**The E**ff**ectiveness of Di**ff**erent 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 <sup>5</sup> and Josep Basora 2,6**


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. 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. Materials and Methods**

#### *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) 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 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.

#### *2.3. Statistical Analysis*

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, 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.

**Figure 1.** Flowchart of the study.

Low-Middle High

**Figure 2.** Adherence to the Mediterranean diet in the different groups of iron supplementation.


**Table 1.** Baseline characteristics of the study population.

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%) 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).



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.



**Table 3.** *Cont.*

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.

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.

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.


**Table 5.** *Cont.*

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.



**Table 6.** *Cont.*

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

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. 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.

**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

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.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *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**


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 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. Materials and Methods**

#### *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*, 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<sup>−</sup>1) or Fe overloaded content (450 mg·kg<sup>−</sup>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<sup>−</sup>1) and serum hepcidin (Hepcidin-25 ng·mL<sup>−</sup>1) were determined using a rat ELISA Kit (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.

#### *2.12. Statistical Analysis*

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

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).

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).



Fe content were significantly different (*<sup>p</sup>* < 0.05, Tukey's test).


**Table 3.** Brain molecular function parameters in brain homogenate (pg mL<sup>−</sup>1) from control and anemic rats (pre-experimental period).

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).


corresponding

 group fed with normal Fe content were significantly different (*<sup>p</sup>* < 0.05, Student's *t* test). MAO,

were significantly different (*<sup>p</sup>* < 0.05, Tukey's test). D Mean values from the

Monoamine oxidase; α-MSH,

α-Melanocyte-stimulating

 hormone.

**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-baseddietswithnormalFecontentorFeoverload(*<sup>n</sup>*=10animalspergroup).

#### *Nutrients* **2019**, *11*, 2394

#### **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 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].

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.

**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.

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

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Iron Homeostasis Disruption and Oxidative Stress in Preterm Newborns**

**Genny Ra**ff**aeli 1,**†**, Francesca Manzoni 1,2,**†**, Valeria Cortesi 1,2, Giacomo Cavallaro 1, Fabio Mosca 1,2 and Stefano Ghirardello 1,\***


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

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].

#### **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

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<sup>+</sup> 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. 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].

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].

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.

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:


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 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]:


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].

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].

#### 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.

#### *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:


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).

#### 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:


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.

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].

#### *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.

**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<sup>+</sup>: 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.

**Funding:** This research received no external funding.

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

#### **List of Abbreviation**



#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Multifactorial Etiology of Anemia in Celiac Disease and E**ff**ect 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 <sup>2</sup> and José Maldonado 3,4,5,6**


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

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).


\* 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).

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 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 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

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. 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.

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 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 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 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.

**Funding:** This research received no external funding.

**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.

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

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Anemia of Inflammation with An Emphasis on Chronic Kidney Disease**

#### **Sajidah Begum <sup>1</sup> and Gladys O. Latunde-Dada 2,\***


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,

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

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].

TNFα, tumor necrosis factorα; TfR1: Erythropoietin receptor 1; IFNɣ, 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

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

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

<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

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

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

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 highdose 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].

#### **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. 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 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 Hif1*α *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.

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).

#### **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.

**Funding:** This research received no external funding.

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

#### **Abbreviations**


#### **References**


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