Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 184–191
ORIGINAL ARTICLE
Associations of oxidative stress status parameters with traditional cardiovascular disease risk factors in patients with schizophrenia
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BOJANA VIDOVIĆ1, ALEKSANDRA STEFANOVIĆ2, SRÐAN MILOVANOVIĆ3,4, BRIŽITA ÐORÐEVIĆ1, JELENA KOTUR-STEVULJEVIĆ2, JASMINA IVANIŠEVIĆ2, MILICA MILJKOVIĆ2 & SLAVICA SPASIĆ2 Departments of 1Bromatology and 2Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, 3Clinic for Psychiatry, Clinical Center of Serbia, Belgrade, and 4Faculty of Medicine, University of Belgrade, Belgrade, Serbia Abstract Background. The purpose of this study was to assess oxidative stress status parameters and their possible associations with traditional cardiovascular risk factors in patients with schizophrenia, as well as their potential for patient-control discrimination. Methods. Fasting glucose, lipid profile and oxidative stress status parameters were assessed in 30 schizophrenic patients with atypical antipsychotic therapy and 60 control subjects. Results. Malondialdehyde (MDA), pro-oxidant/antioxidant balance (PAB) and total anti-oxidant status (TAS) were significantly higher whereas total sulfhydryl (SH) groups were significantly lower in schizophrenic patients vs. control group. Higher serum PAB values showed an independent association with schizophrenia. The addition of PAB to conventional risk factors improved discrimination between healthy control subjects and patients. Conclusion. Increased oxidative stress and changed lipid profile parameters are associated in schizophrenic patients and may indicate risk for atherosclerosis. The serum PAB level may reflect the levels of oxidative stress in schizophrenia and improve discrimination of patients from controls. Key Words: Overweight, cardiovascular disease, antioxidative capacity, lipids, antipsychotic treatment Abbreviations: CVD, cardiovascular disease; ROS, reactive oxygen species; t-C, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; TG, triglycerides; Apo A-I, apolipoprotein A-I; Apo B, apolipoprotein B; MDA, malondialdehyde; TBARS, thiobarbituric acid-reactive substances; SOD, superoxide dismutase; SH, sulfhydryl groups; DTNB, 5,5′-dithiobis (2-nitrobenzoic acid); TAS, total anti-oxidant status; ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid; PAB, pro-oxidant/antioxidant balance; ROC, receiver operating characteristic curves; AUC, area under the curve.
Introduction Schizophrenia is a severe psychiatric disorder characterized by psychotic symptoms, such as hallucinations and delusions, and by deficits in normal cognitive, emotional, and social functioning [1]. Patients with schizophrenia have a reduced life expectancy and increased rates of physical illness compared with the general population [2]. Genetic and lifestyle factors (poor diet, sedentary lifestyle and smoking) as well as disease-specific and antipsychotic treatment contribute to a higher prevalence of cardiovascular disease (CVD) in schizophrenia [3]. Major traditional risk factors for CVD
development, such as obesity, smoking, diabetes, hypertension and altered lipid profile parameters are more common among patients with schizophrenia than in the general population [4]. Oxidative stress defined as an imbalance between the formation of reactive oxygen species (ROS) and clearance of ROS by components of the antioxidant defense system [5], has been identified as a possible element in the pathophysiology of schizophrenia [6]. Impaired antioxidant enzyme activities, reduced levels of antioxidants and increased lipid peroxidation have been reported in drug-naive, first episode
Correspondence: Bojana Vidović, Department of Bromatology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, P. Box 146, 11000 Belgrade, Serbia. Tel: ⫹ 381 11 3951395. Fax: ⫹ 381 11 3972840. E-mail: [email protected] (Received 30 October 2013 ; accepted 7 December 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.873947
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Oxidative stress in schizophrenia and chronically-medicated schizophrenic patients [7]. Accumulating evidence suggests that many inter-related mechanisms increase the production of ROS and/or decrease antioxidant defense in schizophrenic patients [8]. In addition, oxidative stress plays a significant role in the pathogenesis of atherosclerosis-related conditions including CVD [9]. However, there are a limited number of studies examining the relationship between oxidative stress markers and traditional CVD risk factors in patients with schizophrenia. The objectives of this study were to determine the presence of conventional CVD risk factors and evaluate the oxidative stress status, as well as possible associations amongst them in schizophrenic patients. Furthermore, we wanted to explore the possibility of oxidative stress parameters being useful markers for discriminating patients from controls.
Materials and methods Subjects Thirty chronically medicated and stabilized patients diagnosed with schizophrenia according to International Classification of Diseases (ICD-10) criteria (World Health Organization [WHO] 1992) were enrolled from the outpatients’ treatment unit of the Clinic for Psychiatry, Clinical Center of Serbia in Belgrade. All patients were treated with atypical antipsychotic medication. Patients treated with other antipsychotics, combinations of antipsychotic drugs or mood stabilizers were excluded from the study. Control subject (n ⫽ 60) consisted of volunteers who were recruited from the general population and academic community. Both patients and control subjects had similar socioeconomic status and dietary patterns. A complete medical history including physical examination and laboratory tests was obtained from all subjects. None of the subjects included in the study had a history of substance abuse or dependence, serious medical conditions, severe head injury or seizure disorders. None of the control subjects presented a personal or family history of a psychiatric disorder. No participants were being treated with antioxidant supplementation or lipid-lowering drugs. All subjects gave signed informed consent to participate in the study protocol that was conducted in accordance with the Helsinki declaration and was approved by the Ethical Committee of the Clinical Center of Serbia in Belgrade. Sample collection Two samples (each of 10 mL) of venous blood were drawn from the antecubital vein after nighttime fasting (⬎ 10 h). The blood was collected into one EDTA sample tube (for plasma) and one sample tube with separator gel (for serum) before immediate
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centrifugation at 1500 g for 10 min at 4°C. Plasma and serum samples were stored at ⫺80°C in aliquots for up to one month until analysis. Biochemical parameters Fasting glucose and lipid status parameters [total cholesterol (t-C), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), triglycerides (TG), apolipoprotein AI (Apo AI) and apolipoprotein B (Apo B)] were measured in serum by standard laboratory procedures (ILab 300 ⫹ analyzer, Instrumentation Laboratory, Milan, Italy).
Oxidative stress status parameters Malondialdehyde (MDA) concentrations were measured in serum using the thiobarbituric acidreactive substances (TBARS) assay employing the molar absorption coefficient of 1.56 ⫻ 105 M⫺1cm⫺1 and spectrophotometry at 535 nm, previously published by Girotti et al. [10]. Plasma superoxide dismutase (SOD) activities were measured according to the previously-described method by Misra and Fridovich [11]. One unit of SOD activity is defined as the activity that inhibits the auto-oxidation of adrenalin by 50%. The concentrations of sulfhydryl (SH) groups were determined in plasma using 0.2 mmol/L 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) [12]. DTNB reacts with aliphatic thiols (at pH 9.0) producing 1 mole of p-nitrophenol per mole of thiol. p-Nitrophenol was measured at 412 nm. Total anti-oxidant status (TAS) was determined in serum according to Erel’s method [13]. This assay is based on the discoloration of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS⫹) by antioxidants present in serum. The color change was measured using an ILab 300 Plus autoanalyzer. The reaction is calibrated with Trolox (a water-soluble analogue of vitamin E, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and the TAS value of the samples tested is expressed as μmol Trolox equivalent/L. Prooxidant-antioxidant balance (PAB) was measured according to the previously published method [14], modified in our laboratory. The assay is based on 3,3′,5,5′-tetramethylbenzidine and its cation, used as a redox indicator participating in two simultaneous reactions. The standard solutions were prepared by mixing varying proportions of (0–100%) of 250 μmol/L hydrogen peroxide with 10 mmol/L uric acid. The intra-assay and inter-assay coefficients of variation were 6.6% and 7.2%, respectively. Serum PAB was not affected by storage at 4°C for 1 day, or for 1 week at ⫺20°C. PAB is expressed in arbitrary HK units, which represent the percentage of hydrogen peroxide in the standard solution.
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Statistical analysis Data were expressed as mean and standard deviation for normally-distributed variables, and as geometric mean and 95% confidence interval (95% CI) for log-normal variables. Comparisons of continuous variables were performed using the Student’s t-test or Mann-Whitney U test where appropriate. Analysis of categorical variables was carried out using the Chi-square test for contingency tables. A logarithmic transformation of glucose, TG levels, TG/HDL and MDA was performed because of the skewed distribution in analysis using Student’s t-test analysis [15]. To test association between oxidative stress and antioxidative defense parameters and traditional CVD risk factors we applied Spearman’s correlation analyses in control and patient groups. Binary logistic regression analysis was employed to determine parameters with the ability to discriminate patients from controls. Interpretation of the strength (effect size) of positive relationship was calculated by The d Family testing by Kraemer et al. [16]. The control group was used as the reference group and was coded 0, while the schizophrenic patient group was coded 1. Adjustment analysis was performed to correct the influence of BMI, smoking and lipid profile risk factors (t-C, TG, LDL-C and HDL-C) and oxidative stress status parameters (TAS, PAB, SH). For each odds ratio (OR) two-tailed probability values and the 95% confidence interval (CI) were estimated. Receiver operating characteristic (ROC) curves were constructed with the predictive probabilities from different logistic regression models. Pairwise comparisons using the areas under the ROC curve (AUCs) were also performed. By utilizing the Hosmer and Lemeshow rule for logistic models, the discriminative abilities of the models were classified
according to their AUC values as poor (0.5 ⱕ AUC ⬍ 0.7), acceptable (0.7 ⱕ AUC ⬍ 0.8), excellent (0.8 ⱕ AUC ⬍ 0.9) or outstanding (AUC ⱖ 0.9) [17]. All statistical analysis was performed using SPSS for Windows 11.5 (Chicago, IL, USA) and MedCalc (version 11.4 Software, Belgium) software. All statistical tests were considered significant at the 0.05 probability level.
Results The demographic and biochemical data of the study groups are summarized in Table I. There were no significant differences in gender distribution, age and smoking habits between patients with schizophrenia and control subjects (Table I). The patients were treated with various atypical antipsychotics (mean duration of treatment was 26 months, mean neuroleptic dose in chlorpromazine equivalents was 262 ⫾ 193 mg per day). Nine patients (30%) resaved clozapine, 5 patients (17%) were treated with olanzapine and 16 patients (53%) resaved risperidone. As expected, patients with schizophrenia had an elevated BMI compared with the control group. Significantly higher TG concentrations (p ⫽ 0.043) and apo B (p ⬍ 0.001) were evident in the patient group (Table I). In contrast, their HDL-C was lower (p ⬍ 0.001) compared with control subjects. There were no significant differences between the patients and the controls when glucose, t-C, LDL-C and apo A-I were compared. The atherogenic index of serum (TG/HDL-C), AIS, was significantly higher in schizophrenic patients than in the control group (p ⫽ 0.014). Oxidative stress status parameters of the schizophrenic patients and healthy control group are shown
Table I. Demographic and biochemical characteristic of the study groups. Parameter Gender (M/F) Age (years) BMI (kg/m2) Smokers, n (%) Systolic pressure (mm Hg) Diastolic pressure (mm Hg) Glucose (mmol/L)a t-C (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L) TG (mmol/L)a AIS (TG/HDL-C)a Apo A-I (g/L) Apo B (g/L)
Patients with schizophrenia (n ⫽ 30)
Control group (n ⫽ 60)
p1
13/17 38.4 ⫾ 12.08 27.6 ⫾ 4.18 13 (43%) 115.52 ⫾ 15.32 78.1 ⫾ 8.39 5.28 (4.86–5.73) 5.59 ⫾ 1.19 3.66 ⫾ 0.96 1.15 ⫾ 0.28 1.54 (1.28–1.85) 1.37 (1.09–1.71) 1.44 ⫾ 0.33 1.06 ⫾ 0.29
17/43 38.7 ⫾ 11.13 24.5 ⫾ 3.33 22 (37%) 120.33 ⫾ 13.68 79.5 ⫾ 7.79 5.00 (4.88–5.13) 5.70 ⫾ 1.27 3.79 ⫾ 1.08 1.45 ⫾ 0.35 1.22 (1.09–1.37) 0.86 (0.74–1.00) 1.42 ⫾ 0.24 0.79 ⫾ 0.22
0.236 0.912 ⬍ 0.001 0.702 0.138 0.442 0.108 0.298 0.597 ⬍ 0.001 0.043 0.014 0.825 ⬍ 0.001
Data are expressed as mean ⫾ SD. 1Continuous variables were compared using Student’s t-test and categorical variables by the Chi-square test. aGeometric means and 95% confidence interval for log-normal variables (95% CI).
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Table II. Oxidative stress status parameters of the study groups. Parameter (μmol/L)a
MDA PAB (HUK/L)b TAS (μmol/L) SOD (kU/L)b Total SH groups (mmol/L)
Patients with schizophrenia
Control group
p
0.99 (0.91–1.06) 104.1 (97.27–119.98) 596.69 ⫾ 131.51 28 (25–32) 0.49 ⫾ 0.09
0.83 (0.76–0.90) 49.3 (41.87–67.06) 459.13 ⫾ 161.24 30 (29–32) 0.61 ⫾ 0.12
0.006 ⬍ 0.001 ⬍ 0.001 0.167 ⬍ 0.001
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Data are expressed as mean ⫾ SD. aGeometric means and 95% CI (log-normal variable distribution). bMedian and 95% CI for the median.
in Table II. Lipid peroxidation, measured by serum MDA level, was significantly higher in patients with schizophrenia (Table II). Similarly, the serum level PAB in schizophrenic patients was higher (p ⬍ 0.001) than in control subjects. The slightly decreased activity of SOD seen in patients with schizophrenia was not significantly different from the control group. Unexpectedly, patients showed a significantly higher TAS value (p ⬍ 0.001) than the control group. The concentrations of SH groups was significantly lower (p ⬍ 0.001) in the patients group compared with the controls. Spearman’s correlation analyses were performed to test for associations between oxidative stress parameters and BMI, lipid profile parameters and AIS as potentional CVD risk factors (Table III). Antioxidative enzyme activity (SOD) was significantly positively correlated with t-C and LDL-C concentrations in controls, while in patients SOD was significantly positively correlated with t-C and triglyceride concentrations (Table III). The oxidative stress parameter MDA concentration showed significant positive correlations with t-C and LDL-C concentrations only in the controls (Table III). Also, PAB was not significantly correlated with any parameter in either group (Table III). We also performed correlation analyses to test possible association between PAB and other oxidative stress status parameters in patients and controls. PAB was significantly positively correlated with MDA (ρ ⫽ 0.312; p ⫽ 0.012) and significantly negatively correlated with antioxidant enzyme SOD (ρ ⫽ ⫺0.382; p ⫽ 0.037) in patients. No significant correlations were seen in the controls. Binary logistic regression was used to determine whether the measurement of oxidative stress status parameters, which was significantly different between patients with schizophrenia and the control group, had the potential to discriminate patients with schizophrenia from control subjects. The control group was used as the reference group. Unadjusted analysis indicated that oxidative stress status parameters: TAS, SH, PAB had a strong potential to separate patients with schizophrenia from control subjects [TAS: OR (95% CI) ⫽ 0.992 (0.987–0.996), p ⬍ 0.001; PAB: OR (95% CI) ⫽ 0.995
(0.992–0.997), p ⬍ 0.001; SH groups: OR (95% CI) ⫽ 1.761 (0.957–3.240), p ⬍ 0.001]. Following this analysis new logistic regression models were constructed to further test the potential independent association of oxidative stress status parameters with schizophrenia. Serum PAB was the parameter with the strongest ability to separate the patients from controls. Calculated d values for PAB was 0.61. If d Family was between 0.60 and 0.70 then general interpretation of the strength of a relationship could be defined as ‘typical to larger than typical’. The models incorporated adjustments of PAB for conventional cardiovascular risk factors (Model 1) and adjustments of PAB for traditional cardiovascular risk factors with other oxidative stress status parameters (Model 2). The observed discriminative powers of TAS and SH groups were lost after taking into account the conventional CVD risk factors (BMI, smoking, lipid status). In contrast, the association between the level of PAB and schizophrenia remained strong, regardless of the confounding variable. It proved itself to be a potential marker for the discrimination between schizophrenia patients and healthy controls (Table IV). We also investigated the potential benefit of adding PAB measurement to traditional risk factors to discriminate better the schizophrenia patients from healthy subjects. To achieve this, ROC curves were constructed with predictive probabilities from traditional risk factors [see aforementioned logistic regression model (Model 1) with and without PAB]. The addition of PAB significantly increased the AUC for traditional risk factors (p ⫽ 0.008) (Table V, Figure 1). Discussion The results obtained in this study are in agreement with the findings of previous studies which state that traditional CVD risk factors are highly prevalent in schizophrenic patients [3]. In the present study, we observed the presence of a higher percentage of overweight individuals and changed lipid profile parameters towards an atherogenic profile in the patients group (Table I). These results are not unexpected because it is known that metabolic syndrome
0.864 0.210 0.979 0.590 0.218 0.837 0.600 0.211 0.033 ⫺0.240 0.005 ⫺0.102 ⫺0.232 0.039 ⫺0.100 ⫺0.235 ⫺0.119 0.001 ⫺0.187 ⫺0.241 ⫺0.073 ⫺0.037 ⫺0.234 ⫺0.091 ⫺0.14 0.525 0.835 0.827 0.917 0.171 0.936 0.677 0.456 ⫺0.12 0.040 ⫺0.04 ⫺0.02 ⫺0.26 ⫺0.01 0.079 0.926 0.681 0.038 0.046 0.449 0.926 0.025 0.544 0.012 ⫺0.05 0.268 0.259 0.100 0.012 0.294 0.081 0.654 0.409 0.775 0.409 0.112 0.463 0.182 0.113 ⫺0.08 0.159 0.054 ⫺0.16 0.296 ⫺0.14 ⫺0.25 0.296 0.066 0.270 0.002 0.021 0.308 0.675 0.023 0.359 0.259 0.156 0.421 0.319 0.144 ⫺0.06 0.320 0.132 0.505 0.112 0.625 0.033 0.575 0.097 0.022 0.823 0.795 0.497 0.973 0.527 0.326 0.273 0.345 0.286
Table IV. Logistic regression analysis for the association of prooxidant/antioxidant balance (PAB) with schizophrenia (reference is control group).
Unadjusted PAB Model 1 Model 2
BMI Systolic pressure Glucose t-C TG HDL-C LDL-C AIS
OR
95% CI
p
0.995 0.993 0.994
0.992–0.997 0.989–0.997 0.989–0.999
⬍ 0.001 0.001 0.018
Model 1, adjusted for BMI, smoking, t-C, TG, LDL-C and HDL-C. Model 2, adjusted for BMI, smoking, t-C, TG, LDL-C, HDL-C, TAS, SH-group and MDA.
and other cardiovascular risk factors are highly prevalent in patients with schizophrenia [4]. Overweight/ obesity is associated with lifestyle factors (e.g. lack of exercise, poor diet), but also with illness-related (negative, disorganized and depressive symptoms) and treatment-related factors [3]. Weight gain is also an established side-effect of most antipsychotic drugs [18]. Higher triglycerides and decreased HDL-C were also seen in the patients group (Table I). This type of atherogenic dyslipidemia is a well-established risk factor for atherosclerosis development [19]. Although it may not be the main cause, oxidative stress has been suggested to contribute to the pathogenesis and progression of schizophrenia [20,21]. In particular, oxidative damage to lipids, proteins, and DNA as observed in schizophrenia is known to impair cell viability and function, which may subsequently account for the outcome of this pathophysiological condition. The brain is particularly vulnerable to oxidative stress as a result of high rates of oxidative metabolic activity, relatively low level of antioxidants, high levels of oxidizable polyunsaturated fatty acids and high iron content, which plays a role in generating ROS [22]. However, there are limited data on oxidative processes in cerebrospinal fluid and the brain [20]. Most measurements of oxidative stress in patients with schizophrenia have been made on peripheral tissues. But there is speculation that a peripheral indicator may not necessarily reflect the conditions of the oxidative stress parameters in the brain [23]. These factors led us to determine the oxidative stress status parameters in serum and plasma (peripheral tissues) and to establish some association between the general state of oxidative stress and atherogenic dyslipidemia and Table V. The results of receiver operating characteristic (ROC) analysis for discriminating subjects with schizophrenia from the control group.
0.124 0.128 0.189 0.408 ⫺0.06 0.334 0.403 ⫺0.14
0.372 0.217 0.35 0.214 0.167 ⫺0.01 0.002 ⫺0.44 0.665 0.504 0.015 0.258 0.003 0.283 0.300 0.332
0.249 0.265 0.939 0.014 0.004 0.168 0.130 0.073
⫺0.04 ⫺0.09 0.005 0.086 ⫺0.13 0.150 0.130 ⫺0.15
0.129 0.307 0.095 0.396 0.109 0.314 0.423 ⫺0.04
ρ p ρ p ρ p ρ p ρ p ρ ρ p ρ
Controls
p
Controls Patients
Patients
0.404 0.992 0.188 0.088 0.613 0.801 0.106 0.532
p ρ p ρ p
Controls Patients Controls Patients Controls
MDA (μmol/L) Total SH groups (mmol/L) TAS (μmol/L) SOD (kU/L)
Table III. Correlation between antioxidative defense and oxidative stress parameters with conventional CVD risk factors parameters in patients and controls.
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PAB ( HUK/L)
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Model 1 Model 2
AUC
Confidence interval
Std. error
0.821 0.915a,∗
0.718–0.899 0.830–0.966
0.049 0.033
Model 1 – BMI, smoking, t-C, TG, LDL-C and HDL-C. Model 2 – BMI, smoking, t-C, TG, LDL-C, HDL-C and PAB. ∗p ⫽ 0.008. ap values for testing the differences in AUCs between Model 1 and the corresponding additional model, Model 2.
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Oxidative stress in schizophrenia
Figure 1. Comparison of ROCs of Model 1 and Model 2 for discriminating subjects with schizophrenia from the control group. Model 1 – BMI, smoking, t-C, TG, LDL-C and HDL-C. Model 2 – BMI, smoking, t-C, TG, LDL-C, HDL-C and PAB.
overweight which could also be accompanied by increased oxidative stress [9]. Previous studies, that mainly focused on the measurement of peripheral markers of oxidative stress status, have suggested that schizophrenia increases the level of oxidative stress and impaired antioxidative defense, though not consistently [6,20]. Differences in results may be attributed to different clinical symptoms, therapeutic features or duration of the illness [24]. Several factors, such as differences in measurement methods and types of biological materials (red blood cells vs. plasma vs. serum) as well as lifestyle (diet, physical inactivity, levels of smoking) can contribute to the heterogeneity of the results [25,26]. MDA is the final product of lipid peroxidation and is presented as the most commonly-used marker of oxidative stress in schizophrenia [20,21]. Padurariu et al. [27] have demonstrated that lipid peroxidation is positively correlated with the severity of symptoms and inversely with levels of membrane polyunsaturated fatty acids. The result of this study is in agreement with results of the metaanalysis published by Zhang et al. [6] that showed significantly higher levels of MDA in patients with schizophrenia. The antioxidative defense system includes enzymatic and non-enzymatic antioxidants [28]. SOD is a critical antioxidant enzyme responsible for the elimination of superoxide radicals converting them into hydrogen peroxide and molecular oxygen [29]. A decrease in the activity of plasma SOD in the schizophrenic group in comparison to the control group was observed in the present study, but this was not statistically significant. The fact that SOD activity did not change significantly in patients with
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schizophrenia, despite increased lipid peroxidation, may be due to compensative increases in the activity of other antioxidants. Non-enzymatic antioxidant status was studied by estimating TAS and total SH group concentrations. There are several antioxidants, such as proteins, uric acid and ascorbic acid which account for ⬎ 90% of the total antioxidant capacity in human serum [13]. Protein-bound SH groups are very susceptible to oxidation and several studies indicate that levels of protein–SH are correlated negatively with markers of lipid peroxidation and markers of protein oxidative damage [30]. Several studies reported that serum-free SH groups in patients with schizophrenia were significantly lower than in control groups [31,32]. Data from our study showed that levels of total SH groups were significantly lower in the plasma of patients with schizophrenia than those of controls. However, the levels of serum TAS in schizophrenic patients were significantly higher than in the control group (Table II). These findings may be explained as a possible consequence of increased uric acid, which has been reported in schizophrenic patients on atypical antipsychotics treatment [33,34]. Without measuring these parameters this remains a hypothesis. Our study appears to be the first to investigate PAB as an oxidative stress marker in schizophrenia. This method for PAB determination is the first that can measure the balance of oxidants and antioxidants simultaneously in one experiment. This is one of the main advantages of the PAB method in comparison with other assays. It has been calibrated against the most significant known oxidants and antioxidants (mixtures of hydrogen peroxide and uric acid) [14]. In the original paper describing the PAB method [14], results had been compared with widely-utilized and documented methods, ‘gold standards methods’ and showed a great level of similarity with them. Results of previous studies showed that PAB was significantly-correlated with oxidative stress-related assays [14], so PAB may present an adequate measure of oxidative damage. In the present study, it was found that the level of PAB in patients with schizophrenia was significantly higher when compared with control subjects. This huge increase in PAB level in the patients group could be a consequence of the presence of several components of metabolic syndrome in every patient. If we consider the fact that metabolic syndrome and other cardiovascular risk factors are highly prevalent in people with schizophrenia [18], it is possible that increased PAB levels and generally increased oxidative stress in these patients could be the consequence of the presence of metabolic abnormalities. This result is in agreement with the study by Korkamaz et al. [35] who investigated the relationship between conventional risk factors in the metabolic syndrome (hypertension, hyperglycemia,
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obesity...) and PAB levels. The result of Korkamaz study [35] indicates that high PAB levels were both highly sensitive and highly specific for metabolic syndrome. Similar results have been observed in other oxidative stress-related diseases [14,36]. We found that PAB was significantly negatively correlated with antioxidant enzyme SOD and positively correlated with MDA in the patients group while those correlations were not seen in the controls. Those results could be explained by the fact that in the presence of disease, a significant increase in PAB levels is accompanied by a considerable increase in the production of final products of lipid peroxidation and decrease of antioxidant defense. Notably, all our patients were on atypical antipsychotic therapy because some authors reported that chronic administration of typical antipsychotics induces higher oxidative stress by decreasing the activity of antioxidant enzymes and a higher production of lipid peroxidation products [37]. However, another study [38] reported that there is actually no difference in oxidative stress status parameters between patients treated with typical and atypical antipsychotics. According to these authors antipsychotic drugs can normalize the abnormal free radical metabolism in schizophrenia; their pharmacological mechanisms are different but end point effects on oxidative stress could be the same. To further investigate the independent possibility of PAB to discriminate patients from controls we preformed adjustment binary logistic regression analyses (Table IV). The discriminative potential remained significant even after adjustment for conventional risk factors (BMI, smoking and lipid status) as well as after adjustment for combinations of the conventional risk factors and oxidative stress status parameters (MDA, TAS, SH) (Table IV). With this result, we demonstrated an independent association of PAB with the presence of schizophrenia. The logical continuation of our study was to determine the ability of PAB to discriminate between patients and controls since this is the fundamental property of any diagnostic test or indicative system. ROC curve analysis (Table V, Figure 1) revealed that PAB measurement was highly accurate. However, Model 2 that incorporated PAB with traditional CVD risk factors (Table V, Figure 1) demonstrated a significant increase in the clinical accuracy over the model that included measurements of traditional CVD risk factors alone (Model 1). According to these results we suggest that high PAB levels could be a potent parameter for discrimination of schizophrenia patients from controls. The combination of PAB with traditional CVD risk factors facilitates the discrimination of subjects with schizophrenia. ROC curve analysis revealed that the combination of PAB with conventional risk factors (Model 2) had outstanding clinical accuracy
(AUC ⱖ 0.9) and the ability to discriminate patients from control subjects. Because of the cross-sectional design of the study, conclusions concerning the causal relationship between higher PAB levels and the presence of schizophrenia cannot be drawn. In addition, the study sample size was relatively small which could limit the generalization of our results. Also, the limitation of our study is that the patients’ psychiatric status is not reported. Prospective studies including large numbers of patients are needed to elucidate the relationship between PAB and schizophrenia as well as psychopathological state and to assess its possible diagnostic and prognostic value. In conclusion, elevated oxidative stress together with raised BMI and lipid status abnormalities were observed in patients with schizophrenia. The serum PAB level may help to discriminate patients from the control group. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was financially supported by grants from the Ministry of Education, Science and Technological Development, Republic of Serbia (project number 175035 and III 46001).
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ORIGINAL ARTICLE
Associations of oxidative stress status parameters with traditional cardiovascular disease risk factors in patients with schizophrenia
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BOJANA VIDOVIĆ1, ALEKSANDRA STEFANOVIĆ2, SRÐAN MILOVANOVIĆ3,4, BRIŽITA ÐORÐEVIĆ1, JELENA KOTUR-STEVULJEVIĆ2, JASMINA IVANIŠEVIĆ2, MILICA MILJKOVIĆ2 & SLAVICA SPASIĆ2 Departments of 1Bromatology and 2Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, 3Clinic for Psychiatry, Clinical Center of Serbia, Belgrade, and 4Faculty of Medicine, University of Belgrade, Belgrade, Serbia Abstract Background. The purpose of this study was to assess oxidative stress status parameters and their possible associations with traditional cardiovascular risk factors in patients with schizophrenia, as well as their potential for patient-control discrimination. Methods. Fasting glucose, lipid profile and oxidative stress status parameters were assessed in 30 schizophrenic patients with atypical antipsychotic therapy and 60 control subjects. Results. Malondialdehyde (MDA), pro-oxidant/antioxidant balance (PAB) and total anti-oxidant status (TAS) were significantly higher whereas total sulfhydryl (SH) groups were significantly lower in schizophrenic patients vs. control group. Higher serum PAB values showed an independent association with schizophrenia. The addition of PAB to conventional risk factors improved discrimination between healthy control subjects and patients. Conclusion. Increased oxidative stress and changed lipid profile parameters are associated in schizophrenic patients and may indicate risk for atherosclerosis. The serum PAB level may reflect the levels of oxidative stress in schizophrenia and improve discrimination of patients from controls. Key Words: Overweight, cardiovascular disease, antioxidative capacity, lipids, antipsychotic treatment Abbreviations: CVD, cardiovascular disease; ROS, reactive oxygen species; t-C, total cholesterol; LDL-C, LDL-cholesterol; HDL-C, HDL-cholesterol; TG, triglycerides; Apo A-I, apolipoprotein A-I; Apo B, apolipoprotein B; MDA, malondialdehyde; TBARS, thiobarbituric acid-reactive substances; SOD, superoxide dismutase; SH, sulfhydryl groups; DTNB, 5,5′-dithiobis (2-nitrobenzoic acid); TAS, total anti-oxidant status; ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid; PAB, pro-oxidant/antioxidant balance; ROC, receiver operating characteristic curves; AUC, area under the curve.
Introduction Schizophrenia is a severe psychiatric disorder characterized by psychotic symptoms, such as hallucinations and delusions, and by deficits in normal cognitive, emotional, and social functioning [1]. Patients with schizophrenia have a reduced life expectancy and increased rates of physical illness compared with the general population [2]. Genetic and lifestyle factors (poor diet, sedentary lifestyle and smoking) as well as disease-specific and antipsychotic treatment contribute to a higher prevalence of cardiovascular disease (CVD) in schizophrenia [3]. Major traditional risk factors for CVD
development, such as obesity, smoking, diabetes, hypertension and altered lipid profile parameters are more common among patients with schizophrenia than in the general population [4]. Oxidative stress defined as an imbalance between the formation of reactive oxygen species (ROS) and clearance of ROS by components of the antioxidant defense system [5], has been identified as a possible element in the pathophysiology of schizophrenia [6]. Impaired antioxidant enzyme activities, reduced levels of antioxidants and increased lipid peroxidation have been reported in drug-naive, first episode
Correspondence: Bojana Vidović, Department of Bromatology, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, P. Box 146, 11000 Belgrade, Serbia. Tel: ⫹ 381 11 3951395. Fax: ⫹ 381 11 3972840. E-mail: [email protected] (Received 30 October 2013 ; accepted 7 December 2013) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2013.873947
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Oxidative stress in schizophrenia and chronically-medicated schizophrenic patients [7]. Accumulating evidence suggests that many inter-related mechanisms increase the production of ROS and/or decrease antioxidant defense in schizophrenic patients [8]. In addition, oxidative stress plays a significant role in the pathogenesis of atherosclerosis-related conditions including CVD [9]. However, there are a limited number of studies examining the relationship between oxidative stress markers and traditional CVD risk factors in patients with schizophrenia. The objectives of this study were to determine the presence of conventional CVD risk factors and evaluate the oxidative stress status, as well as possible associations amongst them in schizophrenic patients. Furthermore, we wanted to explore the possibility of oxidative stress parameters being useful markers for discriminating patients from controls.
Materials and methods Subjects Thirty chronically medicated and stabilized patients diagnosed with schizophrenia according to International Classification of Diseases (ICD-10) criteria (World Health Organization [WHO] 1992) were enrolled from the outpatients’ treatment unit of the Clinic for Psychiatry, Clinical Center of Serbia in Belgrade. All patients were treated with atypical antipsychotic medication. Patients treated with other antipsychotics, combinations of antipsychotic drugs or mood stabilizers were excluded from the study. Control subject (n ⫽ 60) consisted of volunteers who were recruited from the general population and academic community. Both patients and control subjects had similar socioeconomic status and dietary patterns. A complete medical history including physical examination and laboratory tests was obtained from all subjects. None of the subjects included in the study had a history of substance abuse or dependence, serious medical conditions, severe head injury or seizure disorders. None of the control subjects presented a personal or family history of a psychiatric disorder. No participants were being treated with antioxidant supplementation or lipid-lowering drugs. All subjects gave signed informed consent to participate in the study protocol that was conducted in accordance with the Helsinki declaration and was approved by the Ethical Committee of the Clinical Center of Serbia in Belgrade. Sample collection Two samples (each of 10 mL) of venous blood were drawn from the antecubital vein after nighttime fasting (⬎ 10 h). The blood was collected into one EDTA sample tube (for plasma) and one sample tube with separator gel (for serum) before immediate
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centrifugation at 1500 g for 10 min at 4°C. Plasma and serum samples were stored at ⫺80°C in aliquots for up to one month until analysis. Biochemical parameters Fasting glucose and lipid status parameters [total cholesterol (t-C), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), triglycerides (TG), apolipoprotein AI (Apo AI) and apolipoprotein B (Apo B)] were measured in serum by standard laboratory procedures (ILab 300 ⫹ analyzer, Instrumentation Laboratory, Milan, Italy).
Oxidative stress status parameters Malondialdehyde (MDA) concentrations were measured in serum using the thiobarbituric acidreactive substances (TBARS) assay employing the molar absorption coefficient of 1.56 ⫻ 105 M⫺1cm⫺1 and spectrophotometry at 535 nm, previously published by Girotti et al. [10]. Plasma superoxide dismutase (SOD) activities were measured according to the previously-described method by Misra and Fridovich [11]. One unit of SOD activity is defined as the activity that inhibits the auto-oxidation of adrenalin by 50%. The concentrations of sulfhydryl (SH) groups were determined in plasma using 0.2 mmol/L 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) [12]. DTNB reacts with aliphatic thiols (at pH 9.0) producing 1 mole of p-nitrophenol per mole of thiol. p-Nitrophenol was measured at 412 nm. Total anti-oxidant status (TAS) was determined in serum according to Erel’s method [13]. This assay is based on the discoloration of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS⫹) by antioxidants present in serum. The color change was measured using an ILab 300 Plus autoanalyzer. The reaction is calibrated with Trolox (a water-soluble analogue of vitamin E, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and the TAS value of the samples tested is expressed as μmol Trolox equivalent/L. Prooxidant-antioxidant balance (PAB) was measured according to the previously published method [14], modified in our laboratory. The assay is based on 3,3′,5,5′-tetramethylbenzidine and its cation, used as a redox indicator participating in two simultaneous reactions. The standard solutions were prepared by mixing varying proportions of (0–100%) of 250 μmol/L hydrogen peroxide with 10 mmol/L uric acid. The intra-assay and inter-assay coefficients of variation were 6.6% and 7.2%, respectively. Serum PAB was not affected by storage at 4°C for 1 day, or for 1 week at ⫺20°C. PAB is expressed in arbitrary HK units, which represent the percentage of hydrogen peroxide in the standard solution.
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Statistical analysis Data were expressed as mean and standard deviation for normally-distributed variables, and as geometric mean and 95% confidence interval (95% CI) for log-normal variables. Comparisons of continuous variables were performed using the Student’s t-test or Mann-Whitney U test where appropriate. Analysis of categorical variables was carried out using the Chi-square test for contingency tables. A logarithmic transformation of glucose, TG levels, TG/HDL and MDA was performed because of the skewed distribution in analysis using Student’s t-test analysis [15]. To test association between oxidative stress and antioxidative defense parameters and traditional CVD risk factors we applied Spearman’s correlation analyses in control and patient groups. Binary logistic regression analysis was employed to determine parameters with the ability to discriminate patients from controls. Interpretation of the strength (effect size) of positive relationship was calculated by The d Family testing by Kraemer et al. [16]. The control group was used as the reference group and was coded 0, while the schizophrenic patient group was coded 1. Adjustment analysis was performed to correct the influence of BMI, smoking and lipid profile risk factors (t-C, TG, LDL-C and HDL-C) and oxidative stress status parameters (TAS, PAB, SH). For each odds ratio (OR) two-tailed probability values and the 95% confidence interval (CI) were estimated. Receiver operating characteristic (ROC) curves were constructed with the predictive probabilities from different logistic regression models. Pairwise comparisons using the areas under the ROC curve (AUCs) were also performed. By utilizing the Hosmer and Lemeshow rule for logistic models, the discriminative abilities of the models were classified
according to their AUC values as poor (0.5 ⱕ AUC ⬍ 0.7), acceptable (0.7 ⱕ AUC ⬍ 0.8), excellent (0.8 ⱕ AUC ⬍ 0.9) or outstanding (AUC ⱖ 0.9) [17]. All statistical analysis was performed using SPSS for Windows 11.5 (Chicago, IL, USA) and MedCalc (version 11.4 Software, Belgium) software. All statistical tests were considered significant at the 0.05 probability level.
Results The demographic and biochemical data of the study groups are summarized in Table I. There were no significant differences in gender distribution, age and smoking habits between patients with schizophrenia and control subjects (Table I). The patients were treated with various atypical antipsychotics (mean duration of treatment was 26 months, mean neuroleptic dose in chlorpromazine equivalents was 262 ⫾ 193 mg per day). Nine patients (30%) resaved clozapine, 5 patients (17%) were treated with olanzapine and 16 patients (53%) resaved risperidone. As expected, patients with schizophrenia had an elevated BMI compared with the control group. Significantly higher TG concentrations (p ⫽ 0.043) and apo B (p ⬍ 0.001) were evident in the patient group (Table I). In contrast, their HDL-C was lower (p ⬍ 0.001) compared with control subjects. There were no significant differences between the patients and the controls when glucose, t-C, LDL-C and apo A-I were compared. The atherogenic index of serum (TG/HDL-C), AIS, was significantly higher in schizophrenic patients than in the control group (p ⫽ 0.014). Oxidative stress status parameters of the schizophrenic patients and healthy control group are shown
Table I. Demographic and biochemical characteristic of the study groups. Parameter Gender (M/F) Age (years) BMI (kg/m2) Smokers, n (%) Systolic pressure (mm Hg) Diastolic pressure (mm Hg) Glucose (mmol/L)a t-C (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L) TG (mmol/L)a AIS (TG/HDL-C)a Apo A-I (g/L) Apo B (g/L)
Patients with schizophrenia (n ⫽ 30)
Control group (n ⫽ 60)
p1
13/17 38.4 ⫾ 12.08 27.6 ⫾ 4.18 13 (43%) 115.52 ⫾ 15.32 78.1 ⫾ 8.39 5.28 (4.86–5.73) 5.59 ⫾ 1.19 3.66 ⫾ 0.96 1.15 ⫾ 0.28 1.54 (1.28–1.85) 1.37 (1.09–1.71) 1.44 ⫾ 0.33 1.06 ⫾ 0.29
17/43 38.7 ⫾ 11.13 24.5 ⫾ 3.33 22 (37%) 120.33 ⫾ 13.68 79.5 ⫾ 7.79 5.00 (4.88–5.13) 5.70 ⫾ 1.27 3.79 ⫾ 1.08 1.45 ⫾ 0.35 1.22 (1.09–1.37) 0.86 (0.74–1.00) 1.42 ⫾ 0.24 0.79 ⫾ 0.22
0.236 0.912 ⬍ 0.001 0.702 0.138 0.442 0.108 0.298 0.597 ⬍ 0.001 0.043 0.014 0.825 ⬍ 0.001
Data are expressed as mean ⫾ SD. 1Continuous variables were compared using Student’s t-test and categorical variables by the Chi-square test. aGeometric means and 95% confidence interval for log-normal variables (95% CI).
Oxidative stress in schizophrenia
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Table II. Oxidative stress status parameters of the study groups. Parameter (μmol/L)a
MDA PAB (HUK/L)b TAS (μmol/L) SOD (kU/L)b Total SH groups (mmol/L)
Patients with schizophrenia
Control group
p
0.99 (0.91–1.06) 104.1 (97.27–119.98) 596.69 ⫾ 131.51 28 (25–32) 0.49 ⫾ 0.09
0.83 (0.76–0.90) 49.3 (41.87–67.06) 459.13 ⫾ 161.24 30 (29–32) 0.61 ⫾ 0.12
0.006 ⬍ 0.001 ⬍ 0.001 0.167 ⬍ 0.001
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Data are expressed as mean ⫾ SD. aGeometric means and 95% CI (log-normal variable distribution). bMedian and 95% CI for the median.
in Table II. Lipid peroxidation, measured by serum MDA level, was significantly higher in patients with schizophrenia (Table II). Similarly, the serum level PAB in schizophrenic patients was higher (p ⬍ 0.001) than in control subjects. The slightly decreased activity of SOD seen in patients with schizophrenia was not significantly different from the control group. Unexpectedly, patients showed a significantly higher TAS value (p ⬍ 0.001) than the control group. The concentrations of SH groups was significantly lower (p ⬍ 0.001) in the patients group compared with the controls. Spearman’s correlation analyses were performed to test for associations between oxidative stress parameters and BMI, lipid profile parameters and AIS as potentional CVD risk factors (Table III). Antioxidative enzyme activity (SOD) was significantly positively correlated with t-C and LDL-C concentrations in controls, while in patients SOD was significantly positively correlated with t-C and triglyceride concentrations (Table III). The oxidative stress parameter MDA concentration showed significant positive correlations with t-C and LDL-C concentrations only in the controls (Table III). Also, PAB was not significantly correlated with any parameter in either group (Table III). We also performed correlation analyses to test possible association between PAB and other oxidative stress status parameters in patients and controls. PAB was significantly positively correlated with MDA (ρ ⫽ 0.312; p ⫽ 0.012) and significantly negatively correlated with antioxidant enzyme SOD (ρ ⫽ ⫺0.382; p ⫽ 0.037) in patients. No significant correlations were seen in the controls. Binary logistic regression was used to determine whether the measurement of oxidative stress status parameters, which was significantly different between patients with schizophrenia and the control group, had the potential to discriminate patients with schizophrenia from control subjects. The control group was used as the reference group. Unadjusted analysis indicated that oxidative stress status parameters: TAS, SH, PAB had a strong potential to separate patients with schizophrenia from control subjects [TAS: OR (95% CI) ⫽ 0.992 (0.987–0.996), p ⬍ 0.001; PAB: OR (95% CI) ⫽ 0.995
(0.992–0.997), p ⬍ 0.001; SH groups: OR (95% CI) ⫽ 1.761 (0.957–3.240), p ⬍ 0.001]. Following this analysis new logistic regression models were constructed to further test the potential independent association of oxidative stress status parameters with schizophrenia. Serum PAB was the parameter with the strongest ability to separate the patients from controls. Calculated d values for PAB was 0.61. If d Family was between 0.60 and 0.70 then general interpretation of the strength of a relationship could be defined as ‘typical to larger than typical’. The models incorporated adjustments of PAB for conventional cardiovascular risk factors (Model 1) and adjustments of PAB for traditional cardiovascular risk factors with other oxidative stress status parameters (Model 2). The observed discriminative powers of TAS and SH groups were lost after taking into account the conventional CVD risk factors (BMI, smoking, lipid status). In contrast, the association between the level of PAB and schizophrenia remained strong, regardless of the confounding variable. It proved itself to be a potential marker for the discrimination between schizophrenia patients and healthy controls (Table IV). We also investigated the potential benefit of adding PAB measurement to traditional risk factors to discriminate better the schizophrenia patients from healthy subjects. To achieve this, ROC curves were constructed with predictive probabilities from traditional risk factors [see aforementioned logistic regression model (Model 1) with and without PAB]. The addition of PAB significantly increased the AUC for traditional risk factors (p ⫽ 0.008) (Table V, Figure 1). Discussion The results obtained in this study are in agreement with the findings of previous studies which state that traditional CVD risk factors are highly prevalent in schizophrenic patients [3]. In the present study, we observed the presence of a higher percentage of overweight individuals and changed lipid profile parameters towards an atherogenic profile in the patients group (Table I). These results are not unexpected because it is known that metabolic syndrome
0.864 0.210 0.979 0.590 0.218 0.837 0.600 0.211 0.033 ⫺0.240 0.005 ⫺0.102 ⫺0.232 0.039 ⫺0.100 ⫺0.235 ⫺0.119 0.001 ⫺0.187 ⫺0.241 ⫺0.073 ⫺0.037 ⫺0.234 ⫺0.091 ⫺0.14 0.525 0.835 0.827 0.917 0.171 0.936 0.677 0.456 ⫺0.12 0.040 ⫺0.04 ⫺0.02 ⫺0.26 ⫺0.01 0.079 0.926 0.681 0.038 0.046 0.449 0.926 0.025 0.544 0.012 ⫺0.05 0.268 0.259 0.100 0.012 0.294 0.081 0.654 0.409 0.775 0.409 0.112 0.463 0.182 0.113 ⫺0.08 0.159 0.054 ⫺0.16 0.296 ⫺0.14 ⫺0.25 0.296 0.066 0.270 0.002 0.021 0.308 0.675 0.023 0.359 0.259 0.156 0.421 0.319 0.144 ⫺0.06 0.320 0.132 0.505 0.112 0.625 0.033 0.575 0.097 0.022 0.823 0.795 0.497 0.973 0.527 0.326 0.273 0.345 0.286
Table IV. Logistic regression analysis for the association of prooxidant/antioxidant balance (PAB) with schizophrenia (reference is control group).
Unadjusted PAB Model 1 Model 2
BMI Systolic pressure Glucose t-C TG HDL-C LDL-C AIS
OR
95% CI
p
0.995 0.993 0.994
0.992–0.997 0.989–0.997 0.989–0.999
⬍ 0.001 0.001 0.018
Model 1, adjusted for BMI, smoking, t-C, TG, LDL-C and HDL-C. Model 2, adjusted for BMI, smoking, t-C, TG, LDL-C, HDL-C, TAS, SH-group and MDA.
and other cardiovascular risk factors are highly prevalent in patients with schizophrenia [4]. Overweight/ obesity is associated with lifestyle factors (e.g. lack of exercise, poor diet), but also with illness-related (negative, disorganized and depressive symptoms) and treatment-related factors [3]. Weight gain is also an established side-effect of most antipsychotic drugs [18]. Higher triglycerides and decreased HDL-C were also seen in the patients group (Table I). This type of atherogenic dyslipidemia is a well-established risk factor for atherosclerosis development [19]. Although it may not be the main cause, oxidative stress has been suggested to contribute to the pathogenesis and progression of schizophrenia [20,21]. In particular, oxidative damage to lipids, proteins, and DNA as observed in schizophrenia is known to impair cell viability and function, which may subsequently account for the outcome of this pathophysiological condition. The brain is particularly vulnerable to oxidative stress as a result of high rates of oxidative metabolic activity, relatively low level of antioxidants, high levels of oxidizable polyunsaturated fatty acids and high iron content, which plays a role in generating ROS [22]. However, there are limited data on oxidative processes in cerebrospinal fluid and the brain [20]. Most measurements of oxidative stress in patients with schizophrenia have been made on peripheral tissues. But there is speculation that a peripheral indicator may not necessarily reflect the conditions of the oxidative stress parameters in the brain [23]. These factors led us to determine the oxidative stress status parameters in serum and plasma (peripheral tissues) and to establish some association between the general state of oxidative stress and atherogenic dyslipidemia and Table V. The results of receiver operating characteristic (ROC) analysis for discriminating subjects with schizophrenia from the control group.
0.124 0.128 0.189 0.408 ⫺0.06 0.334 0.403 ⫺0.14
0.372 0.217 0.35 0.214 0.167 ⫺0.01 0.002 ⫺0.44 0.665 0.504 0.015 0.258 0.003 0.283 0.300 0.332
0.249 0.265 0.939 0.014 0.004 0.168 0.130 0.073
⫺0.04 ⫺0.09 0.005 0.086 ⫺0.13 0.150 0.130 ⫺0.15
0.129 0.307 0.095 0.396 0.109 0.314 0.423 ⫺0.04
ρ p ρ p ρ p ρ p ρ p ρ ρ p ρ
Controls
p
Controls Patients
Patients
0.404 0.992 0.188 0.088 0.613 0.801 0.106 0.532
p ρ p ρ p
Controls Patients Controls Patients Controls
MDA (μmol/L) Total SH groups (mmol/L) TAS (μmol/L) SOD (kU/L)
Table III. Correlation between antioxidative defense and oxidative stress parameters with conventional CVD risk factors parameters in patients and controls.
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PAB ( HUK/L)
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Model 1 Model 2
AUC
Confidence interval
Std. error
0.821 0.915a,∗
0.718–0.899 0.830–0.966
0.049 0.033
Model 1 – BMI, smoking, t-C, TG, LDL-C and HDL-C. Model 2 – BMI, smoking, t-C, TG, LDL-C, HDL-C and PAB. ∗p ⫽ 0.008. ap values for testing the differences in AUCs between Model 1 and the corresponding additional model, Model 2.
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Oxidative stress in schizophrenia
Figure 1. Comparison of ROCs of Model 1 and Model 2 for discriminating subjects with schizophrenia from the control group. Model 1 – BMI, smoking, t-C, TG, LDL-C and HDL-C. Model 2 – BMI, smoking, t-C, TG, LDL-C, HDL-C and PAB.
overweight which could also be accompanied by increased oxidative stress [9]. Previous studies, that mainly focused on the measurement of peripheral markers of oxidative stress status, have suggested that schizophrenia increases the level of oxidative stress and impaired antioxidative defense, though not consistently [6,20]. Differences in results may be attributed to different clinical symptoms, therapeutic features or duration of the illness [24]. Several factors, such as differences in measurement methods and types of biological materials (red blood cells vs. plasma vs. serum) as well as lifestyle (diet, physical inactivity, levels of smoking) can contribute to the heterogeneity of the results [25,26]. MDA is the final product of lipid peroxidation and is presented as the most commonly-used marker of oxidative stress in schizophrenia [20,21]. Padurariu et al. [27] have demonstrated that lipid peroxidation is positively correlated with the severity of symptoms and inversely with levels of membrane polyunsaturated fatty acids. The result of this study is in agreement with results of the metaanalysis published by Zhang et al. [6] that showed significantly higher levels of MDA in patients with schizophrenia. The antioxidative defense system includes enzymatic and non-enzymatic antioxidants [28]. SOD is a critical antioxidant enzyme responsible for the elimination of superoxide radicals converting them into hydrogen peroxide and molecular oxygen [29]. A decrease in the activity of plasma SOD in the schizophrenic group in comparison to the control group was observed in the present study, but this was not statistically significant. The fact that SOD activity did not change significantly in patients with
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schizophrenia, despite increased lipid peroxidation, may be due to compensative increases in the activity of other antioxidants. Non-enzymatic antioxidant status was studied by estimating TAS and total SH group concentrations. There are several antioxidants, such as proteins, uric acid and ascorbic acid which account for ⬎ 90% of the total antioxidant capacity in human serum [13]. Protein-bound SH groups are very susceptible to oxidation and several studies indicate that levels of protein–SH are correlated negatively with markers of lipid peroxidation and markers of protein oxidative damage [30]. Several studies reported that serum-free SH groups in patients with schizophrenia were significantly lower than in control groups [31,32]. Data from our study showed that levels of total SH groups were significantly lower in the plasma of patients with schizophrenia than those of controls. However, the levels of serum TAS in schizophrenic patients were significantly higher than in the control group (Table II). These findings may be explained as a possible consequence of increased uric acid, which has been reported in schizophrenic patients on atypical antipsychotics treatment [33,34]. Without measuring these parameters this remains a hypothesis. Our study appears to be the first to investigate PAB as an oxidative stress marker in schizophrenia. This method for PAB determination is the first that can measure the balance of oxidants and antioxidants simultaneously in one experiment. This is one of the main advantages of the PAB method in comparison with other assays. It has been calibrated against the most significant known oxidants and antioxidants (mixtures of hydrogen peroxide and uric acid) [14]. In the original paper describing the PAB method [14], results had been compared with widely-utilized and documented methods, ‘gold standards methods’ and showed a great level of similarity with them. Results of previous studies showed that PAB was significantly-correlated with oxidative stress-related assays [14], so PAB may present an adequate measure of oxidative damage. In the present study, it was found that the level of PAB in patients with schizophrenia was significantly higher when compared with control subjects. This huge increase in PAB level in the patients group could be a consequence of the presence of several components of metabolic syndrome in every patient. If we consider the fact that metabolic syndrome and other cardiovascular risk factors are highly prevalent in people with schizophrenia [18], it is possible that increased PAB levels and generally increased oxidative stress in these patients could be the consequence of the presence of metabolic abnormalities. This result is in agreement with the study by Korkamaz et al. [35] who investigated the relationship between conventional risk factors in the metabolic syndrome (hypertension, hyperglycemia,
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obesity...) and PAB levels. The result of Korkamaz study [35] indicates that high PAB levels were both highly sensitive and highly specific for metabolic syndrome. Similar results have been observed in other oxidative stress-related diseases [14,36]. We found that PAB was significantly negatively correlated with antioxidant enzyme SOD and positively correlated with MDA in the patients group while those correlations were not seen in the controls. Those results could be explained by the fact that in the presence of disease, a significant increase in PAB levels is accompanied by a considerable increase in the production of final products of lipid peroxidation and decrease of antioxidant defense. Notably, all our patients were on atypical antipsychotic therapy because some authors reported that chronic administration of typical antipsychotics induces higher oxidative stress by decreasing the activity of antioxidant enzymes and a higher production of lipid peroxidation products [37]. However, another study [38] reported that there is actually no difference in oxidative stress status parameters between patients treated with typical and atypical antipsychotics. According to these authors antipsychotic drugs can normalize the abnormal free radical metabolism in schizophrenia; their pharmacological mechanisms are different but end point effects on oxidative stress could be the same. To further investigate the independent possibility of PAB to discriminate patients from controls we preformed adjustment binary logistic regression analyses (Table IV). The discriminative potential remained significant even after adjustment for conventional risk factors (BMI, smoking and lipid status) as well as after adjustment for combinations of the conventional risk factors and oxidative stress status parameters (MDA, TAS, SH) (Table IV). With this result, we demonstrated an independent association of PAB with the presence of schizophrenia. The logical continuation of our study was to determine the ability of PAB to discriminate between patients and controls since this is the fundamental property of any diagnostic test or indicative system. ROC curve analysis (Table V, Figure 1) revealed that PAB measurement was highly accurate. However, Model 2 that incorporated PAB with traditional CVD risk factors (Table V, Figure 1) demonstrated a significant increase in the clinical accuracy over the model that included measurements of traditional CVD risk factors alone (Model 1). According to these results we suggest that high PAB levels could be a potent parameter for discrimination of schizophrenia patients from controls. The combination of PAB with traditional CVD risk factors facilitates the discrimination of subjects with schizophrenia. ROC curve analysis revealed that the combination of PAB with conventional risk factors (Model 2) had outstanding clinical accuracy
(AUC ⱖ 0.9) and the ability to discriminate patients from control subjects. Because of the cross-sectional design of the study, conclusions concerning the causal relationship between higher PAB levels and the presence of schizophrenia cannot be drawn. In addition, the study sample size was relatively small which could limit the generalization of our results. Also, the limitation of our study is that the patients’ psychiatric status is not reported. Prospective studies including large numbers of patients are needed to elucidate the relationship between PAB and schizophrenia as well as psychopathological state and to assess its possible diagnostic and prognostic value. In conclusion, elevated oxidative stress together with raised BMI and lipid status abnormalities were observed in patients with schizophrenia. The serum PAB level may help to discriminate patients from the control group. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was financially supported by grants from the Ministry of Education, Science and Technological Development, Republic of Serbia (project number 175035 and III 46001).
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