Research Article • DOI: 10.1515/tnsci-2016-0002 • Translational Neuroscience • 7 • 2016 • 6-11
Translational Neuroscience
Meng-na Lv1, Hong Zhang1,2* , Yi Shu1, Shan Chen3, Yuan-yuan Hu4, Min Zhou5 Department of Neurology, Zhongnan Hospital of Wuhan University, Wuhan 430071, P. R. China 2 Teaching and research section of Neuropsychiatry, Zhongnan Hospital of Wuhan University, Wuhan 430071, P. R. China 3 Department of Pediatric, Union Hospital, Fujian Medical University, Fuzhou 350001, China 4 Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109, USA 5 Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, P. R. China 1
Received 13 August 2015 accepted 23 November 2015
Introduction Autism spectrum disorder (ASD) comprises a family of developmental disabilities characterized by behavioral abnormalities that include language delays, difficulties in social interactions, and repetitive or stereotyped behaviors [1, 2]. The majority of structural brain alterations in individuals with ASD identified in postmortem studies have been found both in cortical and non-cortical regions [3-8]. In the previous studies [9, 10], we examined postmortem brains of ASD individuals and found that cortical thickness values in ASD subjects decreased significantly with age. High spine densities were associated with decreased brain weights and were most commonly found
The neonatal levels of TSB, NSE and CK-BB in autism spectrum DISORDER from Southern China Abstract Background: Autism spectrum disorder (ASD) is a serious neurodevelopmental disorder that impairs a child’s ability to communicate with others. It also includes restricted repetitive behaviors, interests and activities. Symptoms manifest before the age of 3. In the previous studies, we found structural abnormalities of the temporal lobe cortex. High spine densities were most commonly found in ASD subjects with lower levels of cognitive functioning. In the present study, we retrospectively analyzed medical records in relation to the neonatal levels of total serum bilirubin (TSB), neuron-specific enolase (NSE), creatine kinase brain band isoenzyme (CK-BB), and neonatal behavior in ASD patients from Southern China. Methods: A total of 80 patients with ASD (ASD group) were screened for this retrospective study. Among them, 34 were low-functioning ASD (L-ASD group) and 46 were high-functioning ASD (H-ASD group). Identification of the ASD cases was confirmed with a Revised Autism Diagnostic Inventory. For comparison with ASD cases, 80 normal neonates (control group) were selected from the same period. Biochemical parameters, including TSB, NSE and CK-BB in the neonatal period and medical records on neonatal behavior were collected. Results: The levels of serum TSB, NSE and CK-BB in the ASD group were significantly higher when compared with those from the control group (P < 0.01, or P < 0.05). The amounts of serum TSB, NSE and CK-BB in the L-ASD group were significantly higher when compared with those in the H-ASD group (P < 0.01, or P < 0.05). The Neonatal Behavioral Assessment Scale (NBAS) scores in the ASD group were significantly lower than that in the control group (P < 0.05). Likewise, the NBAS scores in the L-ASD group were significantly lower than that in the H-ASD group (P < 0.05). There was no association between serum TSB, NSE, CK-BB and NBAS scores (P > 0.05) in the ASD group. Conclusions: The neonatal levels of TSB, NSE and CK-BB in ASD from Southern China were significantly higher than those of healthy controls. These findings need to be investigated thoroughly by future studies with large sample. Keywords • Autism spectrum disorder • Creatine kinase brain band isoenzyme • Neonatal behavioral assessment • Neuron-specific enolase • Total serum bilirubin
in ASD subjects with lower levels of cognitive functioning. The current high prevalence estimates for ASD suggest that some common perinatal and/or postnatal biological factors during the neonatal period may be responsible for this increased risk, but the relationship between abnormal brain structure and these factors in ASD patient are still unknown. Therefore, identification of biomarkers associated with these alterations may provide further insights into the disease mechanisms. In this study, we retrospectively analyzed hospital medical records in relation to the neonatal levels of total serum bilirubin (TSB), neuron-specific enolase (NSE), creatine kinase brain band isoenzyme (CK-BB), and neonatal behavior in ASD from Southern China.
* E-mail: [email protected] © 2016 Meng-na Lv et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
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Materials and methods Participants A total of 80 patients with ASD (ASD group) were screened for this retrospective study. Out of these 80 patients, 34 were low-functioning ASD (L-ASD group) and 46 were high-functioning ASD (H-ASD group). Identification of the ASD cases was made based upon available medical and psychological hospital records from June 2003 to June 2009, and the diagnosis was confirmed with a Revised Autism Diagnostic Inventory [11]. For comparison with the ASD cases, 80 normal neonates (control group) were also selected from the same period, whose all medical records and psychological documentation were within normal limits.
Translational Neuroscience
The characteristics of neonates in the ASD group and control group are listed in Table 1. This study was carried out in accordance with the Declaration of Helsinki, and approved by the institutional review boards of the local Bioethics Committee in Zhongnan Hospital of Wuhan University.
Laboratory tests Venous blood samples were collected from all neonates in the hospital. Biochemical parameters, including total serum bilirubin (TSB), neuron-specific enolase (NSE) and creatine kinase brain band isoenzyme (CKBB) were routinely measured by an automatic biochemical analyzer, radioimmunoassay and immunoluminometric assay [12, 13]. Serum fasting glucose, total cholesterol, triglycerides, alanine transaminase, aspartate transaminase, total serum protein, serum albumin, and serum prealbumin were also evaluated (data not shown).
Evaluation of neonatal behavior The Neonatal Behavioral Assessment Scale (NBAS) developed by Brazelton et al. [14] was used to evaluate the neonatal behavior within the first week after delivery in the hospital. The NBAS was performed by 1 of 2 examiners blinded to maternal psychiatric and medical status. The NBAS includes 28 behavioral items and 18 reflex items that are administered in a particular sequence. Items of the NBAS are categorized into 7 clusters for scoring purposes; these clusters reduce the dimensionality of data. Clusters include: 1) Habituation, defined as the ability to respond to and inhibit discrete stimuli while asleep, 2) Orientation, defined as the ability to attend to visual and auditory stimuli and the quality of overall alertness, 3) Motor cluster, defined as a measure of motor performance and the quality of movements and muscle tone, 4) Range of state, defined as a measure of infant arousal and state lability, 5) Regulation of state, defined as a measure of the infant’s ability to regulate his or her state in the face of increasing levels of stimulation, 6) Autonomic stability, defined as signs of stress related to homeostatic adjustments of the central nervous system, and 7) Reflexes, defined as the number of abnormal reflexes.
Statistical methods
Correlation analysis
All data were expressed as the mean ± standard deviation (SD). Group differences were analyzed using multiple regression analysis to compare the levels of TSB, NSE, CK-BB and NBAS mean scores. Pearson’s correlation coefficient was used to analyze the association between the levels of TSB and serum NSE, as well as between the levels of TSB and serum CK-BB in the ASD group. The correlation between the levels of serum NSE and NBAS mean scores, and between the levels of serum CK-BB and NBAS mean scores in the ASD group was evaluated in the same way. All statistical analyses were performed with SPSS Statistics for Windows, Version 11.0 (SPSS Inc., Chicago, IL, USA), with a significance level set at a = 0.05.
Pearson’s correlation analysis revealed no association between serum TSB, NSE, CK-BB and NBAS scores (P > 0.05) in the ASD group (Fig. 3, A-E).
Results Comparison of serum TSB, NSE, CK-BB and NBAS scores between the ASD group and the control group in the neonatal period The levels of serum TSB (μmol/L), NSE (μg/L) and CK-BB (mol/L) in the ASD group were significantly higher when compared with those in the control group (P < 0.01, or P < 0.05). The NBAS scores in the ASD group were significantly lower than that in the control group (P < 0.05, Fig. 1).
Comparison of serum TSB, NSE, CK-BB and NBAS scores between the L-ASD group and the H-ASD group in the neonatal period The levels of serum TSB (μmol/L), NSE (μg/L) and CK-BB (mol/L) in the L-ASD group were significantly higher when compared with those in the H-ASD group (P < 0.01, or P < 0.05). The NBAS scores in the L-ASD group was significantly lower than that in the H-ASD group (P < 0.05, Fig. 2).
Discussion ASD is characterized by impairments in verbal and nonverbal communication and social interaction, with onset usually occurring around the first 36 months of childhood. Latest reports estimate that ASD affects approximately 1-2 per 1,000 children, and that male/female ratio is approximately 4/1 [15, 16]. The dramatic increase in reported prevalence has encouraged an intense effort to identify early biological markers [17]. Such markers could allow for earlier identification and therapeutic intervention, contributing to improved prognosis [18]. Several investigators have postulated that exposure to some biological factors during the critical period of rapid brain growth that occurs during the second and third trimester of gestation may lead to abnormal fetal brain development in ASD. One possible biological factor is hyperbilirubinemia. A population-based matched case-control study of 473 children with ASD children born from 1990 to 1999 in Denmark found an almost fourfold risk for ASD in infants who had hyperbilirubinaemia after birth [19]. Another population-based, follow-up study of all children born alive in Denmark between 1994 and 2004 showed that jaundice in the neonatal period increased risk of disorders of psychological development for children born at term. The excess risk of the infantile ASD was 67% [20]. A prospective study from a Canadian group showed an association with ASD in the combined moderate and severe hyperbilirubinemia group [21]. In contrast, a large case-control study nested within the
Table 1. Characteristics of the subjects in the ASD group and the control group (mean ± standard deviation). P value
L-ASD group
H-ASD group
Control group
Male (female)
18 (16)
24 (22)
42 (38)
Gestational age (weeks)
38.1 ±1.0
37.8 ± 0.8
38.4 ± 1.4
> 0.05
Age in days
6.5 ± 1.5
5.6 ± 1.7
6.0 ± 1.6
> 0.05
Birth weight (kg)
3.5 ± 0.9
3.6 ± 0.8
3.4 ± 1.0
> 0.05
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Translational Neuroscience
cohort of singleton term infants born between 1995 and 1998 in Kaiser Permanente Northern California hospital, reported that children with any degree of bilirubin level elevation were not at increased risk of ASD [22]. A retrospective study from India showed that prematurity was
associated with neonate hyperbilirubinemia in ASD [23], but this association was not found in the meta-analysis [24]. In this retrospective study, we analyzed two different ASD groups and showed that the level of TSB in the neonatal period in the ASD group was higher than in the
Figure 1. Serum TSB, NSE, CK-BB and NBAS scores in the ASD group and the control group (mean ± standard deviation) in the neonatal period. The amounts of serum TSB (μmol/L), NSE (μg/L) and CK-BB (mol/L) in the ASD group were significantly higher than those in the control group (*P < 0.05, **P < 0.01). The NBAS scores in the ASD group were significantly lower than in the control group. ASD, autism spectrum disorder; CK-BB, creatine kinase brain band isoenzyme; NBAS, Neonatal Behavioral Assessment Scale; NSE, neuron-specific enolase; TSB, total serum bilirubin.
Figure 2. Serum TSB, NSE, CK-BB and NBAS scores in the neonatal period in the low-functioning ASD (L-ASD) group and the high-functioning ASD (H-ASD) group (mean ± standard deviation). The amounts of serum TSB (μmol/L), NSE (μg/L) and CK-BB (mol/L) in the L-ASD group were significantly higher than those in the H-ASD group (*P < 0.05, **P < 0.01). The NBAS scores in the L-ASD group were significantly lower than those in the H-ASD group (*P < 0.05). CK-BB, creatine kinase brain band isoenzyme; NBAS, Neonatal Behavioral Assessment Scale; NSE, neuron-specific enolase; TSB, total serum bilirubin.
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control group. It was also significantly higher in the L-ASD group in comparison to the H-ASD group. Although the underlying mechanisms are unknown, it is currently thought that hyperbilirubinemia exert toxicity upon the basal ganglia and cerebellum, two brain regions that have been implicated as very important for the development of ASD [25]. Brain neurons contain a number of enolases. NSE is present predominantly in the neuron’s cytoplasm and accounts for about 75% of the total enolase subunits. The clinical interest of using NSE as marker of neuronal damage has been confirmed by several investigators (for review, see [26]). In this retrospective study, we found that the level of serum NSE in the ASD group was significantly higher than in the control group, also with significantly higher values in the H-ASD group as compared with those in the L-ASD group. In the cells, the cytosolic CK enzymes consist of two subunits, which can be either B (brain type) or M (muscle type). There are, therefore, three different isoenzymes: CK-MM, CK-BB and CK-MB. Structural brain damage may produce a leakage of cytoplasmic enzymes into the blood. Therefore, biochemical marker such as CK, especially its brain-specific isoenzyme CK-BB, has been used as indicator of brain damage [27]. Al-Mosalem et al. [28] measured plasma CK in 30 Saudi autistic patients and compared those levels to 30 age-matched control samples; there were 72.35% higher levels of CK in autistic patients. Poling et al. [29] performed a retrospective study including 159 patients with autism not previously diagnosed with metabolic disorders and 94 age-matched controls, and found that serum CK level was abnormally elevated in 22 out of 47 patients with autism. Recently, Ramsey et al. [30] reported higher levels of CK-MB in the youngest ASD group and low levels in the oldest ASD group. In this retrospective study, we found that the level of serum CK-BB in the ASD group was significantly higher than that in the control group, with significantly higher levels in the H-ASD group as compared with the L-ASD group. Developed in 1973, the NBAS, which assesses both cortical and subcortical functions, is used to evaluate neurobehavioral outcomes in
Translational Neuroscience
Figure 3. Correlations between serum TSB, NSE, CK-BB, and NBAS scores in the neonatal period in the ASD group (A-E). A. Correlation between the level of TSB and the
amount of serum NSE (r = 0.1191, P= 0.2926). B. Correlation between the level of TSB with the amount of serum CK-BB (r = 0.1189, P = 0.2935). C. Correlation between the level of TSB and NBAS mean scores (r = 0.0365, P = 0.7479). D. Correlation between the amount of serum NSE and NBAS mean scores (r = 0.0162, P = 0.8864). E. Correlation between the amount of serum CK-BB and NBAS mean scores (r = 0.1292, P = 0.2532). ASD, autism spectrum disorder; CK-BB, creatine kinase brain band isoenzyme; NBAS, Neonatal Behavioral Assessment Scale; NSE, neuron-specific enolase; TSB, total serum bilirubin.
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Translational Neuroscience
the infant. It is a neurobehavioral screening tool, and will identify gross abnormalities or asymmetries. All items are administered when the baby is in the appropriate behavioral state, and there is a sequence to follow. The NBAS seeks to score the baby’s best performance, so that the examiner is working to elicit the best possible reactions from the baby. It provides information about how babies manage to protect their sleep, comfort themselves and organize their sleep and awake states. The NBAS is a reliable and useful instrument, and had been used by other studies of prenatal exposure and infant outcomes [31-33]. In this retrospective study, we found that the NBAS
scores in the ASD group were significantly lower than those in the control group, again with significantly lower scores in the L-ASD group as compared with the H-ASD group. The underlying mechanisms nevertheless remain elusive and require further investigation. Taken together, we proposed that higher levels of TSB may increase brain damage. In turn, increased brain damage may cause elevated levels of serum NSE and serum CK-BB, which may be associated with abnormal brain structure and neonatal behavior in children with ASD from Southern China. Elucidation of molecular mechanisms underlying these changes may help develop novel therapeutic
interventions for neuronal protection in ASD. Unfortunately, the correlation analysis showed no association between serum TSB, NSE, CK-BB and NBAS scores in the ASD group. Therefore, these findings need to be investigated thoroughly by future studies with larger sample sizes.
Acknowledgments Conflict of interest statement: The authors declare that they have no competing interests. The authors would like to thank all patients who participated in this study.
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Med. Pathol., 1995, 16, 210-214 [13] Kruse A., Cesarini K.G., Bach F.W., Persson L., Increases of neuronspecific enolase, S-100 protein, creatine kinase and creatine kinase BB isoenzyme in CSF following intraventricular catheter implantation, Acta Neurochir., 1991, 110, 106-109 [14] Lester B., Als H., Brazelton T., Regional obstetric anesthesia and newborn behavior: a reanalysis toward synergistic effects, Child Dev., 1982, 53, 687-692 [15] Trottier G., Srivastava L., Walker C.D., Etiology of infantile autism. A review of recent advances in genetic and neurological research, J. Psychiatry Neurosci., 1999, 24, 103-115 [16] American Psychiatric Association., American psychiatric association, diagnostic and statistical manual-text revision (DSM IV-TR), American Psychiatric Association, Washington, DC, USA, 2000 [17] Chakrabarti S., Fombonne E., Pervasive developmental disorders in preschool children: confirmation of high prevalence, Am. J. Psychiatr., 2005,162, 1133-1141 [18] Aman M.G., Treatment planning for patients with autism spectrum disorders, J. Clin. Psychiatry, 2005, 66, 38-45 [19] Maimburg R.D., Vaeth M., Schendel D.E., Bech B.H., Olsen J., Thorsen P., Neonatal jaundice: a risk factor for infantile autism?, Paediatr. Perinat. Epidemiol., 2008, 22, 562-568 [20] Maimburg R.D., Bech B.H., Vaeth M., Møller-Madsen B., Olsen J., Neonatal jaundice, autism, and other disorders of psychological development, Pediatrics, 2010, 126, 872-878 [21] Jangaard K.A., Fell D.B., Dodds L., Allen A.C., Outcomes in a population of healthy term and near-term infants with serum bilirubin levels of > or = 325 micromol/L (> or = 19 mg/dL) who were born in Nova Scotia, Canada, between 1994 and 2000, Pediatrics, 2008, 122, 119-124 [22] Croen L.A., Yoshida C.K., Odouli R., Newman T.B. Neonatal hyperbilirubinemia and risk of autism spectrum disorders, Pediatrics, 2005, 115, e135-138 [23] Mamidala M.P., Polinedi A., Praveen Kumar P.T.V., Rajesh N., Vallamkonda O.R., Udani V., et al., Prenatal, perinatal and neonatal
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risk factors of autism spectrum disorder: a comprehensive epidemiological assessment from India, Res. Dev. Disabil., 2013, 34, 3004-3013 [24] Amin S.B., Smith T., Wang H., Is neonatal jaundice associated with autism spectrum disorder: a systematic review, J. Autism Dev. Disord., 2011, 41, 1455-1463 [25] Guinchat V., Thorsen P., Laurent C., Cans C., Bodeau N., Cohen D., Pre-, peri- and neonatal risk factors for autism, Acta Obstet. Gynecol. Scand., 2012, 91, 287-300 [26] Lamers K.J., Vos P., Verbeek M.M., Rosmalen F., van Geel W.J., van Engelen B.G., Protein S-100B, neuron-specific enolase (NSE), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) and blood of neurological patients, Brain Res. Bull., 2003, 61, 261-264 [27] Vázquez M.D., Sánchez-Rodriguez F., Osuna E., Diaz J., Cox D.E., PérezCárceles M.D., et al., Creatine kinase BB and neuron-specific enolase in cerebrospinal fluid in the diagnosis of brain insult, Am. J. Forensic. Med. Pathol., 1995, 16, 210-214 [28] Al-Mosalem O.A., El-Ansary A., Attas O., Al-Ayadhi L., Metabolic biomarkers related to energy metabolism in Saudi autistic children, Clin. Biochem., 2009, 42, 949-957
[29] Poling J.S., Frye R.E., Shoffner J., Zimmerman A.W., Developmental regression and mitochondrial dysfunction in a child with autism, Child Neurol., 2006, 21, 170-172 [30] Ramsey J.M., Guest P.C., Broek J.A., Glennon J.C., Rommelse N., Franke B., et al., Identification of an age-dependent biomarker signature in children and adolescents with autism spectrum disorders, Mol. Autism, 2013, 4, 27 [31] Lester B.M., Als H., Brazelton T.B., Regional obstetric anesthesia and newborn behavior: a reanalysis toward synergistic effects, Child Dev., 1982, 53, 687-692 [32] Engel S.M., Zhu C., Berkowitz G.S., Calafat A.M., Silva M.J., Miodovnik A., et al., Prenatal phtalate exposure and performance on the Neonatal Behavioral Assessment Scale in a multiethnic birth cohort, Neurotoxicology, 2009, 30, 522-528 [33] Engel S.M., Berkowitz G.S., Barr D.B., Teitelbaum S.L., Siskind J., Meisel S.J., et al., Prenatal organophosphate metabolite and organochlorine levels and performance on the Brazelton Neonatal Behavioral Assessment Scale in a multiethnic pregnancy cohort, Am. J. Epidemiol., 2007, 165, 1397-1404
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Translational Neuroscience
Meng-na Lv1, Hong Zhang1,2* , Yi Shu1, Shan Chen3, Yuan-yuan Hu4, Min Zhou5 Department of Neurology, Zhongnan Hospital of Wuhan University, Wuhan 430071, P. R. China 2 Teaching and research section of Neuropsychiatry, Zhongnan Hospital of Wuhan University, Wuhan 430071, P. R. China 3 Department of Pediatric, Union Hospital, Fujian Medical University, Fuzhou 350001, China 4 Department of Biologic and Materials Sciences, University of Michigan School of Dentistry, Ann Arbor, Michigan 48109, USA 5 Joint Sino-US Food Safety Research Center and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, P. R. China 1
Received 13 August 2015 accepted 23 November 2015
Introduction Autism spectrum disorder (ASD) comprises a family of developmental disabilities characterized by behavioral abnormalities that include language delays, difficulties in social interactions, and repetitive or stereotyped behaviors [1, 2]. The majority of structural brain alterations in individuals with ASD identified in postmortem studies have been found both in cortical and non-cortical regions [3-8]. In the previous studies [9, 10], we examined postmortem brains of ASD individuals and found that cortical thickness values in ASD subjects decreased significantly with age. High spine densities were associated with decreased brain weights and were most commonly found
The neonatal levels of TSB, NSE and CK-BB in autism spectrum DISORDER from Southern China Abstract Background: Autism spectrum disorder (ASD) is a serious neurodevelopmental disorder that impairs a child’s ability to communicate with others. It also includes restricted repetitive behaviors, interests and activities. Symptoms manifest before the age of 3. In the previous studies, we found structural abnormalities of the temporal lobe cortex. High spine densities were most commonly found in ASD subjects with lower levels of cognitive functioning. In the present study, we retrospectively analyzed medical records in relation to the neonatal levels of total serum bilirubin (TSB), neuron-specific enolase (NSE), creatine kinase brain band isoenzyme (CK-BB), and neonatal behavior in ASD patients from Southern China. Methods: A total of 80 patients with ASD (ASD group) were screened for this retrospective study. Among them, 34 were low-functioning ASD (L-ASD group) and 46 were high-functioning ASD (H-ASD group). Identification of the ASD cases was confirmed with a Revised Autism Diagnostic Inventory. For comparison with ASD cases, 80 normal neonates (control group) were selected from the same period. Biochemical parameters, including TSB, NSE and CK-BB in the neonatal period and medical records on neonatal behavior were collected. Results: The levels of serum TSB, NSE and CK-BB in the ASD group were significantly higher when compared with those from the control group (P < 0.01, or P < 0.05). The amounts of serum TSB, NSE and CK-BB in the L-ASD group were significantly higher when compared with those in the H-ASD group (P < 0.01, or P < 0.05). The Neonatal Behavioral Assessment Scale (NBAS) scores in the ASD group were significantly lower than that in the control group (P < 0.05). Likewise, the NBAS scores in the L-ASD group were significantly lower than that in the H-ASD group (P < 0.05). There was no association between serum TSB, NSE, CK-BB and NBAS scores (P > 0.05) in the ASD group. Conclusions: The neonatal levels of TSB, NSE and CK-BB in ASD from Southern China were significantly higher than those of healthy controls. These findings need to be investigated thoroughly by future studies with large sample. Keywords • Autism spectrum disorder • Creatine kinase brain band isoenzyme • Neonatal behavioral assessment • Neuron-specific enolase • Total serum bilirubin
in ASD subjects with lower levels of cognitive functioning. The current high prevalence estimates for ASD suggest that some common perinatal and/or postnatal biological factors during the neonatal period may be responsible for this increased risk, but the relationship between abnormal brain structure and these factors in ASD patient are still unknown. Therefore, identification of biomarkers associated with these alterations may provide further insights into the disease mechanisms. In this study, we retrospectively analyzed hospital medical records in relation to the neonatal levels of total serum bilirubin (TSB), neuron-specific enolase (NSE), creatine kinase brain band isoenzyme (CK-BB), and neonatal behavior in ASD from Southern China.
* E-mail: [email protected] © 2016 Meng-na Lv et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
6
Materials and methods Participants A total of 80 patients with ASD (ASD group) were screened for this retrospective study. Out of these 80 patients, 34 were low-functioning ASD (L-ASD group) and 46 were high-functioning ASD (H-ASD group). Identification of the ASD cases was made based upon available medical and psychological hospital records from June 2003 to June 2009, and the diagnosis was confirmed with a Revised Autism Diagnostic Inventory [11]. For comparison with the ASD cases, 80 normal neonates (control group) were also selected from the same period, whose all medical records and psychological documentation were within normal limits.
Translational Neuroscience
The characteristics of neonates in the ASD group and control group are listed in Table 1. This study was carried out in accordance with the Declaration of Helsinki, and approved by the institutional review boards of the local Bioethics Committee in Zhongnan Hospital of Wuhan University.
Laboratory tests Venous blood samples were collected from all neonates in the hospital. Biochemical parameters, including total serum bilirubin (TSB), neuron-specific enolase (NSE) and creatine kinase brain band isoenzyme (CKBB) were routinely measured by an automatic biochemical analyzer, radioimmunoassay and immunoluminometric assay [12, 13]. Serum fasting glucose, total cholesterol, triglycerides, alanine transaminase, aspartate transaminase, total serum protein, serum albumin, and serum prealbumin were also evaluated (data not shown).
Evaluation of neonatal behavior The Neonatal Behavioral Assessment Scale (NBAS) developed by Brazelton et al. [14] was used to evaluate the neonatal behavior within the first week after delivery in the hospital. The NBAS was performed by 1 of 2 examiners blinded to maternal psychiatric and medical status. The NBAS includes 28 behavioral items and 18 reflex items that are administered in a particular sequence. Items of the NBAS are categorized into 7 clusters for scoring purposes; these clusters reduce the dimensionality of data. Clusters include: 1) Habituation, defined as the ability to respond to and inhibit discrete stimuli while asleep, 2) Orientation, defined as the ability to attend to visual and auditory stimuli and the quality of overall alertness, 3) Motor cluster, defined as a measure of motor performance and the quality of movements and muscle tone, 4) Range of state, defined as a measure of infant arousal and state lability, 5) Regulation of state, defined as a measure of the infant’s ability to regulate his or her state in the face of increasing levels of stimulation, 6) Autonomic stability, defined as signs of stress related to homeostatic adjustments of the central nervous system, and 7) Reflexes, defined as the number of abnormal reflexes.
Statistical methods
Correlation analysis
All data were expressed as the mean ± standard deviation (SD). Group differences were analyzed using multiple regression analysis to compare the levels of TSB, NSE, CK-BB and NBAS mean scores. Pearson’s correlation coefficient was used to analyze the association between the levels of TSB and serum NSE, as well as between the levels of TSB and serum CK-BB in the ASD group. The correlation between the levels of serum NSE and NBAS mean scores, and between the levels of serum CK-BB and NBAS mean scores in the ASD group was evaluated in the same way. All statistical analyses were performed with SPSS Statistics for Windows, Version 11.0 (SPSS Inc., Chicago, IL, USA), with a significance level set at a = 0.05.
Pearson’s correlation analysis revealed no association between serum TSB, NSE, CK-BB and NBAS scores (P > 0.05) in the ASD group (Fig. 3, A-E).
Results Comparison of serum TSB, NSE, CK-BB and NBAS scores between the ASD group and the control group in the neonatal period The levels of serum TSB (μmol/L), NSE (μg/L) and CK-BB (mol/L) in the ASD group were significantly higher when compared with those in the control group (P < 0.01, or P < 0.05). The NBAS scores in the ASD group were significantly lower than that in the control group (P < 0.05, Fig. 1).
Comparison of serum TSB, NSE, CK-BB and NBAS scores between the L-ASD group and the H-ASD group in the neonatal period The levels of serum TSB (μmol/L), NSE (μg/L) and CK-BB (mol/L) in the L-ASD group were significantly higher when compared with those in the H-ASD group (P < 0.01, or P < 0.05). The NBAS scores in the L-ASD group was significantly lower than that in the H-ASD group (P < 0.05, Fig. 2).
Discussion ASD is characterized by impairments in verbal and nonverbal communication and social interaction, with onset usually occurring around the first 36 months of childhood. Latest reports estimate that ASD affects approximately 1-2 per 1,000 children, and that male/female ratio is approximately 4/1 [15, 16]. The dramatic increase in reported prevalence has encouraged an intense effort to identify early biological markers [17]. Such markers could allow for earlier identification and therapeutic intervention, contributing to improved prognosis [18]. Several investigators have postulated that exposure to some biological factors during the critical period of rapid brain growth that occurs during the second and third trimester of gestation may lead to abnormal fetal brain development in ASD. One possible biological factor is hyperbilirubinemia. A population-based matched case-control study of 473 children with ASD children born from 1990 to 1999 in Denmark found an almost fourfold risk for ASD in infants who had hyperbilirubinaemia after birth [19]. Another population-based, follow-up study of all children born alive in Denmark between 1994 and 2004 showed that jaundice in the neonatal period increased risk of disorders of psychological development for children born at term. The excess risk of the infantile ASD was 67% [20]. A prospective study from a Canadian group showed an association with ASD in the combined moderate and severe hyperbilirubinemia group [21]. In contrast, a large case-control study nested within the
Table 1. Characteristics of the subjects in the ASD group and the control group (mean ± standard deviation). P value
L-ASD group
H-ASD group
Control group
Male (female)
18 (16)
24 (22)
42 (38)
Gestational age (weeks)
38.1 ±1.0
37.8 ± 0.8
38.4 ± 1.4
> 0.05
Age in days
6.5 ± 1.5
5.6 ± 1.7
6.0 ± 1.6
> 0.05
Birth weight (kg)
3.5 ± 0.9
3.6 ± 0.8
3.4 ± 1.0
> 0.05
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cohort of singleton term infants born between 1995 and 1998 in Kaiser Permanente Northern California hospital, reported that children with any degree of bilirubin level elevation were not at increased risk of ASD [22]. A retrospective study from India showed that prematurity was
associated with neonate hyperbilirubinemia in ASD [23], but this association was not found in the meta-analysis [24]. In this retrospective study, we analyzed two different ASD groups and showed that the level of TSB in the neonatal period in the ASD group was higher than in the
Figure 1. Serum TSB, NSE, CK-BB and NBAS scores in the ASD group and the control group (mean ± standard deviation) in the neonatal period. The amounts of serum TSB (μmol/L), NSE (μg/L) and CK-BB (mol/L) in the ASD group were significantly higher than those in the control group (*P < 0.05, **P < 0.01). The NBAS scores in the ASD group were significantly lower than in the control group. ASD, autism spectrum disorder; CK-BB, creatine kinase brain band isoenzyme; NBAS, Neonatal Behavioral Assessment Scale; NSE, neuron-specific enolase; TSB, total serum bilirubin.
Figure 2. Serum TSB, NSE, CK-BB and NBAS scores in the neonatal period in the low-functioning ASD (L-ASD) group and the high-functioning ASD (H-ASD) group (mean ± standard deviation). The amounts of serum TSB (μmol/L), NSE (μg/L) and CK-BB (mol/L) in the L-ASD group were significantly higher than those in the H-ASD group (*P < 0.05, **P < 0.01). The NBAS scores in the L-ASD group were significantly lower than those in the H-ASD group (*P < 0.05). CK-BB, creatine kinase brain band isoenzyme; NBAS, Neonatal Behavioral Assessment Scale; NSE, neuron-specific enolase; TSB, total serum bilirubin.
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control group. It was also significantly higher in the L-ASD group in comparison to the H-ASD group. Although the underlying mechanisms are unknown, it is currently thought that hyperbilirubinemia exert toxicity upon the basal ganglia and cerebellum, two brain regions that have been implicated as very important for the development of ASD [25]. Brain neurons contain a number of enolases. NSE is present predominantly in the neuron’s cytoplasm and accounts for about 75% of the total enolase subunits. The clinical interest of using NSE as marker of neuronal damage has been confirmed by several investigators (for review, see [26]). In this retrospective study, we found that the level of serum NSE in the ASD group was significantly higher than in the control group, also with significantly higher values in the H-ASD group as compared with those in the L-ASD group. In the cells, the cytosolic CK enzymes consist of two subunits, which can be either B (brain type) or M (muscle type). There are, therefore, three different isoenzymes: CK-MM, CK-BB and CK-MB. Structural brain damage may produce a leakage of cytoplasmic enzymes into the blood. Therefore, biochemical marker such as CK, especially its brain-specific isoenzyme CK-BB, has been used as indicator of brain damage [27]. Al-Mosalem et al. [28] measured plasma CK in 30 Saudi autistic patients and compared those levels to 30 age-matched control samples; there were 72.35% higher levels of CK in autistic patients. Poling et al. [29] performed a retrospective study including 159 patients with autism not previously diagnosed with metabolic disorders and 94 age-matched controls, and found that serum CK level was abnormally elevated in 22 out of 47 patients with autism. Recently, Ramsey et al. [30] reported higher levels of CK-MB in the youngest ASD group and low levels in the oldest ASD group. In this retrospective study, we found that the level of serum CK-BB in the ASD group was significantly higher than that in the control group, with significantly higher levels in the H-ASD group as compared with the L-ASD group. Developed in 1973, the NBAS, which assesses both cortical and subcortical functions, is used to evaluate neurobehavioral outcomes in
Translational Neuroscience
Figure 3. Correlations between serum TSB, NSE, CK-BB, and NBAS scores in the neonatal period in the ASD group (A-E). A. Correlation between the level of TSB and the
amount of serum NSE (r = 0.1191, P= 0.2926). B. Correlation between the level of TSB with the amount of serum CK-BB (r = 0.1189, P = 0.2935). C. Correlation between the level of TSB and NBAS mean scores (r = 0.0365, P = 0.7479). D. Correlation between the amount of serum NSE and NBAS mean scores (r = 0.0162, P = 0.8864). E. Correlation between the amount of serum CK-BB and NBAS mean scores (r = 0.1292, P = 0.2532). ASD, autism spectrum disorder; CK-BB, creatine kinase brain band isoenzyme; NBAS, Neonatal Behavioral Assessment Scale; NSE, neuron-specific enolase; TSB, total serum bilirubin.
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Translational Neuroscience
the infant. It is a neurobehavioral screening tool, and will identify gross abnormalities or asymmetries. All items are administered when the baby is in the appropriate behavioral state, and there is a sequence to follow. The NBAS seeks to score the baby’s best performance, so that the examiner is working to elicit the best possible reactions from the baby. It provides information about how babies manage to protect their sleep, comfort themselves and organize their sleep and awake states. The NBAS is a reliable and useful instrument, and had been used by other studies of prenatal exposure and infant outcomes [31-33]. In this retrospective study, we found that the NBAS
scores in the ASD group were significantly lower than those in the control group, again with significantly lower scores in the L-ASD group as compared with the H-ASD group. The underlying mechanisms nevertheless remain elusive and require further investigation. Taken together, we proposed that higher levels of TSB may increase brain damage. In turn, increased brain damage may cause elevated levels of serum NSE and serum CK-BB, which may be associated with abnormal brain structure and neonatal behavior in children with ASD from Southern China. Elucidation of molecular mechanisms underlying these changes may help develop novel therapeutic
interventions for neuronal protection in ASD. Unfortunately, the correlation analysis showed no association between serum TSB, NSE, CK-BB and NBAS scores in the ASD group. Therefore, these findings need to be investigated thoroughly by future studies with larger sample sizes.
Acknowledgments Conflict of interest statement: The authors declare that they have no competing interests. The authors would like to thank all patients who participated in this study.
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