Relationship of serum ferritin concentrations with metabolic cardiovascular risk factors in men without evidence for coronary artery disease

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1 Atherosclerosis 128 (1997) Relationship of serum ferritin concentrations with metabolic cardiovascular risk factors in men without evidence for coronary artery disease Martin Halle*, Daniel König, Aloys Berg, Joseph Keul, Manfred W. Baumstark Center for Internal Medicine, Department of Rehabilitation, Pre ention and Sports Medicine, Freiburg Uni ersity Hospital, Hugstetter Strasse 55, D Freiburg, Germany Received 24 January 1996; revised 20 September 1996; accepted 1 October 1996 Abstract Elevated serum ferritin concentrations between 200 and 500 g/l have been found to be a strong risk factor for acute myocardial infarction in Finnish men, but the reason for this association is still uncertain. In the Finnish population ferritin concentrations correlated with factors of insulin resistance syndrome. As these factors have been found to be associated with an LDL subfraction phenotype of increased concentrations of small, dense LDL particles, we hypothesized an association between ferritin and an atherogenic LDL subfraction profile, a finding which could be an explanation for the observed relationship between ferritin and atherosclerosis. Therefore we determined serum ferritin levels, metabolic cardiovascular risk factors, and the LDL subfraction phenotype in 93 healthy men without signs for infection or coronary heart disease. We found that men with moderately elevated ferritin levels ( g/l; n=31) had a significantly worse coronary risk profile than men with lower levels ( 200 g/l; n=62). Elevated ferritin concentrations were associated with significantly higher values for serum triglycerides, VLDL cholesterol, VLDL apolipoprotein B (P 0.01), IDL cholesterol, fasting glucose (P 0.05) and uric acid (P 0.01), and lower levels for HDL 2b and HDL 2a cholesterol and apolipoprotein A-I (P 0.05), and lipoprotein(a) (P 0.01). Elevated ferritin levels were, however, not associated with an unfavourable LDL subfraction profile of increased concentrations of small, dense LDL particles Elsevier Science Ireland Ltd. All rights reserved Keywords: Coronary risk factors; Atherosclerosis; Ferritin; LDL subfractions; Insulin resistance syndrome 1. Introduction * Corresponding author. Tel.: ; fax: ; mh@msm1.ukl.uni-freiburg.de A role for serum iron and iron stores, expressed as elevated ferritin levels, in coronary heart disease has been suggested [1 3]. Sullivan [2] even proposed that the higher serum ferritin levels in men compared with premenopausal women are responsible for the higher incidence of coronary heart disease in males. However, the data are still controversial and others failed to show an association [4 6]. However, the Kuopio Ischaemic Heart Disease Risk Factor Study prospectively investigating the incidence of acute myocardial infarction in Eastern Finnish men revealed that particularly men with serum ferritin concentrations 200 g/l had a 2.2-fold higher risk of acute myocardial infarction than men with lower serum ferritin concentrations. After adjustment for other risk factors this association was even stronger in men with additionally elevated serum low density lipoproteins (LDL) cholesterol levels 193 mg/dl [1]. Therefore it was hypothesized that this association might reflect the interaction between iron, oxygen free radicals and lipoproteins leading to the formation of lipid peroxides and oxidized LDL particles [1], progression of atherosclerosis and finally acute myocardial infarction [7]. The oxidation of lipoproteins /97/$ Elsevier Science Ireland Ltd. All rights reserved

2 Atherosclerosis 128 (1997) Relationship of serum ferritin concentrations with metabolic cardiovascular risk factors in men without evidence for coronary artery disease Martin Halle*, Daniel König, Aloys Berg, Joseph Keul, Manfred W. Baumstark Center for Internal Medicine, Department of Rehabilitation, Pre ention and Sports Medicine, Freiburg Uni ersity Hospital, Hugstetter Strasse 55, D Freiburg, Germany Received 24 January 1996; revised 20 September 1996; accepted 1 October 1996 Abstract Elevated serum ferritin concentrations between 200 and 500 g/l have been found to be a strong risk factor for acute myocardial infarction in Finnish men, but the reason for this association is still uncertain. In the Finnish population ferritin concentrations correlated with factors of insulin resistance syndrome. As these factors have been found to be associated with an LDL subfraction phenotype of increased concentrations of small, dense LDL particles, we hypothesized an association between ferritin and an atherogenic LDL subfraction profile, a finding which could be an explanation for the observed relationship between ferritin and atherosclerosis. Therefore we determined serum ferritin levels, metabolic cardiovascular risk factors, and the LDL subfraction phenotype in 93 healthy men without signs for infection or coronary heart disease. We found that men with moderately elevated ferritin levels ( g/l; n=31) had a significantly worse coronary risk profile than men with lower levels ( 200 g/l; n=62). Elevated ferritin concentrations were associated with significantly higher values for serum triglycerides, VLDL cholesterol, VLDL apolipoprotein B (P 0.01), IDL cholesterol, fasting glucose (P 0.05) and uric acid (P 0.01), and lower levels for HDL 2b and HDL 2a cholesterol and apolipoprotein A-I (P 0.05), and lipoprotein(a) (P 0.01). Elevated ferritin levels were, however, not associated with an unfavourable LDL subfraction profile of increased concentrations of small, dense LDL particles Elsevier Science Ireland Ltd. All rights reserved Keywords: Coronary risk factors; Atherosclerosis; Ferritin; LDL subfractions; Insulin resistance syndrome 1. Introduction * Corresponding author. Tel.: ; fax: ; mh@msm1.ukl.uni-freiburg.de A role for serum iron and iron stores, expressed as elevated ferritin levels, in coronary heart disease has been suggested [1 3]. Sullivan [2] even proposed that the higher serum ferritin levels in men compared with premenopausal women are responsible for the higher incidence of coronary heart disease in males. However, the data are still controversial and others failed to show an association [4 6]. However, the Kuopio Ischaemic Heart Disease Risk Factor Study prospectively investigating the incidence of acute myocardial infarction in Eastern Finnish men revealed that particularly men with serum ferritin concentrations 200 g/l had a 2.2-fold higher risk of acute myocardial infarction than men with lower serum ferritin concentrations. After adjustment for other risk factors this association was even stronger in men with additionally elevated serum low density lipoproteins (LDL) cholesterol levels 193 mg/dl [1]. Therefore it was hypothesized that this association might reflect the interaction between iron, oxygen free radicals and lipoproteins leading to the formation of lipid peroxides and oxidized LDL particles [1], progression of atherosclerosis and finally acute myocardial infarction [7]. The oxidation of lipoproteins /97/$ Elsevier Science Ireland Ltd. All rights reserved PII S (96)

3 236 M. Halle et al. / Atherosclerosis 128 (1997) was, however, not determined in the Finnish study, so that it remains a hypothesis that elevated ferritin concentrations act via oxidation of lipoproteins. Therefore it may be possible that not ferritin itself but other factors that are associated with elevated serum ferritin concentrations could be responsible for the increased incidence of acute myocardial infarction. In the Finnish study ferritin concentrations correlated with metabolic cardiovascular risk factors, namely elevated concentrations of serum triglycerides, glucose, systolic blood pressure, apolipoprotein B and reduced concentrations of HDL 2 cholesterol, and low physical fitness [1]. As these factors are known to be closely related to an LDL subfraction phenotype of increased concentrations of small, dense LDL particles [8 14], we hypothesized that ferritin might be associated with an atherogenic LDL subfraction profile [13 15], a finding which would help to explain the relationship between ferritin and atherosclerosis. Therefore, we determined serum ferritin levels in a group of healthy men and compared these with a detailed coronary risk profile including lipoprotein subfractions, lipoprotein(a), factors indicating insulin resistance, serum fibrinogen, body mass index, blood pressure, and maximal oxygen consumption. We were particularly interested in the association of serum ferritin levels with the concentrations of circulating small, dense LDL particles. Besides its role in atherogenesis ferritin is also an acute-phase protein, which is elevated in acute infection or chronic inflammatory disease such as atherosclerosis. Additionally extremely elevated ferritin concentrations are observed in hemochromatosis [16]. As these factors could falsely influence the association of ferritin levels and coronary risk factors, those individuals with evidence of infection or ferritin levels exceeding 500 g/l were excluded from the study. Therefore the present investigation focused on the relationship between moderately elevated ferritin levels with a detailed coronary risk profile in healthy men without infection or coronary heart disease (CHD). 2. Methods 2.1. Study subjects One hundred and seventy-two healthy men were recruited for the study. These subjects were either volunteers from staff and students of the Freiburg University Medical School or were randomly selected from the out-patient clinic of the Department of Rehabilitation, Prevention and Sports Medicine, where they had presented for an elective cardio-pulmonary assessment. A detailed dietary assessment was not performed. Exclusion criteria were intake of more than 0.3 l of beer or 0.25 l of wine on more than 2 days per week, a history of any acute or chronic infection, leucocyte count 9000/mm 3, ferritin levels 500 g/l, drug therapy of any kind, diabetes mellitus, a history of gastrointestinal, hepatic or endocrine disease, symptoms of CHD, pathological stepwise exercise stress testing, or an abnormal physical examination. The study was approved by an institutional review committee and all subjects gave informed consent for participation in the study Anthropometric data and exercise testing Weight, height, blood pressure (BP) and heart rate (HR) were measured after blood was drawn. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. A symptom-limited bicycle exercise stress test was performed with a stepwise increment in work load of 50 W every 3 min with continuous ECG recording. Maximal oxygen consumption (VO 2 max) was estimated according to a nomogram [17] Density gradient ultracentrifugation EDTA plasma was obtained after an overnight fast. VLDL (d g/ml), IDL (d= g/ml), LDL (d= g/ml), and HDL (d= g/ml) were prepared by sequential flotation according to Lindgren [18]. Total LDL were separated into 6 and HDL into 3 density classes by equilibrium density gradient centrifugation as described before [8,19,20]. The density ranges of LDL subfractions as determined by precision refractometry [18] of blank gradients were: LDL-1: g/ml; LDL-2: g/ml; LDL-3: g/ml; LDL-4: g/ml; LDL-5: g/ml; LDL-6: g/ml; HDL 2b : g/ml; HDL 2a : g/ml; and HDL g/ml Chemical analysis Lipids and apolipoproteins In all HDL and LDL subfractions cholesterol and triglycerides were measured by automated (EPOS, Eppendorf, Hamburg, Germany) enzymatic methods (Boehringer Mannheim, Germany; biomérieux, Nürtingen, Germany). The apolipoprotein (apo) A-I, B and A-II were measured by endpoint nephelometry (Behring, Marburg, Germany). The within-assay coefficient of variation for the determination of LDL subfraction concentrations was between 2.2 and 4.5% for cholesterol and between 3.0 and 5.8% for apolipoprotein B, depending on the subfraction.

4 M. Halle et al. / Atherosclerosis 128 (1997) Lipoprotein(a) Lipoprotein(a) (Lp(a)) was determined by ELISA from frozen EDTA plasma with polyclonal anti-apo(a) antibodies (Immunozym Lp(a), Immuno, Germany). The samples were stored at 20 C and were analyzed within 2 months Insulin, free fatty acids and insulin resistance Fasting insulin concentrations were determined by ELISA (Boehringer Mannheim, Germany) and free fatty acids (FFA) by an enzymatic colorimetric method (Wako Chemicals, Neuss, Germany). Insulin resistance (IR) was calculated from fasting blood glucose in (mmol/l) and fasting serum insulin concentrations using a computer-solved homeostasis model assessment method [21]: IR=insulin glucose/ Fibrinogen Fibrinogen was determined by nephelometry that detects immune complexes of fibrinogen and specific antibodies (Behring, Germany) Ferritin Ferritin was measured by ELISA (Boehringer Mannheim Immundiagnostica, Germany) from fresh plasma. The day to day coefficient of variation was 4% Statistical analysis To test the hypothesis that men with serum ferritin levels between 200 and 500 g/l have a more unfavourable coronary risk profile than individuals with lower serum ferritin concentrations, the 172 men fulfilling all inclusion criteria were divided into 2 groups according to their serum ferritin levels (group 1: ferritin 200 g/l, n=141; group 2: ferritin g/l, n=31). When the 2 groups were matched for age and BMI 62 men remained in group 1. The 2 groups were compared by the nonparametric Mann-Whitney U test. Data were analyzed using the Statistical Package for the Social Sciences (SPSS/PC+, SPSS, Chicago IL). All P 0.05 were considered to indicate statistical significance. 3. Results In clinically healthy men, who were matched for age and BMI, a higher ferritin level was not associated with blood pressure, physical fitness, or serum fibrinogen levels. Only uric acid and fasting glucose levels were significantly higher in men with ferritin levels 200 g/l than in individuals with lower levels (Table 1). The mean values for these parameters were, though, still within normal limits. Lp(a) was significantly lower in men with elevated ferritin levels than in men with lower levels, despite a wide S.D. (Table 1). The detailed analysis of the relationship between ferritin and lipoproteins revealed that elevated ferritin levels were associated with an atherogenic lipoprotein profile. In particular concentrations of triglycerides and triglyceride-rich lipoproteins such as VLDL and IDL were higher in men with ferritin levels 200 g/l (Table 2a and b). However, small, dense LDL particles that are closely related to the metabolism of triglyceride-rich particles were not different between the groups. The concentrations of cholesterol and apo B of LDL with the highest density ( g/ml; LDL-6) were 10% higher in men with ferritin levels 200 g/l, but this was not statistically significant (Table 2a and b). In contrast the analysis of the HDL subfraction profile showed that the concentration of HDL 2 cholesterol (30 12 mg/dl versus 22 9 mg/dl, P 0.01) and apo A-I (45 20 mg/dl versus mg/dl, P 0.05) were lower in men with higher ferritin levels. This difference was present for both HDL 2 subfractions HDL 2b and HDL 2a (Table 2a and b). No differences were observed for apo A-II concentrations in total HDL or HDL subfractions (Table 2b). 4. Discussion This study showed that moderately elevated serum ferritin concentrations between 200 and 500 g/l are associated with a distinct lipoprotein risk profile particularly characterized by reduced concentrations of HDL 2 cholesterol and HDL 2 apo A-I in healthy men. Table 1 Differences between ferritin groups (ferritin 200 g/l vs. ferritin 200 g/l) for age, BMI, HR, systolic and diastolic blood pressure (BP sys,bp dia ), VO 2 max, fasting glucose, insulin, IR, free FFA, uric acid, serum fibrinogen and Lp(a) Parameters Ferritin 200 g/l (n=62) Ferritin 200 g/l (n=31) Age (years) BMI (kg/m 2 ) HR (min 1 ) BP sys (mmhg) BP dia (mmhg) VO 2 max/kg (ml/ kg/min) Insulin ( U/ml) Glucose (mg/dl) * IR FFA (mmol/l) Uric acid (mg/dl) ** Fibrinogen (mg/ dl) Lp(a) (mg/dl) ** Values are mean S.D. * P 0.05; ** P 0.01; *** P

5 238 M. Halle et al. / Atherosclerosis 128 (1997) Table 2 Differences between ferritin groups for (a) serum cholesterol, triglycerides and cholesterol concentrations (mg/dl) and (b) apolipoprotein concentrations (apo A-I, apo A-II, apo B) (mg/dl) of VLDL, IDL, total LDL, LDL subfractions (LDL-1 LDL-6), total HDL, and HDL subfractions (HDL 2b, HDL 2a, HDL 3 ) (mg/dl) Ferritin 200 g/l Ferritin 200 g/l (n=62) (n=31) (a) Serum cholesterol Serum triglycerides ** VLDL cholesterol ** IDL cholesterol * LDL cholesterol LDL-1 cholesterol LDL-2 cholesterol LDL-3 cholesterol LDL-4 cholesterol LDL-5 cholesterol LDL-6 choles terol HDL cholesterol ** HDL 2b cholesterol HDL 2a choles ** 13 4* terol HDL 3 cholesterol (b) VLDL apo B ** IDL apo B LDL apo B LDL-1 apo B LDL-2 apo B LDL-3 apo B LDL-4 apo B LDL-5 apo B LDL-6 apo B HDL apo A-I HDL 2b apo A-I HDL 2a apo A * 27 10* I HDL 3 apo A-I HDL apo A-II HDL 2b apo A-II HDL 2a apo A II HDL 3 apo A- II * P 0.05; ** P 0.01; *** P The additional HDL subfractions analysis revealed that elevated ferritin concentrations were associated with both lower HDL 2b and HDL 2a cholesterol concentrations and were not related to HDL 3 particles. As HDL 2 is the more cardioprotective part of HDL [22] this association might be an indirect explanation for the association of ferritin with acute myocardial infarction. However, we could not confirm our hypothesis that elevated ferritin levels are associated with an LDL subfraction profile of increased concentrations of small, dense LDL particles. Although the concentrations of small, dense LDL cholesterol were in mean 10% higher in individuals with elevated ferritin levels compared to those with lower levels, this was not statistically significant. This relationship could have been expected as the LDL subfraction profile of increased concentrations of small, dense LDL particles are particularly expressed in individuals with increased fasting glucose levels and hypertriglyceridemia [9,23], two findings present in men with elevated ferritin concentrations in this study. Two factors might be responsible for the lack of association between ferritin and small, dense LDL particles in our study group. It is known that the LDL subfraction profile is expressed beyond the age of 35 years in obese, unfit individuals with a high insulin resistance [24 26]. Concentrations of all LDL subfractions are further increased in patients with hypercholesterolemia and hypertriglyceridemia [9]. In the present study only young men with good physical fitness without insulin resistance or dyslipoproteinemia were included. Therefore, it remains to be determined whether the association between ferritin and the LDL subfraction profile is present in a population including older, unfit men with severe dyslipoproteinemia. This is particularly important as the association between ferritin and acute myocardial infarction in Finnish men was observed in individuals older than 40 years and was closest in hypercholesterolemic individuals with LDL cholesterol levels 193 mg/dl [1]. The present study could, however, confirm the finding by Salonen et al. [1] that elevated ferritin levels are associated with higher fasting glucose levels and additional factors of insulin resistance syndrome [1]. We also observed higher serum concentrations of triglycerides and uric acid in the individuals with elevated ferritin levels 200 g/l, despite the fact that these values were within normal limits and determined in non-obese, physically fit men. To date it is uncertain why ferritin is associated with factors of insulin resistance syndrome and whether there is possibly a causal relationship. This relationship should, however, particularly be followed as it seems independent of obesity. This finding can be important for understanding the role of ferritin in atherosclerosis, as it is known that elevated glucose levels increase the oxidation of LDL particles [27]. Indeed, Salonen et al. hypothesized that

6 M. Halle et al. / Atherosclerosis 128 (1997) the synergistic association of ferritin and LDL cholesterol concentrations regarding the risk of acute myocardial infarction in their study was promoted through the oxidation of LDL particles [1]. Differences in the oxidative susceptibility of LDL subfractions have been reported, particularly showing that small, dense LDL particles are predominantly vulnerable to oxidative stress [28,29]. An additional reduction of HDL particles can even further increase the oxidation of LDL particles [30]. It may be hypothesized that the combination of elevated ferritin levels with elevated concentrations of serum glucose and low HDL 2 cholesterol concentrations, as found in our study, favours the synthesis of modified LDL particles. As we did not measure LDL oxidation, this hypothesis has to be confirmed in future studies. Oxidation of lipoproteins is also increased during inflammation [30]. The finding that ferritin correlated with leucocyte count and ischemic electrocardiographic findings in Finnish men, two factors that were also associated with increased risk for acute myocardial infarction [1], is a further indicator for the role of oxidation and inflammation in acute myocardial infarction. In the present study, however, the association between ferritin and coronary risk factors was independent of the effect of ferritin as an acute-phase protein, because only healthy, relatively young, non-obese individuals without evidence for acute or chronic infection or atherosclerosis were included. The finding that Lp(a) was lower in men with elevated ferritin levels was unexpected. The inverse relationship between these two risk factors can currently not be explained and makes the role of ferritin as a cardiovascular risk factor even more complex. However, as the metabolism and role of Lp(a) in atherosclerosis is still under investigation, this association should be followed up and both parameters be included in future studies. Although the present study could not further clarify the role of ferritin in atherosclerosis, the study has raised several questions. Nonetheless, the study showed that the relationship of ferritin with several coronary risk factors is even present in a healthy, relatively young population with only moderately elevated serum ferritin levels up to 500 g/l. The study could not confirm the hypothesis that ferritin is associated with an LDL subfraction profile of small, dense LDL particles. Nevertheless, as this investigation was not population based, these results have to be investigated in larger epidemiologic studies particularly including older individuals with low physical fitness and dyslipoproteinemia. To shed some more light on the role of ferritin in CHD, these studies should include other coronary risk factors besides ferritin such as LDL subfractions, LDL oxidation, Lp(a) and parameters of acute and chronic inflammation. Clinical trials should focus on the hypothesis that ferritin could be a parameter distinguishing those individuals with silent from those with active inflammatory atherosclerotic disease. Acknowledgements This study was supported by the German Heart Foundation (Deutsche Herzstiftung), Frankfurt/Main, Germany. The laboratory assistance of S. Jotterand and H. Zurmöhle is greatly appreciated. We also thank B. Spielberger and G. Zöllner for technical assistance and D. Grathwohl for statistical advice. References [1] Salonen JT, Nyyssönen K, Korpela H, Tuomilehto J, Seppänen R, Salonen R. High stored iron levels are associated with excess risk of myocardial infarction in Eastern Finnish men. Circulation 1992;86:803. [2] Sullivan JL. 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