Combined choline and DHA supplementation: a randomized controlled trial

  • Wolfgang BernhardEmail author
  • Katrin Böckmann
  • Christoph Maas
  • Michaela Mathes
  • Julia Hövelmann
  • Anna Shunova
  • Verena Hund
  • Erwin Schleicher
  • Christian F. Poets
  • Axel R. Franz
Original Contribution



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.


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



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





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.

Author contributions

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.


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

Compliance with ethical standards

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.

Supplementary material

394_2019_1940_MOESM1_ESM.docx (40 kb)
Supplementary material 1 (DOCX 40 KB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Wolfgang Bernhard
    • 1
    Email author
  • Katrin Böckmann
    • 1
  • Christoph Maas
    • 1
  • Michaela Mathes
    • 1
  • Julia Hövelmann
    • 1
  • Anna Shunova
    • 1
  • Verena Hund
    • 3
  • Erwin Schleicher
    • 4
  • Christian F. Poets
    • 1
  • Axel R. Franz
    • 1
    • 2
  1. 1.Department of NeonatologyUniversity HospitalTübingenGermany
  2. 2.Center for Pediatric Clinical StudiesUniversity Children’s HospitalTübingenGermany
  3. 3.University Pharmacy DepartmentEberhard-Karls-UniversityTübingenGermany
  4. 4.Department of Internal Medicine IVEberhard-Karls-UniversityTübingenGermany

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