Red Wine Effects Upon Gut Flora and Plasma Levels of Trimethylamine-N-oxide (TMAO) - WineFlora Study

Evaluation of Red Wine Effects Upon Gut Flora and Plasma Levels of Trimethylamine-N-oxide (TMAO) in Patients With Established Atherosclerotic Disease

Recent evidence indicates that Trimethylamine-N-oxide (TMAO) is a pro-atherosclerotic, phosphatidylcholine-dependent metabolite of diet and intestinal flora. Food substrates derive from carnitine and phosphatidylcholine (lecithin), present mainly in eggs, red meat, liver and pork. The intestinal flora pattern that favors the formation of TMAO is very similar to that which predisposes to insulin resistance and obesity: a high proportion between phylum Firmicutes over Bacteroidetes. The intestinal microbiota is sensitive and variable; the use of prebiotics and probiotics can change the relationship between Firmicutes/Bacteroidetes phyla. Red wine (RW), for its composition with polyphenols and possible bactericidal role, may play a role in the intestinal flora modification and could promote proliferation of beneficial bacteria. However, the influence of RW on TMAO is not known. This is the hypothesis to be tested in this trial. METHODS: This is a prospective, crossover, randomized, controlled trial with patients from Heart Institute (InCor), FMUSP and volunteers recruited through press releases. We will evaluate 42 patients, all men, with established atherosclerotic disease. Patients will be evaluated in a crossed manner: each subject receives both treatments, intervention and control (in random order), and they will be divided into 2 groups: A and B. In the first intervention stage, after 2 weeks of washout for all patients , group A receives Red Wine (RW) and group B is the control, abstemious. In the 2nd stage of intervention, after 2 weeks of washout for all patients the groups are inverted: group B receives RW; and group A will be abstemious. In the period with wine intervention, patients will receive 250 mL/day of red wine per day, for 5 days of the week, for 3 weeks. Patients will maintain their usual diet without the use of prebiotics or probiotics, or other polyphenolic derivatives. At the beginning and at the end of each stage, patients will be submitted to serum TMAO and intestinal microbiota evaluation. For the intestinal microbiota evaluation, the new generation sequencing will be used in the highly preserved portion of the 16S subunit of the rRNA gene. The determination of TMAO in plasma will be by liquid chromatography coupled to mass spectrometry. Expected results: It is expected to determine if RW acts on the intestinal flora to the point of influencing plasma TMAO concentration.

Forty two men with established atherosclerotic disease will be studied:after a 2 week washout period, each subject receives both treatments, intervention and control

After a two weeks washout period,in the intervening period with red wine, patients will receive 250 mL / day of red wine per Day, 5 days a week, for 3 weeks.

After a two weeks washout period,in the Abstemious period, patients should not consume any kind of alcohol beverages, for 3 weeks

After a two weeks washout period,in the intervening period with red wine, patients will receive 250 mL / day of red wine per Day, 5 days a week, for 3 weeks.

After a two weeks washout period,in the Abstemious period, patients should not consume any kind of alcohol beverages, for 3 weeks

Gut flora modification assessed by 16S ribosomal rNA gene sequencing and plasma concentration of TMAO changes measured by liquid chromatography coupled to mass spectrometry in 42 patients after ingestion 250 mL of Red Wine 5 days/week, for 3 weeks

Patients will be divided into 2 groups: A and B. In the first intervention stage, after 2 weeks of washout for all patients , group A receives Red Wine (RW) and group B is the control, abstemious. In the 2nd stage of intervention, after 2 weeks of washout for all patients the groups are inverted: group B receives RW; and group A will be abstemious. In the period with wine intervention, patients will receive 250 mL/day of red wine per day, for 5 days of the week, for 3 weeks. Patients will maintain their usual diet without the use of prebiotics or probiotics, or other polyphenolic derivatives. At the beginning and at the end of each stage, patients will be submitted to serum TMAO and intestinal microbiota evaluation. For the intestinal microbiota evaluation, the new generation sequencing will be used in the highly preserved portion of the 16S subunit of the rRNA gene. The determination of TMAO in plasma will be by liquid chromatography coupled to mass spectrometry

Inclusion Criteria: Men, 45-70 years Established atherosclerotic diseases defined as: A. Previous history of CAD: angina or acute myocardial infarction (AMI), myocardial revascularization or angioplasty, angiographic evidence of stenosis ≥50% in at least one of the major coronary arteries B. Cerebrovascular disease: transient ischemic stroke or cerebrovascular accident C. Peripheral arterial disease: clinical evidence of extracoronary atherosclerosis Exclusion Criteria: Acute events in the last 30 days (AMI, troponin elevation, coronary angioplasty or coronary artery bypass grafting) Heart Failure (NYHA functional class ≥ II) Renal Failure (clearance creatinine <30 mL / min by the Cockcroft-Gault formula) Hepatic Failure Gastro-intestinal cancer Intestinal inflammatory diseases Obstructive biliary diseases Prior gastrointestinal surgeries: cholecystectomy or colectomy Use of antibiotics within the last 2 months or during protocol Alcoholism or alcohol intolerance Diabetes mellitus or use of antidiabetics drugs

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Haas EA, Saad MJA, Santos A, Vitulo N, Lemos WJF, Martins AMA, Picossi CRC, Favarato D, Gaspar RS, Magro DO, Libby P, Laurindo FRM, Da Luz PL; WineFlora Study. A red wine intervention does not modify plasma trimethylamine N-oxide but is associated with broad shifts in the plasma metabolome and gut microbiota composition. Am J Clin Nutr. 2022 Dec 19;116(6):1515-1529. doi: 10.1093/ajcn/nqac286.