Discussion
In this single-center, cross-sectional, case-control study, we found that patients with ADHD and ASDs and healthy control children differed significantly regarding vitamin B12 and homocysteine. Patients with ASDs had the lowest vitamin B12 and the highest homocysteine levels. We did not find a significant difference in serum folate levels both in ASD and ADHD group compared to control group. Gender and age of children had no significant effect on vitamin levels.
Our study results support the studies showing low serum vitamin B12 and folate levels in ASD patients.27,28 A recent study conducted in Turkey found lower vitamin B12 serum levels in children diagnosed with ASD compared to controls.29 Similarly, another study from Oman reported lower serum folate and vitamin B12 levels in children diagnosed with ASD.28 As for ADHD, Unal et al. reported that Turkish children with ADHD had significantly lower vitamin B12 levels and that those levels negatively correlated with psychosomatic symptoms and learning problems as reported by teachers.23 This finding was supported and extended to folate in another study by Altun et al.30
Vitamin B12 status in the brain may play an important role in methylation-dependent processes and low serum vitamin B12 levels in ASD patients were interpreted to reflect increased levels of oxidative stress and impaired DNA methylation which can be an important factor in the pathophysiology of ASD.31,32 DNA methylation, an epigenetic regulatory system is known to be important in during pre- and post-natal brain development and alterations in DNA methylation have been shown in patients with ASDs.32,33 In line with these study results, DNA hypomethylation which affect the development of the CNS may be a possible explanation for the relation between vitamin B12 deficiency and autism.8 Lower levels of serum B12 were also found in ADHD group compared to control group. Vitamin B12 is required for a unique process known as dopamine-stimulated phospholipid methylation (PLM) performed by D4 receptor in CNS which is mostly associated with ADHD.34 PLM activity – differs by D4 gene variants – plays an important role in attention and neuronal synchronization and is known to be disturbed in children with both ASDs and ADHD.35 Although cross-sectional nature of our study precludes speculations on causality and may not reflect micro-nutrient levels in the fetal period, deficits in vitamin B12 and folate along with reduced methionine synthaseactivity might be posited to contribute to ADHD and ASD pathogenesis. Further, longitudinal studies exploring this issue are needed.
Patients with ASDs and ADHD in our sample were also found to have higher serum homocysteine levels compared to control group. Reduced vitamin B12 levels may increase the level of homocysteine. Homocysteine is also known to be a powerful excitotoxin, and its metabolic products may cause neuronal damage and disrupt the synthesis of proteins and neurotransmitters which are important for the structural integrity of the brain. As well as high levels of homocysteine were found to play a role in the etiology of various psychopathologies, recent clinical studies also reported higher serum and urine homocysteine levels in children with ASDs when compared to healthy controls.27,36,37 As for ADHD, some studies report elevated levels of homocysteine while others report reduced levels.13,25,30 The differing results may be due to enrollment of children with different comorbidities, diagnosed with separate ADHD subtypes and from variable age ranges. Our results support and extend those findings in showing reduced vitamin B12 and elevated homocysteine levels in patients with ADHD and ASDs.
As another finding in our study, oppositionality and hyperactivity/impulsivity symptoms in ADHD group were related to vitamin B12 and homocysteine levels which partially supported Saha et al’s results.13 Some preliminary studies suggest that vitamin and micronutrient supplements may reduce emotional lability, aggression and oppositional behaviors in children with ADHD and ASDs.38-40
Our results should be evaluated within the context of their limitations. Due to the cross-sectional nature of our study direction of causality could not be ascertained. Children with ASDs often display avoidance/restriction of certain foods due to sensory reactivity and resistance to novelty. This may also cause deficiencies of vitamins in children with ASDs. To control for this possibility, we also evaluated correlations with CARS sensory reactivity item scores but did not apply checklists to parents (ie, ABC, etc.). Also, in the planning stage of our study, we aimed to conduct separate univariate analyses of variance controlling for type I errors as per the Garipardic study but chose to conduct a multivariate analysis of covariance to control for effects of gender and age.19 Our data violated some of the assumptions of MANCOVA but we tried to control for this by using robust measures (ie, Pillai’s trace). Also, our findings may be affected by timing of measurements and may not reflect CNS levels of micronutrients in earlier development. Regardless of those limitations, our study is among the first ones to evaluate vitamin B12, vitamin B9 (folate) and homocysteine levels in children with ASDs and ADHD and compare the results with controls.
Our study results show low B12 and high homocysteine levels are detected in children diagnosed with ASD and ADHD compared to healthy controls. Children with ADHD or ASD often show emotional and behavioral problems. Deficiencies in micronutrients like B12 may play a role in the etiopathogenesis and clinical symptoms of these neurodevelopmental disorders. However, these parameters should be analyzed in a wider population to clarify the effect on the etiology of ADHD and ASD.