Oxidative stress markers in schizophrenic patients

Immuno-analyse et biologie spécialisée (2013) 28, 51—56

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Oxidative stress markers in schizophrenic patients Les marqueurs du stress oxydant chez les patients schizophréniques H. Mabrouk a,∗,b, I. Houas a, H. Mechria c, A. Mechri b,c, W. Douki a,b, L. Gaha b,c, M.F. Najjar a a

Biochemistry-Toxicology Laboratory, University Hospital of Monastir, 5000 Monastir, Tunisia Research Laboratory ‘‘Vulnerability to psychotic disorders LR 05 ES 10’’, Psychiatry Department, University Hospital of Monastir, 5000 Monastir, Tunisia c Psychiatry Department, University Hospital of Monastir, 5000 Monastir, Tunisia b

Received 29 June 2012; accepted 17 October 2012

KEYWORDS Schizophrenia; TBARS; Non-enzymatic antioxidants; Oxidative stress

∗

Summary Objective. — This study aimed to investigate the variations of the plasma TBARS levels (lipid peroxidation marker) and of the non-enzymatic antioxidants (uric acid, bilirubin and albumin) and their associations with the clinical and therapeutic characteristics in schizophrenic patients. Patients and methods. — A case control study included 121 schizophrenic patients and 88 control subjects aged respectively, 37.3 ± 10.3 and 33.5 ± 13,9 years. TBARS was determined by spectrophotometric method based on the reaction between malonedialdehyde (MDA) and thiobarbituric acid (TBA). Plasma uric acid (UA) and total bilirubin (TB) concentrations were determined using Cobas 6000TM and albumin level was determined using Konelab 30TM equipment. Results. — Compared to controls, patients had significantly higher levels of UA (270 ± 68 vs. 220 ± 73; P < 0.0001) and lower levels of TB (4.74 ± 3.58 vs. 14.67 ± 8.01; P < 0.0001). Furthermore, TBARS levels were significantly correlated with albumin. The highest TBARS levels were noted in paranoid sub-type and in patients treated with typical antipsychotics, but without significant differences. Moreover, the risk doubled for an increase in UA was significantly more frequent in schizophrenic patients. Conclusion. — Schizophrenic patients had an increase in TBARS levels and perturbations in their non-enzymatic antioxidant status that contribute to increase the risk of oxidative stress. In addition, our results revealed that there was no association between the increase of TBARS levels, non-enzymatic antioxidants and any clinical or therapeutic characteristics. Therefore, such patients require specific care, particularly with regard to their lipid peroxidation and their non-enzymatic antioxidant. © 2012 Elsevier Masson SAS. All rights reserved.

Corresponding author. E-mail address: hajer [email protected] (H. Mabrouk).

0923-2532/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.immbio.2012.10.005

52

H. Mabrouk et al.

MOTS CLÉS Schizophrénie ; TBARS ; Antioxydants non enzymatiques ; Stress oxydant

Résumé Objectif. — Étudier les variations du taux plasmatique de TBARS (marqueur de la peroxydation lipidique) et des antioxydants non enzymatiques (acide urique, bilirubine et albumine) et leurs associations avec les caractéristiques cliniques et thérapeutiques chez des patients schizophrènes. Patients et méthodes. — Notre étude a concerné 121 patients schizophrènes et 88 sujets témoins, âgés respectivement de 37,3 ± 10,3 et 33,5 ± 13,9 ans. Le dosage du malonedialdhéhyde (MDA), marqueur de la peroxydation lipidique, a été déterminé par une méthode spectrophotométrique basée sur sa réaction avec l’acide thiobarbiturique (TBA). Les concentrations plasmatiques de l’acide urique (AU) et de la bilirubine totale (BT) ont été déterminées sur Cobas 6000TM (Roche Diagnostics). Le dosage de l’albumine a été effectué sur Konélab 30TM (Thermo Electron Corporation). Résultats. — Les concentrations plasmatiques de l’acide urique étaient significativement plus élevées (270 ± 68 vs 220 ± 73 ; p < 0,0001) et celles de la bilirubine plus basses (4,74 ± 3,58 vs 14,67 ± 8,01 ; p < 0,0001) chez les patients schizophrènes par rapport aux témoins. Cependant, les TBARS sont significativement corrélés avec l’albumine. Les concentrations plasmatiques de TBARS étaient plus élevées dans le sous-type paranoïde et chez les patients traités par les antipsychotiques typiques, mais sans différence significative. L’augmentation de l’AU était significativement plus fréquente chez les patients schizophrènes avec un risque multiplié par deux. Conclusion. — Les patients schizophrènes présentent une augmentation des concentrations plasmatiques de TBARS, de l’acide urique et de la bilirubine, qui pourrait être en relation avec la physiopathologie de la schizophrénie. Le suivi clinique et biologique de ces patients et le contrôle régulier de leur statut antioxydant sont à recommander dans le cadre de leur prise en charge globale. © 2012 Elsevier Masson SAS. Tous droits réservés.

Introduction Schizophrenia is a neuropsychiatric disorder that affects approximately 1% of the population [1—3]. One of the factors contributing to the development of schizophrenia is thought to be oxidative damage to neuronal cells [1]. Oxidative stress is the condition that occurs from the imbalance between reactive oxygen species (ROS) and antioxidant systems. Also, brain is particularly vulnerable to oxidative stress as a result of the relatively low levels of antioxidants, high levels of polyunsaturated fatty acids and increased need of oxygen [2—4]. There are strong links between oxidative stress anomalies and the pathophysiology of schizophrenia, indicated by the increased lipid peroxidation products in plasma and red cells, altered levels of both enzymatic and non-enzymatic antioxidants in chronic schizophrenic patients [6,7]. The aim of this study was to investigate the variations of the plasma TBARS (thiobarbituric acid reactive substances) levels as a lipid peroxidation marker, and of three nonenzymatic antioxidants (uric acid, bilirubin and albumin) and their association with clinical and therapeutic characteristics in patients with schizophrenia.

Materials and methods Subjects Our case control study included 121 patients with schizophrenia from the psychiatric department of the University Hospital Fattouma Bourguiba of Monastir. The mean age was 37.3 ± 10.3 years. There were 21 women

(38.2 ± 10.2 years) and 100 men (37.1 ± 10.4 years). Consensus on the diagnosis, according to the Diagnostic And Statistical Manual Of Mental Disorders, fourth edition (DSMIV) criteria [8], was made by psychiatrists. Patients who had other psychiatric illness, epilepsy or mental retardation, suffering from endocrinological or cardiovascular diseases were excluded from the study. The control group consisted of 88 volunteer subjects without psychiatric or endocrinological diagnoses. The mean age was 33.5 ± 13,9 years, with 31 women (44.5 ± 12.2 years) and 57 men (27.6 ± 10.7 years). All participants were interviewed about their age, personal medical history, previous treatments, cigarette and alcohol consumption habits. This study was approved by the local ethical committee and informed consent was obtained from each participant before procedure, all subjects were from Tunisian origin. Demographic, clinical and therapeutic characteristics of the population are shown in Table 1. There were significant differences in the mean age, gender and BMI between the two groups (Table 1).

Samples After 12 hours of an overnight fast, venous blood for each patient was drawn into lithium-heparin tubes, and immediately centrifuged for 15 min at 3000 rpm. The plasma samples were stored at —20 ◦ C until the biochemical analysis.

Biochemical analysis Plasma uric acid (UA) and total bilirubin (TB) concentrations were determined by enzymatic methods using Cobas 6000TM

TBARS and schizophrenia Table 1

53

Demographic and clinical characteristics of the sample population.

Sex ratio (male/female) Age (years ± SD) BMI (kg/m2 ) Duration of illness (years ± SD) Cigarette smoking Smokers Non-smokers Alcohol beverage Consumers Non-consumers Clinical sub-type Disorganized Undifferentiated Paranoid Antipsychotics Typical Atypical Typical + Atypical

Schizophrenic patients(n = 121)

Control group(n = 88)

P value

(4.76) 100/21 37.3 ± 10.3 25.2 ± 3.7 9.1 ± 7.3

(1.83) 57/31 33.5 ± 13,9 25.5 ± 3.8 —

0.005 0.027 0.190

76 (62.8) 45 (37.2)

52 (59.1) 36 (40.9)

0.688

25 (20.7) 96 (79.3)

16 (18.2) 72 (81.8)

0.687

26 (21.5) 58 (47.9) 37 (30.6)

— — —

— — —

103 (85.1) 6 (4.9) 12 (10)

— — —

— — —

Bold data represent significant results.

(Roche Diagnostics) and albumin level was determined by colorimetric technique using Konelab 30TM equipment (Thermo Electron Corporation).

Lipid peroxidation (TBARS) assay Lipid peroxidation was determined by spectrophotometric method based on the reaction between malonedialdehyde (MDA) and thiobarbituric acid (TBA) [9]. Briefly, 150 ␮L of each plasma were mixed with 500 ␮L of 20% trichloroacetic acid (TCA) (Sigma, Germany) and centrifuged at 3000 rpm for 5 min. Then, 250 ␮L of 0,8% TBA (Sigma, Germany) were added to 500 ␮L of the supernatant. The mixture was heated in boiling water for 30 min. After cooling on ice, the resulting chromogen was extracted with 650 ␮L of n-butyl alcohol. The organic phase was separated by centrifugation at 3000 rpm for 5 min and absorbance was recorded at wavelength of 532 nm. MDA solution was made freshly by the hydrolysis of 1,1,3,3-tetraethoxypropane (TEP) (Sigma, Germany) used as standard. The results are expressed as nmol MDA/mL plasma.

Clinical evaluations Body mass index (BMI) was calculated as weight (kg) divided by height meter squared (m2 ). Obesity was defined when BMI ≥ 30 kg/m2 [10].

Statistical analyses Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 11.0 (SPSS. Inc, Chicago. IL). Quantitative variables were presented as mean ± SD and qualitative variables comparisons were performed using ␹2 test. Comparisons between patients and controls were performed using analysis of variance (ANOVA)

after adjustment for potential confounder factors (gender, age, BMI). Correlations were determined using Pearson test. The calculation of the disruption risk of oxidative stress parameters was done using the quartile. The statistical significance level was set at P < 0.05.

Results The schizophrenic patients had significantly higher levels of UA and lower levels of TB compared to the control group (UA = 270 ± 68 vs. 220 ± 73, P < 0.0001; TB = 4.74 ± 3.58 vs. 14.67 ± 8.01, P < 0.0001). There was no significant difference between the groups on levels of albumin and TBARS (albumin = 55.2 ± 19.8 vs. 52.1 ± 8.4, P = 0.176; TBARS = 7.46 ± 4.68 vs. 6.87 ± 2.50, P = 0.293) (Table 2). After adjustment of oxidative stress markers for confounder factors, we noted a significant difference between patients and controls for AU values (P < 0.0001) and BT (P < 0.0001), but no significant variation was observed for albumin (P = 0.069) and TBARS (P = 0.152) (Table 2). In patients, there was no significant difference according to the sub-type of schizophrenia in oxidative stress markers. However, disorganized patients had higher mean values of UA and albumin. Moreover, we found higher mean values of TBARS levels in paranoid patients than other patients (Table 3). We also showed that these oxidative stress markers were not significantly associated with the antipsychotic treatment, since, patients treated with a typical antipsychtic treatment had the highest levels in the TBARS and the lowest levels in TB. Moreover, patients treated with combination of typical and atypical antipsychotics had the highest levels of albumin and the lowest levels of TBARS (Table 3). Disturbances parameters of oxidative stress were significantly more frequents in schizophrenic patients than

54 Table 2

H. Mabrouk et al. Comparisons of oxidative stress markers between schizophrenic patients and controls.

Biological variables

Patients (n = 121)

Uric acid (␮mol/L) Bilirubin (␮mol/L) Albumin (g/L) TBARS (nmol/mL)

270 4.74 55.2 7.46

± ± ± ±

Controls (n = 88)

68 3.58 19.8 4.68

220 14.67 52.2 6.87

± ± ± ±

73 8.01 8.5 2.50

P value

P valuea

< 0.0001 < 0.0001 0.176 0.293

< 0.0001 < 0.0001 0.069 0.152

Bold data represent significant results. a ANOVA test adjusted by gender, age and BMI.

Table 3

Variations of oxidative stress markers in schizophrenic patients according to clinical sub-types and treatments.

Variables

Uric acid (␮mol/L)

Bilirubin (␮mol/L)

Albumin (g/L)

TBARS (nmol/mL)

Clinical sub-types Undifferentiated (n = 58) Paranoid (n = 37) Disorganized (n = 26) P

273 ± 70 256 ± 67 283 ± 65 0.267

5.06 ± 7.72 4.41 ± 3.72 4.51 ± 3.11 0.646

53.72 ± 22.82 53.84 ± 13.72 60.51 ± 19.82 0.309

7.03 ± 4.61 8.12 ± 4.87 7.48 ± 4.62 0.545

Antipsychotics Typical (n = 103) Atypical (n = 6) Typical + Atypical (n = 12) P

269 ± 67 266 ± 89 268 ± 72 0.849

4.62 ± 3.65 5.58 ± 3.24 5.44 ± 3.26 0.655

54.91 ± 19.43 52.56 ± 20.84 59.53 ± 24.13 0.724

7.76 ± 4.80 6.42 ± 4.78 5.21 ± 2.61 0.195

Table 4

Disturbances parameters of oxidative stress parameters.

Parameters

Patients

Controls n

OR

CI 95%

P

n

%

%

UA (␮mol/L) < 226 > 315

31 30

50.8 49.2

45 6

88.2 11.8

7.258 2.418b

[2.700 — 19.508] [1.389 — 4.209]b

< 0.0001 0.002a

TB (␮mol/L) < 2.3 > 6.25

30 30

50 50

0 83

— 100

— —

— —

— —

Albumin (g/L) < 45.2 > 60.2

31 30

50.8 49.2

22 13

62.8 37.2

1.638 1.342b

[0.700 — 3.831] [0.855 — 2.107]b

0.254 0.201a

TBARS (nmol/mL) < 3.41 > 10.63

30 30

50 50

12 0

100 0

— —

30 —

50 —

OR: odds ratio; CI: confident interval 95%; UA: uric acid; TB: total bilirubin. Bold data represent significant results. a ANOVA test adjusted by gender, age and BMI. b Calculation of the disruption risk of oxidative stress parameters was done using the quartile.

controls, with the risk doubled for an increase in UA (Table 4). Only albumin was significantly correlated with TBARS levels (r = 0.211; P = 0.020) (Table 5).

Table 5 Relationship between TBARS and non-enzymatic antioxidants in schizophrenic patients. Biological variables

r

Discussion

Uric acid Total bilirubin Albumin

0.110 0.039 0.211

Schizophrenic patients had higher plasma TBARS levels than control subjects, without significant difference. Ranjekar

Bold data represent significant results.

P 0.230 0.673 0.020

TBARS and schizophrenia et al. [11] did not find an increase in plasma lipid peroxides TBARS in schizophrenic patients, although some other studies have reported an increase of lipid peroxides in schizophrenic patients [1,4,12—14]. Increased MDA levels might be the result of increased free radical production and/or inadequate response of antioxidative defense mechanisms. Since brain tissue is accepted to be vulnerable to free radical damage, it was speculated that the oxidative stress observed in the periphery is even more serious in the brain [5]. Indeed, Gama et al. [7] reported that a high level of TBARS is a sign of peroxidative injury to membrane phospholipids. Neuronal functioning is affected by this injury either by changes in membrane fluidity or by alterations in membrane receptors, which can cause altered neurotransmitter uptake and release, and even cell death. Padurariu et al. [4] have shown increase levels of TBARS in plasma, erythrocytes, leucocytes, and platelets in schizophrenic patients. The elevation of plasma lipid peroxides, an indicator of lipid peroxidation and one of the key indices of membrane pathology, support the hypothesis that a contribution of oxidative stress-mediated cellular membrane pathology may be involved in the pathophysiology of schizophrenia [12,13,16,17]. Among the non-enzymatic antioxidant, we find a significantly higher level of UA and lower level of TB in the schizophrenic patients compared to the control group. Plasma UA level was studied for its strong antioxidant characteristic and it has been suggested that decreased UA provides an additional support to the hypothesis that antioxidant stress in schizophrenia may be due to a defect in the antioxidant system [18]. Several studies [2,16,19,20] had shown lower plasma antioxidants (albumin, bilirubin and uric acid) in schizophrenic patients than controls. Hence, these findings suggest that oxidative stress occurs in the early course of schizophrenia, and may have an important role in pathogenesis and symptomatology of schizophrenia. There is increasing evidence that schizophrenia is associated with abnormalities in the antioxidant defense system and redox signaling [16,19]. Moreover, Akyol et al. [18] have shown a relationship between hyperbilirubenimia and Gilbert’s syndrome in schizophrenia. Hyperbilirubinemia among patients with schizophrenia has been variously explained by an increase in vulnerability of red blood cells membranes, effect of medication or due to stress [18,21]. On the other hand, oxidative stress markers were not associated to the sub-type of schizophrenia. However, disorganized patients had higher mean values of UA and albumin. Moreover, paranoid patients had higher mean values of TBARS than others. These results suggest that there is a difference in oxidative stress in different sub-types of schizophrenia. Zhang et al. [12] reported that paranoid, disorganized and residual schizophrenic patients had higher MDA levels than healthy controls, but there were no significant differences in MDA levels among the schizophrenic subgroups. Akyol et al. [18] studied the TBARS levels in a sample of disorganized, paranoid and residual schizophrenics; only the TBARS level of the residual schizophrenics was significantly higher than the healthy controls. Gama et al. [7] found no difference among different sub-types (paranoid, disorganized and undifferentiated) for the mean serum TBARS concentrations.

55 The exact effect induced by different typical or atypical medications on both antioxidant enzymes and lipid peroxidation levels is still not known [12]. A further finding of our study is that there were no significant differences among the typical and atypical antipsychotic treatments of schizophrenic patients in any parameter tested in this study. This result suggests that long-term treatment with both typical and atypical antipsychotics may produce the similar effects on the activities of the non-enzymatic antioxidants and the levels of lipid peroxidation. Kunz et al. [13] showed a decrease on oxidative stress parameters associated with chronic antipsychotics treatment. The mechanism through which antipsychotics decrease oxidative stress might involve modulation of the molecular regulation of expression of antioxidant enzymes. Antipsychotic drugs have been found to induce the expression of immediate early genes such as c-fos and c-jun transcription factors, growth factors and peptides [17]. These early genes and growth factors can subsequently regulate the expression of antioxidant enzymes, which provide a part of neuroprotective mechanisms associated with growth factors [17]. Ng et al. [22] reported that a differential impact on oxidative stress status may exist between typical and atypical antipsychotic medications. Indeed, higher levels of lipid peroxidation products have been reported in patients treated with typical rather than atypical drugs. However, some studies have reported an increased lipid peroxidation only in rats chronically treated with haloperidol, but not in animals treated with atypical antipsychotics [4,7,15,23]. Reddy et al. [16] reported that plasma antioxidants (albumin, bilirubin and uric acid) were significantly reduced in schizophrenic patients. More importantly, these reductions are observed independently of treatment since patients were neuroleptic-naïve. Yao et al. [24] reported that individual antioxidants (albumin, bilirubin and uric acid) were significantly reduced in plasma of chronic schizophrenic patients, during on and off haloperidol treatment conditions. However, these findings are inconsistent with typical and atypical antipsychotics, which suggests that typical and atypical antipsychotics may not have a direct regulatory effect on oxidative stress in patients with schizophrenia [12]. However, in contrast with other studies [25,26], no correlation was found between plasma TBARS and plasma non-enzymatic antioxidant concentrations in the present study. It should be noted that the antioxidant defense system is a very complex system including non-enzymatic components (albumin, bilirubin, uric acid, etc.). Additional studies are needed to confirm this hypothesis. Several limitations should be noted in our study. First, a larger sample size of groups would be beneficial. Second, our work is a cross-sectional study that does not permit follow-up of biological parameters. Third, the sample of schizophrenic patients may not be representative of more heterogeneous population. Finally, although the classical determination of MDA by complexion with TBA is widely used for assaying lipid peroxides, clinical application is limited because it tends to be highly susceptible to experimental conditions. One alternative approach is to separate interfering substances from MDA-TBA adduct by high-pressure liquid chromatography (HPLC) prior to spectrophotometric measurement. This HPLC procedure provides considerably better sensitivity and specificity, which results in more

56 reliable reference values than any previously published results.

Conclusion Schizophrenic patients had an increase in TBARS levels and perturbations in their non-enzymatic antioxidant status that contribute to increase the risk of oxidative stress. In addition, our results revealed that there was no association between the increase of TBARS levels, non-enzymatic antioxidant and any clinical or therapeutic characteristics. Therefore, such patients require specific care, particularly with regard to their lipid peroxidation and their nonenzymatic antioxidant.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.

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