点击显示 收起
1 From the Institute of Food Research, Norwich Research Park, Norwich, United Kingdom (MAR, SLO, CM, JAH, RF, JRD, GM-N, and SJF-T); the Department of Human Nutrition, University of Otago, Dunedin, New Zealand (A-LMH); and the Norfolk and Norwich University Hospital NHS Trust, Norwich, United Kingdom (GW).
2 Supported by the UK Food Standards Agency, the Biotechnology and Biological Sciences Research Council, and the New Zealand Health Research Council (Fellowship to A-LMH). Corn Flakes and Poptarts without added iron were supplied by the Kellogg Company, Warrington, United Kingdom. 3 Address reprint requests to SJ Fairweather-Tait, Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, Norfolk, United Kingdom. E-mail: sue.fairweather-tait{at}bbsrc.ac.uk.
ABSTRACT
Background: The suggestion that carriers of the HFE C282Y mutation absorb nonheme iron more efficiently than do carriers of the wild type has public health implications for countries where the C282Y mutation is common and foods are fortified with iron.
Objective: We investigated the effect of C282Y heterozygosity on nonheme-iron absorption from a diet high in bioavailable iron and from iron-fortified cereals.
Design: The subjects were recruited from a parallel study investigating the relation between HFE mutations, habitual diet, and iron status. Iron absorption was measured in 15 wild-type carriers and 15 C282Y heterozygotes aged 40 y. Each subject consumed 3 meals of high iron bioavailability (labeled with Fe-57) for 2 d and 2 meals with fortified cereal products (labeled with Fe-54) for the next 3 d. Iron absorption was measured from isotope incorporation into red blood cells 14 d after the last labeled meal and was corrected for utilization of absorbed iron by means of an intravenous infusion of Fe-58.
Results: Absorption of Fe-57 with the high-iron-bioavailability diet was 6.8 ± 6.8% (0.6 ± 0.6 mg/d) in the wild-type carriers and 7.6 ± 3.2% (0.7 ± 0.3 mg/d) in the C282Y heterozygotes. Absorption of Fe-54 with cereal products was 4.9 ± 2.0% (0.7 ± 0.3 mg/d) in the wild-type carriers and 5.3 ± 1.3% (0.8 ± 0.2 mg/d) in the C282Y heterozygotes.
Conclusions: There was no overall significant difference between C282Y heterozygotes and wild-type men in iron absorption from either dietary nonheme iron or fortified cereal products.
Key Words: Iron absorption • HFE mutations • C282Y heterozygotes • iron fortification • dietary iron
INTRODUCTION
For many decades, iron deficiency has been the primary driver of public health policies concerned with iron nutrition. However, with increasing interest in the role of dietary prooxidants in chronic disease and the discovery that the cysteine-to-tyrosine substitution at codon 282 of the HFE gene (C282Y mutation) is a major risk factor for hereditary hemochromatosis (1), the potential health risks associated with an overabundant iron supply are under scrutiny. Up to 1 in 150 persons (2–4) in populations of northern European origin are homozygous for the C282Y mutation of the HFE gene. Hemochromatosis is an autosomal recessive condition in which excess iron is absorbed and accumulated and, if untreated, will cause progressive tissue damage, in particular, cirrhosis of the liver. Although most patients with hemochromatosis are homozygous for the C282Y mutation of the HFE gene (5), many other mutations in the HFE coding sequence have also been identified, the most common of which is the H63D mutation. Compound heterozygotes are heterozygous for both the C282Y mutation and the H63D mutation and account for 2.6% of hemochromatosis patients in the United Kingdom (5).
Fifteen years ago, Lynch et al (6) undertook iron-absorption studies in treated hemochromatosis patients and children or siblings who shared a single HLA haplotype. The inferred heterozygotes (geometric mean serum ferritin: 68 µg/L) and healthy control subjects (geometric mean serum ferritin: 50 µg/L) absorbed a similar amount of nonheme iron from a hamburger meal containing 3.4 mg nonheme iron, but when 20 mg Fe (as ferrous sulfate, with 100 mg ascorbic acid) was added to the meal, the heterozygotes absorbed nearly 3 times the amount of iron as did the control subjects (9.2% and 3.4%, respectively). This observation fueled concern about iron fortification in countries where HFE mutations are relatively common. If heterozygotes partially express the homozygote phenotype and also absorb significantly more iron from fortified foods, they may accumulate iron and be susceptible to diseases associated with inappropriately high levels of body iron. Many breakfast cereals are fortified with iron, but they are rarely eaten as part of a meat-containing meal. The observation that heterozygotes absorb more iron from an iron-fortified hamburger meal (6) may not be applicable to fortified foods that are not consumed with meat, eg, breakfast cereals. The objective of the present study was to measure iron absorption from iron-fortified breakfast cereals and separately from meals containing highly bioavailable iron in C282Y heterozygotes and wild-type men; a smaller group of C282Y/H63D compound heterozygotes, identified as part of a parallel investigation, was also studied.
SUBJECTS AND METHODS
Men aged 40 y were recruited for the study, and a 10-mL blood sample was taken for HFE genotype analysis (7). Subjects with chronic or acute illness or those taking medication regularly, which could affect iron absorption, were excluded. The study was approved by the Norwich District Ethics Committee, and written informed consent was obtained from all subjects. As part of a general diet and health questionnaire, all subjects were asked about their family history of hemochromatosis; none reported a history of this disorder. A 10-mL fasting blood sample was taken from the subjects to exclude those whose biochemical and hematologic indexes fell outside the normal range. Absorption from a diet high in bioavailable iron and containing iron-fortified cereal products was measured in 15 C282Y, 15 wild-type carriers, and 5 C282Y/H63D compound heterozygotes. The 3 genetic groups were matched as closely as possible in terms of age and body mass index (Table 1).
View this table:
TABLE 1. Characteristics of the subjects1
Subjects attended the Human Nutrition Unit at the Institute of Food Research for test meals and blood sampling. On days 1 and 2, they consumed a diet high in bioavailable iron (Table 2), which was extrinsically labeled with a total of 18 mg Fe-57 as ferrous sulfate. On days 3, 4, and 5, a diet containing fortified cereal products extrinsically labeled with a total of 45 mg Fe-54 as ferrous sulfate was consumed (Table 3). Iron absorption was determined by measuring the incorporation of Fe-57 and Fe-54 in red blood cells 14 d after the last test meal. The utilization of absorbed iron was measured from red blood cell incorporation of an intravenous infusion of Fe-58–labeled iron citrate.
View this table:
TABLE 2. Menu for the high-iron-bioavailability diet
View this table:
TABLE 3. Menu for the iron-fortified cereal diet
On day 1, a 25-mL venous blood sample was taken from fasting subjects via an indwelling cannula, and iron-isotope and iron-status indexes were measured (hemoglobin, serum iron, total-iron-binding capacity, serum ferritin, serum transferrin receptor, and C-reactive protein). Breakfast, extrinsically labeled with 3.0 mg Fe-57-enriched ferrous sulfate, was consumed and 1 h later a 400-µg dose of Fe-58 as iron citrate was slowly infused over 3 h. The rate of intravenous infusion of iron was based on the estimated 2 µg/min plasma appearance of iron normally absorbed from the gastrointestinal tract. After the infusion, the subjects consumed a meal extrinsically labeled with 3.0 mg Fe-57-enriched ferrous sulfate. An evening meal extrinsically labeled with 3.0 mg Fe-57–enriched ferrous sulfate was consumed 3–5 h after lunch. A selection of low-iron snacks and water was available from 2 h after the evening meal until 2200. The same procedure was followed on day 2, with the exception of the intravenous infusion.
On days 3, 4, and 5, the subjects consumed a breakfast cereal containing no added iron (Corn Flakes; Kellogg’s, Warrington, United Kingdom) and milk, extrinsically labeled with 7.3 mg Fe-54 as ferrous sulfate. Lunch, containing no added iron, was provided to the subjects within a minimum of 2 h after their breakfast. A further 7.3 mg Fe-54 as ferrous sulfate was consumed with an unfortified cereal-based snack (Kellogg’s Poptart, Warrington, United Kingdom) a minimum of 2 h after lunch. An evening meal, containing no added iron, and snacks were provided from a minimum of 2 h after the cereal snack until 2200. Weak tea was permitted with the unlabeled lunch and evening meals if requested.
Fourteen days after the last labeled meal was consumed (day 19), a 25-mL venous blood sample was taken from fasting subjects for the measurement of iron-status indexes (hemoglobin, serum iron, total-iron-binding capacity, serum ferritin, serum transferrin receptor, and C-reactive protein) and the iron-isotope enrichment of red blood cells. Iron absorption from the test meals was calculated from the isotopic enrichment of red blood cells 14 d after the last test meal, the total iron concentration in whole blood, and an estimate of blood volume from the equation (10):
RESULTS
There were no significant differences in iron status between the genotype groups (Table 1), which thereby minimized differences in iron absorption related to systemic concentrations of iron. The mean iron absorption for each group is shown in Table 4. The results of an analysis of variance of the relation between iron absorption, iron status, and genotype are given in Table 5.
View this table:
TABLE 4. Nonheme-iron absorption from the high-iron-bioavailability diet and the iron-fortified cereal diet1
View this table:
TABLE 5. ANOVA models with absorption of Fe-57 from the high-iron-bioavailability diet and absorption of Fe-54 from cereal products as the response variables
Mean absorption of the Fe-57 added to the high-iron-bioavailability diet was 6.8% (0.6 mg/d), 7.6% (0.7 mg/d), and 8.3% (0.7 mg/d) for the wild-type, C282Y heterozygote, and C282Y/H63D compound heterozygote groups, respectively. Mean absorption of the Fe-54-fortified cereal was 4.9% (0.7 mg/d), 5.3% (0.8 mg/d), and 5.1% (0.8 mg/d) for the wild-type, C282Y heterozygote, and C282Y/H63D compound heterozygote groups, respectively. There was no significant effect of genotype on iron absorption.
Absorption from all nonheme iron in the test meals was calculated by analyzing the total iron content of the test meals and deducting estimated heme iron (Table 6). Mean daily intakes were 15.2 mg/d from the high-iron-bioavailability diet and 16.4 mg/d from the cereal products (breakfast and afternoon snack). Total nonheme-iron absorption was estimated from the percentage absorption of the labeled isotope dose, assuming that the iron from the extrinsic isotope and the nonheme iron from unlabeled meals form a common pool in the gut. Mean (±SD) absorption of nonheme iron from the high-iron-bioavailability diet was 1.0 ± 1.0, 1.2 ± 0.5, and 1.3 ± 1.5 mg/d in the wild-type, C282Y heterozygote, and C282Y/H63D compound heterozygote groups, respectively (Table 4). Mean absorption of nonheme iron from the cereal products was 0.8 ± 0.3, 0.9 ± 0.2, and 0.8 ± 0.4 mg/d in the wild-type, C282Y heterozygote, and C282Y/H63D compound heterozygote groups, respectively (Table 4). There was no effect of genotype on nonheme-iron absorption.
View this table:
TABLE 6. Nutrient content of the test meals
Absorption of nonheme iron from the high-iron-bioavailability diet was highly correlated with absorption of nonheme iron from cereal products (R2 = 0.68, P < 0.001), and there was weak evidence that absorption from the high-iron-bioavailability diet was greater than that from the cereal products (P = 0.062, Wilcoxon’s matched-pairs signed-ranks test). The relation between serum ferritin and absorption of Fe-57 from the high-iron-bioavailability diet and Fe-54 from the cereal products is presented in Figure 1. Linear regression analysis of the log transforms of serum ferritin and the difference between absorption from the 2 diets showed that the subjects with a lower serum ferritin concentration absorbed significantly more nonheme iron from the high-iron-bioavailability diet than from the fortified cereal products (P < 0.001). There was a significant negative correlation between serum ferritin concentration and iron absorption (mg/d) from the high-iron-bioavailability diet in both the wild-type group (R2 = 0.28, P < 0.05) and C282Y (R2 = 0.44, P < 0.01) heterozygotes. This was also the case with the iron-fortified cereals for the wild-type group (R2 = 0.36, P < 0.02), but the correlation was not significant for C282Y heterozygotes (R2 = 0.24, P < 0.1). When the subjects with a serum ferritin concentration > 70 µg/L were excluded from the analysis, the correlation between serum ferritin concentration and iron absorption was much stronger from the high-iron-bioavailability meals (R2 = 0.54, P < 0.001) and the cereal products (R2 = 0.51, P < 0.001), which provides further experimental support for the theory that iron absorption is similar to basal iron requirements when serum ferritin exceeds 70 µg/L (31, 32).
FIGURE 1.. Relation between serum ferritin and the percentage absorption of Fe-57 from a diet with high iron bioavailability (r = –0.54, P < 0.01) and of Fe-54 from cereal products (r = –0.54, P < 0.01). , C282Y heterozygote, high-iron-bioavailability diet; , wild type, high-iron-bioavailability diet; , C282Y heterozygote, iron-fortified cereal products; , wild type, iron-fortified cereal products. Linear regression analysis of the log transformed serum ferritin concentration and the difference between nonheme-iron absorption from the high-iron-bioavailability diet and the iron-fortified cereal products showed a strong negative effect of serum ferritin (P < 0.001).
Mean (± SD) red blood cell utilization of the intravenously administered Fe-58 was 79 ± 4%, 73 ± 8%, and 75 ± 5% in the wild-type, C282Y heterozygote, and compound-heterozygote groups, respectively. Individual values varied between 62% and 88%; the overall mean value was 76 ± 7%. Analysis of variance showed no significant effect of genotype on red blood cell utilization.
An analysis of variance model was used to examine the relation between iron absorption (mg of isotope dose) from the high-iron-bioavailability diet and the iron-fortified cereal products, indexes of iron status (hemoglobin, serum iron, total-iron-binding, transferrin saturation, serum ferritin and soluble transferrin receptor), lean body mass, body surface area, and HFE genotype. The linear regression model (Table 5) indicated that serum ferritin (P < 0.001) and serum iron (P < 0.001) were the only significant predictors of absorption from the high-iron-bioavailability diet. In this model, 2 subjects were identified as outliers (1 wild-type carrier and 1 compound heterozygote, both of whom had low serum ferritin and high iron absorption) and were removed from the analysis. There was weak evidence (P = 0.06) that a third subject, also a wild type, with low serum ferritin and high iron absorption, was an outlier. When this subject was removed from the analysis, genotype became a significant predictor of absorption (P = 0.017), with Tukey’s honestly significant difference test showing that heterozygotes absorbed an average of 0.42 mg/d more Fe-57 than did the wild-type control subjects. Serum ferritin (P < 0.001) and total-iron-binding capacity (P < 0.05) were the only significant predictors of absorption from cereal products.
DISCUSSION
C282Y homozygotes and C282Y/H63D compound heterozygotes are at increased risk of developing hemochromatosis because of poorly controlled iron absorption. Although it is acknowledged that the HFE protein is involved in iron metabolism, its role in the regulation of iron absorption is not well understood (33). A multifunctional role for HFE, dependent on expression levels of proteins involved in iron transport, has been suggested on the basis of the results that indicated that HFE inhibits iron efflux in human colonic carcinoma cells (34). Before the discovery of the HFE gene, Lynch et al (6) identified 22 relatives of hemochromatosis patients, most of whom we assume were C282Y heterozygotes. These subjects absorbed significantly more nonheme iron than did healthy control subjects from a hamburger meal to which 20 mg Fe (as ferrous sulfate) with 100 mg ascorbic acid was added, but absorption of iron from the unfortified meal was not significantly different from that of the control subjects. The additional amount of iron absorbed from the meal was 1.35 mg (2.15 compared with 0.80 mg in the healthy control subjects). With the consumption of high-iron meals over a period of time, this group would have been expected to accumulate higher iron stores than the control subjects, yet the geometric mean serum ferritin concentration of the 22 relatives tested by Lynch et al was 50 µg/L compared with 68 µg/L in the 75 "normal" subjects, indicating that their habitual diet provided no opportunity to acquire additional iron. Serum ferritin concentration has not been reported to be higher in male and female C282Y heterozygotes (35, 36), but male C282Y/H63D compound heterozygotes do have a higher serum ferritin concentration (37, 38).
Hunt and Zeng (39), following the design used by Lynch et al (6), recently reported that 11 C282Y heterozygotes (8 female, 3 male) and 12 wild-type subjects (9 female, 3 male) did not absorb different amounts of nonheme iron from single test meals. They were unable to confirm the finding that C282Y heterozygotes absorbed more nonheme iron from a fortified hamburger meal than did wild-type control subjects. There are many possible explanations for the differences between our results and those of Lynch et al (6). First, the differences in iron absorption between relatives of hemochromatosis patients and control subjects recruited by Lynch et al may not be representative of heterozygotes who do not have relatives with clinical hemochromatosis. Second, the isotope test doses in our study were consumed with 6 meals over a period of 2 (high-iron-bioavailability diet) or 3 (cereal products) d to give an estimate of average absorption and to minimize day-to-day variation associated with single-meal studies (40), whereas Lynch et al (1) measured absorption from a single meal containing a large dose (20 mg) of bioavailable iron. It is possible that short-term differences in absorption seen in a single meal are not reflected in subsequent meals because of the mucosal block effect (41–43).
Although our findings appear to have shown no significant differences between the groups, we cannot exclude the possibility that there is a subtle effect of genotype on iron absorption. The absorption from the high-iron-bioavailability diet data included 2 outliers, who were removed from the analysis, and there was also weak evidence (P = 0.06) of a third outlier. When this subject was removed from the analysis, genotype became a significant predictor of absorption (P = 0.017); heterozygotes absorbed an average of 0.42 mg Fe-57/d more than did the control subjects. Confirmation of a genotype effect would, however, require a sample size of 332 subjects per group to show a significant difference of 0.1 mg/d between groups, based on the pooled SD (0.46) of the heterozygote and wild-type men.
The calculation of iron absorption from isotope incorporation in erythrocytes is usually based on the assumption that 80% of absorbed iron is incorporated into red blood cells. In the present study, we measured the red blood cell incorporation of intravenous Fe-58 to examine the effect of genotype on iron metabolism. Mean red blood cell utilization was close to the mean 80% value previously reported for populations with normal iron stores, and the range of values agrees with those previously reported (44), which indicated that the utilization of absorbed iron in C282Y heterozygotes and C282Y/H63D compound heterozygotes is not significantly different from that of wild-type men.
Our results indicate that the gross degree of regulatory control of nonheme-iron absorption in C282Y heterozygotes is not significantly different from that of wild-type men consuming Western-style diets that include large amounts of enhancers (440 mg ascorbic acid/d and 364 g/d of meat, fish, or poultry) or moderate amounts of additional nonheme iron (15 mg/d). The small number of C282Y/H63D compound heterozygotes in this study made it difficult to draw conclusions about the effects of genotype on the efficiency of iron absorption. However, a small proportion of compound heterozygotes accumulate excess iron, and it is estimated that the genotype is 0.5–1.5% as penetrant as is C282Y homozygosity (1, 45). Our results, the well-established low penetrance of the C282Y homozygous genotype (3, 7, 46–48) and the recent suggestion that hepcidin may play an important role in the regulation of iron absorption (49), suggest that HFE mutations alone may not be sufficient to produce iron overload. Failure to up-regulate hepcidin concentrations, despite hepatic iron overload, has been shown in HFE knockout mice (50) and in HFE-related hemochromatosis (51), and disruption of the normal hepcidin response to iron stores may contribute to iron accumulation.
In conclusion, our results indicate that there is no statistically significant difference in nonheme-iron absorption from a diet high in bioavailable iron and from iron-fortified cereals in C282Y heterozygotes and wild-type control subjects, although there may be a subtle effect of genotype on the efficiency of iron absorption.
ACKNOWLEDGMENTS
SJF-T and MAR designed and coordinated the project. A-LMH was responsible for the dietary aspects. SLO, CM, and GM-N carried out the human study and prepared the samples for stable-isotope measurement by mass spectrometry, which was conducted by JAH. JRD carried out the stable-isotope calculations. RF performed the statistical analysis of the data GW determined the genotypes of the subjects. There were no conflicts of interest for any of the authors.
REFERENCES