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Articles |
1
Bone Metabolic Unit and
2
Department of Clinical Chemistry, Scientific Institute H San Raffaele, Via Olgettina 60, 20132 Milan, Italy.
a Address correspondence to this author at: Unita' Metabolica dell' Osso, Istituto Scientifico San Raffaele, Via Olgettina 60, I-20132 Milan, Italy. Fax 39-02-26433038; e-mail a.rubinacci{at}hsr.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Total and free Dpd were measured by HPLC and by immunosorbent assay, respectively, in two groups of subjects, one (calcium-treated; n = 16) taking calcium and the other not (control; n = 9).
Results: The urinary excretion of total Dpd at 2 and 4 h after oral calcium loading was decreased compared with controls. By contrast, changes in free Dpd were similar in the calcium-treated and control groups, reflecting only circadian rhythm.
Conclusions: Total and free Dpd do not show comparable sensitivity in detecting short-term inhibition of osteoclast activity. The degradation process of peptide-bound to free Dpd could render free Dpd insensitive to acute changes of osteoclast activity.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The total amount of Dpd in urine correlates with indices of resorption in bone biopsies (3) and is of clinical utility in disease states of increased bone resorption such as postmenopausal osteoporosis (4)(5), bone metastasis (6), and Paget disease (7)(8). It is also of clinical utility in monitoring treatments with bone-active agents such as estrogens (9)(10) and bisphosphonates (11)(12). Measurement of total Dpd in urine by HPLC is time- and effort-consuming. An ELISA that recognizes free Dpd has been developed to facilitate the measurements and extend the clinical use of Dpd (13). However, recent studies showing that free Dpd does not always behave as total Dpd in monitoring bone response to bisphosphonate treatment (6)(7)(8)(14) have raised the possibility that the former is affected by factors other than bone resorption. Free and total Dpd are highly correlated in both healthy and diseased subjects (13), but whether the free-to-total cross-link ratio is constant in both normal and high bone turnover states remains controversial (1)(13)(14).
To adequately compare the relative performance of free and total Dpd in detecting changes in bone resorption, a short-term inhibition of osteoclast activity that is not likely to alter the normal degradation pathway of collagen would be desirable. Because short-term oral calcium load induces a mild increment of plasma calcium that, via the afferent loop of the reciprocal causality (15), enhances calcitonin (CT) (16) and reduces parathyroid hormone (PTH) (17)(18)(19)(20)(21) concentrations, thus reducing bone resorption (18)(19)(20)(21). This perturbation of calcium homeostasis could be a challenging state for the free-to-total Dpd comparison in a physiological condition. Therefore, the following study was designed to evaluate free and total Dpd urinary excretion at sequential times after oral calcium loading.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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200 mg) following a generally acknowledged
procedure (22). Serum concentrations of calcium, creatinine,
and PTH were measured before (t0) and
1 (t1) and 3
(t2) h after the load. In nine
subjects, CT concentrations were also measured. Urinary calcium
(CaU, mol/mol creatinine) and total and free Dpd
(µmol/mol creatinine) were measured in urines collected before
(t0) and 2
(t1) and 4
(t2) h after the calcium loading. In
view of the diurnal variation of urinary collagen cross-links
(23)(24), sampling time was strictly controlled
and kept uniform for all subjects. Testing started at 0830. The calcemic response, the reduction of the intact PTH serum concentration, and the calciuric response were defined as described elsewhere (22). The calcemic response equals the serum calcium at t2 minus the serum calcium at t0; the reduction of intact PTH equals the percentage of change of serum PTH concentration at t2 compared with the basal value t0; the calciuric response equals the calcium concentration (CaU/creatinine) at t2 minus the calcium concentration at t0. By analogy with the percentage of PTH reduction, the percentage of CT increment equals the percentage of change of serum CT concentration at t2 compared with the basal value at t0.
Nine adult subjects (seven women and two men; mean age, 28 years; range, 25–35 years) were admitted to the study as a control group. All subjects were placed on the same dietary restrictions as described above, but they did not receive the oral calcium load received by the calcium-treated group. The milk intake was substituted by fruit (one banana) with negligible calcium content. All other procedures were identical.
All of the subjects included in this study (n = 25) had no past or present diseases or medications with potential influence on calcium homeostasis. All of the subjects included in the study gave oral informed consent, and the procedures followed for the study were in accordance with the current revision of the Helsinki Declaration.
assays
Plasma and urinary calcium as well as plasma and urinary
creatinine were measured colorimetrically (Boehringer Mannheim) using a
Hitachi 747 automated analyzer. Intact PTH was measured with an
IRMA (Incstar). The Cotube PTH IRMA assay utilizes the general
principles of the two-site immunometric assay to measure biologically
active, intact PTH. The coefficients of variation (CVs) within the
assay and among assays for PTH values within the reference
interval were both <6%.
Total urinary Dpd (total Dpd) was measured by ion-pair reversed-phase chromatography after hydrolysis (16 h at 105 °C) of the diluted urines (250 µL plus 250 µL of 12 mol/L HCl) and extraction with partition chromatography as described elsewhere (25). Briefly, the hydrolysate was loaded on a 500-mg CF-1 cellulose column, and total Dpd was eluted with 7 mL of distilled water. The analyte was detected fluorometrically (excitation wavelength, 295 nm; emission wavelength, 400 nm) utilizing its natural fluorescence. Within-run precision of the HPLC method was assessed by analysis of 50 replicates of a hydrolyzed urine specimen; the CV was 5%.
Free urinary Dpd (free Dpd) was measured with a competitive enzyme immunoassay (Pyrilinks-D; Metra Biosystems). The assay utilizes a monoclonal anti-Dpd antibody adsorbed on the well wall to capture Dpd. Dpd in the sample competes with conjugated Dpd-alkaline phosphatase for the antibody, and the reaction is detected with a p-nitrophenyl phosphate substrate. Absorbance was measured at 405 nm. The reported within- and among-assay CVs for free Dpd values for normal or increased resorption were both <6%.
The urinary markers were expressed as creatinine ratio. Variations of the plasma and urinary biochemical indices at different times vs basal were measured in the same assay. All indices of mineral metabolism (PTH and plasma and urinary calcium) and glomerular filtration (creatinine) were measured shortly after collection. Total and free Dpd were measured after storage at -20 °C in the dark for a mean of 2 years, under which conditions free and total Dpd are stable (26). Total Dpd matched measurements (n = 10) performed at the time of collection (15.4 ± 5.25 µmol/mol creatinine) and 2 years later (17.32 ± 8.16 µmol/mol creatinine) did not show significant differences.
statistics
Descriptive statistics were calculated using a GraphPad Prism
statistical package (Ver. 2.01 for Microsoft Windows). Data were
expressed as mean ± SE. The differences between times were
assessed by one-way ANOVA for repeated measurement and the Tukey
multiple comparisons test. Differences between assays were calculated
by two-way ANOVA for repeated measurements and with
t0 as covariate (Unistat for Windows,
Ver. 3.0). Differences between groups (calcium-treated vs control) were
assessed by means of a two-way ANOVA for repeated measurements (time)
with one grouping factor (treatment).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. After oral calcium load, a significant calcemic response, a
significant reduction of intact PTH serum concentration, a significant
calciuric response, and a significant CT increment were detected in the
calcium-treated group (Table 2
). The calcemic response, the PTH reduction, and the calciuric
response were not correlated to the age of the subjects. The highest
increment in plasma and urinary calcium concentrations as well as the
highest decrement of PTH concentration and calcitonin increment were
recorded at t2. No significant
calcemic response and PTH reduction were observed in the control group,
whereas the calciuria was significantly (P <0.005) lower at
t2 than at
t0 (Table 2
).
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In the calcium-treated group, the urinary total Dpd and free Dpd
corrected for creatinine, thus controlling for urine dilution, were
decreased in almost all subjects and reached a significant decrement at
t2 (free Dpd, P <0.01 vs
t0; total Dpd, P <0.001 vs
t0; P <0.01 vs
t1; Fig. 1
A). Total and free Dpd decrements were significantly different
(P = 0.045), and their time patterns were different as
shown by the significant interaction (P = 0.0018) of
Dpd fractions with time. The free-to-total Dpd ratio was significantly
(P <0.01) different between
t1 and
t2. Total and free Dpd concentrations
were significantly correlated at all experimental times
(t0, r = 0.88,
P <0.0001; t1,
r = 0.94, P <0.0001;
t2, r = 0.91,
P <0.0001). The individual decrements of total and free Dpd
after oral calcium load were inversely correlated (r =
-0.76, P <0.001; and r = -0.64,
P <0.01, respectively) with their relevant basal values
(r = -0.76, P <0.001; and
r = -0.64, P <0.01, respectively; Fig. 2
, A and B); the higher the basal value, the greater the
reduction obtained. The individual decrements of total Dpd after oral
calcium load were correlated (r = -0.55,
P = 0.026) with the calcemic response, whereas those of
free Dpd were not (Fig. 3
). No significant correlations were found between the
reduction in both Dpd fractions (values at
t2 minus values at
t0) and the other responses of the
mineral metabolism indices, i.e., PTH reduction, calciuric response,
and CT increment.
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In the control group, the urinary concentrations of total and free Dpd
corrected for creatinine, thus controlling for urine dilution, were
decreased in almost all subjects and reached a significant decrement at
t1 and
t2 vs
t0 only for free Dpd (P
<0.05; Fig. 1B
). Total and free Dpd decrements as well as their time
patterns were not significantly different. The ratio of free-to-total
Dpd did not show a significant change. Total and free Dpd
concentrations were significantly correlated at all experimental times
(t0, r = 0.96,
P <0.0001; t1,
r = 0.96, P <0.0001;
t2, r = 0.99,
P <0.0001). The individual decrements over time of free Dpd
were significantly correlated with their relevant values at
t0 (Fig. 2
, C and D), whereas those of
total Dpd were not.
The time patterns of the total Dpd decrements were significantly
different in the treated and control groups as shown by the significant
(P <0.05) interaction of the linear effect of time with
treatment. The decrement of total Dpd at
t2 vs
t0 was higher (-6.23 ± 1.31
µmol/mol creatinine) in the calcium-treated group than in the control
(-2.4 ± 1.2 µmol/mol creatinine) without reaching statistical
significance. However, when the comparison was limited to the subjects
displaying a calcemic response 
0.1 mmol/L
(27)(28), thus to avoid the bias attributable to
calcium malabsorption (29)(30), the difference
became significant (P <0.05). The time patterns of the free
Dpd decrements were not significantly different in the treated and
control groups, even when the comparison was limited to subjects
displaying a calcemic response 0.1 mmol/L. No significant
correlations were found between the reduction in both Dpd fractions in
the treated and control groups and the age of the subjects.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The possibility of a short-term block of osteoclast activity by
perturbing calcium homeostasis offers the opportunity to test markers
of bone resorption in a physiological condition that reduces any
possible bias attributable to variation of posture, diet, age, hormonal
status, and renal clearance. The short-term increment of plasma calcium
induces a transient reduction of the remodeling space by inhibiting
existing osteoclasts (15); it is therefore less prone than
long-term treatments to confounding factors. In fact, estrogens are
involved in the metabolic pathway of several collagens displaying
cross-linkage of their 
chains (31)(32),
whereas bisphosphonates affect both synthetic (33) and
degradative (14) pathways of the cross-links in bone cells
as well as the composition and content of cross-links in rat bone
(34). The calcium loading caused major changes in PTH
(18)(19)(20)(21) and calcitonin concentrations (16), and
these could also lead to associated perturbation of the typical
degradation pathway of collagen either in bone or in other tissues.
However, these effects, if any, might be minimal for the transient
nature of the changes.
This study showed that the reduction in Dpd concentration is a specific effect of the calcemic increments and is not caused by the circadian variation of the cross-links in the urines (23)(24). However, the effect of calcium loading was clearly distinguishable from the circadian variation only when total Dpd fraction changes were considered. On the contrary, the excretion pattern of free Dpd fraction over time after calcium loading was comparable to that related to circadian rhythm. Only when maximal perturbation of calcium homeostasis was achieved, i.e., at 4 h after calcium loading (27), was a higher but not significant decrement of the urinary free Dpd vs control observed. The sensitivity of total Dpd in detecting osteoclast activity inhibition after a physiological perturbation of calcium homeostasis was confirmed by the significant correlations between total Dpd decrement and the bone metabolic state (basal values), and between total Dpd and the calcemic increment and is in agreement with other studies (18)(19)(20)(21). It follows that osteoclast inhibition and the subsequent drop of total Dpd in the urines represents a short-term error correction mechanism in calcium homeostasis and is not merely a phenomenon of circadian rhythm.
The daytime osteoclast activity reduction and the subsequent drop of Dpd fractions in the urines are not necessarily linked to a daytime decrease in PTH. In fact, Dpd fraction decrements in the control group were not accompanied by a PTH reduction. It is, however, interesting to note that by reducing serum PTH concentrations with calcium loading, a further inhibition of osteoclast activity could be achieved that was detected only by the total fraction of Dpd, thus in agreement with others (21). This implies that the amplitude of the observed decrease in total Dpd in the urine represents the cumulative effect of PTH-dependent and -independent modulation of the osteoclast activity. Cortisol (35), posture, age, menopause, and osteopenia (36) have also been investigated and found to have no effect.
Because the physiological perturbation of calcium homeostasis failed to alter the circadian pattern of free Dpd excretion, it follows that total Dpd displays better sensitivity than free Dpd. This is supported by the observation that total Dpd was correlated with the calcemic response, i.e., the amplitude of the calcium homeostasis perturbation, whereas free Dpd was not. It is therefore conceivable that the inability of free Dpd to detect short-term physiological changes in the metabolic state of bone resorption is attributable to a still undefined production of the free Dpd fractions that mask the mild change of bone turnover when its basal activation frequency is relatively low. This view is supported by the observation that the free-to-total Dpd ratio changed at subsequent times after baseline in the calcium-treated group and remained stable in the controls, thus indicating a slightly greater reduction in total Dpd than in free Dpd when bone resorption is inhibited. This observation is in accordance with the relative reductions in free and total Dpd produced by bisphosphonate therapy in pagetic (7)(8) and neoplastic (6) patients. All of these observations confirm the view of Randall et al. (8) that the change in the free-to-total Dpd ratio occurs as a result of the decrease from a state of high bone turnover to one of low turnover and suggests a mechanism of a rate-limiting conversion of conjugated to free cross-links, likely occurring in the kidney (37). It is even likely that the degradation process of peptide-bound to free Dpd is slow, rendering free Dpd insensitive to acute changes of osteoclasts activity.
Because the mean value of total Dpd was higher in the calcium-treated group than in the controls, the free-to-total Dpd ratio was different between the groups. This difference is another point suggesting that the ratio is affected by the metabolic state of bone. It is in fact likely that only total Dpd was able to discriminate the subjects with enhanced activation frequency of bone remodeling as a response to the calcium deprivation before the load. These subjects were present in both groups, but unfortunately, the calcium-treated group had a higher basal mean total Dpd value than the control group. The difference in the basal Dpd between treated and control groups was not attributable to any age-related bias because the total Dpd decrement was not correlated to the age of the subjects. The fact that free Dpd did not discriminate the subjects with activated frequency of bone remodeling or the subjects taking calcium further supports the existence of a rate-limiting process for free Dpd.
In conclusion, this study indicates that total and free Dpd do not show comparable sensitivity in detecting short-term inhibition of osteoclast activity: whereas the former detects both PTH-dependent and -independent changes in osteoclast activity, the latter does not. It is conceivable that renal clearance and the metabolic state of bone likely determines the proportions of free and total Dpd in urine. Furthermore, this study has demonstrated that osteoclast activity is inhibited by physiologic calcemic increments and supports the view that osteoclasts are involved in the short-term error correction mechanism of plasma calcium homeostasis.
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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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  H. W. Vesper, L. M. Demers, R. Eastell, P. Garnero, M. Kleerekoper, S. P. Robins, A. K. Srivastava, G. R. Warnick, N. B. Watts, and G. L. Myers Assessment and Recommendations on Factors Contributing to Preanalytical Variability of Urinary Pyridinoline and Deoxypyridinoline Clin. Chem., February 1, 2002; 48(2): 220 - 235. [Abstract] [Full Text] [PDF]
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).
, P
<0.01 vs T1.
