Abstract
Background/Aim: Oxidative stress in association with metabolic syndrome represents a complex disease entity that has emerged as a significant public health challenge, and it is closely linked to an elevated risk of cardiovascular disease, type 2 diabetes, and even cancer. The objective of this study was to investigate the effectiveness of selenium supplementation in managing oxidative stress while considering a well-balanced diet based on a healthy lifestyle and diet therapy. Patients and Methods: The study included a total of 206 participants divided into three groups: the control group consisting of 35 individuals (17.0%) named LC, the diet therapy group comprising 119 individuals (57.8%) named LD, and the diet therapy group supplemented with selenium consisting of 52 individuals (25.2%) named LD+Se. Various clinical parameters such as body mass index (BMI), weight status, fat mass, visceral fat, and sarcopenia index, as well as paraclinical parameters including the HOMA index, cholesterol, triglycerides, C-reactive protein, and HGZ, were evaluated. Additionally, oxidative stress parameters using the FORD, FORT and MIXT tests were measured. Results: Selenium supplementation, along with FORD and FORT tests, demonstrated effectiveness in individuals with chronic venous disease, with a significantly greater decrease observed in those with chronic venous disease in the LD+Se group. Conclusion: Physiological aging has an important role in triggering or aggravating oxidative stress, and the use of antioxidant products such as selenium can reduce this process.
Metabolic syndrome (MS) and oxidative stress are two distinct, yet challenging issues due to their long-term association with atherosclerosis, which has been identified as a leading cause of global mortality and morbidity in recent research (1). Cardiovascular diseases contribute significantly to annual deaths worldwide (2-4). Furthermore, MS has been found to share low-grade inflammatory mechanisms with mood disorders and neurological conditions (5) like bipolar disorder, major depression (6), and Parkinson’s disease (7). These conditions are characterized by elevated levels of proinflammatory cytokines, acute phase proteins, lipid peroxidation, and oxidized low-density lipoprotein cholesterol (LDL-c), along with reduced antioxidant levels (8).
Biomarkers of oxidative stress are utilized to identify individuals at higher risk of complications associated with metabolic syndrome and to determine appropriate therapies for reducing this burden. These biomarkers encompass molecules reflecting lipid peroxidation, protein and amino acid oxidation, as well as DNA oxidation. Markers used for lipid peroxidation include thiobarbituric acid-reactive substances, malondialdehyde, 4-hydroxy-2-nonenal, and F2 isoproteins (1). Protein oxidation is indicated by protein carbonyls, advanced glycation end products (AGEs), oxidized LDL (ox-LDL), and advanced oxidation proteins (9). DNA oxidation can be assessed through markers such as 8-oxo-2-deoxyguanosine, 5-chlorouracil, and 5-chlorocytosine (10). Other relevant biomarkers associated with metabolic syndrome include adipokines, such as adiponectin and leptin. Reduced levels of adiponectin are linked to the development of metabolic syndrome, while leptin, a hormone involved in energy metabolism, is positively associated with metabolic syndrome and abdominal obesity (11).
Numerous studies have demonstrated that diet type influences the composition of the gut microbiota (12, 13), and so on the metabolism (14, 15). For instance, the Western diet has been associated with decreased microbial diversity and an increased Firmicutes/Bacteroidetes ratio, whereas a diet rich in polyunsaturated fatty acids (PUFAs) exhibits anti-inflammatory effects (16). Substituting saturated fatty acid intake with polyunsaturated fats (PUFAs) and monounsaturated fats (MUFAs), or high-quality carbohydrates like whole grains, may reduce the risk of cardiovascular disease (17, 18).
In this study, oxidative stress was evaluated using the CR3000 analyzer (Callegari, Catellani Group, Parma, Italy), enabling the analysis of oxidative stress from a single drop of capillary blood. The CR3000 analyzer employs two colorimetric assays to assess oxidative stress: the oxygen free radical test (FORT) and the oxygen free radical defense test (FORD) (19). The FORT test measures the levels of reactive oxygen species (ROS) in the blood, with the desired values for healthy individuals being ≤2.3 mmol/l of H2O2. On the other hand, the FORD test measures the antioxidant levels in the blood, and the normal range for Trolox equivalents is 1.07-1.53 mmol/l. Both the FORT test and the FORD test are valuable methods for assessing oxidative stress levels in patients with type 2 diabetes and obesity and have been utilized in research for over 10 years (20).
The FORT method is based on the Fenton reaction. In this method, hydroperoxides react with transition metal ions released from proteins, converting them into alkoxy and peroxy radicals. The radicals generated by this reaction, which are directly proportional to the amount of lipid peroxides, interact with an additive that forms a radical molecule (21). Additionally, the oxygen free radical defense (FORD) assay employs stable and colored preformed radicals, leading to a decrease in absorbance that is proportional to the concentration of antioxidants in the blood (19).
Finally, interventions based on dietary therapy, as well as the combination of dietary therapy and selenium administration, have a significant impact on altering the values of clinical and paraclinical indicators associated with metabolic syndrome. These interventions effectively reduce oxidative stress and enhance the body’s antioxidant function. Figure 1 illustrates the effectiveness of different antioxidants in the human body and their role in neutralizing free radicals.
The efficacy of various antioxidants in the human body and the neutralization of free radicals.
Patients and Methods
Body analysis. The assessment of the body composition was conducted using the INBODY 370 body bio-electrical impedance analyzer (Inbody, Seoul, Republic of Korea) and the results were evaluated using its specialized medical software (Inbody 370s). BIA body analyzers are approved devices by the World Public Health Nutrition Association (WPHNA) known for their high accuracy in determining body composition. The margin of error for these measurements is 0.1 kg.
Paraclinical analysis. Paraclinical evaluations were performed to support the diagnosis and confirm metabolic disorders. Several paraclinical analyses were conducted, such as assessments of the HOMA index, cholesterol, triglycerides, C-reactive protein, and HGZ. These paraclinical analyses were carried out in the laboratory using enzymatic, colorimetric, spectrophotometric methods, and immuno-enzymatic tests. Fasting glucose and insulin levels were used to calculate the homeostatic model of insulin resistance (HOMA-IR) using the following formula: [glucose mg/dl×insulin (IU/l))/405].
Oxidative stress analysis. To assess oxidative balance, colorimetric analysis of oxygen free radicals (FORT) and oxygen free radical defense (FORD) was performed on fingertip capillary blood. The FORT test (FORM, CR 2000, Callegari, Parma, Italy) utilizes the catalytic properties of transition metals, such as iron, in the decomposition of hydroperoxides (ROOH) through the Fenton reaction. The resulting reactive oxygen species interact with an additive (phenylenediamine derivative, 2CrNH®2), forming a stable and colored radical cation that can be measured at 505 nm using a spectrophotometer. The intensity of the color directly correlates with the level of radical compounds and, consequently, the oxidative state of the sample, following the Beer-Lambert law. Results are expressed in FORT units, where 1 FORT unit corresponds to 0.26 mg/l of H2O2. The intra-assay and inter-assay coefficients of variation for this method were 3.7% and 6.2%, respectively.
The FORD test evaluates the plasma antioxidant system by utilizing preformed, stable, and colored radicals. It measures the decrease in absorption, which is proportional to the concentration of antioxidants in the blood, following the Beer-Lambert law. In an acidic environment (pH=5.2) and in the presence of an appropriate oxidant (FeCl3), the chromogen containing 4-amino-N,N-diethylalline sulfate forms a stable and colored radical cation that can be detected photometrically at 505 nm. The antioxidant compounds in the sample reduce the radical cation of the chromogen, resulting in a decrease in color and solution discoloration proportional to their concentration. Absorbance values obtained from the samples are compared to a standard curve generated using Trolox (6-hydroxy-2,5,7,8-tetramethylcroan-2-carboxylic acid) - a cell-permeable derivative of vitamin E commonly used as an antioxidant. The intra-assay and inter-assay coefficients of variation for this test were 4.2% and 6.6%, respectively.
Participants. The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of the University of Oradea (protocol code CEFMF/1 from 31 January 2023 and date of approval). A 12-month prospective study: Out of 2,144 patients assessed, 206 were chosen from those seeking guidance from a nutritionist at the “TELIOS CARE” private medical office in Oradea, Romania. Exclusion criteria encompassed a history of malignant tumors, organ failure, limitations for evaluations due to specific pathologies, and study refusal. Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.
Statistical analysis. In this study, statistical analyses were conducted to investigate the changes in biomarkers over time. Numerical summaries, such as mean±standard deviation, were provided, along with case profile plots, for each biomarker. Graphs illustrating the relative changes from baseline (%) were also generated. No evidence was found to suggest deviations from normality in the distributions of each biomarker at different time points, tested by the Skewness and Kurtosis test.
To model the changes over time, a linear mixed model was utilized. This model incorporated a random effect for each individual and specified an unstructured correlation for repeated measures over time within the same individual. The time of testing was included as a fixed effect, initially as a categorical variable to enable comparisons of mean change at each time point, and subsequently as a continuous variable to compare the slopes of each biomarker over time. Pearson correlations were used to examine relationships between biomarkers.
All statistical analyses were performed using Statistical Product and Service Solutions (SPSS) software (version 20, IBM, Armonk, NY, USA). The significance level was set at p<0.05. Residual plots from the fitted model were employed to assess the model’s assumptions and its fit for each biomarker at each time point.
Results
A total of 206 individuals participated in the study, and they were divided into three groups: the control group (LC) comprised 35 individuals (17.0%) denoted as LC, the diet therapy group (LD) consisted of 119 individuals (57.8%) denoted as LD, and the diet therapy combined with selenium group included 52 individuals (25.2%) denoted as LD+Se, presented in Table I.
Information regarding demographics.
Several risk factors, including smoking, alcohol consumption, chronic venous disease, and stress, measured non-invasively by increased cardiac output and low CO2 partial pressure, were closely followed. The presence of these risk factors was assessed through a patient questionnaire. Their presence was indicated with “1”, while their absence was indicated with “0” for statistical evaluation.
Figure 2A shows that the LD group had the highest number of smokers, with 10 out of 14 individuals falling into this category. Among the groups, the LD+Se group had the highest incidence of alcohol consumption, as depicted in Figure 2B, with 52 individuals recorded. Chronic venous disease, as seen in Figure 2C, was most prevalent in the LD group, with 16 out of 22 individuals affected. Regarding stress levels (Figure 2D), there were no statistically significant differences observed among the groups, although patients in the LD group tended to exhibit slightly higher stress levels.
Graphical representation of risk factors, such as smoking (A), alcohol consumption (B), chronic venous disease (C), and noninvasive stress (D), in the three research groups.
Oxidative stress was assessed using the FORD, FORT and MIXT tests.
Initially, a low FORD test value was observed in 88 individuals (42.7%) at the cohort level. By the end of the study, this number decreased to 30 individuals (14.6%), as shown in Table II. Concerning the FORT test, increased values indicate oxidative stress. Throughout the research period, improvements in FORT test values were observed in 59 individuals, with a decrease in FORT test values. The MIXT test combines the results of the FORT and FORD tests to provide an index of oxidative stress. This index has demonstrated good reliability. In this study, the changes in the MIXT test were also monitored. The final results demonstrated an improvement in the MIXT test among 28 individuals out of the total number of individuals who were monitored, as depicted in Table II. Based on Figure 3, which presents the graphical representation of the oxidative stress parameters in the three research groups, the most significant difference, indicating the greatest improvement, was observed in the LD+Se group for all three parameters: the FORD test (A), the FORT test (B), and the MIXED test (C).
Oxidative stress parameters in the LC, LD and LD+Se groups.
Graphical representation of the differences regarding the FORD (A), FORT (B) and MIXT (C) test in the three study groups.
Moreover, oxidative stress parameters linked to risk factors, especially smoking, were explored using the FORD, FORT, and MIXT tests (Table III). The results showed a significant decline in FORD levels, with 30 out of 88 individuals reaching low levels by the study’s end. Non-smokers experienced more significant improvements in FORD. Elevated FORT values were initially seen in 77 individuals, but only 18 retained high values later. Regarding the MIXT test, 16 individuals among the initial 44 maintained high results. Analyzing the correlation between smoking and the difference in initial and final FORD values, smokers exhibited a larger reduction due to their higher initial toxin levels, depicted in Figure 4.
Evolution of oxidative stress parameters according to smoking, alcohol consumption, chronic venous disease, and chronic stress.
Graphical representation of the differences in the oxidative stress test parameters (the FORD, FORT and MIXT tests) and the risk factors.
Similarly, when examining the correlation between smoking as a risk factor and the difference between the final and initial values of the FORT test, a more significant decrease was observed in smokers. This can be attributed to the higher initial toxin levels in smokers compared to non-smokers, as illustrated in Figure 4. Furthermore, examining the link between smoking as a risk factor and the disparity in final vs. initial MIXED test values among the cohort highlighted a more significant reduction in smokers. This is due to the elevated initial toxin levels in smokers in contrast to non-smokers, as indicated in Figure 4.
Turning to alcohol consumption as a risk factor, the evaluation of oxidative stress parameters through the FORD, FORT, and MIXT tests was conducted. It was observed that a low level of FORD was registered at the end of the study in 30 out of 88 individuals, which indicated a statistically significant difference. Notably, non-drinkers showed a more pronounced improvement. The FORT test (Figure 4) displayed an initial increase in 77 individuals but decreased by the end of the research period, with only 18 individuals exhibiting high values. In terms of the MIXT test, at the end of the study, only 16 individuals out of the initial 44 remained.
Examining the correlation between alcohol consumption as a risk factor and the difference between the final and initial values of the FORT tests at the cohort level, a more substantial decrease was observed in individuals who consumed alcohol. This trend is similar to the correlation seen in the FORD test (low), as depicted in Figure 4. The strong correlation between the FORD and FORT tests supports this observation. Examining the correlation between alcohol consumption as a risk factor and the difference between the final and initial values of the MIXED tests at the cohort level reveals a more substantial decrease in individuals consuming alcohol, attributed to alcoholic liver diseases, as described in Figure 4.
Analyzing the evolution of oxidative stress parameters through the FORD, FORT and MIXT tests, they were evaluated with respect to chronic venous disease as a risk factor. A low level of FORD (Figure 4) was observed at the end of the study in 30 out of 88 individuals, indicating a statistically significant difference, with a more pronounced improvement in those with chronic venous disease. The FORT test initially increased in 77 individuals but decreased by the end of the research period, with only 18 individuals showing high values (Figure 4) (the desired values for healthy individuals are ≤2.3 mmol/l H2O2). Regarding the MIXT test, only 16 individuals remained out of the initial 44 with a high MIXT test. Examining the correlation between chronic venous disease as a risk factor and the difference between the final and initial values of the MIXED tests at the cohort level reveals a more substantial decrease in individuals with chronic venous disease, potentially attributed to the higher level of pro-inflammatory cytokines, as depicted in Figure 4.
Moreover, delving into the changes of oxidative stress parameters using the FORD, FORT, and MIXT tests, they underwent assessment for chronic stress, which was measured as heightened cardiac output along with diminished carbon dioxide partial pressure, acting as a risk determinant. It was found that a low level of FORD was recorded at the end of the study in 30 out of 88 individuals, indicating a statistically significant difference, with a more pronounced improvement observed in individuals without chronic stress. The FORT test initially increased in 77 individuals but decreased by the end of the research period, with only 18 individuals displaying high values. Similarly, the MIXT test showed a decrease, with only 16 individuals remaining out of the initial 44 with a high MIXT test.
Examining the correlation between chronic stress as a risk factor and the difference between the final and initial values of the FORD tests at the cohort level reveals a more substantial decrease in individuals without chronic stress, potentially indicating the direct effect of diet therapy and selenium on oxidative stress, as illustrated in Figure 4. Similarly, analyzing the correlation between chronic stress (measured by increased cardiac output with low PCO2) as a risk factor and the difference between the final and baseline values of the FORT tests at the cohort level shows a more substantial decrease in individuals with chronic stress, comparable to the FORD test (low), as depicted in Figure 4 (the normal range for Trolox equivalents is 1.07-1.53 mmol/l). Lastly, investigating the correlation between chronic stress (increased cardiac output with low PCO2) as a risk factor and the difference between the final and initial values of the MIX tests at the cohort level reveals a more substantial decrease in individuals without chronic stress, as shown in Figure 4.
Risk factors were cumulatively monitored to highlight selenium’s impact on reducing oxidative stress. This was evident through increased baseline values without low FORD at the study’s end, alongside a decrease in patients with low FORD levels, as depicted in Figure 5A. The most significant improvements were noticed in patients with chronic stress but no alcohol consumption, smoking, or chronic venous disease. Notably, the second most substantial enhancement occurred in patients with chronic stress, alcohol consumption, but without smoking or chronic venous disease.
Evolution of FORD, FORT and low MIXT test among individuals who smoke, consume alcohol, have chronic venous disease, and experience chronic stress. STRESS: Chronic stress; CVD: chronic venous disease.
Similarly, risk factors were collectively observed to underscore selenium’s role in diminishing oxidative stress. This was reflected in an increase in initial values without elevated FORT test outcomes by the end of the study, accompanied by fewer patients displaying elevated FORT test results (Figure 5B). The most substantial improvements were observed in patients experiencing chronic stress without alcohol consumption, smoking, or chronic venous disease. The second most notable improvement was seen in patients with chronic stress, alcohol consumption, but without smoking or chronic venous disease.
Discussion
In recent years, there has been a growing understanding of the potential role of oxidative stress in metabolic syndrome (22). While insulin resistance is widely accepted as the primary mechanism underlying metabolic changes in individuals with metabolic syndrome or other conditions (23), mounting evidence highlights the close association between metabolic syndrome, chronic inflammation, and oxidative stress as secondary abnormalities (24).
Sarcopenia, particularly in the elderly population, has been the focus of research due to its correlation with the inflammatory process and frailty. In our study, a body analyzer with a margin of error of 0.1 kg was used to track sarcopenia. Published research supports the use of the MF-BIA method as a diagnostic tool for sarcopenia in older adults, with a specificity of 90% (25). The Asian Working Group on Sarcopenia (2019) has defined sarcopenia as a low skeletal muscle mass index (SMI) of <7.0 kg/m2 for men and <5.7 kg/m2 for women (26), and sarcopenic obesity as a combination of low SMI and a high percentage of body fat (PBF) (SMI and low PBF ≥25% for men and SMI and low PBF ≥30% for women) (27, 28). In our study, we also used the same SMI threshold for healthy individuals. However, references to a high sarcopenic index are scarce in the existing literature. A high sarcopenic index indicates the infiltration of fat mass into the muscles, which can interfere with insulin signaling and contribute to the development of metabolic syndrome (29-31). Although none of our patients had sarcopenia, we followed the sarcopenic index from this perspective. We observed a high initial sarcopenic index, which significantly decreased in the LD+Se group but remained above the lower limits. This improvement in insulin signaling and metabolic syndrome was not as pronounced in the control group, indicating the effectiveness of dietary intervention. Selenium supplementation was particularly effective in the LD+Se group, highlighting its role in improving outcomes.
Within the human body, the gut contains the most extensive immune system, accountable for more than 80% of immune defense responses. It acts as a robust barrier against bacteria, viruses, pathogens, and foreign proteins present in food. The proper digestion, transit, and healthy crossing of food through the intestinal mucosa ensures its good tolerance and contributes to a balanced microbiome. Adequate serum vitamin D levels also hold significance in maintaining a healthy microbiome (32). However, an exaggerated response to risk factors can trigger oxidative stress reactions. Therefore, this aspect was meticulously monitored and subjected to statistical analysis using multiple compatible methods.
In a study conducted in 2021, examining individuals with non-communicable diseases (lifestyle diseases), it was observed that smokers exhibited a higher prevalence of such conditions. Additionally, these smokers reported significantly elevated stress levels compared to non-smokers (33). In our study, the efficiency of selenium supplementation, as well as FORD and FORT tests, was evident in smokers, with a significantly greater decrease observed in non-smokers in the LD+Se group. This highlights the importance of adopting a healthy lifestyle by reducing smoking and alcohol consumption to enhance the antioxidant effect of selenium.
Oxidative stress plays a significant role in the pathogenesis of vascular changes by initiating or exacerbating the underlying biochemical processes associated with metabolic syndrome. Experimental and clinical observations have also implicated oxidative stress as a crucial mechanism in obesity-related metabolic syndrome, diabetes development and its complications, and conditions such as nonalcoholic steatohepatitis (NASH) (34). In our study, chronic venous disease was monitored as a risk factor that can increase excessive rosins secretion and aggravate oxidative stress. Notably, patients with chronic venous disease who received selenium supplementation demonstrated a more substantial reduction in oxidative stress. This finding further supports the link between vascular pathogenesis and oxidative stress and highlights the effectiveness of selenium supplementation in mitigating these effects.
Dysglycemia consists of two components: chronic sustained hyperglycemia (35), which includes chronic postprandial hyperglycemia (36) and chronic hyperglycemia (37), as well as acute spike-to-trough blood glucose fluctuations (38). Both components contribute to the development of diabetic complications (39) through two main mechanisms: excessive protein glycation (40) and activation of oxidative stress (41).
These two mechanisms have been integrated into an elegant theory proposing that the glycemic disturbances observed in diabetic patients activate oxidative stress (42) by causing an overproduction of superoxide by the mitochondrial electron transfer chain (43). This activation, in turn, triggers a cascade of detrimental metabolic events (44) such as increased polyol activity (45), heightened formation of advanced glycation end products (46), activation of protein kinase C and nuclear factor 
B (47), and augmented hexosamine pathway flux (48).
Dyslipidemia is defined as a condition characterized by elevated levels of blood cholesterol and triglycerides, which can increase the risk of cardiovascular disease, stroke, and other health problems (49, 50). Low levels of circulating high-density lipoprotein (HDL), increased clearance of HDL particles, elevated postprandial triglyceride values, and elevated plasma levels of very low-density lipoprotein (VLDL) promote the generation of reactive oxygen species (ROS) in the endothelium (51). In addition to its pro-inflammatory effects, ROS can directly damage lipids, proteins, or DNA and modulate intracellular signaling pathways, such as mitogen-activated protein kinases and redox-sensitive transcription factors, leading to alterations in protein/lipid expression and irreversible oxidative damage (51). In the current study, we monitored the progression of the two components of dyslipidemia with regards to dietary therapy and additional treatment for oxidative stress, specifically selenium supplementation.
Possible factors contributing to oxidative stress in obesity include hyperglycemia, elevated tissue lipid levels, deficiencies in vitamins and minerals, chronic inflammation, hyperleptinemia, increased muscle activity to support excessive weight, endothelial dysfunction, impaired mitochondrial function, and dietary factors (52, 53). In our study, we observed improved results only with dietary therapy compared to the low-calorie (LC) group, with comparable outcomes to the low-calorie diet plus selenium (LD+Se) group in some cases, albeit without significant differences. In comparison to other studies, our study provides new data on the relationship between metabolic syndrome, oxidative stress, and nutritional biomarkers.
Conclusion
Selenium supplementation, combined with FORD and FORT tests, demonstrated notable effectiveness in smokers and individuals with chronic venous disease within the LD+Se group, showcasing the potential benefits of healthier habits. Managing risk factors and employing antioxidant interventions like selenium can effectively counteract physiological aging-triggered oxidative stress, mitigating its impact and promoting overall well-being. Chronic stress significantly contributes to oxidative stress, linking to serious conditions like cardiovascular diseases, diabetes, and cancer.
Acknowledgements
The Authors would like to thank the University of Oradea, for supporting the payment of the publication fee, through an internal project.
Footnotes
Authors’ Contributions
Conceptualization, T.C.G. and A.M.D.; methodology, T.C.G.; software, T.C.G.; validation, T.C.G. and A.M.D.; formal analysis, T.C.G.; investigation, A.M.D.; resources, A.M.D.; data curation, A.M.D.; writing—original draft preparation, A.M.D.; writing—review and editing, T.C.G. and A.F.B.; visualization, T.C.G.; supervision, T.C.G.; project administration, T.C.G.; funding acquisition, T.C.G. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received July 7, 2023.
- Revision received August 12, 2023.
- Accepted August 28, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
