Developing a Biomarker Panel to Assess Choline Nutritional Status

Choline (Cho) is an essential nutrient and most Americans' diets do not achieve the
recommended intake. Diets low in Cho are associated with liver and muscle disease and with
suboptimal fetal development, while diets too high in choline may be associated with
increased risk for heart disease. Cho is a required nutrient and in 1998, an Adequate Intake
(AI) and a Tolerable Upper Limit (UL) for Cho was established In 2016, the US Food and Drug
Administration (FDA) set a Recommended Daily Intake (RDI) for Cho based on the AIs as part of
the new Nutrition Facts label for packaged foods (published in the Federal Register on May
27, 2016; FDA-2012-N-1210-0875, Federal Register Number:2016-11867). The AI/RDI varies by age
and gender, but is 550 mg/d in adolescent and adult men and 425 mg/d in adult women (more in
pregnant and lactating women) and 400 mg/day for adolescent women.

There is no validated biomarker for choline status (the availability of the various forms of
Cho needed to sustain optimal cellular function). Measurement of plasma Cho concentrations is
not adequate as plasma choline is homeostatically regulated. Based on extensive preliminary
and published data this group identified a panel of potential biomarkers that could be used
to assess Cho status, and now they propose studies to validate this biomarker panel against
measures of Cho pool size using isotope dilution. The largest stores of Cho are located in
the liver, and mass resonance spectroscopy (MRS) of liver has been used in the past to assess
Cho status in humans. This method is not practical for use as a biomarker in clinical or
public health practice as it is expensive and the availability of the instrumentation is
limited. Liver biopsy is risky and not practical, making measurement of hepatic Cho and Cho
metabolites concentrations a poor choice for assessing Cho status.

Perhaps there is a panel of biomarkers that together will more accurately and reliably
reflect Cho status. By making measurements in people fed 3 different dietary amounts of Cho
for two weeks at a time, and comparing the biomarker measures to body total Cho pool size
assessed using isotope dilution (a proxy for the availability of the various forms of Cho),
investigators will be able to identify the combination of biomarkers and algorithm for
calculating a Cho status score that best predicts total Cho pool size, and therefore predicts
choline nutritional status (the availability of the various forms of Cho needed to sustain
cellular function). In this pilot study, they are seeking to test a method for using stable
isotope dilution to measure body choline stores, and then ask how this measure correlates
with a panel of biomarkers in plasma and with liver fat measured using Fibroscan®. Using
isotope dilution can provide an estimate of the size of the body pool of Cho. Our proposed
method is conceptually similar to the method for measuring total body water from a bolus dose
of labeled water. Similar methodology was used recently in studies of metabolic flux of Cho
in pregnant women. Isotope dilution is a well-established method used to estimate pool size
for other nutrients, such as vitamin A. Similar to vitamin A, the major storage pools for Cho
are in the liver, and ingested Cho is rapidly absorbed and accumulated by liver. This pilot
study tests a method for using stable isotope dilution to measure body choline stores, and it
correlates this measure with a panel of biomarkers in plasma and with liver fat measured
using Fibroscan®. People who eat diets low in choline should deplete their choline (Cho)
stores, and measurement of Cho pool size using isotope dilution should reflect this
depletion. The investigator will identify a biomarker panel that correlates well with
measured Cho pool size across the range of different degrees of depletion. MRS/MRI scans will
also be performed to investigate correlation between these "gold standard" measures and the
other methods described above.

Participants will be consume meals we provide in two week dietary intervals with 3 different
levels of choline with 2 week washout periods between those dietary intervals. Participants
will receive 100% of the recommended intake (RDI) of Cho (550mg Cho/day); 50% of the RDI of
Cho (275mg/day); and 25% of the RDI of Cho (137.5mg/day). The meal order will be randomly
assigned and all participants will receive all diets at some point in the study. There will
be a minimum of a two week washout between diet intervals. Both participants and researchers
will be blinded to the diet order.

Participants will have brief exercise challenges (Biodex) to assess muscle function as an
additional predictor of choline status.

To validate our isotope dilution and Fibroscan measures participants will also complete
MRI/MRS scans.

Saliva samples, urine, and stool samples will be collected.

Inclusion Criteria:

- Provision of signed and dated informed consent form

- Stated willingness to comply with all study procedures and availability for the
duration of the study

- Male or female, aged 17-70 years

- In good general health as evidenced by medical history, clinical chemistries, physical
exam, and BMI≤ 30

- Women who are included in the study and are of pregnancy potential will have a urine
pregnancy test at the beginning and end of each dietary intervention arm and must be
using birth control during the study.

Exclusion Criteria:

- using drugs or medication known to be damaging to liver or muscle at typically
prescribed doses or that have the potential to alter Cho metabolism (e.g.,
methotrexate);

- history of hepatic, renal, or other chronic systemic disease.

- subjects with liver abnormalities (e.g.cysts) as determined by ultrasound

- current smokers

- consume >2 alcoholic beverages/d or >14/wk

- substance abusers or drug addicted

- eating unusual diet that would interfere with the study

- food allergies, (e.g., soy) or any problems with eating all foods on required study
diet

- using Cho-containing dietary supplements

- women who are breastfeeding, pregnant, or plan to become pregnant due to potential
risk to fetus/child of low choline diet

- performing intense exercise of more than 1 hour a day or other intense muscle building
exercise (such as weightlifting beyond low weight repetitions)

- Actively participating in other research study where required to exercise or ingest
any food, medicine, or supplement in any manner

- claustrophobia

- has a cardiac pacemaker, artificial heart valve, metal plate, pin, or other metallic
implant, intrauterine device, insulin or other drug pump, aneurysm clips, previous
gunshot wound, cochlear implant or other implantable hearing device, employment
history as a metalworker, or permanent cosmetic tattoos (eyeliner, eyebrow)