The Relationship between Selected Bioelements and Depressiveness Associated with Testosterone Deficiency Syndrome in Aging Men

Background and Objectives: Abnormal concentrations of bioelements (magnesium, manganese, chromium, copper, zinc) have been associated with physical and emotional dysfunctions, including depression. This association, however, has not been analyzed in testosterone deficiency syndrome (TDS) or patients with depressiveness, i.e., when individual symptoms do not form the picture of a full-syndrome depressive disorder. This study aimed to assess the relationship between concentrations of selected bioelements and the incidence of depressive symptoms in men aged 50 years and older with a concurrent testosterone deficiency syndrome. Material and Methods: Blood samples were taken from 314 men; the mean age of the population was 61.36 ± 6.38 years. Spectrophotometric method for biochemical analysis of magnesium (Mg), manganese (Mn), chromium (Cr), copper (Cu), and zinc (Zn) was used. The diagnosis of testosterone deficiency syndrome (TDS) was based on the total testosterone (TT), free testosterone (FT), estradiol (E2), and dehydroepiandrosterone sulfate (DHEAS) levels by ELISA. Each participant completed the Androgen Deficiency in Aging Male (ADAM) questionnaire, as well as the Beck Depression Inventory (BDI-Ia) measuring the severity of depressive symptoms. Results: Emotional disturbances manifested as depressive symptoms were diagnosed in 28.7% of all participants and testosterone deficiency syndrome in 49.3%. In the TDS group, the analysis showed a significant correlation between the level of manganese (R = 0.225, p = 0.005) and chromium (R = 0.185, p = 0.021) with the incidence of depression. Conclusions: The results of our study demonstrated a relationship between manganese and chromium concentrations with the incidence of depression in men aged 50 years and older with a concurrent testosterone deficiency syndrome. This may indicate that there is a correlation between these bioelements, as well as emotional disorders manifested as depressive symptoms in aging men with a diagnosed testosterone deficiency.


Introduction
It is estimated that the prevalence of depression and depressiveness, i.e., individual symptoms that do not form the picture of a full-syndrome depressive disorder, increases among people over On the basis of the presence of testosterone deficiency syndrome, participants were assigned to one of two groups. Group I included 155 people with TDS. Group II included 159 people without TDS. Patients in both groups were assigned to a subgroup with and without the depressive disorder, which was diagnosed on the basis of the Beck Depression Inventory (BDI-Ia). Research procedures and analyses were the same for all participants. The study flowchart (according to STROBE guidelines) is shown in Figure 1.
3 course and objectives of the study. The study excluded people undergoing oncological treatment; those with thyroid disease; those receiving neuroleptics, antidepressants, and supplements containing studied bioelements and treated with steroid therapy; those undergoing testosterone replacement therapy; individuals with depression at any stage diagnosed by a psychiatrist; or those diagnosed with TDS.
On the basis of the presence of testosterone deficiency syndrome, participants were assigned to one of two groups. Group I included 155 people with TDS. Group II included 159 people without TDS. Patients in both groups were assigned to a subgroup with and without the depressive disorder, which was diagnosed on the basis of the Beck Depression Inventory (BDI-Ia). Research procedures and analyses were the same for all participants. The study flowchart (according to STROBE guidelines) is shown in Figure 1.

Ethical Considerations
The study was performed in accordance with the Declaration of Helsinki after it was approved by the Bioethical Committee of the Pomeranian Medical University in Szczecin, Poland (2012-12-10 no. KB-0012/159/12). Each patient enrolled in the study signed an informed consent form for . Data regarding education, smoking and marital status, occupational activity, and chronic diseases such as diabetes and hypertension were also collected. Body mass index (BMI) was calculated on the basis of height and weight.

Ethical Considerations
The study was performed in accordance with the Declaration of Helsinki after it was approved by the Bioethical Committee of the Pomeranian Medical University in Szczecin, Poland (2012-12-10 no. KB-0012/159/12). Each patient enrolled in the study signed an informed consent form for participation and study procedures. In terms of personal data protection, the analysis was performed on dehumanized (anonymous) data.

Study Questionnaires
Three types of questionnaires were used in the study: a dedicated questionnaire prepared by the authors, the Morley questionnaire, and the Beck Depression Inventory (BDI-Ia). The first questionnaire included an interview regarding basic sociodemographic data and medical history information, focusing on determining the presence of possible contraindications in participating in the study.
The questionnaire created by J.E. Morley was used to assess the symptoms of testosterone deficiency [23]. It consisted of 10 closed questions that were single choice, to which possible "yes" or "no" answers primarily focused on sex drive, physical activity, exercise tolerance, and well-being. This questionnaire, despite being a very good tool for assessing the occurrence of symptoms associated with TDS, does not provide any information on their severity.
The Beck Depression Inventory (BDI-Ia) was used to measure the severity of depressive symptoms and included 21 multiple-choice questions with a range of disjunctive questions related to current mood, level of self-esteem, relationships with other people, appetite, quality of sleep, and fear for one's health and life [24]. The answers were summarized and compared to the standards outlined in the questionnaire in which a score between 26 and 29 points means high intensity of depressive symptoms, 20-25 points means medium severity, 12-19 points means mild severity, and the result of ≤11 points means no depressive symptoms.

Bioelement Analysis by Spectrophotometry
In patients from both study groups, 9 mL of fasting blood was drawn from a venipuncture into tubes with gel separator and clot activator, which were then centrifuged and stored at −80 • C in 1.5 mL microtubes. Bioelements were analyzed by optical emission spectrometry with inductively coupled plasma (ICP-OES, ICAP 7400 Duo, Thermo Scientific; Waltham, MA, USA). The analysis was carried out in axial and radial orientation. Tested bioelements included magnesium (Mg), manganese (Mn), chromium (Cr), copper (Cu), and zinc (Zn).
Samples of 0.75 ml were thawed to room temperature and microwaved with the MARS 5 system, CEM. Next, the material was transferred to polypropylene tubes, 2 ml of 65% HNO 3 (Suprapur, Merck; Darmstadt, Germany) was added to each tube, and the tubes were left for 30 min. After this time, 1 ml of unstabilized 30% H 2 O 2 solution (Suprapur, Merck) was added. Next, all samples were placed in Teflon vessels and heated at 180 • C for 35 min using a microwave system. After this time, the samples were cooled to room temperature and transferred to 15 ml acid-washed polypropylene tubes. A 25-fold dilution was made before ICP-OES measurement. A volume of 400 µL was taken and enriched with a standard to a final concentration of 0.5 mg/L yttrium 1 ml 1% Triton (Triton X-100, Sigma; Darmstadt, Germany). It was then diluted with 0.075% nitric acid (Suprapur, Merck) to give a volume of 10 ml. The samples were stored at the temperature of 8 • C. A blank sample was prepared by adding 300 µL of nitric acid and diluting it in the same way as the test sample. Multifactor

Hormone Level Determination and Diagnosis of Testosterone Deficiency Syndrome
Total testosterone (TT), free testosterone (FT), estradiol (E2), and dehydroepiandrosterone sulfate (DHEAS) levels were determined by ELISA, using reagents in ready-made kits (DRG Medtec; Warsaw, Poland) at the Medical Analytics Department of the Laboratory Diagnostics Department of Pomeranian Medical University in Szczecin.
Testosterone deficiency syndrome (TDS) was diagnosed on the basis of the results of laboratory tests with a TT result of less than 2.5 ng/ml or 3.5-2.5 ng/ml when clinical symptoms were assessed using the ADAM (Androgen Deficiency in Aging Male) questionnaire, supplemented by affirmative answers to questions proposed by the European Menopause and Andropause Society referring to the less frequent occurrence of morning erections and sexual thoughts and more frequent erectile dysfunction [25].

Statistical Analysis
The normality of quantitative data distribution was assessed using the Shapiro-Wilk test. Quantitative data were evaluated using the Mann-Whitney U test. Qualitative variables were analyzed using the chi-squared test or chi-squared test with Yates correction. Multivariate logistic regression analysis adjusted for age, smoking and marital status, education, occupational activity, body mass index (BMI), and waist-hip ratio (WHR) classifications was performed. The statistical analysis was performed using the Statistica 13 licensed program (StatSoft, Inc. Tulsa, OK, USA). A p-value of ≤0.05 was regarded as statistically significant.

Results
The full characteristics of the study group are presented in Table 1. Among the respondents (average age of 61.36 ± 6.38 years), 90 men were diagnosed with emotional disturbances manifested as depressive symptoms, which constituted 28.7% of all participants. A total of 155 or 49.3% of participants were diagnosed with testosterone deficiency syndrome. The results regarding the parameters included in the analysis are presented in Table 2.
Out of participants from the first study group, an increase in mean manganese (p = 0.005) and chromium (p = 0.022) levels was observed in patients with a known testosterone deficiency syndrome. Legend: Mn-manganese, Zn-zinc, Cu-copper, Cr-chromium, Mg-magnesium, SD-standard deviation. *10 −3 increase to ten to the minus third power.
Out of the participants in the second study group, in patients without known testosterone deficiency syndrome, there were no significant changes in the average values of bioelements depending on the presence of depression. The results are presented in Table 3. Legend: Mn-manganese, Zn-zinc, Cu-copper, Cr-chromium, Mg-magnesium, SD-standard deviation. *10 −3 increase to ten to the minus third power.
The analysis also showed no significant correlation between the selected concentrations of bioelements and depression among patients from the group without known testosterone deficiency syndrome and with TDS. In the TDS group, the analysis showed a significant correlation between manganese (R = 0.225, p = 0.005) and chromium (R = 0.185, p = 0.022) levels with the presence of depression. The results are presented in Table 4. Table 4. Correlation between the concentration of bioelements and depressive symptoms in patients with and without testosterone deficiency syndrome. Logistic regression was performed to analyze the data obtained from participants with depressive symptoms and testosterone deficiency syndrome, which was corrected for age, smoking and marital status, education, occupational activity, BMI, and WHR classifications. The results are shown in Table 5.

Discussion
Our research hypothesis was to show a relationship between bioelement in patients with depression and co-occurring testosterone deficiency. On the basis of logistic regression, the study showed a significant relationship between the increase in manganese and chromium levels with depressive symptoms and the incidence of testosterone deficiency syndrome in men over 50 years of age. The other bioelements tested such as magnesium, selenium, copper, zinc, and molybdenum did not show significant differences in concentration between the control and the examined patients. A significant correlation between depressiveness and manganese concentrations may confirm its neurotoxic effect on the basal ganglia, resulting in cognitive, emotional, and anxiety disorders, as well as on the induction of oxidative stress causing cell death. These observations have been broadly described in scientific works by, among others, Dukhande et al. in 2006 [26].
In addition, other explanations of the pathomechanism of depressive disorders correlated with an increase in manganese concentrations can also be found in the literature. This is due to its possible effect on the glutamatergic system [27][28][29][30][31][32] and gamma-aminobutyric acid (GABA) receptors [33]. There are already works suggesting a negative correlation between manganese concentrations and attenuation of cognitive functions observed among children between 6 and 13 years of age [34]. Our research shows that similar changes can also be seen in another age group-aging men. A similar negative correlation of manganese can also be indicated for the TT concentration, which corresponds to the results of our work [22].
A negative correlation between chromium concentration and the incidence of depressive symptoms was observed in studies conducted by Młyniec et al. in 2014. This can be explained by the effect of this bioelement on the regulation of emotional functions and the ability to remember by regulating neurotransmitters and neuromodulators belonging to the monoaminergic system [18,19]. However, due to the small number of studies regarding the relationship between depressive and cognitive disorders and chromium, and given the results presented in this paper, we consider it important to continue research on this bioelement on a larger and more diverse study group. Future studies should also include patients undergoing major surgery or treated in the intensive care unit to evaluate the role of chromium levels in postoperative cognitive disorders and postoperative delirium [35,36].
In the case of testosterone deficiency syndrome, however, we observed a positive correlation between chromium and free testosterone (FT) levels and negative fractions in the case of sex hormone binding globulin (SHBG), suggesting a positive effect of this bioelement on patients suffering from TDS [22]. In the research by Serefko et al. in 2016, the positive effect of magnesium was described for a number of biochemical pathways in the brain, the GABAergic system, as well as the monoaminergic system, which may lead to a reduction of symptoms in patients suffering from personality disorders, anxiety disorders, and even clinical depression [17]. Rajizadeh et al. in research performed in 2017 examined 60 people suffering from magnesium deficiency and symptomatic depression, demonstrating that taking 500 mg of magnesium for more than 8 weeks has positive results in the treatment of both these disorders and diseases [37].
On the other hand, a proven positive correlation between magnesium concentration and TT concentration may show the significance of this bioelement when determining the pathomechanisms of testosterone deficiency syndrome [38]. In our work, however, we did not find any relationship between depression and magnesium levels in either people with testosterone deficiency syndrome or those without it.
A meta-analysis carried out by Ni et al. in 2018, with results collected from other studies, indicated a significant relationship, one that increases with age, between the incidence of depression symptoms and copper concentration. The authors additionally suggested that this bioelement is a biomarker for a full-blown disease [39]. This was confirmed when analyzing the effect of this bioelement on the glutamatergic, GABAergic and monoaminergic systems [18,19]. However, due to the insufficient amount of research conducted on these relationships, the role of copper in the mechanisms of depressive disorders still remains controversial. Studies by Chang et al. in 2011 showed a negative correlation between serum testosterone level and copper concentrations [40]. However, in our research, no significant relationship was observed between copper and the concentrations of male steroid hormone and depressiveness in aging men.
The relationship between depressive and anxiety disorders and zinc is one of the most extensively described in the literature around issues related to this work [41,42]. In animal studies, supplementation with this bioelement was shown to even have a positive effect on the treatment of clinical depression [18]. It may be related to the zinc involvement in the function of many systems described earlier that regulate cognitive, emotional, and memory functions, i.e., the glutamatergic, GABAergic, and monoaminergic systems [18,19]. Additionally, this element has been shown to modulate the 5-HT receptors (in particular type 5-HT 1A ), as is the case with SSRIs, which reduce receptor sensitivity and inhibit serotonin reuptake in the synaptic cleft. This mechanism has been broadly described by Duboszewska et al. [43]. Moreover, zinc deficiency affects the incidence of inflammatory processes by influencing interleukin (IL)-1β production. This element also helps to reduce oxidative stress that leads to neurodegeneration and cell death in the nervous system [42,44].
Additionally, in the case of testosterone deficiency syndrome, zinc deficiency seems to be one of the components of the pathomechanism due to the negative correlation with SHBG, causing the body's access to free testosterone fractions [22]. Other studies have shown that zinc is inversely correlated with glycemic levels in type II diabetes [45]. In 2007, Spark et al. described the relationship between type II diabetes and a decrease in testosterone levels [46]. Studies by Kelishadi et al. from 2010 proved that zinc deficiency affects an increase in plasma total and low-density lipoprotein (LDL) cholesterol and an increase of BMI [47]. Increases in body fat tissue leading to obesity are also a risk factor for testosterone deficiency syndrome. It is associated with the aromatase enzyme present in this tissue, which is responsible for an irreversible transformation of testosterone into estradiol [48]. However, our research has not confirmed the relationship between zinc and testosterone deficiency syndrome, which may be due to the small study group.

Conclusions
The results of our study showed the relationship between manganese and chromium concentration and the occurrence of depression in men over 50 years of age with a concurrent testosterone deficiency syndrome. This may indicate the relationship between these bioelements, and emotional disorders manifested as depressive symptoms in aging men with a diagnosed testosterone deficiency.

Limitations
The most significant limitation of our study was the ambiguity of the classification criteria of testosterone deficiency syndrome. There are many standards set by various TDS testing organizations around the world. However, in the above study, we chose those accepted by the International Society of Andrology (ISA), International Society for the Study of the Aging Male (ISSAM), European Association of Urology (EAU), European Academy of Andrology (EAA), and American Society of Andrology (ASA) in 2009. An additional difficulty is the limited amount of literature available on bioelements and their impact on the functioning of cognitive processes. Moreover, the power of the investigations was obtained slightly lower than 0.8; therefore, our results need confirmation in a larger patient population with depressive symptoms and TDS. No information related to alcohol consumption, detailed medical history, or physical activity that could affect the results presented in the study was collected. We believe that in order to increase the credibility of the results, the number of study participants should be increased in the future. Free testosterone determination was performed by ELISA, which may cause inaccuracy in the determination of this parameter.