Choline Nutritional Status: Development of a Biomarker Panel

Healthy Clinical Trial

Official title:

Choline Nutritional Status: Development of a Biomarker Panel

Clinical Trial Details — Status: Completed

Administrative data

• NCT number: NCT03726671
• Other study ID #: 17-1982
• Secondary ID: 1R01DK115380-01
• Status: Completed
• Phase: N/A
• Start date: November 1, 2018
• Est. completion date: October 20, 2021

Study information

• Verified date: October 2022
• Source: University of North Carolina, Chapel Hill
• Contact: n/a
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial Summary

People who eat diets low in choline should deplete their choline (Cho) stores, and measurements of Cho pool size using isotope dilution should reflect this depletion. Investigators will identify a biomarker panel that correlates well with measured Cho pool size across the range of different degrees of depletion.The investigators propose that, as body stores of Cho diminish, cells and organs will reach the point when metabolism/function in the cell is altered, and that this will result in a progression of changes in biomarkers that reflect Cho status.

Description:

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 the investigators propose 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. However, the MRS can be utilized to confirm correlations between the biomarker panel and the isotope dilution method. 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). 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 investigators will identify a biomarker panel that correlates well with measured Cho pool size across the range of different degrees of depletion. This study tests a method for using stable isotope dilution to measure body choline stores, and then asks 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. The investigators' 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. MRS/MRI scans will also be performed to investigate correlation between these "gold standard" measures and the other methods described above. Participants will consume meals provided in two week dietary intervals with 3 different levels of choline with a 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. Participants enrolled prior to 3/2020 completed MRI/MRS scans. We have determined that Fibroscan provides adequate measurement of liver fat such that we eliminated the added inconvenience to participants of travel to Winston-Salem for MR scanning. Saliva, stool, and urine samples will be collected.

Recruitment information / eligibility

• Status: Completed
• Enrollment: 101
• Est. completion date: October 20, 2021
• Est. primary completion date: October 20, 2021
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 17 Years to 70 Years

Eligibility

Inclusion Criteria:

• Provision of signed and dated informed consent form
• Weigh 130-177lbs (or if over 177 must have BMI under 28)
• 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 or if over 177lbs with a BMI under 28
• 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 some form of 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
• have been screened for this study between August and March and have not provided proof of flu vaccination prior to enrollment

Related Conditions & MeSH terms

• Healthy

Intervention

Other:
• 25% Cho diet
Subjects will consume meals containing 25% of recommended intake of Choline for 2 weeks. On day 12 of the diet period, subjects will consume 250 mg of Cho in the form of Cho chloride (trimethyl-d9, 9%), Cambridge Isotope Laboratories, Tewksbury, Massachusetts, (USA), as a bolus.
• 50% Cho diet
Subjects will consume meals containing 50% of recommended intake of Choline for 2 weeks. On day 12 of the diet period, subjects will consume 250 mg of Cho in the form of Cho chloride (trimethyl-d9, 9%), Cambridge Isotope Laboratories, Tewksbury, Massachusetts, (USA), as a bolus.
• 100% Cho diet
Subjects will consume meals containing 100% of recommended intake of Choline for 2 weeks. On day 12 of the diet period, subjects will consume 250 mg of Cho in the form of Cho chloride (trimethyl-d9, 9%), Cambridge Isotope Laboratories, Tewksbury, Massachusetts, (USA), as a bolus.

Locations

Country	Name	City	State
United States	UNC Chapel Hill Nutrition Research Institute	Kannapolis	North Carolina

Sponsors (2)

Lead Sponsor	Collaborator
University of North Carolina, Chapel Hill	National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

Country where clinical trial is conducted

United States, 

Outcome

Type	Measure	Description	Time frame	Safety issue
Other	SNPs That Create Inefficiencies in Choline Metabolism Associated With Change in Choline Pool Size and Choline Biomarkers	Exploratory analysis of >2 million SNPs measured on a custom Illumina Expanded Multi-Ethnic genotyping array (Mega-Ex). The same analysis described for Outcome 4 will be applied for Outcome 7. Benjamini-Hochberg method for False Discovery Rate (FDR) correction will be used for multiple testing correction.	At baseline visit
Primary	Ratio of Liver Choline Pool Size by Isotope Dilution	The liver choline pool determined by the dilution of the deuterated choline metabolites formed in liver and released to plasma as measured by isotopic enrichment ratio (IER). The IER for a given metabolite is defined as the concentration of a deuterated metabolite divided by the sum of deuterated and non-deuterated metabolite.	24 hours following administration of choline-d9 on day 12 of respective dietary intervention
Primary	Difference in Choline Deficiency Signature	Plasma choline metabolites (micromolar): choline, dimethylglycine, betaine, phosphatidylcholine, sphingomyelin, trimethylamine-oxide, and homocysteine measured by targeted metabolomic profiling. The signature for choline deficiency is defined by choline <0.793 mmol/L or betaine <34.9 mmol/L. The levels of these metabolites at the end of each intervention will be compared. The association between choline metabolites and choline pool size will be investigated.	At the end of 2 weeks of respective Cho diet
Primary	Comparison of Choline Pool Size Between Participants With and Without Choline Metabolites Signature During Cho Depletion	The 25% Cho arm was selected because only at that intake level is sufficient depletion achieved. Participants with plasma choline <0.793 mmol/L or betaine <34.9 mmol/L were considered as choline depleted (showing signature), participants with plasma choline >=0.793 mmol/L and betaine >=34.9 mmol/L were considered as not choline depleted (not showing signature). Available choline pool size was determined by the dilution of the deuterated choline metabolites formed in liver and released to plasma as measured by isotopic enrichment ratio (IER). The IER for a given metabolite is defined as the concentration of a deuterated metabolite divided by the sum of deuterated and non-deuterated metabolite.	24 hours following administration of choline-d9 on day 12 of 25% Cho diet
Primary	Ion Abundance (Intensity) of Metabolites as Indicators of the Intake of 25%, 50%, or 100% Choline in the Diet. The Ratio of the Intensity of Metabolite Signals for Each Dietary Group Can be Calculated and Correlated With the Level of Choline in the Diet	Metabolomics was conducted on plasma that was collected from individuals at the end of each 2-week diet period. UHPLC High Resolution Mass Spectrometry was used for differential profiling (PMID: 33415121). Supervised Orthogonal Partial Least Squares Discriminant Analysis was used to determine signals that differentiated the 25% choline dietary group from the 100% choline dietary group. Metabolites that differentiated the 25% and 100% choline dietary groups with variable importance to projection (VIP) >1 and p-value < 0.05 are reported. The signals for these metabolites were matched by retention time, exact mass, and MS/MS to standards run on the same platform. Because this is a differential profiling method (not quantitative), the mean and standard deviation of peak intensities detected on the untargeted platform are reported. Results are reported for the selected metabolites for the 25%, 50%, and 100% choline dietary groups. Ratios can be obtained by division of the intensity data.	At the end of 2 weeks of respective Cho diet
Primary	Comparison of Choline Pool Size Between Participants With Different Genotypes in Phosphatidylethanolamine-N-methyltransferase (PEMT) Single Nucleotide Polymorphism (SNP rs12325817)	DNA was collected and evaluated for the presence of the PEMT SNP rs12325817. Genotypes was measured by real time polymerase chain reaction (RT-PCR). The magnitude of changes in choline pool size as measured by IER at the end of each dietary intervention was compared among subjects with different genotypes in the PEMT SNP. Linear mixed model with repeated measures was performed for each group (healthy males, pre- and postmenopausal females) separately to study the genotype effect and genotype x diet interaction effect on choline pool size.	24 hours following administration of choline-d9 on day 12 of respective dietary intervention
Primary	Change in Liver Fat Content Based on CAP Values	Controlled attenuation parameter (CAP) as measured by Fibroscan is an ultrasound-based technique to measure liver fat. This method will be used with other biomarkers to indicate functional signs of choline status.	Day 1 and Day 15 of respective Cho diet
Secondary	Validation of Isotope Dilution Method to Assess Choline Pool Size by Magnetic Resonance Spectroscopy (MRS)	MRS is a direct measurement of liver choline content. Changes in liver choline by MRS should correlate with changes in liver choline inferred by calculation of isotope enrichment ratio (IER) of plasma metabolites. Pearson correlation coefficient used to study the correlation between data generated from the two types of measurements.	At the end of 2 weeks of respective Cho diet