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

Abstract

Introduction:

Several studies have shown altered levels of nitric oxide (NO) and its stable metabolites (NO_x^-) 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 NO_x^- 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 NO_x^- levels (p=0.05).

Conclusion:

We could replicate and extend previous findings showing altered NO_x^- levels in BPD and an influence of NOS3 rs2070744 on NOS3 expression and NO_x^- concentration. Together, these data point to a role of the nitrinergic pathway in BPD.

Keywords

Nitric oxide bipolar disorder attention deficit-hyperactivity disorder genetic polymorphism biomarkers

Introduction

Nitric oxide (NO) is a gaseous messenger molecule with pleiotropic 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 guanylate 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 glutamatergic 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 NO_x^-) 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 bipolar disorder (BPD) have been linked to short alleles of the functional 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 decreased (Reif et al., 2004). Pharmacological inhibition of NOS-I has been shown to have both anxiolytic and antidepressant properties (Volke et al., 2003).

Regarding functional studies on NO, several studies have assessed NO_x^- levels in psychiatric patients. In patients suffering 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-mortem brain samples and the studies analysing NO_x^- levels in ADHD and BPD were conducted by the same group. As a consequence, 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 variation 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 NO_x^- 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 polymerase chain reaction (qRT PCR) to assess the expression levels 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 validated 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 elsewhere (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 control participants were severe medical conditions, severe neurological disorders, mental retardation, current carcinomas and acute and chronic infectious diseases. Exclusion criteria for controls were a history of psychiatric disorder or first-line relatives 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 NO_x^- measurements, 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.

Table 1.

Demographic data.

Age at sampling (years)	n	m	SD
Control	33	34.1	± 11.4
ADHD	14	34.3	± 12.5
Bipolar	41	46.5	± 13.9
SEX (MALE/FEMALE)	n	ratio
Control	15/18	0.83
ADHD	7/7	1.0
Bipolar	14/27	0.52
Bipolar		n
Lithium only MS		2
Antipsychotic only MS		10
Anticonvulsant only MS		4
Combination Li+antipsychotic		2
Combination Li+anticonvulsant		1
Combination Li+antipsychotic+anticonvulsant		1
Combination antipsychotic+anticonvulsant		18
No mood stabilizer		2
Medication not available		1
Plus antidepressant		22
Psychiatric comorbidities
Substance abuse disorder		3
Other (Fibromyalgia)		1
Bipolar I/Bipolar II		16/25
Polarity at sampling
depressive		25
(hypo-)manic		9
mixed		6
rapid cycling		1
ADHD
Stimulant medication		0
Antidepressant		8
Psychiatric Comorbidties
Major Depression		9
Substance abuse disorder		3
Abbreviations: MS=mood stabilizer, Li=Lithium

Measuring NO_x^- 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. NO_x^- levels were determined after filtration with the Nitrite/Nitrate Assay Kit colorimetric from Sigma-Aldrich (Buchs, Switzerland) in triplicates according 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 (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 visualized, 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 individuals and 71 and 58 bp for the minor allele. For rs1799983, amplicons 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 384^TM real-time PCR detection System from Biorad (Hercules, USA). Expression of the target genes (NOS1 and NOS3) and the housekeeping genes (18S rRNA, ACTB, ALAS, GAPDH) was estimated 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 specific. Normalization of the target genes against the housekeeping 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 distribution, 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 analysis 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 NO_x^- levels, r=0.32, and α=0.05. A marginally sufficient power of 1–ß=0.76 was achieved.

Results

NO_x^- concentration in de-proteinized blood serum

NO_x^- 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 antidepressant treatment on NO metabolite concentration, and the subgroup of BPD was tested additionally regarding the influence of treatment with mood stabilizers. There was no significant influence of either treatment on NO_x^- levels. NO_x^- concentrations differed significantly in patients suffering from BPD as compared 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 significant difference between controls and ADHD patients. Bipolar patients nominally showed the highest NO_x^- levels and ADHD patients the lowest. In a post-hoc analysis, BPD patients were analysed stratified by polarity. NO_x^- concentrations were nominally 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 NO_x^- 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 polymorphisms 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, haplotype 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 association with aADHD.

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= 0.349
Exon 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=0.600
NOS3
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=0.138
Intron 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=0.303
rs1799983
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.

Regarding the influence of single marker polymorphisms on NO_x^- levels, NOS3 rs1799983 (G894T) T/T carriers showed significantly higher NO_x^- levels than G/T and G/G carriers on analysis 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 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 NO_x^- 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 haplotype analysis, none of the included haplotypes was associated with altered NO_x^- levels.

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	0.07
G/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.

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 expression 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 genotypes (T/T>C/T>C/C) remained statistically significant after correction 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 severity 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 correlation 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 no significant correlation between NO_x^- concentrations and NOS3 mRNA levels in the whole sample.

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	0.05
C/T	0.39	0.033
T/T	0.45	0.044

Discussion

In this study, we aimed to replicate and extend former studies showing increased NO_x^- 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 NO_x^- levels in a smaller group of aADHD patients. However, the aADHD group did not show significant differences regarding NO_x^- 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 NO_x^- levels in bipolar patients, which replicates and extends previous findings showing elevated NO_x^- 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 NO_x^- 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 comparison with these studies, we also modified NO_x^- determination to refine accuracy of the measurement. First, we de-proteinized the sera before NO_x^- determination, as proteins may disturb photometric 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 reduction 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 measurements with results gained by HPLC determinations (Marzinzig et al., 1997). Finally, we used NO₃^- standards instead of NO₂^- standards, as 90% of the NO_x^- 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 NO_x^- 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 NO_x^- 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 NO_x^- levels. We found the NOS3 rs2070744 SNP to be a genetic determinant of NO_x^- concentrations specifically in bipolar patients. Also we found the NOS3 rs1799983 SNP to be associated with NO_x^- 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 NO_x^- levels, but could show an influence of the haplotypes on NO_x^- concentrations (Sandrim et al., 2007). In the whole sample, NO_x^- 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 published 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 expression 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 previous 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 NO_x^- 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 physiological mechanisms which lead to elevated NO_x^- levels in BPD through differently activated NOS isoforms. According to these models, the activity of NOS might be enhanced through different 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 alterations in the glutamatergic system in bipolar patients (McCullumsmith et al., 2007; Woo et al., 2004; Zarate et al., 2012), therefore elevated NO_x^- 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 NO_x^- levels in peripheral blood. Recent research hints at oxidative 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 evidence 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 we did not genotype our sample regarding polymorphism in the NOS-II gene. Due to the fact that neither the genetic polymorphism 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 NO_x^- levels. The influence on expression level was true for the whole sample, but rs2070744 may be a specific determinant of NO_x^- 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 NO_x^- levels. Apparently, neither NOS1 nor NOS3 expression levels were thus the main source of the elevated NO_x^- 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 NO_x^- 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. NO_x^- 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 disorder-specific pathways.

Limitations

Several methodological limitations have to be considered. First, the number of the bipolar patients in the different polarity subgroups was probably too small to be able to detect differences due to polarity. The sample as a whole was slightly underpowered 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 genotype groups was rather small for the risk variants. Third, bipolar patients were significantly older than the healthy control participants and the ADHD patients, and had a different, but not statistically significant, sex ratio, but as neither the NO_x^- 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 separately, but as the healthy controls and the ADHD patients were not taking mood stabilizers, an influence of mood-stabilizing medication on NO_x^- levels cannot finally be excluded. Further studies investigating larger samples also including drug-naïve bipolar patients would be needed to overcome this methodological weakness.

Footnotes

Acknowledgements

T. Töpner, J. Auer, C. Gagel and I. Reck are credited for excellent technical assistance. Finally, we thank the patients and controls for their participation in this study.

Declaration of Conflicting Interests

The 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.

Funding

The 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 supported by a grant of the German Excellence Initiative to the Graduate School of Life Sciences, University of Würzburg. This work was supported 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 financial support.

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

Abstract

Introduction:

Methods:

Results:

Conclusion:

Keywords

Introduction

Materials and methods

Participants and sample preparation

Measuring NOx- concentration in de-proteinized blood serum

Genotyping

Expression of NOS1 and NOS3 mRNA in blood cells

Statistical analysis

Power analysis

Results

NOx- concentration in de-proteinized blood serum

Genotyping

Quantification of NOS1 and NOS3 mRNA in human leukocyte blood cells

Discussion

Limitations

Footnotes

Acknowledgements

Declaration of Conflicting Interests

Funding

References

Measuring NO_x^- concentration in de-proteinized blood serum

NO_x^- concentration in de-proteinized blood serum