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HFEC282Y homozygotes have an increased risk for developing increased iron stores and related disorders. It is controversial whether dietary iron restrictions should be recommended to such individuals.
To determine whether dietary iron content influences iron stores in HFEC282Y homozygotes as assessed by serum ferritin concentration.
Serum ferritin concentration was measured and a dietary iron questionnaire was completed as part of the evaluation of 213 HFEC282Y homozygotes who were identified through screening of >100,000 primary care patients at five HEmochromatosis and IRon Overload Screening (HEIRS) Study Field Centers in the United States and Canada.
No significant relationships between serum ferritin concentration and dietary heme iron content, dietary nonheme iron content or reports of supplemental iron use were found.
These results do not support recommending dietary heme or nonheme iron restrictions for HFEC282Y homozygotes diagnosed through screening in North America.
Les homozygotes HFEC282Y présentent un risque accru d’emmagasiner des réserves de fer et de présenter des troubles connexes. La décision de recommander des restrictions de fer d’origine alimentaire à ces individus est controversée.
Déterminer si le contenu en fer d’origine alimentaire influe sur les réserves en fer chez les homozygotes HFEC282Y, telles qu’elles sont évaluées par la concentration de ferritine sérique.
Les chercheurs ont mesuré la concentration de ferritine sérique et ont fait remplir un questionnaire sur le fer d’origine alimentaire dans le cadre de l’évaluation de 213 homozygotes HFEC282Y recrutés par dépistage de plus de 100 000 patients en soins primaires de cinq centres d’études HEIR sur le dépistage sur le terrain de l’hémochromatose et de la surcharge en fer aux États-Unis et au Canada.
Les chercheurs n’ont décelé aucune relation significative entre la concentration de ferritine sérique et le contenu en fer hémique d’origine alimentaire, le contenu en fer non hémique d’origine alimentaire ou les déclarations d’utilisation de suppléments de fer.
Ces résultats n’appuient pas la recommandation de restreindre le fer hémique ou non hémique chez les homozygotes HFEC282Y diagnostiqués par dépistage en Amérique du Nord.
Iron is absorbed from the diet as a part of the heme molecule or as ferrous iron not bound to heme (1). Heme is found primarily in such proteins as hemoglobin and myoglobin. Iron not bound to heme is present in vegetables, cereals and other foodstuffs. Iron absorption occurs most efficiently in the duodenum, and heme iron is absorbed more readily than iron not bound to heme (2). Heme crosses from the lumen into the enterocyte (3) by an unknown mechanism, after which iron is removed from the protoporphyrin ring through the action of heme oxygenase (4). Nonheme iron uptake into the enterocyte is mediated by the iron transporter, divalent metal transporter 1 (5). Divalent iron is exported across the basolateral membrane of enterocytes to the portal blood stream by ferroportin 1 (6), and the membrane-associated ferroxidase, hephaestin (7), assists in converting the iron to the ferric form that is bound by plasma transferrin. Several studies indicate that dietary heme iron content is a significant predictor of iron stores among individuals homozygous for the C282Y mutation of the HFE gene on chromosome 6p (8,9). Population studies conducted predominantly among Caucasians and Hispanics have shown that amounts of dietary heme iron, but not those of dietary nonheme iron, are positively associated with serum ferritin (SF) concentration (10,11).
The HEmochromatosis and IRon Overload Screening (HEIRS) Study is a multicentre, National Institutes of Health-sponsored study designed to determine the prevalence of primary iron overload in adult primary care patients of various ethnicities who reside in the United States (US) and Canada (12). More than 100,000 participants were screened by testing for HFEC282Y and HFEH63D mutations and measuring SF concentration and transferrin saturation (TS) (13). Participants with HFEC282Y homozygosity or combined elevations of SF and TS levels were invited to return for clinical follow-up, which included a dietary questionnaire. The purpose of the present research was to determine whether dietary nonheme iron, dietary heme iron and supplemental iron use influence SF concentration in HFEC282Y homozygotes identified in screening programs.
The present study was approved by the institutional review board of each participating institution and written informed consent was obtained from each participant.
A description of the HEIRS Study design has been reported (12). The study was approved by the institutional review board of each participating institution and written informed consent was obtained from each participant. Participants identified through primary care clinics and medical blood drawing laboratories were screened over a two-year period (February 2001 to March 2003) at five HEIRS Field Centres (Washington DC; Birmingham, Alabama; Irvine, California; Portland, Oregon – Honolulu, Hawaii [USA]; and London, Ontario). Both patients and other persons accompanying the patient were potential participants. Eligibility criteria included age ≥25 years and an ability to understand the informed consent. Participants were asked how they heard about the study and whether they had been previously diagnosed with iron overload or hemochromatosis. Race/ethnicity was determined by self-reported answers to two questions: one asking about Hispanic background and one asking for nonexclusive choice of five racial groups: Caucasian, African American, Asian, Pacific Islander and American Indian. Participants affirming Hispanic background were classified as Hispanic. All participants were asked about a history of hemochromatosis or iron overload, liver disease, diabetes, arthritis, congestive heart failure, impotence and infertility before receiving any genetic test results. HFEC282Y alleles were detected from blood spots using a modification of the Invader assay (Third Wave Technologies, USA) that increases the allele-specific fluorescent signal by including 12 cycles of locus-specific polymerase chain reaction before the cleavase reaction (14).
Of 299 HFEC282Y homozygotes identified in the HEIRS Study, 227 were not previously diagnosed with iron overload (13) and, were therefore, eligible for the present study. All were invited to participate in a postinitial screening clinical evaluation and 219 (96%) participated. Of these, 213 were included in the present analysis (216 completed questionnaires but three were excluded for extreme dietary values: see Statistical analysis section below). The clinical evaluations were performed over a two-and-one-half year period from July 2001 to February 2004. The mean ± SD number of days between initial screening and the clinical evaluation was 257±204. Participants in the clinical evaluation gave written informed consent in addition to the consent obtained in the initial screening part of the study. The purpose of the present clinical evaluation was to assess possible signs, symptoms and complications of iron overload, and to collect information regarding dietary and other factors possibly related to iron overload. Medical history, including the use of medications, vitamins, herbal remedies, and other supplements, were recorded on standardized forms and a food frequency questionnaire was completed (15). A limited physical examination was performed to detect signs of liver disease, heart disease, arthropathy or abnormal skin pigmentation. Venous blood was collected, usually in the fasting state.
SF concentration was measured by turbidometric immunoassay (Roche Applied Science/Hitachi 911, USA). Serum iron concentration and unsaturated iron-binding capacity were measured spectrophotmetrically and the TS was calculated. Serum concentrations of glucose, alanine aminotransferase (ALT), aspartate aminotransferase, gamma-glutamyl transferase and C-reactive protein were determined using a Hitachi 911 analyzer (Roche Diagnostics/Boehringer Mannheim Corporation, USA). Serum insulin concentrations were measured using a DPC Immulite Analyzer (Diagnostic Products Corporation, USA). Complete blood counts were measured using the Beckman Coulter GenS (Beckman/Coulter, USA). Participants with hemoglobin level ≤132 g/L (men) or ≤116 g/L (women), or mean corpuscular volume ≤77 fL, underwent analysis of reticulocyte count by flow cytometry, serum concentrations of haptoglobin, lactate dehydrogen-ase, direct and indirect bilirubin (Hitachi 911 analyzer), and hemoglobin electrophoresis with hemoglobin A2 and F quantification (high-performance liquid chromatography methodology). Participants with elevated ALT levels (≥40 IU/L in men or ≥31 IU/L in women) were tested for hepatitis B surface antigen and antibody to hepatitis C virus (Vitros ECi, Ortho-Clinical Diagnostics, Inc, USA).
To estimate dietary iron content and alcohol consumption, participants completed the University of Hawaii Multi-Ethnic Dietary Questionnaire. The correlation of nutrient intake estimated from this questionnaire with information from 24 h recalls has been documented (15). The questionnaire asks about average eating habits over the past year. The questionnaire was analyzed at the University of Hawaii and the results of the analysis provided estimates for average daily total dietary iron and the amount of dietary iron derived from meat, fish and poultry. An estimate for supplemental iron intake was also provided. The iron derived from meat, fish, and poultry was classified as heme iron, and the difference between total dietary iron and dietary heme iron as nonheme iron.
Statistical analyses were performed using SAS version 9.1.3 and Splus 7.0 (SAS Institute, USA) for Windows (Microsoft Corporation, USA). SF concentration was natural log transformed in all analyses. Multivariate linear regression was used to investigate the relationship between natural log SF concentrations and dietary iron content in HFEC282Y homozygotes, after considering potentially confounding factors such as age, sex, ethnicity, phlebotomy history, alcohol consumption, tea consumption, liver function tests and markers of inflammation. The antilog of regression coefficients shows the per cent change in SF concentration for a one unit difference in the independent variable. CIs for the coefficients were computed as the antilog of the geometric mean ± 1.96 × SE.
The primary analysis was multivariate linear regression of natural log SF concentration as a function of dietary nonheme iron, dietary heme iron and supplemental iron in the past two years (expressed as mg/kg/day), calories (kcal/day), age, race (white or nonwhite), male sex, alcohol consumption, C-reactive protein and ALT levels in participants with no history of blood transfusions or phlebotomy therapy. Two participants were excluded from the analysis because they reported extreme values of heme iron and one participant was excluded due to reporting extreme values for supplemental iron.
The characteristics of the study participants (HFEC282Y homozygotes who presented for clinical evaluation who completed a dietary questionnaire, and did not undergo previous quantitative phlebotomy or have >10 units of blood transfused) are summarized in Table 1. HFEC282Y homozygotes in this cohort were predominantly Caucasian, predominantly female and had a low prevalence of hepatitis, HIV infection or inflammatory conditions according to the medical history. Fourteen per cent had a history of malignancy.
The relationship between dietary iron and natural log SF is shown in Figure 1. After adjustment for the factors listed in the Methods section, neither heme nor nonheme iron was significantly associated with natural log SF (Table 2). Known adjustment factors of age, alcohol consumption, ALT and male sex were significantly associated with higher natural log SF.
The present study did not find evidence that dietary iron content, either heme iron or nonheme iron, influences SF concentration in HFEC282Y homozygotes identified by screening primary care patients in North America.
In previous studies by other investigators, the absorption of heme and nonheme iron from an unfortified meal (16) or nonheme iron from an oral iron test dose (17) by hemochromatosis patients diagnosed in medical care was greater than would be predicted from the relationship between iron absorption and SF levels observed in normal volunteers. There was a significant inverse relationship of absorption of nonheme iron with increasing SF. In contrast, heme iron absorption was not significantly related to SF concentration (16). In women in the United Kingdom, HFEC282Y homozygotes had SF concentrations 2.4 times higher than women with wild-type HFE genotype. The association between heme iron intake estimated using food frequency questionnaires and SF concentration was stronger in HFEC282Y homozygotes than in subjects with other HFE genotypes; nonheme iron intake had little effect on SF concentration (9,18,19). In the Netherlands, women >50 years of age, HFEC282Y homozygotes and HFEC282Y/H63D compound heterozygotes had significantly higher SF concentrations than women with other HFE genotypes. HFEC282Y homozygotes and HFEC282Y/H63D compound heterozygotes who consumed relatively high amounts of heme iron had the highest SF concentration (20). It has been proposed that the high rate of expression of iron overload and associated manifestations in hemochromatosis homozygotes diagnosed in medical care in Australia is attributable to the high national rate of meat consumption (21). Red meat consumption was positively associated with higher SF concentrations among adults in Busselton, Australia, regardless of HFE genotype (22,23). Some persons with HFEC282Y homozygosity may be especially susceptible to iron loading from diets in which a high proportion of available iron is present as heme, although they may also absorb increased fractions of nonheme iron.
Other studies have suggested that fortification of food with iron may increase the severity of iron overload in persons with hemochromatosis. In seven treated hemochromatosis patients, the absorption of nonheme iron from a test meal was measured using the extrinsic tag technique to simulate the effects of fortification (24). Doubling of the iron dose produced a proportional increase similar to that in normal subjects (25). In Sweden, fortification before 1995 accounted for 42% of the mean daily dietary iron content, although Swedish hemochromatosis homozygotes had lower iron burdens, on average, than those in Australia (26). Decreased fortification in Sweden since 1995 is expected to decrease the rate of iron accumulation in persons with hemochromatosis (27). It is similarly predicted that iron fortification of wheat flour at the current US levels would accelerate the initial rate of iron loading in persons with hemochromatosis, and that the accelerated evolution of clinical disease in susceptible individuals is directly proportional to the amount of fortification iron added (24,28).
The iron phenotypes of HFEC282Y homozygotes diagnosed in screening are typically less severe than those of hemochromatosis patients diagnosed in medical care. This could account for some differences observed in the present and in previous studies. There are several other limitations to the present study. For example, SF concentration was measured at one point in time and did not address the effects of dietary therapy in a randomized trial. We did not use direct measures of body or liver iron. Dietary iron content in the present study was determined by recall questionnaire; the amounts are, therefore, approximations rather than precise amounts. The questionnaire may not have had sufficient sensitivity to fully reflect the variability in dietary iron content. Nevertheless, these results do not support recommending dietary iron restrictions to patients identified through screening programs.
Formulating an iron-restricted diet is difficult because iron is ubiquitous in fruits, vegetables and meat products and, in the US, all products that contain flour are fortified with iron. Regardless, the concept of dietary iron restriction for management of hemochromatosis and iron overload continues to hold appeal for patients and is often advocated by patient support groups as a way to limit iron accumulation. Dietary iron deficiency has been reported in developing countries; however, in these studies it is difficult to exclude the effects of enteric infections such as Helicobacter pylori and parasitic infestations. Vegan diets have been associated with iron deficiency (29). In persons with hemochromatosis diagnosed in medical care, iron absorption decreases as body iron stores increase (17,30,31). Iron absorption can also be affected by other dietary factors such as tea consumption, alcohol consumption and the use of proton pump inhibitors that suppress gastric acid secretion (32). Iron depletion by periodic phlebotomy removes iron efficiently, whereas dietary manipulation does not remove absorbed iron and has little effect on the rate of iron accumulation. It is likely that less emphasis on an iron-restricted diet could improve the quality of life of patients and simplify long-term management of hemochromatosis.
AUTHOR CONTRIBUTIONS: Victor R Gordeuk participated in planning and conducting the study, data analysis and writing the manuscript. Laura Lovato participated in data analysis and writing the manuscript. James C Barton participated in planning and conducting the study and writing the manuscript. Mara Vitolins participated in data analysis and writing the manuscript. Gordon McLaren participated in planning and conducting the study and writing the manuscript. Ronald T Acton participated in planning and conducting the study and writing the manuscript. Christine McLaren participated in planning and conducting the study and writing the manuscript. Emily L Harris participated in planning and conducting the study and writing the manuscript. Mark Speechley participated in planning and conducting the study and writing the manuscript. John H Eckfeldt participated in planning and conducting the study and writing the manuscript. Sharmin Diaz participated in conducting the study and writing the manuscript. Phyliss Sholinsky participated in planning the study and writing the manuscript. Paul Adams participated in planning and conducting the study and writing the manuscript.
DISCLOSURES: The authors have no conflicts of interest to declare.
FUNDING: The HEIRS Study was initiated and funded by the National Heart, Lung, and Blood Institute, in conjunction with the National Human Genome Research Institute. The study is supported by contracts N01-HC05185 (University of Minnesota); N01-HC05186, N01-CM-07003-74, and Minority CCOP (Howard University); N01-HC05188 (University of Alabama at Birmingham); N01-C05189 (Kaiser Permanente Center for Health Research); N01-HC05190 (University of California, Irvine); N01-HC05191 (London Health Sciences Centre); and N01-C05192 (Wake Forest University). Additional support was provided by the University of Alabama at Birmingham General Clinical Research Center (GCRC) grant M01-RR00032, Howard University GCRC grant M01-RR10284, and the University of California, Irvine UCSD/UCI Satellite GCRC grant M01-RR00827, sponsored by the National Center for Research Resources, National Institutes of Health; Howard University Research Scientist Award UH1-HL03679-05 from the National Heart, Lung and Blood Institute and the Office of Research on Minority Health (VRG); and Southern Iron Disorders Center (JCB, RTA).
PARTICIPATING HEIRS STUDY INVESTIGATORS AND INSTITUTIONS: Field Centres: Birmingham, Alabama – University of Alabama at Birmingham: Dr Ronald T Acton (Principal Investigator), Dr James C Barton (Co-Principal Investigator), Ms Deborah Dixon, Dr Susan Ferguson, Dr Richard Jones, Dr Jerry McKnight, Dr Charles A Rivers, Dr Diane Tucker and Ms Janice C Ware. Irvine, California – University of California, Irvine: Dr Christine E McLaren (Principal Investigator), Dr Gordon D McLaren (Co-Principal Investigator), Dr Hoda Anton-Culver, Ms Jo Ann A Baca, Dr Thomas C Bent, Dr Lance C Brunner, Dr Michael M Dao, Dr Korey S Jorgensen, Dr Julie Kuniyoshi, Dr Huan D Le, Dr Miles K Masatsugu, Dr Frank L Meyskens, Dr David Morohashi, Dr Huan P Nguyen, Dr Sophocles N Panagon, Dr Chi Phung, Dr Virgil Raymundo, Dr Thomas Ton, Professor Ann P Walker, Dr Lari B Wenzel and Dr Argyrios Ziogas. London, Ontario – London Health Sciences Centre: Dr Paul C Adams (Principal Investigator), Ms Erin Bloch, Dr Subrata Chakrabarti, Ms Arlene Fleischhauer, Ms Helen Harrison, Ms Kelly Jia, Ms Sheila Larson, Dr Edward Lin, Ms Melissa Lopez, Ms Lien Nguyen, Ms Corry Pepper, Dr Tara Power, Dr Mark Speechley, Dr Donald Sun and Ms Diane Woelfle. Portland, OR and Honolulu, HI – Kaiser Permanente Center for Health Research, Northwest and Hawaii, and Oregon Health and Science University: Dr Emily L Harris (Principal Investigator), Dr Mikel Aickin, Dr Elaine Baker, Ms Marjorie Erwin, Ms Joan Holup, Ms Carol Lloyd, Dr Nancy Press, Dr Richard D Press, Dr Jacob Reiss, Dr Cheryl Ritenbaugh, Ms Aileen Uchida, Dr Thomas Vogt and Dr Dwight Yim. Washington, DC: Howard University: Dr Victor R Gordeuk (Principal Investigator), Dr Fitzroy W Dawkins (Co-Principal Investigator), Ms Margaret Fadojutimi-Akinsiku, Dr Oswaldo Castro, Dr Debra White-Coleman, Dr Melvin Gerald, Ms Barbara W Harrison, Dr Ometha Lewis-Jack, Dr Robert F Murray, Dr Shelley McDonald-Pinkett, Ms Angela Rock, Dr Juan Romagoza and Dr Robert Williams. Central Laboratory: Minneapolis, Minnesota, University of Minnesota and University of Minnesota Medical Center Fairview: Dr John H Eckfeldt (Principal Investigator and Steering Committee Chair), Ms Susie DelRio-LaFreniere, Ms Catherine Leiendecker-Foster, Dr Ronald C McGlennen, Mr Greg Rynders, Dr Michael Y Tsai and Dr Xinjing Wang. Coordinating Centre: Winston-Salem, North Carolina – Wake Forest University: Dr David M Reboussin (Principal Investigator), Dr Beverly M Snively (Co-Principal Investigator), Dr Roger Anderson, Ms Elease Bostic, Ms Brenda L Craven, Ms Shellie Ellis, Dr Curt Furberg, Mr Jason Griffin, Dr Mark Hall, Mr Darrin Harris, Ms Leora Henkin, Dr Sharon Jackson, Dr Tamison Jewett, Mr Mark D King, Mr Kurt Lohman, Ms Laura Lovato, Dr Joe Michaleckyj, Ms Shana Palla, Ms Tina Parks, Ms Leah Passmore, Dr Pradyumna D Phatak, Dr Stephen Rich, Ms Andrea Ruggiero, Dr Mara Vitolins, Mr Gary Wolgast and Mr Daniel Zaccaro. NHLBI Project Office: Bethesda, Maryland – Ms Phyliss Sholinsky (Project Officer), Dr Ebony Bookman, Dr Henry Chang, Dr Richard Fabsitz, Dr Cashell Jaquish, Dr Teri Manolio and Ms Lisa O’Neill. NHGRI Project Office: Bethesda, Maryland – Dr Elizabeth Thomson. Dr Jean MacCluer, Southwest Foundation for Biomedical Research, also contributed to the design of this study.
DISCLAIMER: The views expressed in the present article do not represent those of the National Heart, Lung, and Blood Institute, the National Institutes of Health, or the Department of Health and Human Services.