REVIEWS. Iron deficiency and cardiovascular disease

Transcription

1 Iron deficiency and cardiovascular disease Stephan von Haehling, Ewa A. Jankowska, Dirk J. van Veldhuisen, Piotr Ponikowski and Stefan D. Anker Abstract Iron deficiency affects up to one-third of the world s population, and is particularly common in elderly individuals and those with certain chronic diseases. Iron excess can be detrimental in cardiovascular illness, and research has now also brought anaemia and iron deficiency into the focus of cardiovascular medicine. Data indicate that iron deficiency has detrimental effects in patients with coronary artery disease, heart failure (HF), and pulmonary hypertension, and possibly in patients undergoing cardiac surgery. Around one-third of all patients with HF, and more than one-half of patients with pulmonary hypertension, are affected by iron deficiency. Patients with HF and iron deficiency have shown symptomatic improvements from intravenous iron administration, and some evidence suggests that these improvements occur irrespective of the presence of anaemia. Improved exercise capacity has been demonstrated after iron administration in patients with pulmonary hypertension. However, to avoid iron overload and T cell activation, it seems that recipients of cardiac transplantations should not be treated with intravenous iron preparations. von Haehling, S. et al. Nat. Rev. Cardiol. advance online publication 21 July 2015; doi: /nrcardio REVIEWS Institute of Innovative Clinical Trials, Department of Cardiology and Pneumology, University of Göttingen Medical School, Robert-Koch- Strasse 40, Göttingen, Germany (S.v.H., S.D.A.). Department of Heart Diseases, Wroclaw Medical University, ulica Weigla 5, Wroclaw, Poland (E.A.J., P.P.). Department of Cardiology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Gronigen, Netherlands (D.J.v.V.). Correspondence to: S.v.H. stephan.von.haehling@ web.de Introduction Iron deficiency is an extremely common nutritional disorder that affects up to 2 billion people worldwide, 1,2 particularly infants, young children, adolescents, elderly individuals, those with chronic inflammatory diseases, and women, in whom menstruation and pregnancy are additional risks. Iron deficiency affects 30 40% of pre-school-age children and pregnant women in industrialized countries, and nearly all individuals in these groups are affected in developing countries. 3 Physiologically, apart from erythropoiesis, iron is involved in many organ systems, with roles in oxygen transport and storage, mitochondrial function, and synthesis and degradation of proteins, lipids, and ribonucleic acids, as well as myocardial and skeletal muscle metabolism, and function of the thyroid gland, central nervous system, and immune system. However, the human body prioritizes the metabolic use of iron in several ways, and erythropoiesis has priority over other functions. Therefore, erythropoiesis remains undisturbed until late in the course of iron depletion. 4 However, the demands for erythropoiesis are high, given that a single red blood cell contains on the order of 1 billion atoms of iron, and anaemia develops quickly when iron stores are depleted. 5 Competing interests S.v.H. has received consulting honoraria from Novartis, Pfizer, Respicardia, Thermo Fisher Scientific, and Vifor Pharma, as well as lecture fees from Amgen and Sorin. E.A.J. has received honoraria for lectures and participation in advisory boards from Vifor Pharma. D.J.v.V. has received board membership fees from Amgen and Vifor Pharma. P.P. is a consultant for, and has received honoraria for speaking from, Amgen and Vifor Pharma, as well as research grants from Vifor Pharma. S.D.A. has received consulting fees and honoraria from Bayer, BRAHMS, Novartis, and Vifor Pharma, consulting fees from Keryx, and research support from Abbott Vascular and Vifor Pharma. Plasma concentrations of thyroid hormones increase in response to cold exposure, and the increase is attenuated in patients who are iron deficient, because of impaired functioning of haem-dependent thyroid peroxidase. 6 Iron deficiency also affects intellectual functioning, and impairs intellectual development in children. The importance of iron for brain function is supported by the finding that receptors for the iron-binding protein transferrin are present on capillary endothelial cells in the brain, and transport of iron across the blood brain barrier is tightly regulated. 7 Prenatal iron supplementation in pregnant women has beneficial effects on the intellectual development of offspring. 8 In elderly individuals, effects of iron deficiency on brain function might be mitigated by the increase in levels of iron in the brain that occurs with advancing age. 9 However, iron deficiency is highly prevalent in those aged >80 years. Couterintuitively, patients with age-related degenerative disorders, such as Alzheimer s disease, present with abnormally high iron levels in the brain. Experimental data from in vitro and animal studies indicate that iron deficiency impairs skeletal muscle energetics by mechanisms ranging from shifts in energy-substrate preferences to subcellular and molecular abnormalities, which result in insufficiencies in o xidative capacity. 10,11 Research has now focused on the roles of iron and iron deficiency in patients with cardiovascular disease. In 2000, the results of the first study aimed at treating anaemia in patients with heart failure (HF) were published, showing that subcutaneous application of erythropoietin, combined with intravenous iron sucrose, improved haemoglobin levels, left ventricular ejection fraction (LVEF), and NYHA classification of HF. 12 The results of subsequent studies have shown NATURE REVIEWS CARDIOLOGY ADVANCE ONLINE PUBLICATION 1

2 Key points Iron deficiency has detrimental effects in patients with coronary artery disease, heart failure (HF), pulmonary hypertension, and possibly in patients undergoing cardiac surgery One-third of all patients with HF and more than one-half of patients with pulmonary hypertension present with iron deficiency Improved exercise capacity, quality of life, or both have been shown after iron administration in patients with iron deficiency and HF or pulmonary hypertension Recipients of cardiac transplantations should probably not be treated with intravenous iron preparations that replenishing iron alone can be beneficial in patients with HF and iron deficiency, irrespective of anaemia. 13 Indeed, a hypothesis published in 1981, suggesting that iron has cardiotoxic effects, 14 has since been called into question, and studies have shown that iron deficiency is important not only in patients with HF, but also in patients with other cardiovascular diseases, such as coronary artery disease (CAD) or pulmonary hypertension, and potentially in those undergoing cardiac surgery. It seems that, although iron can be detrimental in some instances, iron repletion can also be beneficial. In this Review, we present discussions of the diagnostic dilemmas involved in the assessment of human iron metabolism, the effects of iron deficiency in patients with cardiovascular disease, and the selection of individuals who will benefit the most from iron repletion. We also highlight therapeutic possibilities in patients with iron deficiency, with or without anaemia. Pathophysiology of iron metabolism Iron is an essential trace element with unique features. The bivalent, ferrous form (Fe 2+ ) can donate electrons, whereas the trivalent, ferric form ( ) can accept electrons. These features are required for oxygen transport, but potentially cause toxicity by the generation of free radicals. In the human body, iron is bound by proteins that detoxify it. The total amount of iron in the body has been estimated at 3 5 g, with two-thirds contained in haemoglobin. 15 Iron is also bound to ferritin in several cell types, with healthy men storing 800 1,000 mg, and women storing mg iron in this way. These f erritin-bound iron reserves would be sufficient for nearly 3 years in men and 6 months in women at normal rates of bodily iron loss (1 2 mg per day), should the iron supply from external sources cease. 15 The human body does not possess any known mechanism of active iron excretion, but instead regulates iron uptake in the duo denum and upper jejunum. 16 A typical diet in the Western world contains 5 6 mg of iron per 1,000 kcal, 90% of which is nonhaem iron that mostly consists of. This ferric iron is much more poorly absorbed than Fe 2+, which in the form of haem can comprise half of the iron in meat, fish, and poultry. 17 Daily iron intake in developed nations averages mg, 1 2 mg of which is ultimately absorbed, equivalent to the quantity of iron that is lost via blood, skin, urine, or gut mucosa. 17,18 Iron deficiency has been detected using parameters that vary in sensitivity, specificity, and sources of error (Table 1). The definition of iron deficiency that has been used most extensively in cardiology derives from large-scale studies of HF, and includes both absolute and functional iron deficiency; the latter implies that iron is present in stored form, but cannot be mobilized, and functional benefits can result from iron supplementation. In functional iron deficiency, iron can be trapped inside macrophages, enterocytes, and hepatocytes as a result of the downregulation of ferroportin, a transmembrane channel protein that transports intracellular Fe 2+ across the cell membrane (Figure 1). Ferroportin Table 1 Diagnostic tests to assess iron status 92 Parameter Description Reference value in healthy individuals Serum iron Iron contained in serum Men μg/dl; women μg/dl Ferritin Transferrin Transferrin saturation (TSAT) Soluble transferrin receptor Hypochromic red blood cells (HYPO) Reticulocyte haemoglobin content (CHr) Responsible for iron storage inside various cell types Men μg/l; women μg/l Sensitivity Specificity Notes Poor* Poor* Strong circadian rhythm, high levels in the morning, levels rise after meals, levels fall during acute infections 35 48% % Sources of error include inflammatory processes, malign tumours, liver disease, chronic alcohol abuse, and smoking Transport protein with two mg/dl Poor* Poor* Sources of error include inflammatory processes, binding sites for malign tumours, and oral contraception Represents the amount of iron bound to circulating transferrin Transferrin receptors expressed on the cell surface shuttle iron into the cell; proteolysis cleaves them from the cell surface Red blood cells with reduced haemoglobin content Haemoglobin content in red blood cells that are only 1 2 days old *Assessment of each of these tests on its own is not useful % 59 88% 63 78% Reliable test for assessment of iron deficiency; sources of error include malnutrition (can increase transferrin saturation if serum iron is constant) mg/dl 70 81% 59 71% High cost relative to other tests <2.5% 64 78% 77 78% Increased values reflect iron deficiency in erythropoiesis pg 53 78% % Representative of the availability of iron for erythropoiesis a few days before testing 2 ADVANCE ONLINE PUBLICATION

3 Iron intake Haem (Fe 2+ ) Non-haem ( ) Oral administration (Fe 2+ ) Liver ferritin Transferrin receptor Ascorbate Haem Fe 2+ HCP1 DMT1 Haem Ferrireductase Haem oxidase Porphobilinogen Ferroportin Hepcidin Fe 2+ Hephaestin Ferroportin Receptormediated endocytosis Fe 2+ Fe 2+ Gut mucosa cell ferritin Transferrin Macrophage Figure 1 Iron absorption in the gut mucosa and iron storage inside cells of the reticuloendothelial system. In the gut mucosa, haem is transported by HCP1, (which is insoluble above ph 3) is mostly converted to Fe 2+ by ferrireductase activity or reduction by dietary ascorbate, and Fe 2+ is transported by DMT1. Intracellularly, Fe 2+ is released from haem by haem oxidase activity. Fe 2+ is converted to and bound by ferritin for storage, or released via ferroportin and converted to by hephaestin. Hepcidin, a liver-derived acute-phase reactant, binds to ferroportin and inhibits its function in iron absorption and iron mobilization from stored pools. Transferrin binds and transports, and is taken up by receptormediated endocytosis into macrophages for storage (ferritin) and subsequent release (ferroportin). Abbreviations: DMT1, divalent metal transporter 1; HCP1, haem carrier protein 1. activity is negatively regulated by the liver-derived, acute-phase reactant hepcidin. 15,19 Inflammation results in the upregulation of hepcidin expression, inhibiting the m obilization of iron stores. Iron deficiency in patients with HF (and possibly other cardiovascular diseases) can be diagnosed when serum ferritin is <100 μg/l, or when serum ferritin is <300 μg/l and transferrin saturation (TSAT) the percentage of iron binding sites on transferrin occupied by iron is <20%. Bone marrow requires around 20 mg of iron per day for erythropoiesis, most of which is recycled from the degradation of aged red blood cells. However, iron deficiency can negatively affect the development of fatigue, exercise capacity, and quality of life, whether anaemia is present or not (Figure 2). In cardiovascular illness, such effects can add to already present symptoms and reduce quality of life even further. Therapeutic options for the treatment of iron deficiency include oral iron administrations that generally contain Fe Their effectiveness is limited by the small amount of iron that is absorbed in the gut, their metallic taste, and because up to 40% of recipients experience adverse effects, most frequently gastrointestinal discomfort. 21 Parenteral iron preparations have been available for >50 years, and have seen tremendous development over the past 20 years. Currently, six different intravenous iron preparations are available across most European countries iron(iii) hydroxide dextran, iron(iii) gluconate, and iron(iii) hydroxide sucrose which have been on the market for >30 years and iron(iii) hydroxide polymaltose complex (ferric carboxy maltose), ferumoxytol, and iron isomaltoside 1000, which enable higher doses of iron to be administered in one sitting, to replenish iron stores rapidly. 22,23 In the setting of cardiovascular illness, most experience relates to the use of iron sucrose and ferric carboxymaltose, both of which have a good overall safety profile. Iron in CAD In 1981, Sullivan published a hypothesis suggesting that higher levels of stored iron lead to the increased incidence of heart disease in men and postmenopausal women compared with premenopausal women, and that stored iron is a chronic poison in the heart, causing arrhythmias and diffuse myocardial fibrosis. 14 On the basis of this hypothesis, vigorous exercise or phlebotomy have been suggested to correct iron accumulation and prevent ischaemic heart disease. 24 The results from one of the first studies to investigate the role of iron overload in the risk of CAD were published in 1992, and showed that a high level of stored iron demonstrated by elevated serum ferritin is a risk factor for coronary heart disease (CHD). 25 Iron was thought to induce the formation of highly reactive oxygen species and the peroxidation of lipids, thereby promoting atherosclerosis. 26,27 These conclusions have been questioned, and in nine of 11 cross-sectional or case control studies to investigate this hypothesis up to 2002, no association was found between CAD and serum ferritin levels. 27 In a meta-analysis of 12 long-term, prospective studies involving a total of 7,800 patients, 11 studies included adjustments for smoking and standard cardiovascular risk factors. 28 The findings were fairly consistent among different markers of iron status, and did not support the existence of strong epi demiological associations between iron status and CHD. 28 The connection between iron and heart disease was questioned as a result of these findings and others, with the conclusion that resources could be better invested in the investigation of other potential risk factors of heart disease, or in the exploration NATURE REVIEWS CARDIOLOGY ADVANCE ONLINE PUBLICATION 3

4 Iron stores Serum ferritin/tsat Haematopoiesis Iron repletion Iron deficiency Near depletion of iron stores Normal/normal Normal or low/low Low/low Anaemia No No Yes Figure 2 Comparison of different levels of iron storage and their effects on serum markers and haematopoiesis. Abbreviation: TSAT, transferrin saturation. of associations between iron stores and other disease outcomes, such as diabetes mellitus. 29 Furthermore, not all forms of iron are necessarily equivalent, and iron that is not bound to transferrin might be chemically reactive and able to enter cells directly. 29 Subsequent meta-analyses have also found no positive association between body iron content and CAD. 30,31 In a systematic review and meta-analysis of 21 studies, including 9,236 patients with CAD or myocardial infarction (MI) and a total of 156,427 participants, with follow-up duration of 3 17 years, no significant association was found between serum ferritin, total iron-binding capacity, serum iron, and CAD or MI. 30 However, contrary to Sullivan s hypothesis, a significant negative association was identified between transferrin saturation and CAD or MI, leading to the conclusion that high body iron stores could confer protection against development of CHD. 30,31 In contrast to the proposal that iron depletion via blood loss has potential benefit, in a study involving 38,244 men free from diagnosed cardiovascular disease, blood donations were not associated with the risks of MI or fatal CHD. 32 During 4 years of follow-up observation, 328 nonfatal MIs and 131 coronary deaths occurred. The age-adjusted relative risk of MI in the highest category of >30 blood donations, compared with men who never donated blood, was 1.2 (95% CI , P = 0.4). 32 Acceptance of the hypothesis that iron causes heart disease has hampered the development of treatment strategies for symptomatic patients with cardiovascular disease. Researchers are now studying iron deficiency in patients with CAD. An analysis from the Ludwigshafen Risk and Cardiovascular Health Study 33 showed that, among 1,480 patients with stable CAD and 682 individuals without CAD, the risk of having CAD was increased in patients in the lowest quartiles of TSAT, ferritin, and soluble transferrin receptor (stfr) levels. The association between iron depletion and CAD was independent of concomitant anaemia. 33 In a high-risk group of 287 patients with type 2 diabetes and stable CAD, followed up for a mean of 45 ± 19 months, both serum ferritin and stfr strongly predicted 5 year all-cause mortality, independently of other variables including haemoglobin, renal function, and markers of inflammation or neurohormonal activation. 34 Whereas an exponential relationship was observed between stfr levels and mortality (adjusted HR 4.24 for a difference of 1 in the logarithm [base 10] of transferrin levels [mg/l], 95% CI , P = 0.01), the curve describing the relationship between serum ferritin and mortality was U shaped (adjusted HR 7.18 for the lowest quintile versus the middle quintile, 95% CI , P = 0.002). 34 Along with high levels of stfr, both low and high levels of serum ferritin identify patients with type 2 diabetes and clinically overt CAD who have a poor prognosis. 34 Iron status has been investigated in the bone marrow of patients with stable CAD who qualified for CABG surgery. 35 Iron deficiency was defined as depleted extracellular iron stores (grade 0 1 according to the Gale scale), and 10% of erythroblasts containing iron. With this definition, iron deficiency was found in the bone marrow of 48% of patients (63% of anaemic patients and 43% of nonanaemic patients). Among circulating biomarkers of iron deficiency, high levels of serum stfr had the strongest association with depleted iron in bone marrow (area under the curve ± 0.048, the most accurate cut-off being 1.32 mg/l, with 67% sensitivity and 97% specificity). 35 Analysis of the data suggests that iron deficiency is frequent in patients with CAD, regardless of the definition of iron deficiency that is used low ferritin, high stfr, or directly measured depleted iron stores in bone marrow. Iron deficiency increases the likelihood of death, particularly in patients with high-risk profiles, such as diabetics. In men, increased iron stores cannot be viewed as a risk factor for the development of CAD. However, whether replenishing iron stores in patients with CAD and iron deficiency is beneficial or not cannot yet be answered with certainty. Iron in HF In patients with HF, the role of iron deficiency in pathophysiology and treatment is pronounced, and has been investigated thoroughly. 13,16 The studies have focused on patients with HF and reduced ejection fraction, and have included patients with ischaemic or nonischaemic aetiology. In response to iron supplementation, symptomatic improvements and benefits in terms of exercise capacity have been shown to be independent of the presence of anaemia. Prevalence of anaemia and iron deficiency Anaemia is common in patients with HF, with estimated prevalences of % in patients with reduced ejection fraction, and % in patients with preserved ejection fraction. 37,40,41 Overall, anaemia has been estimated to affect one-third of all patients with HF, and its presence has independent predictive value for morbidity and mortality. 43 The pathophysiology of anaemia is complex, and iron deficiency is one important aspect Several studies have been conducted to investigate the prevalence of iron deficiency in patients with chronic HF, using various definitions. 47 In a Canadian study involving 4 ADVANCE ONLINE PUBLICATION

5 12,065 patients with newly diagnosed HF, 17% had anaemia and 21% of these anaemic patients had absolute iron deficiency, determined by hospital discharge data using International Classification of Diseases (ICD) 9 codes, implying depletion of iron stores. 48 In an examination of bone-marrow biopsy samples from 37 anaemic patients with end-stage HF, 27 had iron deficiency in the bone marrow, even though they had normal or nearnorm al serum ferritin levels. 49 Using the broad definition of iron deficiency serum ferritin <100 μg/l or serum ferritin <300 μg/l in combination with TSAT <20% in 546 consecutive patients with clinically stable, chronic HF with reduced ejection fraction (LVEF 26 ± 7%, 82% of patients in NYHA class II or class III), iron deficiency was found in 37% of patients (57% of anaemic and 32% of nonanaemic individuals). 50 Risk factors for the development of iron deficiency included female sex, more-advanced stage of HF, higher levels of N terminal pro-b type natriuretic peptide (NT probnp), and higher serum levels of C reactive protein (CRP). 50 Iron deficiency but not anaemia was an independent predictor of unfavourable outcome. 50 An analysis of data from the US National Health and Nutrition Examination Survey (NHANES) database demonstrated that 352 of 574 patients with HF were iron deficient. 51 Haemoglobin level was an independent predictor of cardiovascular mortality, but iron deficiency was not. 51 Treatment of anaemia and iron deficiency Initial attempts to improve cardiovascular health in patients with HF by correcting anaemia used a combination of erythropoietin and iron sucrose, which produced improvements in NYHA class, haemo globin levels, and LVEF in cohorts of 32 individuals. 12,52 Similar improvements were observed in a larger cohort of 179 patients with HF, 47% of whom were diabetics. 53 The results of these initial, nonblinded studies were subsequently corroborated by the results of single-blinded 54 and double-blinded, randomized, studies with combinations of erythropoietin 54,55 or the synthetic erythropoietin analogue darbepoetin alfa and intravenous iron sucrose or oral iron preparations. Oral ferrous gluconate was less effective at improving anaemia and cardiovascular function than intravenous iron supplementation. 54,55 The results of these studies of the correction of anaemia in patients with HF have demonstrated that symptomatic and functional improvements are possible within time-frames of 3 12 months. In the RED HF trial, 59 2,278 patients with systolic HF and mild-to-moderate anaemia (haemoglobin g/dl) were randomly assigned to receive darbepoetin alfa or placebo. The primary end point of the trial was a composite of death from any cause or hospitalization for worsening HF. Unfortunately, no significant betweengroup differences were observed in the primary or any of the secondary outcomes, in any of the prespecified subgroups of patients. 59 Moreover, an increased rate of thromboembolic events (13.5% versus 10.0%; P = 0.009) and risk of ischaemic stroke (4.5% versus 2.8%; P = 0.03) was observed in patients receiving darbepoetin alfa compared with the placebo group. 59 The disappointing results of the RED-HF study led to a halt in the development of trials of erythropoietin and darbepoetin alfa in the setting of HF, although the reason for the neutral effect remains unknown. Possible explanations include the rapid increase in haemoglobin, the haemoglobin target of 13 g/dl (not justified by evidence), and the rather high threshold for initiation of iron supplementation. The results of the first study into the effects of intravenous iron sucrose supplementation without erythropoietin were published in In this open-label, nonrandomized, noncontrolled study, 16 patients with anaemia (haemoglobin 12 g/dl) in NYHA class II (56%) or class III (44%) were treated for 12 days and followed up for a mean of 92 ± 6 days. 60 At the end of the study, all patients were in NYHA class II, their haemoglobin levels had risen, and their quality-of-life scores had improved. 60 These results were corroborated by those of a double-blind, randomized, trial involving 40 patients with anaemia and symptomatic HF, who were treated with intravenous iron sucrose. 61 In an observer-blinded study, 35 patients with chronic HF were randomly allocated in a 2:1 ratio to receive intravenous iron sucrose or no treatment; iron loading had beneficial effects on exercise capacity, and the benefits were more evident in anaemic than nonanaemic patients. 62 The double-blind, randomized,, multicentre FAIR HF trial 63 was conducted to assess the effect of ferric carboxymaltose in patients with chronic HF. Patients were eligible for inclusion in the trial if they were symptomatic in NYHA class II with an LVEF 40%, or in NYHA class III with an LVEF 45% (Table 2). 63 As the inclusion criteria included haemoglobin levels between 9.5 g/dl and 13.5 g/dl, the presence of anaemia was not a prerequisite. 63 However, the presence of iron deficiency at enrolment was a conditio sine qua non for study inclusion, and was defined as a serum ferritin level of <100 μg/l, or μg/l with TSAT <20%. Randomization was performed at a 2:1 ratio of intravenous ferric carboxymaltose to placebo. Patients in the iron-loading group received 200 mg ferric carboxymaltose weekly during an initial correction phase, until the iron deficit calculated using Ganzoni s formula was corrected. This phase was followed by a maintenance phase during which patients received 200 mg ferric carboxymaltose monthly. 63 After 24 weeks of follow-up in 459 patients, 50% of individuals in the iron-loading group experienced improvement according to the self-reported Patient Global Assessment (the primary end point of the study), compared with 28% in the placebo group (OR 2.51 for being in a better rank, 95% CI , P <0.001). 64 Other parameters of physical well-being, such as NYHA class, 6 min walk distance, and quality of life had also improved significantly in the ferric carboxy maltose treatment group. Improvements in the 6 min walk distance and quality of life were seen as early as 4 weeks after commencement of treatment. Most importantly, this effect was present irrespective of whether patients were anaemic. Rates of death, hospitalization, and adverse events were similar in treatment and control groups. 64 NATURE REVIEWS CARDIOLOGY ADVANCE ONLINE PUBLICATION 5

6 Table 2 Inclusion criteria and study objectives in major trials of iron replacement in patients with HF Variables Clinical trial FAIR-HF 63 CONFIRM-HF 68 EFFECT-HF 71 FAIR-HFpEF ClinicalTrials.gov reference NCT NCT NCT Not yet assigned Diagnosis HFrEF HFrEF HFrEF HFpEF Number of patients Randomization scheme 2:1 (FCM:placebo) 1:1 (FCM:placebo) 1:1 (FCM:standard of care) 1:1 (FCM:placebo) Blinding Double-blind Double-blind Open-label Double-blind Symptoms NYHA class II III NYHA class II III NYHA class II III NYHA class II III LVEF 40% in NYHA class II, 45% in NYHA class III Definition of iron deficiency Serum ferritin <100 ng/ml, or serum ferritin ng/ml with TSAT <20% 45% 45% 45% together with evidence of diastolic dysfunction Serum ferritin <100 ng/ml, or serum ferritin ng/ml with TSAT <20% Serum ferritin <100 ng/ml, or serum ferritin ng/ml with TSAT <20% Serum ferritin <100 ng/ml or serum ferritin ng/ml with TSAT <20% Haemoglobin g/dl <15 g/dl 15 g/dl >9.0 g/dl to 14.0 g/dl Natriuretic peptides Not included BNP >100 pg/ml, NT probnp >400 pg/ml BNP >100 pg/ml, NT probnp >400 pg/ml Duration 24 weeks 52 weeks 24 weeks 52 weeks Primary end point Self-reported PGA score at week 24 and NYHA class at week 24, adjusted for baseline NYHA class Change in 6 min walk distance from baseline to week 24 Change in peak VO 2 from baseline to week 24 BNP >100 pg/ml, NT probnp >400 pg/ml, MR proanp >125 pmol/l Change in 6 min walk distance from baseline to week 24 Abbreviations: BNP, B type natriuretic peptide; FCM, ferric carboxymaltose; HF, heart failure; HFpEF, heart failure with preserved ejection fraction; HFrEF, heart failure with reduced ejection fraction; LVEF, left ventricular ejection fraction; MR proanp, mid-regional atrial natriuretic peptide prohormone; NT probnp, N terminal BNP prohormone; PGA, Patient Global Assessment; TSAT, transferrin saturation. Publication of the results of the FAIR-HF trial in 2009 led to the inclusion of a recommendation in the 2012 ESC guidelines for the management of HF. 65 The guidelines currently give a class Ic recommendation for the assessment of blood biochemistry parameters to enable detection of reversible and treatable causes of HF (such as hypocalcaemia and thyroid dysfunction) and comorbidities (for example iron deficiency). 65 The guidelines cite chronic HF, haemodilution, iron loss or poor utilization, renal failure, chronic disease, and malignancy as causes of anaemia, and recommend performance of a diagnostic work-up and consideration of treatment. On the subject of iron deficiency, the guidelines state that iron therapy can be considered as a treatment in affected patients. 65 A 2011 update of the National Heart Foundation of Australia and Cardiac Society of Australia and New Zealand guidelines for the management of HF contained a grade B recommendation to look for and treat iron deficiency in patients with chronic HF, to reduce symptoms and improve exercise tolerance and quality of life. 66 The 2013 joint HF guidelines of the ACC and AHA did not consider the results of the FAIR HF study to be sufficient to make a recommendation, stating in the absence of a definitive evidence base, the writing committee has deferred a specific treatment r ecommendation regarding anemia. 67 The evidence base for management of HF was substantially strengthened by the results of the doubleblind, multicentre, prospective, randomized, two-arm CONFIRM HF study, 68 which were very similar to those of the FAIR HF trial. In the CONFIRM HF trial, 68 ambulatory patients with symptomatic chronic HF (NYHA class II or class III), with LVEF 45%, elevated BNP or NT probnp, iron deficiency, and haemoglobin <15 g/dl were enrolled (Table 2) and randomly assigned in a 1:1 ratio to treatment with ferric carboxymaltose or placebo for 52 weeks. The dosing regimen was based on patients haemoglobin values and body weight, so that they received 500 2,000 mg of iron (or placebo) within the first 6 weeks after study entry, with an additional 500 mg of iron at each visit in weeks 12, 24, and 36 if ferritin and TSAT measurements indicated the persistence of iron deficiency. 68 The primary end point was the change in the 6 min walk distance from baseline to week 24. In addition, assessments of the change in the 6 min walk distance were performed from baseline to weeks 6, 12, 36, and 52. Quality of life, measured by the Patient Global Assessment score, was determined at weeks 6, 12, 24, 36, and 52. Safety end points included overall safety over the treatment period of 52 weeks. 68,69 The group of patients that received ferric carboxymaltose had a significantly greater 6 min walk distance than patients in the placebo group at week 24, with a difference of 33 ± 11 m (P = 0.002), and this effect was sustained to week Self-reported Patient Global Assessment and fatigue scores were significantly improved with iron-loading after 24 weeks compared with placebo, and the improvement was maintained to week Interestingly, treatment with intravenous ferric carboxymaltose was associated with a significant reduction in the risk of hospitalization for worsening HF (HR 0.39, 95% CI , P = 0.009) ADVANCE ONLINE PUBLICATION

7 Table 3 Treatment studies of IV iron alone in patients with cardiovascular disease Study Study design Intervention and number of patients Heart failure Bolger et al. Open-label, (2006) 60 nonrandomized, noncontrolled Toblli et al. Double-blind, (2007) 61 randomized, Okonko et al. (2008) 62 [FERRIC-HF] Open-label, observer-blinded, randomized, Usmanov et al. Open-label, (2008) 93 nonrandomized, noncontrolled Anker et al. (2009) 64 [FAIR-HF] Ponikowski et al. (2014) 68 [CONFIRM-HF] Double-blind, randomized, Double-blind, randomized, Pulmonary arterial hypertension Viethen et al. Open-label, (2014) 77 nonrandomized, noncontrolled Cardiac surgery Madi-Jebara et al. (2004) 90 Garrido-Martin et al. (2012) 91 Double-blind, randomized, Double-blind, randomized, Anaemia as prerequisite for study inclusion Duration Definition of ID Main results IV iron sucrose (n = 16) Yes 92 ± 6 days Ferritin <400 μg/l Improvements in NYHA class, haemoglobin levels, 6 min walk distance, and quality of life IV iron sucrose (n = 40; n = 20 in treatment group) IV iron sucrose (n = 35; n = 24 in treatment group) Yes 6 months Ferritin <100 μg/l and/or TSAT <20% No 18 weeks Ferritin <100 μg/l, or ferritin <300 μg/l and TSAT <20% Improvements in NT probnp level and 6 min walk distance Improvements in haemoglobin levels and peak oxygen consumption (spiroergometry) IV iron sucrose (n = 32) Yes 26 weeks No ID definition Improvements in NYHA class, haemoglobin levels, and several echocardiography parameters (posterior wall thickness, left ventricular end-diastolic and endsystolic volume and diameter) IV ferric carboxymaltose (n = 459; n = 304 in treatment group) IV ferric carboxymaltose (n = 304; n = 152 in treatment group) IV ferric carboxymaltose (n = 20) IV iron sucrose (n = 120), with or without single dose of EPO (n = 80 in two treatment groups) IV iron sucrose or oral ferrous fumarate (n = 159; n = 107 in two treatment groups) No 24 weeks Ferritin <100 μg/l, or ferritin <300 μg/l and TSAT <20% No 52 weeks Ferritin <100 μg/l, or ferritin <300 μg/l and TSAT <20% No 8 weeks Serum iron <10 μmol/l, ferritin <150 μg/l, and TSAT <15% Improvements in PGA, quality of life, NYHA class, and 6 min walk distance Improvements in PGA, quality of life, NYHA class, 6 min walk distance, and fatigue score; reduced risk of hospitalization for worsening heart failure Improvements in quality of life and 6 min walk distance Yes 30 days No ID definition No benefit with regard to haemoglobin increase or blood transfusion needs; data with regard to exercise capacity or quality of life were not recorded Yes 1 month No ID definition No benefit with regard to haemoglobin increase or blood transfusion needs; data with regard to exercise capacity or quality of life were not recorded Abbreviations: EPO, erythropoietin; ID, iron deficiency; IV, intravenous; NT probnp, N terminal B type natriuretic peptide prohormone; PGA, Patient Global Assessment; TSAT, transferrin saturation. Ongoing trials have been designed to characterise further the effects of iron loading in relation to HF (Table 3). A study (FAIR HF 2) to investigate the effects of intravenous ferric carboxymaltose treatment on morbidity and mortality in patients with HF and reduced ejection fraction, is currently underway. An investigation into the effects of intravenous ferric carboxymaltose in patients with HF and preserved ejection fraction (FAIR HFpEF) is scheduled to commence recruitment in The EFFECT HF trial 71 is in the recruitment phase, and is designed to recruit up to 160 patients with iron deficiency and HF with reduced ejection fraction (Table 2), with a primary end point of effect of ferric carboxymaltose on patient exercise capacity measured as peak VO 2 value. Iron in PAH Discovery of the beneficial effects of iron supplementation in patients with chronic HF led to investigations into its effects in other areas of cardiovascular disease. Pulmonary arterial hypertension (PAH) has many features in common with chronic HF, including subclinical inflammation and clinical symptoms. 72 In a study of the prevalence of iron deficiency in 85 patients with idiopathic PAH and 120 patients with chronic thromboembolic pulmonary hypertension (CTEPH), 73 iron deficiency was defined as serum ferritin levels <10 μg/l, with normal or elevated serum transferrin levels, and a normal CRP value. With this extremely conservative definition of iron deficiency, 30.1% of patients with NATURE REVIEWS CARDIOLOGY ADVANCE ONLINE PUBLICATION 7

8 idiopathic PAH were iron deficient, compared with 4.9% of patients with CTEPH (P <0.0001). The prevalence of iron deficiency was higher in premenopausal women than in postmenopausal women. 73 With a definition based on serum iron <10 μmol/l and TSAT <15% in women and <20% in men, iron deficiency was found in 30 of 70 patients with idiopathic PAH. 74 Patients with iron deficiency had lower exercise capacity as assessed by the 6 min walk test, relative to patients without iron deficiency (390 ± 138 m versus 460 ± 143 m; P <0.05). 74 This effect was irrespective of the presence of anaemia (haemoglobin <12 g/dl in women and <13 g/dl in men). In another study, iron deficiency was defined as an stfr level >4.4 mg/l in women and >5.0 mg/l in men, which is likely to be less influenced by inflammatory activation than the assessment of ferritin would be (Table 1). 75 With this definition, 44.8% of 29 patients with idiopathic PAH presented with iron deficiency, even though anaemia was present in only 13.8% of the overall patient cohort. 75 The average 6 min walk distance of patients with iron deficiency was not reduced compared with that of patients without iron deficiency, but patients with iron deficiency were more likely to present with advanced symptomatic burden (NYHA class III). 75 In addition, in patients with iron deficiency relative to those without, mean pulmonary arterial pressure was higher (63.3 ± 12.2 mmhg versus 38.8 ± 16.7 mmhg; P = 0.04), levels of NT probnp were increased (median, interquartile range; 854 ng/l, 118 1,177 ng/l versus 136 ng/l, ng/l; P = 0.04), and cardiac index was reduced (1.3 ± 0.2 l/min/m 2 versus 2.5 ± 0.4 l/min/m 2 ; P = 0.001). 75 In a study of 98 patients with idiopathic PAH, 63% had stfr levels above the normal range of nmol/l, indicating iron deficiency. 76 Affected patients, relative to those with normal levels of stfr, had lower values of serum ferritin (median, interquartile range; 43.0 μg/l, μg/l versus 66.5 μg/l, μg/l; P <0.001), TSAT (median, interquartile range; 15.6%, % versus 27.0%, %; P <0.001), and hepcidin (median, interquartile range; 14.4 ng/ml, ng/ml versus 53.0 ng/ml, ng/ ml; P <0.001). 76 Erythropoietin levels were significantly elevated in patients with high stfr, compared with normal stfr (median, interquartile range: 32.5 miu/ml, miu/ml versus 12.5 miu/ml, miu/ ml; P <0.001). Importantly, the increased levels of stfr remained predictive of increased mortality after adjustment for age, WHO functional class, and 6 min walk distance (P = 0.01). These results led to the proposal that iron deficiency was a target for therapeutic intervention in idiopathic PAH. 76 In a pilot study, 20 patients with PAH and iron deficiency were treated with intravenous ferric carboxymaltose. 77 The control group consisted of 20 patients with PAH without iron deficiency, who did not receive iron supplementation. 77 Iron deficiency was defined by levels of serum iron <10 μmol/l, serum ferritin <150 μg/l, and TSAT <15%, in the absence of elevated levels of CRP (Table 3). 77 The iron deficit was calculated using Ganzoni s formula, and patients received ferric carboxymaltose as a single bolus infusion of 1,000 mg, not exceeding 15 mg/kg of body weight. The average dose was 925 mg. Patients with iron deficiency who underwent treatment with ferric carboxymaltose showed an increase in 6 min walk distance from ± 28.3 m to ± 25.5 m after 8 weeks (P = 0.007). This effect was associated with an improvement in the patients quality of life, assessed using the SF 36 questionnaire (P = 0.01). 77 Iron in heart transplantation Just as anaemia and iron deficiency have major roles in patients with HF, a similar situation has been demonstrated in heart transplantation recipients. Even though anaemia does not seem to be an independent predictor of death after cardiac transplantation, because its predictive value is lost after adjustment for kidney function, many patients remain anaemic even years after surgery. 78 For example, 41% of 160 heart transplantation patients who were followed up for an average of >8 years were reported to be anaemic. 79 In 267 patients undergoing orthotopic cardiac transplantation, pretransplantation anaemia was present in 26%, and post-transplantation anaemia at 6 weeks in 78%. 80 Survival at 1 year was 70% in individuals who were anaemic before transplantation, and 81% in those who were not (P <0.03). 80 In 105 heart transplantation recipients, iron deficiency was present in 39% after around 8 years of follow-up. 81 Iron administration in cardiac transplantation is a double-edged sword. Animal experiments in several organ-transplantation models have shown that after cardiac death of the donor, reduction of chelatable iron by the use of deferoxamine helps to reduce ischaemia reperfusion injury, prolonging graft viability and function. 18,82 In cardiac transplantation, addition of deferoxamine to the cardioplegic solution has helped to improve cardiac output and reduce apoptosis in the myocardium. 83 In relation to organ rejection, animal models have demonstrated that iron is required for T cell proliferation. 84 The T cell receptor is co-expressed with the transferrin receptor, the expression of which increases around 10 fold after T cell activation. 18,85 Blocking the transferrin receptor by the use of monoclonal antibodies inhibits T cell activation via iron starvation. 86 In patient care after heart transplantation, avoiding iatrogenic iron overload caused by intravenous iron infusions has been suggested to be important for optimizing outcomes. 18 The situation for patients on the waiting list for transplantation is different, because assessment of iron status and treatment of iron deficiency could improve patient outcomes, as it does for patients with HF. 18 Iron in nontransplant cardiac surgery In contrast to heart transplantation, anaemia is an independent predictor of mortality in patients undergoing other cardiac surgery. Prevalence of preoperative anaemia was reported to be 25.2% in a sample of 943 patients undergoing cardiac surgery, and was higher in men than in women (27.6% versus 19.9%; P <0.01). 87 In a prospective, observational cohort study involving 165 anaemic patients undergoing elective cardiac surgery requiring 8 ADVANCE ONLINE PUBLICATION

9 cardiopulmonary bypass, functional iron deficiency was present in 78 patients (48%) preoperatively. 88 Absolute iron deficiency, with serum ferritin <15 μg/l in women and <22 μg/l in men, was present in 26 of these patients. Functional iron deficiency was determined by values >4% for low haemoglobin density, which also encompasses patients with absolute iron deficiency. A high plasma h epcidin concentration was associated with poor outcomes at 30 days, but the presence of iron deficiency itself was not. 88 The predictive value of hepcidin was independent of operative risk, age, haemoglobin concentration, and sex. Mean days alive and out of hospital decreased by 0.8 (95% CI ) for each increase in hepcidin of 10 ng/ml (P = 0.01). 88 In a study of 100 patients undergoing cardiac surgery, 37% had iron deficiency, defined as serum ferritin <80 μg/l, or μg/l with TSAT <20%. 89 The group of patients with iron deficiency had a lower average age and higher female:male ratio than the group without iron deficiency. Relative to patients without iron deficiency, preoperative iron deficiency was associated with lower preoperative haemoglobin levels (P <0.006), higher perioperative transfusion rates during the first week (62% versus 35%; P <0.02), and higher scores of physical fatigue 7 days after surgery (P = 0.01). 89 Studies have also been conducted to investigate the value of oral or intravenous iron administration in patients undergoing cardiac surgery, primarily for the treatment of anaemia. In one double-blind trial, 120 patients were randomly assigned to a postoperative regimen of placebo, intravenous iron sucrose, or intravenous iron sucrose in combination with a single dose of erythropoietin. 90 The iron sucrose dose was calculated according to Ganzoni s formula. No significant differences were observed in haemoglobin increase or transfusion needs between the three groups of patients. 90 Unfortunately, quality of life, mobility, and physical wellbeing were not recorded, so it cannot be determined whether the treated patients benefitted in terms of earlier mobility or less fatigue after surgery. 90 In another study, investigators randomly assigned 159 patients undergoing cardiac surgery requiring cardiopulmonary bypass, in a double-blinded manner, to one of three treatment groups. 91 Group I received 300 mg of intravenous iron(iii) hydroxide sucrose complex per 24 h and one placebo tablet per 24 h during preoperative and postoperative hospitalization, with placebo only for 30 days after discharge. Group II received a placebo infusion while hospitalized, and one tablet of oral ferrous fumarate iron per day during hospitalization and for 30 days after discharge. Group III received oral and intravenous placebo during hospitalization, and oral placebo after discharge. At the end of the study, no differences were found between groups with regard to transfusion needs or haemoglobin levels. The greatest increase in serum ferritin levels was noted in group I, with intravenous iron loading. Physical mobility and well-being were not recorded. 91 On the basis of these studies, no clear recommendation can be given with regard to the correction of iron deficiency in patients undergoing cardic surgery. Conclusions Clinical interest and research into the roles of anaemia and iron deficiency in cardiovascular disease particularly HF has resulted in the inclusion of recommendations relating to both clinical entities in HF guidelines, some of which advocate the correction of iron deficiency to achieve symptomatic improvement. Although results relating to the prognostic value of iron deficiency remain inconclusive, anaemia has been shown unequivocally to be an independent predictor of death in patients with HF. Evidence of symptomatic improvements with iron supplementation in the one-third of patients with HF who are affected by iron deficiency is growing. Assessment of the potential reduction in morbidity and mortality after correction of iron deficiency in patients with HF (currently underway in the FAIR HF 2 trial) is the next step in this research. The roles of iron deficiency and anaemia in other areas of chronic cardiovascular disease, such as CAD, PAH, and cardiovascular surgery require further investigation. In patients with CAD, preliminary evidence suggests that iron deficiency and CAD have a detrimental association, and iron administration by any route can confer benefit. In PAH, more patients seem to be affected by iron deficiency than expected. The results of an open-label, nonrandomized treatment study suggest that intravenous iron administration can help to improve exercise capacity in patients with PAH. The involvement of iron deficiency in patients undergoing cardiac surgery requires further research, because studies have so far focused on anaemia treatment and the requirements for blood transfusions, even though iron administration might have beneficial effects in terms of improved quality of life or earlier mobility after surgery. The situation is entirely different in patients undergoing cardiac transplantation surgery. Before surgery, patients with HF can benefit from intravenous iron treatment, but after surgery it seems to be contraindicated, because administration of large doses of iron might have a role in T cell activation and rejection of the transplanted organ. Iron deficiency is a common problem in patients with cardiovascular conditions. Evidence suggests that correction of iron deficiency can result in clinical benefits. Ongoing and future research should define the groups of patients other than those with HF who are most likely to benefit from iron supplementation, and determine the optimal treatment regimen(s) in these patients. 1. DeMaeyer, E. & Adiels-Tegman, M. The prevalence of anaemia in the world. World Health Stat. Q. 38, (1985). 2. Stoltzfus, R. Defining iron-deficiency anemia in public health terms: a time for reflection. J. Nutr. 131, 565S 567S (2001). 3. Lundqvist, H. & Sjöberg, F. Food interaction of oral uptake of iron / a clinical trial using 59 Fe. Arzneimittelforschung 57, (2007). 4. Andrews, N. C. & Schmidt, P. J. Iron homeostasis. Annu. Rev. Physiol. 69, (2007). 5. Andrews, N. C. Disorders of iron metabolism. N. Engl. J. Med. 341, (1999). 6. Zimmermann, M. B. The influence of iron status on iodine utilization and thyroid function. Annu. Rev. Nutr. 26, (2006). NATURE REVIEWS CARDIOLOGY ADVANCE ONLINE PUBLICATION 9