Association of folic acid and vitamin B12 deficiency in children with iron deficiency anaemia

Objective: To find out the association of folic acid and vitamin B12 deficiency in patients with iron deficiency anaemia in Indian children admitted to a tertiary hospital. Design: Observational cross sectional study. Setting: Tertiary-level hospital from October 2010-September 2011, for a period of one year. Participants: All children aged between 6 months to 13 years, admitted in paediatric ward of this hospital for any complaints, were evaluated for anaemia. All patients with Hemoglobin levels less than the WHO cut off levels for anaemia were included in the study. Those who were sick and those who were on drugs, other conditions causing bone marrow suppression were excluded from the study. Methods: Serum iron, serum iron binding capacity, serum ferritin, serum vitamin B12, and serum folic acid analyses were conducted with autoanalyzers using commercial kits. Results: Out of 975 children who were admitted during the above period, 100 (34 females and 66 males) anemic children were included in the study. 2 patients were excluded as one child was diagnosed with Thalassemia minor and another patient was diagnosed as acute lymphoblastic Leukemia. Hence 98 children were taken up for study. Iron and Folic acid Deficiency observed in 6.12 % (6/98), Iron and vitamin B12 Deficiency in 3.06 % (3/98), Folic acid and vitamin B12 Deficiency in 1.02 % (1/98), Iron, Folic acid and vitamin B12 Deficiency in 1.02 % (1/98) of patients. Conclusions: Iron deficiency anaemia coexist with vitamin B12 and folate deficiency, but their association with iron deficiency anemia was not significant in our study.


Introduction
Anaemia is a major public health problem all over the world especially in developing countries. The third National Family Health Survey (NFHS -3)  found that the prevalence of anaemia among under 5 children approaches 70% even though there is a national programme to control anaemia for many years [1]. The irreparable damage that anemia in childhood can cause particularly to the development of a young child on one hand and the knowledge and mechanism available for its control on the other, makes this silent morbidity completely unacceptable in modern times where we strive for millennium development Goal 4 [2]. Out of all the causes, nutritional anaemias account for the major cause of anaemias. Pregnant women, infants, young children and adolescents are at a higher risk of nutritional anaemia as they have a high demand of nutrients such as iron, folic acid, vitamin B 12 and other nutrients. Nutritional anaemia is seen in almost one billion people all over the world and commonest being iron deficiency anemia [3]. Anaemia in children results in impaired cognitive performance, behavioral and language development and scholastic achievement. Anaemia is also associated with increased mortality and morbidity from infectious diseases [4]. Other causes of anemia include chronic infections, particularly malaria, hereditary hemoglobinopathies and folic acid deficiency. It is worth noting that multiple causes of anemia can coexist in an individual or in a population and contribute to the severity of the anemia.
The term 'nutritional anemia' encompasses all pathological conditions in which the blood hemoglobin concentration drops to an abnormally low level, due to a deficiency in one or several nutrients. The main nutrients involved in the synthesis of hemoglobin are iron, folic acid, and vitamin B 12 . In public health terms, iron deficiency is by far the first cause of nutritional anemia worldwide. Folic acid deficiency is less widespread and is often observed with iron deficiency. Vitamin B 12 deficiency is far rarer and it occurs mainly in vegetarians [5]. There are various studies done to find out the prevalence of iron deficiency anaemias in paediatric population in India and other developing countries [6,7,8,9]. But there is paucity of studies using laboratory measurements to find out the exact cause of nutritional anaemia and to find out the association of folic acid and vitamin B 12 deficiency in patients with iron deficiency anaemia in Indian population [10]. Hence this study was done with the objective to study the association of folic acid and vitamin B 12 deficiency in patients with iron deficiency anaemia in Indian children admitted to a tertiary hospital.

Materials and Methods
This study is an observational cross sectional study, conducted in paediatric ward of Vydehi Institute of Medical Sciences and Research Centre, a tertiary care hospital in southern India from October 2010-September 2011 for a period of one year. Informed consent was taken from the care takers of the patients for this study. Permission was taken from the Ethics committee of the hospital for this study. All patients admitted in paediatric ward of this hospital for any complaints were evaluated for anaemia. Those patients who were less than 6 months of age, sick patients and those patients who were on drugs, other conditions causing bone marrow suppression were excluded from the study. All other patients with Hemoglobin levels less than the WHO cut off levels for anaemia were included in the study. WHO Expert group proposed that anaemia should be considered to exist when Hemoglobin is below the following levels in venous blood. 6 months to 6 years 11 gm / dl, less than 12 g/dL for girls from 6 to 18 yrs and boys from 6 to 14 years, and less than 13 g/dl for boys from 15 to 18 yrs of age [11].
In those patients found to have anaemia, the following laboratory investigations were done like mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), serum ferritin, serum iron, total iron binding capacity (TIBC), serum transferrin (TRF), serum folate assay and serum vitamin B 12 assay. 5 ml of blood was collected in a EDTA tube for the study. Serum hemoglobin was estimated by spectrophotometric method by automated machine, serum ferritin by Chemiluminescent immunoassay, serum iron by Ferrozine_no deproteinization, TIBC by ion exchange resin ferrozine method, serum transferrin (TRF) by rate nephelometry, serum folate assay and vitamin B 12 assay by Chemiluminescent immunoassay. In case they were suffering from acute infective or inflammatory conditions [12] the above investigations were done after 3 afebrile days. The normal values for various study parameters in this study were considered from Nelson Text Book of Pediatrics [13].

Results
100 patients with anaemia as defined by WHO, admitted in the paediatric ward from the period from October 2010-September 2011, were included in the study. The age of the patients ranged from 6 months to 13 years (Table 1). There were 66 (66 %) boys and34 (34 %) girls in the study population. 2 patients were excluded as one child was diagnosed with Thalassemia minor and another patient was diagnosed as Acute lymphoblastic Leukemia. The haemoglobin levels of the study subjects are mentioned as per Table 2, which shows that 69% had Hb levels between 10 -12 g/dl, 15% had 9-10g/dl, 6% between 8-9 g/dl, 5% between 7-8g/dl and 3% less than 7 g/dl. Levels of blood folate, serum B12, serum ferritin, serum Iron, TIBC and serum transferrin levels of patients studied are mentioned as per Table 3 which shows 16.3% had folic acid deficiency, 7.1% had B12deficiency and 43.8% had iron deficiency. Incidence of Iron deficiency anemia in our study was 54.1%, Folic Acid deficiency was seen in 16.3% and Vitamin B12deficiency anemia was seen in 13.2 % (Table 4). Mean Age ± SD: 6.03±4.28     [14] in 2011, studied the micronutrients such as vitamin B 12 , folate, iron and vitamin A concentrations of 396 children in the age group of 12 -23 months in rural Karnataka in south India. They found that 65.6% had at least one micronutrient deficiency and those children between 1-2 years who are breast feeding should be targeted during micronutrient supplementation programs. Ahmed F et al [15] in 2008 studied the prevalence of selected micronutrient deficiencies amongst anaemic adolescent school girls in rural Bangladesh, and they found that 28% of the girls had depleted iron stores, 25% had folic acid deficiency, 89% had vitamin B 2 and 7% had vitamin B 12 deficiencies.
They concluded that there is coexistence of micronutrient deficiencies among anaemic adolescent girls in rural Bangladesh, although they do not suffer from energy deficiency. Of all micronutrients only iron and vitamin B 2 concentrations were found to be related to the Hb concentration. Gadowsky SLet al [16] estimated the prevalence of biochemical iron, folate, and vitamin B 12 depletion among a group of Canadian pregnant adolescents. They found that 22% of the pregnant adolescents had anaemia, 78% had depleted iron stores and 25% had plasma vitamin B 12 values in the sub-optimal range. Metz J [17] reviewed the prevalence of anemia based on biochemical evidence. He found that the overall contribution of vitamin B 12 deficiency to the global burden of anemia was not significant, except in women and their infants and children in vegetarian communities.
He also found that it was unlikely that folate deficiency makes a major contribution to the burden of anemia in developing countries. Iron-deficiency anemia may coexist with vitamin B 12 and especially folate deficiency, and may confound the hematological features of the vitamin deficiencies whose prevalence would then be underestimated. Van [14,19] but its potential contribution as a cause of nutritional anemia in this study was not estimated. Thirdly, quantitative estimation of CRP was not done, which would have identified false positive elevation of serum ferritin. It is possible that ferritin values, especially those in the neighborhood of the cut-off point were increased by acute inflammation [20,21].
Fourthly, the measurements of metabolites such as homocysteine and methylmalonoic acid may have identified the functional deficiencies of vitamin B 12 and folic acid. Finally the sample size of this study is small and hence the results of this study could not be generalized to the whole population even though our hospital catered to patients from all over the country. Hence further studies are recommended to formulate a policy in treating those patients with nutritional anaemias.

Conclusion
Anaemia continues to be a public health program in children in India. Iron deficiency either alone or in combination is the commonest nutritional cause of anaemia. Iron deficiency anemia and its association with folic acid deficiency and vitamin B 12 deficiency were not significant in our study. Pure or mixed folic acid and vitamin B12 deficiency are important but not commonly recognized cause of anaemia in these children.