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Choline Nutritional Status: Development of a Biomarker Panel

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ClinicalTrials.gov Identifier: NCT03726671
Recruitment Status : Recruiting
First Posted : October 31, 2018
Last Update Posted : December 13, 2019
Sponsor:
Collaborator:
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Information provided by (Responsible Party):
University of North Carolina, Chapel Hill

Tracking Information
First Submitted Date  ICMJE October 25, 2018
First Posted Date  ICMJE October 31, 2018
Last Update Posted Date December 13, 2019
Actual Study Start Date  ICMJE November 1, 2018
Estimated Primary Completion Date March 2021   (Final data collection date for primary outcome measure)
Current Primary Outcome Measures  ICMJE
 (submitted: October 29, 2018)
  • Change in Liver Choline Pool Size by Isotope Dilution [ Time Frame: 24h following administration of choline-d9 on day 12 of each dietary intervention ]
    The liver choline pool will be determined by the dilution of the deuterated choline metabolites formed in liver and released to plasma. The fraction of plasma phosphatidylcholine-d9 (PCd9) derived from liver is expressed as the tracer to tracee ratio (TTR), and is millimoles PCd9 (tracer) divided by millimoles of unlabeled PC (tracee): TTR = PCd9/PC. The liver choline pool size is the dose of Chod9 given (2.2 mmoles) divided by the TTR. Choline pool sizes at the end of each intervention will be compared.
  • Biomarkers of Choline Status in Humans [ Time Frame: At the end of 2 weeks of 100% or 50% or 25% Cho diet ]
    Plasma choline metabolites (micromolar): choline, dimethylglycine, betaine, phosphatidylcholine, sphingomyelin, trimethylamine-oxide, and homocysteine will be measured by targeted metabolomic profiling. 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.
  • Untargeted Metabolomics to Validate Choline Status Measured by Isotope Dilution [ Time Frame: At the end of 2 weeks of 100% or 50% or 25% Cho diet ]
    Changes in metabolites measured by untargeted metabolomics methods: carnitine, butyrylcarnitine, isobutyrylcarnitine, isovaleryl-L-carnitine, propionylcarnitine, hippurate, 3-indolepropionic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid, creatinine, myo-inositol, pyridoxylate, erythronic acid, urate, pseudouridine, glutamine, pyroglutamate, valine, glutamyl-valine, glycine, leucine, trans-4-hydroxyproline, and stachydrine, will be studied. Differences in peak sizes at the end of each dietary intervention will be compared using linear mixed model. Peak sizes for an additional ~10,000 plasma and urine metabolites will also be assessed for changes associated with the interventions. Supervised Orthogonal Partial Least Squares Regression will be used to identify a panel of metabolites associated with choline pool size, and assign weight to each metabolite. The selected metabolites will be used to calculate a composite choline status score to predict choline pool size.
  • Phosphatidylethanolamine-N-methyltransferase (PEMT) Single Nucleotide Polymorphisms (SNPs) and their associations with choline pool size [ Time Frame: At the end of 2 weeks of 100% or 50% or 25% Cho diet ]
    DNA will be collected and evaluated for the presence of the various PEMT SNPs. Genotypes will be measured by RT-PCR (real time polymerase chain reaction) and a custom Illumina Expanded Multi-Ethnic genotyping array (Mega-Ex). The magnitude of changes in choline pool size at the end of each dietary intervention will be compared among subjects with different genotypes in PEMT SNPs. Linear mixed model with repeated measures will be performed for each group (healthy men, pre- and postmenopausal women) separately to study the genotype effect and genotype*intervention interaction effect on choline pool size (or choline status score). The P values from this analysis will be adjusted for multiple testing correction.
  • Validation of Fibroscan Method by Magnetic Resonance Imaging (MRI) to Assess Liver Fat Content [ Time Frame: At the end of 2 weeks of 100% or 50% or 25% Cho diet ]
    Controlled attenuation parameter (CAP) as measured by Fibroscan is an ultrasound-based technique to measure liver fat. This method will be validated by MRI where liver fat is normalized to spleen fat.
Original Primary Outcome Measures  ICMJE Same as current
Change History
Current Secondary Outcome Measures  ICMJE
 (submitted: October 29, 2018)
Validation of Isotope Dilution Method to Assess Choline Pool Size by Magnetic Resonance Spectroscopy (MRS) [ Time Frame: At the end of 2 weeks of 100% or 50% or 25% Cho diet ]
MRS is a direct measurement of liver choline content. Changes in liver choline by MRS should correlate with changes in liver choline measured by isotope dilution. Pearson correlation coefficient or non-parametric correlation analyses, such as Spearman correlation and Kendall correlation, will be used to study the correlation between data generated from the two types of measurements.
Original Secondary Outcome Measures  ICMJE Same as current
Current Other Pre-specified Outcome Measures
 (submitted: October 29, 2018)
SNPs that create inefficiencies in choline metabolism associated with change in choline pool size and choline biomarkers [ Time Frame: At baseline visit ]
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.
Original Other Pre-specified Outcome Measures Same as current
 
Descriptive Information
Brief Title  ICMJE Choline Nutritional Status: Development of a Biomarker Panel
Official Title  ICMJE Choline Nutritional Status: Development of a Biomarker Panel
Brief 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.
Detailed 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.

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

Saliva, stool, and urine samples will be collected.

Study Type  ICMJE Interventional
Study Phase  ICMJE Not Applicable
Study Design  ICMJE Allocation: Randomized
Intervention Model: Crossover Assignment
Intervention Model Description:
Healthy volunteers will consume meals, provided by the investigator, in two week intervals with 3 different levels of choline (Cho). Participants will receive 100% of the recommended daily intake (RDI) of Cho (550 mg Cho/day); 50% of the RDI of Cho (275mg/day); and 25% of the RDI of Cho (137.5 mg/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 where participants return to their regular diets. Both participants and researchers will be blinded to the diet order.
Masking: Triple (Participant, Investigator, Outcomes Assessor)
Masking Description:
Participant dietary arm assignment is randomized by a randomization plan created by the study coordinator at www.randomization.com. Each of the 3 hormonal related demographic groups (male, premenopausal females, and postmenopausal females) will have a list of the same order of diets created by the randomizer. Each participant will be assigned upon entry into the study into the next open diet for their group as ordered by the randomizer. No one collecting or processing data will be informed of the choline levels the participant is experiencing in their dietary arms at any given time to attempt to eliminate bias and ensure appropriate data collection. All staff who interact with participants or who are handling samples/data, will not be informed of the code linking dietary choline levels to diet order.
Primary Purpose: Screening
Condition  ICMJE Healthy
Intervention  ICMJE
  • 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
    Other Name: 137.5mg Choline/day
  • Other: 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
    Other Name: 275mg Choline/day
  • Other: 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
    Other Name: 550mg Choline/day
Study Arms  ICMJE
  • Experimental: 25%Cho, 50%Cho, 100%Cho
    Diets containing 137.5mg (25% Cho diet), 275mg (50% Cho diet), and 550mg (100% Cho diet) will be given in that order for two weeks each with 2 weeks of washout between.
    Interventions:
    • Other: 25% Cho diet
    • Other: 50% Cho diet
    • Other: 100% Cho diet
  • Experimental: 25% Cho, 100% Cho, 50% Cho
    Diets containing 137.5mg (25% Cho diet) , 550mg (100% Cho diet), and 275mg Cho (50% Cho diet) will be given in that order for two weeks each with 2 weeks of washout between.
    Interventions:
    • Other: 25% Cho diet
    • Other: 50% Cho diet
    • Other: 100% Cho diet
  • Experimental: 50% Cho, 25% Cho, 100% Cho
    Diets containing 275mg (50% Cho diet), 137.5mg (25% Cho diet), and 550mg Cho (100% Cho diet) will be given in that order for two weeks each with 2 weeks of washout between.
    Interventions:
    • Other: 25% Cho diet
    • Other: 50% Cho diet
    • Other: 100% Cho diet
  • Experimental: 50% Cho, 100% Cho, 25% Cho
    Diets containing 275mg (50% Cho diet), 550mg (100% Cho diet), and 137.5mg Cho (25% Cho diet) will be given in that order for two weeks each with 2 weeks of washout between.
    Interventions:
    • Other: 25% Cho diet
    • Other: 50% Cho diet
    • Other: 100% Cho diet
  • Experimental: 100% Cho, 25% Cho, 50% Cho
    Diets containing 550mg (100% Cho diet), 137.5mg (25% Cho diet), and 275mg Cho (50% Cho diet) will be given in that order for two weeks each with 2 weeks of washout between.
    Interventions:
    • Other: 25% Cho diet
    • Other: 50% Cho diet
    • Other: 100% Cho diet
  • Experimental: 100% Cho, 50% Cho, 25% Cho
    Diets containing 550mg (100% Cho diet), 275mg (50% Cho diet), and 137.5mg Cho (25% Cho diet) will be given in that order for two weeks each with 2 weeks of washout between.
    Interventions:
    • Other: 25% Cho diet
    • Other: 50% Cho diet
    • Other: 100% Cho diet
Publications * Not Provided

*   Includes publications given by the data provider as well as publications identified by ClinicalTrials.gov Identifier (NCT Number) in Medline.
 
Recruitment Information
Recruitment Status  ICMJE Recruiting
Estimated Enrollment  ICMJE
 (submitted: October 29, 2018)
180
Original Estimated Enrollment  ICMJE Same as current
Estimated Study Completion Date  ICMJE March 2021
Estimated Primary Completion Date March 2021   (Final data collection date for primary outcome measure)
Eligibility Criteria  ICMJE

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 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
  • 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)
  • have been screened for this study between August and March and have not provided proof of flu vaccination prior to enrollment
Sex/Gender  ICMJE
Sexes Eligible for Study: All
Ages  ICMJE 17 Years to 70 Years   (Child, Adult, Older Adult)
Accepts Healthy Volunteers  ICMJE Yes
Contacts  ICMJE
Contact: Julie M. Stegall, MSW 704-250-5048 julie_stegall@unc.edu
Contact: Isis Trujillo, PhD 704-250-5041 isis_trujillo@unc.edu
Listed Location Countries  ICMJE United States
Removed Location Countries  
 
Administrative Information
NCT Number  ICMJE NCT03726671
Other Study ID Numbers  ICMJE 17-1982
1R01DK115380-01 ( U.S. NIH Grant/Contract )
Has Data Monitoring Committee No
U.S. FDA-regulated Product
Studies a U.S. FDA-regulated Drug Product: No
Studies a U.S. FDA-regulated Device Product: No
IPD Sharing Statement  ICMJE Not Provided
Responsible Party University of North Carolina, Chapel Hill
Study Sponsor  ICMJE University of North Carolina, Chapel Hill
Collaborators  ICMJE National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Investigators  ICMJE
Principal Investigator: Steven H. Zeisel, MD, PhD UNC Chapel Hill - Nutrition Research Institute
PRS Account University of North Carolina, Chapel Hill
Verification Date December 2019

ICMJE     Data element required by the International Committee of Medical Journal Editors and the World Health Organization ICTRP