Evaluation of Calcium, Vitamin D, Magnesium, and Zinc Absorption in Children

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.