Journal of Psychiatric Research 58 (2014) 167e174
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Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/psychires
Association of altered CuZn superoxide dismutase and cognitive impairment in schizophrenia patients with tardive dyskinesia Jing Qin Wu a, b, c, Da Chun Chen c, Yun-Long Tan c, Shuping Tan c, Zhiren Wang c, Fude Yang c, Jair C. Soares d, Xiang Yang Zhang c, d, * a
School of Biomedical Sciences and Pharmacy, Faculty of Health, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia Schizophrenia Research Institute, Sydney, Australia Psychiatry Research Center, Beijing HuiLongGuan Hospital, Peking University, Beijing, China d Department of Psychiatry and Behavioral Sciences, Harris County Psychiatric Center, The University of Texas Health Science Center at Houston, Houston, TX, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 July 2014 Received in revised form 30 July 2014 Accepted 31 July 2014
Free radical-mediated abnormalities may contribute to the development of tardive dyskinesia (TD) and specific aspects of schizophrenia symptomatology such as cognitive deficits. Superoxide dismutase (SOD), a critical enzyme in the detoxification of superoxide radicals, was found to be abnormal in TD. While most of previous studies focused on the manganese isoform located in mitochondria, this study investigated the activities of isoform CuZnSOD present in the plasma. We recruited 113 male chronic patients with TD (n ¼ 43) and without TD (n ¼ 70) meeting DSM-IV criteria for schizophrenia, and 84 male control subjects. We examined the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), CuZnSOD activity for both the patient and control groups along with total antioxidant status (TAS) and malondialdehyde (MDA) levels in a subset of the cohort. Positive and Negative Symptom Scale (PANSS) and the Abnormal Involuntary Movement Scale (AIMS) were assessed in the patient group. Our results showed lower CuZnSOD activity and TAS levels, but higher MDA levels in patients with TD than those without TD (all p < 0.05). Patients with TD had lower RBANS subscales of Visuospatial/ Constructional (p < 0.05) and attention (p < 0.01) than those without TD. Multiple regression analysis showed that in either TD or non-TD group, CuZnSOD was an independent contributor to the attention index of RBANS (both p < 0.05). These results implicated that TD patients suffered greater oxidative stress and cognitive dysfunction than non-TD patients. Oxidative stress could contribute to both TD development and cognitive impairment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Schizophrenia Tardive dyskinesia Cognition Superoxide dismutase Oxidative stress
1. Introduction Long-term antipsychotic treatment for schizophrenia is associated with the emergence of tardive dyskinesia (TD). TD is a motor syndrome consisting of involuntary, hyperkinetic movements (Lohr et al., 2003) and currently has no well-accepted treatments. For the development of TD, a number of risk factors such as age, gender, duration of treatment, greater cognitive impairment, and diagnosis of diabetes mellitus have been identified (Sachdev, 2000). Among these risk factors, cognitive dysfunction has drawn particular
* Corresponding author. Psychiatry Research Center, Beijing HuiLongGuan Hospital, HuiLongGuan Town, Chang-Ping District, Beijing, 100096, PR China. Tel.: þ86 10 62715511x6464; fax: þ86 10 62912169. E-mail addresses: [email protected], [email protected] (X.Y. Zhang). http://dx.doi.org/10.1016/j.jpsychires.2014.07.028 0022-3956/© 2014 Elsevier Ltd. All rights reserved.
interest since it is a core feature of schizophrenia and greater cognitive impairment has been consistently reported in schizophrenia patients with TD (Byne et al., 1998; Waddington and Youssef, 1986; Waddington et al., 1987; Wegner et al., 1985b). This association has been further confirmed by longitudinal studies showing that cognitive deficits either preceded the onset of TD (Struve and Willner, 1983; Wegner et al., 1985a) or TD was predictive of impaired cognitive function (Waddington and Youssef, 1996) as well as our recent cross sectional study using a larger cohort (Wu et al., 2013). While the etiology and pathophysiology of TD are not well understood, a widely accepted hypothesis is that long-term antipsychotic treatments may eventually lead to increased production of neurotoxic free radicals (Lohr, 1991; Lohr and Browning, 1995; Lohr et al., 2003), resulting in potentially irreversible damage in motor regions. The theoretical framework was first proposed by Cadet
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et al. who suggested that cell membranes may be destabilized by the toxic action of free radicals produced during the chronic use of antipsychotics, which may lead to structural neuropathological changes in the terminals of catecholaminergic and other neurotransmitter systems (Cadet et al., 1986). It was thus surmised that antioxidants may be helpful for the treatment of TD when concomitantly administered with antipsychotics, which was indeed supported by the follow-up study showing that a-tocopherol (vitamin E), a powerful antioxidant, was beneficial in the treatment of some TD patients (Lohr et al., 1988). Furthermore, a role for oxidative stress in the neurotoxic effects of methamphetamine and methylenedioxymethamphetamine, the development of Parkinson's disease, and the degeneration of dopamine system has been elaborated in the literature review (Cadet and Brannock, 1998). The accumulated evidence all pointed towards the involvement of oxidative stress arising from catecholamine oxidation in the degeneration of monoaminergic systems. In addition to damage in motor regions, oxidative stressinduced damage may also contribute to cognitive impairment such as learning and memory (Fukui et al., 2001; Hu et al., 2006; Kapogiannis and Mattson, 2011; Nicolle et al., 2001). A study using the DN-DISC1 mice model has shown that the significant oxidative stress in the prefrontal cortex could have an impact on epigenetic and transcriptional machinery, which points to a mediating condition contributing to cognitive impairment (Johnson et al., 2013). In schizophrenia, the marker of oxidative modification of proteins, protein carbonyl content (PCC), was negatively correlated with working memory (Asevedo et al., 2013) and serum protein oxidative stress was also elevated in siblings of patients with schizophrenia (Massuda et al., 2013). In agreement with these findings, our recent study has reported that total antioxidant status was significantly associated with attention index and RBANS total scores in schizophrenia patients, suggesting that progressive cognitive impairments in schizophrenia may be causally related to impaired antioxidant capacity resulting in increased oxidative stress (Zhang et al., 2012b). As for TD, lower levels of antioxidant markers superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS) in cerebrospinal fluid and peripheral tissues have been reported in schizophrenia patients with TD (Lohr et al., 1990; Peet et al., 1993; Tsai et al., 1998; Yamada et al., 1997). We have also found lower levels of glutathione peroxidase, catalase, total antioxidant status (TAS), and plasma SOD in schizophrenia patients with TD than those without TD (Chen et al., 2010; Zhang et al., 2003a, 2007). Among the antioxidant systems, SOD is one of the critical scavenging enzymes that have been under constant investigation in schizophrenia with elevated levels reported in numerous studies (Condray and Yao, 2011; Galecki et al., 2006; Glazer, 2000a, b; Held et al., 2000; Kay et al., 1987; Palmer et al., 2009; Sharma and Antonova, 2003). SOD has three isoforms: the manganese isoform (MnSOD/SOD2) located in mitochondria, and the copper and zinc (CuZn) isoforms present in the cytoplasm (SOD1) and extracellular space (SOD3). Since cellular metabolism and production of reactive oxygen species occur primarily in the mitochondria, MnSOD has been the major focus of the aforementioned studies and our recent study has also found that MnSOD was an independent contributor to the RBANS total score in schizophrenia patients either with or without TD (Wu et al., 2014). However, research on CuZnSOD is limited even though its involvement in schizophrenia and TD has been observed in previous studies showing marked reduction of CuZnSOD in cerebrospinal fluid of patients with recent-onset schizophrenia (Coughlin et al., 2013) as well as the association between CuZnSOD and dyskinetic movements (Zhang et al., 2003a). Therefore, we conducted the current study by investigating both the cognitive performance assessed by RBANS and the activity of
CuZnSOD in blood from schizophrenia patients stratified by TD presence in a Chinese population. Furthermore, we also evaluated two additional markers, TAS and malondialdehyde (MDA) levels in a subset of our cohort to confirm the oxidative stress status reflected by CuZnSOD. While the former provided an index of total nonenzymatic antioxidants in plasma, the latter reflected the levels of the end product of lipid peroxidation. Given the fact that gender differences exist in cognitive domains and oxidative stress parameters in both schizophrenia patients and controls (Halari et al., 2006; Han et al., 2012; Wisner et al., 2011; Zhang et al., 2012a), we only included male subjects in this study. 2. Methods 2.1. Ethics statement The research protocol was approved by the Institutional Review Board, Beijing Hui-Long-Guan hospital. A psychiatrist explained the research protocol and procedures to the potential subject. The description of the study was tailored to maximize the understanding of the subject using language appropriate to the subject's level of comprehension, and emotional readiness. If the subject was willing to consent to participate in the study the researcher provided an in depth description to the subject and in certain instances, to their parents or guardians. Most of the patients and all the healthy controls provided written informed consent. Only in two patients who also assented to participate in the study, the parents gave their written consent on behalf of the patients under the request of the patients. 2.2. Subjects One hundred and thirteen inpatients with schizophrenia were recruited from Beijing Hui-Long-Guan hospital, a Beijing-cityowned psychiatric hospital. All patients met the following inclusion criteria: 1) age 40e70 years, Han Chinese; 2) confirmed DSMIV diagnosis of schizophrenia; 3) with at least 5 years of illness; 4) had been receiving stable doses of oral antipsychotic drugs for at least 12 months before entry into the study. All patients were of the chronic type, and had been ill for an average 30.2 ± 8.0 years, with current antipsychotic treatment for an average of 4.8 ± 4.1 years. Since admission, all patients received dietetically balanced hospital meals, which were occasionally supplemented by gifts (usually fruit). Patients had the opportunity to exercise for about an hour per day. Antipsychotic drug treatment was mainly monotherapy with clozapine (n ¼ 53), risperidone (n ¼ 21), and typical drugs (perphenazine, pipotiazine, chlorpromazine, sulpiride, and haloperidol; n ¼ 39). A mean daily dose of antipsychotics (Table 1), including both the first- and second-generation antipsychotics, was converted to approximate daily mean chlorpromazine milligram equivalents for each subject using standard guidelines (Lehman et al., 2004; Woods, 2003). Age-, education-, and BMI-matched control subjects (n ¼ 84) were recruited from the local community in Beijing. Current mental status and personal or family history of any mental disorder was assessed by unstructured interviews. None of the healthy control subjects presented a personal or family history of psychiatric disorder. A complete medical history, physical examination and laboratory tests were obtained from patients and control subjects. Any subjects with major medical illness were excluded. None of the subjects met criteria for drug or alcohol abuse or dependence. All subjects are Han Chinese recruited at the same period from the Beijing area. Demographic data for patients and normal controls are summarized in Table 1.
J.Q. Wu et al. / Journal of Psychiatric Research 58 (2014) 167e174 Table 1 Characteristics of controls, schizophrenic patients with and without TD. Characteristic
Normal controls (n ¼ 84)
Age (yrs) 54.0 ± 7.4 Education (yrs) 9.5 ± 3.3 2 25.4 ± 4.3 BMI (kg/m ) Smoker/ 47/37 Nonsmoker Age of onset (yrs) Duration of Illness (yrs) Number of hospitalizations Drugs (clozapine/ risperi-done/ typical drugs) Daily AP dose (mg/day) (CPZ equivalent) Duration of treatment (ms) PANSS total score P subscore N subscore G subscore AIMS total score CuZnSOD (U/ml) 44.3 ± 14.6 TASa (U/ml) 215.3 ± 39.0 a MDA (nmol/ml) 2.5 ± 1.7
F or X2
Patients without TD (n ¼ 70)
Patients with TD (n ¼ 43)
53.8 ± 5.7 9.1 ± 2.2 25.5 ± 5.0 60/10
56.2 ± 6.7 9.5 ± 2.4 23.0 ± 4.9 38/5
24.5 ± 6.6 29.3 ± 7.9
24.5 ± 5.6 31.6 ± 8.1
0.001 0.981 2.071 0.153
3.9 ± 2.6
4.3 ± 2.4
0.666 0.416
33/15/22
20/6/17
1.31
544.8 ± 836.6 399.9 ± 355.5
54.2 ± 57.0 59.6 11.3 22.4 25.8 1.2 57.2 184.1 6.4
± ± ± ± ± ± ± ±
13.0 4.5 6.7 4.8 1.4 14.6 48.9 5.2
61.5 ± 56.3 67.1 14.5 24.2 28.4 7.1 51.0 159.1 9.3
± ± ± ± ± ± ± ±
1.865 0.584 2.026 13.228
11.5 4.8 5.9 5.2 2.5 15.3 42.6 7.2
p value 0.158 0.559 0.136 0.001**
0.519
1.159 0.284
comprised of 12 subtests that are used to calculate 5 age-adjusted index scores and a total score. Test indices are Immediate Memory (comprised of List Learning and Story Memory tasks); Visuospatial/Constructional (comprised of Figure Copy and Line Orientation tasks); Language (comprised of Picture Naming and Semantic Fluency tasks); Attention (comprised of Digit Span and Coding tasks); and Delayed Memory (comprised of List Recall, Story Recall, Figure Recall, and List Recognition tasks). Our group previously translated RBANS into Chinese and the clinical validity and its test-retest reliability established among controls and schizophrenia patients (Zhang et al., 2009). Each subject came in the testing room on a separate day to be introduced to our research center by a research member and a proper training session had been performed for individual to become acclimated to the testing environment and computerized tasks. 2.4. Plasma CuZnSOD activity, TAS, and MDA measurements
0.420 0.519 9.750 13.249 1.933 6.956 248.802 14.655 14.596 16.475
169
**
0.002 0.000** 0.161 0.01* 0.000** 0.000** 0.000** 0.000**
TD: tardive dyskinesia; BMI: body mass index; AP: antipsychotic; CPZ: chlorpromazine; PANSS: Positive and Negative Symptom Scale; P: PANSS positive symptom subscale; N: PANSS negative symptom subscale; G: PANSS general psychopathology subscale; AIMS: Abnormal Involuntary Movement Scale. *p < 0.05; **p < 0.01. a markers assessed in a subset of our cohort with sufficient plasma for additional assays (TD: n ¼ 30, non-TD: n ¼ 34, and healthy controls: n ¼ 40).
2.3. Clinical measures Each subject filled out a detailed questionnaire that recorded general information, sociodemographic characteristics, and medical and psychological conditions. Additional information was collected from available medical records and collateral data (from family and/or treating clinician). Four experienced psychiatrists who were blinded to the clinical status of the patients assessed the severity of tardive dyskinesia using the abnormal involuntary movement scale (AIMS) (Guy, 1976). Diagnosis of TD was based on the Research Diagnostic Criteria (RDC) in DSM-IV, in combination with the criteria of Schooler and Kane (Schooler and Kane, 1982). Dyskinesia was classified as present in a particular subject with an AIMS score of at least three (moderate degree) in any body part or with at least two (mild degree) in two or more body parts. Each item on the AIMS ranges from 0 to 4, and the total AIMS score was calculated by adding items 1e7. The patients with TD were re-evaluated at least 1 month later for TD using the AIMS and diagnosed with TD only if both evaluations consistently revealed the presence of TD. Psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1987) on the same day as the TD evaluation. All raters for the AIMS and PANSS had simultaneously attended a training session in their use before this study started. After training repeated assessments showed a correlation coefficient between raters of greater than 0.84 was maintained for the PANSS and 0.88 for the AIMS total scores. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS, Form A) (Randolph et al., 1998) was individually administered to measure cognitive functioning. The RBANS is
Venous blood was obtained from each subject between 0700 and 0900 AM following an overnight fast. CuZnSOD activity (Zhang et al., 2006, 2013), TAS (Li et al., 2011), and MDA levels (Yagi, 1994; Zhang et al., 2007) were analyzed using established procedures. Our previous reports give a full description of the assays. The interand intra-assay coefficients of variation for CuZnSOD were 8% and 6%. Blood CuZnSOD activity and TAS are expressed as units per milliliter plasma (U/ml). Plasma MDA values were calculated using the extinction coefficient of MDA-thiobarbituric acid complex at 532 nm. MDA results are expressed as nmol/ml. A research assistant blind to the clinical situation assayed all samples. A code number maintained by the primary investigator indicated the identity of subjects. 2.5. Statistical analysis Demographic and clinical variables of TD and non-TD groups were compared using analysis of variance (ANOVA) for continuous variables and chi-squared for categorical variables. We compared RBANS scores among the three groups using ANOVA and Fisher's least significant difference (LSD) test was used to perform post-hoc pairwise comparisons (non-TD versus controls; TD versus controls; TD versus non-TD). When significance was found in ANOVA, the effect of age, education, BMI, smoking status, duration of antipsychotic treatment, type and dose of antipsychotic drugs was tested by adding these variables to the analysis model as covariates. Relationships between variables have been assessed with Pearson's product moment correlation coefficients. Bonferroni corrections were applied to each test to adjust for multiple testing. Multiple regression analysis using RBANS total or the index scores as the dependent variable was carried out to investigate the impact of a range of variables including CuZnSOD level, age, education, BMI, smoking status, duration of antipsychotic treatment, type and dose of antipsychotic drugs, and PANSS total and index scores. SPSS version 17.0 was used to do all statistical analysis. Data are presented as mean and standard deviation (mean ± SD). All p-values are two tailed with significance level set at 0.05. 3. Results Clinical and demographic characteristics for the schizophrenia patients with and without TD along with healthy controls are presented in Table 1. There was no significant difference in any characteristics between patients with TD, without TD, and normal controls (all p > 0.05) except for smoking status (p < 0.01), which was adjusted in the following analyses. Age, education, BMI, and smoking status were not associated with CuZnSOD activity in the
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combined groups, or when the association was examined in TD, non-TD, and control groups respectively (all p > 0.05). Between schizophrenia patients with and without TD, there was no significant difference in the number of hospitalizations, antipsychotic types (between clozapine, risperidone, and typical drugs), daily antipsychotic dose (chlorpromazine equivalent), duration of antipsychotic treatment (all p > 0.05). Patients with TD had significantly higher levels of the general psychopathology (p < 0.05), positive symptom (p < 0.01), and total scores of PANSS (p < 0.01) as well as AIMS total score (p < 0.001) than those without TD. The significant differences in the positive symptom subscore (p < 0.01) and total score of PANSS (p < 0.05) as well as AIMS total score (p < 0.001) remained after controlling for age, BMI, education, duration of antipsychotic treatment, type and dose of antipsychotic drugs. Between clozapine, risperidone, and typical drugs, there was no significant difference in AIMS total score for either the combined patients or separate TD and non-TD groups (p > 0.05). 3.1. CuZnSOD activity in healthy controls and schizophrenia with and without TD CuZnSOD activity was lower in patients with TD than in those without TD (51.0 ± 15.3 U/ml vs. 57.2 ± 14.6 U/ml; F ¼ 4.7, df ¼ 1, 111, p ¼ 0.033). This difference remained significant after covarying for age, education, smoking status, and antipsychotic dose (p ¼ 0.018). Furthermore, CuZnSOD activity was higher in both patient groups than in the control group (44.3 ± 14.6 U/ml; Table 1) (TD vs. controls: p ¼ 0.018; non-TD vs. control: p < 0.001). These differences remained significant after covarying for age, education, BMI, and smoking status (TD vs. controls: p ¼ 0.019; non-TD vs. control: p < 0.001). Between clozapine, risperidone, and typical drugs, there was no significant difference in CuZnSOD activities for either the combined patients or separate TD and non-TD groups (p > 0.05). In the combined patient group, or when the TD and non-TD group were examined separately, CuZnSOD activity was not associated with the duration of antipsychotic treatment, type (between clozapine, risperidone, and typical drugs), or dose of antipsychotic medication (all p > 0.05). In addition, CuZnSOD was not correlated with any subscore or total score of PANSS in either TD or non-TD group alone (all p > 0.05). 3.2. TAS and MDA levels in healthy controls and schizophrenia with and without TD To further confirm the oxidative stress status reflected by CuZnSOD activity, TAS and MDA levels were further assessed in a subset of our cohort with sufficient plasma for additional assays (TD: n ¼ 30, non-TD: n ¼ 34, and healthy controls: n ¼ 40). There was a significant difference in TAS among the patients with TD, without TD, and normal controls (F ¼ 14.6, df ¼ 2,101, p < 0.001). Post hoc tests showed that the patients with TD had lower levels of
TAS than those without TD (159.1 ± 42.6 U/ml vs 184.1 ± 48.9 U/ml, p ¼ 0.024). Both patient groups had lower TAS levels than the control group (215.3 ± 39.0 U/ml; TD vs. controls: p < 0.001; non-TD vs. controls: p ¼ 0.003). There was also a significant difference in MDA among the TD, non-TD, and control groups (F ¼ 16.5, df ¼ 2,101, p < 0.001). Post hoc tests revealed that the patients with TD had higher MDA levels than those without TD (9.3 ± 7.2 nmol/ml vs. 6.4 ± 5.2 nmol/ml, p ¼ 0.024). Both patient groups had higher MDA levels than the control group (2.5 ± 1.7 nmol/ml; TD vs. control: p < 0.001; non-TD vs. control: p ¼ 0.001). 3.3. Cognitive performance in healthy controls and schizophrenia with and without TD The mean and standard deviation of RBANS total and index scores of 43 TD, 70 non-TD, and 84 controls are shown in Table 2. The significant differences between the TD and non-TD patients included the lower RBANS subscales of Visuospatial/Constructional (78.1 ± 16.9 vs. 85.0 ± 15.6, p ¼ 0.024) and attention (74.8 ± 12.7 vs. 82.9 ± 11.5, p ¼ 0.005). After controlling for age, education, BMI, smoking status, duration of illness, and antipsychotic treatment (type, dose and duration of antipsychotic treatment), the differences remained significant for RBANS subscales of Visuospatial/ Constructional (p ¼ 0.032) and attention (p ¼ 0.004). In addition, both patient groups (TD vs. control; non-TD vs. control) scored lower on all subscales (all p < 0.01) except for the Visuospatial/ Constructional index than the controls. These differences remained significant after covarying for age, education, BMI, and smoking (all p < 0.05). 3.4. Correlation between CuZnSOD and cognitive performance In the combined patient group, correlation analysis showed that CuZnSOD activity was negatively associated with the attention index of RBANS (r ¼ 0.31, df ¼ 111, p ¼ 0.001). In either TD or non-TD group, CuZnSOD activity was negatively associated with the attention index of RBANS (non-TD: r ¼ 0.31, df ¼ 68, p ¼ 0.009; TD: r ¼ 0.52, df ¼ 41, p < 0.0005). Furthermore, CuZnSOD activity was also negatively associated with immediate memory index (r ¼ 0.35, p ¼ 0.02) and total score of RBANS (r ¼ 0.32, p ¼ 0.04, both df ¼ 41) in TD patients. Multiple regression analysis showed that in either TD or non-TD group, CuZnSOD (non-TD: b ¼ 0.26, t ¼ 2.0, p < 0.05; TD: b ¼ 0.64, t ¼ 3.77, p < 0.01; Fig. 1) was an independent contributor to the attention index of RBANS. Furthermore, in the TD group the PANSS positive symptom (b ¼ 0.48, t ¼ 2.81, p < 0.01) and CuZnSOD (b ¼ 0.36, t ¼ 2.09, p < 0.05) were independent contributors to the RBANS total score, after controlling for age, BMI, smoking, duration of illness and antipsychotic treatment (type, dose and duration of antipsychotic treatment). However, in the healthy control group, the CuZnSOD activity was not found to be
Table 2 Comparison of total and index scores of the RBANS between schizophrenia patients with TD, without TD, and controls. Normal controls (n ¼ 84) Immediate memory Visuospatial/Constructional Language Attention Delayed memory Total scale
76.2 83.4 97.1 89.4 89.1 82.9
± ± ± ± ± ±
17.6 15.1 9.8 17.5 15.3 13.7
Patients without TD (n ¼ 70) 61.5 85.0 86.6 82.9 70.0 69.9
± ± ± ± ± ±
14.9 15.6 13.2 11.5 19.2 14.1
*Indicates significance of the comparisons between TD and non-TD groups. **p < 0.01, *p < 0.05. a Refers to Bonferroni corrections.
Patients with TD (n ¼ 43) 58.9 78.1 85.7 74.8 70.2 67.1
± ± ± ± ± ±
16.7 16.9* 12.3 12.7** 20.9 14.0
F
p Value
Corrected pa
21.7 2.7 20.3 14.5 26.0 24.6
Contents lists available at ScienceDirect
Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/psychires
Association of altered CuZn superoxide dismutase and cognitive impairment in schizophrenia patients with tardive dyskinesia Jing Qin Wu a, b, c, Da Chun Chen c, Yun-Long Tan c, Shuping Tan c, Zhiren Wang c, Fude Yang c, Jair C. Soares d, Xiang Yang Zhang c, d, * a
School of Biomedical Sciences and Pharmacy, Faculty of Health, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia Schizophrenia Research Institute, Sydney, Australia Psychiatry Research Center, Beijing HuiLongGuan Hospital, Peking University, Beijing, China d Department of Psychiatry and Behavioral Sciences, Harris County Psychiatric Center, The University of Texas Health Science Center at Houston, Houston, TX, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 July 2014 Received in revised form 30 July 2014 Accepted 31 July 2014
Free radical-mediated abnormalities may contribute to the development of tardive dyskinesia (TD) and specific aspects of schizophrenia symptomatology such as cognitive deficits. Superoxide dismutase (SOD), a critical enzyme in the detoxification of superoxide radicals, was found to be abnormal in TD. While most of previous studies focused on the manganese isoform located in mitochondria, this study investigated the activities of isoform CuZnSOD present in the plasma. We recruited 113 male chronic patients with TD (n ¼ 43) and without TD (n ¼ 70) meeting DSM-IV criteria for schizophrenia, and 84 male control subjects. We examined the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), CuZnSOD activity for both the patient and control groups along with total antioxidant status (TAS) and malondialdehyde (MDA) levels in a subset of the cohort. Positive and Negative Symptom Scale (PANSS) and the Abnormal Involuntary Movement Scale (AIMS) were assessed in the patient group. Our results showed lower CuZnSOD activity and TAS levels, but higher MDA levels in patients with TD than those without TD (all p < 0.05). Patients with TD had lower RBANS subscales of Visuospatial/ Constructional (p < 0.05) and attention (p < 0.01) than those without TD. Multiple regression analysis showed that in either TD or non-TD group, CuZnSOD was an independent contributor to the attention index of RBANS (both p < 0.05). These results implicated that TD patients suffered greater oxidative stress and cognitive dysfunction than non-TD patients. Oxidative stress could contribute to both TD development and cognitive impairment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Schizophrenia Tardive dyskinesia Cognition Superoxide dismutase Oxidative stress
1. Introduction Long-term antipsychotic treatment for schizophrenia is associated with the emergence of tardive dyskinesia (TD). TD is a motor syndrome consisting of involuntary, hyperkinetic movements (Lohr et al., 2003) and currently has no well-accepted treatments. For the development of TD, a number of risk factors such as age, gender, duration of treatment, greater cognitive impairment, and diagnosis of diabetes mellitus have been identified (Sachdev, 2000). Among these risk factors, cognitive dysfunction has drawn particular
* Corresponding author. Psychiatry Research Center, Beijing HuiLongGuan Hospital, HuiLongGuan Town, Chang-Ping District, Beijing, 100096, PR China. Tel.: þ86 10 62715511x6464; fax: þ86 10 62912169. E-mail addresses: [email protected], [email protected] (X.Y. Zhang). http://dx.doi.org/10.1016/j.jpsychires.2014.07.028 0022-3956/© 2014 Elsevier Ltd. All rights reserved.
interest since it is a core feature of schizophrenia and greater cognitive impairment has been consistently reported in schizophrenia patients with TD (Byne et al., 1998; Waddington and Youssef, 1986; Waddington et al., 1987; Wegner et al., 1985b). This association has been further confirmed by longitudinal studies showing that cognitive deficits either preceded the onset of TD (Struve and Willner, 1983; Wegner et al., 1985a) or TD was predictive of impaired cognitive function (Waddington and Youssef, 1996) as well as our recent cross sectional study using a larger cohort (Wu et al., 2013). While the etiology and pathophysiology of TD are not well understood, a widely accepted hypothesis is that long-term antipsychotic treatments may eventually lead to increased production of neurotoxic free radicals (Lohr, 1991; Lohr and Browning, 1995; Lohr et al., 2003), resulting in potentially irreversible damage in motor regions. The theoretical framework was first proposed by Cadet
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et al. who suggested that cell membranes may be destabilized by the toxic action of free radicals produced during the chronic use of antipsychotics, which may lead to structural neuropathological changes in the terminals of catecholaminergic and other neurotransmitter systems (Cadet et al., 1986). It was thus surmised that antioxidants may be helpful for the treatment of TD when concomitantly administered with antipsychotics, which was indeed supported by the follow-up study showing that a-tocopherol (vitamin E), a powerful antioxidant, was beneficial in the treatment of some TD patients (Lohr et al., 1988). Furthermore, a role for oxidative stress in the neurotoxic effects of methamphetamine and methylenedioxymethamphetamine, the development of Parkinson's disease, and the degeneration of dopamine system has been elaborated in the literature review (Cadet and Brannock, 1998). The accumulated evidence all pointed towards the involvement of oxidative stress arising from catecholamine oxidation in the degeneration of monoaminergic systems. In addition to damage in motor regions, oxidative stressinduced damage may also contribute to cognitive impairment such as learning and memory (Fukui et al., 2001; Hu et al., 2006; Kapogiannis and Mattson, 2011; Nicolle et al., 2001). A study using the DN-DISC1 mice model has shown that the significant oxidative stress in the prefrontal cortex could have an impact on epigenetic and transcriptional machinery, which points to a mediating condition contributing to cognitive impairment (Johnson et al., 2013). In schizophrenia, the marker of oxidative modification of proteins, protein carbonyl content (PCC), was negatively correlated with working memory (Asevedo et al., 2013) and serum protein oxidative stress was also elevated in siblings of patients with schizophrenia (Massuda et al., 2013). In agreement with these findings, our recent study has reported that total antioxidant status was significantly associated with attention index and RBANS total scores in schizophrenia patients, suggesting that progressive cognitive impairments in schizophrenia may be causally related to impaired antioxidant capacity resulting in increased oxidative stress (Zhang et al., 2012b). As for TD, lower levels of antioxidant markers superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS) in cerebrospinal fluid and peripheral tissues have been reported in schizophrenia patients with TD (Lohr et al., 1990; Peet et al., 1993; Tsai et al., 1998; Yamada et al., 1997). We have also found lower levels of glutathione peroxidase, catalase, total antioxidant status (TAS), and plasma SOD in schizophrenia patients with TD than those without TD (Chen et al., 2010; Zhang et al., 2003a, 2007). Among the antioxidant systems, SOD is one of the critical scavenging enzymes that have been under constant investigation in schizophrenia with elevated levels reported in numerous studies (Condray and Yao, 2011; Galecki et al., 2006; Glazer, 2000a, b; Held et al., 2000; Kay et al., 1987; Palmer et al., 2009; Sharma and Antonova, 2003). SOD has three isoforms: the manganese isoform (MnSOD/SOD2) located in mitochondria, and the copper and zinc (CuZn) isoforms present in the cytoplasm (SOD1) and extracellular space (SOD3). Since cellular metabolism and production of reactive oxygen species occur primarily in the mitochondria, MnSOD has been the major focus of the aforementioned studies and our recent study has also found that MnSOD was an independent contributor to the RBANS total score in schizophrenia patients either with or without TD (Wu et al., 2014). However, research on CuZnSOD is limited even though its involvement in schizophrenia and TD has been observed in previous studies showing marked reduction of CuZnSOD in cerebrospinal fluid of patients with recent-onset schizophrenia (Coughlin et al., 2013) as well as the association between CuZnSOD and dyskinetic movements (Zhang et al., 2003a). Therefore, we conducted the current study by investigating both the cognitive performance assessed by RBANS and the activity of
CuZnSOD in blood from schizophrenia patients stratified by TD presence in a Chinese population. Furthermore, we also evaluated two additional markers, TAS and malondialdehyde (MDA) levels in a subset of our cohort to confirm the oxidative stress status reflected by CuZnSOD. While the former provided an index of total nonenzymatic antioxidants in plasma, the latter reflected the levels of the end product of lipid peroxidation. Given the fact that gender differences exist in cognitive domains and oxidative stress parameters in both schizophrenia patients and controls (Halari et al., 2006; Han et al., 2012; Wisner et al., 2011; Zhang et al., 2012a), we only included male subjects in this study. 2. Methods 2.1. Ethics statement The research protocol was approved by the Institutional Review Board, Beijing Hui-Long-Guan hospital. A psychiatrist explained the research protocol and procedures to the potential subject. The description of the study was tailored to maximize the understanding of the subject using language appropriate to the subject's level of comprehension, and emotional readiness. If the subject was willing to consent to participate in the study the researcher provided an in depth description to the subject and in certain instances, to their parents or guardians. Most of the patients and all the healthy controls provided written informed consent. Only in two patients who also assented to participate in the study, the parents gave their written consent on behalf of the patients under the request of the patients. 2.2. Subjects One hundred and thirteen inpatients with schizophrenia were recruited from Beijing Hui-Long-Guan hospital, a Beijing-cityowned psychiatric hospital. All patients met the following inclusion criteria: 1) age 40e70 years, Han Chinese; 2) confirmed DSMIV diagnosis of schizophrenia; 3) with at least 5 years of illness; 4) had been receiving stable doses of oral antipsychotic drugs for at least 12 months before entry into the study. All patients were of the chronic type, and had been ill for an average 30.2 ± 8.0 years, with current antipsychotic treatment for an average of 4.8 ± 4.1 years. Since admission, all patients received dietetically balanced hospital meals, which were occasionally supplemented by gifts (usually fruit). Patients had the opportunity to exercise for about an hour per day. Antipsychotic drug treatment was mainly monotherapy with clozapine (n ¼ 53), risperidone (n ¼ 21), and typical drugs (perphenazine, pipotiazine, chlorpromazine, sulpiride, and haloperidol; n ¼ 39). A mean daily dose of antipsychotics (Table 1), including both the first- and second-generation antipsychotics, was converted to approximate daily mean chlorpromazine milligram equivalents for each subject using standard guidelines (Lehman et al., 2004; Woods, 2003). Age-, education-, and BMI-matched control subjects (n ¼ 84) were recruited from the local community in Beijing. Current mental status and personal or family history of any mental disorder was assessed by unstructured interviews. None of the healthy control subjects presented a personal or family history of psychiatric disorder. A complete medical history, physical examination and laboratory tests were obtained from patients and control subjects. Any subjects with major medical illness were excluded. None of the subjects met criteria for drug or alcohol abuse or dependence. All subjects are Han Chinese recruited at the same period from the Beijing area. Demographic data for patients and normal controls are summarized in Table 1.
J.Q. Wu et al. / Journal of Psychiatric Research 58 (2014) 167e174 Table 1 Characteristics of controls, schizophrenic patients with and without TD. Characteristic
Normal controls (n ¼ 84)
Age (yrs) 54.0 ± 7.4 Education (yrs) 9.5 ± 3.3 2 25.4 ± 4.3 BMI (kg/m ) Smoker/ 47/37 Nonsmoker Age of onset (yrs) Duration of Illness (yrs) Number of hospitalizations Drugs (clozapine/ risperi-done/ typical drugs) Daily AP dose (mg/day) (CPZ equivalent) Duration of treatment (ms) PANSS total score P subscore N subscore G subscore AIMS total score CuZnSOD (U/ml) 44.3 ± 14.6 TASa (U/ml) 215.3 ± 39.0 a MDA (nmol/ml) 2.5 ± 1.7
F or X2
Patients without TD (n ¼ 70)
Patients with TD (n ¼ 43)
53.8 ± 5.7 9.1 ± 2.2 25.5 ± 5.0 60/10
56.2 ± 6.7 9.5 ± 2.4 23.0 ± 4.9 38/5
24.5 ± 6.6 29.3 ± 7.9
24.5 ± 5.6 31.6 ± 8.1
0.001 0.981 2.071 0.153
3.9 ± 2.6
4.3 ± 2.4
0.666 0.416
33/15/22
20/6/17
1.31
544.8 ± 836.6 399.9 ± 355.5
54.2 ± 57.0 59.6 11.3 22.4 25.8 1.2 57.2 184.1 6.4
± ± ± ± ± ± ± ±
13.0 4.5 6.7 4.8 1.4 14.6 48.9 5.2
61.5 ± 56.3 67.1 14.5 24.2 28.4 7.1 51.0 159.1 9.3
± ± ± ± ± ± ± ±
1.865 0.584 2.026 13.228
11.5 4.8 5.9 5.2 2.5 15.3 42.6 7.2
p value 0.158 0.559 0.136 0.001**
0.519
1.159 0.284
comprised of 12 subtests that are used to calculate 5 age-adjusted index scores and a total score. Test indices are Immediate Memory (comprised of List Learning and Story Memory tasks); Visuospatial/Constructional (comprised of Figure Copy and Line Orientation tasks); Language (comprised of Picture Naming and Semantic Fluency tasks); Attention (comprised of Digit Span and Coding tasks); and Delayed Memory (comprised of List Recall, Story Recall, Figure Recall, and List Recognition tasks). Our group previously translated RBANS into Chinese and the clinical validity and its test-retest reliability established among controls and schizophrenia patients (Zhang et al., 2009). Each subject came in the testing room on a separate day to be introduced to our research center by a research member and a proper training session had been performed for individual to become acclimated to the testing environment and computerized tasks. 2.4. Plasma CuZnSOD activity, TAS, and MDA measurements
0.420 0.519 9.750 13.249 1.933 6.956 248.802 14.655 14.596 16.475
169
**
0.002 0.000** 0.161 0.01* 0.000** 0.000** 0.000** 0.000**
TD: tardive dyskinesia; BMI: body mass index; AP: antipsychotic; CPZ: chlorpromazine; PANSS: Positive and Negative Symptom Scale; P: PANSS positive symptom subscale; N: PANSS negative symptom subscale; G: PANSS general psychopathology subscale; AIMS: Abnormal Involuntary Movement Scale. *p < 0.05; **p < 0.01. a markers assessed in a subset of our cohort with sufficient plasma for additional assays (TD: n ¼ 30, non-TD: n ¼ 34, and healthy controls: n ¼ 40).
2.3. Clinical measures Each subject filled out a detailed questionnaire that recorded general information, sociodemographic characteristics, and medical and psychological conditions. Additional information was collected from available medical records and collateral data (from family and/or treating clinician). Four experienced psychiatrists who were blinded to the clinical status of the patients assessed the severity of tardive dyskinesia using the abnormal involuntary movement scale (AIMS) (Guy, 1976). Diagnosis of TD was based on the Research Diagnostic Criteria (RDC) in DSM-IV, in combination with the criteria of Schooler and Kane (Schooler and Kane, 1982). Dyskinesia was classified as present in a particular subject with an AIMS score of at least three (moderate degree) in any body part or with at least two (mild degree) in two or more body parts. Each item on the AIMS ranges from 0 to 4, and the total AIMS score was calculated by adding items 1e7. The patients with TD were re-evaluated at least 1 month later for TD using the AIMS and diagnosed with TD only if both evaluations consistently revealed the presence of TD. Psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1987) on the same day as the TD evaluation. All raters for the AIMS and PANSS had simultaneously attended a training session in their use before this study started. After training repeated assessments showed a correlation coefficient between raters of greater than 0.84 was maintained for the PANSS and 0.88 for the AIMS total scores. The Repeatable Battery for the Assessment of Neuropsychological Status (RBANS, Form A) (Randolph et al., 1998) was individually administered to measure cognitive functioning. The RBANS is
Venous blood was obtained from each subject between 0700 and 0900 AM following an overnight fast. CuZnSOD activity (Zhang et al., 2006, 2013), TAS (Li et al., 2011), and MDA levels (Yagi, 1994; Zhang et al., 2007) were analyzed using established procedures. Our previous reports give a full description of the assays. The interand intra-assay coefficients of variation for CuZnSOD were 8% and 6%. Blood CuZnSOD activity and TAS are expressed as units per milliliter plasma (U/ml). Plasma MDA values were calculated using the extinction coefficient of MDA-thiobarbituric acid complex at 532 nm. MDA results are expressed as nmol/ml. A research assistant blind to the clinical situation assayed all samples. A code number maintained by the primary investigator indicated the identity of subjects. 2.5. Statistical analysis Demographic and clinical variables of TD and non-TD groups were compared using analysis of variance (ANOVA) for continuous variables and chi-squared for categorical variables. We compared RBANS scores among the three groups using ANOVA and Fisher's least significant difference (LSD) test was used to perform post-hoc pairwise comparisons (non-TD versus controls; TD versus controls; TD versus non-TD). When significance was found in ANOVA, the effect of age, education, BMI, smoking status, duration of antipsychotic treatment, type and dose of antipsychotic drugs was tested by adding these variables to the analysis model as covariates. Relationships between variables have been assessed with Pearson's product moment correlation coefficients. Bonferroni corrections were applied to each test to adjust for multiple testing. Multiple regression analysis using RBANS total or the index scores as the dependent variable was carried out to investigate the impact of a range of variables including CuZnSOD level, age, education, BMI, smoking status, duration of antipsychotic treatment, type and dose of antipsychotic drugs, and PANSS total and index scores. SPSS version 17.0 was used to do all statistical analysis. Data are presented as mean and standard deviation (mean ± SD). All p-values are two tailed with significance level set at 0.05. 3. Results Clinical and demographic characteristics for the schizophrenia patients with and without TD along with healthy controls are presented in Table 1. There was no significant difference in any characteristics between patients with TD, without TD, and normal controls (all p > 0.05) except for smoking status (p < 0.01), which was adjusted in the following analyses. Age, education, BMI, and smoking status were not associated with CuZnSOD activity in the
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combined groups, or when the association was examined in TD, non-TD, and control groups respectively (all p > 0.05). Between schizophrenia patients with and without TD, there was no significant difference in the number of hospitalizations, antipsychotic types (between clozapine, risperidone, and typical drugs), daily antipsychotic dose (chlorpromazine equivalent), duration of antipsychotic treatment (all p > 0.05). Patients with TD had significantly higher levels of the general psychopathology (p < 0.05), positive symptom (p < 0.01), and total scores of PANSS (p < 0.01) as well as AIMS total score (p < 0.001) than those without TD. The significant differences in the positive symptom subscore (p < 0.01) and total score of PANSS (p < 0.05) as well as AIMS total score (p < 0.001) remained after controlling for age, BMI, education, duration of antipsychotic treatment, type and dose of antipsychotic drugs. Between clozapine, risperidone, and typical drugs, there was no significant difference in AIMS total score for either the combined patients or separate TD and non-TD groups (p > 0.05). 3.1. CuZnSOD activity in healthy controls and schizophrenia with and without TD CuZnSOD activity was lower in patients with TD than in those without TD (51.0 ± 15.3 U/ml vs. 57.2 ± 14.6 U/ml; F ¼ 4.7, df ¼ 1, 111, p ¼ 0.033). This difference remained significant after covarying for age, education, smoking status, and antipsychotic dose (p ¼ 0.018). Furthermore, CuZnSOD activity was higher in both patient groups than in the control group (44.3 ± 14.6 U/ml; Table 1) (TD vs. controls: p ¼ 0.018; non-TD vs. control: p < 0.001). These differences remained significant after covarying for age, education, BMI, and smoking status (TD vs. controls: p ¼ 0.019; non-TD vs. control: p < 0.001). Between clozapine, risperidone, and typical drugs, there was no significant difference in CuZnSOD activities for either the combined patients or separate TD and non-TD groups (p > 0.05). In the combined patient group, or when the TD and non-TD group were examined separately, CuZnSOD activity was not associated with the duration of antipsychotic treatment, type (between clozapine, risperidone, and typical drugs), or dose of antipsychotic medication (all p > 0.05). In addition, CuZnSOD was not correlated with any subscore or total score of PANSS in either TD or non-TD group alone (all p > 0.05). 3.2. TAS and MDA levels in healthy controls and schizophrenia with and without TD To further confirm the oxidative stress status reflected by CuZnSOD activity, TAS and MDA levels were further assessed in a subset of our cohort with sufficient plasma for additional assays (TD: n ¼ 30, non-TD: n ¼ 34, and healthy controls: n ¼ 40). There was a significant difference in TAS among the patients with TD, without TD, and normal controls (F ¼ 14.6, df ¼ 2,101, p < 0.001). Post hoc tests showed that the patients with TD had lower levels of
TAS than those without TD (159.1 ± 42.6 U/ml vs 184.1 ± 48.9 U/ml, p ¼ 0.024). Both patient groups had lower TAS levels than the control group (215.3 ± 39.0 U/ml; TD vs. controls: p < 0.001; non-TD vs. controls: p ¼ 0.003). There was also a significant difference in MDA among the TD, non-TD, and control groups (F ¼ 16.5, df ¼ 2,101, p < 0.001). Post hoc tests revealed that the patients with TD had higher MDA levels than those without TD (9.3 ± 7.2 nmol/ml vs. 6.4 ± 5.2 nmol/ml, p ¼ 0.024). Both patient groups had higher MDA levels than the control group (2.5 ± 1.7 nmol/ml; TD vs. control: p < 0.001; non-TD vs. control: p ¼ 0.001). 3.3. Cognitive performance in healthy controls and schizophrenia with and without TD The mean and standard deviation of RBANS total and index scores of 43 TD, 70 non-TD, and 84 controls are shown in Table 2. The significant differences between the TD and non-TD patients included the lower RBANS subscales of Visuospatial/Constructional (78.1 ± 16.9 vs. 85.0 ± 15.6, p ¼ 0.024) and attention (74.8 ± 12.7 vs. 82.9 ± 11.5, p ¼ 0.005). After controlling for age, education, BMI, smoking status, duration of illness, and antipsychotic treatment (type, dose and duration of antipsychotic treatment), the differences remained significant for RBANS subscales of Visuospatial/ Constructional (p ¼ 0.032) and attention (p ¼ 0.004). In addition, both patient groups (TD vs. control; non-TD vs. control) scored lower on all subscales (all p < 0.01) except for the Visuospatial/ Constructional index than the controls. These differences remained significant after covarying for age, education, BMI, and smoking (all p < 0.05). 3.4. Correlation between CuZnSOD and cognitive performance In the combined patient group, correlation analysis showed that CuZnSOD activity was negatively associated with the attention index of RBANS (r ¼ 0.31, df ¼ 111, p ¼ 0.001). In either TD or non-TD group, CuZnSOD activity was negatively associated with the attention index of RBANS (non-TD: r ¼ 0.31, df ¼ 68, p ¼ 0.009; TD: r ¼ 0.52, df ¼ 41, p < 0.0005). Furthermore, CuZnSOD activity was also negatively associated with immediate memory index (r ¼ 0.35, p ¼ 0.02) and total score of RBANS (r ¼ 0.32, p ¼ 0.04, both df ¼ 41) in TD patients. Multiple regression analysis showed that in either TD or non-TD group, CuZnSOD (non-TD: b ¼ 0.26, t ¼ 2.0, p < 0.05; TD: b ¼ 0.64, t ¼ 3.77, p < 0.01; Fig. 1) was an independent contributor to the attention index of RBANS. Furthermore, in the TD group the PANSS positive symptom (b ¼ 0.48, t ¼ 2.81, p < 0.01) and CuZnSOD (b ¼ 0.36, t ¼ 2.09, p < 0.05) were independent contributors to the RBANS total score, after controlling for age, BMI, smoking, duration of illness and antipsychotic treatment (type, dose and duration of antipsychotic treatment). However, in the healthy control group, the CuZnSOD activity was not found to be
Table 2 Comparison of total and index scores of the RBANS between schizophrenia patients with TD, without TD, and controls. Normal controls (n ¼ 84) Immediate memory Visuospatial/Constructional Language Attention Delayed memory Total scale
76.2 83.4 97.1 89.4 89.1 82.9
± ± ± ± ± ±
17.6 15.1 9.8 17.5 15.3 13.7
Patients without TD (n ¼ 70) 61.5 85.0 86.6 82.9 70.0 69.9
± ± ± ± ± ±
14.9 15.6 13.2 11.5 19.2 14.1
*Indicates significance of the comparisons between TD and non-TD groups. **p < 0.01, *p < 0.05. a Refers to Bonferroni corrections.
Patients with TD (n ¼ 43) 58.9 78.1 85.7 74.8 70.2 67.1
± ± ± ± ± ±
16.7 16.9* 12.3 12.7** 20.9 14.0
F
p Value
Corrected pa
21.7 2.7 20.3 14.5 26.0 24.6
