Calcium Absorption, Bone Mass Accumulation, and

Kinetics Increase during Early Pubertal

Development in Girls*

STEVEN A. ABRAMS, KENNETH C. COPELAND, SHEILA K. GUNN,

CAREN M. GUNDBERG, KAREN OERTER KLEIN, AND KENNETH J. ELLIS

U.S. Department of Agriculture/Agricultural Research Service, Children’s Nutrition Research Center

(S.A.A., K.J.E.), Section of Endocrinology (K.C.C., S.K.G.), Department of Pediatrics (S.A.A., K.J.E.),

Baylor College of Medicine, and Texas Children’s Hospital (S.A.A., K.J.E.), Houston, Texas 77030;

Department of Clinical Science, A. I. duPont Hospital for Children (K.O.K.), Wilmington, Delaware

19899; and Department of Orthopedics and Rehabilitation, Yale University School of Medicine

(C.M.G.), New Haven, Connecticut 06510

ABSTRACT

To evaluate the changes in calcium and bone mineral metabolism

associated with early pubertal development, we performed longitudinal measurements of calcium absorption, calcium kinetics, bone

mineral content, and hormonal markers related to puberty in a multiethnic group of girls beginning when they were 7 or 8 yr old. Girls

were Tanner stage 1 (breast) at the start of the study. They were

placed on a 1200 mg/day dietary calcium intake and studied at approximately 6-month intervals until they reached Tanner stage 2

(breast). Results at that time point (PUB) were compared to values

obtained approximately 1 yr earlier (LatePRE) and those 1 yr before

that (EarlyPRE). We found an increase in calcium absorption comparing PUB to LatePRE (n 5 34; 36.6 6 8.7% vs. 30.7 6 9.9%; P 5

0.002). Using whole body, dual energy, x-ray absorptiometry scanning, we found an increase in calcium gain during the LatePRE to

PUB period compared with that during the EarlyPRE to LatePRE

period (135 6 53 vs. 110 6 45 mg/day; P 5 0.04). Calcium kinetic

studies showed a significant increase in the bone calcium deposition

rate (Vo1) during the PUB compared to the LatePRE period. Hormonal and biochemical markers of bone development were also significantly increased at PUB compared to LatePRE. Hormonal activity, as evidenced by the unstimulated LH level, was significantly

correlated with calcium gain between the LatePRE and PUB studies

and the bone calcium deposition rate in the PUB study. These data

demonstrate, using multiple independent methods, an increase in

calcium utilization associated with the earliest physical signs of puberty. (J Clin Endocrinol Metab 85: 1805–1809, 2000)

I

T IS WELL recognized that the pubertal growth spurt is

associated with a rapid increase in calcium gain by the

skeleton. Recent studies have further demonstrated a sharp

decrease in girls in the rate of absorption and skeletal gain

of calcium beginning within 2–3 yr after menarche (1, 2). The

mechanisms associated with these changes are poorly understood, but may relate to changes in calcium (Ca) metabolism during puberty. It has previously been shown that

puberty is associated with higher rates of dietary calcium

absorption and kinetically determined bone calcium deposition rates (Vo1) than those of prepuberty or adulthood

(2–5). However, these earlier reports were cross-sectional in

design and contained minimal information about either the

physical or hormonal status of the girls studied.

Recent dietary guidelines have recommendations that

girls increase their calcium intake beginning at age 9 yr (6).

This is 2 yr younger that that recommended by the previous guidelines, partly because of the significant proportion of 9- and 10-yr-old girls who show physical signs of

pubertal development (7). However, the calcium metabolic changes in this transitional age group are poorly

described. For example, there are no longitudinal data

regarding the relationships between the physical and hormonal changes that occur during early puberty and calcium metabolism.

The identification of physical signs of puberty remains the

current standard for defining the onset of puberty in girls. We

sought to identify whether the transitional period between

prepubertal development and early puberty (as defined by

breast development consistent with Tanner stage 2) is associated with changes in calcium metabolism, and whether

these changes are related to the hormonal changes of puberty. By using multiple techniques to assess mineral utilization and hormonal status, we hypothesized that we could

identify specific hormonal changes related to the increase in

calcium gain and Vo1 in early puberty.

Received September 21, 1999. Revision received December 1, 1999.

Accepted December 17, 1999.

Address all correspondence and requests for reprints to: Steven A.

Abrams, M.D., U.S. Department of Agriculture/Agricultural Research

Service Children’s Nutrition Research Center, 1100 Bates Street, Houston, Texas 77030. E-mail: sabrams@bcm.tmc.edu.

* This work is a publication of the USDA/Agricultural Research

Service Children’s Nutrition Research Center, Department of Pediatrics,

Baylor College of Medicine and Texas Children’s Hospital (Houston,

TX). This project was supported in part with federal funds from the

USDA/Agricultural Research Service under Cooperative Agreement

58-6250-6-001; the NIH, National Center for Research Resources

(NCRR), General Clinical Research for Children Grant RR00188; and

NIH Grant AR-43740. The contents of this publication do not necessarily

reflect the views or policies of the USDA, nor does mention of trade

names, commercial products, or organizations imply endorsement by

the U.S. Government.

0021-972X/00/$03.00/0 Vol. 85, No. 5

The Journal of Clinical Endocrinology & Metabolism Printed in U.S.A.

Copyright © 2000 by The Endocrine Society

1805
Subjects and Methods

Subjects

Seven- and 8-yr-old girls who were Caucasian, African-American, or

Hispanic were eligible for enrollment in this longitudinal study if they

were healthy and were not taking any prescription medications or receiving any mineral supplements. Physical examinations were performed on potential subjects to determine Tanner stage. Thirty-four girls

who were Tanner stage 1 (breast) were enrolled and completed the study

protocol. Initial studies were performed at the time of the initial physical

examination, and follow-up studies were scheduled at 6- to 9-month

intervals. There was some variability in this interval length due to the

subjects’ school schedules and the need for in-patient studies.

Of these 34 girls, 26 remained Tanner stage 1 at follow-up at least 6

months after the initial visit. The other 8 girls were Tanner stage 2 at the

initial follow-up visit. The analysis identified the first visit in which the

girls were Tanner stage 2 (breast) as the index visit (PUB) and went

backwards to identify the visit of approximately 12 months earlier as the

late prepubertal study (LatePRE). For 26 girls who had multiple visits

while Tanner stage 1, we identified a visit of approximately 12 months

before the LatePRE visit as the early prepubertal study (EarlyPRE).

Written informed consent was obtained from a parent or legal guardian for each subject; written assent was obtained from all of the study

subjects. The institutional review board of Texas Children’s Hospital/

Baylor College of Medicine approved this protocol.

Clinical protocol

After an overnight fast (except for water), a heparin lock catheter was

inserted at 0800 h, and 20 mL blood were withdrawn for biochemical and

hormonal analysis [insulin-like growth factor I (IGF-I), LH, FSH, alkaline

phosphatase activity, and osteocalcin]. Subsequently, 2.5 mg/kg (maximum, 100 mg) GnRH were administered iv. Blood was collected for

serum LH and FSH at 30 and 60 min after the infusion. After the GnRH

was infused, each subject was given a breakfast containing approximately 350 mg calcium. Toward the end of breakfast, the subjects were

given 46Ca (0.4 mg/kg) that had been premixed (and allowed to equilibrate in the refrigerator for 12–24 h) with 120 mL milk. The calcium in

this milk was part of the total 350 mg given with breakfast. One hour

after the GnRH infusion, 42Ca (80 mg/kg) was infused over 2 min via the

heparin lock catheter. After infusion, the heparin lock was infused with

3 mL saline, and the hub of the catheter was replaced. Two milliliters of

blood were removed for mass spectrometric analysis at 6, 12, 20, 30, 45,

60, 120, 180, 240, and 480 min after completion of the infusion. Beginning

with breakfast, a complete 24-h urine collection was obtained in 8-h

aliquots.

Subjects were placed on a 1200 mg/day calcium diet at enrollment in

the study. At study entry (1–2 months before the initial calcium kinetic

study), the research dietician provided each girl with an individualized

dietary plan, based on their food preferences, to meet the study calcium

intake. Girls and their families were specifically counseled regarding

ongoing strategies to maintain the girls’ calcium intake at this level using

dietary methods. Multiple methods, including telephone calls and diet

diaries, were used to assess compliance.

A complete physical examination, including Tanner staging, was

performed on each subject at each visit by one of two pediatric endocrinologists who were unaware of the previous assignment of Tanner

stage or any of the study results for that subject.

Analytical methods

Urine and serum samples were prepared for mass spectrometric

analysis as previously described using an oxalate precipitation technique (2–4). Samples were analyzed for isotopic enrichment with a

Finnigan MAT 261 (Bremen, Germany) magnetic sector thermal ionization mass spectrometer. Each sample was analyzed for the ratio of

42Ca/43Ca and 46Ca/43Ca, with correction for fractionation to the reference 44Ca/43Ca ratio. Accuracy and precision of this technique for

natural abundance samples compared to standard data are 0.15% or

better, depending on the ratio being measured.

Dual energy x-ray absortiometry (DXA) methods

Body composition measurements were performed using a QDR-2000

dual energy x-ray absorptiometer (Hologic, Inc., Waltham, MA). The

whole body was scanned in the single beam mode. Total body bone

mineral content and percent body fat were determined as described by

Ellis et al. (8). Whole body bone mineral content measurements were

converted to total body calcium values using 32.2% as the percentage of

calcium in the bone mineral based on our previous evaluation of this

instrument (8), which was recently confirmed (9). The daily rate of

change in total calcium (calcium gain rate, milligrams per day) was

calculated for each set of paired data by dividing the interval difference

in total body calcium by the number of days between DXA measurements for that subject.

Hormonal and biochemical studies

IGF-I, assayed using the acid-ethanol cryoprecipitation technique,

which effectively isolates IGF-I from its binding protein, was measured

by RIA (Diagostics Systems Laboratories, Inc., Webster, TX).

Estradiol was measured using an ultrasensitive assay (10). This technique uses a strain of the yeast Saccharomyces cerevisiae, which has been

genetically engineered to allow for its use in a bioassay measuring very

low levels of estradiol. Specifically, the yeast was transformed with two

plasmids, one encoding the human estrogen receptor and the other an

estrogen-responsive promoter fused to the structural gene for b-galactosidase. Blood samples were obtained while ensuring that the serum

was kept from contact with rubber stoppers (which cross-react with the

assay). An ether-based extraction was performed, and an assay was

performed in which expression of b-galactosidase was measured. A

value of 6.3 pmol/L estradiol equivalent was used to indicate pubertal

levels of estradiol (10). This value represents 2 sd above the mean

prepubertal value.

LH and FSH were measured using time-resolved fluoroimmunoassay

kits from Wallac, Inc. (Gaithersburg, MD). Serum total alkaline phosphatase activity was measured using standard clinical laboratory techniques. Urinary calcium was measured in 8-h pooled samples using

atomic absorption spectrophotometry.

Osteocalcin was determined using a RIA method previously described (11). Total alkaline phosphatase values were determined using

standard clinical methods by a commercial laboratory.

Calculations and modeling

The compartmental model used for calcium kinetics is similar to that

described by Neeret al. (12). The model is based on three sequential pools

before calcium deposition in the deep bone Ca pool. Bone Ca deposition

(Vo1) is the rate of flow of Ca to the final pool. Compartmental modeling

of the data was performed with the aid of the SAAM (simulation,

analysis, and modeling) program (12). Details of this program and its

application to calcium metabolism have been described previously

(3, 12).

Comparisons of values between study time points were made using

repeated measures ANOVA. Differences over time in anthropometric,

biochemical, hormonal, or calcium metabolic parameters were evaluated for the 26 complete datasets, followed by pairwise t tests between

both the EarlyPRE-LatePRE time points (n 5 26 pairs) and the LatePREPUB time points (n 5 34 pairs). The relationship between hormonal and

biochemical parameters and Vo1 or changes in total body calcium were

determined in the cross-sectional analysis using linear and multiple

regression analysis (StatView 4.5 for Macintosh, StatView Corp., Berkeley, CA; and DeltaGraph 3.5 for Macintosh, Deltapoint, Inc., Monterey,

CA). All data are presented as the mean 6 sd.

Results

Mean ages at the time of the 3 studies were 8.3 yr (EarlyPRE), 9.1 yr (LatePRE), and 10.2 yr (PUB), respectively

(Table 1). Of the 26 girls for whom data from all 3 time points

were used, 7 were Caucasian, 8 were African-American, and

11 were Mexican-American. Of the additional 8 girls who did

not have an EarlyPRE study, 2 were Caucasian, 2 were Af1806 ABRAMS ET AL. JCE & M • 2000

Vol 85 • No 5
rican-American, and 4 were Mexican-American. There were

no significant ethnic differences in age, weight, or height

among the girls at the start of the study.

Comparisons between groups for height, weight, and pubertal characteristics are also shown in Table 1. At the

LatePRE time point, few girls showed evidence of puberty

based on LH concentration, but the majority had an estradiol

level greater than 6.3 pmol/L. By the PUB time point, baseline (pre-GnRH administration) LH level was significantly

increased compared to the LatePRE study values, and the

majority of girls showed a pubertal response to GnRH, as

evidenced by a stimulated LH level greater than 5.0 nmol/L

(13).

Changes in biochemical values, hormones, and calcium

metabolic parameters are shown in Table 2. Calcium absorption, bone calcium deposition rate, alkaline phosphatase activity, IGF-I, and osteocalcin all showed significant increases

between the LatePRE study and the PUB study, whereas

none of these increased significantly between the EarlyPRE

and LatePRE studies. Each of these distinctions was shown

both by repeated measures ANOVA using the 26 complete

sets and by paired t tests for 34 datasets (LatePRE vs PUB),

except for calcium absorption. In that case, only the paired

t tests were significantly different.

Calcium gain, determined by calculating a daily rate of

change in total body calcium between DXA studies for each

girl, is shown in Fig. 1. There was a significant increase in

calcium gain (P , 0.05) between the LatePRE and PUB studies (DTBC2) compared with the EarlyPRE and LatePRE studies (DTBC1; 135 6 53 vs. 110 6 45 mg/day; P 5 0.04). Also

shown in this figure is the peak rate of calcium gain of 212

mg/day reported by Martin et al. (14).

The relationships between Vo1 and biochemical/hormonal markers of bone mineral metabolism were evaluated

using linear regression analysis. At the PUB visit (Fig. 2), but

not the EarlyPRE or LatePRE visits, Vo1 was significantly

correlated to serum osteocalcin (r 5 0.49; P 5 0.003 at the PUB

visit; r 5 0.27; P 5 0.18 at the EarlyPRE visit; and r 5 0.13;

P 5 0.46 at the LatePRE visit). Similarly, the relationship

between Vo1 and the total serum alkaline phosphatase activity at that visit was significant (r 5 0.40; P 5 0.02).

The bone calcium deposition rate was significantly correlated to the baseline LH level at the PUB visit (r 5 0.44; P 5

0.01). It was not significantly correlated, however, with the

GnRH-stimulated LH level (r 5 0.26; P 5 0.13). The importance of the LH level in calcium metabolism in early puberty

was further demonstrated by the significant correlation between the DTBC2 and the baseline LH level (r 5 0.50; P 5

0.01). In contrast, the estradiol values were not correlated to

Vo1 at any visit.

A significant relationship was found between DTBC2 and

Vo1 at the PUB study (r 5 0.55; P , 0.001; Fig. 3) and between

DTBC2 and serum alkaline phosphatase activity at the PUB

study (r 5 0.49; P , 0.01). In contrast, DTBC2 was not significantly correlated to serum osteocalcin at the PUB study

(r 5 0.08; P 5 0.65).

Although limited data are available to perform ethnic comparisons, we did not find any significant ethnic differences

among girls for any of the primary study end points. These

included the rate of increase in calcium gain between study

points, the baseline or GnRH-stimulated LH level, the estradiol level, the serum alkaline phosphatase activity, or

Vo1. However, serum osteocalcin was significantly lower

both at the LatePRE and the PUB study in African-American

compared to Caucasian or Hispanic girls (P , 0.01).

Calcium intake values from the 3-day dietary records

showed mean intakes of 1200–1300 mg/day at the study

periods. The group sd of intakes at each time period was

TABLE 2. Calcium metabolic and hormonal changes in girls during early puberty

Early PRE (n 5 26) Late PRE (n 5 34) PUB (n 5 34)

Calcium absorption (%) 33.0 6 7.4 30.7 6 9.9 36.6 6 8.7a

Urinary Ca excretion (mg/day) 89.0 6 47.6 87.2 6 62.6 102.2 6 65.3

Vo1 (mg/day) 1504 6 221 1563 6 285 1952 6 407a

Total APA (IU/L) 233 6 39 244 6 51 297 6 80a

IGF-I (ng/mL) 257 6 101 255 6 112 323 6 117a

Osteocalcin (ng/mL) 21.7 6 7.6 22.1 6 7.1 30.5 6 11.6a

There were no significant differences between early PRE and late PRE for any value. APA, Alkaline phosphatase activity; Vo1, bone calcium

deposition rate. a P , 0.01, late PRE vs. PUB. (by paired t test for 34 girls at both time points).

TABLE 1. Anthropometric and hormonal characteristics of the study subjects

Early PRE (n 5 26) Late PRE (n 5 34) PUB (n 5 34)

Age (yr) 8.3 6 0.7 9.1 6 0.9a 10.2 6 0.8b

Ht (cm) 129.8 6 5.9 133.3 6 6.3a 139.8 6 6.3b

Wt (kg) 28.6 6 6.0 30.4 6 5.5a 35.2 6 5.8b

Tanner stage 2 (pubic) 0/26 3/34 17/34b

Baseline LH (ng/mL) 0.06 6 0.06 0.19 6 0.51 0.77 6 1.17b

Stimulated LH .5 ng/mL (%)c 12 18 59c

Estradiol .6.3 pmol/L (%) 35 83a 83

By group definition, all girls at early PRE and late PRE were Tanner stage 1 (breast), and all girls at PUB were Tanner stage 2 (breast).

One girl was Tanner stage 3 (pubic hair) in the PUB study. a P , 0.01, early PRE vs. late PRE (by paired t test for 26 girls studied at both time points). b P , 0.01, late PRE vs. PUB (by paired t test for 34 girls at both time points). c Peak LH level 30 or 60 min after receiving GnRH iv.

CALCIUM KINETICS IN EARLY PUBERTY 1807
150–190 mg/day. These results were consistent with our

target intake of 1200 mg/day. Because of the narrow range

of intakes and the study design, which did not include a

prolonged in-patient adaptation period, limited interpretation of the exact intake is possible.

Discussion

In a longitudinal multiethnic study, we found a significant

increase in the utilization of calcium associated with the

onset of the physical development of puberty. This change

was evidenced by increased calcium absorption, calcium

gain, and kinetically determined rates of bone calcium deposition. These findings support the importance of advocating

adequate mineral nutrition in girls during early puberty and

are consistent with the new lower age at which it is recommended that calcium intake be increased.

We found that changes in calcium metabolism and utilization were associated with maturation of the hypothalamicpituitary axis, as measured by the GnRH-simulated LH level,

and occurred after initial increases in estradiol in most girls.

Small increases in estradiol are probably the earliest marker

of pubertal changes before physical changes are observed, as

estradiol stimulates these physical changes. Ultrasensitive

estradiol measurements may be a more sensitive indicator of

pubertal onset than LH level (10, 13). In this study we found

that changes in calcium metabolism were concomitant with

physical changes of puberty and were probably the consequence of these hormonal changes. These increases in calcium gain and bone calcium deposition were paralleled by

changes in IGF-I and biochemical markers of bone formation, including alkaline phosphatase activity and serum

osteocalcin.

Few longitudinal data are available regarding this developmental period in humans. Recently, McKay and co-workers (15) reported the changes in total body bone mineral

content in girls using a DXA technique. They did not relate

their results to daily changes in total body calcium. If this

calculation is made from the data presented in their report,

the calculated increments in calcium gain (110 mg/day at age

10 yr and 140 mg/day at age 11 yr) are similar to those we

found in this study. Of note is that they reported a maximum

increment of 260 mg/day at age 12–13 yr. This value is

slightly higher than the peak value of 212 mg/day reported

by Martin et al. (14) for girls. These more recent values for

peak rates of calcium gain are very similar to the estimates

derived from rates of weight change reported by Leitch and

Aikten in the 1950s (16).

Although the increased rate of calcium gain between the

LatePRE and PUB compared to the EarlyPRE and LatePRE

periods in our study was relatively modest, the increases in

kinetic values and absorption probably presage the marked

increase in calcium gain during subsequent pubertal development. It is not possible to identify an exact onset of puberty, and therefore, the increments we identified may be

lower than the maximums achieved in early puberty.

This study was not designed to evaluate the effects of

FIG. 1. Shown are the rates of calcium gain in girls derived from

longitudinal DXA data during early puberty. DTBC1 and DTBC2

represents the daily increase in total body calcium for each girl (mean

and SD for 26 paired studies) between studies performed at early vs.

late prepubertal time points (DTBC1) or between late prepubertal and

pubertal studies (DTBC2). Differences are significant (P 5 0.04). Also

shown is the peak rate of calcium gain derived by Martin et al. (14)

from longitudinal DXA data.

FIG. 2. The serum osteocalcin concentration and the bone calcium

deposition rate, Vo1, were significantly correlated (y 5 17.3x 1 1423;

r 5 0.49; P , 0.01). Values are from the study performed when girls

were Tanner 2 breast (PUB study). Values for African-American girls

are indicated by an open circle. No significant correlation was present

between osteocalcin and Vo1 for the African-American girls. The 95%

prediction intervals for the entire dataset are shown.

FIG. 3. The rate of calcium gain determined by DXA between the late

prepubertal and early pubertal studies (LatePRE-PUB studies) was

significantly correlated with the bone calcium deposition rate, Vo1, at

the PUB study (y 5 4.3x 1 1345; r 5 0.55; P , 0.001).

1808 ABRAMS ET AL. JCE & M • 2000

Vol 85 • No 5
different levels of calcium intake on calcium absorption or

gain. Calcium intakes were maintained at approximately

1200 mg/day with careful dietary monitoring during the

study. It is important to note that even with intakes at this

level, calcium absorption increased at the PUB compared to

the LatePRE time period.

In comparing the results from this study with our previous

evaluation of calcium absorption in pubertal girls with lower

calcium intakes, we found similar fractional calcium absorption on diets of 900 vs. 1200–1300 mg/day (2). Values for

absorption during puberty in this study were slightly higher

than predicted for this intake by O’Brien et al. (17), which

probably reflects the fact that the girls in this study were all

pubertal, whereas those in the O’Brien report were at various

pubertal stages. Nevertheless, the findings of similar fractional absorption at intakes of 900 and 1200–1300 mg/day

support the concept that increasing calcium intake to 1200–

1300 mg/day in early puberty will be advantageous (6)

We had previously demonstrated from cross-sectional

data an increase in fractional absorption of calcium in puberty and a decrease late in puberty. This study supports

these findings and indicates that the increase in absorption

is an early phenomenon during puberty. This may be analogous to the increase in calcium absorption during pregnancy that occurs during the second trimester before the

maximum utilization of calcium by the fetus during the third

trimester (18).

Serum osteocalcin is often used as a marker of bone formation (19). Little is known, however, about its changes in

early puberty or the relationship between osteocalcin and

kinetic measures of bone calcium utilization. In a study involving both adults and adolescent girls (20), a close relationship was found between serum osteocalcin and Vo1 (r 5

0.82; P , 0.001). This correlation is closer than the one we

found relating those two values (r 5 0.49; P 5 0.003). This is

probably due to the much wider range of ages and, therefore,

Vo1 in the previous study. The inclusion in this study of

African-American girls, who had lower osteocalcin values

than the Caucasian and Hispanic girls, may also have decreased the correlation we found. Nonetheless, our study

shows that bone formation markers are significantly related

to kinetically determined bone calcium deposition in early

puberty, although this relationship is not as close as might be

hoped for clinical utility in relating these values, especially

for African-American girls.

We conclude that longitudinal data demonstrate a change

in bone mineral metabolism during early puberty associated

with maturation of the hypothalamic-pituitary axis and

physical changes of breast development. These changes lead

to increases in multiple aspects of calcium metabolism during early puberty, leading to peak rates of calcium gain

during later pubertal development. Dietary guidelines

should reflect the importance of early puberty in mineral

metabolism. As mean ages of LatePRE and PUB were 9.1 and

10.2 yr, respectively, recent dietary guidelines recommending increased calcium intake beginning at age 9 yr are appropriate (6).

Acknowledgments

We acknowledge the assistance of the nursing staff of the Metabolic

Research Unit of the Children’s Nutrition Research Center and the

General Clinical Research Center of Texas Children’s Hospital for caring

for the study subjects; Leslie Loddeke for editorial assistance; Christopher Branner, Lucinda Clarke, Penni Davila, Dalia Galicia, Becky

Gorham, Ian Griffin, M.D., Kimberly A. Larmore, Lily Liang, Roman

Shypailo, Janice Stuff, Ph.D., Lisa Turner, and Courtnay Wilson for

technical and dietary support; Mercedes Villarreal for study recruitment; and E. O’Brian Smith, Ph.D., for biostatistical review.

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