Detection of serum hepcidin in renal failure and inflammation by using ProteinChip System.

Abstract

Hepcidin, a key regulator of iron metabolism, is expressed in the liver, distributed in blood, and excreted in urine. However, to date, no reliable and practical method for measuring the bioactive form of hepcidin in serum has been developed. Here, we used surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) to analyze the distinctive serum proteomic patterns of patients receiving hemodialysis. In the range of 1000 to 15,000 m/z, we found 3 peptides at 2192, 2789, and 2851 m/z that showed a significant correlation with the serum ferritin levels. The molecular sizes of peptides at 2192 and 2789 m/z matched with the reported sizes of hepcidin-20 and -25, respectively, and the serum peptide at 2789 m/z was identified as hepcidin-25 by collision-induced dissociation tandem MS. By using SELDI-TOF MS, we developed a semiquantitative assay for hepcidin-25. In this assay, the level of serum hepcidin-25 correlated well with levels of serum ferritin and serum interleukin-6. Hepcidin-25 was found to accumulate in the serum of patients receiving hemodialysis; this could contribute to the pathogenesis of renal anemia by decreasing the available iron for hematopoiesis. Thus, SELDI-TOF MS would be a clinically useful tool to detect and semiquantify bioactive hepcidin in serum.

Full text

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doi:10.1182/blood-2005-10-4043
Prepublished online April 18, 2006;




Umehara and Isao Ishikawa
Naohisa Tomosugi, Hiroshi Kawabata, Rumi Wakatabe, Masato Higuchi, Hideki Yamaya, Hisanori
ProteinChip System®
Detection of serum hepcidin in renal failure and inflammation by using
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RED CELLS-------------------------------------------------------------------------------------------------------

Detection of serum hepcidin in renal failure and inflammation by
using ProteinChip System®

Naohisa Tomosugi1, Hiroshi Kawabata2, Rumi Wakatabe3, Masato Higuchi4, Hideki

Detection of serum hepcidin in renal failure and inflammation by
using ProteinChip System®
Yamaya1, Hisanori Umehara5, and Isao Ishikawa1
1Division of Nephrology, Department of Internal Medicine, Kanazawa Medical
University, Ishikawa, Japan
2Department of Hematology and Oncology, Graduate School of Medicine, Kyoto
University, Kyoto, Japan
3Ciphergen Biosystems, Biomarker Discovery Center, Yokohama, Japan
4Department of Toxin, Chugai Pharma Co.LTD, Shizuoka, Japan
5Division of Hematology and Immunology, Department of Internal Medicine, Kanazawa
Medical University, Ishikawa, Japan

Running title: DETECTION OF SERUM HEPCIDIN
Regular manuscript

Supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports,
Science and Technology of Japan

One of the authors (RW) is employed by Ciphergen Biosystems.
One of the authors (MH) is employed by Chugai Pharma Co.LTD.
Ciphergen Biosystems and Chugai Pharma Co.LTD did not have any products in the
study.

Reprints:Reprints: Naohisa Tomosugi, Division of Nephrology, Department of Internal Medicine,
Kanazawa Medical University, Uchinada, Kahoku, Ishikawa 920-0293, Japan. ; e-mail:
tomosugi@kanazawa-med.ac.jp

Abstract: 200
Text: 4,478, Figures: 6, Table: 1, Supplementary figures: A-E
References: 32
Scientific section designation: RED CELLS

Blood First Edition Paper, prepublished online April 18, 2006; DOI 10.1182/blood-2005-10-4043
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Abstract

Hepcidin, a key regulator of iron metabolism, is expressed in the liver, distributed
Abstract
in blood, and excreted in urine. However, to date, no reliable and practical method for
measuring the bioactive form of hepcidin in serum has been developed. Here, we
employed surface-enhanced laser desorption ionization time of flight mass spectrometry
(SELDI-TOF MS) to analyze the distinctive serum proteomic patterns of hemodialysis
patients. In the range of 1000 to 15,000 m/z, we found three peptides at 2192, 2789, and
2851 m/z that showed a significant correlation with the serum ferritin levels. The
molecular sizes of peptides at 2192 and 2789 m/z matched with the reported sizes of
hepcidin-20 and -25, respectively, and the serum peptide at 2789 m/z was identified as
hepcidin-25 by collision-induced dissociation tandem MS. By employing SELDI-TOF
MS, we developed a semiquantitative assay for hepcidin-25. In this assay, the level of
serum hepcidin-25 correlated well with those of serum ferritin and serum interleukin-6.
Hepcidin-25 was found to accumulate in the serum of hemodialysis patients; this could
contribute to the pathogenesis of renal anemia by decreasing the available iron for
hematopoiesis. Thus, SELDI-TOF MS would be a clinically useful tool to detect and
semiquantify bioactive hepcidin in serum.

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Introduction ------------------------------------------------------------------------ Introduction ------------------------------------------------------------------------
Current understanding of the regulation of iron metabolism is based on the biology of a
number of critical proteins, including transferrin, transferrin receptors, ferritin, iron
regulatory proteins, divalent metal transporter 1, ferroportin, HFE, hemojuvelin, and
hepcidin.1-6 Among these proteins, plasma transferrin and ferritin are generally
measured in the laboratory as the total iron binding capacity and an indicator of overall
iron storage, respectively. The peptide hepcidin, which is produced by the liver, controls
plasma iron levels by regulating the absorption of food iron from the intestine as well as
the release of iron from macrophages. Further, it is also an acute phase reactant
induced by inflammation and shows antimicrobial activity in vitro.7-9 Moreover, it has
been shown that expression of hepcidin is regulated by anemia and hypoxia.10 Assays
for urine hepcidin have been previously described and these were successfully applied to
clinical cases.8,11,12 However, no reliable and practical assay system for measuring
serum hepcidin has been developed to date.
In hemodialysis patients, renal anemia is one of the major clinical problems, although
it can be partially alleviated by the administration of human recombinant
erythropoietin.13 In addition to the reduced production of erythropoietin in the kidneys,
other factors such as hyperparathyroidism, aluminum toxicity, systemic infection, and
impaired iron metabolism have been postulated as the mechanisms of anemia in these
patients.14
In this study, we employed a new technology, namely, surface-enhanced laser
desorption/ionization time of flight mass spectrometry (SELDI-TOF MS), to analyze
distinctive serum proteomic patterns in hemodialysis patients. SELDI-based
ProteinChip System® (Ciphergen Biosystems, Inc., Freemont, CA) array technology has
been successfully used to detect relevant biomarkers in various diseases such as
immunological conditions, cancer, neurological conditions, cardiovascular conditions,
and diseases of the lacrimal gland.15-18 SELDI technology is based on classical solid
phase extraction chromatography combined with direct laser desorption/ionization
mass spectrometric detection, and it requires minimal amounts of biological fluid
without pretreatment. This technology enables us to evaluate the subtle differences
between disease and control patients with regard to the expression of individual
proteins or groups of proteins in various fluids such as serum, urine, tears, or
cerebrospinal fluid. Further, it has a number of advantages, including high throughput
capability, very high sensitivity for detecting proteins in picomole to femtomole ranges,
high resolution for low molecular weight proteins, i.e., less than 20 kDa, and ease of
operation. By employing this technology, we compared the serum proteomic pattern and
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the level of serum ferritin in hemodialysis patients in order to identify biomarkers
associated with iron metabolism. In this experiment, we found three peptide peaks that
showed a good correlation with the levels of serum ferritin; one of these peaks was
identified as the peak of hepcidin-25. We developed a semiquantitative assay system for
serum hepcidin-25 and applied it to some clinical cases with iron overload, iron
deprivation, and acute and chronic inflammation. This new semiquantitative assay for
serum hepcidin will extend the possibilities for studying iron homeostasis in
physiological as well as pathological settings in man.

-------------------------------------------------------------------------------------------------------------------------
Materials and Methods Materials and Methods

Study participants Study participants
Serum samples were obtained from 40 hemodialysis patients before and after
hemodialysis in the Renal Center of Kanazawa Medical University Hospital and
Koshino Hospital. All the patients received hemodialysis treatment three times a week.
In addition, serum samples were collected from patients with acute pyelonephritis (2
patients), pneumonia (6 patients), and sepsis (8 patients) as well as from 16 healthy
volunteers. Serial serum and urine samples obtained from two patients with acute
pyelonephritis and two hemodialysis patients who received intravenous iron
administration for iron deficiency were also analyzed. Red blood cell (RBC) count,
hemoglobin (Hb), hematocrit (Ht), plasma iron, and ferritin levels were evaluated for
the clinical assessment of iron status and anemia. The serum samples were separated
by centrifugation (3000 rpm) and stored at –800C until assayed. Approval was obtained
from The Kanazawa Medical University institutional review board for these studies,
and informed consent was obtained from all the participants for the analysis of the
protein profiling of serum and/or urine.

SELDI-TOF MS analysis of serum and urine proteins SELDI-TOF MS analysis of serum and urine proteins
For preliminary trials, multiple types of protein chips were used with various surface
characteristics, including weak cation exchange, strong anion exchange, and
immobilized metal affinity capture for protein molecules that bind divalent cationic
copper. We eventually selected the immobilized metal affinity capture ProteinChip®
array (IMAC 30, Ciphergen) for the entire study because of its reproducibility in
detecting protein species in serum and urine. SELDI analysis was performed according
to the manufacturer’ s instructions with some modifications. In brief, serum and urine
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samples were diluted 20 fold and 10 fold, respectively, with a binding buffer (phosphate
buffered saline, pH 7.4). The dilution was determined by preliminary experiments.
Using a bioprocessor, 40 µL of the diluted samples was applied onto different arrays of
an IMAC 30 chip pretreated with 100 mM copper sulfate binding buffer and incubated
at room temperature for 30 minutes with constant horizontal shaking. Unbound
proteins were removed by washing three times with binding buffer for 5 minutes. The
chip arrays were rinsed twice with 400 µL of water, air-dried, and 0.5 µL of
alpha-cyano-4-hydroxy-cinnamic acid (CHCA, Ciphergen) in 50% acetonitrile (ACN)
and 0.5% trifluoroacetic acid was added twice to the array surface. Next, the arrays
were analyzed with the Ciphergen ProteinChip® Reader (model PBS II, Ciphergen).
The proteins with CHCA were ionized and their molecular masses were determined by
TOF analysis. The mass to charge ratio (m/z) of each of the proteins/peptides captured
on the array surface was determined according to the externally calibrated standards:
Arg8-vasopressin (1084.25 Da), somatostatin (1637.9 Da), dynorphin (2147.5 Da),
bovine insulin beta-chain (3495.94 Da), human insulin (5807.65 Da), bovine ubiquitin
(8564.8 Da), and bovine cytochrome C (12,230.9 Da). The mass spectra of the samples
were generated using an average of 80 laser shots at a laser intensity of 180 and a
detector sensitivity of 10. For confirming reproducibility, each sample was applied to
two separate spots on the IMAC 30 chip and the mean value of 24 mass spectra getting
from 12 different positions of each spot was taken as its representative value. Synthetic
hepcidin-25 (Peptide Institute Inc, Osaka, Japan) was used as a marker of MW2789.
The data from the peak intensities were normalized with total ion current by using
Biomarker Wizard (Ciphergen ProteinChip® Software 3.1.1) to compensate for the
variations in sample concentrations loaded onto a spot; the data were represented as
arbitrary units (AU) calculated using this software.
pI value of peptides pI value of peptides
The pI value was evaluated on the cationic ProteinChip® array (CM 10, Ciphergen). Six
aliquots of one serum sample were diluted with Tris-HCl buffer in the pH range of 8 to
10 at intervals of 0.4 pH units.

Purification, characterization, and identification of a biomarker candidate Purification, characterization, and identification of a biomarker candidate
The following purification strategy was determined preliminarily by using protein chip
arrays on a micro scale. Human serum (100 µL) was diluted 4 fold with 50 mM Tris-HCl
buffer, pH 8.0, and loaded onto a CM ceramic hyper DF spin column (BioSepra, Pall
Corporation, East Hills, NY). After fractionation by applying increasing NaCl
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concentrations, the purification progress was monitored using IMAC 30 arrays. The
fraction eluted by 500 mM NaCl was further separated by a reverse-phase high
performance liquid chromatography (HPLC). The fraction at a volume of 300 µL was
applied to the Protein-RP column (250 × 4.6 mm, YMC Co., Ltd, Kyoto, Japan) and
eluted by a 10%ዊ60% ACN linear gradient. The fraction eluted at 46 minutes was
collected and confirmed to contain the purified target peptide without other peptides by
using normal phase arrays(NP20, Ciphergen), which are used for general binding of
peptides via serine, threonine or lysine.
The fraction was reduced with 5 mM dithiothreitol (DTT) in 25 mM ammonium
bicarbonate buffer for 2 minutes at 700C or 50 mM iodoacetamide in 25 mM ammonium
bicarbonate buffer for 30 minutes at room temperature under dark conditions in NP20
arrays to detect the number of internal disulfide bridges. DTT reduction alone or with
iodoacetamide added two hydrogen atoms (2 Da) or two iodoacetamide molecules (114
Da) to form two free sulfhydryl groups for each disulfide bond broken, respectively.
For identification, the peptide was reduced by DTT and analyzed by QSTAR Pulsar i
(Applied Biosystems, Foster City, CA) equipped with a PCI 1000 ProteinChip Array
interface (Ciphergen) for collision-induced dissociation (CID) tandem MS analysis.
Database searches were performed using Mascot.

Development of a semiquantitative assay for serum hepcidin-25 Development of a semiquantitative assay for serum hepcidin-25
Synthetic hepcidin-25 was serially diluted in a range from 4 to 256 nM with distilled
water. Each standard sample was further diluted 20 fold with 19-fold diluted human
serum that had no detectable 2789 m/z peak. The diluted standard samples were
applied to the SELDI-TOF MS to detect the peak at 2789 m/z as described above.
Intra-day precision was determined using four different concentrations of synthetic
hepcidin-25(4, 16, 64 and 256nM) with 8 replicates being used per concentration. The 8
replicates were done on the 8 different chips within-day. This was repeated on 5
separate days to permit an assessment of inter-day precision. Intra- and inter-day
precisions were also determined in the sera from two patients. Precision was assessed
by coefficient of variation(CV) at each point.
Interleukin-6 concentrationInterleukin-6 concentration
The concentrations of serum interleukin (IL-6) were assayed with enzyme-linked
immunosorbent assay (ELISA) kits (Quantikine HS, R&D, Minneapolis, MN) according
to the manufacturer’ s protocol.

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Statistical analysis Statistical analysis
Statistical analysis of paired variables was performed using the parametric paired t test.
To study the linear relationship between continuous variables, Pearson correlation
coefficients were calculated. To compare the inclination of regression lines, analysis of
covariance was used. The comparison of the results from the experimental groups in
intra-and inter assay was carried out by one-way analysis of variance followed by the
Scheffe post hoc test. The p values were calculated based on a two-sided alternative
hypothesis and values less than 0.05 were considered significant.
-------------------------------------------------------------------------------------------------------------------------
Results Results
Screening of serum proteins related to iron metabolism Screening of serum proteins related to iron metabolism
Figure 1 shows representative protein mass spectra of sera in an m/z range of 2000 to
4000 from two hemodialysis patients and two volunteers with normal renal function;
the spectra reveal differences in their peak patterns. From the spectra of 62
participants, the Biomarker Wizard software automatically detected 114 peaks in the
range of 1000 to 15,000 m/z.
To determine the proteins related to iron metabolism, the correlation coefficients
between the intensity of each peak and RBC, Hb, Ht, serum iron, or ferritin were
calculated (Table 1). For serum ferritin, the intensities of only 3 of the 114 peaks— 2192,
2789, and 2851 m/z— showed good correlation coefficients, i.e., greater than 0.8 (r = 0.93,
0.83, and 0.80, respectively). None of the peaks showed a significant correlation with
RBC, Hb, Ht, or serum iron. Correlations of the intensities of these peaks with serum
ferritin were also observed in volunteers with normal renal function (r=0.84, Figure 2).
A significant correlation was also observed between the intensities of the 2192 and 2789
m/z peaks (r = 0.9), implicating that these two molecules may be regulated by the same
mechanism.
By searching through a protein database (SWISS-PROT), we found that the peaks
of 2192 m/z and 2789 m/z almost exactly correspond to the molecular weight of the
oxidative forms of human hepcidin-20 (theoretical molecular weight of the reduced form
in SWISS-PROT: 2199.78 Da) and hepcidin-25 (2797.41 Da), respectively. In a previous
report,8 the molecular weights of hepcidin-20 and hepcidin-25 determined by
matrix-assisted laser desorption ionization (MALDI)-TOF MS were 2191.77 and
2789.40 Da, respectively. In fact, the peak at 2789 m/z was identical to that of the
synthetic hepcidin-25 peptide containing four disulfide bridges. The peak at 2851 m/z
was not found in the database.
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Characteristic evaluation of serum peptides corresponding to the 2192 and 2789 m/z Characteristic evaluation of serum peptides corresponding to the 2192 and 2789 m/z
peaks for pI peaks for pI
Cationic protein chip CM 10 was used for the pI evaluation of 2192 and 2789 m/z peaks.
Tris-HCL buffer in the pH range of 8.0 to 10.0 was used as the diluting and washing
buffer. Both peptides remained bound to the chip at pH 10 (Supplementary Figure A),
suggesting that they were strongly cationic.

Purification of the serum peptide at the 2789 m/z peak— a biomarker candidate Purification of the serum peptide at the 2789 m/z peak— a biomarker candidate
Based on the results of molecular size and characteristics, we predicted that the peptide
corresponding to the peak at 2789 m/z was hepcidin-25. To confirm this, we purified the
peptide from human serum. The target peptide in the adsorption conditions at pH 8.0
was eluted by 500 mM NaCl in 50 mM Tris-HCl buffer on arrays (Supplementary
Figure A and B). These optimized purification conditions were directly applied to
small-scale purification. Diluted serum was applied to the CM ceramic hyper DF spin
column. After equilibration with the same buffer, elution was performed with a stepwise
NaCl gradient from 0 to 500 mM and monitored by profiling on the ProteinChip system.
The target peptide was eluted in the 500 mM NaCl fraction (Supplementary Figure C).
The eluant of 500 mM NaCl was then applied to a reverse-phase HPLC for further
separation. The fraction that eluted at 46 minutes included the target 2789-Da peptide
(Supplementary Figure D-a) and was selected for characterization and identification.
Characteristic evaluation of the peptide at the 2789 m/z peak for disulfide bridges Characteristic evaluation of the peptide at the 2789 m/z peak for disulfide bridges
The effect of DTT on the fraction eluted at 46 minutes is shown in Supplementary
Figure D. The 2789 m/z peak was reduced after DTT treatment and a new peak at 2797
m/z appeared (Supplementary Figure D-b), indicating that the peptide gained 8 Da after
reduction. Furthermore, by DTT treatment with iodoacetamide, the peptide gained
another 456 Da, thereby showing a peak at 3253 m/z (Supplementary Figure D-c).
These results suggested that the peptide at the 2789 m/z peak contained four internal
disulfide bridges.

Identification of the 2789-Da marker candidate as hepcidin-25 Identification of the 2789-Da marker candidate as hepcidin-25
In order to further characterize the candidate marker, the peptide was analyzed by CID
tandem MS via the ProteinChip Interface. The masses of the product ions were applied
to the Mascot search engine (Supplementary Figure E). Search results showed that the
amino acid sequence of this peptide was DTHFPICIFCCGCCHRSKCGMCCKT, and the
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top matching proteins were prohepcidin and hepcidin-25.

Development of a semiquantitative assay system for serum hepcidin-25 Development of a semiquantitative assay system for serum hepcidin-25
Since a practical assay for serum hepcidin-25 has not been reported, we decided to
develop an assay system for this peptide by using SELDI-TOF MS. The evaluation of
the performance characteristics of an assay system for this peptide by using
SELDI-TOF MS was as follows. Linear standard curves were obtained for peak
intensity at 2789 m/z (y=0.24x+2.5, r=0.996) from a set of 8 standard curves when
synthetic peptide was in a range from 4 nM to 256 nM (Figure 3). We found a small
intra-assay precision (42.7, 34.8, 5.0 and 4.6 %) and a considerable inter-assay precision
(33.8, 35.2, 29.4 and 19.6 %) at 4, 16, 64 and 256 nM, respectively. Lower limit of
detection was defined 16 nM(p<0.001) as the lowest concentration that could be
differentiated from zero and had the signal-to-noise values more than 2. The intra-assay
precisions for two patients were 7.5 % (peak intensity at 2789 m/z =53.7ዊ4.0) and 9.2 %
(6.4ዊ0.6), and the inter-assay precisions were 25.7 % (49.2ዊ12.6) and 27.5 % (8.8ዊ2.4),
respectively. Thus, we considered that this assay was not completely quantitative but
could be used as a semiquantitative assay system.
Clearance of hepcidin-25 by hemodialysis Clearance of hepcidin-25 by hemodialysis
Figure 4 shows the changes in the peak intensities at 2192, 2789 (hepcidin-25), and
2851 m/z before and after hemodialysis. The hemodialysis procedure was performed for
approximately 4 h in each patient. The removal rate of blood urea nitrogen measured at
the same time was 71.4 ± 2.4% in these patients. A clear reduction in the intensities of
peaks at 2192, 2789, and 2851 m/z was observed in the majority of patients; however, no
reduction was observed in some patients. In hemodialysis patients, the peak intensity
at 2789 m/z (hepcidin-25) was significantly higher than that in healthy volunteers with
the same levels below 200 ng/ml of ferritin (Figure 2, p<0.001); this indicates that
hepcidin-25 may have an inclination to accumulate in the serum of hemodialysis
patients.

Time course of the level of serum hepcidin-25 after iron loadingTime course of the level of serum hepcidin-25 after iron loading
Figure 5A shows the time course of the serum hepcidin-25 and ferritin level in a
72-year-old woman who was intravenously administered 40 mg of iron twice a week for
8 weeks for severe anemia. The intensity of the peak at 2789 m/z increased gradually
from 2.2 to 48.4 AU with iron administration, and during this period, the level of serum
ferritin increased from 11.3 to 273 ng/mL. On the other hand, in a 34-year-old woman
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who suffered from massive bleeding due to menstruation, the level of ferritin decreased
from 202 to 28 ng/mL during a period of four months, and the peak intensity at 2789 m/z
also reduced gradually from 47.8 to 11.2 AU (Figure 5B). In both cases, no remarkable
changes were found in the peak patterns, except for the peaks at 2192 and 2789 m/z.
The dialysis conditions for these two patients were the same throughout these periods.

Analysis of hepcidin-25 in the serum and urine of a patient with acute pyelonephritis Analysis of hepcidin-25 in the serum and urine of a patient with acute pyelonephritis
Protein profiling of urine and serum from a patient with acute pyelonephritis is shown
in Figure 5C-D. The peak at 2789 m/z was prominent in both urine and serum, and the
peaks at 2192 m/z and 2436 m/z were prominent only in urine before treatment. The
patient was successfully treated with antibiotics for seven days. By day 17, the peak at
2789 m/z (hepcidin-25) decreased from 66 to 32 AU in urine and from 66 to 18 AU in
serum. In urine, the peak intensity at 2192 m/z did not change remarkably during this
period. The levels of ferritin on day 0 and day 17 were 343.7 and 230.6 ng/mL,
respectively.

Association of the serum hepcidin-25 levels with serum IL-6 concentrationsAssociation of the serum hepcidin-25 levels with serum IL-6 concentrations
The serum IL-6 concentrations were less than 30 pg/mL in all the hemodialysis patients,
except one with systemic lupus erythematosus (SLE) who had been treated with
glucocorticoids (Figure 6). In contrast, the serum IL-6 levels in patients with acute
inflammation, including pyelonephritis, pneumonia, and sepsis, were elevated and
showed good correlation with the intensity of the 2789 m/z peak (r=0.60) which should
reflect the serum concentration of hepcidin-25 (Figure 6).

DiscussionDiscussion
In this study, we employed SELDI-TOF MS technology to identify serum proteins that
are regulated by body iron status and found a cluster of peptides at 2192, 2789, and
2851 m/z based on the correlation with the level of serum ferritin. The sizes of two of
these peptides at 2192 m/z and 2789 m/z almost exactly matched with those of
hepcidin-20 and -25 reported by Park et al.8Similar to hepcidin-20 and -25, these
peptides were strongly cationic. We purified the serum peptide corresponding to the
peak at 2789 m/z and identified it as hepcidin-25 by CID tandem MS. Since hepcidin is a
key molecule in iron homeostasis, the measurement of its active form can be a useful
tool for the differential diagnosis of anemia and iron overload in human diseases.19
Therefore, in the present study, we focused on characterizing the peak of hepcidin-25,
and the detailed characterization of the other two peptides was left for future study.
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In this study, we developed a semiquantitative assay system for serum hepcidin-25.
The standard curve shown in Figure 3 revealed that the effective measuring range of
this assay was approximately 16 to 256 nM. This assay appears to be suitable for
detecting high levels of serum hepcidin. Some of the human iron disorders such as
hereditary hemochromatosis are caused by hepcidin deficiency;1,4,19 however, this
assay may not be sufficiently sensitive to detect such abnormally low levels of serum
hepcidin. In this system, the serum expression of hepcidin-25 showed a good
correlation with that of serum ferritin in hemodialysis patients as well as in healthy
volunteers (Figure 2). This correlation was also observed in serial samples from a
patient who received iron supplementation as well as in a patient with repeated
massive hemorrhage (Figure 5A-B). Furthermore, hepcidin-25 in both serum and urine
was very high in a patient with urinary tract infection; after treatment with antibiotics,
the levels of hepcidin-25 in both serum and urine showed a clear decrease (Figure
5C-D). These results are consistent with the current understanding on the regulation
of the expression of hepcidin,1,4,19 implicating that our semiquantitative assay system
may be useful in clinical settings. In addition, this assay system may be a useful tool
for analyzing rapid iron metabolism in the body. For example, Nemeth et al20 have
shown that the stimulatory effects of dietary iron rapidly induce the expression of
hepcidin and reduce iron absorption from the intestine; however, the kinetics of
hepcidin in the serum during this “ mucosal block phenomenon” remains unknown. Our
SELDI-based assay system could be applied to detect and analyze the rapid kinetics of
the bioactive form of serum hepcidin in such phenomena.
Park et al8detected hepcidin-20, -22, and -25 in urine by using MALDI-TOF MS
analysis. Nemeth et al11 successfully measured urine hepcidin by using western blotting
following chromatographic purification Recently, Kemna et al12 reported a
semiquantitative method to analyze urine hepcidin by SELDI technology using
hydrophilic normal phase chips (NP20). However, to date, no reliable and practical
method for quantifying the mature forms of serum hepcidin has been developed,
probably due to the difficulty involved in developing specific antibodies against these
peptides, which have compact-folding structures21 and may also due to the complexity of
the serum matrix that deteriorate the reproducibility and linearity of various assays
including MS. The serum concentration of prohepcidin, an immature form of hepcidin
has been measured by ELISA.22,23 In previous studies,23-25 elevated levels of serum
prohepcidin measured by ELISA were found in renal insufficiency; however, no clear
relationship between serum prohepcidin concentration and body iron status was
observed. Prohepcidin, which is produced in the liver, needs to be processed by specific
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proteases to generate the mature forms of hepcidin that are observed in urine and in the
circulation.8Kemna et al12 analyzed urine samples from patients with iron deficiency
anemia, hereditary hemochromatosis, and sepsis and detected three peaks that
corresponded to the molecular weights of hepcidin-20, -22, and -25. Hepcidin-25, the
major form of mature hepcidin, is cleaved from prohepcidin by convertases, and
hepcidin-20 and -22 may be directly generated from prohepcidin by specific convertases
or indirectly by degradation of hepcidin-25. Hepcidin-25 has both iron-regulatory and
antimicrobial activities; hepcidin-22 has potent antimicrobial activity in vitro, while no
iron-regulatory function of hepcidin-20 has been identified.8,24 In the present study, we
detected the peaks corresponding to hepcidin-20 and -25 in the serum but could not
detect the peak corresponding to hepcidin-22 as a biomarker candidate related to iron
status, although it was detected only in urine of acute pyelonephritis. According to the
report by Park et al,8hepcidin-20 and -25 are the major forms of hepcidin in urine,
whereas hepcidin-22 is a minor form in urine.The same may be true in serum. However,
it is unclear whether urinary hepcidin is filtered from the blood or originates from the
kidney. It has been shown that hepcidin is expressed not only in the liver but also in the
renal tubular cells;26 therefore, it may be released partly from the kidneys in the case of
renal interstitial inflammation.
At present, it is well documented that hepcidin is reduced by anemia and hypoxia,
and induced by inflammation,10 and the association between hepcidin and inflammation
is becoming clearer.27 IL-6 appears to be the major cytokine responsible for the
induction of hepcidin production in inflammation, and IL-1β may be the second cytokine
that induces hepcidin production.27,28 IL-6 induces the expression of hepcidin mRNA in
human hepatocytes, a hepatoma cell line, and mouse hepatocytes in vitro.11,20,29 In the
in vivo model of inflammation induced by turpentine, the expression of hepcidin mRNA
in the liver was increased in the wild-type mice, but not in the IL-6 knockout mice.20 In
human studies, only a few reports have been published regarding the association
between hepcidin and inflammation. These reports showed that urinary excretion of
hepcidin was greatly increased in patients with anemia of inflammation, acute infection
with epididymitis,11 and severe sepsis.12 Nemeth et al20 reported that IL-6 infusion to
human volunteers caused a rapid increase in hepcidin excretion in urine. However, to
date the correlation between hepcidin and IL-6 in serum has not been investigated. In
this study, we showed that the increased levels of serum IL-6 in acute pyelonephritis,
pneumonia, and sepsis were accompanied with increased serum hepcidin levels.
It is well documented that iron overload stimulates the expression of both ferritin
and hepcidin. Similarly, inflammatory cytokines such as IL-6 may also contribute to the
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increase in not only serum hepcidin but also serum ferritin in human diseases. Nemeth
et al11 reported that in anemia of inflammation and sepsis, the urinary hepcidin
excretion correlated well with the serum ferritin concentration. Rogers30 reported that
IL-6 induced the expression of ferritin at the translational level, since the mRNA levels
of ferritin H and L subunits remained unchanged, whereas the synthesis of ferritin
subunits increased. In the case of hepcidin, IL-6 induces its expression at the mRNA
level.19The toxicity of iron in the cellular system is largely attributable to its capacity to
participate in the generation of reactive oxygen species.31 Ferritin captures and buffers
the intracellular labile iron pool to protect the cells,31 while hepcidin may prevent iron
toxicity by inhibiting iron absorption from the intestine. Thus, in the condition of iron
overload, active hepcidin might physiologically play a key role along with ferritin in
maintaining iron homeostasis. However, the precise mechanism of induction of hepcidin
by IL-6 and iron overload remains unclear.19
Low molecular weight proteins/peptides comprise several classes of physiologically
important proteins such as cytokines, chemokines, peptide hormones, and proteolytic
fragments of larger proteins.32 Interestingly, 114 small proteins in the serum with a
molecular weight less than 15,000 Da were detectable even in volunteers with normal
renal function, although these peptides are smaller than the presumed cutoff size for
kidney filtration. Furthermore, the two peptides at 2192 and 2789 m/z (hepcidin-20 and
-25, respectively) were sufficiently cationic to be filtered through the anionic glomerular
basement membrane; this suggests that these peptides may be parts of larger protein
complexes or may have unknown specific retention mechanisms. The intensities of
these two peaks corresponding to hepcidin-20 and -25 in hemodialysis patients were
higher than those in healthy volunteers when adjusted with the levels of serum ferritin
(Figure 2). In addition, hepcidin-25 did not decrease after the hemodialysis procedure in
some patients, indicating that hemodialysis could not sufficiently remove excess
hepcidin-25. Alternatively, these results may reflect the induction of hepcidin
expression due to the hemodialysis procedure itself. In either case, the accumulation of
hepcidin-25 in serum probably contributes, at least in part, to the pathogenesis of renal
anemia in hemodialysis patients by decreasing the available iron for hematopoiesis.

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Acknowledgments Acknowledgments
We thank Dr. Y. Koshino, Ms. S. Miyagishi, M. Fukuta, and Y. Izaki for their excellent

technical support.
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