Evaluation of Calcium, Vitamin D, Magnesium, and Zinc Absorption in Children
Condition: Mineral Absorption
Study type: Interventional

Summary: 
      The goal of our research is to provide data to enhance the development of nutritional
      guidelines, especially as related to mineral nutrition, in children. Using human
      experimentation, we are studying methods of delivering the key minerals of calcium, zinc and
      iron in the diet so as to optimize health outcomes. We will conduct a controlled trial of
      vitamin D supplementation to assess the effects of vitamin D status on calcium absorption in
      small children. We will evaluate the effects of differing intakes of zinc on zinc and copper
      absorption. These studies will utilize stable isotope techniques so as to provide accurate,
      practically applicable information which may be obtained from the study populations in a safe
      manner. These data will have global application and provide a strong basis for evidence-based
      nutritional recommendations to be developed.

      Objective #1: To evaluate the effects of supplemental vitamin D in enhancing calcium
      absorption in healthy children 4 to 8 yrs of age.

      Objective #2: Assess the absorption of magnesium and zinc in healthy children 4 to 8 yrs of
      age.
    

Detailed description: 
      We plan to stratify subjects for randomization to either vitamin D supplements or placebo by
      serum 25-hydroxyvitamin D (25 OHD) into three groups: 0-20 ng/mL, 20-32 ng/mL, and >32 ng/mL.

      There is no group assignment or randomization for the magnesium and zinc aspects of the
      study.

      SCREENING VISIT: At the time of enrollment, subjects and their families will be asked to come
      to the General Clinical Research Center (GCRC) of Texas Children's Hospital for a screening
      visit. Prior to this visit, demographics from the parent/guardian will be recorded including
      the child's approximate height and weight, and the study dietitian will obtain two 24-hour
      dietary recalls from the parent to determine calcium intake for enrollment. During this
      screening visit, informed written consent will be obtained, a medical history taken, and a
      physical examination performed.

      The study dietitian will instruct the parent/guardian and child on the use of a food scale to
      weigh and record dietary intake for a 3-day period (ie, 3-day weighed diet record). The 3-day
      weighed diet record will begin the following day. This method has been shown to provide the
      best estimate of dietary intakes in children (Crawford PB et al, J Am Diet Assoc. 1994;
      94:626-630; Fisher JO et al, Am J Clin Nutr. 2008; 88:407-415). Upon analysis of their
      child's intake, parents will be instructed to maintain a similar nutrient intake throughout
      the study. Compliance will be monitored via 3-day weighed home diet records timed with their
      other study visits. If analysis shows that the child's intake has significantly changed (±
      20% of a nutrient), the parent will be counseled by the study dietitian on readjusting the
      child's intake back to the usual level determined at baseline.

      In addition, the study dietitian will prepare sample menus based on the child's food
      preferences and dietary intake levels and provide them to the parent. Parents can use the
      sample menus to assist them in maintaining the child's usual mineral intake.

      STUDY VISIT 1: Subjects and a parent/guardian will return to the GCRC for a 24-hour overnight
      stay, arriving in the morning for a baseline stable isotope study. We will utilize the
      dual-tracer stable isotope technique as in our previous studies. At this visit subjects will
      receive intravenous isotope doses 1 mg 42Ca, 6 mg 25Mg, and 0.4 mg 70Zn. At the time of the
      IV isotope, we will collect a blood sample (10mL for serum calcium, phosphorus, alkaline
      phosphatase, magnesium, 25OHD, 1,25-dihydroxyvitamin D, hepcidin, ferritin, TIBC, transferrin
      saturation, hemoglobin, hematocrit, and RBC indices). Topical numbing cream or spray to
      minimize pain at the injection site will be offered to all subjects.

      A dual-energy x-ray absorptiometry (DEXA) measurement of total body bone mineral
      content/density and body fat will be made. This is done to use as a covariate in evaluating
      vitamin D levels and calcium absorption.

      A 24-hr urine collection while they stay inpatient will begin with the timing of the first
      isotope. After they are discharged, subjects will be instructed on collecting another 48-hr
      urine collection (72-hr total) at home as well as a final spot urine at 96 hours after the
      first isotope.

      Subjects will receive oral isotopes (20 mcg 46Ca, 12 mg 26Mg, and 2 mg 67Zn) mixed with 120
      mL of calcium and vitamin D-fortified orange juice or milk. The breakfast will be a fixed
      meal providing a total of ~300 mg calcium. Lunch will provide ~300mg calcium; dinner will
      provide ~300mg calcium (totaling ~900mg calcium). All meals at the GCRC will be pre- and
      post-weighed to determine actual intake. After discharge, subjects will record dietary intake
      using another 3-day weighed diet record.

      Approximately one week after Study Visit 1, the results from the serum 25OHD test will be
      back and we will stratify subjects accordingly into three groups: 0-20 ng/mL, 20-32 ng/mL,
      and >32 ng/mL. Subjects will return to the CNRC to be provided supplemental vitamin D3 (1000
      IU) or placebo, given once daily (based on the stratification noted above). Vitamin D/placebo
      will be provided using a softgel or liquid. Subjects will be instructed to take the
      supplement daily for 8 weeks. They will be provided with study calendars to mark daily when
      they remember to take the supplement. Calendars and remaining supplements will be returned to
      the study center for counting to determine compliance.

      Subjects will return urine samples, food scales, and diet records to the study personnel at
      the CNRC. The CNRC driver may also be utilized for the purpose of picking up samples or
      delivering the supplement. Periodic phone calls will be made to the home of the subject to
      monitor compliance with taking the supplement.

      STUDY VISIT 2: Eight (8) weeks after the subjects began taking their supplement, subjects
      will return to the GCRC for a repeat study of absorption. Prior to this visit, subjects will
      receive a food scale and will perform another 3-day weighed diet record to demonstrate
      consistency of diet throughout the study period. Subjects will again arrive in the morning to
      the GCRC for a 24-hr inpatient study visit. They will bring with them all study calendars and
      remaining supplements for compliance monitoring. Study Visit 2 is similar in all aspects of
      Study Visit 1 regarding the calcium and vitamin D portion of the study. Magnesium and zinc
      measurements will not be repeated at Study Visit 2.

      At this visit subjects will receive an intravenous isotope dose of 1 mg 42Ca. At the time of
      the IV isotope, we will collect a blood sample (same labs as in Study Visit 1). Topical
      numbing cream or spray to minimize pain at the injection site will be offered to all
      subjects.

      A 24-hr urine collection while they stay inpatient will begin with the timing of the first
      isotope. After they are discharged, subjects will not be required to continue any additional
      urine collections.

      Subjects will receive 20 mcg 46Ca stable isotope mixed with 120 mL of calcium and vitamin
      D-fortified orange juice or milk. The breakfast will be a fixed meal providing a total of
      ~300 mg calcium. Lunch will provide ~300mg calcium; dinner will provide ~300mg calcium
      (totaling ~900mg calcium). All meals at the GCRC will be pre- and post-weighed to determine
      actual intake.

      Upon discharge, subjects will discontinue the supplementation and the study will be complete.

      In interpreting the results, we will note ethnicity, the season of measurement and
      qualitative descriptions of sun exposure by considering the amount of time spent outside.
      However, we will not specifically assess sun exposure as this is not practical in small
      children. In general, we would not expect large changes in these in Houston during the 8
      weeks of the study, but will ensure that the two studies do not cross a period of major
      change such as having the first study done before summer camp and the second right
      afterwards.

      Urine and serum samples will be prepared for mass spectrometric analysis using an oxalate
      precipitation technique. Samples will be analyzed for isotopic enrichment using a magnetic
      sector ICP-MS. This is a high-speed instrument capable of analysis of the desired ratio with
      precision and accuracy of 0.3-0.5%.

      Contingencies: We do not anticipate any problems with this study that would require changing
      or altering the protocol. Our sample size does not allow us to evaluate gender or ethnicity
      separately to determine differences in effects among these. There is no reason to expect
      differences in vitamin D effects on calcium absorption based on these (Weaver, personal
      communication). Additionally, dietary recommendations reflect the diversity of our population
      and if we identify trends towards specific ethnic or gender effects we can study this further
      in future population studies. This study will look at absorption only as an endpoint. If a
      benefit is found, we will develop a long-term trial evaluating bone mineral outcomes.
      However, this would require larger groups and a full year of study and is beyond the scope of
      this initial study. If no difference in calcium absorption is shown with 1000 IU/d of vitamin
      D, it is extremely unlikely that an effect on bone mineral would be found justifying a
      long-term study.

      Magnesium absorption studies require a full 72-hour urine collection for accuracy. It is not
      practical to keep children this age in-patient for this time so after 24 hours, collections
      will be done at home. We have extensive experience with assisting families in home urine
      collections. There is the possibility of some loss of urine in a 72-hour collection. However,
      in that case we would continue the collection as the method's accuracy is not highly
      sensitive to the loss of a single or a small number of urine specimens in small children.
      Magnesium balance is not regulated by endogenous excretion (similar to calcium) and thus the
      absorption studies will provide adequate information to estimate net balance.

      Subjects may be told their weight and height measurements at each visit to the GCRC. All
      other study related information will be held until the end of the study.
    

Intervention name(s): Vitamin D
Intervention type(s): Dietary Supplement
Intervention description(s): Subjects will be provided supplemental vitamin D3 (1000 IU) or placebo, given once daily (based on the stratification noted above). Vitamin D/placebo will be provided using a softgel or liquid. Subjects will be instructed to take the supplement daily for 8 weeks.
Intervention MeSH terms: Vitamin D
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Double (Participant, Investigator)
Primary purpose: Basic Science
Keywords: Calcium; Vitamin D; Magnesium; Zinc