Plasma and Urine Total Antioxidant Capacity in Patients With Adrenal Tumors

Total antioxidant capacity better characterizes the redox status of the biological system than the determination of individual antioxidants separately. This study is the rst to assess the total antioxidant/oxidant status in the plasma and urine of patients with adrenal tumors. The study group consisted of 60 patients (31 women and 29 men) with adrenal masses, classied into three subgroups: non-functional incidentaloma, pheochromocytoma and Cushing’s/Conn’s adenoma. The number of patients was set a priori based on our previous experiment (α = 0.05, test power = 0.9) Plasma total antioxidant capacity (TAC) was increased in incidentaloma patients, whereas in pheochromocytoma group was decreased. Plasma and urine total oxidant status (TOS) and oxidative stress index (OSI) were signicantly higher in patients with adrenal tumors. Ferric reducing antioxidant potential (FRAP) was decreased in plasma and urine, while DPPH (2,2-diphenyl-1-picrylhydrazyl) antiradical activity only in plasma of patients with adrenal masses. In pheochromocytoma patients, plasma and urine TAC, as well as plasma DPPH and FRAP correlated positively with methanephrine and normethanephrine. Reduced levels of TAC, DPPH and FRAP clearly indicate a reduced ability to scavenge free radicals and thus a lack of effective protection against oxidative stress in patients with adrenal tumors. Therefore, those patients are especially vulnerable to oxidative stress and oxidative damage, which can lead to impaired cellular metabolism. Both plasma and urine redox biomarkers can be used to assess systemic antioxidant status in adrenal tumor patients. are as with signicant differences signicant differences from the pheochromocytoma group; total antioxidant capacity


Introduction
Although malignant adrenal tumors are rare, benign adrenal masses are the most common of all tumors in humans. Typically, they are detected incidentally during diagnostic imaging due to other diseases, hence the term incidentaloma [1]. A adrenal incidentalomas occur in up to 9% of the population [2]. The incidence of adrenal tumors is about 3% in middle age and increases with age up to 10% [3]. Even tough, most of these tumors are benign and nonfunctional, some may cause overproduction of hormones (aldosterone, cortisol or catecholamines) or progress to malignancy [4,5]. Unfortunately, the pathogenesis of adrenal tumors is not fully understood. Currently, it is believed that most of them are caused by genetic abnormalities [6]. Hypoxia-induced factor (HIF-1) deregulation has been involved in the pathogenesis of

Materials And Methods
The study group consisted of 60 patients (31 women and 29 men aged from 50 to 65 years) with adrenal masses diameter > 4 cm and < 8 cm, who were treated using endoscopic adrenalectomy at the First Department of General and Endocrine Surgery at the University Hospital in Bialystok. The diagnosis of patients for adrenal masses was performed in the departments of internal diseases with an endocrine pro le. The patients were classi ed into three subgroups: patients with non-functional incidentaloma (n=20), pheochromocytoma (n=20) and Cushing's/Conn's adenoma (n=20). In the adenoma subgroup Cushing's syndrome was diagnosed in 11 patients and Conn's syndrome in 9 patients. Preoperatively patients with Conn's syndrome received potassium supplementation or spironolactone (aldosterone receptor blocker). Patients with phaeochromocytoma took doxazosin (a selective alpha-1-adrenergic receptor blocker) for 10 to 14 days before surgery to avoid intraoperative hypertensive crisis.
The control group included 60 healthy people (31 women and 29 men aged 50 to 65) whose blood counts and biochemical blood tests (Na +, K +, ALT, AST, creatinine and INR) were within the reference values. The patients of the controls group were treated at the Specialist Dental Clinic at the Medical University of Bialystok.
The study was designed and conducted in accordance with the Guidelines for Good Clinical Practice and the Declaration of Helsinki. The study was also approved by the Bioethics Committee of the Medical University of Bialystok (code of permission: R-I-002/66/2015, APK.002.341.2020). All patients gave their informed consent to participate in this study.
The patients from both study and control groups were quali ed for the study based on a negative medical history concerning: neoplastic diseases, metabolic diseases (osteoporosis, gout, mucopolysaccharidosis, insulin resistance and type 1 diabetes), cardiovascular diseases, autoimmune diseases (ulcerative colitis, Hashimoto's disease and Crohn's disease), diseases of the genitourinary, digestive and respiratory systems, infectious diseases (HIV / AIDS, hepatitis A, B and C), acute in ammation, as well as pregnancy in women. The participants of the study were not abusing alcohol nor smoking. Additional exclusion criteria were taking nonsteroidal anti-in ammatory drugs, glucocorticosteroids, antibiotics and antioxidant supplements (including iron preparations) for three months before collecting material for the study. Patients in all groups were on a diet (2000 kcal, including 55% carbohydrates, 30% fat, and 15% protein) determined by a dietician. The clinical and laboratory characteristics of the control and study groups are shown in Table 1.

Blood and urine collection
All samples from healthy individuals and patients with adrenal mass were collected in a fasting state. The patients declared, that they did not perform intense physical activity twenty-four hours prior to blood sampling. Blood samples were collected into EDTA and serum tubes (SARSTEDT, S-Monovette) and centrifuged at 4°C, 4000 rpm for 10 minutes at. The urine samples were collected in a sterile disposable container from the rst-morning portion of urine from the middle stream immediately after bedtime and centrifuged at 1500 rpm for 5 minutes. In order to protect against oxidation, the supernatant was added (10 µl of 0.5 M BHT / 1 ml of plasma/serum and urine) and stored at -80°C until appropriate determinations were made [9,26].

Laboratory measurements
Serum cortisol before 10 a.m., serum aldosterone, Na + , K + , glucose, and urine methanephrine and normethanephrine, uric acid as well as full blood count were analyzed using an Abbott analyzer (Abbott Diagnostics, Wiesbaden, Germany). The total phenolic content (TPC) was assayed according to the Folin-Ciocalteu method [27].

Redox Assays
All reagents used to perform the redox assays were obtained from Sigma-Aldrich (Nümbrecht, Germany / Saint Louis, MO, USA). The absorbance of the samples was measured using Mindray MR-96 Microplate Reader (Mindray, Nanshan, China). Determinations of all tested parameters were carried out in triplicate samples. The results were standardized to 1 mg of total protein.

Total Oxidant Status (TOS)
In the presence of the oxidants contained in the sample, the level of plasma total oxidant status (TOS) was evaluated bichromatically at 560/800 nm based on the oxidation reaction of Fe 2+ to Fe 3+ [29].

Radical-Scavenging Activity Assay (DPPH)
The antioxidant potential of plasma and urine was also assayed using DPPH (1,1-diphenyl-2picrylhydrazyl) radical and Trolox as a standard [23]. The absorbance of DPPH, after decolorization in the presence of antioxidants, was measured spectrophotometrically at 515 nm [31].

Ferric-Reducing Antioxidant Power (FRAP)
The level of ferric-reducing antioxidant power (FRAP) was assayed using the reduction reaction of Fe 2+ to Fe 3+ an acidic environment. Absorbance of the resulting a colorful ferrous tripyridyltriazine (Fe 3+ -TPTZ) complex was measured colorimetrically at 592 nm [32,33].

Statistical analysis
Statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, USA) and Microsoft Excel 16.49 for MacOS. The Shapiro-Wilk test were used to evaluate the distribution of the results and data were presented as mean ± SD. The homogeneity of variance was checked by Levine's test. The groups were compared using one-way analysis of variance ANOVA with Tukey's post-hoc test. Multiplicity adjusted p value was also calculated. Correlations between biomarkers and clinical parameters were assessed based on the Pearson correlation coe cient. Statistically signi cant value was p ≤ 0.05.
The number of patients was determined a priori based on the previous pilot study (n = 40). The power of the test was assumed as 0.9 and α = 0.05. Variables used for sample size calculation were plasma and urine TAC, TOS and FRAP. The ClinCalc online calculator provided the sample size for one group. The minimum number of patients was 17.  Table 1 demonstrates a comparison of the clinical and laboratory characteristics of the controls and patients with adrenal masses: incidentaloma, pheochromocytoma, and Cushing's/Conn's adenoma. We found greater BMI values and serum glucose concentration in all study subgroups compared to the healthy controls. The PLT content was decreased in patients with incidentaloma and Cushing's/Conn's adenoma than in the controls and patients with pheochromocytoma. Urinary metanephrine and normetanephrine were increased in the pheochromocytoma group than the controls and incidentaloma and Cushing's/Conn's adenoma patients. However, concentration of serum cortisol and aldosterone were higher in Cushing's/Conn's adenoma group as compared to the controls. Patients with incidentaloma had higher serum concentration of UA and content of TPC than the controls.

Discussion
In recent years, many studies have been conducted trying to explain the pathogenesis of cancer. The burden of cancer continues to increase worldwide. According to the World Health Organization (WHO), cancer is the second leading cause of death in the world, accounting for approximately 9.6 million deaths in 2018 [34]. Numerous studies suggest that redox imbalance may be a factor predisposing to cancer development [9,35,36]. However, it has not yet been clari ed in what direction the redox equilibrium is shifted and whether these disorders may be involved in the development of adrenal tumors. This is the rst study to evaluate the total antioxidant potential in patients with adrenal tumors. Additionally, we compared redox status depending on the type of the tumor: incidentaloma, pheochromocytoma and Cushing's/Conn's adenoma.
Antioxidants can act additively or synergistically, and can be absorbed and utilized in the body in different ways [37]. Therefore, the assessment of total antioxidant activity provides more reliable information about the biological system than the assessment of individual antioxidants separately [22,38]. There are many different methods for measuring total antioxidant activity. The contribution of individual antioxidants varies, because the same antioxidants have different reactivity in various methods [39,40]. Moreover, in order to correctly measure total antioxidant activity, it is recommended to perform at least two different tests. These methods use the ability of the test compound or product to scavenge free radicals and / or metal ions involved in the oxidation reaction. It is also important to distinguish between antioxidant and antiradical activity. Antioxidant activity is characterized by the ability to inhibit the oxidation process, while antiradical activity is the ability of compounds to react with free radicals [41]. TAC does not provide information on the nature of the compounds, but is used to evaluate synergistic interactions between antioxidants. Nevertheless, the TAC method measures only part of the antioxidant capacity, i.e. nonenzymatic activity [23,42]. In our study we observed diminished plasma TAC in patients with pheochromocytoma. This may be a result of a decreased plasma concentration of GSH, the major nonenzymatic antioxidant in these patients [21]. Diminished GSH concentrations lead to the intensi cation of the in ammatory process with an increase in the secretion of in ammatory mediators: IL-1β and TNF-α [43,44]. Antioxidants and oxidants react together stoichiometrically, therefore, the assessment of TAC is mainly in uenced by antioxidants present at the highest concentrations. Uric acid and thiol protein groups have the largest share in TAC in human plasma. Uric acid is also the major contributor in urine TAC [45].
This is also con rmed by the results of our study. Nevertheless, we observed a positive correlation between UA and TAC only in the control group.
Other methods for measuring total antioxidant potential include DPPH and FRAP. The DPPH test uses stable 1,1-diphenyl-2-picrylhydrazyl free radical and thus re ects the radical scavenging process or antiradical activity [46]. The FRAP method is based on the reduction of iron ions by antioxidants contained in the sample [47]. The contribution of individual antioxidants to the total antioxidant potential varies depending on the test used. Due to low pH = 3.6, share of GSH and thiol groups in the total antioxidant potential is signi cantly lower in the FRAP assay than in DPPH and TAC methods [23,48]. Therefore, plasma FRAP much better re ect the antioxidant potential of the human body [37].
In our study, we observed decreased plasma DPPH and FRAP in all study groups: incidentaloma, pheochromocytoma and Cushing's/Conn's adenoma. Although we did not directly evaluate the rate of ROS production in our patients, total oxidative potential (TOS) was signi cantly higher in adrenal cancer cases as compared to heathy controls. This parameter expresses the total oxidant content in the biological material and may indicate increased free radical formation in adrenal cancer patients. The question now arises: is there a shift in redox equilibrium in favor of oxidation reactions? For this purpose, we calculated the oxidative stress index (OSI), which is the quotient of total antioxidant potential (TAC) to TOS. OSI was signi cantly higher in all patients with adrenocortical carcinoma and therefor antioxidant / oxidant barrier is shifted towards an increased oxidation process. Thus, in patients with adrenal tumors, oxidative damage to proteins, lipids, and DNA may be exacerbated. Although we observed disturbances in the redox homeostasis in all study groups, they were the most severe in patients with pheochromocytoma. Increased oxidative stress in patients with phaeochromocytoma can be associated with HIF-1 (hypoxia-inducible factor 1) activity. Under hypoxic conditions, HIF-1, by stabilizing HIF-1α, increases the activity of NADPH oxidase, contributing to the ROS overproduction [12,15,49]. Moreover, most patients with adrenal gland tumors are overweight or obese. It is well known that an excessive amount of adipose tissue leads to increased production of ROS [50]. Therefore, the question arises whether the redox disturbances are not the result of increased body weight. Although we have not investigated this directly, it can be speculated that the increased oxidative stress in patients with adrenal tumors may be associated with obesity. It has been described that adipokines secreted by adipose tissue can activate nuclear factor kappa B (NF-κB), which induces the secretion of proin ammatory cytokines (IL-1, IL-6, IL-8), tumor necrosis factor α (TNF-α), as well as impairs the bioavailability of NO and increases the formation of free radicals [50][51][52][53]. Further on, patients with functional adrenal tumors, especially phaeochromocytomas suffer often from impaired lipid and glucose metabolism, and insulin resistance [54], which may be the result of increased production of catecholamines [55], obesity [56], as well as the advantage of the oxidative process over antioxidant.
It should also be noted that the total antioxidant potential may vary depending on the biological uid in which it is measured. Parameters that assess redox homeostasis are usually measured in serum or plasma as a stable environment for systemic biomarkers [57]. Nevertheless, Il'yasova et al. [58] argue that urine is a better biological uid for the evaluation of oxidative stress markers than plasma or serum; and urinary oxidative stress parameters may re ect local and systemic oxidative status [57]. The urine has a lower content of metals and ROS promoters, therefore in the urine there is a lower risk of obtaining results with elevated values of oxidative stress markers [58]. In this study we observed higher TAC, DPPH and FRAP values in the plasma than in urine. However, it was also observed that urine TAC had similar or higher values than in blood plasma [59]. Therefore, it is important to check whether redox biomarkers correlate between different body uids. Antioxidant status measured in body uids generally re ect a local, not a systemic, redox homeostasis [60]. However, we found positive correlations between plasma FRAP and urine FRAP in patients with incidentaloma. In pheochromocytoma subgroup, plasma TAC correlated positively with urine TAC, as well as plasma DPPH and urine DPPH, plasma FRAP and urine FRAP. In Cushing's/Conn's adenoma, plasma TAC highly positively correlated with urine TAC, plasma DPPH with urine DPPH and plasma FRAP with urine FRAP. This indicates that urinary antioxidant status re ects changes in blood and can be used to assess systemic redox imbalances. These hypotheses are also supported by the correlations between plasma/urinary antioxidant status and the classical biomarkers evaluated to assess disease progression: cortisol, metanephrine, and normetanephrine.
In the study groups, both TAC, DPPH, and FRAP generally did not correlate with UA concentration and total polyphenolic content. Thus, as opposed to healthy people, these compounds may be marginally responsible for plasma/urine antioxidant activity. The weakening of the antioxidant barrier may be due to depletion of other low molecular weight antioxidants such as hydrophilic GSH, total thiols, ascorbic acid, and lipophilic α-tocopherol, β-carotene, retinol, and coenzyme Q10 [23,61]. This issue requires further research and may be of great clinical importance.
Although antioxidants are the main defense mechanism against ROS overproduction, the reduced levels of TAC, DPPH and FRAP clearly indicate a reduced ability to scavenge free radicals and thus a lack of effective protection against oxidative stress in patients with adrenal tumors. Therefore, those patients are especially vulnerable to oxidative stress and oxidative damage, which can lead to impaired cellular  Figure 1 Plasma TAC (A), TOS (D) and OSI (G), urine TAC (C), TOS (E) and OSI (H), and plasma/urie index of TAC (C), TOS (F) and OSI (I) of the controls, incidentaloma, pheochromocytoma, and Cushing's/Conn's adenoma patients. Results are presented as mean with standard deviation. * p<0.05, ** p<0.01, *** p < 0.001, **** p < 0.0001 indicate signi cant differences from the controls; ^ p<0.05, ^^ p<0.01, ^^^^ p<0.0001 indicate signi cant differences from the pheochromocytoma group; total antioxidant capacity (TAC), total oxidant status (TOS) and oxidative status index (OSI) Figure 2 Plasma DPPH (A) and FRAP (D), urine DPPH (B) and FRAP (E), and plasma/urine index of DPPH (C) and FRAP (F) of the controls, incidentaloma, pheochromocytoma, and Cushing's/Conn's adenoma patients.