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Perfil nutricional em criança autista
DOI: 10.5020/18061230.2018.6714
PERFIL NUTRICIONAL DE CRIANÇAS PORTADORAS DO TRANSTORNO DO

ESPECTRO AUTISTA
Nutritional profile of children bearing autism spectrum disorder
Perfil nutricional de niños portadores de trastorno del espectro autista
Maria Vanuza Caetano

Instituto Federal de Educação, Ciência e Tecnologia do Ceará - IFCE - Limoeiro do Norte (CE) - Brasil
Daniel Cordeiro Gurgel

Instituto Federal de Educação, Ciência e Tecnologia do Ceará - IFCE - Limoeiro do Norte (CE) - Brasil
RESUMO
Objetivo: Avaliar o estado nutricional e o consumo alimentar de crianças portadoras do transtorno do espectro autista (TEA). Métodos: O
estudo teve abordagem de natureza quantitativa, descritiva, exploratória e transversal. Participaram 26 crianças, de 3 a 10 anos de idade, com
diagnóstico do TEA, de ambos os sexos, atendidas no município de Limoeiro do Norte, Ceará. Os dados foram coletados através de entrevistas,
ordenadas por um questionário sociodemográfico (idade, renda familiar, escolaridade dos participantes, tratamento psicofarmacológico, idade
recebida do diagnóstico do TEA, classificação da CID-10 e histórico clínico); histórico nutricional; aplicação de 3 recordatórios de 24 horas; e
medidas antropométricas (peso, altura, circunferência do braço e as dobras cutâneas tricipital e subescapular), com posterior cálculo do índice
de massa corporal (IMC). Utilizou-se análise descritiva e as variáveis contínuas foram expressas em média ± desvio padrão e coeficiente
de variação. Resultados: Das crianças avaliadas, 10 (38,5%) apresentaram sobrepeso (23,1%, n=6) e obesidade (15,38%, n=4) pelo IMC/I
(Índice de Massa Corporal para Idade), bem como 10 crianças (38,5%) apresentaram risco de sobrepeso. O consumo de energia (EER)
esteve acima do recomendado para 14 (53,85%) dos autistas. Identificou-se inadequação no consumo de vitamina A (77%, n=20), vitamina
B6 (58%, n=15) e cálcio (50%, n=13). Conclusão: As crianças com o TEA demonstram elevados índices de sobrepeso, obesidade e elevada
inadequação na ingestão de vitaminas e minerais.
Descritores: Avaliação Nutricional; Consumo de Alimentos; Transtorno Autístico.
ABSTRACT
Objective: To evaluate the nutritional status and dietary intake of children bearing the autism spectrum disorder (ASD). Methods: The
study had a quantitative, descriptive, exploratory and cross-sectional approach. Twenty-six children aged 3 to 10 years, with diagnosis of
ASD, of both sexes, attended to in the city of Limoeiro do Norte, Ceará State, Brazil, participated in the study. Data was collected through
interviews, guided by a socioeconomic questionnaire (age, family income, participants’ level of education, psychopharmacological treatment,
age at diagnosis of ASD, ICD-10 coding, and clinical history); dietary history, application of three 24-hour recall, and anthropometric
measurements (body weight, height, arm circumference and tricipital and subscapular skinfolds), with a subsequent calculation of the body
mass index (BMI). Descriptive analysis was used and continuous variables were expressed as mean ± standard deviation and coefficient of
variation. Results: Of the children evaluated, 10 (38.5%) were overweight (23.1%, n=6) or obese (15.38%, n=4) according to the BMI-for-
age, and a further 10 children (38.5%) were at risk of overweight. The estimated energy requirement (EER) was above that recommended
in 14 (53.85%) of the autistic patients. The intake of vitamins A (77%, n=20) and B6 (58%, n=15) and calcium intake (50%, n=13) were
inadequate. Conclusion: Children with ASD evidence high overweight and obesity rates, and high inadequacy in vitamins and minerals
intake.
Descriptors: Nutrition Assessment; Food Consumption; Autistic Disorder.
Este artigo está publicado em acesso aberto (Open Access) sob a licença Recebido em: 08/07/2017
Creative Commons, que permite uso, distribuição e reprodução em qualquer Revisado em: 04/12/2017
meio, sem restrições, desde que o trabalho seja corretamente citado. Aceito em: 17/01/2018
Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018 1

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Caetano MV, Gurgel DC
RESUMEN
Objetivo: Evaluar el estado nutricional y el consumo de alimentos de niños portadores de trastorno del espectro autista (TEA). Métodos:
Estudio de abordaje cuantitativo, descriptivo, exploratorio y transversal. Participaron 26 niños entre 3 y 10 años de edad, de ambos los sexos
y con el diagnóstico de TEA que eran asistidos en el municipio de Limoeiro de Norte, Ceará. Se recogieron los datos a través de entrevistas
ordenadas por un cuestionario sociodemográfico (edad, renta familiar, escolaridad de los participantes, tratamiento psicofarmacológico,
edad que recibió el diagnóstico de TEA, clasificación de la CID-10 e historia clínica); el histórico nutricional; la aplicación de 3 recordatorios
de 24 horas; y las medidas antropométricas (el peso, la altura, la circunferencia del brazo y los pliegues cutáneos tricipital y subescapular)
con posterior cálculo del índice de masa corporal (IMC). Se utilizó el análisis descriptivo y las variables continuas se expresaron en media
± desviación típica y coeficiente de variación. Resultados: De entre los niños evaluados, 10 (38,5%) presentaron sobrepeso (23,1%, n=6)
y obesidad (15,38%, n=4) a partir del IMC/E (Índice de Masa Corporal para la edad), así como 10 niños (38,5%) presentaron riesgo
de sobrepeso. El consumo de energía (EER) estuvo por encima del recomendado para 14 (53,85%) de los autistas. Se identificó como
inadecuado el consumo de vitamina A (77%, n=20), vitamina B6 (58%, n=15) y calcio (50%, n=13). Conclusión: Los niños con TEA han
demostrado elevados índices de sobrepeso y obesidad e inadecuada ingesta de vitaminas y minerales.
Descriptores: Evaluación Nutricional; Consumo de Alimentos; Trastorno Autístico.
INTRODUÇÃO
O Transtorno do Espectro Autista (TEA) é um transtorno do desenvolvimento caracterizado por alterações na capacidade
cognitiva e nas interações sociais que pode levar ainda a uma seletividade alimentar. Essa desordem apresenta diversidade
de manifestações clínicas de alta complexidade, as quais podem estar relacionadas com inúmeras interações entre os genes,
fatores epigenéticos e a exposição aos fatores ambientais(1). A etiologia do TEA ainda é desconhecida, apesar de haver inúmeras
hipóteses e já terem se passado mais de 70 anos desde os primeiros estudos e publicações sobre o tema(2).
Na década de 1980-1990, a prevalência estimada era de 4-5/10.000 habitantes, aumentando para 30-60/10.000 na década
1990-2000. Segundo o Centers for Disease Control and Prevention (CDC), em 2015, houve uma prevalência do transtorno de
14,7 por 1000 (1 em 45) crianças com idade de 8 anos, afetando 1 a cada 42 meninos e 1 a cada 189 meninas(3).
De acordo com o censo 2010 do Instituto Brasileiro de Geografia e Estatítica (IBGE)(4), estima-se que haja 454.706 crianças
com transtorno do espectro autista (TEA) no Brasil, com uma taxa de prevalência de uma para 150, na proporção de 3 homens
para 1 mulher. No Ceará, os dados são imprecisos.
Diante desse cenário, os diferentes níveis de atenção poderiam delinear estratégias de promoção da saúde para os portadores
de TEA e seus familiares, com o intuito de proporcionar atenção integral à saúde e promoção da qualidade de vida. Para tanto,
é necessária a participação dos profissionais de saúde e dos gestores de políticas públicas ofertando cuidados básicos de saúde
quanto ao diagnóstico, à prevenção de agravos e às ofertas de reabilitação e cuidados contínuos(5).
As principais intervenções de promoção da saúde apontadas pela literatura, relacionadas aos autistas, consideram aspectos
importantes de seu contexto (motoras, cognitivas, comunicação, expressão, socialização, psíquicas e nutricionais), visando à
prevenção do agravo da deficiência e ao favorecimento de competências sociais para sua autonomia e independência, que visam
melhorar suas vidas em geral(6).
Além das características mais marcantes percebidas nos portadores do transtorno do espectro autista (TEA), relacionadas,
principalmente, ao falho desenvolvimento da linguagem e interação social, ainda há uma série de desordens gastrointestinais que
podem acometer os autistas, como diminuída produção de enzimas digestivas, inflamações da parede intestinal e permeabilidade
intestinal alterada, e todos esses fatores agravam os sintomas dos portadores da doença(7).
A alimentação inadequada e a falta de equilíbrio energético são motivos de especial preocupação, pois a ingestão de
micronutriente está estreitamente relacionada à ingestão de energia, sendo provável que as crianças cujo consumo de energia
seja menor também sofram de deficiências de vitaminas e minerais(8). Dados sugerem que as crianças autistas possuem de duas a
três vezes mais chances de serem obesas do que adolescentes na população em geral(9). Portanto, a atividade física e os cuidados
nutricionais são elementos valiosos na prevenção de doenças, como a obesidade infantil, para manutenção da independência
funcional, participação social e qualidade de vida. O inadequado estado nutricional, a limitada variedade de alimentos e a
gravidade da sintomatologia associada ao TEA podem causar significativo impacto na qualidade de vida dos pacientes, pais e
cuidadores(10).
Desse modo, percebe-se uma relação ainda inconclusiva entre o TEA e os aspectos nutricionais, como o estado nutricional
e comportamento alimentar. Portanto, pesquisas que aprimorem essa relação podem colaborar diretamente para a construção de
evidências de qualidade e, consequentemente, fornecer adequadas estratégias de intervenções para pacientes e familiares. Neste
contexto, o objetivo do trabalho foi avaliar o estado nutricional e o consumo alimentar de crianças portadoras do transtorno do
espectro autista (TEA).
2 Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018

MÉTODOS
Trata-se de um estudo de natureza quantitativa, descritiva, exploratória e transversal, realizado na associação de pais
denominada Diamante Azul, do município de Limoeiro do Norte, Ceará, Brasil, no período de março a junho de 2017.
Incluíram-se na pesquisa crianças com idade entre 3 e 10 anos, de ambos os sexos, com diagnóstico do TEA de acordo
com a classificação de doenças CID-10(11), segundo Manual de Diagnóstico e Estatística de Doenças Mentais da Academia
Americana de Psiquiatria (DSM-IV-TR)(12), e cujo pai ou responsável tenha permitido a participação através da assinatura do
termo de consentimento livre e esclarecido.
Os dados foram coletados através de visita domiciliar, durante os meses de março a junho de 2017, na qual se realizou
entrevistas, ordenadas por um questionário sociodemográfico composto por questões objetivas e subjetivas, histórico nutricional,
três recordatórios de 24 horas (dois dias referente à semana e outro do final de semana), bem como, houve coleta das medidas
antropométricas.
Realizou-se a entrevista após assinatura do Termo do Assentimento do Menor e Termo de Consentimento Livre e Pós-
Esclarecido pelos pais das crianças participantes da pesquisa, sendo acentuados os aspectos pertinentes à autonomia, ao sigilo
e à confidencialidade dos dados.
O questionário sociodemográfico foi aplicado a fim de identificar idade, renda familiar, escolaridade, tratamento
psicofarmacológico, idade recebida do diagnóstico do TEA, classificação da CID-10(11) e histórico clínico dos pacientes.
Avaliou-se a estimativa do consumo de alimentos por meio do inquérito recordatório alimentar de 24 horas de três dias,
todos aplicados de forma presencial na residência do participante. Este método consiste na obtenção de informações verbais,
referente à ingestão alimentar das últimas 24 horas, contendo dados sobre os alimentos e bebidas, bem como peso/tamanho das
porções consumidas(13). Cabe ressaltar que o inquérito aplicado foi respondido pelos responsáveis das crianças. Determinou-
se o valor energético total (VET), bem como o cálculo das quantidades de carboidratos, proteína e lipídios, obedecendo a
recomendação de 45% a 65%, 10% a 30%, e 25% a 35%, respectivamente(14). Neste estudo, considerou-se margem de ingestão
de 10% do recomendado, ou seja, foi considerada adequada a ingestão entre 90% a 110% do recomendado; inferior a 90%,
ingestão abaixo do recomendado; e, superior a 110%, ingestão acima do recomendado.
Utilizou-se a Tabela Brasileira de Composição de Alimentos(15) como referência para obtenção da composição nutricional
dos alimentos. Após os cálculos, compararam-se os valores com as ingestões dietética de referência (DRIs) de acordo com a
necessidade média estimada (EAR), ou Ingestão Adequada (AI), e Limite Superior Tolerável de Ingestão (UL)(16), considerando
sexo e idade de cada paciente. Avaliaram-se os seguintes micronutrientes: cálcio (Ca), magnésio (Mg), fósforo (P), ferro (Fe),
potássio (K), sódio (Na), vitamina A (A), vitamina B1 (B1), vitamina B2 (B2), niacina, Vitamina B6 (B6), vitamina C (C) e
ainda fibras e colesterol.
Os parâmetros antropométricos utilizados foram peso, altura, circunferência do braço e as dobras cutâneas tricipital e
subescapular. Aferiu-se o peso em uma balança eletrônica do tipo plataforma digital (Toledo®), com capacidade de 200kg.
A estatura e a circunferência do braço (CB) foram aferidas utilizando-se fita métrica inextensível (Cescorf®) com precisão
de 1mm. Para aferição da estatura, os participantes eram encostados na parede, descalços com os pés paralelos, os tornozelos
juntos, em posição ereta, os braços ao longo do corpo, com a cabeça posicionada de forma que a parte inferior da órbita ocular
estivesse no mesmo plano do orifício externo do ouvido(17). A circunferência braquial foi realizada no ponto médio entre
o acrômio e o olecrano(18). Para a coleta das dobras cutâneas (tricipital e subescapular), utilizou-se um adipômetro clínico
(Cescorf®) que exerce pressão de 10g/mm2 com precisão de 1 mm.
A partir desses parâmetros, realizou-se, então, a classificação do estado nutricional de cada participante, seguindo os índices
peso para idade (P/I), estatura para idade (E/I), peso para estatura (P/E) e índice de massa corpórea para idade (IMC/I) descritos
pelo SISVAM(19), segundo o escore Z. Para a medida da circunferência braquial, foram calculados os percentuais de adequação
para: circunferência braquial (% CB), circunferência muscular do braço (% CMB), circunferência muscular do braço corrigida
(% CMBc) e dobra cutânea tricipital (% DCT)(20). Para classificar a reserva de adiposidade, utilizaram-se os percentis de DCT
(dobra cutânea tricipital), DCS (dobra Cutânea Sub- escapular) e SDTS (soma das dobras cutâneas tricipital e subescapular)(18).
Utilizou-se o software Statistical Package for Social Sciences, versão 21.0 (SPSS® Inc, Chicago, IL), para análise
descritiva, e as variáveis contínuas foram expressas em média ± desvio padrão e coeficiente de variação.
O estudo recebeu aprovação do Comitê de Ética em Pesquisa (CEP) do Instituto Federal de Educação, Ciência e Tecnologia
do Ceará (IFCE), conforme Parecer n° 2.160.713.
RESULTADOS
Os participantes da pesquisa (n= 26), apresentaram idade média de 7 anos (±2), não estando nenhum participante com idade
inferior aos três anos. O presente estudo identificou que 24 (92,31%) crianças da amostra eram do sexo masculino e apenas
2 (7,69%) do sexo feminino. Todas as crianças participantes da pesquisa possuíam diagnóstico fechado com classificação do
CID-10(11). Destas, 24 (92,4%) possuíam classificação para F84.0 (Autismo infantil) e 2 (7,6 %) se classificaram dentro do

F84.0 mais associações F71.0 (Retardo mental moderado) e F90.0 (Transtornos hipercinéticos). Os dados demonstraram que
50% (n=13) das famílias possuem renda familiar entre 1 a 1,5 salários mínimos.
Os resultados apontaram que 69,2% dos participantes (n=18) faziam tratamento psicofarmacológico. Dentre os fármacos
mais consumidos está a risperidona, com 42,3% (n=11). Além do tratamento psicofarmacológico, 21 (80,8 %) crianças realizam
terapias multiprofissionais com psicólogos, terapeutas ocupacionais e fonoaudiólogos, pelo menos uma vez por semana, como
também toda a amostra frequenta escolas ou creches. (Tabela I).
Tabela I - Descrição dos dados socioeconômicos das crianças de 3 a 10 anos com diagnóstico de transtorno do espectro autista.
Limoeiro do Norte, Ceará, 2017.
Variáveis Categorias n %
Sexo
Feminino 2 7,69
Masculino 24 92,31
Idade
De 3 a 4 anos 4 15,4%
De 5 a 6 anos 8 30,8%
De 7 a 8 anos 8 30,8%
De 9 a 10 anos 6 23,1%
Renda Familiar
< 1 salário mínimo 1 3,8%
1 salário mínimo 13 50,0%
De 1,5 até 2 salários mínimos 8 30,8%
Mais de 2 salários mínimos 4 15,4%
Possui Laudo Diagnóstico
Sim 26 100,0%
Não 0 0,0%
Idade de Diagnóstico
<3 anos 5 19,2%
De 3 a 4 anos 16 61,5%
De 5 a 6 anos 2 7,7%
De 7 a 8 anos 2 7,7%
De 9 a 10 anos 1 3,8%
Classificação no CID-10
F84.0 24 92,4%
F84.0/F71.0 1 3,8%
F84.0/F90.0 1 3,8%
Uso de Medicação
Sim 18 69,2%
Não 8 30,8%
Medicação utilizada
Rispiridona 11 42,3%
Ritalina 4 15,4%
Neuleptiil 3 11,5%
Depaquer 3 11,5%
Outros 5 19,2%
Frequenta escola
Sim 26 100%
Não 0 0%
Terapia multiprofissional
Sim 21 80,8%
Não 5 19,2%
Legenda: F84.0: Autismo infantil; F71.0: Retardo mental moderado; F90.0: Transtornos hipercinéticos
Na avaliação do consumo de energia estabeleceram-se faixas de classificação. Os valores inferiores a 90% da recomendação
foram considerados consumo abaixo da recomendação; de 90% a 110%, adequado; e acima da recomendação para 110%. Os
valores encontrados podem ser observados na Figura 1.

90% 0,88
80%
70% 0,65
60% 0,58
0,54
50%
40% 0,38
0,35
0,31
30%
20%
0,12
10% 0,08
0,04 0,04 0,04
0%
EER CARBOIDRATO PROTEINA LIPÍDIO
ABAIXO ADEQUADO ACIMA
Figura 1 - Descrição entre o percentual de adequação do gasto calórico (EER) e do consumo de macronutrientes em crianças
autistas. Limoeiro do Norte, Ceará, 2017.
Legenda: EER: Necessidade energética
Para avaliação da adequação dos macronutrientes, utilizou-se a recomendação da IOM(14), que indica: 45% a 65% para
carboidrato, 10% a 30% para proteína, e 25% a 35% para lipídios, com faixa etária 4 a 18 anos.
Os resultados para carboidrato (57,69%, n= 15) e proteínas (88,46%, n=23) se encontravam adequados, o que difere para
o valor para lipídios (65%, n=17), que estava abaixo do recomendado. Entretanto, os resultados encontrados para o consumo
de energia (EER) estão acima do recomendado para 14 (53,85%) dos participantes da pesquisa.
A seguir, a Tabela II demonstra a adequação de minerais e vitaminas, respectivamente, encontrados com base nas
recomendações das DRIs(16).
Tabela II - Descrição dos resultados do consumo de minerais e vitaminas em crianças autistas segundo as DRIs (ingestões
dietética de referência). Limoeiro do Norte, Ceará, 2017.
Minerais Vitaminas
Consumo Ca Mg P Fe K Na A B1 B2 Niac. B6 C

(mg) (mg) (mg) (mg) (mg) (mg) (mcg) (mg) (mg) (mg) (mg) (mg)
46,15% 0,00% 80,77% 88,46% 100,00% 26,92% 23,08% 84,62% 69,23% 26,92% 42,31% 69,23%
POS ADE
(n=12) (n=0) (n=21) (n=23) (n=26) (n=7) (n=6) (n=22) (n=18) (n=7) (n=11) (n=18)
POS 50,00% 42,31% 19,23% 7,69% 0,00% 69,23% 76,92% 15,38% 30,77% 19,23% 57,69% 30,77%
INAD (n=13) (n=11) (n=5) (n=2) (n=0) (n=18) (n=20) (n=4) (n=8) (n=5) (n=15) (n=8)
POS 3,85% 57,69% 0,00% 3,85% 0,00% 3,85% 0,00% 0,00% 0,00% 53,85% 0,00% 0,00%
NOC (n=1) (n=15) (n=0) (n=1) (n=0) (n=1) (n=0) (n=0) (n=0) (n=14) (n=0) (n=0)
Legenda: POS ADE: possivelmente adequado; POS INAD: possivelmente inadequado; POS NOC: possivelmente nocivo; Ca: cálcio; Mg:
magnésio; P: fósforo; Fe: ferro; K: potássio; Na: sódio; mg: miligrama; vitamina A; B1: vitamina B1; B2: vitamina B2; Niac.: niacina; B6:
vitamina B6; C; vitamina C; mcg: micrograma
Entre os minerais, identificou-se uma possível inadequação no consumo de cálcio (50%, n=13) e sódio (69,23%, n=18). O
magnésio obteve resultado possivelmente nocivo (57,69%, n=15). Já para o fósforo, ferro e potássio, encontraram-se resultados
possivelmente adequados, com 80,77% (n=21), 88,46% (n=23) e 100% (n=26) respectivamente.

Os dados apresentados para o consumo de vitaminas demostraram uma inadequação para vitamina A (77%, n=20) e B6
(58%, n=15), estando abaixo das recomendações.
Foi encontrado o consumo dentro do recomendado para as vitaminas B1(84,62%, n=22), B2 (69,23%, n=18) e C (69,23%,
n=18), estando apenas a niacina como possivelmente nociva.
Os resultados encontrados para a porcentagem de fibras consumidas pelas crianças foram 10 (38%) adequadas, entretanto
9 (35%) foram nocivas e 7 (27%) inadequadas. O consumo de fibra dietética pode estar associado aos hábitos intestinais dos
participantes à medida que 20 (77%) realizavam a defecação pelo menos uma vez todos os dias, enquadrando-se como 2 ou 3
na Escala de Bristol.
A Tabela III demonstra o estado nutricional dos portadores de TEA e destaca que 10 (38,5%) apresentaram sobrepeso
(23,1%, n=6) e obesidade (15,38%, n=4) pelo IMC/I (Índice de Massa Corporal para Idade), outras 10 crianças (38,5%)
apresentaram risco de sobrepeso e 20 crianças (76,98%) risco de obesidade. Resultado semelhante aos dados referentes ao
parâmetro de IMC/I, o índice peso para estatura (P/E), no qual a soma do percentual de crianças com risco de sobrepeso
(15,38%, n=4), sobrepeso (26,9%, n=7) e obesidade (19,23%, n=5) ultrapassa o percentual de crianças com diagnóstico de
eutrofia (38,46%, n=10).
O índice P/I evidenciou que a maioria das crianças (57,69%, n=15) estava com peso adequado para idade, não deixando de
ser relevantes os valores referentes às crianças que apresentaram peso elevado para idade (38,5%, n=10). A E/I apresentou-se
como adequada em toda a amostra avaliada.
Tabela III - Classificação do estado nutricional de crianças autistas. Limoeiro do Norte, Ceará, 2017.
Classificação do estado nutricional

IMC para idade (IMC/I)
Magreza Eutrofia Risco de sobrepeso Sobrepeso Obesidade
3,85% (n=1) 19,23% (n=5) 38,46% (n=10) 23,08% (n=6) 15,38% (n=4)
Peso para idade (P/I)
Baixo peso para idade Peso adequado para idade Peso elevado para idade
3,8% (n=1) 57,69% (n=15) 38,51% 9 (n=10)
Peso para estatura (P/E)
Eutrofia Risco de sobrepeso Sobrepeso Obesidade
38,46% (n=10) 15,38% (n=4) 26,93% (n=7) 19,23% (n=5)
Estatura para idade (E/I)
Estatura adequada para idade
100% (n=26)
Legenda: n: número de crianças; IMC: Índice de Massa Corporal
O perfil nutricional do público avaliado, segundo os percentuais de %CB, %CMB, %CMBc, %DCT, DCT, DCS e SDTS está
representado na Tabela IV. A classificação nutricional através do %CB, indica que 38,46% (n=10) apresentaram classificação
eutrófica. Entretanto, não pode ser considerado um valor positivo se este for associado aos 42,31% (n=11) referentes a sobrepeso
e obesidade, bem como 19,23% (n=5) de desnutridos moderados e leves. Para reserva de gordura, de acordo com as DCT, DCS
e SDCTS, encontrou-se que o maior número dos pacientes apresenta percentual de gordura em excesso (obesidade), 65,38%
(n=17), 76,92% (n=20) e 76,92% (n=20), respectivamente.

Estes resultados colaboram para a importância da avaliação nutricional (antropométrica, da composição corporal) dentro
da rotina clínica de pacientes com TEA e seus familiares, sempre considerando as características singulares de cada paciente.
Tabela IV - Classificação da composição corporal de crianças autistas. Limoeiro do Norte, Ceará, 2017.
Classificação de composição corporal

Circunferência braquial (%CB)
Desnutrição moderada Desnutrição leve Eutrofia Sobrepeso Obesidade
7,69% (n=2) 11,54% (n=3) 38,46% (n=10) 19,23% (n=5) 23,08% (n=6)
Circunferência muscular do braço (%CMB)
Desnutrição grave Desnutrição moderada Desnutrição leve Eutrofia
11,54% (n=3) 7,69% (n=2) 26,92% (n=7) 53,85% (n=14)
Circunferência muscular do braço corrigida (%CMBc)
Normal Desnutrição leve moderada
73,08% (n=19) 26,92% (n=7)
Dobra cutânea tricipital (%PCT)
Desnutrição grave Desnutrição leve Eutrofia Sobrepeso Obesidade
7,69% (n=2) 3,85% (n=1) 7,69% (n=2) 7,69% (n=2) 73,08% (n=19)
Dobra cutânea tricipital (DCT)
Desnutrido Eutrofia Obesidade
3,85% (n=1) 30,77% (n=8) 65,38%(n=17)
Dobra cutânea subescapular (DCS)
Eutrofia Obesidade
23,08%(n=6) 76,92% (n=20)
Soma das dobras cutâneas tricipital e subescapular (SDTS)
Desnutrido Eutrofia Obesidade
3,85% (n=1) 19,23%(n=5) 76,92%(n=20)
Legenda: n: número de crianças
DISCUSSÃO
No presente estudo, a amostra analisada (n=26) apresentou uma maior prevalência para o sexo masculino, corroborando os
achados de estudos anteriores(13,21), que relatam maior incidência em pesquisas epidemiológicas do autismo, com uma média de
3,5 a 4 meninos para cada 1 menina; como também destacam o autismo como sendo 4 vezes mais comum em garotos do que
em garotas, numa escala de 5/10.000. A média de idade em que as crianças fecharam o diagnóstico foi aos 4 anos, sendo que
nenhuma crianças apresentou idade menor que três anos no presente estudo, fato que se associa à dificuldade de confirmação
do diagnóstico precoce desse transtorno(13). O diagnóstico para o TEA é eminentemente clínico, e deve ser realizado a partir dos
critérios da CID-10(11), através de anamnese com os pais, responsáveis legais e/ou cuidadores, bem como da observação clínica
do comportamento(3).
Os dados avaliados na presente pesquisa mostram que 50 % das famílias possuíam renda familiar entre 1 a 1,5 salários
mínimos. Esse resultado é similar ao observado em outro estudo(22), em que 56% das famílias entrevistadas tinham renda
familiar semelhante.
Encontrou-se elevada prevalência de autistas (69,2%) que fazem tratamento psicofarmacológico na atual pesquisa. Dentre
os fármacos mais utilizados, está a risperidona, com 42,3%, a qual tem evidenciado resultados positivos, que incluem a redução
de agressividade, da irritabilidade e do isolamento; porém, seus efeitos adversos mais comuns são sonolência, tontura, sialorreia
e ganho ponderal. O uso de risperidona está associado também a alterações metabólicas, como aumento da resistência à insulina,
hiperglicemia, hipertensão arterial e dislipidemia(23). Os fármacos de ação no sistema nervoso central não são utilizados com
a finalidade de cura, mas de alívio de sintomas. A participação de 100% da amostra em escolas ou creches auxilia a criar
alternativas para a inclusão social, pois o contato com terapias multiprofissionais amplia suas formas de se expressarem e se
comunicarem(24).
Os resultados do presente estudo se encontravam adequados para carboidrato (57,69%) e proteínas (88,46%), o que
difere para o valor para lipídios (65%), que se encontrava abaixo do recomendado. Vale ressaltar que dietas com quantidades
insuficientes de gordura podem levar a redução da absorção de alguns micronutrientes, como vitaminas lipossolúveis(25).
Entretanto, os resultados encontrados para o consumo de energia (EER) estavam acima do recomendado para 53,85% dos
autistas. Tais achados possivelmente se associam aos frequentes erros alimentares característicos do transtorno, como a
seletividade e a compulsão alimentar. A seletividade alimentar varia de criança para criança, constituindo um problema quando
interfere na rotina diária e social(26). Relatos e testemunhos de pessoas com TEA sugerem que as características sensoriais dos
alimentos, como o odor, a textura, a cor e a temperatura, possam contribuir para a seletividade alimentar(27,28).

É muito comum as crianças autistas possuírem deficiências nutricionais, pois a maioria apresenta uma alimentação
monótona. Porém, mesmo que a criança possua uma dieta variada e adequada nutricionalmente, ela precisa ser capaz de
executar três funções básicas que, infelizmente, não são feitas pela maioria: digerir e quebrar adequadamente o alimento até
uma forma absorvível, absorver os nutrientes através do TGI saudável e converter os nutrientes em uma forma utilizável em
nível celular(25).
As deficiências de micronutrientes mais comuns nos TEA são das vitaminas B1, B3, B5, B6, B9, B12, A e dos minerais
cálcio (Ca), zinco (Zn), selênio (Se) e magnésio (Mg)(29). No atual trabalho, pôde-se observar uma possível inadequação
(50%) de cálcio (Ca), que está diretamente associado a diversas funções orgânicas, como modulação de sinais de transdução,
metabolismo de produção de energia e proliferação celular, estando os sintomas resultantes de sua deficiência associados à:
ansiedade, depressão, hiperatividade, agitação, alucinações, irritabilidade, nervosismo, agressão, estresse crônico, dificuldade
de aprendizagem e perda de memória(30). Em relação à ingestão de vitamina A, o resultado da atual investigação apontou
para uma possível inadequação. Essa deficiência, em crianças, pode causar falha de crescimento e danos oculares, como a
xeroftalmia(30).
Nutrientes como a vitamina B6 são de extrema importância para a metilação, transulfatação e sulfatação, que representam
um conjunto de atividades bioquímicas que não funcionam adequadamente nos portadores com TEA. Quando da limitação
dessas transformações metabólicas, os neurotransmissores não são adequadamente ativados, ocasionando sintomas de ansiedade,
depressão, déficit de atenção e transtorno do sono. Isto associado ao maior consumo de alumínio, mercúrio, glutamato e várias
substâncias artificiais ingeridas na alimentação, favorecem o acúmulo no organismo e proporcionam alterações cerebrais que
acarretam irritabilidade, agressividade, hiperatividade(13). A limitação do consumo desses nutrientes junto à conversão das
enzimas parece ser uma das razões para a deficiência de componentes essenciais para o organismo das crianças autistas, como:
sulfato, cisteína, taurina e glutationa, levando aos prejuízos da maioria dos processos metabólicos encontrados(25).
A ingestão adequada de outras vitaminas, como B1 e niacina, é de grande importância para as crianças autistas, já que sua
deficiência é caracterizada por sinais neurológios, podendo intensificar os sintomas relacionados ao transtorno, já que impede a
conversão do acetaldeído (substância neurotóxica) nas crianças autistas, prejudicando sua eliminação pelo organismo. Isto pode
afetar estruturas cerebrais, vindo a interferir no desenvolvimento neural dos autistas(23).
Além da grande importância associada à avaliação dos micronutrientes, deve ser dada atenção ao papel desempenhado
pelas fibras dietéticas na modulação da saúde intestinal. O consumo adequado de fibras deve proporcionar um funcionamento
normal do intestino, prevenir câncer relacionado à dieta e diminuir a concentração sérica de colesterol para redução de risco
de doença cardiovascular. Sua inadequação pode afetar as funções neurológicas por diversos mecanismos, refletindo em
muitos dos sintomas vistos no TEA(25). Estudos(25,31) demonstraram que a digestão dos alimentos pelas crianças autistas está
prejudicada, normalmente, devido à baixa sulfatação e transulfatação, processos essenciais na digestão, para a integridade da
mucosa intestinal, a destoxificação e o equilíbrio da microbiota, podendo interferir na absorção de micronutrientes e ocasionar
baixos níveis de minerais e vitaminas, entre outros compostos.
Além dos parâmetros relacionados à ingestão alimentar, a avaliação física e antropométrica adequadas podem contribuir
para o melhor entendimento do quadro clínico dos autistas. Desse modo, com relação ao estado nutricional das crianças
portadoras do TEA, trabalhos(13,32) têm sido realizados para avaliar o perfil nutricional dessa população.
Observações clínicas mostram que essas crianças apresentam maior risco de excesso de peso, pois possuem grandes
dificuldades em praticar exercícios físicos de forma estruturada, além do isolamento social, o que possibilita o aumento de
sedentarismo e corrobora os dados desta pesquisa, em que todos os participantes não praticam nenhuma atividade física. Porém,
o sobrepeso e a obesidade são problemas de saúde pública na população em geral, pois a incidência de muitas doenças crônicas
na vida adulta está diretamente ligada à obesidade na infância(33).
Na criança, a obesidade aumenta o risco de problemas em curto e longo prazos, como diabetes, doenças cardiovasculares
e psicossociais. Estudo internacional indica que crianças e adolescentes com TEA podem ser particularmente vulneráveis a
essas alterações ponderais(10). Avaliou-se 30 crianças de uma escola especial de Campo Grande, Brasil e observou-se que
quatro (13,3%) estavam obesas e sete (23,3%) com baixo peso. Como também avaliaram 23 crianças e adolescentes autistas
e observaram que três (13%) apresentavam baixo peso, cinco (21,7%) tinham sobrepeso e seis (26,1%) exibiam obesidade(7).
Embora os métodos de avaliação apresentados estabeleçam um perfil nutricional geral de pacientes portadores de TEA, faz-se
necessária à associação de métodos de avaliação da composição corporal, pois permitem obter um diagnóstico nutricional mais
preciso.
A adequação do percentual da CB é um parâmetro nutricional antropométrico recomendado pela OMS para estimativa da
proteína musculoesquelética total, fornecendo índice de depósito de gordura e massa muscular local, e pode ser considerada
uma medida independente, sendo uma das medidas mais utilizadas na avaliação nutricional(34). O percentual de adequação
da CMB avalia a reserva de tecido muscular sem correção da área óssea (não leva em consideração o diâmetro do osso). O
diagnóstico encontrado para esse parâmetro aponta que 53,85% das crianças com TEA do atual estudo estavam eutróficas.
Em contrapartida, 46,15% apresentavam desnutrição leve à desnutrição grave. A utilização dessa fórmula para o cálculo da

adequação da CMB pode subestimar a perda muscular em até 20 a 25%, uma vez que a área óssea é incluída no cálculo,
mascarando assim a perda corporal.
O diagnóstico apresentado para o percentual de adequação da CMBc, que avalia a reserva de tecido muscular corrigindo a
área óssea (leva em consideração o diâmetro do osso), apresentou-se normal para 78,03% (n=19) das crianças, refletindo mais
adequadamente a verdadeira magnitude das mudanças do tecido muscular do que o percentual de adequação da CMB(20). O estado
nutricional, segundo o percentual de adequação da DCT, revela uma maior prevalência para obesidade com 73,08% (n=19).
Essa dobra cutânea é a mais utilizada na prática clínica para monitoramento do estado nutricional por ser mais representativa
da distribuição de gordura corporal, independentemente da idade e do sexo, pois se correlaciona de forma significativa com o
peso corporal e a massa gorda(18,20). De acordo com a OMS(11), tem-se notado aumento da prevalência de obesidade na infância
e na adolescência, independentemente do método de classificação antropométrico utilizado.
Esse fato demanda atuações voltadas para a promoção da saúde, como uma política de alimentação saudável, ações que
incentivem a atividade física regular e atividades culturais de lazer. Os estudos ainda não determinaram o tratamento ideal que
engloba o contexto nutricional, controle comportamental, medicação, aspectos físicos e educacionais(29). A intervenção dietética
tem como objetivo melhorar a saúde física e bem-estar desses indivíduos, sendo essencial o acompanhamento nutricional junto
às crianças autistas, contribuindo na correção de erros alimentares, bem como na promoção da saúde e da qualidade de vida. O
diagnóstico precoce é o único consenso em todo o mundo no que diz respeito ao autismo(35).
O desenvolvimento de novas pesquisas e a continuação de novos estudos é importante para melhorar a forma de abordagem
profissional; consequentemente, também a qualidade de vida e a saúde desses pacientes. O presente estudo apresenta algumas
limitações, pois a amostra é de uma área limitada do nordeste do país e pode não representar as mesmas características para
outros grupos de crianças com TEA.
CONCLUSÃO
As crianças com o Transtorno do Espectro Autista avaliadas demonstraram elevados índices de sobrepeso e obesidade,
repertório alimentar limitado, elevada inadequação na ingestão de vitaminas (A e B6) e do mineral cálcio, o que pode estar
associado ao alto consumo de alimentos ricos em calorias e pobres em micronutrientes.
CONFLITO DE INTERESSES
Não houve conflito de interesses no presente estudo.
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Endereço para correspondência:

Maria Vanuza Caetano
Rua Estevão Remígio de Freitas, 1145
Bairro: Monsenhor Otávio
CEP: 62930-000 - Limoeiro do Norte - CE - Brasil
E-mail: vanuza.caetano@hotmail.com

relato de caso Seletividade alimentar: uma abordagem nutricional

Selective eating: a nutritional approach
Ana Beatriz de Mello Sampaio1, Thais Lourenço Nogueira1, Ruth Bartelli Grigolon1, Ana Maria Roma1,
Leticia Enrique Pereira1, Karin Louise Lenz Dunker1
RESUMO
A seletividade alimentar é caracterizada por recusa alimentar, pouco apetite e desinteresse

pelo alimento. É um comportamento típico da fase pré-escolar, mas, quando presente em
ambientes familiares desfavoráveis, pode acentuar-se e permanecer até a adolescência. Este
artigo trata de um relato de caso em que o paciente, com diagnóstico de seletividade ali-
mentar, inicia tratamento em serviço especializado de transtornos alimentares aos 14 anos. A
particularidade deste caso é a rápida e boa evolução do quadro, possivelmente decorrente
do desejo próprio de se tratar e do apoio recebido pela família. A análise do caso em questão
aponta para a importância de identificar os casos de seletividade de forma correta e precoce
Palavras-chave para que eles sejam encaminhados o quanto antes a profissionais habilitados no tratamento
Seletividade, alimentação, de distúrbios alimentares nos diferentes estágios de desenvolvimento da infância e adoles-
avaliação nutricional. cência, resultando em melhor prognóstico do quadro.
ABSTRACT
Selective eating is known by food refusal, lack of appetite and interest in food. It is atypical
behavior of preschool, but when present in adverse family environments, can perpetuate
and remain until adolescence. This paper is a case report of a patient with diagnosis of se-
lective eating that seeks for specialized treatment center for eating disorders at 14 years old.
The particularity of this case is the fast and good evolution, possibly due to the desire to treat
himself and the support received by the family. The case points out the importance of identi-
Keywords fying earlier and correctly cases of selectivity so they will be submitted as soon as possible to
Selective, eating, evaluation professionals specialized in treatment of eating disorders in different developmental stages
nutritional. of childhood and adolescence, resulting in a better prognosis framework.
1 Universidade Federal de São Paulo (Unifesp), Departamento de Psiquiatria e Psicologia Médica, Programa de Atenção aos Transtornos
Alimentares (Proata).
Recebido em
14/2/2013
Aprovado em
18/5/2013
Endereço para correspondência: Karin Louise Lenz Dunker

Universidade Federal de São Paulo, Departamento de Psiquiatria
Rua Borges Lagoa, 570, 7o andar – 04038-020 – São Paulo, SP, Brasil
E-mail: kdunker00@yahoo.com.br
relato de caso Seletividade alimentar 165
INTRODUÇÃO porcentagem de proteínas, carboidratos, gorduras, além do

consumo de cálcio, ferro e vitamina A.
A recusa alimentar é um comportamento típico da primei- Utilizou-se a balança digital Filizola com estadiômetro e
ra infância, caracterizado por comportamentos como: fazer capacidade máxima de 200 kg para aferição de peso e esta-
birras, demorar a comer, tentar negociar o alimento que será tura. O estado nutricional foi avaliado a partir do índice de
consumido, levantar da mesa durante a refeição e beliscar ao massa corporal (IMC) e analisado por meio das curvas de
longo do dia. No entanto, parece haver crianças que persis- crescimento da Organização Mundial da Saúde (2006/2007)
tem com comportamentos peculiares até meados da infân- para estatura e IMC para idade e sexo. Consideram-se norma-
cia ou continuam pelas demais fases da vida. Esses compor- lidade os valores encontrados entre o P3 e P85. Nesse caso,
tamentos seriam definidos como seletividade alimentar (SA), o traçado preto indica antes e o cinza, durante o tratamento
que é caracterizada por um consumo alimentar altamente nutricional (vide legenda nas Figuras 1 e 2).
limitado e extrema resistência em experimentar novos ali- O tratamento foi realizado em serviço público especiali-
mentos. Esse tipo de comportamento resulta em uma limita- zado (hospital/escola), sendo parte do protocolo o Termo de
ção das atividades sociais relacionadas à alimentação1,2. Consentimento Livre e Esclarecido dos pacientes.
De acordo com os sistemas de classificação DSM-IV e CID-
10, a SA não está descrita como um diagnóstico específico
de transtorno alimentar na infância, mas ambos associam-na
a uma dificuldade persistente em comer adequadamente, RELATO DE CASO
com falha no ganho de peso ou importante perda ponderal
durante o último mês. Não existem condições fisiopatológi- Encaminhado para tratamento especializado em transtornos
cas ou desordens mentais associadas, não há falta de acesso alimentares, VNS é um paciente de 14 anos, sexo masculino,
aos alimentos e o distúrbio deve ter início antes dos 6 anos1,2. natural de São Paulo-SP, estudante da nona série do ensino
Os dados de prevalência de SA são escassos, mas vêm fundamental, apresenta bom desempenho escolar e mora
sendo caracterizados nas percepções e relatos de pais e cui- com os pais e um irmão de 9 anos.
dadores. Segundo eles, a seletividade seria mais frequente A avaliação psiquiátrica indicou a queixa principal de SA
em crianças de 4 a 24 meses (19% a 50% dos casos)3. A im- com prejuízo na vida social. Observou-se ausência de tristeza e
precisão sobre a definição exata da SA, assim como a varia- de sintomas psicóticos, compulsão alimentar ou desconforto
bilidade de critérios metodológicos utilizados nos estudos, com o peso e forma corporal, mas demonstrou preocupação
dificulta a determinação da prevalência. Não há evidências com o crescimento físico. Relatou que desde os 10 anos de
que relacionem o quadro de SA com comportamentos ca- idade sente-se desconfortável ao comer diante de outras pes-
racterísticos de transtornos alimentares, como: fazer dieta, soas, sugerindo um diagnóstico de fobia social. Foi prescrita
episódios de compulsões alimentares e controle obsessi- sertralina 25 mg/dia inicialmente com aumento posterior, até
vo do peso corporal2. A ausência de dados consistentes de se alcançar a dose de 50 mg/dia, para melhorar o comporta-
prevalência, definição do distúrbio e de suas características, mento social e facilitar o convívio com os amigos. O paciente
principalmente em termos das escolhas alimentares, justifi- foi encaminhado para acompanhamento nutricional na Insti-
ca a descrição do relato de caso de seletividade alimentar, tuição e sugeriu-se acompanhamento psicológico com abor-
fornecendo informações que podem auxiliar profissionais da dagem em terapia cognitivo-comportamental (TCC).
área da saúde na identificação e no tratamento do distúrbio. O primeiro atendimento nutricional incluiu uma inves-
tigação sobre a história alimentar do paciente desde o seu
nascimento: o aleitamento materno exclusivo ocorreu por
METODOLOGIA apenas 5 dias; as frutas foram incluídas na alimentação aos
5 meses e a sopa liquidificada, aos 6 meses, permanecendo
O caso foi escolhido considerando a evolução nutricional como parte da dieta até 1 ano de idade.
positiva. Os dados foram coletados no prontuário clínico e Desde então iniciou recusa da sopa e de novos alimen-
nutricional. tos oferecidos pela mãe e manteve somente o consumo de
A avaliação da dieta quanto à qualidade e quantidade foi leite com Sustagem®, sucos e frutas. A mãe ofereceu vários
realizada por meio do recordatório alimentar de 24h e sua alimentos em diversas formas de apresentação e, após mui-
evolução, acompanhada por meio do Registro Alimentar. Os tas tentativas, ele aceitou cebola em rodelas e batata frita
valores nutricionais foram calculados por meio do programa palito, o que demonstra preferência por alimentos de cor
de análise nutricional (Avanutri) e comparados com os pa- clara, sendo esta a base de suas refeições durante a infância.
drões recomendados pelo National Research Council (NRC) Com o tempo, estabeleceu um cardápio próprio que incluiu
e Standing Committee on the Scientific Evaluation of Dietary batata smile assada, biscoitos, pães, salgadinhos industriali-
Reference Intakes (DRI Committee) para valor calórico total, zados, leite integral, refrigerantes e alguns sucos de frutas.
J Bras Psiquiatr. 2013;62(2):164-70.

166 Sampaio ABM et al. relato de caso
De 4 a 8 anos de idade, quando a família passava o dia Maier et al.6 ressaltam que o número de exposições ao
fora de casa, os pais ofereciam salgadinhos industrializados e novo alimento seria mais determinante para a aceitação do
biscoitos, pois a criança não consumia alimentos desconhe- que a variedade, sendo importante alternar as formas de
cidos. Nesse período recebeu tratamento psicológico espe- apresentação, preparação e textura6-8. A eficácia dessa con-
cífico a SA, no entanto não houve resposta, porque VNS se duta pôde ser observada em crianças de cidades europeias7.
apresentava indiferente ao problema. Neste caso foram oferecidos vários alimentos em diversas
Na consulta nutricional inicial, aos 14 anos e 8 meses de formas de apresentação ou preparação, mas com difícil acei-
idade, observou-se nas curvas de crescimento (Figura 1 e 2) tação. A maneira de oferecer alimentos também foi variada,
que ocorreram algumas variações na evolução da estatura como alternar entre dar comida na boca ou deixar comer
e peso, porém em nenhum momento afastou-se demasia- sozinho, assim como dispor os alimentos em pratos em dife-
damente da média esperada (P50) para o IMC e na estatura rentes locais da casa.
para idade. Encontrava-se no estágio 3 de Tanner4, ou seja, Segundo Bryant-Waugh et al.11, o quadro clínico da SA
em fase anterior à velocidade máxima de crescimento. está normalmente associado ao consumo de alimentos
Não foi encontrada nenhuma alteração significativa nos de uma tonalidade (como branco) e de sabor muito sua-
exames bioquímicos que justificasse a SA. Apesar disso, o ve (como leite, pão, macarrão etc.) ou determinada textu-
paciente fazia uso de suplemento multivitamínico e mineral ra (pastoso ou crocante), com recusa de preparações com
há três anos, prescrito pelo pediatra por causa da limitada alimentos de texturas variadas. Em certos casos é possível
observar preferência por determinadas marcas de alimen-
variedade na alimentação.
tos ou mesmo a temperatura em que são servidos (frios ou
quentes). Além disso, algumas crianças não toleram o odor
de alimentos que não fazem parte de sua restrita lista de
DISCUSSÃO preferências, prejudicando seu convívio com familiares e
amigos no momento das refeições. Neste caso, o paciente
A literatura é escassa em estudos de casos de crianças com
apresentava preferência por alimentos de cor clara, batata,
seletividade alimentar, porém Timimi et al.5 acompanharam
biscoitos, pães e frutas, e alimentos com textura como purês
durante quatro anos 33 crianças seletivas, com idades entre
que ainda não são aceitos.
4 e 14 anos. Dois terços da amostra eram meninos e a mi-
De acordo com a literatura, algumas crianças mostram-se
noria apresentava déficit de crescimento e ganho ponderal,
ansiosas diante de alimentos com molho ou que são fáceis
assim como o paciente deste caso. As crianças eram ansiosas
para se sujar, comportamento que pode estar associado a
e apresentavam sintomas obsessivo-compulsivos com a co-
mães obsessivas por limpeza ou que não permitiram ao filho
mida e outros comportamentos não relacionadas com ela.
usar as mãos para alimentar-se5. Alimentos com molho nun-
As refeições representavam momentos de conflito, especial- ca foram aceitos pelo paciente, mas neste caso não se deve
mente em crianças menores, e quando já não haviam muitas às atitudes inadequadas da mãe.
tentativas para mudanças de hábitos alimentares, situações A família e os cuidadores são fundamentais para a for-
observadas neste caso. mação e a estruturação dos hábitos e condutas alimentares
De acordo com a literatura, a amamentação poderia faci- das crianças. Também são responsáveis por promover o am-
litar a aceitação de novos alimentos, visto que as caracterís- biente das primeiras experiências alimentares que podem
ticas sensoriais do leite materno são influenciadas pela dieta ser decisivas para a aceitação de novos alimentos3,12. Se um
da mãe e permite o primeiro contato da criança com sabores ambiente é agradável e novos alimentos são oferecidos sem
e odores variados6-8. Esse contato possibilitaria a melhora da coação, a criança fica muito mais suscetível a desenvolver
aceitação de novos alimentos quando oferecidos inicialmen- preferência por eles.
te. O curto período de aleitamento materno neste caso im- Neste caso, sempre coube à mãe a responsabilidade pela
possibilitou essa experiência, e até os 5 meses de idade ele alimentação da família, e ela procurou variar a alimentação
só conhecia o sabor do leite artificial. do filho, enquanto o pai pouco se envolvia na questão ali-
Crianças que apresentam atraso na introdução de alimentar e, quando o fazia, era sempre impaciente e rude com
mentos sólidos durante o primeiro ano de vida estão mais o filho, tinha atitudes inadequadas, como deixar o filho sem
propensas a desenvolver comportamentos seletivos ao comer quando ele se recusava a comer o que era oferecido.
longo da infância, sendo estes mais claramente identifica- O paciente relata que não tinha afinidade com o pai e havia
dos ao redor dos 7 anos de idade9,10. Neste caso houve boa pouco diálogo entre eles.
aceitação na introdução das frutas, porém houve recusa de Sabe-se que a desestruturação familiar pode influenciar
alimentos salgados sólidos até 1 ano de idade. Tal compor- a SA em algumas crianças13,14, sendo importante a cons-
tamento poderia predispor à SA, conforme indicam os estu- cientização do problema vivenciado, de forma a impor
dos mencionados. limites sem, contudo, perder o controle. Não podemos afir-
J Bras Psiquiatr. 2013;62(2):164-70.

mar que a família era desestruturada, contudo levava VNS A evolução positiva quanto à qualidade nutricional e à
ao descontrole emocional, situação que não colaborava variedade pode ser observada na tabela 1.
para a melhora do quadro. É importante ressaltar a reco- Conforme avaliação inicial (Tabela 1), identificou-se um
mendação de que os pais devem ponderar suas atitudes e déficit no valor calórico total da dieta consumida em relação
ambos devem participar ativamente no processo de edu- à sua necessidade energética diária. Em relação aos macro-
cação alimentar. nutrientes (proteínas, gorduras e carboidratos), observa-se
Em alguns casos, quando a criança não supre as expecta- que estes estavam adequados, o que não foi observado
tivas dos pais com relação ao consumo alimentar, o descon- entre os micronutrientes (Ca, Fe e vitamina A), que se en-
trole emocional que se instala coloca a criança no controle contravam abaixo do ideal para a faixa etária. Ao longo do
da situação, permitindo que ela mesma decida o que comer, tratamento, verificou-se que o paciente conseguiu aumentar
e não seus responsáveis13,14. É importante ressaltar que crian- seu consumo energético, chegando ao que é recomendado
ças seletivas normalmente apresentam comportamentos para sua idade. Os macronutrientes não sofreram modifica-
fóbicos diante de alimentos que não são de sua preferência, ções relevantes, por outro lado houve aumento evidente no
gerando confrontos e atitudes agressivas para evitar comer consumo de todos micronutrientes, apesar de somente o
o que não querem5. cálcio não ter alcançado o valor de referência. Estudos na-
A ansiedade dos familiares para ver a criança comer le- cionais demonstram que o consumo inadequado de cálcio
varia à substituição de alimentos saudáveis por aqueles de é muito frequente em adolescentes saudáveis16,17. Os resulta-
baixo valor nutritivo, os quais normalmente fazem parte das dos, portanto, indicam que o paciente atualmente apresenta
preferências dos seletivos. Dados demonstram que 45% um perfil nutricional semelhante ao de jovens de sua idade
dos pais gostariam de mudar os hábitos das crianças; para e que as deficiências nutricionais apresentadas inicialmente
isso, 51% oferecem recompensas e 69% persuadem seus fi- na dieta foram corrigidas.
lhos14,15. Com relação a esse tema, a mãe não permitia substi- Suas primeiras experiências com novos alimentos foram
tuir as refeições principais por guloseimas, com exceção dos positivas. Pôde-se observar na tabela 2 que VNS foi exposto a
dias em que a família passava o dia fora de casa. 71 tipos de alimentos e/ou preparações no percurso do trata-
O prejuízo social parece ser uma característica comum mento e, desse total, cerca de 65% (n = 46) foram aceitos e in-
no cotidiano dos indivíduos identificados com seletividade troduzidos na sua rotina alimentar. Diante disso, pode-se afir-
alimentar, especialmente quando a lista de alimentos acei- mar que o paciente vem apresentando boa resposta à terapia
táveis é muito restrita. A criança pode sentir-se intimidada nutricional, com receptividade para incluir novos alimentos.
pelos demais e, à medida que vai crescendo, seu constran- Quanto ao seu estado nutricional, pôde-se observar (Fi-
gimento sobre sua forma de alimentar-se a torna muito re- gura 1) que a seletividade não afetou o crescimento, man-
servada11. Esse foi um dos motivos que levaram o paciente a tendo a estatura dentro da normalidade, dos 9 aos 14 anos.
procurar tratamento. No entanto, verificou-se uma variação de peso ao longo dos
anos, que refletiu em períodos de aparente sobrepeso dos
Evolução do tratamento nutricional 9 aos 13 anos, mas que se normalizou após essa idade, pro-
Na primeira consulta nutricional, foi discutido com o pacien- vavelmente por causa da fase do estirão em que o paciente
te e sua mãe o conceito de neofobia alimentar, para que ele se encontra (Figura 2). Esses dados confirmam o que a lite-
não tenha expectativa de ter sucesso na primeira tentativa ratura aponta sobre o crescimento das crianças identificadas
de experimentar um novo alimento. com SA, o qual não necessariamente fica comprometido18.
Nas consultas que se seguiram, o paciente sempre vinha A restrição na variedade de alimentos consumidos na infân-
acompanhado da mãe, e esta e o paciente eram orientados cia não necessariamente tem impacto no estado nutricional,
quanto à introdução de um novo alimento, variando a tex- uma vez que muitas vezes a criança consome uma quantida-
tura e o preparo. O paciente era orientado a fazer o diário de maior desses alimentos específicos, sendo suficiente para
alimentar, para que se pudesse discutir em cada consulta os suprir suas necessidades energéticas.
alimentos que experimentou, apresentando os que aprovou Nas figuras 1 e 2, pode-se observar que houve estabiliza-
e os que não aprovou. ção em relação ao crescimento, pois o paciente se manteve
O paciente passou por 10 consultas nutricionais durante no P50. Passados 10 meses de tratamento, observa-se que
10 meses. A introdução de novos alimentos foi estimulada a o paciente realmente incluiu os alimentos de que gostou,
cada consulta, e a receptividade do paciente foi surpreenden- incorporando-os na sua rotina alimentar. Persiste ainda a re-
te, com aceitação de vários alimentos na primeira tentativa, sistência em aceitar preparações com consistência pastosa
que ocorreu depois da segunda consulta. Vale salientar que (purê de batatas) e com molho (feijão, estrogonofe). O au-
essa resposta positiva precoce se deu devido à aceitação por mento da variedade de alimentos permitiu melhorar suas
parte do paciente do diagnóstico de SA e à conscientização relações sociais e se envolver em atividades como almoçar
das consequências desse transtorno em suas relações sociais. com os amigos da escola.
J Bras Psiquiatr. 2013;62(2):164-70.

Tabela 1. Evolução do padrão nutricional nas consultas

Valores de
Padrão nutricional * 31/05 14/06 28/06 09/08 30/08 27/09 18/10 08/11
referência**
Valor calórico (kcal) 2.200 2.605 Não fez diário 2.721 2.750 2.972 3.006 3.237 2.932
% proteína 13 13,9 12,4 14,3 14,1 13,8 14,31 10 a 30
% carboidrato 61 58,9 64,2 56,9 54,0 57,5 63,38 45 a 65
% gordura 26 28 23,4 28,7 31,8 28,7 22,31 25 a 35
Cálcio (mg) 155,8 159,2 296,0 189,9 247,1 603,1 577,4 1.300
Ferro (mg) 4,6 6,4 8,1 12,4 13,5 13,4 18,8 11
Vitamina A (µg) 114 488,8 437,6 480,4 536,7 974,6 778,0 900
* Os valores nutricionais foram calculados pelo programa de análise nutricional (AVANUTRI).
** Valores recomendados pelo National Research Council (NRC) for energy e Standing Committee on the Scientific Evaluation of Dietary Reference Intakes (DRI Committee) para valor calórico, porcentagem de proteínas, carboidratos, gordura
e consumo de cálcio, ferro e vitamina A.
Tabela 2. Evolução da introdução dos alimentos nas consultas

14/06 28/06 09/08 30/08 27/09 18/10 08/11
Alimentos Cenoura ralada Beterraba Salada de feijão Arroz de forno com frango Risoto de camarão Salame Espaguete ao molho
introduzidos e Rúcula Agrião branco e milho Macarrão alho e óleo Peito de peru de calabresa
aceitos Camarão empanado Medalhão Salada de espinafre Couve-flor empanada frita Salada de lentilha e Costelinha de Arroz 7 grãos
Salsicha bovino Peixe empanado Macarrão ao pesto cenoura ralada porco Couve refogada
Filé de frango Batata Macarrão Lentilha sem caldo Bolinho de espinafre Acelga Carne de churrasco
Ovo frito assada Almôndega bovina Bolinho de arroz Morango (fraldinha)
Pizza de calabresa Polenta Salmão cru e grelhado Pão de queijo Bisteca de porco
Rolinho primavera Torrada
Uva Manteiga
Pera Requeijão
Abacaxi Esfirra de carne
Alimentos Bife (paillard fino) Brócolis Rabanete Couve-flor cozida Patê de atum com Tomate seco Ameixa
introduzidos e Pepino Purê de batata Vagem maionese Abobrinha Abóbora refogada
não aceitos Laranja Mandioquinha Geleia refogada Espinafre refogado
Mexerica Mandioca frita Capelleti Palmito
Arroz branco Pimentão Carne seca
Manga
Amora
Figura 1. Curvas de referência para estatura para idade e sexo de acordo com as normas da Organização Mundial da Saúde (2006
e 2007).
J Bras Psiquiatr. 2013;62(2):164-70.

Figura 2. Curvas de referência para índice de massa corporal para idade e sexo de acordo com as normas da Organização
Mundial da Saúde (2006 e 2007).
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2008;41(7):626-34.
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2004;104(S1):s57-64.
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ence from birth through weaning: contrasted patterns in two nearby European regions.
Ana Beatriz de Mello Sampaio – Participou da coleta de Appetite. 2007;49(2):429-40.
dados, concepção, análise e interpretação dos dados e da 8. Northstone K, Emmett P, Nethersole F. The effect of age of introduction to lumpy solids
on foods eaten and reported feeding difficulties at 6 and 15 months. J Hum Nutr Diet.
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Ruth Bartelli Grigolon, Ana Maria Roma, Leticia Enri-
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tação dos dados e elaboração do artigo. 10. Pearson N, Biddle SJH, Gorely T. Family correlates of fruit and vegetable consumption in
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Participaram da coordenação, concepção, desenho do estu- 11. Bryant-Waugh R, Markham L, Kreipe RE, Walsh BT. Feeding and eating disorders in child-
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J Autism Dev Disord

DOI 10.1007/s10803-013-1771-5
ORIGINAL PAPER
Feeding Problems and Nutrient Intake in Children with Autism

Spectrum Disorders: A Meta-analysis and Comprehensive Review
of the Literature
William G. Sharp • Rashelle C. Berry • Courtney McCracken •
Nadrat N. Nuhu • Elizabeth Marvel • Celine A. Saulnier •

Ami Klin • Warren Jones • David L. Jaquess
Ó Springer Science+Business Media New York 2013
Abstract We conducted a comprehensive review and Introduction

meta-analysis of research regarding feeding problems and
nutrient status among children with autism spectrum dis- Autism spectrum disorders (ASD) represent a range of
orders (ASD). The systematic search yielded 17 prospec- complex developmental disabilities involving severe
tive studies involving a comparison group. Using rigorous impairments in social interaction and communication
meta-analysis techniques, we calculated the standardized accompanied by behavioral inflexibility, repetitive behav-
mean difference (SMD) with standard error and corre- iors, and/or restricted interests (APA 2000). In addition to
sponding odds ratio (OR) with 95 % confidence intervals the core diagnostic features, children with ASD often
(CI). Results indicated children with ASD experienced present with comorbid ear infections (Konstantareas and
significantly more feeding problems versus peers, with an Homatidis 1987), increased use of antibiotics (Niehus and
overall SMD of 0.89 (0.08) and a corresponding OR of Lord 2006). constipation (Ibrahim et al. 2009), possible
5.11, 95 % CI 3.74–6.97. Nutrient analyses indicated sig- gastroenterological disturbances (Horvath et al. 1999), and
nificantly lower intake of calcium (SMD: -0.65 [0.29]; an array of challenging behaviors, including self-injury,
OR: 0.31, 95 % CI 0.11–0.85) and protein (SMD: -0.58 severe tantrums, feeding problems, aggression, toileting,
[0.25]; OR: 0.35, 95 % CI: 0.14–0.56) in ASD. Future and sleep disturbances (Whiteley 2004; Herzinger and
research must address critical questions regarding the Campbell 2007; Seiverling et al. 2010). Of these concerns,
cause, long-term impact, and remediation of atypical feeding arguably involves the most essential of human
feeding in this population. activities, necessary to assure appropriate development and
sustain life. Chronic feeding problems place children at risk
Keywords Diet Food selectivity Mealtime problems for a number of detrimental medical and developmental
Nutrition Picky eating Pediatric feeding disorders outcomes, including malnutrition, growth retardation,
invasive medical procedures (e.g., placement of a feeding
tube), developmental delays, psychological and social
deficits, and poor academic achievement (Kerwin 1999;
Sharp et al. 2010). Researchers, however, have only
recently begun to systematically investigate eating and
nutrient intake patterns associated with ASD, and many
W. G. Sharp C. McCracken C. A. Saulnier A. Klin questions remain regarding prevalence, consequences, and
W. Jones D. L. Jaquess
remediation of feeding problems in this population.
Department of Pediatrics, Emory University School of Medicine,
Atlanta, GA, USA Lack of research on this topic is remarkable given the
historical link between feeding and ASD. Leo Kanner’s
W. G. Sharp (&) R. C. Berry N. N. Nuhu E. Marvel initial description of the condition cited atypical eating
C. A. Saulnier A. Klin W. Jones D. L. Jaquess
patterns as prominent in his sample and past diagnostic
The Marcus Autism Center, 1920 Briarcliff Road,
Atlanta, GA 30329, USA systems included feeding difficulties as a defining charac-
e-mail: wgsharp@emory.edu teristic (Kanner 1943; Ritvo and Freeman 1978). Further,
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the social and behavioral demands of feeding situations tap In a more recent review, Cermak et al. (2010) identified
into all three areas of difficulty displayed by children with studies investigating food selectivity and nutrient adequacy
ASD. Communication, behavioral flexibility, and social in ASD. The authors identified 817 participants in 16
engagement each play important roles in promoting intake, studies with two foci: food selectivity (nine studies) or
increasing dietary diversity, and assuring the saliency of nutritional status related to dietary intake (four studies);
social reinforcement during meals. Related theories reflect three studies spanned both areas of inquiry. Findings sug-
this connection, with different authors positing different gested food selectivity was a significant problem in ASD;
etiologies, including idiosyncratic focus on detail, behav- however, Cermak et al. cited the lack of a comparison
ioral rigidity, sensory impairments, social skills deficits, group, present in only 6 of the 12 studies, as a key limi-
and/or communication deficits (Cumine et al. 2000; Ahearn tation to drawing definitive conclusions. Findings regard-
et al. 2001). Finally, research regarding feeding problems ing the nutritional status of children with ASD were
in ASD and related dietary vulnerabilities has important equivocal. Four studies involving comparison groups
implications for a growing interest regarding the use of reported conflicting results, with the nutrient intake of
dietary manipulation (e.g., gluten and/or casein free, GFCF children with ASD described as below, above, or at the
diet) in this population, as well as the possible role of same level as typically developing peers. Three remaining
dietary insufficiencies in the pathology of the condition, studies comparing the nutrient intake of children with ASD
such as vitamin D (Cannell 2008). to recommended dietary standards also reported both
Much of what is known regarding feeding patterns in nutrient deficits (e.g., vitamin D) and excesses (e.g., pro-
ASD is based on anecdotal and case reports describing tein); however, no consistent pattern emerged, and lack of
children with ASD as presenting with unusual eating pat- comparison groups precluded conclusions as to whether a
terns, rituals regarding food preparation/presentation, food deviation from recommended levels was unique to ASD.
refusal and/or displaying strong emotional responses to The works of Ledford and Gast (2006) and Cermak et al.
new foods (Cornish 1998; Ahearn et al. 2001). Food (2010) provide an important foundation for understanding
selectivity (by type, texture, and/or presentation) is the feeding concerns and nutritional status of children with
feeding problem most often associated with ASD, typically ASD, offering provisional evidence that feeding problems
involving strong preferences for carbohydrates, snacks, may be endemic in the ASD population. Recent growth in
and/or processed foods while rejecting fruits and vegeta- research into feeding in ASD, combined with the avail-
bles (Ahearn et al. 2001; Schreck et al. 2004; Williams ability of quantitative procedures for synthesizing outcome
et al. 2005). Many past reports, however, documenting this data, present the opportunity for a more detailed analysis of
trend involved children seeking intervention for severe the extant literature. The current review sought to (a) sur-
food selectivity, often in the form of behavioral interven- vey the medical, habilitative, and psychological literature
tion aimed at expanding dietary variety (e.g., Sharp et al. in order to identify studies using empirical methods to
2010), and a more general picture regarding the eating investigate the feeding behaviors and/or nutritional status
patterns and nutritional status of all children with ASD has of children with ASD and (b) summarize the evidence on
yet to emerge. the basis of both descriptive and meta-analytic procedures.
Ledford and Gast (2006) conducted the first literature To address limitations noted in previous reviews, we
review of feeding problems in ASD, identifying seven focused exclusively on prospective research involving a
studies (381 total children) published between 1994 and comparison group to quantify the magnitude of feeding
2004. All studies reported significant feeding difficulties, problems and/or nutrient deficiencies associated with ASD
primarily in the form of food selectivity by type and/or and used this information to develop ASD-specific rec-
texture, with estimates ranging from 46 to 89 % of children ommendations to guide future clinical and research activ-
with ASD with atypical feeding habits. While providing ities in this area.
evidence of widespread feeding problems, large variability
in prevalence estimates reflected wide methodological
variability among the studies. Less than half of the studies Method
included a comparison group, and the primary method of
data collection involved chart audits or study specific Study Identification and Eligibility Criteria
questionnaires. In addition, few studies presented infor-
mation regarding participants’ definitive diagnostic status Following the guidelines outlined by the Preferred
(i.e., autistic disorder, PDD-NOS, Asperger syndrome), Reporting Items for Systematic Reviews and Meta-Anal-
with 85 % having no specific ASD diagnosis and no yses (PRISMA) statement, we searched MedLine, PsychI-
standardized assessment of disability which limits gener- NFO, and PubMed databases (January 1980 and August
alizability of the findings. 2011), reviewed reference lists, and conducted ancestral
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J Autism Dev Disord
and online searches in English language journals for eli- procedures, feeding/nutrient assessment measures, and
gible studies. The search parameters included combinations summary of findings. Characteristics in each of these cate-
of key words regarding the target population (autism, gories were coded using a standardized checklist system. For
autistic, autism spectrum disorders, pervasive develop- feeding behaviors, we categorized item(s) and/or assess-
mental disorder, PDD-NOS, Asperger syndrome), meal- ment measure(s) and their content based in three categories:
time-related variables (diet, dietary intake, eating, feeding, food selectivity (e.g., by type, texture, or presentation),
food selectivity, nutrition, mealtime behaviors, pediatric food refusal (e.g., refusing food by crying, pushing away
feeding disorder), and evaluation methodology (assess- food, leaving the table)/poor oral intake (concerns regarding
ment, mealtime observation, food frequency). total calories or nutrients consumed), and/or behavioral
We focused on prospective studies utilizing a comparison rigidity during meals (e.g., difficulty eating across environ-
group to present quantitative information about feeding ments, insists on rituals at table). If food selectivity was
behaviors and/or nutrient intake in a pediatric population reported, we documented whether the pattern of food intake
(birth to 18 years of age) with ASD and sought to capture a was analyzed (e.g., preference or rejection of certain types).
wide range of children regardless of the presence of feeding For dietary information, data collection focused on the fol-
related difficulties. As a result, we excluded recent program lowing key dietary indicators: vitamin A, C, D, & E, zinc,
evaluations (Laud et al. 2009; Sharp et al. 2011) single- calcium, iron, fiber, fat, protein, carbohydrates, and total
subject designed studies (see Sharp et al. 2010 for a sum- energy (kcal). When available, we also recorded nutritional
mary), and chart reviews of children with ASD evaluated due risk based on the cut point method (Barr et al. 2002), a dif-
to atypical feeding patterns (Williams et al. 2005) in order to ferent approach to assessing dietary status that involves
avoid a known sampling bias. This procedure also excluded calculating an individual’s typical intake of each nutrient,
studies focusing on the impact of dietary manipulation (e.g., identifying the total number of nutrients falling within
GFCF diet) on nutrition or behavioral functioning (e.g., established standards (e.g., estimated average requirement),
Elder et al. 2006). To be included in the review, studies also and determining the proportion of children in each group
needed to meet the following criteria: meeting or not meeting recommended levels. The research
team involved a registered dietician, who was responsible for
1. Evaluated feeding through a standardized, replicable
calculating nutritional risk, as well as selecting and inter-
manner, such as dietary intake (e.g., 3-day food diary),
preting specific dietary indicators. To determine growth
feeding questionnaires [e.g., Children’s Eating Behav-
status, we also recorded anthropometric data (i.e., height,
ior Inventory-Revised (CEBI-R); Archer et al. 1991],
weight, body-mass index) when presented.
Brief Autism Mealtime Behavior Inventory (BAMBI;
Multiple researchers independently coded all studies. The
Lukens and Linscheid 2008), study specific question-
mean inter-rater agreement for categorical data was 97 %
naires involving set questions, and/or mealtime obser-
(range 87–100 %) with a corresponding Kappa of 0.94
vation with a detailed protocol.
(range 0.79–1). The overall intra-class correlation for inter-
2. Included a dependent variable(s) focused on feeding
val and continuous data was 0.93 (range 0.54–1). Coder
behavior (i.e., chronic food selectivity, food refusal/
agreement exceeded the 80 % standard widely adopted and
poor oral intake, and/or behavioral rigidity during
recommended during quantitative synthesis of research
meals), nutritional status, or dietary variety. Data
(Campbell 2003). Due to the wide range of assessment
obtained through these measures was presented in the
methods and item content related to feeding behaviors in
study, either descriptively (e.g., frequencies, percent-
ASD, two members of the research team with expertise in
ages, means) or statistically (e.g., p values, t scores).
autism and pediatric feeding disorders conducted a third
3. Focused on active chronic feeding concerns (i.e., not
level review of all extracted data to determine inclusion
studies or items pertaining to historical concerns alone
status and classify item/scale content based on the criteria
such as feeding during infancy, difficulty transitioning to
outlined above. The mean inter-rater agreement for items to
solids).
analyze was 91 % (range 66–100 % across studies) with a
corresponding Kappa of 0.80 (range 0.33–1).
Variables Coded, Data Extraction, and Reliability
Statistical Analysis
Data were extracted from articles using a three-phase
system. First, all articles identified through the literature To calculate the effect size (ES), we used means (standard
were screened for eligibility criteria. We then extracted deviations) or frequencies (percentages) and, if necessary,
descriptive information, collecting information regarding we estimated the ES from test statistics (e.g., Chi Square,
study descriptors, participant demographic variables, t tests). When summary statistics were not presented, we
composition of the comparison group(s), diagnostic attempted to contact the primary author via email before
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J Autism Dev Disord
using alternative methods and, if unsuccessful, we used outcomes using the funnel plot (Egger et al. 1997), failsafe
exact p values to calculate the ES. If an exact p value was N (Becker 2005), and the trim and fill method (Duval and
not provided, we adopted a conservative approach to esti- Tweedie 2000).
mating a p value closest to the level provided (Lipsey and
Wilson 2001).
The primary goal of the meta-analysis was to determine Results
the overall difference in feeding behavior and/or nutritional
status between children with and without ASD. We, Characteristic of Studies and Participants
therefore, calculated an overall mean study ES when
multiple comparisons had been made (Rosenthal and Rubin The search yields 17 articles meeting inclusion criteria out
1986). In line with these criteria, we combined outcome of a pool of 678 possible studies (see Fig. 1). Sixty-five
variables (e.g., food selectivity, food refusal), resulting in a percent of the articles were published in journals special-
single ES. Likewise, when studies separately presented izing in ASD or related DD, with the Journal of Autism and
individual items, or individual subscales, along with total Developmental Disorders contributing nearly half of the
scale scores, only items or scales pertaining to these criteria studies in this area (see Table 1). Ten articles (88 %) were
were used in the present analysis. For nutrient data, we published since 2000; five since 2010. Compared with
calculated a separate ES estimates for each nutrient across previous reviews, 2 of the 7 studies (29 %) identified by
studies. For studies involving multiple comparison groups, Ledford and Gast (2006) and 6 of the 16 studies (36 %)
we pooled the comparison groups, producing an overall ES. summarized by Cermak et al. (2010) met inclusion criteria.
Separate ES estimates for each comparison group [i.e., Ten articles were unique to this review.
ASD vs. typically developing peers (TD); ASD vs. siblings Feeding assessment methods primarily involved esti-
(SIB); ASD vs. children with other developmental dis- mates of nutrient intake (e.g., 3-day food diary) and
abilities (DD)] were also calculated to identify possible questionnaires specific to the study involving single item
moderator variables using the between groups Q test, with analysis (Table 2). Standardized questionnaires were uti-
a significance level of p \ 0.05. We did not conduct lized in only three studies (18 %). While most studies
additional analysis of potential moderators (e.g., age, sex, broadly assessed feeding behaviors using single items or
diagnostic status) given the lack of descriptive data pre- scales (e.g., eats a narrow range of foods, doesn’t try new
sented in the articles (described below). foods), three studies (Bandini et al. 2010; Emond et al.
Data were entered and analyzed using Comprehensive
Meta-Analysis 2 (Borenstein et al. 2005). We converted all
ES estimates to standardized mean difference (SMD). For Studies identified from
feeding behaviors, a positive SMD (SMD [ 0) indicated search N = 678
more feeding-related concerns in children with ASD com-
pared with the comparison group. We coded nutritional data Excluded on the basis of
so that a negative SMD (SMD \ 0) indicated more nutri- abstract n = 618
tional deficits in children with ASD. The point estimates and
standard error were calculated using a random-effects model Studies with potentially
of meta-analysis (Hunter and Schmidt 2004). We evaluated eligible abstracts n = 60
SMD magnitude using conventional standards (0.2 = small;
0.5 = medium; 0.9 = large; Cohen 1988). To aid in clinical Excluded on the basis of
interpretation of outcomes, we also calculated the corre- full text because study did
sponding odds ratio (OR) with 95 % CIs, with values not assess feeding behaviors
or nutrient intake n = 31
reflecting the odds of a child with ASD having a feeding Studies presenting data on
difficulty compared to child without ASD. feeding behaviors or
nutrient intake N = 29
To assess heterogeneity within subgroups and between
studies, effect sizes and associated 95 % CIs were calcu- Excluded because study did
not include a control group
lated for each subgroup. We also used the Q test to for- or focused on children with
mally determine if heterogeneity was present. To assess the ASD seeking assessment
robustness of our results, we conducted a sensitivity anal- and treatment for feeding
problems n = 12
ysis, which involved repeatedly calculating the effect size Studies included in the
with one study omitted per iteration and comparing the systematic review N = 17
results with the overall study effect. We analyzed the threat
of possible publication bias to the validity of the obtained Fig. 1 Flow diagram of included and excluded studies
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J Autism Dev Disord
Table 1 Summary of articles by journal and year of publication Growth Status

Journal title n %
Seven studies (41 %) involving 426 children presented
Journal of Autism and Developmental Disorders 7 41 information regarding anthropometric parameters [e.g.,
Autism 1 5.9 height, weight, or body mass index (BMI)] compared with
Journal of the American Dietetic Association 1 5.9 typically developing peers. Six studies compared mean
Journal of Developmental and Physical Disabilities 1 5.9 values between groups, finding no statistically significant
Journal of Learning Disabilities 1 5.9 differences in anthropometric parameters. One study ana-
The Journal of Pediatrics 1 5.9 lyzed the percentage of children in each group identified as
Pediatrics 1 5.9 overweight (BMI C85th %) or underweight (BMI \5th %)
Physical & Occupational Therapy in Pediatrics 1 5.9 and reported no difference in the number of children falling
Research in Autism Spectrum Disorders 1 5.9 into these classifications (Bandini et al. 2010).
Research in Developmental Disabilities 1 5.9
Topics in Clinical Nutrition 1 5.9 Dietary Variety
Total 17 100
Year published Ten studies presented detailed food group preferences, six
2010–present 5 29 of which supported past reports indicating children with
2000–2009 10 59 ASD consumed fewer vegetables (Lukens and Linscheid
1980–1989 2 12 2008; Johnson et al. 2008; Martins et al. 2008; Bandini
et al. 2010; Emond et al. 2010) and fruits (Lukens and
Linscheid 2008; Martins et al. 2008; Emond et al. 2010), as
2010; Zimmer et al. 2012) calculated a dietary variety well as demonstrated preference for crispy/crunchy snack
score based on responses to a food frequency question- foods (Schmitt et al. 2008). One study reported signifi-
naire, focusing on the number of foods identified as never cantly fewer accepted foods across all food groups in
consumed. Food selectivity represented the primary type of children with ASD (Schreck et al. 2004), while another
feeding problem assessed among studies, representing study reported lower variety of dairy but equivalent variety
54 % of the items or scales (e.g., eats a narrow variety of of other food groups (Shearer et al. 1982). Raiten and
foods, obsessive eating habits), 21 % of the items or scales Massaro (1986) found no significant difference in food
focused on food refusal or poor oral intake (e.g., disrup- groups consumed, and Herndon et al. (2009) reported
tions/tantrums during meals, throw or spits food), 17 % increased intake of fruit among children with ASD but
assessed behavioral rigidity during meals (e.g., eats only in equivalent intake of other food groups.
specific places, requires specific utensils), and 7 %
involved overlapping content. Overall Measure of ES for Feeding Behaviors
The pool of studies involved a total sample of 881 and Nutritional Intake
children with ASD. Data regarding feeding behaviors were
gathered from 832 (94 %) participants, while 263 children Tables 4 and 5 present ES estimates calculated using ran-
(30 %) from eight studies provided data on micronutrient dom effects models. The overall test for heterogeneity of
intake. Only 29 % of studies presented data regarding study effect sizes was statistically significant (Q = 29.4,
diagnostic status, resulting in 669 participants (76 %) with df = 14, p = 0.009) indicating that the random effects
a nonspecific ASD diagnosis. In terms of comparison model was appropriate. The presences of heterogeneity
groups, most studies (82 %) involved typically developing within subgroups further supported the use of the random
peers or children drawn from the general population, fol- effects model.
lowed by studies involving children with developmental or All studies reported greater levels of feeding concerns
learning disabilities (18 %) or siblings (18 %). Most associated with ASD, regardless of the type of comparison
studies (82 %) reported equivalence between ASD and group or method of assessment. SMD estimates across
comparison groups in terms of age. Two studies (12 %) did studies ranged from 0.48 to 1.56 (Fig. 2) and the overall
not statistically analyze possible age difference across SMD involving all comparison groups was large and sta-
groups, while one study reported that the ASD group was tistically significant (p \ 0.001). Analyses involving indi-
significantly older. In terms of gender, the ASD groups vidual comparison subtypes suggested medium to large
tended to involve a higher ratio of males to females com- differences in feeding problems, ranging from a SMD of
pared to the comparison groups; four studies (24 %) sta- 0.69 (0.19) when the comparison group involved children
tistically analyzed this variable, all reporting higher with DD to 0.97 (0.22) when siblings were compared. The
numbers of males to females in the ASD groups (Table 3). corresponding overall OR involving all comparison groups
123
Table 2 Description of experimental characteristics and assessment methodology by study
Study
123
Bandini Collins Dominick Emond Herndon Johnson Lockner Luckens Martins Matson
et al. et al. et al. et al. et al. et al. et al. and Linsheid et al. et al.
(2010) (2003) (2007) (2010) (2009) (2008) (2008) (2008) (2008) (2009)
Outcomes presented
Feeding behaviors/Food selectivity X X X X X X X X X
Micronutrient analysis of dietary intake X X X X X
Setting
Community wide X X X X X X X
Diagnostic clinic/Early intervention X X X
Other
Feeding measure(s)*
Standardized questionnaires X X
Estimates of nutritional intake X X X X X X
Subtypes: Food diary o o o
24 h recall o o
Food frequency inventory o o o o
Study specific questionnaire X X X X X X X
ASD Diagnostic indicator
Parent report X
ASD rating scale X X
Clinical provider X X X
ADOS X X X
ADI-R X X X
Not specified X X
Cognitive functioning/IQ X Xa Xa
Anthropometric data*
Weight X X
Height X X
BMI X X X
J Autism Dev Disord
Table 2 continued
Study
Nadon Provost Raiten Schmitt Schreck Shearer Zimmer N % of total
et al. et al. and Massaro et al. et al. et al. et al. studies
(2011) (2010) (1986) (2008) (2004) (1982) (2012) (17 total)
J Autism Dev Disord
Outcomes presented
Feeding behaviors/Food selectivity X X X X X X 15 88
Micronutrient analysis of dietary intake X X X 8 47
Setting
Community wide X X X 10 59
Diagnostic clinic/Early intervention X X 5 29
Other X X 2 12
Feeding measure(s)*
Standardized questionnaires X 3 18
Estimates of nutritional intake X X X X X 11 65
Subtypes: Food diary o o o 6 35
24 h recall 2 12
Food frequency inventory o o 6 35
Study specific questionnaire X X X X 11 65
ASD Diagnostic indicator
Parent report
ASD rating scale X 3 18
Clinical provider X X 5 29
ADOS X 4 24
ADI-R X 4 24
Not specified X X X 5 29
Cognitive functioning/IQ 3 18
Anthropometric data*
Weight X X 4 24
Height X X 4 24
BMI X X 5 29
* Subheadings may not add up to 100 % due to multiple measures used in a study
a
Data only presented for ASD group
123
Table 3 Description of participants
Study
Bandini Collins Dominick Emond Herndon Johnson Lockner Luckens Martins Matson
123
et al. et al. et al. et al. et al. et al. et al. and Linsheid et al. et al.
(2010) (2003) (2007) (2010) (2009) (2008) (2008) (2008) (2008) (2009)
ASD group
Sample size 53 107 54 79 46 19 20 68 41 112
ASD diagnosis X X
Autistic disorder 45 72
PDD–NOS 1 40
Asperger syndrome
Age (months) X X X X X X X X X X
Mean 79.2 96 91.2 6, 15, 24, 38, 54* 55.9 39.2 72.8 85.2
SD 25.2 43.3 29.8 13.9 8.9 29.8 34.4
Range 36–216 33–96 24–48 36–60 36–132 36–132 36–192
Gender X X X X X
Male (%) 44 (83 %) 47 (87 %) 44 (96 %) 56 (82 %) 34 (83 %)
Female (%) 9 (17 %) 7 (13 %) 2 (4 %) 12 (18 %) 7 (17 %)
Comparison group
Sample size 53a 331 (DD: 262; SB: 69) 38a 12,901a 31a 15a 20a 40a 55a (TD: 41; SB: 14) 167a
Subtype**
TD X X X X X X X X
DD X X X
SB X X
Age (months) X X X X X X X X
Mean 80.4 DD: 95.8; SIB: 99.4 95.3 6, 15, 24, 38, 54* 59.9 36.4 72.8
SD 28.8 DD: 50.2; SIB: 45.1 33.1 16.5 9.46 29.8
Range DD: 24–216.9; SIB: 24–216 24–48 36–132 TD: 12–48; SIB: 24–132
Gender X X X Xc X
Male (%) 45 (78 %) 21 (71 %) 23 (74 %) 20 (50 %) TD: 23 (56 %) SIB: 7 (50 %)
Female (%) 13 (22 %) 11 (29 %) 8 (26 %) 20 (50 %) TD: 18 (44 %); SIB: 7 (50 %)
J Autism Dev Disord
Table 3 continued
Study
Nadon Provost Raiten Schmitt Schreck Shearer Zimmer Total % of total

et al. et al. and Massaro et al. et al. et al. et al. sample/N studies
(2011) (2010) (1986) (2008) (2004) (1982) (2012) (17 total)
J Autism Dev Disord
ASD group
Sample size 48 24 40 20 138 12 22 881
ASD diagnosis X X X 5 29
Autistic disorder 10 12 22 5 29
PDD–NOS 3 3 18
Asperger syndrome 4 1 6
Age (months) X X X X X X X 17 100
Mean 94.8 51.2 127 99 96 98.4 14 82
SD 30 10.6 52 29 9.6 38.4 13 76
Range 45.6–154.8 36–70 84–120 53–152 11 65
Gender X X X X X X 11 65
Male (%) 44 (92 %) 18 (75 %) 28 (70 %) 20 (100 %) 121 (88 %) 20 (91 %)
Female (%) 4 (8 %) 6 (25 %) 12 (30 %) 14 (10 %) 2 (9 %)
Comparison group
Sample size 48a 24a 34b 18a 298a 12 20a 13,544
Subtype**
TD X X X X X X 14 82
DD 3 18
SB X 3 18
Age (months) X X X X X X X 15 88
Mean 92.4 51.2 105.6 108 100.8 97.2 13 76
SD 34.8 9.8 57.6 7.2 39.6 11 65
Range 37.2–153.6 36–72 84–120 60–144 8 47

Gender Xc X Xc X Xc X 11 65
Male (%) 20 (42 %) 18 (75 %) 19 (56 %) 18 (100 %) 158 (53 %) 10 (45 %)
Female (%) 28 (58 %) 6 (24 %) 15 (44 %) 140 (47 %) 12 (55 %)
TD typically developing, DD other developmental delay, SB siblings

* Longitudinal design
** Subheadings may not add up to 100 % due to multiple groups used in a study
a
Reported matched for age
b
Reported age difference with ASD group
c
Reported higher ratio of males to female in ASD group
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J Autism Dev Disord
Table 4 Effect sizes, 95 % confidence limits and within-group tests for heterogeneity for studies included in the meta-analysis for feeding
behavior problems by comparison groups
ASD versus Number of Random effects model Within-groups
subgroup contributing
studies SMD (SE) OR 95 % confidence limits p value v2 test (Q) p value
LCL UCL
All groups 15 0.89 (0.08) 5.11 3.74 6.97 \0.001

TD 13 0.94 (0.11) 5.49 3.77 7.98 \0.001 29.9 0.003
SB 3 0.98 (0.22) 5.89 2.73 12.71 \0.001 0.45 0.798
DD 2 0.67 (0.19) 3.36 1.69 6.67 0.001 0.012 0.913
TD typically developing, DD other developmental delay, SB siblings
Table 5 Effect sizes, 95 % confidence limits and within-group tests for heterogeneity for studies included in the meta-analysis for nutritional
data
Nutrient Number of Random effects model
contributing
studies SMD (SE) OR 95 % confidence limits p value
LCL UCL
Calcium 8 -0.65 (0.29) 0.31 0.11 0.85 0.022

Carbohydrates 7 -0.02 (0.07) 0.97 0.76 1.24 0.810
Energy 6 0 (0.06) 0.99 0.80 1.25 0.995
Fiber 6 0.09 (0.12) 1.18 0.77 1.78 0.448
Iron 7 0.17 (0.20) 1.35 0.66 2.76 0.414
Protein 7 -0.58 (0.25) 0.35 0.14 0.86 0.021
Total fat 6 0.03 (0.06) 1.05 0.84 1.30 0.690
Vitamin A 6 -0.51 (0.35) 0.39 0.11 1.37 0.143
Vitamin C 7 -0.13 (0.19) 0.98 0.52 1.87 0.507
Vitamin D 6 -0.07 (0.19) 0.88 0.45 1.71 0.703
Vitamin E 5 0.05 (0.17) 1.10 0.61 1.98 0.742
Zinc 6 -0.03 (0.09) 0.95 0.69 1.31 0.758
was 5.11 (95 % CI 3.74–6.97), suggesting that the odds of each study. No study significantly altered the overall mean
having a feeding problem in children with ASD are 5 times ES estimates for feeding behaviors or nutrient intake.
the odds for children without ASD. Visual inspection of the funnel plots indicated no potential
Analyses involving nutritional data suggested children publication bias for outcome related to feeding behaviors
with ASD had significantly lower consumption of calcium or analyses involving calcium or protein. Furthermore, the
(p \ 0.05) and protein (p \ 0.05) compared to TD peers. failsafe N analysis indicated that there would need to be
No other significant differences in nutrient consumption 858 published studies with non-significant findings related
were detected between groups. Bandini et al. (2010) and to feeding behaviors to change the current effect size to
Zimmer et al. (2012) also assessed risk of inadequate non-significant. The failsafe N was 37 for the calcium
nutrient intake using the cut point method; both reporting intake outcome was 33 for the protein intake outcome. This
children with ASD were significantly more likely to have evidence lends credence to the robustness of our findings.
inadequacies compared to TD children (p \ 0.03). Given
the small sample of studies, we did not estimate an ES for
cut point data. Discussion
Sensitivity Analysis, Publication Bias and Reliability This meta-analysis shows a strong association between
of Results feeding difficulties and ASD, corroborating anecdotal
reports and descriptive studies documenting this trend.
Sensitivity analysis involved visual inspection of confi- While previous reviews summarized the literature, this
dence intervals for the overall effect size after removing systematic evaluation of the research base quantifies the
123
J Autism Dev Disord
Study Name OR (95 % CI) OR (95 % CI)
Bandini et al. (2010) 4.91 (2.40 - 10.1)

Collins et al. (2003) 4.08 (1.79 - 9.24)
Dominick et al. (2007) 16.8 (5.76 - 49.1)
Emond et al. (2010) 2.39 (1.79 - 3.19)
Johnson et al. (2008) 4.79 (1.29 - 17.8)
Lockner et al. (2008) 10.5 (1.31 - 84.3)
Luckens & Linsheid (2008) 8.45 (3.93- 18.2)
Martins et al. (2008) 5.84 (2.92 - 11.7)
Matson et al. (2009) 5.18 (2.55 - 10.5)
Nadon et al. (2011) 4.56 1.29 - 16.1)
Provost et al. (2010) 4.48 (0.71- 28.4)
Raiten & Massaro (1986) 3.07 (0.59- 15.8)
Schmitt et al. (2008) 4.28 (0.99- 18.4)
Schreck et al. (2004) 4.72 (3.24 - 6.89)
Zimmer et al. (2012) 10.7 (3.29 - 34.9)
Mean ES 5.11 (3.74 - 6.97)
Fig. 2 Forest plot of feeding problems with 95 % confidence intervals
magnitude of effect. By conventional standards, findings regarding the impact of aberrant feeding patterns on health
reflect a ‘‘large’’ difference in the presence of feeding and developmental in the ASD population.
problems between children with and without ASD, corre- When considering the impact of chronic feeding prob-
sponding with an estimated fivefold increase in the odds of lems, growth and nutrition represent key barometers of
having a feeding problem in this population. Higher rates health status. Findings from the current review, however,
of feeding problems were detected regardless of the make- indicate that feeding problems and subsequent nutritional
up of the comparison group or the assessment methodol- intake deficits do not necessarily translate into greater risk
ogy, providing convergent evidence that feeding problems for compromised growth. All seven studies analyzing
are more likely to occur in children with ASD. This sug- growth parameters reported no significant difference in
gests, at a minimum, assessment of feeding problems in height, weight, and/or BMI between children with and
ASD should be included as part of routine screenings in without ASD. This parallels nutrient data indicating com-
pediatric settings, which would necessitate enhanced parable intake of energy, carbohydrates, and fats when
awareness among caregivers and practitioners regarding compared to typically developing peers. This suggests,
this issue. Encouragingly, the pool of studies included in despite increased feeding problems, children with ASD
this review reflects a relative surge in case–control pro- apparently consume enough volume of food to meet gross
spective research of feeding problems in ASD, with more energy needs and relying exclusively on anthropometric
than a quarter of the studies published since 2010. Despite parameters to assess health status may in fact mask
greater empirical attention in this area, a closer examina- underlying nutritional deficits. It may also explain why
tion suggests a sizable gap between studies of feeding and feeding problems are often overlooked in relation to other
other areas of inquiry. For example, even after removing area of clinical concern in the ASD population, since
our conservative inclusion criteria, the largest source of failure to thrive or a declining growth velocity are the
articles in the current review, Journal of Autism and standard nutritional health indicators (WHO 2006) that
Developmental Disorders, published only nine studies on trigger clinical attention in pediatric settings (Ledford and
feeding problems in ASD between 1980 and 2011; this Gast 2006). Closer examination of nutrient intake, how-
represents 0.3 % of the 2,485 articles published in the ever, indicates significant specific deficits (lower intake of
journal over the same time period. Further, only two calcium and protein) and a higher number of nutritional
studies were published in pediatric journals despite the deficits overall among children with ASD. These patterns
frontline role pediatricians play in screening and identify- may well place this population at risk for long-term med-
ing health concerns among children with ASD. Given the ical complications not captured by broad anthropometrics
significant level of feeding concerns associated with ASD or energy intake. For example, lower levels of calcium,
and the biological and social significance of healthy eating, compounded by the increased need for this nutrient during
greater clinical and research scrutiny in this area are clearly childhood to promote growth of bones, may portend risk of
needed to improve assessment methods, increase access osteomalacia and osteoporosis. This assertion is consistent
to treatment, and develop more definitive conclusions with findings indicating decreased bone cortical thickness
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J Autism Dev Disord
in a group of 75 boys with ASD when compared to peers, deficits (Lukens and Linscheid 2008). With this in mind,
highlighting the need to investigate the calcium intake and caregivers should employ utmost caution when deciding to
bone growth in children with ASD, as well as identify pursue this form of treatment. At a minimum, families
possible etiologies (Hediger et al. 2007). Together, the wishing to pursue a possible dietary intervention should do
available evidence suggests the need to look beyond gross so under the guidance of a healthcare professional (e.g.,
anthropometric parameters, such as incorporating idiosyn- registered dietitian) who can assess the impact of further
cratic analysis of nutritional intake as part of routine restrictions on a child’s nutritional status and work to
medical care in ASD. It will also be important to determine ensure the child’s nutritional needs are met during the
the long-term health burden associated with atypical pat- intervention. Similarly, untested interventions that may
terns of intake on a population level, particularly high secondarily affect nutritional status, such as chelation
consumption of snack and fats in ASD, which may portend therapy, could compound an already risky situation by
increased risk for diet-related diseases (e.g., obesity, car- further reducing the bioavailability of key nutrients, such as
diovascular disease) in adolescence or adulthood. calcium. Clearly, such potential iatrogenic effects should
Two candidates for explaining reduced nutrient intake in be carefully investigated prior to recommending any
ASD are food selectivity and/or elimination diets (e.g., the treatment. To assist caregivers with making an informed
GFCF diet). Only three studies in the current review, decision, pediatric practitioners must screen for preexisting
however, specifically investigated the relationship between feeding concerns, highlight the tenuous empirical support
restricted patterns of intake and nutritional status. Herndon for diet modification as treatments of ASD and review
et al. (2009) reported fewer servings of dairy and that this potential consequences (e.g., further nutritional deficits,
relationship remained after excluding children following a stigmatization, diversion of treatment resources; Mulloy
GFCF diet. Zimmer et al. (2012) excluded children on et al. 2010) and barriers (e.g., resources to purchase spe-
elimination diets and still reported that selective eaters with cialized foods, strategies for ensuring dietary compliance;
ASD had lower intake of calcium, vitamin B12, and vita- Elder 2008) associated with dietary interventions. Addi-
min D, compared to non-selective eaters with ASD and tional research will also be needed to more clearly eluci-
lower intake of protein, calcium, vitamin A, and vitamin D, date the impact of dietary manipulations on growth,
compared with typically developing peers. Finally, Bandini nutrition, and family resources.
et al. (2010) reported children with ASD experienced more The higher rate of feeding concerns in ASD also
nutrient inadequacies than typically developing children, a emphasizes a subsequent need to identify and disseminate
finding that persisted after excluding children on special empirically-supported treatments for feeding problems
diets. Together, there is evidence suggesting that nutri- associated with ASD. At this time, behavioral intervention
tional issues associated with ASD may be related to the represents the only empirically supported treatment for
patterns of food selectivity beyond what could be attributed pediatric feeding disorders Sharp et al. (2010) and there is
to parent-mediated dietary manipulations. Going forward, provisional evidence that these benefits apply to children
it will be important to control for the use of vitamin/min- with ASD. With this said, support for behavioral treatment
eral supplements, which may mask an even greater risk of to expand dietary variety has primarily been documented at
compromised dietary status among children with ASD. day-treatment or inpatient feeding programs (Laud et al.
Provisional evidence suggests higher use of supplements 2009; Sharp et al. 2011). Unfortunately, few inpatient and
among caregivers concerned about increased levels of food day-treatment programs exist, which curtails adequate
selectivity or food refusal (Yu et al. 1997), and parents of access to care. Given the need for feeding intervention in
children with ASD may be more likely to try dietary sup- this population, an important goal moving forward will be
plementation in general (Lockner et al. 2008). to develop additional treatment options, such as organizing
The combination of increased feeding problems and disciplines involved in providing care along clinical service
nutritional concerns raises important questions regarding lines and expanding training and educational opportunities
the use (and possible detrimental impact) of dietary for community providers regarding behavioral strategies
manipulations in the ASD population. Many of these diets for targeting food selectivity. It will also be important to
(e.g., the GFCF) eliminate dairy proteins, placing addi- determine whether intervention to address food selectivity
tional restrictions on a population vulnerable for lower in ASD can be adapted for delivery through less intensive
calcium intake, and provisional evidence suggests that this methods of service delivery, such as outpatient treatment,
may lead to greater deficits in bone development among group therapy, or caregiver training.
children with ASD (Hediger et al. 2007). Elimination diets This review also highlights important areas for future
also target starches and snack foods often identified as research to enhance understanding of feeding problems and
preferred foods among children with ASD, which may nutrient status in ASD (Table 6). More detailed diagnostic
increase the risk for weight loss and further nutritional characterization continues to be needed to better define
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Table 6 Summary of key recommendations for clinical and research nature of feeding problems associated with ASD remain
activities for feeding and ASD elusive. The current study collapsed food selectively and
In clinical settings, healthcare providers are encouraged to: food refusal under the larger umbrella of feeding problems
1. Include assessment of feeding problems as part of routine due to the heterogeneity of item content, which reflects a
medical evaluations more global need to develop consensus regarding the def-
2. Screen for nutritional deficits/excesses in addition to inition of specific feeding concerns (e.g., food refusal vs.
measurement of gross anthropometric parameters food selectivity) in the pediatric feeding disorder literature.
3. Engage in caregiver education regarding tenuous empirical Increased diagnostic clarity would, in turn, aid in the
support for diet modification as treatment of ASD
development of standardized feeding measures.
4. Review potential consequences of pursuing an elimination diet
Finally, findings also raise important questions regarding
with consideration to the child’s unique feeding and nutritional
presentation how best to measure the nutritional status of children with
To enhance the literature moving forward, researchers should seek to: ASD. Idiosyncratic food choices among selective eaters will
1. Include detailed diagnostic characterization (e.g., ADOS, ADI) to likely result in different patterns of nutrient deficiencies
confirm ASD status based on the core foods that comprise an individual’s diet,
2. Further develop assessment methods to quantify feeding which may explain conflicting results among past reports.
problems and nutrient status We recommend that studies present data regarding overall
4. Identify and disseminate empirically-supported treatments for group analysis of nutrient intake, as well as an individual
feeding problems in ASD analysis regarding number of deficiencies using the cut point
5. Determine the long-term health burden associated with atypical method. Research would also benefit from increased stan-
patterns of intake on a population level (e.g., obesity,
cardiovascular disease), as well as the relationship with other
dardization in the measurement of nutritional intake (e.g.,
areas of functioning (e.g., quality of life, gastrointestinal issues) food diary, 24 h recall), consistent documentation of
anthropometric data, and long-term assessment regarding
the stability of dietary patterns over time. Finally,
samples of children with ASD. The dearth of studies in this researchers are also encouraged to extend the net of inquiry
review that provided a well characterized sample utilizing to include additional related outcomes, including quality of
diagnostic measures, like the Autism Diagnostic Observa- life, family functioning, relationship with gastrointestinal
tion Schedule (ADOS; Lord et al. 2000) and Autism issues, impact on developmental and cognitive status, and
Diagnostic Interview (ADI; Lord et al. 1994) that have etiological factors influencing dietary preference in ASD.
been standards of best practice in research for over a
decade, is striking and further emphasizes the limitations of
our knowledge of feeding profiles in ASD. Without stan-
Conclusion
dardized measures across samples, questions regarding the
relationship between ASD symptomatology and feeding
Our results confirm that children with ASD have more
behaviors remain unanswered. The relationship of atypical
feeding problems compared with peers. We also found a
feeding and intellectual status in children with ASD also
trend of lower intake of calcium and protein on a popula-
remains unclear given the lack detailed psychometric data,
tion level, and higher levels of nutritional inadequacies in
but represents an important focus for future research.
ASD, detected via idiosyncratic analyses using the cut
There is also a clear need to develop a frontline feeding
point method. Provisional data suggest food selectivity
screening tool to support research which can also be effi-
contributes to nutritional concerns related to ASD outside
ciently applied during medical appointments. Outcomes
of parent-mediated restrictions. Clinicians are encouraged
summarized in this review primarily involved study-
to increase screening for feeding concerns in children with
specific single-item measures, which limit conclusions
ASD and to use this information when counseling care-
regarding prevalence and topography that can be drawn
givers interested in pursuing an elimination diet.
across studies. Specifically, prevalence rates varied
depending on the content of the item or assessment Conflict of interest This was an unfunded study and no member of
method, with estimates as high as 95 % of a sample the research team has a conflict of interest.
describe as resisting trying new foods (Lockner et al.
2008). This could explain—at least in part—the high var-
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Gastrointestinal Conditions in Children With Autism Spectrum Disorder:

Developing a Research Agenda
Daniel L. Coury, Paul Ashwood, Alessio Fasano, George Fuchs, Maureen Geraghty,
Ajay Kaul, Gary Mawe, Paul Patterson and Nancy E. Jones
Pediatrics 2012;130;S160
DOI: 10.1542/peds.2012-0900N
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Gastrointestinal Conditions in Children With Autism

Spectrum Disorder: Developing a Research Agenda
Autism spectrum disorders (ASDs) are a set of complex neuro- AUTHORS: Daniel L. Coury, MD,a Paul Ashwood, PhD,b
developmental disorders defined behaviorally by impaired social in- Alessio Fasano, MD,c George Fuchs, MD,d Maureen
Geraghty, PhD, RD,e Ajay Kaul, MBBS, MD,f Gary Mawe,
teraction, delayed and disordered language, repetitive or stereotypic PhD,g Paul Patterson, PhD,h and Nancy E. Jones, PhDi
behavior, and a restricted range of interests. ASDs represent a significant aDevelopmental/Behavioral Pediatrics, Nationwide Children’s
public health issue with recent estimates indicating that as many as 1% Hospital, Columbus, Ohio; bDepartment of Medical Microbiology
of children in the United States are diagnosed with an ASD.1,2 Many and Immunology, University of California, Davis, Davis, California;
cPediatrics, University of Maryland, School of Medicine,
individuals with ASDs have symptoms of associated medical conditions,
Baltimore, Maryland; dPediatrics/Pediatric Gastroenterology,
including seizures, sleep problems, metabolic conditions, and gastro- University of Arkansas for Medical Sciences, Little Rock,
intestinal (GI) disorders, which have significant health, developmental, Arkansas; eMedical Dietetics, Abbott Laboratories, Columbus,
social, and educational impacts. Gastrointestinal complaints are a com- Ohio; fPediatrics, Cincinnati Children’s Hospital, Cincinnati, Ohio;
gAnatomy and Neurobiology, University of Vermont, Burlington,
monly reported concern for parents and may be related to problem Vermont; hCalifornia Institute of Technology, Pasadena, California;
behaviors and other medical issues such as dysregulated sleep (ATN and iClinical Programs, Autism Speaks, Los Angeles, California
Annual Registry Report, unpublished data, November 2009).3 Despite the KEY WORDS
magnitude of these issues, potential GI problems are not routinely autism, gastrointestinal disease, research
considered in ASD evaluations. This likely reflects several factors, in- ABBREVIATIONS
cluding variability in reported rates of GI disorders, controversies re- ASD—autism spectrum disorder
GI—gastrointestinal
garding the relationship between GI symptoms and the putative causes 5-HT—serotonin
of autism, the limited verbal capacity of many ASD patients, and the lack
Drs Ashwood, Fasano, Fuchs, Geraghty, Kaul, Mawe, and
of recognition by clinicians that certain behavioral manifestations in Patterson contributed equally to this work.
children with ASDs are indicators of GI problems (eg, pain, discomfort, or This manuscript has been read and approved by all authors.
nausea).4–10 This paper is unique and not under consideration by any other
publication and has not been published elsewhere.
Whether GI issues in this population are directly related to the patho-
www.pediatrics.org/cgi/doi/10.1542/peds.2012-0900N
physiology of autism, or are strictly a comorbid condition of ASD remains
to be determined, but clinical practice and research to date indicate the doi:10.1542/peds.2012-0900N
important role of GI conditions in ASDs and their impact on children as Accepted for publication Aug 8, 2012
well as their parents and clinicians.9 Address correspondence to Daniel L. Coury, MD, Professor of
Pediatrics and Psychiatry, The Ohio State University, Chief,
On November 15, 2009, a symposium addressing these issues was or- Developmental & Behavioral Pediatrics, Nationwide Children’s
ganized as an adjunct to the annual meeting of the North American Hospital, 700 Children’s Dr, Timken G-350, Columbus OH
Society for Pediatric Gastroenterology, Hepatology, and Nutrition. A panel 43205-2696
of international experts presented the latest scientific information on PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275).
pathophysiology, evaluation, and treatment strategies for GI conditions Copyright © 2012 by the American Academy of Pediatrics
in children and adolescents with ASDs. One aim of the meeting was to FINANCIAL DISCLOSURE: The information reviewed in this
article was based on presentations from a symposium and
raise awareness among gastroenterologists and GI researchers of GI
workshop funded by Autism Speaks and cosponsored by the
disorders in the ASD population and to provide clinicians with in- American Academy of Pediatrics GI Subcommittee and the North
formation to improve their clinical practice for these children. The American Society for Pediatric Gastroenterology, Hepatology
symposium addressed 4 major areas of concern for children with ASDs: and Nutrition. The authors have indicated they have no financial
relationships relevant to this article to disclose.
reflux, constipation, diarrhea, and nutrition. Each session reviewed the
state of the evidence, the latest findings on issues such as intestinal
permeability, inflammatory processes, innervation, motility, nutrition,
and the epidemiology, presentation, and clinical management of GI
issues.
The symposium also set the context for a follow-up workshop on No-
vember 16 that focused on identifying and prioritizing the key research
topics for further investigation. The 1-day workshop brought together
S160 COURY et al
SUPPLEMENT ARTICLE
a group of symposium participants and

speakers with a broad range of ex-
pertise in GI and autism clinical prac-
tice, and clinical as well as basic science
research.
This report provides an overview of
findings in GI and ASDs as presented in
the symposium and highlights the key
conclusions from the workshop. Spe-
cifically, the group identified the fol-
lowing 4 topics as priority areas for
further study: epidemiology of GI con-
ditions in ASDs, underlying pathology
(gut-brain connection, immune function,
animal models and genome-microbiome
interaction), treatment and outcome,
and nutrition. A recent consensus report FIGURE 1
Reported prevalence of gastrointestinal disorders in children with ASD.
on GI conditions in ASDs published in
Pediatrics in 20109 put forth 23 rec-
ommendations for the evaluation and
management of GI conditions in ASDs. in children with ASDs and higher rates UNDERLYING BIOLOGY OF GI
Although ASDs are behaviorally defined in all but one study when a control DYSFUNCTION IN ASDs
disorders, current thinking suggests population was used.10
Current State of Knowledge
multiple “autisms” with varying bi- Recommendations The underlying nature of GI dysfunction
ological underpinnings. Some of these
Accurately determining the rates of GI in ASDs and its relationship to etiology
may eventually be identified as having
disorders will require addressing the and ASD symptoms are poorly un-
associated GI symptoms, as has been
significant methodological limitations derstood, and systematic research in
seen with the MET gene. Pending new
of previous studies. These limitations this area has been limited. There is,
evidence on any such relationships, the
include suboptimal or, in some cases, however, emerging evidence relevant to
recommendations in this current arti-
lack of controls, variability in the control ASDs in the areas of immune function,
cle expand upon several of the key
groups used across studies, inclusion the relationship between signaling path-
recommendations made in the con-
of populations of children with ASDs ways of the gut and brain, and genome–GI
sensus statement highlighting areas in
that are heterogeneous, retrospective microbiome interactions. Increasingly,
need of new knowledge.
approaches, bias (selection, referral, evidence supports a combination of
or recall), and/or reliance on parent changes in gut microflora, intestinal
EPIDEMIOLOGY OF GI CONDITIONS report only.22 Developing rigorously permeability, inappropriate immune
IN ASDs designed, prospective population-based response, activation of specific meta-
Several studies have assessed the prev- prevalence studies of defined GI symp- bolic pathways, and behavioral changes
alence and types of GI disorders in chil- toms and disorders in ASD is a high pri- in genetically predisposed individuals.
dren with ASDs. The reported prevalence ority, and a shared data set would enable: Integrating findings across these areas
of any GI disorder in children with ASDs 1. Identification of risk factors includ- into a unifying theory will be critical to
ranges from 9% to 91% (see Fig 1), ab- ing clinical and behavioral indicators understanding the mechanisms and
dominal pain or discomfort ranges from of GI problems manifestations of GI disorders in ASDs.
2% to 41%, constipation from 6% to 45%, 2. Identification of atypical presenta-
diarrhea from 3% to 77%, and persistent Immune Function
tions of GI disorders in ASDs
diarrhea from 8% to 19%3,9,11–21. Al- Many reports have described immune
3. Identification of subpopulations with-
though all the studies have significant abnormalities in ASDs including changes
in ASD that have GI symptoms
methodological limitations, they col- in immune-related gene expression,
lectively indicate unusually high rates 4. Correlation with biomarkers skewed cytokine production, altered
of GI disorders or certain GI symptoms 5. Stratification of treatment groups T-cell function, and enhanced innate
PEDIATRICS Volume 130, Supplement 2, November 2012 S161

immune responses.23,24 Mucosal im- affect gut function and sensitivity.35 neophobia, deficits in social interaction
mune cells make up ∼70% of the Recently, studies involving germ-free and verbal and olfactory communica-
immune cells within the body, and mice inoculated with selective bacte- tion, increased stereotyped and re-
dysfunction in these cells may have ria have made clear that commensal petitive motor behaviors, enhanced
adverse consequences for GI function. and probiotic gut microbes can have anxiety, abnormal pain sensitivity and
Endoscopic analyses of children with an influence on brain function and eye blink conditioning, disturbed sleep
ASD and GI symptoms have revealed the behavior.36 patterns, seizures, and deficits in sen-
presence of a subtle, diffuse inflammation Serotonin (5-HT), a highly prevalent sorimotor gating. Some animal models
of the intestinal tract (reviewed in refs signaling molecule in the gut (80%–95% also exhibit neuropathology that is
9 and 25). Characterizing the nature of of 5-HT receptors are found in the gut), frequently seen in autism such as a
this inflammation remains an area in is critical for GI function, and alter- spatially restricted Purkinje cell deficit,
need of further investigation to fully ations in 5-HT signaling are implicated as well as characteristic neurochemi-
understand and to provide further ev- in a number of GI disorders. In both cal changes (5-HT abnormalities) and
idence of its relationship to GI symp- human and animal studies, altered 5- alterations in the immune status in the
toms in individuals with ASDs. HT signaling has been implicated in brain and periphery.46,49 It is critically
Autoimmune responses in children with inflammatory bowel diseases such as important that these animal models of
ASDs and a familial history of autoim- Crohn disease and ulcerative colitis ASD environmental and genetic risk
munity have been reported.25–28 Among and in functional disorders such as factors also be examined for GI symp-
the most common findings in ASD sub- irritable bowel syndrome and chronic toms. It should be noted that treatment
jects and their mothers is an increased constipation.37–42 Recent studies have with potential probiotic bacteria can
presence of autoantibodies directed demonstrated that 5-HT acts as a normalize anxiety-like behavior in colitis
toward central nervous system pro- proinflammatory modulator in the in- and infection mouse models,50 whereas
teins.29–33 In addition, autoantibodies testinal mucosa,43,44 and that circulat- such treatments in normal mice can
that bind to basement membranes ing 5-HT arising from the gut affects induce anxiolytic-like effects in some
of epithelial cells and colocalize with bone growth, with elevated 5-HT levels tests and anxiogenic effects in other
complement proteins are observed in leading to decreased bone density. tests.51–53
the intestinal mucosa of children with Because both GI and bone disorders Intestinal Permeability and Gut
ASD and GI symptoms.34 It has been have been observed in ASD, altered gut Microbiome
speculated that these autoantibodies 5-HT signaling may be associated with
It has been hypothesized that altered
could indicate the presence of inflam- these changes.45 The 5-HT system
intestinal permeability (“leaky gut”)
matory processes and/or an autoim- within the gut may be an important
may play a pathogenic role in autism.
mune component that could affect factor contributing to GI problems in
Indeed, several studies have reported
the integrity of the mucosal barrier children with ASD (see also Nutrition
impaired intestinal barrier function
and contribute to decreased mucosal below).
in ASD.54,55 Moreover, Altieri and col-
barrier integrity; it is also possible
leagues56 recently reported a signifi-
that they indicate previous mucosal
Animal Models cant (P , .01) elevation in the level of
inflammation.
Several environmental factors that in- a bacterial product in the urine of
crease the risk for the development of young autistic children. This molecule,
Gut–Brain Connection and autistic features, including maternal p-cresol, is produced by bacterial strains
Serotonin Signaling in the Gut infection and valproate exposure, have that are found to be elevated in ASD. ASD
The digestive system has its own com- been used successfully in rodents to children have higher counts and more
plex (enteric) nervous system that generate animal models of these ASD species of Clostridia, Bacteroidetes, and
regulates gut functions such as motility environmental risk factors (reviewed Firmicutes than controls.57,58
and mucosal secretion. It is now well in ref 46). There is also a non-human One critical feature of the GI tract is its
accepted that the gut–brain axis is a 2- primate model involving administra- ability to regulate the trafficking of
way street. For example, in addition to tion of antibrain antibodies to pregnant macromolecules between the environ-
traditional autonomic projections that monkeys47 and activating the maternal ment and the host through a barrier
regulate digestive reflexes, signals immune system.48 These environmen- mechanism. Together with gut-associated
traveling from the gut to the brain in- tal and genetic models exhibit vari- lymphoid tissue and the neuroendocrine
fluence satiety, and stress and anxiety ous characteristics of autism including network, the intestinal epithelial barrier,
S162 COURY et al
SUPPLEMENT ARTICLE
with its intercellular tight junctions, availability is altered in ASD children and nutritional factors affecting nu-
controls the trafficking of macro- with GI symptoms versus age-matched trition.
molecules.59 The protein zonulin is controls with GI symptoms. Another Nutritional status and nutrient intake
a component of intercellular tight junc- critical need is the determination of are inextricably related in children with
tions that is involved in regulating gut unique biomarkers (eg, cytokines, glu- autism. The assessment of nutritional
permeability.60,61 Small-intestinal expo- tathione redox, antibodies, and zonulin) status in ASDs has been recently dis-
sure to bacteria and gluten are 2 of in the plasma, urine, or stool associated cussed in terms of anthropometric,
the more powerful triggers for zonulin- with the development of ASD in high-risk biochemical, and clinical parameters.74
induced tight junction disassembly. En- populations. Current literature that addresses
teric infections have been implicated in overall nutrient intakes in ASD does not
the pathogenesis of several pathologic Animal Models indicate a definitive consensus either
conditions, including allergic, autoim- The work in human subjects will be toward evidence for differing nutri-
mune, and inflammatory diseases, by enhanced by capitalizing on animal tional intake or similar nutritional in-
causing impairment of the intestinal models to investigate the mechanisms take in children with ASD compared
barrier. In addition to bacterial expo- of altered GI function and sensation in with typically developing children.
sure, gliadin, the environmental trigger ASDs. The outcome measures would There is also a body of studies targeted
of celiac disease, has been shown to include the histochemical and immu- specifically on intake and status of
alter the intestinal barrier function by nologic abnormalities seen in subsets nutrients related to bone health.68
releasing zonulin.61 Moreover, there is of ASD children, as well as the changes Problems in comparing existing stud-
increasing evidence supporting an in gut microbial composition observed ies include: (1) lack of adequate control
association of gut microbiota with in ASD. Specifically, animal models for groups in some studies; (2) variations
behavioral abnormalities such as ASD can be used to determine if there is in assessment tools and nutrient
anxiety and emotional reactivity51,62–65 a distinct GI phenotype in autism and if analysis programs; (3) different time
and potentially affecting 5-HT metab- such GI conditions lead to behaviors frames postdiagnosis; and (4) differ-
olism.66 When considered as a whole, associated with ASD. Animal models can ent reference values and “cutoffs”
there is much to learn regarding the also be used to examine potential defining inadequate. Table 1 summa-
integrity of the intestinal mucosa, its therapeutic options, for instance by rizes existing studies and significant
response to various gut microbiota, experimentally altering the gut micro- findings.
and the resultant effects on the body bial composition.53,67 Outcome meas-
systemically. Hediger et al45 reported that dairy-free
ures here would include behavior,
diets and unconventional food prefer-
immune status, neuropathology, as
Recommendations ences could place boys with autism
well as GI motility and sensitivity.
Current evidence indicates the impor- and ASDs at high risk for thinner, less
tance of better characterizing the un- dense bones (based on bone cortical
Nutrition
derlying pathology of GI problems in thickness measures) in comparison with
ASD and determining if there are any Current State of Knowledge a standardized reference based on
unique characteristics of GI dysfunction Several factors affect the nutritional age-matched typical boys. This occurred
specificto autism. Two recommendations status in children with ASDs, with even for those not on dairy-restricted
are made for research in this area. The most falling into 2 main categories: diets, although the differences were
first is to determine whether children (1) medical/nutritional factors and greater for those on casein-free diets.
with ASD differ from typically developing (2) behavioral factors.68–73 Medical/ Several factors have been implicated in
children in terms of the GI microbiome, nutritional factors encompass GI the higher risk for suboptimal bone
metabolites, inflammation, neurotrans- symptoms/problems, food allergies, development in children with ASDs, in-
mitters, immune response, and mucosal metabolic abnormalities, and/or pre- cluding lack of exercise, GI problems,
integrity. Second, it is important to de- existing nutrient deficiencies, as well and compromised vitamin D and cal-
termine if any confirmed differences in as nutrition-related medication side cium intake owing to either sensory/
these factors could be used to identify effects. Behavioral factors include texture issues, idiosyncratic eating
those with or at risk for developing ASD problem eating or idiosyncratic eat- patterns, and restricted diets, partic-
by using appropriate control groups. ing behaviors, sensory-processing ularly the gluten-free, casein-free
For example, 1 critical area would in- difficulties, and family factors.68 In diets.45,68,85 A recent nutrient intake
clude determining if mucosal 5HT this section, we focus on the medical study (based on 3-day food records) in

TABLE 1 Studies Investigating Nutritional Quality in Children with Autism

Study No.of ASD No. of Control/ Dietary Tool Significant Findings
subjects Typical Subjects
Raiten and Massaro, 40 34 7-d Diet Record 1. ASD group had significantly greater intake of protein, carbohydrates,
198675 niacin, thiamin, riboflavin, calcium, phosphorus, and iron (P , .02)
2. No significant difference in vitamin A, vitamin C, or fat.
Ho et al, 199776 54 N/A 3-d Diet Record 1. Only 4 subjects with ASD (7.4%) met recommended servings from all
food groups.
2. All subjects had adequate protein intake, but had lower fat intake
and higher carbohydrate intake than recommended nutrient intake
for Canadians (RNI).
3. 42.6% of subjects were obese.
Cornish, 199877 17 N/A 3-d Dietary Recall 1. Nutrient intakes below RNI levels in 53% of children for one or more of
Food Frequency following: vitamin C, vitamin D, niacin, riboflavin, vitamin B6, calcium,
Questionnaire (FFQ) and zinc.
2. Calcium and niacin levels .200% or RNI in those children who drank
milk.
Lindsay et al, 200678 20 N/A FFQ 1. ,80% Recommended Dietary Allowance (RDA)/dietary reference
intake (DRI) considered inadequate
2. 45% consumed ↓calcium, 30% ↓pantothenic acid, 25% ↓ vitamin D,
40% ↓vitamin K
Levy et al, 200779 52 N/A 3-d Diet Record 1. The ASD subjects met 95%–101% of RDA guidelines for calories,
carbohydrates, and fat
2. ASD subjects overconsumed protein at 211% of RDA with a range of
67%–436% RDA among subjects
3. This study used 77% of RDA consumption as adequate diet
Lockner et al, 200880 20 20 3-d Diet Record 1. Vitamins E and A were the least likely to be met by Estimated Average
Requirement (EAR) for both groups
2. ASD subjects consumed less calcium and fiber, but with no established
EAR, significance was not determined
Schmitt et al, 200881 20 18 3-d Diet Record 1. This study defined adequate consumption as .67% of DRI
2. Both groups consumed ,67% for fiber
3. ASD group consumed ,67% for vitamins E and K
Johnson et al, 200871 19 15 24-h Recall 1. This study considered ,80% of RDAs or DRIs as inadequate
2. ASD group consumed significantly less vitamin K and significantly
fewer food choices from the vegetable group
Herndon et al, 200982 46 31 3-d Diet Record 1. ASD children consumed significantly less calcium but consumed
increasingly more vitamins B6 and E than controls.
2. ASD children consumed significantly more nondairy proteins and
fewer dairy items.
3. Both groups did not meet RDI for fiber, calcium, iron, vitamin D, and
vitamin E
Xia et al, 201083 111 N/A 3-d Diet Record 1. Comparison with Dietary Reference Intakes (DRI) for Chinese children.
2. Vitamin E and niacin exceeded 100% of DRI
3. Intakes were below DRI for vitamins A, B6 and C, Folic acid, calcium and
zinc
Bandini et al, 201071 53 58 Parent Interview 1. For both groups, fiber intake was inadequate as was intake of vitamin D,
vitamin E, and calcium
FFQ 2. For ASD children, inadequate intake of vitamin D and calcium was more
frequent
3-d Diet Diary 3. ASD children had a higher number of nutrients at inadequate intake
levels.
4. A limited food repertoire, but not food refusal, was associated with
higher nutritional inadequacy for both groups
Zimmer et al, 201184 22 22 FFQ 1. ASD children had higher intakes of magnesium
2. ASD children had lower intakes of protein, calcium, vitamin B12, and
vitamin D.
3. Selective eaters with ASD had greater likelihood of inadequate intake
of calcium, vitamin B12, zinc, and vitamin D
4. Selective eaters with ASD compared with nonselective eaters with ASD
had greater likelihood of inadequate intake of calcium.
5. ASD children had less food variety than typically developing children
S164 COURY et al
SUPPLEMENT ARTICLE
children with ASDs ages 3 to 9 years on correcting identified deficiencies address the particular needs of an
revealed that the nutrients commonly and any additional benefits to the ASD population. Evidence-based algo-
analyzed as inadequate were those individual’s ASD symptoms. Further rithms specific for ASD should be de-
important for bone health (vitamins elucidation of the interrelationships veloped for each of the most common
A, D, and K) with 58.3%, 58.3%, and among these variables will assist in diagnoses that would address both
91.7% of the children consuming the establishment of clinical algo- management as well as treatment
intakes ,80% dietary reference in- rithms for categorization and effec- outcomes, tested rigorously, and sub-
take, respectively.86 tive treatment. sequently refined to optimize outcomes
(see this issue). These algorithms will
Collectively to date, these studies in-
provide the basis for comprehensive
dicate a trend for clinically significant TREATMENT AND OUTCOME guidelines and final recommendations
suboptimal nutrient intake in children
Current State of Knowledge provided to clinicians caring for chil-
with ASDs, with particular concern
dren with ASD.
related to bone health nutrients. In As highlighted in the previous sections,
general, nutritional status parameters children with ASDs frequently experi-
need to be refined and tailored for this ence GI symptoms, but their prevalence, Placebo-Controlled Trials
population. nature and, therefore, best treatments The positive impact of treatments aimed
remain elusive. Limited understanding at the GI problems in ASD children is
of the underlying pathology of GI con- widely reported by parents, but there
Recommendations
ditions in ASD limits the scientific ra- is little well-designed, controlled re-
Standardize Nutrient Assessment tionale for many of the interventions search to validate these interventions. It
Nutrient intake studies in ASDs should (eg, antifungal therapy, enzymes, and will also help substantially to stratify
be standardized by: (1) including con- nutritional supplements) aimed at the ASD population to identify specific
trol groups with typical children, (2) correcting these GI dysfunctions.9,10 A subgroups of children who may better
using consistent nutrient assessment possible theory unifying all the factors benefit from interventions. Many of
tools and analysis programs, (3) ac- mentioned above would link changes these approaches involve life-long
counting for the time frame post- in the gut microbiome with GI in- interventions (such as implementa-
diagnosis,and(4)establishing consistent flammation and other immunologic tion of a gluten-free diet), so better
reference values and acceptable cutoffs changes. identifying the individuals likely to re-
for defining inadequate intake. The most common GI diagnoses iden- spond to particular treatments can
tified in children with ASDs include reduce the costs and burden on families
Correlations With Nutritional Status constipation, diarrhea, and gastro- associated with nonefficacious treat-
esophageal reflux, and these are usu- ments. Stratification might be con-
Research should address the relation- ally treated in a standard manner.9,10 ducted by using clinical phenotypes or
ships between baseline nutritional Children with ASDs may not present through the identification and use of
status (by using standardized nutrient with the typical symptoms of a GI dis- specific biomarkers (see Table 2 ).
intake study principles and a thorough order, however, and an alteration of
nutrition assessment) and health status, The identification of specific ASD
their baseline behavior may be the microbiome and metabolic phenotypes
GI symptoms (constipation, diarrhea, only indicator of its existence. There is
flatulence, bloating, and nausea), bone a serious dearth of adequately designed
cortical thickness/bone mineral density, studies on treatments for documented TABLE 2 Biomarkers as Potential Outcome
behavior, sleep latency, food selectivity/ GI disorders and their outcomes, in- Measures
idiosyncratic behaviors, sensory- cluding behavioral changes, in children Biomarker Clinical Significance
processing difficulties (hypersensitiv- with ASDs. Intestinal Leaky gut
ity to certain food textures, tastes, or permeability
smells), and measures of inflammation Calprotectin Intestinal inflammation
(eg, cytokines, c-reactive protein). Recommendations Celiac disease Celiac disease and gluten
serology tests sensitivity
Treatment Guidance Food allergy panel Food allergies
Organic acid testing Vitamin B12 or folic acid
Efficacy of Nutritional Intervention Although general pediatric guidelines deficiencies
Studies should determine the effec- exist for specific GI conditions, the Analysis gut Gut dysbiosis
tiveness of nutritional interventions recommendations may not always microbiota

TABLE 3 Key Takeaway Messages microbiota in ASD, but also on other

Epidemiology metabolic imbalances that may be
• Gastrointestinal conditions in ASD are at least as common as in typically developing population present. This knowledge, in turn, could
• It is less clear whether GI conditions are more common in children with ASD aid in the development of strategies for
• Individuals with ASDs should receive diagnostic evaluations and medical treatments for GI disorders just as
children without ASD do
novel approaches for the prevention/
Biology treatment of ASD.
• Several studies identify abnormalities of the immune system in ASD
• Some GI conditions in ASD may have an immunologic basis
• Some individuals with ASD have abnormalities in serum concentrations of the neurotransmitter serotonin DISCUSSION
• Alterations in serotonin may be associated with anxiety, GI symptoms, and bone density abnormalities
Nutrition Clinical experience, parent concern, and
• Several studies have identified abnormalities of diet and altered bone mineral density, but the cause developing lines of research all make
(dietary preference, allergic issues, or absorptive problems) remains uncertain.
Treatment clear the importance of addressing GI
• No treatments for GI problems specific to children with ASD currently exist. problems and symptoms among chil-
• Individuals with ASD and GI conditions appear to respond to treatment much as people with typical dren and adolescents with ASD. Although
development do.
• Studies that can lead to management that addresses specific challenges of the disorder are still needed. thissymposium and reviewindicatehuge
gaps in evidence, especially to support
interventions to improve the health and
TABLE 4 Key Research Objectives for an Integrated Approach to Addressing Knowledge Gaps GI symptoms in ASD, several promising
1. Determine the pathology of gastrointestinal conditions in ASDs through integrated studies of the gut areas of research bring optimism and
microbiome, metabolism, inflammation, immunity, and mucosal integrity. The outcome measures would focus to the key next steps in in-
include the histochemical and immunologic abnormalities seen in subsets of ASD children, as well as the vestigation and treatment trials. Table 3
changes in gut microbial composition observed in ASDs.
2. Increase the use of animal models to better understand the underlying pathology. Studies manipulating the indicates key messages from this work.
microbiome in animal models may help promote better understanding of these conditions. Subsequent to this symposium, others
3. Identify biomarkers for assessing the status of these conditions and to guide identification and treatment. endorsed many of the same points.9,10
4. Identify biomarkers of nutritional status that will not only guide monitoring of response to treatment but
help identify those requiring intervention.
This article reviews key areas of epi-
5. Identify behavioral phenotypes related to poor nutritional status to better identify and treat this population. demiology, underlying pathology, nu-
6. Develop evidence-based clinical algorithms to help guide clinicians in the evaluation and treatment of trition, and treatment and outcome.
gastrointestinal problems in individuals with ASDs.
Ongoing efforts should also address
how to integrate diverse areas of re-
can also help to define additional di- microbiota composition. These findings search and findings into better un-
agnostic tools, biomarkers, and thera- may have a far-reaching impact not derstanding of the complex activities
peutic interventions such as the use only on our understanding of the role of the GI system in children with ASDs
of probiotics to change the intestinal of specific allergens, leaky gut, and (Table 4).
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S168 COURY et al
Gastrointestinal Conditions in Children With Autism Spectrum Disorder:

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www.nature.com/scientificreports
OPEN Altered gut microbiota and short

chain fatty acids in Chinese children
with autism spectrum disorder
Received: 7 June 2018 Simeng Liu1, Enyao Li2, Zhenyu Sun2, Dongjun Fu3, Guiqin Duan4, Miaomiao Jiang2, Yong Yu1,
Accepted: 21 November 2018 Lu Mei1, Pingchang Yang5, Youcai Tang2,6 & Pengyuan Zheng1
Published: xx xx xxxx
Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impairments
in social interactions and communication, restricted interests and repetitive behaviors. Several studies
report a high prevalence of gastrointestinal (GI) symptoms in autistic individuals. Cumulative evidence
reveals that the gut microbiota and its metabolites (especially short-chain fatty acids, SCFAs) play
an important role in GI disorders and the pathogenesis of ASD. However, the composition of the gut
microbiota and its association with fecal SCFAs and GI symptoms of autistic children remain largely
unknown. In the present study, we sequenced the bacterial 16S rRNA gene, detected fecal SCFAs,
assessed GI symptoms and analyzed the relationship between the gut microbiome and fecal SCFAs in
autistic and neurotypical individuals. The results showed that the compositions of the gut microbiota
and SCFAs were altered in ASD individuals. We found lower levels of fecal acetic acid and butyrate and
a higher level of fecal valeric acid in ASD subjects. We identified decreased abundances of key butyrate-
producing taxa (Ruminococcaceae, Eubacterium, Lachnospiraceae and Erysipelotrichaceae) and an
increased abundance of valeric acid associated bacteria (Acidobacteria) among autistic individuals.
Constipation was the only GI disorder in ASD children in the present study. We also found enriched
Fusobacterium, Barnesiella, Coprobacter and valeric acid-associated bacteria (Actinomycetaceae)
and reduced butyrate-producing taxa in constipated autistic subjects. It is suggested that the gut
microbiota contributes to fecal SCFAs and constipation in autism. Modulating the gut microbiota,
especially butyrate-producing bacteria, could be a promising strategy in the search for alternatives for
the treatment of autism spectrum disorder.
Autism spectrum disorder is characterized by persistent deficits in social interactions and communication com-
bined with restricted, repetitive patterns of behaviors, interests, and activities1. The estimated male to female ratio
of ASD is 4–5:1, and the reported prevalence of ASD is 14.6/1,000 in children at eight years of age2. ASD imposes
a heavy healthy and economic burden on autistic individuals, their families and society. It is well accepted that
both genetic and environmental factors contribute to the etiology of ASD3,4.
Emerging evidence indicates that the composition of the gut microbiota and microbial metabolites are altered
in a wide range of diseases, including ASD5. The human gut harbors trillions of microorganisms and contains
approximately 1014 bacterial genes that belong to approximately 1,000 species6. The gut microbiota commu-
nicates with the central nervous system (CNS) through endocrine, immune, metabolic and neural pathways7.
Communication occurs via a complex bidirectional system known as the “microbiome-gut-brain axis”, which
plays an important role in physiology, brain development, immune and metabolic homeostasis8. Disturbance of
the microbiome-gut-brain axis may be associated with the pathogenesis of ASD.
Short-chain fatty acids (SCFAs) are the functional and vital components in the microbiome-gut-brain axis.
SCFAs mainly consist of acetic acid (AA), propionic acid (PPA), butyrate (BTA) and valeric acid (VA). It is
1
Department of Gastroenterology, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.
2
Department of Children Rehabilitation Medicine, the Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou,
450052, China. 3School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, Henan, 450001, China.
4
Center of Children Psychology and Behavior, the Third Affiliated Hospital of Zhengzhou University, Zhengzhou,
450052, China. 5Brain Body Institute, McMaster University, Hamilton, ON, Canada. 6Department of Pediatrics, the
Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China. Correspondence and requests for
materials should be addressed to Y.T. (email: tangyoucai@hotmail.com) or P.Z. (email: medp7123@126.com)
Scientific REPorTS | (2019) 9:287 | DOI:10.1038/s41598-018-36430-z 1

www.nature.com/scientificreports/
reported that PPA disturbs GI function in a manner similar to the abnormalities in ASD9. Butyrate inhibits his-
tone deacetylases, suppresses intestinal pro-inflammatory macrophage function10, and regulates the blood-brain
barrier (BBB) and gut permeability11, which are altered in ASD12. However, the published data on fecal SCFAs in
autism are inconsistent.
Cumulative evidence demonstrates that gastrointestinal (GI) disorders are common comorbidities in autistic
children. GI symptoms, such as constipation, abdominal pain, diarrhea and flatulence, have a close relation-
ship with ASD13. Furthermore, Adams suggested that GI problems are associated with the severity of autism14.
Campbell revealed genetic evidence supporting the association between GI disorders and ASD15. However, it has
not been determined which GI symptoms are associated with autistic children.
Additionally, the composition of the gut microbiota and its association with fecal SCFAs and GI symptoms
in autistic children remain largely unknown. In this study, we sequenced the V3-V4 hypervariable regions of the
bacterial 16S ribosomal RNA gene, detected fecal SCFAs and assessed the GI symptoms of autistic and neurotyp-
ical children to better understand the role of the gut microbiota in ASD.
Results
The ASD group has a higher incidence of constipation than the NT group. The mean age of autis-
tic and neurotypical children was 4.43 ± 1.47 and 4.28 ± 1.00 years, respectively (P = 0.177). The sex ratio (boys to
girls) of autistic subjects was 25:5 (indicating a male gender bias in autism) and was 16:4 for neurotypical children.
There were eight children (26.67%) with a moderate level of constipation and one child (3.33%) with a severe level
of constipation out of 30 autistic subjects. Constipation was significantly higher in autistic subjects than that in
neurotypical subjects (only 1 out of 20 normal subjects had a moderate level of constipation) (P < 0.05). None of
the patients suffered from diarrhea or abdominal pain. We found no significant differences between autistic and
neurotypical subjects in regard to the average stool consistency, stool smell and flatulence. In addition, there was
no significant difference in the total GI scores between the two groups (Table 1).
Fecal levels of SCFAs in the ASD group distinctly vary from those in the NT group. The concen-
trations of acetic acid, propionic acid, butyric acid and valeric acid in feces are shown in Fig. 1. The acetic acid
level was significantly decreased in autistic subjects (732.4 ± 229.8) compared with that in neurotypical controls
(899.9 ± 204.4, P = 0.011). No significant differences were found in the levels of propionic acid between the two
groups (P = 0.243). Compared with neurotypical children (563.3 ± 186.7), the butyrate concentration was sig-
nificantly decreased in autistic children (413.2 ± 172.3, P = 0.005). However, the level of valeric acid in autistic
children (1,646.9 ± 451.3) was markedly higher than those in neurotypical participants (615.8 ± 221.0, P < 0.001).
Autistic subjects harbor an altered composition of the gut microbiota. We obtained 1,922,441
sequences and 838,859,484 bases from 50 samples with an average of 38,449 sequences per sample. The analysis
of alpha diversity revealed no significant differences using SOBS, Chao and ACE (reflecting species richness).
However, the scores of the Shannon (Fig. 2a) and Shannoneven (Fig. 2b) indexes regarding the OTU levels in
autistic samples decreased compared to those in neurotypical controls, suggesting that autistic children had less
species diversity and evenness. Moreover, we performed principal coordinated analysis of beta diversity of the
weighted UniFrac distance on the OTU data (Fig. 2c). The results revealed that the overall composition of the
ASD gut microbiota was different from that of the NT gut microbiota.
We assessed the mean relative abundances of the taxa in ASD and NT to discriminant the specific alterations
of the microbiota. At the phylum level, Firmicutes were significantly decreased in ASD (51.91%) compared to
those in NT (58.82%), whereas Acidobacteria were considerably increased in ASD (0.01143%) compared to those
in NT (0.00351%) (Fig. 3a). At the family level, there were no significant differences regarding the abundance of
Bacteroidaceae, the most abundant family in the two groups. Furthermore, we observed six bacterial taxa that
displayed different abundances between the two groups. Veillonellaceae and Enterobacteriaceae were enriched in
autistic samples, whereas Ruminococcaceae, Streptococcaceae, Peptostreptococcaceae and Erysipelotrichaceae were
significantly decreased in ASD with respect to those in NT (Fig. 3b).
LEfSe was used to detect significant alterations in the bacterial composition associated with ASD and
NT16. ASD subjects showed a significant increase in Acidobacteria, Enterobacteriaceae and Pseudomonadaceae
apart from Firmicutes. The results also revealed a specific association between Ruminococcaceae and
Peptostreptococcaceae within the order of Clostridiales in NT subjects. Regarding the order of Lactobacillales,
Enterococcaceae and Streptococcaceae were overrepresented in ASD and NT subjects, respectively. In addition,
there were a significant increase of the taxa Veillonellaceae and a significant decrease of Erysipelotrichaceae in ASD
subjects compared to those in NT subjects. Furthermore, Megamonas was enriched in ASD subjects, whereas
Eubacterium and Lachnospiraceae-NC2004-group were enriched in NT subjects at the genus level (Fig. 4).
Correlation of the gut microbiota with SCFAs. Spearman correlation was used to associate the dif-
ferentially abundant taxa with the fecal levels of SCFAs at the family level (shown in Fig. 5). Acidobacteria
(significantly increased in ASD, rs = 0.349, P = 0.013) and Actinomycetaceae (rs = 0.298, P = 0.036) were pos-
itively correlated with valeric acid. Streptococcaceae (rs = 0.368, P = 0.009), Peptostreptococcaceae (rs = 0.378,
P = 0.007), Lactobacillaceae (rs = 0.467, P = 0.001) and Clostridiaceae_1 (rs = 0.441, P = 0.001), which were
enriched in NT subjects, had a strong positive correlation with butyrate. In addition, we also observed a positive
correlation between Family_XIII (rs = 0.281, P = 0.048), Leuconostocaceae (rs = 0.321, P = 0.023) and butyrate.
Desulfovibrionaceae (rs = 0.295, P = 0.038) and Streptococcaceae (rs = 0.281, P = 0.048) were highly correlated
with propionic acid. Moreover, Desulfovibrionaceae (rs = 0.36, P = 0.01) was also positively correlated with the
level of acetic acid.

Autistic Neurotypical p value

Subjects (n) 30 20 −
Age (years) 4.43 ± 1.47 4.28 ± 1.00 n.s.
Gender n.s.
Male 25 (83%) 16 (80%)
Female 5 (17%) 4 (20%)
Constipation 0.035*
0 21 (7%) 19 (95%)
1 8 (26.7%) 1 (5%)
2 1 (3.3%) 0 (0%)
Diarrhea 1.000
0 30 (100%) 20 (100%)
1 0 (0%) 0 (0%)
2 0 (0%) 0 (0%)
Average stool consistency 0.771
0 29 (96.7%) 19 (95%)
1 1 (3.3%) 1 (5%)
2 0 (0%) 0 (0%)
Stool smell 0.783
0 23 (76.7%) 16 (80%)
1 7 (23.3%) 4 (20%)
2 0 (0%) 0 (0%)
Flatulence 0.243
0 28 (93.3%) 20 (100%)
1 2 (6.7%) 0 (0%)
2 0 (0%) 0 (0%)
Abdominal pain 1.000
0 30 (100%) 20 (100%)
1 0 (0%) 0 (0%)
2 0 (0%) 0 (0%)
Totally scores 0.164
0 15 15
1 12 3
2 1 2
3 2 0
Table 1. Characteristics and GI symptom scores of participants. Note: n.s. means not statically significant.
*p < 0.05 vs NT group.
Figure 1. Fecal levels of short-chain fatty acids in participants. AA: acetic acid, PPA: propionic acid, BTA:
butyrate, VA: valeric acid. Values are expressed as the mean ± SD, *p < 0.05 vs NT group; **p < 0.01 vs NT group.
Constipation alters the composition of the gut microbiota in ASD. Constipation was the signifi-
cant GI symptom in ASD among the 6 GI symptoms evaluated in our study and is often reported as a common
comorbidity in ASD. Considering the single constipated NT sample, which showed no statistical significance, we
further compared the composition of the gut microbiota among constipated (ASD-C), non-constipated autistic
(ASD-NC) and non-constipated neurotypical subjects (NT-NC). The species richness and diversity computed

Figure 2. Gut microbiota diversity in ASD and NT subjects. (a) Alpha diversity based on the Shannon index of
the OTU level (b) Alpha diversity based on the Shannoneven index of the OTU level. (c) PCoA of beta diversity
based on the weighted UniFrac analysis of the OTU level. Autistic and neurotypical subjects are colored in red
and green, respectively. *p < 0.05 vs NT group.
Figure 3. Abundances of taxa in ASD and NT participants. The mean relative abundances of taxa at the phylum
(a) and family (b) levels in ASD and NT participants. Red and green bars indicate the mean relative abundances
of taxa in autistic and neurotypical subjects, respectively. *p < 0.05 vs NT group; **p < 0.01 vs NT group.
by analysis of alpha diversity did not show any significant differences among these three groups. When using
the weighted UniFrac distances to calculate beta diversity, it was revealed that the microbiota of ASD-C devi-
ated from those of ASD-NC and NT-NC (Fig. 6a). Order level analysis showed that Clostridiales, Lactobacillales,
and Erysipelotrichales were represented in NT subjects and Fusobacteriales was represented in ASD-C sub-
jects (Fig. 6b). We further analyzed genera with differentially abundant gene expression in ASD-C, ASD-NC
and NT-NC samples using LEfSe. The results revealed a specific association of Fusobacterium, Barnesiella,
Coprobacter, Olsenella, Allisonella and Actinomycetaceae with ASD-C subjects. Holdemanella was overrepresented
in ASD-NC subjects compared to that in ASD-C and NT-NC subjects. The NT-NC microbiome was characterized
by a preponderance of Eubacterium_rectale_group, Streptococcus, Butyricicoccus, Eubacterium_ventriosum_group,
Propionibacterium and Lachnospiraceae-NC2004-group (Fig. 6c).
Discussion
Several studies have noted alterations in the composition of the gut microbiota in ASD patients compared with
neurotypical individuals. In the present study, we found less species diversity and evenness in autistic chil-
dren and the overall structure of the gut microbiota compared with NT children. The results are similar to the
study conducted by Kang17. Several studies demonstrate a significant increase in Firmicutes and a lower abun-
dance of Bacteroidetes, resulting in an increased Firmicutes/Bacteroidetes ratio in autism spectrum disorder.
We observed a considerable reduction of Firmicutes and a small proportion of the Clostridium genus in autis-
tic children, consistent with the study of Finegold18. More specifically, Ruminococcaceae, Peptostreptococcaceae,
Lactobacillales, Streptococcaceae, Eubacterium and Lachnospiraceae-NC2004-group were reduced within the phy-
lum of Firmicutes in autistic subjects. A positive correlation between these bacteria and the fecal concentration of
butyrate was determined in our study. Butyrate is mainly produced by anaerobic microbes from the fermentation
of host-indigestible carbohydrates via the acetyl-coenzyme A (AcCoA) pathway19. Most butyrate-producing bac-
teria belong to Clostridium clusters IV and XIVa, including Ruminococcaceae, Lachnospiraceae, Butyricicoccus and
Eubacterium_rectale_group20,21. Other Clostridium clusters also produce butyrate, including clusters I and XVI.
However, these clusters produce lower levels of butyrate in comparison with Clostridium clusters IV and XIVa22,23.
Moreover, Hakalehto reports that Lactobacilli contributes to the growth of butyrate-producing clostridia24. Apart

Figure 4. Differentially abundant bacterial taxa associated with the ASD and NT groups according to LEfSe
analysis. The Cladogram generated by the LEfSe (from phylum to family level) and LDA scores (genus level)
identify differentially abundant bacterial taxa associated with ASD and NT subjects. (a) Red and green dots
indicate the bacterial taxa enriched in ASD and NT subjects, respectively. (b) Enriched bacterial taxa in ASD
have positive LDA scores (red), and NT enriched bacterial taxa have negative scores (green). Only the taxa
having an LDA > 2.0 are shown in the figure.
from Firmicutes, we found that Erysipelotrichaceae was highly decreased in ASD, and Estaki indicated that
Erysipelotrichaceae is also a key butyrate-produce member25.
Our data also showed a decrease in the fecal butyrate levels in ASD subjects. Butyrate is one of the major
metabolites of the gut microbiome and regulates the activities of the microbiome-gut-brain axis. Butyrate regu-
lates the integrity of gut barrier functions. Peng found that butyrate regulated AMP-activated protein kinase to
reorganize and upregulate the expression of tight junction-associated protein26. Huang reported that butyrate,
acting as a histone deacetylase-1 (HDAC-1) inhibitor, restored the intestinal epithelial barrier function by reg-
ulating the TWIK-related potassium channel-1 (Trek-1)27. Butyrate can strengthen mucosal immunity by its
anti-inflammatory potential. In addition, a previous study indicates that butyrate restores BBB permeability by
increasing histone acetylation and the expression of tight junction proteins28. Moreover, Kartsman discovered
that the treatment with butyrate could regulate social behavior in an autism mouse model. Together, our results
and the information mentioned above demonstrate that butyrate can regulate the activities of the gut-brain axis
in ASD via specific transporters/receptors, regulating immune system and as an HDAC inhibitor.
We also observed increases in the abundance of taxa in the autistic group, including Acidobacteria,
Enterobacteriaceae, Pseudomonadaceae, Veillonellaceae and Megamonas. Acidobacteria was positively corre-
lated with valeric acid, leading to an increase in the fecal levels of valeric acid. Consistent with our research, a
higher level of valeric acid has also been reported in ASD children29. It seems that valeric acid is another critical
factor in autism, although its physiological functions are less reported regard to regulating the activities of the
microbiome-gut-brain axis. Further studies on this point are needed. Enterobacteriaceae and Pseudomonadaceae
are facultative anaerobic or aerobic fermentative Gram-negative bacilli. Most of these bacilli are opportun-
istic pathogens. Enterobacteriaceae overgrow in most inflammation models30. Enterobacteriaceae or their
components (lipopolysaccharides) induce inflammation, aggravate intestinal injury and increase intestinal per-
meability, hypersensitivity, constipation and IBS31. Translocation of lipopolysaccharides into the blood can cause

Figure 5. Correlation of the gut microbiota with the levels of fecal SCFAs. The heatmap shows the correlation
coefficient between bacterial taxa and the level of fecal SCFAs at the family level. *p < 0.05; **p < 0.01.
neuroinflammation and increase the BBB permeability. Lipopolysaccharides are associated with neurological
disorders, such as major depression and Parkinson’s disease32. De Angelis also revealed an elevated abundance of
Enterobacteriaceae in autistic children33.
The results of previous studies on fecal SCFAs are inconsistent in autism. Lower concentrations of SCFAs
were reported in children with ASD by Adams14. In contrast to their study, Wang reported elevated SCFAs in
autistic children29. This inconsistency might be due to the specific alteration of the gut microbiota in different
populations and the technical or samples preparation differences in the different studies. Our results showed
lower concentrations of fecal AA and BTA and a higher concentration of fecal VA in ASD individuals. The level
of fecal SCFAs was mainly determined by the amount of SCFA producing bacteria and complex carbohydrates in
the diet. In our study, all subjects had a normal diet. The lower levels of fecal BTA and higher levels of fecal VA are
probably due to the variance of BTA-producing and VA-producing bacteria. We also found lower levels of fecal
AA and a positive correlation between Desulfovibrionaceae and AA in ASD. Acetate is produced by various types
of bacteria, including Bacteroides, Bifidobacterium and Prevotella34. Acetic acid promotes cholesterol synthesis
in the liver. PPA is mainly produced by Clostridia, Desulfovibrio, Propionibacterium and Bacteroides35. We found
that Desulfovibrionaceae and Streptococcaceae were positively correlated with PPA. PPA can induce ASD-like
behaviors through intracerebroventricular and intraventricular administration36,37. PPA induces impaired social
behavior through regulating neurotransmitters, such as dopamine38. We did not find a significant alteration of
fecal PPA in autistic subjects in our study, probably due to ethnic and dietary structure difference in Chinese
children compared to others.

Figure 6. Constipation alters the composition of the gut microbiota in ASD. (a) PCoA of beta diversity based
on the weighted UniFrac analysis on the OTU level among ASD-C, ASD-NC and NT-NC subjects. (b) The
mean relative abundances of taxa at the order level in ASD-C, ASD-NC and NT-NC participants. (c) LDA scores
indicate differentially abundant bacterial taxa associated with ASD-C, ASD-NC and NT-NC subjects (only the
taxa having an LDA > 2.0 are shown in the figure). Blue, red and green bars indicate the bacterial taxa enriched in
ASD-C, ASD-NC and NT-NC subjects, respectively. *p < 0.05 vs NT group; **p < 0.01 vs NT group.
Despite the report of constipation, diarrhea, abdominal pain, flatulence and other GI disorders in ASD
patients by other researchers, we only observed that constipation was significantly increased in the ASD group
compared to that in the control group. Holingue indicates that constipation is the most prevalent GI symp-
tom in ASD39. We found enriched Fusobacterium, Barnesiella, Coprobacter and valeric acid-associated bacteria
(Actinomycetaceae) and reduced butyrate-producing taxa in ASD-C subjects, suggesting that constipation has a
notable effect on the gut microbial composition within ASD subjects.
In summary, constipation is the GI comorbidity in ASD children in our study. We observed a shifted gut
microbiota and its association with fecal SCFAs in ASD individuals. Therapeutic strategies that modulate the gut
microbiota and SCFAs, especially butyrate-producing bacteria, may have potential to relieve ASD and GI-related
symptoms in ASD.
Materials and Methods

Study participants. We recruited 30 (2.5–18 years old) autistic subjects (ASD) from the Fifth and Third
Affiliated Hospital of Zhengzhou University. Twenty age- and sex-matched healthy volunteers were accepted
as neurotypical controls (NT). The diagnosis of autism was established according to DSM-V (Diagnostic and
Statistical Manual of Mental Disorders, 5th Edition)1 and ICD-10 (International Statistics Classification of Diseases
and Related Health Problems, 10th Revision)40 by two experienced child neuropsychiatrists. Neurotypical controls
were typically developing children, without an autism diagnosis and not directly related to an autistic individual.
The exclusion criteria included a history of nutritional supplements and special diets, presence of significant
physical abnormalities, and neurological disorders of known etiology. Participants in this study were not treated
with antibiotics, antifungals, probiotics or prebiotics for at least three months before sampling. All methods were
carried out in accordance with the relevant guidelines and regulations. All experimental protocols were approved
by the Ethical Committee of the Fifth Affiliated Hospital of Zhengzhou University (No. 2016-1001). Informed
written constent was obtained from the parents and/or legal guardians of the enrolled participants.
Severity scales of GI symptoms. Gastrointestinal symptoms were assessed following a modified version
of the 6-GI Severity Index14, including constipation, diarrhea, average stool consistency, stool smell, flatulence
and abdominal pain.
Fecal sample collection. Fecal samples were collected and transported to our laboratory for processing
within 30 minutes. The 200 mg stool was preserved in fecal bacteria DNA storage tubes (Tinygene Biological
Company, China) and stored at −80 °C for 16S rRNA sequencing. The 600 mg stool was preserved in sterile tubes
and immediately frozen at −80 °C for further SCFA analysis.
Fecal SCFA analysis. The short-chain fatty acids were extracted as described previously41. Briefly, 300 mg
stool was homogenized with 1 ml ddH2O and centrifuged at 12,000 g for 10 min. The supernatant was homog-
enized with 100 μl concentrated HCl and subsequently extracted for 20 min using 5 ml of diethyl ether. After
centrifugation (3,500 rpm, 10 min), we mixed the organic phase with 500 μl NaOH (1 M). Then, the organic phase
was extracted and centrifuged again. The aqueous phase was mixed with 100 μl concentrated HCl and filtered

through a 0.22 μm filter. A high-performance liquid chromatography system (HPLC, Waters Alliance e2695,
Milford, USA) with a UV detector (Waters 2468, Milford, USA) was used with a Venusil ASB C18 (4.6 × 250 mm,
5 μm, China) column at a flow rate of 1 ml/min at 210 nm and 30 °C. The mobile phase consisted of 0.01% H3PO4
in HPLC-grade water (A) and methanol (B). All samples were evaluated in duplicate.
DNA extraction and amplification of the V3-V4 region of the bacterial 16S rRNA gene. Microbial
DNA was extracted from 200 mg fecal samples using the QIAamp Fast DNA Stool Mini Kit (QIAamp, California,
USA). The final DNA concentration and purity were detected by a NanoDrop 2000 UV (Thermo Scientific,
Wilmington, USA). We amplified the bacterial 16S rRNA gene using primers specific for the V3-V4 hypervari-
able regions (338 F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806 R: 5′-GGACTACHVGGGTWTCTAAT-3′)
in a thermocycler PCR system (GeneAmp 9700, ABI, USA). The amplicons were extracted and further purified
using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified with a
™
QuantiFluor -ST (Promega, USA) following the manufacturer’s instructions.
Illumina MiSeq sequencing. The sequencing data were pooled equimolarly and paired-end sequenced
(2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by
Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw reads were deposited in the NCBI Sequence
Read Archive (SRA) database. Raw fastq files were demultiplexed and quality-filtered by Trimmomatic. The reads
were merged by FLASH as described previously42. Operational taxonomic units (OTUs) were clustered with a
97% similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/), and chimeric sequences were iden-
tified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed by the RDP
Classifier algorithm (http://rdp.cme.msu.edu/) against the Silva (SSU123) 16S rRNA database using a confidence
threshold of 70%43.
Bioinformatic analysis. All sequencing data were computed using the R package (V.2.15.3). Rarefaction was
applied to the OTUs to reduce sampling heterogeneity for further alpha and beta diversity calculations. Principal coor-
dinates analysis (PCoA) was used to assess species composition dissimilarity by the weighted UniFrac. The linear discri-
minant analysis effect size (LEfSe), an algorithm for high-dimensional biomarker discovery, was computed to identify
differentially abundant taxa between two groups. The linear discriminant analysis (LDA) effect size (LEfSe) method
was used to estimate the effect of each differentially-abundant taxon and discriminate the most biological one16. The
Wilcoxon rank-sum test (for two groups) or Kruskal-Wallis test (for more than two groups) was used for comparisons.
Additionally, Spearman correlation was used to associate abundant differential taxa with SCFAs.
Statistical analysis. Other data were analyzed using SPSS20.0 software (IBM Corp., Armonk, N.Y., USA).
Comparison of data between autistic and neurotypical subjects was conducted using Student’s t-test (qualitative
data, equal variance), Welch’s t-test (qualitative data, unknown variance) or the Chi-square test (qualitative data).
Data are presented as the mean ± SD. Significance was set at 0.05.
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Acknowledgements
The authors gratefully acknowledge all the participants and their families. We would like to thank Dr. Gabriela
Vallejo Flores (Max Planck Institute for Infection Biology) for insightful discussions. Financial support: This
study was supported by the National Natural Science Foundation of China (No. 81370494 and No. 31471330)
and the Special Scientific Research Fund of Public Welfare Profession of National Health and Family Planning
Commission (No. 201502026).
Author Contributions
S.M.L., P.Y.Z. and Y.C.T. designed the project. S.M.L., Z.Y.S. and M.M.J. collected the clinical samples. E.Y.L. and
G.Q.D. diagnosed autism. Y.Y. and L.M. assessed the GI symptoms. D.J.F. performed the SCFA analysis. S.M.L.
analyzed the data and wrote the manuscript. P.Y.Z., Y.C.T. and P.C.Y. revised the manuscript.
Additional Information
Competing Interests: The authors declare no competing interests.
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Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-
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article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the
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mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the
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© The Author(s) 2019

Liu et al. Translational Psychiatry (2019)9:43

https://doi.org/10.1038/s41398-019-0389-6 Translational Psychiatry
ARTICLE Open Access
Altered composition and function of

intestinal microbiota in autism spectrum
disorders: a systematic review
Feitong Liu1,2,3, Jie Li1, Fan Wu1, Huimin Zheng1,2, Qiongling Peng4 and Hongwei Zhou 1
Abstract
At present, the pathophysiology of autism spectrum disorder (ASD) remains unclear. Increasing evidence suggested
that gut microbiota plays a critical role in gastrointestinal symptoms and behavioral impairment in ASD patients. The
primary aim of this systematic review is to investigate potential evidence for the characteristic dysbiosis of gut
microbiota in ASD patients compared with healthy controls (HCs). The MEDLINE, EMBASE, Web of Science and Scopus
were systematically searched before March 2018. Human studies that compared the composition of gut microbiota in
ASD patients and HCs using culture-independent techniques were included. Independent data extraction and quality
assessment of studies were conducted according to PRISMA statement and Newcastle-Ottawa Scale. Phylogenetic
Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to infer biological functional
changes of the shifted microbiota with the available data in four studies. Sixteen studies with a total sample size of 381
ASD patients and 283 HCs were included in this systematic review. The quality of the studies was evaluated as medium
to high. The overall changing of gut bacterial community in terms of β-diversity was consistently observed in ASD
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patients compared with HCs. Furthermore, Bifidobacterium, Blautia, Dialister, Prevotella, Veillonella, and Turicibacter were
consistently decreased, while Lactobacillus, Bacteroides, Desulfovibrio, and Clostridium were increased in patients with
ASD relative to HCs in certain studies. This systematic review demonstrated significant alterations of gut microbiota in
ASD patients compared with HCs, strengthen the evidence that dysbiosis of gut microbiota may correlate with
behavioral abnormality in ASD patients. However, results of inconsistent changing also existed and further big-
sampled well-designed studies are needed. Generally, as a potential mediator of risk factors, the gut microbiota could
be a novel target for ASD patients in the future.
Introduction Pervasive Development Disorder Not Otherwise Specified

Autism spectrum disorder (ASD) is a complex, perva- (PDD-NOS). According to recent estimate, the prevalence
sive neurobiological disorder, characterized by impaired of ASD is elevating with 1–2% of children currently
social and communication skills, as well as stereotyped diagnosed worldwide2. The etiology of ASD remains
behaviors and restricted patterns of interests1. ASD unclear and appears to involve a complicated interaction
includes autism (AD), Asperger’s Syndrome, and of genetic and environmental factors3,4. By estimate, the
heritability including de novo mutations, common var-
iants, and short nucleotide polymorphisms identified in
Correspondence: Hongwei Zhou (biodegradation@gmail.com)
1 ASD cases altogether accounts for approximately 50% of
State Key Laboratory of Organ Failure Research, Division of Laboratory
Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, the disorder5,6. As well, the possibility for environmental
China
2
risk factors and related medical comorbidities which
Department of Environmental Health, School of Public Health, Southern
Medical University, Guangzhou, China
Full list of author information is available at the end of the article.
These authors contributed equally: Feitong Liu, Jie Li
© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Liu et al. Translational Psychiatry (2019)9:43 Page 2 of 13 43
contribute to core neurobehavioral symptoms of the dis- Materials and methods

order has been highlighted by many studies. Among the Protocol
comorbidities in ASD, gastrointestinal (GI) symptoms are We conducted the systematic review to evaluate the
quite common, such as diarrhea, constipation, and com- altered gut microbiota in ASD patients compared with
mutative diarrhea/constipation, they are also correlated HCs. Available literatures were identified and examined as
with the severity of the neurobehavioral disorder7. The a systematic review but not a meta-analysis due to the
association of ASD with great prevalence of GI symptoms heterogeneity of methods and results. To report this
is spurring an intensive search of the ASD gut microbiota. systematic review, the method was consistent with the
There is growing evidence demonstrating that dis- PRISMA statement guidelines (Preferred Reporting Items
turbances in the pathway underlying the microbiota-gut- for Systematic Reviews and Meta-Analysis), and the pro-
brain axis, especially the disordered gut microbiota, may tocol was registered at PROSPERO (registration number:
result in neurobehavioral and intestinal dysfunction in CRD42017060769).
ASD patients8,9. Gut microbiota makes critical contribu-
tion to maintaining the integrity of intestinal epithelia, Selection criteria
protecting intestinal barrier and preventing bacterial LPS Studies compared gut microbiota in ASD patients and
and other toxins into bloodstream. It has been confirmed HCs were included. The inclusion criteria were as follows:
that systematic inflammation by LPS induced behavioral (1) ASD diagnosis with definite criteria; (2) the age of
impairment and damaged the blood-brain barrier in ani- participants ranged from 2 to 18; (3) detection of gut
mal models10,11. Conversely, gut microbiota reconstitu- microbiota with gut biopsy or fecal samples; (4) metage-
tion with probiotics could alter blood metabolic profiles, nomic sequencing, 16S rDNA sequencing, quantitative
remediate gut permeability and improve ASD-related real-time PCR techniques (qPCR) or FISH. The exclusion
behaviors in mice model12. Moreover, gut microbiota may criteria were as follows: (1) medicated participants; (2)
regulate the central nervous system (CNS) activities failure to provide data for the microbiota; (3) culture-
through neural, immune and endocrine pathways. For dependent methods; (4) intervention studies without
example, gut microbiota can regulate the hypothalamic- initial data or reviews; (5) duplicate publications.
pituitary-adrenal (HPA) axis13, and produce many che-
micals affecting brain function (e.g., serotonin, dopamine, Search strategy and study selection
r-aminobutyric acid, SCFAs, and p-cresol)14,15. A systematic search was conducted using MEDLINE,
ASD individuals vary widely in clinical presentation, EMBASE, Web of Science and Scopus for the studies
severity and treatment response. The complexity is published before March 2018. The reference lists of all
motivating an exploration to identify biological factor identified studies that matched the key search terms were
helps to achieve earlier diagnoses and predict clinical manually searched for relevant trials. The specific search
prognosis. Thus, the gut microbiota in ASD patients has strategy was: (Autism OR autistic OR ASD) AND
gained growing attention as a potential mediator of risk (microbiome OR microbiota OR microflora OR flora). At
factors. So far, several case-control studies aiming to the beginning of study selection, irrelevant articles were
observe the aberrant gut microbiota have been per- excluded via an assessment of the tittle, abstract and
formed16–32. Moreover, a recent open-label study has keywords. The full-text of potentially relevant studies was
indicated that fecal microbiota transfer (FMT) therapy then retrieved. Following the elimination of duplicates,
alters gut ecosystem and improves GI and neurobeha- two independent authors (FL and JL) assessed the articles
vioral symptoms in ASD patients19. All these results have for eligibility considering established criteria detailed
gained an insight into the potential mechanisms of gut above. Any disagreement between authors were resolved
microbiota in ASD. At present, no systematic review has by discussion until consensus was achieved.
addressed the evidence of the altered gut microbiota in
ASD patients focusing on culture-independent methods Data extraction
especially the high-throughput sequencing techniques. Data for gut microbiota in eligible studies were extrac-
These techniques enable the identification of previously ted to the Excel spreadsheet. The following information
unknown bacterial species, thereby provide novel insights was extracted: author; publication year; country of origin;
into the compositional diversity and the functional capa- the characteristics of case and control (including sample
city of gut microbiota. size, mean age, sex ratio, GI symptom and the diagnosis
The aim of this systematic review is to explore the criteria for ASD); the method for microbiota analysis
current evidence for the alteration of gut microbiota in (including sample source, DNA extraction, the informa-
ASD patients compared with HCs using culture- tion of PCR or FISH or sequencing and the referred
independent techniques. database); outcomes (including the differences of overall
microbiota structure and the specific bacteria). Besides, Genomes (KEGG) pathways predicted by using Phyloge-
the raw sequencing data and biom files were also collected netic Investigation of Communities by Reconstruction of
in studies using high-throughput sequencing methods. Unobserved States (PICRUSt)33. As for the study provided
Two authors (FL and JL) independently extracted data with raw sequencing data, we re-clustered the available
from the selected articles, and the data was then cross- data in QIIME with the command of pick_closed_refer-
checked for accuracy (FW). ence_otus.py with the Greengenes reference version 13_8.
The operational taxonomic unit (OTU) picking method is
Quality assessment Usearch61. As for the study provided OTU biome file, we
Studies included in this systematic review were carefully directly used the biome file to analysis.
evaluated for the methodological quality and the risk of
bias by two authors (FL and JL). Study quality was Results
assessed on the basis of Newcastle-Ottawa Scale (NOS) Study selection
for case-control studies. NOS included three domains: In total, 985 records were identified through the elec-
selection, comparability and exposure criteria. The tronic search. 208 duplicate articles and 13 articles that
selection criteria included four aspects: (1) adequate were not published in English were discarded. 44 full-text
definition of the cases; (2) representativeness of the cases; articles were retrieved for eligibility following the exclu-
(3) selection of controls; (4) definition of controls. The sion of tittles and abstracts that were not relevant to the
comparability criteria included comparability of case and research. Three additional papers were identified via
controls according to the design and analysis. The expo- checking the references of relevant articles. The remain-
sure criteria included three aspects: (1) ascertainment of ing 47 full-text papers were further assessed according to
exposure; (2) same method of ascertainment for cases and the fore-mentioned criteria, resulting in the exclusion of
controls; (3) non-response rate. 30 papers due to the following reasons: culture-dependent
method, randomized controlled trials (RCTs) without
Summary measures and secondary bioinformatics analysis initial data, without control group, secondary research;
The overall microbiota structure, including α-diversity, mycology and cell experiment. Following the selection
β-diversity (compositional dissimilarity), the relative process (Fig. 1), 17 articles remained and were included in
abundance of the specific genus and the quantity of spe- the systematic review16–32. Among them, William
cific bacteria by q-PCR or FISH were the primary out- et al.24,25 reported one study in two different articles, so
comes. Moreover, to explore the potential function of gut did Wang et al.29,30. It is also noteworthy that one article23
microbiota in ASD patients, the Linear Discriminant included two group of ASD simultaneously: AD and
Analysis (LDA) effect size (LEfSe) was applied to the PDD-NOS, actually counted as two case-control studies.
relative abundance of Kyoto Encyclopedia of Genes and Thus, a total of 17 articles (16 studies) were included.
Fig. 1 Flow chart of identification, exclusion and inclusion of eligible studies. Flow chart indicates the progression of trials through each stage
of the selection process
Study characteristics Song et al.31 and Helena et al.32 lacked an adequate

Sixteen studies were published in English journals definition of the ASD patients, thus these two studies
between 2005 and 2018. Eight studies were conducted in achieved 3 points in the selection assessment. As for
USA16,17,19,21,22,24,27,31, three in Italy18,23, two in Aus- comparability, Luna et al.17, Inoue et al.20, De Angelis
tralia26,29, one in UK32, one in Japan20 and one in Slova- et al.23 and William et al.24,25 included case and control
kia28. The characteristics and main results of studies were with better design, such as all participants has consistent
outlined in Table 1. A total of 381 ASD patients and 283 GI symptoms at baseline, thus these studies achieved 2
HCs were included, with sample size ranged from 12 to points in the comparability assessment.
104. Of 283 HCs, 107 subjects were healthy siblings21,23,26.
The age of ASD patients ranged from 2–18 and the Heterogeneity
proportion of male ranged from 77.5–100%. Concerning Methodological sources of heterogeneity included the
about GI symptoms, five studies17,19,22,24,27 reported all of type of sample (fecal or biopsy), the temperature samples
the ASD patients had GI symptoms, seven stu- stored, the methods of DNA extraction and the primer
dies16,18,21,26,28,29,32 reported that a part of ASD patients used to PCR. Although the included studies had slightly
had GI symptoms. And three studies20,23 conducted in differences in primer selection, there was significant
ASD patients without any GI symptoms. Different diag- overlap in the key variable and constant regions of the 16S
nostic criteria were used: ADOS, DSM-5, ABD, ADI-R, gene. The bacterial identification platform was also a
and CARS. However, two studies31,32 did not report the possible source of heterogeneity. 16S rDNA sequencing
specific diagnostic criteria. As restricted diet is very can quantitatively identify all bacteria present in one
common in ASD patients, we tried to extract the infor- sample. However, qPCR and FISH only detected the
mation of eating habit in ASD and control group. specific bacteria, which lacked the evaluation of the whole
Whereas, only six studies reported dietary information. community.
Strati et al.18 reported that all subjects of the study went Possible clinical heterogeneity included age, gender,
through a Mediterranean diet. Angelis et al.23 reported type of control (sibling vs non-sibling) and whether had
that the major dietary differences were excluded since GI symptoms. Although age structure varied greatly
each of these pairs of children belonged to the same among studies, extensive overlap was found that age of all
family unit. The remaining four studies consistently participants ranged from two to eighteen. In terms of
reported that some of the ASD patients had special diets, gender, Luna et al.17 and William et al.24 only included
such as casein-free (CF) or gluten-free (GF) diet21,22,27,32. male individuals, the remaining studies included both
In addition, Son et al.21 conducted further nutritional male and female. Besides, Son et al.21, De Angelis et al.23
analysis of one-week food diary, no significant difference and Gondalia et al.26 recruited healthy siblings as control,
between two group with respect to daily intake of mac- whereas other studies recruited normal control from the
ronutrients (calories, protein, fats, carbohydrates, sugars whole population. Considering the impact of GI symp-
or dietary fiber) were noted. toms on composition of gut microbiota, a potential source
The alteration of gut microbiota composition was of heterogeneity may come from the varied baseline of GI
assessed with fecal samples16,18–22,25–31 or gut biopsy16,23. symptoms.
Ten studies16–23,26,27 assessed gut microbiota by high-
throughput molecular approaches: Illumina MiSeq plat- The altered composition of gut microbiota in ASD patients
forms, 454 pyrosequencing or bTEFAP using a 454 FLX In the studies using high-throughput sequencing
Sequencer. Three studies28–31 detected gut microbiota method, the diversity of species in the samples can be
with qPCR and one32 with FISH approach. One study24,25 expressed in many ways, including richness, evenness, and
used both sequencing and qPCR method. Of the studies α-diversity. α-diversity is the number of species and their
adopting 16S rDNA-based method, two studies26,27 did proportion within one sampling site. Of the nine studies
not report which hypervariable region of the 16S rDNA compared α-diversity, three studies demonstrated that α-
was targeted and the remaining studies targeted varied diversity has a significant reduction in ASD patients16,19,22.
sets of regions. Meanwhile, the database used for mapping In contrast, De Angelis et al.23 reported the increased α-
the sequences were GreenGenes16,18–20,24, RDP-227, diversity was found in both AD and PDD-NOS patients
Silva17,21, SSURef22 and GenBank23, while one study26 did compared with their sibling control. β-diversity means the
not report the database used. dissimilarity between communities of two sites or two
samples. In this systematic review, ten studies analyzed β-
Risk of bias diversity (unweighted UniFrac distance, weighted UniFrac
16 studies were identified and assessed as medium (6–7) distances, and Bray-Curtis), six of them consistently
to high (8) quality by the NOS as presented in Table 2. reported that the microbiota of ASD patients clustered
When assessing the quality of selection, the studies of significantly apart from that of HCs16–18,23,27.
Table 1 Characteristics of the trials included
References Case/Control participants ↔Microbiology assessment Outcomes (compared with control)
Study country Sample size Mean age Male ratio Symptom of GI Diagnose of Sample source PCR or FISH Or Referred Outcomes (compared with control)
(Year) (%) (%) ASD DNA extraction Sequencing Database
Kang16 ASD:21 10.1 ± 4.1 15/21 GI symptom ATEC Fecal sample; 16S rRNA V2-3 region; Greengenes ↓α diversity;
2018 CON:23 8.4 ± 3.4 22/23 were more PDD-BI −80 °C; Genome Sequencer database β diversity (P < 0.05);
severe in ASD FLX-Titanium System
USA PowerSoil® DNA Genus:↓Prevotella and Coprococcus
Isolation Kit ↓Faecalibacterium, Haemophilus
Luna17 ASD:14 Age:4-13 14/14 14/14 ADOS Rectal biopsy; 16S rRNA V1V3, V4 Silva database β diversity (P < 0.05);
region; MiSeq Illumina
2017 CON:15 Age:3-18 12/15 15/15 −80 °C; ↑Clostridiales; Clostridium; Lachnoclostridium;
platform Flavonifractor;
USA PowerSoil® DNA ↓Dorea, Blautia, Sutterella;
Isolation Kit Other findings: ↓tryptophan in rectal;
18
Strati ASD: 40 11.1 ± 6.8 31/40 5/50 DSM-5 Fecal sample; V3-V5 Greengenes ↔ α diversity;β diversity (P < 0.05);
2017 CON:40 9.2 ± 7.9 28/40 11/40 ADOS −80 °C; 454 database Phylum:↑Firmicutes/Bacteroidetes
ratio;↓Bacteroidetes;
Italy ABD FastDNA™ SPIN pyrosequence Genus:↑Collinsella, Corynebacterium, Dorea,
Kit and Lactobacillus;
↓Alistipes, Bilophila, Dialister, Parabacteroides
and Veillonella;
Other findings: Candida was more than
double in the autistic;

19
Kang ASD:18 10.8 ± 1.6 16/18 18/18 ADI-R Stool samples 16S rRNA V4 region; Greengenes ↓α diversity;
MiSeq database
2017 CON:20 11.4 ± 2.5 18/20 0/20 PowerSoil® DNA Illumina platform Genus:↓Bifidobacterium;
USA Isolation Kit Other findings: ASD had lower fiber
consumption;
ASD were breastfed significantly shorter time;
20
Inoue ASD:6 age 3–5 NA 0/6 DSM-5 Fecal sample; 16S rRNA V3-4 region; Greengenes Genus:↑Faecalibacterium; ↓Blautia;
MiSeq database
2016 CON:6 age 3–5 NA 0/6 PARS −80 °C; Illumina platform Other findings: number of GO biological
processes associated with response to viruses
Page 5 of 13 43
Japan M-CHAT
Table 1 continued
e QuickGene DNA were enriched: IFN-γ and type-I IFN signaling

Tissue kit pathways.
Son21 ASD:59 10.3 ± 1.8 52/59 25/59 ADOS Fecal sample; 16S rRNA V1V2, V1V3 Silva database ↔α diversity; ↔β diversity;
2015 SIB:44 10.0 ± 1.8 21/44 13/44 ADI-R -80 °C; region; MiSeq Illumina ↔ the relative abundance of any phylum and
(age 7–14) platform genus;
USA ZR Fecal DNA
MiniPre
22
Kang ASD:20 6.7 ± 2.7 18/20 20/20 ADOS Fecal sample; 16S rRNA V2-3 region; SSURef ↓α diversity;
2013 CON:20 8.3 ± 4.4 17/20 0/20 ADI-R −80 °C; QIAamp bTEFAP using a 454 database Genus:↓Prevotella, Coprococcus, unclassified
(age 3–16) FLX Sequencer; Veillonellaceae;
USA ATEC DNA Stool Mini Akkermansia was very high in several autism
PDD-BI Kit subjects;
De Angelis23 AD:10 age 4–10 NA 0/10 ADI-R Fecal sample; 16S rRNA V1-3 region; GenBank ↑α diversity;β diversity(P < 0.05);
2013 SIB:10 age 4–10 NA 0/10 ADOS −80 °C; bTEFAP using a 454 databases Phylum:↓Firmicutes; Fusobacteria and
FLX Sequencer; Verrucomicrobia; Firmicutes/Bacteroidetes
ratio;
Italy CARS FastDNA Pro Soil- ↑Bacteroidetes;
Direct Kit Genus: ↓Faecalibacterium, Oscillospira,
Bifidobacterium, Fusobacterium, Escherichia,

Turicibacter, Eubacterium;
↑Caloramator, Sarcina, Clostridium, Roseburia,
Akkermansia, Shigella, Enterobacter, Dorea;
Other findings: AD fecal samples contained
higher FFA; phenol, 4-(1,1,3,3-
tetramethylbutyl)-phenol, p-cresol; SCFAs was
lower;
23
De Angelis PDD-NOS:10 age 4–10 NA 0/10 ADI-R Fecal sample; 16S rRNA V1-3 region; GenBank ↑α diversity; β diversity(P < 0.05);
2013 SIB:10 age 4–10 NA 0/10 ADOS −80 °C; bTEFAP using a 454 databases Phylum:↓Fusobacteria and Verrucomicrobia;
FLX Sequencer;
Italy CARS FastDNA Pro Soil-
Page 6 of 13 43
Direct Kit
Table 1 continued
Genus: ↓Oscillospira, Bacteroides,

Fusobacterium, Escherichia, Prevotella,
Turicibacter; Clostridium;
↑Faecalibacterium, Ruminococcus, Roseburia,
Alistipes, Dorea;
Other findings: AD fecal samples contained
higher phenol, 4-(1,1,3,3-tetramethylbutyl)-

phenol, p-cresol; SCFAs was lower;
24,25
William ASD:15 4.5 ± 1.3 15/15 15/15 DSM-5 ileal and cecal 16S rRNA V2 region; Greengenes Phylum:↑Firmicutes/Bacteroidetes ratio;
biopsies; −80 °C bTEFAP using a 454 database Firmicutes, Betaproteobacteria; ↓Bacteroidetes;
FLX Sequencer;
2011,2012 CON:7 4.0 ± 1.1 7/7 7/7 ADI-R Quantitative Real-time Family: ↑ Lachnospiraceae and
PCR Ruminococcaceae;
USA Genus:↑Sutterella, Faecalibacterium;Other
findings: Presence of Alcaligenaceae in some
ASD children but absence in controls.
26
Gondalia ASD:51 age 2–12 42/51 28/51 CARS Stool samples bTEFAP using a 454 NA ↔α diversity; ↔β diversity;
2012 SIB:53 age 2–12 19/34 4/53 −20 °C; QIAamp FLX Sequencer; ↔ the relative abundance of any phylum and
Australia DNA stool kit genus;
Finegold27 ASD:11 age 2–13 NA 11/11 CARS Fecal sample; bTEFAP using a 454 RDP-II database ↔ α diversity;β diversity (P < 0.05);
2010 CON:8 age 2–13 5/8 NA -80 °C;QIAamp FLX Sequencer; Phylum:↑Bacteroidetes, Proteobacteria;
USA DNA stool mini kit ↓Firmicutes, Actinobacteira;
Genus:↑Alkaliflexus, Desulfovibrio,
Acetanaerobacterium,
Parabacteroides, Bacteroides;
↓Weissella, Turicibacter, Clostridium,
Anaerofilum, Dialister, Pseudoramibacter,
Ruminococcus, Streptococcus, Anaerovorax;
Page 7 of 13 43
Table 1 continued
Species:↑ Desulfovibrio spp. and Bacteroides

vulgatus;
↓Bifidobacterium longum, Dialister invisus,
Clostridium leptum;
28
Tomova ASD:10 age 2–9 9/10 9/10 ICD-10 Stool samples Quantitative Real-time NA ↑Lactobacillus;
2015 CON:10 age 2–11 10/10 6/10 CARS −80 °C; QIAamp PCR ↑Clostridia cluster l and Desulfovibrio (not
DNA stool kit significant);

Slovakia ADI ↓Bacteroidetes/Firmicutes ratio;
Wang29,30 ASD:23 10.3 ± 0.8 21/23 9/23 CARS Fecal sample; Quantitative Real-time NA ↑Sutterella spp, Ruminococcus torques (p =
PCR 0.08);
2011 CON:9 12 ± 1.3 4/9 1/9 DSM-5 −80 °C; A repeat ↓Bifidobacterium spp; Akkermansia.
bead beating plus muciniphila;
Australia column; Other findings: No differences between
groups in levels of
Faecalibacterium prausnitzii;
31
Song ASD:15 Age Gender NA NA Stool samples TaqMan NA ↑Clostridia bolteae, Clostridia and clusters I
2004 CON:8 matched matched −80 °C; QIAamp Real-time PCR and XI;
DNA stool kit
USA
Helena32 ASD:58 7 ± 3.76 48/58 53/58 NA Fecal sample; FISH NA ↑Clostridium histolyticum group (Clostridium
2005 CON:10 6 ± 2.88 6/10 0/10 −20 °C; clusters I and II);
UK
Page 8 of 13 43
Table 2 Quality assessment of included studies were also reported. Two sequencing studies reported that
the abundance of Akkermansia was elevated in ASD
First author Year Selection Comparability Exposure Total
patients22,23. Inconsistently, one qPCR study30 indicated
Kang16 2018 4 1 2 7
that lower amount of Akkermansia muciniphila was
found in feces of ASD patients than HCs. Dorea18,23 and
Luna17 2017 4 2 2 8
Sutterella25,29 were reported increased significantly in
18
Strati 2017 4 1 2 7 ASD patients, but they were reported decreased in
Kang 19
2017 4 1 2 7 another study17. Faecalibacterium20,23,24 and Rumino-
Inoue20 2016 4 2 2 8
coccus23,24,29 were reported increased in ASD patients by
21
three studies, but they were reported reduced in another
Son 2015 4 1 2 7
study27.
22
Kang 2013 4 1 2 7
De Angelis 23
2013 4 2 2 8 The altered function of gut microbiota in ASD patients
De Angelis23 2013 4 2 2 8
Functional profiles of microbial communities with high-
throughput sequencing were predicted by PICRUSt. Raw
William24,25 2012 4 2 2 8
sequence data was provided in three studies18,21,22 and the
OTU biome file was provided in one study19. Among
26
Gondalia 2012 4 1 2 7
Finegold 27
2010 4 1 2 7 them, three studies18,19,22 indicated the functional mod-
Tomova28 2015 4 1 2 7
ules of gut microbiota in ASD patients were substantially
29,30
different from that in HCs (Fig. 2). In the study of Strati
Wang 2011 4 1 2 7
et al.18 (Fig. 2a), the pathways with the highest five dis-
criminative power in HCs were “Glycan Biosynthesis and
31
Song 2004 3 1 2 6
Helena 32
2005 3 1 2 6 Metabolism”, “Membrane and intracellular structural
molecules”, “Lipopolysaccharide biosynthesis proteins”,
“Pores ion channels” and “Lipopolysaccharide biosynth-
To assess the specific changing of bacteria in ASD esis”. The pathways with the highest discriminative power
patients, we analyzed the phyla grouping at first. Three in ASD patients were “ABC transporters” under Mem-
studies indicated a clear alteration of the gut bacterial brane Transport category, followed by pathways of
community in ASD patients characterized by a higher “Replication, recombination and repair proteins”, “Lysine
Firmicutes/Bacteroidetes ratio in ASD than that in HCs biosynthesis”, “Genetic Information Processing” and
due to a significant increase of Firmicutes and/or a “Signal transduction mechanisms”. In two studies of Kang
reduction of Bacteroidetes18,24,28. Whereas, De Angelis et al. (Figs. 2b, c)19,22, the pathways of “Cell Motility”,
et al.23 and Finegold et al.27 reported totally opposite “Cellular Processes” and “Bacterial motility proteins” had
results. Phylum of Fusobacteria and Verrucomicrobia consistent significant discriminative power in HCs. In
were significantly decreased in patients of ASD (AD and addition, “Bacterial chemotaxis” and “Flagellar assembly”
PDD-NOS) in the data of De Angelis et al.23 Consistently, were also noted. In ASD patients, the functional modules
William et al.24 and Finegold et al.27 both reported that of metabolism were higher than that in HCs, such as
the Proteobacteria was elevated in ASD patients rather “Oxidative phosphorylation” under Energy Metabolism
than HCs. category and “Glycine, serine and threonine metabolism”
Further analysis of the alterations of genus and species under Amino Acid Metabolism. It should be noted that
in ASD, four studies consistently demonstrated a sig- pathways of “Huntingtons disease” and “Amyotrophic
nificantly decrease of Bifidobacterium in patients with lateral sclerosis (ALS)” under Neurodegenerative Diseases
ASD relative to HCs19,23,27,30. Consistent with this, the category as well as “Glutamatergic synapse” under Ner-
abundance of Blautia17,20, Dialister18,27, Prevotella16,22,23, vous System category were also increased in ASD patients.
Turicibacter23,27 and Veillonella18,22 were all decreased. In Whereas, in the study of Son et al.21, there was no dif-
contrast, Lactobacillus18,28, Bacteroides23,27, and Desulfo- ference of functional modules between ASD patients and
vibrio27,28 were all increased in ASD patients rather than HCs, which was accordance with the result of gut
controls. Five studies17,23,28,31,32 all revealed that there was microbiota composition.
a significant increase of Clostridium in ASD. In addition,
De Angelis et al.23 indicated that Oscillopira decreased Discussion
and Roseburia increased in both AD and PDD-NOS This systematic review demonstrated that there was
patients. Meanwhile, they also found that some oppor- consistent evidence for the alterations of gut microbiota
tunistic pathogen such as Enterobacter and Shigella were in ASD patients compared with HCs. Novel culture-
elevated in ASD patients23. Whereas, conflicting results independent techniques that analyzed bacterial DNA
A Autism Control B Autism Control

Glycan Biosynthesis and Metabolism
Membrane and intracellular structural molecules
Lipopolysaccharide biosynthesis proteins Cell Motility
Pores ion channels Cellular Processes
Lipopolysaccharide biosynthesis Bacterial motility proteins
Transport and Catabolism Transcription factors
Citrate cycle (TCA cycle) Flagellar assembly
Lysosome Bacterial chemotaxis
Chaperones and folding catalysts Synthesis and degradation of ketone bodies
Glycosyltransferases Secretion system
Carbon fixation pathways in prokaryotes
Ribosome Biogenesis
Glycosaminoglycan degradation
Two-component system
Human Diseases
Glycosphingolipid biosynthesis - globo series Biosynthesis of ansamycins
Restriction enzyme Thiamine metabolism
Environmental Adaptation
Ubiquinone and other terpenoid-quinone biosynthesis
Glycosphingolipid biosynthesis - ganglio series Plant-pathogen interaction
Ascorbate and aldarate metabolism Excretory System
Circadian rhythm - plant Proximal tubule bicarbonate reclamation
Signal transduction mechanisms Peroxisome
Genetic Information Processing Cell motility and secretion
Lysine biosynthesis Glutamatergic synapse
Replication, recombination and repair proteins Nervous System
ABC transporters Biosynthesis of vancomycin group antibiotics
Toluene degradation
Signaling Molecules and Interaction
Neurodegenerative Diseases
Steroid hormone biosynthesis
Autism Control
C Polyketide sugar unit biosynthesis
Huntingtons disease
Cell Motility
Cellular Processes Glycine, serine and threonine metabolism
Bacterial motility proteins Lipoic acid metabolism
G protein-coupled receptors Glyoxylate and dicarboxylate metabolism
Various types of N-glycan biosynthesis Ascorbate and aldarate metabolism
Flagellar assembly Aminobenzoate degradation
Bacterial chemotaxis Protein folding and associated processing
Nitrogen metabolism Biotin metabolism
Glycan biosynthesis and metabolism Geraniol degradation
Electron transfer carriers Glycosaminoglycan degradation
Linoleic acid metabolism Biosynthesis and biodegradation of secondary metabolites
Tetracycline biosynthesis Lysosome
Polycyclic aromatic hydrocarbon degradation Oxidative phosphorylation
Bisphenol degradation Type I diabetes mellitus
Fatty acid biosynthesis Carbon fixation pathways in prokaryotes
Glycerolipid metabolism
Transport and Catabolism
Glycine, serine and threonine metabolism
Metabolism of Terpenoids and Polyketides Pores ion channels
Oxidative phosphorylation Amino sugar and nucleotide sugar metabolism
Arginine and proline metabolism Amino Acid Metabolism
Energy Metabolism Membrane and intracellular structural molecules
Amino Acid Metabolism Amyotrophic lateral sclerosis (ALS)
Metabolism Metabolism
Fig. 2 Linear discriminative analysis effect size (LEfSe) of statistically significantKEGG pathways between autism and control in the studies.
a Strati et al., b, c two studies of Kang et al. Positive LDA scores (green) are enriched in control while negative LDA scores (red) are enriched in autism
offered a unique, more in-depth look into the gut negative bacteria and includes a variety of opportunistic
microbiome. Overall, the changed structure of gut bac- pathogens. Meanwhile, as microbial signature of dysbiosis in
terial community in terms of β-diversity was observed gut microbiota, Proteobacteria is associated with host
coherently in ASD patients compared with HCs. Con- inflammation36. It is also remarkable that these Gram-
sistently, ASD patients had elevated abundance of Pro- negative bacteria produce a potent toxic factor LPS37. An
teobacteria rather than HCs. In addition, Bifidobacterium, animal study indicated that prenatal LPS exposure reduced
Blautia, Dialister, Prevotella, Veillonella, and Turicibacter the level of glutathione in the brain38. Glutathione is a sig-
were consistently decreased, while Lactobacillus, Bacter- nificant antioxidant and closely related to detoxification in
oides, Desulfovibrio, and Clostridium were increased in the brain39. Thus, the elevated abundance of Proteobacteria
ASD patients relative to HCs. in ASD patients need more attention in future studies. As
Several included studies reported the Firmicutes/Bacter- mentioned, the level of some genera (Bifidobacterium,
oidetes ratio due to the alteration of Firmicutes and Bacter- Blautia, Veillonella and Prevotella) was decreased in ASD
oidetes, but has not reached to any concordant conclusions. patients. Notably, these particular species are known to be
Some studies indicated that elevated Firmicutes /Bacter- versatile carbohydrate metabolizers40. Bifidobacterium is
oidetes ratio was correlated with inflammatory conditions among the first colonizers of human intestinal and one of the
such as inflammation bowel diseases (IBDs)34 and obesity35. dominant groups in the gut microbiota of breast-fed
The elevated Proteobacteria phylum was found in ASD infants41. It can ferment complex polysaccharides to reg-
patients. It is noted that Proteobacteria is a major of Gram- ulate host function and promote health42,43, encouraging
interest in its use as probiotics. Blautia plays an important chemotaxis, and flagellar assembly, which indicated the
role in nutrient assimilation44 and gut maturation in chil- basic physiological maintenance under normal condition.
dren45. The reduction of these beneficial bacteria in ASD Whereas, in ASD patients, the most enhanced functional
patients may be implicated in the pathogenesis of the disease. module of microbiota was metabolism, including amino
On the other hand, overgrowth of Bacteroides, Desulfovibrio, acid metabolism, lipid metabolism, carbohydrate meta-
and Clostridium were also found in ASD patients. Indeed, bolism, energy metabolism, cofactors and vitamins
Bacteroides is an abundant genus at all ages, from infants to metabolism and tetracycline biosynthesis. Functions of
adults46. It is the main producer of propionate in the gut, and xenobiotics degradation such as toluene, aminobenzoate,
the abundance of propionate in feces correlates strongly with polycyclic aromatic hydrocarbon, and bisphenol were also
the abundance of Bacteroides47. Propionate produced by enhanced. Moreover, some functional modules involved
microbiota is used for gluconeogenesis in liver and represents in neurodegenerative diseases in human, such as Hun-
a source of glucose level for the host48. Whereas, a study tington disease and ALS were also raised in ASD patients.
indicated that neurodevelopmental abnormality in ASD So far, a variety of mechanism has been proposed in ASD
patients accompanied with impaired propionic acid meta- associated with the function of gut microbiota, including
bolism49, which may relate to the changing of propionate- immune activation/dysfunction, bacterial-derived toxin
producing bacteria. Desulfovibrio produces LPS as well as (e.g., LPS, phenols, p-cresol, 4-EPS), metabolites aberra-
hydrogen sulfide which could be toxic to intestinal cells tions in fermentation process or products, such as pro-
under certain circumstance27,50. Clostridium has been pionic acid (PPA) and other SCFAs, and the dysregulated
extensively studied in ASD51,52 due to its characteristic of metabolism of free amino acids61. Thus, the functional
producing exotoxins and propionate, which may aggravate analysis of gut microbiota may give some hints to these
the symptoms of ASD53. In addition, some species belonging underlying mechanisms and needs to be studied in the
to the Clostridium produce p-cresol. This chemical meta- future.
bolite could cause the reduction of glutathione and reported
to be a possible urinary biomarker for autism54,55. De Angelis Microbiome reconstitution could be a potential therapy to
et al.23 indicated that Enterobacter and Shigella were ASD patients in future
increased in ASD patients, which were positively correlated Since gut microbiota appears strongly associated with
with the GI symptoms in autism18. These opportunistic ASD, the interests in remodeling gut microbiota with diet,
pathogens have been previously reported to cause or underlie antibiotics, prebiotics, probiotics, and FMT are advan-
human infections such as bacteremia and intra-abdominal cing19,62–65. In ASD children, an open-label study indi-
infection56. However, the high population of Lactobacillus in cated that treatment with 8 weeks oral vancomycin greatly
ASD patients was not expected. Lactobacillus has the ability improved GI symptoms and ASD disorders64. A rando-
of fermenting a series of carbon sources primarily to lactic mized double-blind crossover trial showed that treatment
acid and is widely recognized as probiotics57. Nonetheless, a with probiotics resulted in significant differences in the
recent research indicated that Lactobacillus was more stool consistency compared to placebo and behavior
abundant in T2DM patients than in HCs58. scores compared to baseline65. In addition, Buffington
There were still some conflicting results about the et al. reported that maternal high fat diet (MHFD) can
alterations of Akkermansia, Ruminococcus, Sutterella, and induce abnormal social behavior through mediating the
Faecalibacterium in ASD patients. Akkermansia and dysbiosis of gut microbiota, but reconstituting microbiota
Ruminococcus are mucin-degrading bacterium and asso- with probiotics can correct social deficits in MHFD off-
ciated with the gut permeability59,60. Sutterella can reg- spring66. Thus, targeting the gut microbiota could be a
ulate mucosal metabolism and intestinal epithelial possible treatment for ASD patients in the future.
integrity25,29. Changes of mucus-degrading microbes may
be related with mucus producing, which could impact the The possible factors for contradictory findings
mucosal barrier in the gut. Faecalibacterium is regarded There are numerous confounding factors that may limit
as commensal or even beneficial due to its function of the consensus of the studies analyzed in this systematic
producing anti-inflammation butyrate61. Thus, the varia- review. Geography and dietary habits are main factors that
tion of these genera needs to be further explored with the play a great role in microbiome composition. The studies
pathogenesis of ASD in future studies. included in this systematic review were conducted in dif-
The predicted biological functions of the observed ferent countries. Besides, implementation of restricted diet
microbial community in ASD patients were significantly is very common in ASD subjects, such as GF and CF diet.
different from that in HCs, but varied among studies. In Thus, the enormous variation of dietary habit and its effect
two accordant studies19,22, functional modules of micro- on the gut microbiota may mask the true picture of the
biota in HCs mostly involved in cell motility, cellular differences. Secondly, the selection of control (that is, sib-
processes, bacterial motility proteins, bacterial ling vs non-sibling) may also have subtle influence on gut
microbiota. Krajmalnik et al. reported that neurotypical factor, the gut microbiome could be a novel target for
(NT) siblings of ASD children have altered microbiome ASD patients in the future.
compared to that of unrelated children67. It can be an
Acknowledgements
alternative explanation that why studies21,26 using NT sib- This work was supported by the National Natural Science Foundation of China
ling controls did not find any significant difference, while (grant number: NSFC313220143).
other studies using non-sibling controls did. Thirdly, the
frequent occurrence of GI symptoms in ASD patients may Author details
1
State Key Laboratory of Organ Failure Research, Division of Laboratory
affect the composition of gut microbiota. Hence, the base- Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510282,
line comparability of GI symptoms in case and control, can China. 2Department of Environmental Health, School of Public Health,
facilitate a more robust interpretation of the results. Southern Medical University, Guangzhou, China. 3Department of Food and
Nutritional Sciences, School of Chemistry, Food and Pharmacy, The University
Another important consideration is that biogeographic of Reading, Whiteknights, Reading RG6 6AP, UK. 4Children Healthcare
variation of gut microbiota. The samples were collected Department, Baoan Maternal and Child Health Hospital, Jinan University,
from different anatomic subsites within the gut tract or Shenzhen 518106, China
feces. Zoetendal et al. reported that using biopsies other Conflict of interest
than feces allowed to assess the mucosa-epithelia associated The authors declare that they have no conflict of interest.
microbiota. As biopsies likely established more intimate
interplay with the human intestinal epithelium and immune
Publisher’s note
cells68. Besides, the different results of gut microbiota may Springer Nature remains neutral with regard to jurisdictional claims in
also arise from distinct techniques for DNA isolation, PCR published maps and institutional affiliations.
and sequencing, and small-sample sized studies.
Received: 22 February 2018 Revised: 12 December 2018 Accepted: 2
January 2019
Limitations
Generally, the analyses and results of gut microbiota
were relatively difficult to evaluated. It is probably because
that the gut microbiota is a new research field that cur- References
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nutrients
Article
Role of Milk-Derived Opioid Peptides and Proline
Dipeptidyl Peptidase-4 in Autism
Spectrum Disorders
Beata Jarmołowska 1 , Marta Bukało 1 , Ewa Fiedorowicz 1 , Anna Cieślińska 1, * ,
Natalia Karolina Kordulewska 1 , Małgorzata Moszyńska 2 , Aleksander Światecki ˛ 3 and
Elżbieta Kostyra 1
1 Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury,
Oczapowskiego 1A Street, 10-19 Olsztyn, Poland; bj58@wp.pl (B.J.); marta.bukalo@gmail.com (M.B.);
ewa.kuzbida@uwm.edu.pl (E.F.); natalia.smulska@uwm.edu.pl (N.K.K.);
elzbieta.kostyra@uwm.edu.pl (E.K.)
2 Center for Diagnosis, Treatment and Therapy of Autism at the Regional Children’s Hospital in Olsztyn,
Zolnierska 18 A Street, 10-561 Olsztyn, Poland; magmoszyn@gmail.com
3 Faculty of Biology and Biotechnology, Department of Microbiology and Mycology, University of Warmia
and Mazury, Oczapowskiego 1A Street, 10-19 Olsztyn, Poland; aswiat@uwm.edu.pl
* Correspondence: anna.cieslinska@uwm.edu.pl; Tel.: +48-89-523-36-67; Fax: +48-89-535-20-15

Received: 7 November 2018; Accepted: 28 December 2018; Published: 4 January 2019
Abstract: Opioid peptides released during digestion of dietary proteins such as casein, were suggested
to contribute to autism development, leading to the announcement of opioid excess hypothesis of
autism. This paper examines role of enzyme proline dipeptidyl peptidase-4 (DPPIV; EC 3.4.14.5)
and it is exogenous substrate, β-casomorphin-7 (BCM7) in autism etiology. Our study included
measurements of DPPIV and BCM7 concentrations in serum and urine, which were analyzed with
ELISA assays and activity of DPPIV was measured by colorimetric test. The effect of opioid peptides
from hydrolysed bovine milk on DPPIV gene expression in peripheral blood mononuclear cells
(PBMC) in autistic and healthy children was determined using the Real-Time PCR (Polymerase
Chain Reaction) method. Our research included 51 healthy children and 86 children diagnosed with
autism spectrum disorder (ASD, ICDF84). We determined that the concentration of BCM7 in serum
was significantly, 1.6-fold, higher in the ASD group than in controls (p < 0.0001). Concentration of
DPPIV was found to also be significantly higher in serum from ASD children compared to the control
group (p < 0.01), while we did not notice significant difference in enzymatic activity of serum DPPIV
between the two study groups. We confirmed correlation according to the gender between analyzed
parameters. The inspiration for this study emanated from clinical experience of the daily diet role in
relieving the symptoms of autism. Despite this, we have concluded that milk-derived opioid peptides
and DPPIV are potentially factors in determining the pathogenesis of autism; conducted studies are
still limited and require further research.
Keywords: autism spectrum disorder; β-casomorphin-7; bovine milk; food allergy; proline dipeptidyl
peptidase-4
1. Introduction
Autism Spectrum Disorders (ASDs) are the pervasive developmental disorders characterized by
repetitive, stereotyped patterns of behavior, impaired social interaction, and communication. ASDs are
a combination of complex neurological developmental disorders, which include autistic disorder,
Asperger’s syndrome, and pervasive developmental disorders (PDDs), are not otherwise specified [1].
Nutrients 2019, 11, 87; doi:10.3390/nu11010087 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 87 2 of 13
During the last 40 years, there has globally been 20–30-fold increases in the prevalence of autism.
The prevalence of autism spectrum disorders varies worldwide from 1.4/10,000 children in the Arabian
Peninsula to 185/10,000 children of Asian population. In Europe, the highest prevalence has been
observed in Sweden (115/10,000), while the lowest has been in Croatia (2–3/10,000, respectively) [2].
The occurrence of autism is ranging from three-to-five times higher in males than females [3]. While this
asymmetry in the male-female ratio has been known for many years, the underlying factors remain
largely unknown and this proportion is actually broadly revised [4]. Nowadays, ASD is considered as
a multifactorial disease, promoted by a variety of genetic and environmental factors [5,6].
It has been widely discussed that individuals with ASD may have undiagnosed gastrointestinal
conditions and hypersensitivities, which are the effects of the ingestion of casein and gluten.
This disorders may result in an exacerbation of ASD behaviors (e.g., tantrums, screaming,
and aggression) and inattention to tasks due to the distraction, and because of the pain [7]. According to
the previous hypothesis postulated by Panksepp, disruptions in opioid system are possible because of
the so-called autistic behavior [8] and this opioid theory of autism was also proved by pharmacological
studies [9]. Exogenous opioid peptides released during the digestion of dietary proteins, such as casein
and gluten were suggested to contribute to autism development, leading to the announcement of the
opioid excess hypothesis of autism [10].
One of the most biologically active milk-derived peptides are β-casomorphins (BCMs),
in particular, β-casomorphin-7 (BCM7; YPFPGPI) released from bovine β-casein sequence [11].
BCM7 has the ability to permeate the intestinal barrier and may induce biological effects through
the µ-opioid receptors (MORs) in the immune and nervous systems [12,13]. Moreover, BCMs were
found to have an inflammatory effect on the gastrointestinal system and may contribute to etiology of
food allergy [12,14]. In vitro studies have shown that BCM7 may alter lymphocyte proliferation and
inflammatory markers release [14,15]. Elevated levels of BCM7 have been observed in serum and urine
of ASDs patients in several independent studies [16,17]. The presence of milk-derived opioid peptides
is confirmed in bovine milk [11], breast milk and infant formula [18–20], dairy products including
cheese or yoghurt [13,18,21], and high-protein supplements for sportsmen [22].
The concentration of milk-derived opioid peptides like casomorphins in the human body depends
on several factors: The content in the food, the release from the protein precursors during food
digestion, and proline dipeptidyl peptidase-4 activity (DPPIV, CD26; EC 3.4.14.5). BCMs are identified
as exogenous substrates for DPPIV and inactivated under the influence of the enzyme. Deficiency
of DPPIV and/or its lower enzymatic activity were suggested as possible causes for the increased
level of opioids in patients with autism [23,24]. Moreover, DPPIV is involved in immune response and
nonspecific inflammatory processes and its decreased activity is generally associated with impaired
immune status [25].
Milk-derived opioid peptides should be considered as environmental stressors, which can lead to
disorders in the gut, reduced proteolytic activity, and increased permeability of the intestinal barrier.
These aspects, in correlation with low levels of circulating peptidases and increased blood–brain
barrier permeability, may cause the accumulation of opioid peptides in the blood and the brain [17].
The consequence is opioid-dependent modulation of neurotransmitter system and central nervous
system functioning, which may lead to the development of ASD [26]. Therefore, a currently proposed
treatment is the gluten-free, casein-free (GFCF) diet, supported by numerous scientific reports [27–29],
and information obtained from the parents about a significant improvement in health and even
recovery of children with symptoms of ASD [30].
The aim of this study was determination of BCM7 influence on DPPIV functioning in children
with ASD in comparison to healthy children, which is also according to gender. For this purpose,
we examined content and activity of serum DPPIV, content of BCM7 in serum and urine, and studied
the effect of hydrolysed bovine milk, as a source of opioid peptides, on DPPIV gene expression in
peripheral blood mononuclear cells (PBMC) in both groups.
Nutrients 2019, 11, 87 3 of 13
2. Materials and Method
2.1. Patients and Control Group Characteristic

Our research included 86 children diagnosed with autism spectrum disorder (ASD, ICDF84)
(71 male and 15 female; age range 3–10 years; and age mean 5.4 years), considered in this paper as
research group. The control group consisted of 51 healthy children with no history of behavioral
disorders (32 male and 19 female; age range 3–9 years; and age mean 5.2 years). The patients were
recruited by specialists in the Center for Diagnosis, Treatment and Therapy of Autism at the Regional
Children’s Hospital in Olsztyn, Poland. Diagnosis was based on the International Classification of
Mental and Behavioural Disorders—ICD-10. F84 disease in children was identified on the basis of
interdisciplinary differential diagnosis: Psychiatric examination excluding mental illness; studies
evaluating cognitive parameters in the respondents (Leiter scale—standard IQ from 70 to 107;
Wechsler—standard IQ from 90 to 104); neurological examination—EEG, evaluation of reflexes; speech
therapy—evaluation study of the development of speech; passive and participatory observation lasting
from 6 to 12 months; and analysis of the documentation: Names of parents, the opinions of educational
institutions, and video. Subjects with fever, infections, or skin problems and those taking steroids or
antibiotics were excluded from the study or control group. All subjects gave their written informed
consent for inclusion before they participated in the study. The study was approved by the Local
Bioethics Committee (number 19/2016 from the date of 18 May 2016).
2.2. Examined Substances

The study used pasteurized bovine milk which was commercially available. Prior to the experiments,
milk was hydrolyzed enzymatically to reflect human food digestion. Enzymatic hydrolysis and sample
preparation were previously described and published by Fiedorowicz et al. [12]. In brief, 20 mL of
hydrolysed milk was acidified to pH 4.2, centrifuged at 10,000× g, 25 min, and 4 ◦ C. The middle layer
was collected and ultrafiltrated using 30 kDa and 3 kDa cut-off points membrane tubes (AmiconUltra
30 and 3 kDa, Millipore, Burlington, MA, USA) in the following conditions: 5000× g, 30 min, 4 ◦ C.
The peptide extract obtained from hydrolyzed bovine milk was immediately used for analyses or
stored at −80 ◦ C. Before the in vitro analysis sample was diluted 2.5 times. Examined substances were
sterilized through a 0.22 µm filter (Becon Dickinson, Franklin Lakes, NJ, USA).
2.3. Biological Material

One of the 5–10 mL peripheral blood samples, 2 mL of serum, and 15 mL of urine were collected
from each patient. Samples were obtained by medical staff at the Regional Children’s Hospital in
Olsztyn. Biological material was immediately transported to the laboratory and was used for analysis
or stored at −80 ◦ C.
2.4. Measurement of BCM7 Concentration

ELISA test enabled identification of BCM7 contents in the serum and urine from patients, as well
as in tested peptide extract obtained from hydrolyzed bovine milk. The analysis was performed in
triplicate by means of the method previously described [13]. The results were analyzed in the GraphPad
PRISM version 6.0 (GraphPad Software, Inc., La Jolla, CA, USA) and the BCM7 concentrations were
determined, based on the standard curve for the concentrations 0.001–100 µg/mL.
2.5. Measurement of DPPIV Activity and Concentration in Serum

DPPIV activity was determined with the “direct photometric method”, adapted to 96-well
plates, as was described by Jarmołowska et al. [20]. Briefly, performance of analysis was as follows:
10 µL of test serum, water (blank) or standard (3 mM p-nitroanilide) was added to the reaction
mixture contained 50 µL 0.3 M Gly/NaOH buffer (pH 8.7), 100 µL 3 mM Gly-Pro-p-nitroanilide
Nutrients 2019, 11, 87 4 of 13
p-toluenosulfonate and 50 µL water. Control wells had no serum. After 30 min of incubation at 37 ◦ C,
the reaction was stopped by adding 50 µL ice-cold (4 ◦ C) 1 M acetate buffer (pH 4.2) and 10 µL of test
serum was added to the control wells. The absorbance was measured at 405 nm with a microplate
reader (Asys UVM 340, Biochrom, Holliston, MA, USA). The calculations were made after adjusting
the measurements with the blank. The enzyme activity was calculated as: 100 × (E − C)/S (where E,
C, and S stand for the absorbance of the test, control and standard samples, respectively). One unit of
the enzyme activity was defined as the amount of the enzyme liberating 1 µmol p-nitroanilide/min/L
test serum at 37 ◦ C.
Concentration of DPPIV in serum were evaluated using commercial ELISA kit (BioVendor, Brno,
Czech Republic) according to the manufacturer instructions.
2.6. Analysis of the DPPIV Gene Expression under the Influence of Peptide Extract Obtained from Hydrolysed
Bovine Milk
The blood from the patients was obtained directly into tubes containing K3 ETDA (BD, Biosciences)
and the peripheral blood mononuclear cells (PBMCs) isolation was immediately started. Fresh PBMCs
were then prepared, as previously described by Fiedorowicz et al. [15]. PBMCs were counted by
automatic cell counter—Scepter (MerckMillipore). The cells were seeded into 24-well plates at
0.5 × 106 per well with RPMI-1640 (Sigma, St. Louis, MO, USA) and supplemented with 1% heat
inactivated human AB serum (Sigma), 1% gentamicin (Sigma) and 0.25% phytohaemagglutinin
(PHA, Roche, Basel, Switzerland). After 48 h incubation, 300 µL peptide extracts obtained from
hydrolyzed bovine milk was added to each well, giving a final 5-fold dilution with the culture medium.
The incubation with the examined substance was conducted for five days. PBMCs suspension was
centrifuged (800× g, 20 ◦ C, 5 min), cell residue was rinsed twice with Dulbecco’s Phosphate-Buffered
Saline (DPBS, Invitrogen, Carlsbad, CA, USA) and used for RNA isolation.
RNA was isolated from the PBMCs using TRIzol Reagent (Sigma, St. Louis, USA), as previously
described by Kordulewska et al. [31]. RNA purity was estimated by calculating the ratio between
absorbance at 260 and 280 nm (A260/A280), with 1.8–2.0 results, and stored at −80 ◦ C for
further analysis. The reaction of reverse transcription was carried out using the QuantiTect Reverse
Transcription set (Qiagen, Hilden, Germany) according to the producer instructions. The obtained
cDNA was used as a matrix for the qualitative identification of gene amplification in Real Time
PCR. The 7500 FAST Sequence Detection System equipment and Fast SYBR Green Master Mix reader
by Applied Biosystems (Foster City, CA, USA) were used for the analysis. DPPIV gene and the
housekeeping human β-actin gene (ACTB) were examined, with ACTB used as a reference gene to
normalize differences in total RNA amounts in each sample. Oligonucleotide primers specific to each
gene were designed with Primer-BLAST, and the PCR primers are listed in Table 1.
Table 1. Primers used in Real-Time PCR for the analysis of DPP IV gene expression.
Annealing
Gene Forward Primer Revers Primer Base Pairs
Temperature
ACTB 50 -TCC CTG GAG GAA 50 -AGC ACT GTG
60 ◦ C 194 bp
NM-001101.3 GAG CTA CGA-30 TTG GCG TAC G-3
50 -GAA TTA TCC GGT 50 -GTG ACA TCA
DPPIV NM-001935 60 ◦ C 189 bp
CGG GTT TT-30 CTG CCC ACA TC-30
Reaction was carried out in the following conditions: preliminary polymerase activation 95 ◦ C,
20 s; 40 cycles: 3 s, 95 ◦ C; 60 ◦ C/ACTB/DPPIV, 30 s. The control of the product purity was performed
with the use of electrophoresis in 1.5% agarose gel (Sigma). The results were presented in relative units
in relation to the level of gene expression in native cells, referred to as 1 using the method described by
Pfaffl [32].
Nutrients 2019, 11, 87 5 of 13
2.7. Statistical
Nutrients 2019, 11, Data
x FOR PEER REVIEW 5 of 13
All statistical analyses were performer using GraphPad Prism 6.0 software (GraphPad Software
expression gender differences were analyzed using a two-way Anova test. Correlations were tested
Inc., San Diego, CA, USA). Results have been presented as a mean ± SEM. Student’s t-test was used
using the Spearman test.
for comparison of parametric data—DPPIV activity in serum, while Mann-Whitney U-test for content
3.DPPIV in serum. Kruskal-Wallis test was used for comparison of non-parametric data. Gene expression
Results
gender differences were analyzed using a two-way Anova test. Correlations were tested using the
Spearman
3.1. test. of BCM7 Concentration in Hydrolysed Bovine Milk and Patients’ Serum and
Measurement
Urine
3. Results
Application of the ELISA test confirmed the presence of BCM7 in peptide extract obtained from
3.1. Measurement
hydrolyzed bovine of milk.
BCM7TheConcentration in Hydrolysed
mean concentration Bovine Milk
of BCM7 and
in the Patients’
sample wasSerum and Urine
60 ng/mL (data not
shown).Application of the ELISA test confirmed the presence of BCM7 in peptide extract obtained from
We have
hydrolyzed analyzed
bovine milk.aThe
concentration of BCM7ofinBCM7
mean concentration serum, in which was was
the sample more60significantly
ng/mL (datadifferent (p
not shown).
< 0.0001) in the ASD group (42.96 ± 2.52 ng/mL) than in controls (26.42 ± 1.63 ng/mL)
We have analyzed a concentration of BCM7 in serum, which was more significantly different (Figure 1A). We
showed that in
(p < 0.0001) thethe
higher concentrations
ASD group of serum
(42.96 ± 2.52 BCM7
ng/mL) than(pin< controls
0.001) occurred
(26.42 ±among ASD boys
1.63 ng/mL) (39.52
(Figure 1A).
±We
2.26showed
ng/mL)that
compared to control boys (25.5 ± 1.9 ng/mL). We also noted significant
the higher concentrations of serum BCM7 (p < 0.001) occurred among ASD boys difference (p <
0.05) occurred among ASD girls (59.2 ± 9.04 ng/mL) than in control girls (27.9
(39.52 ± 2.26 ng/mL) compared to control boys (25.5 ± 1.9 ng/mL). We also noted significant ± 2.97) (Figure 1A).
Urine (p
difference BCM7
< 0.05)concentration
occurred amongis presented
ASD girlsin Figure
(59.2 1B.ng/mL)
± 9.04 There than
wereinno significant
control difference
girls (27.9 ± 2.97)
detected in
(Figure 1A). urine levels of BCM7 between two study groups, and also between gender (Figure 1B).
Figure 1. Comparison of serum (A) and urine (B) BCM7 concentration in control and autistic group
Figure
(ASD) 1.(ng/mL
Comparison of serum
± SEM). (A) and
Significant urine (B) BCM7
differences concentration
between in control
groups, divided and autistic
by gender: * p group
< 0.05;
(ASD) (ng/mL
*** p < 0.001. ± SEM). Significant differences between groups, divided by gender: * p < 0.05; *** p <
0.001.
Urine BCM7 concentration is presented in Figure 1B. There were no significant difference detected
3.2. Measurement
in urine of DPPIV
levels of BCM7 Activity
between twoand Concentration
study groups, and in Serum
also between gender (Figure 1B).
3.2. Figure 2A,B ofshow

Measurement serum
DPPIV DPPIV
Activity activity in control
and Concentration in Serumand ASD groups. We did not notice
significant difference in enzymatic activity of serum DPPIV between two study groups (p = 0.262;
FigureFigure 2A,Bsignificantly
2A). The show serum DPPIV activityactivity
lower DPPIV in control
(pand ASDwas
< 0.05) groups. We didinnot
observed notice
girls withsignificant
autism,
difference in enzymatic activity
compared to ASD boys (Figure 2A). of serum DPPIV between two study groups (p = 0.262; Figure 2A).
The DPPIV
significantly lower DPPIV
concentration activity
was found (p < 0.05) was
significantly observed
higher in girls
(p < 0.01) with autism,
in serum from ASDcompared
childrento(1089
ASD
boys (Figure 2A).
± 44.3 ng/mL) compared to the control group (934.0 ± 52.7 ng/mL) (Figure 2B). The concentration of
serum DPPIV was significantly higher (p < 0.01) in autistic (1050 ± 36.86 ng/mL) than in healthy boys
(922.2 ± 65.04 ng/mL). There was also statistical difference between the concentration of serum DPPIV
in ASD girls (1336 ± 144.0 ng/mL) compared to both control girls (953.4 ± 92.14 ng/mL, p < 0.05) and
control boys (p < 0.01) (Figure 2B).
Nutrients 2019, 11, 87 6 of 13

Nutrients 2019, 11, x FOR PEER REVIEW 6 of 13
Figure 2. Comparison of DPPIV enzymatic activity (A) and concentration (B) in serum of control
Figure 2. Comparison of DPPIV enzymatic activity (A) and concentration (B) in serum of control and
and autistic group (ASD) (Figure 2A: U/L ± SEM; Figure 2B: ng/mL ± SEM). Significant differences
autistic group (ASD) (Figure 2A: U/L ± SEM; Figure 2B: ng/mL ± SEM). Significant differences between
between groups, divided by gender: * p < 0.05, ** p < 0.01.
groups, divided by gender: * p < 0.05, ** p < 0.01.
DPPIV concentration was found significantly higher (p < 0.01) in serum from ASD children
3.3. DPPIV
±Figure Gene Expression in
to PBMCs
(1089 44.3 ng/mL) compared
2. Comparison the enzymatic
of DPPIV control group
activity (A)±
(934.0 and52.7 ng/mL) (Figure
concentration (B) in 2B).
serumThe
of concentration
control and
of serum DPPIV
autistic
There werewas
group significantly
no(ASD) (Figurehigher
significant 2A: U/L(p± <
differences 0.01)
SEM; in autistic
in Figure
DPPIV 2B: (1050
ng/mL
gene ±± 36.86
SEM).
expression ng/mL)the
Significant
under than in healthy
differences
influence betweenboys
of extract
(922.2 ± 65.04
groups,
obtained fromng/mL).
divided There was
by gender:
hydrolyzed also statistical
* p < 0.05,
bovine difference
** p There
milk. between the concentration of serum
< 0.01. were also no differences according to gender. DPPIV
inNevertheless,
ASD girls (1336 we±noticed
144.0 ng/mL)
a tendencycompared to both
to increase thecontrol
DPPIVgirls
gene(953.4 ± 92.14
expression ng/mL,
among p <with
girls 0.05)autism
and
3.3.
control DPPIV
boys
(Figure 3). (pGene
< Expression
0.01) (Figure in
2B). PBMCs
There
3.3. DPPIV were
Gene no significant
Expression differences in DPPIV gene expression under the influence of extract
in PBMCs
obtained from hydrolyzed bovine milk. There were also no differences according to gender.
There were no
Nevertheless, wesignificant
noticed adifferences
tendency in
to DPPIV gene
increase theexpression
DPPIV geneunder the influence
expression amongof extract obtained
girls with autism
from hydrolyzed
(Figure 3). bovine milk. There were also no differences according to gender. Nevertheless, we noticed
a tendency to increase the DPPIV gene expression among girls with autism (Figure 3).
Figure 3. Influence of milk-derived opioid peptides on DPPIV gene expression in control and autistic
group (ASD), and divided by gender (relative unit ± SEM).
3.4. Correlation of Examined Parameters between ASD and Control Groups, and with Gender
Figure 3. Influence of milk-derived opioid peptides on DPPIV gene expression in control and autistic
group (ASD),
Figure
Figure 4A,B and divided
3. Influence
show by gender
of milk-derived
correlation (relative
between unit ±activity
opioidDPPIV SEM).
peptides on DPPIV
andgene
BCM7expression in control
concentration and autistic
(Figure 4A), and
DPPIVgroup (ASD), and and
concentration divided by gender
activity in ASD (relative unit ± SEM).
and control groups (Figure 4B). We observed a negative
3.4. Correlation of Examined Parameters between ASD and Control Groups, and with Gender
correlation between concentration of BCM7 and DPPIV activity in serum in the ASD group (p < 0.05).
3.4.Figure
Correlation
Positive of Examined
4A,B show
correlation between Parameters
correlation betweenbetween
concentration DPPIV ASD and
activity
and activity Control
and
DPPIV in Groups,
BCM7 andobserved
with(Figure
concentration
serum was Gender
in 4A),
both,
and DPPIV
control (p concentration
< 0.01) and and
ASD activity
group (p <in ASD
0.001). and control groups (Figure 4B). We observed
Figure 4A,B show correlation between DPPIV activity and BCM7 concentration (Figure 4A), and a negative
correlation between concentration
DPPIV concentration of BCM7
and activity in ASDand and DPPIV
controlactivity
groups in (Figure
serum in theWe
4B). ASD group (pa<negative
observed 0.05).
Positive correlation between concentration and activity DPPIV in serum was observed in
correlation between concentration of BCM7 and DPPIV activity in serum in the ASD group (p < 0.05). both, control
(p < 0.01) and
Positive ASD group
correlation (p < 0.001).
between concentration and activity DPPIV in serum was observed in both,
control (p < 0.01) and ASD group (p < 0.001).

Nutrients 2019, 11, 87 7 of 13
Figure 4. Correlation between DPPIV activity and BCM7 concentration in serum (A) and between
DPPIV activity and concentration in serum (B) in control and ASD group (▲—ASD group, □—control
Figure 4. Correlation
Figure Correlationbetween
betweenDPPIV
DPPIVactivity andand
activity BCM7 concentration
BCM7 in serum
concentration (A) and
in serum (A)between DPPIV
and between
group).
activity and concentration in serum (B) in control and ASD group (N —ASD group, —control
DPPIV activity and concentration in serum (B) in control and ASD group (▲—ASD group, □—control group).
group).
Figure5 summarizes
Figure 5 summarizes all obtained
all obtained results.results. Principal
Principal component component analysis
analysis (PCA) (PCA)
showed showed
significant
significantwithin
differences differences within
the tested the tested
samples. samples. Concentration
Concentration of DPPIV andofBCM7
DPPIVinand BCM7
serum was instatistically
serum was
Figure 5 summarizes all obtained results. Principal component analysis (PCA) showed
statistically
significantly significantly
higher in higher
autistic in autistic
children than children
in the than
control in the
group.control group.
According toAccording
the gender to the gender
correlation
significant differences within the tested samples. Concentration of DPPIV and BCM7 in serum was
correlation
found betweenfound between
DPPIV DPPIV
activity and activityconcentration
BCM7 and BCM7 concentration =in0.418,
serum p=(R0.075),
= 0.418, p =DPPIV
0.075),
statistically significantly higher in autistic children than in in theserum
control(Rgroup. According toand
the gender
and DPPIV activity and concentration in serum (R = 0.503, p = 0.067) in healthy girls, and between
activity andfound
correlation concentration
betweeninDPPIV
serumactivity andpBCM7
(R = 0.503, = 0.067) in healthy girls,
concentration and between
in serum DPPIV
(R = 0.418, p = activity
0.075),
DPPIV
and DPPIVactivity and DPPIVinconcentration
concentration serum in ASD in boys
serum(Rin=ASD boys
0.446, p =(R = 0.446,
0.005). A p = 0.005). A interesting
particularly particularly
and DPPIV activity and concentration in serum (R = 0.503, p = 0.067) in healthy girls, and between
interesting
are ASD arecharacterized
girls, ASD girls, characterized by the correlation
by the correlation between thebetween the concentration
concentration of BCM7, DPPIV
DPPIV activity and DPPIV concentration in serum in ASD boys (R = 0.446, of
p =BCM7,
0.005).DPPIV content,
A particularly
content,
and DPPIVareand DPPIV
expression.expression.
interesting ASD girls, characterized by the correlation between the concentration of BCM7, DPPIV
content, and DPPIV expression.
Figure5.5.Correlations
Figure Correlationsbetween
betweenall
alltested
testedparameters
parametersinincontrol
controland
andautistic
autisticgroup
group(ASD),
(ASD),and
anddivided
divided
by gender (N —boys with ASD, #—girls with ASD, —healthy
by gender (▲—boys with ASD, ○—girls with ASD, □—healthy children).children).
Figure 5. Correlations between all tested parameters in control and autistic group (ASD), and divided
by gender (▲—boys with ASD, ○—girls with ASD, □—healthy children).
Nutrients 2019, 11, 87 8 of 13
4. Discussion
Autism is a developmental disorder, which usually manifests itself in the first years of life.
Children with autism exhibit abnormal behavior in social interaction and communication problems.
As indicated by the global statistics from year-to-year, the incidence of autism increases and is currently
epidemic of this disease should be considered [1,33]. ASD prevalence is strongly associated with the
male gender [3], which is also represented by our sex distribution in the ASD group. The causes of
autism are not fully explained, but it is clear that the development of the disease is affected by genetic
and autoimmune factors, metabolic disorders, and epigenetic changes depending on the environmental
and nutritional factors. In recent years, researchers have focused on the role of the opioid system in
various pathological processes [33].
Numerous studies confirmed, that milk-derived opioid peptides may penetrate the intestinal
barrier and induce biological effects through the opioid receptors of the immune and nervous
system [12,34,35]. We found that the content of BCM7 in serum was significantly higher (p < 0.0001) in
ASD than in the control group (Figure 1A). Elevated level of BCM7 in serum of autistic children was
reported by several authors [9,17], which is consistent with our results. We also noticed a difference
between gender—in ASD girls there was the highest level of BCM7 in serum (p < 0.05) among all
patients (Figure 1A). Statistical difference between ASD girls and boys can be explained as small
number of girls. However, obtaining a similar number of girls and boys in ASD group was difficult
because it is associated with a higher incidence of autism in boys than in girls. Hyperpeptidemia
and increased blood–brain barrier permeability may cause accumulation of BCM7 in the blood and
the brain, leading to the development of ASD [24,26]. We did not observe differences in BCM7
concentration in urine (Figure 1B), whereas Sokolov et al. [17] demonstrated that autistic children have
significantly higher levels of urine BCM7 than healthy children, and the severity of autistic symptoms
were correlated with concentrations of the peptide in the urine. These authors suggested that peptiduria
in autistic children is the potential defect in their proteolytic and/or peptide excretion systems,
consistent with previous studies that infants with delayed psychomotor development had elevated
levels of the postprandial bovine BCM7 compared with a healthy control group. Chronic exposure to
elevated levels of bovine casomorphins may contribute to disorders during early child development,
also including autistic disorders, because these peptides interact with opioid and serotonin receptors,
the known modulators of synaptogenesis [17,24]. Opioid peptides alter not only the mechanism
of neuromodulation in the central nervous system (CNS), but also trigger inflammation and food
allergy [12,36]. It is known that subcutaneous injection of BCM7 causes local pseudo-allergic reactions,
masts cell degranulation, and histamine secretion even in healthy children [37]. β-casomorphin-5
may cause mast cells degranulation in mice, confirming the nature of the allergenic potential of this
peptide [38]. Consequently, consumption of bovine milk containing BCM7 may induce inflammatory
response in intestine by activating Th2 pathway [36], which was described in our previous research [12].
According to this approach, BCM7 consumption by autistic children induces gastrointestinal problems,
such as abnormalities of the bowel mucosa, dysfunctions associated with intestine permeability,
and changes in the gut microbiota [39]. High-protein diet may result in increased BCM7 concentration
in the serum and urine of autistic children (Figure 1A,B). Consequently, milk-derived opioid peptides
may reduce the uptake of cysteine, resulting in a decrease in glutathione synthesis and availability
of the methyl donor S-adenosylmethionine (SAM). The decrease in SAM translates into effects on
global DNA methylation, with epigenetic consequences, which alter neurological disorders such as
autism [40]. Therefore avoiding milk-derived opioid peptides may lead to reduced peptiduria in
children with autism and improves health, including silenced autoimmunity, improved emotional
state and the opportunity to establish social relationships [24,29,41,42].
DPPIV is involved in immune response and nonspecific inflammation processes and its decreased
activity is generally associated with impaired immune status [25]. While we did not observe significant
difference in serum DPPIV activity between autistic group and control group (Figure 2A), deficiency
and/or low enzymatic activity of DPPIV were suggested as possible causes for the presence of elevated
Nutrients 2019, 11, 87 9 of 13
levels of opioids in patients with autism, subsequently worsening autistic symptoms [23,43]. It has
been reported, that DPPIV activity in women is slightly lower than in men [44], which was observed in
the ASD group (Figure 2A). We demonstrated that female autistic children have lower levels of DPPIV
activity than males (p < 0.01), whereas there were no significant differences between genders in control
group (Figure 2B). This dependence is difficult to explain, but in our opinion its mechanisms should be
sought in the biological role of DPPIV. It is known that DPPIV inactivates glucagon-like peptide (GLP)-1,
and glucose-dependent insulinotropic peptide (GIP) exert pivotal functions on metabolic homeostasis
such as glucose-dependent insulin secretion or suppression of excessive glucagon secretion. DPPIV
causes degradation of milk-derived opioid peptides, stromal cell-derived factor 1 (SDF-1), substance P,
and neuropeptide Y (NPY), and also participates in various processes, including immune stimulation,
binding to extracellular matrix, and lipid accumulation [45].
Biological function of DPPIV is revealed not only by its enzymatic activity, but also by the
concentration in the serum. Bashir and Laila [46] concluded that autistic patients have lower levels of
plasma DPPIV than control group, but DPPIV content was not correlated to the severity of autism,
according to CARS scoring results. Another authors did not find any defects in DPPIV in the blood
of 11 autistic children [47]. We demonstrated that concentration of serum DPPIV is significantly
higher (p < 0.01) in ASD children compared to the control group (Figure 2B). Constantly, there was also
significant difference between ASD and control girls (p < 0.05) and ASD and control boys (p < 0.01).
It should be noted that the highest DPPIV content was correlated with the highest serum BCM7
concentration among ASD girls. This dependence is clear to explain, because DPPIV is the only one
enzyme that is able to hydrolyze the BCM7 and increased expression of DPPIV is the obvious response
to the presence of BCM in blood (Figures 1A, 2B and 3).
The presence of DPPIV was confirmed on the surface of T lymphocytes, simultaneously describing
its abnormal expression in patients with various diseases [12,25], but information about changes in the
expression of membrane forms DPPIV are still limited and require further research. It is known that
BCM7 circulating in the bloodstream, can influence of the expression of DPPIV triggering a number
of pathological mechanisms as inflammation and cytokine secretion. Impact of food ingredients on
human physiological mechanisms is difficult to estimate in in vivo conditions, so we decided to use
PBMCs as a model the human immune system. We confirmed the presence of BCM7 in extract obtained
from hydrolysed bovine milk (60 ng/mL) and then we incubated PBMCs with this sample to estimate
if milk-derived opioid peptides alter DPPIV gene expression on immune cells. While the carried
out analysis has not revealed significant changes, a tendency to increase DPPIV gene expression
in ASD girls was observed (Figure 3). Cieślińska et al. [33] indicated correlation in DPPIV gene
expression under the influence of BCM7 and hydrolyzed milk between healthy and ASD-affected
children, but only with genotype GG (polymorphism in DPPIV gene: rs7608798). Our previous studies
revealed that PBMCs DPPIV gene expression was 1.5–2.5 times lower after BCM7 and hydrolyzed milk
stimulation in patients with atopic dermatitis compared to the control group [12]. Therefore, changes
in DPPIV gene expression should be considered as a potential factor with different biological effects
depending on the studied disease.
The casein-free diet and avoidance of milk-derived opioid peptides are crucial in regulation
of the DPPIV content and activity in patients with ASD. In autistic children we noted negative
correlation between DPPIV activity and BCM7 concentration, and positive correlation between DPPIV
concentration and DPPIV activity in serum. We observed positive correlation in both DPPIV activity
and BCM7 concentration, and DPPIV concentration and DPPIV activity in serum among the control
group (Figure 4A,B). According to gender positive correlation was found between DPPIV activity
and DPPIV concentration in serum of ASD boys (Figure 5). In healthy girls, we noted a positive
correlation between DPPIV activity and BCM7 concentration in serum, and DPPIV activity and DPPIV
concentration in serum. (Figure 5). An interesting result of the present study was correlation between
DPPIV content and BCM7 concentration in serum from girls with ASD. We suggest, that despite
higher DPPIV content in serum, and its enzymatic activity per amount of protein was actually lower
Nutrients 2019, 11, 87 10 of 13
in autistic girls than in the other groups. The higher levels of protein could compensate for reduced
overall activity of the enzyme. The overall difference between individual parameters between girls
and boys seems to be interesting, it requires further research and should be considered at the level
of sex hormones. Boys are subjected to higher testosterone activity than girls, which can affect brain
function and anxiety in social situations. In turn, girls are subjected to a higher concentration of
oxytocin, which facilitates social behavior and correlations. The effects of these hormones are revealed
in fetal life and may affect the severity of autistic symptoms in later life [48–50]. Moreover, it has been
observed that TSH level is about 31% lower in ASD boys in comparison to healthy ones [51]. Thyroid
hormones are essential for brain maturation and function throughout life. Thyroid hormone deficiency,
even for short periods, may lead to irreversible brain damage, the consequences of which depends on
the specific timing of onset and duration of thyroid hormone deficiency [52].
To the best of our knowledge, this is the first study that examines the influence of BCM7 on the
role of DPPIV in populations of healthy children and those diagnosed with autism spectrum disorder.
The inspiration for this study emanated from clinical experience of the role daily diet in relieving
the symptoms of autism. This paper considers the role of opioid peptides and DPPIV as potential
factors in determining the pathogenesis of autism in aspect of BCM7 biological activity and recognizes
numerous reports about the effectiveness of elimination diets (casein free) in the treatment of children
with neurological disorders. However, this issue requires further investigation.
Author Contributions: Conceptualization, B.J., E.K., M.B. and E.F.; methodology, B.J., M.B., E.F., A.C. and N.K.K.;
software, A.Ś.; validation, B.J., M.B., E.F. and N.K.K.; investigation, M.B., E.F., A.C. and N.K.K.; writing—original
draft preparation, E.F., A.C. and N.K.K.; writing—review and editing, B.J., M.M. and A.Ś.; visualization, M.B.,
N.K.K., A.C., E.F. and A.Ś.; supervision, E.K. and A.Ś.
Funding: This research received no external funding.
Acknowledgments: There was no funding for this paper.
Conflicts of Interest: None of the authors have reported any financial interests or potential conflicts of interest.
None of the authors has a financial relationship with a commercial entity that has an interest in the subject of
this manuscript.
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© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
J Autism Dev Disord

DOI 10.1007/s10803-017-3340-9
ORIGINAL PAPER
Sensory Sensitivity and Food Selectivity in Children with Autism

Spectrum Disorder
Liem T. Chistol1 · Linda G. Bandini2,3 · Aviva Must4 · Sarah Phillips4 ·
Sharon A. Cermak5 · Carol Curtin6
© Springer Science+Business Media, LLC 2017
Abstract Few studies have compared atypical sensory ASD may be enhanced by including strategies that address
characteristics and food selectivity between children with oral sensory processing.
and without autism spectrum disorder (ASD). We compared
oral sensory processing between children with (n = 53) and Keywords Autism spectrum disorder · Food selectivity ·
without ASD (n = 58), ages 3–11 years. We also examined Sensory sensitivity
the relationships between atypical oral sensory processing,
food selectivity, and fruit/vegetable consumption in children
with ASD. We found that more children with ASD presented Introduction
with atypical sensory processing than children without ASD.
Among children with ASD, those with atypical oral sensory Autism spectrum disorder is a developmental disability
sensitivity refused more foods and ate fewer vegetables than characterized by deficits in communication, social inter-
those with typical oral sensory sensitivity. The findings sug- action, and restrictive, repetitive behavioral patterns that
gest that efforts to address food selectivity in children with impair social, occupational, and daily functioning (Ameri-
can Psychiatric Association 2013). This disorder is also
often associated with sensory processing difficulties, which
includes over- or under-sensitivity to sensory stimuli in the
* Carol Curtin environment. The prevalence of ASD in the United States
carol.curtin@umassmed.edu is currently estimated to affect 1 in 68 children with higher
1 rates among males and non-Hispanic whites (Christensen
Sargent College of Health & Rehabilitation Sciences, Boston
University, 635 Commonwealth Avenue, Boston, MA 02215, et al. 2016). ASD poses a major health and educational chal-
USA lenge for parents and caretakers as it affects many aspects
2
Department of Pediatrics, E.K. Shriver Center, UMass of daily living. Feeding and mealtimes are especially chal-
Medical School, 55 Lake Avenue North, Worcester, lenging for these children and their caretakers (Curtin et al.
MA 01655, USA 2015; Zobel-Lachiusa et al. 2015). Parents and clinicians
3
Department of Health Sciences, Boston University, 635 frequently report that children with ASD are overly selective
Commonwealth Ave., Boston, MA 02215, USA in their eating patterns; they consume less varied diets with
4
Department of Public Health and Community Medicine, very few fruits and vegetables (Ahearn et al. 2001; Bandini
Tufts University School of Medicine, 136 Harrison Ave., et al. 2010; Cermak et al. 2010; Emond et al. 2010; John-
Boston, MA 02111, USA
son et al. 2008; Ledford and Gast 2006; Marí-Bauset et al.
5
Division of Occupational Science and Occupational Therapy 2014; Martins et al. 2008; Ranjan and Nasser 2015; Schreck
at the Herman Ostrow School of Dentistry, University
et al. 2004; Sharp et al. 2013; Zimmer et al. 2012). The lack
of Southern California, 1540 Alcazar Street CHP‑133,
Los Angeles, CA 90089, USA of food variety may put individuals at risk for nutritional
6 inadequacies (Bandini et al. 2010; Ma et al. 2016; Zimmer
Department of Family Medicine & Community Health,
E.K. Shriver Center, UMass Medical School, 55 Lake et al. 2012). One recent review estimated that children with
Avenue North, Worcester, MA 01655, USA ASD have a five-fold risk of feeding problems compared
13
Vol.:(0123456789)
J Autism Dev Disord
to children without ASD (Sharp et al. 2013). While lon- TD children to assess differences in oral sensory processing
gitudinal data are limited, two recent studies suggest that compared to children with ASD. The aims of the study were
food selective behaviors may persist over time (Bandini et al. to: (1) compare oral sensory processing function between
2017; Suarez et al. 2014). Therefore, addressing the underly- children with and without ASD; (2) examine the relationship
ing factors associated with food selectivity may be neces- between atypical oral sensory processing and food selec-
sary to mitigate the comorbidities associated with long-term tivity in children with ASD; and (3) examine the relation-
inadequate nutritional intake in this population. ship between atypical oral sensory processing and fruit and
In recent years, it has been suggested that food selectivity vegetable consumption. We hypothesized that atypical oral
in individuals with ASD may be related to sensory process- sensory processing would be greater in children with ASD,
ing dysfunction, specifically oral sensory sensitivity (Cer- and would be associated with higher levels of food selectiv-
mak et al. 2010; Suarez 2012; Zobel-Lachiusa et al. 2015). ity and with decreased fruit and vegetable variety.
Sensory processing refers to the ability to register, process,
and organize sensory information and to execute appropriate
responses to environmental demands, which may manifest as Methods
over- or under-sensitivity to the stimuli (Dunn 2001). Atypi-
cal sensory processing has been a recognized feature of ASD This study used data from the Children’s Activity and
since the condition was first described by Kanner (1943), Meal Patterns Study (CHAMPS), a cross-sectional study
occurring in 45–95% in persons with ASD (Ben-Sasson conducted in 2007–2008 at the University of Massachu-
et al. 2009; Schreck and Williams 2006; Tomchek and Dunn setts Medical School’s Eunice Kennedy Shriver Center.
2007). Anecdotal reports from parents of children with ASD CHAMPS was designed to assess and compare dietary pat-
and autobiographies of individuals with ASD often attribute terns, mealtimes, and physical activity patterns between chil-
food selectivity to aversions to color, taste, smell, and/or dren with and without ASD. Recruitment, exclusion criteria,
texture, which suggests an underlying sensory component and the procedures for verifying participants’ diagnosis of
to this behavior (Shore 2001; Whiteley et al. 2000; Williams ASD have been previously described (Bandini et al. 2010).
et al. 2000). Several studies have suggested that sensory sen- Participants included 53 children with ASD and 58 TD chil-
sitivity may lead children with ASD to restrict their intake dren ages 3–11 years. The study was approved by the Insti-
to foods of preferred, tolerable, and manageable textures. tutional Review Board at the University of Massachusetts
The texture of foods has consistently been identified as a Medical and parents provided written informed consent.
related aspect of food acceptance (Ahearn et al. 2001; Hub- Parents completed a demographic/medical questionnaire,
bard et al. 2014; Postorino et al. 2015; Schreck and Wil- a Food Frequency Questionnaire (FFQ), and a 3-day food
liams 2006; Schreck et al. 2004; Williams et al. 2005). For record. The Vineland Adaptive Behavior Scales (VABS)
example, using data from the current study, Hubbard et al. were used to characterize children’s adaptive skills and the
(2014) found that texture and consistency of foods are highly Differential Abilities Scales (DAS) were administered to
correlated with parent report of food refusal in a sample assess cognitive ability in children with ASD.
of 53 children with ASD ages 3–11 years. While a small Parents also completed the Sensory Profile (Dunn
number of studies have suggested that there is a significant 1999), a 125-item caregiver questionnaire frequently used
relationship between atypical oral sensory processing and in clinical practice to identify sensory processing chal-
food selectivity in children with ASD, the methodologies lenges in children with ASD. It is a standardized tool that
used to elucidate this relationship have been limited by the compares the child’s score to data from a reference sample
use of parent report of food selectivity rather than the use of of 1200 children without disabilities and identifies the sen-
validated dietary measures. For example, studies have used sory systems that are affected by the child’s sensitivity to
a variety of parent-report tools such as the Eating Profile stimuli and contribute to functional impairments. Parents
(Nadon et al. 2011), the Child Eating Behavior Question- reported their child’s responses to sensory stimuli using a
naire (Kral et al. 2015), the Brief Assessment of Mealtime 5-point Likert scale (always, frequently, occasionally, sel-
Behavior Inventory (BAMBI) (Lane et al. 2014) or a single dom, never). Questions were scored along nine factors of
question about food selectivity on a parent questionnaire sensory processing difficulties including Sensory Seeking,
(Suarez et al. 2014). The inconsistency in measures and the Emotionally Reactive, Low Endurance/Tone, Oral Sensory
lack of a standardized definition of food selectivity limits the Sensitivity, Inattention/Distractibility, Poor Registration,
ability to compare results across studies. Sensory Sensitivity, Sedentary, Fine Motor/Perceptual.
The purpose of the current study was to evaluate the rela- Children scoring within the one standard deviation, or
tionship between oral sensory processing and food selec- within the 84th percentile of the reference sample, are
tivity in children with ASD using quantitative measures of classified as “Typical”, which indicates typical sensory
food selectivity. We also included a comparison group of processing ability for each factor. Children who scored one
13
J Autism Dev Disord
or more standard deviations from the mean of the reference Data Analysis
group (> 85th percentile) were classified as “Atypical”,
which is indicative of a likelihood of sensory processing Differences in participant characteristics between children
difficulty for that factor. Within the Atypical classification, with and without ASD were compared using independent
participants were further classified as having “probable” samples t-tests for continuous variables and Chi square tests
sensory processing issues (corresponding to between one for categorical variables. Differences in sensory processing
and two standard deviations below the mean, or between measures between children with and without ASD were
the 2nd and 16th percentiles) or a “definite” sensory pro- compared using non-parametric Wilcoxon Rank Sum tests.
cessing disorder (two standard deviations below the mean, After dichotomizing Oral Sensory Sensitivity and the Oral
or children scoring in the lowest 2%) (Dunn 1999). Sensory Over-sensitivity scores into Typical and Atypical,
In order to explore the relationship between oral sen- we used independent samples t-tests to compare the mean
sory over-sensitivity and food selectivity, we analyzed a difference in food selectivity measures and fruit and veg-
subset of four questions from the Oral Sensory Sensitivity etable variety between the groups. Linear regression was
factor that measure over-sensitivity or a low tolerance for used to assess the impact of age on the relationship between
taste/smell stimuli (Fig. 1) (Dunn 1999; McIntosh et al. food selectivity measures and Typical vs. Atypical sen-
1999). These items comprised: (1) avoids certain tastes sory sensitivity. Statistical significance was declared when
or food smells that are typically part of children’s diets; p < 0.05. All analyses were conducted in SAS Version 9.3
(2) will only eat certain tastes; (3) limits self to particular (SAS Institute, Cary, NC) and IBM SPSS Statistics Version
food textures/temperatures; and (4) picky eater, especially 21 (Armonk, NY: IBM Corp).
regarding food textures. The subscale is scored using the
same approach as described above. We refer to this sub-
scale as Oral Sensory Over-sensitivity. Results
Relative to food selectivity, we used a quantitative
approach to define this phenomenon that includes two Demographic characteristics of the 53 children with ASD
distinct domains: food refusal and limited food reper- and 58 TD children are presented in Table 1. There were no
toire (Bandini et al. 2010). Briefly, to assess food refusal, significant differences in age, sex, or race/ethnicity between
parents were asked to complete a modified version of the two groups. TD children were more likely to be an only
the Youth/Adolescent Food Frequency Questionnaire child than were children with ASD (26% and 11% respec-
(Rockett et al. 1997). We modified the FFQ to measure tively, p = 0.05). However, whether or not a child was an only
the refusal and frequency of consumption of 131 common child was unrelated to any aspect of food selectivity. Charac-
food items during the past year based on parent report. teristics of food selectivity for the ASD and TD groups are
The food refusal score was expressed as the percentage also shown. Compared to the TD group, children with ASD
of foods refused of those offered to the child. Food rep- had significantly higher levels of food refusal, significantly
ertoire was operationally defined as the absolute number lower levels of food repertoire, and less variety of fruits and
of unique foods the child consumed (including beverages) vegetables.
over 3 days using a 3-day food record. Parents were asked To address the question of group differences in sen-
to complete the 3-day food record for 2 weekdays and 1 sory processing, we examined scores on the Sensory
weekend day, including food consumed at school. In addi- Profile (Table 2). On average, children with ASD scored
tion, we examined fruit and vegetable variety, which was lower on the Sensory Profile than TD children, indicat-
determined by the number of unique fruits and vegetables ing more atypical sensory processing. Scores between
consumed on the FFQ, which included 13 fruits and 20 the two groups differed significantly across all 9 fac-
vegetables. tors. Mean scores in the TD group were classified in the
Fig. 1 Factor 4 oral sensory Oral Sensory Sensitivity

sensitivity (OSS) items on the *55. Avoids certain tastes or food smells that are typically part of children’s diets
Sensory Profile and Taste/Smell
*56. Will only eat certain tastes
Sensitivity (TSS) Questions
on the Short Sensory Profile.
*57. Limits self to particular food textures/temperatures
*Indicates questions on oral *58. Picky eater, especially regarding food textures
over-sensitivity measure 59. Routinely smells nonfood objects
60. Shows strong preference for certain smells
61. Shows strong preference for certain tastes
62. Craves certain foods
63. Seeks out certain tastes or smells
13
J Autism Dev Disord
Table 1 Participant characteristics
Participant characteristics Children with ASD TD children (n = 58) p value
(n = 53)
Age, years: mean (SD) 6.6 (2.1) 6.7 (2.4) 0.75

Sex, male (%) 83% 78% 0.47
Race, white (%) 83% 76% 0.35
Vineland Adaptive Behavior Scales (VABS) Score: mean (SD) 71.1 (12.4) – –
Differential Abilities Scales General Conceptual Ability Score (DAS): 85.8 (22.1) – –
mean (SD)a
Child is an only child (%) 11% 26% 0.05
Food refusal, mean (SD) 41.7 (21.1) 18.9 (15.6) < 0.001
Food repertoire, mean (SD) 19.0 (4.9) 22.5 (4.6) < 0.001
Fruit variety, mean (SD) 6.5 (3.3) 9.4 (2.9) < 0.001
Vegetable variety, mean (SD) 5.9 (4.9) 10.8 (4.8) < 0.001
a
n = 47
Table 2 Comparison Factor Children with ASD (n = 53*) TD Children (n = 58*) p valuea

of Sensory Sensitivity
Questionnaire Subscale Scores Mean (SD) Median Mean (SD) Median
between children with ASD and
TD Children Sensory seeking 54.7 (9.6) 54 71.3 (9.7) 73.0 < 0.001
Emotionally reactive 46.6 (12.4) 47 68.6 (7.2) 70.0 < 0.001
Low endurance/tone 35.0 (9.0) 37.5 43.6 (2.7) 45.0 < 0.001
Oral sensitivity 29.6 (8.4) 30.0 40.6 (5.2) 42.0 < 0.001
Inattention/distractibility 18.6 (4.2) 19.0 28.6 (3.5) 29.0 < 0.001
Poor registration 28.9 (4.5) 29.0 38.0 (2.5) 39.0 < 0.001
Sensory sensitivity 16.4 (3.9) 17.0 19.0 (1.7) 20.0 < 0.001
Sedentary 12.4 (4.4) 13 15.1 (2.9) 15.0 < 0.001
Fine motor/perceptual 8.5 (2.8) 9.0 12.6 (2.6) 13.0 < 0.001
*Sample sizes vary slightly due to missing data

a
Non-parametric Wilcoxon test
typical performance range for all nine subscales, while

mean scores in the ASD group fell in the atypical range
for seven of nine subscales.
Performance classifications for Oral Sensory Sensitivity Table 3 Summary of number (and %) of children in each group in
and Oral Sensory Over-sensitivity for both groups are pre- the typical, probable difference, and definite difference range in the
sented in Table 3. More children with ASD were classified oral sensory sensitivity and taste/smell sensitivity subscales
as Atypical for Oral Sensory Sensitivity and Oral Sensory Factor ASD TD
Over-sensitivity compared to TD children. Among TD chil- n (%) n (%)
dren, only 7% were classified as Atypical based on their
Oral sensory sensitivity
Oral Sensory Sensitivity scores and 9% as Atypical based
Typical 18 (36) 54 (93)
on their Oral Sensory Over-sensitivity scores. Conversely,
Atypical 32 (64) 4 (7)
among children with ASD, 64% were classified as Atypi-
Probable difference 14 (28) 2 (3)
cal based on their Oral Sensory Sensitivity scores and 66%
Definite difference 18 (36) 2 (3)
were classified as Atypical based on their Oral Sensory
Oral sensory over-sensitivity
Over-sensitivity scores. Whereas there was no significant
Typical 17 (34) 53 (91)
difference in mean age between the ASD subgroups for
Atypical 33 (66) 5 (9)
Oral Sensory Sensitivity, those characterized as Typical on
Probable difference 5 (10) 4 (7)
the Oral Sensory Over-sensitivity were slightly older than
Definite difference 28 (56) 1 (2)
those characterized as Atypical [7.5 years (median = 7.9) vs.
13
J Autism Dev Disord
6.2 years (median = 5.5), p = 0.03]. Sex was not significantly 1998; Hazen et al. 2014; Leekam et al. 2007; Nadon et al.
associated with either of these variables. 2011; Rogers et al. 2003; Tomchek and Dunn 2007). In our
In order to evaluate the relationship between typical and study, the majority of children with ASD scored outside the
atypical oral sensory processing with food selectivity and typical range for seven of the nine factors. However, scores
fruit/vegetable variety in children with ASD, we stratified spanned the entire range of performance classifications for
measures of food selectivity among children with Typical the group (typical, probable and definite difference). The
versus Atypical Oral Sensory Sensitivity and Oral Sensory distribution of scores across the three performance catego-
Over-sensitivity (Table 4). Analysis of this association was ries is consistent with data reported by earlier studies, which
limited to children with ASD as the sample size was too supports the increased recognition that children with ASD
small in the Atypical Oral Sensory Sensitivity and Oral are not a homogenous population (Brockevelt et al. 2013;
Sensory Over-sensitivity subgroup among TD children. Tomchek and Dunn 2007; Watling et al. 2001).
Children with ASD who scored in the Atypical vs. Typical The primary objective of this study was to evaluate the
range for Oral Sensory Sensitivity had significantly higher relationship between oral sensory sensitivity and food selec-
levels of food refusal (48.2 vs. 33.2%, respectively, p = 0.01). tivity among children with ASD. We previously reported
Compared to children with ASD who scored in the Typical that children with ASD exhibit more food selectivity than
range for Oral Sensory Over-sensitivity, those who scored their TD peers (Bandini et al. 2010). Various factors can
in the Atypical range had twice the level of food refusal (52 contribute to food selectivity and our aim was to evaluate
vs. 25%, p < 0.001). Fruit variety was significantly lower for the extent that food selectivity relates to atypical oral sen-
children who scored in the Atypical vs. Typical range for sory processing. In the present study, we found that children
Oral Sensory Over-sensitivity (5.3 vs. 8.2, p = 0.003) but with ASD were more likely than TD children to score in the
was not significantly different for those who scored in the Atypical range for both Oral Sensory Sensitivity and Oral
Atypical versus Typical range on the Oral Sensory Sensitiv- Sensory Over-sensitivity. Among children with ASD, those
ity measure. Children who were categorized as Atypical for with Atypical Oral Sensory Sensitivity refused more foods
Oral Sensory Sensitivity and Oral Sensory Over-sensitivity and ate fewer vegetables compared to those with Typical
had significantly lower levels of vegetable variety compared Oral Sensory Sensitivity. Children with ASD with Atypical
to children who scored in the Typical range (4.2 vs. 8.6 and Oral Sensory Over-sensitivity exhibited more food refusal,
4.2 vs. 8.9, p < 0.01), respectively. When age was included a narrower food repertoire, and less variety of fruits and
as a covariate in multivariable linear regression models, the vegetables compared to those with Typical Oral Sensory
statistical significance of the findings remained unchanged. Over-sensitivity. These data suggest that children with ASD
and Oral Sensory Sensitivity, and possibly more so with
Oral Sensory Over-sensitivity, may exhibit food selectivity
Discussion and restrictive eating behaviors. Children with Oral Sensory
Sensitivity may restrict their diet to foods with preferred, tol-
Our results are consistent with findings from other studies erable, or manageable sensory features (Cermak et al. 2010).
that demonstrate that children with ASD had significantly Whole grains, lean protein, fresh fruits, and vegetables are
greater Oral Sensory Sensitivity or Oral Sensory Over-sen- foods that are nutrient-dense, but often characterized by
sitivity compared to children without ASD (Ermer and Dunn strong flavors and textures; our data support the observation
Table 4 Food Selectivity Among Children with ASD with Typical vs. Atypical Oral Sensory Sensitivity and Taste/Smell Sensitivity
Oral sensory sensitivity Oral sensory over-sensitivity
a
Typical (n = 18) Atypical (n = 32) p value Typical (n = 17) Atypical (n = 33) p valuea
Mean (SD) Mean (SD) Mean (SD) Mean (SD)
Food refusalb 33.2 (19.1) 48.2 (20.3) 0.013 24.9 (17.7) 52.0 (16.2) < 0.001
Food repertoirec 19.8 (3.9) 18.3 (5.7) 0.34 20.7 (4.2) 17.9 (5.3) 0.08
Fruit varietyd 7.1 (3.2) 5.9 (3.3) 0.23 8.2 (2.6) 5.3 (3.2) 0.003
Vegetable varietyd 8.6 (5.4) 4.2 (3.6) 0.005 8.9 (4.7) 4.2 (4.0) < 0.001
a
p value from independent samples t-test
b
Percentage of foods refused of those offered
c
Number of unique foods consumed from 3-day food record
d
Unique number of fruits/vegetables consumed based on the FFQ, which included 13 fruits and 20 vegetables
13
J Autism Dev Disord
that children with ASD who exhibit oral sensory sensitiv- Our study is one of the first to report a significant associa-
ity may be less likely to accept these types of foods, which tion between oral sensory processing and variety of fruit and
consequently may put them at risk for inadequate nutrition. vegetables consumed by children with ASD. Children with
Our findings are consistent with studies that have shown a ASD exhibiting Oral Sensory Over-sensitivity were found
correlation between sensory sensitivity and food selectivity, to consume significantly less variety of fruits and vegetables
although prior studies have been limited by the methodol- compared to children with ASD without Oral Sensory Over-
ogy for measurement of food selectivity. Using the Sensory sensitivity. Fruits and vegetables are important components
Profile, Kral et al. (2015) reported that children with ASD of a healthy diet and provide key nutrients for growth and
had significantly higher levels of oral sensory sensitivity development. Previous studies have reported that children
compared to TD children, and, among children with ASD, with ASD consume fewer fruits and vegetables overall (Ban-
those with atypical oral sensory sensitivity had signifi- dini et al. 2010; Emond et al. 2010; Johnson et al. 2008;
cantly greater food neophobia, greater food fussiness, and Martins et al. 2008; Sharp et al. 2013; Suarez and Crinion
increased under-eating due to negative emotions compared 2015), and authors have speculated that avoidance of fruits
to children with ASD and typical oral sensory sensitivity. and vegetables may be related to their flavor, texture, and/
However, food selectivity was based on parent responses or consistency (Cermak et al. 2010; Schreck et al. 2004;
to questions on the Child Eating Behavior Questionnaire Suarez et al. 2014). However, few studies have quantified the
(CEBQ) rather than a quantitative measure of food selectiv- association between sensory sensitivity and the consumption
ity as was used in our research. Similarly, in a study of 95 of fruit and vegetables, and no studies have measured this
children with ASD ages 3–10 years, Nadon et al. (2011) association in children with ASD. The findings of our study
reported that participants with atypical scores on the Short suggest that efforts to increase consumption of fruits and
Sensory Profile (SSP) for Taste/Smell Sensitivity had sig- vegetables in this population may be enhanced by including
nificantly more eating problems than children with typical strategies that address oral sensory processing.
performance, measured using the parent-reported Eating While our study suggests that there may be sensory pro-
Profile. This tool did not directly capture quantitative meas- cessing factors that manifest as food selectivity in the child
ures of food selectivity but rather, evaluated eating prob- with ASD, additional studies are needed to fully elucidate
lems across 6 domains focusing on developmental eating the nature of the relationship. Several studies have proposed
milestones, mealtime behaviors, impact on the daily life of neurobiological mechanisms to explain how oral sensory
the family and feeding problems. Lane et al. (2014) also sensitivity may result in food selective behaviors. Tavassoli
found Taste/Smell Sensitivity as measured with the SSP to and Baron-Cohen (2012) reported differences in taste iden-
be significantly associated with limited variety (r = − 0.73, tification as measured using taste strips between adults with
P < 0.05) and food refusal (r = 0.46, P < 0.01), which were ASD and a control group. Bennetto et al. (2007) found that
measured with the Brief Assessment of Mealtime Behav- individuals with ASD perceived olfaction and taste stimuli
ior Inventory (BAMBI). Scores for limited variety, food differently than those without ASD; however, there were
refusal, and autism-specific behaviors at mealtime on the no differences in detection thresholds using stimuli pre-
BAMBI were based on parent-report of 18 eating behavior sented via electrodes on the tongue. Thus, it is possible that
items using a 5-point Likert scale. While Lane et al. (2014) individuals with Oral Sensory Sensitivity may present with
included 3-day food records, these were used to determine differences in the brain that affect their ability to perceive
nutrient intake and not to quantify food selectivity. Suarez taste. However, other factors may influence food selectivity
et al. (2014) conducted one of the few longitudinal studies as well, such as restricted interests and/or behavioral rigid-
on this topic, reporting that higher levels of hypersensitivity ity which are frequently characteristic of persons with ASD
were associated with consuming fewer variety of foods, and (Johnson et al. 2014; Schreck et al. 2004; Suarez et al. 2014).
that this behavior remained stable over the 20-month study Sharp et al. (2014) suggests that there are medical, biologi-
period. However, food selectivity methodology was again a cal, environmental, parental, and behavioral factors at play
limitation as investigators utilized a questionnaire that asked that influence food selectivity in individuals with ASD.
parents to select a category representing how many foods We acknowledge several limitations in the present study.
their child accepts as part of his or her regular diet (i.e. less The study was advertised as a study about mealtime and
than 5, 6–10, 11–20, 21–30, and 30+). In light of the extant physical activity; this may have resulted in a biased sample,
literature and the findings for the current study, there appears as parents of children with feeding difficulties may have been
to be convincing evidence to confirm that children with ASD more interested in participating in the study. We attempted
with more sensory sensitivity demonstrate higher levels of to minimize this potential bias by recruiting children with
food selectivity. Practitioners working with this population and without autism and emphasizing that we were seeking
should be aware of this relationship, and the potential impact children whether or not their parents had concerns in these
this may have on the quality of the child’s diet. areas in our recruitment. Because the present study was
13
J Autism Dev Disord
cross-sectional in design, we cannot establish a causal rela- food and/or stuffing his mouth (Cermak et al. 2010). No
tionship between sensory sensitivity and food selectivity. In studies to date have examined over- versus under-sensitivity
addition, we determined food selectivity based on the modi- to taste/smell stimuli for children with ASD, although pre-
fied FFQ and 3-day food records, which have limitations vious studies have reported varying frequencies of overall
for use that have been previously discussed (Bandini et al. sensory under-sensitivity and over-sensitivity in this popula-
2010). These tools did not allow us to determine if a child tion (Baranek et al. 2006; Ben-Sasson et al. 2009). Further
was not offered a food by the parent based on the child’s pre- study in this area is warranted.
sumed or historical refusal of that food. Fruit and vegetable The present study assessed the relationship between oral
items were disaggregated on the FFQ based on our desire to sensory processing and food selectivity in a moderate-sized
accurately measure refusal of these foods. However, foods sample of children with ASD with an age-matched control
such as mixed-food entrees were kept aggregated to mini- group. To the best of our knowledge, no similar studies have
mize participant burden; this may have introduced error, used direct measures of food intake to determine food selec-
which would likely be non-differential. The FFQ may not tivity. In contrast to earlier studies on this topic, this study
have accurately captured everything each child ate because quantified food selectivity using validated food measures
the assessment was limited to items found on FFQ. This list and operationalized definitions of food refusal and limited
was limited to fruit and vegetables commonly consumed in food repertoire. Additionally, while most studies proceeded
western diets, although there was an opportunity to write in with unverified diagnoses of ASD, our study ensured that
any foods eaten more than once per week. Also, the 3-day ASD was independently verified using the Autism Diagnos-
food record may not adequately capture the repertoire of a tic Interview-Revised (Rutter et al. 2003) a gold-standard
typical diet, and our coding decisions may have affected our assessment of ASD and ASD severity. Use of two measures
estimation of variety, although it is expected that any errors of oral sensory processing ability (Oral Sensory Sensitivity
introduced in this way would have affected both groups and Oral Sensory Over-sensitivity) also allowed us to dif-
similarly. There were also several limitations of the Sensory ferentiate between overall oral sensory sensitivity (includ-
Profile tool used to assess oral sensory sensitivity in the ing items reflecting both over and under-sensitivity) and
study. Identification of sensory sensitivities were based on specifically oral sensory over-sensitivity, which allow for
parent report rather than observed directly, and may have more specificity when characterizing oral sensory process-
been affected by caregiver bias and observational abilities. ing dysfunction in this population.
Studies using direct observation rather than parent-reported
data would likely address the limitations of this methodol-
ogy, however this approach is not as practical for larger- Conclusion
scale studies. This study also did not collect information
on parental food preferences, and thus it remains unknown Our study shows that more children with ASD present with
whether parents’ food choices and preferences had an influ- atypical sensory characteristics compared to children with-
ence on what was served. Parents whose own food repertoire out ASD. Among children with ASD, those with Atypical
is limited may not offer a wide variety of foods to the family, Oral Sensory Sensitivity refused more foods and ate fewer
which would result in limited food repertoires on the part of vegetables compared to those with Typical Oral Sensory
children. However, our measure of food refusal was based on Sensitivity. Further, Atypical Oral Sensory Over-sensitiv-
food refused from those offered. We observed in this study ity, a measure of sensory hyper-sensitivity for taste and
and in our analyses of these data (Bandini et al. 2010; Cur- smell sensory input, was associated with higher rates of
tin et al. 2015) that children with ASD had higher rates of food refusal, a narrower food repertoire and consumption
food refusal than TD children. In this study, food refusal was of fewer unique fruits and vegetables. Early identification
positively and significantly associated with oral sensory sen- of and intervention for sensory processing abnormalities
sitivities. Finally, lack of representation by a wider range of in children with ASD is critical for addressing problem-
racial/ethnic groups of varying socio-economic status limits atic behaviors associated with eating and mealtimes. These
the generalizability of our findings. findings suggest that children with ASD who have Atypical
Although the current study did not assess oral sensory Oral Sensory Sensitivity and food selectivity may benefit
under-sensitivity, delineating differences between over- and from working with a multidisciplinary team of specialists,
under-sensitivity to oral stimuli may provide insight into including speech pathologists, occupational therapists, and
particular food selective eating behaviors in children with dietitians to improve sensory experiences related to eating
ASD. For example, oral over-sensitivity may result in diffi- and to increase adequacy and variety of the diet. Strategies
culty with textures while oral under-sensitivity, in which the may include changing the texture and consistency of foods
child does not appear to adequately perceive sensations, may to more manageable sensory characteristics, and using a sen-
result in the child consuming large amounts of a particular sory integration approach to decrease sensory sensitivity.
13
J Autism Dev Disord
More importantly, treatment plans should be individualized Baranek, G. T., David, F. J., Poe, M. D., Stone, W. L., & Watson, L.
to the unique sensory characteristics of each child. Future R. (2006). Sensory Experiences Questionnaire: Discriminating
sensory features in young children with autism, developmental
areas of study should focus on the relationship between oral delays, and typical development. Journal of Child Psychology
sensory sensitivity and neurobiological factors and the spe- and Psychiatry, 47(6), 591–601.
cific sensory characteristics of food, as well as differentiate Bennetto, L., Kuschner, E. S., & Hyman, S. L. (2007). Olfaction
between sensory over- and under-sensitivity. Direct observa- and taste processing in autism. Biological Psychiatry, 62(9),
1015–1021.
tions of sensory processing ability may address the limita- Ben-Sasson, A., Hen, L., Fluss, R., Cermak, S. A., Engel-Yeger, B.,
tions of using a parent-reported tool. Longitudinal studies & Gal, E. (2009). A meta-analysis of sensory modulation symp-
are needed to fully elucidate the nature of the relationship toms in individuals with autism spectrum disorders. Journal of
between sensory sensitivity and food selectivity in children Autism and Developmental Disorders, 39(1), 1–11.
Brockevelt, B. L., Nissen, R., Schweinle, W. E., Kurtz, E., & Larson,
with ASD and how it persists over time. K. J. (2013). A comparison of the Sensory Profile scores of
children with autism and an age- and gender-matched sample.
Funding This research was funded by the following Grants: South Dakota Medicine, 66(11), 459, 461, 463–455.
R21HD048989-01A2 (NICHD; Diet, Activity and Obesity in Children Cermak, S. A., Curtin, C., & Bandini, L. G. (2010). Food selectivity
with Autism); UA3MC25735-01-00 (MCHB; MCH Research Network and sensory sensitivity in children with autism spectrum dis-
on Promoting Healthy Weight (HWRN) among Children with Autism orders. Journal of the American Dietetic Association, 110(2),
Spectrum Disorders (ASD) and other Developmental Disabilities); 238–246.
2P30HD004147-33A2 (NICHD; Interdisciplinary Research in Intellec- Christensen, D. L., Baio, J., Van Naarden Braun, K., Bilder, D.,
tual & Developmental Disabilities); and P30DK046200 (NIH; Boston Charles, J., Constantino, J. N., et al. (2016). Prevalence and
Nutrition Obesity Research Center). characteristics of autism spectrum disorder among children
aged 8 years–autism and developmental disabilities monitor-
Author Contributions LTC and LGB conceived of the study with ing network, 11 sites, United States, 2012. MMWR. Surveillance
clinical and scholarly input from CC and SAC. SP performed the sta- summaries: Morbidity and mortality weekly report. Surveillance
tistical analyses with input/consultation from AM. All authors partici- summaries/CDC, 65(3), 1–23.
pated in the writing of the manuscript. CC led the effort to respond to Curtin, C., Hubbard, K., Anderson, S. E., Mick, E., Must, A., &
the reviewers’ critiques and revised the manuscript accordingly, with Bandini, L. G. (2015). Food selectivity, mealtime behavior
input and approval from all the other authors. problems, spousal stress, and family food choices in children
with and without autism spectrum disorder. Journal of Autism
Compliance with Ethical Standards and Developmental Disorders, 45(10), 3308–3315.
Dunn, W. (1999). The sensory profile: User’s manual. San Antonio,
TX: The Psychological Corporation.
Conflict of interest The authors declare that they have no conflict Dunn, W. (2001). The sensations of everyday life: Empirical, theo-
of interest. retical, and pragmatic considerations. The American Journal of
Occupational Therapy, 55(6), 608–620.
Ethical Approval All procedures performed in studies involving Emond, A., Emmett, P., Steer, C., & Golding, J. (2010). Feeding
human participants were in accordance with the ethical standards of symptoms, dietary patterns, and growth in young children with
the institutional and/or national research committee and with the 1964 autism spectrum disorders. Pediatrics, 126(2), e337.
Helsinki declaration and its later amendments or comparable ethical Ermer, J., & Dunn, W. (1998). The sensory profile: A discriminant
standards. analysis of children with and without disabilities. The American
Journal of Occupational Therapy, 52(4), 283–290.
Informed Consent Informed consent was obtained from all indi- Hazen, E. P., Stornelli, J. L., O’Rourke, J. A., Koesterer, K., &
vidual participants included in the study. McDougle, C. J. (2014). Sensory symptoms in autism spec-
trum disorders. Harvard Review of Psychiatry, 22(2), 112–124.
Hubbard, K. L., Anderson, S. E., Curtin, C., Must, A., & Bandini,
L. G. (2014). A comparison of food refusal related to charac-
teristics of food in children with autism spectrum disorder and
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nutrients
Article
Comprehensive Nutritional and Dietary Intervention
for Autism Spectrum Disorder—A Randomized,
Controlled 12-Month Trial
James B. Adams 1, * ID , Tapan Audhya 2 , Elizabeth Geis 1 , Eva Gehn 1 , Valeria Fimbres 1 ,
Elena L. Pollard 1 , Jessica Mitchell 3 , Julie Ingram 1 , Robert Hellmers 4 , Dana Laake 5 ,
Julie S. Matthews 6 , Kefeng Li 7 , Jane C. Naviaux 7 , Robert K. Naviaux 7 , Rebecca L. Adams 1 ,
Devon M. Coleman 1 ID and David W. Quig 8
1 Arizona State University, School for Engineering of Matter, Transport & Energy, Tempe, AZ 85287, USA;
autismstudynurseasu@gmail.com (E.G.); ecgehn@gmail.com (E.G.); Valeria.Fimbres@asu.edu (V.F.);
epollard1025@gmail.com (E.L.P.); julieaingram@yahoo.com (J.I.); thebeckyadams@gmail.com (R.L.A.);
devon.coleman@asu.edu (D.M.C.)
2 Health Diagnostics, South Amboy, NJ 08879, USA; audhyatk@optonline.net
3 Southwest College of Naturopathic Medicine, Tempe, AZ 85282, USA; J.Mitchell@scnm.edu
4 Arizona Allergy Associates, Phoenix, AZ 85004, USA; rhellmers@aol.com
5 Dana Laake Nutrition, Kensington, MD 20895, USA; danalaake@aol.com
6 Nourishing Hope, San Francisco, CA 94117, USA; julie@NourishingHope.com
7 University of California, The Mitochondrial and Metabolic Disease Center, San Diego, CA 92093, USA;
kli@ucsd.edu (K.L.); jnaviaux@ucsd.edu (J.C.N.); naviaux@ucsd.edu (R.K.N.)
8 Doctor’s Data, St. Charles, IL 60174, USA; dquig@DoctorsData.com
* Correspondence: jim.adams@asu.edu; Tel.: +1-480-965-3316
Received: 30 January 2018; Accepted: 10 March 2018; Published: 17 March 2018
Abstract: This study involved a randomized, controlled, single-blind 12-month treatment study
of a comprehensive nutritional and dietary intervention. Participants were 67 children and adults
with autism spectrum disorder (ASD) ages 3–58 years from Arizona and 50 non-sibling neurotypical
controls of similar age and gender. Treatment began with a special vitamin/mineral supplement,
and additional treatments were added sequentially, including essential fatty acids, Epsom salt baths,
carnitine, digestive enzymes, and a healthy gluten-free, casein-free, soy-free (HGCSF) diet. There was
a significant improvement in nonverbal intellectual ability in the treatment group compared to the
non-treatment group (+6.7 ± 11 IQ points vs. −0.6 ± 11 IQ points, p = 0.009) based on a blinded
clinical assessment. Based on semi-blinded assessment, the treatment group, compared to the
non-treatment group, had significantly greater improvement in autism symptoms and developmental
age. The treatment group had significantly greater increases in EPA, DHA, carnitine, and vitamins
A, B2, B5, B6, B12, folic acid, and Coenzyme Q10. The positive results of this study suggest
that a comprehensive nutritional and dietary intervention is effective at improving nutritional
status, non-verbal IQ, autism symptoms, and other symptoms in most individuals with ASD.
Parents reported that the vitamin/mineral supplements, essential fatty acids, and HGCSF diet
were the most beneficial.
Keywords: autism; autism spectrum disorder; vitamins; minerals; essential fatty acids; carnitine;
Epsom salts; digestive enzymes
1. Introduction
Many studies have demonstrated that children and adults with ASD often have significant
nutritional deficiencies, metabolic imbalances, and digestive problems. Several nutritional and dietary
Nutrients 2018, 10, 369; doi:10.3390/nu10030369 www.mdpi.com/journal/nutrients

Nutrients 2018, 10, 369 2 of 43
treatment studies have demonstrated benefits in treating these underlying conditions [1–3]. In the
following sections we discuss specific research related to vitamins, minerals, essential fatty acids,
mitochondrial disorders/carnitine issues, and gastrointestinal disorders (digestive problems and food
sensitivities).
1.1. Vitamins/Minerals
Several studies suggest that customized vitamin/mineral supplementation is beneficial for
children with ASD. Three studies have demonstrated that children with ASD have impaired
methylation, decreased glutathione, and increased oxidative stress [4–6]. Those studies demonstrated
that nutritional supplementation (with methyl-B12, folinic acid, and trimethylglycine) is beneficial.
Several other studies have also demonstrated increased oxidative stress [7–12]. Methylation is
important because that controls epigenetics, and there is evidence that there are many differentially
methylated regions in the brains of children with ASD vs. controls [13].
In 2008/2009 we conducted an extensive comparison of the nutritional and metabolic status of
children with ASD (n = 55) compared to neurotypical children of similar age and gender (n = 44) [14].
Study measurements included vitamins, biomarkers of vitamin status, minerals, plasma amino acids,
plasma glutathione, neurotransmitters, and biomarkers of oxidative stress, methylation, sulfation and
energy production. Many statistically significant differences (p < 0.001) were observed in the ASD
group compared to the neurotypical group, including: low levels of biotin, glutathione, methylation
status (S-adenosylmethionine (SAM) and uridine), ATP, NADH, NADPH, sulfate (free and total),
tryptophan, and GABA; also, high levels of oxidative stress markers and plasma glutamate.
That study was followed by a three-month randomized, double-blind, placebo-controlled
treatment study involving a customized vitamin/mineral supplement [15]. The supplement was found
to be well-absorbed and result in many significant improvements in metabolic status, including SAM,
reduced glutathione, ratio of oxidized glutathione to reduced glutathione (GSSG:GSH), nitrotyrosine,
ATP, NADH, and NADPH. Most of these metabolic biomarkers improved to normal or near-normal
levels. However, although free and total plasma sulfate levels improved, they remained below
normal, suggesting that additional treatments are needed to fully normalize sulfation. That study also
found that the supplement group had significantly greater improvements than the placebo group on
autism-related symptoms on the Parental Global Impressions-Revised Average Change (p = 0.008),
and on the subscores for Hyperactivity (p = 0.003), Tantrumming (p = 0.009), Overall (p = 0.02),
and Receptive Language (p = 0.03).
1.2. Essential Fatty Acids

Several polyunsaturated fatty acids (PUFAs) are either essential or conditionally essential, including
several omega-3 and omega-6 fatty acids. Meta-analyses of many studies have demonstrated that
omega-3 levels are decreased in certain psychiatric disorders including schizophrenia (meta-analysis of
14 studies) [16], ADHD (9 studies) [17]), depression (14 studies) [18], bipolar disorder (six studies) [19],
and dementia (10 studies) [20].
Meta-analyses of many clinical trials have demonstrated benefits of supplementation with omega-3
PUFA’s for schizophrenia (10 trials) [21], ADHD (16 trials) [17], major depression (12 trials) [22],
bipolar depression (5 trials) [23], and possibly dementia (eight of 13 trials positive) [24]. Meta-analyses
findings indicated that EPA was more beneficial than DHA, and that higher levels of EPA were more
beneficial. There is also one study [25] that found omega-3 fatty acid supplementation is very helpful for
infants with Rett’s syndrome, a disorder which often includes autistic symptoms.
PUFA’s may also play a role in some gastrointestinal problems, since they are important for
intestinal membrane function. One epidemiological study found that increasing incidence of Crohn’s
disease correlated very strongly (r = 0.79) with low levels of omega-3 fatty acids [26]. A one-year,
double-blind, placebo-controlled trial of fish oil (2.7 g/day of omega-3 fatty acids) in people with
Crohn’s disease found that subjects taking the fish oil had a significantly reduced relapse rate, with no
Nutrients 2018, 10, 369 3 of 43
significant adverse effects [27]. Gastrointestinal problems are common in ASD [14,28], and PUFA
supplementation may be beneficial for reducing some gastrointestinal problems in children with ASD.
A meta-analysis [29] of fifteen case-control studies (n = 1193) found that, compared with typically
developed individuals, the ASD group had lower eicosapentaenoic acid (EPA), docosahexaenoic acid
(DHA) and arachidonic acid (AA), and a lower ratio of total omega-3 to total omega-6 fatty acids;
these differences were primarily found in studies with children, and not in studies with adolescents or
adults. A meta-analysis [29] of four small randomized controlled trials (n = 107) [30–33] found that
compared with placebo, omega-3 fatty acid supplementation improved social withdrawal (p < 0.02)
and restricted interests and behaviors (p = 0.05), but did not have a significant effect on communication,
irritability, or hyperactivity (all rated per the Aberrant Behavior Checklist). These studies only lasted
6–16 weeks, so were too short to observe full effect, since omega-3 supplementation requires about six
months to reach steady-state levels in erythrocytes, and about 1–1.5 months for half of that change
to occur [34]. These studies used doses of 0.5–1.5 g/day of omega-3 fatty acids. Two other small
randomized studies [35,36] not included in the meta-analysis [29] did not find significant effects on
symptoms despite longer duration (6 months), possibly due to small sizes (under 35 participants
completed each study) or low dose (200 mg DHA) in one study [36].
Overall, it appears that omega-3 fatty acids are decreased in ASD, and that supplementation may
be helpful. Higher doses and longer treatment may result in greater benefit. For example, a treatment
study for Crohn’s disease found that long-term treatment (12 months) may be needed for improvement
in gastrointestinal problems. We hypothesize that children with ASD who do not regularly eat seafood
(the major source of omega-3 fatty acids in most western diets) are more likely to benefit from fish
oil supplementation.
1.3. Sulfate
Sulfur is the fourth most common mineral in the body [37]. Most sulfate is produced in vivo
by metabolism of cysteine [14]. Sulfation is important for many reactions including detoxification,
inactivation of catecholamines, synthesis of brain tissue, sulfation of mucin proteins which line
the gastrointestinal tract, and more. Low free and total plasma sulfate in children with ASD has
been previously reported in three studies [14,38,39], and is consistent with four studies [38,40–42]
which found that children with ASD, compared to controls, had a significantly decreased sulfation
capacity, based on decreased ability to detoxify paracetamol (acetaminophen). The finding of low
plasma sulfate is also consistent with a large study that found high sulfate in the urine of children
with ASD [43], as sulfate wasting in the urine partly explains low levels in the plasma. ATP is
required for the kidneys to resorb sulfate, and one study [14] found that plasma ATP was low in
children with ASD and moderately correlated with levels of free and total plasma sulfate (r = 0.32
and 0.44, respectively), suggesting, that low levels of ATP are a contributor to decreased sulfate in
children with ASD. One study [43], also reported high levels of urinary sulfite in children with ASD,
suggesting that there was a problem of converting sulfite to sulfate in the mitochondria. In 38% of cases
(14/38) urinary sulfite and sulfate levels improved by giving 50 mcg of molybdenum, based upon
molybdenum dependence of the enzyme necessary for converting sulfite to sulfate (sulfite oxidase).
Another study [15] found that a vitamin/mineral supplement (containing molybdenum) was able to
improve, but not normalize, free and total plasma sulfate in children with ASD. Overall, these studies
suggest that sulfate is low in children with ASD, and that vitamin/mineral supplementation is helpful
but additional sources of sulfate (such as Epsom salt baths) are needed.
1.4. Carnitine and Mitochondrial Disorders

Carnitine is a conditionally essential nutrient that is vital in energy production and fatty acid
metabolism. Carnitine carries long-chain fatty acids (fuel) into the mitochondria, and it also carries
potentially toxic organic acids out of the mitochondria and cell so they can be eliminated from
the body. Several studies have suggested that mitochondrial disorders are common in children
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with ASD [44–48]. Note that the term “mitochondrial disorders” is used to denote a generalized
impairment of mitochondrial function, and are generally not as severe as “mitochondrial diseases”,
which involve specific severe genetic abnormalities. One study found decreased levels of carnitine in
children with ASD [49]. A recent double-blind, placebo-controlled 3-month study (n = 30) found that
supplementation with carnitine was beneficial [50]. Specifically, the study found significantly greater
improvements in the Childhood Autism Rating Scale 2 (CARS-2) and Clinical Global Impressions
(CGI) scores in the treatment group compared to the placebo group. In addition, scores significantly
improved in cognition and marginally in speech on the Autism Treatment Evaluation Checklist (ATEC).
L-carnitine therapy significantly increased serum carnitine concentrations, and significant correlations
between changes in serum free-carnitine levels and positive clinical changes were observed. L-carnitine
therapy was generally well-tolerated by study subjects. A second study [51] found similar results.
Overall, the literature suggests that mitochondrial disorders are common in ASD, and that a
combination of therapy with vitamins, minerals, CoEnzyme Q10, essential fatty acids, and L-carnitine
may be helpful in improving mitochondrial function.
1.5. Gastrointestinal Problems, Digestive Enzymes, Limited Diets, and Food Sensitivities
Gastrointestinal problems are common in children with ASD, especially chronic constipation,
diarrhea, abdominal pain, and gastrointestinal inflammation [28,52]. A study by our group [53] found
that those problems are strongly correlated to autism severity (r = 0.59, p < 0.001), suggesting that it
is important to investigate them. As reported, in those with ASD, gastrointestinal problems appear
to be partly due to deficiencies in digestive enzymes, partly due to food sensitivities, and possibly
(as discussed above) due to low levels of omega-3 fatty acids, which in turn, could result in abnormal
gut bacteria [54]. One large study by Horvath et al. evaluated disaccharidase activity from endoscopic
biopsies in 90 children with ASD. They found that 49% had at least one deficient enzyme activity,
and 20% had deficiencies in two or more disaccharidase enzymes “Lactase and maltase deficiencies were
the most frequent, followed by low activity of sucrase, palatinase, and glucoamylase. All of the children
with low enzyme activity had loose stools and/or gaseousness”. Another large study [55] involving
intestinal biopsy samples of 199 children and adults with ASD (ages 22 months to 28 years) found that
many had deficiencies in disaccharidases (enzymes for digesting simple sugars). Specifically, they found
that 62% had deficiencies in lactase, 16% were deficient in sucrase, and 10% were deficient in maltase.
The problems seemed to be equally common in children and adults, suggesting that these problems
are lifelong. An open-label treatment study of 46 children and adults with ASD reported a wide
range of benefits from the use of digestive enzymes [56], but results of randomized controlled trials
are mixed [57,58]. Some studies have suggested that children with ASD have poor diets, leading to
decreased intake of key nutrients [59,60].
Several studies have found that children with ASD have abnormal immune responses to certain
foods, especially glutens (in wheat, rye, barley, oats) and casein (in dairy products) and sometimes
soy. One study [61] found that many children with ASD have food sensitivities. Four studies [62–65]
found that children with ASD had more hypersensitivities to food allergens than did typical children,
and may be related to increased intestinal permeability [65,66]. A large study of 150 children with
ASD found that 87% had IgG antibodies (sensitivity) to gluten, vs. 1% of the age and gender-matched
controls, and 90% had IgG antibodies to casein, vs. 7% of the controls [67].
Several studies suggest that special diets can be beneficial for individuals with ASD. One open-label
study [68] found that an 8-week diet which avoided allergic foods resulted in benefits in an open study
of 36 children with ASD. One long-term open-label study of 70 children with ASD who followed a
gluten-free, casein-free diet for one year or longer found that 81% improved significantly by the third
month, with improvements continuing over the next 12 months. Large improvements were observed
in social isolation, eye contact, mutism, learning skills, hyperactivity, stereotypic activity, and panic
attacks [67]. A single-blind study of 10 children with autism found that 8 benefitted from a gluten-free,
casein-free (GFCF) diet [69]. A 12-week, double-blind, cross-over study of a GFCF diet in 15 children
Nutrients 2018, 10, 369 5 of 43
with ASD did not find significant benefits, but parents reported benefits that were not identified by
the testing [70]. However, a 12-month, randomized, single-blind, placebo-controlled GFCF diet study
involving 54 children with ASD found statistically significant benefits in communication subscores
(Autism Diagnostic Observation Schedule (ADOS) evaluation) in the GFCF diet group compared to
the control group [71]. The parents (who were not blinded) also reported benefits in social interaction,
daily living skills, inattention, and hyperactivity.
Overall, these studies suggest that children with ASD often have deficiencies in lactase and other
digestive enzymes, may have poor diets, often have food sensitivities, especially to gluten and casein, and
hence may benefit from digestive enzymes, healthier diets, and/or gluten-free, casein-free diets [65,66].
1.6. Study Goal

The goal of this study is to investigate a comprehensive nutritional and dietary intervention to
treat children and adults with ASD. Each of these treatments have been previously studied individually
and found to have some benefit, mostly in short-term studies. The goal of this study is to investigate the
effect of the combination of those treatments in a long-term study. Combinations of these treatments are
commonly used for treating children and adults with ASD, their effects are expected to be synergistic,
and longer-term treatment may lead to greater benefits. An unusually wide age range was used in this
study because this study was funded primarily by families participating in our annual walk fundraiser,
and they have requested treatment studies that address all ages. This study is not designed to look
at the effect of individual treatments (most of them have already been investigated individually);
rather the goal is to investigate the effect of a combination of treatments which provide comprehensive,
synergistic nutritional support.
1.7. Hypothesis
A combination of nutritional and dietary interventions will be effective in reducing the symptoms
of autism, reducing gastrointestinal problems, and increasing overall functioning level.
2. Methods
2.1. Study Design and Justification

This was a one-year, single-blinded study, involving a treatment group and a non-treated group
of children and adults with ASD. Single-blind means that the clinical evaluators were blinded, but
the participants were not. The reason for this design is that we wanted to conduct a long-term
study on the effects of a comprehensive set of dietary and nutritional treatments, similar to what
the Autism Research Institute has recommended for years [72]. We believe that length of time is
too long for a double-blind, placebo-controlled format, because too many participants would drop
out. Also, it is extremely difficult to do a double-blind study when making major diet changes,
especially the switch to a “healthy” diet as described below. A single-blind study is still a rigorous
study design for assessments for which the clinical observer is fully blinded, and the additional data
from unblinded participants provides some useful information similar to an open-label study. Since
this was an exploratory new treatment, the choice of sample size was roughly estimated based on
previous studies. The authors confirm that all ongoing and related trials for this drug/intervention are
registered (Clinicialtrials.gov: NCT02059577).
2.2. Participant Enrollment

The study was advertised by email to approximately 2500 ASD families in Arizona, using the
contact list of the Autism Society of Greater Phoenix and the Autism/Asperger’s Research Program at
Arizona State University (ASU). Interested ASD families attended a one-hour informational meeting,
and consenting families joined the study. Neurotypical families were recruited from friends of the
ASD families and professionals who work with ASD families. Participants were recruited for the study
Nutrients 2018, 10, 369 6 of 43
from October 2011 to April 2014. All subjects gave their informed consent for inclusion before they
participated in the study. The study was conducted in accordance with the Declaration of Helsinki,
and the protocol was approved by the Institutional Review Board (IRB) of Arizona State University.
2.3. Enrollment Criteria—ASD Group

1. Diagnosis of autism spectrum disorder (autism, Pervasive Developmental Disorder-Not Otherwise
Specified (PDD-NOS), or Asperger’s) by a psychiatrist, psychologist, or developmental pediatrician.
2. Verification of diagnosis by ASU staff based on the ADOS and/or CARS-2.
3. Age of 2.5–60 years.
4. No major changes in behavioral or medical treatments in the previous two months, and no
intention to make such changes during the 12 months of the study.
5. No usage of nutritional supplements (vitamins, minerals essential fatty acids, carnitine) or special
diets in the previous two months.
2.4. Enrollment Criteria—Neurotypical Group

1. No diagnosed mental disorders, including autism spectrum disorders, Attention Deficit Hyperactivity
Disorder (ADHD), depression, anxiety, etc.
2. No first-degree relatives of individuals with ASD (no siblings or parents).
3. Age of 2.5–60 years.
4. No usage of nutritional supplements (vitamins, minerals, essential fatty acids, carnitine) or special
diets in the previous two months.
Note that there was no exclusion for individuals with ASD with specific metabolic or genetic
disorders. No participants reported unusual genetic or metabolic disorders, but there was no attempt
to screen for those, and it is likely that some may have existed.
2.5. Participants
The characteristics of the study participants are listed in Table 1. The ASD treatment group,
ASD non-treatment group, and the neurotypical controls have similar age distributions (mostly children,
some teens, and a few adults), and similar gender distributions (mostly male). The ASD treatment and
non-treatment group have similar diagnoses (mostly autism). For the ASD group, 100% met the criteria
for ASD per the CARS-2, and 88% met the criteria for ASD per the ADOS (most of the 8 participants
who met only the CARS-2 criteria were high-functioning teens/adults who in the clinical judgement of
the evaluator were clearly on the ASD spectrum, so they were admitted to the study).
Table 1. Participants.
ASD–Treatment ASD–Non–Treatment Neurotypical

Total Participants 37 30 50
Male 30 (81%) 25 (83%) 41 (82%)
Female 7 (19%) 5 (17%) 9 (18%)
Age (years) 10.8 ± 7.0 12.3 ± 10.1 12.2 ± 7.5
Children (ages 3–12) Children n = 28 (76%) Children n = 20 (67%) Children n = 34 (68%)
Teens (ages 13–20) Teens n = 6 (16%) Teens n = 7 (23%) Teens n = 11 (22%)
Adults (ages 20+) Adults n = 3 (8%) Adults n = 3 (10%) Adults n = 5 (10%)
Autism = 29 (83%) Autism = 21 (70%)
Diagnosis Asperger’s = 3 (9%) Asperger’s = 5 (17%)
PDD-NOS = 3 (9%) PDD-NOS = 4 (13%)
Regressive = 13 (36%) Regressive = 9 (31%)
Autism Onset Plateau = 8 (22%) Plateau = 10 (34%)
Early Onset = 15 (42%) Early Onset = 10 (34%)
Asthma 9 (25%) 8 (27%) 8 (16%)
Food Allergies 13 (36%) 3 (10%) 2 (4%)
Other Allergies 19 (51%) 13 (43%) 15 (30%)
Other Health Issues—frequency 15 (41%) 14 (47%) 2 (4%)
Other Health Issues—description (note: these
ADHD-4; sensory problems-3; intellectual disability-2; ADHD-6, cerebral palsy-3, hypotonia-2, learning
are likely under-reported since we only asked
seizures; early puberty; vascular malformation; mood disability-2, depression-2, dysphagia, sensory disorder,
a general question about “other health nocturnal enuresis; Hashimoto’s thyroiditis
disorder; spinal fusion; agenesis of lung; gastritis; reflux, seizures, sleep disorder, sexual prematurity,
conditions”, and some of these symptoms
eczema; apraxia; type 2 diabetes type 1 diabetes, OCD, anxiety
might be viewed as part of autism)
Medications (participants taking one or more allergy-6; psych-4; asthma-3; seizure-2; sleep-2; psych-8; allergy-4; seizure-2; thyroid-2; sleep-2; blood
Allergies-2; asthma-1; thyroid-1
of the different types of medications) diabetes-1; cholesterol-1; laxative-1 pressure-1; diabetes-1; acne-1
Nutrients 2018, 10, 369 7 of 43
2.6. Randomization
After enrollment and ADOS/CARS-2/Reynolds Intellectual Assessment Scales (RIAS)/Severity
of Autism Scale (SAS-Pro) assessment, participants were randomly assigned to either the Treatment
or Non-treatment group. The study coordinator enrolled participants, conducted the randomization,
and assigned participants to interventions. She was not involved in any of the evaluations.
The Treatment group began treatment immediately, whereas the Non-treatment group was asked
not to make any changes in medical, nutritional, therapy, or education treatment for 12 months.
The Non-treatment group was promised that they would receive all the supplements and diet advice
at the end of the study if they made no major changes to any educational interventions for 12 months,
which helped minimize the drop-out rate. Participants were enrolled on a rolling basis, and participants
with similar ages were matched and then randomly assigned to one of the two groups.
2.7. Protocols
2.7.1. Protocol for ASD Treatment Group

Initial evaluation of autism severity and overall functioning level.
Physical examination by the study physician to verify that the participant is in sufficient good
health to participate in the study. Initial blood draw and first-morning urine collection.
Day 0: Vitamin/Mineral supplementation begins.
Day 30: Essential Fatty Acid supplementation begins.
Day 60: Epsom salt baths begin.
Day 90: Carnitine Supplementation begins.
Day 180: Digestive Enzyme supplementation begins.
Day 210: Healthy, casein-free, gluten-free diet begins.
Day 365: Final assessment of autism severity and overall functioning status. Final blood draw
and urine collection.
2.7.2. Protocol for ASD Non-Treatment Group

Initial evaluation of autism severity and overall functioning level.
Physical examination by study physician to verify that participant is in sufficient good health to
participate in the study. Initial blood draw and first-morning urine collection.
Day 365: Verification of no changes in treatment during last 12 months. Final assessment of
autism severity and overall functioning status. Final blood draw and urine collection.
2.7.3. Protocol for Neurotypical Group

Physical examination by study physician to verify that participant is in sufficient good health to
participate in the study. Initial blood draw and first-morning urine collection.
2.8. Biomarker Measurements

Biomarkers in blood and urine were measured at the beginning and end of the study in the
children and adults in the ASD groups, and one time at the start of the study in the neurotypical group.
The samples were sent in a blinded fashion to the laboratories for testing.
Some testing was done by LabCorp, and some by Doctor’s Data. Both commercial laboratories
are approved by the Clinical Laboratory Improvement Amendments (CLIA) program operated by the
US Department of Health and Human Services which oversees approximately 200,000 laboratories in
the US. Samples were taken by courier from our medical office to the local LabCorp testing facility in
Phoenix (refrigerated or on dry ice as appropriate). Samples for Doctor’s Data were shipped overnight,
either with cold packs or on dry ice as appropriate.
Nutrients 2018, 10, 369 8 of 43
LabCorp conducted standard measurements of blood chemistry, Complete Blood Count (CBC)
with differential, ammonia, lactic acid, creatine kinase, and a thyroid panel (TSH, T3, T4).
Doctor’s Data conducted measurements of Red Blood Cell (RBC) elements and urinary iodine
using the same methods as reported in a previous study [14]. Doctor’s Data also measured RBC fatty
acids, C-Reactive Protein, and homocysteine-related metabolites (homocysteine, cysteine, methionine).
RBC fatty acids were measured by gas chromatography using a flame ionization detector. Red Blood
Cells were washed and derivatized to their methyl esters and extracted and separated according to
carbon number.
• High sensitivity C-Reactive Protein was measured using an immunoturbidimetric method using
Kamiya Reagents and analyzed on a Beckman Coulter AU680 Chemistry Analyzer (Brea, CA, USA)
• Homocysteine was measured by LC/MS after reduction using dithiothreitol and derivatization.
Vitamins and carnitine were measured at UC-San Diego by some of our team using liquid
chromatography tandem mass spectrometry (LC-MS/MS) (SCIEX, Redwood City, CA, USA) as previously
described with modifications [73] (PMID 25705365). The absolute concentrations of acylcarnitines were
calculated using stable isotope internal standards. The levels of vitamin and co-factors were normalized
using neurotypical control baseline values and reported as peak area ratios.
2.9. Handgrip Strength

Handgrip strength, an indicator of muscle strength, was assessed by using a pneumatic, adjustable
squeeze pinch-gauge/dynamometer (Baseline Evaluation Instruments; White Plains, NY, USA) by
a study nurse unaware of the treatment status of the subject. This instrument is a reliable and valid
method for obtaining muscle force measurements in children and adults, and takes only a few min.
One of three bulb sizes was used, depending on the participant’s hand size, and the same size was
used at beginning and end of the study. Each participant was shown how to squeeze the bulb with one
hand, and then three measurements were taken, and the highest value was recorded.
2.10. Autism Severity and Overall Functioning Assessments

A large number of assessments of autism severity and overall functioning were used because
we hypothesized that there might be improvements in many different areas. Most assessments were
conducted for the autism treatment and non-treatment groups at the beginning and end of the study.
The PGI-2 was assessed at month, 3, 6, 9, and 12 for the treatment group, and at month 12 for the
non-treatment group. The initial assessments were conducted before randomization, so neither the
participants nor the evaluator knew which group they were in. At the end of the study, the evaluator
first conducted the ADOS and RIAS assessments in a blinded manner. The evaluator then conducted
the CARS-2 and SAS-Pro assessments, which did involve some discussion with the parents (except for
a few high-functioning participants who did not have a parent available). The Vineland was conducted
by phone by a different blinded evaluator. The blinding of the evaluators at the beginning and end of
the study was complete in all cases except for one CARS-2/SAS-Pro assessment at the end of the study
(a participant inadvertently commented about the treatment).
Autism Diagnostic Observation Schedule (ADOS): The ADOS is a 1-h structured interaction
and is one of the primary tools used for clinical diagnosis of autism and autism spectrum disorders.
It involved a blinded evaluation by clinicians certified in ADOS assessment.
Reynolds Intellectual Assessment Scales (RIAS): The RIAS assesses verbal and non-verbal IQ and
memory. It involves a 20–30 min blinded assessment by clinicians using a variety of standardized IQ
and memory activities.
Childhood Autism Rating Scale 2 (CARS-2): The CARS-2 evaluation was conducted after the
ADOS and RIAS evaluations, and was based partly on the participant’s performance and interactions
on those assessments, and partly on specific interactions and interview questions with the participant
Nutrients 2018, 10, 369 9 of 43
and their parents. The clinician was blinded as to the participant’s treatment status, but the participants
and the parents were not, so this is classified as “semi-blinded”.
Severity of Autism Scale (SAS-Pro): The SAS-Pro is a single number on a scale of 0–10 to evaluate
overall severity of autism symptoms. It was evaluated by the professional evaluator after the ADOS,
RIAS, and CARS-2, so it was classified as “semi-blinded”.
All of the ADOS, RIAS, CARS-2, and SAS-Pro evaluations were done by the same professional
evaluator at beginning and end (either EP or JI).
Vineland Adaptive Behavior Scale II (VABS-II): The VABS-II was conducted by a phone interview
with the participant’s parents (or the participants in a few cases for high-functioning adults), so it
was classified as “semi-blinded”. One evaluator (RLA) conducted all the interviews at beginning
and end. The calculated raw scores were then converted into an age equivalent. However, most of
the questions are geared towards younger ages, and there are fewer questions for the older ages,
and a difference of one point for the older participants can result in a jump of more than 1 year of
developmental age. Therefore, for scoring purposes we set a maximum age for the following subscales,
based on the age at which questions became sparse: Receptive—11 years; Communication—12.3 years;
Written—15.3 years; Domestic—15.3 years; Play—19 years; Coping—17.8 years; Gross Motor—6.8 years;
Fine Motor—6.8 years; the other subscales (Personal and Community) had a maximum of 22 years.
Only 2 participants in the treatment group and 2 participants in the non-treatment group had some
scores at the maximum of any subscale, except for the Gross Motor and Fine Motor subscales, which
had 32% and 44% of the treatment and non-treatment group scoring at the maximum for those
subscales. So, although scores for Gross Motor and Fine Motor skills are reported, they need to
be interpreted cautiously.
Parents (or the participants in a few cases for high-functioning adults) completed an initial medical
history form, and at the beginning and end of the study they also completed several questionnaires
to assess autism and related symptoms, including the following: ATEC, Pervasive Developmental
Disorders Behavior Inventory (PDD-BI), Social Responsiveness Scale (SRS), 6-item Gastrointestinal
Severity Index (6-GSI), Short Sensory Profile (SSP), Aberrant Behavior Checklist (ABC), and Medical
History. Also, the Parent Global Impressions-2 (PGI-2) was completed at the end of months 3, 6, 9, and
12, to assess changes during the previous 3 months.
The PGI-2 is introduced here as an expanded version of the PGI-R [14]. The PGI-2 evaluates
changes in 17 areas, and overall, using a 7-point scale ranging from “much worse” to “much better”.
An “Average Change” is computed by computing the average in all 18 scores of the PGI-2, but exempting
areas where initial symptom severity was “none”. The PGI-2 is similar to the CGI, but conducted by a
parent instead of a clinician, and specific to autism. As discussed in a previous paper [15] our experience
indicates it is more reliable to ask parents directly about observed changes than to have them estimate
symptom severity at beginning and end and then compute a difference. Also, the use of a 7-point scale
to detect changes seems to yield a high sensitivity to changes. Note that for each symptom we only
report changes if the participant had the symptom at the start of the study.
2.11. Treatments
2.11.1. Vitamin/Mineral Supplement

This study involved an improved version of the vitamin/mineral supplement which was found
to be beneficial for children and adults with ASD in a previous study [15]. The supplement from that
study was slightly modified based on pre and post measurements of levels of vitamins, minerals, and
other biomarkers [15]. The major changes for this study included: increases of some nutrients (vitamin
D, niacin, pantothenic acid, biotin, selenium, mixed tocopherols) and decreases of others (manganese,
molybdenum, lithium). Also, several new nutrients were added, including Vitamin K, potassium,
carnitine, vanadium, and boron.
Nutrients 2018, 10, 369 10 of 43
Table 2 lists the supplement used in this study, at a dosage for a 60-pound (27 kg) child. The dosage
was adjusted up or down based on bodyweight, to a maximum of 120 pounds (54 kg). The dosage was
slowly increased over 4 weeks to the level listed in Table 2. In most cases the dosage was split into
three doses (breakfast, lunch, dinner), but in a few cases the families preferred to split it into two doses
for convenience (breakfast and dinner).
Table 2. Vitamin/Mineral Supplement.
Ingredients Amount
Vitamin A (85% beta carotene and 15% palmitate, IU) 6500
Vitamin C (from calcium ascorbate, mg) 500
Vitamin D3 (cholecalciferol, IU) 1000
Vitamin E (as alpha-tocopherol, IU) 150
Vitamin K (K1 and K2, mcg) 55
Vitamin B1 (thiamin hydrochloride, mg) 20
Vitamin B2 Riboflavin (mg) 40
Niacin (71% inositol hexanicotinate & 29% niacinamide, mg) 35
Vitamin B6 (50% as P5P pyridoxal 5 phosphate, 50% as pyridoxine hydrochloride, mg) 40
Folate (as folic acid, folinic acid, and L-5-methyltetrahydrofolate, mcg) 600
Vitamin B12 (50% as methylcobalamin & 50% as cyanocobalamin, mcg) 500
Biotin (mcg) 225
Pantothenic Acid (calcium d-pantothenate, mg) 30
Iodine (potassium iodide, mcg) 100
Lithium (mcg) 350
Choline (from choline bitartrate, mg) 250
Inositol (mg) 100
Calcium (mg) 70
Magnesium (magnesium citrate, mg) 100
Zinc (zinc gluconate, mg) 15
Selenium (selenomethionine and sodium selenite, mcg) 40
Manganese (manganese amino acid chelate, mg) 1
Chromium (chromium amino acid chelate, mcg) 70
Molybdenum (sodium molybdate dihydrate, mcg) 100
Potassium (from potassium chloride, mg) 50
MSM (methylsulfonylmethane, mg) 500
Vitamin E as mixed tocopherols (mg) 100
CoQ10 (mg) 50
N-acetyl-cysteine (mg) 45
Acetyl-L-carnitine (mg) 200
Vanadium (mcg) 25
Boron (mcg) 250
This is the dosage for a 60-pound (27 kg) child. Dosage was adjusted up/down by bodyweight.
2.11.2. Essential Fatty Acids

One of the best sources of omega-3 fatty acids is fish oil, and a recent study [74] confirms the
high absorption of omega-3 fatty acids from fish oil. We used a concentrated fish oil supplement,
ProEFA-Xtra by Nordic Naturals, which is a blend of fish oil (for omega-3 fatty acids) and modest
amounts of borage oil (for omega-6 fatty acids). Each capsule contains: 609 mg omega-3 fatty acids
(425 mg EPA, 110 mg DHA, 74 mg other omega-3 fatty acids), 198 mg omega-6 fatty acids (including
128 mg GLA), and 15 mg omega-9 fatty acids. The dosage varied with body weight:
• 30–50 pounds (14–23 kg): 2 capsules/day

• 51–100 pounds (23–45 kg): 3 capsules/day
• 100+ pounds (45+ kg): 4 capsules/day
Initial dosages started at 1 capsule/day, and increased to the above dosage over 2–4 weeks.
2.11.3. Epsom Salt Baths

Epsom salts are magnesium sulfate, and internal research by one of our team (TA) has found
that Epsom salt baths are one of the most effective ways to raise plasma sulfate levels, which are
Nutrients 2018, 10, 369 11 of 43
normally low in people with ASD. Therefore, each participant was asked to take a warm bath for 20 min
2×/week, with 2 cups Epsom salt and a half cup baking soda (which increases absorption of Epsom
salts) added to the bath.
2.11.4. Carnitine
Each participant was given a dosage of 50 mg acetyl-L-carnitine/kg bodyweight-day, to a maximum
of 2 grams/day, the same dosage as used in a previous study [50] involving L-carnitine, as that dosage
was found to be beneficial and well-tolerated. The dosage was gradually increased to the full dosage
over 4 weeks. Half the dosage was given in the morning, and half at dinnertime.
2.11.5. Digestive Enzymes

This study involved the use of a comprehensive digestive enzyme complex for digesting food
proteins (peptidase, protease 4.5, protease 3.0), carbohydrates (lactase, alpha-galactosidase, invertase,
xylanase), starches (amylase, glucoamylase) and fats (lipase)—see Table 3. The dosage was one capsule
for a snack or small adult meal, two capsules for a typical adult meal, and three capsules for a large
adult meal. Compared to other commercial digestive enzymes, this complex is characterized as
“low-medium” in protease activity, “medium” level in carbohydrase and starch-hydrolyzing activity,
and “medium-high” in lipase activity.
Table 3. Digestive Enzyme Ingredients (1 capsule).
Ingredients
Amylase 3500 DU
Peptidase 13,000 HUT
Glucoamylase 50 AGU
Xylanase 7000 XU
Protease 4.5 22,000 HUT
Protease 3.0 35 SAPU
Amylase 1500 DU
Invertase 800 SU
Alpha-galactosidase 100 GalU
Lactase 500 ALU
Lipase 500 FIP
2.11.6. Healthy, Gluten-Free, Casein-Free, Soy-Free Diet

Participants were provided with written instructions about the diet, a 1-h PowerPoint presentation
with audio describing the diet, a 1-h personal consult with one of our nutritionists, and the opportunity
to ask additional questions from the nutritionist or study nurse. The nutritionist provided detailed
advice, but the family made the final decision about meal plans and their degree of adherence to these
guiding principles. The degree of compliance with each of these guiding principles was self-evaluated.
The major guiding principles of the dietary plans included:
1. Adequate intake of a variety of vegetables (including leafy greens) and fruit (preferably whole fruit).
2. Adequate protein quality and intake.
3. Adequate, but not excessive, caloric intake.
4. Minimal consumption of “junk” foods and replacement with healthy snacks.
5. Healthy, gluten-free, casein-free, and soy-free (HGCSF).
6. Avoidance of artificial flavors, colors, and preservatives.
2.12. Statistical Analysis

Different types of statistical analyses were used, depending on the research question being addressed.
For comparison of changes of behavioral symptoms of the treatment vs. non-treatment group, 1-sided
unpaired t-tests assuming unequal variance were used, since our hypothesis was that the treatment
Nutrients 2018, 10, 369 12 of 43
group would improve more than the non-treatment group. For comparing changes in biomarkers,
2-sided unpaired t-tests comparisons assuming unequal variance were used. For individual comparisons,
a p-value of 0.05 or lower was assumed significant. No correction was made for multiple comparisons
sincecomparisons, a p‐value of 0.05 or lower was assumed significant. No correction was made for multiple
in most cases (such as vitamin measurements) there were a large number of significant findings.
comparisons
This was since in study,
an exploratory most cases (such studies
and future as vitamin
canmeasurements)
use our resultsthere were specific
to make a large hypotheses
number of and
significant findings. This was an exploratory study, and future studies can use our results to make
appropriate statistical corrections for multiple hypotheses.
specific hypotheses and appropriate statistical corrections for multiple hypotheses.
2.13. Participant Withdrawals, Removals, and Adverse Effects
2.13. Participant Withdrawals, Removals, and Adverse Effects
Figure 1 displays a flow chart of the study. 67 participants with ASD began the study, and 50
Figure 1 displays a flow chart of the study. 67 participants with ASD began the study, and
neurotypical participants were assessed at baseline only.
50 neurotypical participants were assessed at baseline only.

Figure 1. Study Flowchart.
Figure 1. Study Flowchart.
2.14. 2.14. Treatment Group
Treatment Group
37 families
37 families startedstarted
in thein the Treatment
Treatment group,group,
three three
droppeddropped out,
out, six six disqualified,
were were disqualified,
and 28and 28
completed
completed the study
the study
 one participant dropped after four months because of lack of benefit
•  participant
one droppedafter
one family dropped afterseven
four months because
months due of lack ofbenefit
to insufficient benefitand disinterest in taking
• one supplements
family dropped after seven months due to insufficient benefit and disinterest in
 one participant dropped after four months for unknown reasons
taking supplements
 four participants were disqualified by researchers due to poor compliance with study protocol
• one participant dropped after four months for unknown reasons
(parents were inconsistent in giving supplements due to parental, not child, issues)
• four

participants were disqualified by researchers due to poor compliance with study protocol
two participants (brothers) were disqualified because they discontinued all supplements and
(parents were inconsistent in giving supplements due to parental, not child, issues)
only completed the special diet
• two
participants (brothers) were disqualified because they discontinued all supplements and only
completed the special diet
Nutrients 2018, 10, 369 13 of 43
2.15. Non-Treatment Group

Thirty families started in the Non-treatment group, none dropped out, three were disqualified,
and 27 completed the study without making any major changes in their baseline treatments. This is an
unusually high percentage for a 120 month study, primarily due to the promise of receiving a full year
of free supplements if they waited.
Three participants were disqualified because they made significant changes to their baseline
treatments—one made a major diet change and added three psychiatric medications; one started
a developmental preschool at 10 h/week; one changed their school and home therapy program.
Also, one participant did only part of the final evaluation (ADOS/CARS-2/RIAS/SAS-Pro), but no
parent questionnaires or blood work due to parental issues.
3. Results
3.1. Adverse Effects

A few adverse effects were reported for some treatments.
Vitamin/Minerals: Two participants (brothers ages 7 and 12) had worsening behavior (moderate
severity) that was possibly due to the vitamin/mineral supplement, so after four months they stopped
use of all supplements and only implemented the healthy HGCSF diet (which was beneficial and
resolved severe pica in the seven-year-old). Our measurements found that they both had extremely
low cobalamin (4–5% of normal), and low levels of other nutrients compared to normal children,
including methylcobalmin (40–50% of normal), beta carotene (23–36% of normal), riboflavin (46–52%
of normal). The boy with pica also had low vitamin C (34% of normal), low nictotinic acid (29% of
normal), and low pantothenic acid (35% of normal). The boy without pica also had low levels of
folic acid (34% of normal). So, the very low level of cobalamin, and lower levels of other nutrients,
may have made them very sensitive to nutritional supplements, and/or they may have an underlying
metabolic problem with cobalamin. It seems likely that these nutritional deficiencies also contributed
to the severe pica in one of the boys.
Carnitine: One participant reported that the carnitine made their child feel sick, so they discontinued it.
Digestive Enzymes: One participant was not able to tolerate the digestive enzyme due to intestinal
symptoms, and stopped taking it after one month. One participant developed a facial rash after
extended use of the digestive enzyme, and eventually discontinued it despite reporting improvements
in constipation and behavior.
Healthy HGCSF diet: One parent reported that implementation of the diet in a strict manner
resulted in increased aggression towards peers, inability to problem solve, and increased spinning
behavior, probably due to frustration in regards to removal of favorite foods.
No adverse events were reported with the essential fatty acids or Epsom salt bath.
About 20% of the participants complained about the taste of the unflavored vitamin/mineral
supplement and/or the fish oil, but mixing it with juice helped in most cases, and a capsule form of
the vitamin/mineral supplement was made available to some families who preferred this form of
delivery. A few participants had temporary mild nausea, and one child had loose stools, but lowering
the dosage resolved those concerns.
3.2. Compliance
Compliance with taking the supplements and following the diet was self-reported by participants
at the end of the study. The percentages of families who missed doses 1×/week or less was 85% for
the vitamin/mineral supplement, 89% for the essential fatty acids, 82% for the carnitine, and 78% for
the digestive enzymes. For the vitamin/mineral supplement, one child took only 2/3 of the full dose,
and one child took 3/4 of the full dose. One child did not take the carnitine supplement, and two did
not take the digestive enzymes.
Nutrients 2018, 10, 369 14 of 43
For compliance with a healthy diet, 7% of families reported they had only 60% compliance,
60% For compliance with a healthy diet, 7% of families reported they had only 60% compliance, 60%
reported they had 80% compliance, and 33% reported they had 90% or higher compliance.
Most noncompliance
reported was
they had 80% reported as
compliance, occurring
and at school
33% reported they orhad
with care
90% or providers other thanMost
higher compliance. the
primary parent.
noncompliance was reported as occurring at school or with care providers other than the primary parent.
For compliance with a HGCSF diet, 61% reported < 1 exposure per month, 14% reported one
For compliance with a HGCSF diet, 61% reported < 1 exposure per month, 14% reported one
exposure per week, 18% reported 1–2 exposures/week, and 7% reported reduced intake of gluten,
exposure per week, 18% reported 1–2 exposures/week, and 7% reported reduced intake of gluten, casein,
casein, and soy but did not eliminate it. Families reported that compliance with the diet was the
and soy but did not eliminate it. Families reported that compliance with the diet was the hardest part
hardest part of the treatment protocol to follow.
of the treatment protocol to follow.
3.3. Highlights
3.3. Highlights
Figure 2 provides a summary of the highlights of the study. It plots the behavioral changes that
Figure 2 provides a summary of the highlights of the study. It plots the behavioral changes that
were significantly different between the treatment and non-treatment groups for the major assessments.
were significantly different between the treatment and non‐treatment groups for the major assessments.
Improvement
***
60% ** * * ** *** *** ** **
50%
40%
Percent Change
30%
20%
10%
0%
‐10%
Treatment
‐20%
Non‐Treatment
‐30%
* p<0.05 ** p<0.01 *** p<0.001

Figure 2. Summary of significant changes in major evaluations, for both the treatment and non‐
Figure 2. Summary of significant changes in major evaluations, for both the treatment and
treatment groups. For some scales an increase is an improvement, and for some the opposite is true;
non-treatment groups. For some scales an increase is an improvement, and for some the opposite is
so, here we plot them with improvement being in the same direction on the y‐axis. Note that the %
true; so, here we plot them with improvement being in the same direction on the y-axis. Note that the
change for the PDD‐BI composite is based on the average change in each of the composite subscales.
% change for the PDD-BI composite is based on the average change in each of the composite subscales.
Error bars represent standard deviations.
Error bars represent standard deviations.
3.4. Blinded Evaluations (RIAS, ADOS)
3.4. Blinded Evaluations (RIAS, ADOS)
3.4.1. Reynolds Intellectual Assessment Scales (RIAS)
3.4.1. Reynolds Intellectual Assessment Scales (RIAS)
The treatment group improved significantly more than the non‐treatment group on the Non‐
The treatment group improved significantly more than the non-treatment group on the Non-Verbal
Verbal IQ test (+6.7 ± 11.4 vs. −0.6 ± 10.7, p = 0.009), see Table 4 and Figure 3. There was no significant
IQ test (+6.7 ± 11.4 vs. −0.6 ± 10.7, p = 0.009), see Table 4 and Figure 3. There was no significant
difference on the Verbal IQ test or the Memory test. It is noted that at baseline the treatment group
difference on the Verbal IQ test or the Memory test. It is noted that at baseline the treatment group had
had a lower score than the non‐treatment group on the Non‐Verbal IQ test (p < 0.05), which was a
a lower score than the non-treatment group on the Non-Verbal IQ test (p < 0.05), which was a random
random difference between the groups as a result of the randomization process.
difference between the groups as a result of the randomization process.
Nutrients 2018, 10, 369 15 of 43

RIAS
115 **
Nonverbal Intelligence Index
100 Initial
Final
85
70
55
40
Treatment Non‐Treatment
*p<0.05 **p<0.01 ***p<0.001

Figure 3. Reynolds Intellectual Assessment Scales (RIAS) nonverbal IQ score at the beginning and
Figure 3. Reynolds Intellectual Assessment Scales (RIAS) nonverbal IQ score at the beginning and end
end of the study, for the treatment and non‐treatment groups. RIAS scores are normalized so that 100
of the study, for the treatment and non-treatment groups. RIAS scores are normalized so that 100 is
is an “average” IQ; thus, the average of the ASD groups is substantially lower than the average for
an “average” IQ; thus, the average of the ASD groups is substantially lower than the average for the
the general population. Error bars represent standard deviations.
general population. Error bars represent standard deviations.
Table 4. Professional Evaluations.
Table 4. Professional Evaluations.
Treatment Group (n = 28) Non‐Treatment Group (n = 27) t‐Test
Initial S.D. Group
Final S.D. % Change InitialNon-Treatment
S.D. Final S.D.
Treatment (n = 28) Group (n = 27) % Change t-Test
RIAS
Initial 69.6
Nonverbal Intelligence Index S.D. Final 76.3
23 S.D. % Change
24 +10% Initial85.8 S.D. 24 Final85.3 S.D. 24 % Change
−1% 0.01
Verbal Intelligence Index
RIAS 63.1 24 66.0 26 +5% 77.6 24 81.5 25 +5% n.s.
Composite Memory Index
Nonverbal 71.3 20 75.3 23 +6% 81.0 23 87.9 25 +8% n.s.
69.6 23 76.3 24 +10% 85.8 24 85.3 24 −1% 0.01
Intelligence Index
CARS‐2 39.3 5.6 33.9 7.0 −22% # 38.2 5.2 35.0 5.8 −14% # 0.03
Verbal Intelligence
SAS‐Pro 6.9 1.7 6.0 2.1 +5% −13% 77.6 6.0 24 1.9 81.5 5.6 25 2.3 −6% 0.04
63.1 24 66.0 26 +5% n.s.
Index
t‐test values were considered not significant (n.s.) if they were above a p‐value of 0.1. #‐ the percent change in CARS‐2
Composite
71.3 20 75.3 23 +6% 81.0 23 87.9 25 +8% n.s.
Memory Index
CARS-2 39.3 5.6
calculated based on minimum possible score of 15.
33.9 7.0 −22% # 38.2 5.2 35.0 5.8 −14% # 0.03
SAS-Pro 6.9 1.7 6.0 2.1 −13% 6.0 1.9 5.6 2.3 −6% 0.04
t-test values were considered not significant (n.s.) if they were above a p-value of 0.1.
3.4.2. Autism Diagnostic Observation Schedule (ADOS) #- the percent change in
CARS-2 calculated based on minimum possible score of 15.
There was no significant change on the ADOS scores for either treatment or non‐treatment group.
This assessment is meant for diagnosis, and is relatively insensitive to changes since it is scored on a 3‐
3.4.2. Autism Diagnostic Observation Schedule (ADOS)
point scale.
There was no significant change on the ADOS scores for either treatment or non-treatment group.
This assessment is meant for diagnosis, and is relatively insensitive to changes since it is scored on a
3.5. Semi‐Blinded Evaluations (CARS‐2, SAS‐Pro, Vineland)
3-point scale.
3.5.1. Childhood Autism Rating Scale (CARS‐2)
3.5. Semi-Blinded Evaluations (CARS-2, SAS-Pro, Vineland)
The treatment group improved somewhat more than the non‐treatment group on the CARS‐2,
3.5.1. Childhood Autism Rating Scale (CARS-2)
and the difference was significant (−5.5 ± 5.2 vs. −3.2 ± 3.7, p = 0.03), see Table 4 and Figure 4. These
improvements correspond to a 22% decrease vs. a 14% decrease, respectively (since the CARS‐2 has
The treatment group improved somewhat more than the non-treatment group on the CARS-2,
a minimum score of 15, the percentages are calculated relative to the minimum score of 15).
and the difference was significant (−5.5 ± 5.2 vs. −3.2 ± 3.7, p = 0.03), see Table 4 and Figure 4.
These improvements correspond to a 22% decrease vs. a 14% decrease, respectively (since the CARS-2
has a minimum score of 15, the percentages are calculated relative to the minimum score of 15).
Nutrients 2018, 10, 369 16 of 43
Nutrients 2018, 10, x FOR PEER REVIEW CARS 16 of 44
CARS
*
45
*
45
40
Initial
40
35
Final
Initial
35
30
Final
30
25
*p<0.05 **p<0.01 ***p<0.001

25
Figure 4. CARS‐2 scores at beginning and end of the study. The scale goes from 15 to 60, with scores
*p<0.05 **p<0.01 ***p<0.001
of approximately 27 and above being the cut‐off for ASD. Error bars represent standard deviations.

Figure 4. CARS‐2 scores at beginning and end of the study. The scale goes from 15 to 60, with scores
3.5.2. Severity of Autism Scale—Professional Evaluation (SAS‐Pro)
Figure 4. CARS-2 scores at beginning and end of the study. The scale goes from 15 to 60, with scores of
of approximately 27 and above being the cut‐off for ASD. Error bars represent standard deviations.
approximately 27 and above being the cut-off for ASD. Error bars represent standard deviations.
The treatment group improved somewhat more than the non‐treatment group on the SAS‐Pro
3.5.2. Severity of Autism Scale—Professional Evaluation (SAS‐Pro)
as (−0.93 ± 1.2 vs. −0.33 ± 0.12,
rated by our clinical evaluator, and the difference was significant
p = 0.04), see Table 4 and Figure 5. These changes correspond to a 13% and 6% decrease in SAS‐Pro
3.5.2. Severity of Autism Scale—Professional Evaluation (SAS-Pro)
The treatment group improved somewhat more than the non‐treatment group on the SAS‐Pro
scores, respectively.
as rated by our clinical evaluator, and the difference was significant (−0.93 ± 1.2 vs. −0.33 ± 0.12,
The treatment group improved somewhat more than the non-treatment group on the SAS-Pro
The RIAS was single‐blinded, and the CARS‐2 and SAS‐Pro were semi‐blinded (evaluator was
p = 0.04), see Table 4 and Figure 5. These changes correspond to a 13% and 6% decrease in SAS‐Pro
as ratedblinded,
by our participants
clinical
were not). and
evaluator, For the
the RIAS, higher was
difference numbers mean more
significant ability,
(−0.93 ± 1.2with 100
vs. −being
0.33 ± 0.12,
average for the general population. For the CARS‐2 and SAS‐Pro, higher numbers
p = 0.04), seeThe RIAS was single‐blinded, and the CARS‐2 and SAS‐Pro were semi‐blinded (evaluator was
Table 4 and Figure 5. These changes correspond to a 13% and 6% decrease in SAS-Pro mean worse
problems. #—For the CARS‐2, since the lowest possible scores is a 15, the % change is relative to that
blinded, participants were not). For the RIAS, higher numbers mean more ability, with 100 being
baseline of 15.
average for the general population. For the CARS‐2 and SAS‐Pro, higher numbers mean worse
problems. #—For the CARS‐2, since the lowest possible scores is a 15, the % change is relative to that
baseline of 15. Professional SAS
9 *
Professional SAS
8
9 *
7
8
6 Initial
7
Final
5
6 Initial
4 Final
5
3
4 Treatment Non‐Treatment
*p<0.05 **p<0.01 ***p<0.001
3

Figure 5. SAS scores (as rated by the professional evaluator) at beginning and end of the study. The
Figure 5. SAS scores (as rated by the professional evaluator) at *p<0.05 beginning and end of the study.
scale goes from zero (no symptoms) to 10 (severe autism). Error bars represent standard deviations.
**p<0.01 ***p<0.001

The scale goes from zero (no symptoms) to 10 (severe autism). Error bars represent standard deviations.
Figure 5. SAS scores (as rated by the professional evaluator) at beginning and end of the study. The

scale goes from zero (no symptoms) to 10 (severe autism). Error bars represent standard deviations.
The RIAS was single-blinded, and the CARS-2 and SAS-Pro were semi-blinded (evaluator was
blinded, participants were not). For
the RIAS, higher numbers mean more ability, with 100 being
average for the general population. For the CARS-2 and SAS-Pro, higher numbers mean worse
problems. #—For the CARS-2, since the lowest possible scores is a 15, the % change is relative to that
baseline of 15.
Nutrients 2018, 10, 369 17 of 43
3.5.3. Vineland Adaptive Behavior Scale II (VABS-II)

For the average developmental age of the Communication, Social, and Daily Living domains,
3.5.3. Vineland Adaptive Behavior Scale II (VABS‐II)
the treatment group improved significantly more than the non-treatment group (18.4 ± 16 months
vs. 4.3 ± 16 months, p = 0.008), see Table 5 and Figure 6. The treatment group improved significantly
For the average developmental age of the Communication, Social, and Daily Living domains,
more the treatment group improved significantly more than the non‐treatment group (18.4 ± 16 months vs.
on the Communication, Daily Living Skills, and Social Skills Domains. For the 9 subscales,
4.3 ± 16 months, p = 0.008), see Table 5 and Figure 6. The treatment group improved significantly
the treatment group improved significantly more than the non-treatment group on four of them (Written
more on the Communication, Daily Living Skills, and Social Skills Domains. For the 9 subscales, the
Skills, Domestic Skills, Interpersonal Relationships, Coping Skills) and marginally significant greater
treatment group improved significantly more than the non‐treatment group on four of them (Written
improvement on three others (Receptive Skills and Expressive Skills, and Community Skills), but no
Skills, Domestic Skills, Interpersonal Relationships, Coping Skills) and marginally significant greater
significant difference in Personal Daily Living Skills or Play/Leisure Skills—see Figure 7. For the Gross
improvement on three others (Receptive Skills and Expressive Skills, and Community Skills), but no
Motorsignificant
and Finedifference
Motor subscales both
in Personal groups
Daily had
Living similar
Skills degrees ofSkills—see
or Play/Leisure improvement,
Figure but it is
7. For important
the
Gross Motor and Fine Motor subscales both groups had similar degrees of improvement, but it is
to remember that 32% and 44% of the treatment and non-treatment groups, respectively, were at the
maximumimportant to remember that 32% and 44% of the treatment and non‐treatment groups, respectively,
score, so they could not improve more. Due to a combination of scheduling problems and
were at the maximum score, so they could not improve more. Due to a combination of scheduling
limited parental interest in the lengthy interview, pre and post VABS-II evaluations were completed
problems and limited parental interest in the lengthy interview, pre and post VABS‐II evaluations
on only 60% of the treatment group and 59% of the non-treatment group. However, a comparison of
were completed on only 60% of the treatment group and 59% of the non‐treatment group. However,
their PGI-2 scores shows
a comparison of their little
PGI‐2 difference between
scores shows those who
little difference did and
between did
those notdid
who complete
and did bothnot VABS-II
evaluations, so the limited number of evaluations did not seem to bias the results.
complete both VABS‐II evaluations, so the limited number of evaluations did not seem to bias the
results.
Table 5. Vineland Adaptive Behavior Scales II (VABS-II).
Table 5. Vineland Adaptive Behavior Scales II (VABS‐II).
Treatment Group
Treatment Group (n (n = 19)
= 19) Non-Treatment Group (n = 16)t‐Test
Non‐Treatment Group (n = 16) t-Test
Initial
Initial S.D.
S.D. FinalFinalS.D. S.D. % Change Initial
% Change S.D.
Initial Final
S.D. S.D.
Final % Change
S.D. % Change
Communication 4.5 1.8 5.7 2.5 +27% 5.5 2.9 5.8 2.7 +5% 0.01
Communication
Receptive 4.5
3.3 2.3 1.8 4.8 5.7 2.8 2.5 +43% +27% 3.8 5.5
2.1 2.9
4.5 5.8
2.6 2.7
+17% +5%
0.09 0.01
Receptive
Expressive 3.3
3.6 1.7 2.3 4.8 4.8 2.5 2.8 +34% +43% 4.5 3.8
2.8 2.1
4.9 4.5
2.9 2.6
+9% +17%
0.06 0.09
Expressive
Written 3.6
6.6 2.3 1.7 7.6 4.8 2.7 2.5 +16% +34% 8.2 4.5
4.1 2.8
8.1 4.9
3.1 2.9
−2% +9%
0.03 0.06
Written
Daily Living Skills 6.6
5.3 3.4 2.3 6.9 7.6 3.7 2.7 +31% +16% 7.4 8.2
5.0 4.1
7.7 8.1
4.6 3.1
+4% − 2%
0.007 0.03
Daily Living Skills
Personal 5.3
4.9 3.6 3.4 6.5 6.9 4.3 3.7 +34% +31% 7.5 7.4
5.5 5.0
8.3 7.7
5.6 4.6
+11% +4%
n.s. 0.007
Personal
Domestic 4.9
4.8 3.6 3.6 6.9 6.5 3.7 4.3 +44% +34% 7.0 7.5
4.5 5.5
7.0 8.3
4.3 5.6
−1% +11%
0.002 n.s.
Domestic
Community 4.8
6.1 4.1 3.6 7.1 6.9 3.7 3.7 +18% +44% 8.2 7.0
5.5 4.5
8.4 7.0
4.5 4.3
+2% − 1%
0.07 0.002
Community
Social 6.1
4.4 2.8 4.1 6.2 7.1 3.8 3.7 +39% +18% 5.5 8.2
3.9 5.5
5.9 8.4
3.4 4.5
+8% +2%
0.05 0.07
Social
Interpersonal Relationships 4.4
3.5 2.5 2.8 5.6 6.2 4.2 3.8 +59% +39% 3.9 5.5
3.1 3.9
3.9 5.9
2.6 3.4
+2% +8%
0.01 0.05
Interpersonal Relationships
Play and Leisure Time 3.5
4.3 2.6 2.5 5.4 5.6 2.7 4.2 +25% +59% 5.4 3.9
3.5 3.1
6.9 3.9
4.2 2.6
+28% +2%
n.s. 0.01
Play andCoping Skills
Leisure Time 4.3
5.5 3.9 2.6 7.5 5.4 5.3 2.7 +37% +25% 7.1 5.4
5.8 3.5
6.8 6.9
4.8 4.2
−5% +28%
0.03 n.s.
Coping Skills
Motor Skills 5.5
4.4 1.3 3.9 5.1 7.5 1.0 5.3 +15% +37% 5.1 7.1
1.6 5.8
5.7 6.8
1.0 4.8
+12% −n.s.
5% 0.03
Motor Skills
Gross Motor 4.4
4.0 1.3 1.3 4.5 5.1 1.2 1.0 +13% +15% 4.8 5.1
1.6 1.6
5.6 5.7
1.3 1.0
+16% +12%
n.s. n.s.
Gross Motor
Fine Motor 4.0
4.8 1.6 1.3 5.6 4.5 1.2 1.2 +17% +13% 5.4 4.8
1.6 1.6
5.8 5.6
1.0 1.3
+9% +16%
n.s. n.s.
Fine Motor
Average VABS (excluding motor skills) 4.8
4.7 2.5 1.6 6.3 5.6 3.2 1.2 +32% +17% 6.1 5.4
3.7 1.6
6.5 5.8
3.4 1.0
+6% +9%
0.008 n.s.
Average VABS (excluding motor skills) 4.7 2.5 6.3 3.2 +32% 6.1 3.7 6.5 3.4 +6% 0.008
Units are developmental age in years. t‐test values were considered not significant (n.s.) if they were above a p‐
Units are developmental age in years. t-test values were considered not significant (n.s.) if they were above a p-value of 0.1.
value of 0.1.
Vineland
14.0 * * *
12.0
Developmental Age
10.0
8.0 Initial
6.0 Final
4.0
2.0
0.0
T N T N T N T N
Communication Daily Living Skills Social Skills Average
*p<0.05 **p<0.01 ***p<0.001

Figure 6. Change in the developmental age for the Vineland domains, and the average of the three
Figure 6. Change in the developmental age for the Vineland domains, and the average of the three
domains. “T” refers to the treatment group and “N” refers to the non‐treatment group. Note that the
domains. “T” refers to the treatment group and “N” refers to the non-treatment group. Note that
the physical age of the participants at the start of the study was 10.8 and 12.3 years for the treatment
and non-treatment groups, respectively. So, their developmental age was far below their physical age,
even after a significant increase for the treatment group. Error bars represent standard deviations.
physical age of the participants at the start of the study was 10.8 and 12.3 years for the treatment and
Nutrients 2018, 10, 369
non‐treatment groups, respectively. So, their developmental age was far below their physical age, 18 of 43
even after a significant increase for the treatment group. Error bars represent standard deviations.
Vineland Change in Developmental Age
Difference in Developmental Age (years)
2.5 ** * *
2.0
Treatment
1.5 Group
1.0 Non‐
Treatment
0.5 Group
0.0
‐0.5
Personal
Play and Leisure Time
Average
Community
Expressive
Domestic
Interpersonal relationships
Coping Skills
Written
Receptive
*p<0.05 **p<0.01 ***p<0.001

Figure 7. Vineland Subscale Changes.
Figure 7. Vineland Subscale Changes.
3.6. Unblinded Parent/Self Evaluations
3.6. Unblinded Parent/Self Evaluations
3.6.1. Pervasive Developmental Disorders Behavior Inventory (PDD‐BI)
3.6.1. Pervasive Developmental Disorders Behavior Inventory (PDD-BI)
There was significantly greater improvement on the modified Autism Composite score of the
There
PDD‐BI was significantly
for the treatment greater
group improvement
compared to onthe
thenon‐treatment
modified Autism Composite
group score
(−35 ± 29 of the
vs. −11 PDD-BI
± 17,
p = treatment
for the 0.0002), see Table compared
group 6 and Figure 8. If non-treatment
to the we calculate the (−35
average
group ± 29
of the % vs. −11on
change ±each
17, pof =the
0.0002),
subscales
see Table 6 and that
Figurecompose
8. If wethe Autism
calculate Composite,
the average ofthe
theaverage
% change % on
change
each was 21%
of the and 5%
subscales for compose
that the
treatment and non‐treatment groups, respectively. The treatment group also had significantly greater
the Autism Composite, the average % change was 21% and 5% for the treatment and non-treatment
improvement on most of the PDD‐BI subscales.
groups, respectively. The treatment group also had significantly greater improvement on most of the
PDD-BI subscales.
PDDBI
25
*
20
**
15
10
** ** ** ** * **
5
‐5
‐10 Treatment
Non‐Treatment
‐15
‐20
*p<0.05 **p<0.01 ***p<0.001

Figure 8. Change in PDD‐BI subscale scores. Note that the first seven subscales are for maladaptive
Figure 8. Change in PDD-BI subscale scores. Note that the first seven subscales are for maladaptive
behaviors, so a decrease is beneficial. The last three subscales are for adaptive behaviors, so an
behaviors, so a decrease is beneficial. The last three subscales are for adaptive behaviors, so an increase
increase is beneficial. Error bars represent standard deviations.
is beneficial. Error bars represent standard deviations.
3.6.2. Autism Treatment Evaluation Checklist (ATEC)
There were significantly greater improvements on the total score of the ATEC for the treatment
group compared to the non‐treatment group (−28% vs. −6%, p = 0.00004), see Table 7 and Figure 9.
The treatment group had significantly greater improvements on all four of the subscales.
‐20
*p<0.05 **p<0.01 ***p<0.001

Figure 8. Change in PDD‐BI subscale scores. Note that the first seven subscales are for maladaptive
Nutrients 2018, 10, 369 19 of 43
behaviors, so a decrease is beneficial. The last three subscales are for adaptive behaviors, so an
increase is beneficial. Error bars represent standard deviations.
compared to the non-treatment group (−28% vs. −6%, p = 0.00004), see Table 7 and Figure 9.
groupgroup compared to the non‐treatment group (−28% vs. −6%, p = 0.00004), see Table 7 and Figure 9.
ATEC
45
***
40 *** ** ***
35
30
25
Initial
20
Final
15
10
0
T N T N T N T N
Speech/Communication Sociability Sensory/Cognitive Health/Physical Behavior
Awareness
*p<0.05 **p<0.01 ***p<0.001

Figure 9. The scores for the four ATEC subscales at the beginning and end of the study. “T” refers to
Figure 9. The scores for the four ATEC subscales at the beginning and end of the study. “T” refers
the treatment group and “N” refers to the non‐treatment group. Higher scores represent greater
to theseverity. Error bars represent standard deviations.
treatment group and “N” refers to the non-treatment group. Higher scores represent greater
severity. Error bars represent standard deviations.
3.6.3. Aberrant Behavior Checklist (ABC)

3.6.3. Aberrant Behavior Checklist (ABC)
There were significantly greater improvements on the total score of the ABC for the treatment
There were significantly greater improvements on the total score of the ABC for the treatment
compared to the non-treatment group (−26% vs. −7%, p = 0.001), see Table 8 and Figure 10.
groupgroup compared to the non‐treatment group (−26% vs. −7%, p = 0.001), see Table 8 and Figure 10. The
The treatment group had significantly greater improvements on four of the five subscales.
treatment group had significantly greater improvements on four of the five subscales.
ABC
40 ***
35
30 * *
25
Initial
20
Final
**
15
*
10
0
T N T N T N T N T N
Irritability Lethargy Stereotopy Hyperactivity Inappropriate
Speech
*p<0.05 **p<0.01 ***p<0.001

Figure 10. ABC subscales at beginning and end of the study. “T” refers to the treatment group and
“N” refers to the non‐treatment group. Higher scores represent greater severity. Error bars represent
“N” refers to the non-treatment group. Higher scores represent greater severity. Error bars represent
standard deviations.
3.6.4. Social Responsiveness Scale (SRS)
There were significantly greater improvements on the total score of the SRS for the treatment
group compared to the non‐treatment group (−14% vs. −3%, p = 0.004), see Table 9 and Figure 11. The
treatment group also had significantly greater improvements on four of the five subscales.
Licensed to Thaynara Cavalcanti TLimaN- thaynaralimanutri@hotmail.com
T N T N T N T N
Irritability Lethargy Stereotopy Hyperactivity Inappropriate
Speech
*p<0.05 **p<0.01 ***p<0.001

Nutrients 2018, 10, 369 20 of 43
“N” refers to the non‐treatment group. Higher scores represent greater severity. Error bars represent
group compared to the non-treatment group (−14% vs. −3%, p = 0.004), see Table 9 and Figure 11.
group compared to the non‐treatment group (−14% vs. −3%, p = 0.004), see Table 9 and Figure 11. The
The treatment group also had significantly greater improvements on four of the five subscales.
treatment group also had significantly greater improvements on four of the five subscales.
SRS Total
**
150
135
Initial
120
Final
105
90
75
60
Treatment Delay
*p<0.05 **p<0.01 ***p<0.001

Figure 11. Total SRS scores at the beginning and end of the study. Higher scores indicate greater
Figure 11. Total SRS scores at the beginning and end of the study. Higher scores indicate greater
severity, and 54 is the cut‐off for an ASD diagnosis. Error bars represent standard deviations.
severity, and 54 is the cut-off for an ASD diagnosis. Error bars represent standard deviations.
3.6.5. Short Sensory Profile (SSP)
3.6.5. Short Sensory Profile (SSP)
There were significantly greater improvements on the total score of the SSP for the treatment
There were significantly greater improvements on the total score of the SSP for the treatment
group compared to the non-treatment group (12% vs. 2%, p = 0.0003), see Table 10 and Figure 12.
group compared to the non‐treatment group (12% vs. 2%, p = 0.0003), see Table 10 and Figure 12. The
The treatment group also had significantly greater improvements on five subscales (tactile sensitivity,
treatment group also had significantly greater improvements on five subscales (tactile sensitivity,
taste/smell sensitivity, under-responsiveness/seeks sensation, auditory filtering, and visual/auditory
taste/smell sensitivity, under‐responsiveness/seeks sensation, auditory filtering, and visual/auditory
sensitivity, p < 0.05), and greater but not-significant improvement on the other two subscales.
sensitivity, p < 0.05), and greater but not‐significant improvement on the other two subscales.
SSP Total
***
150
140
130
120 Initial
110 Final
100
90
80
Treatment Delay
*p<0.05 **p<0.01 ***p<0.001

Figure 12. SSP scores at the beginning and end of the study. Note that higher scores represent fewer
Figure 12. SSP scores at the beginning and end of the study. Note that higher scores represent fewer
sensory problems. Error bars represent standard deviations.
sensory problems. Error bars represent standard deviations.
3.6.6. Parent Global Impressions—Revised‐2 (PGI‐2)
Table 11 lists the number of participants in each group who had each symptom. At the end of
12 months, there were significantly greater improvements on the Average Score of the PGI‐2 for the
treatment group compared to the non‐treatment group (1.24 ± 0.74 vs. 0.08 ± 0.54, p < 0.00000001),
see Table 11. The treatment group also had significantly greater improvements on 16 of the 17 individual
Nutrients 2018, 10, 369 21 of 43
3.6.6. Parent Global Impressions—Revised-2 (PGI-2)

Table 11 lists the number of participants in each group who had each symptom. At the end of 12
months, there were significantly greater improvements on the Average Score of the PGI-2 for the treatment
group compared to the non-treatment group (1.24 ± 0.74 vs. 0.08 ± 0.54, p < 0.00000001), see Table 11.
The treatment group also had significantly greater improvements on 16 of the 17 individual areas.
The PGI-2 assesses change in symptoms (from beginning to end of study), using a scale ranging
from −3 (much worse) to zero (no change) to 1 (slightly better), 2 (better), 3 (much better). The table
lists the number of participants who had the symptom at the start of the study.
PGI-2 vs. Time: The PGI-2 was also measured at 3, 6, and 9 months for the treatment group only,
and the Average Score of the PGI-2 is plotted in Figure 13. Most of the improvements in symptoms
occurred during the first three months,
Nutrients 2018, 10, x FOR PEER REVIEW with smaller improvements after that point. 22 of 44
PGI‐R
3
2.5 Treatment
Non‐Treatment
2
1.5
1
***
0.5
0
0 3 6 9 12
‐0.5
*p<0.05 **p<0.01 ***p<0.001

Figure 13. PGI‐R2 scores during the study. The scale goes from −3 (much worse) to 0 (no change) to
Figure 13. PGI-R2 scores during the study. The scale goes from −3 (much worse) to 0 (no change) to 1
1 (slightly better), 2 (better), 3 (much better). Error bars represent standard deviations.
(slightly better), 2 (better), 3 (much better). Error bars represent standard deviations.
3.6.7. 6‐item Gastrointestinal Severity Index (6‐GSI)
3.6.7. 6-item Gastrointestinal Severity Index (6-GSI)
This analysis was limited to the participants who had non‐zero Total Severity Scores on the
This analysis was limited to the participants who had non-zero Total Severity Scores on the 6-GSI
6‐GSI at the start of the study (22 of 28 in the treatment group and 21 of 27 in the non‐treatment
at the start of the study (22 of 28 in the treatment group and 21 of 27 in the non-treatment group).
group). The treatment group improved more than the non‐treatment group on the total severity score
The treatment group improved more than the non-treatment group on the total severity score (−30% vs.
(−30% vs. −10%, p = 0.05). The largest improvements were in constipation, diarrhea, and stool smell,
−10%, p = 0.05). The largest improvements were in constipation, diarrhea, and stool smell, see Table 12.
see Table 12. Also, there were three participants who initially had zero scores on the 6‐GSI, but
Also, there were three participants who initially had zero scores on the 6-GSI, but developed some
developed some GI symptoms at the end of the study (two participants in the treatment group had a
GI symptoms at the end of the study (two participants in the treatment group had a 1 and 2 point
1 and 2 point worsening, respectively, and one participant in the non‐treatment group had a 4‐point
worsening, respectively, and one participant in the non-treatment group had a 4-point worsening).
worsening).
Nutrients 2018, 10, 369 22 of 43
Table 6. Pervasive Developmental Disorders Behavior Inventory (PDD-BI).
Treatment Group (n = 27) Non-Treatment Group (n = 26) t-Test

Initial S.D. Final S.D. % Change Initial S.D. Final S.D. % Change
Maladaptive Behaviors—higher scores mean worse problems
Sensory 24.4 14.1 18.7 12.3 −23% 14.4 11.0 13.7 11.2 −5% 0.001
Ritual 18.9 7.2 13.9 7.6 −27% 15.4 7.8 15.1 8.1 −2% 0.001
SOCPP 20.6 7.8 16.1 8.2 −22% 17.3 6.7 16.3 6.6 −6% 0.008
SEMPP 20.1 7.8 17.3 7.7 −14% 15.2 6.8 14.5 6.5 −5% 0.08
AROUSE 20.7 7.2 16.3 7.8 −21% 17.4 7.2 17.0 7.2 −2% 0.002
FEARS 23.4 9.0 18.7 9.4 −20% 24.4 8.1 23.4 8.8 −4% 0.03
AGG 18.6 12.4 14.2 11.0 −24% 14.0 11.0 14.8 11.2 5% 0.002
Adaptive Behaviors—higher scores mean higher ability
SOCAPP 62.8 22.2 74.2 23.3 +18% 68.2 20.1 73.3 17.3 +8% 0.02
EXPRESS 57.4 24.7 66.1 24.2 +15% 66.8 24.3 70.5 21.0 +5% 0.004
LMRL 27.4 8.9 29.6 8.6 +8% 27.1 8.6 28.1 7.9 +4% 0.07
Modified Autism Composite −56.3 57.7 −91.6 60.7 −21% # −85.2 53.0 −96.0 50.2 −5% # 0.0002
# Since the Autism Composite is not rated on an absolute scale, we report the average of the % changes of each of the subscales of which it is composed, to give a sense of the degree of
change of symptoms.
Table 7. Autism Treatment Evaluation Checklist (ATEC).

Speech Communication 9.4 5.8 6.0 4.8 −36% 7.0 6.5 6.0 5.9 −14% 0.0007
Sociability 16.5 8.2 11.4 7.1 −31% 15.9 5.9 15.2 7.0 −5% 0.003
Sensory/Cognitive Awareness 16.6 6.6 11.7 5.6 −30% 12.3 6.9 11.6 6.8 −6% 0.00002
Health/Physical/Behavior 28.4 11.7 21.7 12.0 −24% 22.9 7.6 22.1 7.6 −4% 0.0009
Total ATEC Score 70.9 25.7 50.8 24.2 −28% 58.2 20.7 54.8 22.7 −6% 0.00004
Higher scores mean worse problems.
Nutrients 2018, 10, 369 23 of 43
Table 8. Aberrant Behavior Checklist.

Irritability 16.0 10.0 12.8 9.2 −20% 12.0 10 11.6 10.0 −4% 0.02
Lethargy/Social Withdrawal 14.9 10.6 10.1 8.5 −32% 14.1 7.6 12.5 7.3 −11% 0.01
Stereotypy 7.6 4.9 5.2 4.1 −31% 6.5 4.8 5.7 4.9 −12% 0.01
Hyperactivity 24.2 13.0 18.3 11.7 −24% 18.5 13 18.0 12.9 −3% 0.0001
Inappropriate Speech 6.4 3.3 5.1 3.1 −21% 4.0 2.9 3.6 2.9 −9% 0.05
Total ABC Score 68.9 33.5 51.4 31.7 −26% 55.0 27 51.4 27.5 −7% 0.001
Higher scores mean worse problems.
Table 9. Social Responsiveness Scale (SRS).

Awareness 15.0 4.1 13.4 4.0 −11% 12.4 2.7 11.7 2.5 −6% n.s.
Cognition 21.4 6.4 18.6 7.4 −13% 18.8 4.1 18.3 4.5 −3% 0.01
Communication 38.8 11.6 33.2 12.4 −14% 34.5 6.8 33.3 8.0 −4% 0.01
Motivation 17.3 6.1 14.5 6.1 −16% 17.6 4.7 16.8 4.7 −5% 0.03
Mannerisms 23.3 6.1 20.0 7.0 −14% 20.7 6.4 20.6 6.8 0% 0.02
Total SRS 115.7 30.6 99.7 33.7 −14% 104.0 18.2 100.6 21.4 −3% 0.01
Higher scores mean worse problems. t-test values were considered not significant (n.s.) if they were above a p-value of 0.1.
Table 10. Short Sensory Profile.

Tactile Sensitivity 22.7 5.9 26.4 5.2 +16% 24.1 4.9 25.2 5.1 +4% 0.007
Taste/Smell Sensitivity 10.5 6.0 11.8 5.3 +12% 11.1 5.4 11.5 5.5 +3% 0.05
Movement Sensitivity 11.2 4.4 11.7 4.0 +4% 10.8 3.5 10.6 3.3 −1% 0.06
Underresponsiveness/Seeks Sensation 17.9 6.9 20.5 5.5 +14% 22.0 7.1 22.3 7.3 +2% 0.006
Auditory Filtering 16.2 4.7 18.9 5.1 +16% 16.2 5.5 16.0 5.3 −1% 0.0003
Low Energy/Weak 19.3 7.5 20.4 7.3 +6% 17.5 7.1 17.9 7.4 +2% n.s.
Visual/Auditory Sensitivity 14.9 4.8 16.9 4.5 +13% 16.0 4.1 16.0 4.2 0% 0.004
Total SSP 113 27 127 27 +12% 118 20 120 21 +2% 0.0003
Higher scores mean less sensory problems. t-test values were considered not significant (n.s.) if they were above a p-value of 0.1.
Nutrients 2018, 10, 369 24 of 43
Table 11. Parent Global Impressions 2 (PGI 2).
Treatment n = 28 Non-Treatment n = 26 t-Test

n for Each Symptom at Start of Study Change S.D. n for Each Symptom at Start of Study Change S.D.
Expressive Language/Speech 27 1.7 0.8 25 0.3 0.9 0.0000006
Receptive Language/Comprehension 26 1.9 0.9 26 0.3 0.7 0.000000003
Play Skills 27 1.5 0.8 24 0.3 0.8 0.0000008
Cognition Thinking 28 1.6 0.9 24 0.4 0.7 0.000003
Attention Focus 28 1.5 1.1 25 0.3 0.9 0.00006
Stools/GI Issues 24 0.9 1.5 19 −0.1 0.6 0.003
Sleep 22 0.9 1.2 16 −0.2 1.2 0.01
Sociability 28 1.4 1.0 25 0.1 0.8 0.000009
Hyperactivity 23 0.9 1.0 16 −0.3 0.6 0.00008
Tantruming 23 1.2 1.3 19 −0.1 1.3 0.003
Eye Contact 27 1.4 0.9 25 0.2 0.8 0.000003
Mood/Happines 24 1.6 1.0 23 0.1 0.9 0.000003
Anxiety 27 0.9 1.3 25 −0.2 1.0 0.002
Stimming/Preservation 28 1.1 1.0 22 −0.1 0.8 0.00001
Sound Sensitivity 26 1.0 1.0 23 0.1 0.5 0.0002
Aggression 21 0.9 1.2 16 −0.3 1.3 0.01
Self-Abusive 16 0.8 1.4 12 −0.2 1.5 0.089
Overall 28 1.68 0.77 26 0.27 0.92 0.0000002
Average 28 1.2 0.7 27 0.1 0.5 0.00000003
Table 12. 6-Item Gastrointestinal Symptom Index (6-GSI).

Constipation 0.6 0.8 0.4 0.5 −43% 1.1 0.8 1.0 0.7 −14% n.s.
Diarrhea 0.3 0.6 0.1 0.3 −67% 0.1 0.2 0.0 0.0 −100% n.s.
Average Stool Consistency 0.4 0.5 0.3 0.5 −13% 0.3 0.5 0.2 0.4 −50% n.s.
Stool Smell 0.7 0.8 0.4 0.6 −44% 0.8 0.9 0.9 1.0 +20% 0.020
Flatulence 1.0 0.8 0.9 0.8 −13% 1.1 0.8 1.0 0.9 −10% n.s.
Abdominal Pain 0.3 0.6 0.3 0.6 −14% 0.4 0.5 0.4 0.6 −13% n.s.
Total Severity Score 3.4 1.8 2.4 1.8 −30% 3.7 1.8 3.3 2.2 −10% 0.050
Since only a subset of participants had GI problems, we only included them in this analysis if they had non-zero Total Severity Scores at the start of the study. t-test values were considered
not significant (n.s.) if they were above a p-value of 0.1.
Nutrients 2018, 10, 369 25 of 43
There was a slight increase in handgrip strength in both groups, consistent with an increase in
There was a slight increase in handgrip strength in both groups, consistent with an increase in
physical development over 12 months, but no significant differences between the two groups, see
physical development over 12 months, but no significant differences between the two groups, see Table 13.
Table 13.
Table 13. Handgrip Strength.
Table 13. Handgrip Strength.
Treatment Group (n = 28) Non‐Treatment Group (n = 25) t‐Test
Initial
Initial S.D.
S.D. Final
Final S.D.
S.D. % Change
% Change Initial
Initial S.D.
S.D. Final
Final S.D. % Change
S.D. % Change
Handgrip Strength
Handgrip Strength 40.3
40.3 26.6
26.6 41.8
41.8 21.5
21.5 +4%
+4% 44.8
44.8 24.9
24.9 46.7
46.7 22.5
22.5 +4%
+4% n.s.
n.s.
t‐test values were considered not significant (n.s.) if they were above a p‐value of 0.1.
3.8. Treatment Effectiveness

3.8. Treatment Effectiveness
At the end of the study, families were asked to rate the estimated effect of each treatment, since the
At the end of the study, families were asked to rate the estimated effect of each treatment, since the
treatments were started at least one month apart from one another, with the caveat that some treatments
treatments were started at least one month apart from one another, with the caveat that some treatments
may take longer than one month to have an effect so effects may overlap. Figure 14 plots the results.
may take longer than one month to have an effect so effects may overlap. Figure 14 plots the results.
The
The highest
highest rated
rated treatments
treatments were
were the
the vitamin/mineral supplement and
vitamin/mineral supplement and the
the essential
essential fatty
fatty acids,
acids,
followed by the Healthy HGCSF diets, followed by the carnitine, digestive enzymes, and Epsom
followed by the Healthy HGCSF diets, followed by the carnitine, digestive enzymes, and Epsom salt
salt baths.
baths.
2.5
2.0
Effectiveness Rating
1.5
1.0
0.5
0.0

Figure 14. Effectiveness of each treatment as rated by parents. This is rated on a scale of −3 (much
Figure 14. Effectiveness of each treatment as rated by parents. This is rated on a scale of −3 (much
worse) to 0 (no effect) to 1 (slightly better) to 2 (better) to 3 (much better). Error bars represent standard
worse) to 0 (no effect) to 1 (slightly better) to 2 (better) to 3 (much better). Error bars represent
deviations.
3.9. Treatment Continuation
3.9. Treatment Continuation
At the end of the study, families were asked which treatments they were going to continue when
At the end of the study, families were asked which treatments they were going to continue when
the study ended. Figure 15 plots the data. The vitamin/mineral supplement and the essential fatty
the study ended. Figure 15 plots the data. The vitamin/mineral supplement and the essential fatty
acids were the most likely to be continued (>85%). 70% of families planned to continue the Epsom
acids were the most likely to be continued (>85%). 70% of families planned to continue the Epsom salt
salt baths, 63% planned to continue the healthy HGCSF diet, and 44% planned to continue the
baths, 63% planned to continue the healthy HGCSF diet, and 44% planned to continue the carnitine
carnitine and digestive enzymes.
and digestive enzymes.
Nutrients 2018, 10, 369 26 of 43

100%
90%
80%
Percntage of Participants
70%
60%
50%
40%
30%
20%
10%
0%

Figure 15. Percentage of participants who plan to continue each treatment.
Figure 15. Percentage of participants who plan to continue each treatment.
3.10. Medical Tests
3.10. Medical Tests
3.10.1. Complete Blood Count (CBC)
3.10.1. Complete Blood Count (CBC)
Most of the CBC measurements did not change significantly. The only significant differences
Most of the CBC measurements did not change significantly. The only significant differences were
were that the treatment
that the treatment group group has a decrease
has a slight slight decrease in
in RBC and RBC and the non‐treatment
the non-treatment group
group did not did not
change
change (−2% vs. 0%, p = 0.04), and the treatment group had a very slight increase in mean corpuscular
(−2% vs. 0%, p = 0.04), and the treatment group had a very slight increase in mean corpuscular volume
volume (MCV) and the non‐treatment group did not change (+1% vs. 0%, p = 0.02), see Table 14.
(MCV) and the non-treatment group did not change (+1% vs. 0%, p = 0.02), see Table 14.
3.10.2. Blood Chemistry Panel (ChemPanel)
3.10.2. Blood Chemistry Panel (ChemPanel)
Most of the ChemPanel measurements did not change significantly, see Table 15. For Blood Urea
Most of the ChemPanel measurements did not change significantly, see Table 15. For Blood Urea
Nitrogen (BUN),
Nitrogen (BUN), the
the treatment
treatment group
group had
had aa small
small decrease
decrease and
and the
the non-treatment
non‐treatment group
group did
did not
not
change (−16% vs. 5%, p = 0.01). For Serum Potassium, there was a slight decrease in the treatment
change (−16% vs. 5%, p = 0.01). For Serum Potassium, there was a slight decrease in the treatment
group and little change in the non‐treatment group (−6% vs. −1%, p = 0.02).
group and little change in the non-treatment group (−6% vs. −1%, p = 0.02).
3.10.3. Body Mass Index (BMI)

3.10.3. Body Mass Index (BMI)
There was no significant difference in the change in the BMI of the treatment group (20.7 ± 5.3 to
There was no significant difference in the change in the BMI of the treatment group (20.7 ± 5.3
20.0 ± 4.6) compared to the change of the non-treatment group (20.4 ± 5.5 to 19.9 ± 5.4).
to 20.0 ± 4.6) compared to the change of the non‐treatment group (20.4 ± 5.5 to 19.9 ± 5.4).
3.10.4. Fatty Acids

3.10.4. Fatty Acids
The treatment group had large increases in eicosapentaenoic acid (EPA) and docosahexaenoic
The treatment group had large increases in eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA) compared to the non-treatment group (EPA, + 525% vs. +22%, p = 1 × 10 −9 , DHA: +83%
acid (DHA) compared to the non‐treatment group (EPA, + 525% vs. +22%, p = 1 × 10 −9, DHA: +83% vs.
vs.
+13%, p = 1 × 10 10−9 ). The treatment group also had a small decrease in arachidonic acid (AA, −20%
+13%, p = 1 ×−9). The treatment group also had a small decrease in arachidonic acid (AA, −20% vs.
vs.
−1%, p = 1 × 10 10−7 ), linoleic acid (−15% vs. −1%, p = 0.0001), dihomo-γ-linolenic acid (DGLA,
−1%, p = 1 −7×), linoleic acid (−15% vs. −1%, p = 0.0001), dihomo‐γ‐linolenic acid (DGLA, −12% vs.
− 12% vs. +2%, p = 0.003), elaidic acid (−26% vs. −12%, p = 0.03), and palmitoleic acid (−13% vs.
+2%, p = 0.003), elaidic acid (−26% vs. −12%, p = 0.03), and palmitoleic acid (−13% vs. +11%, p = 0.01),
+11%, p = 0.01), see Table 16.
see Table 16.
Low initial levels of linoleic acid were inversely correlated with parents reports of Treatment
Low initial levels of linoleic acid were inversely correlated with parents reports of Treatment
Effectiveness of the EFA supplement (r = −0.55, p < 0.001). In other words, the group reporting the
Effectiveness of the EFA supplement (r = −0.55, p < 0.001). In other words, the group reporting the
most improvement on the EFA supplement tended to be those with the lowest initial levels of linoleic
most improvement on the EFA supplement tended to be those with the lowest initial levels of linoleic
acid. There were no other significant correlations of improvement with the EFA supplement and
levels of other PUFAs.
Nutrients 2018, 10, 369 27 of 43
acid. There were no other significant correlations of improvement with the EFA supplement and levels
of other PUFAs.
3.10.5. C-Reactive Protein (CRP)

There was no significant difference between the change in CRP in the treatment and non-
treatment groups.
3.10.6. Vitamins
For the treatment group compared to the non-treatment group there were large and significant
increases in biomarkers for Vitamin B2 (riboflavin, +268% vs. −17%, p = 0.00000002), B5 (pantothenic
acid, +351% vs. +90%, p = 0.0002), folic acid (folic acid, +119% vs. −34%, p = 0.02), and CoQ10H2 (the
reduced form of CoQ10, +72% vs. −19%, p = 0.001; no significant change in the oxidized form, CoQ10).
There was a large increase in one biomarker of vitamin B6 (4-pyridoxic +435% vs. +6%, p = 0.0000006),
but only a small increase in another biomarker (pyridoxine, +25% vs. −33%, p = 0.008). There were
moderate increases in one form of vitamin B12 (cyanocobalamin, +44% vs. −5%, p = 0.006) but not in
another (methylcobalamin), see Table 17. There were no significant changes in the other biomarkers.
3.10.7. RBC Elements

There was a significant increase in selenium (+5% vs. −8%, p = 0.001) and chromium (+18% vs.
−16%, p = 0.05) in the treatment group compared to the non-treatment group, see Table 18. There were
no significant changes in other minerals.
3.10.8. Homocysteine Pathway

For the treatment group compared to the non-treatment group, there was a significantly larger
decrease (improvement) in homocysteine (−29% vs. −7%, p = 0.00002), resulting in normal levels;
see Table 19. There were no significant changes in cysteine or methionine.
3.10.9. Carnitine
There was a significant increase in L-carnitine in the treatment group compared to the
non-treatment group (+20% vs. −5%, p = 0.03), see Table 20. There was a non-significant increase
in acetyl-L-carnitine in the treatment group compared to the non-treatment group (+32% vs. +4%,
n.s.). The parent rating of Treatment Effectiveness for carnitine had a modest, non-significant inverse
correlation (r = −0.29) with initial levels or the level of acetyl-carnitine.
Nutrients 2018, 10, 369 28 of 43
Table 14. Complete Blood Count.

WBC (×103 /uL) 6.86 2.02 6.88 2.41 0% 6.08 1.57 5.79 1.57 −5% n.s.
RBC (×106 /uL) 4.98 0.50 4.89 0.52 −2% 4.92 0.31 4.93 0.28 0% 0.04
Hemogoblin (g/dL) 14.2 1.44 14.1 1.55 −1% 14.1 1.12 14.2 1.10 +1% n.s.
Hematocrit (%) 41.4 3.91 41.2 4.26 −1% 41.3 2.92 41.4 2.73 0% n.s.
MCV (fL) 83.4 5.70 84.4 5.96 +1% 84.0 2.78 84.0 2.94 0% 0.02
MCH (pg) 28.5 2.09 28.8 2.23 +1% 28.6 1.33 28.7 1.33 0% n.s.
MCHC (g/dL) 34.2 0.71 34.1 0.76 −0% 34.1 0.81 34.2 0.96 0% n.s.
RDW (%) 13.7 0.68 13.5 0.71 −1% 13.6 0.77 13.5 0.69 −1% n.s.
Platelets (×103 /uL) 319 75.5 311 87.3 −3% 285 59.7 296 72.9 4% n.s.
Neutrophils (Absolute) (×103 /uL) 3.34 1.46 3.58 1.88 +7% 2.86 1.05 2.70 0.93 −6% n.s.
Lymphs (Absolute) (×103 /uL) 2.65 0.94 2.47 1.03 −7% 2.46 0.93 2.40 0.85 −3% n.s.
Monocytes (Absolute) (×103 /uL) 0.56 0.16 0.58 0.19 +4% 0.50 0.16 0.45 0.14 −11% 0.08
Eos (Absolute) (×103 /uL) 0.27 0.30 0.21 0.21 −24% 0.24 0.17 0.21 0.16 −12% n.s.
Baso (Absolute) (×103 /uL) 0.04 0.05 0.02 0.04 −33% 0.01 0.04 0.01 0.04 0% n.s.
Immature Granulocytes (%) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Immature Grans (Absolute) (×103 /uL) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Table 15. Blood Chemistry.

Glucose, Serum (mg/dL) 87.6 5.63 90.0 8.98 +3% 92.7 28.1 95.6 21.9 +3% n.s.
BUN (mg/dL) 13.5 3.77 11.3 3.77 −16% 13.3 3.29 14.0 3.56 +5% 0.01
Creatinine, Serum (mg/dL) 0.61 0.22 0.62 0.21 0% 0.61 0.18 0.65 0.21 +6% n.s.
BUN/Creatinine Ratio 25.0 11.8 20.3 8.53 −19% 23.4 9.83 24.0 12.1 +3% 0.04
Sodium, Serum (mmol/L) 139 2.39 140 2.64 +1% 139 4.04 139 2.19 0% n.s.
Potassium, Serum (mmol/L) 4.28 0.28 4.01 0.25 −6% 4.09 0.32 4.05 0.30 −1% 0.02
Chloride, Serum (mmol/L) 101 2.60 101 2.64 0% 102 2.75 103 2.68 +1% n.s.
Carbon Dioxide, Serum (mmol/L) 19.7 2.04 20.7 2.36 +5% 20.5 1.82 20.4 2.72 −0% n.s.
Calcium, Serum (mmol/L) 10.1 0.43 9.96 0.41 −2% 10.1 0.49 9.8 0.62 −4% n.s.
Protein, Total, Serum (g/dL) 7.07 0.47 7.22 0.49 +2% 7.04 0.47 7.08 0.51 +1% n.s.
Albunim spp., Serum (g/dL) 4.49 0.40 4.58 0.31 +2% 4.33 0.38 4.54 0.21 +5% n.s.
Globulin, Total (g/dL) 2.58 0.36 2.64 0.37 +2% 2.71 0.58 2.55 0.41 −6% 0.02
A/G Ratio 1.78 0.33 1.77 0.27 −1% 1.70 0.47 1.83 0.28 +7% n.s.
Bilirubin, Total (mg/dL) 0.36 0.23 0.46 0.35 +29% 0.34 0.12 0.34 0.12 0% 0.08
Nutrients 2018, 10, 369 29 of 43
Table 15. Cont.

Alkaline Phosphatase, S (IU/L) 204 90.0 184 87.1 −10% 221 111 200 92.2 −9% n.s.
AST (SGOT) (IU/L) 26.2 6.74 28.4 12.8 +8% 24.7 6.38 23.4 7.43 −6% n.s.
ALT (SGPT) (IU/L) 22.1 10.5 26.1 21.2 +18% 18.9 9.58 20.5 11.9 +9% n.s.
Ammonia, Plasma (ug/dL) 79.3 39.57 76.70 26.06 −3% 64.1 29.5 76.0 23.4 +19% n.s.
Creatine Kinase, Total, Serum (U/L) 117 49.7 111 55.0 −6% 102 38.3 113 56.3 +11% n.s.
Lactic Acid, Plasma (mg/dL) 16.9 10.1 16.7 12.5 −1% 13.0 6.22 13.3 11.4 +2% n.s.
TSH (uIU/mL) 2.30 0.99 2.55 1.30 +11% 2.11 1.07 2.16 0.94 +2% n.s.
Triiodothyronine, Free, Serum (pg/mL) 4.07 0.50 4.12 0.66 +1% 3.95 0.58 4.05 0.63 +3% n.s.
T4, Free (Direct) (ng/dL) 1.33 0.15 1.32 0.14 −1% 1.27 0.19 1.21 0.20 −5% n.s.
Table 16. Red Blood Cell Fatty Acids.
Average—Treatment (n = 25) Average—Non-Treatment (n = 24) t-Test

Initial SD Final SD % Change Initial SD Final SD % Change
Arachidonic Acid (µmol/L) 910 89 727 99 −20% 911 66 899 99 −1% 0.0000001
Dihomo-g-linolenic (µmol/L) 95 22 84 16 −12% 95 22 97 17 +2% 0.003
Docosahexaenoic acid (µmol/L) 175 58 320 78 +83% 183 54 207 54 +13% 0.000000001
Eicosapentaenoic (µmol/L) 21 13 130 60 +525% 17 7.4 20 8.0 +22% 0.000000001
Elaidic (µmol/L) 8.7 1.8 6.4 1.7 −26% 8.3 2.3 7.3 1.7 −12% 0.03
Linoleic (µmol/L) 670 93 568 90 −15% 622 98 617 77 −1% 0.0001
Oleic (µmol/L) 650 77 725 75 +11% 619 35 670 44 +8% n.s.
Palmitelaidic (µmol/L) 0.92 0.23 0.76 0.17 −17% 0.89 0.23 0.74 0.20 −17% n.s.
Palmitic (µmol/L) 1194 108 1197 75 0% 1136 101 1153 65 +1% n.s.
Palmitoleic (µmol/L) 10.9 3.9 9.6 4.4 −13% 11.2 4.2 12.4 5.4 +11% 0.01
Stearic (µmol/L) 885 82 833 40 −6% 872 55 862 55 −1% 0.08
Nutrients 2018, 10, 369 30 of 43
Table 17. Vitamin Levels.
Average—Treatment (n = 27) Average—Non-Treatment (n = 26) t-Test

Vit B2 (riboflavin) 0.69 0.22 2.53 1.36 +268% 1.18 0.68 0.98 0.67 −17% 0.00000001
Vit B3 (niacinamide) 1.31 0.85 1.34 0.87 +2% 0.99 0.43 1.14 0.47 +14% n.s.
Vit B5 (pantothenic acid) 1.01 1.28 4.54 3.21 +351% 0.86 0.65 1.63 1.34 +90% 0.0002
Vit B6 (4-pyridoxic acid) 1.21 1.30 6.45 3.85 +435% 1.21 1.29 1.29 1.07 +6% 0.000001
Vit B6 (pyridoxine) 1.21 0.55 1.51 0.73 +25% 1.26 0.75 0.85 0.42 −33% 0.008
Folic acid 0.65 0.61 1.42 1.07 +119% 1.10 1.25 0.73 1.86 −34% 0.02
B12-(cyanocobalamin) 0.86 0.49 1.24 0.54 +44% 0.94 0.41 0.90 0.26 −5% 0.006
B12-(methylcobalamin) 0.98 0.49 1.08 0.57 +9% 0.96 0.34 0.99 0.44 +3% n.s.
Vit C (ascorbic acid) 0.79 0.32 1.18 0.56 +48% 0.81 0.49 1.01 0.50 +24% n.s.
Vit D3 1.03 0.63 1.12 0.44 +9% 0.85 0.29 1.00 0.37 +18% n.s.
Vit K2 0.97 0.55 1.22 0.47 +26% 0.83 0.54 1.09 0.44 +32% n.s.
Choline 1.08 0.41 0.99 0.30 −8% 1.01 0.27 0.88 0.24 −12% n.s.
Myoinositol 1.25 0.46 1.48 0.74 +19% 0.98 0.27 1.05 0.40 +7% n.s.
CoQ10 (oxidized) 1.35 0.86 1.18 0.64 −13% 1.30 0.65 1.49 0.97 +15% n.s.
CoQ10 (reduced form, CoQ10H2) 0.94 0.62 1.63 0.79 +72% 1.31 0.85 1.05 0.66 −20% 0.001
Vitamin levels as measured by a semi-quantitative metabolomics method, so results are normalized to levels in un-supplemented neurotypical controls (i.e., a level of 1.0 is the average of
the neurotypical controls). t-test values were considered not significant (n.s.) if they were above a p-value of 0.1.
Table 18. RBC Elements.

Ca (µg/g) 18.0 4.0 17.0 3.0 −5% 17.0 4.0 17.0 5.0 +3% n.s.
Mg (µg/g) 46.0 4.0 46.0 5.0 0% 46.0 4.0 48.0 4.0 +3% n.s.
K (mEq/L) 78.0 4.0 78.0 4.0 0% 78.0 5.0 77.0 3.0 −1% n.s.
P (µg/g) 599 36.0 580 43.0 −3% 597 50.0 581 44.0 −3% n.s.
Cu (µg/g) 0.7 0.1 0.7 0.1 +7% 0.7 0.1 0.7 0.1 +4% n.s.
Zn (µg/g) 9.6 1.5 9.6 1.5 0% 9.5 1.1 9.5 1.2 −1% n.s.
Fe (µg/g) 865 47.0 858 30.0 −1% 865 47.0 853 37.0 −1% n.s.
Mn (µg/g) 0.02 0.01 0.02 0.01 +2% 0.02 0.01 0.02 0.01 +5% n.s.
Cr (µg/g) 0.0005 0.0003 0.0006 0.0003 +12% 0.0005 0.0003 0.0004 0.0001 −15% 0.05
Se (µg/g) 0.3 0.03 0.3 0.04 +5% 0.3 0.04 0.2 0.03 −8% 0.001
Nutrients 2018, 10, 369 31 of 43
Table 19. Homocysteine Pathway.

Cysteine (µM/dL) 24.5 4.1 24.1 3.8 −2% 24.4 2.7 24.2 3.1 −1% n.s.
Homocysteine (µM/L) 7.1 2.7 5.0 1.4 −29% 6.1 1.7 5.7 1.4 −7% 0.00002
Methionine (µM/dL) 2.4 0.5 2.4 0.8 0% 2.2 0.5 2.6 0.6 +17% 0.09
Table 20. Carnitine levels in plasma.

L -carnitine 1.10 0.32 1.32 0.41 +20% 1.12 0.31 1.07 0.29 −5% 0.03
Acetyl-L-carnitine 1.05 0.42 1.39 0.47 +32% 0.96 0.53 1.01 0.39 +4% n.s.
Units are normalized to those of neurotypical controls of similar age and gender (i.e., neurotypical group has levels of 1.0). For the treatment group, we exclude the 1 participant who did
not tolerate the carnitine supplement. t-test values were considered not significant (n.s.) if they were above a p-value of 0.1.
Nutrients 2018, 10, 369 32 of 43
3.11. Case Studies

There were also 3 exceptional cases of improvement during the study, all of which occurred in the
treatment group.
3.11.1. Case Study A

Increase in Physical Strength/Endurance/Energy: Participant A was a 9-year-old female with
severe ASD, moderately overweight (BMI = 31.5), and very low strength, endurance, and energy level.
She could not get in/out of the family van, climb stairs, or get up off the floor by herself, and had a
low activity level overall. She could only walk a quarter mile before sitting and refusing to get up,
so a wheelchair was used for outings. Around four months after treatment started her strength and
endurance began to improve significantly, and by 6–12 months into the study she was able to get in/out
of the van, walk up/down stairs, walk two miles, and attend outings without tiring. The wheelchair
was put in storage and no longer needed. Her overall energy level increased substantially to the level
when she was a toddler, and she began to skip around the house. Her diet had been self-limited with
total avoidance of beef and pork products (the main dietary sources of carnitine), and her improvement
seemed to primarily change with the addition of high-dose carnitine at 4 months into the study.
An in-depth assessment of her carnitine status revealed that, averaging over measurements of 37
different types of carnitine (acetyl-carnitine species), her pre-treatment levels averaged only 68% of
normal, and after treatment they averaged 18% above normal. So, low carnitine seems likely to have
contributed to her challenges, and carnitine supplementation seems to have helped.
3.11.2. Case Study B

Complete Resolution of Inability to Urinate: Participant B was a 27-year-old male with severe
ASD and a history of severe urinary retention and occasional kidney stones for three years, requiring
daily catheterization and occasional hospitalization. The cause was unknown and assumed to be
neurological. Previous treatment with Flomax and Bethanecol was ineffective. The daily intermittent
catheterization caused much discomfort to the subject, including bouts of urinary tract infections,
bladder infections and irritation of the external urethral orifice, requiring numerous treatments with
oral antibiotics and antifungals. His parents reported “His quality of life and social activity were
much diminished. In addition, behavioral issues started to emerge, including an obsession with the
constant touching of his genitals (probably initially caused by irritation which then evolved into a
stimulative behavior). This became a huge problem when out in public, around peers and with his
family members”.
As step one of a HGCSF diet, the subject was taken off all dairy products. Approximately four
days after all dairy had been removed from his diet, the subject spontaneously went to the restroom
and urinated on his own. He continued to be able to urinate on his own numerous times a day to
the point where catheterization was no longer required. About three weeks after the subject had
been taken off dairy products, he accidentally ate ice-cream, and the subject immediately ceased
to be able to urinate on his own, returning to the necessity of daily intermittent catheterization.
After approximately four days after eating the ice cream, the subject once again started spontaneously
urinating on his own without assistance. He continued to be able to urinate on his own, eliminating
the need for catheterization. Approximately four months after dairy had been removed from his diet,
he accidentally ate cheese, upon which the subject once again lost the ability to urinate on his own,
requiring intermittent catheterization. After approximately four days the subject started spontaneously
urinating on his own again. The participant remained completely dairy-free for the remainder of the
study, and continued to be able to urinate on his own and catheterization was no longer required,
and there were zero episodes of kidney stones, urinary tract infections, bladder infections or urethral
irritation. His parents reported that “his quality of life has improved dramatically and all behavior
Nutrients 2018, 10, 369 33 of 43
issues, including the constant touching of his genitals, have ceased. His social interactions with his
peers and family members have improved dramatically and he is overall a much happier person”.
3.11.3. Case Study C

Complete resolution of Pica: Participant C was a seven-year-old boy with severe pica. Within one
week of starting the HGCSF diet there was a complete resolution of the pica which continued until the
end of the study. Note that at baseline this boy had low levels of many nutrients compared to typical
children, including: cobalamin (5% of normal), methylcobalamin (49%), beta-carotene (23%), nicotinic
acid (29%), vitamin C (34%), pantothenic acid (35%), and riboflavin (52%). It seems likely that the
severe pica was due to many significant nutritional deficiencies, and possibly a metabolic problem
with cobalamin absorption or conversion.
4. Discussion
4.1. Blinded Evaluations (RIAS)

The significant improvement on the nonverbal IQ test suggests a substantial improvement in
cognitive function. The verbal IQ did not change, perhaps because significant impairments in language
remained. Since the RIAS evaluator was completely blinded, and the RIAS test is highly standardized,
this increase in nonverbal IQ appears to be real. The improvement in nonverbal IQ is consistent with
the much higher ratings of improvement in cognition on the PGI-2 for the treatment group vs. the
non-treatment group (1.57 ± 0.9 vs. 0.38 ± 0.7). The magnitude of the effect is clinically significant: on
the PGI-2 cognition question, 53% of treatment group were rated as better (39%) or much better (14%),
vs. only 8% of the non-treatment group were rated as better, and none were rated much better.
4.2. Semi-Blinded Evaluations (VABS-II, CARS-2, SAS-Pro)

During the 12 months of treatment, the non-treatment group gained only 4 months of development
on the VABS-II, consistent with a major developmental delay. In contrast, the treatment group
gained an average of 18 months of development, with substantial improvements in many areas.
However, their average developmental age still remained well below their biological age, so that they
were still significantly impaired. This is consistent with modest but significant improvements on the
CARS-2 and SAS-Pro.
4.3. Unblinded Parent/Self Evaluations (PDD-BI, ATEC, ABC, SRS, SSP, PGI-2, 6-GSI)
As mentioned previously, these unblinded evaluations are somewhat affected by a “placebo”
effect, so the improvements reported represent an “upper bound” on the actual degree of benefit.
The use of a non-treatment group provides some control for normal developmental improvement over
the 12 months of the study. For the non-treatment group, it is interesting to note that there were some
small improvements in several of the assessments (<10%, see Figure 2). However, the treatment group
improved significantly more than the non-treatment group on all of the ASD/behavioral assessments.
4.4. GI Symptoms
The treatment group had significantly more improvement than the non-treatment group on the
6-GSI, primarily due to improvements in constipation, diarrhea, and stool smell. This is consistent
with the PGI-2 subscale for Stool/GI symptoms, which found that the treatment group improved
much more than the non-treatment group (approximately “slightly better (0.94) vs. “no change (−0.11),
see Table 11. This is also consistent with a significant reduction in constipation symptoms (−48%
vs. −13%, p = 0.003) reported as one question on the ATEC (only evaluating those with initial mild,
moderate, or severe constipation—19 in the treatment group, and 17 in the non-treatment group).
The ATEC did not show a significant difference in diarrhea. Overall, these three assessments suggest
Nutrients 2018, 10, 369 34 of 43
that there was a significant improvement in GI symptoms in the treatment group compared to the
non-treatment group.

Both groups had only a small increase in handgrip strength, consistent with what might be
expected by 12 months of development, but no significant difference between them and not enough to
catch up to their peers.
4.6. Treatment Effectiveness and Continuation

The parents rated the vitamin/mineral supplement (started day 0) and the EFAs (started day 30) as
the two most effective treatments, which is consistent with Figure 14 showing that most improvement
occurred during the first three months of treatment. This is also consistent with those two treatments
being rated by parents as the most likely treatments to continue. There was also a small increase
in the PGI-2 between months 9–12, presumably due to the start of the healthy HGCSF diet on day
210. It should be noted that compliance with the healthy HGCSF diet was lower than for the other
treatments, so benefit might have been somewhat larger if compliance was higher.
4.7. Medical Tests

The CBC, ChemPanel, and BMI results demonstrated that the treatment combination was generally
safe, consistent with the modest number and modest intensity of adverse effects. There was no
significant change in CRP, a general marker of inflammation, but levels at the start of the study were
not significantly different between the ASD and neurotypical groups.
4.7.1. PUFAs
The PUFA test results demonstrate compliance with consumption of the EFA supplement, and
demonstrate a large effect on PUFA levels in RBC. It should be noted that the EFA supplement included
mostly omega-3 fatty acids (609 mg/capsule), but also some omega-6 fatty acids (198 mg/capsule).
The percentage increase in EPA (+525%) was approximately 6 times higher than the increase in DHA
(+83%), which is consistent with the composition of the fish oil used in this study (approximately 4:1
EPA:DHA). Conversely, there were small but significant decreases in linoleic acid, dihomo-γ-linolenic
acid (DGLA), and arachidonic acid (AA); those small decreases may be partly due to competitive
absorption with the omega-3 fatty acids, and partly due to competition for delta-5-saturase and
delta-6-desaturases. Those desaturases are shared among omega-3, -6, and -9 fatty acids, and the
desaturases have a preference in the order of omega 3-> omega-6 > omega-9. So, giving large amounts
of omega-3 decreases the availability of the desaturases for omega-6 and omega-9, so less linoleic
acid is converted to DGLA and AA. The decrease in arachidonic acid likely results in less production
of pro-inflammatory eicosanoids, and that decrease is likely beneficial for some children with ASD.
Similarly, the increase in oleic acid (non-significant) and significant decreases in elaidic and palmitoleic
are also likely partly due to increased competition for the desaturases, so that less oleic is converted to
elaidic and palmitoleic; elaidic and possibly palmitoleic are harmful trans-fats. It is also possible that
the HGCSF diet, which recommended avoiding “junk food”, also helped decrease the level of elaidic
acid, which is the most common trans-fat in hydrogenated vegetable oils.
Overall, the increases in EPA and DHA, and decreases in AA and elaidic acid, are very positive,
and likely reduce inflammation throughout the body.
4.7.2. Vitamins
The treatment group had significant improvements in the level of many vitamins (B2, B5, B6,
folic acid, B12 (as cyanocobalamin), and CoQ10H2 ), but not all vitamins. This suggests that larger doses
and/or more bioavailable forms of the other vitamins are needed to have a significant effect on blood
Nutrients 2018, 10, 369 35 of 43
levels, and larger doses/absorption may in some cases result in greater therapeutic benefit. It also
suggests that the methylcobalamin was not absorbed, and that cyanocobalamin was not converted to
methylcobalamin in plasma, but this conversion can occur intracellularly, and the improvement in
homocysteine (which requires methyl-B12) suggests that this conversion occurred. For future studies
we recommend increasing the level of vitamin D, since the current supplement did not increase levels
significantly despite supplementation levels well above the Recommended Dietary Allowance (RDA)
for vitamin D, and other studies of even higher doses of vitamin D for ASD reported significant
benefits [75,76].
4.7.3. Essential Elements (“Minerals”)

The treatment group had minor increases in selenium and chromium, and no other significant
changes. There were no significant differences in the levels of essential elements between the ASD
and neurotypical group at the start of the study, and only modest dosages of minerals were given in
the vitamin/mineral supplement. Since the body can regulate levels of most essential minerals by
adjusting absorption and excretion based on current mineral levels, it is not surprising that there was
little significant effect of modest mineral supplementation when mineral levels on average are in the
normal range.
4.7.4. Homocysteine
Compared to the non-treatment group, the treatment group had a significant decrease in
homocysteine, resulting in levels similar to (slightly below) the neurotypical group. This is presumably
partly due to the supplementation with folinic acid and vitamin B12, which are enzymatic cofactors for
recycling homocysteine to methionine, and partly due to supplementation with vitamin B6, which is
the enzymatic co-factor for converting homocysteine to cystathionine, and for converting cystathionine
to cysteine.
4.7.5. Carnitine
The carnitine test results demonstrated that both groups started at levels similar to the
neurotypical controls, and the treatment resulted in a significant increase in L-carnitine, and a similar
non-significant increase in acetyl-L-carnitine. Since the carnitine supplementation was primarily
acetyl-L-carnitine (there was a small amount of L-carnitine in the vitamin/mineral supplement),
this suggests inter-conversion between the two forms, as expected. However, the increase in plasma
carnitine was only approximately 25%, which was less compared to another study [50] which used an
equivalent dosage of L-carnitine and found a 70% increase in total carnitine in plasma, and another
study using twice the dosage of L-carnitine found even higher increases in carnitine levels [51].
Comparing the results of the present study with the other two ASD studies suggests that L-carnitine
may be better absorbed than acetyl-L-carnitine, and hence may be more effective. Indeed, L-carnitine
supplementation was found to result in several significant improvements in symptoms in children
with ASD in two other randomized, double-blind, placebo-controlled studies [50,51], whereas the
benefit in this study seemed to be more modest (see Table 20). Also, initial levels of plasma carnitine
were not good predictors of parents’ ratings of Treatment Effectiveness of carnitine—we hypothesize
that intracellular levels may be better predictors.
4.7.6. Digestive Enzymes

The digestive enzymes were not rated as highly as the three top-rated treatments. One randomized,
double-blind study did find that digestive enzymes were helpful for autism [74], but another similar
study did not find significant benefit [57]. So, it may be that the particular blend of digestive enzymes
is important.
Nutrients 2018, 10, 369 36 of 43
4.8. Case Studies

The three case studies suggest that nutritional deficiencies and/or food intolerances can have
significant effects, and the comprehensive nutritional/dietary treatment protocol is a safe and effective
way to identify and treat some intractable problems.
4.9. Age Effects

An evaluation of changes on all the outcome measures suggests that there was no significant
correlation of benefits with age, so children and adults of all ages are likely to benefit from this
combination treatment.
4.10. Developmental History

An evaluation of changes on all the outcome measures for the early-onset group vs. the regression/
plateau group did not reveal significant differences between the groups, but the sample size of
each subgroup is small so only large differences could be observed. So, it appears that individuals
with early-onset autism and regressive autism are both likely to benefit from this comprehensive
nutritional treatment.
4.11. Age and Gender Sub-Analysis

For age, there was a small correlation of adjusted age vs. change in CARS (r = −0.43) and average
PGI-R 2 (r = 0.40), suggesting the older participants improved somewhat more, but there was no
correlation of adjusted age vs. the other measures (CARS, SRS, SSP, ATEC, ABC, PDD-BI, Vineland,
PSAS, RIAS (VIX, NIX, CMX)). So, overall, age was not associated with degree of change, and all ages
(children and adults) seemed to improve approximately the same.
For gender, there were no significant differences on any of the behavioral assessments when
comparing males vs. females. The males improved slightly more on ABC, RIAS (NIX), PDD-BI,
and ATEC, and the females improved slightly more on SSP, PGI-R2, PSAS and Vineland, but these
differences were not significant. The other behavioral assessments (ADOS, SRS, CARS, and RIAS
(CMX, CIX and VIX)) were similar between males and females (less than 5% difference).
4.12. Limitations and Strengths

This study was designed to assess the possible benefits of a comprehensive nutritional and dietary
intervention. A strength of the study is that it was a randomized, controlled study, but a major
limitation of this study is that implementation of a healthy, HGCSF diet does not allow blinding of
participants. The RIAS evaluation was single-blinded, and the CARS and SAS-Pro were semi-blinded
(the evaluators were blinded, the participants were not), so those results are fairly robust. The parent
evaluations certainly are subject to some placebo-effect but provide an upper-bound on possible
benefits. The laboratory measurements were conducted in a blinded manner, so those results should
be reliable.
A strength of this study is the long-term nature (12 months), allowing a fuller determination of
possible benefits and adverse effects, which is important since the effect of nutritional interventions
is likely slower than the effect of pharmaceutical interventions, and since individuals with autism
often use nutritional supplements for long periods. The study was primarily designed to evaluate the
cumulative effect of all of the treatments, but the sequential administration of treatments provided
some insight into the relative merits of the individual treatments. A limitation of this study is its
exploratory nature, so that sample size estimates were not made formally due to lack of information
on the effect size of the combined treatment protocol.
A limitation of this study is that all participants received all treatments, whereas probably
only a subset are likely to benefit from any single intervention (for example, only participants
with low carnitine are likely to benefit from carnitine supplementation). Unfortunately, in many
Nutrients 2018, 10, 369 37 of 43
cases we do not have reliable biomarkers to accurately predict who is likely to benefit from a given
intervention. So, this study used a comprehensive nutritional and dietary intervention approach,
so that all participants were provided with all therapies, even though a given participant might only
benefit from a subset of those interventions. Future studies could try to determine which treatments
were most beneficial, using the results of this study to guide those future studies.
4.13. Relevance to Clinical Practice

The individual treatments used in this study have been used in previous studies, and were generally
well-tolerated with minimal adverse effects. This study demonstrates that the combination of all of
those treatments is feasible for most families, again with minimal adverse effects. The HGCSF diet had
the lowest compliance, but still most families managed to implement the diet successfully. The number
of pills required for all of the supplements was also a concern for some families, but splitting them
up into 2–3 times/day made it manageable for most participants. Figure 15 demonstrates that most
families wanted to continue with most treatments. So, we believe that the treatments used here should
be considered for use in clinical practice for most children and adults with ASD.
4.14. Suggestions for Future Research

The results of this study strongly suggest that future similar studies are warranted, with a focus on
vitamins/minerals, essential fatty acids, and HGCSF diet. A randomized double-blind placebo-controlled
study design could be considered if the diet portion is not included. More extensive biochemical
measurements, including metabolomics, may help determine how to optimize vitamin/mineral
supplements and other treatments. For essential fatty acids, higher doses may be warranted, as even
higher doses are used for improving cardiac health. For carnitine, switching to L-carnitine may improve
benefit. For digestive enzymes, it appears more research is needed to improve them. For Epsom salt
baths, it was previously shown that the vitamin/mineral supplement (with MSM, a source of sulfate)
was only partially able to improve levels of plasma sulfate [15], and biochemical measures are needed
to determine if the addition of the Epsom salt baths was sufficient to meet biological needs for sulfate,
the fourth most abundant mineral in the body.
5. Conclusions
The study results suggest that the comprehensive nutrition/diet protocol was safe and effective.
The nutritional supplements and healthy diet improved nutritional status, and hence presumably
increased the brains ability to function and learn. This is supported by the increase in non-verbal
IQ, and the substantial 18-month increase in developmental ability in communication, daily living
skills, and social skills. Modest improvements in CARS-2 and SAS-Pro suggest some reduction in
autism symptoms, consistent with parent reports of improvements on the PDD-BI, ATEC, and SRS.
Parent reports also suggest improvements in aberrant behaviors (ABC—Irritability, Lethargy/Social
Withdrawal, Stereotypy, and Hyperactivity), sensory processing (SSP), and GI symptoms (6-GSI, PGI-2,
ATEC), and Overall (PGI-2). There was not a significant effect on handgrip strength. The treatment
efficacy seemed to be similar for both genders and all ages, probably because nutritional requirements
are similar for both genders and all ages (after normalizing for caloric intake).
The three unusual case reports, in which three very different long-term problems were greatly
improved, shows the power of comprehensive nutritional interventions in addressing complex,
puzzling medical conditions which may involve one or more nutritional deficiencies.
There were many significant increases in vitamins, essential fatty acids, and carnitine, and an
improvement in homocysteine. The vitamin/mineral supplement and essential fatty acids appeared
to have the most clinical benefit, although other treatments appeared to have some benefit for some
individuals. So, this comprehensive treatment approach is recommended as a promising therapy for
children and adults with ASD, with an emphasis on the vitamin/mineral supplement and essential
fatty acids as probably being the most helpful.
Nutrients 2018, 10, 369 38 of 43
The data also suggests some possible improvements could be made to the treatment combination.
Specifically, it appears that L-carnitine may be better absorbed than acetyl-L-carnitine. Also, although
many vitamins were well-absorbed, larger doses and/or more bioavailable forms of the other vitamins
are needed to have a significant effect on blood levels, and larger doses/absorption may in some cases
result in greater therapeutic benefit. So, it seems likely that the current treatment protocol could be
further improved by making these changes.
Acknowledgments: We thank the Autism Research Institute and the Zoowalk for Autism Research for funding
this research study. We thank Nordic Naturals for providing the essential fatty acid supplement. We thank Yasoo
for providing the vitamin/mineral supplement (now available from www.AutismNRC.org). We thank NOW
for providing the carnitine supplement. We thank Houston Enzymes for designing and providing the digestive
enzyme. We thank Walgreens Pharmacy for supplying the Epsom salts. We especially thank all the ASD families
and neurotypical families who assisted with this study. We thank Aniyka Agrawal, Sara Dessoy, Crystal Gutgsell,
Chiao-May Lee, Rebecca Smith for help with data entry/analysis. We thank Nick Tkacenko, Stephanie Seitz,
Sandra Ezcurra, and Michelle McConnell for help with phlebotomy and blood processing. We thank Marie Adams
for measuring handgrip strength and general nursing assistance.
Author Contributions: All authors read and approved the final manuscript. J.B.A. was the PI of the study,
designed the study, oversaw the study, and led the analysis and writing of the paper. T.A. helped design the
study and the levels of supplements and interpret results. E.Geis was the study nurse, and E.Gehn was the study
coordinator. E.L.P. and J.I. conducted the ADOS, RIAS, CARS, and SAS evaluations. J.M. and R.H. were the
study physicians. D.L. and J.S.M. were the study nutritionists and trained the families in how to implement the
special diet. V.F. led most of the data entry and assisted with the data analysis. R.L.A. conducted the Vineland
assessments. K.L., J.C.N. and R.K.N. conducted the measurements of vitamins and carnitine. D.M.C. led the final
data entry and assisted with the data analysis. D.W.Q. oversaw the measurements at Doctor’s Data and assisted
with interpreting the results.
Conflicts of Interest: J.B.A. is the president of the Autism Nutrition Research Center (ANRC), a non-profit
which provides information to autism families and which produces an improved version of the vitamin/mineral
supplement used in this study. He serves as an unpaid volunteer, and does not receive any royalties from the sale
of the vitamin/mineral supplement. T.A. consults for Health Diagnostics, a commercial testing lab. D.W.Q. works
at Doctor’s Data, a commercial testing lab. The other authors do not have any competing interests.
Abbreviations
6-GSI 6-item Gastrointestinal Severity Index
AA Arachidonic Acid
ABC Aberrant Behavior Checklist
ADHD Attention Deficit Hyperactivity Disorder
ADOS Autism Diagnostic Observation Schedule
AGG Aggressiveness
AROUSE Arousal Regulation Problems
ASD Autism Spectrum Disorder
ASU Arizona State University
ATEC Autism Treatment Evaluation Checklist
ATP Adenosine Triphosphate
BMI Body Mass Index
BUN Blood Urea Nitrogen
CARS-2 Childhood Autism Rating Scale 2
CBC Complete Blood Count
CGI Clinical Global Impressions
CLIA Clinical Laboratory Improvements Amendments
CRP C-Reactive Protein
DGLA Dihomo-Gamma-Linolenic Acid
DHA Docosahexaenoic Acid
EFA Essential Fatty Acids
EPA Eicosapentaenoic Acid
GABA Gamma-Aminobutyric Acid
GFCF Gluten-Free, Casein-Free
GLA Gamma-Linolenic Acid
HGCSF Healthy Gluten-Free Casein-Free Soy Free
IRB Institutional Review Board
LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry
LMRL Learning, Memory, and Receptive Language
Nutrients 2018, 10, 369 39 of 43
MCV Mean Corpuscular Volume

n.s. Not Significant
NADH Nicotinamide Adenine Dinucleotide
NADPH Nicotinamide Adenine Dinucleotide Phosphate
PDD-BI Pervasive Developmental Disorders Behavior Inventory
PDD-NOS Pervasive Developmental Disorder-Not Otherwise Specified
PGI-2 Parent Global Impressions-2
PUFA Polyunsaturdated Fatty Acid
RBC Red Blood Count
RIAS Reynolds Intellectual Assessment Scales
S.D. Standard Deviation
SAM S-Adenosylmethionine
SAS-Pro Severity of Autism Scale
SEMPP Semantic/Pragmatic Problems
SOCPP Social Pragmatic Problems
SRS Social Responsiveness Scale
SSP Short Sensory Profile
VABS-II Vineland Adaptive Behavior Scale II
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© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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PERFIL NUTRICIONAL DE CRIANÇAS PORTADORAS DO TRANSTORNO DO

Maria Vanuza Caetano

Daniel Cordeiro Gurgel

Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018 1

Caetano MV, Gurgel DC

Descriptores: Evaluación Nutricional; Consumo de Alimentos; Trastorno Autístico.

2 Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018

Perfil nutricional em criança autista

Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018 3

Caetano MV, Gurgel DC

4 Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018

Perfil nutricional em criança autista

Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018 5

Caetano MV, Gurgel DC

Classificação do estado nutricional

6 Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018

Perfil nutricional em criança autista

Classificação de composição corporal

Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018 7

Caetano MV, Gurgel DC

8 Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018

Perfil nutricional em criança autista

Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018 9

Caetano MV, Gurgel DC

10 Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018

Perfil nutricional em criança autista

Endereço para correspondência:

Rev Bras Promoç Saúde, Fortaleza, 31(1): 1-11, jan./mar., 2018 11

relato de caso Seletividade alimentar: uma abordagem nutricional

A seletividade alimentar é caracterizada por recusa alimentar, pouco apetite e desinteresse

Endereço para correspondência: Karin Louise Lenz Dunker

INTRODUÇÃO porcentagem de proteínas, carboidratos, gorduras, além do

J Bras Psiquiatr. 2013;62(2):164-70.

J Bras Psiquiatr. 2013;62(2):164-70.

J Bras Psiquiatr. 2013;62(2):164-70.

Tabela 1. Evolução do padrão nutricional nas consultas

Tabela 2. Evolução da introdução dos alimentos nas consultas

J Bras Psiquiatr. 2013;62(2):164-70.

J Bras Psiquiatr. 2013;62(2):164-70.

J Bras Psiquiatr. 2013;62(2):164-70.

J Autism Dev Disord

Feeding Problems and Nutrient Intake in Children with Autism

Nadrat N. Nuhu • Elizabeth Marvel • Celine A. Saulnier •

Ó Springer Science+Business Media New York 2013

Abstract We conducted a comprehensive review and Introduction

J Autism Dev Disord

J Autism Dev Disord

J Autism Dev Disord

J Autism Dev Disord

Table 1 Summary of articles by journal and year of publication Growth Status

Nadon Provost Raiten Schmitt Schreck Shearer Zimmer Total % of total

Range 37.2–153.6 36–72 84–120 60–144 8 47

TD typically developing, DD other developmental delay, SB siblings

J Autism Dev Disord

All groups 15 0.89 (0.08) 5.11 3.74 6.97 \0.001

Calcium 8 -0.65 (0.29) 0.31 0.11 0.85 0.022

J Autism Dev Disord

Study Name OR (95 % CI) OR (95 % CI)

Bandini et al. (2010) 4.91 (2.40 - 10.1)

Fig. 2 Forest plot of feeding problems with 95 % confidence intervals

J Autism Dev Disord

J Autism Dev Disord