Effects of betaine supplementation on inflammatory markers: a systematic review and meta-analysis of randomised controlled trials

Abstract Several studies have suggested that betaine is closely related to inflammatory biomarkers that contribute to the development of metabolic diseases, but the effect remains controversial. This meta-analysis aimed to assess the effects of betaine supplementation on inflammatory markers based on randomised controlled trials (RCTs). PubMed, Web of Science and ResearchGate databases were searched up to March 2023. A total of 6 RCTs with 7 intervention trials involving 277 participants were included. Betaine supplementation led to a slight reduction in levels of circulating IL-1β of 0.65 pg/mL (95% CI, −1.23 to −0.06) with high heterogeneity (I2 = 95%). Betaine produced a small but nonsignificant reduction in levels of circulating CRP (0.33 mg/L; 95% CI, −1.79 to 1.14), IL-6 (0.47 pg/mL; 95% CI, −1.13 to 0.18) and TNF-α (0.25 pg/mL; 95% CI, −0.98 to 0.48). The present meta-analysis does not provide sufficient evidence to conclude that betaine supplementation improved the inflammation state.


Introduction
Inflammation involves a comprehensive array of physiological responses to detrimental stimuli (Kirsch-Volders and Fenech 2022).However, excessive or prolonged accumulation of low-grade systemic inflammation may cause or accelerate the pathogenesis of various chronic metabolic diseases.Accumulating evidence has demonstrated that inhibiting inflammation or controlling the intensity of inflammatory responses can reduce the risk of various diseases, including non-alcoholic steatohepatitis (NASH) and obesity (Cox et al. 2015;Schuster et al. 2018).It is worth mentioning that healthy dietary patterns or nutrient interventions are intended to suppress various low-grade inflammation-associated diseases by inhibiting inflammatory responses (Iddir et al. 2020).
Betaine is a trimethyl derivative of the amino acid glycine, which is found in high levels in foods such as spinach, wheat germ/bran, and sugar beets (Zeisel et al. 2003;Craig 2004;Kempson et al. 2013).Betaine has beneficial effects in various chronic metabolic diseases due to its interesting biological anti-inflammatory and antioxidant activities (Chiu et al. 2010;Veskovic et al. 2019).Animal studies have shown that the potential anti-inflammatory mechanism of betaine is related to the improvement of sulfur-containing amino acid metabolism by reducing oxidative stress and the inhibition of proinflammatory mediators by blocking the nuclear factor kappa B (NF-κB) signalling pathway and weakening the activation of the nucleotide-binding domain leucine-rich-containing family pyrin domaincontaining-3 (NLRP3) inflammasome (Jorgačević et al. 2022).A cross-sectional study suggested that high dietary intake of betaine was associated with lower concentrations of inflammatory markers, including C-reactive protein (CRP), interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α), in healthy individuals (Detopoulou et al. 2008).
Recently, the anti-inflammatory potential of betaine has attracted considerable attention, with randomised controlled trials (RCTs) exploring the correlation between betaine and health.Abdelmalek et al. reported that betaine supplementation did not reduce levels of proinflammatory cytokines IL-6 and TNF-α in NASH patients (Abdelmalek et al. 2009).Another RCT showed that betaine supplementation inhibited the increase in levels of circulating IL-1 beta (IL-1β), IL-6 and TNF-α in professional youth soccer players (Nobari et al. 2021).Given the inconsistency of the results due to small sample sizes and differences in study quality, it is well justified to question whether betaine supplementation had the lowering effects in levels of circulating inflammatory markers.Therefore, the present meta-analysis of RCTs aimed to determine the potential effects of betaine supplementation on inflammatory markers, including circulating CRP, IL-1β, IL-6 and TNF-α, in different populations and health conditions.

Literature search and data sources
This meta-analysis was conducted and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines (Page et al. 2021) (Supplementary Table 1).The protocol was registered in the International Prospective Register of Systematic Reviews (https:// www.crd.york.ac.uk/PROSPERO/;CRD42023443055).A systematic literature search for publications in English was performed in the PubMed, Web of Science and ResearchGate databases through March 2023 using the following combination of search terms: (betaine OR betaine supplementation) AND ("inflammation" OR "system inflammation" OR "inflammatory biomarker", OR "C-reactive protein" OR "highsensitivity C-reactive protein" OR CRP OR hs-CRP, OR"interleukin-1beta" OR IL-1β, OR "interleukin-6" OR IL-6, OR "tumor necrosis factor-alpha" OR TNF-α) AND ("intervention" OR "trial" OR "randomized" OR "cross-over").Studies were not excluded based on language or publication date.Unpublished studies were not taken into consideration.We performed a manual search using the reference lists of recent reviews and original articles.We did not contact the first authors or corresponding authors for additional information on the primary studies.

Study selection
The population, intervention, comparison, outcome, and study design (PICOS) criteria for the inclusion and exclusion of 6 RCTs with 7 intervention arms are presented in Table 1.Studies were selected for inclusion in the present meta-analysis according to the following criteria: (1) RCT design exploring betaine supplementation in participants, including adults and teenagers; (2) inclusion of a placebo-controlled or comparison group; (3) intervention duration of 3 weeks or longer; and (4) net effects of betaine supplementation in levels of circulating CRP, IL-1β, IL-6 or TNF-α and their corresponding standard deviations (SDs) were reported or data was available to calculate these values.If more than one dose of betaine was administered, all doses were included as intervention arms.

Data extraction
Two authors (J.X. and Z.N.) extracted the following information from each included study: the first author's name, publication year, country, study design, sample size, intervention duration, the type and daily dose of betaine, participant characteristics including gender, mean age, and health status, and baseline and net changes in levels of circulating inflammatory markers.To assess the consistency of results, levels of each inflammatory factor were converted to the same unit.If overlapping publications from the same study were identified, the article with more detailed information and longer intervention time was included and recorded.

Quality assessment
We assessed the risk of bias (RoB) to determine the quality of eligible studies using the Cochrane Collaboration's tool (2011) through RevMan software (version 5.4) (Higgins et al. 2011).The overall quality of each RCT was determined to be good (low risk in > 3 domains), fair (low risk in 3 domains) or poor (low risk in < 3 domains), according to the following domains of RoB criteria: selection bias (random sequence generation; allocation concealment), performance bias (blinding of the participants and trial administrators), detection bias (blinding of outcome assessment), attrition bias (incomplete outcome data), and reporting bias (selective outcome reporting).We also performed a modified Jadad scale to assess the methodological quality of each trial according to the descriptions of randomisation, allocation concealment, blinding, withdrawals and dropouts (Clark et al. 1999).When the score was 4 points or higher, the RCT study was considered high-quality, otherwise, it was considered low-quality.

Statistical analysis
The pooled effect size was demonstrated as weighted mean difference (MD) and 95% confidence interval (CI).If the trial was a parallel design, the net changes in each variable in the intervention and control groups were reported as differences between the baseline and final levels of each inflammatory factor.For crossover trials, the net changes were calculated as differences in the post-treatment values of each group (Higgins et al. 2019).If the studies did not report SDs, we calculated SDs according to corresponding standard errors, CIs, or medians and interquartile ranges through a standard formula (Wan et al. 2014;Luo et al. 2018;Cumpston et al. 2019).If a RCT only reported the baseline and endpoint of inflammatory factors, SDs were calculated following the method from Follmann's report (Follmann et al. 1992).
The heterogeneity of the effect size among studies was tested using Cochran's Q test at a significance threshold of p < 0.10.I 2 values, a quantitative measure of heterogeneity, were calculated across studies (Higgins and Thompson 2002).An I 2 value < 50% was deemed to indicate low heterogeneity, whereas an I 2 value > 50% was considered to indicate substantial heterogeneity across trials.A random-effects model was used to calculate the overall effect size in the presence of significant heterogeneity or p < 0.10 on the Q test; otherwise, a fixed-effects model was used (DerSimonian and Laird 1986).We performed a sensitivity analysis to investigate the influence of each individual study on the overall risk estimate by omitting one study in turn.Potential publication bias was evaluated using Begg's funnel plots and Egger's regression test (Egger et al. 1997) by STATA software (version 17.0 StataCorp, College Station, TX, USA).RevMan software (version 5.4) was used for the meta-analysis.p < 0.05 was considered statistically significant, except where otherwise specified.

Literature research
We initially identified 695 publications through the literature search, and 673 were excluded after screening abstracts or titles.After performing a full-text review of the 22 eligible studies, 6 studies with 7 intervention arms were finally included according to the inclusion criteria.The effects of betaine supplementation on inflammation were evaluated in terms of levels of circulating CRP (n = 3), IL-1β (n = 3), IL-6 (n = 4) and TNF-α (n = 4).The flow chart of the search process is presented in Figure 1.

Study characteristics
The characteristics of the selected studies are listed in Table 2 and Supplementary Table 2. Of the 6 RCTs published from 2009 to 2022, two studies were conducted in the USA, and the rest were from Finland, the Czech Republic, Iran, and Poland, respectively.Sample sizes varied from 27 to 63, with a total of 277 participants.Of the 6 RCTs with 7 intervention trials, five trials had a parallel design, and 2 trials had a crossover design; three trials followed a triple-blind design, and the remaining trials had a double-blind design (n = 3) or provided no information on blinding (n = 1).Betaine supplementation differed in these studies.Five studies used betaine as intervention arms (Abdelmalek et al. 2009;Schwab et al. 2011;Grizales et al. 2018;Nobari et al. 2021;Zawieja et al. 2022), and one study received betaine combined with 42 g of white wine (Rajdl et al. 2016).The intervention duration ranged from 3 to 48 weeks, and the effective dose of betaine ranged from 2.0 g to 20.0 g per day.
In terms of participants, three studies enrolled both males and females (Abdelmalek et al. 2009;Schwab et al. 2011;Grizales et al. 2018), and the remaining three studies enrolled only males (Rajdl et al. 2016;Nobari et al. 2021;Zawieja et al. 2022).Four RCTs were conducted in healthy individuals (Schwab et al. 2011;Rajdl et al. 2016;Nobari et al. 2021;Zawieja et al. 2022), and the remaining RCTs were performed in individuals with obesity and prediabetes (n = 1) (Grizales et al. 2018) or non-alcoholic fatty liver disease (n = 1) (Abdelmalek et al. 2009).Two studies reported no serious side effects in the betaine supplementation group (Schwab et al. 2011;Zawieja et al. 2022), one study indicated that NASH patients had gastrointestinal side effects after betaine supplementation (Abdelmalek et al. 2009), one trial reported that betaine can attenuate the adverse effects of alcohol consumption (Rajdl et al. 2016), and two trials did not mention the side effects (Grizales et al. 2018;Nobari et al. 2021).

Quality assessment of included trials
The RoB assessment results are summarised in Figure 2 and Supplementary Table 3.According to the Cochrane scoring system (The Cochrane Collaboration, 2011), 3 studies were defined as high quality, one study with the risks of selection bias, detection bias and attrition bias was judged to be low quality, and the remaining (n = 2) were of fair quality.In addition, the Jadad scale scores of the six studies ranged from 3 to 6 (Supplementary Table 4).Two studies had a  score of 6, one study had a score of 5, two studies had a score of 4 and one study had a score of 3.

Sensitivity analyses
Considering the high heterogeneity in levels of circulating CRP, IL-1β, IL-6 and TNF-α, sensitivity analyses were conducted by omitting one trial in turn to examine the effect of an individual trial on the overall effects.After we excluded the RCT by Nobari et al. (Nobari et al. 2021), betaine supplementation had a null effect in levels of circulating IL-1β (MD = −0.08 pg/mL; 95% CI, −0.19 to 0.03), with no heterogeneity.In terms of CRP and IL-6 levels, elimination of each study did not have a significant effect on the overall influence, suggesting that the effect size was stable.The effect of betaine supplementation in levels of circulating TNF-α appeared to be heavily influenced by the study by Nobari et al. (Nobari et al. 2021).When this study was removed, the I 2 values decreased to 0.

Discussion
The present meta-analysis reviewed six RCTs to comprehensively evaluate the effects of betaine supplementation on circulating inflammatory markers, including CRP, IL-1β, IL-6 and TNF-α in healthy populations or participants with metabolic diseases.
Our results suggested that betaine supplementation reduced the concentration of circulating IL-1β, but these findings exhibited high heterogeneity.When the trial by Nobari et al. (Nobari et al. 2021) was excluded, this effect disappeared, as did the heterogeneity.The present meta-analysis does not support the effects of betaine supplementation on circulating CRP, IL-6 and TNF-α levels, as there were slight but statistically nonsignificant reductions.The present meta-analysis followed the PRISMA guidelines and included relatively high-quality studies with high Jadad scores.However, caution is merited in the interpretation of our findings due to considerable heterogeneity among trials.Regarding circulating CRP, the heterogeneity can be attributed to the limited number of studies.The study by Rajdl et al. had relatively high risks of performance bias and detection bias owing to the lack of blinding information; in addition, the study used a combined intervention with betaine and 42 g of white wine (Rajdl et al. 2016).Thus, the combined intervention may have influenced the effect of betaine supplementation on circulating CRP levels.CRP, which is a key regulator of proinflammatory mediator synthesis and a strong predictor or even a direct cause of many metabolic diseases, is one of the most commonly used markers of systemic inflammation in clinical studies (Avan et al. 2018;Tang et al. 2022).Despite the insufficient evidence in this meta-analysis, betaine may have the potential effect of reducing circulating CRP levels.In a cross-sectional investigation with a total of 3042 healthy adults from the ATTICA study, higher betaine intake of more than 360 mg/d was associated with lower circulating CRP and TNF-α levels (Detopoulou et al. 2008).The possible mechanism of the effects of betaine may be related to the inhibition of inflammatory signalling pathways, including NF-κB signalling and the NLRP3 inflammasome (Zhao et al. 2018).Considering the inconsistency of these results, a well-designed RCT with a large sample size is needed to determine whether betaine can reduce circulating CRP levels.
Regarding other inflammatory factors, our primary result indicated that betaine supplementation exhibited a slight but significant reduction in circulating IL-1β levels of 0.65 pg/mL.Animal studies have demonstrated that betaine can suppress the production of the inflammatory cytokine IL-1β by targeting the phosphatidylinositol 3 kinase/protein kinase B signalling pathway (Huang et al. 2020).Considering the high heterogeneity among studies, the results should be interpreted with caution, and the sensitivity analysis suggested that the decrease in circulating IL-1β levels disappeared after omitting the trial by Nobari et al. (Nobari et al. 2021).Betaine supplementation did not decrease circulating IL-6 and TNF-α levels, which may be attributed to the variation in study characteristics.Although we could not further explore this effect based on study characteristics due to the limited number of studies, the trial by Nobari et al. may have influenced the TNF-α results (Nobari et al. 2021), because I 2 values decreased to 0 after this trial was omitted in the sensitivity analyses.However, the null effect was not changed.Although the present meta-analysis did not identify the sources of heterogeneity influencing the pooled effect in circulating IL-6 levels, the difference in study characteristics might impact the results regarding the effect of betaine supplementation in circulating IL-6 levels.Additionally, the number of RCTs enrolled in the present meta-analysis was limited and insufficient for identifying the sources of heterogeneity by subgroup analysis.
Betaine is an important nutrient for humans obtained from all kinds of foods, and many animal studies have provided evidence that it has an anti-inflammatory effect in various chronic metabolic diseases.In some animal models of non-alcoholic fatty liver disease, betaine inhibited hepatic inflammation by mediating the glutathione/AMPK/NF-κB signalling pathways (Chen et al. 2022).In an animal model of colitis-associated cancer, betaine may have prevented inflammation-associated colon carcinogenesis by reducing reactive oxygen species generation and inhibiting oxidised glutathione concentration (Kim et al. 2014).There is no doubt that large-sample clinical trials are needed to further confirm the anti-inflammatory effect of betaine in the future.
To the best of our knowledge, this is the first to perform a systemic review and meta-analysis of RCTs to assess the potential of betaine supplementation on circulating inflammatory markers, including CRP, IL-1β, IL-6 and TNF-α.Additionally, most of the participants were healthy without apparent inflammatory diseases at the baseline measurement of inflammatory markers.Finally, our results are unlikely to be due to publication bias, according to both Begg's funnel plots and Egger's regression test.Unfortunately, the current study had several limitations that should be considered.First, the limited number of included studies and inadequate sample sizes with varying individuals made it difficult to explore the potential sources of heterogeneity by performing a subgroup analysis.Although we conducted sensitivity analysis to examine the effect of a single trial on the overall effects, the high heterogeneity in levels of circulating inflammatory markers cannot be reasonably and sufficiently explained.Additionally, considering that the studies were conducted in different countries, genetic variation or a gene-diet interaction may have influenced the magnitude of the effect of betaine supplementation on circulating inflammatory markers.

Conclusion
In summary, our findings do not provide sufficient evidence that betaine supplementation had beneficial effects on improving inflammation state.However, our results should be interpreted with caution because of the presence of high heterogeneity.Therefore, further large-scale studies with a targeted population and a longer intervention duration are needed to determine the effects of betaine supplementation on circulating inflammatory markers.

Figure 1 .
Figure 1.flow diagram of the study selection process.

Figure 2 .
Figure 2. risk of bias assessment of the included studies.(a) risk of bias graph, (b) risk of bias summary.

Table 1 .
PIcoS criteria for inclusion and exclusion of studies.

Table 2 .
overview and characteristic of the trials and participants in this meta-analysis.for parallel design, sample size is betaine group vs. control group; Intervention and baseline is betaine group vs. control group.