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Iron is essential for many biological processes, but iron levels must be tightly regulated to avoid harmful effects of both iron deficiency and overload. Here, we perform genome-wide association studies on four iron related biomarkers (serum iron, serum ferritin, transferrin saturation, total iron binding capacity) in the Trøndelag Health Study (HUNT), the Michigan Genomics Initiative (MGI) and the SardiNIA study, followed by their meta-analysis with publicly available summary statistics, analyzing up to 257 953 individuals. We identify 127 genetic loci associated with iron traits. Among 19 novel protein-altering variants, we observe a rare missense variant (rs367731784) in HUNT, which suggests a role for DNAJC13 in transferrin recycling. We further validate the latest genetic risk scores for each biomarker in HUNT (6% variance in serum iron explained) and present linear and non-linear Mendelian randomization analyses of the traits on all-cause mortality. We find evidence of a harmful effect of increased serum iron and transferrin saturation in linear analyses that estimate population-averaged effects. However, there was weak evidence of a protective effect of increasing serum iron at the very low end of its distribution. Our findings contribute to our understanding of the genes affecting iron status and its consequences on human health.

Iron is essential for many biological processes, but iron levels must be tightly regulated to avoid harmful effects of both iron deficiency and overload. Here, we perform genome-wide association studies on four iron-related biomarkers (serum iron, serum ferritin, transferrin saturation, total iron-binding capacity) in the Trøndelag Health Study (HUNT), the Michigan Genomics Initiative (MGI), and the SardiNIA study, followed by their meta-analysis with publicly available summary statistics, analyzing up to 257,953 individuals. We identify 123 genetic loci associated with iron traits. Among 19 novel protein-altering variants, we observe a rare missense variant (rs367731784) in HUNT, which suggests a role for DNAJC13 in transferrin recycling. We further validate recently published results using genetic risk scores for each biomarker in HUNT (6% variance in serum iron explained) and present linear and non-linear Mendelian randomization analyses of the traits on all-cause mortality. We find evidence of a harmful effect of increased serum iron and transferrin saturation in linear analyses that estimate population-averaged effects. However, there was weak evidence of a protective effect of increasing serum iron at the very low end of its distribution. Our findings contribute to our understanding of the genes affecting iron status and its consequences on human health.

Unbound iron binding capacity (UIBC) is more accurate than total iron binding capacity (TIBC) and percent transferrin saturation in diagnosing empty iron stores. It is unknown whether UIBC is more or less accurate than soluble transferrin receptor (sTFR). We obtained public-use data from the U.S. National Health and Nutrition Examination Survey (NHANES) 2005-2006 to compare the accuray of UIBC and sTFR in diagnosing empty iron stores in 2337 women aged 12–49 years. We grouped the women according to CRP less than 5 mg/L and pregnancy (four groups) and used three definitions of empty iron stores: Serum ferritin less than 10, 15, and 20 µg/L. Receiver operating characteristic (ROC) curve analysis was used to estimate the diagnostic accuracy. UIBC showed a better diagnostic accuracy than sTFR in all groups and definitions of empty iron stores, except in nonpregnant women with CRP at least 5 mg/L when empty iron stores were defined as ferritin less than 10 and 15 µg/L. Two differences reached statistical significance: In nonpregnant women without inflammation the area under the ROC curve for UIBC was 0.830 compared to 0.793 for sTFR (p = .007) when empty iron stores were defined as ferritin less than 20 µg/L. The corresponding figures for pregnant women without inflammation were 0.843 for UIBC and 0.739 for sTFR (p = .003). In conclusion, UIBC is a more accurate test than sTFR in diagnosing empty iron stores in women without inflammation.

Iron loading in p.C282Y homozygous HFE hemochromatosis subjects is highly variable, and it is unclear what factors cause this variability. Finding such factors could aid in predicting which patients are at highest risk and require closest follow-up. The degree of iron loading has previously been associated with certain HLA-types and with abnormally low CD8 + cell counts in peripheral blood. In 183 Norwegian, p.C282Y homozygotes (104 men, 79 women) originally found through population screening we determined HLA type and measured total T-lymphocytes, CD4 + and CD8 + cells, and compared this with data on iron loading. In p.C282Y homozygous men, but not in homozygous women, we found that the presence of two HLA-A*03 alleles increased the iron load on average by approximately 2-fold compared to p.C282Y homozygous men carrying zero or one A*03 allele. On the other hand, the presence of two HLA-A*01 alleles, in male subjects, apparently reduced the iron loading. In p.C282Y homozygous individuals, the iron loading was increased if the CD8 + cell number was below the 25 percentile or if the CD4 + cell number was above the 75 percentile. This effect appeared to be additive to the effect of the number of HLA-A*03 alleles. Our data indicate that homozygosity for the HLA-A*03 allele significantly increases the risk of excessive iron loading in Norwegian p.C282Y homozygous male patients. In addition, low CD8 + cell number or high CD4 + cell number further increases the risk of excessive iron loading.

ObjectivesThe overnight dexamethasone (DXM) suppression test (DST) has high sensitivity, but moderate specificity, for diagnosing hypercortisolism. We have evaluated if simultaneous measurement of S-DXM may correct for variable DXM bioavailability and increase the diagnostic performance of DST, and if saliva (sa) is a feasible adjunct or alternative to serum.Design and methodsProspective study of DST was carried out in patients with suspected Cushing’s syndrome (CS) (n = 49), incidentaloma (n = 152) and healthy controls (n = 101). Cortisol, cortisone and DXM were assayed by liquid chromatography–tandem mass spectrometry (LC–MS/MS).ResultsThree hundred and two subjects underwent DST; S-cortisol was ≥50 nmol/L in 83 patients, of whom 11 had CS and 27 had autonomous cortisol secretion. The lower 2.5 percentile of S-DXM in subjects with negative DST (n = 208) was 3.3 nmol/L, which was selected as the DXM cut-off level. Nine patients had the combination of low S-DXM and positive DST. Of these, three had been misdiagnosed as having autonomous cortisol secretion. DST results were highly reproducible and confirmed in a replication cohort (n = 58). Patients with overt CS had significantly elevated post-DST sa-cortisol and sa-cortisone levels compared with controls; 23 of 25 with autonomous cortisol secretion had elevated sa-cortisone and 14 had elevated sa-cortisol.ConclusionsSimultaneous measurement of serum DXM and cortisol reduced false-positive DSTs by 20% and improved the specificity. S-DXM >3.3 nmol/L is sufficient for the suppression of cortisol

Background: Accurate quantification of aldosterone is essential in the diagnosis of primary aldosteronism. Liquid chromatography-tandem mass spectrometry (LC-MSMS) analysis is increasingly being used to improve analytical sensitivity and specificity, since this technology reduces most of the interferences observed with immunological methods. Methods: Serum samples with d7-aldosterone as internal standard were extracted with methyl tert-butyl ether, using liquid-liquid extraction (LLE). Chromatographic separation was performed on a C18 reverse phase column with a methanol-water gradient containing ammonium fluoride. Aldosterone detection was performed on an Agilent 6490 triple quadrupole using electro spray ionisation in positive mode. Results: Multiple reaction monitoring transitions were m/z 361.2–315.1 for aldosterone, and 368.5–323.3 for d7-aldosterone. Chromatographic retention time was 2.7 min. The method’s total CVs at aldosterone concentrations of 45.4 and 1080 pmol/L were 7.0% and 4.8%, respectively. The intra-assay CVs at concentrations of 60.0 and 637 pmol/L were 4.0% and 2.6%, respectively. The method’s LOQ and LOD were 10 and 5 pmol/L, respectively, demonstrating an excellent analytical sensitivity. The upper limit of quantification was set to 5000 pmol/L, corresponding to the highest calibrator concentration. The long-term stability of the method was evident from repeated measurements of external control pools from UKNEQAS over a period of about 3 years, showing CVs between 2.0 and 7.0%. Conclusions: We have described a precise, sensitive and stable LC-MSMS method for the measurement of serum aldosterone. In addition, due to the use of LLE and a short LC-column, the method is simple to perform, with a short chromatographic run time.

Aim. To study the association between blood hemoglobin concentration (b-hemoglobin) and serum ferritin concentration (s-ferritin) in an ambulant patient population without inflammation and with normal kidney function. Methods. In ambulant, adult patients with normal values of s-CRP and s-creatinine, median b-hemoglobin and the fraction with anemia was compared in groups with lower s-ferritin from a level of 100 μg/L. The 10, 50 and 90 percentiles of b-hemoglobin were modelled as functions of s-ferritin using quantile regression. Results. Among 3206 women the entire b-hemoglobin distribution was shifted downwards in patients with s-ferritin less than 20 μg/L. Accordingly, the median b-hemoglobin was statistically significantly lower and the fraction with anemia was higher. In 1246 men the findings were similar, except that the turning point toward lower b-hemoglobin was at a s-ferritin level of 30 μg/L. Conclusions. Low s-ferritin is associated with decreased b-hemoglobin in many more subjects than...

In addition to hepatocellular cancer, HFE C282Y homozygotes are reported to have increased risk of colorectal cancer and breast cancer. This study was done to further explore the cancer risk in C282Y homozygotes.We studied cancer incidence in 292 homozygotes and 62,568 others that participated in the HUNT 2 population screening in 1995-1997. Using Cox proportional hazard models, we estimated cancer hazard ratio as a function of C282Y homozygosity and several screening variables including serum transferrin saturation, alcohol consumption and daily smoking.Cancer was diagnosed in 36 homozygotes, five of which had two cancer diagnoses. The overall cancer incidence was not increased in C282Y homozygotes (hazard ratio 1.10 [95% CI 0.60-2.03] in women and 0.94 [95% CI 0.53-1.66] in men). However, homozygous men had increased risk of colorectal cancer (hazard ratio 3.03 [95% CI 1.17-7.82], p = 0.022) and primary liver cancer (hazard ratio 54.0 [95% CI 2.68-1089], p = 0.009). The risk of breast cancer in homozygous women was not increased (hazard ratio 1.13 [95% CI 0.35-3.72]). Adjusted for other variables including C282Y homozygosity, very low and very high serum transferrin saturation were associated with increased overall cancer incidence.C282Y homozygosity is associated with increased risk of colorectal cancer and hepatocellular cancer in men. In the general population, individuals with a very low or a very high serum transferrin saturation may have increased cancer risk.

It has been suggested that comparison of posttest dexamethasone and cortisol concentrations may improve the evaluation of the dexamethasone suppression test (DST) for Cushing's syndrome. In particular, this would be reasonable if posttest cortisol differs by dexamethasone levels within the range that is usually attained in the DST. Using fractional polynomial regression, we therefore studied the association between posttest 0800 h dexamethasone and cortisol levels in 53 subjects without Cushing's syndrome who were tested with the 1 mg overnight DST. Plasma dexamethasone was associated with plasma cortisol (P5.0 nmol/l, there was no association between dexamethasone and cortisol levels (P=0.55). In conclusion, subjects tested with the 1 mg overnight DST usually attain an 0800 h plasma dexamethasone >5 nmol/l, and plasma cortisol does not differ by plasma dexamethasone in these subjects. This suggests that routine comparison of dexamethasone and cortisol levels may not be a useful approach to improve the performance of the 1 mg DST. However, dexamethasone measurements may identify subjects with inadequately low plasma dexamethasone and may therefore be of value when retesting subjects with possibly false-positive DST results.

Transferrin receptor (TFR) 1 and 2 are expressed in the liver; TFR1 levels are regulated by cellular iron levels while TFR2 levels are regulated by transferrin saturation. The aims of this study were to 1) determine the relative importance of TFR1 and TFR2 in transferrin-bound iron (TBI) uptake by HuH7 human hepatoma cells and 2) characterize the role of metal-transferrin complexes in the regulation of these receptors. TFR expression was altered by 1) incubation with metal-transferrin (Tf) complexes, 2) TFR1 and TFR2 small interfering RNA knockdown, and 3) transfection with a human TFR2 plasmid. TBI uptake was measured using 59Fe-125I-labeled Tf and mRNA and protein expression by real-time PCR and Western blot analysis, respectively. Fe2Tf, Co2Tf, and Mn2Tf increased TFR2 protein expression, indicating that the upregulation was not specifically regulated by iron-transferrin but also other metal-transferrins. In addition, Co2Tf and Mn2Tf upregulated TFR1, reduced ferritin, and increased hypoxia-inducible factor-1α protein expression, suggesting that TFR1 upregulation was due to a combination of iron deficiency and chemical hypoxia. TBI uptake correlated with changes in TFR1 but not TFR2 expression. TFR1 knockdown reduced iron uptake by 80% while TFR2 knockdown did not affect uptake. At 5 μM transferrin, iron uptake was not affected by combined TFR1 and TFR2 knockdown. Transfection with a hTFR2 plasmid increased TFR2 protein expression, causing a 15–20% increase in iron uptake and ferritin levels. This shows for the first time that TFR-mediated TBI uptake is mediated primarily via TFR1 but not TFR2 and that a high-capacity TFR-independent pathway exists in hepatoma cells.

Serum levels of transferrin receptor and erythropoietin were determined in 2 patients with hereditary hemochromatosis undergoing phlebotomy therapy. The objective of the study was to determine changes in serum transferrin receptor and serum erythropoietin occurring during therapy, and to investigate if such changes could be useful to monitor the therapy. The study showed that serum transferrin receptor, and to a lesser extent serum erythropoietin, may be better parameters than serum ferritin as indicators of when phlebotomy should be discontinued. The most sensitive parameter, however, appeared to be the serum transferrin receptor/ferritin ratio.

Background Hemochromatosis is a common disease with a good prognosis, when diagnosed early and treated appropriately. The aim of this overview is to give updated information on hemochromatosis with special focus on biochemical features, diagnosis and treatment. Material and methods This article is based on our own experience and a review of available literature in various databases such as PubMed and Medline. Results Hereditary hemochromatosis is an autosomal recessive disease characterized by iron overload due to increased intestinal iron uptake over many years. Hemochromatosis is often discovered through coincidental detection of high levels of transferrin and/or ferritin. The early symptoms are asthenia and joint pain. About 85 % of patients with hereditary hemochromatosis are homozygote for the C282Y mutation in the HFE: gene, but the majority of homozygotes remain asymptomatic. With ferritin levels > 500 microg/, both hereditary hemochromatosis and iron overload (of unknown cause) are treated with blood-letting. Interpretation The pathogenesis is not fully elucidated but recent reports indicate that the protein hepcidin (produced in the liver) plays a key role in the development of hemochromatosis. Iron overload may also be secondary to other diseases such as thalassemia and other conditions requiring multiple long-term blood transfusions. The goal is to maintain ferritin values at approximately 20 - 50 microg/L.

BACKGROUND The regulation of iron absorption has previously been considered >. Recent research has given us interesting information on the regulation of the iron metabolism and pathological iron overload; the present article aims at providing an overview of these topics. MATERIALS AND METHODS The article is based on a review of literature retrieved from PubMed. RESULTS The peptide hepcidin binds to ferroportin on membranes of enterocytes, macrophages and hepatocytes. The complex is internalised and degraded and this results in decreased export of iron to the circulation, and thus a lower level of plasma iron. Hepcidin production is up-regulated in iron overload and down-regulated with iron deficiency. The liver proteins human haemochromatosis protein (HFE), transferrin receptor 2 (TfR2), haemojuvelin (HJV) and bone morphogenetic protein (BNP) are necessary regulators for activation of the hepcidin synthesis. Lack of or mutations in the genes for these proteins, e.g. the HFE mutation C282Y in primary haemochromatosis, reduces the synthesis of hepcidin. Iron regulatory proteins (IRP) may bind to iron responsive elements (IRE) of ferritin-mRNA and transferritin-mRNA and regulate the protein synthesis. INTERPRETATION Regulation of uptake, utilization, release and storage of iron occurs at the gene level. Hepcidin is currently considered to be the > of the iron balance. Intracellular iron balance is maintained by iron regulating proteins. Synthesis of ferritin increases with high iron levels, while synthesis of TfR1 is reduced. The opposite occurs with a low iron level.

Purpose Cutaneous adverse reactions (cADRs) from carbamazepine (CBZ) have been associated with human leukocyte antigens (HLA). Our aims were to assess the clinical usefulness of HLA-A*31:01 as a predictor of CBZ-induced cADRs in the Norwegian population and to explore whether cADRs from aromatic antiepileptic drugs (AEDs) in general might be linked with a common HLA-A-marker. Materials and methods 86 ethnic Norwegians with a history of non-bullous cADRs from aromatic AEDs were included. 114 subjects tolerant to at least one aromatic AED were used as drug-specific controls. Complete HLA-A genotyping was performed. 1026 blood donors were used as population controls. Results Comparing all cADR subjects with controls and blood donors, there were no statistical differences for any HLA-A allele, except for HLA-A*24 ( p =0.022 vs. controls and p =0.014 vs. blood donors). When comparing tolerant controls with patients having had a cADR to one of the two most used drugs, CBZ ( n =48) and lamotrigine ( n =28), we found no significant associations for CBZ to HLA-A*31:01 or HLA-A*24:02 , but for lamotrigine there was an association with HLA-A*24:02 ( p =0.027). In patients developing cross-reactivity ( n =14) to aromatic AEDs, the presence of HLA-A*31:01 or HLA-A*24:02 was not different compared to patients with a single cARD tolerant to at least one other drug. Conclusion We question the clinical usefulness of HLA-A*31:01 as a marker for CBZ rash in the Norwegian population. A previously suggested protective effect of aromatic AED cross-reactivity from HLA-A*24:02 was not confirmed. The association between HLA-A*24:02 and lamotrigine-induced rash should be further investigated.

We wanted to study the association between blood hemoglobin concentration (b-hemoglobin) and serum ferritin concentration (s-ferritin) in a healthy female population, and compare the findings to those in a previous study of ambulant female patients.We compared median b-hemoglobin and the fraction with anemia in groups of women with s-ferritin from less than 10 μg/L to 100 μg/L. These women, aged 20-55 years, were part of a health screening survey (HUNT 2) where they reported to have 'good' or 'very good' general health and were found to have normal s-creatinine. The s-ferritin values were adjusted to the level of the previous study. The 10, 50 and 90 percentiles of b-hemoglobin were modelled as functions of s-ferritin using quantile regression.Among 2122 healthy females the entire b-hemoglobin distribution was shifted downwards in women with s-ferritin less than 20 μg/L. Accordingly, the median b-hemoglobin was statistically significantly lower. In women with s-ferritin less than 20 μg/L the fraction with anemia was 0.15.Lower s-ferritin is associated with lower b-hemoglobin in many more subjects than those labelled anemic.

In this large cross-sectional population-based study, 51,272 persons responded to a headache questionnaire and were screened for hemochromatosis. Phenotypic hemochromatosis and the C282Y/C282Y genotype were both associated with an 80% increase in headache prevalence evident only among women. The reason for this association is unclear, but one may speculate that iron overload alters the threshold for triggering a headache by disturbing neuronal function.

Hereditary hemochromatosis (HH) is a common genetic disease with iron overload in certain organs, especially the liver. Most cases are homozygous for the C282Y mutation in the HFE gene; a few are C282Y heterozygous, compound C282Y/H63D heterozygous, or have no known mutation. A third mutation, S65C, has been associated with HH, but this finding is disputed. We have studied the clinical significance of various genotypes with the S65C mutation. In a population-based screening for HH in 65,238 persons, 613 had high serum transferrin saturation in two blood samples and were invited for HFE genotyping. In 556 persons with complete data sets, we studied the serum ferritin concentration and the risk of being diagnosed with phenotypic HH in the various genotypic groups. The phenotypic diagnosis was given without knowing the genotypic result. Except for the C282Y homozygotes, no differences in median serum ferritin concentrations were found between the various genotypic groups. However, the C282Y/S65C compound heterozygous group had a higher risk of being diagnosed with phenotypic HH than the wild-type group, as did the C282Y homozygous and the C282Y/H63D compound heterozygous groups. When combined with the C282Y mutation, the S65C mutation is associated with an increased risk of being diagnosed with phenotypic HH.

Treatment with the first-line antiepileptic drug, carbamazepine (CBZ), is associated with adverse cutaneous reactions in up to 10% of patients. One predisposition to these side-effects has been linked to the HLA-A*31:01 allele. HLA-typing is costly and time-consuming. A single nucleotide polymorphism (SNP, rs1061235AT) has been suggested as a marker for the HLA-A*31:01 allele. We sought to develop and validate a simple, fast and inexpensive assay for rs1061235 to apply in the Norwegian population.We designed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the SNP and tested it on a set of 16 samples with known HLA-A alleles.The assay identified all HLA-A*31:01 alleles present, but also marked for HLA-A*33:03. In a second set of 204 samples from Norwegian epilepsy patients with unknown HLA alleles, nine samples heterozygous for the rs1061235 were found. Subsequent HLA-typing showed that one sample was HLA-A*33:01, whereas the other eight were identified as HLA-A*31:01. The remaining 195 samples were correctly identified as neither carrying the rs1061235 SNP nor HLA-A*31:01. The sensitivity and specificity of the rs1061235 SNP test was 100% and 99.5%, respectively. Misinterpretation of the rare HLA-A*33 variants as HLA-A*31:01 has minor consequence, as it only would result in choosing an alternative drug to CBZ.We have designed and validated a simple, fast and inexpensive test for the rs1061235AT SNP as a marker for HLA-A*31:01 in the Norwegian population for potential use in a personalized treatment approach to patients planned to receive CBZ.

The aim of this study was to investigate a possible association between haemochromatosis (HFE) gene mutations and the prevalence of Parkinson's disease. The HFE gene encodes a protein that modulates iron absorption. Several studies have documented increased iron levels in the basal ganglia in patients with Parkinson's disease. In a study on patients with concurrent hereditary haemochromatosis and Parkinson's disease, abnormal deposition of iron in the basal ganglia was suggested as an inductor of Parkinson's disease. In this study, genotype frequencies of the HFE mutations C282Y, H63D and S65C were estimated in 388 patients with Parkinson's disease and compared with frequencies found in comparable studies. No significant differences were found in frequencies between the patients and comparable populations. This study does not indicate increased susceptibility to Parkinson's disease in HFE gene mutation carriers in Norway.

Background : The present literature review is part of the NNR5 project with the aim of reviewing and updating the scientific basis of the 4th edition of the Nordic Nutrition Recommendations (NNR) issued in 2004. Objective : The objective of this systematic literature review was to assess the health effects of different intakes of iron, at different life stages (infants, children, adolescents, adults, elderly, and during pregnancy and lactation), in order to estimate the requirement for adequate growth, development, and maintenance of health. Methods : The initial literature search resulted in 1,076 abstracts. Out of those, 276 papers were identified as potentially relevant. Of those, 49 were considered relevant and were quality assessed (A, B, or C). An additional search on iron and diabetes yielded six articles that were quality assessed. Thus, a total of 55 articles were evaluated. The grade of evidence was classified as convincing (grade 1), probable (grade 2), suggestive (grade 3), and inconclusive (grade 4). Results : There is suggestive evidence that prevention or treatment of iron deficiency (ID) and iron deficiency anemia (IDA) improves cognitive, motoric, and behavioral development in young children, and that treatment of IDA improves attention and concentration in school children and adult women. There is insufficient evidence to show negative health effects of iron intakes in doses suggested by the NNR 4. There is insufficient evidence to suggest that normal birth weight, healthy, exclusively breast-fed infants need additional dietary iron before 6 months of life in the Nordic countries. An iron concentration of 4-8 mg/L in infant formulas seems to be safe and effective for normal birth weight infants. There is probable evidence that iron supplements (1-2 mg/kg/day) given up to 6 months of age to infants with low birth weight (< 2,500 g) prevents IDA and possibly reduce the risk of behavioral problems later on. There is probable evidence that ID and IDA in pregnant women can be effectively prevented by iron supplementation at a dose of 40 mg/day from week 18-20 of gestation. There is probable evidence that a high intake of heme iron, but not total dietary, non-heme or supplemental iron, is associated with increased risk of type 2 diabetes (T2D) and gestational diabetes. Conclusions : Overall, the evidence does not support a change of the iron intakes recommended in the NNR 4. However, one could consider adding recommendations for infants below 6 months of age, low birth weight infants and pregnant women. Keywords: iron; iron deficiency; anemia; child development; pregnancy; diabetes (Published: 12 July 2013) Citation: Food & Nutrition Research 2013. 57 : 21667 - http://dx.doi.org/10.3402/fnr.v57i0.21667 Access the supplementary material to this article – see Supplementary files under Article Tools online. Special Issue: This paper is part of the Nordic Nutrition Recommendations - The NNR5 project . More papers from this issue can be found at http://www.foodandnutritionresearch.net

This paper reports the results from a longitudinal study on iron status and diet among Norwegian and immigrant children living in the same residential area. This was the second part of the study and the children were two years old. All mothers with a normal pregnancy and delivery were invited to participate, and 80 of 95 eligible, accepted. Iron status analyses were carried out for 78 healthy children; 37 Norwegians and 4 1 immigrants (20 of Turkish origin). More immigrant children had depleted iron stores, compared to Norwegians. The median values for haemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), serum ferritin (SF), transferrin saturation (TfS), free erythrocyte protoporphyrin (FEP) and transferrin receptor (TfR) were all within normal ranges. The differences between immigrants and Norwegians were significant except for Hb and SF. There was a rise in MCV and TfS and a decline in TfR among both Norwegian and immigrant children from one to two years, and a decline in SF among the Norwegians. The Norwegian children had a higher intake of soft drinks, dark bread and different iron-rich bread spreads than the immigrants, who had a higher intake of orange juice, and a more frequent use of citrus fruits than the Norwegians. Milk intake was negatively related – and consumption frequency of brown whey cheese was positively related – to iron stores indicated by SF values above or below 15 pg/L. The results suggest that differences in food intake may explain the lower iron status in immigrants compared to Norwegians. More attention should be directed to children's diet and iron status in the second and probably third years of life

Unbound iron binding capacity (UIBC) in serum, which is s-total iron binding capacity (2 times s- transferrin) minus s-iron, may be a more accurate marker of empty iron stores than serum transferrin saturation. Previously we have shown this for healthy females of childbearing age.Now we used receiver operating characteristic (ROC) curve analysis to compare the diagnostic accuracy of s-iron, s-transferrin, s-transferrin saturation and s-UIBC in diagnosing empty iron stores in 29,251 female and 19,652 male outpatients. Empty iron stores were defined as s-ferritin less than 10, 15 or 20 μg/L.At all definitions of empty iron stores s-UIBC had a better diagnostic accuracy than the other tests in both male and female outpatients, with an area under the ROC curve of 0.85-0.97. Also in subpopulations with elevated s-CRP or low b-hemoglobin s-UIBC was more accurate than the other tests. All tests performed better in males than in females, and generally they were more accurate in adults than in children.When diagnosing empty iron stores calculation of s-UIBC is a better way to utilize the information in s-iron and s-transferrin than the calculation of s-transferrin saturation.

Hereditary Hemochromatosis (HH) is a recessively inherited disorder of iron overload occurring commonly in subjects homozygous for the C282Y mutation in HFE gene localized on chromosome 6p21.3 in linkage disequilibrium with the human leukocyte antigen (HLA)-A locus. Although its genetic homogeneity, the phenotypic expression is variable suggesting the presence of modifying factors. One such genetic factor, a SNP microhaplotype named A-A-T, was recently found to be associated with a more severe phenotype and also with low CD8(+)T-lymphocyte numbers. The present study aimed to test whether the predictive value of the A-A-T microhaplotype remained in other population settings. In this study of 304 HH patients from 3 geographically distant populations (Porto, Portugal 65; Alabama, USA 57; Nord-Trøndelag, Norway 182), the extended haplotypes involving A-A-T were studied in 608 chromosomes and the CD8(+) T-lymphocyte numbers were determined in all subjects. Patients from Porto had a more severe phenotype than those from other settings. Patients with A-A-T seemed on average to have greater iron stores (p = 0.021), but significant differences were not confirmed in the 3 separate populations. Low CD8(+) T-lymphocytes were associated with HLA-A*03-A-A-T in Porto and Alabama patients but not in the greater series from Nord-Trøndelag. Although A-A-T may signal a more severe iron phenotype, this study was unable to prove such an association in all population settings, precluding its use as a universal predictive marker of iron overload in HH. Interestingly, the association between A-A-T and CD8(+) T-lymphocytes, which was confirmed in Porto and Alabama patients, was not observed in Nord-Trøndelag patients, showing that common HLA haplotypes like A*01-B*08 or A*03-B*07 segregating with HFE/C282Y in the three populations may carry different messages. These findings further strengthen the relevance of HH as a good disease model to search for novel candidate loci associated with the genetic transmission of CD8(+) T-lymphocyte numbers. Funding: FEDER through the program POFC-COMPETE (COMPETE “FCOMP-01-0124-FEDER-008447”) and portuguese national funds through the FCT (Portuguese Foundation for Science and Technology) grants: PTDC/SAU-GMC/67868/2006, PIC/IC/82785/2007and SFRH/BD/69186/2010; and partly by: 2) INOVA/APRF, USA; 3)The BIIC-Programa Roche/ICBAS/HAS 2008/2010; 4)Southern Iron Disorders Center, Birmingham, Alabama; 5) Nord-Trøndelag Health Study (The HUNT Study), a collaboration between HUNT Research Centre (Faculty of Medicine, Norwegian University of Science and Technology NTNU), Nord-Trøndelag County Council and The Norwegian Institute of Public Health; 6) The Cancer Foundation at St. Olav Hospital, Trondheim; 7) The Throne Holst Foundation, Oslo; 8) The Liaison Committee between the Central Norway Regional Health Authority (RHA) and the Norwegian University of Science and Technology (NTNU) and 9) The Norwegian Haemochromatosis Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The aim of the present study was to determine if human N-terminal half-transferrin (N- fragment), prepared by thermolysin cleavage of diferric transferrin, would bind to the rat hepatocyte transferrin receptor and donate iron to the cell. Competition experiments between 125I-labelled N-fragment and diferric transferrin revealed no receptor binding of the half-transferrin. Still, the N-fragment delivered iron to the cells in amounts approximately 30-fold above what could be accounted for by uptake of the fragment itself. The rate of cellular iron uptake from the fragment was comparable to what is seen with the intact transferrin. The uptake of 125I-labelled N-fragment was not inhibited by excess non-radioactive diferric transferrin. By comparison, the uptake of 59Fe from the N-fragment was inhibited 70% by excess nonradioactive diferric transferrin. This suggests that iron derived from diferric transferrin competes with the iron derived from the N-fragment for a common transport pathway. Although some cellular degradation of the N-fragment occurred, the extent of degradation was too low to explain the amount of iron accumulated by the cells. The results show that the hepatocyte has an effective transferrin-receptor-independent mechanism for accumulation of iron from transferrin.

In an attempt to improve our understanding of the complex interplay between cell compartments and chemical species during cellular uptake of iron from transferrin, we designed a computer simulation program based on current models of receptor-mediated endocytosis and pinocytosis. The program calculates and visualizes, as a function of time, the changes in transferrin, apotransferrin, and iron concentrations occurring in all relevant cellular compartments during cellular iron acquisition from transferrin. Simulation of literature data showed that the program generates results that are in accordance with experimental data. Furthermore, from measurements of the uptake of [carboxyl-14C]dextran we could utilize the program to suggest rate constants characteristic for the pinocytic process in rat reticulocytes. Moreover, simulations indicate that the apparent difference in the iron uptake process observed between reticulocytes and hepatocytes may be explained by the contribution made by pinocytosis to the iron uptake process. Finally, the present program should have potential as an educational tool during introduction to the field of receptor-mediated endocytosis in general and to cellular iron metabolism in particular.

Hereditary hemochromatosis (HH) is a genetic condition characterized by increased iron absorption. Most HH cases are homozygous for the C282Y mutation in the HFE gene, but accurate prevalence data for the Norwegian population is lacking. In population studies, serum transferrin saturation (TS) is commonly used as a screening test. However, the sensitivity and specificity of TS in this setting is not well documented. The purpose of this study was to determine the prevalence of the C282Y mutation in the general population, and to evaluate the diagnostic accuracy of the TS test as a screening criterion for finding C282Y homozygotes.The hemochromatosis screening study in Nord-Trøndelag county, Norway (the HUNT2 study) comprised 65,238 participants. The HUNT biobank contains biological material and data from the participants, and 5000 individuals were randomly selected. Genotyping of the common HFE gene mutations was successful for 4827 samples, from which TS data existed for 4804 individuals. From these data we calculated the population frequency of the C282Y mutation, and the sensitivity and specificity of TS measurements.The prevalence of C282Y homozygosity in the population was 0.75%. Using 55% (men) and 50% (women) as decision limits, the sensitivity of two consecutive elevated TS measurements was 90.0% for men and 55.0% for women, whereas the specificity was 99.6% and 99.4%, respectively.An unbiased estimate of the C282Y homozygote prevalence in Norway is 0.75%. Two measurements of TS is an accurate screening test for C282Y homozygosity in men, but not in women.

In a previous hemochromatosis screening study including a total of 65,238 individuals, 566 persons were genotyped for the C282Y and the H63D mutations. Of these, a total of 433 samples (298 homozygous C282Y and 135 homozygous wild type) were reanalyzed to investigate if the potential presence of the newly described 5569G/A polymorphism had confounded the genotyping results for the C282Y mutation. Genotyping with a polymorphism-insensitive primer pair yielded no samples that altered their genotype. By utilizing the polymorphism-sensitive primer pair and elevated annealing temperatures, 133 samples previously genotyped as heterozygous C282Y were reanalyzed to verify the presence of the polymorphism in the population studied. Out of a total of 266 chromosomes, we found the polymorphism present in 9 chromosomes, yielding an allele frequency of 0.034 in this particular subpopulation. In one of the samples, the polymorphism was present on the same DNA strand as the C282Y mutation. We conclude that in the population studied, the 5569 G/A polymorphism is present, but its presence had no implications for the outcome of the previous genotyping. Nevertheless, we recommend that C282Y genotyping by restriction endonuclease digestion of PCR products in the future should utilize a primer pair that is not influenced by the 5569G/A polymorphism.

Hemochromatosis is a common disease with a good prognosis, when diagnosed early and treated appropriately. The aim of this overview is to give updated information on hemochromatosis with special focus on biochemical features, diagnosis and treatment.This article is based on our own experience and a review of available literature in various databases such as PubMed and Medline.Hereditary hemochromatosis is an autosomal recessive disease characterized by iron overload due to increased intestinal iron uptake over many years. Hemochromatosis is often discovered through coincidental detection of high levels of transferrin and/or ferritin. The early symptoms are asthenia and joint pain. About 85 % of patients with hereditary hemochromatosis are homozygote for the C282Y mutation in the HFE: gene, but the majority of homozygotes remain asymptomatic. With ferritin levels500 microg/, both hereditary hemochromatosis and iron overload (of unknown cause) are treated with blood-letting.The pathogenesis is not fully elucidated but recent reports indicate that the protein hepcidin (produced in the liver) plays a key role in the development of hemochromatosis. Iron overload may also be secondary to other diseases such as thalassemia and other conditions requiring multiple long-term blood transfusions. The goal is to maintain ferritin values at approximately 20 - 50 microg/L.

The regulation of iron absorption has previously been consideredan enigma. Recent research has given us interesting information on the regulation of the iron metabolism and pathological iron overload; the present article aims at providing an overview of these topics.The article is based on a review of literature retrieved from PubMed.The peptide hepcidin binds to ferroportin on membranes of enterocytes, macrophages and hepatocytes. The complex is internalised and degraded and this results in decreased export of iron to the circulation, and thus a lower level of plasma iron. Hepcidin production is up-regulated in iron overload and down-regulated with iron deficiency. The liver proteins human haemochromatosis protein (HFE), transferrin receptor 2 (TfR2), haemojuvelin (HJV) and bone morphogenetic protein (BNP) are necessary regulators for activation of the hepcidin synthesis. Lack of or mutations in the genes for these proteins, e.g. the HFE mutation C282Y in primary haemochromatosis, reduces the synthesis of hepcidin. Iron regulatory proteins (IRP) may bind to iron responsive elements (IRE) of ferritin-mRNA and transferritin-mRNA and regulate the protein synthesis.Regulation of uptake, utilization, release and storage of iron occurs at the gene level. Hepcidin is currently considered to be thekey regulatorof the iron balance. Intracellular iron balance is maintained by iron regulating proteins. Synthesis of ferritin increases with high iron levels, while synthesis of TfR1 is reduced. The opposite occurs with a low iron level.

Associations between and changes in plasma phospholipid fatty acid (FA) concentrations and expression of delta 5 desaturase (D5D), delta 6 desaturase (D6D) and delta 9 desaturase (D9D) in leucocytes were investigated both before and during n-3 FA supplementation for 2 weeks in 20 healthy individuals. Participants were divided into two groups depending on fish intake: one fish meal or less per week and no marine FA supplement (Lowfish, n = 9) and more than one fish meal per week and/or daily oral marine FA supplement (Highfish, n = 11). Before starting supplementation (t = 0), concentrations of n-3 FAs were significantly lower in the Lowfish group compared to the Highfish group. During supplementation in both groups, n-3 FAs increased, whereas n-6 FAs decreased. D5D expression was significantly higher in Lowfish compared to Highfish at t = 0. No difference in D6D or D9D expression was observed. D5D expression was inversely correlated with EPA, DPA, DHA and total n-3 FA, and positively correlated with the ratio total n-6 FA/total n-3 FA at t = 0. Expression of D5D in the Lowfish group as well as D6D in both groups significantly decreased relative to the expression at t = 0 during the first day of supplement. PUFA concentration was generally predicted by its precursor FA and D5D or D6D expression. The correlations mentioned disappeared after 2 weeks of supplementation. This indicates that steady-state FA desaturase expression is associated with plasma phospholipid FA composition. Whether leucocyte desaturase expression may have potential as a marker of PUFA status merits further investigation.

Seasonal iron overload in Svalbard reindeer was studied by light and electron microscopy and by X-ray microanalysis. The hepatic iron overload was of two types. The first type was characterized by massive siderosis of both parenchymal and non-parenchymal cells caused by a diet very rich in iron but low in energy and protein. Hepatocytes contained a moderate amount of free ferritin particles in the cytosol together with numerous siderosomes. This pattern is similar to that seen in primary haemochromatosis and thalassaemia. Kupffer cells contained large quantities of cytosolic ferritin, siderosomes and lysosomes with disintegrating red blood cells as seen in thalassaemia. The second type was characterized by massive non-parenchymal siderosis caused by an energy- and protein-poor diet with normal iron concentration. Hepatocytes contained little cytosolic ferritin and few siderosomes, but there were abundant electron-dense bodies without iron (i.e., autophagosomes). Kupffer cells were as described above. Ferritin was also present within the duodenal mucosa of these animals, located within enterocytes and lamina propria macrophages, as well as in the extracellular space and capillary and lacteal lumina. Ferritin was also present in the acinar cells of submucosal Brunner's glands. Changes consistent with exchange of ferritin particles between different cell types were observed. The role of ferritin as a possible iron transporter in this condition is discussed.

The expression of delta 5 desaturase (D5D), delta 6 desaturase (D6D) and delta 9 desaturase (D9D) was determined by RT-PCR in the human promyelocytic cell line HL60. During 72 h of culture with 10% FBS, D5D and D6D were upregulated 5 to 6-fold, whereas D9D approximately doubled. The addition of fatty acids (FAs) to the culture medium suppressed upregulation of all desaturases. N-3 and n-6 FA appeared to be more effective than n-9 or saturated FA. When FAs were added after 72 h, further upregulation during the next 24 h was suppressed for nearly all desaturases and FAs tested, except for D5D when oleic acid (OA) or stearic acid (SA) was added. In cells cultured with restricted amounts of FBS, desaturase expression increased with decreasing concentrations of FBS. Cellular FA content decreased by 60% in the neutral lipid fraction, whereas that of the phospholipid fraction decreased by 10% during 72 h of culture. The largest decrease occurred in the sum of n-3 and n-6 FA of the neutral lipid fraction, which was reduced by 83%, whereas the content of these FAs in the phospholipid fraction decreased by 32%. The results indicate that when the supply of FA to HL60 cells is limited, the intracellular content of n-3 and n-6 FA decreases and this leads to upregulation of the desaturases, particularly D5D and D6D. Since HL60 cells resemble human leukocytes, the results suggest that desaturase expression in leukocytes may be exploited as a biomarker for FA status.

Normally the body is unable to regulate its own iron content effectively by excretion. Urinary excretion of iron is about 10% of total daily loss.A 40-year-old woman was admitted to hospital because of anaemia that turned out to be caused by iron deficiency. In spite of extensive investigations no cause of her iron deficiency was identified. The urine was then tested for iron and showed excessive urinary loss as the cause of her anaemia. To our knowledge, extensive urinary iron loss as the cause of iron deficiency has not been reported before.

In this large cross-sectional population-based study, 51,272 persons responded to a headache questionnaire and were screened for hemochromatosis. Phenotypic hemochromatosis and the C282Y/C282Y genotype were both associated with an 80% increase in headache prevalence evident only among women. The reason for this association is unclear, but one may speculate that iron overload alters the threshold for triggering a headache by disturbing neuronal function.

Hereditary hemochromatosis (HH) is a common genetic disease leading to accumulation of iron in several organs, most notably the liver. The C282Y/C282Y mutation in the HFE gene is found in most cases. In order to prevent clinical disease and to study the cost and feasibility of screening, a large population was screened.In a Norwegian county, all inhabitants 20 years or older were invited to participate in a population-based health survey programme. Screening for HH was one of several subprojects. Blood samples were obtained from 65,238 persons. Subjects with high serum transferrin saturation in two tests and high serum ferritin were clinically evaluated for HH. All subjects with high serum transferrin saturation in two tests were offered genotyping.HH was newly diagnosed in 92 women and 177 men. Phlebotomy treatment was performed in 64 women and 152 men. Severe organ damage (liver cirrhosis) was ascertained in only 4 men. We found no correlation between serum ferritin and age. The estimated cost was US$ 1.6 per subject screened and US$ 390 per newly discovered HH subject. The estimated prevalence of phenotypical HH not previously known was 0.34% in women and 0.68% in men. The prevalence of the C282Y/C282Y mutation was at least 0.68%.Large-scale screening for HH can be performed at a relatively low cost if combined with a health survey programme. The yield in terms of newly discovered cases is considerable, but few cases were found seriously ill. Better knowledge of the natural course of HH is necessary if we are to be able to estimate the cost-effectiveness of large-scale screening.

In the present paper, we describe an individual, found as part of a screening study, being homozygous for the C282Y mutation and at the same time heterozygous for the H63D mutation in the HFE gene. Identical results were obtained by three different methods, i.e., by PCR-RFLP, by sequencing, and by melting curve analysis. Thus, the common conception that the C282Y and the H63D mutations are mutually exclusive is not valid. Clinical symptoms and laboratory data on the individual were similar to hemochromatosis patients homozygous for the C282Y mutation. The implications of our finding for diagnostic analytical laboratory procedures are briefly discussed.

The lanthanide metal, gadolinium, is currently used in contrast agents for magnetic resonance imaging. We have performed a study of the interaction between isolated rat hepatocytes and 153Gd complexed to diethylene-triamine pentaacetic acid (DTPA) or to DTPA-albumin conjugates. The study shows that isolated hepatocytes are able to take up both types of 153Gd complexes. The 153Gd-DTPA-albumin complexes are apparently taken up by pinocytosis, and possibly receptor-mediated endocytosis and/or adsorptive endocytosis, whereas the uptake mechanism of 153Gd-DTPA is unknown. The 153Gd-DTPA-albumin complexes, but not the 153Gd-DTPA complex, are degraded by the cell. The degradation is inhibited by ammonium chloride. Gadolinium is slowly released back to the medium after loading of the cells with both complex types. In the experiments reported here no evidence of any adverse effects on the hepatocyte resulting from exposure to the 153Gd-complexes were observed.