and LPO Levels of LDL



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  		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 closed circles indicate values before and after withdrawal, respectively.

Coefficients of correlation between the changes ({Delta}) in lag time and the changes in various parameters in the combined subjects of both HTG groups are presented in Table 5Down. Significant correlations were observed between {Delta}lag time and {Delta}TG (P=.023) and between {Delta}lag time and {psi}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 2Down).


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  		Table 5. Correlations Between the Change ({Delta}) in Lag Time and the Changes in Various Parameters in Subjects in Both HTG Groups



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  		Figure 2. Correlations among {Delta}lag time, {Delta}plasma TG, and {Delta}PPD in subjects of both HTG groups. Open and closed circles indicate subjects in the HTG/control group (n=8) and the HTG/withdrawal (n=10) group, respectively. A, Correlation between {Delta}lag time and {Delta}plasma TG; B, correlation between {Delta}lag time and {Delta}PPD; C, correlation between {Delta}PPD and {Delta}plasma TG.


*    Discussion
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 *Discussion
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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 Cooperative Lipoprotein Phenotyping Study by Castelli et al4 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 by Kesaniemi et al.32 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 by Crouse and Grundy.33

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 associated with an increased risk for CAD.6 7 LDL pattern B has often been observed in individuals with increased levels of TGs.8 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 lipase,34 hepatic TG lipase,35 36 and CETP37 affect LDL subclass distribution. Musliner et al38 and Krauss39 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 lipoprotein lipase40 41 and inhibition of CETP42 but does not affect hepatic TG lipase activity.40 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 activity by alcohol withdrawal.42 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 would be needed. However, Jansen et al43 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 after treatment with lipid-lowering agents in HTG patients9 10 11 12 13 14 and after an intensive diet and exercise program in obese subjects.15 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 receptors,44 45 increased binding to arterial wall proteoglycans,46 enhanced uptake by aortic subendothelial cells,47 and so on. Recently, the focus has been on the oxidative susceptibility of small, dense LDL. Increased oxidizability of all LDLs in subjects with pattern B21 and of the small, dense LDL subfraction in healthy subjects20 has been reported. It has also been reported that there are improvements in LDL oxidizability after reductions in plasma TG levels by medication for HTG9 and by diet and exercise in obese subjects.15 Furthermore, Regnström et al48 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 {Delta}lag time and changes in the various parameters in the HTG groups, significant correlations were observed between {Delta}lag time and {Delta}TG (P=.023) and between {Delta}lag time and {Delta}PPD (P=.012). This would confirm the above observation that LDL oxidizability is associated with LDL particle size. A significant correlation between {Delta}lag time and {Delta}TG might be produced as a secondary result due to the correlation between {Delta}PPD and {Delta}TG. Regnström et al48 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 5Up). With regard to HDL, the enzymes associated with it, such as paraoxonase49 and platelet-activating factor acetylhydrolase,50 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, Salonen et al51 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 oxidation52 and decreased LDL oxidizability after consumption of red wine.53 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 reported by Lin et al18 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 lower contents of vitamin E in LDL. Croft et al19 also reported that increased oxidizability of LDL was observed after alcohol ingestion. On the other hand, Suzukawa et al54 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 ß-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.


*    Selected Abbreviations and Acronyms
 
CAD = coronary artery disease
CE(TP) = cholesteryl ester (transfer protein)
FC = free cholesterol
{gamma}-GTP = {gamma}-glutamyl transpeptidase
HDL-C = HDL cholesterol
HTG = hypertriglyceridemic
LDL-C = LDL cholesterol
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.

Received April 1, 1997; accepted August 4, 1997.


*    References
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 *References
 

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