Lifestyle determinants of HDL2- and HDL3-cholesterol levels in a hypercholesterolemic male population

1

Atherosclerosis, 87 (1991) 1-8 0 1991 Elsevier Scientific Publishers Ireland, Ltd. 0021-9150/91/$03.50 ADONIS 002191509100077V

ATHERO

04598

Lifestyle determinants of HDL,- and HDL,-cholesterol in a hypercholesterolemic male population Matti M’Anttbi ‘, Pekka Koskinen I, Vesa Manninen and Jussi K. Huttunen ’

levels

I, Leena Tenkanen

’

’First Department of Medicine,Helsinki University Central Hospital, and ’ National Public Health Institute, Helsinkr (Finland) (Received 29 May, 1990) (Revised, received 15 October, 1990) (Accepted 25 October, 1990)

Summary In this cross-sectional study we investigated the role of lifestyle and other factors in determining serum HDL,and HDL,-cholesterol levels among 82 dyslipidemic (total cholesterol minus HDL-cholesterol 2 5.2 mmol/l) middle-aged participants of the Helsinki Heart Study. Alcohol consumption correlated positively with both subfractions of HDL-cholesterol, while leisure time physical activity had a significant correlation with the HDL,-subfraction only. HDL levels were lower in smokers than in non-smokers but the differences were not statistically significant. Using the multiple linear regression model, alcohol consumption emerged as the only significant factor influencing both HDL cholesterol subfraction levels. Leisure time physical activity had an independent contribution to HDL,-level, but lifestyle variables other than alcohol consumption did not contribute significantly to HDL,-cholesterol level. The model incorporating alcohol consumption, physical activity, smoking and relative body weight explained 13.4% of the variation in HDL, and 17.5% in HDL,-cholesterol.

Key words:

HDL,-cholesterol;

HDL,-cholesterol;

Introduction The negative association between high density lipoprotein (HDL) cholesterol and coronary heart

Correspondence to: Matti Minttti, M.D., First Department of Medicine, Helsinki University Central Hospital, Haartmaninkatu 4, SF 00290 Helsinki, Finland. Tel.: 358-0624988; Fax: 358-O-624998.

Alcohol;

Physical

activity;

Smoking;

Relative

weight

disease has been demonstrated in several populations [l-3]. Controversy still exists over the relative importance of HDL-subfractions (HDL, and HDL,) as predictors of coronary heart disease [4-61. The Helsinki Heart Study demonstrated that elevation of HDL-cholesterol with gemfibrozil made an independent contribution to the reduction in coronary heart disease [7], while a subgroup analysis showed that the main effect of gemfibrozil was on the HDL,-subfraction [S].

2 The lifestyle variables of diet, alcohol consumption, physical activity and smoking influence high density lipoprotein cholesterol (HDL-, HDL,-, and HDL,-) levels [9-121. High dietary intakes of carbohydrate, especially. sucrose and starch, have been associated with low serum levels of HDLcholesterol [13,14]. Alcohol consumption and physical activity increase total HDL-cholesterol, while smoking has been associated with low HDL-cholesterol levels in several studies [15,16]. Intervention studies have further demonstrated that the anticipated changes in HDL-levels occur after manipulation of the corresponding lifestyle variable [17-211. Few studies have examined the joint impact of lifestyle variables on cholesterol levels in the HDL subclasses [11,22,23] and almost all data are. derived from healthy normolipidaemic volunteers. In the present study we examined the association between lifestyle factors and total HDL and HDL subfraction (HDL, and HDL,) cholesterol levels in a dyslipidemic middle-aged male population.

Methods This paper deals with 82 middle-aged men whose lipoprotein profile was analyzed using preparative ultracentrifugation at the beginning of the Helsinki Heart Study. The study design and primary results have been described in detail elsewhere [7,24,25]. In brief, subjects free of coronary heart disease or any other major illness and fulfilling the lipid acceptance criterion of nonHDL-cholesterol 2 5.2 mmol/l (= total cholesterol minus HDL-cholesterol) at two consecutive visits were selected to participate in this 5-year coronary primary prevention trial. The lipid acceptance criterion selected men from the upper end of serum total cholesterol distribution in the screened population (n = 18 965). On the other hand the HDL-cholesterol distribution of the Heart Study participants (n = 4081) was very close to the screened population

PI*

Sera of the first 90 participants fulfilling the acceptance criteria at one study clinic were collected for preparative ultracentrifugation. All determinations were made from fresh sera after an

overnight fast. Eight subjects were excluded from the analyses because of insufficient samples. High density lipoproteins were separated with sequential ultracentrifugation using a Beckman L70 ultracentrifuge with a Type 50 Ti Beckman rotor (Beckman Instruments Inc., Palo Alto, CA, U.S.A.). Chylomicrons were first removed at a density of 1.006 g/ml by centrifuging for 30 min at 20000 x g. VLDL was then separated by centrifugation for 18 h at the same density. The density of the bottom fraction was then increased to 1.063 g/ml with a solution containing NaCl(55 g/l) KBr (109 g/l) and EDTA (0.1 g/l) and the LDL fraction was isolated by spinning at 105 000 X g for 24 h. A sample of the bottom fraction was taken for the determination of total HDL, and thereafter the density of the fraction was increased to 1.125 g/ml with NaCl (153 g/l) and KBr (354 g/l). HDL, was isolated by ultracentrifugation at 105 000 X g for 48 h with a Type TFT 45.6 rotor (Kontron Ltd., Zurich, Switzerland). HDL, was finally isolated by centrifuging at 105 000 X g for 65 h after adjusting the density of the infranatant to 1.210 g/ml. Cholesterol and triglyceride concentrations in the lipoprotein fractions were determined with Boehringer Mannheim GmbH kits NOS. 187313 and 29771. The recoveries for total HDL-cholesterol were 94 * 6%. Lipid determinations by the preparative ultracentrifugation method gave 8% lower levels for HDL-cholesterol compared to routine determinations from the Helsinki Heart Study samples. Lifestyle factors were recorded using standard questionnaires. For alcohol consumption, the modified questionnaire of the Scandinavian Drinking Survey [26] was used. The reported quantities and the number of drinking occasions for spirits, wine and beer were converted to amounts of absolute alcohol and the annual consumption (centilitres per year) was used in the analyses as a continuous variable. Smoking habits were classified using the reported number of cigarettes smoked per day. For the analyses, the subjects were dichotomized to either non-smokers or smokers irrespective of the quantities. All exsmokers had stopped more than 3 months before the beginning of the study and were categorized as non-smokers in the analyses. Spare time and occupational physical activity

3 were recorded with Ccategory scales using the questionnaire adapted from the Gothenburg study [27]. Subjects reporting sedentary lifestyle during spare time (Cat I and II) were considered as physically inactive and those reporting regular exercise (Cat III and IV) as physically active in the analyses. The same categorization was used for self-reported occupational activities. Relative weights were estimated using body mass index (weight/height’). The r-test was used in the comparison of the lipid levels. To estimate the contribution of the selected variables on the lipid levels, multiple linear regressions were calculated using the method of least squares. Class variables were dichotomized for the analyses as described above. All regression coefficients are presented as standardized to allow easy comparison of the relative importances between various lifestyle factors. Due to skewness of the distribution, log values of triglycerides were used in the regression analyses. ReStlIt. The lifestyle characteristics and lipoprotein lipid levels of this study population are given in Table 1. Alcohol consumption was positively correlated with both HDL,- and HDL,-cholesterol levels (Fig. 1). The difference between the lowest ( < 100 cl/yr) and highest ( > 430 cl/yr) tertiles of alcohol consumption was 11.3% for HDL, (P = 0.02) and 19.5% for HDL, (P = 0.04). The univariate regression coefficients were 0.262 (P = 0.02) for HDL, and 0.242 (P = 0.03) for HDL, (Table 2). Spare time physical activity did not influence HDL,-cholesterol. The levels among sedentary and physically active were 0.44 f 0.17 and 0.45 f 0.19 mmol/l respectively, while the corresponding values for HDL, were 0.63 + 0.10 and 0.70 f 0.15 mmol/l (P = 0.008). The difference in total HDL-cholesterol between the sedentary and the active, 1.07 f 0.23 and 1.14 f 0.30, respectively, did not quite reach the level of significance (P = 0.1). The joint effect of spare time physical activity and triglycerides on HDL-cholesterol and its subclasses is shown in Fig. 2. The difference in total and HDL,-cholesterol between active and inactive subjects was slightly, but not significantly,

TABLE

1

LIPID LEVELS AND DISTRIBUTION OF LIFESTYLE VARIABLES IN THE STUDY POPULATION (n = 82) Variable

Mean (SD) or number

HDL-chol. (mmol/l) HDL,-chol. (mmol/l) HDLs-chol. (mmol/l) LDL-chol. (mmol/l) VLDL-chol. (mmol/l) Triglycerides (mmol/l) Body mass (weight/height*) Systolic BP (mm Hg) Diastolic BP (mm Hg) Alcohol consumption (cl/year) Smoking Smokers Ex-smokers Non-smokers Numbers in physical activity categories: I II III IV

1.09 (0.25) 0.44 (0.18) 0.64 (0.12) 4.55 (0.67) 0.64 (0.12) 2.06 (1.29) 26.5 (2.8) 137 (17) 89 (9) median 247 range O-1865 33 23 26 Spare time

Occupational

5 53 23

17 32 29 3

greater in hypertriglyceridemic subjects than in those with low triglyceride levels. Studies of joint effects between spare time physical activity and other lifestyle variables did not produce any meaningful results (data not shown). Occupational HDL-CHOL

I

pco.03

I

t_ [ II Tertife

of alcohol COnSUmPtiOn

Fig. 1. Association between alcohol consumption and HDLcholesterol levels in the study population.

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5 TABLE

3

MULTIPLE REGRESSION COEFFICIENTS WITH AND WITHOUT TRIGLYCERIDES The figures in parenthesis

denote

the values when triglycerides

Variables Dependent

Independent

HDL-chol.

Alcohol Body mass Smoking Physical act. Alcohol Body mass Smoking Physical act. Alcohol Body mass Smoking Physical act.

HDL,-chol.

HDL,-chol.

ON HDL-CHOLESTEROL IN THE MODEL. are incorporated

LEVELS

IN THE

P-value

0.35 (0.34) - 0.23 (- 0.07) -0.14(-0.14) 0.10 (0.08) 0.30 (0.29) -0.25 (-0.08) -0.16 (-0.17) - 0.03 (- 0.06) 0.32 (0.32) -0.12 (-0.04) - 0.03 ( - 0.04) 0.28 (0.27)

0.02 (0.001) 0.04 (0.52) 0.24 (0.44) 0.37 (0.44) 0.01 (0.005) 0.03 (0.48) 0.15 (0.09) 0.76 (0.59) 0.004 (0.004) 0.28 (0.73) 0.76 (0.74) 0.01 (0.01)

Discussion In this dyslipidemic population alcohol sumption was associated with both HDL,

conand

POPULATION

(n = 82)

in the model.

Beta

HDL levels in this population significantly, probably due to the rather narrow age range (40-55 years). When the recorded variables were considered simultaneously in multiple regression analyses, alcohol consumption and body mass emerged as the only significant factors contributing to total HDL-cholesterol variation. When triglycerides were included, the independent contribution of body mass disappeared (Table 3). The correlation coefficient between triglycerides and relative weight in this population was 0.41 (P = 0.001). The whole model explained 29.5% of the variation in total HDL-level. Both alcohol and triglycerides made independent contributions to HDL,cholesterol level (Table 3), with the model explaining 29.6% of the variation. Alcohol consumption and spare time activity significantly influenced HDL,-cholesterol level, while triglycerides were of marginal importance and body mass had no effect at all. The coefficient of determination for the whole model was significantly lower for HDL, (0.209) than for HDL, (0.296) and for total HDL (0.295).

STUDY

R2

0.167 (0.295)

0.134 (0.296)

0.175 (0.209)

HDL,-subfraction cholesterol levels in multivariate analyses. The magnitude of the effect was similar for both fractions. Six and seven percent respectively of the variation in these fractions was explained by alcohol consumption. Previous studies have indicated that the major fraction influenced by moderate alcohol intake is HDL,, even when adjusted for other lifestyle variables [13,20,28]. The effect of alcohol on HDL,cholesterol is more controversial. Clinical studies have suggested that moderate intake does not influence HDL,, whereas chronic consumption of large amounts raises HDL, [28-291. The situation is further illustrated in Table 4 relating our results to previous cross-sectional studies. Physical activity level in our study population was not very high (23 out of 82 clssified as actives) and contributed significantly only to HDL,cholesterol level. Previous studies have indicated that exercise increases HDL, level, while the effect on the HDL, subfraction has been more variable [11,19,30]. Williams et al. [31] have suggested that these inconsistences relate to particle heterogeneity and to different definitions of the HDL-subfractions. The mechanisms are still partially unresolved, but at least the lipoprotein lipase, one of the key enzymes in HDL metabolism is activated [32]. Schwartz [33] compared the lipid changes in obese subjects during weight reduction

6 TABLE

4

COMPARISON OF THE CROSS-SECTIONAL STUDIES EVALUATING THE EFFECTS OF LIFESTYLE HDL-SUBFRACTION CHOLESTEROL LEVELS IN MIDDLE-AGED MALE POPULATIONS

Hafner et al. [22] HDL,-chol. HDL,-chol. Robinson et al. [ll] HDL,-chol. HDL,-chol. Diehl. et al. [23] HDL,-chol. HDL,-chol. Present study HDL,-chol. HDL,-chol. i = increases,

d = decreases,

Alcohol

Exercise

Smoking

HDL separation

i i

_

0 d

Precipitation

i i

0 i

0 0

Precipitation

0 i

0 0

0 0

Ultracentrifugation

i i

0 i

0 0

Ultracentrifugation

0 = insignificant

effect,

FACTORS

ON

- = not analyzed.

induced either by physical exercise or caloric restriction. Physical exercise increased apolipoprotein AI levels and HDL,-cholesterol, while caloric restriction reduced the level of triglycerides and caused enrichment of the HDL particles with cholesterol. Hypertriglyceridemia may modify the effects of exercise on lipoproteins [34] and a recent finding suggests that postprandial hypertriglyceridemia increases HDL, production in exercising muscles [35]. In our hyperlipidemic population the combination of physical inactivity and elevated fasting triglycerides was associated with low HDL-cholesterol values. Spare time physical activity among these subjects resulted in significantly higher HDL-levels with the effect being confined to the HDL,-subfraction. Total HDL and HDL,-cholesterol levels in this population were lower among smokers than nonsmokers (P = 0.10 and P = 0.08, respectively). Interpretation of these data is complicated by the fact that ex-smokers had the lowest levels. Both HDL-subfractions have been reported to be reduced in smokers [ll]. Quitting smoking increases total HDL-level [21] but this effect is not evident when body weight and energy intake are held constant [36]. Body mass had an independent contribution to total HDL and HDL,-cholesterol levels in this population, but the association disappeared when adjusted for serum triglycerides. It is noteworthy

that according to two recent studies HDL, is strongly and independently correlated with the ratio of waist-to-hip circumference, but not with total body fat [12,37]. The data on the association between coronary heart disease and HDL-subfractions is controversial. The severity of the angiographically estimated disease seems to be correlated to HDL, [4], while the number of diseased vessels is more closely correlated to HDL, [5]. The HDL, fraction is reduced in survivors of myocardial infarction [38], and the published prospective data [6] also stresses the major role of HDL,. On the other hand, preliminary epidemiological follow-up data from Finland indicates HDL, as the major HDL subfraction for determining coronary heart disease [39], while in a representative subsample of the Helsinki Heart Study population gemfibrozil produced a statistically significant increment in the HDL,-subfraction only [8]. Furthermore, an inverse association between HDL,-cholesterol and incidence of coronary heart disease has recently been reported in two prospective studies from the UK [40]. In conclusion, alcohol consumption was associated with increased cholesterol levels of both HDL-cholesterol subfractions in 82 middle-aged dyslipidemic men, and this effect was preserved in multiple regression analyses. Spare time physical activity was associated with raised HDL,-cholesterol but was not correlated with HDL,. Smoking

7 was associated with lower HDL-cholesterol, although the differences were not statistically significant. Relative weight did contribute to HDL,variation, but the correlation became insignificant after adjustment for triglyceride levels. The recorded lifestyle variables explained 13.4% of the variation in HDL, and 17.5% in HDL,.

References Gordon, D.J., Probstfield, J.L., Garrison, R.J., Neaton, I.D., Caste& W.P., Knoke, J.D., Jacobs, D.R., Bangdiwalla, S. and Tyroler, H.A., High density lipoprotein cholesterol and cardiovascular disease: four prospective American studies. Circulation, 79 (1989) 8. Manninen, V., Huttunen, J.K., Tenkanen, L., Heinonen, O.P., Mitt&i, M. and Frick, M.H., High-density lipoprotein cholesterol as a risk factor for coronary heart disease in the Helsinki Heart Study. In: N.E. Miller (Ed.), High Density Lipoproteins and Atherosclerosis, II, Excerpta Medica, ICS 826, Amsterdam, 1989, pp. 35-42. Goldbourt, U., Holtzrnan, E. and Neufeld, H.N., Total and high density lipoprotein cholesterol in the serum and risk of mortality: evidence of a threshold effect. Br. Med. J., 290 (1985) 1239. Miller, N.E., Hammet, F., Saltissi, S., Rao, S., VanZeller, H., Coltart, J. and Lewis, B., Relation of angiographically defined coronary artery disease to plasma lipoprotein subfractions and apolipoproteins. Br. Med. J., 282 (1981) 1741. Levy, R.I., Brensike, J.F., Epstein, SE., Kelsey, S.F., Passarnani, E.R., Richardson, J.M., Loh, I.K., Stone, N.J., Aldrich, R.F., Battagliani, J.W., Moriarty, D.J., Fisher, M.L., Friedman, L., Friedewald, W. and Detre, K.M., The influence of change in lipid values induced by cholestyramine and diet on progression of coronary heart disease. Results of the NHLBI Type II coronary intervention study. Circulation, 69 (1984) 325. Gofman, J.W., Young, W. and Tandy, R., lschemic heart disease, atherosclerosis and longevity. Circulation, 34 (1966) 679. Manninen, V., Elo, O., Frick, M.H., Haapa, K., Heinonen, O.P., Heinsahni, P., Helo, P., Huttunen, J.K., Kaitaniemi, P., Koskinen, P., MPenpII, H., Mtiilkiinen, M., MBnttBri, M., Norola, S., Pastemack, A., Pikkarainen, J., Romo, M., Sjoblom, T. and Nikkila, E.A., Lipid alterations and decline in the incidence of coronary heart disease in the Helsinki Heart Study. J. Am. Med. Assoc., 288 (1988) 641. M;inttti, M., Koskinen, P., Manninen, V., Huttunen, J.K., Frick, M.H. and N&kill, EA., Effect of gemfibrozil on the concentration and composition of serum lipoproteins. A controlled study with special reference to initial triglyceride levels. Atherosclerosis, 81 (1990) 11. Wilson, P.W.F., Garrison, R.J., Abbott, R.D. and Caste& W.P., Factors associated with lipoprotein cholesterol levels, Arteriosclerosis, 3 (1983) 273.

10 Patterson, C.C., McCrum, E., McMaster, D., Kerr, M., Sykes, D. and Evans, A.E., Factors influencing total cholesterol and high-density lipoprotein cholesterol concentrations in a population at high coronary risk. Acta Med. Stand. Suppl., 728 (1988) 150. 11 Robinson, D., Ferns, G.A., Bevan, E.A., Stocks, J., Williams, P.T. and Galton, D.J., High density lipoprotein subfractions and coronary risk factors in normal men. Arteriosclerosis, 7 (198) 341. 12 Marti, B:, Suter, E., Riesen, W.F., Tschopp, A. and Wanner, H-U. Anthropometric and Lifestyle correlates of serum lipoprotein and apolipoprotein levels among normal non-smoking men and women. Atherosclerosis, 79 (1989) 111. 13 Williams, P.T., Krauss, R.M., Wood, P.D., Albers, J.J., Dreon, D. and Ellsworth, N., Associations of diet and alcohol intake with high-density lipoprotein subclasses. Metabolism, 34 (1985) 524. 14 Ehnholm, C., Huttunen, J.K., Pietinen, P., Leino, U., Mutanen, M., Kostiainen, E., lacono, J., Dougherty, R. and Puska, P., Effect of a diet low in saturated fatty acids on plasma lipids, lipoproteins and HDL subfractions. Arteriosclerosis, 4 (1984) 265. 15 Criqui, M.H., Wallace, R.B., Heiss, G., Mishkel, M., Schonfeld, G. and Jones, G.T.L., Cigarette smoking and plasma high density lipoprotein cholesterol: The Lipid Research Clinics Program Prevalence Study. Circulation, 62 (1980) Suppl 4, 70. 16 Seidel, D., Cremer, H., Elster, H., Weise, M. and Wieland, H., Influence of smoking on the plasma lipoprotein profile. Klin. Wochenschr., 62 (1984) Suppl. II, 18. 17 Huttunen, J.K., L;insimies, E., Voutilainen, E., Ehnholm, C., Hietanen, E., Penttill, I., Siitonen, 0. and Rauramaa, R., Effect of moderate physical excercise on serum lipoproteins. A controlled clinical trial with special reference to serum highdensity lipoproteins. Circulation, 60 (1979) 1220. 18 Kavanagh, T., Shephard, R.J., Lindley, L.J. and Pieper, M., Influence of exercise and life-style variables upon high density lipoprotein cholesterol after myocardial infarction. Arteriosclerosis, 3 (1983) 249. 19 Rauramaa, R., Salonen, J.T., Kukkonen-Hajula, K., Sepptien, K., Seppala, E., Vapaatalo, H. and Huttunen, J.K., Effects of mild exercise on serum lipoproteins and metabolites of arachidonic acid: a controlled randomised trial in middle-aged men. Br. Med. J., 288 (1984) 603. 20 Haskell, W.L., Camargo, Jr.. C., Williams, P.T., Vranizan, K.M., Krauss, R.M., Lindgren, F.T. and Wood, P.D., The effect of cessation of moderate alcohol intake on serum high-density lipoprotein subfractions. A controlled study. N. Engl. J. Med., 310 (1984) 805. 21 Stubbe, I., Eskilsson, J. and Nilsson-Ehle, P., High-density lipoprotein concentrations increase after stopping smoking. Br. Med. J. 284 (1982) 1511. 22 Hafner, S.M., Applebaum-Bowden, D., Wahl, P.W., Hoover, J.J., Warnick, G.R., Albers, J.J. and Hazzard, W.R., Epidemiological correlates of high density lipoprotein subfractions, apolipoproteins A-l, A-II and D, and lechitin cholesterol acyltransferase. Effects of smoking, alcohol and adiposity. Arteriosclerosis, 5 (1985) 169.

8 23 Diehl, A.K., Fuller, J.H., Mattock, M.B., Salter, A.M., El-Gohari, R. and Keen, H., The relationship of high density lipoprotein subfractions to alcohol consumption, and coronary heart disease. other lifestyle factors, Atherosclerosis, 69 (1988) 145. 24 M%ntt&ri, M., Elo, O., Frick, M.H., Haapa, K., Heinonen, O.P., Heinsalmi, P., Helo, P., Huttunen, J.K., Kaitaniemi, P., Koskinen, P., Mamrinen, V., Maenpii;i, H., MZlkonen, M., Norola, S., Pastemack, A., Pikkarainen, J., Romo, M., Sjoblom, T. and NikkiH, E.A., The Helsinki Heart Study: basic design and randomization procedure. Eur. Heart J., 8 (1987) Suppl. I, 1. 25 Frick, M.H., Elo, O., Haapa, K., Heinonen, O.P., Heinsahni, P., Helo, P., Huttunen, J.K., Kaitaniemi, P., Koskinen, P., Manninen, V., Maenpti, H., Malkilnen, M., Mtintt8ri, M., Norola, S., Pastemack, A., Pikkarainen, J., Romo, M., SjBblom, T. and Nikkila, E.A., Helsinki Heart Study: primary-prevention trial with gemfibrozil in middleaged men ‘with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. N. Engl. J. Med., 317 (1987) 1237. 26 Simpura, J., Scandinavian drinking survey: construction of indices of alcohol intake. Report 46, National Institute for Alcohol Research, Oslo, 1981. 27 Wilhelmsen, L., Tibblin, G., Fodor, J. and WerkB, L., A. multifactorial primary prevention trial in Gothenburg, Sweden. In: Coronary heart disease and physical fitness. O.A. Larsen and R.O. Malmborg (I%.), Munksgaard, Copenhagen, 1971, pp. 267-270. 28 Danielsson, B., Ekman, R., Fex, G., Johansson, B.G., Kristensson, H., Nilsson-Ehle, P. and Wadstein, J., Changes in plasma high density lipoproteins in chronic male alcoholics during and after abuse. Scand. J. Clin. Lab. Invest., 38 (1978) 113. 29 Taskinen, M-R., VXlim&ki, M., N&k%, 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. 30 Wood, P.D., Stefanick, M.L., Dreon, D.M., Frey-Hewitt, B., Garay, S.G., Williams, P.T., Superko, H.R., Fortman, S.P., Albers, J.J., Vranizan, K.M., Ellsworth, N., Terry, R.B. and Haskell, W., Changes in plasma lipids and lipoproteins in overweight men during weight loss through dieting as compared with exercise. N. Engl. J. Med., 319 (1988) 1173.

31 Williams, P.T., Krauss, R.M., Wood, P.D., Lindgren, F.T., Giotas, C. and Vranizan, K.M., Lipoprotein subfractions of runners and sedentary men. Metabolism, 35 (1986) 45. 32 Kuusi, T., Nikkila, E.A., Saarinen, P., Varjo, P. and Laitinen, L.A., Plasma high density lipoproteins HDL, and HDL, and postheparin plasma lipases in relation to parameters of physical fitness. Atherosclerosis, 41 (1982) 209. 33 Schwartz, R.S., The independent effects of dietary weight loss and aerobic training on high density lipoproteins and apolipoprotein A-I concentrations in obese men. Metabolism, 36 (1987) 165. 34 Vega, G.L., Groszek, E., Wolf, R. and Grundy, SM., Influence of polyunsaturated fats on composition of plasma lipoproteins and apolipoproteins. J. Lipid Res., 23 (1982) 811. 35 Ruys, T., Shaik, M., Nordestgaard, B.G., Sturgess, I., Watts, G.F. and Lewis, B., Effects of exercise and fat ingestion on high density lipoprotein production by peripheral tissues. Lancet, ii (1989) 1119. 36 Quensel, M., Soderstrom, A., Agardh, C.D. and NilssonEhle, P., High density lipoprotein concentrations after cessation of smoking: the importance of alterations in diet. Atherosclerosis, 75 (1989) 189. 37 Ostlund, R.E., Staten, M., Kohrt, W.M., Schultz, J. and Malley, M., The ratio of waist-to-hip circumference, plasma insulin level, and glucose intolerance as independent predictors of the HDL, cholesterol level in older adults. N. Engl. J. Med., 322 (1990) 229. 38 Ballantyne, F.C., Clark, R.S., Simpson, H.S. and Ballantyne, D., High density and low density lipoprotein subfractions in survivors of myocardial infarction and in control subjects. Metabolism, 31 (1982) 433. 39 Salonen, J., SeppLinen, K. and Rauramaa, R., Serum high density lipoprotein cholesterol subfractions and the risk of acute myocardial infarction: a population study in Eastern Finland. Circulation, 78 (1988) Suppl. II, 281. 40 Sweetnam, P., Elwood, P., Yamell, J., Bainton, D., Baker, I., Miller, N. and Bolton, C., HDL cholesterol and its subfractions in the Caerphilly and Speedwell Heart Disease Studies. In: N.E. Miller (Ed.), High Density Lipoproteins and Atherosclerosis, II, Excerpta Medica, ICS 826, Amsterdam, 1989, pp. 43-52.