Nutrition For Anemia

Nutrition for
Anemia
Edited by
Javier Diaz-Castro
Printed Edition of the Special Issue Published in Nutrients
www.mdpi.com/journal/nutrients
Nutrition for Anemia
Nutrition for Anemia
Editor
Javier Diaz-Castro
MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin
Editor
Javier Diaz-Castro
University of Granada
Sapin
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MDPI
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This is a reprint of articles from the Special Issue published online in the open access journal
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Nutrition Anemia).
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Contents
About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface to ”Nutrition for Anemia” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Jorge Moreno-Fernandez, Marı́a J. M. Alférez, Inmaculada López-Aliaga

and Javier Dı́az-Castro
Role of Fermented Goat Milk on Liver Gene and Protein Profiles Related to Iron Metabolism
during Anemia Recovery
Reprinted from: Nutrients 2020, 12, 1336, doi:10.3390/nu12051336 . . . . . . . . . . . . . . . . . . 1
Teresa Nestares, Rafael Martı́n-Masot, Ana Labella, Virginia A. Aparicio,

Marta Flor-Alemany, Magdalena López-Frı́as and José Maldonado
Is a Gluten-Free Diet Enough to Maintain Correct Micronutrients Status in Young Patients with
Celiac Disease?
Reprinted from: Nutrients 2020, 12, 844, doi:10.3390/nu12030844 . . . . . . . . . . . . . . . . . . . 15
Takana Mary Silubonde, Jeannine Baumgartner, Lisa Jayne Ware, Linda Malan,
Cornelius Mattheus Smuts and Shane Norris
Adjusting Haemoglobin Values for Altitude Maximizes Combined Sensitivity and Specificity to
Detect Iron Deficiency among Women of Reproductive Age in Johannesburg, South Africa
Shuichi Shibuya, Toshihiko Toda, Yusuke Ozawa, Mario Jose Villegas Yata
and Takahiko Shimizu
Acai Extract Transiently Upregulates Erythropoietin by Inducing a Renal Hypoxic Condition
in Mice
Javier Diaz-Castro, Jorge Moreno-Fernandez, Ignacio Chirosa, Luis Javier Chirosa,

Rafael Guisado and Julio J. Ochoa
Beneficial Effect of Ubiquinol on Hematological and Inflammatory Signaling during Exercise
Chiao-Ming Chen, Shu-Ci Mu, Chun-Kuang Shih, Yi-Ling Chen, Li-Yi Tsai, Yung-Ting Kuo,
In-Mei Cheong, Mei-Ling Chang, Yi-Chun Chen and Sing-Chung Li
Iron Status of Infants in the First Year of Life in Northern Taiwan
Lucı́a Iglesias Vázquez, Victoria Arija, Núria Aranda, Estefanı́a Aparicio, Núria Serrat,
Francesc Fargas, Francisca Ruiz, Meritxell Pallejà, Pilar Coronel, Mercedes Gimeno and
Josep Basora
The Effectiveness of Different Doses of Iron Supplementation and the Prenatal Determinants of
Maternal Iron Status in Pregnant Spanish Women: ECLIPSES Study
Jorge Moreno-Fernández, Inmaculada López-Aliaga, Marı́a Garcı́a-Burgos,

Marı́a J.M. Alférez and Javier Dı́az-Castro
Fermented Goat Milk Consumption Enhances Brain Molecular Functions during Iron
Deficiency Anemia Recovery
v
Genny Raffaeli, Francesca Manzoni, Valeria Cortesi, Giacomo Cavallaro, Fabio Mosca and
Stefano Ghirardello
Iron Homeostasis Disruption and Oxidative Stress in Preterm Newborns
Rafael Martı́n-Masot, Maria Teresa Nestares, Javier Diaz-Castro, Inmaculada López-Aliaga,

Maria Jose Muñoz Alférez, Jorge Moreno-Fernandez and José Maldonado
Multifactorial Etiology of Anemia in Celiac Disease and Effect of Gluten-Free Diet:
A Comprehensive Review
Sajidah Begum and Gladys O. Latunde-Dada

Anemia of Inflammation with An Emphasis on Chronic Kidney Disease
vi
About the Editor
Javier Diaz-Castro is Professor at the Department of Physiology from the University of Granada.
His research has been developed in the area of Physiology and Nutrition, participating in 14
funded research projects. He has published more than 80 research articles, 2 books, and 13 scientific
book chapters. He has presented 60 communications to national scientific conferences and 61
communications to international scientific conferences. He has directed 12 doctoral theses, 40 Final
Master’s Projects, and 52 Final Degree Projects and has been awarded with several scientific awards.
vii
Preface to ”Nutrition for Anemia”
ANEMIA: A PUBLIC HEALTH PROBLEM. The World Health Organization (WHO) reports
that anemia affects around 800 million children and women. In fact, 528.7 million women
and 273.2 million children under the age of 5 were anemic in 2011, and about half of them were also
iron-deficient. Malnutrition and micronutrient malnutrition have serious economic consequences,
with an estimated cost of 2.3% of the world’s gross domestic product (GDP) per year. Investment
in prevention and treatment of micronutrient malnutrition results in improved health status and
reduced infant and maternal mortality. Iron deficiency is the most widespread micronutrient
deficiency in the world, often resulting in chronic iron deficiency or iron deficiency anemia. Taking
into account background, it is important to address the causes of iron deficiency to mitigate the
serious health consequences; however, excessive iron intake or overload can be harmful, potentially
leading to iron overload and blood disorders. In order to implement efficient and feasible strategies as
a solution to iron deficiency anemia, it is important that each country addresses the recommendations
of experts in iron nutrition and the WHO in a systematic way, including legislation and research,
bioavailability and provision of iron fortification, and educate the population about iron deficiency
and conduct tests on individuals using clinical pathways to measure serum or plasma ferritin
concentration as an index of iron deficiency and overload. This important health problem is
addressed in this book, reporting nutritional strategies and the consequences for the organism of
excessive iron consumption, in an attempt to raise awareness of the importance of this health problem
with repercussions on health and economic policies worldwide.
Javier Diaz-Castro
Editor
ix
nutrients
Article
Role of Fermented Goat Milk on Liver Gene and
Protein Profiles Related to Iron Metabolism during
Anemia Recovery
Jorge Moreno-Fernandez 1,2,3 , María J. M. Alférez 1,2 , Inmaculada López-Aliaga 1,2, * and
Javier Díaz-Castro 1,2
1 Department of Physiology, University of Granada, 18071 Granada, Spain; jorgemf@ugr.es (J.M.-F.);
malferez@ugr.es (M.J.M.A.); javierdc@ugr.es (J.D.-C.)
2 Institute of Nutrition and Food Technology “José Mataix Verdú”, University of Granada,
18071 Granada, Spain
3 Nutrition and Food Sciences Ph.D. Program, University of Granada, 18071 Granada, Spain
* Correspondence: milopez@ugr.es; Tel.: +34-958-243880; Fax: +34-958-248959
Received: 6 April 2020; Accepted: 30 April 2020; Published: 8 May 2020
Abstract: Despite the crucial role of the liver as the central regulator of iron homeostasis, no studies
have directly tested the modulation of liver gene and protein expression patterns during iron deficiency
instauration and recovery with fermented milks. Fermented goat milk consumption improves the
key proteins of intestinal iron metabolism during iron deficiency recovery, enhancing the digestive
and metabolic utilization of iron. The aim of this study was to assess the influence of fermented
goat or cow milk consumption on liver iron homeostasis during iron-deficiency anemia recovery
with normal or iron-overload diets. Analysis included iron status biomarkers, gene and protein
expression in hepatocytes. In general, fermented goat milk consumption either with normal or high
iron content up-regulated liver DMT1, FPN1 and FTL1 gene expression and DMT1 and FPN1 protein
expression. However, HAMP mRNA expression was lower in all groups of animals fed fermented
goat milk. Additionally, hepcidin protein expression decreased in control and anemic animals fed
fermented goat milk with normal iron content. In conclusion, fermented goat milk potentiates the
up-regulation of key genes coding for proteins involved in iron metabolism, such as DMT1, and
FPN1, FTL1 and down-regulation of HAMP, playing a key role in enhanced iron repletion during
anemia recovery, inducing a physiological adaptation of the liver key genes and proteins coordinated
with the fluctuation of the cellular iron levels, favoring whole-body iron homeostasis.
Keywords: fermented cow and goat milk; anemia; iron homeostasis; iron repletion; gene and
protein expression
1. Introduction
Iron deficiency anemia (IDA) is a highly prevalent pathology and a medical condition in the
clinical practice, affecting more than two billion people worldwide. This public health problem has
a negative impact on the lives of infants and fertile women worldwide [1]. Daily, in the duodenum
and proximal jejunum, 2 mg of iron is absorbed. The non-heme iron in the diet is in Fe3+ form and it
must be reduced to ferrous form before it can be absorbed by the action of duodenal cytochrome B.
Fe2+ is then transported across the apical membrane of enterocytes by the divalent metal transporter
1 (DMT1). This protein transports some divalent cations including ferrous iron. Iron crosses the
basolateral membrane of intestinal enterocytes by the action of ferroportin, an iron exporter, entering
into systemic circulation. After that, iron can be stored in the liver joined to ferritin, which is an iron
storage protein. The mechanisms regulating systemic iron homeostasis are largely centered on the
Nutrients 2020, 12, 1336; doi:10.3390/nu12051336 1 www.mdpi.com/journal/nutrients

Nutrients 2020, 12, 1336
liver and involve two molecules, hepcidin and ferroportin, that work together to regulate the flow of
iron from cells into the systemic circulation [2].
Due to the key role of iron in many physiological cell functions, including replication, ATP, DNA
synthesis and the heme group in hemoglobin [3], and as a constituent of essential cofactors such as
iron–sulfur (Fe-S) clusters [4], the organism has evolved to conserve body iron stores; however, it does
not have an efficient mechanism for removing excess iron, iron-overload being highly deleterious to
many physiological mechanisms. In addition, defects in molecules related to regulating iron homeostasis
are usually a cause of genetically inherited iron overload, called hereditary hemochromatosis (HH) [2].
Therefore, it is essential to tightly regulate iron homeostasis, a function performed mainly by the
liver [5].
The liver is one of the most functionally and metabolically active organs in the body. In addition
to roles in detoxification, digestion, protein synthesis, gluconeogenesis, and fat metabolism, the
liver also plays a significant role in iron homeostasis. It is responsible for approximately 8% of
plasma iron turnover and it is the major site for iron storage, acting also as the key regulator of
iron homeostasis [6]. The liver synthesizes hepcidin, a peptide which binds to and induces the
internalization of ferroportin [7], reducing the amount of iron released into the bloodstream, therefore
being the major contributor to systemic regulation of iron status [8]. The noteworthy influence of the
liver on body iron regulation can be attributed to the expression of many liver-specific or liver-enriched
proteins, which play an important role in the physiological regulation of iron metabolism [6].
On the other hand, it has been previously reported that fermented goat milk consumption
improves the key proteins of intestinal iron metabolism during IDA recovery, enhancing the digestive
and metabolic utilization of iron [9]. However, in spite of the crucial role of the liver as a central
regulator of iron metabolism, to date, no studies have directly tested the modulation of hepatic gene
and protein expression profiles during anemia recovery, including divalent metal transporter 1 (DMT1),
ferritin light chain 1 (FTL1), ferroportin 1 (FPN1), and hepcidin antimicrobial peptide (HAMP). Taking
into account all these considerations, the aim of this study was to contribute to a better understanding
of iron metabolism during IDA recovery, by studying how fermented goat vs. cow milk (the most
commonly consumed milk worldwide) consumption affects liver iron homeostasis during nutritional
iron repletion in animal models with severe, induced iron-deficiency anemia and overload.
2. Material and Methods
2.1. Animals and Experimental Design

One hundred male Wistar rats aged 3 weeks and weighing about 34.56 ± 6.35 g, purchased
from the University of Granada Laboratory Animal Service (Granada, Spain), were included in this
study. The animals were maintained under standard animal housing conditions with a 12-hour
light/dark cycle (lights on at 9:00 A.M.), temperature (23 ± 2 ◦ C) and humidity (60 ± 5%). All animal
experiments were carried out in accordance with Directive 2010/63/EU on the protection of animals
used for scientific purposes.
In the pre-experimental period (PEP), 100 rats were randomly divided into two groups; a control
group received the AIN-93G diet (n = 50) [10] and an anemic or experimental group received a
low Fe diet (n = 50) for 40 days [11]; deionized water and diet were available ad libitum. In the
experimental period (EP), control and anemic groups were fed for 30 days with either fermented cow
milk or fermented goat milk diet, with normal Fe (4.5 mg/100 g) or high Fe content (45.0 mg/100 g)
(Fe citrate) [12]. Deionized water was available ad libitum, and dietary intake pattern induced by pair
feeding (80% of the average intake) (Figure 1). At the end of the PEP and EP, hematological parameters
and serum iron, total iron binding capacity (TIBC), transferrin saturation, ferritin, serum hepcidin
and transaminases were determined. Furthermore, the liver was removed (at the end of the PEP,
in 10 animals per group and in all the animals in the EP), for measurement of hepatosomatic index
(HSI), and a fraction of the liver was snap-frozen in liquid nitrogen and keep in a −80 ◦ C freezer for
2
Nutrients 2020, 12, 1336
subsequent mineral analysis (liver iron content). Subsequently 1 g of liver was stored overnight at 4 ◦ C
with RNA-later (Thermo Fisher Scientific, Waltham, MA, USA), the solution was removed and stored
at −80 ◦ C until isolation of total RNA.
Figure 1. Experimental design of the study. * 10 animals per group and ** all the animals were
anesthetized, peripheral blood samples from caudal vein were analyzed for hematological and
biochemical parameters and the liver was removed.
2.2. Diets Preparation

Diets were prepared with fermented cow or fermented goat milk. Lactobacillus bulgaricus subsp.
delbrueckii and Streptococcus thermophilus were inoculated to an initial concentration of 1 × 1011 CFU/mL
(10 mL/L inoculum) in goat or cow milk, and then the samples were incubated at 37 ◦ C for approximately
24 h. Subsequently, fermented milk samples were dehydrated by a smooth industrial process to obtain
products with a moisture content ranging between 2.5% and 4.5%. Sufficient amounts of fermented
dehydrated cow or goat milk were utilized in the experimental diets to provide 20% of protein and 10%
of fat. The constituents and nutrient compositions of the experimental diets are presented in Table 1.
3
Nutrients 2020, 12, 1336
Table 1. Composition of experimental diets.
Constituents Pre-Experimental Period Experimental Period

g/100 g Diet Fermented Milk Diets 2
AIN-93G 1 Cow Milk Goat Milk
Protein 20.00 20.50 20.60
Lactose - 29.50 29.10
Fat 10.00 10.00 29.10
Wheat starch 50.00 20.00 20.30
Constant ingredients 3 20.00 20.00 20.00
1 Normal iron content for control rats (4.5 mg Fe/100 g diet) [10], or low iron content (0.5 mg Fe/100 g diet) [11] for
the anemic group. 2 Specific vitamin and mineral premix supplements for fermented goat and cow milk diets were
to meet the recommendations of the AIN-93G for diets with normal iron (4.5 mg Fe/100 g diet) [12] or diets with
high iron content (45 mg Fe/100 g diet) (Raja et al., 1994). 3 Fibre (micronized cellulose) 5%, sucrose 10%, choline
chloride 0.25%, L-cystine 0.25%, mineral premix 3.5%, vitamin premix 1%.
2.3. Hematological Tests

Hematological parameters were measured at the end of PEP and EP using a fully automated
hematology analyzer (Mythic 22CT, C2 Diagnostics, Grabels, France).
2.4. Transferrin Saturation, Serum Iron, Total Iron Binding Capacity (TIBC), Serum Ferritin and
Serum Hepcidin
Transferrin saturation, serum iron and TIBC were determined using Sigma Diagnostics Iron and
TIBC reagents (Sigma-Aldrich Co., St. Louis, MO). Serum ferritin was measured by the Elisa method
using a standard kit (rat ferritin ELISA Kit) supplied by Biovendor GmbH, Heidelberg (Germany).
Hepcidin-25 was determined using a DRG ELISA Kit (DRG Instruments GmbH, Germany).
2.5. Hepatosomatic Index, Hepatic Iron Concentration and Transaminases Analysis

The HSI was determined by the use of the following equation:
HSI = (weight of the liver/weight of the body) × 100
Prior to iron analysis, liver fractions were mineralized by wet digestion in a sand bath (Selecta,
Barcelona, Spain) using nitric acid followed by a mixture of HNO3:HClO4, 1:4 v/v (69%:70%, v/v; Merck,
Darmstadt, Germany) until the total elimination of organic matter. Finally, the samples were diluted
with Milli Q (Millipore S.A., Bedford, MA, USA) ultrapure water. Iron analysis was undertaken using
a PerkinElmer Optima 8300 inductively coupled plasma-optical emission spectrometer (ICP-OES)
(Waltham, MA, USA) with a segmented-array charge-coupled device (SCD) detector. Multi-elemental
Atasol calibration solution (Analytika, Khodlova, Prague) was used to calibrate the apparatus. For the
calibration curve, diluted standards were prepared from concentrated standard solutions. After each
series of 5 samples, an internal standard solution of 10 mg/L was used. The acceptable result was
assessed as 10%. Three replicates of each sample were analyzed.
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by
standard colorimetric and enzymatic methods using a BS-200 Chemistry Analyzer (Shenzhen Mindray
Bio-Medical Electronics Co. Ltd., Shenzhen, China).
2.6. RNA Extraction and Quantitative Real Time PCR

From liver samples, total RNA was extracted with TRIsure lysis reagent (Bioline, Luckenwalde,
Germany) following the manufacturer’s instructions. The RNA quantity and purity were measured
using a spectrophotometer (NanoDrop 1000, Thermo Fisher Scientific, Waltham, Massachusetts, USA)
at 260/280 nm. Reverse transcription was performed on 1 μg of total RNA in a 20 μL reaction to
synthesize complementary DNA (cDNA), using an iScript cDNA Synthesis kit (Bio-Rad). Quantitative
4
Nutrients 2020, 12, 1336
real-time PCR was performed in a total reaction volume of 20 μL using the CFX96 Touch Real-Time PCR
Detection System (Bio-Rad) and SYBR Green detection using Sso Avdvanced Universal SYBR Green
Supermix (Bio-Rad). Primer sequences of divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1),
ferritin (FTL1) and hepcidin antimicrobial peptide (HAMP) for quantitative real-time PCR are shown
in Table 2 and were obtained from Eurofins MWG Biotech (Ebersberg, Germany). The expression
of the target genes was normalized to the housekeeping gene β-actin. All the measurements were
done in duplicate and, to confirm PCR product size, melt curve analysis and gel electrophoresis
were conducted.
Table 2. Primers, annealing temperatures, and product sizes for PCR amplification.
Annealing Size
Gene Direction Primer Sequence (5 →3 )
Temperature (bp)
Forward GGGGTGTTGAAGGTCTCAAA
β-Actin 57 ◦ C 165
Reverse TGTCACCAACTGGGACGATA
Forward GGCATGTGGCACTGTATGTG
DMT1 59 ◦ C 163
Reverse CCGCTGGTATCTTCGCTCAG
Forward GAACAAGAACCCACCTGTGC
FPN1 57 ◦ C 191
Reverse AGGATGGAACCACTCAGTCC
Forward CCTATCTCCGGCAACAGACG
HAMP 59 ◦ C 121
Reverse GGGAAGTTGGTGTCTCGCTT
Forward GCCCTGGAGAAGAACCTGAA
FTL1 59 ◦ C 247
Reverse AGTCGTGCTTCAGAGTGAGG
2.7. Western Blotting and Immunocytochemistry

Liver samples were mechanically homogenized in tissue protein extraction reagent (T-PER)
(Thermo Scientific Inc., Hanover Park, IL, USA) supplemented with a protease inhibitor cocktail
(Sigma-Aldrich, St. Louis, MO, USA) under ice-cold conditions. On 4%–20% Criterion TGX (Tris-Glycine
extended) gels (Mini-PROTEAN TGX Precast Gels; Bio-Rad), 12 μg of total protein was separated
in a vertical electrophoresis tank (Mini-PROTEAN System; Bio-Rad) at 250 V for 20 min Separated
proteins were transferred onto a polyvinylidene difluoride membrane (Bio-Rad) by wet transfer for
60 min at 120 V. Thereafter, the membranes were blocked with 5% dry milk in Tris-buffered saline
(TBS) plus Tween-20 (TTBS) (Bio-Rad) solution for 1 hour at room temperature. After three washes
in TBS, membranes were incubated with rabbit anti-DMT1 polyclonal, dilution 1:400 (Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA), rabbit anti-SLC40A1 polyclonal antibody (FPN1), dilution
1:800, hepcidin polyclonal antibody, dilution 1:500, and mouse anti-β-actin monoclonal, dilution 1:1000
(Abcam, UK) as primary antibodies, in 5% dry milk in TTBS overnight at 4 ◦ C with shaking. β-actin was
used as the loading control. Then, the membranes were washed 3 times in TTBS and incubated for 1 h at
room temperature with the appropriate secondary conjugated antibody Immun-Star Goat Anti-Rabbit
(GAR)-HRP; Bio-Rad Laboratories; 1:40,000 amd ImmunStar Goat Anti-Mouse (GAM)-HRP; 1:80,000 in
TTBS. Immunoblots were detected with a chemiluminescence Luminata forte western HRP Substrate
(Merck KGaA, Darmstadt, Germany) and visualized with chemiluminescence using ImageQuant LAS
4000 (Fujifilm Life Science Corporation, USA). All results were analyzed with Image J software.
2.8. Statistical Analysis

Data are presented as mean ± standard error of the mean (SEM) and statistical analyses were
performed using SPSS 26.0 (SPSS Inc., Chicago, IL, USA). Differences between groups (control versus
anemic during the PEP and normal Fe versus high Fe during the EP) were tested for statistical
significance with Student’s t-test. Following a significant F-test (p <0.05), individual means were tested
5
Nutrients 2020, 12, 1336
by pairwise comparison with Tukey’s multiple comparison test when main effects and interactions
were significant. Two-way analysis of variance (ANOVA) was used to determine the effect of the type
of diet supplied to the animals, anemia, and iron content in the diet. Statistical significance was set at
p < 0.05 for all comparisons.
3. Results
3.1. Effect of Iron Deficiency on Hepatosomatic Index, Liver Iron Content and Serum Levels of Aspartate
Aminotransferase and Alanine Aminotransferase
Iron deprivation impaired all the hematological parameters studied [9], and these parameters are
detailed in Supplementary Table S1. Additionally, body weight and liver iron content were lower in
the anemic group (p < 0.001); however, liver weight remained unchanged, and, as a consequence, HSI
was higher in anemic group (p < 0.05). Transaminases were significantly higher in the anemic group
(p < 0.001) (Table 3).
Table 3. Hepatosomatic index, liver iron content and serum levels of aspartate aminotransferase and
alanine aminotransferase from control and anemic rats in the pre-experimental period (PEP).
Control Group Anemic Group

(n = 20) (n = 20)
Body weight (g) 239.7 ± 3.9 201.15 ± 2.9 **
Liver weight (g) 6.324 ± 0.31 6.129 ± 0.31
HSI (%) 2.55 ± 0.07 2.89 ± 0.09 *
Liver iron content (μg/g dry weight) 615.25 ± 31.10 432.31 ± 24.07 **
AST (UI/L) 103.58 ± 8.93 228.04 ± 18.45 **
ALT (UI/L) 24.57 ± 1.16 52.28 ± 2.73 **
Values are means ± SEM (n = 10). HSI, hepatosomatic index; AST, aspartate aminotransferase; ALT, alanine
aminotransferase. * Significantly different from the control group (*, p < 0.05) Student’s t-test). ** Significantly
different from the control group (**, p < 0.001) Student’s t-test).
3.2. Effect of Fermented Milk-Based Diets on Hepatosomatic Index, Liver Iron Content and Serum Levels of
Aspartate Aminotransferase and Alanine Aminotransferase during Anemia Recovery
After supplying both fermented milk-based diets during a month, all the hematological parameters
were recovered, either with normal Fe or high Fe content. The results were described [9] and are shown
in Supplementary Table S2. Fermented goat milk reduced body weight in control and anemic animals,
either with normal Fe or high Fe content (p < 0.001) compared to animals fed fermented-cow-milk-based
diets. Body weight was significantly lower in anemic animals fed both fermented milks with normal
Fe content (p < 0.01). Body weight was lower a with high Fe content diet than with a normal Fe content
diet in animals fed with fermented-cow-milk-based diets in both groups (control and anemic) (p < 0.05).
HSI was higher in both groups of animals (control and anemic) fed fermented-goat-milk-based diets,
either with normal Fe or high Fe, compared to animals fed fermented-cow-milk-based diets (p < 0.001).
Liver iron content was increased in animals fed with fermented goat milk compared to animals fed with
fermented cow milk with normal Fe content diets (p < 0.01). In contrast, liver iron content was lower
in animals fed fermented goat milk compared to fermented cows’ milk with high Fe content (p < 0.01).
Liver iron content was lower in anemic animals compared to the control group, irrespective of the iron
content in both types of fermented milks (p < 0.01). As expected, dietary high Fe content increased the
liver content of this mineral in control and anemic animals fed fermented cow milk (p < 0.001), this
increase being lower in the animals fed fermented goat milk (p < 0.05). AST and ALT were lower in
rats fed with fermented-goat-milk-based diets compared to rats fed with fermented-cow-milk-based
diets in all experimental conditions (p < 0.01). High Fe content did not affect the transaminases when
supplying both milk-based diets (Table 4).
6
Table 4. Hepatosomatic index, liver iron content and serum levels of aspartate aminotransferase and alanine aminotransferase, from control and anemic rats fed for
30 days with fermented cow- or goat-milk-based diets with normal Fe or high Fe content in the experimental period (EP).
Fermented Cow Milk Fermented Goat Milk 2-WAY ANOVA

Fe
Fe Content Control Group Anemic Group Control Group Anemic Group Diet Anemia
Content
Nutrients 2020, 12, 1336
Normal 365.23 ± 8.61 a 347.21 ± 8.39 A, * 278.98 ± 3.70 b 255.41 ± 2.85 B, * <0.001 <0.01
Body weight (g) <0.05
High 339.42 ± 5.18 a,c 329.22 ± 5.81 A,C 287.27 ± 4.92 b 267.57 ± 4.03 B <0.001 NS 1
Normal 6.528 ± 0.24 a 6.269 ± 0.10 A 8.391 ± 0.23 b 8.521 ± 0.21 B <0.001 NS
Liver weight (g) <0.05
High 6.764 ± 0.2 a 6.555 ± 0.12 A 7.692 ± 0.22 b,c 7.934 ± 0.22 B,C <0.01 NS
Normal 1.84 ± 0.04 a 1.77 ± 0.02 A 2.95 ± 0.03 b 3.27 ± 0.05 B <0.001 NS
HSI (%) NS
High 1.79 ± 0.03 a 1.82 ± 0.03 A 2.65 ± 0.04 b 3.12 ± 0.04 B <0.001 NS
Liver iron content Normal 559.56 ± 28.72 a 401.56 ± 24.50 A, * 666.45 ± 33.21 b 489.32 ± 29.64 B, * <0.01 <0.01
<0.01
(μg/g dry weight) High 832.25 ± 32.56 a,c 782.32 ± 33.55 A,C, * 735.67 ± 29.33 b,c 657.15 ± 29.22 B,C, * <0.01 <0.01
Normal 107.62 ± 4.29 a 80.86 ± 4.25 A, * 67.99 ± 2.75 b 61.11 ± 2.12 B <0.01 <0.05
AST (UI/L) NS
High 82.92 ± 4.15 a,c 78.19 ± 3.82 A 60.43 ± 1.10 b 68.47 ± 2.03 B <0.01 NS
Normal 28.91 ±1.34 a 27.77 ± 3.91 A 23.14 ± 1.9 b 16.49 ± 0.76 B, * <0.01 <0.01
ALT (UI/L) NS
High 24.0.4 ± 1.73 a 19.00 ± 1.21 A 19.47 ± 0.53 b 14.48 ± 0.35 B, * <0.01 <0.01
7
1
Values are means ± SEM (n = 10). NS, not significant. HSI, hepatosomatic index; AST, aspartate aminotransferase; ALT, alanine aminotransferase. * Significantly different from the
control group (p < 0.05) Student’s t-test). a,b Mean values among groups of controls rats fed with different diets; different lower-case letters in the same row indicate a significant difference
by two-way ANOVA (Tukey’s test). A,B Mean values among groups of anemic rats fed with different diets; different upper-case letters in the same row indicate a significant difference by
two-way ANOVA (Tukey’s test). c Mean values of controls rats were significantly different from the corresponding group of rats fed with normal Fe content at p < 0.05 by Student’s t-test.
C Mean values of anemic rats were significantly different from the corresponding group of rats fed with normal Fe content at p < 0.05 by Student’s t-test.
Nutrients 2020, 12, 1336
3.3. Effect of Fermented Milk-Based Diets on Liver Iron Homeostasis during Anemia Recovery
Fermented goat milk up-regulated liver DMT1 gene expression in control and anemic rate fed
with high Fe content (p < 0.01) and in control groups fed with a normal Fe diet, and previously induced
anemia reduced DMT1 expression in the animals fed fermented goat milk with normal Fe content
(p < 0.05) (Figure 2A). Similarly, DMT1-relative protein expression was higher in control and anemic
animals fed fermented goat milk with normal Fe (p < 0.001). Induced anemia increased liver DMT1
protein expression in animals fed both fermented milk with normal Fe content (p < 0.001). High Fe
content increased DMT1 protein expression in all groups of animals fed fermented goat or cow milk
(p < 0.001), except in control animals fed fermented goat milk, which showed a decrease (p < 0.05)
(Figure 2B).
Expression of FPN1 mRNA increased in control and anemic animals fed fermented goat either
with normal Fe (p < 0.001) or high Fe content (p < 0.01) (Figure 2C). Protein expression of FPN1
increased in control and anemic animals fed fermented goat milk with normal Fe content (p < 0.001).
Anemia decreased FPN1 protein expression in animals fed fermented goat milk with normal Fe
(p < 0.01) and increased in animals fed both fermented milks with high Fe content (p < 0.001). High Fe
content increased liver FPN1 protein expression in all groups of animals fed fermented goat or cow
milk (p < 0.001), except control animals fed fermented goat milk, which showed a decrease (p < 0.001)
(Figure 2D).
HAMP mRNA expression was lower in control and anemic animals fed fermented goat milk with
normal Fe (p < 0.05) and also in control and anemic animals fed fermented goat milk with high Fe
content (p < 0.001). Anemia increased HAMP mRNa expression in animals fed fermented cow milk
with high Fe content (p < 0.01). High Fe content increased HAMP gene expression in anemic animals
fed both fermented milks (p < 0.001) and in control groups fed a fermented cow milk diet (p < 0.01)
(Figure 2E). Hepcidin protein expression decreased in control and anemic animals fed fermented goat
milk with normal Fe content (p < 0.001). Anemia decreased hepcidin protein expression in animals fed
fermented cow milk with normal Fe content (p < 0.001) and increased in animals fed fermented goat
milk with normal Fe content (p < 0.05); however, with high Fe content, anemia increased hepcidin
protein expression in the animals fed both fermented milks (p < 0.001). High Fe content increased
hepcidin protein expression in all groups of animals fed fermented cow or goat milk (p < 0.01) but
decreased in control animals fed fermented cow milk (p < 0.001) (Figure 2F).
In general, fermented goat milk induced an up-regulation of mRNA FTL1 expression in control
and anemic animals fed either with normal Fe or high Fe content (p < 0.01). Anemia up-regulated
liver mRNA FTL1 expression in animals fed fermented cow milk with high Fe content (p < 0.01).
High Fe content up-regulated FTL1 mRNA expression in the anemic groups fed both fermented milks
(p < 0.001) and in the control group fed fermented cow milk (p < 0.01) (Figure 3).
8
Nutrients 2020, 12, 1336
Figure 2. mRNA levels (A,C,E) and protein expression levels (B,D,F) of DMT-1, FPN1 and hepcidin in
livers of control and anemic rats, fed normal Fe or high Fe content fermented cow or goat milk-based
diets. Values are represented as mean ± SEM (n = 10). For protein expression, values are expressed as %
vs β-actin. a,b: mean values among groups of control rats fed with different diets; different lower-case
9
Nutrients 2020, 12, 1336
letters in the same row indicate a significant difference by two-way ANOVA (Tukey’s test). A,B: Mean
values among groups of anemic rats fed with different diet; different upper-case letters in the same row
indicate a significant difference by two-way ANOVA (Tukey’s test). * Significantly different (p < 0.05)
from the control group by Student’s t-test. c: Mean values of control rats were significantly different
from the corresponding groups of rats fed with normal Fe content at p < 0.05 by Student’s t-test.
C: Mean values of anemic rats were significantly different from the corresponding group of rats fed
with normal Fe content at p < 0.05 by Student’s t-test. (G) Representative immunoblots. Abbreviations:
cow control (CC), cow anemic (CA), cow control high Fe content (CC+Fe), cow anemic high Fe content
(CA+Fe), goat control (GC), goat anemic (GA), goat control high Fe content (GC+Fe), goat anemic high
Fe content (GA+Fe).
Figure 3. mRNA levels of FTL1 in the liver of control and anemic rats, fed normal Fe or high Fe content
fermented cow- or goat-milk-based diets. Values are represented as mean ± SEM (n = 10). a,b: mean
values among groups of controls rats fed with different diets; different lower case letters in the same
row indicate a significant difference by two-way ANOVA (Tukey’s test). A,B: mean values among
groups of anemic rats fed with different diets; different upper case letters in the same row indicate a
significant difference by two-way ANOVA (Tukey’s test). * Significantly different (p < 0.05) from the
control group by Student’s t-test. c: mean values of controls rats were significantly different from the
corresponding group of rats fed with normal Fe content at p < 0.05 by Student’s t-test. C: mean values
of anemic rats were significantly different from the corresponding group of rats fed with normal Fe
content at p < 0.05 by Student’s t-test.
4. Discussion
Anemia reduced body weight in the PEP, due to the impairment of hematological parameters and
the depletion of the hepatic iron levels. These negatively affect the weight gain of animals during the
growing period, since the hypoxia induced by the lack of iron limits ATP production and decreases
levels of thyroid hormones and ghrelin, inducing cachexia and leading to reductions of lean mass [13].
In a previous study, we reported that serum total bilirubin was increased in hepatocellular damage.
In that study, bilirubin was increased and AST and ALT activities were also found elevated in IDA.
The increase of transaminase release into the bloodstream could be due to the impairment of the
hepatic function induced by the iron deficiency [13,14].
During the EP, body weight was statistically lower in the animals fed fermented goat milk.
This finding can be explained because, as previously reported [15], fermented goat milk consumption
influences adipose tissue depot homeostasis during iron deficiency recovery, reducing adiposity,
increasing leptin elevation and reducing ghrelin, and, therefore, diminishing appetite and increasing
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Nutrients 2020, 12, 1336
basal metabolic rate. Additionally, fermented goat milk consumption reduces adiponectin levels and
shows an inverse correlation with non-esterified fatty acids, indicating increased lipolysis rates in
adipose tissue [15].
Liver iron homeostasis is tightly regulated to maximize the iron supply when anemia is established
and to promote storage when iron status is adequate. Intracellular iron content is regulated by the
relative rate of cellular import, via DMT1, versus export, via FPN1 [16].
Fermented goat milk improves iron metabolism, because, as previously reported [15], it increases
expression of duodenal key proteins in intestinal iron metabolism, improving iron absorption after
induced anemia. However, in spite of the role of the liver as the central regulator of iron homeostasis,
a better characterization of the hepatic events in the pathophysiology of iron deficiency instauration
and recovery leads to new nutritional strategies to improve liver molecular functions related to
iron homeostasis.
After supplying the fermented-milk-based diets, iron repletion was more efficient with fermented
goat milk, a fact that is corroborated by the recovery of hematological parameters and the liver iron
content recorded in the current study. This fact can be explained due to the beneficial increase in
the liver DMT1 iron-transport gene and protein expression, enhancing iron metabolism and storage
compared with fermented cow milk. DMT1 is localized both to the plasma membrane and in the
cytosol of hepatocytes [17]. In addition to its role in endosomal iron export, the presence of DMT1 on
the microvillous membrane of hepatocytes reveals that the liver is capable of absorbing non-heme,
non-transferrin-bound iron. Contrary to the duodenal DMT1, hepatic DMT1 expression parallels iron
status [17], playing a key role by regulating iron homeostasis in the liver. Therefore, the increased liver
DMT1 protein levels faithfully reflect the increased liver iron storage after dietary iron repletion [18]
and, this way, the increase in DMT1 protein expression in anemic animals fed fermented goat milk
reveals that iron repletion is more efficient, a finding that is supported and corroborated by the higher
iron storage in the rats fed fermented goat milk compared with fermented cow milk. As previously
reported, goat milk fat is richer in medium-chain triglycerides, which are oxidized in the mitochondria,
providing fast energy discharge used in several metabolic pathways [19] and thus contributing to
increasing the synthesis of carrier proteins such as DMT1 [20]. On the other hand, goat milk has
more than twice the vitamin A content of cow milk [19], and this vitamin increases liver DMT1
protein expression by posttranscriptional regulation via increased protein translation or decreased
degradation [18].
Hepcidin is a key indicator of iron status and metabolism. This hormone regulates iron levels
and location in response to nutritional status. High hepcidin levels block intestinal iron absorption
and macrophage recycling, causing anemia. Low hepcidin levels favor bone marrow iron supply for
hemoglobin synthesis and erythropohiesis. Multiple factors regulate the expression of hepcidin in the
liver. As expected, low serum hepcidin levels were recorded in the PEP due to the increased expression
of key duodenal proteins involved in iron absorption [20], favoring red blood cell production during
insufficient dietary iron supply. On the other hand, inflammation is a strong inducer of hepcidin
production and release from the liver [21–23], resulting in reduced iron release from stores and
macrophages, thereby reducing iron in the circulation and disrupting iron homeostasis [24]. In the
current study, a down-regulation of liver hepcidin has been recorded in animals consuming fermented
goat milk, a fact that increases the iron efflux from the hepatocytes to the serum, due to the inverse
correlation between hepcidin expression and the FPN1 activity [9,25].
In addition, we have previously reported that fermented goat milk consumption decreased
pro-inflammatory cytokines and increased anti-inflammatory cytokines [25], due to the
anti-inflammatory activities of its biologically active lipid fractions, such as sphingomyelin,
phosphatidylcholine, and phosphatidylethanolamine lipid derivatives [26,27]. Moreover, fermented
goat milk has a higher content in conjugated linoleic acid than fermented cow milk, featuring a
putative role modulating anti-inflammatory responses [25]. All these anti-inflammatory properties of
fermented goat milk would contribute to the decrease in liver hepcidin expression. This decrease in
11
Nutrients 2020, 12, 1336
hepcidin, together with the increase in DMT1 and FPN1 gene and protein liver expression recorded
when fermented goat milk is supplied, would led to the mobilization of iron from hepatic storage to
sustain erythropoiesis and improve iron status, even under Fe-overload conditions. These data indicate
a not only a better hematological recovery but also improved iron mobilization for liver storage to
target organs.
Although, in physiological conditions, hepatocytes have a high capacity for iron storage, the
liver is also the main organ affected by the oxidative stress caused by Fe-overload toxicity due to
its high propensity to induce reactive oxygen species (ROS) [28]. As mentioned above, the increase
in DMT1 and FPN1 gene and protein liver expression recorded with fermented goat milk lead to
lower amounts of iron stored in the hepatocytes during the dietary Fe overload in comparison with
fermented cow milk, reducing the evoked oxidative stress. In this sense, fermented goat milk has
positive effects on enzymatic antioxidant hepatic defense, even in a situation of Fe overload, which
limits the processes of lipid peroxidation in comparison with cow milk, due to the improvement of
zinc bioavailability (with antioxidant capacity) and the better lipid quality in comparison to fermented
cow milk, reducing the generation of ROS [29]. Additionally, these findings are supported by the
previous results [30], reporting a clear diminishment of transaminase release into the bloodstream,
corroborating the hepatoprotective effect of goat milk during Fe overload and reducing hepatocellular
damage, a fact that could avoid, at least partially, the progression of Fe-overload-related diseases [31].
FTL1 is the intracellular iron storage protein that stores and releases iron in a controlled way, and its
expression greatly increases when cellular iron concentrations rise, providing the cell with an enormous
ability to sequester iron. When intracellular iron content is low, iron-responsive element-binding
protein 2 (IRP2) represses FTL1, mRNA translation, while intracellular iron accumulation promotes
IRP2 degradation and allows FTL1 mRNA translation [32]. As previously mentioned, the increased
DMT1 and FPN1 expression levels when fermented goat milk is supplied improve iron storage, also
explaining the higher levels of mRNA FTL1. In addition, Fe overload increased FTL1 gene expression,
probably as a compensatory mechanism to avoid the liver oxidative damage induced by Fe overload,
because intracellular iron redox activity is controlled by ferritin [33].
5. Conclusions
Fermented goat milk consumption potentiates the up-regulation of key genes and proteins
involved in iron metabolism, such as DMT1 and FPN1, and downregulates liver hepcidin, enhancing
and improving iron repletion during anemia recovery. In addition, taking into account the better iron
storage in the liver during anemia recovery, the reduction in the iron accumulation during Fe overload,
and the improvement of the hematological parameters, fermented goat milk consumption induces
a hepatic physiological adaptation of the key genes and proteins that regulate the fluctuation of the
cellular iron levels, favoring whole-body iron homeostasis.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/5/1336/s1,

Table S1: Hematological parameters of control and anemic rats in the pre-experimental period (PEP); Table S2.
Hematological parameters from control and anemic rats fed for 30 days with fermented cow or goat milk-based
diets with normal-Fe content or Fe-overload in the experimental period (EP).
Author Contributions: Research Design, I.L.-A. and J.D.-C.; Methodology, J.M.-F., J.D.-C., I.L.-A., and M.J.M.A.;
Formal Analysis, Investigation and Software, J.M.-F. and J.D.-C.; Writing—Original Draft Preparation J.M.-F.,
I.L.-A., J.D.-C. and M.J.M.A.; Supervision and Project Administration, I.L.-A. All persons designated as authors
qualify for authorship, and all those who qualify for authorship are listed. All authors have read and agreed to the
published version of the manuscript.
Funding: This study is funded by the Excellence Project (P11-AGR-7648) from the Regional Government
of Andalusia.
Acknowledgments: J.M.-F. was supported by two fellowships from the Ministry of Education, Culture and Sport
(Spain), FPU and Traslados Temporales FPU (University of King´s College of London). J.M.-F. is grateful to the
Excellence Program “Nutrición y Ciencias de los Alimentos” from the University of Granada. The authors are
grateful to Susan Stevenson for her efficient support in the revision with the English language.
12
Nutrients 2020, 12, 1336
Conflicts of Interest: The authors declare no conflict of interest.
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
14
nutrients
Article
Is a Gluten-Free Diet Enough to Maintain Correct
Micronutrients Status in Young Patients with
Celiac Disease?
Teresa Nestares 1, *, Rafael Martín-Masot 2 , Ana Labella 3 , Virginia A. Aparicio 1,4 ,
Marta Flor-Alemany 1 , Magdalena López-Frías 1 and José Maldonado 5,6,7,8
1 Department of Physiology and “José MataixVerdú” Institute of Nutrition and Food Technology (INYTA),
University of Granada, 18071 Granada, Spain; virginiaparicio@ugr.es (V.A.A.);
martadelaflor@correo.ugr.es (M.F.-A.); maglopez@ugr.es (M.L.-F.)
2 Pediatric Gastroenterology and Nutrition Unit, Hospital Regional Universitario de Malaga, 29010 Málaga,
Spain; rafammgr@gmail.com
3 Pediatric Clinical Management Unit., “San Cecilio” University Hospital, 18016 Granada, Spain;
alnestares92@gmail.com
4 Sport and Health University Research Institute (iMUDS), 18071 Granada, Spain
5 Department of Pediatrics, University of Granada, 18071 Granada, Spain; jmaldon@ugr.es
6 Biosanitary Research Institute, 18071 Granada, Spain
7 Maternal and Child Health Network, Carlos III Health Institute, 28029 Madrid, Spain
8 Pediatric Clinical Management Unit. “Virgen de las Nieves” University Hospital, 18071 Granada, Spain
* Correspondence: nestares@ugr.es; Tel.: +34-696989989
Received: 28 February 2020; Accepted: 19 March 2020; Published: 21 March 2020
Abstract: The current study assesses whether the use of a gluten-free diet (GFD) is sufficient for
maintaining correct iron status in children with celiac disease (CD). The study included 101 children.
The celiac group (n = 68) included children with CD, with long (> 6 months) (n = 47) or recent
(< 6 months) (n = 21) adherence to a GFD. The control group (n = 43) included healthy children.
Dietary assessment was performed by a food frequency questionnaire and a 3-day food record. Celiac
children had lower iron intake than controls, especially at the beginning of GFD (p < 0.01). The group
CD-GFD >6 months showed a higher intake of cobalamin, meat derivatives and fish compared to that
of CD-GFD <6 months (all, p < 0.05). The control group showed a higher consumption of folate, iron,
magnesium, selenium and meat derivatives than that of children CD-GFD >6 months (all, p < 0.05).
Control children also showed a higher consumption of folate and iron compared to that of children
CD-GFD <6 months (both, p < 0.05). The diet of celiac children was nutritionally less balanced than
that of the control. Participation of dietitians is necessary in the management of CD to guide the GFD
as well as assess the inclusion of iron supplementation and other micronutrients that may be deficient.
Keywords: celiac disease; gluten-free diet; nutritional adequacy; iron deficiency anemia; children
1. Introduction
Celiac disease (CD) occurs in about 1% of people in most populations [1,2]. Diagnosis rates
are increasing, which seems to be due to a true rise in incidence that requires greater awareness
and early detection. In response to unknown environmental factors, it is believed that ingestion of
gluten promotes an immunologically mediated small intestinal enteropathy in genetically susceptible
individuals [3]. The disease primarily affects the small intestine; however, the clinical manifestations
are large, with both intestinal and extra-intestinal symptoms. Patients with CD might also present
with various deficiency states, including anemia, osteopenia or osteoporosis. Upon diagnosis, patients
should be tested, since between 20%–38% of CD patients show nutritional deficiencies. Specifically,

Nutrients 2020, 12, 844
12%–69% display iron deficiency, more frequent in adults than in children [4], and 8%–41% display
Vitamin B12 deficiency, and about 31% of patients with CD present low ferritin concentrations [5].
A lifelong strict gluten-free diet (GFD) is the only available treatment for CD. Improvement and
resolution of symptoms typically occur within days or weeks, and often precede the normalization
of serological markers and of duodenal villous atrophy [6]. About 20% of patients with CD have
persistent or recurrent symptoms despite having a good adherence to a GFD [7], and the nutritional
adequacy of the GFD remains controversial [8], although evidence suggests that GFD is nutritionally
unbalanced [8]. In fact, wheat is a source of iron, folates and B vitamins (thiamin, riboflavin and
niacin) and not only a major source of protein. Indeed, gluten-free products are low in these nutrients
compared to their gluten-containing equivalents [9–11].
An essential micronutrient for adequate erythropoietic function is iron, which is implicated in
oxidative metabolism, enzymatic activities and cellular immune responses [12]. Iron deficiency anemia
(IDA) is a public health problem where children are one of the weaker groups [13]. CD leads to
decreased absorption of many nutrients in duodenum-mucosal damage, including iron [14] (the site of
maximal iron absorption). Therefore, iron malabsorption is usually observed in CD and may be the
presenting clinical feature even in the absence of weight loss or diarrhea [15–17]. Although IDA is
very common in CD, it could persist after the initiation of a GFD [18]. Unfortunately, the relationship
between IDA and CD has been poorly explored.
Therefore, the purpose of the present study was to determine the importance of a GFD in the
normalization of iron metabolism in patients with CD, as well as to test the influence of the time in a
GFD on iron metabolism.
2. Materials and Methods
2.1. Subjects
The study was carried out according to the principles of the Declaration of Helsinki and
its later amendments and approved by the Ethics Committee of the University of Granada (Ref.
201202400000697). The study included 101 children aged 7–18 years old, attending the Gastroenterology,
Hepatology and Child Nutrition Service from the “Virgen de las Nieves” University Hospital in
Granada, Spain.
The control group included 43 healthy children, whose serological screening was detected to be
negative and who had no history of any chronic disease. These children attended this Service due to
minor symptoms related to chronic functional constipation, according to the Rome IV criteria. After
verifying that it was due to transitory gastrointestinal symptoms (functional constipation), they were
included in the control group. The inclusion criteria for the control group were age between 7 and 18
years, absence of serum IgA and IgG anti-transglutaminase (tTG) antibodies, normal weight for the
age, absence of gastrointestinal disorders in the previous year and normal appetite. Children with CD
diagnosed in accordance with the European Society for Pediatric Gastroenterology Hepatology and
Nutrition (ESPGHAN) [19] were included in the CD group (n = 68), which was divided into patients
on a GFD longer than 6 months (GFD > 6, n = 47) and patients following a GFD less than 6 months
(GFD < 6, n = 21). The exclusion criteria for both groups were liver or kidney diseases, acute and
chronic inflammation, inflammatory bowel disease, diabetes, chronic asthma and consumption of
dietary supplements containing substances with antioxidant activity. We also excluded obese patients
(according to the criteria of the International Task Force) [20] and those who did not sign the informed
consent. Written informed consent was obtained from all parents.
2.2. Clinical and Socio-Demographics

Participants’ clinical and socio-demographic characteristics including age, household composition,
parents’ marital status, educational level and smoking habit were assessed by the same group
of researchers.
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When IDA was detected in CD children, elemental iron was administered at doses of 3 to 5 mg
per kilogram of weight per day. When iron deficiency without anemia was detected, prophylactic
doses of elemental iron were prescribed at a dose of 2 mg per kilogram per day in two daily doses for
2–3 months [21].
2.3. Anthropometric Measures

Anthropometric characteristics (weight, height) were assessed in the control and the celiac subjects.
Height was measured to the nearest 5 mm using a stadiometer (Seca 22, Hamburg, Germany). Body
weight was measured using the same mechanical balance (Seca 200, Hamburg, Germany).
2.4. Blood Haematological and Biochemical Anaemia-Related Markers

Venous blood samples were collected into anticoagulated tubes with sodium heparin during
morning hours from fasting patients. In order to obtain plasma, blood samples were centrifuged
at 2500× g at 4 ◦ C for 10 min. Plasma samples were frozen (−80 ◦ C) until measurements. All
hematological and biochemical parameters studied were measured using an automated hematology
analyzer (K-1000D, Sysmex, Tokyo, Japan). The following biochemical parameters were determined:
iron, ferritin, transferrin, thyroid-stimulating hormone (TSH) and thyroxine.
2.5. Dietary Assessment

Dietary intake was assessed through a semi-quantitative 78-item food frequency questionnaire
(FFQ) previously validated in Spain by Mataix et al. [22] and a three-day food record, two on weekdays
and one on the weekend. The diary was carefully explained, face to face, by the same trained dietitian
to the children and their parents and was accompanied by detailed instructions for the compilation
and a photographic atlas including different portion size pictures and a set of household measures [23].
The survey included a daily record of all foods consumed during the different meals (breakfast,
morning snack, lunch, afternoon snack, dinner). For each meal, participants were requested to report
an exhaustive description of the food and the recipes (including cooking methods and sugar or fats
added during the meal preparation), food amount (according to the atlas) and the brands of packaged
foods consumed.
All diaries were analyzed by the same trained dietitian using the Evalfinut software that includes
the Spanish Food Composition Database [24]. The program estimated the energy intake (kilocalories)
and macronutrients (proteins, total fats, saturated fats, carbohydrates, simple sugars and fiber, expressed
in grams) and the percentage of energy provided by each macronutrient. Recommended Energy and
Nutrient Intake Levels for Spanish population [24] were taken as reference values for energy and
nutrient intake and for food group consumption. The composition of GF products was retrieved from
product labels.
2.6. Data Analyses

We employed descriptive statistics (mean, standard deviation) for quantitative variables and
percentage of participants (%) for categorical variables to describe the baseline characteristics of
the study sample. We conducted Student’s t-test to explore differences in the continuous variables.
Furthermore, we assessed differences in categorical variables by using the chi-squared test.
A one-way analysis of covariance (ANCOVA) after adjustment for age, sex and body weight was
employed to assess the differences in hematological and biochemical anemia-related markers between
the celiac and control healthy groups. An ANCOVA after adjustment for age, sex and body weight
was also performed to explore differences in hematological and biochemical anemia-related markers
and dietary behavior between the healthy controls, the patients with a GFD in the last 6 months and
the patients engaged in a GFD less than 6 months. Significant pairwise comparisons with Bonferroni
adjustment were performed to keep the experiment error rate at α = 0.05 and to identify between which
groups the differences were significant (e.g., control healthy group vs. less than 6 months in a GFD).
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Nutrients 2020, 12, 844
The data analyses were conducted with the Statistical Package for Social Sciences (IBM SPSS
Statistics for Windows, Version 20, IBM Corp, Armonk, NY, USA), and statistical significance was set
at p ≤ 0.05.
3. Results
The baseline sociodemographic and anthropometric characteristics of the study sample are shown
in Table 1. A total of 68 children with CD participated in the study (mean age 8.5 ± 4.1 years). More
than half of the participants with CD followed a GFD for more than 6 months and met physical
activity recommendations. The control group included 43 healthy children (mean age 10.3 ± 4.5 years).
There were differences in height (p = 0.006) and weight (p = 0.002) between groups. Regarding the
hematological results, the CD group showed lower levels of hemoglobin, erythrocyte and hematocrit
compared to healthy children (p < 0.05).
Table 1. Anthropometric, clinical and sociodemographic characteristics of the study participants.
Celiac Group Healthy Group

Variable p
(n = 68) (n = 43)
Sex (female, n (%)) 52 (76.5) 20 (46.5) 0.014
Age (years) 8.5 (4.1) 10.3 (4.5) 0.025
Anthropometry
Height (cm) 129.2 (23.5) 142.2 (25.2) 0.006
Weight (kg) 30.5 (12.9) 39.6 (16.4) 0.002
Passive smoker (yes, %) 8.9 25.7 0.031
Time in gluten-free diet (%)
Not started or less than 6 months 32.9 -
More than 6 months 67.1 -
Strictly following gluten-free diet (n, (%)) 55 (85.9) 1 (1.8)
Any other celiac family member (%)
No (or unknown) 35.9 -
Siblings or parents 46.9 -
Cousins or uncles 6.3 -
Various degrees of consanguinity 10.9 -
Meet physical activity recommendations 75.5 88.9 0.161
Values shown as mean (standard deviation) unless otherwise indicated.
Differences in serum hematological and biochemical anemia-related markers between healthy

control children, patients with a GFD in the last 6 months and patients with a GFD less than 6 months are
shown in Table 2. Serum erythrocytes differed between groups (p = 0.016), with pairwise comparisons
showing lower erythrocyte counts in both groups of CD patients compared to the healthy control group
(p < 0.05). Hematocrit also differed between groups (p = 0.031), with pairwise comparisons showing
lower hematocrit concentration in the CD group following the GFD for less than 6 months compared to
the control group (38.6 ± 0.7 versus 41.0 ± 0.4, respectively, p < 0.05). Finally, hemoglobin levels differed
between groups (p < 0.05), being lower in CD groups but without pairwise significant difference.
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Nutrients 2020, 12, 844
Table 2. Differences between healthy control children, patients with a gluten-free diet in the last 6
months and patients with a gluten-free diet less than 6 months on serum hematological and biochemical
anemia-related markers.
Less than 6 More than 6

Healthy Control
Months on Months on
Variable Group p
Gluten-Free Diet Gluten-Free Diet
(n = 43)
(n = 21) (n = 47)
Erythrocytes (millions) 4.74 (0.08) a 4.80 (0.05) b 4.99 (0.05) ab 0.016
Hematocrit (%) 38.6 (0.70) a 40.0 (0.44) 41.0 (0.49) a 0.031
Hemoglobin (mg/dL) 13.2 (0.21) 13.4 (0.14) 13.9 (0.15) 0.046
Erythrocyte corpuscular volume (fL) 82.6 (1.28) 83.2 (0.80) 81.8 (0.89) 0.521
Hemoglobin corpuscular volume (fL) 28.3 (0.47) 28.0 (0.29) 27.9 (0.33) 0.744
RDW 13.3 (0.20) 13.7 (0.13) 13.5 (0.14) 0.130
Iron (μg/dL) 75.7 (8.0) 77.3 (4.5) 89.1 (5.4) 0.226
Ferritin (μg/dL) 44.3 (10.1) 41.9 (5.3) 50.3 (6.5) 0.618
Transferrin (mg/dL) 319.6 (13.3) 292.3 (8.4) 285.5 (11.6) 0.161
TSH (UI/L) 2.42 (0.25) 2.71 (0.17) 2.23 (0.18) 0.150
Thyroxine (μg/dL) 0.97 (0.03) 0.93 (0.02) 0.95 (0.02) 0.588
Values shown as mean (standard error); TSH: thyroid-stimulating hormone; RDW: red blood cell distribution
width. Model adjusted for sex, age and body weight. a,b Common superscript in a same row indicates a significant
difference (p < 0.05) between the groups. Pairwise comparisons were performed with Bonferroni’s adjustment.
Tables 3 and 4 show the total daily energy and the macronutrient and micronutrient intakes in
the three study groups, and the results from daily food group consumption, as collected by the FFQ.
Total energy daily intake did not differ between groups. There were no significant differences for
macronutrient intake. There were some differences for micronutrients involved in iron metabolism;
iron intake was lower for CD groups vs controls (p < 0.006). Selenium and magnesium intake was
lower for GFD > 6 months vs the control group (p < 0.001).
months and patients with a gluten-free diet less than 6 months on dietary intake outcomes.

Healthy Control
Months on Months on
Variable Group p
(n = 43)
(n = 21) (n = 47)
Dietary intake
Energy intake (Kcal/day) 1790 (150.5) 1814 (75.1) 1870 (88.9) 0.861
Fat (g/day) 60.3 (8.4) 78.1 (4.2) 75.9 (5.0) 0.171
Protein (g/day) 68.8 (7.5) 70.0 (3.5) 77.0 (4.2) 0.409
Fiber (g/day) 18.8 (1.7) 14.2 (0.8) 15.7 (1.0) 0.056
Carbohydrates (g/day) 179.3 (19.3) 200.1 (9.94) 212.6 (11.7) 0.376
Folate (μg) 159.8 (17.6) a 159.0 (9.0) b 191.4 (10.6) ab 0.075
Cobalamin (μg) 4.04 (1.99) a 7.50 (1.00) a 6.50 (1.18) 0.289
Calcium (mg) 741.8 (99.7) 732.1 (49.7) 798.1 (58.9) 0.701
Iron (mg) 7.32 (0.99) a 7.61 (0.49) b 10.1 (0.58) ab 0.006
Iodine (μg) 74.4 (9.4) 75.7 (6.3) 88.8 (6.8) 0.313
Magnesium (mg) 174.8 (18.4) 165.6 (9.23) a 206.9 (10.9) a 0.022
Selenium (μg) 52.3 (7.0) 45.5 (3.5) a 68.9 (4.2) a <0.001
Zinc (mg) 6.50 (0.70) 5.81 (0.35) 7.11 (0.42) 0.070
Values shown as mean (standard error); s, servings; m, month; wk, week. Model adjusted for sex, age and body
weight. Upper case a,b letters in the same row indicate a significant pairwise difference (p < 0.05) between groups
with the same letter. Bonferroni’s correction for multiple comparisons was applied to analyze pairwise differences.
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months versus less than 6 months on food frequency outcomes.

Healthy Control
Months on Months on
Variable Group p
(n = 36)
(n = 17) (n = 35)
Food frequency
Chicken (s/m) 9.6 (3.3) 10.9 (2.2) 15.8 (2.3) 0.221
Beef (s/m) 3.7 (0.71) a 3.2 (0.47) a 3.5 (0.47) 0.802
Pork (s/m) 4.9 (1.40) 7.7 (0.94) 6.5 (0.96) 0.256
Cured ham (s/m) 7.1 (2.09) ab 13.7 (1.40) a 13.0 (1.40) b 0.034
Ham (s/m) 7.1 (2.68) a 13.6 (1.79) ab 7.3 (1.81) b 0.030
Tukey(s/m) 10.4 (3.27) 10.3 (2.19) 8.59 (2.21) 0.843
Meat derivatives (s/m) 5.5 (2.81) a 13.5 (1.88) ab 7.0 (1.90) b 0.020
Viscera (s/m) 0.13 (0.19) 0.34 (0.13) 0.11 (0.13) 0.440
White fish (s/m) 3.8 (0.9) a 5.9 (0.7) 7.3 (0.7) a 0.025
Blue fish (s/m) 3.1 (0.7) 4.0 (0.5) 5.1 (0.5) 0.104
Lentils (s/m) 3.5 (0.87) 4.3 (0.58) 3.7 (0.59) 0.661
Grouped fish and seafood (s/m) 14.1 (2.86) a 22.8 (1.89) a 21.4 (1.88) 0.044
Grouped red meat and subproducts (s/m) 28.5 (5.13) a 52.1 (3.43) a 37.4 (3.47) < 0.001
Olive oil (s/m) 50.1 (10.9) 61.7 (7.35) 68.7 (7.43) 0.407
Nuts (s/m) 6.46 (6.73) 9.33 (4.24) 18.4 (4.33) 0.232
Values shown as mean (standard error); s, servings; m, month; wk, week. Model adjusted for sex, age and body
weight. Upper case a,b letters in the same row indicate a significant pairwise difference (p < 0.05) between groups
with the same letter. Bonferroni’s correction for multiple comparisons was applied to analyze pairwise differences.
The differences in dietary habits of the study participants between the control group, the celiac
children following a GFD for at least 6 months and the celiac children following a GFD for less than
6 months are shown in Table 3. The children with CD who followed a GFD for at least 6 months
showed a higher intake of cobalamin, meat derivatives and fish when compared with those patients
who followed a GFD for less than 6 months (all, p < 0.05). The children in the healthy control group
showed a higher consumption of folate, iron, magnesium, selenium and meat derivatives than those
children with CD following a GFD for at least 6 months (all, p < 0.05). The children in the control group
showed a higher consumption of folate and iron compared with the children with CD following a GFD
for less than 6 months (both, p < 0.05).
4. Discussion
The main findings of the present study indicate that patients with CD showed lower levels
of hemoglobin, erythrocyte and hematocrit compared to healthy children, although neither group
presented average values compatible with anemia, nor microcytosis. Regardless of the GFD tracking
time, those patients following a GFD for more than 6 months showed a lower hematocrit than healthy
controls. Moreover, both CD groups had lower iron and folate intake than the healthy controls. In
addition, a lower intake of magnesium and selenium was also observed in patients with CD who had
followed a GFD for more than 6 months compared to the control group. Finally, the children with CD
who had followed a GFD for less than 6 months showed a lower cobalamin intake compared to those
with a greater period of adherence.
In our study, there were 52 (76.5%) celiac females vs 20 (46.5%) healthy females, a sample that
is representative of the real incidence of CD by sex [25] and consistent with other similar ones [26].
Moreover, the groups differed in age and weight, but we controlled all the analyses for such covariates.
It is well known that CD is a cause of anemia as a result of malabsorption of iron, folic acid
and cobalamin [5]. IDA is a frequent finding in patients with CD and adherence to a GFD may be
sufficient to reverse IDA and iron deficiency, although both may persist despite a good adherence to a
GFD [7,23]. In fact, in the present study, erythrocyte count, hemoglobin and hematocrit were lower
in CD patients compared to healthy controls. However, these patients cannot be considered anemic
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Nutrients 2020, 12, 844
(despite hemoglobin was lower in CD groups, these values were within the normal range for its age). It
is possible that the non-presence of IDA in our patients with CD may be due to the fact that, in addition
to the GFD, all children who presented IDA at the time of the diagnosis of CD were prescribed oral
iron supplements (ferrous sulfate). This fact could induce a normalization of both the IDA and ID
indicators in the first months of treatment, despite the fact that dietary iron intake was lower than
recommended [22]. A strict monitoring of the GFD leads to the remission of clinical symptoms, the
normalization of serological markers of CD and the recovery of the histological lesion in the small
intestine, but the IDA and/or the ID may take a long time in recovering, between 6 months and a year
or even longer [17,23]. In addition, it should be taken into account that anemia is present in 22%–63%
of patients with CD at the time of diagnosis [15]; the more severe the villus lesion of the intestine, the
higher the incidence of anemia [27].
In addition to ID, it is known that in CD there are other micronutrient deficiencies (copper, zinc,
folic acid and vitamins A, D, E, K, B6 and B12) as a consequence of the malabsorption [28]. Gluten is
found in cereals rich in the referred micronutrients, so a GFD may predispose to its deficiency [29].
In our study, children with CD who followed a GFD for more than 6 months had a lower intake of
folic acid, magnesium and selenium than the control group. These data are consistent with those
reported in other studies [30–32] that described a lower intake of folic acid, magnesium and selenium,
among other micronutrients. Folate intake in children with CD has been poorly investigated, and
there are data that indicate lower folate intake with a GFD [33]. One of the factors that could also
explain the differences found in the study in relation to micronutrient intake is the pattern of the diet.
However, when we evaluate the frequency of consumption of the different food groups, we only found
differences in the intake of meat derivatives that was higher in patients with CD who had followed the
GFD for at least 6 months, compared with the control group (p < 0.020); these results that have also
been described previously [26].
Daily energy intake does not meet Recommended Daily Intake for Spanish population [24] since
these are around 2000 kcal for children aged 6 to 9 years and 2400 kcal for children aged 10 to 12
years. With respect to food group consumption, as collected by FFQ, CD > 6 consumed more red
meat and subproducts, which has been described in CD by other authors [26]. The daily intake of
carbohydrates and the energy intake provided by carbohydrates were lower in the CD < 6 group,
although not significantly, and in all groups the percentage of energy supplied by carbohydrates
reached the recommendations [24]. Several studies have shown that children have a high risk of
consuming excess fats, and that this problem may be increased in patients with CD, because GF bakery
products have a higher total fat content and saturated fat [34]. This higher fat intake may predispose to
suffer from overweight and obesity, although there are no consistent data to support this fact [35]. In
our study no increase in total fat intake was observed.
Previous studies [36] revealed, through a skin model derived from fibroblasts, the importance
of nutrient restriction in early stages of life, being one of the earliest determining factors that mark
accelerated aging. Fibroblasts have been associated with resistance to multiple forms of cellular stress
and with DNA repair mechanisms, increased proteastasis, and resistance to cellular senescence. In
CD, it is known that there is an uncontrolled activation of the proinflammatory pathway [37] that
maintains the production of free radicals that causes oxidative stress through the increase of reactive
oxygen species and consequent damage to cellular DNA [38], and it has been documented that chronic
inflammation may be involved in at least a third of cancer cases worldwide [39], so it is essential to
ensure a correct balance of micronutrients from childhood.
Another problem that is usually described in children with CD is a low fiber intake due to the
lower content of it in the flours used for the manufacture of GF products [40]. In the present study, all
children covered the recommended daily intakes due to the consumption of GF vegetables and grains
with a fiber content similar to wheat [35]. Data from a study conducted in Spanish children with CD
also showed that fiber intake was adequate [41].
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Nutrients 2020, 12, 844
In general, patients with CD who participated in the present study were well controlled from a
nutritional point of view, and the majority (86%) was aware of the dietary recommendations to be
followed (strict GFD) and the performance of physical activity. It has been described that higher levels
of adherence to a GFD are positively associated with the perceived adoption of healthy behaviors [42].
In fact, among our patients, it seems that low compliance could be related to a lack of nutritional
knowledge and that over time they would acquire more information and there would be greater
adherence to the GFD. This is achieved through medical visits during the follow-up of the disease
and the participation and dedication of the dietitian, essential for the acquisition of knowledge and
obtaining positive reinforcement [43,44].
5. Strengths and Limitations

The present study sample size was relatively small, and, consequently, the present results must
be interpreted with caution. Based on the present findings, it is advisable to recruit a higher number
of participants to render the obtained results with more statistical relevance. Moreover, the groups
differed in age, weight and sex, but we controlled all the analyses for such covariates. On the other
hand, this is the first study exploring the effect of nutritional adequacy of the GFD and the influence of
the time following this GFD on iron status.
6. Conclusions
In conclusion, CD may be associated with iron deficiency, due to malabsorption, but also as a
result of a poor adherence to a balanced GFD; it is not clear what percentage each factor influences. We
believe that the participation of dietitians in the management of the disease is necessary to guide the
GFD, to make the diet more balanced, as well as to fortify it in iron and other micronutrients such as
folate that may be deficient.
Author Contributions: Conceptualization, T.N. and R.M.-M.; Methodology; T.N., R.M.-M. and J.M.; Validation,
T.N.; V.A.A.; R.M.-M.; M.L.-F.; A.L.; M.F.-A. and J.M.; Formal Analysis, V.A.A. and M.F.-A.; Investigation, T.N.;
R.M.-M. and J.M.; Resources, T.N. and M.L.-F.; Data Curation, R.M.-M.; J.M. and A.L.; Writing—original Draft
preparation, T.N.; V.A.A.; J.M. and R.M.-M.; Writing—Review & Editing: T.N.; V.A.A.; and R.M.-M.; Project
Administration, T.N.; Funding Acquisition, T.N. and M.L-F. All authors have read and agreed to the published
version of the manuscript.
Funding: This study was partially funded by the Regional Government of Andalusia, Excellence Research Project
No P12-AGR-2581. This study was also supported by the University of Granada Research and Knowledge Transfer
Fund (PPIT)2016, Excellence Actions Programme: Scientific Units of Excellence (UCEES), and the Regional
Ministry of Economy, Knowledge, Enterprises and University, European Regional Development Funds (ref.
SOMM17/6107/UGR). MFA was additionally funded by the Spanish Ministry of Education, Culture and Sports
(Grant number FPU17/03715).
Acknowledgments: The authors thank the patients for their participation in the current study. We are grateful to
Ana Yara Postigo Fuentes for her assistance with the English language.
Conflicts of Interest: The authors declare no conflicts of interest.
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24
nutrients
Article
Adjusting Haemoglobin Values for Altitude
Maximizes Combined Sensitivity and Specificity to
Detect Iron Deficiency among Women of
Reproductive Age in Johannesburg, South Africa
Takana Mary Silubonde 1,2, *,† , Jeannine Baumgartner 1,3,† , Lisa Jayne Ware 2 , Linda Malan 1 ,
Cornelius Mattheus Smuts 1 and Shane Norris 2,4
1 Centre of Excellence for Nutrition, Faculty of Health Sciences, North-West University, Potchefstroom 2531,
South Africa; jeannine.baumgartner@hest.ethz.ch (J.B.); Linda.Malan@nwu.ac.za (L.M.);
Marius.Smuts@nwu.ac.za (C.M.S.)
2 SAMRC/Wits Developmental Pathways for Health Research Unit, Faculty of Health Sciences,
University of the Witwatersrand, Johannesburg 2000, South Africa; lisa.ware@wits.ac.za (L.J.W.);
Shane.Norris@wits.ac.za (S.N.)
3 Laboratory of Human Nutrition, Department of Health Sciences and Technology, ETH Zurich,
8092 Zurich, Switzerland
4 School of Health and Human Development, University of Southampton, Southampton S016 6YD, UK
* Correspondence: 31260632@student.g.nwu.ac.za
† These authors contributed equally to this work.
Received: 31 January 2020; Accepted: 16 February 2020; Published: 27 February 2020
Abstract: In South Africa, haemoglobin (Hb) is measured to screen for iron deficiency (ID). However,
low levels of Hb are only a late stage indicator of ID. Furthermore, Hb values are generally not
adjusted for altitude even though recommended by WHO. We determined the Hb threshold with the
highest combined sensitivity and specificity for detecting ID among South African women living
at 1700 m above sea level. In a cross-sectional study of 492 18–25-year-old women, we measured
Hb and iron status biomarkers. Using receiver operating characteristic curves, we determined
the Hb threshold with maximum Youden Index for detecting ID. This threshold of <12.35 g/dL
resulted in a 37.2% anaemia prevalence (20.9% IDA), and sensitivity and specificity of 55.7% and
73.9%, respectively. The WHO altitude-adjusted threshold of <12.5 g/dL resulted in a 39% anaemia
prevalence (21.3% IDA), and sensitivity and specificity of 56.8% and 70.8%, respectively. In contrast,
using the unadjusted Hb cut-off of <12 g/dL resulted in a 18.5% anaemia prevalence (12.6% IDA),
and sensitivity and specificity of 35.1% and 88.6%, respectively. In this sample of South African
women of reproductive age an Hb threshold <12.35 g/dL had the highest combined sensitivity and
specificity for detecting ID. The diagnostic performance of this Receiver operating characteristic
curve-determined threshold was comparable to the altitude-adjusted threshold proposed by WHO.
Thus, clinical and public health practice in South Africa should adopt adjustment of Hb for altitude
to avoid underestimation of ID and missing women in need for intervention.
Keywords: anaemia; altitude adjustment; haemoglobin; iron; South Africa; women of

reproductive age
1. Introduction
Iron deficiency (ID) is the most common micronutrient deficiency worldwide and the major
cause of anaemia. Women of reproductive age (WRA) have high iron requirements because of iron
loss through menstruation. An estimated half a billion WRA are affected by anaemia, with ID being

Nutrients 2020, 12, 633
responsible for approximately half the cases [1]. During pregnancy, physiological iron requirements
increase even further to ensure adequate blood volume expansion and optimal placental and foetal
development [2]. However, in low-and middle-income countries, iron intake and/or absorption are
often inadequate to meet demands, resulting in ID and ID anaemia (IDA) [3].
Maternal ID and IDA are associated with serious consequences for the mother and her offspring,
including an increased risk of morbidity and mortality, adverse birth outcomes, and impaired physical
and neurological development in the offspring [4]. In an effort to prevent the adverse health effects
of ID and IDA, the World Health Organization (WHO) recommends intermittent iron-folic acid
supplementation in menstruating women living in settings where the prevalence of anaemia is 20% or
higher [5], and daily oral iron-folic acid supplementation in all pregnant women as part of routine
antenatal care [6]. To identify populations at risk for ID and individuals in need for treatment, accurate
diagnosis is imperative.
In primary health care settings, the measurement of haemoglobin (Hb) concentration is commonly
used to screen individuals who require iron supplementation, e.g., by using inexpensive and easy to
use point-of-care haemoglobinometers [7]. Hb is an iron-containing protein found within red blood
cells (RBCs). Mature human RBCs have a life span of ~120 days, after which they become senescent and
are phagocytosed by macrophages, with iron being recycled [8]. Thus, Hb concentrations only drop
when iron stores are depleted (the late stages of ID) or when iron cannot be mobilized from storage in
hepatocytes or macrophages, e.g., in the presence of inflammation [9,10]. Furthermore, anaemia has a
multifactorial aetiology and can exist without ID [11]. Therefore, measuring Hb concentrations has
generally low sensitivity (ability of a test to correctly identify those with the condition) and specificity
(ability of the test to correctly identify those without the condition) for the detection of ID [12,13].
Nonetheless, due to the lack of a single, inexpensive, and simple measure of iron status, measuring Hb
concentration remains the method of choice for ID screening in primary healthcare, particularly in
low-resource settings.
However, the interpretation of Hb values is not free of challenges. Hb cut-off points to diagnose
anaemia remained relatively unchanged since 1968, and have been defined for different age categories
of children, men, non-pregnant and pregnant women [12]. Since 2001, the WHO further recommended
adjusting Hb concentrations downwards (or correcting cut-off point upwards) in individuals residing
at altitudes higher than 1000 m above sea level [12]. Erythropoiesis (production of RBCs) increases as
a result of chronic hypoxia [14]. Partial pressure of oxygen decreases as altitude increases, resulting
in a lower oxygen saturation of RBCs and an increase in erythropoiesis as an adaptive response [15].
Thus, failure to adjust Hb values for altitude may lead to an underestimation of the anaemia and IDA
prevalence at population level, and missed diagnosis and subsequent treatment at an individual level.
Despite these risks, the adjustment of Hb values for altitude is not a standard practice, neither by
researchers nor by health care professionals in clinical settings, and remains a matter of debate [16,17].
Sharma and colleagues recently re-examined associations of Hb with altitude in a dataset including
68,193 observations among preschool-aged children and WRA from all WHO regions (except South-East
Asia) [18]. The authors confirmed that Hb should be adjusted for altitude, but indicated that current
recommendations may underestimate anaemia for those residing at lower altitudes (<2000 m) and
overestimate anaemia for those residing at higher altitudes (<3000 m). Furthermore, small differences
in Hb concentrations may exist between different ethnic groups. Thus, Karakochuk et al. recently stated
that the validity of adjusting Hb cut-offs in individuals of African origin warrants further research [7].
South Africa occupies the southern tip of Africa, and consists of a high-lying inland plateau
separated from the narrow, low-lying coastal plain by mountainous escarpment. The country’s
mean altitude is approximately 1200 m, and at least 40% of the surface is at a higher elevation [19].
Nonetheless, South African clinical guidelines do not advise adjusting Hb values [20–22], which is
therefore not a general practice in the public healthcare sector. However, in the most recent South
African Demographic and Health Survey (SADHS) conducted in 2016 [23], anaemia prevalence rates
were reported based on altitude-adjusted Hb values, while previous national surveys, such as the
26
Nutrients 2020, 12, 633
2012 South African National Health and Nutrition Examination Survey (SANHANES) [24], reported
unadjusted values. It can be speculated that this may explain the increase in anaemia prevalence in
WRA from 23.1% to 31.5% in the 2012 and 2016 surveys, respectively. Consensus is needed on whether
to adjust Hb values for altitude or not in South African population surveys and in the primary health
care sector.
Therefore, the aim of this study was to establish an Hb threshold (≈adjustment value) that would
maximize the combined sensitivity and specificity for the detection of ID among WRA residing in
Soweto, South Africa, situated at 1700 m above sea level. Our objectives were three-fold: (i) to
determine the Hb cut-off point with the highest combined sensitivity and specificity (maximum Youden
Index) for detecting ID based on ferritin [adjusted and non-adjusted for inflammation] and iron
deficient erythropoiesis (IDE) based on soluble transferrin receptor (sTfR); (ii) to assess and compare
the sensitivity and specificity of the Hb cut-off points adjusted for altitude (as recommended by WHO)
and without adjustment for the detection of ID; and (iii) to determine and compare the prevalence of
anaemia, IDA and ID without anaemia using these different cut-off points.
2.1. Study Setting and Population

For this study, we used data collected between 2018 and 2019 from the Soweto Young Women’s
Survey, a prospective study conducted in Gauteng, South Africa’s most populous province (home
to approximately 26% of the South African population). Data collection took place at the South
African Medical Research Medical Council (SAMRC) Developmental Pathways to Health Research
Unit (DPHRU), located within the Chris Hani Baragwanath Academic Hospital (CHBAH), a tertiary
hospital in Soweto (South Western Township). Generally healthy, 18–25-year-old, non-pregnant
WRA were recruited from Soweto, a historically disadvantaged urban area of 200 km2 in the city of
Johannesburg, Gauteng province. Over 1.3 million people reside in Soweto (6400/km2 ) with 98.5% of
the population being of black African descent. Women were eligible for inclusion if they were aged
18–25 years; proficient in local languages; born in South Africa or neighbouring countries; and if they
had been residing in their home in Soweto for at least 3 months. Women were excluded if they had
a previous diagnosis of type-1 diabetes; cancer or epilepsy; or not able or willing to provide written
informed consent. Due to South Africa’s high prevalence of HIV infection (23.2% of women aged
15–49 years [25]), women who were HIV positive were included in the study for the sample to be a
better representation of the general population. As a result, HIV was self-reported and CD4 and viral
load were not assessed.
A cluster design was employed for recruitment, where each Soweto community centre was a
cluster. Thirty clusters with a radius of 1 km2 each were identified around Soweto using churches
as the midpoint of each cluster. An online search was performed using the Google search engine
to locate the information of all churches in Soweto. Using street address information, geolocations
of each church structure were obtained, and each church was visited by fieldworkers and verified.
The latitude and longitude of the 104 churches identified and verified were then classified using
k-means clustering. The church with the shortest straight line distance to the cluster centroid was
selected for inclusion in the study as it was at the centre of a cluster of churches that was maximally
distant from the other churches in Soweto. An equal number of participants were recruited from two
randomly selected clusters.
During the recruitment process, all households in the selected community clusters were approached
to identify those where young women lived. Once identified, potential participants were visited in
their homes and were informed in their home language about: (i) the objectives of the study; (ii) the
use of the results; and (iii) the risks and benefits of the study. An informed consent form was supplied
to potentially eligible women who were interested in being part of the study. The interested women
were then invited to the study site for the signing of informed consent and data collection.
27
Nutrients 2020, 12, 633
2.2. Ethical Considerations

This study was conducted in accordance with the ethical principles laid down in the Declaration
of Helsinki, and all procedures involving human participants were approved by the Human Research
Ethics Committees of the North-West University, Potchefstroom (NWU-0042919-S1), and the University
of the Witwatersrand, Johannesburg (M171137).
2.3. Measurement of Anthropometric Indicators and Socio-Demographic Information

Weight (kg) and height (cm) of participants were measured in order to calculate body mass index:
BMI = weight (kg)/(height [m])2 . Weight was measured using the Seca 877 Scale (Seca, Hamburg,
Germany) and recorded to the nearest 100 g. Women wore light clothing and removed shoes and
heavy outerwear (e.g., sweaters) before obtaining weight. Height was measured to the nearest 0.1 cm
using a single calibrated Holtain Stadiometer (Holtain Limited, Crymych, UK). Participants were
measured either barefoot or wearing thin socks. Mid upper arm circumference (MUAC) was measured
to the nearest 0.1 cm using a plastic measuring tape. Measurement was taken at the mid-point of the
upper arm, between the acromion process and the tip of the olecranon. A MUAC ≤24 cm was used to
define undernutrition [26]. A questionnaire was administered by a trained research assistant to assess
socio-economic and demographic characteristics of the women. Food insecurity (hunger) was assessed
by a shortened version of the Community Childhood Hunger Identification Project (CCHIP) Index [27].
2.4. Biomarker Analysis

Hb concentrations were measured at the health research unit in capillary blood collected by an
experienced nurse using a calibrated Hb 201+ HemoCue® system (HemoCue Johannesburg, South
Africa). The HemoCue® system is an easy-to-use haemoglobinometer that is commonly used for
determining haemoglobin concentrations in health research and for point-of-care testing in primary
health care settings [7,28]. The South African point-of-care cut-off to diagnose anaemia and ID in WRA
is a finger-prick Hb <12 g/dL [20,22]. Participants diagnosed with anaemia, according to these national
guidelines, were given a referral letter to visit their nearest clinic.
Venous blood samples were drawn into lithium heparin tubes (BD, Plymouth, UK). Plasma was
separated within 1 h after blood collection, and aliquots stored at −20 ◦ C for a maximum of 14 days
until transportation (on dry ice) for storage at −80 ◦ C until analysis. For the analysis of iron status
indices and the inflammation/infection markers C-reactive protein (CRP) and alpha-1-acid glycoprotein
(AGP), the Q-Plex™ Human Micronutrient Array (7-plex, Quansys Bioscience, Logan, UT, USA) was
used [29].
As plasma ferritin concentrations are elevated in the presence of inflammation, ferritin
concentrations were adjusted for inflammation using the correction factors proposed by
Thurnham et al. [30]. For participants in the incubation phase (CRP > 5 mg/L and AGP ≤ 1 g/L),
ferritin values were adjusted using a correction factor of 0.77. For the early convalescence phase
(CRP > 5 mg/L and AGP > 1 g/L), a correction factor of 0.53 was used, while in the late convalescence
phase (CRP ≤ 5 mg/L and AGP > 1 g/L), a correction factor of 0.75 was applied.
Participants were classified as being iron deficient if their inflammation-adjusted plasma ferritin
concentration was <15 μg/L or, if their unadjusted plasma ferritin concentration was <30 μg/L as
recommended by WHO in settings where infection or inflammation is prevalent [31]. However, since
the ferritin cut-off of <15 μg/L is still commonly used for unadjusted ferritin values in the diagnosis
of ID in the public health sector in South Africa (e.g., population surveys), we also reported the ID
prevalence based on unadjusted ferritin values using a cut-off of <15 μg/L. IDA was defined as ferritin
<15 μg/L (adjusted) or <30 μg/L (unadjusted) plus haemoglobin <12 g/dL. Iron deficient erythropoiesis
(IDE) was defined as sTfR ≥8.3 mg/L.
28
Nutrients 2020, 12, 633
2.5. Data and Statistical Analysis

Study data were collected and managed using REDCap electronic data capture tools hosted at
the University of Witwatersrand [32]. Data processing and statistical analysis of data were performed
using Microsoft Office Excel 2010 (Microsoft, Redmond, WA, USA) and SPSS version 25 (SPSS Inc.,
Chicago, IL, USA).
Data were tested for normality by means of visual inspection using Q-Q plots and histograms,
and the Shapiro–Wilk test. Normally distributed data are expressed as means ± SD; non-normally
distributed data are expressed as medians (IQR).
Receiver operating characteristic (ROC) curves were created for the use of Hb in the detection of
ID defined by ferritin (inflammation-adjusted <15 μg/L; unadjusted <30 μg/L) and sTfR (≥8.3 mg/L).
The diagnostic threshold of Hb indicative of ID was selected based on the Youden Index, which is
defined by the formula: sensitivity + specificity − 1. The maximal Youden Index identifies the optimal
threshold when sensitivity and specificity are considered of equal importance, and this maximal
value is the “knee” of the ROC curve [33]. For each ROC curve, the area under the curve (AUC) is
reported. An AUC value of 0.50 indicates completely random predictions while a value of 1 indicates
perfect predictions.
Furthermore, we determined the sensitivity, specificity, and Youden Index for the diagnostic
thresholds indicative of ID (based on inflammation-adjusted and unadjusted ferritin or cut-off values)
when Hb values were adjusted for altitude (Hb value—0.5 g/dL at 1700 m above sea level), which is
equivalent to the use of an Hb cut-off of <12.5 g/dL, as recommended by WHO [12].
3. Results
3.1. Sample Characteristics

The characteristics of the 492 WRA participating in this study are shown in Table 1. All women
were of African descent with a median age of 21 years (IQR 19–23). The majority of the women (61%)
had obtained a high school leaving certificate. The median household size was six residents (IQR 4–8)
and 47% of women reported food insecurity. Half of the women (51%) were nulliparous. The median
BMI was 24.4 (21.2–29.6) kg/m2 , with 22% of women overweight and 24% obese.
3.2. Hematological Indicators

Table 2 presents the inflammatory and iron status indicators in the sample of WRA. Thirty-four
percent of the women had inflammation/infection (CRP > 5 mg/L and/or AGP > 1 g/L) with 14%
of the women in the late convalescence stage. The median adjusted ferritin concentration was
25.9 (8.0–55.1) μg/L, and the median unadjusted ferritin 28.2 (9.3–62.9) μg/L. The prevalence of ID
based on the adjusted and unadjusted ferritin values was 38% and 36%, respectively, when using the
cut-off point of <15 μg/L, but 52% for unadjusted ferritin values with the cut-off point of <30 μg/L.
The prevalence of IDE (sTfR > 8.3 mg/L) was 42%.
Figure 1 shows receiver operating characteristic (ROC) curves for the use of Hb to diagnose ID
(Figure 1a–c) and IDE (Figure 1d). ROC curve analysis resulted in an Hb threshold of 12.35 g/dL
with maximum sensitivity and specificity (Youden Index = 0.30) and an AUC of 0.681 to detect ID
based on an inflammation-adjusted ferritin <15 μg/L (Figure 1a). To detect ID based on an unadjusted
ferritin <30 μg/L (WHO recommendation) or <15 μg/L (current clinical and public health practice),
ROC curve analysis resulted in an Hb threshold of 12.45 g/dL (Figure 1b,c) and an AUC of 0.651 and
0.675, respectively. When using sTfR to define IDE, ROC curve analysis resulted in an Hb threshold of
12.35 g/dL and AUC of 0.633.
29
Nutrients 2020, 12, 633
Table 1. Characteristics of 18–25 year-old women of reproductive age (WRA) residing in Soweto, South
Africa (n = 492).
Characteristics Median (IQR) or n (%)

Age 21 (19–23)
BMI (kg/m2 ) 24.4 (21.2–29.6)
Underweight (<18.5 kg/m2 ) 41 (8)
Normal weight (18.5–24.9 kg/m2 ) 224 (46)
Overweight (25–29.9 kg/m2 ) 110 (22)
Obese (>30 kg/m2 ) 117 (24)
MUAC (cm) 27.6 (24.8–31.5)
Undernutrition (≤24 cm) 94 (19)
HIV positive (self-reported) Yes 22 (4)
Food insecurity
Yes 230 (47)
Household asset score 9 (7–10)
Low (1–5) 38 (8)
Medium (6–9) 308 (63)
High (10–13) 146 (30)
Highest level of education 195 (40)
Primary school or less 297 (61)
High school leaving certificate
Household size (number of people) 6 (4–8)
1–4 154 (32)
5–10 269 (56)
>10 57 (12)
Parity
Nulliparous 253 (51)
Primiparous 192 (39)
Multiparous 47 (10)
BMI = body mass index, MUAC = mid upper arm circumference.
Table 2. Inflammatory and iron status indicators in 18–25 year-old women of reproductive age residing
in Soweto, South Africa (n = 492).
Biomarker Median (IQR) or n (%)

CRP (mg/L) 1.41 (0.44–3.84)
AGP (g/L) 0.86 (0.72–1.02)
Inflammatory status
No inflammation (CRP ≤ 5 mg/L and AGP ≤ 1 g/L) 327 (67)
Incubation (CRP >5 mg/L and AGP ≤ 1 g/L) 57 (12)
Early convalescence (CRP > 5 mg/L and AGP > 1 g/L) 41 (8)
Late convalescence (CRP ≤ 5 mg/L and AGP > 1 g/L) 67 (14)
Inflammation-adjusted ferritin (μg/L) 1 25.9 (8.0–55.1)
Non-ID (ferritin ≥ 15 μg/L) 307 (63)
ID (ferritin < 15 μg/L) 185 (38)
Unadjusted ferritin (μg/L) 28.2 (9.3–62.9)
sTfR (mg/L) 7.5 (5.7–10.5)
Non-IDE (sTfR ≤ 8.3 mg/L) 288 (59)
IDE (sTfR > 8.3 mg/L) 204 (42)
AGP, α-1-acid glycoprotein; CRP, C-reactive protein; ID, iron deficiency; IDA, iron deficiency anaemia; sTfR, serum
transferrin receptor; IDE, iron deficient erythropoiesis. 1 Ferritin values were adjusted for inflammation using the
correction factors suggested by Thurnham et al. [30].
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Nutrients 2020, 12, 633
Figure 1. Receiver operating characteristic curves for the use of haemoglobin to diagnose
iron deficiency in 18–25 year-old non-pregnant women of reproductive age (n = 492) based on:
(a) Inflammation-adjusted ferritin (<15 μg/L), area under the curve (AUC) = 0.681; (b) unadjusted
ferritin (<30 μg/L), AUC = 0.651; (c) unadjusted ferritin (<15 μg/L), AUC = 0.675; and (d) soluble
transferrin receptor (sTfR > 8.3 mg/L), AUC = 0.633. The solid line indicates the haemoglobin cut-off
point with the highest combined sensitivity and specificity (maximum Youden Index), while the dotted
line indicates the unadjusted haemoglobin cut-off point, typically used in the public healthcare sector.
Table 3 shows the diagnostic accuracy of the Hb cut-off points to detect ID based on
inflammation-adjusted and unadjusted ferritin values, and presents the iron and anaemia status
of the participating WRA based on the different Hb cut-off points and ferritin criteria to define ID.
The unadjusted Hb cut-off point of <12.00 g/dL, that is currently used in primary health care, had a
sensitivity of 35.1% and specificity of 88.6% to diagnose ID based on an inflammation-adjusted ferritin
<15 μg/L. It resulted in an anaemia, IDA and ID without anaemia prevalence of 18.5% and 12.6% and
25.0%, respectively. The ROC-curve-determined Hb cut-off point of 12.35 g/dL optimized to diagnose
ID based on an inflammation-adjusted ferritin <15 μg/L had a sensitivity of 55.7% and specificity of
73.9%. It resulted in an anaemia, IDA and ID without anaemia prevalence of 37.2% and 20.9% and
16.7%, respectively. The ROC-curve-determined Hb cut-off point of 12.45 g/dL to determine ID based
on an unadjusted ferritin <30 μg/L had a sensitivity of 51.6% and a specificity of 74.4%. It resulted
in an anaemia, IDA and ID without anaemia prevalence of 39.0% and 26.6% and 25.0%, respectively.
The ROC-curve-determined Hb cut-off point of 12.45 g/dL to determine ID based on an unadjusted
ferritin <15 μg/L had a sensitivity of 56.2% and a specificity of 70.7%, and resulted in an anaemia, IDA
and ID without anaemia prevalence of 39.0% and 20.3% and 14.6%, respectively. The altitude-adjusted
31
Nutrients 2020, 12, 633
Hb cut-off point of <12.5 g/dL, as recommended by WHO, had a sensitivity of 56.8% and a specificity
of 70.8%. It resulted in an anaemia, IDA and ID without anaemia prevalence of 39.0% and 21.3% and
16.3%, respectively.
Table 3. Diagnostic accuracy of the different Hb cut-off points to detect ID based on

inflammation-adjusted and unadjusted ferritin, and iron and anaemia status in 18–25 year-old
non-pregnant women of reproductive age residing in Soweto, South Africa (n = 492).
Diagnostic Performance of Hb
Cut-off Points to Detect ID
Anaemia ID
Total Non-ID &
IDA without without Sensitivity Specificity Youden
Anaemia Non-Anaemic
(n [%]) ID Anaemia (%) (%) Index
(n [%]) (n [%])
(n [%]) (n [%])
Hb <12.00 g/dL (As currently used in SA
primary health care clinics to diagnose ID 91 (18.5) 62 (12.6) 29 (5.9) 123 (25.0) 278 (56.5) 35.1 88.6 0.24
[inflammation-adjusted ferritin <15 μg/L])
Hb <12.35 g/dL (ROC-curve-determined to
diagnose ID [inflammation-adjusted ferritin 183 (37.2) 103 (20.9) 80 (16.3) 82 (16.7) 227 (46.1) 55.7 73.9 0.30
<15 μg/L])
192 (39.0) 131 (26.6) 61 (12.4) 132 (25.0) 177 (36.0) 51.6 74.4 0.26
diagnose ID [unadjusted ferritin <30 μg/L])
192 (39.0) 100 (20.3) 92 (18.7) 72 (14.6) 228 (46.3) 56.2 70.7 0.27
diagnose ID [unadjusted ferritin <15 μg/L])
Hb <12.50 g/dL (Altitude-adjusted based on
WHO recommendations to diagnose ID 192 (39.0) 105 (21.3) 87 (17.7) 80 (16.3) 220 (44.7) 56.8 70.8 0.27
[inflammation-adjusted ferritin <15 μg/L])
Hb, haemoglobin; ID, iron deficiency; ROC, receiver operation characteristics; SA, South Africa; WHO, World
Health Organization.
4. Discussion
The results of this analysis among 18–25 year-old South African WRA living at 1700 m above sea
level showed that the Hb threshold with the highest combined sensitivity and specificity for detecting
both ID (based on inflammation-adjusted ferritin <15 μg/L) and IDE (based on sTfR > 8.3 mg/L) is
<12.35 g/dL. The diagnostic performance of this ROC-curve-determined cut-off value was comparable
to the altitude-adjusted Hb cut-off of <12.5 g/dL proposed by WHO for this altitude. In contrast,
the Hb cut-off of <12.00 g/dL, which is currently used in the South African primary health care setting
without adjusting Hb values for altitude, had a lower sensitivity for the detection of ID than the
ROC-curve-determined and altitude-adjusted cut-offs (35% versus ~56%). According to our analysis,
the current Hb threshold used in primary health care for the detection of anaemia missed more than
half of the anaemia cases detected when using an altitude adjusted Hb threshold (18.5% versus 39%),
and resulted in a markedly lower prevalence of IDA (12.6% versus 21%), and a higher prevalence of ID
without anaemia (25% versus ~16%). The latter indicates the proportion of women with depleted iron
stores who would be missed if measuring Hb to screen for ID.
According to the 2018 Standard Treatment Guidelines and Essential Medicines List for South
Africa, the first step in the diagnosis of ID at primary healthcare level is measuring Hb [22]. Thereby,
a Hb <12 g/dL is considered indicative of anaemia in non-pregnant WRA, which requests a full blood
count to determine the likely aetiology of anaemia based on measured mean corpuscular volume
(MCV; average size and volume of red blood cells). If MCV is normal, systemic disease or haemolysis
are considered likely causes, and if MCV is low then ID is the most likely cause, while high MCV is
indicative of a folate and/or vitamin B12 deficiency [22]. Only an estimated 50% of anaemia cases are
attributable to ID [28], and therefore proper identification of the anaemia cause is necessary to ensure
cause-specific treatment [34]. However, follow-up assessments to confirm the cause of anaemia and
the presence of IDA, including the one described above, are rarely done in primary healthcare clinics
in South Africa.
Furthermore, clinical guidelines do not specify how to interpret Hb values from individuals
residing in higher altitude settings [20–22], which makes up a substantial proportion of South Africa’s
population [35]. The results of this study indicate that the current point-of-care threshold to detect
32
Nutrients 2020, 12, 633
anaemia (<12 g/dL) has poor sensitivity (35.1%) for the detection of ID (88.6% specificity) in women
residing at an altitude of 1700 m above sea level. In our sample, the ROC curve was optimized
at an Hb threshold of <12.35 (AUC = 0.681) using the Youden Index, which defines the maximum
potential effectiveness of Hb to detect ID when equal weight is given to sensitivity and specificity.
Thereby, the ROC curve-determined threshold increased sensitivity to 55.7%, while reducing specificity
to 73.9%. The diagnostic performance of this threshold was comparable to the performance of the
altitude-adjusted Hb threshold (<12.5 g/dL) proposed by WHO, which slightly improved sensitivity
(56.8%) while maintaining a favourable specificity (70.8%). This confirms the findings by Sharma et al.,
who recently re-examined haemoglobin adjustments to define anaemia among WRA residing at
different altitudes and concluded that Hb values (or thresholds) should be adjusted for altitude [18].
Not only can the lack of altitude-adjustment lead to missed diagnosis and treatment of ID in
primary health care clinics, it can also result in an underestimation of the anaemia burden in the
population. At the public health level, WHO recommends intermittent (once, twice, or three times per
week on non-consecutive days) iron and folic acid supplementation of adult women and adolescent girls
in populations where the prevalence of anaemia among WRA is 20% or higher [5]. The recommendation
for blanket iron supplementation emphasizes that sensitivity of Hb as a screening tool to detect ID
is more important than specificity in the public health context. Nonetheless, sensitivity alone does
not provide the basis for informed decisions following positive screening test results because those
positive results could contain many false positive outcomes [36], hence the use of the Youden Index
to define a cut-off with maximum combined sensitivity and specificity. The anaemia prevalence of
18.5% obtained in our sample when using the non-adjusted Hb cut-off of <12.00 g/dL would indicate
that no intervention at the public health level is needed. In contrast, the 39% anaemia prevalence
obtained when using the ROC-curve-determined and the altitude-adjusted cut-offs would indicate
a moderate to borderline severe public health problem that requires intervention. Recent national
surveys have reported discrepant anaemia prevalence rates among South African WRA. The (South
African Demographic Health Survey) SADHS conducted in 2016 reported an anaemia prevalence
in WRA of 31.5% [23], while the South African National Health and Nutrition Examination Survey
(SANHANES) 2012 reported an anaemia prevalence of 23.1% [24]. The latter survey did not adjust Hb
values for altitude and used the HemoCue 201+ system to measure Hb while SANHANES 2012 used
an automated haematology analyser. These and other factors make it impossible to conclude whether
the situation truly got worse or whether the 2012 survey potentially underestimated the anaemia
prevalence [37].
Another challenge lays in the interpretation of ferritin in settings with a high prevalence of
inflammation. Ferritin is not only a marker of iron stores but also an acute-phase protein that
increases during inflammation independently of iron status [38,39]. In this study, 34% of women had
inflammation, highlighting the importance of considering inflammation when interpreting ferritin
values and determining ID prevalence. Possible strategies to interpret ferritin values in settings with
a high inflammatory burden are to raise the cut-off that defines deficiency [31], or to adjust ferritin
values of individuals with inflammation [30,39]. The latter requires the measurement of inflammatory
markers, which is not always feasible. Furthermore, there is currently no consensus on the most
appropriate adjustment method. For the determination of an optimized Hb threshold to detect ID,
we defined ID using different ferritin interpretation approaches. ROC analysis using unadjusted
ferritin cut-off values of either <30 or <15 μg/L to define ID resulted in an optimized Hb cut-off of
<12.45 g/dL. However, the highest Youden Index and AUC for Hb to detect ID was obtained when
ferritin was adjusted for inflammation using the correction factors proposed by Thurnham et al. In the
current study, we used the correction factors proposed by Thurnham et al. [30]. However, for interest
sake we also adjusted ferritin values using the Biomarkers Reflecting Inflammation and Nutritional
Determinants of Anaemia (BRINDA) approach and repeated the ROC curve analysis for Hb to detect
ID based on BRINDA-adjusted ferritin <15 μg/L [40]. This resulted in an even lower median ferritin
concentration of 19.8 (6.2–41.2) μg/L and ID prevalence of 55.3%. Nonetheless, the Hb threshold
33
Nutrients 2020, 12, 633
with optimized combined sensitivity and specificity to detect ID remained at <12.35 g/dL (Youden
Index = 0.281), with a sensitivity and specificity of 52.7% and 74.4%, respectively. Thus, overall,
the different approaches to interpret ferritin did not relevantly affect the diagnostic performance of Hb
to detect ID, but the use of the higher ferritin cut-off of <30 μg/L likely resulted in an overestimation of
ID and IDA.
When interpreting the results of this study, the following limitations must be considered. Firstly,
since we only included data from women residing at one particular altitude, we cannot draw a
definite conclusion on whether the lack in sensitivity of the current point-of-care cut-off is driven by
altitude or by other factors. Thus, future studies should repeat these analyses in South African women
residing at different altitudes. Secondly, we did not perform a full blood count using an automated
haematology analzyer to validate the accuracy of the HemoCue 201+ system and to determine the likely
causes of anaemia using MCV. The HemoCue 201+ system has been used in the most recent SADHS
survey (versus automated haematology analyser in SANHANES) and is being used in most primary
healthcare settings in South Africa to screen for anaemia and IDA. Nonetheless, studies that compared
the HemoCue 201+ system with automated haematology analyzers showed that Hb concentrations
measured by the HemoCue 201+ system were on average 0.1 to 0.4 g/dL higher than when measured
with the haematology analyser [7,37]. This would suggest that if measured with an automated
haematology analyser, the ROC-curve-determined Hb threshold for detecting ID would have been
slightly higher, and therefore getting even closer to the altitude-adjusted threshold recommended
by WHO. Lastly, we did not consider the potential effects of smoking on Hb concentrations in our
analysis, since information on smoking was incomplete. Similar to altitude-exposure, the reduced
oxygen-carrying capacity in smokers caused by exposure to carbon monoxide results in a compensatory
increase in Hb concentrations in individuals smoking ≥1/2 packet per day [40]. Therefore, WHO
recommends adjustment of Hb for smoking, and Sharma et al. showed that adjustments for smoking
and altitude are additive [12]. Thus, we cannot rule out that Hb concentrations of smoking WRA would
have needed even further adjustment, again potentially closing the gap between the ROC-determined
and the altitude-adjusted Hb threshold.
The strength of this study was the assessment of iron status by measuring two different biomarkers
(ferritin and sTfR) using a validated method [29]. However, we do acknowledge that a recent study
comparing the Quansys multiplex immunoassay with reference-type assays found poor precision for
sTfR in comparison with the Roche cobas 6000 clinical analyser [41]. Nonetheless, the Hb threshold
that we obtained by ROC curve analysis for the detection of ID (based on ferritin) was identical to the
one for the detection of IDE (based on sTfR), although the latter mainly served as sensitivity analysis.
In addition, the analysis of both CRP and AGP enabled us to adjust ferritin for inflammation and
perform a sensitivity analysis.
5. Conclusions
In conclusion, the sensitivity and specificity of the optimized Hb cut-off point of 12.35 g/dL
determined by ROC curve analysis for the detection of ID was comparable to the Hb cut-off point
adjusted for altitude (<12.5 g/dL) as recommended by WHO. It further resulted in comparable anaemia,
IDA, and ID without anaemia prevalence rates. In contrast, the use of an unadjusted Hb cut-off
point (or lack of adjusting single Hb values) resulted in an underestimation of the anaemia and
IDA prevalence, and missed detection of ID in 25% of women (versus 16% for altitude-adjusted
cut-off) when analysis of additional iron status indicators is not possible or standard practice. Thus,
this study confirms that clinical and public health practice should adopt the adjustment of Hb values
for altitude according to WHO guidelines to avoid underestimation of the anaemia and IDA burden
in the population and missing individuals in need for intervention. The lack of addressing a large
anaemia burden in WRA may not only compromise their own health, but also the health of their baby
if they become pregnant.
34
Nutrients 2020, 12, 633
Author Contributions: Conceptualization, J.B., L.J.W., C.M.S. and S.N.; methodology, T.M.S., J.B., L.J.W., L.M.,
C.M.S. and S.N.; investigation, T.M.S. and L.J.W.; data curation, L.J.W. and L.M.; formal analysis, T.M.S. and J.B.;
supervision, L.J.W., L.M., C.M.S. and S.N.; resources, C.M.S. and S.N.; project administration, T.M.S. and L.J.W.;
funding acquisition, C.M.S. and S.N.; writing—original draft preparation, T.M.S. and J.B.; writing—review and
editing, T.M.S., J.B., L.J.W., L.M., C.M.S. and S.N. All authors have read and agreed to the published version of
the manuscript.
Funding: The HeLTI study is supported by the South African Medical Research Council, and the Canadian
Institutes of Health Research. Additional funding for this study was provided by the North-West University in
Potchefstroom, South Africa.
Acknowledgments: The authors thank the women who participated in this study, and Cecile Cooke (Centre of
Excellence for Nutrition, North-West University, South Africa) for her assistance with sample management and
laboratory analysis.
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37
nutrients
Article
Acai Extract Transiently Upregulates Erythropoietin
by Inducing a Renal Hypoxic Condition in Mice
Shuichi Shibuya 1,2 , Toshihiko Toda 2 , Yusuke Ozawa 2 , Mario Jose Villegas Yata 3 and
Takahiko Shimizu 1,2, *
1 Aging Stress Response Research Project Team, National Center for Geriatrics and Gerontology,
7-430 Morioka-cho, Obu, Aichi 474-8511, Japan; s-shibuya@ncgg.go.jp
2 Department of Endocrinology, Hematology and Gerontology, Chiba University Graduate School of
Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan; hik_toda-jac@proteome.jp (T.T.);
ozawayusuke3@gmail.com (Y.O.)
3 FRUTA FRUTA, Inc., 3-3 Kandajimbo-cho, Chiyoda-ku, Tokyo 101-0051, Japan; mario@frutafruta.com
* Correspondence: shimizut@ncgg.go.jp; Tel.: +81-562-44-5651; Fax: +81-562-48-2373
Abstract: Acai (Euterpe oleracea Mart. Palmae, Arecaceae) is a palm plant native to the Brazilian
Amazon. It contains many nutrients, such as polyphenols, iron, vitamin E, and unsaturated
fatty acids, so in recent years, many of the antioxidant and anti-inflammatory effects of acai
have been reported. However, the effects of acai on hematopoiesis have not been investigated
yet. In the present study, we administered acai extract to mice and evaluated its hematopoietic
effects. Acai treatment significantly increased the erythrocytes, hemoglobin, and hematocrit contents
compared to controls for four days. Then, we examined the hematopoietic-related markers following
a single injection. Acai administration significantly increased the levels of the hematopoietic-related
hormone erythropoietin in blood compared to controls and also transiently upregulated the gene
expression of Epo in the kidney. Furthermore, in the mice treated with acai extract, the kidneys were
positively stained with the hypoxic probe pimonidazole in comparison to the controls. These results
demonstrated that acai increases the erythropoietin expression via hypoxic action in the kidney.
Acai can be expected to improve motility through hematopoiesis.
Keywords: acai; erythropoiesis; erythropoietin
1. Introduction
To maintain oxygen homeostasis, mammals have hematopoietic regulatory mechanisms, including
erythropoiesis. Erythropoietin (EPO) is a hematological factor mainly expressed in the kidney in
adults [1]. EPO is induced under conditions of reduced oxygen levels, as well as blood loss [2].
The Epo transcription is regulated by hypoxia-inducible transcription factors (HIFs), which have two
oxygen-responsive sites associated with prolyl hydroxylase and lead to degradation by ubiquitination
under normoxia [3]. This evidence demonstrates that the redox states on renal proteins containing HIF
are potential indicators of erythropoiesis in adult mammals.
Acai (Euterpe oleracea Mart. Palmae, Arecaceae) is a large palm plant found in the northern
region of South America, called the “Amazon”, in Brazil. Acai berries have a high polyphenol
content, including anthocyanins, such as cyanidine-3-glucoside (C3Glc), cyanidine-3-diglucoside, and
cyanidin-3-rutinoside, which contribute to antioxidant activity [4]. In several rodent studies, the benefits
of acai intervention have been reported to include improving cardiac dysfunction following myocardial
infarction [5], protection from diet-induced obesity [6] and hepatic steatosis [7], prevention of brain
oxidative damage [8], and modulation of age-related hippocampal inflammation [9]. Acai intake is
also expected to be a useful therapeutic strategy for chronic kidney disease with oxidative stress,

Nutrients 2020, 12, 533
inflammation, and dysbiosis [10]. However, no studies have determined the erythropoietic effect of
acai on renal redox alteration.
In the present study, in order to clarify the erythropoietic action of acai, we administered acai
extract to mice and examined the relationship between the erythropoietic factors and the redox change
in the kidney.
2.1. Animals
C57BL/6NCrSlc mice were purchased from Japan SLC (Shizuoka, Japan) and inbred in our own
cohorts. The animals were housed under a 12-h light/dark cycle and fed an MF diet (Oriental Yeast
Co., Ltd., Tokyo, Japan) ad libitum. The mice were maintained and studied according to the protocols
approved by the Animal Care Committee of the Chiba University.
2.2. Administration
Acai extract (Table 1, Lot. 171115 and 180622) provided by FRUTA FRUTA, Inc. (Tokyo, Japan)
was made by finely grinding whole fruit and filtrating with a #30 strainer. The extract was orally
administered at 10 mL/kg/day via gavage to mice 1 time (n = 8) and for four days (n = 4) at 12–16 weeks
of age. C3Glc (NS380102) was purchased from Nagara Science (Gifu, Japan). ASP1517 (roxadustat,
#15294) was purchased from Cayman Chemical (Ann Arbor, MI, USA). The water-dissolved C3Glc
(50 mg/kg, n = 6) and 0.5% carboxymethyl cellulose-suspended ASP1517 (80 mg/kg, n = 7) were
administered orally once to littermate mice of the acai-treated cohort. The study was performed using
the blood and kidney tissue of animals collected under anesthesia 2–3 h after the final administration.
Table 1. Contents in 100 g of acai extract.
Contents Fatty Acids (%)

Energy intake 82.0 kcal Palmitic acid (C16:0) 22.5
Protein 1.4 g Palmitoleic acid (C16:1) 3.3
Total lipids 6.9 g Stearic acid (C18:0) 1.9
Carbohydrates 2.0 g Oleic acid (C18:1) 61.2
Dietary fiber 3.3 g Linoleic acid (C18:2) 10.6
Total polyphenols 390 mg Linolenic acid (C18:3) 0.5
Iron 1.0 mg
2.3. Histology
To evaluate the tissue hypoxic area, HypoxyprobeTM -1 Omni (Hypoxyprobe, Inc., Burlington,
MA, USA) was used [11]. Mice were sacrificed 15 min after being intraperitoneally injected with
anesthetic and 60 mg/kg of pimonidazole. The kidney was fixed in a 4% paraformaldehyde phosphate
buffered saline (PBS) (Nacalai Tesque, Inc., Kyoto, Japan) and embedded in paraffin. The rehydrated
sections were antigen retrieved with 10 mM citrate buffer (pH 6.0, with 0.05% Tween 20) at 95 ◦ C
for 30 min, washed with PBS containing 0.1% Tween 20 three times, and intrinsic peroxidases were
quenched with 3% H2 O2 for 30 min. We performed blocking with 3% goat serum or Blocking One
Histo (Nacalai Tesque) and then treated samples with 1:200 or 1:20 diluted anti-pimonidazole antibody
(Hypoxyprobe, Inc.). The pimonidazole-positive area was evaluated by the QWin V3 imaging software
program (Leica, Wetzlar, Germany) or a BZ-X710 analyzer (Olympus Co., Tokyo, Japan) using the
ABC staining kit (Vector Labs., Inc., Burlingame, CA, USA) or the FITC-fluorescence method (goat
anti-rabbit antibody #AP307F, Sigma (St. Louis, MO, USA) and Fluoro-KEEPER Antifade Reagent with
DAPI #12745-74; Nacalai Tesque), respectively.
40
Nutrients 2020, 12, 533
2.4. Measurement of EPO

The plasma EPO level was measured using the Mouse Erythropoietin Quantikine ELISA kit
(#MEP00B; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions.
In brief, thawed plasma was diluted 2-fold by calibrator diluents and then incubated on an
antibody-coated microplate with each volume of assay diluents for 2 h using an orbital shaker.
Washed wells were treated with antiserum conjugate for 2 h and with a substrate mixture for 30 min.
The optical density measured at 450 and 540 nm was analyzed with the standards curve using the
4-parameter logistic curve-fit.
2.5. Quantitative Polymerase Chain Reaction (PCR)

Total RNA was extracted from the kidney using the RNAlater (Thermo Fisher Scientific, Waltham,
MA, USA) and the Sepasol-RNA I Super G reagent (Nacalai Tesque) according to the manufacturer’s
instructions. The cDNA was synthesized from 1 mg total RNA using ReverTra Ace qPCR RT Master
Mix (Toyobo, Osaka, Japan). Real-time PCR was performed with the SsoAdvanced SYBR Green
Supermix (Bio-Rad, Hercules, CA, USA) on a Mini-Opticon (Bio-Rad) according to the manufacturer’s
protocols. All data were normalized to the level of the housekeeping gene β-Actin/Actb. The following
primers were used for the analysis: Actb, forward, 5 -GCC CTA GGC ACC AGG GTG TGA-3 , and
reverse, 5 -TCC TCA GGG GCC ACA CGC A-3 ; Epo, forward, 5 -TCA TCT GCG ACA GTC GAG
TTC TG-3 , and reverse, 5 -GGT ATC TGG AGG CGA CAT CAA TTC-3 ; and Vegfa, forward, 5 -GCA
GCT TGA GTT AAA CGA ACG-3 , and reverse, 5 -GGT TCC CGA AAC CCT GAG-3 .
2.6. Hematological Cytometry

The number of red blood cells and leukocytes, hemoglobin levels, and hematocrit levels were
measured by the Oriental Yeast hematology analyzing service (Tokyo, Japan).
2.7. Statistical Analyses

The statistical analyses were performed using the Student’s t-test for comparisons between two
groups and a one-way analysis of variance/Tukey’s test for comparisons of three or more groups.
Differences between the data were considered significant when the p-values were less than 0.05.
All data are expressed as the mean ± standard deviation (SD).
3. Results
3.1. Acai Extract Alters Hematological Parameters

First, to investigate the hematological effects, we administered acai extract and measured the
hematological parameters in mice. Treatment with acai extract significantly increased the erythrocyte
content (RBC), hemoglobin level (HGB), and hematocrit level (HCT) but not the leukocyte content
(WBC) or reticulocyte count (RET) in blood for four days (Table 2). Acai extract also induced no
significant change in the erythrocyte mean cell volume (MCV), mean cell hemoglobin (MCH), mean
cell hemoglobin concentration (MCHC), or platelet content (PLT) (Table 2). These results suggest that
acai induced a hemopoietic effect or suppressed red blood cell degradation.
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Nutrients 2020, 12, 533
Table 2. Effect of acai extract on the hematological parameters in mice (n = 4).
Control 10 g/kg of Acai for Four Days

WBC (/μL) 2500 ± 804 2275 ± 660
RBC (×104 /μL) 876 ± 11 931 ± 12 *
HGB (g/dL) 14.1 ± 0.5 14.9 ± 0.4 *
HCT (%) 47.6 ± 0.6 51.1 ± 1.4 *
MCV (fL) 54.4 ± 0.7 54.9 ± 0.8
MCH (pg) 16.0 ± 0.5 16.0 ± 0.4
MCHC (%) 29.5 ± 0.8 29.3 ± 0.9
PLT (×104 /μL) 95 ± 5 97 ± 13
RET (%) 27.8 ± 4.8 23.0 ± 1.2
Hematological parameters in blood treated with acai extract (10 g/kg) daily for four days orally. WBC: Leukocyte
content; RBC: Erythrocyte content; HGB: Hemoglobin; HCT: Hematocrit; MCV: Mean cell volume; MCH: Mean cell
hemoglobin; MCHC: Mean cell hemoglobin concentration; PLT: Platelet content; RET: Reticulocytes. * P < 0.05 by
t-test.
3.2. Acai Extract Acutely Upregulates the EPO Contents in Blood

EPO is the principle regulator of red blood cell production [12]. In general, the Epo expression is
transiently stimulated by a hypoxic condition [2]. After four days of administration of acai, the renal Epo
expression showed a slight increase (Figure 1A). In this context, we performed a transient experiment,
administering acai extract to mice and measuring the EPO contents in plasma 2–3 h after treatment.
The acai treatment caused a significant increase in the plasma EPO level compared with vehicle control
(Figure 1B). Furthermore, acai upregulated the Epo transcript level in the kidney compared with the
control (Figure 1C). The erythropoiesis inducer roxadustat (ASP1517), which is also used to treat renal
anemia, also upregulated both the EPO contents in plasma and the Epo transcript level in kidney
(Figure 1B,C). In contrast to acai, the administration of C3Glc caused no significant change in either the
EPO contents or the Epo level (Figure 1B,C). Furthermore, the relationship between the plasma EPO
concentration and the kidney Epo transcript level was positive (Figure 1D). These results suggest that
acai extract transcriptionally induced EPO production in the kidney.
Figure 1. Acai extract upregulates both the plasma erythropoietin (EPO) concentration and kidney Epo
expression. (A). The relative Epo transcript level in the kidney after the oral administration of acai extract
42
Nutrients 2020, 12, 533
(10 g/kg) dairy for four days. * P < 0.05 by t-test. (B) The plasma EPO concentration in mice 2–3 h after
the oral administration of acai extract (10 g/kg), C3Glc (50 mg/kg), and ASP1517 (80 mg/kg). (C) Relative
Epo transcript levels in kidney 2–3 h after the oral administration of acai extract (10 g/kg), C3Glc
(50 mg/kg), and ASP1517 (80 mg/kg). (D) Relationship between the plasma EPO concentration and Epo
transcription in kidney. Error bars indicate the standard deviation. * P < 0.05 by an ANOVA/Tukey’s test.
3.3. Acai Extract Induces a Renal Hypoxic Condition

Finally, to investigate the relationship between EPO induction and renal hypoxia under acai
administration, we administered the hypoxic probe pimonidazole to mice and histologically detected
renal hypoxia. By immunostaining with pimonidazole, which accumulates in hypoxic areas, acai extract
was shown to induce renal hypoxia (Figure 2A). A quantitative analysis with the fluorescence intensity
of pimonidazole staining showed that acai created a significantly larger hypoxic area than was seen in
the controls (Figure 2B,C). Furthermore, acai induced renal hypoxia mainly at the corticomedullary
junction where erythropoietin is produced (Figure 2A,B). Along with hypoxia induction, acai also
increased the expression of Vegfa, which located downstream of HIF, in the kidney (Figure 2D).
These results suggest that renal erythropoietin production caused by acai depends on the hypoxic
reaction in the kidney.
Figure 2. Acai extract induces a hypoxic reaction in kidney. (A–C) The pimonidazole-positive hypoxic
area in kidney sections 2–3 h after the oral administration of acai extract (10 g/kg) was detected by
3,3 -diaminobenzidine (A) and fluorescein isothiocyanate (B,C). (D) Relative Vegfa transcript level in
the kidney 2–3 h after acai extract. Scale bar denotes 1 mm. Error bar indicates the standard deviation.
* P < 0.05 by t-test.
4. Discussion
Since EPO, a hematopoietic hormone that controls erythropoiesis, is produced in response to tissue
hypoxia, athletes often incorporate high-altitude training. The induction of EPO expression mainly
involves the transcription factor HIF induced by hypoxia [3]. Under hypoxic conditions, degradation
of HIF is inhibited by ubiquitination due to the suppression of proline hydroxylase, thereby promoting
hematopoiesis [3]. ASP1517 induces the production of erythrocytes via the prevention of HIF
degradation by inhibiting the proline hydroxylase [13]. Actually, the remarkable induction of renal
hypoxia by acai suggests the HIF-mediated Epo and Vegfa upregulation (Figures 1 and 2).
43
Nutrients 2020, 12, 533
In our study, acai administration did not increase RET, which is in contrast to the increases it
induced in erythropoietin and HCT (Table 2). Testosterone supplementation improved anemia in
aged mice without increasing the number of reticulocytes, suggesting the contributions of an increase
in erythrocyte-related genes in spleen and the normalization of iron homeostasis [14]. Since acai
contains a large amount of iron, it can also increase the ferritin and iron contents, leading to iron-related
hematopoiesis. As EPO is transiently induced by hypoxic conditions [2], the upregulation of EPO
by acai may affect acute erythropoiesis. These findings suggest that, in contrast to the HIF-mediated
erythropoiesis mechanism of ASP1517, acai may exert multiple hematopoietic mechanisms. Since the
combination of EPO-stimulating and iron agents is an important anemia management strategy for
patients undergoing hemodialysis [15], acai has potential applications in therapy for various types of
anemia. We need to analyze the mechanisms underlying the hematopoietic effect of acai more strictly
in future studies.
Acai contains large amounts of iron, various polyphenols, and long-chain fatty acids, such as oleate,
palmitate and linoleate (Table 1), suggesting various beneficial effects for anemia. The monounsaturated
oleic acid depresses the cytokine expression in vitro [16]. Stoner et al. reported that acai reduced
the serum IL-5 and IL-8 levels in rats [17]. Anemia in the elderly population is caused by the
increased expression of proinflammatory cytokines [18]. Acai may protect against anemia by reducing
the levels of inflammatory cytokines increased by aging and various stressors. Furthermore, acai
treatment attenuated renal ischemia/reperfusion injury via protection against renal oxidative stress
in diabetic and spontaneously hypertensive rats [19,20]. In a rat study, acai treatment significantly
decreased the 8-isoprostane immunostaining level and thiobarbituric acid reactive substances level
in the kidney [20], suggesting the transition from an oxidative to a reductive condition in the kidney.
These findings show that acai induces erythropoietic action via an altered redox status in the kidney.
Mitochondrial angiotensin II receptors regulate oxygen consumption in kidney mitochondria [21],
suggesting a deep association with the renal status and oxygen consumption. Hernandez-Vargas et al.
reported that the suppressors of cytokine signaling (SOCS) family negatively regulated the angiotensin
II-activated signal pathway, including JNK, resulting in a decreased renal oxygen consumption [22].
AICAR, an AMPK activator, decreased the renal oxygen consumption via the modulation of SOCS1 [23].
In this context, acai may inhibit the SOCS family, leading to the activation of the angiotensin II-mediated
pathway associated with increased oxygen consumption and hypoxia in the kidney.
Polyphenols protect against reactive oxygen species-induced hemolysis via increased red blood
cell integrity associated with the inhibition of lipid peroxidation [24–27]. Similarly, red cabbage
extracts rich in anthocyanins rescue oxidative hemolysis in streptozotocin-induced diabetes [28].
Anthocyanins also exert antisickling activity by stabilizing red blood cells and their membranes
and inhibiting polymerization on hemoglobin S [29,30]. However, C3Glc alone did not alter the
erythropoietin or Epo levels (Figure 2), suggesting that not only polyphenols but also iron and fatty
acids of acai contribute to erythropoiesis.
Acai showed no toxicity in experimental models [31–36] nor any significant differences in the
body weight or food consumption [17,31,32,36], suggesting potential applications in the prevention of
various disease. Conversely, Mn, which is abundant in acai, suppresses Fe absorption, suggesting a
risk for anemia [37]. The kidneys are prone to hypoxia because of their high energy consumption [38].
Chronic renal hypoxia is a final common pathway to end-stage renal failure, resulting in an irreversible
decline in the renal function [39,40]. In a study of hypertensive rats, acai treatment for 45 days decreased
the creatinine contents in the serum and urine, suggesting a protective effect on the kidney [20]. Since we
only performed the acai trials for up to four days, a more detailed evaluation of the effects of longer-term
treatment on the hematopoietic activity and renal function is needed. EPO formulations are presently
banned as a doping substance. The erythropoietic mechanism of acai via EPO must be further clarified,
and its use in sports needs to be carefully discussed.
In summary, acai induces an erythropoietic effect associated with renal hypoxia. These findings
provide valuable insight into the potential utility of acai for future research on hematopoiesis in humans.
44
Nutrients 2020, 12, 533
Author Contributions: S.S., T.T., and T.S. designed the study; S.S. and T.S. wrote the manuscript; S.S., T.T., M.J.V.Y.,
and Y.O. performed the experiments; S.S. analyzed the data; T.S. edited the article; and T.S. coordinated and
directed the project. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by FRUTA FRUTA, Inc.
Conflicts of Interest: This does not alter the authors’ adherence to all Nutrients policies on sharing data
and materials.
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38. Gallagher, D.; Belmonte, D.; Deurenberg, P.; Wang, Z.; Krasnow, N.; Pi-Sunyer, F.X.; Heymsfield, S.B.
Organ-tissue mass measurement allows modeling of REE and metabolically active tissue mass. Am. J. Physiol.
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Rev. Nephrol. 2010, 6, 667–678. [CrossRef]
40. Nangaku, M. Chronic hypoxia and tubulointerstitial injury: A final common pathway to end-stage renal
failure. J. Am. Soc. Nephrol. 2006, 17, 17–25. [CrossRef]
47
nutrients
Article
Beneficial Effect of Ubiquinol on Hematological and
Inflammatory Signaling during Exercise
Javier Diaz-Castro 1,2, *, Jorge Moreno-Fernandez 1,2 , Ignacio Chirosa 3 , Luis Javier Chirosa 3 ,
Rafael Guisado 4 and Julio J. Ochoa 1,2
1 Institute of Nutrition and Food Technology “José Mataix”, University of Granada, Biomedical Research
Centre, Health-Sciencies Technological Park, Avenida del Conocimiento s/n, Armilla, E-18071 Granada,
Spain; jorgemf@ugr.es (J.M.-F.); jjoh@ugr.es (J.J.O.)
2 Department of Physiology, University of Granada, E-18071 Granada, Spain
3 Departament of Physical Education, University of Granada, E-18071 Granada, Spain; ichirosa@ugr.es (I.C.);
lchirosa@ugr.es (L.J.C.)
4 Faculty of Health Sciences, University of Granada, E-18071 Granada, Spain; rguisado@ugr.es
* Correspondence: javierdc@ugr.es; Tel.: +34-958-24-10-00 (ext. 20303)
Abstract: Strenuous exercise (any activity that expends six metabolic equivalents per minute or more
causing sensations of fatigue and exhaustion to occur, inducing deleterious effects, affecting negatively
different cells), induces muscle damage and hematological changes associated with high production of
pro-inflammatory mediators related to muscle damage and sports anemia. The objective of this study
was to determine whether short-term oral ubiquinol supplementation can prevent accumulation of
inflammatory mediators and hematological impairment associated to strenuous exercise. For this
purpose, 100 healthy and well-trained firemen were classified in two groups: Ubiquinol (experimental
group), and placebo group (control). The protocol was two identical strenuous exercise tests with
rest period between tests of 24 h. Blood samples were collected before supplementation (basal value)
(T1), after supplementation (T2), after first physical exercise test (T3), after 24 h of rest (T4), and after
second physical exercise test (T5). Hematological parameters, pro- and anti-inflammatory cytokines
and growth factors were measured. Red blood cells (RBC), hematocrit, hemoglobin, VEGF, NO,
EGF, IL-1ra, and IL-10 increased in the ubiquinol group while IL-1, IL-8, and MCP-1 decreased.
Ubiquinol supplementation during high intensity exercise could modulate inflammatory signaling,
expression of pro-inflammatory, and increasing some anti-inflammatory cytokines. During exercise,
RBC, hemoglobin, hematocrit, VEGF, and EGF increased in ubiquinol group, revealing a possible
pro-angiogenic effect, improving oxygen supply and exerting a possible protective effect on other
physiological alterations.
Keywords: high intensity exercise; ubiquinol; hematological parameters; inflammation; ergogenic effect
1. Introduction
There are multiple beneficial effects associated with regular and planned exercise [1,2], including the
reduction in the age-related changes in nuclear pore complex proteins, protection of the neuromuscular
junction, and the increase in the lives of susceptible motoneurons, preserving neuromuscular integrity
and innervation status [3,4]. In addition, exercise increases blood flow and improves vascular integrity,
enhancing angiogenesis, resulting in reversal of rarefaction and hypertension, and enhancement of
cerebral blood flow and cognition [5].
On the other hand, strenuous exercise can induce a range of adverse effects including oxidative
stress, hematological changes, and inflammatory response involved in activating catabolic pathways
inducing muscle damage [6]. Strenuous exercise (defined as any activity that expends six metabolic

Nutrients 2020, 12, 424
equivalents (METS) per minute or more [7]) is harmful to health [8], because it causes structural
damage to muscle cells indicated by muscle soreness and swelling, prolonged loss of muscle function,
increased free radicals output, induction of the pro-inflammatory signaling, impairment of immune
functions, including the immunoglobulin production or T-cell function, and leakage of muscle proteins
into circulation, among other effects [9,10].
The aerobic energy metabolism during strenuous exercise (established when sensations of fatigue
and exhaustion occur, inducing deleterious effects, affecting negatively different cells) plays a crucial role
on the performance. Hematological changes such as decreases in the hemoglobin (Hb) concentrations
and RBC counts are often found to result from participation in strenuous exercise [11]. In this sense,
in athletes performing high intensity exercise, a high prevalence of “sports anemia” or iron deficiency
anemia induced by strenuous exercise has been reported [12,13], which has been associated with
hematological changes such as a decrease in RBC, hemoglobin, and hematocrit [14,15]. These changes
are suggested to be mainly caused by iron deficiency and a negative iron balance caused by intense
physical exercise [16].
Within the oxygen transport chain, RBC mass is crucial for oxygen supply to working muscles which
could regulate the aerobic performance capacity [17]. However, although during the strenuous exercise
there is a greater erythropoietic activity, an important loss of RBC is featured [14,18]. During exercise
an inflammatory state is featured and a high production of free radicals, both factors affecting iron
metabolism [18] and by a direct effect on the iron reduction due to its affinity for H2 O2 [13]. In addition,
skeletal muscle is a highly regenerative tissue, but muscle repair potential is limited, and inflammatory
signaling contributes to muscle repair [19], however the pro-inflammatory cytokines can also be
deleterious to health. Therefore, these cytokines are both cause and effect of inflammation [20].
Elevated levels of these inflammatory markers not only increase risk for chronic diseases, but also
contribute to disease pathogenesis [21]. In addition, the composition of the serum microbiome is linked
to indices of inflammation, altering immunity [22].
Athletes may be susceptible to a heightened anti-inflammatory state. These results can transiently
suppress immune function and increase the risk of infection [23]. In this sense, reducing inflammation
has been recognized as one of the ways to reduce the risk of chronic disease [24].
During regeneration, reconstitution of muscle fibers, RBC mass, and blood supply is imperative
for full muscular recovery and prevention of muscle atrophy [25]. In this sense, vascular endothelial
growth factor (VEGF) has a key role for angiogenesis and muscle fibers repair in skeletal muscle and
has been shown to be upregulated by a single bout of dynamic exercise [26].
On the other hand, inflammation, angiogenesis, RBC mass, and oxygen supply have a key role on
muscle damage associated to high intensity exercise and other physiological alterations that can affect
physical performance. Therefore, it would be interesting to assess the effect of oral supplementation
with a substance capable of improving hematological parameters and diminishing inflammatory
signaling associated to this performance [27,28], however scarce studies are available about molecules
with these characteristics to support the regeneration process of skeletal muscle after strenuous exercise.
One of these substances could be coenzyme Q10 (CoQ10) [29].
The data available in the scientific literature have provided a direct link between physical
performance and blood and muscle tissue CoQ10 levels [30]. However, most of these studies
are focused mainly on the exercise performance and radical-scavenging activity of CoQ10 during
low intensity exercise [30], with the studies about the influence of CoQ10 supplementation during
the performance of high intensity (strenuous) exercise on the inflammatory signaling, hematology,
and muscle recovery after strenuous exercise being scarce. CoQ10 exists in two forms: Ubiquinone
(oxidized form), the most common form in CoQ10 supplements, and ubiquinol, the reduced and most
active form, which has properties related to bioenergetic and antioxidant activity [31,32], but poorly
studied. Therefore, we aimed to determine whether, a short term oral ubiquinol supplementation may
be efficient ameliorating the pro-inflammatory effects, improving hematological parameters, effects
that could promote skeletal muscles regeneration and oxygen supply after strenuous exercise.
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Nutrients 2020, 12, 424
2.1. Subjects and Supplementation Protocol

This study was a randomized, double-blind, and placebo-controlled trial. One hundred healthy
and well trained, but not on an elite level, firemen of the Fire Department of the City of Granada
were taking part in this study. Participants completed a medical and health history and physical
activity questionnaire (IPAQ-SF) [33] prior to enrolment. All of them were nonsmokers, did not take
any nutritional supplements and did not present febrile/inflammatory clinical symptoms, did not
use immunosuppressive or nephrotoxic drugs, did not use energy, protein, and/or antioxidant
supplements. The firemen were randomly divided into two groups: Ubiquinol group (ubiquinol)
(n = 50), and placebo group (control) (n = 50). The ubiquinol group was supplemented with an oral
dose of 200 mg/day of ubiquinol during two weeks, administrating two brown liquid filled hard
gelatine capsules of 100 mg/day, and subjects assigned to the control group took placebo using the same
scheme. The capsules Kaneka QH ubiquinol (Kaneka Corporation, Osaka, Japan) contained 100 mg
of ubiquinol in a basis of canola oil, diglycerol monooleate, beeswax, and soy lecithin. The placebo
capsules contained the same composition without ubiquinol and were also supplied by Kaneka
(Kaneka Corporation, Osaka, Japan). The study was approved by the Commission of Ethics in Human
Research of the University of Granada (ref. 804). The study has been registered in ClinicalTrials.gov,
with number NCT01940627. Informed consent was obtained from all subjects with written consent to
participate in this study.
2.2. Strenuous Exercise Performance Programme

Characteristics, intensity, and muscle damage (loss of skeletal muscle function and soreness) of
this protocol was previously reported by measuring blood myoglobin and CK [32] and similar increases
have been observed in other strenuous exercise tests [34]. After two-weeks period of ubiquinol or
placebo supplementation, subjects performed the strenuous exercise protocol in order to induce muscle
damage. Prior to the starting of each test, subjects performed a warm-up which was divided into two
phases: General activation phase and specific phase. The protocol consisted of conducting two identical
strenuous exercise tests, with a rest period between tests within 24 h. Circuit weight training (CWT),
characterized by alternating exercises between upper- and lower-body segments performed at stations,
has been widely used in practice settings. The main advantage of this method is that it allows faster
training performance. Moreover, it is commonly used for persons interested in weight management,
although a previous study showed similar effects on body composition and on muscular strength
and size in trained men after CWT and multiple-set resistance training [35]. The rest time between
sets was five minutes to allow recovery and complete the designed workout [35]. Both strenuous
exercise tests consisted of performing a circuit composed of 10 bodybuilding exercises (1. athletic
press; 2. chest press in Smith Machine; 3. seated oar; 4. shoulders press; 5. femoral biceps flexion;
6. chest press in Smith Machine; 7. step with weight; 8. surveyor’s pole chest; 9. shove with weight;
10. quadriceps extension) [32]. In order to establish the minimum magnitude of the load to be displaced
for each subject, one week before strenuous exercise protocol, a session of pre-training was held with
the subjects to conform the load individually in terms of two parameters in each exercise: (a) Scale
OMNI-RES [36] values of perceived exertion between 6–7, and (b) 10 repetitions.
2.3. Blood Sampling

Blood samples were collected from the participants by venous catheter into heparinized tubes
before and immediately after the physical test. Five blood samples and urine were taken: Before
supplementation (basal value) (T1), after supplementation (two weeks) (T2), after first physical exercise
test (T3), after 24 h of rest (T4), after second physical exercise test (T5). One aliquot of blood was
collected in tubes with an EDTA anticoagulant for hematological analysis. The remaining blood was
immediately centrifuged at 1750 g for 10 min at 4 ◦ C in a Beckman GS-6R refrigerated centrifuge
51
Nutrients 2020, 12, 424
(Beckman, Fullerton, CA, USA) to separate plasma from red blood cell pellets. Plasma samples were
immediately frozen and stored at −80 ◦ C until analysis.
2.4. Inflammatory Parameters

Epidermal growth factor (EGF), interferon gamma (IFN-γ), vascular endothelial growth factor
(VEGF), monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor alpha (TNF-α), interleukin
(IL)-1, IL-1ra (receptor agonist), IL-6, IL-10, and IL-15 were determined using the HCYTOMAG-60K
Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore Corporation, Missouri,
USA), based on immunoassays on the surface of fluorescent-coded beads (microspheres), following
the specifications of the manufacturer (50 events per bead, 50 μL sample, gate settings: 8000–15,000,
time out 60 s, melatonin bead set: 34). The plate was read on a LABScan 100 analyzer (Luminex
Corporation, Austin, TX, USA) with xPONENT software for data acquisition. With these biomarkers
we characterize mediators of adaptive immunity, mediators of innate immunity and inflammation,
chemotaxis, haematopoietic mediators and growth factors, allowing us to have an overview of the
various pathways of cytokines in the immune and inflammatory process. Average values for each set
of duplicate samples or standards were within 15% of the mean. Cytokines concentrations in plasma
samples were determined by comparing the mean of triplicate samples with the standard curve for
each assay.
2.5. Hematological Parameters

Hemoglobin (Hb) concentration, red blood cells (RBC), hematocrit, mean corpuscular volume
(MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), red cell distribution
width (RDW), platelets, mean platelets volume (MPV), leukocytes, neutrophils, lymphocytes,
monocytes, eosinophils, basophils of fresh blood samples were measured using an automated
hematology analyzer Mythic 22CT (C2 Diagnostics, Grabels, France).

All data are presented as the mean ± standard error of the mean (SEM). All variables were
tested to see if they followed the criteria of normality and homogeneity of variance using the
Kolmogorov–Smirnoff’s and Levene’s tests, respectively. To compare general characteristics of the
subjects in both experimental groups, unpaired Student’s t-test was used. To assess the effect of the
supplementation and the evolution in the time of each variable studied in each experimental group a
general linear model of variance for repeated measures with an adjustment by means of Bonferroni´s
test has been performed. Bonferroni’s test allowed us to know intra- and inter-subject differences
(effect of time in each group and supplementation in each period, respectively) in a very robust way in
terms of power. A value of p < 0.05 was considered significant. For data analysis we used the SPSS
version 20.0 (SPSS Statistics for Windows, 20.0.0. SPSS INC. Chicago, IL, USA).
3. Results
No statistically significant differences between both groups were found for weight, age, height,
and BMI (Table 1). In addition, no significant differences were recorded between groups for the short
form of the International Physical Activity Questionnaire [32]. The subjects of both experimental groups
were categorized as “Health Enhancing Physical Activity” (HEPA Active): Category 3, the highest
measurement threshold of total physical activity of the questionnaire. In addition, as we have previously
reported [32], the high intensity protocol induces muscle damage based on lactate output (increase of
290% (2.9 ± 0.1 vs. 8.5 ± 0.3 mmol/L) after the first exercise session and an increase of 355% (2.9 ± 0.1
vs. 10.5 ± 0.5 mmol/L) after the second training session, myoglobin increased 358% higher after the
first session (25.7 ± 3.2 vs. 92.1 ± 7.9 ng/mL) and 387% after the second session (25.7 ± 3.2 vs. 99.3 ± 6.8
ng/mL) and creatine kinase (CK-MM) increased 158% after the first exercise test (2.01 ± 0.3 vs. 3.2 ± 0.4
ng/mL) and 196% after the second session (2.01 ± 0.3 vs. 4.0 ± 0.3 ng/mL). We have also reported that
52
Nutrients 2020, 12, 424
ubiquinol supplementation increased plasma CoQ10 levels 522% (1.00 ± 0.06 vs. 5.22 ± 0.41 mmol/L).
The dropout percentage was similar in both groups (24% after finishing the first test and 32% after
finishing the second exercise test) and neither differences were observed between dropout reasons in
both groups [32].
Table 1. Subjects baseline characteristics.
Age Height Weight BMI SBP DBP RHR

(Years) (cm) (kg) (kg/m2 ) (mmHg) (mmHg) (Beats/min)
Ubiquinol 38.9 ± 1.4 175.4 ± 0.8 76.8 ± 1.5 25.0 ± 0.4 137.0 ± 2.2 81.4 ± 1.5 57.4 ± 1.8
Control 38.2 ± 1.2 174.5 ± 1.2 76.3 ± 2.0 25.0 ± 0.5 134.1 ± 2.1 79.1 ± 1.9 57.1 ± 1.5
Data expressed as the mean ± SEM. BMI: Body mass index; SBP: Systolic blood pressure; DBP: Diastolic blood
pressure; RHR: Resting heart rate.
IL-1 was lower in ubiquinol compared with the control group in T3. Regarding the evolution,
IL-1 increased in the ubiquinol and control group in T5 compared with T1, T2, T3, and T4 (Figure 1A).
No differences were observed in IL-1ra due to the supplementation. Regarding the evolution, IL-1ra
increased in the ubiquinol group in T5 compared to T4, T2, and T1 (Figure 1B). No differences were
observed in IL-6 due to the supplementation. Regarding the evolution, IL-6 increased in the ubiquinol
group in T3 and T5 compared with T1, in T3 and T5 compared with T2, and also in T5 compared with
T4, while decreased in T4 compared with T3 (Figure 1C). IL-8 was lower in the supplemented group in
T3 compared with the control group. Regarding the evolution, in the ubiquinol group, IL-8 decreased
in T4 with respect to T2, T3, and T5. In the control group, there was an increase in T3 with respect to
T1, T2, and T4, and also increased in T5 with respect to T1 and T4 (Figure 1D). IL-10 was higher in
ubiquinol compared with the control group in T5 (Figure 1E). Regarding the evolution, IL-10 increased
in the ubiquinol group in T3 compared with T1 and T5, and also increased in T5 with respect to T1 and
T4 while no differences by the evolution of time were observed in the control group. No differences
were observed in IL-15 due to the supplementation. Regarding the evolution, IL-15 decreased in T4
compared with T3 in the control group (Figure 1F).
53
Nutrients 2020, 12, 424
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Figure 1. Effects of exercise and ubiquinol supplementation on plasma cytokines: interleukin (IL)-1
(A), IL-1ra (B), IL-6 (C), IL-8 (D), IL-10 (E), IL-15 (F). Results are expressed as the mean ± SEM. * means
statistically significant differences between groups (p < 0.05). T1: Before supplementation (basal value);
T2: After supplementation (two weeks) and before the first physical test; T3: After first physical exercise
test; T4: After 24 h of rest and before the second physical test; T5: After second physical exercise test.
Different letters in every group indicates significant differences due to the time (control (A, B, C, D, E);
ubiquinol (a, b, c, d, e)) (p < 0.05).
No differences were observed in TNF-α due to the supplementation. Regarding the evolution,
TNF-α increased in the ubiquinol group in T3 compared with T1 and T4. In the control group, TNF-α
increased in T3 compared with the rest of the blood samples and also increased in T5 compared with T1
and T4 (Figure 2A). No differences were observed in IFN-γ due to the supplementation or evolution of
time (Figure 2B). A higher level of EGF was observed in the ubiquinol compared with the control group
in T2 and T3. Regarding the evolution, EGF increased in the ubiquinol group in T2 compared with
T1 and in T3 compared with T1, T4, and T5. In the control group, EGF was higher in T1 with regard
to T1, T2 and also increased in T4 with regard to T1 (Figure 2C). VEGF was higher in the ubiquinol
compared with the control group in T3. VEGF increased in the ubiquinol group in T5 compared
with T1 and T2, while no differences by the evolution of time were observed in the control group
(Figure 2D). MCP-1 decreased in ubiquinol compared with the control group in T3 and T5. Regarding
the evolution, MCP-1 increased in the ubiquinol group in T3 compared with T1, and in T5 compared
with T4, while decreased in T4 compared with T2, and in T4 compared with T3. In the control group,
MCP-1 increased in T2, T3, and T5 compared with T1, increased in T3 compared with T2, and in T5
compared with T4, while showed a decrease in T4 compared with T2, and in T4 and in T5 compared
with T3 (Figure 2E).
54
Nutrients 2020, 12, 424
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factor alpha (TNF-α) (A), interferon gamma (IFN-γ) (B), epidermal growth factor (EGF) (C), vascular
endothelial growth factor (VEGF) (D), monocyte chemotactic protein 1 (MCP-1) (E). Results are
expressed as mean ± SEM. * means statistically significant differences between groups (p < 0.05). T1:
Before supplementation (basal value); T2: After supplementation (two weeks) and before the first
physical test; T3: After first physical exercise test; T4: After 24 h of rest and before the second physical
test; T5: After second physical exercise test. Different letters in every group indicates significant
differences due to the time (control (A, B, C, D, E); ubiquinol (a, b, c, d, e)) (p < 0.05).
RBC increased in the ubiquinol compared with the control group in T3 and T4. Regarding the
evolution, RBC increased in the ubiquinol group in T2 and T3 compared with T1 and T5, while decreased
in the control group in T4 and T5 compared with T1 (Figure 3A). Hemoglobin levels were higher in
ubiquinol compared with the control group in T3 and T4. In the control group, hemoglobin decreased
in T4 compared with T1, T2, T3, and T5 (Figure 3B). Hematocrit increased in the ubiquinol compared
with the control group in T3. Hematocrit decreased in T4 and T5 compared with T1 in the control
group (Figure 3C).
Leukocytes increased in the ubiquinol group in T3 compared with T1 and T4. Neutrophils were
higher in the ubiquinol compared with the control group in T4, also increased in the control group in
T5 compared with T1, T2, and T4 and also in T3 and T5 in regard to T1, T2, and T4 in the ubiquinol
group. Lymphocytes decreased in the ubiquinol group in T4, in T5 compared with T1 and T2 in the
ubiquinol group and in T3, and also in T5 compared with T4 in the control group. Monocytes were
lower in the ubiquinol compared with the control group in T4, also decreased in T2, T3, T4, and T5
compared with T1 in the control and ubiquinol group. Eosinophils decreased in T3 compared with
T1 in the control and ubiquinol group. Basophils decreased in T4 and T5 compared with T1 in the
55
Nutrients 2020, 12, 424
ubiquinol group and increased in T5 compared with T1 and T4 in the control group. No changes were
recorded in platelets during the study, MCV or MCH nor due to exercise and neither to ubiquinol.
MCHC increased in the ubiquinol compared with the control group in T5 and also increased in T4 and
T5 compared with T1 and T2 in the ubiquinol group, while decreased in T3, T4, and T5 compared with
T1 in the control group. No significant changes were recorded in RDW and MPW due to exercise and
neither to ubiquinol (Table 2).
Figure 3. Effects of exercise and ubiquinol supplementation on hematological parameters: Red blood
cells (A), hemoglobin (B), hematocrit (C). Results are expressed as mean ± SEM. * means statistically
significant differences between groups (p < 0.05). T1: Before supplementation (basal value); T2: After
supplementation (two weeks) and before the first physical test; T3: After first physical exercise test; T4:
After 24 h of rest and before the second physical test; T5: After second physical exercise test. Different
letters in every group indicates significant differences due to the time (control (A, B, C, D, E); ubiquinol
(a, b, c, d, e)) (p < 0.05).
56
Nutrients 2020, 12, 424
Table 2. Effects of exercise and ubiquinol supplementation on hematological parameters. Results are
expressed as mean ± SEM. * means statistically significant differences between groups (p < 0.05). T1:
Before supplementation (basal value); T2: After supplementation (two weeks) and before the first
physical test; T3: After first physical exercise test; T4: After 24 h of rest and before the second physical
test; T5: After second physical exercise test. Different letters in every group indicates significant
differences due to the time (control (A, B, C, D, E); ubiquinol (a, b, c, d, e)) (p < 0.05).
T1 T2 T3 T4 T5
Leukocytes
(103 /μL)
Control 5.9 ± 0.2 A 5.9 ± 0.2 A 6.2 ± 0.2 A 5.9 ± 0.2 A 6.2 ± 0.2 A
Ubiquinol 5.8 ± 0.2 a,c 6.1 ± 0.2 a,b,c 6.8 ± 0.3 b 5.9 ± 0.2 c 6.4 ± 0.3 a,b,c
Neutrophils
(%)
Control 52.0 ± 1.1 A,C 52.0 ± 1.1 A,C 57.1 ± 1.3 B 52.0 ± 1.1 C 56.0 ± 1.1 B
Ubiquinol 59.9 ± 1.1 a 54.2 ± 1.0 b 57.9 ± 1.0 c 56.5 ± 1.5 b,c,d, * 60.1 ± 1.5 d
Lymphocytes
(%)
Control 34.7 ± 1.1 A,B,C,D 35.4 ± 0.9 A,C,D 32.1 ± 1.3 B,D 36.8 ± 1.0 C 32.8 ± 1.3 D
Ubiquinol 36.2 ± 1.1 a 34.5 ± 1.1 a,b 32.3 ± 1.0 b,c 33.6 ± 1.3 a,b,c, * 30.6 ± 1.3c
Monocytes
(%)
Control 8.1 ± 0.3 A 7.1 ± 0.3 B 6.6 ± 0.3 B 7.4 ± 0.3 A,B 6.7 ± 0.3 B
Ubiquinol 8.5 ± 0.3 a 7.0 ± 0.3 b 6.5 ± 0.3 b 6.6 ± 0.3 b, * 6.4 ± 0.3 b
Eosinophils
(%)
Control 4.8 ± 0.5 A 4.5 ± 0.6 A,B 3.5 ± 0.5 B 4.1 ± 0.5 A,B 3.8 ± 0.5 A,B
Ubiquinol 4.1 ± 0.4 a,c 3.6 ± 0.3 a,c 2.7 ± 0.3 b 3.5 ± 0.3 c 3.2 ± 0.3 a,b,c
Basophils
(%)
Control 0.4 ± 0.04 A,C 0.6 ± 0.05 B,C,D 0.7 ± 0.04 C,D 0.5 ± 0.04 A,B 0.7 ± 0.07 D
Ubiquinol 0.4 ± 0.03 a 0.7 ± 0.05 b 0.6 ± 0.04 b 0.5 ± 0.04 c 0.6 ± 0.06 b,c
Platelets
(103 /μL)
251.6 ± 12.6
Control 240.0 ± 6.3 A 232.9 ± 6.1 A 248.9 ± 7.9 A 231.9 ± 6.6 A A
Ubiquinol 239.9 ± 5.8 a 239.8 ± 7.0 a 250.3 ± 6.2 a 244.7 ± 6.6 a 244.1 ± 5.6 a
MCV (fL)
Control 90.4 ± 0.5 A 90.5 ± 0.6 A 90.5 ± 0.6 A 90.8 ± 0.7 A 90.5 ± 0.7 A
Ubiquinol 90.4 ± 0.7 a 90.5 ± 0.7 a 90.7 ± 0.7 a 90.4 ± 0.8 a 90.8 ± 0.8 a
MCH (pg)
Control 31.9 ± 0.3 A 32.0 ± 0.3 A 32.3 ± 0.3 A 32.3 ± 0.3 A 32.3 ± 0.3 A
Ubiquinol 32.0 ± 0.3 a 32.1 ± 0.3 a 32.3 ± 0.3 a 32.3 ± 0.3 a 32.7 ± 0.3 a
MCHC
(g/dL)
Control 35.3 ± 0.1 A 35.4 ± 0.1 A,B 35.6 ± 0.1 B 35.6 ± 0.1 B 35.6 ± 0.1 B
Ubiquinol 35.4 ± 0.1 a 35.4 ± 0.1 a 35.6 ± 0.1 a,b 35.7 ± 0.1 b 36.0 ± 0.1 c, *
RDW (%)
Control 13.2 ± 0.1 A 13.1 ± 0.1 A 13.1 ± 0.1 A 13.2 ± 0.1 A 13.2 ± 0.1 A
Ubiquinol 13.3 ± 0.1 a 13.2 ± 0.1 a,b 13.0 ± 0.1 b 13.2 ± 0.1 a,b 13.1 ± 0.1 a,b
MPV (fL)
Control 8.6 ± 0.1 A 8.8 ± 0.1 A 8.7 ± 0.1 A 8.7 ± 0.1 A 8.7 ± 0.1 A
Ubiquinol 8.4 ± 0.1 a 8.6 ± 0.2 a 8.4 ± 0.1 a 8.5 ± 0.1 a 8.5 ± 0.1 a
MCV: Mean corpuscular volume; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular hemoglobin
concentration; RDW: Red cell distribution width; MPV: Mean platelet volume.
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4. Discussion
Regular physical exercise is associated with numerous health benefits including a lower risk of
all-cause mortality [37,38], nevertheless, strenuous exercise specially in amateur athletes, promotes
the generation of oxidative stress and a pro-inflammatory state, which are one of the main reasons
for the muscular aggression observed in high intensity exercise, together with other physiological
alterations such as sports anemia [12], characterized by hematological changes including decrease
in RBC, hemoglobin, and hematocrit [12,13,15]. These changes reduce oxygen supply and energy
production which could regulate the aerobic performance capacity [17], which could lead to a reduction
in the physical performance, incorrect adaptation to training protocols, and other possible physiological
alterations [12]. Taking into account the importance of inflammation in most of these alterations,
supplementation with effective molecules against these alterations could be beneficial. CoQ10 could
be suitable for a muscle-protective supplementation because it has anti-inflammatory and antioxidant
activity and it is intimately involved in energy production [39,40]. However, scarce studies of CoQ10
supplementation investigating its effects during strenuous physical exercise are available in the
scientific literature, especially in the field of the inflammatory signaling and hematological parameters
and virtually nonexistent when referring to supplementation with the reduced form of this molecule
(ubiquinol) [29,39]. Both groups studied were homogeneous in terms of weight, height, blood pressure,
and age. In addition, as commented above, we have previously reported that both groups showed
similar physical nutritional status and characteristics, and that our protocol of exercise features a high
intensity and induced muscle damage [32].
During intense exercise, muscles are damaged and reconstitution of muscle fibers is imperative
for full muscular recovery and prevention of muscle atrophy, being pivotal factors in this process the
inflammatory signaling and an adequate RBC mass and blood supply. The inflammatory response
during high intensity exercise initiates a rapid and sequential invasion of muscle fibers, mediating the
repairing process during recovery from strenuous exercise. Thus, the inflammatory response induced
by muscle damage may be a functionally response to favor muscle regeneration [41].
In response to exercise, skeletal muscle releases pro-inflammatory cytokines exponentially
according to the exercise intensity, duration, mass of muscle recruited, and endurance capacity [42].
Acute bouts of exercise cause transient damage to contracting skeletal muscles, triggering an
inflammatory response that increases the levels of pro-inflammatory cytokines and acute-phase
reactants in the blood [43], such as IL-6 and TNF-α which are pro-inflammatory cytokines primarily
secreted by stimulated immune cells (e.g., monocytes and macrophages) [44], nevertheless, although
IL-6 and TNF-α are pro-inflammatory agents, they can stimulate the production of an anti-inflammatory
cytokine such as IL-10 [45]. In the ubiquinol group, the increase in IL-6 is not preceded by an increase
in tumor necrosis factor alpha (TNF-α) and, most importantly, is followed by increased levels of
anti-inflammatory cytokines, namely IL-1 receptor antagonist (IL-1ra) and IL-10 [45,46], together with
a decrease in IL-8, as well as lower expression of Il-1, IL-15, and MCP-1. In turn, IL-10 inhibits the
synthesis of some pro-inflammatory cytokines such as TNF-α [47], a fact that can be observed especially
after the second physical test. TNF-α seems to have a biphasic effect on muscle: High levels of the
cytokine promote muscle catabolism, probably by a nuclear factor kB (NF-kB) mediated effect, whereas
low levels of TNF-α such as those recorded in the ubiquinol group do not induce NF-kB and stimulate
myogenesis [48].
During strenuous exercise, there is a reduction in RBC, which could imply a decrease in the
oxygen supply to the cells. On the other hand, previous investigations have reported that lower
cardiorespiratory fitness, assessed by maximal oxygen consumption (VO2 max kg−1 ), is associated with
higher basal IL-8 and MCP-1 concentrations [49,50]. High VO2 max kg−1 is correlated with low IL-8
levels [51], suggesting that low IL-8 levels is an important inflammatory parameter that predicts the
level of cardiorespiratory fitness. In the current study, ubiquinol also reduced MCP-1 (key chemokine
that regulates migration and infiltration of monocytes/macrophages in response to inflammation) and
IL-8. Interestingly, at physiological concentration, MCP-1 is the only adipocytokine able to impair
58
Nutrients 2020, 12, 424
insulin signaling and glucose uptake in skeletal muscle and in this sense, a decrease in this biomarker
during physical exercise facilitates insulin-mediated glucose disposal [52]. Therefore, we can assume
that rather than pro-inflammatory, the acute exercise-induced increase in pro-inflammatory cytokines
such as IL-6, in the ubiquinol group, may actually lead to an anti-inflammatory environment [53]
and also an improvement in physical performance due to a higher cardiorespiratory fitness and
glucose consumption.
Regarding the hematological changes associated with strenuous exercise, it is asserted that
exercise performed until exhaustion decreases the number of leukocytes, a fact that can be associated
with metabolic changes such as ischemia that occur during exercise and increased muscle activity
leads to a greater incidence of capillary swelling and leukocyte adherence to venules [54]. However,
as explained below, ubiquinol features a vasodilator activity and pro-angiogenic effect which avoids
capillary swelling and leukocyte adherence to blood vessels, reducing impairment of immune functions
related to strenuous exercise [55], avoiding this reduction in the leukocytes. Moreover, as mentioned
above, ubiquinol reduced MCP-1 that plays an important role in selectively recruiting monocytes and
lymphocytes [56], the reason why we recorded a decrease of these white cells after 24 h of the first
physical test.
As previously mentioned, in elite athletes, a decrease in the percentages of RBC, hemoglobin
and hematocrit is recorded and, in this situation, inflammation is one of the main causes. Even if
there is only a small decrease in RBC, it is important to highlight that during strenuous exercise a
greater demand for oxygen is necessary and therefore even a small decrease in RBC could affect the
performance. Thus, an adequate RBC mass and blood supply is imperative for full muscular recovery,
energy requirements, and prevention of muscle atrophy [25]. In this regard, there are three aspects that
have to be taken into account: The vasodilation, the angiogenesis or generation of new blood vessels,
and the RBC mass circulating, physiological pathways that are closely related to increase blood supply.
In the current study, ubiquinol prevented the decrease in RBC, hemoglobin, and hematocrit after the
first physical test, a fact that would increase oxygen delivering to tissues, especially muscles. During
exercise, oxidative stress induces deleterious structural and functional changes in RBC, however,
ubiquinol prevents those changes in erythrocytes, due to its antioxidant properties [29], avoiding
the impairment in hematological changes such as decreases in Hb and RBC caused by strenuous
exercise [11]. In addition, ubiquinol supplementation also increases NO output [32], a fact that is
beneficial during physical activity, featuring a vasodilator action that helps both exercise performance
and nutrient supply in muscle recovery, as well as improvement in the supply of substrates such as
glucose, together facilitate the regulatory role in the immune system [57]. This can be explained by
the link between VEGF and nitric oxide (NO). VEGF is a critical cytokine involved in angiogenesis,
and NO is a downstream effector. Importantly, recent studies suggest that NO is an essential mediator
of endothelial cells migration and VEGF-induced angiogenesis [58]. Angiogenesis is a crucial process
for effective regeneration not only by providing stable vessels for supporting the metabolic activity of
the regenerated tissue, but also by generating a new population of endothelial and periendothelial cells
that will supply a large array of molecules that sustain myogenesis [25]. In this sense, EGF promotes
growth and migration of vascular smooth muscle cells through activation of EGF receptor, and VEGF
promotes the generation of new blood vessels, essential processes during vascular remodeling and
muscular fibers reconstitution [59,60]. In our study, VEGF increased in the ubiquinol group, revealing
not only a vasodilator action of ubiquinol due to the NO, but also a pro-angiogenic effect, a fact that
would improve nutrient and oxygen supply and muscle recovery after strenuous exercise, supporting
and explaining the possible ergogenic effect of ubiquinol during intense exercise. On the other hand,
ubiquinol also increased EGF, probably due to its antioxidant activity [61]. VEGF and EGF increase
due to the ubiquinol supplementation can be directly linked with the muscle fibers regeneration after
intense exercise.
59
Nutrients 2020, 12, 424
5. Conclusions
In summary, the present study demonstrates a strong correlation between the high intensity
exercise and inflammatory signaling as shown by the overexpression in the pro-inflammatory cytokines.
In addition, the present findings provide evidence that oral supplementation of ubiquinol during
high intensity exercise could modulate the inflammatory signaling associated to exercise by reducing
the overexpression of pro-inflammatory cytokines, together with an increase in anti-inflammatory
cytokines which limits the detrimental, pro-inflammatory actions of strenuous exercise. In addition,
during exercise, RBC, hemoglobin, hematocrit, VEG, NO, and EGF did not decrease in the ubiquinol
group, revealing a possible pro-angiogenic effect, a fact that could improve nutrients and oxygen
supply and therefore muscle recovery after strenuous exercise. Therefore, the knowledge gained from
these findings reveals the benefit of ubiquinol supplement in athletes prior to the performance of
strenuous exercise in order to reduce the undesirable effects of the inflammation signaling during
high intensity exercise, increasing blood supply, reducing the muscle damage and hematological
impairment, and improving skeletal muscle fibers regeneration.
Author Contributions: J.J.O. designed the research proposal and provided funding. J.D.-C. and J.M.-F. conducted
the research and wrote the manuscript. I.C., L.J.C., and R.G. revised the data and the manuscript. All persons
designated as authors qualify for authorship, and all those who qualify for authorship are listed. All authors have
read and agree to the published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments: J.M.-F. was supported by a fellowship from the Ministry of Education, Culture and Sport
(Spain), and is grateful to the Excellence PhD Program “Nutrición y Ciencias de los Alimentos” from the University
of Granada. We would like to thank the Fire Department of Granada for their participation in the current study.
Funding for this work, ubiquinol, and placebo capsules were provided by (Kaneka Corporation, Osaka, Japan).
The authors are grateful to Susan Stevenson for her efficient support in the revision of the English language.
Conflicts of Interest: The investigators and the University of Granada have no direct or indirect interest in the
tested product (Kaneka QH) or in Kaneka Corporation and therefore the authors declare no conflict of interest.
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63
nutrients
Article
Iron Status of Infants in the First Year of Life in
Northern Taiwan
Chiao-Ming Chen 1,† , Shu-Ci Mu 2,3,† , Chun-Kuang Shih 4 , Yi-Ling Chen 3,5 , Li-Yi Tsai 3,6 ,
Yung-Ting Kuo 7,8 , In-Mei Cheong 1 , Mei-Ling Chang 1 , Yi-Chun Chen 4 and Sing-Chung Li 4, *
1 Department of Food Science, Nutrition, and Nutraceutical Biotechnology, Shih Chien University,
Taipei 10462, Taiwan; charming@g2.usc.edu.tw (C.-M.C.); mei11792004@gmail.com (I.-M.C.);
mlchang@g2.usc.edu.tw (M.-L.C.)
2 School of Medicine, Fu-Jen Catholic University, New Taipei City 24205, Taiwan; musc1006@gmail.com
3 Department of Pediatrics, Shin-Kong Wu Ho-Su Memorial Hospital, Taipei 11101, Taiwan;
Ylchen1219@gmail.com (Y.-L.C.); Ly.tsai95@gmail.com (L.-Y.T.)
4 School of Nutrition and Health Sciences, College of Nutrition, Taipei Medical University,
250 Wu-Hsing Street, Taipei 11031, Taiwan; ckshih@tmu.edu.tw (C.-K.S.); yichun@tmu.edu.tw (Y.-C.C.)
5 School of Medicine, Taipei Medical University, Taipei 11031, Taiwan
6 Institute of Environmental and Occupational Health Sciences, College of Public Health, National Taiwan
University, Taipei 10617, Taiwan
7 Department of Pediatrics, Shuang Ho Hospital, Ministry of Health and Welfare, Taipei Medical University,
New Taipei 23561, Taiwan; pedkuoyt@tmu.edu.tw
8 Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University,
Taipei 11031, Taiwan
* Correspondence: sinchung@tmu.edu.tw; Tel.: +886-2-27361661 (ext. 6560)
† These authors contributed equally to this work.
Received: 3 December 2019; Accepted: 31 December 2019; Published: 3 January 2020
Abstract: Iron deficiency (ID) and iron deficiency anemia (IDA) typically occur in developing countries.
Notably, ID and IDA can affect an infant’s emotion, cognition, and development. Breast milk is
considered the best food for infants. However, recent studies have indicated that breastfeeding for
more than six months increases the risk of ID. This study investigated the prevalence of ID and
IDA, as well as the association between feeding type and iron nutritional status in northern Taiwan.
A cross-sectional study was conducted on infants who returned to the well-baby clinic for routine
examination from October 2012 to January 2014. Overall, 509 infants aged 1–12 months completed
the iron nutritional status analysis, anthropometric measurement, and dietary intake assessment,
including milk and complementary foods. The results revealed that 49 (10%) and 21 (4%) infants in
their first year of life had ID and IDA, respectively, based on the World Health Organization criteria.
Breastfed infants had a higher prevalence rate of ID and IDA than mixed-fed and formula-fed infants
(p < 0.001). Regarding biomarkers of iron status, plasma hemoglobin (Hb), ferritin, and transferrin
saturation (%) levels were significantly lower in ID and IDA groups. The prevalence of ID and IDA
were 3.7% and 2.7%, respectively, in infants under six months of age, but increased to 20.4% and 6.6%,
respectively, in infants above six months of age. The healthy group had a higher total iron intake than
ID and IDA groups, mainly derived from infant formula. The total dietary iron intake was positively
correlated with infants’ Hb levels. Compared with formula-fed infants, the logistic regression revealed
that the odds ratio for ID was 2.157 (95% confidence interval [CI]: 1.369–3.399) and that for IDA was
4.196 (95% CI: 1.780–9.887) among breastfed infants (p < 0.001) after adjusted for all confounding
factors (including gestational week, birthweight, sex, body weight percentile, body length percentile,
age of infants, mothers’ BMI, gestational weight gain, education level, and hemoglobin level before
delivery). In conclusion, our results determined that breastfeeding was associated with an increased
the prevalence of ID and/or IDA, especially in infants above six months. This suggests that mothers
who prolonged breastfeed after six months could provide high-quality iron-rich foods to reduce the
prevalence of ID and IDA.

Nutrients 2020, 12, 139
Keywords: infant; breast milk; formula milk; iron deficiency; iron deficiency anemia
1. Introduction
Breast milk is considered the optimal nutrition and healthy food for the first six months of life,
and breastfeeding is typically complemented with other foods from six months of age until at least
12 months for nearly all infants [1]. A reappraisal of the evidence from a recent expert review for the
European Food Safety Authority (EFSA) concluded that for infants across the EU, complementary
foods might be introduced safely between the fourth and sixth month [2]. Studies have determined
that the iron content in breast milk is low, infants fed with solid food before six months or received
iron supplement will decrease the risk of IDA [3]. Anemia is defined as a reduced erythrocyte count
or hemoglobin (Hb) value of 5 percentile below the normal hemoglobin value specified for that
age in healthy individuals [4]. Therefore, the US Department of Agriculture and the Centers for
Disease Control recommend the introduction of complementary food in infants aged between 4 and
6 months [5].
In addition to inadequate iron intake, reduced bioavailability of dietary iron, increased iron
requirements, and chronic blood loss is common causes of iron deficiency (ID). ID is currently the
most common micronutrient deficiencies worldwide, and a World Health Organization (WHO) survey
in 2008 revealed that globally, approximately 293 million (47.4% prevalence) preschool-age children
and approximately 56 million (41.8% prevalence) pregnant women suffer from anemia, among which
approximately 50% cases are attributable to ID [6]. ID is undoubtedly the major cause of most anemia
cases, although other minerals or vitamins can also be responsible for this pathology. Prolonged ID
often leads to iron deficiency anemia (IDA) [7]. The American Academy of Pediatrics determined
that IDA adversely affects an infant’s social and emotional behavior development and cognitive
performance [8]. Approximately 25% of infants in developing countries have IDA, and IDA during
pregnancy increases the risk of preterm delivery and adverse perinatal outcomes, such as maternal
hemorrhage, sepsis, low birth weight, and possibly poor neonatal health [9–11].
Recent studies have indicated that iron content in breast milk is low [12–14], and prolonged use of
breast milk as the main food in infants might increase the risk of ID and IDA [15]. In Taiwan, the Ministry
of Health and Welfare promotes the benefits of breastfeeding for a long period. Although Tsai et al.
reported that IDA was associated with prolonged, predominant breastfeeding, the study sample size
was small, and no relevant risk factors were evaluated [16]. The purpose of the present study was to
investigate the iron status of infants in their first year of life and analyze the relevant influencing factors.
2. Participants and Methods
2.1. Study Subjects

A cross-sectional study was conducted in three hospitals, namely, Shin Kong Wu Ho-Su Memorial
Hospital, Taipei Medical University Hospital, and Shuang Ho Hospital, from October 2012 to
January 2014. Overall, 2804 healthy infants aged 1–12 months who came to the well-baby clinic
for routine vaccination were screened for eligibility. The inclusion criteria for infants and mothers
enrolled were as follows: No systemic diseases (toxemia, hypertension, diabetes mellitus, and heart
disease) during pregnancy, age below one year, healthy and without any disease, as determined by a
pediatrician. Infants with premature birth, congenital diseases (such as heart, lung, liver, and intestinal
diseases), growth disorders, diagnosis of gastrointestinal disorders (nausea, vomiting, pain, flatulence,
diarrhea, and malabsorption), and thalassemia were excluded.
The Ethics Committee of Shin Kong Wu Ho-Su Memorial Hospital and Taipei Medical University
approved this study in accordance with the International Ethical Guidelines for Biomedical Research
Involving Human Subjects and ethical principles of the Declaration of Helsinki (20120901R and
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Nutrients 2020, 12, 139
201308004). Written informed consent was obtained from all participants or legal representatives
before the study procedures were performed.
2.2. Basic Characteristics and Dietary Iron Intake Assessment

The contents of the questionnaire were validated by experts, including the infant’s basic
information, such as gestational weeks, chronological age, birth weight, birth length; anthropometrics,
such as body length, body weight, and head circumference; and dietary intake. Gestational weeks,
chronological age, birth weight, and birth length were recorded per the medical chart. Body length
and body weight were measured using an infantometer and weighing scales. The head circumference
was measured by applying a plastic tape around the forehead (above the eyebrows) and the occipital
protuberance. These measurements were converted to percentiles according to the growth charts
for Taiwanese children released by the Ministry of Health and Welfare. The diet was assessed using
a semi-quantitative frequency method, and the included food items were tofu, chicken, pork, beef,
fish, egg yolk, viscera, vegetables, rice, wheat, baby rice cereal, baby wheat cereal, formula milk,
juice, and puree. Complementary food and formula milk intakes were quantified based on standard
bowls, spoons, and feeding bottles. Iron intake from the complementary food was calculated using the
Nutritional Chamberlain Line, Nutritionist Edition, version 2002 (E-Kitchen Business Corp, Taiwan).
Iron intake from formula milk and baby cereal was calculated according to their nutrient labels.
Iron content in breast milk was obtained through actual measurement. Breast milk intake was obtained
from mothers’ reports. If the mothers were directly breastfeeding their infants, the total volume of
breast milk intake was assessed according to the report of Lyu et al. [17]. Pre-pregnancy body weight
(kg), gestational weight gain (kg), current weight, and education level were obtained from self-reported
data. Current body weight (kg) and height (cm) were measured using an electronic health scale
(Tanita corp., Tokyo, Japan). Complete blood count of the mothers, including Hb level, hematocrit
(Hct) level, and mean corpuscular volume (MCV) before delivery at 37–40 weeks pregnancy were
recorded according to the medical charts.
2.3. Breast Milk and Blood Collection

Breast milk from the mothers’ unelated breast was collected using an electric milking machine
(Lactina, Medela, Switzerland) for 20 min, which was then mixed, dispensed, and ice-cooled at −80 ◦ C
until analysis. Blood samples (4 mL) of infants were collected through an arterial puncture of one arm
into vacutainers with and without anticoagulant ethylenediaminetetra-acetic acid (EDTA) by a trained
technician. Serum was collected after centrifugation at 1400× g for 10 min at 4 ◦ C and immediately
sent to Central Laboratory, Shin-Kong Wu Ho-Su Memorial Hospital, for analysis.
2.4. Biochemical Analyses

Complete blood count was determined using an automatic blood cell analyzer (Biotecnica
Instruments SpA, Roma, Italy). Ferritin was detected using a chemiluminescent immunoassay (Roche
Diagnostics, Lewes, UK). Serum iron was analyzed using the ferrozine method (Siemens Healthcare,
Marburg, Germany). Total iron binding capacity (TIBC) is the ability of transferrin to bind with iron
that was measured by chemistry analyzer using dedicated reagents (Siemens Healthcare, Marburg,
Germany). Transferrin saturation (TS, %) represents the percentage of transferrin bound to iron ions,
calculated by dividing serum iron concentration by TIBC and multiplying the result by 100.
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Nutrients 2020, 12, 139
2.5. Breast Milk Iron Content Analysis

Aliquots of 0.5 mL of the mixed breast milk sample was added to 1.5 mL of 70% nitric acid
and 0.5 mL of 30% hydrogen peroxide, separately. After mixing and allowing the samples resting
for one night, the mixture was digested in a 50 mL polypropylene digestion bottle at 95 ◦ C for 1 h.
After cooling at room temperature, the digested sample was diluted using 50 mL of deionized water.
Subsequently, 1 mL of the dilution was pipetted into a 15 mL centrifuge tube and diluted with 10 mL
of 2% aqueous nitric acid solution to detect the iron content by using inductively coupled plasma mass
spectrometry (ICP-MS) (ThermoFisher Scientific, Bremen, Germany) [18]. The iron content in breast
milk was calculated using a standard curve constructed using pure iron standards for ICP-MS (Merck,
Darmstadt, Germany); with an R value of ≥0.99, coefficient of variation (CV) at 2%, and a recovery rate
of 80–120%.

The WHO defines ID as a serum ferritin level less than 15.0 ng/mL and IDA as serum ferritin
level less than 15.0 ng/mL and Hb less than 10.5 g/dL. We divided the subjects into three groups
according to the WHO definitions: The normal, ID, and IDA groups. All data were confirmed to have
a normal distribution by using the Kolmogorov–Smirnov test. Data are presented as means ± standard
deviations (SDs), median (interquartile range), or percentage. Intergroup differences were determined
using one-way ANOVA, followed by the Scheffé method for post hoc test or nonparametric statistics.
Pearson’s chi-squared test was used to assess categorical variables. The correlation between Hb and
dietary iron intake was determined using the Pearson correlation test. The association between feeding
types and anemia was determined using multivariable logistic regression. All data analyses were
performed using SPSS (version 19; SPSS Inc., Chicago, IL, USA). Differences were considered significant
at p < 0.05.
3. Results
3.1. Participant Characteristics and Infant Anemia Diagnosis

A total of 1368 infants were eligible for this study. However, 779 mothers did not provide consent
to extract their infants’ blood, and therefore, the 589 subjects were ultimately enrolled in this study.
However, blood draws were unsuccessful in 39 infants. Thus, a total of 550 cases were included for
data analysis. Because no introduction of complementary food to infants over six months of age
was considered as abnormal feeding, four infants aged eight months and two aged 12 months were
excluded accordingly. In addition, 35 infants with a WBC count of >0.000/mm3 , suspected of infection,
were excluded. Data on the iron status analysis of 509 infants are presented in Figure 1.
Anemia was defined according to the WHO criteria, and 49 (10%) and 21 infants (4%) were
diagnosed with ID and IDA, respectively, by the physician (Table 1). No statistically significant
intergroup differences were noted in terms of sex gestational age, birthweight, body length, body
weight, and head circumference of infants. The formula-fed type (52.8%) was the predominant
population in the healthy group. The breastfed infants had a significantly higher prevalence of ID
(65.3%) and IDA (85.7%) than the mixed-fed and formula-fed infants (p < 0.001). No significant
intergroup differences were observed in terms of the mother’s age, body mass index (BMI), gestational
weight gain, and education.
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Nutrients 2020, 12, 139
Figure 1. Flowchart of the enrolment of infants.
Table 1. Demographic characteristics of subjects diagnosed with iron deficiency (ID) or iron deficiency
anemia (IDA) in the first year of life a .
Normal ID b IDA
Characteristics p Value
N = 439 N = 49 N = 21
Infant
Number (%) 439 (86) 49 (10) 21 (4)
Male (%) 236 (53.8) 24 (49.0) 12 (57.1) 0.546
Gestational age 38.4 ± 1.5 38.2 ± 1.6 38.2 ± 1.4 0.673
Chronological age 5.9 ± 4.3 9.9 ± 3.6 9.6 ± 3.4 <0.001 *
Birth weight (kg) 3.0 ± 0.5 3.0 ± 0.6 3.0 ± 0.5 0.919
Body length (percentile) 52.9 ± 30.9 55.8 ± 31.4 37.9 ± 31.3 0.118
Body weight (percentile) 51.2 ± 28.3 51.3 ± 30.0 43.9 ± 30.4 0.568
Head circumference (percentile) 56.4 ± 30.2 50.1 ± 31.2 51.9 ± 20.3 0.424
Feeding type c
Breast-fed (%) 127 (28.9) 32 (65.3) 18 (85.7) <0.001 *
Mix-fed (%) 80 (18.2) 6 (12.2) 2 (9.5)
Formula-fed (%) 232 (52.8) 11 (22.4) 1 (4.8)
Mother
Age (year) 32.6 ± 4.2 32.1 ± 3.6 32.4 ± 4.7 0.791
BMI (kg/m2 ) 21.3 ± 3.7 20.8 ± 5.5 20.7 ± 3.8 0.654
Gestational weight gain (kg) 14.3 ± 7.9 16.3 ± 11.9 11.6 ± 5.5 0.125
Education (year) 14.6 ± 2.3 15.0 ± 2.3 15.2 ± 2.0 0.382
a Values are expressed as mean ± SD or n (%); b According to definitions by the World Health Organization,
iron deficiency (ID): Serum ferritin < 15.0 ng/mL, iron deficiency anemia (IDA): Serum ferritin < 15.0 ng/mL and
hemoglobin < 10.5 g/dL.; c Definition of feeding type: Breast-fed means that the dairy products in the infant’s diet
are all breast milk; Mix-fed means that the dairy products in the infant’s diet include breast milk and formula milk;
Formula-fed refers to the dairy products in the infant’s diet only formula milk. * Differences between groups were
tested using one-way ANOVA, followed by the Scheffé method for a post hoc or Chi-square test; p < 0.05 was
considered statistically significant.
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Nutrients 2020, 12, 139
3.2. Analysis of Iron Status

The iron status of the infants and mothers before delivery are presented in Table 2.
Biochemical parameters, ferritin, and TS were significantly low in ID and IDA groups. Although the
mothers of infants with IDA had a slightly lower Hb level before delivery, no statistical difference
was observed.
Table 2. Hematologic data of subjects a .
Normal ID b IDA
Classification p Value c
N = 439 N = 49 N = 21
Infant
Hb (g/L) 11.4 ± 1.3 a 11.6 ± 0.7 a 9.2 ± 1.4 b <0.001
Ferritin (ng/mL) 55.0 (98.4) a 10.2 (5.7) b 5.2 (5.7) b <0.001
TS (%) 22.5 ± 11.3 a 11.5 ± 5.5 bc 7.0 ± 4.5 c <0.001
Maternal (before
delivery)
Hb (g/L) 12.0 ± 1.9 12.1 ± 1.4 11.3 ± 1.4 0.361
Hct (%) 36.2 ± 4.6 36.6 ± 3.2 34.5 ± 3.7 0.333
MCV (fl) 86.8 ± 8.2 87.1 ± 8.4 86.7 ± 8.1 0.584
a Data are presented as mean ± SD or median (interquartile range); b ID, iron deficiency; IDA, iron deficiency
anemia; Hb, hemoglobin; TS, transferrin saturation; Hct, hematocrit; MCV, mean corpuscular volume; c Means in
the column with different superscripts indicate a significant difference (p < 0.05), tested using one-way ANOVA,
followed by the Scheffé method for post hoc test or the Kruskal–Wallis test.
3.3. Iron Intake of Infants

The total iron intake was calculated as the sum of the iron contents in milk and complementary
food, as presented in Table 3. Because complementary food was introduced in only 17 infants, and most
of the complementary foods were cereals and fruit purees for infants aged 4–6 months, data of the
iron intake from complementary food are not shown. During ages 1–6 months, the daily iron intake
from milk in the normal, ID, and IDA groups were 3.43, 0.13, and 0.13 mg, respectively. Moreover,
the normal group had a higher total iron intake (3.49 mg daily) than the ID (0.13 mg daily) and IDA
(0.13 mg daily) groups at ages 1–6 months. The low total iron intake in infants of the ID and IDA
groups could be attributed to exclusive breastfeeding, and both ID and IDA were observed in infants
aged 4–6 months.
Table 3. Iron intake of infants aged 1–6 months and 7–12 months a .
Parameters Normal ID b IDA p Value

1–6 months
Number (%) 307 (93.6) 12 (3.7) 9 (2.7)
Chronological age 3.4 ± 1.8 5.0 ± 1.2 4.8 ± 1.1 0.007 *
Breastfeed (%) 174 (56.7) 12(100) 9 (100) 0.001 *
Iron intake from milk (mg/day) c 3.43 (6.62) 0.13 (0.03) 0.13 (0.17) 0.003 *
Total iron intake (mg/day) d 3.49 (6.81) 0.13(0.18) 0.13(0.17) 0.007 *
7–12 months
Number (%) 132 (73.0) 37 (20.4) 12 (6.6)
Chronological age 11.3 ± 2.9 11.2 ± 2.9 11.3 ± 2.0 0.976
Breastfeed (%) 21 (15.9) 26 (70.3) 11 (91.7) <0.001 *
Iron intake from milk (mg/day) c 5.04 (3.92) 0.14 (2.01) 0.14 (0.01) <0.001 *
Iron intake from complementary food (mg/day) 1.04 (1.00) 1.29 (2.00) 1.20 (2.00) 0.840
Total iron intake (mg/day) 6.47 (4.35) 2.28 (3.02) 1.33 (0.98) <0.001 *
a Data are presented as median (interquartile range); b ID, iron deficiency; IDA, iron deficiency anemia; c Iron intake
from milk was the sum of iron intake from formula milk and breast milk.; d Total iron intake was the sum of iron
intake from milk and complementary food; * Intergroup differences were tested using the Kruskal–Wallis test or chi
square test; p < 0.05 was considered statistically significant.
70
Nutrients 2020, 12, 139
The normal group had the highest iron intake from milk (5.04 mg daily), whereas, the ID and IDA
groups had a low iron intake from milk. This could be attributed to the continuous breastfeeding of the
infants in the ID and IDA groups. No intergroup differences were noted in terms of the iron intake from
complementary food. Therefore, milk was the major source of iron intake in infants aged 7–12 months.
Furthermore, we determined that the prevalence of ID and IDA in infants aged 7–12 months was 20.4%
and 6.6%, respectively, which was significantly higher than 3.7% and 2.7%, respectively, in infants
aged 1–6 months. Our results revealed that infants with ID or IDA before six months were all in aged
4–6 months and breastfed. The medium iron intake in infants aged 4–6 months in the normal, ID and
IDA groups were 5.13 mg (6.54), 0.13 mg (0.18), and 0.13 mg (0.29), respectively. In aged 7–12 months,
infants with IDA had the highest breastfeeding rate, followed by those with ID.
Iron is essential for hematopoiesis. We observed that the total dietary iron intake was positively
correlated with Hb (r = 0.292, p < 0.001) (Figure 2A). We further divided the data into the following
two subgroups: One to six months (Figure 2B) and 7–12 months (Figure 2C). The data revealed that the
correlation between total dietary iron intake and Hb in infants aged 7–12 months was higher than in
those aged 1–6 months (r = 0.390, p < 0.001 vs. r = 0.130, p = 0.028).
Figure 2. Correlations between total dietary iron intake and hemoglobin at 1–12 months (A),
1–6 months (B) and 7–12 months (C).
3.4. Association between Feeding Type and Iron Status

Logistic regression models were employed to identify predictors of ID and IDA. Our data revealed
that feeding type was a major indicator in predicting anemia (Table 4). In model 1, no covariates were
adjusted, and in model 2, gestational week, birthweight, sex, body weight percentile, body length
percentile, and age of infants were adjusted. In model 3, in addition to the aforementioned variables for
infants, mothers’ BMI, gestational weight gain, education level, and Hb concentration before delivery
were also adjusted. Compared with formula-fed infants, breastfed infants had a higher odds ratio (OR)
for ID (OR: 2.715; 95% CI: 1.830–4.030) and IDA (OR: 4.338; 95% CI: 2.108–8.927) in model 1 (p < 0.001).
Furthermore, ORs for ID and IDA significantly differed after adjustment for variables in models 2 and 3.
Table 4. Association between feeding type and iron status.
Variables β SE a OR 95% CI p Value

Model 1 b
ID 0.999 0.201 2.715 1.830–4.030 <0.001 *
IDA 1.467 0.368 4.338 2.108–8.927 <0.001 *
Model 2
ID 0.984 0.201 2.674 1.803–3.966 <0.001 *
IDA 1.361 0.369 3.901 1.893–8.042 <0.001 *
Model 3
ID 0.769 0.232 2.157 1.369–3.399 0.001 *
IDA 1.434 0.437 4.196 1.780–9.887 0.001 *
a SE, standard error of mean; OR, odds ratio; 95% CI, 95% confidence interval; b Model 1: Not adjusted; Model 2:
Adjusted for gestational week, birthweight, sex, body weight percentile, body length percentile, age; Model 3:
Adjusted for gestational week, birthweight, sex, body weight percentile, body length percentile, age of infants, as
well as mothers’ BMI, gestational weight gain, education level, and hemoglobin level before delivery; * p < 0.05.
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Nutrients 2020, 12, 139
4. Discussion
The period of infancy constitutes a critical window of growth and brain development, and thus,
micronutrient deficiencies during this period may have adverse effects on neurocognitive functions.
Iron deficiency is the most prevalent nutritional deficiency among infants in developing countries.
Women of childbearing age are at risk of iron deficiency because of poor iron content in the diet,
increased demand for iron during pregnancy, and iron loss during menstruation and childbirth.
In addition, breastfed infants are vulnerable to developing ID because of rapid growth, depletion of
their iron endowment, and low iron content in breast milk and in some complementary foods. In our
study, among the 509 mother–infant dyads who submitted complete data, 3.7% and 2.7% of the infants
aged below six months had ID and IDA, respectively. However, in infants aged above six months,
the prevalence of ID and IDA rapidly increased to 20.4% and 6.6%, respectively. Analysis of the
maternal hematologic data revealed that anemia was not detected in the mothers during the routine
prenatal examination. The basic characteristics of all infants (Normal, ID, IDA) were similar except for
the chronological age and feeding type.
The prevalence of ID and IDA varies greatly among countries worldwide. In India, the prevalence
of ID in infants aged 3, 4, and 5 months was 5.4%, 21.4%, and 36.4%, respectively, whereas, that of IDA
was 4.6%, 16.7%, and 11.4%, respectively [19]. In Turkey, Germany, and Brazil, the prevalence of ID
in infants aged four months were 19.8%, 6%, and 5.7%, the prevalence of IDA in those infants were
9.5%, 0%, and 3.4%, respectively [20–22]. The prevalence of ID and IDA in infants before six months of
age in our study was concordant with that observed in Germany and Brazil. In Spain, the prevalence
of ID and IDA was 9.6% and 4.3%, respectively, in 12-month-old infants [23]. The prevalence of ID
was 14.0%, and that of IDA was 9.4% in infants aged 9–12 months in Estonia [24]. In Saudi Arabia,
out of the 274 infants aged 6–24 months studied, 126 (51%) were diagnosed as having IDA [25].
Among 619 Korean infants aged 8–15 months old, ID and IDA were diagnosed in 174 (28.1%) and
87 infants (14.0%), respectively [26]. The prevalence of ID and IDA in two cohorts of infants aged nine
months old in China was 2.8% and 20.7% and 12.0% and 31.2%, respectively [27]. The prevalence of ID
and IDA in infants above six months of age in our study was concordant with that observed in China
and Korea.
Breast milk is considered the best food for infants, because it is highly nutritious for infant growth
and contains maternal antibodies that provide defense against pathogens. A recent study indicated
that human breast milk has very little iron (0.5 mg/L at one month and 0.29 mg/L at 3–5 months) [28].
Our data revealed that the average iron content of breast milk from the mothers of the infants was
0.21 ± 0.06 mg/L. Although iron in human breast milk has higher bioavailability, it may not be sufficient
for infants. Therefore, infants’ body iron stores meet most requirements for breast-fed infants during
the first six months of life. A study concluded that a normal, healthy, full-term infant has sufficient
amounts of iron until approximately 4–6 months of age [29]. Our results revealed that the prevalence
of ID and IDA in infants aged 1–6 months was 3.7% and 2.7%, respectively, and these infants were
all in aged 4–6 month and breastfed. Hence, breastfeeding for more than four months can slightly
increase the risk of ID and IDA with lower iron stores.
Studies have indicated that the risk factors for the development of ID included small-for-gestational-
age, infants below 10th percentile weight for gestation, infants of diabetic mothers, very-low-birth
weight preterm neonates (VLBW, <1500 g at birth) and infants with lower iron stores [30,31]. Therefore,
although the iron status of our subjects seems to be more related to iron intake, studies have shown
the smoking, obesity, and childbirth by caesarean section also affected the iron status of infants [32].
These factors were not considered in the present study. In the absence of inflammation, serum ferritin
measurement is the most specific test to determine total iron content stored in the body. Our data
revealed that infants in ID and IDA groups with lower serum ferritin levels reflected depleted iron
stores. Another indicator of iron deficiency was TS. In cases of iron deficiency, serum iron is reduced,
and TIBC is increased, resulting in a substantial reduction in TS. A threshold of 16% is generally used
72
Nutrients 2020, 12, 139
to screen for iron deficiency. Our data revealed that subjects with ID and IDA had a TS below the
threshold and TS in IDA group was lower than that in ID group.
Another possible reason causing iron depletion in infants below six months of age could be
maternal iron deficiency during pregnancy, which results in offspring with inadequate iron storage
in the body. Some studies have found little or no correlation between maternal and neonatal iron
status, whereas, others have suggested that the fetus was vulnerable to maternal ID [33,34]. Mothers of
IDA infants had lower Hb level, although there were no statistical differences. We further analyzed
the correction between infants Hb level and mothers Hb level, and the results were not correlated.
Although the mother’s iron nutrition status during pregnancy may affect the child’s iron nutrition
status, our results revealed that feeding still mainly affects the infant’s iron nutrition status. In addition,
the timing of umbilical cord clamping affects iron stores in the newborn. A study indicated that delay
in cord clamping increases the red blood cell volume and iron stores in infants [35]. Hence, cord
clamping could be a feasible solution to improve the iron status of infants.
The WHO recommends exclusive breastfeeding of infants for the first six months of life. Thereafter,
infants should receive complementary foods with continued breastfeeding up to two years of age or
above. Taiwan’s health policy also follows this recommendation. In this study, among 328 infants
below six months of age, only 17 were introduced to complementary food. Most of these foods were
cereals or fruit juices that had low iron content. Thus, in addition to prenatal storage, formula milk
contributed more iron to the infants below six months of age. Although human milk has a high
bioavailability of iron, the iron content was so low that exclusive breastfeeding increased the risk
of ID and IDA. We determined that the total iron intake was positively correlated with Hb level in
infants below six months of age, thereby indicating that although prenatal storage was crucial to
maintain iron status, additional dietary iron intake can improve the Hb levels and prevent ID and IDA,
especially in infants aged 4–6 months. Evidence from randomized control trials suggests that the rate
of IDA in breastfed infants could be positively altered by the introduction of solids at four months of
age [36]. The American Academy of Pediatrics recommends that exclusively breastfed, full-term infants
receive 1 mg/kg of iron supplements per day from the age of four months [37]. Taiwan’s maternal
and child policy particularly emphasizes the benefits of breastfeeding. The exclusive breastfeeding
rate for infants at six months in Taiwan increased from 20.1% in 2004 to 50.2% in 2011 [38]. Therefore,
introducing complementary food early or providing iron supplementation is imperative for infants
aged 4–6 months who are exclusively breastfed.
Although infants aged 7–12 months were provided complementary foods, the iron intake from
complementary foods was not different among the normal, ID, and IDA groups. Notably, milk was
the major source of iron. Upon further analysis of data, we observed that up to 70% of infants with
ID and 90% of infants with IDA were still breastfed. This result indicated that infants who are still
breastfed for more than six months do not obtain adequate iron from breast milk. This suggests that
mothers who still breastfeed after six months could provide high-quality iron-rich foods to reduce the
prevalence of ID and IDA.
When we reported the iron status of infants to their mothers, most of the mothers presented their
infants had good growth and not aware of the insufficient iron intake and that it increased the risk
of ID or IDA. Conversely, infants who used commercial formulas instead of breast milk had better
iron status because most commercial formulas are iron-fortified. Future health policies should educate
mothers to prepare high-quality iron-rich foods or provide iron supplementation for breastfed infants.
The maximum iron requirement for infants aged 4–12 months is approximately 1 mg/kg or
10 mg/day. The average full-term infant requires 8 mg of iron daily from approximately six months
of age [39]. Our data demonstrated that the median iron intake of infants aged 1–6 months was
3.49 mg/day, and the median iron intake of infants aged 7–12 months was 6.47 mg/day, which could
reach the normal iron nutritional status. Infants with IDA had normal growth and no obvious
symptoms except for pallor were observed, and no infants were suspected of having anemia by
73
Nutrients 2020, 12, 139
caregivers. Therefore, the presentation of iron deficiency is subtle and can be detected accurately only
by medical personnel.
Although our results determined that breastfeeding was associated with an increased the
prevalence of ID and/or IDA, the advantages of breast milk are recognized and unquestionable. Thus,
more attention should be paid to the problem of iron deficiency in infants, and strategies should
be proposed, including improving maternal iron status, introducing high-quality complementary
food early, and even providing iron supplement to breastfed infants. The Pediatrics Association
of Taiwan revised the guidelines for breastfed infants in 2016. The content includes the following:
(1) Encourage full-term infants to start breastfeeding as soon as possible after birth, (2) Continue to
breastfeed until one year of age. After one year of age, the mother may continue to breastfeed the
infants, (3) Breastfed infants should be started on complementary foods at 4–6 months of age. If no
complementary foods are added after four months, oral iron supplementation should be started at
1 mg/kg/day. Iron deficiency affects the development of cognition, nerves, and behavior, some of which
are long-term and irreversible [40]. In this study, all infants with ID and IDA improved iron status and
anemia after medical iron supplementation and regular checkups by pediatricians.
The strength of the present work is that it is the first to look at the association between breastfeeding
and ID and IDA during infancy in Taiwan. The results can be used as a reference for nutrition
policymakers. However, the study had some limitations. First, this was a cross-sectional study that
was unable to clarify the effects of longitudinal nutritional status on the development of ID and IDA.
Second, the sample size was relatively small, and all participants lived in northern Taiwan, which might
limit the generalizability of the results. Therefore, additional large-sample, multicenter studies are
required. Third, we only prioritized examining several clinical iron markers that are more likely related
to iron status. Fourth, the lack of information regarding the initiation of complementary feeding,
that could significantly impact iron status in infants. Nevertheless, further studies are warranted,
including those for following up infants with iron deficiency in the first year to analyze whether it
affects their cognition function and growth, as well as whether early introduction of high-quality
iron-rich complementary foods can improve the iron status of infants.
5. Conclusions
Our data revealed that exclusive breastfeeding increases the prevalence of ID and IDA in infants
aged 4–6 months, and prolonged breastfeed above six months can significantly increase the prevalence
of ID and IDA. Although iron in breast milk has good bioavailability, its low iron content results
in insufficient iron intake in infants and increases the risk of anemia. Hence, health policies should
encourage the early introduction of iron-rich complementary foods and educate mothers to prepare
high-quality complementary foods to reduce the risk of anemia, especially in infants above six months.
In addition, if no complementary foods are added after four months, oral iron supplementation should
be started in breastfeeding infants.
Author Contributions: Conceptualization, S.-C.L. and C.-M.C.; methodology: All; data curation: S.-C.M., Y.-L.C.,
L.-Y.T., Y.-T.K., I.-M.C. and Y.-C.C.; validation: S.-C.M., C.-K.S. and M.-L.C.; drafting of the manuscript: S.-C.L.;
critical revision of the manuscript: C.-M.C. and S.-C.M.; final approval of the version to be published: S.-C.L.
All authors have read and agreed to the published version of the manuscript.
Funding: This study was supported by the Ministry of Health and Welfare (DOH101-HP-4204 and
DOH102-HP-4203).
Acknowledgments: We thank the participants and the medical staff for their participation. This manuscript was
edited by Wallace Academic Editing.
Conflicts of Interest: The authors have no conflicts of interest to declare.
74
Nutrients 2020, 12, 139
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76
nutrients
Article
The Effectiveness of Different Doses of Iron
Supplementation and the Prenatal Determinants of
Maternal Iron Status in Pregnant Spanish Women:
ECLIPSES Study
Lucía Iglesias Vázquez 1 , Victoria Arija 1,2, *, Núria Aranda 1 , Estefanía Aparicio 1 , Núria Serrat 3 ,
Francesc Fargas 4 , Francisca Ruiz 4 , Meritxell Pallejà 2 , Pilar Coronel 5 , Mercedes Gimeno 5 and
Josep Basora 2,6
1 Department of Preventive Medicine and Public Health, Faculty of Medicine and Health Sciences,
Universitat Rovira i Virgili, 43201 Reus, Spain; lucia.iglesias@urv.cat (L.I.V.); nuria.aranda@urv.cat (N.A.);
estefania.aparicio@urv.cat (E.A.)
2 Tarragona–Reus Research Support Unit, Jordi Gol University Institute for Primary Care Research,
43202 Tarragona, Spain; meritxell.palleja@urv.cat (M.P.); josep.basora@urv.cat (J.B.)
3 Clinical Laboratory, University Hospital Joan XXIII, Institut Català de la Salut, Generalitat de Catalunya,
43005 Tarragona, Spain; nserrat.tarte.ics@gencat.cat
4 Sexual and Reproductive Health Service of Reus–Tarragona, Institut Català de la Salut,
Generalitat de Catalunya, 43202 Tarragona, Spain; ffargas.tarte.ics@gencat.cat (F.F.);
paqui.r.d@hotmail.com (F.R.)
5 Meiji Pharma Spain S.A. (formerly Tedec-Meiji Farma S.A.) Alcalá de Henares, 28802 Madrid, Spain;
p.coronel@tedecmeiji.com (P.C.); m.gimeno@tedecmeiji.com (M.G.)
6 CIBERobn (Center for Biomedical Research in Physiopathology of Obesity and Nutrition),
Instituto de Salud Carlos III, 28029 Madrid, Spain
* Correspondence: victoria.arija@urv.cat; Tel.: +97-7759334
Received: 16 September 2019; Accepted: 7 October 2019; Published: 10 October 2019
Abstract: Iron deficiency (ID), anemia, iron deficiency anemia (IDA) and excess iron
(hemoconcentration) harm maternal–fetal health. We evaluated the effectiveness of different doses
of iron supplementation adjusted for the initial levels of hemoglobin (Hb) on maternal iron status
and described some associated prenatal determinants. The ECLIPSES study included 791 women,
randomized into two groups: Stratum 1 (Hb = 110–130g/L, received 40 or 80mg iron daily) and
Stratum 2 (Hb > 130g/L, received 20 or 40mg iron daily). Clinical, biochemical, and genetic information
was collected during pregnancy, as were lifestyle and sociodemographic characteristics. In Stratum 1,
using 80 mg/d instead of 40 mg/d protected against ID on week 36. Only women with ID on week
12 benefited from the protection against anemia and IDA by increasing Hb levels. In Stratum 2,
using 20 mg/d instead of 40 mg/d reduced the risk of hemoconcentration in women with initial serum
ferritin (SF) ≥ 15 μg/L, while 40 mg/d improved SF levels on week 36 in women with ID in early
pregnancy. Mutations in the HFE gene increased the risk of hemoconcentration. Iron supplementation
should be adjusted to early pregnancy levels of Hb and iron stores. Mutations of the HFE gene should
be evaluated in women with high Hb levels in early pregnancy.
Keywords: iron supplementation; pregnancy; randomized controlled trial; serum ferritin; hemoglobin;
iron status; iron stores; HFE gene
1. Introduction
Iron requirements increase during pregnancy. Since dietary sources cannot always prevent
iron deficit, iron supplements are usually prescribed to women who plan to become pregnant.

Nutrients 2019, 11, 2418
However, there is no consensus on the ideal iron dosage during pregnancy. Anemia is the most
common and widespread nutritional disorder globally and a significant public health problem [1,2].
Anemia is attributed to iron deficiency (ID) in half of the cases in the general population [1,3] and in up
to 90% of cases of pregnant women [4]. Studies show that an inadequate iron status during pregnancy
can lead to adverse mother–child outcomes. In the mother, iron deficiency, anemia, and iron deficiency
anemia (IDA) have been associated with preeclampsia, preterm delivery, and even miscarriage, and in
the child with fetal growth restriction, low birth weight and impaired cognitive development [5–10].
Furthermore, some studies have underscored the importance of timing of ID and IDA, since some
long–term consequences, especially regarding the development and functioning of the child’s brain,
are irreversible, even after correcting iron levels [11,12]. As a result, it is essential to maintain good
nutritional care even before getting pregnant, as well as throughout the whole gestation, to ensure an
optimal health status for mother and baby.
In addition to participating as enzymatic cofactor in a wide range of metabolic reactions, iron is
indispensable for the synthesis of hemoglobin (Hb), the synthesis and methylation of DNA, and oxygen
transport [13,14]. The increase in blood volume and the formation of new tissue during pregnancy are
the main mechanisms underlying the increased iron requirements [15–17]. Crucially, iron has a key
role in neuronal proliferation, myelination, and the synthesis of several neurotransmitters during the
development of the fetal brain [11,18]. Despite concerns about the state of prenatal iron, which caused
the launch of public health policies to address iron deficiency [19], it is estimated that in Europe,
around 25% of pregnant women become anemic during pregnancy [2,3,19]. The prevalence of ID
is greater than the prevalence of IDA, and it often develops during the later months of pregnancy,
even in women with sufficient iron stores at the start of the pregnancy [20]. In addition, while diet
and supplementation are the main sources of iron, the maternal iron status is influenced by many
other biological, lifestyle, and even social factors. According to published research, genetic alterations,
ethnicity, obstetric history, toxic habits (i.e., smoking or alcohol), and socioeconomic status (SES) could
have a defining role [21–25].
On the other hand, unnecessary or excessive iron supplementation might generate high levels
of Hb, also known as the risk of hemoconcentration, in the second and third trimesters of pregnancy.
This condition, which affects between 8.7% and 42% of pregnancies in industrialized countries [26,27],
increases oxidative stress and blood viscosity, causing placental infarction and hindering the perfusion
of oxygen and nutrients to the fetus [28–31]. Although hemoconcentration can be as harmful as iron
deficiency for maternal health and children’s health, in clinical practice, iron supplementation is usually
not adjusted to fit iron status.
The primary aim of this study was to evaluate the effectiveness of iron supplements during
pregnancy in different doses adjusted to the Hb levels of the first trimester. As secondary outcomes,
we described the percentage of ID, anemia, IDA, and risk of hemoconcentration in a large sample
of pregnant Spanish women and the prenatal factors associated with maternal iron status at the end
of pregnancy.
2.1. Study Design

The ECLIPSES study [32] was a community randomized controlled trial (RCT) conducted in the
province of Tarragona (Catalonia, Spain) between 2013 and 2017. The 791 participants were contacted
in their primary care centers during the first routine visit with midwives and were included in the trial
according to the following inclusion criteria: over 18 years of age, gestation time ≤12 weeks, no lab
indication of anemia (Hb ≥ 110 g/L on week 12), ability to understand the official State languages
(Spanish or Catalan), and the ability to understand the characteristics of the study. Women with multiple
pregnancy, adverse obstetric history, those who had taken >10 mg iron daily during the three months
prior to week 12 of gestation, and those who reported a previous severe illness (immunosuppression)
78
Nutrients 2019, 11, 2418
or chronic disease that could affect their nutritional status (cancer, diabetes, malabsorption, or liver
disease) were excluded. A signed informed consent was obtained from all participants.
The participants were allocated into two strata according their initial Hb levels on week 12 of
pregnancy, as follows:
(1) Stratum 1: women with initial Hb levels between 110 and 130 g/L were prescribed 40 or
80 mg/d of iron supplementation.
(2) Stratum 2: women with initial Hb levels > 130 g/L were prescribed 40 or 20 mg/d iron
supplementation. Although in clinical practice, only plasma Hb and serum ferritin (SF) levels are
measured, we suspected that women with initial Hb > 130 g/L could have some alteration in the HFE
gene which would predispose them to iron overload.
In addition to the recruitment visit before the 12th week of gestation, the study consisted of three
visits throughout the pregnancy: at the 12th, 24th, and 36th weeks of gestation. Separately, the women
attended routine pregnancy visits with their midwives and obstetricians.
During the first visit (12th week), the midwives delivered the supplements to the participants
according to the intervention group to which they had been assigned. The prescription of each dose
of supplements within the groups was randomized and triple blinded. The laboratories Tedec–Meiji
made the same box for all different doses of supplements, so that the laboratory technicians, the clinical
staff and the researchers did not know the dose of iron received by each woman until the study ended.
Women were advised to take one pill per day until the next visit, at which time they had to return any
left-over pills to evaluate adherence. An independent investigator compared the number of pills left
over with the compliance reported by the participants. Good compliance was considered for women
who reported having forgotten to take the supplement less than twice per week at every visit of the
study. When they reported forgetting two or more times per week in any of the visits, compliance was
considered low.
If women developed anemia in the middle of pregnancy (24th week), they received the usual
treatment for anemia.
The sample size was calculated according to previous data from our research group [9,33],
taking into account the risk of IDA and hemoconcentration during the third trimester of pregnancy as
principal variables [32]. The study was designed in agreement with the Declaration of Helsinki/Tokyo.
All procedures involving human subjects were approved by Clinical Research Ethics Committee of the
Jordi Gol University Institute for Primary Care Research (Institut d’ Investigació en Atenció Primària;
IDIAP), the Pere Virgili Health Research Institute (Institut d’Investigació Sanitària Pere Virgili; IISPV),
and the Spanish Agency for Medicines and Medical Devices (Agencia Española del Medicamento y
Productos Sanitarios; AEMPS). Signed, informed consent was obtained from all women participating
in the study. This clinical trial was registered at www.clinicaltrialsregister.eu as EudraCT number
2012-005480-28 and at www.clinicaltrials.gov with identification number NCT03196882.
2.2. Data Collection
2.2.1. Baseline Data (on Week 12 of Gestation)

Midwives and researchers of the study (dietitians) compiled clinical and obstetrical data from
participants during the first visit. They obtained the following information during the personal
interview and from specific questionnaires: date of birth, weight, height, blood pressure, parity (yes/no),
number of previous children, planned pregnancy (yes/no), previous use of contraceptives (yes/no),
and type of contraceptives. Medical and surgical history and obstetric data were also recorded.
Maternal age was classified as <25 years, 25–34 years, and ≥35 years. Each maternal pre–pregnancy
body mass index (BMI, Kg/cm2 ) was categorized as underweight (BMI < 18.5), normal weight (BMI
18.5–24.9), overweight (BMI 25–29.9), or obese (BMI ≥ 30).
The dietary assessment was obtained using a short food frequency questionnaire (FFQ) validated in
our population [34] and filled by participants at each visit of the study. From this information, we were
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able to calculate the percentage of adherence to the Mediterranean diet [35], considered a high–quality
dietary pattern. In addition, women were asked about their use of multivitamin supplements,
including >10mg iron, which constituted an exclusion criteria for this study.
Lifestyle habits before conception were also recorded, including alcohol intake and smoking.
To assess smoking, we used the Fagerström test [36] and women were classified as smokers and
non-smokers at the first visit of the study. The International Physical Activity Questionnaire (IPAQ) [37]
was used to record the physical activity (PA) of participants. They reported the time spent doing
exercise of different intensity (vigorous, moderate or a walk lasting at least 10 minutes) during the
previous week; the information was recorded as “days per week” in which physical activity of each
intensity was performed, and the “hours” and “minutes” dedicated in each of those days. Women also
reported amount of time spent sitting during a typical day. We used these data to calculate the
metabolic equivalents of task.
Sociodemographic data of participants and their partners were also recorded. The educational
level was classified into four groups: unfinished primary school (<12 years old), primary school
(up to 12 years old), secondary school (up to 18 years old) and higher education, which included
university and vocational studies. Regarding occupational status, women were classified as students,
employed or unemployed. Women in employment were asked about their profession, which was
classified following the Catalan Classification of Occupations (CCO-2011) [38]. All this information
was used to calculate the family’s socioeconomic status (SES).
Regarding ethnicity, five categories were used: Caucasian, Latin American, Asian, Arab, and Black.
Blood samples were taken on week 12 of gestation to perform blood and genetics tests.
Hematological parameters (Hb, mean corpuscular volume (MCV), and hematocrit) and some specific
biochemical markers (serum ferritin (SF) and C–reactive protein (CRP)) were measured, and genetic
mutations of the HFE gene (C282Y, H63D, and S65C) were checked for. The samples were stored in the
BioBank for future use.
2.2.2. Data Recorded during Scheduled Study Visits

Diet and physical activity were also evaluated at 24th and 36th weeks of gestation. In addition,
blood was collected during both visits to analyze routine blood parameters, including Hb levels.
On week 36, SF levels were also measured.
Any adverse effect from the supplementation was recorded and included in the statistical analyses.
2.2.3. Definition of Iron Status

Anemia was defined as Hb < 110 g/L at 12th and 36th weeks and Hb < 105 g/L at 24th week
of gestation. ID was defined as SF < 15 μg/L and IDA as anemia and one of the following criteria:
SF < 15 μg/L or MCV < 70 fL. SF levels ≥ 15 μg/L was considered as non–deficient or normal iron stores.

All statistical analyses were performed for the population by intention to treat (ITT) and
per-protocol. The population by ITT considered all the participants that were initially included
in the study; the per-protocol population, however, consisted only of those participants who complied
with the protocol of the study. In the latter, therefore, we excluded women who developed anemia on
visit 2, at 24 weeks of gestation.
All analyses were performed separating the sample by stratum; i.e., according to the Hb levels in
the first visit of the study. Student’s t-test and ANOVA were used to describe continuous variables
(mean and SD), and the chi-squared test for categorical variables (percentages). Natural logarithm (Ln)
transformation was applied to normalize the distribution of SF, increasing the validity of analyses,
and using the median and interquartile ranges (IQR).
Multivariate regression models (multiple linear regressions and logistic regressions) were used
to assess the effect of different doses of iron supplementation, along with other prenatal predictors,
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on maternal iron status on week 36 of pregnancy. The models were adjusted for the following variables:
maternal age, parity, socioeconomic status, use of hormonal contraception prior to getting pregnant,
planned pregnancy, smoking habit, alcohol intake, pre–pregnancy maternal BMI, gestational weight
gain, Hb on week 12 of gestation, SF on week 12 of gestation, CRP on week 12 of gestation, HFE gene
genotypes, maternal ethnic origin, physical activity as weekly mean of metabolic equivalent of task
(METs), and adherence to Mediterranean diet.
Furthermore, adjusted multivariate regression models were performed for each stratum, separating
women with and without ID in the first trimester in order to explore whether iron supplementation
acted differently according to iron reserves at the beginning of pregnancy. They were adjusted for the
same variables previously mentioned, except for SF on week 12 of gestation. To avoid information
overload, the tables only show the statistically significant regression models.
SPSS (version 25.0 for Windows; SPSS Inc., Chicago, IL, USA) was used for statistical analyses.
Statistical significance was set at p < 0.05.
3. Results
Of the total of 791 pregnant women included in the study at week 12 of pregnancy (529 from
Stratum 1 and 262 from Stratum 2), the data shown in this article are based on the population by ITT,
which consisted of of 534 women with data on week 36 (354 from Stratum 1 and 180 from Stratum 2).
Attrition was due to: voluntary abandonment (22.75%); miscarriage (1.64%); emergence of exclusion
criteria during pregnancy (5.82%), including serious or chronic illness that could affect the nutritional
development (e.g., cancer, diabetes, and malabsorption); and participants lost to follow up (2.28%).
Attrition was proportional in both Strata, as shown in the Flowchart (Figure 1). In the supplementary
materials, we also show the analyses for the per-protocol population, which excluded anemic women
at 24th week of gestation (11.7% in Stratum 1 and 2.7% in Stratum 2).
Table 1 shows the biological, lifestyle, and sociodemographic characteristics of participants at
baseline. Compared with Stratum 1, women from Stratum 2 had a statistically significant higher
baseline weight (64.83 and 67.17 kg, respectively, p = 0.017) and pre–pregnancy BMI (24.66 and 25.82,
respectively, p = 0.001), and had gained significantly less weight during gestation (11.11 and 9.69 kg,
respectively, p = 0.030). These differences did not translate into a significant effect on maternal iron
status in the multivariate analyses. Table 1 also shows a trend (p = 0.075) toward a higher percentage
of women with previous pregnancies in Stratum 1 (62.3%) than in Stratum 2 (55.7%). No significant
differences in baseline characteristics were detected between women who dropped out of the study
and women who reached the end of the intervention (Table S1).
We excluded the S65C mutation in the HFE gene from the multivariate analyses because of its low
prevalence in our sample. For the same reason, subjects who were homozygous and heterozygous for
H63D, together with the combined heterozygote H63D/C282Y, were grouped as “carrier of the H63D
mutation.” We compared, therefore, three categories of mutation of the HFE gene in the multivariate
analyses: wild type (WT/WT), heterozygous for C282Y/WT, and carrier of the H63D mutation. A similar
situation occurred with maternal ethnic origin: we excluded Asian and Black subjects from subsequent
analyses due to the low representation in the studied population, and only three final categories were
considered: Caucasian, Arab, and Latin American.
Since diet is expected to influence iron status, adherence to the Mediterranean diet was compared
among the different study groups (Figure 2), but no significant differences were found.
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Figure 1. Flowchart of the study.
80
% Adherence to Mediterranean diet
70
60
50
40
30
20
10
0
80 mg/d 40 mg/d 40 mg/d 20 mg/d
Stratum 1 Stratum 2
Low-Middle High
Figure 2. Adherence to the Mediterranean diet in the different groups of iron supplementation.
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Table 1. Baseline characteristics of the study population.
Stratum 1 (n = 529) Stratum 2 (n = 262) p

Mean SD Mean SD
Age, years 30.40 5.07 30.21 5.28 0.613
Weight, Kg 64.83 11.31 67.17 13.60 0.017
Pre–pregnancy BMI, Kg/m2 24.66 4.13 25.82 5.08 0.001
Gestational weight gain, Kg 11.11 8.17 9.69 9.47 0.030
% (n) % (n)
Smoking 16.6 (88) 20.2 (53) 0.214
Parity 62.3 (329) 55.7 (146) 0.075
Planned pregnancy 79.8 (422) 80.5 (211) 0.801
Use of hormonal
18.3 (97) 18.1 (47) 0.929
contraception
Pre–pregnancy BMI
Underweight 1.3 (7) 2.3 (6) 0.314
Normal weight 60.9 (322) 51.5 (135) 0.012
Overweight 25.7 (136) 27.9 (73) 0.518
Obesity 12.1 (64) 18.3 (48) 0.018
HFE gene mutation 31.7 (130) 32.53 (68) 0.834
HFE genotype
WT/WT 67.3 (280) 66.2 (141) 0.779
C282Y/WT 3.8 (16) 2.8 (6) 0.506
Carrier of H63D mutation 27.4 (114) 29.1 (62) 0.652
Carrier of S65C mutation 1.4 (6) 1.9 (4) 0.679
Family socioeconomic status
Low 16.4 (87) 15.6 (41) 0.774
Middle 66.7 (353) 67.6 (177) 0.816
High 16.8 (89) 16.8 (44) 0.991
Maternal ethnic origin
Caucasian 82.8 (405) 82.9 (203) 0.991
Asian 0.2 (1) 0.8 (2) 0.221
Arab 7.8 (38) 8.2 (20) 0.853
Black 1.8 (9) 0.4 (1) 0.114
Latin American 7.4 (36) 7.7 (19) 0.849
Adherence to Mediterranean
diet
Low–Middle 64.7 (342) 71.0 (186) 0.075
High 35.3 (187) 29.0 (76) 0.075
BMI: body mass index; WT: wild type. Sample size HFE genotype = 629; sample size maternal ethnic origin = 734.
We also performed a bivariate analysis comparing the percentage of women with and without
risk of hemoconcentration on week 36 of gestation based on their initial Hb levels and HFE genotypes.
As shown in Figure 3, we found that the H63D mutation in the HFE gene was significantly more
prevalent among women from Stratum 2 (initial Hb levels > 130 g/L) who developed iron overload,
compared with women who completed the pregnancy without risk of hemoconcentration (41.4% and
19.8%, respectively, p = 0.045). Similar results were obtained regarding the S65C mutation, which was
observed in 6.9% of women who showed risk of hemoconcentration at the end of gestation, compared to
0.8% of women with normal Hb levels in the last trimester (p = 0.031). On the other hand, women with
wild type (WT) genotype, i.e., without mutations in the HFE gene, were significantly more prevalent in
the group from Stratum 2 who finished the pregnancy without risk of excess iron, than among women
with Hb levels above 130 g/L on week 36 of gestation (74.6% and 51.7%, respectively, p = 0.015).
In Table 2 we describe and compare the blood tests results of women on weeks 12 and 36 of
gestation among the intervention groups; a significant difference (p = 0.042) was observed in SF levels
at week 36 between 80 and 40 mg/d iron in Stratum 1 (median: 17.19, IQR: 11.53, and median: 14.70,
IQR: 9.37, respectively) in the non–adjusted bivariate analyses. Table 2 also shows that the prevalence
of ID on week 36 was significantly higher (p = 0.012) in the group receiving 40 mg iron per day (51%)
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than in women receiving 80 mg daily (38.2%). No other significant differences were observed between
groups regarding prevalence of various iron states, although the risk of hemoconcentration in the third
trimester of pregnancy showed a tendency to be higher among women who received 40 mg daily of
iron (24%) than those receiving 20 mg of iron per day (13.1%). The same results were obtained in the
per–protocol population (Table S2).
Figure 3. Percentage of women with and without risk of hemoconcentration (Hc) on week 36 of
pregnancy, according to their initial hemoglobin (Hb) levels and HFE genotypes.
Multivariate analyses were performed to explore the effectiveness of iron dosages evaluated
in Stratum 1 (80 mg/d and 40 mg/d) and Stratum 2 (40 mg/d and 20 mg/d), as well as the impact
of several possible prenatal determinant factors. The results of the adjusted multivariate analyses
for Stratum 1, summarized in Table 3, show that taking an iron supplement of 40 mg/d instead of
80 mg/d significantly reduced SF levels (p = 0.026) and doubled the risk of ID (p = 0.022) at the end
of pregnancy. In contrast, the intervention with different doses of iron did not significantly change
Hb levels (p = 0.718), the risk of anemia (p = 0.166), or IDA (p = 0.299). SF levels in early pregnancy
were positively associated with Hb levels (β: 1.70; SE: 0.66; p = 0.010) and SF levels (β: 0.60, SE: 0.04,
p < 0.001) in the third trimester. Additionally, maternal age 35 years and above increased SF in week 36
of pregnancy (β: 0.21; SE: 0.07; p = 0.002). Increasing early pregnancy levels of SF showed a protective
effect against ID (OR: 0.29; 95%CI: 0.19–0.45; p < 0.001), anemia (OR: 0.54; 95%CI: 0.32–0.90; p = 0.018),
and IDA (OR: 0.32; 95%CI: 0.17–0.59; p < 0.001). No differences were observed between the iron
dosages evaluated in Stratum 1 in relation to the risk of hemoconcentration at week 36 of gestation
after adjusting for possible confounders (p = 0.481). The adjusted multiple linear regression model
for the risk of hemoconcentration was not statistically significant (p = 0.071). Moreover, in Stratum
1, when the regression models were performed separating women with and without ID on week 12
(Table 4), we observed that only in women with ID, the dose of 80 mg/d instead of 40 mg/d increased
Hb levels in the third trimester (β: 8.81; SE: 2.40; p = 0.001), protecting women against anemia and IDA
(OR: 0.03; 95%CI: 0.01–0.60; p = 0.021, for both cases).
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Table 2. Blood tests results of participants on week 36 of gestation according to supplementation dose.
Stratum 1 Stratum 2
80 g/d 40 g/d p 40 g/d 20 g/d p
12th week
Hemoglobin (g/L) 123.26 (5.32) 123.44 (4.77) 0.689 135.68 (4.59) 136.61 (4.44) 0.098
Serum ferritin (μg/L) 38.95 (26.10) 38.20 (25.05) 0.740 38.50 (28.98) 40.75 (30.00) 0.965
Mean corpuscular volume (fL) 87.08 (6.36) 87.30 (6.63) 0.696 88.53 (3.43) 88.54 (3.74) 0.980
C–reactive protein (mg/L) 0.73 (0.62) 0.74 (0.72) 0.815 0.72 (0.54) 0.70 (0.53) 0.779
Iron deficiency (%) 14.2 (38) 14.2 (37) 0.999 14.4 (19) 12.3 (16) 0.620
36th week
Hemoglobin (g/L) 117.63 (7.55) 117.21 (8.35) 0.622 123.07 (10.19) 121.04 (8.85) 0.157
Serum ferritin (μg/L) 17.19 (11.53) 14.70 (9.38) 0.042 11.10 (8.10) 11.00 (6.80) 0.798
Mean corpuscular volume (fL) 89.31 (6.87) 88.19 (12.06) 0.261 90.23 (4.19) 89.61 (4.11) 0.299
C–reactive protein (mg/L) 0.76 (0.74) 0.71 (0.64) 0.470 0.70 (0.69) 0.75 (0.56) 0.593
Iron deficiency (%) 38.2 (71) 51 (98) 0.012 66 (68) 69.7 (62) 0.590
Iron deficiency anemia (%) 8.5 (15) 9.6 (17) 0.711 7.3 (7) 11.9 (10) 0.291
Anemia (%) 11.9 (21) 13 (23) 0.747 8.3 (8) 11.9 (10) 0.426
Hemoconcentration (%) 6.8 (12) 7.9 (14) 0.684 24 (23) 13.1 (11) 0.063
Continuous variables expressed as means (SD), except for serum ferritin, which is expressed as median (interquartile
range). Categorical variables expressed in percentages (n).
Table 3. The effects of the intervention with iron supplementation (40 or 80 mg/day) throughout
pregnancy on hemoglobin and serum ferritin levels and on the risk of iron deficiency (ID), anemia,
iron deficiency anemia (IDA), and hemoconcentration on the third trimester in women from Stratum 1.
Hemoglobin levels
Independent variables β SE p Model
R2 = − 0.002
a Intervention (0:80 mg/d, 1:40 mg/d) −0.42 0.85 0.622
p = 0.622
b Intervention (0:80 mg/d, 1:40 mg/d) 0.33 0.92 0.718
R2 = 0.031
Hemoglobin on week 12 of pregnancy 0.25 0.10 0.015
p = 0.050
Serum ferritin on week 12 of pregnancy 1.70 0.66 0.010
Serum ferritin levels
R2 = 0.004
p = 0.085
c Intervention (0:80 mg/d, 1:40 mg/d) −0.12 0.05 0.026
Serum ferritin on week 12 of pregnancy 0.60 0.04 <0.001 R2 = 0.436
Maternal age (0:25–34 years, 1:<25 years) 0.05 0.08 0.559 p< 0.001
Maternal age (0:25–34 years, 1:≥35 years) 0.21 0.07 0.002
Iron deficiency (0:no, 1:yes)
Independent variables OR 95% CI p Model
a R2 Nagelkerke = 0.022
Intervention (0:80 mg/d, 1:40 mg/d) 1.69 1.12–2.54 0.012
p = 0.012
c Intervention (0:80 mg/d, 1:40 mg/d) 1.82 1.09–3.03 0.022 R2 Nagelkerke = 0.241
Serum ferritin on week 12 of pregnancy 0.29 0.19–0.45 <0.001 p < 0.001
Anemia (0:no, 1:yes)
p = 0.747
bIntervention (0:80 mg/d, 1:40 mg/d) 1.70 0.80–3.61 0.166
R2 Nagelkerke = 0.146
Planned pregnancy (0:no, 1:yes) 3.57 1.00–12.80 0.050
p = 0.027
Serum ferritin on week 12 of pregnancy 0.54 0.32–0.90 0.018
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Table 3. Cont.
Iron–deficiency anemia (0:no, 1:yes)

p = 0.711
b Intervention (0:80 mg/d, 1:40 mg/d) 1.58 0.67–3.71 0.299 R2 Nagelkerke = 0.19
Serum ferritin on week 12 of pregnancy 0.32 0.17–0.59 <0.001 p = 0.004
Hemoconcentration (0:no, 1:yes)
p = 0.684
b Intervention (0:80 mg/d, 1:40 mg/d) 1.44 0.52–3.97 0.481
Genotype HFE (0:WT/WT, 1: carrier of H63D) 3.28 0.09–6.68 0.026 R2 Nagelkerke = 0.166
Genotype HFE (0:WT/WT, 1: C282Y/WT) 1.93 0.21–18.02 0.566 p = 0.071
Parity (0:no, 1:yes) 0.26 0.09–0.76 0.014
a Crude model. b Adjusted for: iron supplementation dosage, maternal age, use of hormonal contraception,
pre–pregnancy maternal body mass index, gestational weight gain, HFE genotypes, maternal ethnic origin,
hemoglobin on week 12, serum ferritin on week 12, c–reactive protein on week 12, socioeconomic status, weekly mean
METS on week 12, smoking habit, alcohol intake, planned pregnancy, parity, mean caloric intake during pregnancy,
and adherence to a Mediterranean diet. c Adjusted for: model b, except for hemoglobin on week 12.
Table 4. The effect of the intervention with iron supplementation in Stratum 1 (0:80 mg/d, 1:40 mg/d)
throughout pregnancy on maternal iron status on the third trimester, according to their initial iron stores.
SF < 15 μg/L
Hemoglobin levels β SE p Model
R2 = 0.129
Crude model – 7.06 2.43 0.006
p = 0.006
a R2 = 0.32
Adjusted model – 8.81 2.40 0.001
p = 0.003
Iron deficiency (0:no, 1:yes) OR 95% CI p Model
Crude model 3.10 0.93–10.39 0.066
p = 0.060
b R2 Nagelkerke = 0.429
Adjusted model 4.51 0.78–26.08 0.092
p = 0.013
Anemia (0:no, 1:yes) OR 95% CI p Model
Crude model 5.50 1.05–28.75 0.043
p = 0.025
Adjusted model 29.14 1.67–508.56 0.021
p = 0.020
Iron–deficiency anemia (0:no, 1:yes) OR 95% CI p Model
Crude model 5.50 1.05–28.75 0.043
p = 0.025
Adjusted model 29.14 1.67–508.56 0.021
p = 0.020
SF≥15 μg/L
R2 = −0.001
Crude model 0.75 0.88 0.395
p = 0.395
a R2 = 0.035
Adjusted model 0.42 0.96 0.664
p = 0.031
a Adjusted for: iron supplementation dosage, maternal age, use of hormonal contraception, pre–pregnancy maternal
body mass index, gestational weight gain, HFE gene genotypes, maternal ethnic origin, hemoglobin on week
12, c–reactive protein on week 12, socioeconomic status, weekly mean of METS on week 12, smoking habit,
alcohol intake, planned pregnancy, parity, mean caloric intake during pregnancy, and adherence to Mediterranean
diet. b Adjusted for: model a, except for hemoglobin on week 12.
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Similarly, Table 5 shows the results of multivariate analyses performed after selecting women from
Stratum 2. Adjusting for possible confounding factors, we found that a daily iron supplementation of
20 mg as opposed to 40 mg during pregnancy reduced the risk of hemoconcentration by 69% (p = 0.035)
without increasing the risk of any iron deficit states studied at the end of pregnancy. Similarly to
Stratum 1, higher SF levels on week 12 of gestation were positively correlated with SF levels (β: 0.42;
SD: 0.06; p < 0.001) in the last months. Increasing SF levels in early pregnancy protected, therefore,
against ID (OR: 0.36; 95%CI: 0.19–0.68; p = 0.002), anemia and IDA (OR: 0.26; 95%CI: 0.08–0.66;
p = 0.023, for both cases). Furthermore, the analyses showed the effect of maternal age on iron status
on week 36, with women under 25 years presenting reduced SF levels (β: –0.28; SE: 0.11; p = 0.013),
and women 35 years and older at lower risk of ID (OR: 0.37; 95%CI: 0.16–0.91; p = 0.029) than women
between 25 and 34 years of age. It was also found that the middle–high SES, compared with low SES,
protected against anemia and IDA (OR: 0.06; 95%CI: 0.01–0.40; p = 0.003, for both cases) in women
who started pregnancy with Hb levels above 130 g/L. Regarding iron overload, in addition to the
aforementioned effect of the low iron dose, higher Hb levels early in pregnancy and being a carrier of
the H63D mutation significantly increased Hb levels on week 36 (β: 0.72; SE: 0.16, and β: 3.93; SE: 1.74,
respectively) and the risk of hemoconcentration (OR: 1.20; 95%CI: 1.08–1.33, and OR: 3.09; 95%CI:
1.10–8.71, respectively). When the multivariate analyses were applied to the sample of women from
Stratum 2, categorized according their initial iron stores, we found that compared to 20 mg, 40 mg of
iron per day increased SF on week 36 (β: 0.39; SE: 0.15; p = 0.014) only in women with iron deficiency,
while 20 mg/d reduced the risk of hemoconcentration (OR: 0.25; 95%CI: 0.07–0.85; p = 0.027) in women
with initial iron stores within the normal range (Table 6).
Table 5. The effects of the intervention with iron supplementation (40 or 20 mg/day) throughout
pregnancy on hemoglobin and serum ferritin levels and on the risk of ID, anemia, IDA,
and hemoconcentration on the third trimester in women from Stratum 2.
Hemoglobin levels
R2 = 0.004
p = 0.188
b Intervention (0:40 mg/d, 1:20 mg/d) −2.50 1.47 0.092
Genotype HFE (0:WT/WT, 1: carrier of H63D) 3.93 1.74 0.025 R2 = 0.116
Genotype HFE (0:WT/WT, 1: C282Y/WT) 1.34 3.70 0.718 p = 0.003
Hemoglobin on week 12 of pregnancy 0.72 0.16 <0.001
Serum ferritin levels
a R2 = −0.003
Intervention (0:40 mg/d, 1:20 mg/d) 0.02 0.07 0.734
p = 0.734
cIntervention (0:40 mg/d, 1:20 mg/d) 0.01 0.08 0.954
Maternal age (0:25–34 years, 1:<25 years) −0.28 0.11 0.013 R2 = 0.218
Maternal age (0:25–34 years, 1:≥35 years) 0.12 0.10 0.221 p < 0.001
Serum ferritin on week 12 of pregnancy 0.42 0.06 <0.001
Low iron stores (0:no, 1:yes)
p = 0.590
cIntervention (0:40 mg/d, 1:20 mg/d) 1.45 0.69–3.02 0.326
Maternal age (0:25–34 years, 1:<25 years) 3.07 0.78–12.12 0.109 R2 Nagelkerke = 0.229
Maternal age (0:25–34 years, 1:≥35 years) 0.37 0.16–0.91 0.029 p = 0.003
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Table 5. Cont.
Anemia (0:no, 1:yes)

p = 0.426
SES (0:low; 1:middle + high) 0.06 0.01–0.40 0.003
p = 0.002
Iron–deficiency anemia (0:no, 1:yes)
p = 0.291
SES (0:low; 1:middle + high) 0.06 0.01–0.40 0.003
p = 0.002
Hemoconcentration (0:no, 1:yes)
p = 0.060
b Intervention (0:40 mg/d, 1:20 mg/d) 0.31 0.11–0.92 0.035
Hemoglobin on week 12 of pregnancy 1.20 1.08–1.33 0.001 R2 Nagelkerke = 0.282
Genotype HFE (0:WT/WT, 1: carrier of H63D) 3.09 1.10–8.71 0.033 p = 0.004
Genotype HFE (0:WT/WT, 1: C282Y/WT) 0.00 . 0.999
a Crude model. b Adjusted for: iron supplementation dosage, maternal age, use of hormonal contraception,
pre–pregnancy maternal body mass index, gestational weight gain, HFE gene genotypes, maternal ethnic origin,
hemoglobin on week 12, serum ferritin on week 12, c–reactive protein on week 12, socioeconomic status, weekly mean
of METS on week 12, smoking habit, alcohol intake, planned pregnancy, parity, mean caloric intake during pregnancy,
and adherence to a Mediterranean diet. c Adjusted for: model b, except for hemoglobin on week 12.
Table 6. The effect of the intervention with iron supplementation on Stratum 2 (0:40 mg/d, 1:20 mg/d)
throughout pregnancy regarding maternal iron status in the third trimester, according to initial
iron stores.
SF<15 μg/L
Serum ferritin levels β SE p Model
R2 = 0.061
Crude model −0.24 0.14 0.083
p = 0.083
R2 = 0.344
b Adjusted model −0.39 0.15 0.014
p = 0.021
Iron deficiency (0:no, 1:yes) OR 95% CI p Model
Crude model 6.11 0.60–62.23 0.126
p = 0.085
b R2 Nagelkerke = 0.924
Adjusted model 42.09 0.00–50.00 0.614
p = 0.001
SF≥15 μg/L
R2= 0.002
Crude model –1.75 1.54 0.260
p = 0.260
a R2 = 0.184
Adjusted model –2.00 1.56 0.200
p < 0.001
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Table 6. Cont.
Serum ferritin levels β SE p Model

R2 = −0.002
Crude model 0.06 0.08 0.455
p = 0.455
b R2 = 0.077
Adjusted model 0.08 0.09 0.368
p = 0.008
Anemia (0:no, 1:yes) OR 95% CI p Model
Crude model 1.34 0.43–4.20 0.611
p = 0.611
Adjusted model 1.02 0.20–5.10 0.984
p = 0.007
Iron–deficiency anemia (0:no, 1:yes) OR 95% CI p Model
Crude model 1.63 0.50–5.39 0.421
p = 0.417
Adjusted model 1.02 0.20–5.10 0.984
p = 0.007
Hemoconcentration (0:no, 1:yes) OR 95% CI p Model
Crude model 0.46 0.20–1.06 0.068
p = 0.061
Adjusted model 0.25 0.07–0.85 0.027
p = 0.001
a Adjusted for: iron supplementation dosage, maternal age, use of hormonal contraception, pre–pregnancy maternal
body mass index, gestational weight gain, HFE gene genotypes, maternal ethnic origin, hemoglobin on week
12, C–reactive protein on week 12, socioeconomic status, weekly mean of METS on week 12, smoking habit,
alcohol intake, planned pregnancy, parity, mean caloric intake during pregnancy, and adherence to a Mediterranean
diet. b Adjusted for: model a, except for hemoglobin on week 12.
In the multivariate analyses of Stratum 2, the results for the per–protocol and for the ITT
populations were the same (Table S4); for Stratum 1, the regression models for Hb levels, anemia and
IDA lost statistical significance when women who were anemic at mid–pregnancy were removed from
the sample. However, the results about the effects on SF levels and ID were the same as for the ITT
population (Table S3).
4. Discussion
Despite the wealth of research on prenatal iron supplementation, there is a lack of consensus
on the optimal iron dosage in relation to the characteristics of each woman. Consequently, we were
determined to investigate the effectiveness of different doses of iron supplementation on preventing
iron deficiency and excess iron in the last trimester of gestation. To our knowledge, few publications
address the interplay of early maternal iron status and the effect of prenatal iron supplementation [39].
Firstly, we observed that the prevalence of ID found in both strata of our study population
(38.2%–69.70%) was in the range of the European estimates for pregnant women published in the
most recent reports [2,3]; regarding the prevalence of anemia (8.3%–13%) and IDA (7.3%–11.9%),
our results were considerably lower than the estimates of the same reports (24.5% and 35%, respectively).
In relation to the risk of hemoconcentration, we observed that its prevalence (~13%) was similar to
previous reports from Spain by Arija et al. [27] and within the wide range reported in European
countries (8.7% to 42%) [26]. We should underscore that most research focuses on iron deficiency,
and only few studies have described the prevalence of excess iron; consequently, the estimates on iron
overload are less updated and not as established. As expected, we observed a significantly higher
prevalence of risk of hemoconcentration in Stratum 2 (13.1% for 20 mg/d and 24% for 40 mg/d) than in
Stratum 1 (6.8% for 80 mg/d and 7.9% for 40 mg/d) at the end of pregnancy. This difference supports
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Nutrients 2019, 11, 2418
our hypothesis that women with normal–high initial Hb levels were at greater risk of iron overload,
possibly due to the persistent effect that genetic alterations in the HFE gene exert on iron levels [40,41].
Our results also show a higher prevalence of HFE gene mutations in women from Stratum 2 at risk
of hemoconcentration on week 36, as opposed to the higher prevalence of the wild type genotype in
women who finished the pregnancy without that risk (see Figure 2). This highlights the influence of the
genetic alteration in the HFE gene on the risk of iron overload in women with initial Hb levels > 130 g/L.
Moreover, within Stratum 2, we found that the percentage of women at risk of hemoconcentration on
week 36 in the group of 20 mg of iron per day was fifty percent less than in the group receiving 40 mg
daily (13.1% and 24%, respectively, p = 0.063), confirming our hypothesis that low iron doses are the
best option in this case.
To clarify the effectiveness of different doses of prenatal iron supplementation on maternal iron
status, the multivariate analyses were adjusted for several associated variables, including obstetric,
biological, and socioeconomic conditions, as well as HFE gene genotype and iron–related blood
parameters. In this regard, in women from Stratum 1 who began the gestation with Hb levels between
110 and 130 g/L, we observed that a daily dosage of 80 mg iron, as opposed to 40 mg, improved SF
levels (b: 0.12, p = 0.026) and protected against ID (OR: 0.55, p = 0.022) at the end of pregnancy.
Furthermore, when we explored the effect of iron supplementation in women within Stratum 1
according their initial iron reserves, we found that the higher dose of iron (80 mg/d) reduced the risk of
anemia and IDA (OR: 0.03 and p = 0.021, for both cases) during the last months of gestation in women
with iron–deficiency (SF < 15 μg/L, 14.2%) at the start of the pregnancy. In contrast, no significant effect
was observed in women with SF ≥ 15 μg/L on week 12. These results respond to the physiological
regulation of intestinal iron absorption in accordance with iron reserves, by which the body strongly
regulates iron absorption when stores are sufficient [42,43]. On the contrary, and in agreement with
Milman et al. [44], we did not find additional effects of high doses of iron in women with correct iron
reserves at the beginning of the study. We can conclude that the usual prescribed dose of 40 mg daily
would be effective in women with optimal initial iron reserves, but not in women with iron deficiency
in early pregnancy.
On the other hand, in Stratum 2 (initial Hb levels >130 g/L), women who received a daily dosage
of 20 mg iron, compared with the group that received 40 mg, reduced the risk of hemoconcentration
in the third trimester (OR: 0.31, p = 0.035), without increasing the risk of iron deficit. In this case,
we should underscore that the risk of iron overload trebles in carriers of the H63D mutation of the HFE
gene (OR: 3.09, p = 0.033). Accordingly, we would advise to prescribe low doses of iron to women with
normal–high Hb (>130 g/L) levels in early pregnancy. Interestingly, the baseline prevalence of ID was
higher than expected in this group (13.4%); similarly to Stratum 1, the different doses produced different
results regarding iron status, which varied in accordance with the initial iron stores. The protective
effect of 20 mg iron per day against the risk of hemoconcentration (OR: 0.25, p = 0.027) was only
observed in women with sufficient iron reserves in early pregnancy (SF ≥ 15 μg/L).
Based on these findings, we emphasize that iron supplementation during pregnancy should
be adapted to the initial iron status of each woman, assessed not only by Hb levels but also by SF
levels, to prevent both iron deficiency and iron overload at the end of gestation. These conclusions
are in agreement with the valuable contributions of Milman et al. [45,46], Casanueva et al. [47],
and Peña-Rosas and Viteri [26], who advocate adapting prenatal iron supplementation in view that
both iron deficit and hemoconcentration have been associated with negative effects on maternal–child
health [5–10,28–30].
Generally, in clinical practice only Hb levels are measured to monitor maternal iron status during
pregnancy. However, while detecting anemia, Hb levels fail to diagnose ID. Our results show that the
effects of iron supplementation vary as a function of initial iron reserves, indicating the importance of
detecting ID at the beginning of the gestation. We advocate for the routine measurement of SF levels
during antenatal checks. We also underscore that mutations in the HFE gene should be studied in
women with normal–high Hb levels at the beginning of pregnancy to avoid excessive iron supply.
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Nutrients 2019, 11, 2418
Indeed, in relation to this, it is known that there is a racial difference in the prevalence of alterations
in the HFE gene, being greater in the populations of northern Europe than in the Mediterranean
countries [24]. This adds even more weight to the premise that it is necessary to evaluate the individual
characteristics of women to prescribe the most efficient prenatal iron supplementation in each case.
In this study, the multivariate analyses have also revealed some prenatal determinants of maternal
iron status at the end of pregnancy. For instance, high SF levels on week 12 were associated with the
increase of Hb and SF levels on week 36 in both strata, reducing the risk of all iron deficiency states:
71% and 64% lower risk of ID, 68% and 74% lower risk of IDA, and 46% and 74% lower risk of anemia
for Stratum 1 and Stratum 2, respectively (see Tables 3 and 4). The results show that SF levels on week
36 increased with maternal age, and in Stratum 2, maternal age was also linked to the risk of ID in the
third trimester of gestation, although to our knowledge, the underlying mechanism of this association
is not yet elucidated. We found a protective role of middle–high SES against anemia and IDA (OR: 0.06,
p = 0.003, for both cases), specifically in women from Stratum 2. This finding coincides with previous
reports that conclude that low–income status is a risk factor for iron deficiency, presenting as ID,
anemia and IDA, especially in developing countries [23,48,49]. This observation stresses that a low
SES might be associated with less healthy lifestyles and under-attendance to antenatal care [50,51].
Also in agreement with other studies [40,52,53], we found that the H63D mutation in the HFE gene
increased Hb levels (b: 3.93, p = 0.025) and trebled the risk of hemoconcentration (OR: 3.09, p = 0.033)
on week 36. It is well established that mutations in the HFE gene are highly prevalent in Caucasian
populations and that they are linked to iron overload [3,54]. It has been suggested that HFE gene
mutations increase intestinal iron absorption [41,55]. In our study, therefore, the results suggest that
the presence of some mutation in the HFE gene would increase iron absorption in women with initial
Hb levels >130mg/L. Unexpectedly, maternal iron status was not significantly associated with diet in
the multivariate analyses in any strata. Similarly, comparative analyses, including adherence to the
Mediterranean diet failed to show significant differences between different supplementation groups.
This result suggests that the diet was very similar among all the women in the study. Finally, the
trend for a higher percentage of parity in Stratum 1 (62.3%) than in Stratum 2 (55.7%) suggests that
previous births could weaken the iron status of women at the beginning of pregnancy. Interestingly, in
the multivariate analyses parity seemed to reduce by 74% the risk of hemoconcentration in women of
Stratum 1, but the results in the regression model were not statistically significant (p = 0.071).
Understanding that the prenatal iron supplementation has a different effect on maternal iron
status at the end of pregnancy according to initial levels of Hb and SF could contribute to improving
public health policies and to adapting clinical practices to the population groups at risk. Taking into
consideration other associated prenatal determinants of maternal iron status can also improve antenatal
care. In view of the evidence presented in this study, we emphasize firstly, the importance of full iron
reserves before pregnancy, in preparation for the high cost of iron during gestation; and secondly,
we recommend that clinicians adapt iron supplementation to the initial levels of Hb and iron
reserves (see Figure 4). To assess the presence of genetic mutations in the HFE gene in women with
normal–high Hb levels and full iron reserves at the beginning of pregnancy can help to reduce the risk
of hemoconcentration in this group.
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Nutrients 2019, 11, 2418
Figure 4. Adaptation of prenatal iron supplementation according the individual characteristics of

women in the first trimester of pregnancy.
Strengths and Limitations

The main strengths of the current community RCT are the large sample size (n = 791) and the
extensive data collection regarding sociodemographic conditions, clinical information, obstetric data,
and lifestyle, including diet and physical activity. In addition, testing for HFE gene mutations has added
valuable information on the effect of genetic variability on iron metabolism and on the possible impact
of personalized iron supplementation. Methodologically, we were able to evaluate the progression
of iron status by monitoring blood parameters at different stages of pregnancy. However, some
limitations must be taken into account when interpreting the findings of this study. Firstly, the notable
dropout rate, although this is not uncommon in community interventions such as ours, which require
several visits. No woman dropped out due to gastrointestinal side effects, since we used ferrimanitol
ovalbumin instead of ferrous sulfate in our study. Another limitation was the lack of SF measurements
in the 24th week of pregnancy, which would have strengthened the results. Since women gave birth in
hospitals, data on maternal iron status at delivery were not available for inclusion.
5. Conclusions
In conclusion, we advise routine monitoring of Hb and SF during antenatal check–ups. These tools
can be used in clinical practice to prescribe the optimal dose of iron supplements, with the ultimate
aim of achieving the best pregnancy outcomes. In addition, the study of mutations in the HFE
gene in women with normal–high Hb levels at the beginning of pregnancy could reduce the risk of
hemoconcentration. Further studies are needed to assess the effect of mutations in the HFE gene on the
maternal iron status and its interplay with prenatal iron supplementation to determine if there is a
real need to use supplements in these cases. Future studies should also assess whether, in addition to
the benefits for pregnant women, the supplementation with different doses of iron have benefits for
their children.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/10/2418/s1,

Table S1: Baseline characteristics of the population lost trhought the study; Table S2: Biochemical characteristics of
participants at 36th week of gestation according to dose of supplementation (by protocol); Table S3: Effect of the
intervention with iron supplementation (40 or 80 mg/day) through pregnancy on hemoglobin and serum ferritin
levels and on the risk of ID, anemia, IDA and hemoconcentration at third trimester in women from Stratum 1
(by protocol); Table S4: Effect of the intervention with iron supplementation (40 or 20 mg/day) through pregnancy
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Nutrients 2019, 11, 2418
on hemoglobin and serum ferritin levels and on the risk of ID, anemia, IDA and hemoconcentration at third
trimester in women from Stratum 2 (by protocol).
Author Contributions: Conceptualization: V.A. and N.A.; data curation: L.I.V., N.A., E.A., and M.P.;
formal analysis: L.I.V.; funding: V.A., N.A., E.A., N.S., and F.F.; investigation: L.I.V., V.A., N.A., E.A., N.S.,
F.F., F.R., and J.B.; project administration: V.A. and J.B.; resources: V.A., P.C., M.G., and J.B.; supervision: V.A.;
data visualization: L.I.V.; writing (original draft): L.I.V.; writing (review and editing): L.I.V. and V.A.
Funding: This research was funded by the Health Research Fund of the Ministry of Health and Consumer Affairs
(Madrid, Spain) (Instituto de Salud Carlos III, Fondo de Investigación Sanitaria, Ministerio de Sanidad y Consumo)
with the grant number PI12/02777.
Acknowledgments: We thank to Meiji Pharma Spain S.A. –formerly Tedec-Meiji Farma S.A– (Pilar Coronel and
Mercedes Gimeno) for logistic assistance and for providing free iron supplements. Research Group in Nutrition
and Mental Health (NUTRISAM), Universitat Rovira i Virgili (Victoria Arija, Josepa Canals, Estefanía Aparicio,
Núria Aranda, Cristina Bedmar, Carmen Hernández, Lucía Iglesias, Cristina Jardí and Núria Voltas). Sexual and
Reproductive Health Care Services (ASSIR) of Tarragona, Spain (Francesc Fargas, Francisca Ruiz, Gemma March,
Susana Abajo) and team of midwives recruiting for the study (Irene Aguilar, Sònia Aguiles, Rosa Alzúria,
Judit Bertrán, Carmen Burgos, Elisabet Bru, Montserrat Carreras, Beatriz Fernández, Carme Fonollosa, María Leiva,
Demetria Patricio, Teresa Pinto, María Ramírez, Eusebia Romano and Inés Sombreo). Jordi Gol University Institute
for Primary Care Research (IDIAP) (Josep Basora and Meritxell Pallejà from the Research Unit in Tarragona;
and Rosa Morros, Helena Pere and Anna García Sangenis from the Central Unit in Barcelona). Laboratory of the
Institut Català de la Salut (Núria Serrat).
Conflicts of Interest: The authors declare no conflict of interest. The sponsors had no role in the design, execution,
interpretation, or writing of the study.
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nutrients
Article
Fermented Goat Milk Consumption Enhances Brain
Molecular Functions during Iron Deficiency
Anemia Recovery
Jorge Moreno-Fernández 1,2 , Inmaculada López-Aliaga 1,2 , María García-Burgos 1,2 ,
María J.M. Alférez 1,2, * and Javier Díaz-Castro 1,2
1 Department of Physiology, Faculty of Pharmacy, Campus Universitario de Cartuja, E-18071 Granada, Spain;
jorgemf@ugr.es (J.M.-F.); milopez@ugr.es (I.L.-A.); mariagb@ugr.es (M.G.-B.); javierdc@ugr.es (J.D.-C.)
2 Institute of Nutrition and Food Technology “José Mataix Verdú”, University of Granada,
E-18071 Granada, Spain
* Correspondence: malferez@ugr.es; Tel.: +34-958-243883
Received: 30 July 2019; Accepted: 18 September 2019; Published: 7 October 2019
Abstract: Iron deficiency anemia (IDA) is one of the most prevalent nutritional deficiencies worldwide.
Iron plays critical roles in nervous system development and cognition. Despite the known detrimental
consequences of IDA on cognition, available studies do not provide molecular mechanisms elucidating
the role of iron in brain functions during iron deficiency and recovery with dairy components. In
this study, 100 male Wistar rats were placed on a pre-experimental period of 40 days and randomly
divided in two groups: a control group receiving a normal-Fe diet, (45 mg/kg), and an Fe-deficient
group receiving a low-Fe diet (5 mg/kg). At day 40, 10 rats per group were sacrificed to anemia
control, and 80 rats were divided into eight experimental groups fed with fermented goat or cow
milk-based diets, with normal Fe content or Fe overload (450 mg/kg) for 30 days. IDA decreased
most of the parameters related to brain molecular functions, namely dopamine, irisin, MAO-A,
oxytocin, β-endorphin, and α-MSH, while it increased synaptophysin. These alterations result in
an impairment of brain molecular functions. In general, during anemia recovery, fermented goat
milk diet consumption increased dopamine, oxytocin, serotonin, synaptophysin, and α-MSH, and
decreased MAO-A and MAO-B, suggesting a potential neuroprotective effect in brain functions,
which could enhance brain molecular functions.
Keywords: iron deficiency anemia; fermented goat milk; brain molecular functions;
neuroprotective effect
1. Introduction
Despite the global progress achieved in nutrition and science development, iron deficiency (ID)
remains the most prevalent nutritional deficiency worldwide, affecting between 4–6 billion people.
ID is also the main cause of anemia. Two billion people, more than 25% of the world’s population,
has manifestations of iron deficiency anemia (IDA) [1]. Among its many physiological functions, iron
plays key roles in nervous system development and function, via biochemical processes involved in
brain structure and functions. There are several studies on the effects of ID on brain development
and functions [2,3], and on the link between iron status and cognitive function [4,5]. In this sense,
iron is involved in the adequate myelination of the white matter, hippocampus development, and
neurotransmitter homeostasis, with neurophysiological and behavioral outcomes showing relations
to iron status at several life stages [6]. Behavioral performance and brain functions, as measured by
electroencephalography, are sensitive to iron status [5].
Moreover, several studies showed that anemia may be associated with impairment in cerebral blood
flow and oxygen metabolism, impaired cognitive function, confusion, loss of concentration, impaired

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memory, and low mental alertness [7]. Acute anemia results in the slowing of data-processing ability
and memory impairment in humans [8]. In addition, cognitive ability is improved by erythropoietin [9].
Recent studies found several evidences that iron supplementation improved attention and
concentration [5,10], as well as cognitive function [4]. These improvements in psychological symptoms
appear to be mediated by an enhancement of oxygen transport to the brain [11].
In the scientific literature, raised awareness about the effects of nutrition on brain molecular
functions exists, although few studies have examined the cognitive effects of fermented milks on
cognitive function, and none of them linked to iron status. Currently, dairy products are increasingly
consumed to prevent cognitive impairment and dementia [12]. Recent evidence suggests that the
inclusion of dairy products in a balanced diet might have several positive effects on cognitive health in
advanced age [13].
On the other hand, we have reported previously that fermented goat milk consumption is beneficial
in IDA due to the improvement in the key proteins of intestinal iron metabolism, enhancing the
digestive and metabolic utilization of iron, increasing iron deposits in target organs, and favoring the
recovery of hematological parameters [14]. However, despite the known detrimental consequences of
IDA on cognition, research linking iron status to brain functions has largely been ignored, and available
studies are mainly focused on psychological tests evaluating cognitive function, concentration, memory,
and alertness without providing molecular mechanisms elucidating the role of iron in brain functions
during ID and recovery with dairy components. Hence, taking into account all these considerations, we
set out to investigate the impact of fermented goat or cow milk-based diets (with normal or overload
iron content) consumption during IDA recovery on the brain molecular functions in animal models.
2.1. Fermentation and Dehydration of the Milks

Fermented milks were prepared following the previously described method [15].
2.2. Animals
One hundred male Wistar albino breed rats (3 weeks of age and weighing about 34.56 ± 6.35 g)
were included in this study. Experimental procedures were carried out in agreement with the guidelines
about animal welfare and experimentation (Declaration of Helsinki; Directive 2010/63/EU).
Individual, ventilated, and thermos-regulated cages were used to randomly allocate the rats.
The room temperature (22.5 °C), humidity (60%), and a 12-h light-dark cycle were automatically
controlled. Animal weight was taken as a variable factor, and numbers randomly assigned were
generated (Microsoft Excel, 2016 (Microsoft, Redmond, WA, USA). Subsequently, the mean of the
100 weights were compared by a one-way ANOVA. The groups were formed to obtain a probability
level of p = 0.09. The animals of each group had initial weights of 34.28 ± 5.12 g, 34.39 ± 5.01 g,
36.27 ± 4.58 g, 35.23 ± 6.12 g, 36.13 ± 6.37 g, 35.29 ± 6.01 g, 36.13 ± 5.13 g, 37.92 ± 6.21 g, 33.88 ± 6.11 g
and 35.53 ± 5.76 g (p > 0.05), respectively. Diet intake was controlled, pair feeding all the animals, and
bidistilled water was available ad libitum.
2.3. Experimental Design

Figure 1 features the experimental design of the study. During the pre-experimental period (PEP),
(n = 100) rats were divided into two groups: the control group receiving the AIN 93G diet with a
normal Fe diet (n = 50, 44.8 mg/kg by analysis) [16], and the anemic group receiving the same diet,
but with a low Fe content (n = 50, 6.1 mg/kg by analysis), induced experimentally during 40 days
by a method developed previously by our research group [17]. On day 40 of the study, 2 aliquots of
blood were collected from the caudal vein of each rat. One of them had Ethylenediaminetetraacetic
acid (EDTA) to measure all the hematological parameters, and the other one was centrifuged (1500× g,
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4 °C, 15 min) without anticoagulant to separate the serum and subsequent analysis of serum iron, total
iron-binding capacity (TIBC), ferritin, and hepcidin.
Figure 1. The study experimental design. * 10 control and 10 anemic rats were totally bled out by
cannulation of the abdominal aorta. ** 80 animals were totally bled out by cannulation of the abdominal
aorta. AIN-93 G diet, Standard diet from the American Institute of Nutrition (for the growth phase).
When the induction of the anemia period finalized (day 40 of the study), 10 rats per group
were sacrificed, and the remaining 80 animals subsequently started the experimental period (EP) in
which the control (n = 40) and anemic groups (n = 40) were further fed for 30 days with a fermented
cow milk or fermented goat milk-based diet, with normal Fe content (45 mg·kg−1 ) or Fe overloaded
content (450 mg·kg−1 ) to induce chronic Fe overload [18] prepared with fermented cow or goat milk,
as previously reported [14].
At the end of the PEP and the end of EP, 20 and 80 animals respectively were anesthetized
intraperitoneally with sodium pentobarbital (Sigma-Aldrich Co., St. Louis, MO, USA) and totally bled
out. Blood aliquots with EDTA were analyzed to measure the hematological parameters, and the rest
of the blood was centrifuged (1500× g, 4 °C, 15 min) without anticoagulant to separate the red blood
cells from the serum and to determine the parameters related to iron status. The brain was removed
immediately, weighed, and was split into two portions (which included parts of both hemispheres)
and placed on cold saline buffer. Brain samples were homogenized in phosphate-buffered saline (PBS),
pH 7.4 by the homogenizer. Protease inhibitor cocktail (Sigma-Aldrich Co., St. Louis, MO, USA)
was used. The homogenate was centrifuged at 2000× g for 15 min at 4 °C. Thereafter, supernatants
were divided into aliquots, and were stored at −80 ◦ C for further analysis of parameters related
to brain molecular functions. The remaining part was frozen in liquid nitrogen and immediately
stored at −80 ◦ C. This aliquot was used for precipitation with acetonitrile to proceed the neuropeptide
assessment with Milliplex MAP assay.
2.4. Hematological Test

The hematological parameters were measured using an automated hematology analyzer Sysmex
K-1000D (Sysmex, Tokyo, Japan).
2.5. Iron Assessments

Serum iron, total iron-binding capacity (TIBC), and transferrin saturation were determined using
Sigma Diagnostics Iron and TIBC reagents (Sigma, St Louis, MO, USA). Concentrations of serum
ferritin (μg·L−1 ) and serum hepcidin (Hepcidin-25 ng·mL−1 ) were determined using a rat ELISA Kit
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(Biovendor Gmbh, Heidelberg, Germany) for TIBC and another commercial kit for serum ferritin (DRG
Instruments GmbH, Marburg, Germany).
2.6. Dopamine
Dopamine levels in brain homogenate were measured using a commercial enzyme immunoassay
kit (MyBioSource, San Diego, CA, USA). Once the tissues were homogenized, the resulting suspension
was subjected to ultrasonication. After that, the homogenates were centrifugated for 15 min at 1500× g.
Measurements in duplicate were used to determine intra-assay variability.
2.7. Serotonin
The level of serotonin in brain homogenate was determined using a commercially available
enzyme immunoassay ELISA Kit (MyBioSource, San Diego, CA, USA). One hundred microliters of the
homogenate were added to duplicate wells in the ELISA plate, which was then processed according to
the directions of the manufacturer.
2.8. MAO-A and MAO-B

To determine monoamine oxidase A and B (MAO-A and MAO-B) brain homogenate levels, a
commercial rat ELISA kit was performed (Wuhan Fine Biological Technology Co., Ltd., Wuhan, China).
After brain homogenization, the resulting suspension was sonicated with an ultrasonic cell disrupter to
break the cell membranes. After that, the homogenates were centrifugated for 5 min at 5000× g. Then,
the concentration of MAO-A and MAO-B was determined by measuring the absorbance at 450 nm
(Bio-tek, Winooski, VT, USA).
2.9. Irisin
Irisin levels in brain homogenate were measured using a commercial kit (Wuhan Fine Biological
Technology Co., Ltd., Wuhan, China). To further break the cells, the homogenate suspension was
sonicated with an ultrasonic cell disrupter. The homogenates were then centrifuged for 5 min at
5000× g, and absorbance from each sample was measured in duplicate using a spectrophotometric
microplate reader at a wavelength of 450 nm (Bio-tek, Winooski, VT, USA).
2.10. Synaptophysin
Synaptophysin in brain homogenate was measured using a commercial ELISA kit (USCN Life
Science, Euromedex, Souffelweyersheim, France). After addition of the substrate solution, the intensity
of color developed is reverse proportional to the concentration of synaptophysin in the sample. Plates
were read spectrophotometrically (Bio-tek, Winooski, VT, USA) at 450 nm.
2.11. Neuropeptides Assessment

α-Melanocyte-stimulating hormone (α-MSH), β-endorphin, neurotensin, oxytocin, and substance
P on acetonitrile-precipitated brain homogenate extracts were determined, using the RMNPMAG-83K
Milliplex MAP Rat Neuropeptide Magnetic Bead Panel (Millipore Corporation, City, TX, USA).
The plate was read on a LABScan 100 analyzer (Luminex Corporation, Austin, TX, USA) with
xPONENT software (Luminex Corporation, Austin, TX, USA) for data acquisition. Neuropeptides
on acetonitrile-precipitated brain homogenate extracts were determined by comparing the mean of
duplicate samples with the standard curve for each assay.

These analyses were carried out using the SPSS computer program (version 24.0, 2016, SPSS Inc.,
Chicago, IL, USA). To test differences between groups (normal Fe versus low Fe) during the PEP,
Student’s t test was used. Individual means were tested by pairwise comparison with Tukey’s multiple
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comparison test when main effects and their interactions were significant. Two-way ANOVA was
performed to determine the effects of type of diet, anemia, and iron content in the diet. p < 0.05 was set
as significant.
3. Results
Iron deprivation during the pre-experimental period markedly decreased all the hematological
parameters in the anemic group compared to the controls (p < 0.001); meanwhile, red cell distribution
width, platelets count, total iron-binding capacity, and hepcidin were higher (p < 0.001), and the white
blood cell count remained unchanged (Table 1).
Table 1. Hematological parameters from control and anemic rats (pre-experimental period).
Normal-Fe Control Group (n = 50) Low-Fe Anemic Group (n = 50)

Hemoglobin concentration (g·L−1 ) 130.62 ± 2.77 60.46 ± 2.77 *
Red blood cells (1012 ·L−1 ) 7.11 ± 0.22 3.19 ± 0.30 *
Hematocrit (%) 41.13 ± 1.03 13.12 ± 1.33 *
Mean corpuscular volume (fL) 54.92 ± 0.52 37.21 ± 0.37 *
Mean corpuscular hemoglobin (pg) 20.01 ± 0.15 14.22 ± 0.53 *
Mean corpuscular hemoglobin
36.12 ± 0.37 30.56 ± 0.73 *
concentration (g·dL−1 )
Red cell distribution width (%) 16.33 ± 0.45 19.67 ± 0.35 *
Platelets (109 ·L−1 ) 731 ± 70.11 2123 ± 112 *
White blood cells (109 ·L−1 ) 8.32 ± 0.41 8.45 ± 0.59
Lymphocytes (106 ·mL−1 ) 7.76 ± 0.51 5.62 ± 0.77 *
Fe (μg·L−1 ) 1342 ± 97.31 607 ± 55.22 *
Total iron-binding capacity (μg·L−1 ) 2623 ± 179 18002 ± 539 *
Transferrin saturation (%) 49.22 ± 5.86 4.01 ± 0.45 *
Ferritin (μg·L−1 ) 79.54 ± 2.23 49.12 ± 1.58 *
Hepcidin (ng·mL−1 ) 13.25 ± 0.32 15.42 ± 0.68 *
Data are shown as the mean values ± Standard error of the mean. * Significantly different from the control group
(p < 0.001, Student’s t test).
With both fermented milk-based diets, the hematological parameters showed a recovery after
supplying the normal-Fe or Fe-overload fermented milk-based diets (EP). Serum hepcidin decreased
in control and anemic animals fed fermented goat milk (normal iron content or Fe overload) in
comparison with fermented cow milk (p < 0.001). Serum iron increased in the Fe-overload groups in
all experimental conditions (p < 0.01). Fe overload also increased hemoglobin (p < 0.001), hematocrit
(p < 0.01), total iron-binding capacity (p < 0.01), transferrin saturation (p < 0.01), and serum ferritin
(p < 0.01) (Table 2).
IDA decreased most of the parameters related to brain molecular functions, namely dopamine
(p < 0.05), MAO-A, oxytocin, irisin, α-MSH, and β-endorphin (p < 0.001), while it increased
synaptophysin (p < 0.001) (Table 3).
With regard to the brain molecular function parameters studied after IDA recovery (EP), Table 4
shows that 30 days after supplying the fermented milk-based diets, fermented goat milk consumption
increased dopamine in both groups of animals with normal Fe with respect to fermented cow milk
(p < 0.01), while it decreased this parameter in the Fe-overload control animals (p < 0.01). Fermented
goat milk decreased MAO-A in both groups of animals: those in the Fe-overload group (p < 0.001) and
in the control group fed normal Fe (p < 0.05). It also decreased the MAO-B in both groups of animals
fed fermented goat milk with normal Fe (p < 0.001) levels. Synaptophysin increased in all the groups
fed fermented goat milk either, with normal Fe or Fe overload (p < 0.001 for the control groups, and
(p < 0.01 for the anemic groups, except for anemic animals fed fermented goat milk with normal Fe, in
which we observed a reduction (p < 0.01). α-MSH increased in all the groups fed fermented goat milk
(p < 0.001 for normal Fe and p < 0.01 for Fe overload).
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Table 2. Hematological parameters from control and anemic rats fed for 30 days with fermented cow or goat milk-based diets with normal Fe content or Fe overload
(n = 10 animals per group).
Fermented Cow Milk Diet Fermented Goat Milk Diet Two-Way ANOVA
Fe Content
Control Anemic Control
Anemic Group Diet Anemia Fe Content
Group Group Group
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Hemoglobin Normal 127.73 ± 2.35 128.42 ± 2.43 131.91 ± 2.44 130.01 ± 2.39 NS 1 NS
<0.001
concentration (g·L−1 ) Overload 141.97 ± 2.53 C 140.18 ± 2.87 C 141.43 ± 2.77 C 145.62 ± 2.92 BC <0.05 NS
Normal 7.21 ± 0.18 7.16 ± 0.20 7.35 ± 0.22 7.32 ± 0.19 NS NS
Red blood cells (1012 ·L−1 ) <0.05
Overload 7.01 ± 0.19 7.19 ± 0.19 8.12 ± 0.28 AC 7.21 ± 0.22 <0.01 NS
Normal 40.22 ± 1.12 39.22 ± 1.01 41.87 ± 1.41 A 43.05 ± 1.11 B <0.01 NS
Hematocrit (%) <0.01
Overload 39.87 ± 1.25 45.11 ± 2.34 C 44.75 ± 1.51 AC 44.91 ± 1.41 C <0.05 NS
Mean corpuscular volume Normal 57.28 ± 0.54 55.28 ± 0.53 57.41 ± 0.57 55.22 ± 0.54 NS NS
NS
(fL) Overload 56.90 ± 0.60 53.39 ± 0.54 56.62 ± 0.54 56.33 ± 0.51 B <0.05 NS
Normal 935.62 ± 72.11 961.53 ± 67.33 929.21 ± 78.11 937.32 ± 68.53 NS NS
Platelets (109 ·L−1 ) NS
Overload 939.22 ± 71.24 963.29 ± 70.21 936.12 ± 79.76 942.12 ± 70.25 NS NS
Normal 1323 ± 81.88 1347 ± 85.33 1362 ± 88.22 1332 ± 91.13 NS NS
Serum Fe (μg·L−1 ) <0.01
Overload 1576 ± 98.56 C 1592 ± 96.25 C 1552 ± 97.89 C 1569 ± 95.76 C NS NS
Total iron-binding Normal 2782 ± 153 2788 ± 142 2791 ± 143 2786 ± 152 NS NS
102
<0.01
capacity (μg·L−1 ) Overload 3151 ± 167 C 3234 ± 171 C 3241 ± 166 C 3188 ± 169 C NS NS
Normal 46.22 ± 0.91 45.50 ± 0.87 46.79 ± 0.78 46.89 ± 0.91 NS NS
Transferrin saturation (%) <0.01
Overload 47.85 ± 1.21 C 47.62 ± 1.12 C 48.96 ± 1.11 C 48.79 ± 1.07 C NS NS
Normal 83.11 ± 1.56 83.77 ± 1.29 84.31 ± 1.65 82.24 ± 1.82 NS NS
Serum ferritin (μg·L−1 ) <0.01
Overload 86.95 ± 1.88 C 85.98 ± 1.76 C 86.96 ± 1.83 C 87.03 ± 1.79 C NS NS
Serum hepcidin Normal 16.21 ± 0.61 16.37 ± 0.53 14.11 ± 0.58 A 14.33 ± 0.61 B <0.01 NS
NS
(ng·mL−1 ) Overload 16.42 ± 0.58 16.39 ± 0.51 15.07 ± 0.58A 14.22± 0.57B <0.01 NS
1 A
Data are shown as the mean values ± SEM. NS, not significant. Mean values from control group fed with fermented cow milk diet were significantly different (p < 0.05, Tukey’s test).
B Mean values from the anemic group fed with fermented cow milk diet were significantly different (p < 0.05, Tukey’s test). C Mean values from the corresponding group fed with normal
Fe content were significantly different (p < 0.05, Tukey’s test).

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Table 3. Brain molecular function parameters in brain homogenate (pg mL−1 ) from control and anemic
rats (pre-experimental period).
Normal-Fe Control Group (n = 10) Low-Fe Anemic Group (n = 10)

Dopamine 1970.00 ± 180.21 1560.01 ± 90.05 *
Serotonin 9179.0 ± 174.9 9409.0 ± 1813.5
MAO-A 7856.3 ± 225.2 6622.3 ± 212.7 **
MAO-B 267.96 ± 25.85 219.85 ± 10.81
Neurotensin 513.40 ± 48.18 442.61 ± 37.63
Oxytocin 249.71 ± 21.22 181.85 ± 17.86 **
Irisin 21.17 ± 1.19 16.74 ± 0.90 **
Synaptophysin 771.79 ± 36.97 1121.37 ± 28.62 **
α-MSH 616.34 ± 20.52 200.58 ± 44.29 **
β-Endorphin 2431.5 ± 126.0 1308.3 ± 186.6 **
Data are shown as the mean values ± SEM. Significantly different from the control group (* p < 0.05, ** p < 0.001,
Student’s t test). MAO, Monoamine oxidase; α-MSH, α-Melanocyte-stimulating hormone.
Anemia decreased the dopamine in animals fed fermented goat or cow milk with Fe overload
(p < 0.05 and p < 0.001 respectively), MAO-A and MAO-B in the animals fed fermented cow milk
(p < 0.001 for normal Fe and p < 0.05 for Fe overload), and goat milk with Fe overload (p < 0.001 MAO-A
and p < 0.05 MAO-B). Anemia increased neurotensin in the normal-Fe groups fed fermented cow milk
(p < 0.001). Oxytocin increased in the anemic animals fed fermented cow milk with normal Fe (p < 0.01),
and decreased in animals fed both types of fermented milk with Fe overload (p < 0.001). Serotonin
increased in anemic animals fed both milk-based diets with normal Fe (p < 0.001) in comparison with
control animals. Synaptophysin increased in the anemic animals fed fermented cow milk (p < 0.01), and
decreased in the anemic animals fed fermented goat milk (p < 0.001) in both normal and Fe overload.
Anemia decreased α-MSH levels in all the groups fed both milk-based diets either with normal Fe or
Fe overload (p < 0.001). Anemia decreased β-endorphin levels in the animals fed both fermented milks
with normal Fe (p < 0.001) (Table 4).
Fe overload increased dopamine levels in both groups of animals fed fermented cow milk diet
(p < 0.01), and increased MAO-A levels in the control groups fed both milk-based diets (p < 0.001),
while it decreased this parameter in the anemic group fed fermented goat milk (p < 0.001). Fe overload
also increased MAO-B levels in all the groups fed both types of fermented milk (p < 0.01), except for
the anemic groups fed fermented cow milk. Fe overload increased neurotensin in the control group
fed fermented cow milk (p < 0.01), and decreased this parameter in the anemic group fed the same
diet (p < 0.01). For the mice in the Fe-overload groups, oxytocin increased in control animals fed
both fermented milk diets (p < 0.01) and decreased in anemic animals fed both fermented milk diets
(p < 0.001). Fe overload caused a marked reduction in serotonine levels in control and anemic rats fed
both milk-based diets (p < 0.001). Synaptophysin decreased in the control groups and anemic rats fed
fermented cow milk (p < 0.001), and increased in the animals fed fermented goat milk (p < 0.001). Fe
overload increased α-MSH levels in the groups fed fermented cow milk (p < 0.001), and decreased
this hormone in the groups fed fermented goat milk (p < 0.01). β-Endorphin decreased in the control
animals fed fermented cow or goat milk with Fe overload (p < 0.001) (Table 4).
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Table 4. Brain molecular function parameters in brain homogenate (pg mL−1 ) from control and anemic rats fed for 30 days with fermented cow or goat milk-based
diets with normal Fe content or Fe overload (n = 10 animals per group).
Fermented Cow Milk Diet Fermented Goat Milk Diet Two-Way ANOVA
Fe Content
Control Group Anemic Group Control Group Anemic Group Diet Anemia Fe Content
Normal 1210.00 ± 40.01 980.14 ± 30.07 1710.10 ± 16.07 A 1320.11 ± 11.23 B <0.01 NS
Dopamine
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<0.05
Overload 3130.02 ± 22.14 D 1440.21 ± 30.12 CD 1400.02 ± 60.04 A 1260 ± 40.33 C <0.01 < 0.01
Normal 33651.9 ± 3334.5 61341.9 ± 5820.1 C 39278.9 ± 4571.7 82050.5 ± 2995.1 BC <0.05 <0.001
Serotonin <0.001
Overload 26346.4 ± 2943.3 D 29564.3 ± 2806.3 D 20981.4 ± 852.7 D 23688.0 ± 962.7 D NS NS
Normal 8220.1 ± 261.5 7279.3 ± 298.6 C 7390.3 ± 211.9 A 6716.7 ± 483.3 B <0.05 <0.05
MAO-A <0.01
Overload 13935.1 ± 282.3 D 7152.6 ± 498.0 C 8403.7 ± 353.4 AD 4001.8 ± 168.3 BCD <0.001 <0.001
Normal 260.96 ± 10.81 237.89 ± 11.57 C 215.13 ± 10.10 A 208.93 ± 10.07 B <0.001 <0.05
MAO-B <0.01
Overload 327.70 ± 9.56 D 259.96 ± 6.68 C 305.24 ± 9.01 D 267.52 ± 7.90 CD NS <0.01
Normal 317.98 ± 11.94 405.37 ± 18.23 C 336.36 ± 21.37 373.21 ± 10.15 NS <0.05
Neurotensin <0.05
Overload 360.77 ± 12.67 D 345.61 ± 12.80 D 355.79 ± 10.39 341.56 ± 9.98 NS NS
Normal 145.57 ± 9.27 173.42 ± 33.90 C 182.41 ± 7.27 A 177.74 ± 1.87 <0.05 <0.05
Oxytocin <0.001
Overload 217.27 ± 3.58 D 86.95 ± 0.13 CD 225.52 ± 3.94 D 89.85 ± 1.57 CD NS <0.01
Normal 16.75 ± 0.70 15.96 ± 0.35 19.18 ± 1.26 17.24 ± 1.03 NS NS
Irisin NS
Overload 19.32 ± 0.60 16.66 ± 0.85 17.58 ± 0.79 15.64 ± 0.70 NS NS
104
Normal 836.97 ± 31.40 907.33 ± 27.57 C 1091.02 ± 26.92 A 730.09 ± 20.44 BC <0.001 <0.01
Synaptophysin <0.001
Overload 552.01 ± 40.56 D 789.02 ± 49.59 CD 1290.56 ± 51.89 AD 883.40 ± 24.74 BCD <0.001 <0.001
Normal 310.87 ± 14.95 86.28 ± 1.38 C 1162.05 ± 9.07 A 494.28 ± 22.64 BC <0.001 <0.001
α-MSH <0.001
Overload 669.74 ± 33.48 D 251.69 ± 18.04 CD 726.85 ± 21.86 A D 312.54 ± 9.40 BC D <0.01 <0.001
Normal 2426.5 ± 79.4 744.3 ± 39.9 C 2551.9 ± 131.7 671.3 ± 36.4 C NS <0.01 <0.05
β-Endorphin
Overload 741.6 ± 41.3 D 688.6 ± 40.9 1344.2 ± 71.5 AD 640.1 ± 34.0 C <0.05 NS
Data are shown as the mean values ± SEM. NS, not significant. A Mean values from control group fed a fermented cow milk diet were significantly different (p < 0.05, Tukey’s test).
B Mean values from anemic group fed a fermented cow milk diet were significantly different (p < 0.05, Tukey’s test). C Mean values from the corresponding group of anemic animals
were significantly different (p < 0.05, Tukey’s test). D Mean values from the corresponding group fed with normal Fe content were significantly different (p < 0.05, Student’s t test). MAO,
Monoamine oxidase; α-MSH, α-Melanocyte-stimulating hormone.
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4. Discussion
A better characterization of the events in the pathophysiology of IDA and the influence on nervous
system molecular functions and homeostasis during the recovery of this deficiency would led to new
nutritional strategies improving brain molecular functions and the other deleterious effects of the
pathology. The animal model used in the current study simulates physiological deficiencies to iron
and anemia recovery, and therefore argues that the response to brain functions can be modulated by
dietary components.
The results of the current study reveal a clear impairment in some brain molecular functions
due to IDA, because most of the parameters studied were impaired. ID manifests as alterations in
cognitive function, behavior, and mood [19]. Iron is a cofactor of many metabolic processes as well as
the synthesis of aminergic neurotransmitters; it plays a major function in brain development, and has a
key role in myelinogenesis and synaptogenesis. Several mechanisms regarding the influence of iron
deficiency on brain functions have been reported: the decrease in oxygen-carrying capacity of the blood
could result in hypoperfusion of the brain, which could lead to an imbalance in oxidative/antioxidant
status and inflammatory responses, causing neurodegenerative processes [20]. Furthermore, anemia
induces renal changes, leading to lower erythropoietin levels as well as increasing the risk of neural
degeneration, as this hormone has neuroprotective effects in situations of hypoxia [21]. Thus, IDA
induces pathological changes in brain tissue and vessels, leading to reduced oxygen transportation as
well as impaired synaptic functioning [22].
Dopamine levels have been heavily examined in patients suffering from IDA, and it is well
established that this neurotransmitter is implicated in learning, memory, and attention, as well as
several hormonal pathways, stress responses, addiction, and emotional behavior [23]. IDA leads
to diminished central dopaminergic transmission and receptor trafficking, with the D2 receptor
particularly affected [24]. Other evidence of biochemical abnormalities during IDA include decreased
concentrations of thyroid hormones, as we have previously reported [25], increasing the levels
of circulating catecholamines. This involves beta adrenergic receptors and affects the availability
of glucose, impairing synaptic plasticity and changes in dendritic structure that lead to a loss of
neurons [26], indicating that IDA can alter neurotransmitter metabolism.
Surprisingly, milk-based diets have really different effects on dopamine and serotonin, which
are both monoamines that have iron-dependent synthesis pathways. In general, there is consensus
in the scientific literature reporting a negative effect of ID on dopamine functions and synthesis [27],
which coincides with our results during the anemia induction. However, conflicting results exist on
the effects of ID on serotonin levels in rats. ID decreases serotonin due to a down-regulation of several
biosynthesis pathways in young rats [28]. On the contrary, an increase in serotonin levels during ID
has been reported in adults, reflecting a down-regulation of serotonin metabolism [29]. Serotonin
transporters are reduced in the striatum of ID mice [30]. Additionally, gestational ID reduces serotonin
uptake by synaptic vesicles in offspring, which is a process that can be normalized after four weeks of
iron replenishment [31]. However, in other studies, ID had no effect on serotonin levels or metabolism
in newborns or adults [32], and serotonin levels in the prefrontal cortex of the ID rats did not differ
from controls [33]. These reported results in serotonin homeostasis appear conflicting, and hint at
underlying additional mechanisms of the iron–monoamine relationship [2]. Therefore, although ID did
not changed serotonin levels, the differences during iron repletion could be attributed to the different
behavior of serotonin mentioned above, and the enhancement of the digestive and metabolic utilization
of iron in fermented goat milk [14]. Moreover, a limitation of the study is the lack of a regional analysis
of the brain molecules studied, which could help to specify the effect of fermented dairy products on
brain functions, and could explain these results.
It has been reported that irisin promotes the differentiation of human embryonic stem cell-derived
neural cells into neurons, as well as increased mature neuronal and astrocyte markers together with
the improved expression of neurotrophic factors in the brain [34]. Therefore, the decrease of irisin
levels in this study due to IDA reveals impairment in the molecular mechanisms driving neuron
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Nutrients 2019, 11, 2394
homeostasis. Moreover, the decrease recorded in MAO-A can be explained because iron is a key factor
for MAO activity, and monoamine neurotransmitter synthesis requires the iron-dependent enzymes
tyrosine and tryptophan hydroxylase, which is a finding that has been correlated with later behavioral
consequences in juvenile monkeys, influencing brain function [35]. IDA also decreased oxytocin,
which is a hypothalamic neuropeptide involved in regulating social behavior, and has a key role in
physiological conditions and brain diseases. It has been reported that CO exerts an inhibitory tone on
oxytocin secretion [36], and it is well known that CO output is increased during IDA. Additionally,
IDA also decreased α-MSH which has been proven as an anti-inflammatory and neuroprotective
hormone in animal studies [37] and β-endorphin, which has an important role in the development of
the non-synaptic or paracrine communication between neurons [38], revealing the impairment of brain
molecular functions.
On the other hand, synaptophysin increased during IDA. This interesting result can be explained
because synaptobrevin II is a vesicular protein receptor that is essential for neurotransmitter release;
therefore, its correct trafficking to synaptic vesicles is critical to render them fusion-competent.
Synaptophysin binds to synaptobrevin II in the synaptic vesicles and facilitates its retrieval during
endocytosis. Under physiological conditions, the expression of synaptophysin in a 1:2 ratio with
synaptobrevin II is sufficient to fully rescue normal synaptobrevin II trafficking. The balance between
synaptophysin and synaptobrevin II is critical for the exocytotic release of neurotransmitters [39]. Since
as previously mentioned, anemia is associated with an impairment in cerebral blood flow and oxygen
metabolism, as well as low neurotransmitter release [7], the overexpression of synaptophysin could be
a compensatory mechanism to cope with the low neurotransmitter release from the synaptic vesicles
during this condition.
In general, an improvement of nervous system molecular functions has been observed after
anemia recovery with fermented goat milk (including dopamine, oxytocin, serotonin, α-MSH, and
synaptophysin), which can be explained by several factors, including the better recovery of IDA with
this dairy product.
Previous studies of our research group showed an increased expression of some key iron
metabolism proteins such as duodenal cytochrome b, divalent metal transporter 1, and ferroportin 1 in
rats fed fermented goat milk compared to fermented cow milk. These proteins enable overcoming the
effects of IDA, increasing iron bioavailability in target organs and efficient iron repletion after IDA [14],
and have significant implications in the brain molecular mechanisms related to cognitive functions.
On the other hand, synaptophysin is considered a reliable biomarker for synaptic density and
synaptogenesis [40]. Synaptophysin is also correlated with a loss or increase in synaptic densities
in studies of aging and neurodegenerative disorders [41]. In the current study, the production of
synaptophysin was significantly increased in the rats consuming fermented goat milk, because as
previously reported [42], the production of neurotrophic factors, as well as the survival of neuronal
synapses and the retention of cognitive function is increased when the inflammation is suppressed.
In this sense, we have previously reported that in control and anemic rats, interleukin (IL)-1β,
IL-2, IL-12p70, IP-10 and tumour necrosis factor (TNF)-α (pro-inflammatory cytokines) decrease after
fermented goat milk consumption, and levels of IL-4, IL-13 and IL-10 (pro-inflammatory cytokines) [43]
increase. These results are due to the better nutritional characteristics of fermented goat milk, in
comparison with fermented cow milk, playing a potential role of this dairy product as a high nutritional
value food with anti-inflammatory properties. It has also been reported that TNF-α produced by
microglia exacerbates the neurodegenerative diseases [44]. In the brain, microglia mainly regulate
immunological phenomena; as a result, it seems that fermented goat milk may regulate also the
microglial inflammatory response, leading to a suppression of the inflammatory signaling and neuronal
degeneration. Chronic inflammation in the brain exacerbates the pathological condition and cognitive
functions decline in many neurodegenerative processes, which is due to neurotrophic factors that are
suppressed by inflammation and are toxic to neurons [45,46].
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Nutrients 2019, 11, 2394
Increased awareness of the role of oxidative stress in the pathogenesis of neurodegenerative

processes has highlighted the issue of whether oxidative damage is a fundamental step in the
pathogenesis or instead results from disease-associated pathology. Recently, it has been reported
that oxidative damage results in amyloid deposition in the brain, resulting in neuronal cell death
and neurodegenerative diseases [47,48]. In this sense, we have previously reported [49,50] that
fermented goat milk increased some antioxidant enzymes in brain tissue as well as total antioxidant
status and melatonin, even in the case of Fe overload. These increases limit the oxidative damage to
the brain biomolecules (lipids, protein DNA, prostaglandins) and protect the nervous tissue of the
oxidative-induced cell death that induces neurodegenerative processes.
In the current study, iron overload increased dopamine levels in the brain with a fermented cow
milk based-diet, and it has been previously reported that iron accumulation relates to dopamine,
comprising a toxic couple that is reliant on interacting with other biomolecules, and causes selective
neurodegeneration in some areas of the brain [51]. MAO-A levels were increased in control animals
consuming both types of fermented milk with iron overload; an enzyme catalyzes the oxidative
deamination of monoamine neurotransmitters, increasing the production of reactive oxygen species
and oxidative stress, which is potentially a risk factor for neuronal loss and neurodegenerative
disorders [52]. Iron excess also reduced β-endorphin in the control animals, which has several activities
such as analgesic, immunostimulatory, stress busting, and anti-inflammatory, as well as having a key
role in the adequate neurological responses [53]. These deleterious effects of iron overload are in
accordance with previous reports revealing that iron supplementation results in brain accumulation
and subsequent toxicity, increasing oxidative damage up to a level that natural defenses fail, and at
which neuronal apoptotic rates are exacerbated [54].
We have previously reported that except for alanine, in which the differences were not statistically
significant, all other amino acids were higher in fermented goat milk than in fermented cow milk,
including glycine threonine and tyrosine [15], and it has been reported that orally administered
β-lactopeptide of glycine-threonine-tryptophan-tyrosine inhibits the activity of monoamine oxidase in
the brain [55]. It has also been reported that the MAO inhibitor reduces reactive oxygen species and
suppresses some neurodegenerative diseases [56]. In addition, the inhibitory activity of MAO reduces
the activation of nuclear factor kappa B (NF-κB) and suppresses NF-κB-regulated pro-inflammatory
responses [57], which supports the results obtained with the animals fed fermented goat milk. In
the current study, an increase in α-MSH was also recorded in all groups fed fermented goat milk.
α-MSH is a hormone that functions as a neurotransmitter and neuromodulator; it is involved in
significant neuronal circuitry, and is also a mediator of immunity and inflammation. At the molecular
level, these effects of α-MSH are mediated via the inhibition of the activation of transcription factors
such as NF-κB [58], reducing once more the pro-inflammatory responses in the nervous system and
suppressing some pathways that lead to neurodegenerative environments.
5. Conclusions
In conclusion, by using an animal model of severe iron deficiency, the current study has showed a
relation between iron status and brain molecular parameters related to key functions during anemia
instauration and recovery. The alterations recorded in the biomarker-related brain functions studied
may result in impairments in behavioral and cognitive functions during severe iron deficiency anemia.
In addition, the results of the current study reveal that in general, during anemia recovery, a fermented
goat milk-based diet, normal iron, or iron overload inhibits MAO activities, and increases serotonin,
synaptophysin, and α-MSH levels. It also has been implicated in the suppression of inflammatory
responses, an improvement in iron metabolism, and a reduction of evoked oxidative stress, as observed
in previous studies. Taken together, all these results suggest a potential neuroprotective effect of
fermented goat milk, which could enhance brain molecular functions, although further studies are
needed to confirm these findings.
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Nutrients 2019, 11, 2394
Author Contributions: I.L.-A. and J.D.-C. designed the study. J.M.-F., J.D.-C., M.J.M.A. and M.G.-B. performed
the experiments, analyzed the data, and wrote the manuscript. All the authors approved the final version of
the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the
accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated
as authors qualify for authorship, and all those who qualify for authorship are listed.
Funding: This study was supported by the Excellence Research Project (P11-AGR-7648) from the Regional
Government of Andalusia.
Acknowledgments: J.M.-F. and M.G.-B. were supported by a fellowship from the Ministry of Education, Culture
and Sport (Spain), and are grateful to the Excellence Ph.D. Program “Nutrición y Ciencias de los Alimentos”
from the University of Granada. The authors also thank S.S. for her efficient support in the revision with the
English language.
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nutrients
Review
Iron Homeostasis Disruption and Oxidative Stress in
Preterm Newborns
Genny Raffaeli 1,† , Francesca Manzoni 1,2,† , Valeria Cortesi 1,2 , Giacomo Cavallaro 1 ,
Fabio Mosca 1,2 and Stefano Ghirardello 1, *
1 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, NICU, 20122 Milano, Italy;
genny.raffaeli@unimi.it (G.R.); francescamanzoni.unimi@gmail.com (F.M.);
valeria.cortesi92@gmail.com (V.C.); giacomo.cavallaro@policlinico.mi.it (G.C.); fabio.mosca@unimi.it (F.M.)
2 Department of Clinical Sciences and Community Health, Università degli Studi di Milano,
20122 Milano, Italy
* Correspondence: stefano.ghirardello@mangiagalli.it; Tel.: +39-2-5503-2234; Fax: +39-2-5503-221
† Co-first authorship.
Received: 30 April 2020; Accepted: 25 May 2020; Published: 27 May 2020
Abstract: Iron is an essential micronutrient for early development, being involved in several cellular
processes and playing a significant role in neurodevelopment. Prematurity may impact on iron
homeostasis in different ways. On the one hand, more than half of preterm infants develop iron
deficiency (ID)/ID anemia (IDA), due to the shorter duration of pregnancy, early postnatal growth,
insufficient erythropoiesis, and phlebotomy losses. On the other hand, the sickest patients are exposed
to erythrocytes transfusions, increasing the risk of iron overload under conditions of impaired
antioxidant capacity. Prevention of iron shortage through placental transfusion, blood-sparing
practices for laboratory assessments, and iron supplementation is the first frontier in the management
of anemia in preterm infants. The American Academy of Pediatrics recommends the administration
of 2 mg/kg/day of oral elemental iron to human milk-fed preterm infants from one month of age
to prevent ID. To date, there is no consensus on the type of iron preparations, dosages, or starting
time of administration to meet optimal cost-efficacy and safety measures. We will identify the main
determinants of iron homeostasis in premature infants, elaborate on iron-mediated redox unbalance,
and highlight areas for further research to tailor the management of iron metabolism.
Keywords: iron; redox unbalance; prematurity; transfusion; anemia; blood-sparing
1. Iron Homeostasis and Prematurity

Iron is an essential micronutrient that plays a pivotal role in early development, being involved in
hemoglobin synthesis, oxygen delivery, electron transfer, energy metabolism, and cell differentiation [1].
Nowadays, there is consistent evidence about the relationship between in utero iron supply and
subsequent cognitive and neurobehavioral outcomes [2,3]. Iron homeostasis is a balance between
iron absorption, storage, and recycle by erythroid precursors. The total body iron is distributed into
three compartments [4]. The majority of total body iron (at least two-thirds) is within erythrocytes,
in the form of hemoglobin (Hb). A small part (about 15%) is “storage iron”, mostly kept within
ferritin or hemosiderin in liver and spleen, ready to be mobilized [5–7]; the remainder (10%) is
non-heme-non-storage tissue-circulating iron. The latter is bound to serum transferrin, an iron chelator
that keeps iron in a soluble, inert, reduced state, preventing toxic oxidative reactions [7]. Hepcidin is
a peptide hormone acting as a negative feedback regulator of iron metabolism, by modulating
the expression of ferroportin, which is an iron-exporter transmembrane protein of enterocytes and
macrophages. Hepcidin synthesis in the hepatocytes is determined by circulating and stored iron,
inflammation, and erythropoietin. In the event of high iron levels or inflammation, hepatic hepcidin

Nutrients 2020, 12, 1554
release is increased and ferroportin expression is downregulated. Conversely, anemia, hypoxia, and
low iron levels are associated with reduced hepcidin expression, leading to the increased activity of
ferroportin and the mobilization of iron reserves [4,6]. Iron deficiency (ID) is a relevant public health
problem and is the most common single-element deficiency worldwide, affecting around 2 billion
people globally [1,8]. Both pre-existing anemia and increased iron requirements during pregnancy
make pregnant women particularly vulnerable to develop iron deficiency anemia (IDA) [2]. It is
estimated that maternal IDA affects approximately 30–50% of pregnant women in developing countries
and less than 1% in developed countries where iron supplementation is part of routine care [9].
There are no conclusive results about the relationship between maternal and neonatal iron status.
Past studies showed that early maternal sideropenic anemia doubles the risk of preterm delivery
and low birth weight [10,11]. Iron endowment at birth depends on iron stored during gestation,
tightly connected to maternal iron status, and iron received perinatally as Hb, depending on the time
of cord clamping [12]. Adequate iron stores at birth are necessary to satisfy requirements in the first
6–9 months of age, when the neonatal gut is not fully developed to properly regulate iron intake and
breast milk cannot meet the recommended needs [2]. Poor levels of iron at birth, on the contrary,
predict future ID and/or IDA during infancy [12].
In the same way, iron homeostasis can be deeply influenced by prematurity. Iron is mostly
(>66%) transferred during the third trimester of pregnancy [10] so that total iron stores are inversely
related to gestational age. Moreover, many pregnancy complications, such as multiple pregnancies,
obesity, gestational diabetes, and hypertension with intrauterine growth restriction (IUGR) [13],
can also induce impaired iron endowment due to chronic placental insufficiency [10,14]. Iron demand,
as with all nutrients, increases during fetal and neonatal development [15]. In premature infants, the
early onset of erythropoiesis and the fast catch-up growth occurring in the first 6–8 weeks of life require
additional iron, especially in the most immature neonates [16]. Moreover, premature infants’ iron
stores can be further depleted by recurrent phlebotomy and administration of recombinant human
erythropoietin (rHuEPO) in the absence of adequate iron supplementation.
Low iron reserves and increased iron requirement explain why preterm infants are at high risk of
ID/IDA and need iron supplementation [1,14]. It is estimated that between 25% and 85% of premature
newborns develop iron deficiency, usually in the first six months of life [16]. In preterm neonates,
ID can affect the majority of organs, causing poor growth, temperature instability, thyroid dysfunction,
decreased cell-mediated immune response and impaired DNA and collagen synthesis, even before
microcytic and hypochromic anemia appears [16,17]. However, the main concern is the impact of ID
on brain development [9,18].
On the other hand, premature infants are vulnerable to oxidative stress caused by
non-transferrin-bound iron (NTBI) overload due to the immature antioxidant system and the
high transferrin saturation [1,6,19,20]. NTBI can derive from high doses of oral or parental iron
supplementation and recurrent erythrocyte transfusions [14]. The latter is relevant if we consider
that around 40% of very low birth weight (VLBW) and more than 90% of extremely low birth weight
(ELBW) infants receive at least one blood transfusion during hospitalization [21]. Birth, itself, is an
oxidative challenge since there is a rapid increase in the oxygen concentrations compared to the
hypoxic intrauterine environment [22]. Indeed, plasma NTBI concentrations appear to be higher in
the neonatal period if compared to the following ages [19,20]. Iron excess cannot be removed by
physiological pathways and can accumulate and generate free radicals resulting in cellular toxicity.
The same toxic reaction can be produced when the iron is delocalized from its binding protein, as it
happens in the setting of hypoxia [6,23]. As a result, the sickest premature neonates are prone to
develop complications that may share the same etiology, grouped under the expression coined by
Saugstad, “the oxygen radical disease of neonatology” [24]: bronchopulmonary dysplasia (BPD),
retinopathy of prematurity (ROP), necrotizing enterocolitis (NEC), intraventricular hemorrhage (IVH),
periventricular leukomalacia (PVL), and punctate white matter lesions (PWM) [1,21,25].
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2. Iron and Brain Development

Iron is critical for brain development in the fetal and early neonatal period, and major issues are
related to the long-lasting effect of ID on neurodevelopment. Biologically, in the case of negative iron
balance, iron is redistributed following a hierarchical strategy and primarily used by red cells to the
detriment of other tissues: firstly liver, followed by the heart, skeletal muscle and. finally. brain [4,26].
It results that ID can injure the brain even in the absence of IDA [9,27,28]. Indeed, ID can impact
brain functions because several iron-dependent enzymes are essential for neurotransmitter synthesis,
myelination, synaptogenesis, gene expression and neuronal energy production [4,26].
The American Academy of Pediatrics (AAP) has highlighted the relevance of ID and IDA
screening during infancy [2], due to both short- and long-term negative consequences of IDA on motor,
cognitive, social, and behavioral development [3,29,30].
Both timing and duration of ID are critical because the brain’s affected areas are in a crucial
developing phase at the time of ID [9]. In particular, ID during gestation and lactation carries the most
severe effects, as it occurs during the brain’s growth spurt, [9]. Rodent models have shown that early
(from the late fetal period to 24 months of postnatal age) ID can affect the dopaminergic system in
the striatum, and alter gene expression and dendritogenesis in the hippocampus, leading to motor
and memory life-long alterations, respectively [9]. In the same way, the composition of myelin lipids
persists irreversibly altered in adulthood despite iron treatment [9].
Intrauterine ID, as well, can have life-long consequences as proved by the association between
cord ferritin levels below the lowest quartile (<76 mg/L), a marker of in-utero fetal iron status [31],
and language and fine motor skills delay at five years of age [2,32].
Scarce data evaluated the relationship between in-utero ID and long-term neurobehavioral effects
in preterm infants. Premature infants suffering from fetal ID have abnormal auditory brainstem-evoked
responses (ABR), a marker of brain maturation, with longer latencies in the perinatal period [31].
Moreover, anemic preterm infants show altered neurological reflexes at 37 weeks, probably suggesting
impaired myelination or neurotransmitters synthesis [33]. Interestingly, ID in premature infants tends
to express through motor deficits, while cognitive impairment prevails in term infants [16].
Based on preclinical evidence [34], limited benefits were reported for the promotion of mental
or motor development in infants with IDA [35,36]. This result could be explained by the late timing
of the onset of supplementation: ID begins prenatally, and interventions targeting infancy or early
childhood were too late [37]. On the other hand, various studies showed that iron supplementation
in infants at risk of ID had positive effects on motor and cognitive development [38–40]. Similarly,
early (<61 days of life) iron supplementation in premature infants improves neurocognitive outcome
VLBW neonates [41].
The main core of the debate is related to the fact that not only ID can be troublesome, but also iron
excess, as mentioned above, may induce oxidative stress, which contributes to the pathophysiology of
several prematurity-related diseases [25].
Indeed, preterm infants often experience repeated episodes of hyperoxia, hypoxia, and ischemia
resulting in free radicals and reactive oxygen species (ROS) production that these patients cannot
cope with, due to an ineffective anti-oxidant system [25]. Specifically, several antioxidant enzymes,
such as the superoxide dismutase, catalase, and glutathione peroxidase, show a decreased activity in
the immature brain [42].
Iron becomes toxic when not bound to proteins [43,44]. Neonates, particularly if born prematurely,
are prone to generate NTBI as a result of repeated episodes of hypoxia, acidosis, and ischemia in
the perinatal period, when plasma transferrin, ceruloplasmin and total iron bind capacity (TIBC) are
constitutionally low [42]. Indeed, amoeboid microglial cells of the periventricular white matter show
increased intracellular iron concentration after a hypoxic insult that can lead to oligodendrocyte cell
death and axonal swelling [45].
Growing evidence suggests a role for iron in hypoxic-ischemic encephalopathy (HIE).
Indeed, high amounts of NTBI were found in the cerebrospinal fluid (CSF) and the serum of newborns
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after a hypoxic-ischemic injury [23,46]. NTBI concentrations are directly related to the severity of
brain injury [23], and blood NTBI has been considered an early predictive marker of long-term
neurodevelopmental outcomes [20,46]. Iron-mediated radical factors disrupt the blood-brain barrier
and cause endothelial necrosis following a hypoxic-ischemic injury [42].
Similarly, iron-induced neuronal toxicity is a determinant of IVH pathophysiology in the preterm
brain as the free iron released from heme destruction after intracerebral hemorrhage (ICH) contributes
to secondary brain injury and post-hemorrhagic ventricular dilatation [43]. Indeed, while the primary
hit lies in the presence of the hematoma itself, the secondary injury refers to the subsequent release
of neurotoxic iron-related compounds from the hematoma. Specifically, heme oxygenase catabolizes
heme into carbon monoxide, biliverdin, and free iron. The free iron accumulation increases the risk of
oxidative damage to lipids, protein and DNA, by inducing the free radical production by means of
the Fenton reaction [43]. In both HIE and IVH, the potential mechanism by which iron can damage
the neonatal brain has been recently studied and has been named “ferroptosis”, a non-apoptotic
iron-dependent pathway of cell death (Figure 1) [43].

Figure 1. Presumptive molecular pathways of ferroptosis following brain injury in the developing
brain. Excess free iron in the brain may be the result of Hb degradation by HO-1 after intracerebral
hemorrhage. Similarly, a hypoxic-ischemic insult enhances iron liberation from its binding proteins.
Fe2+ , the reactive form of iron, promotes ROS production via the Fenton reaction leading to lipid
peroxidation and membrane damage while the damaged brain releases glutamate. High extracellular
glutamate concentrations inhibit the cystine/glutamate antiporter system xc- thus reducing cellular
cystine levels, necessary for GSH synthesis. Reduced intracellular cystine concentration indirectly
inactivates GPX4, the enzyme responsible for lipid hydroperoxide reduction and GSH consumption.
The accumulation of lipid hydroperoxides in an enriched Fe2+ environment leads to significant lipid
ROS formation that induces membrane permeabilization and ferroptosis [6,47]. Fe2+ : ferrous cation;
GPX4: glutathione peroxidase 4; GSSG: oxidized GSH; GSH: reduced glutathione; Hb: hemoglobin;
HO-1: heme oxygenase 1; LOOH: lipid hydroperoxides; LOH: lipid alcohols; ROS: reactive oxygen
species; TF: transferrin; TfR1: transferrin receptor 1. Adapted from Wang et al. [6].
NTBI can trigger ferroptosis, inducing a process mediated by lipid peroxidation and glutathione
consumption with subsequent hydroxyl radical production via Fenton and Haber–Weiss reactions.
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Free radicals and nitric oxide generated during brain reperfusion following hypoxic events activate a
series of chain reactions that mobilize an increasing amount of iron from its binding proteins and red
cells [48], thus amplifying cell death and brain injury [6]. To support this theory, high concentrations of
malondialdehyde, a product of lipid peroxidation, have been found in CSF of infants with HIE [23].
This pathogenetic mechanism is relevant in the neonatal brain if we consider the large amount
of iron and polyunsaturated fatty acids that constitute the lipidic membrane in the white matter,
readily susceptible to free radical attack [46]. Additionally, CSF is characterized by a low TIBC that
can bind NTBI and an unbalanced relationship between ceruloplasmin and vitamin C that favors the
latter and results in iron oxidation in the active ferrous form [20,49]. Even orally-administered iron can
damage brain: preclinical studies demonstrated Parkinson-like neurodegeneration in adults that have
been fed with great amounts of enteral iron during breastfeeding [16].
Iron chelators have shown a neuroprotective effect in animal studies [23]. rHuEPO likewise
protects against inflammation, apoptosis, and oxidation and decreases unbound iron by stimulating
erythropoiesis [43]. In preclinical models of neonatal brain damage, rHuEPO acts as a neuroprotector,
promoting neurogenesis and neural regeneration after an insult [50]. In clinical practice, rHuEPO is
administered at the dose of 300–500 U/kg for the prevention and treatment of the anemia of
prematurity [51]. Low-doses of rHuEPO has been previously associated with better short-term and
long-term neurological outcomes in both full-term and preterm infants [50,51]. However, the results
from a recent randomized trial, enrolling extremely preterm infants to receive high-dose of rHuEPO
(1000 U/kg) in the first weeks of life, did not improve neurodevelopmental at two years of age [52],
if compared to the placebo group.
3. Iron Status Measurement

Even if bone marrow aspiration is considered the gold standard for ID diagnosis, it has
been replaced in clinical practice by other less invasive laboratory parameters [53], classified in
hematological and non-hematological tests. The former includes Hb, mean corpuscular volume (MCV),
reticulocyte count, and hemoglobin reticulocytes content, designated as the mean cellular hemoglobin
content of reticulocytes (CHr) or reticulocyte hemoglobin equivalent (RET-He). The latter include
serum ferritin (SF), transferrin saturation, soluble transferrin receptor (STfR1), zinc protoporphyrin to
heme ratio (ZnPP/H).
The changes in iron status tests in response to ID, IDA, or iron overload are reported in
Table 1. [14,54,55].
Table 1. Iron status parameters: their response to ID, IDA and iron overload [14,54,55].
Parameter ID IDA Iron Overload

Hb Normal Reduced Normal
MCV Normal Reduced Normal
RET-He/CHr Reduced Reduced Normal
SF Reduced Reduced Increased
Transferrin saturation Reduced Reduced Increased
sTfR1 Increased Increased Reduced
ZnPP/H ratio Increased Increased Reduced
ID, iron deficiency; IDA, iron deficiency anemia; Hb, hemoglobin; MCV, mean corpuscular volume;
RET-HE, reticulocyte hemoglobin equivalent; CHr, mean cellular hemoglobin content of reticulocytes;
SF, serum ferritin; sTfR1, serum transferrin receptor; ZnPP/He ratio, zinc protoporphyrin to
heme ratio.
The assessment of neonatal iron status is a challenging task as neonatal blood sampling requires
a well-trained phlebotomist and is not routinely performed among healthy newborns. For this
reason, neonatologists, instead of “normal values” established from healthy neonates, use “reference
ranges” that include values between the 5th and the 95th percentile derived from newborns with
minor pathology [56].
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Moreover, most hematological parameters are influenced by gestational age and postnatal
developmental changes [56]. As a result, gestational age-specific laboratory markers are needed.
The site of blood collection (venous, arterial, capillary) and the use of different reagents can further
impact on values [57]. Reference ranges for the main iron status parameters in term and preterm
neonates are listed in Table 2.
Table 2. Reference ranges for the main iron status parameters in term and preterm neonates.
Capillary Blood
Cord Blood
(within 72 h from Birth)
Preterm Term Preterm Term
Hb
12.4–19.2 13.3–18.4 14.5–22.5 Lorenz et al. 2013 [57]
(g/dL)
MCV
103–133 97.8–118.5 95–121 Lorenz et al. 2013 [57]
(fL)
Serum ferritin
35–267 40–309 Siddappa et al. 2007 [28]
(μg/L)
STfR1
6.1–13.7 6.4–10.6 Sweet e al. 2001 [58]
(mg/L)
ZnPP/H ratio
55–135.5 49.6–108.4 Juul et al. 2003 [59]
(μmol/mol)
RET-He Löfving et al. 2018 [60]
27.4–36 24.3–36.2 25.5–37.6
(pg) Lorenz et al. 2017 [61]
Hb, hemoglobin; MCV, mean corpuscular volume; sTfR1, serum transferrin receptor; ZnPP/He ratio,
zinc protoporphyrin to heme ratio; RET-HE, reticulocyte hemoglobin equivalent. All are central 95% reference
intervals, except for SF that is central 90% and STfR1 that is the interquartile range.
Given the relevance of iron homeostasis in neonates, especially among premature ones, and the
potential reversibility of pathologic conditions with prompt treatment, the reliable measurement of
iron status is pivotal [57]. However, normative values of the main parameters used for the diagnosis of
ID and IDA in premature infants are still lacking [62].
The iron reduction can be summarized through three stages of increasing severity:
1. Iron depletion: decrease of total iron storage, as reflected by SF, the first laboratory index to
decline in ID;
2. Iron deficient erythropoiesis: initial reduction of Hb concentration due to complete depletion of
iron stores; and
3. Iron deficiency anemia: ID associated with anemia, defined by WHO as Hb level two standard
deviations below the median of a healthy population of the same age and sex [5,14].
Hb is the most frequently used hematologic parameter to screen for ID in infants. In clinical
practice, the terms “anemia” and “IDA” have been interchangeably used as if the ID is the only cause
of anemia [14,63]. However, Hb alone lacks sensibility and specificity since low Hb levels can derive
from several conditions other than ID, such as hemolysis, chronic infections, genetic disease, or other
less common nutrient deficiencies, particularly folate or vitamin B12 [14]. For this reason, to establish
IDA, Hb measurement should be associated with: (1) SF and C reactive protein (CRP), or (2) CHr,
based on AAP recommendations [14].
However, anemia suggests a severe depletion of iron stores. Indeed Hb is a late marker of ID
and is not a reliable indicator of neonatal iron status, especially if measured in newborns suffering
from chronic hypoxia, where stored iron is prioritized to preserve erythropoiesis to the detriment of
other tissues [13].
Since ID alone, even without anemia, may impair neurodevelopment, prompt diagnosis and
treatment of ID are essential. The AAP suggests to screen ID through the measurements of (1) SF and
CRP, or (2) CHr [14].
Serum ferritin estimates total iron body stores; its cord blood concentration steadily increases
throughout gestation [62]. Recently, ferritin concentration has been measured in cord blood in
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neonates from 23 to 41 gestational age [28]. Specifically, SF values below 35 μg/L indicate ID in
preterm infants and correlates with complete depletion of liver iron stores [28]. On the other hand,
SF concentrations >300 μg/L depict iron overload [64]. However, SF is an acute phase reactant and
can reach high concentration during inflammation and infection [2]. As an increase in SF during
co-existing inflammatory processes can mask ID, the AAP suggests the simultaneous measurement of
SF and CRP [2]:
• low SF concentrations regardless of CRP level confirm ID;

• increased or normal SF levels associated with normal CRP concentrations rule out ID; and
• when high or normal SF concentrations are associated with increased CRP levels, iron status
cannot be assessed [14].
Serum ferritin concentrations increase in the early postnatal period due to hemolysis and
delivery itself, so that cord ferritin levels are approximately 1/3 of those in the first 72 h of life [58].
The opposite happens to serum iron concentration, increased in the umbilical vein, as a result of iron
transport from the mother to the fetus [58].
RET-He and CHr quantify iron concentration inside reticulocytes and, therefore, by providing a
real-time assessment of bone marrow iron status, may represent a preventive screening test for ID [63].
Indeed, their detection may anticipate ID diagnosis, if compared to Hb, which is a late marker of
ID, since Hb evaluates the whole red blood cell population. Recent studies suggest a role for CHr
in predicting future IDA even among infants; however, its measurement is not readily available in a
laboratory setting [63]. A linear correlation has been reported between RET-He and CHr [65].
Similarly, STfR1 concentration is related to intracellular iron stores. High STfR1 concentrations are
found in term and preterm neonates as a result of maternal ID, reflecting a poor iron endowment [28].
They are not directly influenced by gestational age [58]. Neonatal reference ranges should be established
to improve its use in clinical practice.
ZnPP/H detects zinc incorporation into protoporphyrin IX in erythrocytes [66]. In case of
insufficient iron delivery to bone marrow and reduced erythropoiesis [62], iron is replaced by zinc,
thus increasing ZnPP/H ratio. Nevertheless, it cannot distinguish whether it is caused by body iron
stores depletion or enhanced rate of erythropoiesis, as it happens in premature infants during the first
weeks of life. ZnPP/H ratio is inversely correlated with gestational age [59,62]. Higher ZnPP/H ratios are
detected in those born to mothers affected by gestational diabetes and IUGR or with chorioamnionitis,
suggesting a potential association with inflammatory or infectious processes [28,59,62].
Brain iron concentrations are hardly measurable and can be quantified only at autopsy. Differently,
serum ferritin can be used as an indirect index of brain ID while cord ferritin as a marker of in-utero
fetal iron status [31]. Neurobehavioral tests are used to investigate multiple brain functions that can
indicate the injured brain area, such as the Bayley Scales for Infant Development, Griffith Development
Scale, Wechsler Preschool and Primary Scale of Intelligence (WPPSI), and the Wechsler Intelligence
Scale for Children (WISC). Nevertheless, they show a low specificity for ID, since other nutrients
deficiencies (e.g., zinc, copper, iodine) can lead to similar neurobehavioral abnormalities [6].
4. Iron Deficiency and Supplementation
4.1. Risk Factors
4.1.1. Maternal Iron Status

In the past, it has been assumed that a poor maternal iron status during pregnancy,
unless determining severe IDA, does not affect fetal or neonatal iron endowment, as testified by
the absence of association between maternal and cord blood Hb level [11]. Placental transferrin
receptors increase in the case of maternal ID to transfer more iron to the fetus [11]. Conversely, it is
known that maternal IDA is associated with preterm delivery and low birth weight, both of which
lead to decreased neonatal iron stores [11].
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However, a direct relationship between cord blood ferritin concentration, maternal Hb, and SF
concentration has been found [11]. These observations may indicate that, even if cord blood Hb
concentrations are within normal values, those born from mother with ID have reduced iron stores
and are more likely to be anemic during infancy [67].
Iron supplementation during pregnancy is beneficial for both the mothers and their infants.
The improvement of maternal iron status, even in women with satisfactory iron stores, prevents ID
in the subsequent pregnancy [11] and reduces maternal fatigue with a positive impact on mental
health, thus producing indirect postnatal benefits on neonatal development [37]. Moreover, iron intake
is associated with higher blood ferritin concentration and better neurodevelopmental outcomes in
their infants [27].
4.1.2. Maternal Comorbidities

Up to 10% of pregnancies in the developed countries are complicated by IUGR, secondary to
placental insufficiency, and structural abnormalities of the placental vessels. These conditions may
impair iron transport to the fetus, while chronic fetal hypoxia induces erythropoietin synthesis and
subsequent iron use for Hb production [13]. Low cord blood ferritin has been found in about 50% of
IUGR infants (<60 ng/mL). Similarly, maternal diabetes mellitus increases fetal metabolism and oxygen
consumption by approximately 30% [62]. Hypoxia stimulates Hb synthesis, which requires 3.47 mg of
iron for 1 g of Hb [4]. This increased demand depletes heart, liver, and brain iron stores with a severity
that is inversely related to maternal glycemic control [4]. Furthermore, the physiological regulatory
mechanism of the placenta seems to be lost. Despite fetal hypoxia, placental transferrin receptor (TfR1)
concentration is low, indicating a decreased receptor response capacity [68].
As a result, IUGR infants and those born to a diabetic mother are at higher risk of brain ID.
At autopsy, the most severe cases showed total iron brain content reduced by 30–40%, and more than
half of them have low ferritin in cord blood [9].
4.1.3. Prematurity
Around 25–85% of premature newborns develop ID, associated or not with IDA, during infancy [16],
usually with earlier onset than in full-term neonates. Indeed, at birth, total body iron concentration
is, on average, 75 mg/kg in term neonates versus 64 mg/kg in preterm neonates [66]. Similarly, in the
latter group, serum iron concentrations and cord SF are lower with higher sTfR1 levels at birth,
when compared to term neonates [62].
Many factors contribute to the negative iron imbalance and can explain why premature infants
are so vulnerable to ID and IDA. Firstly, even if placental iron transfer begins in the first trimester
of pregnancy, approximately 80% of iron accumulates during the last one [68]. Therefore, the more
premature the infant is, the poorer its iron status will be. Postnatally, low iron stores are further
reduced by the rapid “catch up” growth with rapid blood volume expansion and increased Hb demand,
which requires further iron [62]. Moreover, recurrent phlebotomies for diagnostic purposes cause an
extra iron loss in VLBW neonates equal to 6 mg/kg/week, on average [64].
In the past, additional iron was supplied with red blood cells (RBC) transfusions to prevent the
anemia of prematurity. In the last years, more restrictive RBC transfusion policies are encouraged,
particularly among the sickest extremely preterm neonates, because of the associations between early
exposure to RBC transfusions, and increased mortality and short-term morbidities [21,69–71].
4.1.4. Low Birth Weight

SGA neonates show lower iron stores when compared to gestation-matched
appropriate-for-gestational-age (AGA) neonates in cord blood and at four weeks postnatally [72].
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4.1.5. Gender
Gender may influence iron endowment at birth: males have a smaller iron supply at birth reflected
by a significantly lower concentration of Hb, MCV, SF, and higher ZnPP and sTfR1 levels at four,
six, and nine months when compared with females [73]. These features place male infants at increased
risk of ID during infancy. Therefore, to exclude physiological sex-related differences, the construction
of gender-specific reference intervals may be beneficial.
4.1.6. Breastfeeding
As human breast milk contains a very low quantity of iron (0.2–0.4 mg/L), exclusively breastfed
infants are prone to develop ID [26,74]. The incidence of ID in the first six months of life is 6–15% and
12–37% in industrialized and developing countries, respectively [68]. The iron content in human milk
satisfies full-term neonates’ iron demand in the first 4–6 months of life, whereas iron stores of preterm
neonates are depleted within 1–4 months after birth [16]. Despite the low iron content in human milk,
its bioavailability is around 50%, much higher than in formula milk. Indeed lactoferrin, which is an
iron-binding protein contained in breast milk, facilitates iron absorption. In contrast, casein and other
cow milk’s proteins in cow milk-based formula have an inhibitory effect on iron absorption [26].
4.2. Prevention
Recently, placental transfusion techniques, such as delayed cord clamping (DCC) and umbilical
cord milking (UCM), have been implemented in routine neonatal care. DCC consists of delaying
umbilical cord clamping for at least 30–60 s after delivery in both term and preterm infants not
requiring immediate resuscitation [75]. This practice allows the transfer of 25–35 mL/kg of placental
blood to the newborn, thus increasing iron stores by approximately 30% after three minutes of
DCC [26]. Indeed, DCC, compared to immediate cord clamping, is associated with higher hemoglobin
concentration in first weeks after birth in both term and preterm newborns, and lower rates of RBC
transfusions in preterm newborns [75–77]. In the long term, DCC maintains its benefits, as evidenced
by the higher ferritin levels and the lower incidence of ID at six months of age in term neonates [2,75,78].
Indeed, the amount of blood transferred via DCC is crucial in defining iron endowment at birth [12].
As largely illustrated above, ID and brain development are closely linked. DCC has been associated
with improved neurodevelopmental outcome at four years in full-term infants and two years in very
preterm newborns [79,80].
UCM consists of a gentle squeezing of the umbilical cord towards the baby two to five times before
clamping. Similarly to DCC, UCM enhances iron stores in the first weeks after birth in premature
infants as compared to early cord clamping [81]. However, results from a recent randomized clinical
trial comparing UCM vs. DCC in preterm infants born before 32 weeks’ gestation have shown a
significantly higher rate of severe IVH in the UCM group [82].
Phlebotomy is the first cause of anemia in the first weeks of life [83], especially in the most
premature newborns. Therefore, there is increasing attention to minimize blood loss in these patients.
To this goal, the use of low blood volume point-of-care testing, non-invasive monitoring and the
reduction of unnecessary blood sampling are mainstays of prevention [84].
Furthermore, otherwise discarded placental blood has been endorsed as an alternative source for
Neonatal Intensive Care Unit (NICU) admission laboratory tests (complete blood count, blood culture,
blood type, antibody screen, and metabolic screening) [85,86]. Of note, umbilical cord blood seems not
suitable to assess neonatal hemostatic profile, as placental specimens show a procoagulant imbalance
if compared to the neonatal counterparts [87]. Drawing blood directly from the umbilical vessels
avoids invasive and painful neonatal procedures and results, especially in VLBW infants, in higher
hemoglobin concentration, lower rates of RBC transfusions, and need for vasopressors in the first week
of life [88]. Additionally, it appears to be a complementary procedure to DCC to maximize circulating
blood volume.
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4.3. Supplementation
4.3.1. Enteral Iron Supplementation

Enteral supplementation is the preferred route of iron administration. It can be provided
through iron fortified-human milk, iron-fortified formula, or medicinal elemental iron, in the form of
ferrous sulfate or ferrous fumarate. The former has been associated with better absorption, while the
latter produced less oxidative stress in vitro. To our knowledge, no studies have compared the two
preparations in premature infants [89].
Iron gut absorption has a high inter-individual variability ranging from 10% to 50% of the dose
administered and is increased when given with breast milk or with vitamin C [16,57]. Iron intake
depends on:
1. Local factors, such as gastric pH and intestinal mucosal function; and

2. General drivers, such as the source of enteral iron (human or formula milk) and the iron stores of
the infant [90].
It has been hypothesized that iron absorption and release may be impaired due to enterocytes’
immaturity, thus explaining inadequate iron intakes in orally supplemented neonates [91].
The position paper by the European Society for Pediatric Gastroenterology,
Hepatology, and Nutrition (ESPGHAN) recommend an elemental iron intake of 1–2 mg/kg/day
for all premature infants with a birth weight less than 2500 g and of 2–3 mg/kg/day for those
weighing less than 2000 g [92]. Similarly, the Committee on Nutrition of the AAP suggests a daily
iron supplementation of 2 mg/kg for all breastfed preterm infants, from 1 to 12 months of age [93].
An enteral iron dosage >5 mg/kg/day should be avoided in these patients [94].
Iron content in infant formula is 14.6 mg/L and 12 mg/L in standard preterm and term formula,
respectively. Based on a standard daily milk intake of 150 mL/kg, formula-fed neonates receive around
1.8–2.2 mg/kg/day of iron [14]. Although being fed with iron-enriched formula, up to 14% of preterm
infants develop ID in the first year of life. Thus, iron status should be monitored to individualize iron
supplementation of preterm neonates [14].
In case of IDA in preterm newborns, elemental enteral iron supplementation is increased to
3–6 mg/kg per day for three months [93].
The current recommendation may not be adequate to meet the needs of all premature infants.
The most immature neonates, such as ELBW, have a poor iron endowment and may require extra iron
due to the catch-up growth and increased erythropoiesis. Indeed, 15% of infants with birth-weight
<1301 g are iron deficient at two months of age even if supplemented with 4–6 mg/kg of iron since the
second week of age [95]. Nevertheless, the majority of ELBW infants receive 3–5 RBC transfusions
during their hospital stay that improve their iron stores [16]. For these reasons, the iron status of ELBW
infants should be monitored to tailor iron administration.
No gastrointestinal adverse effects have been described after the administration of iron-rich
formula or elemental iron. Hematochezia has been reported in 17% of premature infants exposed to
high doses of medical iron (8–16 mg/kg/day) [90]. However, a causal link with oral iron administration
was not established, and iron supplementation can be resumed after the resolution of symptoms [16].
Concerns were raised regarding the possible interaction in absorption between iron and other
divalent cations since they share the same gut transport mechanism (divalent metal transporter 1, DMT1).
However, iron supplementation at the recommended doses does not interfere with zinc or selenium
uptake, and zinc supplementation does not compromise iron absorption [16,96]. Conversely, it is
known that oral iron alters copper metabolism, although further research is needed [97].
Iron supplementation may induce oxidative stress by promoting ROS production as a result of
increased intra- and extra-cellular free iron concentrations [98], whose underlying mechanism has been
previously mentioned (cfr paragraph 2, Figure 1).
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4.3.2. Parenteral Iron Supplementation

In addition to the oral route, iron can be administered parenterally. Intramuscular administration is
not recommended because it is painful and prone to complications, while intravenous (i.v.) iron appears
to be safe [99]. Although the daily iron intake during the third trimester is about 1.6–2 mg/kg/day,
a dose of 120 μg/kg/day is adequate and, since there is no physiologic regulatory mechanism for iron
excretion, almost the totality of it gets stored in tissues [16]. When compared with the enteral route,
i.v. supplementation is associated with higher SF levels, while inconclusive results are related to its
efficacy in supporting erythropoiesis [99]. Besides, the need for i.v. line in a full enterally-fed infant
makes the i.v. route unreasonable in clinical practice. Moreover, a transient rise of malondialdehyde
(MDA), indicating lipid peroxidation, has been reported after iron infusions, thus suggesting a risk of
oxidative stress [16]
4.4. Risk Groups Requiring Tailored Iron Supplementation

Preterm infants treated with rHuEPO require higher doses of iron because rHuEPO improves
growth, stimulates erythropoiesis, and decreases the number of RBC transfusions at the expense of
tissues iron stores, as reflected by the decrease in SF after the initiation of rHuEPO therapy [90,100].
The AAP recommends an oral iron supplementation of 6 mg/kg/day during rHuEPO therapy [93].
This dose is appropriate to sustain erythropoiesis; however, it may be inadequate to preserve body
iron stores. No differences have been found in the SF levels nor in the hematological response after an
oral iron supplementation at a high dose (16 mg/kg/day) or low dose (8 mg/kg/day) in infants treated
with rHuEPO, as reported by Bader et al. [90].
Ferritin concentration higher than 100 μg/L should be considered the threshold to guide iron
administration during rHuEPO therapy [16].
Despite the lack of specific recommendations, infants with increased SF concentrations (>350 μg/L)
might require personalized iron supplementation. This condition can indicate two different states:
1. Iron overload (e.g., after recurrent erythrocyte transfusions); or

2. Iron sequestration (e.g., in the setting of an inflammatory process).
While the former group is at risk of iron overload associated insults, and should not be
supplemented, the latter can suffer from bone marrow ID and subsequent insufficient erythropoiesis
and thus could benefit from iron administration. Assessing bone marrow iron status by measuring
reticulocyte count and ZnPP/H ratio can support decision making [16].
Nevertheless, one out of four premature neonates with SF levels >95th percentile at hospital
discharge will manifest ID at 6–12 months of age if iron supplementation is discontinued [16].
4.5. Timing of Iron Supplementation and Screening of ID

Iron supplementation should start from four to six weeks of age [16] when serum iron and
ferritin concentration start reducing and iron incorporation in RBC occurs more efficiently [16].
The AAP does not recommend iron supplementation before the second week of age, due to the
immature antioxidant capacity before that age [4]. In contrast, iron supplementation, by two weeks
of age, as suggested by ESPGHAN [94], is associated with decreased rates of RBC transfusions and
incidence of ID at 2–6 months of age when compared with later onset. Only one study evaluated
the long-term effect of early (started at a median age of 14 days) versus late (eight weeks) iron
supplementation reporting better cognitive and motor outcomes at five years of age following the
earlier start [41]. However, this study was underpowered to evaluate improvements in neurocognitive
development [41,101]. Therefore, in the absence of consistent data, considering the potential risk of
iron administration in the first month of life due to iron overload and immaturity of the gastrointestinal
function, caution is required, especially in ELBW infants.
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Iron supplementation should be provided at least up to six months of age when weaning with
iron-rich foods begins [94]. The AAP recommends prolonging iron supplementation until the end of
the first year of life [93].
The iron status of premature infants should be monitored periodically. The frequency and
timing of follow up controls should be scheduled, taking into account the iron status at discharge,
iron supplementation, and type of feeding [16].
5. Iron Overload and Toxicity
5.1. Risk Factors
5.1.1. RBC Transfusions

Transfusion-derived iron is the main cause of iron overload in premature infants. Around 80%
of VLBW and 95% of ELBW infants require at least one RBC transfusion during hospitalization,
with 0.5–1 mg of iron intake for each mL of packed RBC transfused. [16].
Biologically, the exceeding iron cannot be actively excreted by humans. Consequently, after multiple
transfusions, higher serum and ferritin concentrations and liver iron storage can be found in
preterm infants [16].
The pathogenesis of prematurity-related comorbidities is multifactorial. The association between
the rate of erythrocyte transfusions and the incidence of NEC, IVH, ROP, and BPD has been
suggested [21,102–104]. In this context, iron (excess) has been hypothesized to play a causative role
due to its impact on the immune system [105], nitric oxide-induced vasoregulation [106], and oxidative
stress [107]. Specifically, both the preparation and storage of pediatric packed RBCs may predispose to
redox unbalance. Indeed, pediatric RBCs are prepared from adult blood, by replacing most of the plasma
with additive solutions, thus reducing the net amount of iron-binding proteins and antioxidants [108].
During storage, the exposure of blood to shear stress, plastic bags, anticoagulants, and additives
contributes to the increase of extracellular iron and NTBI [109], leading to a rise in MDA [110].
In adults, the storage duration of transfused RBCs does not impact on mortality [111]. In pediatrics,
there is a low level of evidence stemming from observational studies of the association between longer
storage duration of transfused RBCs and worse outcomes [112]. However, data from clinical trials do
not support the use of fresh blood [113,114]. Donor exposure is another relevant issue, especially when
it comes to the smallest and sickest premature infants, requiring multiple transfusions in the early days
of life. The use of satellite bags (small-volume aliquots from the same unit of donor blood) may limit
the donor exposure rate [115,116].
Finally, procedures that require massive blood products exposure, as in the case of exchange
transfusion or extracorporeal membrane oxygenation, further increases the neonatal exposure to
hemolysis, oxidative stress and, hence, the risk of mortality [117,118].
5.1.2. Excessive Iron Supplementation

At routinely-used dosage, parenteral or enteral iron does not produce oxidative stress injury, even
if a transient peak of serum iron level can be registered after administration [16]. It is reported that
even an enteral supplementation at higher doses (up to 18 mg/kg/day) [119] does not cause oxidative
stress damage but, instead, stimulate erythropoiesis, as low ZnPP/H ratio testify [66]. In VLBW infants,
a one-week course of high doses of oral iron (18 mg/kg/day) did not result in a change in oxidative
stress biomarkers nor anti-oxidants levels [89].
However, lower doses (3–6 mg/kg/day) for a longer period (up to nine months of age)
produce an increase in glutathione peroxidase concentrations, a marker of oxidative stress [120].
Moreover, large doses of enteral iron administration have been linked to hemolysis in preterm infants
with vitamin E deficiency [16].
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5.2. Iron Toxicity

Iron excess has been associated with poor growth and interference with zinc and copper
metabolism [26]. The relationship between iron and infection is widely recognized [121]. Previous studies
reported an increased incidence of respiratory tract infections in neonates supplemented with high iron
doses (formula fortified with 20.7 mg iron/L) [120]. Certainly, NTBI acts as a potent pro-oxidant agent
through the generation of ROS.
Recently, a trial showed that iron-fortified foods modulate the intestinal microbiota,
with overgrowth of pathogenic enterobacteria, thus triggering gut inflammation [122].
The consequences of this process are not yet known and deserve to be explored.
Premature infants are vulnerable to iron toxicity due to low TIBC levels and immature anti-oxidant
defenses that facilitates iron oxidation of the ferrous state with the increase of ROS production [16].
Vitamin E and vitamin C are radical-scavenging antioxidants, which are poorly adsorbed until the
34th week of gestation and low activity in the first two weeks of life [4]. Finally, superoxide dismutase
(SOD), which is an enzyme with a key antioxidant role, is lacking in preterm neonates [4,25].
A recent study involving 95 premature neonates found no association between moderate iron
overload, as defined by SF >400 ng/mL but <1000 ng/mL at 34–35 weeks of corrected age and
neurodevelopmental impairment at 8–12 months [119]. However, preterm newborns in the first
weeks of life are prone to develop oxidative stress, due to concomitant predisposing risk factors.
Therefore even modest SF levels may damage the brain [119].
The early identification of biomarkers of oxidative stress could prevent future sequelae and allow
new therapeutic strategies. To date, serum and urinary prostanoids, particularly urinary 8-isoprostane,
and visfatin, an adipocytokine involved in inflammation, have been identified as potential markers of
oxidative stress in premature newborns [25,89,123].
Additionally, the dosage of plasma antioxidants, such as glutathione, vitamin E, and vitamin C
(ascorbic acid), particularly oxidized to total ascorbic acid ratio (DHAA/TAA), has been proposed to
prevent oxidative stress [89]. Recently, serum gamma-glutamyltransferase (GGT) has been found to
have a role in ROS production and glutathione synthesis, and it might be used as a cheap and reliable
marker of oxidative stress [124]. Plasma adenosine appears as a promising biomarker to predict brain
damage; however, its role in the neonatal setting has yet to be demonstrated [25].
6. Concluding Remarks and Future Perspectives

Many preventive measures have already been implemented in clinical practice to tackle ID,
and iron supplementation is recommended for all preterm neonates. However, since iron levels are
influenced by several perinatal factors, a tailored-supplementation based on laboratory iron status
parameters should be encouraged.
For this reason, gestational age-related reference ranges for the main iron-related diagnostic
indices should be established, especially for the most immature neonates. Attention should be paid to
the new ID markers, such as RET-He and CHr, which could anticipate ID diagnosis, thus allowing a
prompt therapy.
Beyond ID and IDA, which have been widely addressed, iron overload is emerging as a ‘new’
issue in the management of sick preterm infants. Future efforts should focus on the early identification
of biomarkers of oxidative stress, which could improve patients’ care by paving the way for innovative
therapeutic targets.
The main risk factors, prevention strategies, and therapies for iron deficiency and overload are
summarized in Figure 2.
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Figure 2. Iron homeostasis in preterm newborns: risk factors, prevention strategies and treatment.
DCC: delayed cord clamping; Dcytb: duodenal cytochrome b; DMT1: divalent metal transporter;
Fe2+ : ferrous cation; Fe3+ : ferric cation; FPN: ferroportin; RBC: red blood cell; rHuEPO: recombinant
human erythropoietin; SGA: small for gestational age; TF: transferrin.
Author Contributions: Conceptualization: G.R., G.C., F.M. (Fabio Mosca), and S.G.; methodology: G.R., F.M.
(Francesca Manzoni), V.C., G.C., F.M. (Fabio Mosca), and S.G.; supervision: G.C., F.M. (Fabio Mosca), and S.G.;
visualization: G.R., F.M. (Francesca Manzoni), and V.C.; writing—original draft preparation: G.R. and F.M.
(Francesca Manzoni); writing—review and editing: V.C., G.C., F.M. (Fabio Mosca), and S.G. All authors have read
and agreed to the published version of the manuscript.
List of Abbreviation
AAP American Academy of Pediatrics
ABR Auditory brainstem-evoked response
AGA Appropriate-for-gestational-age
BPD Bronchopulmonary dysplasia
CHr Reticulocyte hemoglobin content
CRP C reactive protein
CSF Cerebrospinal fluid
DCC Delayed cord clamping
DHAA/TAA Oxidized to total ascorbic acid ratio
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DMT1 Divalent metal transporter 1

ELBW Extremely low birth weight
ESPGHAN The European Society for Pediatric Gastroenterology, Hepatology, and Nutrition
GGT Gamma-glutamyltransferase
Hb Hemoglobin
HIE Hypoxic-ischemic encephalopathy
ICH Intracerebral hemorrhage
ID Iron deficiency
IDA Iron deficiency anemia
IUGR Intrauterine growth restriction
i.v. Intravenous
IVH Intraventricular hemorrhage
MDA Malondialdehyde
MCV Mean corpuscular volume
NEC Necrotizing enterocolitis
NICU Neonatal Intensive Care Unit
NTBI Non-transferrin-bound iron
PVL Periventricular leukomalacia
PWM Punctate white matter lesions
RBC Red blood cells
RET-He Reticulocyte hemoglobin equivalent
rHuEPO Recombinant human erythropoietin
ROP Retinopathy of prematurity
ROS Reactive oxygen species
SF Serum ferritin
SGA Small-for-gestational-age
STfR1 Soluble transferrin receptor
TACO Transfusion-associated circulatory overload
TfR1 Transferrin receptor
TIBC Total iron bind capacity
TRALI Transfusion-related acute lung injury
UCM Umbilical cord milking
VLBW Very low birth weight
ZnPP/H Zinc protoporphyrin to heme ratio
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nutrients
Review
Multifactorial Etiology of Anemia in Celiac
Disease and Effect of Gluten-Free Diet:
A Comprehensive Review
Rafael Martín-Masot 1 , Maria Teresa Nestares 2, *, Javier Diaz-Castro 2 , Inmaculada López-Aliaga 2 ,
Maria Jose Muñoz Alférez 2 , Jorge Moreno-Fernandez 2 and José Maldonado 3,4,5,6
1 Pediatric Clinical Management Unit, Málaga Regional University Hospital, 29010 Málaga, Spain;
rafammgr@gmail.com
2 Department of Physiology and Institute of Nutrition and Food Technology “José MataixVerdú”, University
of Granada, 18071 Granada, Spain; javierdc@ugr.es (J.D.-C.); milopez@ugr.es (I.L.-A.);
malferez@ugr.es (M.J.M.A.); jorgemf@ugr.es (J.M.-F.)
3 Department of Pediatrics, University of Granada, 18071 Granada, Spain; jmaldon@ugr.es
4 Biosanitary Research Institute, 18012 Granada, Spain
5 Maternal and Child Health Network, Institute Carlos III, 28020 Madrid, Spain
6 Pediatric Clinical Management Unit. Virgen de las Nieves University Hospital, 18014 Granada, Spain
* Correspondence: nestares@ugr.es; Tel.: +34-6-9698-9989
Received: 6 September 2019; Accepted: 15 October 2019; Published: 23 October 2019
Abstract: Celiac disease (CD) is a multisystemic disorder with different clinical expressions, from
malabsorption with diarrhea, anemia, and nutritional compromise to extraintestinal manifestations.
Anemia might be the only clinical expression of the disease, and iron deficiency anemia is
considered one of the most frequent extraintestinal clinical manifestations of CD. Therefore,
CD should be suspected in the presence of anemia without a known etiology. Assessment of
tissue anti-transglutaminase and anti-endomysial antibodies are indicated in these cases and, if
positive, digestive endoscopy and intestinal biopsy should be performed. Anemia in CD has a
multifactorial pathogenesis and, although it is frequently a consequence of iron deficiency, it can be
caused by deficiencies of folate or vitamin B12 , or by blood loss or by its association with inflammatory
bowel disease (IBD) or other associated diseases. The association between CD and IBD should be
considered during anemia treatment in patients with IBD, because the similarity of symptoms could
delay the diagnosis. Vitamin B12 deficiency is common in CD and may be responsible for anemia
and peripheral myeloneuropathy. Folate deficiency is a well-known cause of anemia in adults, but
there is little information in children with CD; it is still unknown if anemia is a symptom of the most
typical CD in adult patients either by predisposition due to the fact of age or because biochemical and
clinical manifestations take longer to appear.
Keywords: celiac disease; gluten-free diet; iron deficiency; anemia; micronutrient deficiencies
1. Introduction
Celiac disease (CD) is one of the most frequent genetic diseases, affecting 1% of the world
population. Diagnosed cases are increasing and it seems to be due to the actual increase in the incidence
rather than due to the advancement of diagnostic methods or to the larger awareness of the disease
among the lay population [1,2].
Celiac disease is a systemic disorder, caused by an immune reaction activated by the ingestion of
gluten and related proteins occurring in individuals carrying haplotypes of major histocompatibility
antigen (HLA) class II: more than 90% of celiac patients are HLA-DQ2 haplotype positive, and almost
all of the remaining patients carry HLA-DQ8. Exposure to gluten has a double effect, triggering both

Nutrients 2019, 11, 2557
innate and adaptive immune responses, with symptoms at the intestinal and extra-intestinal levels [3].
The contact of the intestinal mucosa with gluten leads to a characteristic histological lesion, although
not pathognomonic. Their typical histological features are an increase in intraepithelial lymphocytes,
villous atrophy, crypt hyperplasia, and infiltration of inflammatory cells in the lamina propria.
Diagnosis of CD is conducted by combining serological screening tests
(anti-tissue-transglutaminase and anti-endomysial IgA antibodies) and an intestinal biopsy [4].
The duodenal biopsy can be avoided [5] in adolescents and children with symptoms or signs of CD
and with high anti-tissue-transglutaminase antibody levels, positivity for anti-endomysial antibodies,
and presence of HLA DQ21 or HLA-DQ8 heterodimer.
Recent reports have demonstrated that specific miRNAs are modulated in duodenal mucosa
affected by CD. The miRNAs dysregulated during the development of CD could be potentially
involved in the pathogenesis of CD [6]. Overexpression or downregulation of several miRNAs could
potentially stimulate or inhibit pathways related to the pathogenesis of CD. A study has demonstrated
the regulation of circulating miRNA-21 and miRNA-31 expression levels in children with CD and
showed that miR-21 expression level was positively correlated with the anti-tissue-transglutaminase
IgA antibodies [7]. This correlation may indicate that the altered expression of the circulating miRNAs
could be used as potential non-invasive diagnostic and prognostic biomarkers for CD patients.
In addition, Vaira et al. [8] have shown the downregulation of miR-194-5p and the overexpression of
miR-638 in celiac patients with anemia compared with celiac patients with classical symptoms.
Patients with CD could feature various deficiency states, leading to anemia and bone mass loss
and a wide range of digestive and extra-digestive symptoms. Upon diagnosis, nutritional deficiencies
were found in vitamins and minerals; patients should be tested for micronutrient deficiencies, in
particular iron, folic acid, vitamin B12 , vitamin D, copper, and zinc. Celiac disease is a cause of anemia,
usually due to the malabsorption of iron, folic acid, and vitamin B12 [9]. Anemia is mainly due to
the fact of iron deficiency as a consequence of iron malabsorption. Iron malabsorption is usually
observed in CD, being considered a clinical diagnostic feature of CD even in subjects not presenting
the classic digestive symptoms. Iron deficiency anemia (IDA) is a frequent finding in patients with
overt CD (10–20% of cases) [10], despite the fact that they are consuming iron supplements. A recent
meta-analysis found that more than 3% of patients with IDA have histological evidence of CD. This
high percentage of subjects with IDA who are celiac, reinforces the need for screening CD in patients
with IDA [11]. Folate and vitamin B12 malabsorption, nutritional deficiencies, blood loss, inflammation,
development of refractory CD or concomitant Helicobacter pylori infection are other causes of anemia in
such patients [12] (Table 1).
Table 1. Etiology of anemia in celiac disease.
Cause Incidence
12–69% (adults)
Iron deficiency
10–20% (children)
Folic acid deficiency 20–30%
Vitamin B12 deficiency 8–41%
Copper deficiency Very low
Zinc deficiency Very low *
Bad response to the gluten-free diet 23%
Medullary aplasia Very low (12 cases)
Chronic disease 4–17%
* It has been reported that 50% of celiac patients have low serum levels at diagnosis, but it has not been related to
celiac disease (CD).
134
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The mainstay of treatment for CD remains adherence to a gluten-free diet (GFD). In the vast majority
of cases, strict monitoring of GFD leads to the disappearance of clinical symptoms and serological
signs, the recovery of normal histology in the duodenum and the prevention of complications derived
from CD [13]. However, in approximately 20% of celiac patients, symptoms persist despite excluding
gluten from their diet [14].
The aim was to perform a review of recent literature data regarding causes of anemia in CD patients.
For this purpose, we performed a literature search on two databases—PubMed and Embase—using
the Medical Subject Headings (MESH) term “celiac disease” and several keywords referring to the
associated hematological features and nutritional imbalances. Articles identified from this search
strategy were evaluated for relevance to the topic. Clinically significant full-text articles were selected
for their inclusion in this review.
2. Micronutrient Deficiencies and Celiac Disease
2.1. Iron Deficiency

Iron is an essential micronutrient, it is required for adequate erythropoietic function, oxidative
metabolism, enzymatic activities, and cellular immune responses [15]. IDA is a major public
health problem. Iron deficiency anemia occurs when iron loss and body’s requirement for iron
are not met by dietary sources, therefore the iron storage of the organism is depleted. This
pathological process is characterized by the production of smaller red cells because the concentration
of hemoglobin (Hb) is abnormally low [15]. Iron deficiency anemia results in fatigue and diminished
muscular oxygenation, which may affect muscle strength and quality and, subsequently, physical
performance [16]. Celiac disease constitutes one of the groups at highest risk of iron deficiency (ID) [17].
Iron requirements exceed iron intake at some time points throughout life: the first 6–18 months of life
and then, for women, during adolescence and all fertile period. Iron deficiency during the first year of
life occurs at a time point of rapid neural development and when morphological, biochemical, and
bioenergetic alterations may all influence future functioning [18]. The brain is the most vulnerable
organ during critical periods of development [19]. Iron is present in the brain from very early in life,
when it participates in the neural myelination processes [20], learning, and interacting behaviors, and
iron is needed by enzymes involved in the synthesis of serotonin and dopamine neurotransmitters [21].
The most common causes of ID are blood loss and failure of the enterocytes of the proximal
intestine to uptake iron from the diet in patients who have enough dietary iron. Celiac disease leads to
an abnormal immune response, which is followed by a chronic inflammation of the small intestinal
mucosa with progressive disappearance of intestinal villi [22] leading to a decrease in absorption of
many nutrients, including iron [23,24]. Unfortunately, this interesting association between CD and
IDA has been poorly appreciated [25] in spite of the great interest of micronutrient deficiency as a
diagnostic clue in asymptomatic CD, especially for iron and IDA [26].
Celiac disease is an increasingly recognized disorder in Caucasian populations of European
origin. Murray et al. [27] analyzed HLA genotypes and frequencies of CD between Caucasians and
non-Caucasians with ID. The results showed that CD is associated with ID in Caucasians, but CD is rare
among non-Caucasians—even among individuals with features of CD, such as ID. Pirán Arce et al. [28]
evaluated the nutritional status of iron in 44 celiac children by determining biochemical parameters and
their relationship with the intake of this mineral and adherence to the GFD. These authors concluded
that under conditions of adequate iron consumption, iron status is related to the degree of adherence
to the GFD. Although GFD is an effective treatment for CD, IDA remains an occasional finding during
follow-up and correlates to inadequate gluten exclusion [10].
Malabsorption causes should be considered especially in refractory IDA; this malabsorption can
be the only manifestation in subclinical and silent CD [29,30]. The study of Shahriari et al. [11] suggests
serologic screening for CD in patients with refractory IDA to minimize the complications of CD and
repeated iron treatment. A study [31] revealed a significant association between H. pylori infection and
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Nutrients 2019, 11, 2557
IDA in patients with CD, and Samasca et al. [32] recommend performing the screening for H. pylori
infection in patients with CD and ID, but currently there is no evidence to support this recommendation.
Elli et al. [33] evaluated the role of the TMPRSS6 variant rs855791 in GFD treated CD patients with
IDA persistence against non-IDA CD and non-CD subjects. The authors found a significantly higher
percentage of TMPTSS6 mutation in CD patients than in non-CD controls, while no differences were
found between IDA and non-IDA CD patients. Conversely, De Falco et al. [34] investigated the role of
HFE C282Y, H63D, and TMPRSS6 A736V gene variants in the pathogenesis of IDA in CD patients, at
diagnosis and after 1 year of GFD. This study suggests a protective role of HFE in IDA CD patients
and confirms the role of TMPRSS6 in predicting oral iron response modulating hepcidin action on
iron absorption. Iron supplementation therapeutic management in CD could depend on TMPRSS6
genotype that could predict persistent IDA despite iron supplementation and GFD.
Iron enters the enterocytes through an apical divalent metal transporter (DMT-1) (Figure 1).
Sharma et al. [35] have evaluated iron regulatory proteins in celiac patients compared to controls
and iron deficient patients using duodenal biopsies. The results showed that DMT-1, ferroportin,
hephaestin, and transferrin receptor protein mRNA increased, primarily due to the fact of iron
deficiency, while body iron stores were reduced in CD. In contrast, these authors [35] showed that
expression of DMT1 and ferroportin are increased in CD patients with or without ID. In this study,
ferritin expression was also found to be increased in CD, but only in those with ID.
Figure 1. Iron absorption metabolism. Non-heme iron is ultimately taken up from the lumen by
divalent metal transporter (DMT-1) on the microvillus membrane, before joining the labile iron pool in
the cell. Ferric iron has to be reduced to the ferrous form by duodenal cytochrome b (Dcytb) before the
uptake. Ferrous iron in the labile iron pool is then transferred to the circulation by ferroportin (FPN),
which requires hephaestin for oxidation to the ferric form to bind transferrin. Heme iron is taken up
by a specific receptor. Internalized heme iron is degraded by heme-oxygenase, releasing non-heme
iron. The non-heme iron is then transported to the cytoplasm, joining the labile iron pool and is then
transferred to the bloodstream by FPN in the same manner as non-heme iron.
Tolone et al. [36] reported the link between DMT-1 IVS4+44C-AA and anemia in 387 Italian
celiac children and the functional role of the polymorphism. They found that the DMT-1 IVS4+44-AA
genotype confers a four-fold risk of developing anemia, despite the atrophy degree. Anemia in patients
with CD is multifactorial.
Patients with CD may benefit from iron supplementation (iron sulfate), but intolerance to iron
sulfate could reduce the efficacy of this supplementation. Sucrosomial iron, a presentation of ferric
pyrophosphate covered by a phospholipid and sucrester membrane, can be effective in providing
iron supplementation in difficult-to-treat patients with CD and intolerance to iron sulfate, allowing
good intestinal absorption independently of the DMT-1 carrier [37]. A study provides evidence that
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FeralgineTM , a solution of ferrous bisglycinate chelate and sodium alginate, is well absorbed in celiac
patients [38]. Furthermore, it might by suggested that the iron complex might be absorbed regardless
of the presence of DMT-1.
The prevalence of CD in subjects presenting IDA has been described by other authors [39–41] with
different results, due to the probable differences in the study of the designs. Lasa et al. [40] designed a
study to avoid the abovementioned bias. They decided to evaluate all patients diagnosed with IDA by
performing upper endoscopy and duodenal biopsies, and not only those with positive antibodies or
with IDA of unknown origin (after an extensive work-up). Patients with IDA have an increased risk for
CD, up to 25% of these patients may not present any endoscopic sign suggesting villous atrophy [39].
This finding makes routine duodenal biopsy necessary when performing upper endoscopy on IDA
patients. In a systematic review and meta-analysis, Mahadev et al. [3] found that approximately 1 out
of 31 patients with IDA have histologic evidence of CD; this prevalence value justifies the screening of
patients with IDA for CD (Figure 2).
Figure 2. Abbreviated flow chart of the investigation of iron deficiency anemia in celiac disease patients.
2.2. Folate and Vitamin B12 Deficiency

Usually, people suffering from CD can develop folate and vitamin B12 deficiencies as a result of
generalized malabsorption linked to villi atrophy. Both vitamins are essential for normal hematopoiesis
and neurologic function.
Folate absorption occurs primarily in the jejunum, which is commonly affected by CD [10,42].
Several studies in adult celiac patients have shown an increased risk of folate deficiency, which can
reach up to 20–30% of newly diagnosed patients [43,44]. Prior to uptake, folate must be deconjugated
by a brush border membrane peptidase and the intestinal mucosa damage in CD may affect enzyme
activity leading to a folate deficiency. Serum and red cell folate measurements are usually used for the
diagnosis of folate deficiency. Serum folate levels reflect largely folate intake and it is common for
levels to be high in patients with a vitamin B12 deficiency. Red cell folate is not a specific indicator
for folate deficiency, as it can be decreased in patients with vitamin B12 , but red cell folate levels are
less influenced by variations in folate intake. Patients with CD commonly have elevated levels of
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homocysteine which may serve as an important clue for the diagnosis. However, the sensitivity of this
measurement is somewhat less for vitamin B12 deficiency [45].
Vitamin B12 requires formation of a primary complex with intrinsic factor to be absorbed in the
proximal small intestine, and small amounts may also be absorbed by passive transport throughout
the entire intestine. Deficiency of vitamin B12 is common in CD and frequently results in anemia.
Though the terminal ileum is the primary site of absorption of vitamin B12 , García-Manzanares and
Lucendo [44] reported a prevalence of vitamin B12 deficiency between 8% and 41% in patients with
newly diagnosed CD.
The causes of B12 deficiency in CD are still not clear, but they may be related to complications
of small intestinal injury including a decreased gastric acidity, cobalamin intake due to the frequent
finding of bacterial overgrowth, autoimmune gastritis, and decreased efficiency of the intrinsic factor
or even dysfunction of the distal small intestine. Abnormalities in the absorption of folate or vitamin
B12 may result in anemia in children with untreated CD. The range of low folate and low vitamin B12
prevalence were 15.7–18.3% and 4.3–8%, respectively [42,46].
Both folate and vitamin B12 deficiencies can lead to a macrocytic anemia with low values for
hemoglobin or hematocrit, and high mean corpuscular volume levels. Vitamin B12 deficiency should
be considered in patients with CD and hematological and neurological disorders [47]. Vitamin B12
levels measured within the lower range of normal or if they coexist with folic acid deficiency can be
misleading and difficult to interpret. Under these circumstances, high serum levels of methylmalonic
acid may improve the diagnostic accuracy of vitamin B12 deficiency [48].
2.3. Copper and Zinc Deficiency

Micronutrient deficiencies are common in celiac patients. In addition to the abovementioned
deficiencies (i.e., iron, folic acid, and vitamin B12 ), at the time of diagnosis there may be deficiencies for
other vitamins and minerals, in particular copper and zinc [22].
Copper deficiency is a rare complication in CD and its prevalence remains unknown.
This deficiency can lead to anemia, thrombocytopenia, neutropenia, and peripheral neuronal
involvement. In adult celiac patients, peripheral myeloneuropathy has been described along with
hypocupremia with a good clinical response to copper supplementation [49,50]. Halfdanarion et al. [51]
reported five cases of adult celiac patients with copper deficiency; all of them presented neurological
complications and three of them presented hematological abnormalities. Cavallieri et al. [52] recently
described a rare case of myelopathy induced by copper deficiency secondary to undiagnosed CD, and
they have suggested that patients with hypocupremia should be tested for CD.
Likewise, the presence of clinical alterations as a consequence of zinc deficiency is also uncommon
in celiac patients. Fractional zinc absorption is no different between celiac patients and controls, but the
rapid zinc exchange body compartment is lower in CD than in control patients [49]. The mechanism of
zinc depletion and its possible implications are unknown [53].
3. Aplastic Anemia and Celiac Disease

Celiac disease has been linked to various hematological abnormalities [54], such as anemia,
thrombopenia or thrombocytosis, leukopenia, splenic dysfunction, immunoglobulin A deficiency or
lymphoma. Anemia is the most frequent cause of CD. In addition to the various etiologies of anemia in
CD (iron deficiency, due to the micronutrient deficiency or chronic disorders), various cases of aplastic
anemia associated with CD have been described in the literature [55–60], both in pediatric age and
in adulthood. Despite the underlying mechanism of this association being still unknown [55], it has
been suggested that both conditions might share a similar underlying pathophysiological mechanism,
mediated by autoreactive T cells involved in tissue destruction [56] (Table 2). In all cases, the patient
presented with pancytopenia and the diagnosis was achieved by bone marrow biopsy. Pancytopenia
was resolved with the GFD only in some cases [57], while in other cases, the response was only partial
and immunosuppressive treatment was required or even hematopoietic progenitor transplantation.
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Although infrequent, aplastic anemia may be an underdiagnosed entity [56], so it will be necessary
to have the diagnostic suspicion both in the case of pancytopenia without apparent cause and in CD
with pancytopenia. The etiology of anemia may be due to the presence of several factors, such as
autoimmunity or chronic inflammation caused by the CD [60]. Based on the cases reported to date,
it seems that the GFD is not enough to improve pancytopenia; therefore, most patients require other
treatments. Some authors have suggested that the prognosis is better at pediatric age, possibly because
the duration of exposure to chronic inflammation is shorter, and the GFD may probably reverse the
process [57].
Table 2. Characteristics of patients with aplastic anemia and celiac disease.

Study Reported Grey-Davies [51] Salmeron [53] Maheswari [52] Basu [54] Badyal [55] Omar [50]
Number of cases 3 5 1 1 1 1
Bone Bone
Anemia diagnosis Bone marrow biopsy Bone marrow biopsy Bone marrow biopsy marrow Bone marrow biopsy marrow
biopsy biopsy
Age (years) 23, 37, 43 Not reported 13 40 9 6
Villus Villus
Intestinal biopsy Villus atrophy Villus atrophy Villus atrophy Not available
atrophy atrophy
GFD, antithymocyte
GFD, corticotherapy, GFD, GFD, corticotherapy,
globulin, cyclosporine.
Treatment antithymocyte GFD corticotherapy, antithymocyte GFD
hematopoietic cell
globulin, cyclosporine cyclosporine globulin
transplantation
GFD; Gluten Free Diet.
4. Anemia of Chronic Disease

Anemia of chronic disease (ACD) is an old concept in the scientific literature, but current
research on the role of pro-inflammatory cytokines and iron metabolism has yielded more information
about the pathophysiology of this disease. This type of anemia is linked to the deterioration of
the production of erythrocytes associated with chronic inflammatory conditions including cancer,
infections or autoimmune diseases. In addition, recent epidemiological studies have linked ACD
with obesity, aging, and kidney failure. This type of anemia responds to a multifactorial pathogenesis
including four fundamental mechanisms. These mechanisms consist of abnormalities in iron utilization,
decrease in half-life of red blood cells, direct inhibition of hematopoiesis, and relative deficiency of
erythropoietin [61].
Hospitalized patients feature acute or chronic inflammation caused by immune activation and
occurring associated with anemia, ACD being the most common form found in these patients [62].
Under these conditions, erythropoiesis can be directly inhibited by an increase in the production
of inflammatory cytokines inducing changes in iron homeostasis which could be characterized by
reductions in both iron absorption and macrophage iron release [63]. Iron is a fundamental component
of all living cells because iron is a cofactor for mitochondrial respiratory chain enzymes, the citric acid
cycle, DNA synthesis, as well as an essential component for the transport of O2 through the hemoglobin
and myoglobin. In addition, a sufficient amount of iron is important for immune preservation due to
the fact of its role in promoting the growth of immune system cells, as the immune function and iron
metabolism are widely linked.
Anemia of chronic disease is not considered a frequent cause of anemia in celiac patients; in fact,
systemic inflammation, based on the increase in serum levels of acute phase proteins, is rare in CD
patients, although gliadin-dependent activation of mononuclear cells of the mucous lamina propria
causes an overproduction of proinflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6
(IL-6) [64,65]; both cytokines are mediators of ACD [66,67]. These proinflammatory cytokines are key
factors in iron metabolism and in the development of ACD in celiac patients.
Thus, IL-6 inhibits the expression of the transferrin receptor mRNA, stimulates the synthesis
of DMT-1, and is a mediator of hypoferremia in inflammation, which induces the synthesis of the
hepcidin hormone regulating the iron export. An increase in hepcidin synthesis causes an increase
in the degradation of ferroportin and the inhibition of iron release by the enterocyte, which leads
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to the alteration in the iron homeostasis associated with ACD [10,68]. IFN-γ stimulates ferritin
transcription but at the same time inhibits its translation. IFN-γ also inhibits the transferrin receptor
mRNA expression, which blocks the incorporation of iron mediated by the transferrin receptor, but
increases the expression of DMT-1, thereby increasing the uptake and storage of ferrous iron. IFN-γ
also decreases the mRNA of the transmembrane protein ferroportin, which exports iron to the outside
of the cells. Therefore, IFN-γ favors iron retention within monocytes [52].
In this sense, some cytokines such as TNF-alpha, IL-1, and IL-10 are also released into circulation
due to the inflammatory process [69]. These cytokines act on the liver and they contribute to the increase
of hepcidin production, inhibiting the duodenal absorption of dietary iron. DMT-1 expression can also
be induced by these cytokines. The net effect is the uptake of circulating iron in the reticuloendothelial
system. In addition, IL-15 also seems to contribute to this pathway [70]. IL-15 is involved in the
pathophysiology of CD and is partly responsible for sustained inflammation in active disease [71].
Taking into account all the above mentioned inflammatory pathways, CD could contribute to the
development of de novo ACD. GFD is capable of reducing the oxidative state of patients with CD,
although chronic inflammation persists even after two years of GFD. These patients showed a persistent
high level of IFN-γ, IL-1α, interferon-inducible protein 10 (IP-10), and tumor necrosis factor beta
(TNF-β) [23].
Bergamaschi et al. [72] studied anemia in patients with CD, reporting that ACD affected 17% of
the subjects (11 out of 65 patients). Iron status parameters are similar in patients with ACD and those
usually found during inflammatory processes, and an isolated iron deficiency or other pathogenic
mechanisms could not be the explanation for their anemia. Their results reported a defective production
of endogenous erythropoietin, in addition to changes in iron homeostasis, as a pathogenic mechanism
of ACD. Other study conducted by Harper et al. [23] also confirmed that ACD can affect patients with
CD and this fact is not completely unexpected, although these patients generally lack signs of systemic
inflammation. Even though, the mean serum levels of inflammatory cytokines contributing to ACD
(including IL-1β, IL-6, TNF-α, and IFN-γ) increased during active CD [73–76]. Although with a lower
prevalence (3.9%), Berry et al. [77] also reported the presence of ACD in patients with CD.
In light of the observed studies and although ACD is not the most prevalent hematological
disorder in patients with CD, it is necessary to take into account the pathogenesis of CD influence on
its pathogenesis, given the role of iron in inflammatory signaling and in the turnover of epithelial cells.
5. Refractory Anemia to the Gluten-Free Diet

The etiology of persistent refractory anemia is multiple, and it must first be ruled out that it is
due to the poor adherence to a GFD. Other causes of refractory anemia are chronic inflammation or
anemia of chronic disorders, refractory celiac disease (RCD), the higher prevalence of the disease than
expected by the involvement of other intestinal sections or the appearance of other comorbidities [77].
The first suggested finding is that it is a false refractoriness or persistence of anemia because
the adherence to the treatment is not being conducted correctly. GFD is not easy to comply with
nor is it generally well performed [78]. The traditional methods used to monitor the disease have
poor performance, because, for example, with the serological method, for every six examinations we
would detect the transgression in only one of them [79], besides presenting little correlation with villus
atrophy [80]. The immunogenic gluten peptide in feces is postulated as a better tool for assessing diet
adherence [81].
Celiac disease responds in the majority of patients on a GFD in a few weeks [13]. However, despite
the correct adherence to a GFD, villous atrophy, malabsorption, and chronic intestinal inflammation
persist in some patients for 12 months, which defines the RCD [82–84]. This can lead to persistence of
symptoms and signs, including anemia. RCD is considered a rarity in pediatric age and, although its
exact prevalence and incidence in adulthood is unknown, it is an uncommon condition [85]. Due to
the poor response of the disease to treatment at this stage and its prognosis, it is important to correctly
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make the diagnosis [86], which is considered exclusion. The complete histological evaluation of the
entire small intestine is needed for the diagnosis of refractoriness or complications [87].
In a recent study [88], mucosal involvement in patients with RCD was compared to patients with
uncomplicated CD, showing that the involvement was greater in patients’ refractory to treatment,
which may indicate that one of the causes of the persistence of symptoms is precisely the greatest extent
of the disease. In a study [89] conducted in adult patients with CD and persistent IDA, 23% of patients
showed lesions that were detected by video capsule endoscopy (VCE) of the small intestine. In another
recent study conducted in pediatric CD patients [90], patients with anemia at diagnosis showed
significantly larger histological lesions than CD patients without anemia; 92% of the patients recovered
from the anemia after one year of adherence to a GFD. In patients with suspected RCD, especially type
II, the performance of VCE is recommended [91]. Video capsule endoscopy is a relatively safe method
with high sensitivity (approximately 89%) and specificity (approximately 95%) [92] to detect villus
atrophy, and this could help differentiate RCD type I and II [88].
Furthermore, it is important to distinguish patients with uncomplicated CD from those with RCD,
due to the risk of developing complications such as enteropathy associated with T-cell lymphoma
(EATL), adenocarcinoma, jejunoileitis or B-cell lymphoma [93,94]. If left untreated, CD presents an
increased risk of developing long-term tumors, especially of EATL and small bowel adenocarcinoma [95]
compared to the general population. Enteropathy associated with T-cell lymphoma is sometimes
diagnosed due to the signs and symptoms such as perforation, intestinal occlusion or bleeding, and
persistent anemia, which may be an indicator of it. Likewise, ulcerative jejunoileitis is one of the
phenotypic expressions of RCD. The characteristic symptom of this complication is abdominal pain,
in relation to sub-occlusive symptoms, although the disease may present with hemorrhagic symptoms,
perforation or protein-losing enteropathy due to the presence of inflammatory ulcers and strictures in
the entire small intestine. In addition, it is associated with an increased risk of EATL [96].
The presence of other comorbidities, not always associated with CD itself, are linked to persistent
symptoms once adherence to the GFD has been verified, such as microscopic colitis, irritable bowel
syndrome, food allergies, motility disorders or collagen sprinkles [85]. The sprue collagen manifests
itself in the form of refractoriness, and its occasional association with EATL has also been described [97].
The diagnosis is performed by biopsy and pathological analysis.
6. Conclusions and Future Perspectives

Celiac disease is a multisystemic disorder with different forms of clinical expression, from
malabsorption with diarrhea, anemia, and growth retardation in children, to extraintestinal
manifestations, such as those due to the fact of malabsorption and micronutrient deficiencies,
including iron, folic acid, and vitamin B12 . In fact, anemia may be the only clinical expression
of the disease, and IDA is considered one of the most frequent extraintestinal clinical manifestations of
CD. Celiac disease should be suspected in the presence of anemia without known etiology. Therefore,
the determination of tissue anti-transglutaminase antibodies and anti-endomysial antibodies are
indicated in these cases and, if positive, the performance of digestive endoscopy and intestinal biopsy
is recommended.
Anemia in CD has a multifactorial pathogenesis and, although it is more frequently a consequence
of iron deficiency, anemia can also be caused by deficiencies of folate or vitamin B12 , as well as by
blood loss or by its association with inflammatory bowel disease (IBD) or other associated diseases.
The association between CD and IBD should be considered because the similarity of the symptoms
could delay the diagnosis; the possibility of association among both pathologies should always be
taken into account during the treatment of anemia in patients with IBD.
Vitamin B12 deficiency is common in CD and may be responsible for anemia and peripheral
myeloneuropathy. Folate deficiency is a well-known cause of anemia in adults, but there is little
information in children with CD. To date, it is still unknown if anemia is a symptom of the most typical
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CD in adult patients either by predisposition due to the age or because the biochemical and clinical
manifestations take longer to appear.
Iron is a critical micronutrient whose deficiency in CD, in most cases, is a consequence of
malabsorption secondary to the damage of the villi of the intestinal mucosa. However, iron deficiency
in CD may also be a consequence of the reduced expression of different regulatory proteins. Alterations
of iron absorption that could explain the inappropriate response to a GFD. It is known that the iron
transporter DMT1 is positively regulated in CD to counteract iron malabsorption by villus atrophy, and
that the risk of anemia in CD is related to the DMT-1 IVS + 44 AA genotype. A variant of this genotype
can limit the overexpression of the transporter occurring normally prior to iron deficiency, being
ineffective to counteract iron deficiency in the severe stage of the disease. Furthermore, the evaluation
of the TMPRSS6 genotype, which influences iron metabolism through its effects on hepcidin, could be
of clinical importance for the therapeutic management of iron supplementation, because a mutation
can induce a poor response to iron therapy and predict the persistence of IDA despite iron treatment
and GFD.
Author Contributions: Conceptualization, R.M.-M. and M.T.N.; Methodology, R.M.-M.; Resources, R.M.-M.,
M.T.N., J.D.-C., I.L.-A., M.J.M.A., J.M.-F. and J.M.; Writing-Original Draft Preparation, R.M.-M.; Resources,
R.M.-M., M.T.N., J.D.-C., I.L.-A., M.J.M.A., J.M.-F. and J.M.; Writing-Review & Editing, R.M.-M. and M.T.N.;
Supervision, J.M.
Acknowledgments: Jorge Moreno-Fernandez is supported by a fellowship from the Ministry of Education,
Culture and Sport (Spain) and is grateful to the Excellence Ph.D. Program “Nutrición y Ciencias de los Alimentos”
from the University of Granada. The authors also thank Susan Stevenson for her efficient support in the revision
of the English language. We acknowledge Nutraceutical Translations for English-language editing of this review.
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147
nutrients
Review
Anemia of Inflammation with An Emphasis on
Chronic Kidney Disease
Sajidah Begum 1 and Gladys O. Latunde-Dada 2, *
1 Faculty of Life Sciences and Medicine, Henriette Raphael House Guy’s Campus King’s College London,
London SE1 1UL, UK; sajidah.begum@kcl.ac.uk
2 Department of Nutritional Sciences, School of Life Course Sciences, King’s College London.
Franklin-Wilkins-Building, 150 Stamford Street, London SE1 9NH, UK
* Correspondence: yemisi.latunde-dada@kcl.ac.uk; Tel.: +44-20-7848-4256
Received: 28 August 2019; Accepted: 30 September 2019; Published: 11 October 2019
Abstract: Iron is vital for a vast variety of cellular processes and its homeostasis is strictly controlled and
regulated. Nevertheless, disorders of iron metabolism are diverse and can be caused by insufficiency,
overload or iron mal-distribution in tissues. Iron deficiency (ID) progresses to iron-deficiency anemia
(IDA) after iron stores are depleted. Inflammation is of diverse etiology in anemia of chronic disease
(ACD). It results in serum hypoferremia and tissue hyperferritinemia, which are caused by elevated
serum hepcidin levels, and this underlies the onset of functional iron-deficiency anemia. Inflammation
is also inhibitory to erythropoietin function and may directly increase hepcidin level, which influences
iron metabolism. Consequently, immune responses orchestrate iron metabolism, aggravate iron
sequestration and, ultimately, impair the processes of erythropoiesis. Hence, functional iron-deficiency
anemia is a risk factor for several ailments, disorders and diseases. Therefore, therapeutic strategies
depend on the symptoms, severity, comorbidities and the associated risk factors of anemia. Oral
iron supplements can be employed to treat ID and mild anemia particularly, when gastrointestinal
intolerance is minimal. Intravenous (IV) iron is the option in moderate and severe anemic conditions,
for patients with compromised intestinal integrity, or when oral iron is refractory. Erythropoietin
(EPO) is used to treat functional iron deficiency, and blood transfusion is restricted to refractory
patients or in life-threatening emergency situations. Despite these interventions, many patients
remain anemic and do not respond to conventional treatment approaches. However, various novel
therapies are being developed to treat persistent anemia in patients.
Keywords: iron; anemia; kidney; hepcidin; erythropoietin
1. Introduction
Iron is an essential micronutrient required for a number of cellular processes. It is involved in the
structure and function of hemoglobin and myoglobin, as well as in the formation of heme enzymes and
other iron-containing enzymes of the electron transport chain. Iron is necessary for many biological
functions, however, when in excess, toxicity results due to the production of reactive oxygen species
and this leads to the malfunctioning of organs [1]. Iron deficiency (ID) describes a condition in which
the iron stores in the body are reduced but not sufficiently to limit erythropoiesis. If iron deficiency is
severe enough to reduce erythropoiesis, iron-deficiency anemia (IDA) results [2]. In 2016, a systematic
analysis for the Global Burden of Disease Study stated that IDA is one of the five leading causes of years
lived with disability, particularly in women, and thereby highlighted the prevention and treatment
of IDA as a major public health goal [3]. IDA is estimated to affect 1.24 billion people in the world,
comprising mostly children and reproductive women, and particularly, in less-developed economies [4].
Iron deficiency (ID) in the absence of anemia has been suggested to be twice the incidence of IDA [5].
Substantive evidence has revealed that both ID and IDA have deleterious consequences on cognition,

Nutrients 2019, 11, 2424
mental function, work performance, and pregnancy outcomes [6,7]. Furthermore, functional iron
deficiency occurs when iron is sequestered in storage organs during inflammation and infections or
in situations such as increased erythropoiesis either naturally, due to increased Erythropoietin (EPO)
release in response to anemia, or, pharmacologically by erythropoietin-stimulating agents (ESA’s) [8,9].
Anemia describes a state in which there is a reduced erythrocyte count or a reduced level of
hemoglobin within erythrocytes [10]. Anemia can be classified in several ways; which can be based
on etiological factors, such as nutritional, aplastic, hemorrhagic or hemolytic. However, in clinical
practice, classification could be based on the morphology of erythrocytes such as the mean corpuscular
volume (MCV). Based on the MCV, anemia can be described as microcytic (MCV< 82 fL), normocytic
(MCV = 82–98 fL) or macrocytic (MCV >98 fL). The limitation of this classification is that red cell
morphology during hematopoiesis is often not influenced during the early stages of iron deficiency and
a class of anemia type could transverse 2 classification groups. Broadly, however, typical examples of
microcytic anemia are iron deficiency, thalassemic and sideroblastic anemia. Normocytic anemia includes
hemolytic and anemia of chronic disease and folic and vitamin-B12-deficiency anemia are macrocytic.
2. Causes of Iron-Deficiency Anemia

Several factors contribute to the development of iron-deficiency anemia and these are presented
in a recent review [5]. Physiologically, an increased demand for iron which cannot be met from dietary
sources will lead to iron deficiency. This occurs during rapid growth of infants and adolescents,
menstrual blood loss, post blood donation and during the first and second trimesters of pregnancy.
Nutritionally, inadequate iron intake, malnutrition or poor dietary absorption can lead to iron-deficiency
anemia. Pathological causes include decreased absorption and chronic blood loss. Causes of decreased
absorption include gastrectomy, bariatric surgery, duodenal bypass, inflammatory bowel disease
and atrophic gastritis. Causes of chronic blood loss include bleeding of the gastrointestinal tract
(oesophagitis, peptic ulcer, diverticulitis, benign and malignant tumour, hookworm infestation
and hemorrhoids), genitourinary system (heavy menses, menorrhagia, intravascular haemolysis
(paroxysmal nocturnal haemoglobinuria) and systemic bleeding (trauma, hemorragic telangiectasia
and chronic schistosomiasis). Certain classes of drugs have also been implicated in the development of
iron-deficiency anemia and these include glucocorticoids, salicylates, non-steroidal anti-inflammatory
drugs and proton pump inhibitors. Iron-refractory iron-deficiency anemia is an inherited cause of
iron-deficiency anemia. Finally, the availability of iron can be restricted, leading to functional iron
deficiency that is associated with anemia of chronic inflammatory conditions [2,5,11,12]. This is a
literature review on a few other types of anemia that are associated or concomitant with chronic disease
inflammatory conditions. It evaluates the variations in phenotypes, management and discusses the
differences in the therapeutic approaches employed.
3. Anemia of Inflammation or Anemia of Chronic Disease

Inflammation is an immune response to injury and infection. The inflammatory process causes
hypoferremia as an acute-phase response to fight against infection. It involves the secretion of cytokines
to regulate iron redistribution, creating hypoferremia that delays pathogen growth, thereby causing the
invaders to be engulfed by phagocytes. The orchestrated defense system mounted by the host to fight
and fence off pathogens culminates sequentially in tissue iron sequestration, serum iron deficiency,
and anemia. This epitomizes the essentiality of life preservation and survival in a competitive hostile
environment as normal tissue functions (oxygen delivery) are partially or transiently sacrificed to
combat infection [13]. Concomitant with the survival response is the marshalling of the armoury
of the erythropoietic drive to override and inhibit both inflammatory and iron-sensing pathways in
order to attenuate the downregulation of iron absorption by hepcidin [14–16]. Anemia of Chronic
Disease (ACD) thus has a multifactorial etiology and has been estimated to afflict over a billion
individuals globally [4]. It is prevalent in chronic diseases and disorders, such as heart disease, cancer,
inflammatory bowel disease and chronic kidney disease, in which inflammation causes anemia due to
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Nutrients 2019, 11, 2424
increased levels of hepcidin in circulation [4]. The manifestation of the proinflammatory process in
a spectrum results in variation in hepcidin levels and the magnitudes of anemia phenotype. ACD,
therefore, is caused by a complex interplay of proinflammatory cytokines which induce dysregulation
in iron homeostasis, erythroid progenitor cell differentiation, erythropoietin synthesis and red cell
longevity, all culminating in the pathogenesis of anemia [17].
Systemic inflammation induced by infection, trauma, dialysis, malignancy or autoimmune
disorders activate immune cells to produce cytokines such as Interleukins, (IL) ILI, IL6, IL10, interferon
ɣ (IFNɣ ) and tumor necrosis factorα (TNFα). Erythropoiesis is impaired by iron restriction, suppression
of EPO production and shortened life-span of erythroid progenitors, all culminating in iron-deficiency
anemia [17] (Table 1). Thus, ACD is an underlying secondary disorder that is deleterious to the survival
of erythrocytes and erythropoiesis.
Table 1. Features of anemia of inflammation [17].
Cells/Tissue Cytokine Effector Function

Induction of hepcidin expression in the liver. Hepcidin
IL6 and
Hepatocytes inhibits iron efflux from the macrophages and the
lipopolysaccharide (LPS)
duodenum by blocking or degrading ferroportin.
Macrophage
Lipopolysaccharide (LPS)
Increase DMT1 expression and uptake of ferrous iron(Fe2+ ).
and TNFα
Promotes damage of erythrocyte membranes and the
TNFα
stimulation of phagocytosis.
Decrease expression of ferroportin to inhibit iron efflux and
IFNɣ and LPS
accentuated by hepcidin.
Induction of ferritin expression, storage and retention of iron
TNFα-IL1, IL6 and IL10
within macrophages.
Enhances TfR1 expression to promote uptake of
Monocytes IL10
transferrin-bound iron.
Dysregulated erythropoietin receptor EPOR expression and
signalling via blunted expression of Scribble (Scb) and
Kidney TNFα, IFNɣ and IL1 inhibition of erythropoietin (EPO) and erythroferrone (ERFE)
which production. The cytokines also directly inhibit the
differentiation and proliferation of erythroid progenitor cells.
TNFα, tumor necrosis factorα; TfR1: Erythropoietin receptor 1; IFN ɣ , interferon ɣ; DMT1, divalent metal
transporter 1.
4. Anemia of Cancer
Anemia is prevalent in various types of cancer and iron deficiency accounts for a significant
proportion of this comorbidity [18]. The etiology of different tumor types could be multifactorial and
complicated by varying underlying factors, but an overriding cause of anemia is chemotherapy-induced.
Persistent blood loss, coupled with nutritional deficiencies, culminate in dysregulated iron
homeostasis [19]. Features of ID in cancer range from a spectrum of low to high or elevated serum
ferritin, a blend of mild absolute ID and a functional ID (FID) gradient. However, as it is a chronic
disease that is akin to an inflammatory condition, most cancer patients suffer from FID [19]. It was
reported that the prevalence of anemia is about 50% of non-myeloid tumor patients undergoing systemic
therapy in a cohort of Spanish hospitals [20]. Moreover, a Europe-wide study that evaluated routine
practice in chemotherapy-induced anemia (CIA) management showed that 74% of patients exhibited
Hb ≤10 g/dL, including 15% with severe anemia (Hb <8 g/dL). Furthermore 42% of the cancer patients
had low-iron levels (ferritin ≤100 ng/mL) [21]. Anemia thus contributes significantly to disease burden
and reduced quality of life in cancer patients undergoing therapy. It is, therefore, imperative to treat
anemia in the different malignant forms of cancers. Conventional therapeutic approaches include red
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blood transfusion, the administration of erythropoietin-stimulating agents (ESA), intravenous iron

supplementation (IV) and a combination of ESA and IV [22]. While blood transfusion predisposes
patients to thromboembolism and increased mortality [22], ESA administration could be refractory in
cancer patients. The adverse consequences of IV on oxidative stress and tumorigenesis in heterogeneous
cancers are not yet clarified in clinical trials [23]. Recent guidelines and recommendations on the
treatment of cancer-related anemia advocate reduction or avoidance of red blood cells (RBC) transfusions,
intravenous (IV) alone or a combinatorial use of IV to enhance low-dosage ESA administration [23].
Novel approaches to the treatment of functional anemia that typify chronic diseases, including cancer,
are discussed herein later under anemia of chronic kidney disease (ACKD).
5. Anemia of Heart Failure

Anemia is prevalent in patients with heart failure (HF), correlates with severity of the disease
and is responsible for increased morbidity and mortality in patients [24]. It is characterized by
decreased exercise capacity (reduced exercise capacity of 5,6 and worse) by the New York Heart
Association (NYHA) functional classification. There are different causes of anemia in HF, arising from
the heterogeneous manifestations of the disorder [25]. Anemia caused by absolute iron deficiency
in HF may be due to nutritional factors, such as low dietary iron, poor appetite loss, decreased
iron absorption due to gastrointestinal blood loss caused by gut inflammation and consequences of
iatrogenic agents [26]. However, as it is a chronic disease that is akin to an inflammatory condition, most
HF patients suffer from FID [26]. Hemodilution and renal insufficiency are also linked with anemia of
HF. Incidence of iron deficiency in chronic HF patients in Europe is about 50%, compared with 61% in
Asian populations [27]. A study that analysed about 2000 patients in cohorts in Poland, Spain and the
Netherlands reported a prevalence of 32% cases of ID, 12% of IDA and a combination of both as 20% [28].
Anemia thus results significantly in disease burden, increased hospitalizations, and reduced quality of
life in HF patients, as well as decreased functional capacity. Conventional therapy for numerous clinical
trials is intravenous (IV) iron administration, particularly in patients with symptomatic systolic HF.
Intravenous iron infusion has been shown to significantly reduce hospitalizations, improve quality of
life, increase exercise capacity and decrease mortality in HF patients. Therapy of symptomatic diastolic
HF with IV iron has yet to be clarified or confirmed. Recent guidelines and recommendations on the
treatment of HF-related anemia advocate IV iron for symptomatic patients (serum ferritin <100 μg/L,
or ferritin between 100–299 μg/L and transferrin saturation (TSAT) <20%) to improve exercise capacity
and quality of life [29]. Serum ferritin and TSAT mean values could be variable in HD patients due to
confounding factors that are influenced by the magnitude of the symptoms [30]. Such confounders,
complexities and the multifactorial nature of iron metabolism dysregulation [31] possibly account for
the unreliability of serum hepcidin as a clinical marker of iron status in HD patients [32]. The choice of
IV administration of iron compounds varies in different countries.
6. Anemia of Surgery
Anemia is prevalent in patients undergoing major surgery and poses an additional independently
modifiable risk to patients undergoing blood transfusion [33]. Anemia can range from 7–35% in
orthopaedic surgery patients and is associated with high morbidity and mortality [34]. Iron deficiency
due to increased requirements, reduced absorption, increased lysis and losses of red blood cells are
the main causes of preoperative anemia. Moreover, anemia prior to surgery coupled with increased
lysis and losses of red blood cells, iron-sequestration and restricted erythropoiesis lead to over 80%
prevalence in postoperative anemia [35]. Perioperative anemia manifests mostly as absolute and
functional iron-deficiency anemia that are respectively characterized by scarcity and sequestration of
iron in tissues [36]. A chronic inflammatory condition accentuates iron sequestration and FID in the
patients. Of note also is that hematinic deficiencies lead insidiously to latent iron deficiency without
anemia. A large study of hospital patients of diverse surgical procedures (cardiac, gynecological,
colorectal/liver cancer resection) reported the overall prevalence of anemia as 36%. In the anemic
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patients, 62% had absolute iron deficiency, while FID was 10% [37]. Women accounted for more than
twice in number in the cohort. Perioperative and postoperative anemia thus contribute significantly to
disease burden and reduced quality of life, the magnitude of which varies with the different disorders.
Conventional therapeutic approaches advocate the diagnosis and treatment of anemia before any
surgical procedure. Iron deficiency without anemia needs to be treated to replenish iron stores for
preoperative requirements and postoperative anemia challenge [38]. Preoperative oral iron could
be prescribed for mild-to-moderate anemia patients that are tolerant and do not suffer from adverse
gastrointestinal consequences. IV iron supplementation, preferably as a large single dose, is the therapy
guideline in moderate to severe postoperative anemic patients. However, a combination of IV and ESA
is recommended only in severe anemia that is refractive and resistant over a long period of time.
Red blood cell transfusion (RBCT) may be an acute, inevitable option to correct severe anemia
in critically physiologically drained patients after surgery. Guidelines and recommendations for
RBCT are restrictive because of the attendant risk of infection, thromboembolic events, high
morbidity and mortality in the patients [39,40]. Patient blood management (PBM) that involves
evaluation of the hematological status of patients will not only prevent preoperative anemia, but also
reduce intraoperative transfusion risk and postoperative complications [41]. Recent guidelines and
recommendations advocate a preoperative Hb <13 g/dL to be considered as suboptimal in both men
and women and treated before any major surgical procedure [38].
7. Anemia of Inflammatory Bowel Disease (IBD)

Anemia is a common comorbidity of inflammatory bowel disease (IBD). The aetiology of IBD
is multifactorial and the pathogenesis is complicated by varying underlying factors, such as genetic
predisposition, immune dysregulation, loss of mucosal integrity and intestinal microbial composition.
This results in a spectrum of chronic relapsing inflammatory disorders that are characterized by
ulceration and bleeding of the mucosal epithelium. An inflammatory condition in IBD elevates
hepcidin levels in circulation, hence functional iron deficiency ensues; however, chronic intestinal
bleeding results also in absolute iron deficiency. Consequently, negative regulators (erythroferrone,
growth differentiation factor 15 (GDF-15), platelet-derived growth factor-BB (PDGF-BB) and/or
hypoxia-inducible factors (HIFs)) of hepcidin expression dominate to enhance iron absorption from
the gastrointestinal tract [42]. Anemia in IBD could arise from several factors that include intestinal
blood loss, medications and reduced iron absorption that is also a consequence of reduced appetite
during active flaring episodes of the disorder. Moreover, vitamin deficiencies such as those of folate
and vitamin B12 are common due to decreased absorption from abnormal duodenum. Absolute
iron deficiency caused by diminished absorption and depleted iron stores leads to anemia. Other
extenuating consequences of pathological conditions promote acute-phase reactants and cytokines
that impair erythroid differentiation and proliferation [43]. A retrospective cohort study, of patients
diagnosed with Crohn’s disease and ulcerative colitis during the period 1963–2010 was randomly
selected from the population-based IBD cohort of Örebro University Hospital in Sweden and revealed
a mean annual incidence rate of anemia as 15.9 per 100 person-years and a prevalence of 22.6%. Of
this, anemia was 19.3 per 100 person-years and the prevalence was 28.7% in Crohn’s compared with
12.9% and 16.5%, respectively, for ulcerative colitis [44]. In earlier reports, however, anemia incidence
correlated with the disease activity rather than type, although anemia was higher in women [45].
Apart from the consequences of anemia, such as fatigue, headache, dizziness, shortness of breath, or
tachycardia, anemia exerts a significant impact on the quality of life of IBD patients. Disease burden
caused by abdominal pain or diarrhoea is compounded by persistently debilitating chronic fatigue
that is due to anemia [46].
Conventional management and therapeutic approaches recommend anemia screening every
3 months for outpatients with active disease and 6 to 12 months for those in the mild or remission
state [47,48]. The thresholds for diagnosis according to World Health Organization (WHO) guideline
in adult males and non-pregnant women, stipulate haemoglobin (Hb) of <13.0 g/dL, <12.0 g/dL, and
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<11.0 g/dL in pregnant women [48]. Specifically, to screen for anemia in IBD patients [49] TfS <20% and
a serum ferritin concentration <30 g/L (with a serum CRP level within the normal range or a ferritin
concentration of less than 100 g/L with an elevated serum CRP level) are specified. Recommendations
for anemia management in IBD patient care are currently conflicting and remain, thereby, an ongoing
process because of limited evidence from human studies. However, recommendations for oral iron
therapy should be limited to IBD patients with mild anemia and with due considerations given to
doses, duration and the types of iron compounds used. This is with the objective to maximize efficiency
and efficacy while minimizing side effects. In IBD patients with moderate to severe anemia, oral
iron causes gastrointestinal disturbances and is refractory, then, intravenous (IV) iron is the preferred
recommendation [50,51]. Inhibition of iron absorption by hepcidin-induced inflammation is by-passed
by IV iron to replenish iron stores and replete Hb levels in the patients. ESA, in combination with IV,
is prescribed for FID in IBD patients and blood transfusion is an option as an acute measure only in
critically anemic patients [50]. Iron therapy and treatment of the other symptoms of IBD will culminate
in the reduction of disease burden to improve the quality of life of the patients.
8. Anemia of Rheumatoid Arthritis (RA)

Patients with rheumatoid arthritis (RA) may display IDA and anemia of chronic disease (ACD).
IDA could be due to gastrointestinal bleeding, gynaecological blood loss, or urinary bleeding or
chemotherapy-induced [51]. As inflammation is a chronic condition in RA, FID is common. ACD
in RA arises from several factors, including ineffective erythropoiesis inflammatory markers (e.g.,
IL-6 and TNF-α), and disordered iron metabolism. Functional iron deficiency in ACD can be due to
overexpression of iron-regulatory hormone, hepcidin, leading to sequestration into storage sites from
circulation, resulting in hypoferrinemia and iron-restricted erythropoiesis. Although decreased serum
hepcidin levels were reported to correlate with the reduction of disease activity [52], this observation
was not evident in other studies [53,54]. Hepcidin could be suppressed, independently of inflammation.
Therefore, the use of hepcidin as a diagnostic tool in the routine clinical management of this disease
still requires further investigation. Prevalence of anemia in RA has been reported to range from
64–70%, while ACD was observed in 50–60% of patients [55,56]. RA patients have been reported to
have both physical disability and increased mortality [57,58]. Relief in swollen, painful, tender joints,
pain, muscle strength, and energy levels symptoms has been reported because of the resolution of
anemia in RA patients [58].
Since anemia in RA is multifactorial and often associated with other malignancies, it is important
to diagnose the nature and the types of anemia in order to apply the most appropriate treatment
regimen. The safety and the use of EPO in the treatment of ACD in RA are controversial [59]. However,
regarding the vital role of IL-6 in ACD, a recent study reported a significant increase in Hb and Hct
levels after IL-6 receptor inhibitor tocilizumab (TCZ) therapy in anemic and non-anemic patients with
rheumatoid arthritis, compared with other biologic and non-biologic disease-modifying antirheumatic
drugs (DMARDs) [60].
9. Anemia of Chronic Kidney Disease

Chronic kidney disease (CKD) is a condition in which renal function deteriorates over time as
the glomerular filtration rate (GFR) declines progressively. Anemia in CKD is of clinical concern as it
predisposes patients to cardiovascular disease, and is associated with poor quality of life, increased
hospitalizations, impaired cognition and mortality [61]. Anemia is a consequence of chronic kidney
disease (CKD), principally because of the depreciation of and reduced synthesis of erythropoietin. Hence
Glomerular Filtration Rate (GFR) is a predictor of anemia in patients with CKD [62]. The progression
of anemia leads to several debilitating symptoms, such as lethargy, muscle fatigue, and deterioration
of renal function. These culminate ultimately in a high prevalence of cardiovascular diseases, such as
left ventricular hypertrophy and heart failure, which account for a significant number of mortalities
in patients with CKD [63,64]. However, the etiology of anemia in CKD is multifactorial. EPO is an
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anti-apoptotic hormone, produced by the kidneys, which promotes the survival, proliferation and
differentiation of erythrocyte precursors [65,66]. As CKD progresses, renal mass declines, which
reduces EPO production and EPO-deficiency results [66]. The expression of EPO is regulated by
the transcription factor, HIF-2α. EPO is produced by peritubular interstitial fibroblasts in the renal
cortex and outer medulla and not by the renal tubular epithelial cells or peritubular endothelial
cells [67,68], as previously presumed. The regulation of EPO production is by HIF 2α and is modulated
by oxygen pressure in the cells and tissues [69,70]. Ablation of HIF-2α, and not HIF-1α, was shown
to cause anemia that was restored by recombinant EPO [71]. The regulation of erythropoietin by
HIF-2α is also confirmed by increased erythropoietin levels and the ensuing erythrocytosis when the
HIF-2α translation is de-repressed in iron regulatory protein 1 (IRP1) knockout mice [72–75]. Under
normoxia, HIF-2α is hydroxylated by O2 -and iron-dependent HIF prolyl-4-hydroxylases (HIF-PHD)
and targeted for proteasomal degradation in a E3-ligase complex. However, under hypoxic conditions,
HIF-2α is stabilized and is no longer degraded but translocated to the nucleus where it forms a
heterodimer with HIF-β or the aryl hydrocarbon receptor nuclear translocator (ARNT). HIF-2α/β
heterodimers, together with transcriptional coactivators, such as CREB-binding protein (CBP) and
p300, bind to consensus elements in the 5’ or 3’ regions of the gene for the kidney or liver, respectively,
to initiate and increase EPO transcription. Factors such as iron chelators, nitric oxide, ROS or CoCl2
inhibit HIF-PHDs association (increased HIF-2α), which culminates to increase EPO transcription and
production [72]. Conversely, excess iron was shown to decrease levels of HIF 2α and EPO expression
in an erythropoietin-deficient mouse model [76]. Furthermore, kynurenine, a product of L-tryptophan
catabolism is increased in anemia of inflammation and in CKD [77]. Kynurenine activates ARNT and
competes with HIF-2α to prevent its binding to HIF-β, thereby decreasing EPO production. Similarly,
in CKD, a uremic toxin, indoxyl-sulfate, apart from simulating hepcidin expression [78], may also
activate ARNT to suppress EPO production [79]. Consequently, an elevated hepcidin level is caused
by a medley of interacting factors, such as inflammation, excess iron, decreased EPO/erythropoiesis
or metabolites or products of certain processes or pathways of systemic metabolism (Figure 1). The
mechanisms by which each player directly influences EPO production are still not yet clearly defined.
Figure 1. Iron metabolism and the mechanisms of renal anemia. In the enterocyte, duodenal cytochrome
b (DCYTB) and other dietary reducing agents reduce ferric iron (Fe3+ ) to its ferrous (Fe2+ ) state, via the
divalent metal transporter 1 (DMT1). Iron efflux into the circulation occurs via hepcidin-regulated
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ferroportin (FPN). In blood, iron is transported bound to transferrin (TF) to the liver, cells of the
reticulo-endothelial system (RES) and to other tissues and organs. Inflammatory cytokines suppress
erythropoiesis in the bone marrow and stimulate hepcidin production in the liver, which influences
iron absorption and efflux negatively. Decreased GDF11/GDF15 or erythroferrone leads to increased
hepcidin production. Uremic toxins enhance hepcidin expression and modulate the EPO level via
Hif-2α, which also induces the transcription of DCYTB, DMT1, FPN, and TF [72].
In the advanced stages of CKD, regular hemodialysis contributes to absolute iron deficiency. Blood
is lost during the hemodialysis process in the tubing and the apparatus and also, through the numerous
blood samples taken from the patient [80,81]. Under normal physiological conditions, macrophages
engulf senescent erythrocytes and recycle the iron incorporated in hemoglobin [82]. During such blood
losses, this opportunity for iron recycling is lost [77,83]. After blood loss, in healthy individuals, EPO
aids in the absorption of iron, but this is reduced in CKD patients as they suffer from EPO deficiency
as their condition deteriorates. Therefore, in CKD, there is difficulty in replenishing iron stores and
consequently, erythropoiesis is limited [84].
Hepcidin Expression and Function in CKD Patients

Iron availability is the rate-limiting step in the maturation of erythroblasts into erythrocytes [9].
EPO increases the synthesis of erythrocytes in the bone marrow and this leads to a depletion of iron
stores and the reduced availability of iron contributes to anemia in CKD [66,85]. Replenishing these
iron stores in CKD is also more difficult than in healthy subjects, thereby exacerbating the problem [86].
Furthermore, the reduced glomerular filtration rate in CKD results in impaired renal clearance of
hepcidin. Dialysis reduces hepcidin level; however, this rapidly rises again in the interval between
dialysis sessions [85]. Mobilization of iron from hepatocyte and reticuloendothelial stores is restricted,
leading to absolute iron deficiency due to reduced intestinal absorption of iron [87]. This impairs
erythropoiesis, which is iron-dependent and contributes to anemia. CKD patients have a greater
predisposition to infection as long-term hemodialysis exposes the patients repeatedly to pathogens in
the environment [88]. As previously discussed, this inflammatory state promotes increased hepcidin
levels, which contributes to impaired iron absorption and mobilisation [89,90]. Hepcidin produces
these effects by downregulating FPN function and iron efflux into the blood. [9,87,91].
Hepcidin expression is suppressed by erythropoiesis to meet iron demand to support the process
and EPO has been reported to have a direct effect [16,89,92], possibly in conjunction with co-factors,
such as twisted gastrulation protein homolog 1 (TWSG 1), growth differentiation factor 15 (GDF15),
GDF11, and erythroferrone (ERFE). The functions of TWSG-, GDF11 and GDF15 in the inhibition of
erythropoiesis are still controversial. However, erythroblasts synthesise ERFE upon stimulation by
EPO and this inhibits the expression of the hepcidin gene [84,93], particularly under stress. As CKD
progresses, reduced production of EPO results in dwindling erythrocyte production and consequently,
decreased erythroferrone production. This, in turn, leads to increased hepcidin expression, which
reduces iron absorption and decreases iron mobilisation from the stores [93,94]. Reduced iron levels
limit erythrocyte maturation and exacerbate anemia of CKD even further.
The regulation of hepcidin induction at the cellular level and in the liver is both intricate and
complex and involves membrane-bound iron sensors that include the transferrin receptors (TfR) 1 and
2, HFE, and hemojuvelin (HJV). The signal for hepcidin expression is initiated by bone morphogenic
protein (BMP) ligands using the glycosylphosphatidylinositol-anchored membrane protein, HJV, as
a coreceptor that binds to Type I and Type II BMP serine threonine kinase receptors. This induces a
cascade of activation and phosphorylation of the receptors that channel downstream to Suppressor of
Mothers Against Decapentaplegic (SMAD) proteins involved in signalling during hepcidin expression.
A detailed description of the triggers that regulate the hepcidin expression process is reviewed
elsewhere [14,18]. In summary, hepcidin expression is regulated by the BMP6-HJV-SMAD and
IL-6-STAT3 signaling cascade. BMP6 binds the BMP receptors and HJV coreceptor, which causes the
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phosphorylation of SMAD1/5/8. Phosphorylated SMAD proteins associate with SMAD4 and these
complexes traverse the nuclear membrane to bind to the promoter region of the hepcidin gene to induce
hepcidin expression. The inflammatory stimulus, IL-6, binds the IL-6 receptor and activates Janus
kinase 2 (JAK2), which phosphorylates Signal Transducer and Activator of Transcription 3 (STAT3).
Phosphorylated STAT3 translocates into the hepatocyte nucleus to bind the STAT3 responsive element
at the promoter region of the hepcidin gene to induce hepcidin expression.
10. Treatment of Anemia in CKD

To treat anemia in CKD, it is necessary to enhance the synthesis of erythrocytes, as well as ensure
the maintenance of adequate levels of iron for hemoglobin formation [88,95]. The National Institute for
Health and Care Excellence (NICE) recommends the use of either iron or erythropoiesis-stimulating
agents, or both in combination, for the treatment of anemia of CKD [96]. This is aimed at addressing
both absolute and functional iron deficiency that lead to restricted iron access and EPO deficiency [97].
As deficiency in EPO is a major contributory factor to anemia in CKD, recombinant human erythropoietin
(rHuEPO), such as epoetin alpha and epoetin beta, are used in the treatment of anemia in CKD
patients [98]. This initial therapy was brought into clinical practice in the 1980’s and has been found to
successfully treat the signs and symptoms of anemic CKD, such as fatigue, weakness and headaches [99].
In addition, these patients also required less frequent blood transfusions, a further benefit from the
use of ESAs in anemic CKD [99]. However, large randomized control studies including the Normal
Hematocrit Study (NHCT), the Correction of Haemoglobin and Outcomes in Renal Insufficiency
(CHOIR) trial, the Cardiovascular Risk Reduction by Early Anemia Treatment (CREATE) trial and
the Trial to Reduce Cardiovascular Events with Aranesp Therapy (TREAT), which highlighted the
potential harm of high-dose ESA therapy, have challenged these therapeutic claims from utilising
ESAs [100]. It has been emphasized that the complete correction of anemia, and indeed raising
hemoglobin levels above 11 g/dL was associated with adverse outcomes. These include an increased
risk of stroke, cardiovascular incidents, rapid malignant progression in cancer patients and increased
mortality in other patients [61]. Moreover, pure red blood cell aplasia can be induced in rare instances
through the use of ESAs, which, in turn, promotes severe anemia and results in the patient becoming
transfusion-dependent [66]. It was proposed that these adverse outcomes were due to the very high
doses of ESA that are administered [61]. Such high doses of ESA are often prescribed to patients that
are hyporesponsive to ESA therapy to correct their hemoglobin deficits and higher doses are provided
to attain target hemoglobin levels. The results from randomized controlled trials have subsequently
influenced KDIGO guidelines, which recommend that non-dialyzed CKD patients are not administered
ESA if haemoglobin levels are above 10 g/dL. CKD patients on dialysis should receive ESA therapy
when hemoglobin levels lie between 9 g/dL and 10 g/dL. In all adult patients, ESA therapy should be
used to maintain haemoglobin levels no higher than 11.5 g/dL [100].
Evidence from large randomized control studies highlighting the negative health effects of high-
dose ESA administration was the reason for advocating the use of iron therapy as an adjunct to
ESAs. Consequently, iron and lower doses of ESA are currently prescribed to preclude the adverse
outcomes associated with high-dose ESA administration [66]. Moreover, as ESA therapy acts to
increase erythropoiesis, this results in the depletion of the iron pool, causing a relative iron deficiency
for which iron supplementation is recommended as a preventive measure [61]. Inflammation inhibits
erythropoiesis, which influences erythropoietin (EPO) hyporesponsiveness [101] and decreases the
systemic circulation of iron levels by the production of hepcidin [102,103]. Inflammation in CKD, apart
from causing decreases in iron availability via elevated hepcidin levels, also directly aggravates anemia
by suppressing EPO production [104]. Inflammation also decreases the enhancing effect of EPO on
erythropoiesis [105].
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11. Iron Supplementation for the Treatment of Anemia of CKD

CKD patients, as previously explained, suffer from increased blood loss and reduced intestinal
absorption of dietary iron and thus, iron supplementation is important to prevent absolute iron
deficiency. Iron supplementation may be administered through the oral or IV route, nevertheless, both
routes have advantages and disadvantages.
Oral administration using ferrous sulphate is adequate for moderate anemia, and the advantages
include its relative low cost [99]. However, the side effects include constipation, nausea and abdominal
discomfort, as well as reduced patient compliance [98,106]. Additionally, intestinal iron absorption can
be impaired in CKD and the efficacy of oral iron can be variable [99]. Incidentally, sucrosomial iron
(SI), a newly developed oral iron preparation, in a randomized trial in CKD patients, has been shown
to be comparable to IV iron gluconate in elevating hemoglobin levels [107]. The future of oral iron
therapy may involve dietary supplementation with nanoparticles. Nanoparticulate tartrate-modified
Fe (III) poly oxo-hydroxide (Nana Fe (III)) has also been shown to be absorbed by a DMT1-independent
mechanism for replenishing hemoglobin levels in mice without the side effects associated with oral
therapy [108]). The dosage of oral and IV iron in CKD patients are dependent on the presence or
absence of inflammatory status of the gut. In Europe and USA, higher doses of IV iron have been used
in dialysis patients because of higher inflammation status than in those of Japan. Low doses of IV
iron or oral iron have been effective in the Japanese dialysis patients with the same efficacy because
inflammation is minimal [109]. Although compared with Western countries, the Japanese guidelines
for prescription of IV iron in dialysis patients are more conservative, the outcomes, nevertheless, are as
good or better than their American counterparts [110]. Given the potential safety issues with aggressive
IV iron treatment, and lack of well-powered studies to examine safety, a more conservative approach
to iron therapy should be considered in the US [111].
Intravenous administration is highly efficient at replenishing iron stores, enhancing erythropoiesis
and reducing the required ESA dose. This practice is advantageous as high doses of ESA therapy have
been associated with negative clinical outcomes [112]. The pitfalls of IV therapy, however, include the
invasive method of delivery and increased infection risk [99]. Results from studies of IV iron use and
infection have highlighted conflicting results [113,114]. Data from observational, laboratory and animal
studies have also indicated that IV iron treatment promotes oxidative stress, atherosclerotic plaque
development, infection, hypersensitivity responses and increased cardiovascular mortality [113,115].
Studies involving apolipoprotein E (ApoE) knockout mice have highlighted that elevated iron does
not cause atherosclerotic plaque progression, whereas other studies have shown that IV iron sucrose
increases superoxide production and monocyte adhesion to the endothelium, instigating atherosclerotic
plaque formation [116,117]. IV iron has been found to be effective for functional iron-deficiency anemia
in CKD patients with high inflammation but had negative consequences on markers of oxidative stress
that could have clinical implications [111]. Moreover, different preparations of IV iron carry different
risks. Iron dextran carries a higher risk of adverse reactions, including type 1 hypersensitivity reactions,
in comparison to iron sucrose, sodium ferric gluconate and ferric carboxymaltose [118,119]. The
recommended adult doses of iron sucrose and sodium ferric gluconate carry lower risks in CKD [120,121].
The Ferumoxytol for Anemia of CKD Trial (FACT), a randomized, phase 4 study [122], reported
comparable efficacy and safety of ferumoxytol and iron sucrose in patients with CKD undergoing
hemodialysis. However, as clinical trials such as the ferinject assessment in patients with iron-deficiency
anemia and non-dialysis-dependent chronic kidney disease (FIND-CKD) and a randomized trial to
evaluate intravenous and oral iron in chronic kidney disease (REVOKE) were un-unanimous in their
conclusions on IV iron safety, its use remains a subject of continuous debate [112,114]. However, the
use of IV iron should be with caution since iron overload has been detected by MRI in hemodialysis
patients with relatively low serum ferritin levels [123], suggesting that iron overload can occur in CKD
patients receiving standard doses of IV iron. Thus, it was recommended that the dose of IV iron should
be reduced to <250 mg/month to avoid iron overload in CKD patients [124]. Recommendations on
iron management in CKD patient care are an ‘ongoing process’ because of limited research evidence.
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The outcomes of several randomized controlled trials (RCTs) and observational studies are varied
regarding the effectiveness and adverse effects of iron or ESA supplementation. Heterogeneity of
confounders have been associated with the study design and can be due to the type, dosage, duration or
route of iron administration, population size and the inherent variability within the baseline [125,126]
hematological profile of patients.
12. Novel Therapies for the Treatment of Anemia of CKD

Although recent therapies offer benefits to most patients, some patients remain anemic and,
therefore, there is a drive to develop novel therapies to address persistent anemic conditions (Table 2).
12.1. Targeting Hepcidin

High levels of hepcidin recorded in CKD patients act to impair absorption and mobilization
of iron.
Furthermore, the chronic inflammation which manifests in CKD patients results in the production
of pro-inflammatory cytokines including interleukin-6 (IL-6), which has been shown to stimulate the
synthesis of hepcidin. Currently, inhibitors of hepcidin production are being investigated. The two main
pathways involved in regulating hepcidin expression are the BMP6-HJV-SMAD and the IL-6-STAT3
signalling pathways. Studies have revealed the existence of a cross-talk between the two pathways.
In vitro studies have shown that therapies which act to inhibit the BMP pathway by sequestering
ligands for the BMP receptor or antagonizing the BMP receptor also inhibit hepcidin expression via the
inflammatory IL-6-STAT3 signaling pathway. Inhibitors of this pathway currently being investigated
include anti-IL-6 antibodies such as Tocilizumab and IL-6 monoclonal antibodies such as Sultuximab.
The safety of using these drugs needs to be verified as sultuximab, despite causing an increase in
hemoglobin levels, has been associated with increased infection risk [127]. Dorsomorphin is an
inhibitor of the BMP type I serine threonine kinase receptors and targets HJV and IL-6 and thus
also dampens down the inflammation-induced expression of hepcidin. However, Dorsomorphin is
non-selective and also inhibits the action of AMP kinase. Nonetheless, this highlights the potential
therapeutic benefit of developing BMP inhibitors that can have a dual action on both BMP- and
IL-6-mediated hepcidin expression [128]. Other potential antagonists or suppressors of hepcidin
expression include Atorvastatin, TNFα and TGFɣ inhibitors. A 6-month administration of Atorvastatin
to CKD patients in a randomized double-blind crossover study revealed a significant decrease in
serum hepcidin [129]. This was concomitant with improved haematological parameters. Similarly,
Sotatercept and Luspatercept are recombinant soluble activin type-II receptor-IgG-Fc fusion proteins
that were reported to increase red blood cell numbers and hemoglobin levels in humans treated for
renal anemia [119,130]. An anti-inflammatory Pentoxifylline (PTX)—a phosphodiesterase inhibitor
of anti-TNF-alpha activity—has also been proposed as a potential therapy for different disorders,
including anemia, and its use in CKD awaits further research [131].
12.2. Hepcidin-Ferroportin Axis

Currently under investigation are newer potential therapies that will target the hepcidin–
ferroportin axis in the treatment of anemia in CKD. This axis can be targeted at various points.
For example, direct hepcidin antagonists, such as hepcidin antibodies, are currently in clinical trials and
are thought to inhibit the action of hepcidin. These antibodies have been shown to bind both human
and monkey hepcidin and inhibit its action on ferroportin, and as such, enhance the absorption of
dietary iron and promote its mobilization from iron stores for use in erythropoiesis [132]. An additional
direct hepcidin antagonist currently under development is hepcidin RNA interference (RNAi), which
is predicted to inhibit hepcidin gene expression, to promote FPN function and thereby elevate iron
levels [133].
Some therapies are also exploring FPN stabilizers, which make FPN less sensitive to the action
of hepcidin, thereby promoting elevated, optimal iron efflux into circulation. These stabilizers
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tend to reduce hepcidin expression and inhibit its action while preventing FPN degradation, which
aid in the treatment of absolute iron deficiency in anemia of CKD. One example of such an FPN
stabilizer is the anti-ferroportin monoclonal antibody, which prevents the interaction between hepcidin
and FPN [128]. Two other monoclonal antibodies, LY3113593 and LY2928057, targeting BMP6 and
ferroportin, respectively, tested in CKD patients resulted in an increase in haemoglobin and reduction
in ferritin (compared to the placebo) [134]. Serum iron efflux increased through LY2928057 binding
to ferroportin and blocking interactions with hepcidin. In the same vein, LY3113593 blocked BMP6
binding to its receptor to decrease hepcidin expression.
12.3. Targeting Hif1α Inhibitors

The stabilization of HIF via a prolyl hydroxylase inhibitor (HIFα-PHI) system is a novel approach
that may also be an effective therapeutic target in the treatment of anemia of CKD as EPO deficiency
contributes greatly to this condition. HIF1α regulates renal EPO production and erythropoiesis
(described in Section 9). Thus, this approach involves manipulating a physiological regulatory
process rather than the conventional EPO administration. Examples of HIF-PHIs that are in clinical
trials include Vadustat, Daprodustat and Roxadustat [97,135–138]. Phase II clinical trials of HIF1α
stabilizers have concluded the effectiveness and safety for short-term use [128,139]. More recently, a
Phase II, randomized, double-blind, placebo-controlled, trial showed a dose-dependent increase in
Hb compared to placebo in adult CKD patients with anemia after 6 weeks of Desidustat (ZYAN1)
treatment [140]. Desidustat (ZYAN1) is an oral hypoxia-inducible factor prolyl hydroxylase inhibitor
(HIF-PHI) that stimulates erythropoiesis. Another novel hypoxia-inducible factor prolyl hydroxylase
inhibitor, Molidustat, has the potential to treat anemia of CKD by increasing erythropoietin production
and improving iron availability particularly, in non-dialysis patients [141]. However, HIFs have
roles in other biological pathways, including in the expression of vascular endothelial growth factor
(VEGF), which is associated with retinal disease and cancer [142]. Consequently, the long-term safety
of HIF-PHIs, and possibly Hif 2α, needs to be elucidated, in particular, for long-term therapy [143].
12.4. Other Compounds

An additional novel therapeutic strategy in the treatment of anemia in CKD is the development
of engineered lipocalins called anticalins, which are able to bind small hydrophobic molecules such
as hepcidin and thereby inhibit it from carrying out its function [144]. Anticalin PRS-080#22 has also
been shown to sequester hepcidin in a Phase I clinical trial [145]. PRS-080#22 decreased hepcidin and
increased serum iron and transferrin saturation in a dose-dependent manner. In mice and in patients
with deep vein thrombosis (DVT), administration of the anticoagulant, heparin, caused decreased
hepcidin levels and increased mobilization of iron from splenic stores, thereby increasing the circulating
iron level [145].
Heparins have been shown to be inhibitors of hepcidin expression in vitro and in vivo [146]. They
suppress hepcidin expression via the BMP6/SMAD pathway and are, therefore, promising for the
treatment of anemia of CKD that is, in part, exacerbated by high hepcidin levels in the patients [147].
The mechanism by which heparin antagonizes the BMP/SMAD pathways awaits future clarification.
Vitamin D has also been found to reduce hepcidin gene transcription, lower serum levels by
50% in healthy individuals within 24 h, enhance erythropoiesis and reduce inflammation [135,148].
Moreover, in early-stage chronic kidney disease patients, vitamin D3 supplementation decreased
hepcidin level after three months of administration [149]. However, the calcitriol form of vitamin D
did not reduce serum hepcidin concentrations among individuals with mild to moderate CKD [150].
Similarly, in pregnant women, vitamin D3 supplementation did not influence hepcidin, ferritin, or
inflammatory status, indicating no beneficial effect in alleviating iron depletion in the subjects [151].
Further studies are needed to confirm the long-term effect of vitamin D in CKD patients.
160
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Potential New Therapy for Anemia of Chronic Disease in the Future

Recently, the bone-secreted hormone fibroblast growth factor (FGF23) inhibitor has been advocated
for the treatment of anemia in CKD patients. FGF23, apart from its canonical functions in bone
mineralization for the regulation of phosphate vitamin D homoeostasis exerts a pleiotropic role in
iron metabolism in CKD patients [152]. Iron deficiency, inflammation and EPO have been shown to
increase FGF23 protein levels and cleavage [153–155], resulting in an increase in the anemic condition
in CKD. Conversely, studies have also shown that FGF23 may promote anemia, iron deficiency, and
systemic inflammation, particularly in CKD [142,156]. Hence, inhibitors of FGF23 could be employed
to stimulate erythropoiesis and treat anemia in CKD patients. Agoro and others [157] reversed anemia
and iron deficiency in a mouse model of CKD by inhibiting and blocking the FGF23 signalling pathway
with its peptide antagonist. The mice displayed increased erythropoiesis, serum and ferritin levels and
reduced erythroid apoptosis and inflammation. Table 2 summarizes the potential novel therapies for
anemia in CKD.
Table 2. Summary of potential therapies for anemia of chronic kidney disease (CKD).
Name Mode of Action Adverse Effects References

Hepcidin antagonist, e.g., Viral delivery system—risk
hepcidin antibodies Inhibit hepcidin action of random genome
[121,122,132,
RNA Interference Inhibit hepcidin expression integration.
133,144,158]
Atorvastatin, Sotatercept and promote FPN function Unfavorable immunological
Luspatercept responses
Hepcidin binding proteins
Lipocalin, e.g., Anticalin Inhibit hepcidin function Non-specificity [159]
(PRS-080)
Hepcidin production
inhibitors
Inhibit hepcidin expression
BMP inhibitors, e.g., soluble
and the BMP6-HJV-SMAD Unknown [134,160]
HJV, Dorsomorphin,
pathway
Anti-BMP6 monoclonal
antibody
Anti- IL6 monoclonal Inhibits IL-6 STAT3 signaling
Unknown [158]
antibody, e.g., Siltuximab cascade
Bleeding
Decreases hepcidin levels Thrombocytopenia
Heparin Increases mobilisation of Hyperkalaemia [161]
iron stores Alopecia
Osteoporosis
In excess causes nausea,
Decreases hepcidin
Vitamin D vomiting, depression, [148–150]
gene transcription
weakness, and confusion.
FPN stabilizers, e.g.,
anti-ferroportin monoclonal Increase ferroportin action Unknown [130,134]
antibody
HIF-PHDI, e.g., Roxadustat Pulmonary hypertension
Increase endogenous EPO [97,98,134,
Daprodustat, Vadadustatt, Increase in VEGF
expression 136,141]
Molidustat, Tumour progression
Decreases hepcidin levels
Anticalin PRS-080#22 Increases mobilisation of Unknown [144]
iron stores
Stimulates and promotes
FGF23 inhibitor Unknown [153,157]
erythropoeisis
161
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13. Conclusions
Is anemia a symptom, disorder or disease? Iron deficiency occurs insidiously as a symptom
or syndrome over a spectrum of severity, with low hemoglobin as a later manifestation of extreme
deficiency [11]. Absolute anemia, that manifests after the depletion of iron stores, is a clinical disease
condition. However, functional iron-deficiency anemia is a risk factor for several ailments, disorders
and diseases. In general, the etiology of anemia is multifactorial and this necessitates diverse therapeutic
guidelines in the management of the syndrome. Hence, there are variations in guidelines specifications
or consensus statements for the therapy of different stages of iron deficiency and anemia in different
disorders. When dietary iron sources are limiting, iron formulations are prescribed as oral supplements.
Parenteral or intravenous iron therapy becomes the choice of therapy for iron-deficiency anemia for
these patients intolerant or refractory to oral iron administration. However, recommendations to start
therapy vary with different conditions. For example, for IBS, CKD or anemia of heart failure, the
recommendation to commence therapy is based on a wide range of serum ferritin levels (30–299 ng/mL)
and when transferrin saturation is below 20%. A key regulator of iron homeostasis is hepcidin, which
contributes to anemia by reducing the absorption of iron from the diet, as well as through diminishing
the mobilization of iron from iron stores. One of such anemia is that associated with CKD, which
manifests absolute iron deficiency and iron-restricted functional anemia and impaired erythropoiesis.
Current therapy is successful in some patients in alleviating the signs and symptoms of anemia such as
weakness, headache, vertigo and fatigue; however, despite current intervention, the disorder remains
endemic in patients. The current review describes several novel therapies to tackle this devastating
condition and correct elevated hepcidin levels in CKD patients not responding to current interventions.
However, the efficacy, tolerability and side effect profiles of these novel therapies in CKD patients
have not been fully elucidated. It is encouraging that studies are on-going on some of these novel
therapeutic approaches that can be translated into clinical applications.
Author Contributions: S.B. and G.O.L.-D. wrote the review.” Authorship must be limited to those who have
contributed substantially to the work reported.
Abbreviations
ID iron deficiency IDA Iron-deficiency anaemia
GBD Global Burden of Disease CKD chronic kidney disease
ACD Anemia of Chronic Disease IBD inflammatory bowel disease
IV intravenous EPO erythropoietin
ESA erythropoietin stimulating agent rHuEPO recombinant human erythropoietin
FPN ferroportin DMT1 divalent metal transporter
Dcytb duodenal cytochrome b MCV mean corpuscular volume
Hb haemoglobin BMP bone morphogenic protein
HJV hemojuvelin SMAD Mothers Against Decapentaplegic
Signal transducer and activator of
JAK2 Janus kinase 2 STAT 3
transcription 3
GDF 15 growth differentiation factor 15 PDGF-BB platelet-derived growth factor-BB
HIF hypoxia-inducible factor VEGF vascular endothelial growth factor
IL Interleukin FCF23 fibroblast growth factor
Kidney Disease: Improving Global Correction of Haemoglobin and
KDIGO CHOIR
Outcomes Outcomes in Renal Insufficiency
Cardiovascular Risk Reduction by Early Trial to Reduce Cardiovascular Events
CREATE TREAT
Anemia Treatment with Aranesp
Hypoxia-Inducible factor Prolyl
HIF-PHDI
Hydroxylase Inhibitor
162
Nutrients 2019, 11, 2424
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171
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Nutrition For Anemia

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Nutrition for

ISBN 978-3-03936-936-2 (Hbk)

About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Preface to ”Nutrition for Anemia” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Jorge Moreno-Fernandez, Marı́a J. M. Alférez, Inmaculada López-Aliaga

Teresa Nestares, Rafael Martı́n-Masot, Ana Labella, Virginia A. Aparicio,

Javier Diaz-Castro, Jorge Moreno-Fernandez, Ignacio Chirosa, Luis Javier Chirosa,

Jorge Moreno-Fernández, Inmaculada López-Aliaga, Marı́a Garcı́a-Burgos,

Rafael Martı́n-Masot, Maria Teresa Nestares, Javier Diaz-Castro, Inmaculada López-Aliaga,

Sajidah Begum and Gladys O. Latunde-Dada

Received: 6 April 2020; Accepted: 30 April 2020; Published: 8 May 2020

Nutrients 2020, 12, 1336; doi:10.3390/nu12051336 1 www.mdpi.com/journal/nutrients

2. Material and Methods

2.1. Animals and Experimental Design

2.2. Diets Preparation

Table 1. Composition of experimental diets.

Constituents Pre-Experimental Period Experimental Period

2.3. Hematological Tests

2.5. Hepatosomatic Index, Hepatic Iron Concentration and Transaminases Analysis

HSI = (weight of the liver/weight of the body) × 100

2.6. RNA Extraction and Quantitative Real Time PCR

2.7. Western Blotting and Immunocytochemistry

2.8. Statistical Analysis

Control Group Anemic Group

Fermented Cow Milk Fermented Goat Milk 2-WAY ANOVA

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/5/1336/s1,

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Received: 28 February 2020; Accepted: 19 March 2020; Published: 21 March 2020

Nutrients 2020, 12, 844; doi:10.3390/nu12030844 15 www.mdpi.com/journal/nutrients

2. Materials and Methods

2.2. Clinical and Socio-Demographics

2.3. Anthropometric Measures

2.4. Blood Haematological and Biochemical Anaemia-Related Markers

2.5. Dietary Assessment

2.6. Data Analyses

Table 1. Anthropometric, clinical and sociodemographic characteristics of the study participants.

Celiac Group Healthy Group

Diﬀerences in serum hematological and biochemical anemia-related markers between healthy

Less than 6 More than 6

Less than 6 More than 6

Less than 6 More than 6

5. Strengths and Limitations

Received: 31 January 2020; Accepted: 16 February 2020; Published: 27 February 2020

Keywords: anaemia; altitude adjustment; haemoglobin; iron; South Africa; women of

Nutrients 2020, 12, 633; doi:10.3390/nu12030633 25 www.mdpi.com/journal/nutrients

2. Materials and Methods

2.1. Study Setting and Population

2.2. Ethical Considerations

2.3. Measurement of Anthropometric Indicators and Socio-Demographic Information

2.4. Biomarker Analysis

2.5. Data and Statistical Analysis

3.1. Sample Characteristics

3.2. Hematological Indicators

Characteristics Median (IQR) or n (%)

Biomarker Median (IQR) or n (%)

Table 3. Diagnostic accuracy of the diﬀerent Hb cut-oﬀ points to detect ID based on

Received: 18 January 2020; Accepted: 17 February 2020; Published: 19 February 2020

Keywords: acai; erythropoiesis; erythropoietin

Nutrients 2020, 12, 533; doi:10.3390/nu12020533 39 www.mdpi.com/journal/nutrients