Combined choline and DHA supplementation: a randomized controlled trial



Choline and docosahexaenoic acid (DHA) are essential nutrients for preterm infant development. They are metabolically linked via phosphatidylcholine (PC), a constitutive plasma membrane lipid and the major transport form of DHA in plasma. Plasma choline and DHA-PC concentrations rapidly decline after preterm birth. To improve preterm infant nutrition, we evaluated combined compared to exclusive choline and DHA supplementation, and standard feeding.


Randomized partially blinded single-center trial.


Neonatal tertiary referral center in Tübingen, Germany.


24 inborn preterm infants < 32 week postmenstrual age.


Standard nutrition (control) or, additionally, enteral choline (30 mg/kg/day), DHA (60 mg/kg/day), or both for 10 days. Single enteral administration of 3.6 mg/kg [methyl-D9-] choline chloride as a tracer at 7.5 days.

Main outcome measures

Primary outcome variable was plasma choline following 7 days of supplementation. Deuterated and unlabeled choline metabolites, DHA-PC, and other PC species were secondary outcome variables.


Choline supplementation increased plasma choline to near-fetal concentrations [35.4 (32.8–41.7) µmol/L vs. 17.8 (16.1–22.4) µmol/L, p < 0.01] and decreased D9-choline enrichment of PC. Single DHA treatment decreased DHA in PC relative to total lipid [66 (60–68)% vs. 78 (74–80)%; p < 0.01], which was prevented by choline. DHA alone increased DHA-PC only by 35 (26–45)%, but combined treatment by 63 (49–74)% (p < 0.001). D9-choline enrichment showed preferential synthesis of PC containing linoleic acid. PC synthesis via phosphatidylethanolamine methylation resulted in preferential synthesis of DHA-containing D3-PC, which was increased by choline supplementation.


30 mg/kg/day additional choline supplementation increases plasma choline to near-fetal concentrations, dilutes the D9-choline tracer via increased precursor concentrations and improves DHA homeostasis in preterm infants.

Trial registration Identifier: NCT02509728.

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Adequate intake


Arachidonic acid (C20:4)


Bronchopulmonary dysplasia


Docosahexaenoic acid (C22:6)


Linoleic acid (C18:2)


Oleic acid (C18:1)


European Society of Paediatric Gastroenterology::Hepatology and Nutrition


Gestational age


Heated electrospray ionization interface


Liquid chromatography–electrospray ionization interface tandem mass spectrometry


Long-chain polyunsaturated fatty acid


Neonatal intensive care unit








Postmenstrual age


Randomized controlled trial




Specific reaction monitoring


Very low-density lipoproteins




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This study was supported by an institutional grant to Christoph Maas (project no. E.03.27032.1) of the University of Tübingen. The authors would like to thank Alisa Bernhard, Katharina Löhner-Böttcher, Anne-Sophie Meixner, Anita Spiegelberg and Emmanuelle Seyboldt-Allaire for their help in sample as well as clinical data collection, respectively.


This study was funded by an internal Grant of the Eberhard-Karls-University, Medical Faculty, Tübingen (E.03.27032.1).

Author information




ARF, CM, and WB conceptualized and designed this trial. CM obtained an institutional grant for this study, and was involved in sample collection, assessment of clinical data, manuscript revision, and approval as submitted. WB developed the analytical techniques, supervised the analyses, drafted the initial manuscript, and approved the final manuscript as submitted. AS prepared the samples, carried out the mass spectrometric, and gas chromatographic analyses. KB, CM, and ARF recruited patients, supervised the patients’ well-being, and collected the study samples. KB, MM, and JH collected and recorded clinical data, revised the manuscript and approved the final manuscript as submitted. VH prepared and controlled the choline supplement, and ES supervised the fatty acid analysis. CFP supervised the project as the head of department, critically reviewed the manuscript, and approved the final manuscript as submitted. ARF was coordinator of the project. He was responsible for concept and design of the randomized trial, contributed to patient recruitment and supervision and to sample collection and assessment of clinical data, revised the manuscript and approved the final manuscript as submitted.

Corresponding author

Correspondence to Wolfgang Bernhard.

Ethics declarations

Financial disclosure

Axel Franz has received speaker or consultant honoraria from Nestlé, Milupa, and Hipp, all marketing infant formulas. Furthermore, Axel Franz has received grants from Nestlé for the conduct of educational seminars and for the conduct of a clinical study. Furthermore, Axel Franz and Wolfgang Bernhard have received consultant honoraria from Baxter and Fresenius Kabi, all marketing components of parenteral nutrition. All the other authors indicate they have no financial relationships relevant to this article.

Conflict of interest

Based on current and previous work of this group, the University of Tuebingen, Medical Faculty, submitted a patent application for the combined administration of choline, ARA and DHA for prevention of developmental disorders associated with very preterm birth. The inventors of said patent application (WB, ARF) and all the other authors indicate that they do not have any conflict of interest to disclose.

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What is already known on this topic?

  1. 1.

    Choline and docosahexaenoic acid (DHA) are essential nutrients, with an intimate metabolic connection and tightly regulated organ concentrations in the form of phosphatidylcholine (PC).

  2. 2.

    Cellular choline uptake for PC synthesis is proportional to its plasma concentration, but in preterm infants plasma choline rapidly decreases from ~ 40 µmol/L to ~ 20 µmol/L.

  3. 3.

    PC is the major transport form of DHA in plasma, but after preterm delivery DHA-PC decreases compared to the age-matched fetus.

What this study adds

  1. 1.

    Supplementing preterm infants with 30 mg/kg/day choline, in addition to a median supply with 24 mg/kg/day, increased plasma choline from 18(16–22)µmol/L to near-fetal concentrations [35 (33–42) µmol/L].

  2. 2.

    Labeling with D9-choline indicates increased choline and choline metabolite pools in response to choline supplementation and a rapid plasma turnover of choline

  3. 3.

    Combination of docosahexaenoic acid (DHA) supplementation with choline reversed the shift of plasma DHA from neutral lipids to PC, and increased DHA-PC more than DHA alone.

  4. 4.

    Linoleic acid may be a strong competitor of DHA-PC synthesis and overall DHA homeostasis.

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Bernhard, W., Böckmann, K., Maas, C. et al. Combined choline and DHA supplementation: a randomized controlled trial. Eur J Nutr 59, 729–739 (2020).

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  • Arachidonic acid
  • Choline
  • D9-choline
  • Combined supplementation
  • Docosahexaenoic acid
  • Neonate
  • Nutrition
  • PEMT
  • Phosphatidylcholine
  • Phospholipid
  • Plasma
  • Preterm infant
  • Stable isotopes
  • Tandem mass spectrometry