This study aimed to contribute to the growing understanding of immune dysfunction in the pathophysiology of ASD by investigating protein separation patterns in Nigerian children with ASD using cellulose acetate electrophoresis. To achieve this objectives, 16 ASD children diagnosed by a child psychiatrist using DSM-5 were recruited from the Department of Child and Adult Psychiatry, UCH, Ibadan. The anthropometric characteristics of these cases were not significantly different from those of controls. This use of age-matched controls in this study enables reasonable comparison of biochemical parameters between cases and controls with close biological makeup, reducing the influence of confounding variables on our results [22].

The analysis of serum protein profiles revealed interesting findings. While the overall protein profiles between cases and controls were largely similar, significant differences emerged in specific fractions. Children with ASD exhibited significantly higher gamma globulin levels compared to neurotypical children. Interestingly, children with other NDDs in the control group showed significantly lower alpha1 globulin levels compared to the neurotypical group. Similarly, the trend toward lower albumin levels in the positive control group compared to the ASD and typically developing groups raises interesting questions about the role of serum albumin in NDDs. Further studies with larger sample sizes are needed to explore whether these trends could become significant and to understand the underlying mechanisms that may contribute to these differences. These findings highlight the potential for distinct protein profiles associated with different neurodevelopmental conditions. Studies on other psychological disorders support this notion. For example, lower serum total protein and albumin but increased serum alpha1 globulin was observed in patients with major depression [23]. Similarly, increased levels of serum total protein and various globulins (alpha1, alpha2, beta, and gamma) have been associated with psychological stress [24]. In the context of autism, Croonenberghs, Wauters [15] reported a unique protein profile in a Belgian population, characterized by elevated serum total protein, albumin, gamma globulin, and specific IgG subclasses (IgG2 and IgG4). Our study partially aligns with their findings regarding hypergammaglobulinemia in children with ASD compared to controls. However, it contrasts the findings of ElBaz, Zaki [16] who found no significant difference in serum protein levels between autistic children and healthy controls in Egypt.

The underlying mechanisms for the observed hypergammaglobinemia in our ASD group remain unclear. However, several potential explanations can be explored. One possibility involves autoimmune disorders, linked to NDDs, including ASD [4], which is often accompanied by a raised gammaglobulinemia resulting from polyclonal B-cell activation [25]. Apart from antibodies, proteins with gamma mobility (e.g., complement products) could also contribute to the increased gamma globulin [15]. Exposure to viral infections congenitally or during postnatal period have been implicated in the incidence of ASD [4]. Such chronic infectious state or increased susceptibility to infections may also contribute to increased gamma globulin observed in autistic children compared to neurotypical children. Furthermore, autistic children often presents with comorbidities such as gastrointestinal problems characteristic of inflammatory bowel disease [26]. This condition is accompanied with an increase in specific IgG subclasses [27], thereby raising gamma globulin concentration. These potential explanations for hypergammaglobulinemia in ASD require further investigation.

Another unique finding of this study involved the observation of lower alpha2 globulin in children with ASD and related NDDs compared to neurotypical controls. The difference, however, was statistically significant only between the NDD group and neurotypical children. While specific reasons remain unclear, this findings might be related to gastrointestinal problems, particularly Inflammatory Bowel Disease. This condition, commonly associated with NDDs, causes damage to the intestinal lining, resulting in the loss of blood proteins, such as alpha2 globulins [28]. Low alpha2 globulin can also occur in cases of malnutrition, megaloblastic anemia, and Wilson’s disease [29], conditions that may be particularly relevant to neurodiverse children due to their selective dietary habits. This observation contrasts with past research by Cortelazzo, De Felice [30], which found an elevated alpha2 globulins, particularly alpha2 macroglobulin, in ASD patients compared to controls. Alpha2 globulins play a crucial role in regulating immune response. For instance, alpha2 macroglobulin, due to its proteolytic activity, inhibit proteases released by neutrophils during inflammation or by invading microorganisms [31]. Therefore, the observed low alpha2 globulins in our study might indicate potential immune dysregulation in autistic children, warranting further exploration.

Beyond serum analysis, urine analysis offers a valuable non-invasive tool for assessing a wide range of plasma proteins, including low-molecular-weight proteins and various peptides [32]. This method can potentially reveal biomarkers for diseases not primarily affecting the kidneys, as pathological changes in the kidney's microvascular architecture often reflect systemic conditions. In our study, consistent with previous studies reporting higher protein excretion in autistic children [33, 34], we found significantly higher random urine protein levels in the ASD group compared to controls. This finding aligns with the frequent occurrence of gastrointestinal symptoms in autistic children, such as inflammation, diarrhea, and increased gut permeability, which allows smaller proteins and peptides to enter the bloodstream and be excreted in the urine [33].

Interestingly, despite the higher overall protein levels, urinary albumin excretion was similar across ASD and control groups in our study. This suggests that the elevated protein levels in ASD are likely due to the excretion of smaller, non-albumin proteins [34]. Furthermore, our use of cellulose acetate electrophoresis (CAE) for urinary protein separation revealed a normal urine pattern, with only faint albumin bands observed in all groups. This finding indicates that potential protein biomarkers in the urine of ASD children might be below the detection threshold of CAE, a technique known for its limitations in resolving heterogeneous proteins [35]. The higher spot urine protein levels in ASD children do not necessarily point to kidney pathology, as no significant differences were found in urine albumin levels between the ASD, positive control, and negative control groups. Albumin is typically the primary protein excreted in pathological kidney conditions; therefore, the absence of elevated albumin suggests that the increased protein excretion in ASD is likely not due to glomerular damage [36].

Several explanations could explain this observation. First, ASD has been linked to low-grade inflammation in the brain and body [9, 26]. This could lead to increased permeability of the glomerular filtration barrier in the kidneys [37], allowing non-albumin proteins, such as low molecular weight proteins, to pass into the urine [33]. Second, the unique behaviors often observed in ASD—such as hyperactivity, repetitive movements, and heightened stress responses—could contribute to transient increases in protein excretion, such as in exercise-induced proteinuria [38]. Third, differences in protein metabolism or turnover rates in children with ASD, possibly influenced by their selective dietary habits, could result in increased urinary excretion of specific proteins, contributing to the higher overall protein levels [39]. These metabolic variation might be independent of any renal pathology and could be linked to the unique physiological or nutritional profiles often seen in ASD. Nevertheless, the observed difference in overall urine protein levels underscores the need for further investigation into the urine proteome of ASD children using more advanced protein analysis techniques. This could potentially lead to the identification of novel urine protein biomarkers for ASD.

The correlation analysis revealed interesting associations between various protein fractions and urinary measures across the groups. Notably, a significant positive correlation was observed between urine protein and serum globulin in children with NDDs (excluding ASD). Similarly, the autistic group exhibited a significant positive correlation between beta globulin and urine microalbumin. These finding suggests a potential link between overall protein metabolism and urinary protein excretion in these children. Further investigation is warranted to elucidate the specific mechanisms underlying these relationships.