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Clinical measurement of Hepcidin-25 in human serum: Is quantitative mass spectrometry up to the job?

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From its discovery, hepcidin has generated many hopes in terms of diagnosis and management of a wide variety of iron-related diseases. However, in clinical use its accurate quantification remains a challenge due to the limited sensitivity, specificity or reproducibility of the techniques described. In this work, we adapted a highly specific and quantitative mass spectrometry method based on selected reaction monitoring (SRM) to measure hepcidin. Our objective was to adapt the feasibility and reproducibility of the workflow to a clinical environment. Analytical validation was performed according to ISO 15189 norms for determining the limit of detection (LOD, 2 ng/mL), limit of quantification (LOQ, 6 ng/mL), repeatability, reproducibility and linearity (up to 200 ng/mL). Using the serum of patients with various iron-related diseases we compared our SRM detection method to the well-characterized competitive ELISA (cELISA) test. The two methods were commutable (Bland-Altman plot) and we found a positive and significant correlation (r2 = 0.96, Pearson correlation coefficient p < 0.001) between both methods, although the absolute concentration determined is different from factor 5. The validation of our SRM method encourages us to propose it as an alternative approach for accurate determination of hepcidin in human samples for clinical diagnosis, follow-up and management of iron-related diseases.
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Clinical
measurement
of
Hepcidin-25
in
human
serum:
Is
quantitative
mass
spectrometry
up
to
the
job?
Constance
Delabya,b,c,
Jérôme
Vialareta,b,c,
Pauline
Brosa,b,c,
Audrey
Gabellea,b,c,
Thibaud
Lefebvred,e,f,
Hervé
Puyd,e,f,
Christophe
Hirtza,b,c,,
Sylvain
Lehmanna,b,c,
aCHU
Montpellier,
Institut
de
Recherche
en
Biothérapie,
Hôpital
St
Eloi,
Laboratoire
de
Biochimie
Protéomique
Clinique
et
CCBHM,
Montpellier
F-34000,
France
bUniversité
Montpellier
1,
Montpellier
F-34000,
France
cINSERM
U1040,
Montpellier
F-34000,
France
dCentre
de
Recherche
sur
l’Inflammation
(CRI)/UMR
1149
INSERM
Université
Paris
Diderot,
France
eAP-HP,
Centre
Franc¸ais
des
Porphyries,
Hôpital
Louis
Mourier,
178
rue
des
Renouillers,
92701
Colombes
Cedex,
France
fLaboratory
of
Excellence
GR-Ex,
France
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
16
December
2013
Received
in
revised
form
10
February
2014
Accepted
14
February
2014
Keywords:
Hepcidin
Iron
deficiency
Mass
spectrometry
ELISA
a
b
s
t
r
a
c
t
From
its
discovery,
hepcidin
has
generated
many
hopes
in
terms
of
diagnosis
and
man-
agement
of
a
wide
variety
of
iron-related
diseases.
However,
in
clinical
use
its
accurate
quantification
remains
a
challenge
due
to
the
limited
sensitivity,
specificity
or
repro-
ducibility
of
the
techniques
described.
In
this
work,
we
adapted
a
highly
specific
and
quantitative
mass
spectrometry
method
based
on
selected
reaction
monitoring
(SRM)
to
measure
hepcidin.
Our
objective
was
to
adapt
the
feasibility
and
reproducibility
of
the
workflow
to
a
clinical
environment.
Analytical
validation
was
performed
according
to
ISO
15189
norms
for
determining
the
limit
of
detection
(LOD,
2
ng/mL),
limit
of
quantification
(LOQ,
6
ng/mL),
repeatability,
reproducibility
and
linearity
(up
to
200
ng/mL).
Using
the
serum
of
patients
with
various
iron-related
diseases
we
compared
our
SRM
detection
method
to
the
well-characterized
competitive
ELISA
(cELISA)
test.
The
two
methods
were
commutable
(Bland–Altman
plot)
and
we
found
a
positive
and
significant
correlation
(r2=
0.96,
Pearson
correlation
coefficient
p
<
0.001)
between
both
methods,
although
the
absolute
concentra-
tion
determined
is
different
from
factor
5.
The
validation
of
our
SRM
method
encourages
us
to
propose
it
as
an
alternative
approach
for
accurate
determination
of
hepcidin
in
human
samples
for
clinical
diagnosis,
follow-up
and
management
of
iron-related
diseases.
©
2014
The
Authors.
Published
by
Elsevier
B.V.
on
behalf
of
European
Proteomics
Association
(EuPA).
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Abbreviations:
SRM,
selected
reaction
monitoring;
ELISA,
enzyme-linked
immunosorbent
assay;
aa,
aminoacid;
TCA,
trichloroacetic
acid.
Corresponding
authors
at:
CHU
Montpellier,
Institut
de
Recherche
en
Biothérapie,
Hôpital
St
Eloi,
Laboratoire
de
Biochimie
Protéomique
Clinique
et
CCBHM,
Montpellier
F-34000,
France.
Tel.:
+33
603422349.
E-mail
addresses:
c-hirtz@chu-montpellier.fr
(C.
Hirtz),
s-lehmann@chu-montpellier.fr
(S.
Lehmann).
http://dx.doi.org/10.1016/j.euprot.2014.02.004
2212-9685/©
2014
The
Authors.
Published
by
Elsevier
B.V.
on
behalf
of
European
Proteomics
Association
(EuPA).
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Author's personal copy
e
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61
1.
Introduction
Hepcidin
is
a
25
amino-acid
(aa)
cystein-rich
peptide
involved
in
iron
metabolism:
it
regulates
both
intestinal
iron
absorp-
tion
and
macrophage
iron
recycling.
There
are
several
clinical
applications
for
hepcidin
measurements
such
as
iron
overload
disorders
and
anemia,
chronic
inflammation,
kidney
disease
and
cancer
[1,2].
This
peptide
can
also
be
found
in
the
20,
22
and
24-aa
isoforms
[3];
however
the
histopathological
rel-
evance
of
these
isoforms
still
needs
to
be
determined
and
they
are
most
likely
the
result
of
the
degradation
of
the
bioactive
25-aa
isoform.
Since
2001
[4–6],
various
methods
have
been
published
on
accurate
quantification
of
hep-
cidin
[7–9].
Among
them,
we
can
mention
radio-immuno
assay
(RIA),
Enzyme-linked
immunosorbent
assay
(ELISA)
and
more
recently,
mass
spectrometry
analysis
(SELDI-TOF
and
LC–MS/MS).
Immunoassays
are
based
on
the
use
of
anti-hepcidin
antibodies
(therefore
potentially
identifying
the
different
isoforms)
and
mass
spectrometric
approaches
to
specifically
detect
the
different
types
of
hepcidin.
Further-
more,
absolute
quantitation
is
possible
through
normalization
via
a
spiked
internal
standard
of
hepcidin.
These
methods
have
their
limits,
especially
in
regards
to
specificity
(for
the
25
aa-isoform)
and
assay
reproducibility
for
repetitive
quantifi-
cation
of
a
same
sample.
In
addition,
the
different
approaches
differ
greatly
in
terms
of
absolute
values
of
hepcidin.
These
discrepancies
are
specifically
related
to
the
inherent
prop-
erties
of
the
peptide’s
structure,
its
low
immunogenicity,
its
propensity
to
aggregate
as
well
as
the
existence
of
smaller
iso-
forms,
which
could
affect
the
immunochemical
assay
results.
However,
the
various
studies
comparing
these
approaches
have
reported
a
good
correlation
between
the
different
meth-
ods
[7,8].
Recently
and
in
order
to
standardize
the
quantitation
of
hepcidin,
different
methods
have
been
compared
via
inter-
laboratory
studies
[7,8].
These
studies
have
underlined
the
challenges
in
developing
a
reliable
assay
and
recommended
the
use
of
internal
standards
for
MS-based
methods,
using
a
calibrator
mimicking
human
serum
and
setting
a
consensus
on
calibrator
levels.
We
have
developed
a
nano-liquid
chromatography
tandem-mass
spectrometry
(LC–MS/MS)
to
reliably
quantify
hepcidin-25
in
human
serum.
Nano-LC
system
are
generally
not
recommended
in
clinical
applications
since
they
are
known
for
not
being
as
robust
as
micro-LC.
However
in
our
workflow
we
used
a
nano-Chip/MS
system
which
is
known
to
be
more
versatile
and
reliable
than
regular
nano-LC
system.
Firstly,
we
performed
an
analytical
validation
to
test
the
specificity,
reproducibility
and
repeatability
of
LC–MS/MS
as
well
as
determining
its
LOD,
LOQ
and
linearity.
We
then
com-
pared
our
results
to
the
well
characterized
C-ELISA
method
to
quantify
serum
hepcidin-25
in
human
samples
[10].
Despite
discrepancies
in
the
absolute
values
of
hepcidin
measured,
we
showed
a
good
correlation
between
these
two
methods
(r2=
0.96),
thus
validating
our
LC–MS/MS
assay
for
accurate
measurement
of
hepcidin
in
human
serum
samples.
Further-
more,
we
underlined
the
relevance
of
this
measurement
in
iron-deficiency
anemia,
anemia
of
inflammation
and
other
iron-related
diseases.
2.
Materials
and
methods
2.1.
Chemicals
used
Trichloroacetic
acid
(TCA),
ref.
T9159-250G
(Sigma–Aldrich);
Water
ULC–MS,
ref.
23214102,
formic
acid
ULC–MS
(FA)
ref.
069141A8,
acetonitrile
ULC–MS
(ACN)
ref.
01204101
all
from
Biosolve
(Dieuze,
France);
Normal
Goat
serum,
ref.
S-
1000
Clinisciences
(Nanterre,
France);
Protein
LoBind
tube
1.5
mL,
ref.
022431081
Eppendorf
(Le
Pecq,
France);
glass
vial
insert
ref.
5181–1270
and
ProtID-Chip-43
II
ref.
G4240-
62005
both
from
Agilent
Technologies
(Santa
Clara,
CA,
USA).
Human
hepcidin
standard
(DTHFPICIFCCGCCHRSKCGMCCKT)
and
internal
hepcidin
standard
(DTHFPICIFCCGCCHRSKCGM-
CCKT)
[13C6,15 N4]
Arg16 were
purchased
from
Eurogentec
(Seraing,
Belgium)
with
a
purity
>97%
assessed
by
RP-HPLC
and
mass
spectrometry.
Standard
peptides
were
synthetized
in
lyophilized
form
with
the
same
amino
acid
sequence
(25
AA)
and
folding
(4
disulfite
bridges
Cys
7–23;
10–13;
11–19;
14–22)
as
the
endogenous
human
hepcidin-25.
2.2.
Human
samples
and
ELISA
determination
of
hepcidin
In
France,
since
hepcidin
is
already
an
accepted
serum
ana-
lyte
used
in
clinical
settings,
which
is
determined
using
mass
spectrometry
or
validated
competitive
C-ELISA
[10],
there
was
no
need
for
a
specific
authorization
from
an
Ethics
committee
for
this
work.
However,
serum
samples
included
in
this
study
were
part
of
a
biobank
(official
registration
#
DC-2008-417)
and
all
patients
signed
an
informed
consent
form
to
authorize
the
use
of
their
samples
for
research
conducted
in
accordance
with
the
local
Ethics
committee.
2.3.
Liquid
chromatography
(LC)
separation
Nano-LC
separation
was
carried
out
on
a
1290
nano-LC
system
(Agilent
technologies).
Peptides
were
loaded
on
a
ProtID-
Chip-43
(Agilent
technologies)
containing
a
43
mm
×
75
m
analytical
column
and
a
40
nL
trap-column
packed
with
Zor-
bax
300SB-C18 5
m.
The
mobile
phase
was
composed
of
H2O/ACN/FA
(phase
A
97:3:0.1,
v/v/v
and
phase
B
10:90:0.1,
v/v/v).
The
sample
was
loaded
on
the
trapping
column
with
a
flow
rate
of
2.5
l/min
using
the
capillary
pump
to
deliver
an
isocratic
enrichment
phase
composed
of
15%
B.
Further-
more,
7
l
of
flush
volume
was
used
for
cleaning
the
trapping
column
from
un-retained
compound.
The,
trapped
peptides
were
separated
from
the
analytical
column
using
the
nanop-
ump.
A
10-minute
gradient
was
performed,
starting
with
3%
of
solvent
B
and
linearly
ramped
to
100%
in
7
min.
The
column
was
then
washed
for
2
min
and
re-equilibrated
during
1
min
with
97%
of
solvent
A.
2.4.
Multiple
reaction
monitoring
analysis
Mass
spectrometric
detection
was
performed
using
a
6490
triple
quadripole
with
a
nano-ESI
source
operating
in
positive
mode
and
in
SRM
mode
(Agilent
technologies,
Wald-
bronn,
Germany).
The
control
of
the
LC–MS/MS
was
done
Author's personal copy
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with
MassHunter
Software
(Agilent
technologies,
Waldbronn,
Germany).
The
ESI
nano
spray
was
set-up
according
to
the
following
specificities:
capillary
tension
1700–2100
V,
nebu-
lization
gas
flow
11l/min
and
temperature
at
150 C.
We
selected
the
most
abundant
ion
detected
after
standard
pep-
tide
injection.
Precursor
ions
followed
were
559.4
m/z
(z
=
5)
for
the
endogenous
Hepcidin
and
560.6
m/z
(z
=
5)
for
the
heavy
standard.
Precursors
ions
were
transferred
inside
the
first
quadrupole
with
an
accelerator
voltage
of
4
V
while
ion
funnel
RF
high
pressure
was
set
to
180
V
and
low
pressure
to
80
V.
Pre-
cursor
ions
were
fragmented
in
“Product
Ion
Scan”
mode
and
the
5
most
abundant
generated
fragments
were
selected
to
constitute
the
SRM
method
(560.6
1045.5,
985,
766.7,
696.8,
646.2;
559.4
1045,
983,
764.6,
694.8,
645).
Collision
energies
(CE)
were
optimized
as
described
in
Table
1.
2.5.
Preparation
of
Hepcidin-25
standards
Lyophilized
standards
were
resuspended
at
200
ng/L
with
H2O/ACN/FA
(66.2:33.8:0.1,
v/v/v).
The
solution
was
then
sep-
arated
into
aliquots
of
50
L
in
LoBind
tubes
and
stored
at
80 C.
The
internal
standard
was
prepared
at
5
ng/L
using
the
same
protocol.
Isotopically-labeled
hepcidin
was
cho-
sen
as
the
internal
standard
because
of
its
physicochemical
properties
which
are
similar
to
hepcidin.
Both
compounds
exhibited
the
same
behavior
during
the
preparation
and
chro-
matographic
process
while
the
mass
difference
of
10
amu,
provided
by
the
heavy
aa
of
the
internal
standard,
permitted
the
independent
detection
of
each
compound.
Appropriate
dilutions
of
200
ng/L
hepcidin
stock
solutions
(0.125,
1.25,
25
g/mL)
were
made
with
H2O/ACN/FA
(79:20:1,
v/v/v)
to
prepare
the
matrix-based
calibration
curve
(normal
goat
serum)
at
the
concentration
range
of
0–200
ng/mL
(0,
5,
10,
20,
50,
100,
200
ng/mL).
The
internal
standard
was
spiked
in
the
model
matrix
and
biological
sample
at
a
final
concentration
of
100
ng/mL
(Table
2).
2.6.
Hepcidin-25
pre-fractionation
protocol
All
experiments
were
performed
at
4C.
In
a
1.5
mL
LoBind
tube,
50
L
of
serum
sample
was
mixed
with
1
L
of
internal
hepcidin
standard
solution
(5
ng/L),
vortexed
10
s
and
then
1:1
ratio
of
4%
trichloroacetic
acid
(TCA)
solution
was
added.
Samples
were
then
vortexed
a
few
seconds
and
centrifuged
at
17,000
×
g
during
5
minutes
to
obtain
a
clear
supernatant,
fol-
lowed
by
a
new
centrifugation
step
at
17,000
×
g
during
5
min
after
thawing
on
ice.
The
supernatant
was
transferred
into
a
new
LoBind
tube
and
dried
in
a
vacuum
concentrator
(Lab-
conco,
Kansas
city,
USA).
The
samples
were
resuspended
with
10
L
of
H2O/ACN/FA
(20:1:79,
v/v/v)
and
vortexed
at
1000
rpm
for
10
min.
The
LC
vial
was
then
centrifuged
3
min
at
17,000
×
g
at
room
temperature
before
transferring
the
sample
and
mak-
ing
sample
preparations
in
duplicates.
2.7.
Method
validation
To
validate
this
method
we
evaluated
specificity,
linearity,
LOD,
LOQ,
precision
and
accuracy.
Specificity
was
assessed
by
confirming
that
there
were
no
interference
peaks
at
the
same
retention
time
than
those
of
the
analytes
in
the
blank
sample.
Table
1
Optimized
LC-SRM
method
and
selected
compound
targets.
Compound
name ISTD
Precursor
ion
MS1
res
Product
ion
MS2
res
Dwell
time
Fragmentor
Collision
energy
Cell
accelerator
voltage
Polarity
Fragment
DTHFPICIFCCGCCHRSKCGMCCKT
True
560.6
Wide
1045.5
Wide
40
380
19
4
Positive
[y19]2+
DTHFPICIFCCGCCHRSKCGMCCKT
True
560.6
Wide
985
Wide
40
380
19
4
Positive
[y18-H2O]2+
DTHFPICIFCCGCCHRSKCGMCCKT
True
560.6
Wide
766.7
Wide
40
380
11
4
Positive
[y21]3+
DTHFPICIFCCGCCHRSKCGMCCKT
True
560.6
Wide
696.8
Wide
40
380
11
4
Positive
[y19]3+
DTHFPICIFCCGCCHRSKCGMCCKT
True
560.6
Wide
646.2
Wide
40
380
11
4
Positive
[y23]4+
DTHFPICIFCCGCCHRSKCGMCCKT
False
559.4
Wide
1042
Wide
40
380
19
4
Positive
[y19]2+
DTHFPICIFCCGCCHRSKCGMCCKT
False
559.4
Wide
983
Wide
40
380
19
4
Positive
[y18-H2O]2+
DTHFPICIFCCGCCHRSKCGMCCKT
False
559.4
Wide
764.6
Wide
40
380
11
4
Positive
[y21]3+
DTHFPICIFCCGCCHRSKCGMCCKT
False
559.4
Wide
694.8
Wide
40
380
11
4
Positive
[y19]3+
DTHFPICIFCCGCCHRSKCGMCCKT
False
559.4
Wide
645
Wide
40
380
11
4
Positive
[y23]4+
Author's personal copy
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3
(
2
0
1
4
)
60–67
63
Table
2
Appropriate
dilutions
of
light
and
heavy
hepcidin
stock
solutions
to
prepare
the
calibration
curve
using
model
matrix
(Normal
Goat
serum)
in
the
concentration
range
of
0
to
200
ng/mL
(0,
5,
10,
20,
50,
100,
200
ng/mL).
Internal
standard
(Heavy
hepcidin)
was
spiked
in
the
model
matrix
and
in
the
biological
sample
at
a
final
concentration
of
100
ng/mL.
Normal
goat
serum
Heavy
hepcidin
Light
hepcidin
Volume
(L)
Volume
(L)
Initial
concentration
(ng/L)
Final
concentration
(ng/mL)
Volume
(L)
Initial
concentration
(ng/L)
Final
concentration
(ng/mL)
50
1
5
100
0
0.125
0
1
100
2
5
1
100
4
10
1
100
0.8
1.25
20
1
100
2
50
1
100
4
100
1
100
8
200
In
order
to
do
this,
a
0
ng/mL
point
was
added
to
the
calibra-
tion
curve
and
the
software
took
it
into
account
for
computing
it.
Calibration
curves
were
constructed
over
the
0–200
ng/mL
range.
Intra-assay
precision
and
accuracy
were
evaluated
by
analyzing
the
replicates
of
the
calibration
curve
on
the
same
day.
Inter-assay
precision
and
accuracy
were
assessed
by
ana-
lyzing
the
same
quality
control
(QC)
samples
on
series
of
analyses
performed
on
different
days.
The
LOD
has
been
determined
using
the
signal
obtained
with
the
replicates
at
100
ng/mL.
S/N
average
obtained
was
of
148.
LOD
is
calculated
based
on
S/N
=
3.
LOD
of
2
ng/mL
was
obtained.
LOQ
was
deter-
mined
in
the
same
way
but
using
S/N
=
10.
LOQ
of
6
ng/mL
was
obtained.
Accuracy
was
calculated
using
the
Agilent
software
(Masshunter
Quantitative
analysis)
based
on
the
calibration
curve
equation.
Formula
used
was
Accuracy
=
[(calculated
concentration)/(expected
concentration)]
×
100”.
2.8.
Data
treatment
and
statistics
We
used
the
MassHunter
Quantitative
Analysis
software
(Agi-
lent
technologies)
to
conduct
bioinformatics
data
treatment.
All
replicate
results
for
the
calibration
curve
were
loaded
into
the
software
database.
An
automatic
quantification
method
was
used
to
treat
all
data
in
order
to
obtain
the
equation
curve
with
the
best
fit
for
the
experimental
points.
The
two
transi-
tions
559.4
694.8
(light
hepcidin)
and
560.6
696.8
(heavy
hepcidin)
were
used
as
quantifiers
and
were
automatically
detected
on
specific
retention
time
windows.
Other
transitions
were
used
as
qualifiers.
Statistical
analyses
were
performed
with
the
MedCalc
software
(7.3).
Unless
indicated,
we
used
non-parametric
tests
(Kruskal–Wallis)
to
compute
all
available
data.
We
also
used
the
Bland
and
Altman
plot
[11]
and
Dem-
ing
adjusted
regression
curves
[12]
to
test
the
commutability
of
the
methods.
3.
Results
and
discussion
3.1.
Pre-fractionation
optimization
In
order
to
measure
the
hepcidin-25
level
using
LC-SRM
method,
we
adapted
the
pre-fractionation
protocol
from
Murao
et
al.
[13].
Based
on
protein
precipitation,
this
method
is
fast,
simple,
reproducible,
cost-effective
and
overall
very
adapted
to
a
routine
clinical
environment.
TCA
precipitation
showed
a
sufficient
recovery
for
peptides
with
a
molecular
weight
under
3
kDa,
and
allowed
a
direct
injection
of
the
sample
without
evaporation
or
dilution.
With
this
sample
preparation,
we
were
able
to
detect
5
times
more
hepcidin-25
than
with
direct
analysis
(not
shown),
probably
due
to
sam-
ple
simplification
and
consequently
matrix
effect
reduction.
On
a
serum
sample,
the
reproducibility
test
was
calculated
using
an
area
ratio
endogeneous
hepcidin/heavy
hepcidin
and
it
showed
a
coefficient
of
variation
of
1.5%
(see
below).
3.2.
LC-SRM
method
optimization
Firstly,
transmission
of
ion
precursor
through
the
triple
quadrupole
was
optimized.
For
this
purpose,
cell
accelerator
voltage
(CAV)
and
RF
of
ion
funnel
were
optimized
to
increase
ion
transmission
inside
the
Q1.
Optimum
CAV
and
ion
funnel
RF
enabled
a
gain
of
signal
with
respectively
49%
and
30%
com-
pared
to
reference
values.
The
CAV
optimum
value
was
found
at
4
V
(reference
7
V)
and
the
ion
funnel
RF
at
180–80
V
(refer-
ence
210–110
V).
Then
1
L
of
10
g/mL
solution
hepdidin-25
standards
(light
and
heavy)
were
injected
to
determine
the
most
abundant
ion
detected
in
“full-scan
mode”
for
the
light
and
heavy
standards
(Fig.
1A).
The
mass
spectra
showed
a
similar
series
of
ions,
mainly
the
quadruply,
quintuply
and
sextuply
charged
molecular
ions
for
both
standards.
A
highest
S/N
were
found
at
m/z
559.4
(M
+
5H]5+)
for
the
light
standard
ions
and
at
560.6
m/z
(M
+
5
H]5+)
for
the
heavy
standard
ions.
Precursor
ions
were
fragmented
in
“product
ion
scan”
mode
to
observe
the
subsequent
generated
fragments.
Five
transitions
per
peptide
were
followed
and
two
transitions
559.4
694.8
(light
hepcidin)
and
560.6
696.8
(heavy
hepcidin)
were
used
as
quantifiers
(higher
S/N
ratio).
The
sequence
of
the
pep-
tides
chosen
was
located
in
the
unfolded
N
termini
area
of
the
protein
sequence
(y19
to
y23
ions
with
a
mix
of
charge
states)
(Fig.
1B).
Accordingly,
the
SRM
method
was
optimized
for
the
collision
energy
(CE)
transition
by
transition.
This
step
was
performed
automatically
with
“Peptide
Optimizer”
soft-
ware
using
a
ramping
CE
in
order
to
find
the
optimum.
CE
was
a
predominant
parameter
for
the
optimization,
with
an
SRM
Signal
enhancement
of
up
to
6-fold
compared
to
the
method
of
reference.
Optimum
conditions
were
achieved
by
re-suspending
sam-
ples
in
20%
acetonitrile/1%
formic
acid/79%
water
and
storing
Author's personal copy
64
e
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p
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e
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p
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o
t
e
o
m
i
c
s
3
(
2
0
1
4
)
60–67
Fig.
1
(A)
Light
hepdidin-25
standard
chromatograms
obtained
by
nano-HPLC–MS
in
“full-scan
mode”
showing
quadruply,
quintuply
and
sextuply
charged
molecular
ions.
(B)
MS/MS
spectra
of
the
[M
+
5
H]5+ precursor
of
light
hepdidin-25
standard.
In
red,
the
peptide
fragment
chosen
located
in
the
unfolded
N
termini
area
of
the
protein
sequence
(y19
to
y23
ions).
the
sample
in
a
LC
glass
vial.
The
samples
were
then
loaded
with
15%
of
B
phase
(90%
CAN,
0.1%
FA)
at
2.5
l/min
flow
rate
using
the
capillary
pump.
This
increased
the
signal/noise
ratio
about
30-fold
probably
due
to
a
reduction
of
nonspecific
interactions
between
proteins
and
surfaces,
and
a
reduction
in
sample
complexity
linked
to
the
fact
that
in
this
situ-
ation
a
number
of
hydrophilic
peptides
were
eluted
before
the
analysis.
To
clean
the
trapping
column
from
un-retained
compound
we
used
7
l
of
flush
volume
as
well
as
a
10-min
chromatographic
separation
gradient
starting
with
3%
of
sol-
vent
B
and
linearly
ramped
to
100%
in
7
min.
The
column
was
then
washed
for
2
min
and
re-equilibrated
for
1
min
with
97%
of
solvent
A.
Under
these
conditions,
elution
time
of
the
hepcidin-25
peptide
was
3
min
(65%
phase
B).
3.3.
Analytical
validation
and
quantitation
The
specificity
of
the
method
was
evaluated
by
validating
the
absence
of
interference
peaks
in
blank
sample
at
the
retention
time
of
the
analytes
(0
ng/mL
of
hepcidin-25
in
normal
goat
serum).
This
blank
sample
was
integrated
by
the
Mass
Hunter
software
into
the
calibration
curve
per-
formed
using
a
model
matrix
(normal
goat
serum)
free
of
hepcidin-25.
Calibration
curves
were
constructed
over
the
0–200
ng/mL
range
based
on
hepcidin
values
found
with
the
orthogonal
analysis
method.
The
equation
was
linear
when
ignoring
origin
and
weighted
1/y.
r2was
obtained
with
the
fol-
lowing
equation
y
=
1.54636
+
0.02970
and
was
equal
to
0.9665
(Fig.
2B).
Finally,
the
quantitation
of
hepcidin-25
in
cohort
sam-
ples
did
not
exceed
140
ng/mL
therefore
the
calibration
curve
(0–200
ng/mL)
is
perfectly
suitable
for
dosage.
Each
LC-SRM
dosage
was
performed
in
duplicate
in
order
to
calculate
a
coefficient
of
variation.
Intra-assay
precision
and
accuracy
were
assessed
by
analyzing
four
replicates
of
the
cali-
bration
curve
point
on
the
same
day.
Intra-assay
precision
was
around
7%
for
the
50
ng/mL
point
and
28%
for
the
lowest
con-
centration
point
(5
ng/mL).
Intra-assay
accuracy
was
close
to
100%
for
the
20,
50,
100
and
200
ng/mL
points
and
around
80%
for
the
lowest
concentration
points
(5
and
10
ng/mL)
(Table
3).
In
order
to
evaluate
inter-assay
precision
and
accuracy,
we
chose
2
points
on
the
calibration
curve
(50
and
200
ng/mL)
as
quality
control
(QC)
samples
which
were
analyzed
on
four
sep-
arate
days.
Inter-assay
precision
was
around
9–10%,
accuracy
was
close
to
80%
for
the
two
QCs
and
LOD
was
determined
using
the
signal
obtained
with
the
replicates
at
100
ng/mL.
S/N
average
obtained
was
148.
LOD
is
calculated
based
on
S/N
=
3.
LOD
of
2
ng/mL
was
obtained.
LOQ
was
determined
in
the
same
way
but
using
S/N
=
10.
LOQ
of
6
ng/mL
was
obtained.
Author's personal copy
e
u
p
a
o
p
e
n
p
r
o
t
e
o
m
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c
s
3
(
2
0
1
4
)
60–67
65
Fig.
2
(A)
Workflow
for
nano-HPLC–MS
quantification
of
hepdidin-25
in
human
serum
samples.
(B)
Calibration
curves
of
hepdidin-25
using
model
matrix
(normal
goat
serum)
in
the
concentration
range
of
0–200
ng/mL
(0,
5,
10,
20,
50,
100,
200
ng/mL).
The
equation
was
linear
when
ignoring
origin
and
weighted
1/y.
r2,
obtained
with
the
following
equation
y
=
1.054636
+
0.029760,
was
equal
to
0.9665.
However,
one
cannot
exclude
that,
in
some
human
samples,
interfering
compounds
could
affect
the
precision
of
the
assay,
and
additional
validation
to
comply
with
the
ISO
15189
norm
might
be
needed.
3.4.
Comparison
of
LC-SRM
and
C-ELISA
approaches
for
hepcidin
quantitation
Among
the
different
methods
used
to
quantify
bioactive
hepcidin-25
in
serum,
the
competitive
C-ELISA
developed
by
Ganz
et
al.
in
2008
[10]
is
a
very
valuable
method
that
correlates
well
with
a
specific
weak
cation
exchange
(WCX)
time-of-flight
MS
assay
[1].
We
further
analyzed
serum
samples
previously
Table
3
Intra-assay
coefficient
of
variation
and
accuracy
(%).
Light
concentration
(pg/L)
Intra-assay
CV
(%)
Intra-assay
precision
accuracy
(%)
5
28
24
10
21
18
20
8
6
50
7
3
100
17
0
200
13
9
measured
with
this
C-ELISA
method
using
our
MS
protocol
(Fig.
3A).
We
found
a
significant
positive
correlation
(r2=
0.96,
Pearson’s
r
P
<
0.001)
and
the
Bland–Altman
plot
(Fig.
3B)
vali-
dated
the
commutability
of
these
methods.
However,
the
slope
of
the
linear
correlation
(5.0255x)
indicated
that
our
assay
pro-
vided
values,
which
were
five
times
lower
than
those
obtained
with
the
C-ELISA.
This
result
is
likely
related
to
the
fact
that
MS
methods
are
fully
specific
(i.e.
only
the
25aa
isoform
is
quantified)
and
to
the
difference
in
origin
and
purity
of
the
material
used
for
the
standard
curves.
Furthermore,
the
LC-
SRM
CV
for
the
30
patients
in
our
study
was
9%
compared
to
5–19%
in
the
study
previously
described
by
Ganz
et
al.
In
addi-
tion,
our
LOD
is
2
ng/mL
compared
with
a
LOD
at
5.5
ng/mL
for
the
reference
C-ELISA
method.
Finally,
our
method
shows
reproducibility
under
10%,
compared
to
the
reproducibility
of
C-ELISA
which
rises
to
12%.
Thus,
our
experimental
results
underline
the
robustness
and
reliability
of
our
quantitative
assay
for
hepcidin-25,
as
well
as
its
suitability
for
the
relevant
evaluation
of
hepcidin-25
in
human
serum
within
a
clinical
environment.
As
expected,
hepcidin-25
levels
differed
for
patients
suffering
from
iron
deficiency
(mean
value
of
29.87
ng/mL
or
49.97
ng/mL
for
patients
with
ferritin
<
300
ng/mL
and
>300
ng/mL,
respectively)
and
our
results
show
an
appropri-
ate
correlation
between
hepcidin
and
ferritin
(r2=
0.38
or
by
splitting
into
2
groups,
r2=
0.42
and
0.74
for
patients
with
Author's personal copy
66
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m
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c
s
3
(
2
0
1
4
)
60–67
C
Anemia Hepcidin
sTfr/log
ferrin
sTfR/lo
g
ferrin
25
ng/mL
Iron
Deficiency
Anem
ia
Mixed
Anem
ia
Anemia of
Inflamm
aon
≥ 1
≥ 1
< 1
< 1
> 25
ng/mL
Hepcidin levels (ng/mL)
0
20
40
60
80
100
120
140
MRM
800
700
600
500
400
300
200
100
0
ELISA
Bland-A ltman plot
0
100
200
300
400
500
AVE
RAGE of ELISA
an
d MRM
200
180
160
140
120
100
80
60
(ELISA - MRM) / Average %
Mea
n
143,6
-1.96 SD
99,3
+1.96 SD
187,8
BA
Fig.
3
(A)
Comparison
of
hepcidin-25
concentrations
measured
in
serum
samples
from
patient
using
nano
HPLC–MS
and
ELISA.
A
significant
positive
correlation
was
obtained
(r2=
0.96,
Pearson
product-moment
correlation
coefficient
(Pearson’s
r)
P
<
0.001).
(B)
Commutability
of
the
methods
(ELISA
and
SRM)
using
a
Bland–Altman
plot.
The
Deming
adjusted
regression
curve
equation
is:
y
=
11,7691
+
4,9683x.
(C)
Proposed
patient
stratification
scheme
for
biochemical
discrimination
of
iron
deficiency
versus
inflammatory
status
as
set
up
in
this
assay
(note:
the
origin
and
purity
of
the
standard
material
may
modify
this
value).
ferritin
<
300
ng/mL
and
>300
ng/mL,
respectively),
thus
reflecting
the
regulation
of
both
proteins
by
iron
stores.
Moreover,
hepcidin
values
also
differed
in
patients
with
an
inflammatory
status
(mean
value
of
4.64
ng/mL
for
patients
with
CRP
<
10
mg/dL,
vs.
mean
value
of
55.85
ng/mL
for
patients
with
CRP
>
10
mg/dL).
The
values
of
hepcidin
in
our
cohort
were
distributed
between
10
and
100
ng/mL.
Though,
this
assay
allows
biochemical
discrimination
of
iron
defi-
ciency
status
and
inflammatory
status
of
patients
as
proposed
in
the
decision
tree
illustrated
in
Fig.
3C
based
on
the
article
by
Sasu
et
al.
[14].
4.
Conclusion
These
past
10
years,
numerous
assays
relying
on
immuno-
chemical
and
mass
spectrometry
methods
have
been
developed
and
described
to
measure
hepcidin
in
human
sam-
ples
of
blood
and
urine.
The
experimental
method
described
in
the
present
work
matches
the
appropriate
standard
for
clin-
ical
measurement
of
hepcidin-25
in
human
serum.
Hepcidin
levels
obtained
differed
from
a
reference
C-ELISA
assay
which
validated
the
need
for
a
calibrator
mimicking
human
serum
and
establishing
a
consensus
on
calibrator
levels.
This
method
also
has
the
ability
to
detect
the
truncated
20,
22
and
24-aa
isoforms
of
hepcidin
(not
shown)
even
though
the
relevance
of
these
isoforms
for
clinical
applications
still
needs
to
be
established
[15].
More
importantly,
our
method
relies
on
sim-
ple,
robust,
reproducible
and
straightforward
cost-effective
pre-analytical
steps
that
are
specifically
adapted
to
a
clinical
environment,
thus
avoiding
the
need
of
solid
phase
extrac-
tion
for
example.
In
conclusion,
our
approach
is
compatible
with
routine
hepcidin
measurements
in
daily
clinical
practice
and
for
discriminating
between
iron-deficiency
anemia
and
anemia
of
chronic
disease.
Acknowledgments
This
study
was
supported
by
grants
from
Laboratory
of
Excel-
lence
GR-Ex,
reference
ANR-11-LABX-0051.
The
labex
GR-Ex
is
funded
by
the
program
“Investissements
d’avenir”
of
the
Author's personal copy
e
u
p
a
o
p
e
n
p
r
o
t
e
o
m
i
c
s
3
(
2
0
1
4
)
60–67
67
French
National
Research
Agency,
reference
ANR-11-IDEX-
0005-02.
Authors
are
grateful
to
laboratory
of
excellence
GR-Ex
for
access
to
clinical
serum
samples.
The
authors
would
like
to
specially
thank
Agilent
Technologies
for
their
help
within
our
collaboration
and
Bénédicte
Clément
for
editing
the
manuscript.
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e
f
e
r
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n
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... For MS methods, hepcidin was quantified following previously described methodologies [15,16]. Briefly, for center 1 (Montpellier), nano-LC separation (1290-nano-LC system, Agilent Technology) was performed followed by MS detection using a 6490 triple quadrupole with a nano-ESI source operating in positive mode and SRM mode (Agilent Technology, CA, USA) [15]. ...
... For MS methods, hepcidin was quantified following previously described methodologies [15,16]. Briefly, for center 1 (Montpellier), nano-LC separation (1290-nano-LC system, Agilent Technology) was performed followed by MS detection using a 6490 triple quadrupole with a nano-ESI source operating in positive mode and SRM mode (Agilent Technology, CA, USA) [15]. For center 2 (Paris), LC system consisted in an Acquity ultra performance liquid chromatography (Waters, Saint Quentin en Yvelines, France) and MS/MS detection was carried out using a Xevo-TQMS (Waters) with electrospray ion source [16]. ...
... Hepcidin was quantified in the serum of 119 patients, previously included in the HEPCIDANE trial [11]. Three different methods were tested for this assay: HS-assay ELISA, the MS approach developed by the Montpellier center (IRMB, MS-Montpellier) [15] and the MS approach developed by the Paris center (APHP, MS-Paris) [16]. The main The main analytical performance characteristics of the ELISA, MS-Montpellier and MS-Paris methods are indicated: LOD, LOQ, precision, accuracy and quantification range. ...
Article
Full-text available
Aim: To compare methods of quantifying serum hepcidin (based on MS and ELISA) and their ability to diagnose true iron deficiency anemia in critically ill patients. Materials & methods: Serum hepcidin was measured in 119 critically ill patients included in the HEPCIDANE clinical trial, using either an ultra-sensitive ELISA kit (from DRG) or two different MS methods. Results: The results show a good correlation between the different methods studied. The Bland–Altman analysis and the Kappa test for clinical groups show a good or very good agreement between the different tests. Conclusion: ELISA or MS show a satisfactory commutability to quantify serum hepcidin. This is of great importance for the determination of therapeutic strategies in iron deficiency.
... Five patients with RLS and four controls had undetectable hepcidin levels (i.e. below the detection limit 26 ). When hepcidin levels were divided into tertiles, we found that the number of patients in each tertile progressively increased in contrast to controls. ...
... Comparison of methods of quantification of hepcidin. We quantified serum hepcidin levels using two different methods by LC-MS/MS assay as previously reported 9,26 and by ELISA (the current method) in 42 patients with RLS (27 women, median age 63.00 years [34.78-77.22]) and 33 controls (19 women, median age 53.91 years [23.46-74.87]). ...
... In the last few years, different immunochemical and mass spectroscopy-based assays have been used to quantify serum hepcidin-25 level in humans, but its clinical relevance remains to be determined. We previously found using LC-MS/MS in the MRM mode (the reference method for peptide quantification 9,26 ) that serum hepcidin levels were higher in patients with RLS than in controls, and were correlated with RLS severity (nonlinear relationship), and with PLMS. By using an ELISA assay with a monoclonal antibody, another reliable but less expensive method for hepcidin quantification that could be easily implemented in clinical settings 28 , we confirmed the higher serum hepcidin levels in patients with RLS than in controls. ...
Article
Full-text available
The association between restless legs syndrome (RLS) and iron homeostasis remains unclear. We compared serum hepcidin and ferritin levels in patients with RLS and controls, and assessed their relationships with RLS phenotype, drug intake, and history of augmentation syndrome. 102 drug-free RLS patients (age 58.9 [24.5–77.2], 63 females) and 73 controls (age 56.8 [23.46–76.6], 45 females) underwent a polysomnography recording. Hepcidin levels were quantified by ELISA. 34 RLS patients had a second assessment after starting dopaminergic drugs. Ferritin level was low (< 50 µg/l) in 14.7% of patients and 25% of controls, with no between-group differences in the mean values. Hepcidin levels were higher in patients even after adjustment for confounding factors, and excluding participants with low ferritin levels. Ferritin and hepcidin levels were comparable before and after treatment, and between patients with (n = 17) and without history of augmentation. Ferritin and hepcidin levels correlated with age, body mass index, and periodic leg movements. Higher hepcidin levels were associated with older age, older age at RLS onset, less daytime sleepiness and familial RLS. In conclusion, serum hepcidin levels but not ferritin were higher in RLS patients regardless of treatment and history of augmentation. Serum hepcidin may be a more relevant biomarker of RLS than ferritin.
... De par les problèmes d'immunogénécité de l'hep-25, et de par la spécificité nécessaire entre les différentes isoformes, les techniques basées sur de la spectrométrie de masse ont largement été appliquées. Différentes méthodes de pré-fractionnement ont été publiées comme compatible avec une analyse de l'hep-25 : précipitation des protéines [270], ultrafiltration [271], échange de cations [272], séparation sur phase inverse [273]. Ces pré-fractionnements sont couplés à différents modes de détection tels que le SELDI, le MALDI [274], ou la LC-MS [263,[270][271][272][273][275][276][277][278][279]. ...
... Différentes méthodes de pré-fractionnement ont été publiées comme compatible avec une analyse de l'hep-25 : précipitation des protéines [270], ultrafiltration [271], échange de cations [272], séparation sur phase inverse [273]. Ces pré-fractionnements sont couplés à différents modes de détection tels que le SELDI, le MALDI [274], ou la LC-MS [263,[270][271][272][273][275][276][277][278][279]. ...
... Grâce à la sensibilité de ces méthodes analytiques, l'hep-25 a pu être dosé dans différents fluides biologiques comme le sérum [270], l'urine [279], le sang total [281], le DBS [282], et même dans le liquide céphalo-rachidien [277]. Le programme CANFER a représenté 6% des analyses. ...
Thesis
Depuis quelques années, la spectrométrie de masse est considérée comme la méthode de référence en chimie analytique. La « protéomique », concept qui a émergé dans les années 2000, consiste en l’identification et/ou la quantification d’un ensemble de peptides et protéines présentes dans un échantillon donné (cellules, tissus, ou des prélèvements biologiques), à un instant donné. Un champ plus spécifique de ce concept, la « protéomique clinique » concerne plus particulièrement l’étude du protéome pour la recherche d’une part, de marqueurs diagnostiques, pronostiques et de suivi thérapeutique des pathologies humaines et, d’autre part, d’acteurs physiopathologiques pouvant servir de cible thérapeutique. Actuellement, la technologie de choix utilisée pour l’analyse des protéines en biochimie clinique est le dosage immuno-enzymatique de type ELISA qui possède des inconvénients majeurs : pas ou peu multiplexable, une grande variabilité, pas de standardisation interne et l’incapacité à distinguer des protéoformes d'une même protéine. La protéomique ciblée de type Liquid Chromatography/Multiple Reaction Monitoring (LC-MRM) permet de surpasser ces inconvénients car elle est multiplexable (>200 protéines/expérimentations), robuste, permet l’utilisation de standard protéique/peptidique interne, et permet de distinguer une grande variété de modifications post traductionnelles.Ce projet de thèse consiste donc à évaluer et valider des techniques de spectrométrie de masse ciblées de type LC-MRM pour la quantification de protéines d’intérêt cliniques. Dans ce cadre, nous allons présenter trois développements de méthode de protéomique clinique : le phénotypage de l’Apolipoprotéine E, facteur de risque de la maladie d’Alzheimer ; la quantification sérique d’un anticorps monoclonal thérapeutique (Bevacizumab) avec immuno-enrichissement ; et la quantification absolue de l’hepcidine dans le cadre des pathologies liées au métabolisme du fer.
... The attractiveness of MS-based assays stems from the fact that they are able to distinguish hepcidin isoforms. Literature reports of MS-based hepcidin concentrations indicate significant variations 14 , most likely due to the lack of transferable hepcidin gold standards. Intact hepcidin has 4-disulfide linkages 15 , making consistent synthesis of intact hepcidin standard with high purity challenging, and thus degrading quantitation accuracy. ...
... The MS2 spectra for the intact heavy hepcidin-25 is characterized by poor fragmentation as previously observed 19 . This is likely due to the four disulfide linkages which also makes synthesis of a consistent peptide product challenging 20,21 , and in part accounting for the significant variability reported for MS-based assays 14 . Thus, we reasoned that elimination of disulfide linkages via generation of an alkylated hepcidin-25 peptide product may significantly contribute to reducing the challenge associated with synthesis while simultaneously improving the low fragmentation efficiency associated with the disulfide linkages in intact hepcidin. ...
... The LOD and LOQ values are better than what has been previously reported using intact hepcidin as an internal standard. For example, Delaby et al. 14 recently reported a mass spectrometry-based hepcidin-25 assay with analytical validation performed according to ISO15189 norms. In that study they reported LOD of 2 ng/mL and LOQ of 6 ng/mL in serum. ...
Article
Full-text available
Hepcidin, a cysteine-rich peptide hormone, secreted mainly by the liver, plays a central role in iron metabolism regulation. Emerging evidence suggests that disordered iron metabolism is a risk factor for various types of diseases including cancers. However, it remains challenging to apply current mass spectrometry (MS)-based hepcidin assays for precise quantification due to the low fragmentation efficiency of intact hepcidin as well as synthesis difficulties for the intact hepcidin standard. To address these issues we recently developed a reliable sensitive targeted MS assay for hepcidin quantification from clinical samples that uses fully alkylated rather than intact hepcidin as the internal standard. Limits of detection and quantification were determined to be <0.5 ng/mL and 1 ng/mL, respectively. Application of the alkylated hepcidin assay to 70 clinical plasma samples (42 non-cancerous and 28 ovarian cancer patient samples) enabled reliable detection of endogenous hepcidin from the plasma samples, as well as conditioned culture media. The hepcidin concentrations ranged from 0.0 to 95.6 ng/mL across non-cancerous and cancer plasma specimens. Interestingly, cancer patients were found to have significantly higher hepcidin concentrations compared to non-cancerous patients (mean: 20.6 ng/ml for cancer; 5.94 ng/ml for non-cancerous) (p value < 0.001). Our results represent the first application of the alkylated hepcidin assay to clinical samples and demonstrate that the developed assay has better sensitivity and quantification accuracy than current MS-based hepcidin assays without the challenges in synthesis of intact hepcidin standard and accurately determining its absolute amount.
... Our procedure was based on several previously published methods and optimized to achieve high recovery rates (79-88%) associated with low limit of quantification (2 ng/mL) and short time of analysis (8 min). We used a single step extraction like proposed for biologically active peptides with molar mass below 3.0 kDa [25,26]. However, instead of trichloroacetic acid (TCA) as an extraction reagent we used ACN, for which we obtained higher and more reproducible recoveries. ...
... To avoid ionization suppression, reported for TFA application [29], FA was added to the mobile phase. Additionally, thanks to the use of QTOF mass spectrometer, we managed to avoid problems with unstable and inefficient fragmentation of hepcidin, caused by the presence of four disulfide bonds in hepcidin molecule, encountered by Hwang et al. [30], as well as the variability in measurements, reported by Delaby et al. [25]. As described in the latest publication by Moghiep et al. [31], the use of fully alkylated hepcidin instead of an intact peptide may help to solve those problems. ...
Article
Hepcidin is a peptide hormone regulating iron metabolism, the dyshomeostasis of which has been implicated in dementia. Yet, data on hepcidin status in dementia are scanty, limited to Alzheimer's disease (AD) and inconsistent due to methodological problems with its determination using immunoassays and/or lack of homogeneity of evaluated groups. Hepcidin association with vascular dementia (VaD) remains unknown. We proposed a mass spectrometry method of hepcidin quantification in sera and aimed at determining hepcidin systemic status in patients with dementia of AD, VaD, or mixed (MD) pathology, with reference to the degree of cognitive loss and structural changes in the brain as well as at evaluating the diagnostic potential of hepcidin as a biomarker. We found that hepcidin concentrations were significantly elevated in VaD and insignificantly so in AD or MD and that they positively correlated with the Clinical Dementia Rating and inversely with the Mini Mental State Examination. Hepcidin tended to be more pronouncedly elevated in patients with advanced cortical atrophy and white matter lesions. It displayed a biphasic relationship with the Hachinski Ischemic Scale and a good accuracy as dementia but not differential marker. Taken together, our results demonstrated that dementia of vascular and not neurodegenerative pathology is associated with significant elevation of systemic hepcidin. Hepcidin elevation reflects the degree of cognitive loss as well as the severity of structural changes in the brain. If confirmed in a prospective study, hepcidin quantification may hold promise as a diagnostic marker; its accuracy as a differential marker of VaD is insufficient.
... However, hepcidin is currently not a bedside measurement, and the available measurement methods are expensive. In addition, the cutoff levels for hepcidin are unclear, and there is significant intertest variation [16,17]. Therefore, we aim to explore the diagnostic test performance of commonly available iron parameters as well as of hepcidin for the development of AI during an ICU stay. ...
Article
Background: Anemia of inflammation (AI) is the most common cause of anemia in the critically ill, but its diagnosis is a challenge. New therapies specific to AI are in development, and they require accurate detection of AI. This study explores the potential of parameters of iron metabolism for the diagnosis of AI during an ICU stay. Methods: In a nested case-control study, 30 patients developing AI were matched to 60 controls. The iron parameters were determined in plasma samples during an ICU stay. Receiver operating characteristic curves were used to determine the iron parameter threshold with the highest sensitivity and specificity to predict AI. Likelihood ratios as well as positive and negative predictive values were calculated as well. Results: The sensitivity of iron parameters for diagnosing AI ranges between 62 and 76%, and the specificity between 57 and 72%. Iron and transferrin show the greatest area under the curve. Iron shows the highest sensitivity, and transferrin and transferrin saturation display the highest specificity. Hepcidin and ferritin show the lowest specificity. At an actual anemia prevalence of 53%, the diagnostic accuracy of iron, transferrin, and transferrin saturation was fair, with a positive predictive value between 71 and 73%. Combining iron, transferrin, transferrin saturation, hepcidin, and/or ferritin levels did not increase the accuracy of the AI diagnosis. Conclusions: In this explorative study on the use of different parameters of iron metabolism for diagnosing AI during an ICU stay, low levels of commonly measured markers such as plasma iron, transferrin, and transferrin saturation have the highest sensitivity and specificity and outperform ferritin and hepcidin.
Article
Background The peptide hormone hepcidin-25 plays an important role in iron metabolism. Low or high levels of hepcidin-25 are associated with various iron disorders; therefore, hepcidin-25 is an important biomarker. This study describes an easy and fast analytical assay for the quantification of hepcidin-25 with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Methods Sample preparation was performed by protein precipitation with trichloroacetic acid, and injection onto a LC-MS/MS was directly conducted from a LoBind 96-well plate. Results The concentration range covered by the quality control samples, ranged from 0.25 nmol/L (12.3% CV) to 11.9 nmol/L (CV < 9%). Matrix effect was limited (mean recovery of 99.9% with a CV of 6.4%). The assay was validated for serum, EDTA and heparin plasma. An international secondary reference material was used for calibration. The reference interval (90% CL) was estimated for hepcidin-25 by analysing serum and plasma samples from 156 healthy subjects with a lower limit: 0.12 (0.07–0.19) and upper limit: 11.2 nmol/L (9.5–13.0). Conclusions We present a fast and easy assay for the quantification of hepcidin-25 in serum and plasma samples. The assay was successfully used for the detection of various forms of hereditary haemolytic anaemias, to characterize the interplay between erythropoiesis and iron levels.
Article
Iron is an essential element due to its role in a wide variety of physiological processes. Iron homeostasis is crucial to prevent iron overload disorders as well as iron deficiency anemia. The liver synthesized peptide hormone hepcidin is a master regulator of systemic iron metabolism. Given its role in overall health, measurement of hepcidin can be used as a predictive marker in disease states. In addition, hepcidin-targeting drugs appear beneficial as therapeutic agents. In this review, we discuss recent knowledge on analytic techniques (immunochemical, mass spectrometry and biosensors) and therapeutic approaches (hepcidin agonists, stimulators and antagonists). These insights highlight hepcidin as a potential biomarker as well as an aid in the development of new drugs for iron disorders.
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Background: Hepcidin, a 25 amino acid peptide, plays an important role in iron homeostasis. Some hepcidin truncated peptides have antibiotic effects. Results: A new analytical method for hepcidin determination in human plasma using LC-HRMS operating in full-scan acquisition mode has been validated. The extraction consists of protein precipitation and a drying reconstitution step; a 2.1 x 50 mm (idxL) C18 analytical column was used. Detection specificity, stability, accuracy, precision and recoveries were determined. The LOQ/LOD were 0.25/0.1 nM, respectively. More than 600 injections of plasma extracts were performed, allowing evaluation of the assay robustness. Hepcidin-20, hepcidin-22 and a new isoform, hepcidin-24, were detected in patients. Conclusion: The data underscore the usefulness of LC-HRMS for in-depth investigations related to hepcidin levels and pathways.
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Mass spectrometry (MS)-based assays for the quantification of the iron regulatory hormone hepcidin are pivotal to discriminate between the bioactive 25-amino acid form that can effectively block the sole iron transporter ferroportin and other naturally occurring smaller isoforms without a known role in iron metabolism. Here we describe the design, validation and use of a novel stable hepcidin-25(+40) isotope as internal standard for quantification. Importantly, the relative large mass shift of 40 Da makes this isotope also suitable for easy-to-use medium resolution linear time-of-flight (TOF) platforms. As expected, implementation of hepcidin-25(+40) as internal standard in our weak cation exchange (WCX) TOF MS method yielded very low inter/intra run coefficients of variation. Surprisingly, however, in samples from kidney disease patients, we detected a novel peak (m/z 2673.9) with low intensity that could be identified as hepcidin-24 and had previously remained unnoticed due to peak interference with the formerly used internal standard. Using a cell-based bioassay it was shown that synthetic hepcidin-24 was, like the -22 and -20 isoforms, a significantly less potent inducer of ferroportin degradation than hepcidin-25. During prolonged storage of plasma at room temperature, we observed that a decrease in plasma hepcidin-25 was paralleled by an increase in the levels of the hepcidin-24, -22 and -20 isoforms. This provides first evidence that all determinants for the conversion of hepcidin-25 to smaller inactive isoforms are present in the circulation, which may contribute to the functional suppression of hepcidin-25, that is significantly elevated in patients with renal impairment. The present update of our hepcidin TOF MS assay together with improved insights in the source and preparation of the internal standard, and sample stability will further improve our understanding of circulating hepcidin and pave the way towards further optimization and standardization of plasma hepcidin assays.