- Email: [email protected]
Serum nitric oxide metabolite levels and the effect of antipsychotic therapy in schizophrenia
Archives of Medical Research 35 (2004) 401–405
ORIGINAL ARTICLE
Serum Nitric Oxide Metabolite Levels and the Effect of Antipsychotic Therapy in Schizophrenia Fatma Taneli,a S¸ebnem Pırıldar,b Fisun Akdeniz,a Bekir Sami Uyanıka and Zeki Arıa a
Department of Biochemistry and Clinical Biochemistry, Celal Bayar University School of Medicine, Manisa, Turkey b Department of Psychiatry, Ege University School of Medicine, Izmir, Turkey Received for publication February 10, 2004; accepted June 11, 2004 (04/024).
Background. Recently it was proposed that nitric oxide metabolites (NO) may have a role in the pathophysiology of schizophrenia and major depressive disorders. The present study was performed to assess changes in serum nitric oxide metabolite levels in schizophrenic patients compared with healthy controls. Our secondary aim was to further evaluate the impact of psychopharmacologic treatment on circulating NO levels not assessed previously. Methods. Serum NO levels of patients with schizophrenia (n ⫽ 20) before and after 6 weeks of treatment were compared with those of healthy controls (n ⫽ 20). Severity of schizophrenia and response to treatment were assessed with positive and negative symptoms of schizophrenia. NO levels were estimated by Griess method in serum samples. Results. In patients with schizophrenia, pre-treatment serum NO levels were higher than those of control subjects (39.15 ⫾ 18.24 vs. 25.40 ⫾ 5.83 µmol/L, p ⫽ 0.036) and also of post-treatment values (34.41 ⫾ 16.35 vs. 25.40 ⫾ 5.83 µmol/L, p ⫽ 0.049), respectively. However, no significant difference was found between serum NO levels in pre- and posttreatment values. Conclusions. Our findings of increased serum NO levels in schizophrenic patients confirmed the role of NO in the pathophysiology of schizophrenia. However, we found that antipsychotic drugs do not reveal significant effects on serum levels of NO in schizophrenia in a 6-week treatment regimen. Further studies with longer therapy periods may suggest some new clues for novel treatment strategies employing antioxidants and NOS inhibitors in schizophrenia. 쑖 2004 IMSS. Published by Elsevier Inc. Key Words: Antipsychotic, Nitric oxide metabolites, Treatment, Schizophrenia.
Introduction Recently, research has focused on the biochemical, physiologic, and molecular actions of nitric oxide metabolites (NO) in normal physiologic processes as well as during pathologic conditions. NO is an important intercellular physiologic messenger capable of evoking a number of cellular responses (1). Additionally, NO is accepted as an oxygen radical
Address reprint requests to: Fatma Taneli, M.D., Department of Biochemistry and Clinical Biochemistry, Celal Bayar University School of Medicine, Manisa, 45020, Turkey. Phone: (⫹90) (0236) 232-5889, ext. 325; FAX: (⫹90) (0236) 237-0213; E-mails: [email protected] and [email protected]
0188-4409/04 $–see front matter. Copyright d o i : 1 0 .1 0 16 / j . a rc m e d .2 00 4 .0 6 .0 02
and is also recognized as a major messenger molecule involved in regulation of vasodilatation and neurotransmission in brain and peripheral nervous system. It is produced by various cell types to facilitate intracellular communication. NO formation is catalyzed by nitric oxide synthase (NOS), an enzyme present in a variety of cell types (2). NO, a soluble gas produced by the activity of an enzyme found in neurons, is known to affect neurodevelopmental processes in the central nervous system (CNS) (3). Several studies indicate that nitric oxide metabolites are involved in the etiopathogenesis of many neuropsychiatric disorders such as schizophrenia, depression, Alzheimer’s disease, Huntington’s disease, and stroke (4–8). A distorted reported distribution of nicotineamide reported distorted distribution
쑖 2004 IMSS. Published by Elsevier Inc.
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Taneli et al. / Archives of Medical Research 35 (2004) 401–405
of nicotineamide-adenine dinucleotide phosphate-diaphorase (NADPH-d) neurons in various areas of postmortem brains of individuals with schizophrenia (9,10). Given that NADPH-d coexists with NOS, this discovery suggests that there may be an abnormal production of NO in brains of patients with schizophrenia. While the role of neurotransmitters such as serotonin, norepinephrine, dopamine, corticotropin-releasing hormone, and arginine vasopressin has been extensively studied, new evidence suggests a role for the unique neurotransmitter NO (11,12). This highly diffusible and reactive molecule is synthesized by at least three enzyme subtypes of NOS. The commonly known neuronal NOS subtype is localized in areas of the brain related with stress and depression (11). Postmortem analysis of brain specimens of cerebella from patients with schizophrenia revealed elevated NOS concentrations (13). It was suggested that NO plays a role in modulating release of other neurotransmitters acting as a cellular communicator in plasticity and development and/or acting as a vasodilator in regulation of blood flow in the pathophysiology of stress and depression (11). This study examines whether NO production is increased in schizophrenic patients by comparing serum nitric oxide metabolites of schizophrenic patients with healthy controls. Our secondary aim was to further evaluate the impact of psychopharmacologic treatment on circulating NO levels not previously assessed.
dose 4.13 ⫾ 1.06 mg/day, range, 2–6), two received clozapine (mean ⫾ SD, dose 450.00 ⫾ 70.71 mg/day, range, 400– 500), and the remaining three received olanzapine (mean ⫾ SD, dose 25.0 ⫾ 5.0 mg/day, range, 20–30). Mean ⫾ SD age of patients with schizophrenia was 26.8 ⫾ 8.5 years (range, 17–46 years); mean age at onset of psychosis was 23.4 ⫾ 2.9 years, while mean illness duration was 15.1 ⫾ 12.7 months (range, 6–78 months). Mean age ⫾ SD of controls included in the study (7 males and 13 females) was 25.5 ⫾ 5.8 (range, 20–45) years. Patients with conditions possibly affecting their plasma nitrite and nitrate levels, such as alcohol and/or drug abuse and certain diseases including organic brain disorder, pregnancy, or any physical illness as assessed by personal history, or abnormal signs in clinical examination or laboratory data (including complete blood count, serum electrolyte assay, liver function tests, thyroid function tests, urinalysis, urine drug screen, hepatitis, HIV serology, and electrocardiography) were excluded from this study. The study was performed in accordance with the Declaration of Helsinki and was approved by the Celal Bayar University School of Medicine Ethics Committee on September 11, 2003. All participants gave written informed consent to be interviewed and to undergo venipuncture. Venous fasting blood samples of patients and control subjects were collected between 11:00 a.m. and 12:00 p.m. and were stored at ⫺70⬚C until assessment. Blood samples were collected at baseline and after 6 weeks of psychopharmacologic treatment.
Materials And Methods
NO assessment. NO is instantly converted into other molecules; thus, its detection as a native NO molecule is difficult. It can be measured as nitrite and nitrate metabolites. Biochemical assessments of stable NO oxidative metabolites, ⫺ nitrite (NO⫺ 2 ), and nitrate (NO3 ) levels were analyzed by Griess method (15). Sera were deproteinized with Somogy reagent composed of 75 mmol/L ZnSO4 and 55 mmol/L NaOH, and supernatants were used for further analysis. An aliquot of samples was taken for nitrite assessment, produced by diazotization of sulfanilamide and coupling with naphthylethylene diamine. Another aliquot of samples was analyzed for sum of nitrite and nitrate assessments, which were reduced by Cu-coated cadmium granules in glycine-NaOH buffer at pH 9.7 and further produced by diazotization of sulfanilamide and coupling with naphthylethylene diamine; nitrate level is found by the difference between the two aliquots. Absorbance of the reaction was measured at 545 nm with a spectrophotometer (Shimadzu Co., Kyoto, Japan). Standard curve was performed with concentrations of 2–10 µmol/L sodium nitrate. NO data in this study present sum of nitrite and nitrate concentrations that are NO metabolites and were expressed in µmol/L.
Patients. Twenty patients (16 female and 4 male) who had not not taken any psychotropic drugs for a period of at least 2 weeks prior to the study were enrolled. All patients included in the study met DSM-IV (American Psychiatric Association, 1994) criteria for schizophrenia (14). Control group consisted of 20 age- (13 female and 7 male) and sexmatched physically and mentally healthy subjects who volunteered to participate in the study (Table 1). Assessment of clinical situation and treatment. We used positive and negative symptoms of the schizophrenia (PANSS) scale to analyze positive and negative symptoms on each patient with schizophrenia at baseline and after 6 weeks of treatment period. All schizophrenic patients received atypical antipsychotics. Antipsychotics were chosen according to patient tolerance and response observed in their previous episodes. Three patients (two males and one female) who did not respond to antipsychotic treatment at the end of week 6 were excluded from the study because our primary objective was to compare baseline values with values during response-to-treatment. Response-to-treatment was defined as at least 30% improvement in PANSS scores. Ten patients were only in their first episode. Fifteen patients received risperidone (mean ⫾ standard deviation [SD],
Statistical analysis. Statistical analyses were done by SPSS (Statistical Package for the Social Sciences Program) software. Data distributions were evaluated by Kolmogorov and
Serum Nitric Oxide Metabolite Levels in Schizophrenia
403
Table 1. Demographic clinical treatment characteristics in 20 patients with schizophrenia
Antipsychotic Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (3 mg/day) Risperidone (5 mg/day) Risperidone (5 mg/day) Risperidone (2 mg/day) Risperidone (4 mg/day) Risperidone (3 mg/day) Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (6 mg/day) Risperidone (6 mg/day) Clozapine (500 mg/day) Clozapine (400 mg/day) Olanzapine (30 mg/day) Olanzapine(20 mg/day) Olanzapine (25 mg/day)
PANSS
Chlorpromazine equivalent dose (mg) (drug-free period, week)
Positive1/positive2
Negative1/negative2
General1/general2
400 400 400 300 (4) 500 (24) 500 (3) 200 (4) 400 (2) 300 (4) 400 (36) 400 (3) 400 (22) 400 (3) 600 (36) 600 (8) 1,000 (4) 800 (4) 600 (3) 400 (7) 500 (5)
35/12 28/12 40/23 27/12 39/31 33/28 15/13 48/16 33/14 24/17 33/37 31/20 38/16 27/33 34/34 28/21 43/36 16/9 30/10 26/12
20/9 24/25 28/15 16/14 45/32 30/22 24/19 32/15 24/11 37/26 44/21 36/20 35/15 42/40 30/30 30/24 32/28 29/17 31/12 29/19
53/23 46/39 63/37 44/16 76/56 73/55 43/35 67/29 40/23 54/42 42/17 63/43 60/42 44/44 43/41 50/36 67/50 51/30 65/25 57/29
PANSS, positive and negative symptoms of schizophrenia scale.
Smirnov test, while Student t test was used for comparison of patient and control groups and a value of p ⬍0.05 was considered significant. Relationship of nitrite and nitrate levels with PANSS scores were assessed by Spearman correlation.
Results Serum NO levels in study and control groups are depicted in Figure 1. Pre- and post-treatment values of circulating
nitric oxide metabolite levels in patients with schizophrenia were 39.15 ⫾ 18.24 µmol/L and 34.41 ⫾ 16.35, respectively, given as mean ⫾ SD. Serum NO concentrations in control group were 25.40 ⫾ 5.83 µmol/L. Serum NO levels were higher in pre-treatment values of schizophrenic patients than in control subjects (39.15 ⫾ 18.24 vs. 25.40 ⫾ 5.83 µmol/ L, p ⫽ 0.036) and also post-treatment values (34.41 ⫾ 16.35 vs 25.40 ⫾ 5.83 µmol/L, p ⫽ 0.049), respectively. However, in patients with schizophrenia, no significant difference was found in serum NO levels in pre- (39.15 ⫾ 18.24 µmol/L) and post-treatment (34.41 ⫾ 16.35 µmol/L) values. We were unable to obtain any significant correlation between change in nitrite plus nitrate levels and PANSS total/subscale scores.
Discussion
Figure 1. Serum nitric oxide metabolite levels in patients with schizophrenia and control subjects
These data provide evidence that serum NO concentrations increase during schizophrenia. In addition, antipsychotic therapy resulted in insignificant decreases (p ⬎0.05) in circulating NO levels. Our results overlap with studies revealing that significantly increased levels of plasma NO levels were found in schizophrenia patients (16–18). In addition, our results confirm previous reports suggesting that arginine-NO pathway is involved in the pathogenesis of schizophrenia (11). In schizophrenia, Akyol et al. (17) proposed that increased plasma xanthine-oxidase activity and increased NO production by NOS suggest a possible role of NO in the pathophysiologic process of schizophrenia (17,18). Plasma
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Taneli et al. / Archives of Medical Research 35 (2004) 401–405
nitrite levels in the schizophrenic group were significantly higher than those of healthy controls (18). In addition to circulating levels of NO, further intracellular analyses of NO have also been evaluated in red blood cells, platelets, and polymorphonuclear neutrophils (PMNs). Herken et al. (2) found significantly increased levels of redblood-cell nitric oxide metabolite levels in patients with schizophrenia compared with control subjects. Significantly increased NO levels in erythrocytes from neuroleptic-treated NO levels in erythrocytes from neuroleptic-treated schizophrenic patients indicated a possible role of NO in the pathogenesis of schizophrenia. In addition, overall NOS activity was found significantly higher in platelets of drugnaı¨ve schizophrenic patients compared with controls and drug-treated patients with schizophrenia (4). Nitrite content in PMN was reduced to 68%, while plasma and platelet nitrite content in patients with schizophrenia was not significantly changed in comparison with controls (19,20). Our studies indicate significant increases in circulating serum NO levels in schizophrenic patients. NO can produce peroxynitrite (ONOO⫺) by interacting with superoxide radical (O2⫺). This molecule can act as a neurotoxin by means of its interaction with thiol groups of proteins as well as by decomposition into the most potent hydroxyl radical (2). There have been controversies with regard to the function of NO in the brain. Some authors believe that NO elevation in schizophrenic patients suggests that NO may be a protective factor against free radical toxicity. This NO elevation may also be accepted as a compensatory mechanism of the body because NO has been shown to consume O2 and thus protect cellular and extracellular structures from O2⫺ and further free oxygen radicals such as singlet oxygen and hydroxyl radical (21). On the other hand, it has also been suggested that activation of NO synthase and xanthine oxidase of brain cells lead to formation of peroxynitrite, which is highly cytotoxic and can result in profound cellular injury and cell death, providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity (22,23). We believe that NO function is concentration dependent and that excessive increases in NO concentrations may cause deleterious effects by peroxynitrite formation. Thus, our results showed an increase in both pre- and post-treatment values in patients with schizophrenia compared with controls. This study has its limitations because the source of surplus NO production cannot be located exactly and also due to the small number of patients included in the study. However, in experimental studies it was suggested that centrally increased nitrite and nitrate could overflow into plasma (24). Neuronal nitric oxide metabolites synthase (nNOS) catalyzes synthesis of neuronal nitric oxide metabolites from Larginine. Although behavioral and neurochemical studies implicate neuronal nitric oxide metabolites in the pathophysiology of schizophrenia and in actions of standard antipsychotic drugs, Tarazi et al. (25) suggest that nNOS plays
a minimal role in rat brain in mediating long-term actions of newer antipsychotic drugs including olanzapine, risperidone, and quetiapine. In our patients, antipsychotic drugs that included olanzapine, risperidone, and clozapine were used and our post-therapy results of NO levels in schizophrenic patients completely overlap with Tarazi’s data in that antipsychotic drugs play minimal effects on serum NO levels. In conclusion, our findings of increased levels of serum nitric oxide metabolites in schizophrenic patients confirmed the role of NO in the pathophysiology of schizophrenia. However, we found that antipsychotic drugs do not reveal significant effects on serum levels of nitric oxide metabolites in schizophrenia in a treatment regimen of 8 weeks. Further studies with longer therapy periods may suggest some new clues for novel treatment strategies employing antioxidants and NOS inhibitors in schizophrenia.
References 1. Dawson VL, Dawson TM. Nitric oxide actions in neurochemistry. Neurochem Int 1996;29:97–110. ¨ zyurt H, Akyol O. Red blood cell nitric oxide levels 2. Herken H, Uz E, O in patients with schizophrenia. Schizophr Res 2001;52:289–290. 3. Black MD, Selk DE, Hitchock JM, Wettstein JG, Sorensen SM. On the effect of neonatal nitric oxide synthase inhibition in rats: a potential neurodevelopmental model of schizophrenia. Neuropharmacology 1999; 38:1299–1306. 4. Das I, Khan NS, Puri BK, Sooranna SR, de Belleroche J, Hirsch SR. Elevated platelet calcium mobilization and nitric oxide synthase activity may reflect abnormalities in schizophrenic brain. Biochem Biophys Res Commun 1995;212:375–380. 5. Zoroglu SS, Yurekli M, Meram I, Sogut S, Tutkun H, Yetkin O, Sivasli E, Savas HA, Yanik M, Herken H, Akyol O. Pathophysiological role of nitric oxide and adrenomedullin in autism. Cell Biochem Funct 2003;21:55–60. 6. Dieperink E, Willenbring M, Ho SB. Neuropsychiatric symptoms associated with hepatitis C and interferon alpha: a review. Am J Psychiatry 2000;157:867–876. 7. Suzuki E, Nakaki T, Nakamura M, Miyaoka H. Plasma nitrate levels in deficit vs. nondeficit forms of schizophrenia. J Psychiatry Clin Neurosci 2003;28:288–292. 8. Suzuki E, Yoshida Y, Shibuya A, Miyaoka H. Nitric oxide involvement in depression during interferon-alpha therapy. Int J Neuropsychopharmacol 2003;6:415–419. 9. Akbarian S, Bunney WE Jr, Potkin SG, Wigal SB, Hagman JO, Sandman CA, Jones EG. Altered distribution of nicotinamide-adenine dinucleotide phosphate-diaphorase cells in frontal lobe of schizophrenics implies disturbance of cortical development. Arch Gen Psychiatry 1993;50:169–177. 10. Akbarian S, Vinuela A, Kim JJ, Potkin SG, Bunney WE Jr, Jones EG. Distorted distribution of nicotinamide-adenine dinucleotide phosphatediaphorase neurons in temporal lobe of schizophrenics implies anomalous cortical development. Arch Gen Psychiatry 1993;50:178–187. 11. McLeod TM, Lo´pez-Figueroa AL, Lo´pez-Figueroa MO. Nitric oxide, stress, and depression. Psychopharmacol Bull 2001;35:24–41. 12. Fossier P, Blanchard B, Ducroq C, Leprince C, Tauc L, Baux G. Nitric oxide transforms serotonin into an inactive form and this affects neuromodulation. Neuroscience 1999;93:597–603. 13. Karson CN, Griffin WS, Mrak RE, Husain M, Dawson TM, Synder SH, Moore NC, Sturner WQ. Nitric oxide synthase (NOS) in schizophrenia: increases in cerebellar vermis. Mol Chem Neuropathol 1996;27:275– 284.
Serum Nitric Oxide Metabolite Levels in Schizophrenia 14. American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders (DSM-IV). 4th ed. Washington, D.C., USA: American Psychiatric Association;1994. 15. Cortas NK, Wakid NW. Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 1990;36: 1440–1443. 16. Yanik M, Vural H, Kocyigit A, Tutkun H, Zoroglu SS, Herken H, Savas HA, Koylu A, Akyol O. Is the arginine-nitric oxide pathway involved in the pathogenesis of schizophrenia? Neuropsychobiology 2003;47:61–65. 17. Akyol O, Herken H, Uz E, Fadillioglu E, Unal S, Sogut S, Ozyurt H, Savas HA. The indices of endogenous oxidative and antioxidative processes in plasma from schizophrenic patients. The possible role of oxidant/antioxidant imbalance. Prog Neuropsychopharmacol Biol Psychiatry 2002;26:995–1005. 18. Zoroglu SS, Herken H, Yurekli M, Uz E, Tutkun H, Savas HA, Bagci C, Ozen ME, Cengiz B, Cakmak EA, Dogru MI, Akyol O. The possible pathophysiological role of plasma nitric oxide and adrenomedullin in schizophrenia. J Psychiatr Res 2002;36:309–315. 19. Srivastava N, Barthwal MK, Dalal PK, Agarwal AK, Nag D, Srimal RC, Seth PK, Dikshit M. Nitrite content and antioxidant enzyme levels
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in the blood of schizophrenia patients. Psychopharmacology (Berl) 2001;158:140–145. Srivastava N, Barthwal MK, Dalal PK, Agarwal AK, Nag D, Seth PK, Srimal RC, Dikshit M. A study on nitric oxide, beta-adrenergic receptors and antioxidant status in the polymorphonuclear leukocytes from the patients of depression. J Affect Disord 2002;72:45–52. Wink DA, Hanbauer I, Krishna MC, De Graff W, Gamson J, Mitchell JB. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc Natl Acad Sci USA 1993;90:9813– 9817. Virag L, Szabo E, Gergely P, Szabo C. Peroxynitrite-induced cytotoxicity: mechanism and opportunities for intervention. Toxicol Lett 2003; 140–141:113–124. Pagliaro P. Differential biological effects of products of nitric oxide (NO) synthase: it is enough to say NO. Life Sci 2003;3:2137–2149. Suzuki E, Yagi G, Nahali T, Kanba S, Asai M. Elevated plasma nitrate levels in depressive states. J Affect Disord 2001;63:221–224. Tarazi FI, Zhang K, Baldessarini RJ. Long-term effects of newer antipsychotic drugs on neuronal nitric oxide synthase in rat brain. Nitric Oxide 2002;7:297–300.
ORIGINAL ARTICLE
Serum Nitric Oxide Metabolite Levels and the Effect of Antipsychotic Therapy in Schizophrenia Fatma Taneli,a S¸ebnem Pırıldar,b Fisun Akdeniz,a Bekir Sami Uyanıka and Zeki Arıa a
Department of Biochemistry and Clinical Biochemistry, Celal Bayar University School of Medicine, Manisa, Turkey b Department of Psychiatry, Ege University School of Medicine, Izmir, Turkey Received for publication February 10, 2004; accepted June 11, 2004 (04/024).
Background. Recently it was proposed that nitric oxide metabolites (NO) may have a role in the pathophysiology of schizophrenia and major depressive disorders. The present study was performed to assess changes in serum nitric oxide metabolite levels in schizophrenic patients compared with healthy controls. Our secondary aim was to further evaluate the impact of psychopharmacologic treatment on circulating NO levels not assessed previously. Methods. Serum NO levels of patients with schizophrenia (n ⫽ 20) before and after 6 weeks of treatment were compared with those of healthy controls (n ⫽ 20). Severity of schizophrenia and response to treatment were assessed with positive and negative symptoms of schizophrenia. NO levels were estimated by Griess method in serum samples. Results. In patients with schizophrenia, pre-treatment serum NO levels were higher than those of control subjects (39.15 ⫾ 18.24 vs. 25.40 ⫾ 5.83 µmol/L, p ⫽ 0.036) and also of post-treatment values (34.41 ⫾ 16.35 vs. 25.40 ⫾ 5.83 µmol/L, p ⫽ 0.049), respectively. However, no significant difference was found between serum NO levels in pre- and posttreatment values. Conclusions. Our findings of increased serum NO levels in schizophrenic patients confirmed the role of NO in the pathophysiology of schizophrenia. However, we found that antipsychotic drugs do not reveal significant effects on serum levels of NO in schizophrenia in a 6-week treatment regimen. Further studies with longer therapy periods may suggest some new clues for novel treatment strategies employing antioxidants and NOS inhibitors in schizophrenia. 쑖 2004 IMSS. Published by Elsevier Inc. Key Words: Antipsychotic, Nitric oxide metabolites, Treatment, Schizophrenia.
Introduction Recently, research has focused on the biochemical, physiologic, and molecular actions of nitric oxide metabolites (NO) in normal physiologic processes as well as during pathologic conditions. NO is an important intercellular physiologic messenger capable of evoking a number of cellular responses (1). Additionally, NO is accepted as an oxygen radical
Address reprint requests to: Fatma Taneli, M.D., Department of Biochemistry and Clinical Biochemistry, Celal Bayar University School of Medicine, Manisa, 45020, Turkey. Phone: (⫹90) (0236) 232-5889, ext. 325; FAX: (⫹90) (0236) 237-0213; E-mails: [email protected] and [email protected]
0188-4409/04 $–see front matter. Copyright d o i : 1 0 .1 0 16 / j . a rc m e d .2 00 4 .0 6 .0 02
and is also recognized as a major messenger molecule involved in regulation of vasodilatation and neurotransmission in brain and peripheral nervous system. It is produced by various cell types to facilitate intracellular communication. NO formation is catalyzed by nitric oxide synthase (NOS), an enzyme present in a variety of cell types (2). NO, a soluble gas produced by the activity of an enzyme found in neurons, is known to affect neurodevelopmental processes in the central nervous system (CNS) (3). Several studies indicate that nitric oxide metabolites are involved in the etiopathogenesis of many neuropsychiatric disorders such as schizophrenia, depression, Alzheimer’s disease, Huntington’s disease, and stroke (4–8). A distorted reported distribution of nicotineamide reported distorted distribution
쑖 2004 IMSS. Published by Elsevier Inc.
402
Taneli et al. / Archives of Medical Research 35 (2004) 401–405
of nicotineamide-adenine dinucleotide phosphate-diaphorase (NADPH-d) neurons in various areas of postmortem brains of individuals with schizophrenia (9,10). Given that NADPH-d coexists with NOS, this discovery suggests that there may be an abnormal production of NO in brains of patients with schizophrenia. While the role of neurotransmitters such as serotonin, norepinephrine, dopamine, corticotropin-releasing hormone, and arginine vasopressin has been extensively studied, new evidence suggests a role for the unique neurotransmitter NO (11,12). This highly diffusible and reactive molecule is synthesized by at least three enzyme subtypes of NOS. The commonly known neuronal NOS subtype is localized in areas of the brain related with stress and depression (11). Postmortem analysis of brain specimens of cerebella from patients with schizophrenia revealed elevated NOS concentrations (13). It was suggested that NO plays a role in modulating release of other neurotransmitters acting as a cellular communicator in plasticity and development and/or acting as a vasodilator in regulation of blood flow in the pathophysiology of stress and depression (11). This study examines whether NO production is increased in schizophrenic patients by comparing serum nitric oxide metabolites of schizophrenic patients with healthy controls. Our secondary aim was to further evaluate the impact of psychopharmacologic treatment on circulating NO levels not previously assessed.
dose 4.13 ⫾ 1.06 mg/day, range, 2–6), two received clozapine (mean ⫾ SD, dose 450.00 ⫾ 70.71 mg/day, range, 400– 500), and the remaining three received olanzapine (mean ⫾ SD, dose 25.0 ⫾ 5.0 mg/day, range, 20–30). Mean ⫾ SD age of patients with schizophrenia was 26.8 ⫾ 8.5 years (range, 17–46 years); mean age at onset of psychosis was 23.4 ⫾ 2.9 years, while mean illness duration was 15.1 ⫾ 12.7 months (range, 6–78 months). Mean age ⫾ SD of controls included in the study (7 males and 13 females) was 25.5 ⫾ 5.8 (range, 20–45) years. Patients with conditions possibly affecting their plasma nitrite and nitrate levels, such as alcohol and/or drug abuse and certain diseases including organic brain disorder, pregnancy, or any physical illness as assessed by personal history, or abnormal signs in clinical examination or laboratory data (including complete blood count, serum electrolyte assay, liver function tests, thyroid function tests, urinalysis, urine drug screen, hepatitis, HIV serology, and electrocardiography) were excluded from this study. The study was performed in accordance with the Declaration of Helsinki and was approved by the Celal Bayar University School of Medicine Ethics Committee on September 11, 2003. All participants gave written informed consent to be interviewed and to undergo venipuncture. Venous fasting blood samples of patients and control subjects were collected between 11:00 a.m. and 12:00 p.m. and were stored at ⫺70⬚C until assessment. Blood samples were collected at baseline and after 6 weeks of psychopharmacologic treatment.
Materials And Methods
NO assessment. NO is instantly converted into other molecules; thus, its detection as a native NO molecule is difficult. It can be measured as nitrite and nitrate metabolites. Biochemical assessments of stable NO oxidative metabolites, ⫺ nitrite (NO⫺ 2 ), and nitrate (NO3 ) levels were analyzed by Griess method (15). Sera were deproteinized with Somogy reagent composed of 75 mmol/L ZnSO4 and 55 mmol/L NaOH, and supernatants were used for further analysis. An aliquot of samples was taken for nitrite assessment, produced by diazotization of sulfanilamide and coupling with naphthylethylene diamine. Another aliquot of samples was analyzed for sum of nitrite and nitrate assessments, which were reduced by Cu-coated cadmium granules in glycine-NaOH buffer at pH 9.7 and further produced by diazotization of sulfanilamide and coupling with naphthylethylene diamine; nitrate level is found by the difference between the two aliquots. Absorbance of the reaction was measured at 545 nm with a spectrophotometer (Shimadzu Co., Kyoto, Japan). Standard curve was performed with concentrations of 2–10 µmol/L sodium nitrate. NO data in this study present sum of nitrite and nitrate concentrations that are NO metabolites and were expressed in µmol/L.
Patients. Twenty patients (16 female and 4 male) who had not not taken any psychotropic drugs for a period of at least 2 weeks prior to the study were enrolled. All patients included in the study met DSM-IV (American Psychiatric Association, 1994) criteria for schizophrenia (14). Control group consisted of 20 age- (13 female and 7 male) and sexmatched physically and mentally healthy subjects who volunteered to participate in the study (Table 1). Assessment of clinical situation and treatment. We used positive and negative symptoms of the schizophrenia (PANSS) scale to analyze positive and negative symptoms on each patient with schizophrenia at baseline and after 6 weeks of treatment period. All schizophrenic patients received atypical antipsychotics. Antipsychotics were chosen according to patient tolerance and response observed in their previous episodes. Three patients (two males and one female) who did not respond to antipsychotic treatment at the end of week 6 were excluded from the study because our primary objective was to compare baseline values with values during response-to-treatment. Response-to-treatment was defined as at least 30% improvement in PANSS scores. Ten patients were only in their first episode. Fifteen patients received risperidone (mean ⫾ standard deviation [SD],
Statistical analysis. Statistical analyses were done by SPSS (Statistical Package for the Social Sciences Program) software. Data distributions were evaluated by Kolmogorov and
Serum Nitric Oxide Metabolite Levels in Schizophrenia
403
Table 1. Demographic clinical treatment characteristics in 20 patients with schizophrenia
Antipsychotic Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (3 mg/day) Risperidone (5 mg/day) Risperidone (5 mg/day) Risperidone (2 mg/day) Risperidone (4 mg/day) Risperidone (3 mg/day) Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (4 mg/day) Risperidone (6 mg/day) Risperidone (6 mg/day) Clozapine (500 mg/day) Clozapine (400 mg/day) Olanzapine (30 mg/day) Olanzapine(20 mg/day) Olanzapine (25 mg/day)
PANSS
Chlorpromazine equivalent dose (mg) (drug-free period, week)
Positive1/positive2
Negative1/negative2
General1/general2
400 400 400 300 (4) 500 (24) 500 (3) 200 (4) 400 (2) 300 (4) 400 (36) 400 (3) 400 (22) 400 (3) 600 (36) 600 (8) 1,000 (4) 800 (4) 600 (3) 400 (7) 500 (5)
35/12 28/12 40/23 27/12 39/31 33/28 15/13 48/16 33/14 24/17 33/37 31/20 38/16 27/33 34/34 28/21 43/36 16/9 30/10 26/12
20/9 24/25 28/15 16/14 45/32 30/22 24/19 32/15 24/11 37/26 44/21 36/20 35/15 42/40 30/30 30/24 32/28 29/17 31/12 29/19
53/23 46/39 63/37 44/16 76/56 73/55 43/35 67/29 40/23 54/42 42/17 63/43 60/42 44/44 43/41 50/36 67/50 51/30 65/25 57/29
PANSS, positive and negative symptoms of schizophrenia scale.
Smirnov test, while Student t test was used for comparison of patient and control groups and a value of p ⬍0.05 was considered significant. Relationship of nitrite and nitrate levels with PANSS scores were assessed by Spearman correlation.
Results Serum NO levels in study and control groups are depicted in Figure 1. Pre- and post-treatment values of circulating
nitric oxide metabolite levels in patients with schizophrenia were 39.15 ⫾ 18.24 µmol/L and 34.41 ⫾ 16.35, respectively, given as mean ⫾ SD. Serum NO concentrations in control group were 25.40 ⫾ 5.83 µmol/L. Serum NO levels were higher in pre-treatment values of schizophrenic patients than in control subjects (39.15 ⫾ 18.24 vs. 25.40 ⫾ 5.83 µmol/ L, p ⫽ 0.036) and also post-treatment values (34.41 ⫾ 16.35 vs 25.40 ⫾ 5.83 µmol/L, p ⫽ 0.049), respectively. However, in patients with schizophrenia, no significant difference was found in serum NO levels in pre- (39.15 ⫾ 18.24 µmol/L) and post-treatment (34.41 ⫾ 16.35 µmol/L) values. We were unable to obtain any significant correlation between change in nitrite plus nitrate levels and PANSS total/subscale scores.
Discussion
Figure 1. Serum nitric oxide metabolite levels in patients with schizophrenia and control subjects
These data provide evidence that serum NO concentrations increase during schizophrenia. In addition, antipsychotic therapy resulted in insignificant decreases (p ⬎0.05) in circulating NO levels. Our results overlap with studies revealing that significantly increased levels of plasma NO levels were found in schizophrenia patients (16–18). In addition, our results confirm previous reports suggesting that arginine-NO pathway is involved in the pathogenesis of schizophrenia (11). In schizophrenia, Akyol et al. (17) proposed that increased plasma xanthine-oxidase activity and increased NO production by NOS suggest a possible role of NO in the pathophysiologic process of schizophrenia (17,18). Plasma
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nitrite levels in the schizophrenic group were significantly higher than those of healthy controls (18). In addition to circulating levels of NO, further intracellular analyses of NO have also been evaluated in red blood cells, platelets, and polymorphonuclear neutrophils (PMNs). Herken et al. (2) found significantly increased levels of redblood-cell nitric oxide metabolite levels in patients with schizophrenia compared with control subjects. Significantly increased NO levels in erythrocytes from neuroleptic-treated NO levels in erythrocytes from neuroleptic-treated schizophrenic patients indicated a possible role of NO in the pathogenesis of schizophrenia. In addition, overall NOS activity was found significantly higher in platelets of drugnaı¨ve schizophrenic patients compared with controls and drug-treated patients with schizophrenia (4). Nitrite content in PMN was reduced to 68%, while plasma and platelet nitrite content in patients with schizophrenia was not significantly changed in comparison with controls (19,20). Our studies indicate significant increases in circulating serum NO levels in schizophrenic patients. NO can produce peroxynitrite (ONOO⫺) by interacting with superoxide radical (O2⫺). This molecule can act as a neurotoxin by means of its interaction with thiol groups of proteins as well as by decomposition into the most potent hydroxyl radical (2). There have been controversies with regard to the function of NO in the brain. Some authors believe that NO elevation in schizophrenic patients suggests that NO may be a protective factor against free radical toxicity. This NO elevation may also be accepted as a compensatory mechanism of the body because NO has been shown to consume O2 and thus protect cellular and extracellular structures from O2⫺ and further free oxygen radicals such as singlet oxygen and hydroxyl radical (21). On the other hand, it has also been suggested that activation of NO synthase and xanthine oxidase of brain cells lead to formation of peroxynitrite, which is highly cytotoxic and can result in profound cellular injury and cell death, providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity (22,23). We believe that NO function is concentration dependent and that excessive increases in NO concentrations may cause deleterious effects by peroxynitrite formation. Thus, our results showed an increase in both pre- and post-treatment values in patients with schizophrenia compared with controls. This study has its limitations because the source of surplus NO production cannot be located exactly and also due to the small number of patients included in the study. However, in experimental studies it was suggested that centrally increased nitrite and nitrate could overflow into plasma (24). Neuronal nitric oxide metabolites synthase (nNOS) catalyzes synthesis of neuronal nitric oxide metabolites from Larginine. Although behavioral and neurochemical studies implicate neuronal nitric oxide metabolites in the pathophysiology of schizophrenia and in actions of standard antipsychotic drugs, Tarazi et al. (25) suggest that nNOS plays
a minimal role in rat brain in mediating long-term actions of newer antipsychotic drugs including olanzapine, risperidone, and quetiapine. In our patients, antipsychotic drugs that included olanzapine, risperidone, and clozapine were used and our post-therapy results of NO levels in schizophrenic patients completely overlap with Tarazi’s data in that antipsychotic drugs play minimal effects on serum NO levels. In conclusion, our findings of increased levels of serum nitric oxide metabolites in schizophrenic patients confirmed the role of NO in the pathophysiology of schizophrenia. However, we found that antipsychotic drugs do not reveal significant effects on serum levels of nitric oxide metabolites in schizophrenia in a treatment regimen of 8 weeks. Further studies with longer therapy periods may suggest some new clues for novel treatment strategies employing antioxidants and NOS inhibitors in schizophrenia.
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