Environmental and genetic factors are important in the etiology of autism 1. Vitamin D is crucial for several key physiological processes, including brain development, DNA repair, and regulation of many genes. Evidence indicates that prenatal and early postnatal vitamin D deficiency increases autism risk, probably through multiple effects, including impaired brain development and increased de-novo mutations 2. Vitamin D helps protect against oxidative stress, which is a key cause of DNA damage, and also aids in the repair of DNA damage once it occurs 3,4.
Like most steroid hormones, activated vitamin D acts as a molecular switch, activating more than 200 target genes, thereby regulating gene expression through multiple mechanisms 5. Vitamin D may therefore play a major role in the etiology of autism by influencing the expression of genes related to autism. An example of a gene that is implicated in autism and that may have its expression influenced by vitamin D is Slc25a126,7.
Studies suggest that vitamin D availability and metabolism may have notable effects on mental health 8,9. The mechanisms of these effects are not fully understood as yet, but animal studies have shown that low prenatal vitamin D3 in utero can produce abnormal brain development characterized by increased brain size, enlarged ventricles, reduced brain content of nerve growth factor, and distortion in brain shape 10–12.
Neuroimaging findings in autism, although not diagnostic, have consistently revealed enlargement in cerebral volume that affects both gray and white matter, as well as enlarged ventricles 13–15. Neuroimaging findings in autism also include abnormalities in brain chemistry, serotonin synthesis, and brain electrophysiology 13–15. These structural and functional abnormalities resemble those found in animals with prenatal exposure to vitamin D deficiency 10–12.
As noted earlier, there is evidence that vitamin D plays very important roles in regulating hundreds of genes and that vitamin D also has significant roles in modulating inflammatory processes, in promoting normal brain development, and in combating infection – even in aiding embryonic survival and successful implantation 16,17. Thus, if individuals carry etiologic factors that increase their vulnerability to autism, the added vitamin D deficiency may tend to put these individuals at higher developmental jeopardy with increased risk of embryonic, fetal, and childhood brain vulnerability.
Cannell and Hollis 4 have urged the investigation of the clinical usefulness of vitamin D in ameliorating symptoms of various disease conditions, based on its physiological role of regulating gene expression in many different body tissues.
Vitamin supplements are among the most widely recommended medical interventions for autism 18. However, there have been relatively few treatment studies on vitamin supplements in general and vitamin D in particular for children with autism 19.
This paper aimed to assess the effect of vitamin D supplementation on the symptoms of autism in children. 25-Hydroxy-vitamin D [25(OH)D] level was measured at the beginning and at the end of the study in the 21 children aged 2–12 years and compared with that of neurotypical children of similar age and sex. Three measures of autism severity were assessed at the beginning and end of the study period.
Patients and methods
The study was designed as a prospective, randomized, controlled study conducted during the period from January 2009 until June 2010 (a time span of 18 months). The study was conducted with the approval of the Ethics Committee, Faculty of Medicine, Ain Shams University.
Patients were recruited from the Child Psychiatry Clinic, Psychiatry Institute, Faculty of Medicine, Ain Shams University. Informed consent to contribute to the study was signed by their mothers/caregivers. Twenty-one children with autistic disorder completed the trial to test whether supplementation with oral vitamin D3 at a daily dose of 2000 IU for 6 months would improve their autistic symptoms and serum level of 25(OH)D. Neurotypical children were enrolled to provide a reference range for 25(OH)D level.
Children were diagnosed with autistic disorders by a child psychiatrist using DSM-IV-TR criteria 20. None of the children had taken a vitamin/mineral supplement in the last 2 months. There were also no changes in the medical and dietary conditions, behavior, or other treatments over the last 2 months and during the study period. Children with known genetic syndromes, such as trisomy 21, tuberous sclerosis, and other known static or progressive neurologic conditions, and nonambulatory patients (because of the known association with increased bone turnover and deranged calcium homeostasis), and those using drugs that interfere with vitamin D metabolism were excluded from the study.
All patients were kept on behavioral and speech therapy for 30 min three times/week at the rehabilitation department according to the protocol of the Child Psychiatry Unit, Institute of Psychiatry, Ain Shams University.
Forty-two autistic children met the inclusion criteria. Thirty-one agreed to participate. Six children dropped out during the study and another four children were lost to follow-up (Fig. 1). All 21 children who completed the study were Whites and comprised 16 (76%) boys and five (24%) girls ranging in age from 2 to 12 years (the mean age of boys was 5.2 years and that of girls was 5.7 years).
The patients were randomly assigned to one of two groups. The parents or caregivers of group I (10 patients; eight boys and two girls) were instructed to give their infants a daily oral dose of 2000 IU cholecalciferol (D3) throughout the 6-month study period. The caregivers were reviewed on a monthly basis to return the empty vitamin D bottles as a measure of adherence with the study protocol. Also, parents were asked to contact the research team at any time to inform of any difficulties or report adverse effects. Group II included 11 autistic patients (eight boys and three girls) who were not supplemented with vitamin D. A comparison group of 23 apparently healthy children matched for age, ethnicity, and socioeconomic status to the patient group was also selected for the study. They had no previous history of developmental delay or neurologic symptoms; their ages ranged from 2 to 10 years and comprised 14 (60.9%) boys and nine (39.1%) girls. They were recruited from the siblings of the attendants of the Outpatient Pediatrics Clinic, Children’s hospital, Faculty of Medicine, Ain Shams University.
A venous sample was collected to measure 25(OH)D concentration using the ELISA technique. Children were not required to fast. Parents of the supplemented group were asked to give their children a daily oral dose of 2000 IU vitamin D3 in the form of Vi-Drop drops (Medical Union Pharmacuticals, Cairo, Egypt) (2800 IU/1 ml) at 20 drops/day for 6 months. Assessment of autism severity was carried out for all participants once at the beginning and once at the end of the study (6 months). A blood sample was collected again at the end of the study. For the neurotypical children, only steps 1–3 were followed – they did not participate in the treatment portion of the study.
Three tools were used by the same clinician at the beginning and at the end of the study for assessment: the Vineland Adaptive Behavior Scale (VABS) 21 to evaluate the level of functioning; the Childhood Autism Rating Scale (CARS) 22 for evaluating symptom severity; and the Autism Treatment Evaluation Checklist (ATEC) 23 to evaluate response to treatment.
Data were entered into an Excel spreadsheet, were double entered to ensure accuracy, and then uploaded into statistical program for the social sciences (SPSS, version 12; SPSS Inc., Chicago, Illinois, USA) for statistical analysis using statistically appropriate tests. The significance level for all statistical comparisons was set as P value less than 0.5.
As shown in Fig. 1, of the 31 children who agreed to participate in the study, 21 completed the 6-month study. In the vitamin D-supplemented group comprising 15 autistic patients, three dropped out, two because of hyperactivity and reduced sleep, and one because of increased bowel motion and diarrhea. The caregivers attributed these side effects to the vitamin supplementation. Two patients were lost to follow-up after the baseline visit. Regarding the other group of patients who were not supplemented with vitamin D, one dropped out because his parents decided to start a casein-free diet and the other four patients were lost to follow-up. No adverse effects were reported by the other caregivers. Twenty percent of the parents expressed the desire to continue independently with the supplement. Ten percent of the parents chose not to continue, and 80% did not provide this information.
There was no significant difference between the three groups regarding the mean±SD age, which was 2.7±1.9 years for the vitamin D-supplemented group, 5.8±2.9 years for the unsupplemented autistic group, and 5±1.8 years for the neurotypical control group. Mean presupplementation CARS scores for the two autistic groups were 33.9±2.9 and 33±3 (P>0.05), whereas mean VABS was 51.4±16 and 55.7±20 (P>0.05). The mean presupplementation ATEC scores of the two autistic groups were 91.8±20 and 98±16 (P>0.05). Thus, the two autistic groups were matched with regard to the level of social maturity measured by VABS or severity of autistic features measured by CARS and ATEC.
Although the 25(OH)D level in the autistic group was lower, there was no significant difference in the average presupplementation level of 25(OH)D between autistic cases and neurotypical controls, as seen in Table 1. Despite the rise in 25(OH)D level in the supplemented autistic group after supplementation compared with the minimal change in the unsupplemented group, on comparing the values with those of the neurotypical children, no statistically significant difference was found as seen in Table 2.
At the end of the 6-month study period, the supplemented group was seen to have significant increase in 25(OH)D levels. No significant change was found in the nonsupplemented group, as seen in Fig. 2 and Table 3.
Effect of supplement on symptoms
There was no significant difference between the two groups regarding social maturity and autism severity, as seen in Table 4. Yet, on comparing social maturity and symptom severity scores at the time of entry with scores of the same tests after 6 months, significant improvement was seen in both groups in the three scales used, as seen in Table 5 and Figs 3–5.
In recent years, increasing evidence has shown that children with autism spectrum disorders (ASDs) have lower levels of 25(OH)D relative to healthy controls. A recent study showed that there was a significant negative relationship between circulating serum 25(OH)D levels and severity of autism evaluated according to CARS scores (P=0.000). After adjusting for all other possible covariates, the 25(OH)D levels that remained can be seen as an independent predictor of ASD with an adjusted odds ratio of 1.23 (95% confidence interval 1.10–1.37). These results indicate that lower 25(OH)D levels may be independently associated with severity of ASD, and lower serum 25(OH)D levels could be considered an independent risk factor for ASD 24.
In the current study, although the mean 25(OH)D level of the neurotypical children was higher than that of the autistic group, the difference was statistically insignificant. It is worth mentioning that the optimal level of 25(OH)D is an area of debate 25. However, generally 75 and 120 nmol/l are considered the least sufficient normal values for healthy children 26 and autistic children 27, respectively. Therefore, our autistic patients’ mean 25(OH)D level (59 nmol/l) was far lower than the sufficient level. These findings are consistent with those of previous studies 28,29.
There was a nearly one-third elevation in the mean 25(OH)D level in the supplemented group from a mean of 47 nmol/l to a mean of 71 nmol/l, indicating adequate absorption and compliance. Still, the postsupplementation mean was below the sufficient 25(OH)D level.
Both groups showed significant improvement in the three scales used to assess autism severity and social maturity. The difference between the two groups was statistically insignificant. The significant improvement could be explained by the fact that all of our patients were on regular behavioral and speech therapies throughout the 6-month trial, which is the cornerstone of autism treatment 30. Furthermore, the natural course of developmental maturation in children may contribute to a spontaneous improvement in autism symptoms over time, especially in those younger than 5 years 29.
A confounding factor was the initial average level of 25(OH)D in the supplemented group (47±20), which was lower than the unsupplemented group’s initial average value (69±41), which consequently affected the follow-up average levels: 71±35 and 70±36 for the supplemented and unsupplemented groups, respectively. This confounder might have masked a difference in improvement in the two studied groups. Furthermore, the supplement dose failed to normalize the mean 25(OH)D level, a factor that could have contributed to the absence of a significant difference between the two studied autistic groups. Therefore, we hypothesize that a larger dose of vitamin D may be needed for the symptom improvement to be more vivid and fully reflected on the scales examined.
No significant difference was found between the two studied groups in the scales included here to assess severity. Perhaps if other scales more sensitive at detecting changes had been used a significant difference would have been elicited.
Higher vitamin doses may be needed specifically for autistic children to improve their metabolic status compared with neurotypical children 19. In the literature, there are many studies of very high dose vitamin supplementations – namely, B630 and vitamin C 31 – in children with autism, with most showing beneficial effects in reducing autism severity. It is worth trying higher doses of vitamin D based on its beneficial effects on brain functions. A recent study suggested that ASDs are disorders of the immune system that occur in a very early phase of embryonic development. In a background of genetic predisposition and environmental predisposition (probably vitamin D deficiency), an infection (notably a viral infection) could trigger a deranged immune response that, in turn, results in damage to specific areas of the central nervous system. If proven, this hypothesis would have dramatic consequences for strategies aimed at preventing and treating ASD 32.
Limitations of this study
This study is limited by the absence of additional verification for the neurotypical group other than the parental report. A larger sample size is needed for appropriate statistical power for possible significant differences. The formulation of the supplement was not tailored to body weight, and the length of the study may not have been long enough to observe the full effect of the supplement, and longer treatment may result in a larger effect.
The vitamin D supplement was found to be generally well absorbed, resulting in improvement in the 25(OH)D level. The supplement was well tolerated, with few side effects. Both the supplemented and nonsupplemented groups showed improvement in autism severity scores. We hypothesize that larger doses may result in greater improvements.
Conflicts of interest
There are no conflicts of interest.
1. Currenti SA. Understanding and determining the etiology of autism
. Cell Mol Neurobiol 2010; 30:161–171.
2. Bakare MO, Munir KM, Kinney DK. Association of hypomelanotic skin disorders with autism
: links to possible etiologic role of vitamin-D levels in autism
? Hypothesis (Tor) 2011; 9pii: e2.
3. Kinney DK, Barch DH, Chayka B, Napoleon S, Munir KM. Environmental risk factors for autism
: do they help cause de novo genetic mutations that contribute to the disorder? Med Hypotheses 2010; 74:102–106.
4. Cannell JJ, Hollis BW. Use of vitamin D
in clinical practice. Altern Med Rev 2008; 13:6–20.
5. Dusso AS, Brown AJ, Slatopolsky E. Vitamin D
. Am J Physiol Renal Physiol 2005; 289:F8–F28.
6. Palmieri L, Papaleo V, Porcelli V, Scarcia P, Gaita L, Sacco R, et al.. Altered calcium homeostasis in autism
-spectrum disorders: evidence from biochemical and genetic studies of the mitochondrial aspartate/glutamate carrier AGC1. Mol Psychiatry 2010; 15:38–52.
7. Sakurai T, Ramoz N, Barreto M, Gazdoiu M, Takahashi N, Gertner M, et al.. Slc25a12 disruption alters myelination and neurofilaments: a model for a hypomyelination syndrome and childhood neurodevelopmental disorders. Biol Psychiatry 2010; 67:887–894.
8. Yan J, Feng J, Craddock N, Jones IR, Cook EH Jr, Goldman D, et al.. Vitamin D
receptor variants in 192 patients with schizophrenia and other psychiatric diseases. Neurosci Lett 2005; 3801–237–41.
9. Humble MB. Vitamin D
, light and mental health. J Photochem Photobiol B 2010; 101:142–149.
10. Eyles D, Brown J, Mackay-Sim A, McGrath J, Feron F. Vitamin D3 and brain development. Neuroscience 2003; 118:641–653.
11. McGrath JJ, Féron FP, Burne TH, Mackay-Sim A, Eyles DW. Vitamin D3-implications for brain development. J Steroid Biochem Mol Biol 2004; 89–901–5557–560.
12. Eyles DW, Feron F, Cui X, Kesby JP, Harms LH, Ko P, et al.. Developmental vitamin D
deficiency causes abnormal brain development. Psychoneuroendocrinology 2009; 34Suppl 1S247–S257.
13. Courchesne E, Redcay E, Kennedy DP. The autistic brain: birth through adulthood. Curr Opin Neurol 2004; 17:489–496.
14. Hazlett HC, Poe M, Gerig G, Smith RG, Provenzale J, Ross A, et al.. Magnetic resonance imaging and head circumference study of brain size in autism
: birth through age 2 years. Arch Gen Psychiatry 2005; 62:1366–1376.
15. Lainhart JE. Advances in autism
neuroimaging research for the clinician and geneticist. Am J Med Genet C Semin Med Genet 2006; 142C:33–39.
16. Ozkan S, Jindal S, Greenseid K, Shu J, Zeitlian G, Hickmon C, Pal L. Replete vitamin D
stores predict reproductive success following in vitro fertilization. Fertil Steril 2010; 94:1314–1319.
17. Anifandis GM, Dafopoulos K, Messini CI, Chalvatzas N, Liakos N, Pournaras S, Messinis IE. Prognostic value of follicular fluid 25-OH vitamin D
and glucose levels in the IVF outcome. Reprod Biol Endocrinol 2010; 8:91.
18. Golnik AE, Ireland M. Complementary alternative medicine for children with autism
: a physician survey. J Autism
Dev Disord 2009; 39:996–1005.
19. Adams JB, Audhya T, McDonough-Means S, Rubin RA, Quig D, Geis E, et al.. Effect of a vitamin/mineral supplement on children and adults with autism
. BMC Pediatr 2011; 11:111.
20. American Psychiatric Association. Diagnostic and statistical manual of mental disorders, text revision (DSM-IV-TR) 2000:4th ed..Washington, DC: American Psychiatric Association.
21. Schopler E, Reichler RJ, DeVellis RF, Daly K. Toward objective classification of childhood autism
: Childhood Autism
Rating Scale (CARS). J Autism
Dev Disord 1980; 10:91–103.
22. Rimland B, Edelson M. Autism Treatment Evaluation Checklist.
Adams Avenue, San Diego, CA: Autism
Research Institute; 1999; 4812: 92116.
23. Sparrow S, Bella D, Cicchetti D. Vienland scales of adaptive behavior. Survey from manual 1984.Circle Pines, MN: American Guidance Service.
24. Gong ZL, Luo CM, Wang L, Shen L, Wei F, Tong RJ, Liu Y. Serum 25-hydroxyvitamin D levels in Chinese children with autism
spectrum disorders. Neuroreport 2014; 25:23–27.
25. Thacher TD, Clarke BL. Vitamin D
insufficiency. Mayo Clin Proc 2011; 86:50–60.
26. Bordelon P, Ghetu MV, Langan RC. Recognition and management of vitamin D
deficiency. Am Fam Physician 2009; 80:841–846.
27. Cannell JJ, Hollis BW, Zasloff M, Heaney RP. Diagnosis and treatment of vitamin D
deficiency. Expert Opin Pharmacother 2008; 9:107–118.
28. Meguid NA, Hashish AF, Anwar M, Sidhom G. Reduced serum levels of 25-hydroxy and 1,25-dihydroxy vitamin D
in Egyptian children with autism
. J Altern Complement Med 2010; 16:641–645.
29. Humble MB, Gustafsson S, Bejerot S. Low serum levels of 25-hydroxyvitamin D (25-OHD) among psychiatric out-patients in Sweden: relations with season, age, ethnic origin and psychiatric diagnosis. J Steroid Biochem Mol Biol 2010; 1211–2467–470.
30. Matson JL. Determining treatment outcome in early intervention programs for autism
spectrum disorders: a critical analysis of measurement issues in learning based interventions. Res Dev Disabil 2007; 28:207–218.
31. Adams JB, George F, Audhya T. Abnormally high plasma levels of vitamin B6 in children with autism
not taking supplements compared to controls not taking supplements. J Altern Complement Med 2006; 12:59–63.
32. Gentile I, Zappulo E, Militerni R, Pascotto A, Borgia G, Bravaccio C. Etiopathogenesis of autism
spectrum disorders: fitting the pieces of the puzzle together. Med Hypotheses 2013; 81:26–35.