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:

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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 

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