Plasma total antioxidant status and cognitive impairments in schizophrenia

Schizophrenia Research 139 (2012) 66–72

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Plasma total antioxidant status and cognitive impairments in schizophrenia Xiang Yang Zhang a, b,⁎, Da Chun Chen b, Mei Hong Xiu b, Wei Tang c, Feixue Zhang c, LianJing Liu c, Yuanling Chen c, JiaHong Liu c, Jeffrey K. Yao d, Therese A. Kosten a, Thomas R. Kosten a, b,⁎⁎ a

Menninger Department of Psychiatry and Behavioral Sciences, Baylor College of Medicine, Houston, TX, USA Psychiatric Research Center, Beijing Hui-Long-Guan Hospital, Beijing, China Department of Psychiatry, Kangning Hospital, Wenzhou, Zhejiang Province, China d VA Pittsburgh Healthcare System, and Department of Psychiatry, University of Pittsburgh School of Medicine, Western Psychiatric Institute and Clinic, Pittsburgh, USA b c

a r t i c l e

i n f o

Article history: Received 1 February 2012 Received in revised form 4 April 2012 Accepted 6 April 2012 Available online 1 May 2012 Keywords: Schizophrenia Cognition Oxidative stress Psychopathology

a b s t r a c t Oxidative stress-induced damage to neurons may contribute to cognitive deficits during aging and in neurodegenerative disorders. Schizophrenia has a range of cognitive deficits that may evolve from oxidative stress, and this study examines this association of oxidative stress with cognitive deficits in schizophrenia. We recruited 296 chronic schizophrenia patients and 181 healthy control subjects and examined the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) and plasma total antioxidant status (TAS) in both groups. Schizophrenia symptoms were assessed using the positive and negative syndrome scale (PANSS). Our results showed that TAS levels were significantly lower in patients than controls (179.6 ± 81.0 U/ml vs. 194.8 ± 46.0 U/ml, p b 0.05). Cognitive scores on the RBANS and nearly all of its five subscales (all p b 0.001) except for the Visuospatial/Constructional index (p > 0.05) were significantly lower in schizophrenia patients than normal controls. For the patients, TAS was inversely associated with some domains of cognitive deficits in schizophrenia, such as Attention and Immediate Memory. Our findings suggest that oxidative stress may be involved in the pathophysiology of schizophrenia, and its associated cognitive impairment. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Schizophrenia is a psychiatric disorder characterized by a range of cognitive deficits in executive function, attention, working memory, and long-term memory. Disturbances in cognition appear to be core features of the illness (Heinrichs and Zakzanis, 1998; Harvey et al., 2004; Goff et al., 2011). They can occur before the onset of the other symptoms of schizophrenia (Niendam et al., 2003; Harvey, 2009), and generally persistent during the course of the schizophrenia illness (Heaton et al., 2001; Rajji and Mulsant, 2008; Irani et al., 2011). Moreover, outcome measures correlate more closely with the extent of cognitive deficits than with the severity of psychotic symptoms and some aspects of cognitive function are specifically impaired in many individuals (Lewis and Lieberman, 2000). The most notable cognitive difficulties are in memory, language, executive functioning, and attention (Kremen et al., 2001). Recently, numerous studies have demonstrated an association between these deficits and poor

⁎ Correspondence to: X.Y. Zhang, VA Medical Center, Research Building 109, Room 130, 2002 Holcombe Boulevard, Houston, TX 77030, USA. Tel.: + 1 713 791 1414x5824; fax: + 1 713 794 7938. ⁎⁎ Correspondence to: T.R. Kosten, VA Medical Center, Research Building 110, Room 229, 2002 Holcombe Boulevard, Houston, TX 77030, USA. Tel.: + 1 713 794 7032; fax: + 1 713 794 7938. E-mail addresses: [email protected] (X.Y. Zhang), [email protected] (T.R. Kosten). 0920-9964/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2012.04.009

functional outcome, including performance of basic activities of daily living, social skills acquisition, social problem solving, occupational functioning and community outcome (Keefe, 2008), and quality of life (Gold et al., 2002; Green et al., 2004). However, cognitive deficits are generally not responsive to currently available pharmacotherapies and the pathophysiological mechanisms underlying these cognitive deficits are still unclear (Galletly, 2009; Barch, 2010; Goff et al., 2011). Free radicals are highly reactive chemical species generated during normal metabolic processes, and, in excess, can damage lipids, proteins, and DNA, causing cellular dysfunction and even death (Lohr and Browning, 1995; Reddy and Yao, 1996; Yao and Keshavan, 2011). Oxidative stress (OS) involves a disequilibrium between pro-oxidant processes and the antioxidant defense system in favor of the former (Lohr et al., 2003; Yao and Keshavan, 2011). There are potentially multiple pathological consequences of increased oxidative stress, including initiation of lipid peroxidation (Yao and Keshavan, 2011). Under normal physiological conditions, freeradical-induced oxidative stress is combated by a complex antioxidant defense system (Lohr and Browning, 1995; Bitanihirwe and Woo, 2011; Yao and Reddy, 2011). A major contribution to the total antioxidant capacity comes from antioxidant molecules in plasma. Several antioxidant molecules in plasma, such as albumin, uric acid and ascorbic acid account for 85% of the total human antioxidant capacity (Yao et al., 1998); other antioxidants in blood include bilirubin,

X.Y. Zhang et al. / Schizophrenia Research 139 (2012) 66–72

α-tocopherol and β-carotene (Yao et al., 2000; Reddy et al., 2003). Although measuring levels of specific antioxidant molecules can yield valuable information, determination of total antioxidant status (TAS) provides an index of all antioxidants (Yao et al., 1998). Cognitive decline can result from imbalances between local reactive oxygen species (ROS) and antioxidant capacity within the brain (Davies, 2000; Liu et al., 2003; Nagai et al., 2003; Ames, 2006; Corbetta et al., 2008). An unbalanced accumulation of oxidatively modified proteins in the brain potentiates neurodegeneration and impairs cognitive function (Davies, 2000; Radak et al., 2007). Aging in humans, as well as in experimental animals, is associated with a slow deterioration of cognitive performance particularly of learning and memory that increases the risk of neurodegenerative disorders (Kolosova et al., 2006). Oxidative damage has long been proposed to be critically involved in several pathological manifestations of aging. Numerous studies have confirmed that the accumulation of oxidative damage such as oxidized proteins and lipid peroxides in aged mammalian brains underlies the molecular basis of brain aging and neurodegeneration (Kolosova et al., 2006). Oxidative stress-induced damage to the rat synapse in the cerebral cortex and hippocampus during aging may contribute to the deficit of cognitive function as assessed in these models (Fukui et al., 2001). Also, the dramatic decline in learning and memory taking place in early middle-aged mice was associated with a large increase in brain oxidative stress. Finally, antioxidants and low doses of SOD/catalase mimetics protect the nervous system in the brain against oxidative stress during aging and, hence, almost completely reverse cognitive deficits associated with this increase in oxidative stress (Liu et al., 2003; Clausen et al., 2010). Oxidative stress may be involved in the pathophysiology of schizophrenia with altered peripheral activities of antioxidant enzymes (Lohr, 1991; Lohr and Browning, 1995; Mahadik and Mukherjee, 1996; Reddy and Yao, 1996; Yao et al., 2001; Reddy et al., 2003; Zhang et al., 2006). High levels of lipid peroxidation products have been reported in plasma (McCreadie et al., 1995; Mahadik et al., 1998), red blood cells (Herken et al., 2001) and cerebrospinal fluid of schizophrenia patients (Lohr et al., 1990) with significant correlations between psychopathology and the levels of antioxidant enzymes and lipid peroxidation products (Mahadik et al., 1999; Yao et al., 2000; Khan et al., 2002; Arvindakshan et al., 2003; Zhang et al., 2003). Furthermore, some schizophrenia symptoms improve with antioxidant treatments, such as vitamins (Mahadik and Gowda, 1996; Yao et al., 2001), extract of Ginkgo biloba (EGb) (Zhang et al., 2001a, 2001b) or essential polyunsaturated fatty acids (EPUFA) (Peet and Horrobin, 2002; Mahadik and Evans, 2003). Interestingly, one most recent report showed relationships between levels of arachidonic acid (AA) and performance on tests of cognition for schizophrenia patients, with defects in AA signaling associated with deficits in cognition (Condray and Yao, 2011). These findings suggest that oxidative stress may contribute to the pathophysiology of schizophrenia. In the view of cognitive deficits and the marked alterations in oxidative stress existed in schizophrenia, and the important implication of oxidative stress in cognition, it would be of interest to explore the association between cognitive impairments and oxidative stress in schizophrenia. However, to our best knowledge, none has examined this association in patients with schizophrenia. We hypothesized that oxidative stress may be associated with cognitive deficits in schizophrenia. 2. Methods 2.1. Subjects Two hundred ninety-six inpatients (266 men and 30 women) were recruited from the Beijing Hui-Long-Guan Hospital, a Beijing

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City owned psychiatric hospital. All patients met the DSM-IV criteria for schizophrenia as diagnosed by two clinical psychiatrists on the basis of the Structured Clinical Interview for DSM-IV (SCID). Patients were of the chronic type, aged 26–71 years (mean 48.2 ± 10.7), with a mean duration of illness of 25.5 years (SD = 8.6), and a mean education of 8.9 years (SD = 3.3). All patients had been receiving stable doses of oral neuroleptic medications for at least 6 months. Normal controls were recruited from the local community, and included 140 men and 41 women with an average age of 48.1± 8.9 years (range from 26 to 70 years), and 8.2 ± 2.4 years of education. All subjects were Han Chinese and were recruited at the same period from the Beijing area. Both patients and normal subjects had similar socioeconomic status and dietary patterns. A complete medical history and physical examination, laboratory tests, and electrocardiogram were obtained from all subjects. All participant patients were without physical diseases. Neither the schizophrenia patients nor the control subjects suffered from substance abuse/dependence. All subjects gave written, informed consent to participate in the study, which was approved by the Institutional Review Board of Beijing Hui-Long-Guan Hospital. 2.2. Blood sampling and plasma TAS measurements Plasma samples from healthy controls and patients were collected between 7 and 9 AM following an overnight fast. The plasma was separated, aliquoted and stored at −70 °C before use. The TAS levels in plasma were measured by a technician, who was blind to the diagnostic status of subjects. The identity of all subjects was indicated by a code number maintained by the investigator until all biochemical analyses were completed. Our previous report provides a full description of the assays for TAS biomarkers (Li et al., 2011). Briefly, the TAS of fasting plasma was measured as ferric reducing antioxidant potential (FRAP) by using a commercially available kit. In this assay, antioxidants are evaluated as reductants of Fe3 + to Fe2 +, which are chelated by TPTZ to form a Fe 2 +–TPTZ complex absorbing at 593 nm (Benzie and Strain, 1996) and recorded using the Multiskan microplate reader (FlowLabs, McLean, VA, USA). Activity was expressed as Units per milliliter plasma (U/ml). 2.3. Cognitive tests We individually administered the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) (Randolph et al., 1998) to measure cognitive functioning. The RBANS is 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 established its clinical validity and its test–retest reliability among controls and schizophrenia patients (Zhang et al., 2009). A higher RBANS score is associated with better cognitive functioning. 2.4. Statistical analysis Demographic and clinical variables of the patient and healthy control groups were compared using t-test or analysis of variance (ANOVA) for continuous variables and chi-squared for categorical variables. Since the TAS variables were normally distributed in patients and normal controls (Kolmogorov–Smirnov one sample test; both p > 0.05), the principal outcome analysis consisted of oneway analysis of variance (ANOVA). We also compared RBANS scores

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among the patient and control groups using ANOVA. When ANOVA was significant, we tested the effects of sex, age, education, smoking, and body mass index (BMI) by adding these variables to the analysis model as covariates. We assessed relationships between variables with Pearson's product moment correlation coefficients and applied Bonferroni corrections to adjust for multiple testing. We used single regression analysis to assess the correlations of the TAS with cognitive function as assessed on the RBANS total and index scores. We then used multivariate regression analyses (stepwise regression model) to assess associations of TAS with RBANS while adjusting for various potentially confounding variables of age, sex, education, smoking, BMI in both patient and control groups, and clinical variables in the patient group, such as PANSS, years of education, age of onset, hospitalization, antipsychotic treatment, and anticholinergic drugs. SPSS version 15.0 was used to do all statistical analysis. In addition, a power calculation and effect size determination were performed using the G*Power 3.0.10 program (http://www.softpedia.com/get/Science-CAD/G-Power.shtml). Data were presented as mean ± S.D. Statistical significance was defined as p b 0.05.

3. Results Clinical and demographic characteristics for the schizophrenia patients and healthy controls are presented in Table 1. Patients and control groups showed no differences in age, but differed in smoking (p b 0.001), gender (p b 0.001), education (p b 0.05) and BMI (p b 0.05), which were adjusted in the following analyses. Age and TAS levels showed a significantly negative association for both the patients (r = −0.20, p b 0.01) and healthy controls (r = −0.19, p b 0.01). TAS levels for patients also showed a significant association with the duration of antipsychotic treatment (r = − 0.28, p b 0.001), and duration of illness (r = −0.17, p b 0.005); however, TAS levels were not associated with type and dose of antipsychotic drugs (all p > 0.05). In addition, TAS levels were not associated with BMI (p > 0.05). Furthermore, TAS levels did not differ between smoker and nonsmoker subjects in the combined groups, or when the smokers and nonsmokers were examined in patient and control groups separately (all p > 0.05).

Table 1 Demographics of first-episode patients and normal control subjects.

Sex (M/F) Age (years) Education (years) BMI (kg/m2) Smokers Duration of illness (years) Hospitalization Duration of antipsychotic treatment (years) Subtypes of schizophrenia Paranoid type Disorganized type Undifferentiated type Residual type Others PANSS Positive subscale Negative subscale General subscale Total TAS levels (U/ml)

Schizophrenia (n = 296)

Control subjects (n = 181)

F or χ2

df

p

266/30 48.2 ± 10.7 8.9 ± 3.3 25.7 ± 3.4 224 (75.7%) 25.5 ± 8.6 4.6 ± 2.9 3.6 ± 4.1

140/41 48.1 ± 8.9 8.2 ± 2.4 24.2 ± 3.7 93 (51.4%)

13.9 0.05 5.95 18.75 35.2

1 1,475 1,473 1,433 1

b.001 0.82 0.02 b.001 b.001

Plasma TAS levels were lower in patients than in controls (179.6± 81.0 U/ml vs. 194.8 ± 46.0 U/ml, F = 5.28, df= 468, p b 0.05) and not attenuated after covariance for age, sex, education, smoking, and BMI (F= 8.12, df= 6, 460; p b 0.01) and had an effect size of 0.502 with power of 0.99 (Table 1). TAS levels also were significantly lower in female than male patients (105.3 ± 43.6 U/ml vs. 189.1 ± 87.2 U/ml; F= 16.33, df=1,294, pb 0.001). However, we observed no sex difference in TAS levels for healthy controls (p >0.05). TAS levels had a significant negative correlation with the negative symptom subscale of PANSS (r= −0.19, p b 0.01) and the PANSS total score (r= −0.14, p b 0.05), but not with positive symptoms (p> 0.05). Furthermore, we found no significant difference in TAS between subtypes of schizophrenia (F= 1.19, df =4, 291, p= 0.32).

3.2. Cognitive performance in schizophrenia and healthy controls Table 2 shows RBANS total and index scores of 232 patients with schizophrenia and 176 healthy controls. The RBANS scores were significantly lower in patients on all subscales (all p b 0.001) except for the Visuospatial/Constructional index. The difference in RBANS total score remained significant after adjusting for sex, age, smoking, education and BMI. Examination of the RBANS percentile distributions of the index scores reveals a downward shift for patients relative to controls (Table 3). There was more than a 4-point decrease of RBANS total score in patients compared to controls on the 10th, 50th or 90th percentiles. Also, there was more than a 3-point difference or no difference between patients and controls on the 10th, 50th or 90th percentiles with three notable exceptions. At the 90th percentile on Visuospatial/Constructional index, the score for schizophrenia was 112 in comparison with the score of 100 for controls; at the 50th percentile on Visuospatial/Constructional index, the scores were 84 for patients and 75 for controls. At the 10th percentile on Attention, the score for patients was 60 in patients in comparison with the score of 56 for controls. We found no significant difference in RBANS total and its five index scores among schizophrenia patient subtypes (F = 0.51–1.35, df = 4,227, p = 0.25–0.73). Female patients performed better than male patients on delayed memory, immediate memory and the RBANS total scores (all p b 0.05). However, in the normal control group, the males and females showed no significant differences on the RBANS total score or on any of the index scores (all p > 0.05). These results suggested that male patients may have suffered more cognitive impairment than female patients. These RBANS comparisons between schizophrenia patients and healthy controls did not suffer from insufficient statistical power and had a power of 0.86–0.99 across the total score and five indices.

Table 2 Total and index scores on the RBANS in schizophrenia versus controls.

82 (27.7%) 27 (9.1%) 11 (3.7%) 171 (57.8%) 5 (1.7%) 11.0 ± 4.4 24.4 ± 8.4 24.7 ± 5.9 60.1 ± 14.9 179.6 ± 81.0

3.1. Plasma TAS in schizophrenia and healthy controls

194.8 ± 46.0

5.28

1,468

0.03

Index

Schizophrenia (n = 232)

Controls (n = 176)

Adjusted Fa

P

Immediate memory Attention Language Visuospatial/ Constructional Delayed memory Total

53.4 ± 11.9 64.3 ± 14.4 79.4 ± 13.5 75.5 ± 18.1

71.4 ± 16.3 82.8 ± 17.6 94.2 ± 11.2 78.7 ± 14.3

66.3 73.6 56.1 0.7

0.0001 0.0001 0.0001 ns

61.9 ± 16.8 59.8 ± 11.5

84.7 ± 15.1 77.1 ± 13.3

90.4 90.1

0.0001 0.0001

a Adjusted F means that F value was controlled for sex, age, smoking, education and body mass index (BMI).

X.Y. Zhang et al. / Schizophrenia Research 139 (2012) 66–72 Table 3 RBANS index raw score percentile distributions in patients with schizophrenia and normal controls. Percentile Immediate memory

Attention Language Visuospatial/ Constructional

Schizophrenia (n = 232) 99 123 115 90 87 100 75 76 94 50 61 82 25 49 72 10 44 60 5 40 56 1 40 44 Normal controls (n = 176) 99 121 132 90 94 109 75 83 94 50 73 82 25 61 68 10 49 56 5 48 49 1 40 46

Delayed memory

Total

118 103 95 87 79 68 57 47

126 112 100 84 69 60 53 50

117 98 91 75 52 44 40 40

117 93 83 70 59 52 50 44

130 103 105 101 95 87 79 68

116 100 89 75 65 60 58 53

111 101 95 88 78 60 56 40

115 99 86 77 67 56 52 48

3.3. Correlation between TAS and cognitive performance Correlation analysis showed that TAS levels were positively associated with the Visuospatial/Constructional (r = 0.24, df = 1,175, p b 0.05) and Attention indices (r = 0.22, df = 1,175, p b 0.05) for the healthy controls. However, these results did not pass Bonferroni test. For the patients, TAS levels were inversely associated with the Attention index (r = −0.382, df = 1, 231, p b 0.001) and the RBANS total score (r = − 0.271, df = 1,231, p = 0.006) for the patients (Fig. 1). After controlling for sex, age, duration of antipsychotic treatment and duration of illness, partial correlation analyses still showed significant association between TAS levels and Attention (p b 0.01) and the RBANS total score (p b 0.05). Multiple regression analysis showed that education had an independent positive contribution to the RBANS total and its 5 index scores (all p b 0.01) for the healthy controls. For the patients the PANSS negative symptom (t= −3.23, p = 0.002), education (t= 3.07, p = 0.003), and 500

TAS U/ml

r=-0.38 n=231 p<0.001

250

0 30

60

90

120

RBANS Attention Index

TAS U/ml

530 r=–0.27 n=231 p=0.006

280

30 30

60

90

120

RBANS total score Fig. 1. There was a significant negative relationship between plasma total antioxidant status (TAS) levels and both the RBANS Attention index (r = − 3.82, n = 231, p b 0.001) and the RBANS total score (r = − 0.33, df = 231, p = 0.006) in patient group.

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TAS (t= −1.98, p = 0.05) were independent contributors to the RBANS total score, after controlling for sex, age, duration of antipsychotic treatment and duration of illness, with R2 = 0.23. The PANSS negative symptom (t= −4.53, p b 0.001), TAS (t= −3.44, p b 0.001) and education (t= 2.07, p = 0.04) were independent contributors to the Attention Index of RBANS, and the PANSS negative symptom (t= −2.92, p = 0.004) and TAS (t= −2.17, p = 0.03) were independent contributors to the Immediate Memory Index of RBANS, after controlling for age, duration of antipsychotic treatment and duration of illness. 4. Discussion Our study found that 1) TAS levels were significantly decreased in chronic schizophrenia patients; 2) schizophrenia patients had significantly lower than normal cognitive scores on the RBANS and nearly all of its five subscales except for the Visuospatial/Constructional index; 3) TAS was negatively associated with the Attention index and the RBANS total score in schizophrenia patients. To our best knowledge, this is the first report to investigate the relationship between oxidative stress biomarker and cognitive impairments in schizophrenia patients. These findings, if replicated, have numerous implications for the progression and treatment of schizophrenia. Our finding of decreased levels of plasma TAS in chronic schizophrenia patients accords with previous findings in patients on (Virit et al., 2009) and off (Ustundag et al., 2006; Pazvantoglu et al., 2009; Chittiprol et al., 2010) antipsychotic treatment. Our finding of decreased plasma TAS is also consistent with previous findings showing decreased plasma levels of individual antioxidants in schizophrenia (Yao et al., 2000; Reddy et al., 2003). A major contribution to the body's total antioxidant capacity comes from antioxidant molecules in plasma, such as albumin, uric acid and ascorbic acid, which account for >85% of the total antioxidant capacity in human plasma (Miller et al., 1993), and other antioxidants in blood include bilirubin, α-tocopherol and β-carotene (Yao et al., 1998). Although individual antioxidants play a specific role in the antioxidant defense system, the above antioxidant molecules may act cooperatively in vivo to provide synergistic protection against oxidative damage (Miller et al., 1993; Yao et al., 1998). Determination of total antioxidant capacity provides an index of all antioxidants (Selek et al., 2008; Virit et al., 2009). Thus, our finding low level of such antioxidant capacity suggests oxidative stress in schizophrenia, but the underlying mechanisms of decreased TAS in schizophrenia remain complex (Yao et al., 1998). Duration of illness, physical activity, exposure to antipsychotic treatment, or different ethnic origin, lifestyle and dietary patterns can influence TAS. Hence, the role of decreased TAS in the pathophysiology of schizophrenia warrants further investigation. Our finding of a sex difference in TAS levels among the patients, but not among normal controls, may have several etiologies. A correlate of one etiology is that females live longer than males perhaps due to up-regulated expression of antioxidant enzymes via the estrogen receptor. Estrogens and phytoestrogens also activate MAPK, which in turn activates the NFkB signaling pathway and up-regulation of longevity-related genes (Vina et al., 2011). Thus, hormonal status and sex steroids could reduce oxidative stress. The lower TAS levels among females in our study seem to contradict estrogen's upregulation of antioxidant enzymes. However, the lower enzyme levels may simply reflect generally lower levels of oxidative stress due to other actions of these longevity related genes, which warrants further investigation. We found a significant negative relationship of TAS levels with the PANSS total score and negative symptom subscore, but not with the PANSS positive symptom subscore. Interestingly, in our previous study of first-episode and drug naïve patients with schizophrenia, we found a similar inverse correlation between TAS levels and the PANSS negative symptom subscore (p = 0.06) (Li et al., 2011). Thus, TAS and oxidative stress may play a role in schizophrenia patients'

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psychopathology that evolves and becomes more severe as the disease progresses. However, due to the cross-sectional rather than longitudinal design of both studies, we cannot support a causative or longitudinally evolving process as the underlying mechanisms for this association. Our schizophrenia patients had significantly lower cognitive performance than normal patients on nearly all of the RBANS' five subscales except for the Visuospatial/Constructional index. Confounds (i.e. education) only partially accounted for the approximately 17 point difference in the RBANS total score between the schizophrenia patients and normal controls. These deficits are consistent with the majority of studies assessing cognitive performance in schizophrenia patients and in particular with a recent USA study (Dickerson et al., 2004). However, Dickerson's study also found a significant difference on the Visuospatial/Constructional index. This difference may reflect the different demographic and clinical states of the two samples, since Dickerson included female outpatients stabilized on psychotropic medication for as little as 3 weeks. In contrast, our predominantly male inpatient sample was on stable antipsychotics for at least 6 months. Our patients were also older and less educated. While our controls were matched on age, their controls were substantially younger, which likely led to better performance on the RBANS tests (46 vs. 36 years old). The importance of these gender, age and education effects on the RBANS profiles of schizophrenia patients was emphasized in a recent study of this instrument (Loughland et al., 2007). Interestingly, TAS was found to be negatively associated with the Attention index and the RBANS total score in schizophrenia, but the association was opposite to our expected direction. This added risk from OS was beyond the conventional risk factors of negative psychotic symptoms, education and age, suggesting that OS-induced damage may importantly and independently contribute to cognitive impairment in schizophrenia. This is congruent with current scientific evidence relating OS to neurodegenerative disorders, where lipid peroxidation and inefficient antioxidant activity specifically damage neuronal membranes (Markesbery, 1997; Akyol et al., 2002; Jomova et al., 2010). Higher TAS being negatively rather positively associated with impaired cognitive function in schizophrenia is consistent with a previous study showing negative associations between TAS or lipoperoxides and cognitive impairment in older healthy adults (Sánchez-Rodríguez et al., 2006). An imbalance between the production and removal of free radicals can produce DNA damage during aging (Mendoza-Nuñez et al., 1999). In that study and a replication study of old age people those with normal antioxidant levels showed a higher frequency of DNA damage, while subjects with low antioxidant levels showed little DNA damage (Mendoza-Nuñez et al., 1999, 2001). Thus, DNA damage leads to an increase in antioxidant levels in order to prevent further damage and promote restoring mechanisms. Comparatively high antioxidant capacity being associated with more cognitive impairment in schizophrenia needs to be viewed in the context of overall lower TAS in these patients compared to normal controls. Low antioxidant concentrations in our schizophrenia patients appear to reflect a poor balance between free radical production and antioxidant levels such that schizophrenia cannot produce sufficient TAS to match the ordinary needs imposed on TAS by aging (Aejmelaeus et al., 1997; MendozaNúñez et al., 2001). Consequently, TAS may be increased in response to increased oxidative tone that increased oxidative tone can produce OS-induced cognitive impairment, but TAS is increased from a relatively low baseline level that is overall inadequate to match the normal OS processes that occur with aging. This speculative mechanism needs to be explored in future longitudinal investigations about how OS might cause cognitive impairment in schizophrenia. In addition, although our results suggest a close relationship between cognitive functioning in schizophrenia and TAS levels, the correlation between TAS and cognitive performance in schizophrenia could reflect a generalized deficit rather than a causal association between TAS and cognition. Further

research using a longitudinal and prospective study with first-episode and drug naïve patients is needed to clarify the relationship between TAS and cognitive performance in schizophrenia. Several limitations of this study should be noted. First, we measured only TAS, which is a single antioxidant defense parameter. Effective antioxidant protection is provided by the cooperative and sequential actions of antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and non-enzyme antioxidant molecules. The level of this one antioxidant parameter provided only partial insights into free radical-mediated CNS neuronal dysfunction. Second, the cases and controls showed a large difference in male versus female recruitment, which may lead to a possible gender bias in the statistical analysis due to the imbalance in the ratio of males/females in the schizophrenia group (8×) and control group (3×). Although we statistically adjusted for this gender difference in assessing TAS levels and cognitive performance between the patient and control groups, and the data were analyzed by gender stratification, gender was an important association in our findings. However, this gender difference would tend to weaken the worse cognitive performance and lowered TAS levels in schizophrenia patients, since the female patients showed fewer deficits and less decreases in TAS than did the male patients. Nevertheless, further investigations should have a more balanced ratio of male and female subjects in examining TAS levels in schizophrenia. Third, it is still uncertain whether peripheral indices of oxidative stress reflect similar changes in the central nervous system. Fourth, the patient group differed from the control group not only in their illness but also in their medication treatment. Perhaps the medications contributed to the differences in TAS and cognitive function studied in the present study. Fifth, no direct evidence supports a causative linkage between oxidative damage and increased TAS or that OS causes cognitive impairment in schizophrenia. In summary, TAS was decreased in chronic schizophrenia patients, which provides additional evidence that oxidative stress may be involved in the pathophysiology of schizophrenia. These chronic schizophrenia patients also displayed a range of cognitive deficits, and altered TAS was associated with some impaired cognitive domains in these schizophrenia patients. While we hypothesized that oxidative stress constitutes a risk factor for cognitive impairment in schizophrenia, the underlying mechanisms are still unknown and the correlational associations between TAS levels and cognitive impairment were opposite our initial predictions. Whether these data support using antioxidant adjuncts to antipsychotics and/or using augmentation strategies with dietary antioxidants for the improvement of cognitive deficits in schizophrenia remains to be tested. Since we were limited to a chronically ill Chinese population our study will be strengthened with replication in first-episode and drug naïve patients or in another set of samples of a different ancestry, such as Caucasian.

Role of the Funding Source Funding for this study was provided by grants from the Stanley Medical Research Institute (03T-459 and 05T-726), and the Department of Veterans Affairs, VA Senior Research Career Scientist Award (JKY), VISN 16, Mental Illness Research, Education and Clinical Center (MIRECC), United States National Institute of Health K05-DA0454, P50-DA18827 and U01-MH79639. These sources had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. The contents of this article do not represent the views of the VA or the United States Government.

Contributors Xiang Yang Zhang, and Thomas R Kosten were responsible for study design, statistical analysis, manuscript preparation and writing the protocol and the paper. Jeffrey K. Yao, and Therese A Kosten were involved in evolving the ideas and editing the manuscript. Da Chun Chen, Mei Hong Xiu, Wei Tang, Feixue Zhang, LianJing Liu, Yuanling Chen and JiaHong Liu were responsible for clinical data collection and lab experiments. All authors have contributed to and have approved the final manuscript.

X.Y. Zhang et al. / Schizophrenia Research 139 (2012) 66–72 Conflict of interest The authors have no conflicts to disclose. Acknowledgment The authors would like to thank Wu Fang Zhang, Yun Long Tan, Song Chen, Zhi Ren Wang, Bao Hua Zhang, and Gui Gang Yang for all of their hard work and significant contributions toward the study.

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