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Original Studies |
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
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{at}bcm.tmc.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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 Vo+ in early puberty.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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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 µg/kg (maximum, 100 µg) 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 µg/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 µg/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, 3, 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 ß-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 ß-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 Neer et al. (12). The model is based on three sequential pools before calcium deposition in the deep bone Ca pool. Bone Ca deposition (Vo+) 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 = 26 pairs) and the LatePRE-PUB time points (n = 34 pairs). The relationship between hormonal and biochemical parameters and Vo+ 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 ± SD.
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Results |
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). 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 African-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.
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. 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.
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. There was a significant increase in
calcium gain (P < 0.05) between the LatePRE and PUB
studies (
TBC2) compared with the EarlyPRE and LatePRE studies
(TBC1; 135 ± 53 vs. 110 ± 45 mg/day;
P = 0.04). Also shown in this figure is the peak rate
of calcium gain of 212 mg/day reported by Martin et al.
(14).
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), but not the EarlyPRE or LatePRE
visits, Vo+ was significantly correlated to
serum osteocalcin (r = 0.49; P = 0.003 at the PUB
visit; r = 0.27; P = 0.18 at the EarlyPRE visit;
and r = 0.13; P = 0.46 at the LatePRE visit).
Similarly, the relationship between Vo+ and
the total serum alkaline phosphatase activity at that visit was
significant (r = 0.40; P = 0.02).
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TBC2
and the baseline LH level (r = 0.50; P = 0.01). In
contrast, the estradiol values were not correlated to
Vo+ at any visit.
A significant relationship was found between TBC2 and
Vo+ at the PUB study (r = 0.55;
P < 0.001; Fig. 3) and
between TBC2 and serum alkaline phosphatase activity at the PUB
study (r = 0.49; P < 0.01). In contrast, TBC2
was not significantly correlated to serum osteocalcin at the PUB study
(r = 0.08; P = 0.65).
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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 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.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We found that changes in calcium metabolism and utilization were associated with maturation of the hypothalamic-pituitary 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 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 Vo+ (r = 0.82; P < 0.001). This correlation is closer than the one we found relating those two values (r = 0.49; P = 0.003). This is probably due to the much wider range of ages and, therefore, Vo+ 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).
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Acknowledgments |
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Footnotes |
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Received September 21, 1999.
Revised December 1, 1999.
Accepted December 17, 1999.
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References |
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neutron activation facilities. Phys Med Biol.
44:N113–N118.
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