Vitamin K Supplementation Study in Healthy Volunteers (Aim 2B)

The overall purpose of this study is to determine how variation in the CYP4F2 gene modulates the synthesis of vitamin K-dependent clotting factors. We propose that the CYP4F2*3 gene variant increases short- and long-term vitamin K concentrations in the liver by reducing the efficiency of vitamin K metabolism. The investigators will study the effect of vitamin K supplementation on two biomarkers of hepatic vitamin K concentration in groups with defined CYP4F2*3 genotype. Specifically, the investigators will test for an association between our novel biomarkers of long-term (plasma Factor II proteoforms) and short-term (urinary K-Acid catabolites) hepatic vitamin K concentration and CYP4F2*3 following a 10-day period of vitamin K supplementation in healthy volunteers.

Study Overview

• Status: Terminated
• Conditions: Vitamin K Status
• Intervention / Treatment: Genetic: Phase I, Buccal Swab Collection for DNA Isolation; Dietary supplement: Phase II, Vitamin K (Vitacost) Supplementation

Detailed Description

The investigators will study the effect of vitamin K supplementation on two biomarkers of hepatic vitamin K concentration in groups with defined CYP4F2*3 genotype. Specifically, the investigators will test for an association between our novel biomarkers of long-term (plasma Factor II proteoforms) and short-term (urinary K-Acid catabolites) hepatic vitamin K concentration and CYP4F2*3 following a 10-day period of vitamin K supplementation in healthy volunteers.

The investigators will recruit, by posted advertisements at UWMC, male and female healthy volunteers. A two-step selection process will be employed. Subjects will self-select by responding to flyers, and contact the research coordinator. The research coordinator will screen them for eligibility over the phone and if eligible, make an appointment for the first study visit. For the first phase, the Research Coordinator will collect a buccal swab of DNA from ~ 200 eligible candidates; the DNA will be tested for absence or presence of the CYP4F2*3 variant allele. The PI and Research Coordinator will review the genotyping results and then select 14 individuals with either a homozygous CYP4F2*3 genotype or a heterozygous CYP4F2*1/*3 genotype, and a demographically matched group of 14 with a homozygous CYP4F2*1 genotype at the diagnostic locus for the supplementation phase of the study. The study coordinator will then contact these subjects by phone for Phase II participation.

Procedures - Phase I, Buccal Swab Collection for DNA Isolation.

Demographic Questionnaire: Self-reported heritage, age, and sex will be collected through a brief demographic questionnaire.

Buccal Swab: We will collect cheek cells with a cotton swab and isolate DNA and test for the CYP4F2*1 and CYP4F2*3 alleles.

Genotyping: CYP4F2 genotype will be determined by a validated TaqMan assay (ABI/ThermoFisher Scientific), using commercially available DNA hybridization probes to test for the absence or presence of the CYP4F2*3 allele. Subjects who are either a homozygous CYP4F2*3 genotype or a heterozygous CYP4F2*1/*3 genotype, and CYP4F2*3 alleles will be eligible for Phase II.

Procedures - Phase II, Vitamin K Supplementation.

Vitamin K Supplementation: Research participants selected for the supplementation study based on CYP4F2 genotype will be given 1-mg/day phylloquinone (Vitacost; Natures Life K-1 Phylloquinone) for 10 consecutive days. Each dose will be taken in the morning (~ 8 am), with one half pint of 2% milk to facilitate absorption.

Sample Collection: For the supplementation study, a venous blood sample (10 mL EDTA tube) and a spot urine sample will be collected after an overnight fast from d1 through d5 and on d8 and d10 of Vitamin K supplementation for 10 days. Plasma will be isolated and both samples will be stored at -70°C until analysis. Women of child-bearing potential will have a urine pregnancy test of d1 and if positive, will be withdrawn from the study, and all samples and data will be destroyed.

Measurement of Plasma Factor II Proteoforms: Plasma concentration of the 11 individual proteoforms of Factor II will be measured byLC-MS/MS. This data will be used to calculate three metrics of long-term vitamin K status, unFII, ucFII:iFII and inFII:aFII. These represent different mathematical measures of the degree of Factor II undercarboxylation. Prospective analysis of plasma vitamin K1, PIVKA II and ucOC is not planned, but will be held in reserve should the need for that data arise.

Measurement of Urinary K-Acid I and II: Quantitation of K-Acid I and II, and creatinine (Cr) in spot urine samples will be performed, using a validated LC-MS/MS assay that the investigators have recently developed. The total and individual urinary K-Acid/Cr ratio will be computed.

Statistical Analysis Plan: The investigators will conduct an unpaired t-test to compare the mean response to vitamin K supplementation in the two CYP4F2 genotype groups. Response is defined as the area under the vitamin K status value x time curve, corrected for baseline, for the 10-day intervention period. A secondary metric will be the absolute peak change in the vitamin K status ratio. Both short (urinary K-Acid/Cr ratio) and long-term (ucFII:iFII and inFII:aFII) biomarkers will tested separately. Based on pilot data, a sample size of 14 in each genotype group will provide a power of at least 80% to detect a significant effect at the significance 0.05 level, when there is a two-fold difference in the response to supplementation.

• Study Type: Interventional
• Enrollment (Actual): 106
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Kenneth E Thummel, PhD; Phone Number: 206-543-0819; Email: thummel@uw.edu

Study Locations

• United States
  • Washington
    • Seattle, Washington, United States, 98195
      • University of Washington

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years and older (Adult, Older Adult)
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

1. Participants will be males or females 18 years and older.
2. Participants must read and understand English.
3. Participants must be able to provide informed consent.
4. Women not currently pregnant or lactating.

Exclusion Criteria:

1. Participants less than 18 years of age.
2. Participants unable to read and understand English.
3. Participants unable to provide informed consent.
4. Women who are pregnant or lactating.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Basic Science
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: None (Open Label)

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Phase I, Buccal Swab Collection for DNA Isolation; Approximately 200 healthy volunteers recruited. They complete a brief demographic survey and a undergo one-time buccal swab for collection of cheek cells for DNA analysis.	Genetic: Phase I, Buccal Swab Collection for DNA Isolation; Buccal swab will be taken from study participants for DNA isolation.
Experimental: Phase II, Vitamin K (Vitacost) Supplementation; Subjects from Phase I with a homozygous CYP4F2*1 (n=14) or CYP4F2*3 carriers (n=14) are selected to receive daily vitamin K supplementation, for 10-days. Blood and urine samples are collected sequentially, at baseline, and during the supplementation period.	Dietary supplement: Phase II, Vitamin K (Vitacost) Supplementation; 1-mg/day phylloquinone (Vitacost; Natures Life K-1 Phylloquinone) for 10 consecutive days. Each dose will be taken in the morning (~ 8 am), with one half pint of 2% milk to facilitate absorption. Blood and urine samples collection before and during the vitamin K supplementation period.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Short-term Hepatic Vitamin K Status-1	urinary vitamin K-acid/creatinine ratio	10-days
Long-term Hepatic Vitamin K Status	plasma factor II carboxylation state	10-days

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Short-term Hepatic Vitamin K Status-2	Plasma vitamin K acid concentration	10-days

Collaborators and Investigators

• Sponsor: University of Washington
• Collaborators: National Institute of General Medical Sciences (NIGMS)
• Investigators: Principal Investigator: Kenneth E Thummel, PhD, University of Washington

Study record dates

Study Major Dates

• Study Start (Actual): August 25, 2017
• Primary Completion (Actual): July 31, 2023
• Study Completion (Actual): July 31, 2023

Study Registration Dates

• First Submitted: June 22, 2020
• First Submitted That Met QC Criteria: June 25, 2020
• First Posted (Actual): June 29, 2020

Study Record Updates

• Last Update Posted (Estimated): December 19, 2023
• Last Update Submitted That Met QC Criteria: December 18, 2023
• Last Verified: December 1, 2023

More Information

Terms related to this study

• Keywords: pharmacogenetics; vitamin K; blood clotting factors; CYP4F2
• Additional Relevant MeSH Terms: Physiological Effects of Drugs; Molecular Mechanisms of Pharmacological Action; Fibrin Modulating Agents; Micronutrients; Vitamins; Antifibrinolytic Agents; Hemostatics; Coagulants; Vitamin K
• Other Study ID Numbers: STUDY00001593; P01GM116691-02S1 (U.S. NIH Grant/Contract)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No