The iron paradigm of ischemic heart disease

Volume 1 I? Number 5

9.

10.

11.

12.

AIDS and heart disease

death in acquired immunodeficiency syndrome. Arch Path01 Lab Med 1985;109:735. Silver MA, Macher AM, Reichert CM, Levens DL, Parrillo JE, Longo DL, Roberta WC. Cardiac involvement by Kaposi’s sarcoma in acquired immunodeficiency syndrome. Am J Cardiol 1984$3:983. Balasubramayam A, Waxman M, Zaxal JL, Lee MH. Malignant lymphoma of the heart in acquired immune deficiency syndrome. Chest 1986;90:243. D’Cruz IA, Sengupta EE, Abrahams C, Redy HK, Turlapati RV. Cardiac involvement, including tuberculous pericardial effusion complicating acquired immune deficiency syndrome. AM HEART J 1986;112:1100. Sunderam G, McDonald RJ, Maniatis T, Oleshe J, Kapila R, Reichman LB. Tuberculosis as a manifestation of the acquired immunodeficiency syndrome (AIDS). JAMA 1986; 2561362.

13.

14.

15.

16.

17.

Lewis W, Lipsich J, Cammarosano C. Cryptococcal myocarditis in acquired immune deficiency syndrome. Am J Cardiol 1985;55:1240. Lafont A, Wolff M, Marche C, Clair B, Regnier B. Overshelming myocarditis due to Cryptococcus neoformans in an AIDS patient. Lancet 1987;2:1145. Schuster M, Valentine F, Holzman R. Cryptococcal pericarditis in an intravenous drug abuser. J Infect Dis 1985; 153:842. Zugar A, Holzman RS, Simberkoff MS, Rahal JJ. Cryptococcal disease in patients with the acquired immunodeficiency syndrome. Ann Intern Med 1986;104:234. Henochowicz S, Mustafa M, Lawrinson WE, Pistole M, Lindsey J. Cardiac aspergillosis in acquired immune deficiency syndrome. Am J Cardiol 1985;55:1239.

18. Holtz HA, Lavery DP, Dapila R. Actinomycetales infection in the acquired immunodeficiency syndrome. Ann Intern Med 1985;102:203. 19. Raffanti SP, Chiaramida AJ, Sen P, Wright P, Middleton JR, Chiaramida S. Assessment of cardiac function in patients with the acquired immunodeficiency syndrome. Chest 1988; 93:592. in 20. Fink L, Reichek N, Sutton M. Cardiac abnormalities acquired immune deficiency syndrome. Am J Cardiol 1984; 54:1161. 21. Calabrese LH, Proffitt MR, Yen-Leberman B, Hobbs RE, Ratliff NB. Congestive cardiomyopathy and illness related to the acquired immunodeficiency syndrome associated with isolation of retrovirus from myocardium. Ann Intern Med 1987;107:691. 22. Dittrich H, Chow L, Denaro F, Spector S. Human immunodeficiency virus, coxsackievirus, and cardiomyopathy. Ann Intern Med 1988;108:308. 23. Kereiakis DJ, Parmley WW. Myocarditis and cardiomyopathy. AM HEART J 1984;108:1318. 24. Wink K, Schmitz H. Cytomegalovirus myocarditis. AM HEART J 1980;100:667. 25. Baroldi G, Corallo S, Moroni M, Repossini A, Mutinelli MR, Laxzarin A, Antonacci CM, Cristina S, Negri C. Local lymphocytic myocarditis in acquired immunodeficiency syndrome (AIDS): a correlative morphologic and clinical study in 26 consecutive fatal cases. J Am Co11 Cardiol 1988,12:463. 26. Levy WS, Varghese J, Anderson DW, Leiboff RH, Orenstein JM, Virmani R, Bloom S. Myocarditis diagnosed by endomyocardial biopsy in human immunodeficiency virus infection with cardiac dysfunction. Am J Cardiol 1988;62:658.

The iron paradigm of ischemic heart disease Jerome L. Sullivan, MD, PhD. Charleston,

S.C.

In 1981 the hypothesis was proposed that iron depletion protects against ischemic heart disease (IHD).’ This hypothesis was an attempt to explain the well-known difference between the sexes with regard to risk of heart disease and also to account for certain puzzling effects of menopause on the occurrence of heart disease. At that time the Framingham Study2,3 among others4s5 had suggested that the risk of heart disease in women is increased equally by natural menopause, or by either simple hysterectomy or hysterectomy with bilateral oophorectomy. These phenomena appeared incompatible with the From the Laboratory Service, Veterans Administration Medical Center, and the Department of Pathology and Laboratory Medicine, Medical University of South Carolina. Received for publication Sept. 8,1988; accepted Nov. 21, 1988. Reprint requests: Jerome L. Sullivan, MD, PhD, VAMC-113, 109 Bee St., Charleston, SC 29403.

long-held notion that endogenous estrogens are protective. The results of the Framingham Study seemed to implicate uterine rather than ovarian function as the factor that conferred protection against IHD.1~5 These patterns suggested that regular menstrual blood loss might be the protective factor. Regular blood loss has many possible effects. Why consider its effect on iron status as a possible protective factor? The initial stimulus for considering a role for iron was simply that the changes in stored iron as a function of age and sex are quantitatively quite similar to changes in the incidence of IHD as a function of age and sex.” 5 One way of ilhrstrating these relationships is to plot the sex ratios (M/F) of median serum ferritin values6 and the incidence of IHD deaths? as a function of age (Fig. 1). The M/F ratio of typical cholesterol values* 1177

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1

10

20

30

40 AGE (years)

50

1

60

70

60

1. Effect of age on sex ratio of IHD death rates (V),7 median serum ferritin (0) (Data replotted from Fig. 1, Cook JD, et al. Blood 1976;48:449. Reproduced with

Fig.

permission), and total plasma cholesterol (O)8 in representative populations in United States.

is shown to convey an appreciation of scale. The numeric values of the M/F peaks for both ferritin and IHD deaths fall within the same range, and the slopes between the ages of 40 and 70 years are quite similar. This conspicuous correlation is not simply an artifact of these particular data. Plotting other sets of ferritin values and incidences of IHD deaths yields curves that are in essence the same as those in Fig. 1 (data not shown). The correlation of the curves for ferritin values and IHD deaths is striking, but what if anything does it mean? The correlation could be fortuitous. However, since there is as yet no accepted explanation for the sex difference in IHD, this conspicuous correlation at the least compels a more searching inquiry into the role of iron in IHD. One reasonable interpretation suggested by inspection of Fig. 1 is that serum ferritin values may be a powerful risk factor for IHD. A second reason for considering iron as a possible risk factor was the well-known cardiotoxic effect of iron in hemochromatosis’s lo and thalassemia.l’s l2 Two objections arise immediately. The first is that the cardiotoxicity of iron in these disorders is only seen in association with heavy iron overload, with serum ferritin values many times higher than the upper limit of normal. Suggesting that a substance might cause IHD at “normal” levels when it is known that the substance produces heart disease at grossly abnormal concentrations has a clear precedent in medical history. An important early impetus to the development of the cholesterol paradigm of II-ID was familial hypercholesterolemia (FH).13 Individuals who are homozygous for FH have very high

May 1989 Heart Journal

levels of serum cholesterol, six to eight times normal. Those who are homozygous for FH can have a myocardial infarction (MI) in childhood. Heterozygous individuals have somewhat lower cholesterol levels, but these values are still two or three times normal. Heart disease is less severe in heterozygous individuals, with clinically apparent disease usually occurring in the third or fourth decade. Inhabitants of Third World countries generally have very little IHD and low cholesterol values. Inasmuch as people in developed nations have a substantial incidence of IHD, and levels of cholesterol intermediate between those of impoverished Third World subjects on the one hand and FH-heterozygous subjects on the other, it is difficult not to make an intuitive leap and conclude that cholesterol at “normal” levels causes IHD. There is a rather exact analogy between this reasoning and the suggestion that iron “depletion” might offer protection against IHD. The second objection is that there is no obvious connection between the heart lesion in hemochromatosis and atherosclerotic heart disease. This criticism involves two separate issues. The first is in the distinction between atherosclerosis and IHD (as in Oliver’s14 insightful question: “What are we trying to prevent-coronary atherosclerosis or ischaemic heart disease?“). Coronary atherosclerosis is not synonymous with IHD. Consider two hypothetical 40-year-old men with angiographically identical coronary atherosclerosis. Why does one of these men have MI at age 40 and the other die of carcinoma at age 90 without ever having had MI? Why does a large fraction of those in the highest decile for serum cholesterol never have MI, whereas an appreciable number in the lowest decile dies of MI? One answer to these questions may be in the existence of previously undefined factors that increase the vulnerability of the myocardium to ischemia. Endogenous iron, at levels previously considered normal, may be such a factor. The second issue raised by this criticism involves the role of iron in atherogenesis. A role for iron in the development of coronary lesions is not indispensable to the hypothesis that iron depletion protects against IHD.5 Iron depletion could exert a protective effect by mechanisms that do not involve effects on atherogenesis, for example, by decreasing the vulnerability of the myocardium to ischemia. However, it would be premature to reject a role for iron in atherogenesis. Iron depletion could inhibit atherogenesis through a variety of mechanisms including, for example, protection of endothelial

cells from oxygen radical injury15 or inhibition low-density

lipoprotein

modification.16s11

of

Volume 117 Number 5

Iron

A third compelling reason for considering stored iron as a possible risk factor was that a person at risk can easily rid himself of this factor by a safe and well-understood method. Blood donation three times a year lowers a man’s serum ferritin level to that typically seen in a young menstruating woman.’ The possibility of safe preventive therapy suggested that the hypothesis should be carefully considered. Since the initial publication of the hypothesis in 1981, there have been several developments that require review and comment. Most significantly there has emerged a growing body of evidence that iron is intimately involved in ischemic myocardial injury. Remarkably it now appears that the iron chelator deferoxamine can decrease reperfusion injury in the hearts of animals with “normal” iron status. These new data are clearly needed for a more complete definition of the pathophysiology of myocardial injury. However, the significance of these experiments with regard to iron, deferoxamine, and the heart goes beyond explanations of the mechanisms of injury. These data also provide important indirect support for a new paradigm, based on a central role for iron in the development of IHD. This paradigm may explain such diverse epidemiologic phenomena as the sex difference in IHD, the circadian variation in the occurrence of MI, and the primary preventive effects of aspirin. It may also allow formulation of simple primary preventive measures, for example, deliberate depletion of stored iron by regular phlebotomy. The purpose of this article is to review these new developments, comment on new results on the effects of menopause on IHD, and summarize a number of corollary hypotheses. IRON, DEFEROXAMINE,

AND THE HEART

Iron-dependent mechanisms of cellular injury have been studied intensively, especially in recent years. Iron is thought to act as a catalyst in the generation of the hydroxyl radical via the HaberWeiss reaction and to have an important role in lipid peroxidation. These reactions have been thoroughly reviewed by others.18-20 Uncertainties remain, particularly concerning the exact role of iron-dependent processes in cell and tissue injury in vivo. This section will review work on deferoxamine and ischemic myocardial injury and suggest interpretations of these data within the context of the proposed paradigm. There have been several recent reports2*-28 on the effects of deferoxamine in ischemic myocardial injury (Table I). The animals in all of these studies had normal iron status as conventionally defined, that is, no iron-depleting or iron-loading manipulations

and ischemic

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were described. With one partial exception= results of these studies all showed significant protective effects of deferoxamine in acute ischemic myocardial injury. Bernier et al. 24also found that a very low ADP (100 nmol/L with concentration of FeCl, respect to Fe) in the perfusion fluid “increased dramatically the incidence of reperfusion-induced ventricular fibrillation and tachycardia.” The results may have considerable generality, since different experimental systems in three different species were studied. Essentially two general conclusions seem to have been drawn from these studies on deferoxamine and myocardial injury: (1) iron is involved in some forms of ischemic myocardial injury, and (2) deferoxamine may be a clinically useful drug for treatment of cardiac disease (e.g., MI) or as an additive to cardioplegic solutions. It is implicitly assumed that iron, in the form needed to promote injury, is present in vivo in excess. In other words, measurements of total tissue iron levels suggest that enough iron is always potentially available to catalyze injurious reactions at maximal rates. For iron-dependent mechanisms this reactant is assumed to be available in excess at the site of injury. The excess iron may be roughly defined as the pool of iron available for chelation by deferoxamine. This is a rough definition, since some of the injury-promoting iron may not always be available for chelation by deferoxamine. The hypothesis that depletion of iron protects against IHD suggests that: (1) iron status affects the size of the deferoxamine-chelatable, injury-promoting iron pool, (2) vulnerability of the myocardium to ischemic injury is proportional to the size of this iron pool, and (3) in iron depletion both the size of the pool and vulnerability to ischemic myocardial injury approach their minima. Recent findings on the antiinflammatory effects of iron deficiency are consistent with the hypothesis and these implications.29-31 Results of these studies suggest that iron deficiency may exert persistent antiinflammatory effects as strong as those of acute deferoxamine treatment. The proposed iron paradigm of IHD implies that there is a relationship between these findings in animals with apparently normal iron status and earlier clinical observations on the use of deferoxamine in patients with thalassemia.“v12 Patients with transfusion-dependent thalassemia acquire massive iron overload and usually die of intractable congestive heart failure or cardiac arrhythmias in the second or third decade. Survival after the onset of cardiac symptoms is typically a matter of a few months. Regular frequent administration of defel

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I. Studies on the effects of deferoxamine on ischemic myocardial injury in animals with “normal” Reference

Species

Babbs2’

Rat

Myers et a1?2,23

Rabbit

Bernier et alz4

Rat

Bolli et alzs

Dog

Ambrosio et al.26

Rabbit

Menasche et alz’

Rat

Farber et a1.28

Dog

System

Effects

Experimental cardiorespiratory arrest and resuscitation in intact animals Ischemia-reperfusion in isolated hearts Ischemia-reperfusion hearts Ischemia-reperfusion animals Ischemia-reperfusion hearts Ischemia-reperfusion hearts Ischemia-reperfusion animals

roxamine delays the onset of cardiac symptoms and improves chances for survival. Complete depletion of the iron load is apparently not essential for this action. Cardiac improvement can occur after treatment with deferoxamine even in the continued presence of massive iron overload. Deferoxamine may also improve ventricular function in thalassemic patients with active heart failure.32 Thus deferoxamine improves ventricular function in both the failing hearts of patients with thalassemia and the hearts of normal animals after experimental ischemic episodes. These findings suggest that mechanisms of myocardial injury may be similar in the failing myocardium of patients with thalassemia and in the postischemic myocardium in subjects with conventionally normal iron status. In both situations an increased vulnerability to pump failure and lethal arrhythmias and a positive response to treatment with deferoxamine are observed. When iron overload reaches some critical level in patients with thalassemia, their hearts may be continuously subjected to concentrations of excess iron that are only encountered in a normal heart after an ischemic event. The size of the deferoxamine-chelatable, injury-promoting iron pool may be similar in the two situations. Patients with thalassemia acquire a massive overload of iron; however, clearly not all of this iron is continuously available at sites of myocardial injury. The amount of potentially toxic iron at Mllnerable sites within the myocardium in a patient with thalassemia could be comparable in size to that liberated by ischemia within the normal myocardium. In the postischemic myocardium, iron may be liberated from injured myocardial cells, from neutrophils recruited to the site of injury, or from circulating ferritin. Treatment

in isolated in open-chest in isolated in isolated in open-chest

May 1989 Heart Journal

iron status

of deferoxamine

Increased survival at 10 days after arrest Decreased CPK release; little effect on other measures of recovery Lower incidence of arrhythmias Improved recovery of contractile function Improved recovery of several measurements of function Improved recovery of several measurements of function Improved recovery of several measurements of function

with deferoxamine must be frequent and regular in patients with thalassemia probably because the toxic iron pool is constantly replenished from the massive stored iron compartment. Oliver33 has made an eloquent plea for more intensive study of myocardial vulnerability to ischemia. As a target for preventive efforts, ischemic myocardium “is a Cinderella to the ugly sisters of coronary atheroma and the ‘classic’ risk factors,” since it is “the source of pain, arrhythmias, failure, and death.“33 Unfortunately myocardial vulnerability to ischemia cannot be measured or predicted in normal ambulatory individuals. Even if it could be measured no clinically useful methods for decreasing vulnerability have been established. The deferoxamine experiments imply that myocardial vulnerability to ischemia could be decreased by continual administration of deferoxamine in subjects with conventionally normal iron status. This is not proposed as a practical alternative, although it is not immediately obvious that preventive therapy with deferoxamine would be more expensive on a costper-life-saved basis than, for example, long-term cholestyramine therapy. 34 In the context of the proposed iron paradigm, myocardial vulnerability to ischemia could be predicted by measurement of serum ferritin levels and decreased by depletion of the injury-promoting iron pool. MENOPAUSE

AND HEART DISEASE

The hypothesis that iron depletion protects against IHD was formulated to explain the effects of menopause on IHD and related phenomena. The Framingham Study found that menopause or cessation of menses for any reason was associated with a marked and significant increase in the incidence of

Volume 117 Number 5

IHD. A more recent prospective study, the Nurses’ Health Studyt5 found no significant effect of menopause on IHD. In addition to these discrepant findings, there persists in the literature a fallacious interpretation. of national statistics on IHD death rates among women. I wish to propose a possible resolution of the apparent discrepancy between results of the Framingham Study and those of the Nurses’ Health Study and to correct a widespread misinterpretation of plots of IHD deaths in women as a function of age. The relevance to the menopausal question of findings on heart disease expression in women who are heterozygous for FH and results of studies on the use of postmenopausal estrogen will also be discussed. The fallacy of the semilog plot. It has long been recognized that a semilog plot of incidence against age produces a smooth linear increase in IHD with age in women. This phenomenon has been widely interpreted as evidence that menopause has no effect on the incidence of IHD.36-3g As recently as 1987 this finding has been cited as “the strongest evidence against the hypothesis that premenopausal women enjoy some degree of protection”3g against IHD. These investigators have reasoned that the absence of an inflection in the line at the age of menopause excludes such a protective effect. The flaw in this argument is that a straight line in such a semilog plot can be generated by averaging the risks at each age of two distinct populations of women: those who menstruate and those who do not. As a given population of women ages the postmenopausal group is recruited from members of the premenopausal group. An empiric confirmation that a straight line can be generated from two such populations is shown in Fig. 2, in which data from the Framingham Study’ are plotted. Inasmuch as menopausal status was recorded for each woman at each examination, it is possible to plot separately the incidences for premenopausal women, postmenopausal women:, and all women. The points for all women describe a line with no inflection at the age of menopause. Nonetheless, it is apparent that there is a marked menopausal effect if the incidences for pre- and postmenopausal women are plotted separately. Thus a straight, uninflected line in such a plot cannot be regarded as evidence against a menopausal effect on IHD. Fig. 2 also shows an increasing incidence in older premenopausal women. This effect is compatible with the proposed iron paradigm. In the last few years before menopause, menstrual periods often become irregular and scanty. Any decrease in blood loss should have a positive effect on iron balance, as

Iron and ischemic heart disease

0.q

,

,

35-39

40-44

, 45-49 AGE (years)

, 50-54

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Fig. 2. Incidence of cardiovascular disease by age in years, sex, and menopausal status in Framingham Study 20-year follow-up. Rates/lOOO/year are displayed for men (o), all women (o), premenopausal women (VI, and postmenopausal women (A). Rates for pre- and postmenopausal women “less than 40 years” are plotted with the 35 to 39-year-old group. (Data from Tables 1 and 2, Kannel WB, et al. Ann Intern Med 1976;85:447. Reproduced with permission.)

occurs in younger women who take oral contraceptives.40*41 Increasing levels of stored iron in the last few years before menopause may be associated with an increased incidence of IHD and may explain the convergence of the curve for older premenopausal women with those past menopause in Fig. 2. Proposed Framingham

resolution of the discrepancy between Study and the Nurses’ Health Study.

the

The Nurses’ Health Study found that premenopausal women have a lower risk of coronary heart disease than age-matched postmenopausal women.% These findings are discrepant with those of the Framingham Study because the difference did not achieve significance. The discrepancy appears to be in the estimates of the magnitude of the pre- and postmenopausal differences in risk. The source of the discrepancy may be in the composition of the premenopausal reference group in the Nurses’ Health Study. High levels of oral contraceptive use by the premenopausal nurses may have inflated their incidence of coronary heart disease. In 1976, the year in which the Nurses’ Health Study began, oral contraceptives were in widespread use. Oral contraceptives were not available to the Framingham women during the first years of that study in the 1950s and 1960s. Use of oral contraceptives, especially together with smoking, has a significant effect on risk of coronary heart disease.40*42-44In terms of their risk of coronary heart disease, users of

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y5 lx?w s j

MEN

O.l-

iii i!l 8F

-*-----* o-*

20

30

40 AGE (years)

50

60

3. Cumulative probability by decade of myocardial infarction or coronary artery disease death in persons heterozygous for FH (0 ) and their unaffected relatives (0). Upper panel, Probability for men. Lower panel, Probability for women. (From Stone NJ et al. Circulation 1974;49:47. Reproduced with permission.)

Fig.

oral contraceptives would be expected to be similar to women who had recently undergone menopause. The less significant difference in risk of heart disease between pre- and postmenopausal women in the Nurses’ Health Study may be attributable to the larger number of users of oral contraceptives in their population. A reanalysis of risk in the group of premenopausal women who have never used oral contraceptives should produce results that are not discrepant with those of the Framingham Study. The proposed iron paradigm is predicated on a significant difference in risk of IHD between menstruating women who have no reason (such as oral contraceptives use) for increased iron stores and age-matched women whose menses have ceased. Results of the foregoing analysis suggest that such a difference is compatible with observations to date.2-5s35*46In terms of the paradigm, stored iron levels among the nurses would be expected to be lowest in premenopausal nonusers of oral contraceptives, higher in premenopausal users of oral contraceptives, and still higher in postmenopausal women, with a similar pattern of expected incidence of coronary disease in these three groups. FH, heart disease, and menopause. FH is an autosomal dominant disorder associated with grossly abnormal concentrations of cholesterol and lowdensity lipoprotein. There appears to be no sex difference in the lipid phenotype, that is, both male and female subjects who are heterozygous for FH have comparably elevated levels of cholesterol and low-density lipoprotein starting early in life.13

Heart Journal

Remarkably, despite the similarity in lipid phenotype in heterozygous males and females, the sex difference in clinical expression of IHD is preserved.5x46j47Heterozygous females have roughly a lo-year lag in the appearance of clinically evident IHD in comparison to heterozygous males. This pattern is quite similar to that seen in normal men and women.” In one large series,47 the incidence of heart disease in heterozygous individuals was compared with that in their unaffected relatives (Fig. 3). Heterozygous males had a markedly higher incidence of disease than their normal male relatives by age 40. In sharp contrast, the incidence in heterozygous females was essentially indistinguishable from that of their normal female relatives until after age 40. The heterozygous females were clearly susceptible to the heart lesion of FH, since after age 40 they had a progressively higher incidence of disease than their normal female relatives. Despite grossly perturbed lipids, heterozygous females were apparently completely protected from clinically evident heart disease until after the age of menopause. These phenomena are pertinent to the debate on the effect of menopause on heart disease. Data in Fig. 3 strongly support a protective effect of premenopausal status against IHD, The expression of heart disease in women with FH presents a challenge to theories on the sex difference in IHD. Premenopausal women who are heterozygous for FH seem to have a factor that confers a high degree of protection against the cardiac effects of grossly elevated cholesterol levels. It must be regarded as a hopeful finding that any factor could protect so completely against such high levels. After age 40 this protection is lost, presumably without a major change in the lipid phenotype. Theories that propose beneficial alterations in lipids by endogenous estrogens as a protective factor3g,48 must explain how these putative alterations work in the presence of the FH phenotype. The female hormonal milieu seems to have little effect on the lipid phenotype in heterozygous individuals. These findings recall Oliver’s14 question on the distinction between atherosclerosis and IHD.14 Such a high degree of protection could result either from an inhibition of coronary atherosclerosis or from a decrease in myocardial vulnerability to ischemia. There may be little sex difference among individuals who are heterozygous for FH in the degree of coronary atherosclerosis, just as there is no apparent sex difference in the lipid phenotype. Absence of such a sex difference in coronary atherosclerosis would lend support to a possible sex difference in myocardial vulnerability to ischemia.

Volume 117

Iron and ischemic heart disease

Number 5

In commenting on the discrepant results of two large prospective studies on postmenopausal estrogen use and IHD, Bailafls concluded that he “cannot tell from present evidence whether these hormones add to the risk of various cardiovascular diseases, diminish the risk, or leave it unchanged.” He was speaking of the Framingham Study and the Nurses’ Health Study,so but he could just as well have been summarizing the whole body of evidence of the subject. The relevance of these inconclusive studies to the hypothesis that iron depletion protects against IHD is uncertain. Future investigations should include measurements of iron status, especially in postmenopausal women who have not had a hysterectomy. Use of estrogen after menopause may be associated with uterine bleeding sufficient to decrease iron stores. To my knowledge, no previous studies have considered estrogen-related bleeding as a possible protective factor against IHD. Postmenopausal

COROLLARY

estrogen

use.

HYPOTHESES

The core hypothesis of the proposed new paradigm is that iron depletion protects against IHD. A new paradigm, in Kuhn’s51 sense of the word, is proposed because of the broad range of IHD-related phenomena that can be parsimoniously explained in terms of this hypothesis. Previous explanations for some of these diverse observations have been couched in terms of other paradigms, for example, the lipid hypothesis. Other phenomena, such as inheritance patterns for premature MI, circadian variation in the occurrence of MI, or the sex difference in IHD, are not readily accounted for by existing paradigms. An important feature of the core hypothesis is that it can be tested. One experimental approach would be a randomized prospective study of blood donors. With appropriate controls it should be possible to show a protective effect of iron depletion. Sex, iron, and heart disease. The sex difference in IHD is an anomaly that has received relatively little attention, considering the evident strength of male sex as a risk factor. Application of the paradigm to this anomaly has been discussed previously.1T5s52 It has been widely assumed that sex is a nonmodifiable risk factor. This perhaps explains why sex is “the most widely ignored of the major risk factors.“53 The iron paradigm offers an explanation that does not invoke an immutable sex difference. Loss of blood and iron with each menstrual cycle is dependent on a fundamental difference between men and women. But it is clearly not a nonmodifiable difference, since men can also lose blood regularly, for example,

1183

by regular blood donation, without compromising any of the cherished characteristics of their sex. Iron and IHD in the Third World. IHD is much more prevalent among people in developed nations than in those of Third World countries. Those migrating from poor Third World countries to wealthy industrialized areas acquire the greater risk of IHD found in the latter. Even in poorer countries lower prevalences of IHD are seen among the most impoverished groups. It is consistent with the proposed paradigm that iron deficiency is generally most prevalent among those groups with the lowest prevalence of IHD.’ Multiple factors predispose the world’s poor population to iron deficie*y. These include diets low in iron, high-fiber diets that retard iron absorption, and the prevalence of parasites that promote urinary or fecal blood loss. l, 54 These factors are generally lost after migration to areas with high meat consumption and good public sanitation. For the iron paradigm it is iron repletion after migration and not raised cholesterol levels that explains the increased incidence of IHD among migrants. Heterozygous hemochromatosis premature myocardial infarction.

as a risk factor

for

The iron paradigm offers a possible solution to an intriguing problem in the genetics of premature MI. Results of numerous studies have suggested that the risk of premature MI (MI under age 50) may be increased by a heritable factor not associated with conventional risk factors.55-6L Roughly 5 % to 7 % of individuals in the general population are members of high-risk families.61 Some of the high-risk families have a familial predisposition to hypertension or hyperlipidemia. However, the preponderance of evidence suggests no aggregation of conventional risk factors in the majority of the high-risk families. It is significant that the men in these high-risk families are at relatively greater risk than their female relatives.60,64 Exactly such a pattern would be expected if heterozygosity for the hemochromatosis gene conferred an increased risk of premature MI. In terms of the paradigm the increased risk should not be dependent on the traditional risk factors. Women should be relatively protected from the effects of the gene, since they regularly lose iron until menopause. The apparent selectivity for persons under age 50 may be explained as follows: Heterozygous subjects acquire a modest load of stored iron earlier in life than do normal subjects.% As they age normal individuals attain levels of stored iron similar to those seen in younger heterozygous individuals.6s65 Thus as heterozygous individuals age they are at

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relatively less of a disadvantage. Heterozygous persons would be expected to lose the protective effects of low iron stores at an earlier age than unaffected subjects. Finally, it is clear that the hemochromatosis gene occurs in the heterozygous condition frequently enough to account for the observed 5% to 7% of high-risk individuals. For example, heterozygote frequencies of 11.2 % and 13.8% have been found in Utah65 and Sweden,% respectively. A heterozygote frequency greater than the proportion of high-risk individuals is not incompatible with the paradigm. In many individuals who are obligate heterozygotes, iron loads indistinguishable from normal are encountered. 65 According to the paradigm, a heterozygous person with a statistically normal iron load should be at “normal” risk of premature MI, that is, at the same risk as a normal subject with an identical iron load. Circadian variations of Ml and iron. Recent work has shown that the onset of MI is not distributed evenly throughout the day but has a prominent morning peak.67*68 A threefold increase in the frequency of onset at peak (9 AM) as compared with trough (11 PM) periods has been found. Results of other studies have disclosed a similar circadian rhythm in transient myocardial ischemic episodes in patients with stable coronary artery disease.69s70 This circadian variation is identical to that previously observed for serum iron.?l Serum iron levels are two to three times higher in the morning (between 8 AM and 10 AM) than at night. Transferrin does not show appreciable circadian variation. Thus transferrin saturation is also higher in the morning and lower at night. It is noteworthy that the peak-to-trough ratios for both occurrence of MI and serum iron values are similar. Risk of MI and transient ischemia appears to be directly proportional to serum iron or transferrin saturation. Such a pattern would be expected if myocardial vulnerability to ischemia were directly influenced by the level of serum iron or transferrin saturation. An experimental approach to establishing a connection between the circadian rhythms of transient ischemic episodes and serum iron levels might involve monitoring patients with stable coronary disease who receive daily subcutaneous deferoxamine. If these rhythms are related, daily administration of deferoxamine should abolish the circadian rhythm in ischemic myocardial episodes. Cholestyramine, iron, and the Lipid Research Clinics study. Risk of MI is greater for those at the upper

end of the plasma cholesterol distribution than for those at the lower end. Risk of MI increases with increasing levels of plasma cholesterol in persons

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migrating from areas of low to areas of high prevalence of IHD. These observations generally support the cholesterol paradigm but do not constitute conclusive evidence in its favor. What is missing is a demonstration that lowering plasma cholesterol levels in normal subjects is effective in lowering the incidence of MI. Recognizing the importance of this key piece of missing evidence, the Lipid Research Clinics (LRC) investigators undertook a lengthy prospective study of the effects of cholesterol lowering on risk of coronary heart disease.72 Incidence of the primary end point-definite coronary disease death, definite nonfatal myocardial infarction, or both-was assessed in groups receiving cholestyramine or placebo. After an average of 7.4 years of observation, the cumulative incidence of the primary end point was 7% in the cholestyramine group and 8.6% in the control group. The difference was interpreted as significant by the LRC investigators and declared to be the crucial missing evidence. Other investigators have criticized this interpretation noting, for example, that the statistical significance of the main finding is highly questionable.7’ The amount and sometimes emotional tone of the critical response to the LRC study74-80 suggests the beginning of a Kuhnian “crisis”51 for the cholesterol paradigm. The relevance of the iron paradigm to these findings lies in the effects of cholestyramine on iron absorption.8’ Cholestyramine inhibits absorption of iron and its prolonged feeding to rats is associated with dose-dependent depletion of stored iron.s2r83 In designing their study on the effect of cholesterol lowering on the incidence of coronary heart disease, the LRC investigators may have inadvertently selected a method that has more than one effect capable of influencing the primary end point. The levels of stored iron in the two groups at the end of the study cannot be determined from the published evidence. 72 The lack of change in serum iron or hematocrit levels does not rule out a significant decrease in serum ferritin in the experimental group. A large and significant decrease in serum ferritin could have easily occurred in the experimental group without significant change in serum iron or hematocrit values. It is noteworthy that iron binding capacity was significantly greater in the experimental group. This was one of the very few laboratory values that differed significantly between the two groups. Increased iron binding capacity is not uniquely associated, but is compatible, with a significant decrease in stored iron in the experimental group. Given the known effects of cholestyramine on iron absorption and the small increase in iron bind-

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ing capacity, the burden of proof rests with the LRC investigators to demonstrate that there was no significant difference in levels of stored iron between the two groups during their lengthy study. If cholestyramine decreased levels of both cholesterol and stored iron, a lower incidence of disease in the experimental group may logically be as attributable to iron loss as to cholesterol lowering. If cholestyramine did lower levels of stored iron, why was the decrease in the incidence of disease so small? Since the study conditions were not designed to lower iron levels, any decrease in serum ferritin would have been an unintentional benefit. Under their conditions treatment with cholestyramine may have been an inefficient method for depletion of iron in humans. Oral contraceptives, iron, and IHD. The effects of oral contraceptive use on the incidence of IHD are consistent with the notion that the incidence of IHD is proportional to the level of stored iron.40,42-u It is well known that use of oral contraceptives diminishes menstrual blood flow. The predictable increase in stored iron has now been demonstrated.41 In that study, use of oral contraceptives was associated with approximately a twofold increase in serum ferritin levels.4l Young women who regularly take oral contraceptives may have levels of stored iron usually seen in older, perimenopausal women. The iron paradigm suggests that the incidence of IHD is increased by a common mechanism in users of oral contraceptives and in women who have recently undergone menopause, that is, increased iron stores. A persistent increase in the incidence of IHD in women who have discontinued use of oral contraceptiveP is consistent with the iron paradigm. Inasmuch as established iron stores may take a considerable time to deplete after resumption of normal menstrual blood flow, persistence of the effect on the incidence of IHD in some women would be expected.40 Aspirin, iron, and IHD. Regular daily ingestion of even small doses of aspirin, if continued for months or years, may cause a significant decrease in iron stores. Apparently trivial daily blood losses from the gut, not detectable by the patient, could affect iron stores if the treatment is prolonged. Decreased platelet function and minor gastric irritation could easily be associated with gastrointestinal blood loss of an extra 1 to 2 ml/day above baseline,@ amounting to 30 to 60 ml/month and equaling typical menstrual losses. In the primary prevention of MI, aspirin may act in part by causing loss of stored iron.% Fish oils, iron, and IHD. A similar mechanism may explain the effects of fish consumption on the

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incidence of IHD.% Moderate fish intake is associated with significantly increased bleeding times.87 Small increases in bleeding time could result in significant loss of stored iron from occult gastrointestinal blood loss if such increases are sustained. Decreased risk of IHD in association with a moderate intake of fish has not been demonstrated in brief studies but only after lengthy periods of observation-years to decades .a Significantly, for the iron paradigm, that study88 found no relation between moderate consumption of fish and the conventional major risk factors (cholesterol, hypertension, and cigarette smoking). Nonetheless, they observed that moderate consumption of fish was associated with fewer deaths from heart disease. MECHANISMS

The considerations that led to the formulation of the iron paradigm do not allow specification of the mechanisms by which iron depletion protects against IHD. Results of the deferoxamine experiments are consistent with the idea that iron may act directly on the myocardium to increase its vulnerability to ischemia. The paradigm does not necessarily require this specific mechanism, and it does not require that the enhanced vulnerability be mediated by toxic oxygen species. The paradigm does suggest that a closer look at the possible antioxidant effects of iron depletion might be fruitfu1.52,2s-3* Unexpected protective mechanisms could emerge from such studies. Iron depletion or mild iron deficiency could have a net antioxidant effect in vivo.52 An antioxidant effect could be especially important in opposing ischemic myocardial injury, since the heart is relatively poorly endowed with antioxidant enzymes.8g Iron depletion probably decreases the concentration of deferoxamine-chelatable, injury-promoting iron in the myocardium. Iron depletion or mild iron deficiency could also influence myocardial vulnerability to ischemia through at least three additional mechanisms. These are not mutually exclusive. Plasma antioxidant activity. Unsaturated transferrin has strong antioxidant activity.W Iron depletion and iron deficiency increase the plasma concentration of unsaturated transferrin.gl Direct comparisons of available iron binding capacity at potential sites of myocardial injury in iron-depleted humans with that of the deferoxamine-treated hearts in the studies in Table I are not possible with existing data. Transferrin and deferoxamine differ in molecular weight and in other properties that could markedly influence their concentrations and antioxidant activities at sites of myocardial injury. It does seem

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clear that iron-depleted and iron-deficient humans continuously perfuse their own hearts with a plasma significantly enriched in a potent iron-binding antioxidant, that is, unsaturated transferrin. The increased antioxidant activity could have a number of important protective effects, including decreased modification of low-density lipoproteins,16v17 protection of coronary artery endothelium from oxygen radical-mediated injury,15 and greater resistance of the myocardium to acute ischemia. Iron-dependent enzymes. Iron depletion and iron deficiency alter the activities of many iron-dependent enzymes. Some of the iron-dependent enzymes may have significant roles in ischemic myocardial injury. Xanthine oxidase activity, for example, has been shown to be decreased in iron-deficient ratss2 Other examples of potential importance include key enzymes in the metabolism of arachidonic acid. Both cyclooxygenase and lipoxygenase appear to be iron dependent. s3,s4Alterations in their activities by iron depletion could have significant and complex effects on eicosanoid-dependent processes including, for example, the ‘effects of leukotrienes on neutrophil functions. s5 The effects of iron depletion on the activities of these or other relevant irondependent enzymes could decrease myocardial vulnerability to ischemia. Iron-dependent cellular functions. Iron status may regulate the functions of cellular elements involved in myocardial injury. For example, some important neutrophil functions are iron dependent and have been found to be altered in iron deficiency.x,97 Neutrophils probably enhance postischemic injury.s8 Iron depletion or iron deficiency may be associated with significant inhibition of the myocardial damaging effects of neutrophils. CONCLUSION

For many years the cholesterol paradigm has largely defined the problems of interest and the permissible solutions in the study of IHD. Much valuable data have been collected, but the fundamental question of whether lowering cholesterol levels prevents IHD in normolipidemic subjects remains controversial.‘4-80 It is perhaps significant that over the years the debate seems to have shifted to controversy over progressively smaller potential benefits from cholesterol reduction.gg Does anyone now seriously believe that lowering a patient’s cholesterol level to below 200 mg/dl will prevent IHD in the same sense that, for example, lime juice prevents scurvy? And yet there appears to be some factor that completely prevents IHD in menstruating women who are heterozygous for FH in the face of grossly

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elevated cholesterol levels. Dare we hope that this factor might be equally effective in subjects with normal cholesterol levels? The nature of this factor has not been defined, perhaps because it is a problem that cannot be adequately formulated within the cholesterol paradigm. In the iron paradigm, regular menstrual iron loss is the protective factor that prevents IHD in both normal and FH-heterozygous women. The proposed paradigm suggests that excess iron is another of the major contributing causes of IHD. It also offers an alternative way of thinking about the IHD problem. In this way of thinking hemochromatosis serves as a model, just as does FH for the cholesterol paradigm. The paradigm has significant explanatory power and provides a conceptual framework for understanding some previously puzzling phenomena. The hypothesis that iron depletion protects against IHD is of more than theoretic interest, since a convenient form of preventive therapy is suggested. REFERENCES

1. Sullivan JL. Iron and the sex difference in heart disease risk. Lancet 1981;1:1293-4. 2. Kannel WB, Hjortland MC, McNamara PM, Gordon T. Menopause and the risk of cardiovascular disease. The Framingham Study. Ann Intern Med 1976;85:447-52. 3. Hiortland MC. McNamara PM. Kannel WB. Some atherog&c concomitants of menopause: the Framingham Study. Am J Epidemiol 1976;102:304-11. Centerwall BS. Premenopausal hysterectomy and cardiovascular disease. Am J Obstet Gynecol 1981;139:58-61. Sullivan JL. The sex difference in ischemic heart disease. Perspect Biol Med 1983;26:65’7-71. Cook JD, Finch CA, Smith NJ. Evaluation of the iron status of a population. Blood 1976;48:449-55. Division of Vital Statistics, National Center for Health Statistics. Chartbook for the conference on the decline in coronary heart disease motahty. Washington: United States Government Printing Office, 197813-15. 8. Braunwald E. Heart disease. A textbook of cardiovascular medicine. Philadelphia: WB Saunders, 1980:1254. 9. Buja LM, Roberts WC. Iron in the heart. Etiology and clinical significance. Am J Med 1971;51:209-21. 10. Vigorita VJ, Hutchins GM. Cardiac conduction system in hemochromatosis: clinical and pathologic features of six patients. Am J Cardiol 1979;44:418-23. 11. Leon MB, Borer JS, Bacharach SL, et al. Detection of early cardiac dysfunction in patients with severe beta-thalassemia and chronic iron overload. N Engl J Med 1979;301:1143-48. 12. Wolfe L, Olivieri N, SalIan D, Colan S, Rose V, Propper R, Freedman MH, Nathan DG. Prevention of cardiac disease by subcutaneous deferoxamine in patients with thalassemia major. N Engl J Med 1985;312:1690-3. 13. Fredrickson DS, Goldstein JL, Brown MS. The familial hyperlipoproteinemias. In: Standury JB Wyngaarden JB, Fredrickson DS, eds. The metabolic basis of inherited disease. New York: McGraw-Hill, 1978. 14. Oliver MF. What are we trying to prevent-coronary atherosclerosis or ischaemic heart disease? Acta Med &and 1986;712(suppI):7-9. 15. Gannon DE, Varani J, Phan SW, Ward JH, Kaplan J, Till GO, Simon RH, Ryan US, Ward PA. Source of iron in

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neutrophil-mediated killing of endothelial cells. Lab Invest 1987;51:37-44. 16. Heinecke JW, Rosen H, Chait A. Iron and copper promote modification of low density lipoprotein by human arterial smooth muscle cells in culture. J Clin Invest 1984;74: 1890-4. 17. Heinecke JW, Baker L, Rosen H, Chait A. Superoxidemediated modification of low densitv liwnrotein bv arterial smooth muscle cells. J Clin Invest 1986;7’%757-61. ” 18. Aust SD, Svingen BA. The role of iron in enzymatic lipid peroxidation. In: Pryor WA, ed. Free radicals in biology. vol V. New York: Academic Press. 1982:1-28. 19. Halliwell B, Gutteridge JMC. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984,219:1-14. 20. Editorial. Metal chelation therapy, oxygen radicals, and human disease, Lancet 1985;1:143-5. 21. Babbs CF. Role of iron ions in the genesis of reperfusion injury following successful cardiopulmonary resuscitation: preliminary data and a biochemical hypothesis. Ann Emerg Med 1985;14:777-83. 22. Myers CL, Weiss SJ, Kirsh MM, Shlafer M. Involvement of hydrogen peroxide and hydroxyl radical in the “oxygen paradox.” Reduction of enzyme leakage by catalase, allopurino1 or deferoxamine, but not by superoxide dismutase. J Mol Cell Cardiol 1985;17:675-84. 23. Myers CL, Weiss SJ, Kirsh MM, Shepard BM, Shlafer M. Effects of supplementing hypothermic crystalloid cardioplegic solution with catalase, superoxide dismutase, allopurinol, or deferoxamine on functional recovery of globally ischemic and reperfused isolated hearts. J Thorac Cardiovasc Surg 1986;91:281-9. 24. Bernier M, Hearse DJ, Manning AS. Reperfusion-induced arrhythmias and oxygen-derived free radicals. Studies with anti-free radical interventions and a free-radical generating system in the isolated perfused rat heart. Circ Res 1986;58:331-40. 25. Bolli R, Pate1 BS, Zhu W-X, O’Neill PG, Hartley CJ, Charlat ML, Roberts R. The iron chelator desferrioxamine attenuates postischemic ventricular dysfunction. Am J Physiol 1987; 253:H1372-80. 26. Ambrosio G, Zweier JL, Jacobus WE, Weisfeldt ML, Flaherty JT. Improvement of postischemic mvocardial dvsfunction and metabolism induced by administration of deferoxamine at the time of reflow: the role of iron in the pathogenesis of reperfusion injury. Circulation 1987;76:906-15. 27. Menasche P, Grousset C, Gaudel Y, Mouas C, Piwnica A. Prevention of hydroxyl radical formation: a critical concept for improving cardioplegia. Protective effects of deferoxamine. Circulation 1987;76(suppl V):V-180-5. 28. Farber NE, Vercellotti GM, Jacob HS, Pieper GM, Gross GJ. Evidence for a role of iron-catalyzed oxidants in functional and metabolic: stunning in the canine heart. Circ Res 1988;63:351-60. 29. Sullivan JL, Till GO, Ward PA. Iron depletion decreases lung injury after e,ystemic complement activation. Fed Proc 1986;45:452. 30. Andrews FJ, IMorris CJ, Lewis EJ, Blake DR. Effect of nutritional iron deficiency on acute and chronic inflammation. Ann Rheum Dis 1987:46:859-65. 31. Chandler DB, Barton JC, Briggs DD, Butler TW, Kennedy JI, Grizzle WE, Fulmer JD. Effect of iron deficiency on bleomycin-induced lung fibrosis in the hamster. Am Rev Respir Dis 198&137:85-g. 32. Freeman AP, Giles RW, Berdoukas VA, Walsh WF, Choy D, Murray PC. Early left ventricular dysfunction and chelation therapy in thalassemia major. Ann Intern Med 1983;99: 450-4. 33. Oliver MF. Prevention of coronary heart disease-propaganda, promises, problems, and prospects. Circulation 1986;73: 1-9. 34. Renfrew RA. Lipid Research Clinics Program. JAMA 1984; 252:2545.

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35. Colditz GA, Willett WC, Stampfer MJ, Rosner B, Speizer FE, Hennekens CH. Menopause and the risk of coronary heart disease in women. N Engl J Med 1987;316:1105-10. 36. Tracy RE. Sex difference in coronary disease: two opposing views. J Chronic Dis 1966;19:1245-51. 37. Furman RH. Are gonadal hormones (estrogens and androgens) of signific&e in the development of ischemic heart disease? Ann NY Acad Sci 1968149822-33. 38. Heller RF, Jacobs HS. Coronary heart disease in relation to age, sex, and the menopause. Br Med J 1978;1:472-4. 39. Godsland IF, Wynn V, Crook D, Miller NE. Sex, plasma lipoproteins, and atherosclerosis: prevailing assumptions and outstanding questions. AM HEART J 1987;114:1467-1503. 40. Sullivan JL. Oral contraceptives and the risk of myocardial infarction. N Engl J Med 1981;305:1531. 41. Frassinelli-Gunderson EP, Margen S, Brown JR. Iron stores in users of oral contraceptive agents. Am J Clin Nutr 1985;41:703-12. 42. Mann JI, Vessey MP, Thorogood M, Doll R. Myocardial infarction in young women with special reference to oral contraceptive practice. Br Med J 1975;2:241-5. 43. Stadel BV. Oral contracentives and cardiovascular disease. N Engl J Med 1981;305:672-7. 44. Slone D, Shapiro S, Kaufman DW, Rosenberg L, Miettinen OS, Stolley PD. Risk of myocardial infarction in relation to current and discontinued use of oral contraceptives. N Engl J Med 1981;305:4204. 45. Punnonen R, Ikalainen M, Seppala E. Premenopausal hysterectomy and risk of cardiovascular disease. Lancet 1987; 1:1139. 46. Slack J. Risks of ischaemic heart-disease in familial hyperlipoproteinaemic states. Lancet 1969;2:1380-2. 47. Stone NJ, Levy RI, Fredrickson DS, Verter J. Coronary artery disease in 116 kindred with familial type II hyperlipoproteinemia. Circulation 1974;49:476-88. 48. Tikkanen JM, Nikkila EA. Regulation of hepatic lipase and serum lipoproteins by sex steroids. AM HEART J 1987;113: 562-7. 49. Bailar JC. When research results are in conflict. N Engl J Med 1985;313:1080-1. 50. Stampfer MJ, Willett WC, Colditz GA, Rosner B, Speizer FE, Hennekens CH. A prospective study of postmenopausal estrogen therapy and coronary heart disease. N Engl J Med 1985;313:1044-9. 51. Kuhn TS. The structure of scientific revolutions. 2nd ed. Chicago: The University of Chicago Press, 1970. 52. Sullivan JL. Sex, iron, and heart disease. Lancet 1986; 2:1162. 53. Editorial. Sex, hormones, and atherosclerosis. Lancet 1986;2:552-3. 54. Sullivan JL, Vegetarianism, ischemic heart disease, and iron. Am J Clin Nutr 1983;37:882-6. 55. Cambien F, Richard JL, Ducimetiere P. Etude des antecedents famillaux de cardiopathies ischemiques et d’hypertension arterielle en liason avec l’incidence des cardiopathies ischemiques. L’etude prospective Parisienne. Rev Epidomiol Sante Pub1 1980;28:21-37. 56. Nora JJ, Lortscher RH, Spangler RD, Nora AH, Kimberling WJ. Genetic-epidemiologic study of early-onset ischemic heart disease. Circulation 1980;61:503-8. 57. ten Kate LP, Boman H, Daiger SP, Motulsky AG. Familial aggregation of coronary heart disease and its relation to known genetic risk factors. Am J Cardiol 1982;50:945-53. 58. Snowden CB, McNamara PM, Garrison RJ, Feinleib M, Kannel WB, Epstein FH. Predicting coronary heart disease in siblings-a multivariate assessment. The Framingham Heart Study. Am J Epidemiol 1982;115:217-22. 59. Neufeld HN, Goldbourt U. Coronary heart disease: genetic aspects. Circulation 1983;67:943-54. 60. Barrett-Connor E, Khaw KT. Family history of heart attack as an independent predictor of death due to cardiovascular disease. Circulation 1984;69:1065-9.

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61. Williams RR. Population based perspectives of the genetic epidemiology of early coronary disease in Framingham and Utah. In: Rao DC, Elston RC, Kuller LH, et al, eds. Genetic epidemiology of coronary heart disease: Past, present, and future. New York: Alan R. Liss. 198489-91. 62. Hopkins PN, Williams RR, Hunt SC. Magnified risks from cigarette smoking for coronary prone families in Utah. West J Med 1984;141:196-202. 63. Conroy RM, Mulcahy R, Hickey N, Daly L. Is a family history of coronary heart disease an independent coronary risk factor? Br Heart J 1985;53:378-81. 64. Hamsten A, de Faire U. Risk factors for coronary artery disease in families of young men with myocardial infarction. Am J Cardiol 1987;59:14-9. 65. Cartwright GE, Edwards CQ, Kravitz K, Skolnick M, Amos DB, Johnson A, Buskjaer L. Hereditary hemochromatosis. Phenotypic expression of the disease. N Engl J Med 1979; 301:175-g. 66. Olsson KS, Ritter B, Rosen U, Heedman PA, Staugard F. Prevalence of iron overload in central Sweden. Acta Med Stand 1983;213:145-50. 67. Muller JE, Stone PH, Turi ZG, Rutherford JD, Czeisler C, Parker C, Poole WK, Passamani E, Roberta R, Robertson T, Sobel BE, Willerson JT, Braunwald E, MILIS Study Group. Circadian variation in the frequency of acute myocardial infarction. N Engl J Med 1985;313:1315-22. 68. Tofler GH. Brezinski D. Schafer AI. Czeisler CA. Rutherford JD, Willich SN, Gleason RE, Williams GH,‘Muller JE. Concurrent morning increase in platelet aggregability and the risk of myocardial infarction and sudden cardiac death. N Engl J Med 1987;316:1514-18. 69. Rocco MB. Barrv J. Camnbell S. Nabel E. Cook EF. Goldman L, Selwyn’ AP. Circadian variation of transient myocardial ischemia in patients with coronary artery disease. Circulation 1987;75:395-400. 70. Rocco MB, Nabel EG, Selwyn AP. Circadian rhythms and coronary artery disease. Am J Cardiol 1987;59:13C-17C. 71. Hamilton LD, Gubler CJ, Cartwright GE, Wintrobe MM. Diurnal variation in the plasma iron level of man. Proc Sot Exp Biol Med 1950;75:65-8. 72. Lipid Research Clinics Program. The Lipid Research Clinics Coronary Primary Prevention Trial results. JAMA 1984; 251:351-74. 73. Kronmal RA. Commentary on the published results of the Linid Research Clinics Coronarv Primarv Prevention Trial. JAMA 1985;253:2091-3. ” ” 74. Enloe CF. Coronary disease prevention should be individualized. Nutr Today 1984;19:12-14. 75. Ahrens EH. The diet-heart question in 1985: has it really been settled. Lancet 1985;1:1085-7. 76. Oliver MF. Consensus or nonsensus conferences on coronary heart disease. Lancet 1985;1:1087-9. 77. Mann GV. Coronary heart disease-“doing the wrong things.” Nutr Todav 1985:20:12-14. 78. Olso\ RE. Mass intervention vs screening and selective intervention for the prevention of coronary disease. JAMA 1986;255:2204-7. 79. .- Reaven ~~~ GM. Looking at the world through LDL-Cholesterol colored glasses. J N;tr 1986;116:1143-7. 80. Pinckney ER, Smith RL. Statistical analysis of Lipid Research Clinics Program. Lancet 1987;1:503.

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81. Sullivan JL. Lipid Research Clinics Program. JAMA 1984;252:2547. 82. Thomas FB, McCullough FS, Greenberger NJ. Inhibition of the intestinal absorption of inorganic and hemoglobin iron by cholestvramne. J Lab Clin Med 1971:78:70-80. 83. Thomas FB, Salsburey D, Greenberg& NJ. Inhibition of iron absorption by cholestyramine: demonstration of diminished iron stores following prolonged administration. Am J Dig Dis 1972;17:263-9. 84. Graham DY, Smith JL. Aspirin and the stomach. Ann Intern Med 1986;104:390-8. 85. Sullivan JL. Iron, aspirin, and heart disease risk. JAMA 1982;247:751. 86. Sullivan JL. Eicosapentaenoic acid, heart disease, and iron. West J Med 1985;142:559. 87. Houwelingen RV, Nordoy A, van der Beek E, Houtsmuller U, de Metz M, Hornstra G. Effect of a moderate fish intake on blood pressure, bleeding time, hematology, and clinical chemistry in healthy males. Am J Clin Nutr 1987;46:424-36. 88. Kromhout D, Bosschieter EB, de Lezenne Coulander C. The inverse relation between fish consumption and 20-year mortality from coronary heart disease. N Engl J Med 1985; 312:1205-g. 89. Doroshow JH, Locker GY, Myers CE. Enzymatic defenses of the mouse heart against reactive oxygen metabolites: alterations produced by doxorubicin. J Clin Invest 1980;65:12835. 90. Galdston M, Feldman JG, Levytska V, Magnusson B. Antioxidant activity of serum ceruloplasmin and transferrin available iron-binding capacity in smokers and nonsmokers. Am Rev Respir Dis 1987;135:783-7. 91. Herbert V. Recommended dietary intakes (RDI) of iron in humans. Am J Clin Nutr 1987;45:679-86. 92. Kelley MK, Amy NK. Effect of molybdenum-deficient and low iron diets on xanthine oxidase activity and iron status in rats. J Nutr 1984;114:1652-9. 93. Peterson DA, Gerrard JM, Rao GHR, Mills EL, White JG. Interaction of arachidonic acid and heme iron in the synthesis of prostaglandins. Adv Prostaglandin Thromboxane Res 1980;6:157-61. 94. Greenwald JE, Alexander MS, Fertel RH, Beach CA, Wong LK, Bianchine JR. Role of ferric iron in platelet lipoxygenase activity. Biochem Biophys Res Commun 1980;96:817-22. 95. Dahlen S-E, Bjork J, Hedgvist P, Artors K-E, Hammarstrom S, Lindgren J-A, Samuelsson B. Leukotrienes promote plasma leakage and leukocyte adhesion in postcapillary venules: in vivo effects with relevance to the acute inflammatory resnonse. Proc Nat1 Acad Sci USA 1981:78:3887-91. 96. Mackler B, Person R, Ochs H, Finch CA. Iron deficiency in the rat: effects on neutrophil activation and metabolism. Pediatr Res 1984;18:549-51. 97. Moore LL, Humbert JR. Neutrophil bactericidal dysfunction towards oxidant radical sensitive microorganisms during experimental iron deficiency. Pediatr Res 1984;18:684-9. 98. Ernst E, Hammerschmidt DE, Bagge U, Matrai A, Dormandy JA. Leukocytes and the risk of ischemic diseases. JAMA 1987;257:3218-24. 99. Taylor WC, Pass TM, SheparctDS, Komaroff AL. Cholesterol reduction and life expectancy. A model incorporating multiple risk factors. Ann Intern Med 1987;106:605-14.