Modulation of Oxidative Stress Biomarkers by Ascorbic Acid and Creatine Co-Treatment in Male Rabbits”

INTRODUCTION

Oxidative stress is a pathological condition characterized by an imbalance between reactive oxygen species (ROS) production and the capacity of biological systems to detoxify these reactive intermediates or repair the resulting damage [1]. ROS such as superoxide anions, hydrogen peroxide, and hydroxyl radicals are generated as natural byproducts of cellular metabolism, particularly within the mitochondria, and play essential roles in cell signaling and homeostasis[2]. However, excessive ROS accumulation can damage cellular components, including lipids, proteins, and DNA, contributing to various diseases such as cancer, neurodegeneration, cardiovascular disorders, and infertility[3].To counteract oxidative stress, organisms rely on a complex antioxidant defense system comprising both enzymatic components, such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase, as well as non-enzymatic antioxidants like glutathione (GSH), vitamins C and E[4]. The thiobarbituric acid reactive substances (TBARS) assay, which measures malondialdehyde (MDA) levels, is commonly used as a marker of lipid peroxidation and oxidative damage [5]. Ascorbic acid (vitamin C) is a potent water-soluble antioxidant that directly scavenges ROS and restores other antioxidants such as vitamin E to their active forms. It also protects crucial antioxidant enzymes from oxidative inactivation and contributes to the regeneration of glutathione [6]. Numerous animal studies have confirmed its effectiveness in reducing lipid peroxidation and enhancing antioxidant enzyme activities under oxidative stress conditions [7]. Creatine, a naturally occurring nitrogenous compound found primarily in muscle tissue, has traditionally been studied for its role in energy metabolism [8]. However, recent evidence suggests that creatine also possesses antioxidant properties. It can stabilize mitochondrial membranes, enhance ATP buffering, and reduce ROS formation by supporting mitochondrial efficiency [9]. Studies have reported that creatine supplementation mitigates oxidative damage in both muscle and brain tissues [10]. The combined administration of ascorbic acid and creatine may provide a synergistic antioxidant effect by targeting multiple aspects of oxidative stress [11]. While ascorbic acid directly scavenges free radicals, creatine may enhance cellular bioenergetics and reduce mitochondrial ROS generation [12,13]. Despite their individual benefits, limited studies have examined their combined effect on oxidative stress biomarkers in vivo, particularly in rabbit models. Exploring this combination may offer insights into new therapeutic strategies against oxidative stress-related conditions [14]. Therefore, the present study aims to evaluate the modulatory effect of co-treatment with ascorbic acid and creatine on antioxidant enzyme activities (CAT, SOD), glutathione levels (GSH), and lipid peroxidation (TBARS) in male rabbits. This research could provide a scientific basis for the potential synergistic interaction between these two compounds in enhancing antioxidant defense mechanisms.

Materials and Methods

A total of 20 healthy adult male rabbits (Oryctolagus cuniculus), weighing between 1.8 to 2.2 kg, were used in this study. The animals were housed under standard laboratory conditions (temperature 22–25°C, 12-hour light/dark cycle) and were provided with acommercial pellet diet and water ad libitum. The rabbits were randomly divided into four groups (n = 5 per group) as follows: Group I (Control): Received no treatment and served as the negative control. Group II (Ascorbic Acid): Administered ascorbic acid at a dose of 20 mg/kg body weight orally, once daily for 6 weeks. Group III (Creatine – CrS): Administered creatine monohydrate at a dose of 5 mg/kg body weight orally, once daily for 6 weeks. Group IV (Ascorbic Acid + CrS): Co-treated with both ascorbic acid (20 mg/kg) andcreatine (5mg/kg) orally, once daily for6weeks.All substances were freshly prepared in distilled water before administration. Blood samples were collected from the ear vein of all animals every other week throughout the 6-week experimental period. Blood samples were obtained in the morning before access to feed and water and placed immediately on ice. The blood samples were collected in two tubes, one containing heparin to obtain plasma and the other one without anticoagulants to obtain serum. Glutathione S-transferase (GST; EC 2.5.1.18) activity was determined according to [15]. Catalase (CAT; EC 1.11.1.6) activity was determined using the Luck method involving the decomposition of hydrogen peroxide [16]. Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured according to [17]. Plasma thiobarbituric acid-reactive substances (TBARS) were measured by the method of [18]. Statistical Analysis. Data were expressed as mean ± standard error (SE). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test to compare differences between groups. A p-value < 0.05 was considered statistically significant.

Results

The present study assessed the impact of ascorbic acid, creatine (CrS), and their co-administration on key oxidative stress biomarkers in male rabbits. The results provide compelling evidence that these treatments modulate enzymatic antioxidant defenses and lipid peroxidation levels in a statistically significant manner. Catalase is a vital antioxidant enzyme responsible for decomposing hydrogen peroxide into water and oxygen, thereby protecting cells from oxidative damage. In this study, the CAT activity was significantly elevated in the Ascorbic acid + CrS group (1.12 ± 0.043 U/min/ml) compared to the control (0.933 ± 0.050 U/min/ml), as supported by a highly significant ANOVA p-value of 0.000. The combination treatment exhibited a synergistic effect, showing greater enhancement than either agent alone. SOD serves as the first line of defense against oxidative stress by converting superoxide radicals into hydrogen peroxide. SOD activity was significantly increased in all treated groups compared to the control, with the CrS (1.356 ± 0.065 U/ml) and Ascorbic acid + CrS (1.364 ± 0.040 U/ml) groups showing the highest values. The strong statistical significance (p = 0.000) highlights the effectiveness of these interventions in enhancing superoxide scavenging capacity. GSH is a critical non-enzymatic antioxidant that plays a central role in maintaining cellular redox balance. Interestingly, no significant changes were observed in GSH levels across treatment groups (p = 0.882), although slight, non-significant elevations were recorded in the ascorbic acid (6.006 ± 0.205 U/ml) and combination groups (5.97 ± 0.115 U/ml). TBARS is a well-established marker of lipid peroxidation and oxidative tissue damage. The ascorbic acid group exhibited the most pronounced reduction in TBARS levels (3.209 ± 0.051), followed by the combination treatment and the CrS group. The significant reduction in TBARS (p = 0.000) across all treated groups.

Discussion

The present findings demonstrate that both ascorbic acid and creatine, individually and in combination, significantly modulate oxidative stress biomarkers in male rabbits. This modulation was evident through increased enzymatic antioxidant activities and reduced lipid peroxidation, highlighting the potential of these agents as protective interventions against oxidative stress. The significant elevation of catalase (CAT) activity in the ascorbic acid + CrS group compared to the control group suggests a synergistic antioxidant interaction [19]. Catalase plays a critical role in cellular defense by catalyzing the decomposition of hydrogen peroxide, a harmful reactive oxygen species (ROS), into water and oxygen [20]. The observed increase is consistent with studies reporting that ascorbic acid enhances catalase gene expression and activity via modulation of the Nrf2 signaling pathway [21]. Similarly, creatine supplementation has been shown to upregulate CAT activity and reduce oxidative injury in animal models, likely by improving mitochondrial bioenergetics and scavenging free radicals [22]. Superoxide dismutase (SOD), which catalyzes the dismutation of superoxide radicals into hydrogen peroxide, was significantly elevated in all treated groups, with the highest activity observed in the ascorbic acid groups (p = 0.000). This supports the hypothesis that both treatments enhance cellular defense against superoxide radicals, the primary ROS generated during oxidative stress [23]. Previous studies have confirmed the capacity of creatine to improve SOD activity, possibly through mitochondrial stabilization and reduced oxidative load [24]. Likewise, ascorbic acid is a potent electron donor that regenerates SOD and prevents its oxidative inactivation [25]. In contrast, glutathione (GSH) levels did not show significant variation among the treatment groups, though slight non-significant elevations were noted in the ascorbic acid and combination groups. GSH is a non-enzymatic thiol antioxidant that maintains redox homeostasis and detoxifies ROS via glutathione peroxidase activity [26]. The lack of statistical significance may reflect either the limited sample size or compensatory antioxidant responses involving enzymatic systems such as SOD and CAT [27]. A similar outcome was reported by [28], where short-term antioxidant supplementation in rabbits resulted in minimal changes in plasma GSH levels despite marked improvements in enzymatic antioxidants. The TBARS assay, which measures malondialdehyde (MDA) as an index of lipid peroxidation, revealed a pronounced decrease in all treated groups. The lowest TBARS level was seen in the ascorbic acid group, followed by the combination group, indicating a strong lipid-protective effect [29]. This reduction can be attributed to the free radical scavenging activity of ascorbic acid, which interrupts lipid peroxidation chain reactions[19]. Creatine’s lipid membrane-stabilizing effects have also been documented in oxidative models of ischemia and exercise, and may account for the observed reduction in TBARS in CrS-treated rabbits [30].

In conclusion, Ascorbic acid and creatine, particularly in combination, enhance antioxidant enzyme activities and reduce lipid peroxidation in male rabbits. These findings support the potential synergistic use of these agents in mitigating oxidative stress-related disorders.

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