Physically healthy patients with depression have an increased risk of developing cardiovascular disease (CVD).Reference Lichtman, Bigger, Blumenthal, Frasure-Smith, Kaufmann and Lespérance1 A meta-analysis of prospective cohort studies found that major depressive disorder (MDD) was associated with a significantly increased risk of myocardial infarction and coronary death.Reference Wu and Kling2 This association indicates that endothelial dysfunction may be involved in the development of depression.
Nitric oxide (NO) is a small free radical that plays a critical signalling role in physiology and pathophysiology. Producing sufficient amounts of NO to regulate vascular resistance and maintain adequate blood flow is important for healthy functioning of the vascular system. Endothelial nitric oxide synthase (eNOS) is a constitutive NOS form in endothelial cells that contributes significantly to vascular NO levels under physiological conditions.Reference Huang, Huang, Mashimo, Bloch, Moskowitz and Bevan3 Since NO can be rapidly inactivated by haemoglobin or oxidised to forms of nitrogen dioxide such as nitrates and nitrites (NOx), the plasma NOx level is often used as an indicator of NO production in blood vessels.Reference Archer4 eNOS immunoreactivity decreased in a rat model of depression.Reference Demirtaş, Utkan, Karson, Yazır, Bayramgürler and Gacar5 The possible role of NO in depression has been confirmed through the link between an increased risk of coronary heart disease and NO production, and the effects of antidepressants on NO production.Reference Finkel, Laghrissi-Thode, Pollock and Rong6–Reference Rajagopalan, Brook, Rubenfire, Pitt, Young and Pitt8 We previously reported that milnacipran, a serotonin–noradrenaline reuptake inhibitor, increased plasma levels of NOx in patients with MDD using a small sample.Reference Ikenouchi-Sugita, Yoshimura, Hori, Umene-Nakano, Ueda and Nakamura9 The present study aimed to confirm our previous preliminary findings using a larger sample and different antidepressants.
Method
Participants
This study included 100 Japanese in-patients and out-patients who met DMS-IV-TR criteriaReference First, Spitzer, Gibbon and Williams10 for a first episode of MDD without psychotic features and who scored 8 or higher on the 17-item Hamilton Rating Scale for Depression (HRSD-17).Reference Zimmerman, Martinez, Attiullah, Friedman, Toba and Boerescu11 We also enrolled 50 gender- and age-matched healthy controls recruited from a group of Japanese healthcare workers. We screened for illness history using a questionnaire. All the patients were physically healthy, had no history of psychiatric disorders and had not used any psychotropic medications in the past 6 months; they had not taken antidepressants or mood stabilisers for at least 2 weeks prior to the start of the study. All the patients and healthy controls were non-smokers with no history of CVD.
All participants initially underwent electrocardiography and blood tests to rule out cardiovascular and hepatic dysfunction. Severity of depression was evaluated using the HRSD-17. During this study the patients were treated with the antidepressants sertraline, duloxetine or mirtazapine (24, 48 and 28 patients, respectively) and followed for 8 weeks after initiation of treatment. The attending physician determined the type and dosage of antidepressant using a flexible dose design.
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human patients were approved by the Ethics Committee of the University of Occupational and Environmental Health of Japan (Approval No. 09–08). Written informed consent was obtained from all participants.
Blood samples and assay method
All blood samples were taken at 07.00 h before breakfast (at least 12 h after the last dose of medication). We obtained venous blood samples from participants while they were in a supine position. The plasma samples were separated in a centrifuge and stored at −80°C until analysis. Plasma NOx levels were analysed by the Griess method using high-performance liquid chromatography (ENO-20; Eicom, Kyoto, Japan).
Statistical analyses
The clinical and biochemical data of the study participants are expressed as mean (s.d.). The association between HRSD-17 scores and plasma NOx levels was examined using Spearman's correlation coefficient. Differences in NOx levels between baseline and week 8 were examined by Wilcoxon matched-pairs signed-rank test as the plasma NOx levels were not normally distributed in the Kolmogorov–Smirnov test. The changes in plasma NOx levels in the antidepressant groups were compared using the Kruskal–Wallis test. This comparison was also undertaken using analysis of covariance to adjust for gender and age.
We conducted all statistical analyses using Stata 17 (Stata Corp LLC, College Station, Texas, USA). The statistical significance was set at P < 0.05.
Results
Table 1 shows the characteristics of the study population. The HRSD-17 scores of the MDD group significantly decreased from baseline to 8 weeks after treatment with antidepressants. The plasma NOx levels at baseline were significantly lower in the MDD group compared with the control group (P < 0.001, P-adjusted for age and gender) and increased after 8 weeks of treatment. There was a negative correlation between the baseline HRSD-17 scores and plasma NOx levels (r = −0.278, P = 0.017), and between the HRSD-17 scores (week 8 – baseline) and NOx levels (week 8 – baseline) in patients with MDD (r = −0.269, P = 0.021).
Characteristics of the study and control populations
HRSD-17, 17-item Hamilton Rating Scale for Depression; NOx, metabolites of nitric oxide.
a. The P-values are derived from Wilcoxon matched-pairs signed-rank test.
Eight weeks of treatment using duloxetine resulted in significantly increased plasma NOx levels (P = 0.0127). Sertraline and mirtazapine did not significantly change these levels. The changes in plasma NOx levels did not differ among the antidepressant groups (P = 0.1945). The results were similar for gender- and age-adjusted analysis of covariance (P = 0.224).
Discussion
Main findings
This study's findings showed that the plasma NOx levels of patients with MDD were significantly lower than those of healthy controls. Treating the depression with antidepressants recovered the plasma NOx levels. There was a negative correlation between the HRSD-17 score and plasma NOx concentration, confirming our preliminary findings.Reference Ikenouchi-Sugita, Yoshimura, Hori, Umene-Nakano, Ueda and Nakamura9 In contrast, in a study by Suzuki et al, the plasma NOx levels in patients with MDD were reported to be significantly higher than those in healthy controls and to decrease after recovery from MDD.Reference Suzuki, Yagi, Nakaki, Kanba and Asai12 Furthermore, there are several reports that suicide attempts in patients with mood disorders and schizophrenia are associated with elevated plasma NOx levels.Reference Lara, Archer, Baker and Le Mellédo7,Reference Kim, Paik, Lee, Yoon, Han and Lee13 Thus, the effect of plasma NOx levels on patients with MDD remains controversial.
Treatment with duloxetine, but not sertraline and mirtazapine, increased plasma NOx levels at week 8. We previously reported that in patients with MDD, administration of milnacipran, but not paroxetine, increased plasma NOx levels.Reference Ikenouchi-Sugita, Yoshimura, Hori, Umene-Nakano, Ueda and Nakamura9 Taking these findings into account, it is possible that duloxetine, which influences both serotonin transporters and noradrenaline transporters, increases plasma NOx levels. In contrast, paroxetine and sertraline both specifically only affect serotonin transporters and do not change plasma NOx levels. Seya et al demonstrated that norepinephrine induces eNOS activity.Reference Seya, Fukuda, Isobe, Kawakami and Takekoshi14 Graves & Poston also reported that NO is released from the endothelium of rat mesenteric resistance arteries by activation of β1-adrenergic receptors.Reference Graves and Poston15 Thus, antidepressants that influence norepinephrine neurons might play a role in affecting plasma NOx levels, mainly via noradrenergic signalling.
Limitations
This study has some limitations. First, only plasma NOx levels were measured, and not eNOS activity. Second, body weight and body mass index, which influence plasma NOx levels, were not adjusted for. Third, plasma NOx levels in the control group were not assessed after 8 weeks. Fourth, and perhaps most importantly, a small sample size and weak statistical power may have influenced the outcome. Moreover, we used a flexible drug design. Therefore, further studies without these limitations need to be performed to obtain robust results.
In conclusion, NO might be partially involved in the pathophysiology of MDD, and some antidepressants affecting both noradrenaline and serotonin neurons increase plasma NOx levels in patients with MDD.
Data availability
The data that support the findings of this study are available on request from the corresponding author, A.I.
Acknowledgements
We would like to thank Editage (www.editage.com) for English-language editing.
Author contributions
A.I. and R.Y. investigated and drafted the manuscript. N.O., R.F., Y.K. and Y.F. analysed the data. All authors have read and approved the final manuscript.
Funding
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Declaration of interest
None.
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