The Effect of Moderate M. Vslimtiki,

K. Laitinen,

Alcohol Intake on Serum Apolipoprotein Lipoproteins and Lipoprotein (a)

R. Ylikahrit,

C. Ehnholm,

M. Jauhiainen,

J.M. Bard, J.C. Fruchart,

A-I-Containing and M.-R. Taskinen

Two main types of lipoprotein particles are identified within high-density lipoprotein (HDL): those containing both apolipoprotein (apo) A-l and apo A-II (Lp A-I:A-II) and those containing only apo A-l (Lp A-l). To study the effects of prolonged moderate alcohol intake on apo A-l-containing lipoproteins in serum, 50 g/d of ethanol was administered to 10 healthy male volunteers (age, 27 to 45 years) during 3 weeks. The drinking period was preceded and followed by an abstinence period of 3 weeks. The HDL, cholesterol level increased by 17% (P < .Ol) and decreased by 22% (P < .OOl) on and off alcohol, respectively. The HDL, cholesterol increased by 17% (P = NS) during ethanol intake and decreased by 14% during the following abstention (P < .Ol). The serum concentration of apo A-l increased by 17% (P < .OOl) during drinking and came back to the starting level after 2 weeks of abstention. Ethanol intake caused an increase in the serum levels of both Lp A-l and Lp A-I:A-II, the former explaining one third of the total increase of apo A-l. The Lp (a) concentration decreased by 33% (P < .05) during the first week of ethanol intake, but increased back to the starting level until the end of drinking. These data suggest that the increment of the antiatherogenic Lp A-l may be one beneficial effect provided by ethanol with respect to coronary heart disease. Copyright 0 1991 by W.B. Saunders Company

M

ODERATE ALCOHOL intake reduces the degree of coronary atherosclerosis and the risk of myocardial infarction.’ This association has been considered to be due to increase of plasma high-density lipoprotein (HDL) cholesterol concentration by alcoho1.?~4However, this concept is confronted by the fact that moderate alcohol intake mainly elevates plasma HDL,,5.6which has been considered less antiatherogenic than HDL,.‘.’ Consequently, why moderate alcohol intake protects against coronary heart disease is far from clear. It is well established that moderate alcohol intake increases the serum concentrations of apolipoprotein (apo) A-I and apo A-II.6.y I2 Interestingly, several studies have indicated that the power of high apo A-I and A-II to provide protection against coronary heart disease is stronger than that of HDL.13 The distribution of particles within HDL is heterogeneous not only with respect to hydrated density and particle size, but also in relation to apolipoprotein composition.‘4-‘7 It is well recognized that HDL contains at least two types of apo A-I-containing lipoprotein particles that might have different metabolic functions and clinical significance.” One species contains as main protein components both apo A-I and A-II (Lp A-I:A-II), while in the other (Lp A-I) apo A-II is absent.‘5.18-zoLp A-I particles reside within both HDLz and HDL, density range, although the majority is distributed within HDL, density.‘l Recently, data from patients with angiographically defined coronary artery disease suggested that Lp A-I might represent the antiathero-

From the Third and Second Departments of Medicine, Research Unit of Alcohol Diseases, University of Helsinki, Research Laboratories of Finnish State Alcohol Company (Alko Ltd), Helsinki, and Public Health Laboratory, Helsinki, Finland; and the Institat Pasteur, Lille, France. Suppotied by a grant from the Finnish Alcohol Research Foundation. fDeceased. Address reprint requests to M. Viilimiiki, MD, Third Department of Medicine, University Central Hospital of Helsinki, SF-00290 Helsinki, Finland. Copyright 0 1991 by W.B. Saunders Company 00260495/91/4011-0009$03.00/0

1168

genie fraction of HDL.” The antiatherogenic potential of Lp A-I particles may reside in their putative role in reverse cholesterol transport.*3.24 The apolipoprotein (a) is a glycoprotein that circulates in plasma attached to apo B of low-density lipoprotein (LDL) particles, forming lipoprotein (a).25 The serum level of the Lp (a) seems to be a strong independent risk factor for ischemic heart disease.‘6 In contrast to other apolipoproteins (B, A-I, A-II) the concentration of Lp (a) is not easily modulated by drugs or dietary changes.*’ So far there are no data available to determine if moderate ethanol intake affects the concentrations of Lp (a) in serum. The present investigation was undertaken to study the effects of moderate alcohol consumption on the concentrations of both apo A-I-containing lipoproteins and Lp (a) in serum. MATERIALS Subjects

AND METHODS

and Study Design

The study, approved by the Ethical Committee of the Third Department of Medicine, University of Helsinki, was performed on 10 healthy male volunteers aged 27 to 45 (mean. 36) years. Previously, the subjects had been irregular alcohol drinkers with a daily consumption of one to two drinks. No subject was on drug therapy at the time of the study. Three of the subjects were smokers and seven nonsmokers. The liver function of the subjects was normal. They were requested to maintain their regular dietary and exercise patterns during the trial. The purpose and design of the investigation were explained, and formal consent was obtained from all subjects. The subjects had a clinical examination before the trial, which started after 3 weeks of total abstention from alcohol. Thereafter, the subjects were served wine (two subjects), whisky (four subjects), or vodka (four subjects), according to preference, in doses equivalent to 60 g of absolute alcohol per day for 3 weeks. This period was followed by another abstention phase of 3 weeks. Occasional changes of the habitual drink for another alcoholic beverage with exactly the same amount of alcohol were allowed. The timing of the drinking was not scheduled, but in practice the subjects consumed alcohol mainly during evening hours. Alcohol was delivered to the subjects in amounts equivalent to 1 week’s need and the intake was controlled by diet diaries (covering 2 days) taken before and after the drinking period and at the end of the

Metabolism, Vol40, No 11 (November), 1991: pp 1168-1172

ALCOHOL AND ape A-I-CONTAINING

second abstention period. On the basis of the diaries, the mean intake of nonalcoholic calories of the subjects was 2,108 kcal/d throughout the study, with approximately 46% of the total energy as carbohydrate, 38% as fat, and 16% as protein. Every subject continued his usual daily job. Venous blood was obtained for analysis of serum lipids and lipoproteins in the morning after a lo-hour fast. The interval between the samplings varied from 2 days to 3 weeks, and a total of 12 samples was drawn from each subject. The liver function tests and body weight were recorded regularly during the entire experiment. The serum transaminases and y-ghrtamyl-transferase activities remained normal in all subjects. The body weight of the men did not change significantly (82.0 f 2.3 kg and 83.5 f 2.5 kg before and after drinking ethanol, respectively, mean f SEM). Separation of Lipoproteins The lipoproteins were isolated by sequential flotation= in a Beckman L70 ultracentrifuge (Beckman Instruments, Palo Alto, CA) using a type 50.3-Ti Beckman rotor. The very-low-density lipoprotein (VLDL) fraction was separated by ultracentrifugation at a density of 1.006 g/mL for 18 hours at 105,000 x g. The top layer was removed by tube slicing. The density of the bottom layer was adjusted to 1.019 g/mL with KBr (354 g/L) and NaCl (153 g/L) solutions. Intermediate-density lipoprotein (IDL) was isolated after centrifugation for 18 hours at 105,000 x g at the density of 1.019 g/mL from the top layer by tube slicing. The density of the bottom layer was adjusted to 1.063 g/mL with KBr and NaCl mixture. The LDL fraction was isolated by ultracentrifugation for 24 hours at 105,000 x g. The density of the LDL infranatant was increased to 1.125 g/mL with the same KBr-NaCI mixture. The HDL, fraction (density 1.063 to 1.125 g/mL) was isolated after spinning for 48 hours at 38,000 rpm. Finally, the density of the bottom layer was adjusted to 1.210 g/mL with the salt mixture, and the HDL, fraction (density 1.125 to 1.210 g/mL) was isolated after spinning for 48 hours at 105,000 x g. AI1 lipoprotein fractions were dialyzed overnight against 0.15 mol/L NaCl at 4°C. The recoveries

of cholesterol, triglyceride, and phospholipids in the five lipoprotein fractions averaged 89% t 3%, 87% f 3%, and 91% f 4%, respectively. The sums of triglyceride, cholesterol, phospholipids and protein concentrations in VLDL, IDL, LDL, HDI+ and HI& were taken to be equivalent to the mass concentration. Quanti$cation

1169

LIPOPROTEIN

of Apo A-I-Containing

Lipoprotein Particles

Lp A-I, lipoproteins containing apo A-I, but free of apo A-II, were quantified using a differential electroimmunoassay29 marketed by Sebia, Issy les Moulineaux, France. The concentration of Lp A-I:A-II, lipoproteins containing both apo A-I and apo A-II, was estimated by subtracting the concentration of Lp A-I from the concentration of total apo A-I in serum (see below). Lp (a) Measurement The concentration of Lp (a) was measured using the Pharmacia Apolipoprotein (a) RIA 100 assay system. This Lp (a) assay is a solid-phase two-site immunoradiometric assay using two monoclonal antibodies directed toward different epitopes on apo (a).‘” Analytical Methods The concentration of cholesterol (enzymatic calorimetric method, catalogue no. 187313, a kit of Boehringer Diagnostica, Mannheim BmgH, Germany), triglyceride” (Wahlefeld, 1974, catalogue no. 297771, a kit of Boehringer Diagnostica), phospholipids,” and protein” were measured in whole serum and all the lipoprotein fractions. Total apo A-I and A-II were measured by immunotur-

bidimetry using monospecific y-globulins (Boehringer). The interassay coefficients of variation (CV) of the apo A-I and apo A-II methods were 4.3% and 5.3%, respectively. Apo B in whole serum was determined by radial immunoditfusion using a conventional anti-apo B antiserum (Orion Diagnostica, Helsinki, Finland). The CV of this method was 4.7%. Statistical Analyses The data are expressed as means * SEM. ANOVA with repeated measures was first used for studying changes in each variable, first during the alcohol, and second during the following abstinence period. If a statistically significant difference (P < .05) was found, Student’s paired t test (normal distribution) or Wilcoxon matched-pairs test (nonnormal distribution) was used to study the significance of the differences in the same variable between different time points. The first value in the beginning of the alcohol period (O-value) or that of the following abstinence period (value of 3 weeks) was considered as a reference value in comparison to the highest (peaked data) or lowest (data with a nadir) value or values during the alcohol and abstention periods, respectively. If not otherwise expressed, the P values in the following text represent the significances of these paired comparisons. BMDP statistical programs in a VAX/VMS minicomputer were used in the analysis.” RESULTS

Changes in Major Lipoproteins YLDL, IDL, LDL, and HDL The serum levels of total triglyceride and total cholesterol did not change significantly during alcohol drinking or the following abstention (Table 1). Also, the concentrations of the total VLDL, IDL, and LDL remained unchanged (Fig 1). The concentration of total HDL cholesterol increased by 15% (P < .Ol) during drinking and decreased by 16% (P < .OOl) during the following abstention (Table 1). HDL Subfractions The HDL, cholesterol level increased by 17% and decreased by 14% during ethanol intake and withdrawal, respectively, but only the latter change was significant (P < .Ol) (Table 1). The respective changes of the HDL, cholesterol concentration were an increase of 17% (P < .Ol) and a decrease of 22% (P < .OOl) (Table 1). The HDL, mass tended to increase during. alcohol intake (P = .06, ANOVA). Again, a decrease of 18% induced by 1 week’s abstention reached statistical significance (P < .Ol) (Fig 1). lltt: HDL, mass concentration creased by 8% during ethanol Table 1. Concentrations HDL-Cholesterol

both increased and deconsumption and with-

(mg/dL) of Triglyceride, Cholesterol, and

in Whole Serum and of Cholesterol in HDL Subfractions Before Alcohol

After Alcohol

After Abstention

Total triglyceride

140 * 14

119 + 16

136 + 16

Total cholesterol

220 f 16

216 + 14

221 + 18

Total HOL cholesterol

53 * 3

61 * 3*

51 + 2t

HOL, cholesterol

30 f 3

35 z!?3

30 + 3”

HOL, cholesterol

23 + 1

272

21 + It

l*

NOTE. Results are expressed as mean 2 SEM.

lP i

.Ol, tP