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Erythroferrone as a sensitive biomarker to detect stimulation of erythropoiesis

  • Institute for Dopinganalysis and Sportbiochemistry (IDAS Dresden)


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.
Erythroferrone as a sensitive biomarker to detect stimulation
of erythropoiesis
Kelvin Ramirez Cuevas
| Céline Schobinger
| Emeric Gottardo
Sven Christian Voss
| Tiia Kuuranne
| Jean-Daniel Tissot
| Bernard Favrat
Nathan Townsend
| Nicolas Leuenberger
Swiss Laboratory for Doping Analyses,
University Center of Legal Medicine, Lausanne
and Geneva, Centre Hospitalier Universitaire
Vaudois and University of Lausanne,
Anti-Doping Lab Qatar, Sports City Road,
Sports City, P.O. Box 27775, Doha, Qatar
Transfusion interrégionale CRS, site
d'Epalinges, Switzerland
Department of Ambulatory Care and
Community Medicine, University of Lausanne,
Lausanne, Switzerland
Athlete Health and Performance Centre,
Aspetar Orthopaedic and Sports Medicine
Hospital Doha, Qatar
Nicolas Leuenberger, Swiss Laboratory for
Doping Analyses, Ch. Des Croisettes 22, 1066
Epalinges, Switzerland.
Funding information
Fondation Dr Henri Dubois-Ferrière Dinu
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.
blood doping, erythroferrone, immunoassay
The greatest deterrent to blood doping with erythropoiesis stimu-
lating agents (ESAs) is the hematological module of the Athlete
Biological Passport (ABP).
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.
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.
Proteins involved
in erythropoiesis and iron metabolism have been suggested as
potential biomarkers for ESA abuse.
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. © 2019 John Wiley & Sons, Ltd. 1
erythropoietin (rhEPO) administration.
In 2014, erythroferrone
(ERFE) was discovered as a novel erythroid regulator of iron
metabolism in a mouse model.
However, ERFE has been described
as having the same or a similar structure as myonectin.
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.
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
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.
2.1 |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.
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%,
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
(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.
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
(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.
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.
2.2.2 |Clinical study: blood withdrawal and iron
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 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
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
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
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.
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.
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).
4.1 |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
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.
We previ-
ously evaluated this commercially available kit, and found that ERFE
quantification by this method was not reproducible.
ERFE responses
following rhEPO administration were highly variable, and not coher-
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.
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.
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
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).
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.
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.
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.
Some endurance ath-
letes inject iron to maintain a high serum ferritin concentration.
Intravenous iron supplementation improves fatigue and overall mood
in runners without a clinical iron deficiency.
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.
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.
correlate closely (Figure S2), the influence of altitude exposure
on Ret% measurement should also be valuable for ERFE as
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
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.
Nicolas Leuenberger
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iron injection did not affect ERFE
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(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
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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.
... 107 Principal iron consumer in humans, the erythropoietic activity will decrease the hepcidin transcription through erythroferrone (ERFE) suppression. 108 Known as a useful indicator of iron stores, 53 circulating ferritin decreased after blood withdrawal, 48,53 and tends to increase after reinfusion 46 but with lower amplitude. 53 In addition, the decrease in serum ferritin seems to be progressive with rhEPO treatment 47 although not always observed with microdoses. ...
... 53 In addition, the decrease in serum ferritin seems to be progressive with rhEPO treatment 47 although not always observed with microdoses. 49 However, similarly to iron, circulating ferritin can be significantly affected by repeated iron injections 48 and accentuated day-to-day variability has been observed in female athletes compared with the population standards. 115 Furthermore, as a positive acute phase protein (i.e., proteins increasing in response to inflammation), serum ferritin concentration may be modulated in various conditions without changes in iron storage. ...
... 123 Dependent on EPO release from the kidney, ERFE level is consequently increased in the erythroblasts of the bone marrow. 124 Therefore, ERFE tends to increase after various types of ESA injections 48 including microdoses, 47 despite a large interindividual variability. 52 Regarding blood manipulation, an increase in ERFE is generally observed during the blood withdrawal phase, 54 followed by a decrease during the reinfusion phase. ...
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The detection of blood doping represents a current major issue in sports and an ongoing challenge for anti-doping research. Initially focusing on direct detection methods to identify a banned substance or its metabolites, the anti-doping effort has been progressively complemented by indirect approaches. The longitudinal and individual monitoring of specific biomarkers aims to identify non-physiological variations that may be related to doping practices. From this perspective, the identification of markers sensitive to erythropoiesis alteration is key in the screening of blood doping. The current Athlete Biological Passport (ABP) implemented since 2009 is composed of 14 variables (including two primary markers, i.e. hemoglobin concentration and OFF-score) for the hematological module to be used for indirect detection of blood doping. Nevertheless, research has continually proposed and investigated new markers sensitive to an alteration of the erythropoietic cascade, and specific to blood doping. If multiple early markers have been identified (at the transcriptomic level) or developed directly in a diagnostics' kit (at a proteomic level), other target variables at the end of the erythropoietic process (linked with the red blood cell functions) may strengthen the hematological module in the future. Therefore, this review aims to provide a global systematic overview of the biomarkers considered to date in the indirect investigation of blood doping.
... 22 A study in which ERFE was measured in six subjects receiving relatively high doses of rhEpo or analogs, intravenously or subcutaneously, using an assay different from the one used in this study did not suggest that ERFE would be a reliable marker for rhEpo doping, 23 although (while our manuscript was under revision) the same group reported that a different enzyme-linked immunosorbent assay was able to detect increased ERFE levels in the same samples. 24 Conversely, increased erythropoiesis induced by training did not affect ERFE and hepcidin levels in runners. 25 Our results demonstrate that ERFE is sensitive enough to flag even microdose rhEpo, correlates with Epo levels ( Figure 3A, B) and has a detection window longer than that of Epo, thereby indicating that ERFE holds promise as a novel biomarker of doping for implementation in the ABP, although additional studies are required. ...
... 102 Importantly, when six subcutaneous micro-doses of 20 IU × kg bw −1 epoetin α are injected, ERFE levels increase during treatment. 103 In alignment, 11 intravenous injections of epoetin α (20 IU × kg bw −1 ) augmented ERFE levels by ~100% in 19 healthy men and women. 51 A possible concern for hepcidin is that numerous factors normal for an elite athlete such as altitude 102 and iron supplementation 104 affect the systemic levels. ...
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Blood doping is prohibited for athletes but has been a well-described practice within endurance sports throughout the years. With improved direct and indirect detection methods, the practice has allegedly moved towards micro-dosing, i.e., reducing the blood doping regime amplitude. This narrative review evaluates whether blood doping, specifically recombinant human erythropoietin (rhEpo) treatment and blood transfusions are performance-enhancing, the responsible mechanism as well as detection possibilities with a special emphasis on micro-dosing. In general, studies evaluating micro-doses of blood doping are limited. However, in randomized, double-blinded, placebo-controlled trials, three studies find that infusing as little as 130 ml red blood cells or injecting 9 IU × kg bw-1 rhEpo three times per week for four weeks improve endurance performance ~4-6 %. The responsible mechanism for a performance-enhancing effect following rhEpo or blood transfusions appear to be increased O2 -carrying capacity, which is accompanied by an increased muscular oxygen extraction and likely increased blood flow to the working muscles, enabling the ability to sustain a higher exercise intensity for a given period. Blood doping in micro-doses challenges indirect detection by the Athlete Biological Passport, albeit it can identify ~20-60% of the individuals depending on the sample timing. However, novel biomarkers are emerging, and some may provide additive value for detection of micro blood doping such as the immature reticulocytes or the iron regulatory hormones hepcidin and erythroferrone. Future studies should attempt to validate these biomarkers for implementation in real-world anti-doping efforts and continue the biomarker discovery.
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This study investigated the impact of low-volume blood withdrawal on the hematological biomarkers currently considered for anti-doping purposes. After baseline measurement (D - 7), a 140 mL blood withdrawal was completed (D + 0) on 12 healthy volunteers, followed by weekly monitoring for 21 days (D + 7 - 21). Each visit consisted of a full blood count (Sysmex XN-1000) and duplicate blood volume measurements by CO-rebreathing. A significant decrease in total hemoglobin mass (Hbmass) (-2.3%, p = 0.007) and red blood cell volume (RBCV) (-2.8%, p = 0.028) was reported at D + 7. Despite no atypical passport finding (ATPF) when considering the athlete biological passport adaptive longitudinal model, hemoglobin concentration ([Hb]) increased significantly at D + 21 (+3.8%, p = 0.031). Besides, ferritin (FERR) was significantly downregulated at all points following blood withdrawal, with the largest decrease occurring at D + 7 (-26.6%, p < 0.001). Regardless of the presumable effect of blood reinfusion on ABP biomarkers, these results illustrate the challenge of monitoring hematological variables for the detection of low-volume blood withdrawal. Finally, this study outlines the sensitivity of FERR to altered erythropoiesis to support the implementation of iron markers as complementary variables for the longitudinal monitoring of blood doping, despite the potential influence of confounding factors (e.g., iron supplementations).
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Iron-deficiency anemia (IDA) can be grouped under low hepcidin and high erythroferrone (ERFE) anemia. There is a negative correlation between ERFE and hepcidin, irrespective of the type of anemia. ERFE is a mediator of the response to erythropoietic stress, suppressing hepcidin to promote the mobilization of stored iron and the absorption of dietary iron.
Among the substances prohibited by the World Anti-Doping Agency, "peptide hormones, growth factors, related substances, and mimetics" are classified as prohibited both in- and out-of-competition in section S2. This work reviews growth hormone and its releasing peptides, insulin-like growth factor 1 as the main growth factor, insulin, and erythropoietin and other agents that affect erythropoiesis. This review analyzes the prevalence of use among professional athletes and gym clients, the forms of use, dosing, ergogenic effects and effects on physical performance, as well as side effects and anti-doping detection methods.
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In general, the concept of a mechanism in biology has three distinct meanings. It may refer to a philosophical thesis about the nature of life and biology, to the internal workings of a machine-like structure, or to the causal explanation of a particular phenomenon [1]. Understanding the biological mechanisms that justify acute and chronic physiological responses to exercise interventions determines the development of training principles and training methods. A strong understanding of the effects of exercise in humans may help researchers to identify what causes specific biological changes and to properly identify the most adequate processes for implementing a training stimulus [1]. Despite the significant body of knowledge regarding the physiological and physical effects of different training methods (based on load dimensions), some biological causes of those changes are still unknown. Additionally, few studies have focused on natural biological variability in humans and how specific human properties may underlie different responses to the same training intervention. Thus, more original research is needed to provide plausible biological mechanisms that may explain the physiological and physical effects of exercise and training in humans. In this Special Issue, we discuss/demonstrate the biological mechanisms that underlie the beneficial effects of physical fitness and sports performance, as well as their importance and their role in/influences on physical health. A total of 28 manuscripts are published here, of which 25 are original articles, two are reviews, and one is a systematic review. Two papers are on neuromuscular training programs (NMTs), training monotony (TM), and training strain (TS) in soccer players [2,3]; five articles provide innovative findings about testosterone and cortisol [4,5], gastrointestinal hormones [6], spirulina [7], and concentrations of erythroferrone (ERFE) [8]; another five papers analyze fitness and its association with other variables [7,9–12]; three papers examine body composition in elite female soccer players [2], adolescents [6], and obese women [7]; five articles examines the effects of high-intensity interval training (HIIT) [7,10,13–15]; one paper examines the acute effects of different levels of hypoxia on maximal strength, muscular endurance, and cognitive function [16]; another article evaluates the efficiency of using vibrating exercise equipment (VEE) compared with using sham-VEE in women with CLBP (chronic lowback pain) [17]; one article compares the effects of different exercise modes on autonomic modulation in patients with T2D (type 2 diabetes mellitus) [14]; and another paper analyzes the changes in ABB (acid–base balance) in the capillaries of kickboxers [18]. Other studies evaluate: the effects of resistance training on oxidative stress and muscle damage in spinal cord-injured rats [19]; the effects of muscle training on core muscle performance in rhythmic gymnasts [20]; the physiological profiles of road cyclist in different age categories [21]; changes in body composition during the COVID-19 [22]; a mathematical model capable of predicting 2000 m rowing performance using a maximum-effort 100 m indoor rowing ergometer [23]; the effects of ibuprofen on performance and oxidative stress [24]; the associations of vitamin D levels with various motor performance tests [12]; the level of knowledge on FM (Fibromyalgia) [25]; and the ability of a specific BIVA (bioelectrical impedance vector analysis) to identify changes in fat mass after a 16-week lifestyle program in former athletes [26]. Finally, one review evaluates evidence from published systematic reviews and meta-analyses about the efficacy of exercise on depressive symptoms in cancer patients [27]; another review presents the current state of knowledge on satellite cell dependent skeletal muscle regeneration [28]; and a systematic review evaluates the effects of exercise on depressive symptoms among women during the postpartum period [29]
Iron supplementation is not considered as a doping method; however, it can affect the levels of several biomarkers of the hematologic module of the athlete biological passport (ABP), such as the reticulocyte percentage (%RET) and hemoglobin (HGB) level. Thus, iron injection could be a confounding factor in anti‐doping analyses. Previous studies have suggested that the HGB level and the expression levels of reticulocyte‐related‐mRNAs, such as 5’‐aminolevulinate synthase 2 (ALAS2) and carbonic anhydrase 1 (CA1), could be promising biomarkers for the ABP and detectable in dried blood spots (DBSs). Therefore, in this study, we examined the impact of iron injection on the levels of these potential biomarkers in DBSs. Reticulocyte‐related‐mRNAs analyses were performed by RT‐qPCR. Ferritin level in DBS was measured with ELISA method. Notably, there were no significant effects of iron supplementation on the levels of ALAS2 and CA1 mRNAs but by contrast, the %RET and immature reticulocyte fraction (IRF) measured in whole blood increased significantly following iron injection. As expected, iron supplementation increased the ferritin level significantly in both serum and DBS samples. In conclusion, these findings reinforce the specificity of reticulocyte‐related mRNAs in DBSs as biomarkers of blood doping to target in anti‐doping analyses.
Although many studies have shown that supplementation with iron and erythropoiesis‐stimulating agents (ESA) is frequently used for managing chemotherapy‐induced anemia (CIA), optimal combination therapy using these agents together to ameliorate anemia is not well characterized. To assess the effects of ESA combined with oral or intravenous (IV) iron on relieving CIA, PubMed, Cochrane Library, Embase, China National Knowledge Infrastructure (CNKI) were searched for articles. Data collected in the articles were meta‐analyzed using RevMan 5.3 software with a random‐effects model. Our comprehensive search yielded 1666 potentially relevant trials. A total of 41 trials randomizing 4200 patients with CIA fulfilled inclusion criteria, including 34 Chinese articles and 7 English articles. Meta‐analysis showed that treatment with both ESA and iron more effectively improved CIA relative to iron supplementation alone, with increased hemoglobin, hematocrit, red blood cell count and haematopoietic response rate. Subgroup analyses revealed iron administration, both oral and IV iron, improved anemia in ESA‐treated cancer patients with CIA. Our analysis demonstrates that iron supplementation combined with ESA more effectively ameliorates CIA relative to iron supplementation alone, without regard to whether IV or oral iron was used. Together, our findings may contribute to the clinical treatment of CIA using iron therapy with or without ESA. This article is protected by copyright. All rights reserved.
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The stimulation of erythrocyte formation increases the demand for iron by the bone marrow and this in turn may affect the levels of circulating diferric transferrin. As this molecule influences the production of the iron regulatory hormone hepcidin, we hypothesised that erythropoiesis-driven changes in diferric transferrin levels could contribute to the decrease in hepcidin observed following erythropoietin administration. To examine this, we treated mice with erythropoietin and examined diferric transferrin at various time points up to 18 hours. We also investigated the effect of altering diferric transferrin levels on erythropoietin-induced inhibition of Hamp1, the gene encoding hepcidin. We detected a decrease in diferric transferrin levels five hours after erythropoietin injection and prior to any inhibition of hepatic Hamp1 message. Diferric transferrin returned to control levels 12 hours after erythropoietin injection and had increased beyond control levels by 18 hours. Increasing diferric transferrin levels via intravenous iron injection prevented the inhibition of Hamp1 expression by erythropoietin without altering hepatic iron concentration or the expression of Erfe, the gene encoding erythroferrone. These results suggest that diferric transferrin likely contributes to the inhibition of hepcidin production in the period shortly after erythropoietin injection and that, under the conditions examined, increasing diferric transferrin levels can overcome the inhibitory effect of erythroferrone on hepcidin production. They also imply that the decrease in Hamp1 expression in response to an erythropoietic stimulus is likely to be mediated by multiple signals.
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Background: We aimed to identify associations between erythroferrone (ERFE), a regulator of hepcidin 25, and biomarkers of erythropoiesis and iron metabolism. We also aimed to determine the effects of erythropoiesis-stimulating agents (ESA), continuous erythropoietin receptor activator (CERA) and darbepoetin-α (DA) on ERFE production in patients on hemodialysis (HD). Methods: Blood samples were obtained from 59 patients before HD sessions on day 0 (baseline). Twenty patients who were injected with either CERA (N = 10) or DA (N = 10) at the end of the dialysis week (day 0), who had ferritin ≥ 100 ng/mL and/or transferrin saturation ≥ 20%, and hemoglobin > 9 g/dL were selected from among the 59 patients. Blood was sampled serially before HD sessions on days 3, 5, 7 from patients on DA and on the same days plus day 14 from those on CERA. Results: Levels of ERFE correlated inversely with those of hepcidin 25 and ferritin, and positively with those of soluble transferrin receptor. The hepcidin 25: ERFE ratio and hepcidin 25 levels positively correlated with ferritin levels. Levels of ERFE significantly increased from day 3 of treatment with DA and CERA and decreased by days 7 and 14, respectively. Erythropoiesis-stimulating agents concomitantly decreased levels of hepcidin 25 as those of ERFE increased. Conclusion: We identified a novel association between ESA and ERFE in patients on HD. Both DA and CERA increased levels of ERFE that regulated hepcidin 25 and led to iron mobilization from body stores during erythropoiesis.
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Inherited anemias with ineffective erythropoiesis, such as β-thalassemia, manifest inappropriately low hepcidin production and consequent excessive absorption of dietary iron leading to iron overload. Erythroferrone (ERFE) is an erythroid regulator of hepcidin synthesis and iron homeostasis. Erfe expression was highly increased in the marrow and spleen of Hbb(Th3/+) mice (Th3/+), a mouse model of thalassemia intermedia. Ablation of Erfe in Th3/+ mice restored normal levels of circulating hepcidin at 6 weeks of age suggesting that ERFE could be a factor suppressing hepcidin production in β-thalassemia. We examined the expression of Erfe and the consequences of its ablation in thalassemic mice from 3 to 12 weeks of age. The loss of ERFE in thalassemic mice led to full restoration of hepcidin mRNA expression at 3 and 6 weeks of age and significant reduction in liver and spleen iron content at 6 and 12 weeks of age. Ablation of Erfe slightly ameliorated ineffective erythropoiesis as indicated by reduced spleen index, RDW and MCV but did not improve the anemia. Thus, erythroferrone mediates hepcidin suppression and contributes to iron overload in a mouse model of β-thalassemia. Copyright © 2015 American Society of Hematology.
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Purpose To determine the effect of intravenous iron supplementation on performance, fatigue and overall mood in runners without clinical iron deficiency. Methods Fourteen distance runners with serum ferritin 30–100 µg·L−1 were randomly assigned to receive three blinded injections of intravenous ferric-carboxymaltose (2 ml, 100 mg, IRON) or normal saline (PLACEBO) over four weeks (weeks 0, 2, 4). Athletes performed a 3,000 m time trial and 10×400 m monitored training session on consecutive days at week 0 and again following each injection. Hemoglobin mass (Hbmass) was assessed via carbon monoxide rebreathing at weeks 0 and 6. Fatigue and mood were determined bi-weekly until week 6 via Total Fatigue Score (TFS) and Total Mood Disturbance (TMD) using the Brief Fatigue Inventory and Brunel Mood Scale. Data were analyzed using magnitude-based inferences, based on the unequal variances t-statistic and Cohen's Effect sizes (ES). Results Serum ferritin increased in IRON only (Week 0: 62.8±21.9, Week 4: 128.1±46.6 µg·L−1; p = 0.002) and remained elevated two weeks after the final injection (127.0±66.3 µg·L−1, p = 0.01), without significant changes in Hbmass. Supplementation had a moderate effect on TMD of IRON (ES -0.77) with scores at week 6 lower than PLACEBO (ES -1.58, p = 0.02). Similarly, at week 6, TFS was significantly improved in IRON vs. PLACEBO (ES –1.54, p = 0.05). There were no significant improvements in 3,000 m time in either group (Week 0 vs. Week 4; Iron: 625.6±55.5 s vs. 625.4±52.7 s; PLACEBO: 624.8±47.2 s vs. 639.1±59.7 s); but IRON reduced their average time for the 10×400 m training session at week 2 (Week 0: 78.0±6.6 s, Week 2: 77.2±6.3; ES–0.20, p = 0.004). Conclusion During 6 weeks of training, intravenous iron supplementation improved perceived fatigue and mood of trained athletes with no clinical iron deficiency, without concurrent improvements in oxygen transport capacity or performance.
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Unexplained fatigue is often left untreated or treated with antidepressants. This randomized, placebo-controlled, single-blinded study evaluated the efficacy and tolerability of single-dose intravenous ferric carboxymaltose (FCM) in iron-deficient, premenopausal women with symptomatic, unexplained fatigue. Fatigued women (Piper Fatigue Scale [PFS] score ≥5) with iron deficiency (ferritin <50 µg/L and transferrin saturation <20%, or ferritin <15 µg/L) and normal or borderline hemoglobin (≥115 g/L) were enrolled in 21 sites in Austria, Germany, Sweden and Switzerland, blinded to the study drug and randomized (computer-generated randomization sequence) to a single FCM (1000 mg iron) or saline (placebo) infusion. Primary endpoint was the proportion of patients with reduced fatigue (≥1 point decrease in PFS score from baseline to Day 56). The full analysis included 290 women (FCM 144, placebo 146). Fatigue was reduced in 65.3% (FCM) and 52.7% (placebo) of patients (OR 1.68, 95%CI 1.05-2.70; p = 0.03). A 50% reduction of PFS score was achieved in 33.3% FCM- vs. 16.4% placebo-treated patients (p<0.001). At Day 56, all FCM-treated patients had hemoglobin levels ≥120 g/L (vs. 87% at baseline); with placebo, the proportion decreased from 86% to 81%. Mental quality-of-life (SF-12) and the cognitive function scores improved better with FCM. 'Power of attention' improved better in FCM-treated patients with ferritin <15 µg/L. Treatment-emergent adverse events (placebo 114, FCM 209; most frequently headache, nasopharyngitis, pyrexia and nausea) were mainly mild or moderate. A single infusion of FCM improved fatigue, mental quality-of-life, cognitive function and erythropoiesis in iron-deficient women with normal or borderline hemoglobin. Although more side effects were reported compared to placebo, FCM can be an effective alternative in patients who cannot tolerate or use oral iron, the common treatment of iron deficiency. Overall, the results support the hypothesis that iron deficiency can affect women's health, and a normal iron status should be maintained independent of hemoglobin levels. NCT01110356.
Key Points Human serum ERFE shows similar pathophysiological responses to mouse models.
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.
The concentration of hepcidin, a key regulator of iron metabolism, is suppressed during periods of increased erythropoietic activity. The present study obtained blood samples from 109 elite athletes and examined the correlations between hepcidin and markers of erythropoiesis and iron metabolism (i.e., hemoglobin, erythropoietin (EPO), ferritin, erythroferrone (ERFE), and iron concentration). Furthermore, an administration study was undertaken to examine the effect of recombinant human EPO (rhEPO) delta (Dynepo™) on hepcidin concentrations in healthy male volunteers. The effects on hepcidin were then compared with those on reticulocyte percentage (Ret%) and ferritin concentration. There was a significant positive correlation between hepcidin and ferritin, iron, and hemoglobin levels in athletes, whereas hepcidin showed an inverse correlation with ERFE. Administration of rhEPO delta reduced hepcidin levels, suggesting that monitoring hepcidin may increase the sensitivity of the Athlete Biological Passport (ABP) for detecting rhEPO abuse. This article is protected by copyright. All rights reserved.
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.
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.