Effects of alcohol on lipoprotein lipase, hepatic lipase, cholesteryl ester transfer protein, and lecithin:cholesterol acyltransferase in high-density lipoprotein cholesterol elevation

ATHEROSCLEROSIS ELSEVIER

Atherosclerosis I I1 (1994) 99-109

Effects of alcohol on lipoprotein lipase, hepatic lipase, cholesteryl ester transfer protein, and 1ecithin:cholesterol acyltransferase in high-density lipoprotein cholesterol elevation Masato Nishiwaki*a, Toshitsugu Ishikawa”, Toshimitsu Itoa, Hideki Shi%e”, Koji Tomiyasua, Kei Nakajimaa, Kazuo Kondoa, Hideyuki Hashimoto , Kazunori Saitohb, Mitsuhisa Manabeb, Emiko Miyajima”, Haruo Nakamuraa aFirst Deportment of Internal Medicine, Notional Defense Medical College, 3-2 Nomiki, Tokorozowa, Soitomo. 359, Japan bTokyo Research Laboratories. Doiichi Pure Chemical Co., Ltd., 5-5-12 Norihiro, Sumida, Tokyo, 130, Japon

Received 14 January 1994; revision received 27 May 1994; accepted 8 June 1994

Abstract The mechanism whereby alcohol increases high-density lipoprotein cholesterol (HDL-C) levels is unclear. Lipoprotein lipase (LPL), hepatic lipase (HL), cholesteryl ester transfer protein (CETP) and 1ecithin:cholesterol acyltransferase

(LCAT) act on lipoprotein metabolism. The purpose of the present study is to determine which one or what combination of these factors is responsible for the rise in HDL-C levels following alcohol ingestion. After 3 weeks of abstinence, 12 men consumed 0.5 g/kg bw of alcohol per day for 4 weeks; 13 abstaining men served as controls. Mean plasma total cholesterol (TC) levels were unchanged in either group throughout the study. Among the alcohol consumers, plasma triglycerides (TG), HDL-C, apolipoprotein (apo) A-I and A-II levels increased significantly after 3 weeks of alcohol loading but were unchanged in the control group. High-density lipoprotein, cholesterol (HDL,-C) levels increased significantly in the alcohol consumers after 4 weeks of alcohol loading whereas high-density lipoprotein, cholesterol (HDL,-C) levels were unaffected. In the controls, neither HDL,-C nor HDL,-C changed significantly. Post-heparin plasma (PHP) LPL activity and mass increased significantly (P < 0.01) after the alcohol ingestion (controls remained unchanged) without changing LPL specific activity. HL, CETP and LCAT activities were unaffected in both groups. We conclude that of the factors considered, LPL contributed the most to the alcohol-induced rise in HDL-C. Keyword: Alcohol; High-density lipoprotein cholesterol; HDL subfractions; Lipoprotein lipase; Hepatic lipase; Cholesteryl ester transfer protein; Lecithin:cholesterol acyltransferase

1. Introduction

Studies of alcohol withdrawal [I -71, loading 18-141 and cross-sectional surveys [ 1% 171 in* Corresponding author, Tel.: +81 (429) 95151I, ext. 2365, 2366; Fax: +81 (429) 950638.

dicate that alcohol increases high-density lipoprotein cholesterol (HDL-C), an anti-atherogenic lipoprotein which is of interest in terms of an association between the protective effect of alcohol and ischemic heart disease [ 18,191. However, the mechanism whereby an increase in HDL-C levels is induced by alcohol ingestion is not clear. Li-

0021-9150/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0021-9150(94)05314-Z

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poprotein lipase (LPL), hepatic lipase (HL), cholesteryl ester transfer protein (CETP) and lecithin:cholesterol acyltransferase (LCAT) are the four major factors which act on lipoproteins in the circulation. The triglycerides (TG) of very lowdensity lipoprotein (VLDL) and chylomicron are hydrolyzed by LPL, and HDL has been postulated to be produced from the excessive surface lipids of these TG-rich lipoproteins. HL is the other lipase which acts on lipoproteins in the circulation and is reported to accelerate the conversion of HDL2 to HDLJ by the hydrolysis of TG of HDL2 [20,21]. Taskinen et al. [2] studied alcoholic patients and reported elevations of HDL-C and of post-heparin plasma (PHP) activity of LPL in these subjects. Since these elevations had declined to normal levels after alcohol withdrawal, they suggested that the increased LPL activity had raised the HDL-C levels. CETP is a recently purified hydrophobic protein [22-241, which transfers CE from HDL to VLDL and chylomicron, and also transfers TG in the opposite direction [25] (CE-TG exchange). The compositional change of HDLz by the action of CETP, i.e. from rich in CE to rich in TG, helps to increase substrate susceptibility to HL. Recently, Savolainen et al. [6] and Hirano et al. [7] reported the effect of alcohol on CETP activity. They reported a low CETP activity and a high HDL-C level in chronic alcohol drinkers and also confirmed an increase in CETP activity with a concomitant decrease in HDL-C in these subjects after alcohol withdrawal. LCAT is the enzyme which esterifies unesterified cholesterol mainly on the surface of HDL and works for the HDL conversions, i.e. from discoidal HDL to HDL, and from HDL3 to HDL,. Some recent studies have examined the effect of alcohol intake on LCAT activity [26, 271 or on endogenous cholesterol esterification rate [28,29]. Thus, effects of alcohol consumption on LPL, HL, CETP and LCAT levels have each been separately documented as possible mechanisms by which alcohol intake raises HDL-C levels. However, LPL, HL, CETP and LCAT levels may possibly be interrelated. Therefore, the purpose of the present study is to determine which one or what combination of these factors is responsible

for the rise in HDL-C levels following alcohol ingestion. 2. Methods and materials 2.1. Subjects and protocol Healthy normolipidemic Japanese men (n = 25), determined by physical and blood examinations to be free of anemia, diabetes mellitus and liver, kidney and thyroid diseases, served as subjects. Twelve subjects were selected at random to serve as an alcohol ingestion group while the remaining 13 served as controls. The alcohol consuming habits of the subjects was determined after they had been assigned to study groups. Prior to the study, 2 of the individuals in the alcohol ingestion group regularly consumed l-2 alcoholic drinks per day; the remaining 10 in the alcohol ingestion group and all those in the control group drank irregularly, consuming 2-3 alcoholic drinks per week. Members of the alcohol ingestion group abstained from alcohol for 3 weeks before and after a 4 week alcohol load period, whereas the members of the control group abstained from Table 1 Characteristics of the alcohol consumption group and the control group at the end of week 3. Alcohol loading was started at the beginning of week 4.

Age BMI (kg/m2) Number of smokers Total caloric amount (kcal) TC (mg/dl) TG (mg/dl) LDL-C (mg/dl) HDL-C (mg/dl) Apo E isoform 313 314 and 414 312

Alcohol consumption group (n = 12)

Control group (n = 13)

31.4 f 6.6 24.2 f 3.4 1 1960 i 310

31.2 zt 4.3 24.4 + 3.5 2 2060 zt 300

189 f 89 zt 125 f 46.5 f

182 zt 40 82 f 22 123 zt 41 42.6 zt 4.8

8 2 2

25 32 28 13.4

10 2 1

Abbreviations: BMI, body mass index; TC, total cholesterol; TG, triglyceride; LDL, low-density lipoprotein; HDL, highdensity lipoprotein; LPL, lipoprotein lipase; HL, hepatic lipase; CETP, cholesteryl ester transfer protein; LCAT, lecithin:cholesterol acyltransferase.

M. Nishiwaki

et al. /Atherosclerosis

alcohol during the entire 10 weeks. The 4 week alcohol load period (from the beginning of week 4 through the end of week 7) consisted of a 0.5 g/kg bw (30-49 g) alcohol (brandy) intake between 6 and 9 pm, after dinner. At the outset of the experiment, the two groups did not differ with respect to age, body mass index (kg/m*) and plasma lipids (Table 1). Smokers each smoked 10 to 20 cigarettes daily throughout the study. There was no difference between the alcohol and the control groups in the distribution of apolipoprotein (apo) E isoforms as determined by the method of Zannis and Breslow [30]. Informed consent was obtained from each subject at the outset of the study. Blood samples were obtained weekly from weeks 2 through 10. Fasting blood samples were drawn into EDTA-Na2-containing vacuum tubes at 9 am and separated by low speed centrifugation. Plasma samples were immediately subjected to lipoprotein analysis and lipid assay or stored at -35°C for the other assays. Post-heparin plasma was also collected in EDTA-Na,-containing vacuum tubes 15 min after heparin injection (50 U/kg bw) at the end of week 3 and 7 after drawing fasting blood samples; the post-heparin plasma was stored at -70°C for the determination of LPL activity and mass. Every night between 5 and 7 pm, before blood sampling, all subjects ate the same dish (sushi) to minimize the effects of diet. Diet and physical activity of each subject was monitored by a daily interview with a nutritionist. In the alcohol group, the average total caloric intake was 1963 kcallday during the 3 weeks (weeks l-3) before alcohol loading and 2026 kcal/day during the 4 weeks (weeks 4-7) of alcohol loading, while in the control group the average total caloric intake was 2010 kcallday during weeks 1-3 and 2056 kcallday during weeks 4-7. Caloric proportions of the diet during weeks l-3 were 18.1% of the total calories as protein, 26.9% as fat and 55.0% as carbohydrate in the alcohol-consuming group and 17.7% as protein, 24.3% as fat and 58.0% as carbohydrate in the control. There was no significant change in average daily caloric intake or caloric proportion of the diet between weeks l-3 and 4-7 in either group. Transaminases of all subjects stayed within normal limits throughout the study. Results of a 75 g oral glu-

I1 I (1994)

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case tolerance test, administered to all subjects during weeks 3 and 7, revealed no changes stemming from the experimental protocol. Serum ethanol concentrations in the alcohol-consuming group were measured with a gas chromatograph at the end of weeks 3 and 7; none was detected (less than 0.01 mgdl) in any member of the group. 2.2. Analysis of HDL subfractions and lipid assay HDLz and HDLs were separated by sequential ultracentrifugation at 40 000 rev./min in an Hitachi 65 Ti rotor using an Hitachi SCH-55. The density fractions at 1.063- 1.125 and 1.125- 1.21 g/ml, as adjusted by KBr, were collected as HDL2 and HDL3 fractions. Total cholesterol (TC) and TG were assayed enzymatically. HDL-C was measured by the precipitation method using phosphotungsten: dextran-Ca2+ (Daiichi Pure Chemical Co., Tokyo). Apo A-I and apo A-II were measured by immunoturbidimetry. 2.3. Assays of LPL and HL activities and LPL mass LPL and HL activities were measured by the method of Nozaki et al. [3 11. For the preparation of the substrates of these lipases, 166 mg of triolein was mixed with 3.57 ml of gum arabic solution containing 150 mg/ml gum arabic in 0.2 mol/ml Tris-HCl buffer (pH 8.2 for LPL, pH 8.8 for HL), and was sonicated by eight successive 30 s sonications at room temperature using a Branson Sonifier 450 set at output level 6; 150 ~1 (containing 7.5 pmol of triolein) of this solution was used per sample. For LPL assays, the substrate solution was adjusted to contain 7.5 pmol triolein, 22.5 mg of gum arabic, 25 mg of bovine serum albumin (BSA), 140 ~1of pooled human activator serum (10 x concentration), 50 pmol NaCl, and 100 pmol Tris-HCl (pH 8.2) in a total volume of 500 ~1, and was preincubated at 28°C for 60 min. A 100 ~1 volume of post-heparin plasma was also pre-incubated with 100 ~1 of sodium dodecyl sulfate (SDS) solution (100 mM SDS/200 mM Tris, pH 8.2) for 60 min at 26°C. The reaction was initiated by the addition of 10 ~1 of this mixture to the substrate solution and the incubation was carried out at 28°C for 60 min. For HL assays, the substrate solution was adjusted to contain 7.5 Fmol triolein, 22.5 mg of gum

102

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arabic, 25 mg of BSA, 375 pmol of NaCl and 100 pm01 of Tris-HCl (pH 8.8) in a total volume of 500 ~1. The reaction was initiated by the addition of 5 ~1 of post-heparin plasma to the substrate solution and the incubation was carried out at 28°C for 60 min. Both LPL and HL reactions were stopped by the addition of 2.5 ml of isopropanol:nheptane:sulfuric acid (2.5 molfl) (40: 10:2, v/v/v). The mixture was shaken for 10 min in a mechanical shaker. After the addition of 1.5 ml of nheptane and 1 ml of water, the mixture was further shaken for 10 min and then left for 40 min. 1 ml of the upper phase was collected and was evaporated under a stream of nitrogen. The residue was dissolved in 100 ~1 of Triton X-100 solution (50 ml/l). The emulsified oleic acid was determined enzymatically using a NEFA Kit-K (Nihon Shoji, Osaka, Japan). LPL mass was assayed by sandwich enzymelinked immunosorbant assay (ELISA), as described in Kobayashi et al. [32]. In this method, a monoclonal antibody against bovine milk LPL raised in the mouse and a polyclonal antibody against bovine milk LPL raised in chickens are used. A 50 ~1 volume of post-heparin plasma stored at -70°C was used for this assay. 2.4. Assay of LCAT activity Apolipoprotein A-I (apo A-I) was purified by the method of Brewer et al. [33]. Briefly, the 1.063-1.21 g/ml fraction separated by ultracentrifugation was delipidated by chloroforrnmethano1 (2:1, v/v) and was subjected to a Sephacryl S-200 (Pharmacia, Tokyo) column to separate apolipoprotein A-I. SDS gel electrophoresis of the collected apo A-I fraction stained by Coomassie Brilliant Blue R-250 showed no additional band except for apo A-I. However, this fraction contained LCAT activity, so it was heated at 56°C for 30 min in order to inactivate the activity. LCAT activity was measured by a modification of the method of Chen and Albers [34]. Egg yolk lecithin (39 mg), 3.7 mg unlabeled cholesterol and 400 ~1 of [1,2-3H]-cholesterol (2.00 TBq/mmol, New England Nuclear, Boston, MA) were mixed and evaporated under a nitrogen stream. Purified apo A-I (1.4 mg) and 600 mg of sodium cholate were dissolved in 3.5 ml of assay buffer (0.15 M

11 I (1994) 99-109

NaCl/lO mM Tris/l mM EDTA, pH 7.4) and were added to the dried lipids. After incubation at 24°C for 20 ruin with shaking, the mixture was vigorously dialyzed against assay buffer. The mixture was then adjusted to 4 ml with assay buffer and this liposome substrate was used within 2 days. 100 ~1 of liposome, 125 ~1 of 2% human serum albumin and 235 ~1 of assay buffer were mixed and incubated at 37°C for 15 min, after which 25 ~1 of mercaptoethanol and 15 ~1 of plasma were added to the substrates to initiate the reaction. After incubation at 37°C for 15 h, the mixtures were moved onto ice water and 2 ml of pre-chilled methanol were immediately added to stop the reaction. After 2 ml of chloroform and 2 ml of distilled water were added, the mixtures were centrifuged at 2000 rev./min for 20 min at 4°C and then the infranatants were collected and dried under a nitrogen stream. Dried lipid samples were resolved in 100 ~1 of hexane and applied to thin layer chromatographs using petroleum ether:ethyl acetate (85:15, v/v) as a solvent system. Each part corresponding to FC and CE were collected by scraping and the radioactivity was counted in a liquid scintillation counter (Aloka 3500). LCAT activity was expressed as a fractional activity, i.e., a/(a+b) x 100%/h, where a and b are the radioactivities of CE and FC, respectively. LCAT activity was measured in duplicate. 2.5. Assay of CETP activity CETP activity was assayed by modification of the method of Albers et al. [24]. After incubation of [‘4C]-cholesterol (2.0 GBq/mmol, New England Nuclear) and the d > 1.125 g/ml fraction (buffer A: 0.15 M NaCl/lO mM Tris/l mM EDTA/0.03% NNa3, pH 7.4) at 37°C for 24 h, the d > 1.21 g/ml fraction was collected by sequential ultracentrifugation and dialyzed against buffer A. The TC of [14C]-cholesteryl ester-labeled HDL ([‘4C]-CE-HDL) was adjusted to 100 mg/dl. LDL solution was prepared by taking the 1.006- 1.063 g/ml fraction out of the elution from a dextran cellulose column used for LDL apheresis to a homozygote of familial hypercholesterolemia (FH). This LDL fraction was dialyzed against buffer A and adjusted TC to 750 mg/dl. A 40 ~1 plasma sample, 200 ~1 of [i4C]-CE-HDL and 200 ~1 of

M. Nishiwaki et al./

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Atherosclerosis III (1994) 99-109 140

LDL were incubated at 37°C for 10 h. The reaction was stopped by moving the mixture onto ice water and 400 ~1 of chilled plasma was added. After heparin/Mn2+ precipitation, 600 ~1 of the supernatant was collected for radioactivity counting. CETP activity was calculated as (b-s)/b x lOO%/lOh/40 ~1 plasma, where b is the radioactivity of a blank buffer using 0.1% BSA/buffer A, and s is that of the sample. All assays were carried out in duplicate. 2.6. Statistical analysis Analysis of variance (ANOVA) for repeated measures was used to test the significance of the changes in TG, TC, HDL-C, apo A-I and apo A-II levels. The Friedman test was used to analyze the significance of changes in the apo A-I/A-II ratio. From weeks 3 through 10, these variables were measured and analyzed at the end of each week. In case statistical significance (P < 0.05) was found by ANOVA, paired Student’s t-test was further carried out to test the significance of the differences in the mean values between each point and the point of week 3. For the other parameters, significance of differences within and between groups was analyzed by paired or non-paired Student’s ttest. Correlations were assayed by Pearson’s product-moment correlation coefficient. Differences were considered significant if P I 0.05. 3. Results 3.1. Changes in lipid levels Changes in TG, TC, HDL-C, apo A-I and apo A-II levels were analyzed from weeks 3 through 10. Changes in each of the variables were significant (P < 0.01, ANOVA for repeated measures) in the alcohol consumption group, but no significant changes were found in the control group, nor was the change in apo A-I/A-II ratios significant in either group. In the alcohol consumption group, mean plasma TG levels were significantly higher (P < 0.01, t-test) at weeks 6 and 7 than they had been at week 3 (Fig. lA), whereas mean plasma TC levels did not change significantly throughout the study (Fig. 1B). Mean HDL-C levels were significantly higher (P < 0.01) at weeks 6, 7, and 8 than they had been at week 3 (Fig. 1C). Signifi-

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Fig. 1. Changes in triglyceride (A), total cholesterol (B) and high-density lipoprotein cholesterol (C) in the alcohol consumption group ( - 0 - ) and the control group (-A-). Alcohol (0.5 g/kg bw/day) was loaded to the alcohol consumption group from the beginning of week 4 through the end of week 7. *P c 0.05, **P < 0.01, ***P < 0.005 as compared to the measures obtained in that group at the end of week 3.

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Nishiwaki

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nor HDL,-C levels changed significantly over the same period.

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Fig. 2. Changes in apolipoprotein A-I (A) and A-II (B) levels in the alcohol consumption group ( - 0 - ) and the control group (-A-). Alcohol (0.5 g/kg bw/day) was loaded to the alcohol consumption group from the beginning of week 4 through the end of week 7. *P < 0.05, **P c 0.005 as compared to the measures obtained in that group at the end of week 3.

cant increases were also observed in mean apo A-I and A-II levels (Fig. 2A and B) at weeks 56 and 7. 3.2. Changes in HDL subfractions In the alcohol consuming group, mean HDLz-C and HDLJ-C levels measured 22.4 and 26.2 mg/dl, respectively, at the outset of alcohol load (week 3) and 19.2 and 31.6 mg/dl, respectively, at week 7 (after 4 weeks of alcohol loading) (Table 2). Although the HDLz-C level was unchanged, the difference in HDLs-C level was significant (P < 0.01). In the control group, neither mean HDL,-C

3.3. Effects on LPL, HL, CETP and LCAT In the alcohol consuming group, increased PHP LPL activities were observed in 11 out of 12 subjects after 4 weeks of alcohol loading; mean PHP LPL activity and LPL mass measures were significantly higher (P c 0.01) at week 7 than they were at week 3, but there was no significant difference between the specific activity of LPL at week 3 and at week 7 (Table 3). Equivalent measures in the control group were unchanged from weeks 3 to 7. Mean PHP HL, CETP and LCAT activities were not significantly changed in either the alcohol or in the control group from weeks 3 to 7. 3.4. Correlations To assess the association of HDL-C with age, BMI, TC, TG and LPL, HL, LCAT and CETP activities and LPL mass in the state of abstinence, simple correlations between HDL-C and these various factors at the end of week 3 were analyzed in all subjects (n = 25). HDL-C was significantly correlated with BMI, TG, and CETP activity (negatively) but not with age, TC or LPL, HL, or LCAT activity (Table 4). In the alcohol consumption group (n = 12), there was no correlation between changes of HDL-C during the alcohol loading period (weeks 4 through 7) and those of TG, TC, and LPL, HL, LCAT and CETP activities and LPL mass. 4. Discussion In the present study, a moderate amount of alcohol (0.5 g/kg bw/day) was ingested by 12 healthy normolipidernic Japanese men for 4 weeks; a control group (n = 13) abstained from alcohol during the 4-week alcohol loading period. In the alcohol consuming group, mean plasma TG and HDL-C levels increased significantly by the 3rd week of alcohol loading and returned to base levels (the level in the week prior to alcohol loading) a week or two after the withdrawal of alcohol, but mean plasma TC levels were not affected by alcohol loading (Fig. 1). The increase in plasma HDLC level observed in the present study was in accor-

M. Nishiwaki et al. /Atherosclerosis

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I I I (1994) 99- 109

Table 2 Effects of alcohol intake on HDL, and HDL,-cholesterol Alcohol consumption group (n = 12)

HDL,C

group

(n = 13)

Week 3

Week I

Week 3

Week 7

22.4 f II.3

19.2 zt 10.5

17.8 f 8.0

17.2 f 9.9

*

** I

HDL,-C

Control

26.2 zt 3.8

I

I

I

31.6 f 6.6

26.2 f 3.7

26.5 f 4.3

Alcohol (0.5 g/kg bw/day) was loaded to the alcohol consumption group from the beginning of week 4 through the end of week 7. Statistical significance were analyzed by paired cholesterol; HDL$, high-density lipoprotein-3

dance

or non-paired Student’s t-test. Abbreviations: cholesterol. *P < 0.05. **P < 0.01

HDL,-C,

high-density

lipoprotein-2

reported a rise in LPL activity in the adipose tissues. Hence, the increased LPL activity caused by a rise in LPL mass seems to play an important role in the elevation of HDL-C induced by alcohol intake. PHP LPL activity or mass did not contribute to the difference in HDL-C levels of the subjects during abstinence (Table 4). In the state of abstinence, HDL-C level may be affected by other factors such as CETP and apo E isoforms rather than by LPL. Alternatively, the range of HDL-C, PHP LPL activity and PHP LPL mass of our subjects might be small. HDL-C and CETP levels were significantly (negatively) correlated during the 3 week absti-

with

observations in previous studies [1,13-15,19,35-381; however, the increases in plasma TG that we observed have not always been described in the earlier studies.

In the current study, PHP LPL activity and PHP LPL mass increased significantly with a rise in HDL-C in the group consuming alcohol (Table 3). These findings were consistent with results reported previously. Taskinen et al. [2] and Ekman et al. [9] reported an elevation in both HDL-C and PHP LPL activity in chronic alcoholics. In nonalcoholic volunteers taking a moderate amount of alcohol, Schneider et al. [l l] reported an increase in PHP LPL activity and Belfrage et al. [8]

Table 3 Measures of lipoprotein lipase (LPL) and hepatic lipase (HL) activities, LPL mass, and specific activity of LPL, and cholesteryl transfer protein (CETP), and lecithin:cholesterol acyltransferase (LCAT) activities, at weeks 3 and 7 in the alcohol consumption the control groups Measure

Alcohol consumption (n = 12) Week 3

LPL activity (rmol/h/ml) LPL mass (@ml) Specific activity of LPL (nmolIh/ng) HL activity (hmol/h - ml) CETP activity (?UlO h/40 pl plasma) LCAT activity (nmol/h/ml)

5.73 169 35 26.0 21.1 31.1

f + f f f *

0.79 34 8 11.3 4.2 7.9

group

Control

group (n = 13)

Week 7

Week 3

Week 7

6.51 196 34 26.9 20.1 31.5

5.89 173 35 22.6 22.5 29.4

5.79 178 33 24.0 22.1 28.8

jz 1.24* + 34* +z 8 f 10.6 f 4.3 f 3.2

ester and

+z 0.66 f 33 f I f 1.1 f 3.4 +z 7.3

Alcohol (0.5 g/kg bw/day) was loaded to the alcohol consumption group from the beginning *P < 0.01 as compared to the measures obtained in that group at the end of week 3.

zt zt f zt

1.24 36 8 10.5 ?? 4.1 * 5.4

of week 4 through

the end of week 7.

hf. Nishiwaki et al. /Atherosclerosis III (1994) 99-109

106

Table 4 Simple correlations between HDL-C and various factors Factors (n = 25)

r

Age

ns -0.53** ns -0.58*** ns ns ns ns -0.452

BMI TC TG LPL activity LPL mass HL activity LCAT activity CETP activity

Abbreviations: BMI, body mass index; TC, total cholesterol; TG, triglyceride; LPL, lipoprotein lipase; HL, hepatic lipase; LCAT, lecithin:cholesterol acyltransferase; CETP, cholesteryl ester transfer protein. Simple correlation analyses were carried out between the values obtained at the end of week 3. *P < 005 *P < 0.01, ***P < 0.005, ns, not significant. . , ??

nence period at the outset of the present study (Table 4), but there was no detectable effect of alcohol on CETP activity during the alcohol ingestion phase that followed. Recently, Savolainen et al. [6] reported a low CETP activity and a high HDL-C level in alcoholics whose daily alcohol intake averaged 177 g; they also confirmed an increase in CETP activity with a concomitant decrease in HDL-C in these subjects after alcohol withdrawal. In a later study, Hannuksela et al. [39] recognized a decrease in CETP activity and also a fall in its mass after cessation of alcohol in subjects whose daily alcohol intake averaged 154 g. In an alcohol cessation study of subjects who had each consumed 80-150 g of alcohol daily for 22-50 years, Hirano et al. [7] reported that an increased HDL-C and a decreased CETP activity level returned to normal levels after alcohol withdrawal. These findings, which differ from the results in the present study, might stem from differences in the dose, duration and mode of alcohol intake, diet, the susceptibility of the subjects to ingested alcohol and the methods of CETP assay. In fact, sub[6,7,39] consumed jects in these studies considerably greater amounts of alcohol per day, and for a much longer period, than did subjects in the alcohol consuming group of the present study. Thus, it may be that the response of CETP is dose-

dependent and that CETP is not induced by the small dose of alcohol used in our study. With respect to diet, a large amount of ingested alcohol often substitutes for fat and cholesterol in the caloric proportion of the diet in Western people [40]. Quinet et al. [41] reported that high fat intake increased both CETP activity and its mass in rabbits. Thus, in heavy drinkers who tend to decrease fat intake, CETP activity may decrease as an effect of diet and return to normal by the resumption of an ordinary diet after alcohol cessation. In connection with liver damage, since the liver is the main source of CETP, morphological changes such as fatty liver and cirrhosis are likely to affect CETP activity. These changes are considered to alter the effect of alcohol on both CETP and lipoprotein metabolism. In fact, although VBlimHki et al. [35] also observed in an alcohol withdrawal study of chronic alcoholic women that elevations in HDLC and HDL*-C and a decrease in CETP activity returned to control levels after alcohol cessation, they reported that there was no correlation between the changes in HDL,-C and CETP activity whereas CETP activity inversely correlated with alanine aminotransferase. In a related study [42], a remarkable decrease in HDL-C was observed after cessation of alcohol intake even in a patient homozygous for CETP deficiency who had no liver injury. Thus, CETP is not considered to play a key role in alcohol-induced HDL-C elevation in subjects drinking moderately. Alcohol intake did not affect LCAT activity in the present study (Table 3). Therefore, LCAT was not considered to be a contributor to the HDL-C elevation that followed alcohol intake. This result is consistent with that reported by Albers et al. [26], who found no difference between drinkers and non-drinkers in LCAT values, and also with that reported by Haffner et al. [ 171, who found no correlation between LCAT mass and alcohol consumption. Identification of the subfraction of HDL increased by alcohol intake would indirectly suggest the mechanism of alcohol-induced HDL-C elevation. Contaldo et al. [14] reported an increase in HDL,-C, Haskell et al. [3] reported a rise in HDLs-C, and Taskinen et al. [2] and Ekman et al. [9] described an elevation in both HDL+ and

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HDL,-C. Thus, this issue is cloudy. In the current study, the HDL& level increased significantly while the HDL& level remained unchanged (Table 2), thereby supporting the results of Haskell et al. [3]. It seems that dose and duration of alcohol, diet, and perhaps other factors as well can possibly underlie the differences. Taskinen et al. [43] indicated that large amounts of alcohol intake cause a rise in HDL*-C levels whereas moderate amounts of alcohol intake result in an increase of HDL3-C levels. The results of the present study conform to the latter case. However, since LPL is believed to affect the synthesis of HDLZ rather than HDL3, the increased LPL activity and the HDL,-C elevation observed in our study is not SO easily understood unless an increase in the conversion of HDL2 to HDLs [20,21] can be invoked. Since the rise in plasma TG in the present study indicates an alcohol-induced elevation of VLDL, the CE-TG exchange between HDL and VLDL is considered to have increased even though the CETP activity remained unchanged. Patsch et al. [44] reported that the magnitude of TG response to a fat meal correlates positively with fasting TG concentrations and also correlates negatively with HDL* levels. Thus, CE-TG exchange is considered to be emphasized at a postprandial state with the level of fasting TG concentrations. Further, the increased turnover of VLDL in alcoholic men reported in a kinetic study by Sane et al. [45] may also support an increase in CE-TG exchange. CE-TG exchange is considered to increase susceptibility of HDL, to HL and consequently to accelerate the conversion from HDL2 to HDL3. Thus, we propose that although CETP and HL levels were unchanged in the present study, an accelerated conversion from HDL? to HDL3 might possibly cause the increase of HDL3-C. Mean apo A-I and A-II levels were increased significantly by the alcohol load (Fig. 2A and 2B) in the present study. These findings are consistent with previous studies of alcohol withdrawal [46], alcohol loading studies [13,47,48] and crosssectional surveys [ 16,171. Neither the A-I/A-II nor A-I/HDL-C ratios were significantly changed by alcohol in the present study. Thus, an increased LPL activity or an increased CE-TG exchange did not change the proportion of cholesterol, apo A-I

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and apo A-II in HDL. Recently, it has been noted that HDL is separable into at least two apo A-Icontaining particles, i.e., Lp A-I which contains apo A-I without apo A-II, and Lp A-I:A-II which contains both apo A-I and apo A-II [49], and that only the former is believed to play an important role in reverse cholesterol transport [50]. Vllimlki et al. [48] reported that both Lp A-I and Lp A-I:AII levels were increased in healthy male subjects who had consumed 60 g/day of alcohol for 3 weeks. The lack of change in A-I/A-II ratios in our study suggests that there is no solitary increase of Lp A-I or of Lp A-I:A-II, and thus may confirm their results. The mechanism by which Lp A-I and Lp A-I:A-II are regulated in alcohol intake is unknown, and the relations between these particles and the factors affecting lipoprotein metabolism, such as LPL, HL, CETP and LCAT, are of interest. It may be considered that a direct action of the liver is the cause of the increases of apo A-I and A-II. Malmendier and Delcroix [51] reported an increased rate of apo A-I synthesis in a kinetic study of healthy volunteers who took 60-70 g alcohol daily. Amarasuriya et al. [52] also reported an ethanol-induced increased secretion of apo A-I from Hep-G2 cells. In contrast, Hojnacki et al. [53] reported a significant decrease in fractional catabolic rates of apo A-I in monkeys taking as much as 24% of total calories as ethanol for 18 months. Thus, the effect of alcohol on the synthesis of apo A-I remains unclear. An increased synthesis or a decreased catabolism of apo A-I, as effects of alcohol on the liver and other organs or as secondary effects on apo A-I kinetics by means of changing LPL, HL, CETP and LCAT activities, are considered to be possible mechanisms underlying an increase in apo A-I levels during alcohol intake; these possibilities need further study. We conclude that in considering LPL, HL, CETP and LCAT as factors in the alcohol-induced rise in HDL-C, LPL contributed the most by increases of both activity and mass. Acknowledgments

The technical advice of Dr. Yasushi Saito, Dr. Kohji Shirai, Dr. Junji Kobayashi, and Jun Tashiro to measure LPL mass and of Dr. Shuichi

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Nozaki and Dr. Shinji Kihara to measure LPL activity are greatly appreciated. We are also grateful for the helpful comments provided by two anonymous reviewers. References 111Johannson, B.G. and Medhus, A., Increase in plasma lipoproteins in chronic alcoholics after acute abuse, Acta Med. Stand., 195 (1974) 273. VI Taskinen, M.-R., Vllimaki, M., Nikkilii, E.A., Kuusi, T., Ehnholm, C. and Ylikahri, R., High-density lipoprotein subfractions and postheparin plasma lipases in alcoholic men before and after ethanol withdrawal, Metabolism, 31 (1982) 1168. 131 Haskell, W.L., Camargo, C., Williams, P.T., Vranizan, K.M., Krauss, R.M., Lindgren, F.T. and Wood, P.D., The effect of cessation and resumption of moderate alcohol intake on serum high-density lipoprotein subfractions: a controlled study, N. Engl. J. Med., 310 (1984) 808. I41 Viihmiiki, M., Nikkill, E.A., Taskinen, M.-R. and Ylikahri, R., Rapid decrease in high density lipoprotein subfractions and postheparin plasma hpase activities after cessation of chronic alcohol intake, Atherosclerosis, 59 (1986) 147. 151 Harley-Hartung, G.H., Foreyt, J.P., Reeves, R.S., Krock, L.P., Patsch, W., Patsch, J.R. and Gotto, A.M.J., Effects of alcohol dose on plasma lipoprotein subfractions and lipolytic enzyme activity in active and inactive men, Metabolism, 39 (1990) 81. 161 Savolainen, M.J., Hannuksela, M., Sepplnen, S., Kervinen, K. and Kesaniemi, Y.A., Increased high-density lipoprotein cholesterol concentration in alcoholics is related to low cholesteryl ester transfer protein activity, Eur. J. Chn. Invest., 20 (1990) 593. 171 Hirano, K., Matsuzawa, Y., Sakai, N. et al., Polydisperse low-density lipoproteins in hyperalphalipoproteinemic chronic alcohol drinkers in association with marked reduction of cholesteryl ester transfer protein activity, Metabolism, 41 (1992) 1313. PI Belfrage, P., Berg, B., Hagerstrand, I., Nilsson-Ehle, P., Tomqvist, H. and Wiebe, T., Alterations of lipid metabolism in healthy volunteers during long-term ethanol intake, Eur. J. Clin. Invest., 7 (1977) 127. 191 Ekman, R., Fex, G., Johansson, B.G., Nilsson-Ehle, P. and Wadstein, J., Changes in plasma high density lipoproteins and lipolytic enzymes after long-term heavy ethanol consumption, Stand. J. Clin. Lab. Invest., 41 (1981) 709. HOI Hansson, P. and Nilsson-Ehle, P., Acute effects of ethanol and its metabolites on plasma lipids and lipoprotein lipase activity, Ann. Nutr. Metab., 27 (1983) 328. 1111 Schneider, J., Liesenfeld, A., Mordasini, R. et al., Lipoprotein fractions, lipoprotein lipase and hepatic triglyceride lipase during short-term and long-term uptake of

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