# 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 metabolismassociated with early pubertal development, we performed longitudinal measurements of calcium absorption, calcium kinetics, bonemineral 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 wereplaced 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 beforethat (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 50.002). 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 ( V

o 1 ) 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 incalcium 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). Themechanisms 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 calciumabsorption and kinetically determined bone calcium deposition rates ( V

o 

### 1 ) 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 ofpubertal development (7). However, the calcium metabolic changes in this transitional age group are poorly described. For example, there are no longitudinal dataregarding 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 bybreast development consistent with Tanner stage 2) is associated with changes in calcium metabolism, and whetherthese 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 V

o 

### 1 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 ResearchService 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 theywere 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 m g) GnRH were administered iv. Blood was collected for
serum LH and FSH at 30 and 60 min after the infusion. After the GnRHwas 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 m g/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, wasperformed 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 spectrometricanalysis as previously described using an oxalate precipitation technique (2–4). Samples were analyzed for isotopic enrichment with aFinnigan 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 differencein 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 fluoroimmunoassaykits 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 Neer et al. (12). The model is based on three sequential pools before calcium deposition in the deep bone Ca pool. Bone Ca deposition ( V

o 1 ) 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 V

o 1 or changes in total body calcium were determined in the cross-sectional analysis using linear and multipleregression 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 didnot 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 estradiollevel 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 calciummetabolic 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 V

o 

### 1 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, V

o 

### 1 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 V

o 

### 1 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 pubertywas 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

V

o 

### 1 at any visit. A significant relationship was found between DTBC2 and

V

o 

### 1 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 studypoints, the baseline or GnRH-stimulated LH level, the estradiol level, the serum alkaline phosphatase activity, or

V

o 

### 1 . 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 8.7 a Urinary Ca excretion (mg/day) 89.0 6 47.6 87.2 6 62.6 102.2 6 65.3
V

o 1 (mg/day) 1504 6 221 1563 6 285 1952 6 407 a 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; V

o 1 , 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 0.8 b Ht (cm) 129.8 6 5.9 133.3 6 6.3 a 139.8 6 6.3 b Wt (kg) 28.6 6 6.0 30.4 6 5.5 a 35.2 6 5.8 b Tanner stage 2 (pubic) 0/26 3/34 17/34 b Baseline LH (ng/mL) 0.06 6 0.06 0.19 6 0.51 0.77 6 1.17 b

Stimulated LH . 5 ng/mL (%)c 12 18 59c Estradiol . 6.3 pmol/L (%) 35 83 a 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 aprolonged 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, calciumgain, and kinetically determined rates of bone calcium deposition. These findings support the importance of advocating adequate mineral nutrition in girls during early puberty andare 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 withphysical changes of puberty and were probably the consequence of these hormonal changes. These increases in calcium gain and bone calcium deposition were paralleled bychanges 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 markedincrease 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, V

o 1 , 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 V

o 1 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, V

o 1 , 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 lowercalcium 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 variouspubertal 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-sectionaldata 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 absorptionis 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 andkinetic measures of bone calcium utilization. In a study involving both adults and adolescent girls (20), a close relationship was found between serum osteocalcin and Vo 1 (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,

Vo 1 in the previous study. The inclusion in this study of African-American girls, who had lower osteocalcin valuesthan 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 leadto 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 caringfor 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 fortechnical and dietary support; Mercedes Villarreal for study recruitment; and E. O’Brian Smith, Ph.D., for biostatistical review.



### References

1. Bonjour J-P, Theintz G, Buchs B, Slosman D, Rizzoli R. 1991 Critical years

and stages of puberty for spinal and femoral bone mass accumulation during adolescence. J Clin Endocrinol Metab. 73:555–563.

2. Abrams SA, Stuff JE. 1994 Calcium metabolism in girls: current dietary intakes

lead to low rates of calcium absorption and retention during puberty. Am J Clin

Nutr. 60:739–743.

3. Abrams SA, O’Brien KO, Stuff JE. 1996 Changes in calcium kinetics associated

with menarche. J Clin Endocrinol Metab. 81:2017–2020.

4. Abrams SA. 1993 Pubertal changes in calcium kinetics in girls assessed using

42Ca. Pediatr Res. 34:455–459.

5. Jackman LA, Millane SS, Martin BR, et al. 1997 Calcium retention in relation

to calcium intake and postmenarcheal age in adolescent females. Am J Clin

Nutr. 66:327–33.

6. Institute of Medicine, Food and Nutrition Board. 1997 Dietary reference

intakes. Washington DC: National Academy Press.

7. Herman-Giddens ME, Slora EJ, Wasserman RC, et al. 1997 Secondary sexual

characteristics and menses in young girls seen in office practice: a study from the Pediatric Research in Office Settings network. Pediatrics. 99:505–512.

8. Ellis KJ, Shypailo RJ, Hergenroeder A, Perez M, Abrams S. 1996 Total body

calcium and bone mineral content: comparison of dual-energy x-ray absorptiometry (DXA) with neutron activation analysis (NAA). J Bone Miner Res.

11:843–848.

9. Ma R, Ellis KJ, Yasumura S, Shypailo RJ, Pinson RN. 1999 Total body calcium

measurements: comparison of two delayed-g neutron activation facilities. Phys
Med Biol. 44:N113–N118.

10. Klein KO, Baron J, Colli MJ, McDonnell DP, Cutler GB. 1994 Estrogen levels in childhood determined by an ultrasensitive recombinant cell bioassay. J Clin

Invest. 94:2475–2480.

11. Gundberg CM, Hauschka PV, Lian JB, Gallop PM. 1984 Osteocalcin: isolation, characterization, and detection. Methods Enzymol. 107:516–44.

12. Neer R, Berman M, Fisher F, Rosenberg LE. 1967 Multicompartmental analysis of calcium kinetics in normal adult males. J Clin Invest. 46:1364–1378.

13. Oerter KE, Uriarte M, Rose SR, Barnes KM, Cutler GB. 1990 Gonadotropin secretory dynamics during puberty in normal girls and boys. J Clin Endocrinol
Metab. 71:1251–1258.

14. Martin AD, Bailey DA, McKay HA, Whiting S. 1997 Bone mineral and calcium accretion during puberty. Am J Clin Nutr. 66:611–615.

15. McKay HA, Bailey DA, Mirwald RL, Davison S, Faulkner RA. 1998 Peakbone mineral accrual and age at menarche in adolescent girls: a 6-year longitudinal study. J Pediatr. 133:682–687.

16. Leitch I, Aitken FC. 1959 The estimation of calcium requirement: a re-examination. Nutr Abs Rev. 29:393–409.

17. O’Brien KO, Abrams SA, Liang LK, Ellis KJ, Gagel RF. 1996 Increased efficiency of calcium absorption during short periods of inadequate intake in girls. Am J Clin Nutr. 63:579–583.

18. Heaney RP, Skillman TG. 1971 Calcium metabolism in normal human pregnancy. J Clin Endocrinol Metab. 33:661–670.

19. Gundberg CM, Niemann SD, Abrams S, Rosen H. 1998 Vitamin K status and bone health: an analysis of methods for determination of undercarboxylated osteocalcin. J Clin Endocrinol Metab. 83:3258–3266.

20. Weaver CM, Peacock M, Martin BR, et al. 1997 Quantification of biochemicalmarkers of bone turnover by kinetic measures of bone formation and resorption in young healthy females., J Bone Miner Res. 12:1714–1720. 

### CALCIUM KINETICS IN EARLY PUBERTY 1809