Hepcidin is a useful biomarker to evaluate hyperferritinemia associated with metabolic syndrome

MARIANA R. RAUBER DIOGO A. PILGER DAIANE K. CECCONELLO FREDERICO S. FALCETTA NATÁLIA A. MARCONDES GUSTAVO A.M. FAULHABER About the authors

Abstract

Abstract: Investigation of hyperferritinemia in metabolic syndrome patients represents a diagnostic challenge, but it is essential for the identification of individuals with iron overload. Hepcidin negatively regulates iron absorption and release. An increase in hepcidin occurs when iron levels are sufficient or in inflammatory states, conditions often associated with hyperferritinemia. Hemochromatosis causes hyperferritinemia due to iron overload, but frequently has low hepcidin levels. Our aim was to evaluate biochemical and molecular parameters related to iron metabolism in patients with metabolic syndrome. We evaluated 94 patients with metabolic syndrome according to the International Diabetes Federation criteria in a cross-sectional study. Anthropometric data and diagnostic criteria for metabolic syndrome, iron dosage, ferritin, transferrin saturation, hepcidin, and the C282Y and H63D mutations in the HFE hemochromatosis gene were evaluated. Prevalence of hyperferritinemia in the study population was 27.7% and was higher in males (46.2%) than in females (14.5%). Increase in transferrin saturation correlated with mutations in the hemochromatosis gene. Hyperferritinemia was associated to transferrin saturation and hepcidin after logistic regression analysis. In conclusion, hyperferritinemia is a frequent finding in metabolic syndrome patients, most frequently in men; and hepcidin assessment can be useful for the investigation of ferritin increase in those subjects.

Key words
diagnostic screening; ferritin; hepcidin; iron; metabolic syndrome


INTRODUCTION

Metabolic syndrome (MS) affects approximately 20 to 25% of the adult population and is characterized by factors that increase the risk for cardiovascular disease and diabetes, such as abdominal obesity, hyperglycemia associated with insulin resistance, dyslipidemia and hypertension (ZimmetZIMMET P, ALBERTI KG and SERRANO RÍOS M. 2005. A new international diabetes federation worldwide definition of the metabolic syndrome: the rationale and the results. Rev Esp Cardiol 58: 1371-1376. et al. 2005, Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults 2001, WorldWORLD HEALTH ORGANIZATION. DEPARTMENT OF NONCOMMUNICABLE DISEASE SURVEILLANCE. 1999. Definition, diagnosis, and classification of diabetes mellitus and its complications : report of a WHO consultation. Part 1: diagnosis and classification of diabetes mellitus. Geneva: World Health Organization, Dept. of Noncommunicable Disease Surveillance. Avaliable at: https://apps.who.int/iris/bitstream/handle/10665/66040/WHO_NCD_NCS_99.2.pdf;jsessionid=3E917F17F40B31227F6B98E4BDBC9B94?sequence=1
https://apps.who.int/iris/bitstream/hand...
Health Organization 1999, de Carvalho Vidigal et al. 2013DE CARVALHO VIDIGAL F, BRESSAN J, BABIO N and SALAS-SALVADÓ J. 2013. Prevalence of metabolic syndrome in Brazilian adults: a systematic review. BMC Public Health 13: 1198.). Among laboratorial findings, hyperferritinemia is present in roughly 15% of cases (ChenCHEN LY, CHANG SD, SREENIVASAN GM, TSANG PW, BROADY RC, LI CH and ZYPCHEN LN. 2011. Dysmetabolic hyperferritinemia is associated with normal transferrin saturation, mild hepatic iron overload, and elevated hepcidin. Ann Hematol 90: 139-143. et al. 2011).

Hyperferritinemia should be carefully assessed in patients with MS, since only a minority of cases is due to true iron overload (LorcerieLORCERIE B, AUDIA S, SAMSON M, MILLIÈRE A, FALVO N, LEGUY-SEGUIN V, BERTHIER S and BONNOTTE B. 2017. Diagnosis of hyperferritinemia in routine clinical practice. Presse Med 46: e329-e338. et al. 2017, GootGOOT K, HAZELDINE S, BENTLEY P, OLYNYK J and CRAWFORD D. 2012. Elevated serum ferritin - what should GPs know? Aust Fam Physician 41: 945-949. et al. 2012). Other conditions can cause an increase of this protein, such as inflammatory processes, cancer, infections, alcoholism, nonalcoholic steatohepatitis (NASH), and neurodegenerative and chronic liver diseases (Lorcerie et al. 2017, Goot et al. 2012, ChangCHANG JS, LIN SM, HUANG TC, CHAO JC, CHEN YC, PAN WH and BAI CH. 2013. Serum ferritin and risk of the metabolic syndrome: a population-based study. Asia Pac J Clin Nutr 22: 400-407. et al. 2013, VaismanVAISMAN B, MEYRON-HOLTZ EG, FIBACH E, KRICHEVSKY AM and KONIJN AM. 2000. Ferritin expression in maturing normal human erythroid precursors. Br J Haematol 110: 394-401. et al. 2000, ColliCOLLI ML, GROSS JL and CANANI LH. 2011. Mutation H63D in the HFE gene confers risk for the development of type 2 diabetes mellitus but not for chronic complications. J Diabetes Complications 25: 25-30. et al. 2011).

Hepcidin acts as a negative regulator of iron absorption and release, it binds to the iron transporter ferroportin causing its internalization and proteolysis (PrenticePRENTICE AM. 2017. Clinical Implications of New Insights into Hepcidin-Mediated Regulation of Iron Absorption and Metabolism. Ann Nutr Metab 71(Suppl 3): 40-48. 2017). Erythropoiesis promotes a decrease in hepcidin levels, while hepcidin circulating levels can be increased in response to plasma and liver iron, and also by an inflammatory state. Consequently, several clinical conditions can influence hepcidin regulation, such as anemia, iron intake, liver disease, hemochromatosis-related mutations, inflammatory stimuli and infections, among others (GirelliGIRELLI D, NEMETH E and SWINKELS DW. 2016. Hepcidin in the diagnosis of iron disorders. Blood 127: 2809-2813. et al. 2016, Prentice 2017).

Hepcidin could be an important marker to differentiate hyperferritinemia associated with MS, and hyperferritinemia caused by hereditary hemochromatosis, since hemochromatosis causes hyperferritinemia due to iron overload, but frequently has low hepcidin levels (HareHARE DJ. 2017. Hepcidin: a real-time biomarker of iron need. Metallomics 9: 606-618. 2017, DongiovanniDONGIOVANNI P, FRACANZANI AL, FARGION S and VALENTI L. 2011. Iron in fatty liver and in the metabolic syndrome: a promising therapeutic target. J Hepatol 55: 920-932. et al. 2011, TrombiniTROMBINI P, PAOLINI V, PELUCCHI S, MARIANI R, NEMETH E, GANZ T and PIPERNO A. 2011. Hepcidin response to acute iron intake and chronic iron loading in dysmetabolic iron overload syndrome. Liver Int 31: 994-1000. et al. 2011, CardosoCARDOSO EM, STÅL P, HAGEN K, CABEDA JM, ESIN S, DE SOUSA M and HULTCRANTZ R. 1998. HFE mutations in patients with hereditary haemochromatosis in Sweden. J Intern Med 243: 203-208. et al. 1998). The aim of this study was to evaluate biochemical and molecular parameters related to iron metabolism in patients with MS, using hepcidin as a potential biomarker to help identifying patients with hyperferritinemia and iron accumulation secondary to hemochromatosis.

MATERIALS AND METHODS

STUDY DESIGN

This was a cross-sectional study conducted from May, 2013, to November, 2013. A total of 94 patients from the Internal Medicine outpatient program of the Hospital de Clínicas de Porto Alegre were enrolled.

ETHICAL ASPECTS

This study was conducted after approval of the Research Ethics Committee of the Hospital de Clínicas de Porto Alegre (CAAE 11796912.1.0000.5327 and 05770612.5.0000.5327) and participants were included after written informed consent. This study was conducted in accordance with the Declaration of Helsinki and current laws in Brazil.

PATIENT POPULATION

All patients were ≥18 years old, with MS according to criteria of the International Diabetes Federation (IDF) (Zimmet et al. 2005). The exclusion criteria were daily intake of ethanol ≥60 g for men and ≥30 g for women, diagnosis of chronic viral hepatitis, previous diagnosis of hereditary hemochromatosis or more than two blood donations in the preceding year. Patients had weight, height and waist circumference measured and a peripheral blood sample was harvested.

BIOCHEMICAL ANALYSIS

Serum concentrations of iron, ferritin and transferrin saturation were measured by routine methods with Advia 1800® and Advia Centaur (Siemens Diagnostics, Deerfield, USA). The reference values for ferritin were 10 to 291 ng/mL for women and 22 to 322 ng/mL for men, values above the upper limit were considered as hyperferritinemia. Serum glucose, cholesterol, HDL cholesterol and triglycerides values were obtained from medical records.

DETERMINATION OF HEPCIDIN

All serum samples to determine hepcidin levels were stored at -80°C, until measurements in the same assay. Hepcidin was assessed with enzyme-linked immunosorbent assay (ELISA) method through Hepcidin 25 (bioactive) ELISA kit® (DRG Diagnostics, Marburg, Germany), according to the manufacturer instructions (sensitivity of 0.35 ng/mL and linearity of 0.35 to 80 ng/mL).

MOLECULAR ANALYSIS FOR THE PRESENCE OF C282Y AND H63D MUTATIONS IN THE HFE GENE

Immediately after blood collection with EDTA anticoagulant, its DNA was extracted through commercial kit PureLink Genomic DNA Mini Kit® (Invitrogen, Carlsbad, USA) and subsequently the nucleic acid was stored at -80oC. To evaluate the presence of C282Y and H63D mutations in the HFE gene, it was amplified as described by Cardoso et al. (1998). The amplifications were performed on a Veriti thermocycler (Applied Biosystems, Foster City, USA) with annealing temperature of 63°C for C282Y and 50°C for H63D. The products were analyzed by electrophoresis in 1.5% agarose gel impregnated with GelRed®. Then, the amplified products were digested using restriction enzymes (C282Y by SnaBI at 37°C for 1h30min and H63D by BclI for 1h30min at 50°C), in a protocol adapted from Cardoso et al. (1998). Fragment analysis was performed by electrophoresis in 3.0% agarose gel impregnated with GelRed®. Heterozygote and homozygote samples identified by direct DNA sequencing were used as positive controls.

STATISTICAL ANALYSIS

Results were expressed as mean and standard deviation when parametric or medians and quartiles intervals when nonparametric. Spearman and Pearson correlations were done among all quantitative variables. Patients with normal ferritin and hyperferritinemia were compared by Student’s T test or Mann-Whitney test, when applicable. Data analysis was carried out with SPSS 18.0® (Chicago, EUA). The statistical significance level was set at P<0.05.

RESULTS

Most patients consisted of women with a mean age of 62.2 years and BMI of 34.7 and were classified on average as obese. The other parameters were in accordance with the criteria for diagnosis of MS, according to the IDF (Zimmet et al. 2005). Table I summarizes patient’s demographics, clinical and biochemical parameters. None of the included patients were receiving iron supplementation.

TABLE I
Patients demographics (n=94).

There was no patient with homozygosis for C282Y, while 14.9% were heterozygous and 85.1% did not have the mutation. Only 2 (2.1%) patients were homozygous for H63D mutation, whereas 24.5% were heterozygous and 73.4% did not have the mutation. One patient (1.6%) was heterozygous for both analyzed mutations. Transferrin saturation was correlated with the presence of heterozygosity in H63D (P=0.031/ρ=0.225).

Hyperferritinemia prevalence in the study population was 27.7%. Table II shows the correlations between ferritin levels and clinical aspects and laboratory data, there was a significant positive correlation of ferritin with cholesterol levels.

TABLE II
Correlation between ferritin levels and clinical aspects and laboratory data.

Subsequently, patients were divided into two distinct groups: patients with normal ferritin levels and patients with hyperferritinemia (Table III). Among the analyzed diagnostic criteria for MS, only the dosage of triglycerides and HDL cholesterol showed differences between the groups in univariate analysis. Male gender, cholesterol levels, transferrin saturation and hepcidin levels also differed between patients with normal ferritin levels and hyperferritinemia. Presence of high blood glucose levels, BMI and waist circumference values were similar among groups, as well as the presence or absence of C282Y and H63D mutations. After logistic regression analysis, only transferrin saturation and hepcidin levels remained significantly different between the analyzed groups.

TABLE III
Hyperferritinemia and associated factors.

DISCUSSION

Approximately a quarter of the world population has MS, and although only 15% of patients with MS show hyperferritinemia, a large number of patients have this condition (Zimmet et al. 2005, Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults 2001EXPERT PANEL ON DETECTION, EVALUATION, and TREATMENT OF HIGH BLOOD CHOLESTEROL IN ADULTS. 2001. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA 285: 2486-2497., World Health Organization 1999, Chen et al. 2011). Despite the large number of female included, our results indicate the presence of hyperferritinemia in 27.7% of the study population, which are superior to those already described by Chen et al. (2011).

Hyperferritinemia was more common among men, which could be related to a greater prevalence of NASH among this gender (WilliamsWILLIAMS CD, STENGEL J, ASIKE MI, TORRES DM, SHAW J, CONTRERAS M, LANDT CL and HARRISON SA. 2011. Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology 140: 124-131. et al. 2011, BarrosBARROS RK, COTRIM HP, DALTRO CH and OLIVEIRA YA. 2017. Hyperferritinemia in patients with nonalcoholic fatty liver disease. Rev Assoc Med Bras (1992) 63: 284-289. et al. 2017). Women also have lower iron storage due to menstrual loss, which may reflect a lower serum ferritin. This is in agreement with other studies that considered male gender as a risk factor for iron accumulation (Dongiovanni et al. 2011).

Hepcidin was present in higher levels in patients with hyperferritinemia, indicating that synthesis of this hormone is not impaired and is responsive to iron storage (DaherDAHER R and KARIM Z. 2017. Iron metabolism: State of the art. Transfus Clin Biol 24: 115-119. and Karim 2017). Some studies showed the same hepcidin increase pattern in patients with or without MS associated hyperferritinemia when compared with patients with hereditary hemochromatosis and insulin resistance (SantosSANTOS PCJL, CANÇADO RD, TERADA CT and GUERRA-SHINOHARA EM. 2009. Molecular changes associated with hereditary hemochromatosis. Rev Bras Hematol Hemoter 31: 192-202. et al. 2009, AdamsADAMS PC, MCLAREN CE, SPEECHLEY M, MCLAREN GD, BARTON JC and ECKFELDT JH. 2013. HFE mutations in Caucasian participants of the Hemochromatosis and Iron Overload Screening study with serum ferritin level <1000 µg/L. Can J Gastroenterol 27: 390-392. et al. 2013, DatzDATZ C, FELDER TK, NIEDERSEER D and AIGNER E. 2013. Iron homeostasis in the metabolic syndrome. Eur J Clin Invest 43: 215-224. et al. 2013, MartinelliMARTINELLI N et al. 2012. Increased serum hepcidin levels in subjects with the metabolic syndrome: a population study. PLoS ONE 7: e48250. et al. 2012). Hepatic production of hepcidin is directly related to the amount of liver iron, but the increased production of hepcidin in patients with MS is not yet fully understood (BarisaniBARISANI D, PELUCCHI S, MARIANI R, GALIMBERTI S, TROMBINI P, FUMAGALLI D, MENEVERI R, NEMETH E, GANZ T and PIPERNO A. 2008. Hepcidin and iron-related gene expression in subjects with Dysmetabolic Hepatic Iron Overload. J Hepatol 49: 123-133. et al. 2008).

Increased transferrin saturation suggests iron overload due to hemochromatosis, becoming an important tool for the diagnosis (Cardoso et al. 1998). However, in cases of hyperferritinemia associated with MS, transferrin saturation is usually within normal levels, being less useful for investigation (Chen et al. 2011, AvilaAVILA F et al. 2015. Serum Ferritin Is Associated with Metabolic Syndrome and Red Meat Consumption. Oxid Med Cell Longev 2015: 769739. et al. 2015). We observed a significant difference in transferrin saturation between patients with and without hyperferritinemia, but both groups had transferrin saturation levels within the normal range, indicating a small increase in patients with higher amount of available iron. Study of hemochromatosis associated mutations in patients with MS and hyperferritinemia should be performed only on the subset of patients that also have low hepcidin levels and/or increased transferrin saturation levels.

Our study has some limitations. The major one was that patients were not tested for the mutation that affects ferroportin, which presence has similar clinical presentation (hyperferritinemia and hyperhepcidinemia). Besides, we did not assessed the burden of liver iron using magnetic resonance imaging or liver biopsy (Cardoso et al. 1998).

In summary, in our study patients with MS and hyperferritinemia had higher hepcidin and transferrin saturation levels when compared to those with normal ferritin levels. Hyperferritinemia is a common finding in patients with MS and when associated with increased transferrin saturation, one can direct research for mutations in the HFE gene. However, when transferrin saturation levels are within normal ranges additional tests are needed, and hepcidin proved to be a promising tool in the differential diagnosis of hyperferritinemia related to MS.

ACKNOWLEGMENTS

We acknowledge the Incentive Fund for Research and Events of Hospital de Clínicas de Porto Alegre (FIPE/HCPA) for the financial support.

REFERENCES

  • ADAMS PC, MCLAREN CE, SPEECHLEY M, MCLAREN GD, BARTON JC and ECKFELDT JH. 2013. HFE mutations in Caucasian participants of the Hemochromatosis and Iron Overload Screening study with serum ferritin level <1000 µg/L. Can J Gastroenterol 27: 390-392.
  • AVILA F et al. 2015. Serum Ferritin Is Associated with Metabolic Syndrome and Red Meat Consumption. Oxid Med Cell Longev 2015: 769739.
  • BARISANI D, PELUCCHI S, MARIANI R, GALIMBERTI S, TROMBINI P, FUMAGALLI D, MENEVERI R, NEMETH E, GANZ T and PIPERNO A. 2008. Hepcidin and iron-related gene expression in subjects with Dysmetabolic Hepatic Iron Overload. J Hepatol 49: 123-133.
  • BARROS RK, COTRIM HP, DALTRO CH and OLIVEIRA YA. 2017. Hyperferritinemia in patients with nonalcoholic fatty liver disease. Rev Assoc Med Bras (1992) 63: 284-289.
  • CARDOSO EM, STÅL P, HAGEN K, CABEDA JM, ESIN S, DE SOUSA M and HULTCRANTZ R. 1998. HFE mutations in patients with hereditary haemochromatosis in Sweden. J Intern Med 243: 203-208.
  • CHANG JS, LIN SM, HUANG TC, CHAO JC, CHEN YC, PAN WH and BAI CH. 2013. Serum ferritin and risk of the metabolic syndrome: a population-based study. Asia Pac J Clin Nutr 22: 400-407.
  • CHEN LY, CHANG SD, SREENIVASAN GM, TSANG PW, BROADY RC, LI CH and ZYPCHEN LN. 2011. Dysmetabolic hyperferritinemia is associated with normal transferrin saturation, mild hepatic iron overload, and elevated hepcidin. Ann Hematol 90: 139-143.
  • COLLI ML, GROSS JL and CANANI LH. 2011. Mutation H63D in the HFE gene confers risk for the development of type 2 diabetes mellitus but not for chronic complications. J Diabetes Complications 25: 25-30.
  • DAHER R and KARIM Z. 2017. Iron metabolism: State of the art. Transfus Clin Biol 24: 115-119.
  • DATZ C, FELDER TK, NIEDERSEER D and AIGNER E. 2013. Iron homeostasis in the metabolic syndrome. Eur J Clin Invest 43: 215-224.
  • DE CARVALHO VIDIGAL F, BRESSAN J, BABIO N and SALAS-SALVADÓ J. 2013. Prevalence of metabolic syndrome in Brazilian adults: a systematic review. BMC Public Health 13: 1198.
  • DONGIOVANNI P, FRACANZANI AL, FARGION S and VALENTI L. 2011. Iron in fatty liver and in the metabolic syndrome: a promising therapeutic target. J Hepatol 55: 920-932.
  • EXPERT PANEL ON DETECTION, EVALUATION, and TREATMENT OF HIGH BLOOD CHOLESTEROL IN ADULTS. 2001. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA 285: 2486-2497.
  • GIRELLI D, NEMETH E and SWINKELS DW. 2016. Hepcidin in the diagnosis of iron disorders. Blood 127: 2809-2813.
  • GOOT K, HAZELDINE S, BENTLEY P, OLYNYK J and CRAWFORD D. 2012. Elevated serum ferritin - what should GPs know? Aust Fam Physician 41: 945-949.
  • HARE DJ. 2017. Hepcidin: a real-time biomarker of iron need. Metallomics 9: 606-618.
  • LORCERIE B, AUDIA S, SAMSON M, MILLIÈRE A, FALVO N, LEGUY-SEGUIN V, BERTHIER S and BONNOTTE B. 2017. Diagnosis of hyperferritinemia in routine clinical practice. Presse Med 46: e329-e338.
  • MARTINELLI N et al. 2012. Increased serum hepcidin levels in subjects with the metabolic syndrome: a population study. PLoS ONE 7: e48250.
  • PRENTICE AM. 2017. Clinical Implications of New Insights into Hepcidin-Mediated Regulation of Iron Absorption and Metabolism. Ann Nutr Metab 71(Suppl 3): 40-48.
  • SANTOS PCJL, CANÇADO RD, TERADA CT and GUERRA-SHINOHARA EM. 2009. Molecular changes associated with hereditary hemochromatosis. Rev Bras Hematol Hemoter 31: 192-202.
  • TROMBINI P, PAOLINI V, PELUCCHI S, MARIANI R, NEMETH E, GANZ T and PIPERNO A. 2011. Hepcidin response to acute iron intake and chronic iron loading in dysmetabolic iron overload syndrome. Liver Int 31: 994-1000.
  • VAISMAN B, MEYRON-HOLTZ EG, FIBACH E, KRICHEVSKY AM and KONIJN AM. 2000. Ferritin expression in maturing normal human erythroid precursors. Br J Haematol 110: 394-401.
  • WILLIAMS CD, STENGEL J, ASIKE MI, TORRES DM, SHAW J, CONTRERAS M, LANDT CL and HARRISON SA. 2011. Prevalence of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis among a largely middle-aged population utilizing ultrasound and liver biopsy: a prospective study. Gastroenterology 140: 124-131.
  • WORLD HEALTH ORGANIZATION. DEPARTMENT OF NONCOMMUNICABLE DISEASE SURVEILLANCE. 1999. Definition, diagnosis, and classification of diabetes mellitus and its complications : report of a WHO consultation. Part 1: diagnosis and classification of diabetes mellitus. Geneva: World Health Organization, Dept. of Noncommunicable Disease Surveillance. Avaliable at: https://apps.who.int/iris/bitstream/handle/10665/66040/WHO_NCD_NCS_99.2.pdf;jsessionid=3E917F17F40B31227F6B98E4BDBC9B94?sequence=1
    » https://apps.who.int/iris/bitstream/handle/10665/66040/WHO_NCD_NCS_99.2.pdf;jsessionid=3E917F17F40B31227F6B98E4BDBC9B94?sequence=1
  • ZIMMET P, ALBERTI KG and SERRANO RÍOS M. 2005. A new international diabetes federation worldwide definition of the metabolic syndrome: the rationale and the results. Rev Esp Cardiol 58: 1371-1376.

Publication Dates

  • Publication in this collection
    13 May 2019
  • Date of issue
    2019

History

  • Received
    19 Mar 2018
  • Accepted
    14 Sept 2018
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