Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 8, No. 6, 3808-3815 2024 Publisher: Learning Gate DOI: 10.55214/25768484.v8i6.2824 © 2024 by the authors; licensee Learning Gate © 2024 by the authors; licensee Learning Gate * Correspondence: hamedjadoa@yahoo.com Evaluation of neurotransmitters (Gamma-aminobutyric acid and serotonin) and apoptosis factor (Caspase-7) as biomarkers for the diagnosis of autism spectrum disorder Noor Ahmed Hameed1, Shrouk Abd Alrazak Hassan2, Hamid Jaddoa Abbas3* 1,2Medical Laboratory Technology Deptartment, College of Health & Medical, Technology, Southern Technical University, Basrah, Iraq; nourhan4788@gmail.com (N.A.H.) shrouk.albraheem@stu.edu.iq. (S.A.A.H.) 3Al-Faiha’a Teaching Hospital, Al- Zehra’a Medical College, University of Basrah, Basrah, Iraq; hamedjadoa@yahoo.com (H.J.A.) Abstract: Autism spectrum disorder (ASD) is a neurodevelopmental disability characterized by impaired communication and social skills along with restricted interests and repetitive behaviors. Biomarkers such as γ-aminobutyric acid (GABA), serotonin (5-Hydroxytryptamine, 5-HT) and caspase- 7, showing potential for early diagnosis and management of ASD. To evaluate the effectiveness of GABA, serotonin and caspase-7 as novel biomarkers to predict autism at an early stage. A case- control study which started from November 2023 to June 2024, that involved 77 children’s patients, newly diagnosed with autism. The control group was comprised of 61 age- and sex-matched apparently healthy children, they had no history of autism and any other chronic diseases. Serum samples were analyzed for GABA, serotonin and caspase-7 by using enzyme linked immune-sorbent assay (ELISA) technique. The statistical package for social sciences (SPSS- 26) was used for data analyses. The study revealed significantly elevated GAMA and caspase-7 levels; while, no significant differences of serotonin level between the autistic children’s patients and the control group. This study provides a groundbreaking revelation of the biochemical markers (GAMA and caspase-7) and autism spectrum disorder, the myriad metabolic and neurochemical dysfunctions of which form a complex tapestry of ASD. Keywords: 5-Hydroxytryptamine, Autism spectrum disorder, Caspase7, γ-aminobutyric acid. 1. Introduction Autism is a neurodevelopmental disease marked by deficits in communication and social abilities, as well as restricted interests and repetitive activities [1]. Clinical symptoms often emerge by the age of three; however, impairments in speech, social responsiveness, and play may be evident as early as 6 to 12 months of age [2]. The genesis of ASD likely involves environmental variables that induce physiological anomalies in genetically predisposed people. Notwithstanding this fact, the precise etiology remains unclear; ASD is a complex condition [3]. Neurotransmitters play a fundamental role in the causes of autism and the variation of its symptoms and are considered important markers in diagnosing this disorder [4]. Neurotransmitters are pivotal in the etiology of autism and the variability of its symptoms, serving as significant indicators in the diagnosis of this illness [5]. In immature brains, GABA receptors differ from those in adult brains. GABA serves as the principal excitatory neurotransmitter during cerebral development, affecting proliferation, migration, synaptic maturation, differentiation, and apoptosis [6]. Changes in the gabaminergic and glutaminergic systems alter the excitatory/inhibitory balance and may contribute to autistic behaviors and numerous neurodevelopmental problems [7]. Evidence increasingly suggests that the neurophysiological processes underlying neuropsychiatric diseases, including schizophrenia, depression, and anxiety, include disruption of inhibitory neurotransmission [8]. Extensive research has shown that the mailto:nourhan4788@gmail.com mailto:shrouk.albraheem@stu.edu.iq mailto:hamedjadoa@yahoo.com 3809 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 8, No. 6: 3808-3815, 2024 DOI: 10.55214/25768484.v8i6.2824 © 2024 by the authors; licensee Learning Gate neurophysiological basis of epilepsy arises from aberrant excitatory neuronal firing in certain brain areas due to a lack of GABAergic inhibition [9]. GABAergic dysfunction may offer a plausible explanation for the high comorbidity with epilepsy or the heightened vulnerability to epilepsy seen in individuals with autism [10]. Serotonin (5-HT), a neurotransmitter belonging to the monoamine family, regulates neuronal proliferation, migration, and differentiation. It influences cortical plasticity in adults and significantly contributes to early cortical development as a result [11]. Disruptions, even temporary aberrations, of the 5-HT pathway during development can result in enduring changes in brain function and behavior. Autism spectrum disorder is a neurodevelopmental syndrome with increasing evidence of serotonin (5- HT) involvement [12]. Numerous investigations have shown that the serotonergic system encompasses the neurotransmitters most frequently linked to the pathophysiology of autism. Seven families of serotonin receptors (5-HT1–5-HT7) are involved in mediating the effects of this neurotransmitter, which predominantly influences mood, sleep, memory, learning capacity, muscular contraction balance, and endocrine activities [13]. The expression levels and activity of the tissue serotonin transporter (SERT) govern serotonin levels, both intracellularly and extracellularly [14]. The serotonin transporter significantly influences the control of neuronal serotonin levels. Numerous internal and environmental factors can substantially impact the functionality and expression levels of SERT from early embryonic development through adolescence. Reduced serotonin levels may correlate with several characteristics in autistic children, including sleep difficulties and cognitive and social deficits. Other investigations have shown increased levels of SERT and/or serotonin in autistic children relative to controls [11]. On the other hand, research indicated that caspase 7 play a role in neuroinflammation, a key factor in ASD pathology [15]. Caspase 7, an effector caspase (with caspase 3 and 6), participates in apoptotic and inflammation. Caspase 7 is considered to be functionally redundant with caspase 3, which may be directly triggered by caspase 9 to break cellular substrates during apoptosis [16]. In the context of ASD, caspase-7 might be relevant for several reasons [17], although direct studies linking caspase-7 specifically to autism are limited, but there are some potential connections: 1. Apoptosis dysregulation: There is growing evidence that dysregulation of apoptosis may be implicated with neurodevelopmental disorders, including ASD. Abnormal cell death processes during brain development could affect neuronal connectivity, which is a hallmark of ASD [18]. 2. Neuroinflammation: Since caspases can influence inflammation pathways, an imbalance in caspase-7 activity could be linked to the inflammatory processes that may affect brain development and function in ASD [19]. 3. Synaptic development and Plasticity: Emerging research suggests that caspases might be involved in synaptic pruning (the process of eliminating excess neurons and synapses during development) [20]. Disruptions in synaptic pruning have been implicated in ASD, and caspase-7 could potentially play a role in these processes through its action in apoptosis [21]. 2. Material and Methods A case- control study was carried in specialized autism centers; Usraty Center, Tooba Center and Al-Faihaa Teaching Hospital Center, Basra province, Iraq; from November 2023 to June 2024. The study involved newly diagnosed with autism seventy- seven patients, their aged were between 5 and 14 years; who diagnosed by a specialist psychiatrist. Patients who suffer from mental illnesses or other chronic diseases and patients who are below or above 5- 14 years were excluded. Whole blood was collected for determinations of parameters levels by standard methods. IBM SPSS Statistics version 26.0 was used for data analyses, p values greater than 0.05 which the set of statistical significance. 3. Results Table 1, was shown that children with ASD had significant (P value < 0.05) high levels of gamma- aminobutyric acid and caspase 7; and non-significant (P value > 0.05) of serotonin compared with the control. 3810 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 8, No. 6: 3808-3815, 2024 DOI: 10.55214/25768484.v8i6.2824 © 2024 by the authors; licensee Learning Gate Table 1. Anthropometric and biochemical characteristics of study participants. Variables ASD (n=77) Mean ± SD Control (n=61) Mean± SD P. Value Ages (Years) 7.468± 2.25 7.661± 2.517 0.643* Sex (n%) Males 61 (97.2%) 36 (59.01%) 0.086** Females 16 (20.8%) 25 (%) Total 77 (100%) 61 (100%) HB (gm/dL) 11.006± 1.064 11.752± 1.199 0.0001* FBS (mg/dL) 81.469± 10.886 94.325±15.630 0.0001* B. Urea (mg/dL) 26.678± 6.481 25.030± 5.806 0.133* CPK (IU/L) 94.18± 53.36 88.143± 25.402 0.434* LDH (IU/L) 375.208± 212.407 268.696± 271.401 0.012** GABA (pg/ml) 130.545± 106.879 20.149± 34.044 0.0001** CASP7 (ng/ml) 10.126± 8.126 0.497± 0.608 0.0001** Serotonin (ng/ml) 3.825± 24.553 0.320± 0.482 0.228* Table 2 was revealed no significant statistical differences between males’ group and female group for all of the parameters (P values > 0.05). Table 2. Comparison of biochemical characteristics in patients according to sex. Variables ASD -Males (n=61) Mean ± SD ASD - Females (n=16) Mean ± SD P. Value Age (Years) 7.672±2.315 6.688±1.852 0.120* HB (gm/dL) 10.946±1.121 11.238±0.799 0.333* FBS (mg/dL) 80.852±9.783 83.819±14.496 0.325* B. Urea (mg/dL) 26.774±6.763 26.313±5.453 0.802* CPK (IU/L) 92.934±50.540 98.938±64.647 0.692** LDH (IU/L) 377.197±223.117 367.625±171.433 0.874** GABA (pg/ml) 127.051±95.507 143.86±145.436 0.579** CASP7(ng/ml) 10.414±7.807 9.027±9.442 0.574** Serotonin(ng/ml) 4.682±27.567 0.557±0.505 0.553** Table 3 showed ROC curve plotting and corresponding AUC calculations of accuracy parameters of each of the variables revealed that GABA, CASP7and Serotonin were significant identifies of ASD. Table 3. The ROC curve and AUC analyses for the values of serum biomarkers for the diagnosis of ASD. Variables Area under the ROC curve (AUC) P - value (AUC=0.5) Best cut- off criterion Sensitivity (%) Specificity (%) Efficiency GABA (pg/ml) 0.978 0.0001 30.550 98.1 90.2 94.1 CASP7 (ng/ml) 0.971 0.0001 0.9400 94.8 78.8 86.8 Serotonin (ng/ml) 0.798 0.0001 0.203 81.8 61.7 71.7 3811 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 8, No. 6: 3808-3815, 2024 DOI: 10.55214/25768484.v8i6.2824 © 2024 by the authors; licensee Learning Gate Figure 1. Receiver operational curves for GABA. Figure 2. Receiver operational curves for serotonin. 3812 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 8, No. 6: 3808-3815, 2024 DOI: 10.55214/25768484.v8i6.2824 © 2024 by the authors; licensee Learning Gate Figure 3. Receiver operational curves for CASP 7. Table 4. Correlations among the variables for patients. Note: *Spearman’s correlation coefficient. 4. Discussion Autism is a complex developmental disability. Hence much attention has been given to it due to confusion created by faint and vague sources. Studies being conducted today are directing more and more towards a big tent which includes genetic, environmental, and neurological factors that underlie or contribute to the development of autism [22], [23]. However, it has not been ascertained why autism results to its development in the first place. ASD is determined by the symptomatology that can be mild or severe and which involves speech, social and behavioral profiles [24] . Geschwind (2021) notes that this condition is highly heterogeneous in that the presentation of phenotypes is diverse and therefore Variables RBS Urea CK LDH GABA CASP7 Serotonin Hb R* 0.042 0.035 0.414 0.059 -0.054 -0.146 0.074 P value 0.715 0.765 0.001 0.612 0.642 0.206 0.521 RBS R 0.217 -0.029 0.050 -0.099 0.079 0.083 P value 0.058 0.799 0.667 0.390 0.493 0.472 Urea R -0.100 -0.186 -0.030 -0.097 0.185 P value 0.388 0.106 0.797 0.399 0.108 CK R 0.300 0.014 0.142 0.001 P value 0.008 0.901 0.217 0.992 LDH R -0.082 0.004 0.070 P value 0.477 0.975 0.548 GABA R 0.176 -0.245 P value 0.127 0.032 CASP7 R 0.198 P value 0.084 3813 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 8, No. 6: 3808-3815, 2024 DOI: 10.55214/25768484.v8i6.2824 © 2024 by the authors; licensee Learning Gate makes it very difficult to come up with a definite cause, or molecular pathway, associated with the illness. Science is on the move to provide a distinction between the chemical, physical, and hereditary processes that define ASD [25], [26]. Thus, autism spectrum disorder is still a significant priority for scientific research. The move of appreciating the fact that there cannot be a single theory out to explain all the observed cases is the fundamental concept in this case [27]. Gamma-aminobutyric acid is the principal inhibitory neurotransmitter, although excitatory neurotransmitters are also crucial in the human brain; their balanced interplay is essential for neuronal function. Prior studies indicate that GABA levels may be modified in individuals with autism [28]. Approximately 80 percent of neurons in the cerebral cortex, which envelops the brain's surface, convey excitatory impulses predominantly through the release of the neurotransmitter glutamate. The remaining 20 percent, referred to as interneurons, are inhibitory [29] . They function through gamma-aminobutyric acid. The appropriate equilibrium of these two signal types allows neuronal activity in certain situations while suppressing it in others [30]. In the context of ASD, for example, there is a neurological dysfunction of the excitatory/inhibitory balance. One of the reasons for many of the neurological signs observed in the course of the disease is excessively high levels of excitatory signaling [31]. As for GABA, the authors explain that the levels of GABA, which are alleged to be increased in specific autistic children, might indicate the brain's attempt to restore balance by increasing inhibitory transmission in response to a vast amount of excitatory input [32]. The literature on neurotransmitters shows that the two major neurotransmitter systems affected in the autistic brain are the glutamatergic and GABAergic systems. The explanation given is that the GABAergic neurotransmitters overcompensate for the dysfunction of glutamatergic neurotransmitters [33]. Apoptosis is a crucial process in tissue homeostasis which is otherwise popularly referred to as programmed cell death [34]. There was a high caspase-7 level in the children with autism than in normal children, this could probably mean that the turnover or apoptosis is happening faster in autistic children. This may indies to inflammation, immunization, or neurological stress which are parameters associated with ASD. It has been proved that caspase-7 is involved in immune mechanisms that might be dysregulated in children with autism [35]. This is the case given the fact that there is a dearth of recent papers that focus the role of caspase-7 in autistic states. According to the findings of the present study, the results revealed that GABA concentration were higher in both boys and girls with ASD than in children without this disorder in the control group, this agreed with [36]. Batie et al., rightly mentioned, that the biochemical pathways appear to be imbalanced in both boys and girls diagnosed with ASD. This is so yet the behavioral and cognitive symptoms of ASD present in different ways in males’ children and in girls. This conclusion is supported by other related research that found out that metabolic or neurochemical differences that exist between the sexes do not result from autism. In other words, it demonstrated that the fundamental biochemical properties of autism are forcing dynamics that are beyond sex-specific biological differences. Previous studies indicate that serotonin is regarded as the most significant neurotransmitter. Identified in the initial trimester of human central nervous system development, any disruption, including temporary deviations, of the 5-HT system can result in lasting changes in brain function and behavior [37]. In the present study, serotonin levels showed no significant differences between the groups. The serotonin system’s abnormalities may be connected to difficulties in emotional regulation and social interaction, but it does not exhibit the same level of dysregulation as GABA in the context of autism. This difference may be due to genetic factors, prenatal influences, or neuroinflammatory processes that affect GABA more prominently in autistic brains. GABA had significant negative correlations with serotonin. GABA and serotonin often balance each other in terms of neural excitation and inhibition [38]. High GABA levels might correlate with lower serotonin activity in certain regions of the brain. Area under the curve from the ROC curve provided a numerical value that summarizes the overall diagnostic ability of the test or biomarker. A high AUC would suggest that the diagnostic test or biomarker used in the study is effective and could be applied clinically or in further research [39]. The present study was identified the efficiencies of GABA (89.7), serotonin (71.7), caspase-7 (94.1). Al hence, these biomarkers protentional help in diagnosis of ASD and identify kids with the condition at the earliest stage of their complaint [40]. 3814 Edelweiss Applied Science and Technology ISSN: 2576-8484 Vol. 8, No. 6: 3808-3815, 2024 DOI: 10.55214/25768484.v8i6.2824 © 2024 by the authors; licensee Learning Gate 5. Conclusions There is a significant increase in the vital biomarker; GABA and decrease caspase 7; while, no significant statistical difference in serotonin level. The study successfully identified essential biochemical markers (GABA and caspase 7), that could be used for diagnosing and early detection of autism spectrum disorders. Ethical approval: The current research was approved by the ethical consideration committee of the Training and Human Development Unit, Basrah Health Department, Ministry of Health/ Environment, Iraq. Authors contribution: Samah Fadhil Hadi contributed to data collection, writing, and analysis. Shrouk A. Hassan Al. Ibraheem and Hamid Jaddoa Abbas contributed to the manuscript concept, results, analysis, manuscript submission, revision, and galley proof. Acknowledgment: The authors would like to thank all the specialized autism centers; Usraty Center, Tooba Center and Al-Faihaa Teaching Hospital Center, Basra province, Ira. Copyright: © 2024 by the authors. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). References [1] T. Hirota and B. H. King, “Autism spectrum disorder: a review,” Jama, vol. 329, no. 2, pp. 157–168, 2023. [2] A. Genovese and M. G. Butler, “Clinical assessment, genetics, and treatment approaches in autism spectrum disorder (ASD),” Int. J. Mol. Sci., vol. 21, no. 13, p. 4726, 2020. [3] M. 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