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Erythroferrone as a sensitive biomarker to detect stimulation of erythropoiesis

Authors:

Abstract

Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to erythropoietin stimulation. ERFE suppresses hepatic synthesis of the master iron‐regulatory hormone, hepcidin. The impact of erythropoiesis stimulation on ERFE secretion in humans is poorly understood. This paucity of information is due in part to the lack of available means for ERFE quantification in serum samples. In the present study, we tested a new sensitive sandwich immunoassay for human ERFE. We used this assay to demonstrate that injection of various erythropoiesis stimulating agents (ESAs) increased blood ERFE levels in healthy volunteers. After exogenous stimulation of erythropoiesis, ERFE increased up to 8‐fold with a detection window of 13 days. The impact of one unit of blood withdrawal on erythropoiesis stimulation of ERFE was also tested. ERFE significantly increased after blood withdrawal in subjects injected with both iron and saline solution, suggesting that iron supplementation did not mask ERFE increase after blood withdrawal. We assessed the effects of exercise‐induced muscle damage on ERFE by comparing ERFE levels with creatine kinase levels in samples from subjects with heavy exercise loads, and determined that this was not a confounder. The ERFE assay is a sensitive means to investigate the connection between iron metabolism and erythropoiesis in humans, and to detect ESA abuse in the anti‐doping field.
RESEARCH ARTICLE
Erythroferrone as a sensitive biomarker to detect stimulation
of erythropoiesis
Kelvin Ramirez Cuevas
1
| Céline Schobinger
1
| Emeric Gottardo
1
|
Sven Christian Voss
2
| Tiia Kuuranne
1
| Jean-Daniel Tissot
3
| Bernard Favrat
4
|
Nathan Townsend
5
| Nicolas Leuenberger
1
1
Swiss Laboratory for Doping Analyses,
University Center of Legal Medicine, Lausanne
and Geneva, Centre Hospitalier Universitaire
Vaudois and University of Lausanne,
Switzerland
2
Anti-Doping Lab Qatar, Sports City Road,
Sports City, P.O. Box 27775, Doha, Qatar
3
Transfusion interrégionale CRS, site
d'Epalinges, Switzerland
4
Department of Ambulatory Care and
Community Medicine, University of Lausanne,
Lausanne, Switzerland
5
Athlete Health and Performance Centre,
Aspetar Orthopaedic and Sports Medicine
Hospital Doha, Qatar
Correspondence
Nicolas Leuenberger, Swiss Laboratory for
Doping Analyses, Ch. Des Croisettes 22, 1066
Epalinges, Switzerland.
Email: nicolas.leuenberger@chuv.ch
Funding information
Fondation Dr Henri Dubois-Ferrière Dinu
Lipatti
Abstract
Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in
response to erythropoietin stimulation. ERFE suppresses the hepatic synthesis of
the master iron-regulatory hormone, hepcidin. The impact of erythropoiesis stimu-
lation on ERFE secretion in humans is poorly understood. This paucity of infor-
mation is due in part to the lack of available means for ERFE quantification in
serum samples. The present study tested a new sensitive sandwich immunoassay
for human ERFE. This assay was used to demonstrate that injection of various
erythropoiesis stimulating agents (ESAs) increased the blood ERFE levels in
healthy volunteers. After exogenous stimulation of erythropoiesis, ERFE increased
up to 8-fold with a detection window of 13 days. The impact of one unit of
blood withdrawal on erythropoiesis stimulation of ERFE was also tested. ERFE
significantly increased after blood withdrawal in subjects injected with both iron
and saline solution, suggesting that iron supplementation did not mask the ERFE
increase after blood withdrawal. The effects of exercise-induced muscle damage
on ERFE was assessed by comparing ERFE levels with creatine kinase levels in
samples from subjects with heavy exercise loads, and determined that this was
not a confounder. The ERFE assay is a sensitive means to investigate the connec-
tion between iron metabolism and erythropoiesis in humans, and to detect ESA
abuse in the antidoping field.
KEYWORDS
blood doping, erythroferrone, immunoassay
1|INTRODUCTION
The greatest deterrent to blood doping with erythropoiesis stimu-
lating agents (ESAs) is the hematological module of the Athlete
Biological Passport (ABP).
1
The hematological module focuses on
long-term monitoring of specific blood parameters, such as hemo-
globin concentration (Hb) and reticulocyte percentage (Ret%), to
detect abnormal absolute and/or relative changes in individual pro-
files that may indicate doping with ESAs. Stimulation of
erythropoiesis can be also observed after the blood withdrawal
step necessary for autologous blood transfusion, which is an alter-
native means for blood doping.
1
However, the shift towards blood
transfusions and micro-dose injections of ESAs necessitates the
development of new markers that can be integrated into the ABP
hematological module to increase its sensitivity.
2,3
Proteins involved
in erythropoiesis and iron metabolism have been suggested as
potential biomarkers for ESA abuse.
4
An example for this is
hepcidin which is downregulated by recombinant human
Received: 5 July 2019 Revised: 9 October 2019 Accepted: 13 October 2019
DOI: 10.1002/dta.2720
Drug Test Anal. 2019;17. wileyonlinelibrary.com/journal/dta © 2019 John Wiley & Sons, Ltd. 1
erythropoietin (rhEPO) administration.
5
In 2014, erythroferrone
(ERFE) was discovered as a novel erythroid regulator of iron
metabolism in a mouse model.
6
However, ERFE has been described
as having the same or a similar structure as myonectin.
7
In its role
as an iron metabolism regulator, ERFE mediates hepcidin suppres-
sion during increased erythropoietic activity stimulated by endoge-
nous and exogenous EPO, and facilitates compensatory iron
acquisition during recovery from hemorrhage-induced anemia.
8
These observations suggested that ERFE could be a viable bio-
marker for the detection of blood doping. However, because these
experiments were almost exclusively performed in mice, compara-
tive studies in humans are necessary, yet the quantification of
ERFE with a validated immunoassay remains challenging.
Previously, different ERFE immunoassays were tested to inves-
tigate the link between iron metabolism and erythropoiesis in
human.
9,10
However, the data presented suggested that these
assays were not valuable for the detection of ESA abuse in the
antidoping context, mainly due to sensitivity. Furthermore, the
influence of various confounding factors was not explored in these
different studies.
In the present study, testing and validation of a new commercial
available sensitive sandwich immunoassay for human ERFE were per-
formed. The impact of ESA administration and blood withdrawal on
ERFE levels in human subjects, as well as potential confounding fac-
tors such as iron supplementation and exercise-induced muscle dam-
age, were investigated.
2|MATERIAL AND METHODS
2.1 |Human ERFE ELISA
The commercial ERFE (human) Matched Pair Detection Set (AG-
46B-0012-KI01; AdipoGen Life Sciences, Epalinges, Switzerland)
was used per the manufacturer's instructions (https://adipogen.
com/ag-46b-0012-erythroferrone-human-matched-pair-detection-
set.html). Briefly, 100 μL of standard and serum samples was
added to the ELISA plate and detected by absorbance. The limit of
detection was determined by adding three standard deviations to
the mean value of 50 background level (zero concentration of
ERFE) standards. The standard of the ELISA kit is the full-length
erythroferrone recombinant protein with a His-tag expressed in
mammalian cells. This protein has been validated by AdipoGen
Life Sciences and is characterized to be stable for at least 1 year
when stored at 4C. To assess the intra-assay precision, four sam-
ples of known human recombinant ERFE concentration were
assayed in replicate six times. To test the inter-assay precision,
three samples of known human ERFE concentration were assayed
in four separate assays. The limit of detection of the immunoassay
was calculated as an apparent concentration of 40 pg/mL. The
intra- and inter-day variation ranges were 26% and 59%,
respectively.
2.2 |Clinical study samples
2.2.1 |Clinical studies: administration of
erythropoietic agents
Details regarding the participants and the time of serum sample col-
lection of the different clinical studies are described in previous stud-
ies which are summarized below.
For the first generation rhEPO clinical study, six healthy
Caucasian males (mean age, 27.0 years (SD, 4.1); mean body mass
index (BMI), 23.9 kg/m
2
(SD, 2.66)) received a single intravenous
injection of rhEPO delta (5000 IU; Dynepo, Dynepo Shire Pharma-
ceuticals, Basingstoke, UK) on days 1, 3 and 5 of the study as
described previously.
11
For the third generation rhEPO clinical study, six healthy
Caucasian men with a mean age of 23.0 years (SD, 2.97) and a mean
BMI of 23.3 kg/m
2
(SD, 1.48) received a single subcutaneous or intra-
venous injection of 200 mg of CERA (MIRCERA; Roche Pharma AG,
Reinach, Switzerland) as described previously.
12
In another study, six healthy Caucasian males received a single
intravenous injection of the EPO analog hematide/peginesatide
(Affymax, Inc. Palo Alto, CA) at 50 μg/kg as described previously.
13
2.2.2 |Clinical study: blood withdrawal and iron
injection
A randomized, single-blind, placebo-controlled trial was approved by
the Human Research Ethics Committee of the Canton of Vaud in
Switzerland (Protocol: 201600324). Participants were randomized to
receive an i.v. injection of either iron or placebo at a ratio of 1:1 using
R studio software (Version 1.0.44). Volunteers were blinded to the
study treatment by covering blood bags with opaque bags. The
ClinicalTrials.gov identifier is NCT03014921, and the study is entitled
Impact of Iron Injection on Blood Donation: a Randomized and
Controlled Clinical Trial.The study design is shown in Figure S1.
Written informed consent was obtained from each subject prior to
enrolment in the study.
Male Caucasian volunteers aged 2035 years with a ferritin con-
centration 50 μg/L ± 10% and a BMI of 1830 who were eligible for
blood donation according to national regulations were invited to
participate.
The iron supplement used was a 250 mL perfusion of 0.9% NaCl
(B. Braun Medical AG, Crissier, Switzerland) combined with 10 mL of
a 500 mg ferric carboxymaltose complex (Ferinject
®
, Vifor Pharma,
Villars-sur-Glâne, Switzerland). The placebo used in the control group
was a 250 mL perfusion of 0.9% NaCl (Figure S1).
2.2.3 |Clinical study: impact of exercise-induced
muscle damage on serum ERFE levels
Ten healthy, Caucasian middle- and long-distance runners took part in
this study. The subjects provided written informed consent, and the
2RAMIREZ CUEVAS ET AL.
study was approved by the institutional review board (E2015000073)
of ADL Qatar. Serum samples were collected at sea level at various
time points over a period of 2 weeks. To assess the level of muscle
damage, serum samples were analysed for creatine kinase (CK), a cir-
culating protein that is considered an indirect marker for muscle dam-
age. To reflect individual responses to exercise-induced muscle
damage, three samples per subject were included in this study: the
sample with the lowest and the two samples with the highest CK
values. The highest concentration of CK obtained in this study was
1330 IU/L.
2.3 |Clinical chemistry, immunology, and
hematology
All venous blood samples were obtained from an antecubital vein
according to standard procedures. Blood was drawn into 8.5 mL
serum tubes (SST II Advance, BD Vacutainer, ref. 366644). Serum
tubes were centrifuged for 15 min at 1500 rcf after collection and
stored at 20Cor80C until further analysis. Transferrin satura-
tion (TSAT) was measured using an automated Dimension EXL 2000
technology system (Siemens Healthcare Diagnostic SA Zurich, Swit-
zerland) according to the manufacturer's instructions.
The ferritin concentration was measured with a Centaur instru-
ment. Erythropoietin (EPO) was measured in serum using an Immulite
system (Siemens Healthcare Diagnostic SA, Zurich, Switzerland). Crea-
tine kinase was measured on a fully automated Cobas Integra 400 plus
analyser (Roche Diagnostics, Rotkreuz, Switzerland). The reticulocyte
percentage (Ret%) was measured using a fully automated hematology
analyser (Sysmex XN 2000, Sysmex AG, Yverdon-les-Bains, Switzer-
land). The serum hepcidin concentration was measured by liquid chro-
matography coupled with high-resolution mass spectrometry (LC-
HRMS) according to a method described by Leuenberger et al.
5
2.4 |Statistical analyses
A Shapiro test was used to determine the normality of the data. For
normally distributed data, comparisons between and within groups
were performed using an ANOVA followed by post-hoc pairwise com-
parisons (t-test adjusted by the Bonferroni correction and Tukey
HSD). Non-parametric comparisons were performed using the
Kruskal-Wallis and Wilcoxon tests. Correlations were assessed using a
Spearman's correlation test. A value of P< 0.05 was considered signif-
icant. All tests were conducted using R studio software (Version
1.0.44). Data are presented as mean ± SEM, unless otherwise noted.
3|RESULTS
After injection of recombinant EPOs and the analog peginesatide,
ERFE substantially increased in a minimum of 50% in all subjects
(Figure 1AC). In the Dynepo study, ERFE reached the highest
concentrations on day 3 after the second injection. Interestingly, the
responses were dependent on the individual, with two of the six sub-
jects showing only small changes. Additional injections did not further
increase ERFE levels. For peginesatide, ERFE was increased by an
average 8-fold on day 6 from 0.9 to 6.3 ng/mL, with concentrations
decreasing back to the baseline on day 13. In the CERA group, sub-
jects had an average 6-fold increase from 0.64 to 3.9 ng/mL, which
returned to baseline on day 10. A positive significant Spearman's cor-
relation (rho = 0.73; P= 1.34e-08) was observed between the ERFE
concentration and Ret% after stimulation by C.E.R.A. injection
(Figure S2).
Table 1 summarizes the iron (ferritin, TSAT, and hepcidin) and
erythropoiesis variables at baseline and after blood withdrawal pre-
ceded by saline (saline group) or iron (iron group) injection. The clinical
study design is shown in the supporting information (Figure S1). The
baseline concentration of iron markers was significantly higher in the
iron group than in the saline group, presumably due to the 14 day pre-
ceding iron injection. In both groups, ferritin and TSAT were
decreased after blood withdrawal. As expected, EPO was significantly
induced from day +1 up to day +15 to compensate for blood loss due
to withdrawal. By contrast, hepcidin was significantly decreased in the
saline group, but no change was observed in the iron-treated group,
after blood withdrawal.
In the saline group, the ERFE concentration rose on day +1
(Figure 2A). This elevation lasted until day +9, after which the ERFE
concentration returned close to the initial value of the baseline. For
the iron group, a significant ERFE increase was observed from day +3
until day +6 (Figure 2B).
Healthy blood donors (30 females and 40 males) were subjected
to ERFE measurements (Figure S3). The median ERFE concentration
in males and females was 0.57 ng/mL (95% CI, 0.530.65 ng/mL) and
0.48 ng/mL (95% CI, 0.450.55 ng/mL), respectively. The maximum
and minimum concentration in males and females were 0.17 and 1.11
and 0.16 and 1.1 ng/mL, respectively. No significant sex difference in
ERFE concentrations was observed (Figure S3).
To assess the impact of exercise-induced muscle damage, CK
values from human subjects with a high exercise load were compared
with ERFE concentrations (Figure 3A). No significant correlation
(Spearman's rho = 0.30, P= 0.1) was observed between ERFE and CK
concentrations. Also, ERFE concentration was unchanged in iron-
injected subjects (Figure 3B). By contrast, other hematological and
iron biomarkers, such as Ret%, TSAT, ferritin, and hepcidin, were
impacted by iron injection (Table S1).
4|DISCUSSION
4.1 |Impact of ESAs injection and blood
withdrawal on ERFE level
This study investigated whether serum ERFE levels could be used to
detect small changes in erythropoiesis. The impacts of different ESAs
and the withdrawal of one bag of blood (450 mL) on ERFE
RAMIREZ CUEVAS ET AL.3
concentration were investigated. The goal was to test and validate a
new specific and sensitive sandwich immunoassay. Previously, the
association between ERFE and biomarkers of erythropoiesis and iron
metabolism has been evaluated using a commercially available sand-
wich ELISA kit constituting mouse monoclonal antibodies.
10
We previ-
ously evaluated this commercially available kit, and found that ERFE
quantification by this method was not reproducible.
14
ERFE responses
following rhEPO administration were highly variable, and not coher-
ent.
14
Other commercial ELISAs produced with mouse antibodies
were also tested unsuccessfully.
Previously, Ganz et al. developed a rabbit monoclonal antibody-
based sandwich immunoassay for the detection of human ERFE.
9
A
14 ng/mL limit of quantification was reported for this assay. Contrast-
ingly, the limit of quantitation for our validated ELISA was
0.04 ng/mL. However, both assays and standards should be directly
compared in order to draw conclusions.
The response to blood withdrawal was examined in subjects
injected previously with iron or saline solution (placebo). ERFE was
increased in both contexts, reaching a maximum concentration after
6 days. In placebo patients, this ERFE increase was coincident with a
decline in serum hepcidin concentrations, but this correlation was not
present when iron was supplemented before blood withdrawal. Previ-
ously, Mirciov et al. demonstrated that high circulating iron levels
overcome ERFE inhibition of hepcidin in mice.
15
This effect did not
alter the expression of ERFE itself, and seemed linked to the interfer-
ence of di-ferric transferrin. This explanation could be also extrapo-
lated to the present study, due to the high TSAT in our iron injection
group. Our data suggest that, in humans, the inhibitory effect on
hepcidin is not needed when enough circulating iron is available, simi-
lar to mice.
The personalized follow-up of Ret% is the most common indirect
biomarker for the detection of erythropoiesis stimulation in the
FIGURE 1 ERFE levels after injection
of different ESAs. (A) Effect of
recombinant erythropoietin (rEPO) delta
administration on individual (left panel)
and mean (right panel) ERFE
concentrations. (B) Effect of peginesatide
administration on individual (left panel)
and mean (right panel) ERFE
concentrations. (C) Effect of CERA
administration on individual (left panel)
and mean (right panel) ERFE
concentrations. Vertical black lines
represent ESA administration times. D,
days. *P< 0.05, **P< 0.01. Variance was
calculated within groups compared
with D0
4RAMIREZ CUEVAS ET AL.
context of the ABP. Due to the cellular feature of Ret%, analyses
should be performed within approximately 48 hours after collection.
By contrast, many serum biomarkers can be analysed up to 10 years
after storage at 20C. In fact, some samples used in the present
study were collected 10 years ago in previous clinical studies
(Figure 1).
11-13
Although the ERFE concentration significantly corre-
lates with Ret%, the relative increase observed in the ERFE concentra-
tion was higher than that of Ret%. Indeed, after ESA injection
(peginesatide) an increase in ERFE up to 8-fold was observed.
4.2 |Assessment of different confounding factors
on ERFE concentration
ERFE was identified as an erythroid regulator of iron metabolism.
6,8
However, ERFE had earlier been described as myonectin, or C1q
tumor necrosis factor α-related protein isoform 1500 (CTRP15), and in
mice, was highly expressed in muscle tissue.
7
In this context, muscle
ERFE levels were increased after physical activity in rodents. Because
myonectin is produced also in the muscle of humans, serum samples
from a previously conducted exercise study were used to determine if
muscle damage could cause myonectin to potentially leak into the
blood, affecting circulating ERFE. In the present study, no increase of
circulating ERFE was detectable after muscle damage, as indicated by
the CK levels (Figure 3A). These data suggest that this approach is a
specific measurement of circulating ERFE secreted from erythroid ori-
gin after erythropoietic stimulation, rather than regulation of ERFE by
myocyte metabolism. The apparent specificity of our sandwich immu-
noassay is currently under investigation in our laboratory. However, a
difference of ERFE isoforms caused by specific glycosylation in ery-
throid cells or a different secretion of the quaternary structure (mono-
meric vs multimeric proteins) in muscle versus erythroid cells is a
theoretically possible explanation.
A single infusion of iron was reported to improve fatigue, mental
health, cognitive function, and erythropoiesis in iron-deficient women
with normal or borderline Hb concentrations.
16
Some endurance ath-
letes inject iron to maintain a high serum ferritin concentration.
17
Intravenous iron supplementation improves fatigue and overall mood
in runners without a clinical iron deficiency.
18
Iron injection is not
considered to be a doping method, but could potentially interfere with
antidoping biomarkers involved in iron metabolism. In the present
study, we identified that iron injection did not affect the ERFE con-
centration (Figure 3B). As expected, iron injection dramatically
affected ferritin, TSAT and hepcidin (Table S1). Interestingly, increased
Ret%, an actual hematological marker of the ABP, was observed after
iron injection, as previously demonstrated in females.
16
Together,
these data reinforce the specificity of the presented ERFE immunoas-
say for antidoping purposes.
Regarding the assessment of other confounding factors, no
influence of sex on ERFE concentrations was observed (Figure S3).
To ensure the specificity of the ERFE response to ESA administra-
tion, the impact of altitude exposure as a confounding factor on
ERFE should be characterized. Because ERFE and reticulocytes
TABLE 1 Iron and erythropoiesis biomarker analyses after blood withdrawal
Saline (n = 8) Baseline Day 0 Day +1 Day +2 Day +3 Day +6 Day +9 Day +15 Day +30
Ferritin (μg/mL) 50.39 ± 9.42 48.29 ± 8.48 44.51 ± 8.57 41.46 ± 7.86 40.28 ± 7.99* 31.13 ± 5.30* 26.79 ± 4.71** 19.93 ± 3.05** 22.76 ± 4.12**
TSAT (%) 26.98 ± 3.97 27.01 ± 3.02 27.28 ± 3.58 28.71 ± 4.26 25.11 ± 3.07 21.04 ± 3.69 15.70 ± 1.95* 15.31 ± 1.65* 19.08 ± 4.22
Hepcidin (nM) 1.50 ± 0.30 1.45 ± 0.40 1.12 ± 0.39* 0.70 ± 0.20** 0.56 ± 0.14** 0.55± 0.11** 0.43 ± 0.10** 0.39 ± 0.15** 0.43 ± 0.10**
EPO (mUI/mL) 10.01 ± 0.93 11.68 ± 0.97 16.16 ± 2.08** 16.38 ± 2.08** 15.69 ± 1.86** 16.15 ± 1.76** 15.75 ± 1.80** 13.71 ± 1.07** 11.16 ± 0.92
Iron (n = 8) Baseline Day 0 Day + 1 Day + 2 Day + 3 Day + 6 Day + 9 Day + 15 Day + 30
Ferritin (μg/mL) 224.51 ±22.27
##
220.29 ± 20.41 176.56 ± 18.26* 158.99 ± 17.69* 154.29 ± 18.99* 121.50 ± 13.79* 102.01 ± 13.29** 80.69 ± 12.97** 64.69 ± 12.66**
TSAT (%) 34.84 ± 5.64
#
34.81 ± 5.19 38.81 ± 5.43 34.53 ± 4.15 30.12 ± 3.64 34.93 ± 3.32 32.35 ± 5.42 27.66 ± 5.11 23.30 ± 4.06*
Hepcidin (nM) 3.69 ± 1.90
#
3.49 ± 1.62 3.61 ± 1.27 3.40 ± 2.14 1.95 ± 0.99 2.59 ± 1.09 1.99 ± 1.05 3.07 ± 1.49 3.36 ± 2.01
EPO (mUI/mL) 11.81 ± 1.36 10.01 ± 1.81 19.46 ± 1.74** 16.36 ± 1.60** 18.24 ± 1.10** 14.70 ± 1.93* 14.14 ± 1.43* 16.60 ± 2.62* 10.75 ± 1.67
Data are expressed as mean ± SEM. Significant changes relative to baseline (the mean of day 4 and day 1) are indicated by bold text with grey highlighting. Day 0 corresponds to the mean of hours +3, +6
and + 12 after saline or iron injection. Saline, saline-supplemented group; iron, iron-supplemented group. TSAT, transferrin saturation; EPO, erythropoietin. *p< 0.05, **p< 0.01. Variance was calculated within
groups compared with baseline.
#
P< 0.05,
##
P< 0.01. Variance was calculated between groups compared with baseline.
RAMIREZ CUEVAS ET AL.5
correlate closely (Figure S2), the influence of altitude exposure
on Ret% measurement should also be valuable for ERFE as
biomarker.
In conclusion, we tested a new immunological assay for human
ERFE that can sensitively detect the stimulation of erythropoiesis,
including ESA abuse and blood withdrawal. In an antidoping context,
this assay could be a useful tool for retrospective investigations in
which analysis of Ret% is no longer possible due to sample degrada-
tion. Moreover, this assay could be useful to investigate the interac-
tions between erythropoiesis and iron metabolism in human clinical
studies.
ACKNOWLEDGMENTS
The authors thank the staff of Transfusion Interrégionale CRS for their
assistance. The authors thank Prof. Thomas Ganz for providing the
hERFE plasmid. We are grateful to Fondation Dr Henri Dubois-Fer-
rière Dinu Lipatti for financial support.
ORCID
Nicolas Leuenberger https://orcid.org/0000-0001-7106-6304
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FIGURE 3 Muscle damage and
iron injection did not affect ERFE
levels. (A) Effect of muscle damage
(creatine kinase: CK) on ERFE
concentration. (B) ERFE levels in eight
individuals after iron injection
(500 mg of Ferinject
®
)
FIGURE 2 ERFE levels after blood withdrawal. Effect of blood withdrawal on ERFE concentration in (A) saline (B) and iron groups. Vertical
red lines represent blood withdrawal times. D, days; H, hours. *P< 0.05, **P< 0.01. Variance was calculated within groups compared with
baseline (D-1 and D-4)
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Ramirez Cuevas K, Schobinger C,
Gottardo E, et al. Erythroferrone as a sensitive biomarker to
detect stimulation of erythropoiesis. Drug Test Anal. 2019;17.
https://doi.org/10.1002/dta.2720
RAMIREZ CUEVAS ET AL.7
... By suppressing hepcidin, ERFE promotes iron mobilization from storage cells and dietary iron absorption to improve iron availability for erythropoiesis. As a master regulator connecting erythropoiesis and iron metabolism, ERFE evokes much research interests in many research fields such as thalassemia, myelodysplastic syndromes, chronic kidney disease, anti-doping, hematopoietic stem cell transplant (Kautz et al., 2015;Bondu et al., 2019;Hara et al., 2019;Ramirez Cuevas et al., 2020;Pirotte et al., 2021). Reported results demonstrated that ERFE concentrations significantly increased in animals and humans with normal or impaired kidney function after receiving exogenous erythropoiesis-stimulating agents (ESAs) (Hanudel et al., 2018;Ramirez Cuevas et al., 2020;Robach et al., 2021). ...
... As a master regulator connecting erythropoiesis and iron metabolism, ERFE evokes much research interests in many research fields such as thalassemia, myelodysplastic syndromes, chronic kidney disease, anti-doping, hematopoietic stem cell transplant (Kautz et al., 2015;Bondu et al., 2019;Hara et al., 2019;Ramirez Cuevas et al., 2020;Pirotte et al., 2021). Reported results demonstrated that ERFE concentrations significantly increased in animals and humans with normal or impaired kidney function after receiving exogenous erythropoiesis-stimulating agents (ESAs) (Hanudel et al., 2018;Ramirez Cuevas et al., 2020;Robach et al., 2021). These findings suggest ERFE has potential as an early biomarker for erythropoiesis induced by ESAs. ...
... However, the lack of studies about ERFE dynamics would block and mislead clinical trials which aim to investigate the predictive ability of ERFE in ESA treatment. The current studies about the ERFE changes, either basic research in animals or clinical trials in healthy volunteers or patients, used sparse samples and did not fully reveal the dynamics of ERFE (Kautz et al., 2015;Honda et al., 2016;Bondu et al., 2019;Hara et al., 2019;Ramirez Cuevas et al., 2020;Pirotte et al., 2021). A study in 24 males firstly investigated the role of ERFE in humans and demonstrated that ERFE responded to a very low dose of EPO (Robach et al., 2021). ...
Article
Full-text available
Background: Erythroferrone (ERFE) is a hormone identified recently as a master regulator connecting iron homeostasis and erythropoiesis. Serum ERFE concentrations significantly increase in animals and humans with normal or impaired kidney function after receiving exogenous erythropoiesis-stimulating agents (ESAs), which suggests it might be a predictive factor for erythropoiesis. To evaluate whether ERFE is an early, sensitive biomarker for long-term erythropoietic effects of ESAs, we investigated the relationship between ERFE dynamics and time courses of major erythropoietic responses to ESA treatment. Methods: Healthy rats received single dose and multiple doses (thrice a week for 2 weeks) of recombinant human erythropoietin (rHuEPO) at three dose levels (100, 450, and 1350 IU/kg) intravenously. The rHuEPO and ERFE concentrations in plasma were determined at a series of time points after dosing. Erythropoietic effects including red blood cell count and hemoglobin concentrations were continuously monitored for 24 days (single dose) or 60 days (multiple doses). The expansion of erythroblasts in bone marrow was quantified by flow cytometry analysis. Results: ERFE significantly increased within a few hours and return to baseline at 24 h after rHuEPO treatment. The ERFE response was enhanced after repeated treatment, which was consistent with the observed expansion of erythroblasts in the bone marrow. In addition, the dynamics of ERFE showed double peaks at approximately 2 and 10 h after rHuEPO stimulation, and the ERFE baseline displayed a significant circadian rhythm. There was a strong positive correlation between peak values of short-term ERFE responses and the long-term hemoglobin responses. Conclusion: The stimulated release of ERFE is a rapid process within 24 h. The second peak in the ERFE response to rHuEPO suggests the presence of a feedback mechanism counterregulating the ESA stimulation. The early increase of ERFE at 2 h appears to be a predictor of the hemoglobin response at 14 days after single dose of rHuEPO. Under multiple-dose regimen, the enhanced ERFE responses still correlate with the peak hemoglobin responses. The ERFE baseline also exhibits a circadian rhythm.
... In 2014, Kautz et al. described a protein derived from erythroid precursor cellserythroferrone (ERFE) [12]. The authors indicated that increased erythropoietic activity results in ERFE secretion by erythroblasts [12,13]. ERFE suppresses hepatic synthesis of the master iron-regulatory hormone, hepcidin, leading to increased availability of body iron resources for erythropoiesis [14]. ...
... Other studies in animal models have shown that bacterial pathogen-induced inflammatory anaemia, as well as increased erythropoiesis after significant blood loss, lead to increased ERFE synthesis [19]. Suppressing hepcidin expression with ERFE would exaggerate the availability of circulating iron for an increased iron demand for red blood cell haemoglobinisation [13]. In the present study, we observed greater ERFE levels among an examined group of athletes, which may suggest that intense physical effort following a recovery period enhances the erythropoietic activity, and thus ERFE synthesis. ...
Article
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Intense physical activity contributes to an increased demand for red blood cells, which transport oxygen to working muscles. The purpose of this study was to assess the concentration of erythroferrone (ERFE), the novel marker of erythroid activity in athletes, during the beginning of their training season. The study group consisted of 39 athletes aged 23.24 ± 3.77 years. The study was carried out during the athletes’ preparatory period of the training cycle. The control group consisted of 34 healthy men aged 22.33 ± 2.77 years. The erythropoietic activity was evaluated by determining athletes’ concentrations of erythropoietin (EPO) and erythroferrone (ERFE). The level of physical activity was assessed using the International Physical Activity Questionnaire (IPAQ). In the athletes’ group, we observed higher concentrations of EPO (Me = 12.65 mIU/mL) and ERFE (40.00 pg/mL) compared to the control group (EPO: Me = 5.74 mIU/ml, p = 0.001; ERFE: Me = 25.50 pg/mL, p = 0.0034). The average intensity of physical exercise significantly differentiated the participants as far as EPO and ERFE concentrations. These results suggest that intense physical activity, at least at the beginning of the training season, may stimulate EPO production, which increases ERFE release. This seems to be an adaptative mechanism that provides adequate iron for enhanced erythropoiesis.
... Oxidative stress-dependent HIF2α inactivation was also reported as a possible mechanism of iron-induced suppression of renal EPO expression [38], and increased oxidative stress markers in the present study might be related with a reduced mRNA expression of renal Epo in CFA-iron rats. Considering that erythropoiesisstimulating agent inhibits hepcidin expression through ERFE [39,40], these changes might also have contributed to enhanced hepcidin activity. Based on these results, we can assume that the simultaneous regulation of both hepcidin and EPO is required to induce effective erythropoiesis after IV iron supplementation in a clinically relevant short period in patients with ACI. ...
Article
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We examined changes in hepcidin (closely associated with anemia of chronic inflammation (ACI)) and upstream regulatory pathways after intravenous (IV) iron supplementation in an ACI animal model. ACI was induced in male Sprague-Dawley rats by intraperitoneally administering complete Freund’s adjuvant (CFA). Two weeks after starting CFA treatment, ACI rats received IV iron (CFA-iron) or vehicle (CFA-saline). Three days after IV iron treatment, iron profiles, hepcidin levels, and expression of proteins involved in the signaling pathways upstream of hepcidin transcription in the liver were measured. In CFA-treated rats, anemia with a concomitant increase in the levels of serum inflammatory cytokines and reactive oxygen species occurred. In CFA-iron rats, hemoglobin (Hb) concentration was still lower than that in control rats. In CFA-saline rats, hepatic hepcidin and ferritin levels increased compared with those in control rats and were further increased in CFA-iron rats. In CFA-saline rats, NADPH oxidase- (NOX-) 2, NOX-4, and superoxide dismutase levels in the liver were upregulated compared with those in control rats and their levels were further increased in CFA-iron rats. In CFA-saline rats, activities of the IL-6/STAT and BMP/SMAD pathways were enhanced in the liver compared with those in control rats and their levels were further increased in CFA-iron rats, whereas IL-6 expression remained unaffected after IV iron administration. In HepG2 cells, iron caused phosphorylation of STAT-3 and SMAD1/5 and knockdown of STAT-3 and SMAD1/5 using siRNAs reduced iron-induced hepcidin upregulation to levels similar to those in corresponding control cells. Renal erythropoietin expression and serum erythroferrone concentration were lower in CFA-iron rats than those in control rats. In ACI rats, IV iron supplementation did not recover Hb within three days despite an increase in hepatic ferritin levels, which might be attributable to an additional increase in hepcidin levels that was already upregulated under ACI conditions. Both STAT-3 phosphorylation and SMAD1/5 phosphorylation were associated with hepcidin upregulation after IV iron treatment, and this seems to be linked to iron-induced oxidative stress. 1. Introduction Anemia of chronic inflammation (ACI), also referred to as anemia of chronic disease, is the second most common type of anemia after iron deficiency anemia (IDA) in hospitalized patients and is associated with increased morbidity [1, 2]. Inadequate utilization of iron despite sufficient body iron stores leads to the development of ACI, whereas IDA is caused by an absolute iron deficiency [3]. Hepcidin is a key regulator of iron homeostasis and is known to regulate iron metabolism through ferroportin (FPN), the sole known iron exporter [4, 5]. Hepcidin is upregulated by an increase in iron stores in the body and inflammatory conditions and is downregulated by erythropoiesis [4, 5]. Iron-mediated regulation of hepcidin levels acts mainly through the bone morphogenetic protein (BMP)/Sma mothers against the decapentaplegic (SMAD) signaling pathway. Inflammation and reactive oxygen species (ROS) also influence the expression of hepcidin, mainly by triggering the signal transducer and activator of the transcription-3 (STAT-3) pathway [6]. By contrast, erythroferrone (ERFE), secreted by erythropoietin- (EPO-) stimulated erythroblasts during erythropoiesis, suppresses the expression of hepcidin [7, 8]. Considering the increased hepcidin expression and the resultant decrease in FPN expression and iron sequestration in iron storage cells in ACI, the hepcidin/FPN axis is an ideal target for the treatment of ACI [9]. However, currently there is no drug available that can directly regulate this axis. Iron supplementation and blood transfusion are the only clinical treatment regimens for iron deficiency and anemia, regardless of the underlying mechanisms. In the current clinical practice, intravenous (IV) administration of iron is preferred over oral administration because of its superior efficacy in terms of bioavailability and simple administration protocol [10, 11]. Furthermore, the absorption of dietary iron through duodenal enterocytes decreases as hepcidin expression increases. The stable dextran-based iron complex, containing minimal amount of labile iron, is well tolerated by patients even at high doses during IV administration because of its robust and reliable pharmacokinetic properties [12, 13]. Theoretically, however, iron has two opposite effects on erythropoiesis: it activates erythroid differentiation directly but inhibits it indirectly by stimulating hepcidin and enhancing oxidative stress [14]. In this context, recent clinical studies reported various results related to the impact of a high-dose IV iron on hemoglobin (Hb) levels, especially during a short period of time [15–18]. However, there is a paucity of data on changes in hepcidin levels in relation to IV iron supplementation under ACI conditions in both experimental and clinical studies. Therefore, the primary aim of this study was at investigating short-term changes in hepcidin expression and its regulatory pathways in the liver after IV iron supplementation at a clinically used concentration in a relevant animal model of ACI, which resembles its clinical presentation. The secondary aim of this study was at evaluating the concomitant changes in renal Epo expression and serum ERFE concentration, as well as oxidative stress markers, after IV iron supplementation. We also aimed to identify the role of the signaling pathways upstream to hepcidin in HepG2 cells loaded with iron using short interfering RNAs. 2. Materials and Methods 2.1. Animal Preparation All animal procedures were approved by the Committee for the Care and Use of Laboratory Animals, Yonsei University College of Medicine, and were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (USA). Male Sprague-Dawley rats (130−150 g) were housed in a temperature-controlled environment at 23–25°C with a 12/12 h light/dark cycle and were provided ad libitum access to food and water. 2.2. Study Groups and Experimental Models Animals were randomly assigned to three groups: control-saline (), complete Freund’s adjuvant- (CFA-) saline (InvivoGen, San Diego, CA, USA) (), and CFA-iron (iron isomaltoside, Monofer®; Pharmacosmos A/S, Holbaek, Denmark; ). For the establishment of the ACI model, rats were intraperitoneally (IP) administered 0.2 mL of CFA containing 1 mg/mL of heat-killed Mycobacterium tuberculosis mixed with phosphate buffer saline (in the ratio of 1 : 1, thrice across two weeks), whereas the control rats were administered the same volume of saline [19]. Rats that exhibited a decline in Hb by >2 g/dL from baseline after two weeks were designated as ACI rats [20]. ACI rats received 20 mg/kg of IV iron (which is the highest clinical dose currently used) or an equivalent volume of saline. Serum and tissues were harvested from euthanized rats three days after IV iron or saline administration. 2.3. Hematologic Studies Blood samples were subjected to complete blood counts obtained using a Mindray BC-5000 Vet hematology analyzer (Shenzhen Mindray Bio-Medical Electronics, Shenzhen, China). 2.4. Measurement of Iron Parameters Transferrin saturation (TSAT) and total iron-binding capacity (TIBC) were analyzed by an automated chemistry analyzer (Cobas C702; Roche, Mannheim, Germany) at Seoul Clinical Laboratories (Seoul, Korea). 2.5. Immunohistochemistry (IHC) Liver tissue samples were subjected to IHC analysis. The samples were washed in physiological saline, fixed in 10% buffered formalin, and embedded in paraffin. Sections were then stained with rabbit anti-NADPH oxidase- (NOX-) 2 (1 : 100, Novus, Littleton, CO, USA) and anti-NOX-4 (1 : 500, Abcam, Cambridge, MA, USA), followed by staining with chromogen 3,3-diaminobenzidine (Abcam). Slides were viewed with an Olympus IX73P2F microscope (Olympus America, Melville, NY, USA) equipped with an Olympus DP71 digital camera (20×). 2.6. Cell Culture HepG2 cells (human liver cancer cell line) were purchased from the American Type Culture Collection (Rockville, MD, USA) and were maintained in Eagle’s minimal essential medium glutamine supplemented with 10% foetal bovine serum (FBS), 100 unit/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Culture media and supplements were purchased from Gibco (Carlsbad, CA, USA). 2.7. Cell Viability Assay HepG2 cells () were seeded in 96-well culture plates and incubated overnight. The cells were then incubated for 0–2 days in glutamine supplemented with 10% FBS with or without iron, at the concentrations indicated (Figure 1). Finally, cell viability was evaluated using the Cell Counting Kit- (CCK-) 8 assay (Dojindo, Kumamoto, Japan) according to the manufacturer’s protocol. Experiments were performed in triplicate.
... The current study supposed that associated with the prevalence of anemia in HD more than CKD because HD patients were persisted in ESA treatment lead to high expression of ERFE as a regulator of the erythroid mediator from erythroblast. It is a new sensitive biomarker in humans to estimate the relationship between iron metabolism and erythropoiesis and noticed that ESA might have a detrimental effect in the antidoping field Ramirez Cuevas et al., 2020). As a result, hyperactivation of erythropoiesis and accelerated synthesis of the erythroid progenitor, but this undergo intramedullary apoptosis before the differentiation process is completed, therefore can cause anemia. ...
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RESUMO Introdução: A eritroferrona (ERFE) é uma glicoproteína de síntese e liberação de hormônio pelos eritroblastos. Recentemente identificado como um regulador eritropoiético e ativado em resposta ao estímulo da eritropoietina (Epo). Nas doenças renais crônicas (DRC), a anemia é um distúrbio característico devido a uma diminuição da hipossensibilidade eritropoiética à Epo; esses pacientes recomendaram o uso de agentes estimuladores de eritropoiese (AEEs). Objetivos: Este estudo teve como objetivo avaliar o nível sérico de ERFE em pacientes com DRC e investigar os efeitos contínuos do uso de AEE anêmico em longo prazo associado a marcadores de eritropoiese e metabolismo do ferro. Métodos: Sessenta e cinco pacientes com DRC foram divididos em dois grupos, incluindo 30 pacientes em hemodiálise (HD) e 35 pacientes com DRC sem hemodiálise (não-HD), foram comparados a 25 voluntários saudáveis pareados por sexo e idade inscritos no estudo. O nível sérico de ERFE foi medido através de um ensaio imunoenzimático (ELISA).. Resultados: Um aumento significativo nos níveis séricos de ERFE em pacientes em HD da mediana (IQR) de 17,25 (13,4) ng/mL, razão de possibilidades (OR = 10,161), (AUC 0,996) maior do que CKD 4 (6,1) ng/ml, (OR = 6,295), (AUC = 0,984) p <0,001; também, estes estão positivamente correlacionados com o uso de AEE em HD e CKD (r = 1,00 e r = 0,95), respectivamente, em comparação com o grupo saudável 2 (2,1) ng/ml. Os níveis séricos de ERFE foram significativamente negativos em pacientes com DRC e HD. (p <0,05) relacionado à TFG (r =-0,396, e r =-0,68), saturação de transferrina (TS%) (r =-0,842 e r =-0,877), níveis séricos de ferritina (r =-0,865 e r =-0,866), e Ferro (r =-0,860 e r =-0,851), RBC (r =-0,841 e r =-0,843), hemoglobina (Hb) (r =-0,758 e r =-0,796) Conclusões: O presente estudo demonstra que os níveis séricos elevados de ERFE associados à atividade eritropoiética e anemia são maiores em pacientes com DRC com HD e não-HD tratados com AEE do que em pacientes sem AEE. Este estudo sugeriu o uso de ERFE como uma ferramenta de sucesso para a inspeção da atividade eritropoiética na DRC, especialmente aqueles que tomam AEEs para tratar anemia. ABSTRACT Background: Erythroferrone (ERFE) is a glycoprotein hormone synthesis and release by erythroblasts. Recently identified as an erythropoietic regulator and activated in response to stimulating erythropoietin (Epo). In chronic kidney diseases (CKD), anemia is a hallmark disorder due to a decrease in hyposensitive erythropoietic to the Epo; these patients recommended to use of Erythropoiesis-stimulating agents (ESAs). The aim: This study aimed to assess serum ERFE level in patients with CKD and investigate the continuing effects of long-term anemic ESA use associated with markers of erythropoiesis and iron metabolism. Methods: Sixty-five CKD patients divided in two groups, included 30 hemodialyses (HD) and 35 without hemodialysis (non-HD) CKD patients, were compared to 25 healthy voluntaries matched by gender and age enrolled in the current study. Serum ERFE level was measured by an enzyme-linked immunosorbent assay (ELISA). Results: Serum ERFE level was significantly elevated in HD patients median (IQR) about 17.25 (13.4) ng/mL, odds ratio (OR = 10.161), (AUC 0.996) greater than CKD 4(6.1) ng/ml, (OR = 6.295), (AUC = 0.984) p<0.001; also, these are positively correlated with the use of ESA in HD, and CKD (r = 1.00 and r = 0.95) respectively as compared to healthy group 2(2.1) ng/ml. Serum Periódico Tchê Química. ISSN 2179-0302. (2021); vol.18 (n°37) Downloaded from www.periodico.tchequimica.com 136 ERFE levels were significantly negative (p<0.05) in both CKD and HD patients related to GFR (r =-0.396, and r =-0.68), transferrin saturation (TS%) (r =-0.842, and r =-0.877), serum levels of Ferritin (r =-0.865 and r =-0.866), and Iron (r =-0.860, and r =-0.851), RBC (r =-0.841, and r =-0.843), hemoglobin (Hb) (r =-0.758, and r =-0.796). Conclusion: The present study demonstrated that elevated serum ERFE levels associated with erythropoietic activity and anemia are higher in CKD with HD and non-HD patients treated with ESA than in non-ESA patients. This study suggested using ERFE as a successful tool for erythropoietic activity inspection in CKD, especially those taking ESAs to treat anemia.
... The current study supposed that associated with the prevalence of anemia in HD more than CKD because HD patients were persisted in ESA treatment lead to high expression of ERFE as a regulator of the erythroid mediator from erythroblast. It is a new sensitive biomarker in humans to estimate the relationship between iron metabolism and erythropoiesis and noticed that ESA might have a detrimental effect in the antidoping field Ramirez Cuevas et al., 2020). As a result, hyperactivation of erythropoiesis and accelerated synthesis of the erythroid progenitor, but this undergo intramedullary apoptosis before the differentiation process is completed, therefore can cause anemia. ...
Article
Full-text available
ABSTRACT Background: Erythroferrone (ERFE) is a glycoprotein hormone synthesis and release by erythroblasts. Recently identified as an erythropoietic regulator and activated in response to stimulating erythropoietin (Epo). In chronic kidney diseases (CKD), anemia is a hallmark disorder due to a decrease in hyposensitive erythropoietic to the Epo; these patients recommended to use of Erythropoiesis-stimulating agents (ESAs). The aim: This study aimed to assess serum ERFE level in patients with CKD and investigate the continuing effects of long-term anemic ESA use associated with markers of erythropoiesis and iron metabolism. Methods: Sixty-five CKD patients divided in two groups, included 30 hemodialyses (HD) and 35 without hemodialysis (non-HD) CKD patients, were compared to 25 healthy voluntaries matched by gender and age enrolled in the current study. Serum ERFE level was measured by an enzyme-linked immunosorbent assay (ELISA). Results: Serum ERFE level was significantly elevated in HD patients median (IQR) about 17.25 (13.4) ng/mL, odds ratio (OR = 10.161), (AUC 0.996) greater than CKD 4(6.1) ng/ml, (OR = 6.295), (AUC = 0.984) p<0.001; also, these are positively correlated with the use of ESA in HD, and CKD (r = 1.00 and r = 0.95) respectively as compared to healthy group 2(2.1) ng/ml. Serum Periódico Tchê Química. ISSN 2179-0302. (2021); vol.18 (n°37) Downloaded from www.periodico.tchequimica.com 136 ERFE levels were significantly negative (p<0.05) in both CKD and HD patients related to GFR (r =-0.396, and r =-0.68), transferrin saturation (TS%) (r =-0.842, and r =-0.877), serum levels of Ferritin (r =-0.865 and r =-0.866), and Iron (r =-0.860, and r =-0.851), RBC (r =-0.841, and r =-0.843), hemoglobin (Hb) (r =-0.758, and r =-0.796). Conclusion: The present study demonstrated that elevated serum ERFE levels associated with erythropoietic activity and anemia are higher in CKD with HD and non-HD patients treated with ESA than in non-ESA patients. This study suggested using ERFE as a successful tool for erythropoietic activity inspection in CKD, especially those taking ESAs to treat anemia.
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Erythroferrone (ERFE), the erythroid regulator of iron metabolism, inhibits hepcidin to increase iron availability for erythropoiesis. ERFE plays a pathological role during ineffective erythropoiesis as occurs in X-linked sideroblastic anemia (XLSA) and β-thalassemia. Its measurement might serve as an indicator of severity for these diseases. However, for reliable quantification of ERFE analytical characterization is indispensable to determine the assay's limitations and define proper methodology. We developed a sandwich ELISA for human serum ERFE using polyclonal antibodies and report its extensive analytical validation. This new assay showed, for the first time, the differentiation of XLSA and β-thalassemia major patients from healthy controls (p = 0.03) and from each other (p<0.01), showing the assay provides biological plausible results. Despite poor dilution linearity, parallelism and recovery in patient serum matrix, which indicated presence of a matrix effect and/or different immunoreactivity of the antibodies to the recombinant standard and the endogenous analyte, our assay correlated well with two other existing ERFE ELISAs (both R2 = 0.83). Nevertheless, employment of one optimal dilution of all serum samples is warranted to obtain reliable results. When adequately performed, the assay can be used to further unravel the human erythropoiesis-hepcidin-iron axis in various disorders and assess the added diagnostic value of ERFE.
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Erythroferrone (ERFE) is the main erythroid regulator of hepcidin, the homeostatic hormone controlling plasma iron levels and total body iron. When the release of erythropoietin from the kidney stimulates the production of new red blood cells, it also increases the synthesis of ERFE in bone marrow erythroblasts. Increased ERFE then suppresses hepcidin synthesis, thereby mobilizing cellular iron stores for use in heme and hemoglobin synthesis. Recent mechanistic studies have shown that ERFE suppresses hepcidin transcription by inhibiting bone morphogenetic protein signaling in hepatocytes. In ineffective erythropoiesis, pathological overproduction of ERFE by an expanded population of erythroblasts suppresses hepcidin and causes iron overload, even in non‐transfused patients. ERFE may be a useful biomarker of ineffective erythropoiesis and an attractive target for treating its systemic effects.
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Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to stimulation by erythropoietin (EPO). We previously demonstrated that ERFE messenger RNA expression and serum protein concentration increase in mice subjected to hemorrhage or EPO therapy, that ERFE acts on hepatocytes to suppress hepcidin, and that the resulting decrease in hepcidin augments iron delivery for intensified erythropoiesis. We also showed that ERFE contributes to pathological hepcidin suppression and iron overload in mice with nontransfused β-thalassemia. We now report the development and technical validation of a rabbit monoclonal antibody-based sandwich immunoassay for human ERFE. We use this assay to show that blood loss or EPO administration increases serum ERFE concentrations in humans, and that patients with both nontransfused and transfused β-thalassemia have very high serum ERFE levels, which decrease after blood transfusion. The assay should be useful for human studies of normal and disordered erythropoiesis and its effect on iron homeostasis.
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Erythropoietin (EPO) is the main hormone regulating red blood cell (RBC) production. The large-scale production of a recombinant human erythropoietin (rHuEPO) by biotechnological methods has made possible its widespread therapeutic use as well as its misuse in sports. Since the marketing of the first epoetin in 1989, the development has progressed to the third-generation analogs. However, the production of rHuEPO is costly, and the frequent administration of an injectable formula is not optimal for compliance of therapeutic patients. Hence, pharmaceutical industries are currently developing alternative approaches to stimulate erythropoiesis, which might offer new candidates for doping purposes. The hypoxia inducible factors (HIF) pathway is of particular interest. The introduction of new erythropoiesis-stimulating agents (ESAs) for clinical use requires subsequent development of anti-doping methods for detecting the abuse of these substances. The detection of ESAs is based on two different approaches, namely, the direct detection of exogenous substances and the indirect detection, for which the effects of the substances on specific biomarkers are monitored. Omics technologies, such as ironomics or transcriptomics, are useful for the development of new promising biomarkers for the detection of ESAs. Finally, the illicit use of ESAs associates with multiple health risks that can be irreversible, and an essential facet of anti-doping work is to educate athletes of these risks. The aim of this review is to provide an overview of the evolution of ESAs, the research and implementation of the available detection methods, and the side effects associated with the misuse of ESAs.
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Background: Autologous blood transfusion (ABT) is an efficient way to increase sport performance. It is also the most challenging doping method to detect. At present, individual follow-up of haematological variables via the athlete biological passport (ABP) is used to detect it. Quantification of a novel hepatic peptide called hepcidin may be a new alternative to detect ABT. Study design and methods: In this prospective clinical trial, healthy subjects received a saline injection for the control phase, after which they donated blood that was stored and then transfused 36 days later. The impact of ABT on hepcidin as well as haematological parameters, iron metabolism, and inflammation markers was investigated. Results: Blood transfusion had a particularly marked effect on hepcidin concentrations compared to the other biomarkers, which included haematological variables. Hepcidin concentrations increased significantly: 12 hours and 1 day after blood re-infusion, these concentrations rose by 7- and 4-fold, respectively. No significant change was observed in the control phase. Conclusion: Hepcidin quantification is a cost-effective strategy that could be used in an "ironomics" strategy to improve the detection of ABT. This article is protected by copyright. All rights reserved.
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Recovery from blood loss requires a greatly enhanced supply of iron to support expanded erythropoiesis. After hemorrhage, suppression of the iron-regulatory hormone hepcidin allows increased iron absorption and mobilization from stores. We identified a new hormone, erythroferrone (ERFE), that mediates hepcidin suppression during stress erythropoiesis. ERFE is produced by erythroblasts in response to erythropoietin. ERFE-deficient mice fail to suppress hepcidin rapidly after hemorrhage and exhibit a delay in recovery from blood loss. ERFE expression is greatly increased in Hbb(th3/+) mice with thalassemia intermedia, where it contributes to the suppression of hepcidin and the systemic iron overload characteristic of this disease.