A Physiological Study to Determine the Enteral Threonine Requirements in Infants Aged 1 to 6 Months

Brief Title

A Physiological Study to Determine the Enteral Threonine Requirements in Infants Aged 1 to 6 Months

Official Title

A Physiological Study to Determine the Enteral Threonine Requirements in Infants Aged 1 to 6 Months

Brief Summary

      This is the 5th in a series of physiological studies to determine the amino acid requirements
      of infants. There have been 4 studies to determine tyrosine, methionine, threonine and lysine
      requirements in infants when they are fed by parenterally (intravenously). Due to the new
      requirements of Health Canada for preparation of parenteral solutions, the investigators are
      starting the phase of the study that determines the enteral (oral) intake of threonine in 1 -
      6 mo infants in the interim.
    

Detailed Description

      INTRODUCTION

      Threonine is an indispensable AA that must come from dietary sources. It is critical in the
      production of mucins in the gut (Law, Bertolo et al. 2007) and contributes significantly to
      collagen, elastin and tooth enamel formation (Kramsch, Franzblau et al. 1971, Robinson, Lowe
      et al. 1975). The current adequate intake (AI) for threonine in infants proposed by the
      Institute of Medicine, Food and Nutrition Board, in the 2005 Dietary Reference Intakes (DRIs)
      for macronutrients is based on a factorial approach due to the lack of conclusive empirical
      data. Studies that lead to the determination of amino acid (AA) requirements for infants used
      the nitrogen analysis technique and were performed over 20 years ago (Snyderman, Norton et
      al. 1959). Methodological advances have made it possible to determine AA requirements in
      humans with a more precise technique (Zello, Wykes et al. 1995). However, no studies of
      threonine requirement in infants (1- 6mo of age) with the use of the newer stable-isotope
      methods have been reported.

      To accurately determine amino acid requirements, it is necessary to provide a range of amino
      acid intakes. For ethical reasons, infants cannot be fed very low or high test amino acid
      diets for prolonged periods of time. The minimally invasive IAAO method, initially developed
      in adults in our laboratory (Zello, Pencharz et al. 1993) and now used internationally
      (Huang, Hogewind-Schoonenboom et al. 2011) to determine amino acid requirements, overcomes
      this problem. The IAAO method takes less than 24 hours to conduct and has been used safely in
      parenterally fed neonates, enterally fed neonates, children and adults.

      Our laboratory determined the enteral requirement of threonine in piglets (Bertolo, Chen et
      al. 1998) which was 0.42g/kg/d. We are able to extrapolate from this data, and propose that
      the enteral requirement for neonates would be 84 mg/kg/d. In this study we will be
      determining the enteral requirement for the 1 - 6 months old infant and predict that since
      growth slows and protein requirement decreases after the first month of life, the enteral
      requirement of infants will be similar to the DRI's AI of 73 mg/kg/d. Thus, the objective of
      this study is to determine the enteral Threonine requirement in infants from 1 to 6 months of
      age with the IAAO method.

      SUBJECTS AND METHODS

      Subjects: A total of 18 studies will be performed where participants will be randomized to 1
      of 18 test threonine intakes. Infants between the ages of 1 and 6 months will be recruited
      from the in-patient post-surgical population of the Hospital for Sick Children (SickKids),
      Toronto, Canada. Participants may be eligible to participate in more than one study intake
      following enrolment, therefore being randomized to a second threonine intake. All study
      procedures and consent forms will have the approval of the research ethics board at SickKids.
      Permission to enroll the infants in the study will be obtained from the attending surgeon and
      written informed consent will be obtained from one or both parents/guardians. Recruitment of
      patients is expected to begin in December 2015. The study is registered with
      clinicaltrials.gov # NCT02364843.

      Infants will be included in the study if they are between the ages of 1 to 6 months, were
      born between 32 weeks and 43 weeks gestational age, are clinically stable as determined by
      normal blood values and vital signs, and are in-patients who are being fed via nasogastric
      (NG-), jejunal (NJ- or J-), or gastric (G-) feeding tube. Infants will be excluded if they
      are receiving supplemental oxygen, are mechanically ventilated, have any endocrine or genetic
      anomalies which would influence protein and AA metabolism, or are receiving medication which
      would influence protein and AA metabolism (eg. Corticosteroid therapy).

      Experimental design and study diet

      The study design is the minimally invasive IAAO design, which is based on the concept that,
      because AA's are not stored in the body, the intake of dietary essential AAs relative to
      their use for protein synthesis determines whether they are oxidized or incorporated into
      protein. When the intake of one indispensable AA is restricted, the other indispensable AAs
      are in excess and are oxidized because they cannot be incorporated into protein as previously
      described (Zello, Pencharz et al. 1993). Each study will take place over an 18 - 24 hour
      period following confirmation from the infant's clinical dietitian that an appropriate
      enteral intake of protein and non-protein energy from the infant's feeds has been delivered
      over 24 to 48 hours preceding the study day. On the one study day (the duration which will be
      less than 24 hours; see Fig. 1), the infant will receive a formula made from a commercial
      protein free enteral formula (Pro-Phree) which supplies a known amount of carbohydrate, fat,
      minerals, trace elements and vitamins appropriate for an infant and to which an AA mixture
      will be added. Amino acids will be added in the pattern of breast milk as follows in
      mg·kg-1·d-1: Lys 207, Met 48, Leu 288, Ile 165, Val 165, Phe 126, Try 51, His 63, Tyr 156,
      Arg 69, Pro 240, Cys 51, Gly 69, Ala 114, Ser 150, Asp 270, Glu 534 (WHO 2007). Each infant
      will receive a different intake of Threonine per study and the 18 intakes for the 18 infants
      will fall on a continuum between 15 and 130 mg·kg-1·d-1. As the intake of threonine is
      varied, the formulas will be maintained as isonitrogenous by varying intakes of alanine which
      is a dispensable AA. The amino acid mixture will be put through a 0.22 µ filter to ensure it
      is sterile and pathogen free. This is an added safety step that is not done with commercial
      formulas but was done with our parenteral solutions when studying Threonine and Lysine
      requirements in parenterally fed infants (Chapman 2009, 2010). Combining the AA mixture and
      the commercially prepared Pro-Phree formulation will be done in the hospital's Formula Room
      under their standard operating procedure (SOP) guidelines for clinical formula preparation.
      The infants will receive the study formula via their feeding tube, or a combination of tube
      feeds and oral feeds, over the 18 - 24 hour study period as ordered by the clinical
      dietician.

      A research-grade stable isotope tracer, manufactured by Cambridge Isotope Laboratories (CIL),
      Maine, USA, will be used to measure oxidation or protein deposition in response to the
      varying intakes of threonine. The stable isotope is an amino acid is L-[1-13C]Phenylalanine,
      and is administered with the infant's feeds at a dose of 15 µ·kg-1/h-1. This stable isotope
      is found in all humans as 1% of the total percentage of the amino acid phenylalanine and the
      stable isotope itself has been used successfully and without harm when given intravenously to
      infants in 4 previous studies conducted at The Hospital for Sick Children, Toronto Canada
      (Roberts 2001; Courtney Martin 2008; Chapman 2009, 2010).

      We are using a study protocol that provides sufficient time for the infant to reach a steady
      state with the stable 1-13CPhenylalanine isotope above baseline values as determined by our
      infant parenteral studies (Chapman 2009, 2010). We will conduct the first two studies using
      only the phenylalanine stable isotope. If on analysis, we find that we do not see sufficient
      difference between the baseline and steady state levels of the 1-13CPhenylalanine stable
      isotope, then we will introduce a one-time dose of 13C-sodium bicarbonate (NaH13CO2) at 14
      μmol kg-1 administered by NG/G tube to permit us to see a differentiation between the
      infant's naturally occurring 1-13CPhenylalanine and the amount we administer with the
      formula. We do not believe we will need to use the bicarbonate isotope but have added to our
      protocol for approval should its use become necessary. It is used routinely by a group of
      scientists in the Netherlands who also study infant amino acid requirements and who have not
      reported any issues with its administration (Hogewind-Schoonenboom 2014)

      The stable isotope tracers are labelled "For Research Use Only" and are acceptable for use in
      humans of all ages which will be confirmed by the Isotope manufacturing company who will
      provide a Certificate of Analysis with testing for chemical purity, presence, if any, of
      heavy metals (in parts per million) and microbiological testing including pyrogens. Both the
      isotopes will be prepared in the Diet Kitchen of the hospital at the time of preparation of
      the study formula under the same SOP guidelines as clinical diets.

      Sample collection and analysis

      Breath samples and urine will be collected to determine changes in the infants' response to
      the study diet. Three baseline breath and urine samples will be collected prior to the
      introduction of the study diet. Four plateau breath and urine samples will be collected every
      hour beginning 12-18 hours after the introduction of the Research grade stable
      isotope-labeled tracers. Breath samples will be collected using a Carbon Dioxide Analyzer
      Cart which is similar in design and use as a clinical test device called a Calorimeter. Both
      devices use a vented hood system under which the infants sleep or rest and VCO2 is measured.
      Each breath sample will be collected over a 10 minute period by bubbling the breath effluent
      into a reflux condenser and trapping the breath in a test tube. The breath samples will be
      measured by a continuous flow isotope ratio mass spectrometer. Urine samples will be
      collected by placing cotton balls in the infant's diapers and analyzed on a tandem mass
      spectrometer.

      Statistical analysis

      The effect of threonine intake on phenylalanine flux, oxidation, and F13CO2 will be tested by
      using analysis of variance with the PROC GLM procedure (SAS version 9.4). Estimates of the
      mean threonine intake will be derived by breakpoint analysis of the rate of release of 13CO2
      (F13CO2) data with the use of a 2-phase linear regression crossover model. The breakpoint
      will be calculated using the mixed models and regression procedure of SAS with the slope of
      the line not being significantly different from zero. Statistical significance will be
      established at P< 0.05.

      Regression analysis variables are threonine as the independent variable and F13CO2 and
      phenylalanine oxidation as the dependent variables. Selection of the best model will be
      determined by factors relating to fit (significance of the model and r2) and estimates of
      variation about the model (CV and SE of the estimate). The population Recommended Dietary
      Allowance will be estimated by determining the upper 95% confidence limits of the breakpoint
      estimate.
    


Study Type

Interventional


Primary Outcome

Effect of increasing threonine intakes on breath F13CO2 in enterally fed infants ages 1 - 6 months


Condition

Surgical Infants on NG/NJ/J/G-tube Feeds

Intervention

Amino Acid: Threonine

Study Arms / Comparison Groups

 Dietary Threonine Intake
Description:  Physiological establishment of enteral intake of amino acid threonine required by infants ages 1 - 6 mos

Publications

* Includes publications given by the data provider as well as publications identified by National Clinical Trials Identifier (NCT ID) in Medline.

Recruitment Information


Recruitment Status

Dietary Supplement

Estimated Enrollment

18

Start Date

February 2016

Completion Date

September 2021

Primary Completion Date

September 2021

Eligibility Criteria

        Inclusion Criteria:

          -  Between ages 1 - 6 months

          -  Born between 32 and 43 weeks gestational age

          -  Clinically stable (normal vital signs and blood values)

          -  Fed by nasogastric (NG), jejunal (J or NJ), or gastric (G) tube

        Exclusion Criteria:

          -  Infants will be excluded if they are receiving supplemental oxygen

          -  Mechanically ventilated

          -  Have any endocrine or genetic anomalies that affect protein or AA metabolism

          -  On medications that influence protein and AA metabolism (ex: corticosteroid therapy)
      

Gender

All

Ages

1 Month - 6 Months

Accepts Healthy Volunteers

No

Contacts

Lisa Goos, PhD, , 

Location Countries

Canada

Location Countries

Canada

Administrative Informations


NCT ID

NCT02364843

Organization ID

1000048977


Responsible Party

Principal Investigator

Study Sponsor

The Hospital for Sick Children


Study Sponsor

Lisa Goos, PhD, Study Chair, Research Institute, Hospital for Sick Children


Verification Date

October 2020