Serum Biomarkers to Mild Cognitive Deficits in Children and Adolescents

Intellectual disability (ID) is a condition characterized by significant limitations in both cognitive development and adaptive behavior. The diagnosis is made through clinical assessment, standardized tests, and intelligence quotient (IQ). Genetic, inflammation, oxidative stress, and diet have been suggested to contribute to ID, and biomarkers could potentially aid in diagnosis and treatment. Study included children and adolescents aged 6–16 years. The ID group (n = 16) and the control group (n = 18) underwent the Wechsler Intelligence Scale for Children (WISC-IV) test, and blood samples were collected. Correlations between biomarker levels and WISC-IV test scores were analyzed. The ID group had an IQ score below 75, and the values of four domains (IQ, IOP, IMO, and IVP) were lower compared to the control group. Serum levels of FKN, NGF-β, and vitamin B12 were decreased in the ID group, while DCFH and nitrite levels were increased. Positive correlations were found between FKN and the QIT and IOP domains, NGF and the QIT and IMO domains, and vitamin B12 and the ICV domain. TNF-α showed a negative correlation with the ICV domain. Our study identified FKN, NGF-β, and vitamin B12 as potential biomarkers specific to ID, which could aid in the diagnosis and treatment of ID. TNF-α and oxidative stress biomarkers suggest that ID has a complex etiology, and further research is needed to better understand this condition and develop effective treatments. Future studies could explore the potential implications of these biomarkers and develop targeted interventions based on their findings.


Introduction
Intellectual disability (ID) is equivalent to the term intellectual development disorder in the International Classification of Diseases (ICD-11) [1].The symptoms include deficits in intellectual and adaptive functions, in the conceptual, social, and practical domains.The prevalence of this condition in the general population is estimated to be around 1%. [2].ID is mainly characterized by global developmental and behavioral limitations [3], which involves a complex mixture of multiple genetic and environmental factors, interacting in a non-linear and non-additive way [4].
The diagnosis of ID is made by clinical assessment and standardized tests of adaptive and intellectual function that include the domains: of verbal comprehension, working memory, quantitative reasoning, abstract thinking, and cognitive efficiency [2], in addition to the intelligence quotient (IQ), being classified as mild, moderate, severe, and profound.Mild ID does not substantially affect or affect daily activities or cause disability for work or school activities [5].It accounts for approximately 85% of diagnosed cases.[2].Several genetic syndromes, such as Down syndrome (DS), fragile X syndrome, and Prader-Willi syndrome, are associated with ID.Environmental factors that can contribute to ID include prenatal exposure to drugs or alcohol, infections, and malnutrition.However, in many cases, the cause of ID remains unknown [6,7].To date, no specific biomarker has been identified for ID, whereas several biomarkers are currently being proposed for attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder [8].
Neuroinflammation and trauma can trigger peripheral immune cells to enter the brain and activate glial cells, leading to cognitive deficits and neurodegenerative diseases [9].The production of chemokines and cytokines, such as tumor necrosis factor (TNF-α) and interleukins (IL), can induce changes in cytoarchitecture and communication between astrocytes and neurons.Activated astrocytes can release both cytokines and neurotrophic factors, with the latter, such as neural growth factor-β (NGF-β), brain-derived neurotrophic factor (BDNF), and S100, playing a role in resolving inflammatory processes [10].Serum levels of cytokines and neurotrophins serve as biomarkers in patients with schizophrenia and depression and are associated with cognitive dysfunction [11][12][13] Neurotrophins are neuroprotective, promote the growth and differentiation of developing neurons, regulate axonal and dendritic growth, and survival of mature neurons [14] acting in a series of processes and reactions with a high degree of complexity, among them: the consolidation of the behavioral state, the formation of memories (short or lasting), and the development of general learning [15,16].Moreover, the chemokine fractalkine (FKN or CX3CL1), secreted from neurons, binds to CX3C chemokine receptor 1 (CX3CR1), which is expressed mainly in the microglia [17,18].FKN is presently bound to endothelial surface cells and can be cleaved to a soluble form, acting as a chemotactic substance for mononuclear cells [19].FKN has an anti-inflammatory function, maintaining microglia in a non-neurotoxic and non-activated state [20].Modification in FKN communication and activation of the CX3CR1 receptor can generate many progressive neuroinflammatory disorders [17].
The brain's high-energy requirements and low antioxidant capacity make it vulnerable to oxidative stress, which can contribute to neurological changes and cognitive deficits [21].Chronic oxidative stress has been linked to the development of dementia [22,23].Children with autistic spectrum syndrome may experience changes in neurotrophin levels and an increase in oxidative stress [24].Nutritional deficiencies, such as vitamin B12 deficiency, can also affect brain function by increasing homocysteine levels, which can compromise neurovascular function and increase the risk of cognitive impairment [25].Premature children may have reduced levels of substances that oppose oxidative stress, including vitamin E and vitamin A, as well as lower concentrations of functional enzymes SOD and CAT [26,27].Vitamins have positive effects on cognitive domains (memory, learning, processing, and functionality), in addition to raising the antioxidant standard and promoting neuroprotective effects [28,29].The objective of the work was to search for biomarkers that can be used for a better diagnosis and treatment of mild ID in children and adolescents.

Materials and Methods
The study is a case-control type, which evaluated children and adolescents of both sexes, aged between 6 and 16 years, attended by the multidisciplinary team of the Centro Especializado em Reabilitação (CER, Criciúma, SC).The subjects were initially invited, authorizing their participation by signing a Free and Informed Consent Term (FICT).

Inclusion and Exclusion Criteria
Male and female children and adolescents, under the age of 18, who were able to answer the tests available in the survey and who had signed the informed consent form by their parents and/or guardians, were included.Children and adolescents with any type of neurological diagnosis (physical neurological disease) were excluded from the research, including autism, Down syndrome (DS), Rett syndrome, Fragile X syndrome, and cerebral palsy.Failure to complete the informed consent of parents and/or guardians also led to the cancellation of participation in the study.

Neurocognitive Assessment
Children and adolescents were assessed using the Wechsler Intelligence Scale for Children (WISC-IV).The WISC-IV is a psychometric test that checks for deficits in intellectual function, consisting of 10 central subtests (Table 1), which are divided into four domain-specific factors, namely ICV, Verbal Comprehension Index; IOP, Perceptual Organization Index; IMO, Operating Memory Index; and IVP, Processing Speed Index.The Total Intelligence Quotient (IQ) is obtained by calculating the score of the 10 central subtests [30].Those who had scores of two standard deviations or more, below the population mean, with a measurement error margin (generally, + 5 points), in a general score of 65-75 points (70 ± 5) were diagnosed with ID.

Levels of Vitamin B12 and Vitamin D
The samples were sent to Diagnósticos do Brasil laboratory.Results for vitamin B12 that were expressed in picograms per milliliter, vitamin D in nanograms per milliliter, and protein C reactive (PCR) in milligrams per milliliter were evaluated by chemiluminescence (Diagnósticos do Brasil, 2019).

Redox State Markers
Serum samples from children and adolescents in both groups were used to determine serum levels of redox status markers.

GSH levels
GSH was measured after protein precipitation with 1 mL 10% trichloroacetic acid protein to which an 800 mM phosphate buffer, pH 7.4, and 500 µm of 2,2′-dinitro-5-5'-dithiodibenzoic acid was added.(DTNB).Absorbance was read at 412 nm.A standard curve of reduced glutathione was used to calculate GSH levels in samples [31].

NO
The production of NO was estimated spectrophotometrically from the formation of nitrite (NO 2 − ).The serum was incubated with Griess' reagent (1% sulfanilamide in 0.1 mol/L HCl and 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride) at room temperature for 10 min.Then, the absorbance was read at 540 nm using a microplate reader.The nitrite content was calculated based on a standard curve constructed with NaNO 2 [31].

DCFH Oxidation
Serum samples were incubated with 10 µM DCFH-DA at 37 °C for 30 min and then placed at 4 °C.DCFH-DA was deesterified within cells by endogenous esterases to ionized free acid.The oxidation of DCFH to 2′,7′-dichlorofluorescein (DCF-DA) was monitored with excitation and emission at wavelengths of 488 and 525 nm, respectively, using a SpectraMax fluorescence spectrophotometer (California, USA) [31].

Protein Carbonylation
The concentration of carbonylated proteins was measured by the initial formation of hydrazone derivatives of labeled protein using 2,4-dinitrophenylhydrazine (DNPT).These derivatives were extracted sequentially with 10% trichloroacetic acid followed by treatment with ethanol/ethyl acetate, 1:1 (vol/vol), and re-extraction with 10% trichloroacetic acid.Results were shown for each sample read at 370 nm on a spectrophotometer [31].

SOD was measured by inhibition of adrenaline oxidation.
Serum samples were homogenized in 20 mM sodium phosphate buffer + 140 mM KCl pH 7.4 and catalase (0.0024 mg/ mL), glycine buffer (32 °C, pH 10.2), and 5 µL adrenaline were added (60 mM).Readings were taken for 180 s at 10 s intervals and measured in an ELISA reader at 480 nm.Values were expressed in units of SOD per milligram of protein (U/mg of protein) [31].

Statistical Analysis
To determine the distribution of data regarding normality, the Shapiro-Wilk test was used.Quantitative variables were presented as mean and standard deviation.To compare the means of the quantitative variables between the control and

Epidemiologic Data
Data regards to sex, age, and birth data from control and ID patients are shown in Table 1.No significant differences were observed between control and ID patients, indicating that these data did not confound the presented results.

Cognitive Assessment
Children and adolescents in the ID group had a mean score below 75 on the IQ test characterizing ID, with exception to IOP domain which the value was 75.33, and the values of four of the five domains evaluated were lower when compared to the control, namely IQ (p < 0.001), IOP (p = 0.017), IMO (p = 0.001) and IVP (p = 0.024) (Table 2).

Serum Biomarkers
Biochemical analyses of serum biomarkers IL-1β, TNFα, PCR, FKN, IL-4, BDNF, NGF-β, Enolase NSE, S100β vitamin B12, vitamin D, DCFH, nitrite, SOD activity, and GSH levels are described in Table 3. Serum levels of FKN, NGF-β, and vitamin B12 were decreased in the ID group compared to the control group (p < 0.001).DCFH and nitrite levels were increased in the ID group compared to the control group (p < 0.001).The other markers showed no difference between the groups (Table 3).

Correlation of WISC-IV Test and Serum Inflammatory Markers
The correlation in all subjects of study between the quantitative indicators of the WISC-IV test and the serum markers showed a positive correlation between FKN and the QIT and IOP domains (rs = 0.671 and p = 0.002; rs = 0.679 and p = 0.003 respectively) (Table 4).The inflammatory marker TNF-α showed a negative correlation with the quantitative indicator of the ICV test (rs = − 0.470 and p = 0.049).
There was a positive correlation between NGF with QIT and IMO (rs = 0.624 and p < 0.01).Vitamin B12 showed a moderate positive correlation with the ICV cognitive parameter (rs = 0.669 and p < 0.05) (Table 4).The other markers showed no correlation with the WISC-IV test.

Discussion
ID is a condition with unknown etiology that impairs children's functional capacities, posing challenges for their development.Identification of biomarkers is crucial for improving the diagnosis and treatment of ID, particularly for patients who are often neglected by society.In our study, reduced FKN, NGF-β, and B12 vitamin levels were correlated with cognitive impairment in ID, suggesting their potential use as biomarkers for the condition.
The FKN regulates neuron-glia communication, cognitive function, and synaptic plasticity; nonetheless, decreased on FKN pathways are associated with cognitive deficits in animal models, DS, and dementia patients [32].Our study found a decrease in FKN serum levels in ID patients.FKN has controversial data in the brain, where its low activity could be protective or detrimental depending on the disease [33].Additionally, FKN serves as an endogenous neuronal control to the overproduction of iNOS, IL-1β, TNF-α, and IL-6, and FKN-deficient mice have higher microglial activation and decreased neurogenesis [34,35].The low levels of FKN in ID could indicate its involvement in the cognitive deficits of this population.Moreover, a reduction in FKN receptors impaired certain cognitive domains in mice [36].In our study, FKN levels were positively correlated with the QIT and IOP domains in all study participants, suggesting FKN as a potential biomarker for cognitive impairment in ID.
NGF-β has an important role in the in learning, memory and cognitive process [37,38].In aging, a decrease in NGF-β is associated with cognitive deficits, and studies have shown that NGF-β plays an important role in synaptic plasticity and memory pathways [39][40][41][42].Our results showed that ID patients had significantly lower levels of NGF-β compared to the control group, suggesting that this neurotrophin could serve as a specific biomarker for ID.In contrast, NGF-β levels are either increased or not altered in other neurodevelopmental disorders such as autism and epilepsy [43,  44].In our study, we found a positive correlation between NGF-β levels and the QIT and ICV domains across all study participants, further indicating the importance of NGF-β in cognitive function.Furthermore, while the levels of BDNF were not altered, they are recognized as a biomarker for other diseases such as depression and AD [45].Healthy food and adequate vitamin intake are essential for optimal brain performance.Vitamin B12 is a critical nutrient required for normal brain development and function [46].It is necessary for the folate-dependent enzyme, methionine synthase, which is involved in human methylation reactions such as myelin, DNA, and RNA methylation.Additionally, B12 is required for neurotransmitter synthesis and functioning [47].Our study found lower levels of B12 in ID patients.The lower serum B12 concentrations in adolescents is associated with significantly lower fluid intelligence scores, while higher child serum B12 concentrations were correlated with higher scores in attention and short-term memory [48].Moreover, we observed a positive correlation between B12 and ICV domain, indicating the importance of this vitamin to brain function.
The peripheral inflammatory process can affect the CNS and modify cognitive function [49].Microglia hyperactivation is expressed by a pro-inflammatory phenotype in the brain, where it attracts peripheral immune system cells to the CNS causing neuronal apoptosis; this process can affect brain metabolism and cognitive function characterizing neuroinflammation [50] and induce cognitive deficits [51].None of the study participants exhibited any signs of inflammation, including low grade or trauma.Serum levels of TNF-α were similar between the groups, showing once again that ID patients did not present any classic inflammatory process; however, there was a negative correlation between TNF-α levels and the ICV domain when all individuals in the study were evaluated together, showing that this cytokine, despite not being a preponderant factor for the etiology of mild ID, is essential at least for mastering verbal comprehension.High levels of TNF-α decrease cognitive performance [52] and childhood trauma and inflammation confer vulnerability to the development of psychiatric and physical disorders later in life [53].
Oxidative stress, especially during the most sensitive and early stages of brain development, has an adverse impact on brain neurodevelopment [54] and is proposed as a common factor in neurodevelopmental disorders such as schizophrenia and autism [55].Our study found an increase in DCFH and protein carbonylation, indicating oxidative stress in ID patients, without changes in antioxidant defenses.In a cohort study, a relationship between oxidative stress and cognitive decline was demonstrated [56].In children with DS occur an increase in SOD and CAT enzymes and a decrease in GSH levels, which were also observed in attention-deficit/ hyperactivity [57,58].Increased protein carbonylation has been observed in Alzheimer's disease, diabetes, and DS [59], and existing treatments affect the levels of carbonyl groups pointing to this as a possible biomarker for some diseases such as ID [60,61].
In summary, our study identified several potential biomarkers, including FKN, NGF-β, and vitamin B12, that may be useful for improving the diagnosis and treatment of ID.These biomarkers are specific to ID and may provide valuable insights into the underlying mechanisms of the disease.Furthermore, our findings suggest that the complex etiology of ID involves both TNF-α and oxidative stress pathways, indicating the need for further research to fully understand the disease.Overall, our study highlights the importance of identifying reliable biomarkers for ID to improve patient outcomes and advance our knowledge of this debilitating condition.

Table 1
Sex, age, and data from birth of the control and ID patients Student t tests for independent samples were used.The correlation between the quantitative indicators of the WISC IV test with the inflammatory biomarkers and the oxidative stress biomarkers was investigated by calculating the Spearman correlation coefficient.

Table 2
Scores of cognitive domains Mean biomarker concentration, standard deviation (SD), and P-value in control (n = 18) and intellectual disability (ID) (n = 16) ICV Verbal Comprehension Index, IOP Perceptual Organization Index, IMO Operating Memory Index, IVP Processing Speed Index,

Table 4
Correlation between biomarkers and cognitive domains in all subjects of studyCorrelation value between biomarker and cognitive domainICV Verbal Comprehension Index, IOP Perceptual Organization Index, IMO Operating Memory Index, IVP Processing Speed Index, IQ Total Intelligence Quotient * Mean statistic correlation between biomarker and cognitive domain, p < 0.05