J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Perspective

Volume 15, September 2021, pages 3-12

Is the gluten-free and casein-free diet efficacious in the treatment of childhood autism spectrum disorder?

**Table 1.** Hypothetical mechanisms of action of the GFCF diet in ASD
Hypothetical mechanism	Findings
Excess opioid activity	Opioid overactivity leading to decreased social behavior in animal models of ASD (Panksepp, 1979). Possible involvement of opioid receptors in the pathogenesis of ASD (Genuis and Lobo, 2014). Increased (and also decreased) levels of opioid-like peptides, produced through gluten and casein metabolism and entering the blood circulation through a “leaky gut” (D’Eufemia et al., 1996), in the serum, cerebrospinal fluid or urine of individuals with ASD (Pellissier et al., 2018).
Increased autoimmunity	Elevated markers of innate and adaptive immune response (cytokines, cytokine tumor necrosis factor-α etc.) in children with ASD (with regression) (Jyonouchi et al., 2001; Money et al., 1971) leading to a hypothetical “autoimmune phenotype” with environmental stimuli triggering an immune response which exacerbates ASD features. Hypothetical knock-on effects of dietary triggers on molecular pathways with high levels of antibodies against gliadin, casein and the digestive enzyme dipeptidyl peptidase IV, which is involved in the processing of gliadin (Vojdani et al., 2003, 2004a, 2004b).
Oxidative stress and inflammation	Impaired antioxidant defense in the cerebellum of individuals with ASD (Gu et al., 2013). Problems in the metabolism of nitrous oxide leading to increased oxidative stress (Frye and Slattery, 2016; Wrońska-Nofer et al., 2012). Increased oxidative stress triggered by gliadin (Monguzzi et al., 2019). Increased oxidative stress demonstrated by elevated markers of oxidative stress in untreated children with celiac disease (Stojiljković et al., 2007). Possible release of pro-inflammatory cytokines contributing to gastrointestinal inflammation (leading to “leaky gut”) due to oxidative stress associated with low glutathione levels (Elder et al., 2006).
Reactivity of antibodies to gluten products	Possible indication of gluten sensitivity in celiac disease and ASD by presence of antibodies against tissue-transglutaminase (Cervio et al., 2007), transglutaminase 6 (Hadjivassiliou et al., 2013; Józefczuk et al., 2018) and gliadin (Cade et al., 2000; De Magistris et al., 2013; Kawashti et al., 2006; Lau et al., 2013; Vojdani et al., 2003, 2004a, 2004b) Potential for antibodies and other irritants to travel along the gut-brain axis indicated by findings of inflammation and abnormal intestinal permeability in ASD (Ashwood et al., 2003; Ashwood et al., 2004; Jyonouchi et al., 2002; Souza et al., 2012; Torrente et al., 2002).
Gut microbiota perturbations	Possible link between abnormal gut microbiota composition and ASD (Finegold, 2011; Mulle et al., 2013; O’Mahony et al., 2015; Rosenfeld, 2015; Wang et al., 2013). Possible downregulation of gastrointestinal inflammation and intestinal permeability by interaction between GFCF diet and gut microbiota (Doenyas, 2018b).

Table 1. Hypothetical mechanisms of action of the GFCF diet in ASD

Hypothetical mechanism

Findings

Excess opioid activity

Opioid overactivity leading to decreased social behavior in animal models of ASD (Panksepp, 1979). Possible involvement of opioid receptors in the pathogenesis of ASD (Genuis and Lobo, 2014). Increased (and also decreased) levels of opioid-like peptides, produced through gluten and casein metabolism and entering the blood circulation through a “leaky gut” (D’Eufemia et al., 1996), in the serum, cerebrospinal fluid or urine of individuals with ASD (Pellissier et al., 2018).

Increased autoimmunity

Elevated markers of innate and adaptive immune response (cytokines, cytokine tumor necrosis factor-α etc.) in children with ASD (with regression) (Jyonouchi et al., 2001; Money et al., 1971) leading to a hypothetical “autoimmune phenotype” with environmental stimuli triggering an immune response which exacerbates ASD features. Hypothetical knock-on effects of dietary triggers on molecular pathways with high levels of antibodies against gliadin, casein and the digestive enzyme dipeptidyl peptidase IV, which is involved in the processing of gliadin (Vojdani et al., 2003, 2004a, 2004b).

Oxidative stress and inflammation

Impaired antioxidant defense in the cerebellum of individuals with ASD (Gu et al., 2013). Problems in the metabolism of nitrous oxide leading to increased oxidative stress (Frye and Slattery, 2016; Wrońska-Nofer et al., 2012). Increased oxidative stress triggered by gliadin (Monguzzi et al., 2019). Increased oxidative stress demonstrated by elevated markers of oxidative stress in untreated children with celiac disease (Stojiljković et al., 2007). Possible release of pro-inflammatory cytokines contributing to gastrointestinal inflammation (leading to “leaky gut”) due to oxidative stress associated with low glutathione levels (Elder et al., 2006).

Reactivity of antibodies to gluten products

Possible indication of gluten sensitivity in celiac disease and ASD by presence of antibodies against tissue-transglutaminase (Cervio et al., 2007), transglutaminase 6 (Hadjivassiliou et al., 2013; Józefczuk et al., 2018) and gliadin (Cade et al., 2000; De Magistris et al., 2013; Kawashti et al., 2006; Lau et al., 2013; Vojdani et al., 2003, 2004a, 2004b) Potential for antibodies and other irritants to travel along the gut-brain axis indicated by findings of inflammation and abnormal intestinal permeability in ASD (Ashwood et al., 2003; Ashwood et al., 2004; Jyonouchi et al., 2002; Souza et al., 2012; Torrente et al., 2002).

Gut microbiota perturbations

Possible link between abnormal gut microbiota composition and ASD (Finegold, 2011; Mulle et al., 2013; O’Mahony et al., 2015; Rosenfeld, 2015; Wang et al., 2013). Possible downregulation of gastrointestinal inflammation and intestinal permeability by interaction between GFCF diet and gut microbiota (Doenyas, 2018b).

**Table 2.** Summary of findings of GFCF dietary intervention studies in children with ASD
Highly divergent findings of GFCF diet intervention studies
Methodological flaws of many GFCF intervention studies: strong reliance on (unblinded) parental reports as the sole information source regarding ASD symptoms; frequent lack of control procedures (control groups, measurement baselines, control for additional treatments); frequent lack of assessment of treatment fidelity
Positive effects of GFCF diet adherence mainly found in studies conducted without adequate scientific rigor
No consistent pattern of findings following a GFCF diet in more rigorous scientific evaluations
More positive findings reported by case/group studies with longer follow-up periods
Summary: No clear-cut conclusions possible regarding GFCF diet effects in children with ASD

Table 2. Summary of findings of GFCF dietary intervention studies in children with ASD

Highly divergent findings of GFCF diet intervention studies

Methodological flaws of many GFCF intervention studies: strong reliance on (unblinded) parental reports as the sole information source regarding ASD symptoms; frequent lack of control procedures (control groups, measurement baselines, control for additional treatments); frequent lack of assessment of treatment fidelity

Positive effects of GFCF diet adherence mainly found in studies conducted without adequate scientific rigor

No consistent pattern of findings following a GFCF diet in more rigorous scientific evaluations

More positive findings reported by case/group studies with longer follow-up periods

Summary: No clear-cut conclusions possible regarding GFCF diet effects in children with ASD

**Table 3.** Methodological recommendations regarding GFCF studies in ASD (see also Reissmann et al., 2020)
Randomized controlled trial with adequate control condition and control for attention effects (e.g. nutritionist counseling) in control group
Assessment of additional therapies
Sufficient trial duration (>12 months) with multiple follow-up assessments
Ensuring and maintaining treatment fidelity (diet adherence)
Blinded clinician ratings in addition to parent ratings of behavior
Multi-method/multi-rater assessment using standardized assessment tools: clinical measures, rating scales for parents and teachers, neuropsychological and cognitive tests, ecologically valid behavioral observations in natural settings
Sufficiently large sample size (>30) with control of attrition
Risk measurement regarding nutritional status, physical development, bone density etc
Assessment of potential moderating and mediating variables, such as food allergies, gastrointestinal symptoms and behavioral symptoms (e.g. attention, hyperactivity)

Table 3. Methodological recommendations regarding GFCF studies in ASD (see also Reissmann et al., 2020)

Randomized controlled trial with adequate control condition and control for attention effects (e.g. nutritionist counseling) in control group

Assessment of additional therapies

Sufficient trial duration (>12 months) with multiple follow-up assessments

Ensuring and maintaining treatment fidelity (diet adherence)

Blinded clinician ratings in addition to parent ratings of behavior

Multi-method/multi-rater assessment using standardized assessment tools: clinical measures, rating scales for parents and teachers, neuropsychological and cognitive tests, ecologically valid behavioral observations in natural settings

Sufficiently large sample size (>30) with control of attrition

Risk measurement regarding nutritional status, physical development, bone density etc

Assessment of potential moderating and mediating variables, such as food allergies, gastrointestinal symptoms and behavioral symptoms (e.g. attention, hyperactivity)