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. 2026 Oct 7;65(8):275. doi: 10.1007/s00394-026-04131-3

Betaine supplementation modulates mitochondrial and redox-related adaptations in premenopausal women with overweight: a randomized controlled trial

Emilia Zawieja 1, Monika Młodzik-Czyżewska 1, Agata Chmurzynska 1,✉
PMCID: PMC13645886  PMID: 42841999

Abstract

Background & aims

The aim of this study was to investigate the effects of betaine supplementation on mitochondrial and redox-associated parameters in premenopausal women with overweight or obesity. We hypothesized that betaine enhances selected mitochondrial enzyme content and improves antioxidant defense.

Methods

In this randomized controlled trial, premenopausal women with overweight or obesity were assigned to receive anhydrous betaine supplementation (3 g/d) or a placebo condition for eight weeks. Mitochondrial enzyme content was assessed in peripheral blood mononuclear cells (PBMCs), including citrate synthase (CS) and cytochrome c oxidase (COX). Redox-related parameters were evaluated in plasma; these included the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), as well as oxidized glutathione (GSSG) concentration. Preintervention and postintervention measurements were compared between groups.

Results

A significant treatment × time interaction was observed for CS concentration in PBMCs (p = 0.043). The pre-to-post change differed significantly between the betaine and placebo groups (between-group difference in change: 102.3 μg/mg protein, 95% CI 3.19 to 201.4), although the within-group pre–post changes were not statistically significant. No significant treatment × time interaction was observed for COX (p = 0.326). Plasma CAT activity showed a significant treatment × time interaction (p = 0.003, ηp2 = 0.213) and increased significantly only in the betaine group. No significant effects were observed for SOD (p = 0.825), GPx (p = 0.359), or GSSG (p = 0.693).

Conclusions

Betaine supplementation may selectively influence PBMC mitochondrial-associated parameters and circulating antioxidant enzyme activities in premenopausal women with overweight or obesity. The differential change in CS between the betaine and placebo groups and the increase in plasma CAT activity warrant further investigation, particularly to determine their physiological significance and underlying mechanisms.

Trial registration

https://clinicaltrials.gov/study/NCT06344377.

Keywords: Betaine, Mitochondrial function, PBMC, Antioxidant enzymes, Overweight females

Introduction

Mitochondrial oxidative metabolism is central to cellular energy homeostasis, as oxidative phosphorylation provides the majority of adenosine triphosphate. During this process, electron transfer through the respiratory chain inevitably generates reactive oxygen species (ROS), which are subsequently detoxified by antioxidant systems [1]. At physiological levels, mitochondrial ROS act as signaling molecules regulating pathways involved in mitochondrial biogenesis, substrate utilization, and adaptive stress responses. However, when electron transport becomes inefficient or antioxidant capacity is insufficient, excessive ROS production disrupts redox homeostasis, leading to oxidative damage of lipids, proteins, and DNA. This imbalance contributes to impaired mitochondrial enzyme activity, reduced oxidative capacity, and the propagation of inflammatory and metabolic dysfunction. In conditions such as overweight and obesity, where mitochondrial efficiency and substrate flux are often altered, dysregulated mitochondrial oxidation and oxidative stress are thought to play a mechanistic role in the development of insulin resistance and cardiometabolic risk [2, 3].

Peripheral blood mononuclear cells (PBMCs) are a readily accessible population of lymphocytes and monocytes with active mitochondrial metabolism. Assessment of mitochondrial parameters in PBMCs provides a minimally invasive measure of cellular bioenergetics and redox status, making them useful for investigating mitochondrial function in research settings. PBMC mitochondrial respiration and enzyme activities have been shown to vary with physiological and pathological states, including metabolic alterations and inflammatory responses, supporting their potential as biomarkers of metabolic dysfunction and immune dysregulation [4]. Importantly, redox and mitochondrial alterations in PBMCs have been observed specifically in individuals with overweight and obesity. Monserrat-Mesquida et al. [5] reported progressively higher ROS production in PBMCs with increasing body mass index (BMI), together with alterations in antioxidant enzyme activity and a more pro-oxidant and pro-inflammatory circulating profile. Moreover, PBMC mitochondrial and redox parameters appear to be responsive to nutritional and weight-loss interventions. In individuals with obesity, diet-induced weight loss was associated with reduced mitochondrial ROS production, increased glutathione levels, and improvements in mitochondrial respiration and ATP production in PBMCs [6]. Because PBMCs circulate throughout the body and interact with multiple tissues and inflammatory mediators, mitochondrial measures in these cells provide a practical, minimally invasive approach for examining associations between cellular bioenergetic and redox processes [7].

Evidence from human studies regarding the effects of betaine on mitochondrial and redox parameters in circulating immune cells remains limited. In a double-blind crossover study in healthy men, two weeks of betaine supplementation attenuated the increase in lymphocyte apoptosis following exhaustive exercise [8]. However, betaine did not significantly affect lymphocyte mitochondrial transmembrane potential decline or circulating markers of oxidative damage [8]. Thus, although these findings suggest that betaine may influence some cellular stress responses in circulating lymphocytes, evidence for a direct effect on mitochondrial function or oxidative status in humans remains inconclusive. To our knowledge, the effects of betaine supplementation on mitochondrial enzyme markers in human PBMCs have not been directly investigated.

Dietary betaine supplementation may affect mitochondrial function and oxidative stress through multiple interrelated mechanisms [9]. For example, increased cytochrome c oxidase (COX) activity and mitochondrial membrane potential have been reported in mouse hepatocytes exposed to betaine [10]. Although these findings suggest that betaine may modulate mitochondrial bioenergetics in vitro, evidence regarding its effects on COX protein content, particularly in human PBMCs, remains limited. Moreover, betaine increased mitochondrial content in human hepatoblastoma cells HepG2 and healthy mouse hepatocytes AML12 [11]. Experimental studies in Huh7 human hepatocarcinoma cells have also shown that betaine may modulate mitochondrial dynamics by promoting fusion and reducing fission [12]. In cellular models of stress, betaine supplementation in intestinal porcine epithelial cells decreases mitochondrial ROS production and attenuates stress-induced mitochondrial dysfunction, suggesting a direct protective effect on mitochondrial redox balance [13]. These effects are consistent with the broader biological roles of betaine, including its function as a methyl donor in one-carbon metabolism and its involvement in sulfur amino acid metabolism, as well as its reported associations with oxidative and inflammatory processes [14, 15].

Collectively, preclinical findings suggest that betaine may influence mitochondrial and redox-related processes; however, evidence in humans remains limited, particularly in individuals with overweight or obesity. Therefore, the aim of the present study was to investigate the effects of betaine supplementation on plasma redox-related parameters and mitochondrial-associated protein markers in PBMCs.

Materials & methods

Study design

The present study is part of a larger project funded by the Polish National Science Centre (grant number 2020/37/B/NZ9/00256). The study was a randomized, placebo-controlled, double-blind trial in which participants were randomly allocated to two parallel groups—betaine (BET) and placebo (PLA)—and attended three study meetings (T1–T3): one before supplementation (T1), one during (T2), and one after (T3) (Fig. 1).

Fig. 1.

Fig. 1

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Study design

Blood samples were collected fasted in the morning hours during T1 and T3. The supplementation period was eight weeks. The protocol of the primary study was registered prospectively at https://clinicaltrials.gov/study/NCT06344377 (first posted April 2024), though the present study describes the secondary analysis of samples collected during the main study. Data collection took place between September 2024 and April 2025. The study was conducted at the Department of Human Nutrition and Dietetics, Poznań University of Life Sciences, Poland. Simple randomization was performed using studyrandomizer.com by an impartial person and group assignments were unblinded only after the study’s completion. No stratification according to baseline characteristics was applied. The personnel who enrolled and assigned participants to the interventions did not have access to the random allocation sequence. Sealed containers were used to implement the random allocation sequence.

The study received approval from the local ethical committee (Bioethics Committee at Poznań University of Medical Sciences, Poznań, Poland, decision no. 92/24 of 7 March 2024) and written informed consent was obtained from all participants before the study began. The participants and the research team members were blinded to group allocation. All procedures were conducted in accordance with the ethical standards outlined in the Helsinki Declaration.

Patients and the public were not involved in the design, conduct, reporting, or dissemination plans of this trial.

Participants

Participants were recruited via online advertisements in Poznań, Poland. The inclusion criteria were: premenopausal women aged 18–45 years with overweight or obesity (BMI ≥ 25 kg/m2) and a waist circumference > 80 cm. Exclusion criteria were: betaine supplementation within the 3 months preceding enrollment; pregnancy or breastfeeding; intentional restriction of energy intake for the purpose of weight loss; pharmacological treatment for obesity; previous bariatric surgery; and diabetes.

Sample size calculations were performed for a repeated-measures ANOVA (two groups × two repeated measures), assuming an effect size (f) of 0.225. This value was derived from the study by Cholewa et al. [16] (Cohen’s d = 0.91). To account for the absence of an exercise intervention in the current study, the effect size was conservatively reduced by 50% (d = 0.45), corresponding to f = 0.225. With an α level of 0.05 and a statistical power of 90%, the required sample size was estimated at 34 participants.

Of the 53 randomized participants (Fig. 2), 42 attended all three study meetings and complied with the study protocol. Data from 42 participants were thus analyzed; nineteen of these participants were placed in the BET group and the remaining 23 participants were put in the PLA group.

Fig. 2.

Fig. 2

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Consort flow chart

Supplementation

The supplementation period lasted for eight weeks. Anhydrous betaine was supplemented at 3 g/d. Betaine and placebo supplements were provided in identical-looking white capsules. Each capsule contained either 500 mg of betaine or rice flour (placebo). The supplements were supplied by Nutropharma, Lesznowola, Poland. Participants ingested six capsules per day (three in the morning and three in the evening). During the intervention, participants received reminders about the supplementation scheme every two weeks. The capsules were consumed with at least 250 mL of water. Both betaine and placebo capsules were packaged in white bottles, with each bottle containing precisely 168 capsules (the amount necessary to continue supplementation for four weeks: 4 weeks × 7 days × 6 capsules/day = 168 capsules). The participants received one bottle of capsules at T1 (before supplementation began) and the other at T2 (in the middle of the supplementation period), so that the total supplementation period lasted for eight weeks (168 capsules × 2 = 336 capsules). At T2 (mid-supplementation) and T3 (post-supplementation), participants returned the bottles for a compliance examination. Any remaining capsules were counted to assess supplementation compliance. Furthermore, adherence to the supplementation schedule was monitored through patient surveys.

Study meetings

All participants attended three study meetings. At T1, the presupplementation meeting, baseline anthropometric measurements were taken, questionnaires were administered, and biological samples (saliva and blood) were collected. Anthropometric measurements were performed; these included body weight, fat mass percent, waist and hip circumferences, and height. After these procedures, the participants received the first bottle of capsules for the subsequent four weeks with instructions on how to take them. In order to ensure double-blinding, neither the research team members nor the participants were aware of which supplemental group the participants were in (BET or PL).

After four weeks, the participants attended a mid-supplementation meeting (T2), bringing the bottles with any remaining untaken capsules for a compliance assessment. They then received a new bottle with another set of capsules for the next four weeks. The participants received the same supplement (BET or PLA) as at T1; as before, neither the research team members nor the participants knew which were received.

The final post-supplementation meeting (T3) was after an overall period of eight weeks. At T3, the participants brought the remaining capsules for an adherence assessment and blood samples were collected. Body weight and composition was measured at T1, T2 and T3 using air displacement plethysmography (Bod Pod, Cosmed, Italy).

Plasma collection and analysis

Blood samples (T1 and T3) were collected by a qualified person into EDTA-containing tubes (IMPROVACUTER®, Improve, Shanghai, China) in the morning, with the participant in a fasted state. 6 mL of blood was collected from each participant for plasma preparations and 8 mL for PBMC isolation. Plasma was separated by centrifugation at 1500 g for 10 min and stored at − 80 °C until analysis. Plasma was analyzed for the activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as the oxidized glutathione (GSSG). Enzyme activity levels were analyzed using the commercially available kits #CS00009 for SOD (U/mL), #MAK-531 for CAT (U/L) and #MAK-437 for GPx (U/L), following the guidelines (all kits from Sigma Aldrich, Merck, Darmstadt, Germany). The absorbance was measured using a microplate reader and associated software (Infinite Pro 200 with i-control, Tecan, Austria). GSSG (μmol/L) was measured using a commercial kit #E-BC-K097-M (Elabscience, Houston, Texas, USA).

PBMC collection and analysis

PBMCs were isolated immediately after withdrawal. Venous blood was collected from volunteers into EDTA-coated tubes. The 8 mL of whole blood was diluted twofold with PBS and layered onto 3 mL of Lymphoprep phase (density of 1.077 g/mL; STEMCELL Technologies). After centrifuging (at 1200 g for 20 min, RT), PBMCs were collected into new 15 mL polypropylene tubes and washed twice with PBS [17].

The PBMCs were lysed with RIPA buffer (30 min) and proteases inhibitors. After centrifuging (10 min) the supernatant was collected for analysis. Protein content was measured using the BCA method (with Pierce BCA Protein Assay Kits, Thermo Fisher Scientific, USA). CS and COX concentrations were measured using the commercial kits Human CS ELISA Kit and Human COX IV ELISA Kit (Biorbyt, UK), respectively. The absorbance was measured using a microplate reader and associated software (Infinite Pro 200 with i-control, Tecan, Austria). CS and COX concentrations were normalized to total protein content.

Statistical analysis

Pre-specified primary outcomes included CS and COX concentrations in PBMC before and after the intervention in both groups (betaine and placebo). Secondary outcomes included GPx, SOD, CAT activity and GSSG concentration in plasma before and after both trials. Data were analyzed using Statistica v. 13.3 (StatSoft, Poland) and JASP v. 0.96.0. The significance level (α) was set to 0.05. The normality of data distribution was assessed using the Shapiro–Wilk test. Baseline differences between groups (BET vs. PLA) were analyzed using the independent Student’s t-test for normally distributed data and the Mann–Whitney U-test for non-normally distributed data. Data were further analyzed using repeated measures analysis of variance to assess the treatment (BET vs. PLA) × time (pre vs. post) interaction. When a significant treatment × time interaction was observed, pairwise comparisons of estimated marginal means were performed, with Bonferroni adjustment for multiple comparisons. For significant treatment × time interactions, custom contrasts were performed to directly compare the pre-to-post change between the BET and PLA groups, using a difference-in-differences contrast [(BETpost − BETpre) − (PLApost − PLApre)]. Individual, pseudonymized data can be accessed upon request to the corresponding author.

Results

Baseline characteristics

There were no significant baseline differences between the BET and PLA groups (Table 1).

Table 1.

Baseline characteristics of the participants

BET group (n = 19) PLA group (n = 23) p-value
Height (cm) 167.3 ± 5.90 167.0 ± 6.76 0.893
Weight (kg) 86.8 ± 14.5 88.4 ± 13.7 0.691
Waist circumference (cm) 89.1 ± 11.1 89.4 ± 7.72 0.880
Hip circumference (cm) 110.1 ± 7.36 114.6 ± 17.4 0.214
Body fat (%) 45.1 ± 5.70 44.7 ± 6.58 0.839
FFM (kg) 47.1 ± 5.64 48.6 ± 7.38 0.429
BMI (kg/m2) 31.0 ± 4.95 31.6 ± 3.94 0.638
WHR 0.81 ± 0.08 0.79 ± 0.09 0.440
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BET, betaine; BMI, body mass index; FFM, fat-free mass; PLA, placebo; WHR, waist to hip ratio

Antioxidant markers

Differential responses to the intervention were observed across the plasma antioxidant enzymes. There was a significant treatment × time interaction for plasma CAT activity (p = 0.003, ηp2 = 0.213) (Fig. 3A). A significant difference was shown for BETpre (mean ± SD: 43.1 ± 11.1 U/L) versus BETpost (mean ± SD: 53.8 ± 12.7 U/L) (p = 0.013) and no significant differences were shown for PLA group (PLApre mean ± SD: 51.2 ± 10.8 U/L versus PLApost mean ± SD: 47.5  ± 11.8 U/L, p = 1.000). The pre-to-post change differed significantly between the BET and PLA groups (between-group difference in change: 14.40 U/L, 95% CI 5.30 to 23.50; t(38) = 3.20, p = 0.003). There were no significant treatment × time interactions for plasma SOD (p = 0.825, ηp2 = 0.001; mean ± SD: BETpre: 39.3 ± 12.5 U/mL; BETpost: 36.9 ± 8.15 U/mL; PLApre: 43.3 ± 13.0 U/mL; PLApost: 41.9 ± 13.1 U/mL) (Fig. 3B) or GPx (p = 0.359, ηp2 = 0.021; mean ± SD: BETpre: 236 ± 60.7 U/L; BETpost: 229 ± 58.3 U/L; PLApre: 244 ± 60.4 U/L; PLApost: 219 ± 72.3 U/L) activities (Fig. 3C).

Fig. 3.

Fig. 3

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The effects of betaine and placebo supplementation on plasma antioxidant activities. A: plasma catalase activity. B: plasma superoxide dismutase activity. C: plasma glutathione peroxidase activity. Circles represent individual participant values, with connecting lines indicating paired pre- and post-intervention observations. Boxes represent the interquartile range (25th–75th percentiles), the horizontal line indicates the median, and whiskers represent the minimum and maximum values excluding outliers. BET, betaine; CAT, catalase; GPx, glutathione peroxidase; PLA, placebo; SOD, superoxide dismutase; * p < 0.05

There were no significant treatment × time interactions for plasma GSSG concentration (p = 0.693, ηp2 = 0.004; mean ± SD: BETpre: 1.71 ± 0.21 μmol/L; BETpost: 1.73 ± 0.21 μmol/L; PLApre: 1.68 ± 0.30 μmol/L; PLApost: 1.66 ± 0.18 μmol/L) (Fig. 4).

Fig. 4.

Fig. 4

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The effects of betaine and placebo supplementation on oxidized glutathione concentration. Circles represent individual participant values, with connecting lines indicating paired pre- and post-intervention observations. Boxes represent the interquartile range (25th–75th percentiles), the horizontal line indicates the median, and whiskers represent the minimum and maximum values excluding outliers. BET: betaine; GSSG: oxidized glutathione; PLA: placebo

Mitochondrial enzymes in PBMCs

Changes in mitochondrial enzyme concentrations in the PBMCs exhibited a treatment-specific pattern over time. A significant treatment × time interaction was observed for CS concentration (p = 0.043, ηp2 = 0.098). Mean CS concentration changed from 196 ± 141 to 272 ± 237 μg/mg protein in the BET group (p = 0.255) and from 248 ± 255 to 222 ± 216 μg/mg (p = 1.000) protein in the PLA group. The change from pre- to post-intervention differed significantly between the BET and PLA groups (between-group difference in change: 102.3 μg/mg protein, 95% CI 3.19 to 201.4; t(40) = 2.09, p = 0.043). However, the within-group pre–post comparisons were not statistically significant (Fig. 5A). There were no significant treatment × time interactions for COX concentration in PBMCs (p = 0.326, ηp2 = 0.024; mean ± SD: BETpre: 5.40 ± 5.06 ng/mg protein; BETpost: 5.65 ± 6.06 ng/mg protein; PLApre: 4.23 ± 3.68 ng/mg protein; PLApost: 5.44 ± 4.52 ng/mg protein) (Fig. 5B).

Fig. 5.

Fig. 5

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The effects of betaine and placebo supplementation on PBMC citrate synthase and cytochrome c oxidase concentrations. A: PBMC citrate synthase concentration. B: PBMC cytochrome c oxidase concentration. Circles represent individual participant values, with connecting lines indicating paired pre- and post-intervention observations. Boxes represent the interquartile range (25th–75th percentiles), the horizontal line indicates the median, and whiskers represent the minimum and maximum values excluding outliers. BET, betaine; COX, cytochrome c oxidase; CS, citrate synthase; PLA, placebo; PBMC, peripheral blood mononuclear cells; * p < 0.05

Discussion

In the present study, we examined the effects of betaine supplementation on mitochondrial-associated and redox-related parameters in premenopausal women with overweight and obesity. A significant treatment × time interaction was observed for CS concentration in PBMCs, with the pre-to-post change differing significantly between the BET and PLA groups, although the within-group changes did not reach statistical significance. No significant treatment × time effect was observed for COX content (Fig. 6). With regard to redox-related outcomes, betaine supplementation increased plasma CAT activity, whereas no significant effects were observed for SOD, GPx, or GSSG. Collectively, these findings suggest that betaine may selectively influence PBMC mitochondrial-associated parameters and circulating antioxidant enzyme activities. However, the physiological significance and underlying mechanisms of these responses remain uncertain.

Fig. 6.

Fig. 6

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Schematic summary of mitochondrial-associated and redox-related outcomes following eight weeks of betaine supplementation (3 g/d) in premenopausal women with overweight or obesity. ↑ significant increase after betaine supplementation; * significant time x treatment effect; ↔ no significant changes. CAT, catalase; CS, citrate synthase; COX, cytochrome c oxidase; GPx, glutathione peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species; SOD, superoxide dismutase

CS is a mitochondrial matrix enzyme commonly used as an indirect marker of mitochondrial content, most often when assessed by enzymatic activity [18]. It catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate, representing the first step of the tricarboxylic acid cycle. In the present study however, CS protein concentration was measured and the significant treatment × time interaction and between-group difference in the change in CS concentration was reported, which suggests that betaine may influence this mitochondrial-associated marker. However, because the within-group changes were not statistically significant and mitochondrial density or biogenesis was not directly assessed, the present findings cannot establish an increase in mitochondrial content or mitochondrial biogenesis.

Our findings may be interpreted in the context of emerging evidence indicating that betaine modulates mitochondrial dynamics. Previous experimental studies have reported effects of betaine on mitochondrial respiration and COX enzymatic activity, particularly in hepatocyte-derived cellular models [10]. These findings are not directly comparable with the present results because we assessed COX protein content rather than enzymatic activity or mitochondrial respiration, and our measurements were performed in human PBMCs. The absence of a significant effect on COX content therefore does not exclude possible effects of betaine on COX activity. Differences in cell type, experimental model, intervention duration, and outcome assessment may further contribute to the differences between studies.

Mitochondrial respiration is a major source of ROS. Superoxide (O2·⁻), generated primarily at complexes I and III of the mitochondrial electron transport chain, is converted to hydrogen peroxide (H2O2) by SOD. H2O2 can then be reduced to water (H2O) by either the glutathione system or by catalase. In the glutathione system, reduced glutathione (GSH) is oxidized to GSSG by GPx while H2O2 is simultaneously detoxified to H2O. GSSG is subsequently reduced back to GSH by glutathione reductase, maintaining redox cycling. Catalase, in turn, decomposes H2O2 directly into water and oxygen [19].

In this context, the observed increase in CAT activity following betaine supplementation may reflect a selective response among the redox-related parameters assessed. However, an increase in CAT activity does not establish that H2O2 concentrations were elevated or that its overall removal was enhanced, as neither H2O2 levels nor ROS production or clearance were directly measured. Given that no changes were detected in SOD, GPx, or GSSG, the response appears to be selective for CAT among the antioxidant parameters assessed. To our knowledge, this is the first study to report an increase in plasma CAT activity following betaine supplementation in humans without concomitant changes in SOD or GPx activity or GSSG concentration.

Previous studies, particularly in experimental and preclinical models, have demonstrated that betaine enhances antioxidant defense by increasing the activity of key enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, as well as by elevating intracellular glutathione levels [20, 21].

In contrast, our findings indicate a more selective antioxidant response, limited to increased catalase activity without changes in the glutathione-dependent system, suggesting that betaine modulates specific pathways in dependence on the physiological context. This is consistent with evidence that betaine does not act as a classical free radical scavenger but instead exerts its antioxidant effects through metabolic regulation, particularly via the methionine–homocysteine cycle, thereby supporting endogenous redox capacity [22]. Accordingly, its effect on antioxidant enzymes such as SOD, CAT, and GPx appears to be indirect and context-dependent, likely reflecting improved cellular redox status and availability of reducing equivalents, rather than the primary induction of enzymatic activity.

This study has several limitations that should be considered. First, PBMCs were analyzed on the basis of total protein content, without accounting for cell number or specific immune cell subpopulations. This may have affected the interpretation of mitochondrial markers, given the heterogeneity of PBMCs. Moreover, PBMCs may not fully reflect the mitochondrial adaptations that occur in metabolically active tissues, such as skeletal muscle or liver, limiting the generalizability of our findings. Second, we assessed protein levels of CS and COX rather than their enzymatic activities, and we were unable to directly measure mitochondrial respiration, which would have provided more robust insight into functional mitochondrial changes. We also did not assess direct markers of oxidative stress, making it difficult to determine whether the increase in catalase activity reflects a response to elevated ROS or a preventive adaptation. Finally, although GSSG was successfully measured, total glutathione (GSH) could not be reliably quantified, precluding a comprehensive evaluation of the glutathione redox system.

Conclusions

Betaine supplementation in premenopausal women with overweight and obesity was associated with selective changes in mitochondrial-associated and antioxidant parameters. Betaine may affect CS concentration in PBMCs relative to placebo, although the absence of significant within-group changes warrants cautious interpretation. No significant effects were observed on COX content. In parallel, betaine supplementation increased plasma CAT activity without affecting SOD, GPx, or GSSG. Overall, these findings suggest that betaine may selectively influence PBMC mitochondrial-associated parameters and circulating antioxidant enzyme activities; however, the physiological significance and underlying mechanisms of these effects require further investigation.

Author contributions

Agata Chmurzynska and Emilia Zawieja designed the research; Emilia Zawieja, Monika Mlodzik-Czyzewska and Agata Chmurzynska conducted the research; Emilia Zawieja analyzed the data, performed the statistical analysis, and visualized the results; Emilia Zawieja drafted the paper and had primary responsibility for the final content. Monika Mlodzik-Czyzewska and Agata Chmurzynska reviewed and edited the manuscript. All authors have read and approved the final manuscript.

Funding

This work was supported by the National Science Centre, Poland under Grant 2020/37/B/NZ9/00256.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and national research committees and with the 1964 Helsinki Declaration and its later amendments, or comparable ethical standards. The study was approved by Bioethics Committee at Poznań University of Medical Sciences, Poznań, Poland (Decision no. 92/24 of 7 March 2024).

Consent to participate

Informed consent was obtained from all individual participants included in the study.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No datasets were generated or analysed during the current study.

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