Abstract: help differentiate between types of iron disorders.

Abstract: Hepcidin; an
iron regulating hormone, is synthesized by the liver, influences the function
of ferroportin and macrophages. Regulates the iron levels in the body by attaching
to the ferropotin and stopping the efflux od iron into the body. It is studied
to see the effects of certain diseases related with the iron levels, and
measured to help differentiate between types of iron disorders. Its detected
and measured by various techniques, in this paper, four techniques are
discussed from their preparation till their end results. These techniques are matrix
assisted laser desorption/ionization-time of flight mass spectromy MALDI-TOF
MS, sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE,
enzyme-linked immunosorbent assay ELISA, and Real-time reverse transcriptase polymerase chain reaction
RT-PCR

Keywords: Hepcidin,
Iron, Anemia of Inflammation (AI), Hemochromatosis, ELISA, MALDI-TOF, SPS-PAGE,
RT-PCR

Introduction:

Iron, an essential
element in life, is precisely controlled in the body. Its importance lies in
its role in the hemoglobin as an essential factor for the transportation of
oxygen. Although iron is vital, its free form is likely to be involved in
oxidation-reduction reactions, leading to the formation of free radicals and
oxidative stress. Living organisms have developed protein systems to transport
and store it in a readily mobilizable form to avoid iron toxicity (Daher, Manceau, & Karim, 2017) Hepcidin is one of the peptide hormones that helps in the
maintenance of body iron levels. Hepcidins involvement in iron metabolism was
suggested by the observation that its synthesis is induced by dietary iron (Pigeon et al., 2001).

 Human hepcidin is a 25-amino acid (aa) peptide
synthesized in the liver and is the main organ in which hepcidin mRNA is
expressed. Hepcidin exists as a preprohormone 84 amino acids, prohormone 60
amino acids, and hormone 25 amino acids. Twenty- and 22-amino acid
metabolites of hepcidin also exist in the urine. Deletion of 5 N-terminal amino
acids results in loss of function (Pandur et al., 2009). it reduces the amount of circulating iron by preventing its
release from cells, particularly enterocytes and macrophages. this small
peptide is rapidly eliminated in the urine (Daher et al., 2017). it is transported around the body via the circulation bound to
carrier proteins such as ?2-macroglobulin and albumin (Peslova et al., 2009).  Human hepcidin exerts antibacterial and
antifungal activities at 10–30 µM concentrations (Park, Valore, Waring, & Ganz,
2001).

Hepcidin has a
role in the regulation of iron transport; it interacts with the iron exporter
ferropotin, and since its discovery, hepcidin has provided a molecular
explanation of the homeostatic regulation of iron absorption and distribution
and of its malfunction in hemochromatosis and AI. It is researched for the
explanation of many iron disorders when the iron dietary intake is sufficient
to meet the body’s needs, one of these conditions is anemia of inflammation is
a common, typically normocytic normochromic anemia that is caused by an
underlying inflammatory disease (Nemeth & Ganz, 2014), And another condition is hemochromatosis; a disease in which
too much iron builds up in the body.(Whittington & Kowdley, 2002) (Ganz & Nemeth, 2006).

the liver
produces this small 25-amino-acid peptide and secretes it in a way that its
being regulated by inflammation, iron, erythroid activity, and hypoxia (Lane, Huang, & Richardson, 2013). Most of the absorbed dietary iron or the recycled iron from hemoglobin
is used for the development of erythrocytes. erythrocytes production is increased
in response to hypoxia or blood loss. Therefore, Hepcidin’s production is also
homeostatically regulated by hypoxemia and anemia (Nicolas et al., 2002).
When isn’t delivered adequately, erythrocytes are to be more produced by the homeostatic
response to hypoxia. causing hepcidin’s levels to diminish, so its inhibitory
effects will be decreased,

and iron’s
availability from the diet and from the storage pool in hepatocytes and
macrophages will

uptick (Ganz & Nemeth, 2006), (Elizabeta Nemeth et al., 2004).

The only known
iron exporter is ferroportin and it has an essential role in the export of iron
from

cells to blood,
and from one cell type to another (D. M. Ward & Kaplan, 2012). Liver produces hepcidin when iron stores are high or adequate, it
circulates to the small intestine. There, Hepcidin attaches to ferroportin,
which causes it to be to be endocytosed and degraded. then iron exportation
from enterocytes will be decreased; causing the cells to be filled with iron, which
will eventually be shed into the lumen of the intestine. In case of iron stores
are on the low range, the production of hepcidin is suppressed, ferroportin
will be functioning accordingly on the basolateral membranes of the
enterocytes, and iron will be transported from the enterocyte cytoplasm to the
plasma transferrin (E. Nemeth et al., 2004).

transferrin:
Transferrin exerts a crucial function in the maintenance of systemic iron
homeostasis as component of a plasma iron sensing system that modulates
hepcidin expression (Gkouvatsos, Papanikolaou, &
Pantopoulos, 2012).

Hepcidin can be
used to assess cases of iron disorders that are not responding to nutritional
and medical treatments. In this paper four types of techniques are being
discussed for the detection of hepcidin from diverse samples and how each
technique will handle the sample and analyze it. These four techniques are matrix assisted laser desorption/ionization-time of flight
mass
spectromy MALDI-TOF MS, sodium
dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE, enzyme-linked
immunosorbent assay ELISA, and Real-time
reverse transcriptase polymerase chain reaction RT-PCR

MALDI-TOF MS:

Method: In a
study, urine samples were used to measure the hepcidin levels from 56 patients
with colorectal cancer. The method was to dilute urine samples to 10?g
protein/mL in 0.5 mol/L NaCl, 100 mmol/L sodium phosphate (pH 7.0) and applied
to Cu2+ loaded IMAC30 ProteinChip arrays. MALDI spectra was obtained either by
applying diluted urine (20 ?g protein/mL) or urine desalted using ClinProt C8
magnetic beads (BrukerDaltronic) to GoldChips. Spectra were acquired on a PBS
IIc ProteinChip Reader (Ciphergen) using sinapinic acid as the matrix. Spectra
were normalized to total ion current, baselines
subtracted, and peaks picked using Ciphergen ProteinChip software..(D.
G. Ward et al., 2008)

Synthetic hepcidin-25 Peptides International was employed
for peak/assay validation. Immunocapture was performed, Briefly Protein G
sepharose beads loaded with or without rabbit polyclonal anti-hepcicin-25
(Abcam 31877) were incubated with human urine containing hepcidin 20, 22 and
25. The beads were washed extensively with 20 mmol/L ammonium bicarbonate and
the captured proteins eluted with 50% acetonitrile/0.1% trifluoroacetic acid
and analyzed by MALDI (D.
G. Ward et al., 2008)

Method validation: In the study they used a pre-determined
hepcidin positive urine sample and performed MALDI-TOF, they detected two major
forms of hepcidin; the mature hepcidin 25 (m/z 2793.8), and the N-terminally
truncated hepcidin 20 (m/z 2195.3), in the addition to a hepcidin 22 (m/z
2440.5) that corresponds to a urinary degradation product of hepcidin 25. The
demonstration of the appearance of a hepcidin 25 peak was done by spiking a
urine sample devoid of hepcidin with a synthetic human hepcidin peptide. To
determine if MALDI-TOF MS could be used in a semi-quantitative manner the urine
sample was spiked with low endogenous hepcidin and demonstrated a linear relationship
between hepcidin concentration and intensity of the hepcidin 25 peak. As for
the urine samples of the colorectal cancer patients, the urine was desalted
before the performance of MALDI-TOF because it might interfere with mass
spectromy. Only hepcidin 20 and 25 expression level were analyzed, hepcidin 22
wasn’t analyzed because it is a urine specific degradation product of it. Using
a Spearman Rank test, the correlation coefficients were made Desalting
MALDI-TOF MS vs MALDI-TOF MS, correlation coefficient
= 0.56 P < 0.0001 (D. G. Ward et al., 2008). SDS-PAGE: Method: In a study, proteins in a hepatocyte cell line and in primary human hepatocytes were metabolically radiolabeled and selectively immunoprecipitated hepcidin and its precursors to analyze hepcidin processing (Valore & Ganz, 2008). The human cell line HepG2 clone 4246 was used to metabolically label hepcidin. Prior to radiolabeling, cells were depleted of the intracellular cysteine and methionine by incubation for 1–2 hour in cysteine and methionine-free RPMI containing dialyzed 5% FCS (to deplete free amino acids). In T25 vented flasks, cells were labeled by the addition of 100 ?Ci 35SCys-Met (Valore & Ganz, 2008). broad spectrum prolyl-hydroxylase inhibitors DMOG and DPD were added at concentrations of 500 and 10 ?M, respectively, 24 h prior to and during the radiolabeling procedure; in order to determine if hypoxia has affected the hepcidin processing through the HIF pathway (Valore & Ganz, 2008). Radiolabeled cells were washed with PBS then extracted by vigorous pipetting with 1ml ice cold NETT buffer (20mMTris–HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Triton X-100) containing protease inhibitor cocktail. Extracts were incubated on ice for 30 min then cell debris were removed by centrifugation. A volume of 30 ?l rabbit polyclonal antiserum directed to the propiece (aa; 25–59) SVFPQQTGQLAELQPQDRAGARASWMPMFQRRRRR, or to the mature synthetic refolded peptide (aa; 60–84) was added to a 1/5 volume of cleared cell lysate or culture media and was incubated on ice for 30–60 minutes. Protein A-agarose was added as a 50% slurry in PBS in a volume of 40 ?l and mixed overnight at 4 C. Then agarose was washed with PBS and the immunoprecipitate eluted by incubation in 30–40 ?l 3×sample loading buffer (170mMTris–HCl, pH 8.8, 21% wt/vol glycerol, 6% SDS wt/vol, 120 mM dithiothreitol) at 4 C overnight then boiled for 20 min (Valore & Ganz, 2008). SDS-Tricine polyacrylamide gel electrophoresis (PAGE) and fluorography: separation of the Radiolabeled hepcidin was on a 16.5% SD Tricine polyacrylamide gel with a 4% polyacrylamide stacking layer, stained with Coomassie blue, de-stained in 25% methanol/0.4% formaldehyde then soaked in liquid scintillant (1Msodium salicylate, 4% ethylene glycol, 35% ethanol). After drying, the gels were exposed to Kodak BioMax MS X-ray film at ?80 °C (Valore & Ganz, 2008). the hepcidin Primary human hepatocytes were treated with BMP-9 to induce the expression of hepcidin as otherwise the hepcidin peptide concentrations were too low to detect by metabolic radiolabeling. BMP-9 was added approximately 20 h prior to and during amino acid depletion and radiolabeling with 35Smet–cys and 14C-labeled amino acids. As in HepG2 cells, hepcidin is first made as a proprotein, then rapidly cleaved and secreted from the cell. Immunoprecipitation with anti-pro detected prohepcidin in the cell and a cleaved product released to the culture medium during the first hour of radiolabeling. However, the propiece is not detected at either 15 and 45 min of cold chase (Valore & Ganz, 2008). Immunoprecipitation: Immunoprecipitation (IP) uses the specificity of antibodies to isolate target proteins (antigens) out of complex sample mixtures (Kaboord & Perr, 2008). ELISA: Method: in a study that included 32 healthy controls, 7 patients with HJV associated juvenile hemochromatosis, 10 with iron deficiency anemia and 7 with Hodgkin's Lymphoma Manifesting B-symptoms. Human blood serum and hepatocell line were used.  Recombinant hepcidin25-His was expressed in yeast P. pastoris and the biological activity of the isolated monomer was tested. Recombinant hepcidin25-His was used for the immunization of rabbits and the polyclonal antiserum was extensively purified with affinity chromatography. To determine its binding activity against native hepcidin we first performed immunohistochemistry on paraffin embedded mouse liver sections. The antibody showed a strong cytoplasmic staining in hepatocytes that was reduced after preincubation with hepcidin25-His. the specificity of the antibody against the native peptide in serum was shown by Western Blot analysis of TCA-precipitated proteins less than 30 kDa from 10 ml of serum. A single band at 3 kDa was detected. This signal was abolished when the polyclonal antibody was preincubated with the recombinant peptide hepcidin25-His (Koliaraki et al., 2009). Expression and purification of recombinant hepcidin25-His: recombinant hepcidin25-His in its monomeric form were used to develop the assay. Briefly, a P. pastoris clone expressing hepcidin25 with a His-tag at its C-terminal was grown for 24 h at 30°C and then induced for 3 days by methanol 0.5%. Culture supernatant was concentrated and dialyzed with the use of a TFF Prep Scale Ultrafiltration system equipped with a 1 kDa filter. The concentrate was first purified using Ni -NTA metal affinity chromatography and then by size exclusion chromatography with a Peptide Superdex column to isolate the monomer. Immunization procedure: 100 ?g of the recombinant hepcidin25-His diluted into 0.4 mL in PBS were mixed with 0.4 mL of Complete Freund's adjuvant, and injected into New Zealand white rabbits subcutaneously. The serum was tested for antibody activity with ELISA assay. Purification of the polyclonal antibody: Polyclonal antiserum was extensively purified by affinity chromatography. ELISA procedure: 96-well microtiter plates (Costar, Corning, NY) were coated with hepcidin25-His diluted at 0.5 mg/L in PBS, pH 7.4, at 4°C, for 16 hours. Simultaneously, the polyclonal antibody diluted at 0.33 mg/L in 3% BSA in PBS was incubated with an equal amount of the calibrator or diluted serum (8 ?l in 25 ?l PBS per well) for 16 h at 4°C. As calibrator we used hepcidin25-His diluted in PBS (5, 20, 50, 200, 500 ?g/L). Quantification of the recombinant peptide was performed with a fluorescent quantification system. Subsequently, the plates were washed twice with PBS and blocked with 100 ?l of 3% BSA in PBS for 1 h at 37°C. The complexes formed were then added to the coated wells in quadruplicates and incubated for 1 h at 37°C. After 10 washes with PBS containing 0.5 ml/L Tween 20, the plates were incubated with a secondary anti-rabbit antibody conjugated with HRP (DakoCytomation, Carpinteria, CA) diluted 1?2000 in 3% BSA in PBS for 1 h at room temperature. The plates were washed as before, and visualization of the signal was accomplished after addition of 3,3?,5,5? tetramethylbenzidine for 10 min at room temperature. The reaction was stopped after the addition of 0.2 N sulphuric acid and color development was measured photometrically at 450 nm with a microplate reader (Bio-rad Model 680). Construction of the standard curve was then performed with Microsoft Office Excel (Koliaraki et al., 2009). Method validation: The competition ELISA produces a typical calibration curve for the recombinant hepcidin25-His. The analytical limit of detection of the ELISA assay, defined as the concentration corresponding to the mean signal+3 SD of 10 replicates of the zero calibrator was 5.4 ?g/L. The measurement range was from 10–1500 ?g/L. For the statistical analysis of the linearity, reproducibility, and recovery of the hepcidin ELISA assay, 3 serum samples were used ranging from low (22 ?g/L) to high (150 ?g/L) concentrations chosen from the 32 normal sera tested. The intra-assay coefficients of variance (CVs) were 8–15%, evaluated by assaying 12 replicates of each sample in a single assay. The inter-assay CVs were 5–16% as evaluated by 7 subsequent measurements of the test samples. Inter- and intra-assay CV for the standard curve was 8.5% and 2.8%, respectively. Recovery rate was done by adding the calibrator at 7.5, 30 and 75 ?g/L in each serum sample. It was found to range from 99–115% with a mean recovery index of 107%. Mean linearity was estimated at 101% after measuring 3 serial dilutions (1?2, 1?4, 1?8) of the 3 samples. To determine whether the assay was providing biologically meaningful measurements, serum samples from patients with anticipated low and high hepcidin levels were tested and compared to healthy controls. compared to age-matched healthy controls (42.7 ?g/L), hepcidin concentration was significantly lower in 10 patients with iron deficiency anemia (15.7 ?g/L, p<0.010) and 7 patients with juvenile hemochromatosis (12.8 ?g/L, p<0.010), and significantly higher in 7 untreated patients with Hodgkin's lymphoma and B-symptoms (116.7 ?g/L, p<0.010). they have done correlation with an already established method SELDI-TOF MS to better evaluate the ELISA method they've improved, and the samples procedure for SELDI-TOF MS was done in related way to that of ELISA to ensure correlation and comparison between the two methods; Pearson correlation showed a significant correlation between the ELISA assay and the mass-spec method (correlation: 0.863, p=0.027) (Koliaraki et al., 2009). In conclusion, an ELISA assay capable of measuring hepcidin in human serum samples at a range of 10–1500 ?g/L with acceptable precision, linearity, recovery and specificity was developed. This assay is easy to apply and utilizes a small amount of unprocessed serum, shortening the processing time (Koliaraki et al., 2009). RT-PCR: In a study, they analyzed liver samples from patients undergoing hepatic surgery for cancer or receiving liver transplants and analyzed correlations between clinical parameters and liver hepcidin mRNA and urinary hepcidin concentrations. They wanted to check If urinary hepcidin levels correlate with the bodies levels and if it can be used as an indicator of it (Detivaud et al., 2005). polymerase chain reaction (PCR) is a sensitive assay; Only trace amounts of DNA are needed. It amplifies the DNA Exponentially. The main components are: template DNA, primers, nucleotides, and DNA polymerase. The DNA polymerase is the key enzyme that links individual nucleotides together to form the PCR product (Garibyan & Avashia, 2013). Real-time reverse transcriptase polymerase chain reaction (RT-PCR): direct detection of PCR product during the exponential phase of the reaction, combining amplification and detection in a single step. By construction of complementary DNA (cDNA) plasmid clones, standard curves are generated that allow direct quantification of every unknown sample (Overbergh et al., 2003). Method: Total RNAs were extracted using the SV Total RNA Isolation System. to evaluate the hepcidin expression in each sample polymerase chain reactions were performed in triplicate, compared with the RNA18S expression and with a standard set using the qPCR-Core-kit. Primer and probe sequences used for the hepcidin amplification were: forward primer 5'-TCCCACAACAGACGGGACAA-3', reverse 5'-AGCAGCCGCAGCAGAAAAT-3' and FAM/TAMRA probe 5'-CCATGTTCCAGAGGCGAAGGAGGC-3'. The amplified 137-bp PCR fragment was checked by sequencing. For RNA 18S amplification we used the 18S genomic control kit. The PCR was run on ABI PRISM 7000 sequence detection: 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Analysis of Data: For each sample, hepcidin mRNA threshold cycle (Ct) value was normalized with 18S RNA Ct value and compared to value of a standard sample which was a mix of 4 liver samples exhibiting readily detectable level of hepcidin mRNA as previously analyzed by Northern blot. Results were expressed in log 2 of ratio sample versus standard. The Urinary hepcidin assay was performed, and hepcidin concentration in urine expressed as ng hepcidin/mg creatinine. Because the lower detection limit of the urinary hepcidin assay is 1 ng/mg creatinine, samples with lower value were attributed a value of 1 ng/mg creatinine (Detivaud et al., 2005) Hepcidin mRNA and urinary hepcidin levels appeared to be positively correlated and increasing in parallel. This demonstrates that the urinary hepcidin assay can be considered a valid reflection of hepatic hepcidin production. Though these results might not be accurate since they were taken from patients going to surgery and having hepatic problems (Detivaud et al., 2005) In conclusion, urinary hepcidin concentrations significantly correlates with hepatic hepcidin mRNA concentrations, indicating that it is a valid approach to evaluate body's hepcidin expression. it is also found that in humans hepcidin levels correlates with hepatic iron stores and hemoglobin levels and maybe be affected by hepatic dysfunction (Detivaud et al., 2005)   MALDI-TOF MS SDS-PAGE ELISA RT-PCR Sample type urine Hepatocell line Blood and human serum and hepatocell line Urine and blood Sample processing multistep and process Multistep and process Small amount of unprocessed serum multistep and process Time Long time Long time Short time Long time accuracy Accurate - Accurate Accurate precision Precise Precise Precise   Conclusion: out of the four methods ELISA technique seems to be most productive and reliable one, in case of productivity, reproducibility, sample preparation, time, sample variety, and processing steps. A reliable and easy to apply hepcidin quantification method could prove a valuable tool for diagnostic applications, as hepcidin levels may be used for the differential diagnosis of several iron disorders, and with further refinement, hepcidin assays could contribute to the diagnosis and improved understanding of the pathophysiology of iron disorders.