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.
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ABSTRACT |
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cardiovascular diseases; cohort studies; ferritin; proportional hazards models
Abbreviations: Abbreviations: CHD, coronary heart disease; ICD-9, International Classification of Diseases, Ninth Revision.
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INTRODUCTION |
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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 sitehttp://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 19941998 (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 19811998.
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MATERIALS AND METHODS |
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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 430438 inclusive) or death from stroke (ICD-9 codes 430438). CHD cases were defined as an admission to hospital with a diagnosis of CHD (ICD-9 codes 410414 inclusive) or any procedure that indicates CHD (ICD-9 codes 360363, i.e., angioplasty, coronary artery bypass graft, or revascularization) or as death from CHD (ICD-9 codes 410414 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.
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RESULTS |
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DISCUSSION |
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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 5099 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.
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ACKNOWLEDGMENTS |
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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.
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NOTES |
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REFERENCES |
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