Vitamin B12 or Cobalamin (Cbl) is a water-soluble vitamin. In the human body it occurs in 3 different forms: the natural form hydroxocobalamin (OH-Cbl) and in its two active forms methylcobalamin (Me-Cbl) and adenosylcobalamin (Ado-Cbl). Cyanocobalamin is a commercially available pharmacological form.

Cobalamin is synthesised by microorganisms present in the environment and in the intestines of animals. Common dietary sources include meat fish and dairy products or food that has been fermented. The Recommended Daily Allowance (RDA) ranges from 0.4 mcg/day for age< 6 months to 2.4 mcg/day for adults [1]. Cobalamin deficiency may be more common than previously recognised.

Data from the NHANES III study of 3766 US children aged 4-19years identified 1/1255 children with levels<100 pg/ml and 1/200 children <200 pg/ml. B12 Levels<100 pg/ml were considered to be indicative of clinical deficiency for the purpose of the survey (100 pg/ ml=74 pmol/L) [2]. In less developed countries, B12 deficiency may be even higher. A study of rural Mexican children showed 22% had serum B12 levels <103 pmol/L (140 pg/ml), a quarter of these children were thought to have malabsorption due to Giardia lamblia infection or bacterial overgrowth [3].

a. An elevated propionylcarnitine (C3) on Extended Newborn Screening (ENBS) can be a marker for Vitamin B12 deficiency.

b. Vitamin B12 deficiency in breast feed infants is most commonly due to maternal deficiency.

c. Maternal dietary Vitamin B12 deficiency can occur in non-vegan mothers.

d. Vitamin B12 deficiency has heterogeneous range of neurological presentations.

e. Elevations in Methylmalonic Acid (MMA) and Homocysteine (Hct) are more sensitive functional markers of vitamin B12 status than Mean Corpuscular Volume (MCV).

f. Prompt identification of and treatment of infantile Vitamin B12 deficiency is important to prevent irreversible neurological sequelae.

g. Vitamin B12 deficiency should be considered in every infant with developmental delay and Hypotonia.

Cbl is involved in two essential cellular reactions (Figure 1). Firstly Me-Cbl is required for the methylation of methionine to homocysteine paired with the demethylation of methyltetrahydrofolate to tetrahydrofolate. Secondly Ado-Cbl is a cofactor for the conversion of methylmalonyl-CoA to succinyl-CoA. When these cofactors are deficient homocysteine and/or methylmalonate accumulate.

Figure 1: Metabolic reactions catalysed by the active forms of cobalamin.

Me-Cbl is a cofactor for methionine synthase, catalysing the conversion of homocysteine to methionine in the cytoplasm and additionally participating in the recycling of folate. S-Adenosyl methionine is important as a universal methyl group donor in more than 100 organic reactions. These reactions are particularly important for DNA synthesis.

The second reaction, occurring in the mitochondria, is the conversion of L methylmalonyl CoA to succinyl-CoA. Methylmalonyl CoA is formed in the of catabolism of branched chain amino acids, cholesterol and odd chain fatty acids and this is the essential pathway for its metabolism. Decreased activity of methylmalonyl -CoA mutase results in accumulation of methylmalonyl-CoA and its upstream metabolite, prionyl-CoA. Propionylcarnitine (C3) is produced in the conversion of excess prionyl-CoA. C3 is included in tandem mass spectrometry based Extended Newborn Screening (ENBS) programmes although its sensitivity to detect all neonates with B12 deficiency is unknown. Its sensitivity will depends on whether C3 levels reach the local laboratories assigned cut-off when the ENBS sample is collected.

The causes of maternal B12 deficiency includes strict vegan diet, poverty and malnutrition, occult pernicious anaemia, previous gastric bypass surgery, and short gut syndrome [4-10]. Maternal B12 deficiency can be subclinical, they may not be anaemic and their vitamin B12 levels normal or low-normal [11]. Maternal vitamin B12 deficiency, while associated with a strict vegan diet, can also been seen in mothers with sub-optimal nutrition especially those from a lower socioeconomic status, hence the importance of the Paediatrician taking a maternal dietary history in cases of infantile vitamin B12 deficiency.

During pregnancy the placenta actively concentrates cobalamin in foetus resulting in foetal serum levels twice those of maternal serum [12]. Under normal circumstances the term neonate has sufficient stores to last for 6-12 months [13]. Cobalamin deficiency in newborns therefore reflects deficiency in the mother, and an elevated C3 detected on ENBS can be a marker of maternal vitamin B12 deficiency [14,15]. Specific cases of maternal dietary B12 deficiency and subclinical maternal pernicious anaemia have been reported in the medical literature after identification of an elevated C3 on ENBS in their newborn [16-18]. However the natural history of the rise of C3 in deficient infants is unclear. A negative newborn screen should not be relied upon to rule out Vitamin B12 deficiency [14].

It has been shown that mothers with low cobalamin levels have high Homocysteine (Hct) and Methylmalonate (MMA) levels and predictably low cobalamin and high Hct/MMA levels in their newborns, additionally higher birth number also increases the risk of low cobalamin status in these mothers [16,19]. In infants less than 6 months of age, the MMA concentration is inversely related to cobalamin concentrations [19]. This observation underlies the importance of cobalamin as a cofactor for MMA and Hct metabolism (Figure 1).

Cobalamin deficiency is especially important for mothers who choose to breast feed. On average, the cobalamin concentration in breast milk is 0.42 mcg/L [20]. Breast milk B12 concentrations have been found to be lower in women consuming a strict vegetarian diet compared to omnivorous women (0.23 ± 0.09 mcg/L vs. 0.38 ± 0.08 mcg/L). Infants fed breast milk containing less than 0.36 mcg/L had elevated methylmalonate levels. Additionally the milk B12 concentration was inversely proportional to the length of time the vegetarian diet was consumed [21]. Exclusively breast fed infants of deficient mothers are most at risk as most commercially available infant formulas are fortified with cobalamin.

Numerous autosomal recessively inherited inborn errors of Cbl transport or metabolism are known to exist. They exhibit molecular and clinical heterogeneity. However; the following simple algorithms can be utilised to direct the clinician towards the metabolic pathway affected (Figure 2);

a. Low B12 level, elevated MMA and Hct indicate a B12 deficiency or transport problem

b. Normal B12 level, elevated MMA and normal Hct indicate a block in Ado-Cbl metabolism

c. Normal B12 level, normal MMA and elevated Hct indicate a block in Me-Cbl metabolism

Figure 2: Diagnostic Algorithm for Infantile B12 Deficiency: MMA methylmalonic acid, Hct homocysteine, Cbl Colalamin, MB12D maternal B12 Deficiency, → normal, ↑ elevation, ↓decrease.

Early manifestations of cobalamin deficiency in infancy are non specific and thus can lead to a delayed diagnosis; they include failure to thrive, vomiting, irritability, weakness and refusal to weaned [22].

Neurological manifestations are common in infantile B12 deficiency, perhaps reflecting the importance of vitamin B12 in normal brain development and maturation. Reported infantile neurological manifestations of B12 deficiency include poor feeding, Hypotonia, developmental delay, developmental regression, eye movement abnormalities, irritability, chorea, tremor and seizures [23-25]. A key observation is that infants with neurological manifestations of B12 deficiency can still have normal haematological parameters including MCV [15,26], however the biochemical perturbations of elevated MMA and Hct associated with Vitamin B12 Deficiency precede the haematological and clinical manifestations [15,16].

Cobalamin deficiency in older children may present with paraesthesia, ataxia, abnormal movements, glossitis and personality change. Abnormal pigmentation of the dorsum of the fingers, toes and in the axillae, arms and medial thighs has been reported in older children with severe cobalamin deficiency [8,27,28]. Common reported signs are Hypotonia, hyperreflexia and choreoathetoid movements [11]. The presence of seizures at diagnosis seems to predict more severe developmental outcome [10]. Although not all authors have reported long term neurodevelopment follow up of their cases, poor infant developmental outcomes occur in 38% of pernicious anaemia mothers and 50% of vegan mothers [8,29].

Treatment with parenteral hydroxocobalamin results in dramatic improvement in abnormal movements, cessation in seizures, and improved energy and appetite. Cerebral atrophy as demonstrated on MRI has been shown to reverse [8]. Abnormal movements can appear transiently during treatment in some infants- the cause is unknown.

The infantile brain appears to be highly ssusceptible to adverse sequelae manifesting from vitamin B12 deficiency. The precise mechanism of this neurological dysfunction is unclear, but is likely to be multi-factorial in aetiology, with postulations including;

a. Interference with normal myelination

b. Epigenetic causes from deranged s-adenosylmethionine production

c. Aberrant cytokine regulation

The infantile brain is particularly susceptible to the myelination based mechanisms of B12 deficiency as myelination occurs mostly in the first 2 years of life, but is at its peak in the first 6 months of life. Long standing vitamin B12 deficiency has been well documented to result in delayed myelination or demyelination of the brain and spinal cord [30-32]. Deficiency of Ado-Cbl results in impaired enzymatic activity of methylmalonyl-CoA mutase, which in turn leads to an accumulation of prionyl-CoA. This in turns leads an accumulation of C15 and C17 fatty acids into the nerve sheaths resulting in altered myelin with reduced components of phospholipids, sphingomyelin and ethanolamine [33,34]. A deficiency of Me-Cbl, leading to impaired conversion of homocysteine to methionine, ultimately reduces the conversion of methionine into the key metabolite S-Adenosylmethionine (SAM). SAM is an important methyl donor for the conversion of phosphatidylethanolamine to phosphatidylcholine; both of these lipids are key components of myelin [35]. An interesting clinical observation has been the rapid improvement in the central neurological signs in infants with B12 deficiency after the administration of parenteral B12, especially rapid gains in development, improvements in tone and alertness. This rapid clinical improvement cannot be attributed alone to aberrant myelination.

Reduction in the synthesis of s-adenosylmethionine, a key methyl donor for over 100 cellular enzymatic reactions, can impact on the neurological phenotype of B12 deficiency via multiple end points including; a) abnormal synthesis of proteins, lipids and neurotransmitters, b) over stimulation of N-methyl-D-aspartate receptors, c) inhibited DNA synthesis and cell division [36-39]. Reduced availability of SAM in B12 deficiency has been postulated to increase the production of the CNS Cytokine Tumour Necrosis factor-α, which may play a role in demyelination [40-42].

The diagnosis of vitamin B12 deficiency requires a high index of suspicion in children as the symptoms are generally non-specific. The nutritional history from the mother, if asked, may point to nutritional deficiency. Table 1 summarises the clinical features and biochemical features of vitamin B12 deficiency and inborn errors of metabolism of Cbl synthesis. Table 2 provides a first line strategy for investigating an infant and a mother when infantile vitamin B12 deficiency has been identified. Figure 2 provides a simplified diagnostic overview to the infant with documented B12 Deficiency.

Table 1: Clinical and Biochemical Summary of B12 deficiency and Inborn Errors of Metabolism associated with Cobalamin Synthesis.

Table 2: First line investigations in infantile B12 deficiency.

Treatment regimes depend on the cause. Severe B12 deficiency is treated with intramuscular vitamin B12 with doses up to 1000micrograms 2-7 times per week. Treatment effect should be monitored frequently and the dose and frequency adjusted according to improvement in urinary MMA, plasma homocysteine and red cell indices. Hydroxocobalamin is effective and preferred over Cyanocobalamin for correcting defects in cobalamin metabolism such as CblC [42]. Mild dietary deficiency may be treated with oral vitamin B12; introduction of animal foods alone may not be inadequate. A longitudinal children fed a macrobiotic diet up to an average age of 6 years, before adopting a lacto-vegetarian or omnivorous diet showed at adolescence 21% still had elevated MMA concentrations, 15% had macrocytosis and 37% had low cobalamin concentrations (<218 pmol/L) [43]. Assessment of the cognitive performance of the same group of children in adolescence showed a significant association between cobalamin status (cobalamin<229 pmol/l or elevated urinary MMA>0.29 micromol/L) and deficits in cognitive function [44]. Due to the risk of adverse Neurodevelopmental outcomes, treatment with supplementation should be started as early as possible, there is evidence that long term outcome is related to duration and severity of deficiency.

The clinical features associated with vitamin B12 deficiency are non-specific, and hence can lead to a delay in diagnosis. Most clinical information on infantile B12 deficiency has been derived from clinical case reports with an average delay to diagnosis being 4 months [23].

The infant’s brain is highly susceptible to permanent injury associated with vitamin B12 deficiency. The prevention of neurological damage is paramount on the initial clinical suspicion and timely treatment of B12 deficient states. The early clinical signs of infantile B12 deficiency are non-specific, and the haematological parameters traditional associated with B12 deficiency are a late phenomenon in infants. We propose that B12 deficiency should be considered in all infants with developmental delay and Hypotonia for whom an alternate diagnosis is not identified. Early identification and treatment can prevent irreversible brain injury and its profound associated impacts on the health of the child, their family, and their local health system.