2020 年 67 巻 9 号 p. 935-940
There are some studies regarding the presence/absence of oxidative stress in patients with hypogonadism with limited number of parameters. We aimed to investigate the effects of male hypogonadism and its treatment on oxidative stress parameters. Thirteen male patients with hypogonadotropic hypogonadism and 20 healthy subjects were involved in the study. Patients with hypogonadism were evaluated before and after six months of therapy. Markers indicating lipid and protein oxidation, total oxidant status (TOS) and total anti-oxidant capacity (TAC) were evaluated. Control subjects had significantly higher serum testosterone levels in comparison to hypogonadal patients before the treatment period. After the treatment of hypogonadism serum testosterone levels increased significantly. Myeloperoxidase (MPO) activity, levels of advanced oxidation protein products (AOPP), total lipid hydroperoxide and protein carbonyl compounds (PCC) were similar between the control subjects and the patient group before treatment. Pyrrolized protein and TOS were significantly lower and thiol levels and TAC were significantly higher in the control subjects than in patients with hypogonadism. Treatment of hypogonadism resulted in a significant decrease in AOPP levels while a significant increase was determined in TAC. No significant change was found in MPO activity. In conclusion, patients with hypogonadism have an increased status of oxidative stress which is at least partially improved after appropriate therapy.
MALE HYPOGONADISM is the failure of testicular testosterone production which is characterized by absence or diminished secondary sex characteristics. A number of studies have shown that hypogonadism is associated with some metabolic disorders [1]. Epidemiological data showed that serum testosterone concentrations are positively correlated with HDL-cholesterol and negatively correlated with serum triglyceride and LDL-cholesterol levels [2]. Although the mechanisms of metabolic disorders have not been clearly established, insulin resistance and inflammation are considered as an important contributors [3]. On the other hand, increased oxidative stress has a great negative impact on many systems in human body and it may also be involved in the pathophysiology of metabolic disorders seen in patients with hypogonadism.
Oxidative stress is defined as an imbalance between the production of reactive oxygen species (ROS) and the anti-oxidant defense mechanisms. There are several studies that documented the role of oxidative stress in the etiology of diverse clinical conditions including diabetes mellitus, coronary heart disease, malignancies and aging [4, 5]. Oxidative damage is a well-known mechanism of tissue injury and it is mediated by ROS via cellular dysfunction caused by lipid, protein and DNA damage. Previously, it has been shown that estradiol is correlated with total anti-oxidant capacity (TAC) and postmenopausal women have lower TAC than premenopausal women [6]. Similarly, hypogonadal men have lower TAC than eugonadal men. There are some studies regarding the presence/absence of oxidative stress in patients with hypogonadism with limited number of parameters. In this study, we aimed to extensively investigate the effects of male hypogonadism and its treatment on oxidative stress parameters.
Thirteen male patients with hypogonadotropic hypogonadism and 20 healthy subjects with comparable age and body mass index (BMI) were included in this prospective study. Informed consent was obtained from all the patients and control subjects and the protocol was approved by the Ethics Committee of Erciyes University Medical School.
The diagnosis of hypogonadism was based on the presence of unequivocal low testosterone level, presence of classical hypogonadal symptoms and low/normal serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels. In accordance to the Clinical Reference Laboratory of the Center for Disease Control program, biochemical diagnosis of hypogonadism was done in serum testosterone levels lower than 264 ng/dL in patients with hypogonadal symptoms [7, 8]. In the present study, patients with hypogonadotropic hypogonadism had normal prolactin levels, normal pituitary and target hormone levels and normal pituitary magnetic resonance imaging.
Exclusion criteria were (1) diabetes mellitus, (2) smoking, (3) to take anti-oxidant vitamin/supplements, (4) testosterone replacement therapy, (5) to use lipid-lowering/antihypertensive drugs, (6) patients with any chronic diseases. Patients were diagnosed as hypogonadism when they had clinical features of hypogonadism in the presence of low serum testosterone levels. All the patients were studied at two time points: At baseline and six months after hypogonadism treatment. For patients with hypogonadotropic hypogonadism, patients were treated 1,500 unit of human chorionic gonadotropin (Pregnyl®, Organon, Holland) intra muscularly, three times in a week plus 75 unit of follitropin alpha (Gonal F, Merck Serono, Switzerland) subcutaneously, three times in a week.
Anthropometric measurements including weight, height, and waist circumference were done in the study population. Body mass index (BMI) was calculated as the ratio of weight to the square of height (kg/m2). Fasting blood sample was obtained for the determination of blood chemistry including lipid panel [total cholesterol (TC), HDL-C and LDL-C (estimated by the Friedewald Formula)], triglyceride (TG) and also blood urea nitrogen, creatinine, alanine transaminase, aspartate transaminase, glucose and alkaline phosphatase by routine assays with an autoanalyzer (Roche® Cobas c702; Roche Diagnostics GmbH, Mannheim, Germany). After the treatment period, blood sampling (mainly for testosterone and also other parameters) was performed two days after the last (prior to just) injection of chorionic gonadotropin in patients with secondary hypogonadism. Serum FSH, LH, total testosterone, prolactin and SHBG levels were measured by using Roche® Cobas c601 via electro-chemiluminescence immunoassay (ECLIA; Roche Diagnostics GmbH, Mannheim, Germany).
Analytical proceduresPeripheral venous blood samples were collected in tubes with and without anticoagulants (heparin lithium salt or EDTA) and centrifuged to obtain plasma and serum respectively, at the hypogonad and eugonadal period, and also from controls once, for determination of myeloperoxidase (MPO) activity, and advanced oxidation protein products (AOPP), protein carbonyl compounds (PCC), total lipid hydroperoxide (LHP), pyrrolized protein, thiol levels, total oxidant status (TOS) and total anti-oxidan capacity (TAC).
MPO activity was determined in heparinized plasma by the o-dianisidine method based on kinetically measurement of the yellowish-orange product formation rate from the oxidation of o-dianisidine with MPO in the presence of H2O2, at 460 nm. Degrading one μmole of H2O2 per minute at 25°C was defined as one unit of MPO. A molar extinction coefficient of 1.13 × 104 M–1 cm–1of oxidized o-dianisidine was used for the calculation [9]. MPO activity was expressed in units per liter of plasma (U/L). MPO is a pro-inflammatory and pro-oxidative enzyme that generate hypochlorous acid and its inappropriate stimulation of oxidant formation by MPO result in tissue damage.
Total LHP levels in heparinized plasma were assayed by the ferrous oxidation in xylenol orange method of Nourrooz-Zadeh [10]. This method is based on the oxidation of ferrous ions to ferric ions, by peroxides under acidic conditions, complexed by the xylenol orange (ferric ion indicator), generating a blue-purple complex with an absorbance maximum at 550–600 nm and the results are expressed as micromoles per liter of plasma (μmole/L). It is one of the primary endproducts of lipid oxidation and widely used as a marker of oxidative stress.
Serum pyrrolized proteins were determined using Ehrlich’s reagent (p-(dimethylamino) benzaldehyde; DMAB) by the method suggested Hidalgo et al. [11] which is modified by Martinez-Cruz et al. [12]. The principle was based on the spectrophotometric measurement of color intensity of Ehrlich adducts at 570 nm, resulted from the interaction of pyrroles on proteins and DMAB under high temperature and acid conditions. The extinction coefficient of 35,000 M–1 cm–1 of ε-N-pyrrolylnorleucine as standard was used for the evaluation and the results were given as nanomoles per milligram of protein (nmole/mg protein). It is an another example of plasma protein oxidation marker.
Thiol levels in heparinized plasma were determined by the method of Hu et al. [13] based on the thioldisulphide interchange reaction between thiol and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB). The evaluation was performed using a standard curve of glutathione and thiol levels were expressed in micromoles per liter of plasma (μmole/L). Impaired antioxidant defense may also result in oxidative stress and thiols especially derived from albumin are one of the most important members of the plasma antioxidant system.
Levels of serum PCC (Catalog No: MBS773081) and plasma AOPP (Catalog No: MBS770162) were determined by ELISA kits (MyBioSource®, Inc., USA), and expressed as pg/mL and µmole/L, respectively. AOPP derived from plasma proteins, paricularly albumin, is considered a reliable marker to estimate the degree of protein modifications. PCC is a general biomarker of oxidative protein damage with longer half life in circulation and it occurs relatively earlier stage. The TOS (Catalog No: RL0024) and TAC (Catalog No: RL0017) of the serum were measured using colorimetric kits (Rel Assay Diagnostics®, Gaziantep, Türkiye) developed by Erel [14, 15]. The TOS and TAC results were expressed in μmole H2O2 equivalent/L and mmole Trolox equivalent/L, respectively.
StatisticsAll data were recorded on a computer database and analyzed using Number Cruncher Statistical System (NCSS, Kaysville, Utah, USA). Results are expressed as median (25–75%). The variables were assessed for normality by using the Shapiro-Wilk test. Inter-group differences between the patient and control subjects were analyzed by using the Mann-Whitney U test according to distribution of data. The effects of treatment were compared by Wilcoxon-signed ranks test. Differences were considered significant at a p value of < 0.05.
Age and BMI of the patients and control subjects were similar. Baseline demographic characteristics of the patients and the control subjects are given in Table 1. Overall, ten of the patients were treatment naive, the rest of the patients were not using any gonadal replacement therapy for at least two years. Those three patients also had severe hypogonadal symptoms associated with biochemical hypogonadism.
| Parameters | Reference range | Controls (n = 20) | Patients (n = 13) | |
|---|---|---|---|---|
| Before treatment | After treatment | |||
| Age (years) | 27 (25–30) | 26 (18.8–33.5) | ||
| BMI (kg/m2) | 26.8 (25.9–29.5) | 26.4 (21.8–31.4) | 27 (22.3–32.6) | |
| Testosterone (ng/dL) | 280–800 | 420 (373–504) | 16.5 (11.6–31.9)# | 714.6 (316.8–926.5)*• |
| TG (mg/dL) | 35–150 | 102.5 (68.7–129) | 115 (87.5–217.5) | 114 (82.5–187.5) |
| TC (mg/dL) | 160–200 | 178 (155–191.7) | 186 (147.5–230) | 149 (142–183.5)§ |
| LDL-C (mg/dL) | 100–130 | 108.8 (95.5–118.2) | 103 (77–140.8) | 87.2 (70.4–114.8)¥ |
| HDL-C (mg/dL) | 0–90 | 47.1 (41.9–56.5) | 39 (34.5–60) | 42 (33.5–52.5) |
TC, total cholesterol; TG, triglyceride
Data presented as median (25%–75%)
Statistical comparisons:
– The controls vs. after treatment (* p < 0.05)
– Before treatment vs. after treatment (• p < 0.001)
– Before treatment vs. after treatment (¥§ p < 0.05)
– The controls vs. before treatment (#p < 0.001)
The control subjects had significantly (p < 0.001) higher serum testosterone levels in comparison to hypogonadal patients at baseline. After the treatment of hypogonadism serum testosterone levels increased (p < 0.001) and serum total cholesterol and LDL-cholestrol levels decreased significantly (p < 0.05) (Table 1). MPO activity, AOPP, PCC and total LHP levels were similar between the control subjects and the patient group before treatment. Pyrrolized protein and TOS were significantly (p < 0.05) lower and thiol levels and TAC were significantly (p < 0.05) higher in the control subjects than in the patients with hypogonadism (Table 2). Gonadal replacement therapy resulted in a significant (p < 0.05) decrease in AOPP and a significant (p < 0.05) increase was determined in TAC. Although thiol levels increased and TOS decreased, they did not reach a significant level (Table 2). No significant change was found in MPO activity.
| Parameters | Controls (n = 20) | Patients (n = 13) | |
|---|---|---|---|
| Before treatment | After treatment | ||
| MPO (U/L) | 93 (78.3–129) | 90.7 (72.1–120.8) | 85.5 (70.6–112.1) |
| AOPP (μmol/L) | 37.4 (27.1–43.4) | 39.6 (28–49) | 38.6 (22.1–40.3)¥ |
| PCC (pg/mL) | 458.2 (316.5–507) | 456.6 (356.1–486.9) | 520.8 (438.3–542.3)• |
| Total LHP (μmol/L) | 2.3 (1.5–3.6) | 3.6 (2.3–5.8) | 4.6 (2.1–6.1)* |
| Pyrrolized protein (nmol/mg protein) | 0.6 (0.5–0.7) | 0.7 (0.6–0.8)ɸ | 0.6 (0.6–0.7) |
| Thiol (μmol/L) | 364.1 (343.8–404.1) | 318.4 (272–344)ɸ | 322.7 (289.8–404.8) |
| TAC (mmol/L) | 1.1 (1–1.2) | 0.7 (0.5–0.7)ɸ | 0.8 (0.5–1.2)¥ |
| TOS (μmol/L) | 5.8 (5.4–6.8) | 9.3 (6.2–14.8)# | 6.3 (5.4–9.7) |
TAC, total anti-oxidant capacity; TOS, total oxidant status
Data presented as median (25%–75%)
Statistical comparisons:
– The controls vs. after treatment (* p < 0.05)
– The controls vs. before treatment (#ɸ p < 0.05)
– Before treatment vs. after treatment (¥ p < 0.05)
– Before treatment vs. after treatment (• p < 0.01)
In addition to symptoms related to hypogonadism, epidemiological studies have shown several metabolic consequences of hypogonadism such as osteoporosis, obesity, metabolic syndrome and increased cardio-metabolic risk [1, 2, 16, 17]. On the other hand, the underlying mechanisms of this increased cardio-metabolic risk has not been clearly established. Endothelial dysfunction, inflammation and insulin resistance are considered as the main contributors of increased cardiometabolic risk in patients with hypogonadism. It has been shown that oxidative stress play an important role in the pathogenesis of cardiometabolic risk.
The oxidative stress is the result of imbalance between anti-oxidant systems and ROS generation and it mediates diverse protracted conditions including malignancies, coronary artery disease and inflammatory process [5]. ROS are by-products of normal cellular metabolism and it has been shown that ROS inhibits testicular testosterone production by altering the expression and activity of testicular steroidogenic enzymes which is commonly seen during aging process [18, 19]. However, there is limited data regarding oxidative stress in patients with hypogonadism. Mancini et al. [8] investigated the role of gonadotropins in the regulation of systemic anti-oxidants in patients with secondary hypogonadism. They have compared 16 patients with secondary hypogonadism due to pituitary adenoma operation and 10 normogonadal men. The authors revealed that hypogonadism was characterized by lower levels of Coenzyme Q10 (CoQ10), which is a lipidic anti-oxidant. Testosterone replacement of the patients resulted in an increase serum CoQ10 similar to normogonadic patients [8]. However, the main problem of this study is the etiology of secondary hypogonadism in the included patients. Secondary hypothyroidism was detected in some of these patients due to pituitary surgery. This postoperative model seems to be complicated due to the possible involvement of other pituitary hormone axes and possible confounding effects of hypothyroidism. Because, we have previously shown that, as well as chronic hypothyroidism, acute hypothyroidism has also detrimental effects on oxidant/anti-oxidant systems [20].
Haymana et al. [21] investigated oxidative stress parameters in patients with congenital hypogonadotrophic hypogonadism by using catalase, superoxide dismutase, glutathione peroxidase and malondialdehyde levels. Compared to healthy control subjects, the authors found that catalase activities and malondialdehyde levels were significantly higher and glutathione peroxidase activity was significantly lower in hypogonadal patients. Those results exhibited increased oxidative stress in hypogonadal patients. However, in that study the authors did not check the changes of oxidative stress parameters after the treatment of hypogonadism.
Hypogonadism is associated with a hypercoagulable state, an atherogenic lipid profile, an increase in insulin resistance and oxidative stress may underlie in the above mentioned clinical situations [22]. Our results showed that total cholesterol and LDL-cholesterol levels decreased after the treatment of hypogonadism. Apart from the changes in serum lipid levels, to our knowledge, this is the first study investigating protein and lipid oxidation separately in patients with hypogonadism before and after its treatment. In this study, we have investigated the effects of hypogonadism on protein/lipid oxidation, total oxidant capacity as well as thiol levels and total anti-oxidant status as an indicator of anti-oxidant system. Proteins are sensitive to oxidative damage and their function and structures are modified by oxidative damage. AOPP is an another marker formed during oxidative stress by the action of hypochlorus acid which causes oxidative modification of free functional groups of proteins [23]. Similarly, higher pyrrolized protein levels also support the presence of oxidative stress in patients with hypogonadism. Six months of hypogonadism treatment resulted in a decrease in AOPP and pyrrolized protein levels indicate improvement of protein oxidation.
All the patients had secondary hypogonadism without any other pituitary hormone deficiency and the present study includes a relatively homogenous group of patients. The main topic of this study was not to investigate the patients according to their etiologies, rather it was important to be hypogonadal and reversal of oxidative stress by using the treatment of hypogonadism. In general, our results showed that patients with hypogonadism had an increased oxidative stress characterized by increased TOS and decreased TAC and thiol levels. All those parameters, indicating the presence of oxidative stress in patients with hypogonadism, improved after six months of hypogonadism treatment. MPO is accepted as a marker of neutrophil activation and it gives an estimates of the oxidative stress [23]. Although an increased oxidative stress is found in patients with hypogonadism, unchanged MPO activity suggest that hypogonadism does not affect all the mediators of oxidative stress. Additionally, an improvement was not seen in total LHP and PCC which are also the markers of lipid and protein oxidation, respectively. Our results suggest that oxidative stress in hypogonadism is not a situation of “all or none” and, as far as we know, there is no data regarding MPO activity, total LHP and PCC in patients with hypogonadism. On the other hand, in daily practice treatment of hypogonadism does not limited to six months, thus we do not know what happens in longer time of treatment periods.
The role of testosterone on the oxidative system has been shown also in aging male and neurodegenerative disorders. Ahlbom et al. [24] investigated the protective effects of in vitro testosterone treatment on the oxidative stress in cerebellar granule cells. They found that cerebellar granule cells treated with in vitro testosterone are protected from oxidative stress via a mechanism mediated by the androgen receptor. On the other hand, it has been suggested that decline of testosterone levels by aging are associated with elevated ROS which further detoriate the production of testosterone by Leydig cells [25, 26]. It has been shown that low dose testosterone treatment reduced ROS generation in TM3 Leydig cell culture [27] and anti-oxidant supplementation protects TM3 Leydig cells from oxidative stress and increase cell viability [28]. Moreover, the role of anti-oxidants has been shown in idiopathic male infertility [29]. Considering the numerous detrimental effects of oxidative stress in male reproductive system in addition to its metabolic consequences, our results support that oxidative stress may be decreased without anti-oxidant supplementation in hypogonadal patients by gonadal hormone replacement therapy.
In conclusion, in addition to gonadal functions, treatment of hypogonadism has positive effects on serum cholesterol levels and patients with hypogonadism have an increased oxidative stress which is at least partially improved after appropriate therapy. The effects of longer time treatment of hypogonadism should be investigated in future studies.
None of the authors have any potential conflicts of interest associated with this research.
This study was supported by the Scientific Research Unit of the Erciyes University, under the project number TTU-2018-8381.
