Ketogenic diet a potential therapy for autism by reshaping gut bacteria and brain inflammation

By Vijay Kumar MalesuReviewed by Benedette Cuffari, M.Sc.May 9 2024

In a recent study published in the journal Nutrients, researchers explore the impact of a modified ketogenic diet (KD) on gut microbiota, inflammatory markers, and brain-related microribonucleic acid (miRNA) in children with autism spectrum disorder (ASD) and its correlation with improvements in behavioral symptoms.

Study: Ketogenic Diet Induced Shifts in the Gut Microbiome Associate with Changes to Inflammatory Cytokines and Brain-Related miRNAs in Children with Autism Spectrum Disorder. Image Credit: SewCreamStudio / Shutterstock.com

Systemic symptoms of ASD

ASD is characterized by social and communication challenges and repetitive behaviors. Affecting over one in 100 children globally and as many as one in 36 children in the United States, ASD is also associated with immune dysfunction and increased inflammation, with higher levels of pro-inflammatory cytokines correlating with symptom severity.

Children with ASD often experience unique T-cell activation and prevalent gastrointestinal issues, thus suggesting a disrupted gut microbiome. Dietary approaches like the KD, which increases blood ketone levels, have shown promise in improving symptoms by potentially offering neuroprotective benefits through the gut-brain axis. However, the interactions between gut microbiota, ketone bodies, and butyrate require further research to develop effective therapies.

About the study 

The current study recruited participants at Shriner’s Hospital for Children in Honolulu. A registered dietician/nurse customized a KD for ASD metabolic needs by incorporating medium-chain triglyceride (MCT) oil and maintaining gluten restriction.

The maximum daily carbohydrate intake was 20-25 grams, with protein adjusted by weight and age and additional calories provided by fats. MCT oil comprised 20% of energy needs, with caregivers monitoring ketosis through urine tests.

Blood samples were processed to separate plasma and cells, with plasma stored at -80°C. Stool samples were collected using a prescribed system, stored in a stabilization solution, and processed for deoxyribonucleic acid (DNA) and RNA extraction. The analysis included polymerase chain reaction (PCR) amplification, sequencing for microbial diversity, and quantitative PCR (qPCR) for gene expression.

Plasma cytokines and brain-derived neurotrophic factor (BDNF) levels were also measured using a multiplex immunoassay. Moreover, miRNA was extracted from plasma samples using the TaqMan Advanced miRNA complementary DNA synthesis and MagMAX mirVana Total RNA Isolation Kits, which were subsequently processed with the KingFisher DUO Prime system.

The qPCR assays for miRNAs such as miR-125b-5p, miR-132-3p, miR-134-5p, miR-375, and miR-134-3p were performed in triplicate using the StepOnePlus system. These results were normalized against miR-361-5p and analyzed with a two-tailed t-test to assess expression levels. Statistical analyses were conducted to evaluate the significance of post-diet changes.

Study findings 

Throughout the four-month study period, stool and blood samples were provided for analysis before and after the diet. To confirm the physical state of ketosis, plasma levels of ketone bodies were measured through an enzyme-linked immunosorbent assay (ELISA) assay. To this end, a significant increase in the ketone bodies, acetoacetic acid, and hydroxybutyric acid was observed, thus indicating that the KD was effectively altering metabolism.

Sequencing data from stool samples revealed alterations at the phylum level and a significant increase in microbial diversity, as indicated by Shannon and Simpson indices. These changes occurred across various taxonomic levels, with substantial increases in beneficial bacteria like Lactobacillales and decreases in others such as Bacteroidaceae and Ruminococcus. 

In general, reduced pro-inflammatory cytokine levels were observed in individuals who consumed a KD, with significant reductions in interleukin 1β (IL-1β) and IL-12p70 levels. While the anti-inflammatory cytokine IL-10 increased, it was not statistically significant, which might be due to the small sample size. A significant decrease in plasma levels of BDNF, a protein implicated in neuroinflammation and ASD, was also observed.

Significant reductions were observed in miR-134 and miR-132 levels, both of which are associated with BDNF activity in the brain. While the levels of miR-125b remained unchanged, miR-375 levels significantly increased. 

Conclusions

Previous studies have reported improved social interactions in children with ASD due to increased levels of the ketone body β-hydroxybutyrate after following a modified KD. This suggests that ketosis might directly enhance brain activity by altering metabolic pathways, thereby potentially improving sociability in ASD patients.

However, recent findings reveal more extensive effects of KD, including significant changes in the gut microbiome, inflammation levels, and circulating miRNAs associated with BDNF. These results align with existing research indicating that KD could enhance social behavior in ASD by reducing brain inflammation and improving metabolic and gut health.