The Role of the Trabecular Bone Score in the Assessment of Osteoarticular Disorders in Patients with HFE-Hemochromatosis: A Single-Center Study from Poland.

Type 1 hereditary hemochromatosis (HH) is an autosomal, recessive genetic entity with systemic iron overload. Iron homeostasis disorders develop as a result of HFE gene mutations, which are associated with hepcidin arthropathy or osteoporosis and may cause permanent disability in HH patients despite a properly conducted treatment with phlebotomies. In this study, selected parameters of calcium and phosphate metabolism were analyzed in combination with the assessment of bone mineral density (BMD) disorders in patients from northern Poland with clinically overt HFE-HH. BMD was determined by a dual-energy X-ray absorptiometry (DXA) test with the use of the trabecular bone score (TBS) function. The study included 29 HH patients (mean age = 53.14 years) who were compared with 20 healthy volunteers. A significantly lower TBS parameter and serum 25-OH-D3 concentration, a higher concentration of intact parathormone and more a frequent occurrence of joint pain were found in HH patients compared with the control group. In HH patients, the diagnosis of liver cirrhosis was associated with lower serum 25-OH-D3 and osteocalcin concentrations. In HH, DXA with the TBS option is a valuable tool in the early assessment of the bone microarchitecture and fracture risk. A supplementation of vitamin D, monitoring its concentration, should be considered especially in HH patients with liver damage and liver cirrhosis.

Ilex paraguariensis (A. St.-Hil.) leaf infusion decreases iron absorption in patients with hereditary hemochromatosis: a randomized controlled crossover study.

This study proposed to investigate the effect of Ilex paraguariensis infusion on the absorption of non-heme iron in hereditary hemochromatosis (HH) patients with the HFE genotype. A two-way randomized, controlled, crossover trial was conducted on patients, aged 29-69 years, undergoing maintenance therapy. Fourteen HFE-HH patients ingested a meal containing 11.4 mg iron and 200 mL either of water (control) or of Ilex paraguariensis leaf infusion. The beverages were offered in random order, at intervals separated by a washout period of 7 days. Active surveillance showed no adverse effects. Blood samples were drawn shortly before and 1, 2, 3, and 4 h after the meal for serum iron measurement. A significant reduction in the postprandial serum iron was observed for HH patients after intake of the Ilex paraguariensis infusion (area under the curve (AUC) expressed as mean ± SEM: 173.3 ± 44.7 µmol h-1 L-1) compared to water (1449.4 ± 241.5 µmol h-1 L-1) (p < 0.001). In summary, intake of Ilex paraguariensis leaf infusion significantly inhibited the absorption of iron in patients with HH and, therefore, should be considered as a potential adjuvant for iron overload control.

Clinical characteristics of endocrinopathies in Chinese patients with hereditary haemochromatosis.

AIMS: Hereditary haemochromatosis (HH) is a genetic disorder characterised by systemic iron overload and can lead to end-organ failure. However, very few data on this disorder, especially those on endocrine gland involvement in Chinese populations, are currently available. This study aimed to analyse the clinical features of endocrinopathies in patients with HH to generate concern among endocrinologists and improve the management of this disorder. MATERIALS AND METHODS: Chinese patients with HH-related endocrine dysfunction were enrolled at Peking Union Medical College Hospital from January 2010 to December 2018. All clinical data were analysed and summarised. RESULTS: A total of six patients were enrolled in this study, comprising five men and one woman; the average age was 36.5 ± 13.3 years. Mean serum ferritin concentration was 4508.8 ± 1074.3 ng/ml, and median transferrin saturation was 97.9% (96.6%-110.0%). Endocrine gland involvement associated with HH included the pancreas (5/6 patients), the adenohypophysis (5/6 patients) and the bones (1/6 patients); secondary endocrinopathies consisted of diabetes mellitus, hypogonadism, adrenal insufficiency and osteoporosis. Based on phlebotomy and iron chelation therapy, five patients were treated with exogenous insulin preparations, and three patients were treated with exogenous sex hormone replacement therapy. The clinical symptoms of five patients improved, although one patient died of hepatic encephalopathy and multiple organ failure. CONCLUSIONS: HH can cause multiple endocrinopathies. The possibility of HH should be carefully considered in patients with endocrine gland dysfunctions and concomitant elevated serum ferritin levels. Endocrine gland function should also be assessed and followed up in patients with a clear diagnosis of HH.

Coenzyme Q10 deficiency in patients with hereditary hemochromatosis.

AIM: Hereditary hemochromatosis (HH) is a group of inherited disorders that causes a slow and progressive iron deposition in diverse organs, particularly in the liver. Iron overload induces oxidative stress and tissue damage. Coenzyme Q10 (CoQ10) is a cofactor in the electron-transport chain of the mitochondria, but it is also a potent endogenous antioxidant. CoQ10 interest has recently grown since various studies show that CoQ10 supplementation may provide protective and safe benefits in mitochondrial diseases and oxidative stress disorders. In the present study we sought to determine CoQ10 plasma level in patients recently diagnosed with HH and to correlate it with biochemical, genetic, and histological features of the disease. METHODS: Plasma levels of CoQ10, iron, ferritin, transferrin and vitamins (A, C and E), liver tests (transaminases, alkaline phosphatase and bilirubin), and histology, as well as three HFE gene mutations (H63D, S654C and C282Y), were assessed in thirty-eight patients (32 males, 6 females) newly diagnosed with HH without treatment and in twenty-five age-matched normolipidemic healthy subjects with no HFE gene mutations (22 males, 3 females) and without clinical or biochemical signs of iron overload or liver diseases. RESULTS: Patients with HH showed a significant decrease in CoQ10 levels respect to control subjects (0.31 ±â€¯0.03 µM vs 0.70 ±â€¯0.06 µM, p < 0.001, respectively) independently of the genetic mutation, cirrhosis, transferrin saturation, ferritin level or markers of hepatic dysfunction. Although a decreasing trend in CoQ10 levels was observed in patients with elevated iron levels, no correlation was found between both parameters in patients with HH. Vitamins C and A levels showed no changes in HH patients. Vitamin E was significantly decreased in HH patients (21.1 ±â€¯1.3 µM vs 29.9 ±â€¯2.5 µM, p < 0.001, respectively), but no correlation was observed with CoQ10 levels. CONCLUSION: The decrease in CoQ10 levels found in HH patients suggests that CoQ10 supplementation could be a safe intervention strategy complementary to the traditional therapy to ameliorate oxidative stress and further tissue damage induced by iron overload.

Role of liver magnetic resonance imaging in hyperferritinaemia and the diagnosis of iron overload.

Hyperferritinaemia is a frequent clinical problem. Elevated serum ferritin levels can be detected in different genetic and acquired diseases and can occur with or without anaemia. It is therefore important to determine whether hyperferritinaemia is due to iron overload or due to a secondary cause. The main causes of iron overload are intestinal iron hyperabsorption disorders and transfusion-dependent disorders. Iron homeostasis and iron overload are quantified by different diagnostic approaches. The evaluation of serum ferritin and transferrin saturation is the first diagnostic step to identify the cause of hyperferritinaemia. The assessment of liver iron concentration by liver biopsy or magnetic resonance imaging (MRI) may guide the further diagnostic and therapeutic workup. Liver biopsy is invasive and poorly accepted by patients and should only be carried out in selected patients with hereditary haemochromatosis. As a non-invasive approach, MRI is considered the standard method to diagnose and to monitor both hepatic iron overload and the effectiveness of iron chelation therapy in many clinical conditions such as thalassaemia and myelodysplastic syndromes. Accurate evaluation and monitoring of iron overload has major implications regarding adherence, quality of life and prognosis. There are different technical MRI approaches to measuring the liver iron content. Of these, T2 and T2* relaxometry are considered the standard of care. MRI with cardiac T2* mapping is also suitable for the assessment of cardiac iron. Currently there is no consensus which technique should be preferred. The choice depends on local availability and patient population. However, it is important to use the same MRI technique in subsequent visits in the same patient to get comparable results. Signal intensity ratio may be a good adjunct to R2 and R2* methods as it allows easy visual estimation of the liver iron concentration. In this review a group of Swiss haematologists and radiologists give an overview of different conditions leading to primary or secondary iron overload and on diagnostic methods to assess hyperferritinaemia with a focus on the role of liver MRI. They summarise the standard practice in Switzerland on the use of liver iron concentration MRI as well as disease-specific guideline recommendations.

Rescue case of low birth weight infant with acute hepatic failure.

We report a case involving a rescued low birth weight infant (LBWI) with acute liver failure. CASE: The patient was 1594 g and 323/7 gestational wk at birth. At the age of 11 d, she developed acute liver failure due to gestational alloimmune liver disease. Exchange transfusion and high-dose gamma globulin therapy were initiated, and body weight increased with enteral nutrition. Exchange transfusion was performed a total of 33 times prior to living donor liver transplantation (LDLT). Her liver dysfunction could not be treated by medications alone. At 55 d old and a body weight of 2946 g, she underwent LDLT using an S2 monosegment graft from her mother. Three years have passed with no reports of intellectual disability or liver dysfunction. LBWIs with acute liver failure may be rescued by LDLT after body weight has increased to over 2500 g.

Biochemical, radiologic, ultrastructural, and genetic evaluation of iron overload in acute leukemia and iron-chelation therapy.

Iron overload in hereditary hemochromatosis and hematologic malignancy has unfavorable effects on morbidity. Herein, 53 children (age 108.4±58.3 mo, 25 girls and 28 boys) with acute myeloblastic and lymphoblastic leukemia, who received 4 different chemotherapy protocols, were evaluated for iron overload throughout chemotherapy. Iron overload arose: (1) before chemotherapy, which was dependent on neither chemotherapy nor packed red blood cell transfusions and (2) after chemotherapy, which was dependent on the duration and nature of chemotherapy and partially on transfusion of packed red blood cells. Iron overload was documented in 75% of patients with a ferritin level >1000 ng/mL, by liver and heart magnetic resonance imaging, and they were administered iron-chelation therapy with success. Three of 10 radiologically iron-overloaded patients were heterozygous for H63D mutation. Aminolevulinic acid and porphobilinogen levels were normal. Light microscopic examination of the bone marrow revealed increased iron granules in erythroblasts, platelets, and megakaryocytes, iron-laden macrophages, free iron in the matrix, dyshematopoiesis, and apoptotic cells. Electron microscopic examination revealed iron-laden secondary lysosomes and autolysosomes in normoblasts and iron-laden primary granules in promyelocytes, irrelevant to the ferritin level, implying autophagia due to chemotherapy as a source of the excess iron. We think that iron overload, which is an important complication of acute leukemia, should be evaluated separately from "transfusion overload," and the management principles specific to leukemia should be implemented.

Diagnosis and treatment of hereditary hemochromatosis: an update.

Hereditary hemochromatosis is an inherited iron overload disorder caused by inappropriately low hepcidin secretion leading to increased duodenal absorption of dietary iron, most commonly in C282Y homozygous individuals. This can result in elevated serum ferritin, iron deposition in various organs and ultimately end-organ damage, although there is incomplete biochemical and clinical penetrance and variable phenotypic expression of the HFE mutation in hereditary hemochromatosis. An elevated SF >1000 mg/l [corrected] is associated with an increased risk of cirrhosis and mortality in C282Y homozygotes.Conversely, a SF <1000 µg/l is associated with a very low likelihood of cirrhosis, making liver biopsy unnecessary among C282Y homozygotes in the absence of concomitant risk factors for liver disease. Phlebotomy remains the mainstay of treatment and new treatments being studied include erythrocytapheresis and 'mini-hepcidins'. Iron overload is being recognized to play a carcinogenic role in hepatocellular carcinoma and other cancers, possibly supporting iron depletion in these patients.

Effect of hereditary haemochromatosis genotypes and iron overload on other trace elements.

PURPOSE: Hereditary haemochromatosis is a common genetic disorder involving dysregulation of iron absorption. There is some evidence to suggest that abnormal iron absorption and metabolism may influence the status of other important trace elements. In this study, the effect of abnormal HFE genotypes and associated iron overload on the status of other trace elements was examined. METHODS: Dietary data and blood samples were collected from 199 subjects (mean age = 55.4 years; range = 21-81 years). Dietary intakes, serum selenium, copper and zinc concentrations and related antioxidant enzymes (glutathione peroxidase and superoxide dismutase) in subjects with normal HFE genotype (n = 118) were compared to those with abnormal HFE genotype, with both normal iron status (n = 42) and iron overload (n = 39). RESULTS: For most dietary and biochemical variables measured, there were no significant differences between study groups. Red cell GPx was significantly higher in male subjects with normal genotypes and normal iron status compared to those with abnormal genotypes and normal iron status (P = 0.03) or iron overload (P = 0.001). Red cell GPx was also highest in normal women and significantly lower in the abnormal genotype and normal iron group (P = 0.016), but not in the iron overload group (P = 0.078). CONCLUSION: Although it may not be possible to exclude a small effect between the genotype groups on RBC GPx, overall, haemochromatosis genotypes or iron overload did not appear to have a significant effect on selenium, copper or zinc status.

Successful management of neonatal hemochromatosis by exchange transfusion and immunoglobulin: a case report.

Neonatal hemochromatosis (NH) is a rare and severe liver disease of mainly intra-uterine onset, characterized by neonatal liver failure, hepatic and extrahepatic iron accumulation. This leads to an altered iron metabolism with resulting siderosis. The disease represents the most common cause of liver failure in neonates and is also the most common indication for neonatal liver transplantation. We present a case of a newborn diagnosed with NH and life threatening liver failure. Initial treatment consisted of chelation therapy and antioxidants, but lack of laboratory and clinical improvement led to an exchange transfusion followed by the singular substitution of intravenous immunoglobulin (IVIG). Both, exchange transfusion and IVIG were tolerated well and led to an improvement of the general condition of the patient and recovery of liver synthetic function. The subsequent favorable course of the disease is described in this case report.

Dietary iron intake and serum ferritin concentration in 213 patients homozygous for the HFEC282Y hemochromatosis mutation.

BACKGROUND: 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. OBJECTIVE: To determine whether dietary iron content influences iron stores in HFEC282Y homozygotes as assessed by serum ferritin concentration. DESIGN: 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. RESULTS: No significant relationships between serum ferritin concentration and dietary heme iron content, dietary nonheme iron content or reports of supplemental iron use were found. CONCLUSION: These results do not support recommending dietary heme or nonheme iron restrictions for HFEC282Y homozygotes diagnosed through screening in North America.

C282Y hemochromatosis gene mutation and iron parameters in dialysis patients.

BACKGROUND: Hereditary hemochromatosis is an autosomal recessive condition causing excessive intestinal iron absorption related to C282Y hemochromatosis mutation gene. Dialysis patients receive intravenous iron supplements as treatment for anemia. The gene mutation frequency and its influence on iron deposits and intravenous iron response are unknown in these patients. STUDY DESIGN: Prospective observational. SETTING AND PARTICIPANTS: 290 dialysis patients in Gran Canaria, Spain. OUTCOMES AND MEASUREMENTS: The C282Y hemochromatosis mutation gene was studied. Other active players in iron metabolism have not been included in this study. Red cell parameters, serum iron, transferrin and ferritin concentrations were measured every 2 months for 2 years. RESULTS: No differences in allelic and genotypic frequencies between dialysis patients and the general population were detected. Baseline clinical or analytical parameters were similar in C282Y +/- and C282Y -/- patients. Among those who did not need intravenous iron treatment, C282Y+/- patients maintained constant serum ferritin (302.1 ± 216.7 vs. 319.5 ± 300.5 µg/l after 4 months), whereas C282Y-/- patients showed decreased levels during the same period (306.7 ± 212.2 vs. 221.6 ± 167.8 µg/l, p < 0.001). After 4 months of parenteral iron, serum ferritin increased more intensely in C282Y +/- patients than in C282Y -/- patients (934.2 ± 195.8 vs. 658.7 ± 259.9 µg/l, p < 0.001). A multivariance analysis identified the C282Y allele as the most important factor that explains this difference. CONCLUSIONS: Heterozygosity for the C282Y allele of the hemochromatosis mutation gene could be associated with differences in iron parameters in dialysis patients.

The iron-chelating potential of silybin in patients with hereditary haemochromatosis.

Milk thistle contains silybin, which is a potential iron chelator. We aimed to determine whether silybin reduced iron absorption in patients with hereditary haemochromatosis. In this crossover study, on three separate occasions, 10 patients who were homozygous for the C282Y mutation in the HFE gene (and fully treated) consumed a vegetarian meal containing 13.9 mg iron with: 200 ml water; 200 ml water and 140 mg silybin (Legalon Forte); or 200 ml tea. Blood was drawn once before, then 0.5, 1, 2, 3 and 4 h after the meal. Consumption of silybin with a meal resulted in a reduction in the postprandial increase in serum iron (AUC±s.e.) compared with water (silybin 1726.6±346.8 versus water 2988.8±167; P<0.05) and tea (silybin 1726.6±346.8 versus tea 2099.3±223.3; P<0.05). In conclusion, silybin has the potential to reduce iron absorption, and this deserves further investigation, as silybin could be an adjunct in the treatment of haemochromatosis.

Reflex and reflective testing: efficiency and effectiveness of adding on laboratory tests.

BACKGROUND: Laboratory investigations may be added to existing requests either automatically on the basis of algorithms (reflex testing) or by laboratory professionals (reflective testing). The clinical utility of reflex and reflective testing is not fully established. We studied efficiency (number of tests that needs to be added to make a diagnosis) and effectiveness (number of diagnoses) of reflex and reflective testing in selected biochemical scenarios. METHODS: Using fixed rules, we prospectively measured efficiency and effectiveness of reflex and reflective testing in the following scenarios (reflex initiators in parentheses): (1) hypovitaminosis D (hypocalcaemia plus elevated alkaline phosphatase activity); (2) hypomagnesaemia (hypokalaemia or hypocalcaemia); (3) hypothyroidism (high thyroid-stimulating hormone [TSH]); (4) hyperthyroidism (low TSH); (5) haemochromatosis (reflex or reflective addition of iron studies, followed by reflective addition of genetic studies). Separately, using a different data-set, we examined the impact of varying TSH thresholds on outcomes in the biochemical diagnosis of hyper- and hypothyroidism. RESULTS: In patients aged over 55 y, 25-hydroxy-vitamin D <50 nmol/L could be predicted with > or =90% certainty when albumin-adjusted calcium was < or =2.1 mmol/L plus alkaline phosphatase >150 U/L. Higher numbers of tests were needed to make a diagnosis in other scenarios. In general, more diagnoses were made by reflex testing. Outside the euthyroid TSH range, efficiency of diagnosis of hyper- and hypothyroidism became asymptotic, while effectiveness declined. CONCLUSIONS: Near-maximal efficiency of reflex testing can be achieved, depending on the reflex and diagnostic thresholds applied. Reflective and reflex testing are complementary activities, the clinical utility of which depends on the initiators used.

Effect of iron supplementation on iron status during the first week after blood donation.

INTRODUCTION: Frequent blood donations may lead to a negative iron balance. Iron depletion may be prevented by iron supplementation after whole blood donations. The aim of this study was to compare the short time changes in iron status after donation in two groups randomized to iron supplementation or no additional iron. A second objective was to evaluate the effect of iron supplementation in donors having HFE-variants compared to HFE wild types. METHODS: Subjects of both genders (199 women, 200 men) were randomised to receive iron supplementation or no additional iron after donation. Iron status, defined by the concentration of haemoglobin, serum ferritin, soluble transferrin receptor, concentration of haemoglobin in reticulocytes (CHr) and percent hypochrome mature red blood cells, was determined at the start of donation and 8 +/- 2 days after donation. HFE genotyping was performed at reappearance. RESULTS: There was a significant difference between the two study groups on all the iron status parameters. CHr was an efficient, early marker of ongoing synthesis of haemoglobin. Heterozygosity for the HFE variants C282Y and H63D had no statistically significant influence on the iron status. The donor's baseline serum ferritin value may be basis for an individual iron supplementation regimen, as donors with serum ferritin >50 microg/l do not seem to utilize the iron supplementation, but prefer endogenous iron to restore the loss of haemoglobin. CONCLUSION: Iron supplementation had a significant positive impact on the restoration of iron status one week after donation.

Severity of iron overload of proband determines serum ferritin levels in families with HFE-related hemochromatosis: the HEmochromatosis FAmily Study.

BACKGROUND/AIMS: In families of patients with clinically detected hereditary hemochromatosis (HH) early screening has been suggested to prevent morbidity and mortality. Here, we aim to identify determinants for iron overload in first-degree family members of C282Y homozygous probands with clinically detected HH. METHODS: Data on HFE-genotype, iron parameters, demographics, lifestyle factors and health, were collected from 224 Dutch C282Y homozygous patients with clinically diagnosed HH and 735 of their first-degree family members (FDFM), all participating in the HEmochromatosis FAmily Study (HEFAS). RESULTS: The best predictive multivariable model forecasted 45% of variation of the serum ferritin levels. In this model severity of iron overload in the proband significantly predicted serum ferritin levels in FDFM. Other significant determinants in this model consisted of C282Y homozygosity, compound heterozygosity, age at testing for serum ferritin and supplemental iron intake, whereas a low body mass index showed a protective effect. CONCLUSIONS: This study provides a model to assess the risk of development of iron overload for relatives of probands with HH. These results might be instrumental in the development of an optimal strategy for future family screening programs.

Thyroid-stimulating hormone and free thyroxine levels in persons with HFE C282Y homozygosity, a common hemochromatosis genotype: the HEIRS study.

BACKGROUND: Relationships of thyroid and iron measures in large cohorts are unreported. We evaluated thyroid-stimulating hormone (TSH) and free thyroxine (T4) in white participants of the primary care-based Hemochromatosis and Iron Overload Screening (HEIRS) Study. METHODS: We measured serum TSH and free T4 in 176 HFE C282Y homozygotes without previous hemochromatosis diagnoses and in 312 controls without HFE C282Y or H63D who had normal serum iron measures and were matched to C282Y homozygotes for Field Center, age group, and initial screening date. We defined hypothyroidism as having TSH >5.00 mIU/L and free T4 <0.70 ng/dL, and hyperthyroidism as having TSH <0.400 mIU/L and free T4 >1.85 ng/dL. Multivariate analyses were performed using age, sex, Field Center, log(10) serum ferritin (SF), HFE genotype, log(10) TSH, and log(10) free T4. RESULTS: Prevalences of hypothyroidism in C282Y homozygotes and controls were 1.7% and 1.3%, respectively, and of hyperthyroidism 0% and 1.0%, respectively. Corresponding prevalences did not differ significantly. Correlations of log(10) SF with log(10) free T4 were positive (p = 0.2368, C282Y homozygotes; p = 0.0492, controls). Independent predictors of log(10) free T4 were log(10) TSH (negative association) and age (positive association); positive predictors of log(10) SF were age, male sex, and C282Y homozygosity. Proportions of C282Y homozygotes and controls who took medications to supplement or suppress thyroid function did not differ significantly. CONCLUSIONS: Prevalences of hypothyroidism and hyperthyroidism are similar in C282Y homozygotes without previous hemochromatosis diagnoses and controls. In controls, there is a significant positive association of SF with free T4. We conclude that there is no rationale for routine measurement of TSH or free T4 levels in hemochromatosis or iron overload screening programs.

Involvement of ApoE E4 and H63D in sporadic Alzheimer's disease in a folate-supplemented Ontario population.

Dysregulation of iron homeostasis is implicated in Alzheimer's disease (AD). In this pilot study, common variants of the apolipoprotein E (APOE) and HFE genes resulting in the iron overload disorder of hereditary hemochromatosis (C282Y, H63D and S65C) were evaluated as factors in sporadic AD in an Ontario sample in which folic acid fortification has been mandatory since 1998. Laboratory studies also were done to search for genetic effects on blood markers of iron status, red cell folates and serum B12. Participants included 58 healthy volunteers (25 males, 33 females) and 54 patients with probable AD (20 males, 34 females). Statistical analyses were interpreted at the 95% confidence level. Contingency table and odds ratio analyses supported the hypothesis that in females of the given age range, E4 significantly predisposed to AD in the presence but not absence of H63D. In males, E4 significantly predisposed to AD in the absence of H63D, and H63D in the absence of E4 appeared protective against AD. Among E4+ AD patients, H63D was associated with significant lowering of red cell folate concentration, possibly as the result of excessive oxidative stress. However, folate levels in the lowest population quartile did not affect the risk of AD. A model is presented to explain the experimental findings.

Stable isotope labelling and FPLC-ICP-SFMS for the accurate determination of clinical iron status parameters in human serum.

Iron is involved in the function of all living cells and, in fact, many diseases arise from imbalances in iron homeostasis. Therefore, the development of analytical methodologies to improve and automate the measurement of clinical indices of iron status has increased tremendously over the years. This work describes the development of two complementary methodologies to evaluate transferrin (Tf) saturation, total iron-binding capacity (TIBC), unsaturated iron-binding capacity (UIBC) and serum iron based on the use of iron-selective monitoring by inductively coupled plasma mass spectrometry (ICP-MS). The first methodology is based on the saturation of transferrin (Tf) with natural Fe3+ followed by separation of the different sialoforms in an anion exchange column (Mono-Q) to quantify the iron in each Tf sialoform and total Tf by ICP-MS using post-column isotope dilution analysis. In the second part, the saturation is done with an iron tracer (57Fe) and the application of pattern deconvolution analysis permits the direct quantification of the Tf saturation, the serum iron and the unsaturated iron-binding capacity. These strategies are validated by using a reference serum certified for total Tf and tested also in serum samples from individuals suffering from hemochromatosis and Fe-supplemented patients. The results obtained for all the parameters related to Fe status were in good agreement with those obtained by clinical tests. The use of stable isotope labelling in connection with the on-line coupling of fast protein liquid chromatography (FPLC) to ICP-MS allows the accurate determination of several parameters of great clinical relevance in iron homeostasis by means of two independent chromatographic runs. The main advantage of the proposed methodology is the number of parameters that can be simultaneously obtained.