Serum Ferritin and Cardiovascular Disease: A 17-Year Follow-up Study in Busselton, Western Australia

M. W. Knuiman1 , M. L. Divitini1, J. K. Olynyk2, D. J. Cullen3 and H. C. Bartholomew1

1 School of Population Health, The University of Western Australia, Crawley, Western Australia, Australia.
2 School of Medicine, The University of Western Australia, Crawley, Western Australia, Australia.
3 Department of Gastroenterology, Fremantle Hospital, Fremantle, Western Australia, Australia.

Received for publication October 24, 2002; accepted for publication January 22, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The association between serum ferritin level and coronary heart disease (CHD) and stroke events was evaluated in a long-term Western Australia prospective study in 1981–1998. The cohort consisted of the 1,612 men and women aged 40–89 years who participated in the 1981 Busselton Health Survey and who were free of cardiovascular disease at that time. Serum ferritin levels were obtained from serum samples stored frozen since 1981. The outcomes of interest were time to first CHD event (hospital admission or death) and time to first stroke event. Case-cohort sampling was used to reduce costs and preserve serum but still allow efficient analysis. Ferritin assays were performed for 217 CHD cases, 118 stroke cases, and a random sample of 450 of the total cohort. Proportional hazards regression models were used to obtain age-adjusted and multivariate-adjusted hazard ratios for ferritin level in relation to CHD and stroke. The hazard ratio for the highest tertile group compared with the lowest group was 0.96 (95% confidence interval: 0.60, 1.53) for CHD and 1.43 (95% confidence interval: 0.78, 2.64) for stroke. Little or no evidence was found that ferritin level was a risk factor for cardiovascular disease.

cardiovascular diseases; cohort studies; ferritin; proportional hazards models

Abbreviations: Abbreviations: CHD, coronary heart disease; ICD-9, International Classification of Diseases, Ninth Revision.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In 1981, Sullivan (1) proposed that body iron stores are positively related to coronary heart disease (CHD) risk. The theory was that production of free radicals that subsequently modify low density lipoprotein cholesterol was important in the development of atherosclerosis and that iron helps to catalyze the oxidation reactions that produce free radicals (2, 3). Since the 1992 publication of data from Finland showing a positive relation between serum ferritin and risk of acute myocardial infarction in men (4), there has been considerable interest in this theory.

The accumulated epidemiologic evidence has been inconsistent, but most studies do not support the theory (2, 3, 5, 6). Studies have varied in their design and the measure of stored body iron used. Serum ferritin is regarded as the best biochemical measure of body iron stores (7). Most of the cross-sectional and case-control studies used serum ferritin, and a few found a significant association. However, because the disease itself and changed behavior due to disease influence serum ferritin levels, these study findings are not as useful as those from prospective studies. Eight prospective studies have been reported, and two found a significant association, one with CHD in a Finnish study (4) and one with an ultrasound measure of atherosclerosis in an Italian study (8). Most of the prospective studies had a short follow-up, few provided results for women, and few involved more than 100 cases. In addition, despite the iron theory relating to atherosclerosis in general, few studies have examined serum ferritin in relation to other forms of cardiovascular disease such as stroke. Given the inconsistency and limitations of previous studies, it is surprising that there have been suggestions for changes to dietary recommendations and a suggestion of phlebotomy as a means of lowering iron levels (9, 10). Clearly, further long-term prospective studies are needed.

The Busselton Health Study in Western Australia (Internet Web site—http://bsn.uwa.edu.au), which included comprehensive cardiovascular risk factor and disease data, stored serum collection, and hospital admission and mortality follow-up of representative community cohorts, provided an ideal opportunity to examine the relation between serum ferritin levels and cardiovascular disease. A previous analysis investigated the association between serum ferritin and CHD cross-sectionally within the Busselton 1994 survey and also prospectively for that cohort over the period 1994–1998 (11). This paper reports findings from a further prospective analysis based on the 1981 survey cohort that has been followed for CHD and stroke events over the 17-year period 1981–1998.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Study population
This study is based on the 1,612 men and women aged 40–89 years who participated in the 1981 Busselton Health Survey and had no history of diagnosed CHD or stroke at that time. The conduct of the Busselton health surveys has been described previously (12, 13). The target population was all adults on the electoral roll (registration to vote is compulsory in Australia) in the Shire of Busselton; the overall response rate for the 1981 survey was 64 percent.

Measurement methods
Survey participants were asked to complete a comprehensive health and lifestyle questionnaire and to undergo various measurements and tests. Systolic and diastolic blood pressure were measured by mercury sphygmomanometer after 5 minutes of rest in a sitting position. Height was measured by stadiometer for barefooted subjects, and weight was measured with subjects wearing light underclothes. Body mass index was derived as weight (in kilograms) divided by the square of height (in meters). Information on smoking, diabetes, use of antihypertensive medication, and history of stroke was obtained by questionnaire. CHD was determined from the Rose Questionnaire for angina and myocardial infarction and the electrocardiogram together with a self-reported confirmation that their physicians had reported that they had heart disease (14). Serum cholesterol, triglycerides, and hemoglobin measurements were determined from a fasting blood sample at the time of the survey. The sera were frozen and were stored at –70°C. The ferritin assays were performed on the stored sera in 2001 by using a two-step chemiluminometric immunoassay on an Abbott Architect analyzer (Abbott Diagnostics, Melbourne, Australia).

Cardiovascular outcomes
The outcomes of interest were time from the 1981 survey to first CHD event and time to first stroke event, where event meant hospital admission or death. Events up to 1998 were obtained by linkage to the statewide Hospital Morbidity Data System (HMDS) and the annual death list for Western Australia (15). This system covers all admissions to public and private hospitals in Western Australia from 1980 onward. Follow-up of subjects ended with the outcome of interest; on December 31, 1998; or when subjects left Western Australia. Stroke cases occurring during follow-up were defined as an admission to hospital with a diagnosis of stroke (International Classification of Diseases, Ninth Revision (ICD-9), codes 430–438 inclusive) or death from stroke (ICD-9 codes 430–438). CHD cases were defined as an admission to hospital with a diagnosis of CHD (ICD-9 codes 410–414 inclusive) or any procedure that indicates CHD (ICD-9 codes 360–363, i.e., angioplasty, coronary artery bypass graft, or revascularization) or as death from CHD (ICD-9 codes 410–414 inclusive).

Case-cohort sampling
Case-cohort sampling (16) was used to reduce costs and preserve stored serum. A viable blood serum sample was available for about 75 percent of the eligible disease-free cohort. Ferritin assays were conducted for all 217 CHD cases, all 118 stroke cases, and a random sample of 450 participants in the total cohort, which provided the study with 89 percent power to detect a hazard ratio of 1.3 for CHD for a one standard deviation change in a continuous risk factor (17). Because of overlap between CHD/stroke cases and the random subcohort, the total number of assayed sera samples was 626.

Statistical analysis
To validate the measurement of ferritin levels from serum that had been stored frozen for 20 years, we compared the age-sex (mean) profile for the 1981 ferritin measurements and their correlations with other biochemical variables for the random subcohort (a representative sample of the 1981 population) with the 1994 Busselton Health Survey (n = 3,079), for which ferritin levels were determined from fresh serum samples.

The relation between ferritin levels and CHD or stroke was investigated by using proportional hazards regression modeling with an estimation procedure adapted for case-cohort designs (18), implemented in the SAS statistical software package (19) with the macro ROBPHREG (20). Both the univariate (i.e., after controlling for age and gender) and multivariate (i.e., after controlling for age, gender, body mass index, cholesterol, triglycerides, diabetes, hemoglobin, treatment for hypertension, systolic blood pressure, and smoking) effects of ferritin were examined. Because the univariate and multivariate results were similar, only the multivariate results are reported in this paper. Ferritin level was assessed both as a continuous variable (after log transformation) and in tertile groups. Gender-specific tertile cutpoints were obtained from the random subcohort. Associations were expressed as estimated relative risks (hazard ratios) with 95 percent confidence intervals.

Ethics approval
This study was approved by the Human Research Ethics Committee of The University of Western Australia.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Table 1 shows the baseline characteristics of the CHD cases, stroke cases, random subset, and full cohort by sex. The characteristics of the random subcohort were similar to those of the full cohort, indicating that the subjects for whom viable sera were available were representative of the full cohort. In the random subcohort, 42 percent of the subjects were men, the mean age at baseline was 59 years for men and 58 years for women, and the subjects had a cardiovascular risk factor profile typical of Australian populations at that time (21). Mean ferritin level was 213 µg/liter in men and 101 µg/liter in women.


View this table:
[in this window]
[in a new window]
 
TABLE 1. Characteristics* of Busselton Health Study subjects at baseline in 1981, Western Australia
 
Figure 1 shows the mean of log ferritin by sex and age for the random subcohort for the 1981 survey and the 1994 survey. The correlation between log ferritin and hemoglobin was 0.38 for the 1981 subset and 0.41 for the 1994 survey. The similarity of the age/sex profiles and correlations for the two surveys confirms the validity of the ferritin assays for the 1981 survey on 20-year-old sera.



View larger version (18K):
[in this window]
[in a new window]
 
FIGURE 1. Mean log ferritin levels, by sex and age, for men and women in the 1981 and 1994 Busselton Health Surveys, Western Australia.

 
Table 2 shows the estimated multivariate-adjusted hazard ratios for log ferritin as a continuous variable and ferritin tertile groups in relation to CHD and stroke events. Note that the reported hazard ratio for continuous log ferritin is for a change of one, which is approximately the standard deviation of log ferritin (table 1). There was little or no evidence in the sex-specific and overall results to indicate that serum ferritin is an independent risk factor for CHD and/or stroke. No pattern suggestive of a relation was found, and none of the relative risk estimates reached statistical significance at the 5 percent level. The hazard ratios for serum ferritin were not appreciably altered when hemoglobin was excluded from the models.


View this table:
[in this window]
[in a new window]
 
TABLE 2. Ferritin tertiles and log ferritin in relation to CHD* event, stroke event, and CHD or stroke event in the Busselton Health Study, Western Australia, 1981–1998
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This is the second study of ferritin in relation to cardiovascular disease in Busselton. The first was based on the 1994 survey and consisted of a cross-sectional analysis of ferritin levels in relation to history of CHD and a 4-year follow-up analysis for cardiovascular disease in the subset of participants who were CHD free in 1994 (11). The cross-sectional odds ratio and prospective hazard ratio for continuous ferritin were close to one and were not statistically significant (odds ratio = 0.94, 95 percent confidence interval: 0.78, 1.14; hazard ratio = 1.03, 95 percent confidence interval: 0.94, 1.12). This prospective study based on the 1981 survey cohort included long-term follow-up (17 years) and considered CHD (217 cases during follow-up) and stroke (118 cases during follow-up) separately as well as together. It had adequate statistical power to detect relative risks of 1.3 or more (for a one standard deviation change in log ferritin); hence, true relative risks of that magnitude are not supported by these Busselton data. These two studies indicate that serum ferritin is not a significant predictor of cardiovascular events in this population.

It is possible that ferritin levels measured from 20-year-old serum may be an inaccurate indicator of stored iron levels. Comparison of ferritin data from 1981 samples (on stored serum) and from 1994 samples (on fresh serum) indicated that valid measures were obtained from stored frozen sera. However, we did not have data to directly compare ferritin levels from assays on fresh serum and from assays performed later on the same stored serum and could find no other studies that had carried out such a validity comparison. A number of other studies have also successfully obtained ferritin levels from stored serum, including the original positive Finnish study (4) and a more recent analysis from the Rotterdam Study (22). A follow-up paper from the Finnish study reported no association between serum ferritin levels and storage time, supporting the view that levels are maintained with multiyear storage (23).

The majority of studies have involved populations of predominantly European ancestry, and few have studied women. Our Busselton population is also predominantly of Anglo-Celtic ancestry, but our results were similar for women and men. In addition, most studies have focused on CHD. Our negative result for stroke, albeit lacking in power and failing to distinguish between hemorrhagic and ischemic stroke, is suggestive of a negative finding for cerebrovascular disease.

The initial report from the well-conducted Finnish study described a twofold increase in risk of myocardial infarction for men whose serum ferritin levels were higher than 200 µg/liter, which persisted after an additional 2 years of follow-up (4, 24). In our study, the upper-tertile threshold was 233 µg/liter for men. Because the mean level of ferritin for men in our sample was 213 µg/liter compared with 166 µg/liter in the Finnish sample, the threshold values are relatively comparable. However, the relative risk estimate for CHD for men whose ferritin levels were above 233 µg/liter was 0.86 in our study. The only other known prospective study to support the iron and atherosclerosis theory was the Italian Bruneck Study, which found an association with ultrasound measures of carotid atherosclerosis (8). More recently, a Second National Health and Nutrition Examination Survey Mortality Study analysis of serum ferritin in relation to death from cardiovascular diseases failed to find a significant association in (White) men or women, but a possible U-shaped relation was suggested for women (25). The relative risk for women whose ferritin levels were less than 50 µg/liter was double (95 percent confidence interval: 0.9, 4.7) that of women whose ferritin levels were 50–99 µg/liter. Our two lowest ferritin groups were less than or equal to 49 µg/liter and greater than 49 to less than or equal to 122 µg/liter, but we observed a smaller (not greater) risk for the former group. Furthermore, for both men and women, the risk of CHD was greatest in the middle ferritin tertile group; hence, our results did not provide any evidence for a U-shaped trend for men or women.

It is possible that ferritin may play a role through other risk factors such as blood pressure and cholesterol. However, many studies (including our own) have failed to find a positive association both before and after adjusting for a wide range of cardiovascular risk factors, so this possibility also seems unlikely. Few studies have (or have the statistical power to) thoroughly investigated possible interactions (synergy) between serum ferritin and other risk factors, and this association must remain a possibility.

We know of three studies motivated by the iron and CHD theory that have examined the possible benefits of blood donation in relation to protection from cardiovascular disease (2628). Only the study among Finnish men that also reported the original positive association for serum ferritin and CHD found an association between blood donation and CHD (26).

In conclusion, accumulated evidence from prospective studies does not support the iron theory. Further prospective studies, especially studies of women and studies able to investigate interactions/synergies between serum ferritin and other risk factors, are required.


    ACKNOWLEDGMENTS
 
This study was supported by project grant 110213 from the National Health and Medical Research Council of Australia.

The ferritin assays were carried out by PathCentre under the supervision of Dr. John Beilby.

The authors thank the Busselton Population Medical Research Foundation for access to the survey data and specimens and the community of Busselton for their long-standing cooperation and support for the Busselton Health Study.


    NOTES
 
Correspondence to Prof. Matthew W. Knuiman, School of Population Health, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia (e-mail: matthew{at}dph.uwa.edu.au). Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sullivan JL. Iron and the sex difference in heart disease risk. Lancet 1981;1:1293–4.[ISI][Medline]
  2. Sempos CT, Looker AC, Gillum RF. Iron and heart disease: the epidemiologic data. Nutr Rev 1996;54:73–84.[ISI][Medline]
  3. Meyers DG. The iron hypothesis—does iron cause atherosclerosis? Clin Cardiol 1996;19:925–9.[ISI][Medline]
  4. Salonen JT, Nyyssonen K, Korpela H, et al. High stored iron levels are associated with excess risk of myocardial infarction in eastern Finnish men. Circulation 1992;86:803–11.[Abstract]
  5. Sempos CT. Do body iron stores increase the risk of developing coronary heart disease? Am J Clin Nutr 2002;76:501–3.[Free Full Text]
  6. Ma J, Stampfer MJ. Body iron stores and coronary heart disease. Clin Chem 2002;48:601–3.[Free Full Text]
  7. Cook JD, Lipschitz DA, Miles LE, et al. Serum ferritin as a measure of iron stores in normal subjects. Am J Clin Nutr 1974;27:681–7.[ISI][Medline]
  8. Kiechl S, Willeit J, Egger G, et al. Body iron stores and the risk of carotid atherosclerosis: prospective results from the Bruneck study. Circulation 1997;96:3300–7.[Abstract/Free Full Text]
  9. Sullivan JL. Iron versus cholesterol—perspectives on the iron and heart disease debate. J Clin Epidemiol 1996;49:1345–52.[CrossRef][ISI][Medline]
  10. De Valk B, Marx JJM. Iron, atherosclerosis, and ischemic heart disease. Arch Intern Med 1999;159:1542–8.[Abstract/Free Full Text]
  11. Fox CJ, Cullen DJ, Knuiman MW, et al. Effects of body iron stores and haemochromatosis genotypes on coronary heart disease outcomes in the Busselton health study. J Cardiovasc Risk 2002;9:287–93.[CrossRef][ISI][Medline]
  12. Knuiman MW, Cullen KJ, Bulsara MK, et al. Mortality trends, 1965 to 1989, in Busselton, the site of repeated health surveys and interventions. Aust J Public Health 1994;18:129–35.[ISI][Medline]
  13. Knuiman MW, Jamrozik K, Welborn TA, et al. Age and secular trends in risk factors for cardiovascular disease in Busselton. Aust J Public Health 1995;19:375–82.[ISI][Medline]
  14. Welborn TA, Cumpston GN, Cullen KJ, et al. The prevalence of coronary heart disease and associated factors in an Australian rural community. Am J Epidemiol 1969;89:521–36.[ISI][Medline]
  15. Holman CDJ, Bass AJ, Rouse IL, et al. Population-based linkage of health records in Western Australia: development of a health services research linked database. Aust N Z J Public Health 1999;23:453–9.[ISI][Medline]
  16. Prentice RL. A case-cohort design for epidemiologic cohort studies and disease prevention trials. Biometrika 1986;73:1–11.[ISI]
  17. Wacholder S, Gail M, Pee D. Selecting an efficient design for assessing exposure-disease relationships in an assembled cohort. Biometrics 1991;47:63–76.[ISI][Medline]
  18. Barlow WE. Robust variance estimation for the case-cohort design. Biometrics 1994;50:1064–72.[ISI][Medline]
  19. SAS Institute, Inc. Statistical analysis system, version 6.12. Cary NC: SAS Institute, Inc, 1997.
  20. Ichikawa L, Barlow W. ROBPHREG, version 1.0.2: survival analysis macro with robust variance. Seattle, WA: Center for Health Studies, Group Health Cooperative, 1998. (http://lib.stat.cmu.edu/general/robphreg).
  21. Jamrozik K, Hockey R. Trends in risk factors for vascular diseases in Australia. Med J Aust 1989;150:14–18.[ISI][Medline]
  22. Klipstein-Grobusch K, Koster JF, Grobbee DE, et al. Serum ferritin and risk of myocardial infarction in the elderly: the Rotterdam Study. Am J Clin Nutr 1999;69:1231–6.[Abstract/Free Full Text]
  23. Tuomainen TP, Punnonen K, Nyyssonen K, et al. Association between body iron stores and risk of myocardial infarction in men. Circulation 1998;97:1461–6.[Abstract/Free Full Text]
  24. Salonen JT, Nyyssonen K, Salonen R. Body iron stores and the risk of coronary heart disease. (Letter). N Engl J Med 1994;331:1159.[Free Full Text]
  25. Sempos CT, Looker AC, Gillum RE, et al. Serum ferritin and death from all causes and cardiovascular disease: the NHANES II Mortality Study. National Health and Nutrition Examination Study. Ann Epidemiol 2000;10:441–8.[CrossRef][ISI]
  26. Salonen JT, Tuomainen TP, Salonen R, et al. Donation of blood is associated with reduced risk of myocardial infarction. The Kuopio Ischaemic Heart Disease Risk Factor Study. Am J Epidemiol 1998;148:445–51.[Abstract]
  27. Meyers DG, Strickland D, Maloney PA, et al. Possible association of a reduction in cardiovascular events with blood donation. Heart 1997;78:188–93.[Abstract]
  28. Ascherio A, Rimm EB, Giovannucci W, et al. Blood donations and risk of coronary heart disease in men. Circulation 2001;103:52–7.[Abstract/Free Full Text]