Carbon monoxide (CO) correlates with symptom severity, autoimmunity, and responses to probiotics treatment in a cohort of children with autism spectrum disorder (ASD): a post-hoc analysis of a randomized controlled trial

Abstract

Background: Inflammation, autoimmunity, and gut-brain axis have been implicated in the pathogenesis of autism spectrum disorder (ASD). Carboxyhemoglobin (SpCO) as a non-invasive measurement of inflammation has not been studied in individuals with ASD. We conducted this post-hoc study based on our published clinical trial to explore SpCO and its association with ASD severity, autoimmunity, and response to daily Lactobacillus plantarum probiotic supplementation.

Methods: In this study, we included 35 individuals with ASD aged 3-20 years from a previously published clinical trial of the probiotic Lactobacillus plantarum. Subjects were randomly assigned to receive daily Lactobacillus plantarum probiotic (6 × 1010 CFUs) or a placebo for 16 weeks. The outcomes in this analysis include Social Responsiveness Scale (SRS), Aberrant Behavior Checklist second edition (ABC-2), Clinical Global Impression (CGI) scale, SpCO measured by CO-oximetry, fecal microbiome by 16 s rRNA sequencing, blood serum inflammatory markers, autoantibodies, and oxytocin (OT) by ELISA. We performed Kendall's correlation to examine their interrelationships and used Wilcoxon rank-sum test to compare the means of all outcomes between the two groups at baseline and 16 weeks.

Results: Elevated levels of serum anti-tubulin, CaM kinase II, anti-dopamine receptor D1 (anti-D1), and SpCO were found in the majority of ASD subjects. ASD severity is correlated with SpCO (baseline, R = 0.38, p = 0.029), anti-lysoganglioside GM1 (R = 0.83, p = 0.022), anti-tubulin (R = 0.69, p = 0.042), and anti-D1 (R = 0.71, p = 0.045) in treatment group.

Conclusions: The findings of the present study suggests that the easily administered and non-invasive SpCO test offers a potentially promising autoimmunity and inflammatory biomarker to screen/subgroup ASD and monitor the treatment response to probiotics. Furthermore, we propose that the associations between autoantibodies, gut microbiome profile, serum OT level, GI symptom severity, and ASD core symptom severity scores are specific to the usage of probiotic treatment in our subject cohort. Taken together, these results warrant further studies to improve ASD early diagnosis and treatment outcomes.

Trial registration: ClinicalTrials.gov NCT03337035 , registered November 8, 2017.

Keywords: Autism spectrum disorder (ASD); Autoantibodies; Biomarker; Carboxyhemoglobin; Early screening; Inflammation; Microbiome; Probiotics.

Conflict of interest statement

The authors declare no conflicts of interest. The funding sources and free probiotics/control product provider had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

© 2022. The Author(s).

Figures

Fig. 1

Timeline of study design, subject dropouts, and assessed variables

Fig. 2

Overview of proportion of participants with detected positive SpCO and serum inflammatory markers at baseline. For SpCO, a measured percentage of ≥ 2 is denoted as “positive”; for titers of anti-Dopamine Receptor D1, D2L, anti-Lysoganglioside GM1, anti-Tubulin, and activity of CaM Kinase II of baseline, normal levels are denoted as “negative” and both borderline and elevated levels are denoted as “positive;” all remaining indices are denoted based on the absence (“negative”) or presence at detectable levels (“positive”). GFAP and OT were not included due to lack of reference for the determination of cutoff values

Fig. 3

Correlations between SpCO, ASD socio-behavioral severity, and autoantibody titers at baseline and post-intervention with either probiotics (blue) or placebo (red). Shown correlations are based on Kendall’s correlation using a significance cutoff of α = 0.05. a SRS Total T-scores are positively correlated with SpCO for all subjects at baseline. b The absolute change in SpCO between weeks-0 and -16 is positively correlated with anti-lysoganglioside GM1 log-titers in those receiving the probiotic treatment

Fig. 4

Correlations between serum autoantibody titers, inflammatory markers, and ASD severity scores, at baseline and post-intervention. Shown correlations are based on Kendall’s correlation using a significance cutoff of α = 0.05

Fig. 5

GSI change and its correlations with inflammatory marker and ASD severity

Fig. 6

Differentially enriched microbiota between placebo and probiotic groups. Analyses was conducted at (a) baseline and (b) post-treatment for both groups via LEfSe

Fig. 7

Correlations between point measurements and change in Shannon index, OT, GFAP, ASD severity, and autoantibody titers at both baseline and post-intervention