Serum prohepcidin levels and iron parameters in term small-for gestationalage newborns

SERVET OZKIRAZ1, HASAN KILICDAG1, ZEYNEL GOKMEN1, AYSE ECEVIT1,

AYLIN TARCAN1, & NAMIK OZBEK2

1Faculty of Medicine, Department of Neonatology, Baskent University, Ankara, Turkey and 2Department of Pediatric Hematology, Baskent

University, Ankara, Turkey

AbstractAim. To understand the effect of prenatal chronic hypoxia on prohepcidin levels in term newborns.Method. We determined prohepcidin (Pro-Hep) levels in both term appropriate-for-gestational age (AGA) and term small-for-gestational-age (SGA) infants. Uteroplacental insufficiency had exposed all SGA infants to chronic hypoxia. Serum samples werecollected from nine full-term SGA infants. Samples were analyzed for complete blood count, serum iron and ferritinconcentrations, iron-binding capacity, and prohepcidin levels.Results. The mean serum Pro-Hep level was 156.4 + 46.7 ng/ml for SGA infants and 482 + 371.9 ng/ml for 16 healthy termAGA infants (historical controls); this difference was statistically significant. Statistical analyses revealed significant between-groupdifferences for hemoglobin, hematocrit, mean corpuscular volume, red blood cell distribution width, and serum ferritin and Pro-Hep levels.Conclusion. This study showed that compared with AGA infants, Pro-Hep levels were lower in term SGA infants, suggesting thatprenatal chronic hypoxia decreases Pro-Hep synthesis.

Keywords: Hypoxia, newborn, prohepcidin

Introduction

Hepcidin is the principle iron-regulatory hormone that acts on

intestinal iron absorption and iron retention in reticuloen-

dothelial cells [1–3]. The human hepcidin gene, locus 19q13,

encodes a prepropeptide of 84 amino acids that is cleaved to

the 60-amino acid form prohepcidin. Prohepcidin is pro-

cessed to give the 25-amino acid form hepcidin [4]. Detecting

serum concentrations of hepcidin is difficult, but serum

concentrations of Pro-Hep are readily detectable using an

enzyme-linked immunosorbent assay (ELISA).

The molecular mechanisms that control hepcidin expres-

sion are largely unknown. However, many factors such as

inflammation, infection, anemia, and hypoxia have been

reported to influence hepcidin synthesis [5–7]. Nicolas et al.

[6] reported that experimentally induced hypoxia led to a

decrease in hepcidin gene expression. The effect of hypoxia on

hepcidin synthesis in human newborns remains to be

investigated. Intrauterine growth restriction (IUGR) creates

an adverse intrauterine environment to the fetus, to the

inclusion of relative oxygen deprivation. Fetal hypoxia has

been documented in IUGR fetuses by cordocentesis [8], and

IUGR is a known risk factor for increased erythropoiesis, as

evidence by increased numbers of circulating nucleated red

blood cells at birth [9] and elevated erythropoietin (EPO)

levels [10]. Therefore, we aimed to determine Pro-Hep levels

in term small-for-gestational-age (SGA) infants to understand

the effect of prenatal chronic hypoxia.

Materials and methods

This study included nine asymmetric SGA (birth weight

510th percentile for gestational week) full-term newborns

admitted to the Neonatal Intensive Care Unit, Medical

School of Baskent University, Ankara, Turkey. The control

group was composed of patients from our prior study [11]: 16

healthy, appropriate-for-gestational age (AGA) (birth weight

between the 10th and 90th percentiles) full-term newborns

admitted to our well-baby follow-up outpatient clinic.

Uteroplacental insufficiency detected by prenatal Doppler

ultrasound had exposed all of the SGA babies to chronic

hypoxia. We excluded infants exposed to maternal chronic

disease, smoking, diabetes, alcohol or drug abuse, congenital-

genetic malformations, intracranial hemorrhage, infection,

and those who had received blood transfusion. Our local

hospital ethics committee approved the study and written

informed consent was obtained from the subjects’ parents.

Blood sample was obtained from puncture of a vein from

SGA infants between 4th and 10th days of life and in the

control group (historical control group) it was done between

days 4 and 14 of life during a visit to the well-baby outpatient

clinic. Each specimen was analyzed for complete blood count,

(Received 17 December 2010; revised 4 April 2011; accepted 8 April 2011)

Correspondence: Servet Ozkiraz, Faculty of Medicine, Department of Neonatology, Baskent University, Ankara, Turkey.

E-mail: sozkiraz@yahoo.com

The Journal of Maternal-Fetal and Neonatal Medicine, December 2011; 24(12): 1437–1439

� 2011 Informa UK, Ltd.

ISSN 1476-7058 print/ISSN 1476-4954 online

DOI: 10.3109/14767058.2011.581714

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serum iron and ferritin concentrations, iron-binding capacity,

and prohepcidin levels. Prohepcidin was measured using a

stable ELISA (DRG Instruments, Marburg, Germany) with

high reproducibility and sensitivity, which was developed with

the specific N-terminal hepcidin antibody EG (2)-HepN [3].

No cross-reactivity was observed when heterologous peptides

were used [3].

SSPS software (Statistical Package for the Social Sciences,

version 13.0; SSPS Inc., Chicago, IL) was used for all

statistical analyses. The Independent samples t test was used

to compare group results. The Pearson correlation analysis

was used to test relationships between prohepcidin and iron

parameters. Values for P 5 0.05 were considered statistically

significant.

Results

Serum Pro-Hep levels in the control and study groups were

482 + 371.9 and 156.4 + 46.7 ng/ml, respectively, with a

significant difference between groups. Table I shows the

comparisons of the patients’ characteristics and laboratory

findings.

Analyses revealed no significant between-group differences

for serum iron and iron-binding capacity, but significant

between-group differences were found for hemoglobin,

hematocrit, mean corpuscular volume (MCV), red blood cell

distribution width (RDW), and serum ferritin and Pro-Hep

levels. In our previous study, we had reported no correlation

between Pro-Hep levels and iron parameters in healthy term

infants [11]. The current study also showed no correlation

between Pro-Hep levels and iron parameters in SGA term

infants.

Discussion

We have shown that SGA infants have increased fetal

erythropoiesis, as determined by higher hemoglobin, hema-

tocrit, MCV, and RDW levels. A statistically significant

between-group difference was found with regard to serum

Pro-Hep levels. Serum ferritin and Pro-Hep levels were

significantly lower in SGA infants compared with term AGA

infants. Detivaud et al. [12] showed a relationship between

hemoglobin and hepcidin mRNA expression, although their

correlation was not significant. Kulaksiz et al. [3] reported no

correlations between prohepcidin and iron parameters in

patients with hemochromatosis, chronic renal insufficiency, or

renal anemia. Our results also demonstrate that there is a

reverse relationship between hemoglobin, hematocrit, MCV,

RDW, and Pro-Hep, supporting the hypothesis that anemia

and/or hypoxia have an effect on mRNA expression, as has

been reported in mice [6]. However, we also did not find any

significant correlation between iron parameters and serum

Pro-Hep levels, suggesting additional regulatory mechanisms.

We measured Pro-Hep levels (as opposed to the active form,

hepcidin), which might explain why we found no correlations

with any of the iron parameters studied.

We studied serum Pro-Hep levels rather than hepcidin

because measurement of hepcidin requires a reverse tran-

scription step and polymerase chain reaction. However, an

ELISA assay to detect Pro-Hep level is a simple method that is

easy to perform and appropriate for routine work.

The effect of hypoxia on hepcidin gene expression has been

investigated both in vitro and in vivo [6,13]. Human hepatoma

HepG2 and Hep3B cells cultured for 24 h under hypoxic

conditions display a significant reduction in hepcidin gene

expression [6]. Similarly, when mice were housed in

hypobaric hypoxia chambers (simulated altitude of 5500 m)

for 2 and 4 days, hepcidin gene expression was down

regulated [6]. Amarilyo et al. [13] reported that SGA infants

have evidence of increased erythropoiesis, presumably be-

cause of hypoxia-induced EPO production. But, contrary to

EPO upregulation during IUGR, umbilical cord blood Pro-

Hep concentration appears not to be affected by IUGR. They

speculated that the theoretical explanation is that though fetal

renal hypoxia in IUGR leads to EPO production, there might

be no sufficient liver hypoxia to downregulate Pro-Hep

production.

In our study, mean Pro-Hep levels were significantly lower

in term SGA infants when compared with AGA infants.

Hypoxia is the primary signal regulating EPO production by

the liver in the fetus and the kidney in adults. The hypoxia-

induced increase of EPO in the blood stimulates the

formation of red blood cells resulting in an improvement in

oxygen supply. Nicolas et al. [2] showed that injecting EPO

causes a dramatic decrease in liver hepcidin gene expression

in mice. We suggest that the induced erythropoiesis due to

elevated EPO levels in prenatal chronic hypoxia group was the

main reason for downregulation of Pro-Hep. Therefore, it

seems that both stimulation of erythropoiesis and down-

regulation of prohepcidin production are worthwhile for

improving oxygen supply and iron availability.

Table I. Clinical features and laboratory results for term AGA and term SGA infants.

Mean + SD (median); range

Control group (n ¼ 16) Study group (n ¼ 9) P 5 0.05

Gestational age (wk) 38.8 + 0.8 (39); 38–40 38.1 + 1.4 (39); 37–40 0.102

Birth weight (g) 3371.8 + 250.1; (3000–3800) 2187.7 + 208.9; (1880–2440) 0.0001

Sex (male/female) 10/6 7/2

Hemoglobin (g/dl) 14.0 + 1.3 (13.9); 12.2–17.1 19.3 + 1.5 (19.1); 19.3–22.7 0.001

Hematocrit (%) 40.6 + 4.0 (40.6); 35.5–49.6 57.7 + 14.5 (57.0); 53–67 0.001

MCV (fl) 97.1 + 3.8 (98.0); 87–101 105.4 + 7.5 (106.0); 94–114 0.002

RDW (%) 15.7 + 1.2 (15.5); 14.0–18.0 19.1 + 2.1 (19.5); 15.7–21.5 0.001

Iron (mg/dl) 111.4 + 22.4 (108.5); 71–159 92.0 + 30 (125.0); 97–144 0.07

Iron-binding capacity(mg/dl) 86.6 + 27.9 (77.0); 55–157 86.7 + 27.6 (88.0); 45–131 0.99

Ferritin (ng/ml) 385.7 + 164.8 (347.5); 101–706 146.6 + 91.1 (125.5); 55–330 0.001

Pro-Hep (ng/ml) 482.0 + 371.9 (464.4); 76–1240 156.4 + 46.7 (151.1); 93.7–247.6 0.01

1438 S. Ozkiraz et al.

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Declaration of interest: The authors report no conflicts of

interest. The authors alone are responsible for the content and

writing of the paper.

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