Nutritional anemia is a significant public health challenge in India, particularly among the pediatric population. An estimated 58.5% of children under five years are affected by the condition, with infants and toddlers being particularly vulnerable due to their rapid growth and high nutritional requirements.[1] Anemia in this age group is often due to inadequate intake or poor absorption of essential nutrients, such as iron, vitamin B12, and folic acid, which are crucial for hemoglobin synthesis and oxygen transport in the body. Iron deficiency is the most common cause, accounting for nearly 50% of anemia cases worldwide.[2]

Factors such as poverty, malnutrition, poor dietary practices, and frequent infections can impair nutrient absorption and increase the body's iron requirements. Parasitic infections, such as hookworm, can also contribute to anemia by causing chronic blood loss and reducing iron stores in the body. Socioeconomic status, maternal education, diet diversity, and frequent infections can predict the occurrence and severity of nutritional anemia in pediatric populations.

Despite ongoing public health efforts, the prevalence of nutritional anemia in India has remained persistently high, with only marginal improvements in anemia rates reported compared to previous surveys. The Government of India has implemented several programs aimed at reducing anemia, such as the Anemia Mukt Bharat (AMB) initiative, which focuses on improving iron and folic acid supplementation, promoting dietary diversity, and enhancing deworming and infection control measures.[3]

Recent research has highlighted the potential role of novel biomarkers, such as serum hepcidin, in improving the diagnosis and management of anemia. Elevated serum homocysteine levels could indicate an increased risk of developing nutritional anemia in children, especially in low-resource settings where dietary deficiencies are common. Aims of the study to check the levels of homocysteine in moderate to severe cases of nutritional anemia in age group of 3 months to 2 years.

Study setting: The study was conducted at the outpatient as well as the in-patient ward of the department of Pediatrics of GMERS Medical College & Hospital, Gotri, Vadodara. Study Duration: The study was conducted for 12 months, March 2024 to February 2025.

Study type and design: The current study was an institution-based observational study with a cross-sectional design.

Study sample and grouping: The study population consisted of all inborn or out born children in age group of three months to two years having either symptomatic or asymptomatic but moderate to severe nutritional anemia.

Inclusion criteria:

The inclusion criteria for the study population were:

• Children (3m 2yrs) having moderate to severe nutritional anemia.
• Children of either sex.

Exclusion criteria:

The exclusion criteria for the participants that were considered for the current study were as follows:

• Children having anemia other than nutritional anemia.
• Patients on vitamin supplementation or recent blood transfusion (within the past 3 months).
• Children with known metabolic or genetic disorders.
• Children whose parents did not provide written informed consent.

Outcome variables:

The primary outcome was the homocysteine level in moderate and severe nutritional anemia cases. The secondary outcomes included correlations between homocysteine levels and other variables such as hemoglobin, serum ferritin, vitamin B12, and folate levels.

Sample size and sampling technique:

The sample size was calculated using the Cochran’s formula for the calculation of sample sizes based on an anticipated proportion of outcome in the study sample, which, in the present study, was considered to be the proportion of patients having hyper homocysteinemia in moderate to severe anemia. Based on the data reported by Paksoy et al.[4] the anticipated proportion of the outcome was considered as 65%, and at 95% confidence level and 10% absolute allowable error, the sample size ―n‖ was calculated using the formula:

n = [Z 2 X p X (1-p)] / d2

Where, p is the anticipated proportion d is the allowable error and Z is the critical value at 1-α level (95%) of confidence .The calculated sample size was 98.9, which was rounded off to 100 patients.

Data management and analysis:

The collected data were checked for consistency, completeness and entered into Microsoft Excel (MS-EXCEL, Microsoft Corp.) data sheet. Analyzed with the statistical program Statistical Package for the Social Sciences (IBM SPSS, version 22). Data was organized and presented using the principles of descriptive and inferential statistics. The data was categorized and expressed in proportions. The continuous data was expressed as mean ± SD. The data was graphically presented in the form of vertical bars, horizontal bar, pie diagram. Student’s ttest and ANOVA were used for continuous data; Chi-square and Cochran’s Q tests were done for categorical data.

Where analytical statistics were performed, a p-value of <0.05 was considered to be statistically significant for the purpose of the study.

Ethical consideration: The Institutional Ethics Committee of GMERS, Gotri, reviewed and approved the project before it was carried out. All of the participants were informed in their own language about the study and their rights for participation before providing data for the researcher-administered questionnaire. They were informed about the participant’s role and rights, to clarify that their participation was voluntary, the information was treated confidentially, and they could withdraw from the study at any time. After the collection of data, the data was cleaned, anonymized and stored in a password protected spreadsheet for data analysis.

Table 1: Distribution of Demographic data

Table 2: Distribution of study participants according to their general examination findings on admission (n=100)

Table 3: Distribution of study participants according to their anemia related findings on admission (n=100)

Table 4: Distribution of study participants according to their laboratory examination findings on admission (n=100)

Figure1: Distribution of study participants according to type of anemia (n=100)

The age distribution of study participants revealed that the majority (51%) were between 7-12 months of age, followed by 39% in the 13-24 months category. A smaller proportion about 10% comprised infants aged 3-6 months. Among the study participants, 53% were female and 47% male. The majority of participants about 69% resided in urban areas, whereas 31% came from rural settings. General examination findings on admission revealed a mean heart rate of 81.1 ± 2.9 bpm, a respiratory rate of 21.9 ± 6.6 breaths per minute, a systolic blood pressure of 98.9 ± 4.3 mmHg, a diastolic blood pressure of 61 ± 7.1 mmHg and a body temperature of 97.7 ± 0.5°F. The mean serum B12 level was 269.9 ± 101.6 pg/mL, ferritin level was 16.4 ± 5.7 mcg/mL, and serum homocysteine level was 61.6 ± 38.5 μmol/L. Laboratory findings revealed a mean hemoglobin level of 6.4 ± 1.2 g/dL, hematocrit of 36.1 ± 4.7%, mean corpuscular volume (MCV) of 78.5 ± 6.9 fl, platelet count of 3.5 ± 1.5 lakhs/cumm, and total leukocyte count (TLC) of 1.3 ± 0.7 ×10³/cc.  The most prevalent anemia type was microcytic normochromic (82%), followed by macrocytic hypochromic anemia (26%). Serum B12 levels were significantly lower in macrocytic hypochromic anemia cases (158 ± 56.1 pg/mL) compared to microcytic normochromic anemia cases (298.6 ± 88.9 pg/mL, p<0.001). Similarly, serum homocysteine levels were significantly higher in macrocytic hypochromic anemia (98.2 ± 33.4 μmol/L) compared to microcytic normochromic anemia (53.9 ± 35.7 μmol/L, p<0.001). Ferritin levels did not show a significant difference between the two groups.

The present study aimed to assess homocysteine levels in children aged 3 months to 2 years with moderate to severe nutritional anemia and to determine the prevalence of hyperhomocysteinemia. The majority of participants were aged 7–12 months (51%), followed by 13–24 months (39%) and 3–6 months (10%), aligning with national data highlighting the burden of anemia in infants due to rapid growth and dietary inadequacies (Thankachan et al., 2012) [5]. The sex distribution was nearly equal (53% females, 47% males), consistent with previous studies, although some suggest male infants may be more vulnerable to iron deficiency due to higher growth velocity (Rogers et al., 2003) [6]. Most participants (69%) were from urban areas, reflective of the tertiary care hospital setting, though rural children are often at greater risk due to limited healthcare access and poor dietary diversity (Paksoy et al., 2024) [4]. Socioeconomic status and maternal education are also known to influence anemia prevalence, as noted by Coban et al. (2018) [7].

Vital signs were within normal limits, with a mean heart rate of 81.1 bpm, respiratory rate of 21.9 breaths/min, systolic BP of 98.9 mmHg, diastolic BP of 61 mmHg, and temperature of 97.7°F. These findings suggest hemodynamic stability, consistent with the notion that moderate anemia may not always lead to circulatory compromise (Paksoy et al., 2024) [4]. Laboratory data revealed a mean hemoglobin level of 6.4 g/dL, indicating moderate to severe anemia, with a hematocrit level of 36.1%, mean corpuscular volume (MCV) of 78.5 fL, total leukocyte count of 1.3 × 10³/cc, and platelet count of 3.5 lakhs/cumm. These findings suggest microcytic anemia primarily due to nutritional deficiency, rather than underlying hematologic disorders, in line with previous studies (Verma et al., 2017 [8]; Thankachan et al., 2012 [5]).

A high mean serum homocysteine level of 61.6 μmol/L was observed, indicating a significant prevalence of hyperhomocysteinemia. Serum homocysteine levels showed a strong inverse correlation with both vitamin B12 (r = -0.620, p < 0.001) and ferritin (r = -0.543, p < 0.001), highlighting the role of these micronutrient deficiencies in homocysteine metabolism. This supports existing evidence that vitamin B12 and iron deficiencies contribute to elevated homocysteine levels through impaired methylation processes (Ueland et al., 2003 [9]; Rogers et al., 2003 [6]; Ozkale et al., 2015 [10]). Notably, children with macrocytic hypochromic anemia had significantly higher homocysteine levels (98.2 ± 33.4 μmol/L) than those with microcytic normochromic anemia (53.9 ± 35.7 μmol/L, p < 0.001), emphasizing the impact of vitamin B12 deficiency (Ambroszkiewicz et al., 2006) [11]; (Kerr et al., 2009) [12].

The inverse correlation between homocysteine and ferritin levels suggests that iron deficiency may further aggravate hyperhomocysteinemia, even though iron is not directly involved in homocysteine metabolism (Ljungbald et al., 2022) [13]; (Paksoy et al., 2024) [4]. Maternal vitamin B12 status also influences neonatal homocysteine levels, and deficiencies can lead to early-life metabolic disturbances (Coban et al., 2018 [7]; Lipari Pinto et al., 2022 [14]). The prevalence of hyperhomocysteinemia (65%) in this study is in line with previous findings, reinforcing the need for early detection and intervention strategies (Paksoy et al., 2024 [4]; Rozmaric et al., 2020 [15]; Likhita et al., 2024 [16]).

These results underscore the importance of comprehensive nutritional interventions. While current public health programs emphasize iron supplementation, this study suggests the addition of vitamin B12 to effectively manage pediatric anemia. Prior research has shown that iron-fortified diets improve hemoglobin but not vitamin B12 or homocysteine levels, indicating the need for multi-micronutrient strategies (Thankachan et al., 2012 [5]; Verma et al., 2017 [8]).

Clinically, these findings support routine screening for vitamin B12 in anemic children, especially those with macrocytic anemia. Improving maternal nutrition through dietary modification or supplementation could help prevent hyperhomocysteinemia in infants (Lipari Pinto et al., 2022) [14]. The evidence suggests that addressing both infant and maternal B12 deficiencies is critical to reducing the burden of pediatric anemia and its metabolic complications (Coban et al., 2018 [7]; Lipari Pinto et al., 2022 [14]).

This study highlights a high prevalence of hyper homocysteinemia in children with moderate to severe nutritional anemia. Serum homocysteine levels exhibited a significant inverse correlation with vitamin B12, suggesting that vitamin B12 deficiency plays a central role in its elevation. These findings underscore the need for routine homocysteine screening in pediatric anemia management and reinforce the importance of vitamin B12 supplementation alongside iron in public health interventions. Early identification and treatment of hyper homocysteinemia may prevent neurodevelopmental consequences and improve anemia outcomes in young children at risk of nutritional deficiencies.

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