CTX, N-Mid Oscteocalcin5

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Biochemical Markers of Bone Turnover andResponse of Bone Mineral Density to Interventionin Early Postmenopausal Women: An Experience

in a Clinical LaboratoryLa-or Chailurkit,* Boonsong Ongphiphadhanakul, Noppawan Piaseu, Sunee Saetung,

and Rajata Rajatanavin

Background: Markers of bone formation and resorptionmay be useful as early indicators of response to therapy.

Our aim in this study was to investigate the use of bone

markers for monitoring of intervention for bone loss in

early postmenopausal women and to assess the relation-

ships between these markers and changes in bone

mineral density (BMD).

Methods: Subjects were randomly assigned to the fol-lowing groups: a control group; a group receiving cal-

cium alone; groups receiving calcium plus low or con-

ventional doses of conjugated equine estrogen; and

groups receiving calcium plus low or conventional

doses of calcitriol. At baseline and at 1 and 3 monthsafter intervention, we measured serum intact osteocal-

cin, serum N-terminal midfragment osteocalcin, serum

C-terminal telopeptide of type I collagen (CTx), urinary

deoxypyridinoline cross-links, and urinary CTx. The

BMD of the lumbar spine and the femoral neck was

measured at baseline and after 1 and 2 years of inter-

vention.

Results: No marker changed significantly in the controlgroup except urinary CTx, which increased at 3 months.

Serum CTx decreased in all regimens at 1 or 3 months of

intervention. In addition, the changes of all markers at 3

months were inversely associated with the change in the

BMD of the lumbar spine at 1 or 2 years (r 0.144 to0.314), whereas only the changes of bone resorption

markers at 3 months were inversely correlated with the

changes in femoral BMD at 1 or 2 years (r 0.143 to0.366).

Conclusions: Biochemical markers of bone turnoverappear to be of use in assessing early response totherapy. Bone resorption markers, especially serumCTx, are better indicators than bone formation markersfor estimating the response to intervention in earlypostmenopausal women. However, the early changes inbone markers were weakly related to the later changesin BMD. 2001 American Association for Clinical Chemistry

To reduce accelerated bone loss in early postmenopause(15) and to minimize the risk of fracture, a variety ofantiresorptive treatments (e.g., calcium, calcitonin, estro-gens, and bisphosphonates) have been used. A positiveresponse to therapy can be assessed by the measurementof bone mineral density (BMD),1 but a statistically signif-icant change usually requires 1 or more years because ofthe imprecision of the BMD measurement (6, 7 ). Thislimitation may be avoided by use of biochemical markersof bone turnover in the serum or urine, which can showthe effects of intervention sooner. In addition, bone turn-over markers provide a more representative indicator ofoverall skeletal bone loss than the results obtained bymeasuring rates of change in BMD at a single skeletal site.The ability to determine a positive response to therapy inadvance of BMD facilitates the clinical decision to alter apatients regimen or dosage of medication to achieve thedesired antiresorptive effect. Assays for several biochem-

Division of Endocrinology and Metabolism, Department of Medicine,Ramathibodi Hopital, Mahidol University, Rama VI Road, Bangkok 10400,

Thailand.*Author for correspondence. Fax 66-2-201-1715; e-mail [email protected] November 20, 2000; accepted April 5, 2001.

1 Nonstandard abbreviations: BMD, bone mineral density; OC, osteocalcin;

CTx, C-terminal telopeptide of type I collagen; DPD, deoxypyridinolinecross-links; N-mid OC, N-terminal midfragment osteocalcin; and Cr, creati-nine.

Clinical Chemistry 47:610831088 (2001) Endocrinology and

Metabolism

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ical markers of bone turnover are commercially available,but their relative sensitivities are unclear. In this study,we investigated, in a clinical laboratory setting, biochem-ical markers of bone turnover for monitoring of severaltypes of intervention for early postmenopausal bone lossand evaluated whether the responsiveness of biochemicalmarkers is related to the response in BMD.

Materials and Methods

subjects

Subjects consisted of 214 healthy postmenopausal women(time after menopause 6 years). None was taking anymedications that might interfere with bone metabolism.None had diseases known to affect skeletal turnover. Theparticipants were randomly divided into six groups.Group 1 (n 46) included untreated controls. Group 2(n 36) received 750 mg of calcium (calcium carbonatecapsule) supplementation daily. Group 3 (n 34) re-ceived 750 mg of calcium plus 0.3 mg of conjugatedequine estrogen (Wyeth-Ayerst) daily together with 5 mg

of medrogestrone (Wyeth-Ayerst) 12 days per month.Group 4 (n 33) received the same treatment as group 3,except that the estrogen dose was increased to 0.625 mgdaily. Group 5 (n 33) received 750 mg of calcium plus0.25 g of calcitriol (Roche) supplement daily. Group 6(n 32) received 750 mg of calcium plus 0.5 g ofcalcitriol supplement daily. The study was approved bythe Ethical Clearance Committee on Human Rights re-lated to research involving human subjects of the Facultyof Medicine, Ramathibodi Hospital, Mahidol University.Informed consent was obtained from all participantsbefore a course of treatment was initiated.

laboratory assays

Blood was collected between 0700 and 0930 after anovernight fast. Urine was collected over a 24-h period.Blood and urine samples were collected from all subjectsbefore and 1 and 3 months after initial intervention.Serum and urine samples were frozen at 80 and20 C,respectively, until analysis. Serum intact osteocalcin (OC;NovoCalcin; Metra Biosystem), urinary deoxypyridino-line cross-links (DPD; Pyrilinks-D; Metra Biosystem),and urinary C-terminal telopeptide fragment of type Icollagen (CTx; -CrossLaps; Roche Diagnostics, Mann-heim, Germany) were measured by competitive enzymeimmunoassay. Serum N-terminal midfragment OC (N-mid OC; N-MIDTM Osteocalcin; Roche Diagnostics) was

measured by sandwich enzyme immunoassay. SerumCTx (-CrossLaps; Roche Diagnostics) was determined bysandwich electrochemiluminescence immunoassay. Allresults for urinary markers were expressed relative tourinary creatinine (Cr). Blood and urine samples collectedbefore and after intervention were analyzed in the sameassay. The within-assay CVs for serum intact OC (meanconcentration, 6.5 g/L), serum N-mid OC (16.0 g/L),urinary DPD (4.0 mol/mol of Cr), urinary CTx (5.5mol/mol of Cr), and serum CTx (0.41 g/L) were 13%,

3.6%, 5.2%, 4.3%, and 3.4%, respectively, and the between-assay CVs were 11%, 12%, 7.8%, 4.7%, and 4.1%.

bmd measurements

BMD was measured by dual-energy x-ray absorptiometry(Lunar DPX-L; Lunar Corp.). Daily calibration and qualitycontrol were performed regularly according to the man-

ufacturers instructions. The BMD of the anteroposteriorlumbar spine (L2L4) and the femoral neck was measuredfor all subjects at baseline and at 1 and 3 months ofintervention. In vivo CVs for these sites were 1.2% and1.6%, respectively.

statistical analyses

We analyzed the actual values and the percentages ofchange from baseline of the bone markers and BMD.Descriptive results are presented as the mean SE. Thenormality of results was assessed by the KolmogorovSmirnov test. Differences between baseline and after-treatment values were analyzed by repeated-measures

ANOVA for parametric and the Friedman test for non-parametric variables. The Student paired t-test or theWilcoxon rank-sum test was used to test the significancewithin a group if there was a time-related change withinthe group by repeated-measures ANOVA or the Fried-man test. To correct for multiple testing, P was adjustedaccording to the Bonferroni correction. Associations be-tween markers were identified by Spearman correlations.All analyses were performed using SPSS/PC (Release9.0).

Results

In the untreated (control) postmenopausal women in this

study, the mean values for all markers of bone remodelingat 1 or 3 months of treatment were not significantlydifferent from baseline (Table 1). However, the meanvalue of urinary CTx was significantly increased at 3months. In group 2, who received only calcium supple-mentation, the mean values of serum N-mid OC and CTxwere significantly decreased at 3 months and at 1 and 3months of treatment, respectively. In groups 3 and 4, whoreceived low and traditional doses of estrogen, the meanvalues of all bone markers decreased consistently inresponse to treatment. Significant decreases were seen at1 month for serum intact OC, urinary CTx, and serumCTx in group 3 and for urinary DPD, urinary CTx, andserum CTx in group 4. After 3 months of treatment, all

markers in both groups except urinary DPD in group 3were significantly decreased from baseline values. Ingroups 5 and 6, who received low and traditional doses ofcalcitriol, only urinary CTx and serum CTx were signifi-cantly decreased after 1 and 3 months of intervention. Inaddition, in group 6, a significant decrease was found at 3months for serum N-mid OC.

The mean BMD of either the lumbar spine or thefemoral neck of the untreated (control) and the calcium-treated postmenopausal (group 2) groups was slightly

1084 Chailurkit et al.: Bone Markers in Prediction of BMD


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decreased at 1 or 2 years compared with baseline values(Table 2). In contrast, in the estrogen-treated postmeno-pausal groups (groups 3 and 4), the mean BMD of theboth the lumbar spine and the femoral neck was increasedafter 1 or 2 years of intervention, but a significant increase

in the mean BMD from baseline was observed only at thelumbar spine after 1 year of intervention for group 3 andafter 2 years of intervention for group 4. In the calcitriol-treated groups (group 5 and 6), there were no significantchanges of BMD in either the lumbar spine or the femoralneck after intervention compared with baseline.

Associations between the percentages of change inbone markers and the percentages of change in BMD afterintervention are shown in Table 3. At 1 month, there wasno correlation between serum intact OC, N-mid OC, or

urinary DPD and the long-term BMD change in both theanteroposterior lumbar spine and the femoral neck. Thechange in urinary CTx at 1 month was inversely corre-lated only with the change in femoral BMD at 1 year, andthe change in serum CTx at 1 month was inversely

correlated with the lumbar spine BMD at 1 or 2 years andfemoral BMD at 1 year. At 3 months, for the changes ofbone formation markers, serum intact OC and N-mid OCwere inversely correlated with the changes in the BMD ofthe lumbar spine at 1 or 2 years. Only the change in N-midOC was related to the change in femoral BMD only at 1year. With regard to bone resorption markers, the changesof urinary DPD, urinary CTx, and serum CTx at 3 monthswere inversely correlated with the BMD of the lumbarspine or femoral neck at both 1 and 2 years.

Table 1. Responses of biochemical markers of bone turnover to treatments.

Baselinea

After treatmenta

1 month 3 months

Controls

Serum intact OC, g/L 10.47 0.65 10.36 0.58 10.82 0.61

Serum N-mid OC, g/L 20.73 1.42 20.94 1.46 20.79 1.49

Urinary DPD, mol/mol Cr 4.79 0.24 4.74 0.33 5.40 0.31Urinary CTx, mol/mol Cr 5.20 0.35 5.43 0.43 6.51 0.60b

Serum CTx, g/L 0.42 0.02 0.43 0.03 0.45 0.04

Group 2


Serum N-mid OC, g/L 17.55 1.17 17.47 1.31 15.71 1.05b

Urinary DPD, mol/mol Cr 4.76 0.28 4.02 0.23 4.47 0.35

Urinary CTx, mol/mol Cr 5.37 0.50 4.70 0.59 4.39 0.40

Serum CTx, g/L 0.46 0.04 0.37 0.03b 0.36 0.03b

Group 3

Serum intact OC, g/L 9.98 0.66 9.01 0.71b 7.66 0.60b

Serum N-mid OC, g/L 16.27 1.40 14.87 1.27 11.80 0.97b


Urinary CTx, mol/mol Cr 4.21 0.24 3.72 0.30b 3.83 0.32b

Serum CTx, g/L 0.43 0.04 0.24 0.03b 0.18 0.02b

Group 4

Serum intact OC, g/L 8.70 0.73 7.18 0.82 6.47 0.53b

Serum N-mid OC, g/L 16.92 1.54 16.04 1.48 11.68 1.04b

Urinary DPD, mol/mol Cr 5.27 0.46 3.71 0.29b 4.16 0.37b


Serum CTx, g/L 0.44 0.04 0.24 0.03b 0.21 0.03b

Group 5


Serum N-mid OC, g/L 13.97 0.92 14.52 0.84 12.99 0.88



Serum CTx, g/L 0.37 0.04 0.31 0.04b 0.31 0.04b

Group 6


Serum N-mid OC, g/L 17.81 1.63 16.59 1.46 14.66 1.30b



Serum CTx, g/L 0.39 0.03 0.34 0.04b 0.32 0.04b

a Values are the mean SE.b Significantly different from baseline: P0.0167.

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Discussion

In this study, we measured five different commerciallyavailable biochemical markers of bone turnover. Two ofthe markers were bone formation markers (serum intactOC and serum N-mid OC), and the others were boneresorption markers (urinary DPD, and urinary and serumCTx). Our results showed that the bone resorption mark-ers were more sensitive than the bone formation markersin response to different modalities of intervention. This

might be attributable to a more rapid decrease in the boneresorption markers than in the formation markers as aresult of bone remodeling during the early postmeno-pausal period (810).

Among the bone resorption markers, serum CTx was

the most sensitive; it showed a positive response to allregimens at 1 or 3 months of intervention. Urinary CTxwas comparable to serum CTx but slightly less sensitive,whereas urinary DPD was the least sensitive marker. Thelower sensitivities of urinary DPD and urinary CTx mightbe attributable to an inherent biologic variability (8, 11 ). Amuch lower variability and greater changes with antire-sorptive therapy were seen in the serum measurements(12, 13 ). In addition, serum CTx has been reported to be

more specific for bone resorption than other measure-ments (12). Fall et al. (14) also found that the responses ofurinary and serum N-terminal telopeptides of type Icollagen in postmenopausal women receiving hormonereplacement therapy were comparable to those of serum

Table 2. Responses of BMD (g/cm2) to treatments.

Baselinea

After treatmenta

1 year 2 years

Control

Anteroposterior spine 0.9908 0.027 0.9639 0.037 0.9429 0.037

Femoral neck 0.8361 0.023 0.8040 0.025b 0.8047 0.026b

Group 2Anteroposterior spine 1.0579 0.043 1.0553 0.042 1.0432 0.049b

Femoral neck 0.8316 0.032 0.8186 0.032 0.8212 0.035

Group 3

Anteroposterior spine 1.0020 0.041 1.0665 0.028b 1.0399 0.032

Femoral neck 0.8331 0.031 0.8689 0.018 0.8627 0.021

Group 4

Anteroposterior spine 1.0416 0.028 1.0377 0.057 1.0947 0.035b

Femoral neck 0.8754 0.024 0.8120 0.044 0.8807 0.026

Group 5


Femoral neck 0.8371 0.033 0.8347 0.033 0.8361 0.032

Group 6


Femoral neck 0.8339 0.022 0.8381 0.022 0.8356 0.022a Values are the mean SE.b Significantly different from baseline: P0.0167.

Table 3. Correlation between biochemical markers of bone turnover and BMD.

% change from baseline of

biochemical marker of bone

turnover

% change from baseline of BMD

Anteroposterior spine Femoral neck

Marker Month 1 year 2 years 1 year 2 years

Intact OC 1 r 0.025 r 0.044 r 0.076 r 0.010

3 r 0.178a r 0.145a r 0.109 r 0.131

N-mid OC 1 r 0.006 r 0.035 r 0.042 r 0.031

3 r 0.198b r 0.184a r 0.167a r 0.066

DPD 1 r 0.015 r 0.003 r 0.062 r 0.003

3 r 0.205b r 0.144a r 0.247b r 0.162a

Urinary CTx 1 r 0.134 r 0.080 r 0.151a r 0.106

3 r 0.254b r 0.281b r 0.366b r 0.264b

Serum CTx 1 r 0.294b r 0.212b r 0.157a r 0.110

3 r 0.314b r 0.306b r 0.191b r 0.143a

aP0.05 (two-tailed).

bP0.01 (two-tailed).



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and urinary CTx. Therefore, bone resorption markersmight be preferable for follow-up of antiresorptive inter-ventions.

Regarding markers of bone formation, the performanceof both intact OC and N-mid OC (intact OC and N-terminal fragments) in response to intervention in thisstudy was similar. However, previous data have shown

differences in the performance of different assays used indifferent laboratories. This might be attributable to OCconsisting of multiple fragments and the presence ofimmunologic heterogeneity (1517). In addition, at roomtemperature a fraction of serum intact OC is rapidlycleaved into smaller fragments. The large N-terminalmidfragment is the main breakdown product (18). Mea-suring both the intact molecule and the N-terminal mid-fragment in a single assay has been reported to give morerobust and sensitive results (19, 20 ). Therefore, thismarker requires accurate control of both temperature andlength of storage of samples. In the present study, ourserum samples were stored at 80 C immediately after

separation of the whole blood and had never been thaweduntil assayed. This may explain why our two different OCassays showed quite similar responses.

Among the five different regimens for prevention ofearly postmenopausal bone loss, estrogen replacementwas shown to be the most effective modality. Almost allbiochemical markers decreased after 1 or 3 months ofestrogen replacement. Moreover, BMD in the estrogen-treated postmenopausal groups was slightly increasedafter 1 and 2 years of intervention. Estrogen is known tobe a potent inhibitor of osteoclastic activity (21) and toreduce the rate of bone turnover (22, 23 ). Additionally,the lower dose of estrogen appeared to have the same

effect as the traditional dose in decreasing bone remodel-ing. This finding was in accordance with other studiesdemonstrating that in early postmenopausal women, alower dose of estrogen was sufficient to maintain bonemass compared with the traditional dose after 1 or 2 yearsof treatment (2426).

It is also noteworthy that the antiresorptive effect oftreatment, as assessed by the changes in bone turnovermarkers after 3 months of therapy, could be used topredict the subsequent change in BMD (6, 12, 27 ). Ourstudy indicated that the early changes in the bone mark-ers in this study were weakly related to the later changesin BMD. However, bone resorption markers were betterindicators of BMD change than bone formation markers.

Because antiresorptive agents have a direct inhibitoryeffect on bone resorption and a resulting indirect effect onbone formation, the change in bone formation markersmight lag behind the change in bone resorption markersin predicting the change in BMD. Moreover, bone resorp-tion markers probably reflect the overall contribution ofbone turnover throughout the whole skeleton because thechanges of bone resorption markers at 3 months couldpredict skeletal responsiveness in both the spine andfemur in the first year of treatment. In addition, serum

CTx was found to be a better indicator than other mark-ers, providing an earlier indication of response than othermarkers. The change in serum CTx at 1 month couldpredict the long-term change in bone density after inter-vention. The changes in bone formation markers, how-ever, were insufficient to demonstrate a significant re-sponse in hip BMD. This result was in accordance with

the result reported previously by Cosman et al. (28), whohypothesized that this lack of a significant response in hipBMD might be attributable to the higher cancellous bonecontent in the spine than in the hip. A greater proportionof cellular product (OC, alkaline phosphatase, and tar-trate-resistant acid phosphatase) from the total active cellpopulation might be liberated into the circulation fromthe spine than from the hip, which could account for thebetter relationships between these variables and spinalbone turnover vs hip bone turnover.

In conclusion, biochemical markers of bone turnoverappear to be of use in assessing early response to therapy.

Bone resorption markers, especially serum CTx, are betterindicators than bone formation markers for estimating theresponse to intervention in early postmenopausal women.Moreover, in this clinical laboratory setting, changes inserum CTx were a better indicator of the long-termchanges in BMD after therapeutic intervention than wereother markers.

This work was supported by the Thailand Research Fund.We thank Roche Diagnostics for kindly providing theserum N-mid OC and urinary and serum CTx immuno-assays for this study.

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CTX, N-Mid Oscteocalcin5