History of Assay achievements

A major research focus within the Department of Laboratory Medicine of the Radboud University Nijmegen Medical Center is on iron metabolism regulators, and the translation of new findings into diagnostic assays that can be clinically implemented. Recent work focused on the iron-regulatory-protein hepcidin and resulted in two publications in the Blood-journal (Kemna et al., 2005a,b). In one of these studies, an in vivo human endotoxemia model was used to analyse the effects of endotoxemia as an upstream inflammation activator of hepcidin (Kemna et al., 2005a). These studies underscored the important role of hepcidin as the mediator of anemia of inflammation. This prompted us to develop a novel mass spectrometry (MS)-based assay for quantification of hepcidin in urine (Kemna et al., 2005b) and serum (Kemna et al., 2007). In fact, in 2005 we were the first to publish the potential of this MS approach (see: Kemna et al., 2008a), which paved the way for hepcidin measurements in clinical studies.

 

Our recent advances in quantitative serum and urine hepcidin measurements opened novel opportunities for studies on hepcidin in humans resulting in further insights in the clinical implications of hepcidin-mediated regulation of iron metabolism (see references below). Hepcidin measurements are now routinely performed by weak cation exchange chromatography in combination with time-of-flight mass spectrometry on a Microflex LT MALDI-TOF mass spectrometer (Bruker Daltonics). After the first international round robin (Kroot et al., 2009b), we are also the reference laboratory in a second initiative to harmonize hepcidin methods throughout the world.

 

 

 

Methodological Hallmarks

 Reference
Urine hepcidin measurements by time-of-flight mass spectrometry using normal phase chromatography-based SELDI-TOF MS Kemna et al., 2005b

Serum hepcidin measurement by time-of-flight mass spectrometry using IMAC-Cu2+-based SELDI-TOF MS; identification of hepcidin in urine by immunological and tandem MS/MS approaches; assessment of pre-analytical factors

 Kemna et al., 2007

Review on hepcidin assays available for human studies

 Kemna et al., 2008a
Quantitative serum and urine measurements exploiting an internal hepcidin analogue allowing kinetic studies using  IMAC-Cu2+ - and weak cation exchange (WCX)-based TOF MS Swinkels et al., 2008

Quantitative measurements of bioactive hepcidin-25 in hepatocyte growth medium; correlation with hepcidin mRNA expression

Kartikasari et al., 2008

Assessment of (pre)analytical imprecision, between-subject variability, and daily variations in serum and urine hepcidin levels measured by TOF MS

 Kroot et al., 2009a
The first round robin for both urine and plasma showed that the absolute hepcidin concentrations differed widely between methods, and the between-sample variation and the analytical variation of the methods were similar. Importantly, the analytical variation as percentage of the total variance is low for all methods, indicating their suitability to distinguish hepcidin levels of different samples. Ongoing initiatives should facilitate standardization by exchanging calibrators and representative samples. Kroot et al., 2009b
Comparatave analysis of cELISA and MS assays shows that TOF-MS is a valuable special-purpose assay for disorders with variable concentrations of hepcidin isoforms, such as chronic kidney disease. Kroot et al., 2010

Quantitave mass spectrometry analysis of Hep-1and Hep-2 peptides in experimental moude models is in agreement with the known biology of hepcidin mRNA regulators. These measurement can now be implemented in future mouse studies to provide novel insights in post-transcriptional regulation, hepcidin function, and kinetics.

 Tjalsma et al., 2011

Age and gender specific reference ranges of serum hepcidin concentration in a general Dutch population (n=2998).

 Galesloot et al., 2011

Review on the therapeutic potential for modulating hepcidin expression and the diagnostic potential for hepcidin measurements in clinical practice.

 Kroot et al., 2011

BioMedical Hallmarks

2005- 2007

Hepcidin at the crossroads of iron metabolism and host defence in man

 Kemna et al,. 2005a
Diurnal variation of hepcidin levels in urine and serum Kemna et al., 2007

Correspondence of urine and serum hepcidin levels

Kemna et al., 2007

2008
Algorithm roughly predicts serum hepcidin levels in various disorders of iron metabolism Kemna et al., 2008b
Serum hepcidin levels are innately low in C282Y homozygotes and might be predictive for biochemical and clinical penetrance van Dijk et al., 2008

Promises of serum hepcidin analysis for nephrologists

Swinkels & Wetzels 2008

>97% of the freely filtered serum hepcidin can be reabsorbed in the kidney

 Swinkels et al., 2008

The role of hepcidin in the management of non-hemochromatotic iron overload

 Swinkels & Drenth 2008
Dysregulation of iron homeostasis in HO-1 deficiency may be the result of both defective iron recycling and erythroid activity-associated inhibition of hepcidin expression Kartikasari et al., 2008
GDF15 may contribute to the inappropriate suppression of hepcidin in congenital dyserythropoietic anemia patients Tamary et al., 2008
High concentrations of hepcidin that are associated with malaria may contribute to malarial anemia and an impaired erythropoietic response to iron supplementation de Mast et al., 2008
A subgroup of patients with low grade myelodysplastic syndrome may be at increased risk of iron overload over the course of their disease. Murphy et al., 2008
Gastro-intestinal infections do not elevate urinary hepcidin or IL-6 levels in refugee children, nor are they associated with iron deficiency anemia. Cherian et al., 2008

2009

In contrast to hemolysis, IL-6 levels, hepcidin activity and iron status show no cumulative response during consecutive running sessions of healthy male athletes.

 Peeling et al., 2009
Plasma hepcidin concentrations predict interindividual variation in iron absorption in healthy men Roe et al., 2009
Serum hepcidin is one of the promising biomarkers to select those individuals who will benefit from iron supplements in malaria endemic regions. de Mast et al., 2009
Greater running intensities incur more inflammation and hemolysis in healthy male athletes Peeling et al., 2009
In sickle cell disease patients, hepcidin production is suppressed by increased erythropoietic activity, which is counterbalanced by iron stores and (low grade) inflammation Kroot et al., 2009
In vivo supports for the importance of an HFE-independent IL-6-hepcidin axis in the development of hypoferremia and anemia of inflammation van Deuren et al., 2009
The glomerular filtration rate (GFR) in patients with chronic kidney disease (CKD) is negatively associated with both hepcidin-20 and total hepcidin. Peters et al., 2009
Plasma hepcidin is only a modest predictor of dietary iron bioavailability in humans and oral iron loading increases circulating hepcidin. Zimmermann et al., 2009
Patients suffering from iron refractory iron deficiency anaemia (IRIDA) have no apparant specific geographical or ethnic distribution and are sporadic secondary to different mutations of the matriptase-2 gene. Tchou et al., 2009
Increased hepcidin production may represent the missing link between obesity and disrupted iron metabolism Miraglia Del Giudice et al., 2009
Globular filtration rate is not a major determinant of serum hepcidin-25 levels, while hepcidin-20 and hepcidin-22 accumulate in patients with renal impairment Peters et al., 2009

Fractional excretion of hepcidin-25 is not altered in patients with acute kidney injury

 Laarakkers et al., 2009
High-intensity exercise is accompanied by a significant increase in hepcidin levels, which is mediated through an increase in IL-6 and serum iron levels. Peeling et al., 2009

2010
Asymptomatic malaria is associated with increased hepcidin concentrations and anemia. de Mast et al., 2010
On the role of hepcidin in mitochondrial iron accumulation in patients with sideroblastic myelodysplastic syndrome Cuijpers et al., 2010

Anemia of chronic inflammation in patients with Hodgkin's Lymphoma is exaggerated by IL6/hepcidin-mediated iron deprivation, whereas also hepcidin-independent mechanisms contribute to this phenomenon.

 Hohaus et al., 2010
The (change in) hepcidin levels can predicted early and long-term bone marrow response to exogenous EPO. van der Putten et al., 2010
Body mass Index (BMI) reduction is associated with hepcidin reduction in obese children, which potentially improves iron status and absorption. Amato et al., 2010
Dietary iron absorption is ~40% reduced by asymptomatic malaria infection, likely because of low-grade inflammation and subsequent modulation of circulating hepcidin. Cercamondi et al., 2010

Anemic patients with chronic heart failure have lower urine and serum hepcidin levels compared to control subjects.

Divakaran et al., 2010

2011
Hepcidin and macrophage iron correlate with MCP-1 release and vascular damage in high-risk individuals with metabolic alterations Valenti et al., 2011
Maternal hepcidin is not significantly altered as a function of placental malaria infection and/or anemia van Santen et al., 2011
Hepcidin-25 could be an important modulator of anemia in septic patients with systemic inflammation van Eijk et al., 2011
The role of hepcidin in the pathophysiology of hereditary haemochromatosis Bokhoven et al., 2011
Elevations in mouse serum Hep-1 and urine Hep-2 upon intravenous iron or LPS are moderate and vary considerably between mouse strains. Serum Hep-1 is decreased in mouse models for hemochromatosis. Acute liver damage by paracetamol depletes Hep-1 from serum. Tjalsma et al., 2011
Considerations on the use of serum hepcidin measurementson in the future evidence-based clinical guidelines for patients suspected of having hereditary hemochromatosis.

Swinkels & Fleming, 2011

Elevated GDF15 levels in CDAII patients is in-line with its proposed role as erythroid factor that down-regulates hepcidin contributing to iron overload in dyserythropoiesis.

 Casanovas et al., 2011

Age and gender specific reference ranges of serum hepcidin concentration in a general population indicate ferritin as the primary correlate of serum hepcidin concentration.

 Galesloot et al., 2011
Increased GDF15 and decreased serum hepcidin/ferritin ratio may point towards a hepcidin level that is inappropriately low for body iron level during congenital sideroblastic anaemia Cuijpers et al., 2011

Hepcidin-25, but not pro-hepcidin, shows correspondence across biological fluids in male Guatemalan volunteers; the background 'status' of hepcidin activation may be related to the host's iron stores.

Schuemann et al., 2011

Iron stores (reflected by ferritin levels) may upregulate hepcidin in patients without HFE mutations leading to iron compartmentalization into macrophages and subsequent vascular damage in nonalcoholic fatty liver disease.

 Valenti et al., 2011
Carbohydrate ingestion during endurance running does not attenuate the increase in serum hepcidin levels during the post-run recovery period. Sim et al., 2011

Research is needed to address the role of hepcidin in malaria pathogenesis beyond anemia and iron dysregulation.

 van Santen et al., 2011

Serum hepcidin and, to a lesser extent, urine hepcidin, Ret-Hgb, and RBC-Hgb, are potential useful indicators for detecting iron deficiency in RA patients with anemia and active inflammation.

 van Santen et al., 2011

2012

Iron metabolism has a critical role in the regulation of intra-graft alloimmune responses in humans and provide a set of biomarkers, among which hepcidin, to conduct drug-weaning trials in liver transplantation.

 Bohne et al., 2012

Endurance exercise increases the production of hepcidin, presumably through IL-6, which in turn lowers serum iron concentrations.

 Newlin et al., 2012

Low serum hepcidin levels in a family with severe anaemia.

 Kloss-Brandstätter et al., 2012

 

References

Hepcidin assay

Hepcidin regulation & clinical implications