- Open Access
Evaluation of serum 25-Hydroxy vitamin D levels in children with autism Spectrum disorder
© The Author(s). 2019
- Received: 22 June 2018
- Accepted: 14 November 2018
- Published: 17 December 2018
The Correction to this article has been published in Italian Journal of Pediatrics 2019 45:22
Vitamin D plays an important role in etiology of Autism Spectrum Disorders (ASDs). We aimed to evaluate the serum 25 - hydroxyl vitamin D level among children with ASDs in Ahvaz city, Iran.
It was a cross-sectional study which had conducted on 62 subjects in two groups: a case group (n = 31) consisted of ASD children who study in especial schools; and a control group (n = 31) of healthy children who were selected by simple random sampling from regular schools in Ahvaz city, Iran during 2016. Maching between two groups has done regarding Socioeconomic status, type and amount of food intake, place of living and age. The levels of serum 25 - hydroxyl vitamin D were assessed in early morning means fasted state and also measured using ELISA method. Data were analyzed using Statistical Package for Social Sciences (SPSS) version 20. The significant level was considered < 0.05.
In ASD children, the average serum 25-hydroxyvitamine D level was 9.03 ± 4.14 ng/mg. In ASD group, 96.8% (30 subjects) had vitamin D deficiency. In healthy children group, average serum 25-hydroxyvitamine D level was 15.25 ± 7.89 ng/mg. Average serum 25-hydroxyvitamine D level in intervention group was significantly lower than the control group (P > 0.001). Although the parents of patients in control group reported longer exposure to sun (27.42 m per day against 33.06 m per day), no significant difference was observed between these groups in terms of exposure to sun (P < 0.05).
A significant difference was observed between serum 25-hydroxyvitamine D levels between the healthy and ASD children. It is recommended to use vitamin D supplement in children with ASDs under medical care.
- Vitamin D
Vitamin D may play an important role in etiology of Autism Spectrum Disorders (ASDs). Vitamin D is a neuroactive steroid affecting brain development and function. It plays an essential role in myelination, which is important for connectivity in the brain. Studies have shown that decreased vitamin D levels, decreased maternal vitamin D levels during pregnancy, and decreased exposure to solar UVB might increase the risk of ASD .
Despite extensive studies on ASD, the etiology of this disorder is quite unknown and studies are ongoing [2, 3]. ASD has a dominant genetic origin. However, environmental and genetic factors have interaction in the incidence of this disorder [3–5]. The results of studies have shown that in disease etiology, risk factors such as prenatal and postnatal infections [6, 7], and exposure to Valproic Acid of alcohol during pregnancy [8, 9], the age of mother , and abnormal nutritional and metabolic factors  are effective. During the recent years, the incidence of ADSs has been significantly increased. In previous studies, the incidence of this disorder was 10 in 10,000 , whereas the incidence of this disorder is now estimated as 90–250 in 10,000 [12–15]. In addition, in 2010, CDC reported the incidence of autism disorder in the United States as 1 in 68 and this indicates 78% increase in the incidence level compared with 2002 . However, a part of this sudden increase is probably the result of increased awareness and better reports about autism disorder as well as improved diagnostic criteria, but the exact causes for this sudden increase should be determined in future studies . Increased incidence of the disease can impose a heave financial burden on the society. It is estimated that medication costs for each patient will be 40,000 to 60,000 dollar per year . During the past decades, numerous studies were conducted on the role of vitamin D in neuropsychological disorders [18–23]. The findings of these studies showed that vitamin D deficiency is one of the risk factors of evolutional neuropsychological disorders such as schizophrenia  and autism [19, 25–28]. However, studies on the relationship between vitamin D and autism in different parts of the world such as Sweden , Egypt , Saudi Arabia , and China [31, 32] indicate lower 25 (OH) D level in patients with ASD in different ages compared with the control group. Moreover, some studies [33, 34] have shown different findings and no significant difference was observed between serum levels of vitamin D in ADS and control groups. To our knowledge few studies have been conducted in this regard in Iran and no study has been conducted in Ahvaz city, Southwestern Iran. Therefore, the present study aimed to evaluate the serum 25 - hydroxyl vitamin D level among children with ASDs in Ahvaz city, Iran.
It was a cross-sectional study which had conducted on 62 children in two groups: a case group (n = 31) consisted of ASD children who study in especial schools; and a control group (n = 31) of healthy children were selected from regular schools by using simple random sampling approach in Ahvaz city, Iran; 2016.
The two groups were matched in terms of gender, age, weight, height, head circumference, adequate breastfeeding (for at least six months), type and amount of food, socioeconomic status (the ratio of the number of family members to bedrooms was used as a measure of socioeconomic status) [35, 36], average income, family size, and exposure to smokers.
Students with ASDs entered the study by confirming the diagnosis by a neurologist and based on DSM-IV criteria and obtaining written informed consent from the parents. In the control group, the informed consent of parents was among the inclusion criteria, too.
Exclusion Criteria of the study were the presence of epilepsy and the use of vitamin D supplements were considered as exclusion criteria.
Clinical evaluation of patients with autism
Diagnosis of patients with ASD based on medical experience, clinical examination, and two criteria of DSM-IV and ADI-R was confirmed by a neurology expert.
Evaluation of serum 25-hydroxyvitamine D level
The levels of serum 25 - hydroxyl vitamin D were assessed in early morning means fasted state.
An expert nurse collected 5 mL blood of children to measure 25 (OH) D serum levels in a Blood Transfusion Center in Ahvaz city, Khuzestan, Iran. The serum samples were isolated after centrifugation and kept at − 20 °C until the laboratory assessments. The serum 25 (OH) D levels were measured using the ELISA method (Euroimmun kit, Medizinische Labordiagnostika AG, Germany, EQ. 6411–9601).
According to the guidelines of the American Endocrine Association, vitamin D level is defined with the concentration of 25-hydroxyvitamine D3 in blood. Natural, insufficient, and deficient vitamin D levels were determined with 25-hydroxyvitamine D level lower than 20 ng/ml, 21–29 ng/ml, and 30 ng/ml, respectively .
All the tests in this study were performed in Nargess Laboratory in Ahvaz city under the supervision of a doctor of medical laboratory sciences. Tests were performed two times and the averaged values were used in the analyses to increase the accuracy of the results.
Prior permissions from educational authorities, school principals, and class teachers were obtained, and then written informed consent form was taken from the parents’ children participate. The procedures of this study were approved by Independence Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (IR.AJUMS.REC.1394.199) also we have thanks to all subjects and their parents to participate in this study. Parents of children had informed consent to participate in this study.
Kolmogorov-Smirnov test was performed prior to statistical analysis to examine the normality of the variables. The results were presented in the form of statistical tables and numeric indicators. Chi-square test, t-test, and nonparametric test (Mann-Whitney U test) were used to analyze the data. Variable values were expressed as frequency, mean ± standard deviation (SD). Statistical calculations were performed using Statistical Package for Social Sciences version 20 (SPSS Inc., Chicago, IL, USA). For all statistical analyses, P value less than 0.05 was considered as significant.
Demographic characteristics in two groups
ASD N (%)
Healthy children Number (%)
ASD Mean (SD)
Healthy children Mean (SD)
Age of mother at Childbirth
Serum vitamin D level and exposure to direct sun in two groups
ASD Mean (SD)
Healthy children Mean (SD)
serum 25-hydroxyvitamine D level ng/mg
Time of exposure to sun
ASD N (%)
Healthy children N (%)
Deficient of Vitamin D (≤20 ng/ml)
Insufficient of Vitamin D (21–29 ng/ml)
In ASD children, the serum 25-hydroxy vitamin D level was significantly lower than the control group (P > 0.001). In the ASD group, all children showed deficient or insufficient level of serum 25-hydroxy vitamin D (96.8% or 3.2%, respectively).
In both groups, the use of direct daily sun was equal (67.74%). No significant difference was observed between the groups in exposure to direct sun (min/day) (P-0.56) (Table 2).
Recently, the role of vitamin D deficiency is identified as an environmental risk factor for some of autoimmune disorders [37, 38]. A study by Patrick and colleagues showed that vitamin D ma influence some of social behaviors of children with autism. He emphasized that vitamin D is a gene activator that creates tryptophan hydroxylase enzyme. This enzyme converts tryptophan into serotonin in the brain. Therefore, a sufficient level of vitamin D to produce serotonin in the brain than functions as a neuron transmitter improves social behaviors by positive effects on behavior . In a clinical trial study by Feng and colleagues on 37 children with autism, for three months, these children received 150,000 IU as intramuscular injection(monthly) and 400 IU orally (daily). These researchers reported that disease symptoms and behavioral checklist in children (3 years old and older) with autism improved . In most of the studies conducted on ASDs and vitamin D, lower 25-hydroxyvitamine D level in children with autism was taken into consideration. Our findings showed low level 25-hydroxyvitamine D level in the ASD children, compared with the healthy counterparts. Testes et al. reported that the mean of serum 25-hydroxyvitamine D level in children with autism with different ethnicities was lower than the control group by 35 nmol/L . Moreover, Duan et al. showed that serum 25-hydroxyvitamine D in patients with autism was significantly lower than the control group . Bener et al. attributed some biological and lifestyle factors such as birth, kinship, body mass profile, and physical activity and 25-hydroxyvitamine D level with the incidence of autism. Their findings showed that serum 25-hydroxyvitamine D level in ASD children was lower than the control group with similar ethnicity, age, and gender (P = 0.004) . This finding was supported by the study of Meguid et al. . Saad et al.  reported an inverse relationship between the averaged serum 25-hydroxyvitamine D level and severity of ASD (P > 0.001), which was not evaluated in our study. In another study in Saudi Arabia on comparing serum 25-hydroxyvitamine D level and MAG among 50 children with autism (5–12 years old) and 30 healthy children, a significant negative relationship was observed between serum 25-hydroxyvitamine D level and incidence of autism (P > 0.001) . Neumeyer and colleagues reported that the ration of male children with ASD with serum 25-hydroxyvitamine D level lower than 80 nmol/L was higher than healthy subjects (77% against 37% and p = 0.02). However, the results of a study by Molloy  and Esparham  in the United States showed that there is not significant relationship between serum 25-hydroxyvitamine D level in two groups of children with ASD and without ASD. Ugur and colleagues investigated vitamin D3 level of 54 children with autism and 54 healthy children between 3 and 8 years old in Turkey. They did not observe any significant difference t vitamin D3 serum level between these two groups . The results of a study by Hashemzeh and colleagues in Iran showed no significant difference between vitamin D in children with autism and healthy children. Also, no significant relationship was observed between serum vitamin D level and severity of the disease symptoms .
There significant difference was observed between serum 25-hydroxyvitamine D levels in two groups of this study and different studies confirm that and also there was no significant difference between two groups in time of exposure to sun. Therefore it is recommended to use vitamin D supplement in children with ASDs under medical care.
We have thanks to all subjects and their parents to participate in this study.
This study was supported by Deputy of Research in Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
Availability of data and materials
Please contact author for data request.
AA: Carried out the studies. HK: Participated in concept and carried out the studies. ZR: Performed the statistical analysis and help drafted the manuscript. MK: Participated in design of the study and revised the manuscript. SAH: Participated in design of the study. SMM: Participated in concept of study. SY, Co-ordinate of the study. MA: Data collection. NKM: Data collection. MC: Participated in concept and design of the study also revising it critically for important intellectual content; and final approval of the version to be published. All authors read and approved the final manuscript.
Ethics approval and consent to participate
This study was approved by Independence Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (IR.AJUMS.REC.1394.199).
Consent for publication
The authors declare that they have no competing interests.
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- Fernell E, Bejerot S, Westerlund J, Miniscalco C, Simila H, Eyles D, Gillberg C, Humble MB. Autism spectrum disorder and low vitamin D at birth: a sibling control study. Molecular Autism. 2015;6:3.View ArticleGoogle Scholar
- Fahmy SF, Sabri NA, El Hamamsy MH, El Sawi M, Zaki OK. Vitamin D Intake and Sun Exposure in Autistic Children. IJPSR. 2016;7(3):1043–9.Google Scholar
- Esparham AE, Smith T, Belmont JM, Michael Haden BA, Wagner LE, Evans RG, Drisko JA. Nutritional and Metabolic Biomarkers in Autism SpectrumDisorders: An Exploratory Study. Integrative Medicine. 2015;14(2):40–53.PubMedGoogle Scholar
- Chaste P, Leboyer M. Autism risk factors: genes, environment, and gene-environment interactions. Dialogues Clin Neurosci. 2012;14:281–92.PubMedPubMed CentralGoogle Scholar
- Coleman M, Gillberg C. The autisms. Oxford: Oxford University; 2012.Google Scholar
- Patterson PH. Maternal infection and immune involvement in autism. Trends Mol Med. 2011;17:389–94.View ArticleGoogle Scholar
- Ohkawara T, Katsuyama T, Ida-Eto M, Narita N, Narita M. Maternal viral infection during pregnancy impairs development of fetal serotonergic neurons. Brain and Development. 2015;37:88–93.View ArticleGoogle Scholar
- Meador KJ, Loring DW. Prenatal valproate exposure is associated with autism spectrum disorder and childhood autism. J Pediatr. 2013;163:924.View ArticleGoogle Scholar
- Landgren M, Svensson L, Strömland K, Andersson GM. Prenatal alcohol exposure and neurodevelopmental disorders in children adopted from Eastern Europe. Pediatrics. 2010;125:e1178–85.View ArticleGoogle Scholar
- Idring S, Magnusson C, Lundberg M, Ek M, Rai D, Svensson AC, et al. Parental age and the risk of autism spectrum disorders: findings from a Swedish population-based cohort. Int J Epidemiol. 2014;43:107–15.View ArticleGoogle Scholar
- Chakrabarti S, Fombonne E. Pervasive developmental disorders in preschool children. JAMA. 2001;285:3093–9.View ArticleGoogle Scholar
- New Zealand Guidelines Group. What does ASD look like? In a resource to help identify autism Spectrum disorder. Wellington: New Zealand Guidelines Group; 2010.Google Scholar
- Kogan MD, Blumberg SJ, Schieve LA, Boyle CA, Perrin JM, Ghandour RM, Singh GK, Strickland BB, Trevathan E, van Dyck PC. Prevalence of parent-reported diagnosis of autism Spectrum disorder among children in the us, 2007. Pediatrics. 2009;124:1395–403.View ArticleGoogle Scholar
- Ghanizadeh A. A preliminary study on screening prevalence of pervasive developmental disorder in schoolchildren in Iran. J Autism Dev Disord. 2008;38:759–76.View ArticleGoogle Scholar
- Baio J. Prevalence of Autism Spectrum Disorders—Autism and Developmental Disabilities Monitoring Network,14 Sites, United States, 2008; Morbidity and mortality weekly report; Surveillance summaries, vol. 61. Atlanta: Centers for Disease Control and Prevention; 2012. p. 1–19.Google Scholar
- Developmental Disabilities Monitoring Network Surveillance Year 2010 Principal Investigators; Centers for Disease Control and Prevention (CDC). Prevalence of autism spectrum disorder among children aged 8 years— autism and developmental disabilities monitoring network, 11 sites, United States, 2010. MMWR Surveill Summ. 2014;63(2):1–21.Google Scholar
- Hansen SN, Schendel DE, Parner ET. Explaining the increase in the prevalence of autism Spectrum disorders: the proportion attributable to changes in reporting practices. JAMA Pediatr. 2015;169:56–62.View ArticleGoogle Scholar
- Saad K, Abdel-Rahman AA, Elserogy YM, Al-Atram AA, Cannell JJ, Bjørklund G, et al. Vitamin D status in autism spectrum disorders and the efficacy of vitamin D supplementation in autistic children. Nutr Neurosci. 2015;19(8):346–51.View ArticleGoogle Scholar
- Kočovsk E, Fernell E, Billstedt E, Minnis H, Gillberg C. Vitamin D and autism: clinical review. Res Dev Disabil. 2012;33:1541–50.View ArticleGoogle Scholar
- 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;16:641–5.View ArticleGoogle Scholar
- Bener A, Hoffmann GF. Nutritional rickets among children in a sun rich country. Int J Pediatr Endocrinol. 2010;2010:410–502.View ArticleGoogle Scholar
- Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011;96:1911–30.View ArticleGoogle Scholar
- Eyles DW, Burne TH, McGrath JJ. Vitamin D, effects on brain development, adult brain function and the links between low levels of vitamin D and neuropsychiatric disease. Front Neuroendocrinol. 2013;34:47–64.View ArticleGoogle Scholar
- McGrath J. Is it time to trial vitamin D supplements for the prevention of schizophrenia? Acta Psychiatr Scand. 2010;121:321–4.View ArticleGoogle Scholar
- Cannell JJ. Autism and vitamin D. Med Hypotheses. 2008;70:750–9.View ArticleGoogle Scholar
- 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), vol. 9; 2011. p. e2.Google Scholar
- Cannell JJ, Grant WB. What is the role of vitamin D in autism? Dermatoendocrinol. 2013;5:199–204.View ArticleGoogle Scholar
- 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.View ArticleGoogle Scholar
- 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;121(1-2):467–70.View ArticleGoogle Scholar
- Mostafa GA, Al-Ayadhi LY. Reduced serum concentrations of 25-hydroxy vitamin D in children with autism: relation to autoimmunity. J Neuroinflammation. 2012;17:201.Google Scholar
- Duan XY, Jia FY, Jiang HY. Relationship between vitamin D and autism spectrum disorder. Zhongguo Dang Dai Er Ke Za Zhi. 2013;15:698–702.PubMedGoogle Scholar
- Gong ZL, Luo CM, Wang L, Shen L, Wei F, Tong RJ, et al. Serum 25- hydroxyvitamin D levels in Chinese children with autism spectrum disorders. Neuroreport. 2014;25:23–7.View ArticleGoogle Scholar
- Molloy CA, Kalkwarf HJ, Manning-Courtney P, Mills JL, Hediger ML. Plasma 25(OH)D concentration in children with autism spectrum disorder. Developmental Medicine & Child Neurology. 2010;52:969–71.View ArticleGoogle Scholar
- Hashemzadeh M, Moharreri F, Soltanifar A. Comparative study of vitamin D levels in children with autism spectrum disorder and normal children: a case-control study. Journal of Fundamentals of Mental Health. 2015 July-Aug;17(4):197–201.Google Scholar
- C. Ug˘ur, C.K. Gu¨rkan. Serum vitamin D and folate levels in children with autism spectrum disorders. Research in Autism Spectrum Disorders. 2014;8:1641–1647.Google Scholar
- Rossignol DA, Frye RE. Mitochondrial dysfunction in autism spectrum disorders: a systematic review and meta-analysis. Mol Psychiatry. 2012;17(3):290–314.Google Scholar
- Zhang HL, Wu J. Role of vitamin D in immune responses and autoimmune diseases, with emphasis on its role in multiple sclerosis. Neurosci Bull. 2010;26:445–54.View ArticleGoogle Scholar
- Hamza RT, Awwad KS, Ali MK, Hamed AI. Reduced serum concentrations of 25-hydroxy vitamin D in Egyptian patients with systemic lupus erythematosus: relation to disease activity. Med Sci Monit. 2011;17:CR711–8.View ArticleGoogle Scholar
- Patrck RP, Ames BN. Vitamin D hormone regulates serotonin synthesis.Part1: relevance for autism. FASEB J. 2014;28(6):2398–413. https://doi.org/10.1096/FJ.13-246546.View ArticleGoogle Scholar
- Feng J, Shan L, Du L, Wang B, Li H, Wang W, Wang T, Dong H, Yue X, Xu Z, et al. Clinical improvement following vitamin D3 supplementation in autism Spectrum disorder. Nutr Neurosci. 2016;20(5):284–90.View ArticleGoogle Scholar
- Tostes MHFDS, Polonini HC, Gattaz WF, Raposo NRB, Baptista EB. Low serum levels of 25-hydroxyvitamin D (25-OHD) in children with autism. Trends Psychiatry Psychother. 2012;34:161–3.View ArticleGoogle Scholar
- Bener A, Khattab AO, Al-Dabbagh MM. Is high prevalence of vitamin D deficiency evidence for autism disorder? In a highly endogamous population. J Pediatr Neurosci. 2014;9:227–33.View ArticleGoogle Scholar