The purpose of this clinical trial is to investigate in subjects with and without metabolic syndrome how meals of choline- and carnitine-rich foods (eggs and meat) affect the trimethylamine-N-oxide (TMAO) concentration in blood and urine in relation to the gut microbiota composition.

In response to the subjects´ gut microbiota, the concentrations of TMAO in the plasma and urine of subjects with and without metabolic syndrome (MetS) after ingesting choline- and carnitine-rich foods will be compared.

On two occasions, participants will receive after overnight fasting meatballs (170 g) or three hard-boiled eggs. Blood will be collected before ingestion and over 6 hours after test food consumption.

There is a growing interest in the role of the intestinal microbiota in the global metabolism of their host. Trimethylamine-N-oxide (TMAO), a metabolite linked to the gut microbiota, has recently emerged as a risk metabolite for cardiovascular disease.

The gut microbiota composition was reported to be altered in MetS. After ingestion of foods high in trimethylamine moieties, postprandial TMAO in blood may differ between subjects with and without MetS because of their different microbiota profiles.

Aim of the study is to investigate in subjects with and without MetS the postprandial TMAO concentrations in blood and urine in association with the gut microbiota profile after ingestion of choline- and L-carnitine-rich foods.

Thirty-three subjects aged 18-75 years with (n=12) or without MetS (n=21) were recruited.

Subjects received on two occasions, after overnight fasting, either three hard-boiled eggs or 170 g meatballs.

Blood samples were collected (before ingestion of food, and at 30, 60, 120, 240, and 360 minutes after ingestion). A composite urine sample was collected over 6 hours. A feces sample was collected on the day before the first intervention.

Concentrations of TMAO, trimethylamine, betaine, choline, L-carnitine, acetyl-L-carnitine, and creatinine were measured UPLC-MRM-MS. The incremental area under the curve (iAUC) was calculated for each compound.

TMAO, trimethylamine, betaine, choline, L-carnitine, acetyl-L-carnitine, and creatinine were analyzed in composite urine samples using UPLC-MRM-MS.

Gut microbiome analysis was done by full 16S rRNA gene sequencing using the Oxford Nanopore Technology.

Inclusion criteria for all subjects with and without MetS:

• Age = 18-75y
• Plasma creatinine value within the age-related reference range
• No history of acute or chronic diseases (e.g. diabetes or bowel disease)
• No use of vitamin or mineral supplements, no medication with antibiotics 2 months before recruitment and during the study
• Non-smoker
• Not consuming a special diet (vegetarian)
• No pregnancy, planned conception, or lactation
• No participation in another study.

Inclusion criteria for subjects without MetS:

• Normal BMI (18.6 to 29.0 kg / m 2 )
• Blood counts within reference ranges
• Normal ranges for liver enzymes (P-ALAT, P-AST, P-ALP), blood status, plasma triglycerides, plasma HDL cholesterol, plasma cholesterol, plasma glucose

Inclusion criteria for subjects with MetS:

A minimum of three of the following criteria according the National Cholesterol Education Program Adult Treatment Panel (NCEP-ATP III):

• Waist size > 102 cm in men, > 88 cm in women
• Fasting plasma glucose ≥ 5.6 mmol /l
• Triglycerides > 1.7 mmol / l or treatment
• HDL 1.03 mmol / l in men, 1.29 mmol / l in women or treatment
• Blood pressure ≥ 130 / 85 mm Hg or medication. A value of B-HbA1c in the age-related reference range will be used to exclude diabetes.

Exclusion criteria for all research subjects :

• Smoker
• Pregnancy, planned conception, or lactation
• Medical treatment with antibiotics 2 months before recruitment and during the study
• Use of dietary supplements 2 weeks before and during the study
• Use of probiotics 2 months before and during the study
• Following a special diet (eg vegan, vegetarian, weight loss)
• Participation in another study

There is no plan to share individual data as all data will be presented in the main article through tables and figures or in the supplementary material as mean ± standard deviation. However, individual data will be made available upon reasonable request.