Investigation of ED4 supplementation on cardiovascular parameters in rats with elevated systemic TMAO

Investigation of ED4 supplementation on cardiovascular parameters in rats with elevated systemic TMAO | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Investigation of ED4 supplementation on cardiovascular parameters in rats with elevated systemic TMAO Yi-Ting Qiu, Ping-Hsiu Huang, Yu-Wei Chen, Ming-Kuei Shih, Bao-Hong Lee, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6755780/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Nov, 2025 Read the published version in npj Science of Food → Version 1 posted 12 You are reading this latest preprint version Abstract Trimethylamine-N-oxide (TMAO), a gut microbiota-derived metabolite, is strongly linked to metabolic disorders, cardiovascular diseases, and liver injury. This study aimed to evaluate the therapeutic effects of 3-O-butanoylresveratrol (ED4), a resveratrol derivative, in a rat model with elevated systemic TMAO induced via osmotic pump infusion. ED4 supplementation significantly reduced serum triglycerides, cholesterol, low-density lipoprotein cholesterol, and the total cholesterol/high-density lipoprotein cholesterol ratio while improving liver function and reducing hepatic fat accumulation associated with high systemic TMAO levels. Additionally, ED4 restored short-chain fatty acid levels and modulated gut microbiota composition, increasing the abundance of beneficial species such as Romboutsia ilealis and Parabacteroides merdae . Serum metabolomics revealed ED4's role in enhancing energy and amino acid metabolism, contributing to improved metabolic health. These findings demonstrate that ED4 alleviates TMAO-associated risks by improving lipid metabolism, protecting liver function, and reshaping gut microbiota, highlighting its potential as a functional food component for managing cardiovascular disorders. Biological sciences/Biochemistry Biological sciences/Biological techniques Health sciences/Diseases Health sciences/Health care Health sciences/Signs and symptoms TMAO resveratrol derivative ED4 metabolomics gut microbiota lipid metabolism liver function cardiovascular health Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Trimethylamine-N-oxide (TMAO) is a small molecule with both hydrophobic and hydrophilic properties, allowing it to influence protein structure and function as it penetrates cells 1 . TMAO is primarily produced by intestinal microorganisms, which metabolize nutrients present in foods such as liver, red meat, and egg yolk. Key nutrients, such as choline, phosphatidylcholine, and L-carnitine, are metabolized into trimethylamine (TMA), which is subsequently oxidized into TMAO by the liver enzymes flavin-containing monooxygenases 1 and 3 (FMO1 and FMO3) 2 . Extensive research has established a positive correlation between elevated TMAO levels and an increased risk of metabolic disorders, hypertension, and cardiovascular diseases (CVD) in humans 3 . Evidence suggests that TMAO is a critical biomarker for predicting susceptibility to these conditions. For instance, elevated TMAO levels have been linked to a 68% increased risk of thrombotic events, including stroke 4 . Additionally, high TMAO levels are associated with an increased risk of atherosclerosis, heart disease, type 2 diabetes, and obesity, as well as higher overall mortality rates 5 , 6 . Serum TMAO concentrations also increase with age, further underscoring its significance in age-related health risks 6 . These findings highlight the importance of monitoring and managing TMAO levels to mitigate associated health risks. The dynamic balance of intestinal microbiota is influenced by a range of interconnected factors, including genetic predisposition, dietary habits, and psychological state 1 . Numerous studies have demonstrated that lifestyle choices, such as exercise, disease occurrence, and dietary patterns, play a central role in influencing intestinal microbiota composition and its metabolites 7 – 9 . For instance, dietary variations have been shown to significantly impact gut microbiota composition and metabolite production. Research indicates that individuals consuming eggs and red meat exhibit higher serum TMAO levels and a greater abundance of Firmicutes relative to Bacteroidetes , compared to those consuming fruits as a control diet. Conversely, low TMAO producers typically have a gut microbiota dominated by Bacteroidetes , particularly Bacteroidaceae and Prevotellaceae 10 . These insights underscore the intricate relationship between diet, gut microbiota, and metabolic health, emphasizing the importance of understanding these interactions to support overall health and well-being. Resveratrol (RSV) has been widely recognized for its association with numerous health benefits 11 . Studies have shown that RSV modulates the gut microbiota and inhibits TMA production in mice 1 . Furthermore, RSV has been highlighted for its potential to reduce TMAO levels and provide anti-CVD effects, primarily through enhancing metabolic activity and regulating gut microbial composition 12 . However, its low bioavailability remains a significant barrier to its therapeutic applications 11 . To address this, Tain et al. 2021 utilized Steglich esterification to synthesize RSV with butyric acid, producing novel resveratrol butyrate esters (RBEs) 13 . In this modified structure, the 4'-OH hydroxyl group plays a critical role in antioxidant activity. Notably, RBEs have been reported to regulate acetyl-coenzyme A carboxylase and SREBP-1 activity, thereby reducing lipid accumulation in HepG2 cells 14 . Our prior research further demonstrated that RBE effectively regulates oxidative stress and modulates gut microbiota, suggesting a potential role in pathways associated with the development of hypertension 15 . In addition, several in vitro studies by our team have shown that RBE, including its derivatives 3,4'-di-O-butanoylresveratrol (ED2) and 3-O-butanoylresveratrol (ED4), can effectively inhibit TMAO synthesis by modulating TMA metabolism. These findings have been consistently observed in microbial co-incubation models using C. asparagiforme and B. longum 16 . Moreover, ED2 and ED4 have demonstrated efficacy in reducing fatty acid-induced lipid accumulation and mitigating hypertension associated with chronic kidney disease 16 . Among these, ED4 exhibits superior biological activity, particularly in regulating oxidative stress and exerting anti-obesity effects, likely mediated through its influence on gut microbiota and short-chain fatty acid (SCFA) concentrations, when compared to ED2. We speculate that the monoester structure of ED4 contributes to its enhanced bioactivity relative to the diester structure of ED2. Collectively, these findings lead us to hypothesize that ED4 has the potential to improve serum lipoprotein composition and regulate intestinal bacterial proliferation, offering promising therapeutic potential. Moreover, ED4 has demonstrated beneficial effects in reducing TMAO levels and CVD risk factors by modulating the gut microbiota, restoring SCFA levels, and enhancing carnitine metabolism in high-carnitine-fed rats 17 . While numerous studies have highlighted the association between TMAO, metabolic dysregulation, and cardiovascular disease (CVD), most existing animal models rely on dietary carnitine or choline to induce TMAO accumulation. However, the metabolism of these precursors is highly influenced by gut microbiota composition, host metabolic status, and dietary variability, often resulting in inconsistent and poorly controlled TMAO levels. To address this limitation, the present study aimed to establish a metabolically independent and stable TMAO exposure model by delivering TMAO directly via intraperitoneally implanted osmotic pumps. This approach ensures consistent systemic TMAO levels, allowing for a more precise evaluation of its pathophysiological effects. Using this model, we further investigated whether oral supplementation with ED4, a bioactive resveratrol monoester previously shown to modulate oxidative stress, lipid metabolism, and gut microbiota could mitigate TMAO-induced metabolic and cardiovascular disturbances. This study thus provides a novel platform for elucidating TMAO-related mechanisms and exploring the preventive potential of ED4 in CVD. RESULTS AND DISCUSSION An osmotic pressure pump precisely delivers drugs, fluids, or nutrients, ensuring stable administration with minimal intervention 18 . It can be implanted surgically, subcutaneously, intraperitoneally, or intramuscularly and effectively elevates serum TMAO levels 19 . In this experiment, miniature osmotic pumps were implanted into rat peritoneal cavities, delivering TMAO (37 mg/day, 3.86 nM/s) or saline (control) for three weeks. After a one-week induction, rats maintained high TMAO levels and underwent a two-week intervention with RBE monoester derivative (ED4, 20 mg/kg BW/day). Physiological, biochemical, metabolic, and intestinal microbiota indicators were analyzed to assess TMAO exposure effects and ED4's therapeutic potential. Physiological Status This study observed no significant differences in diet or water intake, as well as liver and kidney weights, among the groups (Table 1 ), and no mortality occurred during the four-week trial. Minimally invasive osmotic pump implantation ensured continuous TMAO or saline delivery, while ED4 was administered via gavage without causing physiological distress or mortality. Table 1 Effects of continuous ED4 supplementation on physiological status in rats with elevated systemic TMAO. SPCN TPCN SPCNM TPCNM Food intake (g) 38.88 ± 2.11 a 39.53 ± 1.75 a 38.45 ± 1.61 a 39.55 ± 1.69 a Water intake (mL) 41.74 ± 5.90 a 41.65 ± 5.94 a 41.07 ± 5.80 a 41.84 ± 5.09 a Body weight (g) 419.00 ± 16.02 ab 443.70 ± 21.79 a 360.0 ± 29.32 c 389.30 ± 8.19 b Weight gain (%) 29.28 ± 4.13 b 37.33 ± 8.15 a 13.42 ± 2.68 d 21.39 ± 3.46 c Liver weight/100g BW 3.75 ± 0.49 a 3.19 ± 0.48 a 3.49 ± 0.32 a 3.06 ± 0.32 a Kidney weight/100g BW 0.79 ± 0.09 a 0.80 ± 0.06 a 0.89 ± 0.06 a 0.86 ± 0.08 a SBP (mmHg) 113.67 ± 0.47 b 131.27 ± 2.37 a 107.67 ± 1.60 b 108.17 ± 0.72 b DBP (mmHg) 85.57 ± 7.15 b 99.13 ± 1.46 a 81.97 ± 3.19 b 81.17 ± 8.55 b The data represent the mean ± SEM of the growth characteristics of each group (n = 6). Different superscript lowercase letters in the same row indicate significant differences (p < 0.05). Weight gain (%) = ((final body weight – initial body weight) / final body weight) * 100 SPCN: Saline pump. TPCN: TMAO pump. SPCNM: Saline pump, and tube feeding 20 mg/kg/Day ED4. TPCNM: TMAO pump, and tube feeding 20 mg/kg/Day ED4. High-dose TMAO exposure led to slightly higher BW, significantly greater weight gain, and elevated systolic (SBP) and diastolic blood pressure (DBP) in the TPCN group compared to the SPCN group (Table 1 ). These findings are consistent with previous reports indicating that elevated TMAO levels contribute to increased BW 20 and blood pressure 3 . This may be attributed to TMAO's impairment of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production, leading to reduced endothelium-dependent vasodilation 21 , 22 . These observations suggest that continuous TMAO administration via osmotic pump may contribute to early signs of cardiovascular dysregulation. ED4 supplementation significantly reduced both BW and weight gain in the TPCNM group compared to the TPCN group. Similarly, the SPCNM group exhibited significantly lower BW than the SPCN group, suggesting that ED4 may attenuate weight gain, potentially through a reduction in fat accumulation, whereas elevated TMAO levels appear to promote it. Previous studies have indicated that ED4 may contribute to blood pressure regulation 16 , and the present findings further support this, as ED4 treatment significantly lowered both SBP and DBP in the TPCNM group relative to the TPCN group. Notably, SBP and DBP levels in the TPCNM group were not significantly different from those in the SPCN control group, suggesting that ED4 supplementation normalized blood pressure altered by TMAO. These results highlight the potential of ED4 as a therapeutic agent for mitigating TMAO-induced metabolic and cardiovascular disturbances. Serum Biochemistry Parameters Serum biochemical analyses were performed at weeks 10 and 12 to evaluate lipid metabolism (Fig. 1 A). Following one week of TMAO administration (week 10), the TPCN group showed significantly elevated levels of TG, TC, LDL-C, and VLDL-C compared to the SPCN group. These differences remained evident at week 12. In addition, high-density lipoprotein cholesterol (HDL-C) levels were reduced, and the TC/HDL-C ratio was increased in the TPCN group. These findings are consistent with previous evidence suggesting that TMAO impairs bile acid metabolism through the FXR–SHP–Cyp7a1 axis, thereby promoting atherogenesis 23 . Overall, these results indicate that TMAO exerts a detrimental impact on lipid homeostasis, even in the absence of a high-carnitine diet and with TMAO delivered solely via osmotic pump. Remarkably, ED4 treatment significantly reduced TG, TC, LDL-C, VLDL-C, and the TC/HDL-C ratio in the TPCNM group, while increasing HDL-C levels, restoring lipid profiles comparable to those in the SPCN group. These improvements were accompanied by reduced BW, suggesting that ED4 modulates lipid metabolism and helps prevent fat accumulation. The present findings support ED4’s ability to improve lipid profiles and reduce cardiovascular risk. Recent studies have also shown that ED4 inhibits fatty acid-induced lipid accumulation and prevents hypertension associated with chronic kidney disease 24 . Additional research suggests that ED4 may regulate gut microbiota and modulate the c pathway through the enterohepatic cycle 25 . Liver Fat Accumulation and Liver Injury In addition to its strong association with CVD, TMAO has also been implicated in liver diseases. For instance, an excessive abundance of TMAO is potentially harmful to patients with non-alcoholic fatty liver disease (NAFLD) due to its ability to regulate the synthesis and transport of bile acids 26 . Furthermore, previous studies demonstrated that RBE exhibited superior efficacy in inhibiting fat accumulation in the liver and showed stronger antioxidant and hepatoprotective properties compared to RSV in adipocyte experiments 14 . Based on these findings, this study evaluates whether ED4, a purified RBE, can mitigate elevated systemic TMAO-induced damage by assessing hepatic lipid accumulation and liver injury markers, such as AST and ALT levels. Lipid accumulation in hepatocytes, mainly as triglycerides, is a key feature in the development of NAFLD. It often appears as lipid vacuoles, reflecting disrupted lipid metabolism due to imbalanced synthesis, oxidation, or export. As these vacuoles enlarge, they can impair hepatocyte function and contribute to disease progression, including inflammation and fibrosis 27 . In this study, significant hepatic fat vacuoles were observed in the TMAO-induced TPCN group compared to the SPCN control group (Fig. 2 A). These findings are consistent with previous studies showing that TMAO-treated groups typically exhibit vacuolation associated with fatty liver pathology 28 , 29 . In contrast, the SPCNM and TPCNM groups treated with ED4 exhibited fewer fat vacuoles than the TPCN group. Hepatocytes and liver lobules in these groups appeared neatly arranged, with the liver tissue structure under microscopic observation closely resembling that of the SPCN group. A similar pattern was observed in the fat droplet area analysis (Fig. 2 B), where the TPCN group displayed the largest fat droplet area, while the TPCNM group showed values comparable to those of the SPCN group. Previous research by Nian et al. demonstrated significantly elevated AST and ALT levels in TMAO-treated groups 30 . Similarly, our study found that AST and ALT levels in the TPCN group were significantly higher than those in the SPCN group at both week 10 and week 12 (Fig. 1 B). These results confirm that high-dose TMAO administration via pump leads to substantial hepatic lipid accumulation and liver injury. Importantly, ED4 treatment significantly lowered AST and ALT levels by week 12, highlighting its hepatoprotective effects. This reduction indicates improved liver function and attenuation of TMAO-induced hepatic injury 26 . These findings underscore the effectiveness of ED4 in reducing liver fat accumulation and mitigating liver damage under elevated TMAO exposure. Serum and Feces SCFA SCFAs are gut microbiota-derived metabolites essential for metabolic health, influencing gut barrier integrity, glucose metabolism, and lipid regulation 31 – 33 . SCFAs play a key role in lipid metabolism by regulating adipocyte function, influencing lipolysis and adipogenesis, and thereby improving circulating lipid profiles. Higher SCFA levels are also associated with lower TG-to-HDL ratios and better insulin sensitivity, highlighting their metabolic benefits 34 . Based on the data presented in Table 2 , high-concentration TMAO exposure significantly affects SCFA levels in both serum and feces, as evidenced by markedly lower levels of acetic acid, propionic acid, isobutyric acid, butyric acid, and valeric acid in TMAO-induced rats (TPCN group) compared to controls (SPCN group). However, supplementation with ED4 (SPCNM and TPCNM groups) restored or even elevated these SCFA levels, surpassing those in the SPCN group. Acetic acid improves liver function and lipid metabolism by activating AMPKα, which enhances lipid oxidation, inhibits lipogenesis, and reduces hepatic fat accumulation 35 . These findings suggest that ED4 supplementation mitigates the adverse effects of elevated TMAO on SCFA production, supporting its role in maintaining metabolic health. Table 2 Effects of continuous ED4 supplementation on serum and feces short-chain fatty acids in rats with elevated systemic TMAO. SCFA (µM) SPCN TPCN SPCNM TPCNM Serum Acetic acid 979.24 ± 112.71 ab 604.93 ± 66.63 d 1013.15 ± 9.91 a 846.19 ± 28.85 c Propionic acid 137.52 ± 7.45 a 50.28 ± 2.15 c 116.53 ± 6.31 ab 98.61 ± 13.19 b Isobutyric acid 170.01 ± 6.36 a 82.14 ± 3.95 c 110.52 ± 6.97 b 111.14 ± 11.49 b Butyric acid 132.15 ± 13.34 a 67.23 ± 2.82 d 117.25 ± 8.74 b 91.67 ± 6.80 c Valeric acid 164.86 ± 3.11 a 73.38 ± 2.73 d 130.14 ± 7.72 b 118.58 ± 9.55 c Hexanoic acid 147.22 ± 8.03 a 96.23 ± 2.99 c 128.12 ± 1.21 ab 106.91 ± 9.83 b Heptanoic acid 110.77 ± 9.11 a 61.83 ± 4.21 b 98.64 ± 8.26 a 105.22 ± 7.15 a Octanoic acid 132.08 ± 6.49 a 77.68 ± 6.35 c 116.62 ± 2.43 ab 107.26 ± 19.21 b Feces Acetic acid 2529.88 ± 69.29 a 1807.52 ± 27.60 c 2409.48 ± 3.42 a 2141.50 ± 15.59 b Propionic acid 1732.29 ± 30.68 a 1021.96 ± 45.55 b 1657.32 ± 43.04 a 1737.49 ± 27.47 a Isobutyric acid 1315.22 ± 27.85 a 973.96 ± 32.59 b 1132.09 ± 11.47 a 987.96 ± 63.80 b Butyric acid 710.70 ± 20.95 a 451.04 ± 1.11 d 689.45 ± 19.76 b 568.48 ± 6.64 c Valeric acid 1034.38 ± 19.64 a 879.76 ± 8.37 d 1006.53 ± 18.50 a 983.47 ± 15.43 a Hexanoic acid 999.22 ± 45.70 a 727.75 ± 17.87 c 982.79 ± 15.49 a 845.11 ± 21.41 b Heptanoic acid 734.16 ± 39.76 a 369.64 ± 7.49 c 715.86 ± 16.14 a 633.98 ± 7.15 b Octanoic acid 528.76 ± 57.07 a 327.02 ± 8.35 c 558.82 ± 16.93 a 413.40 ± 7.83 b The data represent the mean ± SEM of the growth characteristics of each group (n = 6). Different superscript lowercase letters in the same row indicate significant differences (p < 0.05). Similarly, fecal SCFA levels were also significantly altered. The TPCN group demonstrated decreased levels of acetic acid, propionic acid, isobutyric acid, butyric acid, and other SCFAs, reflecting the disruption of gut microbial metabolism due to elevated systemic TMAO. Following ED4 supplementation, the SPCNM and TPCNM groups showed partial or complete recovery of these SCFA levels. Remarkably, the fecal propionic acid and butyric acid levels in the TPCNM group were significantly higher than those in the TPCN group, highlighting ED4's potential role in restoring gut microbial function and SCFA production. The study also demonstrated that RBE supplementation effectively restored SCFA levels in rats with high-fat diet-induced obesity. These findings are consistent with previous research highlighting the critical roles of SCFAs, particularly butyric acid and propionic acid, in regulating lipid metabolism, reducing inflammation, and maintaining gut health 31 – 33 . The recovery of SCFA levels observed in the ED4-supplemented groups further supports the hypothesis that ED4 may influence gut microbiota composition and function to mitigate the adverse metabolic effects of elevated TMAO levels. Serum and Urine Creatinine TMAO, a low-molecular-weight nitrogenous waste, is efficiently filtered by the kidneys but is linked to chronic kidney disease (CKD) risk due to its association with creatinine and urea levels 36 . Elevated TMAO induces vasoconstriction, triggering acute hypertension and raising blood pressure 3 , 37 . Increased blood creatinine and reduced urinary creatinine clearance indicate impaired kidney function and hypertension 22 , 38 . As shown in Fig. 3 A and 3 B, blood and urinary creatinine levels increased in the TPCN group by week 10, whereas ED4 supplementation (SPCNM and TPCNM groups) led to reductions in these levels by week 12, approaching those observed in the SPCN group. CCr, a key indicator of renal function, remained relatively unchanged in the TPCN group over time. In contrast, the SPCNM and TPCNM groups exhibited improved CCr compared to the TPCN group, with values comparable to the SPCN control group (Fig. 3 C). Resveratrol supplementation has been shown to significantly reduce blood urea nitrogen (BUN) and creatinine levels while improving glomerular filtration rate (GFR). Additionally, resveratrol administration has been reported to notably lower serum creatinine levels 39 , 40 . Furthermore, RBE has demonstrated protective effects against kidney disease in adenine-treated rats 41 . Based on our findings and previous research, we propose that RBE supplementation, including its ED4 derivative, may hold potential as a renal protective intervention. Serum TMAO After three weeks, serum TMAO levels in the TPCN group were significantly higher than in the SPCN group (Table 3 ), confirming the effective function of the osmotic pump and TMAO absorption into circulation. Moreover, ED4 significantly reduced TMAO in the SPCNM and TPCNM groups, with TPCNM levels approaching those of SPCN. RSV has been shown to modulate gut microbiota, reduce TMA production, and inhibit TMAO levels 42 – 44 . Co-culture studies indicate that both RSV and RBE promote probiotics while suppressing TMA-producing microbes, with RBE effectively lowering TMA levels 2 . Table 3 Effects of continuous ED4 supplementation on serum metabolites in rats with elevated systemic TMAO. Metabolites (mM) SPCN TPCN SPCNM TPCNM TCA cycle Citrate 2.20 ± 0.17 a 1.83 ± 0.20 a 2.3 ± 0.14 a 2.35 ± 0.12 a Succinate 0.35 ± 0.08 a 0.31 ± 0.04 a 0.47 ± 0.10 a 0.38 ± 0.09 a Fumarate 0.12 ± 0.02 a 0.13 ± 0.03 a 0.15 ± 0.04 a 0.13 ± 0.02 a 2-Oxoglutarate 0.50 ± 0.05 a 0.34 ± 0.03 a 0.59 ± 0.11 a 0.36 ± 0.05 a Energy metabolism Pyruvate 0.39 ± 0.06 b 0.45 ± 0.08 b 1.36 ± 0.21 a 1.31 ± 0.19 a Creatine 1.35 ± 0.19 ab 0.82 ± 0.11 b 1.87 ± 0.18 a 1.72 ± 0.25 a Lactate 25.44 ± 2.57 b 25.45 ± 2.72 b 42.07 ± 4.07 a 35.78 ± 2.84 ab Glycerol 7.47 ± 1.35 a 5.77 ± 0.38 a 6.21 ± 0.44 a 5.65 ± 0.28 a Gut microbial Ethanol 0.30 ± 0.04 a 0.38 ± 0.04 a 0.38 ± 0.05 a 0.34 ± 0.04 a Methanol 1.12 ± 0.07 b 1.31 ± 0.08 b 1.92 ± 0.12 a 1.78 ± 0.15 a Formate 0.52 ± 0.03 a 0.53 ± 0.05 a 0.62 ± 0.05 a 0.59 ± 0.03 a Ketones 3-Hydroxybutyrate 7.28 ± 1.23 ab 5.53 ± 0.90 b 13.1 ± 2.48 a 10.78 ± 1.15 ab Acetone 0.98 ± 0.10 a 0.83 ± 0.12 a 1.22 ± 0.20 a 1.12 ± 0.18 a Acetoacetate 1.81 ± 0.22 a 1.31 ± 0.28 a 2.20 ± 0.54 a 2.10 ± 0.45 a Amino acids Alanine 5.45 ± 0.32 a 5.51 ± 0.39 a 4.72 ± 0.44 a 5.15 ± 0.30 a Arginine 2.50 ± 0.23 a 2.02 ± 0.23 a 2.34 ± 0.08 a 2.40 ± 0.07 a Glutamate 1.86 ± 0.15 a 1.98 ± 0.09 a 2.16 ± 0.23 a 2.48 ± 0.21 a Glutamine 7.61 ± 0.46 a 7.77 ± 0.28 a 6.71 ± 0.45 a 7.03 ± 0.44 a Glycine 3.72 ± 0.27 ab 3.80 ± 0.22 a 2.79 ± 0.34 ab 2.68 ± 0.22 b Histidine 0.35 ± 0.04 b 0.38 ± 0.05 b 0.47 ± 0.07 ab 0.63 ± 0.08 a Isoleucine 1.49 ± 0.10 ab 1.25 ± 0.06 b 1.54 ± 0.12 ab 1.75 ± 0.05 a Leucine 2.12 ± 0.16 ab 1.79 ± 0.07 b 2.29 ± 0.18 ab 2.60 ± 0.08 a Lysine 4.40 ± 0.25 a 4.75 ± 0.25 a 4.52 ± 0.38 a 4.44 ± 0.18 a Methionine 0.66 ± 0.04 a 0.74 ± 0.05 a 0.60 ± 0.04 a 0.62 ± 0.04 a Serine 3.51 ± 0.24 a 3.40 ± 0.06 a 3.12 ± 0.28 a 3.25 ± 0.19 a Taurine 2.01 ± 0.24 a 2.13 ± 0.18 a 2.31 ± 0.13 a 2.53 ± 0.22 a Threonine 3.79 ± 0.63 a 5.34 ± 0.60 a 3.65 ± 0.51 a 3.72 ± 0.24 a Tyrosine 1.03 ± 0.06 b 1.08 ± 0.13 b 1.47 ± 0.05 a 1.25 ± 0.04 ab Valine 2.70 ± 0.17 ab 2.39 ± 0.07 b 2.77 ± 0.19 ab 3.05 ± 0.09 a TMA-related Betaine 0.32 ± 0.04 a 0.26 ± 0.01 a 0.36 ± 0.03 a 0.31 ± 0.03 a Carnitine 0.33 ± 0.05 b 0.41 ± 0.02 ab 0.36 ± 0.03 ab 0.48 ± 0.04 a Choline 0.20 ± 0.01 b 0.19 ± 0.02 b 0.29 ± 0.03 a 0.27 ± 0.02 ab O-Acetylcarnitine 0.21 ± 0.01 a 0.19 ± 0.02 a 0.17 ± 0.02 a 0.19 ± 0.04 a Trimethylamine 0.03 ± < 0.01 a 0.04 ± < 0.01 a 0.05 ± 0.01 a 0.04 ± < 0.01 a Trimethylamine N-oxide 1.01 ± 0.08 b 1.41 ± 0.07 a 0.65 ± 0.08 c 1.05 ± 0.07 b Sugar Glucose 132.29 ± 8.70 133.26 ± 5.04 a 111.23 ± 6.90 a 152.50 ± 24.16 a Lactose 2.40 ± 0.30 a 2.17 ± 0.21 a 2.54 ± 0.33 a 1.67 ± 0.23 a Others 2-Hydroxyisobutyrate 0.05 ± 0.01 b 0.08 ± 0.01 b 0.16 ± 0.02 a 0.16 ± 0.02 a 3-Hydroxyisobutyrate 0.24 ± 0.02 bc 0.23 ± 0.01 c 0.37 ± 0.02 a 0.30 ± 0.02 b Acetamide 0.34 ± 0.03 a 0.27 ± 0.02 a 0.30 ± 0.04 a 0.27 ± 0.02 a Allantoin 1.88 ± 0.25 a 1.69 ± 0.16 a 1.93 ± 0.20 a 1.71 ± 0.17 a Galactonate 1.14 ± 0.16 a 1.13 ± 0.22 a 0.91 ± 0.14 a 0.93 ± 0.14 a Glycolate 0.29 ± 0.06 b 0.43 ± 0.04 ab 0.51 ± 0.03 a 0.52 ± 0.04 a Urea 94.36 ± 4.92 b 88.05 ± 5.35 b 115.32 ± 5.98 a 121.82 ± 2.50 a sn-Glycero-3-phosphocholine 0.30 ± 0.04 a 0.24 ± 0.06 a 0.26 ± 0.02 a 0.31 ± 0.08 a The data represent the mean ± SEM of the growth characteristics of each group (n = 6). Different superscript lowercase letters in the same row indicate significant differences (p < 0.05). Our previous study demonstrated that ED4 reduces TMAO levels in a diet-induced model, likely through modulation of the gut microbiota or by promoting TMAO excretion. As TMAO is primarily eliminated via urine within 24 hours 45 , ED4 may facilitate its clearance. In the current study, urinary TMAO levels were slightly higher in the TPCNM group compared to the TPCN group (Fig. S2), though the difference was not statistically significant. While further investigation is required to confirm whether ED4 enhances TMAO excretion, these findings suggest that ED4 has the potential to suppress elevated systemic TMAO levels and mitigate its associated adverse health effects. Serum Metabolomics Serum metabolomics analysis using high-resolution NMR and Chenomx identified 45 metabolites in the serum. Among these metabolites, 18 exhibited significant changes ( p < 0.05) in response to TMAO exposure or ED4 treatment (Table 3 ). The metabolite contents were analyzed and visualized using statistical plots generated with MetaboAnalyst 6.0. In the PCA plot comparing all groups (Fig. 4 A), the model demonstrated an F-value of 11.58, an R-squared of 0.63, and a p -value of 0.001, indicating a statistically significant separation between groups. The primary separation was driven by ED4 supplementation, with SPCN and TPCN exhibiting significant overlap, as did SPCNM and TPCNM. These overlapping groups were distinctly separated from others along PC1, which accounted for 28.65% of the variance. The results further indicated that TMAO exposure had a less pronounced effect on the separation compared to ED4 supplementation. The PLS-DA scores plot (Fig. 4 B) further illustrated the separation among the four experimental groups based on the first two components, which together explained 36.5% of the total variance (Component 1: 23.7%, Component 2: 12.8%). Distinct clustering was observed between SPCN and SPCNM, as well as between TPCN and TPCNM, reflecting clear metabolic differences. The primary separation along Component 1 was driven by ED4 supplementation, while Component 2 captured secondary differences likely due to TMAO exposure. VIP scores from the PLS-DA analysis (Fig. 4 C) highlighted key metabolites contributing to group differentiation. 2-Hydroxyisobutyrate, pyruvate, and glycolate were the most significant contributors, with 2-hydroxyisobutyrate showing elevated levels in ED4-supplemented groups. The heatmap further demonstrated relative concentrations of these metabolites across the groups, emphasizing their roles in the observed separation. The effect of TMAO exposure The VIP scores from the PLS-DA analysis identified glycolate and creatine as the most significant contributors to the separation between SPCN and TPCN (Fig. S3A). The heatmap revealed that TPCN exhibited higher relative glycolate levels but lower creatine concentrations compared to SPCN, emphasizing the metabolic impact of TMAO exposure. Additionally, other metabolites, including 2-hydroxyisobutyrate and sn-glycero-3-phosphate, also contributed significantly to the observed differences. Pathway enrichment values (PEV) (Fig. 4 D) revealed significant perturbations in pathways such as valine, leucine, and isoleucine biosynthesis, butanoate metabolism, and glycine, serine, and threonine metabolism, which were among the top enriched pathways with the highest enrichment ratios and lower p -values ( p < 0.1). The effect of ED4 supplementation ED4 supplementation induced significant metabolic alterations in both SPCN vs. SPCNM and TPCN vs. TPCNM comparisons (Fig. S3B and S3C). For SPCN vs. SPCNM, key differentiating metabolites included 2-hydroxyisobutyrate, pyruvate, and glycolic acid, while for TPCN vs. TPCNM, pyruvate, 2-hydroxyisobutyrate, and creatine were the primary contributors. Moreover, pathway enrichment analysis identified 14 and 20 significantly altered pathways ( p < 0.05) in SPCN vs. SPCNM (Fig. 4 E) and TPCN vs. TPCNM (Fig. 4 F), respectively. In SPCN vs. SPCNM, pathways such as glyoxylate and dicarboxylate metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and phenylalanine metabolism were prominently enriched, reflecting systemic metabolic adjustments. Similarly, in TPCN vs. TPCNM, pathways including valine, leucine, and isoleucine biosynthesis, glycine, serine, and threonine metabolism, and butanoate metabolism showed significant enrichment, emphasizing ED4's role in modulating amino acid and energy metabolism. ED4 supplementation consistently induced metabolic changes across various experimental conditions, with most metabolites showing elevated levels. Our results demonstrate that ED4 exerts a strong influence on metabolic pathways, particularly those involved in amino acid biosynthesis and energy metabolism, in both SPCN and TPCN groups. Specifically, significant increases were observed in energy metabolites (pyruvate, creatine, lactate) and amino acids (histidine, isoleucine, leucine, valine) in the ED4-treated groups. These findings align with previous research showing that RSV may modulate gut microbiota and regulate amino acid and lipid metabolism, increasing amino acid levels and potentially reducing atherosclerosis risk through improved metabolic profiles and its anti-inflammatory and antioxidant effects 46 . Overall, these findings highlight the metabolic benefits of RSV and suggest that ED4, as its bioactive derivative, may improve metabolic health and help prevent CVD by modulating gut microbiota and nutrient pathways. In this study, the levels of BCAAs (leucine, isoleucine, and valine) and their related metabolites (2-hydroxyisobutyrate and 3-hydroxyisobutyrate) were significantly elevated in the ED4-treated group. Prior research also indicates that RSV treatment can markedly increase isoleucine levels under high-fat diet conditions. Studies have demonstrated the association of BCAAs with protein synthesis and muscle metabolism, which can enhance physical performance. For instance, leucine, a key BCAA, is known to activate the mTOR pathway, promoting muscle protein synthesis, increasing muscle mass, and raising basal metabolic rate 47 . Furthermore, the observed increase in BCAAs may reflect their involvement in beneficial metabolic pathways. For instance, prior research has shown that BCAAs can participate in the TCA cycle, contributing to energy production 48 , 49 . In this study, metabolites associated with the TCA cycle showed a slight increase in the ED4-treated group, although the change was not statistically significant. Such metabolic activity may facilitate energy expenditure, helping to maintain body weight and improve metabolic health. Additionally, we observed an increase in serum 2-hydroxyisobutyrate levels. Although its metabolism and pathophysiological role in humans are not yet fully understood, studies have explored the intricate interactions between gut microbiota, BCAAs, and their metabolites, such as 2-hydroxyisobutyrate and 3-hydroxyisobutyrate 50 , 51 . Research has also highlighted the potential beneficial effects of 2-hydroxyisobutyrate, including resistance to oxidative stress, lifespan extension, and anti-aging properties 52 . The relationship between BCAAs and metabolic health is complex and may be influenced by multiple factors 53 , 54 . Therefore, although elevated BCAA and their derivative levels have been associated with certain metabolic disorders, the underlying metabolic and disease mechanisms are multifactorial and cannot be explained by a single parameter. In this context, the observed increases in BCAAs, 2-hydroxyisobutyrate, and 3-hydroxyisobutyrate in the ED4-treated group should not be directly interpreted as indicators of metabolic risk. We propose that the resveratrol derivative ED4 may promote metabolic health through multiple mechanisms, with elevated BCAA levels potentially reflecting enhanced metabolic activity and modulation of gut microbiota. Further studies employing multi-omics approaches are needed to clarify these mechanisms and validate our findings. Intestinal Microbiota Previous studies have linked dietary TMAO production to gut microbiota, where dietary precursors are metabolized into TMA and converted to TMAO in the liver. TMAO’s impact on gut flora is a key CVD marker 1 , underscoring the link between diet, microbiota, and cardiovascular health 55 . While this study used an elevated systemic TMAO model, assessing treatment effects on gut microbiota remains crucial. To this end, fresh fecal samples were collected before sacrifice for microbiota analysis. Microbiota Abundance In the top 10 species-level relative abundance results (Fig. 5 A), Romboutsia ilealis and Parabacteroides goldsteinii DSM 19448 = WAL 12034 showed notable differences between treatments. R. ilealis is a key producer of acetate, butyrate, and propionate, and studies have linked it to reduced liver inflammation. P. goldsteinii , on the other hand, predominantly produces propionate, butyrate, and valerate 56 – 58 . In the ED4-supplemented TPCNM group, the relative abundance of both species was significantly higher than in the TPCN group. These findings align with the observed increases in serum and fecal SCFA levels as well as improvements in markers of liver injury. The study also found that ED4 supplementation (in the SPCNM and TPCNM groups) led to a reduction in Clostridium disporicum compared to the SPCN and TPCN groups. Interestingly, C. disporicum has been previously reported to correlate positively with serum TMAO levels under normal conditions 59 . This discrepancy is hypothesized to arise from differences in the TMAO source, as the current study provided TMAO directly rather than through dietary precursors. In the TPCN group, a significant decrease in the relative abundance of Duncaniella freteri and Ligilactobacillus murinus was observed. However, these microbes showed a recovery in the TPCNM group, indicating that ED4 supplementation could counteract the negative effects of elevated TMAO levels on these species. Duncaniella spp., known for its high butyrate production and anti-inflammatory properties, has been associated with protective effects against colitis. Additionally, previous studies suggest that high doses of RBE can protect against hypertension in offspring, with increased butyrate levels positively correlated with Duncaniella spp. abundance 15 . Consistently, this study demonstrated significantly higher butyric acid levels in the serum and feces of the TPCNM group compared to the TPCN group. Similarly, an increase in Ligilactobacillus murinus abundance has been linked to the alleviation of low-grade endotoxemia, reducing hypertension progression by restoring endothelial vasodilation. Treatment with L. murinus has been shown to lower blood pressure and improve vascular endothelial function in spontaneously hypertensive rats (SHR) 60 . The findings of this study confirm that elevated systemic TMAO levels can alter the composition of specific gut microbiota and highlight that ED4 supplementation induces significant and beneficial changes in these microbial populations. Microbiota α-diversity According to the results of α-diversity indices (menhinick, Pielou evenness, and Shannon entropy) showed no significant difference ( p > 0.05) (Fig. S4). Although the results were not statistically significant for treatment effectiveness, as observed in several other studies 61 , 62 , there were still slight differences in values during the short-term experimental period. Microbiota β-diversity Beta-diversity analysis is used to compare the differences in microbial community composition between different samples or groups. The Bray-Curtis matrix assesses dissimilarity based on species abundance data, where smaller values indicate less variation in species diversity between samples. According to the results shown in Fig. 5 B, the TMAO-induced group (TPCN) exhibited lower species variation compared to the control group (SPCN). After ED4 supplementation, the SPCNM and TPCNM groups showed species variations closer to that of the control group. Based on the PLS-DA plot in the provided Fig. 5 C, the analysis highlights clear separations among the microbial community compositions across the experimental groups. The ED4-supplemented SPCNM group shows the most distinct clustering, indicating a broader distribution of microbial diversity compared to the other groups. In contrast, the TMAO-induced TPCN group exhibits a more condensed clustering pattern, reflecting reduced microbial diversity. The PLS-DA plot also demonstrates that ED4 supplementation in the SPCNM and TPCNM groups significantly shifted microbial distributions toward a pattern more similar to the control group (SPCN), particularly along Component 1, which explains 9.77% of the variance. Component 2 further captured additional variation (8.38%), emphasizing the effects of both TMAO exposure and ED4 intervention on gut microbiota composition. The impact of individual bacterial strains As shown in Fig. 5 D, the relative abundance of Faecalicatena contorta , Parabacteroides merdae , and Adlercreutzia equolifaciens was significantly reduced in the TMAO-induced group (TPCN). However, their relative abundance was markedly increased in the ED4-supplemented groups (SPCNM and TPCNM). F. contorta is potentially involved in bile acid metabolism by influencing CYP7A1 ligand concentrations, effectively converting CA/CDCA into DCA/LCA, which further regulates cholesterol levels and contributes to maintaining lipid metabolism balance 63 . This aligns with the serum cholesterol analysis results observed in this study. The gut bacterium P. merdae has been reported to prevent cardiovascular damage by enhancing BCAA catabolism 64 . Additionally, A. equolifaciens has been negatively associated with carotid atherosclerosis, potentially due to its production of beneficial metabolites, such as butyrate and equol 65 . In summary, ED4 supplementation may influence the gut microbial composition in rats exposed to high systemic TMAO levels by reducing the abundance of TMAO-associated species. It is hypothesized that ED4 modulates gut microbiota to regulate cholesterol levels, thereby promoting lipid metabolism balance, reducing hepatic fat accumulation, and mitigating the risk of CVD. However, the relationship between biochemical indicators, metabolites, and disrupted bacterial communities remains highly complex, requiring further research to better understand their interconnections and underlying mechanisms. CONCLUSIONS This study employed an animal model with continuous intraperitoneal TMAO infusion, successfully elevating systemic TMAO levels and inducing cardiovascular-related abnormalities such as increased body weight, blood pressure, dyslipidemia, and hepatic fat accumulation. ED4 supplementation effectively mitigated these effects by improving serum lipid profiles, reducing hepatic fat, and normalizing liver injury markers. It also enhanced SCFA production, lowered serum TMAO levels, and modulated gut microbiota by reducing TMAO-associated bacterial species. In addition, the observed increases in BCAAs, 2-hydroxyisobutyrate, and 3-hydroxyisobutyrate, along with restored microbial diversity, suggest that ED4 promotes metabolic health through multiple pathways. While the interplay among metabolites, microbiota, and disease markers remains complex, these findings support ED4’s potential as a therapeutic agent against TMAO-related disorders and highlight the need for further validation through multi-omics and clinical studies. MATERIALS AND METHODS Materials The rat low-density lipoprotein cholesterol (LDL-C; CSB-E11704r) and very-low-density lipoprotein cholesterol (VLDL-C; CSB-E17088r) enzyme-linked immunosorbent assay (ELISA) kits were purchased from CUSABIO Technology LLC (Houston, TX, USA). The ZymoBIOMICS® DNA miniprep kit was purchased from Zymo Research Co. (Irvine, CA, USA). All chemicals used in this study were purchased from Sigma-Aldrich® (Merck KGaA, Darmstadt, Germany) and were used without further pre-treatment unless otherwise stated. ED4 was prepared according to the previously published methods of our team 16 . Experimental animal and experimental design The experimental animal study was conducted as described by Hsu et al. 2020 24 with minor modifications. A total of 24 male, 8-week-old Sprague-Dawley (SD) rats (average body weight (BW) 250 g ± 20 g) were purchased from the BiOLASCO Taiwan Co., Ltd. (Taipei, Taiwan). The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of National Kaohsiung University of Science and Technology under code 0109-AAAP-012. This study was conducted over a total period of four weeks of animal experimentation (Fig. S1 ). Following a one-week adaptation period, the rats were randomly assigned to four groups based on BW. The groups (n = 6 per group) were designated as follows: SPCN (negative control: saline pump), TPCN (positive control: TMAO pump), SPCNM (saline pump with ED4 treatment), and TPCNM (TMAO pump with ED4 treatment). Osmotic pumps (Alzet® osmotic pumps, Models 2ML4, DURECTTM Corp., Cupertino, CA, USA) containing either saline or TMAO were surgically implanted into the intraperitoneal cavity of the rats in each group for continuous administration from week 9 to week 12. The administered dose of TMAO was 37 mg/day (3.86 nM/s), as defined by Ufanl et al. (2015) 66 . Rats in the ED4-treated groups received oral gavage of ED4 at a dosage of 20 mg/kg BW/day from week 10 to week 12. All rats were housed in stainless steel suspended wire mesh cages (two rats per cage) under specific pathogen-free conditions, maintained at 22°C ± 1°C with a humidity of 50% ± 5% and a 12-hour light/dark cycle. Throughout the experiment, the animals were provided with ad libitum access to AIN-93M standard purified feed and drinking water. Daily food intake and water consumption were monitored consistently throughout the study, while weekly BW measurements were recorded over the four-week experimental period. In week 10 and week 12 (before sacrifice), blood pressure was measured in conscious rats using the indirect tail-cuff method (BP-2000; Visitech Systems, Inc., Apex, NC, USA). The rats were gently restrained and calmed before placing the sensor cuff on their tails for measurement. Blood samples were collected from the tail vein at week 10 (one week after implantation). At the end of week 12, following a 10-hour fasting period, rats were anesthetized intramuscularly with a cocktail of Zoletil (25 mg/kg) and Rompun (23.32 mg/kg xylazine hydrochloride) in a 1:1 ratio. Blood samples were collected via cardiac puncture, and the harvested organs were weighed, preserved in 10% formalin for histological staining, and stored for further analysis. Blood samples were centrifuged at 3000×g for 10 minutes at 4°C, and the supernatant was collected to obtain serum samples, which were subsequently stored at -80°C until analysis. Determination of serum biochemistry parameters Triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alanine transaminase (ALT), and aspartate transaminase (AST) levels were analyzed using an automated clinical chemistry analyzer (NX-500i, Fuji, Masaaki Takei, Japan). Serum levels of LDL and VLDL were measured using ELISA kits, following the manufacturer's standardized protocols. The absorbance of the samples was measured at a wavelength of 450 nm using an ELISA reader (BioTek, Agilent Technologies, Inc., Santa Clara, CA, USA). Liver histopathology staining Tissue sections fixed in 10% neutral formalin were embedded in paraffin wax, sectioned into slices approximately 5 µm thick, and stained with hematoxylin and eosin (H&E). The stained sections were mounted on slides, and high-resolution images were captured using the MoticEasyScan system. The images were further analyzed with the Motic DS Assistant (4K). Determination of SCFAs The SCFAs were analyzed using gas chromatography–mass spectrometry (GC-MS) following the method described by Wang et al. (2020) 67 . Serum Sample Preparation Serum samples were centrifuged, and 150 µL of the supernatant was collected and acidified with 50 µL of sulfuric acid. Subsequently, 10 µL of 2-ethylbutyric acid was added as an internal standard, followed by the addition of 400 µL of diethyl ether. The solution was then shaken for 15 minutes and centrifuged at 9000×g for 10 minutes at 4°C. The resulting supernatant was used for GC-MS analysis. Fecal Sample Preparation A 0.1 g sample of rat feces was homogenized in 1 mL of deionized water. The subsequent steps were performed in the same manner as described in the section “Serum Sample Preparation” for the serum preparation. GC-MS analysis SCFA analysis was conducted using a DB-FFAP capillary column (30 m × 0.25 mm × 0.25 µm). Helium was used as the carrier gas at a flow rate of 1 mL/min. The column temperature program started at 80°C (held for 1 minute), followed by an increase to 240°C at a rate of 10°C/min, where it was maintained for 12 minutes. The injector, transfer line, ion source (70 eV), and quadrupole temperatures were set to 240°C, 230°C, and 150°C, respectively. The mass spectrometer scanned in the range of 35–550 m/z. The mass spectra of each identified compound were compared against the GC-MS database for confirmation. Creatinine and CCr analysis The analysis was conducted following a modified method by Jen et al. (2002) 68 .The mobile phase A was prepared using 0.05 M ammonium dihydrogen phosphate, with the pH adjusted to 7.4 using 28% ammonia and 6 N phosphorous acid. The sample diluent was prepared by adjusting PBS to a pH of 2.24 using NaOH. For urine samples, 2 µL of urine was diluted 200-fold with 398 µL of PBS. For serum samples, 50 µL of serum was diluted 1:1 with 50 µL of PBS. The diluted samples were centrifuged at 12,000 rpm at 4°C for 10 minutes, and 100 µL of the supernatant was transferred into a vial for HPLC analysis. HPLC analysis was performed under the following conditions: the mobile phase consisted of 0.05 M ammonium dihydrogen phosphate buffer at pH 7.4. The detection wavelength was set to 235 nm, with a total runtime of 13 minutes per injection. The flow rate was maintained at 1 mL/min, and the sample injection volume was 20 µL. A gradient program was applied with a solution ratio of mobile phase A to mobile phase B set at 100:0 throughout the 0–13 minute runtime. The formula for calculating Creatinine Clearance Rate (CCr) in urine is as follows: Serum metabolomics analysis Serum samples collected at week 12 were filtered using Amicon Ultra-3,000 molecular weight (MW) cutoff filters. The filtrate was collected, and 65 µL of a 4.67 mM DSS-D6 internal standard solution (Chenomx Inc., Edmonton, Alberta, Canada) was added. BS buffer was used to adjust the total volume to exactly 650 µL, with precise recording of the actual volume. The pH was adjusted to 6.8 by adding 1 N HCl or NaOH dropwise, as needed. Proton NMR spectra were acquired using a JEOL ECZ600R 600 MHz NMR spectrometer (Tokyo, Japan). Metabolites were identified and quantified using Chenomx NMRSuite 9.02 software (Chenomx Inc., Edmonton, Canada) 69 . Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and KEGG pathway enrichment analysis of serum metabolites across treatments were conducted via MetaboAnalyst 6.0. Next-generation sequencing (NGS) analysis and bioinformatics analysis Fecal sample collection, pre-processing, and bioinformatics analysis followed the methods 17 , with some modifications. Fecal sample collection occurred a day before osmotic pump implantation and a day prior to trial conclusion. Freshly collected feces from each group were stabilized in a preservation buffer and stored at -80°C until analysis. Bacterial DNA extraction was performed using the DNA Miniprep Kit and subsequently analyzed by Biotools Co., Ltd. (Taipei, Taiwan). DNA libraries were generated via PCR amplification of the V3–V4 regions of the 16S rRNA gene, sequenced using Illumina Miseq (Illumina Inc., San Diego, CA, USA). Paired-end reads were trimmed for quality and grouped into operational taxonomic units (OTUs) based on ≥ 97% similarity using the GreenGene Database (v13.8). Analysis of OTUs (relative abundance, heatmaps), alpha diversity metrics (Shannon index, visualized as Venn diagrams), and beta diversity metrics (PCoA, phylogenetic trees) were performed using Basespace, CLC Genomics Workbench, and GraphPad Prism 8. Functional analysis, including LEfSe and PICRUSt predictions, was conducted via Galaxy/Hutlab (2024). Statistical significance was defined as p < 0.05. Statistical analysis Data were expressed as mean ± standard error of the mean (SEM) (n = 6). Statistical analysis was performed using SPSS software (v12.0, IBM Co., Armonk, NY, USA). A one-way ANOVA was used to evaluate differences within groups, while Tukey's post-hoc test was applied for comparisons between groups ( p < 0.05 indicating statistical significance). Declarations DATA AVAILABILITY All relevant data are available from the corresponding author on reasonable request. ACKNOWLEDGMENTS The National Science and Technology Council funded this research, the Republic of China (Grant no. 112-2221-E-992 -002-MY3; 112-2622-E-992-007-; 111-2221-E-328-001-MY3 and 110-2320-B-992-001-MY3) AUTHOR CONTRIBUTIONS Y.T.Q.: Experimental performance; P.H.H.: Experimental performance, manuscript preparation; Y.W.C. and M.K.S.: Experimental design and supervision, manuscript preparation; B.H.L.: Microbiota analysis, experimental supervision; P.P.S.: Experimental design and supervision; C.Y.H.: Experimental design of the whole study, supervision, manuscript preparation; S.Y.C.: Metabolomics analysis, supervision, manuscript preparation. COMPETING INTERESTS The authors declare that there are no conflicts of interest. References Zhen, J. et al. The gut microbial metabolite trimethylamine N-oxide and cardiovascular diseases. Front Endocrinol (Lausanne) 14, 1085041 (2023). Huang, P. H. et al. 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Gut Parabacteroides merdae protects against cardiovascular damage by enhancing branched-chain amino acid catabolism. Nat Metab 4, 1271–1286 (2022). Wang, Z. et al. Gut microbiota, plasma metabolomic profiles, and carotid artery atherosclerosis in HIV infection. Arterioscler Thromb Vasc Biol 42, 1081–1093 (2022). Ufnal, M., Zadlo, A. & Ostaszewski, R. TMAO: A small molecule of great expectations. Nutrition 31, 1317–1323 (2015). Wang, C. Y. et al. Fast quantification of short-chain fatty acids in rat plasma by gas chromatography. J Food Sci 85, 1932–1938 (2020). Jen, J. F., Hsiao, S. L. & Liu, K. H. Simultaneous determination of uric acid and creatinine in urine by an eco-friendly solvent-free high performance liquid chromatographic method. Talanta 58, 711 (2002). Chen, S. Y. et al. Consumption of vitamin D2 enhanced mushrooms is associated with improved bone health. Journal of Nutritional Biochemistry 26, 696–703 (2015). Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial20250520.docx Cite Share Download PDF Status: Published Journal Publication published 21 Nov, 2025 Read the published version in npj Science of Food → Version 1 posted Editorial decision: Revision requested 09 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 29 Jun, 2025 Reviews received at journal 26 Jun, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviewers agreed at journal 23 Jun, 2025 Reviewers agreed at journal 23 Jun, 2025 Reviewers invited by journal 23 Jun, 2025 Editor assigned by journal 23 Jun, 2025 Submission checks completed at journal 02 Jun, 2025 First submitted to journal 27 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6755780","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":475911070,"identity":"9524d28c-9006-468d-bd29-b459307224d2","order_by":0,"name":"Yi-Ting Qiu","email":"","orcid":"","institution":"National Kaohsiung University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Yi-Ting","middleName":"","lastName":"Qiu","suffix":""},{"id":475911071,"identity":"e7f79788-f574-4a8b-8d13-1541ae5b5912","order_by":1,"name":"Ping-Hsiu Huang","email":"","orcid":"","institution":"Jiangsu Food and Pharmaceutical Science College","correspondingAuthor":false,"prefix":"","firstName":"Ping-Hsiu","middleName":"","lastName":"Huang","suffix":""},{"id":475911072,"identity":"1a2954ae-9b8b-46a0-91c9-b5876518c05e","order_by":2,"name":"Yu-Wei Chen","email":"","orcid":"","institution":"National Chung Hsing University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Wei","middleName":"","lastName":"Chen","suffix":""},{"id":475911073,"identity":"ecec2f4c-44cc-4dca-9f1b-a0016439dd49","order_by":3,"name":"Ming-Kuei Shih","email":"","orcid":"","institution":"National Kaohsiung University of Hospitality and Tourism","correspondingAuthor":false,"prefix":"","firstName":"Ming-Kuei","middleName":"","lastName":"Shih","suffix":""},{"id":475911074,"identity":"132c3697-1230-4db7-8de4-16f64e2a3af6","order_by":4,"name":"Bao-Hong Lee","email":"","orcid":"","institution":"National Chiayi University","correspondingAuthor":false,"prefix":"","firstName":"Bao-Hong","middleName":"","lastName":"Lee","suffix":""},{"id":475911076,"identity":"da31324c-3e16-4e38-8f3c-413eea54cba8","order_by":5,"name":"Pei-Pei Sun","email":"","orcid":"","institution":"National Kaohsiung University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Pei-Pei","middleName":"","lastName":"Sun","suffix":""},{"id":475911080,"identity":"36f1cc2e-f289-431f-a181-285fb452c2f9","order_by":6,"name":"Chih-Yao Hou","email":"","orcid":"","institution":"National Kaohsiung University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Chih-Yao","middleName":"","lastName":"Hou","suffix":""},{"id":475911082,"identity":"54b02e1d-46cd-4ace-b140-13b9e812b3fd","order_by":7,"name":"Shin-Yu Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYHACNiC2YWBgZm6AC0kQoSUNqIURooUHosWAkJbDQEysFvkZyc8efNxxPpq/nbGBuXDHYXt7BuaDt3kY/iQ24NBicCPN3HDmmdu5Mw4Dtcw8czixh4Et2ZqHwQC3FokcNmnettu5DSAtvG2HE3gYeMykgVpycWmRnwHU8rftXO58qBZ7Hgb+b3i1MNwAamFsO5C7AaqFsYeBhw2vFoMzz8wke9uSczcCtRzmPZOe2HOYzdhyjoFxPU6HtSc/k/jZZpc77/zhg495d1jbs7c3P7zxpkLOGJe7GAQSEOwD4KhhBtuOUwMDA/8BJA4jLseMglEwCkbBiAYACMdSt2tyTrEAAAAASUVORK5CYII=","orcid":"","institution":"National Pingtung University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Shin-Yu","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-05-27 05:53:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6755780/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6755780/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41538-025-00552-2","type":"published","date":"2025-11-21T15:56:58+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85419842,"identity":"b2aaef87-6d69-4365-b679-3a60f2eff7d7","added_by":"auto","created_at":"2025-06-25 15:25:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":465724,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of continuous ED4 supplementation on serum lipoprotein compositions (A) and liver injure indicators (B) in rats exposed to high levels of systemic TMAO. The data represent the mean ± standard error of the serum biochemical indicators of each group. Different letters (A-B) indicate significant differences (p \u0026lt; 0.05) between groups given in week 10; Different letters (a-c) indicate significant differences (p \u0026lt; 0.05) between groups given in week 12.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755780/v1/502fc14077a0e6226032b460.jpg"},{"id":85419006,"identity":"c81f6596-a72d-493c-aa77-510d23e94280","added_by":"auto","created_at":"2025-06-25 15:17:51","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1951954,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of continuous ED4 supplementation on liver tissue morphology (A) and liver fat vacuoles (B) in rats exposed to high levels of systemic TMAO. The data represent the mean ± standard error of the serum biochemical indicators of each group. *indicate significant differences (p \u0026lt; 0.05) between the groups.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755780/v1/50a6f6b3b77da7943b982c7b.jpg"},{"id":85419008,"identity":"bfc47bc9-42b4-4276-8937-e7ba522c3339","added_by":"auto","created_at":"2025-06-25 15:17:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":297627,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of continuous ED4 supplementation on serum creatinine (A), urine creatinine (B) and creatinine clearance rat (C) in rats exposed to high levels of systemic TMAO. The data represent the mean ± standard error of the serum biochemical indicators of each group. Different letters (A-B) indicate significant differences (p \u0026lt; 0.05) between groups given in week 10; Different letters (a-c) indicate significant differences (p \u0026lt; 0.05) between groups given in week 12.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755780/v1/4235ac4df82e4720cbf32a16.jpg"},{"id":85419010,"identity":"736ecf36-2a8e-4abb-9f7d-5415cd7afdd7","added_by":"auto","created_at":"2025-06-25 15:17:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":747723,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of continuous ED4 supplementation on serum metabolomics in rats exposed to high levels of systemic TMAO. PCA plot of all groups (A), PLS-DA plot for all groups (B), VIP scores from PLD-DA (C), and pathway enrichment values between SPCN and TPCN (D), SPCN and SPCNM (E), and TPCN and TPCNM (F). Pathway enrichment values (PEV) are calculated based on the proportion of significantly regulated metabolites within a pathway relative to all detected metabolites in that pathway. The formula for PEV is as follows: PEV = (Number of significant metabolites in the pathway / Total number of detected metabolites in the pathway) ÷ (Total number of significant metabolites / Total number of detected metabolites).\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755780/v1/e695c48bb13db142c74c4566.jpg"},{"id":85420247,"identity":"393e135e-6001-4b8b-b87e-735c93f527ab","added_by":"auto","created_at":"2025-06-25 15:33:51","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":596680,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of continuous ED4 supplementation on gut microbiota in rats exposed to high levels of systemic TMAO. Top 10 species-level relative abundances (A), β-diversity analysis based on Bray-Curtis distance (B), PLS-DA of microbial community composition (C), and relative abundance of key individual microbial (D).\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6755780/v1/e15d44c2c1947374eb708445.jpg"},{"id":96650071,"identity":"b4a9b33d-4608-4319-bbd2-b069faf56dde","added_by":"auto","created_at":"2025-11-24 16:06:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5301031,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6755780/v1/82f31a69-6035-4e4f-9f03-54d777d6723b.pdf"},{"id":85419843,"identity":"794888ab-311f-4d9e-9f4e-7fd6c2db8354","added_by":"auto","created_at":"2025-06-25 15:25:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":841788,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial20250520.docx","url":"https://assets-eu.researchsquare.com/files/rs-6755780/v1/9f1b9feea43719b9d2301645.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of ED4 supplementation on cardiovascular parameters in rats with elevated systemic TMAO","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTrimethylamine-N-oxide (TMAO) is a small molecule with both hydrophobic and hydrophilic properties, allowing it to influence protein structure and function as it penetrates cells\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. TMAO is primarily produced by intestinal microorganisms, which metabolize nutrients present in foods such as liver, red meat, and egg yolk. Key nutrients, such as choline, phosphatidylcholine, and L-carnitine, are metabolized into trimethylamine (TMA), which is subsequently oxidized into TMAO by the liver enzymes flavin-containing monooxygenases 1 and 3 (FMO1 and FMO3)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Extensive research has established a positive correlation between elevated TMAO levels and an increased risk of metabolic disorders, hypertension, and cardiovascular diseases (CVD) in humans\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Evidence suggests that TMAO is a critical biomarker for predicting susceptibility to these conditions. For instance, elevated TMAO levels have been linked to a 68% increased risk of thrombotic events, including stroke\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Additionally, high TMAO levels are associated with an increased risk of atherosclerosis, heart disease, type 2 diabetes, and obesity, as well as higher overall mortality rates\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Serum TMAO concentrations also increase with age, further underscoring its significance in age-related health risks\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. These findings highlight the importance of monitoring and managing TMAO levels to mitigate associated health risks.\u003c/p\u003e \u003cp\u003eThe dynamic balance of intestinal microbiota is influenced by a range of interconnected factors, including genetic predisposition, dietary habits, and psychological state\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Numerous studies have demonstrated that lifestyle choices, such as exercise, disease occurrence, and dietary patterns, play a central role in influencing intestinal microbiota composition and its metabolites\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. For instance, dietary variations have been shown to significantly impact gut microbiota composition and metabolite production. Research indicates that individuals consuming eggs and red meat exhibit higher serum TMAO levels and a greater abundance of \u003cem\u003eFirmicutes\u003c/em\u003e relative to \u003cem\u003eBacteroidetes\u003c/em\u003e, compared to those consuming fruits as a control diet. Conversely, low TMAO producers typically have a gut microbiota dominated by \u003cem\u003eBacteroidetes\u003c/em\u003e, particularly \u003cem\u003eBacteroidaceae\u003c/em\u003e and \u003cem\u003ePrevotellaceae\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. These insights underscore the intricate relationship between diet, gut microbiota, and metabolic health, emphasizing the importance of understanding these interactions to support overall health and well-being.\u003c/p\u003e \u003cp\u003eResveratrol (RSV) has been widely recognized for its association with numerous health benefits\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Studies have shown that RSV modulates the gut microbiota and inhibits TMA production in mice\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Furthermore, RSV has been highlighted for its potential to reduce TMAO levels and provide anti-CVD effects, primarily through enhancing metabolic activity and regulating gut microbial composition\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, its low bioavailability remains a significant barrier to its therapeutic applications\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. To address this, Tain et al. 2021 utilized Steglich esterification to synthesize RSV with butyric acid, producing novel resveratrol butyrate esters (RBEs)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. In this modified structure, the 4'-OH hydroxyl group plays a critical role in antioxidant activity. Notably, RBEs have been reported to regulate acetyl-coenzyme A carboxylase and SREBP-1 activity, thereby reducing lipid accumulation in HepG2 cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Our prior research further demonstrated that RBE effectively regulates oxidative stress and modulates gut microbiota, suggesting a potential role in pathways associated with the development of hypertension\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition, several in vitro studies by our team have shown that RBE, including its derivatives 3,4'-di-O-butanoylresveratrol (ED2) and 3-O-butanoylresveratrol (ED4), can effectively inhibit TMAO synthesis by modulating TMA metabolism. These findings have been consistently observed in microbial co-incubation models using \u003cem\u003eC. asparagiforme\u003c/em\u003e and \u003cem\u003eB. longum\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Moreover, ED2 and ED4 have demonstrated efficacy in reducing fatty acid-induced lipid accumulation and mitigating hypertension associated with chronic kidney disease\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Among these, ED4 exhibits superior biological activity, particularly in regulating oxidative stress and exerting anti-obesity effects, likely mediated through its influence on gut microbiota and short-chain fatty acid (SCFA) concentrations, when compared to ED2. We speculate that the monoester structure of ED4 contributes to its enhanced bioactivity relative to the diester structure of ED2. Collectively, these findings lead us to hypothesize that ED4 has the potential to improve serum lipoprotein composition and regulate intestinal bacterial proliferation, offering promising therapeutic potential. Moreover, ED4 has demonstrated beneficial effects in reducing TMAO levels and CVD risk factors by modulating the gut microbiota, restoring SCFA levels, and enhancing carnitine metabolism in high-carnitine-fed rats\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile numerous studies have highlighted the association between TMAO, metabolic dysregulation, and cardiovascular disease (CVD), most existing animal models rely on dietary carnitine or choline to induce TMAO accumulation. However, the metabolism of these precursors is highly influenced by gut microbiota composition, host metabolic status, and dietary variability, often resulting in inconsistent and poorly controlled TMAO levels. To address this limitation, the present study aimed to establish a metabolically independent and stable TMAO exposure model by delivering TMAO directly via intraperitoneally implanted osmotic pumps. This approach ensures consistent systemic TMAO levels, allowing for a more precise evaluation of its pathophysiological effects. Using this model, we further investigated whether oral supplementation with ED4, a bioactive resveratrol monoester previously shown to modulate oxidative stress, lipid metabolism, and gut microbiota could mitigate TMAO-induced metabolic and cardiovascular disturbances. This study thus provides a novel platform for elucidating TMAO-related mechanisms and exploring the preventive potential of ED4 in CVD.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003eAn osmotic pressure pump precisely delivers drugs, fluids, or nutrients, ensuring stable administration with minimal intervention\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. It can be implanted surgically, subcutaneously, intraperitoneally, or intramuscularly and effectively elevates serum TMAO levels\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In this experiment, miniature osmotic pumps were implanted into rat peritoneal cavities, delivering TMAO (37 mg/day, 3.86 nM/s) or saline (control) for three weeks. After a one-week induction, rats maintained high TMAO levels and underwent a two-week intervention with RBE monoester derivative (ED4, 20 mg/kg BW/day). Physiological, biochemical, metabolic, and intestinal microbiota indicators were analyzed to assess TMAO exposure effects and ED4's therapeutic potential.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePhysiological Status\u003c/h2\u003e \u003cp\u003eThis study observed no significant differences in diet or water intake, as well as liver and kidney weights, among the groups (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and no mortality occurred during the four-week trial. Minimally invasive osmotic pump implantation ensured continuous TMAO or saline delivery, while ED4 was administered via gavage without causing physiological distress or mortality.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of continuous ED4 supplementation on physiological status in rats with elevated systemic TMAO.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSPCN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTPCN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSPCNM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTPCNM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFood intake (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38.88\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.53\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater intake (mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.74\u0026thinsp;\u0026plusmn;\u0026thinsp;5.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.65\u0026thinsp;\u0026plusmn;\u0026thinsp;5.94\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.07\u0026thinsp;\u0026plusmn;\u0026thinsp;5.80\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e41.84\u0026thinsp;\u0026plusmn;\u0026thinsp;5.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBody weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e419.00\u0026thinsp;\u0026plusmn;\u0026thinsp;16.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e443.70\u0026thinsp;\u0026plusmn;\u0026thinsp;21.79\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e360.0\u0026thinsp;\u0026plusmn;\u0026thinsp;29.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e389.30\u0026thinsp;\u0026plusmn;\u0026thinsp;8.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWeight gain (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.28\u0026thinsp;\u0026plusmn;\u0026thinsp;4.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37.33\u0026thinsp;\u0026plusmn;\u0026thinsp;8.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.42\u0026thinsp;\u0026plusmn;\u0026thinsp;2.68\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.39\u0026thinsp;\u0026plusmn;\u0026thinsp;3.46\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver weight/100g BW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKidney weight/100g BW\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSBP (mmHg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e113.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e131.27\u0026thinsp;\u0026plusmn;\u0026thinsp;2.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e107.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e108.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDBP (mmHg)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.57\u0026thinsp;\u0026plusmn;\u0026thinsp;7.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81.97\u0026thinsp;\u0026plusmn;\u0026thinsp;3.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.17\u0026thinsp;\u0026plusmn;\u0026thinsp;8.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eThe data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of the growth characteristics of each group (n\u0026thinsp;=\u0026thinsp;6).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eDifferent superscript lowercase letters in the same row indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eWeight gain (%) = ((final body weight \u0026ndash; initial body weight) / final body weight) * 100\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eSPCN: Saline pump.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eTPCN: TMAO pump.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eSPCNM: Saline pump, and tube feeding 20 mg/kg/Day ED4.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eTPCNM: TMAO pump, and tube feeding 20 mg/kg/Day ED4.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHigh-dose TMAO exposure led to slightly higher BW, significantly greater weight gain, and elevated systolic (SBP) and diastolic blood pressure (DBP) in the TPCN group compared to the SPCN group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These findings are consistent with previous reports indicating that elevated TMAO levels contribute to increased BW\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and blood pressure\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This may be attributed to TMAO's impairment of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production, leading to reduced endothelium-dependent vasodilation\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. These observations suggest that continuous TMAO administration via osmotic pump may contribute to early signs of cardiovascular dysregulation.\u003c/p\u003e \u003cp\u003eED4 supplementation significantly reduced both BW and weight gain in the TPCNM group compared to the TPCN group. Similarly, the SPCNM group exhibited significantly lower BW than the SPCN group, suggesting that ED4 may attenuate weight gain, potentially through a reduction in fat accumulation, whereas elevated TMAO levels appear to promote it. Previous studies have indicated that ED4 may contribute to blood pressure regulation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and the present findings further support this, as ED4 treatment significantly lowered both SBP and DBP in the TPCNM group relative to the TPCN group. Notably, SBP and DBP levels in the TPCNM group were not significantly different from those in the SPCN control group, suggesting that ED4 supplementation normalized blood pressure altered by TMAO. These results highlight the potential of ED4 as a therapeutic agent for mitigating TMAO-induced metabolic and cardiovascular disturbances.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSerum Biochemistry Parameters\u003c/h3\u003e\n\u003cp\u003eSerum biochemical analyses were performed at weeks 10 and 12 to evaluate lipid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Following one week of TMAO administration (week 10), the TPCN group showed significantly elevated levels of TG, TC, LDL-C, and VLDL-C compared to the SPCN group. These differences remained evident at week 12. In addition, high-density lipoprotein cholesterol (HDL-C) levels were reduced, and the TC/HDL-C ratio was increased in the TPCN group. These findings are consistent with previous evidence suggesting that TMAO impairs bile acid metabolism through the FXR\u0026ndash;SHP\u0026ndash;Cyp7a1 axis, thereby promoting atherogenesis\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Overall, these results indicate that TMAO exerts a detrimental impact on lipid homeostasis, even in the absence of a high-carnitine diet and with TMAO delivered solely via osmotic pump.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRemarkably, ED4 treatment significantly reduced TG, TC, LDL-C, VLDL-C, and the TC/HDL-C ratio in the TPCNM group, while increasing HDL-C levels, restoring lipid profiles comparable to those in the SPCN group. These improvements were accompanied by reduced BW, suggesting that ED4 modulates lipid metabolism and helps prevent fat accumulation. The present findings support ED4\u0026rsquo;s ability to improve lipid profiles and reduce cardiovascular risk. Recent studies have also shown that ED4 inhibits fatty acid-induced lipid accumulation and prevents hypertension associated with chronic kidney disease\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Additional research suggests that ED4 may regulate gut microbiota and modulate the c pathway through the enterohepatic cycle\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eLiver Fat Accumulation and Liver Injury\u003c/h3\u003e\n\u003cp\u003eIn addition to its strong association with CVD, TMAO has also been implicated in liver diseases. For instance, an excessive abundance of TMAO is potentially harmful to patients with non-alcoholic fatty liver disease (NAFLD) due to its ability to regulate the synthesis and transport of bile acids\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Furthermore, previous studies demonstrated that RBE exhibited superior efficacy in inhibiting fat accumulation in the liver and showed stronger antioxidant and hepatoprotective properties compared to RSV in adipocyte experiments\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Based on these findings, this study evaluates whether ED4, a purified RBE, can mitigate elevated systemic TMAO-induced damage by assessing hepatic lipid accumulation and liver injury markers, such as AST and ALT levels.\u003c/p\u003e \u003cp\u003eLipid accumulation in hepatocytes, mainly as triglycerides, is a key feature in the development of NAFLD. It often appears as lipid vacuoles, reflecting disrupted lipid metabolism due to imbalanced synthesis, oxidation, or export. As these vacuoles enlarge, they can impair hepatocyte function and contribute to disease progression, including inflammation and fibrosis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In this study, significant hepatic fat vacuoles were observed in the TMAO-induced TPCN group compared to the SPCN control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These findings are consistent with previous studies showing that TMAO-treated groups typically exhibit vacuolation associated with fatty liver pathology\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In contrast, the SPCNM and TPCNM groups treated with ED4 exhibited fewer fat vacuoles than the TPCN group. Hepatocytes and liver lobules in these groups appeared neatly arranged, with the liver tissue structure under microscopic observation closely resembling that of the SPCN group. A similar pattern was observed in the fat droplet area analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), where the TPCN group displayed the largest fat droplet area, while the TPCNM group showed values comparable to those of the SPCN group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePrevious research by Nian et al. demonstrated significantly elevated AST and ALT levels in TMAO-treated groups\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Similarly, our study found that AST and ALT levels in the TPCN group were significantly higher than those in the SPCN group at both week 10 and week 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results confirm that high-dose TMAO administration via pump leads to substantial hepatic lipid accumulation and liver injury.\u003c/p\u003e \u003cp\u003eImportantly, ED4 treatment significantly lowered AST and ALT levels by week 12, highlighting its hepatoprotective effects. This reduction indicates improved liver function and attenuation of TMAO-induced hepatic injury\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. These findings underscore the effectiveness of ED4 in reducing liver fat accumulation and mitigating liver damage under elevated TMAO exposure.\u003c/p\u003e\n\u003ch3\u003eSerum and Feces SCFA\u003c/h3\u003e\n\u003cp\u003eSCFAs are gut microbiota-derived metabolites essential for metabolic health, influencing gut barrier integrity, glucose metabolism, and lipid regulation\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. SCFAs play a key role in lipid metabolism by regulating adipocyte function, influencing lipolysis and adipogenesis, and thereby improving circulating lipid profiles. Higher SCFA levels are also associated with lower TG-to-HDL ratios and better insulin sensitivity, highlighting their metabolic benefits\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBased on the data presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, high-concentration TMAO exposure significantly affects SCFA levels in both serum and feces, as evidenced by markedly lower levels of acetic acid, propionic acid, isobutyric acid, butyric acid, and valeric acid in TMAO-induced rats (TPCN group) compared to controls (SPCN group). However, supplementation with ED4 (SPCNM and TPCNM groups) restored or even elevated these SCFA levels, surpassing those in the SPCN group. Acetic acid improves liver function and lipid metabolism by activating AMPKα, which enhances lipid oxidation, inhibits lipogenesis, and reduces hepatic fat accumulation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. These findings suggest that ED4 supplementation mitigates the adverse effects of elevated TMAO on SCFA production, supporting its role in maintaining metabolic health.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of continuous ED4 supplementation on serum and feces short-chain fatty acids in rats with elevated systemic TMAO.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSCFA (\u0026micro;M)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSPCN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTPCN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSPCNM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTPCNM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSerum\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e979.24\u0026thinsp;\u0026plusmn;\u0026thinsp;112.71\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e604.93\u0026thinsp;\u0026plusmn;\u0026thinsp;66.63\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1013.15\u0026thinsp;\u0026plusmn;\u0026thinsp;9.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e846.19\u0026thinsp;\u0026plusmn;\u0026thinsp;28.85\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePropionic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e137.52\u0026thinsp;\u0026plusmn;\u0026thinsp;7.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50.28\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e116.53\u0026thinsp;\u0026plusmn;\u0026thinsp;6.31\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e98.61\u0026thinsp;\u0026plusmn;\u0026thinsp;13.19\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsobutyric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e170.01\u0026thinsp;\u0026plusmn;\u0026thinsp;6.36\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e82.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.95\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e110.52\u0026thinsp;\u0026plusmn;\u0026thinsp;6.97\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e111.14\u0026thinsp;\u0026plusmn;\u0026thinsp;11.49\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eButyric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e132.15\u0026thinsp;\u0026plusmn;\u0026thinsp;13.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e117.25\u0026thinsp;\u0026plusmn;\u0026thinsp;8.74\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e91.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.80\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValeric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e164.86\u0026thinsp;\u0026plusmn;\u0026thinsp;3.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e73.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e130.14\u0026thinsp;\u0026plusmn;\u0026thinsp;7.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e118.58\u0026thinsp;\u0026plusmn;\u0026thinsp;9.55\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHexanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e147.22\u0026thinsp;\u0026plusmn;\u0026thinsp;8.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.99\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e128.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e106.91\u0026thinsp;\u0026plusmn;\u0026thinsp;9.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeptanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110.77\u0026thinsp;\u0026plusmn;\u0026thinsp;9.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.83\u0026thinsp;\u0026plusmn;\u0026thinsp;4.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e98.64\u0026thinsp;\u0026plusmn;\u0026thinsp;8.26\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e105.22\u0026thinsp;\u0026plusmn;\u0026thinsp;7.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e132.08\u0026thinsp;\u0026plusmn;\u0026thinsp;6.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e77.68\u0026thinsp;\u0026plusmn;\u0026thinsp;6.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e116.62\u0026thinsp;\u0026plusmn;\u0026thinsp;2.43\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e107.26\u0026thinsp;\u0026plusmn;\u0026thinsp;19.21\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeces\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2529.88\u0026thinsp;\u0026plusmn;\u0026thinsp;69.29\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1807.52\u0026thinsp;\u0026plusmn;\u0026thinsp;27.60\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2409.48\u0026thinsp;\u0026plusmn;\u0026thinsp;3.42\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2141.50\u0026thinsp;\u0026plusmn;\u0026thinsp;15.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePropionic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1732.29\u0026thinsp;\u0026plusmn;\u0026thinsp;30.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1021.96\u0026thinsp;\u0026plusmn;\u0026thinsp;45.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1657.32\u0026thinsp;\u0026plusmn;\u0026thinsp;43.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1737.49\u0026thinsp;\u0026plusmn;\u0026thinsp;27.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsobutyric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1315.22\u0026thinsp;\u0026plusmn;\u0026thinsp;27.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e973.96\u0026thinsp;\u0026plusmn;\u0026thinsp;32.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1132.09\u0026thinsp;\u0026plusmn;\u0026thinsp;11.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e987.96\u0026thinsp;\u0026plusmn;\u0026thinsp;63.80\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eButyric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e710.70\u0026thinsp;\u0026plusmn;\u0026thinsp;20.95\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e451.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e689.45\u0026thinsp;\u0026plusmn;\u0026thinsp;19.76\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e568.48\u0026thinsp;\u0026plusmn;\u0026thinsp;6.64\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValeric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1034.38\u0026thinsp;\u0026plusmn;\u0026thinsp;19.64\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e879.76\u0026thinsp;\u0026plusmn;\u0026thinsp;8.37\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1006.53\u0026thinsp;\u0026plusmn;\u0026thinsp;18.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e983.47\u0026thinsp;\u0026plusmn;\u0026thinsp;15.43\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHexanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e999.22\u0026thinsp;\u0026plusmn;\u0026thinsp;45.70\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e727.75\u0026thinsp;\u0026plusmn;\u0026thinsp;17.87\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e982.79\u0026thinsp;\u0026plusmn;\u0026thinsp;15.49\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e845.11\u0026thinsp;\u0026plusmn;\u0026thinsp;21.41\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeptanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e734.16\u0026thinsp;\u0026plusmn;\u0026thinsp;39.76\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e369.64\u0026thinsp;\u0026plusmn;\u0026thinsp;7.49\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e715.86\u0026thinsp;\u0026plusmn;\u0026thinsp;16.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e633.98\u0026thinsp;\u0026plusmn;\u0026thinsp;7.15\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e528.76\u0026thinsp;\u0026plusmn;\u0026thinsp;57.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e327.02\u0026thinsp;\u0026plusmn;\u0026thinsp;8.35\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e558.82\u0026thinsp;\u0026plusmn;\u0026thinsp;16.93\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e413.40\u0026thinsp;\u0026plusmn;\u0026thinsp;7.83\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eThe data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of the growth characteristics of each group (n\u0026thinsp;=\u0026thinsp;6).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eDifferent superscript lowercase letters in the same row indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eSimilarly, fecal SCFA levels were also significantly altered. The TPCN group demonstrated decreased levels of acetic acid, propionic acid, isobutyric acid, butyric acid, and other SCFAs, reflecting the disruption of gut microbial metabolism due to elevated systemic TMAO. Following ED4 supplementation, the SPCNM and TPCNM groups showed partial or complete recovery of these SCFA levels. Remarkably, the fecal propionic acid and butyric acid levels in the TPCNM group were significantly higher than those in the TPCN group, highlighting ED4's potential role in restoring gut microbial function and SCFA production.\u003c/p\u003e \u003cp\u003eThe study also demonstrated that RBE supplementation effectively restored SCFA levels in rats with high-fat diet-induced obesity. These findings are consistent with previous research highlighting the critical roles of SCFAs, particularly butyric acid and propionic acid, in regulating lipid metabolism, reducing inflammation, and maintaining gut health\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The recovery of SCFA levels observed in the ED4-supplemented groups further supports the hypothesis that ED4 may influence gut microbiota composition and function to mitigate the adverse metabolic effects of elevated TMAO levels.\u003c/p\u003e\n\u003ch3\u003eSerum and Urine Creatinine\u003c/h3\u003e\n\u003cp\u003eTMAO, a low-molecular-weight nitrogenous waste, is efficiently filtered by the kidneys but is linked to chronic kidney disease (CKD) risk due to its association with creatinine and urea levels\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Elevated TMAO induces vasoconstriction, triggering acute hypertension and raising blood pressure\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Increased blood creatinine and reduced urinary creatinine clearance indicate impaired kidney function and hypertension\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, blood and urinary creatinine levels increased in the TPCN group by week 10, whereas ED4 supplementation (SPCNM and TPCNM groups) led to reductions in these levels by week 12, approaching those observed in the SPCN group. CCr, a key indicator of renal function, remained relatively unchanged in the TPCN group over time. In contrast, the SPCNM and TPCNM groups exhibited improved CCr compared to the TPCN group, with values comparable to the SPCN control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResveratrol supplementation has been shown to significantly reduce blood urea nitrogen (BUN) and creatinine levels while improving glomerular filtration rate (GFR). Additionally, resveratrol administration has been reported to notably lower serum creatinine levels\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Furthermore, RBE has demonstrated protective effects against kidney disease in adenine-treated rats\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Based on our findings and previous research, we propose that RBE supplementation, including its ED4 derivative, may hold potential as a renal protective intervention.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSerum TMAO\u003c/h2\u003e \u003cp\u003eAfter three weeks, serum TMAO levels in the TPCN group were significantly higher than in the SPCN group (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), confirming the effective function of the osmotic pump and TMAO absorption into circulation. Moreover, ED4 significantly reduced TMAO in the SPCNM and TPCNM groups, with TPCNM levels approaching those of SPCN. RSV has been shown to modulate gut microbiota, reduce TMA production, and inhibit TMAO levels\u003csup\u003e\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Co-culture studies indicate that both RSV and RBE promote probiotics while suppressing TMA-producing microbes, with RBE effectively lowering TMA levels\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffects of continuous ED4 supplementation on serum metabolites in rats with elevated systemic TMAO.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMetabolites (mM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSPCN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTPCN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSPCNM\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTPCNM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTCA cycle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCitrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSuccinate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFumarate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Oxoglutarate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnergy metabolism\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePyruvate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCreatine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLactate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.57\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.72\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42.07\u0026thinsp;\u0026plusmn;\u0026thinsp;4.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.78\u0026thinsp;\u0026plusmn;\u0026thinsp;2.84\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycerol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGut microbial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEthanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethanol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFormate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKetones\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Hydroxybutyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.90\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetoacetate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmino acids\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAlanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlutamate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlutamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHistidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsoleucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTaurine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThreonine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTyrosine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eValine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTMA-related\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBetaine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCholine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO-Acetylcarnitine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrimethylamine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrimethylamine N-oxide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSugar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e132.29\u0026thinsp;\u0026plusmn;\u0026thinsp;8.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e133.26\u0026thinsp;\u0026plusmn;\u0026thinsp;5.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e111.23\u0026thinsp;\u0026plusmn;\u0026thinsp;6.90\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e152.50\u0026thinsp;\u0026plusmn;\u0026thinsp;24.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOthers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Hydroxyisobutyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Hydroxyisobutyrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetamide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAllantoin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGalactonate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycolate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUrea\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e94.36\u0026thinsp;\u0026plusmn;\u0026thinsp;4.92\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.05\u0026thinsp;\u0026plusmn;\u0026thinsp;5.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e115.32\u0026thinsp;\u0026plusmn;\u0026thinsp;5.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e121.82\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esn-Glycero-3-phosphocholine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eThe data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM of the growth characteristics of each group (n\u0026thinsp;=\u0026thinsp;6).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eDifferent superscript lowercase letters in the same row indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOur previous study demonstrated that ED4 reduces TMAO levels in a diet-induced model, likely through modulation of the gut microbiota or by promoting TMAO excretion. As TMAO is primarily eliminated via urine within 24 hours\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, ED4 may facilitate its clearance. In the current study, urinary TMAO levels were slightly higher in the TPCNM group compared to the TPCN group (Fig. S2), though the difference was not statistically significant. While further investigation is required to confirm whether ED4 enhances TMAO excretion, these findings suggest that ED4 has the potential to suppress elevated systemic TMAO levels and mitigate its associated adverse health effects.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSerum Metabolomics\u003c/h3\u003e\n\u003cp\u003eSerum metabolomics analysis using high-resolution NMR and Chenomx identified 45 metabolites in the serum. Among these metabolites, 18 exhibited significant changes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in response to TMAO exposure or ED4 treatment (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The metabolite contents were analyzed and visualized using statistical plots generated with MetaboAnalyst 6.0. In the PCA plot comparing all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), the model demonstrated an F-value of 11.58, an R-squared of 0.63, and a \u003cem\u003ep\u003c/em\u003e-value of 0.001, indicating a statistically significant separation between groups. The primary separation was driven by ED4 supplementation, with SPCN and TPCN exhibiting significant overlap, as did SPCNM and TPCNM. These overlapping groups were distinctly separated from others along PC1, which accounted for 28.65% of the variance. The results further indicated that TMAO exposure had a less pronounced effect on the separation compared to ED4 supplementation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe PLS-DA scores plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) further illustrated the separation among the four experimental groups based on the first two components, which together explained 36.5% of the total variance (Component 1: 23.7%, Component 2: 12.8%). Distinct clustering was observed between SPCN and SPCNM, as well as between TPCN and TPCNM, reflecting clear metabolic differences. The primary separation along Component 1 was driven by ED4 supplementation, while Component 2 captured secondary differences likely due to TMAO exposure.\u003c/p\u003e \u003cp\u003eVIP scores from the PLS-DA analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) highlighted key metabolites contributing to group differentiation. 2-Hydroxyisobutyrate, pyruvate, and glycolate were the most significant contributors, with 2-hydroxyisobutyrate showing elevated levels in ED4-supplemented groups. The heatmap further demonstrated relative concentrations of these metabolites across the groups, emphasizing their roles in the observed separation.\u003c/p\u003e \u003cp\u003eThe effect of TMAO exposure\u003c/p\u003e \u003cp\u003eThe VIP scores from the PLS-DA analysis identified glycolate and creatine as the most significant contributors to the separation between SPCN and TPCN (Fig. S3A). The heatmap revealed that TPCN exhibited higher relative glycolate levels but lower creatine concentrations compared to SPCN, emphasizing the metabolic impact of TMAO exposure. Additionally, other metabolites, including 2-hydroxyisobutyrate and sn-glycero-3-phosphate, also contributed significantly to the observed differences. Pathway enrichment values (PEV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) revealed significant perturbations in pathways such as valine, leucine, and isoleucine biosynthesis, butanoate metabolism, and glycine, serine, and threonine metabolism, which were among the top enriched pathways with the highest enrichment ratios and lower \u003cem\u003ep\u003c/em\u003e-values (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.1).\u003c/p\u003e \u003cp\u003eThe effect of ED4 supplementation\u003c/p\u003e \u003cp\u003eED4 supplementation induced significant metabolic alterations in both SPCN vs. SPCNM and TPCN vs. TPCNM comparisons (Fig. S3B and S3C). For SPCN vs. SPCNM, key differentiating metabolites included 2-hydroxyisobutyrate, pyruvate, and glycolic acid, while for TPCN vs. TPCNM, pyruvate, 2-hydroxyisobutyrate, and creatine were the primary contributors. Moreover, pathway enrichment analysis identified 14 and 20 significantly altered pathways (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in SPCN vs. SPCNM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and TPCN vs. TPCNM (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), respectively. In SPCN vs. SPCNM, pathways such as glyoxylate and dicarboxylate metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and phenylalanine metabolism were prominently enriched, reflecting systemic metabolic adjustments. Similarly, in TPCN vs. TPCNM, pathways including valine, leucine, and isoleucine biosynthesis, glycine, serine, and threonine metabolism, and butanoate metabolism showed significant enrichment, emphasizing ED4's role in modulating amino acid and energy metabolism.\u003c/p\u003e \u003cp\u003eED4 supplementation consistently induced metabolic changes across various experimental conditions, with most metabolites showing elevated levels. Our results demonstrate that ED4 exerts a strong influence on metabolic pathways, particularly those involved in amino acid biosynthesis and energy metabolism, in both SPCN and TPCN groups. Specifically, significant increases were observed in energy metabolites (pyruvate, creatine, lactate) and amino acids (histidine, isoleucine, leucine, valine) in the ED4-treated groups. These findings align with previous research showing that RSV may modulate gut microbiota and regulate amino acid and lipid metabolism, increasing amino acid levels and potentially reducing atherosclerosis risk through improved metabolic profiles and its anti-inflammatory and antioxidant effects\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Overall, these findings highlight the metabolic benefits of RSV and suggest that ED4, as its bioactive derivative, may improve metabolic health and help prevent CVD by modulating gut microbiota and nutrient pathways.\u003c/p\u003e \u003cp\u003eIn this study, the levels of BCAAs (leucine, isoleucine, and valine) and their related metabolites (2-hydroxyisobutyrate and 3-hydroxyisobutyrate) were significantly elevated in the ED4-treated group. Prior research also indicates that RSV treatment can markedly increase isoleucine levels under high-fat diet conditions. Studies have demonstrated the association of BCAAs with protein synthesis and muscle metabolism, which can enhance physical performance. For instance, leucine, a key BCAA, is known to activate the mTOR pathway, promoting muscle protein synthesis, increasing muscle mass, and raising basal metabolic rate\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Furthermore, the observed increase in BCAAs may reflect their involvement in beneficial metabolic pathways. For instance, prior research has shown that BCAAs can participate in the TCA cycle, contributing to energy production\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In this study, metabolites associated with the TCA cycle showed a slight increase in the ED4-treated group, although the change was not statistically significant. Such metabolic activity may facilitate energy expenditure, helping to maintain body weight and improve metabolic health.\u003c/p\u003e \u003cp\u003eAdditionally, we observed an increase in serum 2-hydroxyisobutyrate levels. Although its metabolism and pathophysiological role in humans are not yet fully understood, studies have explored the intricate interactions between gut microbiota, BCAAs, and their metabolites, such as 2-hydroxyisobutyrate and 3-hydroxyisobutyrate\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Research has also highlighted the potential beneficial effects of 2-hydroxyisobutyrate, including resistance to oxidative stress, lifespan extension, and anti-aging properties\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe relationship between BCAAs and metabolic health is complex and may be influenced by multiple factors\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Therefore, although elevated BCAA and their derivative levels have been associated with certain metabolic disorders, the underlying metabolic and disease mechanisms are multifactorial and cannot be explained by a single parameter. In this context, the observed increases in BCAAs, 2-hydroxyisobutyrate, and 3-hydroxyisobutyrate in the ED4-treated group should not be directly interpreted as indicators of metabolic risk. We propose that the resveratrol derivative ED4 may promote metabolic health through multiple mechanisms, with elevated BCAA levels potentially reflecting enhanced metabolic activity and modulation of gut microbiota. Further studies employing multi-omics approaches are needed to clarify these mechanisms and validate our findings.\u003c/p\u003e\n\u003ch3\u003eIntestinal Microbiota\u003c/h3\u003e\n\u003cp\u003ePrevious studies have linked dietary TMAO production to gut microbiota, where dietary precursors are metabolized into TMA and converted to TMAO in the liver. TMAO\u0026rsquo;s impact on gut flora is a key CVD marker\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, underscoring the link between diet, microbiota, and cardiovascular health\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. While this study used an elevated systemic TMAO model, assessing treatment effects on gut microbiota remains crucial. To this end, fresh fecal samples were collected before sacrifice for microbiota analysis.\u003c/p\u003e \u003cp\u003eMicrobiota Abundance\u003c/p\u003e \u003cp\u003eIn the top 10 species-level relative abundance results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), \u003cem\u003eRomboutsia ilealis\u003c/em\u003e and \u003cem\u003eParabacteroides goldsteinii\u003c/em\u003e DSM 19448\u0026thinsp;=\u0026thinsp;WAL 12034 showed notable differences between treatments. \u003cem\u003eR. ilealis\u003c/em\u003e is a key producer of acetate, butyrate, and propionate, and studies have linked it to reduced liver inflammation. \u003cem\u003eP. goldsteinii\u003c/em\u003e, on the other hand, predominantly produces propionate, butyrate, and valerate\u003csup\u003e\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In the ED4-supplemented TPCNM group, the relative abundance of both species was significantly higher than in the TPCN group. These findings align with the observed increases in serum and fecal SCFA levels as well as improvements in markers of liver injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe study also found that ED4 supplementation (in the SPCNM and TPCNM groups) led to a reduction in \u003cem\u003eClostridium disporicum\u003c/em\u003e compared to the SPCN and TPCN groups. Interestingly, \u003cem\u003eC. disporicum\u003c/em\u003e has been previously reported to correlate positively with serum TMAO levels under normal conditions \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. This discrepancy is hypothesized to arise from differences in the TMAO source, as the current study provided TMAO directly rather than through dietary precursors.\u003c/p\u003e \u003cp\u003eIn the TPCN group, a significant decrease in the relative abundance of \u003cem\u003eDuncaniella freteri\u003c/em\u003e and \u003cem\u003eLigilactobacillus murinus\u003c/em\u003e was observed. However, these microbes showed a recovery in the TPCNM group, indicating that ED4 supplementation could counteract the negative effects of elevated TMAO levels on these species.\u003c/p\u003e \u003cp\u003e \u003cem\u003eDuncaniella\u003c/em\u003e spp., known for its high butyrate production and anti-inflammatory properties, has been associated with protective effects against colitis. Additionally, previous studies suggest that high doses of RBE can protect against hypertension in offspring, with increased butyrate levels positively correlated with \u003cem\u003eDuncaniella\u003c/em\u003e spp. abundance\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Consistently, this study demonstrated significantly higher butyric acid levels in the serum and feces of the TPCNM group compared to the TPCN group.\u003c/p\u003e \u003cp\u003eSimilarly, an increase in \u003cem\u003eLigilactobacillus murinus\u003c/em\u003e abundance has been linked to the alleviation of low-grade endotoxemia, reducing hypertension progression by restoring endothelial vasodilation. Treatment with \u003cem\u003eL. murinus\u003c/em\u003e has been shown to lower blood pressure and improve vascular endothelial function in spontaneously hypertensive rats (SHR)\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. The findings of this study confirm that elevated systemic TMAO levels can alter the composition of specific gut microbiota and highlight that ED4 supplementation induces significant and beneficial changes in these microbial populations.\u003c/p\u003e \u003cp\u003eMicrobiota α-diversity\u003c/p\u003e \u003cp\u003eAccording to the results of α-diversity indices (menhinick, Pielou evenness, and Shannon entropy) showed no significant difference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig. S4). Although the results were not statistically significant for treatment effectiveness, as observed in several other studies\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e, there were still slight differences in values during the short-term experimental period.\u003c/p\u003e \u003cp\u003eMicrobiota β-diversity\u003c/p\u003e \u003cp\u003eBeta-diversity analysis is used to compare the differences in microbial community composition between different samples or groups. The Bray-Curtis matrix assesses dissimilarity based on species abundance data, where smaller values indicate less variation in species diversity between samples. According to the results shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the TMAO-induced group (TPCN) exhibited lower species variation compared to the control group (SPCN). After ED4 supplementation, the SPCNM and TPCNM groups showed species variations closer to that of the control group.\u003c/p\u003e \u003cp\u003eBased on the PLS-DA plot in the provided Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, the analysis highlights clear separations among the microbial community compositions across the experimental groups. The ED4-supplemented SPCNM group shows the most distinct clustering, indicating a broader distribution of microbial diversity compared to the other groups. In contrast, the TMAO-induced TPCN group exhibits a more condensed clustering pattern, reflecting reduced microbial diversity. The PLS-DA plot also demonstrates that ED4 supplementation in the SPCNM and TPCNM groups significantly shifted microbial distributions toward a pattern more similar to the control group (SPCN), particularly along Component 1, which explains 9.77% of the variance. Component 2 further captured additional variation (8.38%), emphasizing the effects of both TMAO exposure and ED4 intervention on gut microbiota composition.\u003c/p\u003e \u003cp\u003eThe impact of individual bacterial strains\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, the relative abundance of \u003cem\u003eFaecalicatena contorta\u003c/em\u003e, \u003cem\u003eParabacteroides merdae\u003c/em\u003e, and \u003cem\u003eAdlercreutzia equolifaciens\u003c/em\u003e was significantly reduced in the TMAO-induced group (TPCN). However, their relative abundance was markedly increased in the ED4-supplemented groups (SPCNM and TPCNM).\u003c/p\u003e \u003cp\u003e \u003cem\u003eF. contorta\u003c/em\u003e is potentially involved in bile acid metabolism by influencing CYP7A1 ligand concentrations, effectively converting CA/CDCA into DCA/LCA, which further regulates cholesterol levels and contributes to maintaining lipid metabolism balance\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. This aligns with the serum cholesterol analysis results observed in this study. The gut bacterium \u003cem\u003eP. merdae\u003c/em\u003e has been reported to prevent cardiovascular damage by enhancing BCAA catabolism\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eA. equolifaciens\u003c/em\u003e has been negatively associated with carotid atherosclerosis, potentially due to its production of beneficial metabolites, such as butyrate and equol\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, ED4 supplementation may influence the gut microbial composition in rats exposed to high systemic TMAO levels by reducing the abundance of TMAO-associated species. It is hypothesized that ED4 modulates gut microbiota to regulate cholesterol levels, thereby promoting lipid metabolism balance, reducing hepatic fat accumulation, and mitigating the risk of CVD. However, the relationship between biochemical indicators, metabolites, and disrupted bacterial communities remains highly complex, requiring further research to better understand their interconnections and underlying mechanisms.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThis study employed an animal model with continuous intraperitoneal TMAO infusion, successfully elevating systemic TMAO levels and inducing cardiovascular-related abnormalities such as increased body weight, blood pressure, dyslipidemia, and hepatic fat accumulation. ED4 supplementation effectively mitigated these effects by improving serum lipid profiles, reducing hepatic fat, and normalizing liver injury markers. It also enhanced SCFA production, lowered serum TMAO levels, and modulated gut microbiota by reducing TMAO-associated bacterial species. In addition, the observed increases in BCAAs, 2-hydroxyisobutyrate, and 3-hydroxyisobutyrate, along with restored microbial diversity, suggest that ED4 promotes metabolic health through multiple pathways. While the interplay among metabolites, microbiota, and disease markers remains complex, these findings support ED4\u0026rsquo;s potential as a therapeutic agent against TMAO-related disorders and highlight the need for further validation through multi-omics and clinical studies.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe rat low-density lipoprotein cholesterol (LDL-C; CSB-E11704r) and very-low-density lipoprotein cholesterol (VLDL-C; CSB-E17088r) enzyme-linked immunosorbent assay (ELISA) kits were purchased from CUSABIO Technology LLC (Houston, TX, USA). The ZymoBIOMICS\u0026reg; DNA miniprep kit was purchased from Zymo Research Co. (Irvine, CA, USA). All chemicals used in this study were purchased from Sigma-Aldrich\u0026reg; (Merck KGaA, Darmstadt, Germany) and were used without further pre-treatment unless otherwise stated. ED4 was prepared according to the previously published methods of our team\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animal and experimental design\u003c/h2\u003e \u003cp\u003eThe experimental animal study was conducted as described by Hsu et al. 2020\u003csup\u003e24\u003c/sup\u003e with minor modifications. A total of 24 male, 8-week-old Sprague-Dawley (SD) rats (average body weight (BW) 250 g\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were purchased from the BiOLASCO Taiwan Co., Ltd. (Taipei, Taiwan). The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of National Kaohsiung University of Science and Technology under code 0109-AAAP-012. This study was conducted over a total period of four weeks of animal experimentation (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Following a one-week adaptation period, the rats were randomly assigned to four groups based on BW. The groups (n\u0026thinsp;=\u0026thinsp;6 per group) were designated as follows: SPCN (negative control: saline pump), TPCN (positive control: TMAO pump), SPCNM (saline pump with ED4 treatment), and TPCNM (TMAO pump with ED4 treatment). Osmotic pumps (Alzet\u0026reg; osmotic pumps, Models 2ML4, DURECTTM Corp., Cupertino, CA, USA) containing either saline or TMAO were surgically implanted into the intraperitoneal cavity of the rats in each group for continuous administration from week 9 to week 12. The administered dose of TMAO was 37 mg/day (3.86 nM/s), as defined by Ufanl et al. (2015)\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Rats in the ED4-treated groups received oral gavage of ED4 at a dosage of 20 mg/kg BW/day from week 10 to week 12.\u003c/p\u003e \u003cp\u003eAll rats were housed in stainless steel suspended wire mesh cages (two rats per cage) under specific pathogen-free conditions, maintained at 22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a humidity of 50% \u0026plusmn; 5% and a 12-hour light/dark cycle. Throughout the experiment, the animals were provided with ad libitum access to AIN-93M standard purified feed and drinking water. Daily food intake and water consumption were monitored consistently throughout the study, while weekly BW measurements were recorded over the four-week experimental period.\u003c/p\u003e \u003cp\u003eIn week 10 and week 12 (before sacrifice), blood pressure was measured in conscious rats using the indirect tail-cuff method (BP-2000; Visitech Systems, Inc., Apex, NC, USA). The rats were gently restrained and calmed before placing the sensor cuff on their tails for measurement. Blood samples were collected from the tail vein at week 10 (one week after implantation). At the end of week 12, following a 10-hour fasting period, rats were anesthetized intramuscularly with a cocktail of Zoletil (25 mg/kg) and Rompun (23.32 mg/kg xylazine hydrochloride) in a 1:1 ratio. Blood samples were collected via cardiac puncture, and the harvested organs were weighed, preserved in 10% formalin for histological staining, and stored for further analysis. Blood samples were centrifuged at 3000\u0026times;g for 10 minutes at 4\u0026deg;C, and the supernatant was collected to obtain serum samples, which were subsequently stored at -80\u0026deg;C until analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of serum biochemistry parameters\u003c/h2\u003e \u003cp\u003eTriglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), alanine transaminase (ALT), and aspartate transaminase (AST) levels were analyzed using an automated clinical chemistry analyzer (NX-500i, Fuji, Masaaki Takei, Japan). Serum levels of LDL and VLDL were measured using ELISA kits, following the manufacturer's standardized protocols. The absorbance of the samples was measured at a wavelength of 450 nm using an ELISA reader (BioTek, Agilent Technologies, Inc., Santa Clara, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLiver histopathology staining\u003c/h2\u003e \u003cp\u003eTissue sections fixed in 10% neutral formalin were embedded in paraffin wax, sectioned into slices approximately 5 \u0026micro;m thick, and stained with hematoxylin and eosin (H\u0026amp;E). The stained sections were mounted on slides, and high-resolution images were captured using the MoticEasyScan system. The images were further analyzed with the Motic DS Assistant (4K).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of SCFAs\u003c/h2\u003e \u003cp\u003eThe SCFAs were analyzed using gas chromatography\u0026ndash;mass spectrometry (GC-MS) following the method described by Wang et al. (2020)\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSerum Sample Preparation\u003c/p\u003e \u003cp\u003eSerum samples were centrifuged, and 150 \u0026micro;L of the supernatant was collected and acidified with 50 \u0026micro;L of sulfuric acid. Subsequently, 10 \u0026micro;L of 2-ethylbutyric acid was added as an internal standard, followed by the addition of 400 \u0026micro;L of diethyl ether. The solution was then shaken for 15 minutes and centrifuged at 9000\u0026times;g for 10 minutes at 4\u0026deg;C. The resulting supernatant was used for GC-MS analysis.\u003c/p\u003e \u003cp\u003eFecal Sample Preparation\u003c/p\u003e \u003cp\u003eA 0.1 g sample of rat feces was homogenized in 1 mL of deionized water. The subsequent steps were performed in the same manner as described in the section \u0026ldquo;Serum Sample Preparation\u0026rdquo; for the serum preparation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGC-MS analysis\u003c/h2\u003e \u003cp\u003eSCFA analysis was conducted using a DB-FFAP capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m). Helium was used as the carrier gas at a flow rate of 1 mL/min. The column temperature program started at 80\u0026deg;C (held for 1 minute), followed by an increase to 240\u0026deg;C at a rate of 10\u0026deg;C/min, where it was maintained for 12 minutes. The injector, transfer line, ion source (70 eV), and quadrupole temperatures were set to 240\u0026deg;C, 230\u0026deg;C, and 150\u0026deg;C, respectively. The mass spectrometer scanned in the range of 35\u0026ndash;550 m/z. The mass spectra of each identified compound were compared against the GC-MS database for confirmation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCreatinine and CCr analysis\u003c/h2\u003e \u003cp\u003eThe analysis was conducted following a modified method by Jen et al. (2002)\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.The mobile phase A was prepared using 0.05 M ammonium dihydrogen phosphate, with the pH adjusted to 7.4 using 28% ammonia and 6 N phosphorous acid. The sample diluent was prepared by adjusting PBS to a pH of 2.24 using NaOH.\u003c/p\u003e \u003cp\u003eFor urine samples, 2 \u0026micro;L of urine was diluted 200-fold with 398 \u0026micro;L of PBS. For serum samples, 50 \u0026micro;L of serum was diluted 1:1 with 50 \u0026micro;L of PBS. The diluted samples were centrifuged at 12,000 rpm at 4\u0026deg;C for 10 minutes, and 100 \u0026micro;L of the supernatant was transferred into a vial for HPLC analysis.\u003c/p\u003e \u003cp\u003eHPLC analysis was performed under the following conditions: the mobile phase consisted of 0.05 M ammonium dihydrogen phosphate buffer at pH 7.4. The detection wavelength was set to 235 nm, with a total runtime of 13 minutes per injection. The flow rate was maintained at 1 mL/min, and the sample injection volume was 20 \u0026micro;L. A gradient program was applied with a solution ratio of mobile phase A to mobile phase B set at 100:0 throughout the 0\u0026ndash;13 minute runtime.\u003c/p\u003e \u003cp\u003eThe formula for calculating Creatinine Clearance Rate (CCr) in urine is as follows:\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" width=\"584\" height=\"43\"\u003e\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSerum metabolomics analysis\u003c/h2\u003e \u003cp\u003eSerum samples collected at week 12 were filtered using Amicon Ultra-3,000 molecular weight (MW) cutoff filters. The filtrate was collected, and 65 \u0026micro;L of a 4.67 mM DSS-D6 internal standard solution (Chenomx Inc., Edmonton, Alberta, Canada) was added. BS buffer was used to adjust the total volume to exactly 650 \u0026micro;L, with precise recording of the actual volume. The pH was adjusted to 6.8 by adding 1 N HCl or NaOH dropwise, as needed. Proton NMR spectra were acquired using a JEOL ECZ600R 600 MHz NMR spectrometer (Tokyo, Japan). Metabolites were identified and quantified using Chenomx NMRSuite 9.02 software (Chenomx Inc., Edmonton, Canada)\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and KEGG pathway enrichment analysis of serum metabolites across treatments were conducted via MetaboAnalyst 6.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eNext-generation sequencing (NGS) analysis and bioinformatics analysis\u003c/h2\u003e \u003cp\u003eFecal sample collection, pre-processing, and bioinformatics analysis followed the methods\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, with some modifications. Fecal sample collection occurred a day before osmotic pump implantation and a day prior to trial conclusion. Freshly collected feces from each group were stabilized in a preservation buffer and stored at -80\u0026deg;C until analysis. Bacterial DNA extraction was performed using the DNA Miniprep Kit and subsequently analyzed by Biotools Co., Ltd. (Taipei, Taiwan). DNA libraries were generated via PCR amplification of the V3\u0026ndash;V4 regions of the 16S rRNA gene, sequenced using Illumina Miseq (Illumina Inc., San Diego, CA, USA).\u003c/p\u003e \u003cp\u003ePaired-end reads were trimmed for quality and grouped into operational taxonomic units (OTUs) based on \u0026ge;\u0026thinsp;97% similarity using the GreenGene Database (v13.8). Analysis of OTUs (relative abundance, heatmaps), alpha diversity metrics (Shannon index, visualized as Venn diagrams), and beta diversity metrics (PCoA, phylogenetic trees) were performed using Basespace, CLC Genomics Workbench, and GraphPad Prism 8. Functional analysis, including LEfSe and PICRUSt predictions, was conducted via Galaxy/Hutlab (2024). Statistical significance was defined as \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) (n\u0026thinsp;=\u0026thinsp;6). Statistical analysis was performed using SPSS software (v12.0, IBM Co., Armonk, NY, USA). A one-way ANOVA was used to evaluate differences within groups, while Tukey's post-hoc test was applied for comparisons between groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating statistical significance).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll relevant data are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe National Science and Technology Council funded this research, the Republic of China (Grant no. 112-2221-E-992 -002-MY3; 112-2622-E-992-007-; 111-2221-E-328-001-MY3 and 110-2320-B-992-001-MY3)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.T.Q.: Experimental performance; P.H.H.: Experimental performance, manuscript preparation; Y.W.C. and M.K.S.: Experimental design and supervision, manuscript preparation; B.H.L.: Microbiota analysis, experimental supervision; P.P.S.: Experimental design and supervision; C.Y.H.: Experimental design of the whole study, supervision, manuscript preparation; S.Y.C.: Metabolomics analysis, supervision, manuscript preparation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhen, J. \u003cem\u003eet al.\u003c/em\u003e The gut microbial metabolite trimethylamine N-oxide and cardiovascular diseases. \u003cem\u003eFront Endocrinol (Lausanne)\u003c/em\u003e 14, 1085041 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, P. 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Y. \u003cem\u003eet al.\u003c/em\u003e Consumption of vitamin D2 enhanced mushrooms is associated with improved bone health. \u003cem\u003eJournal of Nutritional Biochemistry\u003c/em\u003e 26, 696\u0026ndash;703 (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"TMAO, resveratrol derivative, ED4, metabolomics, gut microbiota, lipid metabolism, liver function, cardiovascular health","lastPublishedDoi":"10.21203/rs.3.rs-6755780/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6755780/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTrimethylamine-N-oxide (TMAO), a gut microbiota-derived metabolite, is strongly linked to metabolic disorders, cardiovascular diseases, and liver injury. This study aimed to evaluate the therapeutic effects of 3-O-butanoylresveratrol (ED4), a resveratrol derivative, in a rat model with elevated systemic TMAO induced via osmotic pump infusion. ED4 supplementation significantly reduced serum triglycerides, cholesterol, low-density lipoprotein cholesterol, and the total cholesterol/high-density lipoprotein cholesterol ratio while improving liver function and reducing hepatic fat accumulation associated with high systemic TMAO levels. Additionally, ED4 restored short-chain fatty acid levels and modulated gut microbiota composition, increasing the abundance of beneficial species such as \u003cem\u003eRomboutsia ilealis\u003c/em\u003e and \u003cem\u003eParabacteroides merdae\u003c/em\u003e. Serum metabolomics revealed ED4's role in enhancing energy and amino acid metabolism, contributing to improved metabolic health. These findings demonstrate that ED4 alleviates TMAO-associated risks by improving lipid metabolism, protecting liver function, and reshaping gut microbiota, highlighting its potential as a functional food component for managing cardiovascular disorders.\u003c/p\u003e","manuscriptTitle":"Investigation of ED4 supplementation on cardiovascular parameters in rats with elevated systemic TMAO","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-25 15:17:46","doi":"10.21203/rs.3.rs-6755780/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-09T21:04:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T19:02:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154482874098278271129989804397302771230","date":"2025-06-29T05:15:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-26T09:08:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45127018801149503492857794436160147021","date":"2025-06-24T17:56:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305360686148535028456178155316989330885","date":"2025-06-24T05:01:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72547619347323054120358800719279408391","date":"2025-06-24T00:07:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"252530012294829339052186860281497730603","date":"2025-06-23T21:02:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-23T17:56:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-23T14:11:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-02T06:32:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Science of Food","date":"2025-05-27T05:47:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-science-of-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjscifood","sideBox":"Learn more about [npj Science of Food](http://www.nature.com/npjscifood/)","snPcode":"41538","submissionUrl":"https://submission.springernature.com/new-submission/41538/3","title":"npj Science of Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d49394bd-bee1-46fc-abfa-7810d8aaee23","owner":[],"postedDate":"June 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50536642,"name":"Biological sciences/Biochemistry"},{"id":50536643,"name":"Biological sciences/Biological techniques"},{"id":50536644,"name":"Health sciences/Diseases"},{"id":50536645,"name":"Health sciences/Health care"},{"id":50536646,"name":"Health sciences/Signs and symptoms"}],"tags":[],"updatedAt":"2025-11-24T16:00:32+00:00","versionOfRecord":{"articleIdentity":"rs-6755780","link":"https://doi.org/10.1038/s41538-025-00552-2","journal":{"identity":"npj-science-of-food","isVorOnly":false,"title":"npj Science of Food"},"publishedOn":"2025-11-21 15:56:58","publishedOnDateReadable":"November 21st, 2025"},"versionCreatedAt":"2025-06-25 15:17:46","video":"","vorDoi":"10.1038/s41538-025-00552-2","vorDoiUrl":"https://doi.org/10.1038/s41538-025-00552-2","workflowStages":[]},"version":"v1","identity":"rs-6755780","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6755780","identity":"rs-6755780","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}