The study was conducted with subjects diagnosed with autism spectrum disorder (ASD) according to DSM 5, among the patients who applied to the Child Psychiatry Outpatient Clinic of Ordu University Training and Research Hospital. The healthy control group was composed of volunteers. After these volunteers were evaluated with routine blood tests, they were evaluated by expert psychiatrists. Accordingly, subjects who did not have any problems in their routine tests and history and who did not have a psychiatric diagnosis were included in the healthy control group. The study was approved by Ordu University Clinical Research Ethics Committee (2018/97). In this study, the exclusion criteria were neurological deficit, obesity (BMI> 25), epilepsy, tumor, and history of systemic disease, and the use of psychotropic drugs in the last 6 months. The subjects and their families were informed in detail about the study and written consent forms were obtained. Then, some plasma obtained from blood samples taken during routine blood tests was reserved for this study and stored until measurements were made at -80 degrees.
Sociodemograhic data form
This form was developed by the researchers and contained information about gender, age, number of siblings, residence, history and order of pregnancy, week of pregnancy, form of birth, birth history, birth weight, birth complications, psychomotor development history, duration of breastfeeding, medical history, family history, medications used, special education status, and body mass index.
Childhood Autism Rating Scale
The childhood autism rating scale (CARS) comprises 15 items that are used to generate a total score defining the severity of autism. CARS rates the child on a scale from 1 to 4 in each of 15 areas (relating to people; imitation; emotional response; listening response; body use; object use; adaptation to change; visual response; taste, smell and touch responses; fear or nervousness; verbal communication; nonverbal communication; activity level; level and consistency of intellectual response; and general impressions). A total score of between 30 and 36.5 indicates mild-to-moderate autism, whereas the interval between 37 and 60 denotes severe autism. CARS is scored by observing the child and through interviews with the family (Esnafoglu and Ayyıldız 2017; Schopler et al. 1986; Sucuoglu et al. 1996).
Apo D Measurement
Plasma Apo D level was measured by a commercial kit using ELISA method (Cat. No. E-EL-H0469, Elabscience Biotechnology, Wuhan, China). The kit based on sandwich ELISA method, displayed a sensitivity of 0.38 ng/ml and the detection range was 0.63-40 ng/ml. According to kit protocol, the samples and standards were added to the wells pre-coated with antibody and then the manufacturer’s instructions were followed. After the enzyme-substrate reaction was terminated, the optical density was measured spectrophotometrically at a wavelength of 450 nm (BioTek, ELx800 brand REF ELX508 SN1310149). Apo D levels in the samples was calculated using the standard curve and the results were presented as µg/ml.
Apo E Measurement
A commercial sandwich-ELISA kit was used to measure plasma Apo E level (Cat. No. E-EL-H0470, Elabscience Biotechnology, Wuhan, China). The kit showed a sensitivity of 14.06 ng/ml and the detection range was 23.44-1500 ng/ml. The measurement was carried out in accordance with the kit protocol. Briefly, the samples and standards were added to the wells pre-coated with human Apo E antibody and incubated for 90 minutes at 37℃. Then, the biotinylated Apo E antibody was added to each well and incubated for 60 minutes at 37℃. After the washing process, Avidin-Horseradish Peroxidase conjugate was added to each well and incubated for 30 minutes at 37℃. At the end of the incubation, 3,3',5,5'-Tetramethylbenzidine (TMB) substrate solution was added and after a 15 min incubation period the enzyme-substrate reaction was terminated by the addition of a stop solution. The absorbance of resulting yellow product was measured spectrophotometrically at a wavelength of 450 nm (BioTek, ELx800 brand REF ELX508 SN1310149). Concentration of Apo E in the samples was calculated using the standard curve and the results were presented as µg/ml.
Statistical analyzes were made using the SPSS 22 software program. Normally distributed data were presented as mean ± standard deviation and those not showing normal distribution as median (minimum-maximum). It was examined whether the numerical values were normally distributed using the Sapiro Wilk test. Chi-square test was used for comparison of categorical variables. Student-t test was used for comparison of BMI, Apo D and Apo E numerical values between the groups, as they showed normal distribution. Since the age distribution was not normally distributed, the Mann Whitney U test was used. Spearman and Pearson tests were used in correlation analysis.