Choline and betaine concentrations in plasma predict dietary choline intake in healthy humans: a double-blind randomized control feeding study

Background Choline is an essential nutrient, and insufficient intake negatively affects organs such as the liver, brain, and muscles. In the United States, average choline intake remains below the Adequate Intake (AI) (550 mg/day men, 425 mg/day women). Although conventional dietary assessment tools can identify people who are eating diets low in choline, no metabolite biomarkers have been proven to reliably assess choline intake.

We tested whether plasma concentrations of choline and its metabolites could determine dietary choline intake. We also assessed whether liver elastography (Fibroscan) could detect diet-induced changes in liver fat.

In a double-blind, randomized, crossover feeding study, participants adhered to 3 distinct dietary patterns for 2-wk intervals, delivering approximately 100%, 50%, and 25% of the choline AI. On Day 12 of each dietary arm, in addition to the food supplied, subjects consumed a single bolus of 2.2 mmol trimethyl-d9-choline. Plasma concentrations of choline, betaine and phosphatidylcholine (PtdCho) were measured using mass spectrometry. Targeted assays quantified choline, betaine, phosphatidylcholine and total homocysteine concentrations. Liver fat content was evaluated non-invasively using Fibroscan.

Plasma concentrations of d9-choline, betaine, and their isotopic enrichment ratio (IER) varied with dietary intake (q<0.0001), and PtdCho IER also differed significantly (q=0.001). In targeted analysis, choline and betaine concentrations were highly responsive to dietary choline intake, while PtdCho and tHcy were not. Receiver Operator Characteristic (ROC) analysis showed strong accuracy using plasma choline (AUC=0.81) and betaine (AUC=0.80), with improved accuracy when combined (AUC= 0.83). Fibroscan identified a subset of participants with increased liver fat in response to the 25% AI vs. 100% AI choline diet, though patterns varied among individuals.

Plasma choline and betaine concentrations are robust biomarkers of dietary choline intake under controlled feeding. These findings support targeted metabolite profiling to improve choline intake assessment and reveal induvial variability in liver response to low choline intake. Competing Interest Statement Dr. Steven H. Zeisel has equity in SNP Therapeutics, this paper is not directly related to the companys product. Dr. Isis Trujillo-Gonzalez has received research funding from Balchem Corporation, a manufacturer of choline. Dr. Evan M. Paules is a Balchem postdoctoral fellow. Balchem had no role in the design, analysis or interpretation of the study. Clinical Trial NCT03726671 Funding Statement NIH/NIDDK R01 DK115380 (SHZ), NIH/NIDDK P30 DK056350 (SHZ) Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of UNC Chapel Hill gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes Funding information NIH/NIDDK R01 DK115380 (SHZ), NIH/NIDDK P30 DK056350 (SHZ) Clinical trials registry Choline Nutritional Status: Development of a Biomarker Panel; NCT03726671 (www.clinicaltrials.gov) Data Availability All data produced in the present work are contained in the manuscript Abbreviations - ACho - acetylcholine - AI - adequate intake - ALT - alanine aminotransferase - AST - aspartate aminotransferase - CAP - controlled attenuation parameter - CK - creatine kinase - EREs - estrogen-responsive elements - FDR - false discovery rate - FSH - follicle-stimulating hormone - IER - Isotopic enrichment ratios - MS - mass spectrometry - MTHFR - methylenetetrahydrofolate reductase - PEMT - phosphatidylethanolamine N-methyltransferase - PtdCho - phosphatidylcholine - ROC - receiver operator characteristics - SNPs - single nucleotide polymorphisms - SST - serum-separating tubes - tHcy - total homocysteine