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2.  Serum levels of the hepcidin-20 isoform in a large general population: The Val Borbera study☆ 
Journal of Proteomics  2012;76(5):28-35.
Hepcidin, a 25 amino-acid liver hormone, has recently emerged as the key regulator of iron homeostasis. Proteomic studies in limited number of subjects have shown that biological fluids can also contain truncated isoforms, whose role remains to be elucidated. We report, for the first time, data about serum levels of the hepcidin-20 isoform (hep-20) in a general population, taking advantage of the Val Borbera (VB) study where hepcidin-25 (hep-25) was measured by SELDI-TOF-MS. Detectable amount of hep-20 were found in sera from 854 out of 1577 subjects (54.2%), and its levels were about 14% of hep-25 levels. A small fraction of subjects (n = 30, 1.9%) had detectable hep-20 but undetectable hep-25. In multivariate regression models, significant predictors of hep-20 were hep-25 and age in males, and hep-25, age, serum ferritin and body mass index in females. Of note, the hep-25:hep-20 ratio was not constant in the VB population, but increased progressively with increasing ferritin levels. This is not consistent with the simplistic view of hep-20 as a mere catabolic byproduct of hep-25. Although a possible active regulation of hep-20 production needs further confirmation, our results may also have implications for immunoassays for serum hepcidin based on antibodies lacking specificity for hep-25.
This article is part of a Special Issue entitled: Integrated omics.
Graphical abstract
► Hepcidin, a 25 amino acid hormone, is the key regulator of iron metabolism. ► We measured, for the first time, serum hepcidin-20 at population level by SELDI-TOF-MS. ► Detectable amount of hepcidin 20 were found in more than half of 1577 individuals. ► The Hep25:hep20 ratio was not constant but increased with increasing iron stores. ► Our results point toward a possible active regulation of hepcidin-20 production.
PMCID: PMC3509339  PMID: 22951294
BMI, body mass index; CRP, C-reactive protein; Hep-20, Hepcidin-20; Hep-24, Hepcidin-24; Hep-25, Hepcidin-25; PTH, parathyroid hormone; VB, Val Borbera; Iron metabolism; Hepcidin; Ferritin; SELDI-TOF-MS
3.  Increased Serum Hepcidin Levels in Subjects with the Metabolic Syndrome: A Population Study 
PLoS ONE  2012;7(10):e48250.
The recent discovery of hepcidin, the key iron regulatory hormone, has changed our view of iron metabolism, which in turn is long known to be linked with insulin resistant states, including type 2 diabetes mellitus and the Metabolic Syndrome (MetS). Serum ferritin levels are often elevated in MetS (Dysmetabolic hyperferritinemia - DHF), and are sometimes associated with a true mild-to-moderate hepatic iron overload (dysmetabolic iron overload syndrome - DIOS). However, the pathophysiological link between iron and MetS remains unclear. This study was aimed to investigate, for the first time, the relationship between MetS and hepcidin at population level. We measured serum hepcidin levels by Mass Spectrometry in 1,391 subjects from the Val Borbera population, and evaluated their relationship with classical MetS features. Hepcidin levels increased significantly and linearly with increasing number of MetS features, paralleling the trend of serum ferritin. In multivariate models adjusted for relevant variables including age, C-Reactive Protein, and the HFE C282Y mutation, ferritin was the only significant independent predictor of hepcidin in males, while in females MetS was also independently associated with hepcidin. Overall, these data indicate that the fundamental iron regulatory feedback is preserved in MetS, i.e. that hepcidin tends to progressively increase in response to the increase of iron stores. Due to recently discovered pleiotropic effects of hepcidin, this may worsen insulin resistance and contribute to the cardiovascular complications of MetS.
PMCID: PMC3483177  PMID: 23144745
4.  Reduced serum hepcidin levels in patients with chronic hepatitis C 
Journal of hepatology  2009;51(5):845-852.
Patients with chronic hepatitis C (CHC) often have increased liver iron, a condition associated with reduced sustained response to antiviral therapy, more rapid progression to cirrhosis, and development of hepatocellular carcinoma. The hepatic hormone hepcidin is the major regulator of iron metabolism and inhibits iron absorption and recycling from erythrophagocytosis. Hepcidin decrease is a possible pathophysiological mechanism of iron overload in CHC, but studies in humans have been hampered so far by the lack of reliable quantitative assays for the 25-amino acid bioactive peptide in serum (s-hepcidin).
Using a recently validated immunoassay, we measured s-hepcidin levels in 81 untreated CHC patients and 57 controls with rigorous definition of normal iron status. All CHC patients underwent liver biopsy with histological iron score.
S-hepcidin was significantly lower in CHC patients than in controls (geometric means with 95% confidence intervals: 33.7, 21.5–52.9 vs. 90.9, 76.1–108.4 ng/mL, respectively; p < 0.001). In CHC patients, s-hepcidin significantly correlated with serum ferritin and histological total iron score, but not with s-interleukin-6. After stratification for ferritin quartiles, s-hepcidin increased significantly across quartiles in both controls and CHC patients (chi for trend, p < 0.001). However, in CHC patients, s-hepcidin was significantly lower than in controls for each corresponding quartile (analysis of variance, p < 0.001).
These results, together with very recent studies in animal and cellular models, indicate that although hepcidin regulation by iron stores is maintained in CHC, the suppression of this hormone by hepatitis C virus is likely an important factor in liver iron accumulation in this condition.
PMCID: PMC2761995  PMID: 19729219
Chronic hepatitis C; Hemochromatosis; Hepcidin; Iron overload; Ferritin
5.  Evaluation of Hepcidin Isoforms in Hemodialysis Patients by a Proteomic Approach Based on SELDI-TOF MS 
The hepatic iron regulator hormone hepcidin consists, in its mature form, of 25 amino acids, but two other isoforms, hepcidin-20 and hepcidin-22, have been reported, whose biological meaning remains poorly understood. We evaluated hepcidin isoforms in sera from 57 control and 54 chronic haemodialysis patients using a quantitative proteomic approach based on SELDI-TOF-MS. Patients had elevated serum levels of both hepcidin-25 and hepcidin-20 as compared to controls (geometric means: 7.52 versus 4.69 nM, and 4.06 versus 1.76 nM, resp., P < .05 for both). The clearance effects of a single dialysis session by different dialysis techniques and membranes were also investigated, showing an average reduction by 51.3% ± 29.2% for hepcidin-25 and 34.2% ± 28.4% for hepcidin-20 but only minor differences among the different dialysis modalities. Measurement of hepcidin isoforms through MS-based techniques can be a useful tool for better understanding of their biological role in hemodialysis patients and other clinical conditions.
PMCID: PMC2857619  PMID: 20414466
6.  Clinical, Pathological, and Molecular Correlates in Ferroportin Disease. A Study Of Two Novel Mutations 
Journal of hepatology  2008;49(4):664-671.
Clinico-pathological manifestations of Ferroportin (Fpn) Disease (FD) are heterogeneous, with some patients presenting with iron overload predominantly in macrophages (“M” phenotype), others predominantly in hepatocytes (“H” phenotype). This appears to reflect functional heterogeneity of Fpn mutants, with loss-of-function generally resulting in the M type.
Two unrelated probands with “non-HFE” hemochromatosis were screened for Fpn mutations. Mutants were functionally characterized by immunofluorescence microscopy, evaluation of their ability to bind hepcidin and export iron, and by expressing them in zebrafish.
Two novel Fpn mutations were identified: I152F in patient-1, presenting with typical M phenotype; and L233P in patient-2, presenting with ambiguous features (massive overload in both macrophages and hepatocytes). Molecular studies suggested loss of function in both cases. The I152F, normally localized on cell membrane and internalized by hepcidin, showed a unique “primary” deficit of iron export capability. The L233P did not appropriately traffic to cell surface. Loss of function was confirmed by expressing both mutants in vivo in zebrafish, resulting in iron limited erythropoiesis. Clinical manifestations were likely enhanced in both patients by nongenetic factors (HCV, alcohol).
The combination of careful review of clinico-pathological data with molecular studies can yield compelling explanations for phenotype heterogeneity in FD.
PMCID: PMC2605969  PMID: 18713659
iron overload; ferroportin; hemochromatosis; hepcidin; zebrafish
7.  High resolution preparation of monocyte-derived macrophages (MDM) protein fractions for clinical proteomics 
Proteome Science  2009;7:4.
Macrophages are involved in a number of key physiological processes and complex responses such as inflammatory, immunological, infectious diseases and iron homeostasis. These cells are specialised for iron storage and recycling from senescent erythrocytes so they play a central role in the fine tuning of iron balancing and distribution. The comprehension of the many physiological responses of macrophages implies the study of the related molecular events. To this regard, proteomic analysis, is one of the most powerful tools for the elucidation of the molecular mechanisms, in terms of changes in protein expression levels.
Our aim was to optimize a protocol for protein fractionation and high resolution mapping using human macrophages for clinical studies. We exploited a fractionation protocol based on the neutral detergent Triton X-114. The 2D maps of the fractions obtained showed high resolution and a good level of purity. Western immunoblotting and mass spectrometry (MS/MS analysis) indicated no fraction cross contamination. On 2D-PAGE mini gels (7 × 8 cm) we could count more than five hundred protein spots, substantially increasing the resolution and the number of detectable proteins for the macrophage proteome. The fractions were also evaluated, with preliminary experiments, using Surface Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI-TOF-MS).
This relatively simple method allows deep investigation into macrophages proteomics producing discrete and accurate protein fractions, especially membrane-associated and integral proteins. The adapted protocol seems highly suitable for further studies of clinical proteomics, especially for the elucidation of the molecular mechanisms controlling iron homeostasis in normal and disease conditions.
PMCID: PMC2649903  PMID: 19228399

Results 1-7 (7)