The Cerebrospinal Fluid Concentration of Methyltetrahydrofolate and Serum Folate in Children with Developmental Delay, Regression, and/or Refractory Epilepsy
Keywords: 5–methyltetrahydrofolate, developmental disabilities, disabled children, folic acid
Vitamins are vital nutrients that cannot be synthesized sufficiently by the body and have to be obtained from the diet. Folate; is an important vitamin of B complex group, present naturally in some foods in the polyglutamated form with methyl or formyl as the one-carbon substitution, and synthetically in food fortification and nutrient supplements, known as folic acid., The main functions of folate include involvement in cell metabolism processes, such as DNA replication, repair and methylation, synthesis of nucleotides, amino acids, and some vitamins. Folate insufficiency can occur as a result of insufficient dietary intake or certain genetic variants that disrupts one-carbon metabolism. In addition, there are certain periods and conditions that increase folate requirement by the body such as pregnancy, childhood, specific diseases (like anemia, and malabsorption syndromes), chronic alcoholism, and by using specific medications (like antiepileptic drugs, triamterene, metformin, and sulfasalazine).
Folate deficiency has been associated with cardiovascular diseases, Down's syndrome, neural tube defects, childhood leukemia, and cancers. It is also known to play a role in different neurological problems, like depression, stroke, dementia, and Alzheimer., Folate deficiency is of great importance in children, as it is involved in early brain development and function., Accordingly, cerebral folate deficiency (CFD) has been defined as any neurological syndrome associated with a low cerebrospinal fluid (CSF) level of the active folate metabolite, 5-methyltetrahydrofolate (5MTHF). The low CSF level of 5MTHF can result from decreased transport across the blood-brain barrier by binding of folate receptor antibodies to the folate receptors in the choroid plexus. It has been suggested that prolonged supplementation with folinic acid can significantly improve the clinical symptoms of neurological syndromes associated with CFD.
Children's developmental delay and regression are important childhood disorders, making it one of the common causes of referrals to pediatric clinics. If the association of developmental delay and regression with CFD, suggested in previous case reports, is proven, important steps can be taken towards the improvement of symptoms by supplementation with this vitamin. Furthermore, not only epilepsy is suggested to be associated with folate deficiency, but also antiepileptic drugs predispose the patients to further folate deficiency; therefore, folate deficiency is of great importance in children with refractory epilepsy. According to the issues raised above, in the present study, we aimed to measure the serum level of folate and CSF level of 5MTHF in such children, namely those with refractory epilepsy, developmental delay, and regression to examine the association of such disorders with folate deficiency.
In this cross-sectional study, children aged 6 months to 5 years, who referred to Imam Reza Clinic and Nemazi Hospital, Shiraz, Iran, from February to October 2018 and were known cases of idiopathic refractory epilepsy, developmental delay, and regression were considered as the study population. The protocol of the study was approved by the Ethics Committee of Shiraz University of Medical Sciences (Ethics code: IR.SUMS.MED.REC.1397.338). The study sample size was calculated at 62 considering the prevalence of the disorder at 62.5% and a 95% confidence interval (95% CI), α = 0.05, and d = 7.5%. Inclusion criteria: The children's developmental delay/regression was diagnosed by insufficiency in at least two developmental domains based on the Ages and Stages Questionnaire (ASQ) protocol. The patient's refractory epilepsy was diagnosed as at least one episode of seizure each month in the last 18 months and uncontrolled epilepsy in the past 3 months, despite appropriate treatment with at least two antiepileptic drugs. The global delay was defined as -2 standard deviations (SD) lower values in at least two aspects of ASQ, and regression was determined by the physician's physical examination as a regressive developmental pattern, compared with the previous status. Exclusion criteria: Any child with the developmental delay because of genetic deficits, like Down's syndrome, prenatal disorders (like infection, and preterm birth), and metabolic and endocrine disorders were not included into the study, and only the children with a global developmental delay of unknown etiology were considered for recruitment. Any patient who used any medication interfering with the research objectives was not included in the study (folinic acid, cobalamin, and folic acid).
Then, the children were selected by simple randomization method, using a table of random numbers. Then, the researcher consulted the parents of the selected children, explained the research objectives to them, and asked them to read and sign the written informed consent if they were willing to participate in the study. All principles of Helsinki's declaration on human studies were met throughout the study steps.
After recording the patients' demographic (including age and sex) and clinical characteristics (including the medications used and the treatment protocol), the children were asked to refer at the desired time for CSF and blood sampling. CSF sample was taken through lumbar puncture by an experienced specialist in the presence of the child's parents. First, the patient was laid to the lateral with the knees bent into the stomach. After local anesthesia with EMLATM 5% cream, the spinal needle was inserted into the subdural area at L3-L4 or L4-L5 and 2 cc CSF was taken, mixed with ascorbic acid, and immediately sent to Peyvand Shiraz Laboratory for measurement of 5MTHF, measured by liquid chromatography tandem mass spectrometry (LC-MS/MS), using Human-5-MTHF5-Methyltetrahydrofolate-ELISA-kit (EH4170 2BScientific, England). The sensitivity of this device is <0.09 ng/mL and detection range of 0.15-10 ng/mL; it was calibrated every 6 months with an estimation of 20%. The normal values for 5MTHF according to this kit were as follows: 40-187 nmol/L for children aged 0.5–2 years and 40–150 nmol/L for children aged 2-5 years.
Also, a 5cc blood sample was taken from the patients' left cubits area in the sitting position and sent to Peyvand Shiraz Laboratory immediately, where it was centrifuged and kept at -80°C until measurement of serum folate and cysteine. Serum level of folate was determined by Antaphase II Folate or Folate/B12 radioassay kit (cat. no. 191–1046), 200-test size (BioRad Laboratories). The plasma's homocysteine was assessed by Abbott Homocysteine (HCY) assay (using Fluorescence polarization immunoassay [FIPA] method). The device was calibrated during the week earlier with an error estimation of 15%.
Any patient, for whom a specific reason for the developmental delay was diagnosed during the study period or refused to continue the study, was excluded from the study.
All statistical analyses were performed by the statistical software IBM SPSS Statistics for Windows version 18.0 (IBM Corp. 2009. Armonk, NY: IBM Corp.). First, the descriptive results of quantitative variables were presented as mean ± SD and that of categorical variables by frequency (percentage). One-sample Kolmogorov-Smirnov test was used to determine whether the data were normally distributed and the equality of variances was also confirmed by the Levene's test. The effect and association of the studied variables on/with developmental delay were determined by Poisson or Cox proportional hazards. The significance of the tests was identified by P values <0.05 and two-tailed 95% CI. The absolute risk difference and number needed to treat (NNT) profit/loss were also calculated.
Of 60 patients who completed the study, 33 boys (55%) and 27 girls (45%). Mean ± SD of the studied population was 26.93 ± 19.97 months (28.24 ± 18.72 months in boys and 25.33 ± 21.67 months in girls). Eighteen patients (30%) had refractory epilepsy, 11 (18.3%) had developmental delay, 12 (20%) had refractory epilepsy and developmental delay, and 19 (31.7%) had refractory epilepsy and developmental regression. The results of brain magnetic resonance imaging (MRI) were normal in 47 (78.3%) children and atrophic in 13 (21.7%) children. Mean ± SD of patients' age in the group with normal MRI results was 22.8 ± 18.14 months and that in the group with the atrophic brain was 41.85 ± 19.84 months. The mean number of medications used was 2.9 with an SD of 1.58.
Mean ± SD of serum level of homocysteine was 9.14 ± 8.58 μmol/L (7.76 ± 4.47 in boys and 10.84 ± 11.71 μmol/L in girls); 20% (N = 12) were ≤4.6, 58.3% (N = 35) were 4.6–10.5, and 21.7% (N = 13) were ≥10.5 μmol/L. Mean ± SD of serum level of folate was 11.60 ± 6.89 nmol/L (12.24 ± 6.86 in boys and 10.83 ± 6.99 nmol/L in girls); 20% (N = 12) were ≤4.6, 48.3% (N = 29) were 4.6–18.7%, and 31.7% (N = 19) were ≥18.7 nmol/L. Mean ± SD of CSF level of 5MTHF was 69.23 ± 54.16 nmol/L (62.03 ± 26.55 in boys and 78.03 ± 75.11 nmol/L in girls); 15% (N = 9) were ≤40, 85% (N = 51) were >40 nmol/L.
In the next step, we compared the difference in these parameters based on the categories of the other parameters. In [Table 1], the categories of homocysteine have been compared based on folate and 5MTHF levels separately, and in [Table 2], based on the combination of folate and 5MTHF levels. Also, the frequency of folate levels was categorized based on the combination of homocysteine and 5MTHF levels in [Table 3].
The correlation between variables was tested by Pearson correlation [Table 4]. As indicated, there was a direct and positive association between homocysteine and 5MTHF as homocysteine levels increased by an increase in 5MTHF levels (P < 0.001). There was also a positive and direct association between age and 5MTHF (P = 0.041, [Table 4]).
Of all the diagnosed patients, nine patients received treatment, among whom four showed a dramatically favorable response considering epilepsy and developmental delay.
In the present study, we included 60 children with developmental disabilities, including 18 with refractory epilepsy, 11 with developmental delay, 12 with refractory epilepsy and developmental delay, and 19 with refractory epilepsy and developmental regression. Of these, only 21.7% had an atrophic brain on MRI. According to the evidence, MRI is considered as an appropriate diagnostic tool in children with developmental delay, especially those with neurological signs, in whom volume abnormalities (hypoplasia or atrophy) have been reported as the most common finding, which is consistent with the results of the present study.
Folate is an important nutrient for brain development. According to the results of the present study, the investigated patients had a mean serum folate level of 11.60 ± 6.89 nmol/L; 20% had apparent folate deficiency, defined as serum folate levels ≤4.6 nmol/L. Several human and animal studies have shown the effect of maternal supplementation with folic acid on the child's brain growth.,, The study by Steenweg–de Graaff and colleagues indicated that the maternal serum concentration of folate significantly affected child's postnatal head growth, independent of overall fetal growth. Pickel and colleagues also demonstrated that maternal MTHF and folate deficiencies resulted in increased developmental delays and smaller embryos in mice. MRI assessment of 256 Dutch children aged between 6 and 8 years indicated smaller brain sizes in children whose mothers had a lower plasma folate concentrations in early pregnancy, which was also associated with language performance and visuospatial deficits. These results confirm the important role of folate on the development of the human brain and developmental disorders, as emphasized by the results of the present study. In addition to the significant role of folate in brain development and association of folate deficiency with developmental disorders, antiepileptic drugs can result in further folate deficiency, which adds to the significance of paying attention to serum folate levels in patients with refractory epilepsy, who have used a wide spectrum of antiepileptic drugs, as indicated in the present study.
Another important factor examined in cases with a neurodevelopmental disorder is homocysteine. Homocysteine, a sulfur-containing amino acid, is generated during methionine metabolism. Serum folate is required for the synthesis of methionine from homocysteine. In cases of folate deficiency or enzyme mutations, methionine, involved in the synthesis of hormones, neurotransmitters, nucleic acids, and proteins, particularly in the brain, will reduce, while homocysteine levels rise. Furthermore, increased homocysteine levels can lead to endothelial dysfunction and neuronal damage by promoting mitochondrial dysfunction and apoptotic cell death., Accordingly, rise in homocysteine levels is observed in several medical conditions, such as cardiovascular disorders, cognitive impairment, dementia, Alzheimer's disease, Parkinson's disease, epilepsy, and depression., In the present study, we measured the serum level of homocysteine and the results showed that the mean serum level of homocysteine was 9.14 ± 8.58 μmol/L in children with developmental delay/regression and/or epilepsy. In addition, 21.7% of the studied patients had a homocysteine level ≥10.5 μmol/L. These results are in line with previous case reports indicating elevated serum level of homocysteine, associated with low folate, and B12 levels in children with developmental delay,, which are consistent with the results of the present study, emphasizing on the importance of folate-homocysteine-methionine axis.
In the present study, we also evaluated the CSF level of 5MTHF and the results showed the mean level of 69.23 ± 54.16 nmol/L; 15% showed CFD, defined as CSF level of 5MTHF ≤40 nmol/L. These results indicate that in addition to the role of folate deficiency in brain development, measurement of brain level of 5MTHF is of great significance. Several case reports have indicated the CFD in children with developmental delay, psychomotor regression, seizures, mental retardation, and autistic features., Moretti and colleagues described further that the child's motor skills improved after supplementation with folinic acid. There are three possible mechanisms suggested for CNS folate deficiency; first, reduced plasma levels of 5MTHF and accordingly reduced diffusion into the brain; second, reduced folate carrier on the choroid epithelial cells to pass 5MTHF through the cerebrospinal fluid; and third, displacement of blocking autoantibodies to the folate receptors. Meanwhile, there must also be other mechanisms involved, as CFD has been reported in Aicardi-Goutiere's, Rett syndromes, and in mitochondriopathies., The importance of CNS folate deficiency, as emphasized in the present study, is as much as studies have suggested cerebral folate deficiency syndrome, is associated with mental/motor retardation, epilepsy, and ataxia, presents from about 4 to 6 months of age in children;, and indicates the efficacy of supplementation with folinic acid on improvement of symptoms in several neurological disorders.,
The present study had the main strength of performing LP in an Asian population, as it is not an easy procedure to perform in our country, as we had to obtain the parents' consent, while we did not impose additional costs to the patients. Nevertheless, this study could have some limitations. The most important limitation refers to the nature of the study, as we had no control group (healthy children) to study the association of the laboratory parameters with the neurodevelopmental diseases studied. In addition, this study was a cross-sectional study and we did not follow all the patients to examine changes in the results by time, although we had no intervention. Finally, there may be some variables that we could not be controlled in this study, acting as confounders, although we tried to include the patients by the random method and minimize the effect of confounders by the inclusion criteria. The small sample size was another limitation of this study.
In conclusion, the present study investigated a group of children with a developmental delay/regression, and/or refractory epilepsy showed the importance of folate deficiency and a rise in homocysteine levels in these patients. Additionally, the results showed the important role of CFD in these patients, independent of serum folate deficiency. Therefore, in the patients with neurodevelopmental disorders, it is not only important to measure the serum level of folate, but also CSF level of the active metabolite of folate, 5MTHF. The results of the present study give insight to researchers to follow the improvement of symptoms in such children after oral/intravenous/intramuscular supplementation with folinic acid (15-150 mg/day) or folic acid 5 mg/day (orally) in future studies.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
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Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2], [Table 3], [Table 4]