Prospective Study of Selenium Levels in Toenails and Risk of Coronary Heart Disease in Men

Kazuko Yoshizawa1, Alberto Ascherio1,2, J. Steven Morris3, Meir J. Stampfer1,2,4, Edward Giovannucci1,4, Connie K. Baskett3, Walter C. Willett1,2,4 and Eric B. Rimm1,2,4 

1 Department of Nutrition, Harvard School of Public Health, Boston, MA.
2 Department of Epidemiology, Harvard School of Public Health, Boston, MA.
3 Research Reactor Center, University of Missouri–Columbia, Research Park, MO.
4 Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA.

Received for publication April 18, 2002; accepted for publication November 13, 2002.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Selenium is a trace mineral that plays a role in antioxidant defenses as a component of glutathione peroxidase. Epidemiologic findings on the relation of selenium status to risk of heart disease are inconsistent. Therefore, the authors investigated prospectively the association between toenail selenium levels and risk of coronary heart disease (CHD) in a case-control study nested within the Health Professionals Follow-up Study. Between 1987 and 1992, 470 CHD cases were newly diagnosed. A control matched to each case on age, smoking status, and date of toenail return was chosen. Toenail selenium levels analyzed by neutron activation were not associated with risk of total CHD after adjustment for age and smoking and other CHD risk factors (highest quintile vs. lowest: odds ratio (OR) = 0.86, 95% confidence interval (CI): 0.55, 1.32; p-trend = 0.75). Selenium level was inversely associated with risk of nonfatal myocardial infarction for extreme quintiles (OR = 0.54, 95% CI: 0.31, 0.93; p-trend = 0.07), was less so for fatal CHD (OR = 0.79, 95% CI: 0.39, 1.60; p-trend = 0.61), and was directly associated with coronary revascularization procedures (OR = 2.38, 95% CI: 1.11, 5.09; p-trend = 0.02). Although these findings suggest no overall relation between selenium status and CHD, a specific protective role for myocardial infarction cannot be excluded.

coronary disease; nutrition; selenium

Abbreviations: Abbreviations: CABG, coronary artery bypass graft; CI, confidence interval; OR, odds ratio; PTCA, percutaneous transluminal coronary angioplasty.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Selenium is an essential trace element that exists in a variety of inorganic and organic forms in nature. The predominant dietary forms are selenocysteine and selenomethionine (1). Among its biologic functions, selenium is a component of glutathione peroxidase, which is part of the antioxidant defense against free radicals (24). Selenium deficiency results in a decline in tissue selenium-dependent glutathione peroxidase activity (3, 5). Oxidized low density lipoprotein cholesterol may induce endothelial damage and thus facilitate the atherogenic process by allowing entry of elements from the blood and by allowing adherence of platelets (6). Some investigators have speculated that glutathione peroxidase protects endothelial cells from the effects of oxidized low density lipoprotein cholesterol (7).

Estimation of average dietary selenium intake using available food composition data is not accurate, because the selenium content of foods varies widely and is dependent on the soil content in which the foods are grown (810). The relation of selenium status to risk of heart disease in epidemiologic settings has been investigated through the use of different biomarkers. Findings have been inconsistent (1118). Two of the studies used toenail selenium levels as a biomarker for assessment of selenium status. Toenail selenium levels provide a reliable and practical biomarker of past selenium intake; a single measurement of toenail selenium levels can rank subjects according to long-term selenium intake (19, 20). In a case-control study in the Netherlands (14), lower toenail selenium levels were associated with risk of acute nonfatal myocardial infarction. In a multicenter case-control study carried out in Europe (16), lower toenail selenium levels were associated with acute myocardial infarction, but the association was restricted to current smokers. Selenium levels in serum have also been used as a marker of selenium status. In a study in Finland, lower serum selenium levels were inversely associated with cardiovascular disease death and myocardial infarction (11). In another study, serum selenium concentration was not associated with the development of clinical manifestations of coronary heart disease (21).

Interactions between selenium and vitamin E have been documented. A combined dietary deficiency of selenium and vitamin E is fatal in animals (4). Selenium deficiency in animals produces conditions similar to those induced by vitamin E deficiency, and the effects of selenium deficiency appear to be alleviated by administering additional vitamin E and vice versa. Inverse associations between selenium status and cigarette smoking and alcohol intake have also been reported (22, 23), suggesting that these factors should also be carefully considered when examining relations between selenium and coronary heart disease. Previously, we examined the interaction between selenium and mercury in this cohort and did not find that toenail selenium levels modified the relation between mercury and coronary heart disease (24).

In this analysis, we assessed the association between prediagnostic levels of selenium in toenails and risk of coronary heart disease among men in a large prospective cohort study. We evaluated whether any influence of selenium was modified by cigarette smoking, vitamin E intake, or alcohol intake.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Population
This study used a case-control design nested within the Health Professionals Follow-up Study, a large prospective cohort study of diet and coronary heart disease and cancer among 51,529 US men who were aged 40–75 years in 1986. The population included 29,683 dentists, 10,098 veterinarians, 4,185 pharmacists, 3,745 optometrists, 2,218 osteopathic physicians, and 1,600 podiatrists. The study began in 1986 when all cohort members completed a mailed questionnaire. Every 2 years, follow-up questionnaires are sent to update information on newly diagnosed heart disease. We use the National Death Index to identify deceased cohort members. Over the first 8 years of follow-up, we received questionnaires from or confirmed the deaths of more than 94 percent of eligible participants (25, 26). In 1987, 33,737 sets of toenail clippings were collected from the cohort members and stored for subsequent analysis. Men whose reported daily energy intake from the 1986 semiquantitative food frequency questionnaire was below 800 kcal/day or greater than 4,200 kcal/day or who left 70 or more questions on the questionnaire blank (n = 1,595) were excluded from the baseline population for analysis. Men who reported a diagnosis of cancer (except nonmelanoma skin cancer), myocardial infarction, angina, or stroke, coronary artery bypass graft (CABG), or percutaneous transluminal coronary angioplasty (PTCA) on the 1986 questionnaire were further excluded. Other details related to the study population can be found elsewhere (24). This study was approved by the Institutional Review Board of the Harvard School of Public Health.

Case ascertainment
Cases were men who had fatal coronary heart disease, nonfatal myocardial infarction, CABG, or PTCA after return of the toenail samples in 1987 but before January 31, 1992. A letter was sent to all men who reported an incident myocardial infarction to confirm the event and ask for permission to review medical records. A nonfatal myocardial infarction was considered confirmed by a study physician if it met the World Health Organization criteria (27). A myocardial infarction was classified as "probable" if the medical records could not be obtained but the participant had required hospital admission and the diagnosis was corroborated by supplementary correspondence or telephone interview. Confirmation of CABG or PTCA was based on self-report only; hospital records obtained for a sample of 102 men confirmed the procedure for 96 percent (25). Most deaths were reported by family members, the postal system, colleagues at work, or the National Death Index. Fatal coronary heart disease was considered "confirmed" if it was the underlying cause on the death certificate and a diagnosis of incident coronary heart disease was confirmed by records or interviews. Sudden death was considered fatal coronary heart disease, because most sudden deaths in men result from myocardial infarction (25, 26).

In 5 years of follow-up between 1987 and 1992, 470 participants eligible for this analysis developed coronary heart disease (207 confirmed nonfatal myocardial infarction, 18 probable myocardial infarction, 62 fatal coronary heart disease, 44 sudden death, and 139 CABG or PCTA). Because our main hypothesis was related to low density lipoprotein cholesterol oxidation and thrombosis associated with atherosclerosis, we included all of these coronary endpoints in our primary analysis to maximize statistical power.

Control selection
For each coronary heart disease case, a control subject without coronary heart disease was chosen randomly from the eligible baseline cohort participants who provided toenails, matched on age within 1 calendar year, smoking status (current, past, or never), and date of toenail return within 1 month. Controls also had to be alive at the date of diagnosis of the matched case. In this analysis, we included 442 matched pairs and an additional 28 cases and 23 controls for whom the matched participant was excluded because of missing trace element or covariate data.

Exposure assessment
Selenium levels in toenails
Prior to the analysis, the toenail clippings were washed with deionized water using a sonicator, dried, weighed, and placed into high-density polyethylene vials for irradiation. Case and control specimens were analyzed together in the same analytical batch but in random order, with the case/control status of the toenail specimens unknown to the laboratory personnel.

Selenium levels in the toenails were analyzed by instrumental three-cycle neutron activation analysis at the University of Missouri Research Reactor (Columbia, Missouri). Specimens were irradiated in a high thermal flux to activate an isotope of selenium (Se77m). The analysis required 5 seconds of irradiation, a 15-second decay, and a 25-second counting period. The gamma-ray emissions from the isotope of selenium were quantified. The amount of selenium in the sample was calculated using the MANIP procedure in Canberra/Nuclear Data software (Canberra Industries, Inc., Itasca, Illinois) for summation of the spectra. In each analytical measurement, a reference standard of bovine liver from the National Bureau of Standards (NBS-SRM 1577) was measured to ensure that quality control standards were met. Specimens were measured twice, and those which had a coefficient of variation greater than 10 percent were reanalyzed to confirm the selenium concentration. In a quality control study which included samples with a selenium concentration range similar to that of the men in this study (unpublished data), we found an average percentage coefficient of variation of 3.0 percent for six split samples sent blinded to the research reactor.

Diet
Nutrient intakes were calculated from the 1986 dietary questionnaire. The questionnaire included 131 food items, each with a specified portion size. The cohort members reported the average frequency with which they had consumed each item during the previous year. They chose from nine response categories ranging from never or once per month to six or more times per day. The use of selenium supplements and multivitamins (brand and type, frequency, and duration) was also recorded. The average daily intake of nutrients with and without supplements was calculated by multiplying the frequency of consumption of each food item by the nutrient content of the specified portion size and totaling the nutrient intake for all of the food items. The nutrient composition of all foods was derived using data from the US Department of Agriculture (1976–1989) (28). The nutrient data were adjusted for total energy intake using the residual method (29).

We assessed the validity of the 1986 131-item food frequency questionnaire in this cohort by comparing average daily intake computed from the questionnaire with that obtained from two 1-week dietary records in a random sample of 127 men living in the Boston, Massachusetts, area (30). The correlation coefficients for correlation between the second questionnaire and the average of two 1-week diet records, adjusted for total energy intake and week-to-week variation in diet records, were 0.86 for alcohol (31), 0.65 for the mean of nutrients (30), and 0.63 for the mean of specific foods (32).

Statistical analysis
Differences in mean toenail selenium values between cases and controls were tested using a Wilcoxon rank sum test for unpaired analyses and a signed rank test for paired analyses (33). The association between toenail selenium level and risk of coronary heart disease was expressed as an odds ratio with a 95 percent confidence interval. Selenium values were categorized into quintiles based on the distribution among the controls, and the Mantel extension test was used to assess linear trend with increasing selenium level by using the median value for each quintile. Multiple logistic regression analysis was used to control for body mass index (weight (kg)/height (m)2) (quintiles), alcohol intake (four categories: 0, 0.1–5.0, 5.1–30.0, and >=30.1g/day), age (six categories: <=50, 51–55, 56–60, 61–65, 66–70, and >70 years), and smoking status (never, current, past, and 1–24 and >24 cigarettes/day). In the present analysis, the Pearson correlation between toenail selenium levels and toenail specimen sample weights was 0.12 after logarithmic transformation of the selenium values. Therefore, we included nail sample weight as a continuous variable in the multivariate analysis to control for this extraneous variation. Interactions between selenium and smoking status, vitamin E intake, and alcohol intake were assessed by stratified analysis and through the addition of cross-product terms (three categories for smoking status and continuous values for intakes of vitamin E and alcohol) to the multivariate model.

Unconditional and conditional logistic regression gave similar overall results. In this paper, we give only the results from the former method to increase the power in the subgroup analyses.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Baseline characteristics of the cases (n = 470) and controls (n = 465) are shown in table 1. Median age and smoking status were the same in cases and controls because of matching. However, the proportions of men with a family history of coronary heart disease, hypertension, hypercholesterolemia, or diabetes and the average body mass index in 1986 were all higher among the cases than among the controls. Intake of alcohol was higher in the controls than in the cases. The median toenail selenium level was slightly higher among the cases than among the controls. Mean selenium levels were 0.95 µg/g (standard deviation 0.43) for cases and 0.93 µg/g (standard deviation 0.29) for controls (nonparametric paired t test: p = 0.59 for the 442 matched pairs; Wilcoxon two-sample test: p = 0.47 for the 470 cases and 465 controls).


View this table:
[in this window]
[in a new window]
 
TABLE 1. Baseline characteristics of 470 coronary heart disease cases and 465 controls aged 40–75 years from the Health Professionals Follow-up Study, 1987–1992
 
Baseline characteristics of the controls according to quintile of toenail selenium level are shown in table 2. The range of selenium values was 0.59–4.27 µg/g. The proportion of current smokers in the bottom quintile (20 percent) was much greater than that in the highest quintile (8 percent). Use of selenium supplements (>50 µg/day) was more common among men with higher toenail selenium levels.


View this table:
[in this window]
[in a new window]
 
TABLE 2. Baseline characteristics of 465 controls aged 40–75 years from the Health Professionals Follow-up Study, according to quintile of toenail selenium level, 1987–1992
 
Odds ratios for coronary heart disease according to quintile of toenail selenium level are shown in table 3. Selenium level was not associated with risk of total coronary heart disease after adjustment for age and smoking; comparing the highest quintile with the lowest, the odds ratio was 0.96 (95 percent confidence interval (CI): 0.63, 1.45; p-trend = 0.85). After results were further simultaneously controlled for alcohol intake, body mass index, family history of coronary heart disease, hypertension, hypercholesterolemia, and diabetes, and toenail sample weight, the odds ratio was 0.86 (95 percent CI: 0.55, 1.32), and there was no overall trend (p-trend = 0.75). A test for interaction between smoking status (three categories: current, past, and never) and selenium level (quintiles) was not significant (p = 0.62). Adjustment for vitamin E intake and folate intake did not materially alter the results (odds ratio (OR) = 0.86, 95 percent CI: 0.56, 1.34; p-trend = 0.77). A test for the interaction between vitamin E intake (three categories) and toenail selenium values (quintiles) was not significant (p = 0.42), nor was the interaction between alcohol intake (four categories) and toenail selenium values (p = 0.68).


View this table:
[in this window]
[in a new window]
 
TABLE 3. Age-adjusted and multivariate odds ratios for total coronary heart disease by quintile of baseline toenail selenium level among 935 cases and controls, Health Professionals Follow-up Study, 1987–1992
 
The median toenail selenium level was lower in current smokers (0.79 µg/g) than in past smokers (0.87 µg/g) or never smokers (0.88 µg/g). Odds ratios for coronary heart disease according to selenium level within each stratum of smoking status are shown in table 4. Toenail selenium level was not significantly associated with coronary heart disease risk in any smoking category after multivariate adjustment.


View this table:
[in this window]
[in a new window]
 
TABLE 4. Multivariate odds ratios for coronary heart disease by quintile of toenail selenium level within three categories of smoking status, Health Professionals Follow-up Study, 1987–1992
 
We further examined associations between toenail selenium level and risk of coronary heart disease within categories of vitamin E and alcohol intake (data not shown). There was no evidence of any inverse relation between toenail selenium level and coronary heart disease risk at any level of vitamin E intake. Among men with a vitamin E intake greater than 30 IU/day or among men who consumed more than 30 g of alcohol per day, we found a suggestion of a direct association between higher toenail selenium levels and coronary heart disease; the associations were not statistically significant.

Table 5 shows results within specific categories of coronary heart disease. Selenium level was inversely associated with risk of nonfatal myocardial infarction when the highest quintile was compared with the lowest (OR = 0.54, 95 percent CI: 0.31, 0.93; p-trend = 0.07). The association between the two extreme quintiles of toenail selenium level was weaker and not statistically significant for fatal coronary heart disease (OR = 0.79, 95 percent CI: 0.39, 1.60; p-trend = 0.61) and was directly associated with CABG or PTCA (OR = 2.38, 95 percent CI: 1.11, 5.09; p-trend = 0.02). Further adjustment for vitamin E intake did not change these estimates (OR = 2.41, 95 percent CI: 1.13, 5.16). Because clinical decisions to conduct CABG or PTCA may be higher in states with a low selenium content in their soil (much of the East Coast and parts of the Midwest), we further controlled for estimated selenium content in the subject’s state of residence, but results did not differ.


View this table:
[in this window]
[in a new window]
 
TABLE 5. Multivariate odds ratios for nonfatal myocardial infarction, fatal coronary heart disease, and coronary artery bypass graft/percutaneous transluminal coronary angioplasty by quintile of baseline toenail selenium level, Health Professionals Follow-up Study, 1987–1992
 
When we examined the combined endpoint of nonfatal myocardial infarction and fatal coronary heart disease, the inverse association for selenium level was of borderline significance (OR = 0.64, 95 percent CI: 0.39, 1.02; p-trend = 0.12). After adjustment for aspirin use, the odds ratio was 0.63 (95 percent CI: 0.39, 1.02; p-trend = 0.11). The difference in the effect estimates between total myocardial infarction and CABG or PTCA suggests that selenium may contribute to an antithrombotic process. To examine this further, we stratified the analysis of combined nonfatal myocardial infarction and fatal coronary heart disease by aspirin use. The odds ratio was 0.53 (95 percent CI: 0.29, 0.99; p-trend = 0.07) among nonusers of aspirin (n = 224 cases, n = 307 controls) and 0.85 (95 percent CI: 0.37, 1.98; p-trend = 0.72) among users (n = 107 cases, n = 158 controls). The odds ratio was 0.53 (95 percent CI: 0.30, 0.92; p-trend = 0.05) among alcohol drinkers (n = 244 cases, n = 372 controls) and 1.27 (95 percent CI: 0.47, 3.48; p-trend = 0.51) among nondrinkers (n = 87 cases, n = 93 controls).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In our study, toenail selenium levels were not associated with the risk of total coronary heart disease. This lack of an association cannot be explained by changes in toenail selenium levels after disease diagnoses, because the toenail specimens were collected before the coronary heart disease events occurred. Lack of an association between selenium levels and total coronary heart disease also could be due to a limited range of selenium exposures. However, our population represented men from across the United States, a geographic area with a wide distribution of soil selenium levels (34). We may have had limited power to detect an association if toenails are not a good indicator of selenium levels. However, a sample from each toe taken at one point in time reflects the formation of toenails over a period of approximately 1 year. Longnecker et al. (20) examined the validity of selenium concentration obtained from a single biologic specimen as a surrogate measure of selenium intake and found that toenails served reasonably well as a measure for ranking subjects according to long-term selenium intake; the adjusted Pearson correlation coefficient was 0.67 for the correlation between selenium intake and a single toenail selenium measurement. In addition, nail selenium levels provide an indicator of long-term exposure (10, 19). Among women in the Nurses’ Health Study, Spearman’s r was 0.48 for specimens obtained from the same persons after an interval of 6 years (35). Further evidence that selenium intake varies within the cohort and that toenail selenium levels capture this variation is provided by the observation that in this same cohort toenail selenium was strongly inversely associated with risk of prostate cancer (36).

The selenium levels among current smokers were lower, but smoking did not modify the association between selenium status and risk of total coronary heart disease. Similarly, vitamin E intake and alcohol intake did not modify the association. In a separate analysis, we also found that selenium did not modify the null association between mercury and coronary heart disease (24).

The positive association between selenium level and the risk of CABG or PTCA is not readily explained biologically; the association may be due to chance. Alternatively, men who elect to have surgery for coronary artery disease and are deemed suitable candidates for the procedure may be the healthiest of those men who develop coronary disease. These men may be more likely to be taking supplements (selenium, antioxidants, or others), which could lead to bias. However, this is unlikely to explain our results, because the odds ratio between extreme levels of toenail selenium was still elevated among participants who did not take any antioxidant supplements (n = 42 cases, n = 141 controls; OR = 2.64, 95 percent CI: 0.60, 11.55; p-trend = 0.26). Furthermore, the general characteristics of cases outlined in table 1 were similar for CABG or PTCA, nonfatal myocardial infarction, and fatal coronary heart disease endpoints.

Kok et al. (14) found an inverse association between toenail selenium level and acute nonfatal myocardial infarction. The range of exposures (median values for the first quartile and the fourth quartile were 0.66 µg/g and 0.90 µg/g) was similar to the range in our study (median values for the first quintile and the fifth quintile were 0.71 µg/g and 1.10 µg/g). The findings from this Dutch study, together with ours, suggest the possibility of different biologic roles for selenium in relation to specific coronary heart disease events. For example, selenium could have antithrombotic effects (37) and thus could prevent myocardial infarction but not angina. However, evidence from a small feeding study did not support an important role for low-selenium diets in platelet aggregation (38)). Furthermore, Thiele et al. (39) reported that average serum selenium levels among patients with severe angina pectoris (but without acute cardiac infarction) were significantly lower than levels among controls. In our current study, higher levels of selenium were inversely associated with risk of combined nonfatal myocardial infarction and fatal coronary heart disease, particularly among nonusers of aspirin. This suggests that selenium may have the strongest effects, those that are most beneficial, among men not already taking antiplatelet medication.

In conclusion, we found that toenail selenium levels are not strongly inversely associated with risk of total coronary heart disease. This relation was not modified by smoking status, vitamin E intake, or alcohol intake. However, a specific protective role for myocardial infarction could not be excluded. Further prospective studies are needed to evaluate the relation of selenium status to specific coronary heart disease events.


    ACKNOWLEDGMENTS
 
This work was supported by research grants HL35464 and CA55075 from the National Institutes of Health.

The authors thank Jill Arnold, Alvin Wing, Karen Corsano, Mira Kaufman, Betsy Frost-Hawes, Kerry Pillsworth-Demers, Mitzi Wolff, Laura Sampson, and Hsi-chiang Li for their expert assistance.


    NOTES
 
Correspondence to Dr. Eric B. Rimm, Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115 (e-mail: erimm{at}hsph.harvard.edu). Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Burk RF. Clinical effects of selenium deficiency. In: Prasad AS, ed. Essential and toxic trace elements in human health and disease: an update. New York, NY: Wiley-Liss, Inc, 1993.
  2. Rotruck JT, Pope AL, Ganther HE, et al. Selenium: biochemical role as a component of glutathione peroxidase. Science 1973;179:588–90.[ISI][Medline]
  3. Burk RF. Biological activity of selenium. Ann Rev Nutr 1983;3:53–70.[CrossRef][ISI][Medline]
  4. Halliwell B, Gutteridge JM. Free radicals in biology and medicine. New York, NY: Oxford University Press, 1989.
  5. Burk RF, Lawrence RA. Non selenium-dependent glutathione peroxidase. In: Wendel SH, ed. Functions of glutathione in liver and kidney. Berlin, Germany: Springer, 1978:114–19.
  6. Steinberg D. Antioxidants and atherosclerosis: a current assessment. Circulation 1991;84:1420–5.[ISI][Medline]
  7. Meister A. On the antioxidant effects of ascorbic acid and glutathione. Biochem Pharmacol 1992;44:1905–15.[CrossRef][ISI][Medline]
  8. Levander OA. The need for a measure of selenium status. J Am Coll Toxicol 1986;5:37–44.[ISI]
  9. Levander OA. A global view of human selenium nutrition. Ann Rev Nutr 1987;7:227–50.[CrossRef][ISI][Medline]
  10. Ovaskainen M-L, Virtamon J, Alfthan G, et al. Toenail selenium as an indicator of selenium intake among middle-aged men in an area with low soil selenium. Am J Clin Nutr 1993;57:662–5.[Abstract]
  11. Salonen JT, Alfthan G, Huttunen JK, et al. Association between cardiovascular death and myocardial infarction and serum selenium in a matched-pair longitudinal study. Lancet 1982;2:175–9.[ISI][Medline]
  12. Virtamo J, Rapola JM, Ripatti S, et al. Effect of vitamin E and beta carotene on the incidence of primary nonfatal myocardial infarction and fatal coronary heart disease. Arch Intern Med 1998;158:668–75.[Abstract/Free Full Text]
  13. Kok FJ, de Bruijn AM, Vermeeren R, et al. Serum selenium, vitamin antioxidants, and cardiovascular mortality: a 9-year follow-up study in the Netherlands. Am J Clin Nutr 1987;45:462–8.[Abstract]
  14. Kok FJ, Hofman A, Witteman JC, et al. Decreased selenium levels in acute myocardial infarction. J Am Med Assoc 1989;261:1161–4.[Abstract]
  15. Salvini S, Hennekens CH, Morris JS, et al. Plasma levels of the antioxidant selenium and risk of myocardial infarction among US physicians. Am J Cardiol 1995;76:1218–21.[CrossRef][ISI][Medline]
  16. Kardinaal AF, Kok FJ, Kohlmeier L, et al. Association between toenail selenium and risk of acute myocardial infarction in European men—The Euramic Study. Am J Epidemiol 1997;145:373–9.[Abstract]
  17. Mihailovic MB, Avramovic DM, Jovanovic IB, et al. Blood and plasma selenium levels and GSH-Px activities in patients with arterial hypertension and chronic heart disease. J Environ Pathol Toxicol Oncol 1998;17:285–9.[Medline]
  18. Bor MV, Cevik C, Uslu I, et al. Selenium levels and glutathione peroxidase activities in patients with acute myocardial infarction. Acta Cardiol 1999;54:271–6.[ISI][Medline]
  19. Willett W. Nutritional epidemiology: issues and challenges. Int J Epidemiol 1987;16:312–17.[Abstract]
  20. Longnecker MP, Stram DO, Taylor PR, et al. Use of selenium concentration in whole blood, serum, toenails, or urine as a surrogate measure of selenium intake. Epidemiology 1996;7:384–90.[ISI][Medline]
  21. Miettinen TA, Alfthan G, Huttunen JK, et al. Serum selenium concentration related to myocardial infarction and fatty acid content of serum lipids. Br Med J (Clin Res Ed) 1983;287:517–19.[Medline]
  22. Lloyd B, Lloyd RS, Clayton BE. Effect of smoking, alcohol and other factors on the selenium status of a healthy population. J Epidemiol Community Health 1983;37:213–17.[Abstract]
  23. Hunter DJ, Morris JS, Chute CG, et al. Predictors of selenium concentration in human toenails. Am J Epidemiol 1990;132:114–22.[Abstract]
  24. Yoshizawa K, Rimm EB, Morris JS, et al. Mercury and the risk of coronary heart disease in men. N Engl J Med 2002;347:1755–60.[Abstract/Free Full Text]
  25. Rimm EB, Giovannucci EL, Willett WC, et al. Prospective study of alcohol consumption and risk of coronary disease in men. Lancet 1991;338:464–8.[ISI][Medline]
  26. Rimm EB, Ascherio A, Giovannucci E, et al. Vegetable, fruit, and cereal fiber intake and risk of coronary heart disease among men. JAMA 1996;275:447–51.[Abstract]
  27. Rose GA, Blackburn H. Cardiovascular survey methods. (WHO monograph series no. 58). Geneva, Switzerland: World Health Organization, 1982.
  28. US Department of Agriculture. Composition of foods—raw, processed, and prepared, 1963–1988. (Agricultural handbook no. 8). Washington, DC: US Department of Agriculture, 1989.
  29. Willett WC. Implications of total energy intake for epidemiologic analyses. In: Willett WC, ed. Nutritional epidemiology. New York, NY: Oxford University Press, 1998:273–301.
  30. Rimm EB, Giovannucci EL, Stampfer MJ, et al. Reproducibility and validity of an expanded self-administered semiquantitative food frequency questionnaire among male health professionals. Am J Epidemiol 1992;135:1114–26.[Abstract]
  31. Giovannucci E, Colditz G, Stampfer MJ, et al. The assessment of alcohol consumption by a simple self-administered questionnaire. Am J Epidemiol 1991;133:810–17.[Abstract]
  32. Feskanich D, Rimm EB, Giovannucci EL, et al. Reproducibility and validity of food intake measurements from a semiquantitative food frequency questionnaire. J Am Diet Assoc 1993;93:790–6.[ISI][Medline]
  33. Rosner B. Fundamentals of biostatistics. Boston, MA: Duxbury Press, 1990.
  34. Hunter D. Biochemical indicators of dietary intake. In: Willett WC, ed. Nutritional epidemiology. New York, NY: Oxford University Press, 1998:174–243.
  35. Garland M, Morris JS, Rosner BA, et al. Toenail trace element levels as biomarkers: reproducibility over a 6-year period. Cancer Epidemiol Biomarkers Prev 1993;2:493–7.[Abstract]
  36. Yoshizawa K, Willett WC, Morris SJ, et al. Study of prediagnostic selenium level in toenails and the risk of advanced prostate cancer. J Natl Cancer Inst 1998;90:1219–24.[Abstract/Free Full Text]
  37. Meltzer HM, Mundal HH, Alexander J, et al. Does dietary arsenic and mercury affect cutaneous bleeding time and blood lipids in humans? Biol Trace Elem Res 1994;46:135–53.[ISI][Medline]
  38. van Dokkum W, Van der Torre HW, Schaafsma G, et al. Supplementation with selenium-rich bread does not influence platelet aggregation in healthy volunteers. Eur J Clin Nutr 1992;46:445–50.[ISI][Medline]
  39. Thiele R, Schuffenhauer M, Winnefeld K, et al. Selenium levels in patients with acute myocardial infarct and in patients with severe angina pectoris without myocardial infarct. Med Klin 1995;90(suppl 1):45–8.