http://jop.sagepub.com/Journal of Psychopharmacology

http://jop.sagepub.com/content/29/1/31The online version of this article can be found at:

 DOI: 10.1177/0269881114555251

2015 29: 31 originally published online 15 October 2014J PsychopharmacolBrigitte Schmidt, Susanne Hempel, Julia Volkert, Klaus-Peter Lesch and Andreas Reif

Sarah Kittel-Schneider, Martin Reuß, Andrea Meyer, Heike Weber, Alexandra Gessner, Carolin Leistner, Juliane Kopf,Multi-level biomarker analysis of nitric oxide synthase isoforms in bipolar disorder and adult ADHD

  

Published by:

http://www.sagepublications.com

On behalf of: 

  British Association for Psychopharmacology

can be found at:Journal of PsychopharmacologyAdditional services and information for    

  http://jop.sagepub.com/cgi/alertsEmail Alerts:

 

http://jop.sagepub.com/subscriptionsSubscriptions:  

http://www.sagepub.com/journalsReprints.navReprints:  

http://www.sagepub.com/journalsPermissions.navPermissions:  

What is This? 

- Oct 15, 2014OnlineFirst Version of Record  

- Dec 10, 2014Version of Record >>

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from

Journal of Psychopharmacology2015, Vol. 29(1) 31 –38

© The Author(s) 2014Reprints and permissions: sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0269881114555251jop.sagepub.com

IntroductionNitric oxide (NO) is a gaseous messenger molecule with pleio-tropic functions in humans. Three different nitric oxide synthases (NOS) catalyse NO production from l-arginine in an oxygen-dependent reaction with the by-product l-citrulline. The NOS isoforms are NOS-I encoded by NOS1, NOS-II encoded by NOS2 and NOS-III encoded by NOS3 (Sessa, 1994). NO signalling involves the NO/sGC/PKG pathway (nitric oxide/soluble gua-nylate cyclase/protein kinase G) as well as the nitrosylation of the amino acids cysteine and tyrosine (Daff, 2003). The NO/sGC/PKG pathway is responsible for long-term potentiation in gluta-matergic synapses where NO acts as a retrograde transmitter released at the post-synapse to diffuse to the pre-synapse to strengthen the connection between the neurons (Bohme et al., 1991; Hopper et al., 2004; Kiss and Vizi, 2001). In the case of cysteine, nitrosylation represents a pathway with similarities to phosphorylation, while the nitrosylation of tyrosine is seen as a sign of oxidative damage occurring, for example in ischemia and tumours (Kanwar et al., 2009). As the half-life of NO is too short to directly determine NO production in biological systems, the concentration of the stable metabolites NO2

- and NO3- (summed

up to NOx-) are often measured to serve as surrogate markers to

assess the activity of the NOSs (Tarpey and Fridovich, 2001).Several studies implicated a role of NOS, especially of

NOS-I and NOS-III, in psychiatric diseases such as affective

disorders, adult attention-deficit/hyperactivity disorder (aADHD) and schizophrenia (Kurrikoff et al., 2012; Reif et al., 2006a, 2006b, 2009; Weber et al., 2014). Most interestingly, impulsive behaviours which are a hallmark of aADHD and bipo-lar disorder (BPD) have been linked to short alleles of the func-tional NOS1 exon 1f-VNTR repeat polymorphism (Reif et al., 2009). In accordance with these human studies, genetic deletion of Nos1 in mice resulted in elevated levels of impulsivity and aggression (Nelson et al., 2006), while the aggression level and depression-like behaviour of Nos3 knockout mice were

Multi-level biomarker analysis of nitric oxide synthase isoforms in bipolar disorder and adult ADHD

Sarah Kittel-Schneider1,2#, Martin Reuß3, Andrea Meyer3, Heike Weber4, Alexandra Gessner3, Carolin Leistner3, Juliane Kopf1, Brigitte Schmidt3, Susanne Hempel3, Julia Volkert1,3, Klaus-Peter Lesch3 and Andreas Reif1,2

AbstractIntroduction: Several studies have shown altered levels of nitric oxide (NO) and its stable metabolites (NOx

-) in blood and cerebrospinal fluid of psychiatric patients. The aim of our study was to replicate previous findings and investigate the influence of the nitrinergic system in bipolar disorder and adult attention-deficit/hyperactivity disorder (aADHD) in particular.Methods: The concentrations of NO2

- and NO3- in peripheral blood in a sample of aADHD, bipolar disorder (BPD) and controls were analysed. The sample

was genotyped for a three marker haplotype in the NOS3 gene (rs2070744, rs1799983 and Intron 4 VNTR) and for genetic variants of the NOS1 gene (NOS1 ex 1c, NOS1 ex 1f). Finally, qRT PCR was performed.Results: We found significantly lower NOx

- levels in BPD (p<0.001). rs2070744 T/T-carriers of the whole sample showed increased mRNA expression of NOS3 (p=0.05). Only in BPD an influence of rs2070744 was seen regarding NO metabolite levels; C/C carriers displayed lower NOx

- levels (p=0.05).Conclusion: We could replicate and extend previous findings showing altered NOx

- levels in BPD and an influence of NOS3 rs2070744 on NOS3 expression and NOx

- concentration. Together, these data point to a role of the nitrinergic pathway in BPD.

KeywordsNitric oxide, bipolar disorder, attention deficit-hyperactivity disorder, genetic polymorphism, biomarkers

1 Department of Psychiatry, Psychosomatics and Psychotherapy, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany

2 Comprehensive Heart Failure Center, University of Würzburg, Würzburg, Germany

3 Department of Psychiatry, Psychosomatics and Psychotherapy, University of Würzburg, Würzburg, Germany

4IZKF Microarray Core Unit, University of Würzburg, Würzburg, Germany

Corresponding author:Sarah Kittel-Schneider, Department of Psychiatry, Psychosomatics and Psychotherapy, Johann Wolfgang Goethe University Frankfurt, Frankfurt, Germany. Email: Sarah.Kittel-Schneider@kgu.de

555251 JOP0010.1177/0269881114555251Journal of PsychopharmacologyKittel-Schneider et al.research-article2014

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from

32 Journal of Psychopharmacology 29(1)

decreased (Reif et al., 2004). Pharmacological inhibition of NOS-I has been shown to have both anxiolytic and antidepres-sant properties (Volke et al., 2003).

Regarding functional studies on NO, several studies have assessed NOx

- levels in psychiatric patients. In patients suffer-ing from BPD, schizophrenia and ADHD significant increases of NO2

- and NO3- levels were noticed (Selek et al., 2008a,

2008b; Yao et al., 2004). However, the study investigating schizophrenia patients measured NO metabolites in post-mor-tem brain samples and the studies analysing NOx

- levels in ADHD and BPD were conducted by the same group. As a con-sequence, and limiting the conclusions of these studies, there is an overlap of the analysed samples which becomes evident immediately. To extend and corroborate these studies on NO biomarkers, we therefore investigated the role of genetic varia-tion in NOS1 and NOS3 in the pathogenesis of BPD and aADHD by performing a multi-level analysis. First, we analysed the concentrations of NO2

- and NO3- in peripheral blood serum in a

sample of 78 participants (aADHD, BPD and healthy controls). Second, as a three-marker haplotype in the NOS3 gene (rs2070744, rs1799983 and the NOS3 Intron 4 VNTR) and a single nucleotide polymorphism (SNP) and a variable number tandem repeat (VNTR) in the promoter regions of two of the nine alternative first exons of the NOS1 gene (NOS1 ex1c, NOS1 ex 1f) were associated with neuropsychiatric disorders and impulsive behaviour in earlier studies (Reif et al., 2006a, 2006b, 2011a, 2011b), we genotyped the sample for these genetic variants in order to correlate genetic variance with NOx

- levels and gene expression levels. Finally, to correlate possible changes in these metabolites with alterations in gene expression – and thereby possibly also enabling to discriminate different NOS isoforms – we then performed quantitative real-time poly-merase chain reaction (qRT PCR) to assess the expression lev-els of NOS1 and NOS3.

Materials and methods

Participants and sample preparation

The investigated sample consisted of 88 participants in total: 41 patients suffering from BPD (16 BPD type 1, 25 BPD type 2), 14 patients suffering from aADHD and 33 healthy controls. All participants were from the German Lower Franconia area. Healthy controls were mainly recruited from University Hospital of Würzburg staff. There was no significant difference in sex ratio between the groups (χ² test, p=0.464) but patients with BPD were significantly older than patients with ADHD and healthy controls (ANOVA, p<0.001, post-hoc Bonferroni t-test p=0.009, p<0.001 respectively). All patients fulfilled ICD-10 criteria for BPD or the DSM-IV criteria for aADHD, respectively, assessed by two experienced clinicians (SK, AR; for demographic date see Table 1). In BPD, diagnosis was vali-dated with a semi-structured interview analogous to the Manual for the Assessment and Documentation of Psychopathology (AMDP) interview as well as the Operational Criteria Checklist for Psychotic and Affective Illness (OPCRIT) interview (Fähndrich, 2007). Severity of the symptoms was measured by Montgomery Åsberg Depression Scale (MADRS) and Young Mania Rating Scale (YMRS). aADHD was diagnosed along the DSM-IV criteria as well as based on the results of the Structured

Clinical Interview for DSM Disorders (SCID-I and SCID-II) interviews. Patients also fulfilled the diagnostic criteria for childhood and adult ADHD assessed by the Connors’ Adult ADHD Rating Scale (CAARS) and Wender–Utah Rating Scale (WURS) semi-structured interviews (Christiansen et al., 2012; Retz-Junginger et al., 2002). All patients were part of larger genetic studies, further description of which can be found else-where (Weber et al., 2011). The control participants were screened for absence of psychiatric symptoms with the Mini-Diagnostic Interview for Psychiatric Disorder (Mini-DIPS) (Margraf, 1994). Exclusion criteria for patients as well as con-trol participants were severe medical conditions, severe neuro-logical disorders, mental retardation, current carcinomas and acute and chronic infectious diseases. Exclusion criteria for controls were a history of psychiatric disorder or first-line rela-tives with psychiatric disorders. Blood samples were taken from each participant after fasting for at least 10 hours by using EDTA monovettes for genotype analysis. For NOx

- measure-ments, serum was separated by centrifugation for 15 minutes at 2300 rpm at 4°C before being stored at -80°C until being used. For gene expression analysis blood was taken by using PAXgene tubes (Qiagen, Hilden, Germany). Patients and controls gave written informed consent before participating in this study. The study was approved by the Ethics Committee of the University of Würzburg.

Measuring NOx- concentration in de-

proteinized blood serum

Blood serum samples from all participants were filtrated with a MultiScreen Filter™ from Merck Millipore (Darmstadt, Germany) to remove all proteins. NOx

- levels were determined after filtration with the Nitrite/Nitrate Assay Kit colorimetric from Sigma-Aldrich (Buchs, Switzerland) in triplicates accord-ing to the manufacturer’s protocol. The deproteinized blood serum was mixed with an equal volume of the Kit’s Buffer solution; 80 µL of this mixture and 80 µL of the NO3

- standard solution were used as inputs for photometric measurements. Samples and standards were subsequently treated with a nitrate-reductase (NAD[P]H) and a co-factor (Nitrite/Nitrate Assay Kit, Sigma-Aldrich, Buchs, Switzerland) to reduce NO3

- to NO2

-. Total NO2- was then measured with a reaction relying

on the Grieß assay (Fox, 1979). In this assay, NO2- is added to

sulphanilamide (Grieß A) and an azonium ion is formed; this azonium ion is subsequently linked to naphtylethylendiamine (Grieß B) in an azo-coupling. The absorbance of the resulting pink azo-dye was measured in a photometer (Multiskan® Spektrum, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 540 nm. Total NO2

- concentrations of the samples were estimated from a calibration curve, which was established by linear regression from the standard concentrations and extinctions.

Genotyping

All included participants were genotyped for polymorphisms in the NOS3 gene (Reif et al., 2006b) as published: the T786C SNP (rs2070744) in the promoter region, an intronic 27 base pair VNTR in intron 4 and the non-synonymous G894T SNP

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from

Kittel-Schneider et al. 33

(rs1799983), which generates an amino acid substitution from glutamic acid to aspartate in exon 8. All participants were also genotyped for genetic variants in the NOS1 gene, namely NOS1 exon 1c-SNP rs41279104 (Reif et al., 2011b) and NOS1 ex1f-VNTR exon 1f (Reif et al., 2009).

The protocol for DNA extraction and genotyping can be found elsewhere (Reif et al., 2006a, 2006b). In brief, DNA was extracted from EDTA blood samples from each participant. The amplicons for Intron 4 VNTR determination were directly visu-alized, the possible resulting amplicon lengths were 393 bp (for Allele 4a) 420 bp (for Allele 4b) and 447 bp (Allele 4c). For rs2070744, amplicons were digested with MspI; the resulting fragment lengths were 140 bp and 40 bp for wild-type individu-als and 71 and 58 bp for the minor allele. For rs1799983, ampli-cons were treated with MboI; the resulting fragment lengths were 71 and 59 bp for individuals carrying the minor allele and 129 for the wild-types. NOS1 VNTR exon 1f was determined by

PCR amplification and product size determination as described (Reif et al., 2006a). Rs41279104 was genotyped by standard PCR and subsequent digest with Fnu4HI.

Expression of NOS1 and NOS3 mRNA in blood cells

RNA was extracted from whole blood collected by means of PAXgene™ blood RNA tubes with the PAXgene™ blood RNA Kit according to the manufacturer’s (Qiagen, Hilden, Germany) protocol. 500 ng of RNA was reverse transcribed in cDNA using the iScript™ cDNA Synthesis Kit from Biorad (Hercules, USA). qRT PCR was performed with the CFX 384TM real-time PCR detection System from Biorad (Hercules, USA). Expression of the target genes (NOS1 and NOS3) and the house-keeping genes (18S rRNA, ACTB, ALAS, GAPDH) was esti-mated in independently conducted experiments in triplicates. One reaction consisted of 1 µL reverse transcribed cDNA, 3 µL H2O, 1 µL Primer Mix and 5 µL iQ ™ SYBR™ Green Supermix (Qiagen, Hilden, Germany). As fluorescence had only one peak when temperature was slowly increased, it was concluded that no other unspecific amplicon or adduct of primers was present in the reaction mixture and the primer pairs were therefore spe-cific. Normalization of the target genes against the housekeep-ing genes was performed with the help of geNORM (Vandesompele et al., 2002). Samples were only included if the PCR efficiency was within a 5% range around the medium PCR efficiency.

Statistical analysis

Statistical analysis was performed with SPSS (Version 21, IBM, Armonk, NY, USA). After having checked for normal dis-tribution, ANOVA tests and Student’s t-tests or non-parametric test (Kruskal–Wallis test, Mann–Whitney U test), respectively, were performed for the analysis. Genotype and haplotype anal-ysis was performed with PLINK v. 1.07. Data were corrected for multiple comparisons using Dunnett-C test in the case of unequal variance and Bonferroni-Holmes correction in case of equal variance.

Power analysis

Post hoc power analysis for main hypothesis was computed with G*Power 3.1 (Faul et al., 2007). Taking a one-way ANOVA model for the main hypothesis, a medium effect size as observed for the differences between the diagnosis groups regarding NOx

- levels, r=0.32, and α=0.05. A marginally sufficient power of 1–ß=0.76 was achieved.

Results

NOx- concentration in de-proteinized blood

serum

NOx- levels were not significantly influenced by age, smoking

status, medication or sex. As healthy controls were not taking psychopharmacological medication, only the BPD and aADHD subgroups were analysed regarding the influence of

Table 1. Demographic data.

Age at sampling (years) n m SD

Control 33 34.1 ± 11.4ADHD 14 34.3 ± 12.5Bipolar 41 46.5 ± 13.9SEX (MALE/FEMALE) n ratioControl 15/18 0.83ADHD 7/7 1.0Bipolar 14/27 0.52Bipolar nLithium only MS 2Antipsychotic only MS 10Anticonvulsant only MS 4Combination Li+antipsychotic 2Combination Li+anticonvulsant 1Combination Li+antipsychotic+anticonvulsant

1

Combination antipsychotic+anticonvulsant

18

No mood stabilizer 2Medication not available 1Plus antidepressant 22Psychiatric comorbidities Substance abuse disorder 3Other (Fibromyalgia) 1Bipolar I/Bipolar II 16/25Polarity at sampling depressive 25(hypo-)manic 9mixed 6rapid cycling 1ADHD Stimulant medication 0Antidepressant 8Psychiatric Comorbidties Major Depression 9Substance abuse disorder 3Abbreviations: MS=mood stabi-lizer, Li=Lithium

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from

34 Journal of Psychopharmacology 29(1)

antidepressant treatment on NO metabolite concentration, and the subgroup of BPD was tested additionally regarding the influ-ence of treatment with mood stabilizers. There was no significant influence of either treatment on NOx

- levels. NOx- concentrations

differed significantly in patients suffering from BPD as com-pared with controls as well as aADHD patients (Kruskal–Wallis test, p<0.001, Figure 1). In a subsequently performed post-hoc Mann–Whitney U test, the difference between BPD and ADHD was still significant after correction for multiple comparison with Dunnett-C test (corrected p=0.05) as well as the difference between BPD and controls (corrected p=0.05). There was no sig-nificant difference between controls and ADHD patients. Bipolar patients nominally showed the highest NOx

- levels and ADHD patients the lowest. In a post-hoc analysis, BPD patients were analysed stratified by polarity. NOx

- concentrations were nomi-nally the highest in mania, the lowest in mixed patients and the depressed patients’ levels lay in between, although in every case higher than in controls. Differences between polarity subgroups, however, were not significant. Severity of symptoms measured with MADRS, YMRS and WURS, CAARS, respectively, showed no significant correlation with NOx

- concentrations. Also, no significant differences between bipolar-I and bipolar-II were detected.

Genotyping

In all, 86 participants were genotyped for two SNPs and one VNTR in NOS3 and for two genetic variants of the NOS1 gene (NOS1 ex 1c, NOS1 ex 1f). The previously shown Allele 4c of the VNTR was not detected in this study. All five polymor-phisms were in Hardy–Weinberg equilibrium. None of the investigated polymorphisms was associated with BPD or ADHD (χ² test, df=4, every p>0.1 (see Table 2). Moreover, hap-lotype frequency was determined in BPD and controls. Six of the eight possible haplotypes had a minor allele frequency above the threshold of 0.05 and were therefore included for this analysis. None of the haplotypes was significantly over- or underrepresented in BPD as compared with controls. Due to the small sample size, negative case-control association data were expected. For the same reason, we did not calculate for an asso-ciation with aADHD.

Regarding the influence of single marker polymorphisms on NOx

- levels, NOS3 rs1799983 (G894T) T/T carriers showed sig-nificantly higher NOx

- levels than G/T and G/G carriers on analy-sis of the whole sample (Kruskal–Wallis test, p=0.035); however, this result was no longer statistically significant after correction for multiple comparisons with Dunnett-C test. There was no

Figure 1. NO metabolite levels in bipolar and aADHD patients and healthy controls.NO metabolite levels are illustrated as mean values ± standard deviation in µMol/L. Significant differences between the groups were found by Kruskal–Wallis test (p<0.001). In post-hoc Mann–Whitney U test, the difference between BPD and ADHD was still significant after correction for multiple comparison with Dunnett-C test (corrected p=0.05) as well as the difference between BPD and controls (corrected p=0.05). Level of significance was determined *p≤0.05.

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from

Kittel-Schneider et al. 35

difference between genotypes in NOS3 rs2070744 (T786C) when analysing the whole sample together, but when analysing the diagnosis groups separately, C/C carriers in the bipolar group showed significantly lower NOx

- levels than C/T and T/T carriers (Kruskal–Wallis test, p=0.017) and the difference remained

statistically significant after correction for multiple comparisons with Dunnett-C test (corrected p=0.05) (see Table 3). In the hap-lotype analysis, none of the included haplotypes was associated with altered NOx

- levels.

Quantification of NOS1 and NOS3 mRNA in human leukocyte blood cells

The target genes of the qRT PCR experiments (NOS1 and NOS3) were normalized against four housekeeping genes (18S rRNA, ACTB, ALAS, GAPDH). While no NOS1 mRNA was detected in human leukocytes in either sample, there was considerable expres-sion of NOS3. Age, sex or smoking status as well as medication (treatment with antidepressants and mood stabilizers was analysed in the patients’ groups) did not influence NOS3 expression levels. A single marker analysis performed with Kruskal–Wallis test revealed significant expression differences between rs2070744 (NOS3 T786C) genotypes when analysing the whole sample, with the T/T genotype showing the highest expression of NOS3 (see Table 4) (Kruskal–Wallis, p=0.035). In a subsequently performed post-hoc Mann–Whitney U test, the differences between the geno-types (T/T>C/T>C/C) remained statistically significant after cor-rection for multiple comparison with Dunnett-C test (corrected p=0.05). However, NOS3 expression levels were not significantly different between patient groups and controls (Kruskal–Wallis test, p=0.83). Patients suffering from BPD were then separated into three groups depending on polarity of the mood episode they were in when blood was taken: hypomanic/manic, mixed or depressed. There was, however, no significant difference between polarities (Kruskal–Wallis test, p=0.34), no significant correlation of sever-ity of symptoms in MADRS and YMRS with NOS3 expression in patients with BPD and no correlation of severity of symptoms measured by CAARS and WURS in patients with aADHD. Also, there was no difference in NOS3 expression between bipolar type I and II patients (Kruskal–Wallis test, p=0.521). When the correla-tion of haplotypes to NOS3 expression was investigated, none of the haplotypes had a significant impact on gene expression in a logistic regression analysis performed with Plink. There was also

Table 2. NOS1 and NOS3 genotype frequencies (and percentages) in controls, ADHD and bipolar patients.

Controls ADHD BPD χ² (df=4)

NOS1 Exon 1c A/A 1 (0.03) 0 (0.0) 0 (0.0) A/G 5 (0.16) 1 (0.08) 11 (0.26) G/G 26 (0.81) 12 (0.92) 30 (0.73) p= 0.349Exon 1f L/L 6 (0.21) 5 (0.38) 14 (0.34) S/L 13 (0.46) 6 (0.46) 19 (0.46) S/S 9 (0.32) 2 (0.15) 8 (0.19) p=0.600NOS3 rs2070744 C/C 6 (0.18) 3 (0.23) 4 (0.10) C/T 9 (0.28) 4 (0.31) 23 (0.56) T/T 17 (0.53) 6 (0.46) 14 (0.34) p=0.138Intron 4 VNTR 4a 1 (0.09) 2 (0.17) 1 (0.03) 4a/b 1 (0.09) 3 (0.25) 15 (0.38) 4b 9 (0.81) 7 (0.58) 23 (0.59) p=0.303rs1799983 G/G 14 (0.48) 9 (0.69) 22 (0.54) G/T 14 (0.48) 3 (0.23) 13 (0.32) T/T 1 (0.03) 1 (0.08) 6 (0.15) p=0.125

Genotype distribution was tested by χ² test.

Table 3. Influence of NOS3 polymorphisms on NOx- concentration in the whole sample.

Genotype [NOx]- in µMol/L

SD p-value corrected p-value

rs1799983 G/G 84.8 48.22 0.035 0.07G/T 89.16 64.21 T/T 129.31 44.7 Intron 4 VNTR 4a/ 4a 60.61 22.24 0.568 4a/ 4b 93.17 68.29 4b/ 4b 88.71 49.6 rs2070744 C/C 78.4 77.6 0.223 C/T 95.06 51.89 T/T 86.58 49.34

Influence of genetic variants was analysed with Kruskal–Wallis test and corrected for multiple comparisons with Dunnett-C test if statistical significant. Statistical significance was determined p≤0.05.

Table 4. Influence of NOS3 polymorphisms on NOS3 mRNA expression in the whole sample.

Genotype Mean Q NOS3 SD p-value corrected p-value

rs1799983 G/G 0.39 0.058 0.274 G/T 0.41 0.050 T/T 0.41 0.034 Intron 4 VNTR 4a/ 4a 0.27 0.029 0.121 4a/ 4b 0.34 0.070 4b/ 4b 0.41 0.029 rs2070744 C/C 0.33 0.064 0.035 0.05C/T 0.39 0.033 T/T 0.45 0.044

Influence of genetic variants was analysed with Kruskal–Wallis test and corrected for multiple comparisons with Dunnett-C test if statistical significant. Statistical significance was determined p≤0.05.

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from

36 Journal of Psychopharmacology 29(1)

no significant correlation between NOx- concentrations and NOS3

mRNA levels in the whole sample.

DiscussionIn this study, we aimed to replicate and extend former studies showing increased NOx

- levels in patients suffering from BPD. Apart from BPD, the NO system is probably also involved in aADHD (Aspide et al., 1998; Hoogman et al., 2011; Reif et al., 2009), and therefore we also investigated NOx

- levels in a smaller group of aADHD patients. However, the aADHD group did not show significant differences regarding NOx

- levels compared with controls. This finding is in contrast to previous studies (Ceylan et al., 2010; Selek et al., 2008b) showing higher NO metabolite levels in children and adults with ADHD; however, the methods to measure NO levels differed from our method, because we did not use copperized cadmium granules to reduce nitrate to nitrite but nitrate reductase. Besides, our ADHD sample was rather small, which could also be a reason for the conflicting results.

The main finding of the present study is an increase in NOx-

levels in bipolar patients, which replicates and extends previous findings showing elevated NOx

- levels in acute episode but also euthymic bipolar patients. The BPD patients in our sample were significantly older than the ADHD and control participants, but as age did not have a significant influence on NOx

- levels when tested in all groups separately (Pearson’s correlation, p>0.1), the age difference should not relevantly bias the results. All previous studies on NO metabolites in serum of bipolar patients, however, were conducted at one single institution, and apparently feature a wide overlap of samples (Gergerlioglu et al., 2007; Savas et al., 2002, 2006; Selek et al., 2008a; Yanik et al., 2004). In compari-son with these studies, we also modified NOx

- determination to refine accuracy of the measurement. First, we de-proteinized the sera before NOx

- determination, as proteins may disturb photo-metric measurements. Second, we reduced NO3

- in the sera using nitrate reductase, but not copperized cadmium granules which were used in the earlier studies, as it is unclear whether NO3

- is reduced completely by these granules. Moreover, further reduc-tion to other products such as N2 is possible by using granules (Sorte and Basak, 2010). The reduction method we used has been previously validated by comparing results of photometric meas-urements with results gained by HPLC determinations (Marzinzig et al., 1997). Finally, we used NO3

- standards instead of NO2-

standards, as 90% of the NOx- in the sera is in fact NO3

-. Hence, using NO3

- standards instead of NO2- standards ensures that our

method is less prone to erroneous measurements due to possible incomplete reduction of NO3

-. Interestingly, our NOx- levels were

two times higher than those reported in the previous studies, which might be due to the ultrafiltration method used here.

To evaluate the mechanism underlying altered NOx- levels in

bipolar patients, we investigated the NO system in greater detail using a multi-level approach including genetic and expression data. First, we genotyped the sample for genetic variants of the NOS3 and the NOS1 gene (NOS3 rs2070744, NOS3 rs1799983, NOS3 Intron 4 VNTR, NOS1 rs41279104 and NOS1 ex1f-VNTR) to examine whether there was an influence of genetic variants on NOx

- levels. We found the NOS3 rs2070744 SNP to be a genetic determinant of NOx

- concentrations specifically in bipolar patients. Also we found the NOS3 rs1799983 SNP to be associated with NOx

- concentrations in the whole sample;

however, this finding could not withstand correction for multiple comparisons. Previous studies evaluating the influence of NOS3 genotypes and haplotypes in patients suffering from diabetes mellitus type II and hypertension did not detect an association of single NOS3 genotypes with NOx

- levels, but could show an influence of the haplotypes on NOx

- concentrations (Sandrim et al., 2007). In the whole sample, NOx

- concentrations of rs2070744 C/C-carriers were nominally lower than of the C/T and T/T carriers, but this did not reach a statistically significant level. If the influence of rs2070744 on NO blood metabolites is truly specific for bipolar patients, needs to be further explored with a larger number of cases and controls.

Second, we investigated NOS1 and NOS3 expression in peripheral leukocytes of the participants. NOS3 expression was not significantly altered in BPD and aADHD compared with healthy controls. NOS1 expression could not be detected in peripheral leukocytes at all, which was surprising because pub-lished studies have reported otherwise (Molero et al., 2002; Yoshimura et al., 2012). Analysing the whole sample together, we detected an association of NOS3 rs2070744 and NOS3 expres-sion levels, with T/T carriers showing significantly higher mRNA levels. The C-allele of the T786C polymorphism (rs2070744) has been shown to reduce the promoter activity by approximately 50% (Nakayama et al., 1999) and our results thus replicate previ-ous findings showing that T/T-allele carriers exhibit increased mRNA NOS3 levels (Cattaruzza et al., 2004; Venturelli et al., 2005). Interestingly, this influence of rs2070744 on the mRNA level in the whole sample only led to lower NOx

- concentrations in C/C-carriers in the group of bipolar patients, but not in healthy controls and aADHD patients.

Several models have been proposed to explain the physiolog-ical mechanisms which lead to elevated NOx

- levels in BPD through differently activated NOS isoforms. According to these models, the activity of NOS might be enhanced through differ-ent mechanisms. First, NOS-I and -III are activated by Ca2+ influx through the postsynaptic membrane, via the NMDA receptor (for NOS-I). Several lines of evidence indicate altera-tions in the glutamatergic system in bipolar patients (McCullumsmith et al., 2007; Woo et al., 2004; Zarate et al., 2012), therefore elevated NOx

- levels in BPD might be explained by glutamate-driven postsynaptic Ca2+ influx. According to this glutamate theory, BPD occurs with states of excitotoxicity caused by elevated intracellular Ca2+ causing disruptions in the brain. However, it is hard to envisage how this can then relate to NOx

- levels in peripheral blood. Recent research hints at oxida-tive stress playing a role in the pathogenesis of BPD (Andreazza et al., 2008). Therefore a peripheral elevated NO metabolite concentration might reflect a general imbalance in pathways related to oxidative stress. Second, NO-production can be enhanced by cytokines such as TNF-α. There is growing evi-dence that at least in subgroups of BPD inflammatory pathways are involved (Goldstein et al., 2009). NO under these conditions is mainly produced by the inducible or immunological NOS also known as NOS-II. After its formation, NO subsequently reacts with superoxide to form peroxynitrite which leads to oxidative damage in DNA and cell membranes. Again, this supports the hypothesis that oxidative damage in the brain may contribute to the pathophysiology of BPD. To our best knowledge, to date there has been no evidence of an association with NOS-II genetic polymorphisms and BPD and/or aADHD, which was the reason

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from

Kittel-Schneider et al. 37

we did not genotype our sample regarding polymorphism in the NOS-II gene. Due to the fact that neither the genetic polymor-phism in the NOS-I and NOS-III genes nor the NOS3 expression explains the increased NO metabolite levels in the bipolar patients, NOS-II will be included in future studies. Because the NOS-II is not constitutionally expressed but inducible due to inflammatory stimuli, further studies also should include the measurement of inflammatory markers such as cytokines and antioxidant enzymes.

In summary, we were able to replicate the finding of elevated NO metabolites in bipolar patients. Moreover, we could confirm results from former studies showing that NOS3 rs2070744 has functional consequences on NOS3 expression and NOx

- levels. The influence on expression level was true for the whole sample, but rs2070744 may be a specific determinant of NOx

- levels in bipolar patients. It is possible that the effect of the C/C genotype on mRNA expression can be seen in the whole sample because it directly results from the reduced promoter activity, whereas NO metabolite levels are influenced by many other mechanisms. Some of them, however, seem to be altered in BPD, such as imbalances in oxidative stress pathways and/or inflammatory processes. This might be the reason why only in BPD rs2070744 has an impact on both NOS3 mRNA expression and NOx

- levels. Apparently, neither NOS1 nor NOS3 expression levels were thus the main source of the elevated NOx

- concentration in BPD. Hence, these might be either due to NOS2 or due to functional changes in the NO pathway, such as alterations in calcium levels. Regarding the role of NOx

- levels as a potential biomarker of BPD, it must be considered that the nitrinergic system seems to play a role in the pathogenesis of various psychiatric and somatic disorders. NOx

- levels might therefore not be an ideal state marker for a specific disorder, but our results add evidence for the involvement of the nitrinergic system in the pathophysiology of BPD, and further studies with extended measurement of other oxidative and inflammatory markers might lead to more disor-der-specific pathways.

LimitationsSeveral methodological limitations have to be considered. First, the number of the bipolar patients in the different polarity sub-groups was probably too small to be able to detect differences due to polarity. The sample as a whole was slightly underpow-ered to test the main hypothesis. Also, no euthymic patients were enrolled in the study. Second, the whole sample was too small for case-control association analysis, which is likely the reason that we did not find an association of single markers or haplotypes with ADHD or BPD. Also, the number of participants in the gen-otype groups was rather small for the risk variants. Third, bipolar patients were significantly older than the healthy control partici-pants and the ADHD patients, and had a different, but not statisti-cally significant, sex ratio, but as neither the NOx

- levels nor the NOS3 mRNA levels were significantly correlated with age or sex, this should therefore not bias the results. Finally, influence of different psychopharmacological treatments could be ruled out in the bipolar group and the ADHD groups when analysed sepa-rately, but as the healthy controls and the ADHD patients were not taking mood stabilizers, an influence of mood-stabilizing medication on NOx

- levels cannot finally be excluded. Further studies investigating larger samples also including drug-naïve

bipolar patients would be needed to overcome this methodologi-cal weakness.

AcknowledgementsT. Töpner, J. Auer, C. Gagel and I. Reck are credited for excellent techni-cal assistance. Finally, we thank the patients and controls for their partici-pation in this study.

Declaration of Conflicting InterestsThe authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: AR participated in two non-interventional trials sponsored by Astra Zeneca. All other authors declare that there is no conflict of interest.

FundingThe authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the DFG (Grant RE1632/5 to AR, KFO 125 to AR, and KPL; SFB TRR 58 to AR and KPL; RTG 1252 to AR and KPL), the IZKF of the University of Würzburg (N-27-N to AR, Z3-24 to SK) and the EC (NEWMOOD LSHM-CT-2003-503474 to AR and KPL). JV was sup-ported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. This work was sup-ported by the BMBF (DZHI, project 01EO1004) and the European Commission (EC FP7 project AGGRESSOTYPE (FP7-Health-2013-Innovation-1 602805). The funding sources had no other role than finan-cial support.

ReferencesAndreazza AC, Kauer-Sant’anna M, Frey BN, et al. (2008) Oxidative

stress markers in bipolar disorder: A meta-analysis. J Affect Disord 111: 135–144.

Aspide R, Gironi Carnevale UA, Sergeant JA, et al. (1998) Non-selective attention and nitric oxide in putative animal models of Attention-Deficit Hyperactivity Disorder. Behav Brain Res 95: 123–133.

Bohme GA, Bon C, Stutzmann JM, et al. (1991) Possible involvement of nitric oxide in long-term potentiation. Eur J Pharmacol 199: 379–381.

Cattaruzza M, Guzik TJ, Slodowski W, et al. (2004) Shear stress insensi-tivity of endothelial nitric oxide synthase expression as a genetic risk factor for coronary heart disease. Circ Res 95: 841–847.

Ceylan M, Sener S, Bayraktar AC, et al. (2010) Oxidative imbalance in child and adolescent patients with attention-deficit/hyperactiv-ity disorder. Prog Neuropsychopharmacol Biol Psychiatry 34: 1491–1494.

Christiansen H, Kis B, Hirsch O, et al. (2012) German validation of the Conners Adult ADHD Rating Scales (CAARS) II: Reliability, validity, diagnostic sensitivity and specificity. Eur Psychiatry 27: 321–328.

Daff S (2003) Calmodulin-dependent regulation of mammalian nitric oxide synthase. Biochem Soc Trans 31: 502–505.

Fähndrich ESRD (ed) 2007. Leitfaden zur Erfassung des psychopatholo-gischen Befundes. Halbstrukturiertes Interview anhand des AMDP-Systems. Berlin: Hogrefe.

Faul F, Erdfelder E, Lang AG, et al. (2007) G*Power 3: A flexible statis-tical power analysis program for the social, behavioral, and biomedi-cal sciences. Behav Res Methods 39: 175–191.

Fox BR Jr. (1979) Kinetics and mechanism of the Griess reaction. Anal Chem 51: 1493–1502.

Gergerlioglu HS, Savas HA, Bulbul F, et al. (2007) Changes in nitric oxide level and superoxide dismutase activity during antimanic treatment. Progr Neuropsychopharmacol Biol Psychiatry 31: 697–702.

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from

38 Journal of Psychopharmacology 29(1)

Goldstein BI, Kemp DE, Soczynska JK, et al.( 2009) Inflammation and the phenomenology, pathophysiology, comorbidity, and treatment of bipolar disorder: A systematic review of the literature. J Clin Psy-chiatry 70: 1078–1090.

Hoogman M, Aarts E, Zwiers M, et al. (2011) Nitric oxide synthase gen-otype modulation of impulsivity and ventral striatal activity in adult ADHD patients and healthy comparison subjects. Am J Psychiatry 168: 1099–1106.

Hopper R, Lancaster B and Garthwaite J (2004) On the regulation of NMDA receptors by nitric oxide. Eur J Neurosci 19: 1675–1682.

Kanwar JR, Kanwar RK, Burrow H, et al. 2009. Recent advances on the roles of NO in cancer and chronic inflammatory disorders. Curr Med Chem 16: 2373–2394.

Kiss JP and Vizi ES (2001) Nitric oxide: a novel link between synaptic and nonsynaptic transmission. Trends Neurosci 24: 211–215.

Kurrikoff T, Lesch KP, Kiive E, et al. (2012) Association of a functional variant of the nitric oxide synthase 1 gene with personality, anxiety, and depressiveness. Dev Psychopathol 24: 1225–1235.

Margraf J (1994) Diagnostisches Kurzinterview bei psychischen Störun-gen: Mini-DIPS. Berlin, Heidelberg, New York: Springer Verlag.

Marzinzig M, Nussler AK, Stadler J, et al. (1997) Improved methods to measure end products of nitric oxide in biological fluids: Nitrite, nitrate, and S-nitrosothiols. Nitric Oxide Biol Chem 1: 177–189.

McCullumsmith RE, Kristiansen LV, Beneyto M, et al. (2007) Decreased NR1, NR2A, and SAP102 transcript expression in the hippocampus in bipolar disorder. Brain Res 1127: 108–118.

Molero L, Garcia-Duran M, Diaz-Recasens J, et al. (2002) Expression of estrogen receptor subtypes and neuronal nitric oxide synthase in neutrophils from women and men: Regulation by estrogen. Cardio-vasc Res 56: 43–51.

Nakayama M, Yasue H, Yoshimura M, et al. (1999) T-786–>C muta-tion in the 5’-flanking region of the endothelial nitric oxide syn-thase gene is associated with coronary spasm. Circulation 99, 2864–2870.

Nelson RJ, Trainor BC, Chiavegatto S, et al. (2006) Pleiotropic contribu-tions of nitric oxide to aggressive behavior. Neurosci Biobehav Rev 30: 346–355.

Reif A, Grunblatt E, Herterich S, et al. (2011a) Association of a func-tional NOS1 promoter repeat with Alzheimer’s disease in the VITA cohort. J Alzheimers Dis 23: 327–333.

Reif A, Herterich S, Strobel A, et al. (2006a) A neuronal nitric oxide synthase (NOS-I) haplotype associated with schizophrenia modifies prefrontal cortex function. Mol Psychiatry 11: 286–300.

Reif A, Jacob CP, Rujescu D, et al. (2009) Influence of functional variant of neuronal nitric oxide synthase on impulsive behaviors in humans. Arch Gen Psychiatry 66: 41–50.

Reif A, Kiive E, Kurrikoff T, et al. (2011b) A functional NOS1 promoter polymorphism interacts with adverse environment on functional and dysfunctional impulsivity. Psychopharmacology (Berl) 214: 239–248.

Reif A, Schmitt A, Fritzen S, et al. (2004) Differential effect of endothe-lial nitric oxide synthase (NOS-III) on the regulation of adult neuro-genesis and behaviour. Eur J Neurosci 20: 885–895.

Reif A, Strobel A, Jacob CP, et al. (2006b) A NOS-III haplotype that includes functional polymorphisms is associated with bipolar disor-der. Int J Neuropsychopharmacol 9: 13–20.

Retz-Junginger P, Retz W, Blocher D, et al. (2002) [Wender Utah rating scale. The short-version for the assessment of the attention-deficit hyperactivity disorder in adults]. Nervenarzt 73: 830–838.

Sandrim VC, De Syllos RW, Lisboa HR, et al. (2007) Influence of eNOS haplotypes on the plasma nitric oxide products concentrations in hypertensive and type 2 diabetes mellitus patients. Nitric Oxide 16: 348–355.

Savas HA, Gergerlioglu HS, Armutcu F, et al. (2006) Elevated serum nitric oxide and superoxide dismutase in euthymic bipolar patients: Impact of past episodes. World J Biol Psychiatry 7: 51–55.

Savas HA, Herken H, Yurekli M, et al. (2002) Possible role of nitric oxide and adrenomedullin in bipolar affective disorder. Neuropsy-chobiology 45: 57–61.

Selek S, Savas HA, Gergerlioglu HS, et al. (2008a) The course of nitric oxide and superoxide dismutase during treatment of bipolar depres-sive episode. J Affect Disord 107: 89–94.

Selek S, Savas HA, Gergerlioglu HS, et al. (2008b) Oxidative imbal-ance in adult attention deficit/hyperactivity disorder. Biol Psychol 79: 256–259.

Sessa WC (1994) The nitric oxide synthase family of proteins. J Vasc Res 31: 131–143.

Sorte K and Basak A (2010) Development of a modified copper-cad-mium reduction method for rapid assay of total nitric oxide. Anal Methods 2: 944–947.

Tarpey MM and Fridovich I (2001) Methods of detection of vascular reactive species: Nitric oxide, superoxide, hydrogen peroxide, and peroxynitrite. Circ Res 89: 224–236.

Vandesompele J, De Preter K, Pattyn F, et al. (2002) Accurate normaliza-tion of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034.

Venturelli E, Galimberti D, Lovati C, et al. (2005) The T-786C NOS3 polymorphism in Alzheimer’s disease: Association and influence on gene expression. Neurosci Lett 382: 300–303.

Volke V, Wegener G, Bourin M, et al. (2003) Antidepressant- and anxiolytic-like effects of selective neuronal NOS inhibitor 1-(2-trifluoromethylphenyl)-imidazole in mice. Behav Brain Res 140: 141–147.

Weber H, Kittel-Schneider S, Gessner A, et al. (2011) Cross-disorder analysis of bipolar risk genes: Further evidence of DGKH as a risk gene for bipolar disorder, but also unipolar depression and adult ADHD. Neuropsychopharmacology 36: 2076-2085.

Weber H, Klamer D, Freudenberg F, et al. (2014) The genetic con-tribution of the NO system at the glutamatergic post-synapse to schizophrenia: Further evidence and meta-analysis. Eur Neuropsy-chopharmacol 24: 65-85.

Woo TU, Walsh JP and Benes FM (2004) Density of glutamic acid decarboxylase 67 messenger RNA-containing neurons that express the N-methyl-D-aspartate receptor subunit NR2A in the anterior cin-gulate cortex in schizophrenia and bipolar disorder. Arch Gen Psy-chiatry 61: 649–657.

Yanik M, Vural H, Tutkun H, et al. (2004) The role of the arginine-nitric oxide pathway in the pathogenesis of bipolar affective disorder. Eur Arch Psychiatry Clin Neurosci 254: 43–47.

Yao JK, Leonard S and Reddy RD (2004) Increased nitric oxide radicals in postmortem brain from patients with schizophrenia. Schizophr Bull 30: 923–934.

Yoshimura T, Moon TC, St Laurent CD, et al. (2012) Expression of nitric oxide synthases in leukocytes in nasal polyps. Ann Allergy Asthma Immunol 108: 172–177.

Zarate CA, Jr, Brutsche NE, Ibrahim L, et al. (2012) Replication of ket-amine’s antidepressant efficacy in bipolar depression: A randomized controlled add-on trial. Biol Psychiatry 71: 939–946.

at TEXAS SOUTHERN UNIVERSITY on December 10, 2014jop.sagepub.comDownloaded from