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Articles |
Department of Internal Medicine, Division of Endocrinology and Metabolism, University of Heidelberg, Bergheimerstrasse 58, D-69115 Heidelberg, Germany.
a Author for correspondence. Fax 49-6221-564101; e-mail Markus_Seibel{at}med.uni-heidelberg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Twenty patients with active PD were studied before and after treatment with 2 mg of intravenous ibandronate over a period of 12 months. TAP (by colorimetry), serum bone-specific alkaline phosphatase (BAP; by enzyme immunoassay), serum osteocalcin (OC; by ELISA), serum bone sialoprotein (BSP; by RIA), and urinary total pyridinoline (PYD; by HPLC) and deoxypyridinoline (DPD; by HPLC) were measured as markers of bone turnover.
Results: Before treatment, TAP, BAP, and BSP were increased in
all 20 patients, whereas OC was increased in 10, PYD in 13, and DPD in
15 patients. Three months post treatment, nine patients showed
normalized TAP values, and a 
25% re-increase (i.e., relapse) was
observed in all patients after 12 months. A normalization of BAP was
achieved in six patients only. No significant changes were found for
OC. BSP was decreased significantly at 24 h, and DPD at 48 h
post treatment. A normalization of BSP was found in 8, of PYD in 18,
and of DPD in 16 cases. Both PYD and DPD increased significantly from 9
months post treatment onward.
Conclusions: Most markers of bone turnover show similar long-term changes after treatment of active PD with ibandronate. With regard to cost-effectiveness and assay performance, TAP remains the marker of choice in therapeutic monitoring of PD. However, more specific markers may improve the biochemical assessment of PD in certain situations.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The therapeutic management of PD has been greatly improved by the development of bisphosphonates, which are now considered the treatment of choice (1)(4)(5)(6)(7)(8). Bisphosphonates decrease both bone resorption and formation, but the effect on bone formation occurs somewhat later than the immediate inhibition of osteoclast activity (9)(10)(11). Ibandronate is a new bisphosphonate that shows a 50-fold higher in vitro biological potency than pamidronate, a second-generation bisphosphonate (12)(13).
Serum total alkaline phosphatase (TAP) and urinary hydroxyproline are widely used markers for the diagnosis, therapeutic monitoring, and detection of a relapse in patients with PD (9)(11)(14). However, because both markers are relatively nonspecific indexes of bone turnover, novel markers with higher specificity and sensitivity for skeletal metabolism have been developed. These include the bone-specific isoenzyme of alkaline phosphatase and osteocalcin (OC) as markers of bone formation (15)(16)(17)(18), as well as the hydroxypyridinium cross-links and telopeptide-related epitopes of type I collagen (19)(20), and bone sialoprotein (BSP) (21)(22) as markers of bone resorption.
In PD, specific markers of bone turnover may be particularly helpful when introducing new drugs or therapeutic regimens. It is still unclear, however, whether these new markers improve the diagnostic sensitivity or the assessment of therapeutic response compared with serum TAP. In the present study, we monitored 20 patients with active PD over a period of 12 months with the following aims: (a) to determine the usefulness of novel biochemical indexes in monitoring disease activity after ibandronate treatment; (b) to compare the new biomarkers with serum TAP as the measure of choice for disease activity; and (c) to compare serum BSP values with the urinary excretion of collagen cross-links as the reference markers of bone resorption.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-glutamyltransferase >50 U/L, respectively] were excluded
from the study. Additional exclusion criteria included hematological
disorders (thrombocytopenia, <120 000 cells/µL; neutrocytopenia,
<3000 cells/µL) bone fractures <6 months prior to the
study; aminoglycoside, mitramycin, or calcitonin therapy within the
last 3 months; and treatment with any other bisphosphonate within 6
months before study enrollment or with lasting effects of earlier
treatment (TAP <50% of baseline values). Patients were treated with ibandronate either by intravenous bolus injection (2 mg of ibandronate in 10 mL of 9 g/L NaCl over 5 min) or by continuous infusion (2 mg of ibandronate in 500 mL of 9 g/L NaCl over 24 h) on day 0. Ibandronate (Bondronat®; Boehringer Mannheim) is a [1-hydroxy-3-(methylpenthylamino)-propylidene] bisphosphonate with a molecular weight of 341.23. The in vitro activity of this aminobisphosphonate is ~50-fold higher than pamidronate and ~10 000-fold higher than etidronate (12).
Follow-up visits with individual physical examinations and evaluation of side effects were performed on days 1, 2, 3, 30, 90, 180, 270, and 365 after treatment. Blood and urine samples were obtained on each visit between 0800 and 1000 with subjects in the fasting state.
The study was approved by the local ethics committees and performed in accordance with the current revision of the Declaration of Helsinki of 1975. Written informed consent was obtained from all individuals before the study.
laboratory analyses
Blood samples were centrifuged within 1 h after phlebotomy
(1500g for 10 min) and stored as aliquots at -80 °C
until assayed. Urine specimen were protected from light and stored
within 2 h of collection at -30 °C until analysis.
Serum activity of TAP was determined by an automated colorimetric assay with a BM/Hitachi System 704 analyzer (Boehringer Mannheim) at 37 °C. The assay utilizes p-nitrophenyl phosphate as a substrate, as recommended in the optimized standard method of the Deutsche Gesellschaft für Klinische Chemie (24). Intra- and interassay CVs were <5% at 60–170 U/L (the range for healthy individuals), recalculated to room temperature (25 °C).
An enzyme immunoassay (Alkphase-BTM; Metra Biosystems) was applied to determine serum activity of bone-specific alkaline phosphatase (BAP). This microtiter plate format immunoassay utilizes a plate-coated monoclonal anti-BAP capture antibody, and the activity of the captured enzyme is detected with p-nitrophenyl phosphate as the substrate (25). The reference values for healthy individuals are 10–22 U/L for premenopausal females and 12–23 U/L for males. Intra- and interassay CVs were 3.2–3.5% and 6.2–7.9%, respectively.
Serum OC was measured by a two-site ELISA (N-MID hOsteocalcin ELISA KITTM; Osteometer) (26). The assay is based on the application of two highly specific antibodies against human osteocalcin: a capture antibody that recognizes the midregion (amino acids 20–43), and for detection, a peroxidase-conjugated antibody that recognizes the N-terminal region (amino acids 1–19) of OC. Intra- and interassay CVs were 3.3–10%. The reference values were 15.0–35.0 µg/L for premenopausal females and 8.8–37.6 µg/L for males.
For measuring serum concentrations of BSP, a recently developed RIA (Immundiagnostik Bensheim) was used as described previously (27). Antibodies against human BSP were raised in chickens and purified by affinity chromatography. The intraassay CV was 7.0%, and the interassay CV was 9.1%. Reference values for healthy individuals were 4.7–13.8 µg/L for premenopausal females and 3.4–15.2 µg/L for males.
Urinary total pyridinoline (PYD) and urinary total deoxypyridinoline (DPD) were determined by HPLC, as described previously (19). After complete acid hydrolysis of urine samples at 107 °C for 16 h, the peptide-free forms of PYD and DPD were separated by ion-paired HPLC, and concentrations were quantified by fluorometry using a fully automated method as described by Pratt et al. (28). Calibrators were derived from sheep bone and were a generous gift from Dr. Simon P. Robins (Aberdeen, Scotland). The overall imprecision of the assay for intra- and interassay variability was between 3% and 12%. Urinary concentrations were expressed relative to urinary Cr concentrations. PYD reference values were 17.6–47.3 nmol/mmol Cr for premenopausal females and 10.8–49.1 nmol/mmol Cr for males; DPD reference values were 2.7–12.7 nmol/mmol Cr for premenopausal females and 2.6–9.3 nmol/mmol Cr for males.
statistical analyses
The Statistical Analysis System (SAS) software package was used
for data analysis. To test for group differences before treatment, the
Student t-test for parametric and the Wilcoxon rank-sum test
for nonparametric variables were performed. Follow-up data were
analyzed using repeated-measures ANOVA. Simple Pearson correlations
were performed to determine the strength of association between two
markers. The Fisher exact test was used to test for differences in the
distribution of participants within categories. All statistical
analyses were two-tailed, and P <0.05 was considered
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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baseline laboratory analyses
The baseline values of biochemical markers of bone turnover for
the total group are shown in Table 1
(day 0). Serum and urinary concentrations of all biochemical
markers were similar in both genders, and no statistically significant
sex-related differences could be established (data not shown). In
addition, no differences were seen between marker concentrations
according to the mode of ibandronate application (intravenous bolus vs
24-h infusion). In patients who had never received bisphosphonates
before, mean serum BSP concentrations were significantly lower
(P <0.05) than in patients who previously had been treated
with bisphosphonates. Otherwise, no significant differences were found
between marker baseline concentrations according to pretreatment status
(data not shown).
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Based on the reference values for the respective markers established in
our laboratory (see cutoff values given in Table 1
), serum
concentrations of TAP, BAP, and BSP were increased in all patients
before treatment. In contrast, serum OC and urinary PYD and DPD were
increased in 10 (50%), 13 (65%), and 15 (75%) patients,
respectively.
ibandronate treatment
In the total group (Table 1
and Fig. 1
), 2 mg of ibandronate led to a significant decrease of serum
TAP beginning at 30 days post treatment, and a nadir was reached 90
days post treatment. All 20 patients had evidence of a relapse of
disease activity after 12 months, as defined by a 25% re-increase
over the TAP nadir. Changes in serum BAP concentrations paralleled
those of serum TAP. However, serum BAP values were normalized in only 6
of 20 patients, whereas serum TAP values were normalized in 11 of 20
patients. Mean serum values of TAP and BAP were significantly higher 1
year after treatment compared with 3 months post treatment
(P <0.05). Serum OC concentrations did not show significant
changes in response to ibandronate treatment (Fig. 1
). Regarding
markers of bone resorption, intravenous ibandronate produced a
significant decrease of mean serum BSP concentrations beginning at
24 h post treatment (P <0.05) with a nadir after 30
days (34 ± 11 µg/L). A statistically significant
decrease in the excretion of urinary cross-links was observed beginning
at 48 h post treatment (DPD, P <0.01), with nadirs
after 3 months (both cross-links components). Both PYD and DPD showed
significantly higher urine concentrations 9 and 12 months post
treatment compared with 3 months after ibandronate application
(P <0.05 and <0.01, respectively).
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Analyses of a stratified data set according to baseline serum TAP
concentrations (baseline TAP <450 U/L vs baseline TAP 450 U/L) are
shown in Table 2
. For serum BAP, a significant reduction of mean values was
found in both groups 3 months post treatment. However, none of the
patients with baseline TAP concentrations 450 U/L reached BAP values
within the range for healthy individuals at any time during the study
period. Compared with baseline values, serum BSP was significantly
reduced only in patients with baseline TAP concentrations 450 U/L
(Table 2
). In patients with baseline TAP values <450 U/L, urinary PYD
and DPD values were significantly lower 3 months post treatment
compared with pretreatment concentrations and significantly higher 12
months post treatment compared with 3 months post treatment (Table 2
).
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When we stratified the data set according to the pretreatment status, patients who had never received bisphosphonates in the past showed a more pronounced reduction in all biomarkers except for serum OC. However, statistically significant group differences could be established only for serum BSP before (P <0.05, see above), and 72 h and 30 days after ibandronate application (P <0.001 and <0.01, respectively). Pretreated patients showed a higher increase in serum TAP and BAP 9 and 12 months post treatment compared with patients without pretreatment (TAP, P <0.05 after 9 months; BAP, P <0.05 after 9 and 12 months). No such differences were found for the other bone turnover markers.
A summary of the most relevant changes in biomarkers is presented in
Table 3
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An index of bone formation superior to serum TAP should, in a routine clinical setting, provide a more reliable way to predict the therapeutic outcome or detect nonresponders or a relapse in disease activity at an earlier point of time. A candidate for the latter is the bone-specific isoenzyme of alkaline phosphatase because it potentially reflects osteoblast activity in a more specific way than does TAP (16)(25)(31). Some studies have shown that serum BAP may be a more sensitive indicator of changes in bone turnover during bisphosphonate treatment (15)(16)(32) than serum TAP.
In the present study, the decreases in serum TAP and BAP activity followed an almost identical pattern, with reductions to 47% and 43% of the respective baseline values 3 months after intravenous ibandronate administration. The percentage of normalized values was higher for serum TAP, which may reflect a greater number of false-positive events. The assessment of disease activity may therefore be somewhat improved by serum BAP because this marker discriminates better between truly normalized and still accelerated osteoblast activity (15)(16)(31).
For both serum TAP and BAP, the percentage of decrease was slightly
more pronounced in a group of patients without previous bisphosphonate
treatment. Thus, patients who had received bisphosphonate treatment in
the past may require higher doses of the drug when retreatment is
necessary (33). In the present study, serum BAP seemed to
better reflect these subtle changes in that the differences in minimum
concentrations between the two groups were higher than for serum TAP
(Table 3
).
The occurrence of the re-increase in osteoblast activity seemed to be reflected by serum TAP and BAP in an almost identical manner. Thus, the question of when to start retreatment may in most cases be sufficiently answered by measuring serum TAP activity. BAP measurements may be advantageous in certain clinical situations, e.g., in the presence of liver disease (17). With regard to cost-effectiveness and assay performance, TAP will remain the biochemical marker of choice in most patients with active PD.
As reported here and elsewhere, serum OC is poorly correlated with the disease activity at baseline (e.g., serum TAP concentrations) and after bisphosphonate treatment (34). The reasons for these observations are still unknown. The lack of correlation between OC and TAP may be attributable to the expression of these markers at different stages of osteoblast development (31)(35).
Recently, we have shown that BSP is a new serum marker of bone turnover (21)(22). In patients with hypercalcemia of malignancy, the intravenous administration of pamidronate led to a rapid and highly significant reduction in serum BSP during the first 7 days. These changes were paralleled by a decrease in urinary total DPD but not in serum TAP, which led to the conclusion that the detection of serum BSP most likely reflects processes predominantly associated with bone resorption (22). In the present study, the reduction in serum BSP preceded the changes in urinary cross-links excretion by ~24 h. This difference may be attributable to the fact that BSP plays a role in initiating bone resorption by mediating the attachment of osteoclasts to bone (36), whereas the cross-link excretion reflects ongoing collagen degradation. It is conceivable that the latter is being suppressed somewhat later in the course of bisphosphonate action. In addition, it has been suggested that the antiresorbing effect of bisphosphonates is cell mediated (37), partly by direct action on osteoclasts and partly through osteoblasts, which produce an inhibiting factor of osteoclast recruitment. Because BSP is thought to be an important factor of cell-adhesion processes, the direct action of bisphosphonates on bone cells may explain why changes in serum BSP concentrations precede those in urinary collagen breakdown products.
In this study, we found some evidence that measuring biochemical markers of bone resorption potentially enhances the chance of an early detection of nonresponders to bisphosphonate therapy. Of the three patients who presented with essentially unchanged serum BSP concentrations during the first 3 days (<10% reduction compared with initial values), none reached serum TAP concentrations within the range for healthy individuals during the study period. The same was true for the four patients with unchanged urinary DPD concentrations. The only "classical" nonresponder (defined as a <25% reduction in serum TAP concentration compared with the initial value) did not show any changes in serum BSP and urinary DPD concentrations during early and late follow-up. In summary, these results suggest that markers of bone resorption may identify nonresponders at an earlier time point than serum TAP. However, it needs to be emphasized that in most clinical situations, patients are rarely seen more frequently than at 4-week intervals. Therefore, in the vast majority of patients with PD, the measurement of serum TAP appears to provide sufficient information regarding the detection of nonresponders to bisphosphonate therapy, and the quantification of markers of bone resorption may not be necessary at all.
According to biochemical measurements, the administration of 2 mg of ibandronate is insufficient in normalizing disease activity in most patients with PD. No significant differences in the concentrations of biochemical markers were found regarding the administration mode of 2-mg ibandronate (intravenous bolus injection vs continuous infusion). Recently, Pecherstorfer et al. (13) reported that a single intravenous injection of 3 mg of ibandronate is safe. A bolus injection, if effective in a way comparable to continuous infusion as demonstrated in the present study, may significantly improve the clinical utility of this bisphosphonate. However, the minimum dose of ibandronate required to efficiently suppress bone turnover in patients with PD remains to be determined.
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Acknowledgments |
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  M. E. Kraenzlin, C. A. Kraenzlin, C. Meier, C. Giunta, and B. Steinmann Automated HPLC Assay for Urinary Collagen Cross-links: Effect of Age, Menopause, and Metabolic Bone Diseases Clin. Chem., September 1, 2008; 54(9): 1546 - 1553. [Abstract] [Full Text] [PDF]
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 J. Barrett, E. Worth, F. Bauss, and S. Epstein Ibandronate: A Clinical Pharmacological and Pharmacokinetic Update J. Clin. Pharmacol., September 1, 2004; 44(9): 951 - 965. [Abstract] [Full Text] [PDF]
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H. W. Woitge, E. Horn, A. V. Keck, B. Auler, M. J. Seibel, and M. Pecherstorfer Biochemical Markers of Bone Formation in Patients with Plasma Cell Dyscrasias and Benign Osteoporosis Clin. Chem., April 1, 2001; 47(4): 686 - 693. [Abstract] [Full Text] [PDF]
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