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Journal of Lipid Research, Vol. 48, 1550-1558, July 2007 Beyond HDL-cholesterol increase: phospholipid enrichment and shift from HDL3 to HDL2 in alcohol consumers
* Division of Gastroenterology, Robert-Bosch-Hospital Stuttgart, Stuttgart, Germany Published, JLR Papers in Press, April 12, 2007.
1 To whom correspondence should be addressed. e-mail: christian.schaefer{at}rbk.de
The reduction of cardiovascular mortality associated with moderate alcohol consumption is chiefly thought to be mediated by an increase of high density lipoprotein cholesterol (HDL-CH). This study highlights additional qualitative changes of HDL that might augment this antiatherogenic effect. In 279 healthy men, alcohol and nutrient consumption were evaluated. Groups 1 (n = 62), 2 (n = 172), and 3 (n = 45) comprised subjects with alcohol consumption of 05.0, 5.130.0, and 30.175 g/day, respectively. Lipid analysis was performed in nonfractionated and fractionated plasma, including subfractions HDL2a, HDL2b, and HDL3. No difference in LDL-cholesterol was observed. Compared with group 1, groups 2 and 3 exhibited significant increases of HDL-CH (group 1, 44 ± 10 mg/dl; group 2, 51 ± 11 mg/dl; group 3, 55 ± 11 mg/dl; mean ± SD, P < 0.0005), accompanied by enhanced lipidation of HDL (increase of the HDL2-CH/HDL3-CH ratio). Moreover, phospholipid enrichment of HDL occurred in alcohol consumers, whereas the ratios between other HDL components remained constant. Multivariate analysis revealed alcohol to have the foremost statistical influence on changes of the HDL fraction, followed by body mass index and physical activity level. The increased lipidation of HDL found in alcohol consumers might augment the antiatherogenic effect of HDL-CH increase. In addition, the phospholipid enrichment of HDL might reduce the inflammatory response of atherogenesis.
Supplementary key words low density lipoprotein very low density lipoprotein high density lipoprotein 2 high density lipoprotein 3 triglycerides beer wine body mass index apolipoprotein Abbreviations: apoA-I, apolipoprotein A-I; BMI, body mass index; CE, cholesteryl ester; CETP, cholesteryl ester transfer protein; FC, free cholesterol; HDL-CH, high density lipoprotein-cholesterol; LDL-CH, low density lipoprotein-cholesterol; PAL, physical activity level; PL, phospholipid; PLTP, phospholipid transfer protein; SM, sphingomyelin; TG, triglyceride
Moderate regular alcohol consumption (530 g/day in men, 520 g/day in women) has been associated with a reduction of overall mortality attributable to a decreased incidence of ischemic heart disease and stroke (14). Among the antiatherosclerotic mechanisms discussed, the alcohol-induced increase of HDLs is thought to play a pivotal role in reducing atherosclerosis by increasing reverse cholesterol transport from the vessel walls to the liver (5). This cross-sectional study in healthy working men was performed to further examine the effect of various alcohol consumption patterns on the lipid profile. Our working hypothesis was that moderate alcohol consumption induces not only quantitative, but also qualitative, changes of the lipid fractions, in particular HDL. Using extensive fractionation of plasma lipids and measurement of lipid and apolipoprotein components, we show that alcohol consumption is associated with increases of cholesterol and other components of the HDL fraction. In addition, two significant qualitative changes were identified that might affect atherogenicity: a shift from small, dense HDL3 to lipid-rich HDL2 (mainly HDL2a), and a significant phospholipid (PL) enrichment of HDL in all subfractions. Because not only alcohol consumption but also lifestyle factors, such as nutrition, physical activity, smoking, age, and obesity, may influence lipid metabolism, we performed a thorough interview to characterize the influence of these factors on HDL. Furthermore, it was examined whether consumption patterns (i.e., preference for beer or wine) had differential effects on high density lipoprotein-cholesterol (HDL-CH), because the data from several epidemiological studies had been inconclusive (reviewed in Refs. 6, 7).
Study subjects This study, which followed the guidelines of the Declaration of Helsinki of 1975, as revised in 1983, was approved by the Ethical Review Board of the University of Tübingen. After providing informed consent, 290 healthy male working volunteers, aged 2070 years, were recruited at their work place (63% automobile industry, 12% wine factory, 18% brewery, 7% health care staff and others). At the first date of examination, blood was obtained after an overnight fast for biochemical testing. Soon thereafter, a 1 hour personal computer-based interview was performed by an experienced nutritional scientist to determine the nutritional profile, including alcohol consumption and energy expenditure.
Of the 290 recruited persons, 11 were excluded for various reasons: self-reported previous myocardial infarction or ischemic stroke (n = 2), heavy (
The remaining 279 individuals were grouped according to their amount of daily alcohol consumption. Group 1 comprised 62 persons with an alcohol consumption of
Evaluation of nutrition and physical activity
Laboratory analyses Briefly, VLDL (d < 1.006 g/ml), LDL (d = 1.0191.063 g/ml), and HDL (d = 1.0631.21 g/ml) were isolated by sequential preparative ultracentrifugation from 5 ml of plasma (50.3 Ti rotor, L8-55M centrifuge; Beckman Instruments). Analogously, HDL subfractions were obtained: HDL2b (d = 1.0631.100 g/ml), HDL2a (d = 1.1001.150 g/ml), and HDL3 (d = 1.1501.210 g/ml). Lipids in each (sub)fraction were determined as follows (10). Cholesterol and triglycerides (TGs) were determined enzymatically with the cholesterol oxidase-peroxidase amino phenazone phenol and the glycerol-3-phosphate oxidase-peroxidase amino phenazone phenol methods (Roche Diagnostics, Mannheim, Germany), respectively. Free cholesterol (FC) and PLs were determined enzymatically with the cholesterol oxidase-peroxidase amino phenazone phenol method and by phospholipase D, cholinoxidase, and peroxidase, respectively, with commercially available reagents (Wako Chemicals, Osaka, Japan). The concentration of esterified cholesterol was calculated from the difference of total cholesterol and FC. In addition, apolipoproteins A-I and A-II (apoA-I and apoA-II) were determined immunoturbidimetrically [anti-apoA-I from Greiner (Flacht, Germany) and anti-apoA-II from Kamiya Biomedical (Seattle, WA)]. This comprehensive analysis was performed on each individual sample.
Statistics We also conducted a multivariate analysis of the influence of various factors on HDL components. The multiple linear regression model was calculated according to the formula y = c1x1 + c2x2 + ...cnxn + a (where y is the dependent variable, c1...cn are calculated coefficients, x1...xn are independent variables, and a is the intercept). As independent variables, alcohol consumption, the influence of beer versus wine, anthropometric characteristics, calorie intake, and energy output were chosen. To compare beer versus wine, we determined the percentage of daily beer consumption [RCbeer vs. wine(%)] in relation to the sum of beer and wine consumption, based on alcohol (EtOH) content [EtOHbeer x 100/(EtOHbeer + EtOHwine)]. Drinks other than beer or wine were disregarded, because they were quantitatively unimportant (Table 1 ).
Characteristics of the study population The basic anthropometric data, alcohol consumption, nutritional characteristics, energy expenditure, and essential laboratory parameters are listed in Table 1. There were no significant differences in age and body mass index (BMI) between groups 1 and 2, whereas persons in group 3 differed slightly, but significantly, in age and BMI. The proportion of smokers did not exceed 20% in each group. The comparison of drinking patterns showed a higher proportion of beer drinkers in group 3 and a high proportion of mixed consumption in group 2. According to regional drinking habits, hard liquors and beverages other than beer or wine comprised only a minor portion of the alcohol consumption. The nutritional profile, except for a lower intake of dietary fiber in group 3, and the data for energy expenditure were comparable between all three groups. The evaluation of basic laboratory data showed no differences between groups 1 and 2, whereas in group 3, a significant increase of the markers for alcohol consumption (mean corpuscular volume and -glutamyl transferase) became evident.
Overall lipid analysis and lipid fractions
For HDL-CH, we found a significant increase in persons of groups 2 and 3 compared with group 1 (Table 2). The increase amounted to 16% in group 2 and 25% in group 3. Low density lipoprotein-cholesterol (LDL-CH) levels were nearly identical in all three groups. Because VLDL particles constitute the major vehicle for plasma TG, the differences between the groups resemble those observed for TG levels in nonfractionated plasma.
Changes in the composition of HDL (HDL2 vs. HDL3) In each of the HDL subfractions, the cholesterol content was increased in alcohol consumers (groups 2 and 3) compared with group 1 (Table 3 ). However, differences between the dose responses of the HDL subfractions were observed. Comparing group 2 and group 1, HDL2b-CH was increased by 24% and HDL2a-CH was increased by 25%, whereas HDL3-CH showed an increase of only 10%. In group 3, this dissociation was even more pronounced (HDL2b-CH, 28%; HDL2a-CH, 37%; HDL3-CH, 11% increase vs. group 1).
Translated into ratios of HDL2a+2b-CH to HDL3-CH, and analogous ratios of PLs and TGs, a substantial shift toward the HDL2 subfraction was measured (Table 3).
Increase of the PL component
To assess the atherogenic potency of HDL-CH, it was also of interest to examine the ratio between the core component cholesteryl ester (CE) and FC in HDL and its subfractions. However, these ratios were constant in all three groups. The relations of TG, the other core component, to CE, FC, and apoA-I remained unchanged (data not shown).
Multivariate analysis: influence of various factors on HDL components
Multivariate analysis: qualitative changes Using analogous multivariate models, the influence of alcohol consumption on qualitative changes of the HDL fraction was examined (Table 6 ). The PL/cholesterol ratio showed a highly significant dependence on alcohol consumption. Interestingly, BMI and PAL were also positively associated with PL enrichment in HDL, whereas energy intake had a negative influence.
The relationship between apoA-I and apoA-II in the HDL fraction constitutes an example of alcohol-independent qualitative changes. Age and PAL shifted the ratio toward apoA-I, whereas a high BMI was associated with increased apoA-II. Finally, the HDL2-CH/HDL3-CH ratio was assessed. In this mathematical model, BMI was the only factor with a significant (inverse) effect on this ratio. The influence of alcohol on this ratio did not reach statistical significance (P = 0.06).
This is, to the best of our knowledge, the first cross-sectional study in a large and well-characterized group of healthy working males comparing the influence of nonaddictive alcohol consumption on the composition of HDL and its subfractions HDL2b, HDL2a, and HDL3. Apart from a highly significant alcohol-associated increase of lipids and apolipoproteins in HDL and its subfractions, we observed two qualitative changes that are likely to influence atherogenicity: a lipid enrichment of HDL, as shown by an increase of the HDL2-CH/HDL3-CH ratio, and an increase of the PL/cholesterol ratio in all HDL subclasses. The latter change can be traced back to PL enrichment in the intermediate density HDL2a and in the dense HDL3 subfractions and additional depletion of FC in the buoyant HDL2b subfraction. Several case-control and epidemiological studies indicate that moderate alcohol consumption reduces cardiovascular mortality and that this effect is associated with the increase of HDL-CH (5, 11). In their meta-analysis, Rimm et al. (5) calculated an increase of HDL-CH of 0.134 mg/dl per gram of alcohol of daily consumption for men (for women, it was 0.095 mg/dl per gram of alcohol). In our study, the coefficient reached 0.24 mg/dl per gram of alcohol of daily consumption for healthy male persons. Several factors may account for this difference. Rimm et al. (5) did not adjust their model for BMI and smoking, both of which have decreasing effects on HDL-CH. Also, we could not find evidence for their statement that the effect of alcohol on HDL-CH was more pronounced in subjects with a sedentary lifestyle. In our multivariate analysis, physical activity had an independent and significantly positive influence on HDL-CH levels. The quantitative change of HDL-CH is thought to be the result of increased hepatic production or an increased transport rate of apoA-I and apoA-II (1214). Other data indicate that alcohol might interfere with the activity of the cholesteryl ester transfer protein (CETP), which mediates cholesterol transfer from HDL to LDL (15). Additionally, it has been shown by ex vivo experiments that moderate alcohol consumption increases the efflux of FC from macrophages to HDL, mainly via the ATP binding cassette transporter 1 (16). Large amounts of alcohol, on the other hand, may result in impaired cholesterol efflux (17). One of the major findings of our study is the divergence of the dose relationships between the HDL subclasses. Compared with controls, the alcohol-related increase of cholesterol, PL, and TG in HDL2a was more than twice that of the increase in the HDL3 subclass. The changes in HDL2b were similar to those in HDL2a, but somewhat less pronounced. The shift toward HDL2 indicates a higher HDL lipid load in alcohol consumers (Table 3). This phenomenon is confirmed by other examinations (1821), whereas some groups found a predominant increase of HDL3 (22, 23) or no relevant change in the relation of HDL2 to HDL3 components (11, 24). These discrepancies might be explained by variant study design and the disregard of powerful confounders, such as obesity and smoking. The increased lipidation of HDL, which may be attributable to an increased cholesterol efflux from peripheral cells (16), could indicate an augmentation of reverse cholesterol transport. This can be inferred from studies showing that risk factors such as obesity or hypertriglyceridemia are associated with the opposite change (i.e., a decrease of large HDL2 vs. an increase of small preß HDL or HDL3 particles) (25, 26), which has been confirmed for obesity by the multivariate analysis of our study. Interestingly, the influence of alcohol on the HDL2-CH/HDL3-CH balance seems to "override" the opposing influence of high BMI or increased TGs, as subjects with an alcohol consumption of >30 g/day (group 3) exhibited the highest HDL2-CH/HDL3-CH ratios despite the increased BMI and TG values (Tables 13). It could be argued that an increase of HDL2 cholesterol might be ambiguous, because this fraction is the substrate of cholesterol transfer protein, which shifts CEs to proatherogenic LDL particles. Thus, we examined the balance between CEs and FC in HDL but found no significant change. From a clinical point of view, the cardioprotective relevance of HDL2 is supported by several population studies, such as the Québec and Kuopio trials (27, 28). In other population studies, such as the Physicians' Health Study (29) and the Caerphilly and Speedwell trials (30), HDL3 was the strongest predictor, but risk reduction was shown for HDL2 as well. The other qualitative change of HDL apart from the increase in lipid-rich particles is the alcohol-induced relative increase of the PL components compared with the other HDL components (cholesterol, TG, apoA-I, and apoA-II). This change was observed in all HDL subfractions and was most pronounced in persons with increased alcohol consumption. In the current multivariate analysis, a highly significant association between alcohol consumption and an increase of the PL/cholesterol ratio was detected, although other factors (BMI, energy intake, and physical activity) seemed to be involved in the regulation of this balance as well. The relative increase of the PL component may have important implications concerning the antiatherosclerotic effects of HDL. Because this change occurs in the surface layer of HDL that interacts with cellular receptors or serum components, it may be of particular importance in two respects: the increase of PL may reduce the inflammatory process in the vessel wall, because HDL particles reconstituted with PLs inhibited the cytokine-induced activation of endothelial cells in vitro (31); and PLs are obviously required for an effective cholesterol efflux from peripheral cells to ApoA-I-containing lipoprotein particles (32). In this context, it could be argued that the PL portion might contain compounds associated with an increased atherogenic risk, such as sphingomyelin (SM). However, it is very unlikely that SM plays such a role, for the following reasons. i) HDL-like LpA1 containing SM may be even more effective at effluxing cholesterol from fibroblasts than LpA1 without SM (33). ii) There is evidence from animal experiments that excess SM induces an increase of VLDL-CH and LDL-CH but a decrease of HDL-CH (33, 34). This situation is contrary to that found in the alcohol consumers of our study, so a general increase of atherogenic SM is unlikely. iii) In alcohol-consuming humans, no change of the SM content in platelets occurred, and even a decrease of SM in erythrocyte membrane occurred (35, 36). Despite this circumstantial evidence, further studies are needed to delineate the influence of alcohol on PL composition in HDL. The exact mechanisms leading to the increase of the PL/cholesterol ratio remain unclarified. Several transfer enzymes promote the exchange of lipid components between HDL, LDL, and VLDL. High-level alcohol consumption results in depressed CETP activity, which is associated with an increase of HDL3b (medium HDL3) at the expense of HDL2a. In addition, alcohol-induced phospholipid transfer protein (PLTP) activity, which shifts PL from TG-rich lipoproteins to HDL, appears to increase HDL2b at the expense of HDL3a (large HDL3) (37). The reduction of CETP and the increase of PLTP were confirmed by another study, which also observed an increase in the net PL transfer from apoB-containing lipoproteins to HDL in alcoholics (38). However, none of these enzymes seems to be changed in moderate alcohol consumers (3941). In our study, the analysis of HDL2a and HDL3 composition shows an isolated dose-dependent increase of the PLs compared with apoA-I, whereas the ratio of FC to apoA-I remained unchanged, which argues for a role of PLTP in PL enrichment in these HDL subclasses. In lipid-rich HDL2b, the situation is more complex, because both PL enrichment and FC depletion occur. Another possible mechanism for the PL enrichment is highlighted by an in vitro study showing that ethanol at concentrations found in heavy drinkers inhibits the incorporation of cholesterol into preß-like particles containing apoA-I and phosphatidylcholine, whereas this phenomenon was not observed in particles without this PL (42). Thus, alcohol, at least at high concentrations, might influence the balance between PL and cholesterol at this very early step of HDL assembly. In terms of antiatherogenicity, it is also of interest to study the effect of alcohol consumption on a third feature of HDL, the relation of HDL particles containing only apoA-I, termed LpA1, to particles containing apoA-I and apoA-II (LpA1/A2), because LpA1 has a higher potency than LpA1/A2 in effluxing cellular cholesterol (43, 44). In our study, the concentrations of LpA1 and LpA1/A2 were not measured directly. However, comparison between the groups and multivariate analysis showed no influence of alcohol consumption on the ratio of apoA-I to apoA-II (Tables 4, 6), which allows the conclusion that LpA1 and LpA1/A2 are increased in a similar manner. This conclusion is in accordance with two studies directly showing an alcohol-related increase of both lipoprotein species (24, 45). Yet, other data suggest that alcohol consumption mainly increases LpA1/A2 (18, 46). It is somewhat surprising that the shift from HDL3 to HDL2 does not result in an increase of apoA-I, given the fact that HDL2 consists mainly of LpA1, with most of LpA1/A2 being associated with HDL3. Two phenomena may account for the constancy of the apoA-I/apoA-II ratio despite the relative increase of HDL2 in alcohol consumers (Table 4): i) the ratios of the two apolipoproteins in HDL3 and in HDL2a, the major portion of HDL2, are almost alike; ii) HDL2b, the minor portion of HDL2, showed a slight, although insignificant, decrease in this ratio in group 3, indicating some loss of LpA1 in this subfraction. Apart from the mere alcohol effect, we examined whether beer or wine consumption had a differential effect on the composition of the HDL fraction. The multivariate analysis showed no significant differences between relative beer and wine consumption on all HDL components and on qualitative changes. Thus, our data are in accordance with the general assumption (5) that the increase of HDL-CH is an effect of the alcohol itself, particularly in the range of moderate alcohol consumption. The effect of alcohol must be interpreted in the context of other lifestyle and anthropometric parameters. In our multivariate model, obesity and factors predisposing to it, such as high caloric intake and scarce physical activity, are associated with a reduction in essential HDL components; thus, they counteract the influence of alcohol on HDL (Table 4). Beyond this, obesity tilts the balance between apoA-I and apoA-II toward the latter, resulting in a relative increase of LpA1/A2 at the cost of LpA1, which is an additional indication of impaired reverse cholesterol transport (Table 5). Cigarette consumption had no statistically significant effect, probably because of the low percentage of smokers in this study, whereas age, interestingly, is associated with a "favorable" increase of the apoA-I/apoA-II ratio. In summary, this study supports previous data showing a powerful influence of moderate (530 g/day) and increased (3075 g/day) alcohol consumption on the concentration of HDL-CH. An additional effect, which deserves further research, is the compositional and qualitative change of HDL particles in alcohol-consuming persons, such as PL enrichment, which may inhibit the inflammatory process associated with the formation of atheromatous plaques, and the relative increase of lipid-rich HDL2 versus HDL3, which indicates a more effective reverse cholesterol transport.
This study was supported by Grant B-50 from the Wissenschaftsförderung der Deutschen Brauwirtschaft. The authors thank Ms. I. Friedrich for excellent technical assistance and J. Dippon, Ph.D., University of Stuttgart, for statistical support. The authors thank the companies that supported this work by recruiting study subjects. Manuscript received May 3, 2006 and in revised form December 4, 2006 and in re-revised form February 28, 2007.
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