This double-blind randomized controlled 12-week study was conducted between January and December 2023 at Tsaritsa Yoanna University Hospital, Sofia, Bulgaria. Thirty consecutive male patients with a history of clinical ASCVD were randomized in the study. The study population included patients who presented for a routine cardiology examination. Inclusion and exclusion criteria are listed in Table 1. All patients were screened for eligibility criteria before the study. The patients were invited to participate in the trial after meeting the criteria. All patients were instructed not to consume read meat, fish, eggs, milk products, and energy drinks 2 days before the study and 2 days before the last study visit in order to limit the effect of dietary factors on the TMAO levels. All other study procedures were performed on the same day. The patients were also fasting on the day of the analyses.

The probiotic-to-placebo ratio was 1:1. Interventional procedures lasted for 12 weeks in both groups and the trial was stopped according to the study protocol (Fig. 1).

Figure 1.

Study design. n = number of examined patients.

During the screening, data regarding patient history, concomitant conditions, and therapy were collected. Information on arterial hypertension, diabetes mellitus, dyslipidemia, premature history of cardiovascular diseases, and smoking status for each patient was also gathered. The current European guidelines on cardiovascular disease prevention were used to determine the presence or absence of these risk factors.^[15] The Fourth Universal Definition of Myocardial Infarction type I was utilized to identify patient history of myocardial infarction, e.g. documented history of rise and/or fall in cardiac troponin level with at least one value above the 99th percentile combined with one of the following: symptoms of acute myocardial ischemia, new ischemic electrocardiogram changes or new pathological Q waves, and imaging evidence of ischemic etiology or identification of a coronary thrombus by angiography.^[16]

Information regarding the degree of coronary vessel disease, number and timing of events, and revascularization procedures was also collected.

All patients underwent a standard physical examination and an electrocardiogram. Blood pressure (BP) measurements were performed after 5 minutes of rest in a supine position. Three BP measurements were performed 1–2 minutes apart and a final BP was recorded as the average of the last two measurements. Body mass index was calculated as the patient’s weight (kg) divided by height squared (m²).

Randomization was performed at the Center of Applied Studies and Innovation, Sofia, Bulgaria. Prepared bottles containing probiotic and placebo at a ratio of 1:1 (n=30) were supplied for the Cardiology Clinic, Tsaritsa Yoanna University Hospital ISUL. Each bottle was coded at the microbiology department and was given a unique number. The researchers and participants were blinded to the type of study treatment. The interventional products were assigned to the participants according to their recruitment sequence.

The patients were fasted for laboratory analyses. The following parameters were examined: creatinine levels, lipid profile, glycated hemoglobin (HbA1c) levels, and glucose levels. The glomerular filtration rate was estimated using chronic kidney disease epidemiology collaboration formula and was represented in mL/min/1.73 m². Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated using the following formula: fasting insulin level (µU/L) × fasting glucose level/22.5.

TMAO levels in human plasma samples were assessed at the Central Laboratory of Therapeutic Drug Management and Clinical Pharmacology at Alexandrovska University Hospital in the Faculty of Medicine of the Medical University of Sofia, Bulgaria, using triple quadrupole tandem mass spectrometry (MS) and high-performance liquid chromatography.^[8–10]

TMAO levels were determined in external standardization mode after plasma protein precipitation using acetonitrile. Chromatography was performed using a Kinetex RPC8 column, 50 mm×2.1 mm, with 2.6-µm particle size in mobile phase consisting of 10% methanol and 90% 0.02 mM ammonium fluoride buffer. The chromatographic run time was 2.5 minutes with a retention time for the TMAO peak of ~1.34 minutes. Detection was performed in the electrospray positive ion mode. MS/MS monitoring of the column effluent in the SRM mode was set to follow the predominant transitions, where collision energy = 18 at m/z 76–58. Raw mass chromatogram data were collected and processed using specialized software Xcalibur 1.4. All concentrations were calculated in external standard mode using the six-point calibration curve. Area of the mass spectrometry TMAO peak was used as a quantitative measure. Concentration units were µg/L. TMAO levels were measured two times: on the first day of the study before the interventional treatment and on the last day after 12 weeks of treatment.

Instructions for the interventional treatment containing information for the investigation product/placebo and prescription data were provided to each patient and explained in detail. Patients were instructed to take the study product two times daily: 1 hour after breakfast and 1 hour after evening. Patients were requested to return the study product on the day of the last (second) visit. Compliance was estimated by obtaining confirmation from patients regarding the medication taken and by the number of tablets returned to the center.

Probiotic consisting of 1×10⁹ CFU of L. plantarum GLP3, 75 mg of trans-resveratrol, non-alcoholic red wine extract, and prebiotic inulin was the investigational product. The weight of each capsule, including the placebo, was 500 mg.

Additional informed consent for the microbiome analysis was obtained from all patients. In those who agreed to this assessment (n=21), stool samples were collected before (as controls) and after therapy. Microbiome sequencing analysis, focusing on bacteria from the genus Lactobacillus, was performed in a commercial laboratory (GANZIMMUN Diagnostics GmbH, Germany).

Statistical analyses were performed using SPSS 23 software for Windows. Kolmogorov Smirnov and Shapiro-Wilk tests were used to assess the distribution of variables. Continuous variables with a normal distribution were presented as the mean ± standard deviation. For variables with a skewed distribution, parameters were presented as the median and interquartile range (IQR) in brackets, where IQR is the difference between the upper and lower quartile. Independent sample t-test and Mann-Whitney two-sample tests were used to compare normally and abnormally distributed continuous variables, respectively. Categorical variables were presented as percentages and counts and analyzed using χ² or Fisher’s exact tests. Relationships between continuous normally distributed variables were determined using Pearson’s and Spearman’s correlation coefficients according to their distribution.

Paired sample t-test and Wilcoxon signed-rank tests were applied to compare the effect of interventional procedures on variable levels and to determine the differences between the groups. A two-tailed p value of <0.05 was considered statistically significant.

The mean age for all examined patients was 58.5±8.16 years. Nineteen patients (65.5%) had a history of myocardial infarction, and eight patients (27.6%) had type 2 diabetes mellitus. There was no statistically significant difference in terms of age and risk factor profile between the patient groups. The main clinical and demographic characteristics of the patients are presented in Table 2.

Baseline TMAO levels before the intervention procedures were 284 (139) µg/L in the probiotic group and 176 (120) µg/L in the placebo group, with no statistically significant difference between them (p=0.19). Other evaluated baseline laboratory parameters, including lipid profile, HbA1C level, glucose level, insulin level, and HOMA-IR, are presented in Table 3.

After 12 weeks of the interventional procedure, the patients receiving the probiotic treatment showed a significant decrease in TMAO levels [from 284 (139) µg/L to 202.5 (96.7) µg/L; p=0.044]. There was no significant difference in TMAO levels in the placebo group after the treatment [from 176 (120) µg/L to 178 (150) µg/L; p=0.258] (Fig. 2).

Figure 2.

Differences in the primary outcome (TMAO levels) after 12 weeks of treatment. In the probiotic group (n=14) TMAO levels were decreased after the treatment. In the placebo groups, TMAO levels were not changed after 12 weeks of treatment. The difference in the TMAO levels after the interventional study procedure was statistically significant between the groups (p=0.044). Wilcoxon Rank Sum Test; TMAO: trimethylamine-N-oxide.

There was no statistically significant difference in the levels of total cholesterol, LDL cholesterol, non-HDL cholesterol, and triglycerides after the interventional study procedures in both probiotic and placebo groups. Significant differences in glucose and HOMA-IR index were not observed after 12 weeks of treatment in patients receiving probiotic and placebo treatments (Tables 4, 5).

The study results showed that Lactobacillus spp. % was not statistically significantly changed in the placebo group (2.01 (0.56) before and 0.74 (0.27) after the treatment; p=0.333). In the probiotic group, Lactobacillus spp. % was significantly increased after the intervention procedures (0.6 (0.14) before and 0.79 (0.6) after the treatment; p=0.041).

TMAO is synthesized by conversion of bacterial enzyme trimethylamine (TMA) lyases from L-carnitine, choline, and betaine-containing foods. This leads to the synthesis of TMA, which is the precursor of TMAO. TMA is then converted to TMAO in the liver via the liver enzyme flavin-dependent monooxygenase 3.^[18] TMAO is predominantly excreted by the kidneys.^[19] Therefore, diet changes, microbiota composition, FMO activity, and kidney function play an important role in the TMAO levels. Vegetarian diet is related to decreased TMAO levels as opposed to high-fat or Western diet, which is associated with increases in TMAO.‌^[20] Other therapeutic factors affecting TMAO levels are currently under investigation. These include the inhibition of microbiota enzymes that convert choline and carnitine to TMA.^[21] Balance and diversity in the microbiome are a key factor for healthy host-gut interaction.^{[22, 23]} Thus, intestinal microbiota modulation in order to decrease TMAO levels seems to be a safe and reasonable approach.

Despite the positive results for TMAO reduction after probiotic treatment in animals, studies evaluating the effect of probiotic supplementation on TMAO levels in humans have produced controversial results.^[17] Moreover, the results from meta-analyses including 270 patients from eight randomized trials did not show statistically significant changes in TMAO levels after the probiotic treatment. Nevertheless, it can be concluded that probiotic treatment can decrease TMAO levels in individuals younger than 50 years old.^[24]

The present study results showed a slight statistically significant decrease in TMAO levels in patients receiving the probiotic treatment. In addition, the probiotic treatment group showed significantly increased Lactobacillus spp. levels after the probiotic supplementation. The present study population had a very high CVD risk, and the probiotic was administered for 12 weeks. Another probiotic supplementation trial with the same study period failed to confirm the efficacy of L. casei in decreasing TMAO levels in 30 metabolic syndrome patients.^[25] The pilot trial design, the lack of beneficial effects on LcS in these patient groups, and the influence of nutritional factors were the main explanations for these results.

It is important to state that treatment was administered for different periods of time in most trials assessing the effect of probiotic supplementation on TMAO levels, which may explain the discrepancy in the results. In particular, four or 12 weeks of probiotic supplementation was utilized. The differences in patient population in terms of inclusion criteria are also important. For example, one of the randomized trials comprised very young healthy patients aged 20–25 years who received the probiotic treatment for four weeks. Statistically significant differences were not present in the TMAO levels between the groups. However, more participants in the probiotic groups showed that the TMAO levels decreased after the intervention.^[26]

In addition, the data from human and animal studies suggested that there are only a few probiotic strains that affect the TMAO levels.^[27] These outcomes showed a decrease in TMAO levels after treatment with L. rhamnosus GG, L. plantarum ZDY04, L. amilovorus LAM 1345, L. plantarum LP1145, and Enterobacter aerogenes ZDY01.‌^[28–31] Results from intervention studies using multi-strain probiotic VSL#3 did not show significant results in reducing TMAO levels.^[32]

L. plantarum GLP3 was used in the present trial in combination with resveratrol, which is a natural plant antioxidant with probiotic activity that has also been shown to decrease the TMAO levels.^[33]

The present study did not show a statistically significant difference in the lipid profile or glycemic status of both the probiotic and placebo groups after the interventional procedure.