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      Suitability and safety of L-5-methyltetrahydrofolate as a folate source in infant formula: A randomized-controlled trial

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          Abstract

          L-5-methyltetrahydrofolate is the predominant folate form in human milk but is currently not approved as a folate source for infant and follow-on formula. We aimed to assess the suitability of L-5-methyltetrahydrofolate as a folate source for infants. Growth and tolerance in healthy term infants fed formulae containing equimolar doses of L-5-methyltetrahydrofolate (10.4 μg/ 100 ml, n = 120, intervention group) or folic acid (10.0 μg/ 100 ml, n = 120, control group) was assessed in a randomized, double-blind, parallel, controlled trial. A reference group of breastfed infants was followed. Both formulae were well accepted without differences in tolerance or occurrence of adverse events. The most common adverse events were common cold, poor weight gain or growth, rash, eczema, or dry skin and respiratory tract infection. Weight gain (the primary outcome) was equivalent in the two groups (95% CI -2.11; 1.68 g/d). In line with this, there was only a small difference in absolute body weight adjusted for birth weight and sex at visit 4 (95% CI -235; 135 g). Equivalence was also shown for gain in head circumference but not for recumbent length gain and increase in calorie intake. Given the nature of the test, this does not indicate an actual difference, and adjusted means at visit 4 were not significantly different for any of these parameters. Infants receiving formula containing L-5-methyltetrahydrofolate had lower mean plasma levels of unmetabolized folic acid (intervention: 0.73 nmol/L, control: 1.15 nmol/L, p<0.0001) and higher levels of red cell folate (intervention: 907.0 ±192.8 nmol/L, control: 839.4 ±142.4 nmol/L, p = 0.0095). We conclude that L-5-methyltetrahydrofolate is suitable for use in infant and follow-on formula, and there are no indications of untoward effects.

          Trial registration: This trial was registered at ClinicalTrials.gov ( NCT02437721).

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          A comparison of the two one-sided tests procedure and the power approach for assessing the equivalence of average bioavailability.

          The statistical test of hypothesis of no difference between the average bioavailabilities of two drug formulations, usually supplemented by an assessment of what the power of the statistical test would have been if the true averages had been inequivalent, continues to be used in the statistical analysis of bioavailability/bioequivalence studies. In the present article, this Power Approach (which in practice usually consists of testing the hypothesis of no difference at level 0.05 and requiring an estimated power of 0.80) is compared to another statistical approach, the Two One-Sided Tests Procedure, which leads to the same conclusion as the approach proposed by Westlake based on the usual (shortest) 1-2 alpha confidence interval for the true average difference. It is found that for the specific choice of alpha = 0.05 as the nominal level of the one-sided tests, the two one-sided tests procedure has uniformly superior properties to the power approach in most cases. The only cases where the power approach has superior properties when the true averages are equivalent correspond to cases where the chance of concluding equivalence with the power approach when the true averages are not equivalent exceeds 0.05. With appropriate choice of the nominal level of significance of the one-sided tests, the two one-sided tests procedure always has uniformly superior properties to the power approach. The two one-sided tests procedure is compared to the procedure proposed by Hauck and Anderson.
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            A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity.

            A common mutation in methylenetetrahydrofolate reductase (MTHFR), C677T, results in a thermolabile variant with reduced activity. Homozygous mutant individuals (approximately 10% of North Americans) are predisposed to mild hyperhomocysteinemia, when their folate status is low. This genetic-nutrient interactive effect is believed to increase the risk for neural tube defects and vascular disease. In this communication, we characterize a second common variant in MTHFR (A1298C), an E to A substitution. Homozygosity was observed in approximately 10% of Canadian individuals. This polymorphism was associated with decreased enzyme activity; homozygotes had approximately 60% of control activity in lymphocytes. Heterozygotes for both the C677T and the A1298C mutation, approximately 15% of individuals, had 50-60% of control activity, a value that was lower than that seen in single heterozygotes for the C677T variant. No individuals were homozygous for both mutations. Additional studies of the A1298C mutation, in the absence and presence of the C677T mutation, are warranted, to adequately address the role of this new genetic variant in complex traits. A silent genetic variant, T1317C, was identified in the same exon. It was relatively infrequent (allele frequency 5%) in our study group, but was quite common in a small sample of African individuals (allele frequency 39%). Copyright 1998 Academic Press.
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              Geographical and ethnic variation of the 677C>T allele of 5,10 methylenetetrahydrofolate reductase (MTHFR): findings from over 7000 newborns from 16 areas world wide.

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                Author and article information

                Contributors
                Role: Project administrationRole: SupervisionRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Project administrationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Project administrationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: MethodologyRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: Project administrationRole: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                19 August 2019
                2019
                : 14
                : 8
                : e0216790
                Affiliations
                [1 ] DSM Nutritional Products Ltd., Kaiseraugst, Switzerland
                [2 ] LMU -Ludwig-Maximilians Universität Munich, Dr von Hauner Children’s Hospital, Munich, Germany
                [3 ] HiPP GmbH & Co. Vertrieb KG, Pfaffenhofen, Germany
                [4 ] Clinical Hospital Center "Dr Dragiša Mišović-Dedinje", Belgrade, Serbia
                [5 ] Charité- Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany
                [6 ] Berlin Institute of Health, KKS Charité, Berlin, Germany
                [7 ] HiPP Study Center, Belgrade, Serbia
                University of Ghana, GHANA
                Author notes

                Competing Interests: BT and RS are employees of DSM Nutritional Products Ltd., MG, CH, and BT are employees of Hipp GmbH & Co. BT and RS are employed by DSM Nutritional Products Ltd., MG and CH are employed by HiPP GmbH & Co and BRT is employed by the HiPP Study center. We declare our adherence to PLOS ONE policies on sharing data and materials and are open to sharing data for research purposes upon request, under conditions respecting the EU General Data Protection Regulation and the protection of personal rights of study subjects.

                ¶ Membership of the MEFOLIN Study Group is provided in the Acknowledgments.

                Author information
                http://orcid.org/0000-0002-5345-7165
                Article
                PONE-D-18-29025
                10.1371/journal.pone.0216790
                6699731
                31425504
                a7327484-b4f8-404e-8658-35296e34349e
                © 2019 Troesch et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 October 2018
                : 19 March 2019
                Page count
                Figures: 4, Tables: 4, Pages: 18
                Funding
                Funded by: DSM
                Award Recipient :
                Funded by: DSM
                Award Recipient :
                Funded by: DSM
                Award Recipient :
                Funded by: DSM
                Award Recipient :
                Funded by: DSM
                Award Recipient :
                Funded by: DSM
                Award Recipient :
                Funded by: Hipp GmbH
                Award ID: provision of study intervention
                DSM provided financial support for the research reported here, and the study was also financially supported in part by HiPP & Co. Vertrieb KG. The study formulae were provided free of charge by HiPP GmbH & Co. Vertrieb KG. The funders of the study, DSM and Hipp, provided support in the form of salaries for authors BT, RS, MG, CH and BT, but did not have any additional role in the study data collection and analysis or decision to publish. The specific roles of these authors are articulated in the ‘author contributions’ section.
                Categories
                Research Article
                Physical Sciences
                Chemistry
                Chemical Compounds
                Organic Compounds
                Vitamins
                B Vitamins
                Folic Acid
                Physical Sciences
                Chemistry
                Organic Chemistry
                Organic Compounds
                Vitamins
                B Vitamins
                Folic Acid
                People and Places
                Population Groupings
                Age Groups
                Children
                Infants
                People and Places
                Population Groupings
                Families
                Children
                Infants
                Biology and Life Sciences
                Physiology
                Physiological Parameters
                Body Weight
                Weight Gain
                Medicine and Health Sciences
                Physiology
                Physiological Parameters
                Body Weight
                Weight Gain
                Research and Analysis Methods
                Research Design
                Clinical Research Design
                Adverse Events
                Biology and Life Sciences
                Nutrition
                Diet
                Beverages
                Milk
                Breast Milk
                Medicine and Health Sciences
                Nutrition
                Diet
                Beverages
                Milk
                Breast Milk
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Milk
                Breast Milk
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Milk
                Breast Milk
                Biology and Life Sciences
                Physiology
                Body Fluids
                Milk
                Breast Milk
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Milk
                Breast Milk
                Biology and Life Sciences
                Physiology
                Physiological Parameters
                Body Weight
                Birth Weight
                Medicine and Health Sciences
                Physiology
                Physiological Parameters
                Body Weight
                Birth Weight
                Biology and Life Sciences
                Physiology
                Physiological Parameters
                Body Weight
                Medicine and Health Sciences
                Physiology
                Physiological Parameters
                Body Weight
                Biology and Life Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Anatomy
                Body Fluids
                Blood
                Blood Plasma
                Biology and Life Sciences
                Physiology
                Body Fluids
                Blood
                Blood Plasma
                Medicine and Health Sciences
                Physiology
                Body Fluids
                Blood
                Blood Plasma
                Custom metadata
                Anonimized data can be made available by the authors upon request provided that accountable assurance is provided on strict compliance with protection of personal data of the human subjects included. The ethical committee of the Medical Faculty of LMU (Contact Dr. Beate Henrikus, Beate.Henrikus@ 123456med.uni-muenchen.de ) and the Data Protection Officer of the LMU Medical Center (Gerhard Meyer, datenschutz@ 123456med.uni-muenchen.de ) demand that personal data protection of study participants are ensured in line with the European General Data Protection Regulation, and that study database that could allow potential identification of study participants, as is the case in our data set, shall not be put in the public domain. Rather, upon request by credible researchers after signature of a written agreement ensuring personal data right protection those data shall be shared that are required for the proposed secondary research. Request for data use should be sent to the Director of the Paediatric Clinical Study Center at Univ. of Munich Medical Center, Ms. Aninna Hermann, Annina.Herrmann@ 123456med.uni-muenchen.de .

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