Paola Trombini

Increased serum ferritin is common in men with essential hypertension

Objectives Insulin-resistance-associated hepatic iron overload syndrome (IRHIO) is characterized by high serum ferritin and presence of metabolic alterations that are part of insulin-resistance syndrome (IRS). Thus, clinical conditions characterized by a high prevalence of IRS may also be characterized by a high prevalence of IRHIO. Design and methods We studied 88 consecutive patients with essential hypertension, 62 patients with IRHIO and 102 healthy normotensive controls. Hemochromatosis, other conditions able to induce secondary iron overload or serum ferritin increase unrelated to body iron stores were excluded. Iron indices, metabolic profiles and hepatic tests in hypertensive with or without increased serum ferritin and in IRHIO with and without hypertension were studied. Metabolic variables, serum iron indices, liver function tests and hepatic ultrasound data were analysed. Data were compared by non-parametric tests. Results In men with hypertension, increased serum ferritin was more frequent than in controls (21 versus 0%, P = 0.001). Hypertensive men with increased serum ferritin had more frequent and pronounced metabolic alterations than those with normal serum ferritin, the metabolic abnormalities and serum ferritin being frequently positively correlated. In hypertensive men with increased serum ferritin, metabolic and iron data were similar to those of IRHIO patients with hypertension. Conclusions In males, hypertension is characterized by a higher prevalence of increased iron stores and metabolic abnormalities that are part of the IRHIO syndrome. This finding may have clinical implications due to the increased risk of IRHIO patients to develop hepatic cirrhosis and also for the role of iron in early atherogenesis.

Hepcidin and iron-related gene expression in subjects with dysmetabolic hepatic iron overload

BACKGROUND/AIMS Many patients with hepatic iron overload do not have identifiable mutations and often present with metabolic disorders and hepatic steatosis. Since the pathophysiology of Dysmetabolic Hepatic Iron Overload (DHIO) is still obscure, the aim of this study was to evaluate, in these patients, possible alterations in iron-related molecule expression. METHODS Iron-related gene mRNA levels were determined by quantitative-PCR in liver biopsies of subjects with NAFLD without iron overload and patients with HFE-hemochromatosis, beta-thalassemia major and DHIO. Urinary hepcidin was measured by immunoblotting. RESULTS No alterations in mRNA expression of either iron transporters or exporters were found in DHIO. mRNA and urinary hepcidin levels normalized for the amount of iron overload showed a significantly lower ratio than in controls, although not as low as in hemochromatosis or beta-thalassemia. Differently from what observed in hemochromatosis, hepcidin mRNA did not correlate with urinary hepcidin. CONCLUSIONS Patients with DHIO show appropriate regulation of mRNAs encoding proteins involved in iron uptake and efflux but dysregulation of hepcidin production. The relatively elevated urinary hepcidin can explain the iron phenotype in DHIO (more macrophage iron retention and low/normal transferrin saturation).

α1-antitrypsin mutations in NAFLD: High prevalence and association with altered iron metabolism but not with liver damage

Hyperferritinemia, a common feature of nonalcoholic fatty liver disease (NAFLD), has been associated with steatohepatitis and fibrosis. Heterozygosity for α1‐antitrypsin (AAT) mutations is a cofactor of liver damage, and AAT influences inflammation and iron metabolism. This study evaluated the prevalence of the common AAT PiS/PiZ mutants in 353 patients with NAFLD, 195 of whom had hyperferritinemia, versus 114 matched controls and their influence on iron metabolism and the severity of liver damage in the 212 patients submitted to biopsy. PiS and PiZ alleles were searched for by restriction analysis. Thirty‐eight patients (10.8%) carried non‐MM genotypes versus 4/114 (3.5%) controls (P = .02). Patients carrying AAT mutations had higher ferritin (573 [454–966] vs. 348 [201–648]; P = .001) with similar transferrin saturation. The difference was more evident in males (P < .0001) and significant in patients not carrying HFE genotypes associated with iron overload (P = .015). The prevalence of non‐MM genotypes was higher in patients with hyperferritinemia than in those without (28/195, 14% vs. 10/158, 6%, P = .016), and AAT mutations were associated with higher prevalence of sinusoidal siderosis (17/27, 63% vs. 70/180, 39%; P = .02), and sinusoidal/total iron score (46.3 ± 38% vs. 25.1 ± 35%, P = .01). Although ferritin was independently associated with fibrosis (P = .047), AAT mutations favoring sinusoidal iron deposition did not affect liver damage. In conclusion, AAT mutations are associated with hyperferritinemia and sinusoidal iron accumulation, but not with more severe liver damage in NAFLD. (HEPATOLOGY 2006;44:857–864.)

Measurement of urinary hepcidin levels by SELDI-TOF-MS in HFE-hemochromatosis

INTRODUCTION Insufficient production of hepcidin, the master regulator of iron metabolism, is recognized as the key pathogenetic feature of HFE-related hereditary hemochromatosis (HH). There is a growing interest in measuring the hepcidin levels, which may improve the diagnosis, prognostic evaluation and clinical management of HH. Nevertheless, few investigative tools are available: an immunodot method for urinary hepcidin developed by a single centre (UCLA), not yet ready for large-scale diffusion, and mass spectrometry (MS) based assays, such as surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF-MS). The latter is well suited to small peptides like hepcidin, and can rapidly analyze crude samples with high throughput. This study measured urinary hepcidin levels by SELDI-TOF-MS in a large group of HH patients at diagnosis and during treatment, including both C282Y homozygous and C282Y/H63D compound heterozygotes. METHODS We used a protocol based on PBSIIc mass spectrometer and Normal Phase chips. Urinary samples from 30 control subjects were compared to those obtained from 80 HH patients (57 C282Y homozygotes, 23 C282Y/H63D compound heterozygotes). Eighteen C282Y homozygotes and 11 C282Y/H63D compound heterozygotes were analyzed at diagnosis, the remainder during maintenance phlebotomy. RESULTS C282Y homozygotes either at diagnosis, or after phlebotomy had significantly lower urinary hepcidin levels than controls (P<0.05). C282Y/H63D compound heterozygotes had hepcidin levels at diagnosis higher than controls, while the hepcidin/ferritin ratio was significantly decreased (P<0.001) suggesting inadequate hepcidin production. After phlebotomy, mean hepcidin levels in the compound heterozygotes were significantly lower than in controls (P<0.001). Samples from 12 randomly selected control subjects were sent to UCLA for duplicate measurement by the immunodot method, yielding a significant correlation (rho=0.64; P=0.024). CONCLUSIONS This study supports the use of SELDI-TOF-MS for measuring hepcidin levels in research and clinical applications. Our results in phlebotomized patients suggest that the depletion of iron stores may further exacerbate the HFE-related hepcidin defect.

A time course of hepcidin response to iron challenge in patients with HFE and TFR2 hemochromatosis

Background Inadequate hepcidin production leads to iron overload in nearly all types of hemochromatosis. We explored the acute response of hepcidin to iron challenge in 25 patients with HFE-hemochromatosis, in two with TFR2-hemochromatosis and in 13 controls. Sixteen patients (10 C282Y/C282Y homozygotes, 6 C282Y/H63D compound heterozygotes) had increased iron stores, while nine (6 C282Y/C282Y homozygotes, 3 C282Y/H63D compound heterozygotes) were studied after phlebotomy-induced normalization of iron stores. Design and Methods We analyzed serum iron, transferrin saturation, and serum hepcidin by both enzyme-linked immunosorbent assay and mass-spectrometry at baseline, and 4, 8, 12 and 24 hours after a single 65-mg dose of oral iron. Results Serum iron and transferrin saturation significantly increased at 4 hours and returned to baseline values at 8–12 hours in all groups, except in the iron-normalized patients who showed the highest and longest increase of both parameters. The level of hepcidin increased significantly at 4 hours and returned to baseline at 24 hours in controls and in the C282Y/H63D compound heterozygotes at diagnosis. The hepcidin response was smaller in C282Y-homozygotes than in controls, barely detectable in the patients with iron-depleted HFE-hemochromatosis and absent in those with TFR2-hemochromatosis. Conclusions Our results are consistent with a scenario in which TFR2 plays a prominent and HFE a contributory role in the hepcidin response to a dose of oral iron. In iron-normalized patients with HFE hemochromatosis, both the low baseline hepcidin level and the weak response to iron contribute to hyperabsorption of iron.

Effects of venesections and restricted diet in patients with the insulin-resistance hepatic iron overload syndrome.

Abstract: Goal: We evaluated the effect of venesections and restricted diet on iron and metabolic indices and liver function tests in patients with insulin‐resistance hepatic iron overload (IR‐HIO).

Effects of plasma transfusion on hepcidin production in human congenital hypotransferrinemia

Hepcidin is the key regulator of systemic iron homeostasis. We describe the modulation of hepcidin production induced by plasma transfusions in a patient with congenital hypotransferrinemia that offers a unique model in which to study the mechanism of hepcidin regulation by iron and erythropoiesis. Urinary hepcidin increased from zero at baseline, when hemoglobin and serum transferrin was low, to a maximum of 98 ng/mg creatinine on day 60, and subsequently decreased. Time-course of urinary hepcidin and serum transferrin concentration suggests that hepcidin production is regulated by the combination of marrow iron requirements and iron supply by transferrin.

Expression of hepcidin and other iron-related genes in type 3 hemochromatosis due to a novel mutation in transferrin receptor-2

This brief report describes the decreased hepatic and urinary expression of hepcidin in type 3 hemochromatosis. Transferrin receptor-2 (TFR2) regulates hepatic hepcidin secretion and when mutated causes type-3 hemochromatosis. No functional study is available in humans. We studied a 47 year-old woman with hemochromatosis. TFR2 DNA and its hepatic transcript were directly sequenced. Hepatic expression of hepcidin and other iron-related genes were measured by qRT-PCR. Urinary hepcidin was measured at baseline and after an oral iron challenge (ferrous sulfate, 65 mg) by SELDI-TOF-MS. A novel homozygous TFR2 mutation was identified in the splicing donor site of intron 4 (c.614+4 A>G) causing exon 4 skipping. Hepcidin and hemojuvelin expression were markedly reduced. Urinary hepcidin was lower than normal and further decreased after iron challenge. This is the first description of iron-related gene expression profiles in a TFR2 mutated patient. The decreased hepatic and urinary expression of hepcidin and lack of acute response to iron challenge confirms the primary role of TFR2 in iron homeostasis.

IRON METABOLISM IN THALASSEMIA AND SICKLE CELL DISEASE

There are two main mechanisms by which iron overload develops in thalassemias: increased iron absorption due to ineffective erythropoiesis and blood transfusions. In nontransfused patients with severe thalassemia, abnormal dietary iron absorption increases body iron burden between 2 and 5 g per year. If regular transfusions are required, this doubles the rate of iron accumulation leading to earlier massive iron overload and iron-related damage. Iron metabolism largely differs between thalassemias and sickle cell disease, but chronic transfusion therapy partially normalize many of the disparities between the diseases, making iron overload an important issue to be considered in the management of patients with sickle cell disease too. The present review summarizes the actual knowledge on the regulatory pathways of iron homeostasis. In particular, the data presented indicate the inextricably link between erythropoiesis and iron metabolism and the key role of hepcidin in coordinating iron procurement according to erythropoietic requirement. The role of erythropoietin, hypoxia, erythroid-dependent soluble factors and iron in regulating hepcidin transcription are discussed as well as differences and similarities in iron homeostasis between thalassemia syndromes and sickle cell disease.

Insulin resistance influences iron metabolism and hepatic steatosis in type II diabetes

Dear Sir: We read with interest the article by Mendler et al.1 who described the association of hepatic iron overload, steatosis, and presence of one or more components of the insulin resistance syndrome. They suggested a role for insulin resistance in the development of hepatic iron overload, steatosis, and increased serum ferritin levels, although the mechanisms whereby insulin resistance would induce alterations in iron metabolism remain to be elucidated. Insulin resistance is very common among subjects with metabolic disorders, but its prevalence varies substantially among clinical conditions.2 Type II diabetes is one of the metabolic conditions with the higher rate of insulin resistance and is frequently associated with increased serum ferritin levels.2,3 We have evaluated iron indices in patients with type II diabetes before and after improvement of metabolic control to provide some insights into the relationship between iron and metabolic disorders. From 1989 to 1990, in our Department of Medicine, 10 outpatients with poorly controlled diabetes were enrolled to evaluate the incidence of hepatic steatosis and its reversibility after correction of the glycemic control. Results of this study have not been published. Chronic alcoholism, acute and chronic liver diseases, malignancies, and inflammatory and iron overload disorders were excluded. After baseline evaluation, patients received a strict diet regimen for 12 months in addition to antidiabetic therapy. The study schedule included monthly measurements of mean blood glucose, fructosamine, glycosylated hemoglobin, cholesterol, and triglyceride levels; assessment of uricemia and liver function tests for 12 months; and hepatic ultrasound examination and liver biopsy at baseline and after 12 months. Small aliquots of the serum samples were collected and stored at 280°. The study was approved by the Hospital’s ethical committee. All patients gave their informed consent to the study. Four denied liver biopsy. We then retrospectively evaluated serum iron, transferrin, and ferritin levels of the patients on each sample and hepatic iron concentration (HIC) at baseline and at the end of study. HIC was determined by atomic absorption spectrophotometry (Perkin-Elmer S2380; Norwalk, CT) in deparaffinized specimens. Table 1 shows the main data of the patients at baseline and during the study. Significant improvement of glycemic control was obtained in each patient. At baseline, serum ferritin levels were significantly higher than those in 20 normal age-matched controls (223 6 139 vs. 122 6 66 μg/L; P , 0.01) and significantly decreased at the end of the study. Serum ferritin correlated with triglyceride at baseline (r 5 0.69; P 5 0.026) and with HIC only at the end of the study (r 5 0.87; P 5 0.024). Liver function test results were normal at baseline and did not change during the study. Hepatic steatosis, as defined by ultrasound examination and histology, was present in 80% of the patients at baseline and in 25% after 12 months; it was mild and often associated with very mild necroinflammatory activity. In 5 patients, HIC decreased after 12 months, but did not change in 1 patient. The results suggest the existence of a relationship between glucose metabolism, fatty liver, serum ferritin, and hepatic iron. Hyperinsulinemia, the main manifestation of insulin resistance, favors the accumulation of free fatty acid in the liver and increases the risk of steatosis.4 High serum ferritin levels are common in patients with metabolic disorders and nonalcoholic steatosis or steatohepatitis, and some of them also have increased HIC.4,5 In our patients, serum ferritin levels at baseline were increased to a degree disproportionate to liver iron stores because they significantly decreased after metabolic improvement when the expected correlation with HIC finally appeared. Also, the mild but significant decrease of HIC at the end of the study suggests a possible influence of diabetic-associated metabolic alterations on hepatocellular iron metabolism. The improvement of the glycemic control observed in the patients can be ascribed to increased insulin action in the liver and in the peripheral tissues. The concomitant improvement of steatosis and the decrease of serum ferritin and hepatic iron levels suggest that these alterations are distinct consequences of a common factor (probably insulin resistance in this case) and that they are at least partially reversible by the improvement of metabolic control. Hypertriglyceridemia is another condition typically associated with the insulin resistance syndrome,2 and the correlation observed in our patients at baseline between serum ferritin and triglyceride further supports the hypothesis of a relationship between insulin resistance and alterations in iron metabolism. Table 1. Iron and Metabolic Indices in 10 Patients With Poorly Controlled Type II Diabetes at Baseline and During Treatment