Abstract
Objective
The purpose of this study was to compare the plasma oxytocin levels of children with separation anxiety disorder (SAD) and their mothers with those of healthy controls and to examine the relationship between oxytocin levels and changes in anxiety three months after treatment.
Methods
Thirty children aged 6−12 years with SAD, 30 healthy children, and mothers of both groups were included in the study. All cases were evaluated with semi-structured interview and Clinical Global Impression Scale. All cases and mothers of both groups filled out scales to determine various psychological variables (anxiety, depression, and attachment). The patient group children were re-evaluated with their mothers after three months, following treatment. Plasma oxytocin levels were evaluated from both groups and their mothers before and after treatment.
Results
The plasma oxytocin levels of mothers of children with SAD were significantly lower than those of the controls and increased significantly three months after their children were treated. No difference was found between the plasma oxytocin levels of children with SAD and the control group, and these children’s levels decreased significantly after treatment. A positive correlation was found between changes in the plasma oxytocin levels of children with SAD and the change in anxiety scores.
Conclusion
Our results show that the change in plasma oxytocin levels in both children and mothers after treatment suggests that oxytocin may be important in the etiology of SAD.
Keywords: Separation anxiety, Oxytocin, Selective serotonin reuptake inhibitors, Child, Fluoxetine
INTRODUCTION
Separation anxiety disorder (SAD) is characterized by excessive fear or anxiety associated with being separated from the home or close attachment figures [1]. When separated from their parents, children with SAD feel excessive anxiety concerning their own and their parents’ health and safety, and may experience nightmares involving separation, find it difficult to sleep alone, experience somatic symptoms, and refuse to go to school [1,2]. In order for separation anxiety to be diagnosed in children and adolescents, this period must last for at least four weeks, and the disorder must lead to clinically significant distress or decreased functionality in the societal, academic, and social spheres [1,2]. Separation anxiety is seen in an average of 4% of children and is the most common childhood anxiety disorder, with an average age at onset of seven years [3,4]. Numerous factors play a role in the etiology of anxiety disorders. One of the potential etiological factors for anxiety disorders in children and adolescents is insecure attachment to the care giver on the part of the child [5]. Insecure attachment is frequently seen in mothers with anxiety disorders, and insecure attachment and more frequently anxiety disorder develop in a more pronounced manner in the children of such mothers [6].
Oxytocin is a neuropeptide hormone with an important role in attachment in humans [7,8]. A large proportion of oxytocin is synthesized in magnocellular neurons in the hypothalamic paraventricular and supraoptic nuclei and is transported to the posterior pituitary gland, from where it is subsequently released into the blood. Oxytocin is also released from parvocellular neurons in the paraventri-cular nucleus [8]. Oxytocin performs numerous functions in the body; it supports positive social interaction and exhibits anxiolytic effects [9-12]. Studies have determined a negative correlation between plasma oxytocin levels and anxiety scores [13-15], and significant oxytocin elevation has been observed in cases of secure mother and child interaction [16-18]. Studies examining the relationship between plasma oxytocin levels and anxiety disorders have largely focused on general anxiety symptoms. However, there has been much less investigation of oxytocin levels in specific anxiety disorders [18]. Few studies have investigated the relationship between separation anxiety and oxytocin levels, and even fewer in children and adolescents [19]. One study of 50 children aged 7−16 years reported significantly lower oxytocin in saliva in children with separation anxiety compared to other anxiety disorders, and observed negative correlation between the severity of separation anxiety and oxytocin levels and negative correlation between oxytocin levels in saliva and family accommodation scores [18]. Another study of 41 children with anxiety aged 7−16 years reported a signifi-cantly higher oxytocin response following mother-child interaction (emotional communication, maternal interven-tion, maternal sensitivity, and child engagement) in children with separation anxiety compared to children with other forms of anxiety [20]. In a study of 23 male with social phobia (SP) aged 18−65 years, higher oxytocin levels were found to be significantly associated with higher Liebowitz Social Anxiety Scale scores after controlling for age and years of education [21]. Another study involving 39 individuals with SP and 28 healthy controls deter-mined no significant difference in oxytocin levels between the two groups [22]. In a different study, attachment- based psychotherapy was applied to cases with clinically significant separation anxiety, selected from among patients with anxiety and mood disorders who did not respond to psychiatric interventions and aged 18−70 years. Oxytocin levels were measured before and after therapy, but no significant change was observed [23]. Children and adolescents with separation anxiety experience intense anxiety when separated from their attachment figure [1]. Considering the function of oxytocin on the subject of attachment and the place of insecure attachment in the etiology of anxiety disorder [5], it may be useful to measure oxytocin levels in SAD. However, existing studies have not been performed solely with samples with separation anxiety, but also with heterogeneous groups including other anxiety disorders. In addition, the majority of these studies have included no control groups. To the best of our knowledge, no previous studies have examined oxytocin levels simultaneously in children with separation anxiety and in their parents. Also, to the best of our knowledge, no studies have evaluated changes in pre- and post-treatment oxytocin levels in children with SAD. The fact that several previous studies have also investigated oxytocin levels in saliva may also be regarded as a significant limitation of such research [18,20,23].
In the light of these gaps in the literature and the limitations of existing studies, the aims of the present study were as follows: to compare the plasma oxytocin levels of children with separation anxiety and of their parents with those of a control group, and to examine the relationship between oxytocin levels of children with separation anxiety and of their mothers and changes in anxiety after three months of treatment. We anticipated that oxytocin levels would be lower in children with separation anxiety and in their mothers compared to the healthy control group, that anxiety levels would decrease, while oxytocin levels increased, in children with separation anxiety and their mothers after three months of treatment of the children, that clinical severity would exhibit inverse correlation with oxytocin levels in separation anxiety, and that the degree of improvement of separation anxiety would be correlated with an increase in oxytocin levels.
METHODS
Participants
The research was conducted at the Istanbul University Istanbul Medical Faculty Child and Adolescent Mental Health Clinic, Turkey. The study was approved by the Istanbul University ethical committee (decision no. 69880 dated 23.06.2020), and all procedures were compatible with the Declaration of Helsinki and local laws and regulations. The research was designed as a case-controlled study. Thirty children aged 6−12 years, diagnosed with separation anxiety based on Diagnostic and Statisti-cal Manual of Mental Disorders Fifth Edition (DSM-5), and agreeing to take part in the study, were enrolled in the case group together with their mothers. Exclusion criteria were unwillingness to take part (absence of written or verbal consent to participate on the part of the child or the parent), age other than 6−12 years, presence of accompanying autism spectrum disorder or intellectual disability, parents lacking the intellectual capacity to complete the scales, maternal use of any psychotropic drug, the child and mother experiencing infection when the sample was taken, the mother being pregnant or lactating, the presence of a diagnosed metabolic or endocrinological (such as thyroid disease) disease in the child or mother, the mother using oral contraceptives or receiving hormone therapy for at least the previous one month, the presence of neurological disease capable of causing organic disorders, such as diagnosed epilepsy, in the mother or child, and substance use or dependence, other than smoking. The control group consisted of healthy children consenting to take part, and was subjected to the same inclusion and exclusion criteria (Fig. 1).
Fig. 1.
Sample selection diagram.
Cases presenting to our outpatient clinic and diagnosed with SAD at diagnostic evaluation based on DSM-5 and who did not use selective serotonin reuptake inhibitors (SSRIs) were verbally informed about the study, and volunteers were recruited for it. Diagnosis of SAD was confirmed at diagnostic evaluation based on DSM-5 performed by the first author. Individuals meeting the inclusion criteria and none of the exclusion criteria were then given an informed consent form to ascertain whether they would agree to take part in the study. The mothers of cases agreeing to take part in the study were given contact information and were asked to provide information in the early follicular stage of their menstrual cycle.
Diagnostic and Symptom Assessment
Cases consenting to take part in the study underwent diagnostic interviews using the Kiddie Schedule for Affective Disorders and Schizophrenia Present and Lifetime version for DSM-5 (K-SADS-PL-DSM-5) [24]. All cases diagnosed with SAD according to DSM-5 were diagnosed as SAD when evaluated according to K-SADS-PL-DSM-5. The children were asked to complete the Revised Children’s Anxiety and Depression Scale-Child Version (RCADS- CV), Separation Anxiety Assessment Scale-Children’s Forms (SAAS-CF), and inventory of Experiences in Close Relation-ships-Revised for children and adolescents-Middle Child-hood Scale (ECR-RC) in the presence of a clinician [25- 27]. Children completed the scales in an average of 30 minutes, but were given a five-minute break between scales if they so desired. The mothers completed a sociodemographic data form, the Adult Separation Anxiety Questionnaire (ASAQ), the Hamilton Depression Rating Scale (HDRS), and the Hamilton Anxiety Rating Scale (HARS) in the presence of the interviewer [28-30]. The clinician completed the Clinical Global Impression Scale (CGIS) in order to evaluate and follow up severity of disease [31]. Participants were weighed and measured on the day they provided blood specimens. Height was expressed in meters and weight in kilograms.
The cases were then referred to the clinics where they were evaluated for routine follow-up and treatment. We learned that all patients with SAD were started on fluoxetine, the first alternative in the therapeutic guidelines. Fluoxetine therapy was initiated at a dosage of 5 mg/day. This was increased to 10 mg/day at the end of the sixth week if necessary and to 20 mg/day, by the clinic performing the follow-up, at the end of the third week in cases exhibiting a partial response. Non-specific symptoms such as increased sleep, abdominal pain, and headache were observed in six cases after treatment, but no drug dose modification was applied. Disinhibition was observed in only one case, in which the drug dosage was increase and treatment was maintained. No structured therapy was applied in any case, only drug therapy and supportive therapy being given.
Interviews were held with the patient group and their mothers every month, at which the severity of and improvement in separation anxiety were evaluated using the CGIS. In our study, CGI disease severity and improvement scores were used. The clinician scored the severity of the disease with the CGI before starting the treatment in the group diagnosed with SAD. At the 1st, 2nd, and 3rd months after starting the treatment, the clinician met with the patients and their mothers again, and the severity of the disease noted each month by the CGI severity score. Disease severity and improvement scores were followed by CGI. Patients with CGIS improvement scores of 1, 2, or 3 at the end of the third month were regarded as responding to treatment. The patient group was again contacted after three months and invited for repeat evaluation. All scales were again completed by the children and parents at assessment in the third month.
Clinical global impression scale
The CGIS was developed by Guy [31] in 1976. The CGIS consists of 3 parts that evaluate disease severity, improvement and severity of side effects. The CGIS severity scale, which ranges from 1 to 7, with 1 = normal or not at all ill while 7 = extremely ill, was used to measure the symptom severity. The CGIS improvement scale, which ranges from 1 to 7, with 1: much improved, 7: much worse, was used to measure the symptom severity. The CGIS side effects scale, which ranges from 1 to 4, with 1 = not at all, 4 = affects at a level that negates the benefits of the therapeutic effect, measures the side effects severity.
Kiddie Schedule for Affective Disorders and Schizophrenia Present and Lifetime version for DSM-5
K-SADS-PL version is a semi-structured interview program to evaluate the present, past and lifetime diagnosis status of children and adolescents [32]. With the publication of the DSM-5, the same chart has been updated according to the DSM-5 diagnoses. The Turkish validity and reliability of the DSM-5 version was performed by Ünal et al. [24] in 2019. In the study, CDSG-SH-DSM-5-T was used for psychiatric evaluation and the diagnosis was coded as yes or no, by combining the information obtained from the child and family and the clinician’s observation, considering the duration and number of symptoms and the effect of symptoms on functionality during the interview.
Revised Children’s Anxiety and Depression Scale-Child Version
It was developed by Chorpita et al. [33] in 2005 to measure depression and anxiety symptoms in children and adolescents, and its Turkish validity and reliability were established by Gormez et al. [25] in 2017. The scale can be applied to children and adolescents to measure anxiety and depression symptoms. It is a 4-point Likert- type scale consisting of 47 items (0 = never true, 1 = sometimes true, 2 = often true, 3 = always true). It consists of subscales: SAD (7 items), SP (9 items), generalized anxiety disorder (6 items), panic disorder (9 items), obsessive compulsive disorder (6 items), and major depressive disorder (10 items). In the Turkish version, the internal consistency coefficient was calculated as 0.95 and between 0.75−0.86 for the sub-dimensions [25].
Separation Anxiety Assessment Scale-Children’s Forms
The Separation Anxiety Assessment Scale was first developed by Hahn et al. [34] in 2003, then Eisen and Schaefer [35] developed the parent form in 2005. The Turkish validity and reliability study of the scale was carried out by Teze and Arslan [26] in 2016. The Separation Anxiety Assessment Scale has a parent and child form, the child form was used in the study. The scale was developed to detect separation anxiety symptoms in children. It is a 4-point Likert-type scale consisting of 14 items and scored as 1-never, 2-sometimes, 3-often and 4-always. The lowest score on the scale is 14, and the highest score is 56. It is said that as the score obtained from the scale increases, separation anxiety increases. In the Turkish version of the SAAS-CF, the reliability coefficient of the scale was calculated as 0.83 [26].
Inventory of Experiences in Close Relationships-Revised for children and adolescents-Middle Childhood Scale
Inventory of ECR-RC was developed by Brenning et al. [36] in 2011 to measure the dimensions of attachment to parents in middle childhood. Adaptation of the scale to Turkish was made by Kırımer et al. [27] in 2014. The scale consists of 36 items and is scored as 1-strongly disagree, 2-disagree, 3-strongly disagree, 4-undecided, 5-somewhat agree, 6-agree, 7-strongly agree. Bonding with mother; It was evaluated in two dimensions as anxiety (18 items) and avoidance (18 items). Odd numbered items in the scale belong to anxiety and even numbered items belong to avoidance dimensions. Higher scores in both dimen-sions correspond to more anxious or avoidant attachment to the mother. In the Turkish version, Cronbach’s alpha coefficient was calculated as 0.78 for the anxiety dimension and 0.90 for the avoidance dimension [27].
Adult Separation Anxiety Questionnaire
The ASAQ was developed by Manicavasagar et al. [37] in 2003. Its Turkish validity and reliability were performed by Diriöz et al. [28] in 2012. It is a 4-point Likert-type scale consisting of 27 items, scored as 0 (never), 1 (rarely), 2 (often), 3 (very often). The Cronbach’s alpha coefficient of the Turkish version is 0.93 [28].
Hamilton Anxiety Rating Scale
The HARS was developed by Hamilton [38] in 1959 to determine the symptoms and severity of anxiety, and Turkish validity and reliability were established by Yazıcı et al. [30] in 1998. The scale consists of 14 items. It is scored between 0−4 points by the practitioner, the lowest score on the scale is 0 and the highest score is 56. It consists of 2 subscales, 5 items psychological and 9 items physical.
Hamilton Depression Rating Scale
The HDRS was developed by Hamilton [39] in 1960, and its Turkish validity and reliability were established in 1996 by Akdemir et al. [29]. It is a 17-item scale, the lowest score on the scale is 0, and the highest score is 53. The scale is scored by the clinician. The increase in the score of the scale indicates the increase in depression. In the Turkish version, Cronbach’s alpha coefficient was found to be 0.75 [29].
Blood Samples
Plasma oxytocin levels can exhibit diurnal variation and be affected by factors such as fasting-satiety and the different phases of the menstrual cycle. Fasting blood samples of between 5 ml and 8 ml were therefor collected from the antecubital vein at 09:00−10:00 AM in the early follicular phase of the menstrual cycle of the mothers of the children in the study and from the children for oxytocin level measurement on months 0 and 3. This reduced confusing factors for oxytocin levels. Due to the presence of infection in one child with separation anxiety when blood was scheduled to be collected on the third month, peripheral venous blood was collected one week later for plasma oxytocin level measurement in that case 3. Oxytocin values could not be measured before treatment in three mothers from the SAD group and in four children and two mothers after treatment since these were below the enzyme linked immunosorbent assay (ELISA) kit detection threshold (< 15 pg/ml). Venous blood specimens from the children in the study and their mothers were placed into chilled tubes containing K3ethylenediaminetetraacetic acid-aprotinin and immediately sent to the laboratory. These were than centrifuged at 1,600 × g for 15 minutes at 4°C. The resulting plasma specimens were then stored at −80°C until the day of study. Plasma oxytocin levels were investigated using the competitive ELISA method (Enzo Life Science, Catalog #: ADI-900-15A3).
Statistical Analysis
Statistical analysis was performed on Statistical Package for the Social Sciences version 21.0 software (SPSS 21.0; IBM Co.). Descriptive statistics were expressed as mean ± standard deviation, median values and 27−75% percentiles, frequency distributions, and percentages. The chi- square test (χ2) and Fisher’s exact test were applied to compare categorical variables between two independent groups. A comparison of sex in the SAD and control groups Pearson chi-square test was used. The chi-square test (χ2) and Fisher’s exact test were used to compare K-SADS- PL-DSM-5 profile in children in the SAD and control groups. The Kolmogorov–Smirnov test was used to determine whether or not continuous variables were normally distributed. Student’s ttest was employed in case of normally distributed continuous variables, and Mann–Whitney Utest if the variables were not normally dis-tributed. A comparison of sociodemographic characteristics in the SAD and control groups Mann–Whitney Utest, Student’s ttest were used. A comparison of total RCADS-CV, SAAS-CF score, and total ECR-RC (attach-ment) scores in children and ASAQ, HDRS, and HARS score in mothers in the SAD and control groups Mann–Whitney Utest, Student’s ttest were used. The Mann–Whitney Utest was used to compare plasma oxytocin levels in children and mothers in the SAD and control groups. The paired samples ttest was applied to compare normally distributed continuous variables between two dependent groups, while continuous variables not exhi-biting normal distribution were evaluated using Wilcoxon’s signed rank test. Wilcoxon’s signed rank test was used to compare pre- and post-treatment the CGIS severity scores among children in the SAD group. Since the anxiety levels of patients and mothers will change after treatment, we thought that plasma oxytocin levels would also be af-fected. For this reason, we wanted to see the changes in the plasma oxytocin level of the patients and mothers after the treatment. Wilcoxon’s signed rank test was used to compare pre- and post-treatment plasma oxytocin levels among children and mothers in the SAD group. Correla-tion between continuous variables were investigated using Spearman’s correlation analysis. Correlations between pre-treatment plasma oxytocin level in children with SAD and pre-treatment total RCADS-CV, SAAS-CF score, and total ECR-RC total score; correlations between plasma oxytocin level changes in children with SAD and total RCADS-CV, SAAS-CF score, and total ECR-RC score changes, and between plasma oxytocin level changes in mothers of children with SAD and ASAQ, HDRS, and HARS score changes Spearman’s correlation analysis was used. Age, body mass index (BMI), and RCADS-CV- MDB for children, and HDRS percentile parameters for mothers thought to be capable of affecting biochemical parameters were determined as covariants. Case and control group plasma log-oxytocin levels were compared using the ANCOVA test. pvalues < 0.05 were regarded as significant for all analyses.
RESULTS
A Comparison of Sociodemographic Characteristics in the SAD and Control Groups
Sixty percent of the 30 cases diagnosed with SAD (n = 18) were males and 40% (n = 12) were females. No difference was observed in terms of age and sex compared to the control group. No significant difference was observed between the two groups in terms of sex (χ2 = 0.000, p > 0.05), age (z = −0.510, p = 0.610), maternal age (z = −1.122, p = 0.262), or maternal BMI (t = −0.510, p = 0.612). However, BMI was significantly lower in the control group (z = −2.151, p = 0.031) than in the SAD group. A comparison of sociodemographic characteristics in children in the SAD and control groups are shown in Table 1.
Table 1.
A comparison of sociodemographic characteristics in the SAD and control groups
| Variable | SAD (n = 30) | Control (n = 30) | Statistics | pvalue |
|---|---|---|---|---|
| Sex | ||||
| Female | 12 (40) | 12 (40) | χ2 = 0.000 | 1.000a |
| Male | 18 (60) | 18 (60) | ||
| Age (yr) | 8.83 (7.44−9.81) | 8.67 (7.75−10.79) | z = −0.510 | 0.610b |
| BMI | 17.13 (15.40−19.44) | 19.28 (16.57−22.05) | z = −2.151 | 0.031b,* |
| Maternal age (yr) | 39.5 (33.75−44.50) | 43 (39.0−46.0) | z = −1.122 | 0.262b |
| Maternal BMI | 26.69 ± 5.94 | 26.01 ± 4.25 | t = −0.510 | 0.612c |
Values are presented as number (%), median (25−75%), or mean ± standard deviation.
SAD, separation anxiety disorder; BMI, body mass index.
aPearson chi-square test pvalue; bMann–Whitney Utest pvalue; cStudent’s ttest pvalue.
*p < 0.05.
A Comparison of K-SDAS-PL-DSM-5-T Profiles in the SAD and Control Groups
The clinical diagnostic profile and accompanying diagnoses in the cases diagnosed with SAD and the control group were determined using K-SDAS-PL-DSM-5-T. A comparison of K-SADS-PL-DSM-5 profile in children in the SAD and control groups are shown in Table 2. At least one psychiatric disease was present during evaluation in 73.3% (n = 22) of the SAD group and 40% (n = 12) of the control group (χ2 = 6.787, p = 0.009). The prevalence of specific phobia (χ2 = 4.320, p = 0.038) and OCD (χ2 = 5.455, p = 0.020) was higher in the SAD group. No significant difference was observed between the two groups in terms of other psychiatric disease.
Table 2.
A comparison of K-SADS-PL-DSM-5 profile in children in the SAD and control groups
| Psychiatric disorders | SAD (n = 30) | Control (n = 30) | χ2 | pvalue |
|---|---|---|---|---|
| Present | ||||
| Specific phobias | 8 (26.7) | 2 (6.7) | 4.320 | 0.038a,* |
| Generalized anxiety disorder | 3 (10.0) | 1 (3.3) | 0.612b | |
| Social anxiety disorder | 6 (20.0) | 2 (6.7) | 0.254b | |
| Attention deficit hyperactivity disorder | 10 (33.3) | 8 (26.7) | 0.317 | 0.573a |
| Enuresis | 4 (13.3) | 0 (0.0) | 0.112b | |
| Encopresis | 2 (6.7) | 1 (3.3) | 1.000b | |
| Obsessive compulsive disorder | 9 (30.0) | 2 (6.7) | 5.455 | 0.020a,* |
| Tic disorder | 1 (3.3) | 2 (6.7) | 1.000b | |
| Lifetime | ||||
| Depressive disorders | 0 (0.0) | 1 (3.3) | 1.000b | |
| Specific phobias | 9 (30.0) | 2 (6.7) | 5.455 | 0.020a,* |
| Generalized anxiety disorder | 3 (10.0) | 1 (3.3) | 0.612b | |
| Social anxiety disorder | 6 (20.0) | 3 (10.0) | 0.472b | |
| Attention deficit hyperactivity disorder | 10 (33.3) | 8 (26.7) | 0.317 | 0.573a |
| Enuresis | 4 (13.3) | 3 (10.0) | 1.000b | |
| Encopresis | 3 (10.0) | 1 (3.3) | 0.612b | |
| Obsessive compulsive disorder | 10 (33.3) | 4 (13.3) | 3.354 | 0.067a |
| Tic disorder | 4 (13.3) | 2 (6.7) | 0.671b |
Values are presented as number (%).
K-SADS-PL-DSM-5, Kiddie Schedule for Affective Disorders and Schizophrenia Present and Lifetime Version- for Diagnostic and Statistical Manual of Mental Disorders Fifth Edition; SAD, separation anxiety disorder.
aPearson chi-square test pvalue; bFisher’s exact test pvalue.
*p < 0.05.
A Comparison of Total RCADS-CV, SAAS-CF Score, and Total Inventory of ECR-RC (attachment) Scores in Children and ASAQ, HDRS, and HARS Score in Mothers in the SAD and Control Groups
A comparison of total RCADS-CV, SAAS-CF score, and total inventory of ECR-RC (attachment) scores in children and ASAQ, HDRS, and HARS score in mothers in the SAD and control groups is shown in Table 3. As this table shows, total RCADS-CV Scores were significantly higher in the patient group (z = −5.005, p < 0.001). SAAS-CF score were significantly higher in the patient group (z = −4.284, p < 0.001). HARS scores were significantly higher in the mothers of the patient group (z = −4.156, p < 0.001). But there was no statistically significant difference in total inventory of ECR-RC (attachment) scores between the children in the two groups (z = −1.823, p = 0.074), in HDRS scores between the mother in the two groups (z = −1.750, p = 0.080), in ASAQ scores between the mother in the two groups (z = −1.496, p = 0.140).
Table 3.
A comparison of total RCADS-CV, SAAS-CF score, and total ECR-RC (attachment) scoresin children and ASAQ, HDRS, and HARS score in mothers in the SAD and control groups
| Variable | SAD (n = 30) | Control (n = 30) | Statistics | pvalue |
|---|---|---|---|---|
| SAAS-CF | 35.00 (27.50−39.25) | 21.00 (16.75−26.50) | z = −4.284 | <0.001a,* |
| Mother-HDRS | 12.00 (9.00−15.25) | 9.50 (5.00−14.75) | z = −1.750 | 0.080a |
| Total RCADS-CV | 56.33 ± 19.71 | 30.87 ± 19.71 | t = −5.005 | <0.001b,* |
| Total ECR-RC (attachment) | 139.83 ± 25.95 | 127.70 ± 25.61 | t = −1.823 | 0.074b |
| Mother-HARS | 23.90 ± 8.90 | 14.87 ± 7.91 | t = −4.156 | <0.001b,* |
| Mother-ASAQ | 28.67 ± 13.21 | 23.40 ± 14.04 | t = −1.496 | 0.140b |
Values are presented as median (25−75%) or mean ± standard deviation.
RCADS-CV, Revised Children’s Anxiety and Depression Scale-Child Version; SAAS-CF, Separation Anxiety Assessment Scale-Children’s Forms; ECR-RC (attachment), Inventory of Experiences in Close Relationships-Revised for children and adolescents-Middle Childhood Scale; ASAQ, Adult Separation Anxiety Questionnaire; HDRS, Hamilton Depression Rating Scale; HARS, Hamilton Anxiety Rating Scale; SAD, separation anxiety disorder.
aMann–Whitney Utest pvalue; bStudent’s ttest pvalue.
*p < 0.001.
Pre- and Post-treatment the CGIS Severity Scores among Children in the SAD Group
A comparison of CGIS severity scores before and at the third month after treatment in the SAD group is shown in Table 4. Children’s CGIS severity scores decreased after treatment (z = −4.569, p < 0.001).
Table 4.
A comparison of pre- and post-treatment the CGIS severity scores among children in the SAD group
| Variable | SAD pre-treatment (n = 30) | SAD post-treatment (n = 26) | z | pvalue |
|---|---|---|---|---|
| CGIS | 6.00 (3.00−7.00) | 3.00 (1.00−5.00) | −4.569 | <0.001a,* |
Values are presented as median (25−75%).
CGIS, Clinical Global Impression Scale; SAD, separation anxiety disorder.
aWilcoxon test pvalue.
*p < 0.001.
Plasma Oxytocin Levels in Children and Mothers in the SAD and Control Groups
A comparison of the oxytocin levels of the children and mothers in the two groups is shown in Table 5. As this table shows, there was no statistically significant difference in oxytocin levels between the children in the two groups (z = −0.177, p = 0.859), but maternal oxytocin levels were significantly higher in the control group (z = −4.779, p < 0.001). When 5 people in the control group who were diagnosed with other anxiety disorders were excluded and oxytocin levels were calculated again, there was no statistically significant difference in oxytocin levels between the children in the two groups (z = −0.118, p = 0.906), but maternal oxytocin levels were significantly higher in the control group (z = −4.588, p < 0.001). Of the 30 individuals in the study group, 6 were diagnosed with SP, 3 with generalized anxiety disorder and 9 people with specific phobia. Half of the individuals (n = 15) had received a diagnosis of other anxiety disorders. After excluding individuals with any other anxiety disorders, there was no statistically significant difference in oxytocin levels between the children in the two groups (t = −0.872, p = 0.394), but maternal oxytocin levels were significantly higher in the control group (z = −3,719, p < 0.001). Since age, BMI, and depression levels may affect oxytocin levels, these variables were determined as covariants, and children’s and mothers’ oxytocin levels were again compared using the ANCOVA test [40-42]. No significant difference was again observed in children’s oxytocin levels (F = 0.110, p = 0.741, ηp2 = 0.002), but maternal oxytocin levels were again higher in the control group (F = 44.605, p < 0.001, ηp2 = 0.477) (Fig. 2).
Table 5.
A comparison of plasma oxytocin levels in children and mothers in the SAD and control groups
| Variable | SAD (n = 30, 27) | Control (n = 30) | z | pvalue | ANCOVAa | ||
|---|---|---|---|---|---|---|---|
|
| |||||||
| F | pvalue | ηp2 | |||||
| Child oxytocin (pg/ml) | 168.44 (124.49−241.51) | 170.28 (138.90−198.06) | −0.177 | 0.859b | 0.110 | 0.741b,c | 0.002 |
| Maternal oxytocin (pg/ml) | 70.61 (52.44−105.65) | 178.31 (155.26−248.27) | −4.779 | <0.001b,* | 44.605 | <0.001b,c,* | 0.477 |
Values are presented as median (25−75%).
RCADS-CV-major depressive disorders score for children; HDRS score for mothers.
SAD, separation anxiety disorder; RCADS-CV, Revised Children’s Anxiety and Depression Scale-Child Version; HDRS, Hamilton Depression Rating Scale.
aCovariants: BMI and age-years. bMann–Whitney Utest pvalue. cLogarithmic transformation was performed before ANCOVA.
*p < 0.001.
Fig. 2.
A comparison of plasma oxytocin levels in children and mothers in the SAD and control groups.
SAD, separation anxiety disorder.
Pre- and Post-treatment Plasma Oxytocin Levels among Children and Mothers in the SAD Group
A comparison of oxytocin levels before and at the third month after treatment in the SAD group is shown in Table 6. Children’s oxytocin levels decreased after treatment (z = −2.808, p = 0.005), while mothers’ oxytocin levels increased (z = −3.389, p = 0.001) (Fig. 3).
Table 6.
A comparison of pre- and post-treatment plasma oxytocin levels among children and mothers in the SAD group
| Variable | SAD pre-treatment (n = 30, 27) | SAD post-treatment (n = 22, 24) | z | pvalue |
|---|---|---|---|---|
| Child oxytocin (pg/ml) | 168.44 (124.49−241.51) | 104.47 (64.22−169.60) | −2.808 | 0.005a,* |
| Maternal oxytocin (pg/ml) | 70.61 (52.44−105.65) | 303.75 (186.36−364.36) | −3.389 | 0.001a,* |
Values are presented as median (25−75%).
SAD, separation anxiety disorder.
aWilcoxon test pvalue.
*p < 0.01.
Fig. 3.
A comparison of pre- and post-treatment plasma oxytocin levels among children and mothers in the SAD group.
SAD, separation anxiety disorder.
Correlations between the Pre-treatment Oxytocin Levels and Pre-treatment Total RCADS-CV, SAAS-CF Score, and Total Inventory of ECR-RC (Attachment) Scores of Children with Separation Anxiety Disorder
A negative correlation was found between the pre-treatment oxytocin levels and pre-treatment attachment scores of children with SAD (r = −0.372, p = 0.043). No signifi-cant correlation was observed between pre-treatment plasma oxytocin levels in children with SAD and the RCADS- CV score or pre-treatment total SAAS-CF score (Table 7).
Table 7.
Correlations between pre-treatment plasma oxytocin level in children with SAD and pre-treatment total RCADS-CV, SAAS-CF score, and total ECR-RC total score
| SAD pre-treatment | Pre-treatment plasma oxytocin levels |
|---|---|
| RCADS-CV total score | |
| r | 0.158 |
| p a | 0.405 |
| SAAS-CF total score | |
| r | 0.098 |
| p a | 0.605 |
| ECR-RC total score | |
| r | −0.372 |
| p a | 0.043* |
SAD, separation anxiety disorder; RCADS-CV, Revised Children’s Anxiety and Depression Scale-Child Version; SAAS-CF, Separation Anxiety Assessment Scale-Children’s Forms; ECR-RC (attachment), Inventory of Experiences in Close Relationships-Revised for children and adolescents-Middle Childhood Scale; r, correlation coefficient.
aSpearman correlation analysis pvalue.
*p < 0.05.
Correlations between Plasma Oxytocin Level Changes in Children with SAD and Total RCADS-CV, SAAS-CF Score, and Total Inventory of ECR-RC (Attachment) Scores Changes, and between Plasma Oxytocin Level Changes in Mothers of Children with SAD and ASAQ, HDRS, and HARS Score Changes
Spearman’s correlation analysis revealed significant positive correlation between changes in the plasma oxytocin levels of children with SAD and the change in total RCADS-CV scores (r = 0.525, p = 0.012). No significant positive correlation was observed between plasma oxytocin levels in children with SAD and post-treatment total SAAS-CF score change or total Inventory of ECR-RC (attach-ment) score change. No significant correlation was determined between the change in plasma oxytocin levels in mothers of children with SAD and post-treatment ASAQ, HDRS, or HARS score changes (Table 8).
Table 8.
Correlations between plasma oxytocin level changes in children with SAD and total RCADS-CV, SAAS-CF score, and total ECR-RC score changes, and between plasma oxytocin level changes in mothers of children with SAD and ASAQ, HDRS, and HARS score changes
| SAD | Child plasma oxytocin level change | Maternal plasma oxytocin level change | |
|---|---|---|---|
| RCADS-CV total score change | ASAQ score change | ||
| r | 0.525 | r | −0.010 |
| p a | 0.012* | p a | 0.965 |
| SAAS-CF total score change | HDRS score change | ||
| r | 0.398 | r | −0.173 |
| p a | 0.066 | p a | 0.453 |
| ECR-RC total score change | HARS total score change | ||
| r | 0.135 | r | −0.057 |
| p a | 0.551 | p a | 0.807 |
SAD, separation anxiety disorder; RCADS-CV, Revised Children’s Anxiety and Depression Scale-Child Version; SAAS-CF, Separation Anxiety Assessment Scale-Children’s Forms; ECR-RC (attachment), Inventory of Experiences in Close Relationships-Revised for children and adolescents-Middle Childhood Scale; ASAQ, Adult Separation Anxiety Questionnaire; HDRS, Hamilton Depression Rating Scale; HARS, Hamilton Anxiety Rating Scale; r, correlation coefficient.
aSpearman correlation analysis pvalue.
*p < 0.05.
DISCUSSION
Although the role of oxytocin in attachment and that of insecure attachment in the etiology of SAD are well known, the effect of oxytocin on the biological basis of SAD is still unclear. To the best of our knowledge, our study is the first to examine plasma oxytocin levels and their changes after treatment in children with SAD and their mothers to examine the possible roles of oxytocin on the biological basis of SAD. In literature, previous studies have only examined saliva oxytocin levels in children with separation anxiety and their mothers, and none have included a control group [18,20]. Our study compared the plasma oxytocin levels of children with separation anxiety and of these children’s parents with those of a control group. In addition, the SAD group was re-evaluated after treatment in order to examine the change in oxytocin levels and its relationship with change in anxiety. No difference in plasma oxytocin levels was determined between the children with SAD and the control group. Plasma oxytocin levels decreased significantly after treatment in children with SAD in this study. Significant positive correlation was observed between plasma oxytocin levels in children with SAD and change in total RCADS- CV scores. Plasma oxytocin levels were significantly lower in the mothers of children with SAD in this study than in the control group mothers. Plasma oxytocin levels among mothers in the SAD group also increased on the third month after treatment. In the literature, studies examining the effects of anxiety disorders on oxytocin levels are limited and reveal inconsistent findings. Some studies of patients with anxiety disorder have determined higher plasma oxytocin levels compared to healthy controls [21,43], while others have observed no significant difference [22,44,45]. The majority of studies investigating oxytocin levels have concentrated on general anxiety, and oxytocin in specific anxiety disorders has been less studied [18]. Very few studies have examined separation anxiety and oxytocin levels in children and adults [18,20,23]. One study of children with anxiety, including children with separation anxiety, reported significantly lower oxytocin in saliva in children with separation anxiety compared to other anxiety disorders [17]. To the best of our knowledge, with one exception, studies of oxytocin levels in patients with SAD have not involved control groups [18,20,23,46]. In the study of Uzun et al. [43], oxytocin levels were found to be higher in children with SP compared to healthy controls; however, the menstrual periods of adolescents were not taken into account when measuring oxytocin levels. It was thought that this situation might affect oxytocin levels [43]. Some authors suggest that we consider previous conflicting findings of researchers and that oxytocin has a variable association with state and trait anxiety in children [43]. Some studies have suggested that these inconsistent findings arose from context- dependent factors and personal factors [47]. In our study, no difference in plasma oxytocin levels was determined between the children with SAD and the control group. It is reasonable to assume that children with separation anxiety may have lower oxytocin levels compared to the healthy control group. The fact that no difference was observed in plasma oxytocin levels between children with SAD before treatment and children in the control group may be related to the increased situational anxiety levels of the control group during processes such as psychiatric interview, assessment, and blood collection. However, the fact that state anxiety levels were not investigated in this study does not permit us to elucidate this relationship. Although we took care that such factors such as fasting-satiation, age, menstrual cycle, pregnancy, lactation, hormone use, and infection should not affect plasma oxytocin levels in this study, oxytocin is a rapidly-changing hormone with a short half-life.
In our study, significant positive correlation was observed between plasma oxytocin levels in children with SAD and change in total anxiety scores. The significant positive correlation between plasma oxytocin levels and the change in total RCADS-CV scores in children with SAD in our study may be due to the compensatory increase of oxytocin to reduce anxiety symptoms. Some studies performed in different populations have determined a positive correlation between anxiety scores and oxytocin levels [21,45], while others have determined negative correlation [13,14,48-53] and one study observed no correlation [54]. In a study of adults with SP, Hoge et al. [45] reported no significant difference in oxytocin levels between these and healthy controls. However, severity of SP symptoms were associated with higher oxytocin levels, and the authors hypothesized that high oxytocin levels may be a compensatory mechanism for oxytocin receptor dysfunction. Another study of 23 male diagnosed with SP reported significantly higher oxytocin levels than in healthy controls. After controlling for age and years of education, higher oxytocin levels were significantly associated with higher Liebowitz Social Anxiety Scale scores; this was thought to be caused by an insufficient compensatory attempt by oxytocin to reduce social anxiety symptoms [21]. This all suggests that oxytocin levels may be a useful biomarker in anxiety [13]. These studies, which found a positive correlation between oxytocin levels and anxiety scores, like ours, support our findings. In some studies that found a negative correlation between oxytocin levels and anxiety scores; this was explained by the anxiolitic effect of oxytocin. A study of 473 healthy adults found significantly lower trait anxiety in male with higher oxytocin levels, suggesting that oxytocin may exhibit anxiolytic effects in male [14]. Scantamburlo et al. [49] explained the negative relationship between oxytocin level and anxiety in terms of oxytocin reducing the stress response and the response of the HPA axis to psychogenic stressors. However, Gordon et al. [54] attributed the negative correlation between oxytocin levels and anxiety to the stress- preventing property of oxytocin and to its role in attach-ment. The inconsistent findings in the literature may be due to the rapidly changing nature of oxytocin.
Plasma oxytocin levels decreased significantly after treatment in children with SAD in this study. Significant positive correlation was observed between plasma oxytocin levels in children with SAD and change in total RCADS-CV scores. Few studies in the literature have followed-up children or adults with SAD and examined their oxytocin levels before and after intervention. In one study, an insignificant decrease in salivary oxytocin levels in adult patients with SAD was determined after an attachment-based therapy. The change being statistically insignificant was attributed to the relatively small sample size, and the authors recommended that further studies be performed using oxytocin as a biomarker in patients with SAD [23]. Another study of 38 patients with SP deter-mined a significant decrease in plasma oxytocin levels after the ‘Trust Game’ compared to initial levels. This was attributed to reduced ability of adults with SP to respond to a prosocial test due to a lack of social skills [22]. Another study involving 50 healthy adolescents reported a rapid increase in salivary oxytocin levels following application of a ‘social stress test’ compared to a control group. This was immediately followed by a decrease, and baseline oxytocin was found to be negatively correlated with experienced anxiety and insecurity [15]. The increase in oxytocin in the face of a stress response was attributed to its being an anxiolytic and to compensatory effects [15]. A different study of 41 children with anxiety reported that children with separation anxiety exhibited a significantly higher salivary oxytocin response immediately after a seven-minute mother-child interaction (emotional communication, maternal intervention, maternal sensitivity, and child participation) compared to children with other forms of anxiety [20]. The authors attributed this increase in oxytocin following mother-child interaction to the role of oxytocin in attachment in the context of child anxiety [20]. However, the fact that salivary oxytocin levels were investigated following a short-term intervention in that study sheds no light on the relationship between oxytocin and change in long-term anxiety levels. We hypothesized that the anxiety levels would decrease and oxytocin levels would increase after three months of treatment in children with separation anxiety. In the present study, the significant decrease in children’s plasma oxytocin levels after three months of follow-up and treatment suggests that the increase in oxytocin levels to compensate for anxiety may have decreased in line with the decrease in post- treatment anxiety. The change in plasma oxytocin levels in children after treatment may be due to the anxiolytic effects of fluoxetine or to its pharmacodynamics properties. Studies have shown that fluoxetine can also affect oxytocin levels, although we encountered only one study of its effect on oxytocin levels in humans, which showed that fluoxetine reduced them significantly [55]. Research into the effect of fluoxetine on oxytocin levels has largely taken the form of animal studies [56,57]. Two animal studies in fact reported that fluoxetine did not affect oxytocin levels [58,59]. A neuroanatomical study reported that the distribution of oxytocin-labeled neurons and serotonin transporter immune reactive fibers in the brain overlapped, and suggested that the effects of SSRIs on social relations and anxiety might be mediated by the oxytocinergic system [60].
Plasma oxytocin levels were significantly lower in the mothers of children with SAD in this study than in the control group mothers. Plasma oxytocin levels among mothers in the SAD group also increased on the third month after treatment. We hypothesized that: [1] oxytocin levels in mothers of children with separation anxiety would be lower than in the control group, [2] oxytocin levels would increase in mothers of children with separation anxiety, and [3] children’s anxiety levels would decrease after three months of treatment. Our results were consistent with our hypothesis. These findings suggest that oxytocin compensates for anxiety in children, but may have a different function in mothers. A follow-up study of 127 pregnant reported a significant correlation between separation anxiety, depression, and general anxiety scores and an insecure attachment style measured during pregnancy, together with low oxytocin in the postpartum period [46]. The authors of that study suggested that elevations in affiliative need may be associated with insecure attachment styles, maladaptive interpersonal coping responses, and unsupportive relationships, and that these may lead to or maintain depressed mood, thus resulting in decreased levels of oxytocin [46]. In their study of 62 pregnant females, Levine et al. [16] suggested that higher oxytocin levels throughout pregnancy and in the postpartum period were associated with maternal bonding to the baby, although the underlying cause was unclear, but that this might be associated with anxiety. It has further been postulated that oxytocin levels in adults may also be associated with maternal attachment to the father [54]. Interpreting our findings together with the existing literature, since SAD is known to be associated with insecure attachment, the fact that mothers’ oxytocin levels were lower than those of the control group may be related to insecure attachment between mother and child. A decrease in separation anxiety and attachment to the parent after treatment allows the child to commence differentiation. This in turn ensures a more positive relationship between the child and the mother, with the attachment relationship between mother and child being positively affected. It is possible that oxytocin may have mediated this change. However, maternal attachment to the child was not investigated in this study, and further research into this is now needed.
There are a number of limitations to this study. The sample numbers in both the patient and control groups were small. In addition, since other anxiety disorders such as specific phobia, generalized anxiety disorder, and SP are known to affect plasma oxytocin levels, the presence of these diagnoses in both the patient and control groups can be regarded as a confounding factor for our findings. Maternal anxiety and depression were evaluated using self-report scales rather than semi-structured or structured interviews. Although the members of the patient group were followed-up in an intensive manner, drug use was irregular in a very small number of children. Although infection in children and their mothers was ruled out close to the time when they gave blood, this information was obtained only through histories, and was not checked with acute phase reactants such as CRP in blood tests. Similarly, we learned whether mothers were pregnant or lactating, or using oral contraceptives or on hormone therapy from the mothers’ histories. Blood samples were collected after treatment only from the patient group and their mothers, and not from the control group. The members of the control group were not followed-up because these had no psychological problems. Additionally, the increase in state anxiety in the patient and control groups due to interventions such as psychiatric evaluation and blood collection may have affected plasma oxytocin levels. Although we took care that such factors such as fasting-satiation, age, menstrual cycle, pregnancy, lactation, hormone use, and infection should not affect plasma oxytocin levels in this study, oxytocin is a rapidly-changing hormone with a short half-life.
Although there are limitations in our study, there are many strengths that make our study meaningful. The patient and control groups were matched in terms of age and sex; exclusion of participants and their mothers from the study with any identified metabolic, endocrinological, neurological, recent infections; Exclusion of the mothers of the patients and the control group who were in pregnancy and lactation period, using oral contraceptive medication for at least the last 1 month, or receiving hormone therapy, ensured that the confounding factors were eliminated to a large extent.
Since the plasma oxytocin level can change during the day and can be affected by factors such as fasting, satiety, and different periods of the menstrual cycle, blood was taken from mothers in the early follicular phase of the menstrual cycle and from children between 9:00−10:00 AM in the morning; this situation reduces the confounding factors for oxytocin level and strengthens our study. The fact that our study was not a cross-sectional study, and the patient group was interviewed every month face-to-face to monitor the severity of the disease and whether there was any improvement with the CGI scale, making it easier for them to come to the follow-ups regularly, reducing the frequency of irregular use of the treatment and providing the continuation of our data, strengthened us to be a follow-up study.
In our study, in the control group of 30 people, there were only 5 people diagnosed with other anxiety dis-orders. When the plasma oxytocin levels were calculated by excluding these 5 individuals, it was observed that it did not affect significantly the statistical data, so it was considered okay to include them. When these 5 people in the control group were excluded and oxytocin levels were calculated again, there was no statistically significant difference in oxytocin levels between the children in the two groups (z = −0.118, p = 0.906), but maternal oxytocin levels were significantly higher in the control group (z = −4.588, p < 0.001). In addition, since both mothers and children were included in the study, we decided to include the mothers of these 5 people in the study because they met the inclusion criteria of the study.
Of the 30 individuals in the study group, 6 were diagnosed with SP, 3 with generalized anxiety disorder and 9 people with specific phobia. Half of the individuals (n = 15) had received a diagnosis of other anxiety disorders. After excluding individuals with any other anxiety disorders, there was no statistically significant difference in oxytocin levels between the children in the two groups (t = −0.872, p = 0.394), but maternal oxytocin levels were significantly higher in the control group (z = −3,719 p < 0.001). Specific phobia, generalized anxiety disorder, and SP diagnoses can often accompany patients with SAD. When the plasma oxytocin levels were calculated by excluding these 15 individuals, it was observed that it did not affect significantly the statistical data.
Specific phobia, generalized anxiety disorder, and SP diagnoses can often accompany patients with SAD. Keeping the diagnosis of other anxiety disorders to a minimum in the patient and control groups in our study minimizes the confounding factors in our study.
Finally, although there have been various opinions expressed regarding which is the most reliable method for measuring oxytocin levels, plasma levels obtained from peripheral blood were investigated using ELISA in the present study. Our findings suggest that determining the change in oxytocin levels following treatment may be useful in terms of the follow-up and treatment of these cases. However, further studies involving larger samples and considering plasma and cerebrospinal fluid levels are now needed for a better understanding of the biological basis of oxytocin in SAD.
No difference in plasma oxytocin levels was determined between the children with SAD and the control group in this study. Plasma oxytocin levels decreased significantly after treatment in children with SAD. Plasma oxytocin levels were significantly lower in the mothers of children with SAD in this study than in the control group mothers. Plasma oxytocin levels among mothers in the SAD group also increased on the third month after treatment. The change in plasma oxytocin levels in both children and mothers after treatment suggests that oxytocin may be important in the etiology of SAD. Further studies are now needed to elucidate the relationship between SAD and oxytocin levels.
Acknowledgements
Support for this research came from the Istanbul University Scientific Research Projects unit, which provides financial means with the project code TTU-2020-36927 to Dr. Gülcü Üstün. The authors gratefully recognize the assistance of all patients and controls.
Funding Statement
Funding Financial support for this research came from the Istanbul University Scientific Research Projects unit, which provides financial means with the project code TTU-2020-36927 to Dr. Gülcü Üstün. The funding sources were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
Footnotes
Conflicts of Interest
Financial support for this research came from the Istanbul University Scientific Research Projects unit, which provides financial means with the project code TTU-2020- 36927 to Dr. Gülcü Üstün. The funding sources were not involved in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. Dr. Gümüş and Dr. Soylu have no financial interests to disclose.
Author Contributions
Conceptualization: Nur Seda Gülcü Üstün. Data acquisition: Nur Seda Gülcü Üstün. Formal analysis: Nur Seda Gülcü Üstün, Şefika Nur Gümüş. Funding: Nusret Soylu. Supervision: Nusret Soylu. Writing—original draft: Nur Seda Gülcü Üstün. Writing—review & editing: Nur Seda Gülcü Üstün, Nusret Soylu.
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