Abstract: Hepcidin; aniron regulating hormone, is synthesized by the liver, influences the functionof ferroportin and macrophages.
Regulates the iron levels in the body by attachingto the ferropotin and stopping the efflux od iron into the body. It is studiedto see the effects of certain diseases related with the iron levels, andmeasured to help differentiate between types of iron disorders. Its detectedand measured by various techniques, in this paper, four techniques arediscussed from their preparation till their end results. These techniques are matrixassisted laser desorption/ionization-time of flight mass spectromy MALDI-TOFMS, sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE,enzyme-linked immunosorbent assay ELISA, and Real-time reverse transcriptase polymerase chain reactionRT-PCRKeywords: Hepcidin,Iron, Anemia of Inflammation (AI), Hemochromatosis, ELISA, MALDI-TOF, SPS-PAGE,RT-PCRIntroduction:Iron, an essentialelement in life, is precisely controlled in the body. Its importance lies inits role in the hemoglobin as an essential factor for the transportation ofoxygen. Although iron is vital, its free form is likely to be involved inoxidation-reduction reactions, leading to the formation of free radicals andoxidative stress. Living organisms have developed protein systems to transportand 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 themaintenance of body iron levels. Hepcidins involvement in iron metabolism wassuggested by the observation that its synthesis is induced by dietary iron (Pigeon et al.
, 2001). Human hepcidin is a 25-amino acid (aa) peptidesynthesized in the liver and is the main organ in which hepcidin mRNA isexpressed. Hepcidin exists as a preprohormone 84 amino acids, prohormone 60amino acids, and hormone 25 amino acids. Twenty- and 22-amino acidmetabolites of hepcidin also exist in the urine. Deletion of 5 N-terminal aminoacids results in loss of function (Pandur et al.
, 2009). it reduces the amount of circulating iron by preventing itsrelease from cells, particularly enterocytes and macrophages. this smallpeptide is rapidly eliminated in the urine (Daher et al., 2017). it is transported around the body via the circulation bound tocarrier proteins such as ?2-macroglobulin and albumin (Peslova et al.
, 2009). Human hepcidin exerts antibacterial andantifungal activities at 10–30 µM concentrations (Park, Valore, Waring, & Ganz,2001).Hepcidin has arole in the regulation of iron transport; it interacts with the iron exporterferropotin, and since its discovery, hepcidin has provided a molecularexplanation of the homeostatic regulation of iron absorption and distributionand of its malfunction in hemochromatosis and AI. It is researched for theexplanation of many iron disorders when the iron dietary intake is sufficientto meet the body’s needs, one of these conditions is anemia of inflammation isa common, typically normocytic normochromic anemia that is caused by anunderlying inflammatory disease (Nemeth & Ganz, 2014), And another condition is hemochromatosis; a disease in whichtoo much iron builds up in the body.(Whittington & Kowdley, 2002) (Ganz & Nemeth, 2006). the liverproduces this small 25-amino-acid peptide and secretes it in a way that itsbeing regulated by inflammation, iron, erythroid activity, and hypoxia (Lane, Huang, & Richardson, 2013). Most of the absorbed dietary iron or the recycled iron from hemoglobinis used for the development of erythrocytes. erythrocytes production is increasedin response to hypoxia or blood loss.
Therefore, Hepcidin’s production is alsohomeostatically regulated by hypoxemia and anemia (Nicolas et al., 2002).When isn’t delivered adequately, erythrocytes are to be more produced by the homeostaticresponse to hypoxia. causing hepcidin’s levels to diminish, so its inhibitoryeffects will be decreased, and iron’savailability from the diet and from the storage pool in hepatocytes andmacrophages willuptick (Ganz & Nemeth, 2006), (Elizabeta Nemeth et al., 2004).The only knowniron exporter is ferroportin and it has an essential role in the export of ironfromcells to blood,and from one cell type to another (D. M.
Ward & Kaplan, 2012). Liver produces hepcidin when iron stores are high or adequate, itcirculates to the small intestine. There, Hepcidin attaches to ferroportin,which causes it to be to be endocytosed and degraded. then iron exportationfrom enterocytes will be decreased; causing the cells to be filled with iron, whichwill eventually be shed into the lumen of the intestine. In case of iron storesare on the low range, the production of hepcidin is suppressed, ferroportinwill be functioning accordingly on the basolateral membranes of theenterocytes, and iron will be transported from the enterocyte cytoplasm to theplasma transferrin (E. Nemeth et al.
, 2004).transferrin:Transferrin exerts a crucial function in the maintenance of systemic ironhomeostasis as component of a plasma iron sensing system that modulateshepcidin expression (Gkouvatsos, Papanikolaou, , 2012).Hepcidin can beused to assess cases of iron disorders that are not responding to nutritionaland medical treatments. In this paper four types of techniques are beingdiscussed for the detection of hepcidin from diverse samples and how eachtechnique will handle the sample and analyze it. These four techniques are matrix assisted laser desorption/ionization-time of flightmassspectromy MALDI-TOF MS, sodiumdodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE, enzyme-linkedimmunosorbent assay ELISA, and Real-timereverse transcriptase polymerase chain reaction RT-PCRMALDI-TOF MS:Method: In astudy, urine samples were used to measure the hepcidin levels from 56 patientswith colorectal cancer. The method was to dilute urine samples to 10?gprotein/mL in 0.
5 mol/L NaCl, 100 mmol/L sodium phosphate (pH 7.0) and appliedto Cu2+ loaded IMAC30 ProteinChip arrays. MALDI spectra was obtained either byapplying diluted urine (20 ?g protein/mL) or urine desalted using ClinProt C8magnetic beads (BrukerDaltronic) to GoldChips.
Spectra were acquired on a PBSIIc ProteinChip Reader (Ciphergen) using sinapinic acid as the matrix. Spectrawere normalized to total ion current, baselinessubtracted, and peaks picked using Ciphergen ProteinChip software..(D.G. Ward et al., 2008)Synthetic hepcidin-25 Peptides International was employedfor peak/assay validation.
Immunocapture was performed, Briefly Protein Gsepharose beads loaded with or without rabbit polyclonal anti-hepcicin-25(Abcam 31877) were incubated with human urine containing hepcidin 20, 22 and25. The beads were washed extensively with 20 mmol/L ammonium bicarbonate andthe captured proteins eluted with 50% acetonitrile/0.1% trifluoroacetic acidand analyzed by MALDI (D.
G. Ward et al., 2008)Method validation: In the study they used a pre-determinedhepcidin positive urine sample and performed MALDI-TOF, they detected two majorforms of hepcidin; the mature hepcidin 25 (m/z 2793.
8), and the N-terminallytruncated hepcidin 20 (m/z 2195.3), in the addition to a hepcidin 22 (m/z2440.5) that corresponds to a urinary degradation product of hepcidin 25. Thedemonstration of the appearance of a hepcidin 25 peak was done by spiking aurine sample devoid of hepcidin with a synthetic human hepcidin peptide. Todetermine if MALDI-TOF MS could be used in a semi-quantitative manner the urinesample was spiked with low endogenous hepcidin and demonstrated a linear relationshipbetween hepcidin concentration and intensity of the hepcidin 25 peak.
As forthe urine samples of the colorectal cancer patients, the urine was desaltedbefore the performance of MALDI-TOF because it might interfere with massspectromy. Only hepcidin 20 and 25 expression level were analyzed, hepcidin 22wasn’t analyzed because it is a urine specific degradation product of it. Usinga Spearman Rank test, the correlation coefficients were made DesaltingMALDI-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 inprimary human hepatocytes weremetabolically radiolabeled and selectively immunoprecipitatedhepcidin and its precursors toanalyze hepcidin processing (Valore& Ganz, 2008). The human cell line HepG2 clone 4246 was used tometabolically label hepcidin. Prior to radiolabeling, cells were depleted ofthe intracellular cysteine and methionine by incubation for 1–2 hour incysteine and methionine-free RPMI containing dialyzed 5% FCS (to deplete freeamino acids).
In T25 vented flasks, cells were labeled by the addition of 100?Ci 35SCys-Met (Valore& Ganz, 2008).broad spectrum prolyl-hydroxylaseinhibitors DMOG and DPD were added at concentrations of 500 and 10 ?M,respectively, 24 h prior to and during the radiolabeling procedure; in order todetermine if hypoxia has affected the hepcidin processing through the HIFpathway (Valore& Ganz, 2008).Radiolabeled cells were washed with PBS then extracted byvigorous pipetting with 1ml ice cold NETT buffer (20mMTris–HCl, pH 7.4, 150 mMNaCl, 10 mM EDTA, 1% Triton X-100) containing protease inhibitor cocktail.Extracts were incubated on ice for 30 min then cell debris were removed bycentrifugation. A volume of 30 ?l rabbit polyclonal antiserum directed to thepropiece (aa; 25–59) SVFPQQTGQLAELQPQDRAGARASWMPMFQRRRRR, or to the maturesynthetic refolded peptide (aa; 60–84) was added to a 1/5 volume of clearedcell 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 andmixed overnight at 4 C.
Then agarose was washed with PBS and the immunoprecipitateeluted by incubation in 30–40 ?l 3×sample loading buffer (170mMTris–HCl, pH8.8, 21% wt/vol glycerol, 6% SDS wt/vol, 120 mM dithiothreitol) at 4 Covernight then boiled for 20 min (Valore& Ganz, 2008).SDS-Tricine polyacrylamide gel electrophoresis (PAGE) andfluorography: separation of theRadiolabeled hepcidin was on a 16.5% SD Tricinepolyacrylamide gel with a 4% polyacrylamide stacking layer, stained withCoomassie blue, de-stained in 25% methanol/0.4% formaldehyde then soaked inliquid 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 withBMP-9 to induce the expression of hepcidin as otherwise the hepcidin peptideconcentrations were too low to detect by metabolicradiolabeling.
BMP-9 was added approximately 20 h prior toand during amino acid depletion and radiolabeling with 35Smet–cys and14C-labeled amino acids. As in HepG2 cells, hepcidin is first made as aproprotein, then rapidly cleaved and secreted from the cell. Immunoprecipitationwith anti-pro detected prohepcidin in the cell and a cleaved product releasedto the culture medium during the first hour of radiolabeling. However, thepropiece is not detected at either 15 and 45 min of cold chase (Valore& Ganz, 2008).
Immunoprecipitation: Immunoprecipitation (IP) uses thespecificity of antibodies to isolate target proteins (antigens) out of complexsample mixtures (Kaboord& Perr, 2008).ELISA:Method: in a study that included 32 healthy controls, 7patients with HJV associated juvenile hemochromatosis, 10 with iron deficiencyanemia and 7 with Hodgkin’s Lymphoma Manifesting B-symptoms. Human blood serumand hepatocell line were used. Recombinanthepcidin25-His was expressed in yeast P. pastoris and the biological activityof the isolated monomer was tested. Recombinant hepcidin25-His was used for theimmunization of rabbits and the polyclonal antiserum was extensively purifiedwith affinity chromatography.
To determine its binding activity against nativehepcidin we first performed immunohistochemistry on paraffin embedded mouseliver sections. The antibody showed a strong cytoplasmic staining inhepatocytes that was reduced after preincubation with hepcidin25-His. thespecificity of the antibody against the native peptide in serum was shown byWestern Blot analysis of TCA-precipitated proteins less than 30 kDa from 10 mlof serum. A single band at 3 kDa was detected. This signal was abolished whenthe polyclonal antibody was preincubated with the recombinant peptidehepcidin25-His (Koliarakiet al., 2009).
Expression and purification of recombinant hepcidin25-His: recombinanthepcidin25-His in its monomeric form were used to develop the assay. Briefly, aP. pastoris clone expressing hepcidin25 with a His-tag at its C-terminal wasgrown for 24 h at 30°C and then induced for 3 days by methanol 0.5%. Culturesupernatant was concentrated and dialyzed with the use of a TFF Prep ScaleUltrafiltration system equipped with a 1 kDa filter. The concentrate was firstpurified using Ni -NTA metal affinity chromatography and then by size exclusionchromatography with a Peptide Superdex column to isolate the monomer.Immunization procedure: 100 ?g of the recombinanthepcidin25-His diluted into 0.4 mL in PBS were mixed with 0.
4 mL of CompleteFreund’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 antiserumwas 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 in3% BSA in PBS was incubated with an equal amount of the calibrator or dilutedserum (8 ?l in 25 ?l PBS per well) for 16 h at 4°C. As calibrator we usedhepcidin25-His diluted in PBS (5, 20, 50, 200, 500 ?g/L). Quantification of therecombinant peptide was performed with a fluorescent quantification system.
Subsequently, the plates were washed twice with PBS and blocked with 100 ?l of3% BSA in PBS for 1 h at 37°C. The complexes formed were then added to thecoated wells in quadruplicates and incubated for 1 h at 37°C. After 10 washeswith PBS containing 0.5 ml/L Tween 20, the plates were incubated with asecondary anti-rabbit antibody conjugated with HRP (DakoCytomation,Carpinteria, CA) diluted 1?2000 in 3% BSA in PBS for 1 h at room temperature. The plateswere washed as before, and visualization of the signal was accomplished afteraddition of 3,3?,5,5? tetramethylbenzidine for 10 min at room temperature. Thereaction was stopped after the addition of 0.2 N sulphuric acid and colordevelopment was measured photometrically at 450 nm with a microplate reader(Bio-rad Model 680). Construction of the standard curve was then performed withMicrosoft Office Excel (Koliarakiet al.
, 2009). Method validation: The competition ELISA produces a typicalcalibration curve for the recombinant hepcidin25-His. The analytical limit ofdetection of the ELISA assay, defined as the concentration corresponding to themean signal+3 SD of 10 replicates of the zero calibrator was 5.4 ?g/L. Themeasurement range was from 10–1500 ?g/L. For the statistical analysis of thelinearity, reproducibility, and recovery of the hepcidin ELISA assay, 3 serumsamples were used ranging from low (22 ?g/L) to high (150 ?g/L) concentrationschosen 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 asingle assay. The inter-assay CVs were 5–16% as evaluated by 7 subsequentmeasurements of the test samples.
Inter- and intra-assay CV for the standardcurve was 8.5% and 2.8%, respectively. Recovery rate was done by adding thecalibrator at 7.
5, 30 and 75 ?g/L in each serum sample. It was found to rangefrom 99–115% with a mean recovery index of 107%. Mean linearity was estimatedat 101% after measuring 3 serial dilutions (1?2, 1?4, 1?8) of the 3 samples.To determine whether the assay was providing biologicallymeaningful measurements, serum samples from patients with anticipated low andhigh 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 irondeficiency anemia (15.7 ?g/L, p<0.
010) and 7 patients with juvenilehemochromatosis (12.8 ?g/L, p<0.010), and significantly higher in 7untreated patients with Hodgkin's lymphoma and B-symptoms (116.7 ?g/L,p<0.010).they have done correlation with an already established methodSELDI-TOF MS to better evaluate the ELISA method they've improved, and thesamples procedure for SELDI-TOF MS was done in related way to that of ELISAto ensure correlation and comparison between the two methods; Pearsoncorrelation showed a significant correlation between the ELISA assay and themass-spec method (correlation: 0.
863, p=0.027) (Koliarakiet al., 2009).Inconclusion, an ELISA assay capable of measuring hepcidin in human serum samplesat a range of 10–1500 ?g/L with acceptable precision, linearity, recovery andspecificity was developed. This assay is easy to apply and utilizes a smallamount of unprocessed serum, shortening the processing time (Koliarakiet al.
, 2009).RT-PCR:Ina study, they analyzed liver samples from patients undergoing hepatic surgeryfor cancer or receiving liver transplants and analyzed correlations betweenclinical parameters and liver hepcidin mRNA and urinary hepcidinconcentrations. They wanted to check If urinary hepcidin levels correlate withthe bodies levels and if it can be used as an indicator of it (Detivaudet al., 2005).polymerase chain reaction (PCR) is a sensitive assay; Onlytrace amounts of DNA are needed. It amplifies the DNA Exponentially. The maincomponents are: template DNA, primers, nucleotides, and DNA polymerase. The DNApolymerase is the key enzyme that links individual nucleotides together to formthe PCR product (Garibyan& Avashia, 2013).
Real-time reverse transcriptase polymerase chain reaction(RT-PCR): direct detection of PCR product during the exponential phase of thereaction, combining amplification and detection in a single step. Byconstruction of complementary DNA (cDNA) plasmid clones, standard curves aregenerated that allow direct quantification of every unknown sample (Overberghet al., 2003).Method: Total RNAs were extracted using the SV Total RNAIsolation System.
to evaluate the hepcidin expression in each sample polymerase chainreactions were performed in triplicate, compared with the RNA18S expression andwith a standard set using the qPCR-Core-kit. Primer and probe sequences usedfor the hepcidin amplification were: forward primer5′-TCCCACAACAGACGGGACAA-3′, reverse 5′-AGCAGCCGCAGCAGAAAAT-3’and FAM/TAMRA probe 5′-CCATGTTCCAGAGGCGAAGGAGGC-3′. The amplified 137-bp PCRfragment was checked by sequencing.
For RNA 18S amplification we used the 18Sgenomic control kit. The PCR was run on ABI PRISM 7000 sequence detection: 95°Cfor 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1minute. Analysis of Data: For each sample, hepcidin mRNA thresholdcycle (Ct) value was normalized with 18S RNA Ct value and compared to value ofa standard sample which was a mix of 4 liver samples exhibiting readilydetectable level of hepcidin mRNA as previously analyzed by Northern blot. Resultswere expressed in log 2 of ratio sample versus standard. The Urinary hepcidinassay was performed, and hepcidin concentration in urine expressed as nghepcidin/mg creatinine. Because the lower detection limit of the urinaryhepcidin assay is 1 ng/mg creatinine, samples with lower value were attributeda value of 1 ng/mg creatinine (Detivaudet al., 2005)Hepcidin mRNA and urinary hepcidin levels appeared to bepositively correlated and increasing in parallel. This demonstrates that theurinary hepcidin assay can be considered a valid reflection of hepatic hepcidinproduction.
Though these results might not be accurate since they were takenfrom patients going to surgery and having hepatic problems (Detivaudet al., 2005)In conclusion, urinary hepcidin concentrations significantlycorrelates with hepatic hepcidin mRNA concentrations, indicating that it is avalid approach to evaluate body’s hepcidin expression. it is also found that inhumans hepcidin levels correlates with hepatic iron stores and hemoglobinlevels and maybe be affected by hepatic dysfunction (Detivaudet 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 thefour methods ELISA technique seems to be most productive and reliable one, incase of productivity, reproducibility, sample preparation, time, samplevariety, and processing steps.A reliable and easy to apply hepcidinquantification method could prove a valuable tool for diagnostic applications,as hepcidin levels may be used for the differential diagnosis of several irondisorders, and with further refinement, hepcidinassays could contribute to the diagnosis and improved understandingof the pathophysiology of iron disorders.