Apo D and Apo E Levels in Autism Spectrum Disorders

Apo D is an atypical plasma apolipoprotein and a member of the lipocalin protein superfamily. In recent years, Apo D has been identied as an important factor in the pathology of neurodegenerative and neuropsychiatric diseases. Apo D is highly produced in the brain and acts as a multiligand and multifunctional carrier. Apo D binds to and stabilizes Arachidonic acid in the cell membrane and cytosol. Thus, it suppresses inammation and protects the cell membrane against oxidation. Apo E is important in cholesterol transfer and it has been reported that it may play a role in immune regulation, nerve regeneration, synaptogenesis and neuronal homeostasis. In this study, plasma Apo D and Apo E levels were examined and its possible role in Autism Spectrum Disorders (ASD) pathology was investigated. Thirty-nine subjects with ASD were compared with 30 healthy subjects who typically developed. Accordingly, plasma Apo D and Apo E levels were statistically signicantly lower in the ASD group compared to the healthy control group. According to the results of this study, it can be suggested that low levels of Apo D and Apo E may play a role in ASD pathogenesis.


Introduction
Apolipoproteins are lipoprotein proteins and their important functions include; Helping the dissolution of non-polar lipids, participating in the activation of important enzymes in lipoprotein metabolism, providing lipoprotein uptake and destruction by binding to speci c receptors on the cell surface (Kelley 1997). In addition to their functions in lipid metabolism, the role of some apolipoproteins such as Apolipoprotein E (Apo E) and Apolipoprotein D (Apo D) in the pathology of various diseases has been emphasized in recent years.
Apo D is a 29 kDa glycoprotein of 169 amino acids. Although classi ed as apolipoprotein, Apo D is a member of the lipocalin family. In recent years, Apo D has been identi ed as an important factor in the pathology of neurodegenerative and neuropsychiatric diseases (Rassart et al. 2000; Thomas et al. 2001;2003a;Mahadik et al. 2002). Apo D is highly produced in the brain and functions as a multiligand and multifunctional carrier (Hu et al. 2001). It has been shown that Apo D can carry small hydrophobic molecules such as arachidonic acid (AA), steroid hormone, and cholesterol during the regulation of metabolism or signal transduction (Rassart et al. 2000; Dassati et al. 2014;Martínez et al. 2013). Apo D stabilizes AA in the cell membrane, and also binds to cytosolic free AA and prevents its normal functions. Thus, it restricts eicosanoid synthesis by removing AA from the cyclooxygenase pathway (Thomas and Yao 2007). Most eicosanoids such as prostaglandins and leukotrienes are mediators of in ammation.
In ammation has been reported to play a role in many psychiatric illnesses. It has also been reported that Apo D can protect the membrane against oxidation. Consistent with these functions, changes in Apo D expression have been reported to be associated with many pathological conditions such as breast cancer, prostate cancer, Parkinson's disease, Alzheimer's disease, schizophrenia, and bipolar disorder (Dassati et al. 2014).

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Apo E is a glycoprotein with a molecular weight of ~ 34 kDa and consisting of 299 amino acids (Rall et al. 1982;Weisgraber et al. 1994). In addition to its classical functions such as transport and uptake of lipids, it has been reported to be associated with cytoskeleton and microtubule remodeling, axon growth, synaptic plasticity and cell migration (Mahley 2016;Liu et al. 2013). Due to its wide-ranging role in neuronal signal transduction pathways, the place of Apo E in the pathology of various neurological diseases has started to attract attention. Studies have shown that Apo E is polymorphic and has many isoforms that can be detected by isoelectric focusing. Analysis of Apo E isoforms reveals two types of Apo E gene polymorphism that are genetically determined and cannot be determined. Genetically

Methods
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 de cit, 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 de ning 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. Brie y, 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
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.

Results
There were no statistically signi cant differences between the groups in terms of age, gender, and BMI.
The mean CARS score in the ASD group was 49.05 ± 5.53. Accordingly, disease severity was evaluated as severe in the ASD group. Apo D and E levels were found to be signi cantly lower in the ASD group (Table 1, Figs. 1 and 2). According to the correlation analysis, no signi cant correlations were found between Apo D and E levels and CARS score, age and BMI. The results found are shown in Table 1. Apo D is a soluble carrier protein that mediates the transport of steroid hormones such as progesterone and pregnenolone, as well as lipophilic molecules such as cholesterol and arachidonic acid (Rassart et al. 2000; Dassati et al. 2014). Recent data show that ApoD is not only a lipophilic molecule carrier, but also controls the fate of these ligands by altering their stability and oxidation state. It is particularly interesting that Apo D, which is expressed in neurons and glial cells in the central and peripheral nervous system, binds to arachidonic acid and its derivatives, which play a central role in normal brain functions (Hu et al. 2001;Dassati et al. 2014). Apo D has been shown to act as a catalyst for the reduction of peroxideed eicosanoids and reduce lipid peroxidation in the brain (Thomas et al. 2003b). It has been shown that the Apo D gene in the human brain is upregulated with aging (Perdomo and Dong 2009; Muffat and Walker 2010).
In addition, it has been found that Apo D levels in the nervous system increase in many neurological diseases such as Alzheimer's disease, schizophrenia, and stroke (Dassati et al. 2014). There is increasing evidence that Apo D has a neuroprotective role due to its antioxidant and anti-in ammatory activities. Apo D is an evolutionarily conserved protein and is considered an anti-stress protein because it is induced by oxidative stress and in ammation, and is seen as an effective therapeutic agent against various neuropathologies and even against aging (Dassati et al. 2014). In this study we conducted with ASD patients, it was observed that the plasma apo D level was statistically signi cantly lower compared to the control group. Considering its antioxidant and anti-in ammatory effects, apo D de ciency may have a role in the pathophysiology of ASD.
Apo E, found in all plasma lipoproteins, accounts for approximately 10-20% of VLDL and approximately 1-2% of HDL. Critical for cholesterol transport and lipoprotein particle metabolism, Apo E also plays a role in immune regulation, nerve regeneration, lipolytic enzyme activation, synaptogenesis. Apo E is synthesized in most organs and the second largest production site in the body is the brain (

Conclusion
In this study, it was determined that the levels of Apo D and Apo E in the plasma of ASD patients were signi cantly decreased. Considering the antioxidant and anti-in ammatory effects of Apo D and the role of Apo E in axonal growth and neuronal survival, the decrease in these proteins may play an important role in the pathophysiology of ASD. Approaches that will cause an increase in Apo D and Apo E levels can provide positive improvements in these patients. Yet whether the decrease in circulation re ects the decrease in brain tissue is unknown. This issue should be examined in more detail with both clinical and experimental studies. Figure 1 Apo D distribition in ASD and healthy control group Apo E distribition in ASD and healthy control group