Urinary opioid peptides in children with autism spectrum disorders. A Pilot Study

1Introduction

Autism spectrum disorders (ASD) is a group of neurodevelopmental disorders that involve definite impairments in social interactions, disturbance in language, and a stereotyped pattern of behavior. According to the literature [1], the prevalence of ASD is estimated to be between 1–2%, making it a significant public health concern [1]. Importantly, these core symptoms often co-occur with a range of neurological and psychiatric comorbidities, including epilepsy, intellectual disabilities, ADHD, sleep disturbances, eating issues, self-injurious behavior, and notably, gastrointestinal problems [2–6]. This diversity of symptoms underscores the heterogeneity of children with autism, revealing ASD as a spectrum of neurobiological disorders with overlapping phenotypes and various etiological underpinnings.

Twin studies have provided substantial evidence, indicating a high concordance rate (60–90%) among monozygotic twins compared to a significantly lower rate among dizygotic twins. This high concordance rate strongly suggests a substantial genetic influence on ASD [7]. Nevertheless, the exact etiology and pathogenesis of ASD remain largely elusive. For most children with ASD, no single underlying cause can be identified, and there is a dearth of evidence-based biomedical investigations. The genetic mechanisms responsible for ASD are not fully understood, and we are confronted with a wide array of biochemical abnormalities and potential toxicological factors that may contribute, either individually or in combination with genetic vulnerabilities, to the manifestation of the ASD phenotype.

One long-standing hypothesis in ASD research is the opioid peptide excess [8–11]. The opioid peptide excess theory postulates that exogenous opioid peptides, which exhibit morphine-like activity, are derived from various dietary sources such as dairy and gluten-containing cereals. Dietary peptides are recognized as essential sources of exogenous opioids due to their structural similarities to endogenous opioids. The opioid excess hypothesis suggests that a combination of reduced concentrations and activity of digestive enzymes, including proteases and peptidases like the DPPIV enzyme [12, 13], along with increased intestinal permeability [14–16], may result in an elevated presence of biologically active opioid peptides from food sources. These dietary opioid peptides will not be produced endogenously; hence the level of detected peptides will also depend on the amount of milk protein and gluten consumed in the diet.

The main goal of the present pilot study was to determine whether opioid peptides from food could be detected in the urine applying a novel HPLC-MS analysis in a group of clinically well-characterized children suffering from ASD and compare with healthy children (HC).

2Ethics

Written informed consent was obtained from the parents of all study participants. The pilot study was approved by the Regional Committees for Medical and Health Research Ethics (REK). The study conforms with The Code of Ethics of the World Medical Association (Declaration of Helsinki), printed in the British Medical Journal (18 July 1964).

3Materials and methods

Study design: This study is a pilot study conducted as a case-control study.

4Control subjects (Healthy controls – HC)

Eighteen non-autistic children were included as controls, comprising 10 boys and 8 girls with age between 3 and 17 years (Mean 10.6). The children visited the Department of Pediatrics, Innlandet Hospital Trust for different kinds of medical conditions. They were all admitted to the same outpatient clinic by their general practitioner or pediatrician. They were not interviewed in depth and were not neurologically examined. The exclusion criteria for the control group included neurological and neurodevelopmental diseases or suffering from immune insufficiency and gastrointestinal (GI) symptoms. Children in the control group did not have ADHD or mild intellectual disability (ID) diagnoses and were from the same area as the patients with similar characteristics. They were selected to the study in a similar period of year, their urine tests were mostly taken in the same part of the day, as the subject group.

The mean age in the ASD group was 7.6 years (SD 2.7) and 10,6 years (SD 3.9) in the HC group (p = 0.003). The observed significant age difference between the two groups is important to consider when interpreting the study results and will be discussed in the Discussion part of thispaper.

5Results

Urine from 18 cases with ASD and 18 healthy controls (HC) were sampled and analyzed. Taken as a whole, the ASD group shows no significant differences in the concentration of the investigated peptides compared to the HC group (Table 2). When studying the occurrence/frequency of detected peptides in the two groups, we found that, when considering all the peptides together, only a significant increased BCM-8 in the autism group (p < 0.05, Fisher’s exact test) (Table 3) was found. The reason why we did not find other BMC and GE of significance in the urine besides BCM-8 is uncertain. Low concentration of the other peptides, coupled with the method’s detection limit (LOD) not being sufficiently low to detect them, could be one contributing factor. Other reasons could be separation issues, method optimization and sample handling. This must be addressed in future studies.

6Discussion

This study included a small number of patients simply because it was meant like a pilot study. We were able to detect peptides in urine at very low concentrations (pg/mL) using a highly sensitive method, which was the main goal of our pilot study. The urinary opioid peptide concentration did not reach statistically significant results when comparing the autism group to the control group. However, autistic children more frequently exhibited specific BCM-8 detection compared to the healthy controls (p < 0.05). According to our knowledge, there are no studies that can determine whether elevated levels of opioid peptides lead to more severe autism and gastrointestinal symptoms. The increase in opioid peptides is likely secondary or one of many contributing factors. Autism is a condition with a complex etiology. The questions are interesting, but to ascertain this, a much larger patient cohort is necessary. The same applies if we are to elucidate which symptoms correlate with elevated opioid peptides. It is beyond our capability to provide an answer based on this small pilot study. The primary goal of this pilot study was to determine whether there is indeed an increase in opioid peptides in a patient population comprising children with autism. It is worth mentioning that a single result with p < 0.05 could easily arise by chance at the 5% level when multiple tests were performed. BCM-8, as an opioid agonist with the potential to bind to μ-opioid receptors in the gastrointestinal tract and the brain, is of particular interest. BCMs, in general, are known to delay gastric emptying and inhibit intestinal transit by reducing the frequency and amplitude of intestinal contractions [18, 19]. Notably, in our study,47% of the ASD children reported gastrointestinal (GI) symptoms, while no children in the HC group exhibited these symptoms. Specifically, eight out of nine ASD children experiencing constipation were found to have detected BCMs, a combination that was absent in the healthy group, in line with the exclusion criteria for the HC group. This indicates a significant difference (p < 0.05) in the occurrence of gastrointestinal symptoms and constipation between the autism patient group and the healthy control group and the detection of BCMs. The result indicates a significant association between the occurrence of gastrointestinal symptoms and the detection of beta-casomorphin peptides in urine among autism patients.

Fig. 1

Detected BCM-8 in one ASD patient with Multiple Reaction Monitoring (MRM) (upper 2 illustration) and Internal Standard (lower 2 illustration) (extracted from Agilent MassHunter Quantitative Analysis 10.2).

Fig. 2

Detected BCM-8 in another ASD patient with two MRMs (2 upper row) and two Internal Standards (IS) MRMs (2 lower row) (extracted from Agilent MassHunter Qualitative Analysis 10.0).

Fig. 3

Calibration curve for BCM-8 from 0,025 ng/mL to 1,00 ng/mL (R² = 0,99).

There was a statistically significant difference in age between the two groups (p = 0.003, Independent t-test). Whether this difference may impact the result is uncertain, as there is no data to indicate any age-related changes in opioid peptides. Age can be a confounding factor and may influence the outcomes being studied. The age gap between the ASD and HC groups may have clinical implications. It could reflect differences in developmental stages or the progression of symptoms between individuals with ASD and typically developing individuals. Clinicians and researchers should be aware of these age-related variations when designing interventions or conducting assessments. While the age difference is statistically significant, further investigation is warranted to explore its clinical relevance. Future studies could delve deeper into how age influences the manifestation of symptoms and the presence of peptides in urine among individuals with ASD.

7Conclusions

Our main goal was to investigate if dietary opioid peptides are detectable in urine based on a novel LC-MS method. In our pilot study with few participants, we did not observe a significant increase in peptide concentration in the autism group compared to the control group. However, we did identify a heightened occurrence of BCM-8 in children with ASD when compared to their healthy counterparts. To advance our understanding of the potential link between opioid peptides and autism and gastrointestinal symptoms, larger, meticulously designed, and high-quality clinical trials are imperative. It is our opinion that this study should be followed by a larger and broader investigation. These future investigations should encompass the use of biomarkers and incorporate diet interventions. An extended study with several research groups involved and with a larger number of patients is planned.