Lu et al. BMC Psychiatry 2014, 14:123 http://www.biomedcentral.com/1471-244X/14/123 RESEARCH ARTICLE Open Access Decreased plasma neuroactive amino acids and increased nitric oxide levels in melancholic major depressive disorder Yun-Rong Lu1,2†, Xin-Yan Fu2†, Li-Gen Shi2, Yan Jiang1, Juan-Li Wu2, Xiao-Juan Weng2, Zhao-Pin Wang2, Xue-Yan Wu2, Zheng Lin1, Wei-Bo Liu1, Hui-Chun Li1, Jian-Hong Luo2 and Ai-Min Bao2* Abstract Background: Amino acid neurotransmitters and nitric oxide (NO) are involved in the pathogenesis of major depressive disorder (MDD). Here we want to establish whether changes in their plasma levels may serve as biomarker for the melancholic subtype of this disorder. Methods: Plasma levels of glutamic acid (Glu), aspartic acid (Asp), glycine (Gly), gamma-aminobutyric acid (GABA), and NO were determined in 27 medicine-naïve melancholic MDD patients and 30 matched controls. Seven of the MDD patients participated also in a follow-up study after 2 months’ antidepressant treatment. The relationship between plasma and cerebral-spinal fluid (CSF) levels of these compounds was analyzed in an additional group of 10 non-depressed subjects. Results: The plasma levels of Asp, Gly and GABA were significantly lower whereas the NO levels were significantly higher in melancholic MDD patients, also after 2 months of fluoxetine treatment. In the additional 10 non-depressed subjects, no significant correlation was observed between plasma and CSF levels of these compounds. Conclusion: These data give the first indication that decreased plasma levels of Asp, Gly and GABA and increased NO levels may serve as a clinical trait-marker for melancholic MDD. The specificity and selectivity of this putative trait-marker has to be investigated in follow-up studies. Keywords: Major depressive disorder, Glutamic acid, Aspartic acid, Glycine, Gamma - aminobutyric acid, Nitric oxide Background MDD is thought to be due to the interaction between gen- Amino acid neurotransmitters, i.e. the excitatory neuro- etic, early developmental and environmental factors, lead- transmitters glutamic acid (Glu) and aspartic acid (Asp), ing to an individual’s stress response system becoming and the inhibitory neurotransmitters glycine (Gly) and overly responsive to stressful life events, which causes gamma-aminobutyric acid (GABA), are involved in the this system to go into overdrive [4]. The hypothalamo- pathogenesis of mood disorders such as major depressive pituitary-adrenal (HPA)-axis, the monoamine systems and disorder (MDD) [1,2]. The gaseous neurotransmitter nitric the autonomic nervous system are key regulating systems oxide (NO), which is synthesized from amino acid L- for stress responses and form major pathways for arginine (Arg) by NO synthases with citrulline (Cit) as a symptoms of depression [4,5]. Altered glutamatergic and by-product also plays a significant role in depression [3]. reduced GABAergic and NO neurotransmission were ob- served in depression in a number of brain systems, which * Correspondence: [email protected] may significantly affect the neuronal activity involved in †Equal contributors stress responses and mood regulation [1,2,6]. Changes in 2 Department of Neurobiology; Key Laboratory of Medical Neurobiology of depression in the levels of these amino acids were found Ministry of Health of China; Zhejiang Province Key Laboratory of Neurobiology, Zhejiang University School of Medicine, 866 Yuhangtang not only in different brain regions and cerebrospinal fluid Road, Hangzhou 310058, Zhejiang, P. R. China (CSF), but also in blood and urine [7], which implies that Full list of author information is available at the end of the article © 2014 Lu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lu et al. BMC Psychiatry 2014, 14:123 Page 2 of 7 http://www.biomedcentral.com/1471-244X/14/123 they have the potential to serve as clinically relevant had no suicidal thoughts. The exclusion criteria were, biomarkers or treatment efficacy monitors. Plasma levels apart from BD, as follows: psychiatric co-morbidity in of NO metabolites, i.e. nitrite and nitrate, which reflect the the form of substance abuse disorders, psychotic disor- plasma NO concentrations, were also reported to increase ders, anxiety disorders or mental retardation, chronic in depression [8,9]. So far there are, however, quite some physical illness, or abnormal body mass index (BMI ≤ 18 inconsistent findings concerning the plasma amino acid or BMI ≥ 25). The follow-up protocol was as follow: after levels in depression. Decreased [10], increased [11], or no 2 months of antidepressant treatment (fluoxetine 20-40 changes in plasma Gly [12] were reported in depression. In mg/day) the patients came back to the clinic and their addition, no alteration [10] or increase in plasma Glu [11] symptoms and HAMD scores were evaluated again, was observed in depression. These controversial results together with their blood samples being taken. Seven may be due, at least partly, to the variety of depression sub- (6 males and 1 female) out of the 27 melancholic MDD types studied, and to confounding factors such as different patients whose clinical symptoms had improved and gender-ratios of samples, and/or different treatments. whose HAMD scores were at that moment lower than Furthermore, amino acid transporter systems are located 14 (data not shown) voluntarily joined the follow-up in the blood brain barrier (BBB) and transport amino acids study. The 30 healthy control subjects (15 males and 15 from the blood into the brain and vice versa [13]. It is at females, age range 30 to 65 years) who underwent their present, however, not clear whether there is a relationship yearly physical examination in the same hospital volun- between the plasma and the CSF levels of these amino tarily participated in this study during the same period. acids, although some researchers have proposed that the The exclusion criteria for the controls were as follows: plasma levels of these neuroactive amino acids might, to a abnormal BMI, medication for chronic illnesses or men- certain degree, reflect their brain levels [2,14]. tal disorders. The present study aimed, therefore, to analyze the Ten additional patients (4 males and 6 females, age plasma levels of 4 neuroactive amino acids, i.e. Asp, Glu, range 20 to 68 years) with a range of medical conditions, Gly and GABA, and NO levels calculated as Cit/Arg [15] including stroke, uterus with scar, leukemia, brainstem in the major subgroup of melancholic MDD, medicine- hemorrhage, and subarachnoid haemorrhage, and whose naïve patients in their first depressive episode, in relation CSF and plasma samples were obtained for clinical rea- to their clinical symptoms. In addition, the relationship sons within the same hour, voluntarily participated in between plasma and CSF levels of these amino acids was the present study during their stay in the same hospital. analyzed in a group of non-depressive patients. They were all during their clinical recovery phase. The inclusion criteria for this group were as follows: color- Methods less, transparent CSF, leukocyte cell count < 100 × 106/L, Subjects red cell count < 100 × 106/L. The exclusion criteria for Thirty Chinese Han race MDD patients and 30 age- and this group were as follows: abnormal BMI and/or psy- sex-matched control subjects had been recruited in the chiatric disorders. study by the Department of Psychiatry of the Second The investigation was carried out in accordance with the Affiliated Hospital of Zhejiang University School of latest version of the Declaration of Helsinki. All subjects Medicine. Three out of the 30 MDD patients appeared, signed informed consent forms and the study was ap- however, later to develop bipolar disorder (BD). There- proved by the Medical Ethics Committee of the Second fore, 27 MDD patients (14 medicine-naïve males and 13 Affiliated Hospital of Zhejiang University School of Medi- medicine-naïve females, age range 30 to 68 years) who cine. Venous blood samples or CSF samples were collected were in their first depressive episode participated in the between 6:00 a.m. and 9:00 a.m., and frozen at -80°C until present study. They were diagnosed as melancholic measured. MDD according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) by qualified High performance liquid chromatography (HPLC) with psychiatrists. The Mini International Neuropsychiatric fluorescence detection (FLD) for plasma and CSF amino Interview (Chinese modified version) was used to con- acid analysis firm the DSM-IV diagnosis. The 24-item Hamilton De- The plasma (1 ml) and CSF (1-1.5 ml) samples were pression Scale (HAMD) was used to rate the severity of deproteinized with acetonitrile (1:1 v/v), lyophilized and depression, and a score of 35 or above was considered to diluted with 60% (v/v) methanol before analysis. be severe depression [16]. Seven out of the 27 melan- The plasma or CSF amino acid levels were determined by cholic MDD patients had suicidal thoughts, 7 attempted HPLC, which included pre-column derivatization with o- suicide before they joined the study and 1 actually com- phthalaldehyde (Cat: O120, Pickering Laboratories, Inc. mitted suicide and died after one month of the treat- USA) and 3-mercaptopropionic acid (MPA, CAS: 107-96-0, ment. Twelve out of the 27 melancholic MDD patients Acros Organics, NJ, USA), reverse phase separation with Lu et al. BMC Psychiatry 2014, 14:123 Page 3 of 7 http://www.biomedcentral.com/1471-244X/14/123 Agilent 1100 series HPLC system and FLD (Agilent Tech- No changes were found in plasma Glu levels (P = 0.646).