Impact of inflammation on anemia in children: a cross-sectional study

Abstract

Introduction

The association of inflammation and iron deficiency could be related to up to 40% of anemia in young children.

Objective

To describe the anemia and iron deficiency in children and verify possible associations with dietary practices, nutritional status and inflammatory markers.

Methods

This cross-sectional study was conducted with one hundred and twelve children aged between 6 months and 3 years in Early Childhood Education Centers in Minas Gerais, Brazil. Nutritional status regarding iron and inflammatory markers was assessed using the reference values proposed by the World Health Organization.

Results

Anemia and iron deficiency were observed in 58 (51.8%) and 89 (79.5%), respectively, of children evaluated. Hemoglobin values were directly associated with the z-score of height for age after adjusting for high-sensitivity C-reactive protein values (β-adjusted = 0.375; 95% CI 0.088 to 0.662; p = 0.011). The values of high-sensitivity C-reactive protein correlated directly with RDW (r = 0.202; p = 0.033), ferritin (r = 0.425; p < 0.001) and soluble transferrin receptor (r = 0.446; p < 0.001), and inversely with hemoglobin (r = -0.287; p = 0.002), serum iron (r = -0.580; p < 0.001) and transferrin saturation index (r = -0.528; p < 0.001). The ROC curve shows that RDW (AUC = 0.708; CI 95% 0.612 to 0.803) and soluble transferrin receptor (AUC = 0.588; 95% CI 0.481 to 0.694) were the variables that showed the better level of discrimination of anemia.

Conclusions

The prevalence of anemia was higher than in national studies, and there was a correlation between inflammatory markers and biomarkers of iron nutritional status.

Introduction

Anemia is a disease characterized by a low hemoglobin concentration. Its global prevalence is 24.3% (around 1.92 billion people), making it a public health problem in several countries. Globally, the prevalence of anemia among children under 5 years of age can reach up to 40%, affecting 269 million children [1, 2].

Anemia resulting from iron deficiency is common among children and women, especially in emerging economies [2]. The Brazilian National Survey on Child Nutrition (ENANI-2019) showed a prevalence of anemia in children aged 6 to 59 months of 10.1%, 19.0% among those aged 6 to 23 months, and 3.5% of anemia due to iron deficiency (ID). Although epidemiological data in Brazil over the last few decades point to a drop in the prevalence of anemia in this age group, the need for prevention strategies is emphasized, since the disease causes short- and long-term adverse events [3, 4].

Future complications of anemia and ID include impaired growth and neurodevelopment, damage to the immune system, and increased risk and severity of infections [5]. Primary care monitoring, adherence to the national dietary guidelines, and routine family visits to health services, such as those offered by the Family Health Strategy, are essential to preventing anemia [6].

Exclusive breastfeeding up to the sixth month and complemented by a healthy diet, with an emphasis on offering foods that are a source of highly bioavailable iron, up to the age of two or more represent an essential prevention strategy emphasized in the Food Guide for Brazilian Children under 2 years of age [7]. Currently, the Brazilian Ministry of Health has two programs aimed at preventing ID through prophylactic iron supplementation: the NutriSUS/Brazil – Strategy for infant feeding fortification with multiple micronutrients powders (vitamins and minerals) and the National Program of Iron Supplementation [8].

Inflammation interferes with iron metabolism by increasing hepcidin production, inhibiting ferroportin, reducing iron absorption and inducing oxidative stress. These alterations can lead to anemia and/or ID. Although anemia of inflammation is similar to ID in terms of the biomarkers commonly used for diagnosis, it is a disturbance in iron distribution since the macrophages in the liver, spleen and bone marrow responsible for recycling old red blood cells still retain their iron stores. Up to 40% of the worldwide prevalence of anemia can be associated with inflammation or correspond to a combined form with ID [9, 10].

The relationship between nutritional status, consumption of ultra-processed foods, and excess weight is complex and interconnected. Excess weight exacerbates the risk of ID and anemia due to chronic inflammation and changes in micronutrient absorption. In turn, the mechanisms linking the consumption of ultra-processed foods to ID include low iron intake and iron absorption inhibitors in these foods, such as some additives and preservatives that compromise its absorption. In addition, the high intake of sugars and fats can lead to chronic inflammation, increasing hepcidin production [11, 12].

Given the negative impact of anemia and ID on children's health in the short and long term and the importance of expanding and updating knowledge of their determinants to develop more effective prevention strategies, this study aimed to describe the prevalence of anemia and ID in children and verify possible associations with dietary practices, nutritional status, and inflammatory markers.

Methods

Study design

This cross-sectional study was carried out between May and November 2022 in the three Early Childhood Education Centers (ECEC) in Paraguaçu, south of Minas Gerais (MG), Brazil. The ECECs work full time and offer four meals a day: breakfast, lunch, afternoon snack, and dinner, to meet at least 70% (seventy per cent) of the nutritional needs for energy and priority macronutrients and micronutrients (Vitamin A, Vitamin C, Calcium, and Iron).

The municipality of Paraguaçu-MG is located in the south of Minas Gerais, Brazil, and has an estimated population of 21,723. The Municipal Human Development Index is 0.715, the average infant mortality rate is 7.46 per 1,000 live births, and 83.1% of households have adequate sewage disposal. Located at an altitude of 825 m, Paraguaçu-MG has the following geographic coordinates: Latitude: 21° 31′ 59'' South, Longitude: 45° 45′ 59'' West [13].

Population

This study included 112 children aged between 6 months and 3 years enrolled in the public early childhood education system in Paraguaçu-MG. The municipality has 3 (three) Early Childhood Education Units with 174 children enrolled, aged up to 36 months. All units were invited to participate in the study, and the convenience sample was composed of all duly enrolled children if they met the inclusion and exclusion criteria. Children with chronic diseases (except excess weight), such as neurological, genetic, renal and cardiac diseases, and those with any acute inflammatory diseases or infections at the time of blood collection were excluded from the study. The study was approved by the Human Research Ethics Committee of the Federal University of São Paulo, Brazil, under CAAE number 28398719.0.0000.5505, in compliance with the principles of the Declaration of Helsinki. The parents and guardians agreed to participate and signed the Informed Consent Form (Fig. 1).

Fig. 1

Flowchart for recruiting study participants

Data collection

Data collection consisted of 3 stages: 1st) socioeconomic data, birth history and clinical conditions; 2nd) anthropometric assessment and food intake; and 3rd) biochemical variables.

Socio-economic data, birth history and medical conditions

A structured sociodemographic questionnaire collected information on the family's socioeconomic data, pregnancy and childbirth conditions, and questions related to the children's dietary and morbid history. The questionnaire was developed for this study and is available as a supplementary file.

This study used the Criteria of Economic Classification Brazil (CCEB), established by the Brazilian Business and Research Association, to determine the economic status of the sample family nucleus, which demonstrates the following classifications: A1, B1, B2, C1, C2, and DE [14]. The classes are defined by the CCEB, according to the average family income, being: A1 (USD 4.550,13), B1 (USD 2.152,45), B2 (USD 1.190,94), C1 (USD 675,49), C2 (USD 407,81) and DE (USD 184,60). The interviewee should answer questions about property ownership, family income, and the education level of the head of the household.

The Brazilian Household Food Insecurity Measurement Scale (EBIA) was also applied, as proposed by Kepple and Segall [15]. The EBIA, with 14 items, aims to measure the dimensions of access, availability and use of food and can classify the family into the following categories: 1) food security, when the family/household has regular and permanent access to good quality food in sufficient quantity; 2) mild food insecurity (FI), when there is concern or uncertainty about access to food in the future and inadequate food quality resulting from behavior that aims not to compromise the quantity of food; 3) moderate FI, when there is quantitative reduction in food among adults and/or disruption in eating patterns resulting from lack of food among adults; and 4) severe FI, when there is also quantitative reduction in food among those aged under 18 y, implying a disruption in eating patterns resulting from lack of food among all residents.

Anthropometric assessment

The researchers collected weight data using a pediatric scale (for children under two), a platform scale (for children over two), and height data utilising a child's stadiometer and a vertical anthropometer. The data was collected in an appropriate and comfortable place, with a flat floor and smooth walls, without skirting and with sufficient light, as recommended by the Ministry of Health [16].

Subsequently, using the WHO Anthro Software®, the anthropometric indices BMI for Age (BMI/A) and Height for Age (H/A) were calculated, expressed as z-scores, and classified according to the cut-off points proposed by the World Health Organization (WHO) [17].

Food intake

Food consumption of children aged 0–23 months of age was assessed according to indicators proposed by the United Nations Children's Fund (UNICEF) and the World Health Organization, based on the document "Indicators for Assessing Infant and Young Child Feeding Practices: definitions and Measurement methods" [18]. The indicators used cover the sample's dietary practices, considering questions related to the day before the survey, practices related to breastfeeding, complementary feeding, dietary diversity (intake of foods and beverages from at least five of the eight food groups—breast milk; grains, roots, tubers and plantains; pulses (beans, peas, lentils), nuts and seeds; dairy products (milk, infant formula, yogurt, cheese); flesh foods (meat, fish, poultry, organ meats); eggs; vitamin-A rich fruits and vegetables; and other fruits and vegetables—defined during the previous day), protein intake (consumption of foods with eggs and/or meat in the last day), healthy foods (consumption of the eight food groups mentioned), unhealthy foods (consumption of selected unhealthy foods during the previous day, such as candies, chocolate and other sugar confections, frozen treats like ice cream, gelato, sherbet, sorbet, popsicles or similar confections, chips, crisps, cheese puffs, French fries, fried dough, instant noodles, etc.) and consumption of sugary drinks (consumption of at least one sweet drink the day before, such as soda pop, fruit-flavored drinks, sports drinks, chocolate and other flavored milk drinks, malt drinks, etc.). Dietary patterns were assessed based on the time the child spent at daycare, the food provided and consumed at daycare, and the food consumed at home after returning home.

Assessment of biochemical markers

The parents/guardians took the children to the laboratory on a previously scheduled day and 10 mL of blood was collected by peripheral venipuncture using a vacuum system of children and following the good practices recommended for clinical laboratories.

The samples were processed to obtain serum/plasma by an accredited laboratory (Laboratório Gold LTDA), as well as the blood count and iron, ferritin, High-sensitivity C-reactive protein (hs-CRP) and the Transferrin Saturation Index (TSI). The Soluble Transferrin Receptor (sTfR), Alpha-1 Acid Glycoprotein and hepcidin tests were carried out at the Clinical Analysis Laboratory of the Centro Universitário FMABC (FMABC), Santo André (SP). The samples were safely stored in a freezer at −20ºC and they were transported in an isothermal, rigid, waterproof box, internally lined with smooth material and hermetically sealed at an appropriate temperature, and, after arriving at the laboratory, were kept frozen (−20 °C).

Iron status was assessed using a blood count with blood extension analysis (Impedance method and Optical Microscopy with May Grunwald-Giemsa staining and 1000 × magnification/ Equipment ABX Micros 60 with Horiba reagents), serum iron (colorimetric methods/Equipment AU-DXC, IRON and UIBC reagents (Beckman Coulter)), ferritin (chemiluminescence/Equipment AU-DXI 800, Access Ferritin reagent (Beckman Coulter)) and the calculation of the transferrin saturation index, sTfR (nephelometry methods/ETIMAX Equipment: Diasorin) and hepcidin (electrochemiluminescence/Cobas 8000 Equipment—Roche). The acute phase proteins hsCRP (immunoturbidimetry/AU-DXC Equipment, CRP Latex reagent (Beckman Coulter)) and Alpha-1 Acid Glycoprotein (ELISA/ETIMAX Equipment: Diasorin) were also assessed.

Anemia was considered when hemoglobin < 10.9 g/dL for children under 24 months; and hemoglobin < 11.4 g/dL for children over 24 months. Adjustments in hemoglobin concentrations were made, considering the effect of elevation (altitude) of the place of residence on hemoglobin concentrations [19]. Iron status was classified as: a) normal (normal hemoglobin and normal ferritin and sTfR levels); b) iron deficiency without anemia -ID- (normal hemoglobin and ferritin < 30.0 ng/mL (if CRP > 5 mg/L) or Ferritin < 12.0 ng/mL (if CRP < 5 mg/L), and/or high sTfR, > 1.50 mg/L); c) anemia from iron deficiency (both low hemoglobin and ID); and d) non-iron deficiency anemia (low hemoglobin without ID). Cut-off points for ferritin were < 30.0 ng/mL (if CRP > 5 mg/L) and ferritin < 12.0 ng/mL (if CRP < 5 mg/L) [20]. A sTfR of 1.5 mg/L has been used as the cutoff for ID in numerous studies and was taken in the current study [21].

Statistical analysis

The data was entered and consolidated in an Excel® spreadsheet (Office 365). The analysis was carried out using the Statistical Package for the Social Sciences-SPSS 29.0 statistical package (IBM Corp., Armonk, NY, USA). Continuous variables were tested for normality using the Shapiro–Wilk test and kurtosis values. Those with a parametric distribution were presented as mean and standard deviation, while those with a non-parametric distribution were shown as median and interquartile range. The t-Student test was used to compare variables with a normal distribution and the Mann–Whitney test for those without a normal distribution. Categorical and binary variables were presented as absolute numbers and percentages and compared using the Chi-square test.

The ROC (Receiver Operating Characteristic) curve [22] was used for the discriminatory analysis between the variables studied and anemia. Variables with a p-value < 0.10 in the bivariate analysis were included in the ROC curve.

Finally, to study the association between the biochemical marker results, a correlation analysis was carried out using the Spearman method, considering the absolute values of the variables hemoglobin, RDW, ferritin, serum iron, TSI, sTfR, and the biomarkers of Inflammation (hsCRP and Alpha-1-Acid Glycoprotein). A 5% significance level was adopted.

Results

Table 1 shows the general characteristics of the children included in the study. The average age was 28.1 ± 7.9 months, and 50 (44.6%) were female. Regarding socio-economic characteristics, 25 (22.9%), 60 (53.6%) and 59 (52.6%) of the families received the "Auxílio Brasil" [today, called the Bolsa Família Program] cash transfer program, had a CCEB classification > B2 and were classified as having some degree of food insecurity, respectively. Almost a fifth of the mothers had schooling compatible with primary education (less than 9 years). The median duration of exclusive and total breastfeeding was 6 months (4.0, 6.0) and 10 months (5.5, 14.0), respectively. The introduction of complementary foods from the age of 6 months and iron supplementation in the first two years of life occurred in 74 (66.1%) and 89 (83.2%) of the children (Table 1).

Table 1 Characterization of the variables studied in children enrolled in Municipal Early Childhood Education Centres (n = 112), a municipality south of Minas Gerais, Brazil, 2024

Only 15 (14.4%) of the children were breastfeeding during the assessment. The feeding indicators were more than 70% adequate for frequency, minimum diet, and egg/meat consumption. Diversity, frequency of milk consumption, sugary drinks, and unhealthy foods were between 50 and 70% (Table 1). Overweight and short stature were observed in 39 (36.1%) and 27 (25.0%) of children, respectively.

Anemia and iron deficiency were observed in 58 (51.8%) and 89 (79.5%) of the children (Table 1), with no difference with age (children under and over 2 years old—anemia: 18 (46.2%) vs 40 (54.8%); p = 0.431 and iron deficiency: 31 (79.5%) vs 58 (78.5%); p = 0.600). Among the anemic children, 47 (81.0%) had iron deficiency and 20 (34.5%) had microcytosis and hypochromia. In terms of inflammation markers, 29 children (25.9%) had high hs-CRP values (> 5.0 mg/L), and 80 children (71.4%) had high Alpha-1 Acid Glycoprotein values (> 1.0 g/L).

In the population evaluated, 23 (20.5%) of the children had normal hemoglobin, ferritin and sTfR values; 27 (24.2%) had iron deficiency; 38 (33.9%) had iron deficiency with anemia and 24 (21.4%) had anemia without iron deficiency.

Table 2 presents a comparison of the characteristics of children evaluated with and without anemia. The z-score values of BMI (0.33 ± 1.24 vs 0.79 ± 1.12; p = 0.023) and H/A (−1.34 ± 1.08 vs −0.94 ± 1.28; p = 0.044) were lower, and hs-CRP levels were higher [2.39 mg/L (0.68, 14.5) vs 1.25 (0.20, 3.41); p = 0.008] in the group with low hemoglobin levels (Table 2). Linear regression showed that hemoglobin values were directly associated with score H/A (β crude = 0.421; 95% CI 0.145 to 0.689; p = 0.003), even after adjusting for log hsCRP values (β adjusted = 0.375; 95% CI 0.088 to 0.662; p = 0.011) (Fig. 2). In turn, the association between hemoglobin and BMIZ was not maintained after adjustment for hs-CRP (β adjusted = 0.043; CI 95% −0.101 to 0.187; p = 0.553).

Table 2 Comparison of children assessed with and without anemia, a municipality in southern Minas Gerais, Brazil, 2024

Fig. 2

Association between hemoglobin levels and height-for-age Z score, adjusted for high-sensitivity C-reactive protein values

The group with anemia had lower hemoglobin values (10.4 ± 0.64 g/dL vs. 11.7 ± 0.57 g/dL; p < 0.001), red blood cells (4.23 ± 0.34 mm³ vs. 4.60 ± 0.27 mm³; p < 0.001), MCHC (31.1 ± 0.86 g/dL vs. 31.8 ± 0.87 g/dL; p < 0.001) and hematocrit (33.4 ± 1.9% vs. 37.1 ± 1.9%; p < 0.001); as well as higher levels of RDW (14.7 ± 1.37% vs. 13.8 ± 0.97%; p < 0.001) (Table 2).

The ROC curve shows that RDW (AUC = 0.708; 95% CI 0.612 to 0.803) and sTfR (AUC = 0.588; 95% CI 0.481 to 0.694) were the variables that showed the best discrimination between children with and without anemia (Fig. 3).

Fig. 3

ROC curve of variables associated with anemia

The hsCRP correlated directly with RDW (r = 0.202; p = 0.033), ferritin (r = 0.425; p < 0.001) and sTfR (r = 0.446; p < 0.001), and inversely with hemoglobin (r = −0.287; p = 0.002), serum iron (r = −0.580; p < 0.001) and transferrin saturation index (r = −0.528; p < 0.001). Alpha-1 Acid Glycoprotein correlated inversely with hemoglobin (r = −0.201; p = 0.034), iron (r = −0.432; p < 0.001) and the transferrin saturation index (r = −0.371; p < 0.001), and directly with ferritin (r = 0.375; p < 0.001) and sTfR (r = 0.389; p < 0.001) (Fig. 4).

Fig. 4

Correlation between inflammation biomarkers (High-sensitivity C-reactive protein and Alpha-1 Acid Glycoprotein) with hemoglobin, ferritin, serum iron, transferrin saturation index, soluble transferrin receptor and RDW

A supplementary table was created to demonstrate the prevalence of anemia and iron deficiency after excluding children with increased hs-CRP (Supplementary table).

Discussion

The prevalence of anemia in this study was higher than that reported in the Brazilian National Survey on Child Nutrition (ENANI-2019), which found 19% in the 6–23 months of age group. Globally, it is estimated that 40% of all children between 6 and 59 months are affected by anemia [2]. The high prevalence of anemia and iron deficiency was surprising, given that the preschoolers evaluated attend full-time daycare centers in the Municipal Education Network. This environment promotes care and health, and 83.2% of the mothers reported administering iron medication in prophylactic doses.

It should be noted that the data collection took place immediately after the children returned to the nursery after a long absence due to the SARS-CoV-2 pandemic. A recent study showed the impact of the pandemic on the food insecurity and nutritional risk of children living in developing countries such as Brazil, demonstrating the increase in the prevalence of obesity during the COVID-19 pandemic and, paradoxically, the worsening of nutritional status, with an increase in cases of wasting and stunting [23]. In addition, the global COVID-19 pandemic resulted in worsening access to food for many low- and middle-income countries, a situation that is detrimental to children under 5 years of age, increasing the problem of food and nutritional insecurity for the entire population, and consequently, the consumption of ultra-processed foods and the reduction of breastfeeding practices. In Brazil, food and nutritional insecurity doubled in families with children under 10 years of age – from 9.4% in 2020 to 18.1% in 2022 [24].

During the period of social isolation caused by the COVID-19 pandemic, dietary practices were negatively affected, with an increase in the consumption of ultra-processed foods, sausages and fast foods, as opposed to a decrease in the consumption of fruit, vegetables and greens. These changes in eating habits have particularly affected children, given their absence from face-to-face school activities, as well as the imbalance in household food consumption, which has had repercussions on various nutritional disorders, such as excess weight, risk of short stature for chronological age and dietary deficiencies, such as iron deficiency anemia [25, 26].

Anemia, a complex and multi-causal disease, requires multi-professional care and intersectoral actions to prevent its worsening and possible complications in growth and neurodevelopment. In countries with emerging economies, such as Brazil, anemia can have significant economic consequences through treatment, tests, and hospitalizations, generating financial deficits for the state, favoring the occurrence of other nutritional deficiencies, and aggravating Food and Nutrition Insecurity (FNI) [27].

The dietary indicators surveyed in this study were satisfactory, with adequacy above 70% for frequency, minimum diet and egg and meat consumption. However, around half of the families had some degree of FNI. No associations were found between food consumption indicators and the prevalence of anemia in this sample. With the same method used to measure dietary practices, the study by Zou et al. [28], carried out with children aged 6 to 23 months in central-southern China, showed that a diversified diet made up of different food groups, especially fresh foods, reduced the risk of anemia by 45%.

Among the various determining factors of anemia and its physiological, environmental and social circumstances, inflammation and its alterations in iron metabolism stand out as critical clinical determinants. The immune-mediated inflammatory response, through the induction of pro-inflammatory cytokines, can interfere with different pathways of erythropoiesis, leading to anemia. The overexpression of these inflammatory markers alters iron hemostasis, as they induce the overexpression of hepcidin, resulting in reduced iron bioavailability and erythropoiesis [29, 30].

Using the ROC curve, it was found that the RDW and sTfR values showed the best discrimination between children with and without anemia. RDW can help distinguish iron deficiency from other causes, such as inflammation, and is a practical and routine hematological index used in differential diagnoses of variations in the size and shape of erythrocytes and other hemoglobinopathies [31, 32]. No child with any inflammatory process was excluded from the ROC curve.

The sTfR, in turn, has been characterized as an essential marker in distinguishing between iron deficiency anemia and anemia of inflammation and is a parameter used to assess iron status in the pediatric population. Its concentrations increase in parallel with the decrease in cellular iron levels and may increase slightly during the inflammatory response [33]. Thus, the fact that sTfR shows a good level of discrimination between children with and without anemia suggests the efficacy of this marker in diagnosing iron deficiency anemia [34]. RDW represents the presence of anisocytosis or variation in red blood cell size, which can occur for various reasons, from anemia to chronic diseases. sTfR is characterized as the best biomarker for identifying iron deficiency anemia resulting from the presence of inflammation [35].

We found a correlation between positive acute phase reactant proteins, such as hs-CRP and Alpha-1 Acid Glycoprotein, and biomarkers of iron-related nutritional status. Although the anemia of inflammation is like an iron deficiency in terms of some biomarkers such as serum iron, it occurs as a disturbance in iron distribution since the macrophages in the liver, spleen and bone marrow responsible for recycling old red blood cells retain their iron stores in the presence of inflammation. A study of 1,375 Cuban preschoolers showed that a third had at least one elevated inflammation biomarker that interfered with the prevalence of anemia [36].

Alpha-1 Acid Glycoprotein showed a direct correlation with sTfR and an inverse correlation with hemoglobin, serum iron and TSI. sTfR is the soluble form of the transferrin receptor, a protein that plays a crucial role in iron uptake by cells. Its concentration in the blood reflects the cellular demand for iron and is higher in conditions of iron deficiency [37]. Elevated sTfR concentrations are often associated with iron deficiency anemia. When there is inadequate iron availability, cells increase the expression of sTfR to capture more iron in circulation. This adaptive response attempts to compensate for iron deficiency and restore normal hemoglobin levels [38, 39].

The high prevalence of iron deficiency found in this study, associated with elevated Alpha-1 Acid Glycoprotein, demonstrates the importance of using acute phase proteins that act as markers of inflammation. Alpha-1 Acid Glycoprotein is considered the most reliable indicator of chronic inflammation and may take several days to reach peak levels after the onset of inflammation. Therefore, because it produces a more accurate estimate of the prevalence of inflammation, it should be assessed whenever possible [40]. The high prevalence of inflammation can be explained by the fact that data collection occurred shortly after the period of social isolation caused by the COVID-19 pandemic, leading to an increase in the consumption of ultra-processed foods among children [41, 42].

It has already been well reported in the literature that ferritin correlates directly with inflammatory markers, as we have seen, being, on the one hand, a promoter and, on the other, a regulator of inflammation. The positive link between IL-1β, IL-6, IFN-y, TNF-α, hs-CRP and ferritin shows that inflammatory markers can induce their expression. It has been demonstrated that TNF-α and IL-1β work in synergy with IL-6, raising ferritin concentration. This cascade of events promotes iron retention in the liver and macrophages, resulting in anemia. In 2020, a WHO publication proposed that iron deficiency based on ferritin concentrations should be assessed in conjunction with evaluating positive acute phase reactant proteins, such as hsCRP and Alpha-1 Acid Glycoprotein [19, 43, 44].

Anemia and iron deficiency can compromise children's growth and neuropsychomotor development. Low hemoglobin levels can be associated with changes in Growth Hormone (GH) secretion, caused by inhibition of its protein synthesis, and thus its low sensitivity to the production of IGF-1 (insulin-like growth factor type 1), which can interact negatively with the GH/IGF-1 axis and compromise nutritional status [45,46,47].

As pointed out in this study, in addition to hs-CRP, RDW was significantly associated with anemia, reflecting the involvement of iron. The assessment of changes in erythrocyte biology, provided by the degree of anisocytosis of RDW, shows the morpho structural changes resulting from nutritional deficiencies and the inflammatory process. In addition, high RDW values represent an important prognostic biomarker for short- and long-term diseases resulting from dysregulation of erythrocyte homeostasis, triggering metabolic imbalances such as increased oxidative stress and Inflammation [48,49,50,51].

Hepcidin is the leading systemic regulator of iron homeostasis, coordinating the use and storage of this mineral in the body. Its determination can be used for different anemia diagnoses, such as iron deficiency anemia, complementing the most commonly used indicators of total body iron reserves, such as iron and ferritin [52]. Thus, hepcidin levels are low in absolute iron deficiency and iron deficiency anemia and are seen as a promising tool to be included in the current battery of diagnostic tests for iron status in the population [53].

Even considering that serum hepcidin levels are considerably lower in patients with iron deficiency compared to healthy individuals, no significant difference was found between the groups in this study, as seen in other studies and its association with inflammation [54].

One of the limitations of this study is the high frequency of children with increased hsCRP and Alpha-1 Acid Glycoprotein; thus, no children who presented any inflammatory processes were excluded. The assessment of iron status continues to represent a challenge since the main biomarkers do not have specific cutoff points for age and sex. In addition, their concentration is affected by inflammation. Thus, to perform the classifications in this study, the authors followed the recommendations of the World Health Organization in its supporting documents and other studies present in the literature. Given the complexities of iron metabolism, a complete understanding of the nuances of validated diagnostic tool levels is necessary for them to be effective and practical in the clinical and epidemiological environment [55].

This study found anemia and iron deficiency in 51.8% and 79.5% of the children evaluated. A direct and significant association existed between hemoglobin concentrations and the height-for-age Z score, even after adjusting for hsCRP values. The dietary practices assessed were not associated with the presence of anemia. RDW and sTfR showed the best discriminatory power between the groups with and without anemia.

The prevalence of anemia was found to be higher than in national studies. There was a correlation between inflammatory markers and biomarkers of iron nutritional status, pointing to the importance of nutritional, dietary and child health care and childcare actions, indicating the importance of prior diagnosis of anemia in the child population, and the detection of the main risk factors that can increase its prevalence, including inflammation. Public policies aimed at various interventions to improve children's health and nutrition should be enhanced, creating a care network for the nutritional problems affecting this population and enabling strategies for early diagnosis, intervention and clinical, dietary and nutritional management.

Consistent with the literature, the results presented in this study indicate the importance of prior diagnosis of anemia in the child population, as well as the detection of the main risk factors that can increase its prevalence, including inflammation and reinforces the recommendations of the WHO, emphasizing the global evidence-based recommendations on the use of indicators to assess iron status in the population and the joint assessment with indicators of iron status and acute phase proteins. However, it has some limitations, such as a cross-sectional model, which does not allow us to establish temporality between the outcome investigated, and a cause-effect relationship for the associations observed. It was also carried out in the post-COVID-19 period, leading to possible biases, especially among the inflammation markers assessed, with deleterious effects that are still unknown.