Clinical Biochemistry 44 (2011) 771–778

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Clinical Biochemistry

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Establishment of reference intervals for bone markers in children and adolescents

Yun Huang a, Esther Eapen a, Susan Steele b, Vijaylaxmi Grey a,b,⁎a Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canadab Department of Pediatrics, McMaster University, Hamilton, ON, Canada

⁎ Corresponding author at: Department of PatholoMcMaster University Medical Centre, Rm. 2N30A, 120Canada L8N3Z5. Fax: +905 521 2338.

E-mail address: grey@hhsc.ca (V. Grey).

0009-9120/$ – see front matter © 2011 The Canadian Sdoi:10.1016/j.clinbiochem.2011.04.008

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 7 January 2011Received in revised form 30 March 2011Accepted 11 April 2011Available online 22 April 2011

Keywords:Bone markersVitamin DReference intervalsChildrenAdolescents

Objectives: To determine age and gender specific reference intervals for bone markers.Design and methods: Morning blood samples were collected after overnight fast from 356 healthy

children (6 to 18 year-old) for the determination of bone marker levels, PTH and vitamin D3. Multipleregression analysis was done to assess the effect of factors that could influence the bone marker levels; thecentral 95% reference interval and their 90% CI were calculated.

Results: After excluding samples when BMI-z —scores were b−2 or N+2, both vitamin D3 and PTH levelswere abnormal and from children who used steroids the remainder were partitioned using Tanner stagebased chronological age. As expected the reference intervals show a significant variation with age and gender.All the bone marker levels, except total alkaline phosphatase, were significantly higher in puberty withvitamin D3 N75 nmol/L.

Conclusion: Vitamin D status, age and gender are important for establishing reference intervals of bone

markers in healthy children.

© 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Introduction

Bonemetabolism in children differs from that of adults, as it reflectsboth skeletal growth and remodeling. During remodeling, boneformation and resorption are tightly coupled whereas during skeletalgrowth new bone is formed at a site different to that of resorption. Bonemineralization is dependent on adequate intakes of calcium andphosphate. The absorption of calcium is dependent on the vitamin Dsufficiency. In a vitamin D sufficient state, net calcium absorption isapproximately 30% and during active growth it is 60–80%. In vitamin Ddeficiency, calciumabsorption is reduced andphosphate reabsorption isdecreased resulting in diminished levels of these essential constituentsfor bone mineralization [1].

Traditionally, the assessment of bone health is done usingdensitometry techniques, including the gold standard dual-energyx-ray absorptiometry (DXA). However, these measures do not reflectthe dynamic nature of bone tissue [2]. In contrast, biochemicalmarkers of bone metabolism provide a more dynamic picture of thechange during both bone growth and modeling, and their measure-ment may be useful in the assessment of metabolic bone diseases andgrowth disorders in children [2–4]. Bone markers are classified

gy and Molecular Medicine,0 Main St. W, Hamilton, ON,

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according to the processes they reflect, namely formation andresorption. Bone formation markers are formed during differentphases of osteoblast development and reflect osteoblast function andbone formation. These markers include bone alkaline phosphatase(BAP), osteocalcin, and procollagen 1 peptides. The resorptionmarkers are products of bone type 1 collagen degradation, mainlypeptides and small molecules. Hydroxyproline, pyridinoline, anddeoxypyridinoline are degradation products found in the urine, whileN-terminal and C-terminal crosslinked peptides can be found in bothserum and urine. The difficulty of obtaining serial urine collectionscoupled with the large intra-individual variation limits the utility ofurine markers. The N-terminal and C-terminal crosslinked peptidesare commonly used bone resorption markers [5–7].

The widespread applicability of these markers is however limitedby the availability of normative data. Compared to adults, childrenhave dramatically elevated bone marker levels due to their skeletalgrowth velocity and rapid bone turnover. It has been recognized, thatto correctly interpret values of the markers of bone turnover, both ageand pubertal development are necessary, but very few studies havetaken into account the nutritional status of the healthy child,especially vitamin D on the levels of bone markers. Optimal nutritionis also important for the acquisition of peak bone mass, and there isincreasing awareness of vitamin D insufficiency in northern climateseven in otherwise healthy children [8], which may put children at riskfor suboptimal accretion of bone mineral content. In this report, wedetermined the reference intervals of bone markers of formation(alkaline phosphatase, bone alkaline phosphatase, osteocalcin, andprocollagen N-terminal peptide), and of resorption, Elecsys β-cross-laps in children and adolescents. In addition to age and gender, we

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772 Y. Huang et al. / Clinical Biochemistry 44 (2011) 771–778

examined the effect of nutritional status (BMI, vitamin D3, and PTH)on bone marker levels, and calculated robust pediatric referenceintervals according to the recommendations in CLSI 28-A3 guidelinesto reduce the influence of small sample size.

Materials and methods

This studywas approved by the Research Ethics Board of McMasterUniversity. Children were recruited from child daycare centers,schools, religious institutions, community centers and children ofhospital workers in Hamilton, Ontario, Canada. Inclusion criteria arehealthy boys and girls six to 18 years old. Written informed consentwas obtained from participants or their parents.

Information collection

At the time of the blood draw, general information on healthhistory was collected by a questionnaire including age, gender,prescribed medications and vitamins or other supplements. A selfassessment on puberty development was also done by participants orin the case of the young children by their parents using illustrationsand descriptions of the five Tanner stages as published for breast sizeand pubic hair development for girls and genital size and pubic hairfor boys [9–11]. Participants' height and weight were measured bytrained project nurses or coordinators to the nearest 0.1 kg and0.1 cm. The measurement followed the standard procedures inpediatrics. BMI z-scores were calculated by a web-based program(www.bcm.edu/bodycomplab/mainbodycomp.htm).

Sample collection

Before sample collection, participants were given verbal instruc-tion on overnight fasting by the project coordinators. A blood samplewas collected from each individual by project nurses or phlebotomist.The volume of sample collectedwas 5–10 mL from children older than7 y, and 2–5 mL from children b7 y. To minimize the effects of diurnalvariation on bone marker measurement, samples were collectedbetween 7:00 to 9:00 am. Sample transportation and serum separa-tion were completed within two hours after collection. Aliquots ofserum were stored at −80 °C, equipped with an alarm system tomonitor temperature control. All analyses were completed within oneyear of collection. Sample collection, transportation, separation andstorage were performed according to standard laboratory procedures.

Assays

Total alkaline phosphatase activity (ALP) was determined by acolorimetric assay on the Roche P-modular automated analyzer (RocheScientific). The β-CrossLaps, total type I procollagen N-terminal peptide(TPINP), Osteocalcin, PTH and vitamin D3 measurement were per-formed by chemiluminescence immunoassays (Roche Scientific,Elecsys). The sandwich test principle is used for the β-CrossLaps,TPINP, Osteocalcin, PTH and a competitive binding assay is used forVitamin D3. The Elecsys β-CrossLaps/serum assay quantifies all type Icollagendegradation fragments that contain the isomerizedoctapeptideβ-8AA twice (β-CTx) and has an analytical CV of 5%. The Elecsys N-MIDOsteocalcin assay uses two monoclonal antibodies specifically directedagainst epitopes on the N-MID-fragment and the N-terminal-fragment.Hence, it detects both the stable N-MID-fragment as well as the stillintact osteocalcin and has an analytical CV of 2%. The Elecsys total PINPassay detects both the trimeric structure (derived from the trimericcollagen structure) and themonomeric formpresent inbloodandhas ananalytical CV of 2%. The Elecsys vitamin D assay measures the 25OHvitamin D3 form present in most fortified foods and supplements inCanada and has an analytical CV of 10%. Bone alkaline phosphatase wasmeasured by Ostase BAP, a solid phase monoclonal antibody immu-

noenzymetric method (Immunodiagnostics Systems, Boldon, UK) andwith an analytical CV of 8%.

Data analysis and statistics

Multiple regression analysis was used to identify the covariatesthat may influence bone marker values. The covariates included boththe genetic factors such as age and gender, and environmental factorssuch as BMI-z score, Vitamin D3 and parathyroid hormone (PTH). A 5%level of significance was used to indicate the factors influencing thevalues of bone markers. In data collection, the information of bothchronological age and Tanner stage were obtained for only some ofthe children (60.0%, 6–18 year-old in girls and 52.5%, 6–18 year-old inboys). We used this information to calculate the mean chronologicalage and standard deviation of the children for each Tanner stage. Thedata was then partitioned using the Tanner stage based chronologicalage and gender to reflect the trend of bone growth and development.

Data normal distributionwas determined by Kolmogorov-Smirnovtest. Outliers were detected by Dixon/Reed method. The confirmationof data partition and the calculation of central 95% reference intervaland their 90% CI were performed as the instruction of CLSI 28 A3guidelines [12]. In the partitioning between two adjacent groups, theguidelines recommend if calculated z is more than z* or if the standarddeviation of one group is 1.5 times more than the standard deviationof another group, the two groups need to be partitioned. Otherwise,the two groups are combined [13]. Since most of the sample sizes inour study were less than 120, we applied the robust method forreference interval determination as recommended by the guidelines,using an Excel macro courtesy of A.J. Pesce, P. Horn and D. Lewis to theCALIPER (Canadian Laboratory Initiative for the Establishment ofPediatric Reference Intervals) group. The statistical software used formultiple regression analysis and data description were SPSS 15.0(SPSS Inc., Chicago ILL) and Microsoft EXCEL.

Results

Samples were obtained from a total of 356 children, 182 girls and174 boys, age 6–18 years. Sixteen subjects used anti-asthma medica-tion of whom ten were on inhaled steroids. These subjects wereeliminated from the analysis. One subject on thyroid medication wasalso eliminated from the analysis.

The results of the multiple regression analysis are shown inTable 1. The variable BMI-z score affected the value of osteocalcin butafter removing 53 samples with BMI-z score more than +2 and lessthan −2, there was no significant effect of BMI on the values of bonemarkers (pN0.05). Vitamin D3 and PTH affected the values of TPINP,osteocalcin and β-Crosslaps and BAP. After removing five sampleswith both a low Vitamin D3 (equal to or lower than 50 nmol/L) andhigh PTH (higher than its 97.5th (7.66 pmol/L)), the effect of VitaminD3 and PTH on the levels of bone marker still persisted (pb0.05). Ageand gender also had a significant effect on the bone markers. Theremaining number of participants included for the analysis ofreference intervals of the bone markers is shown in Fig. 1. Themeasurement of five bone markers was not done on all samplesbecause of insufficient sample volume.

Table 2 shows the mean chronological ages for the Tanner stagesbased on the self assessment questionnaire of 50% of the participantsfrom age 6 to age 18. The chronological age groups were initially usedin data partition and adjacent groups were combined unless they metthe criteria for partitioning. The robust reference intervals of five bonemarkerswere then calculated. The results are presented in Table 3 andFig. 2. The values of bone markers changed significantly with differentage and gender.

Overall the vitamin D status was normal in the majority of thesechildren [14]. Sixty percent of the subjects had vitamin D levels75 nmol/L, thirty percent had levels between 50 and 75 nmol/L. The

Table 1Multiple regression analysis of affecting factors on bone markers.

Bone markers Coefficients beta Standard error Significance

Osteocalcin(n=295)

Age −4.84 0.76 b0.001Sex −16.23 5.12 b0.01BMI-z score −5.08 2.03 b0.05VitD3 0.30 0.09 b0.01PTH 7.37 1.86 b0.001

BAP(n=290)

Age −6.23 0.58 b0.001Sex −22.90 3.96 b0.001BMI-z score 0.05 1.56 N0.05VitD3 −0.01 0.07 N0.05PTH 3.48 1.43 b0.05

ALP(n=263)

Age −14.25 1.42 b0.001Sex −47.20 9.65 b0.001BMI-z score 1.78 3.92 N0.05VitD3 −0.08 0.18 N0.05PTH 4.86 3.47 N0.05

TPINP(n=280)

Age −46.70 4.65 b0.001Sex −118.41 31.24 b0.001BMI-z score −2.59 12.74 N0.05VitD3 1.09 0.59 N0.05PTH 25.67 11.57 b0.05

β-CrossLaps(n=281)

Age −0.06 0.01 b0.001Sex −0.34 0.06 b0.001BMI-z score −0.03 0.02 N0.05VitD3 0.00 0.00 b0.05PTH 0.07 0.02 b0.01

ALP: alkaline phosphatase. BAP: bone alkaline phosphatase. TPINP: total type Iprocollagen.N-terminal peptide.

Table 2The relationship between Tanner stage and chronological age of girls and boys.

Gender Tannerstage

Mean of age(year)

SD Tanner stage based chronologicalage group (year)

Girls 1 10.0 2.1 6–102 11.1 1.9 ~113 13.7 2.1 ~144 14.2 2.1 ~155 15.2 1.7 N15

Boys 1 8.7 2.5 6–92 11.2 1.4 ~113 13.6 1.2 ~144 14.7 1.3 ~155 16.6 1.6 N15

773Y. Huang et al. / Clinical Biochemistry 44 (2011) 771–778

effect of vitamin D on the bonemarkers and PTHwas further exploredusing regression analysis and the results are shown in Table 4. At alllevels of vitamin D (low- b50 nmol/L, moderate- 50–75 nmol/L, andhigh- N75 nmol/L) bone markers were significantly associated withage, whereas only ALP, BAP and β-Crosslaps were associated withgender. Furthermorewhenwe compared the effect of vitaminD on thebone marker levels of the different age groups corresponding to pre-puberty (6–10 y), puberty (11–15 y) and late puberty (N15 y) an effectof Vitamin D can be seen during puberty (Fig. 3). At values of vitamin

356 (Total)

345 (for multiple regression

Excludedand 1 par

ExcludedExcluded<50nmol/

Osteocalcin 237

ExcludedOutlier 1

236

BAP 232 ALP 205

ExcludedOutlier 1

ExcOut

231

Data partitioEstablishment of refere

204

Fig. 1. Participant recruitment. ALP: alkaline phosphatase. BAP: bone alkaline phosphatase.analyzed because of insufficient sample volume.

D3N75 nmol/L, all the bone marker levels except ALP were signifi-cantly higher (pb0.01 or 0.001). For all tertiles of vitamin D, the PTHlevels remained within the reference range but when the vitamin D isless than 50 nmol/L, the PTH levels were significantly higher (pb0.05)in all the age categories.

Discussion

In this paper we report on pediatric reference intervals of fourbone markers of formation (osteocalcin, TPINP, BAP and ALP) and oneresorption bone marker (β-CrossLaps). The data was first screened toeliminate samples from individuals with extreme values of BMI z-score, vitamin D and PTH who were otherwise healthy. Thechronological age used for partitioning were based on pubertaldevelopment, as suggested by Tanner stage self assessment. This wasconfirmed by the Harris–Boyd method and reference intervals werecalculated by the robust method. The reference intervals show asignificant variation with age and gender as would be expected withgrowth andmetabolism during childhood and adolescence. Vitamin Dlevels also had a significant effect on the levels of bone markersespecially during puberty.

analysis)

10 participants using steroid ticipant using Synthroid

53 participants with BMI <-2 or >+2 5 participants with both Vitamin D L and PTH >7.66 pmol/L

β-CrossLaps 223

ludedlier 1

ExcludedOutlier 1

ExcludedOutlier 1

n nce intervals

TPINP 222

221 222

TPINP: total type I procollagen N-terminal peptide. A smaller number of samples were

Table 3Pediatric reference intervals of five bone markers.

Bone markers Gender Age Sample size 2.5th percentile 97.5th percentile 90% CI for lowlimit

90% CI for highlimit

Osteocalcin (ng/mL) Girls 6~10 y 33 61.4 136.2 46.0 71.3 129.2 144.5N10–14 y 49 24.1 232.1 19.2 30.7 205.2 254.6N14–15 y 16 17.8 119.6 12.2 21.2 93.4 134.0N15 y 37 21.1 76.7 19.2 23.8 65.4 83.9

Boys 6–9 y 30 56.5 152.1 49.5 63.7 137.8 164.0N9–15 y 51 48.2 226.4 36.5 61.5 207.1 247.7N15 y 20 22.5 151.3 18.8 25.8 115.7 176.0

BAP (U/L) Girls 6–11 y 41 23.5 151.1 17.1 48.6 137.8 158.5N11–14 y 38 20.8 172.3 17.2 23.7 138.7 200.0N14y–15 y 16 12.6 105.8 8.9 18.0 69.4 122.9N15y 37 8.1 43.9 6.4 10.4 37.6 48.3

Boys 6–9 y 30 51.0 164.3 44.2 58.4 135.3 191.4N9–11 y 16 65.6 138.2 53.9 76.2 124.8 147.8N11–15 y 34 45.5 208.4 38.3 55.7 178.8 231.3N15 y 19 13.1 80.0 7.6 19.3 56.3 101.1

ALP (U/L) Girls 6–11 y 36 157 359 149 164 334 384N11–14 y 34 60 401 46 80 351 432N14–15 y 13 50 232 41 61 185 264N15y 28 26 110 18 34 103 121

Boys 6–15 y 75 136 414 127 147 388 438N15 y 18 50 251 38 68 174 292

TPINP (ng/mL) Girls 6–10 y 33 411 1022 386 446 956 1081N10–11 y 10 0 1451 0 350 1087 1688N11–14 y 36 109 1346 92 138 1122 1441N14–15 y 15 38 510 18 56 365 574N15 y 30 49 277 43 56 183 328

Boys 6–11 y 45 407 1079 385 441 983 1146N11–14 y 27 339 1399 253 381 1237 1454N14–15 y 6 0 1217 n/a n/a n/a n/aN15 y 19 61 718 50 74 533 831

β-CrossLaps (ng/mL) Girls 6–10 y 33 0.82 2.06 0.61 1.00 1.98 2.15N10–14 y 48 0.49 2.76 0.39 0.65 2.37 3.07N14–15 y 15 0.12 1.73 0.00 0.35 1.45 1.83N15 y 30 0.00 1.59 0.00 0.00 1.32 1.80

Boys 6–9 y 29 1.05 2.38 0.95 1.14 2.19 2.52N9–15 y 48 1.00 2.90 0.93 1.12 2.73 3.03N15 y 19 0.50 2.43 0.41 0.57 1.85 2.76

ALP: alkaline phosphatase. BAP: bone alkaline phosphatase. TPINP: total type I procollagen N-terminal peptide.

774 Y. Huang et al. / Clinical Biochemistry 44 (2011) 771–778

The selection of representative population is one of the key steps inthe establishment of a proper reference interval. The results of multipleregression suggest that BMI z-score influences the osteocalcin levels andwhen the extreme values BMI z-score, b−2 or N+2 were eliminated,this effect was lost. The relationship between adiposity and bone hasbeen suggested in several studies. Increasing adiposity has beensuggested as a risk factor for fracture in children [14] and recent studiespoint to possible role of osteocalcin in energy metabolism [15]. Morerecently Dimitri et al. demonstrated that bone formation relative toresorption is reduced in obese children, and osteoprotegerinmay play afundamental role in lower bone mass of obese children [16].

Age, gender and pubertal stage also influenced the bone markerresults, as has been shown by others [2]. Our initial approach togrouping the data was to classify them according to Tanner stage. Ourresults showed that the pubertal development in boys and girls beganat similar age and boys reached adult pubertal stage one year laterthan girls, similar to that observed by others [17,18]. The results showthat the bone markers vary with pubertal growth, with values beinghigher in early puberty and the values for girls declined earlier thanfor the boys. The pattern of change differs for each bone marker andmay represent the array of physiological factors that affect theseparameters. This increase in bone marker level associated with thepubertal growth spurt has been described by others [19,20].

The PTH levels were within normal (b6.9 pmol/L) in prepubertal,pubertal and late puberty but at higher vitamin D levels (N75 nmol/L)the PTHvaluewas significantly lower than that at lower vitaminD levels(b50 nmol/L). For youngpre-pubertal children, the PTH levelswere also

significantly higher with vitamin D levels between 50 and 75 nmol/L.The relationship of vitamin D and bonemarkers have remained unclear[21,22]. In adults higher levels of vitamin D result in lower boneturnover. In children both bone acquisition and bone remodeling occurespecially during active growth in puberty. Cashman (22) suggestedthat higher levels of vitamin D may be important to improving bonehealth. In our study, during puberty the bone marker levels weresignificantly higher (pb0.01 for osteocalcin, TPINP, and β-Crosslaps andpb0.05 for BAP) when the vitamin D levels were N75 nmol/L. This mayreflect the importance of vitamin D in assuring peak bone massacquisition. During the pubertal years, calcium needs are increased andvitamin D sufficiency will be important to allow for optimal calciumabsorption [1]. The higher levels of bonemarkers when vitamin D levelsare greater than 75 nmol/L may therefore reflect the increasedavailability of calcium for optimal bone accretion. These resultsemphasize the importance of vitaminD status on the reference intervalsfor bonemarkers. This is significant if bonemarkers are going to be usedto assess skeletal health and prevent secondary effects such asosteoporosis that may occur in certain malabsorption syndromes, orgrowth hormone deficiency.

One of the challenges in the establishment of reference interval inchildren is the difficulty in obtaining the 120 subjects as set out in theCLSI guideline. To overcome this we used the robust method for thecalculation of central 95th percentile in reference intervals in thisstudy. It involves an iterative process in which observations are downweighted according their distance from the center and can generatereliable results with small sample size [23].

775Y. Huang et al. / Clinical Biochemistry 44 (2011) 771–778

In summary, we established pediatric bone marker referenceintervals which might better reflect bone growth and metabolism inchildhood and adolescence. The results suggest that vitamin D status isimportant to establish normative data especially during puberty.

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Fig. 2. Left: Scatterplot of the bone markers values. Right: Tanner based chronological age chPartition criteria were done according to the CLSI guidelines and the Harris Boydmethod [12,phosphatase, C —Alkaline Phosphatase, D —TPINP: total type I procollagen N-terminal pept

Acknowledgments

This study was supported by a grant from the McMaster Children'sHospital Research Institute, the Gillian LuxtonMemorial Fund, and the

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anges of bone marker levels. Data were expressed by mean±standard deviation (SD).13]. Groups were divided when: SD1N1.5 SD2 or zNz*. A—osteocalcin, B—Bone alkalineide, E —β-CrossLaps.

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Table 4The association of bone markers values with age and gender at different vitamin D concentration.

Vitamin D b50 nmol/L Vitamin D 50–75 nmol/L Vitamin D N75 nmol/L

Coefficients beta Standard error Significance Coefficients beta Standard error Significance Coefficients beta Standard error Significance

Osteocalcin n=48 n=67 n=121Age −8.78 1.85 b0.001 −5.73 1.20 b0.001 −3.19 1.18 0.008Gender 24.19 12.38 N0.05 21.52 10.83 N0.05 9.89 7.77 N0.05

TPINP n=36 n=66 n=119Age −61.59 14.31 b0.001 −58.43 6.66 b0.001 −45.36 6.71 b0.001Gender 267.89 87.72 b0.01 86.10 53.96 N0.05 42.79 44.11 N0.05

ALP n=33 n=59 n=112Age −18.16 5.63 b0.01 −17.31 2.30 b0.001 −14.71 1.85 b0.001Gender 89.40 30.73 b0.01 57.64 18.62 b0.01 25.46 12.32 b0.05

BAP n=47 n=66 n=118Age −9.63 2.07 b0.001 −7.48 0.90 b0.001 −5.39 0.78 b0.001Gender 36.73 11.85 b0.01 30.26 7.34 b0.001 13.48 5.17 b0.05

β-CrossLaps n=37 n=65 n=120Age −0.09 0.03 b0.01 −0.08 0.01 b0.001 −0.04 0.01 b0.01Gender 0.47 0.15 b0.01 0.31 0.11 b0.01 0.25 0.09 b0.01

776 Y. Huang et al. / Clinical Biochemistry 44 (2011) 771–778

Fig. 3. The influence of vitamin D status on the levels of PTH and bonemarkers. White bar: vitamin D b50 nmol/L; striped bar: vitamin D 50–75 nmol/L; black bar: vitamin D N75 nmol/L.Bonemarkervalues:mean±standarddeviation (SD)a:whencompared to vitaminDb50 and50–75 nmol/L group, pb0.001;b:when compared tovitaminDb50 nmol/L, pb0.01; c:whencompared to vitamin D 50–75 nmol/L group, pb0.05. ALP: alkaline phosphatase. BAP: bone alkaline phosphatase. TPINP: total type I procollagen N-terminal peptide.

777Y. Huang et al. / Clinical Biochemistry 44 (2011) 771–778

Department of Pathology and Molecular Medicine. We thank Dr. M.J.McQueen and the technical staff at both the clinical trials researchlaboratory, and McMaster University Medical Centre, Hamilton forassistance with the analysis. Roche Diagnostics supplied the reagentsfor the Osteocalcin, TPINP, β-Crosslaps, vitamin D3, and PTH. We aregrateful to all the community volunteers who participated in thisstudy.

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