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Enzymes and Protein Markers |
1
Osteometer BioTech A/S, Osteopark, Herlev Hovedgade 207, DK-2730 Herlev, Denmark.
2
Center for Clinical and Basic Research, Ballerup Byvej
222, DK-2750 Ballerup, Denmark.
a Author for correspondence. Fax 45 44 94 89 40;
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Until now only two immunoassays detecting C-terminal telopeptide
fragments of type I collagen in serum samples have been described
(8)(9). Especially, the amino acid sequence
EKAHDGGR
(
CTX),1
found in the C-terminal telopeptides of the 1 chain of type
I collagen, where the aspartic acid residue (D) is ß-isomerized, has
proven to be a specific and sensitive marker of bone resorption in
serum (9). This was demonstrated using a competitive ELISA
with an antiserum specific for the amino acid sequence AHD-ß-GGR.
Additionally, an immunoassay for measurement in serum of collagen type
I N-telopeptides has also been reported (10).
Here we report new progress in the development of immunoassays for measuring bone resorption markers in serum, the Serum CrossLapsTM One Step ELISA. With this new assay we present the first application of monoclonal antibodies (mAbs) for the quantitative determination in serum of degradation products from C-terminal telopeptides of type I collagen. This ELISA method is based on the use of two highly specific mAbs against the amino acid sequence AHD-ß-GGR. The molecules measured in the assay consist of two chains of EKAHD-ß-GGR (ßCTX) that are covalent cross-linked via the lysine residues. Our aim was to obtain a reliable, easily performing assay with a high potential for assessing the rate of bone resorption.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CTX, were purchased from KJ Ross-Petersen ApS.
Buffers for the Serum CrossLaps One Step ELISA were as follow:
phosphate-buffered saline (PBS: 1.5 mmol/L
KH2PO4, 8.5 mmol/L
Na2HPO4·2H2O, 2.7 mmol/L KCl, 137
mmol/L NaCl, pH 7.4); calibrator buffer (PBS containing 10 g/L bovine
serum albumin (BSA), 0.18 g/L Bronidox, 0.03 g/L bromphenol blue, pH
7.4); incubation buffer (7.5 mmol/L KH2PO4,
42.5 mmol/L Na2HPO4·2H2O, 2.7
mmol/L KCl, 137 mmol/L NaCl, 10 g/L BSA, 0.1 g/L Tween 20, 0.18 g/L
Bronidox, 0.03 g/L bromphenol blue, pH 5.75); and washing buffer (25
mmol/L Tris, 50 mmol/L NaCl, 1 g/L Tween 20, pH 7.2).
preparation of mAbs
Production of mAbs.
Female Balb/C x CF1 mice (8–12
weeks of age) were immunized intraperitoneally with 200 µL of a
emulsion of complete Freund's adjuvant and ßCTX conjugated to
thyroglobulin (100 mg/L) by a carboiimide procedure
(11). The conjugate and the adjuvant were mixed in
equal volumes. Immunizations were repeated six times every 2 weeks
using incomplete Freund's adjuvant. Three days before fusion the mice
were boosted intraperitoneally with 100 µL of ßCTX conjugated to
thyroglobulin (100 mg/L). Spleen cells and ATCC P3-X63-Ag8.653
(12) myeloma cells were fused with 500 mL/L polyethylene
glycol (PEG 4000 GK) as described previously, except that human
endothelial culture supernatant (HECS, Costar) was used instead of
feeder cells (12).
Screening of mAbs.
Supernatants from hybridoma cells were
diluted in 300 mmol/L Tris, 10 g/L BSA, 5 g/L Tween 20, pH 8.0, and
incubated in microtiter wells (Nunc) coated with either ßCTX
conjugated to BSA by a glutaraldehyde procedure (13) or with
collagenase-treated collagen from human bone (14). Bound
antibodies were then detected using peroxidase-conjugated rabbit
anti-mouse immunoglobulins (Jackson ImmunoResearch Laboratories).
Hybridomas producing antibodies against both ßCTX and
collagenase-treated collagen were then cloned at least twice with
limiting dilutions and propagated. The mAbs were purified using Protein
A chromatography in accordance with the manufacturer's instructions
(Pharmacia). The subclass of each mAb was determined using the
IsoStripTM Mouse Monoclonal Antibody Isotyping
Kit (Boehringer Mannheim GmbH).
characterization of serum CrossLaps ONE STEP ELISA
BINDING SPECIFICITY
The binding specificity of the Serum CrossLaps One Step ELISA was
investigated using genuine cross-linked urinary fragments containing
either two ßCTX (ßCTX-X-ßCTX), one CTX and one ßCTX
(CTX-X-ßCTX), or two CTX (CTX-X-CTX) epitopes. The three
different genuine cross-linked fragments (cross-link, galactosyl
pyridinoline) were extracted from urine as by immunoaffinity
chromatography and further purified by reversed-phase HPLC as described
previously by Fledelius et al. (15). The fragments
(CTX-X-CTX, CTX-X-ßCTX, and ßCTX-X-ßCTX) were mixed in
equal amounts and separated by reversed-phase HPLC. The fragments were
eluted from a Delta Pak C18 column (3.9 x 150 mm;
particle size, 5 µm; pore size, 30 nm; Waters) with a 0–125 g/L
acetonitrile gradient containing 1 g/L trifluoroacetic acid over 45 min
at a flow rate of 1 mL/min. The effluent was monitored for fluorescent
material at 380 nm (emission) using 297 nm light for excitation (Waters
470 Scanning Fluorescence detector; Waters). The effluent was
fractionated (fraction size, 0.3 mL), freeze-dried, redissolved in PBS,
and assayed in the Serum CrossLaps One Step ELISA.
size exclusion chromatography of serum fragments of the c-terminal
telopeptide 1 chain of type i collagen
Immunoaffinity chromatography.
mAb F1103 was coupled to a
CNBr-activated Sepharose 4B matrix (Pharmacia) in accordance with the
manufacturer's instructions. Eighty milliliters of undiluted human
serum was applied to the column. The column was washed with PBS, and
bound material was eluted with 10 g/L trifluoroacetic acid and
freeze-dried. The recovery of immunoreactive fragments was >75%.
HPLC size exclusion chromatography.
The immunopurified
fraction was redissolved in 0.2 mol/L NH4HCO3
and applied to a Superdex Peptide HR 10/30 column (Pharmacia)
equilibrated with 0.2 mol/L NH4HCO3. Fractions
were collected in volumes of 0.5 mL. Each fraction was freeze-dried,
redissolved in PBS, and assayed in the Serum CrossLaps One Step ELISA.
Calibration of the HPLC column.
For the calibration of this
HPLC size exclusion column, the following molecules were used as mass
markers: kyotorphin (337 Da), CTX (860 Da), purified urinary antigen
(2036 Da) (15), aprotinin (6500 Da), and cytochrome C
(13 337 Da). When the elution time (minutes) was plotted against the
molecular mass (log10 scale), a calibration curve with an
r value of -0.997 was obtained (data not shown).
peroxidase labeling of mAbs
Horseradish peroxidase (Boehringer Mannheim GmbH) was conjugated
to Protein A-purified mAb F12 as described by Nakane and Kawaoi
(16).
biotinylation of mAbs
Conjugation.
A 2 g/L solution of Protein A-purified mAb F1103
(in PBS) was mixed with a solution of 0.3 mol/L
Na2CO3, 0.7 mol/L NaHCO3, pH
9.6, in the ratio 10:1.
Biotinamidocaproate-N-hydroxysuccimide ester (BxNHS)
dissolved in dimethyl sulfoxide (4 g/L) was then added in the ratio of
5 mol BxNHS:1 mol IgG. The mixture was incubated at room temperature
for 2 h with end-over-end rotation. The reaction was stopped by
the addition of 0.2 mL of 0.2 mol/L ethanolamine and incubated for
1 h at room temperature. The solution was dialyzed (cutoff value:
12 000–14 000, Spectra/Por®; Spectrum Medical
Industries) twice against 5 L of PBS for 2 days at 4 °C. Turbidity
was removed by sterile filtration using 0.22 µm disposable syringe
filter holders (Minisart NML, Satorius). Aliquots were stored at
-20 °C.
Determination of mol BxNHS/mol IgG.
The degree of
biotinylation was determined by a HABA kit from Pierce Chemicals and
performed in accordance with the manufacturer's instructions. Briefly,
the principle of this procedure is based on the formation of a complex
between the HABA reagent (4-hydroxyazobenzene-2'-carboxylic acid) and
avidin. When added, biotin will displace the HABA reagent, which will
decrease the absorbance at 500 nm, and the unknown amount of biotin can
be determined.
preparation of calibrators for the elisa
Desalting of urinary antigens.
C8 Sep-Pak
Cartridge columns (Waters) were activated with 800 g/L methanol and
equilibrated with 1 g/L trifluoroacetic acid. Urine from healthy
adults, containing 10 g/L trifluoroacetic acid, was applied to the
columns. The bound material was washed with 1 g/L trifluoroacetic acid,
eluted with 400 g/L acetonitrile containing 1 g/L trifluoroacetic acid,
freeze-dried, reconstituted in PBS, and stored at -20 °C. The
desalted stock solution was quantified in an assay similar to that
described previously by Bonde et al. (9). Briefly, this
assay was a competitive ELISA using polyclonal antibodies specific for
AHD-ß-GGR. Additionally, the assay used the synthetic peptide ßCTX,
both for immobilization to the microtiter wells and for
standardization. The exact concentration of the ßCTX peptide was
determined by quantitative amino acid analysis and expressed in pmol/L.
Calibrators.
The desalted urinary antigens were diluted in the
calibrator buffer. A line of calibrators was prepared covering a range
from 500 to ~15 000 pmol/L. The calibrators were stored in brown
glass vials at 4 °C. In this formulation the antigens were
unaffected by stress treatment at 35 °C for 3 weeks.
serum CrossLaps ONE STEP ELISA
Fifty microliters of calibrators, controls, or unknown serum
samples were pipetted into appropriate microtiter wells coated with
streptavidin (MicroCoat), followed by 150 µL of a mixture of the
biotinylated mAb F1103 and the peroxidase-conjugated mAb F12 in
incubation buffer. The contents of the wells were incubated for 2
h at 20 °C on a mixing apparatus (330 rpm). The wells were then
emptied and washed five times using the washing buffer. Then 100 µL
of a 3,3',5,5'-tetramethylbenzidine solution (Kierkegaard & Perry) was
added to each well and incubated for 15 min in darkness on a mixing
apparatus (330 rpm). The color reaction was stopped by addition of 100
µL of 0.18 mol/L H2SO4 per well.
The absorbance was measured at 450 nm, with 650 nm as the reference
wavelength, using a microtiter plate reader (Emax Easy
Microtiter Reader, Molecular Devices).
known interfering substances
Interfering substances such as hemoglobin, bilirubin, lipids, and
ascorbic acid were added to serum samples in accordance to the
procedures described by Glick et al. (17). A stock solution
of hemoglobin was prepared from fresh hemolysate, whereas the remaining
substances were commercially available. Ditaurobilirubin, conjugated
bilirubin, was purchased from Porphyrin Products, Inc.;
IntraLipid®, 20% was from from Pharmacia & Upjohn A/S;
and ascorbic acid was from Merck.
assays for comparison studies
Urine CrossLaps ELISA.
The commercially available CrossLaps
ELISA from Osteometer BioTech A/S (Herlev, Denmark) was used for the
method comparison. The CrossLaps ELISA uses an antiserum (rabbit)
specific for the amino acid sequence AHD-ß-GGR to measure breakdown
products from C-terminal telopeptides of type I collagen in urine.
Creatinine was determined by Cobas Mira (Roche).
ICTP RIA.
The commercially available Telopeptide ICTP
[I] RIA from Orion Diagnostics was used for
head-to-head comparison of the clinical performance. The ICTP RIA uses
an antiserum from rabbits for the measurement in serum of the
carboxy-terminal cross-linked telopeptides of type I collagen.
participants
Blood samples from 65 premenopausal (38 ± 5 years, mean
± SD) and 169 postmenopausal (67 ± 7 years, mean ± SD)
women were used to determine the reference intervals. All the women
were healthy and were not receiving treatments known to affect calcium
metabolism.
Serum and urine samples (n = 638), originating from a more comprehensive study (Alexandersen et al., submitted for publication), were used for the method comparison study toward the urine CrossLaps ELISA. A subpopulation of 22 early postmenopausal women (57 ± 3 years, mean ± SD) undergoing hormone replacement therapy was selected for a head-to-head comparison between the Serum CrossLaps One Step ELISA and the ICTP RIA. Briefly, serum samples from postmenopausal women receiving either a sequential combination of 17ß-estradiol (2 mg) and norethisteronacetat (1 mg) (n = 10) or a placebo treatment (n = 12) were measured at baseline and after 12 months of therapy.
statistical methods
SAS Institute procedures were used for statistical analysis
(18). The significance of the mean difference between groups
was assessed using the Student t-test for unpaired data. The
significance of changes within groups was determined by the Student
t-test for paired data. To assess longitudinal changes, we
calculated the values for each group and expressed the changes as a
percentage of the initial values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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form of the peptide, CTX (data not shown).
Biotinylation.
Approximately 2 mol of BxNHS were incorporated
per mol of mAb F1103. In experiments where this ratio exceeded 10 mol
BxNHS/mol F1103, destruction of the IgG was observed (data not shown).
determination of the specificity
The HPLC separation of three major urinary degradation products
derived from the C-terminal telopeptide 1 chain of type I collagen
is illustrated in Fig. 1
. The detection by fluorescence revealed the presence of the
three fragments in similar quantities. The results from the
immunological profile indicate that the Serum CrossLaps One Step ELISA
is specific for the measurement of degradation fragments of C-terminal
telopeptide 1 chain of type I collagen, characterized by containing
cross-linked diisomerized ßCTX peptides (ßCTX-X-ßCTX). The two
other breakdown products, containing either no isomerization
(CTX-X-CTX) or one isomerization (CTX-X-ßCTX) were not
detected by the ELISA.
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investigation of the size of serum fragments
Fig. 2
shows that the majority of the degradation fragments of
C-terminal telopeptide 1 chain of type I collagen, which are found
in serum and can be measured with the Serum CrossLaps One Step ELISA,
have molecular masses in the range of 1000–10 000 Da. Additionally,
the data indicate that ~50% of the immunoreactive material has a
molecular mass <3000 Da.
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assay development and performance
The application of biotinylated mAb F1103 as the capture antibody
in streptavidin-coated microtiter wells, together with
peroxidase-conjugated mAb F12 in the Serum CrossLaps One Step ELISA,
yielded a calibration curve that was approximately linear in the range
500–16 000 pmol/L when plotted on a linear scale (Fig. 3
).
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The detection limit, defined as the concentration corresponding to 2 SD
above the mean of 21 determinations of the zero calibrator, was 80
pmol/L. The imprecision (CV) was assessed by the measurement of three
serum samples in 12 consecutive analytical runs and 21 determinations
of each of the three samples in the same analytical run; the overall CV
was <8% (Table 1
) .
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Serum samples from different populations (children, patients with renal
failure, and healthy adults) were used to evaluate the linearity of
serum samples. Samples were diluted in increments of 20% in calibrator
buffer; the sensitivity of the assay to any serum matrix effects was
then investigated (Table 2
). Because the mean observed:expected ratio after dilution was
101% ± 2% (mean ± SD), we concluded that there was no
interference from the serum matrix and that the assay detected
degradation products from type I collagen C-terminal telopeptides in
various serum samples with similar affinity. In support of this
observation, the mean observed:expected recovery ratio was found to be
101% ± 4% (mean ± SD; Table 3
). This result was obtained by mixing various calibrator
solutions containing CrossLaps antigens isolated from urine with
different serum samples in equal volumes. The mixtures were then
measured in the Serum CrossLaps One Step ELISA (Table 3
).
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Serum samples from 638 healthy subjects were assayed in the Serum
CrossLaps One Step ELISA, and the corresponding urine samples were
measured in the CrossLaps ELISA and corrected for creatinine. Although
different kinds of specimens were used, the two assays were highly
correlated (r = 0.856; Fig. 4
). Additionally, a high comparability was found between serum
and plasma samples taken in parallel. Both EDTA- and heparin-treated
plasma from nine volunteers correlated strongly with serum samples,
giving r values of 0.973 and 0.949, respectively (data not
shown).
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The stability during storage of the antigens measured in the Serum
CrossLaps One Step ELISA was examined. Sera from 10 healthy subjects
were isolated within 2 h of blood collection. Aliquots of serum
were incubated for various times at 4 °C and 20 °C and then
frozen at -20 °C. The mean recovery after 7 days of storage was
93% ± 11% and 60% ± 17% (mean ± SD) for 4 °C and
20 °C, respectively. The data indicate that the CrossLaps antigens
are very stable at 4 °C and resistant to ~2 days of storage at
room temperature (Fig. 5
).
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Another important issue for routine clinical settings is the stability of the antigens during repeated freezing and thawing of serum samples. Sera from seven healthy subjects were collected and exposed to seven freeze-thaw cycles over a period of 5 days, and the serum CrossLaps concentration was then measured after 1, 2, 3, 5, and 7 cycles. No significant change in the mean concentration could be observed. The recovery, expressed as a percentage of the initial value, was 97% ± 5% (mean ± SD) after cycle 7 (data not shown).
known interfering substances
Apparently, the concentrations of serum CrossLaps antigens were
not susceptible to the presence of well-known interferents. These
agents, known to be potential interferents in clinical chemistry
analyses, were added to serum samples. The highest concentrations
tested were as follows: ditaurobilirubin, 600 g/L; hemoglobin,
10 000g/L; IntraLipid, 1000 g/L; and ascorbic acid, 100 mg/L. The
serum concentrations were then measured in the Serum CrossLaps One Step
ELISA. No interference was found at any of the concentrations tested.
clinical performance
The individual values obtained in the Serum CrossLaps One Step
ELISA in groups of pre- and postmenopausal women are shown in Fig. 6
. An increase of 69% (P <0.001) in serum CrossLaps
concentrations was observed after menopause. The mean serum
concentration of CrossLaps was 1748 ± 740 pmol/L (mean ±
SD) in the premenopausal group and 2952 ± 1325 pmol/L (mean
± SD) in the postmenopausal group. The t-score between the
two groups (the difference between the mean of pre- and postmenopausal
concentrations, expressed in number of SD from the mean of the
premenopausal group) was 1.63.
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Serum samples from postmenopausal women undergoing hormone replacement
therapy were measured in the Serum CrossLaps One Step ELISA to assess
the ability of the assay for monitoring antiresorptive treatment (Fig. 7
). The mean serum concentration for the actively treated group
decreased after 12 months of therapy by ~75%, thereby returning to a
premenopausal concentration. Compared with baseline, the mean serum
concentration for the placebo-treated group was unchanged after 12
months and was significantly different from the corresponding
concentration of the actively treated group. In contrast, the ICTP RIA,
another serum assay measuring collagen fragments, lacked the capability
to detect a significant difference between the two groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-aspartate,
compared with other type I collagen-containing tissues. However, this
remains to be demonstrated by measurement of the degree of
ß-isomerization of collagen in various tissues and by determination
of the equilibrium constants for the spontaneously occurring
isomerization reaction.
Three types of breakdown products (ßCTX-X-ßCTX, CTX-X-ßCTX,
and CTX-X-CTX) liberated from the C-terminal telopeptide 1
chain of type I collagen were used to evaluate the binding specificity
of the Serum CrossLaps One Step ELISA. We found that this newly
developed ELISA procedure was specific for degradation fragments of
collagen type I characterized by the presence of cross-linked
diisomerized ßCTX peptides. This is in accordance with the inability
of the two mAbs used in the assay to measure the linear form of CTX.
The assay enables exclusive detection of the ßCTX-X-ßCTX fragments
derived from mature bone tissue. This would be relevant in a population
of relatively high age, such as the one investigated in this study,
where a large proportion of the collagen molecules in the bone matrix
is likely to be ß-isomerized.
The procedure applied in the present study to investigate the size of the CrossLaps-reactive antigens in serum provides a reasonable estimate of the molecular mass of the collagen fragments measured in the Serum CrossLaps One Step ELISA. The findings indicate that the masses of the detectable antigens are similar but slightly higher than the major degradation products quantified in the urinary CrossLaps ELISA, where the major immunoreactive fragments have molecular masses of ~2000 Da (15). This observation was supported by the data from the method comparison, where a relatively high correlation (r = 0.856) was found. These findings could suggest that the serum and urinary CrossLaps assays measure similar and related populations of antigens but also that differences in specificity are likely to exist. A more detailed study of CrossLaps antigens in serum is currently being performed to answer these questions.
Various studies have shown that the circulating concentrations of biochemical markers of bone resorption increase by age, mainly because of the change in menopausal status (22)(23). This was also shown in the present study, where the passing of menopause produced a 69% increase in mean serum concentration. Approximately 37% of the samples from the group of postmenopausal women were >3228 pmol/L, corresponding to the mean concentration of the premenopausal group plus 2 SD. It is likely that this subpopulation of postmenopausal women might belong to the group that is characterized by losing substantial amounts of bone mineral, "the fast bone losers" (24). This issue was further investigated in the study of Christgau et al. (25), where Serum CrossLaps One Step ELISA was applied, to assess the future bone loss in a population of postmenopausal women; the findings suggested that high concentrations of serum CrossLaps for early postmenopausal women were associated with accelerated future bone loss. Future studies should reveal if serum CrossLaps measurements early in the menopause also have a predictive value for later development of fractures in the skeleton.
One of the most important features for biochemical markers for bone turnover is their ability to monitor the effect of antiresorptive therapy, such as hormone replacement therapy. In serum samples from postmenopausal women, the Serum CrossLaps One Step ELISA detected a significant reduction to premenopausal concentrations after 12 months of therapy. This observation, together with the findings in the study of Christgau et al. (25), further supports that this new ELISA procedure reflects changes in bone metabolism known to occur during antiresorptive therapies.
Another serum assay for C-terminal telopeptide fragments of type I
collagen, the ICTP assay (8)(26), has been
described previously; this marker, however, has shown only a very
limited response to bisphosphonate (27)(28) and
hormone replacement therapy (29). This feature was also
demonstrated in the present study, where the ICTP RIA failed to detect
a significant effect of hormone replacement therapy after 12 months of
treatment. The epitope recognized by the ICTP antibodies has recently
been characterized and includes a phenylalanine-rich region of the
C-terminal telopeptides of two 1 chains located between the triple
helical domain and the lysine-derived trivalent pyridinoline/pyrrole
cross-linkers. The latter is not directly involved in the binding of
the ICTP antibodies, but the antibodies must connect to the three
constituent chains for successful detection (30). This
phenylalanine-rich region is located farther upstream from the
ß-isomerization-susceptible CTX epitope of the C-terminal
telopeptides. These data indicate that the ICTP assay measures another
population of antigens, which probably are larger than the fragments
detected by the CrossLaps assays.
We have described the development and characterization of a two-site
ELISA procedure that uses two mAbs for the quantitative determination
in serum of degradation products derived from the C-terminal
telopeptide 1 chain of type I collagen. Only fragments characterized
by the presence of cross-linked diisomerized ßCTX peptides
(ßCTX-X-ßCTX) were detected. This specificity ensures the specific
measurement of degradation fragments that derive from matured bone
tissue. The Serum CrossLaps One Step ELISA provides results in <2.5 h
and with high technical precision (CV <8%). Furthermore, the
CrossLaps antigen in serum is highly stable, being resistant to storage
for >24 h at room temperature or to more than seven repeated
freeze-thaw cycles. Additionally, this new ELISA procedure demonstrated
a relative high discriminatory power between populations of pre- and
postmenopausal women, as well as a high potential for monitoring the
effect of antiresorptive treatment for postmenopausal women.
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Acknowledgments |
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Footnotes |
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CTX, EKAHDGGR amino acid sequence; ßCTX, EKAHD-ß-GGR amino acid sequence; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BSA, bovine serum albumin; and BxNHS, biotinamidocaproate-N-hydroxysuccimide ester. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. M. Crofton, N. Evans, M. R.H. Taylor, and C. V. Holland Serum CrossLaps: Pediatric Reference Intervals from Birth to 19 Years of Age Clin. Chem., April 1, 2002; 48(4): 671 - 673. [Full Text] [PDF]
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 V. Parikka, P. Lehenkari, M.-L. Sassi, J. Halleen, J. Risteli, P. Harkonen, and H. K. Vaananen Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts Endocrinology, December 1, 2001; 142(12): 5371 - 5378. [Abstract] [Full Text] [PDF]
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F. Paglia, S. Dionisi, S. De Geronimo, R. Rosso, E. Romagnoli, N. Raejentroph, A. Ragno, M. Celi, J. Pepe, E. D'Erasmo, et al. Biomarkers of Bone Turnover after a Short Period of Steroid Therapy in Elderly Men Clin. Chem., July 1, 2001; 47(7): 1314 - 1316. [Full Text] [PDF]
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P Garnero, M Piperno, E Gineyts, S Christgau, P D Delmas, and E Vignon Cross sectional evaluation of biochemical markers of bone, cartilage, and synovial tissue metabolism in patients with knee osteoarthritis: relations with disease activity and joint damage Ann Rheum Dis, June 1, 2001; 60(6): 619 - 626. [Abstract] [Full Text] [PDF]
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P. Garnero, O. Borel, and P. D. Delmas Evaluation of a Fully Automated Serum Assay for C-Terminal Cross-Linking Telopeptide of Type I Collagen in Osteoporosis Clin. Chem., April 1, 2001; 47(4): 694 - 702. [Abstract] [Full Text] [PDF]
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P. Szulc, F. Munoz, B. Claustrat, P. Garnero, F. Marchand, F. Duboeuf, and P. D. Delmas Bioavailable Estradiol May Be an Important Determinant of Osteoporosis in Men: The MINOS Study J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 192 - 199. [Abstract] [Full Text]
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S. L. Greenspan, H. N. Rosen, and R. A. Parker Early Changes in Serum N-Telopeptide and C-Telopeptide Cross-Linked Collagen Type 1 Predict Long-Term Response to Alendronate Therapy in Elderly Women J. Clin. Endocrinol. Metab., October 1, 2000; 85(10): 3537 - 3540. [Abstract] [Full Text]
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