The study is a case-control type, which evaluated children and adolescents of both sexes, aged between 6-16 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). The project was approved by the Human Research Ethics Committee of the Universidade do Extremo Sul Catarinense, based on Resolution 466/12 of the National Health Council. The DI group was composed of 16 children and adolescents and the control group was composed of 18 children and adolescents, who were equated to the DI group according to age, gender, BMI, and socioeconomic/cultural conditions, preferably choosing relatives or other CER patients-UNESC who did not have a diagnosis of ID.
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 [26]. 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.
Biochemical analyzes
Participants had 20 ml of blood collected that were centrifuged and the serum frozen at -20ºC and used for further analysis. Serum samples from children in both groups were used to determine serum levels of various markers by ELISA method according to the manufacturer's recommendations. Nerve growth factor beta (NGF-β) (R&DS, Inc., DY556), Interleukin 1 beta (IL-1β) (LIFE TECHNOLOGIES, 88-7013-76), IL-4 (LIFE TECHNOLOGIES,88-7044-77), Tumor necrosis factor alpha (TNF-α) (LIFE TECHNOLOGIES, 88-7324-76), FKN (R&DS, Inc., DY537), Brain derived neurotrophic factor (BDNF) (R&DS, Inc., DY248), S100 (Merck, EZHS100B-33K) Neuron-specific enolase (NSE) (R&DS, Inc., DY5169-05).
Levels of Vitamin B12 and Vitamin D.
The samples were sent to Diagnósticos do Brasil laboratory. Results for vitamin B12 were expressed in pg/mL vitamin D in ng/ml and protein C reactive (PCR) in mg/mL 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.
Reduced Glutathione (GSH)
GSH was measured after protein precipitation with 1mL 10% trichloroacetic acid protein to which an 800 mM phosphate buffer, pH 7.4 and 500 µm of 2,2'-dinitro-5-5'-dithio-dibenzoic acid was added. (DTNB). Absorbance was read at 412nm. A standard curve of reduced glutathione was used to calculate GSH levels in samples [27].
Nitric Oxide (NO)
The production of NO was estimated spectrophotometrically from the formation of nitrite (NO2-). 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 NaNO2 [27].
DCFH oxidation
Serum samples were incubated with 10 µM DCFH-DA at 37°C for 30 minutes 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) [27].
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 [27].
Superoxide dismutase (SOD)
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 180s at 10s intervals and measured in an ELISA reader at 480nm. Values were expressed in units of SOD per milligram of protein (U/mg of protein) [27].
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 DI groups, Student t tests for independent samples were used. The association between qualitative variables and the Control and DI groups was investigated using Pearson's chi-square test or Fisher's exact test. 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.