ChoCar trial; The role of gut microbiota in choline and carnitine metabolism on vascular inflammation in metabolic syndrome

A role for gut microbiota in influencing metabolic pathways and as such playing
a role in the etiology of metabolic disorders has been suggested . Whether and
how the interplay between gut microbiota and metabolism can lead to
cardiovascular diseases (CVD) is to be explored. Recently, the direct
participation of gut microbiota in CVD pathogenesis was reported in the setting
of a diet rich in phosphatidylcholine (PC) e.g. eggs, meat and other animal
products, the major dietary source of choline. This study showed that gut
microbiota-mediated catabolism of the choline moiety of PC produces
trimethylamine (TMA), which in the liver is further metabolized to the
proatherogenic species trimethylamine-N-oxide (TMAO), as well as the
PC-metabolites betaine and choline. Plasma levels of TMAO, choline and betaine
were associated with increased cardiovascular disease risk. Using orally
gavaged choline- and PC-rich diets in mice and deuterium labeled PC (d9-PC) in
humans, it was demonstrated that intestinal microbiota play an obligatory role
in formation of TMA and consequently TMAO produced via induction of hepatic
flavin-containing monooxygenases (FMO). Moreover, dietary supplementation with
choline resulted in increased plasma TMAO as well as accelerated macrovascular
inflammation in atherosclerosis-prone ApoE-knockout mice, underscoring
causality. Interestingly, this pro-atherosclerotic phenomenon was not seen when
mice were treated with broad spectrum antibiotics eliminating almost all gut
microbiota. Importantly, supplementation of diet with the gut microbiota
dependent metabolite TMAO accelerated atherosclerosis and macrovascular wall
inflammation in the apolipoprotein E (ApoE)-knockout mice, consistent with TMAO
playing a role in atherosclerosis.
Interestingly, the group of Hazen et al. also found that carnitine, abundant in
red meat and that contains a trimethylamine moiety similar to choline, when
metabolized also leads to increased TMAO-levels. Indeed in mice, oral carnitine
supplementation caused increased atherosclerosis, an effect that was again
completely diminished following suppression of intestinal microbiota with oral
antibiotics. Moreover, antibiotic treatment followed by oral d3-labelled
carnitine challenge was found to diminish plasma TMA and TMAO levels in mice
and humans; a finding that was also observed in humans with a vegetarian (low
red meat consumption) background (10). Apparently, proceeding dietary habits
influence the capability of microbiota in the colon to produce TMA. As with
choline, these results were confirmed by an observational study in humans,
relating plasma carnitine levels to cardiovascular disease. Specifically, the
association of high plasma carnitine levels with prevalent CVD and incident
myocardial infarction (MI)/stroke and death risks were only observed with high
TMAO levels as well * that is, subjects with high carnitine but low TMAO levels
did not have increased cardiac risks. These results are consistent with TMAO
playing a direct pro-atherogenic role, and intestinal microbiota playing an
obligatory role in the formation of TMA (and thus TMAO) from
trimethylamine-containing macronutrients such as PC, choline and carnitine.

Aforementioned studies did however not directly look at gut microbiota
composition in relation to choline and carnitine metabolism. We recently showed
beneficial effects of lean donor fecal transplantation on glucose metabolism in
subjects with metabolic syndrome (MetS), which was mediated by specific changes
in fecal bacterial species. Moreover, metabolic syndrome has recently been
shown to be linked to increased aortic inflammation on PET-CT imaging. Thus,
the aim of our study is to investigate the effect of allogenic (lean
preferentially vegetarian donor) compared to autologous (own feces) microbial
transplantation, derived from fecal samples, on gut microbiota composition and
carnitine/choline metabolism as well as mascrovascular wall inflammation in
obese males with metabolic syndrome.

In this study we aim to investigate whether infusion of intestinal microbiota
from lean (vegetarian) donors has differential effect on choline (d6-labeled
choline) and carnitine (d3-labeled carnitine) metabolism and macrovascular
inflammation (18F-FDG PET-CT scan) in obese subjects with metabolic syndrome
and to associate changes in fecal gut microbiota after lean (vegetarian) donor
microbial transplantation with above mentioned parameters.

Primary objective:
To determine changes in postprandial choline and carnitine metabolism at
baseline and 2 weeks after lean donor microbial transplantation.

Secondary objective:
To compare choline and carnitine metabolism in the donors versus the recipients

Tertiary objective:
To evaluate the relation between changes in plasma choline and carnitine
(metabolism) in relation to macrovascular inflammation

Fourth objective:
To evaluate associations between plasma choline and carnitine (metabolism) in
relation to adipose tissue inflammation (subcutaneous fat biopsy).

This is a double blind single center randomized controlled trial.
Patients will be randomized by sealed envelopes to the following 2 treatment
arms:
1. single allogenic (lean vegetrian donor) fecal transplantation (at baseline)
2. single autologous (own) fecal transplantation (at baseline)

Obese males are recruited via newspaper advertisements and screened for
criteria of the MetS (3 or more out of 5 criteria according to updated NCEP
guidelines including at least increased fasting plasma glucose > 5.6 mmol/l )
(13). Medication and supplement use (including vitamin/choline/carnitine
supplements, energy drinks and carnitine-enriched soymilk) or a history of
cardiovascular disease or cholescystectomy are exclusion criteria. When a
potential subject is eligible, appointments will be made for all experiments to
be completed during the study.

Donors are lean (BMI between 20-25 kg/m2) healthy males, preferentially
vegetarian, and are also recruited via newspaper advertisements. As with the
recipients, medication and use of food supplements (including
vitamin/choline/carnitine supplements, energy drinks and carnitine-enriched
soymilk) are exclusion criteria.

Both donor and recipient will be subjected to a postprandial (Nutridrink
ingestion) oral choline/carnitine challenge. These tests can be done at the
same time. The participants will be provided with a capsule of synthetic
d3-carnitine and another capsule of d6-choline. The capsules will be taken at
the same time simultaneously with Nutridink ingestion and plasma samples are
taken at predefined time points to study plasma choline and carnitine
metabolism. Moreover, an FDG PET-CT scan will be performed to assess level of
macrovascular inflammation, as subjects with metabolic syndrome are
characterized by an increased TBR signal as compared to control subjects.

Study program:
Week -1, Day 1 * 7 (lean vegetarian donors and metabolic syndrome subjects)
The preparation for week 0 starts at home with recording the dietary habits for
the duration of one week within an online dietary booklet. Subjects are asked
to adhere to their personal diet, without changing it throughout the study.

Week 0, Study day 1 (lean vegetarian donors and metabolic syndrome subjects)
At day 1, the first fat biopsy and dietary d6-choline/d3-carnitine challenge
test (CCCT) are performed. Subjects and donors are asked to come to the
clinical research unit after an overnight fast. After a subcutaneous fat biopsy
and baseline blood sample, subjects are given a capsule containing 250mg of
d6-choline and another capsule containing 250mg d3-carnitine to be ingested
together with one bottle of Nutridrink (150cc). Hereafter serial blood samples
are withdrawn for the duration of 6 hours. In addition, 24 hours of urine is
collected and all participants are asked to obtain two tubes with feces from
one fecal sample. At the end of the experiment, subjects are offered a meal to
their choice.

Week 0, Study day 2 (lean vegetarian donors and metabolic syndrome subjects)
At Day 2, 18F-FDG PET-CT scan imaging of the aortic arch and carotid arteries
is performed (12), followed by duodenal tube positioning via Coretrack.
Afterwards bowel lavage will take place via the tube over 4 hours, which is
followed by the actual treatment of either infusion of lean vegetarian
donor-derived microbial solution (allogenic) or placebo (autologous feces). The
placement of the duodenal tube and the transplantation will only take place in
the recipients. For the infusion of the fecal transplant material, both
recipient and donor are asked to provide the study physician with a fresh fecal
sample to enable double blind treatment.

Week 1, Study day 3 (metabolic syndrome subjects)
One week after the treatment, the first and only control visit is planned.
Subjects status will be checked by a short interview, physical examination as
well as a blood withdrawal for safety parameters. Also, subjects are asked to
collect 24h urine in advance, which can be given to the study physician at the
visit.

Week 1, day 1 * 7 (metabolic syndrome subjects)
The preparation for week 2 starts at home with recording the dietary habits for
the duration of one week within an online dietary booklet. Subjects are asked
to adhere to their personal diet, without changing it throughout the study.

Week 2, Study day 4 (metabolic syndrome subjects)
Two weeks after the treatment, the fat biopsy and CCCT is repeated, including
the collection of two fecal samples and 24h urine.

Week 2, Study day 5 (metabolic syndrome subjects)
18F-FDG PET-CT scan imaging of the aortic arch en carotid arteries is
performed. After the scan, subjects are allowed to leave the hospital and the
study is completed.

In general, subjects are asked to be in a fasting state for all visits
throughout the study, meaning they must have refrained from eating and drinking
minimally 10 hours before start of the experiment.

Protocol lean vegetarian donors:
When eligible, donors are asked to also perform the fat biopsy and CCCT once,
including obtaining 24hrs of urine and collection of 2 fecal samples as well as
a PET-CT. This is to compare choline and carnitine metabolism between donors
and recipients in relation to vascular and adipose tissue inflammatory status,
both before and after treatment. As with recipients, donors are asked to write
down their dietary habits the week before the CCCT is performed. Afterwards,
donors will be randomly paired to one recipient. At the day of the
transplantation, donors and metabolic syndrome subjects are both asked to
provide a fecal sample to enable the treatment

Patients will be treated with either allogenic or autologous microbial
transplantion by duodenal tube after bowel lavage.

Both the donor and the subject will deliver a fresh feces sample (150-250g on
average) at the day of infusion (produced within 6 hours before use). After
collection in a special container, feces will be stored at 4 degrees Celcius.
Time of collection will be written down.
After having given a fecal sample to the study physician, subjects will be
getting a duodenal tube placed via Coretrack device, which positioning will be
checked via an abdominal X-ray. During this process and after randomization,
one of both fecal samples will be mixed with 500cc saline solution (0.9% NaCl)
until fully homogenised. Before mixing starts, a sample will be taken of the
total amount of feces for later analysis. After mixing, the feces solution is
poured through a sieve incorporated into a funnel to remove all debris and
obtain a homogenous solution. The solution is poured into a 500cc sterile glass
bottle. Thereafter, the bottle will be kept on refrigerator temperature (4
degrees Celcius) until the patients is finished with bowel lavage. When the
intestines of the participant are completely cleared of fecal material, the
treatment will take place. After treatment, subjects are offered a meal and are
allowed to go home.

No adverse effects are expected in this study. However, subjects are asked for
a time investment, frequent study visits, blood withdrawals as well as PET-CT
scans and abdominal X-ray. In addition, they are subjected to behavioural
changes, such as a dietary restriction, ingestion of choline and carnitine
isotopes and the collection of faecal stool samples and urine. The isotopes do
not confer any health risks.
The advantage for subjects is that their general health will be evaluated by
physical examination and laboratory measurements. Based on their study results
subjects will be given an advice with respect to their cardiovascular risk
profile. In case abnormalities are found, subjects are referred to their
general practitioner or to our outpatient clinic.