Major depressive disorder (MDD) is an important disease that affects millions of people worldwide and can significantly impair quality of life. It can also increase health expenditures and impose a serious burden on the economy directly or indirectly. Although MDD is one of the most common psychiatric disorders in children and adolescents, its etiopathogenesis is still unclear. Currently, no single system or mechanism can explain all aspects of the pathophysiology of MDD. Existing neurochemical studies on the subject focus more on monoaminergic neurotransmitters such as serotonin, noradrenaline, and dopamine. However, the pathophysiology of the disorder cannot be explained only by the monoamine hypothesis, and drugs acting on monoamines do not show the desired effect in all MDD patients [1]. This situation leads researchers to evaluate different areas in the etiopathogenesis of MDD.

Metabolism of L-arginine and polyamines continues to be of interest to researchers in neuropsychiatric disorders. Arginine is a positively charged amino acid. It is metabolized in the human body in three main pathways [2]. In one of these pathways, L-arginine is cleaved into urea and ornithine by the enzyme arginase; ornithine later forms putrescine in a reaction catalyzed by the enzyme ornithine decarboxylase (ODC) [3]. In the second pathway, nitric oxide (NO) is formed from L-arginine via the enzyme nitric oxide synthase (NOS) [4]. In the third pathway, L-arginine is converted to agmatine via arginine decarboxylase (ADC). Agmatine forms other polyamines such as putrescine, spermidine, and spermine by the enzyme agmatinase (Fig. 1) [4].

Although there are different usages in the literature, the term polyamine is primarily used in practice to refer to putrescine, spermidine, and spermine. Some sources include agmatine among the polyamines, while others do not. Polyamines are low molecular weight aliphatic amines and are fully protonated at human body pH. Agmatine is first hydrolyzed to putrescine. Putrescine then forms spermidine and spermine (Fig. 1) [5].

Agmatine, as a biogenic amine, has a wide range of functions including regulation of cell cycle, cell differentiation and proliferation, DNA/RNA stabilization, regulation of innate immunity, and anti-inflammatory response [6,7]. The neuroprotective activity of agmatine was first described by Gilad and Gilad [8], and this finding was later supported by the findings of various studies using experimental models [9,10]. It has been suggested that agmatine can be considered to be a neurotransmitter due to its neurochemical properties and effects on central nervous system receptors [11].

Polyamines such as putrescine, spermidine, and spermine, like agmatine, are molecules with important roles in maintaining the basic functions of the nervous system. They are involved in many physiological functions, including cell growth and proliferation, synthesis of proteins and nucleic acids, differentiation of immune cells, and regulation of inflammatory processes [6]. Polyamines can function as various ionotropic membrane receptor modulators such as nicotinic receptors, gamma-aminobutyric acid (GABA) receptors, and N-methyl-D-aspartate (NMDA) receptors. Thus, they regulate ion flux by modulating ion channels and may modulate some intracellular signaling pathways [12,13]. Modulation of glutamate signaling by polyamines affects a wide range of functional processes in the brain, from the regulation of neuronal and glial excitability to memory and aging [14]. Polyamines also interact with nucleic acids and proteins. By binding to DNA, they affect both the structure and stability of DNA [12].

In the literature, it is stated that agmatine might be an endogenous neuromodulator of mental stress and may play a role in some stress-related diseases such as depression and anxiety disorders [15]. Some researchers suggest that endogenous agmatine is induced in response to stress/inflammation [16]. Various preclinical studies have shown that agmatine might be an endogenous molecule with antidepressant effects [17,18].

Similar to the “agmatine-stress relationship” hypothesis mentioned above, some studies in the literature also mention a mechanism called “polyamine stress response” (PSR) [19]. According to the PSR, a dysregulation in polyamine synthesis occurs when a stressful stimulus is encountered. PSR can be adaptive and/or maladaptive [19,20]. It is stated that PSR is a molecular mechanism involving adaptive and/or maladaptive brain responses to stressful events; in this context, proper regulation of polyamine metabolism might be critical for an appropriate response to stressors [19]. Similar to agmatine, putrescine has been shown to have antidepressant effects in some preclinical studies [21].

Agmatine has also been shown to play a regulatory role in different steps of L-arginine/polyamine metabolism. The major synthesis pathway of polyamines in vertebrates is the one that starts with the formation of ornithine from L-arginine via the enzyme arginase. Putrescine is then formed from ornithine by catalysis of the enzyme ODC [4]. ODC is one of the rate-limiting enzymes in this polyamine biosynthesis pathway [22]. The degradation of ODC is regulated by an endogenous protein called antizyme. Antizyme protein binds to ODC, accelerating its degradation and inhibiting its activity [23]. Antizyme protein also regulates the transport of polyamines via the polyamine transporter [22]. In a preclinical study, agmatine was shown to induce antizyme protein [24]. In addition, agmatine decreases the synthesis of NO, which has been suggested to play a role in the pathophysiology of major depression, by inhibiting NOS [25].

It has been suggested that agmatine and its metabolites might be associated with many psychiatric disorders, including MDD [26-28]. However, more research is needed in this field due to both the lack of a clear mechanism and the insufficient level of evidence of the current findings. As far as can be seen in the literature, there is no study evaluating blood levels of agmatine, putrescine, spermidine, and spermine in adolescents with MDD. There is a very limited number of clinical studies on the subject conducted with adult patients. When these studies were analyzed, it was observed that agmatine was evaluated in three studies [27,29,30] and putrescine in only one study [27].

Considering this gap in the literature on the subject, the aim of this study was to evaluate the blood levels of agmatine, putrescine, spermidine, and spermine in adolescents diagnosed with MDD and to compare the findings with healthy controls.

The study was conducted between April 2020 and April 2022 at Firat University Faculty of Medicine, Child and Adolescent Mental Health and Diseases Outpatient Clinic. The study protocol was approved by Firat Univer-sity Non-Interventional Research Ethics Committee (20.04.2020-389943). The study was supported by the decision of Firat University Scientific Research Projects Coordination Unit with the number TF.20.17. All procedures were performed in accordance with the principles listed in the Declaration of Helsinki.

Participants

The MDD group was composed of patients who presented to the outpatient clinic with depressive symptoms and met the Diagnostic and Statistical Manual of Mental Disorders, 5th edition (DSM-5) MDD diagnostic criteria. The control group was composed of children from volunteer families, taking into account the age and gender distribution of the MDD group. A total of 89 subjects, including 45 cases diagnosed with MDD and 44 healthy control subjects aged 13−17 years, were included in the study.

In the MDD group, those with comorbid psychiatric diagnosis on psychiatric evaluation, those who had used any psychotropic medication in the last 6 months, those who had regularly used any medication/nutritional supplement in the last 6 months, and those with acute/chronic systemic diseases were excluded from the study. In the control group, participants who had any psychiatric diagnosis in psychiatric evaluation, who had regularly used any medication/nutritional supplement within the last six months, and who had acute/chronic systemic diseases were excluded from the study. The mental capacity of the participants was assessed clinically. According to the detailed history and clinical evaluation, subjects who had no problems in basic adaptive skills such as academic achievement, learning, understanding/comprehension, problem solving, association, reasoning, abstract thinking, planning, and neuromotor development according to their age were included in the study.

Clinical Evaluation

Written informed consent was obtained from the parents of all participants. Sociodemographic data were recorded on the sociodemographic data form. The Schedule for Affective Disorders and Schizophrenia for School Age Children-Present and Lifetime Version, DSM-5 November 2016-Turkish Adaptation (K-SADS-PL-DSM-5-T) was used to assess the diagnosis of MDD and other comorbid diagnoses. Depressive symptoms of the participants were evaluated with the Beck Depression Inven-tory (BDI) and anxiety symptoms were evaluated with the Spielberger State-Trait Anxiety Inventory (STAI-S, STAI-T). After psychiatric evaluation, serum levels of agmatine, putrescine, spermidine and spermine were analyzed in all participants.

Tools Used in Clinical Evaluation

Sociodemographic data form

The sociodemographic form was designed by the researchers. Various sociodemographic characteristics of the child such as age, date of birth, sex, height-weight, body mass index, whether girls menstruate or not, place of residence, number of siblings, order of children, educational status, academic achievement score, grade level, age of learning to read and write, smoking-alcohol-substance use, and history of suicide attempt were ques-tioned. In addition, information about the age of the parents, monthly income level of the family, type of family, educational level of the parents, occupation of the parents, smoking-alcohol use, and history of psychiatric illness in the extended family were obtained.

K-SADS-PL-DSM-5-T

This form was developed to determine the presence of previous and current psychopathology in children and adolescents and adapted to DSM-5 criteria by Kaufman et al. [31]. The form consists of three parts. It is a semi-structured interview tool. The validity and reliability of the form were done for the Turkish adaptation of the form [32].

BDI

This scale is used to measure physical, emotional, cognitive, and motivational symptoms in patients with depression. There are 21 items in this scale, which objectively determines the severity of depression symptoms; all of these items have four choices. The scale was developed by Beck et al. [33], and the validity and reliability of the form for the Turkish version was made by Hisli [34].

STAI-S, STAI-T

This inventory was developed by Spielberger et al. [35] to determine the state and trait anxiety levels of individuals. This inventory was included in our study in order to determine the anxiety levels of our subjects. The scale was adapted into Turkish by Öner and LeCompte [36].

Biochemical Evaluation

All participants were referred to the Biochemistry Laboratory of Firat University Hospital for blood collection after clinical evaluation. After fasting for at least 12 hours, 5 ml of venous blood were collected in plain gel biochemistry tubes and allowed to clot for 15 minutes. The blood samples were then centrifuged at 4,000 rpm for 10 minutes and separated into serum. Afterwards, serum were collected in Eppendorf tubes and stored at −80°C until the day of the study. Derivatization and preliminary preparations were performed for all parameters. For serum agmatine level, high performance liquid chromatography (HPLC) system (SPD-M20A, RID-20A; Shimadzu) pump (LC 20 AD; Shimadzu) and UV-Fluorescence absorbance were used with excitation and emission wavelengths set to 350 and 450 nm bands, respectively, and HPLC run time set to 15 minutes. For serum putrescine, spermidine, and spermine levels, HPLC system (SPD-M20A, RID-20A) using pump (LC 20 AD) and UV-Fluorescence absorbance was set to 254 nm band and HPLC run time was 35 minutes. The levels of agmatine, putrescine, spermidine, and spermine were calculated in ng/ml in parallel with the chromatograms of serum samples and in comparison with the running standards.

Statistical Analysis

SPSS 22 package program (IBM Co.) was used for the analyses. Chi-square analysis (Pearson chi-square) was used to compare categorical data between groups. Fisher’s exact test was used if more than 20% of the expected values were less than 5. Compliance of continuous data with the normal distribution was evaluated using the Kolmogorov-Smirnov test. Independent samples t test was used for the comparison of variables conforming to normal distri-bution and Mann-Whitney Utest was used for the comparison of variables not conforming to normal distribution. Pearson Correlation test was used to examine the correlation between continuous variables with normal distribution, and Spearman Correlation test was used for those that did not show normal distribution. Statistical significance level was accepted as p < 0.05 in the analyses.

Sociodemographic and Clinical Data

A total of 89 subjects, 45 with MDD and 44 healthy controls, were included in our study. No significant difference was found between the groups in terms of age, sex, body mass index, smoking, menstruation status in female subjects, parental age, parental education, parental employment status, family type, family income level, place of residence, and family history of psychiatric illness.

While 17.8% of the patients (n = 8) in the MDD group had a history of suicide attempt, no history of suicide attempt was found in the control group (p = 0.006).

Sociodemographic and clinical data of the groups are presented in Table 1.

Biochemical Data

In the MDD group, agmatine and spermine levels were significantly higher (p < 0.001; p < 0.001, respectively); putrescine and spermidine levels were significantly lower (p < 0.001; p = 0.034, respectively). Biochemical data are presented in Table 2.

In the MDD group, there was a significant negative correlation between putrescine and spermidine levels and BDI scores (p = 0.048; p = 0.007, respectively), and a significant positive correlation between spermine levels and BDI scores (p = 0.022). There was no significant correlation between agmatine levels and BDI scores (p = 0.442). Correlation analyses are presented in Table 3.

When the blood parameters of the patients with and without suicide attempt in the MDD group were evaluated, no significant difference was found between the groups in terms of serum agmatine, putrescine, spermidine, and spermine levels (p = 0.065; p = 0.054; p = 0.672; p = 0.390, respectively) (Table 4).

In this study, we aimed to evaluate agmatine and polyamines levels in healthy controls and adolescents diagnosed with MDD. Furthermore, we investigated the severity of MDD symptoms with agmatine and polyamines levels. Our study found that agmatine and spermine levels were significantly higher, and putrescine and spermidine levels were significantly lower in the MDD group compared to the control group.

To our knowledge, there is no study evaluating blood levels of agmatine, putrescine, spermidine and spermine in adolescents with MDD in the literature. In a study conducted by Halaris et al. [29], 16 adult patients with depression and 8 healthy control subjects were evaluated, and plasma agmatine concentrations were significantly higher in patients with depression than in healthy controls. In another study conducted in adulthood, it was reported that plasma agmatine levels in patients with MDD tended to be higher than the control group, but this difference did not reach statistical significance [30]. In the study conducted by Ozden et al. [27], adult subjects with MDD were studied; agmatine levels were found to be significantly higher and putrescine levels were found to be significantly lower in subjects with MDD compared to controls. In general, our findings seem to be compatible with the limited literature on adult patients with depres-sion.

Polyamine metabolism might be related to the etiopathogenesis of MDD through various processes. A review of the literature reveals that the pathophysiology of depression has been hypothesized to be related to “impaired stress response”. The close relationship between stressful life events and depressive episodes is one of the well-studied topics in the pathophysiology of MDD [37]. Hypothalamic-pituitary-adrenal (HPA) axis-related abnormalities such as increased cortisol levels and dexamethasone suppression test positivity found in depressive patients are important biological findings supporting that stress may play a role in MDD [38]. Agmatine may be an endogenous neuromodulator of mental stress and may have a role in stress-related diseases such as depression and anxiety disorder. According to some researchers, endogenous agmatine is induced in response to stress/inflammation [15]. It has been suggested that agmatine acts as a “pendulum” in times of stress/trauma and plays a role in restoring homeostasis together with arginine metabolism; this hypothesis has been named the “pendulum hypothesis” [39]. Agmatine is formed as a result of decarboxylation of L-arginine by a reaction catalyzed by the enzyme ADC [25]. According to the pendulum hypothesis, ADC levels are low in normal healthy tissue at the time of trauma. In this case, low agmatine levels cause a rapid increase in NO production through iNOS activation immediately after trauma. In this way, defense mechanisms are activated along with vasodilation. If high NO levels are sustained, NMDA channels remain open for a long period of time. As a result, there is a continuous calcium influx into the cell and a neurotoxic process occurs. Therefore, the increase in NO levels should be maintained at a level and duration that can provide tissue defense but does not result in neurotoxicity. It has been suggested that ADC/agmatine synthesis might be induced some time after the onset of stress/inflammation in order to prevent toxicity and control NO levels; thus, increased agmatine synthesis may create a protective mechanism both through decreased NO levels by inhibiting NOS and decreased glutamate release by blocking NMDA receptors [15]. In our study, the high agmatine levels found in patients with MDD may be related to this protective mechanism that is stated to occur in response to stress.

Similar to the “agmatine-stress relationship” hypothesis mentioned above, some publications in the literature also mention a mechanism called “PSR” [19,40]. In the PSR, a dysregulation in polyamine synthesis occurs when a stressful stimulus is encountered [8,19]. When exposed to emotional, physical, or hormonal stressors, some second messenger mechanisms activate the PSR, and transient changes in the levels of polyamines such as putrescine, spermidine, and spermine occur in relation to the intensity and magnitude of stress. Severe sustained stress leads to accumulation of brain polyamines. More longer term, polyamine synthesis is inhibited and brain polyamines are depleted, which may result in altered emotional reactivity to stressors [8,40]. It has been suggested that PSR is a molecular mechanism involving adaptive and/or maladaptive brain responses to stressful events, and in this context, proper regulation of polyamine metabolism may be critical for an appropriate response to stressors [19]. It has been stated that PSR is associated with stress-induced activation of the HPA axis, and this is involved in the pathophysiology of depression [27,41]. Altered stress-coping mechanisms may play a role in the etiology of MDD by affecting neuronal communication in brain regions involved in the stress response and control of emotions. In a review, literature findings pointing to dysregulation of the polyamine system as a central factor in the homeostatic response to stress and the etiology of MDD were examined. They concluded in this review paper that stress-mediated hyperactivity observed in brain regions associated with depression may cause changes in polyamine metabolism, and as a result, altered polyamine levels may lead to various damaging effects through modulation of synaptic transmission [42]. In our study, putrescine and spermidine levels were significantly lower in the MDD group compared to healthy controls, whereas spermine levels were significantly higher. Considering the findings in the literature indicating the relationship between depression and stress, it can be suggested that the significant differences in polyamine levels found in the MDD group in our study support the PSR-related assumption in the literature and the dysregulation in polyamine metabolism. However, it is clear that further studies are needed for a more reliable interpretation.

It has been reported that abnormalities related to the glutamatergic signaling system in the brain may also play a role in the etiology of MDD [43]. Dysfunction in glutamatergic neurotransmission in MDD might be associated with abnormal expression of glutamate receptors [44]. It has been reported that dysfunction mediated especially by NMDA receptors may contribute to the neurobiology of MDD [45]. It is known that agmatine binds to NMDA receptors and shows an antagonist effect on these receptors [22]. In hippocampal cell culture, agmatine may protect hippocampal neurons from NMDA/glutamate-induced excitotoxicity through its antiapoptotic or NMDA receptor-blocking properties [46]. Based on this information, one could speculate that the high agmatine levels we found in patients with MDD in our study might also indicate a protective mechanism against NMDA/glutamate-mediated cell damage.

Similarly, polyamines have been reported to be important moderators of glutamate receptors. They can alter the functioning of NMDA receptors, and as ligands of these receptors, they can show activator or inhibitor properties. They can increase NMDA receptor activity by playing a role in the opening of ion channels, while they (especially spermine) can also block open NMDA chan-nels. Spermine may also exert inhibitory effects on NMDA receptor subunits by decreasing their sensitivity and affinity for glutamate [47]. Polyamines may also interact with ionotropic kainate and AMPA glutamate receptors and contribute to changes in membrane excitability [13]. Modulation of glutamate signaling by polyamines affects a wide range of functional processes in the brain, from the regulation of neuronal and glial excitability to memory and aging [14]. Considering all these data in the literature, it may be considered that the differences in agmatine and polyamine levels found in the MDD group in our study may represent a neuromodulatory/neuroprotective mechanism that may occur in response to stress-related damage. However, further studies are needed to elucidate the basis of the possible mechanism, which receptors mediate it, the cause-effect relationship, and to interpret the findings more accurately.

In the correlation analyses in our study, there was a significant negative correlation between blood putrescine and spermidine levels and BDI scores in the MDD group. However, there was a significant positive correlation between blood spermine levels and BDI scores in the MDD group. According to these findings, the severity of depression increased as putrescine and/or spermidine levels decreased, whereas the severity of depression increased as blood spermine levels increased. However, the correlation coefficients we found indicate that there is a weak relationship between the variables. There was no statistically significant correlation between BDI scores and agmatine levels. To our knowledge, there are limited studies on the subject in the literature. Consistent with our study, Ozden et al. [27] also found a negative correlation between blood putrescine levels and depression symptom severity in a study conducted with adult MDD cases, but no relationship was found between agmatine levels and depression severity. Similarly, in the study conducted by Piletz et al. [30], no correlation was found between agmatine levels and Hamilton Depression Rating Scale scores. These concordant findings are exciting for future longitudinal studies with larger samples.

In our study, we observed that blood levels of agmatine, putrescine, spermidine, and spermine did not differ between the groups when the cases with a history of suicide attempt in the MDD group and the cases without suicide attempt were evaluated. When the literature is examined, it is seen that there are some postmortem studies examining polyamine levels in the brain tissues of patients with completed suicide attempts with or without MDD. In a study by Chen et al. [48], polyamine levels were evaluated in postmortem brain tissues of subjects with completed suicide attempts. Sixteen male subjects with MDD who had completed suicide, 13 non-depressed male subjects with completed suicide, and 13 control subjects (male subjects who died from accidents or natural causes) were compared. In that study, brain putrescine and spermidine levels were found to be significantly higher in the MDD group than in the control group, but did not differ significantly from the non-depressed group. In another study by Chen et al. [49], cerebral cortex agmatine levels of 14 male subjects who died by suicide and met the criteria for MDD, 13 male subjects who died by suicide but did not meet the criteria for MDD, and 13 control subjects (male subjects who died by accident or natural causes) were evaluated postmortem. In that study, agmatine levels were found to be significantly lower in both suicide groups than in the control group regardless of the presence of MDD. Since these studies were postmortem studies, taking and studying postmortem tissue samples requires very sensitive work and time, and the reflection between blood levels of polyamines and brain levels is not yet clearly known, the results of these studies cannot be compared with the results of our study. However, considering the strong relationship between suicidal behavior and depression, we would like to underline again the need for further research on the subject.

Our study should be interpreted together with its strengths and limitations. As far as can be seen in the literature, there is no study evaluating blood levels of agmatine, putrescine, spermidine, and spermine in adolescents with MDD. As far as is known, the basic metabolic pathways of agmatine and the enzymes involved in these metabolic pathways do not differ between children and adults; however, the activities of these processes can vary due to factors like age, genetics, and diet as far as we can see, no study has been found in the literature that directly compares the agmatine pathways of children and adults. The current body of research suggests that the existing studies on agmatine and polyamines in patients with depression have generally focused on adult age. These studies in the adult age group provide a basis for understanding the activity and metabolism of the agmatine and polyamine pathways in the child/adolescent age group. However, if the results of adult studies are to be applied to the pediatric depression, it is crucial to carefully consider the developmental, physiological, and metabolic differences between adults and children/adolescents. Given these limitations, further research with the pediatric age group is warranted. Our study, being the first of its kind, is thought to be very important in terms of drawing attention to the gap in the literature on the subject and providing ideas for new studies. In our study, K-SADS-PL-DSM-5-T, which is a semi-structured interview, was used to diagnose all cases. While forming the sample of the study, the ages of the subjects were kept within as narrow a range as possible. Inclusion and exclusion criteria were determined by taking into consideration the confounding factors that were thought to affect the findings of the study as much as possible. In addition, a healthy control group was also used in our study. All these features are thought to increase the methodological strength of the study. In our study, the agmatine pathway of arginine metabolism was studied, but other pathways could not be evaluated, which is a limitation of our study. Studies in which all pathways involved in arginine metabolism are evaluated simultaneously will provide much more holistic results. Another limitation is that our study was a cross-sectional study, and our sample size was relatively small. In our study, only blood levels of agmatine, putrescine, spermidine and spermine were measured. A review of the literature reveals that agmatine can cross the blood-brain barrier [50]. Intravenous administration led to a rapid increase in agmatine concentration in brain [51]. However, there is currently no clear evidence as to the extent to which serum levels of agmatine reflect its levels in the brain. More research is needed on this subject. For this reason it is important to make measurements in the brain tissues, especially cerebrospinal fluid. As mentioned above, agmatine may be an endogenous neuromodulator of stress. However, our study did not reveal a significant correlation between agmatine levels and BDI scores. More research is needed on this subject. In addition to all these, evaluation of enzyme activities involved in the polyamine pathway will allow a much more reliable interpretation of the current findings.

In conclusion, the findings of our study indicate that there are changes in blood levels of agmatine, putrescine, spermidine, and spermine in adolescents with MDD. These changes we detected seem to be worthy of further investigation. Evaluation of the polyaminergic transmission system in MDD with larger samples and longitudinal studies in the future may help to better understand the disorder and perhaps lead to development of more effective treatment methods.

This article is derived from the thesis of Dr. Şükrü Kaan Öztürk, which was conducted under the supervision of Assoc. Prof. Dr. Kemal Utku Yazici. The authors thank Naci Omer Alayunt for his support on biochemical analysis, and also thank the cases and their parents who participated in this study.

No potential conflict of interest relevant to this article was reported.

Conceptualization: Kemal Utku Yazici, Şukru Kaan Ozturk, Ipek Percinel Yazici, Bilal Ustundag. Data acquisition: Kemal Utku Yazici, Şukru Kaan Ozturk, Ipek Percinel Yazici. Formal analysis: Kemal Utku Yazici, Ipek Percinel Yazici, Bilal Ustundag. Writing—original draft: Kemal Utku Yazici, Şukru Kaan Ozturk, Ipek Percinel Yazici. Writing—review & editing: Kemal Utku Yazici, Şukru Kaan Ozturk, Ipek Percinel Yazici, Bilal Ustundag.