Trimethylamine-N-oxide Production and Metabolism

Trimethylamine-N-oxide (TMAO) is a carbon-containing organic compound formed from dietary
precursors including TMAO (high in fish), choline (high in eggs) and carnitine (high in
beef). However, TMAO production is highly variable (Zhang AQ et al., 1999), appears to be
influenced by genetics (Cashman JR et al., 2001) and gut microbiome (Wang Z et al., 2011;
Koeth RA et al., 2013), and is linked to heart disease in cardiac patients (Wang Z et al.,
2011) and colorectal cancer among post-menopausal women (Bae S et al., 2015). At present,
very little is known about the metabolic fate of TMAO and how it is used within the human
body (Bain MA et al., 2005). This study sought to (i) quantify the effects of eggs, beef and
fish on TMAO biomarkers in plasma, muscle, urine and stool; (ii) examine the metabolic fate
of supplemental TMAO labeled isotopically with deuterium; and (iii) determine whether TMAO
production is a function of the gut microbiome.

To accomplish these objectives, a randomized, controlled cross-over study was conducted in
healthy male participants (n=40). The study incorporated four arms comprised of study meals
representing animal sources of TMAO (egg, beef and fish) along with a fruit control. The
study meals were (i) 3 whole hard-boiled eggs; (ii) 6 oz beef (Philly-Gourmet Beef Patties);
(iii) 6 oz fish (cod fillet); and (iv) 2 single-serve packages of Mott's natural applesauce.
Each meal was served with one cup of water, administered in a single day and separated by a
1-week washout period. For the fruit control, 50 mg deuterium-labeled methyl-d9-TMAO
(d9-TMAO; Cambridge Isotopes) was added to one cup of water for oral consumption to enable
the tracing of the metabolic fate of TMAO, and to assess its bioavailability and clearance.

Baseline blood sample was obtained by a phlebotomist using a standard venipuncture
procedure, and participants collected their baseline urine sample. They also turned in
self-collected baseline 24 h urine and stool samples. Following the consumption of the study
meal, serial blood samples were collected at 15, 30 min and 1, 2, 4 and 6 h, while urine
samples were collected throughout the 6 h study period. At 4.5 h, participants were provided
a fixed fruit snack (i.e., applesauce) and water. On the day that participants consumed the
d9-TMAO tracer, participants collected their urine throughout the next 24 h and their stool
at the next bowel movement. In addition, a subset of this group (n=6) were invited to
undergo a muscle biopsy procedure 6 h after the fruit + d9-TMAO tracer consumption.

Inclusion Criteria (main study):

- Healthy men of age 21-50 y

- BMI of 20-29.9 kg/m2 who are willing to comply with the study protocol (consumption
of study meals and sample collections)

Inclusion Criteria (sub-study involving muscle biopsy):

- Healthy participants who are able to undergo or watch medical procedures

Exclusion Criteria (main study):

- Men over 50 y of age

- BMI ≥ 30 kg/m2

- Women, vegetarians, smokers, individuals with gastrointestinal diseases or
complaints, chronic illnesses or other metabolic diseases (including
trimethylaminuria), abnormal laboratory values, and those taking nutritional
supplements, antibiotics or probiotics within 2 months of recruitment.

Exclusion Criteria (sub-study involving muscle biopsy):

- Men with history of a negative or allergic reaction to local anesthetics

- Tendency toward easy bleeding or bruising, on medications that may increase the
chance of bleeding or bruising (e.g., Aspirin, Coumadin, Anti-inflammatories, Plavix)

- Currently on any immunosuppressive medications (e.g., glucocorticoid steroids,
chemotherapy), with disease pathologies that would impair the healing process (e.g.,
diabetes, cancer, keloids, hereditary healing disorders, jaundice, alcoholism,
HIV/AIDS)