Evaluation of Calcium, Vitamin D, Magnesium, and Zinc Absorption in Children
Evaluation of Calcium, Vitamin D, Magnesium, and Zinc Absorption in Children
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.
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.
Inclusion Criteria:

 -  Delivery at > or = 35.0 weeks gestation with birth weight > or = 2.5 kg

 -  BMI: 10th - 90th percentile for age and gender

 -  Ages 4.0 to 8.9 years

 -  Ethnic distribution of greater Houston area

 -  Usual calcium intake 600-1200 mg/d based on two 24-hour dietary recalls by phone

 -  Any regularly taken (daily) vitamins and minerals must be stopped at least 1 month
 before starting the study

 Exclusion Criteria:

 -  History of any chronic illness.

 -  Regularly taken (daily) medications (except seasonal allergies).

 -  Avoidance of dairy products or meat in the diet.