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Nutrition 29 (2013) 537–541

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Nutrition

journal homepage: www.nutr i t ionjrnl .com

Applied nutritional investigation

Low folate and vitamin B12 nourishment is common in Omani childrenwith newly diagnosed autism

Yahya M. Al-Farsi M.D., D.Sc. a, Mostafa I. Waly M.P.H., Ph.D. b,g, Richard C. Deth Ph.D. c,Marwan M. Al-Sharbati Ph.D. d, Mohamed Al-Shafaee M.D. a, Omar Al-Farsi M.Sc. a,Maha M. Al-Khaduri M.D. e, Ishita Gupta M.Sc. f, Amanat Ali Ph.D. b, Maha Al-Khalili M.Sc. a,Samir Al-Adawi Ph.D. d, Nathaniel W. Hodgson Ph.D. c, Allal Ouhtit M.Ph., Ph.D. f,*aDepartment of Family Medicine and Public Health, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Sultanate of OmanbDepartment of Food Science and Nutrition, College of Agriculture and Marine Sciences, Sultan Qaboos University, Muscat, Sultanate of OmancDepartment of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts, USAdDepartment of Behavioral Medicine, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Sultanate of OmaneDepartment of Obstetrics and Gynecology, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Sultanate of OmanfDepartment of Genetics, College of Medicine and Health Sciences, Sultan Qaboos University, Muscat, Sultanate of OmangDepartment of Nutrition, High Institute of Public Health, Alexandria University, Alexandria, Egypt

a r t i c l e i n f o

Article history:Received 24 March 2012Accepted 5 September 2012

Keywords:AutismFolateMethylationNutritionOmanVitamin B12

This study was supported by the Internal Grant FundHis Majesty’s Strategic Fund (SR/MED/FMCO/11/01),Health Sciences, Sultan Qaboos University. A researcResearch Institute provided partial support for this st* Corresponding author. Tel.: þ96-8-2414-3464; fax

E-mail address: aouhtit@squ.edu.om (A. Ouhtit).

0899-9007/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.nut.2012.09.014

a b s t r a c t

Objective: Arab populations lack data related to nutritional assessment in children with autismspectrum disorders (ASDs), especially micronutrient deficiencies such as folate and vitamin B12.Methods: To assess the dietary and serum folate and vitamin B12 statuses, a hospital-based case–control study was conducted in 80 Omani children (40 children with ASDs versus 40 controls).Results: The ASD cases showed significantly lower levels of folate, vitamin B12, and relatedparameters in dietary intake and serum levels.Conclusion: These data showed that Omani children with ASDs exhibit significant deficiencies infolate and vitamin B12 and call for increasing efforts to ensure sufficient intakes of essentialnutrients by childrenwith ASDs to minimize or reverse any ongoing impact of nutrient deficiencies.

� 2013 Elsevier Inc. All rights reserved.

Introduction

A survey of the available literature suggests that nutritionaland dietary interventions are considered routine treatment fordevelopmental disorders including autism [1,2]. However, thestudy and implementation of such measures have been limitedto the industrialized and affluent countries of North America andWestern Europe, with few exceptions [3,4]. Developing coun-tries, and especially Arab countries, lack relevant data becauseautism is not a common theme of research [5].

Autism spectrum disorders (ASDs) are neurodevelopmentaldisorders commonly characterized by debilitating and intran-sigent symptoms in three categories: difficulties with reciprocal

(IG/AGR/Food/10/01) andCollege of Medicine andh grant from the Autismudy.: þ96-8-2441-3300.

ll rights reserved.

social interactions, impaired communication skills, anddisturbed emotional functioning that often manifests as stereo-typical and non-goal–directed behaviors [6].

Oman, an Arab country located on the southern tip of theArabian Peninsula adjacent to the Asian and African continentswith a population nearing 3.5 million, presents fertile ground toexplore the nutritional and dietary deficits in children withdevelopmental disorders [7]. In Oman, a preliminary epidemio-logic survey has suggested that 0.14 per 1000 Omani children (0–14 y old) have ASDs [8].

Issues of child welfare are generally a significant concern fordomestic social policy. Oman, like other emerging economies, iswitnessing the co-occurrence of undernutrition and obesity in itspopulation [9]. Evidence also suggests that the country has yet toovercome persistent malnutrition. Alasfoor et al. [10] reportedthat, nationwide, approximately 7% of children exhibit wasting,11% exhibit stunting, and 18% were classified as underweight. Amore recent study replicating that study has suggested thatthese indices of malnutrition may be even higher [11]. However,these studies limited their sample selection to preschool children

Y. M. Al-Farsi et al. / Nutrition 29 (2013) 537–541538

in mainstream education. Thus, children with ASDs wereunlikely to be accounted for by these assessments of malnutri-tion indices because such children are unlikely to be enrolled inmainstream schools in Oman [12]. To address the potential datagap regarding children with developmental disorders, Al-Farsiet al. [13] examined the nutritional status of 128 children withASDs in Oman and found that approximately 9% of the partici-pants were malnourished according to the indices of under-weight, wasting, and stunting. There is evidence that thevariation in levels of two important nutrients, folate and vitaminB12 [14–16], is strongly relevant for ASD. These two nutrientshave been suggested to play a vital role in cognitive functions[17] similar to those often found to be disturbed in children withASDs [14,18–20].

Building on these emerging data showing that Arab pop-ulations tend to have folate and vitamin B12 deficiencies, weinitially conducted a preliminary study on screening the folateand vitamin B12 status andwe reported that a low status of thesetwo vitamins was associated with hyperhomocysteinemia inOmani autistic children [21]. In the present study, we performeda more comprehensive investigation and compared dietary andserum folate and vitamin B12 statuses between children withASDs and control subjects matched for age, gender, ethnicity, andsociodemographic status.

Materials and methods

The present case–control study was designed to determine the differentialvariation in folate or vitamin B12 levels in preschool-age Omani children (3–5 yold) with and without diagnosed ASDs. The study was conducted from December2009 to August 2010 at Sultan Qaboos University Hospital (SQUH), the mainreferral hospital for all autism cases in Oman.

Participants

The study participants included 80 Omani children (40 ASD cases and 40control subjects of overall similar age and gender). The study was approved bythemedical research ethics committee of Sultan Qaboos University. All caregiversof the participants provided their informed consent to be included in the study.

Ascertainment of an ASD diagnosis was made according to the ChildhoodAutism Rating Scale [22], which was developed using gold-standard criteriabased on the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition,Text Revision [23]. Accordingly, all participants fulfilled the eligibility for a diag-nosis of an ASD, exhibiting symptoms within the triad of typical autistic traits:communication impairment, social deficits, and ritualistic interests. Controlsubjects were randomly selected from eligible outpatients at the Department ofChild Health at SQUH. Eligible subjects included children 3 to 5 y old who werenot known to have any overt neurodevelopmental or behavioral disturbancesand were not previously or currently considered malnourished. Eligible diag-noses included trauma, routine physical examination, dental problems, anddermatologic problems.

Study protocol

Each participant’s mother was interviewed one on one. The interviewquestions were designed to elicit information regarding the child’s develop-mental milestones, sociodemographic background, and dietary history includingvitamin supplementation, in addition to other risk factors that might indicatea predisposition to autism, including pregnancy complications, preterm delivery,breast-feeding, and a family history of autism.

To overcome the problem of reverse causalitydthat autistic children mighthave changed their dietary habits after diagnosisdonly recently diagnosed caseswere included, for which the dietary intake questionnaire was administeredwithin 10 to 14 d of confirming the diagnosis. The retrospective dietary intake offolate and vitamin B12 was ascertained using the Reduced Dietary Questionnaireof Block et al. [24], which is a semiquantitative tool to gauge food intakefrequency. The psychometric properties of the Reduced Dietary Questionnairewere found to be adequate for the present cohort.

Ina food diary, the participants’mothers were asked to report the food itemsthe child had consumed during the year preceding the study and whether theyhad changed their diet from their usual routine during the previous 6 mo.

Subsequently, all food diaries were analyzed using standard computer-assistedprocedures (ESHA Research, Salem, OR, USA).

Laboratory measurements

Blood samples were collected from each of the 40 cases and 40 controlsmatched for age, sex, and weight (within�0.5 kg) to estimate the levels of serumfolate and vitamin B12. For blood sampling, 10 mL of venous blood was collectedfrom the median cubital vein. Blood samples were drawn into a plain tube byvenipuncture. After centrifugation, the serum was transferred to a micro-centrifuge tube and stored at �80�C before the folate and vitamin B12measurements.

We used the AxSYM Instrument (Abbott Diagnostics, Worcester, Massachu-setts) for the quantitative determination of serum levels of folate and vitaminB12. The cutoff values were 3 to 20 ng/mL for folate and 250 to 1250 pg/mL forvitamin B12. Hypersegmented neutrophils on the peripheral blood smear weredefined approximately as more than 5% of neutrophils with at least five lobes ormore than 1% of neutrophils with at least six lobes.

Serum levels of homocysteine and methionine were measured using high-performance liquid chromatography and electrochemical detection. Serumsamples were blownwith nitrogen and 10 mL was injected into an ESA CoulArrayhigh-performance liquid chromatographic system with a BDD analytical cellelectrochemical detector system (model 5040) equipped with an Agilent EclipseXDB-C8 (3 � 150 mm, 3.5 mm) reverse-phase C8 column. A dual mobile phasegradient elution was used, consisting of a mobile phase containing sodiumphosphate 25 mmol/L and 1-octanesulfonic acid 1.4 mmol/L, adjusted to pH 2.65with phosphoric acid, with the second mobile phase containing 50% acetonitrile.The systemwas run at a flow rate of 0.6 mL/min at ambient temperaturewith thefollowing gradients: 0 to 9 min 0% B, 9 to 19 min 50% B, and 19 to 30 min 50% B.Peak area analysis was provided by CoulArray 3.06 software (ESA, Chelmsford,Massachusetts) based on the standard curves generated for each compound.

Statistical analyses

Chi-square analyses were used to evaluate the statistical significance ofdifferences among portions of the categorical data. The non-parametric Fisherexact test (two-tailed) was used instead of the chi-square test for small sampleswhere the expected frequency was less than 5 in any of the 2 � 2 table cells.Unpaired Student’s t test was used to ascertain any significant differencesbetween the mean values of two continuous variables, and the Mann–Whitneytest was used for non-parametric tests. The odds ratios (ORs) for ASDs werecomputed for the bottom andmiddle thirds compared with the top third of folateand vitamin B12 concentrations based on the distributions in the control group.The ORs and 95% confidence intervals (CIs) obtained from logistic regressionmodeling were taken as measurements of association between ASD and levels offolate and vitamin B12. All statistical analyses were performed using SAS 9.1 (SASInstitute, Cary, NC, USA); a cutoff P value <0.05 was the threshold of statisticalsignificance for all tests.

Results

Eighty children, including 40 cases and 40 controls, wereanalyzed. The age was identical in cases and controls. Selectedsociodemographic characteristics of all subjects at the time ofenrollment in the study are listed in Table 1. In the two groups,most participant families lived in urban areas. The groups werealso comparable in the distribution of maternal age, mother’soccupation, and family income. Compared with the controlgroup, mothers of participants in the case group tended to havehigher illiteracy rates and folic acid supplementation duringpregnancy and a higher consumption of fortified flour and bread;however, these differences were not statistically significant(P > 0.05).

Our results showed that children with ASD had consistentlylower levels of folate and vitamin B12 in their diet and in theirserum (Table 2). The level of homocysteine was significantlyincreased in ASD cases by 68% (P ¼ 0.004), whereas the level ofmethionine was significantly decreased by 15% (P ¼ 0.05;Table 2). Because homocysteine is the substrate for the folate-and vitamin B12-dependent enzyme methionine synthase andmethionine is the product, these results are indicative of a func-tional deficit in methionine synthase activity in children with

Table 1Sociodemographic characteristics of autistic case and control groups, Oman, 2010

Characteristics Cases(n ¼ 40)

Controls(n ¼ 40)

P

Gender 1.00Male 20 (50.0%) 20 (50.0%)Female 20 (50.0%) 20 (50.0%)

Age (y) 4.8 � 0.3 4.8 � 0.3 1.00Weight (kg) 15.8 � 3.1 18.34 � 2.4Area of residence 0.19Urban 34 (85.0%) 30 (75.0%)Rural 6 (15.0%) 10 (25.0%)

Monthly family income (Omani riyals) 0.29<500 7 (17.5%) 4 (10.0%)500–1000 15 (37.5%) 20 (50.0%)>1000 18 (45.0%) 18 (45.0%)

Educational level of mother 0.06Illiterate 5 (12.5%) 3 (7.5%)Basic education 23 (57.5%) 30 (75.0%)Finished high school 12 (30.0%) 7 (17.5%)

Occupation of mother 0.38Working 11 (27.5%) 8 (20.0%)Housewife/retired 29 (72.5%) 32 (80.0%)

Non-compliant intake of folic acid duringpregnancy

32 (80.0%) 35 (87.5%) 0.12

Use of unfortified flour and bread 38 (95.5%) 36 (90.0%) 0.71

Values are presented as number (percentage) or mean � SD.

Table 2Comparison of dietary and serum folate and vitamin B12 levels and their relatedparameters in autistic cases and controls, Oman, 2010

Parameters Cases(n ¼ 40)

Controls(n ¼ 40)

P

Dietary folate (mg/d) 136.3 (5.2) 230.5 (3.7) 0.04Dietary vitamin B12 (mg/d) 1.3 (0.9) 2.2 (0.8) 0.02Serum folate (mg/L) 2.1 (0.3) 7.3 (0.4) 0.001Serum vitamin B12 (pg/mL) 183.6 (12.3) 341.2 (27.4) 0.001Homocysteine (mmol/L) 6.59 (0.6) 3.92 (0.5) 0.004Methionine (mmol/L) 259.6 (14.1) 303.5 (16.8) 0.05Hemoglobin (g/dL) 11.3 (0.7) 12.4 (0.8) 0.43Anemia 15 (37.5) 3 (7.5) 0.04Mean corpuscular volume 92 (6) 83 (9) 0.21Megaloblastosis 4 (10.0) 0 d

Hypersegmented neutrophils 2 (5.0) 0 d

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ASD in Oman, consistent with lower serum levels of its vitamincofactors.

Table 3 lists the ORs and 95% CIs for ASD by tertile of folateand vitamin B12 levels after adjusting for age, gender, and othersocioeconomic variables. The OR for being in the ASD groupamong subjects with folate levels in the lowest tertile comparedwith those in the highest tertile was 1.7 (95% CI 0.3–5.2). The ORfor being in the ASD group among subjects with vitamin B12levels in the lowest tertile compared with those in the highesttertiles was 1.6 (95% CI 0.8–8.3). A statistically significant trendtoward membership in the ASD group was found in participantsin the lowest tertiles for folate and vitamin B12 (P < 0.01,P ¼ 0.04, respectively).

Table 3Odds ratios and 95% confidence intervals for autism spectrum disorder bytertiles* of total folate and vitamin B12 levels, Oman, 2010

Variable OR 95% CI P for trend

Folic acid (mg/L) <0.01I. >20.0 1.0 d

II. 3.0–20.0 1.2 (0.4–4.1)III. <3.0 1.7 (0.3–5.2)

Vitamin B12 (pg/mL) 0.04I. >1250 1.0 d

II. 250–1250 0.9 (0.6–7.1)III. <250 1.6 (0.8–8.3)

CI, confidence interval; OR, odds ratio* Tertiles based on the distribution of the controls.

Discussion

The present study shows that there is a differential variationin two nutrients, folate and vitamin B12, in Omani children withASD compared to those defined as control subjects. A significantdecrease was found in the mean values of dietary and serumlevels of folate and vitamin B12 between childrenwith and thosewithout ASD. A decrease in the activity of methionine synthase,for which folate and vitamin B12 are required cofactors, wasobserved, indicating that their dietary and serum deficits havefunctional consequences. On the whole, the present data suggestpervasive and persistent deficiencies of folate and vitamin B12 inthe studied children with ASD.

Even in the general population, the prevalence of folate andvitamin B12 deficiencies in Oman is unknown. However, severalreports have indicated that folate and vitamin B12 deficienciesmay be common in Arab populations generally [25]. A study thatcompared an Arab population with its Western Europe coun-terpart showed that the dietary intake of folate in Arab men andwomen was below the recommended nutrient intakes [26].Another study from Israel showed deficiencies in folate andvitamin B12 in Arab patients with Alzheimer’s disease [27]. Ourprevious study showed that folate deficiency was associatedwith adult type 2 diabetes in Oman [28].

Folate deficiency has been implicated in the manifestation ofvarious clinical disorders [28]. Folate deficiency is characterizedby hypersegmentation of neutrophils, megaloblastosis, andanemia. Megaloblastic anemia can be masked by concurrentiron-deficiency anemia or thalassemia. Folate deficiency hasbeen shown to arrest children’s development, even in theabsence of anemia [29]. Serum folate concentration, althoughtypically low in patients with folate-deficient megaloblasticanemia, is primarily a reflection of a short-term folate balance.The World Health Organization has recommended empirictreatment for severely malnourished children with folate defi-ciency [30]. In Oman, prompt treatment of folate deficiencyshould be routinely prioritized for children with ASDs. Childrenwith autism or a pervasive developmental disorder-not other-wise specified exhibit selective eating habits and very oftenexhibit feeding difficulties [31,32]. Children with autism alsoexhibit greater difficulties with feeding and at meal timescompared with non-autistic children [33,34]. Parents of autisticchildren find it difficult to feed their children, probably becauseof pica, a disorder in which children tend to be picky eaters,prefer to eat non-edible objects, and resist consuming solid food[34]. Although the underlying mechanisms are not very wellunderstood, some autistic children tend to have motor delaysresulting in difficulties while eating and swallowing, whereassome have gastrointestinal problems (constipation or diarrhea)resulting in an aversion to food [35]. Deficiencies in variousnutrients have been reported to be associated with autism,including but not limited to vitamin D [36], calcium and vitaminE [37], and vitamins B6, B9, and B12. One possible explanation forthe relation between folate and/or vitamin B12 deficiencies andASD involves DNA hypomethylation [38].

Vitamin B12 deficiency has been shown to occur in vegetariansand children who are breast-fed by vegetarian mothers [39,40].

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An existing vitamin B12 deficiency is known to inhibit the releaseof vitamin B12 found in subsequently consumed food, which maybe another factor contributing to the predicament of childrenwithASDs [41]. Vitamin B12 deficiency has been shown to precipitatemegaloblastic anemia, atrophic glossitis, neuropathy, and demy-elination of the central nervous system [42]. The fact that vitaminB12 deficiency has been linked to several neurologic disorders isfurther evidence of its vital importance [43]. The question remainsas to whether such adverse effects of vitamin deficiency may alsobe observed in childrenwith ASDs. The present study implies thatincreased intakes of several vitamins and essential nutrientsshould be promoted. Feeding difficulties in autism can be allevi-ated through the supplementation of essential nutrients such asvitamins B6 and B12, which has been recommended by somestudies [44]. In addition, anecdotal and clinical evidence supportsa more active role by health care professionals in the case ofnutritional deficiencies in autistic children because problems aresometimes overlooked owing to picky eating being a commonproblem in neurotypical and autistic children around the agewhen autism is usually diagnosed (reviewed by Rempel et al.[45]).

In general, Oman has been highly proactive with suchinitiatives. National supplementation programs targetingmicronutrient deficiencies were started two decades ago. In1990, a supplementation program began providing all pregnantwomen with a daily dose of folic acid 5 mg and iron 200 mgthroughout the course of their pregnancies [46]. In 1997, anothernational programwas initiated to fortify relevant food consumedin the country with folic acid 5 mg/kg and iron 30 ppm [47]. Bothprograms have been reasonably successful. In 1993, it was foundthat 97% of all pregnant women attending health centers hadreceived the supplements, although their compliance rate withthe supplementation recommendations had reached 77% [48]. In2004, a national survey showed that the initiative for folatefortification in flour and bread had achieved 81% coverage of allhouseholds in Oman, with an average flour consumption of 116g/person per day [46]. The question remains as to whether sucha proactive initiative has reversed known folate and vitamin B12deficiencies in Omani children with autism. From the presentdata, compared with the control group, children with autismshowed significantly decreased levels of folate and vitamin B12in their dietary intake and serum. Therefore, concerted effortneeds to be directed toward such a vulnerable population.

Several limitations of this study must be acknowledged. First,the participants were drawn from a clinical population, namelythose seeking consultation at an urban tertiary care unit. It is wellknown that those seeking care at a hospital are not necessarilya suitable proxy for the community at large. Therefore,community studies with a broader sampling would be prereq-uisite to generalizing the present findings. Second, althoughmost of the measurements used were objective, psychosocialhistories and demographic information were narrated by theparticipants’ caregivers. It is not clear whether any unidentifiedconfounders exist owing to any possible bias of this approach.Third, the present study was restricted to children of preschoolage with ASD; therefore, the present results should be inter-pretedwithin the confines of this age group. Fourth, although thepresent study indicates a greater folate and vitamin B12 defi-ciency in children with ASD compared with control subjects, theobserved association is not temporal and therefore does notestablish a causal link. It remains unclear whether the observeddeficiencies are a consequence of pathologic processes central todevelopmental disorders or a compensatory adaptation. Thecondition of the ASD cases may have rendered them incapable of

following dietary guidelines with respect to the nutrientsinvestigated, or under-consumptionmay have been secondary tothe primary pathologic processes inherent in developmentaldisorders such as ASD.

In summary, the present study showed that Omani childrenwith ASDs exhibit significant deficiencies in folate and vitaminB12 compared with their non-ASD counterparts, defininga nutritional basis for this pervasive developmental disorder inOman. Our findings underscore the importance of mobilizingavailable health and educational resources to ensure the suffi-cient intake of relevant trace elements and other essentialnutrients by children with ASDs and to minimize or reverse anyongoing impact of nutrient deficiencies. Further studies of thistype should be encouraged to translate the present findings intoappropriate policy recommendations.

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