Study of the Association between Thiols and Oxidative Stress Markers in Children with Obesity

Obesity has reached epidemic proportions, and the World Health Organization defined childhood overweight and obesity as a noncommunicable disease that represents the most serious public health challenges of the twenty-first century. Oxidative stress, defined as an imbalance between oxidants and antioxidants causing an impairment of the redox signals, is linked to the development of metabolic diseases. In addition, reactive oxygen species generated during metabolic disorder could increase inflammation, causing the development of insulin resistance, diabetes, and cardiovascular disease. We analyze serum levels of cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), and other markers of oxidative stress, such as thiobarbituric acid reactive substances (T-BARS), 8-isoprostane, and protein carbonyl in our children with obesity. Total antioxidant status was also determined. We found lower GSH and Cys-Gly levels, and higher Hcy and oxidative stress markers levels. We also found a positive correlation between Body Mass Index (BMI), Cys, GSH, and Hcy levels, between insulin and Cys levels, and between BMI and the homeostasis model assessment-estimated insulin resistance (HOMA-IR) with 8-isoprostane levels. Finally, we found a correlation between age and GSH and Cys levels. The deficiency of GSH could be restored by dietary supplementation with GSH precursors, supplying an inexpensive approach to oppose oxidative stress, thus avoiding obesity complications.


Introduction
Disruption of normal metabolic processes could lead to redox imbalance and the initiation of several pathophysiological changes collectively termed metabolic syndrome, caused by overweight and obesity. Obesity has reached epidemic proportions, and the World Health Organization defined childhood overweight and obesity as a noncommunicable disease that represents the most serious public health challenges of the twenty-first century [1]. Indeed, in the last 30 years, childhood obesity has increased constantly in both low-and high-income countries [2][3][4]. Obesity in children and adolescents is defined as a BMI greater than or equal to the 95th percentile by age and sex of the child or a BMI of more than 30.0 kg/m 2 among older adolescents (that is the definition of obesity in adults).
Oxidative stress, defined as an imbalance between oxidants and antioxidants causing an impairment of the redox signals, is related to chronic inflammation and to the evolution Nutrients 2022, 14, 3637 2 of 10 of metabolic diseases [5]. Oxidative stress is higher in children with obesity, where it could boost the evolution of complications [6,7]. Insulin resistance is frequently linked to obesity, and it could contribute as a fount of oxidative stress [8]. Additionally, reactive oxygen species (ROS) generated during metabolic disorder could increase inflammation by damaging redox signaling pathways and modifying gene expression of inflammatory cytokines, chemokines, and growth factors, thus causing the progress of several diseases, such as insulin resistance, diabetes, and cardiovascular disease [9].
As several studies have highlighted the clinical impact of thiol-disulfide hemostasis on obesity [10][11][12][13][14][15], we analyze thiol levels in our cohort of children with obesity. Moreover, we investigate serum markers of oxidative stress in order to verify if ROS overproduction could contribute to the development of complications in obesity. We thus determine the serum levels of cysteine (Cys), cysteinyl-glycine (Cys-Gly), homocysteine (Hcy), and glutathione (GSH), and other markers of oxidative stress, such as thiobarbituric acid reactive substances (T-BARS), 8-isoprostane, and protein carbonyl. Moreover, we also analyze the total antioxidant, as Trolox equivalents, in our cohort of children with obesity.

Samples
Blood was collected into Vacutainer Tubes (Becton Dickinson, Franklin Lakes, NJ, USA); plasma and/or serum was obtained immediately by the centrifugation of the blood at 2000 g for 5 min. and stored at −80 • C until analysis within a maximum of one month from the collection. Technical staff collected clinical data and informed consent from participants, and the corresponding samples were labeled and analyzed as described below. To maintain confidentiality, participants were labeled with de-identified barcodes, and all data were collected and stored in a locked room with limited access.

Total Antioxidant Levels and Oxidative Stress Markers
Total antioxidant, thiobarbituric acid reactive substances (T-BARS), 8-isoprostane, and protein carbonyl levels were analyzed by a commercial kit following the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI, USA).

Other Laboratory Assays
Biochemical parameters, such as glucose, insulin, C-reactive protein (CRP), total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides, alanine aminotransferase (ALT), and aspartate aminotransferase (AST), were measured on samples collected upon admission by using the Roche Cobas 8000/c702 module (Roche Diagnostics, Basel, Switzerland). Insulin and glucose levels were used for the calculation of the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) [17].

Statistical Analysis
Statistical analysis was performed using GraphPad Prism v9.0 (GraphPad Software, San Diego, CA, USA). To test for normal distribution of data, both the histogram and the Kolmogorov-Smirnov test of normality were used. If the data were normally distributed, Nutrients 2022, 14, 3637 4 of 10 parametric tests such as analysis of variance with Bonferroni post hoc test (in the case of more than two variables), or t-test (in the case of two variables) were used. In order to test the differences between different groups, the Mann-Whitney U test was used. A value of p < 0.05 was considered statistically significant in all statistical analyses; a value of p < 0.01 was considered extremely statistically significant. Spearman's rank correlation between the features extracted from each patient was also performed.

Discussion
The existing correlation between inflammation and obesity has been demonstrated [18]. Consistent with these previous findings, we found that CRP levels were significantly higher in our children with obesity. In addition, we also found elevated HOMA-IR values

Discussion
The existing correlation between inflammation and obesity has been demonstrated [18]. Consistent with these previous findings, we found that CRP levels were significantly higher in our children with obesity. In addition, we also found elevated HOMA-IR values compared to those reported for pediatric subjects without increased metabolic risk, confirming that obesity is a risk factor for metabolic disorders [21]. Moreover, we found high LDL and low HDL cholesterol levels. These results support the link between obesity and cardiovascular risk through increased fasting plasma LDL cholesterol, low HDL cholesterol, and elevated blood glucose and insulin levels [19].
Recently, it was demonstrated that the thiol/disulfide homeostasis antioxidant parameters were lower in obese children, with consequent higher oxidant parameters and a shift of the thiol/disulfide balance toward disulfide formation [10]. These findings do not corroborate in adult women with obesity, in which the oxidative damage is demonstrated only in plasma proteins with antioxidant function [11]. Thiol unbalance could thus be an early event culminating in the loss of function of antioxidant plasma proteins, probably due to their irreversible oxidation caused by the cumulating ROS in the environment.
Several studies have highlighted the clinical impact of homocysteine on obesity [12][13][14][15]. This is corroborated in this study, as we found that Hcy is significantly higher in patients with obesity with respect to healthy children. It was extensively demonstrated that high Hcy levels, which are correlated with lower HDL cholesterol in blood in volunteers and animal models, have been associated with an increased risk for cardiovascular diseases [22]. In humans, the relation between methionine and Hcy is dependent on vitamins B6-B12 and folic acid status and on the supply of other amino acids. However, lowering Hcy by itself is not sufficient for decreasing the risk of cardiovascular disease progression [23], as several other mechanisms are involved. First, the connection of one carbon metabolism and protein biosynthesis editing mechanisms has been demonstrated. Thus, Hcy and its metabolites could affect epigenetic control of gene expression, which underlies the pathology of several human diseases [24]. Therefore, Hcy is the hallmark of this imbalance [25]. Second, a decrease in the concentration of intracellular glutathione, a tripeptide involved in redox homeostasis, is also implicated [26]. Glutathione has indeed a fundamental role in the protection from free radicals, and its reduction could cause a chain of dangerous reactions eventually terminating in oxidative stress and presumably in metabolic syndrome [27]. Glutathione reduction could be caused by a reduced glutathione synthesis, an augmented utilization, or a coalescence of both mechanisms. Accordingly, we found dramatically lower levels of GSH in our children with obesity. Our previous studies report an excess of GSH utilization in obesity [14,15], and Nguyen and collaborators [28] suggest that GSH deficiency can promote fat accumulation. They found that mice with GSH deficiency had significantly higher total body fat, and that restoring GSH led to significant fat reductions, suggesting a role for GSH in maintaining fat homeostasis. Consistent with these findings, Chen and colleagues [29] showed that genetic manipulations induce severe hepatic depletion of GSH, resulting in excessive accumulation of fat in the liver of mice, thus suggesting that GSH deficiency could promote fat accumulation. In agreement with these findings, we showed a 50% decrement of GSH levels in the serum of our patients with obesity compared to our control population. This result agrees with our previous findings in the blood of children with obesity, in which we evidenced a depletion of Tot GSH, which is indicative of a defect in the thiol synthesis [14]. This is sustained by the fact that the significantly lower levels of Cys-Gly, an intermediate of the GSH synthesis, are not offset by an equivalent rise of Cys. This could be due to the impaired glycine status found in children with obesity [30]. This finding is also corroborated by Okekunle and colleagues [31], who demonstrated that plasma glycine levels are steadily lower in patients with obesity and T2DM compared to controls (−11% and −15%, respectively). It was earlier demonstrated that glycine availability may be the limiting factor for glutathione synthesis [32]. Indeed, glycine levels in tissues are lower than the Michaelis-Menten constant (Km) of glutathione synthase so that the accessibility of glycine could be excessively low to sustain a satis-Nutrients 2022, 14, 3637 8 of 10 factory synthesis rate of GSH. This is particularly true in metabolic diseases, in which oxidative stress is increased [33]. It has been hypothesized that oxidative stress, defined as the imbalance between oxidants and antioxidants with an increase of oxidants, could impair intracellular redox signals, leading to chronic inflammation and the development of metabolic diseases [5]. The involvement of oxidative stress in the development of obesity in adults has been extensively studied [34], but less information is available in children.
Here we demonstrated a rise in oxidative stress parameter in children with obesity, along with lower total antioxidants. Accordingly, enhanced ROS formation and the increase in oxidatively modified proteins have been previously demonstrated in both overweight and obese youths, and have been implicated in the progress of obesity-related disorders [35].
The deficiency of GSH and the gained oxidative stress evidenced by our study may have cumulative effects; indeed, low GSH levels could intensify the oxidative damage, either of its unavailability as a direct scavenger of free radicals, or of its function as a cofactor for the antioxidant glutathione-dependent enzymes (glutathione peroxidase (GPx), glutathione transferase (GST), and glutathione reductase (GR)). However, as also sustained by the latest literature [34][35][36][37][38][39], glutathione and glycine reduction could be repaired by dietary supplementation with precursors, thus allowing a reasonable approach to neutralize oxidative stress and prevent obesity complications. Furthermore, it was recently demonstrated that diet could increase serum concentrations of chronic inflammation and oxidative stress markers. Early dietary interventions and supplementations could thus decrease chronic inflammation and oxidative stress in children and adolescents [40].

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the Bambino Gesù Children's Hospital on 9 September 2020 (Registration Number: 2160_OPBG_2020).

Informed Consent Statement:
Informed consent was obtained from the parents of each child after illustrating the purpose and the nature of the study and written informed consent has been obtained from the parents of each child to publish this paper.

Data Availability Statement:
The authors confirm that the data supporting the findings of this study are available within the article. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request. The data are not publicly available due to privacy and ethical reasons.

Conflicts of Interest:
The authors declare no conflict of interest.