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Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder
Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder Ali Emhan a, Salih Selek b,n, Hüseyin Bayazıt c, İbrahim Fatih Karababa c, Mahmut Katı c, Nurten Aksoy d a
Clinic of Psychiatry, Ministry of Health, Diyarbakır Education and Research Hospital, Diyarbakır, Turkey Harris County Psychiatric Center, University of Texas Health Science Center at Houston, TX, USA c Department of Psychiatry, Faculty of Medicine, Harran University, Sanliurfa, Turkey d Department of Biochemistry, Faculty of Medicine, Harran University, Sanliurfa, Turkey b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 November 2014 Received in revised form 30 July 2015 Accepted 2 November 2015
Generalized anxiety disorder (GAD) is a prevalent psychiatric disorder. The exact causes of GAD still unknown, in addition to neurochemical and neuroanatomic disorders, genetic and environmental factors are discussed in etiology. In our study we aimed to evaluate the oxidative metabolism's status and investigate the role of oxidative metabolites in GAD. Blood samples were taken from enrolled subjects in appropriate way and total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were studied in Harran University Biochemistry Labs. Results were compared between groups. The patients' TOS and OSI levels were significantly higher than control group. The patients' TAS levels were significantly lower than controls'. According to our findings, oxidative stress mechanism might have a role in GAD pathophysiology. In the future, total antioxidants may be used as a biologic marker in GAD etiology but more research is needed. & 2015 Published by Elsevier Ireland Ltd.
Keywords: Generalized anxiety disorder Oxidative stress Total antioxidant status Total oxidant status
1. Introduction Generalized anxiety disorder (GAD) is a clinical picture characterized by exaggerated feeling of worry related with various daily events and occurring almost all day long. In addition to anxiety, these patients experience unrest, tiredness, difficulty in concentrating, getting nervous rapidly, muscular straining and insomnia (Pine and McClure, 2005a). The prevalence of GAD is reported to be 3–6% in the general population with being two times more frequent in women than in men; it has been reported that it is the most frequent distress disorder among the patients referred to the primary care institutions (Wittchen et al., 2002). Although many biological and psychological factors have been considered to play a role in the etiology of GAD, none of these has been found to be a definite cause. In order to make a definite diagnosis, we need a well-documented history and thorough psychological examination as for the other psychiatric disorders (American Psychiatric Association, 2000). Recently, studies have been conducted to investigate the role of oxidative metabolism in the etiology of anxiety disorders (Atmaca, et al., 2004; Ersan et al., 2006; Tezcan et al., 2003). n
Corresponding author. E-mail address: [email protected] (S. Selek).
Oxidants (or pro-oxidants/free radicals) arise from the reactions of degradation involving oxygen as waste molecules. Having some beneficial effects (such as phagocytosis of monocytes and neutrophils), oxidants involve in reactions with many compounds including DNA, protein, and polyunsaturated fats of the cell membrane phospholipids. Among these reactions, particularly those affecting and damaging DNA pave the way for the development of various diseases. The substances called antioxidants diminish the deleterious effects caused by these oxidative products. There is a balance between the oxidants and antioxidants in a living body, and damage occurs when such balance is lost in favor of oxidants, a case called “oxidative stress” in the literature (Haddad, 2004). As described, though several studies have been carried out to investigate the role of oxidative metabolism in the pathophysiology of anxiety disorders, there is no evaluation of the total antioxidant level (TAL), total oxidant level (TOL) or any specific compound (e.g., malondialdehyde, catalase, superoxide dismutase etc.) in the generalized anxiety disorder (Atmaca et al., 2004; Ersan et al., 2006; Tezcan et al., 2003; Wittchen et al., 2002). Therefore, in this present cross-sectional case-control study, we aimed to determine whether the mechanism of oxidative stress plays a role in the pathophysiology of GAD and examine the diagnostic testing performance of total oxidants and total antioxidants in the etiology of GAD.
http://dx.doi.org/10.1016/j.psychres.2015.11.001 0165-1781/& 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: Emhan, A., et al., Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder. Psychiatry Research (2015), http://dx.doi.org/10.1016/j.psychres.2015.11.001i
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2. Methods
2.3. Measurement and estimation of variables
2.1. Study method and subjects
2.3.1. Total antioxidant level (TAL) measurement The total antioxidant status of the plasma was measured using a novel automated colorimetric measurement method for TAS developed by Erel (2004). In this method the hydroxyl radical, the most potent biological radical, is produced by the Fenton reaction, and reacts with the colorless substrate O-dianisidine to produce the dianisyl radical, which is bright yellowish-brown in color. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the re-action mix are suppressed by the antioxidant components of the plasma, preventing the color change and thereby providing an effective measure of the total antioxidant status of the plasma. The assay results are expressed as mmol Trolox Eq/L, and the precision of this assay is excellent, being lower than 3% (Cao and Prior, 1998).
This study included 40 patients, aged between 18 and 65 years, referred consecutively to the Outpatient Clinic of the Medical Faculty of the Harran University, who had had a diagnosis of GAD according to DSM-IV, and gave a written informed consent to participate in the study. The study was approved by the local ethics committee (the Medical Faculty of the Harran University). The patients' previous medical records and follow-up information were reviewed, and the socio-demographic variables including age, gender, comorbidities, the medications used, and the smoking status were recorded. The exclusion criteria of the study were as follows: pregnancy, serious systemic disease, epilepsy, dementia, disorders of alcohol and drug misuse, depression, panic disorders, obsessive compulsive disorder, schizophrenia, bipolar disorder and personality disorders (Axis II pathology), mild to moderate mental retardation, history of serious head trauma, and inadequate sampling. On the day when serum samples were drawn, they were administered a Hamilton Anxiety Rating Scale, Clinical Global Impression, and completed a socio-demographic questionnaire prepared for the study by one psychiatrist (AE). The control group consisted of 40 healthy volunteers. These volunteers' socio-demographic variables including age and gender as well as the smoking status were also recorded. Inadequate sampling was determined as an exclusion criterion. Blood samples were drawn from the antecubital vein following 12 h fasting. Thereafter, blood samples were poured into tubes, centrifuged at 3000 rpm for 5 min to separate plasma, and stored on ice to be processed within 6 h at most. The separated plasmas were stored at 80 °C for determining total antioxidant level (TAL) and total oxidant level (TOL). The plasma TAL and TOL levels were measured to calculate the oxidative stress index (OSI) in the Biochemistry Laboratory of the Harran University. The patients were gender-matched with the control group. 2.2. Data collection tools 2.2.1. Socio-demographic form Each patient was applied a routine follow-up and adverse effect scale and a socio-demographic form which has been prepared for recording demographics, height, body weight, smoking status and the laboratory results. Data from the patients were screened and recorded. 2.2.2. Hamilton anxiety rating scale (HAM-A) On the day of serum sampling, this scale was administered to all subjects participated in the study by an investigator (AE) in the Department. Used for determining the severity of anxiety and the distribution of symptoms, this scale comprises a total of 14 questions interrogating both somatic and psychic symptoms and compassing sub-dimensions. A five-point Likert-type measurement is provided. A total score is obtained by summing the points of items. The validity and reliability of its Turkish version has been conducted by Yazıcı et al. (1998). 2.2.3. Clinical global impression scale (CGI) CGI is a 3-item scale to evaluate the severity of disease, the improvement, and therapeutic efficacy index. The therapeutic efficacy index has two subsets as therapeutic effect and adverse effect (Guy, 1976). CGI was applied to assess the severity of comorbidities when blood samples were taken. For remission, the comorbidities having a CGI severity subscore r2 were considered as in remission.
2.3.2. Total oxidant level (TOL) measurement The total oxidant status of the plasma was measured using a novel automated colorimetric measurement method for TOS developed by Erel, (2005). In this method oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmol H2O2 Eq/L). 2.3.3. Oxidative stress index (OSI) measurement Oxidative stress index (OSI) was calculated as the total oxidant level (TAL)/total antioxidant level (TOL) ratio (Kosecik et al., 2005). 2.4. Statistical assessment of data Statistical analyses were performed using “SPSS 15.0 for Windows” software. Following the descriptive statistics, a Q–Q diagram was graphed in order to assess the distribution of the parameters investigated. A t-test was used for the paired group comparison of the data showing normal distribution, and Chisquare test for comparing the ratios. Spearman's correlation test was used for correlation analysis. A P value of o 0.05 was accepted as significant.
3. Results The demographical data of the patients and control subjects are given in Table 1. Ages, gender, duration of illness were similar among groups (p4 0.05, for all comparison). (Table 1). TAS, TOS and OSI levels in patients and controls were summarized in Table 2. TAS, TOS and OSI levels were statistically higher in GAD patients compared to healthy controls. (P 40.05 for all comparison). Table 1 Sociodemographic and clinical characteristics of the patients and controls.
Age (mean7 SD) Gender: female/male (n) Duration of illness (years): median
Patients
Controls
P value
42.48 ( 712.03) 26/14 5 (1–30)
41.5 ( 7 12.67) 26/14
0.993
Please cite this article as: Emhan, A., et al., Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder. Psychiatry Research (2015), http://dx.doi.org/10.1016/j.psychres.2015.11.001i
A. Emhan et al. / Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 2 TAS, TOS and OSI levels in patients and controls.
n Total antioxidant status (μmol Trolox Eqv./L)
GAD (mean7 SD)
Controls (mean7 SD)
39
39
0.8438 7 (0.20543)
0.9421 7 (0.19002)
40
40
20.03787 (4.46215)
13.6230 7 (3.47888)
39
39
2.57267 (1.07470)
1.50977 (0.54855)
P value
t¼ 2.192
p ¼0.031 N Total oxidant status (μmol H2O2 Eqv. / L)
t¼ 7.170
p o0.001 N Oxidative stress index
t¼ 5.502
p o0.001 n: number of cases SD: standard deviation
4. Discussion The first finding from this study was the higher TOS level in the patients with GAD than in the control groups. There are ample data suggesting that total oxidant levels are increased in the psychiatric disorders. TOS level has been found high in two studies conducted with the patients with schizophrenia and schizoaffective disorder each (Boskovic et al., 2011; Bulbul et al., 2014). Similarly, TOS level has been found high in the studies each with the patients with major depression (Cumurcu et al., 2009), bipolar disorder (Selek et al., 2011), adult attention deficit hyperactivity disorder (Selek et al., 2012), and obsessive–compulsive disorder (Kandemir et al., 2013). These studies are controlled ones and have used the same biochemical analysis methods. In addition to TOS, changes have also been observed in the specific oxidative parameters. In a study with schizophrenia, the levels of MDA, a product resulted from lipid peroxidation, xanthine oxidase and nitric oxide have been observed to increase compared to the controls (Akyol et al., 2002). In a study by Savaş et al. (2002), the NO level has been found to be higher in the patients with bipolar disorder than in the controls, while in the some recent studies the NO level has been found higher during the euthymic, manic and depressive periods of the bipolar disorder (Gergerlioglu et al., 2007; Savas et al., 2006; Selek et al., 2008a). Similarly, Sarandol et al. (2007) and Bulut et al. (2007) have found the increased MDA levels in 96 patients with major depression and in adult attention deficit hyperactivity disorder, respectively. In the anxiety disorders including GAD, changes have been observed in the specific oxidative parameters. For instance, the levels of MDA have been elevated in both plasma and serum in two separate studies with obsessive–compulsive disorder (OCD) (Chakraborty et al., 2009; Ozdemir et al., 2009), while the levels of NO have been found higher in another study with OCD (Atmaca et al., 2005). The MDA levels and the SOD, CAT and GPX activities have been studied two times in the subjects with social phobia by the same study group in 2004 and 2008, and there have been significant elevations compared to the control group (Atmaca et al., 2004, 2008). Of two separate studies with the patients diagnosed of panic disorder, one has shown that the plasma MDA levels, and the SOD and GPX activities have been increased, and the CAT activity has been unchanged (Kuloglu et al., 2002a), whereas the other has shown that the xanthine oxidase level has been increased, and the NO level and the SOD activity have been unchanged (Herken et al., 2006). In a study with 14 patients and 14 healthy individuals evaluating the oxidative metabolism in post-traumatic stress
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disorder, the MDA level, and the SOD, CAT and GPX activities have been found unchanged, a finding attributed to the ability of body to properly detoxify the rapidly occurring free radicals (Tezcan et al., 2003). As mentioned, there are number of studies on the changes in the oxidative metabolism caused by the psychiatric disorders, but as to GAD, a separate diagnostic category in DSM, no studies have been conducted on changes in either total oxidative stress or specific oxidative parameter. However, in a study investigating the urinary excretion of oxidative metabolites in 31 subjects with a high Hospital Anxiety and Depression Scale (HADS) score who had had a family member diagnosed of advanced cancer and had experienced excessive anxiety because of giving their relatives healthcare, the MDA/creatinine ratio has been found higher in the excessive anxiety group than in the control group (Ratnakar et al., 2008). In a study by Arranz et al. (2007) 33 female patients with a high beck anxiety scale score had significantly higher stimulated and unstimulated neutrophil superoxide anion levels compared to the controls (Arranz et al., 2007). Some of the rats exposed to chronic mild stress may be considered a model of anxiety disorder. Several studies have been carried out to examine the levels of oxidative metabolism products in these experimental animals. In their study, Şahin and Gümüşlü (2004) have found that the postmortem brain tissue of the rats chronically exposed to immobility stress contained low levels of MDA and TBARS (thiobarbituric acidreactive substances), another marker for oxidative stress, and high SOD, CAT activities but low GPX activity (Sahin and Gümüşlü, 2004). In a similar study with rats, Kamper et al. (2009) have found the serum levels of MDA to be high generally, that of NO and the GPX activity to be high in the female rats but unchanged in the male rats, and the SOD activity to be unchanged in both genders (Kamper et al., 2009). In their study, Ciobica et al. (2010) have shown that there are no changes in the GPX and SOD levels, whereas there is a marked increase in the MDA level (Ciobica et al., 2010). As a conclusion, our study results revealed an increase in the levels of oxidants, thus supporting the data from the previous studies. At the present day, the effect of increased oxidants on the psychiatric disorders represents one of the hottest topics. Increased oxidant levels may lead to the destruction of the main intracellular constituents and functional compounds (Akyol et al., 2004). DNA destruction is the most interested issue. There are studies suggesting that the oxidative stress is associated with the DNA destruction in the psychiatric disorders (Andreazza et al., 2007). Increased oxidant levels in GAD may cause the DNA destruction, leading to the psychiatric disorder, although an association between the DNA destruction and the oxidative stress has not yet been shown in GAD. There is no clear causal relationship between the psychiatric disorders and the increased oxidant levels. It is not clearly known whether the increased oxidants cause psychiatric disorders or vice versa. However, the current data suggest that oxidants may lead to psychiatric disorders. For instance, the fact that there are evidences of oxidative stress even during the remission period of the bipolar disorder, and that the oxidant levels decrease with therapy in some disease (Savas et al., 2006; Yu et al., 2003). Since our present study is cross-sectional, it is difficult to reach a definitive judgment about the course of GAD, but in the light of previous data, it might be mentioned that the increased oxidant levels play a role in the pathophysiology of GAD. There is a need for further studies. One of our finding related to the oxidants was that the increased oxidant levels were not associated with a clinical evaluation score. Therefore, it may be suggested that oxidants is not associated with the disease severity, but rather with the presence or absence of the disease. The second important finding from our study was that TAS is decreased in GAD. There are ample of data suggesting that total
Please cite this article as: Emhan, A., et al., Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder. Psychiatry Research (2015), http://dx.doi.org/10.1016/j.psychres.2015.11.001i
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A. Emhan et al. / Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
antioxidant levels are decreased in the psychiatric disorders. For instance, the TAS level has been found decreased in patients with schizophrenia (Akyol et al., 2002), three separate studies including those with major depression (Cumurcu et al., 2009; Gałecki et al., 2009; Sarandol et al., 2007), one study including those with bipolar disorder (Savas et al., 2006), and one study including those with attention deficit hyperactivity disorder (Akyol et al., 2002). In addition to TAS, changes have also been observed in the specific oxidative parameters. For instance, it has been shown that the Vitamin E (Ersan et al., 2006) and selenium levels (Chakraborty et al., 2009) are decreased in the patients with OCD, while the level of glutathione and that of Vitamin E are decreased in the patients with major depression (Kodydková et al., 2009), and in two separate studies (Maes et al., 2000; Owen et al., 2005), respectively. Conversely, TAS has been found to be unchanged in a study with schizoaffective disorder (Bulbul et al., 2014), whereas it has been found to be increased in 4 studies each with the patients with bipolar disorder (Selek et al., 2011), with adult attention deficit hyperactivity disorder (Selek et al., 2012), with obsessive– compulsive disorder (Kandemir et al., 2013; Selek et al., 2008b), and with attention deficit hyperactivity disorder (Ersoy et al., 2008). As mentioned above, whereas there has been no study that evaluated the antioxidant metabolism related to GAD, in a study by Arranz et al. (2007) TAL has been found to be significantly lower in 33 female patients with anxiety and having a high score according to the Beck Anxiety Scale than in the control subjects (Arranz et al., 2007); similarly, the level of glutathione has been found to be apparently low in the brain tissue of the rats chronically exposed to immobility stress (Sahin and Gümüşlü, 2004). In support of many studies mentioned above, our study indicated that the TAS values were significantly lower in the patient group than in the control group. Specific increases in the antioxidant efficacy observed in other psychiatric disorders in which oxidative stress has been evidenced have been considered as the reactive increases in order to compensate the oxidative stress (Kandemir et al., 2013; Selek et al., 2012). The fact in our study that oxidants were increased, whereas antioxidants were mildly decreased might be interpreted that the respective patients were under stress, metabolism did not give an adequate reactive response to the increased oxidants, and there was an impairment of the responsive systems, or a yet-unknown variable would decrease the TAS in the medium. The third finding from this study was the increased OSI in the patients with GAD. OSI is an index obtained by dividing TOS by TAS, and used as a measure for the general evaluation of oxidative stress (Kosecik et al., 2005). Using OSI, we can assess two dimensions of the oxidative metabolism, e.g. antioxidants and oxidants, and see how comparable antioxidants are against oxidants. An increased OSI value compared to the controls shows that the balancing antioxidant system cannot generate an adequate response, and the balance is impaired in the direction of oxidants. The fact in our study that the balancing TAS values were decreased instead of being increased in the patients with GAD indicates a defect in the antioxidant system, and the organism of these patients cannot strike a balance of the oxidative metabolism. In other words, there is a marked oxidative stress in that disorder. As a matter of fact, Cumurcu et al. (2009), and Ersoy et al. (2008) have shown that oxidants are increased, while antioxidants are decreased with OSI being increased in two similar biochemical-designed studies with major depression and panic disorder, respectively. Other finding related to OSI was that the increased OSI was not associated with a clinical evaluation score. Therefore, it may be suggested that OSI is not associated with the disease severity, but rather with the presence or absence of the disease. Brain is sensitive to oxidative stress and any oxidative imbalance may pave the way for the occurrence of the disease
(Halliwell, 2006). There are studies suggesting the sensitivity of brain to the oxidative stress and its relation to other psychiatric disorders (Savaş et al., 2002). In aerobic cells, reactive oxygen species are generated as a result of mitochondrial activity. How much mitochondrial activity increases, reactive oxygen species will be produced so much. Increased oxidant levels may lead to the destruction of the main intracellular constituents and functional compounds (Akyol et al., 2004). There are many studies indicating the role of neuronal damage mediated by the free radicals in the pathophysiology of schizophrenia and depression (Bilici et al., 2001; Kuloglu et al., 2002b; Mukerjee et al., 1996). DNA destruction is the most studied issue and there are studies suggesting that the oxidative stress is associated with the DNA destruction in the psychiatric disorders (Frey et al., 2007). It was demonstrated that increased reactive oxygen species induce DNA damage, especially mitochondrial DNA (Demple and Harrison, 1994). Increased oxidant levels in GAD may cause the DNA destruction, leading to the psychiatric disorder, although an association between the DNA destruction and the oxidative stress has not yet been shown in GAD. Since the size of our sample is small and there is no relationship between the therapeutic options and the oxidative balance, it is difficult to generalize our findings to all patients. As a conclusion, oxidative balance is impaired in GAD. We are not sure that whether this oxidative imbalance is the cause or result of GAD. We think oxidative imbalance plays an important role in the pathophysiology of GAD.
Acknowledgment Study was funded by Harran University Board of Scientific Research Projects (Funding number: 1114). Dr. Selek received travel grants from American Psychiatric Association, Society of Biological Psychiatry, GSK, Astra-Zeneca, M Nevzat and Sanovel Drug Companies.
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Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder Ali Emhan a, Salih Selek b,n, Hüseyin Bayazıt c, İbrahim Fatih Karababa c, Mahmut Katı c, Nurten Aksoy d a
Clinic of Psychiatry, Ministry of Health, Diyarbakır Education and Research Hospital, Diyarbakır, Turkey Harris County Psychiatric Center, University of Texas Health Science Center at Houston, TX, USA c Department of Psychiatry, Faculty of Medicine, Harran University, Sanliurfa, Turkey d Department of Biochemistry, Faculty of Medicine, Harran University, Sanliurfa, Turkey b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 November 2014 Received in revised form 30 July 2015 Accepted 2 November 2015
Generalized anxiety disorder (GAD) is a prevalent psychiatric disorder. The exact causes of GAD still unknown, in addition to neurochemical and neuroanatomic disorders, genetic and environmental factors are discussed in etiology. In our study we aimed to evaluate the oxidative metabolism's status and investigate the role of oxidative metabolites in GAD. Blood samples were taken from enrolled subjects in appropriate way and total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were studied in Harran University Biochemistry Labs. Results were compared between groups. The patients' TOS and OSI levels were significantly higher than control group. The patients' TAS levels were significantly lower than controls'. According to our findings, oxidative stress mechanism might have a role in GAD pathophysiology. In the future, total antioxidants may be used as a biologic marker in GAD etiology but more research is needed. & 2015 Published by Elsevier Ireland Ltd.
Keywords: Generalized anxiety disorder Oxidative stress Total antioxidant status Total oxidant status
1. Introduction Generalized anxiety disorder (GAD) is a clinical picture characterized by exaggerated feeling of worry related with various daily events and occurring almost all day long. In addition to anxiety, these patients experience unrest, tiredness, difficulty in concentrating, getting nervous rapidly, muscular straining and insomnia (Pine and McClure, 2005a). The prevalence of GAD is reported to be 3–6% in the general population with being two times more frequent in women than in men; it has been reported that it is the most frequent distress disorder among the patients referred to the primary care institutions (Wittchen et al., 2002). Although many biological and psychological factors have been considered to play a role in the etiology of GAD, none of these has been found to be a definite cause. In order to make a definite diagnosis, we need a well-documented history and thorough psychological examination as for the other psychiatric disorders (American Psychiatric Association, 2000). Recently, studies have been conducted to investigate the role of oxidative metabolism in the etiology of anxiety disorders (Atmaca, et al., 2004; Ersan et al., 2006; Tezcan et al., 2003). n
Corresponding author. E-mail address: [email protected] (S. Selek).
Oxidants (or pro-oxidants/free radicals) arise from the reactions of degradation involving oxygen as waste molecules. Having some beneficial effects (such as phagocytosis of monocytes and neutrophils), oxidants involve in reactions with many compounds including DNA, protein, and polyunsaturated fats of the cell membrane phospholipids. Among these reactions, particularly those affecting and damaging DNA pave the way for the development of various diseases. The substances called antioxidants diminish the deleterious effects caused by these oxidative products. There is a balance between the oxidants and antioxidants in a living body, and damage occurs when such balance is lost in favor of oxidants, a case called “oxidative stress” in the literature (Haddad, 2004). As described, though several studies have been carried out to investigate the role of oxidative metabolism in the pathophysiology of anxiety disorders, there is no evaluation of the total antioxidant level (TAL), total oxidant level (TOL) or any specific compound (e.g., malondialdehyde, catalase, superoxide dismutase etc.) in the generalized anxiety disorder (Atmaca et al., 2004; Ersan et al., 2006; Tezcan et al., 2003; Wittchen et al., 2002). Therefore, in this present cross-sectional case-control study, we aimed to determine whether the mechanism of oxidative stress plays a role in the pathophysiology of GAD and examine the diagnostic testing performance of total oxidants and total antioxidants in the etiology of GAD.
http://dx.doi.org/10.1016/j.psychres.2015.11.001 0165-1781/& 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: Emhan, A., et al., Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder. Psychiatry Research (2015), http://dx.doi.org/10.1016/j.psychres.2015.11.001i
A. Emhan et al. / Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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2. Methods
2.3. Measurement and estimation of variables
2.1. Study method and subjects
2.3.1. Total antioxidant level (TAL) measurement The total antioxidant status of the plasma was measured using a novel automated colorimetric measurement method for TAS developed by Erel (2004). In this method the hydroxyl radical, the most potent biological radical, is produced by the Fenton reaction, and reacts with the colorless substrate O-dianisidine to produce the dianisyl radical, which is bright yellowish-brown in color. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the re-action mix are suppressed by the antioxidant components of the plasma, preventing the color change and thereby providing an effective measure of the total antioxidant status of the plasma. The assay results are expressed as mmol Trolox Eq/L, and the precision of this assay is excellent, being lower than 3% (Cao and Prior, 1998).
This study included 40 patients, aged between 18 and 65 years, referred consecutively to the Outpatient Clinic of the Medical Faculty of the Harran University, who had had a diagnosis of GAD according to DSM-IV, and gave a written informed consent to participate in the study. The study was approved by the local ethics committee (the Medical Faculty of the Harran University). The patients' previous medical records and follow-up information were reviewed, and the socio-demographic variables including age, gender, comorbidities, the medications used, and the smoking status were recorded. The exclusion criteria of the study were as follows: pregnancy, serious systemic disease, epilepsy, dementia, disorders of alcohol and drug misuse, depression, panic disorders, obsessive compulsive disorder, schizophrenia, bipolar disorder and personality disorders (Axis II pathology), mild to moderate mental retardation, history of serious head trauma, and inadequate sampling. On the day when serum samples were drawn, they were administered a Hamilton Anxiety Rating Scale, Clinical Global Impression, and completed a socio-demographic questionnaire prepared for the study by one psychiatrist (AE). The control group consisted of 40 healthy volunteers. These volunteers' socio-demographic variables including age and gender as well as the smoking status were also recorded. Inadequate sampling was determined as an exclusion criterion. Blood samples were drawn from the antecubital vein following 12 h fasting. Thereafter, blood samples were poured into tubes, centrifuged at 3000 rpm for 5 min to separate plasma, and stored on ice to be processed within 6 h at most. The separated plasmas were stored at 80 °C for determining total antioxidant level (TAL) and total oxidant level (TOL). The plasma TAL and TOL levels were measured to calculate the oxidative stress index (OSI) in the Biochemistry Laboratory of the Harran University. The patients were gender-matched with the control group. 2.2. Data collection tools 2.2.1. Socio-demographic form Each patient was applied a routine follow-up and adverse effect scale and a socio-demographic form which has been prepared for recording demographics, height, body weight, smoking status and the laboratory results. Data from the patients were screened and recorded. 2.2.2. Hamilton anxiety rating scale (HAM-A) On the day of serum sampling, this scale was administered to all subjects participated in the study by an investigator (AE) in the Department. Used for determining the severity of anxiety and the distribution of symptoms, this scale comprises a total of 14 questions interrogating both somatic and psychic symptoms and compassing sub-dimensions. A five-point Likert-type measurement is provided. A total score is obtained by summing the points of items. The validity and reliability of its Turkish version has been conducted by Yazıcı et al. (1998). 2.2.3. Clinical global impression scale (CGI) CGI is a 3-item scale to evaluate the severity of disease, the improvement, and therapeutic efficacy index. The therapeutic efficacy index has two subsets as therapeutic effect and adverse effect (Guy, 1976). CGI was applied to assess the severity of comorbidities when blood samples were taken. For remission, the comorbidities having a CGI severity subscore r2 were considered as in remission.
2.3.2. Total oxidant level (TOL) measurement The total oxidant status of the plasma was measured using a novel automated colorimetric measurement method for TOS developed by Erel, (2005). In this method oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (μmol H2O2 Eq/L). 2.3.3. Oxidative stress index (OSI) measurement Oxidative stress index (OSI) was calculated as the total oxidant level (TAL)/total antioxidant level (TOL) ratio (Kosecik et al., 2005). 2.4. Statistical assessment of data Statistical analyses were performed using “SPSS 15.0 for Windows” software. Following the descriptive statistics, a Q–Q diagram was graphed in order to assess the distribution of the parameters investigated. A t-test was used for the paired group comparison of the data showing normal distribution, and Chisquare test for comparing the ratios. Spearman's correlation test was used for correlation analysis. A P value of o 0.05 was accepted as significant.
3. Results The demographical data of the patients and control subjects are given in Table 1. Ages, gender, duration of illness were similar among groups (p4 0.05, for all comparison). (Table 1). TAS, TOS and OSI levels in patients and controls were summarized in Table 2. TAS, TOS and OSI levels were statistically higher in GAD patients compared to healthy controls. (P 40.05 for all comparison). Table 1 Sociodemographic and clinical characteristics of the patients and controls.
Age (mean7 SD) Gender: female/male (n) Duration of illness (years): median
Patients
Controls
P value
42.48 ( 712.03) 26/14 5 (1–30)
41.5 ( 7 12.67) 26/14
0.993
Please cite this article as: Emhan, A., et al., Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder. Psychiatry Research (2015), http://dx.doi.org/10.1016/j.psychres.2015.11.001i
A. Emhan et al. / Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 2 TAS, TOS and OSI levels in patients and controls.
n Total antioxidant status (μmol Trolox Eqv./L)
GAD (mean7 SD)
Controls (mean7 SD)
39
39
0.8438 7 (0.20543)
0.9421 7 (0.19002)
40
40
20.03787 (4.46215)
13.6230 7 (3.47888)
39
39
2.57267 (1.07470)
1.50977 (0.54855)
P value
t¼ 2.192
p ¼0.031 N Total oxidant status (μmol H2O2 Eqv. / L)
t¼ 7.170
p o0.001 N Oxidative stress index
t¼ 5.502
p o0.001 n: number of cases SD: standard deviation
4. Discussion The first finding from this study was the higher TOS level in the patients with GAD than in the control groups. There are ample data suggesting that total oxidant levels are increased in the psychiatric disorders. TOS level has been found high in two studies conducted with the patients with schizophrenia and schizoaffective disorder each (Boskovic et al., 2011; Bulbul et al., 2014). Similarly, TOS level has been found high in the studies each with the patients with major depression (Cumurcu et al., 2009), bipolar disorder (Selek et al., 2011), adult attention deficit hyperactivity disorder (Selek et al., 2012), and obsessive–compulsive disorder (Kandemir et al., 2013). These studies are controlled ones and have used the same biochemical analysis methods. In addition to TOS, changes have also been observed in the specific oxidative parameters. In a study with schizophrenia, the levels of MDA, a product resulted from lipid peroxidation, xanthine oxidase and nitric oxide have been observed to increase compared to the controls (Akyol et al., 2002). In a study by Savaş et al. (2002), the NO level has been found to be higher in the patients with bipolar disorder than in the controls, while in the some recent studies the NO level has been found higher during the euthymic, manic and depressive periods of the bipolar disorder (Gergerlioglu et al., 2007; Savas et al., 2006; Selek et al., 2008a). Similarly, Sarandol et al. (2007) and Bulut et al. (2007) have found the increased MDA levels in 96 patients with major depression and in adult attention deficit hyperactivity disorder, respectively. In the anxiety disorders including GAD, changes have been observed in the specific oxidative parameters. For instance, the levels of MDA have been elevated in both plasma and serum in two separate studies with obsessive–compulsive disorder (OCD) (Chakraborty et al., 2009; Ozdemir et al., 2009), while the levels of NO have been found higher in another study with OCD (Atmaca et al., 2005). The MDA levels and the SOD, CAT and GPX activities have been studied two times in the subjects with social phobia by the same study group in 2004 and 2008, and there have been significant elevations compared to the control group (Atmaca et al., 2004, 2008). Of two separate studies with the patients diagnosed of panic disorder, one has shown that the plasma MDA levels, and the SOD and GPX activities have been increased, and the CAT activity has been unchanged (Kuloglu et al., 2002a), whereas the other has shown that the xanthine oxidase level has been increased, and the NO level and the SOD activity have been unchanged (Herken et al., 2006). In a study with 14 patients and 14 healthy individuals evaluating the oxidative metabolism in post-traumatic stress
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disorder, the MDA level, and the SOD, CAT and GPX activities have been found unchanged, a finding attributed to the ability of body to properly detoxify the rapidly occurring free radicals (Tezcan et al., 2003). As mentioned, there are number of studies on the changes in the oxidative metabolism caused by the psychiatric disorders, but as to GAD, a separate diagnostic category in DSM, no studies have been conducted on changes in either total oxidative stress or specific oxidative parameter. However, in a study investigating the urinary excretion of oxidative metabolites in 31 subjects with a high Hospital Anxiety and Depression Scale (HADS) score who had had a family member diagnosed of advanced cancer and had experienced excessive anxiety because of giving their relatives healthcare, the MDA/creatinine ratio has been found higher in the excessive anxiety group than in the control group (Ratnakar et al., 2008). In a study by Arranz et al. (2007) 33 female patients with a high beck anxiety scale score had significantly higher stimulated and unstimulated neutrophil superoxide anion levels compared to the controls (Arranz et al., 2007). Some of the rats exposed to chronic mild stress may be considered a model of anxiety disorder. Several studies have been carried out to examine the levels of oxidative metabolism products in these experimental animals. In their study, Şahin and Gümüşlü (2004) have found that the postmortem brain tissue of the rats chronically exposed to immobility stress contained low levels of MDA and TBARS (thiobarbituric acidreactive substances), another marker for oxidative stress, and high SOD, CAT activities but low GPX activity (Sahin and Gümüşlü, 2004). In a similar study with rats, Kamper et al. (2009) have found the serum levels of MDA to be high generally, that of NO and the GPX activity to be high in the female rats but unchanged in the male rats, and the SOD activity to be unchanged in both genders (Kamper et al., 2009). In their study, Ciobica et al. (2010) have shown that there are no changes in the GPX and SOD levels, whereas there is a marked increase in the MDA level (Ciobica et al., 2010). As a conclusion, our study results revealed an increase in the levels of oxidants, thus supporting the data from the previous studies. At the present day, the effect of increased oxidants on the psychiatric disorders represents one of the hottest topics. Increased oxidant levels may lead to the destruction of the main intracellular constituents and functional compounds (Akyol et al., 2004). DNA destruction is the most interested issue. There are studies suggesting that the oxidative stress is associated with the DNA destruction in the psychiatric disorders (Andreazza et al., 2007). Increased oxidant levels in GAD may cause the DNA destruction, leading to the psychiatric disorder, although an association between the DNA destruction and the oxidative stress has not yet been shown in GAD. There is no clear causal relationship between the psychiatric disorders and the increased oxidant levels. It is not clearly known whether the increased oxidants cause psychiatric disorders or vice versa. However, the current data suggest that oxidants may lead to psychiatric disorders. For instance, the fact that there are evidences of oxidative stress even during the remission period of the bipolar disorder, and that the oxidant levels decrease with therapy in some disease (Savas et al., 2006; Yu et al., 2003). Since our present study is cross-sectional, it is difficult to reach a definitive judgment about the course of GAD, but in the light of previous data, it might be mentioned that the increased oxidant levels play a role in the pathophysiology of GAD. There is a need for further studies. One of our finding related to the oxidants was that the increased oxidant levels were not associated with a clinical evaluation score. Therefore, it may be suggested that oxidants is not associated with the disease severity, but rather with the presence or absence of the disease. The second important finding from our study was that TAS is decreased in GAD. There are ample of data suggesting that total
Please cite this article as: Emhan, A., et al., Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder. Psychiatry Research (2015), http://dx.doi.org/10.1016/j.psychres.2015.11.001i
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A. Emhan et al. / Psychiatry Research ∎ (∎∎∎∎) ∎∎∎–∎∎∎
antioxidant levels are decreased in the psychiatric disorders. For instance, the TAS level has been found decreased in patients with schizophrenia (Akyol et al., 2002), three separate studies including those with major depression (Cumurcu et al., 2009; Gałecki et al., 2009; Sarandol et al., 2007), one study including those with bipolar disorder (Savas et al., 2006), and one study including those with attention deficit hyperactivity disorder (Akyol et al., 2002). In addition to TAS, changes have also been observed in the specific oxidative parameters. For instance, it has been shown that the Vitamin E (Ersan et al., 2006) and selenium levels (Chakraborty et al., 2009) are decreased in the patients with OCD, while the level of glutathione and that of Vitamin E are decreased in the patients with major depression (Kodydková et al., 2009), and in two separate studies (Maes et al., 2000; Owen et al., 2005), respectively. Conversely, TAS has been found to be unchanged in a study with schizoaffective disorder (Bulbul et al., 2014), whereas it has been found to be increased in 4 studies each with the patients with bipolar disorder (Selek et al., 2011), with adult attention deficit hyperactivity disorder (Selek et al., 2012), with obsessive– compulsive disorder (Kandemir et al., 2013; Selek et al., 2008b), and with attention deficit hyperactivity disorder (Ersoy et al., 2008). As mentioned above, whereas there has been no study that evaluated the antioxidant metabolism related to GAD, in a study by Arranz et al. (2007) TAL has been found to be significantly lower in 33 female patients with anxiety and having a high score according to the Beck Anxiety Scale than in the control subjects (Arranz et al., 2007); similarly, the level of glutathione has been found to be apparently low in the brain tissue of the rats chronically exposed to immobility stress (Sahin and Gümüşlü, 2004). In support of many studies mentioned above, our study indicated that the TAS values were significantly lower in the patient group than in the control group. Specific increases in the antioxidant efficacy observed in other psychiatric disorders in which oxidative stress has been evidenced have been considered as the reactive increases in order to compensate the oxidative stress (Kandemir et al., 2013; Selek et al., 2012). The fact in our study that oxidants were increased, whereas antioxidants were mildly decreased might be interpreted that the respective patients were under stress, metabolism did not give an adequate reactive response to the increased oxidants, and there was an impairment of the responsive systems, or a yet-unknown variable would decrease the TAS in the medium. The third finding from this study was the increased OSI in the patients with GAD. OSI is an index obtained by dividing TOS by TAS, and used as a measure for the general evaluation of oxidative stress (Kosecik et al., 2005). Using OSI, we can assess two dimensions of the oxidative metabolism, e.g. antioxidants and oxidants, and see how comparable antioxidants are against oxidants. An increased OSI value compared to the controls shows that the balancing antioxidant system cannot generate an adequate response, and the balance is impaired in the direction of oxidants. The fact in our study that the balancing TAS values were decreased instead of being increased in the patients with GAD indicates a defect in the antioxidant system, and the organism of these patients cannot strike a balance of the oxidative metabolism. In other words, there is a marked oxidative stress in that disorder. As a matter of fact, Cumurcu et al. (2009), and Ersoy et al. (2008) have shown that oxidants are increased, while antioxidants are decreased with OSI being increased in two similar biochemical-designed studies with major depression and panic disorder, respectively. Other finding related to OSI was that the increased OSI was not associated with a clinical evaluation score. Therefore, it may be suggested that OSI is not associated with the disease severity, but rather with the presence or absence of the disease. Brain is sensitive to oxidative stress and any oxidative imbalance may pave the way for the occurrence of the disease
(Halliwell, 2006). There are studies suggesting the sensitivity of brain to the oxidative stress and its relation to other psychiatric disorders (Savaş et al., 2002). In aerobic cells, reactive oxygen species are generated as a result of mitochondrial activity. How much mitochondrial activity increases, reactive oxygen species will be produced so much. Increased oxidant levels may lead to the destruction of the main intracellular constituents and functional compounds (Akyol et al., 2004). There are many studies indicating the role of neuronal damage mediated by the free radicals in the pathophysiology of schizophrenia and depression (Bilici et al., 2001; Kuloglu et al., 2002b; Mukerjee et al., 1996). DNA destruction is the most studied issue and there are studies suggesting that the oxidative stress is associated with the DNA destruction in the psychiatric disorders (Frey et al., 2007). It was demonstrated that increased reactive oxygen species induce DNA damage, especially mitochondrial DNA (Demple and Harrison, 1994). Increased oxidant levels in GAD may cause the DNA destruction, leading to the psychiatric disorder, although an association between the DNA destruction and the oxidative stress has not yet been shown in GAD. Since the size of our sample is small and there is no relationship between the therapeutic options and the oxidative balance, it is difficult to generalize our findings to all patients. As a conclusion, oxidative balance is impaired in GAD. We are not sure that whether this oxidative imbalance is the cause or result of GAD. We think oxidative imbalance plays an important role in the pathophysiology of GAD.
Acknowledgment Study was funded by Harran University Board of Scientific Research Projects (Funding number: 1114). Dr. Selek received travel grants from American Psychiatric Association, Society of Biological Psychiatry, GSK, Astra-Zeneca, M Nevzat and Sanovel Drug Companies.
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Please cite this article as: Emhan, A., et al., Evaluation of oxidative and antioxidative parameters in generalized anxiety disorder. Psychiatry Research (2015), http://dx.doi.org/10.1016/j.psychres.2015.11.001i