The Effect of Vitamin D and Calcium on Bone in Pediatric HIV
Condition: HIV Infection; Bone Mass
MeSH terms: HIV Infections
Study type: Interventional
Phase: Phase 2/Phase 3

Summary: 
      We have observed that vitamin D deficiency, as evidenced by low serum 25(OH)D concentrations,
      is common in children and adolescents with HIV infection. To determine whether vitamin D and
      calcium supplementation improve bone mineral content (BMC) and bone mineral density (BMD) in
      HIV-infected children and adolescents, we propose a double-blind, randomized,
      placebo-controlled trial comparing supplementation with oral vitamin D and calcium to
      placebo.

      The specific aims of this project are to:

        1. Determine the effect of vitamin D and calcium supplementation on bone mineral accrual in
           HIV-infected children.

           We hypothesize that BMC and BMD will increase to a greater extent in HIV-infected
           children who receive supplementation with vitamin D and calcium. This hypothesis will be
           tested by comparing changes in BMC and BMD, measured by dual energy x-ray absorptiometry
           (DXA), after one and two years of treatment in HIV-infected children and adolescents
           receiving vitamin D and calcium supplementation compared to those receiving placebo.

        2. Determine the effect of HIV infection and vitamin D and calcium supplementation on
           indices of mineral metabolism and markers of bone turnover.

           We hypothesize that indices of mineral metabolism and markers of bone formation and
           resorption will return toward normal in HIV-infected children and adolescents who are
           randomized to receive vitamin D and calcium supplementation. We will test these
           hypotheses by comparing longitudinal changes in indices of mineral metabolism and bone
           turnover markers in HIV-infected children and adolescents receiving vitamin D and
           calcium supplement versus those receiving placebo

        3. Evaluate if vitamin D stores are a determinant of bone mass in HIV infected children and
           adolescents receiving HAART.

      We hypothesize that vitamin D stores, as assessed by serum 25-hydroxyvitamin D levels, are an
      important determinant of bone mass in HIV-infected children and adolescents receiving HAART.
      We will test this hypothesis by evaluating whether measurements of bone mass are associated
      with vitamin D stores, as measured by serum 25-hydroxyvitamin D levels and other indices of
      mineral metabolism, in treated HAART-treated HIV-infected children and adolescents.
    

Detailed description: 
      A.1.a. Introduction This is a prospective, randomized, double-blind clinical trial designed
      to evaluate the effect of 24 months of supplementation with vitamin D3 and calcium on bone
      mass accrual in HIV-infected children and adolescents.

      A.1.b. Subject recruitment i. HIV-infected subjects on will be enrolled by Co-Investigators
      from among HIV-infected children and adolescents who receive treatment at participating sites
      who meet inclusion and exclusion criteria.

      A.1.c. Measurements:

      Blood for measurement of serum 25(OH)D levels will be obtained from all HIV-infected children
      and adolescents by the participating clinical sites. Samples will be analyzed and the results
      known in advance of the cross-sectional study visit. Serum 25(OH)D levels will be used to
      select participants. The following measures will be performed:

      Medical history, including medications Pregnancy test for females of child bearing capacity
      Height and weight Assessment of pubertal stage Assessment of dietary intake of calcium and
      vitamin D Assessment of physical activity Determination of total body bone mineral content
      (TBBMC), total body bone mineral density (TBBBMD), spinal BMC and BMD, and bone age
      radiograph Fasting morning blood draw for indices of mineral metabolism: serum calcium,
      phosphate, albumin, total alkaline phosphatase activity, 25(OH)D, 1,25(OH)2D, iPTH, urinary
      calcium and creatinine.

      Markers of bone turnover: Serum N-telopeptide (N-tx, a marker of resorption), bone specific
      alkaline phosphatase (BALP) and osteocalcin (markers of formation) These measures will be
      performed in the SLRHC Pediatric Body Composition Unit.

      A.2.a. Study design:

      The double-blind, randomized trial will compare the effect of 24 months of supplementation
      with vitamin D3 and calcium to placebo on bone mineral accrual. Sixty HIV-infected children
      and adolescents, aged 8-16, will be randomized to receive vitamin D3 plus calcium or placebo.
      The primary outcome variables, BMC and BMD, will be measured before, and 1 and 2 years after
      randomization. Serial measurements of serum 25(OH)D, 1,25(OH)2D, PTH, markers of bone
      turnover will be assessed before and at defined intervals during the study.

      A.2.b. Treatment arms:

      Eligible subjects will be assigned by randomization to receive either:

        1. Vitamin D3 (100,000 IU or 2.5 mg) administered orally every 60 days and elemental
           calcium 500 mg twice daily; or

        2. Vitamin D3 placebo administered orally every 60 days plus calcium placebo twice daily.

           A.2.c. Entry and randomization visit:

           All eligible subjects will have the initial study entry evaluation performed at the
           SLRHC Pediatric Body Composition Unit by virtue of their participation in the
           cross-sectional study (Section D.2.f). As the qualifying serum 25(OH)D level will be
           known prior to the cross-sectional study visit, study participants eligible for the
           randomized clinical trial can be randomly assigned (see Methods) during the
           cross-sectional study visit to one of the two treatment arms; in such cases, the
           cross-sectional visit will also function as the entry/baseline visit. Each subject will
           be given the first dose of vitamin D3/placebo and the first 30 day supply of
           calcium/placebo will be dispensed. females of child bearing capacity

           A.2.d. Follow-up Visits:

           Participants will be seen monthly during the first year of the study and every two
           months during the second year. Entry/baseline, 12-month and 24-month visits will take
           place at the SLRHC Body Composition Unit. All other visits will take place at the site
           where usual care is received. Vitamin D3/placebo will be administered during study
           visits every 2 months. At monthly visits during the first year and every 2 months during
           the second year after randomization, a 30 or 60 day supply of calcium/placebo will be
           dispensed. Adherence to calcium supplementation will be assessed at each visit.

           A.2.e. 12 and 24 month study visits:

           These visits will take place at the SLRHC Body Composition Unit. At each visit, the
           following tests will be performed:

           Medical history, including medications Pregnancy test for females of child bearing
           capacity Height and weight Assessment of pubertal stage Assessment of dietary intake of
           calcium and vitamin D Assessment of physical activity Determination of TBBMC, TBBBMD,
           spinal BMC and BMD Bone age radiograph

           A.3. METHODS

           A.3.a. Randomization:

           Randomization of HIV-infected children to treatment arms will be performed by
           computer-generated random numbers (SAS version 8.2 for Windows, SAS Institute, Cary,
           NC). Randomization will stratify by sex and age (e.g., > or < 14 years) in order to
           ensure that treatment groups are balanced with respect to these variables. Block
           randomization will be used to ensure that the number of subjects in each arm remains
           approximately similar over time. A randomization sequence will be generated by the study
           biostatistician and communicated to the research pharmacist who will dispense active or
           placebo medication as subjects are enrolled.

           A.3.b. Adherence:

           Adherence to calcium/placebo will be assessed at each visit through pill counts and use
           of blister packages. Adherence to vitamin D/placebo will be ensured by supervised
           administration during defined study visits.

           A.3.c. Measurement of bone mineral status in children and adolescents:

           Bone mineral status in HIV-infected subjects will be determined by whole body and lumbar
           spine DXA scans. These will be performed at the SLRHC Pediatric Body Composition Unit
           using the Hologic Delphi-A Scanner.

           Measurement of bone mineral content in grams by DXA correlates well with total body
           calcium (Ellis 1996). To date, this laboratory has provided bone mineral and body
           composition data using DXA in more than 13,000 subjects for more than 50 research
           projects and clinical trials.

           The cv for bone mineral density in human subjects in our laboratory is 0.8%. The Hologic
           Delphi

           A.4.d. Assessment of skeletal age:

           Skeletal age will be determined by standardized bone age radiographs of the left wrist
           which will be performed at the SL site Department of Radiology on the same day as the
           DXA scans. The sex-specific standards for bone age using plain films of the hand and
           wrist from the Atlas of Greulich and Pyle will be used as the standard reference
           (Greulich and Pyle 1959). A single pediatric radiologist, who will be blinded to the age
           and treatment status of the subjects, will read all bone age radiographs.

           Follow up bone age radiographs will not be performed on subjects found on entry to have
           achieved skeletal maturity (i.e. epiphyseal fusion).

           Maturation has profound effects on bone mineral accrual. Prior to the appearance of the
           physical signs of puberty the only available method of assessing relative maturity or
           "biological age" is to measure skeletal or "bone age" by standardized x-rays. Skeletal
           age continues to be of use, even after the appearance of the physical signs of puberty,
           because the rate of progression of bone maturation with puberty varies between
           individuals. It is particularly variable in children with chronic disease whose general
           health status and rates of growth and pubertal progress may "catch-up" or "catch-down"
           in response to changes in treatment or in disease activity. In children with chronic
           disease, bone age may be a better indicator of maturational status than chronological
           age, size, or pubertal stage. Bone age therefore an important co-variate in this
           protocol.

           A.3.e. Assessment of pubertal status:

           Standardized physical examination performed by study investigators (Drs. Stephen Arpadi
           and Mary Horlick) after explanation of the procedure and rationale to subjects and
           family members to determined the level of sexual maturation. These will be performed
           during study visits to the SLRHC Pediatric Body Composition Unit using the method of
           Tanner (Tanner 1970, Tanner 1971). Repeat assessments will not be performed on subjects
           determined on entry to have experienced sexual maturation (i.e. Tanner Stage 5).

           Because of the normal wide variability in timing and pace of puberty, standardized
           serial assessment of pubertal status of study participants will be performed to help
           distinguish between the effects of puberty and the effects of study treatments,
           increased age, height and weight on bone mineral accrual. Measurement of this variable
           is potentially of great importance in studies of children with chronic illnesses such as
           HIV-infection where delays in sexual maturation are common (de Martino 2001).

           Changes in bone mineral mass by DXA at the time of puberty and its accompanying growth
           spurt have been reported in both males and females (Glastre 1990, Gilsanz 1991, Rico
           1993, Mora 1999). However, the reference standards generated by DXA manufacturers and
           recent longitudinal studies are age- and sex-specific only (Bachrach 1999). Reference
           standards based on age or puberty only are not helpful in evaluating patients with
           disorders, such as HIV infection, which affect growth, timing of puberty, and may
           independently affect bone mineral acquisition (Leonard 1999, Thearle 2000, Arpadi 2002).

           A.3.f. Procedure for assessment of pubertal status:

           The evaluation of puberty in this project will follow exactly the protocol of the BMDCS
           project. The impact of pubertal stage on bone mineral accrual will be explained to all
           participants at the entry study visit, so that they and their families will understand
           the purpose of the physical examination for pubertal status. The pediatric
           endocrinologist (MH), following the protocol established for the multicenter BMDCS
           project, will evaluate pubertal stage. For girls, breast development will be assessed by
           visual criteria, but also by palpation in the earliest stages of puberty (Marshall and
           Tanner 1971, Biro 1992). For boys, testicular size will be assessed using the ovoids of
           the Prader orchidometer (Zachman 1974, Biro 1995). For both boys and girls, pubic hair
           development will be assessed by the criteria of Tanner (Marshall and Tanner 1970, 1971).

           A.3.g. Procedure for measuring height and weight:

           Standing height will be measured to the nearest 0.1cm using a wall-mounted stadiometer
           (Holtain, Crosswell, Wales). Three separate measurements will be obtained at each time
           point specified and the results averaged. Weight will be obtained with subjects in
           hospital gown and measured to the nearest 0.1 kg (Weight Tronix, New York, NY).

           A.3.h. Assessment of modifiable behavioral factors that influence bone mineral accrual:

           We will use a food frequency questionnaire (FFQ) for dietary data collection to provide
           an estimate of nutritional intake for this study. The availability of standardized
           information on dietary intake also will permit investigation of the relation between
           food/nutrient intake and bone accretion during growth in HIV-infected children.

           Weight-bearing physical activity will be determined using the published self-report tool
           of Slemenda et al (1991). Although several studies support the validity of measures of
           children's self-reports of physical activity, only Slemenda's study reported the use of
           self-report of childhood weight-bearing physical activity (Sallis 1993, Slemenda 1991).

           A.3.i. Procedures for dietary intake and physical activity questionnaires:

           Following the protocol established for the multicenter BMDCS project, subjects will
           complete a FFQ (with the help of their parent or guardian if they are under 14 years).
           The questionnaire will take about 20 minutes and will be completed at the time of the
           visit. A picture containing various serving sizes will be included with each FFQ.
           Subjects are asked to indicate which picture best matches their serving size. Subjects
           will be reminded to indicate how much they usually ate - not how much was served.

           All participants will be administered a questionnaire asking them to recall the number
           of hours during the past week that they were involved in each of several physical
           activities. The investigator will prompt the participants to recall their activity as
           accurately as possible. The child may ask the parent to help. About ten minutes is
           needed to complete the questionnaire.

           The FFQ, BLOCKIDS (NutritionQuest, University of Berkeley, Berkeley, CA), is comprised
           of foods identified by NHANES III as important for this age and demographic group. Of
           relevance to this project, measurements include dietary intake of Vitamin D and calcium.
           The FFQ has been validated in African American children ages 8-10 years (Block 2000) for
           some macro and micronutrients (e.g. correlation for calcium = 0.64). Additional
           validation studies are planned as part of the BMDHC project.

           A.3..j. Procedures for biochemical analyses:

           General laboratory tests will be performed in the clinical chemistry laboratories of
           study sites including CBCs, lymphocyte subsets (CD4, CD8), HIV viral load, renal and
           liver function tests. Sera for specialized biochemistries (PTH, vitamin D metabolites,
           OC, BSAP, NTx) is aliquoted, frozen immediately after collection, and transferred to
           SLRHC where they will be stored at -70 degrees. The frozen samples will be analyzed in
           the Core laboratory of the Irving Center for Clinical Research at Columbia University.
           All samples from individual subjects will be analyzed in batches in the same assay to
           reduce inter-assay variability. All of the planned assays are currently in place in the
           Core Laboratory.

           Serum: Kits for intact PTH, vitamin D metabolites, and osteocalcin will be purchased
           from Corning-Nichols Laboratory (San Juan Capistrano, CA).

           Parathyroid hormone: PTH is currently being measured by a chemiluminescent method that
           is now known to detect the 7-84 fragment of PTH as well as the intact form (1-84)of the
           molecule (72). Intra-assay and inter-assay variability are 3.4% and 5.6% respectively.
           The normal adult range for this assay is 10-65 pg/ml.

           Vitamin D metabolites: After extraction of serum samples, 25OHD will be measured in a
           radio-binding assay. The intra-assay and inter-assay variability are 7.5% and 9.6%
           respectively. The normal range is 9-52 ng/ml. Serum 1,25(OH)2D will be assayed in a
           radioreceptor assay. The normal adult range is 15-60 pg/ml. The intra-assay and
           inter-assay variability are 7.6% and 9.8% respectively.

           Osteocalcin: Osteocalcin is measured by RIA according to the method of Gundberg et al.
           The intra-assay and inter-assay variability are 4.3% and 5.7% respectively. The normal
           range is 3.4-11.7 ng/ml for adult men, 2.4-10.0 ng/ml for adult premenopausal women.

           Bone Specific Alkaline Phosphatase (BSAP): BSAP is measured by a solid phase, two-site
           immunoradiometric assay that specifically measures the skeletal isoenzyme of alkaline
           phosphatase in serum. Kits are purchased from Hybritech Incorporated (San Diego, CA).
           The intra-assay and inter-assay variability are 4.2% and 7.2% respectively. The normal
           range is 8.0 - 16.6 ng/ml for adult women and 7.2 - 15.8 ng/ml for adult men.

           Serum N-telopeptide (Osteomark, Ostex, Seattle, WA) is measured using a
           competitive-inhibition enzyme-linked immunosorbent assay that utilizes microwells as the
           solid phase onto which NTx has been adsorbed. Assay values are reported in nmoles Bone
           Collagen Equivalents (BCE) per liter. The normal range is 6.2 - 19.0 in adult women and
           5.4 to 24.2 in adult men. The intra-assay and inter-assay variability are 4.6% and 6.9%
           respectively.

           A.3.l. Immunologic and virologic measures of HIV disease:

           CD-4 +lymphocyte number and CD4% values for subjects will be obtain from results
           performed in Clinical site laboratories as part of patient care. Quantitative viral load
           assays -HIV RNA determined by polymerase chain reaction method performed as part of
           clinical care at collaborating clinical sites will be collected used for this study.

           A.3.m. Procedure for safety monitoring and toxicity management:

           Urine pregnancy test will be administered to all females of child-bearing capacity at
           each SLRHC visit and immediately before any radiological studies are performed. All
           females of child bearing capacity will be questioned if they are pregnant prior to urine
           testing and DEXA scanning.

           Monitoring for hypercalcemia, hypercalciuria, and hypocalemia: In order to maintain
           blinding of subject treatment assignment, laboratory reports will be reviewed by Dr.
           Shane, a Co-investigator, located at New York-Presbyterian Hospital, which is physically
           apart from any of the clinical research facilities. This is also the location for the
           GCRC where specialized laboratory assays will be performed.

           Serum calcium and albumin and spot morning fasting urine will be monitored on a monthly
           basis during the first year on study, and every 2 months during the second year. These
           assays will be performed through Quest Diagnostics Laboratory as convenience samples
           (i.e., run when collected).

           Clinical or laboratory abnormalities considered by the investigator to be related to
           study drug will be managed as follows:

           If hypercalcemia (>10.5 mg/dl, albumin-corrected) develops, the dose of calcium will be
           reduced by 250 mg/day and the levels rechecked one week later. If the abnormality does
           not resolve, weekly stepwise reductions in oral calcium will be followed until a normal
           level is achieved or supplemental calcium is discontinued. Vitamin D3 or placebo will be
           withheld until serum calcium returns to normal.

           Subjects with persistent abnormality will be referred to the treating physician. Vitamin
           D3 or placebo will be withheld until serum calcium returns to normal.

           If severe hypercalcemia develops (>12.0 mg/dl), all calcium supplements and Vitamin
           D/placebo will be discontinued immediately and the subjects will be withdrawn from the
           study.

           Hypercalceria If hypercalciuria develops (urinary calcium /creatinine ratio >0.25 on
           spot urine specimens), the subject will have a 24 hour urine collection performed for
           confirmation. For confirmed hypercalciuria (>4mg/kg/24 hr), the dose of calcium will be
           reduced by 250mg and a spot urine repeated one week later. If the abnormality does not
           resolve, weekly stepwise reductions in calcium will be followed until hypercalceria
           resolves or supplemental calcium is discontinued. If necessary, vitamin D3 or placebo
           will be withheld until urine calcium returns to normal.

           If hypocalcemia develops (< 8.4 mg/dl, corrected for albumin), blood will be drawn for
           Blood UreaNitrogen, serum electrolytes, creatinine, 25-OHD, PTH, Creatinine, Magnesium,
           and Albumin.

           A subject with confirmed hypocalemia associated with vitamin D deficiency (25-OHD<10
           ng/dl and PTH >73 will be withdrawn from the study and referred to their medical
           provider for treatment and withdrawn from the study

           Monitoring for vitamin D deficiency

           During the first year on study, serum for measurement of 25-OH and iPTH will be drawn
           monthly and every 2 months during the second study year. Each subject's specimens will
           be frozen immediately after collection, and transferred to SLRHC where they will be
           stored at -70 degrees. At one year intervals the each subjects' samples will be assayed
           in "batched " fashion. On the basis of these annual determinations, subjects with
           persistent vitamin D deficiency (25-OHD<10ng/l and PTH>73) that lasts throughout both
           winter and summer months, will be withdrawn from the study and referred to their medical
           provider.

           We anticipate low incidence of these particular abnormalities. In a prior study in which
           the same dose of vitamin D3 (100,000 IU) was administered every 60 days for 6 months,
           Guillemant et al. reported no instances of hypercalcemia in French adolescent males,
           ages 13-16, with seasonal vitamin D deficiency (Guillemant 2000). In addition, in an
           on-going study conducted by Dr. Shane (Prevention of Osteoporosis After Cardiac
           Transplantation, ROI AR 46124-04), among the 74 subjects receiving the metabolically
           active form of Vitamin D, calcitriol (0.25 ug BID), there were only 4 episodes of
           urinary calcium excretion >400 mg/g creatinine. Besides being on calcitriol, these
           subjects were receiving corticosteroids, which also increase urinary calcium excretion.
           Corticosteroids are not a permitted co-administered medication in the present study.

           We do not anticipate Vitamin D3 will have an adverse effect on HIV infection or
           antiretroviral medications. However, HIV-infected subjects who experience an otherwise
           unexplained decline in immune status, based on CD4 count or CD4%, or an increase in
           measure of HIV viral load believed by the treating physician to be possibly related to
           study medications will be withdrawn from study participation. Measurements of CD4 count,
           CD%, and HIV viral load will be measured at the clinical care sites as per standard of
           care. Results will be monitored by the treating physician.

           Subjects who have decline in TBBMC z-score of >0.5 from baseline at the one year
           on-treatment DXA will be discontinued from study and referred for additional clinical
           evaluation by the treating physician. In addition, if low impact or repeated fractures
           occur, a clinical evaluation by the treating physician, safety officer and Study PI will
           determine if the subject should remain on study.Monitoring DXA scan results of study
           subjects will be performed by Dr. Horlick, a Co-Investigator and Director of the
           Pediatric Body Composition Laboratory at St. Luke's-Roosevelt.

           Finally, because this is a placebo-controlled trial involving children, we have elected
           to include an interim data analysis to be performed on 12 month on-treatment data (Human
           Subjects, Section E.). A Data Safety and Monitoring Board comprised of experts in
           biostatistics, pediatric HIV disease, pediatric endocrinology, and pediatric bone
           acquisition will be established as per NIH guidelines to monitor study results for
           safety and efficacy, to establish criteria for early termination of the study in
           consultation with the Principal and Co-Investigators, and to determine if and when these
           criteria have been fulfilled.

           A.3.n Data analysis and power calculations Specific Aim 1: To determine the effect of
           calcium and vitamin D3 supplementation on bone mineral accrual in HIV-infected children
           and adolescents receiving HAART.

           The primary hypothesis of this study is that BMC and BMD will increase in HIV-infected
           subjects randomized to receive vitamin D3 plus calcium compared to subjects receiving
           placebo. We will test this hypothesis by comparing TBBMC, TBBMD, SBMC, and SBMD measured
           by total body and spine DXA scans performed at baseline and at 1 and 2 years
           post-randomization in the 2 study arms using an intention-to-treat analysis.

           A repeated measures analysis of covariance will be used to analyze the bone accrual
           data. Differences in bone mineral content between the treatment and placebo groups will
           be compared at 1 and 2 years while controlling for any possible differences between the
           groups at entry to the trial. The between-group main effect (vitamin D3 and calcium
           versus placebo) will test whether there is an overall difference between the groups,
           while the within-group x time interaction effect will test whether the difference
           between the groups is constant over the two follow-up years. The magnitude of change in
           the vitamin D3 and calcium group will also be compared against normative data from the
           BMDCS to determine how many children in each group achieve levels within normal limits
           (95 % CI) using the algorithm presented in preliminary studies section C.5.

           Based on our prior pilot data, the mean yearly change in TBBMC in 30 HIV-infected
           children between the ages of 7-14 years was 120 g with a standard deviation of 84. A
           difference in SD between boys and girls was noted. In order to maintain conservative
           estimates we chose SD observed in girls. This can be assumed to be the level of yearly
           change that will be observed in the placebo group. Based on data from the Pediatric
           Rosetta Project (NIDDK 37352), a cross-sectional study of >1300 children, the mean
           yearly change in TBBMC in healthy African-American and Hispanic children ages 7-14 years
           is 195 g.

           The effect size (d) comparing a mean yearly change of 120 g in the placebo group versus
           a change of 195 g in the Vitamin D3 and calcium group is therefore 0.89. Twenty-one
           children in each group will be sufficient to detect an effect size of this magnitude
           with alpha = 0.05 and beta = 80%. If the sample size is increased to 30 children in each
           group, power will be sufficient to detect a difference of 61 gm between the vitamin D3
           and calcium group and the placebo group with alpha = 0.05 and power = 80%. This is
           comparable to the difference observed in a clinical trial of healthy adolescent girls
           (mean age, 11.9 years) who received either calcium (110% of RDA) or placebo (Lloyd
           1996).

           Differences in the rate of sexual maturation during the study between treatment groups
           may "unbalance" the groups with respect to a factor of significance to the bone mass
           outcomes. To address this we have included a standardized measurement pubertal stage for
           use as a covariate in the analyses.

           Specific Aim #2: Determine the effect of HIV infection and vitamin D and calcium
           supplementation on indices of mineral metabolism and markers of bone turnover.

           We hypothesize that indices of mineral metabolism are abnormal (low serum 25(OH)D and
           elevated PTH) and biochemical markers of bone formation and resorption are increased in
           HIV-infected children on HAART compared to normal controls and that indices of mineral
           metabolism and markers of bone formation and resorption will return toward normal in
           HIV-infected children and adolescents who are randomized to receive vitamin D and
           calcium supplementation. We will test this hypothesis first by comparing markers of bone
           turnover (osteocalcin, BALP and N-Tx) in HIV-infected and healthy children and
           adolescents enrolled in the cross-sectional study using independent samples t-test to
           compare means.

           Longitudinal changes in bone turnover markers in HIV-infected children and adolescents
           receiving vitamin D and calcium supplement will also be compared to those receiving
           placebo. Levels of vitamin D metabolites and markers of bone turnover ((osteocalcin,
           BALP and N-Tx) taken at intervals will be analyzed using a mixed model regression to
           model trends in these measures over time to evaluate dependent inter-relationships
           between these variables.

           Specific Aim #3: Evaluate vitamin D stores in HIV-infected children and adolescent
           receiving HAART.

           We hypothesize that vitamin D stores are decreased in children with HIV infection
           compared to healthy controls.

           We will test this by comparing serum levels of vitamin D metabolites and indices of
           mineral metabolism (25(OH)D 1,25(OH)2D) and PTH) in HIV-infected children and
           adolescents receiving HAART compared to healthy controls. Data for testing this will be
           obtained from the cross-sectional study. The HIV-infected group will consist of a sample
           of HIV+ children screened for recruitment into the study while the healthy group will
           consist of similar aged children recruited from the same neighborhood (including healthy
           siblings of the HIV positive children). Screening levels of serum vitamin D metabolites
           and PTH in the HIV-positive children will be compared with levels in the healthy
           children using an independent samples t-test.

           The main variable of interest is 25(OH)D as it best reflects vitamin D stores. Based on
           prior pilot data, the mean (SD) expected 25(OH) among HIV-infected subjects is 21 (11)
           ng/ml. The sample size in the present study has 80% power to detect change in 25(OH) D
           of 8 ng/ml with alpha=0.05. Analysis of covariance based on a general linear regression
           model will then be used to determine whether any possible group differences remain after
           controlling for other factors such as race, sex, estimated dietary intake of Vitamin D
           and sunlight exposure (using month of study as surrogate for sunlight exposure).

           Specific Aim #4: Evaluate if vitamin D stores are a determinant of bone mass in HIV-
           infected children and adolescents receiving HAART.

           Our hypothesis is that among HIV-infected children and adolescents receiving HAART,
           vitamin D stores, as measured by serum 25(OH)D, is a significant determinant of bone
           mass.

           We will assess the relationship these variables have to TBBMC in HIV-infected children
           and adolescents. This will be conducted through analysis of data obtained from a sample
           of at least 60 HIV-infected children and adolescent being screened for the enrollment in
           the longitudinal clinical trial. Within the HIV positive group, linear regression will
           be used to determine whether HIV-related factors including antiviral medications,
           markers of immunodeficiency (CD4count), HIV viral load, and levels of cytokines play a
           role in predicting serum vitamin D levels and bone mass. Additional co-variates that
           will be included in the model include age, sex, race, height, weight and pubertal stage.

           These analyses are especially critical measurements for inclusion in this clinical trial
           and in the analyses as these will allow for development of potential mechanistic
           pathways for both the etiology of abnormal bone mass and intermediate response variables
           involved in amelioration.

           Using multiple regression modeling we will assess the relationships of TNF, IL-6 vitamin
           D levels and TBBMC. It is estimated that 50% of the HIV positive children who are
           screened will meet the inclusion criteria for the trial and that approximately 100 to
           120 HIV positive children will be available for the cross sectional comparison and so an
           equal number of healthy children will be also recruited. With a total of 200 children,
           the sample size will be large enough to add up to 18 covariates in addition to the
           grouping factor (HIV-positive versus healthy) and test for a 5% increase in R2, with
           alpha = 0.05 and power = 80 %. A sample of 100 HIV-positive children alone will allow
           for a regression analysis that includes up to 9 predictors which collectively explain at
           least 15% of the variance of the outcome variable (TBBMC, etc.) with alpha = 0.05 and
           power = 80 %.
    

Intervention name(s): cholecalciferol plus calcium carbonate
Intervention type(s): Dietary Supplement
Intervention description(s): oral calciferol 100,000 IU by mouth bimonthly and calcium carbonate (1 gr) by mouth daily
Other intervention name(s): Vitamin D3
Intervention MeSH terms: Vitamin D; Cholecalciferol; Calcium Carbonate; Calcium
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: Pediatric HIV infection, bone mineral content, bone mineral density, Vitamin D, calcium Dual x-ray absorptiometry