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Risk of acute coronary events according to serum concentrations of enterolactone: a prospective population-based case-control study Meri Vanharanta, Sari Voutilainen, Timo A Lakka, Manon van der Lee, Herman Adlercreutz, Jukka T Salonen Summary Background The lignan enterolactone, produced by the intestinal microflora from dietary precursors, has been implicated in protection against cancer. We investigated the association of serum enterolactone concentration with the risk of acute coronary events in a prospective nested casecontrol study in middle-aged men from eastern Finland. Methods Enterolactone was measured by time-resolved fluoroimmunoassay in serum from 167 men who had an average 7·7 years of follow-up to an acute coronary event and from 167 control men. Both cases and controls were from a cohort of 2005 men who had no clinical coronary heart disease (CHD) at baseline. The controls were matched for age, examination year, and residence. Acute coronary events were registered prospectively. Findings The mean baseline serum enterolactone concentration was lower among the cases than the controls (18·2 [SD 21·1] vs 23·5 [18·2] nmol/L, p=0·001). The men in the highest quarter of the enterolactone distribution (>30·1 nmol/L) had a 58·8% (95% CI 24·1–77·6, p=0·005) lower risk of acute coronary events than men in the lowest quarter. After adjustment for the nine most strongly predictive risk factors, men in the highest enterolactone quarter had a 65·3% (11·9–86·3, p=0·03) lower risk than men in the lowest quarter. Interpretation Healthy men with high serum concentrations of enterolactone had a lower risk of acute coronary events than men with lower concentrations. These findings support the hypothesis that plant-dominated fibre-rich food lowers the risk of CHD. Lancet 1999; 354: 2112–15 Introduction Lignans have been postulated to have a role in protection against both cancer and coronary heart disease (CHD).1 Mammalian lignans are synthesised when plant lignans (phyto-oestrogens) are modified by intestinal bacteria.2 The most abundant mammalian lignan is enterolactone. There is evidence that enterolactone influences metabolism of sex hormones and proliferation of malignant cells, inhibits the human aromatase enzyme, and inhibits the metabolism of growth-promoting steroid hormones.1,3–5 These effects of enterolactone provide potential mechanisms for a postulated preventive influence in CHD. Many common foodstuffs, such as seeds, cereals (rye, barley, and wheat), berries, and some vegetables, contain plant lignans.1 However, most mammalian lignan precursors may still be unknown.1 We aimed to test the hypothesis that a high serum enterolactone concentration is associated with a low risk of acute coronary events in middle-aged men with no previous atherosclerotic vascular diseases. Methods Study population The Kuopio Ischaemic Heart Disease Risk Factor Study is a population-based study of risk factors for CHD, atherosclerosis, and other related outcomes.6 The study protocol was approved by the Research Ethics Committee of the University of Kuopio. The study population is a random sample of men living in the city of Kuopio or neighbouring rural communities, stratified and balanced in four strata: exactly 42 years, 48 years, 54 years, or 60 years old at the baseline examination. The baseline examinations were carried out between 1984 and 1989. Of 3235 eligible men, 2682 (82·9%) participated. Men with prevalent CHD at baseline (n=677) were excluded. Prevalent CHD was defined as a history of acute coronary events or angina pectoris, angina pectoris on effort, or use of glyceryl trinitrate tablets at least once a week.7 Thus, this study is based on a cohort of 2005 men without CHD. Design and procedures Research Institute of Public Health (M Vanharanta BSc, S Voutilainen MSc, T A Lakka DrMedSci, Prof J T Salonen DrMedSci) and Department of Public Health and General Practice (Prof J T Salonen), University of Kuopio, Kuopio; and Institute for Preventive Medicine, Nutrition and Cancer, Folkhälsan Research Center and Department of Clinical Chemistry, University of Helsinki, Helsinki (M van der Lee BSc, Prof H Adlercreutz DrMedSci), Finland Correspondence to: Prof Jukka T Salonen, Research Institute of Public Health, University of Kuopio, PO 1627, 70211 Kuopio, Finland (e-mail: [email protected]) 2112 Acute coronary events that occurred between 1984 and 1992 were registered as part of the multinational MONICA (Monitoring of Trends and Determinants in Cardiovascular Disease) Project.8 Data on coronary events between 1993 and 1996 were obtained by record linkage from the national computerised hospitaldischarge registry. The diagnostic classification used was identical to that of the MONICA project. The cohort was followed up on average for 10 years. According to the diagnostic classification of the events,8 there were 81 definite and 55 possible acute myocardial infarctions and 31 typical long episodes of chest pain. The cases were 167 men who had their first acute coronary event by the end of 1996. To ensure the comparability of the controls, one control was matched for each case according to age (42, 48, 54, 60), examination year (1984–1989) and place of residence (the same municipality out of ten), and were drawn from the same cohort as the cases. The follow-up time for the 334 participants varied between 1 month (to event) and 12·8 years. Serum samples THE LANCET • Vol 354 • December 18/25, 1999 n* Cases (n=167) Controls (n=167) p Age (years)† 334 54·2 (3·9) 54·2 (3·9) Physical characteristics Height (cm) Weight (kg) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) ·· 332 334 332 332 171·7 (6·8) 80·9 (13·3) 140 (17) 92 (11) 173·3 (6·1) 80·3 (10·8) 134 (17) 88 (10) 0·02 0·74 0·001 0·002 Lifestyle Smokers Pack-years Alcohol consumption (g/week) 334 328 334 72 (43%) 12·9 (18·8) 83 (126) 45 (27%) 7·5 (15·2) 60 (119) 0·005 0·01 0·04 Diabetes 334 20 (12%) 5 (3%) 0·003 Maximum oxygen uptake in exercise test (mL kg21 min21) 284 28·4 (6·2) 31·6 (8·1) <0·001 Biochemistry Serum enterolactone (nmol/L) Serum apolipoprotein B (mg/L) Serum HDL cholesterol (mmol/L) Serum HDL2 cholesterol (mmol/L) Serum LDL cholesterol (mmol/L) Serum triglycerides (mmol/L) Serum ferritin (mg/L) Serum transferrin receptor to ferritin ratio Plasma fibrinogen (g/L) Plasma ascorbic acid (mg/L) Blood glucose (mmol/L) Urinary excretion of nicotine metabolites (mg/24 h) Dietary intake Calcium (g/day) Sodium (g/day) Iron (mg/day) Potassium (g/day) Magnesium (g/day) 334 316 328 324 326 320 334 330 18·2 (21·1) 521 (586) 1·21 (0·27) 0·77 (0·26) 4·40 (1·04) 1·37 (0·79) 190 (176) 19·0 (23·3) 23·5 (18·2) 0·001 391 (533) <0·001 1·31 (0·30) 0·003 0·86 (0·28) 0·003 4·18 (1·05) 0·02 1·29 (0·71) 0·32 151 (143) 0·02 25·3 (41·0) 0·06 276 328 334 234 3·1 (0·6) 7·0 (4·1) 5·03 (1·38) 8·18 (10·5) 3·0 (0·5) 0·1 8·2 (4·2) 0·006 4·73 (0·68) 0·38 4·40 (8·03) <0·001 324 324 324 324 324 1·4 (0·5) 3·4 (1·4) 18·7 (5·9) 4·57 (1·13) 0·44 (0·11) 1·4 (0·5) 3·3 (1·1) 18·5 (5·5) 4·58 (1·06) 0·44 (0·10) 0·24 0·35 0·83 0·98 0·98 Data are mean (SD) or number of participants. *Total for whom data were available. †Matched variable. Table 1: Risk factors drawn at baseline were stored at –20ºC on average for 11·9 years (range 8·4–14·2) from sampling to enterolactone measurements. The measurement method was based on time-resolved fluoroimmunoassay with a europium chelate as a label,9 with slight modifications: serum was diluted only 1 in 2 instead of 1 in 11 with buffer before hydrolysis, and there were two ethyl-ether extractions (each with 1·5 mL). 59-O-carboxymethylyenterolactone was synthesised and coupled to bovine serum albumin and used as antigen to immunise rabbits. The tracer with the europium chelate was synthesised from the same 59-derivative of enterolactone. After enzymic hydrolysis and ether extraction, the immunoassay was carried out with the Victor 1420 multilabel counter (Wallac, Turku, Finland). No antiserum cross-reactivity with available lignans, isoflavonoids, or flavonoids could be detected. The working range of the assay was 1·5–540 nmol/L. Coefficients of variation within and between assays at three different concentrations varied from 4·5% to 9·2% and from 5·9% to 9·1%, respectively. The correlation coefficient for 44 samples between this method and our reference method (isotope dilution gas-chromatography and mass spectrometry) was 0·82 (p=0·0004), the mean values being 31·2 nmol/L for the reference assay and 31·0 nmol/L for the fluoroimmunoassay. The reproducibility coefficient for a single measurement of serum enterolactone was 0·55 (95% CI 0·41–0·69).10 Demographic variables, medical history and medications, family history of diseases, ischaemic findings in the baseline exercise test, smoking, and blood pressure were assessed as described previously.7,11 The collection of blood samples and the measurement of serum lipids, lipoproteins, and ferritin, plasma fibrinogen and ascorbic acid, maximum oxygen uptake,11 24 h urinary excretion of nicotine metabolites, and sodium excretion have been described previously.7,12–15 Diabetes was defined as a previous diagnosis of diabetes or fasting blood glucose concentrations of 6·7 mmol/L or higher. The consumption of foods was assessed at the time of blood sampling with an instructed 4-day food recording by household measures.16 The instructions were given and the completed food records were checked by a nutritionist. The intake of nutrients and total energy intake were estimated with Nutrica software. The databank of Nutrica mainly uses Finnish values for the nutrient values of foods. Statistical analysis Differences in risk factors between the cases and controls were tested for significance with Wilcoxon’s signed-rank test or with McNemar’s test for two related samples; those between quarters of the serum enterolactone distribution were tested with KruskalWallis ANOVA. Odds ratios for acute coronary events, adjusted for risk factors, were estimated by stepwise and forced conditional multivariate logistic-regression modelling in paired data. 95% CI were estimated on the assumption of asymptotic normality of estimates of the Egret for Windows software (version 1.0). Missing values in urinary nicotine excretion were imputed by means of linear regression on the basis of the self-reported number of cigarettes smoked daily. The self-reported number of cigarettes daily had a correlation with the urinary nicotine excretion of 0·57 before imputation and 0·72 after the imputation. Missing values in other covariates were replaced by means, separately for cases and controls. Results The distributions of the major known coronary risk factors in the cases and controls are shown in table 1. The mean serum enterolactone concentration was 25·1% lower among the cases than among controls (95% CI 4·1–46·2], p=0·001 for difference). In a univariate logistic model, the risk of acute coronary events was decreased on average by 1·4% per unit (nmol/L) of serum enterolactone (0·2–2·6, p=0·016). The Model 1* Model 2† Odds ratio (95% CI) p Odds ratio (95% CI) p Enterolactone Fourth quarter (>30·10 nmol/L) Third quarter (15·11–30·10 nmol/L) Second quarter(7·21–15·10 nmol/L) 0·33 (0·14–0·76) 0·69 (0·31–1·57) 1·11 (0·50–2·45) 0·01 0·38 0·80 0·35 (0·14–0·88) 0·75 (0·31–1·81) 1·22 (0·51–2·96) 0·03 0·53 0·51 Other risk factors Serum apolipoprotein B (per 100 mg/L) Dietary iron intake (mg/day) Family history of coronary heart disease (yes vs no) Ischaemic findings on exercise test (yes vs no) Dietary calcium intake (per 100 mg/day) Urinary excretion of nicotine metabolites (mg/day) Diabetes (yes vs no) Systolic blood pressure (per 10 mm Hg) Maximum oxygen uptake (L kg21 min21) ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· ·· 1·31 (1·12–1·53) 1·12 (1·05–1·21) 2·80 (1·40–5·46) 3·23 (1·36–7·66) 0·89 (0·83–0·97) 1·05 (1·01–1·10) 5·82 (1·36–24·98) 1·26 (1·02–1·54) 0·94 (0·88–0·99) <0·001 0·001 0·004 0·008 0·01 0·02 0·02 0·03 0·03 *A logistic model including three highest enterolactone quarters. †A stepwise model (p for entry 0·05); the second and third enterolactone quarters are forced into the model. Table 2: Strongest risk factors for acute coronary events in conditional multivariate logistic regression models THE LANCET • Vol 354 • December 18/25, 1999 2113 There was significant heterogeneity in systolic and diastolic blood pressure between quarters of the enterolactone distribution (table 3). The mean concentrations of total, HDL, and LDL cholesterol and apolipoprotein B did not differ significantly between these quarters (table 3). Serum enterolactone concentration had a positive correlation (r=0·08, p=0·15) with fibre intake. The correlation was stronger (r=0·12, p=0·03) when fibre intake was expressed in relation to bodyweight. The dietary intake of fibre had, however, no consistent association with the risk of acute coronary events. Discussion Unadjusted and risk-factor-adjusted odds ratios for acute events according to quarters of baseline serum enterolactone concentration decrease in risk was 2·3% per nmol/L serum enterolactone (0·7–3·7, p=0·004) after adjustment for the nine other most strongly predictive risk factors (table 2, model 2). The covariates were selected by stepwise analysis (p<0·05 for entry). Men with high serum enterolactone (above median, 15·11 nmol/L) had a 52·0% (9·4–74·6, p=0·02) lower risk of acute coronary events than men with lower serum enterolactone concentrations. To examine the dose-response relation, we grouped the participants into quarters of the serum enterolactone distribution. Men in the highest quarter (>30·10 nmol/L) had a 58·8% (24·1–77·6, p=0·005) lower risk of acute coronary events than men in the lowest quarter (figure). After adjustment for the nine most strongly predictive risk factors (table 2, model 2), men in the highest quarter had a 65·3% (11·9–86·3, p=0·03) lower risk of acute coronary events than men in the lowest quarter. The linear trend in risk of acute coronary events across quarters of the enterolactone distribution was significant (p=0·01). To test the association between enterolactone and the risk of acute coronary events in men with neither clinical nor subclinical CHD, we excluded 37 pairs because electrocardiography showed ischaemia on the exercise test for either the case or the control. The association of enterolactone with the risk of acute coronary events was stronger than for all pairs. The unadjusted odds ratio for men in the highest quarter of enterolactone was 0·33 (0·14–0·76, p=0·01) compared with those in the lowest quarter. Since smokers are under increased oxidative stress, the impact of antioxidative nutrients on CHD is expected to be greater in smokers. In an unadjusted unpaired logistic regression analysis, the relative benefit in the highest quarter was 79·3% (37·8–93·1, p=0·005) among smokers and 47·0% among non-smokers (216·5 to 75·9, p=0·11). The difference was not significant. Enterolactone is a diphenolic compound of dietary origin that has been associated with a reduced risk of cancer.1,17,18 A role of enterolactone in protection against CHD has been suggested previously.1 Although enterolactone may be simply a biomarker of healthy fibre-rich food, there are mechanisms through which enterolactone itself could actively protect against cardiovascular diseases. Plasma enterolactone concentration depends on the intake of lignan-containing food and the activity of the intestinal microflora. For example, antibiotic treatment decreases the production of enterolactone for weeks. There is an increasing amount of evidence that the initiation of atherosclerosis is related to the oxidative modification of LDL. Lipid peroxidation is associated with accelerated atherosclerotic progression.19 Like all phenolic compounds, enterolactone is likely to inhibit lipid peroxidation.1 Cigarette smoke increases lipid peroxidation.19,20 Because smokers are under high oxidative stress, they are likely to benefit from additional antioxidants,21 such as enterolactone. We found higher relative benefits from high serum enterolactone for smokers than for non-smokers. Intracellular enterolactone could also protect against lipid peroxidation. Because of the interaction between lignans and the sex-hormone-binding globulin (SHBG), enterolactone is carried to the target cells.22 Enterolactone passes freely into the cell4 and could protect against oxidative damage due to intracellular free radicals. Evidence from both basic research and cross-sectional human population studies suggests that increased freeradical stress is associated with hypertension.23 Also, low plasma concentrations of vitamin C and selenium have been associated with high blood pressure.!5 We found higher blood pressure in men with low serum enterolactone concentrations than in those with higher values. Lignans have weak oestrogenic activity, as a result of the 2,3-dibenzylbutane structure, which resembles synthetic oestrogens.24 High amounts of lignans together with other similar compounds entering the portal circulation stimulate SHBG production.3 This is one mechanism through which lignans could protect against atherosclerosis Characteristic First quarter Second quarter Third quarter Fourth quarter p for heterogeneity Systolic blood pressure (mm Hg)* Diastolic blood pressure (mm Hg)* Serum total cholesterol (mmol/L)† Serum LDL cholesterol (mmol/L)† 142 (2) 93 (1) 6·13 (0·13) 4·25 (1·13) 136 (2) 89 (1) 6·13 (0·13) 4·34 (1·03) 134 (2) 88 (1) 6·27 (0·13) 4·39 (1·02) 137 (2) 90 (1) 6·22 (0·13) 4·19 (0·99) 0·026 0·017 0·82 0·52 Serum HDL cholesterol (mmol/L)† Serum apolipoprotein B (mg/L)† 1·24 (0·03) 478 (59) 1·28 (0·03) 350 (59) 1·26 (0·03) 447 (58) 1·28 (0·03) 0·74 519 (59) 0·22 *Adjusted for age, examination year, alcohol intake, 24 h urinary sodium excretion, and dietary magnesium intake. †Adjusted for age, examination year, and alcohol intake. Table 3: Mean (SE) blood pressure and serum lipid concentrations in quarters of distribution of serum enterolactone concentrations from ANOVA 2114 THE LANCET • Vol 354 • December 18/25, 1999 and CHD. Low SHBG concentrations are associated with smaller and denser LDL particles,25 which in turn have been associated with increased CHD risk.26 The oestrogenic effect of lignans on liver SHBG production may parallel an effect on LDL synthesis resulting in larger LDL particles that are more resistant to oxidation. In a female population during a 12-year follow-up period, low plasma SHBG concentrations were associated with increased CHD mortality,27 but in men the evidence is controversial.28 Our finding of high blood pressure in men with low serum enterolactone concentrations needs to be retested. We confirmed the previous finding that fibre intake correlates only weakly with enterolactone concentration in body fluids.1 Even though a high dietary intake of both total fibre and cereal fibre has been associated with a lower risk of CHD in several prospective studies,29,30 we found no such association. Nevertheless, we cannot rule out the possibility that there are other compounds in fibre-rich foods that might add to the protective effect of enterolactone or even be the protective nutrients, enterolactone merely being a biomarker of a protective diet. Our findings must be confirmed by further epidemiological studies, and randomised trials testing the effects of diets and supplements high in plant and mammalian lignans will be warranted. A diet high in enterolactone precursors has no conceivable harmful effects. Eventually, these further studies could justify changes in the current recommendations for a healthy diet. 8 9 10 11 12 13 14 15 16 17 18 Contributors Meri Vanharanta undertook most of the data analyses and drafted the paper, Sari Voutilainen contributed to the data analysis and checked the food record data, Timo Lakka classified coronary events, Manon van der Lee carried out the enterolactone assays, Herman Adlercreutz developed and supervised the enterolactone assays, and Jukka Salonen designed the KIHD investigation and initiated this study. All investigators contributed to the writing of the report. Acknowledgments We thank Kristiina Nyyssönen for supervising part of the chemical analyses; Esko Taskinen, Juha Venäläinen, Riitta Salonen, Hannu Litmanen, and Rainer Rauramaa for supervising exercise tests; Jaakko Tuomilehto, and Kalevi Pyörälä for the access to the Finmonica coronary registry data; Plonja Kuiper for participating in the enterolactone assays; and Sudhir Kurl and Hannu Mykkänen for useful comments. This study was supported by research grants from the National Heart, Lung, and Blood Institute of the USA (grant HL 44199 to George A Kaplan) and the Academy of Finland (grants 41471, 1041086 and 2041022 to Jukka T Salonen). References Adlercreutz H, Mazur W. Phyto-oestrogens and western diseases. A n nM e d 1997; 29: 95–120. 2 Setchell KDR, Lawson AM, Borriello SP, et al. Lignan formation in man: microbial involvement and possible roles in relation to cancer. Lancet 1981; ii: 4–7. 3 Adlercreutz H. Höckerstedt K, Bannwart C, et al. Effect of dietary components, including lignans and phytoestrogens, on enterohepatic circulation and liver metabolism of estrogens and on sex hormone binding globulin (SHBG). J Steroid Biochem 1987; 27: 1135–44. 4 Adlercreutz H, Bannwart C, Wähälä K, et al. Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. J Steroid Biochem Mol Biol 1993; 44: 147–53. 5 Evans BAJ, Griffiths K, Morton MS. Inhibition of 5a-reductase in genital skin fibroblasts and prostate tissue by dietary lignans and isoflavonoids. J Endocrinol 1995; 147: 295–302. 6 Salonen JT. Is there a continuing need for longitudinal epidemiologic research? The Kuopio Ischaemic Heart Disease Risk Factor Study. Ann Clin Res 1988; 20: 46–50. 7 Salonen JT, Nyyssönen K, Korpela H, Tuomielhto J, Seppänen R, Salonen R. High stored iron levels are associated with excess risk of 19 20 21 22 23 24 25 1 THE LANCET • Vol 354 • December 18/25, 1999 26 27 28 29 30 myocardial infarction in eastern Finnish men. Circulation 1992; 86: 803–11. Tuomilehto J, Arstila M, Kaarsalo E, et al. Acute myocardial infarction (AMI) in Finland: baseline data from the FINNMONICA AMI register in 1983–1985. Eur Heart J 1992; 13: 577–87. Adlercreutz H, Wang GJ, Lapcik O, et al. Time-resolved fluoroimmunoassay for plasma enterolactone. Anal Biochem 1998; 265: 208–15. Zeleniuch-Jacquotte A, Adlercreutz H, Akhmedkhanov A, Toniolo P. Reliability of serum measurements of lignans and isoflavonoid phytoestrogens over a two-year period. Cancer Epidemiol Biomarkers Prev 1998; 7: 885–89. Lakka TA, Venäläinen JM, Raurama R, Salonen R, Tuomilehto J, Salonen JT. Relation of leisure-time physical activity and cardiorespiratory fitness to the risk of acute myocardial infarction in men. N Engl J Med 1994; 330: 1549–54. Salonen JT, Salonen R, Seppänen K, Rauramaa R, Tuomilehto J. HDL, HDL2, and HDL3 subfraction, and the risk of acute myocardial infarction: a prospective population study in eastern Finnish men. Circulation 1991; 84: 129–39. Nyyssönnen K, Parvainen MT, Salonen R, Tuomilehto J, Salonen JT. Vitamin C deficiency and risk of myocardial infarction: prospective population study of men from eastern Finland. BMJ 1997; 314: 634–38. Puhakainen EVJ, Barlow RD, Salonen JT. An automated colorimetric assay for urine nicotine metabolites: a suitable alternative to cotinine assays for the assessment of smoking status. Clin Chim Acta 1987; 170: 255–62. Salonen JT, Salonen R, Ihanainen M, et al. Blood pressure, dietary fats, and anxioxidants. Am J Clin Nutr 1988; 48: 1226–32. Ihanainen M, Salonen R, Seppänen R, Salonen JT. Nutrition data collection in the Kuopio Ischemic Heart Disease Risk Factor Study: nutrient intake of middle-aged Eastern Finnish men. Nutr Res 1989; 9: 597–604. Ingram D, Sanders K, Kolybaba M, Lopez D. Case-control study of phyto-oestrogens and breast cancer. Lancet 1997; 350: 990–94. Adlercreutz H, Fotsis T, Heikkinen R, et al. Excretion of the lignans enterolactone and enterodiol and of equol in omnivorous and vegetarian women and in women with breast cancer. Lancet 1982; ii: 1295–99. Salonen JT, Ylä-Herttuala S, Yamamoto R, et al. Autoantibody against oxidised LDL and progression of carotid atherosclerosis. Lancet 1992; 339: 883–89. Frei B, Forte TM, Ames BN, Cross CE. Gas phase oxidants of cigarette smoke induce lipid peroxidation and changes in lipoprotein properties in human blood plasma. Biochem J 1991; 277: 133–38. Steinberg DM, Chait A. Antioxidant vitamin supplementation and lipid peroxidation in smokers. Am J Clin Nutr 1998; 68: 319–27. Martin ME, Haourigui M, Pelissero C, Benassayag C, Nunez EA. Interactions between phytoestrogens and human sex steroid protein. Life Sci 1996; 58: 429–36. Kumar KV, Das UN. Are free radicals involved in the pathobiology of human essential hypertension? Free Radic Res Commun 1993; 19: 59–66. Setchell KDR, Adlercreutz H. Mammalian lignans and phytoestrogen: recent studies on their formation, metabolism and biological role in health and disease. In: IR Rowland, ed. Role of gut flora in toxicology and cancer. London: Academic Press, 1988: 315–43. Haffner SM, Laakso M, Miettinen H, Mykkänen L, Karhapää P, Rainwater DL. Low levels of sex hormone-binding globulin and testosterone are associated with smaller, denser low density lipoprotein in normoglycemic men. J Clin Endocrinol Metab 1996; 81: 3697–701. Austin MA, Breslow JL, Hennekens CH, Buring JE, Willet WC, Krauss RM. Low density lipoproteins subclass patterns, and risk of myocardial infarction. JAMA 1988; 260: 1917–21. Lapidus L, Lindstedt G, Lundberg PA, Bengtsson C, Gredmark T. Concentrations of sex-hormone binding globulin and corticosteroid binding globulin in serum in relation to cardiovascular risk factors and to 12-year incidence of cardiovascular disease and overall mortality in postmenopausal women. Clin Chem 1986; 32: 146–52. Pugeat M, Moulin P, Cousin P, et al. Interrelations between sex hormone-binding globulin (SHBG), plasma lipoproteins and cardiovascular risk. J Steroid Biochem Molec Biol 1995; 53: 567–72. Pietinen P, Rimm EB, Korhonen P, et al. Intake of dietary fiber and risk of coronary heart disease in a cohort of Finnish men. Circulation 1996; 94: 2720–27. Kushi LH, Lew RA, Stare FJ, et al. Diet and 20-year mortality from coronary heart disease. N Engl J Med 1985; 312: 811–18. 2115